Download - Journal of Cell and Molecular Biology - Haliç Üniversitesi
Download - Journal of Cell and Molecular Biology - Haliç Üniversitesi
Download - Journal of Cell and Molecular Biology - Haliç Üniversitesi
Create successful ePaper yourself
Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong><br />
<strong>Molecular</strong> <strong>Biology</strong><br />
Volume 7 · No 2 & Volume 8 · No 1 · June 2010<br />
http://jcmb.halic.edu.tr<br />
Sensitivity <strong>of</strong> the human chromosomes to EMS<br />
Y chromosome microdeletions in spontaneous abortions<br />
Genetic diversity <strong>of</strong> Penicillium species
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong><br />
<strong>Molecular</strong> <strong>Biology</strong><br />
Volume 7 No 2 & Volume 8 No 1<br />
June 2010<br />
İstanbul-TURKEY
<strong>Haliç</strong> University<br />
Faculty <strong>of</strong> Arts <strong>and</strong> Sciences<br />
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong><br />
Founder<br />
Gündüz GEDİKOĞLU<br />
President <strong>of</strong> Board <strong>of</strong> Trustee<br />
Rights held by<br />
Sait SEVGENER<br />
Deputy Rector<br />
Correspondence Address:<br />
The Editorial Office<br />
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong><br />
<strong>Haliç</strong> <strong>Üniversitesi</strong><br />
Fen-Edebiyat Fakültesi,<br />
Kaptan Paşa Mah. Darülaceze Cad. No: 14 34384<br />
Okmeydanı-Şişli, İstanbul -Turkey<br />
Phone: +90 212 220 96 96<br />
E-mail: jcmb@halic.edu.tr<br />
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong> is<br />
indexed in ULAKBIM, EBSCO, DOAJ,<br />
EMBASE, CAPCAS, EM<strong>Biology</strong>, <strong>and</strong><br />
Index COPERNICUS<br />
ISSN 1303-3646<br />
Printed at MART Printing House<br />
Adil Meriç ALTINÖZ, İstanbul, Turkey<br />
Tuncay ALTUĞ, İstanbul, Turkey<br />
Canan ARKAN, Munich, Germany<br />
Aglaia ATHANASSIADOU, Patras, Greece<br />
Şehnaz BOLKENT, İstanbul, Turkey<br />
Nihat BOZCUK, Ankara, Turkey<br />
A. Nur BUYRU, İstanbul, Turkey<br />
Kemal BÜYÜKGÜZEL, Zonguldak, Turkey<br />
H<strong>and</strong>e ÇAĞLAYAN, İstanbul, Turkey<br />
İsmail ÇAKMAK, İstanbul, Turkey<br />
Adile ÇEVİKBAŞ, İstanbul, Turkey<br />
Beyazıt ÇIRAKOĞLU, İstanbul, Turkey<br />
Fevzi DALDAL, Pennsylvania, USA<br />
Zihni DEMİRBAĞ, Trabzon, Turkey<br />
Mustafa DJAMGÖZ, London, UK<br />
Aglika EDREVA, S<strong>of</strong>ia, Bulgaria<br />
Ünal EGELİ, Bursa, Turkey<br />
Advisory Board<br />
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong><br />
<strong>Molecular</strong> <strong>Biology</strong><br />
Published by<br />
<strong>Haliç</strong> University<br />
Faculty <strong>of</strong> Arts <strong>and</strong> Sciences<br />
Editor<br />
Nagehan ERSOY TUNALI<br />
Associate Editor<br />
Mehmet Ali TÜFEKÇİ<br />
Editorial Board<br />
Baki YOKEŞ<br />
Burcu Irmak YAZICIOĞLU<br />
Kürşat ÖZDİLLİ<br />
Aslı BAŞAR<br />
Deniz ERGEÇ, İstanbul, Turkey<br />
Nermin GÖZÜKIRMIZI, İstanbul, Turkey<br />
Ferruh ÖZCAN, İstanbul, Turkey<br />
Füsun GÜMÜŞEL, İstanbul, Turkey<br />
C<strong>and</strong>an JOHANSEN, İstanbul, Turkey<br />
Asım KADIOĞLU, Trabzon, Turkey<br />
Maria V. KALEVITCH, Pennsylvania, USA<br />
Valentine KEFELİ, Pennsylvania, USA<br />
Meral KENCE, Ankara, Turkey<br />
Uğur ÖZBEK, İstanbul, Turkey<br />
Sevtap SAVAŞ, Toronto, Canada<br />
Müge TÜRET SAYAR, İstanbul, Turkey<br />
İsmail TÜRKAN, İzmir, Turkey<br />
Mehmet TOPAKTAŞ, Adana, Turkey<br />
Meral ÜNAL, İstanbul, Turkey<br />
Selma YILMAZER, İstanbul, Turkey<br />
Ziya ZİYLAN, İstanbul, Turkey
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong><br />
CONTENTS Volume 7 No 2 & Volume 8 No 1 June 2010<br />
Review Article<br />
DNA repetitive sequences-types, distribution <strong>and</strong> function: A review<br />
S.R. RAO, S. TREVEDI, D. EMMANUEL, K. MERITA <strong>and</strong> M. HYNNIEWTA<br />
Research Articles<br />
Genetic diversity <strong>of</strong> Penicillium species isolated from various sources in Sarawak,<br />
Malaysia<br />
H.A. ROSLAN, C.S. NGO <strong>and</strong> S. MUID<br />
The sensitivity <strong>of</strong> the human chromosomes to ethyl methanesulfonate (EMS)<br />
S. BUDAK-DİLER <strong>and</strong> M. TOPAKTAŞ<br />
Protective effect <strong>of</strong> pomegranate peel ethanol extract against ferric nitrilotriacetate<br />
induced renal oxidative damage in rats<br />
M.M. AHMED <strong>and</strong> S.E. ALI<br />
<strong>Molecular</strong> <strong>and</strong> cytogenetic evaluation <strong>of</strong> Y chromosome in spontaneous abortion cases<br />
G. KOÇ, K. ULUCAN, D. KIRAÇ, D. ERGEÇ, T. TARCAN <strong>and</strong> A.İ. GÜNEY<br />
Do simple sequence repeats in replication, repair <strong>and</strong> recombination genes <strong>of</strong><br />
mycoplasmas provide genetic variability?<br />
S. TRIVEDI<br />
S<strong>of</strong>tware Review<br />
Tc<strong>of</strong>fee ©: Multipurpose sequence alignments program<br />
A. MANSOUR<br />
UCSC: Genome Browser for genomic sequences<br />
A. MANSOUR<br />
Dotlet©: powerful <strong>and</strong> easy strategy for pairwise comparisons<br />
A. MANSOUR<br />
Instructions for Authors 85<br />
1<br />
13<br />
25<br />
35<br />
45<br />
53<br />
71<br />
75<br />
81
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong> 7(2) & 8(1): 1-11, 2010 Review Article<br />
<strong>Haliç</strong> University, Printed in Turkey.<br />
http://jcmb.halic.edu.tr<br />
DNA repetitive sequences-types, distribution <strong>and</strong> function: A review<br />
Satyawada Rama RAO *,1 , Seema TRIVEDI 2 , Deepika EMMANUEL 2 , Keisham MERITA 1<br />
<strong>and</strong> Marlykynti HYNNIEWTA 1<br />
1 Cytogenetics <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong> Laboratory, Department <strong>of</strong> Biotechnology <strong>and</strong> Bioinformatics, North-<br />
Eastern Hill University, Permanent Campus, Mawkynroh, Umnsing,<br />
Shillong- 793022, Meghalaya (INDIA)<br />
2 Department <strong>of</strong> Zoology, Jai Narain Vyas University, Jodhpur- 342005, Rajasthan (INDIA)<br />
(* author for correspondence; srrao22@yahoo.com)<br />
Received: 21 September 2009; Accepted: 14 May 2010<br />
Abstract<br />
The development <strong>and</strong> use <strong>of</strong> molecular markers for the detection <strong>and</strong> exploitation <strong>of</strong> DNA polymorphism is<br />
one <strong>of</strong> the most significant developments in the field <strong>of</strong> molecular genetics. DNA based molecular markers<br />
have acted as versatile tools <strong>and</strong> have found their own position in various fields like taxonomy, physiology,<br />
embryology, genetic engineering etc. A major step forward in genetic identification is the discovery that<br />
about 30-90% <strong>of</strong> the genome is constituted by regions <strong>of</strong> repetitive DNA which are highly polymorphic in<br />
nature. Microsatellites are multilocus probes creating complex b<strong>and</strong>ing patterns <strong>and</strong> are usually non-species<br />
specific occurring ubiquitously. They form an ideal marker system <strong>and</strong> are dominant fingerprinting markers<br />
<strong>and</strong> co-dominant STMS (sequence tagged microsatellites) markers. Microsatellites markers have been used<br />
successfully to determine the degree <strong>of</strong> relatedness among individuals or groups <strong>of</strong> accessions to clarify the<br />
genetic structure or partitioning <strong>of</strong> variation among individuals, accessions, populations <strong>and</strong> species.<br />
Repetitive sequences have been widely used for examining genome <strong>and</strong> species relationships by in situ <strong>and</strong><br />
by Southern hybridization.<br />
Keywords: Satellites, microsatellites, minisatellites, retroposons <strong>and</strong> proretroviral transposons<br />
Tekrarlı DNA dizileri-tipleri, dağılımları ve fonksiyonları<br />
Özet<br />
DNA polimorfizmlerinin tayini ve kullanılması için moleküler belirteçlerin geliştirip kullanılması moleküler<br />
genetik alanındaki en önemli ilerlemelerden bir tanesidir. DNA tabanlı moleküler belirteçler çok amaçlı<br />
kullanım araçlarıdır ve taksonomi, fizyoloji, embriyoloji, genetik mühendisliği gibi çeşitli alanlar arasında<br />
kendi yerlerini bulmuşlardır. Genetik tayine doğru en büyük adım, genomun hemen hemen %30-90’ının<br />
tekrarlanan, doğada yüksek or<strong>and</strong>a polimorfik olan DNA dizilerinden oluştuğunun keşfidir. Mikrosatelitler<br />
kompleks şerit paterni oluşturan multilokus problardır ve genellikle sıkça bulunup türe spesifik olmazlar.<br />
Bunlar ideal belirteç sistemini oluştururlar ve dominant parmakizi belirteçleri ve kodominant STMS<br />
belirteçleridirler (sequence tagged microsatellites–dizi işaretli mikrosatelitler). Mikrosatellit belirteçler,<br />
bireyler arasında veya katılan gruplar arasında genetik yapının ya da bireyler, gruplar, populasyonlar ve türler<br />
arasındaki varyasyonun grupl<strong>and</strong>ırılmasının aydınlatılması için, yakınlık derecesinin saptanması amaçlı<br />
olarak başarı ile kullanılmıştır. Tekrarlanan diziler, genom ve türlerin ilişkilerinin in situ ve Southern<br />
hibridizasyonu ile incelenmesi için yaygın olarak kullanılmaktadır.<br />
Anahtar Sözcükler: Satelit, mikrosatelitler, minisatelitler, retropozonlar ve proretroviral transpozonlar
2<br />
Satyawada Rama RAO et. al.<br />
Introduction<br />
The analysis <strong>of</strong> genetic diversity <strong>and</strong> relatedness<br />
between or within different species, populations<br />
<strong>and</strong> individuals is a central task for many<br />
disciplines <strong>of</strong> biological science. Classical<br />
strategies <strong>of</strong> evaluating genetic variability are<br />
comparative anatomy, morphology, embryology<br />
<strong>and</strong> physiology. These are complemented by<br />
analysis <strong>of</strong> chemical constituents like plant<br />
secondary compounds or with specific characterization<br />
<strong>of</strong> macromolecules <strong>and</strong> allozymes. In recent<br />
years, focus has been shifted to the development <strong>of</strong><br />
molecular markers based on DNA or protein<br />
polymorphism. The importance <strong>of</strong> these studies lies<br />
in exploitation <strong>of</strong> uniqueness <strong>of</strong> DNA sequences<br />
that facilitate research in diverse disciplines such as<br />
taxonomy, phylogeny, ecology, genetics <strong>and</strong> plant<br />
breeding.<br />
Establishing an individual's identity is one <strong>of</strong><br />
the uses <strong>of</strong> DNA sequence information that<br />
highlight uniqueness <strong>of</strong> a particular sample. The<br />
methodology focuses on ways to reduce complexity<br />
<strong>of</strong> DNA into simple patterns that are representative<br />
<strong>of</strong> the sample. This type <strong>of</strong> analysis is called<br />
fingerprinting, pr<strong>of</strong>iling, genotyping or identity<br />
testing. Jeffreys et al. (1985) introduced this term to<br />
describe a method for the simultaneous detection <strong>of</strong><br />
variable DNA loci by hybridization <strong>of</strong> specific<br />
multilocus probes with electrophoretically separated<br />
restriction fragments. DNA fingerprinting is<br />
useful for forensic identification, determination <strong>of</strong><br />
family relationship, linkage mapping, antenatal<br />
diagnosis, localization <strong>of</strong> disease loci, determination<br />
<strong>of</strong> genetic variation, molecular archaeology<br />
<strong>and</strong> epidemiology (Watkins, 1988; Donis-Keller et<br />
al., 1987; L<strong>and</strong>egren et al., 1988; Paabo, 1989;<br />
Golenberg et al., 1990). <strong>Molecular</strong> markers have<br />
been used for identification <strong>of</strong> individuals, clones,<br />
close relatives, paternity testing or in studies <strong>of</strong><br />
reproductive behavior <strong>and</strong> mating success.<br />
Repetitive sequences as molecular markers<br />
A repeat is recurrence <strong>of</strong> a pattern whereby DNA<br />
exhibits recurrence <strong>of</strong> many features. The number<br />
<strong>of</strong> occurrences <strong>of</strong> a pattern is called copy number.<br />
The number <strong>of</strong> copies in a particular t<strong>and</strong>em repeat<br />
region is termed region copy number. The term<br />
genome copy number refers to number <strong>of</strong> copies <strong>of</strong><br />
t<strong>and</strong>em or interspersed repeats in genome.<br />
The repetitive DNA family(ies) may be widely<br />
distributed in a taxonomic family or a genus, or<br />
may be specific for a species or chromosome.<br />
Repeats may occur in specific locations in a<br />
genome, e.g. in telomeric regions or scattered<br />
throughout the genome. They may acquire large<br />
scale variation in the sequence <strong>and</strong> copy number<br />
over evolutionary time-scale. The repetitive<br />
elements are under different evolutionary constraints<br />
as compared to the genes. Hybrid<br />
polyploids are excellent models for studying<br />
evolution <strong>of</strong> repetitive sequences (Kubis et al.,<br />
1998). These variations are the basis <strong>of</strong> utilization<br />
<strong>of</strong> repetitive sequences for taxonomic <strong>and</strong><br />
phylogenetic studies (Smith <strong>and</strong> Flavell, 1974).<br />
There are many classifications <strong>of</strong> repetitive<br />
DNA based on characteristics measured by<br />
different techniques but consolidation <strong>of</strong> these<br />
systems defines five broad classes: satellites,<br />
microsatellites <strong>and</strong> minisatellites, retroposons <strong>and</strong><br />
proretroviral transposons. The classification<br />
scheme makes a distinction between repetitive<br />
regions exhibiting t<strong>and</strong>em repetition <strong>and</strong> interspersed<br />
repetition but is not precise since each class<br />
retains the characteristics <strong>of</strong> both. Some <strong>of</strong> these<br />
repeats are described as follows:<br />
Moderately repetitive DNA includes reiterations<br />
<strong>of</strong> genes like tRNA, rRNA, hemoglobin etc. that<br />
retain similar or nearly similar sequences due to<br />
duplication. Some <strong>of</strong> these duplications result in<br />
pseudogenes <strong>and</strong> may have many copies in the<br />
genome. Some repetitive DNA sequences are<br />
transposable elements since they ct not to enhance<br />
the success <strong>of</strong> the cell (or organism) they reside in,<br />
behave selfishly <strong>and</strong> also accumulate to the levels<br />
restricted only by the resources available to them.<br />
The selfish DNA hypothesis <strong>of</strong> Doolittle <strong>and</strong><br />
Sapienza, (1980) assumes that repetitive DNA can<br />
behave in a selfish manner because it is not<br />
functional. Indeed, there is some evidence that its<br />
presence can result in losses <strong>of</strong> fitness <strong>of</strong> the host<br />
cell due to mutations caused by transposable<br />
elements. However, some moderately-repetitive<br />
DNA has functions for example, in directing<br />
chromosome movement in eukaryotes (Vogt,<br />
1990). Variations in selfish DNA have the potential<br />
for evolutionary changes, especially when it<br />
changes without having any deleterious effects on<br />
the organism (Flavell et al., 1977). Susumo Ohno<br />
(1970) asserted that "natural selection merely<br />
modified while redundancy created". Duplication<br />
<strong>of</strong> genes can thus be internal source <strong>of</strong> novelty in
the genome. If repetitive DNA is transposable, it<br />
may create novel genes. Repetitive DNA is<br />
therefore the "Research & Development"<br />
laboratory <strong>of</strong> genome, creating both redundancy<br />
<strong>and</strong> novel sequences that may prove valuable for<br />
genome. However, these repetitive sequences are<br />
generally not used for DNA fingerprinting.<br />
T<strong>and</strong>em <strong>and</strong> interspersed repeats<br />
T<strong>and</strong>em repetitions are consecutive head-to-tail,<br />
direct, repetition <strong>of</strong> a pattern due to local<br />
duplication. Interspersed repetitions are recurrence<br />
<strong>of</strong> patterns that may or may not be proximal,<br />
formed by either non-local duplication or multiple<br />
introductions <strong>of</strong> the same or similar extraneous<br />
DNA segments. These repeats are dispersed<br />
throughout the genome <strong>and</strong> have no restriction on<br />
the relative positions <strong>of</strong> identical occurrences<br />
occurring in t<strong>and</strong>em locations. Research indicates<br />
that interspersed repeats are inserts since they<br />
resemble either processed RNAs i.e. retroposons, or<br />
viruses i.e. proretroviral transposons. In addition, a<br />
suspected target sequence for insertion occurs at<br />
both ends <strong>of</strong> these repeats as expected for a circular<br />
DNA crossover insertion. Furthermore, some<br />
repeats actively move within the genome, such as<br />
jumping genes in maize.<br />
DNA repeat patterns also classify as direct,<br />
indirect, complement, reverse complement or<br />
palindrome. A direct or forward repeat is the<br />
.<br />
DNA repetitive sequences<br />
recurrence <strong>of</strong> a pattern on the same str<strong>and</strong> in the<br />
same nucleotide order; e.g. ACCG recurs as<br />
ACCG. An indirect, inverse or reverse repeat recurs<br />
on the same str<strong>and</strong> but the order <strong>of</strong> the nucleotides<br />
is reverse, e.g. the indirect recurrence <strong>of</strong> ACCG is<br />
GCCA. Complement repeats are repeats where the<br />
nucleotides are complemented according to Watson<br />
Crick pairing, e.g. the complement <strong>of</strong> ACCG is<br />
TGGC. A reverse complement repeat recurs on the<br />
same str<strong>and</strong> but, the nucleotides are complemented<br />
<strong>and</strong> the order <strong>of</strong> the nucleotides is reversed; e.g. the<br />
reverse complement <strong>of</strong> ACCG is CGGT. In DNA,<br />
most repetitions occur as forward or reverse<br />
complement repeats <strong>and</strong> rarely as reverse or<br />
complement repeats (Grumbach <strong>and</strong> Tahi, 1994).<br />
Palindrome is a combination <strong>of</strong> two consecutive<br />
occurrences in opposite orientations <strong>and</strong> read the<br />
same when read from left to right or vice-versa.<br />
Repetitive DNA sequences, divided into highrepeat<br />
satellite DNA which replicates thous<strong>and</strong>s or<br />
millions <strong>of</strong> times <strong>and</strong> "moderate-repeat" minisatellite<br />
<strong>and</strong> microsatellite DNA which replicates<br />
tens to perhaps a thous<strong>and</strong> times, account for<br />
varying proportions <strong>of</strong> the genome <strong>of</strong> multicellular<br />
eukaryotes. An example <strong>of</strong> representa-tive data<br />
from eukaryotes has been given in Table 1.<br />
Prokaryotes contain little or no repetitive<br />
sequences. Noncoding repetitive DNA varies from<br />
one group <strong>of</strong> organisms to another; individual to<br />
individual <strong>and</strong> therefore used as DNA fingerprinting<br />
tool.<br />
Table 1. Proportion <strong>of</strong> repetitive sequences <strong>of</strong> genomic DNA in different eukaryotes.<br />
High repeat<br />
Moderate repeat<br />
Non repetitive<br />
T<strong>and</strong>em repeats <strong>and</strong> Satellite DNA<br />
Drosophila Xenopus Mouse Tobacco<br />
13%<br />
13%<br />
74%<br />
As repeats were discovered in different locations<br />
exhibiting different copy numbers, new terms arose<br />
such as satellite, minisatellite <strong>and</strong> microsatellite.<br />
Some researchers refer to all types <strong>of</strong> satellites as<br />
t<strong>and</strong>em repeats <strong>and</strong> describe a specific t<strong>and</strong>em<br />
repeat region according to its location within the<br />
genome, its periodicity, pattern structure <strong>and</strong> copy<br />
3%<br />
43%<br />
54%<br />
10%<br />
20%<br />
70%<br />
5%<br />
65%<br />
30%<br />
number. These repeats were first identified on a<br />
cesium chloride buoyant density gradient as peaks<br />
separate from the primary DNA peak. The separate<br />
or satellite peaks were composed <strong>of</strong> array <strong>of</strong> highly<br />
conserved t<strong>and</strong>em repeats localized to heterochromatic<br />
regions <strong>of</strong> chromosomes like<br />
centromeres (Schueler et al., 2001). The structure<br />
<strong>of</strong> a t<strong>and</strong>em repeat region has well-conserved<br />
3
4<br />
Satyawada Rama RAO et. al.<br />
pattern but varies in size from less than 20 bp to<br />
several thous<strong>and</strong> bp.<br />
Structural <strong>and</strong> functional roles<br />
T<strong>and</strong>em repeats play significant structural <strong>and</strong><br />
functional roles. They occur in abundance in<br />
structural areas such as telomeres, centromeres <strong>and</strong><br />
histone binding regions. They play a regulatory role<br />
near genes <strong>and</strong> perhaps even within genes.<br />
Transcription<br />
The precise role <strong>of</strong> t<strong>and</strong>em repeats in transcription<br />
regulation is not known. Since nucleosomes can<br />
repress or enhance transcription initiation <strong>and</strong><br />
elongation (Hartzog <strong>and</strong> Winston, 1997; Kornberg<br />
<strong>and</strong> Lorch, 1999) repeats may influence<br />
transcription by affecting nucleosome positioning<br />
<strong>and</strong> stability. Tighter bonds between the histone<br />
complex <strong>and</strong> repeats restrict access for RNA<br />
polymerase <strong>and</strong> regulatory proteins (Dai &<br />
Rothman-Denes, 1999). This may happen by<br />
changing the degree <strong>and</strong> direction <strong>of</strong> DNA<br />
supercoiling or forming alternative DNA structures<br />
such as cruciforms <strong>and</strong> hairpins (Shlyakntenko et<br />
al., 1998; Ohyama, 2001). T<strong>and</strong>em repeats having<br />
an alternating purine (R=A or G) pyrimidine (Y=C<br />
or U/T) pattern forms Z-DNA (Yang et al., 1996)<br />
<strong>and</strong> repeats with a RRY or a YRY pattern form<br />
triplex DNA structures (Grabcyzk <strong>and</strong> Usdin,<br />
2000). The degree <strong>of</strong> repression is directly<br />
proportional to repeat length.<br />
Centromeric <strong>and</strong> subtelomeric satellite DNA<br />
families<br />
The t<strong>and</strong>em satellite DNA sequences exhibit<br />
characteristic chromosomal locations, usually at<br />
subtelomeric (or intercalary repetitive sequences)<br />
<strong>and</strong> centromeric regions (Heslop-Harrison et al.,<br />
2003; Jiang et al., 2003). Satellite DNA families<br />
may arise de novo due to molecular mechanisms<br />
like unequal crossing over, rolling circle<br />
amplification, replication slippage <strong>and</strong> mutation.<br />
Satellite DNA have variable repeat unit length<br />
(sometimes equivalent to micro or minisatellite<br />
length), <strong>of</strong>ten forming arrays spanning up to 100<br />
Mb (Charlesworth et al., 1994; Kubis et al., 1998;<br />
Schmidt <strong>and</strong> Heslop-Harrison, 1998; Vergnaud <strong>and</strong><br />
Denoeud, 2000). However, satellite repeat<br />
monomer lengths <strong>of</strong> 140 – 180 bp <strong>and</strong> 300 – 360<br />
bp, corresponding to the length <strong>of</strong> the mono <strong>and</strong><br />
dinucleosomes are most the common (Hemleben,<br />
1990; Traut, 1991; Macas et al., 2002).<br />
Centromeric t<strong>and</strong>em repeats ranging from 150-<br />
200 bp in length (Henik<strong>of</strong>f et al., 2001) are<br />
essential components <strong>of</strong> a functional centromere. A<br />
functional centromere has been defined as the DNA<br />
sequence which interacts with the kinetochore<br />
where the interaction between centromere-kinetochore<br />
appears to be mediated by DNA-protein<br />
recognition process (Jiang et al., 2003). The core<br />
sufficient for centromeric function is an alpha<br />
satellite about 3 Mbp long having a 171 bp pattern<br />
recurring in a t<strong>and</strong>em fashion. (Schueler et al.,<br />
2001; Zhong et al., 2002).<br />
A highly repetitive 180 bps centromeric satellite<br />
DNA family constituting between 2-5% <strong>of</strong> the<br />
Arabidopsis thaliana genome is the key component<br />
<strong>of</strong> its centromere/kinetochore complex (Nagaki et<br />
al., 2003a,b). These repeats are occasionally<br />
interrupted by the Athila retrotransposons, although<br />
the latter are mainly clustered in pericentromeric<br />
regions (Heslop-Harrison et al., 1999; Nagaki et al.,<br />
2003a,b). Similarly, centromeric DNA in several<br />
plants species including rice, maize, wheat, Beta<br />
species <strong>and</strong> Zingeria biebersteiniana mainly<br />
contain satellite sequence repeats <strong>and</strong> retrotransposons<br />
(Gindullis et al., 2001; Kishii et al.,<br />
2001; Kumekawa et al., 2001; Saunders <strong>and</strong><br />
Houben, 2001; Cheng et al., 2002; Nagaki et al.,<br />
2003a,b). A high monomer divergence is observed<br />
within several centromeric repetitive DNA families<br />
thereby indicating presence <strong>of</strong> chromosome<br />
specific variant sequences (Harrison <strong>and</strong> Helsop-<br />
Harrison, 1995; Nagaki et al., 1998; Helsop-<br />
Harrison et al., 2003). For example, chromosome<br />
specific 180 bp satellite repeat variants in<br />
Arabidopsis thaliana may be explained by the<br />
possibility that either the repeat sequences on each<br />
chromosome have been homogenizes independently<br />
or specific variants <strong>of</strong> the satellite sequence have<br />
been amplified on each chromosome (Heslop-<br />
Harrison et al., 1999).<br />
The subtelomeric regions also contain repetitive<br />
sequences (review in Pryde et al., 1997). Not all<br />
species have the same structure but all have<br />
structures containing t<strong>and</strong>em repeats, interspersed<br />
repeats or both (Pryde et al., 1997). Degenerate<br />
TTAGGG repeats enable alignment other subtelomeric<br />
regions allowing sequence exchange<br />
between subtelomeres (Flint et al., 1997).
Minisatellite <strong>and</strong> Microsatellite DNA<br />
Hypervariable regions, also known as variable<br />
number <strong>of</strong> t<strong>and</strong>em repeats (VNTRs) classified as<br />
minisatellites <strong>and</strong> microsatellites are regions that<br />
contain a variable copy number. These repeats are<br />
found throughout the genome (Vogt, 1990) but<br />
rarely within genes. Most regions contain short to<br />
moderate region copy number (Jeffreys, 1985).<br />
DNA fingerprinting capitalizes on the differences<br />
between alleles at specific VNTR loci. Various<br />
human diseases are attributed to high copy numbers<br />
associated with some VNTR locus.<br />
Minisatellites are characterized by moderate<br />
length patterns, usually less than 50 bp (Jeffreys,<br />
1985) with an array <strong>of</strong> 0.5 - 30kb. Two types <strong>of</strong><br />
variability are observed, viz., one displays copy<br />
number variation with each replication event<br />
whereas the other displays distinct alleles within a<br />
population such that different alleles contain<br />
different copy numbers.<br />
Microsatellites, also known as simple sequence<br />
repeats (SSRs) or simple t<strong>and</strong>em repeats (STRs)<br />
have a short well-conserved pattern length <strong>of</strong> 2 to 6<br />
bp <strong>and</strong> region copy number <strong>of</strong> 10 to 40 pattern<br />
copies. Microsatellites have been found in noncentromeric<br />
regions, many <strong>of</strong> them being located<br />
either near or within genes.<br />
Automatic identification <strong>and</strong> characterization <strong>of</strong><br />
t<strong>and</strong>em repeats is crucial as genome projects<br />
generate an ever-increasing quantity <strong>of</strong> sequence<br />
data. T<strong>and</strong>em repeats increase the complexity <strong>of</strong><br />
genome sequence analysis algorithms. For instance,<br />
the process <strong>of</strong> generating full chromosome<br />
sequences <strong>of</strong>ten utilizes the sequence assembly<br />
procedure; a procedure that stitches short, similar<br />
fragments together to reconstruct a larger sequence.<br />
The consecutive recurrence <strong>of</strong> a pattern associated<br />
with t<strong>and</strong>em repeats confuses this process. Some<br />
commercially available algorithms avoid<br />
assembling t<strong>and</strong>em repeat regions. Others <strong>of</strong>ten<br />
assemble moderate-sized t<strong>and</strong>em repeat regions<br />
improperly. At present, algorithms are being<br />
developed for h<strong>and</strong>ling t<strong>and</strong>em repeat regions.<br />
The mechanism responsible for minisatellite <strong>and</strong><br />
simple sequence polymorphisms<br />
Minisatellites <strong>and</strong> simple sequences are <strong>of</strong>ten<br />
characterized by high mutation rates (up to 5%),<br />
which may involve either internal heterogeneity <strong>of</strong><br />
repeats or their number. Mutation rates also show<br />
positive correlation with the total size <strong>of</strong> the array<br />
DNA repetitive sequences<br />
<strong>of</strong> repeats. In accordance with these observations,<br />
high molecular weight b<strong>and</strong>s within a multilocus<br />
fingerprint are <strong>of</strong>ten more variable than b<strong>and</strong>s<br />
occurring in the low molecular weight range. The<br />
molecular basis <strong>of</strong> both minisatellites <strong>and</strong> simple<br />
sequence variability is still debatable. Possible<br />
mechanism include replication slippage, transposition,<br />
recombinational events <strong>and</strong>/or unequal<br />
exchange between sister chromatids or between<br />
homologous chromosomes <strong>and</strong> gene conversion<br />
(reviewed by Jarman <strong>and</strong> Wells, 1989; Jeffreys et<br />
al., 1990; Richards <strong>and</strong> Sutherl<strong>and</strong> 1992; Wolff et<br />
al., 1991.)<br />
The slippage hypothesis implicates mispairing<br />
<strong>of</strong> slipped-str<strong>and</strong> during the replication process.<br />
Str<strong>and</strong> slippage may happen due to shift in origin <strong>of</strong><br />
replication especially during lagging str<strong>and</strong><br />
synthesis. Str<strong>and</strong> slippage <strong>and</strong> mismatch appear to<br />
be nucleotide specific. Differential activities <strong>of</strong><br />
mismatch pair <strong>of</strong> (CAG)n repeats occur but not <strong>of</strong><br />
(CTG)n repeats. Certain factors like the length <strong>of</strong><br />
the repeats <strong>and</strong> replication direction play a role in<br />
destabilizing (CAG)n (CTG)n repeat. Such<br />
positioning effects results in loop formation due to<br />
st<strong>and</strong> slippage <strong>and</strong> results in expansion or reduction<br />
<strong>of</strong> repeat during replication.<br />
Several lines <strong>of</strong> evidence have lent support to<br />
the recombination hypothesis:<br />
• A variety <strong>of</strong> minisatellite core sequences<br />
share homology <strong>of</strong> the bacterial recombination<br />
signal chi.<br />
• Minisatellite - like sequences have been<br />
found at sites <strong>of</strong> meiotic crossing over.<br />
• Both minisatellite <strong>and</strong> macrosatellites<br />
behave as recombinational hot spots in<br />
transfected mammalian cells.<br />
Wolff et al., (1991) observed no exchange <strong>of</strong><br />
flanking markers in a newly created minisatellite<br />
allele, thus ruling out unequal exchange between<br />
homologous chromosomes as a mutational mechanism.<br />
In human minisatellite locus, MS32<br />
(reviewed by Jeffreys et al., 1985), 5’ end <strong>of</strong> the<br />
array has a strong mutation bias, suggesting<br />
existence <strong>of</strong> a mutational hot spot. Some mutant<br />
alleles contain segments from both parental alleles,<br />
providing evidence for interallelic exchange. It is<br />
suggested that the major mutational process<br />
involves nonreciprocal transfer <strong>of</strong> repeats from a<br />
donor allele to the 5’ end <strong>of</strong> a recipient allele.<br />
Therefore, recombinational processes as well as<br />
replication slippage may contribute to the creation<br />
<strong>of</strong> minisatellite <strong>and</strong> simple sequence variability.<br />
However, other (yet unidentified) mechanisms may<br />
5
6<br />
Satyawada Rama RAO et. al.<br />
also be involved, especially in case <strong>of</strong> the explosive<br />
amplification <strong>of</strong> microsatellite like trinucleotide<br />
repeats associated with human genetic diseases <strong>and</strong><br />
polymorphism. Structural analysis <strong>of</strong> mutated vs.<br />
parental alleles may help to gain more information<br />
about the mutational mechanisms. In this respect,<br />
transgenic systems will be informative, since<br />
successive deletion <strong>of</strong> the flanking DNA will allow<br />
precise location <strong>of</strong> mutational hot spots.<br />
Retroposons<br />
Retroposons resemble processed RNAs <strong>and</strong><br />
transpose passively via RNA intermediate (Weiner,<br />
1986). Each element is composed <strong>of</strong> an A-rich tail<br />
at the 3' end <strong>and</strong> short target site duplications<br />
(direct repeats <strong>of</strong> 5-21 bp) flanking the repeat<br />
(Rabin, 1985). Two main subclasses dominate this<br />
class:<br />
Short Interspersed Elements (SINEs)<br />
These are distributed throughout the non-<br />
centromeric regions <strong>of</strong> genome (over 100,000<br />
copies per genome) (Weiner, 1986). A SINE<br />
contains one or more RNA polymerase III,<br />
promoter sites <strong>and</strong> an A-rich region. One subfamily<br />
is composed <strong>of</strong> a head-to-tail catenation <strong>of</strong> two<br />
promoter site, A-rich region pairs (Weiner, 1986).<br />
Both subfamilies are flanked by short direct repeats<br />
<strong>of</strong> 5 to 21 bp. Primate specific Alu sequence (5 to 9<br />
kbp) is a SINE with two promoter sites <strong>and</strong> a<br />
dimer. The uniqueness <strong>of</strong> Alu sequences provides a<br />
wonderful tool for separating primate DNA from<br />
that <strong>of</strong> other species. SINEs present challenges to<br />
sequence assembly due to their high genome copy<br />
number (300,000 to 500,000 copies) (Rogers,<br />
1985).<br />
Long Interspersed Elements (LINEs)<br />
LINEs are composed open reading frames (ORFs)<br />
followed by a 3' A-rich region having 20,000 to<br />
50,000 copies per genome (Hutchison et al., 1989;<br />
Weiner, 1986). Direct repeats <strong>of</strong> 6-15 bp flank the<br />
element. L1 family (primary LINE family) is 6 to 7<br />
kbp long. The consensus structure <strong>of</strong> the family is<br />
well defined but not well conserved because L1<br />
element can deviate significantly from the structure<br />
such that entire structural components are deleted<br />
or duplicated (Weiner, 1986).<br />
Proretroviral transposons<br />
Proretroviral transposons are mobile elements that<br />
transpose via RNA intermediate (Varmus <strong>and</strong><br />
Brown, 1989). Their structure <strong>and</strong> content<br />
resembles integrated viruses <strong>and</strong> <strong>of</strong>ten contain<br />
genes encoding viral products, e.g. protease,<br />
reverse transcriptase <strong>and</strong> integrase (Boefe <strong>and</strong><br />
Corces, 1989). The LTRs contain transcriptional<br />
signals for initiating <strong>and</strong> terminating transcripts, a<br />
promoter, an enhancer <strong>and</strong> a polyadenylation signal<br />
(Temin, 1985; Schmid et al., 1990). Inverse repeats<br />
exist at the ends <strong>of</strong> each LTR <strong>and</strong> always begin<br />
with the bases, TG, <strong>and</strong> end with CA (Temin,<br />
1985). The two LTRs <strong>and</strong> the genes are flanked by<br />
4 to 6 bp direct repeats.<br />
Other recurring genetic features<br />
DNA contains many recurring features that do not<br />
classify as t<strong>and</strong>em or interspersed repeat. A gene<br />
cluster is a group <strong>of</strong> proximal genes having similar<br />
sequence <strong>and</strong> <strong>of</strong>ten, similar structure but, different<br />
function. There may be requirement for multiple<br />
copies <strong>of</strong> functional genes tRNA or rRNA genes.<br />
Copies <strong>of</strong> promoters <strong>and</strong> other regulatory regions<br />
associated with many genes also do not classify as<br />
repetitive DNA.<br />
Telomeres<br />
Telomeric DNA is G-rich consisting <strong>of</strong> the<br />
3′overhang <strong>and</strong> adjacent t<strong>and</strong>em repeat with wide<br />
variation in length across species (reviewed in<br />
Blackburn, 1991; Hemann <strong>and</strong> Greider, 1999). For<br />
example, length <strong>of</strong> telomere TTAGGG repeats in<br />
humans is 5 to15 kbp but in mouse (Mus musculus)<br />
it is ~50 kbp. Yeast, Saccharomyces cerevisiae, has<br />
irregular pattern <strong>of</strong> TG1-3 <strong>and</strong> repeat length <strong>of</strong><br />
~300 bp. A recent model suggests that this region<br />
does a d-loop-t-loop by having the 3′ overhang<br />
invade the t<strong>and</strong>em repeat (Griffith et al., 1999).<br />
This invasion forms a triplex DNA structure, dloop,<br />
<strong>and</strong> encloses a large segment <strong>of</strong> duplex DNA<br />
in a terminal loop or t-loop. Telomere length <strong>and</strong><br />
size <strong>of</strong> loops is species specific (Shore, 2001).<br />
Universal presence <strong>of</strong> this structure across species<br />
is not clear though there may be telomeres that are<br />
unable to form a t-loop (Griffith et al., 1999).
Nucleosomes<br />
Periodicity <strong>of</strong> di-nucleotides (TATA-tetrads) or<br />
t<strong>and</strong>em repeat with a 10 bp pattern <strong>of</strong> 5’<br />
TATAA(A/C)CG(T/C)C 3’ b<strong>and</strong> DNA <strong>and</strong> form<br />
association with histone proteins (Widlund et al.,<br />
1997). However, t<strong>and</strong>em repeats may increase or<br />
decrease nucleosome stability. For example, a<br />
t<strong>and</strong>em repeat having a CAG (=CTG) pattern<br />
located close to a nucleosome increases its stability<br />
(Wang et al., 1994; Wang <strong>and</strong> Griffith, 1995;<br />
Godde <strong>and</strong> Wolffe, 1996). On the other h<strong>and</strong>,<br />
t<strong>and</strong>em repeat CGG (=CCG) has no impact unless<br />
it is methylated. Methylated CGG (=CCG) with a<br />
limited copy number increase the nucleosome<br />
stability while those with large copy numbers<br />
decrease nucleosome stability (Godde et al., 1996;<br />
Wang <strong>and</strong> Griffith, 1996).<br />
T<strong>and</strong>em repeats in genes<br />
T<strong>and</strong>em repeat hypervariability enables<br />
identification <strong>of</strong> genes e.g. antifreeze gene <strong>and</strong><br />
several degenerative diseases. Repeats may help in<br />
stability <strong>of</strong> transcripts or proteins but repeat<br />
expansions <strong>and</strong> instability (particularly <strong>of</strong><br />
trinucleotide repeats) lead to neurological disorders<br />
<strong>and</strong> cancer (Ashley <strong>and</strong> Warren, 1995; Mitas,<br />
1997). Long stretch <strong>of</strong> CAG repeats translated into<br />
polyglutamine tracts result in a gain-<strong>of</strong>-function,<br />
possibly a toxin (Perutz et al., 1994; Baldi et al.,<br />
1999). CGG, AGG <strong>and</strong> TGG repeats form<br />
quadriplex <strong>and</strong> GAA repeats form triplex structures<br />
that can block or reduce transcription <strong>and</strong> DNA<br />
replication (Sinden, 1999). CGG repeats also<br />
destabilize nucleosomes (Sinden, 1999) due to CpG<br />
hypermethylation leading to promoter repression<br />
<strong>and</strong> lack <strong>of</strong> gene expression (Nelson 1995, Baldi et<br />
al., 1999). On the other h<strong>and</strong>, CTG repeats stabilize<br />
nucleosomes <strong>and</strong> block replication forks in E. coli<br />
(Sinden, 1999).<br />
Evolution<br />
Repeats have a role in genome evolution <strong>and</strong><br />
possibly in C-value paradox. Variation in nuclear<br />
DNA amount in higher plants species exemplifies<br />
this. The variation (>2500 fold) in 1C DNA content<br />
in angiosperms ranges from 0.05 picograms in<br />
Cardamine amara to 127.4 picograms in Fritillaria<br />
assyriaca (Bennett, 1985). Part <strong>of</strong> such variation is<br />
due to the numerical changes in chromosomes but<br />
in many, there is substantial variation resulting<br />
DNA repetitive sequences<br />
from amplification or deletion <strong>of</strong> DNA sequences.<br />
Chromosomes <strong>of</strong> many monocot <strong>and</strong> dicot species<br />
contain fast reassociating highly repetitive fraction,<br />
slow reassociating middle repetitive fraction <strong>and</strong><br />
single copy sequences (Britten <strong>and</strong> Kohne, 1968;<br />
Smith <strong>and</strong> Flavell, 1974; Flavell et al., 1977;<br />
Katsiotis et al., 2000). These sequences may be<br />
dispersed repetitive sequences including transposeable<br />
elements or t<strong>and</strong>em repeats. The retroelement<br />
class forms sometimes upto 50% component <strong>of</strong><br />
plant genomes (Guidet et al., 1991; Heuros et al.,<br />
1993; Kubis et al., 1998; Bennetzen, 2000;<br />
Katsiotis et al., 2000; Linares et al., 2000; Ananiev<br />
et al., 2002).<br />
References<br />
Ananiev EV, Vales MI, Phillips RL <strong>and</strong> Rines HW.<br />
Isolation <strong>of</strong> A/D <strong>and</strong> C genome specific<br />
dispersed <strong>and</strong> clustered repetitive DNA<br />
sequences from Avena sativa. Genome. 45: 431-<br />
441, 2002.<br />
Ashley CT <strong>and</strong> Warren ST. Trinucleotide repeat<br />
expansion <strong>and</strong> human disease. Annu Rev Genet.<br />
29: 703-728, 1995.<br />
Baldi P, Brunak S, Chauvin Y <strong>and</strong> Pedersen AG.<br />
Structural basis for triplet repeat disorders: a<br />
computational analysis. Bioinformatics. 15:<br />
918-929, 1999.<br />
Bennett MD. Interspecific variation in DNA<br />
amount <strong>and</strong> the nucleotypic dimension. In Plant<br />
genetics (UCLA symposium on molecular <strong>and</strong><br />
cellular biology), Freeling M (Ed). New York:<br />
Alan R. Liss. 283-302, 1985.<br />
Bennetzen JL. Transposable element contributions<br />
to plant gene <strong>and</strong> genome evolution. Plant Mol<br />
Biol. 42: 251-269, 2000.<br />
Blackburn EH. Telomeres. Trends Biochem Sci. 16:<br />
378–381, 1991.<br />
Boefe JD <strong>and</strong> Corces VG. Transcription <strong>and</strong><br />
reverse transcription <strong>of</strong> retroposons. Annu Rev<br />
Microbiol. 43: 403-434, 1989.<br />
Britten RJ <strong>and</strong> Koehne DE. Repeated sequences in<br />
DNA. Science. 161: 529-540, 1968.<br />
Charlesworth B, Sniegowski P <strong>and</strong> Stephan W. The<br />
evolutionary dynamics <strong>of</strong> repetitive DNA in<br />
eukaryotes. Nature. 371: 215-220, 1994.<br />
7
8<br />
Satyawada Rama RAO et. al.<br />
Cheng Z, Dong F, Langdon T, Ouyang S, Buell<br />
CR, Gu M, Blattner FR <strong>and</strong> Jiang J. Functional<br />
rice centromeres are marked by a satellite repeat<br />
<strong>and</strong> a centromere-specific retrotransposon.<br />
Plant <strong>Cell</strong>. 14: 1691-1704, 2002.<br />
Dai X <strong>and</strong> Rothman-Denes LB. DNA structure <strong>and</strong><br />
transcription. Curr Opin Microbiol. 2: 126-130,<br />
1999.<br />
Donis-Keller H, Green P, Helms C, Cartinhour S,<br />
Weiffenbach B, Stephens K, Keith TP, Bowden<br />
DW, Smith DR, L<strong>and</strong>er ES , Botstein D, Akots<br />
G, Rediker KS, Gravius T, Brown VA, Rising<br />
MB, Parker C, Powers JA, Watt DE, Kauffman<br />
ER, Bricker A, Phipps P, Muller-Kahle H,<br />
Fulton TR, Ng S, Schumm JW, Braman JC,<br />
Knowlton RG, Barker DF, Crooks SM, Lincoln<br />
SE, Daly MJ <strong>and</strong> Abrahamson J. A genetic<br />
linkage map <strong>of</strong> the human genome. <strong>Cell</strong>. 51:<br />
319-337, 1987.<br />
Doolittle WF <strong>and</strong> Sapienza C. Selfish genes, the<br />
phenotype paradigm <strong>and</strong> genome evolution.<br />
Nature. 284: 601-603, 1980.<br />
Flavell RB, Rimpau J <strong>and</strong> Smith DB. Repeated<br />
sequence DNA relationships in four cereal<br />
genomes. Chromosoma. 63: 205-222, 1977.<br />
Flint J, Bates GP, Clark K, Dorman A, Willingham<br />
D, Roe BA, Micklem G, Higgs DR <strong>and</strong> Louis<br />
EJ. Sequence comparison <strong>of</strong> human <strong>and</strong> yeast<br />
telomeres identifies structurally distinct<br />
subtelomeric domains. Hum Mol Genet. 6:<br />
1305-1314, 1997.<br />
Gindullis F, Desel C, Galasso I <strong>and</strong> Schmidt T. The<br />
large-scale organization <strong>of</strong> the centromeric<br />
region in Beta species. Genome Res. 11: 253-<br />
265, 2001.<br />
Godde JS <strong>and</strong> Wolffe AP. Nucleosome assembly<br />
on CTG triplet repeats. J Biol Chem. 271:<br />
15222-15229, 1996.<br />
Godde JS, Kass SU, Hirst MC <strong>and</strong> Wolffe AP.<br />
Nucleosome assembly on methylated CGG<br />
triplet repeats in the fragile X mental retardation<br />
gene 1 promoter. J Biol Chem. 271: 24325-<br />
24328, 1996.<br />
Golenberg EM, Giannasi DE, Clegg MT, Smiley<br />
CJ, Durbin M, Henderson D <strong>and</strong> Zurawski G.<br />
Chloroplast DNA sequence from a miocene<br />
Magnolia species. Nature. 344: 656-658, 1990.<br />
Grabczyk E <strong>and</strong> Usdin K. Alleviating transcript<br />
insufficiency caused by Friedreich’s ataxia<br />
triplet repeats. Nucl Acids Res. 28: 4930-4937,<br />
2000.<br />
Griffith JD, Comeau L, Rosenfield S, Stansel RM,<br />
Bianchi A, Moss H <strong>and</strong> de Lange T.<br />
Mammalian telomeres end in a large duplex<br />
loop. <strong>Cell</strong>. 97: 503-514, 1999.<br />
Grumbach S <strong>and</strong> Tahi F. A new challenge for<br />
compression algorithms: genetic sequences. J<br />
Inform Process Management. 30: 875-886,<br />
1994.<br />
Guidet F, Rogowsky PM, Taylor C, Song W <strong>and</strong><br />
Langridge P. Cloning <strong>and</strong> characterization <strong>of</strong> a<br />
new rye-specific repeated sequence. Genome.<br />
34: 81-87, 1991.<br />
Harrison GE <strong>and</strong> Heslop-Harrison JS. Centromeric<br />
repetitive DNA in the genus Brassica. Theor<br />
Appl Genet. 90: 157-165, 1995.<br />
Hartzog GA <strong>and</strong> Winston F. Nucleosomes <strong>and</strong><br />
transcription: recent lessons from genetics. Curr<br />
Opin Genet Dev. 7: 192-198, 1997.<br />
Hemleben V. Molekularbiologie der Pflanzen.<br />
UTB. Gustav Fischer Verlag, Stuttgart. 1990.<br />
Hemann MT <strong>and</strong> Greider CW. G-str<strong>and</strong> overhangs<br />
on telomeres in telomerase deficient mouse<br />
cells. Nucl Acids Res. 27: 3964-3969, 1999.<br />
Henik<strong>of</strong>f S, Ahmad K <strong>and</strong> Malik HS. The<br />
Centromere Paradox: Stable inheritance with<br />
rapidly evolving DNA. Science. 293: 1098-<br />
1102, 2001.<br />
Heslop-Harrison JS, Murata M, Ogura Y,<br />
Schwarzacher T <strong>and</strong> Motoyoshi F.<br />
Polymorphisms <strong>and</strong> genomic organization <strong>of</strong><br />
repetitive DNA from centromeric regions <strong>of</strong><br />
Arabidopsis chromosomes. Plant <strong>Cell</strong>. 11: 31-<br />
42, 1999.<br />
Heslop-Harrison JS, Br<strong>and</strong>es A <strong>and</strong> Schwarzacher<br />
T. T<strong>and</strong>emly repeated DNA sequences <strong>and</strong><br />
centromeric chromosomal regions <strong>of</strong><br />
Arabidopsis species. Chromosome Res. 11: 241-<br />
253, 2003.<br />
Hueros GY <strong>and</strong> Ferrer E. A structural <strong>and</strong><br />
evolutionary analysis <strong>of</strong> a dispersed repetitive<br />
sequence. Plant Mol Biol. 22: 635-643, 1993.
Hutchison III CA, Hardies SC, Loeb DD, Shehee<br />
WR <strong>and</strong> Edgell MH. LINEs <strong>and</strong> related<br />
retroposons: Long interspersed repeated<br />
sequences in the eukaryotic genome. In Mobile<br />
DNA. Berg DE <strong>and</strong> Howe MM (Ed). American<br />
Society for Microbiology, Washington D.C.,<br />
593-617, 1989.<br />
Jarman AP <strong>and</strong> Wells RA. Hypervariable<br />
minisatellites, recombinators or innocent<br />
byst<strong>and</strong>ers? Trends Genet. 5: 367-371, 1989.<br />
Jeffreys AJ, Wilson V <strong>and</strong> Thein SL. Hypervariable<br />
“minisatellite” regions in human DNA. Nature.<br />
314: 67-73, 1985.<br />
Jeffreys AJ, Neumann R <strong>and</strong> Wilson V. Repeat unit<br />
sequence variation in minisatellites: a novel<br />
source <strong>of</strong> DNA polymorphism for studying<br />
variation <strong>and</strong> mutation by single molecule<br />
analysis. <strong>Cell</strong>. 60: 473-485, 1990.<br />
Jiang J, Birchler JA, Parrott WA <strong>and</strong> Dawe RK. A<br />
molecular view <strong>of</strong> plant centromeres. Trends<br />
Plant Sci. 8: 570-575, 2003.<br />
Katsiotis A, Loukas M <strong>and</strong> Heslop-Harrison JS.<br />
Repetitive DNA, genome <strong>and</strong> species<br />
relationships in Avena <strong>and</strong> Arrhenatherum<br />
(Poaceae). Ann Bot. 86: 1135–1142, 2000.<br />
Kishii M, Nagaki K <strong>and</strong> Tsujimoto H. A t<strong>and</strong>em<br />
repetitive sequence located in the centromeric<br />
region <strong>of</strong> common wheat (Triticum aestivum)<br />
chromosomes. Chromosome Res. 9: 417-428,<br />
2001.<br />
Kornberg RD <strong>and</strong> Lorch Y. Twenty-five years <strong>of</strong><br />
the nucleosome fundamental particle <strong>of</strong> the<br />
eukaryote chromosome. <strong>Cell</strong>. 98: 285-294,<br />
1999.<br />
Kubis S, Schmidt T <strong>and</strong> Heslop-Harrison JS.<br />
Repetitive DNA elements as a major component<br />
<strong>of</strong> plant genomes. Ann Bot. 82: 45-55, 1998.<br />
Kumekawa N, Hosouchi T, Tsuruoka H <strong>and</strong> Kotani<br />
H. The size <strong>and</strong> sequence organization <strong>of</strong> the<br />
centromeric region <strong>of</strong> Arabidopsis thaliana<br />
chromosome 4. DNA Res. 8: 285–290, 2001.<br />
L<strong>and</strong>egren U, Kaiser R, Caskey CT <strong>and</strong> Hood L.<br />
DNA diagnostics-molecular techniques <strong>and</strong><br />
automation. Science. 242: 229-237, 1988.<br />
Linares C, Irigoyen ML <strong>and</strong> Fominaya A.<br />
Identification <strong>of</strong> C-genome chromosomes<br />
involved in intergenomic translocations in<br />
DNA repetitive sequences<br />
Avena sativa L., using cloned repetitive DNA<br />
sequences. Theor Appl Genet. 100: 353-360,<br />
2000.<br />
Macas J, Meszaros T <strong>and</strong> Nouzova M. PlantSat: a<br />
specialized database for plant satellite repeats.<br />
Bioinformatics. 18: 28-35, 2002.<br />
Mitas M. Trinucleotide repeats associated with<br />
human disease. Nucl Acids Res. 25: 2245-2254,<br />
1997.<br />
Nagaki K, Song J, Stupar RM, Parokonny AS,<br />
Yuan Q, Ouyang S, Liu J, Hsiao J, Jones KM,<br />
Dawe RK, Buell CR <strong>and</strong> Jiang J. <strong>Molecular</strong> <strong>and</strong><br />
cytological analyses <strong>of</strong> large tracks <strong>of</strong><br />
centromeric DNA reveal the structure <strong>and</strong><br />
evolutionary dynamics <strong>of</strong> maize centromeres.<br />
Genetics.163: 759-770, 2003a.<br />
Nagaki K, Talbert PB, Zhong CX, Dawe RK,<br />
Henik<strong>of</strong>f S <strong>and</strong> Jiang J. Chromatin<br />
immunoprecipitation reveals that the 180-bp<br />
satellite repeat is the key functional DNA<br />
element <strong>of</strong> Arabidopsis thaliana centromeres.<br />
Genetics. 163: 1221-1225, 2003b.<br />
Nagaki K, Tsujimoto H <strong>and</strong> Sasakuma T. A novel<br />
repetitive sequence <strong>of</strong> sugar cane, SCEN<br />
family, locating on centromeric regions.<br />
Chromosome Res. 6: 295-302.<br />
Nelson DL (1995). The fragile X syndromes.<br />
Seminars in <strong>Cell</strong> <strong>Biology</strong>. 6: 5-11, 1998.<br />
Ohyama T. Intrinsic DNA bends: an organizer <strong>of</strong><br />
local chromatin structure for transcription.<br />
BioEssays. 23: 708–715, 2001.<br />
Paabo S. Ancient DNA: extraction,<br />
characterization, molecular cloning, <strong>and</strong><br />
enzymatic amplification. Proc Natl Acad Sci<br />
U.S.A. 86(6): 1939-1943, 1989.<br />
Perutz MF, Johnson T, Suzuki M <strong>and</strong> Finch JT.<br />
Glutamine repeats as polar zippers: their<br />
possible role in inherited neurodegenerative<br />
diseases. Proc Natl Acad Sci U.S.A. 91: 5355-<br />
5358, 1994.<br />
Pryde FE, Gorham HC <strong>and</strong> Louis EJ. Chromosome<br />
ends: all the same under their caps. Curr Opin<br />
Genet Dev. 7: 822-828, 1997.<br />
Rabin M. In "Discovering Repetitions in Strings,"<br />
Combinatorial Algorithms on Words.<br />
9
10<br />
Satyawada Rama RAO et. al.<br />
Apostolico <strong>and</strong> Galil (Ed). NATO ASI Series.<br />
12: 279-288, 1985.<br />
Richards RI <strong>and</strong> Sutherl<strong>and</strong> GR. Simple repeat<br />
DNA is not replicated simply. Nat Genet. 6:<br />
114-116, 1994.<br />
Rogers JH. Long interspersed sequences in<br />
mammalian DNA: Properties <strong>of</strong> newly identified<br />
specimens. Biochim Biophys. 824: 113-120,<br />
1985.<br />
Saunders VA <strong>and</strong> Houben A. The pericentromeric<br />
heterochromatin <strong>of</strong> the grass Zingeria<br />
biebersteiniana (2n = 4) is composed <strong>of</strong><br />
Zbcen1-type t<strong>and</strong>em repeats that are<br />
intermingled with accumulated dispersedly<br />
organized sequences. Genome. 44: 955-961,<br />
2001.<br />
Schmid CW, Wong EF <strong>and</strong> Deka N. Single copy<br />
sequences in galago DNA resembles a repetitive<br />
human retrotransposon-like family. J Mol Evol.<br />
31: 92-100, 1990.<br />
Schmidt T <strong>and</strong> Heslop-Harrison JS. Genomes,<br />
genes <strong>and</strong> junk: the large-scale organization <strong>of</strong><br />
plant chromosomes. Trends Plant Sci. 3: 195-<br />
199, 1998.<br />
Schueler MG, Higgins AW, Rudd MK, Gustashaw<br />
K <strong>and</strong> Willard HF. Genomic <strong>and</strong> genetic<br />
definition <strong>of</strong> a functional human centromere.<br />
Science. 294: 109-115, 2001.<br />
Shlyakhtenko LS, Potaman VN, Sinden RR <strong>and</strong><br />
Lyubchenko YL. Structure <strong>and</strong> dynamics <strong>of</strong><br />
supercoil-stabilized DNA cruciforms. J Mol<br />
Biol. 280: 61-72, 1998.<br />
Shore D. Telomeric chromatin: replicating <strong>and</strong><br />
wrapping up chromosome ends. Curr Opin<br />
Genet Dev. 11: 189-198, 2001.<br />
Sinden RR. Human genetics ’99: trinucleotide<br />
repeats - Biological implications <strong>of</strong> the DNA<br />
structures associated with disease-causing<br />
triplet repeats. Am J Hum Genet. 64: 346-353,<br />
1999.<br />
Smith DB <strong>and</strong> Flavell RB. The relatedness <strong>and</strong><br />
evolution <strong>of</strong> repeated nucleotide sequences in<br />
the genomes <strong>of</strong> some Gramineae species.<br />
Biochem Genet. 12: 243-256, 1974.<br />
Susumu O. Evolution by gene duplication.<br />
Springer-Verlag, New York, NY. ISBN 0-04-<br />
575015-7, 1970.<br />
Temin HM. Reverse transcription in the eukaryotic<br />
genome: retroviruses, pararetroviruses, retrotransposons,<br />
<strong>and</strong> retrotranscripts. Mol Biol Evol.<br />
2: 455-468, 1985.<br />
Traut W. Chromosomen Klassische <strong>and</strong> molekulare<br />
Cytogenetik. Springer-Verlag, Berlin, 1991.<br />
Varmus HE <strong>and</strong> Brown PO. Retroviruses. In<br />
Mobile DNA. Berg D E <strong>and</strong> Howe M M (Ed)<br />
ASM Publications, New York. 35-56, 1989.<br />
Vergnaud G <strong>and</strong> Denoeud F. Minisatellites:<br />
Mutability <strong>and</strong> genome architecture. Genome<br />
Res.10: 899-907, 2000.<br />
Vogt P. Potential genetic functions <strong>of</strong> t<strong>and</strong>em<br />
repeated DNA sequence blocks in the human<br />
genome are based on a highly conserved<br />
“chromatin folding code”. Hum Genet. 84: 301-<br />
336, 1990.<br />
Wang YH, Amirhaeri S, Kang S, Wells RD <strong>and</strong><br />
Griffith JD. Preferential nucleosome assembly<br />
at DNA triplet repeats from the myotonic<br />
dystrophy gene. Science. 265: 669-671, 1994.<br />
Wang YH <strong>and</strong> Griffith J. Exp<strong>and</strong>ed CTG triplet<br />
blocks from the myotonic dystrophy gene create<br />
the strongest known natural nucleosome<br />
positioning elements. Genomics. 25: 570-573,<br />
1995.<br />
Wang YH <strong>and</strong> Griffith J. Methylation <strong>of</strong> exp<strong>and</strong>ed<br />
CCG triplet repeat DNA from fragile X<br />
syndrome patients enhances nucleosome<br />
exclusion. J Biol Chem. 271: 22937-22940,<br />
1996.<br />
Watkins PC. Restriction Fragment Length<br />
Polymorphism (RFLP): application in human<br />
chromosome mapping <strong>and</strong> genetic disease<br />
research. Biotechniques. 6: 310-320, 1988.<br />
Weiner AM, Deininger PL <strong>and</strong> Efstratiadis A.<br />
Nonviral retroposons: genes, pseudogenes, <strong>and</strong><br />
transposable elements generated by the reverse<br />
flow <strong>of</strong> genetic information. Annu Rev Biochem.<br />
55: 631-661, 1986.<br />
Widlund HR, Cao H, Simonsson S, Magnusson E,<br />
Simonsson T, Nielsen PE, Kahn JD, Crothers<br />
DM <strong>and</strong> Kubista M. Identification <strong>and</strong><br />
characterization <strong>of</strong> genomic nucleosomepositioning<br />
sequences. J Mol Biol. 267: 807-<br />
817, 1997.
Wolff RK, Plaeke R, Jeffreys AJ <strong>and</strong> White R.<br />
Unequal crossing over between homologous<br />
chromosomes is not the major mechanism<br />
involved in the generation <strong>of</strong> new alleles at<br />
VNTR loci. Genomics. 5: 382-384, 1991.<br />
Yang CF, Kim JM, Molinari E <strong>and</strong> DasSarma S.<br />
Genetic <strong>and</strong> topological analyses <strong>of</strong> the bop<br />
promoter <strong>of</strong> Halobacterium halobium:<br />
stimulation by DNA supercoiling <strong>and</strong> non-B-<br />
DNA structure. J Bacteriol. 178: 840-845,<br />
1996.<br />
Zhong CX, Marshall JB, Topp C, Mroczek R, Kato<br />
A, Nagaki K, Birchler JA, Jiang J <strong>and</strong> Dawe<br />
RK. Centromeric retroelements <strong>and</strong> satellites<br />
interact with Maize kinetochore protein<br />
CENH3. Plant <strong>Cell</strong>. 14: 2825-2836, 2002.<br />
DNA repetitive sequences<br />
11
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong> 7(2) & 8(1): 13-23, 2010 Research Article<br />
<strong>Haliç</strong> University, Printed in Turkey.<br />
http://jcmb.halic.edu.tr<br />
Genetic diversity <strong>of</strong> Penicillium species isolated from various sources<br />
in Sarawak, Malaysia<br />
Hairul Azman ROSLAN *, 1 , Chua Suk NGO 1 <strong>and</strong> Sepiah MUID 2<br />
1 Department <strong>of</strong> <strong>Molecular</strong> <strong>Biology</strong>, Faculty <strong>of</strong> Resource Science <strong>and</strong> Technology, Universiti Malaysia<br />
Sarawak, 94300 Kota Samarahan, Sarawak Malaysia<br />
2 Department <strong>of</strong> Plant Sciences <strong>and</strong> Environmental Ecology, Faculty <strong>of</strong> Resource Science <strong>and</strong><br />
Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak Malaysia<br />
(* author for correspondence; rhairul@frst.unimas.my )<br />
Received: 26 December 2008; Accepted 30 December 2009<br />
Abstract<br />
Borneo Isl<strong>and</strong> is one <strong>of</strong> the megadiversity centres <strong>of</strong> the world <strong>and</strong> contain vast amount <strong>of</strong> flora <strong>and</strong> fauna.<br />
The Penicillium species are among the most commonly occurring <strong>and</strong> economically important members <strong>of</strong><br />
micro-fungi family. In this study, morphological <strong>and</strong> r<strong>and</strong>om amplification polymorphic DNA (RAPD)<br />
molecular methods were used to group <strong>and</strong> determine genetic variability <strong>and</strong> relationship among twenty<br />
Penicillium isolates from various locations in Western part <strong>of</strong> Borneo Isl<strong>and</strong> that was maintained in the pure<br />
culture collection <strong>of</strong> University Malaysia Sarawak. Comparison between morphological <strong>and</strong> molecular<br />
method using M13 <strong>and</strong> OPD10 primers were undertaken <strong>and</strong> showed that in some cases, the groupings <strong>of</strong><br />
isolates based on morphological method were consistent with molecular groupings with a few exceptions.<br />
<strong>Molecular</strong> analysis also indicated genotype variability between the isolates with little correlation with either<br />
the origin <strong>of</strong> soil or geographical location.<br />
Keywords: Penicillium species, morphology, RAPD, M13, variation<br />
Malezya Sarawak’ta Farklı Kaynaklardan Elde Edilen Penicillium Türlerinin<br />
Genetik Çeşitliliği<br />
Özet<br />
Borneo adası dünyanın en çok çeşitliliğe sahip merkezlerinden bir tanesidir ve büyük miktarda flora ve<br />
faunaya sahiptir. Penicillium (küf) türleri mikro-mantar ailesinin en sık rastlanan ve ekonomik olarak önemli<br />
üyeleri arasındadır. Bu çalışmada Borneo’nun batı bölgelerinden elde edilip Malezya Sarawak<br />
<strong>Üniversitesi</strong>’ndeki saf kültür koleksiyonlarında muhafaza edilen yirmi Penicillium izolatını gruplamak ve<br />
aralarındaki genetik çeşitliliği ve ilişkiyi belirlemek için, morfolojik ve polimorfik DNA’nın rastgele<br />
amplifikasyonu (RAPD) metodu kullanılmıştır. M13 ve OPD10 primerleri kullanılarak morfolojik ve<br />
moleküler metodlar arasında kıyaslama yapılmış ve birkaç istisna ile bazı durumlarda, izolatların morfolojik<br />
metodlara dayanarak grupl<strong>and</strong>ırılmalarının moleküler grupl<strong>and</strong>ırılmalar ile uyumlu olduğu gösterilmiştir.<br />
Moleküler analiz aynı zam<strong>and</strong>a izolatlar arasında, toprağın kaynağı veya coğrafi bölge ile az korelasyon<br />
göstermesine rağmen, genotip varyasyonu göstermiştir.<br />
Anahtar Sözcükler: Penicillium türleri, morfoloji, RAPD, M13, varyasyon
14<br />
Hairul Azman ROSLAN et al.<br />
Introduction<br />
Fungi <strong>and</strong> bacteria are the principal decomposers<br />
that release carbon, nitrogen <strong>and</strong> other elements<br />
that otherwise would become tied up in organic<br />
matter (Carlile et al., 2001). Fungi play an<br />
important role in decomposing forest litter or dung,<br />
fruits or other organic materials. Farms fruits <strong>and</strong><br />
crops are vulnerable to fungal attack <strong>and</strong> 10% to<br />
50% <strong>of</strong> the world’s harvested fruit is lost each year<br />
due to fungal attack (Campbell <strong>and</strong> Reece, 2002).<br />
However, fungi also have a number <strong>of</strong> practical<br />
uses for humans. The distinctive flavours <strong>of</strong> certain<br />
kinds <strong>of</strong> cheeses, including Roqueorti <strong>and</strong> blue<br />
cheese, come from the fungi used to ripen them.<br />
The s<strong>of</strong>t drink industry uses Aspergillus niger to<br />
produce citric acid. Beside that, a family <strong>of</strong><br />
unicellular fungi, Saccharomyces cerevisiae is the<br />
most important fungi used in the food industries<br />
such as in baking, alcohol brewing <strong>and</strong> wine<br />
making. Apart from food industries, fungi are<br />
medically valuable as antibiotic producers used to<br />
treat infections (Thom, 1945). The Penicillium spp<br />
are among the most commonly occurring <strong>and</strong><br />
economically important members <strong>of</strong> micr<strong>of</strong>ungi<br />
family. Although much is known about Penicillium<br />
physiology <strong>and</strong> mycotoxin chemistry, one <strong>of</strong> the<br />
main challenges is in the area <strong>of</strong> rapid <strong>and</strong> reliable<br />
identification <strong>of</strong> Penicillium in many settings<br />
including community health care, occupational<br />
health <strong>and</strong> food safety (Scott, 1977; Cruz-Perez et<br />
al., 2001; Meklin et al., 2004; Portnoy et al., 2004).<br />
Sarawak is one <strong>of</strong> the centres <strong>of</strong> mega- if not gigadiversity<br />
region <strong>and</strong> possesses a vast potential <strong>of</strong><br />
undiscovered organisms including Penicillium spp.<br />
We have isolated a number <strong>of</strong> Penicillium from<br />
various location <strong>and</strong> sources within Sarawak. Here<br />
we report the genotyping <strong>of</strong> Penicillium spp from<br />
UNIMAS pure culture collections.<br />
Materials <strong>and</strong> Methods<br />
Collection <strong>and</strong> maintenance <strong>of</strong> fungal isolates<br />
Twenty Penicillium isolates were obtained from<br />
Universiti Malaysia Sarawak (UNIMAS) culture<br />
collection. The fungi collection was isolated from<br />
various sources in Sarawak such as from mangrove<br />
soil, leaf litter, peat soil, soy sauce, karas <strong>and</strong><br />
rambutan (Table 1). A map <strong>of</strong> Sarawak state is<br />
shown in Figure 1, indicating the sampling<br />
locations. Fungi from stock culture were recultured<br />
on Malt Extract Agar (MEA) <strong>and</strong> Czapek<br />
Yeast Agar (CYA) in Petri dishes. Each isolate was<br />
inoculated at three-points on each media in petri<br />
dishes. The inoculated plates were kept at room<br />
temperature (22-25ºC) for seven days.<br />
Figure 1. Map <strong>of</strong> Sarawak state in Malaysia indicating sampling sites. 1: Karangas<br />
Forest, 2: Mixed Dipterocarp Forest, 3: Riverine Forest, Samunsam; 4: Sematan; 5:<br />
Kuching; 6: Kampung Bako; 7:Bako Isl<strong>and</strong>; 8: Kota Samarahan; 9: Bintulu
Genetic diversity <strong>of</strong> Penicillium species in Sawarak, Malaysia 15<br />
Table 1 List <strong>of</strong> fungal collection, the substrate it was extracted from <strong>and</strong> location <strong>of</strong> the fungal (*numbers in<br />
superscript indicate origin <strong>of</strong> isolate corresponding to Figure 1)<br />
Fungal<br />
UFI<br />
Substrate Origin<br />
isolates (Unimas Fungi Index)<br />
P1 1433 Mangrove soil<br />
6<br />
Kampung Bako<br />
P2 1435 Mangrove soil<br />
6<br />
Kampung Bako<br />
P3 0646 Leaf litter<br />
1<br />
Karangas Forest, Samunsam<br />
P4 0687 Karas<br />
8<br />
Samarahan<br />
P5 1439 Soy sauce<br />
8<br />
Samarahan<br />
P6 1443 Soy sauce<br />
8<br />
Samarahan<br />
P7 1434 Mangrove soil<br />
6<br />
Kampung Bako<br />
P8 1440 Soy sauce<br />
5<br />
Kuching<br />
P9 0338 Leaf litter<br />
2<br />
Mixed Dipterocarp Forest, Samunsam<br />
P10 1445 Karas<br />
8<br />
Samarahan<br />
P11 1446 Peat soil<br />
8<br />
Samarahan<br />
P12 1436 Mangrove soil<br />
4<br />
Sematan<br />
P13 1438 Soy sauce<br />
8<br />
Samarahan<br />
P14 0630 Leaf litter<br />
1<br />
Karangas Forest, Samunsam<br />
P15 1437 Peat soil<br />
9<br />
Bintulu<br />
P16 0650 Leaf litter<br />
3<br />
Riverine Forest, Samunsam<br />
P17 1441 Mangrove soil<br />
7<br />
Bako Isl<strong>and</strong><br />
P18 1447 Rambutan<br />
8<br />
Samarahan<br />
P19 1442 Mangrove soil<br />
7<br />
Bako Isl<strong>and</strong><br />
P20 1444 Soy sauce<br />
Morphological study<br />
A small tuft <strong>of</strong> mycelium <strong>and</strong> conidiophores were<br />
lifted from a fairly young section <strong>of</strong> the colony <strong>and</strong><br />
placed on a drop <strong>of</strong> acid fuschin on a glass slide. A<br />
cover slip was gently lowered on the specimen.<br />
Slides were sealed with Canada balsam.<br />
Identification <strong>of</strong> the fungi was based on culture<br />
characteristics <strong>and</strong> conidiophore structure. Cultural<br />
characteristics such as colony colour, texture,<br />
colony growth, exudates, odour, zonation <strong>and</strong><br />
pigmentation were examined. Conidiophore<br />
structure that includes its length, phialides, branching<br />
system <strong>and</strong> conidia were also examined. Images<br />
were taken by using Nikon digital camera. Notes <strong>of</strong><br />
International Mycological Institute (IMI)<br />
descriptions were used as reference for the<br />
identification.<br />
<strong>Molecular</strong> study<br />
Isolation <strong>of</strong> DNA<br />
DNA was extracted using a rapid extraction method<br />
as introduced by Taylor <strong>and</strong> Natvig (1987). Genetic<br />
5 Kuching<br />
material was also taken directly from mycelia<br />
growing on CYA using clean, autoclaved tips. The<br />
genetic materials were then mixed with 25-30μl <strong>of</strong><br />
Tris-EDTA (TE) buffer <strong>and</strong> then vortexed. The<br />
DNA was kept in -20ºC until required.<br />
RAPD-PCR amplification<br />
Two PCR primers, M13 (5’-<br />
TTATGTAAACGACGGCCAGT -3’) <strong>and</strong> OPD10<br />
(5’-GTGATCGCAG-3’), were used to amplify 20<br />
Penicillium isolates. A negative control was<br />
included in each amplification process. The PCR<br />
mixture used for the RAPD in this study consisted<br />
<strong>of</strong> 2.5μl <strong>of</strong> 10X PCR buffer (Vivantis), 2.5µl <strong>of</strong><br />
2mM dNTPs (Vivantis), 10 pmol/μl M13 or 5<br />
pmol/μl OPD-10, 2 U Taq polymerase (Vivantis)<br />
<strong>and</strong> 2.5μl DNA template. Sterile distilled water was<br />
added to total up the PCR reaction volume to 25 µl.<br />
Amplification was conducted using Biometra T-<br />
Gradient (Biometra) with the following temperature<br />
pr<strong>of</strong>ile (Table 2):
16<br />
Hairul Azman ROSLAN et al.<br />
Table 2. PCR amplification parameters using the M13 primers <strong>and</strong> OPD-10<br />
Separation <strong>of</strong> DNA fragments by gel<br />
electrophoresis<br />
Parameters Temperature <strong>and</strong> Reaction time<br />
Initial denaturation 94ºC for 3 minutes<br />
Denaturation 84ºC for 30 seconds<br />
Annealing 46ºC / 30ºC for 1 minute (M13 /<br />
OPD10)<br />
Extension 72ºC for 2 minutes<br />
Number <strong>of</strong> cycles 35<br />
Final extension 72ºC for 7 minutes<br />
The amplicons were separated using 1.3 % (w/v)<br />
agarose gel electrophoresis in 1X TAE (Tris-Acetic<br />
acid-EDTA) buffer. The electrophoresis was<br />
performed at 100V for 90 minutes. The gel was<br />
visualised using ethidium bromide under UV<br />
transilluminator <strong>and</strong> documented using Gel<br />
Documentation System (BioRad).<br />
Construction <strong>of</strong> phylogenetic relationships<br />
Each individual RAPD b<strong>and</strong> was considered as<br />
equivalent independent characters <strong>and</strong> all the b<strong>and</strong>s<br />
were scored as present or absent for each isolate.<br />
B<strong>and</strong>ing patterns were converted into binary tables.<br />
The data was analyzed using genetic data analysis<br />
s<strong>of</strong>tware, Numerical Taxonomy <strong>and</strong> Multivariate<br />
Analysis System (NTSYSpc) version 2.2. The data<br />
was quantified by similarity index, Jij = Cij / (ni +<br />
nj – Cij), where Jij= the number <strong>of</strong> individuals i <strong>and</strong><br />
j, ni= the number <strong>of</strong> b<strong>and</strong>s in individual i, nj= the<br />
number <strong>of</strong> b<strong>and</strong>s in individual j. A dendogram was<br />
generated using Unweighted Pair-Group Method<br />
with Arimethrical Averages (UPGMA) as<br />
described by Sneath <strong>and</strong> Sokal (1973).<br />
Results <strong>and</strong> Discussion<br />
Morphological groupings <strong>of</strong> Penicillium isolates<br />
All the isolates were initially identified based on<br />
the cultural characteristics <strong>and</strong> structure <strong>of</strong><br />
conidiophores using stereo <strong>and</strong> compound<br />
microscopes. Among the 20 isolates, 4 isolates<br />
were grouped as Clade 1, 2 isolates were grouped<br />
as Clade 2, 3 isolates were grouped as Clade 3, 2<br />
isolates were grouped as Clade 4, 4 isolates were<br />
grouped as Clade 5, 2 isolates were grouped as<br />
Clade 6, <strong>and</strong> one isolate each for Clade 7, Clade 8<br />
<strong>and</strong> Clade 9 respectively. Table 3 shows the clades<br />
based on morphological characters, isolate name,<br />
substrate it was found <strong>and</strong> origin <strong>of</strong> the isolates.<br />
The detailed morphological classifications <strong>of</strong><br />
selected isolates are presented in Figures 2 to 7<br />
below representing Clade 1 to Clade 6.
Table 3 Morphological groupings <strong>of</strong> Penicillium isolates.<br />
Genetic diversity <strong>of</strong> Penicillium species in Sawarak, Malaysia 17<br />
Clade Fungal isolates Substrate Origin<br />
1 P1 Mangrove soil Kampung Bako<br />
P7 Mangrove soil Kampung Bako<br />
P14 Leaf litter Karangas Forest, Samunsam<br />
P16 Leaf litter Riverine Forest, Samunsam<br />
2 P2 Mangrove soil Kampung Bako<br />
P12 Mangrove soil Sematan<br />
3 P3 Leaf litter Karangas Forest, Samunsam<br />
P9 Leaf litter Mixed Dipterocarp Forest,<br />
Samunsam<br />
P15 Peat soil Bintulu<br />
4 P4 Karas Samarahan<br />
P13 Soy sauce Samarahan<br />
5 P5 Soy sauce Samarahan<br />
P8 Soy sauce Kuching<br />
P17 Mangrove soil Bako Isl<strong>and</strong><br />
P19 Mangrove soil Bako Isl<strong>and</strong><br />
6 P6 Soy sauce Samarahan<br />
P20 Soy sauce Kuching<br />
7 P10 Karas Samarahan<br />
8 P11 Peat soil Samarahan<br />
9 P18 Rambutan Samarahan<br />
Figure 2. Clade 1 Penicillium P7 isolate. (a) Colony surface on CYA, (b) Colony reverse on<br />
CYA colour, (c) Colony surface on MEA, (d) Colony reverse on MEA, (e) Conidiophore<br />
structure (Bi-Asymmetrical), (f) Conidia globose shape
18<br />
Hairul Azman ROSLAN et al.<br />
Figure 3. Clade 2 Penicillium P2 isolate. (a) Colony surface on CYA, (b) Colony reverse on<br />
CYA, (c) Colony surface on MEA, (d) Colony reverse on MEA, (e) Conidiophore structure at<br />
100X magnification (Monoverticillata), (f) Conidia globose shape<br />
Figure 4. Clade 3 Penicillium P15 isolate. (a) Colony surface on CYA, (b) Colony reverse on<br />
CYA, (c) Colony surface on MEA, (d) Colony reverse on MEA, (e) Conidiophore structure at<br />
100X magnification, arrow showing swollen apex, (f) Conidiophore structure at 40X<br />
magnification (Monoverticillata), (g) Conidia globose shape
Genetic diversity <strong>of</strong> Penicillium species in Sawarak, Malaysia 19<br />
Figure 5. Clade 4 Penicillium P13 isolate. (a) Colony surface on CYA, (b) Colony reverse on<br />
CYA, (c) Colony surface on MEA, (d) Colony reverse on MEA, (e) Conidiophore structure at<br />
100X magnification (f) Conidiophore structure at 40X magnification (Bi-asymmetrical), (g)<br />
Conidia globose shape<br />
Figure 6. Clade 5 Penicillium P17 isolate. (a) Colony surface on CYA, (b) Colony reverse on<br />
CYA, (c) Colony surface on MEA, (d) Colony reverse on MEA, (e) Conidiophore structure at<br />
100X magnification, arrow showing lanceolate phialides, (f) Conidiophore structure at 40X<br />
magnification (Bi-asymmetrical), (g) Conidia globose shape
20<br />
Hairul Azman ROSLAN et al.<br />
Figure 7. Clade 6 Penicillium P6 isolate. (a) Colony surface on CYA, (b) Colony reverse on<br />
CYA, (c) Colony surface on MEA, (d) Colony reverse on MEA, (e) Conidiophore structure at<br />
40X magnification (Terverticillata), (f) Conidia elliptical shape.<br />
<strong>Molecular</strong> groupings <strong>of</strong> Penicillium isolates<br />
Single, simple repetitive PCR primers have been<br />
designed to amplify the microsatelite regions <strong>of</strong><br />
chromosomal DNA. In most applications these<br />
primers have provided similar levels <strong>of</strong> specificity<br />
to those seen with RAPD, <strong>and</strong> the results have been<br />
used to group fungi at species level (Meyer et al.,<br />
1992; Schlick et al., 1994; Bridge et al., 1997).<br />
Two sets <strong>of</strong> primers were used, M13 <strong>and</strong> OPD-10<br />
primers to analyse the variations between the 20<br />
isolates <strong>of</strong> Penicillium spp. Six isolates were<br />
excluded from the molecular study because either<br />
the DNA could not be isolated or amplification was<br />
not reproducible. Each sample was repeated at least<br />
two times to determine its reproducibility <strong>and</strong><br />
consistency. B<strong>and</strong>s were scored for each primer<br />
based on the presence (1) or absence (0) <strong>of</strong><br />
amplicon migration in the gel. Figure 8 <strong>and</strong> Figure<br />
9 represent the RAPD pr<strong>of</strong>ile <strong>of</strong> M13 <strong>and</strong> OPD-10<br />
respectively. Figure 10 is a dendogram generated<br />
from M13 data.
Genetic diversity <strong>of</strong> Penicillium species in Sawarak, Malaysia 21<br />
Figure 8. RAPD b<strong>and</strong> pr<strong>of</strong>ile generated using M13 primer visualized on 1.3% (v/v) agarose. The<br />
lane markings correspond to the isolate number. Lane M: 1kbp DNA ladder (Fermentas) <strong>and</strong><br />
Lane N: 100bp DNA ladder (Seegene)<br />
Figure 9. RAPD b<strong>and</strong> pr<strong>of</strong>ile generated using OPD10 primer visualized on 1.3% (v/v) agarose<br />
gel. The lane markings correspond to the isolate number. Lane M: 1kbp DNA ladder (Fermentas)<br />
<strong>and</strong> Lane N: 100bp DNA ladder (Seegene)
22<br />
Hairul Azman ROSLAN et al.<br />
Figure 10. Dendrogram showing relationships among 14 isolates <strong>of</strong> Penicillium species. Genetic<br />
distances were obtained using M13 primer.<br />
The study compared the classification<br />
generated from morphological data <strong>and</strong> molecular<br />
data. Comparison <strong>of</strong> the two datasets indicated that<br />
the RAPD b<strong>and</strong>ing patterns were generally<br />
consistent with morphological data. Combination <strong>of</strong><br />
morphological <strong>and</strong> molecular data can be used to<br />
increase the confidence that the isolates were<br />
grouped correctly. Previous study carried out by<br />
Lutzoni <strong>and</strong> Vilgalys (1995) integrated molecular<br />
<strong>and</strong> morphological data sets in order to estimate<br />
fungal phylogenies in lichenized <strong>and</strong> nonlichenized<br />
Omphalina species. They found that<br />
homogeneity testing <strong>of</strong> the 28S large subunit<br />
ribosomal DNA sequences <strong>and</strong> the morphological<br />
characters showed that the two data sets were<br />
sampling the same phylogenetic history. In this<br />
study, the dendrogram generated from<br />
amplification with M13 primer gives approximately<br />
79% correlation with morphological data as 11 out<br />
<strong>of</strong> 14 isolates were observed to give similar<br />
groupings. As in the case <strong>of</strong> OPD10, there was<br />
approximately 69% correlation with morphological<br />
data as 9 out <strong>of</strong> 13 isolates were observed to<br />
correlate with morphological groupings. <strong>Molecular</strong><br />
analysis has shown that two isolates that were<br />
initially grouped in different cluster based on the<br />
morphological characterization, appeared to be<br />
identical at the genetic levels when characterized<br />
with RAPD analysis. For isolates P5 (peat soil) <strong>and</strong><br />
P19 (mangrove soil), showed minor differences in<br />
their morphological characteristics but showed to<br />
be identical at the genetic levels.<br />
The dendogram generated from M13 primer<br />
(Figure 10) also showed little correlation between<br />
isolates isolated from the same soil type for<br />
example isolates isolated from mangrove soil P1<br />
<strong>and</strong> P2 are grouped into Clade 1 while P2 <strong>and</strong> P12<br />
in Clade 5. Apart from that, geographical origin<br />
also showed little correlation as seen in isolates<br />
isolated from soy sauce from Samarahan area P13<br />
<strong>and</strong> P5 found in Clade 6 <strong>and</strong> Clade 7 respectively,<br />
indicating a wide variation <strong>of</strong> Penicillium that can<br />
be found throughout the sampling area. Spatial<br />
variations in micr<strong>of</strong>ungi communities is quite<br />
common <strong>and</strong> have been shown to be attributed to<br />
various factors such as soil chemistry, plant<br />
composition such as the alpine <strong>and</strong> birch
communities (Lumley et al., 2001; Mclean <strong>and</strong><br />
Huhta 2002; Bellis et al., 2007).<br />
Conclusion<br />
In this study, most isolates showed correlation <strong>and</strong><br />
consistency in morphological <strong>and</strong> molecular data.<br />
<strong>Molecular</strong> analysis was also able to show that in the<br />
instance <strong>of</strong> P5 <strong>and</strong> P19 to be genetically identical<br />
when characterized with RAPD compared to<br />
morphological analysis. The study also indicated<br />
that the isolates showed considerable genotypic<br />
variations within Penicillium spp isolated from a<br />
wide area in Sarawak <strong>and</strong> little correlation to both<br />
the type <strong>of</strong> soil they originated from <strong>and</strong> also<br />
geographical location.<br />
Acknowledgement<br />
This work is supported by Unimas Fundamental<br />
Research Grant.<br />
References<br />
Bellis TD, Kernaghan G, Widden P. Plant<br />
community influences on soil micr<strong>of</strong>ungal<br />
assemblages in boreal mixed-wood forests.<br />
Mycologia. 99(3):356-367. 2007.<br />
Bridge PD, Prior C, Sagbohan J, Lomer CJ, Carey<br />
M <strong>and</strong> Buddie A. <strong>Molecular</strong> characterization <strong>of</strong><br />
isolates from locusts <strong>and</strong> grasshoppers.<br />
Biodiversity <strong>and</strong> Conservation. 6:177-189.<br />
1997.<br />
Campbell NA, Reece JB. <strong>Biology</strong> sixth edition. pp<br />
616-630. 2002.<br />
Carlile MJ, Watkinson SC <strong>and</strong> Graham WG. The<br />
Fungi. Second edition. San Diego, California:<br />
Academic Press; 2001.<br />
Cruz-Perez P, Buttner MP <strong>and</strong> Stetzenbach LD.<br />
Specific detection <strong>of</strong> Stachybotrys chartarum in<br />
pure culture using quantitative polymerase<br />
chain reaction. Mol. <strong>Cell</strong>. Probes. 15 (23), pp.<br />
129–138. 2001.<br />
Lumley TC, Gignac LD <strong>and</strong> Currah RS.<br />
Micr<strong>of</strong>ungus communities <strong>of</strong> white spruce <strong>and</strong><br />
trembling aspen logs at different stages <strong>of</strong> decay<br />
in disturbed <strong>and</strong> undisturbed sites in the boreal<br />
mixedwood region <strong>of</strong> Alberta. Canadian J Bot.<br />
79:76–92. 2001.<br />
Lutzoni F <strong>and</strong> Vilgalys R. Integration <strong>of</strong><br />
morphological <strong>and</strong> molecular data sets in<br />
estimating fungal phylogenies. Canadian J Bot.<br />
73(suppl. 1):S49-659. 1995.<br />
Genetic diversity <strong>of</strong> Penicillium species in Sawarak, Malaysia 23<br />
Mclean MA <strong>and</strong> Huhta V. Micr<strong>of</strong>ungal community<br />
structure in anthropogenic birch st<strong>and</strong>s in<br />
central Finl<strong>and</strong>. <strong>Biology</strong> <strong>and</strong> Fertility <strong>of</strong> Soils.<br />
35:1–12. 2002.<br />
Meklin T, Haugl<strong>and</strong> RA, Reponen T, Varma M,<br />
Lummus Z, Bernstein D, Wymer LJ <strong>and</strong> Vesper<br />
SJ. Quantitative PCR analysis <strong>of</strong> house dust can<br />
reveal abnormal mold conditions. J. Environ.<br />
Monit. 6 pp. 615–620. 2004.<br />
Meyer W, Moraywetz R, Borner T <strong>and</strong> Kubicek<br />
CP. The use <strong>of</strong> DNA fingerprint analysis in the<br />
classification <strong>of</strong> some species. Curr Genet.<br />
21:27-30. 1992.<br />
Portnoy JM, Barnes CS <strong>and</strong> Kennedy K. Sampling<br />
for indoor fungi. J. Allergy Clin. Immunol. 113<br />
pp. 189–198. 2004.<br />
Schlick A, Kuhls K, Meyer W, Lieckfeldt E,<br />
Borner T <strong>and</strong> Messner K. Fingerprinting reveals<br />
gamma-ray induced mutations in fungal DNA:<br />
implications for identification <strong>of</strong> patent strains<br />
<strong>of</strong> Trichoderma harzianum. Curr Genet. 26: 74-<br />
78. 1994.<br />
Scott PM. Penicillium mycotoxins. In "Mycotoxic<br />
Fungi, Mycotoxins, Mycotoxicoses, an<br />
Encyclopedic H<strong>and</strong>book. Vol. 1. Mycotoxigenic<br />
Fungi' eds. T.D. Wyllie <strong>and</strong> L.G. Morehouse.<br />
New York: Marcel Dekker. pp. 283-356. 1977.<br />
Sneath PHA <strong>and</strong> Sokal RR. Numerical Taxonomy.<br />
W.H. Freeman, San Francisco; 1973.<br />
Taylor JW <strong>and</strong> Natvig D. Isolation <strong>of</strong> fungal DNA.<br />
In: Fuller, M.S. <strong>and</strong> Jaworski, A. (eds)<br />
Zoosporic Fungi in Teaching <strong>and</strong> Research.<br />
South-eastern Publishing Corporation, Athens,<br />
Georgia. pp. 252-258. 1987.<br />
Thom C. Mycology presents penicillin. Mycologia.<br />
37:460-475. 1945.
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong> 7(2) & 8(1): 25-34, 2010 Research Article<br />
<strong>Haliç</strong> University, Printed in Turkey.<br />
http://jcmb.halic.edu.tr<br />
The sensitivity <strong>of</strong> the human chromosomes to ethyl methane<br />
sulfonate (EMS)<br />
Songül BUDAK DİLER *,1 , Mehmet TOPAKTAŞ 2<br />
1 University <strong>of</strong> Niğde, Department <strong>of</strong> Science <strong>and</strong> Letters, Niğde, 51200, Turkey<br />
2 University <strong>of</strong> Cukurova, Department <strong>of</strong> Science <strong>and</strong> Letters, Adana, 01330 Turkey<br />
(* author for correspondence; budakdiler@gmail.com)<br />
Received: 03 March 2009; Accepted 19 March 2010<br />
Abstract<br />
The aim <strong>of</strong> this study was to determine the chromosomal susceptibility to breakages by the mutagen Ethyl<br />
methanesulfonate (EMS). For this reason, human peripheral blood lymphocytes were treated with varying<br />
concentrations <strong>of</strong> EMS (5x10 -4 M, 10 -3 M <strong>and</strong> 2x10 -3 M) for 24 <strong>and</strong> 48 hours. The percentages <strong>of</strong> chromosomal<br />
fragmentations in EMS-treated <strong>and</strong> untreated (control) cells were found to be statistically significant. In<br />
addition, the extent <strong>of</strong> breakages <strong>of</strong> the same chromosomes correlated with the concentrations <strong>of</strong> the chemical.<br />
The chromosomes that were fragmented most as a result <strong>of</strong> EMS-treatment in descending order were 1, 2, 6,<br />
4, X, 7, 3, 5, 9, <strong>and</strong> 8.<br />
Keywords: Ethyl methanesulfonate (EMS), human lymphocytes, chromosome damage, lymphocyte culture.<br />
Etil Metansulfonat (EMS)’ye İnsan Kromozomlarının Hassasiyeti<br />
Özet<br />
Bu çalışmanın amacı, mutajen Etil metansulfonat (EMS)’nin insan kromozomlarında oluşturduğu kromozom<br />
kırıklarını incelemek ve en çok kırılan kromozomları saptamaktır. Bu amaç için hücreler, 5x10 -4 M, 10 -3 M ve<br />
2x10 -3 M konsantrasyonlardaki EMS ile 24 ve 48 saat muamele edilmiştir. 24 ve 48 saat EMS ile muamele<br />
edilen insan periferal lenfositlerinde kontrolde ve aynı dozda, farklı kromozomlarda görülen kromozom<br />
kırılma yüzdeleri bir birleriyle karşılaştırılmış ve aralarındaki farkın istatistik bakımında önemli olduğu<br />
bulunmuştur. Ayrıca aynı kromozomun farklı dozlardaki kırılma yüzdeleri kontroldeki kırılma yüzdeleriyle<br />
karşılaştırılmış, aradaki farkın önemli olduğu saptanmıştır. EMS ile muamele sonucu en fazla kromozom<br />
kırılması 1., 2., 6., 4., X, 7., 3., 5., 9., ve 8. kromozomlarda tespit edilmiştir.<br />
Anahtar Sözcükler: Etil metansulfonat (EMS), insan kromozomları, kromozom kırığı, lenfosit kültürü.<br />
Introduction<br />
Ethyl methanesulfonate (EMS) is a colorless liquid.<br />
When heated to decomposition, EMS emits toxic<br />
fumes <strong>of</strong> sulfur oxides. EMS is reasonably<br />
anticipated to be a human carcinogen based on<br />
sufficient evidence <strong>of</strong> carcinogenicity in<br />
experimental animals. EMS is used experimentally<br />
as a mutagen, teratogen, <strong>and</strong> brain carcinogen <strong>and</strong><br />
as a research chemical (IARC 1974, IARC 1987,<br />
HSDB 2000, Merck The Merck Index 1989). When<br />
administered as a single intraperitoneal injection,<br />
EMS induced lung tumors in male mice <strong>and</strong> lung<br />
adenomas in mice <strong>of</strong> both sexes. Three<br />
intraperitoneal injections <strong>of</strong> EMS in arachis oil<br />
induced lung <strong>and</strong> kidney tumors in male mice. In a<br />
similar study, EMS induced renal carcinomas in
26<br />
Songül Budak DİLER <strong>and</strong> Mehmet TOPAKTAŞ<br />
female rats <strong>and</strong> a variety <strong>of</strong> benign <strong>and</strong> malignant<br />
tumors, including lung carcinomas, in rats <strong>of</strong> both<br />
sexes (Ueo et al., 1981).<br />
The tests sister chromatid exchange (SCE) <strong>and</strong><br />
chromosome aberrations (CA) are used to assess<br />
the genotoxicicity <strong>of</strong> mutagenic <strong>and</strong> carcinogenic<br />
chemicals (Perry <strong>and</strong> Evans, 1975). It was also<br />
established that in fish cells, EMS increased SCE<br />
<strong>and</strong> CA in a concentration dependent manner but<br />
had no effect on their replicative index (RI)<br />
(Maddock et al., 1986). Furthermore, Adhikari <strong>and</strong><br />
Grover showed that EMS caused CA in the rat bone<br />
marrow cells (Adhikari <strong>and</strong> Grover, 1988). In 1990,<br />
it was established that EMS enhanced SCE in the<br />
peripheral leukocytes <strong>of</strong> humans in a concentration<br />
dependent manner <strong>and</strong> decreased the RI in a doseindependent<br />
manner (Topaktaş <strong>and</strong> Speit, 1990).<br />
The sensitivities <strong>and</strong> clastogenicities <strong>of</strong> human<br />
chromosomes with high gene density (1, 19 <strong>and</strong> 20)<br />
<strong>and</strong> with low gene density (4 <strong>and</strong> 18) to<br />
combinations <strong>of</strong> EMS <strong>and</strong> cytosine arabinoside<br />
(Ara-C) were measured <strong>and</strong> the high gene density<br />
chromosomes were found to be sensitive (Surralles<br />
et al.,1997). Human peripheral blood lymphocytes<br />
were treated with EMS (1,5x10 -4 M <strong>and</strong> 1,5x10 -3 M)<br />
<strong>and</strong> MMS (1,5x10 -5 M <strong>and</strong> 1,5x10 -4 M) <strong>and</strong> the<br />
treatment resulted in enhanced SCE compared to<br />
untreated corresponding cultures (Harish et al.,<br />
1998). it was demonstrated that the chemical EMS<br />
enhanced SCE in whole blood <strong>and</strong> lymphocyte<br />
cultures (During, 1985). In addition, EMS<br />
treatment at various concentrations (5x10 -4 M, 10 -<br />
3 M <strong>and</strong> 2x10 -3 M) <strong>of</strong> human peripheral blood<br />
lymphocytes enhanced CA (Rencüzoğullari <strong>and</strong><br />
Topaktaş, 2000). Therefore, we aimed at<br />
investigating the degree <strong>of</strong> sensitivity <strong>of</strong> human<br />
chromosomes to EMS by employing the<br />
mutagenicity tests mentioned above.<br />
Materials <strong>and</strong> Methods<br />
In this study, we used peripheral blood <strong>and</strong><br />
lymphocytes from healthy donors, two males (23<br />
<strong>and</strong> 24 years old), <strong>and</strong> two females (23 years old).<br />
EMS (Sigma M-0880) was used as a test substance.<br />
The preparation <strong>of</strong> chromosome was performed in<br />
accordance with Evans 1984. In addition, this study<br />
was prepared according to IPCS guidelines<br />
(Albertini et al., 2000). Whole blood (0.2 ml) from<br />
four healthy donors (two male <strong>and</strong> two female,<br />
nonsmokers, aged: 23 <strong>and</strong> 24) was added to 2.5 ml<br />
chromosome medium B (Biochrom, F5023). The<br />
cultures were incubated at 37°C for 72 h. The cells<br />
were treated with concentrations <strong>of</strong> 5x10 -4 M, 10 -3 M<br />
<strong>and</strong> 2x10 -3 M <strong>of</strong> EMS for 24 h (EMS was added 48<br />
h after initiating the culture) <strong>and</strong> 48 h (EMS was<br />
added 24 h after initiating the culture). The test<br />
substance EMS was dissolved in ethanol (50%).<br />
There was clear evidence that ethanol was not a<br />
bacterial or mammalian cell mutagen in vitro<br />
assays for chromosome aberration. Reported tests<br />
for chromosome aberration induction in vivo were<br />
all negative <strong>and</strong> only a minority <strong>of</strong> micronucleus<br />
tests were positive (Phillips <strong>and</strong> Jenkinson, 2001).<br />
Colchicine (0.06µg/ml, Sigma C 9754) was added<br />
for the last 2 h <strong>of</strong> culture. To collect the cells, the<br />
cultures were centrifuged (1200 rpm, 15 min),<br />
treated with hypotonic solution (0.4% KCl) for 13<br />
min at 37 o C, <strong>and</strong> then fixed in cold methanol:<br />
glacial acetic acid (3: 1) for 20 min at room<br />
temperature. The treatment with fixative was<br />
repeated three times. Then the cells were spread on<br />
glass slides <strong>and</strong> air dried. The slides were stained<br />
with giemsa (5%). Well spread metaphases per<br />
donor were examined 1000× magnification for<br />
occurrence <strong>of</strong> different types <strong>of</strong> chromosome<br />
aberration (CA). 100 metaphase cells with<br />
chromosomes aberrations were examined in each<br />
treated groups <strong>and</strong> control groups. Karyotyping was<br />
performed using Olympus BX50 microscope <strong>and</strong><br />
Cytovision 3.00 Windows NT Applied Imaging<br />
s<strong>of</strong>tware. For statistical analysis, the ONE WAY<br />
ANOVA <strong>and</strong> DUNCAN test was used <strong>and</strong> the<br />
results were tabulated.<br />
Results<br />
In this study, the sensitivity <strong>of</strong> human<br />
chromosomes to EMS was revealed by observing<br />
the chromosomal breakages in each <strong>of</strong> the<br />
chromosomes. The percentage <strong>of</strong> chromosomal<br />
fragmentation varied among chromosomes in the<br />
control groups. In the control groups (24h),<br />
chromosomes 1, 2, 6, X, <strong>and</strong> 4 were found to be<br />
sensitive to first degree fragmentation whilst<br />
chromosomes 22, 20, 19, 18, <strong>and</strong> 11 were<br />
insensitive (Table 1). In the solvent control groups<br />
(ethanol, 50%) while chromosomes 2, 1, 6, 3 <strong>and</strong> 4<br />
were sensitive to first degree fragmentation,<br />
chromosomes 22 <strong>and</strong> 18 were completely<br />
insensitive (Table 1).<br />
In cells that had been treated with 5x10 -4 M <strong>of</strong><br />
EMS for 24 h, chromosomes 1 <strong>and</strong> 2 were the most<br />
sensitive <strong>and</strong> chromosomes 22, 19 <strong>and</strong> 20 were the<br />
least sensitive to fragmentation (Table 1). In those<br />
that were treated with 10 -3 M <strong>of</strong> EMS for 24 h,<br />
chromosomes 1 <strong>and</strong> 2 were the most fragile while<br />
chromosomes 17, 11, 18, 21, 22, 19 <strong>and</strong> 20 were
the least sensitive (Table 1) (Figure1). Karyotyping<br />
<strong>and</strong> fragmentation <strong>of</strong> human chromosomes 1, 2,<br />
<strong>and</strong> 10. At the 2x10 -3 M concentration <strong>of</strong> EMS for<br />
the same duration, chromosomes 6 <strong>and</strong> 1 were<br />
sensitive to first degree while chromosomes X, 2, 5,<br />
<strong>and</strong> 4 were less fragile. In the same cultures,<br />
chromosomes 18, 19, 22, 20 <strong>and</strong> 21 remained<br />
completely insensitive to the chemical (Table 1). In<br />
cultures that had been treated for 24 h with varying<br />
concentrations <strong>of</strong> EMS, <strong>and</strong> in all concentrations<br />
tested, the fragmentation observed in chromosomes<br />
2, 5-11, 13 <strong>and</strong> 16-8 was higher than that in the<br />
control group <strong>and</strong> in the solvent control group. On<br />
the other h<strong>and</strong>, the breakages <strong>of</strong> chromosomes 3, 12<br />
<strong>and</strong> 19 were significant only in comparison to the<br />
control groups. While the 4th chromosome showed<br />
significant fragmentation at all <strong>of</strong> the concentrations<br />
tested in comparison to the controls, this was<br />
significant only at 5x10 -4 M <strong>and</strong> 10 -3 M EMS<br />
concentrations in comparison to the solvent<br />
controls. The fragmentation <strong>of</strong> chromosomes 14, 15<br />
<strong>and</strong> X at EMS concentrations <strong>of</strong> 5x10 -4 M <strong>and</strong> 2x10 -<br />
3 M was significantly higher than the control groups<br />
<strong>and</strong> the solvent control groups. On the other h<strong>and</strong>,<br />
there was no increase in breakages <strong>of</strong> chromosomes<br />
1, 20 – 22 as a result <strong>of</strong> EMS treatment (Table 1).<br />
As a result <strong>of</strong> treatment with EMS for 24 hours, the<br />
Chromosomal fragmentation by EMS<br />
chromosomal fragmentation observed in<br />
comparison to control groups <strong>and</strong> solvent control<br />
groups was found to be dose independent (Table 1).<br />
In the solvent control groups chromosomes 2<br />
<strong>and</strong> 1 are the most susceptible to breakages. There<br />
was no breakages observed on chromosomes 22, 21,<br />
20, 18 <strong>and</strong> 17 (Table 2).In cultures that had been<br />
treated with 5x10 -4 M <strong>of</strong> EMS for 48 h,<br />
chromosomes 1, 2 <strong>and</strong> 4 were the most sensitive to<br />
fragmentation. The least sensitive were<br />
chromosomes 20, 18 <strong>and</strong> 17, with chromosome 22<br />
never showing fragmentation (Table 2). There was<br />
no statistical significance in the observed breakages<br />
among chromosomes treated with 10 -3 M <strong>of</strong> EMS<br />
for 48 h (Table 2, Figure 2). At 2x10 -3 M<br />
concentration <strong>of</strong> EMS chromosomes 1 <strong>and</strong> 2 are<br />
susceptible to the first degree, with the least<br />
sensitive being 22 <strong>and</strong> 20 (Table 2). In cultures<br />
treated with different concentrations <strong>of</strong> EMS for 48<br />
h, there was significant fragmentation <strong>of</strong><br />
chromosomes 10 <strong>and</strong> 11 while at these concentrations<br />
chromosomes 17, 18 <strong>and</strong> 21 showed more<br />
fragmentation in comparison to the control group.<br />
On the other h<strong>and</strong>, chromosomes 1-9, 12-14, 19, 20,<br />
22 <strong>and</strong> X did not any show any significant<br />
fragmentation the control group (Table 2).<br />
27
28<br />
Songül Budak DİLER <strong>and</strong> Mehmet TOPAKTAŞ<br />
Figure 1. Karyotyping <strong>and</strong> fragmentation <strong>of</strong> Human Chromosomes 1, 2, <strong>and</strong> 10 (10 -3 M EMS Treatment for<br />
24h,♀).
Chromosomal fragmentation by EMS<br />
Table 1. Comparison <strong>of</strong> percentages <strong>of</strong> chromosomal fragmentations <strong>of</strong> human peripheral lymphocytes that<br />
were treated with different concentrations <strong>of</strong> EMS for 24 h.<br />
Chromoso<br />
me<br />
Control group Solvent Control<br />
group<br />
5x10 -4 M 10 -3 M 2x10 -3 M Sig<br />
1 3.0±1.0a 3.7±1.0ab 11.7±2.0a 9.2±1.4a 9.0±2.7ab<br />
2 2.5±0.9abB 4.2±1.4aB 10.7±0.9aA 8.7±1.9aA 8.0±0.4abcA **<br />
3 1.0±0.7bcdefB 3.0±1.4abcdAB 5.0±0.7bcdA 5.0±0.4bcdA 4.7±1.1cdefA *<br />
4 1.7±0.7abcdC 2.5±0.5abcdBC 7.0±1.4bcA 6.7±0.4bcA 6.5±0.9abcdAB ***<br />
5 1.5±0.6abcdeB 1.0±0.7cdefB 5.0±1.0bcdA 5.0±0.7bcdA 6.5±0.9abcdA **<br />
6 2.2±0.4abB 3.0±1.0abcB 7.0±0.7bA 7.2±0.4bA 9.2±0.7aA ***<br />
7 1.2±0.4abcdeB 2.2±0.7abcdeB 5.0±0.7bcdA 6.7±0.9bcdA 4.2±0.7defA ***<br />
8 1.2±0.2abcdeB 1.2±0.2abcdefB 4.2±0.4bcdeA 5.5±0.8bcdeA 3.7±0.4defA ***<br />
9 1.2±0.4abcdeB 1.0±0.7cdefB 4.5±0.6bcdeA 5.5±0.6bcdeA 3.5±0.5defghA **<br />
10 0.2±0.2efB 0.7±0.4cdefB 3.7±0.6bcdefA 4.5±0.2bcdefA 3.5±0.9defghA ***<br />
11 0.0±0.0fC 0.5±0.5fC 2.2±0.8efghB 4.7±0.4efghA 2.7±0.7efghAB ***<br />
12 0.7±0.2bcdefB 1.5±0.5abcdefA<br />
B<br />
3.0±0.4defgA 4.0±0.7defgA 3.0±1.0efghAB *<br />
13 0.5±0.2defB 1.2±0.4bcdefB 4.0±1.2bcdefA 3.5±0.2bcdefA 5.2±0.7bcdeA **<br />
14 0.7±0.4cdefB 0.7±0.4cdefB 3.7±1.1cdefA 2.2±0.6cdefgA<br />
B<br />
3.0±0.5efgA *<br />
15 0.2±0.2efB 0.2±0.2fB 3.0±0.7defgA 1.2±0.7defgA<br />
B<br />
3.0±0.7efgA **<br />
16 1.0±0.7bcdefB 0.2±0.2fB 3.5±0.8defA 3.5±0.5defA 3.5±0.2defgA ***<br />
17 0.2±0.2efB 0.2±0.2fB 2.0±0.7efghA 1.5±0.2efghA 2.0±0.4efghA **<br />
18 0.0±0.0fB 0.0±0.0fB 1.2±0.6ghıA 0.7±0.2ghıA 1.2±0.2ghıjA **<br />
19 0.0±0.0fB 0.5±0.2fAB 0.7±0.2hıA 1.0±0.0hıA 1.0±0.4hıjA *<br />
20 0.0±0.0f 0.2±0.2f 0.2±0.2ı 0.7±0.2ı 0.7±0.4ıj<br />
_<br />
21 0.2±0.2ef 1.0±0.7cdef 1.5±0.2fgh 2.2±0.6fgh 0.5±0.2j<br />
22 0.0±0.0f 0.0±0.0f 0.7±0.4hı 1.0±0.5hı 0.7±0.5ıj<br />
X 2.0±0.5abcdB 1.7±0.8abcdefB 5.2±0.6bcdA 6.0±0.4bcdAB 8.0±1.0abcA ***<br />
Sig. *** *** *** *** ***<br />
Key: *** P
30<br />
Songül Budak DİLER <strong>and</strong> Mehmet TOPAKTAŞ<br />
Figure 2. Karyotyping <strong>and</strong> fragmentation <strong>of</strong> human chromosomes 1, 2, 3, 8, 9 <strong>and</strong> 21 (10 -3 M EMS Treatment<br />
for 48h, ♀).
Chromosomal fragmentation by EMS<br />
Table 2. Comparison <strong>of</strong> percentages <strong>of</strong> chromosomal fragmentations <strong>of</strong> human peripheral lymphocytes that<br />
were treated with different concentrations <strong>of</strong> EMS for 48 h.<br />
Chromosome Control<br />
group<br />
Solvent<br />
Control<br />
group<br />
5x10 -4 M 10 -3 M 2x10 -3 M Sig<br />
1 3.0±1.0a 4.7±2.1ab 8.0±1.9a 8.5±2.9 10.2±1.3a _<br />
2 2.5±0.9ab 5.7±1.3a 6.7±2.0a 8.0±2.8 8.2±1.8ab _<br />
3 1.0±0.7bcdef 2.0±0.7cdefg 3.7±2.2cde 3.2±1.4 4.0±0.9cdef _<br />
4 1.7±0.7abcd 3.2±0.4abc 6.5±1.5ab 5.2±2.0 5.5±0.6bcd _<br />
5 1.5±0.6abcde 2.7±0.4bcd 4.0±0.4abcd 2.5±1.3 3.0±1.0def _<br />
6 2.2±0.4abc 3.2±0.4abc 5.2±0.7abc 6.0±2.0 7.2±0.8abc _<br />
7 1.2±0.4abcde 1.2±0.9efghıj 4.0±0.7abcd 3.2±1.1 3.7±0.6def _<br />
8 1.2±0.2abcde 2.0±0.4bcdef 2.7±0.2bcde 2.7±1.1 3.5±0.2def _<br />
9 1.2±0.4abcde 1.5±0.2cdefgh 2.5±0.2cde 2.5±0.8 3.2±0.2def _<br />
10 0.2±0.2efB 1.2±0.2cdefgh<br />
AB<br />
1.2±0.6efgAB 2.5±1.0A 3.2±0.4defA *<br />
11 0.0±0.0fB 1.2±0.4defghı<br />
AB<br />
1.7±0.4defA 2.7±1.1A 3.5±0.6defA **<br />
12 0.7±0.2bcdef 0.5±0.5hıj 1.7±0.4def 2.2±0.7 2.7±0.7def _<br />
13 0.5±0.2def 1.7±0.4cdefg 2.5±0.6cdef 3.2±1.7 4.7±1.0bcde _<br />
14 0.7±0.4cdef 1.2±0.2cdefghı 1.2±0.6efg 2.2±1.0 0.3±0.7def _<br />
15 0.2±0.2efB 0.2±0.2ıjB 1.0±0.4efgAB 1.0±0.4AB 2.5±0.9efgA *<br />
16 1.0±0.7bcdef 0.7±0.4fghıjB 2.7±0.6cdeA 2.5±0.9AB 4.0±0.7cdeA *<br />
B<br />
B<br />
17 0.2±0.2efB 0.0±0.0jB 0.2±0.2fgB 2.0±0.7A 2.5±0.6efgA **<br />
18 0.0±0.0fC 0.0±0.0jC 0.2±0.2fgBC 1.0±0.4B 2.2±0.6efgh<br />
A<br />
***<br />
19 0.0±0.0f 0.5±0.2fghıj 1.0±0.0efg 1.0±0.7 1.7±0.8fgh _<br />
20 0.0±0.0f 0.0±0.0j 0.5±0.5fg 1.0±0.5 0.7±0.4h _<br />
21 0.2±0.2efB 0.0±0.0jB 1.0±0.5efgAB 1.5±0.6A 2.2±0.4efgh<br />
A<br />
**<br />
22 0.0±0.0f 0.0±0.0j 0.0±0.0g 1.5±0.6 1.0±0.7gh _<br />
X 2.0±0.5abcd 2.2±0.4bcde 4.7±1.2abcd 4.7±1.7 5.0±0.5bcde _<br />
Sig *** *** ***<br />
_<br />
***<br />
31
32<br />
Songül Budak DİLER <strong>and</strong> Mehmet TOPAKTAŞ<br />
Discussion<br />
The sensitivity <strong>of</strong> human chromosomes to EMS<br />
treatment was measured by determining the<br />
breakages <strong>of</strong> each chromosome. In cultures treated<br />
with different concentrations <strong>of</strong> EMS for 24 h <strong>and</strong><br />
48 h, the chromosomal breakage percentages were<br />
statistically significant compared to control groups.<br />
In addition, the percentage <strong>of</strong> fragmentation <strong>of</strong> each<br />
chromosome at different concentrations <strong>of</strong> EMS<br />
was significantly higher the controls. 24h EMS<br />
treatment caused more fragmentation than 48h<br />
treatment due to repair <strong>of</strong> damaged cells after 24h<br />
treatment (Franke et al., 2006). Similar findings<br />
also reported by several groups (Çakmak et al.,<br />
2004, Rencüzoğulları et al., 2004, Bayram <strong>and</strong><br />
Topaktaş 2008).<br />
In the control groups, chromosomes 1, 2, 6, X,<br />
4 <strong>and</strong> 5 are susceptible to breakages to the first<br />
degree. As can be seen, those chromosomes that are<br />
tend to (natural) breakages in the control groups are<br />
also susceptible to fragmentation in EMS-treated<br />
cultures.<br />
These results illustrate that the clastogens<br />
cause more fragmentation <strong>of</strong> those chromosomes<br />
that are prone to natural breakages. It has been<br />
proposed that there may be a correlation between<br />
the length <strong>of</strong> chromosomes <strong>and</strong> degree <strong>of</strong><br />
susceptibility to breakages. However, in this study,<br />
in cultures treated with EMS, exception to this<br />
assumption was discovered. For instance,<br />
chromosome 3 fell into the second degree<br />
susceptibility category in both untreated <strong>and</strong> EMS<br />
treated cultures. This finding suggests that<br />
chromosomal susceptibility to fragmentation may<br />
correlate with its length as well as its composition.<br />
Some investigators have discovered mutations<br />
in some <strong>of</strong> the chromosomes derived from some<br />
malignant cells, which we have found to be<br />
sensitive to EMS treatment. The chromosomes we<br />
found to be susceptible (chromosomes 1, 2, 3, 4, 5,<br />
6, X, 7 <strong>and</strong> 8) were also found to be sensitive in<br />
other studies. For instance, Bayani et al. showed by<br />
spectral karyotyping that chromosomes 8, 7 <strong>and</strong> 20<br />
were fragmented <strong>and</strong> rearranged in bone marrow<br />
malignancies (Bayani et al.,2003). In cell lines<br />
derived from stomach cancers, found that the p arm<br />
<strong>of</strong> chromosome 17 showed partial deletion whilst<br />
the q arm demonstrated partial duplication (Chun et<br />
al., 2000). Selzer et al. studied neroblastomas <strong>and</strong><br />
their cell line derivatives, <strong>and</strong> discovered that there<br />
was a loss in 3p <strong>and</strong> 11q whilst 17q showed<br />
enlargement (Selzer et al., 2005). Gorunova et al.<br />
showed that in gull bladder carcinomas,<br />
chromosome 7 was the most frequently rearranged<br />
one, followed by chromosomes 1, 3, 11, 6, 5 <strong>and</strong> 8.<br />
(Gorunova et al., 1999). Morrissette et al.<br />
discovered aberrations <strong>of</strong> chromosome 18 in<br />
patients with partial mosaic tiresome (Morrissette et<br />
al., 2005).<br />
From these findings, it can be deduced that the<br />
EMS test may prove to be indicative in some types<br />
<strong>of</strong> cancer. Honma et al. compared the mutagenic<br />
<strong>and</strong> cytotoxic response <strong>of</strong> the p53 tumor suppressor<br />
gene in normal cells (TK6) <strong>and</strong> in cells with a<br />
mutated p53 gene (WTK-1), both <strong>of</strong> which were<br />
derived from he same ancestor. These two cell lines<br />
were subjected to treatment with X-rays, EMS,<br />
MMS <strong>and</strong> MMC. They found that the WTK-1 cells<br />
were more resistant to induced cytotoxicity than the<br />
TK6 cells, whiles their thymidine kinase (tk) gene<br />
was more susceptible to mutation due to loss <strong>of</strong><br />
heterozygosity (LOH). These studies shows that<br />
EMS can cause malignancies not only by the<br />
cytogenetically specified chromosomal fragmentations<br />
but also by alterations at the genetic level<br />
(Honma et al., 1997).<br />
In our study EMS caused chromosomal<br />
breakages that are similar to the ones described by<br />
these investigators. It can be argued that EMS may<br />
constitute a risk factor in malignant transformations<br />
due to its effect on chromosomal stability.<br />
Acknowledgments<br />
This study was supported by the C.U. Research<br />
Fund. Project No. FBE2002D117.<br />
References<br />
Adhikari N <strong>and</strong> Grover IS. Genotoxic effects <strong>of</strong><br />
same systemic pesticides: In vivo chromosomal<br />
aberrations in bone marrow cells in rats.<br />
Environmental <strong>and</strong> <strong>Molecular</strong> Mutagenesis 12:<br />
235-242, 1988.<br />
Albertini RJ, Anderson D, Douglas GR et al., IPCS<br />
Guidelines for the Monitoring <strong>of</strong> Genotoxic<br />
Effects <strong>of</strong> Carcinogenes in Humans. Mutat Res<br />
463: 111-172, 2000.<br />
Bayani J, Zielenska M, P<strong>and</strong>ita A et al. Spectral<br />
Karyotyping Identifies Recurrent Complex<br />
Rearrangements <strong>of</strong> Chromosomes 8, 17 <strong>and</strong> 20
in Osteosarcomas. Genes Chromosomes Cancer<br />
36(1): 7-16, 2003.<br />
Bayram S <strong>and</strong> Topaktaş M. Confirmation <strong>of</strong> the<br />
Chromosome Damaging effects <strong>of</strong> Lamivudine<br />
in in vitro Human Peripheral Blood<br />
Lymphocytes. Environmantal <strong>and</strong> <strong>Molecular</strong><br />
Mutagenesis 49: 328-333, 2008.<br />
Çakmak T, Topaktaş M <strong>and</strong> Kayraldiz A. The<br />
Induction <strong>of</strong> Chromosomal Aberration by Tetra<br />
Antibiotic in Bone Marrow <strong>Cell</strong>s <strong>of</strong> Rats in vivo.<br />
Russian <strong>Journal</strong> <strong>of</strong> Genetics 40(8): 867-870,<br />
2004.<br />
Chun YH, Kil JI, Suh YS et al. Characterization <strong>of</strong><br />
Chromosomal Aberrations in Human Gastric<br />
Carcinoma <strong>Cell</strong> Lines Using Chromosome<br />
Painting. Cancer Genet. Cytogenet. 119(1): 18 –<br />
25, 2000.<br />
During R. Vergleichende Untersuchungen zur<br />
Induktion von Schwester-chromatidaustauschen<br />
(SCEs) in Menschlichen Lymphozyten in vitro<br />
Nach Kultivierung von Vollblut oder Isolierten<br />
Lymphozyten. Dissertation zu Erlangung des<br />
Doktorgrades der Medizin der Fakültät für<br />
Theoretische Medizin der Universität Ulm, 80s.<br />
1985.<br />
Evans HJ. Human peripheral blood lymphocytes<br />
fort he analysis <strong>of</strong> chromosome aberrations in<br />
mutagen tests, H<strong>and</strong>book <strong>of</strong> Mutagenicity Test<br />
Procedures: Kilbey, B.J., Legator, M., Nichols,<br />
W., Ramel, C. (eds.), Second edition, Elsevier<br />
Science Publishers, BV. pp. 405-427, 1984.<br />
Franke SI, Pra D, Giulian R et al. Influence <strong>of</strong><br />
orange juice in the levels <strong>and</strong> in the<br />
genotoxicity <strong>of</strong> iron <strong>and</strong> copper. Food-Chem-<br />
Toxicol 44(3): 425-35, 2006.<br />
Gorunova L, Parada LA, Limon J et al. Nonr<strong>and</strong>om<br />
Chromosomal Aberrations <strong>and</strong> Cytogenetic<br />
Heterogeneity in Gallbladder Carcinomas.<br />
Genes Chromosomes Cancer 26(4): 312-321,<br />
1999.<br />
Harish SK, Guruprasad KP, Mahmood R et al.<br />
Adaptive Response to Low Dose <strong>of</strong> EMS or<br />
MMS in Human Peripheral Blood Lymphocytes.<br />
Indian J Exp Biol 36: 1147-50, 1998.<br />
Honma M, Hayashi M, S<strong>of</strong>uni T. Cytotoxic <strong>and</strong><br />
Mutagenic Responses to X-Rays <strong>and</strong> Chemical<br />
Mutagens in Normal <strong>and</strong> p53-Mutated Human<br />
Lymphoblastoid <strong>Cell</strong>s. Mutat. Res. 4;374(1):<br />
89-98. 1997.<br />
HSDB Hazardous Substances Data Base. National<br />
Library <strong>of</strong> Medicine.<br />
Chromosomal fragmentation by EMS<br />
http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?<br />
HSDB, 2000.<br />
IARC Same Anti-thyroid <strong>and</strong> Related Substances,<br />
Nitr<strong>of</strong>urans <strong>and</strong> Industrial Chemicals. IARC<br />
Monographs on the Evaluation <strong>of</strong> Carcinogenic<br />
Risk <strong>of</strong> Chemicals to Humans, Lyon, France:<br />
International Agency for Research on Cancer,<br />
vol.7. pp 326, 1974.<br />
IARC. Overall Evaluations <strong>of</strong> Carcinogenicity.<br />
IARC Monographs on the Evaluation <strong>of</strong><br />
Carcinogenic Risk <strong>of</strong> Chemicals to Humans,<br />
Lyon, France: International Agency for<br />
Research on Cancer. Supplement 7. pp 440,<br />
1987.<br />
Maddock ML, Northrup H, Ellingham TJ.<br />
Induction <strong>of</strong> Sister-Chromatid Exchange <strong>and</strong><br />
Chromosomal Aberrations in Hematopoietic<br />
Tissue <strong>of</strong> a Marine Fish Following ın vivo<br />
Exposure to Genotoxic Carcinogens. Mutat Res<br />
29: 145-147, 1986.<br />
Merck The Merck Index, 11th ed. Rahway, NJ:<br />
Merck&Company, Inc. 1989.<br />
Morrissette JJ, Medne L, Bentley T et al. A Patient<br />
with Mosaic Partial Trisomy 18 Resulting From<br />
Dicentric Chromosome Breakage. Am J. Med.<br />
Genet. A. 30;137(2): 208-212, 2005.<br />
Perry P <strong>and</strong> Evans HJ. Cytological Detection <strong>of</strong><br />
Mutagen-Carcinogen Exposure by Sister<br />
Chromatid Exchange. Nature 258: 121-125,<br />
1975.<br />
Phillips BJ <strong>and</strong> Jenkinson P. Is Ethanol Genotoxic?<br />
A Review <strong>of</strong> the Published Data. Mutagenesis<br />
16(2): 91-101, 2001.<br />
Rencüzoğullari E <strong>and</strong> Topaktaş M. Chromosomal<br />
Aberrations in Cultured Human Lymphocytes<br />
Treated with the Mixtures <strong>of</strong> Carbosulfan, Ethyl<br />
Carbamate <strong>and</strong> Ethyl Methanesulfonate.<br />
Cytologia 65: 83-92, 2000.<br />
Rencüzoğulları E, İla HB, Kayraldiz A et al. The<br />
Genotoxic Effect <strong>of</strong> the New Acaricide<br />
Etoxazole. Russian <strong>Journal</strong> <strong>of</strong> Genetics 40(11):<br />
1300-1304, 2004.<br />
Selzer RR, Richmond TA, P<strong>of</strong>ahl NJ et al. Analysis<br />
<strong>of</strong> Chromosome Breakpoints in Neuroblastoma<br />
at Sub-kilobase Resolution Using Fine-tiling<br />
Oligonucleotide Array CGH. Genes<br />
Chromosomes Cancer 44(3): 305-19, 2005.<br />
Surralles J, Sebastian S <strong>and</strong> Natarajan AT.<br />
Chromosomes with High Gene Density are<br />
Preferentially Repaired in Human <strong>Cell</strong>s.<br />
Mutagenesis 12: 437-442, 1997.<br />
33
34<br />
Songül Budak DİLER <strong>and</strong> Mehmet TOPAKTAŞ<br />
Topaktaş M <strong>and</strong> Speit G. Sister Chromatid<br />
Exchange (SCE) Test Make Use <strong>of</strong> Detemine in<br />
the Mutagen <strong>and</strong> Carcinogen. C.U. Sağlık Bil<br />
Der 5: 73-84, 1990.<br />
Ueo HR, Takaki HY, K. Sugimachi. Mammary<br />
carcinoma induced by oral administration <strong>of</strong><br />
ethyl methanesulphonate. Determination <strong>of</strong><br />
some <strong>of</strong> the parameters affecting tumor<br />
induction. Carcinogenesis 2(12): 1223-8, 1981.
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong> 7(2) & 8(1): 35-43, 2010 Research Article<br />
<strong>Haliç</strong> University, Printed in Turkey.<br />
http://jcmb.halic.edu.tr<br />
Protective effect <strong>of</strong> pomegranate peel ethanol extract against ferric<br />
nitrilotriacetate induced renal oxidative damage in rats<br />
Mahgoub Mohammed AHMED *, 1 <strong>and</strong> Safaa Eid ALI 2<br />
1<br />
<strong>Molecular</strong> Drug Evaluation Department, National Organization for Drug Control <strong>and</strong> Research<br />
(NODCAR), Giza, Egypt<br />
2<br />
Food Technology Research Inst., Agricultural Research Center (ARC), Giza, Egypt<br />
(* author for correspondence; dr_mahgoub1@yahoo.com )<br />
Received: 18 December 2009; Accepted: 02 April 2010<br />
Abstract<br />
Pomegranate is an important source <strong>of</strong> bioactive compounds. The nephroprotective effect <strong>of</strong> pomegranate<br />
peel ethanol extract against ferric nitrilotriacetate (Fe-NTA)-induced renal oxidative stress was studied. The<br />
results showed that Fe-NTA enhances renal lipid peroxidation with reduction in renal glutathione content,<br />
antioxidant enzymes, viz., glutathione peroxidase, catalase, glutathione reductase <strong>and</strong> phase-II metabolizing<br />
enzyme, glutathione-S-transferase. It also enhances serum urea <strong>and</strong> creatinine. Treatment <strong>of</strong> rats orally with<br />
pomegranate peel extract (100 <strong>and</strong> 200 mg/kg/day, for seven days) resulted in significant decrease in lipid<br />
peroxidation <strong>and</strong> serum urea <strong>and</strong> creatinine levels. Renal glutathione content, glutathione-S-transferase <strong>and</strong><br />
antioxidant enzymes were also recovered to a significant level (P
36<br />
Mahgoub Mohammed AHMED <strong>and</strong> Safaa Eid ALI<br />
Introduction<br />
Pomegranate (Punica granatum L., Punicaceae), is<br />
one <strong>of</strong> the oldest known drug. It is mentioned in the<br />
Ebers papyrus <strong>of</strong> Egypt written in about 1550 BC<br />
(Ross, 1999). Dried fruit peel is used for diarrhea<br />
<strong>and</strong> to treat respiratory <strong>and</strong> urinary tract infections.<br />
Also, pomegranate fruit peel exerted diverse<br />
pharmacological functions as antioxidant activity<br />
(Yunfeng et al., 2006 <strong>and</strong> Thring et al., 2009),<br />
antifertility effect (Gujraj et al., 1960), cytotoxic<br />
activity (Sato, 1990 <strong>and</strong> Kulkarni et al., 2007),<br />
hepatoprotective activity (Murthy, 2002) <strong>and</strong><br />
hypoglycemic activity (Dhawan et al., 1977 <strong>and</strong><br />
Hontecillas et al., 2009). Also, pomegranate peel<br />
ethanol extract (500 mg/kg b.wt.) has ameliorative<br />
effect against chlorpyrifos-ethyl-induced oxidative<br />
stress in rats (Ahmed <strong>and</strong> Zaki, 2009). Pomegranate<br />
peel contains substantial amounts <strong>of</strong> polyphenols<br />
such as ellagic tannins, ellagic acid <strong>and</strong> gallic acid<br />
(Naser et al., 1996).<br />
Iron is the most abundant metal in the human<br />
body. Although iron is an essential nutritional<br />
element for all life forms, iron overload may lead to<br />
various diseases (De Freitas <strong>and</strong> Meneghini, 2001).<br />
The iron complex <strong>of</strong> the chelating agent<br />
nitrilotriacetic acid is nephrotoxic (Khan <strong>and</strong><br />
Sultana, 2005). Intraperitoneal injection <strong>of</strong> Fe-NTA<br />
induces renal proximal tubular damage associated<br />
with oxidative damage that eventually leads to a<br />
high incidence <strong>of</strong> renal cell carcinoma in rodents<br />
after repeated administration (Okada <strong>and</strong><br />
Midorikawa, 1982). Intraperitoneally injected <strong>of</strong><br />
ferric nitrilotriacetate (Fe-NTA) is absorbed into<br />
portal vein through mesothelium <strong>and</strong> passes into<br />
circulation via the liver (Umemura et al., 1990).<br />
The low molecular weight Fe-NTA is easily filtered<br />
through the glomeruli into the lumen <strong>of</strong> the renal<br />
proximal tubules where Fe 3+ -NTA is reduced to<br />
Fe 2+ -NTA by the glutathione degradation products<br />
cysteine or cysteinylglycine (Taso <strong>and</strong> Curthoys,<br />
1980). In the brush border surface <strong>of</strong> the renal<br />
proximal convoluted tubules, γ-glutamyl<br />
transpeptidase hydrolyses glutathione to<br />
cysteinylglycine that is rapidly degraded to cysteine<br />
<strong>and</strong> glycine by dipeptidase (Khan <strong>and</strong> Sultana,<br />
2005). Cysteinylglycine <strong>and</strong> cysteine are the<br />
proposed thiol reductants that reduce Fe 3+ -NTA to<br />
Fe 2+ -NTA. The auto-oxidation <strong>of</strong> Fe 2+ -NTA<br />
generates superoxide radicals (O .- 2) which<br />
subsequently potentiate the iron catalysed Haber-<br />
Weiss reaction to produce hydroxyl radical (OH . ),<br />
leading to induction <strong>of</strong> lipid peroxidation <strong>and</strong><br />
oxidative DNA damage (Umemura et al, 1990 <strong>and</strong><br />
Khan <strong>and</strong> Sultana, 2005).<br />
For the present study, we prepared the ethanol<br />
extract (80%) <strong>of</strong> the pomegranate peel which<br />
exerted the highest antioxidant effect in vitro. The<br />
objective <strong>of</strong> the study was to determine the possible<br />
effect <strong>of</strong> prophylactic treatment with pomegranate<br />
peel extract on Fe-NTA induced renal oxidative<br />
damage in rats.<br />
Materials <strong>and</strong> methods<br />
Plant material<br />
Pomegranate fruit peel purchased from local market<br />
was dried <strong>and</strong> powdered before extraction.<br />
Plant extract<br />
Powdered plant material (500g) was repeatedly<br />
extracted with 2000 ml solvents <strong>of</strong> increasing<br />
polarity starting with benzene, chlor<strong>of</strong>orm, ethyl<br />
acetate, ethanol (80%) <strong>and</strong> distilled water. The<br />
percolation time for each solvent was 24h. The<br />
extracts were filtered, concentrated <strong>and</strong> freeze<br />
dried. The residues yielded for each solvent were<br />
stored at 4 o C. The ethanol extract (80%) was used<br />
for further study after preliminary in vitro tests viz.<br />
lipid peroxidation, deoxyribose <strong>and</strong> DPPH assays.<br />
Chemicals<br />
Ferric nitrate, NTA disodium salt, reduced<br />
glutathione, 1-chloro-2,4-dinitrobenzene (CDNB),<br />
bovine serum albumin, 1,2-dithio-bis-nitrobenzoic<br />
acid (DTNB) <strong>and</strong> thiobarbituric acid (TBA) were<br />
obtained from Sigma Chemical (St Louis, USA).<br />
All solvents used were HPLC grade (Merck,<br />
Darmstadt, Germany).<br />
Total phenolics<br />
Total phenolics in the pomegranate peel ethanol<br />
extract were determined according to<br />
Jayaparakashsa et al. (2001) using Folin-Ciocalteu<br />
reagent. Four hundred microlitres <strong>of</strong> sample were<br />
taken in test tubes; 1.0 ml <strong>of</strong> Folin–Ciocalteu<br />
reagent (diluted 10-fold with distilled water) <strong>and</strong><br />
0.8 ml <strong>of</strong> 7.5% sodium carbonate were added. The<br />
tubes were mixed <strong>and</strong> allowed to st<strong>and</strong> for 30 min
<strong>and</strong> the absorption at 765 nm was measured against<br />
a blank, which contained 400 µl <strong>of</strong> ethanol in place<br />
<strong>of</strong> sample. The total phenolic content was<br />
expressed as gallic acid equivalents in mg/g <strong>of</strong><br />
ethanol extract.<br />
Animals<br />
Albino male rats (170±30 g) were used in the<br />
present study. The rats were obtained from the<br />
animal house <strong>of</strong> the National Organization for Drug<br />
Control <strong>and</strong> Research (NODCAR), Egypt. The<br />
animals were kept under st<strong>and</strong>ard laboratory<br />
conditions <strong>of</strong> light/dark cycle (12/12h) <strong>and</strong><br />
temperature (25±2˚C). The rats were allowed food<br />
<strong>and</strong> water ad libitum. They were provided with a<br />
nutritionally adequate st<strong>and</strong>ard laboratory diet.<br />
Animals’ diet<br />
The basal diet consists <strong>of</strong> casein 10%, cotton seed<br />
oil 4%, salt mixture 4%, vitamin mixture 1%,<br />
carbohydrates (sucrose, starch 1:1) 80.8% <strong>and</strong><br />
choline chloride 0.2% (American Institute <strong>of</strong><br />
Nutrition, 1980).<br />
Preparation <strong>of</strong> Fe-NTA solution<br />
The Fe-NTA solution was prepared as described in<br />
Deiana et al. (2001) <strong>and</strong> Khan <strong>and</strong> Sultana (2005),<br />
ferric nitrate <strong>and</strong> NTA disodium salt were dissolved<br />
in distilled water to form a 300 <strong>and</strong> 600 mM<br />
solution, respectively. The two solutions were<br />
combined in a volume ratio <strong>of</strong> 1:2 with magnetic<br />
stirring at room temperature <strong>and</strong> the pH was<br />
adjusted to 7.4 with sodium bicarbonate.<br />
Experimental design<br />
Thirty albino rats were r<strong>and</strong>omly allocated to five<br />
groups <strong>of</strong> six rats each:<br />
Group 1 received only saline injection<br />
intraperitoneally at a dose <strong>of</strong> 10 ml/kg body weight.<br />
Group 2 received only a single intraperitoneal<br />
injection <strong>of</strong> Fe-NTA solution at a dose <strong>of</strong> nine mg<br />
Fe/kg body weight (Athar <strong>and</strong> Iqbal 1998).<br />
Group 3 received pomegranate peel extract by<br />
gavage once daily for seven days at a dose <strong>of</strong> 100<br />
mg body weight, p.o. (Parmar <strong>and</strong> Kar, 2008).<br />
Group 4 received pomegranate peel extract once<br />
daily for seven days at a dose <strong>of</strong> 200 mg/kg body<br />
weight, p.o. (Parmar <strong>and</strong> Kar, 2008).<br />
Pomegranate peel extract against renal oxidative damage 37<br />
After the last treatment with pomegranate peel<br />
extract, the animals <strong>of</strong> group 2, 3 <strong>and</strong> 4 received a<br />
single intraperitoneal injection <strong>of</strong> Fe-NTA at a dose<br />
level <strong>of</strong> 9mg Fe/kg body weight.<br />
Group 5 received pomegranate peel extract orally<br />
once daily for seven days at a dose <strong>of</strong> 200 mg/kg<br />
body weight (Parmar <strong>and</strong> Kar, 2008). We used the<br />
high dose <strong>of</strong> pomegranate peel ethanol extract (200<br />
mg/kg b.w. p.o.) to study its effect on kidney.<br />
All rats were sacrificed 12 h after the treated<br />
with Fe-NTA. Blood was collected <strong>and</strong> the<br />
separated serum was used for the estimation <strong>of</strong><br />
creatinine (Bartless et al., 1972) <strong>and</strong> urea (Patton<br />
<strong>and</strong> Crouch, 1977).<br />
Post-mitochondrial supernatant <strong>and</strong> microsomal<br />
fraction preparation<br />
Kidneys were removed quickly <strong>and</strong> washed in cold<br />
isotonic saline. The kidneys were homogenized in<br />
50 mM phosphate buffer (pH 7) using an electronic<br />
homogenizer to prepare 10% w/v homogenate. The<br />
homogenate was centrifuged at 3000 rpm for 10<br />
min at 4 o C by cooling ultracentrifuge (model Sigma<br />
3K 30) to separate the nuclear debris. The aliquot<br />
so obtained was used at 12000 rpm for 20 min at<br />
4 o C to obtain post-mitochondrial supernatant<br />
(PMS), which was used as a source <strong>of</strong> enzymes<br />
(Khan <strong>and</strong> Sultana, 2005). A portion <strong>of</strong> the PMS<br />
was centrifuged for 60 min at 34000 rpm at 4 o C.<br />
The pellet was washed with phosphate buffer (50<br />
mM pH 7).<br />
Estimation <strong>of</strong> reduced glutathione (GSH) in PMS<br />
Reduced GSH in mitochondria was determined by<br />
measuring the rate <strong>of</strong> formation <strong>of</strong> chromophoric<br />
product in a reaction between 5,5́-dithiobis-2-<br />
(nitrobenzoic acid) (DTNB) <strong>and</strong> free sulphydryl<br />
groups, such as GSH, at 412 nm as described by<br />
Ellman (1959).<br />
Estimation <strong>of</strong> Lipid peroxidation (LPO) in<br />
micrososmal fraction<br />
The measurement <strong>of</strong> microsomal fraction lipid<br />
peroxide by a colorimetric reaction with<br />
thiobarbituric acid was done as described by<br />
Okhawa et al. (1979), <strong>and</strong> the determined lipid<br />
peroxide is referred to as malondialdehyde. Briefly,<br />
in a test tube, 2.5 ml <strong>of</strong> 20% trichloroacetic acid<br />
solution <strong>and</strong> 1ml <strong>of</strong> 0.67% thiobarbituric acid<br />
solution were added to the samples. The color <strong>of</strong><br />
thiobarbituric acid pigment was developed in a
38<br />
Mahgoub Mohammed AHMED <strong>and</strong> Safaa Eid ALI<br />
water bath at 100 ◦ C for 30 min. After cooling with<br />
tap water to room temperature, 4ml n-butanol was<br />
added <strong>and</strong> shaken vigorously. After centrifugation,<br />
the color <strong>of</strong> butanol layer was measured at 535 nm.<br />
Assay for glutathione-S-transferase (GST) activity<br />
in PMS<br />
Glutathione-S-transferase activity was assayed by<br />
the method <strong>of</strong> Habig et al. (1974). The method is<br />
based on the rate <strong>of</strong> conjugate formation between<br />
GSH <strong>and</strong> 1-chloro-2,4-dinitrobenzene (CDNB).<br />
The absorbance change was recorded at 340 nm<br />
<strong>and</strong> the enzyme activity calculated as nmol CDNB<br />
conjugates formed/min/mg protein.<br />
Assay for glutathione peroxidase (GPx) activity in<br />
PMS<br />
Glutathione peroxidase activity was assayed by the<br />
method <strong>of</strong> Moh<strong>and</strong>as et al. (1984). The change in<br />
absorbance was recorded spectrophotometrically at<br />
340 nm. GPx activity was expressed as nmol<br />
NADPH oxidized/min/mg protein.<br />
Assay for glutathione reductase (GR) activity in<br />
PMS<br />
Glutathione reductase activity was determined by<br />
the method <strong>of</strong> Carlberg <strong>and</strong> Mannervik (1975). GR<br />
was assayed by following the oxidation <strong>of</strong> NADPH<br />
at 340 nm at 37 o C. GR activity was expressed as<br />
nmol NADPH oxidized/min/mg protein.<br />
Assay for catalase (CAT) activity in PMS<br />
CAT activity measurement in PMS was measured<br />
by the method <strong>of</strong> Takahara et al. (1960). The<br />
reduction rate <strong>of</strong> H2O2 was followed at 240 nm for<br />
30 s at room temperature. CAT activity was<br />
expressed in nmol H2O2 consumed/min/mg protein.<br />
Assay for glucose-6-phosphate dehydrogenase<br />
(GPD) activity in PMS<br />
The activity <strong>of</strong> glucose-6-phosphate dehydrogenase<br />
was determined according to the method <strong>of</strong> Zaheer<br />
et al. (1965). The changes in absorbance were<br />
recorded at 340 nm <strong>and</strong> enzyme activity was<br />
calculated as nmol NADP reduced/min/mg protein.<br />
Estimation <strong>of</strong> protein concentration<br />
The protein concentration in all samples was<br />
determined by the method <strong>of</strong> Lowry et al. (1951).<br />
Statistical analysis<br />
The results are expressed as Mean±SEM. The<br />
collected data were statistically analyzed by the<br />
least significant differences (LSD) at the level 5%<br />
<strong>of</strong> the probability procedure according to Snedecor<br />
<strong>and</strong> Cochran (1980).<br />
Results<br />
Effect <strong>of</strong> pomegranate peel extract on renal toxicity<br />
markers<br />
The effect <strong>of</strong> pre-treatment <strong>of</strong> rats with<br />
pomegranate peel extract on Fe-NTA-induced<br />
enhancement in the level <strong>of</strong> serum creatinine <strong>and</strong><br />
urea are shown in Table (1). Fe-NTA treatment<br />
leads to about 147% <strong>and</strong> 303% enhancement in the<br />
values <strong>of</strong> creatinine <strong>and</strong> urea, respectively, as<br />
compared with saline-treated group. Prophylaxis<br />
with pomegranate peel extract at both doses<br />
resulted in 28-45% <strong>and</strong> 48-88% reduction in the<br />
values <strong>of</strong> serum creatinine <strong>and</strong> urea respectively as<br />
compared with Fe-NTA-treated group.<br />
Effect <strong>of</strong> pomegranate peel extract on glutathione<br />
metabolism<br />
Table (2) shows the effect <strong>of</strong> pretreatment <strong>of</strong><br />
rats with pomegranate peel extracts on Fe-NTAmediated<br />
renal glutathione content <strong>and</strong> on the<br />
activities <strong>of</strong> its metabolizing enzymes, viz,<br />
glutathione-S-transferase <strong>and</strong> glutathione reductase.<br />
Treatment with Fe-NTA alone resulted in the<br />
depletion <strong>of</strong> renal glutathione <strong>and</strong> reduction in the<br />
activities <strong>of</strong> glutathione-S-transferase <strong>and</strong><br />
glutathione reductase by 48%, 55% <strong>and</strong> 46%<br />
respectively, as compared with saline-treated<br />
group. However, pretreatment <strong>of</strong> animals with<br />
pomegranate peel extract at 100 <strong>and</strong> 200 mg/kg<br />
body weight resulted in the recovery by 79-83%,<br />
46-73% <strong>and</strong> 40-72% respectively, as compared<br />
with Fe-NTA-treated group.
Pomegranate peel extract against renal oxidative damage 39<br />
Table 1. Effect <strong>of</strong> pomegranate peel ethanol extract on Fe-NTA-induced enhancement <strong>of</strong> serum<br />
creatinine <strong>and</strong> urea in rats Values Mean±SEM (n=6 animals). a p
40<br />
Mahgoub Mohammed AHMED <strong>and</strong> Safaa Eid ALI<br />
Table 3. Effect <strong>of</strong> pomegranate peel ethanol extract on Fe-NTA-induced reduction in the activity <strong>of</strong><br />
renal antioxidant enzymes (CAT, GPx <strong>and</strong> GPD) <strong>and</strong> enhancement in the level <strong>of</strong> microsomal lipid<br />
peroxidation (LPO) in rats Values are Mean±SEM (n=6 animals). a p
<strong>of</strong>ten metabolized to proximate toxicants by phase I<br />
enzymes, e.g., cytochrome P450 which catalyze<br />
oxidative reactions. The oxidized metabolites <strong>of</strong><br />
potentially toxic xenobiotics are then detoxified by<br />
Phase II metabolizing enzymes into the forms that<br />
are relatively inert <strong>and</strong> more easily excreted<br />
(Talalay et al., 1995).<br />
GSH depletion increases the sensitivity <strong>of</strong> organ<br />
to oxidative <strong>and</strong> chemical injury. Studies with a<br />
number <strong>of</strong> models show that the metabolism <strong>of</strong><br />
xenobiotics <strong>of</strong>ten produced GSH depletion<br />
(Mitchell et al., 1973 <strong>and</strong> Ahmed <strong>and</strong> Zaki, 2009).<br />
The depletion <strong>of</strong> GSH, also, seems to be the prime<br />
factor that permits lipid peroxidation in the Fe-<br />
NTA treated group. Pretreatment <strong>of</strong> pomegranate<br />
peel extract reduced the depletion <strong>of</strong> GSH levels<br />
<strong>and</strong> provided protection to the kidney. The<br />
protection <strong>of</strong> GSH is by forming the substrate for<br />
GPx activity that can react directly with various<br />
aldehydes produced from the peroxidation <strong>of</strong><br />
membrane lipid. Pomegranate peel extract<br />
pretreatment also reduced the elevated levels <strong>of</strong><br />
serum urea <strong>and</strong> ceatinine that are marker<br />
parameters <strong>of</strong> kidney toxicity.<br />
In conclusion, we can say that, the high<br />
antioxidant <strong>and</strong> nephropreventive effect <strong>of</strong> the<br />
pomegranate peel extract appeared to be attributed<br />
to its high phenolics content. The mechanism <strong>of</strong><br />
action <strong>of</strong> pomegranate peel extract may be through<br />
induction <strong>of</strong> various antioxidant <strong>and</strong> phase II<br />
enzymes, <strong>and</strong> scavenging reactive oxygen species.<br />
Thus our data suggest that pomegranate peel<br />
ethanol extract is a potent nephropreventive agent.<br />
Further work is required for the isolation <strong>and</strong><br />
characterization <strong>of</strong> individual phenolic compounds<br />
present in peel ethanol extract <strong>and</strong> to determine the<br />
mechanisms involved in the nephropreventive<br />
effect <strong>of</strong> pomegranate peel extract.<br />
References<br />
Ahmed MM <strong>and</strong> Zaki NI. Assessment the<br />
ameliorative effect <strong>of</strong> pomegranate <strong>and</strong> rutin on<br />
chlorpyrifos-ethyl-induced oxidative stress in<br />
rats. Nature <strong>and</strong> Science. 7(10): 49-61, 2009.<br />
Ahmed MM. Biochemical studies on<br />
nephroprotective effect <strong>of</strong> carob (Ceratonia<br />
siliqua L.) growing in Egypt. Nature <strong>and</strong><br />
Science. 8 (3): 41-47, 2010.<br />
Ajaikumar K.B, Asheef, M, Babu BH <strong>and</strong><br />
Padikkala J. The inhibition <strong>of</strong> gastric mucosal<br />
injury by Punica granatum L. (pomegranate)<br />
Pomegranate peel extract against renal oxidative damage 41<br />
methanolic extract. <strong>Journal</strong> <strong>of</strong> Ethnopharma.<br />
96: 171–176, 2005.<br />
American Institute <strong>of</strong> Nutrition. Report <strong>of</strong> the<br />
American Institute <strong>of</strong> Nutrition. Ad Hoc<br />
Committee J Nutr. 110: 1340-1348, 1980.<br />
Aruoma OI. Methodological considerations for<br />
characterizing potential antioxidant actions <strong>of</strong><br />
bioactive components in plant foods. Mutation<br />
Research. (523-524): 9-20, 2003.<br />
Athar M <strong>and</strong> Iqbal M. Ferric nitrilotriacetate<br />
promotes N-diethylnitrosamine-induced renal<br />
tumorigenesis in the rat: implications for the<br />
involvement <strong>of</strong> oxidative stress.<br />
Carcinogenesis. 19 (6):1133-1139, 1998<br />
Ayrton AD, Lewis DF, Waker R. <strong>and</strong> Ioannides C.<br />
Antimutagenicity <strong>of</strong> ellagic acid towards the<br />
food mutagen IQ: investigation into possible<br />
mechanisms <strong>of</strong> action. Food <strong>and</strong> Chemical<br />
Toxicology, 3D: 289-295, 1992.<br />
Bartles H, Bohmer M <strong>and</strong> Heieri C. Serum<br />
keratinin bestmmung ohno enteeissen. Clinical<br />
Chimca Acta. 37: 139-197, 1972.<br />
Bu-Abbas A, Clifford MN, Walker R <strong>and</strong> Ioannides<br />
C. Marker antimutagenic potential <strong>of</strong> aquous<br />
green tea extracts: Mechanism <strong>of</strong> action.<br />
Mutagenesis. 9: 325-331, 1994.<br />
Carlberg I <strong>and</strong> Mannervik B. Glutathione level in<br />
rat brain. J Biological Chemistry. 250: 4480-<br />
4575, 1975.<br />
De Flora S <strong>and</strong> Ramel C. Mechanism <strong>of</strong> inhibition<br />
<strong>of</strong> mutagenesis <strong>and</strong> carcinogenesis: classification<br />
<strong>and</strong> overview. Mutation Research. 202:<br />
285-306, 1988.<br />
De Freitas JM <strong>and</strong> Meneghini R. Iron <strong>and</strong> its<br />
sensitive balance in the cell. Mutation Research.<br />
475: 153-159, 2001.<br />
Deiana M, Aruma OI, Rosa A, Crobu V, Piga R<br />
<strong>and</strong> Derri MA. The effect <strong>of</strong> ferricnitrilotriacetic<br />
acid on the pr<strong>of</strong>ile <strong>of</strong><br />
polyunsaturated fatty acids in the kidney <strong>and</strong><br />
liver <strong>of</strong> rats. Toxicol Letters. 123: 125-133,<br />
2001.<br />
Dhawan BN, Patnaik GK, Rastogi RP, Singh KK<br />
<strong>and</strong> T<strong>and</strong>on JS. Screening <strong>of</strong> Indian plants for<br />
biological activity. Indian J. Exp. Biol. 15: 208-<br />
219, 1977.<br />
Ellman GL. Tissue sulfhydryl groups. Arch<br />
Biochem Biophys. 82: 70-77, 1959.<br />
Gil MI, Tomas-Barberan FA, Hess Pierce B,<br />
Holcr<strong>of</strong>t, DM. <strong>and</strong> Kader AA. Antioxidant
42<br />
Mahgoub Mohammed AHMED <strong>and</strong> Safaa Eid ALI<br />
activity <strong>of</strong> pomegranate juice <strong>and</strong> its<br />
relationship with phenolic composition <strong>and</strong><br />
processing. <strong>Journal</strong> <strong>of</strong> Agricultural <strong>and</strong> food<br />
chemistry. 48: 4581-4589, 2000.<br />
Goldstein RS <strong>and</strong> Mayor GH. The nephrotoxicity<br />
<strong>of</strong> cisplatin. Life Sci. 32: 685-690, 1983.<br />
Gujraj ML, Varma DR <strong>and</strong> Sareen KN. Oral<br />
contraceptives. Part 1. Preliminary observations<br />
on the antifertility effects <strong>of</strong> some indigenous<br />
drugs. Indian J Med Res. 48: 46-51, 1960.<br />
Habig WH, Pabst MJ <strong>and</strong> Jakoby WB. Glutathione-<br />
S-transferase. The first enzymatic step in<br />
mercapturic acid formation. J Biol Chem. 249:<br />
7130-7139, 1974.<br />
Halliwell, B. <strong>and</strong> Gutteridge, J.M.C. Iron <strong>and</strong> free<br />
radicals: two aspects <strong>of</strong> antioxidant protection.<br />
Trends Biochem. Sci. 11: 372-375, 1986.<br />
Hontecillas R, O'Shea M, Einerh<strong>and</strong> A, Diguardo<br />
M, Bassaganya-Riera J. Activation <strong>of</strong> PPAR<br />
gamma <strong>and</strong> alpha by punicic acid ameliorates<br />
glucose tolerance <strong>and</strong> suppresses obesity-related<br />
inflammation. J Am Coll Nutr. 28(2):184-95,<br />
2009.<br />
Khan N <strong>and</strong> Sultana S. Chemomodulatory effect <strong>of</strong><br />
Ficus racemosa extract against chemically<br />
induced renal carcinogenesis <strong>and</strong> oxidative<br />
damage response in Wistar rats. Life Sciences.<br />
77(11):1194-210, 2005.<br />
Kulkarni AP, Mahal HS, Kapoor S, Aradhya SM.<br />
In vitro studies on the binding, antioxidant, <strong>and</strong><br />
cytotoxic actions <strong>of</strong> punicalagin. J Agric Food<br />
Chem. 55(4):1491-500, 2007.<br />
Li Y, Guo C, Yang J, Wei J, Xu J <strong>and</strong> Cheng S.<br />
Evaluation <strong>of</strong> antioxidant properties <strong>of</strong><br />
pomegranate peel extract in comparison with<br />
pomegranate pulp extract. Food Chemistry. 96:<br />
254-260, 2006.<br />
Lowry OH, Roseborough. NJ, Farr AL, <strong>and</strong><br />
R<strong>and</strong>all RL. Protein measurement with phenol<br />
reagent. <strong>Journal</strong> <strong>of</strong> Biological Chemistry. 193<br />
(1): 265-275, 1951.<br />
Mitchell JR, Jollow DJ, Potter WZ, Gillete JR <strong>and</strong><br />
Brodie BB. Acetaminophen induced hepatic<br />
necrosis. Protective role <strong>of</strong> glutathione. J.<br />
Pharmacol. Exp. Ther. 187: 211-215, 1973.<br />
Moh<strong>and</strong>as M, Marshall JJ, Duggin, GG, Hovath JS<br />
<strong>and</strong> Tiller D. Differential distribution <strong>of</strong><br />
glutathione <strong>and</strong> glutathione related enzymes in<br />
rabbit kidney. Cancer Research. 44: 586-5091,<br />
1984.<br />
Murthy KN, Jayaprakasha GK <strong>and</strong> Singh RP.<br />
Studies on antioxidant activities <strong>of</strong> pomegranate<br />
peel extract using in vivo models. J Agri Food<br />
Chem. 50: 4791-4795, 2002.<br />
Nasr CB, Ayed N <strong>and</strong> Metche M. Quantitative<br />
determination <strong>of</strong> polyphenolic content <strong>of</strong><br />
pomegranate peel. Zeitschrzfi fur lebensmittel<br />
unterschung und forschung. 203: 374-378,<br />
1996.<br />
Ohkawa H, Ohishi N <strong>and</strong> Nagi K. Assay <strong>of</strong> lipid<br />
peroxides in animal tissue by thiobarbituric acid<br />
reaction. Anal Biochem. 95: 251-358, 1979.<br />
Okada S <strong>and</strong> Midorikawa O. Induction <strong>of</strong> rat renal<br />
adenocarcinoma by ferric nitrilotriacetate (Fe-<br />
NTA). Japanese Archives <strong>of</strong> International<br />
Medicine. 29: 485-491, 1982.<br />
Parmar HS, Kar A. Medicinal values <strong>of</strong> fruit peels<br />
from Citrus sinensis, Punica granatum, <strong>and</strong><br />
Musa paradisiaca with respect to alterations in<br />
tissue lipid peroxidation <strong>and</strong> serum<br />
concentration <strong>of</strong> glucose, insulin, <strong>and</strong> thyroid<br />
hormones. J Med Food. 11(2):376-381, 2008.<br />
Patton CJ <strong>and</strong> Crouch SR. Spectrophotometric <strong>and</strong><br />
kinetics investigation <strong>of</strong> the Berthelot reaction<br />
for the determination <strong>of</strong> ammonia. Analytical<br />
Chemistry. 49: 464-469, 1977.<br />
Ross IA. Medicinal plants <strong>of</strong> the world. Humana<br />
Press, Totowa, New Jersey. 273-281, 1999.<br />
Sato A. Cancer chemotherapy with oriental<br />
medicine. 1. Antitumor activity <strong>of</strong> crude drugs<br />
with human tissue cultures, in In vitro<br />
screening. Int J Orient Med. 15 (4): 171-183,<br />
1990.<br />
Snedecor GW <strong>and</strong> Cochran WG. Statistical<br />
methods. 7th ed . IOWA Stat Univ. Press, IOWA,<br />
USA. 420, 1980.<br />
Takahara S, Hamilton BM, Nell JV, Ogura, Y <strong>and</strong><br />
Nishimura ET. Hypocatalasemia, a new genetic<br />
carrier state. J Clin Invest. 29: 610-619, 1960.<br />
Talalay P, Fahey JW, Holtzclaw WD, Prestera, T<br />
<strong>and</strong> Zhang Y. Chemoprotection against cancer<br />
by phase II enzyme induction. Toxicology<br />
Letters. (82-83): 173-179, 1995.<br />
Thring TS, Hili P, Naughton DP. Anti-collagenase,<br />
anti-elastase <strong>and</strong> anti-oxidant activities <strong>of</strong><br />
extracts from 21 plants. BMC Complementary<br />
<strong>and</strong> Alternative Medicine. 9: 17-27, 2009.<br />
Tsao B <strong>and</strong> Cuthoys NP. The absolute asymmetry<br />
<strong>of</strong> prientation <strong>of</strong> gamma glutamyl<br />
transpeptidase <strong>and</strong> amino-peptidase on the<br />
external surface <strong>of</strong> the rat renal brush border
membrane. J Biological Chemistry. 255: 7708-<br />
7711, 1980.<br />
Umemura T, Sai K, Takagi, A, Hasegawa R <strong>and</strong><br />
Kurokawa Y. Oxidative DNA damage, lipid<br />
peroxidation <strong>and</strong> nephrotoxicity induced in the<br />
Pomegranate peel extract against renal oxidative damage 43<br />
kidney after ferric nitilotriacetate administration.<br />
Cancer Letter. 54(1-2): 95-100, 1990.<br />
Zaheer N, Tiwari KK <strong>and</strong> Krishnan PS. Exposure<br />
<strong>and</strong> solubilization <strong>of</strong> hepatic mitochondrial<br />
shunt dehydrogenases. Archive <strong>of</strong> Biochemistry<br />
<strong>and</strong> Biophysics. 109: 646-648, 1965.
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong> 7(2) & 8(1): 45-52, 2010 Research Article<br />
<strong>Haliç</strong> University, Printed in Turkey.<br />
http://jcmb.halic.edu.tr<br />
<strong>Molecular</strong> <strong>and</strong> cytogenetic evaluation <strong>of</strong> Y chromosome in<br />
spontaneous abortion cases<br />
Gülşah KOÇ 1 , Korkut ULUCAN *,1 , Deniz KIRAÇ 2 , Deniz ERGEÇ 1 , Tufan TARCAN 3<br />
<strong>and</strong> A. İlter GÜNEY 1 .<br />
1 Marmara University, Faculty <strong>of</strong> Medicine, Department <strong>of</strong> Medical Genetics, Istanbul, Turkey.<br />
2 Yeditepe University, Faculty <strong>of</strong> Medicine, Department <strong>of</strong> Biochemistry, Istanbul Turkey.<br />
3 Marmara University, Faculty <strong>of</strong> Medicine, Department <strong>of</strong> Urology, Istanbul, Turkey.<br />
(* author for correspondence; korkutulucan@hotmail.com)<br />
Received: 21 April 2010; Accepted: 05 May 2010<br />
Abstract<br />
Infertility is defined as not being able to get pregnant despite having frequent, unprotected sex for at least a year.<br />
Several conditions contribute to infertility <strong>and</strong> 50% is considered to be caused by a male-related factor.<br />
Spontaneous abortion (SAB) is noninduced embryonic or fetal death or passage <strong>of</strong> products <strong>of</strong> conception before<br />
the 20th week <strong>of</strong> pregnancy <strong>and</strong> is the most common complication <strong>of</strong> early pregnancy. SAB can occur by<br />
teratogenic factors, advanced maternal age, genetic factors such as Y chromosome microdeletions <strong>and</strong><br />
chromosomal anomalies. In order to investigate the etiology <strong>of</strong> recurrent pregnancy loss (RPL) <strong>and</strong> to develop an<br />
appropriate therapeutic strategy, it is necessary to ascertain the molecular <strong>and</strong> cytogenetic basis <strong>of</strong> these defects. In<br />
this study, we aimed to reveal the relations between male infertility, Y chromosome microdeletions <strong>and</strong> SAB.<br />
Thirty couples with a spontaneous abortion history <strong>and</strong> thirty fertile men were recruited to the study. All the<br />
women were 46, XX <strong>and</strong> men were 46, XY. We couldn’t detect any Y chromosome microdeletion that could be the<br />
reason for SAB. In order to evaluate effect <strong>of</strong> chromosome anomalies <strong>and</strong> Y chromosome microdeletions on SAB,<br />
further studies with increased number <strong>of</strong> cases <strong>and</strong> controls need to be carried on.<br />
Keywords: Infertility, spontaneous abortion, Y chromosome microdeletions.<br />
Spontan düşük vakalarında Y kromozomunun moleküler ve sitogenetik incelemesi<br />
Özet<br />
Çiftlerin çocuk sahibi olma isteklerine ve düzenli cinsel ilişkiye rağmen, bir yıl içerisinde gebelik elde<br />
edilmemesine infertilite (kısırlık) adı verilmektedir. İnfertiliteye etki eden birçok faktör bulunmaktadır ve bunların<br />
%50’sinde etken erkek infertilitesidir. Gebeliğin ilk 20 haftası içinde, dışarıdan herhangi bir müdahale olmadan,<br />
doğal nedenlerle, embriyo veya fetus ve eklerinin tamamının veya bir kısmının uterus kavitesi dışına atılması<br />
olayına spontan düşük (abortus) denilmektedir ve gebeliğin erken döneminde en çok gözlenen komplikasyondur.<br />
Spontan düşükler, teratojenik faktörler, ileri anne yaşı gibi nedenlerin yanında, Y kromozomu mikrodelesyonları ve<br />
kromozomal anomaliler gibi genetik faktörlere bağlı olarak da oluşabilmektedir. Tekrarlayan gebelik kayıplarının<br />
etyolojisini belirlemek ve uygun bir tedavi yöntemi geliştirmek için bu defektlerin moleküler ve sitogenetik<br />
temellerinin incelenmesi gerekmektedir. Bu çalışmada, erkek infertilitesi, Y kromozom mikrodelesyonları ve<br />
spontan düşükler arasındaki ilişkinin ortaya çıkarılması amaçlanmıştır. Spontan düşük hikayesi bulunan 30 çift ve<br />
fertil 30 erkek çalışmaya dahil edilmiştir. Çalışmaya dahil olan bireylerin kromozom analizi sonuçlarına göre, tüm<br />
kadınlar 46,XX ve erkekler ise 46,XY‘dir. Çalışmamızda spontan düşüklere neden olabilecek herhangi bir Y<br />
kromozom mikrodelesyonu belirlenememiştir. Kromozom anomalilerinin ve Y kromozomu mikrodelesyonlarının<br />
spontan düşükler üzerindeki etkisinin değerlendirilebilmesi için vaka ve kontrol sayılarının arttırılarak başka<br />
çalışmalar yapılması gerekmektedir.<br />
Anahtar Sözcükler: İnfertilite, spontan düşük, Y kromozom mikrodelesyonu.
46<br />
Gülşah Koç et. al.<br />
Introduction<br />
Infertility is the inability <strong>of</strong> being pregnant after<br />
one year <strong>of</strong> unprotected sexual intercourse.<br />
Infertility comprises up to 15% <strong>of</strong> couples <strong>of</strong><br />
reproductive age in which 50% is caused by a male<br />
factor (Noordam <strong>and</strong> Repping, 2006). Several<br />
factors contribute to male infertility, such as gene<br />
defects, hormonal milieu, chromosomal aberrations<br />
<strong>and</strong> genital infections (Stipoljev et al., 2006).<br />
Genetic factors are considered to affect almost 30%<br />
<strong>of</strong> severe male infertility cases (Noordam <strong>and</strong><br />
Repping, 2006). The diagnosis <strong>of</strong> male infertility<br />
include anamnesis, physical examination, semen<br />
analysis, hormonal screening <strong>and</strong> genetic factors <strong>of</strong><br />
somatic cells (Stipoljev et al., 2006).<br />
Spontaneous abortion (SAB) is the expulsion <strong>of</strong><br />
an embryo or fetus due to accidental trauma or<br />
natural causes before approximately 22 nd week <strong>of</strong><br />
gestation. It effects up to 15% clinically recognized<br />
pregnancies <strong>and</strong> considered to be the most common<br />
adverse outcome <strong>of</strong> pregnancy. Although several<br />
studies tried to explain the etiology <strong>of</strong> SAB, the<br />
results are still controversial. Beside the teratogenic<br />
factors <strong>and</strong> advanced maternal age, genetic factors<br />
such as Y chromosome microdeletions <strong>and</strong><br />
chromosomal anomalies are considered to be the<br />
main reason <strong>of</strong> SAB (Dewan et al., 2006; Pryor et<br />
al., 1997).<br />
Y chromosome is essential not only for human<br />
sex determination but also for maintenance <strong>of</strong> sex<br />
cells <strong>and</strong> sex cell development. Y chromosome<br />
(Yq) microdeletions represent the most frequent<br />
molecular genetic cause <strong>of</strong> severe infertility,<br />
observed with a prevalence <strong>of</strong> 10-15% in nonobstructive<br />
azoospermia <strong>and</strong> severe oligozoospermia<br />
(Sinclair et al.,1990). The regions<br />
responsible for male infertility <strong>of</strong> Y chromosome<br />
are located on the long arm <strong>of</strong> chromosome <strong>and</strong> are<br />
termed as AZFa, AZFb, AZFc (AZF: Azoospermia<br />
Factor) ( Burgoyne, 1998) (Stouffs et al., 2009).<br />
The AZFa locus is located on proximal Yq11<br />
(Yq11.21), while AZFb <strong>and</strong> AZFc are located on<br />
distal Yq11 (Yq11.23). These AZF genes code<br />
RNA binding proteins <strong>and</strong> may be involved in the<br />
regulation <strong>of</strong> gene expression, RNA metabolism,<br />
RNA packaging <strong>and</strong> RNA transportation from<br />
nucleus to cytoplasm (Li et al., 2008). Deletions <strong>of</strong><br />
these regions result in spermatogenic arrest <strong>and</strong> are<br />
associated with oligozoospermia, azoospermia <strong>and</strong><br />
also with a extended testis histological pr<strong>of</strong>ile range<br />
from Sertoli cell only (SCO), maturation arrest <strong>and</strong><br />
hypospermatogenesis (Vollrath, 1992) (Vogt et al.,<br />
1996) (Briton-Jones <strong>and</strong> Haines, 2000).<br />
The prevalence <strong>of</strong> the Y chromosome<br />
microdeletions in the proximal AZFc region was<br />
found higher in men from recurrent pregnancy loss<br />
(RPL) couples than from fertile or infertile couples.<br />
Although these patients are from a tertiary referral<br />
center that may not reflect the population<br />
informations, one may consider proximal AZFc<br />
region detecting in the evaluation <strong>of</strong> RPL couples<br />
when all other tests fail to reveal the etiology<br />
(Dewan et al., 2006).<br />
Before performing a molecular test, cytogenetic<br />
analysis is necessary for an accurate approach to<br />
elucidate the causes <strong>of</strong> spontaneous abortion.<br />
Chromosomal anomalies which may cause male<br />
infertility can be determined by cytogenetic<br />
techniques. It is also known that approximately<br />
50% <strong>of</strong> recurrent spontaneous abortions in the first<br />
trimester is caused by chromosomal anomalies.<br />
Besides these, recent data show that Y chromosome<br />
microdeletions can also be a major factor in these<br />
cases. These findings suggest a potential relation<br />
between RPL <strong>and</strong> microdeletions in AZF regions.<br />
In order to investigate the etiology <strong>of</strong> RPL <strong>and</strong><br />
to develop an appropriate therapeutic strategy, it is<br />
necessary to ascertain the molecular <strong>and</strong><br />
cytogenetic basis <strong>of</strong> these defects. So in this study,<br />
we aimed to reveal the relations between male<br />
infertility, Y chromosome microdeletions <strong>and</strong><br />
recurrent spontaneous abortions.<br />
Material <strong>and</strong> methods<br />
Patient <strong>and</strong> Control Groups<br />
Thirty couples that applied to Marmara University,<br />
Department <strong>of</strong> Urology <strong>and</strong> Kartal Education <strong>and</strong><br />
Research Hospital with a spontaneous abortion<br />
history were recruited to the study. Thirty fertile<br />
men, at least having one child, were examined as<br />
the control group. Written informed consent was<br />
taken from all cases.<br />
Chromosome Analyses from Peripheral Blood<br />
<strong>Cell</strong> Culture<br />
Lymphocytes from 400 µl peripheral blood were<br />
cultured for 72 hours at 37ºC culture medium<br />
containing 8.5 ml RPMI, 1.5 ml fetal bovine serum,
200 µl L-Glutamin, 20 µl penicillin- streptomycin<br />
<strong>and</strong> 200 µl phytohaemagglutinin. After incubation<br />
at 37ºC for 72 hours, 200 µl Colchicine was added<br />
to arrest the cells at metaphase. Following an<br />
additional incubation at 37ºC for 30 minutes <strong>and</strong><br />
centrifugation at 20ºC for 8 min. at 1500 rpm the<br />
supernatant was removed. The pellet was resuspended<br />
with up to 10 ml hypotonic solution<br />
(0.4% KCl solution) vortexed immediately. All the<br />
samples were kept at 37ºC for 20 minutes <strong>and</strong><br />
again centrifuged at the same condition. After<br />
removing supernatant from the samples, the pellet<br />
which contains cells at metaphase, was<br />
homogenised. Fixative solution (methanol <strong>and</strong><br />
acetic acid mixed with 3:1 ratio) was added <strong>and</strong> the<br />
tubes were vortexed for the fixation <strong>of</strong><br />
chromosomes. Then samples were centrifuged after<br />
adding up to 5 ml <strong>of</strong> fixative solution. Supernatant<br />
was discarded from the samples <strong>and</strong> fresh fixative<br />
solution was added to the tubes. This procedure<br />
was repeated until the samples were clarified.<br />
According to the cell density, up to 0.5 ml fixative<br />
solution was added to the samples. Then samples<br />
were homogenized <strong>and</strong> cells were lied onto slide<br />
glasses, which were kept at 4ºC in distilled water<br />
till they are used. After spreading the cells on the<br />
slides, the samples were dried at room temperature<br />
<strong>and</strong> kept overnight at 60ºC.<br />
Y chromosome microdeletions in spontaneous abortions<br />
Karyotyping<br />
GTG (Giemsa-Trypsin) b<strong>and</strong>ing technique was<br />
performed. When the b<strong>and</strong>ing <strong>of</strong> the chromosomes<br />
was not successful, the protocol was repeated.<br />
After staining, at least 20 metaphase plaques were<br />
analysed for each sample (Figure 1).<br />
Detection <strong>of</strong> Y chromosome microdeletions<br />
DNA isolation from blood<br />
DNA was extracted from 200 µl peripheral blood by<br />
using High Pure PCR Template Preparation Kit<br />
(Roche-Germany) according to the manufacturer’s<br />
protocol.<br />
Multiplex polymerase chain reaction<br />
(multiplex PCR)<br />
Table 1. Primers used for multiplex PCR <strong>and</strong> the length <strong>of</strong> amplicons.<br />
MIX1 Amlicon<br />
length (bp)<br />
MIX2 Amlicon<br />
length (bp)<br />
For detection <strong>of</strong> Y chromosome microdeletions,<br />
isolated DNA was amplified by multiplex PCR. AB<br />
ANALITICA–The AZF Extension Kit, which is<br />
recommended by European Andrology Association<br />
was used in multiplex PCR. By using this kit, 13<br />
different regions could be investigated at the same<br />
time by performing 3 multiplex PCRs for each<br />
sample. Three primer sets, each containing primers<br />
that is unique to ZFX/Y locus which also exist in X<br />
chromosome are shown in Table 1.<br />
MIX3 Amlicon<br />
length (bp)<br />
ZFX/Y 495 ZFX/Y 495 DBY 689<br />
SRY 472 SRY 472 ZFX/Y 495<br />
sY 254 380 sY 95 303 SRY 472<br />
sY 86 320 sY 117 262 sY 84 326<br />
sY 127 274 sY 125 200 sY 134 301<br />
sY 255 120 DFFRY 155<br />
47
48<br />
Gülşah Koç et. al.<br />
In addition to the mixtures which are found in<br />
the AZF Extension Kit, 0.3µl Taq DNA polymerase<br />
<strong>and</strong> 8µl DNA sample were added to each tube<br />
during multiplex PCR. The conditions <strong>of</strong> PCR<br />
amplification were as follows: a denaturation step at<br />
94˚C for 5 min followed by 35 cycles at 94˚C for 1<br />
min, 60˚C for 1 min, 72˚C for 1 min <strong>and</strong> a final<br />
extension at 72˚C for 7 min <strong>and</strong> stop at 4˚C. After<br />
multiplex PCR, products were electrophoresed on<br />
2% agarose gel.<br />
Results<br />
Karyotyping<br />
Figure 1. Karyotype analyses <strong>of</strong> a male (46, XY) patient.<br />
After performing lymphocyte cell culture,<br />
metaphase plaques were analyzed for the detection<br />
<strong>of</strong> karyotypes <strong>of</strong> patient <strong>and</strong> control groups.<br />
According to karyotype analyses, all the males <strong>and</strong><br />
females were found as 46, XY <strong>and</strong> 46, XX<br />
respectively in the patient group, whereas all the<br />
males were found as 46, XY in the control group<br />
(Figure 1).<br />
Detection <strong>of</strong> Y chromosome microdeletions<br />
After multiplex PCR, PCR products were examined<br />
by electrophoresis on 2% agarose gel. Y<br />
chromosome microdeletions were not found in<br />
patient <strong>and</strong> control groups.
Y chromosome microdeletions in spontaneous abortions<br />
Figure 2. Multiplex PCR analyses <strong>of</strong> Y chromosome microdeletions (M: 50 bp ladder (Fermentas,<br />
Germany); Mix1a, Mix1b <strong>and</strong> Mix1c: 3 sets <strong>of</strong> PCR reactions that amplify different loci on Y chromosome<br />
for sample a; Mix1b, Mix2b, Mix3b for sample b; boxes indicate the region <strong>and</strong> the length <strong>of</strong> the amplicons.<br />
Discussion<br />
Chromosomal abnormalities, including translocations<br />
<strong>and</strong> deletions, are higher in infertile men <strong>and</strong><br />
are recognized as one <strong>of</strong> the main causes <strong>of</strong><br />
spontaneous abortions with an estimated frequency<br />
<strong>of</strong> 50–70% (Svetlana et al., 2005)<br />
In couples experiencing RPL, the incidence <strong>of</strong><br />
chromosomal translocations is higher than the<br />
incidence present in newborn series (De Braekeleer<br />
<strong>and</strong> Dao 1991). There is also evidence which<br />
indicates that the presence <strong>of</strong> translocations<br />
changes the spermatogenic process. It has been<br />
found that the incidence <strong>of</strong> reciprocal translocation<br />
carriers is seven times more than in newborn series.<br />
As a general rule reciprocal translocation carriers<br />
produce more unbalanced sperm than normal or<br />
balanced sperm. The proportion <strong>of</strong> unbalanced<br />
forms depends on the characteristics <strong>of</strong> the<br />
reorganization. Also deletions which remove Y<br />
chromosomal genes required for spermatogenesis<br />
may effect infertility <strong>and</strong> susceptibility <strong>of</strong> RPL<br />
(Byrne <strong>and</strong> Ward, 1994) (Simpson, 1981). As the<br />
severity <strong>of</strong> the spermatogenic defect increases, the<br />
frequency <strong>of</strong> the microdeletions also increases.<br />
In this study, primarily, cytogenetic evaluation<br />
was performed from peripheral blood samples <strong>of</strong><br />
the couples in spontaneous abortion cases. 30<br />
couples who had a spontaneous abortion history<br />
were karyotyped to detect the chromosome<br />
anomalies. According to karyotype analyses, all the<br />
women <strong>and</strong> men were found to be 46, XX <strong>and</strong> 46,<br />
XY, respectively. In our study we couldn’t detect<br />
any numerical <strong>and</strong> structural chromosome<br />
anomalies that can be detected by karyotype<br />
analyses. Other genetic abnormalities such as Y<br />
chromosome microdeletions may effect spermatogenesis,<br />
fertilization <strong>and</strong> post-zygotic metabolism<br />
<strong>and</strong> may influence male infertility <strong>and</strong> RPL.<br />
49
50<br />
Gülşah Koç et. al.<br />
So we used multiplex PCR for the detection <strong>of</strong><br />
microdeletions on the long arm <strong>of</strong> the Y<br />
chromosome.<br />
In this study, “AB ANALITICA–The AZF<br />
Extension Kit” used for the analysis <strong>of</strong><br />
microdeletions rather than AZF-MX Extension kit.<br />
Diagnostic sensitivity is considered to be the<br />
capacity <strong>of</strong> the device to correctly identify the<br />
deleted samples with reference to AZF locus under<br />
investigation. The results obtained from an<br />
experimental investigation show that the diagnostic<br />
sensitivity <strong>of</strong> the system is 100%.<br />
The kit is in premix format as all the reagents<br />
for the amplification are pre-mixed <strong>and</strong> aliquoted in<br />
single dose tubes in which only additional Taq<br />
polymerase <strong>and</strong> the extracted DNA should be<br />
added. This premix format allows the reduction <strong>of</strong><br />
the manipulation in preamplification steps, with<br />
considerable time saving for the operator, the<br />
repeated freezing/thawing <strong>of</strong> reagents (that could<br />
alter the products’ performances) is avoided <strong>and</strong>,<br />
above all, this form minimizes the risk <strong>of</strong> sample<br />
contamination <strong>and</strong> the risk <strong>of</strong> false positive results.<br />
The amplified regions <strong>of</strong> the Y chromosome are<br />
not polymorphic <strong>and</strong> are well known to be deleted<br />
specifically in men affected by oligo/azoospermia<br />
according to the known, clinically relevant<br />
microdeletion pattern (Viswambharan, 2007).<br />
Based on the experience <strong>of</strong> many laboratories <strong>and</strong><br />
the results <strong>of</strong> external quality control <strong>and</strong><br />
considering the multiplex PCR format, the first<br />
choice <strong>of</strong> STS primers recommended in the first<br />
version <strong>of</strong> the guidelines remains basically valid.<br />
These primers include the regions:<br />
For AZFa: sY84, sY86<br />
For AZFb: sY127, sY134<br />
For AZFc: sY254, sY255<br />
The usage <strong>of</strong> this primer set will enable the<br />
detection <strong>of</strong> almost all the clinically relevant<br />
deletions <strong>and</strong> <strong>of</strong> over 95% <strong>of</strong> the deletions reported<br />
in the literature in the three AZF regions <strong>and</strong> is<br />
sufficient for routine analysis (Simoni, 2004).<br />
In this study, the set <strong>of</strong> PCR primers as best<br />
choice for the diagnosis <strong>of</strong> microdeletion <strong>of</strong> the<br />
AZFa, AZFb <strong>and</strong> AZFc region (sY14 (SRY),<br />
ZFX/ZFY, sY84, sY86, sY127, sY134, sY254,<br />
sY255) used in multiplex PCR reactions. We<br />
couldn’t detect any Y chromosome microdeletions<br />
in AZFa, AZFb <strong>and</strong> AZFc regions.<br />
Genes that are located on Y chromosome <strong>and</strong><br />
responsible from spermatogenesis have a mosaic<br />
structure at somatic <strong>and</strong>/or germ cells. When<br />
leukocytes from blood were used, usually the<br />
results can not be suitable for Y chromosome<br />
microdeletion analysis because there may have<br />
been deletions in germ cells (Martin, 2008).<br />
There may be a mosaicism between<br />
seminiferous tubules in terms <strong>of</strong> the expression <strong>of</strong><br />
genetic material. Some seminiferous tubules have<br />
aplasia whereas some tubules can be normal or<br />
mutant arrest at testes. In the identification <strong>of</strong><br />
deletions this situation may show different<br />
outcomes when cells from blood or semen were<br />
used. When fibroblasts or leukocytes are used in<br />
genetic analysis, the proportion <strong>of</strong> a detection <strong>of</strong> Y<br />
chromosome microdeletion is slightly low because<br />
the deletions occuring in germ line cells have an<br />
independent nature from other tissues.<br />
In this study, we used peripherial blood<br />
leukocytes for the detection <strong>of</strong> Y chromosome<br />
microdeletions, however we couldn’t find any<br />
deletions. But the possibility <strong>of</strong> having deletions in<br />
germline cells shouldn’t be omitted. We are looking<br />
forward to extend our study by adding spermial Y<br />
chromosome microdeletion analysis from the same<br />
individuals.<br />
Dewan et al. (2006) reported the relation<br />
between RPL <strong>and</strong> proximal AZFc deletions <strong>and</strong><br />
found a significant correlation. Although, they<br />
detected proximal Y chromosome AZFc<br />
microdeletions in 14 <strong>of</strong> 17 patients (82%), they<br />
couldn’t find any deletion in control group.<br />
Karaer et al. (2008) reported 43 infertile men<br />
among which 7 <strong>of</strong> them have sY 220 (AZFb)<br />
deletions (16%) <strong>of</strong> the 4 examined region, stating<br />
the importance <strong>of</strong> AZF deletions in the aetiology <strong>of</strong><br />
RPL.<br />
In the previous studies, sequenced tagged site<br />
(STS) numbers which were selected for detection<br />
<strong>of</strong> Y chromosome microdeletions are different from<br />
each other. After physical mapping <strong>of</strong> Y<br />
chromosome, more than 300 STS were produced. It<br />
was stated that, analysing <strong>of</strong> low number <strong>of</strong> STS<br />
can be insufficient for detection <strong>of</strong> deletion regions<br />
also high number <strong>of</strong> STS can give false-positive<br />
results as polymorphic regions may identified as<br />
deletions (Simoni, 2001).<br />
One <strong>of</strong> the most important criteria for the<br />
detection <strong>of</strong> Y chromosome microdeletions is the<br />
selected STS. For this reason, European Andrology<br />
Association <strong>and</strong> European <strong>Molecular</strong> Genetics
Quality Network improved a st<strong>and</strong>ardization to<br />
distinguish the differences <strong>of</strong> deletion proportions<br />
between different laboratories. So they proposed 6<br />
STS for detecting <strong>of</strong> AZFa, AZFb ve AZFc regions.<br />
In the present study, although 13 STS including 6<br />
STS which were suggested by European <strong>Molecular</strong><br />
Genetics Quality Network were analyzed, we<br />
couldn’t detect any microdeletions on Y<br />
chromosome. We propose the evolution <strong>of</strong> the<br />
results by increasing the analysed STS.<br />
Due to limited knowledge <strong>of</strong> the metabolism<br />
<strong>and</strong> the progress <strong>of</strong> the genes on Y chromosome,<br />
we can not predict the answers <strong>of</strong> the questions<br />
including Y chromosome microdeletion’s effect on<br />
RPL. For this reason researches should be focused<br />
on the relationship <strong>of</strong> Y chromosome microdeletions,<br />
male infertility <strong>and</strong> RPL.<br />
References<br />
Briton-Jones C, Haines CJ. Microdeletions on the<br />
long arm <strong>of</strong> the Y chromosome <strong>and</strong> their<br />
association with male-factor infertility. HKMJ<br />
6: 184-9, 2000.<br />
Burgoyne PS. The mammalian Y chromosome: A<br />
new perpective. Bioassays, 20:3636, 1998.<br />
Byrne J.L.B, Ward K. Genetic Factors in Recurrent<br />
Abortion. Clinical Obstetircs <strong>and</strong> Gynecology,<br />
37 (3): 693-704, 1994.<br />
De Braekeleer M, Dao TN. Cytogenetic studies in<br />
male infertility: a review. Hum Reprod. 6:245-<br />
50, 1991.<br />
Dewan S, Puscheck EE, Coulam CB, Wilcox AJ,<br />
Jeyendran RS. Y-chromosome microdeletions<br />
<strong>and</strong> recurrent pregnancy loss Andrology<br />
Laboratory Services Inc., Chicago, Illinois,<br />
USA. Fertil Steril.;85(2):441-5, 2006.<br />
Genetics <strong>and</strong> male infertility. Verh K Acad<br />
Geneeskd Belg.; 71(3):115-39. Review. Dutch.,<br />
2009.<br />
Karaer A, Karaer K, Ozaksit G, Ceylaner S, Percin<br />
EF. Y chromosome azoospermia factor region<br />
microdeletions <strong>and</strong> recurrent pregnancy loss.<br />
Am J Obstet Gynecol.;199(6):662.e1-5, 2008.<br />
Li Z., Haines CJ., HanY. Micro-deletions <strong>of</strong> the<br />
human Y chromosome <strong>and</strong> their relationship<br />
with male infertility. J. Genet. Genomics 35,<br />
193−199, 2008.<br />
Y chromosome microdeletions in spontaneous abortions<br />
Martin R.H. Cytogenetic determinants <strong>of</strong> male<br />
fertility. Human Reproduction Update, 14 (4):<br />
379-390, 2008.<br />
Noordam M.J., Repping S. The human Y<br />
chromosome: a masculine chromosome, Current<br />
Opinion in Genetics & Development, 16: 225-<br />
232, 2006.<br />
Pryor JL, Kent-First M, Muallem A. Prospective<br />
analysis <strong>of</strong> Y chromosome microdeletions in<br />
200 consecutive male infertility patients. N Engl<br />
J Med 336: 534-539, 1997<br />
Simoni M. <strong>Molecular</strong> diagnosis <strong>of</strong> Y chromosome<br />
microdeletions in Europa: state-<strong>of</strong>-the-art <strong>and</strong><br />
quality control. Human Reprod, 16(3): 402-409,<br />
2001.<br />
Simoni M., Bakker E., Krausz C. EAA/EMQN<br />
best practice guidelines for molecular diagnosis<br />
<strong>of</strong> y-chromosomal microdeletions. international<br />
journal <strong>of</strong> <strong>and</strong>rology, 27:240–249, 2004.<br />
Simpson J.L. Antenatal Diagnosis <strong>of</strong> Cytogenetics<br />
Abnormalities. Clinical Obstetrics <strong>and</strong><br />
Gynecology, 24: 1024-1039, 1981<br />
Sinclair AH, Berta P, Palmer MS, Hawkins JR,<br />
Griffits BL, Smith MJ. A gene from the human<br />
sex-determining region encodes a protein with<br />
homology to a conserved DNA-binding motif.<br />
Nature, 346:240-4, 1990.<br />
Stipoljev F., Vujisic S., Parazajder J., Hafner D.,<br />
Jezˇek D., Sertic J. Cytogenetic analysis <strong>of</strong><br />
azoospermic patients: karyotype comparison <strong>of</strong><br />
peripheral blood lymphocytes <strong>and</strong> testicular<br />
tissue European <strong>Journal</strong> <strong>of</strong> Obstetrics &<br />
Gynecology <strong>and</strong> Reproductive <strong>Biology</strong> 124<br />
197–203, 2006.<br />
Stouffs K, V<strong>and</strong>ermaelen D, Tournaye H, Liebaers<br />
I, Van Steirteghem A, Lissens W.<br />
Svetlana G. Vorsanova, Alexei D. Kolotii, Ivan Y.<br />
Iourov, Viktor V. Monakhov, Elena A.<br />
Kirillova, Ilia V. Soloviev, <strong>and</strong> Yuri B. Yurov.<br />
Evidence for High Frequency <strong>of</strong> Chromosomal<br />
Mosaicism in Spontaneous Abortions Revealed<br />
by Interphase FISH Analysis <strong>Journal</strong> <strong>of</strong><br />
Histochemistry & Cytochemistry. 53(3): 375-<br />
380, 2005.<br />
Viswambharan N, Suganthi R, Simon A M,<br />
Manonayaki S. Male infertility: polymerase<br />
chain reaction-based deletion mapping <strong>of</strong> genes<br />
on the human chromosome. Singapore Med J;<br />
48 (11): 1140.2007.<br />
51
52<br />
Gülşah Koç et. al.<br />
Vogt, PH, Edelman A, Kirsch S, Henegariu O,<br />
Hirschmann P, Keisewetter. Human Y<br />
chromosome azospermic factors (AZF) mapped<br />
to different subregions in Yq11. Hum Mol<br />
Genet 5: 933-45, 1996.<br />
Vollrath D, Foote S, Hilton A. The human Y<br />
chromosome: A 43 interval map based on<br />
naturally occuring deletions. Science. 258:52-9,<br />
1992.
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong> 7(2) & 8(1): 53-70, 2010 Research Article<br />
<strong>Haliç</strong> University, Printed in Turkey.<br />
http://jcmb.halic.edu.tr<br />
Do simple sequence repeats in replication, repair <strong>and</strong> recombination<br />
genes <strong>of</strong> mycoplasmas providing genetic variability?<br />
Seema TRIVEDI<br />
Computational <strong>Biology</strong> Lab., Department <strong>of</strong> Zoology, JN Vyas University, Jodhpur<br />
(Rajasthan), India<br />
(author for correspondence; svtrived@hotmail.com)<br />
Received: 25 December 2009; Accepted: 14 May 2010<br />
Abstract<br />
Simple sequence repeats (SSRs) or microsatellites are mono to hexa-nucleotide t<strong>and</strong>em repeats <strong>of</strong> DNA that<br />
are ubiquitous in intergenic regions <strong>and</strong> coding regions <strong>of</strong> genomes. SSRs may be essential for any genome<br />
as these repeats are found even in organisms like mycoplasmas (class Mollicutes) that have small genome<br />
size. Mollicutes cause different diseases in vertebrates including humans, insects <strong>and</strong> plants. Antibiotics have<br />
been developed against membrane proteins <strong>and</strong> replication related proteins like gyrase <strong>and</strong> topoisomerase.<br />
However, some pathogens have developed immunity against these drugs. Mycoplasmas can evade host<br />
immune response in which SSRs variations in membrane proteins play an important role. The present study<br />
seeks presence <strong>of</strong> di- to penta-nucleotide repeats in genes associated with DNA replication, repair <strong>and</strong><br />
recombination in thirteen mycoplasma genomes. Association <strong>of</strong> SSRs with these genes may have potential to<br />
provide variability for evading antibiotics against replication, repair <strong>and</strong> recombination proteins. In the<br />
present study SSRs are present in few genes but among the repeats found, maximum repeats are present in<br />
methylase, DNA polymerase, excinuclease <strong>and</strong> topoisomerase genes. Maximum number <strong>of</strong> repeat types are<br />
dinucleotides but present only in M. pulmonis. Pentanucleotide repeats are present in three mycoplasmas but<br />
tetranucleotide repeats are present in eight mycoplasmas.<br />
Keywords: Microsatellites, Mycoplasma, repair, replication, simple sequence repeats<br />
Mikoplazmanın replikasyon, tamir ve rekombinasyon genlerindeki basit dizi tekrarları<br />
genetik çeşitlilik mi sağlıyor?<br />
Özet<br />
Basit dizi tekrarları (SSR) veya mikrosatelitler, genomun intergenik bölgelerinde ve kodlanan bölgelerinde<br />
sıkça rastlanan, DNA’nın birden (mono) altıya (hekza) kadar ard arda gelen nükleotit tekrarlarıdır. Bu<br />
tekrarlar mikoplazma (Mollicutes sınıfı) gibi küçük genom büyüklüğüne sahip bir organizmada bile<br />
bulunduğundan, SSR’ler herhangi bir canlı için elzem olabilir. Mollicutes, insan, böcek ve bitkileri de içeren<br />
omurgalılarda çeşitli hastalıklara yol açarlar. Zar proteinlerine ve giraz, topoizomeraz gibi replikasyonla<br />
alakalı proteinlerine karşı antibiyotikler geliştirilmiştir. Ancak, bazı patojenler bu ilaçlara karşı bağışıklık<br />
kazanmışlardır. Zar proteinlerindeki SSR varyasyonlarının mikoplazmaların konakçı hücrenin bağışıklık<br />
tepkisinden kaçmalarında önemli bir rolü olabilir. Bu çalışma, onüç mikoplazma genomunda DNA<br />
replikasyonu, tamiri ve rekombinasyonu ile ilgili genlerdeki ikiliden (di-) beşliye (penta-) kadar olan<br />
nükleotit tekrarlarını araştırır. Bu genlerin SSR’ler ile ilişkisi, replikasyon, tamir ve rekombinasyon<br />
proteinlerine karşı geliştirilen antibiyotiklerden kaçmaları için çeşitlilik sağlamada potansiyele sahip olabilir.<br />
Bu çalışmada, SSR’ler birkaç gende mevcuttur, ama bulunan tekrarlar arasında en fazla tekrar metilaz, DNA<br />
polimeraz, ekzinükleaz ve topoizomeraz genlerinde vardır. Tekrar tiplerinden en fazla sayıda bulunanlar<br />
dinükleotitlerdir ancak sadece M. Pulmonis’de bulunur. Pentanükleotit tekrarları üç mikoplazmada, ama<br />
tetranükleotit tekrarları sekiz mikoplazmada mevcuttur.<br />
Anahtar Sözcükler: Mikrosatelitler, mikoplazma, tamir, replikasyon, basit dizi tekrarları
54<br />
Seema TRIVEDI<br />
Introduction<br />
Simple sequence repeats (SSRs) or microsatellites<br />
are mono to hexa-nucleotide t<strong>and</strong>em repeats <strong>of</strong><br />
DNA that are ubiquitous in all genomes studied<br />
so far. SSRs are present not only in intergenic<br />
regions <strong>of</strong> a genome, but may also be found in<br />
introns <strong>and</strong> exons <strong>of</strong> coding regions (Karlin et al.,<br />
1997; Bachtrog et al., 1999; Bachtrog et al., 2000;<br />
Butcher et al., 2000; Chambers <strong>and</strong> MacAvoy,<br />
2000; Toth et al., 2000). It is possible that SSRs<br />
are essential part <strong>of</strong> any genome as these repeats<br />
are present even in organisms like mycoplasmas<br />
that have small genome size (Hancock, 1996a).<br />
These repetitive DNA may be involved in<br />
different functions like chromatin organization,<br />
gene regulation, evading host-immune responses,<br />
recom bination hotspots <strong>and</strong> facilitating genome<br />
rearrangements, affecting protein structure thus<br />
possibly protein-protein interactions etc. (Mrázek,<br />
2006). Interestingly, it has also been found that<br />
SSRs may provide variability to host for evading<br />
pathogens. This is reported in case <strong>of</strong> house<br />
finches (Carpodacus mexicanus) that show<br />
multilocus heterozygosity that could result in<br />
reduced susceptibility to Mycoplasma<br />
gallisepticum infection (Hawley et al., 2005).<br />
Similarly, Porcine C3 gene (high homology with<br />
human C3) involved in phagocytosis, inflamema<br />
tion <strong>and</strong> immunoregulation to destroy infectious<br />
micro-organisms, possess Tn SSR in the<br />
3’flanking region which may be helpful in<br />
resisting infections (Mekchay et al., 2003).<br />
Mycoplasmas (class Mollicutes) are parasites<br />
or commensals that may cause different diseases<br />
in vertebrates including humans, insects <strong>and</strong><br />
plants. Repetitive sequences like RepMp <strong>and</strong><br />
MgPar elements are involved in homologous<br />
recombination <strong>of</strong> parts <strong>of</strong> P1, P40, P90 <strong>and</strong> P110<br />
proteins <strong>of</strong> Mycoplasma pneumoniae <strong>and</strong> Myco<br />
plasma genitalium. Recombination events<br />
mediated with help <strong>of</strong> MPN490- <strong>and</strong> MG339encoded<br />
proteins (RecA homo logs) in genes <strong>of</strong><br />
immunogenic adhesion proteins result in<br />
variations that may play eminent roles in immune<br />
evasive strategies (Sluijter et al., 2009). Repeated<br />
sequences, DR-1 <strong>and</strong> DR-2, within the putative<br />
cytadhesin pvpA gene <strong>of</strong> M. gallisepticum are<br />
present in isolates from Chinese poultry farms.<br />
Approximately 30 or more proline residue repeats<br />
<strong>and</strong> 7-10 repeats <strong>of</strong> the tetrapeptide motif may<br />
affect functionality <strong>of</strong> PvpA as an adhesin molecule<br />
(Jiang et al., 2009). Similarly, insertion sequences<br />
(IS3, IS4 <strong>and</strong> IS30) in Mycoplasma bovis<br />
(Lysnyansky et al., 2009) <strong>and</strong> variable-number<br />
t<strong>and</strong>em-repeats (VNTRs) associated with coding<br />
sequences that can provide genetic diversity are<br />
present in Mycoplasma hyopneumoniae strains,<br />
Mycoplasma agalactiae type strain PG2,<br />
Mycoplasma mycoides subspecies mycoides. These<br />
IS <strong>and</strong> VNTRs possibly code for amino acid repeats<br />
<strong>and</strong> can affect cell adhesion <strong>and</strong> interactions with the<br />
host immune system (de Castro et al., 2006;<br />
McAuliffe et al., 2007; McAuliffe et al., 2008).<br />
T<strong>and</strong>em amino acid repeats in M. pneumoniae<br />
RepMP1-containing genes <strong>and</strong> Myco plasma<br />
pulmonis Vsa proteins may have regulatory functions<br />
(Simmons <strong>and</strong> Dybvig, 2007; Musatovova et al.,<br />
2008). Amino acid repeats that may affect antigenic<br />
response are present in haemagglutinins (immuno<br />
genic, variably expressed, surface proteins) <strong>of</strong> M.<br />
synoviae (VlhA) (Bencina, 2002).<br />
Though SSRs have been investigated in<br />
mycoplasmas, the focus <strong>of</strong> studies has not been<br />
replication, repair <strong>and</strong> recombination genes. This<br />
study was undertaken to seek answer to questions<br />
like whether replication, repair <strong>and</strong> recombination<br />
genes in mycoplasmas are associated with SSRs.<br />
How can SSRs association with these genes be<br />
beneficial to these organisms? The present study on<br />
SSRs in replication, repair <strong>and</strong> recombination genes<br />
in thirteen mycoplasmas may indicate mutational<br />
hotspots that may help these organisms evade<br />
antibiotics against these genes. On the other h<strong>and</strong>,<br />
these mutational hotspots may prove to be target sites<br />
for developing drugs to prevent such evasive<br />
strategies <strong>of</strong> the organisms.<br />
Method<br />
Obtaining sequences <strong>and</strong> genome information<br />
Replication, repair <strong>and</strong> recombination related gene<br />
sequences (total numbers given in Table-1) <strong>of</strong><br />
thirteen myco plasmas (Mycoplasma arthritidis<br />
158L3-1, Mycoplasma capricolum subsp capricolum<br />
California kid ATCC 27343, Mycoplasma<br />
gallisepticum strain R, Mycoplasma genitalium G-37,<br />
Mycoplasma hyopneumoniae 232, Mycoplasma<br />
hyopneumoniae 7448, Mycoplasma hyopneumoniae<br />
J, Mycoplasma mobile 163K, Mycoplasma mycoides<br />
SC PG1, Mycoplasma penetrans HF-2, Mycoplasma<br />
pneumoniae M129, Mycoplasma pulmonis UAB
CTIP <strong>and</strong> Mycoplasma synoviae 53) were<br />
downloaded from “The Comprehensive Microbial<br />
Resource” (CMR http://cmr.jcvi.org/cgi-bin/<br />
CMR/CmrHomePage.cgi). Total genome length,<br />
CG richness, coding percentage <strong>and</strong> number <strong>of</strong><br />
Table 1. List <strong>of</strong> mycoplasmas <strong>and</strong> genome details<br />
Organism<br />
Genome<br />
length<br />
nt<br />
Genome<br />
CG%<br />
Coding<br />
%<br />
SSRs in Mycoplasma replication genes<br />
protein coding genes for each Mycoplasma were<br />
downloaded from NCBI (http://www.ncbi.nlM.<br />
nih.gov/sites/entrez?db=genome&cmd=Retrieve&do<br />
pt=Overview&list_uids=) (Table 1).<br />
Total<br />
protein<br />
genes<br />
Total<br />
replication<br />
<strong>and</strong> repair<br />
genes<br />
% <strong>of</strong><br />
genes<br />
with<br />
repeats<br />
Mycoplasma<br />
arthritidis 158L3-1 820453 30 88 631 32 3.13<br />
Mycoplasma<br />
capricolum subsp<br />
capricolum California<br />
kid ATCC 27343<br />
Mycoplasma<br />
1010023 23 88 812 45 8.89<br />
gallisepticum strain R<br />
Mycoplasma<br />
996422 31 87 726 30 3.33<br />
genitalium G-37<br />
Mycoplasma<br />
580076 31 90 475 28 3.57<br />
hyopneumoniae 232<br />
Mycoplasma<br />
892758 28 89 691 34 2.94<br />
hyopneumoniae 7448<br />
Mycoplasma<br />
920079 28 85 657 35 2.86<br />
hyopneumoniae J<br />
Mycoplasma mobile<br />
897405 28 87 657 34 2.94<br />
163K<br />
Mycoplasma mycoides<br />
777079 24 90 633 39 5.13<br />
SC PG1<br />
Mycoplasma penetrans<br />
1211703 23 81 1016 51 1.96<br />
HF-2<br />
Mycoplasma<br />
1358633 25 88 1037 76 2.63<br />
pneumoniae M129<br />
Mycoplasma pulmonis<br />
816394 40 87 689 36 5.56<br />
UAB CTIP 963879 26 89 782 53 9.43<br />
Mycoplasma synoviae<br />
53 799476 28 88 659 36 8.33<br />
55
56<br />
Seema TRIVEDI<br />
SSR search<br />
Replication, repair <strong>and</strong> recombination related<br />
gene sequences <strong>of</strong> all thirteen mycoplasmas were<br />
subjected to repeat search programme SPUTNIK<br />
(http://espressos<strong>of</strong>tware.com/sputnik/index.html).<br />
SPUTNIK looks for di-, tri-, tetra- <strong>and</strong> pentanucleotide<br />
repeats; tolerates insertions, mismatch<br />
es <strong>and</strong> deletions but these affect the overall score.<br />
Repeats found through this search were not<br />
grouped i.e. the 5’ to 3’ or vice-a versa <strong>and</strong> the<br />
complements were not put together. For example,<br />
dinucleotide repeat CA was not grouped with AC,<br />
TG or GT. Hence, each repeat was treated as<br />
separate motif.<br />
SSR CG richness <strong>and</strong> length<br />
Length <strong>of</strong> an SSR was measured in nucleotides<br />
(base pairs). However, repeat length given as<br />
output from SPUTNIK was adjusted to nearest<br />
divisible value i.e. dinucleotide repeat length<br />
should be divisible by 2, trinucleotide by 3,<br />
tetranucleotide repeat by 4 <strong>and</strong> pentanucleotide<br />
repeat by 5. For example, if repeat length for<br />
dinucleotide repeats was indicated as 11nt by<br />
SPUTNIK output; this was adjusted to 10nt, for<br />
trinucleotide repeats if the length was 14nt it was<br />
adjusted to 15nt or if it was 16nt or 17nt it was<br />
considered as 15nt.<br />
Statistical analysis<br />
For statistical analysis, SPSS (Version 16.0) was<br />
used. One way ANOVA followed by Tukey’s<br />
HSD at 95% confidence level was done to seek<br />
significant difference in between the thirteen<br />
species repeat number as well different replica<br />
tion, repair <strong>and</strong> recombination genes.<br />
Pearson’s ‘r’ two tailed correlation analysis <strong>of</strong><br />
genome size <strong>and</strong> CG richness, sequence lengths<br />
<strong>of</strong> replication, repair <strong>and</strong> recombination genes <strong>and</strong><br />
CG richness vis-à-vis repeat numbers, length <strong>and</strong><br />
.<br />
CG richness <strong>of</strong> total repeats <strong>and</strong> repeat types was<br />
done.<br />
Results<br />
Total SSRs, repeat types <strong>and</strong> motifs in thirteen<br />
mycoplasmas replication, repair <strong>and</strong> recombination<br />
genes<br />
Figure 1 shows total numbers <strong>of</strong> SSRs <strong>and</strong> repeat<br />
types present in replication, repair <strong>and</strong> recombination<br />
genes in genomes <strong>of</strong> thirteen mycoplasmas where M.<br />
pulmonis has the maximum number <strong>of</strong> repeats<br />
followed by M. capricolum <strong>and</strong> M. synoviae (5, 4<br />
<strong>and</strong> 3 respectively).<br />
Among repeat types, dinucleotides (five) are<br />
present only in M. pulmonis. Maximum numbers <strong>of</strong><br />
tri nucleotides are in M. capricolum. Presence <strong>of</strong><br />
other repeat types in replication, repair <strong>and</strong><br />
recombination genes <strong>of</strong> mycoplasmas are given in<br />
Figure 1.<br />
There is diversity in repeat motifs found in the<br />
present study among which maximum frequency<br />
(four) is <strong>of</strong> tetranucleotide repeat ATTT followed by<br />
dinucleotide repeat motifs AG (Table-2).<br />
Total replication, repair <strong>and</strong> recombination genes<br />
associated with SSRs in mycoplasmas<br />
Methylase genes have maximum number <strong>of</strong> total<br />
repeats (eight) followed by DNA polymerase,<br />
excinuclease <strong>and</strong> topoisomerase genes (three in each)<br />
among replication, repair <strong>and</strong> recombination genes in<br />
thirteen mycoplasmas (Figure 2). Among repeat<br />
types total dinucleotide followed by tetra nucleotide<br />
repeats as well as the motifs AG <strong>and</strong> ATTT have<br />
maximum frequency (all in methylase genes)<br />
(Figures 2 <strong>and</strong> 3).<br />
Comparison <strong>of</strong> total repeats <strong>and</strong> repeat types in<br />
replication, repair <strong>and</strong> recombination genes <strong>of</strong> each<br />
Mycoplasma is given in Figure 4 <strong>and</strong> motifs in Table<br />
3, where once again methylase genes in M. pulmonis<br />
have maximum repeats.
SSRs in Mycoplasma replication genes<br />
Figure 1. Total SSRs <strong>and</strong> SSR types in thirteen mycoplasmas replication, repair <strong>and</strong> recombination genes.<br />
Di: Dinucleotides, Tri: Trinucleotides, Tetra: Tetranucleotides, Penta: Pentanucleotides, Total: Total Repeats.<br />
Figure 2. Total SSRs <strong>and</strong> SSR types in total replication, repair <strong>and</strong> recombination genes <strong>of</strong> mycoplasmas. Di:<br />
Dinucleotides, Tri: Trinucleotides, Tetra: Tetranucleotides, Penta: Pentanucleotides, Total: Total Repeats.<br />
57
58<br />
Seema TRIVEDI<br />
Table 2. SSR motifs <strong>and</strong> motif numbers in total replication, repair <strong>and</strong> recombination genes <strong>of</strong> thirteen<br />
mycoplasmas. Di: Dinucleotide, Tri: Trinucleotide, Tetra: Tetranucleotide, Penta: Pentanucleotide.<br />
Repeat<br />
Name<br />
Di<br />
Tri<br />
Tetra<br />
Penta<br />
Length <strong>of</strong> repeats<br />
Organism Motifs<br />
AG CA GA<br />
M. pulmonis 3 1 1<br />
Gr<strong>and</strong> Total 3 1 1<br />
AAG AAT AGA GAA TGA TGT<br />
M. arthritidis 1<br />
M. capricolum 1 1 1<br />
M. mobile 1<br />
M. penetrans 1<br />
M. synoviae 1<br />
Gr<strong>and</strong> Total 1 2 1 1 1 1<br />
AATT ATTT TTTA TTTC<br />
M. capricolum 1<br />
M. genitalium 1<br />
M. hyopneumoniae 232 1<br />
M. hyopneumoniae 7448 1<br />
M. hyopneumoniae J 1<br />
M. mobile 1<br />
M. mycoides 1<br />
M. penetrans 1<br />
Gr<strong>and</strong> Total 1 4 2 1<br />
AAAAT AAAGC AATTG ACCAA CAAAC<br />
M. gallisepticum strain R 1<br />
M. pneumoniae 1 1<br />
M. synoviae 1 1<br />
Gr<strong>and</strong> Total 1 1 1 1 1<br />
Repeats in thirteen mycoplasmas are not very<br />
long as maximum repeat lengths are 28nt <strong>and</strong><br />
26nt which are in methylase genes (dinucleotide<br />
CA <strong>and</strong> GA respectively in M. pulmonis)<br />
followed by 24nt which are in methylase <strong>and</strong><br />
gyrase genes (dinucleotide AG in M. pulmonis<br />
<strong>and</strong> trinucleotide AAG in M. arthritidis<br />
respectively) (Figure 5 <strong>and</strong> Table 4).<br />
CG richness <strong>of</strong> SSRs<br />
There is variation (0% to 50%) in CG richness <strong>of</strong><br />
SSRs in replication, repair <strong>and</strong> recombination genes<br />
<strong>of</strong> mycoplasmas (Table 5). CG% differs in the same<br />
gene group among different Mycoplasma. For<br />
example, DNA polymerase shows different CG<br />
richness in different Mycoplasma (0% in M.<br />
synoviae, 33.33% in M. mobile <strong>and</strong> 40% in M.<br />
pneumoniae).
SSRs in Mycoplasma replication genes<br />
Figure 3. SSR motifs in total replication, repair <strong>and</strong> recombination genes in mycoplasmas. legends represent<br />
the motifs as per the bar chart.<br />
59
60<br />
Seema TRIVEDI<br />
Figure 4. Total SSRs <strong>and</strong> SSR Types In Replication, Repair <strong>and</strong> Recombination Genes In Thirteen Mycoplasmas. Di: Dinucleotides, Tri:<br />
Trinucleotides, Tetra: Tetranucleotides, Penta: Pentanucleotides, Total: Total Repeats.
SSRs in Mycoplasma replication genes<br />
Table 3. Number <strong>of</strong> SSR motifs in replication, repair <strong>and</strong> recombination genes <strong>of</strong> thirteen<br />
mycoplasmas.<br />
Organism Motif<br />
Conserved<br />
Hypothetical Protein<br />
DNA Polymerase<br />
Endonuclease<br />
Excinuclease<br />
M. arthritidis AAG 1<br />
Gyrase<br />
Ligase<br />
Methylase<br />
Methyltransferase<br />
P35 Lipoprotein<br />
Homolog<br />
AGA 1<br />
M. capricolum<br />
TGA<br />
TGT 1<br />
1<br />
TTTA 1<br />
M. gallisepticum ACCAA 1<br />
M. genitalium AATT 1<br />
M. hyopneumoniae 232 ATTT 1<br />
M. hyopneumoniae 7448 ATTT 1<br />
M. hyopneumoniae J ATTT 1<br />
M. mobile<br />
GAA<br />
ATTT<br />
1<br />
1<br />
M. mycoides TTTA 1<br />
M. penetrans<br />
AAT<br />
TTTC 1<br />
1<br />
M. pneumoniae<br />
AAAGC<br />
CAAAC 1<br />
1<br />
AG 3<br />
M. pulmonis<br />
CA 1<br />
GA 1<br />
AAT 1<br />
M. synoviae<br />
AAAAT 1<br />
AATTG 1<br />
Replication Initiator<br />
SSBP<br />
Topoisomerase<br />
61
62<br />
Seema TRIVEDI<br />
Figure 5. Lengths <strong>of</strong> total SSRs <strong>and</strong> SSR type in total replication, repair <strong>and</strong> recombination genes <strong>of</strong> mycoplasmas. SSR lengths in<br />
number <strong>of</strong> nucleotides (nt).
SSRs in Mycoplasma replication genes<br />
Table 4. Repeat lengths (nt) in total SSR types <strong>and</strong> SSR motifs in replication, repair <strong>and</strong> recombination genes<br />
<strong>of</strong> thirteen mycoplasmas. Di: Dinucleotides, Tri: Trinucleotides, Tetra: Tetranucleotides, Penta:<br />
Pentanucleotides, Total: Total Repeats.<br />
.<br />
Gene Organism SSR Motif<br />
SSR<br />
Length<br />
Conserved Hypothetical Protein M. synoviae Penta AATTG 10<br />
DNA Polymerase<br />
M. mobile Tri GAA 12<br />
M. pneumoniae Penta CAAAC 10<br />
M. synoviae Tri AAT 12<br />
Endonuclease M. penetrans Tetra TTTC 12<br />
Excinuclease<br />
M. capricolum Tri AGA 12<br />
M. pneumoniae Penta AAAGC 15<br />
M. synoviae Penta AAAAT 10<br />
Gyrase M. arthritidis Tri AAG 24<br />
Ligase M. genitalium Tetra AATT 12<br />
Methylase<br />
M. hyopneumoniae 232 Tetra ATTT 12<br />
M. hyopneumoniae 7448 Tetra ATTT 12<br />
M. hyopneumoniae J Tetra ATTT 12<br />
M. pulmonis Di GA 26<br />
CA 28<br />
14<br />
AG 16<br />
24<br />
Methyltransferase M. mobile Tetra ATTT 12<br />
P35 Lipoprotein Homolog M. penetrans Tri AAT 18<br />
Replication Initiator M. capricolum Tri TGT 12<br />
SSBP M. capricolum Tri TGA 12<br />
Topoisomerase<br />
M. capricolum Tetra TTTA 12<br />
M. gallisepticum Penta ACCAA 10<br />
M. mycoides Tetra TTTA 12<br />
63
64<br />
Seema TRIVEDI<br />
Table 5. CG richness <strong>of</strong> SSRs <strong>and</strong> motifs in replication, repair <strong>and</strong> recombination genes in thirteen<br />
mycoplasmas. Di: Dinucleotide, Tri: Trinucleotide, Tetra: Tetranucleotide, Penta: Pentanucleotide<br />
SSR<br />
CG%<br />
0<br />
Gene Name Repeat Motif Organism<br />
SSR<br />
Number<br />
DNA Polymerase Tri AAT M. synoviae 53 1<br />
Excinuclease Penta AAAAT M. synoviae 53 1<br />
Ligase Tetra AATT M. genitalium G-37 1<br />
Methylase Tetra ATTT M. hyopneumoniae 232 1<br />
M. hyopneumoniae 7448 1<br />
M. hyopneumoniae J 1<br />
Methyltransferase Tetra ATTT M. mobile 163K 1<br />
P35 Lipoprotein<br />
Homolog<br />
Tri AAT<br />
M. penetrans HF-2 1<br />
Topoisomerase Tetra TTTA M. capricolum 1<br />
M. mycoides 1<br />
20 Conserved<br />
Hypothetical Protein<br />
Penta AATTG<br />
M. synoviae 53 1<br />
25 Endonuclease Tetra TTTC M. penetrans HF-2 1<br />
33.33 DNA Polymerase Tri GAA M. mobile 163K 1<br />
Excinuclease Tri AGA M. capricolum 1<br />
Gyrase Tri AAG M. arthritidis 158L3-1 1<br />
Replication Initiator Tri TGT M. capricolum 1<br />
40<br />
SSBP Tri TGA M. capricolum 1<br />
DNA Polymerase Penta CAAAC M. pneumoniae M129 1<br />
Excinuclease Penta AAAGC M. pneumoniae M129 1<br />
Topoisomerase Penta ACCAA M. gallisepticum strain R 1<br />
50 Methylase Di AG M. pulmonis 3<br />
CA M. pulmonis 1<br />
Discussion<br />
SSRs in genomes <strong>of</strong> virus, prokaryotes <strong>and</strong><br />
eukaryotes are present not only in intergenic<br />
regions but also in introns <strong>and</strong> exons <strong>of</strong> coding<br />
sequences (Hancock, 1996a <strong>and</strong> b; Karlin et al.,<br />
1997; Bachtrog et al., 1999; Bachtrog et al., 2000;<br />
Butcher et al., 2000; Toth et al., 2000; Trivedi,<br />
2003, 2004, 2006; Mrázek et al., 2007). Studies in<br />
different genomes have also shown associations<br />
GA M. pulmonis 1<br />
<strong>of</strong> SSRs with replication, repair <strong>and</strong> recombination<br />
genes, housekeeping genes or membrane proteins<br />
(Trivedi, 2003; Mrázek et al., 2007; Guo <strong>and</strong><br />
Mrázek, 2008). Antigenic variations facilitated by<br />
SSRs in host-adapted pathogens like mycoplasmas<br />
have been reported (Guo <strong>and</strong> Mrázek, 2008).<br />
However, studies have so far not focused on
association <strong>of</strong> SSRs with replication, repair <strong>and</strong><br />
recombination genes.<br />
Total repeats<br />
Mycoplasmas have small genomes possibly due<br />
to genome reduction. It is possible that some<br />
SSRs may have played a role in Mycoplasma<br />
evolution (Hancock, 1996a; Rocha <strong>and</strong><br />
Blanchard, 2002) which could be due to<br />
mutational bias towards SSR reduction instead <strong>of</strong><br />
expansion if not maintained by selection (Metzgar<br />
et al., 2002).<br />
The present study shows few repeats in total<br />
replication, repair <strong>and</strong> recombination genes which<br />
corresponds to studies in whole genome studies<br />
on mycoplasmas (Mrázek et al., 2007) but with<br />
some differences from the reported number <strong>of</strong><br />
repeats in these genes in M. hyopneumoniae (Guo<br />
<strong>and</strong> Mrázek, 2008). This difference is possibly<br />
due to difference in the algorithms used in the<br />
present study <strong>and</strong> the analysis criteria. ANOVA<br />
does not show any significant difference between<br />
the thirteen mycoplasmas nor between genes <strong>of</strong><br />
each Mycoplasma in the present study. Besides<br />
this, present study does not show any correlation<br />
<strong>of</strong> genome length with any parameter as indicated<br />
in the material <strong>and</strong> method section. This is<br />
consistent with some studies (Hancock, 2002;<br />
Lim et al., 2004) but contrary to studies done<br />
earlier in eukaryotes <strong>and</strong> prokaryotes (Hancock,<br />
1996b; Primmer et al., 1997; Achaz et al., 2002;<br />
Trivedi, 2004 <strong>and</strong> 2006; Mrázek et al., 2007).<br />
Though the focus <strong>of</strong> present study is on specific<br />
genes, this lack <strong>of</strong> correlation <strong>of</strong> genome size may<br />
indicate confirmation <strong>of</strong> the fact that genome size<br />
is not exp<strong>and</strong>ing <strong>and</strong> possibly SSRs (in total<br />
genomes) that are so few in number are playing a<br />
role in genome reduction in Mycoplasma<br />
(Hancock 1996a; Mrázek et al., 2007; Guo <strong>and</strong><br />
Mrázek, 2008).<br />
Genome CG% show significant positive<br />
correlation only with penta nucleotide number<br />
<strong>and</strong> length. Similarly, significant positive<br />
correlation <strong>of</strong> average CG% <strong>of</strong> total replication,<br />
repair <strong>and</strong> recombination genes with pentanucleo<br />
tide lengths (P< 0.05, 0.587*) is seen in the<br />
present study. There is significant positive<br />
correlation (P< 0.01, 0.931**) <strong>of</strong> total SSR<br />
number with percentage <strong>of</strong> total replication, repair<br />
<strong>and</strong> recombination genes with repeats. Total SSR<br />
numbers show significant positive correlation<br />
with total dinucleotide number (P< 0.01, 0.700**)<br />
<strong>and</strong> total tetranucleotides show significant<br />
SSRs in Mycoplasma replication genes<br />
negative correlation with pentanucleotide number<br />
(P< 0.05, -0.659*).<br />
This data is not sufficient to draw conclusion that<br />
positive correlation <strong>of</strong> percentage <strong>of</strong> genes having<br />
repeats with total repeats may be an indication <strong>of</strong><br />
probable increase in repeat number in these genes in<br />
other pathogens as well. It would be interesting to<br />
investigate repeats in those micro-organisms as well<br />
against which antibiotics are being used. Antibiotic<br />
treatment would alter environment <strong>and</strong> these<br />
pathogens may develop different strategies to evade<br />
these challenges. These evasive strategies may<br />
involve the membrane proteins or replication, repair<br />
<strong>and</strong> recombination related genes or gene products<br />
depending on the target proteins <strong>of</strong> antibiotics.<br />
SSR average length<br />
Many organisms studied so far have shown<br />
differences in not only abundance <strong>of</strong> SSRs but also in<br />
tolerance for length <strong>of</strong> SSRs. For example, archaea<br />
like Methanosarcina mazei strain Goe1,<br />
Methanobacterium thermoautotrophicum DH,<br />
Helicobacter pylori have some SSRs that are long<br />
compared to other species (Field <strong>and</strong> Wills, 1998;<br />
Trivedi, 2006). Among vertebrates, cold-blooded<br />
vertebrates like turtles have long repeats (Chambers<br />
<strong>and</strong> MacAvoy, 2000); whereas humans have longer<br />
SSRs compared to homologues in chimps (Cooper et<br />
al., 1998).<br />
Long SSRs have not been found in the present<br />
study including other studies in M. penetrans, M.<br />
mobile, <strong>and</strong> M. synoviae. Short A(n) <strong>and</strong> T(n) repeats<br />
are abundant in M. hyopneumoniae <strong>and</strong> M. pulmonis<br />
(Mrázek, 2006). The present study shows significant<br />
positive correlation between total SSR average<br />
length with dinucleotide number <strong>and</strong> length as well<br />
as tetranucleotide CG% (P< 0.05, 0.563*, 0.563* <strong>and</strong><br />
P< 0.01, 1.000** respectively). Dinucleotide average<br />
length has significant positive correlation with total<br />
SSR as well as dinucleotide numbers <strong>and</strong> CG% <strong>of</strong><br />
total SSRs (P< 0.01, 0.700**, 1.000** <strong>and</strong> 0.870**<br />
respectively). Significant positive correlation <strong>of</strong><br />
trinucleotide length is seen with trinucleotide number<br />
but negative correlation with pentanucleotide CG%<br />
(P< 0.05, 0.672* <strong>and</strong> P< 0.01,-1.000** respectively).<br />
Tetranucleotide average length has positive<br />
correlation with tetranucleotide number but negative<br />
correlation with pentanucleotide number as well as<br />
length (P< 0.01, 1.000**, P< 0.05, -0.659* <strong>and</strong> P<<br />
0.01, -0.688** respectively). There is positive<br />
correlation <strong>of</strong> pentanucleotide average length with<br />
pentanucleotide number (P< 0.01, 0.963**).<br />
65
66<br />
Seema TRIVEDI<br />
Reports have suggested that in prokaryote<br />
genomes, significantly long SSRs are generally<br />
rare but association <strong>of</strong> long mono-, di-, tri-, <strong>and</strong><br />
tetranucleotides are mostly restricted to hostadapted<br />
pathogens. Besides this, a number <strong>of</strong> long<br />
SSRs are also associated with housekeeping<br />
genes, including rRNA <strong>and</strong> tRNA genes, genes<br />
encoding ribosomal proteins, amino acyl-tRNA<br />
synthetases, chaperones, <strong>and</strong> important metabolic<br />
enzymes. Statistically significant associa tions<br />
between SSRs <strong>and</strong> gene functional classifications<br />
suggest that most long SSRs are not related to a<br />
particular cellular function or process (Guo <strong>and</strong><br />
Mrázek, 2008). This conclusion may be true for<br />
many organisms <strong>and</strong> particularly prokaryotes, but<br />
pilot study on silkworm genes has indicated<br />
biased association <strong>of</strong> SSRs with genes associated<br />
with replication <strong>and</strong> repair process though length<br />
<strong>of</strong> SSRs was not focus <strong>of</strong> the study (Trivedi,<br />
2003). In Mycobacterium leprae long SSRs are<br />
mostly associated with pseudogenes <strong>and</strong> may be<br />
contributing to gene loss following the adaptation<br />
to an obligate pathogenic lifestyle. The authors<br />
speculate that LSSRs may have played a similar<br />
role in genome reduction <strong>of</strong> other host-adapted<br />
pathogens (Guo <strong>and</strong> Mrázek, 2008). Similarly in<br />
M. gallisepticum SSRs are biased towards<br />
deletion unlike eukaryotic genomes where they<br />
are biased towards expansion (Metzgar et al.,<br />
2002).<br />
Among prokaryotes, it is proposed that the<br />
differences could be due to functional nature <strong>of</strong><br />
these repeats or because some <strong>of</strong> these genomes<br />
lacks mismatch repair (Field <strong>and</strong> Wills, 1998).<br />
However, the latter reason does not apply to<br />
eukaryotes <strong>and</strong> particularly to differentces<br />
between chimpanzee <strong>and</strong> human SSRs.<br />
SSR average CG%<br />
Total SSRs CG% show positive correlation with<br />
total SSR <strong>and</strong> dinucleotide number (P
epeats. Frame-shift mutations caused in either di-<br />
or tetra-nucleotide (<strong>of</strong> the present study) repeats<br />
in these genes may facilitate altered methylation<br />
patterns <strong>and</strong> influence gene expression thus may<br />
indirectly contribute to antigenic variations that<br />
could help in evasion <strong>of</strong> host immune response<br />
(Rocha <strong>and</strong> Blanchard, 2002; Mrázek, 2006; Guo<br />
<strong>and</strong> Mrázek, 2008).<br />
DNA polymerase, excinuclease, gyrase <strong>and</strong><br />
topoisomerase<br />
Among replication, repair <strong>and</strong> recom bination<br />
genes; M. mobile, M. pneumoniae, M. synoviae<br />
have repeats in DNA polymerase. Excinuclease<br />
genes have SSRs in M. capricolum, M.<br />
pneumoniae <strong>and</strong> M. synoviae <strong>and</strong> topoisomerase<br />
genes have SSRs in M. capricolum, M.<br />
gallisepticum <strong>and</strong> M. mycoides. However, gyrase<br />
genes have SSR only in M. arthritidis 158L3-1<br />
<strong>and</strong> single str<strong>and</strong> binding proteins (SSBP) only in<br />
M. capricolum. SSRs association with DNA<br />
polymerase, gyrase <strong>and</strong> SSBP genes besides other<br />
essential as well as housekeeping genes has also<br />
been reported in other prokaryotes including<br />
mycoplasmas. Guo <strong>and</strong> Mrázek (2008) speculate<br />
that variation in essential genes like replication,<br />
repair <strong>and</strong> recombination genes (except<br />
methylase) in prokaryotes may not be helpful but<br />
possibly these SSRs affect transcription initiation.<br />
However, there is a possibility that these SSRs<br />
are once again providing most <strong>of</strong> these pathogens<br />
opportunity to evade antibiotics. This hypothesis<br />
gets its support from the fact that many<br />
prokaryotes including mycoplasmas are becoming<br />
resistant to fluoroquinolones that are broadspectrum<br />
antibiotics like <strong>of</strong>loxacin (OFX),<br />
cipr<strong>of</strong>loxacin (CFX) <strong>and</strong> sparfloxacin (SFX)<br />
targeted against proteins like topoisomerase II<br />
family, DNA gyrase <strong>and</strong> topoisomerase IV. Some<br />
mycoplasmas have developed resistance against<br />
these drugs due to mutations in target regions <strong>of</strong><br />
these enzymes (Bébéar et al., 1998). Therefore it<br />
is suggested that those drugs would be more<br />
effective that would target at least two<br />
proteins/genes simultaneously to block <strong>of</strong> slow<br />
down DNA replication. This is because two<br />
mutations simultaneously in two genes to evade<br />
antibiotics would be rare <strong>and</strong> hence it may be<br />
more effective as it has been suggested in studies<br />
done on Staphylococcus aureus (Fournier et al.,<br />
2000).<br />
Similarly, there are mutants against antibiotics<br />
that target Mycoplasma ribosomes (Pereyre et al.,<br />
SSRs in Mycoplasma replication genes<br />
2002). It is possible that SSRs in the rRNA genes<br />
(Guo <strong>and</strong> Mrázek, 2008) (but not part <strong>of</strong> this study)<br />
may be providing genetic variability to the pathogen<br />
to evade altered living environment due to antibiotics<br />
against their ribosomes.<br />
Functions <strong>of</strong> SSRs<br />
SSRs may play diverse roles in genomes <strong>of</strong> different<br />
organisms. Some <strong>of</strong> these repeats act as contingency<br />
loci in association with families <strong>of</strong> surface antigens<br />
in pathogens, affect DNA supercoiling, gene<br />
expression or recombination hotspots. M.<br />
gallisepticum major surface protein pMGA<br />
expression switching from pMGA1.1 to pMGA1.2 is<br />
associated with (GAA)(12) repeat <strong>and</strong> variations in<br />
length (Glew et al., 2000; Liu et al., 2000; Liu et al.,<br />
2002). M. pneumoniae, M. genitalium, Ureaplasma<br />
urealyticum <strong>and</strong> M. pulmonis have recombination<br />
potentials in different genomic regions. In particular<br />
M. pulmonis has illegitimate recombination at the vsa<br />
locus. M. pneumoniae <strong>and</strong> M. genitalium adhesins<br />
have large distant repeats that may be responsible for<br />
homologous recombination for variation (Rocha <strong>and</strong><br />
Blanchard, 2002). However, M. hyopneumoniae have<br />
fewer repeats compared to other mycoplasma.<br />
Therefore, it is intriguing how M. hyopneumoniae<br />
evades host immune response <strong>and</strong> establishes chronic<br />
infection in absence <strong>of</strong> repeats (Minion et al., 2004).<br />
Not all SSRs may be related with these functions.<br />
For example, repeats in M. hyopneumoniae may not<br />
be contingency loci as these appear independent <strong>of</strong><br />
location upstream or downstream <strong>of</strong> genes. Further,<br />
among 3 M. hyopneumoniae strains study shows that<br />
the An <strong>and</strong> Tn repeats may be rarely involved in<br />
genome rearrangements (Mrázek, 2006).<br />
Conclusion<br />
The diversity <strong>of</strong> repeat numbers <strong>and</strong> motifs may be<br />
due to diversity <strong>of</strong> host environments <strong>and</strong> selection<br />
pressures that different mycoplasmas live in. It is<br />
also possible that SSRs are present in regions <strong>of</strong><br />
those segments where the amino acid coded by SSRs<br />
may help in flexibility <strong>of</strong> protein fold (disordered<br />
region). This may enable the protein to be more<br />
flexible in terms <strong>of</strong> protein-protein interactions <strong>and</strong><br />
may have multiple partners, hence making them<br />
possible hubs <strong>of</strong> protein interaction networks<br />
(Mrázek, 2006). However, in the present study, the<br />
possible role <strong>of</strong> SSRs in these genes/proteins<br />
involves DNA binding capacity. Any mutation in<br />
these SSRs or other regions <strong>of</strong> the protein may affect<br />
67
68<br />
Seema TRIVEDI<br />
DNA-protein interaction <strong>and</strong> hence affect<br />
replication efficiency. Since there are antibiotics<br />
targeted against replication enzymes, these SSRs<br />
may be involved in providing platform for small<br />
mutations that are not large enough to totally<br />
affect functions <strong>of</strong> DNA replication, repair <strong>and</strong><br />
recombination but are efficient enough to evade<br />
the antibiotics.<br />
Acknowledgements<br />
The author is grateful to Department <strong>of</strong><br />
Biotechnology (DBT, Ministry <strong>of</strong> Science <strong>and</strong><br />
Technology, India) for funding this work.<br />
References<br />
Achaz G, Rocha EPC, Netter P <strong>and</strong> Coissac E.<br />
Origin <strong>and</strong> fate <strong>of</strong> repeats in bacteria. Nucleic<br />
Acids Res. 30: 2987-2994, 2002.<br />
Bachtrog D, Agis M, Imh<strong>of</strong> M <strong>and</strong> Schlotterer C.<br />
Microsatellite varia bility differs between<br />
dinucleotide repeat motifs - evidence from<br />
Drosophila melanogaster. Mol Biol Evol. 17:<br />
1277-1285, 2000.<br />
Bachtrog D, Weiss S, Zangerl B, Brem G <strong>and</strong><br />
Schlotterer C. Distribution <strong>of</strong> dinucleotide<br />
microsatellites in the D. melanogaster<br />
genome. Mol Biol Evol. 16: 602-610, 1999.<br />
Bébéar CM, Renaudin H, Charron A, Bové JM,<br />
Bébéar C <strong>and</strong> Renaudin J. Alterations in<br />
Topoisomerase IV <strong>and</strong> DNA gyrase in<br />
quinolone-resistant mutants <strong>of</strong> Mycoplasma<br />
hominis obtained In Vitro. Antimicrob Agents<br />
Chemotherapyapy. (42)9:2304-2311, 1998.<br />
Bencina D. Haemagglutinins <strong>of</strong> pat-hogenic avian<br />
mycoplasmas. Avian Pathol. 6: 535-47, 2002.<br />
Butcher RD, Hubbard SF <strong>and</strong> Whitfield WG.<br />
2000. Microsatellite frequency <strong>and</strong> size<br />
variation in the part henogenetic parasitic<br />
wasp Venturia canescens (Gravenhorst)<br />
(Hymen optera: Ichneumonidae). Insect Mol<br />
Biol. 9: 375-384.<br />
Chambers KG <strong>and</strong> MacAvoy ES. Microsatellites:<br />
Consensus <strong>and</strong> cont roversy. Comp Biochem<br />
Physio (Part B). 126: 455-476, 2000.<br />
Cooper G, Rubinsztein DC <strong>and</strong> Amos W.<br />
Ascertainment bias cannot entirely account for<br />
human microsatellites being longer than their<br />
chimpanzee homologues. Hum Mol Genet. 7:<br />
1425-9, 1998.<br />
de Castro LA, Rodrigues Pedroso T, Kuchiishi SS,<br />
Ramenzoni M, Kich JD, Zaha A, Henning<br />
Vainstein M <strong>and</strong> Bunselmeyer Ferreira H.<br />
Variable number <strong>of</strong> t<strong>and</strong>em amino acid repeats in<br />
adhesion-related CDS products in Mycoplasma<br />
hyopneu moniae strains. Vet Microbiol. (4): 258-<br />
69, 2006.<br />
Fabre E, Dujon B <strong>and</strong> Richard GF. Transcription <strong>and</strong><br />
nuclear transport <strong>of</strong> CAG/CTG trinucleotide<br />
repeats in yeast. Nucleic Acids Res. 30(16): 3540-<br />
7, 2002.<br />
Field D <strong>and</strong> Wills C. Abundant microsatellite<br />
polymorphism in Saccharomyces cerevisiae, <strong>and</strong><br />
the different distributions <strong>of</strong> micro satellites in<br />
eight prokaryotes <strong>and</strong> S. cerevisiae, result from<br />
strong mutation pressures <strong>and</strong> a variety <strong>of</strong><br />
selective forces. Proc Natl Acad Sci USA. 95:<br />
1647–1652, 1998.<br />
Fournier B, Zhao X, Lu T, Drlica K <strong>and</strong> Hooper DC.<br />
Selective targeting <strong>of</strong> Topoisomerase IV <strong>and</strong><br />
DNA gyrase in Staphylococcus aureus: different<br />
patterns <strong>of</strong> quinolone-induced inhi bition <strong>of</strong> DNA<br />
synthesis. Antimicrob Agents Chemotherapy.<br />
44(8): 2160–2165, 2000.<br />
Glew MD, Browning GF, Markham PF <strong>and</strong> Walker<br />
ID. pMGA phenotypic variation in Mycoplasma<br />
gallisep ticum occurs in vivo <strong>and</strong> is mediated by<br />
trinucleotide repeat length variation. Infect<br />
Immun. 68(10): 6027-33, 2000.<br />
Guo X <strong>and</strong> Mrázek J. Long simple sequence repeats<br />
in host-adapted pathogens localize near genes<br />
encoding antigens, housekeeping genes, <strong>and</strong><br />
pseudogenes. J Mol Evol. 67(5): 497-509, 2008.<br />
Hancock JM <strong>and</strong> Santibanez-Koref MF.<br />
Trinucleotide expansion diseases in the context <strong>of</strong><br />
micro- <strong>and</strong> minisatellite evolu tion. EMBO J. 17:<br />
5521-5524, 1998.<br />
Hancock JM. Genome size <strong>and</strong> accumulation <strong>of</strong><br />
simple sequence repeats: implications <strong>of</strong> new data<br />
from genome sequencing projects. Genetica. 115:<br />
93-103, 2002.<br />
Hancock JM. Simple sequences <strong>and</strong> the exp<strong>and</strong> ing<br />
genome. Bioessays. 18: 421-425, 1996b.<br />
Hancock JM. Simple sequences in a “minimal”<br />
genome. Nat Genet. 14: 14-15, 1996a.<br />
Hartenstine MJ, Goodman MF <strong>and</strong> Petruska J. Base<br />
stacking <strong>and</strong> even/odd behavior <strong>of</strong> hairpin loops<br />
in DNA triplet repeat slippage <strong>and</strong> expansion<br />
with DNA polymerase. J Biol Chem. 275: 18382-<br />
18390, 2000.
Hawley DM, Sydenstricker KV, Kollias GV <strong>and</strong><br />
Dhondt AA. Genetic diversity predicts<br />
pathogen resistance <strong>and</strong> cell-mediated<br />
immunocompetence in house finches. Biol<br />
Lett. 1(3): 326-9, 2005.<br />
Jiang HX, Chen JR, Yan HL, Li XN, Chen ZL<br />
<strong>and</strong> Zeng ZL. <strong>Molecular</strong> variability <strong>of</strong> DR-1<br />
<strong>and</strong> DR-2 within the pvpA gene in<br />
Mycoplasma gallisepticum isolates. Avian Dis.<br />
53(1): 124-8, 2009.<br />
Karlin S, MrázekJ <strong>and</strong> Campbell AM. Compo<br />
sitional biases <strong>of</strong> bacterial genomes <strong>and</strong><br />
evolutionary impli cations. J Bacteriol. 179:<br />
3899-3913, 1997.<br />
Katti MV, Ranjekar PK <strong>and</strong> Gupta VS.<br />
Differential distribution <strong>of</strong> simple sequence<br />
repeats in eukaryotic genome sequences. Mol<br />
Biol Evol. 18: 1161-1167, 2001.<br />
Kruglyak S, Durrett R, Schug D <strong>and</strong> Aquadro CF.<br />
Distribution <strong>and</strong> abun dance <strong>of</strong> micro satellites<br />
in the yeast genome can be explained by a<br />
balance between slippage events <strong>and</strong> point<br />
mutations. Mol Biol Evol. 17(8): 1210-1219,<br />
2000.<br />
Li Y, Korol AB, Fahima T, Beiles A <strong>and</strong> Nevo E.<br />
Microsatellites: genomic distribution, puta tive<br />
functions <strong>and</strong> mutational mechanisms: a<br />
review. Molec Eco. 11: 2453-2465, 2002.<br />
Lim S, Notley-McRobba L, Lim M <strong>and</strong> Carter<br />
DA. A comparison <strong>of</strong> the nature <strong>and</strong><br />
abundance <strong>of</strong> micro satellites in 14 fungal<br />
genomes. Fungal Genet Biol. 41: 1025-1036,<br />
2004.<br />
Liu L, Dybvig K, Panangala VS, van Santen VL<br />
<strong>and</strong> French CT. GAA trinucleotide repeat<br />
region regulates M9/pMGA gene expression<br />
in Mycoplasma gallisepticum. Infect Immun.<br />
68(2): 871–876, 2000.<br />
Liu L, Panangala VS <strong>and</strong> Dybvig K. Trinucleotide<br />
GAA repeats dictate pMGA gene expression<br />
in Mycoplasma gallisepticum by affect ing<br />
spacing between flanking regions. J Bacteriol.<br />
184(5): 1335–1339, 2002.<br />
Lysnyansky I, Calcutt MJ, Ben-Barak I, Ron Y,<br />
Levisohn S, Methé BA <strong>and</strong> Yogev D.<br />
<strong>Molecular</strong> characterization <strong>of</strong> newly identified<br />
IS3, IS4 <strong>and</strong> IS30 insertion sequence-like<br />
elements in Mycoplasma bovis <strong>and</strong> their<br />
possible roles in genome plasticity. FEMS<br />
Micro Biol Lett. 294(2): 172-82, 2009.<br />
SSRs in Mycoplasma replication genes<br />
McAuliffe L, Ayling RD <strong>and</strong> Nicholas RA.<br />
Identification <strong>and</strong> charac terization <strong>of</strong> variablenumber<br />
t<strong>and</strong>em -repeat markers for the molecular<br />
epidemiological analysis <strong>of</strong> Mycoplasma<br />
mycoides subspecies mycoides SC. FEMS Micro<br />
Biol Lett. 276(2): 181-8, 2007.<br />
McAuliffe L, Churchward CP, Lawes JR, Loria G,<br />
Ayling RD <strong>and</strong> Nicholas RA. VNTR analysis<br />
reveals un expected genetic diversity within<br />
Mycoplasma agalactiae, the main causative agent<br />
<strong>of</strong> contagious agalactia. BMC Microbiol. 8: 193,<br />
2008.<br />
Mekchay S, Ponsuksili S, Schell<strong>and</strong>er K <strong>and</strong><br />
Wimmers K. Association <strong>of</strong> the porcine C3 gene<br />
with haemolytic complement activity in the pig.<br />
Genet Sel Evol. 35(Suppl 1): S83-96, 2003.<br />
Metzgar D, Byt<strong>of</strong> J <strong>and</strong> Wills C. Selection against<br />
frameshift muta tions limits microsatellite<br />
expansion in coding DNA. Genome Res. 10: 72-<br />
80, 2000.<br />
Metzgar D, Liu L, Hansen C, Dybvig K <strong>and</strong> Wills C.<br />
Domain-level differences in micro satellite distri<br />
bution <strong>and</strong> content result from different relative<br />
rates <strong>of</strong> insertion <strong>and</strong> deletion mutations. Genome<br />
Res. 12(3): 408-13, 2002.<br />
Minion FC, Lefkowitz EJ, Madsen ML, Cleary BJ,<br />
Swartzell SM <strong>and</strong> Mahairas GG. The genome<br />
sequence <strong>of</strong> Mycoplasma hyopneumoniae strain<br />
232, the agent <strong>of</strong> swine mycoplasmosis. J<br />
Bacteriol. 186(21): 7123-33, 2004.<br />
Mrázek J, Guo X <strong>and</strong> Shah A. Simple sequence<br />
repeats in prokaryotic genomes. Proc Natl Acad<br />
Sci USA. 104(20): 8472-7, 2007.<br />
Mrázek J. Analysis <strong>of</strong> distribution indicates diverse<br />
functions <strong>of</strong> simple sequence repeats in<br />
Mycoplasma genomes. Mol Biol Evol. 23(7):<br />
1370-85, 2006.<br />
Musatovova O, Kannan TR <strong>and</strong> Baseman JB.<br />
Genomic analysis reveals Mycoplasma<br />
pneumoniae repetitive element 1-mediated<br />
recombination in a clinical isolate. Infect Immun.<br />
76(4): 1639-48, 2008.<br />
Pereyre S, Gonzalez P, De Barbeyrac B, Darnige A,<br />
Renaudin H, Charron A, Raherison S, Bébéar C<br />
<strong>and</strong> Bébéar CM. Mutations in 23S rRNA account<br />
for intrinsic resistance to macrolides in<br />
Mycoplasma hominis <strong>and</strong> Mycoplasma<br />
fermentans <strong>and</strong> for acquired resistance to<br />
macrolides in M. hominis. Antimicrob Agents<br />
Chemotherapy. 46(10): 3142-50, 2002.<br />
69
70<br />
Seema TRIVEDI<br />
Primmer CR, Raudsepp T, Chowdhary BP,<br />
Moller AP <strong>and</strong> Ellegren H. Low frequency <strong>of</strong><br />
microsatellites in the avian genome. Genome<br />
Res. 7: 471-482, 1997.<br />
Rocha EP, Blanchard A. Genomic repeats,<br />
genome plasticity <strong>and</strong> the dynamics <strong>of</strong><br />
Mycoplasma evolution. Nucleic Acids Res.<br />
30(9): 2031-42, 2002.<br />
Simmons WL <strong>and</strong> Dybvig K. Bi<strong>of</strong>ilms protect<br />
Mycoplasma pulmonis cells from lytic effects<br />
<strong>of</strong> complement <strong>and</strong> gramicidin. Infect Immun.<br />
75(8): 3696-9, 2007.<br />
Sluijter M, Spuesens EB, Hartwig NG, van<br />
Rossum AM <strong>and</strong> Vink C. The Mycoplasma<br />
pneumoniae MPN490 <strong>and</strong> Mycoplasma<br />
genitalium MG339 genes encode reca<br />
homologs that promote homologous DNA<br />
str<strong>and</strong> exchange. Infect Immun. 77(11): 4905-<br />
11, 2009.<br />
Toth G, Gaspari Z <strong>and</strong> Jurka J. Microsatellites in<br />
different eukaryo tic genomes: survey <strong>and</strong><br />
analysis. Genome Res. 10: 967-981, 2000.<br />
Trivedi S. Comparison <strong>of</strong> simple sequence repeats<br />
in 19 Archaea. Genet Mol Res. 5(4): 741-72,<br />
2006.<br />
Trivedi S. Do Microsatellites Have Biased<br />
Associations? The Nucleus. 46(1,2): 61-76,<br />
2003.<br />
Trivedi S. Microsatellites (SSRs): Puzzles within<br />
puzzle. Indian J Biotech. 3: 331-347, 2004.
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong> 7(2) & 8(1): 71-72, 2010 S<strong>of</strong>tware Review<br />
<strong>Haliç</strong> University, Printed in Turkey.<br />
http://jcmb.halic.edu.tr<br />
Tc<strong>of</strong>fee ©: Multipurpose sequence alignments program<br />
Authors: C. NOTREDAME, L. HOLME D.G. HIGGINS, J. HERINGA, O.<br />
O'SULLIVAN, K SUHRE, C. ABERGEL<br />
License: Open source freeware<br />
Tc<strong>of</strong>fee at a glance<br />
T-C<strong>of</strong>fee st<strong>and</strong>s for Tree based Consistency<br />
Objective Function for Alignment Evaluation. It is<br />
a recent program for making multiple sequence<br />
alignments. It yields more accurate alignments at<br />
the cost <strong>of</strong> a slightly longer running time than other<br />
programs. Although it uses a progressive alignment<br />
method like ClustalW, it compares segments across<br />
the entire sequence set, which means aligning all<br />
the sequences at the same time. The main<br />
difference between Tc<strong>of</strong>fee <strong>and</strong> ClustalW is that<br />
Tc<strong>of</strong>fee doesn’t directly use substitution matrices to<br />
align sequences. It has many different applications<br />
<strong>and</strong> modules such as the main EXPRESSO for<br />
aligning sequences <strong>and</strong> structures, CORE for<br />
evaluating the accuracy <strong>of</strong> an alignment, Mc<strong>of</strong>fee<br />
for combining many alternative multiple sequence<br />
alignments into one. Briefly, Tc<strong>of</strong>fee could be<br />
described as a collection <strong>of</strong> tools for computing,<br />
evaluating <strong>and</strong> manipulating multiple alignments <strong>of</strong><br />
DNA, RNA <strong>and</strong> protein sequences <strong>and</strong> structures.<br />
Tc<strong>of</strong>fee <strong>and</strong> its contribution to research<br />
Recently, many researchers in the field <strong>of</strong><br />
molecular biology have used Tc<strong>of</strong>fee modules in<br />
building, computing, evaluating <strong>and</strong> manipulating<br />
multiple sequence alignments during their original<br />
research. Although Tc<strong>of</strong>fee has been produced <strong>and</strong><br />
developed few years ago, it has been cited in many<br />
peer reviewed articles. However the number <strong>of</strong><br />
citations is still not comparable to ClustalW. Beside<br />
its quick ability to produce results, the open source<br />
freeware license <strong>and</strong> its efficient multifunctional<br />
modules make Tc<strong>of</strong>fee one <strong>of</strong> the most useful<br />
programs for making multiple sequence alignments.<br />
Advantages <strong>and</strong> disadvantages <strong>of</strong> Tc<strong>of</strong>fee<br />
Advantages<br />
- It produces more accurate alignments than the<br />
other methods.<br />
- It is equipped with many different tools <strong>and</strong><br />
modules such as CORE, Mc<strong>of</strong>fee <strong>and</strong><br />
-<br />
EXPRESSO for structure alignment, evaluation<br />
<strong>and</strong> combining alignments.<br />
Tc<strong>of</strong>fee can deal with many input formats,<br />
including FASTA, Swiss-Prot <strong>and</strong> PIR (Protein<br />
Information Resource).<br />
- Tc<strong>of</strong>fee produces sequence alignment in various<br />
formats so that it can be used as an input for<br />
another program. It also produces a colorized<br />
alignment where every residue appears on a<br />
background that indicates the quality <strong>of</strong> this<br />
alignment in (.html) <strong>and</strong> (.pdf) format.<br />
- It can produce true phylogenetic tree in Newick<br />
format by using the Neighbor Joining method.<br />
- It can work with list <strong>of</strong> DNA, RNA or protein<br />
sequences.<br />
- Tc<strong>of</strong>fee can evaluate the quality <strong>of</strong> any multiple<br />
sequence alignments using CORE server.<br />
Disadvantages<br />
- It takes longer time to align multiple sequences<br />
than other programs.<br />
- It has been cited in limited number <strong>of</strong> peer<br />
reviewed journals compared to ClustalW.<br />
However, this number is increasing rapidly<br />
every day.
72<br />
S<strong>of</strong>tware design<br />
T-C<strong>of</strong>fee is an open source freeware. It can<br />
generate multiple sequence alignment for a given a<br />
set <strong>of</strong> sequences (Protein, RNA or DNA). The latest<br />
version <strong>of</strong> T-C<strong>of</strong>fee is 5.65. It runs on UNIX or<br />
Micros<strong>of</strong>t Windows/Cywin. Version 2.00 <strong>and</strong><br />
higher can combine sequences <strong>and</strong> structures. It<br />
uses bioperl in the design. The interface is self<br />
explanatory with no complicated terms <strong>and</strong><br />
expression. EXPRESSO aligns the structures using<br />
SAP, a program from Taylor <strong>and</strong> Orengo, <strong>and</strong> it<br />
aligns sequences <strong>and</strong> structures using FUGUE, a<br />
threading package from Kenji Mizuguchi<br />
(developed in Tom Blundell’s lab at Cambridge<br />
University). CORE server on www.tc<strong>of</strong>fee.org can<br />
evaluate the quality <strong>of</strong> any multiple sequence<br />
alignments with any <strong>of</strong> the most common formats<br />
(MSF, ALN, FASTA, <strong>and</strong> PIR).<br />
Limitations in use<br />
- The input for Tc<strong>of</strong>fee is limited to a maximum<br />
number <strong>of</strong> sequences <strong>of</strong> 50 <strong>and</strong> the maximum<br />
length <strong>of</strong> sequences <strong>of</strong> 2000.<br />
- The data will remain available on the server for<br />
only nine days. Then it will be deleted.<br />
- It is very important to cite the Tc<strong>of</strong>fee authors<br />
when using its resources. For instance, if you<br />
use the local version <strong>of</strong> Tc<strong>of</strong>fee, cite the<br />
following paper:-<br />
Notredame, D. Higgins, J. Heringa . T-C<strong>of</strong>fee: A<br />
novel method for multiple sequence alignments.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Molecular</strong> <strong>Biology</strong>, Vol 302, pp205-<br />
217,2000.<br />
Otherwise, cite the paper that corresponds to the<br />
server you have been using (click on the "cite"<br />
button associated with every server on<br />
www.tc<strong>of</strong>fee.org).<br />
New features, flavors <strong>and</strong> tools <strong>of</strong> Tc<strong>of</strong>fee<br />
Alignment<br />
TCOFFEE (Regular or advanced): Computes a<br />
multiple sequence alignment <strong>and</strong> the associated<br />
phylogenetic tree.<br />
EXPRESSO (3DC<strong>of</strong>fee) (Regular or advanced):<br />
This server computes structure based Multiple<br />
Sequence Alignments.<br />
MCOFFEE (Regular or advanced): Computes a<br />
multiple sequence alignment <strong>and</strong> the associated<br />
phylogenetic tree by combining the output <strong>of</strong><br />
several multiple sequence alignment packages<br />
(PCMA, Poa, Mafft, Muscle, T-C<strong>of</strong>fee,<br />
ClustalW, ProbCons, DialignT).<br />
COMBINE (Regular or advanced): combines two<br />
(or more) multiple sequence alignments into a<br />
single one.<br />
RCOFFEE (Regular or advanced): Multiple<br />
Sequence Alignment <strong>of</strong> Non Coding RNA<br />
Sequences using RNAplfold predicted<br />
secondary structures.<br />
Evaluation<br />
CORE (Regular or advanced): evaluates your<br />
Alignment <strong>and</strong> outputs a colored version where<br />
bad portions are in blue <strong>and</strong> the good ones in<br />
red. Your alignment must contain at least four<br />
sequences<br />
iRMSD-APDB (Regular or advanced): Evaluates<br />
your Multiple Sequence Alignment using<br />
APDB which estimates the proportion <strong>of</strong><br />
columns correctly aligned in a pairwise or a<br />
multiple alignment <strong>of</strong> sequences with known<br />
structures.<br />
List <strong>of</strong> Tc<strong>of</strong>fee servers availability around<br />
the world<br />
- www.tc<strong>of</strong>fee.org<br />
- http://tc<strong>of</strong>fee.vital-it.ch/cgibin/Tc<strong>of</strong>fee/tc<strong>of</strong>fee_cgi/index.cgi<br />
- http://www.es.embnet.org/Services/MolBio/tc<strong>of</strong>fee/<br />
- http://www.ebi.ac.uk/t-c<strong>of</strong>fee/<br />
Ahmed MANSOUR<br />
Genetics Department,<br />
Faculty <strong>of</strong> Agriculture,<br />
Zagazig University, Egypt<br />
(author for correspondence; amansour@zu.edu.eg)<br />
Received: 27 March 2008; Accepted: 11 December 2009
Tc<strong>of</strong>fee © : Çok amaçlı dizi hizalama programı<br />
Yazarlar: C. NOTREDAME, L. HOLME D.G. HIGGINS, J. HERINGA, O.<br />
O'SULLIVAN, K SUHRE, C. ABERGEL<br />
Lisans: Açık kaynak, ücretsiz yazılım<br />
Tc<strong>of</strong>fee’ye kısa bir bakış<br />
T-c<strong>of</strong>fee "Ağaç Bazlı Tutarlılık Uyum Amaç<br />
Fonksiyonunun Değerlendirilmesi" anlamına<br />
gelmektedir. Çoklu dizi hizalaması yapmak için<br />
kullanılan yeni bir programdır. Biraz daha uzun<br />
çalışma zamanı pahasına diğer programlardan daha<br />
kesin hizalama sağlamaktadır. ClustalW gibi<br />
progresif bir program kullanmasına rağmen aynı<br />
zam<strong>and</strong>a hizalanmış bütün sekansları ifade eden<br />
bütün sekans setiyle karşılaştırmaktadır. Tc<strong>of</strong>fee<br />
sekansları hizalamak için matris yer değişimini<br />
direkt olarak kullanmamasıdır. Tc<strong>of</strong>fee ve<br />
ClustalW arasındaki ana fark Tc<strong>of</strong>fee'nin sekansları<br />
hizalamak için matris yer değişimini direkt olarak<br />
kullanmamasıdır.. Başlıca dizi ve yapıların<br />
hizalanması için EXPRESSO, bir hizalamanın<br />
kesinliğini değerlendirmek için CORE, birçok<br />
alternatif çoklu dizi hizalamalarını tek olarak<br />
birleştirmek için Mc<strong>of</strong>fee gibi çok farklı<br />
uygulamalar ve modüllere sahiptir. Kısaca, Tc<strong>of</strong>fee<br />
DNA, RNA ve protein dizileri ve yapılarının çoklu<br />
dizi hizalamasını kullanma, hesaplama ve<br />
değerlendirme için bir alet topluluğuymuş gibi<br />
tanımlanabilir.<br />
Tc<strong>of</strong>fee ve araştırmaya katkısı<br />
Son zamanlarda, moleküler biyoloji alanındaki<br />
birçok araştırmacı orijinal araştırmaları süresince<br />
çoklu dizi hizalaması yapmak, manipüle etmek,<br />
hesaplamak ve değerlendirmek için Tc<strong>of</strong>fee<br />
modüllerini kullanmaktadır. Tc<strong>of</strong>fee birkaç yıl önce<br />
üretilmiş ve geliştirilmiş olmasına rağmen, birçok<br />
derlenmiş eşdüzey makalede atıfta bulunulmuştur.<br />
Ancak atıf sayısı hala ClustalW ile karşılaştırılamaz.<br />
Sonuçları hızlı elde etme özelliğine ilaveten,<br />
73<br />
açık kaynak ücretsiz yazılım lisansı ve çok<br />
fonksiyonlu etkili modülleri, Tc<strong>of</strong>fee’yi çoklu dizi<br />
hizalaması için en kullanışlı programlardan biri<br />
yapar.<br />
Tc<strong>of</strong>fee’nin avantajları ve dezavantajları<br />
Avantajları<br />
- Diğer metodlardan daha kesin karşılaştırmalar<br />
ortaya koyar.<br />
- Yapı hizalama, hesaplama ve hizalamaları<br />
birleştirme için CORE, Mc<strong>of</strong>fee ve EXPRESSO<br />
gibi birçok farklı aletle ve modülle donatılmıştır.<br />
- Tc<strong>of</strong>fee; FASTA, Swiss-Prot ve PIR (Protein<br />
Information Resource, Protein bilgi kaynağı) da<br />
dahil birçok input formatını düzenlemek için<br />
açabilir.<br />
- Tc<strong>of</strong>fee çeşitli fomatlarda dizi hizalaması yapar.<br />
Bundan dolayı başka bir program için bir input<br />
olarak kullanılabilir. Ayrıca (.html) ve (pdf)<br />
formatında bu hizalamanın kalitesini belirten bir<br />
arka plan üzerinde her kalıntının göründüğü<br />
yerde renklendirilmiş bir hizalama yapar.<br />
- Neighbor Joining metodunu kullanarak Newick<br />
formatında doğru filogenetik ağaç oluşturabilir.<br />
- DNA, RNA veya Protein dizileri listesiyle<br />
çalışabilir.<br />
- Tc<strong>of</strong>fee CORE sunucusunu kullanarak herhangi<br />
çoklu dizi hizalamasının kalitesini değerlendirebilir.<br />
Dezavantajları<br />
- Çoklu dizileri karşılaştırmada diğer<br />
programlardan daha uzun zaman alır.<br />
- ClustalW’e göre sınırlı sayıda derlenmiş<br />
eşdüzey dergide atıfta bulunulmuştur. Ancak bu<br />
sayı her gün hızlıca artmaktadır.
74<br />
Yazılım Dizaynı<br />
T-C<strong>of</strong>fee bir açık kaynak ücretsiz yazılımdır.<br />
Verilen bir dizi seti (Protein, DNA ya da RNA) için<br />
çoklu dizi hizalaması oluşturmaktadır. Tc<strong>of</strong>fee’nin<br />
en son versiyonu 5.65’tir. UNIX ya da Micros<strong>of</strong>t<br />
Windows/Cywin ile çalışır. Sürüm 2.00 ve üzeri<br />
yapıları ve dizileri birleştirebilir. Bu dizaynda<br />
Bioperl kullanır. Arabirim karmaşık olmayan terim<br />
ve anlatımlarla kendiliğinden anlaşılır.<br />
EXPRESSO, Taylor ve Orengo tarafından yazılan<br />
bir program olan SAP’ı kullanarak yapıları hizaya<br />
sokar. Dizileri ve yapıları Kenji Mizuguchi’den bir<br />
dizi paketi (Cambridge üniversitesinde Tom<br />
Blundell’s Laboratuarında geliştirilen) olan<br />
FUGUE’yi kullanarak hizaya sokar.<br />
www.tc<strong>of</strong>fee.org üzerinde CORE sunucusu en<br />
yaygın formatların (MSF, ALN, FASTA ve PIR)<br />
herhangi biriyle çoklu dizi hizalama kalitesini<br />
değerlendirebilir.<br />
Kullanımda sınırlamalar<br />
- Tc<strong>of</strong>fee için input maksimum dizi sayısı 50 ve<br />
maksimum dizi uzunluğu 2000’e kadar<br />
sınırl<strong>and</strong>ırılmıştır.<br />
- Veriler sunucu üzerinde sadece dokuz gün<br />
kullanılabilir olarak kalacaktır. Sonra<br />
silinecektir.<br />
- Kaynaklarını kullanırken Tc<strong>of</strong>fee yazarlarına<br />
atıfta bulunmak önemlidir. Örneğin,<br />
Tc<strong>of</strong>fee’nin sınırlı sürümünü kullanırsanız<br />
belirtilen makaleye atfedin:-<br />
Notredame, D. Higgins, J. Heringa . T-C<strong>of</strong>fee: A<br />
novel method for multiple sequence alignments.<br />
<strong>Journal</strong> <strong>of</strong> <strong>Molecular</strong> <strong>Biology</strong>, Vol 302, pp205-<br />
217, 2000.<br />
Aksi takdirde, kull<strong>and</strong>ığınız sunucuya karşılık<br />
gelen makaleye atıfta bulunun<br />
(www.tc<strong>of</strong>fee.org üzerindeki her sunucuyla<br />
alakalı “cite” tuşuna tıklayarak).<br />
Tc<strong>of</strong>fee’nin aygıtları, belirli özellikleri ve<br />
yeni özellikler<br />
Hizalama<br />
TCOFFEE (normal ya da gelişmiş düzeyde): Çoklu<br />
dizi hizalama ve ilişkilendirilmiş filogenetik<br />
ağacı hesaplar.<br />
EXPRESSO (3DC<strong>of</strong>fee) (normal ya da gelişmiş<br />
düzeyde): Bu sunucu yapı temelli Çoklu Dizi<br />
Hizalamalarını hesaplar.<br />
MCOFFEE (normal ya da gelişmiş düzeyde):<br />
Birkaç çoklu dizi hizalama paketininin (PCMA,<br />
Poa, Mafft, Muscle, T-C<strong>of</strong>fee, ClustalW,<br />
ProbCons, DialignT) output’unu (çıkış)<br />
birleştirerek çoklu dizi hizalama ve ilişkilendirilmiş<br />
filogenetik ağacı hesaplar.<br />
COMBINE (normal ya da gelişmiş düzeyde): iki<br />
(veya daha fazla) çoklu dizi hizalamalarını tek<br />
bir tanesinde birleştirir.<br />
RCOFFEE (normal ya da gelişmiş düzeyde):<br />
RNAplfold tarafından oluşturulan tahmini<br />
ikincil yapıları kullanarak Kodlanmayan RNA<br />
Dizilerinin Çoklu Dizi Hizalaması.<br />
Değerlendirme<br />
CORE (normal ya da gelişmiş düzeyde):<br />
Hizalamanızı ve kötü kısımlarının mavi iyi<br />
olanların kırmızı olduğu renkli çıktılarınızı<br />
değerlendirir. Hizalamanız en azından dört dizi<br />
içermelidir.<br />
iRMSD-APDB (normal ya da gelişmiş düzeyde):<br />
İkili olarak doğru bir şekilde hizalanmış ya da<br />
yapıları bilinen dizilerin çoklu hizalamasında<br />
sütunların oranını tahmin eden APDB<br />
kullanarak Çoklu Dizi Hizalamasını değerlendirir.<br />
Dünya çapında kullanılabilir Tc<strong>of</strong>fee<br />
sunucuları listesi<br />
- www.tc<strong>of</strong>fee.org<br />
- http://tc<strong>of</strong>fee.vital-it.ch/cgibin/Tc<strong>of</strong>fee/tc<strong>of</strong>fee_cgi/index.cgi<br />
- http://www.es.embnet.org/Services/MolBio/tc<strong>of</strong>fee/<br />
- http://www.ebi.ac.uk/t-c<strong>of</strong>fee/<br />
Ahmed MANSOUR<br />
Genetik Bölümü,<br />
Ziraat Fakültesi,<br />
Zagazig <strong>Üniversitesi</strong>, Mısır
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong> 7(2) & 8(1): 75-77, 2010 S<strong>of</strong>tware Review<br />
<strong>Haliç</strong> University, Printed in Turkey.<br />
http://jcmb.halic.edu.tr<br />
UCSC: Genome Browser for genomic sequences<br />
Authors: UCSC Genome Bioinformatics Group led by David Haussler <strong>and</strong> Jim Kent,<br />
Center for Biomolecular Science & Engineering, University <strong>of</strong> California<br />
License: Free for academic, non-pr<strong>of</strong>it, <strong>and</strong> personal use. A license is required for<br />
commercial use.<br />
Genome browser at a glance<br />
Genome Browser is a tool for collecting all relevant<br />
genomic sequence data in one site <strong>and</strong> provides<br />
rapid, reliable, <strong>and</strong> simultaneous display <strong>of</strong> any<br />
requested portion <strong>of</strong> genomes at any scale in a<br />
graphical design. The UCSC Genome Browser<br />
resource contains the reference (or <strong>of</strong>ficial) public<br />
DNA sequences <strong>and</strong> working draft assemblies for<br />
human <strong>and</strong> a large collection <strong>of</strong> other genomes.<br />
There are a number <strong>of</strong> tools within this site that<br />
provides access to the sequences themselves, <strong>and</strong><br />
many other useful genome features to add context<br />
to the genomic information. Researchers can use<br />
this site to find genes <strong>and</strong> gene predictions,<br />
expression information, SNPs <strong>and</strong> variations, crossspecies<br />
comparative data, <strong>and</strong> many more.<br />
Moreover, the UCSC provide the ability to search<br />
for markers <strong>and</strong> sequences, to extract annotations<br />
for specific regions or for the whole genome, <strong>and</strong> to<br />
act as a central starting point for genomic research.<br />
It also provides a portal to the ENCODE project.<br />
UCSC genome browser contribution to<br />
research<br />
<strong>Molecular</strong> biologists use UCSC genome browser<br />
modules during their original research tools such<br />
"Genome Browser", "Gene Sorter", 'Blat", "Table<br />
Browser", "VisiGene" <strong>and</strong> "Genome Graphs" all <strong>of</strong><br />
which allow the user to navigate, sort, blast,<br />
visualize <strong>and</strong> analyzes genomic data for reliable<br />
annotation.<br />
Moreover, researchers can learn more about<br />
the object (e.g., known genes, conservation, or<br />
SNPs, etc.) via researching by simply position the<br />
mouse over information line <strong>and</strong> click, then a new<br />
web page will appear with important details <strong>and</strong><br />
information. The Page Index box usually includes<br />
sequences, microarray data, mRNA secondary<br />
structure, protein domain structure, homologues in<br />
other species, gene ontology descriptions, mRNA<br />
descriptions <strong>and</strong> pathways, etc. This wealth <strong>of</strong><br />
information is more than enough for a molecular<br />
biologist as a start point for genomic research.<br />
Colours language in UCSC genome browser<br />
Colors have important meanings in UCSC genome<br />
browser. For instance, the Black color in gene track<br />
indicates a protein data bank (PDB) structure entry<br />
for this genome fragment. Dark blue indicates<br />
NCBI-reviewed sequence, while light blue<br />
corresponds to provisional sequences. In addition,<br />
SNP types are also color-coded. More information<br />
about any specific color representation <strong>and</strong><br />
annotation or descriptive information can be<br />
obtained by clicking the hyperlink <strong>of</strong> the track.<br />
Advantages <strong>and</strong> disadvantages <strong>of</strong> UCSC<br />
genome browser<br />
Advantages<br />
- UCSC Genome Browser is very easy to use <strong>and</strong><br />
free <strong>of</strong> charge online.<br />
- UCSC uses the same interface <strong>and</strong> display for<br />
each <strong>of</strong> the species listed.<br />
- In addition, UCSC can run on (almost) any<br />
computer that has access to internet <strong>and</strong> can<br />
return the results online or by e-mail.<br />
- The Genome Viewer page provides several<br />
options to make changes<br />
- Text search strategy can be used by typing in<br />
gene name, gene symbol, or ID, etc.<br />
- "Automatic Zoom" <strong>and</strong> "Recenter Action" are<br />
h<strong>and</strong>y features to automatically re-center the<br />
image where you click
76<br />
- The Custom Track hyperlink on the UCSC<br />
home page allows the user to create custom<br />
tracks <strong>of</strong> his data.<br />
Limitations<br />
- The number <strong>of</strong> available genomes are limited,<br />
especially plant genomes.<br />
- Different species have different annotation<br />
tracks depending on the availability <strong>of</strong> data<br />
assembly.<br />
- "Automatic Zoom" can only zoom three folds.<br />
- Not all the genes have the same levels <strong>of</strong> detail,<br />
<strong>and</strong> not every species has all the information.<br />
- BLAT allows to paste up to 25,000 bases,<br />
10,000 amino acids, <strong>and</strong> up to a total <strong>of</strong> 25<br />
sequences in the common FASTA<br />
- Custom tracks are only persistent for 8 h <strong>and</strong><br />
needs to be re-done after 8 h if you have not<br />
downloaded the file.<br />
- The configuration options <strong>of</strong> the output format<br />
<strong>of</strong>fered by Protein Duster program are limited.<br />
UCSC genome viewer tracks<br />
The Genome Viewer section features have the<br />
following tracks:<br />
(1) Mapping <strong>and</strong> Sequencing Tracks;<br />
(2) Phenotype <strong>and</strong> Disease Associations;<br />
(3) Genes <strong>and</strong> Gene Prediction Tracks;<br />
(4) mRNA <strong>and</strong> EST Tracks;<br />
(5) Expression <strong>and</strong> Regulation;<br />
(6) Comparative Genomics, Variation, <strong>and</strong> Repeats;<br />
(7) ENCODE Regions <strong>and</strong> Genes;<br />
(8) ENCODE Transcript Levels;<br />
(9) ENCODE Chromatin Immunoprecipitation;<br />
(10) ENCODE Chromosome, Chromatin, <strong>and</strong> DNA<br />
Structure; <strong>and</strong> ENCODE Comparative.<br />
Different Genome browsers online<br />
UCSC genome browser (http://genome.ucsc.edu/)<br />
Ensembl genome browser<br />
(http://www.ensembl.org)<br />
VISTA (http://genome.lbl.gov)<br />
NCBI MapViewer<br />
(http://www.ncbi.nlm.nih.gov/mapview/)<br />
ECR Browser (http://ecrbrowser.dcode.org)<br />
Combo<br />
(http://www.broad.mit.edu/annotation/argo/)
Genomics <strong>and</strong> ENCODE Variation<br />
UCSC Genome Browser website tools<br />
TOOL NAME FUNCTION<br />
Genome Browser<br />
Gene Sorter<br />
Zooms <strong>and</strong> scrolls over chromosomes, showing the work <strong>of</strong> annotators<br />
worldwide<br />
Shows expression, homology <strong>and</strong> other information on groups <strong>of</strong> genes<br />
that can be related in many ways.<br />
Blat Quickly maps your sequence to the genome.<br />
Table Browser Provides convenient access to the underlying database<br />
VisiGene<br />
Lets you browse through a large collection <strong>of</strong> in situ mouse <strong>and</strong> frog<br />
images to examine expression patterns<br />
Genome Graphs Allows you to upload <strong>and</strong> display genome-wide data sets<br />
77<br />
Ahmed MANSOUR<br />
Genetics Department,<br />
Faculty <strong>of</strong> Agriculture,<br />
Zagazig University, Egypt<br />
(author for correspondence; amansour@zu.edu.eg)<br />
Received: 01 September 2008; Accepted: 11 December 2009
78<br />
UCSC: Genomik diziler için Genom Tarayıcısı<br />
Yazarlar: David Haussler ve Jim Kent liderliğinde UCSC Genom Biyoenformatiği grubu,<br />
Biyomoleküler Bilim ve Mühendislik Merkezi, Kaliforniya <strong>Üniversitesi</strong><br />
Lisans: Akademik, kişisel ve kar amacı gütmeyen durumlarda kullanımı ücretsizdir. Ticari<br />
amaçla kullanımı için lisans gereklidir.<br />
Bir bakışta Genom Tarayıcı<br />
Genom tarayıcı bir genomik dizi ile alakalı tüm<br />
bilgileri bir sayfada toplayan ve genomun herhangi<br />
bir bölgesinin istenilen her ölçüde görsel bir dizayn<br />
ile güvenilir ve hızlı bir şekilde görüntülenmesini<br />
sağlayan bir araçtır. UCSC genom tarayıcısı<br />
kaynağı, insan genomu ve diğer birçok organizma<br />
genomu için resmi, referans veya taslak DNA<br />
dizilerini içermektedir. Sayfada dizilere erişim<br />
sağlayan araçların yanı sıra bu dizilere dair<br />
genomik enformasyona ilave yapabilen birçok<br />
özellik de vardır. Araştırmacılar bu sayfayı bilinen<br />
gen dizilerine, tahmini gen dizilerine,<br />
ekspresyonları ile ilgili bilgilere, türler arası<br />
karşılaştırmalı bilgiye, tek nükleotid<br />
polimorfizmlerine ve varyasyonlarına ve daha<br />
birçok bilgiye erişmek amacıyla kullanabilirler.<br />
UCSC ayrıca markör dizi aranmasına, belirli bir<br />
bölge veya tüm genom hakkında açıklama elde<br />
edilmesine olanak sağlayarak genom<br />
araştırmalarında başlangıç noktası olarak işlev<br />
görmektedir. ENCODE projesine de bağlantı<br />
sağlamaktadır.<br />
UCSC Genom Tarayıcının Araştırmalara<br />
Katkısı<br />
Moleküler biyologlar UCSC genom tarayıcı<br />
modüllerini dizileri düzenlemeye, BLAST araması<br />
yapmaya, görüntülemeye ve güvenilir bilgi için<br />
genomik verilerin analiz edilmesine olanak<br />
sağlayan ''Genome Browser'', ''Gene Sorter'',<br />
''BLAT'', ''Table Browser'',''VisiGene'' ve ''Genome<br />
Graphs'' gibi esas araştırma araçları ile beraber<br />
kullanmaktadırlar. Buna ek olarak araştırmacılar,<br />
araştırma konuları ile (örneğin; bilinen genler,<br />
korunum, tek nükleotid polimorfizmleri vb) ilgili<br />
ek bilgilere fare imlecinin ''information'' kutusunun<br />
üzerine getirilip tıklanmasıyla açılan ve önemli<br />
bilgi ve detayları barındıran yeni sayfa aracılığı ile<br />
ulaşabilirler. Sayfa indeks kutusu genellikle dizileri,<br />
mikroarray verilerini, ikincil mRNA yapısını,<br />
protein yapılarını, diğer türlerle olan homolojileri,<br />
gen ontolojisi ile ilgili açıklamaları ve araştırılan<br />
gen ile alakalı yolakları içerir. Bu bilgi zenginliği,<br />
genomik araştırmaya başlangıç noktası olarak bir<br />
moleküler biyolog için fazlasıyla yeterlidir.<br />
UCSC Genom Tarayıcıda Renklerin Dili<br />
UCSC genom tarayıcıda renklerin önemli anlamları<br />
vardır. Örneğin gen girdisindeki siyah renk, protein<br />
bilgi bankasında (PDB) ilgili genom parçası için<br />
protein yapısına dair bir girdi olduğunu belirtir.<br />
Koyu mavi renk NCBI tarafından onaylanmış<br />
dizileri belirtirken açık mavi doğruluğu<br />
onaylanmamış dizileri belirtir. Tek nükleotid<br />
polimorfizmi çeşitleri de renklerle kodlanmıştır.<br />
Belirli bir renk koduna dair bilgi, girdinin üst<br />
bağlantısına tıklanarak elde edilebilir.<br />
UCSC Genom Tarayıcının Avantajları ve<br />
Dezavantajları<br />
Avantajları<br />
- UCSC Genom Tarayıcıyı kullanmak kolay ve<br />
bedavadır.<br />
- Listelenen bütün türler için aynı arayüz ve<br />
görünüm kullanılmaktadır.<br />
- Ayrıca UCSC, internet erişimi olan neredeyse tüm<br />
bilgisayarlarda çalıştırılabilir ve sonuçlar internet<br />
üzerinden veya e-posta yoluyla elde<br />
edilebilmektedir.<br />
- ''Genome Viewer'' sayfası, değişiklik yapmak için<br />
birçok olanak sağlamaktadır.<br />
- Gen ismi, sembolü veya kodu girilerek metin<br />
araması yapılabilmektedir.<br />
- "Automatic Zoom" ve "Recenter Action" özellikleri<br />
tıklanan noktada görüntüyü ortalamak için
oldukça kullanışlıdır.<br />
- UCSC ana sayfasındaki ''Custom Track'' üst<br />
bağlantısı kullanıcıya konu ilgili kendi kişisel<br />
girdilerini oluşturma imkanı vermektedir.<br />
Dezavantajlar<br />
- Erişilebilir genomların özellikle de bitki<br />
genomlarının sayısı oldukça sınırlıdır.<br />
- O türe dair bilgilerin erişilebilirliğine bağlı olarak,<br />
farklı türlere ilişkin farklı açıklama girdileri<br />
bulumaktadır.<br />
- "Automatic Zoom" özelliği görüntüyü yalnızca 3<br />
kat yaklaştırabilmektedir.<br />
- Her gen için aynı bilgi detayı ve her tür için bütün<br />
bilgiler yoktur.<br />
- ''BLAT'' 25000 baz, 10000 amino asit ve FASTA<br />
formatında 25 sekansın yüklenmesine izin<br />
verebilmektedir.<br />
- Kişisel girdiler yalnızca sekiz saat için geçerlidir<br />
ve dosya indirilmediği takdirde yeniden<br />
oluşturulmaları gerekmektedir.<br />
- ''Protein Duster'' yazılımı tarafından sunulan çıktı<br />
formatlarının yapıl<strong>and</strong>ırma ayarları sınırlıdır.<br />
UCSC Genom Görüntüleyici Girdileri<br />
Genom Görüntüleyici bölümünde aşağıdaki girdi<br />
79<br />
kategorileri bulunmaktadır:<br />
(1) Haritalama ve Dizileme Girdileri;<br />
(2) Fenotip ve Hastalık İlişkileri;<br />
(3) Genler ve Tahmini Gen Girdileri;<br />
(4) mRNA ve EST girdileri;<br />
(5) Ekspresyon ve Regülasyon;<br />
(6) Karşılaştırmalı Genomik, Çeşitlilik ve<br />
Tekrarlar;<br />
(7) ENCODE Bölge ve Genleri;<br />
(8) ENCODE Transkript Seviyeleri;<br />
(9) ENCODE Kromatin İmmünoprespitasyon;<br />
(10) ENCODE Kromozom, Kromatin ve DNA<br />
yapısı; ve Karşılaştırmalı ENCODE<br />
Çeşitli Genom Tarayıcıları<br />
UCSC genome browser (http://genome.ucsc.edu/)<br />
Ensembl genome browser<br />
(http://www.ensembl.org)<br />
VISTA (http://genome.lbl.gov)<br />
NCBI MapViewer<br />
(http://www.ncbi.nlm.nih.gov/mapview/)<br />
ECR Browser (http://ecrbrowser.dcode.org)<br />
Combo<br />
(http://www.broad.mit.edu/annotation/argo/)
80<br />
UCSC Genom Tarayıcı web sayfası araçları<br />
ARAÇ İŞLEVİ<br />
Genome Browser<br />
Gene Sorter<br />
Kromozom bölgelerini yakınlaştırmak, gezinmek, açıklamaları<br />
görebilmek.<br />
Çeşitli şekillerde ilişkilendirilebilecek gen gruplarına dair ekspresyon,<br />
homoloji ve diğer bilgileri göstermek.<br />
Blat Araştıılan diziyi genoma hızlı bir şekilde haritalamak.<br />
Table Browser Temel veritabanına erişim sağlamak.<br />
VisiGene<br />
Ekspresyon kalıplarının incelenebilmesi için in situ fare ve kurbağa<br />
görüntülerini taramak.<br />
Genome Graphs Genom çapında verileri yüklemek ve görüntülemek.<br />
Ahmed MANSOUR<br />
Genetik Bölümü,<br />
Ziraat Fakültesi,<br />
Zagazig <strong>Üniversitesi</strong>, Mısır
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong> 7(2) & 8(1): 81-82, 2010 S<strong>of</strong>tware Review<br />
<strong>Haliç</strong> University, Printed in Turkey.<br />
http://jcmb.halic.edu.tr<br />
Dotlet©: powerful <strong>and</strong> easy strategy for pairwise comparisons<br />
Authors: Marco PAGNI & Thomas JUNIER, form the Swiss Institute <strong>of</strong> Bioinformatics in<br />
Epalinges, Switzerl<strong>and</strong>.<br />
License: Freeware<br />
Dotlet at a glance<br />
For a molecular biologist, making dot plots are the<br />
simplest means for comparing two sequences. This<br />
is because dot or matrix plots provide an easy <strong>and</strong><br />
powerful means <strong>of</strong> sequence analysis (Junier <strong>and</strong><br />
Pagni, 2000). For instance, it is very useful in<br />
searching out regions <strong>of</strong> similarity between two<br />
sequences <strong>and</strong> repeats regions within a single<br />
sequence. In this regard, Dotlet is one <strong>of</strong> the most<br />
user-friendly dot-plot programs available over the<br />
internet. Dotlet program is a very convenient tool<br />
for making dot plots because <strong>of</strong> it is free <strong>of</strong> charge,<br />
easy to use, doesn’t need any installation, <strong>and</strong> can<br />
run on any computer that has access to internet.<br />
Recently, it has been extensively used to build<br />
pairwise comparisons in many peer-reviewed<br />
scientific articles.<br />
Dotlet history<br />
The authors described the reason why they wrote<br />
dotlet as they needed a diagonal plot tool during<br />
their practical sessions in bioinformatics, in<br />
December 1998, at the Institute <strong>of</strong> Biochemistry in<br />
Switzerl<strong>and</strong>. At that time, they needed a program<br />
that would run in a web browser based on the<br />
World-Wide Web. To my knowledge, this program<br />
was the first dot plots s<strong>of</strong>tware based on the<br />
internet then.<br />
Dotlet contribution to research<br />
Many researchers in the field <strong>of</strong> <strong>Molecular</strong> biology<br />
have used Dotlet modules in building Pairwise<br />
Comparisons during their original research. With<br />
many hundreds <strong>of</strong> citations, Dot let is widely cited<br />
bioinformatic programs in biology. The freeware<br />
license <strong>and</strong> its efficient modules beside its quick<br />
ability to produce results make it one <strong>of</strong> the most<br />
popular programs for making Pairwise<br />
Comparisons nowadays.<br />
Advantages <strong>and</strong> disadvantages <strong>of</strong> Dot plot<br />
Advantages<br />
- Dotlet is very easy to use <strong>and</strong> free <strong>of</strong> charge.<br />
- Dotlet is an internet-based program that runs on<br />
(almost) any computer that has access to the<br />
internet.<br />
- Dotlet is not a server but a Java applet which<br />
means that everything Dotlet does, it does on<br />
your own computer <strong>of</strong>fline.<br />
- Dotlet is safe to use because each sequence you<br />
compare with Dotlet stays on your computer<br />
- Dotlet can compare DNA, RNA <strong>and</strong> protein<br />
sequences.<br />
Disadvantages<br />
- It needs some training <strong>and</strong> good experience to<br />
interpret Dotlet results. In other words one must<br />
learn how to fine-tune Dotlet to yield an<br />
informative dot plot<br />
- Dotlet cannot work with long sequences (more<br />
than 10,000 amino acids or nucleotides).<br />
- The speed <strong>of</strong> the program depends on your own<br />
computer. The faster your computer, the faster<br />
Dotlet runs. Differences become apparent with<br />
sequences longer than 1.000 residues.<br />
S<strong>of</strong>tware Design<br />
- Dotlet is an open source freeware. It can<br />
generate pairwise comparison for a given a<br />
couple <strong>of</strong> sequences <strong>of</strong> proteins or DNA. It has<br />
been built as a JAVA applet. The Dotlet source<br />
code is available free <strong>of</strong> charge for academic<br />
users.
82<br />
The distribution is in<br />
ftp://ftp.isrec.isb-sib.ch/pub/s<strong>of</strong>tware/java/dotlet.<br />
Limitations in use<br />
Dotlet is an ideal pairwise comparisons tool for<br />
sequences with lengths <strong>of</strong> less than 10,000 amino<br />
acids or nucleotides. So, it can be helpful for most<br />
proteins sequences but is restricted to small DNA<br />
sequences.<br />
Dotlet availability online<br />
(IBM users): http://myhits.isb-sib.ch/cgi-bin/dotlet<br />
(Mac users):<br />
http://www.isrec.isb-sib.ch/java/dotlet/Dotlet.html<br />
Other online dot plot programs<br />
Dnadot<br />
This program can use a range <strong>of</strong> 100,000 long<br />
characters <strong>of</strong> either proteins or DNA sequences <strong>and</strong><br />
it has been designed using Java language. Dnadot is<br />
available online on the following URL:<br />
http://arbl.cvmbs.colostate.edu/molkit/dnadot/<br />
http://www.vivo.colostate.edu/molkit/dnadot/<br />
Dotter<br />
This program can use as long as 100.000 characters<br />
<strong>of</strong> either proteins or DNA sequences like Dnadot,<br />
However, it is designed to use Unix, Linux <strong>and</strong><br />
Windows as a platform. It is available online on the<br />
following URL:<br />
http://www.cgr.ki.se/cgr/groups/sonnhammer/Dotte<br />
r.html<br />
Dottup<br />
Although this program is also using the same range<br />
<strong>of</strong> DNA as the previous program, it can also be<br />
used for complete genomes. It also uses Unix <strong>and</strong><br />
Linux as a platform. It is available online as a<br />
useful integrated module in emboss package.<br />
URL: http://emboss.sourceforge.net/<br />
References<br />
Junier T. <strong>and</strong> Pagni M. (2000) Dotlet: diagonal<br />
plots in a web browser. Bioinformatics.<br />
16(2):178-9.<br />
Ahmed MANSOUR<br />
Genetics Department,<br />
Faculty <strong>of</strong> Agriculture,<br />
Zagazig University, Egypt<br />
(author for correspondence; amansour@zu.edu.eg)<br />
Received: 01 September 2008; Accepted: 11 December 2009
Dotlet©: İkili karşılaştırmalar için güçlü ve kolay strateji<br />
Yazarlar: Marco PAGNI & Thomas JUNIER, the Swiss Institute <strong>of</strong> Bioinformatics,<br />
Epalinges, Switzerl<strong>and</strong><br />
Lisans: Ücretsiz<br />
Kısa Bir Bakışla Dotlet<br />
Bir moleküler biyolog için, iki diziyi eşleştirmenin<br />
en basit yolu dot blot yapmaktır. Çünkü dot ya da<br />
matriks blotlar dizi analizini kolay ve güçlü şekilde<br />
sağlar. Örneğin, iki dizi arasındaki benzer<br />
bölgelerin ve bir dizideki tekrarlayan bölgelerin<br />
araştırılmasında çok kullanışlıdır. Bu bakımdan<br />
Dotlet internet yoluyla ulaşılabilen dot-plot<br />
programlarının en kullanıcı dostu olanıdır. Dotlet<br />
programı ücretsiz ve kolay kullanımlı olduğu,<br />
kuruluma ihtiyacı olmadığı ve internete girilebilen<br />
her bilgisayarda çalışabildiği için dot plotları<br />
yapmak için çok uygun bir araçtır. Son günlerde<br />
çok sayıda benzer bilimsel makalede ikili<br />
eşleştirmeleri yapmak için yaygın olarak<br />
kullanılmaktadır.<br />
Dotletin Tarihi<br />
Yazarlar, dotlet programını yazmalarının sebebini<br />
Aralık 1998’de İsviçre’deki Biyokimya<br />
Enstitüsünde kendi biyoinformatik çalışmaları<br />
sırasında diyagonal bir plota ihtiyaç duymaları<br />
olarak açıkladılar. Bu arada world-wide web tabanlı<br />
web tarayıcısında çalışabilen bir programa da<br />
ihtiyaçları vardı. Bildiğim kadarıyla bu program o<br />
zamanlarda internet tabanlı ilk dot plot yazılımıydı.<br />
Dotletin Araştırmalara Katkısı<br />
Moleküler biyolojinin bu sahasındaki çoğu<br />
araştırmacı kendi orijinal araştırmaları süresince<br />
ikili karşılaştırmaların yapıl<strong>and</strong>ırılmasında Dotlet<br />
modüllerini kullanmıştır. Yapılan yüzlerce atıfla<br />
Dotlet biyolojideki biyoinformatik programları<br />
arasındaki yerini almıştır. Bu günlerde ücretsiz<br />
yazılım lisansı ve onun etkili modülleri dışında<br />
83<br />
sonuç üretme yeteneğinin çabukluğu onu, ikili<br />
karşılaştırmaları yapmak için en popüler program<br />
haline getirmiştir.<br />
Dot Plot’ın Avantajları ve Dezavantajları<br />
Avantajları<br />
‐ Dotlet’in kullanımı kolay ve ücretsizdir.<br />
‐ Dotlet internet tabanlı bir program olup<br />
internete<br />
çalışabilir.<br />
bağlanabilen her bilgisayarda<br />
‐ Dotlet sunucu bir program değildir ama<br />
bilgisayarınız kapalıyken Dotlet’in yapabildiği<br />
her şeyi yapan bir Java uygulamasıdır.<br />
‐ Dotlet’in kullanımı Dotlet ile karşılaştırdığınız<br />
her dizi bilgisayarınızda kaldığı için güvenlidir.<br />
‐ Dotlet DNA, RNA ve protein dizilerini<br />
karşılaştırabilir.<br />
Dezavantajları<br />
‐ Dotlet sonuçlarını yorumlamak için biraz<br />
alıştırma ve iyi bir deneyim gerekmektedir.<br />
Başka bir deyişle, bilgi verici bir dot plot<br />
sağlamak için Dotlet’e nasıl ince ayar<br />
yapılacağı öğrenilmelidir.<br />
‐ Dotlet uzun diziler (10,000 amino asit ya da<br />
nükleotitten daha fazlası) ile çalışmayabilir.<br />
‐ Programın hızı bilgisayarınıza bağlıdır.<br />
Bilgisayarınız ne kadar hızlıysa dotlet o kadar<br />
hızlı çalışır. 1000 rezidüden daha uzun diziler<br />
ile belirgin farklılıklar oluşabilir.<br />
Yazılım Dizaynı<br />
Dotlet açık kaynaklı bedava bir yazılımdır. Verilen<br />
bir çift protein ya da DNA dizisi için ikili<br />
karşılaştırmalar üretebilir. Java uygula-macığı<br />
olarak yapıl<strong>and</strong>ırılmaktadır. Dotlet kaynak kodu<br />
akademik kullanıcılar için ücretsiz olarak<br />
erişilebilir.
84<br />
Dağıtım:<br />
ftp://ftp.isrec.isb-sib.ch/pub/s<strong>of</strong>tware/java/dotlet<br />
Kullanım Sınırlamaları<br />
Dotlet 10,000 amino asit ya da nükleotitten daha<br />
kısa uzunluktaki diziler için ideal bir ikili<br />
karşılaştırma aracıdır. Böylece çoğu protein dizileri<br />
için yardımcı olabilir ama ufak DNA dizileri için<br />
sınırlanmıştır.<br />
Çevrimiçi Dotlet Erişilebilirliği<br />
IBM users): http://myhits.isb-sib.ch/cgibin/dotlet<br />
(Mac users): http://www.isrec.isbsib.ch/java/dotlet/Dotlet.html<br />
Diğer Çevrimiçi Dot Plot Programları<br />
Dnadot<br />
Bu program protein ya da DNA dizilerinin 100,000<br />
uzunluğundaki bir dizi karakterini kullanabilir ve<br />
Java dilini kullanarak dizayn edilebilir. Dnadot’a<br />
aşağıdaki URL ile çevrimiçi ulaşılabilir:<br />
http://arbl.cvmbs.colostate.edu/molkit/dnadot/<br />
http://www.vivo.colostate.edu/molkit/dnadot/<br />
Dotter<br />
Bu program da protein ya da DNA dizilerinin<br />
100,000 uzunluğundaki bir dizi karakterini Dnadot<br />
gibi kullanabilir. Bununla birlikte, platform olarak<br />
Unix, Linux ve Windows kullanılarak dizayn<br />
edilebilir. Aşağıdaki URL ile çevrimiçi erişilebilir:<br />
http://www.cgr.ki.se/cgr/groups/sonnhammer/Dotte<br />
r.html<br />
Dottup<br />
Bu program önceki program gibi DNA’nın aynı<br />
aralığını kullanmasına rağmen tüm genom için de<br />
kullanılabilir. Platform olarak Unix ve Linux<br />
kullanabilir. “Emboss” paketinde kullanışlı<br />
birleştirilmiş bir modül olarak çevrimiçi ulaşılabilir.<br />
URL: http://emboss.sourceforge.net/<br />
Kaynaklar<br />
Junier T. ve Pagni M. (2000) Dotlet: web<br />
tarayıcısındaki diyagonal plotlar. Biyoinformatik.<br />
16(2):178-9.<br />
Ahmed MANSOUR<br />
Genetik Bölümü,<br />
Ziraat Fakültesi,<br />
Zagazig <strong>Üniversitesi</strong>, Mısır
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong> - GUIDELINES for AUTHORS<br />
(Revised: May 17, 2010)<br />
General<br />
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong><br />
(J<strong>Cell</strong>MolBiol) is an international journal which<br />
covers original works in the field <strong>of</strong> cell biology,<br />
molecular biology, genetics, microbiology,<br />
neurobiology, bioinforma-tics <strong>and</strong> related topics.<br />
The <strong>of</strong>ficial language <strong>of</strong> the journal is English,<br />
but manuscripts in Turkish are accepted as well.<br />
Conditions for publication<br />
This journal publishes research articles, review<br />
articles, short communications, book/s<strong>of</strong>tware<br />
reviews, case reports <strong>and</strong> letters to the editor.<br />
Research articles: Only original contributions will<br />
be accepted which have not been published<br />
previously. Manuscripts should not exceed 15<br />
papers <strong>of</strong> printed text, including tables, figures <strong>and</strong><br />
references<br />
Review articles: Reviews <strong>of</strong> recent developments in<br />
a research field <strong>and</strong> ideas will be accepted.<br />
Manuscripts should not exceed 15 papers <strong>of</strong> printed<br />
text. Illustrations are encouraged.<br />
Short communications: These include small-scale<br />
investigations or innovative methods, techniques,<br />
clinical trials <strong>and</strong> epidemiological studies. It should<br />
not exceed 3 pages.<br />
Letters to editor: These include opinions, news <strong>and</strong><br />
suggestions. Letters should not exceed 2 papers <strong>of</strong><br />
printed text.<br />
Case Reports: These include individual<br />
observations based on small numbers <strong>of</strong> subjects.<br />
This type <strong>of</strong> research cannot indicate causality but<br />
may indicate areas for further research.<br />
REVISED<br />
May 17, 2010<br />
Manuscripts should be submitted on a CD or by email<br />
to:<br />
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Biology</strong><br />
<strong>Haliç</strong> University<br />
Faculty <strong>of</strong> Arts <strong>and</strong> Sciences,<br />
Department <strong>of</strong> <strong>Molecular</strong> <strong>Biology</strong> <strong>and</strong> Genetics<br />
Kaptan Paşa Mah. Darülaceze Cad. No: 14<br />
Okmeydanı-Şişli 34384, İstanbul-TÜRKİYE<br />
Tel: +90 212 220 96 96 Ext. 155<br />
E-Mail: jcmb@halic.edu.tr<br />
Book/s<strong>of</strong>tware reviews: Short but concise<br />
description <strong>of</strong> the book/s<strong>of</strong>tware, not exceeding a<br />
page. Book/s<strong>of</strong>tware reviews are not peer reviewed.<br />
Presentation<br />
Papers should be typed clearly, double-spaced with<br />
3 cm wide margins.<br />
Manuscripts should be prepared using Word<br />
Processor.<br />
Cover Letter: You may briefly explain your work<br />
<strong>and</strong> its contribution to present knowledge.<br />
Title Page: The first page <strong>of</strong> your manuscript<br />
should be a title page containing the type <strong>of</strong> paper;<br />
the title; all authors' full names, <strong>and</strong> affiliations;<br />
<strong>and</strong> the corresponding author's contact address<br />
(including phone <strong>and</strong> fax numbers) <strong>and</strong> e-mail<br />
address. The title should be as short as possible, but<br />
should give adequate information regarding the<br />
contents. Authors should also state a running title<br />
<strong>of</strong> no more than 50 characters including spaces.<br />
All pages must be numbered.<br />
Full Paper<br />
The full paper should be divided into the following<br />
parts in the order indicated:<br />
85
86<br />
Abstract: A brief, informative abstract, not<br />
exceeding 200 words, should be provided in<br />
English <strong>and</strong> in Turkish. For authors who are not<br />
native Turkish speakers, J<strong>Cell</strong>MolBiol can provide<br />
the Turkish abstract.<br />
Keywords: Immediately following the abstract,<br />
authors should provide 5 keywords or phrases that<br />
reflect the content <strong>of</strong> the article.<br />
Introduction should include theory, hypotheses,<br />
prior work<br />
Material <strong>and</strong> methods may include subheadings<br />
Results: If the study consists <strong>of</strong> different parts,<br />
subheadings in this section should be consistent<br />
with subheadings in the methods.<br />
Discussion<br />
Acknowledgements should precede the list <strong>of</strong><br />
references<br />
References: Papers cited in the manuscript should<br />
be listed in alphabetical order according to the first<br />
author's surname.<br />
Tables <strong>and</strong> Figures<br />
Tables <strong>and</strong> figures should be embedded within<br />
the text in their appropriate positions.<br />
Each table should be accompanied by a short<br />
instructive title line plus an explanatory caption at<br />
the top. Indicate footnotes within tables by<br />
superscript letters <strong>and</strong> type footnotes below the<br />
table.<br />
Electronically submitted figures are preferred<br />
in *.jpg or *.tiff (min. 300 dpi) formats. Each figure<br />
should be supplied with a short instructive title line.<br />
Do not give magnification on scales in the figure<br />
titles; instead draw bar scales directly on the<br />
figures.<br />
All the tables <strong>and</strong> figures must be referred to<br />
within the text.<br />
Units, Abbreviations <strong>and</strong> Scientific Names<br />
Only SI units should be used. Current<br />
abbreviations can be used without explanation,<br />
others must be explained.<br />
All acronyms/abbreviations must be explained<br />
in parenthesis after their first occurrence. If many<br />
unfamiliar acronyms/abbreviations are used, please<br />
REVISED<br />
May 17, 2010<br />
compile them in an "Abbreviations" section at the<br />
end <strong>of</strong> the paper.<br />
Latin expressions should be underlined or typed<br />
in italics.<br />
Referencing<br />
Citation in the text should take the form: (Smith<br />
<strong>and</strong> Robinson,1990).<br />
If several papers are cited by the same author in<br />
the same year, they should be lettered in sequence<br />
(1990a), (1990b), etc. When papers are by more<br />
then three authors they should be cited as (Smith et<br />
al.,1990).<br />
In the list, references must be placed in<br />
alphabetical order. The following models for the<br />
reference list cover all situations. The punctuation<br />
given must be exactly followed.<br />
Redford IR. Evidence for a general relationship<br />
between the induced level <strong>of</strong> DNA double<br />
str<strong>and</strong> breakage <strong>and</strong> cell killing after Xirradiation<br />
<strong>of</strong> mammalian cells. Int J Radiat<br />
Biol. 49: 611- 620, 1986.<br />
Tccioli CE, Cottlieb TM <strong>and</strong> Blund T. Product <strong>of</strong><br />
the XRCCS gene <strong>and</strong> its role in DNA repair <strong>and</strong><br />
V(D)J recombination. Science. 265: 1442-1445,<br />
1994<br />
Ohlrogge JB. Biochemistry <strong>of</strong> plant acyl carrier<br />
proteins. The Biochemistry <strong>of</strong> Plants: A<br />
Comprehensive Treatise. Stumpf PK <strong>and</strong> Conn<br />
EE (Ed). Academic Press, New York. 137-157,<br />
1987.<br />
Weaver RF. <strong>Molecular</strong> <strong>Biology</strong>. WCB/Mc Graw-<br />
Hill.1999.<br />
Brown LA. How to cope with your supervisor. PhD<br />
Thesis. University <strong>of</strong> New Orleans, 2005.<br />
Web document with no author: Leafy seadragons<br />
<strong>and</strong> weedy seadragons 2001. retrieved<br />
November 13, 2002, from http:// www.<br />
windspeed.net.au/jenny/seadragons/<br />
Web document with author: Dawson J, Smith L <strong>and</strong><br />
Deubert K. Referencing, not plagiarism.<br />
Retrieved October 31, 2002 from http:<br />
//studytrekk.lis.curtin.edu.au/<br />
Only papers published or in press should be<br />
cited in the literature list. Unpublished results,<br />
including submitted manuscripts <strong>and</strong> those in<br />
preparation, should be indicated as unpublished<br />
data in the text.
Submission Policies <strong>and</strong> Authorship<br />
Upon submission <strong>of</strong> a manuscript, it is accepted<br />
that all co-authors have approved the contents <strong>of</strong><br />
the manuscript <strong>and</strong> its submission by the<br />
corresponding author, <strong>and</strong> that the corresponding<br />
author is authorized to represent all co-authors in<br />
pre-publication discussions with J<strong>Cell</strong>MolBiol.<br />
The corresponding author is responsible for<br />
ensuring that all the contributors to the relevant<br />
work are listed as authors <strong>and</strong> that all authors have<br />
aggreed to the manuscript’s content <strong>and</strong> its<br />
submission to the J<strong>Cell</strong>MolBiol. In case the <strong>Journal</strong><br />
happens to be aware <strong>of</strong> an authorship dispute,<br />
authorship must be approved in writing by all <strong>of</strong> the<br />
parties.<br />
Cost<br />
There are no submission fees or page charges.<br />
Criteria for the Selection <strong>of</strong> Manuscripts<br />
Manuscripts should meet the following criteria: the<br />
study conducted is material is original <strong>and</strong> ethical,<br />
the writing is clear; the study methods are<br />
appropriate, the data are valid, the conclusions are<br />
reasonable <strong>and</strong> supported by the data; the<br />
information is important; <strong>and</strong> the topic is<br />
interesting to our readership.<br />
Editorial Processes<br />
Researchers may request informal feedback from<br />
the editors in a particular manuscript. The<br />
presubmission process aids in the submission<br />
decision for authors<br />
When J<strong>Cell</strong>MolBiol receives a manuscript, the<br />
Editor <strong>and</strong> Associate Editor will first decide<br />
whether the manuscript meets the formal criteria<br />
specified with “Guidelines for Authors” <strong>and</strong><br />
whether it fits within the scope <strong>of</strong> the <strong>Journal</strong>. In<br />
case <strong>of</strong> doubt on the basis <strong>of</strong> initial review, the<br />
editor will consult other members <strong>of</strong> the Editorial<br />
Board.<br />
Manuscripts that are found suitable for peer<br />
review will be assigned to two expert reviewers.<br />
Reviewers may either be Editorial Board members<br />
or external experts selected by the Editorial Board.<br />
The corresponding author is notified by e-mail<br />
when the editor decides to send a paper for review.<br />
The reviewers will have up to three weeks to<br />
review the submitted article. After peer review, the<br />
REVISED<br />
May 17, 2010<br />
editor will contact the author. If the author is<br />
required to submit a revised version, the revised<br />
version has to be submitted by the author within<br />
two weeks. Otherwise, the manuscript will be<br />
removed from the manuscript submission queue<br />
<strong>and</strong> will be considered rejected.<br />
In cases where the referees have requested welldefined<br />
changes to the manuscript, editors may<br />
request a revised manuscript that addresses to<br />
referees’ concerns. The revised version is sent back<br />
to the original referees for re-review. In cases<br />
where the referees’ concerns are more wideranging,<br />
editors may reject the manuscript. The<br />
revised manuscript should be accompanied by a<br />
cover letter that includes a point-by-point response<br />
to referees’ comments <strong>and</strong> an explanation <strong>of</strong> how<br />
the manuscript has been changed.<br />
As a matter <strong>of</strong> policy, we do not suppress<br />
referees’ reports, any comments directed to authors<br />
are transmitted regardless <strong>of</strong> what we may think <strong>of</strong><br />
the content. On rare occasions, we may edit a report<br />
to remove <strong>of</strong>fensive language or comments to<br />
reveal confidentiality.<br />
The final decision to accept or reject a<br />
manuscript will be made by the Editor. If it<br />
becomes apparent that there are serious problems<br />
with the scientific content or with violations <strong>of</strong> our<br />
publishing policies, the Editor also reserves the<br />
right to reject a paper even after it has been<br />
accepted<br />
After acceptance, the Editor may make further<br />
changes to the text <strong>and</strong> figures so that the<br />
manuscript is readable <strong>and</strong> clear. Page pro<strong>of</strong>s will<br />
be sent to the corresponding author via email for<br />
checking before publication. Corresponding authors<br />
are sent pro<strong>of</strong>s <strong>and</strong> are welcome to discuss<br />
proposed changes with the Editor, but<br />
J<strong>Cell</strong>MolBiol reserves the right to make the final<br />
decision about the style. Corrected pro<strong>of</strong>s should be<br />
sent back to the Editor within three days <strong>of</strong> receipt,<br />
otherwise the Editor reserves the rights to correct<br />
the pro<strong>of</strong>s himself <strong>and</strong> to send the material for<br />
publication.<br />
Appeals<br />
Authors have the right to ask the Editor to<br />
reconsider a rejection decision, which is considered<br />
an appeal. Decisions are reversed only if the Editor<br />
is convinced that the original decision was a serious<br />
mistake. If an appeal merits further consideration,<br />
the Editor may send the author’s response or the<br />
revised paper to one or more referees, or Editor<br />
87
88<br />
may ask one referee to comment on the concerns<br />
raised by another referee.<br />
Advance Online Publication<br />
J<strong>Cell</strong>MolBiol provides Advance Online Publication<br />
<strong>of</strong> articles, which benefit authors with an earlier<br />
publication date <strong>and</strong> allows the readers’ access to<br />
accepted papers several weeks before they appear<br />
in print<br />
Ethical Issues<br />
For manuscripts reporting experiments on live<br />
vertebrates or higher invertebrates, authors must<br />
declare that the study was approved by the<br />
institutional ethics committee. Papers describing<br />
investigations on human subjects must include a<br />
statement that informed consent was obtained from<br />
all subjects.<br />
Plagiarism<br />
If portions <strong>of</strong> the manuscript have already been<br />
published by the author on other journals or<br />
websites, J<strong>Cell</strong>MolBiol Editorial Board needs to<br />
know which portions <strong>of</strong> the manuscript have been<br />
previously published <strong>and</strong> where. The author should<br />
include a note in the cover letter indicating which<br />
portions have been published elsewhere.<br />
In case <strong>of</strong> any suspicion on scientific misconduct or<br />
dishonesty in research, J<strong>Cell</strong>MolBiol reserves the<br />
right to forward any submitted manuscript to an<br />
appropriate authori-ty for investigation.<br />
Copyright Notice<br />
It is the responsibility <strong>of</strong> the submitting author to<br />
ensure that the authorship <strong>of</strong> the paper reflects the<br />
contributions <strong>of</strong> the authors to the work described,<br />
<strong>and</strong> that all listed authors have agreed to the<br />
submission <strong>of</strong> the manuscript in its current form.<br />
Conditions <strong>of</strong> publication in J<strong>Cell</strong>MolBiol are<br />
that the paper has not already been published<br />
elsewhere; that it is not currently being considered<br />
for publication else-where; all persons designated<br />
as authors should qualify for authorship, <strong>and</strong> all<br />
those who qualify should be listed. If accepted,<br />
<strong>Haliç</strong> University <strong>and</strong> J<strong>Cell</strong>MolBiol have the<br />
exclusive license to publish.<br />
J<strong>Cell</strong>MolBiol is freely available to individuals<br />
<strong>and</strong> institutions. Copies <strong>of</strong> this <strong>Journal</strong> <strong>and</strong> articles<br />
in this journal may be distributed for research or for<br />
REVISED<br />
May 17, 2010<br />
educational purposes free <strong>of</strong> charge. However,<br />
commercial use <strong>of</strong> articles contained herein is<br />
prohibited without the written consent <strong>of</strong> the editor.<br />
Publication Agreement<br />
The corresponing author is required to assign the<br />
Publication Agreement Form in order to publish the<br />
submitted manuscript in J<strong>Cell</strong>MolBiol.
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong><br />
Review Article<br />
<strong>Molecular</strong> <strong>Biology</strong><br />
DNA repetitive sequences-types, distribution <strong>and</strong> function: A review<br />
S.R. RAO, S. TREVEDI, D. EMMANUEL, K. MERITA <strong>and</strong> M. HYNNIEWTA<br />
Research Articles<br />
Genetic diversity <strong>of</strong> Penicillium species isolated from various sources in Sarawak, Malaysia<br />
H.A. ROSLAN, C.S. NGO <strong>and</strong> S. MUID<br />
The sensitivity <strong>of</strong> the human chromosomes to ethyl methanesulfonate (EMS)<br />
S. BUDAK-DİLER <strong>and</strong> M. TOPAKTAŞ<br />
Protective effect <strong>of</strong> pomegranate peel ethanol extract against ferric nitrilotriacetate<br />
induced renal oxidative damage in rats<br />
M.M. AHMED <strong>and</strong> S.E. ALI<br />
<strong>Molecular</strong> <strong>and</strong> cytogenetic evaluation <strong>of</strong> Y chromosome in spontaneous abortion cases<br />
G. KOÇ, K. ULUCAN, D. KIRAÇ, D. ERGEÇ, T. TARCAN <strong>and</strong> A.İ. GÜNEY<br />
Do simple sequence repeats in replication, repair <strong>and</strong> recombination genes <strong>of</strong> mycoplasmas<br />
provide genetic variability?<br />
S. TRIVEDI<br />
S<strong>of</strong>tware Reviews<br />
Tc<strong>of</strong>fee ©: Multipurpose sequence alignments program<br />
A. Mansour<br />
UCSC: Genome Browser for genomic sequences<br />
A. Mansour<br />
Dotlet©: powerful <strong>and</strong> easy strategy for pairwise comparisons<br />
A. Mansour<br />
Instructions for authors<br />
see page 1<br />
see page 13<br />
see page 25<br />
see page 35<br />
see page 45<br />
see page 53<br />
see page 71<br />
see page 75<br />
see page 81<br />
see page 85