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‹STANBUL

1998

VOLUME 2 • NO. 2 • 2003 • ISSN 1303-3646

HAL‹Ç UNIVERSITY

FACULTY OF ARTS AND SCIENCES

Journal of Cell and

Molecular Biology


Haliç University

Faculty of Arts and Sciences

Journal of Cell and Molecular Biology

Founder

Prof. Dr. Gündüz GED‹KO⁄LU

President of Board of Trustee

Rights held by

Prof. Dr. Ferruh KORKUT

Rector

Correspondence Address:

The Editorial Office

Journal of Cell and Molecular Biology

Haliç Üniversitesi, Fen-Edebiyat Fakültesi,

Ahmet Vefik Pafla Cad., No: 1, 34280, F›nd›kzade,

‹stanbul-Turkey

Phone: 90 212 530 50 24

Fax: 90 212 530 35 35

E-mail: jcmb@halic.edu.tr

Journal of Cell and Molecular Biology is

indexed in EBSCO database.

Summaries of all articles in this journal are

available free of charge from www.halic.edu.tr

ISSN 1303-3646

Printed at Yaflar Printing House

Igor ALEXANDROV, Dubna, Russia

Çetin ALGÜNEfi, Edirne, Turkey

Aglaia ATHANASSIADOU, Patros, Greece

fiehnaz BOLKENT, ‹stanbul, Turkey

Nihat BOZCUK, Ankara, Turkey

‹smail ÇAKMAK, ‹stanbul, Turkey

Adile ÇEV‹KBAfi, ‹stanbul, Turkey

Beyaz›t ÇIRAKO⁄LU, ‹stanbul, Turkey

Ayfl›n ÇOTUK, ‹stanbul, Turkey

Fevzi DALDAL, Pennsylvania, USA

Zihni DEM‹RBA⁄, Trabzon, Turkey

Mustafa DJAMGOZ, London, UK

Aglika EDREVA, Sofia, Bulgaria

Ünal EGEL‹, Bursa, Turkey

Advisory Board

Journal of Cell and

Molecular Biology

Published by Haliç University

Faculty of Arts and Sciences

Editor

Atilla ÖZALPAN

Associate Editor

Narç›n PALAVAN ÜNSAL

Editorial Board

Çimen ATAK

Atok OLGUN

P›nar ÖZKAN

Nihal BÜYÜKUSLU

Nefle AKIfi

Kürflat ÖZD‹LL‹

Damla BÜYÜKTUNÇER

Özge EM‹RO⁄LU

Mehmet Ali TÜFEKÇ‹

Merve ALO⁄LU

Asl› BAfiAR

Secretary

Burçin SARGIN

Aynur GÜREL, ‹zmir, Turkey

Candan JOHANSEN, ‹stanbul, Turkey

As›m KADIO⁄LU, Trabzon, Turkey

Valentine KEFEL‹, Pennsylvania, USA

Michael P. MADAIO, Pennsylvania, USA

Göksel OLGUN, Edirne, Turkey

U¤ur ÖZBEK, ‹stanbul, Turkey

Zekiye SULUDERE, Ankara, Turkey

‹smail TÜRKAN, ‹zmir, Turkey

Mehmet TOPAKTAfi, Adana, Turkey

Meral ÜNAL, ‹stanbul, Turkey

Mustafa YAT‹N, Boston, USA

Ziya Z‹YLAN, ‹stanbul, Turkey


Journal of Cell and Molecular Biology

CONTENTS Volume 2, No. 2, 2003

Review articles

E-cadherin molecular mechanism in prostate cancer

E-cadherinlerin prostat kanserinde moleküler mekanizmas›

D. Büyüktunçer, S. Arisan, K. Özdilli 57-64

A general view: Structure and function of the subunits of EE.. ccoollii RNA polymerase

Genel bak›fl: E. coli RNA polimeraz›n alt birimlerinin yap›lar› ve fonksiyonlar›

N. Büyükuslu 65-77

Research papers

Homocysteine potentiates esterase-induced contraction on rat aorta iinn vviittrroo:

A risk factor for atherosclerosis

Homosisteinin s›çan aortunda esteraz ile artt›r›lan in vitro kas›lmay› güçlendirmesi:

Athereosclerosis oluflumu için bir risk faktörü

F.B.H. Gurib, A.H. Subratty 79-83

Cytoembryological studies on PPaaeeoonniiaa ppeerreeggrriinnaa L.

Paeonia peregrina’da sitoembriyolojik çal›flmalar

R. Öztürk, M. Ünal 85-89

Immunogenicity and specificity of SSaallmmoonneellllaa ttyypphhiimmuurriiuumm outer membrane

antigens

Salmonella typhimurium d›fl membran antijenlerinin immunojenite ve özgüllü¤ü

N. Ak›fl, O. Sayhan, A. Karaçavufl, K. Töreci 91-97

Polymerase chain reaction is a good diagnostic tool for MMyyccoobbaacctteerriiuumm

ttuubbeerrccuulloossiiss in urine samples

‹drar örneklerinde Mycobacterium tuberculosis tan›s› için polimeraz zincir

reaksiyonunun kullan›m›

S. Arisan, N.C. Sönmez, Ö.O. Çak›r, E. Ergenekon 99-103

Cytological investigations in some important tree species of Rajasthan VI.

Radiation induced chromosome aberrations in AAnnooggeeiissssuuss ppeenndduullaa and AA.. llaattiiffoolliiaa

Rajastan IV için baz› önemli a¤aç türlerinde sitolojik araflt›rmalar: Anogeissus pendula

ve A. latifolia’ da ›fl›nlama ile artan kromozom aberasyonlar›

A. Kumar, S.R. Rao, N.S. Shekhawat 105-111

Stimulation of regeneration by magnetic field in soybean (GGllyycciinnee mmaaxx L. Merrill)

tissue cultures

Soya (Glycine max L. Merrill) doku kültürlerinde rejenerasyonun manyetik alan

taraf›ndan stimulasyonu

Ç. Atak, Ö. Emiro¤lu, S. Alikamano¤lu, A. Rzakoulieva 113-119

Letters to editor 121-124

Book reviews 125-127

Instructions to authors 129-130

Volume content 131-132

Author index 133


Haliç University

Faculty of Arts and Sciences

Journal of Cell and Molecular Biology

Founder

Prof. Dr. Gündüz GED‹KO⁄LU

President of Board of Trustee

Rights held by

Prof. Dr. Ferruh KORKUT

Rector

Correspondence Address:

The Editorial Office

Journal of Cell and Molecular Biology

Haliç Üniversitesi, Fen-Edebiyat Fakültesi,

Ahmet Vefik Pafla Cad., No: 1, 34280, F›nd›kzade,

‹stanbul-Turkey

Phone: 90 212 530 50 24

Fax: 90 212 530 35 35

E-mail: jcmb@halic.edu.tr

Journal of Cell and Molecular Biology is

indexed in EBSCO database.

Summaries of all articles in this journal are

available free of charge from www.halic.edu.tr

ISSN 1303-3646

Printed at Yaflar Printing House

Igor ALEXANDROV, Dubna, Russia

Çetin ALGÜNEfi, Edirne, Turkey

Aglaia ATHANASSIADOU, Patros, Greece

fiehnaz BOLKENT, ‹stanbul, Turkey

Nihat BOZCUK, Ankara, Turkey

‹smail ÇAKMAK, ‹stanbul, Turkey

Adile ÇEV‹KBAfi, ‹stanbul, Turkey

Beyaz›t ÇIRAKO⁄LU, ‹stanbul, Turkey

Ayfl›n ÇOTUK, ‹stanbul, Turkey

Fevzi DALDAL, Pennsylvania, USA

Zihni DEM‹RBA⁄, Trabzon, Turkey

Mustafa DJAMGOZ, London, UK

Aglika EDREVA, Sofia, Bulgaria

Ünal EGEL‹, Bursa, Turkey

Advisory Board

Journal of Cell and

Molecular Biology

Published by Haliç University

Faculty of Arts and Sciences

Editor

Atilla ÖZALPAN

Associate Editor

Narç›n PALAVAN ÜNSAL

Editorial Board

Çimen ATAK

Atok OLGUN

P›nar ÖZKAN

Nihal BÜYÜKUSLU

Nefle AKIfi

Kürflat ÖZD‹LL‹

Damla BÜYÜKTUNÇER

Özge EM‹RO⁄LU

Mehmet Ali TÜFEKÇ‹

Merve ALO⁄LU

Asl› BAfiAR

Secretary

Burçin SARGIN

Aynur GÜREL, ‹zmir, Turkey

Candan JOHANSEN, ‹stanbul, Turkey

As›m KADIO⁄LU, Trabzon, Turkey

Valentine KEFEL‹, Pennsylvania, USA

Michael P. MADAIO, Pennsylvania, USA

Göksel OLGUN, Edirne, Turkey

U¤ur ÖZBEK, ‹stanbul, Turkey

Zekiye SULUDERE, Ankara, Turkey

‹smail TÜRKAN, ‹zmir, Turkey

Mehmet TOPAKTAfi, Adana, Turkey

Meral ÜNAL, ‹stanbul, Turkey

Mustafa YAT‹N, Boston, USA

Ziya Z‹YLAN, ‹stanbul, Turkey


Journal of Cell and Molecular Biology 2: 57-64, 2003.

Haliç University, Printed in Turkey.

E-cadherin molecular mechanism in prostate cancer

Damla Büyüktunçer 1 , Serdar Arisan 2 *, Kürflat Özdilli 1

1 Haliç University, Faculty of Arts and Sciences, Molecular Biology and Genetics Deparment, Ahmet

Vefik Pafla Cad. No:1 F›nd›kzade-Istanbul 34280, Turkey; 2 fiiflli Etfal State Hospital, 1. Urology Clinics,

Istanbul-Turkey (*author for correspondence)

Received 3 March 2003; Accepted 5 May 2003

Abstract

Prostate cancer is the most commonly diagnosed noncutaneous malignancy in men in USA. In the year 2002,

according to the health statistics 189,000 men in the United States are expected to be diagnosed with the disease and

30,200 men are expected to die of it. Mortality in prostate carcinoma is associated with metastasis and metastasis in

prostate carcinoma is usually associated with perineural invasion, for it is the preferred mechanism of prostate cancer

metastasis. Tumor markers are biological molecules that indicate the presence of malignancy. They are potentially

useful in cancer screening, aiding diagnosis, assessing prognosis, predicting in advance a likely response to therapy,

and monitoring patients before and after diagnosis. E-cadherin is a promising tumor marker for malignancies in lots

of cancer types. Also, the cadherin functional implication in tumor malignancy is an exciting research area in tumor

biology, and it is expected to give some insights into how tumors acquire an invasive and metastatic phenotype.

Therefore, there are many investigations about E-cadherin activity and its future usage in clinics.

KKeeyy wwoorrddss:: Prostate cancer, tumor markers, e-cadherins, CAMs, catenin

E-cadherinlerin prostat kanserinde moleküler mekanizmas›

Özet

Prostat kanseri oldukça s›k teflhis edilen erkek solid tümörlerindendir. 2002 y›l›nda, sa¤l›k istatistiklerine göre

189,000 kiflinin Amerika Birleflik Devletlerinde bu hastal›ktan ötürü teflhis edildi¤i ve 30,200 tanesininde ölece¤i

tahmin edilmektedir. Prostat kanserinde ölüm metastaz ile iliflkili olup, metastaz perinöral invasyon ile

iliflkilendirilmektedir. Tümör belirteçleri biyolojik moleküller olup malign durumlar› ortaya ç›karmaktad›r.

Özellikle kanser taramalar›nda tan› ve tedavi aç›s›ndan potansiyel avantajlar› söz konusudur. E-cadherin kanser

terapilerini takip aç›s›ndan umut vaadeden bir tümör belirtecidir. Bunun yan› s›ra cadherinlerin fonksiyonel tümör

oluflumunda araflt›r›lmas› gündemde olan konulardan bir tanesidir. Özellikle invasiv veya metastatik tümörlerin

fenotipik belirlemesinde önem kazanm›fllard›r. Bundan ötürü e-cadherin aktivitesinin klinik aç›dan kullan›labilirli¤i

üzerine birçok araflt›rma yap›lmaktad›r.

AAnnaahhttaarr ssöözzccüükklleerr:: Prostat kanseri, tümör mark›rlar, e-cadherinler, CAMs, katenin

What is E-cadherin?

The majority of human cancers originates from

epithelial cells. Normal epithelia is organized by a

number of specific intercellular junctions, including

tight junctions, adherens-type junctions and

desmosomes, which are intimately interconnected with

the actin and intermediate filament cytoskeleton. This

57


58 Damla Büyüktunçer et al.

Figure 1: Molecular pathway of E-cadherin molecules in metastatic prostate cells.

association with the cytoskeletal network is necessary

for stable cell–cell adhesion and for the integration of

cell–cell contacts with the changes in morphology that

are characteristic of epithelial cells (e.g. cuboidal cell

shape, polarised phenotype). Cell–cell adhesion is an

essential component of epithelial morphology and

function. Epithelial cells adhere tightly to their

neighbours, and several specialised adhesive structures

ensure the appropriate integrity and tensile strength of

epithelial sheets. Over the past few years, many

different laboratories have addressed the questions of

how cell-cell adhesion is regulated and how the

epithelial phenotype is generated. Although the

regulatory processes are not fully understood, several

signalling pathways that are activated by cell-cell

adhesion have been identified (DeMarzo et al., 1999;

Umbas et al., 1997; Behrens, 1999; Takeichi, 1991).

Among the many types of cell-cell adhesion

molecules, cadherins play a critical role in establishing

adherens-type wuctions by mediating Ca 2+ -dependent

cell-cell adhesion (Takeichi, 1995; Huber et al., 1996;

Yagi et al., 2000). Cadherin-based cell-cell adhesion is

critically involved in early embryonic morphogenesis,

as exemplified by the early embryonic lethality of

mice lacking E-cadherin, a prototype classical

cadherin (Riethmacher, 1995; Larue et al., 1994). One

of the most studied molecules involved in cell-cell

adhesion is E-cadherin, a 120 kDa transmembrane

glycoprotein. The cytoplasmatic moiety of E-cadherin

binds to β- and γ-catenin, which are linked to the

cytoskeleton via α-catenin, while the extracellular

moiety is a calcium-dependent receptor responsible for

homophilic (E-cadherin/E-cadherin) interactions

(Takeichi, 1995).

Cadherin-mediated cell-cell adhesion is

accomplished by homophilic protein-protein


interactions of extracellular cadherin domains in a

zipper-like fashion. The intracellular domain of

classical cadherins interacts with various proteins,

collectively termed catenins, which assemble the

cytoplasmic cell adhesion complex (CCC) that is

critical for the formation of extracellular cell-cell

adhesion. β-catenin and γ-catenin (also called

plakoglobin) bind to the same conserved site at Ecadherin’s

C-terminus in a mutually exclusive way

(Nathke et al., 1994; Ozawa et al., 1984; Witcher et al.,

1996), whereas p120ctn interacts with multiple sites in

E-cadherin’s cytoplasmic tail, including its

juxtamembrane region (Yap et al., 1998; Ozawa, 1998).

β-catenin and γ-catenin bind directly to α-catenin,

which links the CCC to the actin cytoskeleton. While

the dual role of β-catenin and γ-catenin in cell adhesion

and Wnt-signaling has been extensively studied, the

functions of p120ctn are poorly understood. p120ctn

has been implicated both in cell-cell adhesion and in

cell migration (Anastasiadis and Reynolds, 2000), and

recent studies suggest that p120ctn promotes cell

migration by recruiting and activating Rho-family

GTPases (Noren et al., 2000) (Figure 1).

Why prostate cancer is too important

Prostate cancer is the most commonly diagnosed

noncutaneous malignancy in men in USA. In the year

2002, according to the health statistics 189,000 men in

the United States are expected to be diagnosed with the

disease and 30,200 men are expected to die of it.

Incidence varies greatly, with African Americans

having the highest incidence in the world (224 cases

per 100,000 population). The incidence of prostate

cancer in African Americans stands in stark contrast to

the incidence in white Americans (150 per 100,000)

and that in men in Western Europe (39.6 per 100,000),

Japan (8.5 per 100,000), and China (1.1 per 100,000)

(American cancer soc. reports, 2002; Robbins et al.,

1998). Mortality in prostate carcinoma is associated

with metastasis and metastasis in prostate carcinoma is

usually associated with perineural invasion, for it is the

preferred mechanism of prostate cancer metastasis.

One of the current theory states this form of metastasis

follows the path of least resistance offered by the

perineural sheath. Understanding specific mechanisms

of this carcinoma/nerve interaction is key to potential

therapeutics targeted to this process. Death from

prostate cancer is the result of metastasis and local

Prostate cancer and e-cadherins 59

spread, not from organ-confined disease. Hence,

understanding the mechanism of prostate cancer

spread and metastasis is the key to treating this disease

successfully and increasing survivability (Jiang et al.,

1994).

Tumor markers are biological molecules that

indicate the presence of malignancy. They are

potentially useful in cancer screening, aiding

diagnosis, assessing prognosis, predicting in advance a

likely response to therapy, and monitoring patients

before and after diagnosis (Mason et al., 2002; Tomita

et al., 2000). Because of low prevalance of most

cancers in the general population and the limited

sensivity and spesificity of avaible markers, these tests

alone are generally of little value in screening for

cancer in healthy subjects. Currently, however,

prostate spesific antigen (PSA) in combination with

digital rectal examination (DRE) are undergoing

evaluation as screening modalities and case findings

for prostate cancer (Mettlin et al., 1994). Because of a

lack of sensitivity and spesificity markers are rarely of

use in early diagnosis of cancer. Also they can be used

to monitor the disease during therapy. The goal of

future research should be to develop the most specific,

cheap and easy markers for common cancer types as

prostate cancer (Arisan, 2003).

The natural history of prognostic factors involved

in prostate cancer are not clearly defined. Hence,

molecular parameters that are able to accurately assess

the aggressiveness and the metastatic potential of the

cancer are urgently needed (Arisan, 2003, Gao et al.,

1997; Pettaway, 1998). Since vascular invasion and the

spread of prostate tumor cells to the blood represent

preliminary steps in the metastatic process, bloodborne

detection of circulating prostate epithelial cells

(CPC) could be an early marker of invasiveness

(Gomella et al., 1997). The recent development of

sensitive molecular techniques evidenced such cells in

the blood and urine of patients with localised prostate

cancer, and has been proposed as a new staging

modality. One of the prominent features of the

development and progression of prostate cancer is the

development of abnormalities in cell adhesion in

prostate epithelium and prostate cancer cells. These

abnormalities extend to intercellular adhesion

structures and cell–matrix adhesion molecules.

The metastatic process consists of a complex

pattern of sequential steps whose primary event

requires the detachement of cells from the primary

tumor. The metastatic cascade is composed of a


60 Damla Büyüktunçer et al.

number of separate but important steps, including cell

adhesion ability decreament. Disruption of normal

cell-cell adhesion in transformed cells contributes

directly to tumor cells’ enhanced migration and

proliferation, leading to invasion and metastasis. Most

prostate cancer deaths are due to metastatic disease,

but it is not significant means to combat metastasis in

prostate cancer (Vleminckx et al., 1991; Birchmeier

and Behrens et al., 1999).

Is e-cadherin a tumor supressor gene?

The concept of the tumor suppressor gene has been

extended to genes which are subject to frequent

downregulation in cancer, suggestive of an important

tumor-suppressing activity despite the lack of evidence

for mutation. Examples include cell adhesion

molecules (CAMs) which play important roles in

tissue development and epithelial cell differentiation.

When downregulated, CAMs may be involved in

oncogenic processes through inactivation of cell

adhesion-mediated growth control pathways. Ecadherin

is a member of a family of Ca 2+ dependent

homophilic CAMs involved in developmental

morphogenesis and maintenance of the epithelial

phenotype (Tomita et al., 2000). E-cadherin expression

correlates with epithelial differentiation whereas loss

of E-cadherin expression promotes epithelial

dedifferentiation and invasiveness of human

carcinoma cells. Restoration of wild-type E-cadherin

function prevents invasiveness of epithelial tumor cells

(Hatta and Takeichi, 1986). Absence of E-cadherin

immunostaining has been shown in many carcinoma

types including mouse squamous cell carcinoma of the

skin, human infiltrating basal cell carcinoma, head and

neck, breast, colorectal, gastric, and other carcinomas

(Edelman et al., 1983). Loss of expression of Ecadherin

which is located on chromosome band 16q22

may be associated with a biallelic mutation mechanism

characteristic of tumor suppressor gene inactivation.

Potentially inactivating point mutations of E-cadherin

associated with LOH have been identified in gastric

cancer cell lines derived from signet ring and diffuse

stomach cancers, both of which are poorly

differentiated forms of gastric carcinoma (Doherty and

Walsh, 1996). DNA methylation in mammalian cells

occurs at the 5-position of cytosine within the CpG

dinucleotide. This reaction is catalysed by the DNA

methyltransferase (DNMT) enzymes. More in general,

DNA methylation often causes the downregulation of

tumor suppressor genes (such as pRb, p15 INK4a,

p16INK4a) in cancer cells by changing chromatin

structure, thereby making the DNA inaccessible for

transcription factors and RNA polymerase II. Also,

downregulation of E-cadherin expression is often

accompanied by methylation of 5' CpG island of Ecadherin

in lung, liver, bladder and gastric carcinoma

cell lines. The picture that emerges from the analysis

of all of these studies suggests that promoter

hypermethylation is the main mechanism involved in

promoter silencing of E-cadherin in those tumors,

although not the only one. The data presented in the

two papers confirm that loss of heterozygosity (LOH)

and/or point mutation (or even other mechanisms) also

contributes to the downregulation of E-cadherin. The

mechanistic question remains as to whether aberrant

promoter methylation in those tumors is a causal and

not consequent to malignant transformation. Future

studies, probably investigating the status of E-cadherin

promoter methylation at different stages (e.g.

premalignant versus malignant lesions) might help in

addressing this question (Bird, 2002, Di Croce et al,

2002, Robertson, 2002, Tsao et al., 2003; Chen et al.,

2003).

Further suggestions of the mechanisms by which

E-cadherin is inactivated in cancer are analogous to

those observed for classical tumor suppressor genes.

The role of E-cadherin in prostate cancer was initially

investigated in the Dunning R-3327 rat prostatic cell

line. E-cadherin mRNA and protein expression was

found in well and poorly differentiated lines with low

invasive potential, while all established cell lines with

high invasive potential had no detectable levels of Ecadherin

mRNA (El-Hariry et al., 2001). In human

prostate cancer, E-cadherin immunostaining was

reduced or absent in 46 of 92 primary and metastatic

cases whereas benign non-malignant tissue stained

uniformily positive (De Luca et al., 1999).

E-cadherin molecular mechanism in cancer

It has long been known that cell-cell adhesion is

dramatically changed during the development of

malignant tumors. In particular, in most if not all

cancers of epithelial origin, E-cadherin-mediated cellcell

adhesion is lost concomitant with progression

towards malignancy, and it has been proposed that the

loss of E-cadherin-mediated cell-cell adhesion is a


prerequisite for tumor cell invasion and metastasis

formation (Birchmeier and Behrens, 1994). Multiple

mechanisms are found to underlie the loss of Ecadherin

function during tumorigenesis: mutations or

deletions of the E-cadherin gene itself, mutations in

the β-catenin gene, transcriptional repression of the Ecadherin

gene, for example by hypermethylation or

chromatin rearrangements in the E-cadherin promoter

region and, finally, aberrant tyrosine phosphorylation

of the components of the CCC (Hirohashi, 1998).

Recent reports have highlighted that the DNA binding

protein Snail acts as a strong repressor of E-cadherin

gene expression in tumor cells, thus inducing tumor

malignancy (Batlle et al., 2000, Cano et al., 2000,

Poser et al., 2001; Yokoyama et al., 2001).

The observation that E-cadherin function is

frequently lost in malignant tumors prompted an

examination of the functional role of E-cadherin in

tumor progression. Using tumor cell lines in culture,

several groups demonstrated that re-establishing the

functional cadherin complex, for example by forced

expression of E-cadherin, resulted in a reversion from

an invasive to a benign, epithelial tumor cell phenotype

(Birchmeier and Behrens, 1994, Hirohashi, 1998,

Battle et al., 2000, Cano et al., 2000, Poser et al., 2001,

Yokoyama et al., 2001; Vleminckx et al., 1991).

Although these experiments clearly demonstrated a

critical role for E-cadherin in the suppression of tumor

invasion in cultured cells, it remained elusive whether

the loss of E-cadherin mediated cell adhesion is a prerequisite

for tumor progression or whether it is instead

a consequence of de-differentiation during tumor

progression. It is recently shown that expression of Ecadherin

is lost during the transition from welldifferentiated

adenoma to invasive carcinoma in a

transgenic mouse model of pancreatic β-cell

tumorigenesis (RIP1TAG2). Maintenance of Ecadherin

expression during β-cell tumorigenesis

resulted in arrest of tumor development at the adenoma

stage. By contrast, expression of a dominant negative

E-cadherin induced early invasion and metastasis.

These results demonstrate that loss of E-cadherinmediated

cell-cell adhesion is one the rate-limiting

steps in the progression from adenoma to carcinoma in

vivo and highlight the role of E-cadherin as a

suppressor of tumor invasion. However, several

questions remain to be answered. Tumor invasion is the

result of a sequence of multiple cellular events,

involving not only changes in cell-cell adhesion but

also in cell-matrix adhesion, cell migration, proteolytic

Prostate cancer and e-cadherins 61

degradation of extracellular matrix and so forth.

Therefore, it is difficult to envisage that the loss of Ecadherin-mediated

cell adhesion per se is sufficient to

confer an invasive phenotype to tumor cells. It seems

more likely that E-cadherin downregulation results in

the activation of specific signaling pathways which, in

turn, trigger tumor cell invasion. One of the obvious

candidates for activating such signaling pathways is βcatenin.

Besides being a component of the CCC, βcatenin

plays a key role in Wnt-mediated signal

transduction. β-catenin is usually sequestered in the Ecadherin

adherens junction or in tight-junction

complexes. Non-sequestered, free β-catenin is rapidly

phosphorylated by glycogen synthase kinase 3b (GSK-

3b) in the adenomatous polyposis coli (APC)/GSK-

3b/axin complex and subsequently degraded by the

ubiquitin-proteasome pathway. If the tumor suppressor

APC is non-functional, as is the case in many coloncancer

cells, or GSK-3b activity is blocked by activated

Wnt signaling, β-catenin accumulates at high levels in

the cytoplasm. Subsequently, it translocates to the

nucleus, where it binds to a member of the TCF/LEF-1

family of transcription factors and modulates

expression of TCF/LEF-1-target genes. Target genes of

TCF/β catenin that could be relevant for tumor

progression include the proto-oncogene c-Myc and

cyclin D1 (Perl et al., 1998, Bienz and Clevers, 2000;

Polakis, 2000). Future investigation should focus on the

relationship between E-cadherin downregulation and

β-catenin signaling during tumor progression, in

particular addressing the issue of whether the loss of Ecadherin

results in the activation of the Wnt signaling

pathway thus endowing tumor cells with an invasive

phenotype. Not much attention has been devoted to the

role of the cytoskeleton upon the loss of E-cadherinmediated

cell-cell adhesion and the induction of tumor

malignancy. Cadherin-based adhesion complexes are

functionally linked to the dynamics of actin and

microtubule cytoskeletal structures (Vasioukhin and

Fuchs, 2001; Chausovsky, 2000). Thus, it can be

anticipated that the loss of E-cadherin mediated cell

adhesion leads to dramatic cytoskeletal

rearrangements. Cellular factors that connect cadherin

function with cytoskeletal organization are likely to

play a key role in the structural alterations following

the downregulation of cadherin-mediated cell adhesion.

Small GTPases of the Rho family are obvious

candidates for future investigation, since besides

controlling the actin cytoskeleton they are known to

modulate cadherin activity (Braga, 2000, Kaibuchi et


62 Damla Büyüktunçer et al.

al., 1999; Gumbiner, 2000). Interestingly, among the

molecules linking small GTPases with cadherin

function is IQGAP1, a protein whose dysregulation has

been proposed to correlate with malignancy in gastric

cancer (Takemoto et al., 2001; Sugimoto et al., 2001).

In conclusion, the cadherin switch and its functional

implication in tumor malignancy is an exciting research

area in tumor biology, and it is expected to give some

insights into how tumors acquire an invasive and

metastatic phenotype. However, several issues need to

be addressed before considering the cadherin switch as

a crucial step in tumor progression. Thus far, the

cadherin switch in vivo has only been described during

the development of malignant melanoma and prostate

carcinoma. Further studies on other tumor types are

required to establish whether a switch in cadherin

expression is a common mechanism underlying tumor

progression. In vitro observations on various tumor cell

lines suggest that this might indeed be the case. In

addition, although the classical cadherin family

comprises at least 30 members, not many attempts have

been made to investigate the expression of cadherins

other than E- or N-cadherin in different tumor types.

Such systematic studies might provide evidence of

tumor specific cadherin repertoires, thus raising the

possibility that many more members of the cadherin

family are involved in cadherin switches and that the

cadherins involved in the switch vary in a tumorspecific

manner. Forced expression or genetic ablation

of particular cadherin genes during embryonic

development or in appropriate mouse models of

tumorigenesisesis or other disease will help in

unraveling the functional role of the cadherin switch

(es) in physiological and pathological processes.

Extensive investigations on the functional relevance of

the cadherin switch in vivo may not only provide

additional insights into the molecular mechanisms

underlying tumor progression, but may also allow the

identification of novel molecular targets for anti-cancer

therapy.

References

American Cancer Society: Cancer Facts and Figures-2002.

Atlanta, Ga: American Cancer Society, 2002.

Anastasiadis PZ and Reynolds AB. The p120 catenin family:

complex roles in adhesion, signaling and cancer. J Cell

Sci. 113: 1319-1334, 2000.

Ar›san S. Prostate cancer and importance of tumor marker

studies. J of Cell and Molecular Biology. 2: 49-51, 2003.

Batlle E, Sancho E, Franci C, Dominguez D, Monfar M,

Baulida J, Garcia A and De Herreros. The transcription

factor snail is a repressor of E-cadherin gene expression

in epithelial tumor cells. Nat Cell Biol. 2: 84-89, 2000.

Behrens J. Cadherins and catenins: Role in signal

transduction and tumor progression. Ca Metast Rev. 18:

15-30, 1999.

Bienz M and Clevers H. Linking colorectal cancer to Wnt

signaling. Cell. 103: 311-320, 2000.

Birchmeier W and Behrens J. Cadherin expression in

carcinomas: Role in the formation of cell junctions and

the prevention of invasiveness. Biochim Biophys Acta.

1198: 11-26, 1994.

Bird A. DNA methylation patterns and epigenetic memory.

Genes Dev. 16: 6-21, 2002.

Braga V. Epithelial cell shape: cadherins and small GTPases.

Exp Cell Res. 261: 83-90, 2000.

Cano AMA, Perez-Moreno I, Rodrigo A, Locascio MJ,

Blanco MG, del Barrio F, Portillo MA, Nieto R. The

transcription factor snail controls epithelialmesenchymal

transitions by repressing E-cadherin

expression, Nat Cell Biol. 2: 76-83, 2000.

Chausovsky A, Bershadsky AD and Borisy GG. Cadherin

mediated regulation of microtubule dynamics. Nat Cell

Biol. 2: 797-804, 2000.

Chen CL, Liu SS, Ip SM, Wong CL, Ty NG and Hys N.

E-cadherin expression is silenced by DNA methylation in

cervical cancer cell lines and tumors. Eur J Cancer. PII:

S0959-8049 (02) 00175-2, 2003.

DeLuca SM, Gerhart J, Cochran E, Simak E, Blitz J,

Mattiacci-Paessler M, Knudsen K and George-Weinstein

M. Hepatocyte growth factor/scatter factor promotes a

switch from Eto N-cadherin in chick embryo epiblast

cells. Exp Cell Res. 251: 3-15, 1999.

DeMarzo AM, Knudsen B, Chan-Tack K and Epstein JI. Ecadherin

expression as a marker of tumor aggressiveness

in routinely processed radical prostatectomy specimens.

Urology. 53: 707-713, 1999.

Di Croce L, Raker VA and Corsaro M. Methyltransferase

recruitment and DNA hypermethylation of target

promoters by an oncogenic transcription factor. Science.

295: 1079-1082, 2002.

Doherty P and Walsh FS. CAM-FGF Receptor Interactions:

A model for axonal growth. Mol Cell Neurosci. 8: 99-

111, 1996.

Edelman GM, Gallin WJ, Delouvee A, Cunningham BA and

Thiery JP. Early epochal maps of two different cell

adhesion molecules. Proc Natl Acad Sci. USA 80: 4384-

4388, 1983.

El-Hariry I, Pignatelli M and Lemoine NR. FGF-1 and

FGF-2 modulate the E-cadherin/catenin system in

pancreatic adenocarcinoma cell lines. Br J Cancer. 84:

1656-1663, 2001.

Gao X, Porter AT and Grignon DJ. Diagnostic and

prognostic markers for human prostate cancer. Prostate.

31: 264-281, 1997.


Gomella LG, Raj GV and Moreno JG. Reverse transcriptase

polymerase chain reaction for prostate specific antigen in

the management of prostate cancer. J Urol. 158:

326-337, 1997.

Gumbiner BM. Regulation of cadherin adhesive activity. J

Cell Biol. 148: 399-404, 2000.

Hatta K, Takeichi M. Expression of N-cadherin adhesion

molecules associated with early morphogenetic events in

chick development. Nature. 320: 447-449, 1986.

Hirohashi S. Inactivation of the E-cadherin-mediated cell

adhesion system in human cancers. Am J Pathol. 153:

333-339, 1998.

Huber O, Korn R, McLaughlin J, Ohsugi M, Herrmann BG

and Kemler R. Nuclear localization of beta-catenin by

interaction with transcription factor LEF-1. Mech Dev.

59: 3-10, 1996.

Jiang WG, Puntis MCA and Hallett MB. The molecular and

cellular basis of cancer invasion and metastasis and its

implications for treatment. Br J Surg. 81: 1576-90, 1994.

Kaibuchi K, Kuroda S, Fukata M and Nakagawa M.

Regulation of cadherin-mediated cell-cell adhesion by

the Rho family GTPases. Curr Opin Cell Biol. 11: 591-

596, 1999.

Larue L, Ohsugi M, Hirchenhain J and Kemler R. E-cadherin

null mutant embryos fail to form a trophectoderm

epithelium. Proc Natl Acad Sci. USA 91: 8263-8267,

1994.

Mason MD, Davies G and Jiang WG. Cell adhesion

molecules and adhesion abnormalities in prostate cancer.

Critical Reviews in Oncology/Hematology. 41: 1-28,

2002.

Mettlin C, Murphy GP and Lee F. Characteristics of prostate

cancer detected in the American Cancer Society-

National Prostate Cancer Detection Project. Journal of

Urology. 152: 1737-1740, 1994.

Nathke IS, Hinck L, Swedlow JR, Papkoff J and Nelson WJ.

Defining interactions and distributions of cadherin and

catenin complexes in polarized epithelial cells. J Cell

Biol. 125: 1341-1352, 1994.

Noren NK, Liu BP, Burridge K and Kreft B. p120 catenin

regulates the actin cytoskeleton via Rho family GTPases.

J Cell Biol. 150: 567-580, 2000.

Ozawa M. Identification of the region of alpha-catenin

that plays an essential role in cadherin-mediated cell

adhesion. J Biol Chem. 273: 29524-29529, 1998.

Ozawa M, Baribault H and Kemler R. The cytoplasmic

domain of the cell adhesion molecule uvomorulin

associates with three independent proteins structurally

related in different species. EMBO J. 8: 1711-1717,

1989.

Perl AK, Wilgenbus P, Dahl U, Semb H and Christofori G. A

causal role for E-cadherin in the transition from adenoma

to carcinoma. Nature. 392: 190-193, 1998.

Pettaway CA, Prognostic markers in clinically localized

prostate cancer. Tech Urol. 4: 35-42, 1998.

Prostate cancer and e-cadherins 63

Polakis P. Wnt signaling and cancer. Genes Dev. 14:

1837-1851, 2000.

Poser I, Dominguez D, De Herreros AG, Varnai A,

Buettner R and Bosserhoff AK. Loss of E-cadherin

expression in melanoma cells involves up-regulation of

the transcriptional repressor Snail. J Biol Chem. 276:

24661-24666, 2001.

Riethmacher D, Brinkmann V and Birchmeier C. A targeted

mutation in the mouse E-cadherin gene results in

defective preimplantation development. Proc Natl Acad

Sci. USA 92: 855-859, 1995.

Robbins AS, Whittemore AS and Van Den Eeden SK. Race,

prostate cancer survival, and membership in a large

health maintenance organization. Journal of the National

Cancer Institute. 90: 986-990, 1998.

Robertson KD. DNA methylation and chromatin-unraveling

the tangled web. Oncogene. 21: 5361-5379, 2002.

Sugimoto N, Imoto I, Fukuda Y, Kurihara N, Kuroda S,

Tanigami A, Kaibuchi K, Kamiyama R and Inazawa J.

IQGAP1, a negative regulator of cell-cell adhesion, is

upregulated by gene amplification at 15q26 in gastric

cancer cell lines HSC39 and 40A. J Hum Genet. 46:

21-25, 2001.

Takeichi M. Cadherin cell adhesion receptors as a

morphogenetic regulator. Science. 251: 1451-1455,

1991.

Takeichi M. Morphogenetic roles of classic cadherins. Curr

Opin Cell Biol. 7: 619-627, 1995.

Takemoto H, Doki Y, Shiozaki H, Imamura H,

Utsunomiya T, Miyata H, Yano M, Inoue M, Fujiwara Y

and Monden M. Localization of IQGAP1 is inversely

correlated with intercellular adhesion mediated by Ecadherin

in gastric cancers. Int J Cancer. 91: 783-788,

2001.

Tomita K, van Bokhoven A, van Leenders GJLH, Ruijter

ETG, Jansen CFJ, Bussemakers MJG and Schalken JA.

Cadherin switching in human prostate cancer

progression. Cancer Res. 60: 3650-3654, 2000.

Tomita K, Van Bokhoven A, Van Leenders GJ, Ruijter ET,

Jansen CF, Bussemakers MJ and Schalken JA. Cadherin

switching in human prostate cancer progression. Cancer

Res. 60: 3650-3654, 2000.

Tsao SW, Liu Y and Wang X. The association of E-cadherin

expression and the methylation status of the E-cadherin

gene in nasopharyngeal carcinoma cells. Eur J Cancer.

PII: S0959-8049 (02) 00494-X, 2003.

Umbas R, Isaacs WB, Bringuier PP, Xue Y, Debruyne FM

and Schalken JA. Relation between aberrant a-catenin

expression and loss of E-cadherin function in prostate

cancer. Int J Cancer. 74: 374-377, 1997.

Vasioukhin V and Fuchs E. Actin dynamics and cell-cell

adhesion in epithelia, Curr Opin Cell Biol. 13: 76-84,

2001.

Vleminckx K, Vakaet Jr L, Mareel M, Fiers W and van

Roy F. Genetic manipulation of E-cadherin expression


64 Damla Büyüktunçer et al.

by epithelial tumor cells reveals in invasion suppressor

role. Cell. 66:107-119, 1991.

Vleminckx KL, Vakaet J, Mareel M, Fiers W and van Roy F.

Genetic manipulation of E-cadherin expression by

epithelial tumor cells reveals an invasion suppressor role.

Cell. 66: 107-119, 1991.

Witcher LL, Collins R, Puttagunta S, Mechanic SE,

Munson M, Gumbiner B and Cowin P. Desmosomal

cadherin binding domains of plakoglobin. J Biol Chem.

271: 10904-10909, 1996.

Yagi T and Takeichi M. Cadherin superfamily genes:

functions, genomic organization, and neurologic

diversity. Genes Dev. 14: 1169-1180, 2000.

Yap AS, Niessen CM and Gumbiner BM. The

juxtamembrane region of the cadherin cytoplasmic tail

supports lateral clustering, adhesive strengthening, and

interaction with p120ctn. J Cell Biol. 141: 779-789, 1998.

Yokoyama K, Kamata N, Hayashi E, Hoteiya T, Ueda N,

Fujimoto R and Nagayama M. Reverse correlation of Ecadherin

and snail expression in oral squamous cell

carcinoma cells in vitro. Oral Oncol. 37: 65-71, 2001.


Journal of Cell and Molecular Biology 2: 65-77, 2003.

Haliç University, Printed in Turkey.

A general view: Structure and function of the subunits of EE.. ccoollii RNA

polymerase

Nihal Büyükuslu

Haliç University, Faculty of Arts and Sciences, Department of Molecular Biology and Genetics, 34280,

F›nd›kzade, ‹stanbul, Turkey

Received 9 June 2003; Accepted 23 June 2003

Abstract

The DNA-dependent RNA polymerases are widespread throughout nature. E. coli RNA polymerase, one of the most

well characterized polymerase, consists of two major forms, core enzyme with subunit stoichiometry of α 2ββ' and

holoenzyme which contains an additional σ subunit to core enzyme. E. coli RNA polymerase plays a central role in

transcription. While the core enzyme catalyses the elongation and termination of transcription, to initiate core

enzyme needs to combine with σ subunit. The three dimensional structure of this multimeric enzyme revealed a

thumb-like projection. Using the electron microscope, Tichelar and Heel (1990) proposed a model that is in

agreement with both β and β' together constituting a V-like structure and α dimer associates at the short ends, while

σ is positioned within the concave side of the core, next to the dimer.

In this review, the structure and related functions of the subunits of E. coli DNA-dependent RNA polymerase is

presented based on several researches and reviews. Considering biochemical and genetic studies on the RNA

polymerase of E. coli, a genetic walk on the subunits is summarized.

KKeeyy wwoorrddss:: E. coli RNA polymerase, α subunit, β subunit, β' subunit, σ factors

Genel bak›fl: EE.. ccoollii RNA polimeraz›n alt birimlerinin yap›lar› ve fonksiyonlar›

Özet

DNA-ba¤›ml› RNA polimerazlar do¤ada yayg›n olarak bulunurlar. En iyi karakterize edilen polimerazlardan biri

olan E. coli RNA polimeraz iki ana yap›dan oluflur, α 2ββ' stokiyometrisi ile çekirdek enzim ve çekirdek enzime σ alt

biriminin eklenmesi ile oluflan holoenzim. E. coli RNA polimeraz transkripsiyonda önemli bir rol oynar. Çekirdek

enzim transkripsiyonun uzamas› ve sonlanmas›n› katalizlerken, transkripsiyonun bafllamas› için çekirdek enzime σ

alt biriminin eklenmesi gerekmektedir. Bu çok altbirimli enzimin üç boyutlu yap›s› el ayas›na benzer bir yap›

gösterir. Elektron mikroskobu kullanarak Tichelar ve Heel (1990)’in önerdikleri modele göre β ve β' alt birimleri

birlikte V fleklinde yap› oluflturmakta ve α dimeri k›sa uçlarla birleflmektedir, σ ise çekirde¤in konkav k›sm›nda

dimere komflu olarak yer almaktad›r.

Bu derlemede çeflitli araflt›rmalar ve derlemeler baz al›narak E. coli DNA-ba¤›ml› RNA polimeraz›n alt birimlerinin

yap›lar› ve fonksiyonlar› sunulmufltur. E. coli RNA polimeraz›n üzerinde yap›lan biyokimyasal ve genetik

incelemelere dayan›larak alt birimleri üzerinde genetik bir yürüyüfl özetlenmifltir.

AAnnaahhttaarr ssöözzccüükklleerr:: E. coli RNA polimeraz, α alt birimi, β alt birimi, β' alt birimi, σ faktörleri

65


66 Nihal Büyükuslu

Structure and function of DNA-dependent RNA

polymerase of EE.. ccoollii

Transcription in wide range organisms uses a

homologous family of multisubunit DNA-dependent

RNA polymerases. The complex, multimeric DNAdependent

RNA polymerases are highly conserved

throughout nature, suggesting a common evolutionary

origin. A considerable body of research has allowed

the identification of potential functional regions within

several of the subunits.

The DNA-dependent RNA polymerase plays a

central role in transcription. During transcription in E.

coli, RNA polymerase is involved following steps:

RNA polymerase i) locates specific promoter

sequences in the DNA template; ii) melts a small

region containing the transcription start site; iii)

initiates RNA synthesis; iv) elongates the transcript,

and finally v) terminates and releases the RNA

product. Each step in this process is regulated by

interaction between the polymerase, the DNA, the

nascent RNA and some regulatory proteins and

ligands.

RNA polymerase consists of two major forms, core

enzyme which elongates and terminates transcription

and holoenzyme, which contains an additional σ

subunit responsible for the initiation of transcription.

The core enzyme consists of two α subunits, one β and

one β' subunit with relative molecular masses of

36.511; 150.616; 155.159 kD, respectively. The ω

subunit (10.237 kD) is also associated with RNA

polymerase. The subunits of RNA polymerase are

shown in Table 1.

Table 1: The subunits of RNA polymerase of E. coli.

Subunit Size Size Gene Function

aa kD

Figure 1: Three-dimensional structure of E. coli RNA

holo-polymerase by electron microscopy (Darst et al,

1989).

Three-dimensional structure of E. coli RNA

polymerase by electron microscopy revealed a thumblike

projection, similar to the active site cleft of DNA

polymerase I (Figure 1) (Darst et al., 1989). Polyakov

et al. (1995) supported the thumb-like projection

surrounding a groove or channel about 25Å in

diameter. The thumb of E. coli RNA polymerase

holoenzyme defines a deep but open groove on the

surface of the enzyme while in E. coli core RNA

polymerase the thumb forms part of a ring that

completely enclose the channel. A recent study

(Vassylyev et al., 2002) reported the crystal structure

of a bacterial RNA polymerase holoenzyme from

Thermus thermophilus at 2.6Å resolution. In the

structure, two amino-terminal domains of the σ

subunit form a V-shaped structure near the opening of

Alpha (α) 329 36.511 rpoA Required for assembly of the enzyme; interacts with some regulatory

proteins; also involved in catalysis

Beta (β) 1342 150.616 rpoB Catalysis of RNA synthesis (initiation and elongation); recognition of

terminators; binding of substrate ribonucleoside 5'-triphosphates; binding

of product RNA; stringent control; autogeneous regulation of ββ'

synthesis; binding of rifampicin and streptolydigin

Beta'(β') 1407 155.159 rpoC Binds to the DNA template; binds to sigma subunits

Sigma (σ) 613 70.263 rpoD Promotion of core enzyme maturation; recognition of regular promoters

Omega (ω) 91 10.237 rpoZ Required to restore denatured RNA polymerase in vitro to its fully

functional form.


the upstream DNA-binding channel of the active site

cleft. The carboxy-terminal domain of σ is near the

outlet of the RNA-exit channel, about 57Å from the Nterminal

domains. The extended linker domain forms a

hairpin protruding into the active site cleft, then

stretching through the RNA-exit channel to connect

the N- and C-terminal domains. The holoenzyme

structure provides insight into the structural

organization of transcription intermediate complexes

and into the mechanism of transcription initiation.

The subunits of EE.. ccoollii RNA polymerase

The α subunit

The α subunit of E. coli DNA-dependent RNA

polymerase is encoded by the rpoA gene and is

composed of 329 amino acid residues. The α subunit

maps within the large cluster of ribosomal genes

located at the 72-minute region of the E. coli

chromosome. Genetic and biochemical studies

indicate the α subunit carries out three critical

functions; subunit assembly, promoter recognition by

direct sequence-specific protein-DNA interaction,

transcription activation by a set of activator proteins.

Limited proteolysis experiments showed that the α

subunit consists of N-terminal domain comprised of

amino acids 8-241, a C-terminal domain comprised of

amino acids 249-329, and an unstructured and/or

flexible interdomain linker. Although the α N-terminal

region contains determinants for interaction with the

remainder of RNA polymerase and the α C-terminal

region contains determinants for interaction with

transcription activator proteins (Hayward et al., 1991;

Igarashi et al., 1991; Igarashi and Ishihama, 1991) the

C-terminal 85 amino acids of RNA polymerase a

constitute an independently folded domain that is

shown to be capable of dimerisation and sequencespecific

DNA binding. Furthermore, a C-terminal

deleted α mutant consisting of the N-terminal 235

amino acid residues retains the ability to form α

mutant core enzyme both in vitro (Igarashi et al., 1991;

Igarashi and Ishihama, 1991) and in vivo (Hayward et

al., 1991). For detailed mapping of the N-terminal

assembly domain, Kimura et al. (1994) made a set of

N-terminal and internal mutants and found that the

minimum region of the α subunit required for core

enzyme assembly is located between residues 21 and

235. The temperature-sensitive mutant, rpoA112 at

E. coli RNA polymerase 67

position 45, blocks RNA polymerase assembly

(Igarashi et al., 1990; Ishihama et al., 1980; Kawakami

and Ishihama, 1980). In support of this, Thomas and

Glass (1991) have found that expression of a truncated

a derivative comprising the N-terminal 230 amino

acids results in complementation of the Mel phenotype

of the rpoA341 mutant confirming that this region of α

contains all that is necessary for assembly in vivo. The

substituted deletion derivatives of the same region

were shown to be capable of complementing the Nterminal

αTs mutation suggesting that the presence but

not necessarily the nature of the sequences C-terminal

to residue 287 serve as a molecular ‘scaffold’

facilitating the formation of the correct conformation

without being intrinsically required for function per se

(Zou C. Personal communication)

Recent functional mapping within this assembly

domain by making a series of insertion mutants having

two extra amino acids at every 20 residues, led to a

proposal for the functional organization of the Nterminal

assembly domain of the α subunit. The region

around residue 80 is involved in binding both β and β':

the region including residues 180 and 200 plays a role

in β' binding and in dimerisation, more than one

contact site widely distributed within this assembly

domain are involved or alternatively multiple sites

form a single contact surface (Kimura and Ishihama,

1995). Indeed, a region near residue 80 exists with a

high content of hydrophobic amino acid residues that

appears to be involved in binding both β and β'.

Furthermore, mutations near the C-terminal proximal

region in the assembly domain affect β' binding.

Although the α subunit has been assigned a role

mainly in the assembly of the multisubunit complex,

much evidence suggests that α is also involved in

interactions with transcriptional regulators and plays a

central role in the resulting control of polymerase

activity. In E. coli RNA polymerase, the C-terminal

region of the α subunit is claimed to be the contact site

for the UP element of the rrnB P1 promoter that is

required for the transcription activation of the target

ribosomal RNA gene. Transcription and DNase I

footprinting results with RNA polymerases containing

C-terminal deletions in the α subunit suggest that UPlike

elements which interact with the α subunit might

play a role in transcription at promoters that have

sequences rich in (A+T) in upstream promoter

elements (Ross et al., 1993). An analysis of the Cterminal

domain of the E. coli RNA polymerase α

subunit (αCTD) by nuclear magnetic resonance


68 Nihal Büyükuslu

spectroscopy showed that the structure of αCTD is

compactly folded and comprised of four helices and

two long loops at the ends of the domain. A chemical

shift perturbation experiment was performed to

observe which residues of αCTD are involved in the

interaction with UP promoter elements. The residues

affected by perturbation were attributable to amides of

most of the residues from Glu261 to Ile275 and from

Thr292 to Ile303 that are located in helix 1, the Nterminal

end of the helix 4, and the preceding loop.

The C-terminal region of the α subunit is

responsible for contact with cis-acting UP element as

well as with trans-acting transcription factors. One of

the well characterized transcription factors, cAMP-

CRP, binds to specific (22bp) sites on DNA, the

position of the site(s) depending on the particular

promoters. The cAMP receptor protein, CRP, controls

the initiation of transcription of several genes

especially those involved in carbon-source utilization.

Activation of transcription by the upstream CRP

molecule is blocked by the HL159 substitution,

suggesting that the upstream-bound CRP makes a

direct contact with RNA polymerase. Footprinting

experiments indicated that RNA polymerase contacts

the promoter DNA between the two CRP-binding sites,

most likely due to interactions involving the C-terminal

part of the α subunit (Attey et al., 1994). Although

many CRP-dependent promoters carry a single CRPbinding

site, centered around –40, –60 or –70, a

number of promoters carry multiple CRP-binding sites.

In order to accommodate direct contacts between both

CRP dimers and the two α subunits in ternary

complexes at the ML1 promoter, Busby et al. (1994)

proposed a model that the α subunits are sandwiched

between CRP and RNA polymerase. Since the α dimer

is able to bind directly to DNA (Ross et al., 1993) it

seems probable that the α subunits are responsible for

the upstream RNA polymerase contacts and that the α

dimer bridges the two CRP dimers. In contrast, at class

II promoters, α binds just upstream of the CRP dimer

and makes contact with via activating region I.

Although the CRP dimer contains an activating region

I in each subunit, it is only the activating region I in the

upstream of the CRP dimer that makes contact with

RNA polymerase during transcription initiation. These

results suggest that α makes contact with the upstream

subunit of the CRP dimer whilst the downstream

subunit is likely to make alternative contacts with other

parts of RNA polymerase (Attey et al., 1994).

The ß subunit

The second largest subunit of E. coli RNA polymerase

is composed of 1342 amino acids and is highly

conserved throughout evolution (Sweetser et al., 1987;

Iwabe et al., 1991; Ovchinnikov et al., 1981). The β

subunit alone has no apparent function like the other

subunits. When assembled into the RNA polymerase

complex, β subunit has been shown to be involved in

most of the catalytic functions of RNA polymerase,

including nucleotide binding (Jin and Gross, 1991;

Mustaev et al., 1991), transcription initiation,

elongation and termination (Mustaev et al., 1991; Jin

and Gross, 1988; Kashlev et al., 1990; Landick et al.,

1990; Lee and Goldfarb 1991; Jin 1994), interactions

with both the σ subunit (Glass et al., 1986;1988) and

the NusA proteins (Jin and Gross, 1988; Sparkowski

and Das, 1992). Mutations conferring resistance to

rifampicin define several clustered residues in the

central and amino terminal parts of the β subunit

(Lisitsyn et al., 1984; Severinov et al., 1993;

Ovchinnikov et al., 1981; 1983; Jin and Gross 1988).

Sequence similarities among the subunits of

different organisms implied three main domains; Nterminal

domain, middle domain and C-terminal

domain, and two dispensable regions centered around

residues 300 and 1000. The conserved regions are

thought to be important for function and structure of β

subunit and the homology of these regions among

organisms reveal the evolution of genetic information

flow.

Deletions in the N-terminal region of subunit of E.

coli RNA polymerase between the residues 166-328

and 186-433 showed no obvious effect on function in

vitro, suggesting that this region is dispensable for

minimal function. The ∆(166-328) alteration was also

found to be non-lethal in vivo (Severinov et al., 1994).

A 69-residue segment between the residues 339-409 of

β subunit is widespread among prokaryotes indicating

that this region might play a structural or functional

role. Indeed, a recent study showed that the β subunit

residues 186-433 and 436-445 are commonly used by

σ 54 and σ 70 RNA polymerase holoenzyme for open

promoter complex formation (Wigneshweraraj et al.,

2002). In E. coli this region also contains the paf32

alteration (Severinov et al., 1994) due to a contact site

with the Alc protein, a site-specific termination factor

encoded by bacteriophage T4 that acts as a block to the

transcription of host genes (Kashlev et al., 1993). In

support of this, Landick et al. (1990) identified a series


of substitutions in that region that affects transcription

termination in vivo. All known mutations resulting in

rifampicin resistance map in the β subunit (Halling et

al., 1978; Miller et al., 1994) and lie between residues

512-573 (Kashlev et al., 1990; Landick et al., 1990; Jin

and Gross, 1988; Lisitsyn et al., 1984; Severinov et al.,

1993). Because the rifampicin resistant mutants in both

regions showed the same phenotypes in vivo and in

vitro, Severinov et al. (1993) have suggested that these

two regions perform a common catalytic function in the

core enzyme. Furthermore, cross-linking studies with

initiating substrate analogues implied that the

rifampicin region in the middle domain of β (residue

515-540) is placed a few angstroms away from

Lys1065 and His1237 of the C-terminal domain despite

their separation in the linear sequence of β by more

than 500 amino acids (Severinov et al., 1995). The

intragenic suppression data also supported the idea that

β subunit residues 529, 1237, 564 and 142 might

interact with each other in the folded ternary structure

of the enzyme. Part of the conserved domain 3 of σ 70

and the positions –3 and –4 of the template DNA strand

are in contact with γ-phosphate of the initiating

nucleotide indicating a close proximity to catalytic

center of the enzyme (Severinov et al., 1994; Mustaev

et al., 1994). Footprinting experiments revealed contact

between the Lys1065 in the β subunit and 5' end of the

nascent RNA chain (Krummel and Chamberlin, 1992;

Mustaev et al., 1993).

The rpoB gene of E. coli RNA polymerase is

involved in streptolydigin resistance as well as in

rifampicin resistance. Four contiguous amino acids

have been mapped as the target for streptolydigin

resistance. Although this region (543-546) lies in close

proximity to the Rif cluster, there does not appear to be

any functional overlap in terms of drug resistance

between the Rif R and Stl R regions since Stl R mutants are

not altered in their sensitivity to rifampicin (Heisler et

al., 1993). Moreover, mutations at amino acids 543-

546 do not appear to affect the functioning of RNA

polymerase in vivo. A histidine-tagged mutation at

amino acid 540 was able to transcribe in the presence

of Stl. These results suggest that the Stl R region is

dispensable for RNA polymerase activity and is

probably looped out or surface exposed and there is a

structural effect between the Stl R region in β and the

nearby catalytic center of the subunit. A fine mapping

of amber mutations in the β gene by virtue of the

unique MaeI restriction site created by this subset of

nonsense mutations revealed that the deletion of 31

amino acids between 618-649 is assembled into a

holoenzyme form capable of transcriptional initiation

in vivo (Buyukuslu et al., 1997). Although this region

common to the eubacterial chloroplast subgroup of β

homologous it may not responsible for assembly and

initiation of transcription.

Another region that is dispensable for normal β

function is placed at residues 940-1040 (Borukhov et

al., 1991; Severinov et al., 1992). This region is also

missing from B. subtilis, the homologous RNA

polymerase subunits from mycobacteri, chloroplast,

eukaryotes and archaebacteria (Miller et al., 1994;

Honore et al., 1993; Borukhov et al., 1991). Nene and

Glass (1984) identified a part of the C-terminal region

that is largely inessential, and removal of 62 residues

(965-1143) resulted in a wild type phenotype other

than a slightly slower growth on minimal medium.

In vitro reconstitution studies (Glass et al., 1986;

1988) suggest that the region between the residues

1180-1339 is necessary for holoenzyme formation.

Furthermore, after deletion of the C-terminal region of

E. coli β retained the ability to associate with β' and α

subunits indicating that this region is important in

binding σ. A recent study using trans-dominant

mutations in the 3' terminal region of the rpoB gene

defined highly conserved essential GEME motif. The in

vitro properties of the trans dominant-negative mutants

in the GEME motif (1271→1274) emphasize the

functional significance of this region, and contain

residues that are in close vicinity of the active centre of

the enzyme directly involved in catalysis (Cromie et al.,

1999). Supporting the functionality of GEME motif,

alanine substitution of three of the four GEME residues

(i.e. RFGEME→AFGAAA) greatly reduced the

affinity of RNA polymerase for substrates, without

affecting promoter binding or the maximal enzymatic

rate (Polyakov et al., 1999). Moreover, intragenic

suppression of trans-dominant lethal substitutions in the

GEME motif resulted that the GEME and HLVDDK

(1237→1242) regions are present as α-helices in

holoenzyme, and that functional cooperativity is

through one particular face of each helix (Malik et al.,

1999). In fact, the region 1243→1304 has been shown

to contact the nascent RNA in the elongation complex

(Gusarov and Nudler, 1999) indicating a possible

catalytic site for both β and β' subunits.

The ß' subunit

E. coli RNA polymerase 69

The β' subunit of E. coli RNA polymerase is composed


70 Nihal Büyükuslu

of 1407 amino acid residues and is encoded by rpoC

gene (Ovchinnikov et al., 1982; Squires et al., 1981).

The early studies suggested that the β' subunit is

essential for cell growth in the presence of a

temperature sensitive lethal rpoC mutant (Panny et al.,

1974). Due to its positive charge, the largest subunit

can bind nonspecifically to DNA in the absence of the

other subunits (Zillig et al., 1970) and is also able to

bind other polyanionic molecules, including heparin

(Fukuda and Ishihama, 1974). Some of β' mutations

has been found to cause defects in the assembly of

RNA polymerase (Toketo and Ishihama, 1976).

Recently, Naryshkina et al. (2001) indicated that the β'

subunit of E. coli RNA polymerase is not required for

interaction with initiating nucleotide but is necessary

for interaction with rifampicin. Recent studies

provided new insight into the structural and functional

characteristics of β' subunit, especially its role in

termination transcription.

To identify regions of the β' subunit of RNA

polymerase that are potentially involved in

transcription elongation and termination Weilbaecher

et al. (1994) have characterised amino acid

substitutions in the β' subunit that alter expression of

the genes preceded by terminators in vivo. Based on

the location and properties of these substitutions they

suggested a hypothesis that some intervals in β and β'

defined by termination-altering substitutions may

contact the DNA template or RNA transcript near the

active site (residues 490-570 in β, 300-400 and 700-

800 in β' ).

Among the conserved regions of β' subunit, a

strongly basic region is involved in zinc-binding.

Deletions in this N-terminal region of the β' subunit

gave rise a lethal phenotype. A study by Clerget et al.

(1995) indicated that deletions in the zinc-binding

domain could block factor-independent antitermination

and increase termination in coliphage

HK022 DNA, indicating a nucleic acid-polymerase

contact for termination.

A region of the β' subunit shows similarity to E.

coli DNA polymerase I. Substitutions in this region

identified a portion of β' that contacts the DNA

template, RNA transcript, or both, immediately

upstream from the active site in RNA polymerase

(Weilbaecher et al., 1994), although a conserved

region which is the location of all known amanitinresistance

substitutions in Pol II may fold in two

distinct segments that are separated by a surfaceexposed

region. These segments could interact to form

a functional site on the enzyme.

Substitutions in region 4 altered the ratio of

transcripts initiated at two adjacent start sites of a Pol II

promoter (Hekmatpanat and Young, 1991). Moreover,

cross-link mapping showed an interaction between E.

coli RNA polymerase and 8-azido AMP at the 3' end of

the nascent transcript occurring within the region

spanning Met-932 and Trp-1020 of the β' subunit

(Borukhov et al., 1991). Single substitutions

downstream of this region exhibited a strong effect in

vitro at some terminators indicating a direct effect on

termination, or a retention of residual transcription

factors during purification. In addition, the involvement

of the β subunit in transcription termination has been

suggested based on the isolation of rpoC mutations that

suppress rho201 (Jin and Gross, 1989). An Arg-rich

domain of NusA containing nusA1 and nusA11 has also

been reported to interact with a regulatory component

for transcription termination involving the β' subunit

(Ito et al., 1991). Epitope mapping of monoclonal

antibodies directed against the β' subunit of E. coli

RNA polymerase indicated that the region 817-876 may

be important in enzyme assembly or subunit-subunit

interaction and the region located between amino acids

1047-1093 may be involved in the catalytic function of

RNA polymerase (Luo and Krakow, 1992). A G to C

transition leading to the substitution of aspartate for

glycine at amino acid residue 1033 in the β' subunit

affects chromosomal replication control in E. coli

(Petersen and Flemming, 1991).

190 amino acid-long region centered around

position 1050 of the 1407 amino acid-long β' subunit

of E. coli RNA polymerase is absent from homologues

in eukaryotes, archaea, and many bacteria. Moreover,

in chloroplasts the corresponding region can be more

than 900 amino acids long. The deletion mutagenesis

of this hypervariable region revealed that long

deletions mimicking β' of Gram positive bacteria

failed to assemble into RNA polymerase. Short, 40-60

amino acids-long deletions spanning β' residues 941-

1130 assembled into active RNA polymerase in vitro.

It is proposed that mutations in functionally

dispensable region of β' inhibit transcript cleavage and

elongation by distorting the flanking conserved

segment in the active center supporting the previous

results (Zakharova et al., 2003).

Substitutions in the C-terminal region of β' appear

to affect elongation of transcription. Furthermore, one

conditional lethal and two suppressor substitutions in

the yeast PolII homologue of β' occur in the conserved


egion, supporting the idea that they may affect

transcription in the same way (Weilbaecher et al.,

1994).

The σ subunit

In eubacteria, initiation of transcription is mediated by

the σ subunit of RNA polymerase. However, free σ 70 is

not able to bind specifically at promoter DNA sites

(Wellmann and Meares, 1991) due to the

autoinhibition of σ 70 DNA binding activity by the Nterminal

domain of σ 70 (Dombroski et al., 1992;1993).

Although σ 70 , the major σ factor of E. coli, is required

for initiation at most promoters, alternative σ factors

are also responsible for transcription from other

classes of promoters. Based upon characterisation,

identification and sequence analysis, σ factors can be

classified in two broad classes. One family is similar to

the originally identified E. coli σ 70 subunit, the other is

similar to the 54 kD E. coli σ subunit.

Before beginning a comparative analysis of σ

families it is appropriate to discuss the structure and

function of the σ factor. Sequence alignments of the σ

family of proteins show similarities suggestive of

common ancestry to reflect the various functional roles

of σ factors in the cell (Gribskov and Burges, 1986;

Stragier et al., 1985). In an expanded alignment of σ,

four regions of high conservation have been identified

(Lonetto et al., 1992). Regions 2 and 4 are the most

conserved regions and tend to be very basic. Regions 1

and 3 exhibit lower conservation and are acidic.

Considering the alignment of the σ 70 family of

sequences, it is possible to divide σ factors into three

groups. The first group is comprised of primary σ

factors that exhibit very high similarity and are

responsible for most RNA synthesis in exponentially

growing cells. Group two proteins are quite similar in

sequence to the primary σ, but are dispensable for cell

growth. The third group contains the alternative σs,

which is responsible for transcription of specific

regulons.

Analysis of conserved regions of σ 70 factors

indicated that the four conserved regions could be

further divided into subregions. The N-terminal

regions of the major σ factors from various bacteria

contain approximately 30% identical amino acid, these

can be separated into two subregions: 1.1 and 1.2.

Region 1.1 is rather poorly conserved and is present

only in primary σs and E. coli Sig(38). Region 1.2 has

several residues that are conserved among all primary

E. coli RNA polymerase 71

and alternative σs expect M. xanthus SigB and S.

typhimurium FliA. Although the role of region 1.2 has

not been ascribed yet, the high conservation and a 14

amino acid deletion at position 330 that reduces the

stability of σ 70 at both low and high temperature (Hu

and Gross, 1983) imply that this region is structurally

and functionally important.

Region 2 has been divided into four subregions;

Region 2.1 and 2.3 are rich in aromatic amino acid

residues. A deletion in region 2.1 reduces the binding

of E. coli σ 70 to core RNA polymerase suggesting that

this region of σ involved in binding to core enzyme

(Lesley and Burgess, 1989). There is evidence that

regions 2.1 and 2.3 are involved in promoter

unwinding in the region from –9 to +3 during

formation of the open promoter complex (Siebenlist et

al., 1980; Kirkegaard et al., 1983). Supporting this

idea, a mutation in region 2.3 of SigE, and in the same

region of B. subtilis SigA, interrupt the process of

DNA strand separation during transcription initiation

(Jones and Moran, 1992). Region 2.2 is the most

conserved region among all groups of σ. Region 2.4 is

highly conserved among primary σ, and implicated in

recognition of the –10 region, while a helix-turn-helix

motif located in subregion 4.2 of σ is involved in

recognising the –35 consensus sequence (Daniels et

al., 1990; Khan and Ditta, 1991; Siegele et al., 1989;

Tatti et al., 1991; Waldburger et al., 1990; Zuber et al.,

1989). More recently it was shown that conformation

of conserved domains of σ 70 (region 1, 2.4 and 4.2) was

affected by the core enzyme (Callaci et al., 1998).

Supporting that luminescence resonance energy

transfer measurements showed that E. coli RNA

polymerase induced major changes in σ 70 involves a

movement of the conserved region 1 by~20Å and the

conserved region 4.2 by~15Å with respect to

conserved region 2. This movement of DNA-binding

domains of σ 70 is thought to be an important

mechanism by which the ability of σ 70 to recognize

promoter DNA is regulated (Callaci et al., 1999).

Region 3 is divided into two subregions; 3.1 is more

conserved with a weak resemblance to the helix-turnhelix

and 3.2 is largely acidic and highly conserved

among group 1 σs, but is weakly conserved among the

group 3 σs. A 25 amino acid deletion in region 3.2 of E.

coli σ causes an affinity reduction of this mutant

protein to core enzyme indicating a possible secondary

core-binding site (Lonetto et al., 1992).

Region 4 consists of two subregions; 4.1 is an

amphipatic α-helix and 4.2 is highly conserved among


72 Nihal Büyükuslu

the primary σ factors and has a sequence similarity to

the HTH-DNA binding motif (Brennan and Matthews,

1989; Gribskov and Burgess, 1986; Helmann and

Chamberlin, 1988; Straiger et al., 1989). A spacer

region of variable length and sequence lies between the

two subregions. It appears likely that the conserved

HTH motif in subregion 4.2 may directly contact the

–35 promoter region. Although two mutations, R584C

and R588H, in this region of the E. coli σ 70 factor have

been implicated in recognition of the –35 promoter

sequence, a set of C-truncated σ 70 lacking the –35

recognition domain can activate class II promoters (the

pstS and P1gal), indicating that necessary contact or

activation sites for the two activators lie at different

positions in a segment of σ 70 extending from region 3.2

to the upstream helix within region 4.2 (Kumar et al.,

1994; Makino et al., 1993). Further evidence to support

this suggestion came from the study by Jin et al.,

(1995). Using protein-protein photo-crosslinking, they

showed that the CRP site for galP1, a class 2 promoter,

contacts with amino acids 530-539 of σ 70 region.

All RNA polymerase holoenzymes containing σ 70 -

related σs appear to function in a similar manner.

However, σ 54 (σN), encoded by rpoN, is quite distinct,

both structurally and functionally, from the σ 70 family.

Among 17 sequenced rpoN genes, there is a high

degree of conservation in the protein primary

sequence, and three distinct regions can be identified

(Merrick et al., 1987). Region I is the N-terminal

region is suggested to be α-helical; it is rich in leucine

and glutamine residues. Region II varies between 60 to

110 residues in length, with essentially no sequence

conservation. The C-terminal region III is highly

conserved and contains two motifs; a potential helixturn-helix

(HTH) motif and the ‘RpoN box’.

σ 54 and σ 70 participate in different transcription

mechanisms but bind the same core RNA polymerase

(Gralla, 1991). Recent considerations suggest that σ 54

should contain a minimum of two different elements

Table 2: The E. coli sigma factors

Sigma factor Gene Function

for recognition of the –24 and –12 promoter regions.

C-terminal deletions near to the HTH motif abolished

DNA binding (Sasse-Dwight and Gralla, 1990).

Indeed, specific amino acid substitutions in this region

showed a similar phenotype (Coppard and Merrick,

1991). Merrick and Chambers (1992) found a specific

HTH mutation in K. pneumoniae σN (R363K)

suppressed down mutations at position –13 in the

glnAp2 promoter, suggesting that the conserved HTH

region is involved in recognition of promoter-proximal

sequences. Physical studies indicate that amino acids

in a region from Asn312 to Arg345 in K. pneumoniae

σN, just upstream of the HTH, crosslink to DNA in the

presence or absence of core enzyme (Cannon et al.,

1994). In contrast to the σ 70 family, activation of Eσ 54

is not dependent on the presence of the C-terminal

domain (Lee et al., 1993). Transcription by Eσ 54

appears to be controlled by a mechanism that requires

the use of an activator protein and ATP to catalyze

formation of open complexes (Popham et al., 1989).

Deletions in both region I and region III prevent opencomplex

formation. During open-complex formation,

region II has been proposed to play a role in triggering

conformational changes for DNA melting (Sasse-

Dwight and Gralla, 1990).

The importance of σ S has been increasingly

recognised in recent years. This sigma factor appears

only as cells enter the stationary phase of growth. It is

responsible for transcription of all of the genes whose

products are required during stationary phase

(Ishihama, 2000).

Two families of σ factors have been characterised

in detail. A number of different σ factors belonging to

sigma families are presented in Table 2 according to

the origin, genetic locus, and presumed function.

The ω subunit

The ω subunit, encoded rpoZ, is associated with both

σ 70 rpoD Housekeeping function

σ 54 RpoN (ntrA, glnF) Nitrogen-regulated gene transcription

σ 32 RpoH Heat-shock gene transcription

σ S RpoS Gene expression in stationary phase cells

σ F RpoF Expression of flagellar operons

σ FecI fecI Regulates the fec genes for iron dicitrate transport


core and holoenzyme (Burgess, 1969). The omega

subunit was for many years considered a curiosity

since no function could be ascribed to it. Therefore, the

available literature on the ω subunit as regards to its

structure and function is rare. The ω subunit is not

required for the function of the transcriptional

apparatus both in vivo and in vitro. Strain lacking ω,

that is, a rpoZ null mutant is viable, suggesting a nonessential

nature of the protein or that there might also

be a redundancy in function. The only known

phenotype ascribed to the rpoZ null mutant is a slower

growth time (Mukherjee et al., 1999). However, it is

now known that omega is necessary to restore

denatured RNA polymerase in vitro to its fully

functional form. It may function by binding

simultaneously to the N-terminus and C-terminus of

the β' subunit. The omega subunit is a part of the

Thermus aquaticus enzyme whose structure was

recently determined (Murakami et al., 2002).

A study by Mukharjee and Chatterji (1999) showed

that ω-less holoenzyme has lesser affinity towards the

DNA template and external addition of ω destabilizes

the open complex for both the wild-type and ω-less

enzyme. The ω-less core enzyme interacts with the σ 70

subunit to expose the –35 recognition domain (domain

4·2) unlike that observed in the wild-type interaction.

Thus the absence of the ω subunit leads to the

formation of an enzyme which has altered DNA

binding and σ 70 binding properties. Circular dichroic

measurements also indicate a major conformational

alteration of both holo and core RNA polymerase in

the presence and absence of the ω subunit.

References

Attey A, Belyaeva T, Savery N, Hoggett J, Fujita N,

Ishihama A and Busby S. Interactions between the cyclic

AMP receptor protein and the α subunit of RNA

polymerase at the E. coli galactose operon P1 promoter.

Nucl Acid Res. 22: 4375-4380, 1994.

Borukhov S, Lee J and Goldfarb A. Mapping of a contact for

the RNA 3' terminus in the largest subunit of RNA

polymerase. J Biol Chem. 266: 23932-23935, 1991.

Brennan RT and Matthews BW. The helix-turn-helix DNA

binding motif. J Biol Chem. 264: 1903-1906, 1989.

Burgess RR. Separation and characterization of the subunits

of RNA polymerase. J Biol Chem. 244: 2168-2176,

1969.

Busby S, West D, Lawes M, Webster C, Ishihama A and

Kolb A. Transcription activation by the E. coli cyclic-

E. coli RNA polymerase 73

amp protein-receptors bound in tandem at promoters can

interact synergistically. J Mol Biol. 241: 341-352, 1994.

Büyükuslu N, Trigwell SM, Lim PP, Fujita N, Ishihama A,

Ralphs N and Glass RE. Physical mapping of a

collection of MaeI-generating mutations in the β gene of

E. coli RNA polymerase and the functional effect of

internal deletions constructed through their

manipulation. Genes and Function 1: 119-129, 1997.

Callaci S, Heyduk E and Heyduk T. Conformational changes

of E. coli RNA polymerase σ 70 factor induced by binding

to the core enzyme. J Biol Chem. 273: 329995-33001,

1998.

Callaci S, Heyduk E and Heyduk T. Core RNA polymerase

from E. coli induces a major change in the domain

arrangement of the σ 70 subunit. Molecular Cell. 3: 229-

238, 1999.

Cannon W, Claveriemartin F, Austin S and Buck M.

Identification of a DNA-contacting surface in the

transcription factor σ 54 . Mol Microbiol. 11: 227-236,

1994.

Clerget M, Jin DJ and Weisber RA. A zinc-binding region in

the β' subunit of RNA polymerase is involved in

antitermination of early transcription of phage HK022. J

Mol Biol. 248: 768-780, 1995.

Coppard JR and Merrick MJ. Casette mutagenesis implicates

a helix-turn-helix motif in promoter recognition by the

novel RNA-polymerase σ-factor σ 54 . Mol Microbiol. 5:

1309-1317, 1991.

Cromie K, Ahmad K, Malik T, Büyükuslu N and Glass RE.

Trans-dominant mutations in the 3'-terminal region of

the rpoB gene define highly conserved, essential residues

in the β subunit of RNA polymerase: the GEME motif.

Genes to Cells. 4: 145-159, 1999.

Daniels D, Zuber R and Losick R. Two amino acids in an

RNA polymerase σ factor involved in the recognition of

adjacent base pairs in the –10 region of a cognate

promoter. Proc Nat Acad Sci. USA 87: 8075-8079, 1990.

Darst SA, Edwards AM and Kornberg RD. Threedimensional

structure of E. coli RNA polymerase

holoenzyme determined by electron crystallography.

Nature. 340: 730-732, 1989.

Dombroski AJ, Walter WA, Record MT, Jr. Siegele DA and

Gross CA. Polypeptides containing highly conserved

regions of transcription initiation factor sigma 70 exhibit

specificity of binding to promoter DNA. Cell. 70: 501-

512, 1992.

Dombroski AJ, Walter WA and Gross CA. Amino-terminal

amino acids modulate sigma-factor DNA-binding

activity. Genes Dev. 7: 2446-2455, 1993.

Fukuda R and Ishihama A. Subunits of RNA polymerase in

function and structure. V. Maturation in vitro of core

enzyme from E. coli. J Mol Biol. 87: 523-540, 1974.

Glass RE, Honda A and Ishihama A. Genetic studies on the

β subunit of E. coli RNA polymerase. IX The role of the

carboxy-terminus in enzyme assembly. Mol Gen Genet.


74 Nihal Büyükuslu

203: 492-495, 1986.

Glass RE, Ralphs NT, Fujita N and Ishihama A. Assembly

of amber fragments of the β subunit of E. coli RNA

polymerase. Eur J Biochem. 176: 403-407, 1988.

Gralla JD. Transcriptional control-lessons from an E. coli

promoter data base. Cell. 66: 415-418, 1991.

Gribskov M and Burgess RR. σ factors from E. coli, B.

subtilis, phage SP01 and phage T4 are homologous

proteins. Nucl Acid Res. 14: 6745-6763, 1986.

Gusarov I and Nudler E. The mechanism of intrinsic

transcription termination. Mol Cell. 3: 495-504, 1999.

Halling SM, Burtis KC and Doi RH. β' subunit of bacterial

RNA polymerase is responsible for streptolydigin

resistance in Bacillus subtilis. Nature. 272: 827-842,

1978.

Hayward RS, Igarashi K and Ishihama A. Functional

specialization within the α subunit of E. coli RNA

polymerase. J Mol Biol. 221: 23-29, 1991.

Heisler LM, Suzuki H, Landick R and Gross CA. Four

contiguous amino-acids define the target for

streptolydigin resistance in the β subunit of E. coli RNA

polymerase. J Biol Chem. 268: 25369-25375, 1993.

Hekmetpanah DS and Young RA. Mutations in a conserved

region of RNA polymerase II influence the accuracy of

mRNA start site selection. Mol Cell Biol. 11: 5781-

5791, 1991.

Helmann JD and Chamberlin MJ. Structure and function of

bacterial σ factors. Ann Rev Biochem. 57: 839-872,

1988.

Honore NT, Bergh S, Chanteau S, Doucet-Populaire F,

Eiglmeier K, Garnier T, Geroges C, Lanois P,

Limpaipoon T, Newton S, Niang K, Portillo P,

Ramesh GR, Reddi P, Ridel PR, Sittisombut N,

Wu-Hunter S and Cole ST. Nucleotide sequence of the

first cosmid from the Mycobacterium leprae genome

project: Structure and function of the Rif-Str regions.

Mol Microbiol. 7: 207-214,1993.

Hu JC and Gross CA. Marker rescue with plasmids bearing

deletions in rpoD identifies a dispensible part of E. coli

σ factor. Mol Gen Genet. 199: 7-13, 1983.

Igarashi K, Fujita N and Ishihama A. Sequence analyses of

two temperature-sensitive mutations in the α subunit

gene (rpoB) of E. coli RNA polymerase. Nucl Acid Res.

18: 5945-5948, 1990.

Igarashi K, Hanamura A, Makino K, Aiba H, Mizuno T,

Nakata A and Ishihama A. Functional map of the α

subunit of E. coli RNA polymerase: Two modes of

transcription activation by positive factors. Proc Natl

Acad Sci. USA 88: 8958-8962, 1991.

Igarashi K and Ishihama A. Bipartite functional map of the

E. coli RNA polymerase α subunit: Involvement of the

C-terminal region in transcription activation by cAMP-

CRP. Cell. 65: 1015-1022, 1991.

Ishihama A. Functional Modulation of Escherichia coli RNA

polymerase. Annu Rev Microbiol. 54: 499-518, 2000.

Ishihama A, Shimamoto N, Aiba H, Kawakami K,

Nashimoto H, Tsugawa A and Uchida N. Temperaturesensitive

mutations in the α subunit gene of E. coli RNA

polymerase. J Mol Biol. 137: 137-150, 1980.

Ito K, Egawa K and Nakamura Y. Genetic interaction

between the β' subunit of RNA polymerase and the

arginine-rich domain of E. coli nusA protein. J

Bacteriol. 173: 1492-1501, 1991.

Iwabe N, Kuma KK, Kishino H, Hasegawa M and Miyata T.

Evolution of RNA polymerases and branching patterns

of the three major groups of archaebacteria. J Mol Evol.

32: 70-78, 1991.

Jin DJ. Slippage synthesis at the galp2 promoter of E. coli

and regulation by UTP concentration and cAMP-centerdot-cAMP

protein. J Biol Chem. 269: 17221-17227,

1994.

Jin DJ and Gross CA. RpoB8, a rifampicin-resistant

termination-proficient RNA polymerase, has an

increased Km for purine nucleotides during transcription

elongation. J Biol Chem. 266: 11178-14485, 1991.

Jin DJ and Gross CA. Three rpoBC mutations that suppress

the termination defects of rho mutants also affect the

functions of nusA mutants. Mol Gen Genet. 216: 269-

275, 1989.

Jin DJ and Gross CA. Mapping and sequencing of mutations

in the E. coli rpoB gene led to rifampicin resistance. J

Mol Biol. 202: 45-48, 1988.

Jin R, Sharif KA and Krakow JS. Evidence for contact

between the cyclic AMP receptor protein and the σ 70

subunit of E. coli RNA polymerase. J Biol Chem. 270:

19231-19216, 1995.

Jones CH and Moran CP. Mutant σ factor blocks transition

between promoter binding and initiation of transcription.

Proc Nat Acad Sci. USA 89: 1958-1962, 1992.

Kashlev M, Lee J, Zalenskaya K, Nikivorov V and

Goldfarb A. Blocking of the initiation to elongation

transition by a transdominant RNA polymerase

mutation. Science. 1006-1009, 1990

Kashlev M, Martin E, Polyakov A, Severinov K and

Nikiforov A. Histidine-tagged RNA-polymerasedissection

of the transcription cycle using

immobilized enzyme. Gene. 130: 9-14, 1993.

Kawakami K and Ishihama A. Defective assembly of

ribonucleic acid polymerase subunits in a temperaturesensitive

α subunit mutant of E. coli. Biochem. 19: 3491-

3495, 1980.

Khan D and Ditta G. Molecular structure of FixJ: Homology

of the transcriptional activator domain with the –35

binding domain of σ factors. Mol Microbiol. 5: 987-997,

1991.

Kimura M, Fujita N and Ishihama A. Functional map of the

α subunit of E. coli RNA polymerase-deletion analyses

of the amino-terminal assembly. J Mol Biol. 242: 107-

115, 1994.

Kimura M and Ishihama A. Functional map of the α subunit


of E. coli RNA polymerase-insertion analyses of the

amino-terminal assembly. J Mol Biol. 248: 756-767,

1995.

Kirkegaard K, Buc H, Spassky A and Wang JC. Mapping of

single-stranded regions in duplex DNA at the sequence

level: single-strand-specific cytosine methylation in

RNA polymerase-promoter complexes. Proc Natl Acad

Sci. USA 80: 2544-2548, 1983.

Kumar A, Grimes B, Fujita N, Makino K, Malloch RA,

Hayward RS and Ishihama A. Role of the σ 70 subunit of

E. coli RNA polymerase in transcription activation. J

Mol Biol. 235: 405-413, 1994.

Krummel B and Chamberlin MJ. Structural analysis of

ternary complexes of E. coli RNA polymerase:

deoxyribonuclease I footprinting of defined complexes.

J Mol Biol. 225: 239-250, 1992.

Landick R, Stewart J and Lee DN. Amino acid changes in

conserved regions of the β subunit of E. coli RNA

polymerase alter transcription pausing and termination.

Genes Dev. 4: 1623-1636, 1990.

Lee J and Goldfarb A. lac repressor acts by modifying the

initial transcribing complex so that it cannot leave the

promoter. Cell. 66: 793-798, 1991.

Lee HS, Ishihama A and Kustu S. The C-terminus of the asubunit

of RNA-polymerase is not essential for

transcriptional activation of σ 54 holoenzyme. J

Bacteriol. 175: 2479-2582, 1993.

Lesley SA and Burgess RR. Characterisation of the E. coli

transcription factor σ 70 : localisation of a region involved

in the interaction with core RNA polymerase. Biochem.

28: 7728-7734, 1989.

Lisitsyn NA, Sverdlov ED, Moiseyeva EP, Danilevskaya ON

and Nikiforov VG. Mutation to rifampicin resistance at

the beginning of the RNA polymerase β subunit gene in

E. coli. Mol Gen Genet. 196: 173-174, 1984.

Lonetto M, Gribskov M and Gross C. The σ 70 family:

Sequence conservation and evolutionary relationships.

J Bacteriol. 174: 3843-3849, 1992.

Lou J and Krakow JS. Characterization and epitope mapping

of monoclonal antibodies directed against the β' subunit

of Escherichia coli RNA polymerase. J Mol Chem. 267:

18175-18181, 1992.

Makino K, Amemura M, Kim SK, Nakata A and

Shinagawa H. Role of the σ 70 subunit of E. coli RNA

polymerase in transcription activation by activator

protein phoB in E. coli. Genes Dev. 7: 149-160, 1993.

Malik T, Ahmad K, Büyükuslu N, Cromie K, and Glass RE.

Intragenic suppression of trans-dominant lethal

substitutions in the conserved GEME motif of the β

subunit of RNA polymerase: evidence for functional

cooperativity within the C-terminus. Genes to Cells. 4:

501-515, 1999.

Merrick M and Chambers S. The helix-turn-helix motif of σ 54

is involved in recognition of the –13 promoter element.

J Bacterial. 174: 7221-7226, 1992.

E. coli RNA polymerase 75

Merrick M, Gibbins J and Toukdarian A. The nucleotide

sequence of the σ factor gene ntr (rpoN) of Azobacter

vinelandii: analysis of conserved sequences in NtrA

proteins. Mol Gen Genet. 210: 323-330, 1987.

Miller LP, Crawford JT and Shinnick TM. The rpoB gene of

mycobacterium-tuberculosis. Antimicrobial Agents and

Chemotherapy. 38: 805-811, 1994.

Mukherjee K and Chatterji D. Alteration in template

recognition by Escherichia coli RNA polymerase

lacking the ω subunit: A mechanistic analysis through

gel retardation and foot-printing studies. J Bio Sci.

24: 453-461, 1999

Mukherjee K, Nagai H, Shimamoto N and Chatterji D.

GroEL is involved in activation of E. coli RNA

polymerase devoid of σ subunit in vivo. Eur J Biochem.

266: 228-235, 1999.

Murakami KS, Masuda S and Darst S. Structural basis of

transcription initiation: RNA polymerase holoenzyme at

4Å resolution. Science. 296(5571): 1280-1284, 2002.

Mustaev A, Kashlev M, Polyakov J, Lebedev A,

Zalenskataya K, Grachev M, Goldfarb A and

Nikiforov V. Mapping of the priming substrate contacts

in the active-centre of E. coli RNA polymerase. J Biol

Chem. 266: 23927-23931, 1991.

Mustaev A, Kashlev M, Zaychikov E, Grachev M, and

Goldfarb A. Active-center rearrangement in RNA

polymerase initiation. J Biol Chem. 268: 19185-19187,

1993.

Mustaev A, Zaychikov E, Severinov K, Kashlev M,

Polyakov A, Nikiforov V and Goldfarb A. Topology of

the RNA-polymerase active center probed by chimeric

rifampicin-nucleotide compounds. Proc Natl Acad Sci.

USA 91: 12036-12040, 1994.

Naryshkina T, Mustaev A, Darst SA and Severinov K. The β'

subunit of E. coli RNA polymerase is not required for

interaction with initiating nucleotide but is necessary for

interaction with rifampicin. J Biol Chem. 276: 13308-

13313, 2001.

Nene V and Glass RE. Genetic studies on the β subunit of

E. coli RNA polymerase IV Structure-function

correlates. Mol Gen Genet. 194: 166-172, 1984.

Ovchinnikov YA, Monastyrskaya GS, Guriev VV,

Chertov OY, Modyanov NN, Grinkevich VA,

Makarova IA, Marchenko TV, Polovnikova IN,

Lipkin VM and Sverdlov ED. The primary structure of

E. coli RNA polymerase nucleotide-sequence of the

rpoB gene and amino acid sequence of the subunit. Eur

J Biochem. 116: 621-629, 1981.

Ovchinnikov YA, Monastyrskaya GS, Guriev SO, Kalinina

NF, Sverdlov ED, Gragerov AI, Bass IA, Kiver IF,

Moiseyeva EP, Igumnov VN, Mindlin SZ, Nikiforov VG

and Khesin RB. RNA polymerase rifampicin resistance

mutations in E. coli: Sequence changes and dominance.

Mol Gen Genet. 190: 344-348, 1983.


76 Nihal Büyükuslu

Ovchinnikov YA, Monastryskaya GS, Gubanov VV,

Guryev SO, Salomanita IS, Shuvaeva TM, Lipkin VM

and Sverdlov ED. The primary structure of E. coli RNA

polymerase. Nucleotide sequence of the rpoC gene

and amino acid sequence of the b' subunit. Nucl Acid

Res. 10: 4035-4044, 1982.

Panny SR, Heil A, Mazus B, Palm P, Zillig W, Mindlin SZ.

Ilyina TS and Khesin RB. A temperature sensitive

mutation of the β' subunit of DNA-dependent RNA

polymerase from E. coli T116. FEBS letter. 48: 241-245,

1974.

Petersen SK and Flemming GH. A missense mutation in the

rpoC gene affects chromosomal replication control in

E. coli. J Bacteriol. 173: 5200-5206, 1991.

Polyakov A, Nikiforov V and Goldfarb A. Disruption of

substrate binding site in E. coli RNA polymerase by

lethal alanine substitutions in carboxy terminal domain

of the β subunit. FEBS letter. 444: 189-194, 1999.

Polyakov A, Severinova E and Darst SA. 3-dimensional

structure of E. coli core RNA-polymerase-promoter

binding and elongation conformations of the enzyme.

Cell. 83: 365-373, 1995.

Popham DL, Szeto D, Keener J and Kustu S. Function of a

bacterial activator protein that binds to transcriptional

enhancers. Science. 243: 629-635, 1989.

Ross W, Gosink KK, Salomon J, Igarashi K, Zou C,

Ishihama A, Severinov K and Gourse RL. A third

recognition element in bacterial promoters-DNAbinding

by α subunit of RNA polymerase. Science. 262:

1407-1413, 1993.

Sasse-Dwight σ and Gralla JD. Role of eukaryotic-type

functional domains found in the prokaryotic enhancer

receptor factor σ 54 . Cell 62: 945-954, 1990.

Severinov K, Fenyo D, Severinova E, Mustaev A, Chait BT

and Goldfarb DS. The σ-subunit conserved region-3 is

part of 5'-face of active-center of E. coli RNApolymerase.

J Biol Chem. 269: 20826-20828, 1994.

Severinov K, Mustaev A, Kashlev M, Borukhov S,

Nikiforov V and Goldfarb A. Dissection of the β subunit

in the E. coli RNA polymerase into domains by

proteolytic cleavage. J Biol Chem. 267: 12813-12819,

1992.

Severinov K, Mustaev A, Severinov E, Kozlov M and

Darst SA. The β subunit rif-cluster-I is only angstroms

away from the active center of E. coli RNA-polymerase.

J Biol Chem. 270: 29428-29432, 1995.

Severinov K, Soushko M, Goldfarb A and Nikiforov V.

Rifampicin region revisited- new rifampicin-resistant

and streptolydigin-resistant mutants in the β subunit of

E. coli RNA polymerase. J Biol Chem. 268: 14820-

14825, 1993.

Siebenlist U, Simpson R and Gilbert W. E. coli RNA

polymerase interacts homologously with two different

promoters. Cell. 20: 269-272, 1980.

Siegele DA, Hu JC, Walter WA and Gross CA. Altered

promoter recognition by mutant forms of the σ 70 subunit

of E. coli RNA polymerase. J Mol Biol. 206: 591-603,

1989.

Sparkovski J and Das A. Simultaneous gain and loss of

functions caused by a single amino acid substitution in

the β subunit of E. coli RNA polymerase: Suppression of

NusA and rho mutations and conditional lethality.

Genetics. 130: 411-428, 1992.

Straiger S, Kunkel B, Kroos L, and Losick R. Chromosomal

rearrangement generating a composite gene for a

developmental σ factor. Science. 243: 507-512, 1989.

Straiger P, Parsot C, and Bouvier J. Two functional domains

conserved in major and alternate bacterial σ factors.

FEBS letters. 187: 11-15, 1985.

Squires CH, Defelice M, Wessler SR and Calvo JM. Physical

characterisation of the ilvH1 operon of E. coli K12. J

Bacteriol. 147: 797-804, 1981.

Sweetser D, Nonet M and Young RA. Prokaryotic and

eukaryotic RNA polymerases have homologous core

subunits. Proc Natl Acad Sci. USA 84: 1192-1196, 1987.

Tatti KM, Jones CH and Moran PC. Genetic evidence for

interaction of σE with the spoIIID promoter in B.

subtilis. J Bacteriol. 173: 7828-7833, 1991.

Thomas M and Glass RE. E. coli rpoA mutation which

impairs transcription of positively regulated systems.

Mol Microbiol. 5: 2719-2725, 1991.

Tichelar W and Heel MV. Characteristic views of E. coli

RNA polymerase core enzyme in the scanning

transmission electron microscope. J Structural Biol.

103: 180-184, 1990.

Toketo M and Ishihama A. Biosynthesis of RNA polymerase

in E. coli IV. Accumulation of intermediates in mutants

defective in the subunit assembly. J Mol Biol. 102: 297-

310, 1976.

Vassylyev DG, Sekine S, Laptenko O, Lee J,

Vassylyeva MN, Borukhov σ and Yokoyama S. Crystal

structure of a bacterial RNA polymerase holoenzyme at

2.6Å resolution. Nature. 417: 712-719, 2002.

Waldburger C, Gardella T, Wang R and Suskind MM.

Changes in conserved region 2 of E. coli σ 70 affecting

promoter recognition. J Mol Biol. 215: 267-276, 1990.

Weilbaecher R, Hebron C, Feng GH and Landick R.

Termination-altering amino-acid substitutions in the β'

subunit of Escherichia coli RNA-polymerase identify

regions involved in RNA chain elongation. Genes Dev. 8:

2913-2927, 1994.

Wellman A and Meares CF. Footprint on the sigma protein: a

re-examination. Biochem Biophys Res Comm. 177:

140-144, 1991.

Wigneshweraraj SR, Nechaev S, Severinov K and Buck M.

The β subunit residues 186-433 and 436-445 are

commonly used by σ 54 and σ 70 RNA polymerase

holoenzyme for open promoter complex formation. J

Mol Biol. 319: 1067-1083, 2002.


Zakharova N, Bass A, Arsenieva E, Nikiforov V and

Severinov K. (submitted for publication). Mutations in

and monoclonal antibody binding to evolutionary

hypervariable region of E. coli RNA polymerase β'

subunit inhibit transcript cleavage and transcript

elongation. J Biol Chem. 2003.

Zillig W, Zechel K, Rabussay D, Schachner M, Sethi VS,

Palm P, Heil A and Seifert W. On the role of different

subunits of DNA-dependenet RNA polymerase from

E. coli in the transcription process. Cold Spring Harbor

Symp Quant Biol. 35: 47-58, 1970.

Zuber P, Healy J, Carter HL, Cutting S, Moran CP and

Losick R. Mutation changing the specificity of an RNA

polymerase σ factor. J Mol Biol. 206: 605-614, 1989.

E. coli RNA polymerase 77


Journal of Cell and Molecular Biology 2: 79-83, 2003.

Haliç University, Printed in Turkey.

Homocysteine potentiates esterase-induced contraction on rat aorta iinn

vviittrroo: A risk factor for atherosclerosis

Fatmah Bibi Housnah Gurib and Anwar Hussein Subratty*

Department of Health and Medical Sciences, Faculty of Science, University of Mauritius, Reduit,

Mauritius (*author for correspondence)

Received 24 December 2002; Accepted 5 June 2003

Abstract

The present study was designed to investigate the effect of homocysteine on N-α-l-tosyl arginine methyl esterinduced

contraction of rat thoracic aorta in vitro. Aorta was isolated and mounted in an organ bath containing Krebs

solution. The effect of TAME was tested on intact aortic strips by challenging with different concentrations of TAME

(10 -15 -10 -1 M). The effects of homocysteine were also investigated on TAME-induced contraction on rat aorta strips.

Our data showed that the concentration dependent TAME-induced contraction were more prominent when rat aortic

strips were pre-incubated with homocysteine.

KKeeyy wwoorrddss:: homocystine, TAME, endothelial dysfunction, nitric oxide, atherosclerosis

Homosisteinin s›çan aortunda esteraz ile artt›r›lan iinn vviittrroo kas›lmay› güçlendirmesi:

Athereosclerosis oluflumu için bir risk faktörü

Özet

Bu çal›flmada, homosistein etkisi alt›nda N-α-l-tosil arginin metil ester (TAME) ile artt›r›lm›fl s›çan thoracic

aortunda in vitro kas›lmay› nas›l etkiledi¤i araflt›r›lm›flt›r. Aort izole edilerek Krebs çözeltisi içeren bir organ

banyosuna yerlefltirilmifltir. De¤iflik konsantrasyonlarda (10 -15 -10 -1 M) TAME çözeltisinde kesik aort stripleri test

edilmifltir. TAME etki ettirilmifl bu striplerde ayr›ca homosistein etkisi araflt›r›lm›flt›r. Bulunan sonuçlara göre

konsantrasyona ba¤l› TAME etkili kas›lman›n önceden homosistein ile inkübe edilen striplerde görülen kas›lmaya

göre daha göze çarp›c› oldu¤u saptanm›flt›r.

AAnnaahhttaarr ssöözzccüükklleerr:: Homosistein, TAME, endotelyal fonksiyon bozuklu¤u, nitrik oksit, atherosclerosis

Introduction

Homocysteine is derived from the metabolism of the

essential amino acid methionine which is found in

greatest concentrations in animal proteins. In humans,

dietary animal protein results in a transient rise in

plasma homocysteine levels, which peaks at 8 hours

and may persist for up to 24 hours (Guttormsen et al.,

1994). Homocysteine concentrations are determined

by genetic and nutritional factors. Mutations in the

genes for enzymes involved in homocysteine

metabolism as well as deficiencies of vitamins B6, B12

and folic acid, are associated with

hyperhomocysteinemia (McCully, 1996).

Evidence indicates that chronic

hyperhomocysteinemia is an independent risk factor of

atherosclerosis (Stuhlinger et al., 2001). Chronic

elevations of plasma homocysteine concentration has

79


80 Fatmah Bibi Housnah Gurib and Anwar Hussein Subratty

been shown to be associated with stroke, peripheral

vascular disease, and myocardial infarction (Boushey

et al., 1995). Like hypercholesterolaemia,

hyperhomocysteinemia is caused bye both genetic and

dietary factors and contributes to vascular disease in a

large number of patients (Stampfer and Malinow,

1995). Elevated homocysteine concentrations are

found in almost one-third of all patients with

atherosclerosis and levels only 12 % above the upper

limit of normal (15 µmol/L) are associated with a 3fold

increase in the risk of acute myocardial infarction

(Nygard et al., 1997).

Increasing evidence suggests that the effect of

elevated homocysteine are mediated through

endothelium dysfunction. In children with

cystathionine-β-synthase deficiency and severe

hyperhomocysteinemia and in adults with moderate

hyperhomocysteinemia, chronically elevated

homocysteine concentrations are associated with

impaired endothelium-dependent vasodilatation

(Tawakol et al., 1997). In primates, elevated

homocysteine concentrations following methionineenriched

diet for 4 weeks are associated with vascular

endothelium dysfunction (Lentz et al., 1996).

Similarly, in normal human subjects, high-dose oral

methionine (100mg/kg), which increases plasma

homocysteine by 3-to 4-old, is accompanied by a

reciprocal fall in brachial artery flow-mediated dilation

(Chambers et al., 1998).

It has been shown that homocysteine would reduce

the biological activity of nitric oxide (NO) in

endothelial cells (Staler et al., 1993). Reduced NO

could behave as a trigger mechanism in the

development and progression of tissue injury because

NO modulates vascular tone, inhibits platelet

activation, and attenuates adherence to endothelial

(Furchgott and Zadwadski, 1980).

N-α-tosyl L-arginine methyl ester (TAME)esterase

has been demonstrated to be an enzyme,

involved in the sequence of events leading to the

activation of the kinin-kallikrein system (Subratty &

Moonsamy, 1998). Furthermore, it has been reported

that TAME-esterase induced contraction in toad ileal

strips in vitro was mediated via a NO-cyclic GMP

pathway (Subratty & Hossany, 1999).

TAME-esterase has also been described to be a

possible new cardiovascular risk factor among

smokers (Subratty et al., 2000) and evidence tend to

show that TAME-esterase activity has a significant

contribution to contraction of smooth muscles in vitro.

We have recently reported that calcium antagonists

improve TAME-esterase ‘blunted’ endothelialdependent

relaxation in vitro (Gurib and Subratty,

2001). We have also reported that there is a possible

contribution of tyrosine kinases during TAME-esterase

induced contractions in aorta (Gurib and Subratty,

2002). It has also been reported that TAME-esterase

induced contraction in rat aorta in vitro is mediated

through release of prostaglandin(s) as a result of

endothelial dysfunction (Gurib and Subratty, 2002).

Based on the previous work, the present study was

undertaken to investigate the possible effects of

homocysteine on TAME-induced contractions of rat

aorta in vitro.

Materials and Methods

Experimental design and surgical procedure

Adult male Sprague-Dawley rats weighing between

50-100 g were killed by a severe blow to the head. The

thoracic aorta was isolated and mounted in an organ

bath containing 25 ml of Krebs-Henseleit buffer (mM)

NaCI 118; KCI 4.7; CaCI2.H2O 2.5; MgSO47H2O 1.2;

NaHCO3 25; EDTA 9.7 mg/I and glucose (2 g/I). The

pH of the buffer was adjusted to 7.45.

To prevent blood clot formation in the dissected

aorta, 2 ml of heparin (5,000 IU/l) was added to the

buffer in a petri dish. The tissue bath solution was

maintained at 37°C in a thermostated water bath. A gas

mixture of 95% and 5% CO2 was continuously

bubbled in the buffer. Two stainless steel hooks were

inserted into the aorta lumen, one was fixed while the

other was connected to transducer. Contractile

responses were recorded via an isometric force

transducer connected to a multipen recorder

(Rikadenki Model R50; Japan). Aorta strips were

allowed to equilibrate in the medium for 20 min and

maintained under an optimal tension of 2 g.

Effects of N-a-tosyl L-arginine methyl ester TAME on

rat aorta strips

For studying the effects of TAME (Sigma, UK) on

aortic strips, a 10 -1 M stock solution of TAME was

prepared by dissolving 0.38 g of TAME in 10 ml of

distilled water. Aliquots of this stock solution were used

to make serial dilutions ranging from 10 -1 to 10 -15 M.

Twelve aorta strips from 12 rats were used in this


series of experiments. Each strip was challenged with

100 µl of TAME, beginning with the lowest

concentration (10 -15 M).

The procedure was repeated in order of increasing

concentration to establish a cumulative dose-responce

curve after stabilization of the strip following any

contractile responses or after 3 minutes in case of no

observed changes. The final concentration in the bath

was 4 x 10 -3 M.

Effect of homocysteine on TAME-induced contraction

on rat aortic strips

In addition to TAME, the effects of homocysteine were

also studied on seven rat aorta strips. In this series of

experiments, aortic strips were pre-incubated with 500

µl homocysteine (5.0 mmol/l) for 20 minutes before

being challenged with 100 µl of TAME, beginning

with the lowest concentration (10 -15 M).

Control experiments

In each series of experiments, a parallel control strip

was included from the same aorta. Control aorta strips

were challenged with 100 µl of buffer solution added

at 3-minute intervals during the experiments

investigating the effect of TAME. For tests strips 100

ml of different dilutions of TAME were added at

increasing concentrations and the contractile

responses, if any, were recorded as described above.

Data analysis

All results are expressed as mean ± SE. Manipulation

and statistical analyses of the data were done using

Excel software. Statistical differences between means

were assessed by one way analysis of variance

(ANOVA). Two-way ANOVA was used for analyzing

the difference between two concentration-response

curves. Once a significant difference was detected,

Student’s t-test was used to determine the

homocysteine concentration at which significant

differences were present. P values less than 0.05 were

considered as statistically significant.

Results

The results of the present study demonstrated that

TAME had a dose-dependent effect causing

Homocystein effect on rat aorta 81

contractions of aorta strips in vitro (Figure 1A). There

was no effect by the application of buffer solution

alone (Figure 1C). Contractile responses obtained with

TAME following preincubation of strips homocysteine

were presented in Figure 1B. The same set of data was

expressed as a percentage of maximal response in

TAME

TAME

TAME

n = 12

TAME

n = 7

Figure 1: Tracings showing effects of homocysteine on

TAME-induced contractions on rat aorta in vitro.

Figure 2. EC50 (effective concentration of

pharmacological agent producing half the maximal

response) was calculated by linear interpolation for

n Homocysteine + TAME

♦ TAME

Distilled water / buffer solution

TAME concentration (10g)

Figure 2: Effects of homocysteine on TAME-induced contractions

on rat aorta in vitro.


82 Fatmah Bibi Housnah Gurib and Anwar Hussein Subratty

Table 1: Mean EC 50 values for endothelium-intact aorta strips treated with homocysteine. In statistical evaluation of EC 50 data,

each of two contraction data was against its own control data.

Chemical agents No. of strips Mean EC 50 values (M) P-value

TAME (10 -15 – 10 -1 M) 12 4.0 x 10 -14 < 0.05

TAME (10 -15 – 10 -1 M) pre-incubated

with Homocysteine (5.0 mmol/l) 7 2.8 x 10 -14 > 0.05

each pharmacological agent applied on the rat aorta

strips, using respective cumulative concentration

curves (Table 1). The results showed that TAMEesterase

induced contractions were amplified (p>0.05),

when rat intact aorta strips were pre-incubated with

homocysteine.

Discussion

Hyperhomocysteinemia has long been recognized as

an independent risk factor for the pathogenesis of

arteriosclerosis and venous thrombosis (Schlaich et al.,

2000). Hyperhomocysteinemia, characterized by

accelerated atherosclerosis, is believed to induce

endothelial cell injury and promote atherothrombosis

by supporting the generation of hydrogen peroxide

(Upchurch et al., 1997). Despite the clinical

significance of homocysteine, however, the molecular

mechanisms of homocysteine-induced arteriosclerosis

have not been completely elucidated (Lee & Wang,

1999). It is well established that endothelial

dysfunction, which is characterized by loss of control

in NO, production occurs in a variety of cardiovascular

disorders. A decrease in level of NO leads to a cascade

of pathophysiologic events resulting in neutrophil

infiltration into inflamed tissue a process which is

regulated by adhesion molecules (Lefer and Lefer,

1996).

Stuhlinger et al. (2001) have reported that when

endothelial or nonvascular cells were exposed to DLhomocysteine

or to its precursor L-methionine,

dimethylarginine (ADMA) concentration in the cell

culture medium increased in a dose- and timedependent

fashion. This effect was associated

with the reduced activity of dimethylarginine

dimethylaminohydrolase (DDAH), the enzyme that

degrades ADMA. Furthermore, homocysteine-induced

accumulation of ADMA was associated with reduced

NO synthesis in endothelial cells and segments of pig

aorta. The antioxidant pyrrolidine dithiocarbamate

preserved DDAH activity and reduced ADMA

accumulation. Moreover, homocysteine reduced the

activity of recombinant human DDAH in a cell free

system in a dose-dependent manner by direct

interaction between homocysteine and DDAH. It was

concluded that homocysteine post-translationally

inhibits enzymatic activity of newly synthesized and

causing ADMA to accumulate and inhibit nitric oxide

synthesis.

The findings from the present study showed that

homocysteine amplified the effect on TAME-esterase

induced contractile response on rat aorta. One

plausible mechanism for these observed effects could

be due to homocysteine causing endothelial

dysfunction by down regulating the synthesis of NO.

From the literature it is evident that homocysteinemia

inhibits the important role of NO in preventing

endothelial dysfunction (Schlaich et al., 2000). This is

probably due to the reduced endothelial basal NO

release in the arterial aviculture, and may explain the

known effect of homocysteine to impair endothelialmediated

NO-dependent vasodilatation.

In conclusion, our study further demonstrates the

potential role of TAME in the cascade of events

leading to entothelial dysfunction. However further

work is needed to establish a relationship between the

cardiovascular effects of NO, plasma homocysteine

levels and cardiovascular diseases, our data are

complementary to the more traditional NO-induced

stimulation of guanylate cycles.

References

Boushey CJ, Beresford SA, Omenn GS and Motulsky AG.

A quantitative assessment of plasma homocysteine as a

risk factor for vascular disease. Probable benefits of

increasing folic acid intakes. JAMA, 274: 1049-57, 1995.

Furchgott RF and Zawadski JV. The obligatory role of

endothelial cells in the relaxation of arterial smooth

muscle. Nature. 288: 373-376, 1980.

Gurib FBH and Subratty AH. Involvement of Kinin

Kallikrein and Prostaglandin in TAME-esterase induced


contractions of rat aorta in vitro. Univ of Maur Res Journ.

(in pres) 2002.

Gurib FBH and Subratty AH. Calcium antagonists improve

TAME-esterase blunted endothelial-dependent

relaxation. Indian J Pharmacol. 33: 384-385, 2001.

Guttormsen AB, Refsum H and Ueland PM. The interaction

between nitrous oxide and cobalamin. Biochemical

effects and clinical consequences. Acta Anaesthesiol

Scand. 38: 753-6, 1994.

Lee ME and Wang H. Homocysteine and hypomethylation.

A novel link to vascular disease. Trends Cardiovasc Med.

9: 49-54, 1999.

Lefer AM and Lefer DJ. The role of nitric oxide and cell

adhesion molecules on the microcirculation in ischemiareperfusion.

Cardiovasc Res. 32: 743-752, 1996.

Lentz SR, Sobey CG, Piegors DJ, Bhopatkar MY, Faraci FM,

Malinow MR and Heistad DD. Vascular dysfunction in

monkeys with diet-induced hyperhomocyst(e)inemia. J

Clin Invest. 98: 1 24-9, 1996.

McCully KS. Homocysteine and vascular disease. Nat Med.

2: 386-9,1996.

Nygard O, Nordrehaug JE, Refsum H, Ueland PM,

Farstad M and Vollset SE. Plasma homocysteine levels

and mortality in patients with coronary artery disease.

Engl J Med. 337: 4 230-6, 1997.

Stamler JS, Osborne JA, Jaraki O, Rabbani LE, Mullins M,

Singel D and Loscalzo J. Adverse vascular effects of

homocysteine are modulated by endothelial-derived

relaxing factor and related oxides of nitrogen. J Clin

Invest. 91: 1 308-18, 1993.

Schlaich MP, John S, Jacobi J, Lackner KJ and Schmieder

RE. Mildly elevated homocysteine concentrations

impair endothelial dependent vasodilation in

hypercholesterolemic patients. Atheroscler. 153: 383-9,

2000.

Stampfer MJ and Malinow MR. Can lowering homocysteine

levels reduce cardiovascular risk? N Engl J Med. 332:

328-9, 1995.

Stuhlinger MC, Tsao PS, Her JH, Kimoto M, Balint RF and

Cooke JP. Homocysteine impairs the nitric oxide

synthase pathway: Role of asymmetric dimethylarginine.

Circulation. 21: 2569-75, 2001.

Subratty AH and Hossany R. Does TAME induced

contraction involve an endothelial dependent nitric

oxide-cyclic GMP mediated pathway? Indian J Exp Biol.

37: 406, 1999.

Subratty AH and Moonsamy J. Is TAME a potent constrictor

of non-airway smooth muscles? Indian J Exp Biol.

36: 618, 1998.

Subratty AH, Mooradun A and Jowaheer V. TAME-esterase.

A new cardiovascular risk factor in smokers. Indian J

Exp Biol. 38: 610, 2000.

Tawakol A, Omland T, Gerhard M, Wu JT and Creager MA.

Hyperhomocyst(e)inemia is associated with impaired

endothelial-dependent vasodilation in humans.

Circulation. 95: 5 1119-21, 1997.

Homocystein effect on rat aorta 83


Journal of Cell and Molecular Biology 2: 85-89, 2003.

Haliç University, Printed in Turkey.

Cytoembryological studies on PPaaeeoonniiaa ppeerreeggrriinnaa L.

Recep Öztürk1 , Meral Ünal2 *

1 ‹stanbul University, Department of Biology, Botany Section, Süleymaniye 34460, ‹stanbul-Turkey;

2Marmara University, Department of Biology, Göztepe 81040, ‹stanbul-Turkey

(*author for correspondence)

Received 29 April 2003; Accepted 18 June 2003

Abstract

The anther wall of Paeonia peregrina consists of epidermis, endothecium, 1-2 middle layers and a single-layered

tapetum. In the secretory tapetum, multinucleate cells are resulted from acytokinetic mitosis and restitution nuclei

are frequently produced in these cells. Meiotic division is regular in the pollen mother cells (PMC) of diploid and

tetraploid forms. The ovules are anatropous, bitegmic and crassinucellate. The female gametophyte is of Polygonum

type. In the mature embryo sac the egg cell, two synergids a secondary nucleus and three antipodal cells were

observed. Leucoplasts were seen in all developmental phases up to mature embryo sac.

KKeeyy wwoorrddss:: Paeonia peregrina, tapetum, microsporogenesis, megasporogenesis, megagametogenesis

PPaaeeoonniiaa ppeerreeggrriinnaa’da sitoembriyolojik çal›flmalar

Özet

Paeonia peregrina’n›n anter çeperi epidermis, endotesyum, ara tabaka ve tapetumdan oluflur. Salg› tipi tapetumda

çok nukleuslu hücreler asitokinetik mitoz bölünmeler sonucunda oluflur ve bu hücrelerde s›k s›k restitüsyon

nukleuslar›na rastlan›r. Diploid ve tetraploid bitkilerin polen ana hücrelerinde mayoz bölünme düzenlidir. Tohum

taslaklar› anatrop, bitegmik ve krassinusellat tiptedir. Difli gametofit geliflimi Polygonum tiptedir. Olgun embriyo

kesesinde bir yumurta hücresi, iki sinergit, sekonder nukleus ve üç antipot hücresi bulunur. Olgun embriyo kesesine

kadar tüm geliflim evrelerinde niflasta tanelerine rastlanm›flt›r.

AAnnaahhttaarr ssöözzccüükklleerr:: Paeonia peregrina, tapetum, mikrosporogenez, megasporogenez, megagametogenez

Introduction

According to the classical systems of angiosperm

classification the genus Paeonia belongs to the family

Ranunculaceae. However, many authors have pointed

out that Paeonia is distinct from Ranunculaceae in

respect to their vascular anatomy, floral anatomy,

basic chromosome number and size and morphology

of the chromosomes (Bhojwani and Bhatnagar, 1979).

Embryological data also support the view of

removing Paeonia from Ranunculaceae to a separate

family, Paeoniacea. Bhojwani and Bhatnagar (1979)

made a table showing the embryological differences

between the two families. The most peculiar

embryological feature of this genus is its embryogeny

(Yakovlev and Yoffe, 1957, 1965; Murgai, 1959; Cave

et al., 1961; Wang, 1985, Dane and Olgun, 1997).

The aim of this paper is to study the embryological

features of Paeonia peregrina L.

85


86 Recep Öztürk and Meral Ünal

Material and Methods

The flower buds were collected from the natural

population at ‹spartakule and were fixed in aceticalcohol

(1:3). Sections were cut at 7-10 µm and stained

with Regaurd’s haematoxylin. The aceto-orsein squash

method was also used in the study of

microsporogenesis. KI+I was applied to the sections to

identify starch grains in the embryo sacs.

Results

The anther wall and the divisions in the tapetal cells

The anther of Paeonia peregrina is tetrasporangiate.

The anther wall consists of epidermis, endothecium,

middle layers and tapetum. The single –layered

epidermal cells become stretched during the

maturation of anther. Endothecial cells do not show

any thickenings. The cells of middle layers are crushed

during the development. The cells of secretory tapetum

are large and have dense cytoplasm. They are

uninucleate in the early stage of development but the

mitotic division is not followed by cytokinesis (Figure

1 a-e). As a result, two-nucleated tapetal cells are

Figure 1: Nuclear divisions in the tapetal cells of Paeonia

peregrina. a. Prophase; b. Metaphase; c. Anaphase; d.

Telophase; e. Binucleate cell; f.-h. Irregular anaphase; i.

Prophase in binucleate cell; j. Metaphase in binucleate cell ;

k,l. Anaphase in binucleate cell; m,n. Fournucleate cell; o,

Nuclear fusion.

produced. Diploid chromosome number is 10 but

tapetal cells with 20 chromosomes were also seen. The

second mitotic division was observed resulting fournucleated

cells (Figure 1 i-n). Very often, due to the

fusion of nuclei become polyploid (Figure 1 o). The

following irregularities in the first and second mitoses

were noticed: lagging chromosomes, chromosome

bridges, fragments, unequal distribution of

chromosomes (Figure 1 f-h, k,l). Nuclear fusions were

frequently occurred resulting to the occurrence of

large, irregular shaped giant nuclei with many

nucleoli. Multinucleate tapetum degenerates at the

time of separation of the microspores from each other.

Microsporogenesis

In the majority of pollen mother cells (PMC) the

meiotic division (microsporogenesis) undergoes

normally (Figure 2 a-p). 5 bivalents were observed at

diakinesis (Figure 2 g). Cytokinesis is of simultaneous

type. The microspore tetrads are of isobilateral and

tetrahedral type. The microspores in tetrad are

surrounded by a thick callose wall.

In some PMCs 10 bivalents were seen at diakinesis

(Figure 3 a). Thus, both diploid and tetraploid

chromosome number were counted. Some meiotic

irregularities such as chromosome bridges, univalents

and multivalents, lagging chromosomes, fragments

were recorded in less number of PMCs of the diploid

and tetraploid forms (Figure 3 b,c ) As a result of

irregularities pentads and hexads were detected.

Megasporogenesis and Megagametogenesis

The ovary has 16 anatropous ovules placed along the

ventral wall. The ovules are bitegmic and

crassinucellate.

The archesporium is multicellular. The 3 or 4

archesporial cells differentiate in nucellus and become

prominent with their dense cytoplasm and large nuclei

(Figure 4 a). One of them functions as a megaspore

mother cell (MMC) and undergoes meiosis

(megasporogenesis). Before meiosis the size of MMC

increases. 5 bivalents were counted at metaphase I

(n=5) (Figure l b). Cytokinesis is of the successive

type. As a result of first meiosis, the two dyad cells are

produced; the chalazal one is larger than the other

(Figure 4 c) The megaspore tetrad is linear or T-shaped

(Figure 4 d)

The chalazal megaspore is functional while the


Figure 2: Meiosis in the pollen mother cells of diploid

Paeonia peregrina. a; Interphase; b,c. Leptotene; d.

Zygotene; e. Pachytene, f. Diplotene; g. Diakinesis; h.

Metaphase I; i. Anaphase I; j. Telophase I k. Interkinesis; l.

Metaphase II; m. Anaphase II; n,o. Telophase II; p.

Microspore tetrad.

Figure 3: Meiosis in the pollen mother cells of tetraploid

Paeonia peregrina. a. Diakinesis with multivalents; b,c.

Metaphase I with univalents and multivalents: d. Anaphase I.

other three degenerate (Figure 4 e). 2, 4 and 8

nucleated embryo sacs are formed by three successive

mitotic divisions (megagametogenesis) (Figure 4 f-h).

In mature embryo sac the egg apparatus consists of the

egg cell and the two synergids. These three cells show

a triangular arrangement.

The two polar nuclei fuse in the early stage to form

a secondary nucleus. In mature embryo sac the

secondary nucleus lies near the egg apparatus (Figure

4 i). There are 3 antipodals on the chalazal pole. They

are equal in size and have dense cytoplasm. The

antipodals are the smallest cells of the embryo sac.

Vascular bundles were observed in nusellus and

integuments in P. peregrina. The presence of vascular

bundles in integuments is a primitive character. Starch

grains were also noticed during the developmental

stages, more conspicuous in mature embryo sac.

Discussion

Embryology of Paeonia peregrina 87

In the tapetal cells one or two mitotic divisions

resulting in two to four nuclei which may fuse to form

large polyploid nucleus were recorded in the anthers of

P. peregrina. Acytokinetic mitosis and restitutional

mitosis are the responsible mechanisms in the

formation of polyploid tapetal cells in this species.

Marquardt et al. (1968) reported 16n ploidy level

resulting from three mitoses in the tapetum of P.

tenuifolia.

In the present study, both diploid (2n=2x=10) and

tetraploid (2n=4x=20) PMCs were observed. Meiosis

is regular in most of PMCs but irregular in some

others.The regular meiosis results in the formation of

isobilateral microspore tetrads. The regular meiotic

division in PMCs were observed in some species of

Paeonia; P. anomola, P. wittmanniana, P. californica,

P. brownii (Stebbins and Ellerton, 1939, Walters,

1956). Meiotic irregularities were recorded in the

PMCs of the species P. suffriticosa, P. mlokosewitschi,

P. tenuifolia (Hicks and Stebbins,1934; Dane, 1997).

Dane (1997) observed some meiotic irregularities such

as univalents, tetravalents, chromosome bridges,

laggards in diploid and tetraploid forms of P.

tenuifolia.

The ovules are anatropous, bitegmic and

crassinucellate in P. peregrina. Archesporium is

multicellular, but only one of them differentiates as a

megaspore mother cell. Megasporogenesis is regular.

The development of embryo sac conforms to the


88 Recep Öztürk and Meral Ünal

Figure 4: Megasporogenesis and megagametogenesis in Paeonia peregrina. a. Archesporial cells; b. Megaspore mother cell in

Metaphase I; c.Dyad cells; d. 4 megaspores; e. Functional megaspore; f. 2-nucleate embryo sac.

Polygonum type. Multicellular archesporium is a

family character of the Paeoniaceae. Dane (1997)

observed the multicellular archesporium developed

into multiple megaspore mother cells giving rise to

banches of megaspore tetrads in P. tenuifolia. As a

result, 2 or 3 embryo sacs were seen in one ovule in

this species.

In all developmental stages up to mature embryo


sac leucoplasts were seen in P. peregrina. MMC,

megaspore tetrad, functional megaspore contain starch

grains. They were also found in 2-, 4-, 8-nucleated

embryo sac and mature embryo sac. Starch grains were

located in the outer integument cells, the integumentary

tapetum, the nucellus, around secondary nucleus and in

mature embryo sac of P. tenuifolia (Dane, 1997) but

only in the integument tapetum of P. anomala

(Yakovlev, 1957). Willemse and Went (1984) reported

that the embryo sacs in the majority of angiosperms

lack starch grains but it is abundant in some species.

Vascular bundles which are rarely found were

determined in the nucellar tissue in P. peregrina and

these bundles were observed to be tracheids.

Acknowledgement

We are greatly indebted and thankfull to Prof. Dr. Jale

Tören for providing the necessary research facilities,

useful suggestions and encouragement and for her

invaluable contribution to us.

References

Bhojwani SS and Bhatnagar SP. The embryology of

Angiosperms. Vikas Publishing House PVT, LTD, New

Delhi, Bombay. 1979.

Cave MS, Arnott JH and Cook SA. Embryogeny in the

California Paeonies with reference to their taxonomic

position. Amer Jour Bot. 48: 397-404, 1961.

Dane F. Cytological and Cytoembryological studies on

Paeonia tenuifolia L. Tr J of Botany. 21: 291-3033,

1997.

Dane F and Olgun G. The embryogeny of Paeonia tenuifolia

(Paeoniaceae). Bocconea. 5:557-562, 1997.

Hicks GS and Stebbins GL. Meiosis in some species and a

hybrid of Paeonia. Amer Jour Bot. 21: 228-240, 1934.

Marquardt H, Barth OM and Rahden U von.

Zytophotometrishe und elektronenmikroskopische

Beobachtungen über die tapetumzelleni in den antheren

von Paeonia tenuifolia. Protoplasma. 65: 407-421, 1968.

Murgai P. The development of the embryo in Paeonia -a

reinvestigation-. Phytomorphology. 9: 275-277, 1959.

Stebbins GLJ and Ellerton E. Structural hybridity in Paeonia

californica and Paeonia brownie. Genet. 38: 1-33, 1939.

Wang FX. The early development of embryo and endosperm

of Paeonia lactiflora. Acta Bot Sin. 27: 7-12, 1985.

Walters JL. Spontaneous meiotic chromosome break in

natural population of Paeonia californica. Amer Jour

Bot. 43: 342-353, 1956.

Embryology of Paeonia peregrina 89

Willemse MT and Went JL van. The female gametophyte. In:

Embryology of Angiosperms. Johri BM (Ed). Springer

Verlag Berlin, Heidelberg. 159-196, 1984.

Yakovlev MS and Yoffe MD. On some peculiar features in

the embryogeny of Paeonia L. Phytomorphology. 7: 74-

87, 1957.

Yakovlev MS and Yoffe MD. Embriologija nekotoryh

predstavitelej roda Paeonia. In: Morfologija cvetka i

reproduktivnyj process u pokrytosemennnyh rastenij.

Yakovlev MS (Ed). Moskva, Leningrad. 140-176, 1965.


Journal of Cell and Molecular Biology 2: 91-97, 2003.

Haliç University, Printed in Turkey.

Immunogenicity and specificity of SSaallmmoonneellllaa ttyypphhiimmuurriiuumm outer

membrane antigens

Nefle Ak›fl 1 *, Osman Sayhan 2 , Ali Karaçavufl 3 and Kurtulufl Töreci 3

1 Haliç University, Faculty of Arts and Sciences, Department of Molecular Biology and Genetics, 34280,

F›nd›kzade, Istanbul, Turkey; 2 Marmara University, Faculty of Medicine, Department of Basic Medical

Sciences, Istanbul, Turkey; 3 University of Istanbul, ‹stanbul Faculty of Medicine, Department of Basic

Medical Sciences, Istanbul, Turkey (*author for correspondence)

Received 21 May 2003; Accepted 26 June 2003

Abstract

S. typhimurium was acetone dried, negative stained, and its outer membrane was evaluated by transmission electron

microscopy. White Albino rabbits were immunized against acetone derived S. typhimurium, and the immune sera

were collected. Outer membrane antigens were extracted from acetone dried and veronal buffer treated S.

typhimurium, S. typhi, S. paratyphi-B, E. coli, and S. flexineri. S. typhimurium extract was further fractionated.

Immunogenicity and cross reactivity of each antigenic fraction were evaluated by ELISA and immunodiffusion

using the immune sera and commercially available or in-house product sera. Molecular sizes of proteins in fractions

were evaluated using SDS-PAGE. A S. typhimurium specific 70 kDa antigen was detected. However, highest

antigenicity was detected in 36-43 kDa common protein fraction. These antigenic fractions should have utility in

medical microbial immunology.

KKeeyy wwoorrddss:: Salmonella typhimurium, bacterial antigens, rabbit antibodies, outer membrane, Enterobacteriaceae

SSaallmmoonneellllaa ttyypphhiimmuurriiuumm d›fl membran antijenlerinin immunojenite ve özgüllü¤ü

Özet

S. typhimurium asetonla kurutuldu, negatif boyand› ve transmisyon elektron mikroskopu ile d›fl membran›

incelendi. Sa¤l›kl› beyaz Albino tavflanlar asetonla kurutulmufl bakteri ile ba¤›fl›kland› ve ba¤›fl›k serum topland›.

Asetonla kurutulmufl S. typhimurium, S. typhi, S. paratyphi-B, E. coli, ve S. flexineri'den veronal tamponla muamele

edilerek d›fl membran antijenleri ekstrakte edildi. S. typhimurium ekstrat› daha ileri fraksiyonlara ayr›ld›.

Haz›rlanan ba¤›fl›k serum ve firmalardan al›nan veya laboratuvarda haz›rlanm›fl ba¤›fl›k serumlar kullan›larak her

bir antijenik fraksiyonun immunojenite ve çapraz reaksiyonu ELISA ve immunodifuzyon yöntemleriyle incelendi.

Fraksiyonlardaki proteinlerin moleküler büyüklü¤ü SDS-PAGE ile tayin edildi. S. typhimurium'a özgü 70 kDa

büyüklü¤ünde bir antijen saptand›. Di¤er yandan, 36-43 kDa ortak proteinler tafl›yan fraksiyon en yüksek

antijeniteyi gösterdi. Bu fraksiyonlar t›bbi mikrobiyal immunolojide kullan›m alan› bulabilecektir.

AAnnaahhttaarr ssöözzccüükklleerr:: Salmonella typhimurium, bakteriyal antijenler, tavflan antikorlar›, d›fl membran,

Enterobacteriaceae

91


92 Nefle Ak›fl et al.

Introduction

The incidence of S. typhimurium infections in recent

years in Turkey was increased, and high child

mortality due to gastrointestinal infections was

detected (‹çgen et al., 2002; Willke et al., 2002). Since

common laboratory techniques does not support the

needs of specialists for efficient treatment and follow

up the cases, more accurate or complementary

approaches started to evolve to fulfill the needs

(Jongerius-Gortemaker et al., 2002; Bacarese-

Hamilton et al., 2002; Prager et al., 2003). One of them

is to detect specific bacterial markers in patient

materials using immunochemical techniques (Ak›fl et

al., 1990; Skov et al., 2002). Although, the members of

Enterobacteriaceae carry rich antigens and

immunogens, and they are relatively easy to derive,

isolation of specific components has been more

difficult. Herein we report S. typhmurium specific and

group specific protein markers, which should have

utility in medical microbial immunology and

diagnostic laboratories. The methodologies used to

isolate membrane antigens should be useful for

investigation in multiple areas.

Material and Methods

Microorganisms

Salmonella typhimurium (KEUN-886 (B) S1-28-8 "K

form"); Salmonella paratyphi-B (KUEN 1256 S1-21-

47 "H form"); Salmonella typhi (KUEN1253 (B) S1-

27-74 "H form"); E. coli (ATCC 11,229, 21. 4. 1988)

and Shigella flexineri (KUEN 1286 (B) S5-4-12, tip

2a) were provided from KUKENS (University of

Istanbul, Istanbul, Turkey).

Electron microscopy

S. typhimurium was acetone-dried and examined as

described (Ak›fl et al., 1989).

Rabbits and sera

Female Wistar Albino and Black rabbits were

purchased from Institute for Experimental Medical

Research (University of ‹stanbul, Turkey). All animal

experimentation described in the manuscript were

conducted in accord with the Departmental Guide for

the Care and Use of Laboratory Animals. In

preparation of immune sera acetone dried S.

typhimurium was sterilized using ethylene oxide and

0.2 mg of bacteria was suspended in 0.4 ml volume of

serum physiologic. Rabbits were inoculated

intradermal 17 times in four sessions with 2-6 weeks

intervals. 2-6 immunization were applied in each

session with 4-6 d intervals. Complete Freund’s

adjuvant was used in the first sessions’ immunizations.

The sites for injections were neck in the first and

fourth sessions, and ventral lumber area in the others.

Sera were collected with 3 d intervals after each

session was completed, and were tested for specific

antibody by immunodiffusion. The highest serum titers

were detected after 4 th session, and blood was collected

from the ear vein of the animals. Sera were separated

and aliquots were kept at -20°C (Çetin et al., 1973).

Control sera were in-house product immune sera

against S. typhi (O- and H-sera), S. paratyphi-B (Oand

H-sera), S. typhimurium (H-sera), Proteus OX2 and

-OX19 which were prepared according to standard

protocols (Kaufman et al., 1960) and polyclonal sera

against E. coli 1-4 and against Shigella (mixture of S.

flexineri and S. dysenterie) were purchased

(Wellcome, Oxford, UK).

Immunodiffusion

2.5 mm thick 0.85% agarose gel (buffer: 0.24% TRIS;

0.15% Glycine; 0.29% EDTA; 0.01% NaN3 (Merck,

San Diego, CA); pH 7.2) on slides were used to open

holes 0.2 mm in diameter at 4 mm distance by sucking.

Sera and fra-TXY for all bacteries were loaded in holes

in proximity. The slides were incubated in humidity

chamber for 3 d, first day at 37°C, second day at room

temperature, and last day in refrigerator. The

precipitated immune complex bands in between holes

were visualized under luminated light, and pictures

were taken. None of the reagents were diluted except

that LPS (Difco, Bruxelles, Belgium) was used at 5

mg/ml concentration based on the results of

optimization study. Some results were confirmed by

counterimmunodiffusion. In these experiments, 0.9%

indubiose agarose gel (buffer: 1.05% sodium

barburate; 0.17% barbital; 0.15% calcium lactate; pH

8.6) was used and preceded as described above.

Antigens were loaded in holes at cathode pole and the

electrophoresis was completed under 22 mA.


Antigens

Bacteria were cultured and acetone dried, and

antigenic fractions (Fra-TXY per bacteria) were

extracted from five bacteria as described (Ak›fl et al.,

1990). Briefly, Fra-T was obtained as supernatant by

solubilization of bacteria in veronal buffer; Fra-X and

Fra-Y were obtained by solubilization of the pellet at

either at 95°C for or pH 2, respectively. Pooling of the

three extracts were named as Fra-TXY. Addition to

Fra-T, Fra-X, Fra-Y fractions for S. typhimurium, its

outer membrane antigens were further fractionated as

described (Barber et al., 1966). Protein-polysaccharide

complexes were obtained from the supernatant of

Fra-T following protein precipitation using

trichloroaceticacid at 10% final concentration, and

dialyzed against distillated water (Fra-A). The pellet

was solved at natural pH and then divided for further

extraction. One aliquot was precipitated and the

supernatant was obtained (Fra-B) as described above.

Another aliquot was precipitated using ammonium

sulphate and proceeded as described above (Fra-C).

Polysaccharide solution was obtained from Fra-A

following hydrolysis of the fraction by boiling at

100°C for 90 min at final 5N acetic acid solution. The

supernatant was dialyzed and kept (Fra-PS,

polysaccharide fraction). Acid soluble flagella fraction

was obtained by treatment of acetone-dried bacteria

with HCl solution at pH 2 for 30 min. The supernatant

was removed following ultra centrifugation at 100.000

g (Fra-D). The pellet was incubated with PBS and the

supernatant was kept (Fra-ACF). A bacterial

polysaccharide was purchased (Difco) and water

solution was used in experiments (5 mg/ml).

SDS-PAGE

The samples were run on polyacrylamide gels in

different concentrations varied between 7-14% in

TRIS-buffer as described (Ak›fl et al., 1990).

ELISA

Protein concentration of antigen solutions was

determined by spectroscopy prior to the enzymelinked-immuno-sorbent-assay

(ELISA). Optimal

antigen concentration and sera dilution were used in

the tests. Microplates (Greiner, Frickenhausen,

Germany) were coated with 100 ml antigen solution in

PBS with overnight incubation in refrigerator, blocked

Antibody response to S. typhimurium outer membrane antigens 93

with 2% BSA at 37°C for 2 hours, and stained with

immunoglobulin in sera at 37°C for 1 h. After

conjugation with anti-rabbit antibody (Institute

Table 1: Protein content of fractions.

Fraction mg/g a mg/g b

Fra-X 12

Fra-Y 4.1

Fra-A 18.89

Fra-B 2.55

Fra-C 8.36

Fra-D 50.62

Fra-ACF 29.96

Fra-T 48

Fra-TXY (S. typhi) 42.4

Fra-TXY (S. paratyphi-B) 83.76

Fra-TXY (E. coli) 55.2

Fra-TXY (S. flexineri) 105.6

a Protein (mg)/1 g acetone-dried bacteria

b UV absorbing material (mg)/1 g acetone-dried bacteria

Results for protein content of each antigenic fraction were

presented. The fraction names were enlisted at the left, and protein

contets of the fractions in two different expressions were indicated

at right colums.

Pasteur, Paris, France) and washing using 1% Tween

20-PBS, color development was measured at 20 min.

All assays were performed with multiple aliquots

and confirmed. For ELISA a minimum of a two-fold

difference was judged to be positive.

Results

In electron microscopic examination of bacteria, the

outer membrane has been found compact in 95% of the

bacteria, and pillus and flagellar components were

visible. During the immunization and at the time of

blood drawn the animals appeared grossly normal.

Animals developed high titer serum antibodies as

detected by immunodiffusion (data not shown).

The protein yield of different bacteria and different

fractions varied as shown in Table 1. In cross

examination of immune sera against Fra-TXY from

various bacteria and commercial polysaccharide, a

strong cross reaction was detected against S.

paratyphi-B antigenic fraction by immunodiffusion.

Relatively weak cross reaction between S.

typhimurium antigen and sera against S. typhi, and vice


94 Nefle Ak›fl et al.

Figure 1: Protein band profiles in SDS-PAGE. The fractions were run in parallel and the band profiles were

shown in picture. The sizes were indicated on profiles. The name of each fraction was indicated at the

bottom of each profile.

versa, were observed (data not shown). The results

were confirmed by ELISA. In further investigation by

ELISA, cross reactions between commercial sera

against S. flexineri and Fra-TXY antigen for S.

typhimurium and S. paratyphi-B were observed.

14 antigenic fractions were evaluated by SDS-

PAGE (Figure 1). Shifting of bands at 30 kDa to 31-42

kDa was observed when the denaturation condition

was shifted to 100°C for 5 min from 60°C for 10 min.

This was obvious particularly for Fra-C suggests this

Figure 2: ELISA results obtained with S. typhimurium fractions. Each column presents OD values obtained

with each fraction, and the name of each fraction was indicated at the bottom of each column. OD values

were differentially dotted on diagram, and each dot indicates a different sera. See Figure 3 for description

of the sera.


Figure 3: ELISA results obtained with Fra-TXY fractions

for five bacteria. Each column presents OD values obtained

with each fraction, and the name of each fraction was

indicated at the bottom of each column. OD values were

differentially dotted on diagram, and each dot indicates a

different sera. Description of the sera was shown at the

bottom of the diagram.

fraction contained outer membrane proteins, porins. A

shift was also observed in Fra-X from 25-29 kDa to

33-35 kDa. Presence of polysaccharides in Fra-A was

examined by hydrolysis. Band at 36 kDa shifted to 38-

42 kDa after hydrolysis suggested protein was tightly

bound to polysaccharide (Arockiasamy et al., 2000).

A 70 kDa S. typhimurium specific antigen was

detected. 36-43 kDa proteins were common among

five bacteria examined and could be responsible from

cross reactions. 38 kDa band in Fra-PS; 36 kDa band

in Fra-A; 34 and 51 kDa in Fra-D, 41-41 kDa in Fra-

B, and 43 kDa in Fra-C were clear bands suggests they

can be used in ELISA experiments. 51 kDa in Fra-D

would be expected to be phase-1 pillus antigen (Prager

et al., 2003).

ELISA test was optimized to obtain around 1.000

optic density (OD) for positive and around 0.100 OD

for negative control. In this sensitivity level it was

expected that high dilution antigen and sera would

not allow non-specific cross reactions. In ELISA,

polysaccharide fraction gave highest cross reaction

with other bacterial sera suggests family specific

epitopes dominate in this chemical structure (Figure 2).

Immune sera showed dramatically higher affinity

Antibody response to S. typhimurium outer membrane antigens 95

to the S. typhimurium fractions than other sera

did, suggests some antigenic epitopes were lost

during preparation of O- and H-antigens (Figure 3).

Conservation of original epitopes on acetone-dried

bacteria was shown elsewhere (Ak›fl et al., 1990). Fra-

A showed highest antigenicity in ELISA whereas Fra-

D the lowest one.

Discussion

There are many advantages of using immunochemical

techniques in diagnostic microbiology laboratories,

particularly in evaluation of post-vaccination sera in

big size. The fractions described here should be

particularly useful for examination of Salmonella vs.

unrelated bacteria. Isolation of pure antigenic fractions

has been more difficult than for polysaccharide

fractions. We used multiple step isolation strategy to

obtain pure fractions, and the methods should be

reproducible by others. Although not all of the isolation

methods were rigorously tested, the overall approach

was relatively simple, straightforward and yielded

unequivocal results. It will be briefly summarized.

Nine fractions were extracted successfully from S.

typhimurium. In SDS-PAGE a 70 kDa specific antigen

was detected in Fra-T, however it was not isolated in

this study. 70 kDa protein seems a candidate antigen

for using in immunochemical assays. Developing and

using immunochemical techniques detecting

diagnostic markers for gastroenteritidis, such as

ELISA, have diagnostic value particularly in countries

where gastroenteritis is endemic (Sood et al., 2002;

Purwaningsih et al., 2002; Handojol et al., 2000). For

this reason, description and isolation of species and

serovar specific antigens is important. Many have been

described and were successfully used by others (Veling

et al., 2001; Solano et al., 2000; Arockiasamy et al.,

2000).

S. typhimurium showed high cross reactivity to S.

paratyphi-B, and relatively lower to S. typhi. 36-43

kDa antigens were common antigens to the bacteria

tested, and could have diagnostic value in detection of

family specific antigens in patient material. Fra-A

showed pure band and highest score in ELISA

suggested this fraction is a good candidate for this

purpose. Others described cross reaction between

Salmonella species (Valdirieso-Garcia et al., 2001),

and used family specific antigens in ELISA tests

(Jauho et al., 2000; Wang et al., 2000).


96 Nefle Ak›fl et al.

Strong immune response against acetone-dried

bacteria, similar to natural infection, was shown

previously using Fra-TXY as antigen in ELISA (Ak›fl

et al., 1990). In this study, fractions extracted from

acetone-dried S. typhimurium under mild conditions

were also found displaying strong antigenicity

suggests antigens in these fractions conserved original

epitopes during the extraction procedure.

Shift in bacterial wall protein bands in SDS-PAGE,

particularly those among polysaccharide linked outer

membrane proteins has been described (Sood et al.,

2002). When the chemical bound in proteinpolysaccharide

complex was strong, hydrolysis of

polysaccharide residues would not change the location

of bands in SDS-PAGE. On the other hand, outer

membrane proteins would easily shift. Both of the cases

were evident in different fractions of this study. The

protein content of a cell could also affect the chemical

construction and rigidity of outer membrane. Among

the bacteria, which were used in this study, dramatic

difference was detected in protein content. This could

be due to intrinsic properties of each bacterium.

Antibodies in homolog and heterolog O- and Hsera,

which were prepared according to classical

Grubel-Widal test, did fail to show high affinity to

antigens, whereas in-house product hyperimmune sera

reacted strongly. It is possible that some antigenic

epitopes might be modified during the preparation of

O- and H-antigens and might cause the weak reaction.

On the other hand, S. typhimurium H-sera showed

selective affinity to protein fractions as expected,

however S. paratyphi-B O- and H-sera was not

selective for protein or polysaccharide fractions. The

value of classical Widal test has been discussed

(Willke et al., 2002), and cross species polysaccharides

in O-antigen were described (Wang et al., 2000). In

these studies and others, Widal test was described as a

supportive test rather than being an accurate test due to

low sensitivity and specifity, however description of

antigens according to Kaufmann-White schema has

been continued for years (Popoff et al., 2001). As a

results, new techniques have replaced the place of

Widal test today. Nevertheless additional studies will

be required to determine whether these sera are

specific to pure protein or polysaccharide antigens.

Acknowledgement

We thank Selim Badur; KUKENS, The Electron

Microscopy Facility, Animal Husbandry and Ultra

Centrifugation Facility of the Istanbul School of

Medicine for their technical assistance. This study was

supported by self-renewed divisional fund of the

Microbiology Division.

References

Ak›fl N, Andl C, Zhou D. A mixed hemadsorption assay for

detection of cell surface binding anti-tumor antibodies in

human sera. J Immunol Methods. 261: 119-127, 2002.

Ak›fl N, Badur S. Investigation of specific antibodies in

enterobacterial infections using enzyme-immuno-assay.

‹nfeksiyon Dergisi. 4: 533-539, 1990.

Ak›fl N, Karaçavufl A, Badur S. Asetonla kurutulmufl

Salmonella typhimurium’un elektron mikroskobunda

de¤ifliminin incelenmesi. In: IX. Ulusal

Elektronmikroskopi Kongresi. Kongre kurulu (Ed).

‹stanbul T›p Fakültesi, ‹stanbul. 269-270, 1989.

Arockiasamy A, Krishnaswamy S. Purification of integral

outer-membrane protein OmpC, a surface antigen from

Salmonella typhi for structure-function studies: a method

applicable to enterobacterial major outer-membrane

protein. Anal Biochem. 15 283(1): 64-70, 2000.

Barber C, Vladoianu IR, Dimache GH. Contrubutions to the

study of Salmonella: Imunological specifity of proteins

separated from S. typhi. Immunology. 11:287-294, 1966.

Bacarese-Hamilton T, Bistoni F, Crisanti A. Protein

microarrays: From diagnosis to whole proteome scale

analysis of the immune response against pathogenic

microorganisms. Biotechniques. Supp, 24-29, 2002.

Çetin ET. Seroloji deneyleri. In: Genel ve Pratik

Mikrobiyoloji. 3 rd Ed. Sermet Matbaas›, ‹stanbul. 359-

360, 1973.

Handojo I, Dewi R. The diagnostic value of the ELISA-Th

test for the detection of typhoid fever in children.

Souteast Asian J Trop Med Public Health. 31: 702-707,

2000.

‹çgen B, Gürakan GC, Özcengiz G. Characterization of local

isolates of Enterobacteriaceae from Turkey. Microbiol

Res. 157: 133-238, 2002.

Jauho ES, Boas U, Wiuff C, Wredstrom K, Pedersen B,

Andresen LO, Heegaard PM, Jakobsen MH. New

technology for regiospecific covalent coupling of

polysaccharide antigens in ELISA for serologic

detection. J Immunol Methods. 242: 133-143, 2000.

Jongerius-Gortemaler BG, Gioverde RL, van Knagen

F, Berguerff AA. Surface plasmon resonance

(BIOCORE) detection of serum antibodies against

Salmonella enteritidis and S. typhimurium. J

Immunol Methods. 266: 33-44, 2002

Kaufmann F, Ludertz O, Stierlin H, Westphal O. Zur

Immunochemie der O-antigene von Enterobacteriaceae.

Zbl Bact Hyg. 1 st Abt Orig A178: 442, 1960


Popoff MY, Beckemuhl J, Brenner FW, Gheesling LL.

Supplement 2000 (no 44) to the Kauffmann-White

scheme. Res Microbiol. 152: 907-909, 2001.

Prager R, Strutz U, Fruth A, Tschape H. Subtyping of

pathogenic E. coli strains using flagellar (H)-antigens:

Serotyping versus fliC polymorphism. Int J Med

Microbiol. 292: 477-486, 2003

Purwaningsih S, Handojol I, Prihatin I, Probohoesodo Y.

Diagnostic value of dot-enzyme-immunoassay test to

detect outer membrane protein antigen in sera of patients

with thyroid fever. Southern Asian J Trop Med Public

Health. 32: 507-512, 2002.

Skov MN, Feld NC, Carsten B, Madsen M. The serologic

response to Salmonella enteritidis and S. typhimurium in

experimentally infected chickens, followed by an

indirect lipopolysaccharide enzyme-linked

immunoserbent-assay and bacteriologic examinations

through a one-year period. Avian Dis. 46: 265-273, 2002.

Solano C, Galindo J, Sesma B, Alvarez M, Solsona MJ,

Gamazo C. ELISA with a Salmonella enteritidis antigen

for differentating infected from vaccinated poultry. Vet

Res. 31: 491-497, 2000.

Sood A, Kaur IR. Electrophoretic analysis of S. typhi and

other bacteria. Indian J Med Sci. 56: 265-269, 2002.

Valdirieso-Garcia A, Riche E, Abubakar O, Maddell TE,

Brooks BW. A double antibody sandwich enzyme-linked

immunosorbent assay for the detection of Salmonella

using biotinylated monoclonal antibodies. J Food Prot.

64: 1166-1171, 2001.

Veling J, van Zijderveld FG, van Zijderveld-van Bemmel

AM, Schukken YH, Barkema HW. Evaluation of two

enzyme-linked immunosorbent assay for detecting

Salmonella enterica subsp. enterica serovar Dublin

antibodies in bulk milk. Clin Diagn Lab Immunol.

8: 1049-1055, 2001.

Wang L, Reeves PR. The Escherichia coli O111 and

Salmonella enterica O35 gene clusters: Gene clusters

encoding the same colitose-containing O antigen are

highly conserved. J Bacteriol. 182 (18): 5256-5261,

2000.

Willke A, Ergönül O, Bayar B. Widal test in diagnosis of

typhoid fever in Turkey. Clin Diagn Lab Immunol.

9: 938-941, 2002.

Antibody response to S. typhimurium outer membrane antigens 97


Journal of Cell and Molecular Biology 2: 99-103, 2003.

Haliç University, Printed in Turkey.

Polymerase chain reaction is a good diagnostic tool for MMyyccoobbaacctteerriiuumm

ttuubbeerrccuulloossiiss in urine samples

Serdar Arisan, N. Cem Sönmez*, Ömer Onur Çak›r, Erbil Ergenekon

fiiflli Etfal Research and Training Hospital, 1 st Urology Clinics, fiiflli, Istanbul, Turkey (*author for

correspondence)

Received 3 June 2003; Accepted 27 June 2003

Abstract

Tuberculosis is a major public health problem in developing countries. Tuberculosis of the urinary tract is not

uncommon in all over world, and it continues to be an important clinical problem, mainly because of its nonspecific

clinical presentation and variable radiographic appearance, which often mimic other pathologic lesions. Detection of

suspects bacterial population; direct smears can be used but they are often negative and do not differentiate

tuberculosis from nontuberculous mycobacterium. Culture, which is more sensitive, takes 6 to 8 weeks because of

the slow growth rate of mycobacterium. Polymerase chain reaction (PCR) is a technique used to amplify extremely

small amounts of a specific genomic sequence rapidly. The presence of an extremely small number of bacteria can

thus be detected within 24 to 48 hours. Therefore PCR is a promising tool for rapid detection of urinary tract

tuberculosis in urine samples.

KKeeyy wwoorrddss:: PCR, Mycobacterium tuberculosis, diagnosis

‹drar örneklerinde MMyyccoobbaacctteerriiuumm ttuubbeerrccuulloossiiss tan›s› için polimeraz zincir

reaksiyonunun kullan›m›

Özet

Tuberküloz geliflmekte olan ülkelerde oldukça önemli bir sa¤l›k sorunudur. Uriner sistemde meydana gelen

tuberküloz ise pek yayg›n de¤ildir. Ancak spesifik olmayan klinik bulgular› yüzünden ve de¤iflken radyolojik

görünümü ve di¤er patolojik lezyonlar› taklit etti¤i için önemli bir klinik problem halini alm›flt›r. fiüpheli bakterial

populasyonlar›n tan›s› do¤rudan smear alarak gerçeklefltirilebilmektedir. Ancak al›nan sonuçlar s›kl›kla negatif veya

tüberküloz tipi olmayan mikobakterileri tan›mlamaktad›r. Polimeraz zincir reaksiyonu (PCR) küçük miktarlarda

genomlar› spesifik genom dizilerinde ço¤altmay› hedefleyen bir araç olarak kullan›lmaktad›r. ‹drarda çok az

miktarda bulunan bakteriler 24-48 saat içerisinde bu yolla teflhis edilebilmektedir. Bundan ötürü, PCR uriner sistem

tuberküloz tan›s›nda oldukça umut vadeden bir araçt›r.

AAnnaahhttaarr ssöözzccüükklleerr:: PCR, Mycobacterium tuberculosis, tan›

Introduction

Tuberculosis is a major public health problem in

developing countries. Despite the decline in incidence

seen in the 1980s, resurgence has occurred and 8% to

15% of patients with pulmonary tuberculosis go on to

develop urinary tract tuberculosis (UTB). The

estimated involvement is 400 per 100,000 populations.

99


100 Serdar Arisan et al.

(Styblo, 1980; Peterson, 1994). Tuberculosis of the

urinary tract is not uncommon in all over world, and it

continues to be an important clinical problem, mainly

because of its nonspecific clinical presentation and

variable radiographic appearance, which often mimic

other pathologic lesions (Figure 1).

The earliest urographic change occurs in the minor

calices, and caliceal dilation is the first sign. However,

it is often so minimal that the diagnosis can be

extremely difficult (Ney and Friendenberg, 1981). A

slight loss of sharpness of the calix, which represents

mucosal edema, is another subtle initial sign (Elkin,

1990). As the disease progresses, the calix appears

moth-eaten or feathery (Taylor, 1939). An instance of

calcification in renal tuberculosis was found in 7% of

cases by Crenshaw (1930) and in 14.4% of cases by

Gow (1965). Urethral strictures have been reported in

about 10% to 56% of patients, and bladder

involvement is found in one third of cases of UTB

(Royalance et al., 1970).

Early diagnosis of the disease allows

administration of antitubercular treatment at a stage at

which it may be curative. However, more often than

not, the diagnosis is delayed because of a delay in

presentation and in making a definitive diagnosis, with

the consequence that a number of patients present with

nonfunctioning kidneys, ureteral stricture, and

shrunken bladders. These changes can be avoided if

the diagnosis is made early and treated effectively.

The diagnostic criterion for UTB is the isolation of

Mycobacterium tuberculosis from urine. This is not

easy to achieve, as the discharge of organisms into the

urine is sporadic and, more importantly, involves few

organisms (Gow et al., 1984; Gow, 1992). Direct

smears are often negative and do not differentiate

tuberculosis from nontuberculous mycobacterium.

Culture, which is more sensitive, takes 6 to 8 weeks

because of the slow growth rate of mycobacterium

(Manjunanth et al., 1991). This study conducted by the

fiiflli Etfal Research and Training Hospital in 29

patients suspected of having urinary tract tuberculosis,

the incidence of a positive urine culture for

Mycobacterium tuberculosis among histological

positive cases was 15% (Colabawalla, 1990). In the

literature, the majority of molecular TB detection

protocols is based on the PCR and have been

developed "in house". PCR is a technique used to

amplify extremely small amounts of a specific

genomic sequence rapidly. The presence of an

extremely small number of bacteria can thus be

detected within 24 to 48 hours (Noel-Brisson et al.,

1989; 1991). The high sensitivity of PCR is

particularly useful in paucibacillary situations such as

non-pulmonary tuberculosis. The aims of this study

were to reveal the importance of PCR analysis for

rapid and sharp results for detection of Mycobacterium

tuberculosis in urine samples.

Figure 1: A Ziehl-Neelsen acid-fast stain of some sputum

positive for tuberculosis. Arrows indicate mycobacteria,

which appear as red rods.

Materials and methods

Patients

In the first step of study, urine samples were collected

from 29 patients with suspected urinary tract

tuberculosis between 2000-2002 years in fiiflli Etfal

Research and Training Hospital 1 st Urology Clinics.

These urine specimens used for both routine culture

and PCR techniques.

DNA extraction

100 ml urine samples were used in 2 hours after taken

and concentrated to 2 ml. Bacterial DNA extraction

and PCR for amplification of the Mycobacterium

tuberculosis complex (Yamaguchi et al., 1989) were

carried out according to a previously published

protocol (Seth et al., 1996). DNA was extracted with

QIAamp DNA Mini Kit (Qiagen).

PCR analysis

The urine specimens were prepared for PCR

amplification according to the following protocol.


Sediments were washed three times with an equal

volume of Tris-EDTA buffer (10 mM Tris-HCL, 1 mM

EDTA; pH: 8.0) at 5000-x g for 5 min. The resulting

pellet was resuspended in 0.25 mL of Tris-EDTA

buffer and then boiled for 20 min. After centrifuged at

5000-x g for 5 min, 5 µl of the supernatant was

analyzed by PCR in a 50 µl reaction mixture. The PCR

reaction mixture contained 50 mM KCL, 10 mM

Tris-HCl, pH: 8.3, 1.5 mM MgCl2, 200 µM

deoxynucleoside triphosphates (dNTP), 2.5 U Taq

polymerase (Boehringer Mannheim, Germany) and

0.5 µM (each) of the primers. The primer sets used to

amplify the 123-bp IS 6110 gene fragment consisted of

TBC1 (CCT GCG AGC GTA GGC GTC GG) and

TBC2 (CTC GTC CAG GGC CGC TTC GG)

(Eisenach et al., 1991; Bennedsen et al., 1996 and

Desjardin et al., 1998-Qiagen). The reaction mixture

was subjected to 30 cycles of amplification (95°C, 30

sec; 68°C, 30 sec; 72°C, 30 sec) followed by a 5 min

extension at 72°C (Thermocycler; Techne). 15 µl of

the amplification products were analyzed by

electrophoresis in an ethidium bromide stained 2%

agarose gel (Sambrock et al., 1989).

Results

Total of 29 urine specimens were collected from 15

women and 14 men. Their average ages for women 35

and for men were 42. 9 patient were positive for

tuberculosis by both test, while remain of the study

group were negative. 4 urine samples could not

evaluated by PCR due to the presence of inhibitory

substances of nature. The bladder biopsy was

diagnostic of tuberculosis in 5 (20%) of the 24 patients

biopsied. Fine needle aspiration cytological

examination was done in 1 patient with epididymal

swelling and was suggestive of tuberculosis. The

urinary PCR was falsely positive in 1 (4%) patients

(Table 1).

Mycobacterium tuberculosis organisms were found

to be excreted intermittently in the urine of infected

patients. 7 men patients were showing testicular

Table 1: The comparision of PCR and routine culture results

Culture Staining PCR + PCR -

11 positive 11 positive 10 1

14 negative 14 negative 2 14

swelling, perianal sinus or genital ulcer. 2 women and

3 men patients had secondary coliform infection. 1

woman and 5 men had gross hematuria. Microscopic

hematuria was present 9 men and 11 women. All

patients had frequent urination, initially during the

day; later, at night. Dysuria, frank pain, suprapubic

pain, blood or pus in the urine presented in all patients.

Cystourethroscopy was performed in 29 patients. The

bladder revealed evidence of chronic cystitis in 24

patients. After detailed consultation other fungal

infection for 2 patient, pyonephrosis for 1 patient,

calyceal diverticula for 2 patients and bladder cancer

for 1 patient were detected. Complete blood cell count,

sedimentation rate, serum chemistry were investigated

for all suspect patients. The erythrocyte sedimentation

rate was elevated in 16 patients. Chest and spine

radiographs showed negative results for all patients.

Two patients had abnormal renal parameters. Kidney,

urethra and bladder radiographs reveal calcifications in

the kidney for 4 patients. Calcifications were

intraluminal. Bladder calcification was not obtained

from radiographic results. There was no HIV positive

patient. All positive patients were consulted by

infection consultants and treated with INH, Rifampin,

pyrazinamide and ethambutol for first 2 months and

rifampin and INH treatment were continued for

resistance to either agent exists.

Discussion

Mycobacterium diagnosis by PCR 101

Tuberculosis is still rampant in underprivileged

societies and continues to take a heavy toll. It can

affect any organ system in the body and produce

protean manifestations. UTB occurs in 8% to 20% of

patients with pulmonary tuberculosis, with a

prevalence of 400 per 100,000 population (Styblo,

1980; Peterson, 1994). The most common presenting

symptoms in patients with UTB are irritative voiding

symptoms and hematuria, with the reported incidence

in world literature at around 60% and 50%,

respectively (Narayan, 1982). The speed and

simplicity of amplification techniques can greatly

assist TB diagnosis, therapy and epidemiology. This

improvement will continue with the rapid advances in

the tools available in molecular biology. No long series

has been reported on the sensitivity and specificity of

urinary PCR in UTB. Although, PCR test has been

extensively studied and proved highly sensitive,

specific and rapid. In various studies, data show

sensitivity ranging from 87-100% (usually >90%) and

specificity from 92,2-99,8 % (usually >95%). This


102 Serdar Arisan et al.

compares to cultures (37%), bladder biopsies (47%)

and intravenous pyelogram (IVP) examinations. Along

an accurate clinical assessment, PCR is the best

available tool to avoid delay in starting treatment

because it takes only 6 hours (Davies et al., 1999).

False-negative findings may result from the presence

of inhibitors; non-homogeneous distribution of

bacteria in the specimen so that the fraction tested does

not contain mycobacterium; or low numbers of

mycobacterium in the specimen, which decreases the

probability of the presence of organisms in the fraction

analyzed by PCR (Missirliu et al., 1996; Kolk et al.,

1992). False-positive results with PCR may be caused

by contamination due to the presence of amplicons or

Mycobacterium tuberculosis complex bacilli or DNA

(van Vollenhoven et al., 1996).

Most PCR assays employed for the diagnosis of

tuberculosis do not distinguish between infections

caused by Mycobacterium bovis and Mycobacterium

tuberculosis. The species-specific PCR assays

developed for the differentiation of Mycobacterium

tuberculosis and Mycobacterium bovis have been

invalidated due to false negative results owing to

absence of specific target sequences such as mtp40 in

some Mycobacterium tuberculosis strains (Gillespie

and McHugh, 1997 and Cousins, 1992).

In conclusion, in this study PCR results were same

with routine culture results. Therefore, PCR is a rapid

tool to detect Mycobacterium tuberculosis in urine

samples for UTB patients.

Acknowledgement

We thank to E. Damla Büyüktunçer for editorial

comments and advisory critics.

References

Bennedsen J, Thomsen VO and Pfyffer GE. Utility of PCR

in diagnosis pulmonary tuberculosis. J Clin Microbiol.

34: 1407-1411, 1996.

Brisson-Noel A, Gicquel B and Lecossier D. Rapid diagnosis

of tuberculosis by amplification of mycobacterial DNA

in clinical samples. Lancet. 2: 1069-1071, 1989.

Brisson-Noel A. Aznar C and Churean C. Diagnosis of

tuberculosis by DNA amplification in clinical practice

evaluation. Lancet. 338: 364-366, 1991.

Colabawalla BN. Reflections on urogential tuberculosis.

Indian J Urol. 6: 51-59, 1990.

Cousins DV, Wilton SD and Francis BR. Use of polymerase

chain reaction for rapid diagnosis of tuberculosis. J Clin

Microbiol. 30 : 255-258, 1992.

Crenshaw JL. Renal tuberculosis with calcification. J Urol.

23: 515-533, 1930.

Davies AP, Newport LE, Billington OJ, and Gillespie SH.

Length of time to laboratory diagnosis of

Mycobacterium tuberculosis infection: comparison of inhouse

methods with reference laboratory results. Journal

of Infection. 39: 205-208, 1999.

Desjardin LE, Chen Y and Perkins MD. Comparison of the

ABI 7700 (TaqMan) system and competitive PCR for

quantification of IS1660 DNA in sputum during

treatment of tuberculosis. J Clin Microbiol. 36:

1964-1968, 1998.

Eisenach KD, Sifford MD and Cave MD. Detection of

Mycobacterium tuberculosis in sputum samples using a

polymerase chain reaction. Am Rev Respir. 144:

1160-1163, 1991.

Elkin M. Urogenital tuberculosis. In: Clinical Urography

Pollack HM (Ed). WB Saunders, Philadelphia. 1020-

1052, 1990.

Gillespie SH and McHugh TD. Monitoring the therapy of

pulmonary tuberculosis by nested polymerase chain

reaction. Journal of Infection. 35: 324-325, 1997.

Gow JG. Renal calcification in genito-urinary tuberculosis.

Br J Surg. 52: 283-288, 1965.

Gow JG and Barbosa S. Genito-urinary tuberculosis-a study

of 1117 cases over a period of 34 years. Br J Urol. 56:

449-455, 1984.

Gow JG. Genito urinary tuberculosis. In: Campbell's

Urology. 6th ed. Walsh PC, Retik AB and Stamey TA

(Ed). WB Saunders, Philadelphia. 951-981, 1992.

Kolk AH, Schuitema ARJ and Kuijper S. Detection of

Mycobacterium tuberculosis in clinical samples by using

polymerase chain reaction and a non-radioactive

detection system. J Clin Microbiol. 30: 2567-2575, 1992.

Manjunath N, Shankar P and Rajan L. Evaluation of a

polymerase chain reaction for the diagnosis of

tuberculosis. Tubercle. 72: 21-27, 1991.

Missirliu A, Gasman D and Vogt B. Genito-urinary

tuberculosis: Rapid diagnosis using the polymerase chain

reaction. Eur Urol. 30: 523-524, 1996.

Narayan AS. Overview of renal tuberculosis. Urology. 19:

231-236, 1982.

Ney C and Friedenberg RM. Tuberculosis of the kidney. In:

Radiographic Atlas of the Genito-urinary System.

Ney C and Friedenberg RM (Ed). JB Lippincott,

Philadelphia. 373-409, 1981.

Peterson JC. Tuberculosis of the kidney. In: Renal

Pathology, 2nd ed. Tisher CC and Brenner BM (Ed).

JB Lippincott, Philadelphia. 895-905, 1994.

Royalance J, Penry B and Davies R. Radiology in

management of urinary tract tuberculosis. Br J Urol. 42:

679-687, 1970.

Sambrook J, Fritsch EF, and Maniatis T. Molecular Cloning:

A Laboratory Manual, 2nd ed. Cold Spring


Harbor Laboratory, New York. 1989.

Seth P, Ahuja GK and Bhanu NV. Evaluation of polymerase

chain reaction for rapid diagnosis of clinically suspected

tuberculous meningitis. Tubercle Lung Dis. 77: 353-357,

1996.

Styblo K. Recent advances in epidemiological research in

tuberculosis. Adv Tubercular Res. 20: 1-63, 1980.

Taylor HK. Renal tuberculosis: pathogenesis and roentgen

findings. AJR Am J Roentgenol. 42: 700-708, 1939.

Van VollenHoven P, Heyns CF and de Beer PM. Polymerase

chain reaction in the diagnosis of urinary tract

tuberculosis. Urol Res. 24: 107-111, 1996.

Yamaguchi R, Matsuo K and Yamazaki A. Cloning

characterization of the gene for immunogenic protein

MPB 64 of Mycobacterium bovis BCG. Infect Immun.

57: 283-288, 1989.

Mycobacterium diagnosis by PCR 103


Journal of Cell and Molecular Biology 2: 105-111, 2003.

Haliç University, Printed in Turkey.

Cytological investigations in some important tree species of Rajasthan

VI. Radiation induced chromosome aberrations in AAnnooggeeiissssuuss ppeenndduullaa

and AA.. llaattiiffoolliiaa

Arun Kumar 1 , S. Ramo Rao 1 * and N.S. Shekhawat 2

1 Cytogenetics and Molecular Biology Laboratory, Department of Botany, J. N. V. University, Jodhpur-

342001, India; 2 Biotechnology Unit, Department of Botany, J. N. V. University, Jodhpur-342001, India

(*author for correspondence)

Received 11 June 2003; Accepted 10 July 2003

Abstract

Irradiation of air-dried seeds of Anogeissus pendula and A. latifolia, the two multipurpose hard wood trees, produces

various categories of chromosome aberrations in the root tips. Both chromosome and chromatid types of aberrations

were observed in different cells and at times even with in the same cell. A high degree of polymorphism was

exhibited by the resultant chromosomes after irradiation. The observations clearly showed distinct variations with

respect to percentage values of abnormal cells; altered chromosome; chromosome-interchanges; chromosomeintrachanges;

chromatid-intrachanges and unclassified types. The efficacy of the ionizing radiations in inducing

various categories of chromosome aberrations and their possible role in genetic improvement through mutation

breeding has been discussed in detail.

KKeeyy wwoorrddss:: Anogeissus pendula, A. latifolia, γ-rays, induced aberrations, chromosome/chromatid types

Rajastan IV için baz› önemli a¤aç türlerinde sitolojik araflt›rmalar: AAnnooggeeiissssuuss ppeenndduullaa

ve AA.. llaattiiffoolliiaa’ da radyasyonun oluflturdu¤u kromozom aberasyonlar›

Özet

Çok özellikli sert a¤açlar olan Anogeissus pendula ve A. latifolia’n›n havada kurutulmufl tohumlar›n›n ›fl›nlanmas›

sonucunda kök uçlar›nda kromozom aberasyonlar› meydana gelmektedir. Hem kromozom hem de kromatid

tiplerinde meydana gelen bu aberasyonlar, de¤iflik hücrelerde veya ayn› zamanda ayn› hücrede gözlenebilmektedir.

Yüksek derecede polimorfizmlerin ›fl›nlama sonucunda meydana geldi¤i saptanm›flt›r. Gözlemler sonucunda, de¤iflik

oranlarda anormal hücre oluflumlar›, ayn› kromozomda ya da farkl› kromozomlar aras›nda parça de¤ifliklikleri,

kardefl kromatid de¤iflimleri ve s›n›fland›r›lamayan anormallikler saptanm›flt›r. ‹yonlaflt›r›c› radyasyonun etkisi ile,

de¤iflik kromozom aberasyonlar›n›n meydana gelmesi ve bunun ›slah mutasyonu aç›s›ndan muhtemel rolü detayl› bir

flekilde tart›fl›lm›flt›r.

AAnnaahhttaarr ssöözzccüükklleerr:: Anogeissus pendula, A. latifolia, γ-›fl›nlar›, aberasyonlar, kromozom/kromatid tipleri

Introduction

Studies on biological effects of ionizing radiations and

their utility in inducing gene and/or chromosome

105

mutations have been a topic of immense interest for

cytologists ever since Muller (1927) and Stadler

(1930) reported artificial induction of mutations in

plants and animals respectively. Although a great deal


106 Arun Kumar et al.

of efforts were made to explore the utility of ionizing

radiations for genetic improvement in a good number

of crob plants like wheat (Sears, 1956); rice (Rutger,

1983; 1991); maize (Novak, 1991) and several

horticultural species (Broertjes and Vanharten, 1978).

On contrary, forest tree species have received very

little attention in this regard. The Indian desert (Thar)

encompassing vast areas of Rajasthan province, that

include both arid and semi-arid regions, frequently

experience harsh and inhospitable conditions for both

flora and fauna. Yet it is bestowed with some of the

nature’s unique forms of phyto-diversity including

hard wood tree species such as A. pendula Edgew and

A. latifolia (Roxb. Ex DC). Wall ex Guill & Perr. of

family Combretaceae. These species are popular

among local populace as multipurpose trees yielding

fodder, timber and fuel wood, besides being an

important component of the arid ecosystem.

Sustainable utilization duly supported by genetic

improvement programs in these tree species, by and

large, are tacking. γ-rays have been effectively utilized

in genetic improvement of horticultural tree species

such as Malus sps. (apple) by Nybom and Koch (1965)

to enhance the fruit shape, size and quality. Some

earlier reports are also available on γ-ray induced

chromosome aberrations in gymnospermous tree

species viz. Picea abies, P. glauca, Pinus pinea etc.

(Tarlau, 1957; Sparrow, 1961; Bavilacqua and

Vidakavic, 1963; Rudolph, 1965; Laura, 1966; Roy et

al., 1972). Sparrow and Miksche (1961) also observed

changes in nuclear volume and DNA content following

exposure to γ-radiation in higher plants while Ganchev

(1975) used them for mutation breeding experiments

in Populus. However such studies are conspicuous by

their complete absence in tropical tree species in

general and arid zone tree species in particular. Hence

the role of induced mutations in development of novel

variant strains for achieving genetic improvement is

yet to be explored in case of these taxa. Therefore, the

present studies are the first attempt to collect, collate

and interpret the data for various categories of

chromosome aberrations induced by γ-rays in two

species of Anogeissus viz. A. pendula and A. latifolia.

Materials and Methods

Air dried seeds of A. pendula JNVU/RI 2001 and A.

latifolia BSJO 19571 were exposed to 1,3,6,9,12,15,25

and 50 kR γ-rays from a 60 Co source (dose rate: 11.8

rad/sec at room temperature) made available by

Defense laboratory, Defense Research and

Development Organization (DRDO), Govt. of India,

Jodhpur. Control (unirradiated) and the irradiated

seeds from each dose point were allowed to germinate

on most filter paper in corning petri plates kept in 25 ±

2°C in dark. About 1 cm long root tips pretreated for 3

hours with 0.025% colchicine (Himedia) were fixed in

freshly prepared 1:3 acetic ethanol solution.

Subsequently the root tips were stained in leuco-basic

fuchsin and squashed in 1% aceto-carmine.

A minimum of ten slides from as many root tips

were prepared for each dose point and only three slides

showing maximum mitotic cell division were scored

for various types of aberrations. Various categories of

aberrations were recognized as per the classification

Table 1: Effect of γ-radiation on chromosomes in root tip cells of A. pendula JNVU/R1 2001 and A. latifolia BSJO 19571.

S.No. Parameters A. pendula JNVU/R1 2001 A. latifolia BSJO 19571

Aberrations Maximum Minimum Maximum Minimum

% (dose) % (dose) % (dose) % (dose)

1. Abnormal cells 61.90 (6kR) 40.91 (12kR) 82.85 (15kR) 31.82 (1kR)

2. Aberrations/cell 1.00 (50kR) 0.41 (12kR) 1.57 (15kR) 0.36 (1kR)

3. Altered chromosomes 5.75 (50kR) 2.38 (1kR) 8.57 (15kR) 2.27 (1kR)

4. (i) Total chromosome changes 4.77 (6kR) 1.71 (12kR) 7.29 (50kR) 1.90 (1kR)

(ii) Chromosome intrachanges 4.37 (6kR) 1.19 (1kR) 6.25 (50kR) 1.52 (1kR)

(iii) Chromosome intrachanges 0.99 (50kR) 0.18 (9kR) 2.02 (15kR) 0.38 (1kR)

5. Chromatid intrachanges 1.19 (50kR) 0.56 (6kR) 1.38 (6kR) 0.38 (1kR)

6. Unclassified types 0.64 (25kR) 0.18 (3kR) 0.83 (9kR) 0.20 (3kR)


Radiation induced chromosome aberrations 107

Figure 1-18: Mitotic karyotype of Anogeissus. A. pendula JNVU/RI 2001 (1). A. latifolia BSJO 19571 (2). Various types of

aberrations in A. pendula JNVU/RI 2001 and A. latifolia BSJO 19571 at different dose points (3-18). 1kR, 3kR, 6kR,

asymmetrical incomplete distal interchanges (3-5). 9kR, dicentric chromosome (6). 12kR, centric ring and unclassified type (7).

15kR, dicentric chromosome, interstitial deletions and single minutes (8). 25kR, dicentric chromosome and single minutes (9).

50kR, asymmetrical incomplete, distal interchanges, centric ring, single and double minutes (10, 11). 1kR, terminal deletions and

dicentric chromosomes (12). 3kR, U type interstitial deletions, single minutes and acentric fragments (13). 6kR, dicentric

chromosomes and single minutes (14). 12kR, dicentric chromosomes and unclassified types (15). 15kR, dicentric chromosomes

(16). 25kR, single and double minutes, translocations, acentric fragments and unclassified types (17). 50kR, dicentric

chromosomes and acentric fragments (18).


108 Arun Kumar et al.

proposed by Savage (1975). Those types which did not

fall in to any of the categories were included under

unclassified types. The frequency of each aberration

type was expressed in terms of percentage.

Results

Normal karyotypes

The somatic chromosome number and karyotypic

details of A. pendula JNVU/RI 2001 and A. latifolia

BSJO 19571 have been detailed elsewhere (Kumar and

Rao, 2002) and illustrated for ready reference in

Figures 1-2.

Chromosome abnormalities

The data regarding various types of abnormalities is

summarized in Table 1 and illustrated in Figures 3-18.

Abnormal cells

The frequency of abnormal cells with altered

chromosome complements in A. pendula JNVU/RI

2001 ranged between 40.91 in root tip cells irradiated

with 12kR to 61.90 per cent in 6kR dose points,

whereas in A. latifolia BSJO 19571 these values

ranged between 31.82 in 1kR to 82.85 per cent in

15kR.

Aberrations / cell

In A. pendula JNVU/RI 2001 the average number of

aberrations per cell was highest (1.00) in the root tip

cells irradiated with 50kR and that of lowest (0.41) in

12kR respectively. Whereas in A. latifolia BSJO 19571

the highest (1.57) mean of aberrations per cell was

observed in 15kR and the lowest (0.36) in 1kR.

Altered chromosomes

An altered chromosome (with centromere) could be

the outcome of one, two or more aberrations. Hence

the number of aberrations might not necessarily be

equal to the number of altered chromosomes.

Therefore, the percentage frequency of altered

chromosomes is estimated that ranged from 2.38 in

1kR to 5.75 per cent in 50kR in A. pendula JNVU/RI

2001, whereas in A. latifolia BSJO 19571 the

percentage frequency of altered chromosomes ranged

from 2.27, 1kR to as much as 8.57 in 15kR dose

points.

Chromosome type aberrations

In A. pendula JNVU/RI 2001 the percentage frequency

of total chromosome changes including inter and intraarm

types ranged from 1.71 in 12kR to 4.77 at 6kR

while in A. latifolia BSJO 19571 these values ranged

from 1.90 in 1kR to 7.29 in 50kR dose points.

The percentage frequency of chromosome

interchanges (symmetrical and asymmetrical types) in

A. pendula JNVU/RI 2001 ranged from 1.19 in 1kR to

4.37 in 6kR while these values in A. latifolia BSJO

19571 ranged from 1.52 in 1kR and 6.25 per cent in

50kR.

The percentage frequency of chromosome

intrachanges (interarm, intraarm) ranged from 0.18 in

9 kR to 0.99 in 50kR dose points in A. pendula

JNVU/RI 2001. However these values in A. latifolia

BSJO 19571 were recorded as 0.38 in 1kR and a

maximum of 2.02 per cent in 15kR.

Chromatid type aberrations

This category of aberrations involve sister

(intrachanges) or non-sister (interchanges) chromatids

of chromosomes. Depending on the rejoining pattern

of lesion segments in chromatids, they may be

asymmetrical (U type) or symmetrical (X type) types.

In the present observations on both A. pendula and A.

latifolia exhibited only intrachanges whereas

interchanges and complex types were not encountered.

Hence only the chromatid intrachanges involving both

X and U types have been reported.

In terms of percentage frequency, chromatid

intrachanges in A. pendula JNVU/RI 2001 ranged

from 0.56 in 6kR to 1.19 in 50kR whereas in A.

latifolia BSJO 19571 these values were observed as

0.38 in 1kR and a maximum of 1.83 per cent in 6kR.

Unclassified type aberrations

Due to complexity and ambiguity of a few structures

of chromosomes, following breakages and fusions,

certain aberration types could not be explained, and

therefore they have been grouped under unclassified

ones. Such aberrations were observed in all the dose

points excepts in 1kR and 6kR in A. pendula JNVU/RI


2001 and the frequency of percentage unclassified

types ranged 0.18 percent in 3 kR to 0.64 percent in

25kR wherever recorded. However, in A. latifolia

BSJO 19571 the unclassified types, except in 1, 6,

12kR were observed in all does points whose

percentage frequency ranged from 0.20 percent in 3kR

to 0.83 in 9kR dose points.

Discussion

Induction of variations at an higher rate and making

use of them for the betterment of plants has been the

most preferred strategy of plant breeders. Such studies

have already been carried out in various crop plant and

horticultural species. Several mutants in crop plants

have been developed with useful characters like

enhanced yield, shortened flowering and ripening

periods, adaptability, growth habit, shattering and

shedding resistance, tolerance to drought, heat and

high salinity, diseases and pest resistance as well as

improvement in the quality of starch, protein, fats and

oils (Rutger, 1983, 1991; Novak, 1991; Broertjes and

Vanharten, 1978). However forest tree species are

different from crop plants in terms of their size,

longevity and more importantly the breeding pattern

adapted by them. In general trees species are cross

pollinated and are mostly heterozygous besides being

unisexual in some cases. Such special problems

require appropriate technology which basically relies

on the genome-size and its pattern of organization on

one hand, while their sensitivity to different types of

mutagens on the other. Hence it is not surprising that

only a handful of papers have been published dealing

with induced variations in woody taxa using physical

or chemical mutagens (Tarlau, 1957; Sparrow, 1961;

Sparrow and Miksche, 1961; Bavilacqua and

Vidakavic, 1963; Rudolph, 1965; Laura, 1966; Roy et

al., 1972).

From the present observations detailed above it is

clear that following irradiation with γ-rays the air dried

seeds of A. pendula JNVU/RI 2001 and A. latifolia

BSJO 19571, many of the structural changes of

mitotic chromosomes (chromosome interchanges,

intrachanges, chromatid interchanges and unclassified

types) are produced which could be scored with

reasonable clarity. So far, most of the reports

concerning the induced aberrations are resticted to

easy and popular materials like Tradescantia (Savage,

1975), Phlox (Verma and Raina, 1982; Rao, 1990),

Radiation induced chromosome aberrations 109

Vicia faba (Evans, 1961; Kihlman, 1975), Allium cepa

(Rieger and Michaelis, 1967; Singh et al., 1998).

However, the present studies are indicative that if not

all, at least many of the major aberration types can be

scored even in case of tropical woody tree species.

Savage (1975) has expressed a view, subsequently

supported by other workers as well (Verma and Raina,

1982; Rao, 1990), that the metabolic and physical

status of the cells at the time of irradiation directly

influence the type of aberrations. Depending upon the

status of the cell (G1 and S opposed to G2), both

chromosome and chromatid aberration are possible to

occur. The cells at G2 on irradiation are expected to

produce exclusively chromatid type of aberrations.

However, during the present investigation, not a single

cell exclusively with chromatid aberrations, has been

observed in either of the two species studied. From this

we may conclude that most of these cells at the time of

irradiation were at G1, very and few at S and none at G2

phase.

Chromosome interchanges outnumbered

intrachanges which may be due to preference of lesion

segments producing exchange events between

chromosomes rather than within the chromosome

during the jeoining process. Besides it may also result

if fewer number of chromosomes exhibit more than

one-break events. Similar observations were earlier

reported in a number of plants viz. Vicia faba (Evans,

1961) and Phlox drummondii (Verma and Raina, 1982;

Rao, 1990). An identical trend was observed in both A.

pendula and A. latifolia which are presently

investigated.

Among chromatid aberrations only intrachanges

have been encountered in both the species. In case of

chromatid intrachanges, it is relatively easy to rejoin

after one or two lesions per chromatid within a

chromosome. However, the complete absence of

complex types does indicate that the genome exhibits

highly conserved nature, not providing much leverage

for exchange of segments leading to new linkages

between gene loci.

The low frequency of unclassified types observed

at almost all dose points in both A. pendula and A.

latifolia does not carry much significance unless the

nature of their origin is ascertained.

As in case of several crop plants, forest trees

especially arid zone taxa have several specific

problems for widening their genetic base either

for environmental adaptability and/or genetic

improvement. In such cases radiation induced


110 Arun Kumar et al.

aberrations have a potential role (Kapoor, 1980) to

play for the improvement of tree species. This may

help in development of certain mutant lines/strains

which will perform well under sub-optimal conditions

and may grow even in non-conventional areas and

there by enhance their utilization in agro-forestry and

social forestry programs. Since the present

investigations confirm the possibility of inducing wide

range of chromosome variations at high doses of γradiations

in Anogeissus species, which are known to

experience some of the distinct problems such as

limited distribution, poor adaptability, ultra low

percentage of seed viability, lack of vegetative

propagation methods, die back disease and extensive

branching of trunk in A. pendula, are fit cases for

genetic improvement through mutation breeding

programs. Therefore an integrated mutation breeding

program with a high dose quantum of radiation (above

50kR) in combination with various types of chemical

mutagens (Verma and Raina, 1982; 1990) may be

attempted to bring in novel variations in the genus

Anogeissus.

Acknowledgement

The present work has been carried out with financial

support (SP/SO/A–34/95) from the Department of

Science and Technology, Government of India, New

Delhi. Thanks are due to Dr. SP. Bohra, Head,

Department of Botany, J.N.V. University, Jodhpur for

facilities, Dr. SK. Chaudhary, Defense laboratory

(DRDO), Jodhpur for use of facilities in irradiating the

seeds.

References

Broertjes C and Vanharten AM. Application of Mutation

Breeding Methods in the Improvement of Vegetatively

Propagated Crops. Elsevier Scientific Pub Co,

New York. 1978.

Bavilacqua B and Vidakovic M. Effect of gamma rays on the

chromosomes of somatic cells in Picea abies Karst. Silv

Genet. 12: 41-46, 1963.

Evans HJ. Chromatid aberrations induced by gamma

irradiations I. The structure and frequency of chromatid

interchanges in diploid and tetraploid cells of Vicia faba.

Genetics. 46: 259-275, 1961.

Ganchev P. Hybridization and experimantel mutagenesis in

some species of Populus Sect. Leuce as methods of

genetic study and selection of new form.

Gorskostopanska Nauka. 12: 3-15, 1975.

Kapoor ML. Role of radioisotopes in forestry research. Proc

2 nd Forestry Conf. Dehradun, India. 1980.

Kihlman BA. Root tips of Vicia faba for the study of the

induction of chromosomal aberrations. Mutation Res. 31:

401-402, 1975.

Kumar A and Rao SR. Cytological investigations in some

important tree Species of Rajasthan I.

Karyomorphological investigations in the genus

Anogeissus DC. Guill, Perr & A Rich. Silv Genet. 51:

100-104, 2002.

Laura MP. Influence of conditions of storage of

irradiated pine seeds on cytogenetic aberrations in

meristem cells. Citologija. 8: 558-563, 1966.

Muller HJ. Effects of X-ray radiation on genes and

chromosomes. Anat Rec. 19: 37-174, 1927.

Navok FJ. In vitro mutation system for crop improvement.

Proceedings of International Symposium on

Contribution of Mutation Breeding to Crop

Improvement. IAEA, Vienna, Austria. 1991.

Nybom N and Koch A. Induced mutations and breeding

methods in vegetatively propagated plants. The use of

induced mutations in plant breeding. Rad Bot. 5: 661-

678, 1965.

Rao SR. Cytogenetics of Phlox and Vigna. Ph. D. thesis.

University of Jodhpur, Jodhpur, India. 1990.

Rieger R and Michaelis A. Die chromosomen aberrationen.

In: Genetik-Grundlagen. Ergebnisse und Problemes in

Einzeldarstellungen. Stubbe H. (Ed). Beitrag 6 Jena

VEB Gustav Fischer, Germany. 1967.

Roy RM, Donini B and Brunori A. Biochemical and

cytological studies on developing cotyledons of Pinus

pinea following X-ray irradiation of dry seeds. Rad Bot.

12: 249-260, 1972.

Rudolph TD. The effect of gamma irradiation of pollen

on seed characteristics in white spruce. Rad Bot. 5:

185-191, 1965.

Rutger FJ. Application for induced and spontaneous

mutations in rice breeding and genetics. In:

Advances in Agronomy. Brady NC (Ed). Academic

Press, Inc., San diego CA, USA. 1983.

Rutger FJ. Mutations breeding of rice in California and

United States of America. Proceedings of International

Symposium on Contributions of Plant Mutation Breeding

to Crop Improvement. IAEA, Vienna, Austria. 1991.

Savage JRK. Radiation induced chromosomal

aberrations in the plant Tradescantia: Doseresponse

curves I. Preliminary considerations. Rad

Bot. 15: 87-140, 1975.

Singh A, Rao SR, Singh R and Chacharkar MP.

Identification and dose estimation of irradiated

onions by chromosomal studies. J Food Sci

Technol. 35: 47-50, 1998.

Stadler LJ. Some genetic effects of X-rays in plants. J Hered.

21: 3-19, 1930.


Sears ER. The transfer of leaf rust resistance from Aegilops

umbellata. Proc Brookhaven Symp Biol. 9: 1-21, 1956.

Sparrow AH. Types of ionizing radiation and their

cytogenetic effects. NAS-NRC. 891: 55-119, 1961.

Sparrow AH. and Miksche JP. Correlation of nuclear volume

and DNA content with higher plant tolerance to chronic

radiation. Science. 134: 282-283, 1961.

Taralau HW. Beitrag zur kenntnis der variabilitat der Fichte

II. Die wirkung von Gamma-Strahlung auf Picea abies.

Bot Not. 110: 442-454, 1957.

Verma RC and Raina SN. Phlox drummondii in the study of

radiation induced chromosomal aberrations. Nucleus. 25:

176-184, 1980.

Verma RC and Raina SN. NMU induced translocation and

inversion in Phlox drummondii. Cytologia. 47: 609-614,

1982.

Verma RC and Raina SN. Cytogenetics of Crotalaria IV.

Induced translocation lines in C. Juncea. Nucleus. 33:

11-14, 1990.

Radiation induced chromosome aberrations 111


Journal of Cell and Molecular Biology 2: 113-119, 2003.

Haliç University, Printed in Turkey.

Stimulation of regeneration by magnetic field in soybean (GGllyycciinnee mmaaxx

L. Merrill) tissue cultures

Çimen Atak 1 *, Özge Emiro¤lu 1 , Sema Alikamano¤lu 2 , Aytekin Rzakoulieva 3

1Haliç University, Faculty of Arts and Sciences, Department of Molecular Biology and Genetics,

‹stanbul, Turkey; 2University of ‹stanbul, Faculty of Science, Department of Biology, ‹stanbul, Turkey;

3JINR, Laboratory of Nuclear Problems, Magnetic Research Group, Dubna, Russia (*author for

correspondence)

Received 16 June 2003; Accepted 23 June 2003

Abstract

The determination of the effects of magnetic field on tissue cultures will be helpfull for the magnetic field treatments.

In this study which was begun with this purpose, the seeds belonging to J 357 soybean variety were used. The

soybean seeds were germinated in sterile conditions and cultures were initiated from the shoot tips. The explants in

petri dishes were exposed to 2.9-4.6 mT magnetic fields for 2.2, 6.6, and 19.8 seconds periods. Shoot and root

formation rate, fresh weights and chlorophyll quantities of regenerated shoots from control and treated explants were

determined. While the shoot formation was 61.91% in control group, this rate increased in all magnetic field

experiments and this rate raised up to 86.96% and 74.36% respectively in the explants which were exposed to

magnetic field at 2.2 and 6.6 seconds periods. Again, while the percentage of root formation in control was 14.29%,

this rate raised up to 26.08% and 35.90% respectively in these which were exposed to magnetic field at the same

periods. When the fresh weights were determined, the fresh weights of seedlings regenerated from treatment explants

from 6.6 seconds significantly increased in accordance with control (P


114 Çimen Atak et al.

Introduction

Nowadays, the changes determined at the living

systems which were exposed to different magnetic

field strength and periods of magnetic field (MF) and

electromagnetic field (EMF) with the lowest frequency

have also been drawn the attention of the biologists,

molecular biologists and chemists as physicists. For

recent 30 years, studies have been done concerning the

effects of magnetic field on microorganism, tissue, cell

and subcellular structures of the plants and animals

(Polk et al., 1995).

The researchers have shown that magnetic field

changed the characteristics of cell membrane, effected

the cell reproduction and caused some changes in cell

metabolism. At the same time, it was put forward that

magnetic field effected the growth characteristic and

various cellular functions like mRNA quantity, gene

expression, protein biosynthesis and enzyme activities

and caused the changes concerning the various

functions at the organ and tissue levels (Stein et al.,

1992; Goodman et al., 1995).

It was also shown that MF effected the various

characteristics of the plants like germination of seeds,

root growth rate, seedling growth, reproduction and

growth of the meristem cells and chlorophyll

quantities. In addition to, there were magnetic field

studies done with yield and yield parameters of crops

like cereal, sunflower and soybean. In these studies the

crop yield were increased (Pittman,1972;

Gubbels,1982; Vakharia et al., 1991; Pietruszowski,

1993; Namba et al., 1995; Atak et al., 1997; Özalpan et

al., 1999; Yurttafl et al., 1999; Reina et al., 2001;

Oldaçay, 2002).

While the studies are going on the subject of

interaction of magnetic field with the living systems,

some important unsolved problems have been seen. In

magnetic field studies, it has been known that

magnetic field effect never increased linearly with

the increase of strength and period of exposed to

magnetic field. Consequently, a simple dose-respons

relationship has not been found for magnetic field

frequency, intensity or duration of exposure. Also,

some uncertainties have been seen during the

interaction of the living systems with magnetic field

and at the beginning of the magnetic field effect

mechanism. So, the studies regarding this subject have

been carried on (Rafferty et al.,1992; Goodman et al.,

1995).

It is important to carry on the determination of the

biological effects formed in plants by magnetic field

studies in vitro conditions. In the present study, we

investigated the effects of magnetic field on the

regeneration of soybean (Glycine max L. Merrill)

shoot tip cultures.

Material and Methods

Plant material

The J 357 soybean seeds used for this research

provided from Samsun Blacksea Agriculture Research

Institute. Seeds were surface-sterilized for 1 minute in

70% ethanol and soaked in 20% commercial bleach

(commercial bleach contains about 5% sodium

hypochloride) for 20 minutes. Seeds were rinsed three

times in sterile distilled water. Sterile seeds were left

for germination at 27°C for 5 days in petri dishes

having 0.8% agar.

Plant tissue culture

The soybean explants were prepared from young

seedlings (5 day-old). The explants were incubated on

the medium consisted of inorganic nutrients as

Gamborg’s medium and vitamins, plus 30 g/l sucrose,

0.8% agar, 40 mg/l adenine sulfate, 0.1 g/l glutamine

(6.8 mM) and 0.1 mg/l 2.4-D (Wright et al., 1987; Atak

et al., 1994; 1999).

Explants were exposed to magnetic field in 15x100

mm petri dishes. After the exposed of magnetic field,

explants were immediately transferred to fresh

medium. In these experiments, each treatment had 3

parallels and 15 explants were employed for each

parallel. Explants for shoot tip cultures were incubated

on the medium. At this culture on the 28 th day the fresh

weights of regenerated plants were determined. We

applied Duncan’s multiple range test as the method to

compare the fresh weight of the groups exposed to

magnetic field and control.

Magnetic field experiment

At the magnetic field experiment, we used 10 magnets

of 0.45x0.065x0.022 m dimensions (Figure 1). In

JINR Laboratories, these magnets prepared by

magnetic field group were mounted onto the belt

system which rotated with a rate of 1 m/second in our

laboratories. The height of the magnets from belt


L = 2,2m (Carrying belt length)

h = 0,060m (Distance between sample and magnets)

d = 0,15m (Distance between magnets)

n = 10 (Magnet number)

v= 1 m/s (Passing velocity from magnetic field)

Figure 1: Magnetic field system plan formed at laboratory.

Figure 3: The root formation of the explants exposed to 2.9-

4.6 mT magnetic field at various periods in accordance with

days.

Figure 5: Shoot and root development of the explants

exposed to MF with control and 6.6 second period.

system was 0.060 m. Explants have been passed at 2.9-

4.6 mT magnetic flux density 1, 3 and 9 times at 2.2,

6.6 and 19.8 seconds.

Chlorophyll content

Magnetic field effect on regeneration 115

Figure 2: The regeneration of the explants exposed to 2.9-

4.6 mT magnetic field at various periods in accordance with

days.

Figure 4: Regeneration of explants exposed to MF at various

periods.

Chloroplasts were extracted from the leaves of 28-dayplants.

An extraction of leaf pigments was done with

80% acetone and the absorbance was measured at 663

and 645 nm with UV-160 Schmadzu

spectrophotometer. Chlorophyll a, chlorophyll b and

total chlorophyll quantities were calculated in

accordance with Arnon method (Arnon, 1949).


116 Çimen Atak et al.

Table 1: Shoot and root regeneration percentages and fresh weights formed on 28 th day the shoot tip explants exposed to 2.9-4.6

mT magnetic field at various periods.

Periods exposed to Shoot Regeneration (%) Root Regeneration (%) Avarage Fresh Weight (g)

2.9-4.6 mT MF (second)

Results

Control 61.90 14.29 0.088b*

2.2 86.96 26.08 0.111ab

6.6 74.36 35.90 0.153a

19.8 67.39 19.57 0.076bc

*Means which were not shown with the same letter, are significantly different by Duncan’s multiple range tests (P


cultures. Shoot formation percentage of the explants

exposed to magnetic field with 2.2 second period

found higher in accordance with those which exposed

to long period in this magnetic field flux density.

Investigations of low magnetic field effects on

biological systems were shown that natural

geomagnetic field (g.m.f) has an important role on the

normal functions of the plant cells. At the magnetic

screen (m.s.) conditions which g.m.f. was 10 5 -10 6 fold

screening by a constant component, peas, lentil and

flax seeds were grown. At this conditions, it was

shown with experiments that the germination of the

seeds decreased and the seedling growth inhibitated

according to the g.m.f. Also, cell reproduction cycle

was noted through lengthening of the G1 phase in these

plants, and RNA and protein synthesis were affected

(Govorun et al., 1992; Fomicheva et al., 1992a; 1992b).

Belyavskaya (2001) has determined that

phytoferritin in plastids of peas root meristem cells at

magnetic screen conditions has decreased.

By the magnetic field experiments with plants, it

has been shown that magnetic field has effected the

seed germination, the root elongation and seedling

growth (Vakharia et al., 1991; Namba et al., 1995;

Atak et al., 1997; Özalpan et al., 1999; Muraji et al.,

2000; Atak et al., 2000; Oldaçay, 2002).

Iimoto et al. (1996) showed that a magnetic flux

density of 4 mT had beneficial effect on the growth and

enhancement of CO2 uptake of potato plantlets in vitro.

During our research which are carried out in vitro

conditions, at the leaves belonging to regenerated

soybean shoots, chlorophyll a, chlorophyll b and total

chlorophyll quantities have been determined.

Regenerated shoots and chlorophyll contents were

increase in those which were passed through MF with

2.2 second period. Tian et al. (1991) used magnetic

field in his investigations. When he was irrigating rice

plants with magnetic water, leaf chlorophyll content

was increased. Atak et al. (2000) have determined that

magnetic field of 3.8-4.8 mT increased the chlorophyll

content in Paulownia species according to control. The

determined increases at chlorophyll a, chlorophyll b

and total chlorophyll contents especially appeared in

the expose to magnetic field for a short time.

Furthermore, chlorophyll content has been

investigated in sunflower plants exposed to magnetic

field of 3.8-4.8 mT. While Nantio variety of sunflower

chlorophyll quantities were increased, AS 508 variety

has not shown any effect on chlorophyll quantities

according to control (Oldaçay, 2002).

Magnetic field effect on regeneration 117

Our results show that fresh weight of regenerated

plants from the explants exposed to magnetic field

with 6.6 second period was higher according to the

control. This increase in the fresh weight has been

found significant (P


118 Çimen Atak et al.

chloroplast development nutrient metabolism. Auxins

have been used in order to induce the rooting on the

produced shoots. Apart from this, they also increased

the growth of shoot tip explants at the beginning. Cell

division has been arranged the interaction of auxin and

cytokinins together. Each of these have been effecting

the different phases of the cell cycle. While auxins

effect on DNA replication, cytokinins are effective on

some events causing mitosis. Cell can not enter mitosis

when cytokinin are not available (George, 1996;

Coenen et al., 1997; Kubo et al., 2000).

In summary, shoot regeneration and chloroplast

rate were increased at the explants exposed to

magnetic field with 2.2 second period and root

formation rate and fresh weight were also increased at

the explants exposed to magnetic field with 6.6 second

period in accordance with control. As a consequence,

the increase in cytokinin and auxin synthesis may be

induced by MF and in vitro conditions this plant’s

characteristics have been differently affected by MF

exposure of duration.

References

Arnon DI. Copper enzymes in isolated chloroplasts

Polyphenoloxidase in Beta vulgaris. Plant Physiology.

4, 1-15, 1949.

Atak C, Danilov V, Yurttas B, Yalç›n S, Mutlu D and

Rzakoulieva A. Effects of magnetic field on soybean

(Glycine max L.Merrill) seeds. Com JINR. Dubna, 1-

13, 1997.

Atak Ç and Alikamano¤lu S. Soya fasulyesi (Glycine max L.

Merril) meristem kültürlerinde gama radyasyonunun

bitki rejenerasyonuna etkisi. 12. Ulusal Biyoloji Kongre

Kitab›. 5: 202-207, 1994.

Atak Ç, Alikamano¤lu S and Yalç›n S. Induced mutation and

radiation sensitivity in vitro culture of soybean (Glycine

max L. Merrill). Turkish Journal of Nuclear Science. 26:

2; 69-88, 1999.

Atak Ç, Danilov V, Yurttafl B, Yalç›n S, Mutlu D and

Rzakoulieva A. Effect of magnetic field on Paulownia

seeds. Com JINR. Dubna. 1-14, 2000.

Belyavskaya NA. Ultrastructure and calcium balance in

meristem cells of pea roots exposed to extremely low

magnetic field. Adv Space Res. 28: 4, 645-650, 2001.

Coenen C and Lomax TL. Auxin- cytokinin interactions in

higher plants; old problems and new tools. Trend in

Plant Science Reviews. 2: 9, 1997.

Formicheva VM, Govorun RD and Danilov VT. Proliferative

activity and cell reproduction in the root meristem of pea

lentil and flax in the conditions of screening the

geomagnetic field. Biophysics. 37: 645-648, 1992a.

Formicheva VM, Zaslavskii VA, Govorun RD and

Danilov VT. Dynamics of RNA and protein synthesis in

the cel1s of the root meristems of the pea, lentil and flax.

Biophysics. 37: 649-656, 1992b .

George EF. Plant Propagation by Tissue Culture. Part: 1-2,

Exegetics Limited, England. 1996.

Goodman EM, Greenabaum B and Morron TM. Effects of

electromagnetic fields on molecules and cells.

lnternational Review of Cytology. 158: 279-325, 1995

Govorun RD, Danilov VI, Formicheva VM, Belyavskaya NA

and Yu Zinchenko S. Influence of fluctuation of the

geomagnetic field and its screening on the early phases

of the development of higher plants. Biophysics. 37: 639-

664, 1992.

Gubbels GH. Seedling growth and yield response of flax,

buckwheat, sunflower and field pea after preseeding

magnetic treatment. Can J Plant Sci. 62: 61-64, 1982.

Iimoto M, Watanebe K and Fujiwara K. Effects of magnetic

flux density and direction of the magnetic field on

growth and CO2 exchange rate of potato plantles in vitro.

Acta Horticulturae. 440: 606-610, 1996.

Kuba M and Kakimoto T. The cytokinin hyposensitive genes

of Arabidopsis negatively regulate the cytokinin

signalling pathway for cell division and chloroplast

evelopment. The Plant Journal. 23(1): 385-394, 2000.

Liu ZH, Hsiao JC and Pan YW. Effect of naphthalene acetic

acid on endogenous indole-3-acetic acid, peroxidase and

auxin oxidase in hypocotyl cutting of soybean during

root formation. Bot Bull Acad Sin. 37 (4): 247-253, 1996.

Liu ZH,Wang WC and Yen YS. Effect of hormone treatment

on root formation and endogenous indole-3-acetic acid

and poliamine levels of Glycine max. Bot Bull Acad Sin.

39: 113-118, 1998.

Maruji M, Nishimura M, Tatebe W and Fuji T. Effect of

altenating magnetic field on growth of the primary root

of corn. IEEE Transactions on Magnetics. 28: 1996-

2000, 1992.

Mize CW and Chun WY. Analysing treatment means in plant

tissue culture research. Plant Cell Tissue and Organ

Culture. 13: 201-217, 1988.

Muraji M, Asai T and Tatebe W. Primary root growth of Zea

mays seedlings grown in an alternating magnetic field of

different frequencies. Bioelectrochemistry and

Bioenergetics. 44: 271-273, 1998.

Namba K, Sasao A and Shibusawa S. Effect of magnetic

field on germination and plant growth. Acta

Horticulture. 399: 143-147, 1995.

Oldaçay S. Gama radyasyonu ile ›fl›nlanm›fl ayçiçe¤i

(Helianthus annuus L.) çeflitlerinin üzerine manyetik

alan›n etkisi. Doktora Tezi. ‹Ü. Fen Bilimleri Enstitüsü.

2002.

Özalpan A, Atak C, Yurttas B, Alikamanoglu S, Canbolat Y,

Borucu H, Danilov V and Rzakoulieva A. Magnetik

alan›n soya (Glycine max L.Merrill) verimi üzerine

etkisi. Türk Biyofizik Derne¤i, Xl. Ulusal Kongresi.

Program ve Bildiri Özetleri. 60, 1999.


Phillips JL. Effects of electromagnetic field exposure on

gene transcription. Journal of Cellular Biochemistry. 51:

381-386, 1993.

Phillips JL, Haggaren W, Thomas WJ, Ishida-Jones T and

Adey WR. Magnetic field-induced changes in spesific

gene transcription. Biochimica et Biophysica Acta. 1132:

140-144, 1992.

Pietruszowski S. Effect of magnetic seed treatment on seed

treatment on yields of wheat. Seed Sci and Technol. 21:

621-626, 1993.

Pittman UJ. Biomagnetic responses in potatoes. Can J Plant

Sci. 52: 727-733, 1972.

Polk C and Postow E. Biological Effects of Electromagnetic

Fields. Second Edition, CRC Press. 1995.

Rafferty CN, Phillips RD and Guy AN. Dosimetry

workshop: Extremely-low-frequency electric and

magnetic fields. Bioelectromagnetics Supplement. 1: 1-

10, 1992.

Reina FG, Pascual LA and Fondora IA. Influence of

stationary magnetic field on water relations in Lettuce

seeds. Part II. Experimental Results.

Bioelectromagnetics. 22: 596-602, 2001.

Stange BC, Rowland RE, Rapley BI and Podd JV. ELF

Magnetic field increase aminoacid uptake into Vicia faba

L. roots and alter ion movement across the plasma

membrane. Bioelectromagnetics. 33: 347-354, 2002.

Stein GS and Lian JB. Regulation of cell cycle and growth

control. Bioelectromagnetics Supplement. 1: 247-265,

1992.

Tian WX, Kuang YL and Mei ZP. Effect of magnetic water

on seed germination, seedling growth and grain yield of

rice. Field Crop Abstracts. 044-07228, 1991.

Vakharia DN, Davariya RL and Parameswaran M.

Influence of magnetic treatment on groundnut yield and

attributes. lndian J Plant Physiol. XXXIV: 131-136,

1991.

Wright MS, Ward DV, Hinchee MA, Carnes MG and

Kaufman RJ. Regeneration of soybean (Glycine max L.

Merrill) from cultured primary leaf tissue. Plant Cell

Reports. 6: 83-89, 1987.

Yurttafl B, Atak C, Gökdo¤an G, Canbolat Y, Danilov V and

Rzakoulieva A. Magnetik alan›n ayçiçek bitkisindeki

(Helianthus annuus L.) olumlu etkisinin saptanmas›.

Türk Biyofizik Derne¤i, Xl. Ulusal Kongresi. Program ve

Bildiri Özetleri. 59, 1999.

Zhou J, Li C, Yao G, Chrang H and Chang Z. Gene

expression of cytokinin receptors in HL 60 cells exposed

to a 50 Hz magnetic field. Bioelectromagnetics. 23: 339-

346, 2002.

Magnetic field effect on regeneration 119


Letters to editor

Gene transfer for the treatment of

neoplasms

Neoplazma tedavilerinde gen transferleri

Biotechnology and gene technology are recognized by

experts as invaluable and unique tools to find solutions

to or improve many problems in health, agriculture

and management of the environment, and are regarded

as a driving economic force in the 21 th century. Despite

the great potential of gene therapy to become a new

treatment modality in future medicine, there are still

many limitations to overcome before this gene

approach can pass to the stage of human trial. The

foremost obstacle is the development of a safe,

efficient, and efficacious vector system for in vivo

gene application. Gene therapy is the transfer of

deoxyribonucleic acid (DNA) into a cell to achieve the

expression of a particular therapeutic protein. The

ability to genetically alter a somatic cell offers

intriguing therapeutic possibilities for the treatment of

metabolic, infectious, and neoplastic diseases.

Although clinical validation of the effectiveness of

gene therapy is lacking, a variety of malignant

disorders may someday be treated with gene therapy.

Methods of gene transfer: The gene that is

transferred to a cell is termed the transgene. The

efficient delivery of therapeutic genes and appropriate

gene expression are the crucial issues for clinically

relevant gene therapy. Gene transfer may be

accomplished either directly in vivo or through the

manipulation of cells ex vivo followed by reinstillation.

A gene delivery vehicle, or vector, that

may be of viral or non-viral origin, is generally used to

carry the genetic material.

Viral vectors are the predominant agents used

currently. Viruses have evolved the intrinsic ability to

enter cells and control its machinery to support their

own survival and replication and to promote

expression of the transgene. Retrovirus, lentivirus,

adenovirus, adeno-associated virus, poxvirus and

herpesvirus are employed in more than 70% of clinical

gene therapy trials. So far, viral vectors have been

mainly used because of their inherently high

121

transfection efficiency of gene. This ability made them

desirable for engineering virus vector systems for the

delivery of therapeutic genes. However, there are some

problems to be resolved for the clinical applications,

such as the pathogenicity and immunogenicity of viral

vectors themselves. In most instances, a portion of the

viral genome is deleted to render it replication

defective to reduce pathogenicity. Many achievements

have been made in vector safety, the retargeting of

virus vectors and improving the expression properties

by refining vector design and virus production.

Nonviral vector mediated gene transfer, compared

to viral vector mediated one, is a promising tool for the

safe delivery of therapeutic DNA in genetic and

acquired human diseases. Nonviral methods use DNA

either alone (naked plasmid) or in conjunction with

liposomes. The efficiency and toxicity of the nonviral

vectors used depended on the type of vector, the

DNA/vector ratio, the type of cell, and the presence of

serum. There is no clearly superior vector for all

applications; each method has particular advantages

and is suited to specific applications. Nonviral vectors,

although less efficient at introducing and maintaining

foreign gene expression, have the profound advantage

of being non-pathogenic and nonimmunogenic.

Therefore, many research trials with nonviral vectors

have been performed to enhance their efficiency to a

level comparable to the viral vector. Liposomal

formulations have been developed to improve the level

of efficiency of plasmid-gene transfer. The advantage

of liposomes (Lps) is that the potential toxicity of

viruses is avoided. Vector administration can be

repeated because of their low toxicity. Unfortunately,

Lps have variable formulations, and reproducibility is

troublesome. The initial enthusiasm for liposomal

gene delivery in vivo has not been substantiated.

None of the available modalities is clearly superior

for gene delivery since each has slightly different

features and certain drawbacks. It should be readily

attainable in high titers and be able to accomplish a

high magnitude of transgene expression. The duration

of transgene expression required varies with the

clinical application. For neoplastic diseases, transient

expression may suffice to stimulate an immune

response or directly eradicate a tumor. In fact, an


122

immunogenic vector may actually serve as an adjuvant

for an immune response. Second, it is often desirable

for uptake of the vector to occur only in target cells

(specific cellular entry-selective delivery) which is

often cumbersome or impractical. Third, expression of

the vector should be restricted to the target tissue. The

last characteristic of an ideal vector is inducible

transgene expression. Transcriptional regulatory

elements that respond to endogenous factors or

exogenous substances would likely provide more

physiologic transgene activity and avoid the potential

cellular toxicity of transgene overexpression.

Therapeutic strategies for cancer: There are three

general approaches to the treatment of cancer by using

gene transfer: immunostimulation, cytotoxicity, and

gene correction. Most investigators have used

immune-based strategies for human trials. The early

results have not been impressive although most studies

were designed to test the feasibility and safety of the

techniques and vectors. Perhaps a combination of

approaches in which, for example, an immunologic

strategy using a cytokine gene plus the transfer of a

suicide gene are used in conjunction with

chemotherapy or radiation will prove most successful

in cancer therapy.

Most human tumors are weakly immunogenic;

therefore, methods that enhance an immune reaction

(immunostimulation) are attractive. Most approaches

attempt to generate a specific T-lymphocyte response.

Unfortunately, in many animal models, treatment is

successful only if it is administered prior to or early

after tumor inoculation. One way to initiate an immune

system is through the expression of cytokines in or

near the tumor. In this way, the toxicity of systemic

cytokine delivery may be avoided. A variety of

immunostimulatory cytokines have received attention,

including interleukins (IL-2,4,6,7, and 12), interferon γ

(INFγ), and granulocytemacrophage colonystimulating

factor (GMCSF). The lack of

immunogenicity of most tumors may be attributed in

part to their inability to provide the necessary

additional improvable signals to activate the immune

system (antitumor immune responses) with

adhesion/co-stimulatory molecules (B7-1, B7-2) have

been shown to bind to receptors on T-lymphocytes.

Gene transfer of tumor antigens has great potential for

the treatment of neoplastic disease. A cancer vaccine is

particularly attractive since the impediments of gene

delivery are bypassed. A number of tumor antigens are

under investigation. There are tumor-specific antigens

such as the MAGE and GAGE antigens in melanoma.

Differentiation antigens exist normally and include

those derived from tyrosinase, CEA, and prostatespecific

antigen (PSA). Oncogene or tumor suppressor

gene products may also serve as antigens. Mucins have

also been targeted. The introduction of allogeneic

major histocompatibility antigens into tumor cells is

another strategy for inducing an antitumor response.

Several methods have been devised to accomplish

direct tumor cytotoxicity using gene transfer. The most

widely studied are “suicide” gene transfer and the use

of replication-competent viruses. A “suicide” gene

thymidine kinase gene of herpesvirus and cytosine

deaminase gene are the examples of this issue. Another

approach has been to enhance the effects of

chemotherapy (e.g. transfer of the carboxylesterase

gene). Replication-competent adenovirus and

herpesvirus are being investigated and attractive

agents for the treatment of tumors.

Gene correction involves restoring the normal

genetic composition of a tumor cell. The major

disadvantage of this approach is that gene transfer

must occur in all or at least a high proportion of tumor

cells to be therapeutic. Furthermore, the correction

must be stable. In tumors with mutant p53, gene

transfer of the wildtype gene has been developed and

may have particular relevance for primary and

secondary liver tumors. Another approach is to

interfere with specific cellular messenger RNA.

In conclusion, although thousands of patients have

been involved in clinical trials for gene therapy, using

hundreds of different protocols, true success has been

limited. A major limitation of gene therapy, especially

when nonviral vectors are used, is the poor efficiency

of DNA delivery to the nucleus; a crucial step to

ensure ultimate expression of the therapeutic gene

product. There are several difficulties in effective gene

transfer that must be overcome; these include

developing optimal vectors, targeting gene delivery,

and regulating transgene expression. In addition, the

immunogenicity of the vector and the transgene must

be manipulated to avoid toxicity and to allow effective

gene expression. The potential of gene therapy seems

limitless. However, developed strategies and

performed investigations should be regarded as

preliminary, and therapeutic efficacy awaits clinical

validation.

Hakan Mustafa Köksal

fiiflli Etfal Research and Training Hospital,

1. General Surgery Department, fiiflli, ‹stanbul


Conference Report:

Functional Genomics and Disease,

Prague, Czech Republic,

May 14-17, 2003

Konferans raporu:

Fonksiyonel genomikler ve hastal›klar,

Prag, Çek Cumhuriyeti,

14-17 May›s 2003

The structure of DNA, perhaps the most important

biological advance of the 20 th century, and the human

genome project, have affected our view of biology.

The year 2003 is the celebrity year for genomics, with

the 50 th anniversary of the puplication of the DNA

double helix (April 1953) and the announcement of the

completion of the genome sequence (April 2003). The

determination of structure revealed how genes encode

information and how they are replicated; the

complexion of the human genome sequence now

offers the prospect of disease prediction, prevention

and cure, as well as a deeper understanding of our own

evolution.

It is estimated that around 40% of the open reading

frames in a fully sequenced organism have no known

function at the biochemical level and are unrelated to

any known gene, while at the level of phenotype the

proportion with known properties is much less.

Consequently, a shift of emphasis is now occurring

from genome mapping and sequencing to

determination of genome function. This is the area

known as functional genomics. Functional genomics

has emerged from employing major innovative

technologies for genome-wide analysis supported by

information technology. These activities depend both

on experimental and computational methods. While

high throughput experimental technologies generate

data on gene expression, protein structure, protein

interactions, etc., powerful information systems are

required for the efficient management of experimental

data, integration of information that is distributed in

heterogeneous sources and establishment of computer

assisted experimental strategies.

The European Science Foundation has organised

their first major European conference, which are

focused on the implications of functional genomics

research for understanding and therapy of human

diseases. The elucidation of the human genome and the

studies of gene function, which are being carried out

123

throughout the world, have profoundly affected the

way in which disease mechanisms and treatments are

discovered. Functional Genomics and Disease 2003

included all major areas and bring together experts

from Europe and other parts of the world. The meeting

was held in Prague, the capital of the Czech Republic.

The symposia’s were based on, infection and host /

parasite interaction, hereditary disease, model systems

and knockout, transcriptome, proteome and proteinprotein

interactions, oncology, mitochondrial diseases,

biobanking, intellectual property, ethics,

bioinformatics, cell and gene therapy, advances in

microarray, pharmacogenomics and drug discovery,

complex disease and neurological disease.

Almost five hundred delegates discussed that the

human genome project has produced a high number of

data, with specific questions. Multiple uncertainties

must be solved before large-scale genome wide

investigations are undertaken. Such uncertainties can

be grouped as choosing the populations to study

(isolated or out bred), the phenotypes to be included,

the marker types to be employed, and selection of

variants for genotyping.

With the development of genomics, target

prioritisation is also becoming more and more

important. Hybrigenics has developed intergrated

biological experiment-based and in silico-based

technology platforms that allow target identification,

selection, prioritisation and validation of drug targets.

Target identification is based on the mapping of the

protein-protein interaction networks starting from a

given set of proteins. This step potentially reveals

various novel proteins involved in specific pathways.

Bioinformatics pre-validation tools are then applied in

order to sort out and filter information on proteins and

protein interaction domains. As a result, potential

targets are thus prioritized for further validation.

Biological analysis and validation are then performed

in human cells using various approaches including

dominant negative mutants, overexpressed proteins

and siRNA silecing experiments. This approach has

been applied to inhouse drug discovery programs that

include AIDS and Cancer projects. P. Legrain from

Hybrigenics, France, has presented examples of

protein networks, pathway description and biological

validation of protein functions, in particular for some

proteins involved in the Tgfbeta pathway.

J. E. Celis from Institute of Cancer Biology and

Danish Centre for Translational Research in Breast

Cancer has talked about current proteomic strategies as

applied to bladder and breast cancer. Cancer, which


124

affects a significant fraction of the population, has

become a prime target for the new technologies.

Indeed, tools for the high throughput analysis of genes,

proteins and their complex networks are being used to

identify potential targets for drug discovery as well as

markers for early detection, recurrence, progression,

response to treatment and development of novel

therapies.

In the biobanking session, A. Cambon-Thomsen,

France, has emphasized of whether biobanking for

genomics or genomics for biobanking. The use of

human biological samples as biobanks occurs in a

variety of situations from research and technological

development to medical diagnosis and therapeutic

activities. The concept of biobanks includes the

biological samples themselves (of different kinds

corresponding to variable conservation conditions, but

sources of nucleic acids), the attached databases and a

certain level of openness, availability and exchanges

for different kinds of studies. In the same session, J.

Straus from Max Planck Institute for Intellectual

Property, Germany has presented the constitutional

backgrounds of patenting genomic related functions

with a European perspective. He has argued that any

advanced should be taken under law protection before

it has been puplished. On the contrary, T. Hubbard

from Wellcome Trust Sanger Institute Cambridge, UK

has presented a new economic model for global

healthcare R&D based on puplic domain contributions

(referencing the human genome project) rather than

commercial based Intellectual Property (IP) driven

models.

Apart from the ninety speakers, more than three

hundred posters were also presented on the conference

primarily focusing on microarray based experiments

and gene chip developments. Arround seven industry

satellite symposia’s were arranged by companies such

as Invitrogen, Affymetrix, Lion Bioscience+IBM and

others.

The outcome of the conference has been specifially

around the roadmap that the scientists should choose in

the forthcoming years. The main event driving this

roadmap is the benefits of the human genome

becoming clear in this 50 th anniversary year of DNA

P›nar U. Onganer

Department of Biological Sciences,

Neuroscience Solutions to Cancer Research Group,

Imperial College of Science, Technology & Medicine,

London, U.K.


Book reviews

Leland WK. CHUNG, William B. ISAACS ve

Jonathan W. SIMONS (Eds), Prostat Kanseri:

Biyoloji, Genetik ve Yeni Tedaviler, Humana Press,

531 sayfa, ISBN: 0896038688.

Kitab›n içeri¤i de¤iflik disiplinlerden uzman kiflilerin

prostat kanseri üzerinde yap›lan son çal›flmalar›n

okuyucuya sunulmas› için düzenlenmifltir. Girifl

k›sm›ndan sonra, kitap üç ana bölümden oluflmaktad›r.

‹lk bölümde, prostat kanseri geneti¤i üzerinde

çal›flmalar anlat›lm›flt›r. ‹kinci bölümde prostat

kanserinin biyolojik yönü ve son bölümde ise

uygulanan terapiler ve yeni terapötik ajanlar üzerine

son çal›flmalardan bahsedilmektedir. Genetik ile

ilgili k›s›mda ailesel prostat kanseri ve prostat

kanserinde gen ekspresyonlar› ve array çal›flmalar›n›n

önemine yer verilmifltir. Bu bölümde ayn› zamanda

genetik alterasyonlar, moleküler epidemiyoloji ve

prostat kanserinde etkin androjen reseptörlerin

polimorfizmlerinden de bahsedilmifltir.

Kanser biyolojisi ile ilgili ikinci bölümde temel

biyoloji ve prostat kanseri büyüme ve gelifliminde rol

alan regülatör mekanizmalardan bahsedilmektedir. Bu

bölümde ayr›ca, hücre-hücre ve hücre-matriks

etkileflimleri, hücresel sinyal mekanizmalar› ve prostat

kanseri tan›s›nda önemli hücre adezyonundan sorumlu

e-cadherin ve integrinler gibi, moleküllerden,

bahsedilmektedir. Tirozin kinazlar›n yeni ümit

vaadedici terapi modeli olabilece¤i üzerinde son

çal›flmalara yer verilmifltir. Bu bölümün sonuna do¤ru

prostat kanserinde stroma-epitel iliflkisi ve bunlar›n

mikroçevrede tümörlerin yay›l›m› ile ilgili çal›flmalar

anlat›lm›flt›r.

Terapiler üzerine haz›rlanan son k›s›mda ise, çok

iyi bilinen önleme, cerrahi müdahale ve radyasyon

terapileri üzerinde durulmufltur. Ayn› zamanda bu

bölümde deneysel protokoller anlat›lm›fl ve vitamin D

analoglar› gibi baz› terapötik önem tafl›yan

moleküllerin deneysel prostat kanseri üzerinde

kullan›mlar›na iliflkin literatürlere yer verilmifltir. Bu

bölümde, önemli kemoterapatik maddeler ile yap›lan

deneyler ve bunun sonuncunda da doktorlar için en

etkin ve aktif mekanizman›n anlafl›lmas› üzerine

125

çal›flmalar yer almaktad›r. Kitab›n ilk yazar› Chung,

Virginia Üniversitesi, T›p Fakültesinde görev ald›¤›

süreçte bu kitab› yazm›fl ve kitap prostat kanseri

çal›flmalar›na çok emek vermifl ve akademik t›p için

bir model bilim adam› olan Donald Coffey’e (PhD)

adanm›flt›r.

Sonuç olarak, bu kitab› prostat kanseri

çal›flmalar›nda deneysel veya klinik anlam›nda görev

alan tüm araflt›rmac›lara tavsiye ediyorum. Bu kitap,

prostat kanserinin moleküler mekanizmalar›n›n ve

uygulamalar›n anlafl›labilmesi için iyi bir rehber

olacakt›r.

Turhan Çaflkurlu

fiiflli Etfal Araflt›rma ve E¤itim Hastanesi,

1. Üroloji Klini¤i,

fiiflli, ‹stanbul

Leland WK. CHUNG, William B. ISAACS and

Jonathan W. SIMONS (Eds), Prostate Cancer:

Biology, Genetics & the New Therapeutics, Humana

Press, 531 pp, ISBN: 0896038688.

The main strength of this book is that experts from

several disciplines have contributed their thoughts on

recent advances in prostate cancer. After introduction,

the book has three main chapters; the first explains the

genetics of prostate cancer, the second biologic view

of the prostate cancer, and the third treatment

approaches and new therapeutics. The section of

genetics includes chapters on familial prostate cancer

and on the study of gene expression in prostate cancer

with the use of gene-array libraries and their usage.

This section also covers genetic alterations, molecular

epidemiology, and polymorphisms of androgen

receptors in prostate cancer.

A section on cancer biology is devoted to basic

biology and regulatory mechanisms controlling

prostate cancer growth, progression and in this section

cell-cell and cell-matrix interactions and cellular

signaling pathways, important cell adhesion molecules


126

for diagnosis i.e. e-cadherin and integrins are told, in

prostate cancer. The chapter on tyrosine kinases have

been shown as a new promising therapy in prostate

cancer, as they have for the treatment of other

malignant conditions. The chapter on stromalepithelial

interaction in prostate cancer is also

important, since there is a growing recognition of the

role of the microenvironment in the progression of

tumors.

The section on treatment includes all general and

well known prevention, surgical therapy, and radiation

therapy models. This section also includes chapters on

experimental protocols, mainly for the treatment of

advanced prostate cancer; antiprogression agents such

as vitamin D analogues may hold special promise for

the treatment of prostate cancer. The chapter on trials

of chemopreventive agents is particularly important,

since many patients self-prescribe these natural and

synthetic medications, and physicians need to have a

better understanding of their mechanisms of action and

efficacy. The first author Chung is affiliated with the

University of Virginia School of Medicine and they

dedicate this book to Donald Coffey, Ph.D., who has

made important contributions to the field and has

trained many researchers in prostate cancer. He is a

role model for many in academic medicine.

In conclusion, I recommend this book to

researchers who study prostate cancer, whether at the

bench or at the bedside, and to clinicians who treat

patients with prostate cancer. It is a good guide to

understand the molecular mechanism of prostate

cancer and treatments.

Turhan Çaflkurlu

fiiflli Etfal Research and Training Hospital,

1 st Urology Clinics,

fiiflli, ‹stanbul

Valentine I. KEFEL‹ and Maria V. KALEV‹TCH,

Bitkilerde Do¤al Büyüme ‹nhibitörleri,

Fitohormonlar ve Çevre, Editor: Bruno

BORSARI, Kluwer Academics, Hollanda, 340 sayfa,

ISBN: 1-4020-1069, 2003.

Bu kitap yazarlar›n yaflamlar› boyunca inhibitörler,

bitki hormonlar› ve sürdürülebilir ziraat için pratik

yaklafl›mlarla ilgili olarak yapt›klar› çal›flmalara bir

ithaf› temsil etmektedir. Çal›flmalar›, aktif

moleküllerin çeflitli gruplar›n›n ifllevleri, bunlar›n bitki

büyümesine olan do¤rudan etkileri ve ayn› zamanda

onlar›n çevre ile olan iliflkisi üzerine yo¤unlaflm›flt›r.

Kitab›n ana fikri do¤al büyüme inhibitörleri ve bitki

hormonlar› aras›nda bir de¤erin tayin edilmesi

gereklili¤ine olan ihtiyac› ortaya ç›karmaktad›r. Bu

yaklafl›m bunlar›n biyokimyasal yollar›n›, bitkinin

fizyolojik ifllevleri üzerine olan etkilerinin ve bitki

ontogenezinde stres faktörü olarak etkilerinin daha iyi

anlafl›lmas›n› sa¤lam›flt›r. Böylece bitki fizyolojisi

çal›flmalar›nda daha lojistik yaklafl›m›n gereklili¤i

ortaya konmaktad›r. Bu kitapta bitki-toprak iliflkisi ile

farkl› alelokimyasallar ve bunlar›n bitki büyümesine

etkileri tart›fl›lm›flt›r.

Kitap afla¤›daki bölümleri içermektedir: Bitkide

büyüme ve geliflim düzeninin sistemi; bitki

genomunun kontrolü alt›nda do¤al bitki büyüme

inhibitörleri ve fitohormonlar; yaprak büyüme ve

geliflimi s›ras›ndaki do¤al inhibitörler ve

fitohormonlar; hücre ve organ uzamas› olaylar›nda

do¤al bitki inhibitörleri ve bitki hormonlar›; dormansi

s›ras›nda fenolik inhibitörler ve absisik asit; bitkilerde

ve absise olan yapraklardaki do¤al bitki

inhibitörlerinin katabolizmas›; alelopatojenler ve

herbisitler olarak do¤al büyüme inhibitörleri ve stres

koflullar›; dokunulmam›fl bitkilerde ve izole hücre,

organ ve dokularda do¤al bitki inhibitörleri ve

fitohormonlar; do¤al büyüme inhibitörleri ve

biyotestler; sonuç; referanslar; sözlük; indeks.

Bu kitap lisans, lisansüstü ö¤rencileri ve

araflt›r›c›lar için önemli bir baflvuru kayna¤›

oluflturmaktad›r. Ayr›ca bu kitab› akademik ve

araflt›rma kütüphanelerine önemli bir katk› olarak

tavsiye ederim.

Narçin Palavan-Ünsal

Haliç Üniversitesi,

Moleküler Biyoloji ve Genetik Bölümü

Valentine I. KEFEL‹ and Maria V. KALEV‹TCH,

Natural Growth Inhibitors and Phytohormones in

Plants and Environment, Editor: Bruno

BORSARI, Kluwer Academics, NL, 340 pp, ISBN:

1-4020-1069, 2003.

This book represents the authors lifetime dedication to

the study of inhibitors and phytohormones as well as

its practical applications for achieving a more


sustainable agriculture. Their work focuses on the

functions of various groups of active molecules, their

direct effect upon plant growth, but also implications

for their impact upon the surrounding environment are

explored. The main idea of the book evolved for the

need to determine a balance among natural growth

inhibitors and phytohormones. This approach was

pursued through a better understanding of their

biochemical pathways, their effects on plants

physiological functions, and their influence upon

stress factors on plant ontogenesis. Therefore, this

effort proposes a more logistic approach to the study of

plant physiology, in which the plant soil interactions

are discussed, with a profound description of different

allelochemicals and their effects on plants growth.

This book contains following chapters: System of

growth and development regulation in the plant;

natural growth inhibitors and phytohormones under

the control of the plant’s genome; natural inhibitors

and phytohormones during leaves growth and

development; natural growth inhibitors and

phytohormones in the process of cells and organs

elongation; phenolic inhibitors and abscisic acid

during dormancy; catabolism of natural growth

inhibitors in plant and abscised leaves; natural growth

inhibitors as allelopathogens and botanical herbicides;

natural growth inhibitors and stress conditions; natural

growth inhibitors and phytohormones in the intact

plants and isolated cells, organs and tissues; natural

growth inhibitors and biotests; conclusion; references;

glossary; index.

This book will be excellent application source both

for undergraduate and graduate students and a

reference for researchers in biology. I also recommend

this book to major academic and research libraries as a

welcome addition to their collection of plant biology

literature.

Narçin Palavan-Ünsal

Haliç University,

Department of Molecular Biology and Genetics

127


Instructions for authors

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The Editorial Office

Journal of Cell and Molecular Biology,

Haliç Üniversitesi,

Fen-Edebiyat Fakültesi,

Ahmet Vefik Pafla Cad. No :1

34280, F›nd›kzade,

‹stanbul-Turkey

4. Each submitted manuscript will be assessed by a

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129

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130

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2. In the list, references must be placed in alphabetical

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exactly followed.

Redford IR. Evidence for a general relationship

between the induced level of DNA double-strand

breakage and cell killing after X-irradiation of

mammalian cells. Int J Radiat Biol. 49: 611- 620,

1986.

Taccioli CE, Cottlieb TM and Blund T. Ku 80: Product

of the XRCCS gene and its role in DNA repair and

V (D) J recombination. Science. 265: 1442-1445,

1994.

Ohlrogge JB. Biochemistry of plant acyl carrier

proteins. In: The Biochemistry of Plants: A

Comprehensive Treatise. Stumpf PK and Conn EE

(Ed). Academic Press, New York. 137-157, 1987.

Weaver RF. Molecular Biology. WCB/Mc

Graw-Hill.1999.

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free of charge.


Volume contents

Volume 2, No. 1

Dedication

Review articles

Polyamines in plants: An overview

Bitkilerde poliaminler: Genel bir bak›fl

R. Kaur-Sawhney, A.F. Tiburcio, T. Altabella, A.W. Galston 1-12

Phenolic cycle in plants and environment

Bitkilerde fenolik döngü ve çevre

V.I. Kefeli, M.V. Kalevitch, B. Borsari 13-18

Research papers

The short-term effects of single toxic dose of citric acid in mice

Farelerde sitrik asidin tek toksik dozunun k›sa süreli etkileri

T. Aktaç, A. Kabo¤lu, E. Bakar, H. Karakafl 19-23

Characterisation of RRPPPP77 mutant lines of the col-5 ecotype of AArraabbiiddooppssiiss tthhaalliiaannaa

Arabidopsis thaliana’n›n col-5 ekotipinden elde edilen mutant hatlardan RPP7

geninin karakterizasyonu

C. Can, M. Özaslan, E.B. Holub 25-30

The effect of mmeettaa-topolin on protein profile in radish cotyledons

Meta-topolinin turp kotiledonlar›nda protein profiline etkisi

S. Ça¤, N. Palavan-Ünsal 31-34

The effect of electromagnetic fields on oxidative DNA damage

Elektromanyetik alan›n oksidatif DNA hasar› üzerindeki etkisi

S. ‹fller, G. Erdem 35-38

Chromosomes of a balanced translocation case evaluated with atomic force

microscopy

Dengeli translokasyon vakas›nda kromozomlar›n atomik güç mikroskobu ile

de¤erlendirilmesi

Z. Y›lmaz, M.A. Ergun, E. Tan 39-42

Effect of epirubicin on mitotic index in cultured L-cells

Epirubisinin kültürdeki L-hücrelerinde mitotik indekse etkisi

G. Özcan Ar›can, M. Topçul 43-48

Letter to editor 49-51

Book reviews 53

Instructions to authors 55-56

131


132

Volume 2, No. 2

Review articles

E-cadherin molecular mechanism in prostate cancer

E-cadherinlerin prostat kanserinde moleküler mekanizmas›

D. Büyüktunçer, S. Arisan, K. Özdilli 57-64

A general view: Structure and function of the subunits of EE.. ccoollii RNA polymerase

Genel bak›fl: E. coli RNA polimeraz›n alt birimlerinin yap›lar› ve fonksiyonlar›

N. Büyükuslu 65-77

Research papers

Homocysteine potentiates esterase-induced contraction on rat aorta iinn vviittrroo:

A risk factor for atherosclerosis

Homosisteinin s›çan aortunda esteraz ile artt›r›lan in vitro kas›lmay› güçlendirmesi:

Athereosclerosis oluflumu için bir risk faktörü

F.B.H. Gurib, A.H. Subratty 79-83

Cytoembryological studies on PPaaeeoonniiaa ppeerreeggrriinnaa L.

Paeonia peregrina’da sitoembriyolojik çal›flmalar

R. Öztürk, M. Ünal 85-89

Immunogenicity and specificity of SSaallmmoonneellllaa ttyypphhiimmuurriiuumm outer membrane

antigens

Salmonella typhimurium d›fl membran antijenlerinin immunojenite ve özgüllü¤ü

N. Ak›fl, O. Sayhan, A. Karaçavufl, K. Töreci 91-97

Polymerase chain reaction is a good diagnostic tool for MMyyccoobbaacctteerriiuumm

ttuubbeerrccuulloossiiss in urine samples

‹drar örneklerinde Mycobacterium tuberculosis tan›s› için polimeraz zincir

reaksiyonunun kullan›m›

S. Arisan, N.C. Sönmez, Ö.O. Çak›r, E. Ergenekon 99-103

Cytological investigations in some important tree species of Rajasthan VI.

Radiation induced chromosome aberrations in AAnnooggeeiissssuuss ppeenndduullaa and AA.. llaattiiffoolliiaa

Rajastan IV için baz› önemli a¤aç türlerinde sitolojik araflt›rmalar: Anogeissus pendula

ve A. latifolia’ da ›fl›nlama ile artan kromozom aberasyonlar›

A. Kumar, S.R. Rao, N.S. Shekhawat 105-111

Stimulation of regeneration by magnetic field in soybean (GGllyycciinnee mmaaxx L. Merrill)

tissue cultures

Soya (Glycine max L. Merrill) doku kültürlerinde rejenerasyonun manyetik alan

taraf›ndan stimulasyonu

Ç. Atak, Ö. Emiro¤lu, S. Alikamano¤lu, A. Rzakoulieva 113-119

Letters to editor 121-124

Book reviews 125-127

Instructions to authors 129-130

Volume content 131-132

Author index 133


Author index

Ak›fl N. 91

Aktafl T. 19

Alikamano¤lu S. 113

Altabella T. 1

Arisan S. 49, 57, 99

Atak Ç. 113

Bakar E. 19

Borsari B. 13

Büyüktunçer D. 57

Büyükuslu N. 65

Can C. 25

Ça¤ S. 31

Çak›r ÖO. 99

Çaflkurlu T. 125

Emiro¤lu Ö. 113

Erdem G. 25

Ergenekon E. 99

Ergun MA. 39

Galston AW. 1

Gurib FBH. 79

Holub EB. 25

‹fller S. 35

Kabo¤lu A. 19

Kalevitch MV. 13

Karaçavufl A. 91

Karakafl H. 19

Kefeli VI. 13

Köksal HM. 121

Kumar A. 105

Onganer PU. 123

Özaslan M. 25

Özcan Ar›can G. 43

Özdilli K. 57

Öztürk R. 85

Palavan-Ünsal N. 31, 126

Rao SR. 105

Rzakoulieva A. 113

Sawhney RK. 1

Sayhan O. 91

Shekhawat NS. 105

Sönmez NC. 99

Subratty AH. 79

Tan E. 39

Tiburcio AF. 1

Topçul M. 43

Töreci K. 91

Ünal M. 85

Y›lmaz Z. 39

133


Journal of Cell and Molecular Biology

CONTENTS Volume 2, No. 2, 2003

Review articles

E-cadherin molecular mechanism in prostate cancer

E-cadherinlerin prostat kanserinde moleküler mekanizmas›

D. Büyüktunçer, S. Arisan, K. Özdilli 57-64

A general view: Structure and function of the subunits of E. coli RNA polymerase

Genel bak›fl: E. coli RNA polimeraz›n alt birimlerinin yap›lar› ve fonksiyonlar›

N. Büyükuslu 65-77

Research papers

Homocysteine potentiates esterase-induced contraction on rat aorta in vitro:

A risk factor for atherosclerosis

Homosisteinin s›çan aortunda esteraz ile artt›r›lan in vitro kas›lmay› güçlendirmesi:

Athereosclerosis oluflumu için bir risk faktörü

F.B.H. Gurib, A.H. Subratty 79-83

Cytoembryological studies on Paeonia peregrina L.

Paeonia peregrina’da sitoembriyolojik çal›flmalar

R. Öztürk, M. Ünal 85-89

Immunogenicity and specificity of Salmonella typhimurium outer membrane

antigens

Salmonella typhimurium d›fl membran antijenlerinin immunojenite ve özgüllü¤ü

N. Ak›fl, O. Sayhan, A. Karaçavufl, K. Töreci 91-97

Polymerase chain reaction is a good diagnostic tool for Mycobacterium

tuberculosis in urine samples

‹drar örneklerinde Mycobacterium tuberculosis tan›s› için polimeraz zincir

reaksiyonunun kullan›m›

S. Arisan, N.C. Sönmez, Ö.O. Çak›r, E. Ergenekon 99-103

Cytological investigations in some important tree species of Rajasthan VI.

Radiation induced chromosome aberrations in Anogeissus pendula and A. latifolia

Rajastan IV için baz› önemli a¤aç türlerinde sitolojik araflt›rmalar: Anogeissus pendula

ve A. latifolia’da ›fl›nlama ile artan kromozom aberasyonlar›

A. Kumar, S.R. Rao, N.S. Shekhawat 105-111

Stimulation of regeneration by magnetic field in soybean (Glycine max L. Merrill)

tissue cultures

Soya (Glycine max L. Merrill) doku kültürlerinde rejenerasyonun manyetik alan

taraf›ndan stimulasyonu

Ç. Atak, Ö. Emiro¤lu, S. Alikamano¤lu, A. Rzakoulieva 113-119

Letters to editor 121-124

Book reviews 125-127

Instructions to authors 129-130

Volume content 131-132

Author index 133

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