Comparative chromosome painting in fish using human sex ...

Comparative chromosome painting in fish using
human sex chromosome probes J
N.S. NAGPURE, B. KUSHWAHA, SATISH K. SRNASTAVA,
A. HALDER AND A.G. PONNIAH
Nottom1 Bureau of Faah Genehc Rosaumrs, Canal Rang Road, Teitbagh, P 0
Ddkwha Lucknow - 226 002, lndm
ABSTRACT
The shldy wae undden w )dent@ la. chmmo.ome~ in Chnn~
pwaotva
wlnp human aci ehrmmome spafie w t prok Although he mbsa mvid
dem mx chromoliomaa in human memphve aprsld. no hybndruhon usnris
warn detd on any dtha chrommww m C puwta~ua mlha read* of the
pmasot study mdicatcd that hum sax ehrmollorna pahot probas lrsn not
w&l for d e a g ae. ehmmoaoma ~n Cknw punrlotur
Introduction
Sex determinauan is very mm~lex
phenomenon iofishsa and u detamned
by pneW enwoamentai factors. In gc
net~eally sex determined Alhes male
het~mgamety, female hetemgamety and
multrple ssx chmmowmes are known. Sa
far, sex chromosamea have been desrrikd
mfm apeoes by kmyotypngand
chmmoaome banding, smee sex chrw*
somes are not morpholcgidy differenttated
in many fil apenee. The recently
developed fluoreaeeocs m srtu hybribzatlon
(FISH1 teehmque hse been found
promising m ~dentilicahan of sex c b
mmomes m humane and Wer mom.
mala. Tbs technique has aha been utilid
to tdentify aex chmmwomes in few
fish* like platy &h, medaka, tilapia and
sdmorud sped@ IWIbp. 2001, FWhpa
and Read, 19961 Funber. FISH can be
used for mmparative tytqeonic ~tudiea
w lnveahgate tbe m o t md dhtribuoon
of conserved DNA aagments not ody bs
tween eloaely related species but alao
hetween drstantlv related soecles
bosomal DNA ~~DNAJ sequences have
been vasd w iaate the NOR, m drstantly
related spenes, for example human and
maws ~ibsamal DNA sequence8 wvld
loeata NORa m fishes (Rab el d., 1990,
Fischsr d d , 2000, Rnam el 01.. 2W01
Sdarly d would b mtarssting if aar
ehmmdwme spafie DNA sequences ob
ad fmm higher m a l a car be uhhwd
for mterspenfic mmpan~oo far he
moiogou sequences md eventual identifiutlan
ofaex chromosome m fiahsa.
The present atudy waa therefore und
d e n wtb a nsw to tdennh. wx chm
mesamesm6ah byw~erehmm(~~~me
spenfic pant probes &om human betug.
An atr breathmg teleaat. Chonna

Punetnhnlw~bhnsdmodsltishfmh wator and agsm dehydrated m wnea oi
study slnw dplold chrmnolame numbar ethanol .a menhond abwe
s lesser and sex chrommomes have not
been as far reparced(Mannaand Rmd,
H9b*tk"L
1973. S h a and Amd. 19811 Sa
In abtu hybnduatton af fish and huehrommwes
~dentfied on the basia oi man chromosome wps oerformed mul.
comparative FISH, can be utllucd far
asangeeUs from mmahlre or adult fieh
and aho for sennp of sprm IPhilLpe,
2W11
tail conr!etlng of 3.6-pl hybridization
Matariplrr nnd methods
buffer. 0.6~1 each ofX and Y praba md
0.6~1 tnpla &Ued water waa a~~hed
Chromo~omrpnpamtbnr
mumlip. mdaeded mth rubkcamat
Danaturationoiprobe md duamoms
Wms performed mmulrameaualy on a hot
plate at 74'C far 5 muur. Slides were m.
abated wnnight in mom chamber at
37'C. The mbkr eement and mver ahp
were removed end the slldsa were
waahed by mnrbahngm aolutian of0.35
Chrsmaaome spreads m Channa
pumtolw (n=lOI were pnplvedimm hdnay
tissue, wing eolctunnshypotomc.
methanol - acebe aad - mr drpg tseh.
mque Metaphaaeehmm-rs fmm the
normal human mda were obta~ned by
ualnsstandard lymphwe ruifure techniques
and chrme~lmerl~desasre pre
pared by above-mentioned technique
The chmmosome slides to be ued form
arb hybndisahoo wen -red at -2O'C
Whole chmmoaome pslnt IWCP) probes
for human Xand Y chmmaaame, dmctly
labelled with two duTamnt fluomphara.
ware prauredfmmVym. Frantm, for the
pre~snt study
The ahdea were thawed lust befm
use and treated wth 70% acsbe acid for
2 minutes, followed by dehydration in
senna of 70, 90 and 100% ethanol for 3
mui, each, at m m temprat-
h'P40 lauppl~ad by Vpls lac. France) at
74 * 1'C far 3 mutea fallowed by w-
tahng m aoluuon dNP (0.1%) at rmm
tWFemtm for 23 m um. Slidevert
than dehydrated m seriw of ethanol at
roam temperature. Dried slidea were
atained and mounted with l0pl DAPI-11
with antifade followed by waling with
tnmaparentrub~~ m d the*
ofmversltp.
Detection and oirualization of
h9ridpmbe~.
The slidea wore obaerved under
PnIrrcrtmmL.
The dehydrated audned ~Ldem we18
incubated In 1.25% ao&e ppm soluboo
Nikon fluomseant rmmgmp wing ormge'pn
ator Tbe FISH imase~wrn
captd by wled CCD camera and mnvertcd
O computer lmw by CyLmaion
far 20 rmnuree at 37'C followed by wash. Unu 4.1 aabm (Apphed un-) for
ins m howh hate buffered &s IPBSi dvna '
and ddob~c-dided water ~hdca am
mcubated for lomutea at 4'C m M y RB.ult4 rind
prepared precooled solutlon of Coutenr~mmgbyDAP1f~dLt.ted
pardomaldehyde 10.87%) prepared m ident~ficat~on of chremosomea of C.
PBS Slzdes were then removed and pvnetotva and all the apeimeu wan
warhed with PBS foUowed by distilled found to possess diploid chromosome

peen ngnd, wMuls wall chmmoaame
with red ngnal mheatad presence of Y
ehmmosome The FISH signals could be
d e w even m human mterphaee ceUa
(Rg 21 which confirmed the autability
of lhi8 techque to charatterire sea at
cellular level Mer, hybnhzatlon of
patot probea to human X and Y met.
aphaae chromosomes ~nsured the ear.
rectness of the methodology.
number of 2n= 32 (Fig 11 x hieh la con.
slsteat mth earlter repam (Manna and
Prsnad. 1973, Sharma snd Agamal,
1981) Fiuorescent slpnala were not ot.
served on any chromosomes of C
pumtatlia, wbchlnhcatedthat hyhdi.
zatlan of WCP probes to C punetotus
ehmmwmes could not take pie, due
to absence ofhomologovs DNAsequancea
between fish and human sex chram*
aom8s. since both the apeclss are
phyuogenehcally hatant. On the other
hand, chmmoeamesfromnormal human
male. when hybnhdwwlth same probe.
lluoresant ngDais were obsemed m all
Fyl 2 Maupk h a m e apnd d hu
man mde lhonog X and Y xr ~hrn
mororne patnted with nuortecent
pmba
the metaphase spreads lhg 21 wh~rh
Ymed ae pmhve eantrd XdvOmwme
m humm metaphue spread exbb~tad
-. -. -.- .-. . . quences ae chmmosomc a p d c probea
m p a r fiPh Tefmodon nlgmvzndis and
Oeddechdus Lbro (Fiachsr st 01.. 2000,
Rossl ef 01 , 2000) Similarly. the
IITAGGN) n. sequ~n-a found at human
blomerss have been found soaaew~d 10
many vertebrate apenea (Mewe rt a!.,
19891, heow Lelomenr probea have also
been erarmned m Ocdnlrchiivs laboa in
whch teiomem6 embitad swon~double
signals (Roarnet01,2WO1 However, lack
of h*nhzatlon of human %r chrome
aome probes m C punefotus ehromoaomes
as obsewld in the present study
inheated Ihat humao sex chiomoaoms
punt probes were not useful for deteet-
Ing aex chromosamss In Chonnn
punetntus
Acknowledgement
The authors are graMul w the Head,
De~srtmsntofMadtcal Gsnetles. Sway
Gandh~ Post Graduate Mdcd Inatltuh,
Lucknow. far provlhng labarstory faciil.
Ilea and valuable svggemons
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