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c I C 2AATTCCAGG GGTTGGGG TGCGCCTTTTCCAAGGCAG CCCTGGG TrTGCGCAGG GACGCG

GCTGCTCTGGGCGTGGTTCCGGGAMCGCAGCGGCGCCGACCCTGGGTCTCGCACATTCT

TCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGA

A l A2

CGCTTCCTGCTCCGCCCCTAAGTCGGGMGGTTCCTTGCGGTTCGCGGCGTGCCGGACGT4

D 2 D i

GACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCMD l

TGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCG

TGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCMGCCTCCGGAGCGCACGTCGGCAGTCG

I n iGCTCCCTCGT TGACCGMTCACCGACCTC TC TCCCCAGCTGTA 1 T TCCAAAATGTCG CTT TCT AAC AAG CTG A CG CTGGACAAGCTG GACGTT

r 4F 2

F i g .1 .S e q u en ce o ft h eCp G- 3 7 8 i s l a n dc o n t a i n i n gt h e p r o -- 3 1 8 mo t e ra n df i r s te x o no ft h e- 2 5 8 h u m a n P GK -1g e n e .A r -r o w s i n d i c a t et h eo l i g o n u -

c l e o t i d ep r i m e r p o s i t i o n s- 1 9 8 a n dt h ed i r e c t i o no f r e a d i n g .

T h e a s t e r i s ki n d i c a t e st h e- 1 3 8 m a j o rt r a n s c r i p t i o ns t a r t

s i t e ." I n i "m a r k st h et r a n s l a -- 7 8 t i o ns t a r tp o i n t .- 1 8

+ 4 3

+ 9 8

+ 1 3 7

Fl

P v uI Ip r o b e ,t h es e q u e n c ed e f i n e db y p r i m -e rs e tF b ec am ev i s ib l e ( F i g .2 B ) .A ss h o w n ,b o t h s e q u e n c e sc o u l d b e r e a du n a m b i g -u o u s l y ;t h ec o r r e c tc a l l sw o u l db em a d ef o rat o t a lo f 3 3 6 n u c l e o t i d e s ,e v e n i f t h e s e -q u e n c ew e r en o t k n o w n .5 -M e t h y l c y t o s i n ea p p e a r sa sa g a p i n t h e

c v t o s i n el a d d e ro f a Ma x a m - G i l b e r ts e -q u e n c i n gg e l .To t e s tt h ep r o c e d u r ea s a na s s a yf o rme t h y l a t e dc y t o s i n e s ,we s t u d i e dh u m a n - h a m s t e rh y b r i d c e l ll i n e st h a tc o n -t a i n e de i t h e ra n i n a c t i v eo r a n a c t i v eh u m a nX c h r o m o s o m e( 1 7 ) .P r i m e rs e tA ( F i g .1 )a l l o w e dr e a d i n go f t h es e q u e n c et o w a r dt h et r a n s c r i p t i o ns t a r ts i t e ,a n d t h ed a t ai n d i c a t et h a tt h eh u m a ni n a c t i v eX c h r o m o s o m ei sm e t h y l a t e da t a l l1 7 Cp G d i n u c l e o t i d e si nt h ei n v e s t i g a t e dr e g i o n( F i g .3 , l a n eX i ) ,wh e r e a st h ea c t i v eXic h r o m o s o m ei s u n -m e t h y l a t e da t t h es a m e1 7 s i t e s( F i g .3 , l a n eXa ) .By c o m p a r i s o nw i t h c l o n edDNA, we

f o u n dt h a tHeLa DNA h a sn o d e t e c t a b l em e t h y l a t i o na t 5 2 Cp G d i n u c l e o t i d e sa n a -l y z e di n t h e PGK-1 - a s s o c i a t e dC pG - r i c hi s l a n d ,a r e s u l tc o n s i s t e n tw i t h t h i sc e l ll i n en o t h a v i n ga n i n a c t i v eX c h r o m o s o m e .A l t h o u g ht h e b a n d p a t t e r n ss h o w ni n

F i g s .2 a n d3 a r er e p r o d u c i b l e ,b a n di n t e n s i -t i e sv a r yf r o mf r a gm e n tt of r a gme n tw i t h i nas in g l e l an e ;a f ew b a n d sa r ew e a ko r e v e nm i s s i n go n s h o r te x p o s u r e s .F o r e x am p l e ,t h r e eb a n d si na r e g i o no fa b o u t1 0 0n u c l e o -t i d e sa r en o t s e e n i n t h es e q u e n c es h o w ni nF i g .3 .B a n di n t e n s i t ym a yv a r yb e c a u s e( i )t h ec h e m i c a lr e a c t i v i t yo f i n d i v i d u a lb a s e sm a yd i f f e r ;( i i )s m a l le f f e c t so f s e q u e n c eo nam p l i f i c a t i o ne f f i c i e n c ym a yp l a ys o m er o l e ,a l t h o u g hPCR c a n b e v e r yq u a n t i t a t i v ea n dr e p r o d u c i b l e[4% SD t h r o u g h2 5 c y c l e s( 1 8 ) ] ;( i i i )c e r t a i ns e q u e n c e sm a yn o t b ee x t e n d e dt o a b l u n te n d a s e f f i c i e n t l yi np r i m e re x t e n s i o np r i o r t ol i g a t i o n ;( i v )t h el i g a t i o ns t e pm a y i n t r o d u c es o m e b i a s .O v e r e x p o s e da u t o r a d i o g r a m sc a n o f t e nb eu s e d t o o b t a i ns e q u e n c ei nf o r ma ti o n e v enf r o mw e a kb a n d s .T h i si st h ec a s ef o rm o s to f t h es i t e st h a twe h a v es t u d i e d ,s u c ha st h e

Cp G s i t ea t n u c l e o t i d ep o s i t i o n- 1 1 9 . T h ei n t e n s i t i e so f o n l yt h ew e a k b a n d s w e r ei n c r e a s e db y i n c r e a s in g t h et i m e f o rl i g a t i o no r t h et e m p e r a t u r eo f t h ef i r s tp r i m e re x t e n -s i o nr e a c t i o n( 1 9 ) .T h u s ,t h es t e p sb e f o r et h ePCR m a yb e v e r ye f f i c i e n tf o rs t r o n gb a n d s .E x p o n e n t i a la m p l i f i c a t i o nw o r k ss ow e l l

t h a tb a n dd e t e c t i o np r o b a b l yn o l o n g e rl i m -i t ss e n s i t i v i t y.C o m p a r i n gb a n d i n t e n s i t i e sw i t h t h e s t a n d a r dg e n o m i cs e q u e n c i n gm e t h o da p p l i e dt o t h e s a m ePGK-1 s e -q u e n c es t u d i e dw i t h p r i m e rs e tD , we e s t i -

G C G CG A T CG A T C

- 3 1 3 -