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Isolation and characterization of DNA probes from the short arm of the human X chromosome which detect… Starr, Terence 1986

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ISOLATION AND CHARACTERIZATION OF DNA PROBES FROM THE SHORT ARM OF THE HUMAN X CHROMOSOME WHICH DETECT RESTRICTION FRAGMENT LENGTH POLYMORPHISMS by T e r e n c e S t a r r B . S c , Simon F r a s e r U n i v e r s i t y , 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MEDICAL GENETICS We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIV^RSITY^ilF^RITISH^-OOLUMBIA May 1986 (c) T e r e n c e S t a r r , 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of V \ ^ A A C Q - S 3^ 15L^ ^^ JJ<A e S The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) i i A b s t r a c t The i s o l a t i o n and c h a r a c t e r i z a t i o n of DNA probes which d e t e c t RFLPs from a small r e g i o n of the human genome, Xp, has been the focus of t h i s t h e s i s . T h i r t y X s p e c i f i c DNA probes were i s o l a t e d from a flow s o r t e d l i b r a r y c o n s t r u c t e d from a 49,XXXXY c e l l l i n e . These probes were confirmed to be from the X chromosome by h y b r i d i z a t i o n to dot b l o t s c o n t a i n i n g DNA from mouse, human, and a somatic c e l l h y b r i d which c o n t a i n e d the X chromosome as i t s o n l y human DNA. H y b r i d i z a t i o n to somatic c e l l h y b r i d s c o n t a i n i n g d i f f e r e n t r e g i o n s of the X chromosome l o c a l i z e d 9 of the probes to Xp. A f t e r t e s t i n g 185 probe-enzyme combinations t h r e e of the Xp probes ( l o c a l i z e d to the d i s t a l 2/3 of Xp) were found to d e t e c t RFLPs. One probe, pTS316, was shown not t o be i n h e r i t e d i n a Mendelian f a s h i o n . The presence of one of the polymorphic bands d e t e c t e d by t h i s probe c o r r e l a t e d with the phenomenon of X chromosome i n a c t i v a t i o n . i i i TABLE OF CONTENTS ABSTRACT i i L I S T OF TABLES v L I S T OF FIGURES v i ACKNOWLEDGEMENTS v i i INTRODUCTION 1 . The Human L i n k a g e Map 1 2. G e n e r a l Uses o f RFLPs 2.1 RFLPs as D i a g n o s t i c M a r k e r s 3 2.2 RFLPs i n P o p u l a t i o n S t u d i e s 5 3. The Xp L i n k a g e Map 6 MATERIALS AND METHODS 1. F l o w S o r t e d Human X L i b r a r y 1.1 D e s c r i p t i o n o f L i b r a r y 10 1.2 A m p l i f i c a t i o n o f L i b r a r y 10 2. S u b c l o n i n g o f L sHX I n s e r t s i n t o P l a s m i d V e c t o r s 2.1 DNA D i g e s t i o n 11 2.2 L i g a t i o n 11 2.3 T r a n s f o r m a t i o n 12 3. S o m a t i c C e l l C u l t u r e s 3.1 L y m p h o b l a s t C e l l L i n e s 12 3.2 F i b r o b l a s t and Rodent-Human H y b r i d C e l l L i n e s 13 4. DNA I s o l a t i o n 4.1 L sHX DNA 15 4.2 P l a s m i d DNA 15 4.3 C e l l C u l t u r e s 19 4.4 Whole B l o o d Samples 19 5. DNA E l e c t r o p h o r e s i s 20 6. N i c k T r a n s l a t i o n 21 7. H y b r i d i z a t i o n 7.1 C o l o n y H y b r i d i z a t i o n 22 7.2 Dot B l o t H y b r i d i z a t i o n 23 7.3 S o u t h e r n H y b r i d i z a t i o n 24 8. Common R e a g e n t s , S o l u t i o n s , and B u f f e r s 25 i v RESULTS AND DISCUSSION 1. I s o l a t i o n o f S i n g l e Copy DNA Sequences 1.1 D e t e c t i o n of R e p e t i t i v e DNA 28 1.2 P l a s m i d V e c t o r s 30 1.3 I s o l a t i o n of X S p e c i f i c Probes 32 2. D e t e c t i o n of Xp S p e c i f i c Probes 2.1 Arm L o c a l i z a t i o n of X Probes 38 2.2 C h a r a c t e r i z a t i o n of Probe pTSl 1 9 42 3. D e t e c t i o n of RFLPs 3.1 C h a r a c t e r i z a t i o n of 4 New P o l y m o r p h i c Markers 49 3.2 RFLP Frequency on the X Chromosome 51 4. P e d i g r e e A n a l y s i s 4.1 F a m i l y S e g r e g a t i o n of RFLPs 57 4.2 C h a r a c t e r i z a t i o n of Probe pTS264 i n Two DMD F a m i l i e s 59 4.3 Non-Me.ndelian S e g r e g a t i o n of the pTS31 6 RFLP 59 5. G e n e r a l D i s c u s s i o n 70 6. C o n c l u s i o n s 73 REFERENCES .75 V L I S T OF TABLES T a b l e 1. DNA E l e c t r o p h o r e s i s 21 T a b l e 2. C h a r a c t e r i z a t i o n o f R e c o m b i n a n t P l a s m i d s 31 T a b l e 3. H y b r i d i z a t i o n o f Xp p r o b e s t o H i n d l l l d i g e s t s o f DNA e x t r a c t e d f r o m c e l l l i n e s c o n t a i n i n g v a r i o u s r e g i o n s o f t h e human X chromosome 46 T a b l e 4. Summary o f P o l y m o r p h i c P r o b e s 51 T a b l e 5. Enzymes Used t o D e t e c t RFLPs 53 T a b l e 6. D i s t r i b u t i o n o f TS316 Bands i n M a l e s and F e m a l e s 63 v i L I S T OF FIGURES F i g u r e 1. S c r e e n i n g f o r R e p e t i t i v e DNA 35 F i g u r e 2. D e t e r m i n a t i o n o f Chromosomal O r i g i n o f Rec o m b i n a n t I s o l a t e s U s i n g Dot B l o t s 36 F i g u r e 3. D e t e r m i n a t i o n o f Chromosomal O r i g i n o f R e c o m b i n a n t I s o l a t e s U s i n g S o u t h e r n B l o t s 37 F i g u r e 4. Arm L o c a l i z a t i o n o f X P r o b e s by Dosage 44 F i g u r e 5. DNA R e t a i n e d i n C e l l L i n e s Used f o r Arm L o c a l i z a t i o n B l o t s 45 F i g u r e 6. Arm L o c a l i z a t i o n o f X S p e c i f i c P r o b e s U s i n g S o m a t i c C e l l H y b r i d s 47 F i g u r e 7. H y b r i d i z a t i o n o f P r o b e pTS119 t o DNA From S o m a t i c C e l l H y b r i d s C o n t a i n i n g R e g i o n s o f t h e X Chromosome .....48 F i g u r e 8. D e t e c t i o n o f RFLPs 55 F i g u r e 9. Genomic O r g a n i z a t i o n o f Xp P r o b e s D e t e c t i n g RFLPs 56 F i g u r e 10. B g l l l P o l y m o r p h i s m D e t e c t e d W i t h P r o b e pTS247 64 F i g u r e 11. X b a l P o l y m o r p h i s m D e t e c t e d W i t h P r o b e pTS264 65 F i g u r e 12. A v a i l P o l y m o r p h i s m D e t e c t e d W i t h P r o b e pTS316 66 F i g u r e 13. P v u l l P o l y m o r p h i s m D e t e c t e d W i t h P r o b e pTS119 67 F i g u r e 14. C o - s e g r e g a t i o n o f DMD and pTS264 68 F i g u r e 15. H y b r i d i z a t i o n o f P r o b e pTS316 t o C e l l L i n e s W i t h D i f f e r e n t Numbers o f X Chromosomes 69 v i i Acknowedgements I w o u l d l i k e t o t h a n k Bob S h u k i n , B r u c e W i l s o n , and F r a n c e s Lee f o r t h e i r e x c e l l e n t t e c h n i c a l a s s i s t a n c e ; Ann M a r i e H o w e l l , L i n d a H a r r i s , Dr. Ann Rose, and Dr. S t e p h e n Wood f o r t h e i r h e l p f u l comments i n p r e p a r i n g t h i s m a n u s c r i p t ; Dr. L . K u n k e l f o r p r o v i d i n g t h e human X l i b r a r y ; D r. J . Hamerton f o r p r o v i d i n g t h e 835K3 and 422K6 h y b r i d c e l l l i n e s ; D r s . M. H a r r i s and D. J u r i l o f f f o r p r o v i d i n g m i c e ; my s u p e r v i s o r D r . S t e p h e n Wood f o r h i s p a t i e n c e and l e a d e r s h i p ; my mom and dad f o r t h e i r c o n s t a n t s u p p o r t ; and e s p e c i a l l y t o my w i f e T r u d y f o r h e r u n d e r s t a n d i n g , d e d i c a t i o n , and e n c o u r a g e m e n t . - 1 -INTRODUCTION 1. THE HUMAN LINKAGE MAP The human genome contains up to 50,000 genes (M cKusick and Ruddle, 1977) and i s estimated to be 3,000 centimorgans (cM) i n length, where 1 cM represents a physical distance of 1x1 0^ base pairs on average (Renwick, 1969). In order to better understand human genetics a comprehensive human linkage map i s e s s e n t i a l . Before the era of molecular genetics M cKusick (1975) had catalogued more than 1,200 l o c i , most of which were known by the occurrence of rare disorders. Since the advent of recombinant DNA technology i n the early 1970's, t h i s number has r i s e n to 3,400 (M cKusick, 1983). However only 400 of these have been mapped due to the paucity of highly polymorphic marker l o c i (Skolnick et a l , 1984; McAlpine et a l , 1985). The systematic construction of a human linkage map must therefore r e l y on an al t e r n a t i v e approach. Botstein et a l (1980) proposed that a linkage map could be generated using DNA probes to define marker l o c i which are polymorphic i n DNA sequences. The polymorphic DNA sequences could be d i r e c t l y assayed as r e s t r i c t i o n fragment length polymorphisms (RFLPs) after DNA digestion with r e s t r i c t i o n enzymes and Southern b l o t t i n g (Southern 1975). They hypothesized that i f RFLPs could be s e l e c t i v e l y positioned every 20 cM then approximately 150 polymorphic l o c i would be required to span the entire genome. Any new locus to be mapped would be a maximum of 10 cM away from a known marker and amenable to genetic analysis. -2-As i t i s n o t y e t p o s s i b l e t o c h o s e a t w i l l p r o b e s o f p r e c i s e l o c a t i o n , more f e a s i b l e a p p r o a c h e s a r e r e q u i r e d . S k o l n i c k and W h i t e (1982) d i s c u s s e d s t r a t e g i e s f o r d e t e c t i n g RFLPs by c o n s i d e r i n g t y p e s o f p r o b e s , r e s t r i c t i o n enzymes, and t h e number o f i n d i v i d u a l s t o be t e s t e d . The methods now i n u s e d i f f e r p r i m a r i l y i n t h e s o u r c e o f DNA and i n t h e number o f r e s t r i c t i o n enzymes u s e d . D e t e r m i n i n g w h i c h s o u r c e o f p r o b e s t o u s e depends upon t h e e x p e r i m e n t a l g o a l . G r o u p s i n t e r e s t e d i n f o c u s i n g on a c hromosomal r e g i o n , mapping a chromosome o r f i n d i n g a RFLP l i n k e d t o a d i s e a s e l o c u s , have u t i l i z e d DNA l i b r a r i e s c o n s t r u c t e d f r o m f l o w s o r t e d chromosomes ( D r a y n a e t a l , 1984) o r f r o m s o m a t i c c e l l h y b r i d s ( B i s h o p e t a l , 1 9 8 3 ) . O t h e r g r o u p s i n t e r e s t e d i n t h e genome i n g e n e r a l ( F e d e r e t a l , 1985) have u t i l i z e d more c o m p l e t e human l i b r a r i e s s u c h as t h a t o f M a n i a t i s ( M a n i a t i s e t a l , 1 9 7 8 ) . The c o n s t r u c t i o n o f t h e human l i n k a g e map i s w e l l underway w i t h t h e l o c a t i o n o f a p p r o x i m a t e l y 1500 l o c i f i r m l y e s t a b l i s h e d (de l e C h a p e l l e , 1 9 8 5 ) . The u s e f u l n e s s o f h a v i n g e v e n a p a r t i a l l y c o m p l e t e d map h a s r e c e n t l y b e e n d e m o n s t r a t e d by t h e m apping o f t h e c y s t i c f i b r o s i s gene. C y s t i c f i b r o s i s i s one o f t h e most common g e n e t i c d i s e a s e s i n C a u c a s i a n p o p u l a t i o n s a f f e c t i n g 1 i n 2,000 l i v e b i r t h s ( W a i n w r i g h t e t a l , 1 9 8 5 ) . D e s p i t e an enormous amount o f e f f o r t by many r e s e a r c h e r s , n o t h i n g was known a b o u t t h e gene o r t h e gene p r o d u c t . U s i n g t h e a v a i l a b l e RFLPs r e s e a r c h g r o u p s had b e e n a b l e t o e x c l u d e t h e gene f r o m a p p r o x i m a t e l y 40% o f t h e genome (Newmark, 1 9 8 5 ) . T s u i e t a l (1985) were t h e f i r s t g r o u p t o r e p o r t t h e d i s c o v e r y o f a -3-l i n k e d RFLP t o c y s t i c f i b r o s i s and o t h e r r e s e a r c h g r o u p s (White e t a l , 1985a; W a i n w r i g h t e t a l , - 1 9 8 5 ) were q u i c k l y a b l e t o i d e n t i f y c l o s e l y l i n k e d RFLPs once t h e chromosomal l o c a t i o n was known. The c l o s e l y l i n k e d RFLPs may now be u s e d f o r d i a g n o s t i c p u r p o s e s and as t h e s t a r t i n g p o i n t t o i s o l a t e t h e gene. 2. G e n e r a l U ses o f RFLPs 2.1 RFLPs as D i a g n o s t i c M a r k e r s RFLPs a r e g e n e r a t e d by DNA b a s e s u b s t i t u t i o n s , i n s e r t i o n s , d e l e t i o n s , and r e a r r a n g e m e n t s . They a r e i n h e r i t e d as c o - d o m i n a n t g e n e t i c m a r k e r s and may be u s e d f o r p r e n a t a l d i a g n o s i s and c a r r i e r d e t e c t i o n o f i n d i v i d u a l s a t r i s k f o r g e n e t i c d i s e a s e s . I n g e n e r a l RFLPs a r e u s e f u l a s a l i n k e d m a r k e r f o r t h e d e f e c t i v e gene and c a n o n l y be u s e d p r e d i c t i v e l y i n f a m i l y s t u d i e s . The f i r s t u s e o f an RFLP as a d i a g n o s t i c m arker was by Kan and Dozy (1978) i n t h e s t u d y o f s i c k l e c e l l a n e m i a . T h e s e r e s e a r c h e r s showed a c o r r e l a t i o n between s i c k l e - c e l l anemia and a 13.0 kb H p a l f r a g m e n t when S o u t h e r n b l o t s were h y b r i d i z e d w i t h a B - g l o b i n p r o b e . Normal i n d i v i d u a l s i n h e r i t e d a 7.6 o r a 7.0 kb f r a g m e n t , w h i l e 87% o f i n d i v i d u a l s w i t h t h e s i c k l e - c e l l p h e n o t y p e had t h e 13.0 kb v a r i a n t . T h i s v a r i a n t was shown t o be i n h e r i t e d i n a M e n d e l i a n f a s h i o n and s e g r e g a t e d w i t h t h e s i c k l e - c e l l t r a i t i n i n f o r m a t i v e f a m i l i e s . Thus t h e 13 kb H p a l f r a g m e n t c o u l d be u s e d f o r l i n k a g e a n a l y s i s o f t h e s i c k l e - c e l l _4-g ene. By a v o i d i n g t h e r i s k y p r o c e d u r e o f f e t a l b l o o d s a m p l i n g (by u s i n g DNA i s o l a t e d f r o m f e t a l a m n i o t i c c e l l s ) t h i s work p a v e d t h e way f o r p r e n a t a l t e s t i n g . S i n c e t h e n RFLPs have been e x t e n s i v e l y u s e d w i t h a v a s t number o f g e n e t i c d i s e a s e s i n c l u d i n g many i n w h i c h t h e p r i m a r y b i o c h e m i c a l d e f e c t i s n o t y e t known. T h i s e m p h a s i z e s one o f t h e m a j o r a d v a n t a g e s RFLPs p o s s e s s . A l i n k e d RFLP c a n be u s e d d i a g n o s t i c a l l y e v e n when n o t h i n g i s known a b o u t t h e gene o r gene p r o d u c t , a s i s t h e c a s e w i t h Duchenne m u s c u l a r d y s t r o p h y (DMD) ( H a r p e r e t a l , 1 9 8 3 ) . However, t h e u s e o f RFLPs as d i a g n o s t i c m a r k e r s r e q u i r e s c a r e f u l a n a l y s i s t o a v o i d some common o b s t a c l e s . S i n c e an RFLP i s n o t t h e c a u s a t i v e a g e n t o f a d i s e a s e b u t i s a l i n k e d m a r k e r , t h e r e e x i s t s t h e p o s s i b i l i t y o f a r e c o m b i n a t i o n e v e n t o c c u r r i n g b etween t h e m a r k e r and t h e d i s e a s e l o c u s d u r i n g m e i o s i s . The p r o b a b i l i t y o f s u c h an e v e n t depends upon t h e g e n e t i c d i s t a n c e b e t w e e n t h e two s i t e s . An example i s t h e G8 p r o b e and H u n t i n g t o n s D i s e a s e . I n t h i s c a s e , t h e l i n k e d m arker i s 5 map u n i t s f r o m t h e H u n t i n g t o n s l o c u s ( G u s e l l a e t a l , 1983.). Thus, t h e r e e x i s t s t h e p o s s i b i l i t y o f an i n c o r r e c t d i a g n o s i s 5 t i m e s o u t o f 100. T h i s c a n be d r a s t i c a l l y r e d u c e d by h a v i n g 2 l i n k e d m a r k e r s , one on e i t h e r s i d e o f t h e gene, t h u s l i m i t i n g t h e e r r o r i n d i a g n o s i s t o t h e f r e q u e n c y o f a d o u b l e c r o s s - o v e r e v e n t . Of c o u r s e , t i g h t l y l i n k e d m a r k e r s a r e much more v a l u a b l e t h a n l o o s e l y l i n k e d o n e s . I n some c a s e s , s u c h as p h e n y l k e t o n u r i a (PKU), t h e RFLPs a r e f o u n d w i t h i n t h e gene (Woo e t a l , 1983), t h e r e b y f o r a l l p r a c t i c a l p u r p o s e s e l i m i n a t i n g t h e c o m p l i c a t i o n s p r o d u c e d by r e c o m b i n a t i o n . -5-A n o t h e r d i f f i c u l t y e n c o u n t e r e d when u s i n g RFLPs d e a l s w i t h t h e i n f o r m a t i o n c o n t e n t o f a m a r k e r . D i a l l e l i c s y s t e m s a r e c o n f i n e d by t h e r e s t r a i n t t h a t c r i t i c a l f a m i l y members may be homozygous f o r t h e same a l l e l e t h u s r e n d e r i n g no i n f o r m a t i o n . T h i s i s more p r o n o u n c e d when t h e m i n o r a l l e l e i s i n f r e q u e n t l y f o u n d i n a p o p u l a t i o n , r e s u l t i n g i n few i n f o r m a t i v e m a t i n g s . To c i r c u m v e n t t h i s , a d d i t i o n a l RFLPs d e t e c t e d by t h e same p r o b e a r e s o u g h t t o i n c r e a s e t h e p o l y m o r p h i s m i n f o r m a t i o n c o n t e n t o f a m a r k e r . J e f f r e y s e t a l (1985) have r e p o r t e d a number o f m u l t i a l l e l i c RFLPs c o n t a i n i n g h y p e r v a r i a b l e tandem r e p e a t s w h i c h y i e l d c o r r e s p o n d i n g l y h i g h h e t e r o z y g o s i t i e s . The i n c l u s i o n o f many p r o b e s o f t h i s t y p e i n t o t h e l i n k a g e map w i l l be v e r y b e n e f i c i a l . 2.2 RFLPs i n P o p u l a t i o n S t u d i e s P o p u l a t i o n g e n e t i c i s t s h a v e a l s o f o u n d RFLPs as u s e f u l t o o l s t o a d d r e s s e x p e r i m e n t a l q u e s t i o n s . U s i n g a H p a l p o l y m o r p h i s m Kan and Dozy (1980) showed t h a t t h e s i c k l e - c e l l a l l e l e ( B s ) f o u n d i n A m e r i c a n b l a c k s o r i g i n a t e d i n West A f r i c a and t h a t i t a r o s e i n d e p e n d e n t l y o f a n o t h e r B - g l o b i n m u t a t i o n , h e m o g l o b i n C. They were a b l e t o u s e t h i s d a t a t o p o s t u l a t e t h a t t h e s e m u t a t i o n s o r i g i n a t e d i n a s m a l l g e o g r a p h i c a r e a i n Ghana. A n t o n a r a k i s e t a l (1982) u s i n g a number o f RFLPs f r o m t h e B - g l o b i n c l u s t e r p r e s e n t e d e v i d e n c e t h a t a B E - g l o b i n m u t a t i o n a r o s e t w i c e i n S o u t h e a s t A s i a . C h a k r a v a r t i e t a l (1984) were a b l e t o d e m o n s t r a t e t h a t a h o t s p o t f o r m e i o t i c r e c o m b i n a t i o n e x i s t s i n t h e B - g l o b i n c l u s t e r . By u s i n g RFLPs f r o m t h i s r e g i o n t h e y showed t h e r e e x i s t s 2 c l u s t e r s o f n o n - r a n d o m l y a s s o c i a t e d -6-DNA p o l y m o r p h i s m s , one 5' and t h e o t h e r 3' t o t h e B - g l o b i n s t r u c t u r a l g ene. They s u b s e q u e n t l y d e m o n s t r a t e d t h a t t h e r e was no s i g n i f i c a n t l i n k a g e d i s e q u i l i b r i u m between t h e two c l u s t e r s and were a b l e t o l o c a l i z e t h e r e c o m b i n a t i o n a l h o t s p o t t o a 9.1 kb r e g i o n i m m e d i a t e l y 5' t o t h e B - g l o b i n gene. 3. The Xp L i n k a g e Map, The s h o r t arm o f t h e X chromosome (Xp) c o n t a i n s 6xl-07 bp (1/3 t h e s i z e o f t h e e n t i r e X) and r e p r e s e n t s 2% o f t h e human genome. The g e n e t i c d i s t a n c e o f Xp i s e s t i m a t e d t o be 90 cM ( D r a y n a and W h i t e , 1985) s i m i l a r t o t h e l o n g arm ( X q ) . I f t h e gene d e n s i t y i s r e l a t i v e l y c o n s t a n t t h e n b a s e d on t h e e s t i m a t e o f 50,000 genes i n t h e h a p l o i d genome i t w o u l d be e x p e c t e d t h a t t h e Xp arm e n c o d e s a p p r o x i m a t e l y 1,000 g e n e s . A s s u m i n g t h a t t h e genes were u n i f o r m l y d i s t r i b u t e d t h e n t h e gene d e n s i t y on t h e Xp arm w o u l d be one gene e v e r y 0.09 cM (60,000 b p ) . T h e r e i s l i t t l e e v i d e n c e y e t a v a i l a b l e t o i n d i c a t e t h e a c t u a l gene d e n s i t y t h r o u g h o u t t h e genome. E v i d e n c e o f gene c l u s t e r i n g has b e e n shown f o r b o t h t h e B - g l o b i n c l u s t e r o n chromosome 11 and t h e a - g l o b i n c l u s t e r on chromosome 16 ( r e v i e w e d by K a r l s o n and N e i n h u i s , 1 9 8 5 ) . O t h e r e v i d e n c e has shown t h a t genes s u c h as F a c t o r V I I I ( G i t s c h i e r e t a l , 1984) a r e i n e x c e s s o f 100 kb i n l e n g t h . Thus t h e e s t i m a t e o f one gene e v e r y 60,000 bp c a n n o t be v a l i d a t e d . By 1953 t h i r t y f i v e X - l i n k e d genes were known. I n t h e 30 y e a r s s i n c e t h e n M c ( K u s i c k (1 983) has c a t a l o g u e d 115 genes a s s i g n e d t o t h e X. C o n f i r m e d l o c i ( genes and a r b i t r a r y DNA s e q u e n c e s ) on t h e X chromosome now number a t l e a s t 250 (Shows e t a l , 1984; G o o d f e l l o w e t a l , 1985) w i t h g r e a t e r t h a n 45 a s s i g n e d t o t h e Xp arm. S e v e n t e e n genes have been a s s i g n e d t o Xp, a p p r o x i m a t e l y 2% o f t h e e s t i m a t e d number. O n l y 2 o f t h e s e g e n e s , OTC ( H o r w i c h e t a l , 1984) and MIC2 ( C u r r y e t a l , 1 9 8 4 ) h a v e b e e n c l o n e d . R e c e n t e v i d e n c e by Worton (Ray e t a l , 1985) and K u n k e l (Monaco e t a l , 1985) s u g g e s t s t h a t a n o t h e r gene, t h e Duchenne m u s c u l a r d y s t r o p h y gene l o c a t e d a t Xp21, has a l s o b e e n c l o n e d . F o r t y - e i g h t a r b i t r a r y DNA s e q u e n c e s h ave b e e n a s s i g n e d t o Xp. Of t h e s e 3 9 d e t e c t RFLPs however, 7 a r e n o t i n c l u d e d i n t h e Xp l i n k a g e map due t o t h e i r r e c e n t i s o l a t i o n o r due t o t h e r a r i t y o f t h e m i n o r a l l e l e . I n i t i a l e f f o r t s t o c l o n e t h e DMD gene as w e l l as p r o v i d e g e n e t i c m a r k e r s f o r l i n k a g e a n a l y s i s has l e d t o a w e l l d e v e l o p e d l i n k a g e map o f Xp. The DMD l o c u s was t h o u g h t t o be l o c a t e d a t Xp21 due t o t h e e x p r e s s i o n o f t h e d i s e a s e i n f e m a l e s w i t h X:autosome t r a n s l o c a t i o n s i n v o l v i n g t h e Xp21 r e g i o n ( J a c o b s e t a l , 1981; V e r e l l e n - D u m o u l i n e t a l , 1 9 8 4 ) . W i l l i a m s o n ' s g r o u p was t h e f i r s t t o e s t a b l i s h l i n k a g e t o DMD w i t h an a r b i t r a r y c l o n e d p r o b e (RC8) t h a t had b e en mapped t o t h e Xp21 t o Xp22.3 r e g i o n ( M u r r a y e t a l , 1 9 8 2 ) . T h i s g r o u p a l s o e s t a b l i s h e d l i n k a g e t o DMD w i t h a s e c o n d p r o b e (L1.28) w h i c h had b e e n l o c a l i z e d t o t h e Xp11 r e g i o n ( D a v i e s e t a l , 1 9 8 3 ) . T h i s c o n f i r m e d t h e c y t o g e n e t i c p l a c e m e n t o f t h e DMD l o c u s a t Xp21 and -8-s p u r r e d o t h e r g r o u p s t o t e s t s i m i l a r m e t h o d o l o g i e s . A l d r i d g e e t a l (1984) w h i l e d e v e l o p i n g a s c r e e n i n g s t r a t e g y t o d e t e c t h i g h f r e q u e n c y X chromosome p o l y m o r p h i s m s mapped 3 RFLPs t o t h e Xp arm: pB24, p99-6, and pD2 w h i c h were a l l mapped bet w e e n Xp21 and X p t e r . L i k e w i s e H o f k e r e t a l (1985) i s o l a t e d and mapped 2 more RFLPs (754 a t Xp11.3 t o Xp21, and 782 a t Xp22.2 t o Xp22.3) w h i l e s e a r c h i n g f o r m a r k e r s l i n k e d t o DMD. T h r e e s e p a r a t e g r o u p s i s o l a t e d 3 i n d e p e n d e n t RFLPs t h a t h a v e b e e n mapped t o Xp: p r o b e C7 i s o l a t e d by Mandel ( D o r k i n e t a l , 1985) has been l o c a l i z e d t o t h e r e g i o n Xp21.2 t o Xp21.3; t h e p r o b e d i c 5 6 i s o l a t e d by K u n k e l ( D r a y n a and W h i t e , 1985) has b e e n l o c a l i z e d t o t h e Xp22.3 r e g i o n ; t h e p r o b e p58-1 ( B r u n s e t a l , 1982) maps t o t h e Xp11 r e g i o n . R o z e n e t a l , (1985) u s i n g a cDNA c l o n e o f t h e OTC gene i d e n t i f i e d 2 RFLPs l i n k e d t o DMD ( D a v i e s e t a l , 1 9 8 5 ) w h i c h have b e e n mapped t o t h e Xp11.4 t o Xp21 r e g i o n . The pERT p o l y m o r p h i c p r o b e s ( K u n k e l e t a l , 1985) have b e e n mapped t o t h e Xp21 r e g i o n and a r e t h o u g h t t o be l o c a t e d w i t h i n t h e DMD l o c u s (Monaco e t a l , 1 9 8 5 ) . Thus s u b s t a n t i a l p r o g r e s s i n c l o n i n g t h e DMD l o c u s as w e l l as mapping t h e Xp arm has been made i n t h e 3 y e a r s s i n c e t h e f i r s t Xp RFLP was r e p o r t e d . As i t s t a n d s t o d a t e , t h e l i n k a g e map o f Xp c o n t a i n s 13 g e n e t i c m a r k e r s i n g e n e r a l u s e ; 10 a r b i t r a r y DNA s e q u e n c e s , 2 genes d e t e c t i n g RFLPs, and t h e b l o o d g r o u p marker Xg. T h e s e m a r k e r s a r e c l o s e enough t o e a c h o t h e r t h a t any new X p - l i n k e d l o c u s c a n be mapped t o w i t h i n 10 cM o f a t l e a s t 2 m a r k e r s . As t h e number o f mapped m a r k e r s i n c r e a s e s e v e n t u a l l y an unambiguous gene o r d e r w i l l become known a l l o w i n g p l a c e m e n t o f any m a r k e r o n t o t h i s p o r t i o n o f t h e map. - 9 -The work presented i n t h i s t h e s i s was undertaken i n order t o : 1) develop methodologies f o r r a p i d l y i s o l a t i n g probes from the X chromosome; 2) i s o l a t e new X p - l i n k e d polymorphic markers; 3) c o n t r i b u t e t o the g e n e r a t i o n of an Xp-linkage map by r e g i o n a l l y l o c a l i z i n g any newly i s o l a t e d polymorphic probes; and 4) p r o v i d e a d d i t i o n a l markers f o r d i a g n o s t i c use. MATERIALS AND METHODS 1. F l o w S o r t e d Human X L i b r a r y 1.1 D e s c r i p t i o n o f L i b r a r y The f l o w s o r t e d human X l i b r a r y (L sHX) was a g i f t o f Dr. L o u i s K u n k e l ( K u n k e l e t a l , 1 9 8 2 ) . I n b r i e f , m e t a p h a s e chromosomes f r o m a 49,XXXXY l y m p h o b l a s t c e l l l i n e were i s o l a t e d s t a i n e d w i t h 33258 H o e c h s t dye and s o r t e d a c c o r d i n g t o f l u o r e s c e n c e i n t e n s i t y . The s o r t e d m a t e r i a l was e s t i m a t e d t o c o n t a i n 30 t o 60% X chromosome DNA b a s e d on t h e r e l a t i v e i n c r e a s e o f f l u o r e s c e n t i n t e n s i t y i n t h e r e g i o n e x p e c t e d f o r t h X chromosome. DNA was i s o l a t e d f r o m a chromosome f r a c t i o n e n r i c h e d f o r t h e X and c l o n e d i n t o t h e H i n d l l l s i t e o f t h e lambda phage C h a r o n 21 A. The r e s u l t i n g 60,000 phage, r e p r e s e n t i n g 80 t o 90% o f t h e X chromosome, were a m p l i f i e d , p u r i f i e d i n a C s C l g r a d i e n t , and s t o r e d a t 4°C. 1.2 A m p l i f i c a t i o n o f L i b r a r y ( M a n i a t i s e t a l , 1982) F i v e ml o f L u r i a b r o t h (L b r o t h ) was i n o c u l a t e d w i t h E s c h e r i c h i a c o l i , s t r a i n LE 392. The c e l l s were i n c u b a t e d a t 3 7 ° C f o r 18 h o u r s . A 1.1 ml a l i q u o t o f t h i s c u l t u r e was i n f e c t e d w i t h 110 u l o f L sHX phage ( d i l u t e d t o 1x10^ p l a q u e f o r m i n g u n i t s ( p f u ) w i t h L d i l u t i o n b u f f e r (L d i l ) ) , and i n c u b a t e d a t 3 7 ° C f o r 10 m i n u t e s . The i n f e c t e d c e l l s were m i x e d w i t h 3 ml o f t o p a g a r o s e , p o u r e d o n t o L b r o t h p l a t e s , and -11 -i n c u b a t e d o v e r n i g h t a t 3 7 ° C. F i v e ml o f L d i l was p o u r e d on t o p o f e a c h c o n f l u e n t l y l y s e d p l a t e , and i n c u b a t e d a t 4 ° C f o r 18 h o u r s . The L d i l o v e r l a y was c o l l e c t e d , c e n t r i f u g e d a t 10,000 rpm f o r 10 m i n u t e s , and t h e s u p e r n a t a n t (phage s t o c k ) was c o l l e c t e d and s t o r e d a t 4° C. 2. S u b c l o n i n g o f L sHX I n s e r t s i n t o P l a s m i d V e c t o r s ( D a v i s e t a l , 1980) 2.1 R e s t r i c t i o n Enzyme D i g e s t i o n To e a c h o f 4 ug o f L sHX phage DNA and 1.6 ug o f p l a s m i d DNA was a d d e d 4.0 u l o f 10X c o r e b u f f e r , 4.0 u l o f 10X BSA, 10 u n i t s o f t h e r e s t r i c t i o n enzyme H i n d l l l ( B R L ) , and d i s t i l l e d w a t e r t o b r i n g t h e t o t a l r e a c t i o n volume t o 40 u l . The m i x t u r e was i n c u b a t e d a t 37° C f o r 2 h o u r s , t h e n a t 6 5 ° C f o r 10 m i n u t e s . A 10 u l a l i q u o t was removed and e l e c t r o p h o r e s e d as d e s c r i b e d ( s e c t i o n 5) t o c h e c k t h a t t h e d i g e s t i o n was c o m p l e t e . 2.2 L i g a t i o n A 10.0 u l a l i q u o t o f H i n d l l l d i g e s t e d L sHX DNA was m i x e d w i t h e i t h e r 10.0 u l o f H i n d l l l d i g e s t e d pUC13, pUC19 ( Y a n i s h - P e r r o n e t a l , 1 9 8 5 ) , o r p i S L 1 3 ( S e e d , 1983) p l a s m i d DNA. To t h i s was a d d e d 4.0 u l o f 10X l i g a t i o n b u f f e r (500 mM T r i s , 100 mM M g C l ) , 4.0 u l o f 10X BSA, 4.0 u l o f 10 mM ATP, 4.0 u l o f 0.1M d i t h i o t h r e i t o l , 0.2 u l o f T4 l i g a s e , 4.0 u l o f d i s t i l l e d w a t e r and i n c u b a t e d a t 1 5 ° C f o r 18 h o u r s . -12-2.3 T r a n s f o r m a t i o n Ten u l o f t h e a b o v e l i g a t i o n r e a c t i o n was m i x e d and i n c u b a t e d a t 0° C f o r 60 m i n u t e s w i t h 100 u l o f c o m p e t e n t E .  c o l i , s t r a i n JM83 ( f o r u s e w i t h pUC v e c t o r s ) o r s t r a i n FP3 c e l l s ( f o r u s e w i t h p i S L 1 3 ) . A f t e r i n c u b a t i o n a t 4 2 ° C f o r 10 m i n u t e s , 1 ml o f s t e r i l e L b r o t h was a d d e d and i n c u b a t e d a t 3 7 ° C f o r 45 m i n u t e s . A 50 u l a l i q u o t was p l a t e d on t o X - g a l p l a t e s , i n c u b a t e d a t 3 7 ° C f o r 18 h o u r s , and s t o r e d a t 4° C. G l y c e r o l ( 50% v : v ) was a d d e d t o t h e r e m a i n d e r o f t h e t r a n s f o r m a t i o n r e a c t i o n and s t o r e d a t - 2 0 ° C. The t r a n s f o r m a t i o n e f f i c i e n c y was 2 x 10^ t r a n s f o r m a n t s p e r ug o f p l a s m i d . 3. S o m a t i c C e l l C u l t u r e s Most c e l l l i n e s u s e d i n t h i s work were p u r c h a s e d f r o m t h e Human G e n e t i c M u t a n t C e l l R e p o s i t o r y I n s t i t u t e f o r M e d i c a l R e s e a r c h (Camden, New J e r s e y ) and a r e d e n o t e d by GM numbers. The r e m a i n d e r were g i f t s f r o m c o l l e a g u e s and a r e n o t e d i n t h e acknowledgement s e c t i o n . 3.1 L y m p h o b l a s t C e l l L i n e s G r o w i n g l y m p h o b l a s t o i d c e l l s were m a i n t a i n e d i n RPMI 1640 medium a t 3 7 ° C f o r 3 t o 7 d a y s . Dense c u l t u r e s were r e p e a t e d l y s p l i t 1:3 (V:V) w i t h RPMI u n t i l g r e a t e r t h a n 400 ml were c o l l e c t e d . The c e l l s were c e n t r i f u g e d a t 5,000 rpm f o r 10 m i n u t e s , washed i n 0.85% N a C l , p e l l e t e d , and r e s u s p e n d e d i n 5 ml -13-o f h i g h TE. To t h i s was added, by r a p i d i n j e c t i o n u s i n g a s y r i n g e , 5 ml o f l y s i s b u f f e r . The l y s e d c e l l s were s t o r e d a t 4° C u n t i l r e q u i r e d f o r DNA i s o l a t i o n . RPMI Medium RPMI 1640 medium (Gibco L a b o r a t o r i e s C at. No. 430-1800) 15% F e t a l C a l f Serum 3% Soduim B i c a r b o n a t e (7.5%) L y s i s B u f f e r 100 mM T r i s (pH 8) 40 mM EDTA 0.2% Sodium L a u r y l S u l f a t e (SDS) 3.2 F i b r o b l a s t and Rodent-Human H y b r i d C e l l L i n e s Growing rodent-human s o m a t i c c e l l h y b r i d o r f i b r o b l a s t c e l l l i n e s were m a i n t a i n e d i n MEM medium a t 37° C u n t i l a complete monolayer formed (75 cm^) i n 3 t o 7 days. To s p l i t the c u l t u r e t h e monolayer was r i n s e d t w i c e w i t h 1X Hanks s o l u t i o n , t h e n i n c u b a t e d a t 37° C f o r a p p r o x i m a t e l y 5 minutes w i t h a t r y p s i n s o l u t i o n . The c e l l s were s p l i t 1:3, t r a n s f e r r e d t o new f l a s k s c o n t a i n i n g f r e s h MEM medium, and m a i n t a i n e d a t 37° C u n t i l a new monolayer formed. To c o l l e c t t h e c e l l s f o r l y s i s t h e f l a s k was r i n s e d t w i c e w i t h 1X Hanks, i n c u b a t e d a t 37° C f o r 5 minutes w i t h a t r y p s i n s o l u t i o n , and c e n t r i f u g e d a t 2500 rpm f o r 10 m i n u t e s . The p e l l e t was washed w i t h 0.85% N a C l , resuspended i n 5 ml o f h i g h TE, and l y s e d w i t h 5 ml o f l y s i s b u f f e r . The l y s e d c e l l s were s t o r e d a t 4° C u n t i l r e q u i r e d f o r DNA i s o l a t i o n . -14-MEM Medium Minimum E s s e n t i a l Medium (Gibco L a b o r a t o r i e s Cat. No. 410-1500) 15% F e t a l C a l f Serum 3% Sodium Bicar b o n a t e (7.5%) 1X Hanks S o l u t i o n Hanks Balanced S a l t S o l u t i o n (Gibco L a b o r a t o r i e s Cat. No. 450-1200) T r y p s i n S o l u t i o n 5% B a c t o - T r y p s i n ( D i f c o L a b o r a t o r i e s ) 9 5% 1X Hanks S o l u t i o n L y s i s B u f f e r 100 mM T r i s (pH 8) 40 mM EDTA 0.2% Sodium L a r y l Sulphate (SDS) 4. DNA I s o l a t i o n ( D a v i s e t a l , 1980) 4.1 I s o l a t i o n o f Phage DNA 4.1.1 PEG P r e c i p i t a t i o n o f Phage S t o c k To 77 ml o f phage s t o c k ( s e c t i o n 1.2) was a d d e d 7.7 g o f p o l y e t h y l e n e g l y c o l (PEG) and 4.5 g N a C l a t room t e m p e r a t u r e . The m i x t u r e was g e n t l y i n v e r t e d u n t i l a l l t h e s a l t d i s s o l v e d , t h e n i n c u b a t e d a t 4° C f o r 3 h o u r s . The r e s u l t i n g w h i t e p r e c i p i t a t e was c e n t r i f u g e d a t 10,000 rpm f o r 10 m i n u t e s , and r e s u s p e n d e d i n 9.0 ml o f L d i l . To t h i s 6.75 g o f CsCL was a d d e d , m i x e d , t r a n s f e r r e d t o a 12 ml h e a t s e a l a b l e t u b e , and c e n t r i f u g e d a t 37,000 rpm f o r 66 h o u r s i n a Beckman T i 5 0 r o t o r . The phage band was removed w i t h a 1 ml s y r i n g e , t r a n s f e r r e d t o a 1.5 ml m i c r o f u g e t u b e , and s t o r e d a t 4° C. 4.1.2 Formamide E x t r a c t i o n o f L sHX Phage DNA A 50 u l a l i q u o t o f L sHX p u l l e d f r o m a C s C l g r a d i e n t ( s e c t i o n 4.1.1) was m i x e d w i t h 5 u l o f 2 M T r i s , 0.2 M EDTA (pH 8 . 5 ) , 50 u l f o r m a m i d e , and l e f t a t room t e m p e r a t u r e f o r 60 m i n u t e s . To t h i s , 50 u l w a t e r , 300 u l 95% e t h a n o l was a dded, i n v e r t e d u n t i l m i x e d , t h e n c e n t r i f u g e d a t 12,000 rpm f o r 2 s e c o n d s . The p r e c i p i t a t e d DNA was d i s s o l v e d i n 50 u l o f 1X T E . 4.2 P l a s m i d DNA 4.2.1 DNA I s o l a t i o n by a M i n i P r e p P r o c e d u r e R e c o m b i n a n t p l a s m i d c l o n e s t r a n s f o r m e d i n t o t h e a p p r o p r i a t e E . c o l i h o s t ( s e c t i o n 2) were t r a n s f e r r e d t o 10 ml o f L b r o t h c o n t a i n i n g 15 t o 50 ug/ml a m p i c i l l i n and i n c u b a t e d a t 3 7 ° C -16-f o r 18 h o u r s . The c u l t u r e was c e n t r i f u g e d a t 2500 rpm f o r 5 m i n u t e s and r e s u s p e n d e d i n 1 ml o f l y s i s b u f f e r . The m i x t u r e was t r a n s f e r r e d t o a 1.5 ml m i c r o f u g e t u b e and i n c u b a t e d a t room t e m p e r a t u r e f o r 10 m i n u t e s . To t h i s 20 u l o f 10% SDS was added, m i x e d , i n c u b a t e d a t 2 0 ° C f o r 10 m i n u t e s , and c e n t r i f u g e d a t 12,000 rpm f o r 10 m i n u t e s . The s u p e r n a t a n t was d e c a n t e d i n t o a 1.5 ml t u b e , m i x e d w i t h 5 u l o f d i e t h y l p y r o c a r b o n a t e (DEP), and i n c u b a t e d a t 7 0 ° C f o r 15 m i n u t e s . A f t e r h e a t i n g , 100 u l o f 5 M p o t a s s i u m a c e t a t e was added, m i x e d , c o o l e d i n an i c e - w a t e r b a t h f o r 30 m i n u t e s , and c e n t r i f u g e d a t 12,000 rpm f o r 10 m i n u t e s . The s u p e r n a t a n t was d e c a n t e d i n t o a new m i c r o f u g e t u b e , m i x e d w i t h 1/10 volume o f 4 M ammonium a c e t a t e , 1 volume o f i s o p r o p a n o l , and c e n t r i f u g e d a t 12,000 rpm f o r 10 m i n u t e s . The p e l l e t was a i r d r i e d , d i s s o l v e d i n 100 u l o f 1X TE, and s t o r e d a t 4° C. I f n e c e s s a r y f o r r e s t r i c t i o n enzyme d i g e s t i o n t h e p l a s m i d DNA was e x t r a c t e d o n c e w i t h p h e n o l , once w i t h Sevag ( S e c . 8 ) , p r e c i p i t a t e d w i t h 4 M ammonium a c e t a t e and i s o p r o p a n o l , and d i s s o l v e d i n 1X T E . The a v e r a g e y i e l d was 5 t o 10 ug o f p l a s m i d DNA. L y s i s B u f f e r 50 mM T r i s 50 mM EDTA 15% S u c r o s e 5 mg/ml l y s o z y m e (SIGMA C h e m i c a l s G r a d e I ) added j u s t p r i o r t o u s e . -17-4.2.2 DNA I s o l a t i o n by a L a r g e S c a l e P r o c e d u r e R e c o m b i n a n t p l a s m i d c l o n e s , t r a n s f o r m e d i n t o t h e a p p r o p r i a t e E . c o l i h o s t s ( s e c t i o n 2 ) , were t r a n s f e r r e d t o 10 ml o f L b r o t h c o n t a i n i n g 15 t o 50 ug/ml a m p i c i l l i n and i n c u b a t e d a t 3 7 ° C f o r 18 h o u r s . The c u l t u r e was t r a n s f e r r e d t o f r e s h L b r o t h (250 t o 1000 ml) c o n t a i n i n g a m p i c i l l i n and i n c u b a t e d 3 t o 24 h o u r s a t 37° C. To t h i s was a d d e d 0.6 ml o f c h l o r a m p h e n i c o l (80 mg/ml i n e t h a n o l ) f o r e v e r y 250 ml o f L b r o t h c u l t u r e and i n c u b a t e d a t 37 ° C f o r 18 h o u r s . The c e l l s were c e n t r i f u g e d a t 7,000 rpm f o r 10 m i n u t e s , and t h e p e l l e t was r e s u s p e n d e d i n 5 ml o f l y s i s b u f f e r . The m i x t u r e was i n c u b a t e d a t 2 0 ° C f o r 5 m i n u t e s , t h e n 20 ml o f 0.2 M NaOH, 1% SDS was added, m i x e d , and c o o l e d i n an i c e - w a t e r b a t h f o r 10 m i n u t e s . To t h i s 10 ml o f s o l u t i o n I was m i x e d , c o o l e d on i c e f o r 10 m i n u t e s , and c e n t r i f u g e d a t 19,000 rpm f o r 20 m i n u t e s . The s u p e r n a t a n t was d e c a n t e d i n t o a new t u b e and m i x e d w i t h an e q u a l volume o f i s o p r o p a n o l . T h i s was l e f t a t 2 0 ° C f o r 1 t o 18 h o u r s t h e n spun a t 2,500 rpm f o r 10 m i n u t e s . The p e l l e t was d i s s o l v e d i n 5 ml o f 1X TE, mixed w i t h 0.65 ml e t h i d i u m b r o m i d e (10 mg/ml), 7.5 ml o f w a t e r s a t u r a t e d C s C l , t r a n s f e r r e d t o a 12 ml h e a t s e a l a b l e t u b e , and c e n t r i f u g e d a t 60,000 rpm f o r 18 h o u r s a t 1 5 ° C. The p l a s m i d b and was removed f r o m t h e g r a d i e n t u s i n g a 1 ml s y r i n g e and t r a n s f e r r e d t o a new t u b e . The DNA was r e p e a t e d l y e x t r a c t e d w i t h an e q u a l volume o f w a t e r s a t u r a t e d b u t a n o l u n t i l t h e aqueous l a y e r was f r e e o f e t h i d i u m b r o m i d e . The aqueous l a y e r was m i x e d w i t h 2 v o l u m e s o f w a t e r , 6 volume s o f 95% e t h a n o l , and l e f t a t 2 0 ° C f o r 1 t o 18 h o u r s . The DNA was c e n t r i f u g e d a t -18-2,500 rpm f o r 10 m i n u t e s and d i s s o l v e d i n 1 ml o f 1X T E . The a v e r a g e y i e l d was 500 t o 1,000 ug o f p l a s m i d DNA. L y s i s B u f f e r 25 mM T r i s (pH 8) 10 mM EDTA 50 mM s u c r o s e 5 mg/ml l y s o z y m e (SIGMA C h e m i c a l s G r a d e I ) a d d e d j u s t p r i o r t o u s e S o l u t i o n I 60 ml 5 M P o t a s s i u m A c e t a t e 11.5 ml G l a c i a l A c e t i c A c i d 28.5 ml Water 4.2.3 I s o l a t i o n o f P l a s m i d DNA I n s e r t s P l a s m i d DNA, 5 t o 10 ug, was d i g e s t e d t o c o m p l e t i o n w i t h t h e a p p r o p r i a t e r e s t r i c t i o n enzyme (as o u t l i n e d by t h e m a n u f a c t u r e r ) and e l e c t r o p h o r e s e d as d e s c r i b e d ( s e c t i o n 5 ) . The DNA bands were v i s u a l i z e d w i t h l o n g wave u l t r a v i o l e t (U.V.) l i g h t and t h e a p p r o p r i a t e band was t r a n s f e r r e d t o d i a l y s i s t u b i n g . Enough 1X TE was a d d e d t o c o v e r t h e g e l and t h e DNA was e l e c t r o p h o r e s e d . The b u f f e r f r o m w i t h i n t h e t u b i n g was removed, t r a n s f e r r e d t o a 1.5 ml m i c r o f u g e t u b e , c e n t r i f u g e d a t 12,000 rpm t o p e l l e t any r e s i d u a l a g a r o s e , and t r a n s f e r r e d t o a new t u b e . The DNA was p r e c i p i t a t e d w i t h 4 M ammonium a c e t a t e and i s o p r o p a n o l as d e s c r i b e d ( s e c t i o n 4.2.1) and d i s s o l v e d i n 50 u l o f 1X T E . -19-4.3 C e l l C u l t u r e s The l y s e d c e l l s ( s e c t i o n 3.1, 3.2) were v o r t e x e d w i t h an e q u a l volume of p h e n o l u n t i l a u n i f o r m w h i t e e m u l s i o n was produced and mixed o v e r n i g h t a t room t e m p e r a t u r e . The e m u l s i o n was c e n t r i f u g e d a t 2,500 rpm f o r 10 m i n u t e s , then th e aqueous phase was removed and t r a n s f e r r e d t o a new t u b e . The i n t e r f a c e was r e - e x t r a c t e d w i t h an e q u a l volume of h i g h TE, c e n t r i f u g e d , and t h e aqueous l a y e r s combined. The combined aqueous l a y e r s were e x t r a c t e d once w i t h p h e n o l , then once w i t h sevag. A t each e x t r a c t i o n c a r e was t a k e n t o l e a v e t h e i n t e r f a c e b e h i n d . The DNA was p r e c i p i t a t e d by m i x i n g w i t h 1/10 volume of 4 M ammonium a c e t a t e and t h e n l a y e r i n g 1 volume of i s o p r o p a n o l on t o p . The aqueous and i s o p r o p a n o l l a y e r s were mixed by g e n t l e i n v e r s i o n u n t i l h i g h m o l e c u l a r w e i g h t DNA c o u l d be seen t o p r e c i p i t a t e . The DNA was removed w i t h a g l a s s r o d , r i n s e d w i t h 70% e t h a n o l , t r a n s f e r r e d t o a m i c r o f u g e t u b e , and d i s s o l v e d i n 1X TE. The y i e l d s v a r i e d from 250 t o 5,000 ug depending on the number of c e l l s o r i g i n a l l y c o l l e c t e d . 4.4 DNA From Whole B l o o d Samples One volume (10 t o 30 ml) of f r e s h o r thawed b l o o d was mixed w i t h 5 volumes of s o l u t i o n I , i n c u b a t e d a t 37° C f o r 10 m i n u t e s , c e n t r i f u g e d a t 2,500 rpm f o r 10 m i n u t e s , t h e n a l l but 5 ml o f t h e s u p e r n a t a n t was drawn o f f by s u c t i o n . The remainder was mixed w i t h 10 ml of 0.85% NaCl and c e n t r i f u g e d a t 2,500 rpm f o r 10 m i n u t e s . The p e l l e t was d i s s o l v e d i n 2 ml o f h i g h TE, t h e n l y s e d by r a p i d i n j e c t i o n o f 2 ml o f l y s i s b u f f e r ( s e c t i o n 4.2.2). The l y s e d c e l l s were t r e a t e d as d e s c r i b e d p r e v i o u s l y -20-( s e c t i o n 4 . 3 ) . The a v e r a g e y i e l d f r o m 20 ml o f f r e s h b l o o d was 500 ug o f DNA. S o l u t i o n I 90% 0.155 M NH 4C1 10% 0.17 M T r i s (pH 7.6) 4.5 Genomic DNA From Mouse L i v e r ( K u n k e l e t a l , 1977) Two f r e s h l y d i s s e c t e d l i v e r s f r o m f e m a l e m i c e were h o m o g e n i z e d i n 50 ml o f 0.1 M N a C l , 25 mM T r i s (pH 8 . 0 ) , 50 mM EDTA. The homogenate was m i x e d w i t h 5.5 ml o f 10% SDS, 1 ml o f p r o t e a s e K (5 mg/ml), and i n c u b a t e d a t 37° C f o r 2 h o u r s . T h i s was p h e n o l e x t r a c t e d and t h e DNA p e l l e t e d as d e s c r i b e d ( s e c t i o n 4 . 3 ) . The p e l l e t was d i s s o l v e d i n 25 ml o f 30 mM s o d i u m a c e t a t e , 2 mM EDTA and i n c u b a t e d a t 37° C f o r 2 h o u r s w i t h 625 u l o f RNase A (1 mg/ml). The DNA was p r e c i p i t a t e d a s d e s c r i b e d ( s e c t i o n 4 . 3 ) . 5. DNA E l e c t r o p h o r e s i s R e s t r i c t i o n enzyme d i g e s t e d genomic and p l a s m i d DNA was s i z e f r a c t i o n a t e d by means o f a g a r o s e g e l e l e c t r o p h o r e s i s . A g a r o s e , d i s s o l v e d i n 1X TBE b u f f e r (0.4 t o 1.2% W:V) c o n t a i n i n g 1 ug e t h i d i u m b r o m i d e p e r ml o f g e l , was p o u r e d i n t o a h o r i z o n t a l m o l d and a l l o w e d t o s o l i d i f y a t room t e m p e r a t u r e . The s o l i d i f i e d g e l s were submerged i n 1X TBE b u f f e r and t h e DNA e l e c t r o p h o r e s e d a t v a r i o u s v o l t a g e s as o u t l i n e d i n T a b l e 1. -21 -T a b l e 1. DNA E l e c t r o p h o r e s i s Band S i z e s E x p e c t e d % g e l s g e l s i z e volume v o l t a g e t i m e (mm) (ml) ( h r s ) s i n g l e genomic f r a g m e n t s 0.8 150x200 130 30 18-24 b e tween 0-30 kb m u l t i p l e g e nomic f r a g m e n t s 0.5 150x250 150 40-45 1 8-24 > 15 kb m u l t i p l e genomic f r a g m e n t s 1.2 150x200 130 30 18-24 < 1 kb p l a s m i d s 0.8 60x100 20 80 2 p l a s m i d i n s e r t s 0.8 150x200 150 40 18-24 6. N i c k T r a n s l a t i o n ( M a n i a t i s e t a l , 1982) To a 1.5 ml m i c r o f u g e t u b e t h e f o l l o w i n g were added on i c e : 250 ng DNA, 60 u C i d A T P ( a - 3 2 P New E n g l a n d N u c l e a r 10 mCi/ml, 3,000 C i / m m o l e ) , 2.5 u l o f n i c k t r a n s l a t i o n b u f f e r , 2.5 u l o f a m i x t u r e o f u n l a b e l e d n u c l e o t i d e s , 1.0 u l o f DNase I (0.1 u g / m l ) , 1.0 u l DNA p o l y m e r a s e I , and d i s t i l l e d w a t e r t o b r i n g t h e t o t a l r e a c t i o n volume t o 25 u l . The m i c r o f u g e t u b e was t r a n s f e r r e d t o a l e a d c o n t a i n e r , i n c u b a t e d a t 15° C f o r 2 h o u r s , t h e n s t o p p e d by a d d i n g 65 u l o f n i c k t r a n s l a t i o n s t o p b u f f e r . The i n c o r p o r a t e d n u c l e o t i d e s were s e p a r a t e d f r o m t h e u n i n c o r p o r a t e d n u c l e o t i d e s on a Sephadex G-25 medium column ( S n u t c h 1984) by -22-c e n t r i f u g i n g a t 2,000 rpm f o r 2 m i n u t e s . The e l u t e d f r a c t i o n c o n t a i n i n g t h e r a d i o a c t i v e DNA was t r a n s f e r r e d t o a m i c r o f u g e t u b e , b o i l e d f o r 10 m i n u t e s , and r a p i d l y c o o l e d i n an i c e - w a t e r b a t h j u s t p r i o r t o u s e . T y p i c a l s p e c i f i c a c t i v i t i e s o b t a i n e d were i n t h e o r d e r o f 1 t o 4x10^ cpm/ug DNA. N i c k T r a n s l a t i o n B u f f e r 0.5 M T r i s (pH 8) 0.5X BSA 0.1 M M g C l 2 U n l a b e l e d N u c l e o t i d e s 0.17 mM d e o x y r i b o t h y m i d i n e t r i p h o s p h a t e 0.17 mM d e o x y r i b o c y t i d i n e t r i p h o s p h a t e 0.17 mM d e o x y r i b o g u a n o s i n e t r i p h o s p h a t e N i c k T r a n s l a t i o n S t o p B u f f e r 0.02 M T r i s (pH 8) 1 mg/ml Salmon Sperm DNA ( b o i l e d and s o n i c a t e d ) 0.2% SDS 7. DNA H y b r i d i z a t i o n 7.1 C o l o n y H y b r i d i z a t i o n E . c o l i c e l l s , s t r a i n JM83, c o n t a i n i n g r e c o m b i n a n t p l a s m i d s ( s e c t i o n 2) were t r a n s f e r r e d t o n i t r o c e l l u l o s e l a y e r e d on X - g a l L b r o t h p l a t e s , and i n c u b a t e d a t 3 7 ° C o v e r n i g h t . The n i t r o c e l l u l o s e f i l t e r was s o a k e d t w i c e i n 0.5 M NaOH f o r 3 -23-m i n u t e s , t h e n o n c e f o r 5 m i n u t e s i n 1 M T r i s (pH 7 . 5 ) , and once f o r 5 m i n u t e s i n 1.5 M N a C l , 0.5 M T r i s (pH 7 . 5 ) . The f i l t e r s were b a k e d a t 8 0 ° C f o r two h o u r s and s t o r e d a t 4° C u n t i l r e q u i r e d f o r h y b r i d i z a t i o n . The n i t r o c e l l u l o s e f i l t e r s were p r e h y b r i d i z e d i n 10 ml o f 5X SSPE, 0.3% SDS f o r 1 t o 18 h o u r s a t 6 2 ° C i n s e a l e d p l a s t i c b a g s . The n i c k t r a n s l a t e d p r o b e ( s e c t i o n 6) was d e n a t u r e d by b o i l i n g p r i o r t o u s e and 1x10^ cpm/100cm2 o f f i l t e r was ad d e d t o t h e b a g . H y b r i d i z a t i o n was c a r r i e d o u t a t 6 2 ° C f o r 18 h o u r s . The f i l t e r s were removed and washed t w i c e a t 62° C f o r 30 m i n u t e s i n 0.1X SSC, 0.5% SDS , r i n s e d a t 20° C f o r 5 m i n u t e s i n 0.1X SSC, t h e n a i r d r i e d . The d r i e d f i l t e r s were e n c l o s e d i n p l a s t i c wrap and a u t o r a d i o g r a p h e d f o r 1 t o 3 d a y s a t room t e m p e r a t u r e u s i n g Kodak XRP-1 f i l m and DuPont i n t e n s i f y i n g s c r e e n s . 7.2 Dot B l o t H y b r i d i z a t i o n ( L a u e t a l , 1984) E. c o l i c e l l s s t r a i n JM83 o r FP3 c o n t a i n i n g r e c o m b i n a n t p l a s m i d s ( s e c t i o n 2) were t r a n s f e r r e d t o 1 ml o f L b r o t h c o n t a i n i n g a m p i c i l l i n (50 ug/ml f o r pUC13 o r 15 ug/ml f o r p i S L l 3 r e c o m b i n a n t s ) and i n c u b a t e d a t 37° C f o r 18 h o u r s . A 100 u l a l i q u o t was t r a n s f e r r e d t o a 1.5 ml m i c r o f u g e t u b e c o n t a i n i n g 100 u l o f 0.3 M NaOH, 2 M N a C l , b o i l e d f o r 15 m i n u t e s , t h e n c o o l e d i n an i c e - w a t e r b a t h f o r 10 m i n u t e s . The e n t i r e 200 u l was d o t t e d on t o n i t r o c e l l u l o s e ( s o a k e d i n 1 M ammonium a c e t a t e ) u s i n g a BRL h y b r i - d o t m a n i f o l d , and r i n s e d w i t h 200 u l o f 10X SSC. The f i l t e r s were b a k e d and h y b r i d i z e d a s p r e v i o u s l y d e s c r i b e d ( s e c t i o n 7 . 1 ) . -24-7.3 S o u t h e r n H y b r i d i z a t i o n 7.3.1 Gene S c r e e n as Membrane S u p p o r t R e s t r i c t i o n enzyme d i g e s t e d DNA (5 t o 10 ug) was e l e c t r o p h o r e s e d ( s e c t i o n 5) and t r a n s f e r r e d t o Gene S c r e e n membrane as f o l l o w s : t h e a g a r o s e g e l was s o a k e d a t room t e m p e r a t u r e i n 0.25 M H C l f o r 15 m i n u t e s ; 0.2 M NaOH, 0.6 M N a C l f o r 30 m i n u t e s ; and 0.025 M Na2HP04/NaH 2P04 (pH 6.5) f o r 60 m i n u t e s . The g e l was c a r e f u l l y p l a c e d on t o p o f a Whatman 3MM w i c k s o a k i n g i n t h e a b o v e sodium p h o s p h a t e b u f f e r . A p i e c e o f Gene S c r e e n membrane c u t t o t h e s i z e o f t h e g e l was p l a c e d on t o p , f o l l o w e d by 2 p i e c e s o f Whatman 3MM, and 4 t o 5 i n c h e s o f p a p e r t o w e l s . The DNA was a l l o w e d t o t r a n s f e r f o r 18 t o 24 h o u r s . The g e l was r e s t a i n e d w i t h e t h i d i u m b r o m i d e t o v i s u a l i z e t h e amount o f t r a n s f e r . The membrane was b a k e d a t 8 0 ° C f o r 2 h o u r s and s t o r e d a t 4 ° C u n t i l r e q u i r e d f o r h y b r i d i z a t i o n . The Gene S c r e e n f i l t e r s were p r e h y b r i d i z e d f o r 18 h o u r s a t 42 ° C i n 10 ml o f t h e f o l l o w i n g : 50% for m a m i d e , 10X D e n h a r t s s o l u t i o n , 1.0 M N a C l , 0.1% sodium p y r o p h o s p h a t e , 1% SDS, 10% d e x t r a n s u l p h a t e , and 100 ug/ml s a l m o n sperm DNA. To t h i s 1x10^ cpm (50 t o 100 ng) o f f r e s h l y n i c k - t r a n s l a t e d p r o b e ( s e c t i o n 6) was added, and i n c u b a t e d f o r 18 h o u r s a t 42° C. The f i l t e r s were washed i n : 0.3 M N a C l , 0.06 M T r i s (pH 8.0) f o r 10 m i n u t e s a t 2 0 ° C; 0.3 M N a C l , 0.06 M T r i s (pH 8 . 0 ) , 0.002 M EDTA (pH 8 . 0 ) , 1% SDS f o r 60 m i n u t e s a t 6 2 ° C; 0.006 M T r i s ( p H 8 . 0 ) , 0.0002 M EDTA (pH 8.0) f o r 15 m i n u t e s a t 2 0 ° C. The f i l t e r s were a i r d r i e d and a u t o r a d i o g r a p h e d as p r e v i o u s l y d e s c r i b e d ( s e c t i o n 7 . 1 ) . -25-7.3.2 N i t r o c e l l u l o s e o r N y t r a n as Membrane S u p p o r t R e s t r i c t i o n enzyme d i g e s t e d DNA (5 t o 10 ug) was e l e c t r o p h o r e s e d as p r e v i o u s l y d e s c r i b e d ( s e c t i o n 5 ) . The DNA was t r a n s f e r r e d t o e i t h e r n i t r o c e l l u l o s e o r N y t r a n as f o l l o w s . The g e l was s o a k e d a t room t e m p e r a t u r e i n : 0.25 M H C l f o r 15 m i n u t e s ; 0.5 M NaOH, 1.5 M N a C l f o r 45 m i n u t e s ; 1.0 M ammonium a c e t a t e f o r 30 m i n u t e s . The DNA was t r a n s f e r r e d t o t h e membrane by c a p i l l a r y a c t i o n as d e s c r i b e d ( s e c t i o n 7.3.1) u s i n g 10X SSC as t h e t r a n s f e r b u f f e r . The f i l t e r s were b a k e d a t 80° C f o r a minimum o f 1 h o u r and s t o r e d a t 4° C u n t i l r e q u i r e d f o r h y b r i d i z a t i o n . The n i t r o c e l l u l o s e f i l t e r s were h y b r i d i z e d and washed as d e s c r i b e d ( s e c t i o n 7 . 1 ) . The N y t r a n f i l t e r s were p r e h y b r i d i z e d f o r 1 t o 18 h o u r s a t 4 2 ° C i n t h e f o l l o w i n g b u f f e r : 50% f o r m a m i d e , 6x SSC, 5X D e n h a r t s s o l u t i o n , 1% SDS, and 100 ug/ml d e n a t u r e d s a l m o n sperm DNA. To t h i s , 2x1 0^ cpm (50 t o 100 ng) o f f r e s h l y n i c k - t r a n s l a t e d DNA ( s e c t i o n 6) was added, and i n c u b a t e d a t 4 2 ° C f o r 18 h o u r s . The f i l t e r s were washed and a u t o r a d i o g r a p h e d as p r e v i o u s l y d e s c r i b e d ( s e c t i o n 7 . 1 ) . 8. Common R e a g e n t s , S o l u t i o n s , and B u f f e r s  1X TE 10 mM T r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e Hydro c h l o r i d e ( T r i s ) 1 mM E t h y l e n e d i a m i n e t e t r a - a c e t i c a c i d , d i s o d i u m (EDTA) -26-H i g h TE 100 mM T r i s (pH 8) 40 mM EDTA L B r o t h 0.5 g Y e a s t E x t r a c t ( D i f c o L a b o r a t o r i e s ) 1.0 g T r y p t o n e ( D i f c o L a b o r a t o r i e s ) 0.5 g N a C l w a t e r t o 100 ml X - G a l P l a t e s 0.5 g Y e a s t E x t r a c t 1.0 g T r y p t o n e 0.5 g N a C l 1.1 g B a c t o - a g a r ( D i f c o L a b o r a t o r i e s ) 5 mg A m p i c i l l i n 4 mg 5 - B r o m o - 4 - c h l o r o - 3 - i n d o l y l B - D - g a l a c t o p y r a n o s i d e (SIGMA C h e m i c a l s ) 16 mg i s o p r o p y l B - D - g a l a c t o p y r a n o s i d e (SIGMA C h e m i c a l s ) w a t e r t o 100 ml 1 OX C o r e B u f f e r 0.5 M T r i s (pH 8) 0.1 M M g C l 2 0.5 M N a C l -27-1X TBE B u f f e r 1 mM T r i s (pH 8) 90 mM B o r i c A c i d 2.5 mM EDTA 100X D e n h a r t s S o l u t i o n 4% F i c o l l t y p e 400 4% P o l y v i n y l P y r r o l i d o n e 4% BSA S e v a g C h l o r o f o r m : I s o a m y l A l c o h o l (24:1 ) L D i l 10 mM T r i s (pH 7.5) 10 mM M g C l 2 -28-RESULTS AND DISCUSSION 1. I s o l a t i o n o f S i n g l e Copy DNA S e q u e n c e s From t h e X Chromosome The X chromosome i s a p p r o x i m a t e l y 200x10^ b a s e p a i r s (bp) i n l e n g t h and r e p r e s e n t s 6% o f t h e human genome ( C a r r a n o e t a l , 1 9 7 9 ) . I n o r d e r t o f a c i l i t a t e t h e s e a r c h f o r X s p e c i f i c p r o b e s , an X chromosome f l o w s o r t e d l i b r a r y (LsHX) was u s e d ( M a t e r i a l s and Methods s e c t i o n 1 . 1 ) . T h i s l i b r a r y h as been e s t i m a t e d t o c o n t a i n 30 t o 60% X chromosomal m a t e r i a l , i n c r e a s i n g t h e p r o b a b i l i t y o f i s o l a t i n g an X s p e c i f i c p r o b e by 5 t o 10 f o l d . 1 .1 D e t e c t i o n o f R e p e t i t i v e DNA The human genome i s c o m p r i s e d o f 20 t o 30% r e p e t i t i v e DNA s e q u e n c e s ( J e l i n e k and Schmid, 1 9 8 2 ) : h i g h l y r e p e t i t i v e s e q u e n c e s i n t h e n e i g h b o r h o o d o f 500,000 c o p i e s p e r h a p l o i d genome and m i d d l e r e p e t i t i v e s e q u e n c e s i n t h e r a n g e o f 10^-10^3 c o p i e s . Genomic S o u t h e r n b l o t s , when h y b r i d i z e d w i t h p r o b e s c o n t a i n i n g m i d d l e t o h i g h l y r e p e t i t i v e s e q u e n c e s , a r e d i f f i c u l t t o a n a l y z e due t o i n t e n s e b a c k g r o u n d s i g n a l . A l t h o u g h t h e r e a r e methods t o c i r c u m v e n t t h i s p r o b l e m i t was d e c i d e d t o a v o i d t h i s d i f f i c u l t y a l t o g e t h e r by e l i m i n a t i n g any r e p e t i t i v e p r o b e s f r o m f u t u r e u s e . To do t h i s e a c h r e c o m b i n a n t p l a s m i d c o n t a i n i n g a human i n s e r t was grown i n l i q u i d medium u n d e r a m p i c i l l i n s e l e c t i o n . An a l i q u o t o f e a c h c u l t u r e was l y s e d i n a l k a l i and t h e e n t i r e c e l l u l a r l y s a t e was d o t t e d o n t o n i t r o c e l l u l o s e ( s e c t i o n 7 . 2 ) . The n i t r o c e l l u l o s e f i l t e r was h y b r i d i z e d w i t h - ^ P - l a b e l e d human genomic DNA. T h i s method c o u l d e a s i l y d i s t i n g u i s h h i g h l y r e p e t i t i v e DNA from low r e p e t i t i v e or s i n g l e copy DNA, as i s shown i n F i g . 1 and summarized i n Ta b l e 2. F i g . 1 shows t h e r e s u l t s of one ex p e r i m e n t . On t h i s f i l t e r 71 recombinant c l o n e s and 3 c o n t r o l s a r e h y b r i d i z e d w i t h 3 2 p _ ] _ a j - ) e i e ( j human genomic DNA. The dot a t p o s i t i o n 67, c o n t a i n i n g an A i u sequence (R. Poon p e r s o n a l c o m m u n i c a t i o n ) , gave a s t r o n g h y b r i d i z a t i o n s i g n a l . The dot a t p o s i t i o n 68 c o n t a i n e d p i S L 1 3 DNA and gave no d e t e c t a b l e s i g n a l . The dot a t p o s i t i o n 69 c o n t a i n e d the s i n g l e copy HPRT gene ( J o l l y e t a l , 1983) and a l s o gave no d e t e c t a b l e s i g n a l . The newly i s o l a t e d recombinant c l o n e s a t p o s i t i o n s 23, 43, 44, 48, 53, 60, 61 , 65, 82, 83, h y b r i d i z e d t o genomic 3 2 p _ i _ a b e l e d human DNA and a r e thus c o n s i d e r e d t o c o n t a i n r e p e t i t i v e DNA. The r e m a i n i n g 58 f a i l e d t o h y b r i d i z e and were t h e r e f o r e c o n s i d e r e d t o be e i t h e r s i n g l e copy DNA o r c o n t a i n low r e p e t i t i v e DNA. P l a s m i d DNA from each c l o n e d e v o i d of r e p e t i t i v e DNA was i s o l a t e d , e l e c t r o p h o r e s e d ( s e c t i o n 4.2.1, 5 ) , and So u t h e r n b l o t t e d t o n i t r o c e l l u l o s e . Each Sout h e r n b l o t c o n t a i n e d 0.5 t o 1.0 ug o f p l a s m i d DNA and was h y b r i d i z e d w i t h 3 2 p _ ] _ a j - , e i e c j human DNA. T h i s was done f o r two r e a s o n s ; f i r s t , t o c o n f i r m t h a t each c l o n e c o n t a i n e d an i n s e r t , and s e c o n d l y , as a more s e n s i t i v e t e s t t o d e t e c t r e p e t i t i v e DNA sequences. Of t h e 111 r e p e t i t i v e c l o n e s i d e n t i f i e d 4% (4/111) were d e t e c t e d by t h i s t e s t . Any probe c o n t a i n i n g a r e p e t i t i v e sequence was not f u r t h e r c h a r a c t e r i z e d . -30-1.2 P l a s m i d v e c t o r s The human i n s e r t s f r o m L sHX were s u b c l o n e d i n t o t h e p l a s m i d v e c t o r s p i S L l 3 , pUC13, o r pUC19. The v e c t o r p i S L l 3 i s 1.3 kb i n l e n g t h and c o n t a i n s t h e supF gene and a p o l y l i n k e r i n s e r t e d i n t o t h e l a c Z a l p h a c o m p l e m e n t a t i o n segment. T r a n s f o r m e d E . c o l i c e l l s , s t r a i n FP3 were s e l e c t e d by t h e i r r e s i s t a n c e t o a m p i c i l l i n , and r e c o m b i n a n t p l a s m i d s by t h e i r c o l o r . C o l o n i e s c o n t a i n i n g n o n - r e c o m b i n a n t p l a s m i d s t u r n a b l u e c o l o r , and t h o s e c o n t a i n i n g r e c o m b i n a n t p l a s m i d s r e m a i n w h i t e . The i n i t i a l r e a s o n f o r u s i n g p i S L l 3 was t h a t t h i s v e c t o r was d e v e l o p e d f o r t h e r a p i d s c r e e n i n g o f genomic l i b r a r i e s u s i n g a r e c o m b i n a t i o n - b a s e d a p p r o a c h ( S e e d , 1 9 8 3 ) . Once a human i n s e r t f r o m t h e L sHX l i b r a r y h ad been shown t o d e t e c t an RFLP, i t wo u l d be e a s i e r t o i s o l a t e a l a r g e r human segment from a c o s m i d l i b r a r y u s i n g a r e c o m b i n a n t c l o n e . By f i n d i n g a d d i t i o n a l RFLPs more i n f o r m a t i o n c o u l d be e x t r a c t e d f r o m a l a r g e r p r o b e , t h u s i n c r e a s i n g t h e PIC ( p o l y m o r p h i s m i n f o r m a t i o n c o n t e n t ) v a l u e o f a g i v e n l o c u s . The mai n d i f f i c u l t y e n c o u n t e r e d w i t h p i S L l 3 r e c o m b i n a n t s was t h e i r s l o w g r o w t h r a t e u n d e r a n t i b i o t i c s e l e c t i o n . The F P 3 / p i S L l 3 s y s t e m i s a two s t e p s y s t e m . I n o r d e r f o r t h e FP3 c e l l s t o grow i n t h e p r e s e n c e o f a m p i c i l l i n , t h e s u p F gene p r o d u c t , e n c o d e d on t h e p i S L l 3 v e c t o r , i s f i r s t r e q u i r e d . The su p F gene p r o d u c t s u p p r e s s e s t h e amber m u t a t i o n i n t h e a m p i c i l l i n gene on t h e FP3 c e l l s ' r e s i d e n t P3 p l a s m i d . Once t h e m u t a t i o n i s s u p p r e s s e d t h e n t h e a m p i c i l l i n r e s i s t a n c e gene p r o d u c t i s made, c o n f e r r i n g r e s i s t a n c e t o t h e c e l l . T h i s r e s u l t s i n t h e p o s s i b l e p r e f e r e n c e o f p i S L l 3 r e c o m b i n a n t s t o -31-c o n t a i n s m a l l e r human i n s e r t s s i n c e t h e y c a n r e p l i c a t e f a s t e r , g i v i n g a b e t t e r o v e r a l l r e s i s t a n c e t o a m p i c i l l i n ( s e e T a b l e 2 ) . Of t h e 95 s i n g l e copy i n s e r t s s u b c l o n e d i n t o p i S L l 3 , 82% (78/95) were s m a l l e r t h a n 500 bp i n s i z e w i t h t h e m a j o r i t y o f t h e s e e s t i m a t e d t o c o n t a i n i n s e r t s l e s s t h a n 100 bp i n l e n g t h . I n s e r t s o f 500 bp o r l e s s were n o t f u r t h e r c h a r a c t e r i z e d , as t h e y were c o n s i d e r e d t o o s m a l l t o be u s e f u l as p r o b e s f o r s c r e e n i n g genomic S o u t h e r n b l o t s . The r e m a i n d e r o f t h e i n s e r t s f r o m t h e L sHX l i b r a r y were s u b c l o n e d i n t o pUC13 o r pUC19. B o t h v e c t o r s c o n t a i n t h e a m p i c i l l i n r e s i s t a n c e gene and a p o l y l i n k e r i n s e r t e d i n t o t h e l a c Z a l p h a c o m p l e m e n t a t i o n segment. The s e l e c t i o n s y s t e m f o r r e c o m b i n a n t p l a s m i d s was t h e same as f o r p i S L l 3 . However, t h e s e r e c o m b i n a n t s had t h e a d v a n t a g e o f s t r o n g e r g r o w t h i n t h e p r e s e n c e o f a m p i c i l l i n . Of t h e 136 s i n g l e copy human i n s e r t s s u b c l o n e d i n t o pUC13 o r pUC19, 38% (52/136) were s m a l l e r t h a n 500 bp. T a b l e 2 C h a r a c t e r i z a t i o n o f R e c o m b i n a n t P l a s m i d s C l o n e s R e p e a t s I n s e r t s > 500 bp I n s e r t s < 500 bp p i S L l 3 113 18 18 77 pUC1 3 85 34 30 21 PUC19 144 59 54 31 T o t a l 342 111 102 1 29 -32-1.3 I s o l a t i o n o f X S p e c i f i c P r o b e s Of t h e 102 s i n g l e c o p y p r o b e s o f u s a b l e l e n g t h an e s t i m a t e d 40 t o 70% o f t h e s e w o u l d be d e r i v e d f r o m t h e a u t o s o m e s . F o r t h e p u r p o s e s o f t h i s work a u t o s o m a l DNA was c o n s i d e r e d t o be c o n t a m i n a t i n g DNA and t h u s i t was n e c e s s a r y t o d i f f e r e n t i a t e b etween X and non-X DNA. Two methods were emp l o y e d t o do t h i s . The f i r s t p r o c e d u r e u t i l i z e d d o t b l o t s . E a c h u n i q u e p r o b e was l a b e l e d w i t h 32p and h y b r i d i z e d t o a p a n e l o f d o t s t h a t c o n t a i n e d DNA f r o m t h e f o l l o w i n g c e l l l i n e s ; human ( H ) , mouse (M), and a mouse-human s o m a t i c c e l l h y b r i d (MHX) t h a t c o n t a i n e d t h e X chromosome as i t s o n l y human DNA ( t h e d e t a i l s o f t h e ro d e n t - h u m a n h y b r i d s a r e d i s c u s s e d i n s e c t i o n 2 ) . S t r o n g h y b r i d i z a t i o n t o t h e human and MHX h y b r i d d o t , b u t n o t t o t h e mouse d o t , w o u l d be i n d i c a t i v e o f an X s p e c i f i c p r o b e . On t h e o t h e r hand, s t r o n g h y b r i d i z a t i o n t o t h e human b u t n o t t o t h e mouse o r MHX d o t w o u l d be i n d i c a t i v e o f an a u t o s o m a l p r o b e . F i g . 2 shows t h e r e s u l t s f o r one s e t o f e x p e r i m e n t s . P a n e l s a, b, and c, a r e a u t o r a d i o g r a p h s o f known c o n t r o l s . P a n e l s a and b a r e h y b r i d i z e d w i t h t h e Xp p r o b e s RC8 ( M u r r a y e t a l , 1982) and L1.28 ( B a k k e r e t a l , 1983) r e s p e c t i v e l y . T h e s e g i v e t h e e x p e c t e d r e s u l t s o f s t r o n g h y b r i d i z a t i o n t o t h e human and t h e MHX d o t s . P a n e l c was h y b r i d i z e d w i t h t h e a u t o s o m a l p r o b e D8MGV1 (Wood e t a l , 1 9 8 6 ) . T h i s h y b r i d i z e s s t r o n g l y t o t h e human d o t o n l y . P a n e l s d t o s a r e a u t o r a d i o g r a p h s o f s i n g l e c o p y p r o b e s f r o m t h e L sHX l i b r a r y . F i v e o f t h e s e g i v e t h e p a t t e r n e x p e c t e d f o r an a u t o s o m a l p r o b e ( p a n e l d t o h ) , whereas 11 c o u l d be t e n t a t i v e l y a s s i g n e d t o t h e X chromosome ( p a n e l i t o s ) . -33-The s e c o n d a p p r o a c h e m p l o y e d t o d i s t i n g u i s h X p r o b e s f r o m non-X p r o b e s u t i l i z e d two rodent-human s o m a t i c c e l l h y b r i d s . T h i s method was p o t e n t i a l l y more p o w e r f u l s i n c e i t c o u l d a l s o d i s t i n g u i s h Xp p r o b e s f r o m Xq p r o b e s . I n o t h e r e x p e r i m e n t s ( s e c t i o n 2) i t was shown t h a t t h e DNA i s o l a t e d f r o m t h e c e l l l i n e 835 K3, a hamster-human s o m a t i c c e l l h y b r i d c o n t a i n i n g t h e X c e n t o X p t e r r e g i o n , had b e e n c o n t a m i n a t e d w i t h p l a s m i d DNA. The p r e s e n c e o f p l a s m i d DNA i n t h i s DNA p r e p a r a t i o n r u l e d o u t t h e u s e o f d o t b l o t s s i n c e t h e r e was homology between t h e l a b e l e d v e c t o r DNA and t h e c o n t a m i n a t i n g p l a s m i d DNA. Thus e v e r y d o t c o n t a i n i n g DNA f r o m 835 K3 w o u l d g i v e a p o s i t i v e s i g n a l when h y b r i d i z e d w i t h p r o b e s t h a t c o n t a i n e d v e c t o r DNA. T h i s e m p h a s i z e s one o f t h e common d i f f i c u l t i e s e n c o u n t e r e d when u s i n g d o t b l o t s i n so much as t h e p o s i t i v e s i g n a l o b t a i n e d c a n n o t be d i s t i n g u i s h e d b etween b a c k g r o u n d and homologous b i n d i n g . To c i r c u m v e n t t h i s p r o b l e m r a t h e r t h a n i s o l a t e t h e human i n s e r t s f r o m t h e a p p r o x i m a t e l y 70 c l o n e s t o be t e s t e d i t was d e c i d e d t o h y b r i d i z e e a c h u n i q u e p r o b e t o S o u t h e r n b l o t s t h a t c o n t a i n e d DNA f r o m t h e MHX and 835 K3 c e l l l i n e s . S i n c e e a c h b l o t c o n t a i n e d o n l y two l a n e s i t was more p r u d e n t t o u s e t h i s t y p e o f S o u t h e r n b l o t t h a n t o p r e p a r e f r e s h 835 K3 DNA o r t o i s o l a t e 70 p l a s m i d i n s e r t s . E a c h u n i q u e p r o b e was l a b e l e d w i t h 32p a n ( j h y b r i d i z e d t o a S o u t h e r n b l o t c o n t a i n i n g t h e above m e n t i o n e d H i n d l l l d i g e s t e d DNA ( F i g . 3 ) . P r o b e s o r i g i n a t i n g f r o m t h e X chromosome c o u l d be d i s t i n g u i s h e d f r o m non-X p r o b e s by t h e i r h y b r i d i z a t i o n t o t h e MHX DNA. X p r o b e s w o u l d h y b r i d i z e , a t t h e e x p e c t e d H i n d l l l b and -34-s i z e , and non-X p r o b e s w o u l d n o t . Xp p r o b e s c o u l d be d i s t i n g u i s h e d f r o m Xq p r o b e s by t h e h y b r i d i z a t i o n p a t t e r n t o t h e 835 K3 c e l l l i n e . The Xp p r o b e s w o u l d h y b r i d i z e t o t h i s DNA and t h e Xq p r o b e s w o u l d n o t . Thus i f a p r o b e h y b r i d i z e d t o b o t h l a n e s i t c o u l d be t e n t a t i v e l y a s s i g n e d t o Xp; i f i t o n l y h y b r i d i z e d t o t h e MHX l a n e t h e n i t o r i g i n a t e d f r o m Xq; and i f a p r o b e f a i l e d t o h y b r i d i z e t o e i t h e r l a n e i t was assumed t o be a u t o s o m a l i n o r i g i n and was n o t f u r t h e r c h a r a c t e r i z e d . T h i s m e t h o d o l o g y c o u l d n o t d i s c e r n a u t o s o m a l p r o b e s f r o m p r o b e s w h i c h f a i l e d t o h y b r i d i z e due t o t e c h n i c a l r e a s o n s . F i g . 3 p a n e l (a) was h y b r i d i z e d w i t h t h e p r o b e pTS234 and g i v e s t h e non-X p a t t e r n ; p a n e l (b) was h y b r i d i z e d w i t h pTS228 and i s i n d i c a t i v e o f an Xq d e r i v e d p r o b e ; p a n e l ( c ) was h y b r i d i z e d w i t h pTS247 and i s i n d i c a t i v e o f an Xp d e r i v e d p r o b e . The band common t o a l l t h e b l o t s a t 2.7 kb i n t h e 835 K3 l a n e i s c o n t a m i n a t i n g p l a s m i d DNA. U t i l i z i n g b o t h methods 31/102 u n i q u e p r o b e s were t e n t a t i v e l y a s s i g n e d t o t h e X chromosome. -35-FIGURE 1 SCREENING FOR R E P E T I T I V E DNA T o t a l c e l l u l a r l y s a t e s , c o n t a i n i n g r e c o m b i n a n t p l a s m i d c l o n e s , were d o t t e d o n t o n i t r o c e l l u l o s e and h y b r i d i z e d w i t h 3 2 P - l a b e l e d human genomic DNA. D o t s 22-66, 70-93 a r e newly i s o l a t e d r e c o m b i n a n t c l o n e s d e r i v e d f r o m L sHX; d o t 67 c o n t a i n s an A l u r e p e t i t i v e s e q u e n c e ; d o t 68 c o n t a i n s p i S L l 3 ; d o t 69 c o n t a i n s t h e HPRT gene. FIG.1 23 53 i rd in cn I m 67 o © 82 83 43 44 60 61 48 65 - 3 6 -FIGURE 2 DETERMINATION OF CHROMOSOMAL ORIGIN OF RECOMBINANT  ISOLATES USING DOT BLOTS 0.5 ug o f human ( H ) , mouse (M), and a mouse-human X h y b r i d (MHX) DNA were d o t t e d o n t o n i t r o c e l l u l o s e and h y b r i d i z e d w i t h t h e 3 2 P - l a b e l e d p r o b e s ; (a) RC8, (b) L1.28, ( c ) D8MGV1, ( d - s ) i s o l a t e d p r o b e s f r o m t h e L sHX l i b r a r y . FIG.2 M : H x M H 1  b c i (D M : H x M H d f g h M : H x • • • • • • • # . • • • M •* • • • • ! ' * • I H • # • # # • • * • + ) • i j k I m n o p q r s -37-FIGURE 3 DETERMINATION OF CHROMOSOMAL ORIGIN OF RECOMBINANT  ISOLATES USING SOUTHERN BLOTS 10 ug o f MHX and 835 K3 DNA, d i g e s t e d t o c o m p l e t i o n w i t h H i n d l l l , was S o u t h e r n b l o t t e d and h y b r i d i z e d w i t h i s o l a t e d p r o b e s f r o m t h e L sHX l i b r a r y ; (a) pTS234; (b) pTS228; ( c ) pTS247. The band a t 2.7 kb i n t h e 835 K3 l a n e common t o e a c h b l o t i s due c o n t a m i n a t i n g p l a s m i d DNA. -37 a-8 3 5 K 3 | MHX 8 3 5 K 3 % M H X mm 2] P CO 8 3 5 K 3 MHX 1 1 O I ro • I I • • o o - 3 8 -2. DETECTION OF Xp S P E C I F I C PROBES 2.1 Arm L o c a l i z a t i o n o f X P r o b e s Of t h e 31 p r o b e s c o n s i d e r e d t o o r i g i n a t e f r o m t h e X chromosome, 9 l o c a l i z e d t o t h e Xp arm. A s u s p e c t e d t e n t h Xp p r o b e (pTS119) was shown t o be i n h e r i t e d i n an a u t o s o m a l p a t t e r n ( d i s c u s s e d l a t e r and i n s e c t i o n 4 ) . Two methods were u s e d t o e s t a b l i s h t h e s e r e s u l t s . The i n i t i a l method r e l i e d on o b s e r v i n g d i f f e r e n t h y b r i d i z a t i o n i n t e n s i t i e s c o r r e s p o n d i n g t o t h e amount o f X c hromosomal m a t e r i a l p r e s e n t . F o r example, X p r o b e s h y b r i d i z e d t o DNA f r o m m a l e s (46,XY) and f e m a l e s (46,XX) s h o u l d show a r a t i o o f h y b r i d i z a t i o n s i g n a l o f 1:2 b e c a u s e o f t h e s e c o n d X chromosome p r e s e n t i n f e m a l e s . T h i s o b s e r v a t i o n i s due t o d o s a g e . The c e l l l i n e s u s e d f o r t h e d o s a g e s t u d i e s were: male ( 4 6 , X Y ) ; f e m a l e ( 4 6 , X X ) ; GM88, a 4 6 , X i ( X q ) isochromosome c e l l l i n e w h i c h has 3 c o p i e s o f Xq and one c o p y o f Xp; and GM3384, a 49,XXXXY c e l l l i n e . T h e r e f o r e a d i f f e r e n t h y b r i d i z a t i o n p a t t e r n , most n o t a b l y i n t h e X i X q c e l l l i n e (3:1 r a t i o f o r c o m p a r i n g Xq p r o b e s t o Xp p r o b e s ) , w o u l d be e x p e c t e d f o r p r o b e s o r i g i n a t i n g f r o m t h e Xp o r Xq arms. T h i s p a n e l w o u l d a l s o d i f f e r e n t i a t e , by means o f d o s a g e , between X p r o b e s and non-X p r o b e s . I n i t i a l l y d o t b l o t s were i n v e s t i g a t e d . 0.5 ug o f d i g e s t e d DNA f r o m t h e above c e l l l i n e s were d o t t e d on t o n i t r o c e l l u l o s e a nd h y b r i d i z e d w i t h t h e c o n t r o l s RC8 (an Xp p r o b e ) and D8MGV1 (a chromosome 8 p r o b e ) . The e x p e c t e d d o s i n g p a t t e r n was n o t f o u n d f o r e i t h e r p r o b e . T h i s was a t t r i b u t e d t o t h e i n a b i l i t y o f d o t -39-b l o t s t o d i s t i n g u i s h b etween b a c k g r o u n d n o i s e and homologous b i n d i n g , e v e n when i s o l a t e d i n s e r t s were u s e d as p r o b e s . T h i s f a c t o r l i m i t s t h e u s e o f d o t b l o t s t o p r o c e d u r e s w h i c h y i e l d q u a l i t a t i v e b u t n o t q u a n t i t a t i v e a n s w e r s . S i n c e d o t b l o t s p r o v e d i n s u f f i c i e n t , and t h e s o m a t i c c e l l h y b r i d l i n e s c o n t a i n i n g t h e d i f f e r e n t r e g i o n s o f t h e X chromosome were n o t y e t a v a i l a b l e , S o u t h e r n b l o t s c o n t a i n i n g d i g e s t e d DNA f r o m t h e abo v e d o s a g e p a n e l were made. The p r o b e s RC8 and D8MGV1 were 2 2 p _ ] _ a ) o e ] _ e ( j a n c j h y b r i d i z e d as shown i n F i g . 4. P a n e l (a) shows t h e r e s u l t s f o r RC8 and g i v e s t h e p a t t e r n e x p e c t e d f o r an Xp p r o b e . P a n e l (b) shows t h e r e s u l t s f o r D8MGV1 and g i v e s t h e e x p e c t e d p a t t e r n f o r an a u t o s o m a l p r o b e . P a n e l ( c ) shows t h e r e s u l t s f o r t h e r e c o m b i n a n t c l o n e pTS73 and g i v e s t h e p a t t e r n o f an Xp p r o b e . The u s e o f d o s a g e t o d i s t i n g u i s h between Xq and Xp p r o b e s was s u f f i c i e n t p r o v i d i n g t h e h y b r i d i z a t i o n worked w e l l , t h e b a c k g r o u n d was low, t h e amount o f DNA l o a d e d p e r l a n e was e q u a l , and t h e bands were t i g h t . I f any o f t h e s e c o n d i t i o n s were n o t met, t h e n i t was d i f f i c u l t t o c o n f i d e n t l y a n a l y z e t h e r e s u l t s . S i n c e t h e s o u r c e o f p r o b e s was f r o m DNA p r e p a r e d by a m i n i p r e p p r o c e d u r e ( M a t e r i a l s and Methods S e c . 4.2), w h i c h o f t e n gave h i g h b a c k g r o u n d s a n d / o r weak s i g n a l , t h e n i t was f r e q u e n t l y n e c e s s a r y t o r e p e a t e x p e r i m e n t s t o i n s u r e r e p r o d u c i b i l i t y . To overcome t h i s d i f f i c u l t y s o m a t i c c e l l h y b r i d s , c o n t a i n i n g v a r i o u s r e g i o n s o f t h e X chromosome, were employed t o d i s t i n g u i s h Xp p r o b e s f r o m Xq p r o b e s . -40- . There were 3 rodent-human s o m a t i c c e l l h y b r i d s used t o d i f f e r e n t i a t e Xp and Xq probes ( F i g . 5 ) : a mouse-human X h y b r i d (MHX) t h a t c o n t a i n e d t h e e n t i r e X chromosome as i t s o n l y human DNA; a hamster-human h y b r i d (835 K3) t h a t c o n t a i n e d the Xcen t o X p t e r r e g i o n ; and a hamster-human h y b r i d (422 K6) t h a t c o n t a i n e d the X q t e r t o Xp11 r e g i o n (Wieacker e t a l , 1984). These h y b r i d c e l l l i n e s had been p r e v i o u s l y i n s p e c t e d c y t o g e n e t i c a l l y by o t h e r l a b o r a t o r i e s (Dr. H. Hoehn, Dr. J . Hamerton) t o c o n f i r m the p r e s e n c e o f t h e above mentioned X chromosome r e g i o n s . I t i s known t h a t s o m a t i c c e l l h y b r i d s a r e u n s t a b l e and may te n d t o l o s e chromosomes, t h e r e f o r e i t i s i m p o r t a n t t o r e a f f i r m the k a r y o t y p e a t the time o f each DNA i s o l a t i o n . T h i s was not done a t t h i s t i me but i n s t e a d m o l e c u l a r t e c h n i q u e s were used t o c o n f i r m t h a t t h e documented X chromosome r e g i o n s were r e t a i n e d . S i n c e l o s s o f t h e human X chromosome r e g i o n s from t h e h y b r i d s was t h e o n l y c o n c e r n , i t was f e l t t h a t c o n f i r m a t i o n of r e t e n t i o n o f human X chromosomal m a t e r i a l would s u f f i c e f o r t h e i r use as a means of X probe l o c a l i z a t i o n . T h i s was a c c o m p l i s h e d by h y b r i d i z i n g Xp probes o f known o r i g i n t o Sou t h e r n b l o t s c o n t a i n i n g DNA from t h e s e h y b r i d s . The probes RC8, L1.28, and 58-1, have been mapped and shown t o span the s h o r t arm of t h e X chromosome (Bakker e t a l , 1985; Drayna and White, 1985). The probe RC8 maps w i t h i n t h e Xp22 r e g i o n near the t e l o m e r e . The probe L1.28 maps w i t h i n t h e Xp11.4 r e g i o n c l o s e t o t h e m i d d l e of the Xp arm. The probe 58-1 maps w i t h i n t h e Xp11 r e g i o n near the centromere. Each probe was l a b e l e d w i t h 32p ancj h y b r i d i z e d t o S o u t h e r n b l o t s c o n t a i n i n g DNA from t h e f o l l o w i n g c e l l t y p e s : mouse, MHX, 422 K6, 835 K3, 46,XX. B l o t s c o n t a i n i n g H i n d l l l d i g e s t e d DNA from t h e s e c e l l l i n e s a r e r e f e r r e d t o as arm l o c a l i z a t i o n b l o t s . As shown i n F i g . 6 t h e probe RC8 (a) and L l . 2 8 (b) h y b r i d i z e d t o t h e MHX, 835 K3, and 46,XX l a n e s as e x p e c t e d . The probe 58-1 (c) h y b r i d i z e d t o the MHX, 422 K6, 835 K3, and 46,XX l a n e s as e x p e c t e d . The p r e s e n c e of t h e s e 3 l o c i i n t h e h y b r i d c e l l l i n e s s u p p o r t s the ass u m p t i o n t h a t the h y b r i d c e l l l i n e s r e t a i n e d the a p p r o p r i a t e X chromosome r e g i o n s . T h i s d a t a however, does not e l i m i n a t e t h e p o s s i b i l i t y of s m a l l i n t e r s t i t i a l o r t e l o m e r i c d e l e t i o n s . Each of t h e 9 t e n t a t i v e l y i d e n t i f i e d Xp probes were 3 2 p _ i a b e l e d and h y b r i d i z e d t o arm l o c a l i z a t i o n b l o t s . A t y p i c a l r e s u l t i s shown i n F i g . 6 ( d ) . The probe pTS247 h y b r i d i z e s t o t h e MHX, 835 K3, and 46,XX l a n e s i n d i c a t i v e o f a probe from t h e Xp11 t o X p t e r r e g i o n . The r e s u l t s of t h e r e m a i n i n g probes a r e summarized i n T a b l e 3. To f u r t h e r s u b l o c a l i z e t h e 7 Xp probes a s s i g n e d t o t h e Xp11 t o X p t e r r e g i o n , each was - ^ p ^ a k ^ e ^ a n ( j h y b r i d i z e d t o DNA from a male w i t h an i n t e r s t i t i a l d e l e t i o n a t Xp21 (Franke e t a l , 1985) (see F i g . 5 GM 7947). A l l 7 probes h y b r i d i z e d t o t h i s i n d i v i d u a l s DNA, as w e l l as a 46,XX c o n t r o l , thus e x c l u d i n g t h e s e probes from t h e Xp21 r e g i o n . -42-2.2 C h a r a c t e r i z a t i o n of Probe pTSl19 One probe, pTSl1-9, was i n i t i a l l y l o c a l i z e d to the Xcen to Xp11 r e g i o n . T h i s probe i s a 1.8 kb H i n d l l l i n s e r t subcloned i n t o the v e c t o r piSL13. H y b r i d i z a t i o n of the 3 2 P - l a b e l e d recombinant plasmid to dot b l o t s c o n t a i n i n g DNA from mouse, MHX, and 46,XX gave a p a t t e r n i n d i c a t i v e of an X probe. Subsequent h y b r i d i z a t i o n to arm l o c a l i z a t i o n b l o t s ( F i g . 7a) showed a common band i n a l l c e l l l i n e s at 1.9 kb, and a band at 1.7 kb i n the 46,XY and 46,XX l a n e s . The i n t e n s i t y of the band a t 1.9 kb was g r e a t e r than the band a t 1.7 kb, suggesting t h a t the 1.7 kb band was e i t h e r due to an a r t i f a c t or some minor homology. Since the c e l l l i n e s 46,XY and 46,XX were the only l i n e s which were known to c o n t a i n autosomal DNA i t was concluded t h a t the band at 1.7 kb was due to some minor homology of the probe to one of the autosomes. Thus the band at 1.9 kb was c o n s i d e r e d to be the band of i n t e r e s t , and was i n t e r p r e t e d to o r i g i n a t e from the Xpcen to Xp11 r e g i o n . L a t e r when a n a l y z i n g f a m i l y b l o t s ( F i g . 13 s e c t i o n 4) probe pTS119 proved to be i n h e r i t e d i n an autosomal f a s h i o n . F u r t h e r a n a l y s i s with d i f f e r e n t f a m i l i e s , s e g r e g a t i n g f o r the BamHI or PvuII polymorphism d e t e c t e d with t h i s probe, confirmed i t s autosomal o r i g i n . To c l e a r up t h i s paradox, the human i n s e r t from probe pTSl19 was i s o l a t e d , l a b e l e d with 32p f a n c j h y b r i d i z e d to an arm l o c a l i z a t i o n b l o t ( F i g . 7b). T h i s time o n l y the 1.7 kb band i n the 46,XX lane c o u l d be d e t e c t e d . T h e r e f o r e , i t appeared t h a t the band at 1.9 kb ( F i g . 7a) was due to an a r t i f a c t , homologous to the p i S L l 3 v e c t o r , which had been i n t r o d u c e d on to the arm l o c a l i z a t i o n -43-b l o t . F u r t h e r c h a r a c t e r i z a t i o n o f t h i s a r t i f a c t was n o t a t t e m p t e d . No a t t e m p t t o d e t e r m i n e t h e a u t o s o m a l l o c a t i o n o f t h i s p r o b e was made. However, s i n c e t h e m a j o r a u t o s o m a l c o n t a m i n a n t s i n t h e L sHX l i b r a r y a r e f r o m chromosome 7 and chromosome 8, t h e human i n s e r t o f p T S l 1 9 c o u l d o r i g i n a t e f r o m one o f t h e s e two chromosomes. - 4 4 -FIGURE 4 ARM LOCALIZATION OF X PROBES BY DOSING 10 ug o f DNA was S o u t h e r n b l o t t e d and h y b r i d i z e d w i t h t h e p r o b e s : (a) RC8; (b) D8MGV1; ( c ) pTS73. DNA s a m p l e s : (M) mouse; (MHX) a mouse-human s o m a t i c c e l l h y b r i d t h a t c o n t a i n s t h e X chromosome as i t s o n l y human DNA; (46,XY) male; (46,XX) f e m a l e ; ( 4 6 , X i ( X q ) ) an isochromosome c e l l l i n e t h a t c o n t a i n s 3 c o p i e s o f Xq and 1 c o p y o f Xp; (4X) 49,XXXXY. -44a-CO M H X -M -X Y - , x x - I X i X q - I 4X- I 9 CO b M H X - j M -X Y - J X X - | X i X q - j 4 X - | X Y -X X -X i X q -4 X -l CD -45-FIGURE 5 DNA RETAINED IN CELL LINES USED FOR ARM LOCALIZATION BLOTS P r o b e s t e n t a t i v e l y a s s i g n e d t o t h e X chromosome were h y b r i d i z e d t o S o u t h e r n b l o t s c o n t a i n i n g DNA f r o m t h e s e c e l l l i n e s . -46-T a b l e 3. H y b r i d i z a t i o n o f Xp p r o b e s t o H i n d l l l d i g e s t s o f DNA e x t r a c t e d f r o m c e l l l i n e s c o n t a i n i n g v a r i o u s r e g i o n s o f t h e human X chromosome. CELL LINES HUMAN X PLASMID PROBE pTS CHROMOSOME 70 73 217 247 264 279 283 293 316 46 XX e n t i r e X + + + + + + + + + MHX e n t i r e X + + + + + + + + + 835 K3 X p c e n - X p t e r + + + + + + + + + 422 K6 Xqter-Xp11 - + - - - + -Mouse none GM7947 d e l Xp21 + + + + + + + + + Human H i n d l l l f r a g m e n t (kb) 3.0 0.9 1 .9 4.0 2.9 4.5 1 .7 1.0 3.5 +, p r e s e n c e o f a h y b r i d i z i n g H i n d l l l f r a g m e n t o f t h e same s i z e a s t h e p r o b e t e s t e d -, a b s e n c e o f any h y b r i d i z i n g H i n d l l l f r a g m e n t o f t h e same s i z e a s t h e p r o b e t e s t e d -47-FIGURE 6. ARM LOCALIZATION OF X S P E C I F I C PROBES USING  SOMATIC CELL HYBRIDS 10 ug o f DNA was S o u t h e r n b l o t t e d and h y b r i d i z e d w i t h t h e p r o b e s : (a) RC8; (b) L1.28; ( c ) 58-1; (d) pTS247. DNA s a m p l e s : (M) mouse; (MHX) a mouse-human s o m a t i c c e l l h y b r i d t h a t c o n t a i n s t h e X chromosome as i t s o n l y human DNA; (422 K6) a hamster-human s o m a t i c c e l l h y b r i d t h a t c o n t a i n s t h e X q t e r t o Xp11 r e g i o n ; (835 K3) a hamster-human s o m a t i c c e l l h y b r i d t h a t c o n t a i n s t h e X c e n t o X p t e r r e g i o n ; (46,XX) f e m a l e . I XX -988 -XHI/M - IAI | -XX I -988 -ZZP | - XHIAI - IAI | XX | -988 J -XHIAI | XX I -9 8 8 I -| -XH1AI - IAI -48-FIGURE 7 HYBRIDIZATION OF PROBE p T S l 1 9 TO DNA FROM SOMATIC CELL HYBRIDS CONTAINING VARIOUS REGIONS OF THE X CHROMOSOME 10 ug o f DNA, d i g e s t e d t o c o m p l e t i o n w i t h H i n d l l l , was S o u t h e r n b l o t t e d and h y b r i d i z e d w i t h t h e p r o b e s : (a) pTS119; (b) i s o l a t e d human i n s e r t f r o m p T S l 1 9 ; DNA s a m p l e s : (M) mouse; (MHX) a mouse-human s o m a t i c c e l l h y b r i d t h a t c o n t a i n s t h e X chromosome as i t s o n l y human DNA; (422 K6) a hamster-human s o m a t i c c e l l h y b r i d t h a t c o n t a i n s t h e X q t e r t o Xp11 r e g i o n ; (835 K3) a hamster-human s o m a t i c c e l l h y b r i d t h a t c o n t a i n s t h e X c e n t o X p t e r r e g i o n ; (46,XX) f e m a l e . -48a-F1G. 7 -1 .9 - 1 . 7 -1|| m >• N X CO X CN X CO "<t I I X X X X CM CM LO CO CO -49-3 . DETECTION OF RFLPs 3.1 C h a r a c t e r i z a t i o n o f jl New P o l y m o r p h i c M a r k e r s To d e t e c t R FLPs, S o u t h e r n b l o t s c o n t a i n i n g d i g e s t e d DNA f r o m 5 f e m a l e s and 1 m a l e , were h y b r i d i z e d w i t h t h e 9 Xp p r o b e s . As shown i n T a b l e 5, 185 d i f f e r e n t probe-enzyme c o m b i n a t i o n s were u s e d t o d e t e c t 3 R F L P s . T h i s r e p r e s e n t s 2189 t o 2369 bp s c r e e n e d , w h i c h was c a l c u l a t e d u s i n g t h e f o r m u l a (N + 1 ) b ( C o o p e r and S c h m i d t k e , 1984), where N s t a n d s f o r t h e number o f b a n d s d e t e c t e d on t h e a u t o r a d i o g r a p h , and b s t a n d s f o r t h e number o f bp i n t h e enzyme r e c o g n i t i o n s i t e . S i n c e some enzyme r e c o g n i t i o n s i t e s f a l l w i t h i n o t h e r s , f o r example Sau3a (GATC) and BamHI (GGATCC), t h e n some c u t s i t e s a r e n o t i n d e p e n d e n t o f e a c h o t h e r . T h i s f a c t o r i s n o t c o n s i d e r e d i n t h e above f o r m u l a . To c o r r e c t f o r t h i s , a l l s u c h p o t e n t i a l o v e r l a p s were e x a m i n e d and t h e maximum and minimum number o f i n d e p e n d e n t bp were c a l c u l a t e d . T h i s y i e l d e d a r a n g e o f t o t a l b a s e p a i r s s c r e e n e d as r e p o r t e d a b o v e . The a u t o s o m a l p r o b e p T S H 9 was a l s o u s e d t o s c r e e n f o r RFLPs u s i n g t h e same p a n e l o f f e m a l e and male DNA ( a t t h i s s t a g e PTS119 was c o n s i d e r e d t o be f r o m X p ) . Twenty one probe-enzyme c o m b i n a t i o n s were t e s t e d , r e p r e s e n t i n g 360 bp s c r e e n e d , and 2 i n d e p e n d e n t RFLPs were d e t e c t e d . -50-F i g . 8 shows t h e probe-enzyme c o m b i n a t i o n s w h i c h d e t e c t e d R F L P s . The p r o b e pTS247 d e t e c t s a B g l l l p o l y m o r p h i s m ( F i g . 8 a ) . The p r o b e i s 4.0 kb i n l e n g t h and d e t e c t s p o l y m o r p h i c b a n d s o f 25 o r 20 k b . The m i n o r a l l e l e f r e q u e n c y was f o u n d t o be 0.26 w i t h 10/38 i n d e p e n d e n t chromosomes t e s t e d h a v i n g t h e m i n o r 25 kb a l l e l e . The p r o b e pTS264 d e t e c t s an X b a l p o l y m o r p h i s m ( F i g . 8 b ) . The p r o b e i s 2.9 kb i n l e n g t h and d e t e c t s p o l y m o r p h i c bands o f 4.3 o r 1.8 kb w i t h a common band o f 5.0 k b . The m i n o r a l l e l e f r e q u e n c y was f o u n d t o be 0.10 w i t h 5/52 i n d e p e n d e n t chromosomes t e s t e d h a v i n g t h e m i n o r 1.8 kb a l l e l e . The p r o b e pTS316 d e t e c t s an A v a i l p o l y m o r p h i s m ( F i g . 8 c ) . The p r o b e i s 3.5 kb i n l e n g t h and d e t e c t s p o l y m o r p h i c bands o f 6.5 o r 6.0 k b . The m i n o r band f r e q u e n c y was f o u n d t o be 0.43 w i t h 20/47 i n d e p e n d e n t chromosomes t e s t e d h a v i n g t h e m i n o r 6.0 kb b a n d . T h i s p o l y m o r p h i s m has b e e n shown t o be s e x s p e c i f i c s i n c e o n l y f e m a l e s have t h e 6.0 kb band ( d i s c u s s e d i n s e c t i o n I V ) . The p r o b e p T S l 1 9 i s 1.8 kb i n l e n g t h and d e t e c t e d 2 i n d e p e n d e n t p o l y m o r p h i s m s . The BamHI p o l y m o r p h i c bands ( F i g . 8d) a r e a t 15 kb o r 12 kb w i t h a common band o f 1.6 k b . The m i n o r a l l e l e f r e q u e n c y was f o u n d t o be 0.18 w i t h 4/22 i n d e p e n d e n t chromosomes t e s t e d h a v i n g t h e m i n o r 12 kb a l l e l e . The P v u I I p o l y m o r p h i c bands ( F i g . 8e) a r e a t 21 kb o r 16 kb. The m i n o r a l l e l e f r e q u e n c y was f o u n d t o be 0.24 w i t h 14/58 i n d e p e n d e n t chromosomes t e s t e d h a v i n g t h e m i n o r 21 kb a l l e l e . -51 -The r e s u l t s f o r a l l RFLPs f o u n d a r e summarized i n T a b l e 4. E a c h Xp s p e c i f i c p r o b e o n l y d e t e c t s a s i n g l e RFLP. B o t h t h e p r o b e s pTS247 and pTS264 were t e s t e d w i t h 20 and 19 enzymes r e s p e c t i v e l y . T h e r e f o r e , i t i s p r o b a b l e t h a t t h e p o l y m o r p h i s m s a r e n o t due t o m a j o r DNA r e a r r a n g e m e n t s b u t a r e due t o a l t e r a t i o n s a t t h e enzyme r e c o g n i t i o n s i t e . The p r o b e pTS316 was t e s t e d w i t h o n l y 8 enzymes and t h e p o s s i b i l i t y o f a genomic r e a r r a n g e m e n t c a n n o t be e l i m i n a t e d . An i n t e r p r e t a t i o n o f how t h e RFLPs a r e a r r a n g e d i n t h e genome i s shown i n F i g . 9. T a b l e 4. Summary o f P o l y m o r p h i c P r o b e s PROBE ENZYME BANDS (kb) BAND FREQUENCY PTS119 Bam HI 1 5/1 2 0.82/0.18 PTS119 P v u I I 1 6/21 0.76/0.24 PTS247 B q l l l 20/25 0.76/0.24 PTS264 X b a l 4.3/1.9 0.90/0.10 PTS316 A v a i l 6.5/6.0 0.57/0.43 3.2 RFLP F r e q u e n c y on t h e X Chromosome The d a t a p r e s e n t e d h e r e y i e l d s a RFLP f r e q u e n c y o f a p p r o x i m a t e l y 1/800 bp s c r e e n e d f o r t h e Xp p r o b e s . T h i s i s i n marked c o n t r a s t t o a v a l u e o f 1/180 f o r t h e s i n g l e a u t o s o m a l p r o b e c h a r a c t e r i z e d . T h i s compares t o t h e v a l u e o f 1/100 bp s c r e e n e d f o u n d i n t h e B - g l o b i n c l u s t e r on chromosome 11 ( J e f f r e y s 1 9 7 9 ) . H o f k e r e t a l (1985) have shown t h e f r e q u e n c y o f p r o b e s d e t e c t i n g RFLPs on t h e X chromosome t o be l o w e r t h a n t h e a u t o s o m e s . T h e s e r e s e a r c h e r s f o u n d a RFLP f r e q u e n c y o f 1/1000 bp s c r e e n e d when u s i n g t h e L sHX l i b r a r y as t h e s o u r c e o f X p r o b e s . They a l s o f o u n d a RFLP f r e q u e n c y o f 1/200 bp s c r e e n e d when u s i n g a u t o s o m a l DNA as t h e s o u r c e o f p r o b e s . -53-T a b l e 5. ENZYMES USED TO DETECT RFLPs Enzyme Number o f P r o b e s Number o f F r a g m e n t s Number o f T e s t e d O b s e r v e d bp S c r e e n e d A l u l 1 6 1 0 1 6-64 A v a l 5 6 55 A v a i l 8 1 3 105 BamHI 2 9 1 0 11 4 B g l l l 2 6 1 3 114 C l a l 3 5 5 60 D d e l 6 9 60 E c o R I 9 1 2 1 26 E c o R I I 4 8 54 EcoRV 8 10 1 08 H i n d l l l 1 9 9 1 08 H i n d i 8 1 3 1 05 H i n f I 8 10 72 K p n l 4 8 14 1 32 M s p l 9 14 92 P s t I 9 12 1 26 P v u I I 1 9 14 138 R s a l 4 6 1 3 48-76 S a d 1 9 11 120 S a i l 3 5 6 66 T a b l e 5. c o n t . Enzyme Number o f P r o b e s T e s t e d Number o f F r a g m e n t s O b s e r v e d Number o f bp S c r e e n e d S a u 3 A 2 Sau96 S s t l l T a q I 3 X b a l X h o l 3 7 3 3 9 9 8 16 6 3 16 11 9 40-92 36 24 48-100 1 20 102 TOTAL 185 273 2189-2369 s u p e r s c r i p t i n d i c a t e s o v e r l a p p i n g r e c o g n i t i o n s e q u e n c e s -55-FIGURE 8 DETECTION OF RFLPs S o t h e r n b l o t s c o n t a i n i n g 10 ug o f DNA d i g e s t e d w i t h t h e f o l l o w i n g enzymes: (a) B g l l l (b) X b a l ; ( c ) A v a i l ; (d) BamHI; (e) P v u I I ; were h y b r i d i z e d w i t h t h e p r o b e s : (a) pTS247; (b) pTS264; ( c ) pTS316; (d,e) p T S H 9 . DNA s a m p l e s : (46,XY) male; (46,XX) f e m a l e . -56-FIGURE 9 GENOMIC ORGANIZATION OF Xp PROBES DETECTING RFLPs The p o s s i b l e genomic map o f t h e 3 Xp s p e c i f i c p r o b e s w h i c h d e t e c t e d R F L P s . No a t t e m p t t o o r i e n t t h e l o c u s e i t h e r p r o x i m a l l y o r d i s t a l l y w i t h r e s p e c t t o t h e p o l y m o r p h i c s i t e has been made. (a) B g l l l p o l y m o r p h i s m d e t e c t e d by p r o b e pTS247; (b) X b a l p o l y m o r p h i s m d e t e c t e d by p r o b e pTS264; ( c ) A v a i l p o l y m o r p h i s m d e t e c t e d w i t h p r o b e pTS316. R e s t r i c t i o n enzyme c o d e : (H) H i n d l l l ; (B) B g l l l ; (A) A v a i l ; (X) X b a l . -56a-F I G . 9 PTS247 H H JI L J {V ? 20kb 5kb 2kb PTS264 H X H J I I I T {V f 5.0kb 1.9kb 2.4kb 2 k b PTS316 A _J_ H JL H _L T A I 6.0kb 0.5kb 2kb -57-4. P e d i g r e e A n a l y s i s 4.1 F a m i l y S e g r e g a t i o n o f RFLPs The d i s t i n g u i s h i n g f e a t u r e s o f f a m i l y s t u d i e s w i t h X - l i n k e d p o l y m o r p h i c p r o b e s a r e : 1) a l l m a l e s a r e h e m i z y g o u s ; 2) o n l y f e m a l e s c a n be h e t e r o z y g o u s ; 3) sons do n o t i n h e r i t t h e i r f a t h e r s a l l e l e , and 4) d a u g h t e r s must a l w a y s i n h e r i t t h e same band t h a t t h e i r f a t h e r s h a v e . L a r g e 3 - g e n e r a t i o n Mormon p e d i g r e e s (White e t a l , 1985b) c o n s i s t i n g o f a l l f o u r g r a n d p a r e n t s , b o t h p a r e n t s , and a minimum o f 6 c h i l d r e n were u s e d t o s t u d y t h e s e g r e g a t i o n o f t h e 4 p r o b e s d e t e c t i n g R F L P s . X chromosome RFLPs must be i n h e r i t e d i n an X - l i n k e d p a t t e r n . O n l y f a m i l i e s i n w h i c h t h e g e n e r a t i o n I I f e m a l e i s h e t e r o z y g o u s f o r t h e p o l y m o r p h i s m a r e i n f o r m a t i v e . E a c h o f t h e 4 p r o b e s d e t e c t i n g a RFLP were h y b r i d i z e d t o S o u t h e r n b l o t s c o n t a i n i n g d i g e s t e d DNA o f 9 c o u p l e s f r o m 6 Mormon p e d i g r e e s . S o u t h e r n b l o t s o f t h e i n f o r m a t i v e f a m i l i e s were made, and h y b r i d i z e d w i t h t h e a p p r o p r i a t e ^^•'P-lahelecl p r o b e . F i g . 10 shows p e d i g r e e K-13 40 h y b r i d i z e d w i t h p r o b e pTS247 w h i c h d e t e c t s a B g l l l p o l y m o r p h i s m . The p a t t e r n o f B g l l l r e s t r i c t i o n f r a g m e n t s i n t h i s f a m i l y i s c o n s i s t e n t w i t h X - l i n k e d i n h e r i t a n c e . The g e n e r a t i o n I I f e m a l e (GM7019) i s h e t e r o z y g o u s f o r t h e 25 kb and 20 kb a l l e l e s . She has i n h e r i t e d t h e m i n o r 25 kb a l l e l e f r o m h e r f a t h e r (GM7022) and t h e m a j o r 20 kb a l l e l e f r o m h e r mother (GM7056). B o t h o f h e r d a u g h t e r s (GM7062, GM7053) a r e homozygous f o r t h e m i n o r a l l e l e . Of t h e 4 s o n s , 2 -58- . • i n h e r i t e d t h e m i n o r a l l e l e (GM7008, GM7027), and 2 i n h e r i t e d t h e m a j o r a l l e l e (GM7040,GM7342). F i g . 11 shows p e d i g r e e K1329 h y b r i d i z e d w i t h t h e p r o b e pTS264 w h i c h d e t e c t s !a X b a l p o l y m o r p h i s m . The p a t t e r n o f X b a l r e s t r i c t i o n f r a g m e n t s i n t h i s f a m i l y i s c o n s i s t e n t w i t h X - l i n k e d i n h e r i t a n c e . The g e n e r a t i o n I I f e m a l e (GM6997) i s h e t e r o z y g o u s f o r t h e 4.3 and 1.9 kb a l l e l e s . She has i n h e r i t e d t h e m i n o r 1.9 kb a l l e l e f r o m h e r mother (GM70 45) and t h e m a j o r a l l e l e f r o m h e r f a t h e r (GM6986). One o f h e r d a u g h t e r s (GM6981) i s homozygous f o r t h e m a j o r 4.3 kb a l l e l e , and t h e o t h e r (GM6980) i s h e t e r o z y g o u s . A l l 4 o f h e r s o n s (GM7018, GM7036, GM7047, GM7433) i n h e r i t e d t h e m a j o r a l l e l e . F i g . 12 shows p e d i g r e e K1340 h y b r i d i z e d w i t h t h e p r o b e pTS316 w h i c h d e t e c t s an A v a i l p o l y m o r p h i s m . The p a t t e r n o f A v a i l r e s t r i c t i o n f r a g m e n t s i n t h i s f a m i l y i s c o n s i s t e n t w i t h X - l i n k e d i n h e r i t a n c e . The g e n e r a t i o n I I f e m a l e (GM7019) has b o t h t h e 6.5 and 6.0 kb b a n d s . Both' d a u g h t e r s (GM7062, GM7053) have t h e 6.5 and 6.0 kb band, and t h e 4 s o n s (GM7008, GM7040, GM7342, GM7027) have t h e m a j o r 6.5kb ban d . F i g . 13 shows t h e p e d i g r e e K1345 h y b r i d i z e d w i t h t h e p r o b e PTS119 w h i c h d e t e c t s a P v u I I p o l y m o r p h i s m . A n a l y s i s o f t h e r e s t r i c t i o n p a t t e r n i n t h i s f a m i l y i s n o t c o n s i s t e n t w i t h X - l i n k e d i n h e r i t a n c e . The i n d i v i d u a l s t o n o t e a r e t h e 4 h e t e r o z y g o u s m a l e s (GM7349, GM7354, GM7353, GM7351) w h i c h have b o t h t h e 21 and 16 kb a l l e l e s . S i n c e n o r m a l m a l e s have a s i n g l e X chromosome t h e y c a n n o t be h e t e r o z y g o u s f o r an X - l i n k e d m a r k e r . T h e r e f o r e i t was c o n c l u d e d t h a t t h e human i n s e r t f r o m -59-p r o b e p T S l 1 9 was n o t f r o m t h e X chromosome. A n a l y s i s o f t h e i n h e r i t a n c e p a t t e r n i n t h i s f a m i l y , as w e l l as o t h e r f a m i l i e s s e g r e g a t i n g f o r t h e P v u I I and BamHI p o l y m o r p h i s m s d e t e c t e d by t h i s p r o b e , was c o n s i s t e n t w i t h t h i s p r o b e o r i g i n a t i n g f r o m an a u tosome. 4.2 C h a r a c t e r i z a t i o n o f P r o b e pTS264 i n Two DMD F a m i l i e s S o u t h e r n b l o t s o f f a m i l i e s s e g r e g a t i n g f o r Duchenne m u s c u l a r d y s t r o p h y (DMD) were h y b r i d i z e d w i t h t h e p r o b e pTS264. Two o f t h e s e f a m i l i e s c o - s e g r e g a t e d f o r b o t h t h e TS264 marker and t h e Duchenne l o c u s . I n one f a m i l y ( F i g . 14a) t h e o b l i g a t e c a r r i e r mother (8210) i s h e t e r o z y g o u s f o r t h e 4.3 and 1.9 kb a l l e l e s . Her 2 a f f e c t e d s o n s (8209, 8210) have i n h e r i t e d t h e 4.3 kb a l l e l e . I n t h e o t h e r f a m i l y ( F i g . 14b) t h e o b l i g a t e c a r r i e r m o ther (8407) i s h e t e r o z y g o u s f o r b o t h t h e 4.3 and 1.9 kb a l l e l e . The DMD l o c u s i n t h i s f a m i l y i s c o - s e g r e g a t i n g w i t h t h e m i n o r 1.9 a l l e l e . T h i s c a n be d e d u c e d from h e r o b l i g a t e c a r r i e r mother (8405) who i s h e t e r o z y g o u s and has i n h e r i t e d t h e 4.3 a l l e l e f r o m h e r u n a f f e c t e d f a t h e r . The p r o b a n d (8410) has i n h e r i t e d t h e 1.9 kb a l l e l e and h i s u n a f f e c t e d b r o t h e r (8409) has i n h e r i t e d t h e 4.3 kb a l l e l e . Of t h e f i v e s c o r a b l e m e i o s i s i n t h e s e two f a m i l i e s t h e r e has b een no r e c o m b i n a t i o n between t h e DMD l o c u s and t h e m a r k e r pTS264. 4.3 N o n - M e n d e l i a n S e g r e g a t i o n o f t h e pTS316 RFLP The p r o b e pTS316 has b e e n l o c a l i z e d t o t h e Xp11 t o X p t e r r e g i o n ( s e c t i o n 2) and d e t e c t s an A v a i l p o l y m o r p h i s m . Of t h e 64 i n d i v i d u a l s t y p e d w i t h t h i s p r o b e , e v e r y f e m a l e has b o t h bands -60-and e v e r y male has o n l y t h e 6.5 kb band ( T a b l e 6 ) . S t a t i s t i c a l c a l c u l a t i o n s u s i n g t h e C h i s q u a r e d t e s t (shown below) i n d i c a t e w i t h a 99.9% p r o b a b i l i t y t h a t t h e l o c u s r e p r e s e n t e d by p r o b e TS316 i s n o t i n H a r d y - W e i n b e r g e q u i l i b r i u m w i t h i n t h e p o p u l a t i o n u s e d i n t h i s s t u d y . C h i t e s t H Q : t h e A v a i l p o l y m o r p h i s m d e t e c t e d by t h e p r o b e pTS316 i s i n H a r d y - W e i n b e r g e q u i l i b r i u m i n t h i s p o p u l a t i o n . 6.5 6.5/6.0 6.0 m a l e s 1 0 ( 5 . 7 ) - 0 ( 4 . 3 ) f e m a l e s 0 (6.5) 20 (9.8) 0 (3.7) X 2 = ( 1 0 - 5 . 7 ) 2 / 5 . 7 + ( 0 - 6 . 5 ) 2 / 6 . 5 + ( 2 0 - 9 . 8 ) 2 / 9 . 8 + ( 0 - 4 . 3 ) 2 / 4 . 3 + ( 0 - 3 . 7 ) 2 / 3 . 7 = 28.36 c r i t i c a l v a l u e : a.001' ^-2 = 13.816 28.36 >_ 13.816 t h e r e f o r e H Q i s r e j e c t e d and t h e p o l y m o r p h i s m d e t e c t e d by pTS316 i s n o t i n H a r d y - W e i n b e r g e q u i l i b r i u m . Many f a c t o r s w h i c h p e r t u r b t h i s e q u i l i b r i u m d i r e c t l y b e a r on t h e r e p r o d u c t i v e f i t n e s s o f i n d i v i d u a l s , l e t h a l i t y b e i n g t h e most e x t r e m e c a s e . No i n d i v i d u a l has b e e n shown t o i n h e r i t o n l y t h e 6.0kb ban d . The p r e s e n c e o f o n l y t h e 6.0kb b a n d ( s ) w o u l d be s e l e c t e d a g a i n s t i f t h i s band were l i n k e d t o a l e t h a l m u t a t i o n . -61 -The p o s s i b i l i t y t h a t t h e 6.0kb band i s l i n k e d t o a r e c e s s i v e i l e t h a l c a n be e l i m i n a t e d s i n c e t h i s p r e d i c t s t h a t f e m a l e s w i t h o n l y t h e 6.5kb band w o u l d be n o r m a l . As T a b l e 6 i n d i c a t e s no f e m a l e has o n l y t h e 6.5kb ba n d . A l t e r n a t i v e l y , t h e TS316 l o c u s c o u l d be t i g h t l y l i n k e d t o two l e t h a l m u t a t i o n s i n t r a n s t o e a c h o t h e r . I f t h e TS316-6.5 kb band i s on a chromosome w i t h a f e m a l e s p e c i f i c l e t h a l and t h e TS316-6.0 kb band i s on a chromosome w i t h a r e c e s s i v e l e t h a l t h e n f e m a l e s homozygous f o r e i t h e r chromosome and m a l e s w i t h t h e TS316-6.0 kb chromosome wo u l d be s e l e c t e d a g a i n s t . F a m i l y s t u d i e s h a v e i n d i c a t e d t h a t t h i s l o c u s i s s e g r e g a t i n g a t y p i c a l l y . S t a t i s t i c a l c a l c u l a t i o n s u s i n g t h e C h i s q u a r e d t e s t (shown b e l o w ) i n d i c a t e w i t h a 99.9% p r o b a b i l i t y t h a t t h e l o c u s r e p r e s e n t e d by p r o b e TS316 i s n o t s e g r e g a t i n g i n a M e n d e l i a n f a s h i o n . C h i t e s t H Q : t h e A v a i l p o l y m o r p h i s m d e t e c t e d by t h e p r o b e pTS316 i s s e g r e g a t i n g i n a M e n d e l i a n f a s h i o n . 6^5 6.5/6.0 6.0 m a l e s 16 (8) - 0 (8) f e m a l e s 0 (6) 12 (6) 0 (0) - 6 2 -X 2 = ( 1 6 - 8 ) 2 / 8 + ( 0 - 8 ) 2 / 8 + ( 0 - 6 ) 2 / 6 + ( 1 2 - 6 ) 2 / 6 = 2 8 . 0 c r i t i c a l v a l u e : a # Q 0 1 ' d f 2 = 1 3 . 8 1 6 2 8 . 0 > 13.816 t h e r e f o r e H 0 i s r e j e c t e d and t h e p o l y m o r p h i s m d e t e c t e d by pTS31 6 i s s e g r e g a t i n g a t y p i c a l l y A n o t h e r h y p o t h e s i s , c u r r e n t l y u n d e r i n v e s t i g a t i o n , i s t h a t t h e 6.0kb band c o r r e l a t e s w i t h t h e phenomenon o f X chromosome i n a c t i v a t i o n . I t has l o n g been known t h a t mammalian X chromosomes u n d e r g o i n a c t i v a t i o n a s a mechanism o f d o s a g e c o m p e n s a t i o n ( L y o n , 1 9 6 1 ) . A l t h o u g h t h i s phenomenon has b e e n w e l l e s t a b l i s h e d , l i t t l e i s known o f t h e m e c h a n i c s a t t h e m o l e c u l a r l e v e l . The o b s e r v a t i o n s t h a t t h e X chromosome u n d e r g o e s i n a c t i v a t i o n and r e a c t i v a t i o n , and t h a t t h e i n a c t i v e X i s s t a b l y m a i n t a i n e d t h r o u g h r e p e a t e d c e l l d i v i s i o n s a r e s u g g e s t i v e o f a r e v e r s i b l e DNA s e q u e n c e m o d i f i c a t i o n . I t i s , t h e r e f o r e , p o s s i b l e t h a t t h e o b s e r v e d A v a i l bands a r e a s s o c i a t e d w i t h t h e a c t i v e and i n a c t i v e f o r m s o f t h e X chromosome. To t e s t t h i s h y p o t h e s i s pTS31 6 was h y b r i d i z e d t o DNA from a 49,XXXXY c e l l l i n e . S i n c e i n d i v i d u a l s a n e u p l o i d f o r t h e X chromosome p r e s e r v e one a c t i v e X chromosome p e r a u t o s o m a l s e t ( J a c o b s e t a l , 1979; R a s t a n e t a l , 1 9 8 0 ) , t h e 49,XXXXY i n d i v i d u a l i s e x p e c t e d t o have 3 i n a c t i v e X chromosomes and one a c t i v e X. As shown i n F i g . 1 5 , t h e 6 . 0 kb band, w h i c h i s n e v e r p r e s e n t i n n o r m a l m a l e s , i s p r e s e n t i n 3 c o p i e s and t h e 6 . 5 kb band i s p r e s e n t i n one. T h e s e r e s u l t s s u g g e s t t h a t p r o b e TS316 may be v e r y u s e f u l f o r t h e s t u d y o f X chromosome i n a c t i v a t i o n . -63-T a b l e 6. D i s t r i b u t i o n o f TS316 Bands i n M a l e s and F e m a l e s BAND SIZES 46XY 46XX 6.5 29 0 6.5/6.0 35 6.0 0 0 -64-FIGURE 10 B g l l l POLYMORPHISM DETECTED WITH PROBE pTS247 10 ug o f DNA f o r m e a c h f a m i l y member o f p e d i g r e e K1340 was d i g e s t e d w i t h B g l l l , S o u t h e r n b l o t t e d , and h y b r i d i z e d w i t h t h e 3 2 - P - l a b e l e d p r o b e pTS247 ION) II FIGURE 11 X b a l POLYMORPHISM DETECTED WITH PROBE pTS264 10 ug o f DNA f r o m e a c h f a m i l y member o f p e d i g r e e K1329 was d i g e s t e d w i t h X b a l , S o u t h e r n b l o t t e d , and h y b r i d i z e d w i t h t h e 3 2 - P - l a b e l e d p r o b e pTS264. FIG.11 • - T - O D - T - O a 6 A A A 6 r5 4.3 i I 1 . 9 --66-FIGURE 12 A v a i l POLYMORPHISM DETECTED WITH PROBE pTS316 10 ug o f DNA f r o m e a c h f a m i l y member o f p e d i g r e e K1340 was d i g e s t e d w i t h A v a i l , S o u t h e r n b l o t t e d , and h y b r i d i z e d w i t h t h e 3 2 - P - l a b e l e d p r o b e pTS316. F I G . 1 2 i I 6.5_ 6.0- = t3 « — — -67-FIGURE 13 P v u I I POLYMORPHISM DETECTED WITH PROBE p T S l 1 9 10 ug o f DNA f r o m e a c h f a m i l y member o f p e d i g r e e K1345 was d i g e s t e d w i t h P v u I I , S o u t h e r n b l o t t e d , and h y b r i d i z e d w i t h t h e 3 2 - P - l a b e l e d p r o b e p T S H 9 -67a--68-FIGURE 14 CO-SEGREGATION OF DMD AND pTS264 5.0 ug o f X b a l d i g e s t e d DNA was S o u t h e r n b l o t t e d and h y b r i d i z e d w i t h t h e p r o b e pTS264 -68a--69-F i g u r e 15. H y b r i d i z a t i o n o f P r o b e pTS316 t o C e l l L i n e s W i t h D i f f e r e n t Numbers o f X Chromosomes 10 ug o f DNA, d i g e s t e d t o c o m p l e t i o n w i t h H i n d l l l , was S o u t h e r n b l o t t e d and h y b r i d i z e d w i t h t h e p r o b e pTS316: (a) 46,XY; (b) 49,XXXXY: ( c ) 46,XX. -69a-F I G . 15 6.5 6 . 0 C B A 5. G e n e r a l D i s c u s s i o n The a v a i l a b i l i t y o f f l o w s o r t e d l i b r a r i e s , e n r i c h e d f o r s p e c i f i c chromosomes, has a f f o r d e d r e s e a r c h e r s t h e o p p o r t u n i t y t o f o c u s upon a s m a l l r e g i o n o f t h e genome. T h i s w i l l f a c i l i t a t e t h e c o n s t r u c t i o n o f a human l i n k a g e map by r e d u c i n g t h e t i m e and e f f o r t r e q u i r e d t o e s t a b l i s h l i n k a g e between m a r k e r s . As more RFLPs a r e i s o l a t e d and mapped t h e n s m a l l l i n k a g e g r o u p s w i l l c o n v e r g e t o p r o d u c e a f u n c t i o n a l map o f a chromosome o r chromosomal r e g i o n . Such a map i s n e a r c o m p l e t i o n f o r t h e X chromosome ( D r a y n a and W h i t e , 1 9 8 5 ) . A l t h o u g h f l o w s o r t e d l i b r a r i e s a r e h i g h l y e n r i c h e d f o r s p e c i f i c chromosomes t h e y a r e n o t f r e e o f b a c k g r o u n d DNA and t h u s i t i s f i r s t n e c e s s a r y t o c o n f i r m t h e chromosomal o r i g i n o f any p r o b e i s o l a t e d . T h i s c a n be e a s i l y a c c o m p l i s h e d f o r p r o b e s i s o l a t e d f r o m l i b r a r i e s c o n t a i n i n g a s i n g l e human chromosome i n a r o d e n t b a c k g r o u n d . P r o b e s i s o l a t e d f r o m l i b r a r i e s c o n t a i n i n g o t h e r human chromosomes as t h e b a c k g r o u n d DNA r e q u i r e a d d i t i o n a l s c r e e n i n g t o s p e c i f y t h e chromosomal o r i g i n . P a r t o f t h e work p r e s e n t e d i n t h i s t h e s i s d e s c r i b e s t h e u s e o f d o t b l o t s i n c o n j u n c t i o n w i t h s o m a t i c c e l l h y b r i d s as an i d e a l way t o q u i c k l y c o n f i r m t h e chromosomal o r i g i n o f p r o b e s r a n d o m l y i s o l a t e d f r o m a l i b r a r y c o n s t r u c t e d f r o m a 49,XXXXY c e l l l i n e . T h i s m e t h o d o l o g y c a n be a p p l i e d t o any f l o w s o r t e d l i b r a r y and w i l l a i d i n r e d u c i n g t h e e f f o r t r e q u i r e d t o i s o l a t e chromosome s p e c i f i c p r o b e s . T h e r e a r e 48 a r b i t r a r y DNA p r o b e s a s s i g n e d t o Xp ( G o o d f e l l o w 1 9 8 5 ) . The i n c l u s i o n o f t h e 9 Xp s p e c i f i c p r o b e s r e p o r t e d i n t h i s work b r i n g s t h e t o t a l number o f c l o n e d Xp p r o b e s t o 57. M o r e o v e r t h e r e a r e 19 r e p o r t e d Xp s p e c i f i c R F L P s . T h e s e a r e s u b l o c a l i z e d as f o l l o w s : 10 a r e f o u n d i n t h e Xp22 t o X p t e r r e g i o n ; 7 a r e f o u n d i n t h e Xp21 r e g i o n ; and 2 a r e f o u n d i n t h e Xp11 r e g i o n . The 3 n e w l y i s o l a t e d X s p e c i f i c RFLPs d e s c r i b e d i n t h i s s t u d y h a v e been l o c a l i z e d t o t h e d i s t a l 2/3 o f Xp. They c a n be e x c l u d e d f r o m t h e r e g i o n X c e n t o Xp11. I n a d d i t i o n t h e y c a n be e x c l u d e d f r o m a s m a l l r e g i o n c e n t e r e d a r o u n d Xp21 ( s e e f i g . 5 GM7947) where DMD i s l o c a t e d . R o u y e r e t a l (1986) have shown t h a t a r e g i o n (Xp22.3 t o X p t e r ) o f X-Y homology e x i s t s i n w h i c h r e c o m b i n a t i o n t a k e s p l a c e . P r o b e s i s o l a t e d f r o m t h i s r e g i o n b e h a v e as i f t h e y were i n h e r i t e d i n an a u t o s o m a l f a s h i o n . S i n c e t h e RFLPs i s o l a t e d i n t h i s r e p o r t behave i n an X - l i n k e d manner t h e y c a n be e x c l u d e d f r o m t h e Xp22.3 r e g i o n d i s t a l t o t h e p r o b e DXYS17. DXYS17 i s t h e most p r o x i m a l p s e u d o - a u t o s o m a l p r o b e i s o l a t e d . T h e r e f o r e t h e p r o b e s pTS247, pTS264, and pTS316 c a n be a s s i g n e d t o one o f two r e g i o n s : t h e r e g i o n Xp11.4 t o t h e p r o x i m a l b r e a k p o i n t o f GM7947 a t Xp21, o r t h e r e g i o n f r o m t h e d i s t a l b r e a k p o i n t o f GM79 47 t o Xp22.3 p r o x i m a l t o t h e p r o b e DXYS17. The p r o b e pTS316 h y b r i d i z e s t o two A v a i l f r a g m e n t s . B o t h o f t h e s e f r a g m e n t s a r e a l w a y s p r e s e n t i n X - i n a c t i v a t e d c e l l s . E v i d e n c e t o d a t e s u g g e s t s t h a t t h e 6.0 kb band c o r r e l a t e s w i t h t h e phenomenon o f X chromosome i n a c t i v a t i o n . T h i s i s t h e f i r s t d e s c r i p t i o n o f a p r o b e s p e c i f i c f o r i n a c t i v a t e X chromosomes. I n -72-o r d e r t o t e s t t h e s p e c i f i c i t y f u r t h e r pTS316 c o u l d be h y b r i d i z e d t o s o m a t i c c e l l h y b r i d s w h i c h c o n t a i n e d e i t h e r an a c t i v e o r i n a c t i v e human X chromosome. The a c t i v e X c e l l l i n e i s e x p e c t e d t o g i v e a s i n g l e band a t 6.5 kb and t h e i n a c t i v e X c e l l l i n e s h o u l d show a s i n g l e band a t 6.0 kb i f t h e X - i n a c t i v a t i o n h y p o t h e s i s i s c o r r e c t . B e c a u s e t h e a c t i v e and i n a c t i v e f o r m o f t h e X chromosome c a n be s e e n c y t o l o g i c a l l y , t h e s e p r e d i c t i o n s c a n be v e r i f i e d . S i n c e t h e c l o n e d p r o b e d o e s n ' t i n c l u d e t h e A v a i l s i t e , r e c o v e r y o f l a r g e phage c l o n e s f r o m a v a i l a b l e l i b r a r i e s w o u l d be v a l u a b l e . By h a v i n g t h e c l o n e d A v a i l s i t e t h e m o l e c u l a r n a t u r e o f t h i s p o l y m o r p h i s m c o u l d be i n v e s t i g a t e d . F u r t h e r m o r e t h i s s i t e m i g h t show c r o s s homology t o o t h e r mammalian X chromosomes t h a t a r e i n v o l v e d i n X i n a c t i v a t i o n and may i n f a c t be v e r y v a l u a b l e i n c o n t r i b u t i n g t o a b e t t e r u n d e r s t a n d i n g o f t h e phenomenon o f X-chromosome i n a c t i v a t i o n . 6. CONCLUSIONS 1 . T h i r t y X s p e c i f i c p r o b e s were i s o l a t e d f r o m a phage l i b r a r y t h a t had b e e n e n r i c h e d f o r t h e X chromosome by f l o w s o r t i n g . N i n e o f t h e s e p r o b e s were shown t o o r i g i n a t e f r o m t h e Xp arm. Two l o c a l i z e d t o t h e Xcen t o Xp11 r e g i o n and 7 l o c a l i z e d t o t h e Xp11 t o X p t e r r e g i o n . A l l o f t h e 7 p r o b e s , l o c a l i z e d t o t h e d i s t a l 2/3 o f Xp, were e x c l u d e d f r o m t h e Xp21 r e g i o n . 2. F o u r p r o b e s , i s o l a t e d f r o m t h e L sHX l i b r a r y , d e t e c t i n g 5 i n d e p e n d e n t RFLPs were f o u n d . T h r e e p r o b e s pTS247, pTS264, and pTS316 were shown t o be X - l i n k e d and o r i g i n a t e f r o m t h e Xp11 t o X p t e r r e g i o n . The f o u r t h p r o b e , p T S l 1 9 , d e t e c t e d 2 i n d e p e n d e n t p o l y m o r p h i s m s and was shown t o o r i g i n a t e f r o m one o f t h e a u t o s o m e s . 3. An RFLP f r e q u e n c y o f a p p r o x i m a t e l y 1/800 bp s c r e e n e d , f o r t h e X - l i n k e d p r o b e s , and 1/180 bp f o r t h e s i n g l e a u t o s o m a l p r o b e was f o u n d . T h e s e r e s u l t s a r e i n agreement w i t h H o f k e r e t a l (1985) t h a t t h e X chromosome i s l e s s p o l y m o r p h i c t h a n t h e a u t o s o m e s . 4. The X - l i n k e d p r o b e pTS316, w h i c h was l o c a l i z e d t o t h e Xp11 t o X p t e r r e g i o n and d e t e c t s an A v a i l p o l y m o r p h i s m , i s s e g r e g a t i n g a t y p i c a l l y . T h i s p o l y m o r p h i s m i s s e x s p e c i f i c w i t h a l l f e m a l e s showing b o t h f o r m s o f t h e m a r k e r . T h i s has been -74-p o s t u l a t e d t o c o r r e l a t e w i t h t h e phenomenon o f X chromosome i n a c t i v a t i o n . F u r t h e r a n a l y s i s i s r e q u i r e d i n o r d e r t o s u b s t a n t i a t e t h i s h y p o t h e s i s . -75-REFERENCES A l d r i d g e , J . , L . K u n k e l , G.Bruns, U . T a n t r a v a h i , M . L a l a n d e , T . B r e w s t e r , E.Moreau, M . W i l s o n , W.Bromely, T . R o d e r i c k , S . A . L a t t . 1984. A s t r a t e g y t o r e v e a l h i g h - f r e q u e n c y RFLPs a l o n g t h e human X chromosome. Am J Hum G e n e t 36:546-564. A n t o n a r a k i s , S.E., C.D.Boehm, P . G i a r d i n a , and H . H . K a z a z i a n . 1982. Nonrandom a s s o c i a t i o n o f p o l y m o r p h i c r e s t r i c t i o n s i t e s i n t h e b - g l o b i n gene c l u s t e r . PNAS 79:137-141 A n t o n a r a k i s , S.E., C.D.Boehm, G . R . S e r g e a n t , C . E . T h e i s e n , G . J . D o v e r , H . H . K a z a z i a n . 1984. O r i g i n o f t h e B s - g l o b i n gene i n b l a c k s : t h e c o n t r i b u t i o n o f r e c u r r e n t m u t a t i o n o r gene c o n v e r s i o n o r b o t h . PNAS 81: 853 B a k k e r , E . , P . W i e a c k e r , G . C . B e v e r s t o c k and P . L . P e a r s o n . 1983. R e c o m b i n a n t DNA t e c h n i q u e s f o r mapping t h e human X chromosome. C l i n . G e n e t . 23:225. B a k k e r , E., N.Goor, K.Wrogmann, L . K u n k e l , W.A.Fenton, D . M a j o o r - K r a k a u e r , M.Jahoda, G.vanOmmen, M.H.Hofker, J . L . M a n d e l , K . E . D a v i e s , H . F . W i l l a r d , L . S a n d k u y l , A . E s s e n , E . S . S a c h s , P . L . P e a r s o n . 1985. P r e n a t a l d i a g n o s i s and c a r r i e r d e t e c t i o n o f Duchenne m u s c u l a r d y s t r o p h y w i t h c l o s e l y l i n k e d R F L P s . L a n c e t M a r c h 23;655 B i s h o p , C . E . , G . G u e l l a e n , D . G e l d w e r t h , R.Voss, M . F e l l o u s , and J . W e i s s e n b a c h . 1983. S i n g l e - c o p y DNA s e q u e n c e s s p e c i f i c f o r t h e human Y chromosome. N a t u r e 303:831-832 B o t s t e i n , D . , R . L . W h i t e , M . S k o l n i c k and R.W.Davis. 1980. C o n s t r u c t i o n o f a g e n e t i c l i n k a g e map i n man u s i n g r e s t r i c t i o n f r a g m e n t l e n g t h p o l y m o r p h i s m s . Am.J.Hum.Genet. 32:314-331. B r u n s , G . , J . G u s e l l a , C.Keys, A . L e a r y , D.Housman, and P . G e r a l d . 1982: I s o l a t i o n o f X-chromosome DNA s e q u e n c e s . Adv Exp Med B i o l 154:60-72 C h a k r a v a r t i , A., K.H.Buetow, S.E.Antonarakis, P.G.Waber, C.D.Boehm, and H.G.Kazazian. 1984. Nonuniform recombination w i t h i n the human B - g l o b i n gene c l u s t e r . Am J Genet 36: 1239 de l a Chapelle,A., 1985: The 1985 human gene map and human gene mapping i n 1985. Human gene mapping 8, Cytogenet C e l l Genet 40.: 1 -8 Cooper,D. and J.Schmidtke. 1984: DNA r e s t r i c t i o n fragment l e n g t h polymorphisms and h e t e r o z y g o u s i t y i n the human genome. Hum Genet. 66:1-16. Curry,C., R.Magenis, M.Brown, J.Lanm, T . T s a i , P.O'Lague, P.Goodfellow, T.Mohandas, E.Bergner, and L .Shapiro. 1984: I n h e r i t e d c h o n d r o d y s p l a s i a punctata due to a d e l e t i o n of the t e r m i n a l s h o r t arm of an X chromosome. N Engl J Med 311 -.1010-1015. - 7 6 -D a v i e s , K . E . , P . S . H a r p e r and R . W i l l i a m s o n . 1983. C l o n e d gene p r o b e s f o r c a r r i e r d e t e c t i o n i n m u s c u l a r d y s t r o p h y . L a n c e t i i : 1 0 8 . D a v i e s , K . E . , P . B r i a n d , V . I o n a s e s c u , G . I o n a s e s c u , R . W i l l i a m s o n , C.Brown, C . C a v a r d and L . C a t h e l i n e a u . 1985. Gene f o r OTC: c h a r a c t e r i z a t i o n and l i n k a g e t o DMD. N u c l . A c i d Res. 13:155-165. D a v i e s , K . E . , P . L . P e a r s o n , P . S . H a r p e r , J.M.Murray, T . O ' B r i e n , M . S a r f a r a z i and R . W i l l i a m s o n . 1983. L i n k a g e a n a l y s i s o f two c l o n e d DNA s e q u e n c e s f l a n k i n g t h e Duchenne m u s c u l a r d y s t r o p h y l o c u s on t h e s h o r t arm o f t h e human X chromosome. N u c l . A c i d R e s . 11:2303-2312. Davis,R.W., D . B o t s t e i n and J . R . R o t h . 1980. A manual f o r g e n e t i c e n g i n e e r i n g . A d v a n c e d b a c t e r i a l g e n e t i c s . C o l d S p r i n g H a r b o r . New Y o r k . de M a r t i n v i l l e , B . , L . K u n k e l , G.Bruns F . M o r l e , M.Koenig, J . L . M a n d e l , A . H o r w i c h , S . A . L a t t , J . F . G u s e l l a , D.Housman and U . F r a n c k e . 1985. L o c a l i z a t i o n o f DNA s e q u e n c e s i n t h e r e g i o n o f Xp21 o f t h e human X chromosome: S e a r c h f o r m o l e c u l a r m a r k e r s c l o s e t o t h e Duchenne m u s c u l a r d y s t r o p h y l o c u s . Am.J.Hum.Genet. 37_:235-249. D o r k i n s , H . , C . J u n i e n , J . L . M a n d e l , K.Wrogemann, J . P . M o i s o n , M . M a r t i n e z , J . M . o l d , S.Bundy, M.Swartz, N . C a r p e n t e r , D . H i l l , M . L i n d l o f , A.de l a C h a p e l l e , P . L . P e a r s o n , and K . E . D a v i e s . 1985. S e g r e g a t i o n a n a l y s i s o f a m a r k e r l o c a l i z e d Xp21.2-Xp21.3 i n Duchenne and B e c k e r m u s c u l a r d y s t r o p h y f a m i l i e s . Hum Ge n e t 71: 1 03 Drayna,D., K . D a v i e s , D . H a r t l e y , J . - L . M a n d e l , G.Camerino, R . W i l l i a m s o n and R.White. 1984. G e n e t i c mapping o f t h e human X chromosome by u s i n g r e s t r i c t i o n f r a g m e n t l e n g t h p o l y m o r p h i s m s . P r o c . N a t l . A c a d . S c i . 81:2836-2839. D r a y n a , D e n n i s , and Ray W h i t e . 1985. The g e n e t i c l i n k a g e map o f t h e human X chromosome. S c i e n c e 230: 753 F e d d e r , J . , L.Yen, E.Wijsman, L.Wang, L . W i l k i n s , J . S c h r o d e r , N . S p u r r , H.Cann, M.Blumenberg, and L . C a v a l l i - S f o r z a . 1985. A s y s t e m a t i c a p p r o a c h f o r d e t e c t i n g h i g h f r e q u e n c y r e s t r i c t i o n f r a g m e n t l e n g t h p o l y m o r p h i s m s u s i n g l a r g e genomic p r o b e s . Am J Hum Genet 37:635 F r a n c k e , U . , H.D.Ochs, B.de M a r t i n v i l l e J . G i a c a l o n e , V . L i n d g r e n , C . D i s t e c h e , R.A.Pagon, M.H.Hofker, G.-J.B.van Ommen, P . L . P e a r s o n and R.J.Wedgwood. 1985. M i n o r Xp21 chromosome d e l e t i o n i n a male a s s o c i a t e d w i t h e x p r e s s i o n o f Duchenne m u s c u l a r d y s t r o p h y , c h r o n i c g r a n u l o m a t o u s d i s e a s e , r e t i n i t i s p i g m e n t o s a and McLeod syndrome. Am.J.Hum.Genet. 37:250-267. H o r w i c h , A . , W.Fenton, >K.Williams, F . K a l o u s e k , J . K r a u s , R . D o o l i t t l e , W . K o n i g s b e r , and L . E . R o s e n b e r g . 1984: S t r u c t u r e and e x p r e s s i o n o f a co m p l e m e n t a r y DNA f o r t h e n u c l e a r c o d e d p r e c u r s o r o f human m i t o c h o n d r i a l o r n i t h i n e t r a n s c a r b o m y l a s e . S c i e n c e 224:1068-1074. G a r t l e r , S . M . , and A . D . R i g g s . 1983. Mammalian X-chromosome i n a c t i v a t i o n . Ann Rev Gen e t 1983. 17: 155 G i t s c h i e r , J . , W.Wood, T . G o r a l k a , K.Wion, E.Chen, D . E a t o n , G.Vehar, D.Capon, and R.M.Lawn. 1984: C h a r a c t e r i z a t i o n o f t h e human f a c t o r V I I I gene. N a t u r e 312:326. G o o d f e l l o w , P . , K . E . D a v i e s , H.Ropers. 1985: R e p o r t o f t h e co m m i t t e e on t h e g e n e t i c c o n s t i t u t i o n o f t h e X and Y chromosomes. Human gene mapping 8, C y t o g e n e t C e l l G enet 40:296 G u s e l l a , J . F . , N.Wexler, P . C o n n e a l l y , S . N a y l o r , M.Anderson, R . T a n z i , P . W a t k i n s , K . O t t i n a , M . W a l l a c e , A . S a k a g u c h i , A.Young, I . S h o u l s t o n , E . B o n i l l a , and J . M a r t i n . 1983: A p o l y m o r p h i c DNA mar k e r g e n e t i c a l l y l i n k e d t o H u n t i n g t o n ' s d i s e a s e . N a t u r e 306:234. Hanauer,A., M . L e v i n , R . H e i l i g , D . D a e g e l e n , A.Kahn, and J . M a n d e l . 1983: I s o l a t i o n and c h a r a c t e r i z a t i o n o f cDNA c l o n e s f o r human s k e l e t a l m u s c l e a l p h a - a c t i n . N u c l . A c i d s r e s . 11:3503-3516 H a r p e r , P . S . , T . O ' B r i e n , J.M.Murray, K . E . D a v i e s , P . P e a r s o n and R . W i l l i a m s o n . 1983. The u s e o f l i n k e d DNA p o l y m o r p h i s m s f o r g e n o t y p e p r e d i c t i o n i n f a m i l i e s w i t h Duchenne m u s c u l a r d y s t r o p h y . J.Med.Genet. 20:252-254. Hofker,M.H., M.C.Wapenaar, N.Goor, E . B a k k e r , G.-J.B.van Ommen and P . L . P e a r s o n . 1985. I s o l a t i o n o f p r o b e s d e t e c t i n g r e s t r i c t i o n f r a g m e n t l e n g t h p o l y m o r p h i s m s f r o m X c h r o m o s o m e - s p e c i f i c l i b r a r i e s : p o t e n t i a l u s e f o r d i a g n o s i s o f Duchenne m u s c u l a r d y s t r o p h y . Hum.Genet. 70 :1 48-156. H o r w i c h , A . L . , W.A.Fenton and K . R . W i l l i a m s . 1984. S t r u c t u r e and e x p r e s s i o n o f a c o m p l e m e n t a r y DNA f o r t h e n u c l e a r c o d e d p r e c u r s o r o f human m i t o c h o n d r i a l o r n i t h i n e t r a n s c a r b a m y l a s e . S c i e n c e 224:1068-1074. I n g l e , C , R . W i l l i a m s o n , A.de l a C h a p e l l e , R.R.Herva, K . H a a p a l a , G . B a t e s , H . F . W i l l a r d , P . P e a r s o n and K . E . D a v i e s . 1985. Mapping DNA s e q u e n c e s i n a human X chromosome d e l e t i o n w h i c h e x t e n d s a c r o s s t h e r e g i o n o f t h e Duchenne m u s c u l a r d y s t r o p h y m u t a t i o n . Am.J.Hum.Genet. 37:451-462. J a c o b s , P . A . , A.Matsuyama, I.Buchanan, and C . W i l s o n . 1979: L a t e r e p l i c a t i n g X c h r o m o s o m e s i n human t r i p l o i d y . Am J Hum G e n e t . 31:446-457 -78-. J a c o b s , P . A . P.A.Hunt, M.Mayer and R . D . B a r t . 1981. Duchenne m u s c u l a r d y s t r o p h y (DMD) i n a f e m a l e w i t h an X-autosome t r a n s l o c a t i o n : F u r t h e r e v i d e n c e t h a t t h e DMD l o c u s i s a t Xp21. Am.J.Hum.Genet. 33:513-518. J e f f r e y s , A . F . , 1979. DNA s e q u e n c e v a r i a n t s i n t h e g_9, g_a, and b - g l o b i n g e n e s o f man. C e l l 18:1-10. J e f f r e y s , A . J . , V . W i l s o n , S . L . T h e i n . 1985. H y p e r v a r i a b l e " m i n i s a t e l l i t e " r e g i o n s i n human DNA. N a t u r e 314:67 J e l i n e k , W . R . , and C.W.Schmid. 1982. R e p e t i t i v e s e q u e n c e s i n e u k a r y o t i c DNA and t h e i r e x p r e s s i o n . Ann Rev B iochem. 1982 51: 813 J o l l y , D.J., H.Okayama, P . B e r g , A . C . E s t y , D . F i l u p l a , P . B o h l e n , G.G.Johnson, J . E . S h i v e l y , T . H u n k a p i l a r , and T . F r i e d m a n n . 1983. I s o l a t i o n and c h a r a c t e r i z a t i o n o f a f u l l - l e n g t h cDNA f o r human h y p o x a n t h i n e p h o s p h o r i b o s y l t r a n s f e r a s e . PNAS 80: 477 Kan,Y.W., and A.M.Dosy. 1978. A n t e n a t a l d i a g n o s i s o f s i c k l e - c e l l a n a e mia by DNA a n a l y s i s o f a m n i o t i c f l u i d c e l l s . L a n c e t 2:910-912. Kan,Y.W., and A.M.Dosy. 1980. E v o l u t i o n o f t h e h e m o g l o b i n S and C genes i n w o r l d p o p u l a t i o n s . S c i e n c e 209: 388 K a r l s o n . S . , and A . N i e n h u i s . 1985. D e v e l o p m e n t a l r e g u l a t i o n o f human g l o b i n g e n e s . Ann. Rev. G e n e t . 54:1071-1108. K u n k e l , L . , K . S m i t h , S . B o y e r , D . B o r g a o n k a r , D . W a c h t e l , O . M i l l e r , W.Breg, H.Jones, and J . R a r y . 1977: A n a l y s i s o f human Y-chromosome s p e c i f i c r e i t e r a t e d DNA i n chromosome v a r i a n t s . PNAS 7_4:1 245-1249 K u n k e l , L . M . , U . T a n t r a v a h i , M . E i s e n h a r d and S . A . L a t t . 1982. R e g i o n a l l o c a l i z a t i o n on t h e human X o f DNA segments c l o n e d f r o m f l o w s o r t e d chromosomes. N u c l e i c A c i d s Res. 10:1557-1578. K u n k e l , L . M . , A.P.Monaco, W . M i d d l e s w o r t h , H.D.Ochs and S . A . L a t t . 1985a. S p e c i f i c c l o n i n g o f DNA f r a g m e n t s a b s e n t f r o m t h e DNA o f a male p a t i e n t w i t h an X chromosome d e l e t i o n . P r o c . N a t l . A c a d . S c i . 82:4778-4782. Lau, Y . F . , A.M.Dosy, J.C.Huang, Y.W.Kan. 1984. A r a p i d s c r e e n i n g t e s t f o r a n t e n a t a l s e x d e t e r m i n a t i o n . L a n c e t J a n 7, 1984: 14 Law,D.j., P.M. F r o s s a r d and D . L . R u c k n a g e l . 1984. H i g h l y s e n s i t i v e and r a p i d gene mapping u s i n g m i n i a t u r i z e d b l o t h y b r i d i z a t i o n : a p p l i c a t i o n t o p r e n a t a l d i a g n o s i s . Gene 2_8:153-158. Lindenbaum,R.H., G . C l a r k e , C . P a t e l , M . M o n c r i e f f and J.T.Hughes. 1979. M u s c u l a r d y s t r o p h y i n an X;1 t r a n s l o c a t i o n f e m a l e s u g g e s t s t h a t Duchenne l o c u s i s on X chromosome s h o r t arm. J.Med.Genet. 16:389-392. -79-Lyon,M.F. 1961: Gene a c t i o n i n t h e X-chromosome o f t h e mouse (Mus m u s c u l u s ) N a t u r e 1 90:372 . M a n i a t i s , T . , R . H a r d i s o n , E . L a c y , J . L a u e r , C . O ' C o n n e l l , and D.Quon. 1978: The i s o l a t i o n o f S t r u c t u r a l genes f r o m l i b r a r i e s o f e u c a r y o t i c DNA. C e l l 15:687-701 M a n i a t i s , T . E . F r i t s c h , and J.Sambrook. 1982: M o l e c u l a r c l o n i n g . C o l d S p r i n g H a r b e r L a b o r a t o r y 1982. New y o r k . M c A l p i n e , P . , T.Shows, R . M i l l e r , and A . P a k s t i s . 1985: The 1985 c a t a l o g u e o f mapped genes and r e p o r t o f t h e n o m e n c l a t u r e c o m m i t t e e . Human gene mapping 8, C y t o g e n e t C e l l Genet 40:1-8 M c K u s i c k , V . A . , 1975. M e n d e l i a n i n h e r i t a n c e i n man. 4^ n E d . 1975. The J o h n H o p k i n s U n i v e r s i t y P r e s s , B a l t i m o r e , M a r y l a n d . M c K u s i c k , V . A . , and F . H . R u d d l e . 1977. The s t a t u s o f t h e gene map o f t h e human chromosomes. S c i e n c e 196: 390 M c K u s i c k , V . A . , 1983. M e n d e l i a n i n h e r i t a n c e i n man. 6 t n E d . 1983. The J o h n H o p k i n s U n i v e r s i t y P r e s s , B a l t i m o r e , M a r y l a n d . M i l l e r , O . J . , D.Drayna, and P . G o o d f e l l o w . 1984. R e p o r t o f t h e c o m m i t t e e on t h e g e n e t i c c o n s t i t u t i o n o f t h e X and Y chromosomes. Human gene mapping 7, C y t o g e n e t i c s and C e l l G e n e t i c s 37:176 Monaco,A.P., C . J . B e r t e l s o n , W . M i d d l e s w o r t h , C . - A . C o l l e t t i , J . A l d r i d g e , K . H . F i s c h b e c k , R . B a r t l e t t , M . A . P e r i c a k - V a n c e , A.D.Roses and L .M.Kunkel. 1985. D e t e c t i o n o f d e l e t i o n s s p a n n i n g t h e Duchenne m u s c u l a r d y s t r o p h y l o c u s u s i n g a t i g h t l y l i n k e d DNA segment. N a t u r e 316:842-845. Murray,J.M., K . E . D a v i e s , P . S . H a r p e r , L . M e r e d i t h , C . R . M u e l l e r and R . W i l l i a m s o n . 1982. L i n k a g e r e l a t i o n s h i p o f a c l o n e d DNA s e q u e n c e on t h e s h o r t arm o f t h e X chromosome t o Duchenne m u s c u l a r d y s t r o p h y . N a t u r e 300: 69-71. Newmark, P., 1985. T e s t i n g f o r c y s t i c f i b r o s i s . N a t u r e 318:309 R a s t a n , T . , M.Kaufmann, M.Handyside, and M.F.Lyon. 1980: X-chromosome i n a c t i v a t i o n i n e x t r a - e m b r y o n i c membranes o f d i p l o i d p a r t h e n o g e n e t i c mouse embryos d e m o n s t r a t e d by d i f f e r e n t i a l s t a i n i n g . N a t u r e 288:172 Ray, P.N., B . B e l f a l l , C . D u f f , C.Logan, V.Kean, M.W.Thompson, J . S y l v e s t e r , J . G o r s k i , R . S c h m i c k e l , and R.G.Worton. 1985. C l o n i g o f t h e b r e a k p o i n t o f an X;21 t r a n s l o c a t i o n a s s o c i a t e d w i t h Duchenne m u s c u l a r d y s t r o p y . N a t u r e 318:672 R o u y e r , F . , M.Simmler, C . J o h n s s o n , G.Vergnaud, H.Cooke, and J . W e i s s e n b a c h . 1986: A g r a d i e n t o f s e x l i n k a g e i n t h e p s e u d o a u t o s o m a l r e g i o n o f t h e human sex chromosomes. N a t u r e 319:291 -80-Rozen,R., J.Fox, W.A.Fenton, A.L.Horwich and L.E.Rosenberg. 1985. Gene d e l e t i o n and r e s t r i c t i o n fragment l e n g t h polymorphisms a t the human o r n i t h i n e transcarbamylase l o c u s . Nature 313:815-817. Seed B. P u r i f i c a t i o n of genomic sequences from bacteriophage l i b r a r i e s by recombination and s e l e c t i o n i n v i v o . 1983. N u c l e i c A c i d s Research 11: 2427 Shows,T.B., P.J.McAlpine, and R . L . M i l l e r . 1984. The 1983 c a t a l o g u e of mapped human g e n e t i c markers and r e p o r t of the nomenclature committee. Human gene mapping 7, C y t o g e n e t i c s and C e l l G e n e t i c s 37:340 Skolnick,M., and R.White. 1982. S t r a t e g i e s f o r d e t e c t i n g and c h a r a c t e r i z i n g r e s t r i c t i o n fragment l e n g t h polymorphisms (RFLPs). Human Gene Mapping 6. Cytogenet C e l l Genet 32:58-67. Skolnick,M.H., H . F . W i l l a r d , and L.A.Menlove. 1984. Report of the committee on human gene mapping by recombinant DNA t e c h n i q u e s . Human gene mapping 7, C y t o g e n e t i c s and C e l l G e n e t i c s 37:210 Snutch,T.P., 1984. A molecular and g e n e t i c a n a l y s i s of the heat shock response of C a e n o r h a b d i t i s elegans. PHd t h e s i s August 1984, Simon F r a s e r U n i v e r s i t y , Burnaby, B r i t i s h Columbia. Southern,E.M. 1975. D e t e c t i o n of s p e c i f i c sequences among DNA fragments separated by g e l e l e c t r o p h o r e s i s . J . M o l e c . B i o l . 98:503-517. T s u i , L . C , M.Buchwald, D.Barker, J.Braman, R.Knowlton, J.Schumm, H.Eiberg, J.Mohr, D.Kennedy, N . P l a v s i c , M.Zsiga, D.Markiewicz, G. Akots, V.Brown, C.Helms, T.Gravius, C.Parker, K.Rediker, and H. D o n i s - K e l l e r . 1985. C y s t i c f i b r o s i s l o c u s d e f i n e d by a g e n e t i c a l l y l i n k e d polymorphic DNA marker. Science 230:1054 Verellen-Dumoulin, Ch., M.Freund, R.De Meyer et a l 1984. E x p r e s s i o n of an X - l i n k e d muscular dystrophy i n a female due to t r a n s l o c a t i o n i n v o l v i n g Xp21 and non-rndom i n a c t i v a t i o n of the normal X chromosome. Hum.Genet. 67:115-119. Wainwright, B.J., P.Scambler, J.Schmidtke, E.Watson, H.Y.Law, M . F a r r a l , H.Cooke, H.Eiberg, and R.Williamson. 1985. L o c a l i z a t i o n of c y s t i c f i b r o s i s to human chromosome 7cen-q22. Nature 318:384 White, R., S.Woodward, M.Leppert, P.O'Connell, M.Hoff., J.Herbst, J . L a l o u e l , M.Dean, and G.Vande Woude. 1985. A c l o s e l y l i n k e d g e n e t i c marker f o r c y s t i c f i b r o s i s . Nature 318:382-384 White,R., M.Leppert, D.T.Bishop, D.Barker, J.Berkowitz, C.Brown, P.Callahan, T.Holm and L . J e r o m i n s k i . 1985. C o n s t r u c t i o n of l i n k a g e maps with DNA markers f o r human chromosomes. Nature 313:101-105. -81-W i e a c k e r , P . , K . E . D a v i e s , H.J.Cooke, P . L . P e a r s o n , R . W i l l i a m s o n , S . B h a t t a c h a r y a , J.Zimmer and H.-H.Ropers. 1984. Toward a c o m p l e t e l i n k a g e map o f t h e human X chromosome: R e g i o n a l a s s i g n m e n t o f 16 c l o n e d s i n g l e - c o p y DNA s e q u e n c e s e m p l o y i n g a p a n e l o f s o m a t i c c e l l h y b r i d s . Am.J.Hum.Genet. 36:265-276. Woo, S.L., A . L i d s k y , F . G u t t l e r , T . C h a n d r a , and K.Robson. 1983. C l o n e d human p h e n y l a l a n i n e h y d r o x y l a s e gene a l l o w s p r e n a t a l d i a g n o s i s and c a r r i e r d e t e c t i o n o f c l a s s i c a l p h e n y l k e t o n u r i a . N a t u r e 306:151 Wood,S., R.Poon, D . C . R i d d l e , N . J . R o y l e , J . L . H a m e r t o n . 1986: A Dna m a r k e r f o r human chromosome 8 t h a t d e t e c t s a l l e l e s o f d i f f e r i n g s i z e s . C y t o g e n e t C e l l G enet I n p r e s s . Y a n i s c h - P e r r o n , C., J . V i e i r a , and J . M e s s i n g . 1985. Improved M13 phage c l o n i n g v e c t o r s and h o s t s t r a i n s : n u c l e o t i d e s e q u e n c e s o f t h e Ml3mp18 and p U C l 9 v e c t o r s . Gene, 33:103-119. 

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