Proc. Nati. Acad. Sci. U S A Vol. 9 1 , p p. 5843-5847, June 1994 Medical Sciences Molecular basis fo r H blood group i n Bombay (Oh) a n d para-Bombay individuals (ABO blood group/Secretor blood group/oligosaccharide biosynthesis/fucosyltransferase/surface antigen) ROBERT J . KELLY*, LINDA K . ERNST*, ROBERT D . LARSENt, JANE G . BRYANT , JUDY S. ROBINSON§, AND JOHN B . LOWE*t¶ *Howard Hughes Medical Institute a n tDepartment of Pathology, University o f Michigan, Medical Science Research Building I Room 3510, 1150 West Drive, An n Arbor M I 48109-0650; §American Red Cross Reference Laboratory, 2425 Park Road, Charlotte, N C 28203; a n d tUniversity o f Kentucky Blood Bank, Room HA628, University o f Kentucky Medical Center, 8 0 0 Rose Street, Lexington, KY 40536 Communicated b y Robert L . Hill, December 9, 1993 ABSTRACT T h e penultimate step i n t h e biosynthesis o f the human A B O blood group oligosaccharide antigens i s cat- alyzed b y a-(1,2)-fucosyltransferase(s) (GDP-L-fucose:flD- galactoside 2-a-L-fucosyltransferase, E C 2.4.1.69), whose e x - pression i s determined b y t h e H a n d Secretor (SE) blood group loci (also known a s FUTI a n d FUT2, respectively). These enzymes construct Fucal -) 2Galh3-linkages, known a s H determinants, which a r e essential precursors t o the A a n d B antigens. Erythrocytes from individuals with the rare Bombay a n d para-Bombay blood group phenotypes a r e deficient i n H determinants, a n d thus A a n d B determinants, as a conse- quence o f apparent homozygosity fo r null alleles a t t h e H locus. W e report a molecular analysis o f a human a-(1,2)- fucosyltransferasegene, thought t o correspond t o t h e H blood group locus, i n a Bombay pedigree a nd a para-Bombay pedi- gree. W e find inactivating point mutations i n t h e coding regions o f both alleles f this gene i n each H-deficient individual. These results define t h e molecular basis f o r H blood group antigen deficiency in Bombay a n d para-Bombay phenotypes, provide compelling evidence that this gene represents t he human H blood group locus, a n d strongly support a hypothesis that t h e H a n d SE loci represent a-(1,2)-fucosyltransferase genes. Candidate sequences fo r t h e human S E locus are identified b y low-stringency Southern blot hybridization anal- yses, using a probe derived from t h e H a-(1,2)-fucosyltrans- ferase gene. The antigens determined b y t h e human A B O blood group locus a r e oligosaccharide molecules constructed b y t h e s e - quential action o f specific glycosyltransferases. T h e final step i n this pathway i s catalyzed b y allelic glycosyltransferases encoded b y th e A B O locus. These enzymes require a pre- cursor oligosaccharide substrate (Fucal -* 2Galf-) known a s t he H blood group antigen, constructed b y a-(1,2)-fucosyl- transferase(s) [a(1,2)FTs; GDP-L-fucose:43D-galactoside 2-a-L-fucosyltransferase, E C 2.4.1.69]. Genetic a n d bio- chemical observations a re consistent with a hypothesis that t h e H a n d Secretor (SE) blood group loci (also known as FUTI a d FUT2, respectively) correspond to distinct a(1,2)FT genes with tissue-specific expression patterns a n d close genetic linkage o n chromosome 1 9 (1-6). Under this two-locus model (2), the human H bloodgroup locus deter- mines expression of t h e H antigen ( a s well a s A and/or B antigens) i n t h e erythroid lineage, whereas t h e S E locus controls H expression (and thus A o r B antigen expression) i n a variety o f secretory epithelia a n d i n saliva (Table 1 ) . Nearly a l l humans maintain atleast o n e functional H allele (1). Roughly 8 0 % of these (Secretors) also maintain at least on e functional S E allele, whereas t h e remainder (non- Table 1 . Phenotypes a n d predicted genotypes a t t he human H a n d SE blood group loci (see ref. 2 ) Phenotype Secretory Trivial name Erythroid epithelia Genotype Secretor H positive H positive H/H, SE/- Non-Secretor H positive H negative H/H, se/se para-Bombay (h/h H H positive SE/- Bombay (h/h non-Secretor) H negative H negative h/h, se/se Secretors) a r e homozygous f o r null alleles a t t h e S E locus. Individuals o f t h e rare Oh (Bombay) phenotype ( 1 , 7 ) lack H determinants i n a l l tissues a n d a r e thought to b e homozygous f r null alleles a t both loci (Table 1 ) (2). Other rare individ- uals, o f t h e para-Bombay phenotype (1 , 8 ) , synthesize H determinants in their secretory epithelia b u t n o t in the erythroid lineage (1). These persons a r e believed t o b e homozygous for null alleles a t t he H locus ( 1 , 2 ) b u t appar- ently have a t least o n e functional S E allele. T h e basis f o r null alleles a t these loci ha s n o t been defined, n o r h a s t h e two-locus hypothesis confirmed a t th e molecular level. W e have previously isolated a human a(1,2)FT cDNA whose cognate enzyme is kinetically similar to that o f the H a(1,2)FT a n d whose chromosomal location (number 1 9 ) is syntenic t o the H blood group locus (9). confirm th e hypothesis that this cDNA corresponds to t h e H locus, a n d t o identify t h e molecular basis f o r deficiency o f H determinants i n presumed h / h individuals, w e determined t h e genomic structure a n d sequence of a wild-type allele a n d compared i t t o alleles taken from H-deficient individuals. Inactivatingpoint mutations were identified within th e coding portions ofeach such allele, including alleles i n individuals with wild-type S E locus-determined H antigen expression. These results yield a molecular explanation f o r A BH blood group deficiency in Bombay a n d para-Bombay individuals, provide genetic support f or assignment of this a(l,2)FT gene t o t h e H locus, a nd indicate that t he S E locus represents a n a(1,2)FT gene distinct from th e H locus. MATERIALS A N D METHODS Cloning o f a(1,2)FT Alleles. Genomic DNA sequences corresponding t o a previously described human a(1,2)FT cDNA [pCDM7-a(1,2)FT; ref. 9 ] were isolated from phage genomic DNA libraries prepared from leukocyte DN A iso- lated from a n H-positive individual an d from a n H-deficient Abbreviation: a(1,2)FT, a-(1,2)-fucosyltransferase. T o whom reprint requests should b e addressed. 5843 T h e publication costs o f this article were defrayed i n part b y page charge payment. This article must therefore b e hereby marked advertisement in accordance with 1 8 U.S.C. §1734 solely t o indicate this fact.
Transcript
P r o c . N a t i . A c a d .
S c i . USA
V o l . 9 1 , p p . 5 8 4 3 - 5 8 4 7 , J u n e 1 9 9 4
M e d i c a l S c i e n c e s
M o l e c u l a r b a s i s f o r H b l o o d
g r o u p
i n
Bombay ( O h )
a n d
p a r a - B o m b a y
i n d i v i d u a l s
(ABO b l o o d
g r o u p / S e c r e t o r b l o o d g r o u p / o l i g o s a c c h a r i d e
b i o s y n t h e s i s / f u c o s y l t r a n s f e r a s e / s u r f a c e a n t i g e n )
ROBERT J .
K E L L Y * ,
LINDA K .
E R N S T * ,
ROBERT
D .
L A R S E N t ,
JANE G .
B R Y A N T ,
JUDY S .
R O B I N S O N § ,
AND JOHN B . LOWE*t¶
* H o w a r d H u g h e s M e d i c a l I n s t i t u t e a n
t D e p a r t m e n t
o f
P a t h o l o g y , U n i v e r s i t y
o f
M i c h i g a n ,
M e d i c a l S c i e n c e R e s e a r c h
B u i l d i n g I
Room
3 5 1 0 ,
1 1 5 0 W e s t
D r i v e ,
An n
A r b o r
MI
4 8 1 0 9 - 0 6 5 0 ;
§ A m e r i c a n R e d C r o s s
R e f e r e n c e
L a b o r a t o r y ,
2 4 2 5 P a r k
R o a d ,
C h a r l o t t e ,
NC
2 8 2 0 3 ;
a n d
t U n i v e r s i t y
o f
K e n t u c k y
B l o o d
B a n k ,
Room
H A 6 2 8 ,
U n i v e r s i t y
o f
K e n t u c k y
M e d i c a l
C e n t e r ,
8 0 0 R o s e
S t r e e t ,
L e x i n g t o n ,
KY 4 0 5 3 6
C o m m u n i c a t e d
b y
R o b e r t L .
H i l l ,
D e c e m b e r
9 ,
1 9 9 3
ABSTRACT
T h e p e n u l t i m a t e
s t e p
i n
t h e
b i o s y n t h e s i s
o f
t h e
h u m a n AB O b l o o d g r o u p o l i g o s a c c h a r i d e
a n t i g e n s
i s
c a t -
a l y z e d b y a - ( 1 , 2 ) - f u c o s y l t r a n s f e r a s e ( s ) ( G D P - L - f u c o s e : f l D -
g a l a c t o s i d e
2 - a - L - f u c o s y l t r a n s f e r a s e ,
EC
2 . 4 . 1 . 6 9 ) ,
w h o s e e x -
p r e s s i o n
i s d e t e r m i n e d
b y
t h e
H a n d
S e c r e t o r
( S E )
b l o o d
g r o u p
l o c i ( a l s o k n o w n a s FUTI a n d F U T 2 ,
r e s p e c t i v e l y ) .
T h e s e
e n z y m e s
c o n s t r u c t F u c a l
-)
2 G a l h 3 - l i n k a g e s , k n o w n a s H
d e t e r m i n a n t s ,
w h i c h
a r e e s s e n t i a l
p r e c u r s o r s
t o t h e A a n d B
a n t i g e n s .
E r y t h r o c y t e s
f r o m i n d i v i d u a l s w i t h t h e r a r e
B o m b a y
a n d
p a r a - B o m b a y
b l o o d
g r o u p p h e n o t y p e s
a r e d e f i c i e n t i n H
d e t e r m i n a n t s ,
a n d t h u s A a n d
B
d e t e r m i n a n t s ,
a s
a c o n s e -
q u e n c e
o f
a p p a r e n t
h o m o z y g o s i t y
f o r n u l l a l l e l e s a t t h e H l o c u s .
W e r e p o r t
a m o l e c u l a r
a n a l y s i s
o f
a human
a - ( 1 , 2 ) -
f u c o s y l t r a n s f e r a s e g e n e , t h o u g h t
t o
c o r r e s p o n d
t o t h e H
b l o o d
g r o u p l o c u s ,
i n
a
B o m b a y p e d i g r e e
a n d
a
p a r a - B o m b a y p e d i -
g r e e .
W e f i n d
i n a c t i v a t i n g p o i n t m u t a t i o n s
i n
t h e
c o d i n g r e g i o n s
o f b o t h a l l e l e s
f
t h i s
g e n e
i n
e a c h H - d e f i c i e n t
i n d i v i d u a l .
T h e s e
r e s u l t s d e f i n e t h e
m o l e c u l a r b a s i s f o r
H
b l o o d
g r o u p
a n t i g e n
d e f i c i e n c y
i n
B o m b a y
a n d
p a r a - B o m b a y
p h e n o t y p e s , p r o v i d e
c o m p e l l i n g
e v i d e n c e
t h a t t h i s
g e n e r e p r e s e n t s
t h e human
H
b l o o d
g r o u p
l o c u s ,
a n d
s t r o n g l y s u p p o r t
a
h y p o t h e s i s
t h a t
t h e
H a n d
SE l o c i
r e p r e s e n t a - ( 1 , 2 ) - f u c o s y l t r a n s f e r a s e
g e n e s .
C a n d i d a t e
s e q u e n c e s
f o r t h e human SE l o c u s a r e
i d e n t i f i e d b y l o w - s t r i n g e n c y
S o u t h e r n
b l o t
h y b r i d i z a t i o n
a n a l -
y s e s ,
u s i n g
a
p r o b e
d e r i v e d f r o m
t h e
H
a - ( 1 , 2 ) - f u c o s y l t r a n s -
f e r a s e
g e n e .
T h e
a n t i g e n s d e t e r m i n e d
b y
t h e h u m a n
AB O b l o o d
g r o u p
l o c u s
a r e
o l i g o s a c c h a r i d e
m o l e c u l e s c o n s t r u c t e d
b y
t h e
s e -
q u e n t i a l
a c t i o n o f
s p e c i f i c g l y c o s y l t r a n s f e r a s e s .
T h e
f i n a l
s t e p
i n
t h i s
p a t h w a y
i s
c a t a l y z e d
b y
a l l e l i c
g l y c o s y l t r a n s f e r a s e s
e n c o d e d
b y
t h e
AB O
l o c u s . T h e s e
e n z y m e s
r e q u i r e
a
p r e -
c u r s o r
o l i g o s a c c h a r i d e
s u b s t r a t e
( F u c a l
- *
2 G a l f - )
k n o w n a s
t h e
H b l o o d
g r o u p a n t i g e n ,
c o n s t r u c t e d
b y a - ( 1 , 2 ) - f u c o s y l -
t r a n s f e r a s e ( s ) [ a ( 1 , 2 ) F T s ; G D P - L - f u c o s e : 4 3 D - g a l a c t o s i d e
2 - a - L - f u c o s y l t r a n s f e r a s e ,
EC
2 . 4 . 1 . 6 9 ] .
G e n e t i c
a n d b i o -
c h e m i c a l
o b s e r v a t i o n s a r e c o n s i s t e n t w i t h a
h y p o t h e s i s
t h a t
t h e H a n d
S e c r e t o r ( S E ) b l o o d
g r o u p
l o c i
( a l s o k n o w n a s
FUTI a d
F U T 2 , r e s p e c t i v e l y ) c o r r e s p o n d t o d i s t i n c t
a ( 1 , 2 ) F T g e n e s
w i t h
t i s s u e - s p e c i f i c
e x p r e s s i o n p a t t e r n s a n d
c l o s e
g e n e t i c
l i n k a g e
o n
c h r o m o s o m e
1 9 ( 1 - 6 ) . U n d e r
t h i s
t w o - l o c u s
m o d e l
( 2 ) , t h e h u m a n
H
b l o o d g r o u p l o c u s d e t e r -
m i n e s
e x pr e ss i o n o f t h e H
a n t i g e n ( a s w e l l a s A a n d / o r B
a n t i g e n s )
i n
t h e
e r y t h r o i d l i n e a g e ,
w h e r e a s t h e SE l o c u s
c o n t r o l s
H
e x p r e s s i o n ( a n d
t h u s A o r B
a n t i g e n
e x p r e s s i o n )
i n a
v a r i e t y o f s e c r e t o r y e p i t h e l i a a n d i n
s a l i v a ( T a b l e 1 ) .
N e a r l y
a l l h u m a n s
m a i n t a i n a t l e a s t o n e
f u n c t i o n a l H a l l e l e
( 1 ) .
R o u g h l y 8 0 % o f t h e s e
( S e c r e t o r s ) a l s o m a i n t a i n
a t l e a s t
o n e
f u n c t i o n a l SE
a l l e l e , w h e r e a s t h e r e m a i n d e r ( n o n -
T a b l e 1 . P h e n o t y p e s
a n d
p r e d i c t e d g e n o t y p e s
a t
t h e h u m a n H
a n d SE b l o o d
g r o u p l o c i ( s e e r e f . 2 )
P h e n o t y p e
S e c r e t o r y
T r i v i a l name
E r y t h r o i d e p i t h e l i a G e n o t y p e
S e c r e t o r
H
p o s i t i v e
H
p o s i t i v e H/H, SE/-
N o n - S e c r e t o r H p o s i t i v e H n e g a t i v e H/H,
s e / s e
p a r a - B o m b a y
( h / h
H
H
p o s i t i v e SE/-
Bombay
( h / h
n o n - S e c r e t o r )
H
n e g a t i v e
H
n e g a t i v e h / h , s e / s e
S e c r e t o r s )
a r e
h o m o z y g o u s
f o r n u l l a l l e l e s
a t t h e SE
l o c u s .
I n d i v i d u a l s
o f t h e r a r e O h ( B o m b a y ) p h e n o t y p e ( 1 , 7 )
l a c k H
d e t e r m i n a n t s i n a l l t i s s u e s a n d a r e t h o u g h t t o b e
h o m o z y g o u s
f r n u l l a l l e l e s a t b o t h
l o c i
( T a b l e 1 )
( 2 ) .
O t h e r r a r e
i n d i v i d -
u a l s , o f t h e p a r a - B o m b a y p h e n o t y p e ( 1 ,
8 ) , s y n t h e s i z e H
d e t e r m i n a n t s
i n
t h e i r
s e c r e t o r y e p i t h e l i a b u t n o t i n t h e
e r y t h r o i d l i n e a g e ( 1 ) .
T h e s e
p e r s o n s
a r e b e l i e v e d
t o
b e
h o m o z y g o u s f o r
n u l l
a l l e l e s
a t t h e
H
l o c u s
( 1 , 2 ) b u t
a p p a r -
e n t l y h a v e a t
l e a s t o n e f u n c t i o n a l SE a l l e l e .
T h e b a s i s f o r n u l l
a l l e l e s
a t
t h e s e l o c i h a s
n o t b e e n d e f i n e d ,
n o r h a s t h e
t w o - l o c u s
h y p o t h e s i s c o n f i r m e d a t
t h e
m o l e c u l a r l e v e l . W e h a v e
p r e v i o u s l y i s o l a t e d a h u m a n
a ( 1 , 2 ) F T
cDNA w h o s e
c o g n a t e e n z y m e i s k i n e t i c a l l y
s i m i l a r
t o
t h a t
o f t h e H
a ( 1 , 2 ) F T
a n d w h o s e c h r o m o s o m a l
l o c a t i o n
( n u m b e r 1 9 )
i s
s y n t e n i c
t o t h e H b l o o d g r o u p l o c u s ( 9 ) .
c o n f i r m
t h e
h y p o t h e s i s
t h a t t h i s
cDNA c o r r e s p o n d s t o t h e H
l o c u s , a n d t o
i d e n t i f y
t h e m o l e c u l a r
b a s i s f o r d e f i c i e n c y o f
H
d e t e r m i n a n t s i n
p r e s u m e d
h / h
i n d i v i d u a l s ,
we d e t e r m i n e d t h e
g e n o m i c
s t r u c t u r e a n d
s e q u e n c e o f a
w i l d - t y p e a l l e l e a n d
c o m p a r e d
i t
t o a l l e l e s t a k e n
f r o m
H - d e f i c i e n t
i n d i v i d u a l s .
I n a c t i v a t i n g p o i n t m u t a t i o n s w e r e
i d e n t i f i e d w i t h i n t h e
c o d i n g
p o r t i o n s
o f e a c h s u c h
a l l e l e , i n c l u d i n g
a l l e l e s
i n i n d i v i d u a l s
w i t h w i l d - t y p e SE l o c u s - d e t e r m i n e d
H
a n t i g e n e x p r e s s i o n .
T h e s e r e s u l t s
y i e l d a m o l e c u l a r
e x p l a n a t i o n f o r A B H
b l o o d
g r o u p
d e f i c i e n c y
i n B o m b a y
a n d
p a r a - B o m b a y i n d i v i d u a l s ,
p r o v i d e
g e n e t i c
s u p p o r t f o r a s s i g n m e n t
o f
t h i s
a ( l , 2 ) F T
g e n e
t o t h e H
l o c u s ,
a n d i n d i c a t e t h a t t h e SE l o c u s
r e p r e s e n t s a n
a ( 1 , 2 ) F T
g e n e
d i s t i n c t f r o m t h e H
l o c u s .
M A T E R I A LS
AN D
M E T H O D S
C l o n i n g
o f
a ( 1 , 2 ) F T A l l e l e s .
G e n o m i c DNA s e q u e n c e s
c o r r e s p o n d i n g
t o
a
p r e v i o u s l y d e s c r i b e d h u m a n a ( 1 , 2 ) F T
cDNA
[ p C D M 7 - a ( 1 , 2 ) F T ;
r e f . 9 ] w e r e i s o l a t e d f r o m
p h a g e
g e n o m i c DNA
l i b r a r i e s p r e p a r e d
f r o m
l e u k o c y t e DNA i s o -
l a t e d f r o m a n
H - p o s i t i v e
i n d i v i d u a l a n d
f r o m a n H - d e f i c i e n t
A b b r e v i a t i o n :
a ( 1 , 2 ) F T , a - ( 1 , 2 ) - f u c o s y l t r a n s f e r a s e .
T o
w h o m
r e p r i n t r e q u e s t s s h o u l d b e
a d d r e s s e d .
5 8 4 3
T h e
p u b l i c a t i o n c o s t s o f t h i s a r t i c l e w e r e
d e f r a y e d
i n p a r t b y p a g e
c h a r g e
p a y m e n t .
T h i s
a r t i c l e m u s t t h e r e f o r e b e
h e r e b y
m a r k e d
a d v e r t i s e m e n t
i n a c c o r d a n c e w i t h 1 8 U . S . C .
§ 1 7 3 4 s o l e l y
t o i n d i c a t e t h i s
f a c t .
5 8 4 4
M e d i c a l S c i e n c e s : K e l l y e t a l .
i n d i v i d u a l ( X ; Bombay p h e n o t y p e ) ( 1 0 ) .
L i b r a r i e s w e r e
s c r e e n e d
b y
p l a q u e h y b r i d i z a t i o n ( 1 1 ) u s i n g
a 1 . 2 - k b
H i n f l
f r a g m e n t i s o l a t e d f r o m t h e i n s e r t
i n p C D M 7 - a ( 1 , 2 ) F T ( 9 ) .
A d j a c e n t E c o R I f r a g m e n t s o f 2 . 7 a n d . 5 k b f r o m a s i n g l e
p h a g e w e r e s u b c l o n e d i n t o t h e u n i q u e E c o R I s i t e i n t e v e c t o r
pWE15 ( S t r a t a g e n e ) . B o t h s t r a n d s o f t h e s e f r a g m e n t s w e r e
s e q u e n c e d
b y u s i n g o l i g o n u c l e o t i d e p r i me r s c o r r e s p o n d i n g t o
t h e i n s e r t
s e q u e n c e .
T h e c o d i n g s e q u e n c e s o f t h e t w o a l l e l e s
( Y 1 a n d Y 2 ) f r o m
a p a r a - B o m b a y i n d i v i d u a l w e r e g e n e r a t e d
w i t h t h e PCR ( 1 1 ) ,
u s i n g
p r i m e r p a i r s f l a n k i n g t h e m u t a t i o n
i n Y 2 ( F i g .
1 ) .
O n e
p a i r o f t h e s e p r i m e r s a m p l i f i e s a
s e q u e n c e
b e t w e e n a p o s i t i o n
w i t h i n i n t r o n a t a l o c a t i o n 1 8 7 - 1 5 7
n t 5 ' t o t h e A o f t h e
i n i t i a t i o n c o d o n (GCGCAAGCTTCTCAICCCTiAAAC.
CTAGGACTCAGGCTCT)
a n d a t n t 1 0 3 5 - 1 0 6 5 w i t h i n t h e
c o d i n g
s e q u e n c e ( t a k i n g
t h e A r e s i d u e o f t h e
i n i t i a t i o n
c o d o n
a s n t 1 ,
n u m b e r e d
a s i n
r e f . 9 ; GCGCTCTAGAE[CAT-
TAATGCCCACCCACTCGGGCAG)
a t a s i t e 3 '
t o t h e
Y 2
m u t a t i o n .
T h e o t h e r p r i m e r p a i r a m p l i f i e s
a
s e q u e n c e
b e -
t w e e n n t
6 5 7 a n d 6 8 5 o f
t h e
c o d i n g s e q u e n c e ( 5 '
t o t e
Y 2
m u t a t i o n ; GCGCAAGCTTGCGCCGTGGGGACTATCTG-
CAGGTTATGCC)
a n d b e t w e e n n t 1 1 3 5 a n d 1 1 6 6
w i t h i n
t h e
3 ' u n t r a n s l a t e d
r e g i o n
(GCGCTCTAGACAGGCCTCT-
GAAGCCACGTACTGCTGGCTC).
G e n o m i c DNA w a s
s u b j e c t e d t o 3 0 c y c l e s
o f
a m p l i f i c a t i o n ( d e n a t u r a t i o n
a t
9 4 T C
a
1 k b
exon 1 i n t r o n
5
u t
f o r
1 . 5
m i n ;
a n n e a l i n g / e x t e n s i o n f o r 2 . 5
m i n
a t 7 2 0 C ) . F r a g -
m e n t s g e n e r a t ed w i th t h e s e p r i m e r p a i r s w e r e s u b c l o n e d i n t o
pTZ19R
( P h a r m a c i a L K B ) . C l o n e s c o r r e s p o n d i n g t o
a l l e l e
Y ,
a n d t o a l l e l e Y 2 w e r e d i s t i n g u i s h e d b y d i g e s t i o n w i t h B s t N I
( c l e a v e s Y 2 , n o t w i l d t y p e
o r Y 1 ,
a t t h i s p o s i t i o n ) . M u l t i p l e
c l o n e s c o r r e s p o n d i n g t o e a c h a l l e l e w e r e s e q u e n c e d
t o d i s -
t i n g u i s h PCR e r r o r s f r o m a c t u a l s e q u e n c e
p o l y m o r p h i s m s .
T h e s e a n a l y s e s i d e n t i f i e d t h e m u t a t i o n i n
t h e Y 1 a l l e l e c o r -
r e s p o n d i n g t o a m i n o a c i d r e s i d u e 1 6 4 a n d i n d i c a t e d
t h a t t h e
Y 1
a n d Y 2 c o d i n g r e g i o n s a r e o t h e r w i s e i d e n t i c a t o
t h e
w i l d - t y p e
c o d i n g r e g i o n .
E x p r e s s i o n V e c t o r C o n s t r u c t i o n a n d A n a l y s i s .
E x p r e s s i o n
v e c t o r s c o n t a i n i n g s i n g l e o r m u l t i p l e m u t a t i o n s w e r e a s s e m -
b l e d w i t h r e s t r i c t i o n f r a g m e n t e x c h a n g e p r o c e d u r e s ( 1 1 ) ,
u s i n g r e s t r i c t i o n s i t e s i n t h e 6 . 5 - k b E c o R I f r a g m e n t t h a t
e n c o m p a s s e s t h e t w o
a ( 1 , 2 ) F T
e x o n s
( F i g . 1 ) , c l o n e d i n t o
t h e
E c o R I s i t e
o f v e c t o r pWE15 ( 1 2 ) . D e t a i l s o f v e c t o r c o n s t r u c -
t i o n s a r e a v a i l a b l e f r o m
t h e
a u t h o r s . C O S - 1 c e l l s
w e r e
t r a n s f e c t e d
b y
a D E A E - d e x t r a n p r o c e d u r e ( 1 1 ) . A c o n t r o l
v e c t o r (pCDM7-CAT) e n c o d i n g c h l o r a m ph e n i c o l a c e t y l -
t r a n s f e r a s e [ d e r i v e d
f r o m
pSV2-CAT ( 1 1 )
a n d
pCDM7 ( 9 ) ]
w a s c o t r a n s f e c t e d t o a l l o w n o r m a l i z a t i o n f o r t r a n s f e c t i o n
e f f i c i e n c y .
C e l l e x t r a c t s w e r e
p r e p a r e d 7 2
h r
a f t e r t r a n s f e c -
t i o n
( 9 )
a n d
w e r e
u s e d i n
a ( 1 , 2 ) F T a c t i v i t y a s s a y s ( 1 2 )
p e r f o r m e d
i n
d u p l i c a t e
u n d e r
l i n e a r
r e a c t i o n c o n d i t i o n s .
e x o n
2
c o d i n g r e g i o n
:
X.
1 .
y
K
X
(
7 ~
r
oL
B o m b a y
X 1
w i l d - t y p e
...t t t t t t t t
aagacag...
B o m b a y
.
.
. t t t t t t t t o a a g a c a g . . .
X 2 w i l d - t y p e
... . t g c t g g g a t t a c a g g
...
B o m b a y
...t g c t g g g g t t a c a g g ...
X 3 w i l d - t y p e
B o m b a y
...g c c t c a g t g c a t t t g ...
...
g c c t c a g g g c a t t t g
...
B s t N I
X 4 w i l d - t y p e
...
G C T
G C C
T A C
C T G G C T
...
* - -
A
A
Y 3 1 6 L
A ...
A / u l
B o m b a y
... G C T
G C CTAC
TG GCT
... A
A
S t o p
p a r a - B o m b a y
A I u l
Y 1
w i l d - t y p e
. . . C T G A A G
C T C
T C T
G G C . . .
* - -
L K
L 1 6 4 S
G ...
p a r a - B o m b a y
. . . C T G
A A G C A C T C T
G G C
. . .
.. .
L K H
S G . . .
B s t N I
Y 2
w i l d - t y p e
... A C C T C C
C A G G G C G A T ...
T
S Q 2 7 6 G
D ...
B f a l
p a r a - B o m b a y
...
A C C T C C
J A G
G G C G A T
.. .
...
T
S
S t o p
.. .
X 5
w i l d - t y p e
..
. g t t g g c c g g g a t g g t ...
B o m b a y
...
g t t g g c c a g g a t g g t
X 6 w i l d - t y p e
...
t t t t t t t
a a t t a t t t . . .
B o m b a y
...
t t t t t t t t a a t t a t t t
...
F I G . 1 . S t r u c t u r e o f w i l d - t y p e
a n d
m u t a n t
a ( 1 , 2 ) F T
a l l e l e s .
( a )
S c a l e a n d l i n e a r
map
o f t h e
g e n e . C o d i n g p o r t i o n
o f
exon
2 i s d e n o t e d
b y
t h e s o l i d a r e a .
N o n c o d i n g p o r t i o n s
o f exons 1 a n d
2
ar e s h a d e d . P o s i t i o n s o f
DNA
sequence
d i f f e r e n c e s b e t w e e n a
w i l d - t y p e
a l l e l e
a n d a l l e l e s
i s o l a t e d
f r o m H - d e f i c i e n t i n d i v i d u a l s
( X 1 - X 6 , B o m b a y ;
Y 1 a n d
Y 2 , p a r a - B o m b a y )
are
i n d i c a t e d a b o v e t h e s c h e m a t i c .
R e g i o n
i n d i c a t e d
b y
A b e l o w
t h e
map c o r r e s p o n d s t o t r a n s c r i p t i o n
