Volume 12 Number 24 1984
Nucleic Acids Research
Complete nudeotlde sequence of the marine H-2K* gene. Comparison of three H-2K locus aDeles
Bernd Arnold + , Hans-Gerhard BurgertS, Alan L.Archibaldt and Sune KvistS*
European Molecular Biology Laboratory, Postfach 10.2209, D-6900 Heidelberg, FRG
Received 2 November 1984; Revised and Accepted 30 November 1984
ABSTRACT
We h a v e d e t e r m i n e d the DNA s e q u e n c e o f the H - 2 K k g e n e o f the
m o u s e m a j o r h i s t o c o m p a t i b i 1 i t y c o m p l e x ( M H C ) . C o m p a r i s o n o n the
n u c l e o t i d e and p r o t e i n level o f t h r e e H - 2 K a l l e l e s ( K k , K b and
K d ) r e v e a l s a h i g h d e g r e e o f h o m o l o g y , in p a r t i c u l a r b e t w e e n the
K b and K k a l l e l e s . D i f f e r e n c e s b e t w e e n the two l a t t e r a n t i g e n s
a r e a l m o s t e x c l u s i v e l y c o n f i n e d t o the a l a n d a 2 d o m a i n s . A t
n i n e p o s i t i o n s i n the e x t r a c e l l u l a r p a r t o f the m o l e c u l e s w e h a v e
f o u n d a l l e l e - s p e c i f i c a m i n o a c i d s . I n t e r e s t i n g l y , 78J o f t h e s e
r e s i d u e s a r e e i t h e r p o l a r or c a r r y h y d r o x y l - g r o u p s . T h i s m a k e s
it l i k e l y t h a t t h e y a r e e x p o s e d o n the s u r f a c e o f the m o l e c u l e s
and m i g h t then b e p a r t o f a n t i g e n i c d e t e r m i n a n t s . W e h a v e a l s o
i d e n t i f i e d p o t e n t i a l l y al lei e - s p e c i f i c n u c l e o t i d e s e q u e n c e s o f
the K g e n e s w h i c h m i g h t b e used a s s p e c i f i c DNA p r o b e s .
INTRODUCTION
The murine major histocompatibi1ity complex (MHC) encodes the
t r a n s p l a n t a t i o n a n t i g e n s H - 2 K , D and L ( 1 , 2 ) . T h e s e c l a s s I a n t i g e n s c o n s i s t o f a h e a v y c h a i n (MW 4 5 0 0 0 ) n o n - c o v a l e n t l y a s s o c i a t e d
w i t h 6 2 - " i i c r o g l o b u l i n (MW 1 2 0 0 0 ) . T h e h e a v y c h a i n is a n i n t e g r a l
m e m b r a n e p r o t e i n w i t h the l a r g e s t p a r t o f the m o l e c u l e e x p o s e d o n
the cell s u r f a c e and the r e m a i n i n g o n e f o u r t h o f the p r o t e i n s p a n n i n g the p l a s m a m e m b r a n e and p r o t r u d i n g o n the c y t o p l a s m i c s i d e .
T h e e x t r a c e l l u l a r p a r t o f the m o l e c u l e c a n b e d i v i d e d i n t o t h r e e
d o m a i n s ( a l , a 2 and a 3 ) e a c h c o m p r i s i n g a p p r o x i m a t e l y 9 0 a m i n o
a c i d s . T h e p r i m a r y s t r u c t u r e o f the p r o t e i n has b e e n d e t e r m i n e d
for the H - 2 K m o l e c u l e ( 3 ) . One o f the m o s t s t r i k i n g f e a t u r e s o f
the c l a s s I a n t i g e n s is t h e i r high d e g r e e o f p o l y m o r p h i s m .
More
than 5 0 a l l e l e s a t e a c h o f the K and D loci h a v e s o far b e e n i d e n tified ( 2 ) .
C IR L PreM Limited, Oxford, England.
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The class I histocorapatibi1ity antigens play an important
r o l e in t h e T cell m e d i a t e d immune r e s p o n s e . D u r i n g viral i n fection cytotoxic T lymphocytes show a specificity n o t directed
against the viral proteins themselves b u t against t h e viral antigens in t h e c o n t e x t of s e l f - d e t e r m i n a n t s c o n t a i n e d on H - 2 class I
a n t i g e n s . T h e role of the transplantation antigens seems to be
to r e s t r i c t t h e r e c o g n i t i o n so that only t h e i n f e c t e d c e l l s a r e
lysed. This p h e n o m e n o n has been called H - 2 r e s t r i c t i o n of cytolytic T lymphocytes ( 4 , 5 , 6 ) .
During the past few years the understanding of the molecular
biology of the H - 2 complex has advanced d r a m a t i c a l l y . By using
recombinant D N A technology, several laboratories have isolated
and c h a r a c t e r i z e d class I genes (7,8,9,10,11) a n d a m o l e c u l a r m a p
of t h e H - 2 c o m p l e x h a s been c o n s t r u c t e d ( 1 2 ) .
The H - 2 class I genes so far analysed display a high degree
of h o m o l o g y . ' T h e y all have 8 exons which c o r r e l a t e w i t h t h e d o mains of the corresponding antigen. The first exon encodes the
signal s e q u e n c e , known to b e of i m p o r t a n c e f o r t r a n s l o c a t i o n of
the newly s y n t h e s i z e d p o l y p e p t i d e through t h e m e m b r a n e of t h e
rough endoplasmic reticulum ( 1 3 ) . Exons 2,3 and 4 encode the
t h r e e e x t r a c e l l u l a r d o m a i n s o f t h e a n t i g e n , a l , a 2 a n d ct3, r e s pectively. Exon 5 encodes the transmembrane domain, whereas
exons 6, 7 and 8 code for t h e c y t o p l a s m i c d o m a i n and t h e 3 1 u n t r a n s l a t e d r e g i o n . T h e o v e r a l l l e n g t h o f a n H - 2 c l a s s I g e n e is
approximately 5000 nucleotides (for a review see 1 4 ) .
Two alleles of the H - 2 K locus, K and K , have already been
c h a r a c t e r i z e d ( 1 1 , 1 0 ) . In t h i s r e p o r t w e d e s c r i b e t h e 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 a third allele of t h e H - 2 K l o c u s , t h e K
g e n e . T h i s a l l o w s us to m a k e a c o m p a r i s o n , on t h e p r o t e i n as well
a s on t h e D N A l e v e l , o f t h r e e a l l e l e s f r o m a n H - 2 c l a s s I l o c u s .
MATERIALS AND METHODS
Isolation of lambda phages containing H-2 sequences and
Southern blot hybridization
The H-2 lambda phage library w a s a gift from H. Lehrach and
A.-M. F r i s c h a u f . T h e library w a s constructed from D N A of the
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strain B10.BR partially digested with the restriction enzyme
S a u 3 A 1 a n d c l o n e d i n t o t h e Bara H I s i t e o f t h e E M B L 3 v e c t o r ( 1 5 ) .
Phage clones containing genomic DNA were identified by plaque hybridization ( 1 6 ) . Phage DNA was prepared as described ( 1 7 ) .
Southern blot analyses of phage DNA were carried out as described
e l s e w h e r e (10) .
DNA r e s t r i c t i o n m a p p i n g and s e q u e n c e a n a l y s i s
Restriction maps of lambda phage clones were constructed either
a s p r e v i o u s l y d e s c r i b e d ( 1 8 ) o r b y u s i n g t w o or s e v e r a l r e s t r i c tion e n z y m e s in c o n s e c u t i v e r e a c t i o n s . F o r DNA s e q u e n c e a n a l y s i s
we have used the s u b c l o n i n g d e l e t i o n method ( 1 9 ) . O v e r l a p p i n g
c l o n e s w e r e s e q u e n c e d b y t h e p r o c e d u r e o f M a x a m and G i l b e r t ( 2 0 ) .
Cell c u l t u r e and D N A - m e d i a t e d g e n e t r a n s f e r
T h e i n t r o d u c t i o n o f t h e H - 2 K g e n e s I n t o t h e f i b r o b l a s t cell l i n e
IT 2 2 - 6 w a s d o n e a s p r e v i o u s l y d e s c r i b e d ( 2 1 , 2 3 ) . S e l e c t i o n o f
clones expressing H-2K antigens w a s done using the neophosphotransferase gene system ( 2 2 ) . These experiments have been p u b lished in detail e l s e w h e r e ( 2 3 ) .
Other methods
Immunoprecipitations and sodium dodecyl sulphate polyacrylamide
gel e l e c t r o p h o r e t i c a n a l y s e s w e r e p e r f o r m e d e s s e n t i a l l y as d e s cribed before ( 2 4 ) . For the detection o f the H-2Kk antigen the
m o n o c l o n a l antibody H 1 0 0 - 2 7 R 5 5 w a s used ( 2 5 ) .
Materials
R e s t r i c t i o n e n z y m e s and D N A m o d i f y i n g e n z y m e s w e r e p u r c h a s e d from
Boehringer, Mannheim, FRG. (a- P)dNTPs (lOmCi/ml, 3000C1/mmol) ,
(y- P ) A T P ( l O m C i / m l , 5 0 0 0 C i / m m o l ) a n d 3 5 S - m e t h i o n i n e w e r e f r o m
Amersham, U . K . GeneScreen filters were from New England N u c l e a r .
Protein A-sepharose was from Pharmacia Fine Chemicals (Uppsala,
S w e d e n ) . Geneticin (G-418) w a s purchased from GIBCO.
RESULTS
Isolation o f genomic lambda clones containing H-2 specific
sequences
A genomic l i b r a r y , constructed from l i v e r DNA o f the mouse s t r a i n B10.BR
(H-2 k ) and the vector EMBL 3 ( 1 5 ) , was screened w i t h the cDNA probe pH-2 d -4
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This probe has i t s 51 end at a position corresponding to the 3' end of
(26).
exon 3 of the K antigen and continues through the a3 domain (exon 4 ) , the
membrane and the cytoplasmic domains and ends after a stretch of approximately
50 adenosine residues at the 3' end.
Since i t contains the highly conserved
region of the a3 domain, we expected i t to recognize most, i f not a l l , H-2
class I genes in the mouse genome.
Approximately, 5x10
individual lambda
clones were screened, of which 112 were found to hybridize specifically to
the H-2 probe.
The number of clones screened corresponds to roughly 3 times
the haploid mouse genome.
The 35-40 genes detected per haploid genome are 1n
agreement with data reported before (12).
From 40 of the 112 clones picked, DNAs were prepared and analysed in pools
(5-10 individual clones per pool) for their a b i l i t y to hybridize to an H-2K
locus specific probe (pH-2 -5b).
This probe originates from the 3' untransla-
ted region of the IT gene as has been characterized previously (27).
DNAs from the different pools were subjected to digestion with the res t r i c t i o n enzyme Bgl I I , the fragments were separated on an agarose g e l , transferred to a GeneScreen f i l t e r and hybridized with the radioactively labelled
probe pH 2 -5b (27).
dizing fragment.
tively.
Two of the six pools examined contained a single hybri-
The sizes of these fragments were 3.2 Kb and 2.4 Kb, respec-
In a Southern blot experiment with total genomic B10.BR DNA we were
unable to detect these two bands, most l i k e l y for the following reasons:
F i r s t , each band seems to represent a single copy gene and secondly, the probe
pH-2^5b contains approximately 75S A+T residues which makes hybridization
experiments with total DNA d i f f i c u l t .
DNA from the individual clones of pools
4 and 2 were then analysed in a similar way.
Two clones, termed 6D and 7A,
were shown to contain DNA sequences complementary to the probe pH-2 -5b.
These two clones were considered to be candidates for the authentic H-2K
gene
and were further analyzed by two independent techniques, described below.
R e s t r i c t i o n mapping and DNA-mediated gene t r a n s f e r of H-2K genes
The r e s t r i c t i o n maps f o r clones 6D and 7A r e v e a l a high degree
of homology but d i f f e r
region (Figure 1).
individual
at t h r e e p o s i t i o n s
This i n d i c a t e s
H-2 genes.
1n the 3'
flanking
t h a t the clones c o n t a i n two
By u s i n g 3 1 and 5' H-2 s p e c i f i c DNA probes
i n Southern b l o t e x p e r i m e n t s , we could show t h a t clone 6D does
not c o n t a i n a complete H-2 gene.
This clone does not
hybridize
t o the 5' s p e c i f i c probe c o n t a i n i n g a sequence encoding the a l
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1
7A
R H
R
60
R H
R
K
KR
Xh
B
27-2
R H
R
K
KR Xh
B
HK
K RXh B
1
B
1
B
r
B
3'
Figure 1 .
K K
Restri cti on maps of
H B
R
5'
the lambda c l o n e s
7A, 6D and
the cosmid c l o n e 2 7 - 2 .
The vertical arrows denote the three differences between clones
7A a n d 6 D . T h e h o r i z o n t a l b a r s u n d e r n e a t h e a c h o f t h e r e s t r i c tion maps show the extent o f the H-2 g e n e . F o r clone 6D the 5 '
end o f t h e g e n e i s m i s s i n g a n d t h i s i s i n d i c a t e d b y t h e d a s h e d
bar. LA and RA mean the left and the right arms, r e s p e c t i v e l y ,
of t h e l a m b d a p h a g e D N A . T h e s i g n ^ d e n o t e s l a m b d a p h a g e o r c o s mid D N A s e q u e n c e s . E n z y m e s u s e d w e r e : B , B a m H I ; H , H i n d I I I ; K ,
K p n I; R , E c o R I ; X h , X h o I.
domain of t h e H - 2 K g e n e . Also the homology between the t w o
c l o n e s i n d i c a t e s t h a t t h e 5 1 end o f t h e g e n e in c l o n e 6 D is m i s sing. This is the r e s u l t o f the cloning p r o c e d u r e used. T h e H - 2
g e n e in c l o n e 6 D c o n t a i n s a l l t h e s e q u e n c e s e n c o d i n g e x o n 4 t o
exon 8 as well as the 3 1 n o n - c o d i n g r e g i o n b u t lacks the first
three exons upstream o f the B a m HI site. C l o n e 7A c o n t a i n s a comp l e t e H - 2 g e n e a c c o r d i n g t o r e s t r i c t i o n m a p p i n g and S o u t h e r n b l o t
analysis .
In o r d e r t o find a c o m p l e t e g e n e c o r r e s p o n d i n g t o t h e g e n e in
clone 6 D , w e examined the other clones originally selected to contain H-2 s e q u e n c e s . None o f these clones contained a c o m p l e t e
gene corresponding to clone 6D or a gene which with certainty
c o u l d p r o v i d e t h e a u t h e n t i c 5 ' e n d o f t h e g e n e in c l o n e 6 D .
W e t h e n e x a m i n e d a c o s m i d c l o n e , 2 7 - 2 (28)(generously p r o v i d e d b y
M. S t e i n m e t z ) w h i c h o r i g i n a t e s f r o m a c o s m i d l i b r a r y o f D N A f r o m
the m o u s e s t r a i n A K R ( H - 2 k ) . C l o n e 2 7 - 2 w a s s e l e c t e d on the basis
of i t s h y b r i d i z a t i o n t o t h e K - l o c u s s p e c i f i c p r o b e ( p H - 2 - 5 b ) a n d
therefore could be considered a candidate for c o n t a i n i n g the H-2Kk
gene. By comparing the restriction m a p o f cosmid clone 2 7 - 2 with
clone 6D and 7 A , 1t is c l e a r that clones 2 7 - 2 and 6 D a r e Identical
a t t h e i r 3" e n d s a n d t h a t t h e f o r m e r , in a d d i t i o n , c o n t a i n s t h e
sequences u p s t r e a m o f the B a m HI site of c l o n e 6 0 ( F i g u r e 1 ) .
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By u s i n g D N A - m e d i a t e d g e n e t r a n s f e r e x p e r i m e n t s w i t h e a c h
the clones we could
r i s e to c e l l s
d e m o n s t r a t e t h a t o n l y D N A of c l o n e 2 7 - 2
expressing
the a u t h e n t i c H - 2 K
gene
ted c e l l s w e r e a n a l y s e d w i t h b o t h s o d i u m d o d e c y l
(23).
gave
Transfec-
sulphate
l a m i d e gel e l e c t r o p h o r e s i s and f l u o r e s c e n c e a c t i v a t e d
of
polyacry-
cell
sorter
a n a l y s i s.
From
lowing:
the e x p e r i m e n t s described
a b o v e , we c o n c l u d e the f o l -
(i) C l o n e 27-2 c o n t a i n s the H-2K
g e n e of t h e A K R
mouse.
L.
( i i ) C l o n e 6D p r o b a b l y c o n t a i n s
mouse B1O.BR.
(i 1 1 ) C l o n e 7A c o n t a i n s
plete H-2 gene bearing
quence.
t h e 3 1 end of the H - 2 K
at its 3' end
the H-2K
T h i s c l o n e c o n t a i n s an H - 2 g e n e f r o m
b u t it is n o t t h e a u t h e n t i c H - 2 K k
Amino acid
clones
of t h r e e H - 2 K c l a s s
27-2 was recloned
I s i t e o f pBR 322 by u s i n g Sal
containg
locus specific
the B1O.BR
I linkers.
t h e 1 0 . 5 Kb f r a g m e n t w e r e s e l e c t e d
K
In o r d e r
(19).
responding
aligned with
exon-intron
(23).
deletion
t h o s e of t h e K
boundaries
b
8 e x o n s of v a r y i n g
and K
d
genes
gene shows extensive
(Figure 3 ) .
lengths separated
It is e n c o d e d
by i n t r o n s .
Two
is f o u n d a n o t h e r 27 n u c l e o t i d e s
(29).
We have compared
the
and K
homoby
The
TATA
codonATG
upstream.
Both
to p l a y an i m p o r t a n t r o l e
Both s e q u e n c e s a r e l o c a t e d
in t h e K
leotide homology
and
transcrip-
u p s t r e a m of t h e g e n e .
these sequences have been postulated
gene regulation
t h e DNA
( 1 1 , 1 0 ) to i d e n t i f y
55 n u c l e o t i d e s u p s t r e a m of t h e i n i t i a t i o n
and the C C A A T - b o x
position
along
( F i g u r e s 2 and 3 ) .
t i o n p r o m o t i n g e l e m e n t s are f o u n d
b o x is l o c a t e d
positions
The DNA s e q u e n c e was d e t e r m i n e d
T h e n u c l e o t i d e s e q u e n c e of t h e K
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constructed
to t h e s u b c l o n i n g
s i t e at d i f f e r e n t
elsewhere
logy with other H-2 class I genes
tical
the
W e h a v e used t h e C l a I l i n k e r t o i n t r o d u c e t h e c o r -
restriction
as d e s c r i b e d
into
Plasmid
to d e t e r m i n e t h e n u c l e o t i d e s e q u e n c e of t h e
g e n e , c l o n e 2 7 - 2 - 8 6 was subjected
method
I genes
and for o n e of
these, c l o n e 2 7 - 2 - 8 6 , a partial r e s t r i c t i o n map was
(Figure 2 ) .
se-
genome
gene.
and n u c l e o t i d e c o m p a r i s o n
T h e 1 0 . 5 Kb E c o RI f r a g m e n t of c o s m i d
u n i q u e Sal
g e n e of t h e
t h e D N A s e q u e n c e of a c o m -
at a l m o s t
in
iden-
genes.
the t h r e e H - 2 K a l l e l e s w i t h r e s p e c t to n u c -
in d i f f e r e n t r e g i o n s of t h e g e n e s (Table I ) . Exons
Nucleic Acids Research
10 5 Kb
H R H H
H H R H
R
R
H R R R R
10 Kb
Figure 2.
R e s t r i c t i o n m a p o f t h e 1 0 . 5 Kb f r a g m e n t c o n t a i n i n g t h e
H - 2 K k gene of cosmid 2 7 - 2 .
T h e 1 0 . 5 Kb E c o R I f r a g m e n t w a s c l o n e d i n t o t h e S a l I s i t e o f
p B R 3 2 2 by u s i n g Sal I l i n k e r s . F i l l e d b o x e s d e n o t e e x o n s a n d o p e n
boxes denote introns and flanking sequences.
T h e dashed b o x shows
the 3 1 u n t r a n s l a t e d r e g i o n .
T h e horizontal arrow underneath the
detailed r e s t r i c t i o n m a p indicates t h e r e g i o n for which the DNA
sequence has been d e t e r m i n e d .
E n z y m e s u s e d w e r e a s in F i g u r e 1
and in a d d i t i o n : B g , Bgl I I ; S, S m a I; S I , S a l I; X , X b a I.
two and three (en cod i ng t h e al a n d a 2 d o m a i n s ) d i s p l a y
gree of homology
(88-921) whereas
most extensive homology
slightly
the cytoplasmic
(97-100%).
more homologous
The K
to e a c h o t h e r
and K
the lowest d e exons show the
genes are
t h a n to t h e K
There a r e only nine nucleotide d i f f e r e n c e s
between
K
exons 4 to 8 and they differ by only 13 n u c l e o t i d e s
of t h e a 3 d o m a i n
( e x o n f o u r ) to t h e t e r m i n a t i o n
and K
are 9 9 * homologous
total
sequences
of 1089 nucleotides)
servation
in c o d i n g
as w e l l
in this r e g i o n .
a n d K in
from t h e start
codon.
(13 differences
gene.
Thus, K
o u t of a
T h i s d e g r e e of c o n -
as in n o n - c o d i n g
sequences
is r e m a r -
kable.
The overall
antigens
the K
and K
to t h e K
tween K
h o m o l o g y on t h e p r o t e i n
level
between
the three
is f o r K b - K k 9 0 X , f o r K d - K k 8 6 % a n d f o r K b - K d 8 3 t .
antigens are much more similar
antigen.
O f t h e total
of 35 a m i n o acid
a n d K , 27 a r e c o n t a i n e d
(Figure 4 ) .
Only two differences
(Figure 4, positions
191 and 2 2 5 ) .
a r e found
This
differences be-
in t h e a 3 d o m a i n
is a s u r p r i s i n g l y l o w
that K
tions and K
h a v e 12 s u b s t i t u t i o n s
Amino acids unique at certain
than
in t h e al a n d a 2 d o m a i n s
number, considering
and K
and K
to each other
Thus,
h a v e 11 a m i n o acid
positions
in t h e s a m e
substituregion.
for each allele are found
9479
Nucleic Acids Research
120
240
Mo
6ao
Hat Ala rro Cys Hat Leu Un LOT Lao L«a Ala Ala Ala Lao Ala Pro Tbr Cln Thr Arg Ala C
Ijmo I
ATCO^CCCTCCATCCTCCTCCTCCTCTTCCCCCCCaCCTCOCCCCCACTCACAlXCCCCCSC CTCACT>rmrirmC<)irTr«iA<T.O
I i c m 1050
f w H - . m v r » r i r m v f u f w m i i t •• iv. T*ww.jw/-.^f^rni i n m irrimmTTrTTTiiTT™*'':1'1''''*TV"irr'v"rfTrAflnirftrnr"yi''yrr"r- '170
Cioo 2
l y Pro Wa tor U a Arg Tyr Pta# Hia Tbr Ala Val h r Arg Pro Cly Leu Cly Lya
Ont-I U. 1 UreAOCTCCCOCTCTCACCCCCCCCCCCraXAfl CCCaCATTCCCTCAWTATTTCCACACCCCCCTCTCCCCCCTXCCCCTCCCCAAC
12*9
Pro Art Pha I l a l « r Tal Cly Tyr Val A*p Aap Tbr Cla Tim V«l Arg Pba Aap S«r Aap Ala Cla A M Pro Arg Tyr Cla Pro Arg Val
CttC«TTCATCTCTCTCOCCTACCTCCACCACACCCiCTTCCTCCCCTTCCACAaCA£
1 3
«
Arg Trp h e CID CID Tal Clu Pro Clu Tyr Trp Cla Arg A n Thr Clo II* Al« Lya Cly Am Cla Cla 11* Pb* Arg Val A n L«a Arg
OKTCCATCC*CCA£CTCCAG<XCatfTATTCCCACCttAACACCCACATCCCCA^
'**'
Thr Ala U n Arg Tyr Tyr Aao Clo far Ala Cly C
ACCCCCCTCCCCTACTACAACCAGACCCCCCCC CCICLACTCAaxUXGlU>4«^tfCTrfcrrjtrrXi^A^
1557
Exoa 3
1? t*r
CC TCT
1*74
Hia Thr Pba Cla Arg Hat TyT Cly Cya Clo Val Cly far Aap Trp Arg L n U u Arg Cly Tyr Glu Cla Tyr Ala Tyr Aap Cly Cya A«p
CA£ ACC TTC CAA CCC ATC TAC CCC TCT OAC CTO CCC TCC CAC TOO CCC CTC CTC CCC 00C TAC CAC CAC TAC CCA TAC CAC CCC TCC GAT
17*4
Tyr H a Ala L«n Asa Cla Aap Lau Lya Thr Trp Thr Ala Ala Aap fet Ala Ala Lav I l a Tbr Lya l i a Lya Trp Clo Clo Ala Cly A*p
TACATCaxCTCAJk£CAAC4£CTCAAAAroTCCACC(?CCCCWATCCCCCCCCTC
IU4
Ala Clu Arg Aap Arg Ala Tyr Lau Clu Cly Thr Cya Val Cla Trp L«a Arg Arg Tyr Leu Cla U a Cly A n Al* Thr U a Pro Axg Thr
CCA CAC ACA CAC CCC CCC TAC CTC GAC CCC ACC TCC CTC CAC TCC CTC CCC ACA TAC CTC CAC CTC CCC AAC CCC ACC CTC CCC CCC ACA
IM4
/v^.^TTtNT^n^tftft^pryi/^fY^^^^ . • | ,. . • | | | . ^f-pr^^^^A^-t^vT^^^TTT^^fvvv^^
21 S3
2303
ACTOACTCCTinCCrCCTXaCTCTCTCACCCTrTACACC
T
f
l
^
a
i
c
l
C
T
r
T
r
r
f
t
T
1
r
TTAnr-AnVft1! I1 I1 ' I ' * f r ^ r . f n r ^ i f i T t v i m .-w TAfaH'ft^^^Vrf^AfT^MTTTCTACAArrrTrrtaAflaATACiTTCTCACACATCCCTCCcihii-iuiCOG
T
myrrTfYTia!iifTf.
2423
Liiatmcna:
2663
TAAi.l H I M 1111LMHLLMJJXUAIU.1IATTT
2783
2»3
2»
X123
nTCCAATT>»iirrcTCjcrACcuuni
Ti'.n-.^mirttTf.rirrrirr.
. T «V'.trvv-iY-.t-fr.^.»vv-..t-.iv-^-.r.tvv-rrm-^irtv-.iTIJI.1 III rjCTrJCTnrTCrjrTYinCCrr
r.,
rCACOCCCTCOCACTCAIIllllUua IAfaraCTTACTCAIUJI.UllUACTTCCACTCACACTT
ICTTACT
••^•'••'-rCACOCCCTCOCACTCAIIllllUua
. . i . • IIU.II .tv-..tvwr./^.f..TaxrCACTCTCAATCCTCTCACTTTCCACCATCATirA<yrrTA^
3143
3263
""»» 3
3
35O3
3*23
ap gar Pro Lyi Ala Bla Tal Thr Arg Hia gar Arg Pro Clo Uf LyB Tal Tbr Lao Arg Cya
ATTCCCCAAUCCCCATCTCACCCSTCACACCAGACCTCAACATAAACrCACCCTCACCTCC
3720
Trp Ala Lao Gly Pba Tyr Pro Ala Aap I l a Thr Lau Tor Trp Glo Lao Aco Cly Clo Clo Lao Tbr Clo Aap Mat Clu Lao Val Glo Thr
TGCCCCCTGCCCTTCTACCCrCCTCACATCACCnCAaTiaUCTTCAATOSCCACCACnCACCCACCACATGCACCTTGTCCACACC
3»IO
Arg Pro U a Oly Aap Gly Thr Pba Clo Lya Trp U a tar Val Val Val Pro U u Cly Lya Cla Clo Tyr Tyr Thr Cya U a Val Tyr U s
ACCCCTCCACaCCATCCAACCTTCCACAACTCCCUTCTCTCCTCCTCCCTCTTaKAAGCAGCACTAITACACATCCCATCnTACCAT
3*00
Cla Cly L«o Pro Clu Pro Lau Tbr Lao Arg Trp C
CACCCGCTCCCTCA6CCCCTCACCCTCACATCGC r ' l ' " " " ' " " " ' T ' mia'irjrj'ii'xmraamrfrarjm 11 It l\M-lrirrr^CADCniomcccr
400*
Exon i
"•"•"-^
,m»tw.^»
i.
l o Pro Pro Pro gar Tbr Val tar Aao Thr Val I l a I l a Ala Val Lao Val
,n iCCCTCCTCCATCCACTCTCTCCAACACOCTAATCATTCCTCTTCTCCTT
4111
Tal U a Cly U a U a I l a Val Tbr Cly U a Val Val Ala Pba Val Hat Lys Hst Arg Art Art Aao Tbr G
GTCCTTCCACCTOCAATACTCACTCCACCTCTCCTCCCTTTTCTCATCAACATCACAACG
ACA AAC ACA C CTafTllinntTWMTTCrCACTTTT
r r n f t i n i 111 11 IAGAIJI'I'IW T;T1flT1ATrftaTC17TOrtirifAffirftrAnfvv'A/'rtTPnT*''^'tril'fftftrrrnm''ir-ri'i
^ u f f " iccmrTrrrTA^n-M.tAgiTTT
4207
4327
tm >
ly Cly Lya Cly Cly Asp Tyr Ua Lao Ua Pro C
itntnunritir.
M - . I - CT CCA AAA CCA CCC CAC TAT CCT CTC CCT CCA G CTTACTCTCrmriraJ HUILlIUUCCArtTTCCACTCAA
4434
i7Trmi^TT:.TYra.nt-r.-r<3^.T^.T..T^?r-rrv-r^..-.^
I l l 11 li.l 4 3 M
lioo 7
l y t a r Clo Tar t a r Aa* Lao g a r Lao Pro Asp Cys Lya 7
TTTACCCTAC OC TCC CAC ACC TCT CAT CTC TCT CTC CCA CAT TCT AAA C CTAACACTCTAfiCCTCTCATTCCCCACCCCCAATCTCCaCATCATTCCCTTTCACC
l
•>oa »
a l Urt 'al Bit Xsp Pro BiM ST Lm U s Tr»
w . t ^ t n r f i | l . . i w l ' t t « Y ; . i i i i i •• i.^^rr-./-^ JQ A T Q CTT CAT OAC CCT CAT TCT CTA CCC TCA ACACACCTCCCTCCACTCCA
CTOuar
T
f
t
r
'
nTnrrrmmrjutkicrTrmMn:
i-i-rr^-..Tr-rrmT«-./T.icj'ic1£ii.i»ii iL.
9480
IirimTru-u-rcTtfrjc
463*
4767
4U7
M07
3127
J247
3U7
Nucleic Acids Research
T A B L E I: N u c l e o t i d e s e q u e n c e h o m o l o g y * o f e x o n s
and i n t r o n s o f t h r e e H - 2 K a l l e l e s
Alleles
compared
I
II
III
IV
V
VI-VI
94
90
91
98
96
100
K k -K d
95
88
92
94
96
97
K d -K b
92
90
89
94
91
97
I
II
III
IV
V
VI
VII
95
95
_§
100
100
100
96
d
95
96
94
95
96
98
98
K d -K b
93
96
_§
95
96
98
98
vk
Exon:
vb
Introns:
^k ..b
k
K -K
II
T h e h o m o l o g y is g i v e n a s p e r c e n t a g e
§ Sequence information for the third intron of the H-2KL
g e n e is n o t c o m p l e t e
in n i n e p o s i t i o n s
in t h e t h r e e e x t r a c e l l u l a r d o m a i n s
Five of these a r e contained
(Figure 4 ) .
in t h e al d o m a i n and of t h e s e all e x -
c e p t o n e a r e p r e s e n t in t h e h y p e r v a r i a b l e c l u s t e r a t p o s i t i o n s
62-83.
Another three a r e scattered
in t h e a 2 d o m a i n at p o s i t i o n s
9 9 , 1 5 6 and 1 7 3 . T h e a 3 d o m a i n c o n t a i n s
the last
alleie-specific
r e s i d u e at pos1tion 1 9 1 .
The K
and K
antigens each have two glycosylation
o n e in t h e al d o m a i n a t p o s i t i o n 8 6 a n d t h e s e c o n d
main at position
176 (Figure 4 ) . T h e K
sites,
in t h e a 2 d o -
a n t i g e n h a s an a d d i t i o -
nal c a r b o h y d r a t e g r o u p in t h e a 3 d o m a i n a t r e s i d u e 2 5 6 . A t t h i s
position
the K
and K
polypeptides
contain
tyrosine
residues.
F i g u r e 3. C o m p l e t e D N A s e q u e n c e of t h e H - 2 K k g e n e .
T h e s e q u e n c e w a s d e t e r m i n e d a s p r e v i o u s l y d e s c r i b e d ( 1 9 ) in c o m b i n a t i o n with the M a x a m and G i l b e r t p r o c e d u r e ( 2 0 ) . The deduced
a m i n o a c i d s e q u e n c e is s h o w n o n t o p o f t h e D N A s e q u e n c e .
Transcriptional promoting elements are underlined.
The first nine
a m i n o a c i d s for e x o n 8 a r e g i v e n in i t a l i c s b e c a u s e o f t h e u n c e r t a i n t y o v e r w h i c h a c c e p t o r s i t e is u s e d .
9481
Nucleic Acids Research
etl-OCKAIB
Oly Pro a l l h i K g b g T)i pha nit Thr Ala val Sar Arg Pro Gly Lao Gly Lya Pro Arg Pb« II*
-
v«l
-
-
-
-
-
-
-
-
-
Glu
-
-
Tyr Hat
• • r i v a l Cly Tyr val Asp Aap Thr Gin ph« val Arg Pha Asp Bar Aap Ala Gla Aan Pro Arg Tyr Glu
Pro Arg val Arg Trp Hat Olu oln val Gla pro Glu Tyr Trp Glu ArglAanlThr GlnlllalAla Lya Gly
-
Ala Pro
-
-
Olu Oly
70
-
-
SO
Arg
*
-
-
Sar
»0
Aan Glu Gln| Ila|pna Arg val|Ian| Lau Arg Thr Ala Lau Arg Tyr Tyr ASQ Gin Sar Ala Gly
Lau - aiy
- Ly. - Gin - Ly. -
Gly Mr Hla Thr Pha Qln Arg Hat Tyr Qly Cya Glu Val Gly tar Aap Trp Arg Lau Lau Arg Gly Tyr
-
lla
-
val i l « s«r
120
130
Glu Gin Tyr Ala Tyr Aap Gly Cya Aap Tyr l l a Ala Lau Asn Glu Aap Lau Lya Thr Trp Thr Ala Ala
Gin
-
Pha
140
150
Aap nat Ala Ala Lau Ila Thr Lya Hla Lya Trp Glu cln Ala Gly Aap Ala Glu Arg Aap Arg Ala Tyr
1(0
170
¥fc
180
Lau GlD Gly Thr Cya Val Glo Trp Lau Ar? Axg Tyr Lau Gin Lau Gly Aan Ala Thr Lau Pro Arg Thr
ac3-D0KAX>
113
|»0
200
Aap S*r Fro Lya Ala Hla Val Thrl ArglBla Bar Arg Pro Gla Aap Lya Val Thr Laa Arg Cf
-
-
-
-
-
-
-
-
-
-
Bla
I Tyr I -
-
Pro
-
-
-
-
-
-
5«r Glu val Asp
-
-
-
-
-
-
-
-
-
Trp Ala
-
-
-
-
-
-
210
220
L*u Olj Fha Tyr Pro Ala Asp Iltt Thr L*v Thr Trp Git. Lea Aan Oly Gla Gla L«u Thr Glo Asp Mat
Glu Lau Val Glu Thr Arg Pro Ala Gly Aap Gly Thr Pba Gin Lya Trp Ala Sar val Val val Pro Lao
2t0
270
Gly Lya Cla Gin Tyr Tyr Thr Cya Bla val Tyr Kla Gin Qly Lau pro Glu Pro Lsu Thr Lau Arg Trp
"la
9482
-
Lys
Nucleic Acids Research
T h e c o d o n s u s e d h e r e f o r t y r o s i n e ( T A T ) and a s p a r a g i n e
fer by o n l y o n e n u c l e o t i d e , m o s t l i k e l y c h a n g e d
(AAT) dif-
by a s i n g l e
muta-
ti o n .
Identification
By c o m p a r i n g
of K a 11 e l e - s p e c i f i c
sequences
p r o t e i n and n u c l e o t i d e s e q u e n c e s o f t h e t h r e e
genes we have identified
sequences
unique for each
K allele.
Allele specific sequences
are very useful
be u s e d f o r t h e s y n t h e s i s
of u n i q u e o l i g o m e r i c D N A p r o b e s .
as s u c h i n f o r m a t i o n
have found one such s e q u e n c e for the H-2K
amino acids 62-66
(Figure 5 A ) .
l o n g and d i f f e r s
by 7 and 5 n u c l e o t i d e s
alleles, respectively.
a2 domain, residues
19 n u c l e o t i d e s
A K
g e n e in t h e al
T h i s s e q u e n c e is 15
from the H-2K
(30).
Residues
and K
in
the
This sequence
1 9 1 - 1 9 8 of the a3
antigen define a sequence potentially
allele.
domain,
nucleotides
s p e c i f i c s e q u e n c e is f o u n d
number 94-100 (Figure 5 B ) .
can
We
is
l o n g and h a s a l r e a d y b e e n u s e d a s p r o b e to i d e n t i -
fy K b s p e c i f i c s e q u e n c e s
of t h e K
H-2
It is d i s t i n c t f r o m t h e K
nucleotides, respectively
and K
domain
u n i q u e for t h e K
sequences
in 9 a n d
8
o u t o f 24 ( F i g u r e 5 C ) .
DISCUSSION
The DNA c o n t a i n i n g
the a u t h e n t i c
H-2K
gene was
t w o i n b r e d m o u s e s t r a i n s B 1 0 . B R and A K R / J .
to c o n t a i n a s e c o n d
shown
to c o n t a i n a n H - 2 K - l i k e g e n e ( B . A r n o l d , A . A r c h i b a l d
cells
( H - 2 q ) , a f i b r o b l a s t cell
J DNA) directed
monstrated
antigen
K
results).
When introduced
i n t o IT
(7A) was
antigen.
antibodies specific
and
22-6
l i n e , the DNA of c l o n e 27-2
the e x p r e s s i o n of the H - 2 K k
by u s i n g m o n o c l o n a l
(23).
T h i s c l o n e of D N A
the
was
found
S. K v i s t , u n p u b l i s h e d
H-2K gene.
Isolated from
The B 1 0 . B R DNA
(AKR/
This was de-
for t h e
H-2K
W e b e l i e v e t h a t c l o n e 6D c o n t a i n s the 3' end of the H-2
g e n e of t h e B 1 0 . B R
s t r a i n for the f o l l o w i n g r e a s o n s .
First,
it
F i g u r e 4.
A m i n o acid c o m p a r i s o n of the t h r e e e x t r a c e l l u l a r
d o m a i n s of three H-2K a i l e l i c a n t i g e n s .
On t o p t h e s e q u e n c e o f t h e H - 2 K * a l l e l e is s h o w n .
H o m o l o g y 1s
i n d i c a t e d by a d a s h .
The boxed amino a d d s are a l i e l e - s p e c i f 1c.
The two stars indicate the g l y c o s y l a t i o n
sites and the a r r o w at
p o s i t i o n 256 d e n o t e s the a d d i t i o n a l g l y c o s y l a t i o n s i t e of the
d
H-2K
antigen.
9483
Nucleic Acids Research
A-
62
Kk
B
66
5' CGG AAC ACG CAG ATC
Kb
---
G-G
--A
---
-AA
Kd
GA-
C-G
--A
---
-GA
-
T
94
Kb
100
CT A T T
CAG GTG ATC T C T GG ?
Kk
-G T-C
--A
K<*
-G T - C ---
^-
5'
-AC
--
CG- - - G -TC
--
CG-
--G
191
Kd
198
5' TAT CAC CCC AGA T C T CAA G T T GAT 3'
Kk
CG-
AG-
---
C--
G--
-A-
A-A
Kb
C--, ---
AG-
---
C--
G--
-A-
A-A
Figure 5. Allele specific sequences for the H-2K locus.
D a s h e s i n d i c a t e h o m o l o g y to t h e s e q u e n c e s h o w n o n t o p . T h e
n u m b e r s r e f e r t o t h e a m i n o a c i d p o s i t i o n s 1n t h e a n t i g e n .
carries the K-locus specific sequence at its 31 end and can therefore be assigned to the K-locus. Secondly, Its restriction m a p
is i d e n t i c a l t o t h a t o f c l o n e 2 7 - 2 , w h i c h c o n t a i n s t h e a u t h e n t i c
L.
H-2K g e n e . T h i r d l y , partial DNA sequence a n a l y s i s of clone 6D
confirms its identity with the gene in c l o n e 2 7 - 2 .
The DNA s e q u e n c e of the K gene of c l o n e 2 7 - 2 was
was determint
determined
and shows an e x t r a o r d i n a r i l y high d e g r e e of h o m o l o g y to the H-2K,b
gene. The d i f f e r e n c e s are a l m o s t e x c l u s i v e l y c o n f i n e d to exons
2 and 3. It has been shown that the introns of the K and K
genes d i s p l a y a higher degree of homology than the exons of these
genes ( 1 1 ) . The same is true when these genes a r e compared to
the H - 2 K k s e q u e n c e . T h e introns of all three H-2K a l l e l e s a r e 95981 h o m o l o g o u s w h e r e a s the c o r r e s p o n d i n g figure for the exons is
92-94*.
R e c e n t l y , the complete amino acid s e q u e n c e of the al domain
of the K a n t i g e n has been d e t e r m i n e d ( 3 1 ) . This sequence is
identical to the s e q u e n c e d e d u c e d from the K gene presented
h e r e , e x c e p t for one amino acid. C o m p a r i s o n of the araino acid
s e q u e n c e s of the three H-2K alleles r e v e a l s that the K k and K b
antigens are much m o r e similar to each other than to the K antig e n . The d i f f e r e n c e s between the two former proteins are con9484
Nucleic Acids Research
fined
to t h e al and a 2 d o m a i n s w h e r e
so h a v e d i v e r g e d
most
of t h e t h r e e e x t r a c e l l u l a r
for each K allele
n o t l e s s t h a n 14 a r e c h a r g e d
nonhydrophobic
sines are i n c l u d e d ) .
residues
We would
m u t a g e n e s i s , will
re-
tyro-
e x p e c t t h e s e a m i n o a c i d s to be e x thereby accessible
Future studies, using site
clarify whether
in a n t i g e n i c d e t e r m i n a n t s
c e l l s or
to i n -
directed
these residues are directly
for a n t i b o d i e s
or c y t o l y t i c
inT
not.
We have identified
the H-2K a l l e l e s .
nucleotides
specific nucleotide
Such s e q u e n c e s
c a n be used as p r o b e s
from a n o t h e r .
p e c t to b o t h
The K
genes within
sequences
or i d e n t i c a l
it m i g h t be p o s s i b l e
the q u e s t i o n
to d i s t i n g u i s h
sequence
of w h e t h e r
(H-2
sequences.
and H - 2
one of t h e s e g e n e s
(32,33,34,35).
probes might help elucidate
gene
gene
Kd
H-2
, respectively)
be t h e
probes
case
to s t u d y
( f o r i n s t a n c e in t h e D ,
the unique s e q u e n c e
m
of
oligo-
is u n i q u e w i t h r e s -
If t h i s w o u l d
In s u c h a s t u d y t h e d o n o r g e n e f o r t h e K
identified
one H-2
F o r t h e H - 2 K k and
(30).
to u s e t h e al1 e l e - s p e c i f i c
Qa or T l a r e g i o n s ) h a s d o n a t e d
ready been
as s y n t h e s i z e d
( F i g u r e 5 ) w e d o n o t k n o w if o t h e r
the same h a p l o t y p e
carry similar
sequences for each
are useful
a 11 e l e - s p e c i f 1 c
locus and h a p l o t y p e
a 11 e l e - s p e c i f i c
termed
(Figure 4 ) .
or p o l a r
( i . e . w h e n s e r i n e s and
on t h e s u r f a c e of t h e p r o t e i n a n d
teract with other molecules.
volved
do-
( A r g , L y s , A s n , A s p , G i n , G l u and H i s ) a n d as m a n y as 21
are clearly
posed
positions
unique residues
Of t h e s e 27 a m i n o a c i d s
sidues
al-
extensively.
In n i n e a m i n o acid
m a i n s we have found
the n u c l e o t i d e s e q u e n c e s
to t h e K g e n e .
m u t a n t gene has a l -
Further
analyses with
the mechanisnts(s) of the
such
phenomenon
conversion.
ACKNOWLEDGEMENTS
W e t h a n k M . S t e i n m e t z for t h e c o s m i d
g e n e of the A K R / J
strain.
27-2 containing
We are grateful
to A . - M .
and H . L e h r a c h f o r t h e g i f t o f t h e B 1 0 . B R D N A
technical
a s s i s t a n c e we thank G. KUblbeck
F o r e x c e l l e n t h e l p in p r e p a r i n g
H . S e i f e r t a n d C. D r o z .
the
Frischauf
library.
and C.
For
Raynoschek.
the m a n u s c r i p t we thank
H.-G.B. was supported
H-2Kk
I. B e n n e r ,
by the g r a n t
Do
9485
Nucleic Acids Research
199 14-2 f r o m the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t . A . L . A . w a s
s u p p o r t e d by the A g r i c u l t u r a l and Food R e s e a r c h C o u n c i l , L o n d o n .
"•"Present address: German Cancer Research Center, Im Neuenheimer Feld 280, D-6900 Heidelberg,
FRG
'Present address: Swiss Institute for Experimental Cancer Research, Ch. des Boveresses, CH1066 Epalingcs s/Lausanne, Switzerland
•To whom correspondence should be addressed
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