GENOMICS 14, 852-856 (1992)
The L-Isoaspartyl/D-Aspartyl Protein Methyltransferase Gene (PCMT1) Maps to Human Chromosome 6q22,3-6q24 and the Syntenic Region of Mouse Chromosome 10 DUNCAN C. MACLAREN,*'1 CLARE M. O'CONNOR, t YU-RONG XIA,¢'§ MARGARETEMEHRABIAN,::I:'§ IVANA KLISAK,~ ROBERTS. SPARKES,~ STEVENCLARKE,* AND ALDONS J. LUSlS$'§ *Department of Chemistry and Biochemistry and the Molecular Biology Institute, SDepartment of Medicine, School of Medicine, and §Department of Microbiology and the Molecular Biology Institute, University of Cafifornia, Los Angeles, California 90024; and tWorcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, Massachusetts 01545
ReceivedJuly 9, 1992; revised August 2T,, 1992 We have mapped the genes for the human and mouse L-isoaspartyl/D-aspartyl protein carboxyl methylt r a n s f e r a s e (EC 2 . 1 . 1 . 7 7 ) u s i n g c D N A p r o b e s . W e det e r m i n e d t h a t t h e h u m a n g e n e is p r e s e n t i n c h r o m o some 6 by Southern blot analysis of DNA from a panel o f m o u s e - h u m a n s o m a t i c c e l l h y b r i d s . In situ h y b r i d i z a t i o n s t u d i e s a l l o w e d u s to c o n f i r m t h i s i d e n t i f i c a t i o n a n d f u r t h e r l o c a l i z e t h e h u m a n g e n e ( P C M T 1 ) to the 6q22.3-6q24 region. By analyzing the presence of an EcoRI polymorphism in DNA from backcrosses of C57BL/6J and Mus spretus strains of mice, we locali z e d t h e m o u s e g e n e (Pcmt-1) to c h r o m o s o m e 1 0 , at a p o s i t i o n 8 . 2 _+ 3 . 5 c M p r o x i m a l to t h e M y b l o c u s . T h i s r e g i o n o f t h e m o u s e c h r o m o s o m e is h o m o l o g o u s to t h e h u m a n 6 q 2 4 r e g i o n . © 1992 AcademicPress, Inc.
INTRODUCTION Three classes of protein carboxyl methyltransferases, distinguished by their methyl-acceptor substrate specificity, h a v e b e e n f o u n d in p r o c a r y o t i c a n d e u c a r y o t i c c e l l s ( C l a r k e , 1985; O t a a n d C l a r k e , 1990; B a t t e n a n d O ' D e a , 1990). W e a r e i n t e r e s t e d i n t h e w i d e l y d i s p e r s e d type II enzyme, which catalyzes the transfer of a methyl g r o u p f r o m S - a d e n o s y l - L - m e t h i o n i n e t o t h e free c a r boxyl groups of D-aspartyl and L-isoaspartyl residues. These methyl-accepting residues result from the spontaneous deamidation, isomerization, and racemization of normal L-aspartyl and L-asparaginyl residues (Geiger a n d C l a r k e , 1987) a n d r e p r e s e n t s i t e s o f c o v a l e n t d a m age t o a g i n g p r o t e i n s . T h e r e c o g n i t i o n o f t h e a b n o r m a l residues by this methyltransferase (O'Connor and Yutzey, 1987; A s w a d a n d J o h n s o n , 1987; L o w e n s o n a n d C l a r k e , 1992) is p r o p o s e d t o b e a f i r s t s t e p i n t h e e v e n tual repair or degradation of these proteins (McFadden 1 To whom correspondence should be addressed at UCLA Chemistry and Biochemistry, 405 Hilgard Avenue, Los Angeles, CA 900241569. 0888-7543/92 $5.00 Copyright © 1992 by AcademicPress, Inc. All rights of reproduction in any form reserved.
a n d C l a r k e , 1987; J o h n s o n et al., 1987a,b; O a l l e t t i et al., 1988). The mammalian L-isoaspartyl/D-aspartyl methyltransferases characterized to date are cytosolic monom e r s o f a b o u t 25 k D a ( C l a r k e , 1985). F o r t h e h u m a n enzyme, cDNA and Southern blot analysis indicated t h a t t h e t w o m a j o r i s o z y m e s o f t h i s e n z y m e ( O t a et al., 1988) c a n a r i s e b y a l t e r n a t i v e s p l i c i n g o f a s i n g l e g e n e p r o d u c t ( I n g r o s s o et al., 1991; M a c L a r e n et al., 1992). I n this study, human and mouse methyltransferase cDNAs ( M a c L a r e n et al., 1992; R o m a n i k et al., 1992) w e r e u s e d a s p r o b e s t o d e t e r m i n e t h e c h r o m o s o m a l loci o f t h e m e t h y l t r a n s f e r a s e gene. O u r r e s u l t s i n d i c a t e t h a t t h e h u m a n m e t h y l t r a n s f e r a s e g e n e is l o c a t e d i n t h e q 2 2 . 3 q24 r e g i o n o f h u m a n c h r o m o s o m e 6. T h e m o u s e g e n e m a p s t o m o u s e c h r o m o s o m e 10, 8.2 _+ 3.5 c M p r o x i m a l t o t h e M y b locus, a n d in a r e g i o n s y n t e n i c t o t h e q24 l o c a t i o n o f t h e h u m a n m e t h y l t r a n s f e r a s e gene. MATERIALS A N D METHODS
Somatic cell hybrid analysis--human. A panel of 14 mouse-human somatic cell hybrid clones, derived by fusion of normal male fibroblasts (IMR 91) with thymidine kinase-deficient mouse B82 cells, was constructed and analyzed for chromosome content as described previously (Mohandas et al., 1986). The hybrids contained varying complements of human chromosomes as determined by karyotyping, and have been used for the chromosomal assignment of a large number of genes. DNA was isolated from the hybrids as well as from the parental lines using sodium dodecyl sulfate (SDS) and proteinase K followed by phenol-chloroform extraction and ethanol precipitation. Following cleavage with EcoRI, about 5 ttg of the DNA from each sample was separated by 1% agarose/Tris-acetate gel electrophoresis, transferred to nylon filters, and cross-linked. The filters were probed with the 1182-bp human methyltransferase cDNA insert pRK1 (MacLaren et al., 1992), composed of an incomplete coding region of 324 bases and 844 bases of 3'-untranslated region. The insert was excised from the pBluescriptSK- vector (Stratagene, La Jolla, CA) using EcoRI restriction endonuclease and radiolabeled using the random priming method (Feinberg and Vogelstein, 1983) with [a-SSp]dCTP to a specific activity of 109 cpm/ttg. Filter hybridization was performed using 107 cpm/ ml in hybridization buffer (0.5 M sodium phosphate, pH 7.0, 7% SDS, 1% bovine serum albumin, and 1 mM EDTA) at 65°C for 24 h. Filters were washed twice for 20 min in 2X SSC (lX SSC is 150 mMNaC1, 15 852
PROTEIN
METHYLTRANSFERASE
GENE
TABLE
MAPPING
IN HUMAN
AND
MOUSE
853
1
S e g r e g a t i o n of M e t h y l t r a n s f e r a s e Gene w i t h H u m a n C h r o m o s o m e s in M o u s e - H u m a n Somatic Cell Hybrids Human Hybrid clone
PCMT1
1
2
3
4
5
6
7
8
84-2 84-4 84-7 84-20 84-21 84-25 84-26 84-27 84-30 84-34 84-35 84-37 84-38 84-39
+ + + + + + + + + + + -
+ +
+ +
-
(+)
+ + +
+ + + +
+ +
+ + + -
+ + + + + + -
+ + + +
+ + + + +
+ + (+) + +
6
3
No. of discordant hybrids
+ -
+
+ + +
+
-
+ +
7
9
7
+ + + + + + +
+ + + (+) + (+)
+ + + + + + + +
2
5
0
+ + +
9
10
-
+
-
+ + +
-
+ +
-
+
11
6
chromosome
11
12
13
14
15
16
17
18
19
20
21
22
X
Y
+ + (+) + + + + -
+ + (+) + (+) + + (+) + + + -
+ + -
+ + + + +
+ + -
+ +
+ +
+ -
+ +
+ + + + +
+ + + + + + -
+ + + +
-
+ +
-
+ + + + + + + (+) + + (+) -
(+) -
+
+ + + -(+) +
+
+
+ + + + + + + +
-
+
+ + + + + + + + + + + + + +
+ -
+ +
-
(+) + -
6
4
8
4
6
8
3
5
6
4
8
6
12
11
Note. A ( + ) i n d i c a t e s p r e s e n c e o f t h e c h r o m o s o m e i n 1 0 - 3 0 % o f t h e m e t a p h a s e s a n a l y z e d . m M s o d i u m c i t r a t e , p H 7.0) c o n t a i n i n g 0 . 1 % S D S a t 6 5 ° C . T h e f i n a l w a s h w a s d o n e t w i c e u n d e r l o w s t r i n g e n c y c o n d i t i o n s o f 1X S S C , 0 . 1 % SDS at 50°C. Autoradiograms were prepared at -70°C by exposing Kodak XAR-5 film to the filters.
m e t a p h a s e c h r o m o s o m e s u s i n g a h y b r i d i z a t i o n m i x t u r e c o n t a i n i n g 0.1 # g p r o b e / m l ( M e h r a b i a n et al., 1 9 8 6 ; H a r p e r a n d S a u n d e r s , 1 9 8 1 ; C a n nizzaro and Emanuel, 1984). The slides were exposed for 2 weeks, and all silver grains on or touching chromosomes were scored.
I n situ hybridization to chromosomes--human. The methyltransferase cDNA insert was labeled by random oligonucleotide priming with 3H-labeled deoxynucleotides to a specific activity of about l0 s cpm/#g. The probe was hybridized to G-banded human lymphocyte
Interspecific backcross analysis--mouse. I n t e r s p e c i f i c b a c k c r o s s analysis was used for chromosomal assignment and to generate a genetic linkage map between the mouse methyltransferase gene and seve r a l m a r k e r s p r e v i o u s l y l o c a l i z e d t o m o u s e c h r o m o s o m e 10. T h e mouse methyltransferase locus was mapped using a mouse cDNA probe and Southern analysis of DNAs from N 2 progeny of an interspecific b a c k c r o s s . T h e m o u s e m e t h y l t r a n s f e r a s e c D N A p r o b e is a 1 5 8 0 b p EcoRI-bounded i n s e r t c o n t a i n i n g a l l 9 - b p 5 ' - u n t r a n s l a t e d r e g i o n , the complete 684-bp coding region, and a 777-bp 3'-untranslated reg i o n ( R o m a n i k et al., 1 9 9 2 ) t h a t w a s r a d i o l a b e l e d a s d e s c r i b e d f o r t h e h u m a n p r o b e . T h e i n t e r s p e c i f i c b a c k c r o s s [ ( C 5 7 B L / 6 J x M. spretus)F 1 X C 5 7 B L / 6 J ] w a s d o n e a s d e s c r i b e d b y B u c h b e r g et al. ( 1 9 8 8 , 1 9 8 9 ) i n t h e l a b o r a t o r y o f A. J . L u s i s . B r e e d i n g p a i r s o f M. spretus domesticus ( S t r a i n C 5 7 B L / 6 J ) w e r e o b t a i n e d f r o m J a c k s o n L a b o r a t o r i e s ( B a r H a r b o r , M E ) a n d t h o s e o f M. spretus ( S p a i n ) f r o m D r . M i chael Potter (National Institute of Health, Bethesda, MD). The backcross was performed by crossing F 1females, resulting from the cross of f e m a l e C 5 7 B L / 6 J m i c e w i t h m a l e M. spretus m i c e , w i t h m a l e C 5 7 B L / 6J mice. Genomic DNA was extracted from spleen and kidney of the p a r e n t a l , F1, a n d b a c k c r o s s (N2) m i c e , d i g e s t e d w i t h s e v e r a l r e s t r i c tion enzymes, and analyzed by Southern hybridization with the mouse methyltransferase probe to determine an informative restriction fragment length polymorphism (RFLP). Hybridization and washes were done in the same manner as for the human clone, with the exception that the final wash was done under more stringent conditions (65°C and 0.2X SSC, 0.1% SDS). The mouse cDNA probe identified an EcoRI 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 v a r i a n t o f 3.8 k b u n i q u e t o t h e M . spretus D N A , a n d t h e s e g r e g a t i o n o f t h e M. spretus allele w a s f o l l o w e d i n t h e 61 N~ m i c e . C o m p a r i s o n o f t h e m e t h y l t r a n s f e r a s e s e g r e g a t i o n patterns with known segregation patterns was used for chromosomal assignment. A linkage map of the methyltransferase locus was created by followi n g t h e s e g r e g a t i o n o f t h r e e o t h e r c h r o m o s o m e 10 m a r k e r s i n t h e 61 N 2 a n i m a l s . T h e s e m a r k e r s w e r e Myb ( m y e l o b l a s t o s i s p r o t o - o n c o g e n e ; S h e n - O n g et al., 1 9 8 4 ) , Pah ( p h e n y l a l a n i n e h y d r o x y l a s e ; S a i k i et al., 1 9 8 8 ) , a n d Rrm2-4ps ( r i b o n u c l e o t i d e r e d u c t a s e M 2 f o u r t h p s e u d o g e n e , C l o n e 10; T h e l a n d e r a n d B e r g , 1 9 8 6 ; A. J . L u s i s , u n p u b l i s h e d ) . T h e c h r o m o s o m e a s s i g n m e n t o f Rrm2-4ps is n o v e l . A c D N A c l o n e f o r the M2 subunit of mouse ribonucleotide reductase designated "clone
25 24 23
P
22.3 22.2 22.1 21.3 21.2 21.1 12 11.2 11.1 11.1 11.2 12 13 14 15 16.1 16.2 16.3 21
q
22.1 22.2 22.3 23.1 23.2 23.3 24
O0
00000
0 0 0 0 0 0
25.1 25.2 25.3 26 27 F I G . 1 . I d i o g r a m o f in situ h y b r i d i z a t i o n o f m e t h y l t r a n s f e r a s e c D N A t o G - b a n d e d h u m a n m e t a p h a s e c h r o m o s o m e 6. N o o t h e r c h r o mosome had significant accumulation of grain density (data not shown).
854
M A C L A R E N E T AL.
TABLE 2 Segregation Patterns and Linkage Analysis 1 with Myb, Rrm2-4ps, and Pah on Mouse some 10
of PcmtChromo-
Segregation p a t t e r n s Genotype
Pcmt-1
Myb
Rrm2-4ps
Pah
Number
B S S S S B B B S S S
B S B S S S B B B B S
B S B B S S S B S B B
B S B B B S S S S S S
16 21 1 4 5 2 8 1 1 1 1 61
Total: Linkage analysis Linkage interval
r
Pcmt-l-Myb Pcmt-l-Rrm2-4ps Pcmt-l-Pah Myb-Rrm2-4ps Myb-Pah Rrm2-4ps-Pah
5/61 17/61 21/61 14/61 20/61 8/61
cM ± SE 8.2 27.9 34.4 23.0 32.8 13.1
± ± ± ± ± ±
3.5 5.7 6.1 5.4 6.0 4.3
content of the hybrids showed zero discordancies with chromosome 6. There were two or more discordancies with each of the remaining chromosomes. Therefore, we conclude t hat the human L-isoaspartyl/D-aspartyl protein methyltransferase gene resides on human chromosome 6. T he gene was further localized by examining the in situ hybridization of methyltransferase cDNA to human metaphase chromosomes. T he distribution of silver grains over metaphase chromosomes was scored in 205 cells, 47 of which had grains. A significant accumulation of grains, 23%, occurred in the q22.3-q24 region of chromosome 6 (Fig. 1). No other significant accumulations occurred on any other chromosome. These results confirm the somatic cell hybrid studies and indicate th a t the methyltransferase gene resides on human chromosome 6q22.3-q24.
Mouse Methyltransferase Gene Mapping Comparison of the methyltransferase segregation patterns with approximately 200 known loci typed in the interspecific backcross mice (A. J. L usi s, C. Warden, and M. Mehrabian, unpublished) indicates th a t the mouse methyltransferase gene (Pcmt-1) is on mouse
Human Chromosome 6q
/\/ manLoci
Note. Alleles inherited in the backcrosses are denoted as either B or S to indicate their origin from either C 5 7 B L / 6 J or Mus Spretus respec-
16
tively, r, r e c o m b i n a t i o n frequency; cM, centimorgans; SE, s t a n d a r d error ~/P(1 - P ) / N .
132(q21), A6F(q21)
21 10" was obtained from Dr. Paul Berg, S t a n f o r d University (Thelander and Berg, 1986). Using this cDNA, multiple p o l y m o r p h i s m s were identified, including loci on c h r o m o s o m e s 4, 12 and 13. S o u t h e r n analysis of D N A digested with HindIII yielded major hybridizing b a n d s of 22, 12.5, and 3.5 kb for strain C 5 7 B L / 6 J and b a n d s of 10.0, 6.6, 6.2, and 3.9 kb for M. spretus. D N A from F1 hybrids contained b o t h parental bands. T h e presence or absence of the 6.6-kb M. spretus b a n d segregated with c h r o m o s o m e 10 markers, and the gene has been designated Rrm2-4ps in accordance with n o m e n c l a t u r e of previously m a p p e d Rrm2-related gene loci.
}S1(q21-22) N (q21)
Human Methyltransferase Gene Mapping Th e chromosomal location of the human L-isoaspartyl/D-aspartyl protein methyltransferase gene (designated P C M T 1 for protein carboxyl methyltransferase 1) was determined by examining a panel of hum an-m ouse somatic cell hybrids using the pRK1 (MacLaren et al., 1992) cDNA probe and Southern blot analysis. H u m a n chromosome presence in the EcoRI-digested DNA from these hybrids was indicated by the presence of a 5.8-kb fragment (data not shown). Eleven of the 14 hybrids were positive for the presence of this band (Table 1). Correlation of this band with the human chromosome
cM 38
Mouse Loci Hkq
37 36 35 34 33 32
Ros.1
31 30
29 Fyn
22 22 0•0•022
fB (q22-23) ~GR1 (q23-24)
23 23 23 R (q24-27) 'MT1(q22.3-24).
00000024
RESULTS
Mouse Chromosome 10
AG6E(q26), F2R(q25-27)
• 25 25 • 25
28 27 26 25 24 23 22 21 2O 19 18 17 16 15 14 13 12 11 10
Pcmt-1 Locus Probability Distribution
26 27
Telomere
Centromere
F I G . 2. C o m p a r i s o n of the loci on h u m a n c h r o m o s o m e 6 (Ziegler et al., 1991) syntenic with those on mouse c h r o m o s o m e 10 (Taylor et al., 1991). T h e dots on the G-banded idiogram of h u m a n c h r o m s o m e 6q represent the in situ hybridization results. T h e n u m b e r s along the mouse c h r o m o s o m e are the recombinational distances in centimorgans. Loci t h a t are bold-faced have been m a p p e d on b o t h chromosomes. T h e L-isoaspartyl/D-aspartyl m e t h y l t r a n s f e r a s e is given the abbreviation P C M T 1 for the h u m a n a n d Pcmt-1 for the mouse.
PROTEIN METHYLTRANSFERASE GENE MAPPING IN HUMAN AND MOUSE c h r o m o s o m e 10. T h e l o c a t i o n o n c h r o m o s o m e 10 w a s determined by following the segregation of Pcmt-1 with t h r e e c h r o m o s o m e 10 m a r k e r s (Myb, R r m 2 - 4 p s , a n d P a h ) in t h e b a c k c r o s s m i c e ( T a b l e 2). E a c h b a c k c r o s s a n i m a l a p p e a r e d t o b e e i t h e r h o m o z y g o u s for t h e C 5 7 B L / 6 J a l l e l e o r h e t e r o z y g o u s for t h e C 5 7 B L / 6 J a n d M . s p r e t u s a l l e l e s a t e a c h locus. T h e f r e q u e n c y o f t h e C 5 7 B L / 6 J a n d M . s p r e t u s a l l e l e s for a l l t h e loci e x a m i n e d in t h e N2 p r o g e n y d i d n o t d i f f e r s i g n i f i c a n t l y f r o m t h e 1:1 r a t i o e x p e c t e d f o r a l l e l e s t r a n s m i t t e d i n a n o r m a l M e n d e l i a n f a s h i o n f r o m t h e h e t e r o z y g o u s F1 p a r e n t t o t h e N2 o f f s p r i n g . T h e m e t h y l t r a n s f e r a s e g e n e r e s i d e s 8.2 __ 3.5 c M p r o x i m a l ( t o w a r d t h e c e n t r o m e r e ) t o t h e M y b locus. DISCUSSION As part of an effort to better understand the functions of L-isoaspartyl/D-aspartyl carboxyl methyltransferase, we have mapped the gene for the enzyme to human chrom o s o m e 6 q 2 2 . 3 - q 2 4 a n d 8.2 _+ 3.5 c M p r o x i m a l t o t h e M y b l o c u s o n m o u s e c h r o m o s o m e 10. A s i l l u s t r a t e d i n Fig. 2, o u r r e s u l t s e x t e n d a l a r g e r e g i o n o f s y n t e n y b e t w e e n h u m a n c h r o m o s o m e 6 a n d m o u s e c h r o m o s o m e 10. I f t h e o r d e r o f t h e g e n e s i n t h e r e g i o n is c o n s e r v e d b e t w e e n m o u s e a n d h u m a n , t h e r e s u l t s i n t h e m o u s e suggest that the methyltransferase gene resides on or near h u m a n c h r o m o s o m e 6q24 (Fig. 2). We were interested in comparing the map position of the protein methyltransferase gene with those of two o t h e r g e n e s i n v o l v e d in t h e a g i n g p r o c e s s . W e r n e r s y n drome, a human autosomal recessive condition characterized by premature aging, has recently been associated with markers on human chromosome 8 in a Japanese f a m i l y s t u d y ( G o t o et al., 1992). T h i s r e s u l t s u g g e s t s t h a t d e f e c t s i n t h i s m e t h y l t r a n s f e r a s e d o n o t a c c o u n t for t h e observed phenotype in this population. On the other h a n d , a d e f e c t r e s u l t i n g i n m o u s e c a t a r a c t s (Cat Fr) h a s b e e n m a p p e d t o c h r o m o s o m e 10 ( M u g g l e t o n - H a r r i s et al., 1987). M u t a t i o n s i n t h i s g e n e r e s u l t i n t h e a c c u m u l a t i o n o f a b n o r m a l p r o t e i n s i n t h e e y e l e n s ( G a r b e r et al., 1984), a p h e n o t y p e s i m i l a r t o w h a t w o u l d b e e x p e c t e d for m e t h y l t r a n s f e r a s e l o s s ( M c F a d d e n a n d C l a r k e , 1986). H o w e v e r , t h e Cat Fr g e n e m a p s a t t h e d i s t a l e n d o f c h r o m o s o m e 10 a n d is t h u s a l s o n o t a s s o c i a t e d w i t h t h e Li s o a s p a r t y l / D - a s p a r t y l m e t h y l t r a n s f e r a s e locus. J u s t i c e et al. (1990) s h o w a h i g h t r a n s m i s s i o n r a t i o d i s t o r t i o n o n c h r o m o s o m e 10 f r o m M y b t o t h e Ros-1 l o c u s a n d b e y o n d for a s a m p l e o f 108 m i c e . O u r r e s u l t s showed no such transmission ratio distortion with the m e t h y l t r a n s f e r a s e gene, n o r , c o n t r a r y t o p r e v i o u s f i n d i n g s ( J u s t i c e et al., 1990), w i t h t h e M y b locus, e v e n t h o u g h we u s e d t h e s a m e s u b s t r a i n o f m i c e . ACKNOWLEDGMENTS This work was supported by USHHS National Institutional Research Service Award T32 GM08375 to D.C.M., National Institutes of Health Grant AG08109 to C.M.O., National Institutes of Health
855
Grant GM26020 to S.C., and National Institutes of Health Grants HL42488 and HL28481 to A.J.L.
REFERENCES Aswad, D. W., and Johnson, B. A. (1987). The unusual substrate specificity of eukaryotic protein carboxyl methyltransferases. Trends Biochem. Sci. 12: 155-158. Barten, D. M., and O'Dea, R. F, (1990). The function of protein carboxylmethyltransferase in eucaryotic cells. Life Sci. 47: 181-194. Buchberg, A. M., Bedigian, H. G., Taylor, B. A., Brownell, E., Ihle, J. N., Nagata, S., Jenkins, N. A., and Copeland, N. G. (1988). Localization of Evi-2 to chromosome 11: Linkage to other proto-oncogene and growth factor loci using interspecific backcross mice. Oncogene Res. 2: 149-165. Buchberg, A. M., Brownell, E., Nagata, S., Jenkins, N. A., and Copeland, N. G. (1989). A comprehensive genetic map of murine chromosome 11 reveals extensive linkage conservation between mouse and human. Genetics 122: 153-161. Cannaizzaro, C. A., and Emanuel, B. S. (1984). An improved method for G banding chromosomes after in situ hybridization. Cytogenet. Cell Genet. 38: 308-309. Clarke, S. (1985). Protein carboxyl methyltransferases: Two distinct classes of enzymes. Annu. Rev. Biochem. 54: 479-506. Feinberg, A. P., and Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. Galletti, P., Ciardiello, A., Ingrosso, D., Di Donato, A., and D'Alessio, G. (1988). Repair of isopeptide bonds by protein carboxyl o-methyltransferase: Seminal ribonuclease as a model system. Biochemistry 27: 1752-1757. Garber, A. T., Goring, D., and Gold, R. J. M. (1984). Characterization of abnormal proteins in the soluble lens proteins of Cat Frasermice. J. Biol. Chem. 259: 10376-10379. Geiger, T., and Clarke, S. (1987). Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. J. Biol. Chem. 262: 785-794. Goto, M., Rubenstein, M., Weber, J., Woods, K., and Drayna, D. (1992). Genetic linkage of Werner's syndrome to five markers on chromosome 8. Nature 355: 735-738. Harper, M. E., and Saunders, G. F. (1981). Localization of single copy DNA sequences on G-banded chromosomes by in situ hybridization. Chromosoma 83: 431-439. Ingrosso, D., Kagan, R. M., and Clarke, S. (1991). Distinct C-terminal sequences of isozymes I and II of the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase. Biochem. Biophys. Res. Commun. 175(1): 351-358. Johnson, B. A., Langmack, E. L., and Aswad, D. W. (1987a). Partial repair of deamidation-damaged calmodulin by protein carboxyl methyltransferase. J. Biol. Chem. 262(25): 12283-12287. Johnson, B. A., Murray, E. D. J., Clarke, S., Glass, D. B., and Aswad, D. W. (1987b). Protein carboxyl methyltransferase facilitates conversion of atypical L-isoaspartyl peptides to normal L-aspartyl peptides. J. Biol. Chem. 262: 5622-5629. Justice, M. J., Siracusa, L. D., Gilbert, D. J., Heisterkamp, N., Groffen, J., Chada, K., Silan, C. M., Copeland, N. G., and Jenkins, N. A. (1990). A genetic linkage map of mouse chromosome 10: Localization of eighteen molecular markers using a single interspecific backcross. Genetics 125:855 866. Lowenson, J. D., and Clarke, S. (1992). Recognition of D-aspartyl residues in polypeptides by the erythrocytes L-isoaspartyl/D-aspartyl protein methyltransferase. J. Biol. Chem. 267: 5986-5995. MacLaren, D. C., Kagan, R. M., and Clarke, S. (1992). Alternative splicing of the human isoaspartyl protein carboxyl methyltransferase leads to the generation of a C-terminal -RDEL sequence in isozyme II. Biochem. Biophys. Res. Commun. 185: 277-283.
856
MACLAREN ET AL.
McFadden, P. N., and Clarke, S. (1986). Protein carboxyl methyltransferase and methyl acceptor proteins in aging and cataractous tissue of the human eye lens. Mech. Ageing Dev. 34: 91-105. McFadden, P. N., and Clarke, S. (1987). Conversion of isoaspartyl peptides to normal peptides: Implications for the cellular repair of damaged proteins. Proc. Natl. Acad. Sci. USA 84: 2595-2599. Mehrabian, M., Sparkes, R. S., Mohandas, T,, Klisak, I., Schumaker, V, N., Heinzmann, C., Zollman, S., Ma, Y., and Lusis, A. J. (1986). Human apolipoprotein B: Chromosomal mapping and DNA polymorphisms of hepatic and intestinal species. Somat. Cell Mol. Genet. 12: 245-254. Mohandas, T., Heinzmann, C., Sparkes, R. S., Wasmuth, J., Edwards, P., and Lusis, A. J. (1986). Assignment of human 3-hydroxy-3methylglutaryl coenzyme A reductase gene to q13-q23 region of chromosome 5. Somat. Cell Mol. Genet. 12: 245-254. Muggleton-Harris, A. L., Festing, M. F. W., and Hall, M. (1987). A gene location for the inheritance of the Cataract Fraser (Cat Fr) mouse congenital cataract. Genet. Res. 49: 235-238. O'Connor, C. M., and Yutzey, K. E. (1987). Enhanced carboxyl methylation of membrane-associated hemoglobin in human erythrocytes. J. Biol. Chem. 2 6 3 : 1386-1390. Ota, I. M., Gilbert, J. M., and Clarke, S. (1988). Two major isozymes of the protein D-aspartyl/L-isoaspartyl methyltransferase from human erythrocytes. Biochem. Biophys. Res. Commun. 151: 11361143.
Ota, I. M., and Clarke, S. (1990). The function and enzymology of protein D-aspartyl/L-isoaspartyl methyltransferases in eukaryotic and prokaryotic cells. In "Protein Methylation" (W. K. Paik and S. Kim, Eds.), pp. 179-194, CRC Press, Boca Raton, FL. Romanik, E. A., Ladino, C. A., Killoy, L. C., D'Ardenne, S. C., and O'Connor, C. M. (1992). Genomic organization and tissue expression of the murine gene encoding the protein ~-aspartate methyltransferase. Gene 118: 217-222. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491. Shen-Ong, G. L. C., Potter, M., Mushinski, J, F., Lavu, S., and Reddy, E. P. (1984). Activation of the c-myb locus by viral insertional mutagenesis in plasmacytoid lymphosarcomas. Science 2 2 6 : 10771080. Taylor, B. A., Justice, M. J., and Reeves, R. (1991). Mouse chromosome 10. Mamrn. Genome 1: $146-S157. Thelander, L., and Berg, P. (1986). Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase. Mol. Cel. Biol. 6: 3433-3442. Ziegler, A., Field, L. L., and Sakaguchi, A. Y. (1991). Report of the committee on the genetic constitution of chromosome 6. Cytogenet. Cell Genet. 58: 295-336.
The L-Isoaspartyl/D-Aspartyl Protein Methyltransferase Gene (PCMT1) Maps to Human Chromosome 6q22,3-6q24 and the Syntenic Region of Mouse Chromosome 10 DUNCAN C. MACLAREN,*'1 CLARE M. O'CONNOR, t YU-RONG XIA,¢'§ MARGARETEMEHRABIAN,::I:'§ IVANA KLISAK,~ ROBERTS. SPARKES,~ STEVENCLARKE,* AND ALDONS J. LUSlS$'§ *Department of Chemistry and Biochemistry and the Molecular Biology Institute, SDepartment of Medicine, School of Medicine, and §Department of Microbiology and the Molecular Biology Institute, University of Cafifornia, Los Angeles, California 90024; and tWorcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, Massachusetts 01545
ReceivedJuly 9, 1992; revised August 2T,, 1992 We have mapped the genes for the human and mouse L-isoaspartyl/D-aspartyl protein carboxyl methylt r a n s f e r a s e (EC 2 . 1 . 1 . 7 7 ) u s i n g c D N A p r o b e s . W e det e r m i n e d t h a t t h e h u m a n g e n e is p r e s e n t i n c h r o m o some 6 by Southern blot analysis of DNA from a panel o f m o u s e - h u m a n s o m a t i c c e l l h y b r i d s . In situ h y b r i d i z a t i o n s t u d i e s a l l o w e d u s to c o n f i r m t h i s i d e n t i f i c a t i o n a n d f u r t h e r l o c a l i z e t h e h u m a n g e n e ( P C M T 1 ) to the 6q22.3-6q24 region. By analyzing the presence of an EcoRI polymorphism in DNA from backcrosses of C57BL/6J and Mus spretus strains of mice, we locali z e d t h e m o u s e g e n e (Pcmt-1) to c h r o m o s o m e 1 0 , at a p o s i t i o n 8 . 2 _+ 3 . 5 c M p r o x i m a l to t h e M y b l o c u s . T h i s r e g i o n o f t h e m o u s e c h r o m o s o m e is h o m o l o g o u s to t h e h u m a n 6 q 2 4 r e g i o n . © 1992 AcademicPress, Inc.
INTRODUCTION Three classes of protein carboxyl methyltransferases, distinguished by their methyl-acceptor substrate specificity, h a v e b e e n f o u n d in p r o c a r y o t i c a n d e u c a r y o t i c c e l l s ( C l a r k e , 1985; O t a a n d C l a r k e , 1990; B a t t e n a n d O ' D e a , 1990). W e a r e i n t e r e s t e d i n t h e w i d e l y d i s p e r s e d type II enzyme, which catalyzes the transfer of a methyl g r o u p f r o m S - a d e n o s y l - L - m e t h i o n i n e t o t h e free c a r boxyl groups of D-aspartyl and L-isoaspartyl residues. These methyl-accepting residues result from the spontaneous deamidation, isomerization, and racemization of normal L-aspartyl and L-asparaginyl residues (Geiger a n d C l a r k e , 1987) a n d r e p r e s e n t s i t e s o f c o v a l e n t d a m age t o a g i n g p r o t e i n s . T h e r e c o g n i t i o n o f t h e a b n o r m a l residues by this methyltransferase (O'Connor and Yutzey, 1987; A s w a d a n d J o h n s o n , 1987; L o w e n s o n a n d C l a r k e , 1992) is p r o p o s e d t o b e a f i r s t s t e p i n t h e e v e n tual repair or degradation of these proteins (McFadden 1 To whom correspondence should be addressed at UCLA Chemistry and Biochemistry, 405 Hilgard Avenue, Los Angeles, CA 900241569. 0888-7543/92 $5.00 Copyright © 1992 by AcademicPress, Inc. All rights of reproduction in any form reserved.
a n d C l a r k e , 1987; J o h n s o n et al., 1987a,b; O a l l e t t i et al., 1988). The mammalian L-isoaspartyl/D-aspartyl methyltransferases characterized to date are cytosolic monom e r s o f a b o u t 25 k D a ( C l a r k e , 1985). F o r t h e h u m a n enzyme, cDNA and Southern blot analysis indicated t h a t t h e t w o m a j o r i s o z y m e s o f t h i s e n z y m e ( O t a et al., 1988) c a n a r i s e b y a l t e r n a t i v e s p l i c i n g o f a s i n g l e g e n e p r o d u c t ( I n g r o s s o et al., 1991; M a c L a r e n et al., 1992). I n this study, human and mouse methyltransferase cDNAs ( M a c L a r e n et al., 1992; R o m a n i k et al., 1992) w e r e u s e d a s p r o b e s t o d e t e r m i n e t h e c h r o m o s o m a l loci o f t h e m e t h y l t r a n s f e r a s e gene. O u r r e s u l t s i n d i c a t e t h a t t h e h u m a n m e t h y l t r a n s f e r a s e g e n e is l o c a t e d i n t h e q 2 2 . 3 q24 r e g i o n o f h u m a n c h r o m o s o m e 6. T h e m o u s e g e n e m a p s t o m o u s e c h r o m o s o m e 10, 8.2 _+ 3.5 c M p r o x i m a l t o t h e M y b locus, a n d in a r e g i o n s y n t e n i c t o t h e q24 l o c a t i o n o f t h e h u m a n m e t h y l t r a n s f e r a s e gene. MATERIALS A N D METHODS
Somatic cell hybrid analysis--human. A panel of 14 mouse-human somatic cell hybrid clones, derived by fusion of normal male fibroblasts (IMR 91) with thymidine kinase-deficient mouse B82 cells, was constructed and analyzed for chromosome content as described previously (Mohandas et al., 1986). The hybrids contained varying complements of human chromosomes as determined by karyotyping, and have been used for the chromosomal assignment of a large number of genes. DNA was isolated from the hybrids as well as from the parental lines using sodium dodecyl sulfate (SDS) and proteinase K followed by phenol-chloroform extraction and ethanol precipitation. Following cleavage with EcoRI, about 5 ttg of the DNA from each sample was separated by 1% agarose/Tris-acetate gel electrophoresis, transferred to nylon filters, and cross-linked. The filters were probed with the 1182-bp human methyltransferase cDNA insert pRK1 (MacLaren et al., 1992), composed of an incomplete coding region of 324 bases and 844 bases of 3'-untranslated region. The insert was excised from the pBluescriptSK- vector (Stratagene, La Jolla, CA) using EcoRI restriction endonuclease and radiolabeled using the random priming method (Feinberg and Vogelstein, 1983) with [a-SSp]dCTP to a specific activity of 109 cpm/ttg. Filter hybridization was performed using 107 cpm/ ml in hybridization buffer (0.5 M sodium phosphate, pH 7.0, 7% SDS, 1% bovine serum albumin, and 1 mM EDTA) at 65°C for 24 h. Filters were washed twice for 20 min in 2X SSC (lX SSC is 150 mMNaC1, 15 852
PROTEIN
METHYLTRANSFERASE
GENE
TABLE
MAPPING
IN HUMAN
AND
MOUSE
853
1
S e g r e g a t i o n of M e t h y l t r a n s f e r a s e Gene w i t h H u m a n C h r o m o s o m e s in M o u s e - H u m a n Somatic Cell Hybrids Human Hybrid clone
PCMT1
1
2
3
4
5
6
7
8
84-2 84-4 84-7 84-20 84-21 84-25 84-26 84-27 84-30 84-34 84-35 84-37 84-38 84-39
+ + + + + + + + + + + -
+ +
+ +
-
(+)
+ + +
+ + + +
+ +
+ + + -
+ + + + + + -
+ + + +
+ + + + +
+ + (+) + +
6
3
No. of discordant hybrids
+ -
+
+ + +
+
-
+ +
7
9
7
+ + + + + + +
+ + + (+) + (+)
+ + + + + + + +
2
5
0
+ + +
9
10
-
+
-
+ + +
-
+ +
-
+
11
6
chromosome
11
12
13
14
15
16
17
18
19
20
21
22
X
Y
+ + (+) + + + + -
+ + (+) + (+) + + (+) + + + -
+ + -
+ + + + +
+ + -
+ +
+ +
+ -
+ +
+ + + + +
+ + + + + + -
+ + + +
-
+ +
-
+ + + + + + + (+) + + (+) -
(+) -
+
+ + + -(+) +
+
+
+ + + + + + + +
-
+
+ + + + + + + + + + + + + +
+ -
+ +
-
(+) + -
6
4
8
4
6
8
3
5
6
4
8
6
12
11
Note. A ( + ) i n d i c a t e s p r e s e n c e o f t h e c h r o m o s o m e i n 1 0 - 3 0 % o f t h e m e t a p h a s e s a n a l y z e d . m M s o d i u m c i t r a t e , p H 7.0) c o n t a i n i n g 0 . 1 % S D S a t 6 5 ° C . T h e f i n a l w a s h w a s d o n e t w i c e u n d e r l o w s t r i n g e n c y c o n d i t i o n s o f 1X S S C , 0 . 1 % SDS at 50°C. Autoradiograms were prepared at -70°C by exposing Kodak XAR-5 film to the filters.
m e t a p h a s e c h r o m o s o m e s u s i n g a h y b r i d i z a t i o n m i x t u r e c o n t a i n i n g 0.1 # g p r o b e / m l ( M e h r a b i a n et al., 1 9 8 6 ; H a r p e r a n d S a u n d e r s , 1 9 8 1 ; C a n nizzaro and Emanuel, 1984). The slides were exposed for 2 weeks, and all silver grains on or touching chromosomes were scored.
I n situ hybridization to chromosomes--human. The methyltransferase cDNA insert was labeled by random oligonucleotide priming with 3H-labeled deoxynucleotides to a specific activity of about l0 s cpm/#g. The probe was hybridized to G-banded human lymphocyte
Interspecific backcross analysis--mouse. I n t e r s p e c i f i c b a c k c r o s s analysis was used for chromosomal assignment and to generate a genetic linkage map between the mouse methyltransferase gene and seve r a l m a r k e r s p r e v i o u s l y l o c a l i z e d t o m o u s e c h r o m o s o m e 10. T h e mouse methyltransferase locus was mapped using a mouse cDNA probe and Southern analysis of DNAs from N 2 progeny of an interspecific b a c k c r o s s . T h e m o u s e m e t h y l t r a n s f e r a s e c D N A p r o b e is a 1 5 8 0 b p EcoRI-bounded i n s e r t c o n t a i n i n g a l l 9 - b p 5 ' - u n t r a n s l a t e d r e g i o n , the complete 684-bp coding region, and a 777-bp 3'-untranslated reg i o n ( R o m a n i k et al., 1 9 9 2 ) t h a t w a s r a d i o l a b e l e d a s d e s c r i b e d f o r t h e h u m a n p r o b e . T h e i n t e r s p e c i f i c b a c k c r o s s [ ( C 5 7 B L / 6 J x M. spretus)F 1 X C 5 7 B L / 6 J ] w a s d o n e a s d e s c r i b e d b y B u c h b e r g et al. ( 1 9 8 8 , 1 9 8 9 ) i n t h e l a b o r a t o r y o f A. J . L u s i s . B r e e d i n g p a i r s o f M. spretus domesticus ( S t r a i n C 5 7 B L / 6 J ) w e r e o b t a i n e d f r o m J a c k s o n L a b o r a t o r i e s ( B a r H a r b o r , M E ) a n d t h o s e o f M. spretus ( S p a i n ) f r o m D r . M i chael Potter (National Institute of Health, Bethesda, MD). The backcross was performed by crossing F 1females, resulting from the cross of f e m a l e C 5 7 B L / 6 J m i c e w i t h m a l e M. spretus m i c e , w i t h m a l e C 5 7 B L / 6J mice. Genomic DNA was extracted from spleen and kidney of the p a r e n t a l , F1, a n d b a c k c r o s s (N2) m i c e , d i g e s t e d w i t h s e v e r a l r e s t r i c tion enzymes, and analyzed by Southern hybridization with the mouse methyltransferase probe to determine an informative restriction fragment length polymorphism (RFLP). Hybridization and washes were done in the same manner as for the human clone, with the exception that the final wash was done under more stringent conditions (65°C and 0.2X SSC, 0.1% SDS). The mouse cDNA probe identified an EcoRI 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 v a r i a n t o f 3.8 k b u n i q u e t o t h e M . spretus D N A , a n d t h e s e g r e g a t i o n o f t h e M. spretus allele w a s f o l l o w e d i n t h e 61 N~ m i c e . C o m p a r i s o n o f t h e m e t h y l t r a n s f e r a s e s e g r e g a t i o n patterns with known segregation patterns was used for chromosomal assignment. A linkage map of the methyltransferase locus was created by followi n g t h e s e g r e g a t i o n o f t h r e e o t h e r c h r o m o s o m e 10 m a r k e r s i n t h e 61 N 2 a n i m a l s . T h e s e m a r k e r s w e r e Myb ( m y e l o b l a s t o s i s p r o t o - o n c o g e n e ; S h e n - O n g et al., 1 9 8 4 ) , Pah ( p h e n y l a l a n i n e h y d r o x y l a s e ; S a i k i et al., 1 9 8 8 ) , a n d Rrm2-4ps ( r i b o n u c l e o t i d e r e d u c t a s e M 2 f o u r t h p s e u d o g e n e , C l o n e 10; T h e l a n d e r a n d B e r g , 1 9 8 6 ; A. J . L u s i s , u n p u b l i s h e d ) . T h e c h r o m o s o m e a s s i g n m e n t o f Rrm2-4ps is n o v e l . A c D N A c l o n e f o r the M2 subunit of mouse ribonucleotide reductase designated "clone
25 24 23
P
22.3 22.2 22.1 21.3 21.2 21.1 12 11.2 11.1 11.1 11.2 12 13 14 15 16.1 16.2 16.3 21
q
22.1 22.2 22.3 23.1 23.2 23.3 24
O0
00000
0 0 0 0 0 0
25.1 25.2 25.3 26 27 F I G . 1 . I d i o g r a m o f in situ h y b r i d i z a t i o n o f m e t h y l t r a n s f e r a s e c D N A t o G - b a n d e d h u m a n m e t a p h a s e c h r o m o s o m e 6. N o o t h e r c h r o mosome had significant accumulation of grain density (data not shown).
854
M A C L A R E N E T AL.
TABLE 2 Segregation Patterns and Linkage Analysis 1 with Myb, Rrm2-4ps, and Pah on Mouse some 10
of PcmtChromo-
Segregation p a t t e r n s Genotype
Pcmt-1
Myb
Rrm2-4ps
Pah
Number
B S S S S B B B S S S
B S B S S S B B B B S
B S B B S S S B S B B
B S B B B S S S S S S
16 21 1 4 5 2 8 1 1 1 1 61
Total: Linkage analysis Linkage interval
r
Pcmt-l-Myb Pcmt-l-Rrm2-4ps Pcmt-l-Pah Myb-Rrm2-4ps Myb-Pah Rrm2-4ps-Pah
5/61 17/61 21/61 14/61 20/61 8/61
cM ± SE 8.2 27.9 34.4 23.0 32.8 13.1
± ± ± ± ± ±
3.5 5.7 6.1 5.4 6.0 4.3
content of the hybrids showed zero discordancies with chromosome 6. There were two or more discordancies with each of the remaining chromosomes. Therefore, we conclude t hat the human L-isoaspartyl/D-aspartyl protein methyltransferase gene resides on human chromosome 6. T he gene was further localized by examining the in situ hybridization of methyltransferase cDNA to human metaphase chromosomes. T he distribution of silver grains over metaphase chromosomes was scored in 205 cells, 47 of which had grains. A significant accumulation of grains, 23%, occurred in the q22.3-q24 region of chromosome 6 (Fig. 1). No other significant accumulations occurred on any other chromosome. These results confirm the somatic cell hybrid studies and indicate th a t the methyltransferase gene resides on human chromosome 6q22.3-q24.
Mouse Methyltransferase Gene Mapping Comparison of the methyltransferase segregation patterns with approximately 200 known loci typed in the interspecific backcross mice (A. J. L usi s, C. Warden, and M. Mehrabian, unpublished) indicates th a t the mouse methyltransferase gene (Pcmt-1) is on mouse
Human Chromosome 6q
/\/ manLoci
Note. Alleles inherited in the backcrosses are denoted as either B or S to indicate their origin from either C 5 7 B L / 6 J or Mus Spretus respec-
16
tively, r, r e c o m b i n a t i o n frequency; cM, centimorgans; SE, s t a n d a r d error ~/P(1 - P ) / N .
132(q21), A6F(q21)
21 10" was obtained from Dr. Paul Berg, S t a n f o r d University (Thelander and Berg, 1986). Using this cDNA, multiple p o l y m o r p h i s m s were identified, including loci on c h r o m o s o m e s 4, 12 and 13. S o u t h e r n analysis of D N A digested with HindIII yielded major hybridizing b a n d s of 22, 12.5, and 3.5 kb for strain C 5 7 B L / 6 J and b a n d s of 10.0, 6.6, 6.2, and 3.9 kb for M. spretus. D N A from F1 hybrids contained b o t h parental bands. T h e presence or absence of the 6.6-kb M. spretus b a n d segregated with c h r o m o s o m e 10 markers, and the gene has been designated Rrm2-4ps in accordance with n o m e n c l a t u r e of previously m a p p e d Rrm2-related gene loci.
}S1(q21-22) N (q21)
Human Methyltransferase Gene Mapping Th e chromosomal location of the human L-isoaspartyl/D-aspartyl protein methyltransferase gene (designated P C M T 1 for protein carboxyl methyltransferase 1) was determined by examining a panel of hum an-m ouse somatic cell hybrids using the pRK1 (MacLaren et al., 1992) cDNA probe and Southern blot analysis. H u m a n chromosome presence in the EcoRI-digested DNA from these hybrids was indicated by the presence of a 5.8-kb fragment (data not shown). Eleven of the 14 hybrids were positive for the presence of this band (Table 1). Correlation of this band with the human chromosome
cM 38
Mouse Loci Hkq
37 36 35 34 33 32
Ros.1
31 30
29 Fyn
22 22 0•0•022
fB (q22-23) ~GR1 (q23-24)
23 23 23 R (q24-27) 'MT1(q22.3-24).
00000024
RESULTS
Mouse Chromosome 10
AG6E(q26), F2R(q25-27)
• 25 25 • 25
28 27 26 25 24 23 22 21 2O 19 18 17 16 15 14 13 12 11 10
Pcmt-1 Locus Probability Distribution
26 27
Telomere
Centromere
F I G . 2. C o m p a r i s o n of the loci on h u m a n c h r o m o s o m e 6 (Ziegler et al., 1991) syntenic with those on mouse c h r o m o s o m e 10 (Taylor et al., 1991). T h e dots on the G-banded idiogram of h u m a n c h r o m s o m e 6q represent the in situ hybridization results. T h e n u m b e r s along the mouse c h r o m o s o m e are the recombinational distances in centimorgans. Loci t h a t are bold-faced have been m a p p e d on b o t h chromosomes. T h e L-isoaspartyl/D-aspartyl m e t h y l t r a n s f e r a s e is given the abbreviation P C M T 1 for the h u m a n a n d Pcmt-1 for the mouse.
PROTEIN METHYLTRANSFERASE GENE MAPPING IN HUMAN AND MOUSE c h r o m o s o m e 10. T h e l o c a t i o n o n c h r o m o s o m e 10 w a s determined by following the segregation of Pcmt-1 with t h r e e c h r o m o s o m e 10 m a r k e r s (Myb, R r m 2 - 4 p s , a n d P a h ) in t h e b a c k c r o s s m i c e ( T a b l e 2). E a c h b a c k c r o s s a n i m a l a p p e a r e d t o b e e i t h e r h o m o z y g o u s for t h e C 5 7 B L / 6 J a l l e l e o r h e t e r o z y g o u s for t h e C 5 7 B L / 6 J a n d M . s p r e t u s a l l e l e s a t e a c h locus. T h e f r e q u e n c y o f t h e C 5 7 B L / 6 J a n d M . s p r e t u s a l l e l e s for a l l t h e loci e x a m i n e d in t h e N2 p r o g e n y d i d n o t d i f f e r s i g n i f i c a n t l y f r o m t h e 1:1 r a t i o e x p e c t e d f o r a l l e l e s t r a n s m i t t e d i n a n o r m a l M e n d e l i a n f a s h i o n f r o m t h e h e t e r o z y g o u s F1 p a r e n t t o t h e N2 o f f s p r i n g . T h e m e t h y l t r a n s f e r a s e g e n e r e s i d e s 8.2 __ 3.5 c M p r o x i m a l ( t o w a r d t h e c e n t r o m e r e ) t o t h e M y b locus. DISCUSSION As part of an effort to better understand the functions of L-isoaspartyl/D-aspartyl carboxyl methyltransferase, we have mapped the gene for the enzyme to human chrom o s o m e 6 q 2 2 . 3 - q 2 4 a n d 8.2 _+ 3.5 c M p r o x i m a l t o t h e M y b l o c u s o n m o u s e c h r o m o s o m e 10. A s i l l u s t r a t e d i n Fig. 2, o u r r e s u l t s e x t e n d a l a r g e r e g i o n o f s y n t e n y b e t w e e n h u m a n c h r o m o s o m e 6 a n d m o u s e c h r o m o s o m e 10. I f t h e o r d e r o f t h e g e n e s i n t h e r e g i o n is c o n s e r v e d b e t w e e n m o u s e a n d h u m a n , t h e r e s u l t s i n t h e m o u s e suggest that the methyltransferase gene resides on or near h u m a n c h r o m o s o m e 6q24 (Fig. 2). We were interested in comparing the map position of the protein methyltransferase gene with those of two o t h e r g e n e s i n v o l v e d in t h e a g i n g p r o c e s s . W e r n e r s y n drome, a human autosomal recessive condition characterized by premature aging, has recently been associated with markers on human chromosome 8 in a Japanese f a m i l y s t u d y ( G o t o et al., 1992). T h i s r e s u l t s u g g e s t s t h a t d e f e c t s i n t h i s m e t h y l t r a n s f e r a s e d o n o t a c c o u n t for t h e observed phenotype in this population. On the other h a n d , a d e f e c t r e s u l t i n g i n m o u s e c a t a r a c t s (Cat Fr) h a s b e e n m a p p e d t o c h r o m o s o m e 10 ( M u g g l e t o n - H a r r i s et al., 1987). M u t a t i o n s i n t h i s g e n e r e s u l t i n t h e a c c u m u l a t i o n o f a b n o r m a l p r o t e i n s i n t h e e y e l e n s ( G a r b e r et al., 1984), a p h e n o t y p e s i m i l a r t o w h a t w o u l d b e e x p e c t e d for m e t h y l t r a n s f e r a s e l o s s ( M c F a d d e n a n d C l a r k e , 1986). H o w e v e r , t h e Cat Fr g e n e m a p s a t t h e d i s t a l e n d o f c h r o m o s o m e 10 a n d is t h u s a l s o n o t a s s o c i a t e d w i t h t h e Li s o a s p a r t y l / D - a s p a r t y l m e t h y l t r a n s f e r a s e locus. J u s t i c e et al. (1990) s h o w a h i g h t r a n s m i s s i o n r a t i o d i s t o r t i o n o n c h r o m o s o m e 10 f r o m M y b t o t h e Ros-1 l o c u s a n d b e y o n d for a s a m p l e o f 108 m i c e . O u r r e s u l t s showed no such transmission ratio distortion with the m e t h y l t r a n s f e r a s e gene, n o r , c o n t r a r y t o p r e v i o u s f i n d i n g s ( J u s t i c e et al., 1990), w i t h t h e M y b locus, e v e n t h o u g h we u s e d t h e s a m e s u b s t r a i n o f m i c e . ACKNOWLEDGMENTS This work was supported by USHHS National Institutional Research Service Award T32 GM08375 to D.C.M., National Institutes of Health Grant AG08109 to C.M.O., National Institutes of Health
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Grant GM26020 to S.C., and National Institutes of Health Grants HL42488 and HL28481 to A.J.L.
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