96
BBAEXP 90282
Bioch#nica et Biophysica Acre, ! 129 (1991) 96-99 © 1991 Elsevier Science Publishers B.V. All rights reserved 0167-478! / 9 !/$03.50
Short Sequence-Paper
Cloning of cDNA encoding rat TCP-1 T a k a s h i M o r i t a i, H i r o s h i K u b o t a i, G a b r i e l G a c h e l i n 2, M a s a m i Nozaki i a n d Aizo M a t s u s h i r o t Department of Microbial Genetics, Research Institute j'or Microbial Diseases, Osaka University, Osaka (Japan) and 2 Unlit; de Biologic Moleculaire ~ha Gent; hlstitut Pastern; Paris (France)
(Received 16 September 1991)
K~:ywords: Mouse T-complex: Molecular chaperone: (Ratttas suwvegicus )
We have isolated and sequenched a cDNA encoding a rat homolog of the mouse t-complex polypeptide 1 (TCP-I). Its deduced gene product is a polypeptide of 556 amino acids, with a predicted M r of 60341. The similarity between mouse Tcp.i and the rat homolog is about 94.0% at the nucleotide level and 97.1% at the amino acid level showing the evolutionary conservation of this protein. The similarity of the amino acid sequence of the rat TCP-I is not significantly biased to any of those from wild (TCP-IB) or from t-haplotype mice (TCP-IA). From a comparison of deduced amino acid sequences of eukaryotic TCP-I proteins, we found highly conserved domains. Southern blot analysis revealed that there are at least two similar sequences to Tcp.i in the rat, one is a structural gene and the other seems to be a processed pseudogene.
The mouse t-complex is a tertiary inverted region of chromosome 17 including sets of dominant and recessive mutations, which may cause defects in embryonic development, sperm production and function [1-3]. The t-hapiotype mice are wide spread by their selective advantage of high transmission ratio distortion. Mouse TCP-I is encoded in the t-complex and is suited to the analysis of the evolutionary origin of the t-haplotype, because it exists as two electrophoretic forms, TCP-1B and TCP-IA; TCP-IA is encoded by all complete t-
The sequence data in this paper have been submitted to the EMBL/Genbank under the accession number D90345. Correspondence: T. Morita, Research Institute for Microbial Diseases, Osaka University. 3-1, Yamadaoka, Suita, Osaka, Japan.
haplotype chromosomes and TCP-IB by all inbred strains tested [4]. Willison et al. have isolated their cDNAs [5] as well as human homolog [6,7]. The human sequence has some of the features of the mouse, which are characteristic of the t-haplotype (TCP-IA) rather than TCP-IB. Therefore, it is proposed that Tcp-l" is the ancestral type and that the mouse t-chromosome is the 'wild type' [6]. We analysed the rat Tcp-I sequence to examine whether it has homology to mouse TCP-IA. We also compared the amino acid sequence of TCP-I from different species and predicted the secondary structure to understand the role of the polypeptide. We extracted RNA from Fischer rat testes (Rattus norvegicus) and used it to synthesize cDNA. The cDNA was cloned into the lambda gtl0 phage vector. Recombinant phages (8.10 4) from the library were screened with a mouse Tcp.l cDNA probe [8]. Thirteen positive clones were further screened using a synthetic oligonucleotide which corresponds to the 5' leader sequence of mouse Tcp-I cDNA. A phage clone which gave a strong positive signal by autoradiography was purified.
97 The DNA insert (1.9 kb) was subcloned into the plasmid vector pUC8 and characterized by nucleotide sequencing. The nucleotide sequence of rat Tcp-1 eDNA is shown in Fig. 1. The 1831 bp cDNA contained an ORF that codes for a polypeptide of 556 amino acid with a M r of 60341. The initiation codon at nucleotides, 1-3 was located in GAAGATG which is highly homologous with other Tcp-I genes, i.e. human, Drosophila and mouse [5,7,9]. At nucleotide position 1669-1671, a translation stop signal was found and at nucleotide 1731-1736, the possible polyadenylation signal, ATTAAA. At 17 nucleotides down stream from the signal, apoly A tail was attached. The similarity of the nucleotide sequences between rat and human is 89.8% and that between rat and hamster, 91.5% [10]. The similarity between rat and wild types mouse is 94.0% and between rat and thaplotype mouse is 93.8%, which confirms the close relationship between rat and mouse species. However, the homology of amino acid sequence between rat and human is higher (97.7%) than those between rat and t-haplotype mouse (97.1%) and wild mouse (97.5%). This indicates that the substitution of amino acids in the mouse Tcp-I is rapid [8]. At the positions where nucleotides differ between wild and t-haplotype mouse, the rat amino acid sequence resembles the wild type at 5 positions (amino acid positions, 148, 157, 200, 336, 551 in Fig. 2) and the t-haplotype at 5 positions (25, 45, 306, 415, 439), suggesting a random resemblance. We therefore speculate that each of the Tcp-I genes encoding TCP-1A and TCP-1B evolved rapidly from their common ancestor. TCP-1 has homology with the 'Chaperonin' family of heat-shock proteins [10,11], which assists in the
-77 I 76 1 51 226 301 376 451 526 601 676 751 826 901 976 1051 1126 1201 12"/6 1351 1426 1501 1 576 1651 1741
ATG GAG GGT CCT TTG GCC TCT ATT GCC AAC G&T GTA ACT ATT ACT T G T G R A CTG G C T G A C CTG AAA AAT GCT GAT RAG GAA GCA GTG CGA G C T G C T A A G ACA T C C GTG CTT GCT GTT AAA C A C G G G A G A A G T CAG C T C ItAG A G A A T A G T T CA(; GTG GTT ATT ACA CAG /tAG AT(: CTG GCA GTG GAA GCT GGT GCC AGT AT(: CTG TCT ACA GTC GTA CAG GAG AGG AT(: TTA CGA GGG GCA AGA GTT TTG GAG TCA TAT GCG ACC AGT &TG ACA CTG GCA GTG RAT GTT AAC (:CA GAA CGT GCG GGG GTG TTT GAA CGG ATT GAT GAT CTG TCT GGA GCC CTT G&T AACGAGCTGTCG ( A ) n
Fig. 1. Nucleotide sequence of the rat
proper folding of proteins and their assembly into oligomeric protein complexes, in order to determine the domain structure, we compared the amino acid sequence between the homologs of TCP-1 from rat, Drosophila and yeast [9,12]. Though the species were much diverged, the amino acid sequences were conserved among them. The region of perfect amino acid matches was distributed throughout the polypeptide (Fig. 2). The homology at the N-terminal (40-204 in Fig. 2) is extraordinary and the C-terminal (368-508) also has a highly conserved long sequence. These regions might be involved in oligomeric formation or in its function as a molecular 'chaperon'. The genomic DNA from Fischer rat liver was digested with the restriction enzyme Tagl and analysed by Southern blotting (Fig. 3). When mouse Tcp-I eDNA was used as the probe, we detected one faint (9 kb) and one intense band of 2.4 kb. The 9 kb band is monomorphic but the 2.4 kb band is polymorphic depending on the rat strain. We hybridized the same blot with a labeled PCR product (400 bp), corresponding to mouse intron 8 (our unpublished results) and found that the 2.4 kb band hybridized with it. However, the longer (9 kb) band did not. Thus the rat genome would have at least two Tcp-I sequences; one would be a structural gene and the other might be a processed pseudogene without an intron. The mouse t-complex consists of a large chromosomal segment linked to the major histocompatibility complex (MHC). The human Tcp-! gene and MHC genes are also on the same chromosome, although the locations are different [6]. Furthermore, the TCP-IB and MHC genes of cattle were linked with each other on bovine chromosome 23 [13]. However, the two bands of the rat T~7~-Iare not assigned on chromosome 20 (Mori et al. personal com-
GGCCGCTATCOGGTCTCAGAGCGGA(:CTATCGCTTAGTTGTCTGCGTTGGTAGTGAGATTTGCTTCGTTTCCTGAAG T C C G T G T T C G G G G A C C G C A G C A C T G G G G A A G C G A T C CGC TCC C A G A A C G T T ATG G C T GCA A T T G T T A R A A G T TL"I' C T T G G G C C A G T T G G C TTG GAT AAA ATG TTG GTG GAT GAT ATT GGT R A T G A T G G G G C C A C C A T T CTG A A A T T A T T G G A G GTG GAA CAT CCT GCA GCC AAA GTT CTG C T G CAP, G A C A R A G A A G T T G G A G A T G G A A C T A C C TCA GTG GTA ATC ATT GCA GL"G GAG CTT G A G C T A G T C A A G CAG A A A A T T C A T C C G A C A T C A GTT ATT AGT GGC TAT CGT CTT GCC TGC T A T A T C A A T G A G A A C C T A A T T A T C A A C A C A G A C GAA CTT GGA AGA GAC TGT CTG ATC hAT A T G T C T T C C A A A A T T A T T G G A A T A A A T G G T G A T TTC TTC GCT AAT AT(; GTG GTA GAC GCT T A C A C A G A T A T A CGA G G C CA(; C C T C G C T A T C C A GTC AAT TCT GTT AAT ATT CTA AAA GCC A T A G A A A G C AT(; C T G A T C A A T G G C T A T G C A C T C .%AC TGT GTG GTT GGA TCT CAG GGC ATG A A T G C A A A A A T T G C T T G T C T T G A C T T C A G C C T G CAG AAA ACA AAA AT(; AAG CTT GGT GTA GAC CCT GAG AAA CTG GAC CAG ATT AGA CA(; AGA GAA TCC GAT ATC ACC AAG GAG AGA ATT ACT GGT GCC AAC GTT ATT CTA ACC ACC GGT GGG ATT GAT GAC AT(; TGT CTC; AAG TAT TTT AT(; GCT GTT At..;(; AGA GTT TTA AAA AGA GAC CTC AAA CGT ATT GCA AAA GCT TCT GG~ GCA CTG GCC AAT TTG GAA GGT GAA GAG ACT TTT GAG GCA ACA AT(; TT(; GGA CAA GCG GAA GAG AFT TGT GAC G&T GAG CTG ATC TTA ATC AAA AAT ACT AAG GCT CGT ACG TCA GCT TCA ATT AAC GAC TTC ATG TGT GAT GAA ATG GAA CGC TCC TTA CAT GAC GCT CTT TGT GTG GTG AAG AAG TCT GTG GTC CCA GGT GGA GGT GCT GTA G/t,A GCG GCC TTG TCT ATA TAC CTT GAA AAC GGA TCT CGG GAA CAG CTT GCT ATT GCA GAG TTT GCA AGA TCT CT(; CTT GTG ATT CCT AAT GCT GCC CRA GAC TCC ACT GAC ~ GTT GCC AAG TTA AGA GCT TJl~ CAC RAT GAG GCT CAA AAA AAT CTA RAG TGG ATT GGT ~ GAT TTG GTC CAT GGG AAA CCA CGA GAC AAT AAG CAA CCA ACC ATA GTT AAA GTA AAG AGC ~ RAG TTT GCA ACA GAG GCT GCA ATC ACC AT,;~ CTT ATA AAA TTA CAC CCA GAA AGC AAA GAC GAC AAA CAC GGA GGT TAT GAA AAT GCT GTT CAC GAC TGA TTGGACTTCCCTTTTATTTATGACAGTGTTGGGTGTRATGTCTTAGCCTTGGGTGTCTCAC TT~--T-~.~GTAC
Tcp-! cDNA. The polyadenylation signal is underlined. The numbers on the righl refer to the nucleotidc sequence beginning from the adenine residue in the start codon.
98 of Medicine, Kyoto University) for gifts of rat DN, and unpublished results. This work was supported by Grant-in-Aid for Scientific Research (No. 0161850~ from the Ministry of Education, Science and Culture Japan.
munication) on which the rat MHC resides [14]. Thus, in the rat, the Tcp-I gene is not linked to the MHC locus. We thank Drs. J. Yamada, T. Serikawa, T. Yamada and M. Mori (Institute of Laboratory Animals. Faculty
75
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~. . . . ~ ,, ,~ ~, ..~,.~ ~,~ ,~.a ~ S~,T K~,~K L ~ V V ~ T ~ E K~ l) O ~ O ~ E ~ S l)~,-T K ~ I 0 K!)~L A T ~ A N~ I ~ T T G 3 0 0 ."" ~''~ K~KM~.~,,~t~'VL~|~N~OK!,~EA~A~iLI)~t:~TK~I NM~:i~LGT~VN~Vi~VSG R~AM~ I N~l)~:l~E O~E O ~ - K ~ A G~:|~VL ~:~V K K ~ l l) A ~ A O ~ V ~ T T K
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Fig. 2. Comparison of the amino acid sequences of TCP-I homologs. The rat TCP-! sequence (RA) was compared with those from Drosophila (DS), yeast (YS), wild type mouse (MW), t"-"-haplotype mouse (MT) and human (HU). Dashes denote gaps in the sequence. The common amino acids among rat, Drosophila and yeast are shaded. The different amino acids between wild type mouse and I '~32 haplotype mouse are boxed. The arbitrary numbers on the right refer to the amino acid sequence of TCP-! proteins. The arrows indicate the highly conserved regions.
99
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References
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Fig. 3. Southern Idol analysis (ff rat Tq~-! gene. Ten micrograms of genomic DNAs from Fischer rat (lane I and 4), SHR rat (lane 3 and 6) and the rat cell line ACI (lane 2 and 5), were digested with Ta,ql and elcctrophoresed oil a (),6";i agarose gel. Tile DNAs were hlotled onto nitrocellulose filters, then hybridized with a mouse eDNA probe (a) and an intron probe (b). The size markers are sh~wn at the right.
I Bennett, D. (1975) (.'ell h, 441-454. 2 Silver, L.M. (1985) Annu. Roy. Genet. 19, 179-2()8. 3 ltammer, M.F., Schimenti, J. and Silver, L.M. (1080) Proc. Natl. Acad. Sci. USA 86, 3261-3265. 4 Silver, L.M., Artzt, K. and Bennett, K. (1970)(.:'ell 17, 275-284. 5 Willison, K.R., Dudley, K. and Potter, J. (1986) Cell 44, 727-738. 6 Willison, K., Kelly, A., Dudley, K., G~odfcllow. K., Spurt, N., Groves, V., Gorman. P., Sheer, D. and Trowsdale, J. (1987) EMBO J. h, 1967-1974. 7 Kirchhoff, C. and Willison, K. (1000) Nucleic Acids Res. 18. 4247. 8 Kubota, H., Morita, T.. Nagata, T., Takemoto. Y., Nozaki, M.. Gachelin, G. and Matsushiro, A, (1991) Gene, in press. 9 U,'sic, D. and Ganetzky, B. (1988) Gene 68, 267-274. 10 Ahmad, S. and Gupta, R.S. (1990) Biochim. Biophys. Acta I()M7, 253-255. II Gupta, R.S. (190()) Biochem. Int. 2(I, 833--841. 12 Ursic, I). and Culhertson, M. (1901) Mol. ('ell. Biol. II, 2¢'~20264O. 13 Andersstm, L. (1988) J. Ileredily 70, I-5. 14 Levan, G.. Klinga, K., Szpircr, C. and Szpircr. J. (199()) In: Genetic Maps. (O'Bricn, S.J. cd.). pp. 4.8(~-4.87. ('old Spring ilarhor Lahoratol~ Press, ('old Spring Ilarhor. U.S.A.
BBAEXP 90282
Bioch#nica et Biophysica Acre, ! 129 (1991) 96-99 © 1991 Elsevier Science Publishers B.V. All rights reserved 0167-478! / 9 !/$03.50
Short Sequence-Paper
Cloning of cDNA encoding rat TCP-1 T a k a s h i M o r i t a i, H i r o s h i K u b o t a i, G a b r i e l G a c h e l i n 2, M a s a m i Nozaki i a n d Aizo M a t s u s h i r o t Department of Microbial Genetics, Research Institute j'or Microbial Diseases, Osaka University, Osaka (Japan) and 2 Unlit; de Biologic Moleculaire ~ha Gent; hlstitut Pastern; Paris (France)
(Received 16 September 1991)
K~:ywords: Mouse T-complex: Molecular chaperone: (Ratttas suwvegicus )
We have isolated and sequenched a cDNA encoding a rat homolog of the mouse t-complex polypeptide 1 (TCP-I). Its deduced gene product is a polypeptide of 556 amino acids, with a predicted M r of 60341. The similarity between mouse Tcp.i and the rat homolog is about 94.0% at the nucleotide level and 97.1% at the amino acid level showing the evolutionary conservation of this protein. The similarity of the amino acid sequence of the rat TCP-I is not significantly biased to any of those from wild (TCP-IB) or from t-haplotype mice (TCP-IA). From a comparison of deduced amino acid sequences of eukaryotic TCP-I proteins, we found highly conserved domains. Southern blot analysis revealed that there are at least two similar sequences to Tcp.i in the rat, one is a structural gene and the other seems to be a processed pseudogene.
The mouse t-complex is a tertiary inverted region of chromosome 17 including sets of dominant and recessive mutations, which may cause defects in embryonic development, sperm production and function [1-3]. The t-hapiotype mice are wide spread by their selective advantage of high transmission ratio distortion. Mouse TCP-I is encoded in the t-complex and is suited to the analysis of the evolutionary origin of the t-haplotype, because it exists as two electrophoretic forms, TCP-1B and TCP-IA; TCP-IA is encoded by all complete t-
The sequence data in this paper have been submitted to the EMBL/Genbank under the accession number D90345. Correspondence: T. Morita, Research Institute for Microbial Diseases, Osaka University. 3-1, Yamadaoka, Suita, Osaka, Japan.
haplotype chromosomes and TCP-IB by all inbred strains tested [4]. Willison et al. have isolated their cDNAs [5] as well as human homolog [6,7]. The human sequence has some of the features of the mouse, which are characteristic of the t-haplotype (TCP-IA) rather than TCP-IB. Therefore, it is proposed that Tcp-l" is the ancestral type and that the mouse t-chromosome is the 'wild type' [6]. We analysed the rat Tcp-I sequence to examine whether it has homology to mouse TCP-IA. We also compared the amino acid sequence of TCP-I from different species and predicted the secondary structure to understand the role of the polypeptide. We extracted RNA from Fischer rat testes (Rattus norvegicus) and used it to synthesize cDNA. The cDNA was cloned into the lambda gtl0 phage vector. Recombinant phages (8.10 4) from the library were screened with a mouse Tcp.l cDNA probe [8]. Thirteen positive clones were further screened using a synthetic oligonucleotide which corresponds to the 5' leader sequence of mouse Tcp-I cDNA. A phage clone which gave a strong positive signal by autoradiography was purified.
97 The DNA insert (1.9 kb) was subcloned into the plasmid vector pUC8 and characterized by nucleotide sequencing. The nucleotide sequence of rat Tcp-1 eDNA is shown in Fig. 1. The 1831 bp cDNA contained an ORF that codes for a polypeptide of 556 amino acid with a M r of 60341. The initiation codon at nucleotides, 1-3 was located in GAAGATG which is highly homologous with other Tcp-I genes, i.e. human, Drosophila and mouse [5,7,9]. At nucleotide position 1669-1671, a translation stop signal was found and at nucleotide 1731-1736, the possible polyadenylation signal, ATTAAA. At 17 nucleotides down stream from the signal, apoly A tail was attached. The similarity of the nucleotide sequences between rat and human is 89.8% and that between rat and hamster, 91.5% [10]. The similarity between rat and wild types mouse is 94.0% and between rat and thaplotype mouse is 93.8%, which confirms the close relationship between rat and mouse species. However, the homology of amino acid sequence between rat and human is higher (97.7%) than those between rat and t-haplotype mouse (97.1%) and wild mouse (97.5%). This indicates that the substitution of amino acids in the mouse Tcp-I is rapid [8]. At the positions where nucleotides differ between wild and t-haplotype mouse, the rat amino acid sequence resembles the wild type at 5 positions (amino acid positions, 148, 157, 200, 336, 551 in Fig. 2) and the t-haplotype at 5 positions (25, 45, 306, 415, 439), suggesting a random resemblance. We therefore speculate that each of the Tcp-I genes encoding TCP-1A and TCP-1B evolved rapidly from their common ancestor. TCP-1 has homology with the 'Chaperonin' family of heat-shock proteins [10,11], which assists in the
-77 I 76 1 51 226 301 376 451 526 601 676 751 826 901 976 1051 1126 1201 12"/6 1351 1426 1501 1 576 1651 1741
ATG GAG GGT CCT TTG GCC TCT ATT GCC AAC G&T GTA ACT ATT ACT T G T G R A CTG G C T G A C CTG AAA AAT GCT GAT RAG GAA GCA GTG CGA G C T G C T A A G ACA T C C GTG CTT GCT GTT AAA C A C G G G A G A A G T CAG C T C ItAG A G A A T A G T T CA(; GTG GTT ATT ACA CAG /tAG AT(: CTG GCA GTG GAA GCT GGT GCC AGT AT(: CTG TCT ACA GTC GTA CAG GAG AGG AT(: TTA CGA GGG GCA AGA GTT TTG GAG TCA TAT GCG ACC AGT &TG ACA CTG GCA GTG RAT GTT AAC (:CA GAA CGT GCG GGG GTG TTT GAA CGG ATT GAT GAT CTG TCT GGA GCC CTT G&T AACGAGCTGTCG ( A ) n
Fig. 1. Nucleotide sequence of the rat
proper folding of proteins and their assembly into oligomeric protein complexes, in order to determine the domain structure, we compared the amino acid sequence between the homologs of TCP-1 from rat, Drosophila and yeast [9,12]. Though the species were much diverged, the amino acid sequences were conserved among them. The region of perfect amino acid matches was distributed throughout the polypeptide (Fig. 2). The homology at the N-terminal (40-204 in Fig. 2) is extraordinary and the C-terminal (368-508) also has a highly conserved long sequence. These regions might be involved in oligomeric formation or in its function as a molecular 'chaperon'. The genomic DNA from Fischer rat liver was digested with the restriction enzyme Tagl and analysed by Southern blotting (Fig. 3). When mouse Tcp-I eDNA was used as the probe, we detected one faint (9 kb) and one intense band of 2.4 kb. The 9 kb band is monomorphic but the 2.4 kb band is polymorphic depending on the rat strain. We hybridized the same blot with a labeled PCR product (400 bp), corresponding to mouse intron 8 (our unpublished results) and found that the 2.4 kb band hybridized with it. However, the longer (9 kb) band did not. Thus the rat genome would have at least two Tcp-I sequences; one would be a structural gene and the other might be a processed pseudogene without an intron. The mouse t-complex consists of a large chromosomal segment linked to the major histocompatibility complex (MHC). The human Tcp-! gene and MHC genes are also on the same chromosome, although the locations are different [6]. Furthermore, the TCP-IB and MHC genes of cattle were linked with each other on bovine chromosome 23 [13]. However, the two bands of the rat T~7~-Iare not assigned on chromosome 20 (Mori et al. personal com-
GGCCGCTATCOGGTCTCAGAGCGGA(:CTATCGCTTAGTTGTCTGCGTTGGTAGTGAGATTTGCTTCGTTTCCTGAAG T C C G T G T T C G G G G A C C G C A G C A C T G G G G A A G C G A T C CGC TCC C A G A A C G T T ATG G C T GCA A T T G T T A R A A G T TL"I' C T T G G G C C A G T T G G C TTG GAT AAA ATG TTG GTG GAT GAT ATT GGT R A T G A T G G G G C C A C C A T T CTG A A A T T A T T G G A G GTG GAA CAT CCT GCA GCC AAA GTT CTG C T G CAP, G A C A R A G A A G T T G G A G A T G G A A C T A C C TCA GTG GTA ATC ATT GCA GL"G GAG CTT G A G C T A G T C A A G CAG A A A A T T C A T C C G A C A T C A GTT ATT AGT GGC TAT CGT CTT GCC TGC T A T A T C A A T G A G A A C C T A A T T A T C A A C A C A G A C GAA CTT GGA AGA GAC TGT CTG ATC hAT A T G T C T T C C A A A A T T A T T G G A A T A A A T G G T G A T TTC TTC GCT AAT AT(; GTG GTA GAC GCT T A C A C A G A T A T A CGA G G C CA(; C C T C G C T A T C C A GTC AAT TCT GTT AAT ATT CTA AAA GCC A T A G A A A G C AT(; C T G A T C A A T G G C T A T G C A C T C .%AC TGT GTG GTT GGA TCT CAG GGC ATG A A T G C A A A A A T T G C T T G T C T T G A C T T C A G C C T G CAG AAA ACA AAA AT(; AAG CTT GGT GTA GAC CCT GAG AAA CTG GAC CAG ATT AGA CA(; AGA GAA TCC GAT ATC ACC AAG GAG AGA ATT ACT GGT GCC AAC GTT ATT CTA ACC ACC GGT GGG ATT GAT GAC AT(; TGT CTC; AAG TAT TTT AT(; GCT GTT At..;(; AGA GTT TTA AAA AGA GAC CTC AAA CGT ATT GCA AAA GCT TCT GG~ GCA CTG GCC AAT TTG GAA GGT GAA GAG ACT TTT GAG GCA ACA AT(; TT(; GGA CAA GCG GAA GAG AFT TGT GAC G&T GAG CTG ATC TTA ATC AAA AAT ACT AAG GCT CGT ACG TCA GCT TCA ATT AAC GAC TTC ATG TGT GAT GAA ATG GAA CGC TCC TTA CAT GAC GCT CTT TGT GTG GTG AAG AAG TCT GTG GTC CCA GGT GGA GGT GCT GTA G/t,A GCG GCC TTG TCT ATA TAC CTT GAA AAC GGA TCT CGG GAA CAG CTT GCT ATT GCA GAG TTT GCA AGA TCT CT(; CTT GTG ATT CCT AAT GCT GCC CRA GAC TCC ACT GAC ~ GTT GCC AAG TTA AGA GCT TJl~ CAC RAT GAG GCT CAA AAA AAT CTA RAG TGG ATT GGT ~ GAT TTG GTC CAT GGG AAA CCA CGA GAC AAT AAG CAA CCA ACC ATA GTT AAA GTA AAG AGC ~ RAG TTT GCA ACA GAG GCT GCA ATC ACC AT,;~ CTT ATA AAA TTA CAC CCA GAA AGC AAA GAC GAC AAA CAC GGA GGT TAT GAA AAT GCT GTT CAC GAC TGA TTGGACTTCCCTTTTATTTATGACAGTGTTGGGTGTRATGTCTTAGCCTTGGGTGTCTCAC TT~--T-~.~GTAC
Tcp-! cDNA. The polyadenylation signal is underlined. The numbers on the righl refer to the nucleotidc sequence beginning from the adenine residue in the start codon.
98 of Medicine, Kyoto University) for gifts of rat DN, and unpublished results. This work was supported by Grant-in-Aid for Scientific Research (No. 0161850~ from the Ministry of Education, Science and Culture Japan.
munication) on which the rat MHC resides [14]. Thus, in the rat, the Tcp-I gene is not linked to the MHC locus. We thank Drs. J. Yamada, T. Serikawa, T. Yamada and M. Mori (Institute of Laboratory Animals. Faculty
75
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I HPTSVI $GYRLACKEAVRYI NENL I I:~DDE I HPTSVI SGYRLACKEAVRYI NE~L I I E I H P T S V I $ G Y R L A C K E A V R Y I NEI~L I V N T D E
I1 L A~:'~ Y T O I A ~ V ~ S I K V~\~K~A S~l T l) r R~O'~"A L A~...T O N S K~E I K~P V K A ~ : ~ V i ~ ! K
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~. . . . ~ ,, ,~ ~, ..~,.~ ~,~ ,~.a ~ S~,T K~,~K L ~ V V ~ T ~ E K~ l) O ~ O ~ E ~ S l)~,-T K ~ I 0 K!)~L A T ~ A N~ I ~ T T G 3 0 0 ."" ~''~ K~KM~.~,,~t~'VL~|~N~OK!,~EA~A~iLI)~t:~TK~I NM~:i~LGT~VN~Vi~VSG R~AM~ I N~l)~:l~E O~E O ~ - K ~ A G~:|~VL ~:~V K K ~ l l) A ~ A O ~ V ~ T T K
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TI) P E K L O 0 1 R O R E S I ) I T K E R IOKI LATGANVI PEKLI)QIRQRESDITKER IQKI LATGANVI TI) P E K L I ) O I R O R E S D I T K E R I Q K I L A T G A N V I
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Fig. 2. Comparison of the amino acid sequences of TCP-I homologs. The rat TCP-! sequence (RA) was compared with those from Drosophila (DS), yeast (YS), wild type mouse (MW), t"-"-haplotype mouse (MT) and human (HU). Dashes denote gaps in the sequence. The common amino acids among rat, Drosophila and yeast are shaded. The different amino acids between wild type mouse and I '~32 haplotype mouse are boxed. The arbitrary numbers on the right refer to the amino acid sequence of TCP-! proteins. The arrows indicate the highly conserved regions.
99
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References
5 6
kb 4
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Fig. 3. Southern Idol analysis (ff rat Tq~-! gene. Ten micrograms of genomic DNAs from Fischer rat (lane I and 4), SHR rat (lane 3 and 6) and the rat cell line ACI (lane 2 and 5), were digested with Ta,ql and elcctrophoresed oil a (),6";i agarose gel. Tile DNAs were hlotled onto nitrocellulose filters, then hybridized with a mouse eDNA probe (a) and an intron probe (b). The size markers are sh~wn at the right.
I Bennett, D. (1975) (.'ell h, 441-454. 2 Silver, L.M. (1985) Annu. Roy. Genet. 19, 179-2()8. 3 ltammer, M.F., Schimenti, J. and Silver, L.M. (1080) Proc. Natl. Acad. Sci. USA 86, 3261-3265. 4 Silver, L.M., Artzt, K. and Bennett, K. (1970)(.:'ell 17, 275-284. 5 Willison, K.R., Dudley, K. and Potter, J. (1986) Cell 44, 727-738. 6 Willison, K., Kelly, A., Dudley, K., G~odfcllow. K., Spurt, N., Groves, V., Gorman. P., Sheer, D. and Trowsdale, J. (1987) EMBO J. h, 1967-1974. 7 Kirchhoff, C. and Willison, K. (1000) Nucleic Acids Res. 18. 4247. 8 Kubota, H., Morita, T.. Nagata, T., Takemoto. Y., Nozaki, M.. Gachelin, G. and Matsushiro, A, (1991) Gene, in press. 9 U,'sic, D. and Ganetzky, B. (1988) Gene 68, 267-274. 10 Ahmad, S. and Gupta, R.S. (1990) Biochim. Biophys. Acta I()M7, 253-255. II Gupta, R.S. (190()) Biochem. Int. 2(I, 833--841. 12 Ursic, I). and Culhertson, M. (1901) Mol. ('ell. Biol. II, 2¢'~20264O. 13 Andersstm, L. (1988) J. Ileredily 70, I-5. 14 Levan, G.. Klinga, K., Szpircr, C. and Szpircr. J. (199()) In: Genetic Maps. (O'Bricn, S.J. cd.). pp. 4.8(~-4.87. ('old Spring ilarhor Lahoratol~ Press, ('old Spring Ilarhor. U.S.A.
