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Cloning of a human cDNA encoding a protein with high homology to yeast methionyl-tRNA synthetase
GENE AN INTERNATIONAL J O U R N A L GENES AND GENOMES
ELSEVIER
ON
Gene 178 (1996)187-189
Short communication
Cloning of a human cDNA encoding a protein with high homology to yeast methionyl-tRNA synthetase 1 Hermann Lage *, Manfred Dietel Institute of Pathology, CharitY, Humboldt University Berlin, Schumannstrasse 20/21, D-IOI 17 Berlin, Germany Received 23 January 1996; revised 10 April 1996; accepted 12 April 1996
Abstract
A composite 2779-bp cDNA that encodes human cytoplasmic methionyl-tRNA synthetase (MetRS) has been constructed from partial cDNA clones derived from the gastric carcinoma cell line EPG85-257RNOV and the nucleotide sequence has been determined. The open reading frame (ORF) encodes a 900 amino acid (aa) protein with a predicted molecular mass of 101 kDa. Northern blotting analysis of total RNA extracted from human gastric carcinoma cells demonstrated a single band with a mobility corresponding to a size of 3.0 kb. Keywords: EPG85-257RNOV; RACE; MXR9; Gastric carcinoma; ~. library screening
Methionyl-tRNA synthetase (MetRS) catalyses the ligation of methionine (Met) to transfer RNA in protein translation. Hence the availability of MetRS is essential for protein synthesis in cells of all species. Several MetRS genes have been cloned from procaryotes, e.g. Escherichia coli (Dardel et al., 1984). In eucaryotic cells, the yeast cytoplasmic MetRS gene (Fasiolo et al., 1981) and the yeast mitochondrial MetRS gene (Tzagoloff et al., 1989) have been cloned. A human 1506-bp cDNA clone ( M X R 9 ) with high homology to the yeast cytoplasmic MetRS gene was obtained by screening a ~. cDNA library prepared from the human gastric carcinoma cell line EPG85-257RNOV, which was established in our laboratory (Dietel et al., 1990). The procedure of cDNA synthesis, cloning into ~-vector and library screening was described previously (Lage and Dietel, 1995). The determined nt sequence ( E M B L accession No. Z49216) shows 61.8% of identity to the yeast cytoplasmic MetRS gene in a 641-bp segment by using the FASTA program * Corresponding author. Tel.:(+49-30) 2802-3167/2261;Fax: (+49-30) 2802-3407;e-mail: [email protected] 1On request, the authors will supply detailed experimental evidence for the conclusion reached in this Short communication. Abbreviations: aa, amino acid(s); bp, base pair(s); kb, kilobase(s) or 1000 bp; kDa, kilodalton; MetRS, methionyl-tRNA synthetase; nt, nucleotide(s); ORF, open reading frame; RACE, rapid amplificationof cDNA ends. 0378-1119/96/$15.00© 1996ElsevierScienceB.V. All rights reserved PII S0378-1119(96)00313-7
(Pearson and Lipman, 1988). Northern blotting using the human cell line EPG85-257RNOV revealed that the cDNA clone M X R 9 hybridized to a single transcript. The size of this transcript was estimated to be 3.0 kb. This analysis showed that M X R 9 was not a full-length clone. To obtain the complete sequence a Y-RACE was performed using cDNA prepared from the human gastric carcinoma cell line EPG85-257RNOV. The sequence of the used Y-RACE primer was 5'-GCTTAGGCAGGTCCAGAAACAG-3'. The resulting 1322-bp cDNA clone ( M X R 9 R A C E ) contains the start of transcription and a 7-bp Y-untranslated region. Northern analysis revealed that M X R 9 R A C E hybridized to a single 3.0-kb transcript like the original cDNA clone M X R 9 . Using the FASTA program reveals that the composite 2779-bp human MetRS cDNA (Fig. 1) shows 60.6% identity in a conserved region of 1230bp. This region is located between human nt 765 and nt 1994 corresponding to aa 253 and aa 662 of the protein sequence. The open reading frame (ORF) begins from an ATG at nucleotide 8 and ends with a TAA ochre codon for translation termination at nt 2707. The protein deduced from the MetRS cDNA would consist of 900 aa with a predicted molecular mass of 101 kDa and a pI of 5.73. The predicted protein sequence shows an extensive homology to a highly conserved 403 aa containing region in the middle of the yeast molecule. This region between aa 192 and aa 594 represents the putative nucleotide-binding domain in the yeast
H. Lage, M. Dietel/Gene 178 (1996) 187-189
188
C G G C G A A A T G A G A C T G T T C G T G A G T G A T ~ I'GTcc,-G~SGTT G'-TTGCCGGTGCT G G C CGC C G C C G G G A G A G C C C ~ C A G A G CAGAGC~TGCT CAT C A G C A C T G T A G G C C C G G A A G A T T G 120 M R L F V S K G V P G C L P V L A A A G ?, A R G R A E V L I S T V G P E D C 38 TGTGGTC<~CGTT CCTGACCCGGCCTAAGGTCC'CT,STCT T G C A S G T G G A T A G C G G C A A C T A C C T CT TCT 7 C A C T A G T G C A A T C T G C C G A T A T T T T T TTT TGT T A T C T G G C T G G G A G C A A G A 240 V V P F L T Z P F V P V L Q V D S G N Y L F S T S A I C R Y F F L L S G W E Q D 78 T G A C C T C A C T A A C C A G T G G C T G G A A T G G G A A G ~ G A C A G A 3 c T G C A G C C A G C T T T G T C T G C T C C C C T G T A C T A T T T A G T G G T C C A A G G C A A G A A G G G G G A A G A T GT T C T T G G T T C A G T G C G 360 D L T N Q W L E W E A T E L Q P A L S A P L Y Y L V V Q G K K G E D V L G S V R [18 G A G A G C C C T G A C T C A C A T T G A C C A C A G - T T G A G T CGT C A G A A C T G T CCTT T C C T G G C T G G G G A G A C A G A A T CT C T A G C C G A C A T T GT T T T GT G G G G A G C C CAAT A C C C A T T A C T G C A A G A 480 R A L T H [ D H S L S R Q N C P F L A C7 E T E S L A D I V L W G A Q Y P L L Q D ].58 TCCCGCCTACCT CCCTGAGGAGCTGAGTGCCCTGCACAGCTGGT TCCAGACACT 3AGTACCCAGGAACCATGTCAGCGAGCTGCAGAGACTGTACTGAAACAGCAAGGTGTCCTGGCTCT 600 P A Y L P E E L S A L H S W F Q T L S T Q E P C Q R A A E T V L K Q Q G V L A L 198 C C G G C C T T A C C T C C A A A A G C A G C C C CAG C'-CAGCr"? CG C T G A G G G A A G G G C TOT CAC C A A T G A G C C T G A G G A G G A G G A G C T G G C T AC CCT AT C T G A G G A G G A G A T T G C T AT G G C T GT T A C 720 R P Y L Q K Q P k P S P A E O R A V T N E P E E E E L A T L S E E E I A M A V T 238 T G C T T G G G A G A A G G G C C T A G A A A G T T T,3CP ?'?C G C T G C G G C C C C A G C A G A A T C C A G T G T T G C C T G T G G C T G G A G A A A G G A A T G T G C T CAT C A C C A G T G C C C T CCCT T A C G T C A A C A A T G T 840 A W E K G L E S L P P L Z P Q Q N P V L P V A G E R N V L I T S A L P Y V N N V 278 C C C C C A C C T T G G G A A C A T C A T T G G T T G T G T G C T C A G T G C C G A T G T CT T T G C C A G G T A C T C T C G C C T C C G C C A G T G G A A C A C C C T C TAT CT GT GT G G G A C A G A T G A G T AT G G T A C A G C A A C 960 P H L G N I I G c V L S A D V F A R Y S P, L R Q W N T L Y L C G T D E Y G T A T 318 A S A G A C C A A G G C T CT G G A G G A G G G A C T A A C C C CC C A G G A G A T CTGCGAC;~hGTACCACATCAT C C A T G C T G A C A T C T A C C G C T G G T T T A A C A T T T CGT T T G A T A T T T T T G G T C G C A C C A C 1080 E T K A L E E G L T E Q E I C D K Y H I I H A D I Y Z W F N I S F D I F G R T T 358 C A C T C C A C A G C A G A C C A A A A T C A C C C A G G A C A T T T T C ?AGCAGT T G C T G A A A C G A G G T T T T G T G C T G C A A G A T A C T G T G G A G C A A C T G C G A T G T G A G C A C T G T G C T C G C T T C C T G G C T G A 120 T P Q Q T K i T W D [ F Q Q L L K R G F V L Q D T V E Q L R C E H C A R F L A D 398 C CGCT T C G T G G A G G G C G T GT G T C C C T T C T GT C~SCT AT 3AGGA~93 C T C G G G G T G A C C A G TG T G A C A A G T G T G G C A A G C T CAT CAAT G C T G T C G A G C T T A A G A A G C CT C A G T G T A A A G T C T G 1320 R F V E G V C P F C G Y E E A R O D Q C D K C G K L I N A V E L K K P Q C K V C 438 C C G A T C A T G C C C T G T GGT G C A G T C G A G C C A G C A C C T G T T T C T G G A C C T &C C T A A G C T G G A G A A G C G A C T G G A G G A G T G G T T G G G G A G G A C A T T GC C T G G C A G T G A C T G G A C A C C CAAT G C 1440 R S C P V V Q S S Q H L F L D L P K L E K R L E E W L G R T L P G S D W T P N A 478 C C A G T TTAT C A C C C G T T C T T G G C T T C G G G A T G G C C T C A A G C C A C G C T G C A T A A C C C G A G A C C T C A A A T G G G G A A C C C C T G T A C C C T T A G A A G G T ~ T T G A A G A C A A G G T A T T C T A T G T CTG 1560 Q F I T R S W L R D O L K P R C I T R D L K W G T P V P L E G F E D K V F Y V W 518 G T T T G A T G C C A C T AT T G G C T A T C T GT ? SAT C A C A G C C A A C T A C A C A G A C C A G T GJA~AGAGAT GGT G G A A G A A C C C A G A G C A A G T G G A C C T G T A T C A G T T CAT GGC C A A A G A C A A T GT T C C 1680 F D A T I G Y L S I T A N Y T D Q W E R W W K N P E Q V D L Y Q E M A Z D N V P 558 T T T C C A T A G C T T A G T C T T T CCT T GC T C A G C C CT A G G A G C T,3AC~SATAACT A T A C C T T GGT CAG C C A C C T CAT T GC T A C A G A G T A C C T G A A C T A T G A G G A T G G G A A A T T C T C T A A G A G C CG 1800 F H S L V F P C S A L G A E D N Y T L V S H L I A T E Y L N Y E D G K F S K S R 598 CGGT G T G G G A G T G T T T G G G G A C A T G G C C C A G G A C A C G G G G A T C C C T G C T O A C A T C T G G C G C T T C TAT C T G C T G T A C A T T C G G C C T G A G G G C C A G G A C A G T GCT T T C T C C T G G A C G G A C C T 19212 G V G V F G D M A Q D T G I P A D I W R F Y L L Y I R P E G Q D S A F S W T D L 638 G C T G C T G A A G A A T A A T T C T G A G C T G C T T A A C A A C C T G G G C A A C T T CAT C A A C A G A G C T G G G A T G T T T G T G T C T A A G T T CT T T G G G G G C T A T G T G C C T G A G A T G G T G C T C A C C C C T G A T G A !048 L L K N N S E L L N N L G N F I N F A G M F V S K F F G G Y V P E M V L T P D D 678 TCAGCGCCTGCTGGGCCATGT CACCCTGGAG6 TCCAGCACTAT CACCAGCTACT TGAGAAGGT TCGGAT CCGGGATGCCTTGCGCAGTATCCTCACCATATCT CGACATGGCAACCAATA 2160 Q R L L G H V T L E L Q H Y H Q L L E W V R I R D A L R S I L T I S R H G N Q Y 718 TAT T C A G G T G A A T G A G C C CT G G A A G C G G A T TAAAGGCAGT,3AC~SC T G A C A G G C A A C G G G C A G G A A C A G T G A C T G G C T T GG C A G T G A A T A T A G C T G C C T TGCT C T C T G T C A T G C T T C A G C C 2280 I Q V N E P W K R i F O S E A D R Q R A G T V T G L A V N I A A L L S V M L Q P 758 T T A C A T G C C C A C G G T T A G T G C C A C A A T ,['CASGCCCAGCTGCAGCT C C C A C C T C C A G C C T G C A G T AT CC T GC T G A C A A A C T T C C T G T G T A C C T T A C C A G C A G G A C A C C A G A T T G G C A C A G T 2400 Y M P T V S A T I Q A Q L Q L P P F A C S I L L T N F L C T L P A G H Q i O T V 798 CAG T C C C T TGT T C C A A A A A T T G G A A A A T G A C C A G A T T G A A A G T T T A A G G C A G C G C T T T G G A G G G G G C C A G G C A A A A A C G T C C C C G A A G C C A G CAGT T GT A G A G A C T G T T A C A A C A G C C A A 252¢ S P L F Q K L E N D ~ I E S L R Q P F G G G Q A K T S P K P A V V E T V T T A k" 838 G C C A C A G C A G A T A C A A G C G C T GAT G G A T G A A G T GAC2uAAACAAGGAAACAT T GT " ' C G A G A A C T G A A A G C A C A A A A G G C A G A C A A G A A C G A G G T T G C T G C G G A G G T G G C G A A A C T CT T G G A 2648 P Q Q I Q A L M D E V T E Q G N I V R E L K A Q K A D K N E V A A E V A K L L D 878 T C T A A A G A A A C A G T T G G C T G TAGCT G A G G G G A A A C C C C CT O A A G C C CC T A A A G G C A A G A A G A A A A A G T A A A A G A C C T T G G C T C A T A G A A A G T CACT T T A A T A G A T A G G G A C A G T A A T A A A 276'3 90 0 L K K Q L A V A E O ~ P I: E A P bf G K Y K K 2"=79 T A A A T G T A C A A T CT C T A T A
Fig. 1. Nucleotide sequence of the human cytoplasmic methionyl-tRNA synthetase c D N A obtained from clones MXR9 and MXR9RACE and deduced amino acid sequence. The sequence obtained from clone MXR9 is underlined. This sequence begins at nt 1274 and ends at nt 2779. The sequence used for the construction of a 5'-RACE primer is double underlined. This sequence begins at nt 1345 and ends at nt 1366. The sequence obtained from clone MXR9RACE is not indicated. This sequence begins at nt 1 and ends at nt 1344. There are two overlapping polyadenylation signal sequences which are double underlined, too. The first one begins at nt 2755 and ends at nt 2760. The second one begins at nt 2759 and ends at nt 2764. The complete sequence has been submitted to the EMBL database and will appear under accession No. X94754.
261 AGERNVLITSALPYVNNVPHLGNIIGCVLSADVFARYSRLRQWNTLYLCGTDEYGTATETKALEEGLTPQ 192 PNERNILITSALPYVNNVPHLGNIIGSVLSADIFARYCKGRNYNALFICGTDEYGTATETKALEEGVTPR
(human) (yeast)
331 EICDKYHIIHADIYRWFNISFDIFGRTTTPQQTKITQDIFQQLLKRGFVLQDTVEQLRCEHCARFLADRF 262 QLCDKXHKIHSDVYKWFQIGFDYFGRTTTDKQTEIAQHIFTKLNSNGYLEEQSMKQLYCPVHNSYLADRY
(human) (yeast)
401 VEGVCPFCGYEEARGDQCDKCGKLINAVELKKPQCKVCRSCPVVQSSQHLFLDLPKLEKRLEEWLGRTLP 332 VEGECPKCHYDDARGDQCDKCGALLDPFELINPRCKLDDASPEPKYSDHIFLSLDKLESQISEWVEKASE
(human) (yeast)
471 GSDWTPNAQFITRSWLRDGLKPRCITRDLKWGTPVPLEGFEDKVFYVWFDATIGYLSITANYTDQWERWW 402 EGNWSKNSKTITQSWLKDGLKPRCITRDLVWGTPVPLEKYKDKVLYVWFDATIGYVSITSNYTKEW]
(human) (yeast)
541 KNPEQVDLYQFMAKDNVPFHSLVFPCSALGAEDNYTLVSHLIATEYLNYEDGKFSKSRGVGVFGDMAQDT
(human)
472
(yeast)
NNPEHVSLYQFMGKDNVPFHTWFPGSQLGTEENWTMLHHLNTTEYLQYENGKFSKSRGVGVFGNNAQDS
611 GIPADIWRFYLLYIRPEGQDSAFSWTDLLLKNNSELLNNLGNFINKAGMFVSK 542 GISPSVWRYYLASVRPESSDSHFSWDDEVARNNSELLANLGNFVNRLIKFVNA
(human) (yeast)
Fig. 2. Similarities of putative nucleotide-binding domains of yeast cytoplasmic and human cytoplasmic MetRS aa sequences. Residues that are identical in both sequences are indicated by asterisks while dots indicate conservative aa changes. For clarity, the human sequence is limited to residues 261-663 and the yeast sequence to residues 192 594.
H. Lage, h/L Dietel/Gene 178 (1996) 187-189
molecule (Walter et al., 1983). The homology to the putative human nucleotide-binding region between residues 261 and 663 includes 62.5% of aa identities and 25.3% of conservative replacements (Fig. 2). The overall identity of the human and yeast molecule is 33.4%. Furthermore there are well-established functional consensus sequences which are present in the human molecule. For example the 332KMSKS336 E. coli sequence within the nucleotide binding domain of MetRS is proposed to be a signature sequence for a class of aminoacyltRNA synthetases specific to the aliphatic hydrophobic class of amino acids (Meinnel et al., 1990). This pattern is present in the cytoplasmic yeast molecule (525KFSKS 529) and the cytoplasmic human molecule (594KFSKS598). Outside the nucleotide-binding domain the 53°LYNRIDS35 E. coli sequence which is proposed to be part of the specificity of the MetRS by guiding the Y-acceptor stem of tRNA towards the active center (Meinnel et al., 1990) is homologous to the 73°LFQKID735 yeast sequence and the 8°2LFQRID 8°7 human sequence. These data suggest that the well known functional sites of yeast and human MetRS are evolutionarily conserved at a high degree.
189
References Dardel, F., Fayat, G. and Blanquet, S. (1984) Molecular cloning and primary structure of the Escherichia coli methionyl-tRNA synthctase gene. J. Bacteriol. 160, 1115-1122. Dietel, M., Arps, H., Lage, H. and Niendorf, A. (1990) Membrane vesicle formation due to acquired mitoxantrone resistance in human gastric carcinoma cell line EPG85-257. Cancer Res. 50, 6100-6106. Fasiolo, F., Bonnet, J. and Lacroute, F. (1981) Cloning of the yeast methionyl-tRNA synthetase gene. J. Biol. Chem. 56, 2324-2328. Lage, H. and Dietel, M. (1995) Molecular cloning of several cDNAs of a novel overexpressed mitoxantrone resistance-associated gene in gastric carcinoma cells. Proc. Am. Ass. Cancer Res. 36, 323. Meinnel, T., Mechulam, Y., Dardel, F., Schmitter, J.M., Hountondji, C., Brunie, S., Dessen, P., Fayat, G. and Blanquet, S. (1990) Methionyl-tRNA synthetase from E. coli a review. Biochimie 72, 625-632. Pearson, W.R. and Lipman, D.J. (1988) Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444-2448. Tzagoloff, A., Vambutas, A. and Akai, A. (1989) Characterization of MSM1, the structural gene for yeast mitochondrial methionyl-tRNA synthetase. Eur. J. Biochem. 179, 365-371. Walter, P., Gangloff, J., Bonnet, J., Boulanger, Y., Ebel, J.P. and Fasiolo, F. (1983) Primary structure of the Saccharomyces cerevisiae gene for methionyl-tRNA synthetase. Proc. Natl. Acad. Sci. USA 80, 2437-2441.
ELSEVIER
ON
Gene 178 (1996)187-189
Short communication
Cloning of a human cDNA encoding a protein with high homology to yeast methionyl-tRNA synthetase 1 Hermann Lage *, Manfred Dietel Institute of Pathology, CharitY, Humboldt University Berlin, Schumannstrasse 20/21, D-IOI 17 Berlin, Germany Received 23 January 1996; revised 10 April 1996; accepted 12 April 1996
Abstract
A composite 2779-bp cDNA that encodes human cytoplasmic methionyl-tRNA synthetase (MetRS) has been constructed from partial cDNA clones derived from the gastric carcinoma cell line EPG85-257RNOV and the nucleotide sequence has been determined. The open reading frame (ORF) encodes a 900 amino acid (aa) protein with a predicted molecular mass of 101 kDa. Northern blotting analysis of total RNA extracted from human gastric carcinoma cells demonstrated a single band with a mobility corresponding to a size of 3.0 kb. Keywords: EPG85-257RNOV; RACE; MXR9; Gastric carcinoma; ~. library screening
Methionyl-tRNA synthetase (MetRS) catalyses the ligation of methionine (Met) to transfer RNA in protein translation. Hence the availability of MetRS is essential for protein synthesis in cells of all species. Several MetRS genes have been cloned from procaryotes, e.g. Escherichia coli (Dardel et al., 1984). In eucaryotic cells, the yeast cytoplasmic MetRS gene (Fasiolo et al., 1981) and the yeast mitochondrial MetRS gene (Tzagoloff et al., 1989) have been cloned. A human 1506-bp cDNA clone ( M X R 9 ) with high homology to the yeast cytoplasmic MetRS gene was obtained by screening a ~. cDNA library prepared from the human gastric carcinoma cell line EPG85-257RNOV, which was established in our laboratory (Dietel et al., 1990). The procedure of cDNA synthesis, cloning into ~-vector and library screening was described previously (Lage and Dietel, 1995). The determined nt sequence ( E M B L accession No. Z49216) shows 61.8% of identity to the yeast cytoplasmic MetRS gene in a 641-bp segment by using the FASTA program * Corresponding author. Tel.:(+49-30) 2802-3167/2261;Fax: (+49-30) 2802-3407;e-mail: [email protected] 1On request, the authors will supply detailed experimental evidence for the conclusion reached in this Short communication. Abbreviations: aa, amino acid(s); bp, base pair(s); kb, kilobase(s) or 1000 bp; kDa, kilodalton; MetRS, methionyl-tRNA synthetase; nt, nucleotide(s); ORF, open reading frame; RACE, rapid amplificationof cDNA ends. 0378-1119/96/$15.00© 1996ElsevierScienceB.V. All rights reserved PII S0378-1119(96)00313-7
(Pearson and Lipman, 1988). Northern blotting using the human cell line EPG85-257RNOV revealed that the cDNA clone M X R 9 hybridized to a single transcript. The size of this transcript was estimated to be 3.0 kb. This analysis showed that M X R 9 was not a full-length clone. To obtain the complete sequence a Y-RACE was performed using cDNA prepared from the human gastric carcinoma cell line EPG85-257RNOV. The sequence of the used Y-RACE primer was 5'-GCTTAGGCAGGTCCAGAAACAG-3'. The resulting 1322-bp cDNA clone ( M X R 9 R A C E ) contains the start of transcription and a 7-bp Y-untranslated region. Northern analysis revealed that M X R 9 R A C E hybridized to a single 3.0-kb transcript like the original cDNA clone M X R 9 . Using the FASTA program reveals that the composite 2779-bp human MetRS cDNA (Fig. 1) shows 60.6% identity in a conserved region of 1230bp. This region is located between human nt 765 and nt 1994 corresponding to aa 253 and aa 662 of the protein sequence. The open reading frame (ORF) begins from an ATG at nucleotide 8 and ends with a TAA ochre codon for translation termination at nt 2707. The protein deduced from the MetRS cDNA would consist of 900 aa with a predicted molecular mass of 101 kDa and a pI of 5.73. The predicted protein sequence shows an extensive homology to a highly conserved 403 aa containing region in the middle of the yeast molecule. This region between aa 192 and aa 594 represents the putative nucleotide-binding domain in the yeast
H. Lage, M. Dietel/Gene 178 (1996) 187-189
188
C G G C G A A A T G A G A C T G T T C G T G A G T G A T ~ I'GTcc,-G~SGTT G'-TTGCCGGTGCT G G C CGC C G C C G G G A G A G C C C ~ C A G A G CAGAGC~TGCT CAT C A G C A C T G T A G G C C C G G A A G A T T G 120 M R L F V S K G V P G C L P V L A A A G ?, A R G R A E V L I S T V G P E D C 38 TGTGGTC<~CGTT CCTGACCCGGCCTAAGGTCC'CT,STCT T G C A S G T G G A T A G C G G C A A C T A C C T CT TCT 7 C A C T A G T G C A A T C T G C C G A T A T T T T T TTT TGT T A T C T G G C T G G G A G C A A G A 240 V V P F L T Z P F V P V L Q V D S G N Y L F S T S A I C R Y F F L L S G W E Q D 78 T G A C C T C A C T A A C C A G T G G C T G G A A T G G G A A G ~ G A C A G A 3 c T G C A G C C A G C T T T G T C T G C T C C C C T G T A C T A T T T A G T G G T C C A A G G C A A G A A G G G G G A A G A T GT T C T T G G T T C A G T G C G 360 D L T N Q W L E W E A T E L Q P A L S A P L Y Y L V V Q G K K G E D V L G S V R [18 G A G A G C C C T G A C T C A C A T T G A C C A C A G - T T G A G T CGT C A G A A C T G T CCTT T C C T G G C T G G G G A G A C A G A A T CT C T A G C C G A C A T T GT T T T GT G G G G A G C C CAAT A C C C A T T A C T G C A A G A 480 R A L T H [ D H S L S R Q N C P F L A C7 E T E S L A D I V L W G A Q Y P L L Q D ].58 TCCCGCCTACCT CCCTGAGGAGCTGAGTGCCCTGCACAGCTGGT TCCAGACACT 3AGTACCCAGGAACCATGTCAGCGAGCTGCAGAGACTGTACTGAAACAGCAAGGTGTCCTGGCTCT 600 P A Y L P E E L S A L H S W F Q T L S T Q E P C Q R A A E T V L K Q Q G V L A L 198 C C G G C C T T A C C T C C A A A A G C A G C C C CAG C'-CAGCr"? CG C T G A G G G A A G G G C TOT CAC C A A T G A G C C T G A G G A G G A G G A G C T G G C T AC CCT AT C T G A G G A G G A G A T T G C T AT G G C T GT T A C 720 R P Y L Q K Q P k P S P A E O R A V T N E P E E E E L A T L S E E E I A M A V T 238 T G C T T G G G A G A A G G G C C T A G A A A G T T T,3CP ?'?C G C T G C G G C C C C A G C A G A A T C C A G T G T T G C C T G T G G C T G G A G A A A G G A A T G T G C T CAT C A C C A G T G C C C T CCCT T A C G T C A A C A A T G T 840 A W E K G L E S L P P L Z P Q Q N P V L P V A G E R N V L I T S A L P Y V N N V 278 C C C C C A C C T T G G G A A C A T C A T T G G T T G T G T G C T C A G T G C C G A T G T CT T T G C C A G G T A C T C T C G C C T C C G C C A G T G G A A C A C C C T C TAT CT GT GT G G G A C A G A T G A G T AT G G T A C A G C A A C 960 P H L G N I I G c V L S A D V F A R Y S P, L R Q W N T L Y L C G T D E Y G T A T 318 A S A G A C C A A G G C T CT G G A G G A G G G A C T A A C C C CC C A G G A G A T CTGCGAC;~hGTACCACATCAT C C A T G C T G A C A T C T A C C G C T G G T T T A A C A T T T CGT T T G A T A T T T T T G G T C G C A C C A C 1080 E T K A L E E G L T E Q E I C D K Y H I I H A D I Y Z W F N I S F D I F G R T T 358 C A C T C C A C A G C A G A C C A A A A T C A C C C A G G A C A T T T T C ?AGCAGT T G C T G A A A C G A G G T T T T G T G C T G C A A G A T A C T G T G G A G C A A C T G C G A T G T G A G C A C T G T G C T C G C T T C C T G G C T G A 120 T P Q Q T K i T W D [ F Q Q L L K R G F V L Q D T V E Q L R C E H C A R F L A D 398 C CGCT T C G T G G A G G G C G T GT G T C C C T T C T GT C~SCT AT 3AGGA~93 C T C G G G G T G A C C A G TG T G A C A A G T G T G G C A A G C T CAT CAAT G C T G T C G A G C T T A A G A A G C CT C A G T G T A A A G T C T G 1320 R F V E G V C P F C G Y E E A R O D Q C D K C G K L I N A V E L K K P Q C K V C 438 C C G A T C A T G C C C T G T GGT G C A G T C G A G C C A G C A C C T G T T T C T G G A C C T &C C T A A G C T G G A G A A G C G A C T G G A G G A G T G G T T G G G G A G G A C A T T GC C T G G C A G T G A C T G G A C A C C CAAT G C 1440 R S C P V V Q S S Q H L F L D L P K L E K R L E E W L G R T L P G S D W T P N A 478 C C A G T TTAT C A C C C G T T C T T G G C T T C G G G A T G G C C T C A A G C C A C G C T G C A T A A C C C G A G A C C T C A A A T G G G G A A C C C C T G T A C C C T T A G A A G G T ~ T T G A A G A C A A G G T A T T C T A T G T CTG 1560 Q F I T R S W L R D O L K P R C I T R D L K W G T P V P L E G F E D K V F Y V W 518 G T T T G A T G C C A C T AT T G G C T A T C T GT ? SAT C A C A G C C A A C T A C A C A G A C C A G T GJA~AGAGAT GGT G G A A G A A C C C A G A G C A A G T G G A C C T G T A T C A G T T CAT GGC C A A A G A C A A T GT T C C 1680 F D A T I G Y L S I T A N Y T D Q W E R W W K N P E Q V D L Y Q E M A Z D N V P 558 T T T C C A T A G C T T A G T C T T T CCT T GC T C A G C C CT A G G A G C T,3AC~SATAACT A T A C C T T GGT CAG C C A C C T CAT T GC T A C A G A G T A C C T G A A C T A T G A G G A T G G G A A A T T C T C T A A G A G C CG 1800 F H S L V F P C S A L G A E D N Y T L V S H L I A T E Y L N Y E D G K F S K S R 598 CGGT G T G G G A G T G T T T G G G G A C A T G G C C C A G G A C A C G G G G A T C C C T G C T O A C A T C T G G C G C T T C TAT C T G C T G T A C A T T C G G C C T G A G G G C C A G G A C A G T GCT T T C T C C T G G A C G G A C C T 19212 G V G V F G D M A Q D T G I P A D I W R F Y L L Y I R P E G Q D S A F S W T D L 638 G C T G C T G A A G A A T A A T T C T G A G C T G C T T A A C A A C C T G G G C A A C T T CAT C A A C A G A G C T G G G A T G T T T G T G T C T A A G T T CT T T G G G G G C T A T G T G C C T G A G A T G G T G C T C A C C C C T G A T G A !048 L L K N N S E L L N N L G N F I N F A G M F V S K F F G G Y V P E M V L T P D D 678 TCAGCGCCTGCTGGGCCATGT CACCCTGGAG6 TCCAGCACTAT CACCAGCTACT TGAGAAGGT TCGGAT CCGGGATGCCTTGCGCAGTATCCTCACCATATCT CGACATGGCAACCAATA 2160 Q R L L G H V T L E L Q H Y H Q L L E W V R I R D A L R S I L T I S R H G N Q Y 718 TAT T C A G G T G A A T G A G C C CT G G A A G C G G A T TAAAGGCAGT,3AC~SC T G A C A G G C A A C G G G C A G G A A C A G T G A C T G G C T T GG C A G T G A A T A T A G C T G C C T TGCT C T C T G T C A T G C T T C A G C C 2280 I Q V N E P W K R i F O S E A D R Q R A G T V T G L A V N I A A L L S V M L Q P 758 T T A C A T G C C C A C G G T T A G T G C C A C A A T ,['CASGCCCAGCTGCAGCT C C C A C C T C C A G C C T G C A G T AT CC T GC T G A C A A A C T T C C T G T G T A C C T T A C C A G C A G G A C A C C A G A T T G G C A C A G T 2400 Y M P T V S A T I Q A Q L Q L P P F A C S I L L T N F L C T L P A G H Q i O T V 798 CAG T C C C T TGT T C C A A A A A T T G G A A A A T G A C C A G A T T G A A A G T T T A A G G C A G C G C T T T G G A G G G G G C C A G G C A A A A A C G T C C C C G A A G C C A G CAGT T GT A G A G A C T G T T A C A A C A G C C A A 252¢ S P L F Q K L E N D ~ I E S L R Q P F G G G Q A K T S P K P A V V E T V T T A k" 838 G C C A C A G C A G A T A C A A G C G C T GAT G G A T G A A G T GAC2uAAACAAGGAAACAT T GT " ' C G A G A A C T G A A A G C A C A A A A G G C A G A C A A G A A C G A G G T T G C T G C G G A G G T G G C G A A A C T CT T G G A 2648 P Q Q I Q A L M D E V T E Q G N I V R E L K A Q K A D K N E V A A E V A K L L D 878 T C T A A A G A A A C A G T T G G C T G TAGCT G A G G G G A A A C C C C CT O A A G C C CC T A A A G G C A A G A A G A A A A A G T A A A A G A C C T T G G C T C A T A G A A A G T CACT T T A A T A G A T A G G G A C A G T A A T A A A 276'3 90 0 L K K Q L A V A E O ~ P I: E A P bf G K Y K K 2"=79 T A A A T G T A C A A T CT C T A T A
Fig. 1. Nucleotide sequence of the human cytoplasmic methionyl-tRNA synthetase c D N A obtained from clones MXR9 and MXR9RACE and deduced amino acid sequence. The sequence obtained from clone MXR9 is underlined. This sequence begins at nt 1274 and ends at nt 2779. The sequence used for the construction of a 5'-RACE primer is double underlined. This sequence begins at nt 1345 and ends at nt 1366. The sequence obtained from clone MXR9RACE is not indicated. This sequence begins at nt 1 and ends at nt 1344. There are two overlapping polyadenylation signal sequences which are double underlined, too. The first one begins at nt 2755 and ends at nt 2760. The second one begins at nt 2759 and ends at nt 2764. The complete sequence has been submitted to the EMBL database and will appear under accession No. X94754.
261 AGERNVLITSALPYVNNVPHLGNIIGCVLSADVFARYSRLRQWNTLYLCGTDEYGTATETKALEEGLTPQ 192 PNERNILITSALPYVNNVPHLGNIIGSVLSADIFARYCKGRNYNALFICGTDEYGTATETKALEEGVTPR
(human) (yeast)
331 EICDKYHIIHADIYRWFNISFDIFGRTTTPQQTKITQDIFQQLLKRGFVLQDTVEQLRCEHCARFLADRF 262 QLCDKXHKIHSDVYKWFQIGFDYFGRTTTDKQTEIAQHIFTKLNSNGYLEEQSMKQLYCPVHNSYLADRY
(human) (yeast)
401 VEGVCPFCGYEEARGDQCDKCGKLINAVELKKPQCKVCRSCPVVQSSQHLFLDLPKLEKRLEEWLGRTLP 332 VEGECPKCHYDDARGDQCDKCGALLDPFELINPRCKLDDASPEPKYSDHIFLSLDKLESQISEWVEKASE
(human) (yeast)
471 GSDWTPNAQFITRSWLRDGLKPRCITRDLKWGTPVPLEGFEDKVFYVWFDATIGYLSITANYTDQWERWW 402 EGNWSKNSKTITQSWLKDGLKPRCITRDLVWGTPVPLEKYKDKVLYVWFDATIGYVSITSNYTKEW]
(human) (yeast)
541 KNPEQVDLYQFMAKDNVPFHSLVFPCSALGAEDNYTLVSHLIATEYLNYEDGKFSKSRGVGVFGDMAQDT
(human)
472
(yeast)
NNPEHVSLYQFMGKDNVPFHTWFPGSQLGTEENWTMLHHLNTTEYLQYENGKFSKSRGVGVFGNNAQDS
611 GIPADIWRFYLLYIRPEGQDSAFSWTDLLLKNNSELLNNLGNFINKAGMFVSK 542 GISPSVWRYYLASVRPESSDSHFSWDDEVARNNSELLANLGNFVNRLIKFVNA
(human) (yeast)
Fig. 2. Similarities of putative nucleotide-binding domains of yeast cytoplasmic and human cytoplasmic MetRS aa sequences. Residues that are identical in both sequences are indicated by asterisks while dots indicate conservative aa changes. For clarity, the human sequence is limited to residues 261-663 and the yeast sequence to residues 192 594.
H. Lage, h/L Dietel/Gene 178 (1996) 187-189
molecule (Walter et al., 1983). The homology to the putative human nucleotide-binding region between residues 261 and 663 includes 62.5% of aa identities and 25.3% of conservative replacements (Fig. 2). The overall identity of the human and yeast molecule is 33.4%. Furthermore there are well-established functional consensus sequences which are present in the human molecule. For example the 332KMSKS336 E. coli sequence within the nucleotide binding domain of MetRS is proposed to be a signature sequence for a class of aminoacyltRNA synthetases specific to the aliphatic hydrophobic class of amino acids (Meinnel et al., 1990). This pattern is present in the cytoplasmic yeast molecule (525KFSKS 529) and the cytoplasmic human molecule (594KFSKS598). Outside the nucleotide-binding domain the 53°LYNRIDS35 E. coli sequence which is proposed to be part of the specificity of the MetRS by guiding the Y-acceptor stem of tRNA towards the active center (Meinnel et al., 1990) is homologous to the 73°LFQKID735 yeast sequence and the 8°2LFQRID 8°7 human sequence. These data suggest that the well known functional sites of yeast and human MetRS are evolutionarily conserved at a high degree.
189
References Dardel, F., Fayat, G. and Blanquet, S. (1984) Molecular cloning and primary structure of the Escherichia coli methionyl-tRNA synthctase gene. J. Bacteriol. 160, 1115-1122. Dietel, M., Arps, H., Lage, H. and Niendorf, A. (1990) Membrane vesicle formation due to acquired mitoxantrone resistance in human gastric carcinoma cell line EPG85-257. Cancer Res. 50, 6100-6106. Fasiolo, F., Bonnet, J. and Lacroute, F. (1981) Cloning of the yeast methionyl-tRNA synthetase gene. J. Biol. Chem. 56, 2324-2328. Lage, H. and Dietel, M. (1995) Molecular cloning of several cDNAs of a novel overexpressed mitoxantrone resistance-associated gene in gastric carcinoma cells. Proc. Am. Ass. Cancer Res. 36, 323. Meinnel, T., Mechulam, Y., Dardel, F., Schmitter, J.M., Hountondji, C., Brunie, S., Dessen, P., Fayat, G. and Blanquet, S. (1990) Methionyl-tRNA synthetase from E. coli a review. Biochimie 72, 625-632. Pearson, W.R. and Lipman, D.J. (1988) Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444-2448. Tzagoloff, A., Vambutas, A. and Akai, A. (1989) Characterization of MSM1, the structural gene for yeast mitochondrial methionyl-tRNA synthetase. Eur. J. Biochem. 179, 365-371. Walter, P., Gangloff, J., Bonnet, J., Boulanger, Y., Ebel, J.P. and Fasiolo, F. (1983) Primary structure of the Saccharomyces cerevisiae gene for methionyl-tRNA synthetase. Proc. Natl. Acad. Sci. USA 80, 2437-2441.