Tbe EMBO Journal vol.3 no.8 pp.1825- 1830, 1984 Polypeptide chain elongation factor lei (EF-la) from yeast: nucleotide sequence of one of the two genes for EF-la from Saccharomyces cerevisiae Shigekazu Nagata, Koji Nagashima, Yasuko Tsunetsugu- otic translational machineries, we have screened yeast Yokota, Katsuya Fujimural, Masazumi Miyazakil and chromosomal DNA for homologous sequences with E. coli Yoshito Kaziro tufB and succeeded in cloning a gene for yeast mitochondrial Institute of Medical Science, University of Tokyo, Minatoku, Tokyo 108, EF-Tu (mtEF-Tu) (Nagata et al., 1983). The gene designated and lInstitute of Molecular Biology, School of Science, Nagoya University, as tuiM contained a 1311 nucleotide long continuous reading Nagoya 464, Japan frame coding for a protein of 437 amino acids. The derived Communicated by F.Chapeville amino acid sequence of tu]Mwas 66/o homologous to that of E. coli tufA. Messenger RNA for yeast cytosolic polypeptide chain elong- Here, we describe the cloning of the yeast chromosomal ation factor lca (EF-loe) was partially purified from Saccharo- gene coding for cytosolic EF-lee. Yeast cytosolic EF-lae has myces cerevisiae. Double-stranded complementary DNA been purified to homogeneity, and the antibody against yeast (cDNA) was synthesized and cloned in Escherichia coli with EF-lct prepared (unpublished). Messenger RNA for EF-1e pBR327 as a vector. Recombinant plasmid carrying yEF-la was partially purified, and cDNA for EF-lee, was prepared cDNA was identified by cross-hybridization with the E. coli and cloned on plasmid pBR327. Two chromosomal genes for tufB gene and the yeast mitochondrial EF-Tu gene (tufM) EF-lIc were then isolated from the yeast gene library. The two under non-stringent conditions. A yeast gene library was then genes for EF-lc are not closely linked to each other and the screened with the EF-lee cDNA and several clones containing tufM gene was not found in the neighborhood of any of the the chromosomal gene for EF-lax were isolated. Restriction EFJcr genes. The sequence of one of the two EFJa genes was analysis of DNA fragments of these clones as well as the determined, and the deduced amino acid sequence was 70- Southern hybridization of yeast genomic DNA with labelled 80% homologous to the partial amino acid sequences re- EF-lae cDNA indicated that there are two EF-lee genes in S. ported for EF-lIc from rabbit reticulocytes (Slobin and cerevisiae. The nucleotide sequence of one of the two EF-la Moller, 1976) and Artemia salina (Amons et al., 1983). The genes (designated as EFJaA) was established together with its nucleotide sequence for yeast EF-lIa was -78% homologous 5'- and 3' -flanking sequences. The sequence contained 1374 to the partial nucleotide sequence of A. salina EF-la cDNA nucleotides coding for a protein of 458 amino acids with a (Van Hemert et al., 1983). calculated mol. wt. of 50 300. The derived amino acid se- quence showed homologies of 31% and 32% with yeast mito- chondrial EF-Tu and E. coli EF-Tu, respectively. Results Key words: elongation factor lee/EF-la gene sequence/Sac- Cloning ofyeast EF-Ja cDNA charomyces cerevisiae. Since EF-lee is one of the most abundant proteins in eukary- otic cells (Slobin, 1980), it is expected that the mRNA for EF- lc is also an abundant species among the total cellular Introducdon mRNAs. Therefore, we first purified the EF-lee mRNA by Prokaryotic polypeptide chain elongation factor Tu (EF-Tu) measuring its activity in an in vitro translation system using and its eukaryotic counterpart EF-la promoting the GTP- the antisera against yeast EF-Ice. When 600 ug mRNA ob- dependent binding of aminoacyl-tRNA to the A site of ribo- tained from 10 g of wet cells was fractionated by sucrose den- somes have been purified from various sources and their sity gradient centrifugation, the EF-lee mRNA activity was structure and function extensively studied (for a review, see recovered as a single peak at the position corresponding to Kaziro, 1978). EF-Tu from Escherichia coli, comprising 393 18S rRNA. The active fractions were combined and re-chro- amino acid residues (Arai et al., 1980) is encoded by two matographed on an oligo(dT)-cellulose column and -20 Ag nearly identical but unlinked genes on the E. coli chromo- of the partially purified EF-lIc mRNA was obtained. The some, designated as tufA (73 min) and tufB (89 min) (Jas- purity of EF-loe mRNA at this stage was estimated to be kunas et al., 1975). Both tufA (Shibuya et al., 1979) and tufB - 3-44%, as judged from the amount of the 35S radioactivity (Miyajima et al., 1979) have been cloned and their sequences in the immunoprecipitates. determined (Yokota et al., 1980; An and Friesen, 1980). Double-stranded cDNA was prepared using the above par- Eukaryotic cells possess two independent translational tially purified mRNA, and joined to plasmid pBR327. After apparatuses; one functioning in the cytosolic fraction in con- transformation of E. coli SK1592 with the hybrid DNA, 2800 junction with 80S ribosomes, and the other in the mitochon- tetracycline-resistant colonies were obtained. Identification of dria with 70S ribosomes. The protein components of the the clone harboring EF-lIc cDNA was carried out as follows. mitochondrial translational apparatus including all the ribo- Firstly, 132 clones strongly hybridizing with the labelled somal proteins, aminoacyl-tRNA synthetases and soluble cDNA prepared by reverse transcription of the partially translational factors are encoded by nuclear genes, syn- purified preparation of EF-lae mRNA were selected out of thesized in the cytoplasmic fraction, and transported into the 1152 transformants by in situ colony hybridization (Hanahan mitochondria. and Meselson, 1980). Then plasmid DNA from 48 of the In an attempt to study the genetic organization of eukary- above clones were prepared (Holmes and Quigley, 1981), IRL Press Limited, Oxford, England. 1825 S.Nagata et al. A) : 3 ' i E B C H C H C H BC CE BH E BaP CP CH L II IV_lim ili - ILLLLL p'fEF35 pYEF20,29 pYEF5 . pYFF4 6 _V pYEF32 _1 pYEF45 B) VW H E B CBa B P E CE BH HP B H B )I ..II I I I III pYEF1 1 Kb pvEF2,13 pYEF4 3 Flg. 1. Restriction maps of recombinant plasmids carrying the EF-la genes. The solid bars indicate the EF-la genes and the direction of transcription is from left to right. Restriction sites are abbreviated as: B, BglII site; Ba, BamHI site; C, ClaI site; E, EcoRI site; H, HindIII site; P, PstI site. kb denotes kilobases. (A) Maps of yeast DNA segments carrying the EFlaA gene. An additional site was present for each ClaI and HindIII, but was not mapped. (B) Maps of yeast DNA segments carrying the EFlaB gene. electrophoresed on a 1% agarose gel, and transferred to a nitrocellulose membrane. Southern hybridization was carried out under non-stringent conditions (50!7 formamide, at 20°C) using a mixture of the 1.5-kb HpaI fragment of E. coli tufB (Yokota et al., 1980) and the 1.0-kb PvuII fragment of yeast tufM (Nagata et al., 1983), which were labelled by nick- translation. One plasmid which gave a positive response was designated as pKN3 1. Fig. 2. Southern hybridization analysis of yeast DNA. 20 jg of yeast DNA The 700-nucleotide long cDNA in pKN31 contained a was digested with several restriction enzymes and electrophoresed on 0.8% single ClaI and EcoRI site, and DNA sequence analysis was agarose gel. The DNA was transferred to a nitroceliulose membrane filter carried out using the unique ClaI site. The nucleotide se- and hybridized with the nick-translated PstI fragment of pKN31. Restriction enzymes used were EcoRI (ane 1), BgIII ne 2), BamHI (ane quence corresponded to the position from 100 to 492 in Fig- 3), HindlII Oane 4) and PstI lane 5). 5'-3P-labelled HindIII-digested ure 4. The amino acid sequence derived from that nucleotide lambda DNA was used as a size marker (ane M). sequence revealed an extensive coincidence with the partial amino acid sequence of EF-Ia from S. carlsbergensis which restriction map, these clones could be classified into two was determined with a large fragment obtained by limited groups. In the first group, the DNA inserts from the six proteolysis (data not shown). Twenty eight out of 30 amino clones pYEF35, 20, 5, 46, 32 and 45 overlapped each other acid residues were identical; at two positions (67 and 69 in and covered the range of 19.5 kb of the chromosomal DNA. Figure 4) arginine was replaced by lysine. The difference in The EF-lc gene in this group was designated as EFJoA. The amino acid sequences may be due to the difference of the second group consisting of pYEFI, 2, and 43, covered 17 kb yeast strains used for cDNA cloning (S. cerevisiae) and for of the contiguous chromosomal DNA on which the second preparation of EF-Ia (S. carlsbergensis). From this result, we EF-Ia gene designated as EFJoaB was present. conclude that pKN31 is a clone carrying the 5'-half of the As can be seen in Figure 1, the restriction maps of the two cDNA for yeast EF-Ia. genes are very similar within the structural genes for EF-la, Cloning of the chromosomal gene for EF-Joe while the restriction maps of the 5 '- and 3'-flanking regions are different. These results suggest that the two EF-lc genes Carlson and Botstein (1982) have prepared a recombinant plasmid library by insertion of the partial Sau3A digests of are located in the different chromosomal loci of S.