Both genes for EF-1α in Candida albicans are translated

In previous work, we showed that Candida albicans has two genes, TEF-1 and TEF-2, which encode identical polypeptides for the highly conserved, essential, protein synthesis factor EF-1 alpha (Breviario et al., 1988). This result prompted questions as to whether C. albicans preferentially uses one of the genes over the other and whether both genes are actually translated into protein. Gene-specific sequence differences in the untranslated portion of each gene made it possible to prepare gene-specific oligonucleotide hybridization probes. Results with the probes showed that the relative steady-state mRNA levels of the two genes were equivalent and that the mRNA for each gene was present in active translation complexes.     

Human elongation factor 1 beta: cDNA and derived amino acid sequence.

From a cDNA library in lambda gt11 derived from poly (A+)RNA of human ovarian granulosa cells a cDNA clone lambda HGP34, containing an EcoRI insert of 829 bp, was identified. After subcloning of the insert into pUC18, the clone pHGP34 was obtained and sequenced. The derived amino acid sequence, corresponding to a protein of 225 amino acids, shows a high degree of homology to elongation factor 1 beta (EF-1 beta) of Artemia salina (57%) and known peptide sequences of Xenopus laevis EF-1 beta (86%). We therefore assume that the protein coded for by pHGP34 represents human EF-1 beta. Northern analysis reveals an EF-1 beta specific mRNA of 900 bp. Southern analysis indicates that EF-1 beta in the human genome, like EF-1 alpha, appears to be specified by more than one gene. A high degree of sequence homology for EF-1 beta specific sequences is observed for bovine, rat and mouse species.     

42Sp48 in previtellogenic Xenopus oocytes is structurally homologous to EF-1 alpha and may be a stage-specific elongation factor

We have isolated the cDNA for 42Sp48 and EF-1 alpha from mixed stage oocytes and tailbud (stage 22) Xenopus laevis cDNA libraries by use of the cDNA for human elongation factor-1 alpha (EF-1 alpha) as probe. The nucleotide and deduced amino acid sequences of the entire coding region of 42Sp48 and EF-1 alpha cDNA were established. The proposed functional homology of the proteins is reflected in highly conserved amino acid sequences (91% identity), while the large number of silent mutations at the gene level may serve to prevent recombination at their loci. 42Sp48 is apparently encoded by two genes in Xenopus, while no sequences corresponding to 42Sp48 could be found in murine or human genomic DNA. 42Sp48 has been proposed to act as a stage-specific elongation factor in Xenopus. Comparison of the deduced amino acid sequences of 42Sp48 and EF-1 alpha with that of elongation factor Tu from E. coli, for which the three-dimensional structure including that of the GTP binding sites have been determined, supports this hypothesis.     

Sequence analysis and expression of the two genes for elongation factor 1 alpha from the dimorphic yeast Candida albicans.

Two Candida albicans genes that encode the protein synthesis factor elongation factor 1 alpha (EF-1 alpha) were cloned by using a heterologous TEF1 probe from Mucor racemosus to screen libraries of C. albicans genomic DNA. Sequence analysis of the two clones showed that regions of DNA flanking the coding regions of the two genes were not homologous, verifying the presence of two genes, called TEF1 and TEF2, for EF-1 alpha in C. albicans. The coding regions of TEF1 and TEF2 differed by only five nucleotides and encoded identical EF-1 alpha proteins of 458 amino acids. Both genes were transcribed into mRNA in vivo, as shown by hybridization of oligonucleotide probes, which bound specifically to the 3' nontranslated regions of TEF1 and TEF2, respectively, to C. albicans total RNA in Northern (RNA) blot analysis. The predicted EF-1 alpha protein of C. albicans was more similar to Saccharomyces cerevisiae EF-1 alpha than to M. racemosus EF-1 alpha. Furthermore, codon bias and the promoter and termination signals of the C. albicans EF-1 alpha proteins were remarkably similar to those of S. cerevisiae EF-1 alpha. Taken together, these results suggest that C. albicans is more closely related to the ascomycete S. cerevisiae than to the zygomycete M. racemosus.     

Three genes under different developmental control encode elongation factor 1-alpha in Xenopus laevis.

We have cloned cDNAs encoding two variants of the elongation factor for protein synthesis in Xenopus laevis, called EF-1 alpha. One of these (42Sp50) is expressed exclusively in immature oocytes. It is one of two protein components of a 42S RNP particle that is very abundant in previtellogenic oocytes. The 42S RNP particle consists of various tRNAs, 5S RNA, 42Sp50 and a 5S RNA binding protein (42Sp43). A major function served by 42Sp50 appears to be the storage of tRNAs for later use in oogenesis and early embryogenesis. The second EF-1 alpha variant (EF-1 alpha O) is expressed mainly in oocytes but transiently in early embryogenesis as well. Its mRNA cannot be detected after neurulation in somatic cells. EF-1 alpha O is closely related to a third EF-1 alpha (EF-1 alpha S), discovered originally by Krieg et al. (1). EF-1 alpha S is expressed at low levels in oocytes but actively in somatic cells. The latter two proteins are very similar to known eukaryotic EF-1 alpha from other organisms and presumably function in their respective cell types to support protein synthesis.     

Characterization of the statin-like S1 and rat elongation factor 1 alpha as two distinctly expressed messages in rat.

Previously, we reported a rat S1 protein that is antigenically related to statin, a nonproliferating cell-specific marker; however, it shares high homology with the known human elongation factor-1 alpha (EF-1 alpha). To differentiate S1 from rat EF-1 alpha and to study their respective regulation for expression, a rat EF-1 alpha cDNA clone was isolated and characterized. The nucleotide and deduced amino acid sequences of this partial rat EF-1 alpha cDNA are compared with that of human and mouse as well as with rat S1. Both their messages were detected in rat brain by EF-1 alpha- or S1-specific probes. However, the mRNA encoding EF-1 alpha is more abundant than that encoding S1. S1 and EF-1 alpha expression were investigated in the parotid and submandibular glands of untreated rats and those treated with isoproterenol, a proliferation-inducing catecholamine. Quantitative solution hybridization demonstrated a dramatic reduction (approximately 68%) in the S1 mRNA following isoproterenol injection in proliferation-responsive parotid glands and a mild reduction (approximately 20%) of S1 steady-state messages in the proliferation-refractile submandibular glands. A slight increase or no changes of EF-1 alpha levels in both parotid and submandibular glands following isoproterenol treatment are also observed. Therefore, the EF-1 alpha and S1 genes are different genes, both expressed and regulated in vivo, but in differential quantitative and qualitative patterns.     

Cloning, nucleotide sequence, and expression of one of two genes coding for yeast elongation factor 1 alpha

One gene coding for yeast cytoplasmic elongation factor 1 alpha (EF-1 alpha) was isolated by colony hybridization using a cDNA probe prepared from purified EF-1 alpha mRNA. A recombinant plasmid, pLB1, with a 6-kilobase yeast DNA insert, was found by hybrid selection and translation experiments to carry the entire gene. The nucleotide sequence of the gene with its 5'- and 3'-flanking regions was determined. The 5' and 3' ends of EF-1 alpha mRNA were localized by the S1 nuclease mapping technique. The cloned gene, called TEF1, encodes a protein of 458 amino acids (Mr = 50,071) in a single, uninterrupted reading frame. The amino acid sequence shows a strong homology with several domains of Artemia salina EF-1 alpha cytoplasmic factor, as evidenced by diagonal dot matrix analysis. Protein sequence homology is comparatively much lower with the yeast mitochondrial elongation factor. S1 nuclease mapping of the mRNA, hybridization analysis of chromosomal DNA using intragenic or extragenic DNA probes, and gene disruption experiments demonstrated the existence of two genes coding for the cytoplasmic elongation factor EF-1 alpha/haploid genome. The presence of an intact chromosomal TEF1 gene is not essential for growth of haploid yeast cells.     

Polypeptide chain elongation factor 1 alpha (EF-1 alpha) from yeast: nucleotide sequence of one of the two genes for EF-1 alpha from Saccharomyces cerevisiae.

Messenger RNA for yeast cytosolic polypeptide chain elongation factor 1 alpha (EF-1 alpha) was partially purified from Saccharomyces cerevisiae. Double-stranded complementary DNA (cDNA) was synthesized and cloned in Escherichia coli with pBR327 as a vector. Recombinant plasmid carrying yEF-1 alpha cDNA was identified by cross-hybridization with the E. coli tufB gene and the yeast mitochondrial EF-Tu gene (tufM) under non-stringent conditions. A yeast gene library was then screened with the EF-1 alpha cDNA and several clones containing the chromosomal gene for EF-1 alpha were isolated. Restriction analysis of DNA fragments of these clones as well as the Southern hybridization of yeast genomic DNA with labelled EF-1 alpha cDNA indicated that there are two EF-1 alpha genes in S. cerevisiae. The nucleotide sequence of one of the two EF-1 alpha genes (designated as EF1 alpha A) was established together with its 5'- and 3'-flanking sequences. The sequence contained 1374 nucleotides coding for a protein of 458 amino acids with a calculated mol. wt. of 50 300. The derived amino acid sequence showed homologies of 31% and 32% with yeast mitochondrial EF-Tu and E. coli EF-Tu, respectively.     

Structure and expression of elongation factor 1 alpha in tomato.

A full-length cDNA clone, LeEF-1, has been isolated from tomato for the alpha subunit of elongation factor 1 (EF-1 alpha), a polypeptide which plays a central role in protein synthesis. The 448 amino acid protein encoded by this cDNA appears highly homologous to other EF-1 alpha s having a high degree of similarity (75-78%) to EF1 alpha previously described from both lower eukaryotes and animals. Southern analysis indicated that EF-1 alpha belongs to a small multigene family of 4-8 members in tomato. The pattern of expression of EF-1 alpha mRNA in various tomato tissues was analyzed by Northern analysis, in vitro translation and in situ hybridization. EF-1 alpha mRNA is an abundant species and higher levels of mRNA were found in developing tissues such as young leaves and green fruit compared to the mRNA levels observed in older tissues. The increased levels of EF-1 alpha mRNA therefore appear to correlate with higher levels of protein synthesis in developing tissues.     

Identification of two genes coding for the translation elongation factor EF-1 alpha of S. cerevisiae.

The translation elongation factor EF-1 alpha of the yeast Saccharomyces cerevisiae is coded for by two genes, called TEF1 and TEF2. Both genes were cloned. TEF1 maps on chromosome II close to LYS2. The location of TEF2 is unknown. TEF2 alone is sufficient to promote growth of the cells as shown with a strain deleted for TEF1. TEF1 and TEF2 were originally identified as two strongly transcribed genes, which most likely code for an identical or nearly identical protein as judged from S1 nuclease protection experiments with mRNA-DNA hybrids. The DNA sequence analysis of TEF1 allowed the prediction of the protein sequence. This was shown, by a search in the Dayhoff protein data bank, to represent the translation elongation factor EF-1 alpha due to the striking similarity to EF-1 alpha from the shrimp Artemia. A search for TEF1 homologous sequences in several yeast species shows, in most cases, duplicated genes and a much higher sequence conservation than among genes encoding amino acid biosynthetic enzymes.     

The primary structure and the functional domains of an elongation factor-1 alpha from Mucor racemosus

We have determined the complete nucleotide sequence for TEF-1, one of three genes coding for elongation factor (EF)-1 alpha in Mucor racemosus. The deduced EF-1 alpha protein contains 458 amino acids encoded by two exons. The presence of an intervening sequence located near the 3' end of the gene was predicted by the nucleotide sequence data and confirmed by alkaline S1 nuclease mapping. The amino acid sequence of EF-1 alpha was compared to the published amino acid sequences of EF-1 alpha proteins from Saccharomyces cerevisiae and Artemia salina. These proteins shared nearly 85% homology. A similar comparison to the functionally analogous EF-Tu from Escherichia coli revealed several regions of amino acid homology suggesting that the functional domains are conserved in elongation factors from these diverse organisms. Secondary structure predictions indicated that alpha helix and beta sheet conformations associated with the functional domains in EF-Tu are present in the same relative location in EF-1 alpha from M. racemosus. Through this comparative structural analysis we have predicted the general location of functional domains in EF-1 alpha which interact with GTP and tRNA.     

Purification and characterization of a germ cell-specific form of elongation factor 1 alpha (EF-1 alpha) from Xenopus laevis.

Elongation factor 1 alpha (EF-1 alpha) was purified to homogeneity from full-grown oocytes of Xenopus laevis. This protein is encoded by a gene previously shown to be expressed in male and female germ cells, and repressed in somatic cells. The purified protein was identified with EF-1 alpha on criteria of molecular mass, cross-reaction with antibodies raised against Artemia salina EF-1 alpha, affinity for guanine nucleotides, and ability to promote the mRNA-dependent binding of aminoacyl tRNA to 80S ribosomes.     

Structural and functional properties of thesaurin a (42Sp50), the major protein of the 42 S particles present in Xenopus laevis previtellogenic oocytes

Thesaurin a is one of two protein components of a 42 S ribonucleoprotein particle that is very abundant in previtellogenic oocytes of Xenopus laevis. The primary function of the 42 S particle is the long-term storage of 5 S RNA and aminoacyl-tRNA. Thesaurin a is homologous to eukaryotic elongation factor 1 alpha (EF-1 alpha) and to prokaryotic elongation factor Tu (EF-Tu). Sequence comparison with EF-1 alpha and EF-Tu of different species indicates that thesaurin a is rather distantly related to all eukaryotic elongation factors. In spite of this, the secondary structure of thesaurin a, deduced from hydrophobic cluster analysis, is remarkably similar to that of EF-1 alpha and EF-Tu. The binding and catalytic properties of thesaurin a are also similar but not identical to those of EF-1 alpha. Like EF-1 alpha, purified thesaurin a binds tRNA, GDP, and GTP. Unlike EF-1 alpha, thesaurin a binds discharged tRNA more tightly than charged tRNA, and GTP more tightly than GDP. Thesaurin a also hydrolyzes GTP and catalyzes the mRNA-dependent binding of aminoacyl-tRNA to 80 S ribosomes. The functional properties of the 42 S particle are in general agreement with those of purified thesaurin a. In particular, the 42 S particle contains GTP and efficiently transfers aminoacyl-tRNA to 80 S ribosomes without addition of exogenous elongation factor.     

Apis mellifera cytoplasmic elongation factor 1 alpha (EF-1 alpha) is closely related to Drosophila melanogaster EF-1 alpha

Using low stringency hybridisation with a Drosophila melanogaster EF-1 alpha gene fragment we have isolated a genomic DNA clone encoding elongation factor 1 alpha (EF-1 alpha) from Apis mellifera. The hybridising Apis mellifera sequence could be delineated to two small EcoRI fragments that were also revealed by genomic Southern hybridisation. By comparison with the corresponding Drosophila melanogaster data the complete translational reading frame has been deduced. It is interrupted by two intervening sequences of 220 and about 790 nucleotides. Comparison with known eucaryotic EF-1 alpha sequences further confirms that certain amino acid sequences seem to be invariable within the EF-1 alpha protein family.     

Two forms of elongation factor 1 alpha (EF-1 alpha O and 42Sp50), present in oocytes, but absent in somatic cells of Xenopus laevis

We have purified and partially sequenced the EF-1 alpha protein from Xenopus laevis oocytes (EF-1 alpha O). We show that the two cDNA clones isolated by Coppared et al. (Coppard, N. J., K. Poulsen, H. O. Madsen, J. Frydenberg, and B. F. C. Clark. 1991. J. Cell Biol. 112:237-243) do not encode 42Sp50, as claimed by these authors, but two very similar forms of EF-1 alpha O (EF-1 alpha O and EF-1 alpha O1). 42Sp50 is the major protein component of a 42S nucleoprotein particle that is very abundant in previtellogenic oocytes of X. laevis, 42Sp50 differs from EF-1 alpha O not only by its amino acid sequence, but also by several properties already reported. In particular, 42Sp50 has a low EF-1 alpha activity. It is distributed uniformly in the cytoplasm of previtellogenic oocytes, in contrast to EF-1 alpha O which is concentrated in a small region of the cytoplasm, known as the mitochondrial mass or Balbiani body.