Genes coding for the elongation factor EF‐1_α in Artemia

Abstract

The elongation factor EF-1, is one of the most abundant proteins in eukaryotic cells, where it catalyzes the binding of aminoacyl-tRNA to ribosomes. The genes coding for this protein in the brine shrimp Artemia were analyzed by gene cloning, electron microscopy and chromosomal blot hybridization. There arc only a few (about four) copies of one type of gene per haploid genome. These genes contain five exons divided over lo4 base pairs. Local rearrangements give rise to a number of gene variants. Cross-hybridizations of Artemia cDNA probes with yeast and Drosophila DNA revealed two different yeast EF-1, genes and one or two different Drosophila genes, respectively.Nucleotide sequencing revealed signals for synthesis and processing of EF-1, transcripts as well as the exact location of exons. One interruption in the coding sequence corresponds closely to a splice junction in the gene coding for the homologous chloroplast protein EF-Tu from Euglena gracilis, presumably of prokaryotic origin. The first exon in the chloroplast gene codes for the region of EF-Tu that is homologous to regions of the elongation factor EF-G and of the initiation factor IF2, respectively.During the evolution of the diverse forms of life, basic principles of protein biosynthesis have been conserved. In eukaryotes, the attachment of aminoacyl-tRNA to ribosomes is catalyzed by the elongation factor EF-la, which is homologous to prokaryotic EF-Tu [l]. Both EF-1, and EF-Tu are present in much larger amounts than other protein components of the protein biosynthesis machinery [2, 31. In fact, EF-Tu in Escherichia coli accounts for 5% of the cellular proteins [3, 41, while the amount of EF-1, in eukaryotic cells is comparable to the abundancy of the major cytoskeletal proteins [5 -71.To gain insight into how the active biosynthesis of EF-1, is achieved we describe here the analysis of genomic EF-1, clones and hybridizations of EF-1, cDNA probes to genomic DNA. Previously, we proposed that EF-1, from the brine shrimp Artemia was coded for by between one and four different genes [8] MATERIALS AND METHODS MaterialsArtemia cysts were purchased from Mountain Hartz Corporation (Ontario, Canada). For the isolation of Artemia DNA, free swimming nauplii were homogenized in 4 vol. 10 mM Tris/Cl, 5 mM MgC12, pH 8.0, at 4°C and filtered. Subsequently, glycogen was removed by repeated centrifugation of nuclei through l .5 M sucrose in the same buffer. DNA was purified from nuclei by lysis in 10 mM Tris/Cl, 10 mM EDTA, 2% (w/v) SDS, repeated phenol and phenol/ chloroform extractions, isopropanol precipitation at room temperature, RNase and proteinase K treatment, followed by a second round of phenolic extractions and isopropanol precipitation. Drosophila DNA was a gift of Dr T. J. M.Hulsebos. Nitrocellulose Southern blots of yeast DNA were donated by Dr C. M. T. Molenaar. Construction and screening ojan Artemia gene libraryArtemia DNA (0.5 mg) was digested with 130U MboI (Biolabs) for 2 h and fractionated on a sucrose gradient as described [12]. After dephospho...

Evidence for Three Major Clades within the Snapping Shrimp Genus Alpheus Inferred from Nuclear and Mitochondrial Gene Sequence Data

“…It is one of the most abundant proteins in eukaryotic cells, in which it catalyzes the binding of charged tRNAs to the ribosome during translation. A potential problem with the usefulness of EF-1␣ as a phylogenetic marker in shrimp was the apparent occurrence of two loci [as reported in several other arthropods (France et al, 1999;Duda and Palumbi, 1999;Lenstra et al, 1986;Hovemann et al, 1988)] and the possibility of pseudogenes [e.g., as in humans (Uetsuki et al, 1989)]. We believe that the sequences found in this study represent two functional loci, since our sequences were amplified from cDNA, suggesting that all sequences are from genes that are expressed.…”

Properties of the elongation factor 1α in the thermoacidophilic archaebacterium Sulfolobus solfataricus

“…The amino acid composition of aEF-lcc is shown in Table 2 compared with those of archaebacterial EF-1 a from Methanococcus vannielii [6], EF-Tu from E. coli [29], eubacterial thermophilic EF-Tu from Thermus thermophilus [30] and eukaryotic EF-la from Artemia salina [31]. On the basis of an M , of 49000, aEF-la is composed of 438 amino acid residues/ molecule.…”

Expression of a gene for mouse eucaryotic elongation factor Tu during murine erythroleukemic cell differentiation.

“…and human cDNA clones and the 70 bases of 5'-nontranslated sequence in the Artemia sp. cDNA clone (14,31). The homologous region in the intronless yeast eEF-Tu gene is also 320 bases long, although the length of the 5'-nontranslated sequence is unknown (4).…”

“…Subsequent hydrolysis of GTP and release of the factor as eEF-Tu-GDP allows its reuse throughout the elongation process (reviewed reference in 13). The functional importance of eEF-Tu in 'roteii1 synthesis has stimulated research on the structure and functional domains of the protein itself (12,29,30,33) as well as on the identification and expression of the genes for etF-Tu (2,4,14,15,19,25,31,32).…”

“…(14,31), and humans (2). All exhibit a high degree of amino acid and nucleotide sequence conservation among these eucaryotes.…”