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 c...

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Bibliographic Details
Published in:The Journal of biological chemistry 1985-03, Vol.260 (5), p.3090-3096
Main Authors: Cottrelle, P, Thiele, D, Price, V L, Memet, S, Micouin, J Y, Marck, C, Buhler, J M, Sentenac, A, Fromageot, P
Format: Article
Language:English
Subjects:
Base Sequence
Biological and medical sciences
Biotechnology
Cloning, Molecular
Codon
DNA - analysis
DNA Restriction Enzymes - metabolism
Endonucleases - metabolism
Fundamental and applied biological sciences. Psychology
Gene Expression Regulation
Genetic engineering
Genetic technics
Methods. Procedures. Technologies
Nucleic Acid Hybridization
Peptide Elongation Factor 1
Peptide Elongation Factors - genetics
Saccharomyces cerevisiae - genetics
Single-Strand Specific DNA and RNA Endonucleases
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)89477-7