Molecular cloning, primary structure and disruption of the structural gene of aldolase from Saccharomyces cerevisiae
Open Access
- 3 March 1989
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 180 (2), 301-308
- https://doi.org/10.1111/j.1432-1033.1989.tb14648.x
Abstract
A yeast cDNA genetic library in a bacteriophage expression vector was screened using an antiserum reacting with fructose 1,6-bisphosphate aldolase from Saccharomyces cerevisiae. Radio-labelled probes of selected immunopositive clones were used for screening of a yeast genomic library. From the genomic clones a yeast/Escherichia coli shuttle plasmid was constructed containing on a 1990-base-pair fragment the entire structural gene FBA1 coding for yeast aldolase. The primary structure of the FBA1 gene was determined. An open reading frame comprises 1077 base pairs coding for a protein of 359 amino acids with a predicted molecular mass of 39608 Da. As observed for other strongly expressed yeast genes, codon usage is extremely biased. The 810 base pairs at the 5′ end and the 90 base pairs at the 3′ end of the coding region of the cloned FBA1 gene are sufficient for normal expression and show characteristic elements present in the noncoding sequences of other yeast genes. Aldolase is the major protein in yeast cells transformed with a high-copy-number plasmid containing the FBA1 gene. The aldolase gene was disrupted by insertion of the yeast URA3 gene into the coding region of one FBA1 allele in a homozygous diploid ura3 strain. The haploid offsprings with the defective aldolase allele fbal::URA3lack aldolase enzymatic activity and fail to grow in media containing as a carbon source metabolites of only one side of the aldolase reaction.This publication has 39 references indexed in Scilit:
- Stability and effects of culture conditions on the cellular content and the activity of a phosphatidylcholine/phosphatidylinositol transfer protein from Saccharomyces cerevisiaeBiochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1986
- The major promoter element of rRNA transcription in yeast lies 2 kb upstreamCell, 1984
- [12] One-step gene disruption in yeastMethods in Enzymology, 1983
- TECHNOLOGICAL EXAMINATION OF LOW‐FIRED TERRACOTTA STATUES FROM AYIA IRINI, KEAArchaeometry, 1982
- Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proceedings of the National Academy of Sciences, 1979
- Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase IJournal of Molecular Biology, 1977
- Screening λgt Recombinant Clones by Hybridization to Single Plaques in SituScience, 1977
- Characterization of purified poly(adenylic acid)-containing messenger ribonucleic acid from Saccharomyces cerevisiaeBiochemistry, 1977
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970
- Molecular characteristics of yeast aldolaseBiochemistry, 1969