Summary
In this study we present the characterization of the temperature-sensitive mutant allele cdc9-1 encoding DNA ligase, of Saccharomyces cerevisiae strain A364A by DNA sequencing. Comparison with the published wild-type sequence from strain SKI revealed 13 nucleotide exchanges between these two sequences, which are derived from non-isogenic genetic backgrounds. Only four of these changes, distributed over the whole coding region, lead to amino acid exchanges in the protein chain. Our analysis of the sequence of the wild-type CDC9 allele from strain A364A revealed differences from the isogenic cdc9-1 allele in only two nucleotides: one silent change and one leading to a single amino acid exchange. The latter is therefore responsible for the temperature-sensitive phenotype. A mosaic protein, in which a region carrying this amino acid exchange has been inserted in place of the corresponding part of CDC9 from the non-isogenic strain SKI, is not temperature sensitive. The exchange of a longer stretch of DNA leading to atteration of three amino acids of the protein compared with the original sequence of SKI is required to obtain a temperature-sensitive DNA ligase in this strain, while in strain A364A a single amino acid change is sufficient for expression of a temperature-sensitive protein.
Similar content being viewed by others
References
Barker DG, Johnston LH (1983) Saccharomyces cerevisiae CDC9, a structural gene for yeast DNA ligase which complements Schizosaccharomyces pombe cdc 17. Eur J Biochem 134:315–319
Barker DG, White JHM, Johnston LH (1985) The nucleotide sequence of the DNA ligase (CDC9) from Saccharomyces cerevisiae: a gene, which is cell cycle regulated and induced in response to DNA damage. Nucleic Acids Res 13:8232–8337
Hartwell LH, Mortimer RK, Culotti J, Culotti M (1973) Genetic control of the cell division cycle in yeast: V. Genetic analysis of cdc mutants. Genetics 74:267–286
Klebe RJ, Harriss JV, Sharp DZ, Douglas MG (1983) A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene 25:333–341
Sanger F (1981) Determination of the nucleotide sequence in DNA. Science 214:1205–1210
Unternährer S, Pridmore D, Hinnen A (1991) A new system to amplify 2 μm plasmid copy number in Saccharomyces cerevisiae. Mol Microbiol 5:1539–1549
Author information
Authors and Affiliations
Additional information
Communicated by C. Hollenberg
Rights and permissions
About this article
Cite this article
Unternährer, S., Hinnen, A. Temperature sensitivity of the cdc9-1 allele of Saccharomyces cerevisiae DNA ligase is dependen on specific combinations of amino acids in the primary structure of the expressed protein. Molec. Gen. Genet. 232, 332–334 (1992). https://doi.org/10.1007/BF00280014
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00280014