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Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae : Isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures
Author(s) -
Wickner Reed B.,
Koh Theodore J.,
Crowley Joan C.,
O'Neil Judy,
Kaback David B.
Publication year - 1987
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.320030108
Subject(s) - biology , gene , genetics , plasmid , microbiology and biotechnology , ura3 , open reading frame , chromosome , dna , molecular cloning , peptide sequence
MAK16 is an essential gene on chromosome I defined by the thermosensitive lethal mak16 – 1 mutation. MAK16 is also necessary for M double‐stranded RNA replication at the permissive temperature for cell growth. As part of an effort to clone all the DNA from chromosome I, plasmids that complemented both the temperature‐sensitive growth defect, and the M 1 replication defects of mak16 – 1 strains were isolated from a plasmid YCp50: Saccharomyces cerevisiae recombinant DNA library. The two plasmids analysed contained overlapping inserts that hybridized proportionally to strains carrying different dosages of chromosome I. Furthermore, integration of a fragment of one of these clones occurred at a site linked to ade1 , confirming that this clone was derived from the appropriate region of chromosome I. An open reading frame adjacent to MAK16 potentially coding for a 468 amino acid protein was defined by sequence analysis. 185 amino acids of this open reading frame were replaced with a 1·2 kb fragment carrying the S. cerevisiae URA3 gene by a one‐step gene disruption. The resulting strains grew at a rate indistinguishable from the wild type at 20°C, 30°C, or 37°C, but could not grow at 8°C. The deleted region is thus essential only at 8°C, and we name this gene LTE1 (low temperature essential).