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.
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 M1 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 ade 1, 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 degrees C, 30 degrees C, or 37 degrees C, but could not grow at 8 degrees C. The deleted region is thus essential only at 8 degrees C, and we name this gene LTE1 (low temperature essential).
Wickner
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RB
Section on Genetics of Simple Eukaryotes, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Koh
T J
TJ
Crowley
J C
JC
O'Neil
J
J
Kaback
D B
DB
D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D013696:Temperature; D014170:Transformation, Genetic
D004271:DNA, Fungal