Functional analysis of HO gene in delayed homothallism in Saccharomyces cerevisiae wy2.
Saccharomyces cerevisiae wy2 exhibits a novel life cycle, with delayed homothallism caused by a defective HO gene. In this strain, gradual diploidization occurs during successive subcultures. Three amino acids of wy2 HO were different from those of wild-type (wt) HO, which included a nonsense mutation (TAG) from Trp-292 and two amino acid changes of His-475 to Leu and Glu-530 to Lys. The ho gene of heterothallic strain CG379 was also sequenced in this study. Four amino acids of ho were different from those of HO. Among different amino acids in wy2 HO and ho, the alteration of His-475 to Leu was common between them. His-475 in HO was previously suggested to be involved in the DNA binding. We constructed a variety of chimeric HO genes by exchanging the corresponding restriction fragments generated from the wt HO, wy2 HO and ho genes. These results and the site-directed mutagenesis studies allowed us to draw the following conclusions: (a) Gly-223 is essential for HO activity; (b) mutation of His-475 to Leu significantly reduces the HO activity; (c) amber mutation (TAG) in wy2 HO car be suppressed inefficiently.
Ekino
K
K
Department of Agricultural Chemistry, Kyushu University, Fukuoka, Japan.
Kwon
I
I
Goto
M
M
Yoshino
S
S
Furukawa
K
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D004252:DNA Mutational Analysis; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D013997:Time Factors
D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C530845:HO protein, S cerevisiae; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific