A chemically synthesized α-neo-endorphin (αNE) gene was fused to the PH05 gene which encodes repressible acid phosphatase (APase) of Saccharomyces cerevisiae. Three kinds of genes encoding APase-αNE chimeric proteins were cloned in the yeast. Nucleotide sequence analysis revealed that C-terminal amino acids comprising 6 to 17% of the intact enzyme were replaced by αNE. The APase-αNE chimeric proteins were expressed at the level of approximately 1 × 106 molecules per cell under the derepressed conditions. The radioimmunoassay for αNE revealed that most of the APase-αNE chimeric proteins was not present in the periplasmic fraction but in the protoplast fraction.
ABSTRACT Mutants defective in mating-type conversion were isolated from ascospores of a perfect homothallism strain having the HO HMRa HMLα genotype. Eighty mutants, including 11 temperature-dependent mutants showing a or α mating potency, were isolated from 10,050 colonies derived from spores mutagenized with ethyl methanesulfonate. Of the 80 mutants, 48 were tested by crossing with an Ho HMRa HMLα heterothallic and an HO HMRa HMLα homothallic strain as standards. The results allowed their division into seven classes. Mutants in the first class were due to mutation of HO to an ho allele. The second class of mutants, csm, lacked the HO function as the result of a mutation unlinked with the HO locus. Mutants in the third and fourth classes were defective in HMRa and HMLα gene functions, respectively. Mutants in the fifth and sixth classes were due to a mutation at the mating-type locus showing iwensitivity to the function of the homothallic genes. Mutants in the seventh class lacked the expression of mating type as the result of a mutation unlinked to the mating-type locus. All the temperature-dependent mutants lost the mating potency at permissive temperature (25°). One belonging to the last class of mutants was inferred to be a mutation at a locus necessary for the expression of a mating type at restricted temperature (35°).
The influence of 3'-noncoding region (ncr) of the cDNA derived from an eukaryotic mRNA on the preceding structure gene expression in E. coli was investigated using human immune interferon (GIF) and human tumor necrosis factor (TNF) cDNAs. Two sets of gene, with (ncr+) and without (ncr-) 3'-noncoding region, have been expressed in E. coli. In both cases, the level of ncr+ gene expression was five times lower than that of ncr- gene. When these ncrs were placed just downstream of various structure genes, the same effects were also observed. The RNA brot analysis showed that the ncr+ genes were transcribed into shorter and much less mRNA than ncr- genes. These results suggest that these ncrs accelerate the degradation of the mRNA by either causing the premature termination of transcription or the generation of sequence on the transcripts hypersensitive to E. coli endoribonucleases.
The nucleotide sequence of the PHO5 gene of the yeast, Saccharomyces cerevisiae, which encodes repressible acid phosphatase (APase) was determined. Comparison of N-terminal amino acid sequence deduced from the nucleotide sequence with that of the purified repressible APase revealed the existence of a putative signal peptide in the precursor protein. The signal peptide was shown to contain 17 amino acid residues and its structural features were quite similar to those of higher eukaryotic and prokaryotic signal peptides. The nucleotide sequence of 5' and 3' noncoding flanking regions of the PHO5 gene are also discussed.
