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We have isolated a new mutant of Saccharomyces cerevisiae that exhibits a glucose-derepression resistant (and sucrose-non-fermentor) phenotype. This mutant was obtained by screening for overproduction of alpha-amylase in a strain containing the mouse alpha-amylase gene under the control of the PGK promoter. The mutation designated pop2 (PGK promoter directed over production). The pop2 mutant overproduced amylase 5-6 fold and displayed several other pleiotropic defects: (1) resistance to glucose derepression, (2) temperature-sensitive growth, (3) failure of homozygous diploid cells to sporulate and (4) reduced amount of reserve carbohydrates. We mapped pop2 to chromosome XIV, distal to lys9 and SUP28, indicating that POP2 is a newly-identified locus. We isolated the POP2 gene from two yeast strains of different genetic backgrounds, S288C and A364A, and determined their nucleotide sequences. The predicted amino acid sequence of the POP2 protein contains three glutamine-rich region, a proline-rich region and a serine/threonine-rich region, characteristic of many transcription factors. Steady state levels of RNA transcribed from the PGK-amylase fusion gene and from endogenous PGK gene in stationary-phase pop2 cells were 5- to 10-fold higher than those observed in wild-type cells, showing that the pop2 mutation affects transcription of the PGK gene transcription.

An RGRI gene product is required to repress expression of glucose-regulated genes in Saccharomyces cerevisiae. The abnormal morphology of rgrl cells was studied. Scanning and transmission electron microscopic observations revealed that the cell wail of the daughter cell remained attached to that of mother cell. We cloned the RGRI gene by complementation and showed that the cloned DNA was tightly linked to the chromosomal RGRI locus. The cloned RGRI gene suppressed all of the phenotypes caused by the mutation and encoded a 3.6-kilobase poly(A)' RNA. The RGRI gene is located on chromosome XII, as determined by pulsed-field gel electrophoresis, and we mapped rgrl between gal2 and pep3 by genetic analysis. rgrl was shown to be a new locus. We also determined the nucleotide sequence of RGRI, which was predicted to encode a 123-kilodalton protein. The null mutation resulted in lethality, indicating that the RGRI gene is essential for growth. On the other hand, a carboxy-terminal deletion of the gene caused phenotypes similar to but more severe than those caused by the original mutation. The amount of reserve carbohydrates was reduced in rgrl cells. Possible functions of the RGRI product are discussed.Glucose regulates the expression of many genes in Saccharomyces cerevisiae (for a review, see reference 5). One of them, the SUC2 gene, which encodes invertase, is repressed by glucose (2, 3). Recently, we reported the isolation of a new mutation, rgrl, which affects expression of the SUC2 gene (21). A recessive rgrl-l mutation which caused overexpression of mouse a-amylase under the control of the SUC2 promoter was isolated, and the RGRI gene was found to be required for glucose repression. The rgrl mutation affected several cellular functions. Cells were resistant to glucose repression, temperature sensitive for cell growth, and sporulation deficient and showed abnormal cell morphology. Expression of the SUC2 gene in rgrl strains was resistant to glucose repression, and SUC2 expression was increased under glucose-derepressing conditions. In this report, we describe studies of the morphology of rgrl cells, the cloning and molecular analysis of the RGRI gene, and meiotic linkage analysis of rgrl. We constructed deletion mutations to determine the phenotypes of strains lacking a functional RGRI gene product and determined the nucleotide sequence of the gene. The RGRI gene affected accumulation of reserve carbohydrates. MATERIALS AND METHODSStrains and genetic methods. The strains of S. cerevisiae used in this study are listed in Table 1. All strains were derived from S288C. Crossing, sporulation, and tetrad analysis were carried out by standard genetic methods (23). The permissive and restrictive temperatures were 24 and 37°C, respectively. The phenotype of pep3 strains was scored as described elsewhere (12). The transformation of yeast was performed by the LiOAc-method of Ito et al. (11). Escherichia coli HB101 and JM109 were employed as hosts for * Corresponding author.constructing and propagating plasmids. The transform...

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