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Y Nogi

Publications and source records attributed to Y Nogi.

At least 55 records · Page 3Linked to original sources
PubMed1991

Purification and characterization of the yeast negative regulatory protein GAL80.

Transcription of the GAL genes encoding the enzymes responsible for galactose metabolism in the yeast Saccharomyces cerevisiae is regulated through an interplay of two regulatory proteins, GAL4 and GAL80. GAL4 binds to upstream activating sequences of GAL (UASG) and activates their transcription in yeast growing in the presence of galactose. GAL80 binds to GAL4 and inhibits the activation function of GAL4 in yeast growing without galactose. We have purified GAL80 in its native form as a protein that reacts with an antiserum raised against a synthetic peptide of 18 amino acid residues in the GAL80 sequence. Purification was performed through ammonium sulfate precipitation, streptomycin precipitation, DEAE-cellulose column chromatography, and gel filtration. From 50 g of wet cells, a final sample of 2.3 mg with a purity of more than 80% was obtained. The molecular size of the purified protein in both the native and denatured states was estimated to be approximately 50 kDa, indicating that GAL80 exists as a monomer in yeast cells. The amino-terminal residue of GAL80 was found to be acetylmethionine. The purified protein was shown to bind GAL4. We have also purified mutant GAL80 proteins encoded by two different alleles of gal80 known to be incapable of inhibiting the function of GAL4. These proteins were, in fact, unable to bind GAL4.

Chromatography, Gel↗
PubMed1990

Strain-specific lethal effect of the adenovirus E1a protein on Saccharomyces cerevisiae.

Various adenovirus E1a proteins, including 13S protein, 12S protein and three other derivatives of 13S protein with deletions were expressed in Saccharomyces cerevisiae. Both the C-terminal 67 residues and the 13S unique domain are required for the nuclear targeting in yeast. The N-terminus containing multiple functional domains appears to be involved in the G1 arrest of diploid yeast and two other regions, the region containing amino acid residues between 122 and 139, and the 67 residues of the C-terminus are required for the lethal effect on haploid yeast. The latter effect, however, is dependent on strains. Thus, the yeast system may be utilized for functional dissection of E1a protein by further analyzing metabolic consequences.

Adenoviridae↗
PubMed1990

Yeast Gal11 protein mediates the transcriptional activation signal of two different transacting factors, Gal4 and general regulatory factor I/repressor/activator site binding protein 1/translation upstream factor.

GAL11 was first identified as a gene required for full expression of some of the galactose-inducible genes in the yeast Saccharomyces cerevisiae. A null mutation within the GAL11 locus causes defects in mating, growth on nonfermentable carbon sources, and sporulation of gal11 homozygotes. The mating defect was observed only in MAT alpha gal11 strains. Northern hybridization analysis revealed that a gal11 mutation impaired transcription of alpha-specific genes (MF alpha 1 and STE3) but not of an a-specific gene (STE2). Furthermore, this mutation reduced expression of the MAT alpha locus, suggesting that a deficiency in MAT alpha 1 protein is responsible for the reduced expression of alpha-specific genes. Since general regulatory factor I (GRFI)/repressor/activator site binding protein 1 (RAP1)/translation upstream factor (TUF) is believed to be an activator of MAT alpha expression, we examined whether PYK1, which is known to be regulated by GRFI/RAP1/TUF, is also affected by the gal11 mutation. It was determined that the level of PYK1 message was significantly lowered by the mutation. The requirement for functional GAL11 in transcriptional activation was bypassed when either the upstream activating sequence of galactose-inducible genes or of PYK1 was placed very close to the TATA box, suggesting that one of the Gal11 protein functions is to mediate the activation signal of Gal4 and GRFI/RAP1/TUF, when the respective binding site is situated at the naturally occurring distance from the TATA box.

Blotting, Northern↗
PubMed1989

Functional domains of a negative regulatory protein, GAL80, of Saccharomyces cerevisiae.

To study the functional domains of a transcriptional repressor encoded by the GAL80 gene of Saccharomyces cerevisiae, we constructed various deletion and insertion mutations in the GAL80 coding region and determined the ability of these mutations to repress synthesis of galactose-metabolizing enzymes as well as the capacity of the mutant proteins to respond to the inducer. Two regions, from amino acids 1 to 321 and from amino acids 341 to 423, in the total sequence of 435 amino acids were required for repression. The internal region from amino acids 321 to 340 played a role in the response to the inducer. The 12 amino acids at the carboxy terminus were dispensable for normal functioning of the GAL80 protein. Using indirect immunofluorescence and subcellular fractionation techniques, we also found that two distinct regions (amino acids 1 to 109 and 342 to 405) within the putative repression domain were capable of directing cytoplasmically synthesized Escherichia coli beta-galactosidase to the yeast nucleus. In addition, three gal80 mutations were mapped at amino acid residues 183, 298, and 310 in the domain required for repression. On the basis of these results, we suggest that the GAL80 protein consists of a repression domain located in two separate regions (amino acid residues 1 to 321 and 341 to 423) that are interrupted by an inducer interaction domain (residues 322 to 340) and two nuclear localization domains (1 to 109 and 342 to 405) that overlap the repression domains.

Biological Transport↗
PubMed1989

Expression of a human P-450IIC gene in yeast cells using galactose-inducible expression system.

A cDNA of a human liver cytochrome P-450, corresponding to P-450 human-2, was expressed in Saccharomyces cerevisiae cells by the use of a galactose-inducible expression vector containing the GAL7 promoter and terminator. In Western blots using anti-P-450 human-2 IgG, a single band, which exhibited mobility identical to that of authentic P-450 human-2 purified from human liver, was detected in microsomes of the yeast cells. The amount synthesized in yeast was estimated to be approximately 1% of the total cell protein, and approximately 25% of the cytochrome existed in the holoenzyme state. Microsomes from the P-450 human-2-producing yeast showed a catalytic activity towards benzo(a)pyrene, and the activity was significantly enhanced by the addition of purified NADPH-cytochrome P-450 reductase. The yeast microsomes also catalyzed (S)-mephenytoin 4-hydroxylation but not the demethylation. The present results indicate that the yeast cells containing P-450 human-2 cDNA synthesize a functionally active form of the enzyme, the chemical and catalytic properties of which are identical to those of the human liver preparation.

Antigen-Antibody Reactions↗
PubMed1988

GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae.

Normal function of the GAL11 gene is required for maximum production of the enzymes encoded by GAL1, GAL7, and GAL10 (collectively termed GAL1,7,10) in Saccharomyces cerevisiae. Strains bearing a gal11 mutation synthesize these enzymes at 10 to 30% of the wild-type level in the induced state. In a DNA-RNA hybridization experiment, the gal11 effect was shown to be exerted at the transcription level. Yeast cells bearing the gal11 mutation were shown to grow on glycerol plus lactate more slowly than the wild type. We isolated recombinant plasmids carrying the GAL11 gene by complementation of the gal11 mutation. When the GAL11 locus was disrupted by insertion of the URA3 gene, the resulting yeast cells (gal11::URA3) exhibited phenotypes almost identical to those of the gal11 strains, with respect to both galactose utilization and growth on nonfermentable carbon sources. Deficiency of Gal4, the major transcription activator for GAL1,7,10, was epistatic over the gal11 defect. The Gal11 deficiency lowered the expression of GAL2 but not that of MEL1 or GAL80; expression of these genes is also known to be dependent on GAL4 function. We determined the nucleotide sequence of GAL11, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). An alpha-helix-beta-turn-alpha-helix structure was found in a distal part of the predicted amino acid sequence. A possible role of the GAL11 product in the regulation of galactose-inducible genes is discussed.

Amino Acid Sequence↗
PubMed1987

Autogenous regulation of the Saccharomyces cerevisiae regulatory gene GAL80.

We have suggested previously from Northern blot analysis that transcription of the negative regulatory gene GAL80 was controlled positively by another regulatory gene GAL4, and negatively by GAL80 itself, in similar way to GAL1, GAL7 and GAL10 genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae. To study further the controlled expression of GAL80, we have exploited the gene fusion technique. We constructed gene fusions consisting of 5' fragments of GAL80 and a 5' truncated lacZ of Escherichia coli, and introduced the GAL80'-'lacZ fusions into wild-type yeast or various GAL4 or GAL80 mutants using multiple-copy or single-copy plasmid vectors. We then studied beta-galactosidase activity in the resultant transformants under uninduced, induced or glucose-repressed conditions. Expression of the GAL80'-'lacZ fusions was clearly under the control of Gal4/Gal80. Next we constructed GAL7'-'lacZ fusions, whose upstream activating sequence (UAS) from GAL7 was replaced with a GAL80 fragment containing a UAS-like sequence located in the 5' flanking region of GAL80. Synthesis of beta-galactosidase directed by the hybrid genes was inducible by galactose exactly like the original GAL7'-'lacZ fusion with a UAS from GAL7. Finally we constructed a GAL7-GAL80 hybrid gene, in which the entire 5' flanking region was derived from GAL7. When the chromosomal GAL80 gene in wild-type yeast was replaced with the hybrid gene, the uninduced level, but not the induced level, of the GAL10-encoded enzyme (uridine diphosphoglucose-4-epimerase) was significantly increased.

Galactose↗
PubMed1986

GAL3 gene product is required for maintenance of the induced state of the GAL cluster genes in Saccharomyces cerevisiae.

The activities of the first three enzymes for galactose catabolism normally become detectable within 15 min after the addition of galactose into a culture of the yeast Saccharomyces cerevisiae. In S. cerevisiae with a recessive mutation termed gal3, a longer-than-normal lag is observed before the appearance of the enzyme activities (O. Winge and C. Roberts, C. R. Trav. Lab. Carlsberg Ser. Physiol. 24:263-315, 1948). I isolated two S. cerevisiae mutants with temperature-sensitive defects in the GAL3 gene. Temperature shift experiments with one of those mutants led to the conclusion that the GAL3 function is required not only for the initiation of enzyme induction but also for the maintenance of the induced state in galactose-nonfermenting S. cerevisiae because of a defect in any of the genes for the galactose-catabolizing enzymes, such as gal1 or gal10. In contrast, the GAL3 function is phenotypically dispensable in galactose-metabolizing S. cerevisiae. Thus, the normal catabolism of galactose can substitute for the GAL3 function.

Enzyme Induction↗
PubMed1986

Duplicate upstream activating sequences in the promoter region of the Saccharomyces cerevisiae GAL7 gene.

We constructed a series of deletions in the 5' noncoding region of the Saccharomyces cerevisiae GAL7 gene, fused them to the Escherichia coli gene lacZ, and introduced them into yeasts by using a multicopy vector. We then studied the effect of the deletions on beta-galactosidase synthesis directed by the gene fusions in media with various carbon sources. This analysis identified a TATA box and two upstream activating sequences as necessary elements for galactose-controlled GAL7 transcription. Two upstream activating sequences exhibiting 71% homology with each other were located 255 and 168 base pairs, respectively, upstream of the GAL7 transcription start point. Each sequence consists of 21 base pairs, displaying an approximate rotational symmetry with a core consensus sequence of GAA--AGCTGCTTC--CGCG. At least one of the two sequences is required for galactose induction and also for glucose repression of the GAL7'-lac'Z gene. Analysis with host regulatory mutants delta gal14 and delta gal180 suggests that these sequences are the site at which the GAL4 product exerts its action to activate the GAL7 gene. We also observed that a deletion lacking both upstream activation sequences allowed the gene fusion to be expressed in the absence of galactose at about 10% of the fully induced level of the intact fusion. This constitutive expression depended on the presence of the TATA box of GAL7 in cis but not on a functional GAL4 gene. The level of the uncontrolled expression was decreased by increasing the distance between the TATA box and the pBR322 sequence in the vector plasmid.

Base Sequence↗
PubMed1985

Primary structure of the Saccharomyces cerevisiae GAL7 gene.

We present the nucleotide sequence of a 1599-base pair (bp) DNA fragment containing the entire GAL7 gene that encodes galactose-1-phosphate uridyltransferase of Saccharomyces cerevisiae. The deduced peptide was composed of 364 amino acid residues. The expected molecular weight was 42,005 daltons, which agreed with the observed value for the purified enzyme. The 3'-end of the GAL7 transcript mapped at a position 82 bp downstream from the UAA termination codon by the S1 nuclease protection experiment. We constructed a GAL7'-lac'Z fusion on various types of yeast plasmid vectors. The fused gene on any type of vector was induced by galactose and repressed by glucose as for the GAL7 gene on the chromosome. The response of GAL7'-lac'Z fusion to gal4 delta and gal80 delta regulatory mutations was also similar to the response of the chromosomal GAL7 gene. By using various deletions in the 5'-flanking region of the gene fusion, we delimited the sequence essential for galactose controlled expression with a 180 bp-fragment of DNA lying 92 bp upstream of the transcription initiation site.

Amino Acid Sequence↗
PubMed1984

Nucleotide sequence of the yeast regulatory gene GAL80.

The GAL80 gene in Saccharomyces cerevisiae encodes a negative regulatory protein for the set of inducible genes involving metabolism of galactose and melibiose. We have determined the nucleotide sequence of GAL80 and its flanking regions and assigned the 5' end of its mRNA to the sequence. The deduced coding sequence for GAL80 protein contains 1305 nucleotides and the calculated molecular weight of the peptide chain is 48309. The 5' end of the GAL80 mRNA maps about 67 nucleotides upstream from the translation initiating ATG. We have also determined the nucleotide sequence of uninducible alleles GAL80S-0, GAL80S-1 and GAL80S-2, and found single base substitution in each of these mutant genes which would lead to alteration of amino acid in GAL80 protein.

Alleles↗
PubMed1984

Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. II. The isolation and dosage effect of the regulatory gene GAL80.

The galactose analogue 2-deoxygalactose was found to inhibit the growth of a mutant strain of Saccharomyces cerevisiae constitutively producing the set of galactose utilization enzymes. Based on this fact, the yeast GAL80 gene negatively regulating the expression of the genes encoding those enzymes was isolated for its ability to confer 2-deoxygalactose resistance on a strain carrying a recessive mutation in that gene. The GAL80 gene was located within a 3.0 kb fragment in the cloned DNA. When the isolated gene was incorporated into a multi-copy plasmid, the induced level of three enzymes encoded by the gene cluster GAL7-GAL10-GAL1 in the host chromosome was lowered. Such a gene dosage effect of GAL80 was further pronounced if sucrose, a sugar causing catabolite repression, was added to the growth medium. The ratio of the enzyme activity of the yeast bearing multiple copies of GAL80 to that of the yeast bearing its single copy significantly varied with the enzyme. From these results we suggest that the intracellular inducer interacts with the GAL80 product and that GAL80 molecules directly bind the GAL cluster genes with an affinity different from one gene to another.

Base Sequence↗
PubMed1983

Nucleotide sequence of the transcriptional initiation region of the yeast GAL7 gene.

The GAL7 gene of Saccharomyces cerevisiae encodes Gal-1-P uridylyl transferase, the second enzyme of Leloir pathway for the galactose catabolism. We have determined the sequence of 1003 base pairs surrounding and upstream of the transcriptional initiation site of the GAL7 gene. The region sequenced also encompasses the 3' end of GAL10 gene. The 5' end of GAL7 mRNA was determined on the DNA sequence by the S1 nuclease- and exonuclease VII mapping, which is located 21 to 22 base pairs upstream from the translation initiating ATG codon. The primary structure of the GAL7 5' flanking region has many features common to those of multicellular eukaryotic genes. The 3' end of GAL10 mRNA was also determined by the mapping technique with the single-strand specific nucleases to be about 600 base pairs upstream from the 5' end of GAL7 mRNA.

Base Composition↗
PubMed1983

Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. Isolation and characterization of the regulatory gene GAL4.

The GAL4 gene positively regulating the expression of the gene cluster GAL7-GAL10-GAL1 in the yeast Saccharomyces cerevisiae was isolated for its ability to suppress a recessive mutation in that gene. When the isolated gene was incorporated into a multi-copy plasmid, the GAL cluster genes in the host chromosome partially escaped the normal control; a yeast that harbors the plasmid bearing the GAL4 gene synthesized the galactose-metabolizing enzymes encoded by the GAL cluster genes at a low but significant level in the absence of galactose. If the GAL7 gene was amplified along with GAL4 on the multi-copy plasmid, the constitutive synthesis of Gal-1-P uridylyl transferase encoded by GAL7 was further pronounced and the enzyme activity reached the level of the fully induced wild-type yeast. Such an escape synthesis of the GAL enzymes was not detected if GAL4 or both GAL4 and GAL7 were carried by a single-copy plasmid. The results suggest that the escape synthesis of GAL enzymes observed in the GAL4-amplified yeast was a consequence of overproduction of the GAL4 protein. The GAL80 gene negatively regulating the GAL cluster genes was also isolated, and when amplified together with GAL4, no escape synthesis of the GAL enzymes was observed, suggesting that the balanced synthesis of two regulatory proteins was essential to maintain the repressed state of the GAL cluster genes.

DNA, Fungal↗
PubMed1980

The enzymes of the galactose cluster in Saccharomyces cerevisiae. II. Purification and characterization of uridine diphosphoglucose 4-epimerase.

Uridine diphosphoglucose 4-epimerase (EC 5.1.3.2) of Saccharomyces cerevisiae was purified to homogeneity with a yield of 30%. The purification procedure involved ammonium sulfate precipitation, streptomycin treatment, chromatography on diethylaminoethyl cellulose and hydroxylapatite, and Bio-Gel A-0.5m gel filtration. With the purified enzyme preparation, Km and Vmax values for uridine diphosphogalactose were determined and found to be 0.22 mM and 1.26 mmol/h/mg of protein, respectively. The value of Vmax corresponds to a turnover rate of 3890 molecules of uridine diphosphogalactose converted to uridine diphosphoglucose/min/enzyme molecule. The pH optimum of the enzyme was found to be between 6.8 and 8.0. Amino acid analysis was carried out on the final preparation. Based on the result, the partial specific volume was calculated to be 0.74 ml/g. The NH2-terminal residue of the enzyme was studied by two different methods and found to be threonine. The molecular weight and subunit composition were determined by the combination of the sucrose density gradient centrifugation and gel filtration under nondissociating conditions, and by polyacrylamide gel electrophoresis under dissociating conditions. The results indicated that the enzyme has a molecular weight of 183,000, consisting of two identical subunits. Each molecule of the native enzyme contained 1 molecule of NAD+.

Amino Acids↗