Sequence and nuclear localization of the Saccharomyces cerevisiae HAP2 protein, a transcriptional activator

1987 ◽  
Vol 7 (2) ◽  
pp. 578-585
Author(s):  
J L Pinkham ◽  
J T Olesen ◽  
L P Guarente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Fusion Gene ◽  
Protein A ◽  
Acid Protein ◽  
Genes Encoding ◽  
Beta Galactosidase ◽  
Amino Acid Protein ◽  
Operator Site ◽  
Direct Activator

Activation of the CYC1 upstream activation site (UAS2) and other Saccharomyces cerevisiae genes encoding respiratory functions requires the products of the regulatory loci HAP2 and HAP3. We present here the DNA sequence of the yeast HAP2 gene and an initial investigation into the function of its product. The DNA sequence indicated that HAP2 encoded a 265-amino-acid protein whose carboxyl third was highly basic. Also found in the sequence was a polyglutamine tract spanning residues 120 to 133. Several experiments described herein suggest that HAP2 encodes a direct activator of transcription. First, a bifunctional HAP2-beta-galactosidase fusion gene was localized to the yeast nucleus. Second, a lexA-HAP2 fusion gene was capable of activating transcription when bound to a lexA operator site. The additional requirement for the HAP3 product in activation is discussed.

scholarly journals Sequence and nuclear localization of the Saccharomyces cerevisiae HAP2 protein, a transcriptional activator.

1987 ◽  
Vol 7 (2) ◽  
pp. 578-585 ◽  
Author(s):  
J L Pinkham ◽  
J T Olesen ◽  
L P Guarente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Fusion Gene ◽  
Protein A ◽  
Acid Protein ◽  
Genes Encoding ◽  
Beta Galactosidase ◽  
Amino Acid Protein ◽  
Operator Site ◽  
Direct Activator

Activation of the CYC1 upstream activation site (UAS2) and other Saccharomyces cerevisiae genes encoding respiratory functions requires the products of the regulatory loci HAP2 and HAP3. We present here the DNA sequence of the yeast HAP2 gene and an initial investigation into the function of its product. The DNA sequence indicated that HAP2 encoded a 265-amino-acid protein whose carboxyl third was highly basic. Also found in the sequence was a polyglutamine tract spanning residues 120 to 133. Several experiments described herein suggest that HAP2 encodes a direct activator of transcription. First, a bifunctional HAP2-beta-galactosidase fusion gene was localized to the yeast nucleus. Second, a lexA-HAP2 fusion gene was capable of activating transcription when bound to a lexA operator site. The additional requirement for the HAP3 product in activation is discussed.

Isolation, DNA sequence, and regulation of a Saccharomyces cerevisiae gene that encodes DNA strand transfer protein alpha

1991 ◽  
Vol 11 (5) ◽  
pp. 2576-2582
Author(s):  
A B Clark ◽  
C C Dykstra ◽  
A Sugino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Specific Activity ◽  
Intragenic Recombination ◽  
Strand Transfer ◽  
Homologous Pairing ◽  
Spore Viability ◽  
Transfer Protein ◽  
Acid Protein ◽  
Dna Strand ◽  
Amino Acid Protein

DNA strand transfer protein alpha (STP alpha) from meiotic Saccharomyces cerevisiae cells promotes homologous pairing of DNA without any nucleotide cofactor in the presence of yeast single-stranded DNA binding protein. This gene (DNA strand transferase 1, DST1) encodes a 309-amino-acid protein with a predicted molecular mass of 34,800 Da. The STP alpha protein level is constant in both mitotic and meiotic cells, but during meiosis the polypeptide is activated by an unknown mechanism, resulting in a large increase in its specific activity. A dst1::URA3/dst1::URA3 mutant grows normally in mitotic media; however, meiotic cells exhibit a greatly reduced induction of both DNA strand transfer activity and intragenic recombination between his1 heteroalleles. Spore viability is normal. These results suggest that DST1 is required for much of the observed induction of homologous recombination in S. cerevisiae during meiosis but not for normal sporulation.

Trans-acting regulatory mutations that alter transcription of Saccharomyces cerevisiae histone genes

1987 ◽  
Vol 7 (12) ◽  
pp. 4204-4210
Author(s):  
M A Osley ◽  
D Lycan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Fusion Gene ◽  
Chromosome Replication ◽  
Lacz Fusion ◽  
Histone Genes ◽  
Promoter Elements ◽  
Regulatory Mutations ◽  
Cycle Dependent ◽  
Beta Galactosidase

Using a Saccharomyces cerevisiae strain containing an integrated copy of an H2A-lacZ fusion gene, we screened for mutants which overexpressed beta-galactosidase as a way to identify genes which regulate transcription of the histone genes. Five recessive mutants with this phenotype were shown to contain altered regulatory genes because they had lost repression of HTA1 transcription which occurs upon inhibition of chromosome replication (D. E. Lycan, M. A. Osley, and L. Hereford, Mol. Cell. Biol. 7:614-621, 1987). Periodic transcription was affected in the mutants as well, since the HTA1 gene was transcribed during the G1 and G2 phases of the cell cycle, periods in the cell cycle when this gene is normally not expressed. A similar loss of cell cycle-dependent transcription was noted for two of the three remaining histone loci, while the HO and CDC9 genes continued to be expressed periodically. Using isolated promoter elements inserted into a heterologous cycl-lacZ fusion gene, we demonstrated that the mutations fell in genes which acted through a negative site in the TRT1 H2A-H2B promoter.

The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter

1991 ◽  
Vol 11 (6) ◽  
pp. 3229-3238
Author(s):  
M Bun-Ya ◽  
M Nishimura ◽  
S Harashima ◽  
Y Oshima
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Gene ◽  
Amino Acid Residues ◽  
Sugar Transporters ◽  
Pi Transport ◽  
Constitutive Synthesis ◽  
A Site ◽  
Mutant Cells ◽  
Hydropathy Analysis ◽  
Beta Galactosidase

The PHO84 gene specifies Pi-transport in Saccharomyces cerevisiae. A DNA fragment bearing the PHO84 gene was cloned by its ability to complement constitutive synthesis of repressible acid phosphatase of pho84 mutant cells. Its nucleotide sequence predicted a protein of 596 amino acids with a sequence homologous to that of a superfamily of sugar transporters. Hydropathy analysis suggested that the secondary structure of the PHO84 protein consists of two blocks of six transmembrane domains separated by 74 amino acid residues. The cloned PH084 DNA restored the Pi transport activity of pho84 mutant cells. The PHO84 transcription was regulated by Pi like those of the PHO5, PHO8, and PHO81 genes. A PHO84-lacZ fusion gene produced beta-galactosidase activity under the regulation of Pi, and the activity was suggested to be bound to a membrane fraction. Gene disruption of PHO84 was not lethal. By comparison of nucleotide sequences and by tetrad analysis with GAL80 as a standard, the PHO84 locus was mapped at a site beside the TUB3 locus on the left arm of chromosome XIII.

scholarly journals Expression of the Xylulose 5-Phosphate Phosphoketolase Gene, xpkA, from Lactobacillus pentosus MD363 Is Induced by Sugars That Are Fermented via the Phosphoketolase Pathway and Is Repressed by Glucose Mediated by CcpA and the Mannose Phosphoenolpyruvate Phosphotransferase System

2002 ◽  
Vol 68 (2) ◽  
pp. 831-837 ◽  
Author(s):  
Clara C. Posthuma ◽  
Rechien Bader ◽  
Roswitha Engelmann ◽  
Pieter W. Postma ◽  
Wolfgang Hengstenberg ◽  
...  
Keyword(s):  
Protein A ◽  
Pyruvate Decarboxylase ◽  
Thiamine Pyrophosphate ◽  
Phosphotransferase System ◽  
Knockout Mutant ◽  
Lactobacillus Pentosus ◽  
Acid Protein ◽  
Phosphoketolase Pathway ◽  
Calculated Mass ◽  
Amino Acid Protein

ABSTRACT Purification of xylulose 5-phosphate phosphoketolase (XpkA), the central enzyme of the phosphoketolase pathway (PKP) in lactic acid bacteria, and cloning and sequence analysis of the encoding gene, xpkA, from Lactobacillus pentosus MD363 are described. xpkA encodes a 788-amino-acid protein with a calculated mass of 88,705 Da. Expression of xpkA in Escherichia coli led to an increase in XpkA activity, while an xpkA knockout mutant of L. pentosus lost XpkA activity and was not able to grow on energy sources that are fermented via the PKP, indicating that xpkA encodes an enzyme with phosphoketolase activity. A database search revealed that there are high levels of similarity between XpkA and a phosphoketolase from Bifidobacterium lactis and between XpkA and a (putative) protein present in a number of evolutionarily distantly related organisms (up to 54% identical residues). Expression of xpkA in L. pentosus was induced by sugars that are fermented via the PKP and was repressed by glucose mediated by carbon catabolite protein A (CcpA) and by the mannose phosphoenolpyruvate phosphotransferase system. Most of the residues involved in correct binding of the cofactor thiamine pyrophosphate (TPP) that are conserved in transketolase, pyruvate decarboxylase, and pyruvate oxidase were also conserved at a similar position in XpkA, implying that there is a similar TPP-binding fold in XpkA.

scholarly journals Molecular mechanism of autophagy in yeast, Saccharomyces cerevisiae

1999 ◽  
Vol 354 (1389) ◽  
pp. 1577-1581 ◽  
Author(s):  
Y. Ohsumi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Conjugation System ◽  
Yeast Saccharomyces Cerevisiae ◽  
Whole Process ◽  
Unique Protein ◽  
Acid Protein ◽  
Major Route ◽  
Lytic Compartment ◽  
Amino Acid Protein

Bulk degradation of cytosol and organelles is important for cellular homeostasis under nutrient limitation, cell differentiation and development. This process occurs in a lytic compartment, and autophagy is the major route to the lysosome and/or vacuole. We found that yeast, Saccharomyces cerevisiae , induces autophagy under various starvation conditions. The whole process is essentially the same as macroautophagy in higher eukaryotic cells. However, little is known about the mechanism of autophagy at a molecular level. To elucidate the molecules involved, a genetic approach was carried out and a total of 16 autophagy–defective mutants ( apg ) were isolated. So far, 14 APG genes have been cloned. Among them we recently found a unique protein conjugation system essential for autophagy. The C–terminal glycine residue of a novel modifier protein Apg12p, a 186–amino–acid protein, is conjugated to a lysine residue of Apg5p, a 294–amino–acid protein, via an isopeptide bond. We also found that apg7 and apg10 mutants were unable to form an Apg12p–Apg5p conjugate. The conjugation reaction is mediated via Apg7p, E1–like activating enzyme and Apg10p, indicating that it is a ubiquitination–like system. These APG genes have mammalian homologues, suggesting that the Apg12 system is conserved from yeast to human. Further molecular and cell biological analyses of APG gene products will give us crucial clues to uncover the mechanism and regulation of autophagy.

scholarly journals Suppression of mutations in two Saccharomyces cerevisiae genes by the adenovirus E1A protein.

1995 ◽  
Vol 15 (6) ◽  
pp. 3227-3237 ◽  
Author(s):  
H A Zieler ◽  
M Walberg ◽  
P Berg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Single Gene ◽  
Adenovirus E1a ◽  
C Terminus ◽  
Acid Protein ◽  
Conserved Region ◽  
Mutant Phenotypes ◽  
Yeast Genes ◽  
Amino Acid Protein

The protein products of the adenoviral E1A gene are implicated in a variety of transcriptional and cell cycle events, involving interactions with several proteins present in human cells, including parts of the transcriptional machinery and negative regulators of cell division such as the Rb gene product and p107. To determine if there are functional homologs of E1A in Saccharomyces cerevisiae, we have developed a genetic screen for mutants that depend on E1A for growth. The screen is based on a colony color sectoring assay which allows the identification of mutants dependent on the maintenance and expression of an E1A-containing plasmid. Using this screen, we have isolated five mutants that depend on expression of the 12S or 13S cDNA of E1A for growth. A plasmid shuffle assay confirms that the plasmid-dependent phenotype is due to the presence of either the 12S or the 13S E1A cDNA and that both forms of E1A rescue growth of all mutants equally well. The five mutants fall into two classes that were named web1 and web2 (for "wants E1A badly"). Plasmid shuffle assays with mutant forms of E1A show that conserved region 1 (CR1) is required for rescue of the growth of the web1 and web2 E1A-dependent yeast mutants, while the N-terminal 22 amino acids are only partially required; conserved region 2 (CR2) and the C terminus are dispensable. The phenotypes of mutants in both the web1 and the web2 groups are due to a single gene defect, and the yeast genes that fully complement the mutant phenotypes of both groups were cloned. The WEB1 gene sequence encodes a 1,273-amino-acid protein that is identical to SEC31, a protein involved in the budding of transport vesicles from the endoplasmic reticulum. The WEB2 gene encodes a 1,522-amino-acid protein with homology to nucleic acid-dependent ATPases. Deletion of either WEB1 or WEB2 is lethal. Expression of E1A is not able to rescue the lethality of either the web1 or the web2 null allele, implying allele-specific mutations that lead to E1A dependence.

scholarly journals Repression of the genes for lysine biosynthesis in Saccharomyces cerevisiae is caused by limitation of Lys14-dependent transcriptional activation

1994 ◽  
Vol 14 (10) ◽  
pp. 6411-6418
Author(s):  
A Feller ◽  
E Dubois ◽  
F Ramos ◽  
A Piérard
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Activation Function ◽  
Lysine Biosynthesis ◽  
Binding Motif ◽  
Terminal Portion ◽  
Rich Domain ◽  
Acid Protein ◽  
Amino Acid Protein

The product of the LYS14 gene of Saccharomyces cerevisiae activates the transcription of at least four genes involved in lysine biosynthesis. Physiological and genetic studies indicate that this activation is dependent on the inducer alpha-aminoadipate semialdehyde, an intermediate of the pathway. The gene LYS14 was sequenced and, from its nucleotide sequence, predicted to encode a 790-amino-acid protein carrying a cysteine-rich DNA-binding motif of the Zn(II)2Cys6 type in its N-terminal portion. Deletion of this N-terminal portion including the cysteine-rich domain resulted in the loss of LYS14 function. To test the function of Lys14 as a transcriptional activator, this protein without its DNA-binding motif was fused to the DNA-binding domain of the Escherichia coli LexA protein. The resulting LexA-Lys14 hybrid protein was capable of activating transcription from a promoter containing a lexA operator, thus confirming the transcriptional activation function of Lys14. Furthermore, evidence that this function, which is dependent on the presence of alpha-aminoadipate semialdehyde, is antagonized by lysine was obtained. Such findings suggest that activation by alpha-aminoadipate semialdehyde and the apparent repression by lysine are related mechanisms. Lysine possibly acts by limiting the supply of the coinducer, alpha-aminoadipate semialdehyde.

MEI4, a meiosis-specific yeast gene required for chromosome synapsis

1992 ◽  
Vol 12 (3) ◽  
pp. 1340-1351
Author(s):  
T M Menees ◽  
P B Ross-MacDonald ◽  
G S Roeder
Keyword(s):  
Fusion Gene ◽  
Blot Hybridization ◽  
Coding Region ◽  
Yeast Saccharomyces Cerevisiae ◽  
Type Locus ◽  
Chromosome Synapsis ◽  
Acid Protein ◽  
Mutant Strains ◽  
Rad50 Gene ◽  
Amino Acid Protein

The MEI4 gene product is required for meiotic induction of recombination and viable spore production in the yeast Saccharomyces cerevisiae. DNA sequence analysis shows that the MEI4 gene encodes a 450-amino-acid protein bearing no homology to any previously identified protein. The MEI4 coding region is interrupted by a small intron located near the 5' end of the gene. Efficient splicing of the MEI4 transcript is not dependent on the MER1 protein, which is required for splicing the transcript of another meiotic gene, MER2. Expression of a mei4::lacZ fusion gene is meiosis-specific and depends on both heterozygosity at the mating-type locus and nutrient limitation. Northern (RNA) blot hybridization analysis suggests that MEI4 gene expression is regulated at the level of transcription. A functional MEI4 gene is not required for meiotic induction of transcription of the MER1, MER2, MEK1, RED1, SPO11, or RAD50 gene. Cytological analysis of mei4 mutant strains during meiotic prophase demonstrates that the chromosomes form long axial elements that fail to undergo synapsis. The meiosis II division is delayed in mei4 strains.

The DAL81 gene product is required for induced expression of two differently regulated nitrogen catabolic genes in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (2) ◽  
pp. 1161-1166 ◽  
Author(s):  
P A Bricmont ◽  
J R Daugherty ◽  
T G Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Amino Acid ◽  
The Other ◽  
Loss Of Function ◽  
Induced Expression ◽  
Catabolic Genes ◽  
Acid Protein ◽  
Steady State Levels ◽  
Amino Acid Protein

We demonstrate that the DAL81 gene, previously thought to be specifically required for induced expression of the allantoin pathway genes in Saccharomyces cerevisiae, functions in a more global manner. The data presented show it to be required for utilization of 4-aminobutyrate as a nitrogen source and for 4-aminobutyrate-induced increases in the steady-state levels of UGA1 mRNA. The DAL81 gene encodes a 970-amino-acid protein containing sequences homologous to the Zn(II)2Cys6 motif and two stretches of polyglutamine residues. Deletion of sequences homologous to the Zn(II)2Cys6 motif did not result in a detectable loss of function. On the other hand, loss of one of the polyglutamine stretches, but not the other, resulted in a 50% loss of DAL81 function.

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