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 DAPP1: a dual adaptor for phosphotyrosine and 3-phosphoinositides

1999 ◽  
Vol 342 (1) ◽  
pp. 7-12 ◽  
Author(s):  
Simon DOWLER ◽  
Richard A. CURRIE ◽  
C. Peter DOWNES ◽  
Dario R. ALESSI
Keyword(s):  
Amino Acid ◽  
Sh2 Domain ◽  
Ph Domain ◽  
Phosphorylated Proteins ◽  
High Affinity ◽  
C Terminus ◽  
Acid Protein ◽  
N Terminus ◽  
Src Homology ◽  
Amino Acid Protein

We have identified a novel 280 amino acid protein which contains a putative myristoylation site at its N-terminus followed by an Src homology (SH2) domain and a pleckstrin homology (PH) domain at its C-terminus. It has been termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1). DAPP1 is widely expressed and exhibits high-affinity interactions with PtdIns(3,4,5)P3 and PtdIns(3,4)P2, but not with other phospholipids tested. These observations predict that DAPP1 will interact with both tyrosine phosphorylated proteins and 3-phosphoinositides and may therefore play a role in regulating the location and/or activity of such proteins(s) in response to agonists that elevate PtdIns(3,4,5)P3 and PtdIns(3,4)P2.

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.

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.

scholarly journals TEC1, a gene involved in the activation of Ty1 and Ty1-mediated gene expression in Saccharomyces cerevisiae: cloning and molecular analysis.

1990 ◽  
Vol 10 (7) ◽  
pp. 3541-3550 ◽  
Author(s):  
I Laloux ◽  
E Dubois ◽  
M Dewerchin ◽  
E Jacobs
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Molecular Analysis ◽  
Mating Type ◽  
Gene Activation ◽  
Adjacent Gene ◽  
Transcript Levels ◽  
Acid Protein ◽  
Amino Acid Protein

Ty and Ty-mediated gene expression observed in haploid cells of Saccharomyces cerevisiae depends on several determinants, some of which are required for the expression of haploid-specific genes. We report here the cloning and molecular analysis of TEC1. TEC1 encodes a 486-amino-acid protein that is a trans-acting factor required for full Ty1 expression and Ty1-mediated gene activation. However, mutation or deletion of the TEC1 gene had little effect on total Ty2 transcript levels. Our analysis provides clear evidence that TEC1 is not involved in mating or sporulation processes. Unlike most of the proteins involved in Ty and adjacent gene expression, the product of TEC1 has no known cellular function. Although there was no mating-type effect on TEC1 expression, our results indicate that the TEC1 and the a/alpha diploid controls on Ty1 expression are probably not cumulative.

scholarly journals 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.

The SIR1 gene of Saccharomyces cerevisiae and its role as an extragenic suppressor of several mating-defective mutants

1991 ◽  
Vol 11 (4) ◽  
pp. 2253-2262
Author(s):  
E M Stone ◽  
M J Swanson ◽  
A M Romeo ◽  
J B Hicks ◽  
R Sternglanz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Gene Product ◽  
Open Reading Frame ◽  
Histone H4 ◽  
Reading Frame ◽  
Acid Protein ◽  
Extragenic Suppressor ◽  
Mating Type Genes ◽  
Amino Acid Protein

The SIR1 gene product of Saccharomyces cerevisiae is one of several proteins involved in repressing transcription of the silent mating-type genes. Strains with mutations in the genes coding for these proteins are defective in mating due to derepression of the silent loci. We have found that overexpression of the SIR1 gene suppresses the mating defects of several of these mutants, including nat1 and ard1 mutants (the products of these two genes are responsible for N-terminal acetylation of a subset of yeast proteins), certain sir3 mutants, and a histone H4 mutant. The SIR1 gene has been sequenced and found to contain an open reading frame coding for a 678-amino-acid protein.

scholarly journals The SIR1 gene of Saccharomyces cerevisiae and its role as an extragenic suppressor of several mating-defective mutants.

1991 ◽  
Vol 11 (4) ◽  
pp. 2253-2262 ◽  
Author(s):  
E M Stone ◽  
M J Swanson ◽  
A M Romeo ◽  
J B Hicks ◽  
R Sternglanz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Gene Product ◽  
Open Reading Frame ◽  
Histone H4 ◽  
Reading Frame ◽  
Acid Protein ◽  
Extragenic Suppressor ◽  
Mating Type Genes ◽  
Amino Acid Protein

The SIR1 gene product of Saccharomyces cerevisiae is one of several proteins involved in repressing transcription of the silent mating-type genes. Strains with mutations in the genes coding for these proteins are defective in mating due to derepression of the silent loci. We have found that overexpression of the SIR1 gene suppresses the mating defects of several of these mutants, including nat1 and ard1 mutants (the products of these two genes are responsible for N-terminal acetylation of a subset of yeast proteins), certain sir3 mutants, and a histone H4 mutant. The SIR1 gene has been sequenced and found to contain an open reading frame coding for a 678-amino-acid protein.

TEC1, a gene involved in the activation of Ty1 and Ty1-mediated gene expression in Saccharomyces cerevisiae: cloning and molecular analysis

1990 ◽  
Vol 10 (7) ◽  
pp. 3541-3550
Author(s):  
I Laloux ◽  
E Dubois ◽  
M Dewerchin ◽  
E Jacobs
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Molecular Analysis ◽  
Mating Type ◽  
Gene Activation ◽  
Adjacent Gene ◽  
Transcript Levels ◽  
Acid Protein ◽  
Amino Acid Protein

Ty and Ty-mediated gene expression observed in haploid cells of Saccharomyces cerevisiae depends on several determinants, some of which are required for the expression of haploid-specific genes. We report here the cloning and molecular analysis of TEC1. TEC1 encodes a 486-amino-acid protein that is a trans-acting factor required for full Ty1 expression and Ty1-mediated gene activation. However, mutation or deletion of the TEC1 gene had little effect on total Ty2 transcript levels. Our analysis provides clear evidence that TEC1 is not involved in mating or sporulation processes. Unlike most of the proteins involved in Ty and adjacent gene expression, the product of TEC1 has no known cellular function. Although there was no mating-type effect on TEC1 expression, our results indicate that the TEC1 and the a/alpha diploid controls on Ty1 expression are probably not cumulative.

scholarly journals Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by Sphingomonas paucimobilis SYK-6

2005 ◽  
Vol 187 (15) ◽  
pp. 5067-5074 ◽  
Author(s):  
Daisuke Kasai ◽  
Eiji Masai ◽  
Keisuke Miyauchi ◽  
Yoshihiro Katayama ◽  
Masao Fukuda
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Terminal Region ◽  
Ring Cleavage ◽  
Sphingomonas Paucimobilis ◽  
Acid Protein ◽  
Demethylation Pathway ◽  
Dioxygenase Gene ◽  
Amino Acid Protein

ABSTRACT Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The Km for gallate and the V max were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

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