Biomedical subjects

S Irniger

Publications and source records attributed to S Irniger.

15 recordsLinked to original sources

PubMed2001

Induction of jlbA mRNA synthesis for a putative bZIP protein of Aspergillus nidulans by amino acid starvation.

The jlbA (jun-like hZIP) gene of Aspergillus nidulans was isolated. The deduced amino acid motif of the C-terminal region of jlhA encodes a putative DNA-binding site composed of a basic amino acid domain and an adjacent leucine zipper motif. This region shares highest similarities to the C-terminal DNA-binding domain and the basic zipper (bZIP)-motifs of transcription factors like CPCA from A. niger, Gcn4p from Saccharomyces cerevisiae, human JUNB and c-JUN. The putative jlbA protein contains a PEST-rich region (an instability region rich in the amino acids proline, glutamic acid, serine and threonine) described to be implicated in protein stability. The jlbA mRNA formation is elevated up to 40-fold upon amino acid starvation induced by the addition of the false feedback inhibitor 3-amino-1,2,4-triazole. This induction is partially dependent and partially independent on the presence of the transcription factor CPCA. Therefore jlbA is a novel gene of A. nidulans which is transcriptionally activated by amino acid starvation conditions.

Amino Acid Sequence↗

PubMed2000

Yeast Ran-binding protein Yrb1p is required for efficient proteolysis of cell cycle regulatory proteins Pds1p and Sic1p.

Ubiquitin-dependent proteolysis of specific target proteins is required for several important steps during the cell cycle. Degradation of such proteins is strictly cell cycle-regulated and triggered by two large ubiquitin ligases, termed anaphase-promoting complex (APC) and Skp1/Cullin/F-box complex (SCF). Here we show that yeast Ran-binding protein 1 (Yrb1p), a predominantly cytoplasmic protein implicated in nucleocytoplasmic transport, is required for cell cycle regulated protein degradation. Depletion of Yrb1p results in the accumulation of unbudded G(1) cells and of cells arrested in mitosis implying a function of Yrb1p in the G(1)/S transition and in the progression through mitosis. Temperature-sensitive yrb1-51 mutants are defective in APC-mediated degradation of the anaphase inhibitor protein Pds1p and in degradation of the cyclin-dependent kinase inhibitor Sic1p, a target of SCF. Thus, Yrb1p is crucial for efficient APC- and SCF-mediated proteolysis of important cell cycle regulatory proteins. We have identified the UBS1 gene as a multicopy suppressor of yrb1-51 mutants. Ubs1p is a nuclear protein, and its deletion is synthetic lethal with a yrb1-51 mutation. Interestingly, UBS1 was previously identified as a multicopy suppressor of cdc34-2 mutants, which are defective in SCF activity. We suggest that Ubs1p may represent a link between nucleocytoplasmic transport and ubiquitin ligase activity.

Anaphase↗

PubMed2000

Two different modes of cyclin clb2 proteolysis during mitosis in Saccharomyces cerevisiae.

Sister chromatid separation and mitotic exit are triggered by the anaphase-promoting complex (APC/C) which is a multi-subunit ubiquitin ligase required for proteolytic degradation of various target proteins. Cdc20 and Cdh1 are substrate-specific activators of the APC/C. It was previously proposed that Cdh1 is essential for proteolysis of the yeast mitotic cyclin Clb2. We show that Clb2 proteolysis is triggered by two different modes during mitosis. A fraction of Clb2 is degraded during anaphase in the absence of Cdh1. However, a second fraction of Clb2 remains stable during anaphase and is degraded in a Cdh1-dependent manner as cells exit from mitosis. Most of cyclin Clb3 is degraded independently of Cdh1. Our data imply that degradation of mitotic cyclins is initiated by a Cdh1-independent mechanism.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2000

Glucose and ras activity influence the ubiquitin ligases APC/C and SCF in Saccharomyces cerevisiae.

In budding yeast, the Ras/cAMP pathway is involved in the coordination of cell growth and cell division. Glucose-rich medium stimulates Ras/cAMP signaling, which causes an increase in the critical cell size for cell cycle entry. Here we show that glucose and activated Ras proteins also influence the function of the anaphase-promoting complex (APC/C), a ubiquitin-protein ligase required for sister chromatid separation and mitotic exit. We found that apc10-22 and other mutants defective in the APC/C are suppressed by reduced Ras signaling activity, by a deletion of the RAS2 gene, by a cdc25 mutation, by elevated levels of PDE2, or by growth without glucose. Viability of these mutants is also enhanced by decreased Cdk1 activity. In contrast, a constitutively activated RAS2(Val19) allele or shifts to glucose medium are deleterious to apc10-22 mutants. Remarkably, cdc34-2 mutants, which are impaired in SCF function, are differently affected with respect to Ras activity. Viability of cdc34-2 mutants at elevated temperatures is dependent on glucose and the RAS2 gene. We conclude that glucose and Ras proteins influence the APC/C and the SCF complex in an opposite manner. These ubiquitin ligases might represent novel targets for modulating cell division in response to growth conditions.

Cell Cycle Proteins↗

PubMed1997

Sequence requirements of the bidirectional yeast TRP4 mRNA 3'-end formation signal.

The yeast TRP4 3'-end formation signal functions in both orientations in an in vivo test system. We show here that the TRP4 3'-end formation element consists of two functionally different sequence regions. One region of approximately 70 nucleotides is located in the untranslated region between the translational stop codon and the major poly(A) site. The major poly(A) site is not part of this region and can be deleted without a decrease in TRP4 3'-end formation. 5'and 3'deletions and point mutations within this region affected 3'-end formation similarly in both orientations. In the center of this region the motif TAGT is located on the antisense strand. Point mutations within this motif resulted in a drastic reduce of 3'-end formation activity in both orientations. A second region consists of the 3'-end of the TRP4 open reading frame and is required for 3'-end formation in forward orientation. A single point mutation in a TAGT motif of the TRP4 open reading frame abolished TRP4 mRNA 3'-end formation in forward orientation and had no effect on the reverse orientation.

Base Sequence↗

PubMed1997

The anaphase-promoting complex is required in G1 arrested yeast cells to inhibit B-type cyclin accumulation and to prevent uncontrolled entry into S-phase.

Inactivation of B-type cyclin dependent kinases due to ubiquitin-mediated cyclin proteolysis is necessary for the exit from mitosis. In Saccharomyces cerevisiae, proteolysis is initiated at the onset of anaphase and remains active until Cln1 and Cln2 cyclins appear in late G1 of the subsequent cell cycle. A large particle called the anaphase-promoting complex (APC) which is composed of the TPR proteins Cdc16p/Cdc23p/Cdc27p and other proteins is required for B-type cyclin ubiquitination in both anaphase and during G1 phase. The APC has an essential role for the separation of sister chromatids and for the exit from mitosis, but until now it was unclear whether the persistence of APC activity throughout G1 had any physiological role. We show here that the APC is needed in G1 arrested cells to inhibit premature appearance of B-type cyclins and to prevent unscheduled initiation of DNA replication. When pheromone arrested cells of cdc16 and cdc23 mutants were shifted to the restrictive temperature, they underwent DNA replication in the presence of pheromone. DNA replication also occurred in a G1 arrest induced by G1 cyclin (Cln) depletion, indicating that mutant cells with a defective APC initiate DNA replication without the Cln G1 cyclins, which are normally needed for the onset of S-phase. Degradation of Clb2p, Clb3p and Clb5p depends on the APC. This suggests that accumulation of any one of the six B-type cyclin proteins could account for the precocious replication of cdc16 and cdc23 mutants.

Anaphase↗

PubMed1995

Genes involved in sister chromatid separation are needed for B-type cyclin proteolysis in budding yeast.

B-type cyclin destruction is necessary for exit from mitosis and the initiation of a new cell cycle. Through the isolation of mutants, we have identified three essential yeast genes, CDC16, CDC23, and CSE1, which are required for proteolysis of the B-type cyclin CLB2 but not of other unstable proteins. cdc23-1 mutants are defective in both entering and exiting anaphase. Their failure to exit anaphase can be explained by defective cyclin proteolysis. CDC23 is required at the metaphase/anaphase transition to separate sister chromatids, and we speculate that it might promote proteolysis of proteins that hold sister chromatids together. Proteolysis of CLB2 is initiated in early anaphase, but a fraction of CLB2 remains stable until anaphase is complete.

Anaphase↗

PubMed1994

Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle.

It is thought that DNA replication and mitosis in yeast are triggered by oscillations in the level of G1-specific (CLN1 and CLN2) and G2-specific (CLB1-CLB4) cyclins, which determine the substrate specificity of the CDC28 protein kinase. It is not understood how the time and order of appearance of different cyclin types are determined. We show here that CLB2 proteolysis, which is important for transition from mitosis to G1, is not confined to a narrow window at the end of mitosis as previously thought but continues until reactivation of CDC28 by CLN cyclins toward the end of the subsequent G1 period. Thus, cell cycle-regulated proteolysis prevents accumulation of G2-specific CLB cyclins during G1 and thereby ensures that the CLN-associated forms of the CDC28 kinase are activated without interference from CLB cyclins. Accumulation of CLN cyclins leads to inactivation of CLB cyclin proteolysis, which is a precondition for subsequent activation of G2-specific B-type cyclins.

Anaphase-Promoting Complex-Cyclosome↗

PubMed1994

Saturation mutagenesis of a polyadenylation signal reveals a hexanucleotide element essential for mRNA 3' end formation in Saccharomyces cerevisiae.

The cis-acting signal sequences required for mRNA 3' end formation are highly conserved and well characterized in higher eukaryotes. However, the situation in the yeast Saccharomyces cerevisiae is still unclear. Several sequences have been proposed which share only limited similarities. One difficulty in identifying yeast polyadenylylation signals might be the presence of redundant signal sequences in the 3' region of yeast genes. To circumvent this problem we have analyzed the heterologous 3' region from cauliflower mosaic virus which contains a yeast polyadenylylation signal. We have performed a saturation mutagenesis of the key element TAG-TATGTA, which is a condensed version of the polyadenylylation signal TAG ... TATGTA ... (TTT) which had previously been proposed. Each of the nine nucleotides was replaced by the three other possible nucleotides and all resulting 1-bp mutants were tested for their capacity to specify mRNA 3' end formation in yeast cells. The first three nucleotides of this condensed sequence are not required, but mutagenesis of the other six nucleotides had distinct effects on mRNA 3' end formation. All mutants that were significantly functional had the sequence TAYRTA, and the sequence TATATA had the best capacity for mRNA 3' end formation. The two thymidine residues at the first and fifth positions are the most essential nucleotides in this sequence. Our results suggest that a degenerate hexanucleotide is essential for mRNA 3' end formation in yeast. This is reminiscent of the conserved polyadenylylation signal in higher eukaryotes, AATAAA.

Base Sequence↗

PubMed1993

Messenger RNA 3'-end formation of a DNA fragment from the human c-myc 3'-end region in Saccharomyces cerevisiae.

We have tested the functioning of the human c-myc polyadenylation signal in Saccharomyces cerevisiae. A DNA fragment containing the two AATAAA polyadenylation signals of the c-myc gene was inserted into a plasmid designed for the in-vivo testing of polyadenylation signals in yeast. The c-myc fragment had a partial capacity for directing mRNA 3'-end formation in yeast. The 3'-endpoints were 50-100 bp distant from the mRNA 3'-ends mapped in humans. This human DNA fragment is therefore unspecifically functional in yeast, indicating that other sequence elements than the human polyadenylation signal, AATAAA, are necessary for 3'-end formation.

Base Sequence↗

PubMed1992

The yeast actin intron contains a cryptic promoter that can be switched on by preventing transcriptional interference.

We show that the single intron of the actin gene of the yeast Saccharomyces cerevisiae contains a cryptic promoter for transcription of the second exon. This promoter is inactive in the normal actin gene, but can be activated when the actin gene promoter is deleted. An identical activation was induced by placing efficient transcriptional terminators at position 61 of the 309 bp intron. In all cases transcripts with identical 5' ends close to the boundary of the intron and the second exon were produced. These results indicate that the cryptic promoter in the actin intron is occluded in the normal actin gene by transcriptional interference with the actin gene promoter. Transcription initiation near the intron/exon 2 boundary is enabled by protection from traversing polymerases, that initiated transcription at the upstream located actin gene promoter. A partial promoter protection using leaky terminators resulted in small amounts of transcripts initiated from the cryptic promoter. Although we do not know any function of the cryptic promoter in actin gene expression, it is tentative to speculate that the cryptic intron promoter might be a relict of a promoter that was functional earlier in evolution.

Actins↗

PubMed1992

Cloning, primary structure and regulation of the ARO4 gene, encoding the tyrosine-inhibited 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, two differently regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (DAHPS; EC 4.1.2.15) isoenzymes carry out the first step in the shikimate pathway. Mutations in both genes are necessary to cause aromatic amino acid (aa) auxotrophy, since one isoenzyme alone is sufficient to produce enough DAHP for normal growth of the cells. The phenylalanine-inhibited DAHPS is encoded by the previously isolated and characterized ARO3 gene. Here, we report the cloning and characterization of the ARO4 gene, encoding the second DAHPS, which is inhibited by tyrosine. The aa sequence of the ARO4 gene product reveals 76% similarity to the ARO3-encoded isoenzyme and 66 and 73% to the three DAHPS isoenzymes from Escherichia coli. ARO4 gene expression is regulated by the general control system of aa biosynthesis. As in the case of the ARO3 gene, a single GCN4-recognition element in the promoter is responsible for derepression of the ARO4 gene under aa starvation conditions. However, in contrast to the situation in the isogene, ARO3, GCN4 does not contribute to the basal level of ARO4 transcription under nonderepressing conditions.

3-Deoxy-7-Phosphoheptulonate Synthase↗

PubMed1992

Different sequence elements are required for function of the cauliflower mosaic virus polyadenylation site in Saccharomyces cerevisiae compared with in plants.

We show that the polyadenylation site derived from the plant cauliflower mosaic virus (CaMV) is specifically functional in the yeast Saccharomyces cerevisiae. The mRNA 3' endpoints were mapped at the same position in yeast cells as in plants, and the CaMV polyadenylation site was recognized in an orientation-dependent manner. Mutational analysis of the CaMV 3'-end-formation signal revealed that multiple elements are essential for proper activity in yeast cells, including two upstream elements that are situated more than 100 and 43 to 51 nucleotides upstream of the poly(A) addition site and the sequences at or near the poly(A) addition site. A comparison of the sequence elements that are essential for proper function of the CaMV signal in yeast cells and plants showed that both organisms require a distal and a proximal upstream element but that these sequence elements are not identical in yeast cells and plants. The key element for functioning of the CaMV signal in yeast cells is the sequence TAGTATGTA, which is similar to a sequence previously proposed to act in yeast cells as a bipartite signal, namely, TAG ... TATGTA. Deletion of this sequence in the CaMV polyadenylation signal abolished 3'-end formation in yeast cells, and a single point mutation in this motif reduced the activity of the CaMV signal to below 15%. These results indicate that the bipartite sequence element acts as a signal for 3'-end formation in yeast cells but only together with other cis-acting elements.

Aldehyde Dehydrogenase↗

PubMed1991

Different classes of polyadenylation sites in the yeast Saccharomyces cerevisiae.

This report provides an analysis of the function of polyadenylation sites from six different genes of the yeast Saccharomyces cerevisiae. These sites were tested for their ability to turn off read-through transcription into the URA3 gene in vivo when inserted into an ACT-URA3 fusion gene. The 3' ends of all polyadenylation sites inserted into the test system in their natural configuration are identical to the 3' ends of the chromosomal genes. We identified two classes of polyadenylation sites: (i) efficient sites (originating from the genes GCN4 and PHO5) that were functional in a strict orientation-dependent manner and (ii) bidirectional sites (derived from ARO4, TRP1, and TRP4) that had a distinctly reduced efficiency. The ADH1 polyadenylation site was efficient and bidirectional and was shown to be a combination of two polyadenylation sites of two convergently transcribed genes. Sequence comparison revealed that all efficient unidirectional polyadenylation sites contain the sequence TTTTTAT, whereas all bidirectional sites have the tripartite sequence TAG...TA (T)GT...TTT. Both sequence elements have previously been proposed to be involved in 3' end formation. Site-directed point mutagenesis of the TTTTTAT sequence had no effect, whereas mutations within the tripartite sequence caused a reduced efficiency for 3' end formation. The tripartite sequence alone, however, is not sufficient for 3' end formation, but it might be part of a signal sequence in the bidirectional class of yeast polyadenylation sites. Our findings support the assumption that there are at least two different mechanisms with different sequence elements directing 3' end formation in yeast.

Base Sequence↗

PubMed1989

Localization and functional analysis of the regulated promoter from the Streptomyces glaucescens mel operon.

The transcription initiation site of the mel operon from Streptomyces glaucescens, determined by S1 mapping and primer elongation experiments, lies 32 to 34 bp upstream of the translation initiation codon of the first open reading frame. A total of 172 to 219 bp upstream of the transcription start point are necessary for a fully active and regulated mel promoter. Deletion analysis, gel retardation assays and DNAse I footprint experiments facilitated division of the promoter into three functional domains, which include the RNA polymerase recognition site up to nucleotides -33 to -42, the binding region of a protein of assumed regulatory function between nucleotides -65 and -93, and an upstream activator site, located between positions -158 and -219.

DNA Mutational Analysis↗