Biomedical subjects

Martin Kupiec

Publications and source records attributed to Martin Kupiec.

At least 19 recordsLinked to original sources

PubMed2006

Opposite effects of tor1 and tor2 on nitrogen starvation responses in fission yeast.

The TOR protein kinases exhibit a conserved role in regulating cellular growth and proliferation. In the fission yeast two TOR homologs are present. tor1(+) is required for starvation and stress responses, while tor2(+) is essential. We report here that Tor2 depleted cells show a phenotype very similar to that of wild-type cells starved for nitrogen, including arrest at the G(1) phase of the cell cycle, induction of nitrogen-starvation-specific genes, and entrance into the sexual development pathway. The phenotype of tor2 mutants is in a striking contrast to the failure of tor1 mutants to initiate sexual development or arrest in G(1) under nitrogen starvation conditions. Tsc1 and Tsc2, the genes mutated in the human tuberous sclerosis complex syndrome, negatively regulate the mammalian TOR via inactivation of the GTPase Rheb. We analyzed the genetic relationship between the two TOR genes and the Schizosaccharomyces pombe orthologs of TSC1, TSC2, and Rheb. Our data suggest that like in higher eukaryotes, the Tsc1-2 complex negatively regulates Tor2. In contrast, the Tsc1-2 complex and Tor1 appear to work in parallel, both positively regulating amino acid uptake through the control of expression of amino acid permeases. Additionally, either Tsc1/2 or Tor1 are required for growth on a poor nitrogen source such as proline. Mutants lacking Tsc1 or Tsc2 are highly sensitive to rapamycin under poor nitrogen conditions, suggesting that the function of Tor1 under such conditions is sensitive to rapamycin. We discuss the complex genetic interactions between tor1(+), tor2(+), and tsc1/2(+) and the implications for rapamycin sensitivity in tsc1 or tsc2 mutants.

Amino Acid Transport Systems↗

PubMed2006

A role for the yeast cell cycle/splicing factor Cdc40 in the G1/S transition.

The CDC40 (PRP17) gene of S. cerevisiae encodes a splicing factor required for multiple events in the mitotic and meiotic cell cycles, linking splicing with cell cycle control. cdc40 mutants exhibit a delayed G(1)/S transition, progress slowly through S-phase and arrest at a restrictive temperature in the G(2) phase. In addition, they are hypersensitive to genotoxic agents such as methylmethane sulfonate (MMS) and Hydroxyurea (HU). CDC40 has been suggested to control cell cycle through splicing of intron-containing pre-mRNAs that encode proteins important for cell cycle progression. We screened a cDNA overexpression library and isolated cDNAs that specifically suppress the HU/MMS-sensitivity of cdc40 mutants. Most of these cDNAs surprisingly encode chaperones, translation initiation factors and glycolytic enzymes, and none of them is encoded by an intron-containing gene. Interestingly, the cDNAs suppress the G(1)/S transition delay of cdc40 cells, which is exacerbated by HU, suggesting that cdc40 mutants are HU/MMS-sensitive due to their S-phase entry defect. A role of Cdc40p in passage through G(1)/S (START) is further supported by the enhanced temperature sensitivity and G(1)/S transition phenotype of a cdc40 strain lacking the G(1) cyclin, Cln2p. We provide evidence that the mechanism of suppression by the isolated cDNAs does not (at least solely) involve up-regulation of the known positive START regulators CLN2, CLN3, DCR2 and GID8, or of the large and small essential ribonucleotide reductase (RNR) subunits, RNR1 and RNR2. Finally, we discuss possible mechanisms of suppression by the cDNAs that imply cell cycle regulation by apparently unrelated processes, such as splicing, translation initiation and glycolysis.

Cell Cycle Proteins↗

PubMed2006

Multiple knockout analysis of genetic robustness in the yeast metabolic network.

Genetic robustness characterizes the constancy of the phenotype in face of heritable perturbations. Previous investigations have used comprehensive single and double gene knockouts to study gene essentiality and pairwise gene interactions in the yeast Saccharomyces cerevisiae. Here we conduct an in silico multiple knockout investigation of a flux balance analysis model of the yeast's metabolic network. Cataloging gene sets that provide mutual functional backup, we identify sets of up to eight interacting genes and characterize the 'k robustness' (the depth of backup interactions) of each gene. We find that 74% (360) of the metabolic genes participate in processes that are essential to growth in a standard laboratory environment, compared with only 13% previously found to be essential using single knockouts. The genes' k robustness is shown to be a solid indicator of their biological buffering capacity and is correlated with both the genes' environmental specificity and their evolutionary retention.

Computer Simulation↗

PubMed2005

Quantitative analysis of genetic and neuronal multi-perturbation experiments.

Perturbation studies, in which functional performance is measured after deletion, mutation, or lesion of elements of a biological system, have been traditionally employed in many fields in biology. The vast majority of these studies have been qualitative and have employed single perturbations, often resulting in little phenotypic effect. Recently, newly emerging experimental techniques have allowed researchers to carry out concomitant multi-perturbations and to uncover the causal functional contributions of system elements. This study presents a rigorous and quantitative multi-perturbation analysis of gene knockout and neuronal ablation experiments. In both cases, a quantification of the elements' contributions, and new insights and predictions, are provided. Multi-perturbation analysis has a potentially wide range of applications and is gradually becoming an essential tool in biology.

Algorithms↗

PubMed2005

Evidence for abundant transcription of non-coding regions in the Saccharomyces cerevisiae genome.

BACKGROUND: Recent studies in a growing number of organisms have yielded accumulating evidence that a significant portion of the non-coding region in the genome is transcribed. We address this issue in the yeast Saccharomyces cerevisiae. RESULTS: Taking into account the absence of a significantly large yeast EST database, we use microarray expression data collected for genomic regions erroneously believed to be coding to study the expression pattern of non-coding regions in the Saccharomyces cerevisiae genome. We find that at least 164 out of 589 (28%) such regions are expressed under specific biological conditions. In particular, looking at the probes that are located opposing other known genes at the same genomic locus, we find that 88 out of 341 (26%) of these genes support antisense transcription. The expression patterns of these antisense genes are positively correlated. We validate these results using RT-PCR on a sample of 6 non-coding transcripts. CONCLUSION: 1. The yeast genome is transcribed on a scale larger than previously assumed. 2. Correlated transcription of antisense genes is abundant in the yeast genome. 3. Antisense genes in yeast are non-coding.

Computational Biology↗

PubMed2005

The Elg1 replication factor C-like complex: a novel guardian of genome stability.

The remarkable stability of the eukaryotic genome is achieved by the activity of many overlapping surveillance and repair mechanism. Two protein complexes with resemblance to replication factor C (RFC) have been recently described, that play important roles in maintaining the stability of the genome. These RFC-like complexes (RLCs) share four common subunits (Rfc2-5) and each carry a unique large subunit (Rad24 or Ctf18) replacing the Rfc1 subunit of the replication complex. Work in several laboratories has recently uncovered a novel yeast gene, ELG1, which seems to play a central role in keeping the genome stable. elg1 mutants exhibit increased rates of spontaneous recombination and gross chromosomal rearrangements during vegetative growth. In addition, they lose chromosomes at an enhanced rate, show hyper-transposition of natural repeated elements and exhibit elongated telomeres. The Elg1 protein also associates with the Rfc2-5 subunits of replication factor C (RFC) to form a third RFC-like complex (RLC). Genetic and biochemical data indicate that the Elg1, Ctf18 and Rad24 RLCs work in three separate pathways important for maintaining the integrity of the genome and for coping with various genomic stresses. ELG1 is evolutionarily conserved and may play an important role in preventing the onset of cancer in humans. The Elg1 function is thus clearly required for maintaining genome stability during normal growth, and its absence has severe genetic consequences.

Carrier Proteins↗

PubMed2005

Integrative analysis of genome-wide experiments in the context of a large high-throughput data compendium.

Biological systems are orchestrated by heterogeneous regulatory programs that control complex processes and adapt to a dynamic environment. Recent advances in high-throughput experimental methods provide genome-wide perspectives on such regulatory programs. A considerable amount of data on the behavior of model systems in a variety of conditions is rapidly accumulating. Still, the dominant paradigm is to analyze new genome-wide experiments separately from any other extant data, for example, by clustering the new data alone. Here we introduce a new methodology for analyzing the results of a new functional genomic study vis-à-vis a large compendium of previously published results from heterogeneous experimental techniques. We demonstrate our methodology on Saccharomyces cerevisiae, using a compendium of some 2000 experiments from 60 different publications. Most importantly, we show how the integrated analysis reveals unexpected connections among biological processes, and differentiates between novel and known effects in the analyzed experiments. Such characterization is impossible when new data sets are studied in isolation. Our results exemplify the power of the integrative approach in the analysis of genomic scale data sets and call for a paradigm shift in their study.

Algorithms↗

PubMed2005

Cell cycle-dependent regulation of double-strand break repair: a role for the CDK.

DNA Double-Strand Breaks (DSBs) are dangerous lesions that can lead to genomic instability and to cell death. Eukaryotic cells repair DSBs either by nonhomologous end joining (NHEJ) or by homologous recombination (HR). Recent work has allowed to study the ability of yeast cells to repair a single, chromosomal DSB, at different stages of the cell cycle. Yeast cells repair the broken chromosome during the G(1) stage only by NHEJ, whereas HR is the mechanism of choice during the rest of the cell cycle. HR does not require duplicated chromatids or passage through S-phase. Control over the fate of the broken chromosome is exerted by Clb-CDK activity, which is required to carry out the first step of HR, ssDNA resection. Similar results in other organisms suggest that this control is a conserved feature in all eukaryotes.

Cell Cycle↗

PubMed2004

The yeast VPS genes affect telomere length regulation.

Eukaryotic cells invest a large proportion of their genome in maintaining telomere length homeostasis. Among the 173 non-essential yeast genes found to affect telomere length, a large proportion is involved in vacuolar traffic. When mutated, these vacuolar protein-sorting (VPS) genes lead to telomeres shorter than those observed in the wild type. Using genetic analysis, we characterized the pathway by which VPS15, VPS34, VPS22, VPS23 and VPS28 affect the telomeres. Our results indicate that these VPS genes affect telomere length through a single pathway and that this effect requires the activity of telomerase and the Ku heterodimer, but not the activity of Tel1p or Rif2p. We present models to explain the link between vacuolar traffic and telomere length homeostasis.

Homeostasis↗

PubMed2004

The CDK regulates repair of double-strand breaks by homologous recombination during the cell cycle.

DNA double-strand breaks (DSBs) are dangerous lesions that can lead to genomic instability and cell death. Eukaryotic cells repair DSBs either by nonhomologous end-joining (NHEJ) or by homologous recombination. We investigated the ability of yeast cells (Saccharomyces cerevisiae) to repair a single, chromosomal DSB by recombination at different stages of the cell cycle. We show that cells arrested at the G1 phase of the cell cycle restrict homologous recombination, but are able to repair the DSB by NHEJ. Furthermore, we demonstrate that recombination ability does not require duplicated chromatids or passage through S phase, and is controlled at the resection step by Clb-CDK activity.

Animals↗

PubMed2004

Regulation of leucine uptake by tor1+ in Schizosaccharomyces pombe is sensitive to rapamycin.

TOR protein kinases are key regulators of cell growth in eukaryotes. TOR is also known as the target protein for the immunosuppressive and potentially anticancer drug rapamycin. The fission yeast Schizosaccharomyces pombe has two TOR homologs. tor1+ is required under starvation and a variety of stresses, while tor2+ is an essential gene. Surprisingly, to date no rapamycin-sensitive TOR-dependent function has been identified in S. pombe. Herein, we show that S. pombe auxotrophs, in particular leucine auxotrophs, are sensitive to rapamycin. This sensitivity is suppressed by deletion of the S. pombe FKBP12 or by introducing a rapamycin-binding defective tor1 allele, suggesting that rapamycin inhibits a tor1p-dependent function. Sensitivity of leucine auxotrophs to rapamycin is observed when ammonia is used as the nitrogen source and can be suppressed by its replacement with proline. Consistently, using radioactive labeled leucine, we show that cells treated with rapamycin or disrupted for tor1+ are defective in leucine uptake when the nitrogen source is ammonia but not proline. Recently, it has been reported that tsc1+ and tsc2+, the S. pombe homologs for the mammalian TSC1 and TSC2, are also defective in leucine uptake. TSC1 and TSC2 may antagonize TOR signaling in mammalian cells and Drosophila. We show that reduction of leucine uptake in tor1 mutants is correlated with decreased expression of three putative amino acid permeases that are also downregulated in tsc1 or tsc2. These findings suggest a possible mechanism for regulation of leucine uptake by tor1p and indicate that tor1p, as well as tsc1p and tsc2p, positively regulates leucine uptake in S. pombe.

Alleles↗

PubMed2004

The compact chromatin structure of a Ty repeated sequence suppresses recombination hotspot activity in Saccharomyces cerevisiae.

Recombination between repeated DNA sequences can have drastic consequences on the integrity of the genome. Repeated sequences are abundant in most eukaryotes, yet the mechanism that prevents recombination between them is currently unknown. Ty elements, the main family of dispersed repeats in Saccharomyces cerevisiae, exhibit low levels of exchange. Other regions in the genome have relatively high rates of meiotic recombination (hotspots). We show that a Ty element adjacent to the HIS4 recombination hotspot substantially reduces its activity, eliminating local DSB formation. We demonstrate that the Ty has a closed (nuclease-insensitive) chromatin configuration that is also imposed on the flanking DNA sequences. The compact chromatin structure is determined by sequences at the N terminus of the Ty. Increased binding of the Rap1 protein to the hotspot restores both open chromatin conformation and DSB formation. The chromatin configuration of Ty elements precludes initiation of recombination, thus preventing potentially lethal exchanges between repeated sequences.

Alcohol Oxidoreductases↗

PubMed2004

A genome-wide screen for Saccharomyces cerevisiae deletion mutants that affect telomere length.

Telomeres are nucleoprotein structures present at the ends of eukaryotic chromosomes that play a central role in guarding the integrity of the genome by protecting chromosome ends from degradation and fusion. Length regulation is central to telomere function. To broaden our knowledge about the mechanisms that control telomere length, we have carried out a systematic examination of approximately 4,800 haploid deletion mutants of Saccharomyces cerevisiae for telomere-length alterations. By using this screen, we have identified >150 candidate genes not previously known to affect telomere length. In two-thirds of the identified mutants, short telomeres were observed; whereas in one-third, telomeres were lengthened. The genes identified are very diverse in their functions, but certain categories, including DNA and RNA metabolism, chromatin modification, and vacuolar traffic, are overrepresented. Our results greatly enlarge the number of known genes that affect telomere metabolism and will provide insights into how telomere function is linked to many other cellular processes.

Base Sequence↗

PubMed2004

The Saccharomyces cerevisiae gene CDC40/PRP17 controls cell cycle progression through splicing of the ANC1 gene.

The timing of events in the cell cycle is of crucial importance, as any error can lead to cell death or cancerous growth. This accurate timing is accomplished through the activation of specific CDC genes. Mutations in the CDC40/PRP17 gene cause cell cycle arrest at the G2/M stage. It was previously found that the CDC40 gene encodes a pre-mRNA splicing factor, which participates in the second step of the splicing reaction. In this paper we dissect the mechanism by which pre-mRNA splicing affects cell cycle progression. We identify ANC1 as the target of CDC40 regulation. Deletion of the ANC1 intron relieves the cell cycle arrest and temperature sensitivity of cdc40 mutants. Furthermore, we identify, through point mutation analysis, specific residues in the ANC1 intron that are important for its splicing dependency on Cdc40p. Our results demonstrate a novel mechanism of cell cycle regulation that relies on the differential splicing of a subset of introns by specific splicing factors.

Alleles↗

PubMed2004

Site-specific genomic (SSG) and random domain-localized (RDL) mutagenesis in yeast.

BACKGROUND: A valuable weapon in the arsenal available to yeast geneticists is the ability to introduce specific mutations into yeast genome. In particular, methods have been developed to introduce deletions into the yeast genome using PCR fragments. These methods are highly efficient because they do not require cloning in plasmids. RESULTS: We have modified the existing method for introducing deletions in the yeast (S. cerevisiae) genome using PCR fragments in order to target point mutations to this genome. We describe two PCR-based methods for directing point mutations into the yeast genome such that the final product contains no other disruptions. In the first method, site-specific genomic (SSG) mutagenesis, a specific point mutation is targeted into the genome. In the second method, random domain-localized (RDL) mutagenesis, a mutation is introduced at random within a specific domain of a gene. Both methods require two sequential transformations, the first transformation integrates the URA3 marker into the targeted locus, and the second transformation replaces URA3 with a PCR fragment containing one or a few mutations. This PCR fragment is synthesized using a primer containing a mutation (SSG mutagenesis) or is synthesized by error-prone PCR (RDL mutagenesis). In SSG mutagenesis, mutations that are proximal to the URA3 site are incorporated at higher frequencies than distal mutations, however mutations can be introduced efficiently at distances of at least 500 bp from the URA3 insertion. In RDL mutagenesis, to ensure that incorporation of mutations occurs at approximately equal frequencies throughout the targeted region, this region is deleted at the same time URA3 is integrated. CONCLUSION: SSG and RDL mutagenesis allow point mutations to be easily and efficiently incorporated into the yeast genome without disrupting the native locus.

Genes, Fungal↗

PubMed2004

Revealing modularity and organization in the yeast molecular network by integrated analysis of highly heterogeneous genomewide data.

The dissection of complex biological systems is a challenging task, made difficult by the size of the underlying molecular network and the heterogeneous nature of the control mechanisms involved. Novel high-throughput techniques are generating massive data sets on various aspects of such systems. Here, we perform analysis of a highly diverse collection of genomewide data sets, including gene expression, protein interactions, growth phenotype data, and transcription factor binding, to reveal the modular organization of the yeast system. By integrating experimental data of heterogeneous sources and types, we are able to perform analysis on a much broader scope than previous studies. At the core of our methodology is the ability to identify modules, namely, groups of genes with statistically significant correlated behavior across diverse data sources. Numerous biological processes are revealed through these modules, which also obey global hierarchical organization. We use the identified modules to study the yeast transcriptional network and predict the function of >800 uncharacterized genes. Our analysis framework, SAMBA (Statistical-Algorithmic Method for Bicluster Analysis), enables the processing of current and future sources of biological information and is readily extendable to experimental techniques and higher organisms.

Amino Acids↗

PubMed2004

New insights into the mechanism of homologous recombination in yeast.

Genome stability is of primary importance for the survival and proper functioning of all organisms. Double-strand breaks (DSBs) arise spontaneously during growth, or can be created by external insults. Repair of DSBs by homologous recombination provides an efficient and fruitful pathway to restore chromosomal integrity. Exciting new work in yeast has lately provided insights into this complex process. Many of the proteins involved in recombination have been isolated and the details of the repair mechanism are now being unraveled at the molecular level. In this review, we focus on recent studies which dissect the recombinational repair of a single broken chromosome. After DSB formation, a decision is made regarding the mechanism of repair (recombination or non-homologous end-joining). This decision is under genetic control. Once committed to the recombination pathway, the broken chromosomal ends are resected by a still unclear mechanism in which the DNA damage checkpoint protein Rad24 participates. At this stage several proteins are recruited to the broken ends, including Rad51p, Rad52p, Rad55p, Rad57p, and possibly Rad54p. A genomic search for homology ensues, followed by strand invasion, promoted by the Rad51 filament with the participation of Rad55p, Rad57p and Rad54p. DNA synthesis then takes place, restoring the resected ends. Crossing-over formation depends on the length of the homologous recombining sequences, and is usually counteracted by the activity of the mismatch repair system. Given the conservation of the repair mechanisms and genes throughout evolution, these studies have profound implications for other eukaryotic organisms.

DNA Damage↗

PubMed2004

Multi-knockout genetic network analysis: the Rad6 example.

A novel and rigorous Multi-perturbation Shapley Value Analysis (MSA) method has been recently presented [12]. The method addresses the challenge of defining and calculating the functional causal contributions of elements of a biological system. This paper presents the first study applying MSA to the analysis of gene knockout data. The MSA identifies the importance of genes in the Rad6 DNA repair pathway of the yeast S. cerevisiae, quantifying their contributions and characterizing their functional interactions. Incorporating additional biological knowledge, a new functional description of the Rad6 pathway is provided, predicting the existence of additional DNA polymerase and RFC-like complexes. The MSA is the first method for rigorously analyzing multi-knockout experiments, which are likely to soon become a standard and necessary tool for analyzing complex biological systems.

Algorithms↗