Satellite DNA-based artificial chromosomes for use in gene therapy.

PubMed

Hadlaczky, G

2001-04-01

Satellite DNA-based artificial chromosomes (SATACs) can be made by induced de novo chromosome formation in cells of different mammalian species. These artificially generated accessory chromosomes are composed of predictable DNA sequences and they contain defined genetic information. Prototype human SATACs have been successfully constructed in different cell types from 'neutral' endogenous DNA sequences from the short arm of the human chromosome 15. SATACs have already passed a number of hurdles crucial to their further development as gene therapy vectors, including: large-scale purification; transfer of purified artificial chromosomes into different cells and embryos; generation of transgenic animals and germline transmission with purified SATACs; and the tissue-specific expression of a therapeutic gene from an artificial chromosome in the milk of transgenic animals.

Industrial Relevance of Chromosomal Copy Number Variation in Saccharomyces Yeasts

PubMed Central

Gorter de Vries, Arthur R.; Pronk, Jack T.

2017-01-01

ABSTRACT Chromosomal copy number variation (CCNV) plays a key role in evolution and health of eukaryotes. The unicellular yeast Saccharomyces cerevisiae is an important model for studying the generation, physiological impact, and evolutionary significance of CCNV. Fundamental studies of this yeast have contributed to an extensive set of methods for analyzing and introducing CCNV. Moreover, these studies provided insight into the balance between negative and positive impacts of CCNV in evolutionary contexts. A growing body of evidence indicates that CCNV not only frequently occurs in industrial strains of Saccharomyces yeasts but also is a key contributor to the diversity of industrially relevant traits. This notion is further supported by the frequent involvement of CCNV in industrially relevant traits acquired during evolutionary engineering. This review describes recent developments in genome sequencing and genome editing techniques and discusses how these offer opportunities to unravel contributions of CCNV in industrial Saccharomyces strains as well as to rationally engineer yeast chromosomal copy numbers and karyotypes. PMID:28341679

Quantitative analysis of chromosome condensation in fission yeast.

PubMed

Petrova, Boryana; Dehler, Sascha; Kruitwagen, Tom; Hériché, Jean-Karim; Miura, Kota; Haering, Christian H

2013-03-01

Chromosomes undergo extensive conformational rearrangements in preparation for their segregation during cell divisions. Insights into the molecular mechanisms behind this still poorly understood condensation process require the development of new approaches to quantitatively assess chromosome formation in vivo. In this study, we present a live-cell microscopy-based chromosome condensation assay in the fission yeast Schizosaccharomyces pombe. By automatically tracking the three-dimensional distance changes between fluorescently marked chromosome loci at high temporal and spatial resolution, we analyze chromosome condensation during mitosis and meiosis and deduct defined parameters to describe condensation dynamics. We demonstrate that this method can determine the contributions of condensin, topoisomerase II, and Aurora kinase to mitotic chromosome condensation. We furthermore show that the assay can identify proteins required for mitotic chromosome formation de novo by isolating mutants in condensin, DNA polymerase ε, and F-box DNA helicase I that are specifically defective in pro-/metaphase condensation. Thus, the chromosome condensation assay provides a direct and sensitive system for the discovery and characterization of components of the chromosome condensation machinery in a genetically tractable eukaryote.

Quantitative Analysis of Chromosome Condensation in Fission Yeast

PubMed Central

Petrova, Boryana; Dehler, Sascha; Kruitwagen, Tom; Hériché, Jean-Karim; Miura, Kota

2013-01-01

Chromosomes undergo extensive conformational rearrangements in preparation for their segregation during cell divisions. Insights into the molecular mechanisms behind this still poorly understood condensation process require the development of new approaches to quantitatively assess chromosome formation in vivo. In this study, we present a live-cell microscopy-based chromosome condensation assay in the fission yeast Schizosaccharomyces pombe. By automatically tracking the three-dimensional distance changes between fluorescently marked chromosome loci at high temporal and spatial resolution, we analyze chromosome condensation during mitosis and meiosis and deduct defined parameters to describe condensation dynamics. We demonstrate that this method can determine the contributions of condensin, topoisomerase II, and Aurora kinase to mitotic chromosome condensation. We furthermore show that the assay can identify proteins required for mitotic chromosome formation de novo by isolating mutants in condensin, DNA polymerase ε, and F-box DNA helicase I that are specifically defective in pro-/metaphase condensation. Thus, the chromosome condensation assay provides a direct and sensitive system for the discovery and characterization of components of the chromosome condensation machinery in a genetically tractable eukaryote. PMID:23263988

Industrial Relevance of Chromosomal Copy Number Variation in Saccharomyces Yeasts.

PubMed

Gorter de Vries, Arthur R; Pronk, Jack T; Daran, Jean-Marc G

2017-06-01

Chromosomal copy number variation (CCNV) plays a key role in evolution and health of eukaryotes. The unicellular yeast Saccharomyces cerevisiae is an important model for studying the generation, physiological impact, and evolutionary significance of CCNV. Fundamental studies of this yeast have contributed to an extensive set of methods for analyzing and introducing CCNV. Moreover, these studies provided insight into the balance between negative and positive impacts of CCNV in evolutionary contexts. A growing body of evidence indicates that CCNV not only frequently occurs in industrial strains of Saccharomyces yeasts but also is a key contributor to the diversity of industrially relevant traits. This notion is further supported by the frequent involvement of CCNV in industrially relevant traits acquired during evolutionary engineering. This review describes recent developments in genome sequencing and genome editing techniques and discusses how these offer opportunities to unravel contributions of CCNV in industrial Saccharomyce s strains as well as to rationally engineer yeast chromosomal copy numbers and karyotypes. Copyright © 2017 Gorter de Vries et al.

Chromosome and mitotic spindle dynamics in fission yeast kinesin-8 mutants

NASA Astrophysics Data System (ADS)

Crapo, Ammon M.; Gergley, Zachary R.; McIntosh, J. Richard; Betterton, M. D.

2014-03-01

Fission yeast proteins Klp5p and Klp6p are plus-end directed motors of the kinesin-8 family which promote microtubule (MT) depolymerization and also affect chromosome segregation, but the mechanism of these activities is not well understood. Using live-cell time-lapse fluorescence microscopy of fission yeast wild-type (WT) and klp5/6 mutant strains, we quantify and compare the dynamics of kinetochore motion and mitotic spindle length in 3D. In WT cells, the spindle, once formed, remains a consistent size and chromosomes are correctly organized and segregated. In kinesin-8 mutants, spindles undergo large length fluctuations of several microns. Kinetochore motions are also highly fluctuating, with kinetochores frequently moving away from the spindle rather than toward it. We observe transient pushing of chromosomes away from the spindle by as much as 10 microns in distance.

Chromosome dynamics in the yeast interphase nucleus.

PubMed

Heun, P; Laroche, T; Shimada, K; Furrer, P; Gasser, S M

2001-12-07

Little is known about the dynamics of chromosomes in interphase nuclei. By tagging four chromosomal regions with a green fluorescent protein fusion to lac repressor, we monitored the movement and subnuclear position of specific sites in the yeast genome, sampling at short time intervals. We found that early and late origins of replication are highly mobile in G1 phase, frequently moving at or faster than 0.5 micrometers/10 seconds, in an energy-dependent fashion. The rapid diffusive movement of chromatin detected in G1 becomes constrained in S phase through a mechanism dependent on active DNA replication. In contrast, telomeres and centromeres provide replication-independent constraint on chromatin movement in both G1 and S phases.

Sex Chromosome Evolution in Amniotes: Applications for Bacterial Artificial Chromosome Libraries

PubMed Central

Janes, Daniel E.; Valenzuela, Nicole; Ezaz, Tariq; Amemiya, Chris; Edwards, Scott V.

2011-01-01

Variability among sex chromosome pairs in amniotes denotes a dynamic history. Since amniotes diverged from a common ancestor, their sex chromosome pairs and, more broadly, sex-determining mechanisms have changed reversibly and frequently. These changes have been studied and characterized through the use of many tools and experimental approaches but perhaps most effectively through applications for bacterial artificial chromosome (BAC) libraries. Individual BAC clones carry 100–200 kb of sequence from one individual of a target species that can be isolated by screening, mapped onto karyotypes, and sequenced. With these techniques, researchers have identified differences and similarities in sex chromosome content and organization across amniotes and have addressed hypotheses regarding the frequency and direction of past changes. Here, we review studies of sex chromosome evolution in amniotes and the ways in which the field of research has been affected by the advent of BAC libraries. PMID:20981143

De novo formed satellite DNA-based mammalian artificial chromosomes and their possible applications.

PubMed

Katona, Robert L

2015-02-01

Mammalian artificial chromosomes (MACs) are non-integrating, autonomously replicating natural chromosome-based vectors that may carry a vast amount of genetic material, which in turn enable potentially prolonged, safe, and regulated therapeutic transgene expression and render MACs as attractive genetic vectors for "gene replacement" or for controlling differentiation pathways in target cells. Satellite-DNA-based artificial chromosomes (SATACs) can be made by induced de novo chromosome formation in cells of different mammalian and plant species. These artificially generated accessory chromosomes are composed of predictable DNA sequences, and they contain defined genetic information. SATACs have already passed a number of obstacles crucial to their further development as gene therapy vectors, including large-scale purification, transfer of purified artificial chromosomes into different cells and embryos, generation of transgenic animals and germline transmission with purified SATACs, and the tissue-specific expression of a therapeutic gene from an artificial chromosome in the milk of transgenic animals. SATACs could be used in cell therapy protocols. For these methods, the most versatile target cell would be one that was pluripotent and self-renewing to address multiple disease target cell types, thus making multilineage stem cells, such as adult derived early progenitor cells and embryonic stem cells, as attractive universal host cells.

Designing of plant artificial chromosome (PAC) by using the Chlorella smallest chromosome as a model system.

PubMed

Noutoshi, Y; Arai, R; Fujie, M; Yamada, T

1997-01-01

As a model for plant-type chromosomes, we have been characterizing molecular organization of the Chlorella vulgaris C-169 chromosome I. To identify chromosome structural elements including the centromeric region and replication origins, we constructed a chromosome I specific cosmid library and aligned each cosmid clones to generate contigs. So far, more than 80% of the entire chromosome I has been covered. A complete clonal physical reconstitution of chromosome I provides information on the structure and genomic organization of plant genome. We propose our strategy to construct an artificial chromosome by assembling the functional chromosome structural elements identified on Chrorella chromosome I.

Human Artificial Chromosomes with Alpha Satellite-Based De Novo Centromeres Show Increased Frequency of Nondisjunction and Anaphase Lag

PubMed Central

Rudd, M. Katharine; Mays, Robert W.; Schwartz, Stuart; Willard, Huntington F.

2003-01-01

Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes. PMID:14560014

Human artificial chromosomes with alpha satellite-based de novo centromeres show increased frequency of nondisjunction and anaphase lag.

PubMed

Rudd, M Katharine; Mays, Robert W; Schwartz, Stuart; Willard, Huntington F

2003-11-01

Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.

Process for Assembly and Transformation into Saccharomyces cerevisiae of a Synthetic Yeast Artificial Chromosome Containing a Multigene Cassette to Express Enzymes That Enhance Xylose Utilization Designed for an Automated Platform.

PubMed

Hughes, Stephen R; Cox, Elby J; Bang, Sookie S; Pinkelman, Rebecca J; López-Núñez, Juan Carlos; Saha, Badal C; Qureshi, Nasib; Gibbons, William R; Fry, Michelle R; Moser, Bryan R; Bischoff, Kenneth M; Liu, Siqing; Sterner, David E; Butt, Tauseef R; Riedmuller, Steven B; Jones, Marjorie A; Riaño-Herrera, Néstor M

2015-12-01

A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains. © 2015 Society for Laboratory Automation and Screening.

Initiation at closely spaced replication origins in a yeast chromosome.

PubMed

Brewer, B J; Fangman, W L

1993-12-10

Replication of eukaryotic chromosomes involves initiation at origins spaced an average of 50 to 100 kilobase pairs. In yeast, potential origins can be recognized as autonomous replication sequences (ARSs) that allow maintenance of plasmids. However, there are more ARS elements than active chromosomal origins. The possibility was examined that close spacing of ARSs can lead to inactive origins. Two ARSs located 6.5 kilobase pairs apart can indeed interfere with each other. Replication is initiated from one or the other ARS with equal probability, but rarely (< 5%) from both ARSs on the same DNA molecule.

Improving industrial yeast strains: exploiting natural and artificial diversity

PubMed Central

Steensels, Jan; Snoek, Tim; Meersman, Esther; Nicolino, Martina Picca; Voordeckers, Karin; Verstrepen, Kevin J

2014-01-01

Yeasts have been used for thousands of years to make fermented foods and beverages, such as beer, wine, sake, and bread. However, the choice for a particular yeast strain or species for a specific industrial application is often based on historical, rather than scientific grounds. Moreover, new biotechnological yeast applications, such as the production of second-generation biofuels, confront yeast with environments and challenges that differ from those encountered in traditional food fermentations. Together, this implies that there are interesting opportunities to isolate or generate yeast variants that perform better than the currently used strains. Here, we discuss the different strategies of strain selection and improvement available for both conventional and nonconventional yeasts. Exploiting the existing natural diversity and using techniques such as mutagenesis, protoplast fusion, breeding, genome shuffling and directed evolution to generate artificial diversity, or the use of genetic modification strategies to alter traits in a more targeted way, have led to the selection of superior industrial yeasts. Furthermore, recent technological advances allowed the development of high-throughput techniques, such as ‘global transcription machinery engineering’ (gTME), to induce genetic variation, providing a new source of yeast genetic diversity. PMID:24724938

Chromosome VIII disomy influences the nonsense suppression efficiency and transition metal tolerance of the yeast Saccharomyces cerevisiae.

PubMed

Zadorsky, S P; Sopova, Y V; Andreichuk, D Y; Startsev, V A; Medvedeva, V P; Inge-Vechtomov, S G

2015-06-01

The SUP35 gene of the yeast Saccharomyces cerevisiae encodes the translation termination factor eRF3. Mutations in this gene lead to the suppression of nonsense mutations and a number of other pleiotropic phenotypes, one of which is impaired chromosome segregation during cell division. Similar effects result from replacing the S. cerevisiae SUP35 gene with its orthologues. A number of genetic and epigenetic changes that occur in the sup35 background result in partial compensation for this suppressor effect. In this study we showed that in S. cerevisiae strains in which the SUP35 orthologue from the yeast Pichia methanolica replaces the S. cerevisiae SUP35 gene, chromosome VIII disomy results in decreased efficiency of nonsense suppression. This antisuppressor effect is not associated with decreased stop codon read-through. We identified SBP1, a gene that localizes to chromosome VIII, as a dosage-dependent antisuppressor that strongly contributes to the overall antisuppressor effect of chromosome VIII disomy. Disomy of chromosome VIII also leads to a change in the yeast strains' tolerance of a number of transition metal salts. Copyright © 2015 John Wiley & Sons, Ltd.

Artificial Neural Network for the Prediction of Chromosomal Abnormalities in Azoospermic Males.

PubMed

Akinsal, Emre Can; Haznedar, Bulent; Baydilli, Numan; Kalinli, Adem; Ozturk, Ahmet; Ekmekçioğlu, Oğuz

2018-02-04

To evaluate whether an artifical neural network helps to diagnose any chromosomal abnormalities in azoospermic males. The data of azoospermic males attending to a tertiary academic referral center were evaluated retrospectively. Height, total testicular volume, follicle stimulating hormone, luteinising hormone, total testosterone and ejaculate volume of the patients were used for the analyses. In artificial neural network, the data of 310 azoospermics were used as the education and 115 as the test set. Logistic regression analyses and discriminant analyses were performed for statistical analyses. The tests were re-analysed with a neural network. Both logistic regression analyses and artificial neural network predicted the presence or absence of chromosomal abnormalities with more than 95% accuracy. The use of artificial neural network model has yielded satisfactory results in terms of distinguishing patients whether they have any chromosomal abnormality or not.

Improving industrial yeast strains: exploiting natural and artificial diversity.

PubMed

Steensels, Jan; Snoek, Tim; Meersman, Esther; Picca Nicolino, Martina; Voordeckers, Karin; Verstrepen, Kevin J

2014-09-01

Yeasts have been used for thousands of years to make fermented foods and beverages, such as beer, wine, sake, and bread. However, the choice for a particular yeast strain or species for a specific industrial application is often based on historical, rather than scientific grounds. Moreover, new biotechnological yeast applications, such as the production of second-generation biofuels, confront yeast with environments and challenges that differ from those encountered in traditional food fermentations. Together, this implies that there are interesting opportunities to isolate or generate yeast variants that perform better than the currently used strains. Here, we discuss the different strategies of strain selection and improvement available for both conventional and nonconventional yeasts. Exploiting the existing natural diversity and using techniques such as mutagenesis, protoplast fusion, breeding, genome shuffling and directed evolution to generate artificial diversity, or the use of genetic modification strategies to alter traits in a more targeted way, have led to the selection of superior industrial yeasts. Furthermore, recent technological advances allowed the development of high-throughput techniques, such as 'global transcription machinery engineering' (gTME), to induce genetic variation, providing a new source of yeast genetic diversity. © 2014 The Authors. FEMS Microbiology Reviews published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

Refined human artificial chromosome vectors for gene therapy and animal transgenesis

PubMed Central

Kazuki, Y; Hoshiya, H; Takiguchi, M; Abe, S; Iida, Y; Osaki, M; Katoh, M; Hiratsuka, M; Shirayoshi, Y; Hiramatsu, K; Ueno, E; Kajitani, N; Yoshino, T; Kazuki, K; Ishihara, C; Takehara, S; Tsuji, S; Ejima, F; Toyoda, A; Sakaki, Y; Larionov, V; Kouprina, N; Oshimura, M

2011-01-01

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis. PMID:21085194

NET1 and HFI1 genes of yeast mediate both chromosome maintenance and mitochondrial rho(-) mutagenesis.

PubMed

Koltovaya, N A; Guerasimova, A S; Tchekhouta, I A; Devin, A B

2003-08-01

An increase in the mitochondrial rho(-) mutagenesis is a well-known response of yeast cells to mutations in numerous nuclear genes as well as to various kinds of stress. Despite extensive studies for several decades, the biological significance of this response is still not fully understood. The genetic approach to solving this enigma includes a study of genes that are required for the high incidence of spontaneous rho(-) mutants. We have obtained mutations of a few nuclear genes of that sort and found that mutations in certain genes, including CDC28, the central cell-cycle regulation gene, result in a decrease in spontaneous rho(-) mutability and simultaneously affect the maintenance of the yeast chromosomes and plasmids. Two more genes resembling CDC28 in this respect are identified in the present work as a result of the characterization of four new mutants. These two genes are NET1 and HFI1 which mediate important regulatory protein-protein interactions in the yeast cell. The effects of four mutations, including net1-srm and hfi1-srm, on the maintenance of the yeast mitochondrial genome, chromosomes and plasmids, as well as on the cell's sensitivity to ionizing radiation, are also described. The data presented suggest that the pleiotropic srm mutations determining coordinate changes in the fidelity of mitotic transmission of chromosomes, plasmids and mtDNA molecules identify genes that most probably operate high up in the hierarchy of the general genetic regulation of yeast. Copyright 2003 John Wiley & Sons, Ltd.

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast

PubMed Central

Sanchez, Joseph C.; Kwan, Elizabeth X.; Raghuraman, M. K.; Brewer, Bonita J.

2017-01-01

A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways—DNA replication and ribosome biogenesis. PMID:29036220

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast.

PubMed

Sanchez, Joseph C; Kwan, Elizabeth X; Pohl, Thomas J; Amemiya, Haley M; Raghuraman, M K; Brewer, Bonita J

2017-10-01

A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.

Acentric chromosome ends are prone to fusion with functional chromosome ends through a homology-directed rearrangement

PubMed Central

Ohno, Yuko; Ogiyama, Yuki; Kubota, Yoshino; Kubo, Takuya; Ishii, Kojiro

2016-01-01

The centromeres of many eukaryotic chromosomes are established epigenetically on potentially variable tandem repeats; hence, these chromosomes are at risk of being acentric. We reported previously that artificially created acentric chromosomes in the fission yeast Schizosaccharomyces pombe can be rescued by end-to-end fusion with functional chromosomes. Here, we show that most acentric/functional chromosome fusion events in S. pombe cells harbouring an acentric chromosome I differed from the non-homologous end-joining-mediated rearrangements that result in deleterious dicentric fusions in normal cells, and were elicited by a previously unidentified homologous recombination (HR) event between chromosome end-associated sequences. The subtelomere repeats associated with the non-fusogenic ends were also destabilized in the surviving cells, suggesting a causal link between general subtelomere destabilization and acentric/functional chromosome fusion. A mutational analysis indicated that a non-canonical HR pathway was involved in the rearrangement. These findings are indicative of a latent mechanism that conditionally induces general subtelomere instability, presumably in the face of accidental centromere loss events, resulting in rescue of the fatal acentric chromosomes by interchromosomal HR. PMID:26433224

Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome.

PubMed

Shen, Yue; Wang, Yun; Chen, Tai; Gao, Feng; Gong, Jianhui; Abramczyk, Dariusz; Walker, Roy; Zhao, Hongcui; Chen, Shihong; Liu, Wei; Luo, Yisha; Müller, Carolin A; Paul-Dubois-Taine, Adrien; Alver, Bonnie; Stracquadanio, Giovanni; Mitchell, Leslie A; Luo, Zhouqing; Fan, Yanqun; Zhou, Baojin; Wen, Bo; Tan, Fengji; Wang, Yujia; Zi, Jin; Xie, Zexiong; Li, Bingzhi; Yang, Kun; Richardson, Sarah M; Jiang, Hui; French, Christopher E; Nieduszynski, Conrad A; Koszul, Romain; Marston, Adele L; Yuan, Yingjin; Wang, Jian; Bader, Joel S; Dai, Junbiao; Boeke, Jef D; Xu, Xun; Cai, Yizhi; Yang, Huanming

2017-03-10

Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP -mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain. Copyright © 2017, American Association for the Advancement of Science.

Novel method to load multiple genes onto a mammalian artificial chromosome.

PubMed

Tóth, Anna; Fodor, Katalin; Praznovszky, Tünde; Tubak, Vilmos; Udvardy, Andor; Hadlaczky, Gyula; Katona, Robert L

2014-01-01

Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs) was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe's disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest.

Deep functional analysis of synII, a 770 kb synthetic yeast chromosome

PubMed Central

Gao, Feng; Gong, Jianhui; Abramczyk, Dariusz; Walker, Roy; Zhao, Hongcui; Chen, Shihong; Liu, Wei; Luo, Yisha; Müller, Carolin A.; Paul-Dubois-Taine, Adrien; Alver, Bonnie; Stracquadanio, Giovanni; Mitchell, Leslie A.; Luo, Zhouqing; Fan, Yanqun; Zhou, Baojin; Wen, Bo; Tan, Fengji; Wang, Yujia; Zi, Jin; Xie, Zexiong; Li, Bingzhi; Yang, Kun; Richardson, Sarah M.; Jiang, Hui; French, Christopher E.; Nieduszynski, Conrad A.; Koszul, Romain; Marston, Adele L.; Yuan, Yingjin; Wang, Jian; Bader, Joel S.; Dai, Junbiao; Boeke, Jef D.; Xu, Xun; Cai, Yizhi; Yang, Huanming

2017-01-01

Herein we report the successful design, construction and characterization of a 770 kb synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels, including phenomics, transcriptomics, proteomics, chromosome segregation and replication analysis to provide a thorough and comprehensive analysis of a synthetic chromosome. Our “Trans-Omics” analyses reveal a modest but potentially significant pervasive up-regulation of translational machinery observed in synII is mainly caused by the deletion of 13 tRNAs. By both complementation assays and SCRaMbLE, we targeted and debuged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the HOG response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain. PMID:28280153

Autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast.

PubMed

Matsuhara, Hirotada; Yamamoto, Ayumu

2016-01-01

Autophagy is a conserved intracellular degradation system, which contributes to development and differentiation of various organisms. Yeast cells undergo meiosis under nitrogen-starved conditions and require autophagy for meiosis initiation. However, the precise roles of autophagy in meiosis remain unclear. Here, we show that autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast. Autophagy-defective strains bearing a mutation in the autophagy core factor gene atg1, atg7, or atg14 exhibit deformed nuclear structures during meiosis. These mutant cells require an extracellular nitrogen supply for meiosis progression following their entry into meiosis and show delayed meiosis progression even with a nitrogen supply. In addition, they show frequent chromosome dissociation from the spindle together with spindle overextension, forming extra nuclei. Furthermore, Aurora kinase, which regulates chromosome segregation and spindle elongation, is significantly increased at the centromere and spindle in the mutant cells. Aurora kinase down-regulation eliminated delayed initiation of meiosis I and II, chromosome dissociation, and spindle overextension, indicating that increased Aurora kinase activity may cause these aberrances in the mutant cells. Our findings show a hitherto unrecognized relationship of autophagy with the nuclear structure, regulation of cell cycle progression, and chromosome segregation in meiosis. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Construction of trypanosome artificial mini-chromosomes.

PubMed Central

Lee, M G; E, Y; Axelrod, N

1995-01-01

We report the preparation of two linear constructs which, when transformed into the procyclic form of Trypanosoma brucei, become stably inherited artificial mini-chromosomes. Both of the two constructs, one of 10 kb and the other of 13 kb, contain a T.brucei PARP promoter driving a chloramphenicol acetyltransferase (CAT) gene. In the 10 kb construct the CAT gene is followed by one hygromycin phosphotransferase (Hph) gene, and in the 13 kb construct the CAT gene is followed by three tandemly linked Hph genes. At each end of these linear molecules are telomere repeats and subtelomeric sequences. Electroporation of these linear DNA constructs into the procyclic form of T.brucei generated hygromycin-B resistant cell lines. In these cell lines, the input DNA remained linear and bounded by the telomere ends, but it increased in size. In the cell lines generated by the 10 kb construct, the input DNA increased in size to 20-50 kb. In the cell lines generated by the 13 kb constructs, two sizes of linear DNAs containing the input plasmid were detected: one of 40-50 kb and the other of 150 kb. The increase in size was not the result of in vivo tandem repetitions of the input plasmid, but represented the addition of new sequences. These Hph containing linear DNA molecules were maintained stably in cell lines for at least 20 generations in the absence of drug selection and were subsequently referred to as trypanosome artificial mini-chromosomes, or TACs. Images PMID:8532534

Mutation Rates across Budding Yeast Chromosome VI Are Correlated with Replication Timing

PubMed Central

Lang, Gregory I.; Murray, Andrew W.

2011-01-01

Previous experimental studies suggest that the mutation rate is nonuniform across the yeast genome. To characterize this variation across the genome more precisely, we measured the mutation rate of the URA3 gene integrated at 43 different locations tiled across Chromosome VI. We show that mutation rate varies 6-fold across a single chromosome, that this variation is correlated with replication timing, and we propose a model to explain this variation that relies on the temporal separation of two processes for replicating past damaged DNA: error-free DNA damage tolerance and translesion synthesis. This model is supported by the observation that eliminating translesion synthesis decreases this variation. PMID:21666225

Assignment of xeroderma pigmentosum group C(XPC) gene to chromosome 3p25

DOE Office of Scientific and Technical Information (OSTI.GOV)

Legerski, R.J.; Liu, P.; Li, L.

1994-05-01

The human gene XPC (formerly designated XPCC), which corrects the repair deficiency of xeroderma pigmentosum (XP) group C cells, was mapped to 3p25. A cDNA probe for Southern blot hybridization and diagnostic PCR analyses of hybrid clone panels informative for human chromosomes in general and portions of chromosome 3 in particular produced the initial results. Fluorescence in situ hybridization utilizing both a yeast artificial chromosome DNA containing the gene and XPC cDNA as probes provided verification and specific regional assignment. A conflicting assignment of XPC to chromosome 5 is discussed in light of inadequacies in the exclusive use of microcell-mediatedmore » chromosome transfer for gene mapping. 12 refs., 3 figs.« less

Drought-tolerant rice germplasm developed from an Oryza officinalis transformation-competent artificial chromosome clone.

PubMed

Liu, R; Zhang, H H; Chen, Z X; Shahid, M Q; Fu, X L; Liu, X D

2015-10-29

Oryza officinalis has proven to be a natural gene reservoir for the improvement of domesticated rice as it carries many desirable traits; however, the transfer of elite genes to cultivated rice by conventional hybridization has been a challenge for rice breeders. In this study, the conserved sequence of plant stress-related NAC transcription factors was selected as a probe to screen the O. officinalis genomic transformation-competent artificial chromosome library by Southern blot; 11 positive transformation-competent artificial chromosome clones were subsequently detected. By Agrobacterium-mediated transformation, an indica rice variety, Huajingxian 74 (HJX74), was transformed with a TAC clone harboring a NAC gene-positive genomic fragment from O. officinalis. Molecular analysis revealed that the O. officinalis genomic fragment was integrated into the genome of HJX74. The transgenic lines exhibited high tolerance to drought stress. Our results demonstrate that the introduction of stress-related transformation-competent artificial chromosome clones, coupled with a transgenic validation approach, is an effective method of transferring agronomically important genes from O. officinalis to cultivated rice.

Spatial organization of the budding yeast genome in the cell nucleus and identification of specific chromatin interactions from multi-chromosome constrained chromatin model.

PubMed

Gürsoy, Gamze; Xu, Yun; Liang, Jie

2017-07-01

Nuclear landmarks and biochemical factors play important roles in the organization of the yeast genome. The interaction pattern of budding yeast as measured from genome-wide 3C studies are largely recapitulated by model polymer genomes subject to landmark constraints. However, the origin of inter-chromosomal interactions, specific roles of individual landmarks, and the roles of biochemical factors in yeast genome organization remain unclear. Here we describe a multi-chromosome constrained self-avoiding chromatin model (mC-SAC) to gain understanding of the budding yeast genome organization. With significantly improved sampling of genome structures, both intra- and inter-chromosomal interaction patterns from genome-wide 3C studies are accurately captured in our model at higher resolution than previous studies. We show that nuclear confinement is a key determinant of the intra-chromosomal interactions, and centromere tethering is responsible for the inter-chromosomal interactions. In addition, important genomic elements such as fragile sites and tRNA genes are found to be clustered spatially, largely due to centromere tethering. We uncovered previously unknown interactions that were not captured by genome-wide 3C studies, which are found to be enriched with tRNA genes, RNAPIII and TFIIS binding. Moreover, we identified specific high-frequency genome-wide 3C interactions that are unaccounted for by polymer effects under landmark constraints. These interactions are enriched with important genes and likely play biological roles.

Gateway Vectors for Efficient Artificial Gene Assembly In Vitro and Expression in Yeast Saccharomyces cerevisiae

PubMed Central

Giuraniuc, Claudiu V.; MacPherson, Murray; Saka, Yasushi

2013-01-01

Construction of synthetic genetic networks requires the assembly of DNA fragments encoding functional biological parts in a defined order. Yet this may become a time-consuming procedure. To address this technical bottleneck, we have created a series of Gateway shuttle vectors and an integration vector, which facilitate the assembly of artificial genes and their expression in the budding yeast Saccharomyces cerevisiae. Our method enables the rapid construction of an artificial gene from a promoter and an open reading frame (ORF) cassette by one-step recombination reaction in vitro. Furthermore, the plasmid thus created can readily be introduced into yeast cells to test the assembled gene’s functionality. As flexible regulatory components of a synthetic genetic network, we also created new versions of the tetracycline-regulated transactivators tTA and rtTA by fusing them to the auxin-inducible degron (AID). Using our gene assembly approach, we made yeast expression vectors of these engineered transactivators, AIDtTA and AIDrtTA and then tested their functions in yeast. We showed that these factors can be regulated by doxycycline and degraded rapidly after addition of auxin to the medium. Taken together, the method for combinatorial gene assembly described here is versatile and would be a valuable tool for yeast synthetic biology. PMID:23675537

Highly stable maintenance of a mouse artificial chromosome in human cells and mice.

PubMed

Kazuki, Kanako; Takehara, Shoko; Uno, Narumi; Imaoka, Natsuko; Abe, Satoshi; Takiguchi, Masato; Hiramatsu, Kei; Oshimura, Mitsuo; Kazuki, Yasuhiro

2013-12-06

Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) display several advantages as gene delivery vectors, such as stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Previously, we showed that a MAC vector developed from a natural mouse chromosome by chromosome engineering was more stably maintained in adult tissues and hematopoietic cells in mice than HAC vectors. In this study, to expand the utility for a gene delivery vector in human cells and mice, we investigated the long-term stability of the MACs in cultured human cells and transchromosomic mice. We also investigated the chromosomal copy number-dependent expression of genes on the MACs in mice. The MAC was stably maintained in human HT1080 cells in vitro during long-term culture. The MAC was stably maintained at least to the F8 and F4 generations in ICR and C57BL/6 backgrounds, respectively. The MAC was also stably maintained in hematopoietic cells and tissues derived from old mice. Transchromosomic mice containing two or four copies of the MAC were generated by breeding. The DNA contents were comparable to the copy number of the MACs in each tissue examined, and the expression of the EGFP gene on the MAC was dependent on the chromosomal copy number. Therefore, the MAC vector may be useful not only for gene delivery in mammalian cells but also for animal transgenesis. Copyright © 2013 Elsevier Inc. All rights reserved.

Yeast diversity associated to sediments and water from two Colombian artificial lakes

PubMed Central

Silva-Bedoya, L.M.; Ramírez-Castrillón, M.; Osorio-Cadavid, E.

2014-01-01

In Colombia, knowledge of the yeast and yeast-like fungi community is limited because most studies have focused on species with clinical importance. Sediments and water represent important habitats for the study of yeast diversity, especially for yeast species with industrial, biotechnological, and bioremediation potential. The main purpose of this study was to identify and compare the diversity of yeast species associated with sediment and water samples from two artificial lakes in Universidad del Valle (Cali-Colombia). Yeast samplings were performed from fifteen sediment samples and ten water samples. Grouping of similar isolates was initially based on colony and cell morphology, which was then complemented by micro/mini satellite primed PCR banding pattern analysis by using GTG5 as single primer. A representative isolate for each group established was chosen for D1/D2 domain sequencing and identification. In general, the following yeast species were identified: Candida albicans, Candida diversa, Candida glabrata, Candida pseudolambica, Cryptococcus podzolicus, Cryptococcus rajasthanensis, Cryptococcus laurentii, Williopsis saturnus, Hanseniaspora thailandica, Hanseniaspora uvarum, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Torulaspora delbrueckii, Torulaspora pretoriensis, Tricosporon jirovecii, Trichosporon laibachii and Yarrowia lypolitica. Two possible new species were also found, belonging to the Issatchenkia sp. and Bullera sp. genera. In conclusion, the lakes at the Universidad del Valle campus have significant differences in yeast diversity and species composition between them. PMID:24948924

Evolutionary Role of Interspecies Hybridization and Genetic Exchanges in Yeasts

PubMed Central

Dujon, Bernard

2012-01-01

Summary: Forced interspecific hybridization has been used in yeasts for many years to study speciation or to construct artificial strains with novel fermentative and metabolic properties. Recent genome analyses indicate that natural hybrids are also generated spontaneously between yeasts belonging to distinct species, creating lineages with novel phenotypes, varied genetic stability, or altered virulence in the case of pathogens. Large segmental introgressions from evolutionarily distant species are also visible in some yeast genomes, suggesting that interspecific genetic exchanges occur during evolution. The origin of this phenomenon remains unclear, but it is likely based on weak prezygotic barriers, limited Dobzhansky-Muller (DM) incompatibilities, and rapid clonal expansions. Newly formed interspecies hybrids suffer rapid changes in the genetic contribution of each parent, including chromosome loss or aneuploidy, translocations, and loss of heterozygosity, that, except in a few recently studied cases, remain to be characterized more precisely at the genomic level by use of modern technologies. We review here known cases of natural or artificially formed interspecies hybrids between yeasts and discuss their potential importance in terms of genome evolution. Problems of meiotic fertility, ploidy constraint, gene and gene product compatibility, and nucleomitochondrial interactions are discussed and placed in the context of other known mechanisms of yeast genome evolution as a model for eukaryotes. PMID:23204364

A Study for the Feature Selection to Identify GIEMSA-Stained Human Chromosomes Based on Artificial Neural Network

DTIC Science & Technology

2001-10-25

neural network (ANN) has been adopted for the human chromosome classification. It is important to select optimum features for training neural network...Many studies for computer-based chromosome analysis have shown that it is possible to classify chromosomes into 24 subgroups. In addition, artificial

Control of mitotic chromosome condensation by the fission yeast transcription factor Zas1.

PubMed

Schiklenk, Christoph; Petrova, Boryana; Kschonsak, Marc; Hassler, Markus; Klein, Carlo; Gibson, Toby J; Haering, Christian H

2018-05-07

Although the formation of rod-shaped chromosomes is vital for the correct segregation of eukaryotic genomes during cell divisions, the molecular mechanisms that control the chromosome condensation process have remained largely unknown. Here, we identify the C 2 H 2 zinc-finger transcription factor Zas1 as a key regulator of mitotic condensation dynamics in a quantitative live-cell microscopy screen of the fission yeast Schizosaccharomyces pombe By binding to specific DNA target sequences in their promoter regions, Zas1 controls expression of the Cnd1 subunit of the condensin protein complex and several other target genes, whose combined misregulation in zas1 mutants results in defects in chromosome condensation and segregation. Genetic and biochemical analysis reveals an evolutionarily conserved transactivation domain motif in Zas1 that is pivotal to its function in gene regulation. Our results suggest that this motif, together with the Zas1 C-terminal helical domain to which it binds, creates a cis/trans switch module for transcriptional regulation of genes that control chromosome condensation. © 2018 Schiklenk et al.

Speciation driven by hybridization and chromosomal plasticity in a wild yeast.

PubMed

Leducq, Jean-Baptiste; Nielly-Thibault, Lou; Charron, Guillaume; Eberlein, Chris; Verta, Jukka-Pekka; Samani, Pedram; Sylvester, Kayla; Hittinger, Chris Todd; Bell, Graham; Landry, Christian R

2016-01-11

Hybridization is recognized as a powerful mechanism of speciation and a driving force in generating biodiversity. However, only few multicellular species, limited to a handful of plants and animals, have been shown to fulfil all the criteria of homoploid hybrid speciation. This lack of evidence could lead to the interpretation that speciation by hybridization has a limited role in eukaryotes, particularly in single-celled organisms. Laboratory experiments have revealed that fungi such as budding yeasts can rapidly develop reproductive isolation and novel phenotypes through hybridization, showing that in principle homoploid speciation could occur in nature. Here, we report a case of homoploid hybrid speciation in natural populations of the budding yeast Saccharomyces paradoxus inhabiting the North American forests. We show that the rapid evolution of chromosome architecture and an ecological context that led to secondary contact between nascent species drove the formation of an incipient hybrid species with a potentially unique ecological niche.

The A-Like Faker Assay for Measuring Yeast Chromosome III Stability.

PubMed

Novoa, Carolina A; Ang, J Sidney; Stirling, Peter C

2018-01-01

The ability to rapidly assess chromosome instability (CIN) has enabled profiling of most yeast genes for potential effects on genome stability. The A-like faker (ALF) assay is one of several qualitative and quantitative marker loss assays that indirectly measure loss or conversion of genetic material using a counterselection step. The ALF assay relies on the ability to count spurious mating events that occur upon loss of the MATα locus of haploid Saccharomyces cerevisiae strains. Here, we describe the deployment of the ALF assay for both rapid and simple qualitative, and more in-depth quantitative analysis allowing determination of absolute ALF frequencies.

Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells.

PubMed

Hong, Y K; Kim, D H; Beletskii, A; Lee, C; Memili, E; Strauss, W M

2001-04-01

Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells. Copyright 2001 Academic Press.

Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene.

PubMed

Mollereau, C; Simons, M J; Soularue, P; Liners, F; Vassart, G; Meunier, J C; Parmentier, M

1996-08-06

Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo. Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated. The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin. The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant. Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene. By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p21), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL. Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p21 region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig.

Chromosomal localization and structure of the human type II IMP dehydrogenase gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Glesne, D.; Huberman, E.; Collart, F.

1994-05-01

We determined the chromosomal localization and structure of the gene encoding human type II inosine 5{prime}-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205), an enzyme associated with cellular proliferation, malignant transformation, and differentiation. Using polymerase chain reaction (PCR) primers specific for type II IMPDH, we screened a panel of human-Chinese hamster cell somatic hybrids and a separate deletion panel of chromosome 3 hybrids and localized the gene to 3p21.1{yields}p24.2. Two overlapping yeast artificial chromosome clones containing the full gene for type II IMPDH were isolated and a physical map of 117 kb of human genomic DNA in this region of chromosome 3 wasmore » constructed. The gene for type II IMPDH was localized and oriented on this map and found to span no more than 12.5 kb.« less

Mps1 and Ipl1/Aurora B act sequentially to correctly orient chromosomes on the meiotic spindle of budding yeast.

PubMed

Meyer, Régis E; Kim, Seoyoung; Obeso, David; Straight, Paul D; Winey, Mark; Dawson, Dean S

2013-03-01

The conserved kinases Mps1 and Ipl1/Aurora B are critical for enabling chromosomes to attach to microtubules so that partner chromosomes will be segregated correctly from each other, but the precise roles of these kinases have been unclear. We imaged live yeast cells to elucidate the stages of chromosome-microtubule interactions and their regulation by Ipl1 and Mps1 through meiosis I. Ipl1 was found to release kinetochore-microtubule (kMT) associations after meiotic entry, liberating chromosomes to begin homologous pairing. Surprisingly, most chromosome pairs began their spindle interactions with incorrect kMT attachments. Ipl1 released these improper connections, whereas Mps1 triggered the formation of new force-generating microtubule attachments. This microtubule release and reattachment cycle could prevent catastrophic chromosome segregation errors in meiosis.

Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

PubMed Central

Kakui, Yasutaka; Sunaga, Tomonari; Arai, Kunio; Dodgson, James; Ji, Liang; Csikász-Nagy, Attila; Carazo-Salas, Rafael; Sato, Masamitsu

2015-01-01

Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism. PMID:26108218

Capturing Chromosome Conformation

NASA Astrophysics Data System (ADS)

Dekker, Job; Rippe, Karsten; Dekker, Martijn; Kleckner, Nancy

2002-02-01

We describe an approach to detect the frequency of interaction between any two genomic loci. Generation of a matrix of interaction frequencies between sites on the same or different chromosomes reveals their relative spatial disposition and provides information about the physical properties of the chromatin fiber. This methodology can be applied to the spatial organization of entire genomes in organisms from bacteria to human. Using the yeast Saccharomyces cerevisiae, we could confirm known qualitative features of chromosome organization within the nucleus and dynamic changes in that organization during meiosis. We also analyzed yeast chromosome III at the G1 stage of the cell cycle. We found that chromatin is highly flexible throughout. Furthermore, functionally distinct AT- and GC-rich domains were found to exhibit different conformations, and a population-average 3D model of chromosome III could be determined. Chromosome III emerges as a contorted ring.

Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology.

PubMed

Moralli, Daniela; Monaco, Zoia L

2015-02-01

De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.

Anniversary of the discovery/isolation of the yeast centromere by Clarke and Carbon.

PubMed

Bloom, Kerry

2015-05-01

The first centromere was isolated 35 years ago by Louise Clarke and John Carbon from budding yeast. They embarked on their journey with rudimentary molecular tools (by today's standards) and little knowledge of the structure of a chromosome, much less the nature of a centromere. Their discovery opened up a new field, as centromeres have now been isolated from fungi and numerous plants and animals, including mammals. Budding yeast and several other fungi have small centromeres with short, well-defined sequences, known as point centromeres, whereas regional centromeres span several kilobases up to megabases and do not seem to have DNA sequence specificity. Centromeres are at the heart of artificial chromosomes, and we have seen the birth of synthetic centromeres in budding and fission yeast and mammals. The diversity in centromeres throughout phylogeny belie conserved functions that are only beginning to be understood. © 2015 Bloom. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Structural maintenance of chromosome complexes differentially compact mitotic chromosomes according to genomic context

PubMed Central

Schalbetter, S. A.; Goloborodko, A.; Fudenberg, G.; Belton, J.-M.; Miles, C.; Yu, M.; Dekker, J.; Mirny, L.; Baxter, J.

2017-01-01

Structural Maintenance of Chromosomes (SMC) protein complexes are key determinants of chromosome conformation. Using Hi-C and polymer modeling, we study how cohesin and condensin, two deeply conserved SMC complexes, organize chromosomes in the budding yeast Saccharomyces cerevisiae. The canonical role of cohesin is to co-align sister chromatids whilst condensin generally compacts mitotic chromosomes. We find strikingly different roles for the two complexes in budding yeast mitosis. First, cohesin is responsible for compacting mitotic chromosome arms, independently of sister chromatid cohesion. Polymer simulations demonstrate this role can be fully accounted for through cis-looping of chromatin. Second, condensin is generally dispensable for compaction along chromosome arms. Instead it plays a targeted role compacting the rDNA proximal regions and promoting resolution of peri-centromeric regions. Our results argue that the conserved mechanism of SMC complexes is to form chromatin loops and that distinct SMC-dependent looping activities are selectively deployed to appropriately compact chromosomes. PMID:28825700

RPA facilitates telomerase activity at chromosome ends in budding and fission yeasts

PubMed Central

Luciano, Pierre; Coulon, Stéphane; Faure, Virginie; Corda, Yves; Bos, Julia; Brill, Steven J; Gilson, Eric; Simon, Marie-Noelle; Géli, Vincent

2012-01-01

In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends. PMID:22354040

RPA facilitates telomerase activity at chromosome ends in budding and fission yeasts.

PubMed

Luciano, Pierre; Coulon, Stéphane; Faure, Virginie; Corda, Yves; Bos, Julia; Brill, Steven J; Gilson, Eric; Simon, Marie-Noelle; Géli, Vincent

2012-04-18

In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends.

Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis.

PubMed

Medhi, Darpan; Goldman, Alastair Sh; Lichten, Michael

2016-11-18

The budding yeast genome contains regions where meiotic recombination initiates more frequently than in others. This pattern parallels enrichment for the meiotic chromosome axis proteins Hop1 and Red1. These proteins are important for Spo11-catalyzed double strand break formation; their contribution to crossover recombination remains undefined. Using the sequence-specific VMA1 -derived endonuclease (VDE) to initiate recombination in meiosis, we show that chromosome structure influences the choice of proteins that resolve recombination intermediates to form crossovers. At a Hop1-enriched locus, most VDE-initiated crossovers, like most Spo11-initiated crossovers, required the meiosis-specific MutLγ resolvase. In contrast, at a locus with lower Hop1 occupancy, most VDE-initiated crossovers were MutLγ-independent. In pch2 mutants, the two loci displayed similar Hop1 occupancy levels, and VDE-induced crossovers were similarly MutLγ-dependent. We suggest that meiotic and mitotic recombination pathways coexist within meiotic cells, and that features of meiotic chromosome structure determine whether one or the other predominates in different regions.

Centromere Destiny in Dicentric Chromosomes: New Insights from the Evolution of Human Chromosome 2 Ancestral Centromeric Region.

PubMed

Chiatante, Giorgia; Giannuzzi, Giuliana; Calabrese, Francesco Maria; Eichler, Evan E; Ventura, Mario

2017-07-01

Dicentric chromosomes are products of genomic rearrangements that place two centromeres on the same chromosome. Due to the presence of two primary constrictions, they are inherently unstable and overcome their instability by epigenetically inactivating and/or deleting one of the two centromeres, thus resulting in functionally monocentric chromosomes that segregate normally during cell division. Our understanding to date of dicentric chromosome formation, behavior and fate has been largely inferred from observational studies in plants and humans as well as artificially produced de novo dicentrics in yeast and in human cells. We investigate the most recent product of a chromosome fusion event fixed in the human lineage, human chromosome 2, whose stability was acquired by the suppression of one centromere, resulting in a unique difference in chromosome number between humans (46 chromosomes) and our most closely related ape relatives (48 chromosomes). Using molecular cytogenetics, sequencing, and comparative sequence data, we deeply characterize the relicts of the chromosome 2q ancestral centromere and its flanking regions, gaining insight into the ancestral organization that can be easily broadened to all acrocentric chromosome centromeres. Moreover, our analyses offered the opportunity to trace the evolutionary history of rDNA and satellite III sequences among great apes, thus suggesting a new hypothesis for the preferential inactivation of some human centromeres, including IIq. Our results suggest two possible centromere inactivation models to explain the evolutionarily stabilization of human chromosome 2 over the last 5-6 million years. Our results strongly favor centromere excision through a one-step process. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Mitotic Chromosome Biorientation in Fission Yeast Is Enhanced by Dynein and a Minus-end–directed, Kinesin-like Protein

PubMed Central

Spiridonov, Ilia S.; McIntosh, J. Richard

2007-01-01

Chromosome biorientation, the attachment of sister kinetochores to sister spindle poles, is vitally important for accurate chromosome segregation. We have studied this process by following the congression of pole-proximal kinetochores and their subsequent anaphase segregation in fission yeast cells that carry deletions in any or all of this organism's minus end–directed, microtubule-dependent motors: two related kinesin 14s (Pkl1p and Klp2p) and dynein. None of these deletions abolished biorientation, but fewer chromosomes segregated normally without Pkl1p, and to a lesser degree without dynein, than in wild-type cells. In the absence of Pkl1p, which normally localizes to the spindle and its poles, the checkpoint that monitors chromosome biorientation was defective, leading to frequent precocious anaphase. Ultrastructural analysis of mutant mitotic spindles suggests that Pkl1p contributes to error-free biorientation by promoting normal spindle pole organization, whereas dynein helps to anchor a focused bundle of spindle microtubules at the pole. PMID:17409356

Comparative genomics of wild type yeast strains unveils important genome diversity

PubMed Central

Carreto, Laura; Eiriz, Maria F; Gomes, Ana C; Pereira, Patrícia M; Schuller, Dorit; Santos, Manuel AS

2008-01-01

Background Genome variability generates phenotypic heterogeneity and is of relevance for adaptation to environmental change, but the extent of such variability in natural populations is still poorly understood. For example, selected Saccharomyces cerevisiae strains are variable at the ploidy level, have gene amplifications, changes in chromosome copy number, and gross chromosomal rearrangements. This suggests that genome plasticity provides important genetic diversity upon which natural selection mechanisms can operate. Results In this study, we have used wild-type S. cerevisiae (yeast) strains to investigate genome variation in natural and artificial environments. We have used comparative genome hybridization on array (aCGH) to characterize the genome variability of 16 yeast strains, of laboratory and commercial origin, isolated from vineyards and wine cellars, and from opportunistic human infections. Interestingly, sub-telomeric instability was associated with the clinical phenotype, while Ty element insertion regions determined genomic differences of natural wine fermentation strains. Copy number depletion of ASP3 and YRF1 genes was found in all wild-type strains. Other gene families involved in transmembrane transport, sugar and alcohol metabolism or drug resistance had copy number changes, which also distinguished wine from clinical isolates. Conclusion We have isolated and genotyped more than 1000 yeast strains from natural environments and carried out an aCGH analysis of 16 strains representative of distinct genotype clusters. Important genomic variability was identified between these strains, in particular in sub-telomeric regions and in Ty-element insertion sites, suggesting that this type of genome variability is the main source of genetic diversity in natural populations of yeast. The data highlights the usefulness of yeast as a model system to unravel intraspecific natural genome diversity and to elucidate how natural selection shapes the yeast genome

Analysis of the temporal program of replication initiation in yeast chromosomes.

PubMed

Friedman, K L; Raghuraman, M K; Fangman, W L; Brewer, B J

1995-01-01

The multiple origins of eukaryotic chromosomes vary in the time of their initiation during S phase. In the chromosomes of Saccharomyces cerevisiae the presence of a functional telomere causes nearby origins to delay initiation until the second half of S phase. The key feature of telomeres that causes the replication delay is the telomeric sequence (C(1-3)A/G(1-3)T) itself and not the proximity of the origin to a DNA end. A second group of late replicating origins has been found at an internal position on chromosome XIV. Four origins, spanning approximately 140 kb, initiate replication in the second half of S phase. At least two of these internal origins maintain their late replication time on circular plasmids. Each of these origins can be separated into two functional elements: those sequences that provide origin function and those that impose late activation. Because the assay for determining replication time is costly and laborious, it has not been possible to analyze in detail these 'late' elements. We report here the development of two new assays for determining replication time. The first exploits the expression of the Escherichia coli dam methylase in yeast and the characteristic period of hemimethylation that transiently follows the passage of a replication fork. The second uses quantitative hybridization to detect two-fold differences in the amount of specific restriction fragments as a function of progress through S phase. The novel aspect of this assay is the creation in vivo of a non-replicating DNA sequence by site-specific pop-out recombination. This non-replicating fragment acts as an internal control for copy number within and between samples. Both of these techniques are rapid and much less costly than the more conventional density transfer experiments that require CsCl gradients to detect replicated DNA. With these techniques it should be possible to identify the sequences responsible for late initiation, to search for other late replicating

Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

PubMed Central

Medhi, Darpan; Goldman, Alastair SH; Lichten, Michael

2016-01-01

The budding yeast genome contains regions where meiotic recombination initiates more frequently than in others. This pattern parallels enrichment for the meiotic chromosome axis proteins Hop1 and Red1. These proteins are important for Spo11-catalyzed double strand break formation; their contribution to crossover recombination remains undefined. Using the sequence-specific VMA1-derived endonuclease (VDE) to initiate recombination in meiosis, we show that chromosome structure influences the choice of proteins that resolve recombination intermediates to form crossovers. At a Hop1-enriched locus, most VDE-initiated crossovers, like most Spo11-initiated crossovers, required the meiosis-specific MutLγ resolvase. In contrast, at a locus with lower Hop1 occupancy, most VDE-initiated crossovers were MutLγ-independent. In pch2 mutants, the two loci displayed similar Hop1 occupancy levels, and VDE-induced crossovers were similarly MutLγ-dependent. We suggest that meiotic and mitotic recombination pathways coexist within meiotic cells, and that features of meiotic chromosome structure determine whether one or the other predominates in different regions. DOI: http://dx.doi.org/10.7554/eLife.19669.001 PMID:27855779

Adaptive response to chronic mild ethanol stress involves ROS, sirtuins and changes in chromosome dosage in wine yeasts.

PubMed

Adamczyk, Jagoda; Deregowska, Anna; Skoneczny, Marek; Skoneczna, Adrianna; Kwiatkowska, Aleksandra; Potocki, Leszek; Rawska, Ewa; Pabian, Sylwia; Kaplan, Jakub; Lewinska, Anna; Wnuk, Maciej

2016-05-24

Industrial yeast strains of economic importance used in winemaking and beer production are genomically diverse and subjected to harsh environmental conditions during fermentation. In the present study, we investigated wine yeast adaptation to chronic mild alcohol stress when cells were cultured for 100 generations in the presence of non-cytotoxic ethanol concentration. Ethanol-induced reactive oxygen species (ROS) and superoxide signals promoted growth rate during passages that was accompanied by increased expression of sirtuin proteins, Sir1, Sir2 and Sir3, and DNA-binding transcription regulator Rap1. Genome-wide array-CGH analysis revealed that yeast genome was shaped during passages. The gains of chromosomes I, III and VI and significant changes in the gene copy number in nine functional gene categories involved in metabolic processes and stress responses were observed. Ethanol-mediated gains of YRF1 and CUP1 genes were the most accented. Ethanol also induced nucleolus fragmentation that confirms that nucleolus is a stress sensor in yeasts. Taken together, we postulate that wine yeasts of different origin may adapt to mild alcohol stress by shifts in intracellular redox state promoting growth capacity, upregulation of key regulators of longevity, namely sirtuins and changes in the dosage of genes involved in the telomere maintenance and ion detoxification.

Saccharomyces interspecies hybrids as model organisms for studying yeast adaptation to stressful environments.

PubMed

Lopandic, Ksenija

2018-01-01

The strong development of molecular biology techniques and next-generation sequencing technologies in the last two decades has significantly improved our understanding of the evolutionary history of Saccharomyces yeasts. It has been shown that many strains isolated from man-made environments are not pure genetic lines, but contain genetic materials from different species that substantially increase their genome complexity. A number of strains have been described as interspecies hybrids, implying different yeast species that under specific circumstances exchange and recombine their genomes. Such fusing usually results in a wide variety of alterations at the genetic and chromosomal levels. The observed changes have suggested a high genome plasticity and a significant role of interspecies hybridization in the adaptation of yeasts to environmental stresses and industrial processes. There is a high probability that harsh wine and beer fermentation environments, from which the majority of interspecies hybrids have been isolated so far, influence their selection and stabilization as well as their genomic and phenotypic heterogeneity. The lessons we have learned about geno- and phenotype plasticity and the diversity of natural and commercial yeast hybrids have already had a strong impact on the development of artificial hybrids that can be successfully used in the fermentation-based food and beverage industry. The creation of artificial hybrids through the crossing of strains with desired attributes is a possibility to obtain a vast variety of new, but not genetically modified yeasts with a range of improved and beneficial traits. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Characterization of a microdissection library from human chromosome region 3p14

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bardenheuer, W.; Szymanski, S.; Lux, A.

1994-01-15

Structural alterations in human chromosome region 3p14-p23 resulting in the inactivation of one or more tumor suppressor genes are thought to play a pathogenic role in small cell lung cancer, renal cell carcinoma, and other human neoplasms. To identify putative tumor suppressor genes, 428 recombinant clones from a microdissection library specific for human chromosome region 3p14 were isolated and characterized. Ninety-six of these (22.5%) were human single-copy DNA sequences, 57 of which were unique sequence clones. Forty-four of these were mapped to the microdissected region using a cell hybrid mapping panel. Within this mapping panel, four probes detected two newmore » chromosome breakpoints that were previously indistinguishable from the translocation breakpoint t(3;8) in 3p14.2 in hereditary renal cell carcinoma. One probe maps to the homozygously deleted region of the small cell lung cancer cell line U2020. In addition, microdissection clones have been shown to be suitable for isolation of yeast artificial chromosomes. 52 refs., 3 figs., 2 tabs.« less

Bacterial Artificial Chromosome Libraries of Pulse Crops: Characteristics and Applications

PubMed Central

Yu, Kangfu

2012-01-01

Pulse crops are considered minor on a global scale despite their nutritional value for human consumption. Therefore, they are relatively less extensively studied in comparison with the major crops. The need to improve pulse crop production and quality will increase with the increasing global demand for food security and people's awareness of nutritious food. The improvement of pulse crops will require fully utilizing all their genetic resources. Bacterial artificial chromosome (BAC) libraries of pulse crops are essential genomic resources that have the potential to accelerate gene discovery and enhance molecular breeding in these crops. Here, we review the availability, characteristics, applications, and potential applications of the BAC libraries of pulse crops. PMID:21811383

Construction and characterization of a bacterial artificial chromosome library for hexaploid wheat line 92R137

USDA-ARS?s Scientific Manuscript database

For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137, a bacterial artificial chromosome (BAC) library, including two sub libraries, was constructed using the genomic DNA of 92R137 digested with restriction enzymes HindIII and BamHI. The BAC library was compos...

Scc2 regulates gene expression by recruiting cohesin to the chromosome as a transcriptional activator during yeast meiosis

PubMed Central

Lin, Weiqiang; Jin, Hui; Liu, Xiuwen; Hampton, Kristin; Yu, Hong-Guo

2011-01-01

To tether sister chromatids, a protein-loading complex, including Scc2, recruits cohesin to the chromosome at discrete loci. Cohesin facilitates the formation of a higher-order chromosome structure that could also influence gene expression. How cohesin directly regulates transcription remains to be further elucidated. We report that in budding yeast Scc2 is required for sister-chromatid cohesion during meiosis for two reasons. First, Scc2 is required for activating the expression of REC8, which encodes a meiosis-specific cohesin subunit; second, Scc2 is necessary for recruiting meiotic cohesin to the chromosome to generate sister-chromatid cohesion. Using a heterologous reporter assay, we have found that Scc2 increases the activity of its target promoters by recruiting cohesin to establish an upstream cohesin-associated region in a position-dependent manner. Rec8-associated meiotic cohesin is required for the full activation of the REC8 promoter, revealing that cohesin has a positive feedback on transcriptional regulation. Finally, we provide evidence that chromosomal binding of cohesin is sufficient for target-gene activation during meiosis. Our data support a noncanonical role for cohesin as a transcriptional activator during cell differentiation. PMID:21508318

Bacterial Artificial Chromosome Libraries for Mouse Sequencing and Functional Analysis

PubMed Central

Osoegawa, Kazutoyo; Tateno, Minako; Woon, Peng Yeong; Frengen, Eirik; Mammoser, Aaron G.; Catanese, Joseph J.; Hayashizaki, Yoshihide; de Jong, Pieter J.

2000-01-01

Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) libraries providing a combined 33-fold representation of the murine genome have been constructed using two different restriction enzymes for genomic digestion. A large-insert PAC library was prepared from the 129S6/SvEvTac strain in a bacterial/mammalian shuttle vector to facilitate functional gene studies. For genome mapping and sequencing, we prepared BAC libraries from the 129S6/SvEvTac and the C57BL/6J strains. The average insert sizes for the three libraries range between 130 kb and 200 kb. Based on the numbers of clones and the observed average insert sizes, we estimate each library to have slightly in excess of 10-fold genome representation. The average number of clones found after hybridization screening with 28 probes was in the range of 9–14 clones per marker. To explore the fidelity of the genomic representation in the three libraries, we analyzed three contigs, each established after screening with a single unique marker. New markers were established from the end sequences and screened against all the contig members to determine if any of the BACs and PACs are chimeric or rearranged. Only one chimeric clone and six potential deletions have been observed after extensive analysis of 113 PAC and BAC clones. Seventy-one of the 113 clones were conclusively nonchimeric because both end markers or sequences were mapped to the other confirmed contig members. We could not exclude chimerism for the remaining 41 clones because one or both of the insert termini did not contain unique sequence to design markers. The low rate of chimerism, ∼1%, and the low level of detected rearrangements support the anticipated usefulness of the BAC libraries for genome research. [The sequence data described in this paper have been submitted to the GenBank data library under accession numbers AQ797173–AQ797398.] PMID:10645956

Genome sequence of the lager brewing yeast, an interspecies hybrid.

PubMed

Nakao, Yoshihiro; Kanamori, Takeshi; Itoh, Takehiko; Kodama, Yukiko; Rainieri, Sandra; Nakamura, Norihisa; Shimonaga, Tomoko; Hattori, Masahira; Ashikari, Toshihiko

2009-04-01

This work presents the genome sequencing of the lager brewing yeast (Saccharomyces pastorianus) Weihenstephan 34/70, a strain widely used in lager beer brewing. The 25 Mb genome comprises two nuclear sub-genomes originating from Saccharomyces cerevisiae and Saccharomyces bayanus and one circular mitochondrial genome originating from S. bayanus. Thirty-six different types of chromosomes were found including eight chromosomes with translocations between the two sub-genomes, whose breakpoints are within the orthologous open reading frames. Several gene loci responsible for typical lager brewing yeast characteristics such as maltotriose uptake and sulfite production have been increased in number by chromosomal rearrangements. Despite an overall high degree of conservation of the synteny with S. cerevisiae and S. bayanus, the syntenies were not well conserved in the sub-telomeric regions that contain lager brewing yeast characteristic and specific genes. Deletion of larger chromosomal regions, a massive unilateral decrease of the ribosomal DNA cluster and bilateral truncations of over 60 genes reflect a post-hybridization evolution process. Truncations and deletions of less efficient maltose and maltotriose uptake genes may indicate the result of adaptation to brewing. The genome sequence of this interspecies hybrid yeast provides a new tool for better understanding of lager brewing yeast behavior in industrial beer production.

Genome Sequence of the Lager Brewing Yeast, an Interspecies Hybrid

PubMed Central

Nakao, Yoshihiro; Kanamori, Takeshi; Itoh, Takehiko; Kodama, Yukiko; Rainieri, Sandra; Nakamura, Norihisa; Shimonaga, Tomoko; Hattori, Masahira; Ashikari, Toshihiko

2009-01-01

This work presents the genome sequencing of the lager brewing yeast (Saccharomyces pastorianus) Weihenstephan 34/70, a strain widely used in lager beer brewing. The 25 Mb genome comprises two nuclear sub-genomes originating from Saccharomyces cerevisiae and Saccharomyces bayanus and one circular mitochondrial genome originating from S. bayanus. Thirty-six different types of chromosomes were found including eight chromosomes with translocations between the two sub-genomes, whose breakpoints are within the orthologous open reading frames. Several gene loci responsible for typical lager brewing yeast characteristics such as maltotriose uptake and sulfite production have been increased in number by chromosomal rearrangements. Despite an overall high degree of conservation of the synteny with S. cerevisiae and S. bayanus, the syntenies were not well conserved in the sub-telomeric regions that contain lager brewing yeast characteristic and specific genes. Deletion of larger chromosomal regions, a massive unilateral decrease of the ribosomal DNA cluster and bilateral truncations of over 60 genes reflect a post-hybridization evolution process. Truncations and deletions of less efficient maltose and maltotriose uptake genes may indicate the result of adaptation to brewing. The genome sequence of this interspecies hybrid yeast provides a new tool for better understanding of lager brewing yeast behavior in industrial beer production. PMID:19261625

Cloning of a very virulent plus, 686 strain of Marek’s disease virus as a bacterial artificial chromosome

USDA-ARS?s Scientific Manuscript database

Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek’s disease virus (MDV) is a highly oncogenic herpe...

Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer

PubMed Central

Duffy, Supipi; Fam, Hok Khim; Wang, Yi Kan; Styles, Erin B.; Kim, Jung-Hyun; Ang, J. Sidney; Singh, Tejomayee; Larionov, Vladimir; Shah, Sohrab P.; Andrews, Brenda; Boerkoel, Cornelius F.; Hieter, Philip

2016-01-01

Somatic copy number amplification and gene overexpression are common features of many cancers. To determine the role of gene overexpression on chromosome instability (CIN), we performed genome-wide screens in the budding yeast for yeast genes that cause CIN when overexpressed, a phenotype we refer to as dosage CIN (dCIN), and identified 245 dCIN genes. This catalog of genes reveals human orthologs known to be recurrently overexpressed and/or amplified in tumors. We show that two genes, TDP1, a tyrosyl-DNA-phosphdiesterase, and TAF12, an RNA polymerase II TATA-box binding factor, cause CIN when overexpressed in human cells. Rhabdomyosarcoma lines with elevated human Tdp1 levels also exhibit CIN that can be partially rescued by siRNA-mediated knockdown of TDP1. Overexpression of dCIN genes represents a genetic vulnerability that could be leveraged for selective killing of cancer cells through targeting of an unlinked synthetic dosage lethal (SDL) partner. Using SDL screens in yeast, we identified a set of genes that when deleted specifically kill cells with high levels of Tdp1. One gene was the histone deacetylase RPD3, for which there are known inhibitors. Both HT1080 cells overexpressing hTDP1 and rhabdomyosarcoma cells with elevated levels of hTdp1 were more sensitive to histone deacetylase inhibitors valproic acid (VPA) and trichostatin A (TSA), recapitulating the SDL interaction in human cells and suggesting VPA and TSA as potential therapeutic agents for tumors with elevated levels of hTdp1. The catalog of dCIN genes presented here provides a candidate list to identify genes that cause CIN when overexpressed in cancer, which can then be leveraged through SDL to selectively target tumors. PMID:27551064

Analysis and Dynamics of the Chromosomal Complements of Wild Sparkling-Wine Yeast Strains

PubMed Central

Nadal, Dolors; Carro, David; Fernández-Larrea, Juan; Piña, Benjamin

1999-01-01

We isolated Saccharomyces cerevisiae yeast strains that are able to carry out the second fermentation of sparkling wine from spontaneously fermenting musts in El Penedès (Spain) by specifically designed selection protocols. All of them (26 strains) showed one of two very similar mitochondrial DNA (mtDNA) restriction patterns, whereas their karyotypes differed. These strains showed high rates of karyotype instability, which were dependent on both the medium and the strain, during vegetative growth. In all cases, the mtDNA restriction pattern was conserved in strains kept under the same conditions. Analysis of different repetitive sequences in their genomes suggested that ribosomal DNA repeats play an important role in the changes in size observed in chromosome XII, whereas SUC genes or Ty elements did not show amplification or transposition processes that could be related to rearrangements of the chromosomes showing these sequences. Karyotype changes also occurred in monosporidic diploid derivatives. We propose that these changes originated mainly from ectopic recombination between repeated sequences interspersed in the genome. None of the rearranged karyotypes provided a selective advantage strong enough to allow the strains to displace the parental strains. The nature and frequency of these changes suggest that they may play an important role in the establishment and maintenance of the genetic diversity observed in S. cerevisiae wild populations. PMID:10103269

Multiple α-Glucoside Transporter Genes in Brewer’s Yeast

PubMed Central

Jespersen, Lene; Cesar, Lene B.; Meaden, Philip G.; Jakobsen, Mogens

1999-01-01

Maltose and maltotriose are the two most abundant fermentable sugars in brewer’s wort, and the rate of uptake of these sugars by brewer’s yeast can have a major impact on fermentation performance. In spite of this, no information is currently available on the genetics of maltose and maltotriose uptake in brewing strains of yeast. In this work, we studied 30 brewing strains of yeast (5 ale strains and 25 lager strains) with the aim of examining the alleles of maltose and maltotriose transporter genes contained by them. To do this, we hybridized gene probes to chromosome blots. Studies performed with laboratory strains have shown that maltose utilization is conferred by any one of five unlinked but highly homologous MAL loci (MAL1 to MAL4 and MAL6). Gene 1 at each locus encodes a maltose transporter. All of the strains of brewer’s yeast examined except two were found to contain MAL11 and MAL31 sequences, and only one of these strains lacked MAL41. MAL21 was not present in the five ale strains and 12 of the lager strains. MAL61 was not found in any of the yeast strains. In three of the lager strains, there was evidence that MAL transporter gene sequences occurred on chromosomes other than those known to carry MAL loci. Sequences corresponding to the AGT1 gene, which encodes a transporter of several α-glucosides, including maltose and maltotriose, were detected in all but one of the yeast strains. Homologues of AGT1 were identified in three of the lager strains, and two of these homologues were mapped, one to chromosome II and the other to chromosome XI. AGT1 appears to be a member of a family of closely related genes, which may have arisen in brewer’s yeast in response to selective pressure. PMID:9925567

cDNA cloning, tissue distribution, and chromosomal localization of myelodysplasia/myeloid leukemia factor 2 (MLF2).

PubMed

Kuefer, M U; Look, A T; Williams, D C; Valentine, V; Naeve, C W; Behm, F G; Mullersman, J E; Yoneda-Kato, N; Montgomery, K; Kucherlapati, R; Morris, S W

1996-07-15

A fusion gene between nucleophosmin (NPM) and myelodysplasia/myeloid leukemia factor 1 (MLF1) is formed by a recurrent t(3;5)(q25.1;q34) in myelodysplastic syndrome and acute myeloid leukemia. Here we report the identification of a novel gene, MLF2, which contains an open reading frame of 744 bp encoding a 248-amino-acid protein highly related to the previously identified MLF1 protein (63% similarity, 40% identity). In contrast to the tissue-restricted expression pattern of MLF1, the MLF2 messenger RNA is expressed ubiquitously. The MLF2 gene locus was mapped by fluorescence in situ hybridization to human chromosome 12p13, a chromosomal region frequently involved in translocations and deletions in acute leukemias of lymphoid or myeloid lineage. In a physical map of chromosome 12, MLF2 was found to reside on the yeast artificial chromosome clone 765b9. Southern blotting analysis of malignant cell DNAs prepared from a series of acute lymphoblastic leukemia cases with translocations involving chromosome arm 12p, as well as a group of acute myeloid leukemias with various cytogenetic abnormalities, failed to reveal MLF2 gene rearrangements.

Feasibility of physical map construction from fingerprinted bacterial artificial chromosome libraries of polyploid plant species

PubMed Central

2010-01-01

Background The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC) clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF) technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy. Results The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the construction of single-chromosome

Isolation of a yeast artificial chromosome contig spanning the Greig cephalopolysyndactyly syndrome (GCPS) gene region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vortkamp, A.; Gessler, M.; Le Paslier, D.

1994-08-01

Disruption of the zinc finger gene GLI3 has been shown to be the cause of Greig cephalopolysyndactyly syndrome (GCPS), at least in some GCPS translocation patients. To characterize this genomic region on human chromosome 7p13, we have isolated a YAC contig of more than 1000 kb including the GLI3 gene. In this contig the gene itself spans at least 200-250 kb. A CpG island is located in the vicinity of the 5{prime} region of the known GLI3 cDNA, implying a potential promoter region. 28 refs., 3 figs., 1 tab.

Coincidence of synteny breakpoints with malignancy-related deletions on human chromosome 3

PubMed Central

Kost-Alimova, Maria; Kiss, Hajnalka; Fedorova, Ludmila; Yang, Ying; Dumanski, Jan P.; Klein, George; Imreh, Stefan

2003-01-01

We have found previously that during tumor growth intact human chromosome 3 transferred into tumor cells regularly looses certain 3p regions, among them the ≈1.4-Mb common eliminated region 1 (CER1) at 3p21.3. Fluorescence in situ hybridization analysis of 12 mouse orthologous loci revealed that CER1 splits into two segments in mouse and therefore contains a murine/human conservation breakpoint region (CBR). Several breaks occurred in tumors within the region surrounding the CBR, and this sequence has features that characterize unstable chromosomal regions: deletions in yeast artificial chromosome clones, late replication, gene and segment duplications, and pseudogene insertions. Sequence analysis of the entire 3p12-22 revealed that other cancer-associated deletions (regions eliminated from monochromosomal hybrids carrying an intact chromosome 3 during tumor growth and homozygous deletions found in human tumors) colocalized nonrandomly with murine/human CBRs and were characterized by an increased number of local gene duplications and murine/human conservation mismatches (single genes that do not match into the conserved chromosomal segment). The CBR within CER1 contains a simple tandem TATAGA repeat capable of forming a 40-bp-long secondary hairpin-like structure. This repeat is nonrandomly localized within the other tumor-associated deletions and in the vicinity of 3p12-22 CBRs. PMID:12738884

Use of bacterial artificial chromosomes in generating targeted mutations in human and mouse cytomegaloviruses.

PubMed

Borst, Eva Maria; Benkartek, Corinna; Messerle, Martin

2007-05-01

Cloning of cytomegalovirus (CMV) genomes as bacterial artificial chromosomes (BAC) in E. coli and their manipulation using the techniques of bacterial genetics has greatly facilitated the construction of CMV mutants. This unit describes easily applicable procedures that allow rapid introduction of any kind of targeted mutation into BAC-cloned CMV genomes. Protocols for the reconstitution of virus from isolated BAC DNA, preparation of a virus stock, and isolation and characterization of viral DNA are also included. Special emphasis is laid on description of critical steps and thorough characterization of the altered BACs.

Identifying sites of replication initiation in yeast chromosomes: looking for origins in all the right places.

PubMed

van Brabant, A J; Hunt, S Y; Fangman, W L; Brewer, B J

1998-06-01

DNA fragments that contain an active origin of replication generate bubble-shaped replication intermediates with diverging forks. We describe two methods that use two-dimensional (2-D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificial Saccharomyces cerevisiae chromosomes. The first method uses 2-D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid. A variant form of 2-D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments. Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restriction map.

Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast

PubMed Central

Li, Ping; Jin, Hui; Yu, Hong-Guo

2014-01-01

During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. PMID:25103240

A Three-Dimensional Model of the Yeast Genome

NASA Astrophysics Data System (ADS)

Noble, William; Duan, Zhi-Jun; Andronescu, Mirela; Schutz, Kevin; McIlwain, Sean; Kim, Yoo Jung; Lee, Choli; Shendure, Jay; Fields, Stanley; Blau, C. Anthony

Layered on top of information conveyed by DNA sequence and chromatin are higher order structures that encompass portions of chromosomes, entire chromosomes, and even whole genomes. Interphase chromosomes are not positioned randomly within the nucleus, but instead adopt preferred conformations. Disparate DNA elements co-localize into functionally defined aggregates or factories for transcription and DNA replication. In budding yeast, Drosophila and many other eukaryotes, chromosomes adopt a Rabl configuration, with arms extending from centromeres adjacent to the spindle pole body to telomeres that abut the nuclear envelope. Nonetheless, the topologies and spatial relationships of chromosomes remain poorly understood. Here we developed a method to globally capture intra- and inter-chromosomal interactions, and applied it to generate a map at kilobase resolution of the haploid genome of Saccharomyces cerevisiae. The map recapitulates known features of genome organization, thereby validating the method, and identifies new features. Extensive regional and higher order folding of individual chromosomes is observed. Chromosome XII exhibits a striking conformation that implicates the nucleolus as a formidable barrier to interaction between DNA sequences at either end. Inter-chromosomal contacts are anchored by centromeres and include interactions among transfer RNA genes, among origins of early DNA replication and among sites where chromosomal breakpoints occur. Finally, we constructed a three-dimensional model of the yeast genome. Our findings provide a glimpse of the interface between the form and function of a eukaryotic genome.

cDNA cloning, tissue distribution, and chromosomal localization of myelodysplasia/Myeloid Leukemia Factor 2 (MLF2)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuefer, M.U.; Valentine, V.; Behm, F.G.

A fusion gene between nucleophosmin (NPM) and myelodysplasia/myeloid leukemia factor 1 (MLF1) and myelodysplasia/myeloid leukemia factor 1 (MLF1) is formed by a recurrent t(3;5)(q25.1;q34) in myelodysplastic syndrome and acute myeloid leukemia. Here we report the identification of a novel gene, MLF2, which contains an open reading frame of 744 bp encoding a 248-amino-acid protein highly related to the previously identified MLF1 protein (63% similarity, 40% identity). In contrast to the tissue-restricted expression pattern of MLF1, and MLF2 messenger RNA is expressed ubiquitously. The MLF2 gene locus was mapped by fluorescence in situ hybridization to human chromosome 12p13, a chromosomal regionmore » frequently involved in translocations and deletions in acute leukemias of lymphoid or myeloid lineage. In a physical map of chromosome 12, MLF2 was found to reside on the yeast artificial chromosome clone 765b9. Southern blotting analysis of malignant cell DNAs prepared from a series of acute lymphoblastic leukemia cases with translocations involving chromosome arm 12p, as well as a group of acute myeloid leukemias with various cytogenetic abnormalities, failed to reveal MLF2 gene rearrangements. 19 refs., 2 figs.« less

Analysis of yeast prp20 mutations and functional complementation by the human homologue RCC1, a protein involved in the control of chromosome condensation.

PubMed

Fleischmann, M; Clark, M W; Forrester, W; Wickens, M; Nishimoto, T; Aebi, M

1991-07-01

Mutations in the PRP20 gene of yeast show a pleiotropic phenotype, in which both mRNA metabolism and nuclear structure are affected. srm1 mutants, defective in the same gene, influence the signal transduction pathway for the pheromone response. The yeast PRP20/SRM1 protein is highly homologous to the RCC1 protein of man, hamster and frog. In mammalian cells, this protein is a negative regulator for initiation of chromosome condensation. We report the analysis of two, independently isolated, recessive temperature-sensitive prp20 mutants. They have identical G to A transitions, leading to the alteration of a highly conserved glycine residue to glutamic acid. By immunofluorescence microscopy the PRP20 protein was localized in the nucleus. Expression of the RCC1 protein can complement the temperature-sensitive phenotype of prp20 mutants, demonstrating the functional similarity of the yeast and mammalian proteins.

Construction of human artificial chromosome vectors by recombineering.

PubMed

Kotzamanis, George; Cheung, Wing; Abdulrazzak, Hassan; Perez-Luz, Sara; Howe, Steven; Cooke, Howard; Huxley, Clare

2005-05-23

Human artificial chromosomes (HACs) can be formed de novo by transfection of large fragments of cloned alphoid DNA into human HT1080 cells in tissue culture. In order to generate HACs carrying a gene of interest, one can either co-transfect the alphoid DNA and the gene of interest, or one can clone both into a single vector prior to transfection. Here we describe linking approximately 70 kb of alphoid DNA onto a 156-kb BAC carrying the human HPRT gene using Red homologous recombination in the EL350 Escherichia coli host [Lee et al., Genomics 73 (2001) 56-65]. A selectable marker and EGFP marker were then added by loxP/Cre recombination using the arabinose inducible cre gene in the EL350 bacteria. The final construct generates minichromosomes in HT1080 cells and the HPRT gene is expressed. The retrofitting vector can be used to add the approximately 70 kb of alphoid DNA to any BAC carrying a gene of interest to generate a HAC vector. The method can also be used to link any unrelated BAC or PAC insert onto another BAC clone. The EL350 bacteria are an excellent host for building up complex vectors by a combination of homologous and loxP/Cre recombination.

Microgravitational effects on chromosome behavior (7-IML-1)

NASA Technical Reports Server (NTRS)

Bruschi, Carlo

1992-01-01

The effects of the two major space-related conditions, microgravity and radiation, on the maintenance and transmission of genetic information have been partially documented in many organisms. Specifically, microgravity acts at the chromosomal level, primarily on the structure and segregation of chromosomes, in producing major abberations such as deletions, breaks, nondisjunction, and chromosome loss, and to a lesser degree, cosmic radiation appears to affect the genic level, producing point mutations and DNA damage. To distinguish between the effects from microgravity and from radiation, it is necessary to monitor both mitotic and meiotic genetic damage in the same organism. The yeast Saccharomyces cerevisiae is used to monitor at high resolution the frequency of chromosome loss, nondisjunction, intergenic recombination, and gene mutation in mitotic and meiotic cells, to a degree impossible in other organisms. Because the yeast chromosomes are small, sensitive measurements can be made that can be extrapolated to higher organisms and man. The objectives of the research are: (1) to quantitate the effects of microgravity and its synergism with cosmic radiation on chromosomal integrity and transmission during mitosis and meiosis; (2) to discriminate between chromosomal processes sensitive to microgravity and/or radiation during mitosis and meiosis; and (3) to relate these findings to anomalous mitotic mating type switching and ascosporogenesis following meiosis.

Mutation analysis of the chromosome 14q24.3 dihydrolipoyl succinyltransferase (DLST) gene in patients with early-onset Alzheimer disease.

PubMed

Cruts, M; Backhovens, H; Van Gassen, G; Theuns, J; Wang, S Y; Wehnert, A; van Duijn, C M; Karlsson, T; Hofman, A; Adolfsson, R

1995-10-13

Linkage analysis studies have indicated that the chromosome band 14q24.3 harbours a major gene for familial early-onset Alzheimer's disease (AD). Recently we localized the chromosome 14 AD gene (AD3) in the 6.4 cM interval between the markers D14S289 and D14S61. We mapped the gene encoding dihydrolipoyl succinyltransferase (DLST), the E2k component of human alpha-ketoglutarate dehydrogenase complex (KGDHC), in the AD3 candidate region using yeast artificial chromosomes (YACs). The DLST gene is a candidate for the AD3 gene since deficiencies in KGDHC activity have been observed in brain tissue and fibroblasts of AD patients. The 15 exons and the promoter region of the DLST gene were analysed for mutations in chromosome 14 linked AD cases and in two series of unrelated early-onset AD cases (onset age < 55 years). Sequence variations in intronic sequences (introns 3, 5 and 10) or silent mutations in exonic sequences (exons 8 and 14) were identified. However, no AD related mutations were observed, suggesting that the DLST gene is not the chromosome 14 AD3 gene.

Molecular Characterization of the Pericentric Inversion That Causes Differences Between Chimpanzee Chromosome 19 and Human Chromosome 17

PubMed Central

Kehrer-Sawatzki, Hildegard; Schreiner, Bettina; Tänzer, Simone; Platzer, Matthias; Müller, Stefan; Hameister, Horst

2002-01-01

A comparison of the human genome with that of the chimpanzee is an attractive approach to attempts to understand the specificity of a certain phenotype's development. The two karyotypes differ by one chromosome fusion, nine pericentric inversions, and various additions of heterochromatin to chromosomal telomeres. Only the fusion, which gave rise to human chromosome 2, has been characterized at the sequence level. During the present study, we investigated the pericentric inversion by which chimpanzee chromosome 19 differs from human chromosome 17. Fluorescence in situ hybridization was used to identify breakpoint-spanning bacterial artificial chromosomes (BACs) and plasmid artificial chromosomes (PACs). By sequencing the junction fragments, we localized breakpoints in intergenic regions rich in repetitive elements. Our findings suggest that repeat-mediated nonhomologous recombination has facilitated inversion formation. No addition or deletion of any sequence element was detected at the breakpoints or in the surrounding sequences. Next to the break, at a distance of 10.2–39.1 kb, the following genes were found: NGFR and NXPH3 (on human chromosome 17q21.3) and GUC2D and ALOX15B (on human chromosome 17p13). The inversion affects neither the genomic structure nor the gene-activity state with regard to replication timing of these genes. PMID:12094327

Bacterial Artificial Chromosome Clones of Viruses Comprising the Towne Cytomegalovirus Vaccine

PubMed Central

Cui, Xiaohong; Adler, Stuart P.; Davison, Andrew J.; Smith, Larry; Habib, EL-Sayed E.; McVoy, Michael A.

2012-01-01

Bacterial artificial chromosome (BAC) clones have proven invaluable for genetic manipulation of herpesvirus genomes. BAC cloning can also be useful for capturing representative genomes that comprise a viral stock or mixture. The Towne live attenuated cytomegalovirus vaccine was developed in the 1970s by serial passage in cultured fibroblasts. Although its safety, immunogenicity, and efficacy have been evaluated in nearly a thousand human subjects, the vaccine itself has been little studied. Instead, genetic composition and in vitro growth properties have been inferred from studies of laboratory stocks that may not always accurately represent the viruses that comprise the vaccine. Here we describe the use of BAC cloning to define the genotypic and phenotypic properties of viruses from the Towne vaccine. Given the extensive safety history of the Towne vaccine, these BACs provide a logical starting point for the development of next-generation rationally engineered cytomegalovirus vaccines. PMID:22187535

Bacterial artificial chromosome clones of viruses comprising the towne cytomegalovirus vaccine.

PubMed

Cui, Xiaohong; Adler, Stuart P; Davison, Andrew J; Smith, Larry; Habib, El-Sayed E; McVoy, Michael A

2012-01-01

Bacterial artificial chromosome (BAC) clones have proven invaluable for genetic manipulation of herpesvirus genomes. BAC cloning can also be useful for capturing representative genomes that comprise a viral stock or mixture. The Towne live attenuated cytomegalovirus vaccine was developed in the 1970s by serial passage in cultured fibroblasts. Although its safety, immunogenicity, and efficacy have been evaluated in nearly a thousand human subjects, the vaccine itself has been little studied. Instead, genetic composition and in vitro growth properties have been inferred from studies of laboratory stocks that may not always accurately represent the viruses that comprise the vaccine. Here we describe the use of BAC cloning to define the genotypic and phenotypic properties of viruses from the Towne vaccine. Given the extensive safety history of the Towne vaccine, these BACs provide a logical starting point for the development of next-generation rationally engineered cytomegalovirus vaccines.

To pair or not to pair: chromosome pairing and evolution.

PubMed

Moore, G

1998-04-01

Chromosome pairing in wild-type wheat closely resembles the process in both yeast and Drosophila. The recent characterisation of a mutant Ph1 wheat and the observation that chromosome pairing in the absence of Ph1 more closely resembles that of mammals and maize has shed light on the evolution of chromosome pairing in the cereals.

Centromere pairing – tethering partner chromosomes in meiosis I

PubMed Central

Kurdzo, Emily L; Dawson, Dean S

2015-01-01

In meiosis, homologous chromosomes face the obstacle of finding, holding onto and segregating away from their partner chromosome. There is increasing evidence, in a diverse range of organisms, that centromere–centromere interactions that occur in late prophase are an important mechanism in ensuring segregation fidelity. Centromere pairing appears to initiate when homologous chromosomes synapse in meiotic prophase. Structural proteins of the synaptonemal complex have been shown to help mediate centromere pairing, but how the structure that maintains centromere pairing differs from the structure of the synaptonemal complex along the chromosomal arms remains unknown. When the synaptonemal complex proteins disassemble from the chromosome arms in late prophase, some of these synaptonemal complex components persist at the centromeres. In yeast and Drosophila these centromere-pairing behaviors promote the proper segregation of chromosome partners that have failed to become linked by chiasmata. Recent studies of mouse spermatocytes have described centromere pairing behaviors that are similar in several respects to what has been described in the fly and yeast systems. In humans, chromosomes that fail to experience crossovers in meiosis are error-prone and are a major source of aneuploidy. The finding that centromere pairing is a conserved phenomenon raises the possibility that it may play a role in promoting the segregation fidelity of non-exchange chromosome pairs in humans. PMID:25817724

Fission yeast strains with circular chromosomes require the 9-1-1 checkpoint complex for the viability in response to the anti-cancer drug 5-fluorodeoxyuridine.

PubMed

Shamim, Hossain Mohammad; Minami, Yukako; Tanaka, Daiki; Ukimori, Shinobu; Murray, Johanne M; Ueno, Masaru

2017-01-01

Thymidine kinase converts 5-fluorodeoxyuridine to 5-fluorodeoxyuridine monophosphate, which causes disruption of deoxynucleotide triphosphate ratios. The fission yeast Schizosaccharomyces pombe does not express endogenous thymidine kinase but 5-fluorodeoxyuridine inhibits growth when exogenous thymidine kinase is expressed. Unexpectedly, we found that 5-fluorodeoxyuridine causes S phase arrest even without thymidine kinase expression. DNA damage checkpoint proteins such as the 9-1-1 complex were required for viability in the presence of 5-fluorodeoxyuridine. We also found that strains with circular chromosomes, due to loss of pot1+, which have higher levels of replication stress, were more sensitive to loss of the 9-1-1 complex in the presence of 5-fluorodeoxyuridine. Thus, our results suggest that strains carrying circular chromosomes exhibit a greater dependence on DNA damage checkpoints to ensure viability in the presence of 5-fluorodeoxyuridine compared to stains that have linear chromosomes.

Fluorescence in situ hybridization evaluation of chromosome deletion patterns in prostate cancer.

PubMed

Huang, S F; Xiao, S; Renshaw, A A; Loughlin, K R; Hudson, T J; Fletcher, J A

1996-11-01

Various nonrandom chromosomal aberrations have been identified in prostate carcinoma. These aberrations include deletions of several chromosome regions, particularly the chromosome 8 short arm. Large-scale numerical aberrations, reflected in aberrant DNA ploidy, are also found in a minority of cases. However, it is unclear whether prostate carcinomas contain aberrations of certain chromosome regions that are deleted frequently in other common types of cancer. In this study, we performed dual-color fluorescence in situ hybridization on intact nuclei from touch preparations of 16 prostate cancers. Chromosome copy number was determined using pericentromeric probes, whereas potential chromosome arm deletions were evaluated using yeast artificial chromosome (YAC) and P1 probes. Two YAC probes targeted chromosome 8 short arm regions known to be deleted frequently in prostate cancer. Other YACs and P1s were for chromosome regions, including 1p22, 3p14, 6q21, 9p21, and 22q12, that are deletion targets in a variety of cancers although not extensively studied in prostate cancer. Hybridization efficiencies and signal intensities were excellent for both repeat sequence (alpha-satellite) and single, copy (YAC and P1) fluorescence in situ hybridization probes. Of 16 prostate cancers, 11 had clonal aberrations of 1 or more of the 13 chromosome regions evaluated, and 10 cases (62.5%) had 8p deletions, including 4 cases with 8p deletion in virtually all cells and aneuploidy in only a subset of those deleted cells. Deletions at 3p14, 6q21, and 22q12 were identified in 2, 1, and 1 case, respectively, and each of those cases had a similarly sized cell population with 8p deletion. These studies confirm 8p deletion in the majority of prostate carcinomas. 8p deletions appear to be early events in prostate tumorigenesis, often antedating aneuploidy. Fluorescence in situ hybridization strategies incorporating pericentromeric and single-copy regional chromosome probes offer a powerful and

Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome

PubMed Central

Erliandri, Indri; Fu, Haiqing; Nakano, Megumi; Kim, Jung-Hyun; Miga, Karen H.; Liskovykh, Mikhail; Earnshaw, William C.; Masumoto, Hiroshi; Kouprina, Natalay; Aladjem, Mirit I.; Larionov, Vladimir

2014-01-01

In human chromosomes, centromeric regions comprise megabase-size arrays of 171 bp alpha-satellite DNA monomers. The large distances spanned by these arrays preclude their replication from external sites and imply that the repetitive monomers contain replication origins. However, replication within these arrays has not previously been profiled and the role of alpha-satellite DNA in initiation of DNA replication has not yet been demonstrated. Here, replication of alpha-satellite DNA in endogenous human centromeric regions and in de novo formed Human Artificial Chromosome (HAC) was analyzed. We showed that alpha-satellite monomers could function as origins of DNA replication and that replication of alphoid arrays organized into centrochromatin occurred earlier than those organized into heterochromatin. The distribution of inter-origin distances within centromeric alphoid arrays was comparable to the distribution of inter-origin distances on randomly selected non-centromeric chromosomal regions. Depletion of CENP-B, a kinetochore protein that binds directly to a 17 bp CENP-B box motif common to alpha-satellite DNA, resulted in enrichment of alpha-satellite sequences for proteins of the ORC complex, suggesting that CENP-B may have a role in regulating the replication of centromeric regions. Mapping of replication initiation sites in the HAC revealed that replication preferentially initiated in transcriptionally active regions. PMID:25228468

Genome Sequence of Saccharomyces carlsbergensis, the World’s First Pure Culture Lager Yeast

PubMed Central

Walther, Andrea; Hesselbart, Ana; Wendland, Jürgen

2014-01-01

Lager yeast beer production was revolutionized by the introduction of pure culture strains. The first established lager yeast strain is known as the bottom fermenting Saccharomyces carlsbergensis, which was originally termed Unterhefe No. 1 by Emil Chr. Hansen and has been used in production in since 1883. S. carlsbergensis belongs to group I/Saaz-type lager yeast strains and is better adapted to cold growth conditions than group II/Frohberg-type lager yeasts, e.g., the Weihenstephan strain WS34/70. Here, we sequenced S. carlsbergensis using next generation sequencing technologies. Lager yeasts are descendants from hybrids formed between a S. cerevisiae parent and a parent similar to S. eubayanus. Accordingly, the S. carlsbergensis 19.5-Mb genome is substantially larger than the 12-Mb S. cerevisiae genome. Based on the sequence scaffolds, synteny to the S. cerevisae genome, and by using directed polymerase chain reaction for gap closure, we generated a chromosomal map of S. carlsbergensis consisting of 29 unique chromosomes. We present evidence for genome and chromosome evolution within S. carlsbergensis via chromosome loss and loss of heterozygosity specifically of parts derived from the S. cerevisiae parent. Based on our sequence data and via fluorescence-activated cell-sorting analysis, we determined the ploidy of S. carlsbergensis. This inferred that this strain is basically triploid with a diploid S. eubayanus and haploid S. cerevisiae genome content. In contrast the Weihenstephan strain, which we resequenced, is essentially tetraploid composed of two diploid S. cerevisiae and S. eubayanus genomes. Based on conserved translocations between the parental genomes in S. carlsbergensis and the Weihenstephan strain we propose a joint evolutionary ancestry for lager yeast strains. PMID:24578374

Centromere-Like Regions in the Budding Yeast Genome

PubMed Central

Lefrançois, Philippe; Auerbach, Raymond K.; Yellman, Christopher M.; Roeder, G. Shirleen; Snyder, Michael

2013-01-01

Accurate chromosome segregation requires centromeres (CENs), the DNA sequences where kinetochores form, to attach chromosomes to microtubules. In contrast to most eukaryotes, which have broad centromeres, Saccharomyces cerevisiae possesses sequence-defined point CENs. Chromatin immunoprecipitation followed by sequencing (ChIP–Seq) reveals colocalization of four kinetochore proteins at novel, discrete, non-centromeric regions, especially when levels of the centromeric histone H3 variant, Cse4 (a.k.a. CENP-A or CenH3), are elevated. These regions of overlapping protein binding enhance the segregation of plasmids and chromosomes and have thus been termed Centromere-Like Regions (CLRs). CLRs form in close proximity to S. cerevisiae CENs and share characteristics typical of both point and regional CENs. CLR sequences are conserved among related budding yeasts. Many genomic features characteristic of CLRs are also associated with these conserved homologous sequences from closely related budding yeasts. These studies provide general and important insights into the origin and evolution of centromeres. PMID:23349633

Fission yeast cdc24(+) encodes a novel replication factor required for chromosome integrity.

PubMed

Gould, K L; Burns, C G; Feoktistova, A; Hu, C P; Pasion, S G; Forsburg, S L

1998-07-01

A mutation within the Schizosaccharomyces pombe cdc24(+) gene was identified previously in a screen for cell division cycle mutants and the cdc24(+) gene was determined to be essential for S phase in this yeast. We have isolated the cdc24(+) gene by complementation of a new temperature-sensitive allele of the gene, cdc24-G1. The DNA sequence predicts the presence of an open reading frame punctuated by six introns which encodes a pioneer protein of 58 kD. A cdc24 null mutant was generated by homologous recombination. Haploid cells lacking cdc24(+) are inviable, indicating that cdc24(+) is an essential gene. The transcript of cdc24(+) is present at constant levels throughout the cell cycle. Cells lacking cdc24(+) function show a checkpoint-dependent arrest with a 2N DNA content, indicating a block late in S phase. Arrest is accompanied by a rapid loss of viability and chromosome breakage. An S. pombe homolog of the replicative DNA helicase DNA2 of S. cerevisiae suppresses cdc24. These results suggest that Cdc24p plays a role in the progression of normal DNA replication and is required to maintain genomic integrity.

Fission yeast cdc24(+) encodes a novel replication factor required for chromosome integrity.

PubMed Central

Gould, K L; Burns, C G; Feoktistova, A; Hu, C P; Pasion, S G; Forsburg, S L

1998-01-01

A mutation within the Schizosaccharomyces pombe cdc24(+) gene was identified previously in a screen for cell division cycle mutants and the cdc24(+) gene was determined to be essential for S phase in this yeast. We have isolated the cdc24(+) gene by complementation of a new temperature-sensitive allele of the gene, cdc24-G1. The DNA sequence predicts the presence of an open reading frame punctuated by six introns which encodes a pioneer protein of 58 kD. A cdc24 null mutant was generated by homologous recombination. Haploid cells lacking cdc24(+) are inviable, indicating that cdc24(+) is an essential gene. The transcript of cdc24(+) is present at constant levels throughout the cell cycle. Cells lacking cdc24(+) function show a checkpoint-dependent arrest with a 2N DNA content, indicating a block late in S phase. Arrest is accompanied by a rapid loss of viability and chromosome breakage. An S. pombe homolog of the replicative DNA helicase DNA2 of S. cerevisiae suppresses cdc24. These results suggest that Cdc24p plays a role in the progression of normal DNA replication and is required to maintain genomic integrity. PMID:9649516

Yeasts in nectar of an early-blooming herb: sought by bumble bees, detrimental to plant fecundity.

PubMed

Herrera, Carlos M; Pozo, María I; Medrano, Mónica

2013-02-01

Through their effects on physicochemical features of floral nectar, nectar-dwelling yeasts can alter pollinator behavior, but the effect of such changes on pollination success and plant reproduction is unknown. We present results of experiments testing the effects of nectar yeasts on foraging patterns of captive and free-ranging bumble bees, and also on pollination success and fecundity of the early-blooming, bumble bee-pollinated Helleborus foetidus (Ranunculaceae). Under controlled experimental conditions, inexperienced Bombus terrestris workers responded positively to the presence of yeasts in artificial sugar solutions mimicking floral nectar by visiting proportionally more yeast-containing artificial flowers. Free-ranging bumble bees also preferred yeast-containing nectar in the field. Experiments conducted in two different years consistently showed that natural and artificial nectars containing yeasts were more thoroughly removed than nectars without yeasts. Experimental yeast inoculation of the nectar of H. foetidus flowers was significantly associated with reductions in number of pollen tubes in the style, fruit set, seed set, and mass of individual seeds produced. These results provide the first direct evidence to date that nectar yeasts can modify pollinator foraging patterns, pollination success, and the quantity and quality of seeds produced by insect-pollinated plants.

Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast.

PubMed

Li, Ping; Jin, Hui; Yu, Hong-Guo

2014-10-01

During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. © 2014 Li et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

PP2A(Cdc55)'s role in reductional chromosome segregation during achiasmate meiosis in budding yeast is independent of its FEAR function.

PubMed

Kerr, Gary W; Wong, Jin Huei; Arumugam, Prakash

2016-07-26

PP2A(Cdc55) is a highly conserved serine-threonine protein phosphatase that is involved in diverse cellular processes. In budding yeast, meiotic cells lacking PP2A(Cdc55) activity undergo a premature exit from meiosis I which results in a failure to form bipolar spindles and divide nuclei. This defect is largely due to its role in negatively regulating the Cdc Fourteen Early Anaphase Release (FEAR) pathway. PP2A(Cdc55) prevents nucleolar release of the Cdk (Cyclin-dependent kinase)-antagonising phosphatase Cdc14 by counteracting phosphorylation of the nucleolar protein Net1 by Cdk. CDC55 was identified in a genetic screen for monopolins performed by isolating suppressors of spo11Δ spo12Δ lethality suggesting that Cdc55 might have a role in meiotic chromosome segregation. We investigated this possibility by isolating cdc55 alleles that suppress spo11Δ spo12Δ lethality and show that this suppression is independent of PP2A(Cdc55)'s FEAR function. Although the suppressor mutations in cdc55 affect reductional chromosome segregation in the absence of recombination, they have no effect on chromosome segregation during wild type meiosis. We suggest that Cdc55 is required for reductional chromosome segregation during achiasmate meiosis and this is independent of its FEAR function.

Agrobacterium tumefaciens Integrates Transfer DNA into Single Chromosomal Sites of Dimorphic Fungi and Yields Homokaryotic Progeny from Multinucleate Yeast

PubMed Central

Sullivan, Thomas D.; Rooney, Peggy J.; Klein, Bruce S.

2002-01-01

The dimorphic fungi Blastomyces dermatitidis and Histoplasma capsulatum cause systemic mycoses in humans and other animals. Forward genetic approaches to generating and screening mutants for biologically important phenotypes have been underutilized for these pathogens. The plant-transforming bacterium Agrobacterium tumefaciens was tested to determine whether it could transform these fungi and if the fate of transforming DNA was suited for use as an insertional mutagen. Yeast cells from both fungi and germinating conidia from B. dermatitidis were transformed via A. tumefaciens by using hygromycin resistance for selection. Transformation frequencies up to 1 per 100 yeast cells were obtained at high effector-to-target ratios of 3,000:1. B. dermatitidis and H. capsulatum ura5 lines were complemented with transfer DNA vectors expressing URA5 at efficiencies 5 to 10 times greater than those obtained using hygromycin selection. Southern blot analyses indicated that in 80% of transformants the transferred DNA was integrated into chromosomal DNA at single, unique sites in the genome. Progeny of B. dermatitidis transformants unexpectedly showed that a single round of colony growth under hygromycin selection or visible selection of transformants by lacZ expression generated homokaryotic progeny from multinucleate yeast. Theoretical analysis of random organelle sorting suggests that the majority of B. dermatitidis cells would be homokaryons after the ca. 20 generations necessary for colony formation. Taken together, the results demonstrate that A. tumefaciens efficiently transfers DNA into B. dermatitidis and H. capsulatum and has the properties necessary for use as an insertional mutagen in these fungi. PMID:12477790

An automated microplate-based method for monitoring DNA strand breaks in plasmids and bacterial artificial chromosomes

PubMed Central

Rock, Cassandra; Shamlou, Parviz Ayazi; Levy, M. Susana

2003-01-01

A method is described for high-throughput monitoring of DNA backbone integrity in plasmids and artificial chromosomes in solution. The method is based on the denaturation properties of double-stranded DNA in alkaline conditions and uses PicoGreen fluorochrome to monitor denaturation. In the present method, fluorescence enhancement of PicoGreen at pH 12.4 is normalised by its value at pH 8 to give a ratio that is proportional to the average backbone integrity of the DNA molecules in the sample. A good regression fit (r2 > 0.98) was obtained when results derived from the present method and those derived from agarose gel electrophoresis were compared. Spiking experiments indicated that the method is sensitive enough to detect a proportion of 6% (v/v) molecules with an average of less than two breaks per molecule. Under manual operation, validation parameters such as inter-assay and intra-assay variation gave values of <5% coefficient of variation. Automation of the method showed equivalence to the manual procedure with high reproducibility and low variability within wells. The method described requires as little as 0.5 ng of DNA per well and a 96-well microplate can be analysed in 12 min providing an attractive option for analysis of high molecular weight vectors. A preparation of a 116 kb bacterial artificial chromosome was subjected to chemical and shear degradation and DNA integrity was tested using the method. Good correlation was obtained between time of chemical degradation and shear rate with fluorescence response. Results obtained from pulsed- field electrophoresis of sheared samples were in agreement with those obtained using the microplate-based method. PMID:12771229

Development of a quantitative pachytene chromosome map and its unification with somatic chromosome and linkage maps of rice (Oryza sativa L.).

PubMed

Ohmido, Nobuko; Iwata, Aiko; Kato, Seiji; Wako, Toshiyuki; Fukui, Kiichi

2018-01-01

A quantitative pachytene chromosome map of rice (Oryza sativa L.) was developed using imaging methods. The map depicts not only distribution patterns of chromomeres specific to pachytene chromosomes, but also the higher order information of chromosomal structures, such as heterochromatin (condensed regions), euchromatin (decondensed regions), the primary constrictions (centromeres), and the secondary constriction (nucleolar organizing regions, NOR). These features were image analyzed and quantitatively mapped onto the map by Chromosome Image Analyzing System ver. 4.0 (CHIAS IV). Correlation between H3K9me2, an epigenetic marker and formation and/or maintenance of heterochromatin, thus was, clearly visualized. Then the pachytene chromosome map was unified with the existing somatic chromosome and linkage maps by physically mapping common DNA markers among them, such as a rice A genome specific tandem repeat sequence (TrsA), 5S and 45S ribosomal RNA genes, five bacterial artificial chromosome (BAC) clones, four P1 bacteriophage artificial chromosome (PAC) clones using multicolor fluorescence in situ hybridization (FISH). Detailed comparison between the locations of the DNA probes on the pachytene chromosomes using multicolor FISH, and the linkage map enabled determination of the chromosome number and short/long arms of individual pachytene chromosomes using the chromosome number and arm assignment designated for the linkage map. As a result, the quantitative pachytene chromosome map was unified with two other major rice chromosome maps representing somatic prometaphase chromosomes and genetic linkages. In conclusion, the unification of the three rice maps serves as an indispensable basic information, not only for an in-depth comparison between genetic and chromosomal data, but also for practical breeding programs.

Unstable transpositions of his4 in yeast.

PubMed Central

Greer, H; Fink, G R

1979-01-01

Unstable transpositions in yeast have been selected in which the his4C gene from chromosome III is inserted into chromosome XII. This event is associated with the generation of a recessive lethal mutation, resulting from the integration of his4C into an essential gene. Strains with these transpositions are viable as diploids or aneuploids for chromosome XII. The event that generates the transpositions does not lead reciprocally to a deletion on chromosome III, implying that synthesis of a new copy of his4C and subsequent transposition may have occurred. The his4C transpositions are unstable and give rise to C- segregants at a high frequency, as a result of either precise excision of the his4C gene (restoring function of the gene into which insertion had occurred) or chromosome loss. PMID:386353

Radiation-induced mitotic and meiotic aneuploidy in the yeast Saccharomyces cerevisiae.

PubMed

Parry, J M; Sharp, D; Tippins, R S; Parry, E M

1979-06-01

A number of genetic systems are described which in yeast may be used to monitor the induction of chromosome aneuploidy during both mitotic and meiotic cell division. Using these systems we have been able to demonstrate the induction of both monosomic and trisomic cells in mitotically dividing cells and disomic spores in meiotically dividing cells after both UV light and X-ray exposure. The frequency of UV-light-induced monosomic colonies were reduced by post-treatment with photoreactivity light and both UV-light- and X-ray-induced monosomic colonies were reduced by liquid holding post-treatment under non-nutrient conditions. Both responses indicate an involvement of DNA-repair mechanisms in the removal of lesions which may lead to monosomy in yeast. This was further confirmed by the response of an excision-defective yeast strain which showed considerably increased sensitivity to the induction of monosomic colonies by UV-light treatment at low doses. Yeast cultures irradiated at different stages of growth showed variation in their responses to both UV-light and X-rays, cells at the exponential phase of growth show maximum sensitivity to the induction of monosomic colonies at low doses whereas stationary phase cultures showed maximum induction of monosomic colonies at high does. The frequencies of X-ray-induced chromosome aneuploidy during meiosis leading to the production of disomic spores was shown to be dependent upon the stage of meiosis at which the yeast cells were exposed to radiation. Cells which had proceeded beyond the DNA synthetic stage of meiosis were shown to produce disomic spores at considerably lower radiation doses than those cells which had only recently been inoculated into sporulation medium. The results obtained suggest that the yeast sustem may be suitable for the study of sensitivities of the various stages of meiotic cell division to the induction of chromosome aneuploidy after radiation exposure.

Normalization of a chromosomal contact map.

PubMed

Cournac, Axel; Marie-Nelly, Hervé; Marbouty, Martial; Koszul, Romain; Mozziconacci, Julien

2012-08-30

Chromatin organization has been increasingly studied in relation with its important influence on DNA-related metabolic processes such as replication or regulation of gene expression. Since its original design ten years ago, capture of chromosome conformation (3C) has become an essential tool to investigate the overall conformation of chromosomes. It relies on the capture of long-range trans and cis interactions of chromosomal segments whose relative proportions in the final bank reflect their frequencies of interactions, hence their spatial proximity in a population of cells. The recent coupling of 3C with deep sequencing approaches now allows the generation of high resolution genome-wide chromosomal contact maps. Different protocols have been used to generate such maps in various organisms. This includes mammals, drosophila and yeast. The massive amount of raw data generated by the genomic 3C has to be carefully processed to alleviate the various biases and byproducts generated by the experiments. Our study aims at proposing a simple normalization procedure to minimize the influence of these unwanted but inevitable events on the final results. Careful analysis of the raw data generated previously for budding yeast S. cerevisiae led to the identification of three main biases affecting the final datasets, including a previously unknown bias resulting from the circularization of DNA molecules. We then developed a simple normalization procedure to process the data and allow the generation of a normalized, highly contrasted, chromosomal contact map for S. cerevisiae. The same method was then extended to the first human genome contact map. Using the normalized data, we revisited the preferential interactions originally described between subsets of discrete chromosomal features. Notably, the detection of preferential interactions between tRNA in yeast and CTCF, PolII binding sites in human can vary with the normalization procedure used. We quantitatively reanalyzed the

Chromosome integrity at a double-strand break requires exonuclease 1 and MRX

PubMed Central

Nakai, Wataru; Westmoreland, Jim; Yeh, Elaine; Bloom, Kerry; Resnick, Michael A.

2010-01-01

The continuity of duplex DNA is generally considered a prerequisite for chromosome continuity. However, as previously shown in yeast as well as human cells, the introduction of a double-strand break (DSB) does not generate a chromosome break (CRB) in yeast or human cells. The transition from DSB to CRB was found to be under limited control by the tethering function of the RAD50/MRE11/XRS2 (MRX) complex. Using a system for differential fluorescent marking of both sides of an endonuclease-induced DSB in single cells, we found that nearly all DSBs are converted to CRBs in cells lacking both exonuclease 1 (EXO1) activity and MRX complex. Thus, it appears that some feature of exonuclease processing or resection at a DSB is critical for maintaining broken chromosome ends in close proximity. In addition, we discovered a thermal sensitive (cold) component to CRB formation in an MRX mutant that has implications for chromosome end mobility and/or end-processing. PMID:21115410

The dynamic three-dimensional organization of the diploid yeast genome

PubMed Central

Kim, Seungsoo; Liachko, Ivan; Brickner, Donna G; Cook, Kate; Noble, William S; Brickner, Jason H; Shendure, Jay; Dunham, Maitreya J

2017-01-01

The budding yeast Saccharomyces cerevisiae is a long-standing model for the three-dimensional organization of eukaryotic genomes. However, even in this well-studied model, it is unclear how homolog pairing in diploids or environmental conditions influence overall genome organization. Here, we performed high-throughput chromosome conformation capture on diverged Saccharomyces hybrid diploids to obtain the first global view of chromosome conformation in diploid yeasts. After controlling for the Rabl-like orientation using a polymer model, we observe significant homolog proximity that increases in saturated culture conditions. Surprisingly, we observe a localized increase in homologous interactions between the HAS1-TDA1 alleles specifically under galactose induction and saturated growth. This pairing is accompanied by relocalization to the nuclear periphery and requires Nup2, suggesting a role for nuclear pore complexes. Together, these results reveal that the diploid yeast genome has a dynamic and complex 3D organization. DOI: http://dx.doi.org/10.7554/eLife.23623.001 PMID:28537556

Chromosomal location and gene paucity of the male specific region on papaya Y chromosome.

PubMed

Yu, Qingyi; Hou, Shaobin; Hobza, Roman; Feltus, F Alex; Wang, Xiue; Jin, Weiwei; Skelton, Rachel L; Blas, Andrea; Lemke, Cornelia; Saw, Jimmy H; Moore, Paul H; Alam, Maqsudul; Jiang, Jiming; Paterson, Andrew H; Vyskot, Boris; Ming, Ray

2007-08-01

Sex chromosomes in flowering plants evolved recently and many of them remain homomorphic, including those in papaya. We investigated the chromosomal location of papaya's small male specific region of the hermaphrodite Y (Yh) chromosome (MSY) and its genomic features. We conducted chromosome fluorescence in situ hybridization mapping of Yh-specific bacterial artificial chromosomes (BACs) and placed the MSY near the centromere of the papaya Y chromosome. Then we sequenced five MSY BACs to examine the genomic features of this specialized region, which resulted in the largest collection of contiguous genomic DNA sequences of a Y chromosome in flowering plants. Extreme gene paucity was observed in the papaya MSY with no functional gene identified in 715 kb MSY sequences. A high density of retroelements and local sequence duplications were detected in the MSY that is suppressed for recombination. Location of the papaya MSY near the centromere might have provided recombination suppression and fostered paucity of genes in the male specific region of the Y chromosome. Our findings provide critical information for deciphering the sex chromosomes in papaya and reference information for comparative studies of other sex chromosomes in animals and plants.

Form and function of topologically associating genomic domains in budding yeast.

PubMed

Eser, Umut; Chandler-Brown, Devon; Ay, Ferhat; Straight, Aaron F; Duan, Zhijun; Noble, William Stafford; Skotheim, Jan M

2017-04-11

The genome of metazoan cells is organized into topologically associating domains (TADs) that have similar histone modifications, transcription level, and DNA replication timing. Although similar structures appear to be conserved in fission yeast, computational modeling and analysis of high-throughput chromosome conformation capture (Hi-C) data have been used to argue that the small, highly constrained budding yeast chromosomes could not have these structures. In contrast, herein we analyze Hi-C data for budding yeast and identify 200-kb scale TADs, whose boundaries are enriched for transcriptional activity. Furthermore, these boundaries separate regions of similarly timed replication origins connecting the long-known effect of genomic context on replication timing to genome architecture. To investigate the molecular basis of TAD formation, we performed Hi-C experiments on cells depleted for the Forkhead transcription factors, Fkh1 and Fkh2, previously associated with replication timing. Forkhead factors do not regulate TAD formation, but do promote longer-range genomic interactions and control interactions between origins near the centromere. Thus, our work defines spatial organization within the budding yeast nucleus, demonstrates the conserved role of genome architecture in regulating DNA replication, and identifies a molecular mechanism specifically regulating interactions between pericentric origins.

3D organization of synthetic and scrambled chromosomes.

PubMed

Mercy, Guillaume; Mozziconacci, Julien; Scolari, Vittore F; Yang, Kun; Zhao, Guanghou; Thierry, Agnès; Luo, Yisha; Mitchell, Leslie A; Shen, Michael; Shen, Yue; Walker, Roy; Zhang, Weimin; Wu, Yi; Xie, Ze-Xiong; Luo, Zhouqing; Cai, Yizhi; Dai, Junbiao; Yang, Huanming; Yuan, Ying-Jin; Boeke, Jef D; Bader, Joel S; Muller, Héloïse; Koszul, Romain

2017-03-10

Although the design of the synthetic yeast genome Sc2.0 is highly conservative with respect to gene content, the deletion of several classes of repeated sequences and the introduction of thousands of designer changes may affect genome organization and potentially alter cellular functions. We report here the Hi-C-determined three-dimensional (3D) conformations of Sc2.0 chromosomes. The absence of repeats leads to a smoother contact pattern and more precisely tractable chromosome conformations, and the large-scale genomic organization is globally unaffected by the presence of synthetic chromosome(s). Two exceptions are synIII, which lacks the silent mating-type cassettes, and synXII, specifically when the ribosomal DNA is moved to another chromosome. We also exploit the contact maps to detect rearrangements induced in SCRaMbLE (synthetic chromosome rearrangement and modification by loxP -mediated evolution) strains. Copyright © 2017, American Association for the Advancement of Science.

Nuclear envelope expansion is crucial for proper chromosomal segregation during a closed mitosis.

PubMed

Takemoto, Ai; Kawashima, Shigehiro A; Li, Juan-Juan; Jeffery, Linda; Yamatsugu, Kenzo; Elemento, Olivier; Nurse, Paul

2016-03-15

Here, we screened a 10,371 library of diverse molecules using a drug-sensitive fission yeast strain to identify compounds which cause defects in chromosome segregation during mitosis. We identified a phosphorium-ylide-based compound Cutin-1 which inhibits nuclear envelope expansion and nuclear elongation during the closed mitosis of fission yeast, and showed that its target is the β-subunit of fatty acid synthase. A point mutation in the dehydratase domain of Fas1 conferred in vivo and in vitro resistance to Cutin-1. Time-lapse photomicrography showed that the bulk of the chromosomes were only transiently separated during mitosis, and nucleoli separation was defective. Subsequently sister chromatids re-associated leading to chromosomal mis-segregation. These segregation defects were reduced when the nuclear volume was increased and were increased when the nuclear volume was reduced. We propose that there needs to be sufficient nuclear volume to allow the nuclear elongation necessary during a closed mitosis to take place for proper chromosome segregation, and that inhibition of fatty acid synthase compromises nuclear elongation and leads to defects in chromosomal segregation. © 2016. Published by The Company of Biologists Ltd.

Fission yeast dam1-A8 mutant is resistant to and rescued by an anti-microtubule agent

DOE Office of Scientific and Technical Information (OSTI.GOV)

Griffiths, Karen; Masuda, Hirohisa; Dhut, Susheela

2008-04-11

The Dam1/DASH outer kinetochore complex is required for high-fidelity chromosome segregation in budding and fission yeast. Unlike budding yeast, the fission yeast complex is non-essential, however it promotes bipolar microtubule attachment in conjunction with microtubule-depolymerising kinesin-8 Klp5 and Klp6. Here, we screened for dam1 temperature sensitive mutants in a klp5 null background and identified dam1-A8 that contains two amino acid substitutions in the C-terminus (H126R and E149G). dam1-A8klp5 mutant cells display massive chromosome missegregation with lagging chromosomes and monopolar attachment of sister chromatids to one SPB (spindle pole body). Unexpectedly contrary to a deletion mutant that is hypersensitive to microtubule-destabilisingmore » drugs, dam1-A8 is resistant and furthermore the temperature sensitivity of dam1-A8klp5 is rescued by addition of these drugs. This indicates that the hyper-stabilised rigidity of kinetochore-spindle mal-attachments is the primary cause of lethality. Our result shows that fine-tuning of Dam1 activity is essential for chromosome bi-orientation.« less

Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system.

PubMed

Pyeon, Hye-Rim; Nah, Hee-Ju; Kang, Seung-Hoon; Choi, Si-Sun; Kim, Eung-Soo

2017-05-31

Heterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes. To validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem

Visualization of yeast chromosomal DNA

NASA Technical Reports Server (NTRS)

Lubega, Seth

1990-01-01

The DNA molecule is the most significant life molecule since it codes the blue print for other structural and functional molecules of all living organisms. Agarose gel electrophoresis is now being widely used to separate DNA of virus, bacteria, and lower eukaryotes. The task was undertaken of reviewing the existing methods of DNA fractionation and microscopic visualization of individual chromosonal DNA molecules by gel electrophoresis as a basis for a proposed study to investigate the feasibility of separating DNA molecules in free fluids as an alternative to gel electrophoresis. Various techniques were studied. On the molecular level, agarose gel electrophoresis is being widely used to separate chromosomal DNA according to molecular weight. Carl and Olson separate and characterized the entire karyotype of a lab strain of Saccharomyces cerevisiae. Smith et al. and Schwartz and Koval independently reported the visualization of individual DNA molecules migrating through agarose gel matrix during electrophoresis. The techniques used by these researchers are being reviewed in the lab as a basis for the proposed studies.

Replication dynamics of the yeast genome.

PubMed

Raghuraman, M K; Winzeler, E A; Collingwood, D; Hunt, S; Wodicka, L; Conway, A; Lockhart, D J; Davis, R W; Brewer, B J; Fangman, W L

2001-10-05

Oligonucleotide microarrays were used to map the detailed topography of chromosome replication in the budding yeast Saccharomyces cerevisiae. The times of replication of thousands of sites across the genome were determined by hybridizing replicated and unreplicated DNAs, isolated at different times in S phase, to the microarrays. Origin activations take place continuously throughout S phase but with most firings near mid-S phase. Rates of replication fork movement vary greatly from region to region in the genome. The two ends of each of the 16 chromosomes are highly correlated in their times of replication. This microarray approach is readily applicable to other organisms, including humans.

Chromosomal changes during experimental evolution in laboratory populations of Escherichia coli.

PubMed

Bergthorsson, U; Ochman, H

1999-02-01

Short-term rates of chromosome evolution were analyzed in experimental populations of Escherichia coli B that had been propagated for 2,000 generations under four thermal regimens. Chromosome alterations were monitored in 24 independent populations by pulsed-field gel electrophoresis of DNA treated with five rare-cutting restriction enzymes. A total of 11 changes, 8 affecting chromosome size and 3 altering restriction sites, were observed in these populations, with none occurring in strains cultured at 37 degreesC. Considering the changes detected in these experimental populations, the rate of chromosome alteration of E. coli is estimated to be half of that observed in experimental populations of yeast.

Development of canine herpesvirus based antifertility vaccines for foxes using bacterial artificial chromosomes.

PubMed

Strive, Tanja; Hardy, Christopher M; French, Nigel; Wright, John D; Nagaraja, Nitin; Reubel, Gerhard H

2006-02-13

Using bacterial artificial chromosome (BAC) technology, a canine herpesvirus (CHV)-based recombinant vaccine vector was produced for the development of an antifertility vaccine for foxes. Infectious viruses were recovered following transfection of canid cells with a BAC plasmid carrying the complete CHV genome. In vitro growth characteristics of BAC-derived viruses were similar to that of wildtype (wt)-CHV. Two recombinant antigens, fox zona pellucida protein subunit 3 (fZPC) and enhanced green fluorescent protein (EGFP) as control antigen, were inserted into thymidine kinase (TK) locus of the CHV genome and shown to be efficiently expressed in vitro. Inoculation of foxes with transgenic CHVs induced CHV specific antibodies, but was innocuous and failed to elicit transgene-specific antibody responses. Infectious virus or viral DNA was not detected in mucosal secretions or tissues of vaccinated foxes. The CHV-BAC system proved to be a quick and reliable method to manipulate the CHV genome. It will help to readily apply changes in the vector design in order to improve virus replication in vivo.

Naturally occurring minichromosome platforms in chromosome engineering: an overview.

PubMed

Raimondi, Elena

2011-01-01

Artificially modified chromosome vectors are non-integrating gene delivery platforms that can shuttle very large DNA fragments in various recipient cells: theoretically, no size limit exists for the chromosome segments that an engineered minichromosome can accommodate. Therefore, genetically manipulated chromosomes might be potentially ideal vector systems, especially when the complexity of higher eukaryotic genes is concerned. This review focuses on those chromosome vectors generated using spontaneously occurring small markers as starting material. The definition and manipulation of the centromere domain is one of the main obstacles in chromosome engineering: naturally occurring minichromosomes, due to their inherent small size, were helpful in defining some aspects of centromere function. In addition, several distinctive features of small marker chromosomes, like their appearance as supernumerary elements in otherwise normal karyotypes, have been successfully exploited to use them as gene delivery vectors. The key technologies employed for minichromosome engineering are: size reduction, gene targeting, and vector delivery in various recipient cells. In spite of the significant advances that have been recently achieved in all these fields, several unsolved problems limit the potential of artificially modified chromosomes. Still, these vector systems have been exploited in a number of applications where the investigation of the controlled expression of large DNA segments is needed. A typical example is the analysis of genes whose expression strictly depends on the chromosomal environment in which they are positioned, where engineered chromosomes can be envisaged as epigenetically regulated expression systems. A novel and exciting advance concerns the use of engineered minichromosomes to study the organization and dynamics of local chromatin structures.

Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

NASA Astrophysics Data System (ADS)

Miles, Jeff; Formosa, Tim

1992-02-01

We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

Cloning a balanced t(9;11)(p24;q23.1) chromosomal translocation breakpoint segregating with bipolar affective disorder in a small pedigree

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duggan, D.J.; Baysal, B.E.; Gollin, S.M.

A small multigenerational pedigree was previously identified in which a balanced 9;11 chromosomal translocation was cosegregating with bipolar affective disorder. We hypothesize that genes or gene regulatory sequences disrupted by the translocation are contributing to bipolar affective disorder in a dominant fashion. The general strategy involves (1) using somatic cell hybrids containing the derivative 9 or 11 chromosomes to identify the closest chromosome 9 and 11 flanking markers, (2) using the nearest markers as PCR and hybridization probes to isolate both normal DNA (YAC) and patient DNA (cosmid) adjacent to and incorporating the translocation breakpoint, and (3) identifying expressed sequencesmore » in the genomic DNA that may be disrupted by the translocation. From a fusion of the translocation patient cell line and a recipient hamster cell line, somatic cell hybrids were isolated which contain either the human derivative 9 or derivative 11 chromosome. Using PCR-based STS assays with these hybrids, the location of the translocation breakpoint was localized to an estimated 500 kb region at chromosome 11 band q23.1 and a 1 cM region in 9 band p24 (more telomeric than originally reported). From a large set of CEPH and Roswell Park yeast artificial chromosomes (YACs), six chromosome 11 YACs spanning the 11q23.1 breakpoint have now been identified. A combination of pulsed field gel eletrophoresis and YAC mapping has narrowed the chromosome 11 region to less than 430 kb. Current efforts are focused on generating new chromosome 11 probes within the flanking markers, mapping these probes back to the der(9) and der(11) containing hybrids and the chromosome 11 YAC mapping panel. As the region is physically narrowed, we will identify candidate genes whose expression may be altered by this t(9:11) translocation.« less

Construction of a Llama Bacterial Artificial Chromosome Library with Approximately 9-Fold Genome Equivalent Coverage

PubMed Central

Airmet, K. W.; Hinckley, J. D.; Tree, L. T.; Moss, M.; Blumell, S.; Ulicny, K.; Gustafson, A. K.; Weed, M.; Theodosis, R.; Lehnardt, M.; Genho, J.; Stevens, M. R.; Kooyman, D. L.

2012-01-01

The Ilama is an important agricultural livestock in much of South America. The llama is increasing in popularity in the United States as a companion animal. Little work has been done to improve llama production using modern technology. A paucity of information is available regarding the llama genome. We report the construction of a llama bacterial artificial chromosome (BAC) library of about 196,224 clones in the vector pECBAC1. Using flow cytometry and bovine, human, mouse, and chicken as controls, we determined the llama genome size to be 2.4 × 109 bp. The average insert size of the library is 137.8 kb corresponding to approximately 9-fold genome coverage. Further studies are needed to further characterize the library and llama genome. We anticipate that this new library will help facilitate future genomic studies in the llama. PMID:22811594

Depletion of a Drosophila homolog of yeast Sup35p disrupts spindle assembly, chromosome segregation, and cytokinesis during male meiosis.

PubMed

Basu, J; Williams, B C; Li, Z; Williams, E V; Goldberg, M L

1998-01-01

In the course of a genetic screen for male-sterile mutations in Drosophila affecting chromosome segregation during the meiotic divisions in spermatocytes, we identified the mutation dsup35(63D). Examination of mutant testes showed that chromosome misbehavior was a consequence of major disruptions in meiotic spindle assembly. These perturbations included problems in aster formation, separation, and migration around the nuclear envelope; aberrations in spindle organization and integrity; and disappearance of the ana/telophase central spindle, which in turn disrupts cytokinesis. The dsup35(63D) mutation is caused by a P element insertion that affects, specifically in the testis, the expression of a gene (dsup35) encoding the Drosophila homolog of the yeast Sup35p and Xenopus eRF3 proteins. These proteins are involved in the termination of polypeptide synthesis on ribosomes, but previous studies have suggested that Sup35p and closely related proteins of the same family also interact directly with microtubules. An affinity-purified antibody directed against the product of the dsup35 gene was prepared; interestingly, this antibody specifically labels primary spermatocytes in one or two discrete foci of unknown structure within the nucleoplasm. We discuss how depletion of the dsup35 gene product in spermatocytes might lead to the global disruptions in meiotic spindle assembly seen in mutant spermatocytes.

Contrasting evolutionary genome dynamics between domesticated and wild yeasts

PubMed Central

Yue, Jia-Xing; Li, Jing; Aigrain, Louise; Hallin, Johan; Persson, Karl; Oliver, Karen; Bergström, Anders; Coupland, Paul; Warringer, Jonas; Lagomarsino, Marco Consentino; Fischer, Gilles; Durbin, Richard; Liti, Gianni

2017-01-01

Structural rearrangements have long been recognized as an important source of genetic variation with implications in phenotypic diversity and disease, yet their detailed evolutionary dynamics remain elusive. Here, we use long-read sequencing to generate end-to-end genome assemblies for 12 strains representing major subpopulations of the partially domesticated yeast Saccharomyces cerevisiae and its wild relative Saccharomyces paradoxus. These population-level high-quality genomes with comprehensive annotation allow for the first time a precise definition of chromosomal boundaries between cores and subtelomeres and a high-resolution view of evolutionary genome dynamics. In chromosomal cores, S. paradoxus exhibits faster accumulation of balanced rearrangements (inversions, reciprocal translocations and transpositions) whereas S. cerevisiae accumulates unbalanced rearrangements (novel insertions, deletions and duplications) more rapidly. In subtelomeres, both species show extensive interchromosomal reshuffling, with a higher tempo in S. cerevisiae. Such striking contrasts between wild and domesticated yeasts likely reflect the influence of human activities on structural genome evolution. PMID:28416820

Nonselective enrichment for yeast adenine mutants by flow cytometry

NASA Technical Reports Server (NTRS)

Bruschi, C. V.; Chuba, P. J.

1988-01-01

The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

Characterization of Three Maize Bacterial Artificial Chromosome Libraries toward Anchoring of the Physical Map to the Genetic Map Using High-Density Bacterial Artificial Chromosome Filter Hybridization1

PubMed Central

Yim, Young-Sun; Davis, Georgia L.; Duru, Ngozi A.; Musket, Theresa A.; Linton, Eric W.; Messing, Joachim W.; McMullen, Michael D.; Soderlund, Carol A.; Polacco, Mary L.; Gardiner, Jack M.; Coe, Edward H.

2002-01-01

Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density filter sets from all three libraries, made using different restriction enzymes (HindIII, EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage λ. The results indicate that the libraries are of high quality with low contamination by organellar and λ-sequences. The use of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6× coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 × Mo17 genetic map and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three BACs. Twenty-two single-copy number core markers identified an average of 7.4 ± 3.3 positive clones, consistent with the expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction. PMID:12481051

Damage-induced ectopic recombination in the yeast Saccharomyces cerevisiae.

PubMed

Kupiec, M; Steinlauf, R

1997-06-09

Mitotic recombination in the yeast Saccharomyces cerevisiae is induced when cells are irradiated with UV or X-rays, reflecting the efficient repair of damage by recombinational repair mechanisms. We have used multiply marked haploid strains that allow the simultaneous detection of several types of ectopic recombination events. We show that inter-chromosomal ectopic conversion of lys2 heteroalleles and, to a lesser extent, direct repeat recombination (DRR) between non-tandem repeats, are increased by DNA-damaging agents; in contrast, ectopic recombination of the naturally occurring Ty element is not induced. We have tested several hypotheses that could explain the preferential lack of induction of Ty recombination by DNA-damaging agents. We have found that the lack of induction cannot be explained by a cell cycle control or by an effect of the mating-type genes. We also found no role for the flanking long terminal repeats (LTRs) of the Ty in preventing the induction. Ectopic conversion, DRR, and forward mutation of artificial repeats show different kinetics of induction at various positions of the cell cycle, reflecting different mechanisms of recombination. We discuss the mechanistic and evolutionary aspects of these results.

Preferential inclusion of extrachromosomal genetic elements in yeast meiotic spores.

PubMed

Brewer, B J; Fangman, W L

1980-09-01

During meiosis and sporulation in the yeast Saccharomyces cerevisiae, extrachromosomal traits are efficiently transmitted to haploid spores. Although the pattern of inheritance of chromosomal traits reflects the mechanism of regular chromosomal segregation in meiosis, it is not known what processes are reflected by the efficient inheritance of extrachromosomal traits. Because extrachromosomal genetic elements in yeast are present in multiple copies, perpetuation of an extrachromosomal trait could occur by the passive envelopment of a subset of copies or by an active sequestering of all or a subset of copies within the four spores. We show that only subsets of the four extrachromosomal nucleic acids commonly found in yeast are transmitted through meiosis--55% of mitochondrial DNA copies, 82% of the 2-micron DNA plasmids, and about 70% of the L and M double-stranded RNAs. However, electron micrographs of serial sections through yeast asci indicate that the four spore enclose only 30% of the total ascus material. Thus these extrachromosomal elements are preferentially included within the spores, indicating that their inheritance is not a random process. Transmission of mitochondrial DNA can be accounted for by the observed enclosure of 52% of the mitochondrial volume within the spores. The high transmission frequencies of the double-stranded RNAs (which exist as virus-like particles in the cytoplasm) and 2-micron DNA must indicate that either these nucleic acids are actively recruited from the cytoplasm by some mechanism or they are associated in some way with the nucleus during meiosis.

Chromosome congression by kinesin-5 motor-mediated disassembly of longer kinetochore microtubules

PubMed Central

Gardner, Melissa K; Bouck, David C.; Paliulis, Leocadia V.; Meehl, Janet B.; O’Toole, Eileen T.; Haase, Julian; Soubry, Adelheid; Joglekar, Ajit P.; Winey, Mark; Salmon, Edward D.; Bloom, Kerry; Odde, David J.

2008-01-01

Summary During mitosis, sister chromatids congress to the spindle equator and are subsequently segregated via attachment to dynamic kinetochore microtubule (kMT) plus-ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chromosome congression. Here we find in budding yeast that the widely-conserved kinesin-5 sliding motor proteins, Cin8p and Kip1p, mediate chromosome congression by suppressing kMT plus-end assembly of longer kMTs. Of the two, Cin8p is the major effector and its activity requires a functional motor domain. In contrast, the depolymerizing kinesin-8 motor Kip3p plays a minor role in spatial regulation of yeast kMT assembly. Our analysis identified a model where kinesin-5 motors bind to kMTs, move to kMT plus ends, and upon arrival at a growing plus-end promote net kMT plus-end disassembly. In conclusion, we find that length-dependent control of net kMT assembly by kinesin-5 motors yields a simple and stable self-organizing mechanism for chromosome congression. PMID:19041752

Isolation and characterization of chromosome-gain and increase-in-ploidy mutants in yeast.

PubMed

Chan, C S; Botstein, D

1993-11-01

We have developed a colony papillation assay for monitoring the copy number of genetically marked chromosomes II and III in Saccharomyces cerevisiae. The unique feature of this assay is that it allows detection of a gain of the marked chromosomes even if there is a gain of the entire set of chromosomes (increase-in-ploidy). This assay was used to screen for chromosome-gain or increase-in-ploidy mutants. Five complementation groups have been defined for recessive mutations that confer an increase-in-ploidy (ipl) phenotype, which, in each case, cosegregates with a temperature-sensitive growth phenotype. Four new alleles of CDC31, which is required for spindle pole body duplication, were also recovered from this screen. Temperature-shift experiments with ipl1 cells show that they suffer severe nondisjunction at 37 degrees. Similar experiments with ipl2 cells show that they gain entire sets of chromosomes and become arrested as unbudded cells at 37 degrees. Molecular cloning and genetic mapping show that IPL1 is a newly identified gene, whereas IPL2 is allelic to BEM2, which is required for normal bud growth.

Isolation and Characterization of Chromosome-Gain and Increase-in-Ploidy Mutants in Yeast

PubMed Central

Chan, CSM.; Botstein, D.

1993-01-01

We have developed a colony papillation assay for monitoring the copy number of genetically marked chromosomes II and III in Saccharomyces cerevisiae. The unique feature of this assay is that it allows detection of a gain of the marked chromosomes even if there is a gain of the entire set of chromosomes (increase-in-ploidy). This assay was used to screen for chromosome-gain or increase-in-ploidy mutants. Five complementation groups have been defined for recessive mutations that confer an increase-in-ploidy (ipl) phenotype, which, in each case, cosegregates with a temperature-sensitive growth phenotype. Four new alleles of CDC31, which is required for spindle pole body duplication, were also recovered from this screen. Temperature-shift experiments with ipl1 cells show that they suffer severe nondisjunction at 37°. Similar experiments with ipl2 cells show that they gain entire sets of chromosomes and become arrested as unbudded cells at 37°. Molecular cloning and genetic mapping show that IPL1 is a newly identified gene, whereas IPL2 is allelic to BEM2, which is required for normal bud growth. PMID:8293973

Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis

PubMed Central

Chatterjee, Gautam; Sankaranarayanan, Sundar Ram; Guin, Krishnendu; Thattikota, Yogitha; Padmanabhan, Sreedevi; Siddharthan, Rahul; Sanyal, Kaustuv

2016-01-01

The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species—Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast. PMID:26845548

Adaptation and major chromosomal changes in populations of Saccharomyces cerevisiae.

PubMed

Adams, J; Puskas-Rozsa, S; Simlar, J; Wilke, C M

1992-07-01

Thirteen independent populations of Saccharomyces cerevisiae (nine haploid and four diploid) were maintained in continuous culture for up to approximately 1000 generations, with growth limited by the concentration of organic phosphates in medium buffered at pH 6. Analysis of clones isolated from these populations showed that a number (17) of large-scale chromosomal-length variants and rearrangements were present in the populations at their termination. Nine of the 16 yeast chromosomes were involved in such changes. Few of the changes could be explained by copy-number increases in the structural loci for acid phosphatase. Several considerations concerning the nature and frequency of the chromosome-length variants observed lead us to conclude that they are selectively advantageous.

The Budding Yeast Nucleus

PubMed Central

Taddei, Angela; Schober, Heiko; Gasser, Susan M.

2010-01-01

The budding yeast nucleus, like those of other eukaryotic species, is highly organized with respect to both chromosomal sequences and enzymatic activities. At the nuclear periphery interactions of nuclear pores with chromatin, mRNA, and transport factors promote efficient gene expression, whereas centromeres, telomeres, and silent chromatin are clustered and anchored away from pores. Internal nuclear organization appears to be function-dependent, reflecting localized sites for tRNA transcription, rDNA transcription, ribosome assembly, and DNA repair. Recent advances have identified new proteins involved in the positioning of chromatin and have allowed testing of the functional role of higher-order chromatin organization. The unequal distribution of silent information regulatory factors and histone modifying enzymes, which arises in part from the juxtaposition of telomeric repeats, has been shown to influence chromatin-mediated transcriptional repression. Other localization events suppress unwanted recombination. These findings highlight the contribution budding yeast genetics and cytology have made to dissecting the functional role of nuclear structure. PMID:20554704

Metagenomic chromosome conformation capture (meta3C) unveils the diversity of chromosome organization in microorganisms

PubMed Central

Marbouty, Martial; Cournac, Axel; Flot, Jean-François; Marie-Nelly, Hervé; Mozziconacci, Julien; Koszul, Romain

2014-01-01

Genomic analyses of microbial populations in their natural environment remain limited by the difficulty to assemble full genomes of individual species. Consequently, the chromosome organization of microorganisms has been investigated in a few model species, but the extent to which the features described can be generalized to other taxa remains unknown. Using controlled mixes of bacterial and yeast species, we developed meta3C, a metagenomic chromosome conformation capture approach that allows characterizing individual genomes and their average organization within a mix of organisms. Not only can meta3C be applied to species already sequenced, but a single meta3C library can be used for assembling, scaffolding and characterizing the tridimensional organization of unknown genomes. By applying meta3C to a semi-complex environmental sample, we confirmed its promising potential. Overall, this first meta3C study highlights the remarkable diversity of microorganisms chromosome organization, while providing an elegant and integrated approach to metagenomic analysis. DOI: http://dx.doi.org/10.7554/eLife.03318.001 PMID:25517076

Novel brewing yeast hybrids: creation and application.

PubMed

Krogerus, Kristoffer; Magalhães, Frederico; Vidgren, Virve; Gibson, Brian

2017-01-01

The natural interspecies Saccharomyces cerevisiae × Saccharomyces eubayanus hybrid yeast is responsible for global lager beer production and is one of the most important industrial microorganisms. Its success in the lager brewing environment is due to a combination of traits not commonly found in pure yeast species, principally low-temperature tolerance, and maltotriose utilization. Parental transgression is typical of hybrid organisms and has been exploited previously for, e.g., the production of wine yeast with beneficial properties. The parental strain S. eubayanus has only been discovered recently and newly created lager yeast strains have not yet been applied industrially. A number of reports attest to the feasibility of this approach and artificially created hybrids are likely to have a significant impact on the future of lager brewing. De novo S. cerevisiae × S. eubayanus hybrids outperform their parent strains in a number of respects, including, but not restricted to, fermentation rate, sugar utilization, stress tolerance, and aroma formation. Hybrid genome function and stability, as well as different techniques for generating hybrids and their relative merits are discussed. Hybridization not only offers the possibility of generating novel non-GM brewing yeast strains with unique properties, but is expected to aid in unraveling the complex evolutionary history of industrial lager yeast.

X Chromosome Evolution in Cetartiodactyla

PubMed Central

Proskuryakova, Anastasia A.; Kulemzina, Anastasia I.; Makunin, Alexey I.; Kukekova, Anna V.; Lynn Johnson, Jennifer; Lemskaya, Natalya A.; Beklemisheva, Violetta R.; Roelke-Parker, Melody E.; Bellizzi, June; Ryder, Oliver A.; O’Brien, Stephen J.; Graphodatsky, Alexander S.

2017-01-01

The phenomenon of a remarkable conservation of the X chromosome in eutherian mammals has been first described by Susumu Ohno in 1964. A notable exception is the cetartiodactyl X chromosome, which varies widely in morphology and G-banding pattern between species. It is hypothesized that this sex chromosome has undergone multiple rearrangements that changed the centromere position and the order of syntenic segments over the last 80 million years of Cetartiodactyla speciation. To investigate its evolution we have selected 26 evolutionarily conserved bacterial artificial chromosome (BAC) clones from the cattle CHORI-240 library evenly distributed along the cattle X chromosome. High-resolution BAC maps of the X chromosome on a representative range of cetartiodactyl species from different branches: pig (Suidae), alpaca (Camelidae), gray whale (Cetacea), hippopotamus (Hippopotamidae), Java mouse-deer (Tragulidae), pronghorn (Antilocapridae), Siberian musk deer (Moschidae), and giraffe (Giraffidae) were obtained by fluorescent in situ hybridization. To trace the X chromosome evolution during fast radiation in specious families, we performed mapping in several cervids (moose, Siberian roe deer, fallow deer, and Pere David’s deer) and bovid (muskox, goat, sheep, sable antelope, and cattle) species. We have identified three major conserved synteny blocks and rearrangements in different cetartiodactyl lineages and found that the recently described phenomenon of the evolutionary new centromere emergence has taken place in the X chromosome evolution of Cetartiodactyla at least five times. We propose the structure of the putative ancestral cetartiodactyl X chromosome by reconstructing the order of syntenic segments and centromere position for key groups. PMID:28858207

Genomic diversity of Saccharomyces cerevisiae yeasts associated with alcoholic fermentation of bacanora produced by artisanal methods.

PubMed

Álvarez-Ainza, M L; Zamora-Quiñonez, K A; Moreno-Ibarra, G M; Acedo-Félix, E

2015-03-01

Bacanora is a spirituous beverage elaborated with Agave angustifolia Haw in an artisanal process. Natural fermentation is mostly performed with native yeasts and bacteria. In this study, 228 strains of yeast like Saccharomyces were isolated from the natural alcoholic fermentation on the production of bacanora. Restriction analysis of the amplified region ITS1-5.8S-ITS2 of the ribosomal DNA genes (RFLPr) were used to confirm the genus, and 182 strains were identified as Saccharomyces cerevisiae. These strains displayed high genomic variability in their chromosomes profiles by karyotyping. Electrophoretic profiles of the strains evaluated showed a large number of chromosomes the size of which ranged between 225 and 2200 kpb approximately.

DNA Damage and Genomic Instability Induced by Inappropriate DNA Re-replication

DTIC Science & Technology

2007-04-01

Conway, A., Lockhart, D. J., Davis, R. W., Brewer , B. J., and Fangman, W. L. (2001). Replication dynamics of the yeast genome. Science 294, 115–121... Brewer , B. J. (2001). An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint. Mol. Cell 7, 705–713. Vas, A., Mok, W., and...replication in yeast cells. We have demonstrated that re-replication induces a rapid and significant decrease in cell viability and a cellular DNA damage

An unconventional interaction between Dis1/TOG and Mal3/EB1 in fission yeast promotes the fidelity of chromosome segregation.

PubMed

Matsuo, Yuzy; Maurer, Sebastian P; Yukawa, Masashi; Zakian, Silva; Singleton, Martin R; Surrey, Thomas; Toda, Takashi

2016-12-15

Dynamic microtubule plus-ends interact with various intracellular target regions such as the cell cortex and the kinetochore. Two conserved families of microtubule plus-end-tracking proteins, the XMAP215, ch-TOG or CKAP5 family and the end-binding 1 (EB1, also known as MAPRE1) family, play pivotal roles in regulating microtubule dynamics. Here, we study the functional interplay between fission yeast Dis1, a member of the XMAP215/TOG family, and Mal3, an EB1 protein. Using an in vitro microscopy assay, we find that purified Dis1 autonomously tracks growing microtubule ends and is a bona fide microtubule polymerase. Mal3 recruits additional Dis1 to microtubule ends, explaining the synergistic enhancement of microtubule dynamicity by these proteins. A non-canonical binding motif in Dis1 mediates the interaction with Mal3. X-ray crystallography shows that this new motif interacts in an unconventional configuration with the conserved hydrophobic cavity formed within the Mal3 C-terminal region that typically interacts with the canonical SXIP motif. Selectively perturbing the Mal3-Dis1 interaction in living cells demonstrates that it is important for accurate chromosome segregation. Whereas, in some metazoans, the interaction between EB1 and the XMAP215/TOG family members requires an additional binding partner, fission yeast relies on a direct interaction, indicating evolutionary plasticity of this critical interaction module. © 2016. Published by The Company of Biologists Ltd.

Ends-in Vs. Ends-Out Recombination in Yeast

PubMed Central

Hastings, P. J.; McGill, C.; Shafer, B.; Strathern, J. N.

1993-01-01

Integration of linearized plasmids into yeast chromosomes has been used as a model system for the study of recombination initiated by double-strand breaks. The linearized plasmid DNA recombines efficiently into sequences homologous to the ends of the DNA. This efficient recombination occurs both for the configuration in which the break is in a contiguous region of homology (herein called the ends-in configuration) and for ``omega'' insertions in which plasmid sequences interrupt a linear region of homology (herein called the ends-out configuration). The requirements for integration of these two configurations are expected to be different. We compared these two processes in a yeast strain containing an ends-in target and an ends-out target for the same cut plasmid. Recovery of ends-in events exceeds ends-out events by two- to threefold. Possible causes for the origin of this small bias are discussed. The lack of an extreme difference in frequency implies that cooperativity between the two ends does not contribute to the efficiency with which cut circular plasmids are integrated. This may also be true for the repair of chromosomal double-strand breaks. PMID:8307337

The Yeast Polo Kinase Cdc5 Regulates the Shape of the Mitotic Nucleus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walters, Alison D.; May, Christopher K.; Dauster, Emma S.

Abnormal nuclear size and shape are hallmarks of aging and cancer. However, the mechanisms regulating nuclear morphology and nuclear envelope (NE) expansion are poorly understood. In metazoans, the NE disassembles prior to chromosome segregation and reassembles at the end of mitosis. In budding yeast, the NE remains intact. The nucleus elongates as chromosomes segregate and then divides at the end of mitosis to form two daughter nuclei without NE disassembly. The budding yeast nucleus also undergoes remodeling during a mitotic arrest; the NE continues to expand despite the pause in chromosome segregation, forming a nuclear extension, or "flare," that encompassesmore » the nucleolus. The distinct nucleolar localization of the mitotic flare indicates that the NE is compartmentalized and that there is a mechanism by which NE expansion is confined to the region adjacent to the nucleolus. Here we show that mitotic flare formation is dependent on the yeast polo kinase Cdc5. This function of Cdc5 is independent of its known mitotic roles, including rDNA condensation. High-resolution imaging revealed that following Cdc5 inactivation, nuclei expand isometrically rather than forming a flare, indicating that Cdc5 is needed for NE compartmentalization. Lastly, even in an uninterrupted cell cycle, a small NE expansion occurs adjacent to the nucleolus prior to anaphase in a Cdc5-dependent manner. Our data provide the first evidence that polo kinase, a key regulator of mitosis, plays a role in regulating nuclear morphology and NE expansion.« less

The Yeast Polo Kinase Cdc5 Regulates the Shape of the Mitotic Nucleus

DOE PAGES

Walters, Alison D.; May, Christopher K.; Dauster, Emma S.; ...

2014-11-20

Abnormal nuclear size and shape are hallmarks of aging and cancer. However, the mechanisms regulating nuclear morphology and nuclear envelope (NE) expansion are poorly understood. In metazoans, the NE disassembles prior to chromosome segregation and reassembles at the end of mitosis. In budding yeast, the NE remains intact. The nucleus elongates as chromosomes segregate and then divides at the end of mitosis to form two daughter nuclei without NE disassembly. The budding yeast nucleus also undergoes remodeling during a mitotic arrest; the NE continues to expand despite the pause in chromosome segregation, forming a nuclear extension, or "flare," that encompassesmore » the nucleolus. The distinct nucleolar localization of the mitotic flare indicates that the NE is compartmentalized and that there is a mechanism by which NE expansion is confined to the region adjacent to the nucleolus. Here we show that mitotic flare formation is dependent on the yeast polo kinase Cdc5. This function of Cdc5 is independent of its known mitotic roles, including rDNA condensation. High-resolution imaging revealed that following Cdc5 inactivation, nuclei expand isometrically rather than forming a flare, indicating that Cdc5 is needed for NE compartmentalization. Lastly, even in an uninterrupted cell cycle, a small NE expansion occurs adjacent to the nucleolus prior to anaphase in a Cdc5-dependent manner. Our data provide the first evidence that polo kinase, a key regulator of mitosis, plays a role in regulating nuclear morphology and NE expansion.« less

Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice.

PubMed

Schmouth, Jean-François; Castellarin, Mauro; Laprise, Stéphanie; Banks, Kathleen G; Bonaguro, Russell J; McInerny, Simone C; Borretta, Lisa; Amirabbasi, Mahsa; Korecki, Andrea J; Portales-Casamar, Elodie; Wilson, Gary; Dreolini, Lisa; Jones, Steven J M; Wasserman, Wyeth W; Goldowitz, Daniel; Holt, Robert A; Simpson, Elizabeth M

2013-10-14

The next big challenge in human genetics is understanding the 98% of the genome that comprises non-coding DNA. Hidden in this DNA are sequences critical for gene regulation, and new experimental strategies are needed to understand the functional role of gene-regulation sequences in health and disease. In this study, we build upon our HuGX ('high-throughput human genes on the X chromosome') strategy to expand our understanding of human gene regulation in vivo. In all, ten human genes known to express in therapeutically important brain regions were chosen for study. For eight of these genes, human bacterial artificial chromosome clones were identified, retrofitted with a reporter, knocked single-copy into the Hprt locus in mouse embryonic stem cells, and mouse strains derived. Five of these human genes expressed in mouse, and all expressed in the adult brain region for which they were chosen. This defined the boundaries of the genomic DNA sufficient for brain expression, and refined our knowledge regarding the complexity of gene regulation. We also characterized for the first time the expression of human MAOA and NR2F2, two genes for which the mouse homologs have been extensively studied in the central nervous system (CNS), and AMOTL1 and NOV, for which roles in CNS have been unclear. We have demonstrated the use of the HuGX strategy to functionally delineate non-coding-regulatory regions of therapeutically important human brain genes. Our results also show that a careful investigation, using publicly available resources and bioinformatics, can lead to accurate predictions of gene expression.

BAC to degeneration bacterial artificial chromosome (BAC)-mediated transgenesis for modeling basal ganglia neurodegenerative disorders.

PubMed

Lu, Xiao-Hong

2009-01-01

Basal ganglia neurodegenerative disorders, such as Parkinson's disease (PD) and Huntington's disease (HD), are characterized by not only spectrum of motor deficits, ranging form hypokinesia to hyperkinesia, but also emotional, cognitive, and psychiatric manifestations. The symptoms and pathogenic mechanism of these disorders should be viewed as dysfunctions of specific cortico-subcortical neurocircuits. Transgenic approaches using large genomic inserts, such as bacterial artificial chromosome (BAC)-mediated transgenesis, due to its capacity to propagate large-size genomic DNA and faithful production of endogenous-like gene expression pattern/lever, have provided an ideal basis for the generation of transgenic mice as model for basal ganglia neurodegenerative disorders, as well as the functional and structural analysis of neurocircuits. In this chapter, the basic concepts and practical approaches about application of BAC transgenic system are introduced. Existent major BAC transgenic mouse models for PD and HD are evaluated according to their construct, face, and predicative validity. Finally, considerations, possible solutions, and future perspectives of using BAC transgenic approach to study basal ganglia neurodegenerative disorders are discussed.

Telomerase-mediated life-span extension of human primary fibroblasts by human artificial chromosome (HAC) vector

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shitara, Shingo; Kakeda, Minoru; Nagata, Keiko

2008-05-09

Telomerase-mediated life-span extension enables the expansion of normal cells without malignant transformation, and thus has been thought to be useful in cell therapies. Currently, integrating vectors including the retrovirus are used for human telomerase reverse transcriptase (hTERT)-mediated expansion of normal cells; however, the use of these vectors potentially causes unexpected insertional mutagenesis and/or activation of oncogenes. Here, we established normal human fibroblast (hPF) clones retaining non-integrating human artificial chromosome (HAC) vectors harboring the hTERT expression cassette. In hTERT-HAC/hPF clones, we observed the telomerase activity and the suppression of senescent-associated SA-{beta}-galactosidase activity. Furthermore, the hTERT-HAC/hPF clones continued growing beyond 120 daysmore » after cloning, whereas the hPF clones retaining the silent hTERT-HAC senesced within 70 days. Thus, hTERT-HAC-mediated episomal expression of hTERT allows the extension of the life-span of human primary cells, implying that gene delivery by non-integrating HAC vectors can be used to control cellular proliferative capacity of primary cultured cells.« less

[Detection of linear chromosomes and plasmids among 15 genera in the Actinomycetales].

PubMed

Ma, Ning; Ma, Wei; Jiang, Chenglin; Fang, Ping; Qin, Zhongjun

2003-10-01

Bacterial chromosomes and plasmids are commonly circular, however, linear chromosomes and plasmids were discovered among 5 genera of the Actinomycetales. Here, we use pulsed field gel electrophoresis to study the genomes of 19 species which belong to 15 genera in the Actinomycetales. All chromosomes of 19 species are linear DNA, and linear plasmids with different sizes and copy numbers are detected among 5 species. This work provide basis for investigating the possible novel functions of linear replicons beyond Streptomyces and also helps to develop Actinomycetales artificial linear chromosome.

Synthetic genome engineering forging new frontiers for wine yeast.

PubMed

Pretorius, Isak S

2017-02-01

Over the past 15 years, the seismic shifts caused by the convergence of biomolecular, chemical, physical, mathematical, and computational sciences alongside cutting-edge developments in information technology and engineering have erupted into a new field of scientific endeavor dubbed Synthetic Biology. Recent rapid advances in high-throughput DNA sequencing and DNA synthesis techniques are enabling the design and construction of new biological parts (genes), devices (gene networks) and modules (biosynthetic pathways), and the redesign of biological systems (cells and organisms) for useful purposes. In 2014, the budding yeast Saccharomyces cerevisiae became the first eukaryotic cell to be equipped with a fully functional synthetic chromosome. This was achieved following the synthesis of the first viral (poliovirus in 2002 and bacteriophage Phi-X174 in 2003) and bacterial (Mycoplasma genitalium in 2008 and Mycoplasma mycoides in 2010) genomes, and less than two decades after revealing the full genome sequence of a laboratory (S288c in 1996) and wine (AWRI1631 in 2008) yeast strain. A large international project - the Synthetic Yeast Genome (Sc2.0) Project - is now underway to synthesize all 16 chromosomes (∼12 Mb carrying ∼6000 genes) of the sequenced S288c laboratory strain by 2018. If successful, S. cerevisiae will become the first eukaryote to cross the horizon of in silico design of complex cells through de novo synthesis, reshuffling, and editing of genomes. In the meantime, yeasts are being used as cell factories for the semi-synthetic production of high-value compounds, such as the potent antimalarial artemisinin, and food ingredients, such as resveratrol, vanillin, stevia, nootkatone, and saffron. As a continuum of previously genetically engineered industrially important yeast strains, precision genome engineering is bound to also impact the study and development of wine yeast strains supercharged with synthetic DNA. The first taste of what the future

8-Methoxypsoralen photoinduced plasmid-chromosome recombination in Saccharomyces cerevisiae using a centromeric vector.

PubMed Central

Meira, L B; Henriques, J A; Magaña-Schwencke, N

1995-01-01

The characterization of a new system to study the induction of plasmid-chromosome recombination is described. Single-stranded and double-stranded centromeric vectors bearing 8-methoxypsoralen photoinduced lesions were used to transform a wild-type yeast strain bearing the leu2-3,112 marker. Using the SSCP methodology and DNA sequencing, it was demonstrated that repair of the lesions in plasmid DNA was mainly due to conversion of the chromosomal allele to the plasmid DNA. Images PMID:7784218

Mitochondrial and cytoplasmic isoleucyl-, glutamyl- and arginyl-tRNA synthetases of yeast are encoded by separate genes.

PubMed

Tzagoloff, A; Shtanko, A

1995-06-01

Three complementation groups of a pet mutant collection have been found to be composed of respiratory-deficient deficient mutants with lesions in mitochondrial protein synthesis. Recombinant plasmids capable of restoring respiration were cloned by transformation of representatives of each complementation group with a yeast genomic library. The plasmids were used to characterize the complementing genes and to institute disruption of the chromosomal copies of each gene in respiratory-proficient yeast. The sequences of the cloned genes indicate that they code for isoleucyl-, arginyl- and glutamyl-tRNA synthetases. The properties of the mutants used to obtain the genes and of strains with the disrupted genes indicate that all three aminoacyl-tRNA synthetases function exclusively in mitochondrial proteins synthesis. The ISM1 gene for mitochondrial isoleucyl-tRNA synthetase has been localized to chromosome XVI next to UME5. The MSR1 gene for the arginyl-tRNA synthetase was previously located on yeast chromosome VIII. The third gene MSE1 for the mitochondrial glutamyl-tRNA synthetase has not been localized. The identification of three new genes coding for mitochondrial-specific aminoacyl-tRNA synthetases indicates that in Saccharomyces cerevisiae at least 11 members of this protein family are encoded by genes distinct from those coding for the homologous cytoplasmic enzymes.

Dicentric chromosomes: unique models to study centromere function and inactivation.

PubMed

Stimpson, Kaitlin M; Matheny, Justyne E; Sullivan, Beth A

2012-07-01

Dicentric chromosomes are products of genome rearrangement that place two centromeres on the same chromosome. Depending on the organism, dicentric stability varies after formation. In humans, dicentrics occur naturally in a substantial portion of the population and usually segregate successfully in mitosis and meiosis. Their stability has been attributed to inactivation of one of the two centromeres, creating a functionally monocentric chromosome that can segregate normally during cell division. The molecular basis for centromere inactivation is not well understood, although studies in model organisms and in humans suggest that genomic and epigenetic mechanisms can be involved. Furthermore, constitutional dicentric chromosomes ascertained in patients presumably represent the most stable chromosomes, so the spectrum of dicentric fates, if it exists, is not entirely clear. Studies of engineered or induced dicentrics in budding yeast and plants have provided significant insight into the fate of dicentric chromosomes. And, more recently, studies have shown that dicentrics in humans can also undergo multiple fates after formation. Here, we discuss current experimental evidence from various organisms that has deepened our understanding of dicentric behavior and the intriguingly complex process of centromere inactivation.

Dicentric chromosomes: unique models to study centromere function and inactivation

PubMed Central

Stimpson, Kaitlin M.; Matheny, Justyne E.

2013-01-01

Dicentric chromosomes are products of genome rearrangement that place two centromeres on the same chromosome. Depending on the organism, dicentric stability varies after formation. In humans, dicentrics occur naturally in a substantial portion of the population and usually segregate successfully in mitosis and meiosis. Their stability has been attributed to inactivation of one of the two centromeres, creating a functionally monocentric chromosome that can segregate normally during cell division. The molecular basis for centromere inactivation is not well under-stood, although studies in model organisms and in humans suggest that genomic and epigenetic mechanisms can be involved. Furthermore, constitutional dicentric chromosomes ascertained in patients presumably represent the most stable chromosomes, so the spectrum of dicentric fates, if it exists, is not entirely clear. Studies of engineered or induced dicentrics in budding yeast and plants have provided significant insight into the fate of dicentric chromosomes. And, more recently, studies have shown that dicentrics in humans can also undergo multiple fates after formation. Here, we discuss current experimental evidence from various organisms that has deepened our understanding of dicentric behavior and the intriguingly complex process of centromere inactivation. PMID:22801777

Genome Sequence of the Yeast Clavispora lusitaniae Type Strain CBS 6936

PubMed Central

Klopp, Christophe; Biteau, Nicolas; Fitton-Ouhabi, Valérie; Dementhon, Karine; Accoceberry, Isabelle; Sherman, David J.; Noël, Thierry

2017-01-01

ABSTRACT Clavispora lusitaniae, an environmental saprophytic yeast belonging to the CTG clade of Candida, can behave occasionally as an opportunistic pathogen in humans. We report here the genome sequence of the type strain CBS 6936. Comparison with sequences of strain ATCC 42720 indicates conservation of chromosomal structure but significant nucleotide divergence. PMID:28774979

A kidney injury molecule-1 (Kim-1) gene reporter in a mouse artificial chromosome: the responsiveness to cisplatin toxicity in immortalized mouse kidney S3 cells.

PubMed

Kokura, Kenji; Kuromi, Yasushi; Endo, Takeshi; Anzai, Naohiko; Kazuki, Yasuhiro; Oshimura, Mitsuo; Ohbayashi, Tetsuya

2016-10-01

Kidney injury molecule-1 (Kim-1) has been validated as a urinary biomarker for acute and chronic renal damage. The expression of Kim-1 mRNA is also activated by acute kidney injury induced by cisplatin in rodents and humans. To date, the measurement of Kim-1 expression has not fully allowed the detection of in vitro cisplatin nephrotoxicity in immortalized culture cells, such as human kidney-2 cells and immortalized proximal tubular epithelial cells. We measured the augmentation of Kim-1 mRNA expression after the addition of cisplatin using immortalized S3 cells established from the kidneys of transgenic mice harboring temperature-sensitive large T antigen from Simian virus 40. A mouse Kim-1 gene luciferase reporter in conjunction with an Hprt gene reporter detected cisplatin-induced nephrotoxicity in S3 cells. These two reporter genes were contained in a mouse artificial chromosome, and two luciferases that emitted different wavelengths were used to monitor the respective gene expression. However, the Kim-1 reporter gene failed to respond to cisplatin in A9 fibroblast cells that contained the same reporter mouse artificial chromosome, suggesting cell type-specificity for activation of the reporter. We report the feasibility of measuring in vitro cisplatin nephrotoxicity using a Kim-1 reporter gene in S3 cells. © 2016 The Authors. The Journal of Gene Medicine Published by John Wiley & Sons, Ltd.

Detection of maltose fermentation genes in the baking yeast strains of Saccharomyces cerevisiae.

PubMed

Oda, Y; Tonomura, K

1996-10-01

The presence of any one of the five unlinked MAL loci (MAL1, MAL2, MAL3, MAL4 and MAL6) confers the ability to ferment maltose on the yeast Saccharomyces cerevisiae. Each locus is composed of three genes encoding maltose permease, alpha-glucosidase and MAL activator. Chromosomal DNA of seven representative baking strains has been separated by pulse-field gel electrophoresis and probed with three genes in MAL6 locus. The DNA bands to which all of the three MAL-derived probes simultaneously hybridized were chromosome VII carrying MAL1 in all of the strains tested, chromosome XI carrying MAL4 in six strains, chromosome III carrying MAL2 in three strains and chromosomes II and VIII carrying MAL3 and MAL6, respectively, in the one strain. The number of MAL loci in baking strains was comparable to those of brewing strains.

Monitoring Recombination During Meiosis in Budding Yeast.

PubMed

Owens, Shannon; Tang, Shangming; Hunter, Neil

2018-01-01

Homologous recombination is fundamental to sexual reproduction, facilitating accurate segregation of homologous chromosomes at the first division of meiosis, and creating novel allele combinations that fuel evolution. Following initiation of meiotic recombination by programmed DNA double-strand breaks (DSBs), homologous pairing and DNA strand exchange form joint molecule (JM) intermediates that are ultimately resolved into crossover and noncrossover repair products. Physical monitoring of the DNA steps of meiotic recombination in Saccharomyces cerevisiae (budding yeast) cultures undergoing synchronous meiosis has provided seminal insights into the molecular basis of meiotic recombination and affords a powerful tool for dissecting the molecular roles of recombination factors. This chapter describes a suit of electrophoretic and Southern hybridization techniques used to detect and quantify the DNA intermediates of meiotic recombination at recombination hotspots in budding yeast. DSBs and recombination products (crossovers and noncrossovers) are resolved using one-dimensional electrophoresis and distinguished by restriction site polymorphisms between the parental chromosomes. Psoralen cross-linking is used to stabilize branched JMs, which are resolved from linear species by native/native two-dimensional electrophoresis. Native/denaturing two-dimensional electrophoresis is employed to determine the component DNA strands of JMs and to measure the processing of DSBs. These techniques are generally applicable to any locus where the frequency of recombination is high enough to detect intermediates by Southern hybridization. © 2018 Elsevier Inc. All rights reserved.

Brewing characteristics of haploid strains isolated from sake yeast Kyokai No. 7.

PubMed

Katou, Taku; Kitagaki, Hiroshi; Akao, Takeshi; Shimoi, Hitoshi

2008-11-01

Sake yeast exhibit various characteristics that make them more suitable for sake brewing compared to other yeast strains. Since sake yeast strains are Saccharomyces cerevisiae heterothallic diploid strains, it is likely that they have heterozygous alleles on homologous chromosomes (heterozygosity) due to spontaneous mutations. If this is the case, segregation of phenotypic traits in haploid strains after sporulation and concomitant meiosis of sake yeast strains would be expected to occur. To examine this hypothesis, we isolated 100 haploid strains from Kyokai No. 7 (K7), a typical sake yeast strain in Japan, and compared their brewing characteristics in small-scale sake-brewing tests. Analyses of the resultant sake samples showed a smooth and continuous distribution of analytical values for brewing characteristics, suggesting that K7 has multiple heterozygosities that affect brewing characteristics and that these heterozygous alleles do segregate after sporulation. Correlation and principal component analyses suggested that the analytical parameters could be classified into two groups, indicating fermentation ability and sake flavour. (c) 2008 John Wiley & Sons, Ltd.

Cross-referencing yeast genetics and mammalian genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hieter, P.; Basset, D.; Boguski, M.

1994-09-01

We have initiated a project that will systematically transfer information about yeast genes onto the genetic maps of mice and human beings. Rapidly expanding human EST data will serve as a source of candidate human homologs that will be repeatedly searched using yeast protein sequence queries. Search results will be automatically reported to participating labs. Human cDNA sequences from which the ESTs are derived will be mapped at high resolution in the human and mouse genomes. The comparative mapping information cross-references the genomic position of novel human cDNAs with functional information known about the cognate yeast genes. This should facilitatemore » the initial identification of genes responsible for mammalian mutant phenotypes, including human disease. In addition, the identification of mammalian homologs of yeast genes provides reagents for determining evolutionary conservation and for performing direct experiments in multicellular eukaryotes to enhance study of the yeast protein`s function. For example, ESTs homologous to CDC27 and CDC16 were identified, and the corresponding cDNA clones were obtained from ATTC, completely sequenced, and mapped on human and mouse chromosomes. In addition, the CDC17hs cDNA has been used to raise antisera to the CDC27Hs protein and used in subcellular localization experiments and junctional studies in mammalian cells. We have received funding from the National Center for Human Genome Research to provide a community resource which will establish comprehensive cross-referencing among yeast, human, and mouse loci. The project is set up as a service and information on how to communicate with this effort will be provided.« less

Construction of BAC Libraries from Flow-Sorted Chromosomes.

PubMed

Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

2016-01-01

Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.

Enrichment of dynamic chromosomal crosslinks drive phase separation of the nucleolus

PubMed Central

Hult, Caitlin; Adalsteinsson, David; Vasquez, Paula A.; Lawrimore, Josh; Bennett, Maggie; York, Alyssa; Cook, Diana; Yeh, Elaine; Forest, Mark Gregory

2017-01-01

Abstract Regions of highly repetitive DNA, such as those found in the nucleolus, show a self-organization that is marked by spatial segregation and frequent self-interaction. The mechanisms that underlie the sequestration of these sub-domains are largely unknown. Using a stochastic, bead-spring representation of chromatin in budding yeast, we find enrichment of protein-mediated, dynamic chromosomal cross-links recapitulates the segregation, morphology and self-interaction of the nucleolus. Rates and enrichment of dynamic crosslinking have profound consequences on domain morphology. Our model demonstrates the nucleolus is phase separated from other chromatin in the nucleus and predicts that multiple rDNA loci will form a single nucleolus independent of their location within the genome. Fluorescent labeling of budding yeast nucleoli with CDC14-GFP revealed that a split rDNA locus indeed forms a single nucleolus. We propose that nuclear sub-domains, such as the nucleolus, result from phase separations within the nucleus, which are driven by the enrichment of protein-mediated, dynamic chromosomal crosslinks. PMID:28977453

Enrichment of dynamic chromosomal crosslinks drive phase separation of the nucleolus.

PubMed

Hult, Caitlin; Adalsteinsson, David; Vasquez, Paula A; Lawrimore, Josh; Bennett, Maggie; York, Alyssa; Cook, Diana; Yeh, Elaine; Forest, Mark Gregory; Bloom, Kerry

2017-11-02

Regions of highly repetitive DNA, such as those found in the nucleolus, show a self-organization that is marked by spatial segregation and frequent self-interaction. The mechanisms that underlie the sequestration of these sub-domains are largely unknown. Using a stochastic, bead-spring representation of chromatin in budding yeast, we find enrichment of protein-mediated, dynamic chromosomal cross-links recapitulates the segregation, morphology and self-interaction of the nucleolus. Rates and enrichment of dynamic crosslinking have profound consequences on domain morphology. Our model demonstrates the nucleolus is phase separated from other chromatin in the nucleus and predicts that multiple rDNA loci will form a single nucleolus independent of their location within the genome. Fluorescent labeling of budding yeast nucleoli with CDC14-GFP revealed that a split rDNA locus indeed forms a single nucleolus. We propose that nuclear sub-domains, such as the nucleolus, result from phase separations within the nucleus, which are driven by the enrichment of protein-mediated, dynamic chromosomal crosslinks. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genome Sequence of the Yeast Clavispora lusitaniae Type Strain CBS 6936.

PubMed

Durrens, Pascal; Klopp, Christophe; Biteau, Nicolas; Fitton-Ouhabi, Valérie; Dementhon, Karine; Accoceberry, Isabelle; Sherman, David J; Noël, Thierry

2017-08-03

Clavispora lusitaniae , an environmental saprophytic yeast belonging to the CTG clade of Candida , can behave occasionally as an opportunistic pathogen in humans. We report here the genome sequence of the type strain CBS 6936. Comparison with sequences of strain ATCC 42720 indicates conservation of chromosomal structure but significant nucleotide divergence. Copyright © 2017 Durrens et al.

Nectar yeasts warm the flowers of a winter-blooming plant

PubMed Central

Herrera, Carlos M.; Pozo, María I.

2010-01-01

Yeasts are ubiquitous in terrestrial and aquatic microbiota, yet their ecological functionality remains relatively unexplored in comparison with other micro-organisms. This paper formulates and tests the novel hypothesis that heat produced by the sugar catabolism of yeast populations inhabiting floral nectar can increase the temperature of floral nectar and, more generally, modify the within-flower thermal microenvironment. Two field experiments were designed to test this hypothesis for the winter-blooming herb Helleborus foetidus (Ranunculaceae). In experiment 1, the effect of yeasts on the within-flower thermal environment was tested by excluding them from flowers, while in experiment 2 the test involved artificial inoculation of virgin flowers with yeasts. Nectary temperature (Tnect), within-flower air temperature (Tflow) and external air temperature (Tair) were measured on experimental and control flowers in both experiments. Experimental exclusion of yeasts from the nectaries significantly reduced, and experimental addition of yeasts significantly increased, the temperature excess of nectaries (ΔTnect = Tnect − Tair) and the air space inside flowers in relation to the air just outside the flowers. In non-experimental flowers exposed to natural pollinator visitation, ΔTnect was linearly related to log yeast cell density in nectar, and reached +6°C in nectaries with the densest yeast populations. The warming effect of nectar-dwelling yeasts documented in this study suggests novel ecological mechanisms potentially linking nectarivorous microbes with winter-blooming plants and their insect pollinators. PMID:20147331

Structural features of the rice chromosome 4 centromere.

PubMed

Zhang, Yu; Huang, Yuchen; Zhang, Lei; Li, Ying; Lu, Tingting; Lu, Yiqi; Feng, Qi; Zhao, Qiang; Cheng, Zhukuan; Xue, Yongbiao; Wing, Rod A; Han, Bin

2004-01-01

A complete sequence of a chromosome centromere is necessary for fully understanding centromere function. We reported the sequence structures of the first complete rice chromosome centromere through sequencing a large insert bacterial artificial chromosome clone-based contig, which covered the rice chromosome 4 centromere. Complete sequencing of the 124-kb rice chromosome 4 centromere revealed that it consisted of 18 tracts of 379 tandemly arrayed repeats known as CentO and a total of 19 centromeric retroelements (CRs) but no unique sequences were detected. Four tracts, composed of 65 CentO repeats, were located in the opposite orientation, and 18 CentO tracts were flanked by 19 retroelements. The CRs were classified into four types, and the type I retroelements appeared to be more specific to rice centromeres. The preferential insert of the CRs among CentO repeats indicated that the centromere-specific retroelements may contribute to centromere expansion during evolution. The presence of three intact retrotransposons in the centromere suggests that they may be responsible for functional centromere initiation through a transcription-mediated mechanism.

Chromosomal context and replication properties of ARS plasmids in Schizosaccharomyces pombe.

PubMed

Pratihar, Aditya S; Tripathi, Vishnu P; Yadav, Mukesh P; Dubey, Dharani D

2015-12-01

Short, specific DNA sequences called as Autonomously Replicating Sequence (ARS) elements function as plasmid as well as chromosomal replication origins in yeasts. As compared to ARSs, different chromosomal origins vary greatly in their efficiency and timing of replication probably due to their wider chromosomal context. The two Schizosaccharomyces pombe ARS elements, ars727 and ars2004, represent two extremities in their chromosomal origin activity - ars727 is inactive and late replicating, while ars2004 is a highly active, early-firing origin. To determine the effect of chromosomal context on the activity of these ARS elements, we have cloned them with their extended chromosomal context as well as in the context of each other in both orientations and analysed their replication efficiency by ARS and plasmid stability assays. We found that these ARS elements retain their origin activity in their extended/altered context. However, deletion of a 133-bp region of the previously reported ars727- associated late replication enforcing element (LRE) caused advancement in replication timing of the resulting plasmid. These results confirm the role of LRE in directing plasmid replication timing and suggest that the plasmid origin efficiency of ars2004 or ars727 remains unaltered by the extended chromosomal context.

6q deletion detected by fluorescence in situ hybridization using bacterial artificial chromosome in chronic lymphocytic leukemia.

PubMed

Dalsass, Alessia; Mestichelli, Francesca; Ruggieri, Miriana; Gaspari, Paola; Pezzoni, Valerio; Vagnoni, Davide; Angelini, Mario; Angelini, Stefano; Bigazzi, Catia; Falcioni, Sadia; Troiani, Emanuela; Alesiani, Francesco; Catarini, Massimo; Attolico, Immacolata; Scortechini, Ilaria; Discepoli, Giancarlo; Galieni, Piero

2013-07-01

Deletions of the long arm of chromosome 6 are known to occur at relatively low frequency (3-6%) in chronic lymphocytic leukemia (CLL), and they are more frequently observed in 6q21. Few data have been reported regarding other bands on 6q involved by cytogenetic alterations in CLL. The cytogenetic study was performed in nuclei and metaphases obtained after stimulation with a combination of CpG-oligonucleotide DSP30 and interleukin-2. Four bacterial artificial chromosome (BAC) clones mapping regions in bands 6q16, 6q23, 6q25, 6q27 were used as probes for fluorescence in situ hybridization in 107 CLL cases in order to analyze the occurrence and localization of 6q aberrations. We identified 11 cases (10.2%) with 6q deletion of 107 patients studied with CLL. The trends of survival curves and the treatment-free intervals (TFI) of patients with deletion suggest a better outcome than the other cytogenetic risk groups. We observed two subgroups with 6q deletion as the sole anomaly: two cases with 6q16 deletion, and three cases with 6q25.2-27 deletion. There were differences of age, stage, and TFI between both subgroups. By using BAC probes, we observed that 6q deletion has a higher frequency in CLL and is linked with a good prognosis. In addition, it was observed that the deletion in 6q16 appears to be the most frequent and, if present as the only abnormality, it could be associated with a most widespread disease. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Multiple determinants controlling activation of yeast replication origins late in S phase.

PubMed

Friedman, K L; Diller, J D; Ferguson, B M; Nyland, S V; Brewer, B J; Fangman, W L

1996-07-01

Analysis of a 131-kb segment of the left arm of yeast chromosome XIV beginning 157 kb from the telomere reveals four highly active origins of replication that initiate replication late in S phase. Previous work has shown that telomeres act as determinants for late origin activation. However, at least two of the chromosome XIV origins maintain their late activation time when located on large circular plasmids, indicating that late replication is independent of telomeres. Analysis of the replication time of plasmid derivatives containing varying amounts of chromosome XIV DNA show that a minimum of three chromosomal elements, distinct from each tested origin, contribute to late activation time. These late determinants are functionally equivalent, because duplication of one set of contributing sequences can compensate for the removal of another set. Furthermore, insertion of an origin that is normally early activated into this domain results in a shift to late activation, suggesting that the chromosome XIV origins are not unique in their ability to respond to the late determinants.

Confinement-Induced Glassy Dynamics in a Model for Chromosome Organization

NASA Astrophysics Data System (ADS)

Kang, Hongsuk; Yoon, Young-Gui; Thirumalai, D.; Hyeon, Changbong

2015-11-01

Recent experiments showing scaling of the intrachromosomal contact probability, P (s )˜s-1 with the genomic distance s , are interpreted to mean a self-similar fractal-like chromosome organization. However, scaling of P (s ) varies across organisms, requiring an explanation. We illustrate dynamical arrest in a highly confined space as a discriminating marker for genome organization, by modeling chromosomes inside a nucleus as a homopolymer confined to a sphere of varying sizes. Brownian dynamics simulations show that the chain dynamics slows down as the polymer volume fraction (ϕ ) inside the confinement approaches a critical value ϕc. The universal value of ϕc∞≈0.44 for a sufficiently long polymer (N ≫1 ) allows us to discuss genome dynamics using ϕ as the sole parameter. Our study shows that the onset of glassy dynamics is the reason for the segregated chromosome organization in humans (N ≈3 ×109, ϕ ≳ϕc∞), whereas chromosomes of budding yeast (N ≈108, ϕ <ϕc∞) are equilibrated with no clear signature of such organization.

Engineering tolerance to industrially relevant stress factors in yeast cell factories.

PubMed

Deparis, Quinten; Claes, Arne; Foulquié-Moreno, Maria R; Thevelein, Johan M

2017-06-01

The main focus in development of yeast cell factories has generally been on establishing optimal activity of heterologous pathways and further metabolic engineering of the host strain to maximize product yield and titer. Adequate stress tolerance of the host strain has turned out to be another major challenge for obtaining economically viable performance in industrial production. Although general robustness is a universal requirement for industrial microorganisms, production of novel compounds using artificial metabolic pathways presents additional challenges. Many of the bio-based compounds desirable for production by cell factories are highly toxic to the host cells in the titers required for economic viability. Artificial metabolic pathways also turn out to be much more sensitive to stress factors than endogenous pathways, likely because regulation of the latter has been optimized in evolution in myriads of environmental conditions. We discuss different environmental and metabolic stress factors with high relevance for industrial utilization of yeast cell factories and the experimental approaches used to engineer higher stress tolerance. Improving stress tolerance in a predictable manner in yeast cell factories should facilitate their widespread utilization in the bio-based economy and extend the range of products successfully produced in large scale in a sustainable and economically profitable way. © FEMS 2017.

Engineering tolerance to industrially relevant stress factors in yeast cell factories

PubMed Central

Deparis, Quinten; Claes, Arne; Foulquié-Moreno, Maria R.

2017-01-01

Abstract The main focus in development of yeast cell factories has generally been on establishing optimal activity of heterologous pathways and further metabolic engineering of the host strain to maximize product yield and titer. Adequate stress tolerance of the host strain has turned out to be another major challenge for obtaining economically viable performance in industrial production. Although general robustness is a universal requirement for industrial microorganisms, production of novel compounds using artificial metabolic pathways presents additional challenges. Many of the bio-based compounds desirable for production by cell factories are highly toxic to the host cells in the titers required for economic viability. Artificial metabolic pathways also turn out to be much more sensitive to stress factors than endogenous pathways, likely because regulation of the latter has been optimized in evolution in myriads of environmental conditions. We discuss different environmental and metabolic stress factors with high relevance for industrial utilization of yeast cell factories and the experimental approaches used to engineer higher stress tolerance. Improving stress tolerance in a predictable manner in yeast cell factories should facilitate their widespread utilization in the bio-based economy and extend the range of products successfully produced in large scale in a sustainable and economically profitable way. PMID:28586408

A Cytoplasmic Dynein Heavy Chain Is Required for Oscillatory Nuclear Movement of Meiotic Prophase and Efficient Meiotic Recombination in Fission Yeast

PubMed Central

Yamamoto, Ayumu; West, Robert R.; McIntosh, J. Richard; Hiraoka, Yasushi

1999-01-01

Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination. PMID:10366596

[The yeast biofilm in human medicine].

PubMed

Růzicka, Filip; Holá, Veronika; Votava, Miroslav

2007-08-01

In recent years, the role of Candida yeasts as causative agents of nosocomial infections has increased. One of the important virulence factors contributing to the development of such infections is biofilm production. This virulence factor enables yeast to colonize both native surfaces and artificial implants. The most common sources of infection are patients themselves, in particular the gastrointestinal tract and skin. The vectors of exogenous yeast infections are predominantly the hands of the health personnel and contaminated medical instruments. The adhesion of yeasts to the implant surfaces is determined both by implant surface and yeast characteristics. This is followed by proliferation and production of microcolonies and extracellular matrix. The final biofilm structure is also influenced by the production of hyphae and pseudohyphae. The entire process of biofilm production is controlled by numerous regulatory systems, with the key role being played by the quorum sensing system. Like the adhered bacterial cultures, candidas growing in the form of a biofilm are highly resistant to antimicrobial therapy. Resistance of yeast biofilms to antifungals is a complex process with multiple contributing factors. These are especially increased gene expression (e.g. genes encoding the so called multidrug efflux pumps), limited penetration of substances through the extracellular matrix, inhibited cell growth and altered microenvironment in deeper biofilm layers. The concentrations of antifungals able to effectively affect the biofilm cells exceed, by several orders of magnitude, the values of conventionally determined MICs. High biofilm resistance results in ineffective antifungal therapy of biofilm infections. Therefore, if possible, the colonized implant should be removed. Conservative therapy should involve antifungals with a proven effect on the biofilm (e.g. caspofungin). The most effective measure in fighting biofilm infections is prevention, especially adhering to

Yeast: An Overlooked Component of Bactrocera tryoni (Diptera: Tephritidae) Larval Gut Microbiota.

PubMed

Deutscher, Ania T; Reynolds, Olivia L; Chapman, Toni A

2017-02-01

Yeasts, often in hydrolyzed form, are key ingredients in the larval and adult diets of tephritid fruit fly colonies. However, very little is known about the presence or role of yeasts in the diets of tephritid fruit flies in nature. Previous studies have identified bacteria but not detected yeasts in the gut of Queensland fruit fly, Bactrocera tryoni (Froggatt), one of Australia's most economically damaging insect pests of horticultural crops and of significant biosecurity concern domestically and internationally. Here we demonstrate that cultivable yeasts are commonly found in the gut of B. tryoni larvae from fruit hosts. Analysis of the ITS1, 5.8S rRNA gene, and ITS2 sequences of randomly selected isolates identified yeasts and yeast-like fungi of the genera Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Pichia, and Starmerella. The prevalence of these yeasts in fruits suggests that larvae consume the yeasts as part of their diet. This work highlights that yeasts should be considered in future tephritid larval gut microbiota studies. Understanding tephritid-microbial symbiont interactions will lead to improvements in artificial diets and the quality of mass-reared tephritids for the sterile insect technique. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation

PubMed Central

Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J.; Fox, Catherine A.

2016-01-01

The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

Multiple Pairwise Analysis of Non-homologous Centromere Coupling Reveals Preferential Chromosome Size-Dependent Interactions and a Role for Bouquet Formation in Establishing the Interaction Pattern

PubMed Central

Lefrançois, Philippe; Rockmill, Beth; Xie, Pingxing; Roeder, G. Shirleen; Snyder, Michael

2016-01-01

During meiosis, chromosomes undergo a homology search in order to locate their homolog to form stable pairs and exchange genetic material. Early in prophase, chromosomes associate in mostly non-homologous pairs, tethered only at their centromeres. This phenomenon, conserved through higher eukaryotes, is termed centromere coupling in budding yeast. Both initiation of recombination and the presence of homologs are dispensable for centromere coupling (occurring in spo11 mutants and haploids induced to undergo meiosis) but the presence of the synaptonemal complex (SC) protein Zip1 is required. The nature and mechanism of coupling have yet to be elucidated. Here we present the first pairwise analysis of centromere coupling in an effort to uncover underlying rules that may exist within these non-homologous interactions. We designed a novel chromosome conformation capture (3C)-based assay to detect all possible interactions between non-homologous yeast centromeres during early meiosis. Using this variant of 3C-qPCR, we found a size-dependent interaction pattern, in which chromosomes assort preferentially with chromosomes of similar sizes, in haploid and diploid spo11 cells, but not in a coupling-defective mutant (spo11 zip1 haploid and diploid yeast). This pattern is also observed in wild-type diploids early in meiosis but disappears as meiosis progresses and homologous chromosomes pair. We found no evidence to support the notion that ancestral centromere homology plays a role in pattern establishment in S. cerevisiae post-genome duplication. Moreover, we found a role for the meiotic bouquet in establishing the size dependence of centromere coupling, as abolishing bouquet (using the bouquet-defective spo11 ndj1 mutant) reduces it. Coupling in spo11 ndj1 rather follows telomere clustering preferences. We propose that a chromosome size preference for centromere coupling helps establish efficient homolog recognition. PMID:27768699

Physical limits on kinesin-5–mediated chromosome congression in the smallest mitotic spindles

PubMed Central

McCoy, Kelsey M.; Tubman, Emily S.; Claas, Allison; Tank, Damien; Clancy, Shelly Applen; O’Toole, Eileen T.; Berman, Judith; Odde, David J.

2015-01-01

A characteristic feature of mitotic spindles is the congression of chromosomes near the spindle equator, a process mediated by dynamic kinetochore microtubules. A major challenge is to understand how precise, submicrometer-scale control of kinetochore micro­tubule dynamics is achieved in the smallest mitotic spindles, where the noisiness of microtubule assembly/disassembly will potentially act to overwhelm the spatial information that controls microtubule plus end–tip positioning to mediate congression. To better understand this fundamental limit, we conducted an integrated live fluorescence, electron microscopy, and modeling analysis of the polymorphic fungal pathogen Candida albicans, which contains one of the smallest known mitotic spindles (<1 μm). Previously, ScCin8p (kinesin-5 in Saccharomyces cerevisiae) was shown to mediate chromosome congression by promoting catastrophe of long kinetochore microtubules (kMTs). Using C. albicans yeast and hyphal kinesin-5 (Kip1p) heterozygotes (KIP1/kip1∆), we found that mutant spindles have longer kMTs than wild-type spindles, consistent with a less-organized spindle. By contrast, kinesin-8 heterozygous mutant (KIP3/kip3∆) spindles exhibited the same spindle organization as wild type. Of interest, spindle organization in the yeast and hyphal states was indistinguishable, even though yeast and hyphal cell lengths differ by two- to fivefold, demonstrating that spindle length regulation and chromosome congression are intrinsic to the spindle and largely independent of cell size. Together these results are consistent with a kinesin-5–mediated, length-dependent depolymerase activity that organizes chromosomes at the spindle equator in C. albicans to overcome fundamental noisiness in microtubule self-assembly. More generally, we define a dimensionless number that sets a fundamental physical limit for maintaining congression in small spindles in the face of assembly noise and find that C. albicans operates very close to

Comparison of the chromosome maps around a resistance hot spot on chromosome 5 of potato and tomato using BAC-FISH painting.

PubMed

Achenbach, Ute C; Tang, Xiaomin; Ballvora, Agim; de Jong, Hans; Gebhardt, Christiane

2010-02-01

Potato chromosome 5 harbours numerous genes for important qualitative and quantitative traits, such as resistance to the root cyst nematode Globodera pallida and the late blight fungus, Phytophthora infestans. The genes make up part of a "hot spot" for resistances to various pathogens covering a genetic map length of 3 cM between markers GP21 and GP179. We established the physical size and position of this region on chromosome 5 in potato and tomato using fluorescence in situ hybridization (FISH) on pachytene chromosomes. Five potato bacterial artificial chromosome (BAC) clones with the genetically anchored markers GP21, R1-contig (proximal end), CosA, GP179, and StPto were selected, labeled with different fluorophores, and hybridized in a five-colour FISH experiment. Our results showed the location of the BAC clones in the middle of the long arm of chromosome 5 in both potato and tomato. Based on chromosome measurements, we estimate the physical size of the GP21-GP179 interval at 0.85 Mb and 1.2 Mb in potato and tomato, respectively. The GP21-GP179 interval is part of a genome segment known to have inverted map positions between potato and tomato.

High-resolution FISH on super-stretched flow-sorted plant chromosomes.

PubMed

Valárik, M; Bartos, J; Kovárová, P; Kubaláková, M; de Jong, J H; Dolezel, J

2004-03-01

A novel high-resolution fluorescence in situ hybridisation (FISH) strategy, using super-stretched flow-sorted plant chromosomes as targets, is described. The technique that allows longitudinal extension of chromosomes of more than 100 times their original metaphase size is especially attractive for plant species with large chromosomes, whose pachytene chromosomes are generally too long and heterochromatin patterns too complex for FISH analysis. The protocol involves flow cytometric sorting of metaphase chromosomes, mild proteinase-K digestion of air-dried chromosomes on microscopic slides, followed by stretching with ethanol:acetic acid (3 : 1). Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from barley, wheat, rye and chickpea, hybridised with 45S and 5S ribosomal DNAs and the [GAA]n microsatellite, the [TTTAGGG]n telomeric repeat and a bacterial artificial chromosome (BAC) clone as probes. FISH signals on stretched chromosomes were brighter than those on the untreated control, resulting from better accessibility of the stretched chromatin and maximum observed sensitivity of 1 kbp. Spatial resolution of neighbouring loci was improved down to 70 kbp as compared to 5-10 Mbp after FISH on mitotic chromosomes, revealing details of adjacent DNA sequences hitherto not obtained with any other method. Stretched chromosomes are advantageous over extended DNA fibres from interphase nuclei as targets for FISH studies because they still retain chromosomal integrity. Although the method is confined to species for which chromosome flow sorting has been developed, it provides a unique system for controlling stretching degree of mitotic chromosomes and high-resolution bar-code FISH.

X-chromosome tiling path array detection of copy number variants in patients with chromosome X-linked mental retardation

PubMed Central

Madrigal, I; Rodríguez-Revenga, L; Armengol, L; González, E; Rodriguez, B; Badenas, C; Sánchez, A; Martínez, F; Guitart, M; Fernández, I; Arranz, JA; Tejada, MI; Pérez-Jurado, LA; Estivill, X; Milà, M

2007-01-01

Background Aproximately 5–10% of cases of mental retardation in males are due to copy number variations (CNV) on the X chromosome. Novel technologies, such as array comparative genomic hybridization (aCGH), may help to uncover cryptic rearrangements in X-linked mental retardation (XLMR) patients. We have constructed an X-chromosome tiling path array using bacterial artificial chromosomes (BACs) and validated it using samples with cytogenetically defined copy number changes. We have studied 54 patients with idiopathic mental retardation and 20 controls subjects. Results Known genomic aberrations were reliably detected on the array and eight novel submicroscopic imbalances, likely causative for the mental retardation (MR) phenotype, were detected. Putatively pathogenic rearrangements included three deletions and five duplications (ranging between 82 kb to one Mb), all but two affecting genes previously known to be responsible for XLMR. Additionally, we describe different CNV regions with significant different frequencies in XLMR and control subjects (44% vs. 20%). Conclusion This tiling path array of the human X chromosome has proven successful for the detection and characterization of known rearrangements and novel CNVs in XLMR patients. PMID:18047645

Initiation preference at a yeast origin of replication.

PubMed

Brewer, B J; Fangman, W L

1994-04-12

Replication origins in the yeast Saccharomyces cerevisiae are identified as autonomous replication sequence (ARS) elements. To examine the effect of origin density on replication initiation, we have analyzed the replication of a plasmid that contains two copies of the same origin, ARS1. The activation of origins and the direction that replication forks move through flanking sequences can be physically determined by analyzing replication intermediates on two-dimensional agarose gels. We find that only one of the two identical ARSs on the plasmid initiates replication on any given plasmid molecule; that is, this close spacing of ARSs results in an apparent interference between the potential origins. Moreover, in the particular plasmid that we constructed, one of the two identical copies of ARS1 is used four times more frequently than the other one. These results show that the plasmid context is critical for determining the preferred origin. This origin preference is also exhibited when the tandem copies of ARS1 are introduced into a yeast chromosome. The sequences responsible for establishing the origin preference have been identified by deletion analysis and are found to reside in a portion of the yeast URA3 gene.

Genome Diversity and Evolution in the Budding Yeasts (Saccharomycotina)

PubMed Central

Dujon, Bernard A.; Louis, Edward J.

2017-01-01

Considerable progress in our understanding of yeast genomes and their evolution has been made over the last decade with the sequencing, analysis, and comparisons of numerous species, strains, or isolates of diverse origins. The role played by yeasts in natural environments as well as in artificial manufactures, combined with the importance of some species as model experimental systems sustained this effort. At the same time, their enormous evolutionary diversity (there are yeast species in every subphylum of Dikarya) sparked curiosity but necessitated further efforts to obtain appropriate reference genomes. Today, yeast genomes have been very informative about basic mechanisms of evolution, speciation, hybridization, domestication, as well as about the molecular machineries underlying them. They are also irreplaceable to investigate in detail the complex relationship between genotypes and phenotypes with both theoretical and practical implications. This review examines these questions at two distinct levels offered by the broad evolutionary range of yeasts: inside the best-studied Saccharomyces species complex, and across the entire and diversified subphylum of Saccharomycotina. While obviously revealing evolutionary histories at different scales, data converge to a remarkably coherent picture in which one can estimate the relative importance of intrinsic genome dynamics, including gene birth and loss, vs. horizontal genetic accidents in the making of populations. The facility with which novel yeast genomes can now be studied, combined with the already numerous available reference genomes, offer privileged perspectives to further examine these fundamental biological questions using yeasts both as eukaryotic models and as fungi of practical importance. PMID:28592505

The development and characterisation of a bacterial artificial chromosome library for Fragaria vesca

PubMed Central

Bonet, Julio; Girona, Elena Lopez; Sargent, Daniel J; Muñoz-Torres, Monica C; Monfort, Amparo; Abbott, Albert G; Arús, Pere; Simpson, David W; Davik, Jahn

2009-01-01

Background The cultivated strawberry Fragaria ×ananassa is one of the most economically-important soft-fruit species. Few structural genomic resources have been reported for Fragaria and there exists an urgent need for the development of physical mapping resources for the genus. The first stage in the development of a physical map for Fragaria is the construction and characterisation of a high molecular weight bacterial artificial chromosome (BAC) library. Methods A BAC library, consisting of 18,432 clones was constructed from Fragaria vesca f. semperflorens accession 'Ali Baba'. BAC DNA from individual library clones was pooled to create a PCR-based screening assay for the library, whereby individual clones could be identified with just 34 PCR reactions. These pools were used to screen the BAC library and anchor individual clones to the diploid Fragaria reference map (FV×FN). Findings Clones from the BAC library developed contained an average insert size of 85 kb, representing over seven genome equivalents. The pools and superpools developed were used to identify a set of BAC clones containing 70 molecular markers previously mapped to the diploid Fragaria FV×FN reference map. The number of positive colonies identified for each marker suggests the library represents between 4× and 10× coverage of the diploid Fragaria genome, which is in accordance with the estimate of library coverage based on average insert size. Conclusion This BAC library will be used for the construction of a physical map for F. vesca and the superpools will permit physical anchoring of molecular markers using PCR. PMID:19772672

Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity.

PubMed

Clevenger, Kenneth D; Ye, Rosa; Bok, Jin Woo; Thomas, Paul M; Islam, Md Nurul; Miley, Galen P; Robey, Matthew T; Chen, Cynthia; Yang, KaHoua; Swyers, Michael; Wu, Edward; Gao, Peng; Wu, Chengcang C; Keller, Nancy P; Kelleher, Neil L

2018-03-20

The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-C T and the internal C-domains as BenZ-C 1 and BenZ-C 2 . Unexpectedly, we also uncovered evidence suggesting BenY-C T or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal C T domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities.

Genomics and Biochemistry of Saccharomyces cerevisiae Wine Yeast Strains.

PubMed

Eldarov, M A; Kishkovskaia, S A; Tanaschuk, T N; Mardanov, A V

2016-12-01

Saccharomyces yeasts have been used for millennia for the production of beer, wine, bread, and other fermented products. Long-term "unconscious" selection and domestication led to the selection of hundreds of strains with desired production traits having significant phenotypic and genetic differences from their wild ancestors. This review summarizes the results of recent research in deciphering the genomes of wine Saccharomyces strains, the use of comparative genomics methods to study the mechanisms of yeast genome evolution under conditions of artificial selection, and the use of genomic and postgenomic approaches to identify the molecular nature of the important characteristics of commercial wine strains of Saccharomyces. Succinctly, data concerning metagenomics of microbial communities of grapes and wine and the dynamics of yeast and bacterial flora in the course of winemaking is provided. A separate section is devoted to an overview of the physiological, genetic, and biochemical features of sherry yeast strains used to produce biologically aged wines. The goal of the review is to convince the reader of the efficacy of new genomic and postgenomic technologies as tools for developing strategies for targeted selection and creation of new strains using "classical" and modern techniques for improving winemaking technology.

The molecular karyotype of the megabase chromosomes of Trypanosoma brucei stock 427.

PubMed

Melville, S E; Leech, V; Navarro, M; Cross, G A

2000-12-01

We present the molecular karyotype of the megabase chromosomes of Trypanosoma brucei stock 427, clone 221a. This cloned stock is most commonly used in research laboratories in genetic manipulation experiments and in studies of antigenic variation. Using 116 previously characterised chromosome-specific markers, we identify 11 diploid pairs of megabase chromosomes and detect no loss of synteny in EST and gene marker distribution between this stock and the genome project reference stock TREU 927/4. Nevertheless, the chromosomes of 427 are all larger than their homologues in 927, except chromosomes IIa and IXa. The greatest size variation is seen in chromosome I, the smallest of which is 1.1 Mb (927-Ia) and the largest 3.6 Mb (427-Ib). The total nuclear DNA content of both stocks has been estimated by comparison of the mobility of T. brucei and yeast chromosomes. Trypanosomes of stock 427 contain approximately 16.5 Mb more megabase chromosomal DNA than those of stock 927. We have detected the presence of bloodstream-form expression-site-associated sequences on eight or more megabase chromosomes. These sequences are not found on the same chromosomes in each stock. We have determined the chromosomal band location of nine characterised variant surface glycoprotein genes, including the currently expressed VSG 221. Our results demonstrate both the stability of the T. brucei genome, as illustrated by the conservation of syntenic groups of genes in the two stocks, and the polymorphic nature of the genomic regions involved in antigenic variation. We propose that the chromosomes of stock 427 be numbered to correspond to their homologues in the genome project reference stock TREU 927/4.

Construction of an infectious clone of canine herpesvirus genome as a bacterial artificial chromosome.

PubMed

Arii, Jun; Hushur, Orkash; Kato, Kentaro; Kawaguchi, Yasushi; Tohya, Yukinobu; Akashi, Hiroomi

2006-04-01

Canine herpesvirus (CHV) is an attractive candidate not only for use as a recombinant vaccine to protect dogs from a variety of canine pathogens but also as a viral vector for gene therapy in domestic animals. However, developments in this area have been impeded by the complicated techniques used for eukaryotic homologous recombination. To overcome these problems, we used bacterial artificial chromosomes (BACs) to generate infectious BACs. Our findings may be summarized as follows: (i) the CHV genome (pCHV/BAC), in which a BAC flanked by loxP sites was inserted into the thymidine kinase gene, was maintained in Escherichia coli; (ii) transfection of pCHV/BAC into A-72 cells resulted in the production of infectious virus; (iii) the BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus CHV/BAC by co-infection with CHV/BAC and a recombinant adenovirus that expressed the Cre recombinase; and (iv) a recombinant virus in which the glycoprotein C gene was deleted was generated by lambda recombination followed by Flp recombination, which resulted in a reduction in viral titer compared with that of the wild-type virus. The infectious clone pCHV/BAC is useful for the modification of the CHV genome using bacterial genetics, and CHV/BAC should have multiple applications in the rapid generation of genetically engineered CHV recombinants and the development of CHV vectors for vaccination and gene therapy in domestic animals.

Replication and meiotic transmission of yeast ribosomal RNA genes.

PubMed

Brewer, B J; Zakian, V A; Fangman, W L

1980-11-01

The yeast Saccharomyces cerevisiae has approximately 120 genes for the ribosomal RNAs (rDNA) which are organized in tandem within chromosomal DNA. These multiple-copy genes are homogeneous in sequence but can undergo changes in copy number and topology. To determine if these changes reflect unusual features of rDNA metabolism, we have examined both the replication of rDNA in the mitotic cell cycle and the inheritance of rDNA during meiosis. The results indicate that rDNA behaves identically to chromosomal DNA: each rDNA unit is replicated once during the S phase of each cell cycle and each unit is conserved through meiosis. Therefore, the flexibility in copy number and topology of rDNA does not arise from the selective replication of units in each S phase nor by the selective inheritance of units in meiosis.

Rapid divergence and expansion of the X chromosome in papaya

PubMed Central

Gschwend, Andrea R.; Yu, Qingyi; Tong, Eric J.; Zeng, Fanchang; Han, Jennifer; VanBuren, Robert; Aryal, Rishi; Charlesworth, Deborah; Moore, Paul H.; Paterson, Andrew H.; Ming, Ray

2012-01-01

X chromosomes have long been thought to conserve the structure and gene content of the ancestral autosome from which the sex chromosomes evolved. We compared the recently evolved papaya sex chromosomes with a homologous autosome of a close relative, the monoecious Vasconcellea monoica, to infer changes since recombination stopped between the papaya sex chromosomes. We sequenced 12 V. monoica bacterial artificial chromosomes, 11 corresponding to the papaya X-specific region, and 1 to a papaya autosomal region. The combined V. monoica X-orthologous sequences are much shorter (1.10 Mb) than the corresponding papaya region (2.56 Mb). Given that the V. monoica genome is 41% larger than that of papaya, this finding suggests considerable expansion of the papaya X; expansion is supported by a higher repetitive sequence content of the X compared with the papaya autosomal sequence. The alignable regions include 27 transcript-encoding sequences, only 6 of which are functional X/V. monoica gene pairs. Sequence divergence from the V. monoica orthologs is almost identical for papaya X and Y alleles; the Carica-Vasconcellea split therefore occurred before the papaya sex chromosomes stopped recombining, making V. monoica a suitable outgroup for inferring changes in papaya sex chromosomes. The papaya X and the hermaphrodite-specific region of the Yh chromosome and V. monoica have all gained and lost genes, including a surprising amount of changes in the X. PMID:22869742

Checkpoint independence of most DNA replication origins in fission yeast

PubMed Central

Mickle, Katie L; Ramanathan, Sunita; Rosebrock, Adam; Oliva, Anna; Chaudari, Amna; Yompakdee, Chulee; Scott, Donna; Leatherwood, Janet; Huberman, Joel A

2007-01-01

Background In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2). Results Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in

Replication stress-induced chromosome breakage is correlated with replication fork progression and is preceded by single-stranded DNA formation.

PubMed

Feng, Wenyi; Di Rienzi, Sara C; Raghuraman, M K; Brewer, Bonita J

2011-10-01

Chromosome breakage as a result of replication stress has been hypothesized to be the direct consequence of defective replication fork progression, or "collapsed" replication forks. However, direct and genome-wide evidence that collapsed replication forks give rise to chromosome breakage is still lacking. Previously we showed that a yeast replication checkpoint mutant mec1-1, after transient exposure to replication impediment imposed by hydroxyurea (HU), failed to complete DNA replication, accumulated single-stranded DNA (ssDNA) at the replication forks, and fragmented its chromosomes. In this study, by following replication fork progression genome-wide via ssDNA detection and by direct mapping of chromosome breakage after HU exposure, we have tested the hypothesis that the chromosome breakage in mec1 cells occurs at collapsed replication forks. We demonstrate that sites of chromosome breakage indeed correlate with replication fork locations. Moreover, ssDNA can be detected prior to chromosome breakage, suggesting that ssDNA accumulation is the common precursor to double strand breaks at collapsed replication forks.

Biological Dual-Use Research and Synthetic Biology of Yeast.

PubMed

Cirigliano, Angela; Cenciarelli, Orlando; Malizia, Andrea; Bellecci, Carlo; Gaudio, Pasquale; Lioj, Michele; Rinaldi, Teresa

2017-04-01

In recent years, the publication of the studies on the transmissibility in mammals of the H5N1 influenza virus and synthetic genomes has triggered heated and concerned debate within the community of scientists on biological dual-use research; these papers have raised the awareness that, in some cases, fundamental research could be directed to harmful experiments, with the purpose of developing a weapon that could be used by a bioterrorist. Here is presented an overview regarding the dual-use concept and its related international agreements which underlines the work of the Australia Group (AG) Export Control Regime. It is hoped that the principles and activities of the AG, that focuses on export control of chemical and biological dual-use materials, will spread and become well known to academic researchers in different countries, as they exchange biological materials (i.e. plasmids, strains, antibodies, nucleic acids) and scientific papers. To this extent, and with the aim of drawing the attention of the scientific community that works with yeast to the so called Dual-Use Research of Concern, this article reports case studies on biological dual-use research and discusses a synthetic biology applied to the yeast Saccharomyces cerevisiae, namely the construction of the first eukaryotic synthetic chromosome of yeast and the use of yeast cells as a factory to produce opiates. Since this organism is considered harmless and is not included in any list of biological agents, yeast researchers should take simple actions in the future to avoid the sharing of strains and advanced technology with suspicious individuals.

The price of independence: cell separation in fission yeast.

PubMed

Martín-García, Rebeca; Santos, Beatriz

2016-04-01

The ultimate goal of cell division is to give rise to two viable independent daughter cells. A tight spatial and temporal regulation between chromosome segregation and cytokinesis ensures the viability of the daughter cells. Schizosaccharomyces pombe, commonly known as fission yeast, has become a leading model organism for studying essential and conserved mechanisms of the eukaryotic cell division process. Like many other eukaryotic cells it divides by binary fission and the cleavage furrow undergoes ingression due to the contraction of an actomyosin ring. In contrast to mammalian cells, yeasts as cell-walled organisms, also need to form a division septum made of cell wall material to complete the process of cytokinesis. The division septum is deposited behind the constricting ring and it will constitute the new ends of the daughter cells. Cell separation also involves cell wall degradation and this process should be precisely regulated to avoid cell lysis. In this review, we will give a brief overview of the whole cytokinesis process in fission yeast, from the positioning and assembly of the contractile ring to the final step of cell separation, and the problems generated when these processes are not precise.

Structural and functional partitioning of bread wheat chromosome 3B.

PubMed

Choulet, Frédéric; Alberti, Adriana; Theil, Sébastien; Glover, Natasha; Barbe, Valérie; Daron, Josquin; Pingault, Lise; Sourdille, Pierre; Couloux, Arnaud; Paux, Etienne; Leroy, Philippe; Mangenot, Sophie; Guilhot, Nicolas; Le Gouis, Jacques; Balfourier, Francois; Alaux, Michael; Jamilloux, Véronique; Poulain, Julie; Durand, Céline; Bellec, Arnaud; Gaspin, Christine; Safar, Jan; Dolezel, Jaroslav; Rogers, Jane; Vandepoele, Klaas; Aury, Jean-Marc; Mayer, Klaus; Berges, Hélène; Quesneville, Hadi; Wincker, Patrick; Feuillet, Catherine

2014-07-18

We produced a reference sequence of the 1-gigabase chromosome 3B of hexaploid bread wheat. By sequencing 8452 bacterial artificial chromosomes in pools, we assembled a sequence of 774 megabases carrying 5326 protein-coding genes, 1938 pseudogenes, and 85% of transposable elements. The distribution of structural and functional features along the chromosome revealed partitioning correlated with meiotic recombination. Comparative analyses indicated high wheat-specific inter- and intrachromosomal gene duplication activities that are potential sources of variability for adaption. In addition to providing a better understanding of the organization, function, and evolution of a large and polyploid genome, the availability of a high-quality sequence anchored to genetic maps will accelerate the identification of genes underlying important agronomic traits. Copyright © 2014, American Association for the Advancement of Science.

A selective sweep of >8 Mb on chromosome 26 in the Boxer genome.

PubMed

Quilez, Javier; Short, Andrea D; Martínez, Verónica; Kennedy, Lorna J; Ollier, William; Sanchez, Armand; Altet, Laura; Francino, Olga

2011-07-01

Modern dog breeds display traits that are either breed-specific or shared by a few breeds as a result of genetic bottlenecks during the breed creation process and artificial selection for breed standards. Selective sweeps in the genome result from strong selection and can be detected as a reduction or elimination of polymorphism in a given region of the genome. Extended regions of homozygosity, indicative of selective sweeps, were identified in a genome-wide scan dataset of 25 Boxers from the United Kingdom genotyped at ~20,000 single-nucleotide polymorphisms (SNPs). These regions were further examined in a second dataset of Boxers collected from a different geographical location and genotyped using higher density SNP arrays (~170,000 SNPs). A selective sweep previously associated with canine brachycephaly was detected on chromosome 1. A novel selective sweep of over 8 Mb was observed on chromosome 26 in Boxer and for a shorter region in English and French bulldogs. It was absent in 171 samples from eight other dog breeds and 7 Iberian wolf samples. A region of extended increased heterozygosity on chromosome 9 overlapped with a previously reported copy number variant (CNV) which was polymorphic in multiple dog breeds. A selective sweep of more than 8 Mb on chromosome 26 was identified in the Boxer genome. This sweep is likely caused by strong artificial selection for a trait of interest and could have inadvertently led to undesired health implications for this breed. Furthermore, we provide supporting evidence for two previously described regions: a selective sweep on chromosome 1 associated with canine brachycephaly and a CNV on chromosome 9 polymorphic in multiple dog breeds.

Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance.

PubMed

Garcia Sanchez, Rosa; Solodovnikova, Natalia; Wendland, Jürgen

2012-08-01

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus-like strains. Lager yeasts are particularly adapted to low-temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make-up of lager yeast spore clones, we introduced molecular markers to analyse mating-type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18° Plato at 18-25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. Copyright © 2012 John Wiley & Sons, Ltd.

[Yeast colonization of urinary catheters and the significance of biofilm formation].

PubMed

Růžička, Filip; Holá, Veronika; Mahelová, Martina; Procházková, Alena

2012-08-01

Urinary catheters are colonized by a wide range of microorganisms, including numerous yeasts. The catheters are usually colonized by more microbial species forming a community - multispecies biofilm. Catheter colonization usually does not affect the patient's clinical status in any significant way. On the other hand, the biofilm can become a source of endogenous infection and its presence can affect functionality of the catheter and formation of urinary stones. Material a A total of 721 urinary catheters were studied. Microorganisms were released from catheters by sonication and subsequently cultured. Their identification was performed with the use of common phenotypic tests, as well as using MALDI TOF. Yeasts whose identification was ambiguous were recognized by sequencing. Biofilm formation was assessed by growth in a microtiter plate. Yeast colonization was proved in 244 urinary catheters. However, a total of 274 yeast strains were isolated. Most of them occurred together with other yeast species and/or bacteria on the catheters, producing multispecies biofilm there. The most frequent species was Candida albicans (a total of 144 isolated strains), followed by Candida glabrata (41), Candida tropicalis (41) and Candida parapsilosis sensu stricto (14). Other isolated species were as follows: Candida kefyr (10), Candida krusei (9), Candida fabianii (6), Candida lusitaniae (5), Candida dubliniensis (3) and Saccharomyces cerevisiae (one case). Most of the yeasts rather readily formed a firmly adhering biofilm layer on artificial surfaces.

Purifying Selection Maintains Dosage-Sensitive Genes during Degeneration of the Threespine Stickleback Y Chromosome

PubMed Central

White, Michael A.; Kitano, Jun; Peichel, Catherine L.

2015-01-01

Sex chromosomes are subject to unique evolutionary forces that cause suppression of recombination, leading to sequence degeneration and the formation of heteromorphic chromosome pairs (i.e., XY or ZW). Although progress has been made in characterizing the outcomes of these evolutionary processes on vertebrate sex chromosomes, it is still unclear how recombination suppression and sequence divergence typically occur and how gene dosage imbalances are resolved in the heterogametic sex. The threespine stickleback fish (Gasterosteus aculeatus) is a powerful model system to explore vertebrate sex chromosome evolution, as it possesses an XY sex chromosome pair at relatively early stages of differentiation. Using a combination of whole-genome and transcriptome sequencing, we characterized sequence evolution and gene expression across the sex chromosomes. We uncovered two distinct evolutionary strata that correspond with known structural rearrangements on the Y chromosome. In the oldest stratum, only a handful of genes remain, and these genes are under strong purifying selection. By comparing sex-linked gene expression with expression of autosomal orthologs in an outgroup, we show that dosage compensation has not evolved in threespine sticklebacks through upregulation of the X chromosome in males. Instead, in the oldest stratum, the genes that still possess a Y chromosome allele are enriched for genes predicted to be dosage sensitive in mammals and yeast. Our results suggest that dosage imbalances may have been avoided at haploinsufficient genes by retaining function of the Y chromosome allele through strong purifying selection. PMID:25818858

The genetic architecture of low-temperature adaptation in the wine yeast Saccharomyces cerevisiae.

PubMed

García-Ríos, Estéfani; Morard, Miguel; Parts, Leopold; Liti, Gianni; Guillamón, José M

2017-02-14

Low-temperature growth and fermentation of wine yeast can enhance wine aroma and make them highly desirable traits for the industry. Elucidating response to cold in Saccharomyces cerevisiae is, therefore, of paramount importance to select or genetically improve new wine strains. As most enological traits of industrial importance in yeasts, adaptation to low temperature is a polygenic trait regulated by many interacting loci. In order to unravel the genetic determinants of low-temperature fermentation, we mapped quantitative trait loci (QTLs) by bulk segregant analyses in the F13 offspring of two Saccharomyces cerevisiae industrial strains with divergent performance at low temperature. We detected four genomic regions involved in the adaptation at low temperature, three of them located in the subtelomeric regions (chromosomes XIII, XV and XVI) and one in the chromosome XIV. The QTL analysis revealed that subtelomeric regions play a key role in defining individual variation, which emphasizes the importance of these regions' adaptive nature. The reciprocal hemizygosity analysis (RHA), run to validate the genes involved in low-temperature fermentation, showed that genetic variation in mitochondrial proteins, maintenance of correct asymmetry and distribution of phospholipid in the plasma membrane are key determinants of low-temperature adaptation.

Application of bacterial artificial chromosome array-based comparative genomic hybridization and spectral karyotyping to the analysis of glioblastoma multiforme.

PubMed

Cowell, John K; Matsui, Sei-Ichi; Wang, Yong D; LaDuca, Jeffrey; Conroy, Jeffrey; McQuaid, Devin; Nowak, Norma J

2004-05-01

Identification of genetic losses and gains is valuable in analysis of brain tumors. Locus-by-locus analyses have revealed correlations between prognosis and response to chemotherapy and loss or gain of specific genes and loci. These approaches are labor intensive and do not provide a global view of the genetic changes within the tumor cells. Bacterial artificial chromosome (BAC) arrays, which cover the genome with an average resolution of less than 1 MbP, allow defining the sum total of these genetic changes in a single comparative genomic hybridization (CGH) experiment. These changes are directly overlaid on the human genome sequence, thus providing the extent of the amplification or deletion, reflected by a megabase position, and gene content of the abnormal region. Although this array-based CGH approach (CGHa) seems to detect the extent of the genetic changes in tumors reliably, it has not been robustly tested. We compared genetic changes in four newly derived, early-passage glioma cell lines, using spectral karyotyping (SKY) and CGHa. Chromosome changes seen in cell lines under SKY analysis were also detected with CGHa. In addition, CGHa detected cryptic genetic gains and losses and resolved the nature of subtle marker chromosomes that could not be resolved with SKY, thus providing distinct advantages over previous technologies. There was remarkable general concordance between the CGHa results comparing the cell lines to the original tumor, except that the magnitude of the changes seen in the tumor sample was generally suppressed compared with the cell lines, a consequence of normal cells contaminating the tumor sample. CGHa revealed changes in cell lines that were not present in the original tumors and vice versa, even when analyzed at the earliest passage possible, which highlights the adaptation of the cells to in vitro culture. CGHa proved to be highly accurate and efficient for identifying genetic changes in tumor cells. This approach can accurately

Meiotic Nuclear Oscillations Are Necessary to Avoid Excessive Chromosome Associations.

PubMed

Chacón, Mariola R; Delivani, Petrina; Tolić, Iva M

2016-11-01

Pairing of homologous chromosomes is a crucial step in meiosis, which in fission yeast depends on nuclear oscillations. However, how nuclear oscillations help pairing is unknown. Here, we show that homologous loci typically pair when the spindle pole body is at the cell pole and the nucleus is elongated, whereas they unpair when the spindle pole body is in the cell center and the nucleus is round. Inhibition of oscillations demonstrated that movement is required for initial pairing and that prolonged association of loci leads to mis-segregation. The double-strand break marker Rec25 accumulates in elongated nuclei, indicating that prolonged chromosome stretching triggers recombinatory pathways leading to mis-segregation. Mis-segregation is rescued by overexpression of the Holliday junction resolvase Mus81, suggesting that prolonged pairing results in irresolvable recombination intermediates. We conclude that nuclear oscillations exhibit a dual role, promoting initial pairing and restricting the time of chromosome associations to ensure proper segregation. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Genome rearrangements and pervasive meiotic drive cause hybrid infertility in fission yeast

PubMed Central

Zanders, Sarah E; Eickbush, Michael T; Yu, Jonathan S; Kang, Ji-Won; Fowler, Kyle R; Smith, Gerald R; Malik, Harmit Singh

2014-01-01

Hybrid sterility is one of the earliest postzygotic isolating mechanisms to evolve between two recently diverged species. Here we identify causes underlying hybrid infertility of two recently diverged fission yeast species Schizosaccharomyces pombe and S. kambucha, which mate to form viable hybrid diploids that efficiently complete meiosis, but generate few viable gametes. We find that chromosomal rearrangements and related recombination defects are major but not sole causes of hybrid infertility. At least three distinct meiotic drive alleles, one on each S. kambucha chromosome, independently contribute to hybrid infertility by causing nonrandom spore death. Two of these driving loci are linked by a chromosomal translocation and thus constitute a novel type of paired meiotic drive complex. Our study reveals how quickly multiple barriers to fertility can arise. In addition, it provides further support for models in which genetic conflicts, such as those caused by meiotic drive alleles, can drive speciation. DOI: http://dx.doi.org/10.7554/eLife.02630.001 PMID:24963140

Genome rearrangements and pervasive meiotic drive cause hybrid infertility in fission yeast.

PubMed

Zanders, Sarah E; Eickbush, Michael T; Yu, Jonathan S; Kang, Ji-Won; Fowler, Kyle R; Smith, Gerald R; Malik, Harmit Singh

2014-06-24

Hybrid sterility is one of the earliest postzygotic isolating mechanisms to evolve between two recently diverged species. Here we identify causes underlying hybrid infertility of two recently diverged fission yeast species Schizosaccharomyces pombe and S. kambucha, which mate to form viable hybrid diploids that efficiently complete meiosis, but generate few viable gametes. We find that chromosomal rearrangements and related recombination defects are major but not sole causes of hybrid infertility. At least three distinct meiotic drive alleles, one on each S. kambucha chromosome, independently contribute to hybrid infertility by causing nonrandom spore death. Two of these driving loci are linked by a chromosomal translocation and thus constitute a novel type of paired meiotic drive complex. Our study reveals how quickly multiple barriers to fertility can arise. In addition, it provides further support for models in which genetic conflicts, such as those caused by meiotic drive alleles, can drive speciation.DOI: http://dx.doi.org/10.7554/eLife.02630.001. Copyright © 2014, Zanders et al.

Measuring mitotic spindle dynamics in budding yeast

NASA Astrophysics Data System (ADS)

Plumb, Kemp

In order to carry out its life cycle and produce viable progeny through cell division, a cell must successfully coordinate and execute a number of complex processes with high fidelity, in an environment dominated by thermal noise. One important example of such a process is the assembly and positioning of the mitotic spindle prior to chromosome segregation. The mitotic spindle is a modular structure composed of two spindle pole bodies, separated in space and spanned by filamentous proteins called microtubules, along which the genetic material of the cell is held. The spindle is responsible for alignment and subsequent segregation of chromosomes into two equal parts; proper spindle positioning and timing ensure that genetic material is appropriately divided amongst mother and daughter cells. In this thesis, I describe fluorescence confocal microscopy and automated image analysis algorithms, which I have used to observe and analyze the real space dynamics of the mitotic spindle in budding yeast. The software can locate structures in three spatial dimensions and track their movement in time. By selecting fluorescent proteins which specifically label the spindle poles and cell periphery, mitotic spindle dynamics have been measured in a coordinate system relevant to the cell division. I describe how I have characterised the accuracy and precision of the algorithms by simulating fluorescence data for both spindle poles and the budding yeast cell surface. In this thesis I also describe the construction of a microfluidic apparatus that allows for the measurement of long time-scale dynamics of individual cells and the development of a cell population. The tools developed in this thesis work will facilitate in-depth quantitative analysis of the non-equilibrium processes in living cells.

Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector.

PubMed

Do, Ltk; Wittayarat, M; Terazono, T; Sato, Y; Taniguchi, M; Tanihara, F; Takemoto, T; Kazuki, Y; Kazuki, K; Oshimura, M; Otoi, T

2016-12-01

The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 μs. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC-transchromosomic embryos, when the duration of fusion was prolonged to 60 μs. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells. © 2016 Blackwell Verlag GmbH.

Size and Content of the Sex-Determining Region of the Y Chromosome in Dioecious Mercurialis annua, a Plant with Homomorphic Sex Chromosomes.

PubMed

Veltsos, Paris; Cossard, Guillaume; Beaudoing, Emmanuel; Beydon, Genséric; Savova Bianchi, Dessislava; Roux, Camille; C González-Martínez, Santiago; R Pannell, John

2018-05-29

Dioecious plants vary in whether their sex chromosomes are heteromorphic or homomorphic, but even homomorphic sex chromosomes may show divergence between homologues in the non-recombining, sex-determining region (SDR). Very little is known about the SDR of these species, which might represent particularly early stages of sex-chromosome evolution. Here, we assess the size and content of the SDR of the diploid dioecious herb Mercurialis annua , a species with homomorphic sex chromosomes and mild Y-chromosome degeneration. We used RNA sequencing (RNAseq) to identify new Y-linked markers for M. annua. Twelve of 24 transcripts showing male-specific expression in a previous experiment could be amplified by polymerase chain reaction (PCR) only from males, and are thus likely to be Y-linked. Analysis of genome-capture data from multiple populations of M. annua pointed to an additional six male-limited (and thus Y-linked) sequences. We used these markers to identify and sequence 17 sex-linked bacterial artificial chromosomes (BACs), which form 11 groups of non-overlapping sequences, covering a total sequence length of about 1.5 Mb. Content analysis of this region suggests that it is enriched for repeats, has low gene density, and contains few candidate sex-determining genes. The BACs map to a subset of the sex-linked region of the genetic map, which we estimate to be at least 14.5 Mb. This is substantially larger than estimates for other dioecious plants with homomorphic sex chromosomes, both in absolute terms and relative to their genome sizes. Our data provide a rare, high-resolution view of the homomorphic Y chromosome of a dioecious plant.

Chromosomal microarray analysis as the first-tier test for the identification of pathogenic copy number variants in chromosome 9 pericentric regions and its challenge.

PubMed

Wang, Jia-Chi; Boyar, Fatih Z

2016-01-01

Chromosomal microarray analysis (CMA) has been recommended and practiced routinely in the large reference laboratories of U.S.A. as the first-tier test for the postnatal evaluation of individuals with intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies. Using CMA as a diagnostic tool and without a routine setting of fluorescence in situ hybridization with labeled bacterial artificial chromosome probes (BAC-FISH) in the large reference laboratories becomes a challenge in the characterization of chromosome 9 pericentric region. This region has a very complex genomic structure and contains a variety of heterochromatic and euchromatic polymorphic variants. These variants were usually studied by G-banding, C-banding and BAC-FISH analysis. Chromosomal microarray analysis (CMA) was not recommended since it may lead to false positive results. Here, we presented a cohort of four cases, in which high-resolution CMA was used as the first-tier test or simultaneously with G-banding analysis on the proband to identify pathogenic copy number variants (CNVs) in the whole genome. CMA revealed large pathogenic CNVs from chromosome 9 in 3 cases which also revealed different G-banding patterns between the two chromosome 9 homologues. Although we demonstrated that high-resolution CMA played an important role in the identification of pathogenic copy number variants in chromosome 9 pericentric regions, the lack of BAC-FISH analysis or other useful tools renders significant challenges in the characterization of chromosome 9 pericentric regions. None; it is not a clinical trial, and the cases were retrospectively collected and analyzed.

Spindle checkpoint–independent inhibition of mitotic chromosome segregation by Drosophila Mps1

PubMed Central

Althoff, Friederike; Karess, Roger E.; Lehner, Christian F.

2012-01-01

Monopolar spindle 1 (Mps1) is essential for the spindle assembly checkpoint (SAC), which prevents anaphase onset in the presence of misaligned chromosomes. Moreover, Mps1 kinase contributes in a SAC-independent manner to the correction of erroneous initial attachments of chromosomes to the spindle. Our characterization of the Drosophila homologue reveals yet another SAC-independent role. As in yeast, modest overexpression of Drosophila Mps1 is sufficient to delay progression through mitosis during metaphase, even though chromosome congression and metaphase alignment do not appear to be affected. This delay in metaphase depends on the SAC component Mad2. Although Mps1 overexpression in mad2 mutants no longer causes a metaphase delay, it perturbs anaphase. Sister kinetochores barely move apart toward spindle poles. However, kinetochore movements can be restored experimentally by separase-independent resolution of sister chromatid cohesion. We propose therefore that Mps1 inhibits sister chromatid separation in a SAC-independent manner. Moreover, we report unexpected results concerning the requirement of Mps1 dimerization and kinase activity for its kinetochore localization in Drosophila. These findings further expand Mps1's significance for faithful mitotic chromosome segregation and emphasize the importance of its careful regulation. PMID:22553353

Spindle checkpoint-independent inhibition of mitotic chromosome segregation by Drosophila Mps1.

PubMed

Althoff, Friederike; Karess, Roger E; Lehner, Christian F

2012-06-01

Monopolar spindle 1 (Mps1) is essential for the spindle assembly checkpoint (SAC), which prevents anaphase onset in the presence of misaligned chromosomes. Moreover, Mps1 kinase contributes in a SAC-independent manner to the correction of erroneous initial attachments of chromosomes to the spindle. Our characterization of the Drosophila homologue reveals yet another SAC-independent role. As in yeast, modest overexpression of Drosophila Mps1 is sufficient to delay progression through mitosis during metaphase, even though chromosome congression and metaphase alignment do not appear to be affected. This delay in metaphase depends on the SAC component Mad2. Although Mps1 overexpression in mad2 mutants no longer causes a metaphase delay, it perturbs anaphase. Sister kinetochores barely move apart toward spindle poles. However, kinetochore movements can be restored experimentally by separase-independent resolution of sister chromatid cohesion. We propose therefore that Mps1 inhibits sister chromatid separation in a SAC-independent manner. Moreover, we report unexpected results concerning the requirement of Mps1 dimerization and kinase activity for its kinetochore localization in Drosophila. These findings further expand Mps1's significance for faithful mitotic chromosome segregation and emphasize the importance of its careful regulation.

Chromatin Folding, Fragile Sites, and Chromosome Aberrations Induced by Low- and High- LET Radiation

NASA Technical Reports Server (NTRS)

Zhang, Ye; Cox, Bradley; Asaithamby, Aroumougame; Chen, David J.; Wu, Honglu

2013-01-01

We previously demonstrated non-random distributions of breaks involved in chromosome aberrations induced by low- and high-LET radiation. To investigate the factors contributing to the break point distribution in radiation-induced chromosome aberrations, human epithelial cells were fixed in G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome in separate colors. After the images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multimega base pair scale. Specific locations of the chromosome, in interphase, were also analyzed with bacterial artificial chromosome (BAC) probes. Both mBAND and BAC studies revealed non-random folding of chromatin in interphase, and suggested association of interphase chromatin folding to the radiation-induced chromosome aberration hotspots. We further investigated the distribution of genes, as well as the distribution of breaks found in tumor cells. Comparisons of these distributions to the radiation hotspots showed that some of the radiation hotspots coincide with the frequent breaks found in solid tumors and with the fragile sites for other environmental toxins. Our results suggest that multiple factors, including the chromatin structure and the gene distribution, can contribute to radiation-induced chromosome aberrations.

Whole-Genome Sequencing of Sake Yeast Saccharomyces cerevisiae Kyokai no. 7

PubMed Central

Akao, Takeshi; Yashiro, Isao; Hosoyama, Akira; Kitagaki, Hiroshi; Horikawa, Hiroshi; Watanabe, Daisuke; Akada, Rinji; Ando, Yoshinori; Harashima, Satoshi; Inoue, Toyohisa; Inoue, Yoshiharu; Kajiwara, Susumu; Kitamoto, Katsuhiko; Kitamoto, Noriyuki; Kobayashi, Osamu; Kuhara, Satoru; Masubuchi, Takashi; Mizoguchi, Haruhiko; Nakao, Yoshihiro; Nakazato, Atsumi; Namise, Masahiro; Oba, Takahiro; Ogata, Tomoo; Ohta, Akinori; Sato, Masahide; Shibasaki, Seiji; Takatsume, Yoshifumi; Tanimoto, Shota; Tsuboi, Hirokazu; Nishimura, Akira; Yoda, Koji; Ishikawa, Takeaki; Iwashita, Kazuhiro; Fujita, Nobuyuki; Shimoi, Hitoshi

2011-01-01

The term ‘sake yeast’ is generally used to indicate the Saccharomyces cerevisiae strains that possess characteristics distinct from others including the laboratory strain S288C and are well suited for sake brewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sake yeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeated losses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the two strains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNA-mediated recombination of Ty elements. The present study advances our understanding of the functional and evolutionary genomics of the sake yeast. PMID:21900213

Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast

PubMed Central

Gergely, Zachary R.; Crapo, Ammon; Hough, Loren E.; McIntosh, J. Richard; Betterton, Meredith D.

2016-01-01

Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8–deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8–induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen. PMID:27146110

Regional centromeres in the yeast Candida lusitaniae lack pericentromeric heterochromatin

PubMed Central

Kapoor, Shivali; Zhu, Lisha; Froyd, Cara; Liu, Tao; Rusche, Laura N.

2015-01-01

Point centromeres are specified by a short consensus sequence that seeds kinetochore formation, whereas regional centromeres lack a conserved sequence and instead are epigenetically inherited. Regional centromeres are generally flanked by heterochromatin that ensures high levels of cohesin and promotes faithful chromosome segregation. However, it is not known whether regional centromeres require pericentromeric heterochromatin. In the yeast Candida lusitaniae, we identified a distinct type of regional centromere that lacks pericentromeric heterochromatin. Centromere locations were determined by ChIP-sequencing of two key centromere proteins, Cse4 and Mif2, and are consistent with bioinformatic predictions. The centromeric DNA sequence was unique for each chromosome and spanned 4–4.5 kbp, consistent with regional epigenetically inherited centromeres. However, unlike other regional centromeres, there was no evidence of pericentromeric heterochromatin in C. lusitaniae. In particular, flanking genes were expressed at a similar level to the rest of the genome, and a URA3 reporter inserted adjacent to a centromere was not repressed. In addition, regions flanking the centromeric core were not associated with hypoacetylated histones or a sirtuin deacetylase that generates heterochromatin in other yeast. Interestingly, the centromeric chromatin had a distinct pattern of histone modifications, being enriched for methylated H3K79 and H3R2 but lacking methylation of H3K4, which is found at other regional centromeres. Thus, not all regional centromeres require flanking heterochromatin. PMID:26371315

Rapid depletion of budding yeast proteins by fusion to a heat-inducible degron.

PubMed

Sanchez-Diaz, Alberto; Kanemaki, Masato; Marchesi, Vanessa; Labib, Karim

2004-03-02

One effective way to study the biological function of a protein in vivo is to inactivate it and see what happens to the cell. For proteins that are dispensable for cell viability, the corresponding gene can simply be deleted from its chromosomal locus. The study of essential proteins is more challenging, however, because the function of the protein must be inactivated conditionally. Here, we describe a method that allows the target protein to be depleted rapidly and conditionally, so that the immediate effects on the cell can be examined. The chromosomal locus of a budding yeast gene is modified so that a "heat-inducible degron cassette" is added to the N terminus of the encoded protein, causing it to be degraded by a specific ubiquitin-mediated pathway when cells are shifted from 24 degrees to 37 degrees C. Degradation requires recognition of the degron cassette by the evolutionarily conserved Ubr1 protein, which is associated with a ubiquitin-conjugating enzyme. To promote rapid and conditional depletion of the target protein, we use a yeast strain in which expression of the UBR1 gene can be either repressed or strongly induced. Degron strains are constructed by a simple "one-step" approach using the polymerase chain reaction.

Chromosome Doubling of Microspore-Derived Plants from Cabbage (Brassica oleracea var. capitata L.) and Broccoli (Brassica oleracea var. italica L.)

PubMed Central

Yuan, Suxia; Su, Yanbin; Liu, Yumei; Li, Zhansheng; Fang, Zhiyuan; Yang, Limei; Zhuang, Mu; Zhang, Yangyong; Lv, Honghao; Sun, Peitian

2015-01-01

Chromosome doubling of microspore-derived plants is an important factor in the practical application of microspore culture technology because breeding programs require a large number of genetically stable, homozygous doubled haploid plants with a high level of fertility. In the present paper, 29 populations of microspore-derived plantlets from cabbage (Brassica oleracea var. capitata) and broccoli (Brassica oleracea var. italica) were used to study the ploidy level and spontaneous chromosome doubling of these populations, the artificial chromosome doubling induced by colchicine, and the influence of tissue culture duration on the chromosomal ploidy of the microspore-derived regenerants. Spontaneous chromosome doubling occurred randomly and was genotype dependent. In the plant populations derived from microspores, there were haploids, diploids, and even a low frequency of polyploids and mixed-ploidy plantlets. The total spontaneous doubling in the 14 cabbage populations ranged from 0 to 76.9%, compared with 52.2 to 100% in the 15 broccoli populations. To improve the rate of chromosome doubling, an efficient and reliable artificial chromosome doubling protocol (i.e., the immersion of haploid plantlet roots in a colchicine solution) was developed for cabbage and broccoli microspore-derived haploids. The optimal chromosome doubling of the haploids was obtained with a solution of 0.2% colchicine for 9–12 h or 0.4% colchicine for 3–9 h for cabbage and 0.05% colchicine for 6–12 h for broccoli. This protocol produced chromosome doubling in over 50% of the haploid genotypes for most of the populations derived from cabbage and broccoli. Notably, after 1 or more years in tissue culture, the chromosomes of the haploids were doubled, and most of the haploids turned into doubled haploid or mixed-ploidy plants. This is the first report indicating that tissue culture duration can change the chromosomal ploidy of microspore-derived regenerants. PMID:26734028

Chromosome Doubling of Microspore-Derived Plants from Cabbage (Brassica oleracea var. capitata L.) and Broccoli (Brassica oleracea var. italica L.).

PubMed

Yuan, Suxia; Su, Yanbin; Liu, Yumei; Li, Zhansheng; Fang, Zhiyuan; Yang, Limei; Zhuang, Mu; Zhang, Yangyong; Lv, Honghao; Sun, Peitian

2015-01-01

Chromosome doubling of microspore-derived plants is an important factor in the practical application of microspore culture technology because breeding programs require a large number of genetically stable, homozygous doubled haploid plants with a high level of fertility. In the present paper, 29 populations of microspore-derived plantlets from cabbage (Brassica oleracea var. capitata) and broccoli (Brassica oleracea var. italica) were used to study the ploidy level and spontaneous chromosome doubling of these populations, the artificial chromosome doubling induced by colchicine, and the influence of tissue culture duration on the chromosomal ploidy of the microspore-derived regenerants. Spontaneous chromosome doubling occurred randomly and was genotype dependent. In the plant populations derived from microspores, there were haploids, diploids, and even a low frequency of polyploids and mixed-ploidy plantlets. The total spontaneous doubling in the 14 cabbage populations ranged from 0 to 76.9%, compared with 52.2 to 100% in the 15 broccoli populations. To improve the rate of chromosome doubling, an efficient and reliable artificial chromosome doubling protocol (i.e., the immersion of haploid plantlet roots in a colchicine solution) was developed for cabbage and broccoli microspore-derived haploids. The optimal chromosome doubling of the haploids was obtained with a solution of 0.2% colchicine for 9-12 h or 0.4% colchicine for 3-9 h for cabbage and 0.05% colchicine for 6-12 h for broccoli. This protocol produced chromosome doubling in over 50% of the haploid genotypes for most of the populations derived from cabbage and broccoli. Notably, after 1 or more years in tissue culture, the chromosomes of the haploids were doubled, and most of the haploids turned into doubled haploid or mixed-ploidy plants. This is the first report indicating that tissue culture duration can change the chromosomal ploidy of microspore-derived regenerants.

The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsukamoto, Yuta; Katayama, Chisako; Shinohara, Miki

Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our datamore » strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions.« less

[Identification of C(2)M interacting proteins by yeast two-hybrid screening].

PubMed

Yue, Shan-shan; Xia, Lai-xin

2015-11-01

The synaptonemal complex (SC) is a huge structure which assembles between the homologous chromosomes during meiotic prophase I. Drosophila germ cell-specific nucleoprotein C(2)M clustering at chromosomes can induce SC formation. To further study the molecular function and mechanism of C(2)M in meiosis, we constructed a bait vector for C(2)M and used the yeast two-hybrid system to identify C(2)M interacting proteins. Forty interacting proteins were obtained, including many DNA and histone binding proteins, ATP synthases and transcription factors. Gene silencing assays in Drosophila showed that two genes, wech and Psf1, may delay the disappearance of SC. These results indicate that Wech and Psf1 may form a complex with C(2)M to participate in the formation or stabilization of the SC complex.

GTSE1 tunes microtubule stability for chromosome alignment and segregation by inhibiting the microtubule depolymerase MCAK

PubMed Central

Bendre, Shweta; Hall, Conrad; Lin, Yu-Chih

2016-01-01

The dynamic regulation of microtubules (MTs) during mitosis is critical for accurate chromosome segregation and genome stability. Cancer cell lines with hyperstabilized kinetochore MTs have increased segregation errors and elevated chromosomal instability (CIN), but the genetic defects responsible remain largely unknown. The MT depolymerase MCAK (mitotic centromere-associated kinesin) can influence CIN through its impact on MT stability, but how its potent activity is controlled in cells remains unclear. In this study, we show that GTSE1, a protein found overexpressed in aneuploid cancer cell lines and tumors, regulates MT stability during mitosis by inhibiting MCAK MT depolymerase activity. Cells lacking GTSE1 have defects in chromosome alignment and spindle positioning as a result of MT instability caused by excess MCAK activity. Reducing GTSE1 levels in CIN cancer cell lines reduces chromosome missegregation defects, whereas artificially inducing GTSE1 levels in chromosomally stable cells elevates chromosome missegregation and CIN. Thus, GTSE1 inhibition of MCAK activity regulates the balance of MT stability that determines the fidelity of chromosome alignment, segregation, and chromosomal stability. PMID:27881713

Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity

PubMed Central

Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A.; Bradford, William D.; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S.; Li, Rong

2015-01-01

Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein−based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. PMID:25823586

Testing chromosomal phylogenies and inversion breakpoint reuse in Drosophila. The martensis cluster revisited.

PubMed

Prada, Carlos F; Delprat, Alejandra; Ruiz, Alfredo

2011-02-01

The chromosomal relationships of the four martensis cluster species are among the most complex and intricate within the entire Drosophila repleta group, due to the so-called sharing of inversions. Here, we have revised these relationships using comparative mapping of bacterial artificial chromosome (BAC) clones on the salivary gland chromosomes. A physical map of chromosome 2 of Drosophila uniseta (one of the cluster members) was generated by in situ hybridization of 82 BAC clones from the physical map of the Drosophila buzzatii genome (an outgroup that represents the ancestral arrangement). By comparing the marker positions, we determined the number, order, and orientation of conserved chromosomal segments between chromosome 2 of D. buzzatii and D. uniseta. GRIMM software was used to infer that a minimum of five chromosomal inversions are necessary to transform the chromosome 2 of D. buzzatii into that of D. uniseta. Two of these inversions have been overlooked in previous cytological analyses. The five fixed inversions entail two breakpoint reuses because only nine syntenic segments and eight interruptions were observed. We tested for the presence of the five inversions fixed in D. uniseta in the other three species of the martensis cluster by in situ hybridization of eight breakpoint-bearing BAC clones. The results shed light on the chromosomal phylogeny of the martensis cluster, yet leave a number of questions open.

Plant-Derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae.

PubMed

Naseri, Gita; Balazadeh, Salma; Machens, Fabian; Kamranfar, Iman; Messerschmidt, Katrin; Mueller-Roeber, Bernd

2017-09-15

Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harboring cognate cis-regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver/reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC-EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF-DNA binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.

HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe.

PubMed

Benko, Zsigmond; Elder, Robert T; Li, Ge; Liang, Dong; Zhao, Richard Y

2016-01-01

HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability. A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.

Evaluation of an automated karyotyping system for chromosome aberration analysis

NASA Technical Reports Server (NTRS)

Prichard, Howard M.

1987-01-01

Chromosome aberration analysis is a promising complement to conventional radiation dosimetry, particularly in the complex radiation fields encountered in the space environment. The capabilities of a recently developed automated karyotyping system were evaluated both to determine current capabilities and limitations and to suggest areas where future development should be emphasized. Cells exposed to radiometric chemicals and to photon and particulate radiation were evaluated by manual inspection and by automated karyotyping. It was demonstrated that the evaluated programs were appropriate for image digitization, storage, and transmission. However, automated and semi-automated scoring techniques must be advanced significantly if in-flight chromosome aberration analysis is to be practical. A degree of artificial intelligence may be necessary to realize this goal.

Organization of Synthetic Alphoid DNA Array in Human Artificial Chromosome (HAC) with a Conditional Centromere

PubMed Central

Kouprina, Natalay; Samoshkin, Alexander; Erliandri, Indri; Nakano, Megumi; Lee, Hee-Sheung; Fu, Haiging; Iida, Yuichi; Aladjem, Mirit; Oshimura, Mitsuo; Masumoto, Hiroshi; Earnshaw, William C.; Larionov, Vladimir

2012-01-01

Human artificial chromosomes (HACs) represent a novel promising episomal system for functional genomics, gene therapy and synthetic biology. HACs are engineered from natural and synthetic alphoid DNA arrays upon transfection into human cells. The use of HACs for gene expression studies requires the knowledge of their structural organization. However, none of de novo HACs constructed so far has been physically mapped in detail. Recently we constructed a synthetic alphoidtetO-HAC that was successfully used for expression of full-length genes to correct genetic deficiencies in human cells. The HAC can be easily eliminated from cell populations by inactivation of its conditional kinetochore. This unique feature provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This work describes organization of a megabase-size synthetic alphoid DNA array in the alphoidtetO-HAC that has been formed from a ~50 kb synthetic alphoidtetO-construct. Our analysis showed that this array represents a 1.1 Mb continuous sequence assembled from multiple copies of input DNA, a significant part of which was rearranged before assembling. The tandem and inverted alphoid DNA repeats in the HAC range in size from 25 to 150 kb. In addition, we demonstrated that the structure and functional domains of the HAC remains unchanged after several rounds of its transfer into different host cells. The knowledge of the alphoidtetO-HAC structure provides a tool to control HAC integrity during different manipulations. Our results also shed light on a mechanism for de novo HAC formation in human cells. PMID:23411994

Preimplantation diagnosis of repeated miscarriage due to chromosomal translocations using metaphase chromosomes of a blastomere biopsied from 4- to 6-cell-stage embryos.

PubMed

Tanaka, Atsushi; Nagayoshi, Motoi; Awata, Shoichiro; Mawatari, Yoshifumi; Tanaka, Izumi; Kusunoki, Hiroshi

2004-01-01

To evaluate the safety and accuracy of karyotyping the blastomere chromosomes at metaphase in the natural cell cycle for preimplantation diagnosis. A pilot study. A private infertility clinic and a university laboratory. Eleven patients undergoing IVF and preimplantation diagnosis. Intact human embryos at the 4- to 6-cell stage and human-mouse heterokaryons were cultured and checked hourly for disappearance of the nuclear envelope. After it disappeared, the metaphase chromosomes were analyzed by fluorescence in situ hybridization. Percentage of analyzable metaphase plates and safety and accuracy of the method. The success rate of electrofusion to form human-mouse heterokaryons was 87.1% (27/31), and analyzable chromosomes were obtained from 77.4% (24/31) of the heterokaryons. On the other hand, disappearance of the nuclear envelope occurred in 89.5% (17/19) of the human embryos and it began earlier than that in the heterokaryons. Analyzable chromosomes were obtained and their translocation sites were identified in all blastomeres biopsied from the 17 embryos. After the biopsy, 67.0% of the embryos could develop to the blastocyst stage. The natural cell cycle method reported herein requires frequent observation, but it is safe, with no artificial effects on the chromosomes and without loss of or damage to blastomeres, which occurred with the electrofusion method. Using the natural cell cycle method, we could perform preimplantation diagnosis with nearly 100% accuracy.

Multifunctional centromere binding factor 1 is essential for chromosome segregation in the human pathogenic yeast Candida glabrata.

PubMed

Stoyan, T; Gloeckner, G; Diekmann, S; Carbon, J

2001-08-01

The CBF1 (centromere binding factor 1) gene of Candida glabrata was cloned by functional complementation of the methionine biosynthesis defect of a Saccharomyces cerevisiae cbf1 deletion mutant. The C. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins. CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 x 10(9) M(-1)). Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S. cerevisiae. We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay. Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function. Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates. Deletion of the CBF1 gene proved to be lethal in C. glabrata. C. glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline. DAPI (4',6'-diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G(2)/M transition of the cell cycle. Thus, we conclude that Cbf1p is required for chromosome segregation in C. glabrata.

Relationships between chromosome structure and chromosomal aberrations

NASA Astrophysics Data System (ADS)

Eidelman, Yuri; Andreev, Sergey

An interphase nucleus of human lymphocyte was simulated by the novel Monte Carlo tech-nique. The main features of interphase chromosome structure and packaging were taken into account: different levels of chromatin organisation; nonrandom localisation of chromosomes within a nucleus; chromosome loci dynamics. All chromosomes in a nucleus were modelled as polymer globules. A dynamic pattern of intra/interchromosomal contacts was simulated. The detailed information about chromosomal contacts, such as distribution of intrachromoso-mal contacts over the length of each chromosome and dependence of contact probability on genomic separation between chromosome loci, were calculated and compared to the new exper-imental data obtained by the Hi-C technique. Types and frequencies of simple and complex radiation-induced chromosomal exchange aberrations (CA) induced by X-rays were predicted with taking formation and decay of chromosomal contacts into account. Distance dependence of exchange formation probability was calculated directly. mFISH data for human lymphocytes were analysed. The calculated frequencies of simple CA agreed with the experimental data. Complex CA were underestimated despite the dense packaging of chromosome territories within a nucleus. Possible influence of chromosome-nucleus structural organisation on the frequency and spectrum of radiation-induced chromosome aberrations is discussed.

Replication-dependent and independent mechanisms for the chromosome-coupled persistence of a selfish genome

PubMed Central

Liu, Yen-Ting; Chang, Keng-Ming; Ma, Chien-Hui; Jayaram, Makkuni

2016-01-01

The yeast 2-micron plasmid epitomizes the evolutionary optimization of selfish extra-chromosomal genomes for stable persistence without jeopardizing their hosts’ fitness. Analyses of fluorescence-tagged single-copy reporter plasmids and/or the plasmid partitioning proteins in native and non-native hosts reveal chromosome-hitchhiking as the likely means for plasmid segregation. The contribution of the partitioning system to equal segregation is bipartite- replication-independent and replication-dependent. The former nearly eliminates ‘mother bias’ (preferential plasmid retention in the mother cell) according to binomial distribution, thus limiting equal segregation of a plasmid pair to 50%. The latter enhances equal segregation of plasmid sisters beyond this level, elevating the plasmid close to chromosome status. Host factors involved in plasmid partitioning can be functionally separated by their participation in the replication-independent and/or replication-dependent steps. In the hitchhiking model, random tethering of a pair of plasmids to chromosomes signifies the replication-independent component of segregation; the symmetric tethering of plasmid sisters to sister chromatids embodies the replication-dependent component. The 2-micron circle broadly resembles the episomes of certain mammalian viruses in its chromosome-associated propagation. This unifying feature among otherwise widely differing selfish genomes suggests their evolutionary convergence to the common logic of exploiting, albeit via distinct molecular mechanisms, host chromosome segregation machineries for self-preservation. PMID:27492289

Dominant Epistasis Between Two Quantitative Trait Loci Governing Sporulation Efficiency in Yeast Saccharomyces cerevisiae

PubMed Central

Bergman, Juraj; Mitrikeski, Petar T.

2015-01-01

Summary Sporulation efficiency in the yeast Saccharomyces cerevisiae is a well-established model for studying quantitative traits. A variety of genes and nucleotides causing different sporulation efficiencies in laboratory, as well as in wild strains, has already been extensively characterised (mainly by reciprocal hemizygosity analysis and nucleotide exchange methods). We applied a different strategy in order to analyze the variation in sporulation efficiency of laboratory yeast strains. Coupling classical quantitative genetic analysis with simulations of phenotypic distributions (a method we call phenotype modelling) enabled us to obtain a detailed picture of the quantitative trait loci (QTLs) relationships underlying the phenotypic variation of this trait. Using this approach, we were able to uncover a dominant epistatic inheritance of loci governing the phenotype. Moreover, a molecular analysis of known causative quantitative trait genes and nucleotides allowed for the detection of novel alleles, potentially responsible for the observed phenotypic variation. Based on the molecular data, we hypothesise that the observed dominant epistatic relationship could be caused by the interaction of multiple quantitative trait nucleotides distributed across a 60--kb QTL region located on chromosome XIV and the RME1 locus on chromosome VII. Furthermore, we propose a model of molecular pathways which possibly underlie the phenotypic variation of this trait. PMID:27904371

Msc1 acts through histone H2A.Z to promote chromosome stability in Schizosaccharomyces pombe.

PubMed

Ahmed, Shakil; Dul, Barbara; Qiu, Xinxing; Walworth, Nancy C

2007-11-01

As a central component of the DNA damage checkpoint pathway, the conserved protein kinase Chk1 mediates cell cycle progression when DNA damage is generated. Msc1 was identified as a multicopy suppressor capable of facilitating survival in response to DNA damage of cells mutant for chk1. We demonstrate that loss of msc1 function results in an increased rate of chromosome loss and that an msc1 null allele exhibits genetic interactions with mutants in key kinetochore components. Multicopy expression of msc1 robustly suppresses a temperature-sensitive mutant (cnp1-1) in the centromere-specific histone H3 variant CENP-A, and localization of CENP-A to the centromere is compromised in msc1 null cells. We present several lines of evidence to suggest that Msc1 carries out its function through the histone H2A variant H2A.Z, encoded by pht1 in fission yeast. Like an msc1 mutant, a pht1 mutant also exhibits chromosome instability and genetic interactions with kinetochore mutants. Suppression of cnp1-1 by multicopy msc1 requires pht1. Likewise, suppression of the DNA damage sensitivity of a chk1 mutant by multicopy msc1 also requires pht1. We present the first genetic evidence that histone H2A.Z may participate in centromere function in fission yeast and propose that Msc1 acts through H2A.Z to promote chromosome stability and cell survival following DNA damage.

Msc1 Acts Through Histone H2A.Z to Promote Chromosome Stability in Schizosaccharomyces pombe

PubMed Central

Ahmed, Shakil; Dul, Barbara; Qiu, Xinxing; Walworth, Nancy C.

2007-01-01

As a central component of the DNA damage checkpoint pathway, the conserved protein kinase Chk1 mediates cell cycle progression when DNA damage is generated. Msc1 was identified as a multicopy suppressor capable of facilitating survival in response to DNA damage of cells mutant for chk1. We demonstrate that loss of msc1 function results in an increased rate of chromosome loss and that an msc1 null allele exhibits genetic interactions with mutants in key kinetochore components. Multicopy expression of msc1 robustly suppresses a temperature-sensitive mutant (cnp1-1) in the centromere-specific histone H3 variant CENP-A, and localization of CENP-A to the centromere is compromised in msc1 null cells. We present several lines of evidence to suggest that Msc1 carries out its function through the histone H2A variant H2A.Z, encoded by pht1 in fission yeast. Like an msc1 mutant, a pht1 mutant also exhibits chromosome instability and genetic interactions with kinetochore mutants. Suppression of cnp1-1 by multicopy msc1 requires pht1. Likewise, suppression of the DNA damage sensitivity of a chk1 mutant by multicopy msc1 also requires pht1. We present the first genetic evidence that histone H2A.Z may participate in centromere function in fission yeast and propose that Msc1 acts through H2A.Z to promote chromosome stability and cell survival following DNA damage. PMID:17947424

Asy2/Mer2: an evolutionarily conserved mediator of meiotic recombination, pairing, and global chromosome compaction

PubMed Central

Tessé, Sophie; Bourbon, Henri-Marc; Debuchy, Robert; Budin, Karine; Dubois, Emeline; Liangran, Zhang; Antoine, Romain; Piolot, Tristan; Kleckner, Nancy; Zickler, Denise; Espagne, Eric

2017-01-01

Meiosis is the cellular program by which a diploid cell gives rise to haploid gametes for sexual reproduction. Meiotic progression depends on tight physical and functional coupling of recombination steps at the DNA level with specific organizational features of meiotic-prophase chromosomes. The present study reveals that every step of this coupling is mediated by a single molecule: Asy2/Mer2. We show that Mer2, identified so far only in budding and fission yeasts, is in fact evolutionarily conserved from fungi (Mer2/Rec15/Asy2/Bad42) to plants (PRD3/PAIR1) and mammals (IHO1). In yeasts, Mer2 mediates assembly of recombination–initiation complexes and double-strand breaks (DSBs). This role is conserved in the fungus Sordaria. However, functional analysis of 13 mer2 mutants and successive localization of Mer2 to axis, synaptonemal complex (SC), and chromatin revealed, in addition, three further important functions. First, after DSB formation, Mer2 is required for pairing by mediating homolog spatial juxtaposition, with implications for crossover (CO) patterning/interference. Second, Mer2 participates in the transfer/maintenance and release of recombination complexes to/from the SC central region. Third, after completion of recombination, potentially dependent on SUMOylation, Mer2 mediates global chromosome compaction and post-recombination chiasma development. Thus, beyond its role as a recombinosome–axis/SC linker molecule, Mer2 has important functions in relation to basic chromosome structure. PMID:29021238

Mating-type switching by chromosomal inversion in methylotrophic yeasts suggests an origin for the three-locus Saccharomyces cerevisiae system

PubMed Central

Hanson, Sara J.; Byrne, Kevin P.; Wolfe, Kenneth H.

2014-01-01

Saccharomyces cerevisiae has a complex system for switching the mating type of haploid cells, requiring the genome to have three mating-type (MAT)–like loci and a mechanism for silencing two of them. How this system originated is unknown, because the three-locus system is present throughout the family Saccharomycetaceae, whereas species in the sister Candida clade have only one locus and do not switch. Here we show that yeasts in a third clade, the methylotrophs, have a simpler two-locus switching system based on reversible inversion of a section of chromosome with MATa genes at one end and MATalpha genes at the other end. In Hansenula polymorpha the 19-kb invertible region lies beside a centromere so that, depending on the orientation, either MATa or MATalpha is silenced by centromeric chromatin. In Pichia pastoris, the orientation of a 138-kb invertible region puts either MATa or MATalpha beside a telomere and represses transcription of MATa2 or MATalpha2. Both species are homothallic, and inversion of their MAT regions can be induced by crossing two strains of the same mating type. The three-locus system of S. cerevisiae, which uses a nonconservative mechanism to replace DNA at MAT, likely evolved from a conservative two-locus system that swapped genes between expression and nonexpression sites by inversion. The increasing complexity of the switching apparatus, with three loci, donor bias, and cell lineage tracking, can be explained by continuous selection to increase sporulation ability in young colonies. Our results provide an evolutionary context for the diversity of switching and silencing mechanisms. PMID:25349420

Mating-type switching by chromosomal inversion in methylotrophic yeasts suggests an origin for the three-locus Saccharomyces cerevisiae system.

PubMed

Hanson, Sara J; Byrne, Kevin P; Wolfe, Kenneth H

2014-11-11

Saccharomyces cerevisiae has a complex system for switching the mating type of haploid cells, requiring the genome to have three mating-type (MAT)-like loci and a mechanism for silencing two of them. How this system originated is unknown, because the three-locus system is present throughout the family Saccharomycetaceae, whereas species in the sister Candida clade have only one locus and do not switch. Here we show that yeasts in a third clade, the methylotrophs, have a simpler two-locus switching system based on reversible inversion of a section of chromosome with MATa genes at one end and MATalpha genes at the other end. In Hansenula polymorpha the 19-kb invertible region lies beside a centromere so that, depending on the orientation, either MATa or MATalpha is silenced by centromeric chromatin. In Pichia pastoris, the orientation of a 138-kb invertible region puts either MATa or MATalpha beside a telomere and represses transcription of MATa2 or MATalpha2. Both species are homothallic, and inversion of their MAT regions can be induced by crossing two strains of the same mating type. The three-locus system of S. cerevisiae, which uses a nonconservative mechanism to replace DNA at MAT, likely evolved from a conservative two-locus system that swapped genes between expression and nonexpression sites by inversion. The increasing complexity of the switching apparatus, with three loci, donor bias, and cell lineage tracking, can be explained by continuous selection to increase sporulation ability in young colonies. Our results provide an evolutionary context for the diversity of switching and silencing mechanisms.

A Fine Physical Map of the Rice Chromosome 4

PubMed Central

Zhao, Qiang; Zhang, Yu; Cheng, Zhukuan; Chen, Mingsheng; Wang, Shengyue; Feng, Qi; Huang, Yucheng; Li, Ying; Tang, Yesheng; Zhou, Bo; Chen, Zhehua; Yu, Shuliang; Zhu, Jingjie; Hu, Xin; Mu, Jie; Ying, Kai; Hao, Pei; Zhang, Lei; Lu, Yiqi; Zhang, Lei S.; Liu, Yilei; Yu, Zhen; Fan, Danlin; Weng, Qijun; Chen, Ling; Lu, Tingting; Liu, Xiaohui; Jia, Peixin; Sun, Tongguo; Wu, Yongrui; Zhang, Yujun; Lu, Ying; Li, Can; Wang, Rong; Lei, Haiyan; Li, Tao; Hu, Hao; Wu, Mei; Zhang, Runquan; Guan, Jianping; Zhu, Jia; Fu, Gang; Gu, Minghong; Hong, Guofan; Xue, Yongbiao; Wing, Rod; Jiang, Jiming; Han, Bin

2002-01-01

As part of an international effort to completely sequence the rice genome, we have produced a fine bacterial artificial chromosome (BAC)-based physical map of the Oryza sativa japonica Nipponbare chromosome 4 through an integration of 114 sequenced BAC clones from a taxonomically related subspecies O. sativa indica Guangluai 4 and 182 RFLP and 407 expressed sequence tag (EST) markers with the fingerprinted data of the Nipponbare genome. The map consists of 11 contigs with a total length of 34.5 Mb covering 94% of the estimated chromosome size (36.8 Mb). BAC clones corresponding to telomeres, as well as to the centromere position, were determined by BAC-pachytene chromosome fluorescence in situ hybridization (FISH). This gave rise to an estimated length ratio of 5.13 for the long arm and 2.9 for the short arm (on the basis of the physical map), which indicates that the short arm is a highly condensed one. The FISH analysis and physical mapping also showed that the short arm and the pericentromeric region of the long arm are rich in heterochromatin, which occupied 45% of the chromosome, indicating that this chromosome is likely very difficult to sequence. To our knowledge, this map provides the first example of a rapid and reliable physical mapping on the basis of the integration of the data from two taxonomically related subspecies. [The following individuals and institutions kindly provided reagents, samples, or unpublished information as indicated in the paper: S. McCouch, T. Sasaki, and Monsanto.] PMID:11997348

Generic Features of Tertiary Chromatin Structure as Detected in Natural Chromosomes

PubMed Central

Müller, Waltraud G.; Rieder, Dietmar; Kreth, Gregor; Cremer, Christoph; Trajanoski, Zlatko; McNally, James G.

2004-01-01

Knowledge of tertiary chromatin structure in mammalian interphase chromosomes is largely derived from artificial tandem arrays. In these model systems, light microscope images reveal fibers or beaded fibers after high-density targeting of transactivators to insertional domains spanning several megabases. These images of fibers have lent support to chromonema fiber models of tertiary structure. To assess the relevance of these studies to natural mammalian chromatin, we identified two different ∼400-kb regions on human chromosomes 6 and 22 and then examined light microscope images of interphase tertiary chromatin structure when the regions were transcriptionally active and inactive. When transcriptionally active, these natural chromosomal regions elongated, yielding images characterized by a series of adjacent puncta or “beads”, referred to hereafter as beaded images. These elongated structures required transcription for their maintenance. Thus, despite marked differences in the density and the mode of transactivation, the natural and artificial systems showed similarities, suggesting that beaded images are generic features of transcriptionally active tertiary chromatin. We show here, however, that these images do not necessarily favor chromonema fiber models but can also be explained by a radial-loop model or even a simple nucleosome affinity, random-chain model. Thus, light microscope images of tertiary structure cannot distinguish among competing models, although they do impose key constraints: chromatin must be clustered to yield beaded images and then packaged within each cluster to enable decondensation into adjacent clusters. PMID:15485905

Molecular and physiological characteristics of a grape yeast strain containing atypical genetic material.

PubMed

Cappello, M S; Poltronieri, P; Blaiotta, G; Zacheo, G

2010-11-15

The knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae and Saccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from "Primitivo" grape (http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic material from a non-Saccharomyces species. The study was focused on the analysis of chromosomal and mitochondrial gene sequences (ITS and 26S rRNA, SSU and COXII, ACTIN-1 and TEF), 2D-PAGE mitochondrial proteins, and spore viability. The results allowed us to formulate the hypothesis that in the MSH199 isolate a DNA containing an rDNA sequence from Hanseniaspora vineae, a non-Saccharomyces yeast, was incorporated through homologous recombination in the grape environment where yeast species are propagated. Moreover, physiological characterization showed that the MSH199 isolate possesses high technological quality traits (fermentation performance) and glycerol production, resistance to ethanol, SO₂ and temperature) useful for industrial application. Copyright © 2010 Elsevier B.V. All rights reserved.

Prevalence and consequences of chromosomal abnormalities in Canadian commercial swine herds.

PubMed

Quach, Anh T; Revay, Tamas; Villagomez, Daniel A F; Macedo, Mariana P; Sullivan, Alison; Maignel, Laurence; Wyss, Stefanie; Sullivan, Brian; King, W Allan

2016-09-12

Structural chromosome abnormalities are well known as factors that reduce fertility rate in domestic pigs. According to large-scale national cytogenetic screening programs that are implemented in France, it is estimated that new chromosome abnormalities occur at a rate of 0.5 % in fertility-unproven boars. This work aimed at estimating the prevalence and consequences of chromosome abnormalities in commercial swine operations in Canada. We found pig carriers at a frequency of 1.64 % (12 out of 732 boars). Carrier pigs consistently showed lower fertility values. The total number of piglets born for litters from carrier boars was between 4 and 46 % lower than the herd average. Similarly, carrier boars produced litters with a total number of piglets born alive that was between 6 and 28 % lower than the herd average. A total of 12 new structural chromosome abnormalities were identified. Reproductive performance is significantly reduced in sires with chromosome abnormalities. The incidence of such abnormal sires appears relatively high in populations without routine cytogenetic screening such as observed for Canada in this study. Systematic cytogenetic screening of potential breeding boars would minimise the risk of carriers of chromosome aberrations entering artificial insemination centres. This would avoid the large negative effects on productivity for the commercial sow herds and reduce the risk of transmitting abnormalities to future generations in nucleus farms.

Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae

PubMed Central

Feng, Quanzhou; Weber, Scott A.; Li, Shizhong

2018-01-01

Haploid laboratory strains of Saccharomyces cerevisiae are commonly used for genetic engineering to enable their xylose utilization but little is known about the industrial yeast which is often recognized as diploid and as well as haploid and tetraploid. Here we report three unique signature pathway expression patterns and gene interactions in the centre metabolic pathways that signify xylose utilization of genetically engineered industrial yeast S. cerevisiae NRRL Y-50463, a diploid yeast. Quantitative expression analysis revealed outstanding high levels of constitutive expression of YXI, a synthesized yeast codon-optimized xylose isomerase gene integrated into chromosome XV of strain Y-50463. Comparative expression analysis indicated that the YXI was necessary to initiate the xylose metabolic pathway along with a set of heterologous xylose transporter and utilization facilitating genes including XUT4, XUT6, XKS1 and XYL2. The highly activated transketolase and transaldolase genes TKL1, TKL2, TAL1 and NQM1 as well as their complex interactions in the non-oxidative pentose phosphate pathway branch were critical for the serial of sugar transformation to drive the metabolic flow into glycolysis for increased ethanol production. The significantly increased expression of the entire PRS gene family facilitates functions of the life cycle and biosynthesis superpathway for the yeast. The outstanding higher levels of constitutive expression of YXI and the first insight into the signature pathway expression and the gene interactions in the closely related centre metabolic pathways from the industrial yeast aid continued efforts for development of the next-generation biocatalyst. Our results further suggest the industrial yeast is a desirable delivery vehicle for new strain development for efficient lignocellulose-to-advanced biofuels production. PMID:29621349

Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae.

PubMed

Feng, Quanzhou; Liu, Z Lewis; Weber, Scott A; Li, Shizhong

2018-01-01

Haploid laboratory strains of Saccharomyces cerevisiae are commonly used for genetic engineering to enable their xylose utilization but little is known about the industrial yeast which is often recognized as diploid and as well as haploid and tetraploid. Here we report three unique signature pathway expression patterns and gene interactions in the centre metabolic pathways that signify xylose utilization of genetically engineered industrial yeast S. cerevisiae NRRL Y-50463, a diploid yeast. Quantitative expression analysis revealed outstanding high levels of constitutive expression of YXI, a synthesized yeast codon-optimized xylose isomerase gene integrated into chromosome XV of strain Y-50463. Comparative expression analysis indicated that the YXI was necessary to initiate the xylose metabolic pathway along with a set of heterologous xylose transporter and utilization facilitating genes including XUT4, XUT6, XKS1 and XYL2. The highly activated transketolase and transaldolase genes TKL1, TKL2, TAL1 and NQM1 as well as their complex interactions in the non-oxidative pentose phosphate pathway branch were critical for the serial of sugar transformation to drive the metabolic flow into glycolysis for increased ethanol production. The significantly increased expression of the entire PRS gene family facilitates functions of the life cycle and biosynthesis superpathway for the yeast. The outstanding higher levels of constitutive expression of YXI and the first insight into the signature pathway expression and the gene interactions in the closely related centre metabolic pathways from the industrial yeast aid continued efforts for development of the next-generation biocatalyst. Our results further suggest the industrial yeast is a desirable delivery vehicle for new strain development for efficient lignocellulose-to-advanced biofuels production.

Kinetic Analysis of a Molecular Model of the Budding Yeast Cell Cycle

PubMed Central

Chen, Katherine C.; Csikasz-Nagy, Attila; Gyorffy, Bela; Val, John; Novak, Bela; Tyson, John J.

2000-01-01

The molecular machinery of cell cycle control is known in more detail for budding yeast, Saccharomyces cerevisiae, than for any other eukaryotic organism. In recent years, many elegant experiments on budding yeast have dissected the roles of cyclin molecules (Cln1–3 and Clb1–6) in coordinating the events of DNA synthesis, bud emergence, spindle formation, nuclear division, and cell separation. These experimental clues suggest a mechanism for the principal molecular interactions controlling cyclin synthesis and degradation. Using standard techniques of biochemical kinetics, we convert the mechanism into a set of differential equations, which describe the time courses of three major classes of cyclin-dependent kinase activities. Model in hand, we examine the molecular events controlling “Start” (the commitment step to a new round of chromosome replication, bud formation, and mitosis) and “Finish” (the transition from metaphase to anaphase, when sister chromatids are pulled apart and the bud separates from the mother cell) in wild-type cells and 50 mutants. The model accounts for many details of the physiology, biochemistry, and genetics of cell cycle control in budding yeast. PMID:10637314

SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes.

PubMed

Shen, Yue; Stracquadanio, Giovanni; Wang, Yun; Yang, Kun; Mitchell, Leslie A; Xue, Yaxin; Cai, Yizhi; Chen, Tai; Dymond, Jessica S; Kang, Kang; Gong, Jianhui; Zeng, Xiaofan; Zhang, Yongfen; Li, Yingrui; Feng, Qiang; Xu, Xun; Wang, Jun; Wang, Jian; Yang, Huanming; Boeke, Jef D; Bader, Joel S

2016-01-01

Synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) generates combinatorial genomic diversity through rearrangements at designed recombinase sites. We applied SCRaMbLE to yeast synthetic chromosome arm synIXR (43 recombinase sites) and then used a computational pipeline to infer or unscramble the sequence of recombinations that created the observed genomes. Deep sequencing of 64 synIXR SCRaMbLE strains revealed 156 deletions, 89 inversions, 94 duplications, and 55 additional complex rearrangements; several duplications are consistent with a double rolling circle mechanism. Every SCRaMbLE strain was unique, validating the capability of SCRaMbLE to explore a diverse space of genomes. Rearrangements occurred exclusively at designed loxPsym sites, with no significant evidence for ectopic rearrangements or mutations involving synthetic regions, the 99% nonsynthetic nuclear genome, or the mitochondrial genome. Deletion frequencies identified genes required for viability or fast growth. Replacement of 3' UTR by non-UTR sequence had surprisingly little effect on fitness. SCRaMbLE generates genome diversity in designated regions, reveals fitness constraints, and should scale to simultaneous evolution of multiple synthetic chromosomes. © 2016 Shen et al.; Published by Cold Spring Harbor Laboratory Press.

SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes

PubMed Central

Shen, Yue; Stracquadanio, Giovanni; Wang, Yun; Yang, Kun; Mitchell, Leslie A.; Xue, Yaxin; Cai, Yizhi; Chen, Tai; Dymond, Jessica S.; Kang, Kang; Gong, Jianhui; Zeng, Xiaofan; Zhang, Yongfen; Li, Yingrui; Feng, Qiang; Xu, Xun; Wang, Jun; Wang, Jian; Yang, Huanming; Boeke, Jef D.; Bader, Joel S.

2016-01-01

Synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) generates combinatorial genomic diversity through rearrangements at designed recombinase sites. We applied SCRaMbLE to yeast synthetic chromosome arm synIXR (43 recombinase sites) and then used a computational pipeline to infer or unscramble the sequence of recombinations that created the observed genomes. Deep sequencing of 64 synIXR SCRaMbLE strains revealed 156 deletions, 89 inversions, 94 duplications, and 55 additional complex rearrangements; several duplications are consistent with a double rolling circle mechanism. Every SCRaMbLE strain was unique, validating the capability of SCRaMbLE to explore a diverse space of genomes. Rearrangements occurred exclusively at designed loxPsym sites, with no significant evidence for ectopic rearrangements or mutations involving synthetic regions, the 99% nonsynthetic nuclear genome, or the mitochondrial genome. Deletion frequencies identified genes required for viability or fast growth. Replacement of 3′ UTR by non-UTR sequence had surprisingly little effect on fitness. SCRaMbLE generates genome diversity in designated regions, reveals fitness constraints, and should scale to simultaneous evolution of multiple synthetic chromosomes. PMID:26566658

Yeast Based Sensors

NASA Astrophysics Data System (ADS)

Shimomura-Shimizu, Mifumi; Karube, Isao

Since the first microbial cell sensor was studied by Karube et al. in 1977, many types of yeast based sensors have been developed as analytical tools. Yeasts are known as facultative anaerobes. Facultative anaerobes can survive in both aerobic and anaerobic conditions. The yeast based sensor consisted of a DO electrode and an immobilized omnivorous yeast. In yeast based sensor development, many kinds of yeast have been employed by applying their characteristics to adapt to the analyte. For example, Trichosporon cutaneum was used to estimate organic pollution in industrial wastewater. Yeast based sensors are suitable for online control of biochemical processes and for environmental monitoring. In this review, principles and applications of yeast based sensors are summarized.

A highly efficient targeted recombination system for engineering linear chromosomes of industrial bacteria Streptomyces.

PubMed

Pan, Hung-Yin; Chen, Carton W; Huang, Chih-Hung

2018-04-17

Soil bacteria Streptomyces are the most important producers of secondary metabolites, including most known antibiotics. These bacteria and their close relatives are unique in possessing linear chromosomes, which typically harbor 20 to 30 biosynthetic gene clusters of tens to hundreds of kb in length. Many Streptomyces chromosomes are accompanied by linear plasmids with sizes ranging from several to several hundred kb. The large linear plasmids also often contain biosynthetic gene clusters. We have developed a targeted recombination procedure for arm exchanges between a linear plasmid and a linear chromosome. A chromosomal segment inserted in an artificially constructed plasmid allows homologous recombination between the two replicons at the homology. Depending on the design, the recombination may result in two recombinant replicons or a single recombinant chromosome with the loss of the recombinant plasmid that lacks a replication origin. The efficiency of such targeted recombination ranges from 9 to 83% depending on the locations of the homology (and thus the size of the chromosomal arm exchanged), essentially eliminating the necessity of selection. The targeted recombination is useful for the efficient engineering of the Streptomyces genome for large-scale deletion, addition, and shuffling.

A new yeast gene with a myosin-like heptad repeat structure.

PubMed

Kölling, R; Nguyen, T; Chen, E Y; Botstein, D

1993-03-01

We isolated a gene encoding a 218 kDa myosin-like protein from Saccharomyces cerevisiae using a monoclonal antibody directed against human platelet myosin as a probe. The protein sequence encoded by the MLP1 gene (for myosin-like protein) contains extensive stretches of a heptad-repeat pattern suggesting that the protein can form coiled coils typical of myosins. Immunolocalization experiments using affinity-purified antibodies raised against a TrpE-MLP1 fusion protein showed a dot-like structure adjacent to the nucleus in yeast cells bearing the MLP1 gene on a multicopy plasmid. In mouse epithelial cells the yeast anti-MLP1 antibodies stained the nucleus. Mutants bearing disruptions of the MLP1 gene were viable, but more sensitive to ultraviolet light than wild-type strains, suggesting an involvement of MLP1 in DNA repair. The MLP1 gene was mapped to chromosome 11, 25 cM from met1.

Cleavage of the SUN-domain protein Mps3 at its N-terminus regulates centrosome disjunction in budding yeast meiosis

PubMed Central

Koch, Bailey A.; Han, Xuemei

2017-01-01

Centrosomes organize microtubules and are essential for spindle formation and chromosome segregation during cell division. Duplicated centrosomes are physically linked, but how this linkage is dissolved remains unclear. Yeast centrosomes are tethered by a nuclear-envelope-attached structure called the half-bridge, whose components have mammalian homologues. We report here that cleavage of the half-bridge protein Mps3 promotes accurate centrosome disjunction in budding yeast. Mps3 is a single-pass SUN-domain protein anchored at the inner nuclear membrane and concentrated at the nuclear side of the half-bridge. Using the unique feature in yeast meiosis that centrosomes are linked for hours before their separation, we have revealed that Mps3 is cleaved at its nucleus-localized N-terminal domain, the process of which is regulated by its phosphorylation at serine 70. Cleavage of Mps3 takes place at the yeast centrosome and requires proteasome activity. We show that noncleavable Mps3 (Mps3-nc) inhibits centrosome separation during yeast meiosis. In addition, overexpression of mps3-nc in vegetative yeast cells also inhibits centrosome separation and is lethal. Our findings provide a genetic mechanism for the regulation of SUN-domain protein-mediated activities, including centrosome separation, by irreversible protein cleavage at the nuclear periphery. PMID:28609436

Replication-dependent and independent mechanisms for the chromosome-coupled persistence of a selfish genome.

PubMed

Liu, Yen-Ting; Chang, Keng-Ming; Ma, Chien-Hui; Jayaram, Makkuni

2016-09-30

The yeast 2-micron plasmid epitomizes the evolutionary optimization of selfish extra-chromosomal genomes for stable persistence without jeopardizing their hosts' fitness. Analyses of fluorescence-tagged single-copy reporter plasmids and/or the plasmid partitioning proteins in native and non-native hosts reveal chromosome-hitchhiking as the likely means for plasmid segregation. The contribution of the partitioning system to equal segregation is bipartite- replication-independent and replication-dependent. The former nearly eliminates 'mother bias' (preferential plasmid retention in the mother cell) according to binomial distribution, thus limiting equal segregation of a plasmid pair to 50%. The latter enhances equal segregation of plasmid sisters beyond this level, elevating the plasmid close to chromosome status. Host factors involved in plasmid partitioning can be functionally separated by their participation in the replication-independent and/or replication-dependent steps. In the hitchhiking model, random tethering of a pair of plasmids to chromosomes signifies the replication-independent component of segregation; the symmetric tethering of plasmid sisters to sister chromatids embodies the replication-dependent component. The 2-micron circle broadly resembles the episomes of certain mammalian viruses in its chromosome-associated propagation. This unifying feature among otherwise widely differing selfish genomes suggests their evolutionary convergence to the common logic of exploiting, albeit via distinct molecular mechanisms, host chromosome segregation machineries for self-preservation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cytogenetic mapping of a novel locus for type II Waardenburg syndrome.

PubMed

Selicorni, Angelo; Guerneri, Silvana; Ratti, Antonia; Pizzuti, Antonio

2002-01-01

An Italian family in which Waardenburg syndrome type II (WS2) segregates together with a der(8) chromosome from a (4p;8p) balanced translocation was studied. Cytogenetic analysis by painting and subtelomeric probe hybridization positioned the chromosome 8 breakpoint at p22-pter. Fluorescence in situ hybridization analysis with yeast artificial chromosomes from a contig spanning the 8p21-pter region refined the breakpoint in an interval of less than 170 kb between markers WI-3823 and D8S1819. The only cloned gene for WS2 is that for microphtalmia (MITF) on chromosome 3p. In this family, MITF mutations were excluded by sequencing the whole coding region. The 8p23 region may represent a third locus for WS2 (WS2C).

Yeast derivatives and wheat germ in the adult diet modulates fecundity in a tephritid pest.

PubMed

Goane, L; Pereyra, P M; Castro, F; Ruiz, M J; Juárez, M L; Segura, D F; Vera, M T

2018-05-22

Anastrepha fraterculus (Wiedemann), a pest of great economic importance in South America, needs urgently to be controlled by environmentally friendly methods such as the sterile insect technique for which mass rearing of insects is required. Because oogenesis takes place during the adult stage, mass-rearing facilities should provide the females a diet that maximizes egg production at the lowest cost. Accordingly, we investigated the effect of artificial protein sources in the adult diet (yeast derivatives of different cost but with similar amino acids profiles, and the addition of wheat germ) on fecundity. Additionally, we evaluated different ratios of yeast derivatives or wheat germ on ovary maturation, fecundity, and fertility as well as their association with the nutrient content of females. Females fed hydrolyzed yeast and yeast extract attained the highest fecundity level, and those fed brewer's yeast the lowest. Reducing the amount of hydrolyzed yeast, an expensive protein source, in the diet negatively affected fecundity and ovary maturation. Increasing the amount of brewer's yeast, a low-cost protein source, did not favor fecundity. The addition of wheat germ in the adult diet improved fecundity regardless of the yeast derivate considered. Percentage of egg hatch was not affected by the diet. Nutrient content of A. fraterculus females varied according to the adult diet provided and mating status. Our findings provide novel baseline information to understand the role of nutrition on reproductive performance of A. fraterculus females and are discussed in the context of resource allocation. They also provide valuable advances in the search for cost-effective adult diets at fruit fly mass rearing facilities.

[Genetic system for maintaining the mitochondrial human genome in yeast Yarrowia lipolytica].

PubMed

Isakova, E P; Deryabina, Yu I; Velyakova, A V; Biryukova, J K; Teplova, V V; Shevelev, A B

2016-01-01

For the first time, the possibility of maintaining an intact human mitochondrial genome in a heterologous system in the mitochondria of yeast Yarrowia lipolytica is shown. A method for introducing directional changes into the structure of the mitochondrial human genome replicating in Y. lipolytica by an artificially induced ability of yeast mitochondria for homologous recombination is proposed. A method of introducing and using phenotypic selection markers for the presence or absence of defects in genes tRNA-Lys and tRNA-Leu of the mitochondrial genome is developed. The proposed system can be used to correct harmful mutations of the human mitochondrial genome associated with mitochondrial diseases and for preparative amplification of intact mitochondrial DNA with an adjusted sequence in yeast cells. The applicability of the new system for the correction of mutations in the genes of Lys- and Leu-specific tRNAs of the human mitochondrial genome associated with serious and widespread human mitochondrial diseases such as myoclonic epilepsy with lactic acidosis (MELAS) and myoclonic epilepsy with ragged-red fibers (MERRF) is shown.

Real-Time Quantitative PCR (QPCR) and Reverse Transcription-QPCR for Detection and Enumeration of Total Yeasts in Wine▿

PubMed Central

Hierro, Núria; Esteve-Zarzoso, Braulio; González, Ángel; Mas, Albert; Guillamón, Jose M.

2006-01-01

Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage. PMID:17088381

New family of pectinase genes PGU1b-PGU3b of the pectinolytic yeast Saccharomyces bayanus var. uvarum.

PubMed

Naumov, G I; Shalamitskiy, M Yu; Naumova, E S

2016-03-01

Using yeast genome databases and literature data, we have conducted a phylogenetic analysis of pectinase PGU genes from Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and hybrid taxon S. pastorianus (syn. S. carlsbergensis). Single PGU genes were observed in all Saccharomyces species, except S. bayanus. The superfamily of divergent PGU genes has been documented in S. bayanus var. uvarum for the first time. Chromosomal localization of new PGU1b, PGU2b, and PGU3b genes in the yeast S. bayanus var. uvarum has been determined by molecular karyotyping and Southern hybridization.

Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity.

PubMed

Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A; Bradford, William D; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S; Li, Rong

2015-03-30

Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein-based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. Copyright © 2015 Zhu et al.

Extracellular Polysaccharides Produced by Yeasts and Yeast-Like Fungi

NASA Astrophysics Data System (ADS)

van Bogaert, Inge N. A.; de Maeseneire, Sofie L.; Vandamme, Erick J.

Several yeasts and yeast-like fungi are known to produce extracellular polysaccharides. Most of these contain D-mannose, either alone or in combination with other sugars or phosphate. A large chemical and structural variability is found between yeast species and even among different strains. The types of polymers that are synthesized can be chemically characterized as mannans, glucans, phosphoman-nans, galactomannans, glucomannans and glucuronoxylomannans. Despite these differences, almost all of the yeast exopolysaccharides display some sort of biological activity. Some of them have already applications in chemistry, pharmacy, cosmetics or as probiotic. Furthermore, some yeast exopolysaccharides, such as pullulan, exhibit specific physico-chemical and rheological properties, making them useful in a wide range of technical applications. A survey is given here of the production, the characteristics and the application potential of currently well studied yeast extracellular polysaccharides.

Compartmentalization of the yeast meiotic nucleus revealed by analysis of ectopic recombination.

PubMed

Schlecht, Hélène B; Lichten, Michael; Goldman, Alastair S H

2004-11-01

As yeast cells enter meiosis, chromosomes move from a centromere-clustered (Rabl) to a telomere-clustered (bouquet) configuration and then to states of progressive homolog pairing where telomeres are more dispersed. It is uncertain at which stage of this process sequences commit to recombine with each other. Previous analyses using recombination between dispersed homologous sequences (ectopic recombination) support the view that, on average, homologs are aligned end to end by the time of commitment to recombination. We have undertaken further analyses incorporating new inserts, chromosome rearrangements, an alternate mode of recombination initiation, and mutants that disrupt nuclear structure or telomere metabolism. Our findings support previous conclusions and reveal that distance from the nearest telomere is an important parameter influencing recombination between dispersed sequences. In general, the farther dispersed sequences are from their nearest telomere, the less likely they are to engage in ectopic recombination. Neither the mode of initiating recombination nor the formation of the bouquet appears to affect this relationship. We suggest that aspects of telomere localization and behavior influence the organization and mobility of chromosomes along their entire length, during a critical period of meiosis I prophase that encompasses the homology search.

Yeast Infection (Vaginal)

MedlinePlus

Yeast infection (vaginal) Overview A vaginal yeast infection is a fungal infection that causes irritation, discharge and intense itchiness ... symptoms Causes The fungus candida causes a vaginal yeast infection. Your vagina naturally contains a balanced mix of yeast, including ...

Construction and Analysis of Siberian Tiger Bacterial Artificial Chromosome Library with Approximately 6.5-Fold Genome Equivalent Coverage

PubMed Central

Liu, Changqing; Bai, Chunyu; Guo, Yu; Liu, Dan; Lu, Taofeng; Li, Xiangchen; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

2014-01-01

Bacterial artificial chromosome (BAC) libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12), consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger. PMID:24608928

Chromosome

MedlinePlus

... St Louis, MO: Elsevier; 2017:chap 69. Taber's Medical Dictionary Online. Chromosome. www.tabers.com/tabersonline/view/Tabers-Dictionary/753321/all/chromosome?q=Chromosome&ti=0 . Accessed June 11, 2017.

Asy2/Mer2: an evolutionarily conserved mediator of meiotic recombination, pairing, and global chromosome compaction.

PubMed

Tessé, Sophie; Bourbon, Henri-Marc; Debuchy, Robert; Budin, Karine; Dubois, Emeline; Liangran, Zhang; Antoine, Romain; Piolot, Tristan; Kleckner, Nancy; Zickler, Denise; Espagne, Eric

2017-09-15

Meiosis is the cellular program by which a diploid cell gives rise to haploid gametes for sexual reproduction. Meiotic progression depends on tight physical and functional coupling of recombination steps at the DNA level with specific organizational features of meiotic-prophase chromosomes. The present study reveals that every step of this coupling is mediated by a single molecule: Asy2/Mer2. We show that Mer2, identified so far only in budding and fission yeasts, is in fact evolutionarily conserved from fungi (Mer2/Rec15/Asy2/Bad42) to plants (PRD3/PAIR1) and mammals (IHO1). In yeasts, Mer2 mediates assembly of recombination-initiation complexes and double-strand breaks (DSBs). This role is conserved in the fungus Sordaria However, functional analysis of 13 mer2 mutants and successive localization of Mer2 to axis, synaptonemal complex (SC), and chromatin revealed, in addition, three further important functions. First, after DSB formation, Mer2 is required for pairing by mediating homolog spatial juxtaposition, with implications for crossover (CO) patterning/interference. Second, Mer2 participates in the transfer/maintenance and release of recombination complexes to/from the SC central region. Third, after completion of recombination, potentially dependent on SUMOylation, Mer2 mediates global chromosome compaction and post-recombination chiasma development. Thus, beyond its role as a recombinosome-axis/SC linker molecule, Mer2 has important functions in relation to basic chromosome structure. © 2017 Tessé et al.; Published by Cold Spring Harbor Laboratory Press.

Interaction of the Saccharomyces cerevisiae RING-domain protein Nse1 with Nse3 and the Smc5/6 complex is required for chromosome replication and stability.

PubMed

Wani, Saima; Maharshi, Neelam; Kothiwal, Deepash; Mahendrawada, Lakshmi; Kalaivani, Raju; Laloraya, Shikha

2018-06-01

Genomic stability is maintained by the concerted actions of numerous protein complexes that participate in chromosomal duplication, repair, and segregation. The Smc5/6 complex is an essential multi-subunit complex crucial for repair of DNA double-strand breaks. Two of its subunits, Nse1 and Nse3, are homologous to the RING-MAGE complexes recently described in human cells. We investigated the contribution of the budding yeast Nse1 RING-domain by isolating a mutant nse1-103 bearing substitutions in conserved Zinc-coordinating residues of the RING-domain that is hypersensitive to genotoxic stress and temperature. The nse1-103 mutant protein was defective in interaction with Nse3 and other Smc5/6 complex subunits, Nse4 and Smc5. Chromosome loss was enhanced, accompanied by a delay in the completion of replication and a modest defect in sister chromatid cohesion, in nse1-103. The nse1-103 mutant was synthetic sick with rrm3∆ (defective in fork passage through pause sites), this defect was rescued by inactivation of Tof1, a subunit of the fork protection complex that enforces pausing. The temperature sensitivity of nse1-103 was partially suppressed by deletion of MPH1, encoding a DNA-helicase. Homology modeling of the structure of the budding yeast Nse1-Nse3 heterodimer based on the human Nse1-MAGEG1 structure suggests a similar organization and indicates that perturbation of the Zn-coordinating cluster has the potential to allosterically alter structural elements at the Nse1/Nse3 interaction interface that may abrogate their association. Our findings demonstrate that the budding yeast Nse1 RING-domain organization is important for interaction with Nse3, which is crucial for completion of chromosomal replication, cohesion, and maintenance of chromosome stability.

Modeling the cost-effectiveness of insect rearing on artificial diets: A test with a tephritid fly used in the sterile insect technique.

PubMed

Pascacio-Villafán, Carlos; Birke, Andrea; Williams, Trevor; Aluja, Martín

2017-01-01

We modeled the cost-effectiveness of rearing Anastrepha ludens, a major fruit fly pest currently mass reared for sterilization and release in pest control programs implementing the sterile insect technique (SIT). An optimization model was generated by combining response surface models of artificial diet cost savings with models of A. ludens pupation, pupal weight, larval development time and adult emergence as a function of mixtures of yeast, a costly ingredient, with corn flour and corncob fractions in the diet. Our model revealed several yeast-reduced mixtures that could be used to prepare diets that were considerably cheaper than a standard diet used for mass rearing. Models predicted a similar production of insects (pupation and adult emergence), with statistically similar pupal weights and larval development times between yeast-reduced diets and the standard mass rearing diet formulation. Annual savings from using the modified diets could be up to 5.9% of the annual cost of yeast, corn flour and corncob fractions used in the standard diet, representing a potential saving of US $27.45 per ton of diet (US $47,496 in the case of the mean annual production of 1,730.29 tons of artificial diet in the Moscafrut mass rearing facility at Metapa, Chiapas, Mexico). Implementation of the yeast-reduced diet on an experimental scale at mass rearing facilities is still required to confirm the suitability of new mixtures of artificial diet for rearing A. ludens for use in SIT. This should include the examination of critical quality control parameters of flies such as adult flight ability, starvation resistance and male sexual competitiveness across various generations. The method used here could be useful for improving the cost-effectiveness of invertebrate or vertebrate mass rearing diets worldwide.

Modeling the cost-effectiveness of insect rearing on artificial diets: A test with a tephritid fly used in the sterile insect technique

PubMed Central

Birke, Andrea; Williams, Trevor; Aluja, Martín

2017-01-01

We modeled the cost-effectiveness of rearing Anastrepha ludens, a major fruit fly pest currently mass reared for sterilization and release in pest control programs implementing the sterile insect technique (SIT). An optimization model was generated by combining response surface models of artificial diet cost savings with models of A. ludens pupation, pupal weight, larval development time and adult emergence as a function of mixtures of yeast, a costly ingredient, with corn flour and corncob fractions in the diet. Our model revealed several yeast-reduced mixtures that could be used to prepare diets that were considerably cheaper than a standard diet used for mass rearing. Models predicted a similar production of insects (pupation and adult emergence), with statistically similar pupal weights and larval development times between yeast-reduced diets and the standard mass rearing diet formulation. Annual savings from using the modified diets could be up to 5.9% of the annual cost of yeast, corn flour and corncob fractions used in the standard diet, representing a potential saving of US $27.45 per ton of diet (US $47,496 in the case of the mean annual production of 1,730.29 tons of artificial diet in the Moscafrut mass rearing facility at Metapa, Chiapas, Mexico). Implementation of the yeast-reduced diet on an experimental scale at mass rearing facilities is still required to confirm the suitability of new mixtures of artificial diet for rearing A. ludens for use in SIT. This should include the examination of critical quality control parameters of flies such as adult flight ability, starvation resistance and male sexual competitiveness across various generations. The method used here could be useful for improving the cost-effectiveness of invertebrate or vertebrate mass rearing diets worldwide. PMID:28257496

Navigating yeast genome maintenance with functional genomics.

PubMed

Measday, Vivien; Stirling, Peter C

2016-03-01

Maintenance of genome integrity is a fundamental requirement of all organisms. To address this, organisms have evolved extremely faithful modes of replication, DNA repair and chromosome segregation to combat the deleterious effects of an unstable genome. Nonetheless, a small amount of genome instability is the driver of evolutionary change and adaptation, and thus a low level of instability is permitted in populations. While defects in genome maintenance almost invariably reduce fitness in the short term, they can create an environment where beneficial mutations are more likely to occur. The importance of this fact is clearest in the development of human cancer, where genome instability is a well-established enabling characteristic of carcinogenesis. This raises the crucial question: what are the cellular pathways that promote genome maintenance and what are their mechanisms? Work in model organisms, in particular the yeast Saccharomyces cerevisiae, has provided the global foundations of genome maintenance mechanisms in eukaryotes. The development of pioneering genomic tools inS. cerevisiae, such as the systematic creation of mutants in all nonessential and essential genes, has enabled whole-genome approaches to identifying genes with roles in genome maintenance. Here, we review the extensive whole-genome approaches taken in yeast, with an emphasis on functional genomic screens, to understand the genetic basis of genome instability, highlighting a range of genetic and cytological screening modalities. By revealing the biological pathways and processes regulating genome integrity, these analyses contribute to the systems-level map of the yeast cell and inform studies of human disease, especially cancer. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Trypanosome outer kinetochore proteins suggest conservation of chromosome segregation machinery across eukaryotes

PubMed Central

D’Archivio, Simon

2017-01-01

Kinetochores are multiprotein complexes that couple eukaryotic chromosomes to the mitotic spindle to ensure proper segregation. The model for kinetochore assembly is conserved between humans and yeast, and homologues of several components are widely distributed in eukaryotes, but key components are absent in some lineages. The recent discovery in a lineage of protozoa called kinetoplastids of unconventional kinetochores with no apparent homology to model organisms suggests that more than one system for eukaryotic chromosome segregation may exist. In this study, we report a new family of proteins distantly related to outer kinetochore proteins Ndc80 and Nuf2. The family member in kinetoplastids, KKT-interacting protein 1 (KKIP1), associates with the kinetochore, and its depletion causes severe defects in karyokinesis, loss of individual chromosomes, and gross defects in spindle assembly or stability. Immunopurification of KKIP1 from stabilized kinetochores identifies six further components, which form part of a trypanosome outer kinetochore complex. These findings suggest that kinetochores in organisms such as kinetoplastids are built from a divergent, but not ancestrally distinct, set of components and that Ndc80/Nuf2-like proteins are universal in eukaryotic division. PMID:28034897

Distinct Domestication Trajectories in Top-Fermenting Beer Yeasts and Wine Yeasts.

PubMed

Gonçalves, Margarida; Pontes, Ana; Almeida, Pedro; Barbosa, Raquel; Serra, Marta; Libkind, Diego; Hutzler, Mathias; Gonçalves, Paula; Sampaio, José Paulo

2016-10-24

Beer is one of the oldest alcoholic beverages and is produced by the fermentation of sugars derived from starches present in cereal grains. Contrary to lager beers, made by bottom-fermenting strains of Saccharomyces pastorianus, a hybrid yeast, ale beers are closer to the ancient beer type and are fermented by S. cerevisiae, a top-fermenting yeast. Here, we use population genomics to investigate (1) the closest relatives of top-fermenting beer yeasts; (2) whether top-fermenting yeasts represent an independent domestication event separate from those already described; (3) whether single or multiple beer yeast domestication events can be inferred; and (4) whether top-fermenting yeasts represent non-recombinant or recombinant lineages. Our results revealed that top-fermenting beer yeasts are polyphyletic, with a main clade composed of at least three subgroups, dominantly represented by the German, British, and wheat beer strains. Other beer strains were phylogenetically close to sake, wine, or bread yeasts. We detected genetic signatures of beer yeast domestication by investigating genes previously linked to brewing and using genome-wide scans. We propose that the emergence of the main clade of beer yeasts is related with a domestication event distinct from the previously known cases of wine and sake yeast domestication. The nucleotide diversity of the main beer clade more than doubled that of wine yeasts, which might be a consequence of fundamental differences in the modes of beer and wine yeast domestication. The higher diversity of beer strains could be due to the more intense and different selection regimes associated to brewing. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mutation in fission yeast phosphatidylinositol 4-kinase Pik1 is synthetically lethal with defect in telomere protection protein Pot1.

PubMed

Sugihara, Asami; Nguyen, Luan Cao; Shamim, Hossain Mohammad; Iida, Tetsushi; Nakase, Mai; Takegawa, Kaoru; Senda, Mitsuhisa; Jida, Shohei; Ueno, Masaru

2018-02-19

Fission yeast Pik1p is one of three phosphatidylinositol 4-kinases associated with the Golgi complex, but its function is not fully understood. Deletion of pot1 + causes telomere degradation and chromosome circularization. We searched for the gene which becomes synthetically lethal with pot1Δ. We obtained a novel pik1 mutant, pik1-1, which is synthetically lethal with pot1Δ. We found phosphoinositol 4-phosphate in the Golgi was reduced in pik1-1. To investigate the mechanism of the lethality of the pot1Δ pik1-1 double mutant, we constructed the nmt-pot1-aid pik1-1 strain, where Pot1 function becomes low by drugs, which leads to telomere loss and chromosome circularization, and found pik1-1 mutation does not affect telomere resection and chromosome circularization. Thus, our results suggest that pik1 + is required for the maintenance of circular chromosomes. Copyright © 2018 Elsevier Inc. All rights reserved.

A close relative of the nuclear, chromosomal high-mobility group protein HMG1 in yeast mitochondria.

PubMed Central

Diffley, J F; Stillman, B

1991-01-01

ABF2 (ARS-binding factor 2), a small, basic DNA-binding protein that binds specifically to the autonomously replicating sequence ARS1, is located primarily in the mitochondria of the yeast Saccharomyces cerevisiae. The abundance of ABF2 and the phenotype of abf2- null mutants argue that this protein plays a key role in the structure, maintenance, and expression of the yeast mitochondrial genome. The predicted amino acid sequence of ABF2 is closely related to the high-mobility group proteins HMG1 and HMG2 from vertebrate cell nuclei and to several other DNA-binding proteins. Additionally, ABF2 and the other HMG-related proteins are related to a globular domain from the heat shock protein hsp70 family. ABF2 interacts with DNA both nonspecifically and in a specific manner within regulatory regions, suggesting a mechanism whereby it may aid in compacting the mitochondrial genome without interfering with expression. Images PMID:1881919

Microtubule Depolymerization as a Driver for Chromosome Motion

NASA Astrophysics Data System (ADS)

McIntosh, Richard

2014-03-01

Microtubules (MTs) are rigid polymers of the protein, tubulin, which function as intracellular struts. They are also tracks along which motor enzymes can run, carrying cargo to specific cellular locations. Most MTs are dynamic; they assemble and disassemble rapidly, particularly during cell division when the cell forms the ``mitotic spindle,'' a machine that organizes the duplicated chromosomes into a planar disk, then pulls the duplicate copies apart, moving them to opposite ends of the cell. This process is necessary for the daughter cells to have a full complement of DNA. The mitotic spindle is a labile framework that exerts several kinds of forces on the chromosomes to move them in well organized ways. It contains many motor enzymes that contribute to spindle formation, but genetic evidence shows that the motors that attach to chromosomes and might contribute to chromosome motion are dispensable for normal mitosis. Apparently MT dynamics can also serve as a motor and is an important source of force for chromosome motion. We have studied this process and find that MTs can be coupled to a load by specific spindle proteins so that MT depolymerization can exert substantial force. With the yeast protein, Dam1, a single MT can generate 30 pN, about 5-fold more than is generated by a motor enzyme like kinesin or myosin. The resulting motions are processive, so a depolymerizing MT can carry its load for many micrometers. However, Dam1 is found only in fungi. We have therefore sought other proteins that can serve as analogous couplers. Several MT-dependent motor enzymes can do the job in ways that do not require ATP, their normal source of energy. Some non-motor MT-associated proteins will also work, e.g., the kinetochore proteins NDC80 and CENP-F. Data will be presented that show the strengths and weaknesses of each coupler, allowing some generalization about how the mitotic machinery works. Supported by NIH GM033787.

Chromosome Transfer Induced Aneuploidy Results in Complex Dysregulation of the Cellular Transcriptome in Immortalized and Cancer Cells

PubMed Central

Upender, Madhvi B.; Habermann, Jens K.; McShane, Lisa M.; Korn, Edward L.; Barrett, J. Carl; Difilippantonio, Michael J.; Ried, Thomas

2016-01-01

Chromosomal aneuploidies are observed in essentially all sporadic carcinomas. These aneuploidies result in tumor-specific patterns of genomic imbalances that are acquired early during tumorigenesis, continuously selected for and faithfully maintained in cancer cells. Although the paradigm of translocation induced oncogene activation in hematologic malignancies is firmly established, it is not known how genomic imbalances affect chromosome-specific gene expression patterns in particular and how chromosomal aneuploidy dysregulates the genetic equilibrium of cells in general. To model specific chromosomal aneuploidies in cancer cells and dissect the immediate consequences of genomic imbalances on the transcriptome, we generated artificial trisomies in a karyotypically stable diploid yet mismatch repair-deficient, colorectal cancer cell line and in telomerase immortalized, cytogenetically normal human breast epithelial cells using microcell-mediated chromosome transfer. The global consequences on gene expression levels were analyzed using cDNA arrays. Our results show that regardless of chromosome or cell type, chromosomal trisomies result in a significant increase in the average transcriptional activity of the trisomic chromosome. This increase affects the expression of numerous genes on other chromosomes as well. We therefore postulate that the genomic imbalances observed in cancer cells exert their effect through a complex pattern of transcriptional dysregulation. PMID:15466185

Multiplex engineering of industrial yeast genomes using CRISPRm.

PubMed

Ryan, Owen W; Cate, Jamie H D

2014-01-01

Global demand has driven the use of industrial strains of the yeast Saccharomyces cerevisiae for large-scale production of biofuels and renewable chemicals. However, the genetic basis of desired domestication traits is poorly understood because robust genetic tools do not exist for industrial hosts. We present an efficient, marker-free, high-throughput, and multiplexed genome editing platform for industrial strains of S. cerevisiae that uses plasmid-based expression of the CRISPR/Cas9 endonuclease and multiple ribozyme-protected single guide RNAs. With this multiplex CRISPR (CRISPRm) system, it is possible to integrate DNA libraries into the chromosome for evolution experiments, and to engineer multiple loci simultaneously. The CRISPRm tools should therefore find use in many higher-order synthetic biology applications to accelerate improvements in industrial microorganisms.

The map-based genome sequence of Spirodela polyrhiza aligned with its chromosomes, a reference for karyotype evolution.

PubMed

Cao, Hieu Xuan; Vu, Giang Thi Ha; Wang, Wenqin; Appenroth, Klaus J; Messing, Joachim; Schubert, Ingo

2016-01-01

Duckweeds are aquatic monocotyledonous plants of potential economic interest with fast vegetative propagation, comprising 37 species with variable genome sizes (0.158-1.88 Gbp). The genomic sequence of Spirodela polyrhiza, the smallest and the most ancient duckweed genome, needs to be aligned to its chromosomes as a reference and prerequisite to study the genome and karyotype evolution of other duckweed species. We selected physically mapped bacterial artificial chromosomes (BACs) containing Spirodela DNA inserts with little or no repetitive elements as probes for multicolor fluorescence in situ hybridization (mcFISH), using an optimized BAC pooling strategy, to validate its physical map and correlate it with its chromosome complement. By consecutive mcFISH analyses, we assigned the originally assembled 32 pseudomolecules (supercontigs) of the genomic sequences to the 20 chromosomes of S. polyrhiza. A Spirodela cytogenetic map containing 96 BAC markers with an average distance of 0.89 Mbp was constructed. Using a cocktail of 41 BACs in three colors, all chromosome pairs could be individualized simultaneously. Seven ancestral blocks emerged from duplicated chromosome segments of 19 Spirodela chromosomes. The chromosomally integrated genome of S. polyrhiza and the established prerequisites for comparative chromosome painting enable future studies on the chromosome homoeology and karyotype evolution of duckweed species. © 2015 IPK Gatersleben. New Phytologist © 2015 New Phytologist Trust.

Links between nucleolar activity, rDNA stability, aneuploidy and chronological aging in the yeast Saccharomyces cerevisiae.

PubMed

Lewinska, Anna; Miedziak, Beata; Kulak, Klaudia; Molon, Mateusz; Wnuk, Maciej

2014-06-01

The nucleolus is speculated to be a regulator of cellular senescence in numerous biological systems (Guarente, Genes Dev 11(19):2449-2455, 1997; Johnson et al., Curr Opin Cell Biol 10(3):332-338, 1998). In the budding yeast Saccharomyces cerevisiae, alterations in nucleolar architecture, the redistribution of nucleolar protein and the accumulation of extrachromosomal ribosomal DNA circles (ERCs) during replicative aging have been reported. However, little is known regarding rDNA stability and changes in nucleolar activity during chronological aging (CA), which is another yeast aging model used. In the present study, the impact of aberrant cell cycle checkpoint control (knock-out of BUB1, BUB2, MAD1 and TEL1 genes in haploid and diploid hemizygous states) on CA-mediated changes in the nucleolus was studied. Nucleolus fragmentation, changes in the nucleolus size and the nucleolus/nucleus ratio, ERC accumulation, expression pattern changes and the relocation of protein involved in transcriptional silencing during CA were revealed. All strains examined were affected by oxidative stress, aneuploidy (numerical rather than structural aberrations) and DNA damage. However, the bub1 cells were the most prone to aneuploidy events, which may contribute to observed decrease in chronological lifespan. We postulate that chronological aging may be affected by redox imbalance-mediated chromosome XII instability leading to both rDNA instability and whole chromosome aneuploidy. CA-mediated nucleolus fragmentation may be a consequence of nucleolus enlargement and/or Nop2p upregulation. Moreover, the rDNA content of chronologically aging cells may be a factor determining the subsequent replicative lifespan. Taken together, we demonstrated that the nucleolus state is also affected during CA in yeast.

Toward functional genomics in bacteria: Analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus

PubMed Central

Rondon, Michelle R.; Raffel, Sandra J.; Goodman, Robert M.; Handelsman, Jo

1999-01-01

As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes. PMID:10339608

A Dispensable Chromosome Is Required for Virulence in the Hemibiotrophic Plant Pathogen Colletotrichum higginsianum

PubMed Central

Plaumann, Peter-Louis; Schmidpeter, Johannes; Dahl, Marlis; Taher, Leila; Koch, Christian

2018-01-01

The hemibiotrophic plant pathogen Colletotrichum higginsianum infects Brassicaceae and in combination with Arabidopsis thaliana, represents an important model system to investigate various ecologically important fungal pathogens and their infection strategies. After penetration of plant cells by appressoria, C. higginsianum establishes large biotrophic primary hyphae in the first infected cell. Shortly thereafter, a switch to necrotrophic growth occurs leading to the invasion of neighboring cells by secondary hyphae. In a forward genetic screen for virulence mutants by insertional mutagenesis, we identified mutants that penetrate the plant but show a defect in the passage from biotrophy to necrotrophy. Genome sequencing and pulsed-field gel electrophoresis revealed that two mutants were lacking chromosome 11, encoding potential pathogenicity genes. We established a chromosome loss assay to verify that strains lacking this small chromosome abort infection during biotrophy, while their ability to grow on artificial media was not affected. C. higginsianum harbors a second small chromosome, which can be lost without effects on virulence or growth on agar plates. Furthermore, we found that chromosome 11 is required to suppress Arabidopsis thaliana plant defense mechanisms dependent on tryptophan derived secondary metabolites. PMID:29867895

The gene for pancreatic polypeptide (PPY) and the anonymous marker D17S78 are within 45 kb of each other on chromosome 17q21

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chandrasekharappa, S.C.; King, S.E.; Lee, Y.H.

1994-05-15

A gene for early-onset breast and ovarian cancer (BRCA1) has been localized to a small region of chromosome 17q21. A combination of genetic linkage studies, radiation-reduced hybrid analysis, and physical mapping by FISH has identified several genes/markers that lie in this interval. Among these are the gene encoding pancreatic polypeptide (PPY) and a polymorphic marker at locus D17S78. Efforts to construct a physical map of this region by isolating a large number of yeast artificial chromosome (YAC) and cosmid clones demonstrate that PPY and D17S78 are present within the same cosmid clone, and therefore no farther than 45 kb apart.more » This observation takes on particular significance since it excludes a recently described BRCA1 candidate gene from the interval defined by meiotic mapping. Although PPY and D17S78 were found to be no farther than 45 kb apart, identification of a smaller fragment that hybridizes to both probes would indicate that these two are much closer. The probe p131 and the gene PPY were previously mapped to 17q21-q23 and to the proximal long arm of chromosome 17, respectively. The demonstration of the close proximity of these markers should allow them to be treated as a single locus in terms of long-range genomic mapping of this region, and the genomic clones isolated should serve as useful resources for the identification of the BRCA1 gene. Analysis of a large number of a familial and spordic breast and ovarian cancers has identified frequent loss of heterozygosity near the BRCA1 locus. A recent report has suggested the responsible interval lies just telomeric to PPY, and a suggested candidate gene (MCD) for BRCA1 was found to be somatically rearranged in two of several hundred sporadic breast tumors.« less

Protein aggregation induced during glass bead lysis of yeast

PubMed Central

Papanayotou, Irene; Sun, Beimeng; Roth, Amy F.; Davis, Nicholas G.

2013-01-01

Yeast cell lysates produced by mechanical glass bead disruption are widely used in a variety of applications, including for the analysis of native function, e.g. protein–protein interaction, enzyme assays and membrane fractionations. Below, we report a striking case of protein denaturation and aggregation that is induced by this lysis protocol. Most of this analysis focuses on the type 1 casein kinase Yck2, which normally tethers to the plasma membrane through C-terminal palmitoylation. Surprisingly, when cells are subjected to glass bead disruption, non-palmitoylated, cytosolic forms of the kinase denature and aggregate, while membrane-associated forms, whether attached through their native palmitoyl tethers or through a variety of artificial membrane-tethering sequences, are wholly protected from denaturation and aggregation. A wider look at the yeast proteome finds that, while the majority of proteins resist glass bead-induced aggregation, a significant subset does, in fact, succumb to such denaturation. Thus, yeast researchers should be aware of this potential artifact when embarking on biochemical analyses that employ glass bead lysates to look at native protein function. Finally, we demonstrate an experimental utility for glass bead-induced aggregation, using its fine discrimination of membrane-associated from non-associated Yck2 forms to discern fractional palmitoylation states of Yck2 mutants that are partially defective for palmitoylation. PMID:20641011

Live-cell imaging of nuclear-chromosomal dynamics in bovine in vitro fertilised embryos.

PubMed

Yao, Tatsuma; Suzuki, Rie; Furuta, Natsuki; Suzuki, Yuka; Kabe, Kyoko; Tokoro, Mikiko; Sugawara, Atsushi; Yajima, Akira; Nagasawa, Tomohiro; Matoba, Satoko; Yamagata, Kazuo; Sugimura, Satoshi

2018-05-10

Nuclear/chromosomal integrity is an important prerequisite for the assessment of embryo quality in artificial reproductive technology. However, lipid-rich dark cytoplasm in bovine embryos prevents its observation by visible light microscopy. We performed live-cell imaging using confocal laser microscopy that allowed long-term imaging of nuclear/chromosomal dynamics in bovine in vitro fertilised (IVF) embryos. We analysed the relationship between nuclear/chromosomal aberrations and in vitro embryonic development and morphological blastocyst quality. Three-dimensional live-cell imaging of 369 embryos injected with mRNA encoding histone H2B-mCherry and enhanced green fluorescent protein (EGFP)-α-tubulin was performed from single-cell to blastocyst stage for eight days; 17.9% reached the blastocyst stage. Abnormalities in the number of pronuclei (PN), chromosomal segregation, cytokinesis, and blastomere number at first cleavage were observed at frequencies of 48.0%, 30.6%, 8.1%, and 22.2%, respectively, and 13.0%, 6.2%, 3.3%, and 13.4%, respectively, for abnormal embryos developed into blastocysts. A multivariate analysis showed that abnormal chromosome segregation (ACS) and multiple PN correlated with delayed timing and abnormal blastomere number at first cleavage, respectively. In morphologically transferrable blastocysts, 30-40% of embryos underwent ACS and had abnormal PN. Live-cell imaging may be useful for analysing the association between nuclear/chromosomal dynamics and embryonic development in bovine embryos.

Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.

PubMed

Chen, Chao; Zhao, Xinqing; Jin, Yingyu; Zhao, Zongbao Kent; Suh, Joo-Won

2014-11-01

Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by λ-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates

Stochastic simulations of a synthetic bacteria-yeast ecosystem

PubMed Central

2012-01-01

Background The field of synthetic biology has greatly evolved and numerous functions can now be implemented by artificially engineered cells carrying the appropriate genetic information. However, in order for the cells to robustly perform complex or multiple tasks, co-operation between them may be necessary. Therefore, various synthetic biological systems whose functionality requires cell-cell communication are being designed. These systems, microbial consortia, are composed of engineered cells and exhibit a wide range of behaviors. These include yeast cells whose growth is dependent on one another, or bacteria that kill or rescue each other, synchronize, behave as predator-prey ecosystems or invade cancer cells. Results In this paper, we study a synthetic ecosystem comprising of bacteria and yeast that communicate with and benefit from each other using small diffusible molecules. We explore the behavior of this heterogeneous microbial consortium, composed of Saccharomyces cerevisiae and Escherichia coli cells, using stochastic modeling. The stochastic model captures the relevant intra-cellular and inter-cellular interactions taking place in and between the eukaryotic and prokaryotic cells. Integration of well-characterized molecular regulatory elements into these two microbes allows for communication through quorum sensing. A gene controlling growth in yeast is induced by bacteria via chemical signals and vice versa. Interesting dynamics that are common in natural ecosystems, such as obligatory and facultative mutualism, extinction, commensalism and predator-prey like dynamics are observed. We investigate and report on the conditions under which the two species can successfully communicate and rescue each other. Conclusions This study explores the various behaviors exhibited by the cohabitation of engineered yeast and bacterial cells. The way that the model is built allows for studying the dynamics of any system consisting of two species communicating with one

Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha.

PubMed

Tikhomirova, L P; Ikonomova, R N; Kuznetsova, E N

1986-01-01

For the transformation of the yeast Hansenula polymorpha we have constructed a set of hybrid plasmids carrying the LEU2 gene of Saccharomyces cerevisiae as a selective marker and fragments of mitochondrial DNA of Candida utilis and H. polymorpha or chromosomal DNA fragments of H. polymorpha as replicator sequences. The replication properties of chimeric plasmids in the yeast H. polymorpha were investigated. We showed that for plasmids propagated autonomously in this yeast the plasmid monomers could be detected in the transformants only during the immediate time after the transformation event. Further growth under selective conditions led to the selection of polymeric forms of plasmid DNA as it was clearly shown for transformants carrying cosmid pL2 with mtDNA fragment of C. utilis. Such transformants carrying polymerized plasmids showed a remarkably increased stability of the transformed phenotype. Cosmid pL2 was able to shuttle between Escherichia coli, S. cerevisiae and H. polymorpha, whereas plasmids with DNA fragments from H. polymorpha did not transform S. cerevisiae effectively.

The YeastGenome app: the Saccharomyces Genome Database at your fingertips.

PubMed

Wong, Edith D; Karra, Kalpana; Hitz, Benjamin C; Hong, Eurie L; Cherry, J Michael

2013-01-01

The Saccharomyces Genome Database (SGD) is a scientific database that provides researchers with high-quality curated data about the genes and gene products of Saccharomyces cerevisiae. To provide instant and easy access to this information on mobile devices, we have developed YeastGenome, a native application for the Apple iPhone and iPad. YeastGenome can be used to quickly find basic information about S. cerevisiae genes and chromosomal features regardless of internet connectivity. With or without network access, you can view basic information and Gene Ontology annotations about a gene of interest by searching gene names and gene descriptions or by browsing the database within the app to find the gene of interest. With internet access, the app provides more detailed information about the gene, including mutant phenotypes, references and protein and genetic interactions, as well as provides hyperlinks to retrieve detailed information by showing SGD pages and views of the genome browser. SGD provides online help describing basic ways to navigate the mobile version of SGD, highlights key features and answers frequently asked questions related to the app. The app is available from iTunes (http://itunes.com/apps/yeastgenome). The YeastGenome app is provided freely as a service to our community, as part of SGD's mission to provide free and open access to all its data and annotations.

Rif1 is a global regulator of timing of replication origin firing in fission yeast

PubMed Central

Hayano, Motoshi; Kanoh, Yutaka; Matsumoto, Seiji; Renard-Guillet, Claire; Shirahige, Katsuhiko; Masai, Hisao

2012-01-01

One of the long-standing questions in eukaryotic DNA replication is the mechanisms that determine where and when a particular segment of the genome is replicated. Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication and may affect the site selection and timing of origin firing. We identified rif1Δ, a null mutant of rif1+, a conserved telomere-binding factor, as an efficient bypass mutant of fission yeast hsk1. Extensive deregulation of dormant origins over a wide range of the chromosomes occurs in rif1Δ in the presence or absence of hydroxyurea (HU). At the same time, many early-firing, efficient origins are suppressed or delayed in firing timing in rif1Δ. Rif1 binds not only to telomeres, but also to many specific locations on the arm segments that only partially overlap with the prereplicative complex assembly sites, although Rif1 tends to bind in the vicinity of the late/dormant origins activated in rif1Δ. The binding to the arm segments occurs through M to G1 phase in a manner independent of Taz1 and appears to be essential for the replication timing program during the normal cell cycle. Our data demonstrate that Rif1 is a critical determinant of the origin activation program on the fission yeast chromosomes. PMID:22279046

Mapping of the locus for autosomal dominant amelogenesis imperfecta (AIH2) to a 4-Mb YAC contig on chromosome 4q11-q21

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaerrman, C.; Holmgren, G.; Forsman, K.

1997-01-15

Amelogenesis imperfecta (Al) is a clinically and genetically heterogeneous group of inherited enamel defects. We recently mapped a locus for autosomal dominant local hypoplastic amelogenesis imperfecta (AIH2) to the long arm of chromosome 4. The disease gene was localized to a 17.6-cM region between the markers D4S392 and D4S395. The albumin gene (ALB), located in the same interval, was a candidate gene for autosomal dominant AI (ADAI) since albumin has a potential role in enamel maturation. Here we describe refined mapping of the AIH2 locus and the construction of marker maps by radiation hybrid mapping and yeast artificial chromosome (YAC)-basedmore » sequence tagged site-content mapping. A radiation hybrid map consisting of 11 microsatellite markers in the 5-cM interval between D4S409 and D4S1558 was constructed. Recombinant haplotypes in six Swedish ADAI families suggest that the disease gene is located in the interval between D4S2421 and ALB. ALB is therefore not likely to be the disease-causing gene. Affected members in all six families share the same allele haplotypes, indicating a common ancestral mutation in all families. The AIH2 critical region is less than 4 cM and spans a physical distance of approximately 4 Mb as judged from radiation hybrid maps. A YAC contig over the AIH2 critical region including several potential candidate genes was constructed. 35 refs., 4 figs., 1 tab.« less

Fluorescent in situ hybridization shows DIPLOSPOROUS located on one of the NOR chromosomes in apomictic dandelions (Taraxacum) in the absence of a large hemizygous chromosomal region.

PubMed

Vašut, Radim J; Vijverberg, Kitty; van Dijk, Peter J; de Jong, Hans

2014-11-01

Apomixis in dandelions (Taraxacum: Asteraceae) is encoded by two unlinked dominant loci and a third yet undefined genetic factor: diplosporous omission of meiosis (DIPLOSPOROUS, DIP), parthenogenetic embryo development (PARTHENOGENESIS, PAR), and autonomous endosperm formation, respectively. In this study, we determined the chromosomal position of the DIP locus in Taraxacum by using fluorescent in situ hybridization (FISH) with bacterial artificial chromosomes (BACs) that genetically map within 1.2-0.2 cM of DIP. The BACs showed dispersed fluorescent signals, except for S4-BAC 83 that displayed strong unique signals as well. Under stringent blocking of repeats by C0t-DNA fragments, only a few fluorescent foci restricted to defined chromosome regions remained, including one on the nucleolus organizer region (NOR) chromosomes that contains the 45S rDNAs. FISH with S4-BAC 83 alone and optimal blocking showed discrete foci in the middle of the long arm of one of the NOR chromosomes only in triploid and tetraploid diplosporous dandelions, while signals in sexual diploids were lacking. This agrees with the genetic model of a single dose, dominant DIP allele, absent in sexuals. The length of the DIP region is estimated to cover a region of 1-10 Mb. FISH in various accessions of Taraxacum and the apomictic sister species Chondrilla juncea, confirmed the chromosomal position of DIP within Taraxacum but not outside the genus. Our results endorse that, compared to other model apomictic species, expressing either diplospory or apospory, the genome of Taraxacum shows a more similar and less diverged chromosome structure at the DIP locus. The different levels of allele sequence divergence at apomeiosis loci may reflect different terms of asexual reproduction. The association of apomeiosis loci with repetitiveness, dispersed repeats, and retrotransposons commonly observed in apomictic species may imply a functional role of these shared features in apomictic reproduction, as is

BuGZ is required for Bub3 stability, Bub1 kinetochore function, and chromosome alignment

PubMed Central

Toledo, Chad M.; Herman, Jacob A.; Olsen, Jonathan B.; Ding, Yu; Corrin, Philip; Girard, Emily J.; Olson, James M.; Emili, Andrew; DeLuca, Jennifer G.; Paddison, Patrick J.

2014-01-01

Summary During mitosis, the spindle assembly checkpoint (SAC) monitors the attachment of kinetochores (KTs) to the plus ends of spindle microtubules (MTs) and prevents anaphase onset until chromosomes are aligned and KTs are under proper tension. Here, we identify a SAC component, BuGZ/ZNF207, from an RNAi viability screen in human Glioblastoma multiforme (GBM) brain tumor stem cells. BuGZ binds to and stabilizes Bub3 during interphase and mitosis through a highly conserved GLE2p-binding sequence (GLEBS) domain. Inhibition of BuGZ results in loss of both Bub3 and its binding partner Bub1 from KTs, reduction of Bub1-dependent phosphorylation of centromeric histone H2A, attenuation of KT-based Aurora kinase B activity, and lethal chromosome congression defects in cancer cells. Phylogenetic analysis indicates that BuGZ orthologs are highly conserved among eukaryotes, but are conspicuously absent from budding and fission yeasts. These findings suggest BuGZ has evolved to facilitate Bub3 activity and chromosome congression in higher eukaryotes. PMID:24462187

Drosophila Regulate Yeast Density and Increase Yeast Community Similarity in a Natural Substrate

PubMed Central

Stamps, Judy A.; Yang, Louie H.; Morales, Vanessa M.; Boundy-Mills, Kyria L.

2012-01-01

Drosophila melanogaster adults and larvae, but especially larvae, had profound effects on the densities and community structure of yeasts that developed in banana fruits. Pieces of fruit exposed to adult female flies previously fed fly-conditioned bananas developed higher yeast densities than pieces of the same fruits that were not exposed to flies, supporting previous suggestions that adult Drosophila vector yeasts to new substrates. However, larvae alone had dramatic effects on yeast density and species composition. When yeast densities were compared in pieces of the same fruits assigned to different treatments, fruits that developed low yeast densities in the absence of flies developed significantly higher yeast densities when exposed to larvae. Across all of the fruits, larvae regulated yeast densities within narrow limits, as compared to a much wider range of yeast densities that developed in pieces of the same fruits not exposed to flies. Larvae also affected yeast species composition, dramatically reducing species diversity across fruits, reducing variation in yeast communities from one fruit to the next (beta diversity), and encouraging the consistent development of a yeast community composed of three species of yeast (Candida californica, C. zemplinina, and Pichia kluvyeri), all of which were palatable to larvae. Larvae excreted viable cells of these three yeast species in their fecal pools, and discouraged the growth of filamentous fungi, processes which may have contributed to their effects on the yeast communities in banana fruits. These and other findings suggest that D. melanogaster adults and their larval offspring together engage in ‘niche construction’, facilitating a predictable microbial environment in the fruit substrates in which the larvae live and develop. PMID:22860093

Long-read sequencing data analysis for yeasts.

PubMed

Yue, Jia-Xing; Liti, Gianni

2018-06-01

Long-read sequencing technologies have become increasingly popular due to their strengths in resolving complex genomic regions. As a leading model organism with small genome size and great biotechnological importance, the budding yeast Saccharomyces cerevisiae has many isolates currently being sequenced with long reads. However, analyzing long-read sequencing data to produce high-quality genome assembly and annotation remains challenging. Here, we present a modular computational framework named long-read sequencing data analysis for yeasts (LRSDAY), the first one-stop solution that streamlines this process. Starting from the raw sequencing reads, LRSDAY can produce chromosome-level genome assembly and comprehensive genome annotation in a highly automated manner with minimal manual intervention, which is not possible using any alternative tool available to date. The annotated genomic features include centromeres, protein-coding genes, tRNAs, transposable elements (TEs), and telomere-associated elements. Although tailored for S. cerevisiae, we designed LRSDAY to be highly modular and customizable, making it adaptable to virtually any eukaryotic organism. When applying LRSDAY to an S. cerevisiae strain, it takes ∼41 h to generate a complete and well-annotated genome from ∼100× Pacific Biosciences (PacBio) running the basic workflow with four threads. Basic experience working within the Linux command-line environment is recommended for carrying out the analysis using LRSDAY.

Molecular analysis of recombination in a family with Duchenne muscular dystrophy and a large pericentric X chromosome inversion.

PubMed Central

Shashi, V.; Golden, W. L.; Allinson, P. S.; Blanton, S. H.; von Kap-Herr, C.; Kelly, T. E.

1996-01-01

It has been demonstrated in animal studies that, in animals heterozygous for pericentric chromosomal inversions, loop formation is greatly reduced during meiosis. This results in absence of recombination within the inverted segment, with recombination seen only outside the inversion. A recent study in yeast has shown that telomeres, rather than centromeres, lead in chromosome movement just prior to meiosis and may be involved in promoting recombination. We studied by cytogenetic analysis and DNA polymorphisms the nature of meiotic recombination in a three-generation family with a large pericentric X chromosome inversion, inv(X)(p21.1q26), in which Duchenne muscular dystrophy (DMD) was cosegregating with the inversion. On DNA analysis there was no evidence of meiotic recombination between the inverted and normal X chromosomes in the inverted segment. Recombination was seen at the telomeric regions, Xp22 and Xq27-28. No deletion or point mutation was found on analysis of the DMD gene. On the basis of the FISH results, we believe that the X inversion is the mutation responsible for DMD in this family. Our results indicate that (1) pericentric X chromosome inversions result in reduction of recombination between the normal and inverted X chromosomes; (2) meiotic X chromosome pairing in these individuals is likely initiated at the telomeres; and (3) in this family DMD is caused by the pericentric inversion. Images Figure 2 Figure 5 Figure 6 Figure 7 PMID:8651300

Yeast Reporter Assay to Identify Cellular Components of Ricin Toxin A Chain Trafficking.

PubMed

Becker, Björn; Schnöder, Tina; Schmitt, Manfred J

2016-12-06

RTA, the catalytic A-subunit of the ribosome inactivating A/B toxin ricin, inhibits eukaryotic protein biosynthesis by depurination of 28S rRNA. Although cell surface binding of ricin holotoxin is mainly mediated through its B-subunit (RTB), sole application of RTA is also toxic, albeit to a significantly lower extent, suggesting alternative pathways for toxin uptake and transport. Since ricin toxin trafficking in mammalian cells is still not fully understood, we developed a GFP-based reporter assay in yeast that allows rapid identification of cellular components required for RTA uptake and subsequent transport through a target cell. We hereby show that Ypt6p, Sft2p and GARP-complex components play an important role in RTA transport, while neither the retromer complex nor COPIB vesicles are part of the transport machinery. Analyses of yeast knock-out mutants with chromosomal deletion in genes whose products regulate ADP-ribosylation factor GTPases (Arf-GTPases) and/or retrograde Golgi-to-ER (endoplasmic reticulum) transport identified Sso1p, Snc1p, Rer1p, Sec22p, Erv46p, Gea1p and Glo3p as novel components in RTA transport, suggesting the developed reporter assay as a powerful tool to dissect the multistep processes of host cell intoxication in yeast.

Transcription of a protein-coding gene on B chromosomes of the Siberian roe deer (Capreolus pygargus)

PubMed Central

2013-01-01

Background Most eukaryotic species represent stable karyotypes with a particular diploid number. B chromosomes are additional to standard karyotypes and may vary in size, number and morphology even between cells of the same individual. For many years it was generally believed that B chromosomes found in some plant, animal and fungi species lacked active genes. Recently, molecular cytogenetic studies showed the presence of additional copies of protein-coding genes on B chromosomes. However, the transcriptional activity of these genes remained elusive. We studied karyotypes of the Siberian roe deer (Capreolus pygargus) that possess up to 14 B chromosomes to investigate the presence and expression of genes on supernumerary chromosomes. Results Here, we describe a 2 Mbp region homologous to cattle chromosome 3 and containing TNNI3K (partial), FPGT, LRRIQ3 and a large gene-sparse segment on B chromosomes of the Siberian roe deer. The presence of the copy of the autosomal region was demonstrated by B-specific cDNA analysis, PCR assisted mapping, cattle bacterial artificial chromosome (BAC) clone localization and quantitative polymerase chain reaction (qPCR). By comparative analysis of B-specific and non-B chromosomal sequences we discovered some B chromosome-specific mutations in protein-coding genes, which further enabled the detection of a FPGT-TNNI3K transcript expressed from duplicated genes located on B chromosomes in roe deer fibroblasts. Conclusions Discovery of a large autosomal segment in all B chromosomes of the Siberian roe deer further corroborates the view of an autosomal origin for these elements. Detection of a B-derived transcript in fibroblasts implies that the protein coding sequences located on Bs are not fully inactivated. The origin, evolution and effect on host of B chromosomal genes seem to be similar to autosomal segmental duplications, which reinforces the view that supernumerary chromosomal elements might play an important role in genome

Recent advances in yeast cell-surface display technologies for waste biorefineries.

PubMed

Liu, Zhuo; Ho, Shih-Hsin; Hasunuma, Tomohisa; Chang, Jo-Shu; Ren, Nan-Qi; Kondo, Akihiko

2016-09-01

Waste biorefinery aims to maximize the output of value-added products from various artificial/agricultural wastes by using integrated bioprocesses. To make waste biorefinery economically feasible, it is thus necessary to develop a low-cost, environment-friendly technique to perform simultaneous biodegradation and bioconversion of waste materials. Cell-surface display engineering is a novel, cost-effective technique that can auto-immobilize proteins on the cell exterior of microorganisms, and has been applied for use with waste biofinery. Through tethering different enzymes (e.g., cellulase, lipase, and protease) or metal-binding peptides on cell surfaces, various yeast strains can effectively produce biofuels and biochemicals from sugar/protein-rich waste materials, catalyze waste oils into biodiesels, or retrieve heavy metals from wastewater. This review critically summarizes recent applications of yeast cell-surface display on various types of waste biorefineries, highlighting its potential and future challenges with regard to commercializing this technology. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of a novel interspecific hybrid yeast from a metagenomic spontaneously inoculated beer sample using Hi-C.

PubMed

Smukowski Heil, Caiti; Burton, Joshua N; Liachko, Ivan; Friedrich, Anne; Hanson, Noah A; Morris, Cody L; Schacherer, Joseph; Shendure, Jay; Thomas, James H; Dunham, Maitreya J

2018-01-01

Interspecific hybridization is a common mechanism enabling genetic diversification and adaptation; however, the detection of hybrid species has been quite difficult. The identification of microbial hybrids is made even more complicated, as most environmental microbes are resistant to culturing and must be studied in their native mixed communities. We have previously adapted the chromosome conformation capture method Hi-C to the assembly of genomes from mixed populations. Here, we show the method's application in assembling genomes directly from an uncultured, mixed population from a spontaneously inoculated beer sample. Our assembly method has enabled us to de-convolute four bacterial and four yeast genomes from this sample, including a putative yeast hybrid. Downstream isolation and analysis of this hybrid confirmed its genome to consist of Pichia membranifaciens and that of another related, but undescribed, yeast. Our work shows that Hi-C-based metagenomic methods can overcome the limitation of traditional sequencing methods in studying complex mixtures of genomes. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Characterization of chromosomal architecture in Arabidopsis by chromosome conformation capture

PubMed Central

2013-01-01

Background The packaging of long chromatin fibers in the nucleus poses a major challenge, as it must fulfill both physical and functional requirements. Until recently, insights into the chromosomal architecture of plants were mainly provided by cytogenetic studies. Complementary to these analyses, chromosome conformation capture technologies promise to refine and improve our view on chromosomal architecture and to provide a more generalized description of nuclear organization. Results Employing circular chromosome conformation capture, this study describes chromosomal architecture in Arabidopsis nuclei from a genome-wide perspective. Surprisingly, the linear organization of chromosomes is reflected in the genome-wide interactome. In addition, we study the interplay of the interactome and epigenetic marks and report that the heterochromatic knob on the short arm of chromosome 4 maintains a pericentromere-like interaction profile and interactome despite its euchromatic surrounding. Conclusion Despite the extreme condensation that is necessary to pack the chromosomes into the nucleus, the Arabidopsis genome appears to be packed in a predictive manner, according to the following criteria: heterochromatin and euchromatin represent two distinct interactomes; interactions between chromosomes correlate with the linear position on the chromosome arm; and distal chromosome regions have a higher potential to interact with other chromosomes. PMID:24267747

Copy Number Variation in Fungi and Its Implications for Wine Yeast Genetic Diversity and Adaptation

PubMed Central

Steenwyk, Jacob L.; Rokas, Antonis

2018-01-01

In recent years, copy number (CN) variation has emerged as a new and significant source of genetic polymorphisms contributing to the phenotypic diversity of populations. CN variants are defined as genetic loci that, due to duplication and deletion, vary in their number of copies across individuals in a population. CN variants range in size from 50 base pairs to whole chromosomes, can influence gene activity, and are associated with a wide range of phenotypes in diverse organisms, including the budding yeast Saccharomyces cerevisiae. In this review, we introduce CN variation, discuss the genetic and molecular mechanisms implicated in its generation, how they can contribute to genetic and phenotypic diversity in fungal populations, and consider how CN variants may influence wine yeast adaptation in fermentation-related processes. In particular, we focus on reviewing recent work investigating the contribution of changes in CN of fermentation-related genes in yeast wine strains and offer notable illustrations of such changes, including the high levels of CN variation among the CUP genes, which confer resistance to copper, a metal with fungicidal properties, and the preferential deletion and duplication of the MAL1 and MAL3 loci, respectively, which are responsible for metabolizing maltose and sucrose. Based on the available data, we propose that CN variation is a substantial dimension of yeast genetic diversity that occurs largely independent of single nucleotide polymorphisms. As such, CN variation harbors considerable potential for understanding and manipulating yeast strains in the wine fermentation environment and beyond. PMID:29520259

Rapid and efficient introduction of a foreign gene into bacterial artificial chromosome-cloned varicella vaccine by Tn7-mediated site-specific transposition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Somboonthum, Pranee; Koshizuka, Tetsuo; Okamoto, Shigefumi

2010-06-20

Using a rapid and reliable system based on Tn7-mediated site-specific transposition, we have successfully constructed a recombinant Oka varicella vaccine (vOka) expressing the mumps virus (MuV) fusion protein (F). The backbone of the vector was our previously reported vOka-BAC (bacterial artificial chromosome) genome. We inserted the transposon Tn7 attachment sequence, LacZ{alpha}-mini-attTn7, into the region between ORF12 and ORF13 to generate a vOka-BAC-Tn genome. The MuV-F expressing cassette was transposed into the vOka-BAC genome at the mini-attTn7 transposition site. MuV-F protein was expressed in recombinant virus, rvOka-F infected cells. In addition, the MuV-F protein was cleaved in the rvOka-F infected cellsmore » as in MuV-infected cells. The growth of rvOka-F was similar to that of the original recombinant vOka without the F gene. Thus, we show that Tn7-mediated transposition is an efficient method for introducing a foreign gene expression cassette into the vOka-BAC genome as a live virus vector.« less

Mapping yeast origins of replication via single-stranded DNA detection.

PubMed

Feng, Wenyi; Raghuraman, M K; Brewer, Bonita J

2007-02-01

Studies in th Saccharomyces cerevisiae have provided a framework for understanding how eukaryotic cells replicate their chromosomal DNA to ensure faithful transmission of genetic information to their daughter cells. In particular, S. cerevisiae is the first eukaryote to have its origins of replication mapped on a genomic scale, by three independent groups using three different microarray-based approaches. Here we describe a new technique of origin mapping via detection of single-stranded DNA in yeast. This method not only identified the majority of previously discovered origins, but also detected new ones. We have also shown that this technique can identify origins in Schizosaccharomyces pombe, illustrating the utility of this method for origin mapping in other eukaryotes.

Gametocidal chromosomes enhancing chromosome aberration in common wheat induced by 5-azacytidine.

PubMed

Su, W-Y; Cong, W-W; Shu, Y-J; Wang, D; Xu, G-H; Guo, C-H

2013-07-08

The gametocidal (Gc) chromosome from Aegilops spp induces chromosome mutation, which is introduced into common wheat as a tool of chromosome manipulation for genetic improvement. The Gc chromosome functions similar to a restriction-modification system in bacteria, in which DNA methylation is an important regulator. We treated root tips of wheat carrying Gc chromosomes with the hypomethylation agent 5-azacytidine; chromosome breakage and micronuclei were observed in these root tips. The frequency of aberrations differed in wheat containing different Gc chromosomes, suggesting different functions inducing chromosome breakage. Gc chromosome 3C caused the greatest degree of chromosome aberration, while Gc chromosome 3C(SAT) and 2C caused only slight chromosome aberration. Gc chromosome 3C induced different degrees of chromosome aberration in wheat varieties Triticum aestivum var. Chinese Spring and Norin 26, demonstrating an inhibition function in common wheat.

Prions in Yeast

PubMed Central

Liebman, Susan W.; Chernoff, Yury O.

2012-01-01

The concept of a prion as an infectious self-propagating protein isoform was initially proposed to explain certain mammalian diseases. It is now clear that yeast also has heritable elements transmitted via protein. Indeed, the “protein only” model of prion transmission was first proven using a yeast prion. Typically, known prions are ordered cross-β aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in understanding cellular control of prion propagation, prion structure, mechanisms of de novo prion formation, specificity of prion transmission, and the biological roles of prions. This review summarizes what has been learned from yeast prions. PMID:22879407

Brownian dynamics simulation of fission yeast mitotic spindle formation

NASA Astrophysics Data System (ADS)

Edelmaier, Christopher

2014-03-01

The mitotic spindle segregates chromosomes during mitosis. The dynamics that establish bipolar spindle formation are not well understood. We have developed a computational model of fission-yeast mitotic spindle formation using Brownian dynamics and kinetic Monte Carlo methods. Our model includes rigid, dynamic microtubules, a spherical nuclear envelope, spindle pole bodies anchored in the nuclear envelope, and crosslinkers and crosslinking motor proteins. Crosslinkers and crosslinking motor proteins attach and detach in a grand canonical ensemble, and exert forces and torques on the attached microtubules. We have modeled increased affinity for crosslinking motor attachment to antiparallel microtubule pairs, and stabilization of microtubules in the interpolar bundle. We study parameters controlling the stability of the interpolar bundle and assembly of a bipolar spindle from initially adjacent spindle-pole bodies.

Evolution of the dispersed SUC gene family of Saccharomyces by rearrangements of chromosome telomeres.

PubMed Central

Carlson, M; Celenza, J L; Eng, F J

1985-01-01

The SUC gene family of Saccharomyces contains six structural genes for invertase (SUC1 through SUC5 and SUC7) which are located on different chromosomes. Most yeast strains do not carry all six SUC genes and instead carry natural negative (suc0) alleles at some or all SUC loci. We determined the physical structures of SUC and suc0 loci. Except for SUC2, which is an unusual member of the family, all of the SUC genes are located very close to telomeres and are flanked by homologous sequences. On the centromere-proximal side of the gene, the conserved region contains X sequences, which are sequences found adjacent to telomeres (C. S. M. Chan and B.-K. Tye, Cell 33:563-573, 1983). On the other side of the gene, the homology includes about 4 kilobases of flanking sequence and then extends into a Y' element, which is an element often found distal to the X sequence at telomeres (Chan and Tye, Cell 33:563-573, 1983). Thus, these SUC genes and flanking sequences are embedded in telomere-adjacent sequences. Chromosomes carrying suc0 alleles (except suc20) lack SUC structural genes and portions of the conserved flanking sequences. The results indicate that the dispersal of SUC genes to different chromosomes occurred by rearrangements of chromosome telomeres. Images PMID:3018485

Microarray-Based Comparative Genomic Hybridization Using Sex-Matched Reference DNA Provides Greater Sensitivity for Detection of Sex Chromosome Imbalances than Array-Comparative Genomic Hybridization with Sex-Mismatched Reference DNA

PubMed Central

Yatsenko, Svetlana A.; Shaw, Chad A.; Ou, Zhishuo; Pursley, Amber N.; Patel, Ankita; Bi, Weimin; Cheung, Sau Wai; Lupski, James R.; Chinault, A. Craig; Beaudet, Arthur L.

2009-01-01

In array-comparative genomic hybridization (array-CGH) experiments, the measurement of DNA copy number of sex chromosomal regions depends on the sex of the patient and the reference DNAs used. We evaluated the ability of bacterial artificial chromosomes/P1-derived artificial and oligonucleotide array-CGH analyses to detect constitutional sex chromosome imbalances using sex-mismatched reference DNAs. Twenty-two samples with imbalances involving either the X or Y chromosome, including deletions, duplications, triplications, derivative or isodicentric chromosomes, and aneuploidy, were analyzed. Although concordant results were obtained for approximately one-half of the samples when using sex-mismatched and sex-matched reference DNAs, array-CGH analyses with sex-mismatched reference DNAs did not detect genomic imbalances that were detected using sex-matched reference DNAs in 6 of 22 patients. Small duplications and deletions of the X chromosome were most difficult to detect in female and male patients, respectively, when sex-mismatched reference DNAs were used. Sex-matched reference DNAs in array-CGH analyses provides optimal sensitivity and enables an automated statistical evaluation for the detection of sex chromosome imbalances when compared with an experimental design using sex-mismatched reference DNAs. Using sex-mismatched reference DNAs in array-CGH analyses may generate false-negative, false-positive, and ambiguous results for sex chromosome-specific probes, thus masking potential pathogenic genomic imbalances. Therefore, to optimize both detection of clinically relevant sex chromosome imbalances and ensure proper experimental performance, we suggest that alternative internal controls be developed and used instead of using sex-mismatched reference DNAs. PMID:19324990

Screening of a Brassica napus bacterial artificial chromosome library using highly parallel single nucleotide polymorphism assays

PubMed Central

2013-01-01

Background Efficient screening of bacterial artificial chromosome (BAC) libraries with polymerase chain reaction (PCR)-based markers is feasible provided that a multidimensional pooling strategy is implemented. Single nucleotide polymorphisms (SNPs) can be screened in multiplexed format, therefore this marker type lends itself particularly well for medium- to high-throughput applications. Combining the power of multiplex-PCR assays with a multidimensional pooling system may prove to be especially challenging in a polyploid genome. In polyploid genomes two classes of SNPs need to be distinguished, polymorphisms between accessions (intragenomic SNPs) and those differentiating between homoeologous genomes (intergenomic SNPs). We have assessed whether the highly parallel Illumina GoldenGate® Genotyping Assay is suitable for the screening of a BAC library of the polyploid Brassica napus genome. Results A multidimensional screening platform was developed for a Brassica napus BAC library which is composed of almost 83,000 clones. Intragenomic and intergenomic SNPs were included in Illumina’s GoldenGate® Genotyping Assay and both SNP classes were used successfully for screening of the multidimensional BAC pools of the Brassica napus library. An optimized scoring method is proposed which is especially valuable for SNP calling of intergenomic SNPs. Validation of the genotyping results by independent methods revealed a success of approximately 80% for the multiplex PCR-based screening regardless of whether intra- or intergenomic SNPs were evaluated. Conclusions Illumina’s GoldenGate® Genotyping Assay can be efficiently used for screening of multidimensional Brassica napus BAC pools. SNP calling was specifically tailored for the evaluation of BAC pool screening data. The developed scoring method can be implemented independently of plant reference samples. It is demonstrated that intergenomic SNPs represent a powerful tool for BAC library screening of a polyploid genome

Screening of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae.

PubMed

Zhang, Chenlu; Acosta-Sampson, Ligia; Yu, Vivian Yaci; Cate, Jamie H D

2017-01-01

The economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

Isogenic transgenic homozygous fish induced by artificial parthenogenesis.

PubMed

Nam, Y K; Cho, Y S; Kim, D S

2000-12-01

As a model system for vertebrate transgenesis, fish have many attractive advantages, especially with respect to the characteristics of eggs, allowing us to produce isogenic, transgenic, homozygous vertebrates by combining with chromosome-set manipulation. Here, we describe the large-scale production of isogenic transgenic homozygous animals using our experimental organism, the mud loach Misgurnus mizolepis, by the simple process of artificial parthenogenesis in a single generation. These isogenic fish have retained transgenic homozygous status in a stable manner during the subsequent 5 years, and exhibited increased levels of transgene expression. Furthermore, their isogenic nature was confirmed by cloned transgenic homozygous offspring produced via another step of parthenogenic reproduction of the isogenic homozygous transgenic fish. These results demonstrate that a combination of transgenesis and artificial parthenogenesis will make the rapid utilization of genetically pure homozygous transgenic system in vertebrate transgenesis possible.

The Chromosomal Association of the Smc5/6 Complex Depends on Cohesion and Predicts the Level of Sister Chromatid Entanglement

PubMed Central

Jeppsson, Kristian; Carlborg, Kristian K.; Nakato, Ryuichiro; Berta, Davide G.; Lilienthal, Ingrid; Kanno, Takaharu; Lindqvist, Arne; Brink, Maartje C.; Dantuma, Nico P.; Katou, Yuki; Shirahige, Katsuhiko; Sjögren, Camilla

2014-01-01

The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation, also inhibits resolution of sister chromatid intertwinings (SCIs) by the topoisomerase Top2. The cohesin-related Smc5/6 complex (Smc5/6) instead accumulates on chromosomes after Top2 inactivation, known to lead to a buildup of unresolved SCIs. This suggests that cohesin can influence the chromosomal association of Smc5/6 via its role in SCI protection. Using high-resolution ChIP-sequencing, we show that the localization of budding yeast Smc5/6 to duplicated chromosomes indeed depends on sister chromatid cohesion in wild-type and top2-4 cells. Smc5/6 is found to be enriched at cohesin binding sites in the centromere-proximal regions in both cell types, but also along chromosome arms when replication has occurred under Top2-inhibiting conditions. Reactivation of Top2 after replication causes Smc5/6 to dissociate from chromosome arms, supporting the assumption that Smc5/6 associates with a Top2 substrate. It is also demonstrated that the amount of Smc5/6 on chromosomes positively correlates with the level of missegregation in top2-4, and that Smc5/6 promotes segregation of short chromosomes in the mutant. Altogether, this shows that the chromosomal localization of Smc5/6 predicts the presence of the chromatid segregation-inhibiting entities which accumulate in top2-4 mutated cells. These are most likely SCIs, and our results thus indicate that, at least when Top2 is inhibited, Smc5/6 facilitates their resolution. PMID:25329383

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae.

PubMed

Yarimizu, Tohru; Nonklang, Sanom; Nakamura, Junpei; Tokuda, Shuya; Nakagawa, Takaaki; Lorreungsil, Sasithorn; Sutthikhumpha, Surasit; Pukahuta, Charida; Kitagawa, Takao; Nakamura, Mikiko; Cha-Aim, Kamonchai; Limtong, Savitree; Hoshida, Hisashi; Akada, Rinji

2013-12-01

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.

Pleiotropic functions of the yeast Greatwall-family protein kinase Rim15p: a novel target for the control of alcoholic fermentation.

PubMed

Watanabe, Daisuke; Takagi, Hiroshi

2017-06-01

Rim15p, a Greatwall-family protein kinase in yeast Saccharomyces cerevisiae, is required for cellular nutrient responses, such as the entry into quiescence and the induction of meiosis and sporulation. In higher eukaryotes, the orthologous gene products are commonly involved in the cell cycle G 2 /M transition. How are these pleiotropic functions generated from a single family of protein kinases? Recent advances in both research fields have identified the conserved Greatwall-mediated signaling pathway and a variety of downstream target molecules. In addition, our studies of S. cerevisiae sake yeast strains revealed that Rim15p also plays a significant role in the control of alcoholic fermentation. Despite an extensive history of research on glycolysis and alcoholic fermentation, there has been no critical clue to artificial modification of fermentation performance of yeast cells. Our finding of an in vivo metabolic regulatory mechanism is expected to provide a major breakthrough in yeast breeding technologies for fermentation applications.

Micromanipulation studies of chromosome movement. II. Birefringent chromosomal fibers and the mechanical attachment of chromosomes to the spindle

PubMed Central

1979-01-01

The degree of mechanical coupling of chromosomes to the spindles of Nephrotoma and Trimeratropis primary spermatocytes varies with the stage of meiosis and the birefringent retardation of the chromosomal fibers. In early prometaphase, before birefringent chromosomal fibers have formed, a bivalent can be displaced toward a spindle pole by a single, continuous pull with a microneedle. Resistance to poleward displacement increases with increased development of the chromosomal fibers, reaching a maximum at metaphase. At this stage kinetochores cannot be displaced greater than 1 micrometer toward either spindle pole, even by a force which is sufficient to displace the entire spindle within the cell. The abolition of birefringence with either colcemid or vinblastine results in the loss of chromosome-spindle attachment. In the absence of birefringent fibers a chromosome can be displaced anywhere within the cell. The photochemical inactivation of colcemid by irradiation with 366-nm light results in the reformation of birefringent chromosomal fibers and the concomitant re-establishment of chromosome attachment to the spindle. These results support the hypothesis that the birefringent chromosomal fibers anchor the chromosomes to the spindle and transmit the force for anaphase chromosome movement. PMID:479316

Mps1 promotes chromosome meiotic chromosome biorientation through Dam1.

PubMed

Meyer, Régis E; Brown, Jamin; Beck, Lindsay; Dawson, Dean S

2018-02-15

In budding yeast meiosis, homologous chromosomes become linked by chiasmata and then move back and forth on the spindle until they are bioriented, with the kinetochores of the partners attached to microtubules from opposite spindle poles. Certain mutations in the conserved kinase, Mps1, result in catastrophic meiotic segregation errors but mild mitotic defects. We tested whether Dam1, a known substrate of Mps1, was necessary for its critical meiotic role. We found that kinetochore-microtubule attachments are established even when Dam1 is not phosphorylated by Mps1, but that Mps1 phosphorylation of Dam1 sustains those connections. But the meiotic defects when Dam1 is not phosphorylated are not nearly as catastrophic as when Mps1 is inactivated. The results demonstrate that one meiotic role of Mps1 is to stabilize connections that have been established between kinetochores and microtubles by phosphorylating Dam1. © 2018 Meyer et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Construction of a nurse shark (Ginglymostoma cirratum) bacterial artificial chromosome (BAC) library and a preliminary genome survey.

PubMed

Luo, Meizhong; Kim, Hyeran; Kudrna, Dave; Sisneros, Nicholas B; Lee, So-Jeong; Mueller, Christopher; Collura, Kristi; Zuccolo, Andrea; Buckingham, E Bryan; Grim, Suzanne M; Yanagiya, Kazuyo; Inoko, Hidetoshi; Shiina, Takashi; Flajnik, Martin F; Wing, Rod A; Ohta, Yuko

2006-05-03

Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC) library for the nurse shark, Ginglymostoma cirratum. The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 x 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6-28 primary positive clones per probe of which 50-90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible. We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome.

Genetic interactions between the chromosome axis-associated protein Hop1 and homologous recombination determinants in Schizosaccharomyces pombe.

PubMed

Brown, Simon David; Jarosinska, Olga Dorota; Lorenz, Alexander

2018-03-17

Hop1 is a component of the meiosis-specific chromosome axis and belongs to the evolutionarily conserved family of HORMA domain proteins. Hop1 and its orthologs in higher eukaryotes are a major factor in promoting double-strand DNA break formation and inter-homolog recombination. In budding yeast and mammals, they are also involved in a meiotic checkpoint kinase cascade monitoring the completion of double-strand DNA break repair. We used the fission yeast, Schizosaccharomyces pombe, which lacks a canonical synaptonemal complex to test whether Hop1 has a role beyond supporting the generation of double-strand DNA breaks and facilitating inter-homolog recombination events. We determined how mutants of homologous recombination factors genetically interact with hop1, studied the role(s) of the HORMA domain of Hop1, and characterized a bio-informatically predicted interactor of Hop1, Aho1 (SPAC688.03c). Our observations indicate that in fission yeast, Hop1 does require its HORMA domain to support wild-type levels of meiotic recombination and localization to meiotic chromatin. Furthermore, we show that hop1∆ only weakly interacts genetically with mutants of homologous recombination factors, and in fission yeast likely has no major role beyond break formation and promoting inter-homolog events. We speculate that after the evolutionary loss of the synaptonemal complex, Hop1 likely has become less important for modulating recombination outcome during meiosis in fission yeast, and that this led to a concurrent rewiring of genetic pathways controlling meiotic recombination.

Genetic dissection of ethanol tolerance in the budding yeast Saccharomyces cerevisiae.

PubMed

Hu, X H; Wang, M H; Tan, T; Li, J R; Yang, H; Leach, L; Zhang, R M; Luo, Z W

2007-03-01

Uncovering genetic control of variation in ethanol tolerance in natural populations of yeast Saccharomyces cerevisiae is essential for understanding the evolution of fermentation, the dominant lifestyle of the species, and for improving efficiency of selection for strains with high ethanol tolerance, a character of great economic value for the brewing and biofuel industries. To date, as many as 251 genes have been predicted to be involved in influencing this character. Candidacy of these genes was determined from a tested phenotypic effect following gene knockout, from an induced change in gene function under an ethanol stress condition, or by mutagenesis. This article represents the first genomics approach for dissecting genetic variation in ethanol tolerance between two yeast strains with a highly divergent trait phenotype. We developed a simple but reliable experimental protocol for scoring the phenotype and a set of STR/SNP markers evenly covering the whole genome. We created a mapping population comprising 319 segregants from crossing the parental strains. On the basis of the data sets, we find that the tolerance trait has a high heritability and that additive genetic variance dominates genetic variation of the trait. Segregation at five QTL detected has explained approximately 50% of phenotypic variation; in particular, the major QTL mapped on yeast chromosome 9 has accounted for a quarter of the phenotypic variation. We integrated the QTL analysis with the predicted candidacy of ethanol resistance genes and found that only a few of these candidates fall in the QTL regions.

Chromosome Abnormalities

MedlinePlus

... chromosome has attached to another at the centromere. Inversions: A portion of the chromosome has broken off, ... individual and was not inherited from the parents. Inversion: A portion of the chromosome has broken off, ...

The gametocidal chromosome as a tool for chromosome manipulation in wheat.

PubMed

Endo, T R

2007-01-01

Many alien chromosomes have been introduced into common wheat (the genus Triticum) from related wild species (the genus Aegilops). Some alien chromosomes have unique genes that secure their existence in the host by causing chromosome breakage in the gametes lacking them. Such chromosomes or genes, called gametocidal (Gc) chromosomes or Gc genes, are derived from different genomes (C, S, S(l) and M(g)) and belong to three different homoeologous groups 2, 3 and 4. The Gc genes of the C and M(g) genomes induce mild, or semi-lethal, chromosome mutations in euploid and alien addition lines of common wheat. Thus, induced chromosomal rearrangements have been identified and established in wheat stocks carrying deletions of wheat and alien (rye and barley) chromosomes or wheat-alien translocations. The gametocidal chromosomes isolated in wheat to date are reviewed here, focusing on their feature as a tool for chromosome manipulation.

A limited number of Y chromosome lineages is present in North American Holsteins.

PubMed

Yue, Xiang-Peng; Dechow, Chad; Liu, Wan-Sheng

2015-04-01

Holsteins are the most numerous dairy cattle breed in North America and the breed has undergone intensive selection for improving milk production and conformation. Theoretically, this intensive selection could lead to a reduction of the effective population size and reduced genetic diversity. The objective of this study was to investigate the effective population size of the Holstein Y chromosome and the effects of limited Y chromosome lineages on male reproduction and the future of the breed. Paternal pedigree information of 62,897 Holstein bulls born between 1950 and 2013 in North America and 220,872 bulls evaluated by multiple-trait across-country genetic evaluations of Interbull (Uppsala, Sweden) were collected and analyzed. The results indicated that the number of Y chromosome lineages in Holsteins has undergone a dramatic decrease during the past 50 years because of artificial selection and the application of artificial insemination (AI) technology. All current Holstein AI bulls in North America are the descendants of only 2 ancestors (Hulleman and Neptune H) born in 1880. These 2 ancestral Y-lineages are continued through 3 dominant pedigrees from the 1960s; namely, Pawnee Farm Arlinda Chief, Round Oak Rag Apple Elevation, and Penstate Ivanhoe Star, with a contribution of 48.78, 51.06, and 0.16% to the Holstein bull population in the 2010s, respectively. The Y-lineage of Penstate Ivanhoe Star is almost eliminated from the breed. The genetic variations in the 2 ancestral Y-lineages were evaluated among 257 bulls by determining the copy number variations (CNV) of 3 Y-linked gene families: PRAMEY, HSFY, and ZNF280BY, which are spread along the majority (95%) of the bovine Y chromosome male-specific region (MSY). No significant difference was found between the 2 ancestral Y-lineages, although large CNV were observed within each lineage. This study suggests minimal genetic diversity on the Y chromosome in Holsteins and provides a starting point for investigating

Yeast for virus research

PubMed Central

Zhao, Richard Yuqi

2017-01-01

Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are two popular model organisms for virus research. They are natural hosts for viruses as they carry their own indigenous viruses. Both yeasts have been used for studies of plant, animal and human viruses. Many positive sense (+) RNA viruses and some DNA viruses replicate with various levels in yeasts, thus allowing study of those viral activities during viral life cycle. Yeasts are single cell eukaryotic organisms. Hence, many of the fundamental cellular functions such as cell cycle regulation or programed cell death are highly conserved from yeasts to higher eukaryotes. Therefore, they are particularly suited to study the impact of those viral activities on related cellular activities during virus-host interactions. Yeasts present many unique advantages in virus research over high eukaryotes. Yeast cells are easy to maintain in the laboratory with relative short doubling time. They are non-biohazardous, genetically amendable with small genomes that permit genome-wide analysis of virologic and cellular functions. In this review, similarities and differences of these two yeasts are described. Studies of virologic activities such as viral translation, viral replication and genome-wide study of virus-cell interactions in yeasts are highlighted. Impacts of viral proteins on basic cellular functions such as cell cycle regulation and programed cell death are discussed. Potential applications of using yeasts as hosts to carry out functional analysis of small viral genome and to develop high throughput drug screening platform for the discovery of antiviral drugs are presented. PMID:29082230

A comparative study of retrotransposons in the centromeric regions of A and B chromosomes of maize.

PubMed

Theuri, J; Phelps-Durr, T; Mathews, S; Birchler, J

2005-01-01

Bacterial Artificial Chromosomes (BACs) derived from the B chromosome, based on homology with the B specific sequence, were subcloned and sequenced. Analysis of DNA sequence data indicated the presence of 23 common retroelements, as well as novel sequences of B chromosome origin. Generally, where the same retrotransposon type was observed in both A and B chromosomes, there were more copies per unit of sequence in the B centromeric region (the major site of B repeat) than in the A centromere, except for Huck-1. Based on previous estimates of the age of the major burst of transposition into the maize genome, the oldest retrotransposons (Ji-6 and Tekay, approximately 5.0 and 5.2 million years ago, respectively) were found in the B centromere region only, while the next two oldest (Huck-1 and Opie-1) were found in both the A and B sequences. Phylogenetic analysis of Opie retroelements from both A and B centromeres indicated that some of the B Opie centromeric sequences share a more recent common ancestor with A Opie retroelements than they do with other B Opie centromeric sequences. These results imply that the supernumerary maize B chromosome has coexisted with the A chromosomes during that period of transposition. They also support the hypothesis that the B chromosome had its origins from A chromosome elements, or that alternative origins, such as being donated to the maize genome in a wide species cross, preceded six million years ago, because the spectrum of retrotransposons in the two chromosomes is quite similar.

Long G2 accumulates recombination intermediates and disturbs chromosome segregation at dysfunction telomere in Schizosaccharomyces pombe

DOE Office of Scientific and Technical Information (OSTI.GOV)

Habib, Ahmed G.K.; Masuda, Kenta; Yukawa, Masashi

Protection of telomere (Pot1) is a single-stranded telomere binding protein which is essential for chromosome ends protection. Fission yeast Rqh1 is a member of RecQ helicases family which has essential roles in the maintenance of genomic stability and regulation of homologous recombination. Double mutant between fission yeast pot1Δ and rqh1 helicase dead (rqh1-hd) maintains telomere by homologous recombination. In pot1Δ rqh1-hd double mutant, recombination intermediates accumulate near telomere which disturb chromosome segregation and make cells sensitive to microtubule inhibitors thiabendazole (TBZ). Deletion of chk1{sup +} or mutation of its kinase domain shortens the G2 of pot1Δ rqh1-hd double mutant andmore » suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of that double mutant. In this study, we asked whether the long G2 is the reason for the TBZ sensitivity of pot1Δ rqh1-hd double mutant. We found that shortening the G2 of pot1Δ rqh1-hd double mutant by additional mutations of wee1 and mik1 or gain of function mutation of Cdc2 suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of pot1Δ rqh1-hd double mutant. Our results suggest that long G2 of pot1Δ rqh1-hd double mutant may allow time for the accumulation of recombination intermediates which disturb chromosome segregation and make cells sensitive to TBZ. - Ηighlights: • We show link between long G2 and accumulation of toxic recombination intermediates. • Accumulation of recombination intermediates at telomere results in TBZ sensitivity. • Activation of DNA damage checkpoint worsens cells' viability in presence of TBZ.« less

Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC)

PubMed Central

2011-01-01

Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool. PMID:21314965

L-arabinose fermenting yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Min; Singh, Arjun; Suominen, Pirkko

An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. A yeast strain engineered to metabolize arabinose through a novel pathway is also disclosed. Methods of producing ethanol include utilizing these modified yeast strains.

L-arabinose fermenting yeast

DOEpatents

Zhang, Min; Singh, Arjun; Suominen, Pirkko; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric

2014-09-23

An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. A yeast strain engineered to metabolize arabinose through a novel pathway is also disclosed. Methods of producing ethanol include utilizing these modified yeast strains.

Prevention of Yeast Spoilage in Feed and Food by the Yeast Mycocin HMK

PubMed Central

Lowes, K. F.; Shearman, C. A.; Payne, J.; MacKenzie, D.; Archer, D. B.; Merry, R. J.; Gasson, M. J.

2000-01-01

The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts. PMID:10698773

Chromosome rearrangements and the evolution of genome structuring and adaptability.

PubMed

Crombach, Anton; Hogeweg, Paulien

2007-05-01

Eukaryotes appear to evolve by micro and macro rearrangements. This is observed not only for long-term evolutionary adaptation, but also in short-term experimental evolution of yeast, Saccharomyces cerevisiae. Moreover, based on these and other experiments it has been postulated that repeat elements, retroposons for example, mediate such events. We study an evolutionary model in which genomes with retroposons and a breaking/repair mechanism are subjected to a changing environment. We show that retroposon-mediated rearrangements can be a beneficial mutational operator for short-term adaptations to a new environment. But simply having the ability of rearranging chromosomes does not imply an advantage over genomes in which only single-gene insertions and deletions occur. Instead, a structuring of the genome is needed: genes that need to be amplified (or deleted) in a new environment have to cluster. We show that genomes hosting retroposons, starting with a random order of genes, will in the long run become organized, which enables (fast) rearrangement-based adaptations to the environment. In other words, our model provides a "proof of principle" that genomes can structure themselves in order to increase the beneficial effect of chromosome rearrangements.

Centromere proteins CENP-C and CAL1 functionally interact in meiosis for centromere clustering, pairing, and chromosome segregation.

PubMed

Unhavaithaya, Yingdee; Orr-Weaver, Terry L

2013-12-03

Meiotic chromosome segregation involves pairing and segregation of homologous chromosomes in the first division and segregation of sister chromatids in the second division. Although it is known that the centromere and kinetochore are responsible for chromosome movement in meiosis as in mitosis, potential specialized meiotic functions are being uncovered. Centromere pairing early in meiosis I, even between nonhomologous chromosomes, and clustering of centromeres can promote proper homolog associations in meiosis I in yeast, plants, and Drosophila. It was not known, however, whether centromere proteins are required for this clustering. We exploited Drosophila mutants for the centromere proteins centromere protein-C (CENP-C) and chromosome alignment 1 (CAL1) to demonstrate that a functional centromere is needed for centromere clustering and pairing. The cenp-C and cal1 mutations result in C-terminal truncations, removing the domains through which these two proteins interact. The mutants show striking genetic interactions, failing to complement as double heterozygotes, resulting in disrupted centromere clustering and meiotic nondisjunction. The cluster of meiotic centromeres localizes to the nucleolus, and this association requires centromere function. In Drosophila, synaptonemal complex (SC) formation can initiate from the centromere, and the SC is retained at the centromere after it disassembles from the chromosome arms. Although functional CENP-C and CAL1 are dispensable for assembly of the SC, they are required for subsequent retention of the SC at the centromere. These results show that integral centromere proteins are required for nuclear position and intercentromere associations in meiosis.

Establishing biorientation occurs with precocious separation of the sister kinetochores, but not the arms, in the early spindle of budding yeast.

PubMed

Goshima, G; Yanagida, M

2000-03-17

Sister kinetochores are bioriented toward the spindle poles in higher eukaryotic prometaphase before chromosome segregation. We show that, in budding yeast, the sister kinetochores are separated in the very early spindle, while the sister arms remain associated. Biorientation of the separated kinetochores is achieved already after replication. Mtw1p, a homolog of fission yeast Mis12 required for biorientation, locates at the centromeres in an Ndc10p-dependent manner. Mtw1p and the sequences 1.8 and 3.8 kb from CEN3 and CEN15, respectively, behave like the precociously separated kinetochores, whereas the sequences 23 and 35 kb distant from CEN3 and CEN5 previously used as the centromere markers behave like a part of the arm. Mtw1p and Ndc10p are identically located except for additional spindle localization of Ndc10p. A model explaining small centromeres and early spindle formation in budding yeast is proposed.

Chromosomes

MedlinePlus

... Sheets A Brief Guide to Genomics About NHGRI Research About the International HapMap Project Biological Pathways Chromosome Abnormalities Chromosomes Cloning Comparative Genomics DNA Microarray Technology DNA Sequencing Deoxyribonucleic Acid ( ...

Proline as a stress protectant in yeast: physiological functions, metabolic regulations, and biotechnological applications.

PubMed

Takagi, Hiroshi

2008-11-01

Proline is an important amino acid in terms of its biological functions and biotechnological applications. In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. However, it has been shown that proline levels are not increased under various stress conditions in the yeast Saccharomyces cerevisiae cells. Proline is believed to serve multiple functions in vitro such as protein and membrane stabilization, lowering the T (m) of DNA, and scavenging of reactive oxygen species, but the mechanisms of these functions in vivo are poorly understood. Yeast cells biosynthesize proline from glutamate in the cytoplasm via the same pathway found in bacteria and plants and also convert excess proline to glutamate in the mitochondria. Based on the fact that proline has stress-protective activity, S. cerevisiae cells that accumulate proline were constructed by disrupting the PUT1 gene involved in the degradation pathway and by expressing the mutant PRO1 gene encoding the feedback inhibition-less sensitive gamma-glutamate kinase to enhance the biosynthetic activity. The engineered yeast strains successfully showed enhanced tolerance to many stresses, including freezing, desiccation, oxidation, and ethanol. However, the appropriate cellular level and localization of proline play pivotal roles in the stress-protective effect. These results indicate that the increased stress protection is observed in yeast cells under the artificial condition of proline accumulation. Proline is expected to contribute to yeast-based industries by improving the production of frozen dough and alcoholic beverages or breakthroughs in bioethanol production.

Chromosomal location and genetic mapping of the mismatch repair gene homologs MSH2, MSH3, and MSH6 in rye and wheat

PubMed

Korzun; Borner; Siebert; Malyshev; Hilpert; Kunze; Puchta

1999-12-01

The efficiency of homeologous recombination is influenced by mismatch repair genes in bacteria, yeast, and mammals. To elucidate a possible role of these genes in homeologous pairing and cross-compatibility in plants, gene probes of wheat (Triticum aestivum) specific for the mismatch repair gene homologues MSH2, MSH3, and MSH6 were used to map them to their genomic positions in rye (Secale cereale). Whereas MSH2 was mapped to the short arm of chromosome 1R, MSH3 was mapped to the long arm of chromosome 2R and MSH6 to the long arm of chromosome 5R. Southern blots with nullisomic-tetrasomic (NT) lines of wheat indicated the presence of the sequences on the respective homeologous group of wheat chromosomes. Additionally, an MSH6-specific homologue could also be detected on homoeologous group 3 of wheat. However, in the well-known, highly homoeologous pairing wheat mutant ph1b the MSH6-specific sequence is not within the deleted part of chromosome 5BL, indicating that the pairing phenotype is not due to a loss of one of the mismatch repair genes tested.

Plant chromosomes from end to end: telomeres, heterochromatin and centromeres.

PubMed

Lamb, Jonathan C; Yu, Weichang; Han, Fangpu; Birchler, James A

2007-04-01

Recent evidence indicates that heterochromatin in plants is composed of heterogeneous sequences, which are usually composed of transposable elements or tandem repeat arrays. These arrays are associated with chromatin modifications that produce a closed configuration that limits transcription. Centromere sequences in plants are usually composed of tandem repeat arrays that are homogenized across the genome. Analysis of such arrays in closely related taxa suggests a rapid turnover of the repeat unit that is typical of a particular species. In addition, two lines of evidence for an epigenetic component of centromere specification have been reported, namely an example of a neocentromere formed over sequences without the typical repeat array and examples of centromere inactivation. Although the telomere repeat unit is quite prevalent in the plant kingdom, unusual repeats have been found in some families. Recently, it was demonstrated that the introduction of telomere sequences into plants cells causes truncation of the chromosomes, and that this technique can be used to produce artificial chromosome platforms.

Generation of an approximately 2.4 Mb human X centromere-based minichromosome by targeted telomere-associated chromosome fragmentation in DT40.

PubMed

Mills, W; Critcher, R; Lee, C; Farr, C J

1999-05-01

A linear mammalian artificial chromosome (MAC) will require at least three types of functional element: a centromere, two telomeres and origins of replication. As yet, our understanding of these elements, as well as many other aspects of structure and organization which may be critical for a fully functional mammalian chromosome, remains poor. As a way of defining these various requirements, minichromosome reagents are being developed and analysed. Approaches for minichromosome generation fall into two broad categories: de novo assembly from candidate DNA sequences, or the fragmentation of an existing chromosome to reduce it to a minimal size. Here we describe the generation of a human minichromosome using the latter, top-down, approach. A human X chromosome, present in a DT40-human microcell hybrid, has been manipulated using homologous recombination and the targeted seeding of a de novo telomere. This strategy has generated a linear approximately 2.4 Mb human X centromere-based minichromosome capped by two artificially seeded telomeres: one immediately flanking the centromeric alpha-satellite DNA and the other targeted to the zinc finger gene ZXDA in Xp11.21. The chromosome retains an alpha-satellite domain of approximately 1. 8 Mb, a small array of gamma-satellite repeat ( approximately 40 kb) and approximately 400 kb of Xp proximal DNA sequence. The mitotic stability of this minichromosome has been examined, both in DT40 and following transfer into hamster and human cell lines. In all three backgrounds, the minichromosome is retained efficiently, but in the human and hamster microcell hybrids its copy number is poorly regulated. This approach of engineering well-defined chromosome reagents will allow key questions in MAC development (such as whether a lower size limit exists) to be addressed. In addition, the 2.4 Mb minichromosome described here has potential to be developed as a vector for gene delivery.

Mislocalization of centromeric histone H3 variant CENP-A contributes to chromosomal instability (CIN) in human cells

PubMed Central

Shrestha, Roshan L.; Ahn, Grace S.; Staples, Mae I.; Sathyan, Kizhakke M.; Karpova, Tatiana S.; Foltz, Daniel R.; Basrai, Munira A.

2017-01-01

Chromosomal instability (CIN) is a hallmark of many cancers and a major contributor to tumorigenesis. Centromere and kinetochore associated proteins such as the evolutionarily conserved centromeric histone H3 variant CENP-A, associate with centromeric DNA for centromere function and chromosomal stability. Stringent regulation of cellular CENP-A levels prevents its mislocalization in yeast and flies to maintain genome stability. CENP-A overexpression and mislocalization are observed in several cancers and reported to be associated with increased invasiveness and poor prognosis. We examined whether there is a direct relationship between mislocalization of overexpressed CENP-A and CIN using HeLa and chromosomally stable diploid RPE1 cell lines as model systems. Our results show that mislocalization of overexpressed CENP-A to chromosome arms leads to chromosome congression defects, lagging chromosomes, micronuclei formation and a delay in mitotic exit. CENP-A overexpressing cells showed altered localization of centromere and kinetochore associated proteins such as CENP-C, CENP-T and Nuf2 leading to weakened native kinetochores as shown by reduced interkinetochore distance and CIN. Importantly, our results show that mislocalization of CENP-A to chromosome arms is one of the major contributors for CIN as depletion of histone chaperone DAXX prevents CENP-A mislocalization and rescues the reduced interkinetochore distance and CIN phenotype in CENP-A overexpressing cells. In summary, our results establish that CENP-A overexpression and mislocalization result in a CIN phenotype in human cells. This study provides insights into how overexpression of CENP-A may contribute to CIN in cancers and underscore the importance of understanding the pathways that prevent CENP-A mislocalization for genome stability. PMID:28596481

Multigene phylogeny and taxonomic revision of yeasts and related fungi in the Ustilaginomycotina.

PubMed

Wang, Q-M; Begerow, D; Groenewald, M; Liu, X-Z; Theelen, B; Bai, F-Y; Boekhout, T

2015-06-01

The subphylum Ustilaginomycotina (Basidiomycota, Fungi) comprises mainly plant pathogenic fungi (smuts). Some of the lineages possess cultivable unicellular stages that are usually classified as yeast or yeast-like species in a largely artificial taxonomic system which is independent from and largely incompatible with that of the smut fungi. Here we performed phylogenetic analyses based on seven genes including three nuclear ribosomal RNA genes and four protein coding genes to address the molecular phylogeny of the ustilaginomycetous yeast species and their filamentous counterparts. Taxonomic revisions were proposed to reflect this phylogeny and to implement the 'One Fungus = One Name' principle. The results confirmed that the yeast-containing classes Malasseziomycetes, Moniliellomycetes and Ustilaginomycetes are monophyletic, whereas Exobasidiomycetes in the current sense remains paraphyletic. Four new genera, namely Dirkmeia gen. nov., Kalmanozyma gen. nov., Golubevia gen. nov. and Robbauera gen. nov. are proposed to accommodate Pseudozyma and Tilletiopsis species that are distinct from the other smut taxa and belong to clades that are separate from those containing type species of the hitherto described genera. Accordingly, new orders Golubeviales ord. nov. with Golubeviaceae fam. nov. and Robbauerales ord. nov. with Robbaueraceae fam. nov. are proposed to accommodate the sisterhood of Golubevia gen. nov. and Robbauera gen. nov. with other orders of Exobasidiomycetes. The majority of the remaining anamorphic yeast species are transferred to corresponding teleomorphic genera based on strongly supported phylogenetic affinities, resulting in the proposal of 28 new combinations. The taxonomic status of a few Pseudozyma species remains to be determined because of their uncertain phylogenetic positions. We propose to use the term pro tempore or pro tem. in abbreviation to indicate the single-species lineages that are temporarily maintained.

An artificial transport metabolon facilitates improved substrate utilization in yeast.

PubMed

Thomik, Thomas; Wittig, Ilka; Choe, Jun-Yong; Boles, Eckhard; Oreb, Mislav

2017-11-01

Efficient substrate utilization is the first and most important prerequisite for economically viable production of biofuels and chemicals by microbial cell factories. However, production rates and yields are often compromised by low transport rates of substrates across biological membranes and their diversion to competing pathways. This is especially true when common chassis organisms are engineered to utilize nonphysiological feedstocks. Here, we addressed this problem by constructing an artificial complex between an endogenous sugar transporter and a heterologous xylose isomerase in Saccharomyces cerevisiae. Direct feeding of the enzyme through the transporter resulted in acceleration of xylose consumption and substantially diminished production of xylitol as an undesired side product, with a concomitant increase in the production of ethanol. This underlying principle could also likely be implemented in other biotechnological applications.

To Nick or Not to Nick: Comparison of I-SceI Single- and Double-Strand Break-Induced Recombination in Yeast and Human Cells

PubMed Central

Katz, Samantha S.; Gimble, Frederick S.; Storici, Francesca

2014-01-01

Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, or chromosomal rearrangements. By contrast, generation of a DNA single-strand break (SSB), or nick, can stimulate gene correction without the problems of DSB repair because the uncut DNA strand acts as a template to permit healing without alteration of genetic material. Here, we examine the ability of a nicking variant of the I-SceI endonuclease (K223I I-SceI) to stimulate gene targeting in yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK-293) cells. K223I I-SceI is proficient in both yeast and human cells and promotes gene correction up to 12-fold. We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick. We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells. These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy. PMID:24558436

Effect of wine yeast monoculture practice on the biodiversity of non-Saccharomyces yeasts.

PubMed

Ganga, M A; Martínez, C

2004-01-01

The objective of this work was to study the effect of the use of Saccharomyces cerevisiae monocultures over the biodiversity of non-Saccharomyces yeasts in wine-producing areas in Chile. Microvinifications were carried out with grape musts of two areas. In one of them, the fermentation is carried out mainly in a spontaneous manner, whereas in the other the musts are inoculated with commercial yeasts. The isolated yeasts were identified by the internal transcribed (ITS)/restriction fragment length polymorphism technique. In the industrial production area less variability of yeast genera was observed as compared with the traditional area, an observation that is greatest at the end of the fermentation. Furthermore, a study of the production of extracellular enzymes was done. The majority of the yeasts showed at least one of the activities assayed with the exception of beta-glycosidase. The results suggest that in the industrialized area the diversity of yeasts is less in the traditional area. Likewise, the potentiality of the non-Saccharomyces yeasts as enzyme producers with industrial interest has been confirmed. This study shows the negative effect of the use of monocultures over the biodiversity of yeasts in wine-producing regions.

A new chromosome was born: comparative chromosome painting in Boechera.

PubMed

Koch, Marcus A

2015-09-01

Comparative chromosome painting is a powerful tool to study the evolution of chromosomes and genomes. Analyzing karyotype evolution in cruciferous plants highlights the origin of aberrant chromosomes in apomictic Boechera and further establishes the cruciferous plants as important model system for our understanding of plant chromosome and genome evolution. Copyright © 2015 Elsevier Ltd. All rights reserved.

Detection and quantitation of chromosomal mosaicism in human blastocysts using copy number variation sequencing.

PubMed

Ruttanajit, Tida; Chanchamroen, Sujin; Cram, David S; Sawakwongpra, Kritchakorn; Suksalak, Wanwisa; Leng, Xue; Fan, Junmei; Wang, Li; Yao, Yuanqing; Quangkananurug, Wiwat

2016-02-01

Currently, our understanding of the nature and reproductive potential of blastocysts associated with trophectoderm (TE) lineage chromosomal mosaicism is limited. The objective of this study was to first validate copy number variation sequencing (CNV-Seq) for measuring the level of mosaicism and second, examine the nature and level of mosaicism in TE biopsies of patient's blastocysts. TE biopy samples were analysed by array comparative genomic hybridization (CGH) and CNV-Seq to discriminate between euploid, aneuploid and mosaic blastocysts. Using artificial models of TE mosaicism for five different chromosomes, CNV-Seq accurately and reproducibly quantitated mosaicism at levels of 50% and 20%. In a comparative 24-chromosome study of 49 blastocysts by array CGH and CNV-Seq, 43 blastocysts (87.8%) had a concordant diagnosis and 6 blastocysts (12.2%) were discordant. The discordance was attributed to low to medium levels of chromosomal mosaicism (30-70%) not detected by array CGH. In an expanded study of 399 blastocysts using CNV-Seq as the sole diagnostic method, the proportion of diploid-aneuploid mosaics (34, 8.5%) was significantly higher than aneuploid mosaics (18, 4.5%) (p < 0.02). Mosaicism is a significant chromosomal abnormality associated with the TE lineage of human blastocysts that can be reliably and accurately detected by CNV-Seq. © 2015 John Wiley & Sons, Ltd.

The CENP-A N-Tail Confers Epigenetic Stability to Centromeres via the CENP-T Branch of the CCAN in Fission Yeast

PubMed Central

Folco, H. Diego; Campbell, Christopher S.; May, Karen M.; Espinoza, Celso A.; Oegema, Karen; Hardwick, Kevin G.; Grewal, Shiv I. S.; Desai, Arshad

2014-01-01

Summary In most eukaryotes, centromeres are defined epigenetically by presence of the histone H3 variant CENP-A [1-3]. CENP-A containing chromatin recruits the constitutive centromere-associated network (CCAN) of proteins, which in turn directs assembly of the outer kinetochore to form microtubule attachments and ensure chromosome segregation fidelity [4-6]. While the mechanisms that load CENP-A at centromeres are being elucidated, the functions of its divergent N-terminal tail remain enigmatic [7-12]. Here, we employ the well-studied fission yeast centromere [13-16] to investigate the function of the CENP-A (Cnp1) N-tail. We show that alteration of the N-tail did not affect Cnp1 loading at centromeres, outer kinetochore formation, or spindle checkpoint signaling, but nevertheless elevated chromosome loss. N-Tail mutants exhibited synthetic lethality with an altered centromeric DNA sequence, with rare survivors harboring chromosomal fusions in which the altered centromere was epigenetically inactivated. Elevated centromere inactivation was also observed for N-tail mutants with unaltered centromeric DNA sequences. N-tail mutants specifically reduced localization of the CCAN proteins Cnp20/CENP-T and Mis6/CENP-I, but not Cnp3/CENP-C. Overexpression of Cnp20/CENP-T suppressed defects in an N-tail mutant, suggesting a link between reduced CENP-T recruitment and the observed centromere inactivation phenotype. Thus, the Cnp1 N-tail promotes epigenetic stability of centromeres in fission yeast, at least in part via recruitment of the CENP-T branch of the CCAN. PMID:25619765

Y-chromosome evolution: emerging insights into processes of Y-chromosome degeneration.

PubMed

Bachtrog, Doris

2013-02-01

The human Y chromosome is intriguing not only because it harbours the master-switch gene that determines gender but also because of its unusual evolutionary history. The Y chromosome evolved from an autosome, and its evolution has been characterized by massive gene decay. Recent whole-genome and transcriptome analyses of Y chromosomes in humans and other primates, in Drosophila species and in plants have shed light on the current gene content of the Y chromosome, its origins and its long-term fate. Furthermore, comparative analysis of young and old Y chromosomes has given further insights into the evolutionary and molecular forces triggering Y-chromosome degeneration and into the evolutionary destiny of the Y chromosome.

Undetected sex chromosome aneuploidy by chromosomal microarray.

PubMed

Markus-Bustani, Keren; Yaron, Yuval; Goldstein, Myriam; Orr-Urtreger, Avi; Ben-Shachar, Shay

2012-11-01

We report on a case of a female fetus found to be mosaic for Turner syndrome (45,X) and trisomy X (47,XXX). Chromosomal microarray analysis (CMA) failed to detect the aneuploidy because of a normal average dosage of the X chromosome. This case represents an unusual instance in which CMA may not detect chromosomal aberrations. Such a possibility should be taken into consideration in similar cases where CMA is used in a clinical setting. © 2012 John Wiley & Sons, Ltd.

Ribosomal DNA stability is supported by many 'buffer genes'-introduction to the Yeast rDNA Stability Database.

PubMed

Kobayashi, Takehiko; Sasaki, Mariko

2017-01-01

The ribosomal RNA gene (rDNA) is the most abundant gene in yeast and other eukaryotic organisms. Due to its heavy transcription, repetitive structure and programmed replication fork pauses, the rDNA is one of the most unstable regions in the genome. Thus, the rDNA is the best region to study the mechanisms responsible for maintaining genome integrity. Recently, we screened a library of ∼4800 budding yeast gene knockout strains to identify mutants defective in the maintenance of rDNA stability. The results of this screen are summarized in the Yeast rDNA Stability (YRS) Database, in which the stability and copy number of rDNA in each mutant are presented. From this screen, we identified ∼700 genes that may contribute to the maintenance of rDNA stability. In addition, ∼50 mutants had abnormally high or low rDNA copy numbers. Moreover, some mutants with unstable rDNA displayed abnormalities in another chromosome. In this review, we introduce the YRS Database and discuss the roles of newly identified genes that contribute to rDNA maintenance and genome integrity. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Chromosomes, conflict, and epigenetics: chromosomal speciation revisited.

PubMed

Brown, Judith D; O'Neill, Rachel J

2010-01-01

Since Darwin first noted that the process of speciation was indeed the "mystery of mysteries," scientists have tried to develop testable models for the development of reproductive incompatibilities-the first step in the formation of a new species. Early theorists proposed that chromosome rearrangements were implicated in the process of reproductive isolation; however, the chromosomal speciation model has recently been questioned. In addition, recent data from hybrid model systems indicates that simple epistatic interactions, the Dobzhansky-Muller incompatibilities, are more complex. In fact, incompatibilities are quite broad, including interactions among heterochromatin, small RNAs, and distinct, epigenetically defined genomic regions such as the centromere. In this review, we will examine both classical and current models of chromosomal speciation and describe the "evolving" theory of genetic conflict, epigenetics, and chromosomal speciation.

A Novel System for Simultaneous or Sequential Integration of Multiple Gene-Loading Vectors into a Defined Site of a Human Artificial Chromosome

PubMed Central

Suzuki, Teruhiko; Kazuki, Yasuhiro; Oshimura, Mitsuo; Hara, Takahiko

2014-01-01

Human artificial chromosomes (HACs) are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV) into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system) via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding EGFP, Venus, and TdTomato into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming. PMID:25303219

A novel system for simultaneous or sequential integration of multiple gene-loading vectors into a defined site of a human artificial chromosome.

PubMed

Suzuki, Teruhiko; Kazuki, Yasuhiro; Oshimura, Mitsuo; Hara, Takahiko

2014-01-01

Human artificial chromosomes (HACs) are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV) into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system) via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding EGFP, Venus, and TdTomato into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming.

Yeast cell differentiation: Lessons from pathogenic and non-pathogenic yeasts.

PubMed

Palková, Zdena; Váchová, Libuše

2016-09-01

Yeasts, historically considered to be single-cell organisms, are able to activate different differentiation processes. Individual yeast cells can change their life-styles by processes of phenotypic switching such as the switch from yeast-shaped cells to filamentous cells (pseudohyphae or true hyphae) and the transition among opaque, white and gray cell-types. Yeasts can also create organized multicellular structures such as colonies and biofilms, and the latter are often observed as contaminants on surfaces in industry and medical care and are formed during infections of the human body. Multicellular structures are formed mostly of stationary-phase or slow-growing cells that diversify into specific cell subpopulations that have unique metabolic properties and can fulfill specific tasks. In addition to the development of multiple protective mechanisms, processes of metabolic reprogramming that reflect a changed environment help differentiated individual cells and/or community cell constituents to survive harmful environmental attacks and/or to escape the host immune system. This review aims to provide an overview of differentiation processes so far identified in individual yeast cells as well as in multicellular communities of yeast pathogens of the Candida and Cryptococcus spp. and the Candida albicans close relative, Saccharomyces cerevisiae. Molecular mechanisms and extracellular signals potentially involved in differentiation processes are also briefly mentioned. Copyright © 2016 Elsevier Ltd. All rights reserved.

L-arabinose fermenting yeast

DOEpatents

Zhang, Min; Singh, Arjun; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric; Suominen, Pirkko

2010-12-07

An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. Methods of producing ethanol include utilizing these modified yeast strains. ##STR00001##

Nitrile Metabolizing Yeasts

NASA Astrophysics Data System (ADS)

Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing

Candida pruni sp. nov. is a new yeast species with antagonistic potential against brown rot of peaches.

PubMed

Zhang, Dian-peng; Lu, Cai-ge; Zhang, Tao-tao; Spadaro, Davide; Liu, De-wen; Liu, Wei-cheng

2014-07-01

Brown rot caused by Monilinia spp. is among the most important postharvest diseases of commercially grown stone fruits, and application of antagonistic yeasts to control brown rot is one promising strategy alternative to chemical fungicides. In this research, new yeast strains were isolated and tested for their activity against peach brown rot caused by Monilinia fructicola. Three yeast strains were originally isolated from the surface of plums (cv Chinese Angelino) collected in the north of China. In artificially wounded inoculation tests, the yeast reduced the brown rot incidence to 20 %. The population of the yeast within inoculated wounds on peaches significantly increased at 25 °C from an initial level of 5.0×10(6) to 4.45×10(7) CFU per wound after 1 day. The antagonistic strains were belonging to a new species of the genus Candida by sequence comparisons of 26 S rDNA D1/D2 domain and internal transcribed spacer region. The strains are most closely related to C. asparagi, C. musae and C. fructus on the basis of the phylogenetic trees based on the D1/D2 region of 26S rDNA. However, the strains are notably different from C. asparagi, C. musae and C. fructus, in morphological and physiological characteristics. Therefore, the name Candida pruni is proposed for the novel species, with sp-Quan (=CBS12814T=KCTC 27526T=GCMC 6582T) as the type strain. Our study showed that Candida pruni is a novel yeast species with potential biocontrol against brown rot caused by M. fructicola on peaches.

A Slowed Cell Cycle Stabilizes the Budding Yeast Genome.

PubMed

Vinton, Peter J; Weinert, Ted

2017-06-01

During cell division, aberrant DNA structures are detected by regulators called checkpoints that slow division to allow error correction. In addition to checkpoint-induced delay, it is widely assumed, though rarely shown, that merely slowing the cell cycle might allow more time for error detection and correction, thus resulting in a more stable genome. Fidelity by a slowed cell cycle might be independent of checkpoints. Here we tested the hypothesis that a slowed cell cycle stabilizes the genome, independent of checkpoints, in the budding yeast Saccharomyces cerevisiae We were led to this hypothesis when we identified a gene ( ERV14 , an ER cargo membrane protein) that when mutated, unexpectedly stabilized the genome, as measured by three different chromosome assays. After extensive studies of pathways rendered dysfunctional in erv14 mutant cells, we are led to the inference that no particular pathway is involved in stabilization, but rather the slowed cell cycle induced by erv14 stabilized the genome. We then demonstrated that, in genetic mutations and chemical treatments unrelated to ERV14 , a slowed cell cycle indeed correlates with a more stable genome, even in checkpoint-proficient cells. Data suggest a delay in G2/M may commonly stabilize the genome. We conclude that chromosome errors are more rarely made or are more readily corrected when the cell cycle is slowed (even ∼15 min longer in an ∼100-min cell cycle). And, some chromosome errors may not signal checkpoint-mediated responses, or do not sufficiently signal to allow correction, and their correction benefits from this "time checkpoint." Copyright © 2017 by the Genetics Society of America.

Genome dynamics and evolution in yeasts: A long-term yeast-bacteria competition experiment

PubMed Central

Katz, Michael; Knecht, Wolfgang; Compagno, Concetta; Piškur, Jure

2018-01-01

There is an enormous genetic diversity evident in modern yeasts, but our understanding of the ecological basis of such diversifications in nature remains at best fragmented so far. Here we report a long-term experiment mimicking a primordial competitive environment, in which yeast and bacteria co-exist and compete against each other. Eighteen yeasts covering a wide phylogenetic background spanning approximately 250 million years of evolutionary history were used to establish independent evolution lines for at most 130 passages. Our collection of hundreds of modified strains generated through such a rare two-species cross-kingdom competition experiment re-created the appearance of large-scale genomic rearrangements and altered phenotypes important in the diversification history of yeasts. At the same time, the methodology employed in this evolutionary study would also be a non-gene-technological method of reprogramming yeast genomes and then selecting yeast strains with desired traits. Cross-kingdom competition may therefore be a method of significant value to generate industrially useful yeast strains with new metabolic traits. PMID:29624585

Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.

PubMed

Tao, Zhihua; Gao, Peng; Liu, Hung-Wen

2009-12-15

Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

Genomic structural variation contributes to phenotypic change of industrial bioethanol yeast Saccharomyces cerevisiae.

PubMed

Zhang, Ke; Zhang, Li-Jie; Fang, Ya-Hong; Jin, Xin-Na; Qi, Lei; Wu, Xue-Chang; Zheng, Dao-Qiong

2016-03-01

Genomic structural variation (GSV) is a ubiquitous phenomenon observed in the genomes of Saccharomyces cerevisiae strains with different genetic backgrounds; however, the physiological and phenotypic effects of GSV are not well understood. Here, we first revealed the genetic characteristics of a widely used industrial S. cerevisiae strain, ZTW1, by whole genome sequencing. ZTW1 was identified as an aneuploidy strain and a large-scale GSV was observed in the ZTW1 genome compared with the genome of a diploid strain YJS329. These GSV events led to copy number variations (CNVs) in many chromosomal segments as well as one whole chromosome in the ZTW1 genome. Changes in the DNA dosage of certain functional genes directly affected their expression levels and the resultant ZTW1 phenotypes. Moreover, CNVs of large chromosomal regions triggered an aneuploidy stress in ZTW1. This stress decreased the proliferation ability and tolerance of ZTW1 to various stresses, while aneuploidy response stress may also provide some benefits to the fermentation performance of the yeast, including increased fermentation rates and decreased byproduct generation. This work reveals genomic characters of the bioethanol S. cerevisiae strain ZTW1 and suggests that GSV is an important kind of mutation that changes the traits of industrial S. cerevisiae strains. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

PubMed Central

Schmeisser, Falko; Weir, Jerry P

2007-01-01

Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors. PMID:17501993

The Precarious Prokaryotic Chromosome

PubMed Central

2014-01-01

Evolutionary selection for optimal genome preservation, replication, and expression should yield similar chromosome organizations in any type of cells. And yet, the chromosome organization is surprisingly different between eukaryotes and prokaryotes. The nuclear versus cytoplasmic accommodation of genetic material accounts for the distinct eukaryotic and prokaryotic modes of genome evolution, but it falls short of explaining the differences in the chromosome organization. I propose that the two distinct ways to organize chromosomes are driven by the differences between the global-consecutive chromosome cycle of eukaryotes and the local-concurrent chromosome cycle of prokaryotes. Specifically, progressive chromosome segregation in prokaryotes demands a single duplicon per chromosome, while other “precarious” features of the prokaryotic chromosomes can be viewed as compensations for this severe restriction. PMID:24633873

A single cyclin–CDK complex is sufficient for both mitotic and meiotic progression in fission yeast

PubMed Central

Gutiérrez-Escribano, Pilar; Nurse, Paul

2015-01-01

The dominant model for eukaryotic cell cycle control proposes that cell cycle progression is driven by a succession of CDK complexes with different substrate specificities. However, in fission yeast it has been shown that a single CDK complex generated by the fusion of the Cdc13 cyclin with the CDK protein Cdc2 can drive the mitotic cell cycle. Meiosis is a modified cell cycle programme in which a single S-phase is followed by two consecutive rounds of chromosome segregation. Here we systematically analyse the requirements of the different fission yeast cyclins for meiotic cell cycle progression. We also show that a single Cdc13–Cdc2 complex, in the absence of the other cyclins, can drive the meiotic cell cycle. We propose that qualitatively different CDK complexes are not absolutely required for cell cycle progression either during mitosis or meiosis, and that a single CDK complex can drive both cell cycle programmes. PMID:25891897

Transcription Factors Encoded on Core and Accessory Chromosomes of Fusarium oxysporum Induce Expression of Effector Genes

PubMed Central

van der Does, H. Charlotte; Schmidt, Sarah M.; Langereis, Léon; Hughes, Timothy R.

2016-01-01

Proteins secreted by pathogens during host colonization largely determine the outcome of pathogen-host interactions and are commonly called ‘effectors’. In fungal plant pathogens, coordinated transcriptional up-regulation of effector genes is a key feature of pathogenesis and effectors are often encoded in genomic regions with distinct repeat content, histone code and rate of evolution. In the tomato pathogen Fusarium oxysporum f. sp. lycopersici (Fol), effector genes reside on one of four accessory chromosomes, known as the ‘pathogenicity’ chromosome, which can be exchanged between strains through horizontal transfer. The three other accessory chromosomes in the Fol reference strain may also be important for virulence towards tomato. Expression of effector genes in Fol is highly up-regulated upon infection and requires Sge1, a transcription factor encoded on the core genome. Interestingly, the pathogenicity chromosome itself contains 13 predicted transcription factor genes and for all except one, there is a homolog on the core genome. We determined DNA binding specificity for nine transcription factors using oligonucleotide arrays. The binding sites for homologous transcription factors were highly similar, suggesting that extensive neofunctionalization of DNA binding specificity has not occurred. Several DNA binding sites are enriched on accessory chromosomes, and expression of FTF1, its core homolog FTF2 and SGE1 from a constitutive promoter can induce expression of effector genes. The DNA binding sites of only these three transcription factors are enriched among genes up-regulated during infection. We further show that Ftf1, Ftf2 and Sge1 can activate transcription from their binding sites in yeast. RNAseq analysis revealed that in strains with constitutive expression of FTF1, FTF2 or SGE1, expression of a similar set of plant-responsive genes on the pathogenicity chromosome is induced, including most effector genes. We conclude that the Fol

Physical determinants of bipolar mitotic spindle assembly and stability in fission yeast

NASA Astrophysics Data System (ADS)

Betterton, Meredith; Blackwell, Robert; Edelmaier, Christopher; Sweezy-Schindler, Oliver; Lamson, Adam; Gergely, Zachary; O'Toole, Eileen; Crapo, Ammon; Hough, Loren; McIntosh, J. Richard; Glaser, Matthew

Mitotic spindles use an elegant bipolar architecture to segregate duplicated chromosomes with high fidelity. Bipolar spindles form from a monopolar initial condition; this is the most fundamental construction problem that the spindle must solve. Microtubules, motors, and crosslinkers are important for bipolarity, but the mechanisms necessary and sufficient for spindle assembly remain unknown. Here we describe a physical model that exhibits de novo bipolar spindle formation. We began with previously published data on fission-yeast spindle-pole-body size and microtubule number, kinesin-5 motors, kinesin-14 motors, and passive crosslinkers. Our model results agree quantitatively with our experiments in fission yeast, thereby establishing a minimal system with which to interrogate collective self assembly. By varying features of our model, we identify a set of functions essential for the generation and stability of spindle bipolarity. When kinesin-5 motors are present, their bidirectionality is essential, but spindles can form in the presence of passive crosslinkers alone. We also identify characteristic failed states of spindle assembly, which are avoided by creation and maintenance of antiparallel microtubule overlaps. DMR-0847685, DMR-1551095, DMR-1420736, K25GM110486, R01GM104976, R01GM033787.

Activation of a yeast replication origin near a double-stranded DNA break.

PubMed

Raghuraman, M K; Brewer, B J; Fangman, W L

1994-03-01

Irradiation in the G1 phase of the cell cycle delays the onset of DNA synthesis and transiently inhibits the activation of replication origins in mammalian cells. It has been suggested that this inhibition is the result of the loss of torsional tension in the DNA after it has been damaged. Because irradiation causes DNA damage at an undefined number of nonspecific sites in the genome, it is not known how cells respond to limited DNA damage, and how replication origins in the immediate vicinity of a damage site would behave. Using the sequence-specific HO endonuclease, we have created a defined double-stranded DNA break in a centromeric plasmid in G1-arrested cells of the yeast Saccharomyces cerevisiae. We show that replication does initiate at the origin on the cut plasmid, and that the plasmid replicates early in the S phase after linearization in vivo. These observations suggest that relaxation of a supercoiled DNA domain in yeast need not inactivate replication origins within that domain. Furthermore, these observations rule out the possibility that the late replication context associated with chromosomal termini is a consequence of DNA ends.

A new chromosome race of Calycadenia pauciflora (Asteraceae: Heliantheae-Madiinae) from Butte County, California.

PubMed

Carr, G D; Carr, R L

2000-10-01

We initiated a biosystematic study of a recently discovered population of Calycadenia pauciflora in order to evaluate its cytogenetic relationship to previously characterized chromosome races of that species. Cytogenetic analyses of six or more artificially produced individuals of each of the five possible interracial hybrid combinations indicated that the new race (designated Wurlitzer) is differentiated from the other races (Elegans, Healdsburg, Pauciflora, Ramulosa, and Tehama) by the equivalent of 2-4 reciprocal chromosome translocations and in one instance apparently a pericentric inversion. Mean pollen stainability in the hybrids ranged from 13 to 26%. The floral and vegetative features of the new race are very similar to those of races Pauciflora, Ramulosa, and Tehama of C. pauciflora. We ascribe the apparent lack of single-step cytogenetic events in the evolution of the races of C. pauciflora to one or more of the following: (1) (in some cases) the occurrence of saltational chromosome reorganization; (2) extinction of or failure to detect intermediate populations in C. pauciflora; and (3) an insufficient consideration of the possibility of the existence of intermediate races in the closely related species, C. fremontii. We conclude that the C. fremontii-C. pauciflora alliance is one of the most complex and potentially instructive examples of diploid chromosome evolution in plants.

Effects of Anticancer Drugs on Chromosome Instability and New Clinical Implications for Tumor-Suppressing Therapies.

PubMed

Lee, Hee-Sheung; Lee, Nicholas C O; Kouprina, Natalay; Kim, Jung-Hyun; Kagansky, Alex; Bates, Susan; Trepel, Jane B; Pommier, Yves; Sackett, Dan; Larionov, Vladimir

2016-02-15

Whole chromosomal instability (CIN), manifested as unequal chromosome distribution during cell division, is a distinguishing feature of most cancer types. CIN is generally considered to drive tumorigenesis, but a threshold level exists whereby further increases in CIN frequency in fact hinder tumor growth. While this attribute is appealing for therapeutic exploitation, drugs that increase CIN beyond this therapeutic threshold are currently limited. In our previous work, we developed a quantitative assay for measuring CIN based on the use of a nonessential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene. Here, we used this assay to rank 62 different anticancer drugs with respect to their effects on chromosome transmission fidelity. Drugs with various mechanisms of action, such as antimicrotubule activity, histone deacetylase inhibition, mitotic checkpoint inhibition, and targeting of DNA replication and damage responses, were included in the analysis. Ranking of the drugs based on their ability to induce HAC loss revealed that paclitaxel, gemcitabine, dactylolide, LMP400, talazoparib, olaparib, peloruside A, GW843682, VX-680, and cisplatin were the top 10 drugs demonstrating HAC loss at a high frequency. Therefore, identification of currently used compounds that greatly increase chromosome mis-segregation rates should expedite the development of new therapeutic strategies to target and leverage the CIN phenotype in cancer cells. ©2016 American Association for Cancer Research.

New yeasts-new brews: modern approaches to brewing yeast design and development.

PubMed

Gibson, B; Geertman, J-M A; Hittinger, C T; Krogerus, K; Libkind, D; Louis, E J; Magalhães, F; Sampaio, J P

2017-06-01

The brewing industry is experiencing a period of change and experimentation largely driven by customer demand for product diversity. This has coincided with a greater appreciation of the role of yeast in determining the character of beer and the widespread availability of powerful tools for yeast research. Genome analysis in particular has helped clarify the processes leading to domestication of brewing yeast and has identified domestication signatures that may be exploited for further yeast development. The functional properties of non-conventional yeast (both Saccharomyces and non-Saccharomyces) are being assessed with a view to creating beers with new flavours as well as producing flavoursome non-alcoholic beers. The discovery of the psychrotolerant S. eubayanus has stimulated research on de novo S. cerevisiae × S. eubayanus hybrids for low-temperature lager brewing and has led to renewed interest in the functional importance of hybrid organisms and the mechanisms that determine hybrid genome function and stability. The greater diversity of yeast that can be applied in brewing, along with an improved understanding of yeasts' evolutionary history and biology, is expected to have a significant and direct impact on the brewing industry, with potential for improved brewing efficiency, product diversity and, above all, customer satisfaction. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Y-chromosome microdeletions are not associated with SHOX haploinsufficiency.

PubMed

Chianese, C; Lo Giacco, D; Tüttelmann, F; Ferlin, A; Ntostis, P; Vinci, S; Balercia, G; Ars, E; Ruiz-Castañé, E; Giglio, S; Forti, G; Kliesch, S; Krausz, C

2013-11-01

assess whether the SHOX duplications found in the two men with Y-chromosome microdeletions and a normal karyotype represent a neutral polymorphism or are actually associated with the presence of the microdeletion. Men suffering from infertility due to the presence of Y-chromosome microdeletions can resort to artificial reproductive technology (ART) to father their biological children. However, infertile couples must be aware of the risks implied and this makes genetic counseling a crucial step in the patient's management. This study does not confirm previous alarming data that showed an association between Y-chromosome microdeletions and SHOX haploinsufficiency. Our results imply that deletion carriers have no augmented risk of SHOX-related pathologies (short stature and skeletal anomalies) and indicate that there is no need for radical changes in genetic counseling of Yq microdeletion carriers attempting ART, since the only risk established so far for their male offspring remains impaired spermatogenesis. This work was supported by the Italian Ministry of University (grant PRIN 2010-2012 to C.K.), Tuscan Regional Health Research Program ('Progetto Salute 2009') to G.F., the Spanish Ministry of Health (grant FIS-11/02254) and the European Union 'Reprotrain' Marie Curie Network (project number: 289880 to C.K.). The authors declare that no conflicting interests exist.

Vaginal yeast infection

MedlinePlus

Yeast infection - vagina; Vaginal candidiasis; Monilial vaginitis ... Most women have a vaginal yeast infection at some time. Candida albicans is a common type of fungus. It is often found in small amounts in the ...

Artificial activation of toxin-antitoxin systems as an antibacterial strategy.

PubMed

Williams, Julia J; Hergenrother, Paul J

2012-06-01

Toxin-antitoxin (TA) systems are unique modules that effect plasmid stabilization via post-segregational killing of the bacterial host. The genes encoding TA systems also exist on bacterial chromosomes, and it has been speculated that these are involved in a variety of cellular processes. Interest in TA systems has increased dramatically over the past 5 years as the ubiquitous nature of TA genes on bacterial genomes has been revealed. The exploitation of TA systems as an antibacterial strategy via artificial activation of the toxin has been proposed and has considerable potential; however, efforts in this area remain in the early stages and several major questions remain. This review investigates the tractability of targeting TA systems to kill bacteria, including fundamental requirements for success, recent advances, and challenges associated with artificial toxin activation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Schizosaccharomyces japonicus: the fission yeast is a fusion of yeast and hyphae.

PubMed

Niki, Hironori

2014-03-01

The clade of Schizosaccharomyces includes 4 species: S. pombe, S. octosporus, S. cryophilus, and S. japonicus. Although all 4 species exhibit unicellular growth with a binary fission mode of cell division, S. japonicus alone is dimorphic yeast, which can transit from unicellular yeast to long filamentous hyphae. Recently it was found that the hyphal cells response to light and then synchronously activate cytokinesis of hyphae. In addition to hyphal growth, S. japonicas has many properties that aren't shared with other fission yeast. Mitosis of S. japonicas is referred to as semi-open mitosis because dynamics of nuclear membrane is an intermediate mode between open mitosis and closed mitosis. Novel genetic tools and the whole genomic sequencing of S. japonicas now provide us with an opportunity for revealing unique characters of the dimorphic yeast. © 2013 The Author. Yeast Published by John Wiley & Sons Ltd.

The Malleable Nature of the Budding Yeast Nuclear Envelope: Flares, Fusion, and Fenestrations.

PubMed

Meseroll, Rebecca A; Cohen-Fix, Orna

2016-11-01

In eukaryotes, the nuclear envelope (NE) physically separates nuclear components and activities from rest of the cell. The NE also provides rigidity to the nucleus and contributes to chromosome organization. At the same time, the NE is highly dynamic; it must change shape and rearrange its components during development and throughout the cell cycle, and its morphology can be altered in response to mutation and disease. Here we focus on the NE of budding yeast, Saccharomyces cerevisiae, which has several unique features: it remains intact throughout the cell cycle, expands symmetrically during interphase, elongates during mitosis and, expands asymmetrically during mitotic delay. Moreover, its NE is safely breached during mating and when large structures, such as nuclear pore complexes and the spindle pole body, are embedded into its double membrane. The budding yeast NE lacks lamins and yet the nucleus is capable of maintaining a spherical shape throughout interphase. Despite these eccentricities, studies of the budding yeast NE have uncovered interesting, and likely conserved, processes that contribute to NE dynamics. In particular, we discuss the processes that drive and enable NE expansion and the dramatic changes in the NE that lead to extensions and fenestrations. J. Cell. Physiol. 231: 2353-2360, 2016. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Correlation of Meiotic DSB Formation and Transcription Initiation Around Fission Yeast Recombination Hotspots.

PubMed

Yamada, Shintaro; Okamura, Mika; Oda, Arisa; Murakami, Hiroshi; Ohta, Kunihiro; Yamada, Takatomi

2017-06-01

Meiotic homologous recombination, a critical event for ensuring faithful chromosome segregation and creating genetic diversity, is initiated by programmed DNA double-strand breaks (DSBs) formed at recombination hotspots. Meiotic DSB formation is likely to be influenced by other DNA-templated processes including transcription, but how DSB formation and transcription interact with each other has not been understood well. In this study, we used fission yeast to investigate a possible interplay of these two events. A group of hotspots in fission yeast are associated with sequences similar to the cyclic AMP response element and activated by the ATF/CREB family transcription factor dimer Atf1-Pcr1. We first focused on one of those hotspots, ade6-3049 , and Atf1. Our results showed that multiple transcripts, shorter than the ade6 full-length messenger RNA, emanate from a region surrounding the ade6-3049 hotspot. Interestingly, we found that the previously known recombination-activation region of Atf1 is also a transactivation domain, whose deletion affected DSB formation and short transcript production at ade6-3049 These results point to a possibility that the two events may be related to each other at ade6-3049 In fact, comparison of published maps of meiotic transcripts and hotspots suggested that hotspots are very often located close to meiotically transcribed regions. These observations therefore propose that meiotic DSB formation in fission yeast may be connected to transcription of surrounding regions. Copyright © 2017 by the Genetics Society of America.

Repetitive telomeric sequences in chromosomal translocations involving chromosome 21

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qu, J.; Dallaire, L.; Fetni, R.

Telomeres perform key functions in maintaining chromosome integrity. In some structural rearrangements the structure and polymorphism in human telomeres may play a significant role. However, of all the telomeric and subtelomeric sequences, only the terminal TTAGGG repeats are believed essential for telomere function. During the course of a study on the role of telomere structure and polymorphism in chromosomal rearrangements observed in families referred for prenatal diagnosis, we studied three cases in which chromosome 21 was involved. Repetitive TTAGGG sequences for all human chromosomes were used as probes (Oncor). Case 1, a de novo cryptic translocation (2;21) was initially identifiedmore » as monosomy 21 in a child with psychomotor delay and mild dysmorphism. Using a cosmid probe specific for region 21q22.3 and whole chromosome 21 specific painting probe, the long arm of 21 was found on the short arm of chromosome 2 with an interstitial telomere at the breakpoint junction. All the cells were monosomic for 21pter{yields}q21. Case 2 is a familial (19;21) translocation. GTG-banding and FISH with a satellite probe showed no apparent loss of material at the end of either 19q or 21q, with an interstitial telomere at the fusion site of the two intact chromosomes. In case 3, a four generation reciprocal (20;21) translocation, there was no interstitial telomere. The persistence of an interstitial telomere is a relatively rare event which can now be observed with in situ hybridization. Its study may lead to a better understanding of the dynamics of translocations and of chromosome imbalance.« less

Effects of cryoprotectants on the viability and activity of freeze dried recombinant yeasts as novel oral drug delivery systems assessed by an artificial digestive system.

PubMed

Blanquet, Stéphanie; Garrait, Ghislain; Beyssac, Erick; Perrier, Céline; Denis, Sylvain; Hébrard, Géraldine; Alric, Monique

2005-09-01

The aim of this study was to investigate, in a gastric-small intestinal system TIM-1, the effect of cryoprotectants on the survival of freeze-dried Saccharomyces cerevisiae expressing the heterologous P450 73A1 and their ability to convert trans-cinnamic acid into p-coumaric acid. Yeasts were lyophilized in suspensions of trehalose, maltose, lactose, or a milk proteins/trehalose mix. Freeze-dried or native yeasts and trans-cinnamic acid were introduced simultaneously into TIM-1 at the beginning of digestion. Yeast survival rate was evaluated by cell counting in the ileal effluents. P450 73A1 activity was followed by HPLC assay of p-coumaric acid. Freeze-dried yeasts showed high tolerance to digestive conditions. Nevertheless, their survival rate was lower than that of non-dried cells (around 80% whatever the protective agent vs. 96%). The ability of recombinant freeze-dried S. cerevisiae to perform a bioconversion reaction in the digestive tract was shown with all the protectants. The highest trans-cinnamic acid conversion rate (24 vs. 41% for native yeasts) was obtained with the milk proteins/trehalose mix. These results show that freeze-drying might be considered for the pharmaceutical formulation of new drug delivery systems based on orally administered recombinant yeasts and that TIM-1 could be a helpful tool for the pre-screening of oral dosage forms.

Marine yeast isolation and industrial application.

PubMed

Zaky, Abdelrahman Saleh; Tucker, Gregory A; Daw, Zakaria Yehia; Du, Chenyu

2014-09-01

Over the last century, terrestrial yeasts have been widely used in various industries, such as baking, brewing, wine, bioethanol and pharmaceutical protein production. However, only little attention has been given to marine yeasts. Recent research showed that marine yeasts have several unique and promising features over the terrestrial yeasts, for example higher osmosis tolerance, higher special chemical productivity and production of industrial enzymes. These indicate that marine yeasts have great potential to be applied in various industries. This review gathers the most recent techniques used for marine yeast isolation as well as the latest applications of marine yeast in bioethanol, pharmaceutical and enzyme production fields. © 2014 The Authors FEMS Yeast Research published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

Fragile DNA Motifs Trigger Mutagenesis at Distant Chromosomal Loci in Saccharomyces cerevisiae

PubMed Central

Saini, Natalie; Zhang, Yu; Nishida, Yuri; Sheng, Ziwei; Choudhury, Shilpa; Mieczkowski, Piotr; Lobachev, Kirill S.

2013-01-01

DNA sequences capable of adopting non-canonical secondary structures have been associated with gross-chromosomal rearrangements in humans and model organisms. Previously, we have shown that long inverted repeats that form hairpin and cruciform structures and triplex-forming GAA/TTC repeats induce the formation of double-strand breaks which trigger genome instability in yeast. In this study, we demonstrate that breakage at both inverted repeats and GAA/TTC repeats is augmented by defects in DNA replication. Increased fragility is associated with increased mutation levels in the reporter genes located as far as 8 kb from both sides of the repeats. The increase in mutations was dependent on the presence of inverted or GAA/TTC repeats and activity of the translesion polymerase Polζ. Mutagenesis induced by inverted repeats also required Sae2 which opens hairpin-capped breaks and initiates end resection. The amount of breakage at the repeats is an important determinant of mutations as a perfect palindromic sequence with inherently increased fragility was also found to elevate mutation rates even in replication-proficient strains. We hypothesize that the underlying mechanism for mutagenesis induced by fragile motifs involves the formation of long single-stranded regions in the broken chromosome, invasion of the undamaged sister chromatid for repair, and faulty DNA synthesis employing Polζ. These data demonstrate that repeat-mediated breaks pose a dual threat to eukaryotic genome integrity by inducing chromosomal aberrations as well as mutations in flanking genes. PMID:23785298

Yeast ecology of Kombucha fermentation.

PubMed

Teoh, Ai Leng; Heard, Gillian; Cox, Julian

2004-09-01

Kombucha is a traditional fermentation of sweetened tea, involving a symbiosis of yeast species and acetic acid bacteria. Despite reports of different yeast species being associated with the fermentation, little is known of the quantitative ecology of yeasts in Kombucha. Using oxytetracycline-supplemented malt extract agar, yeasts were isolated from four commercially available Kombucha products and identified using conventional biochemical and physiological tests. During the fermentation of each of the four products, yeasts were enumerated from both the cellulosic pellicle and liquor of the Kombucha. The number and diversity of species varied between products, but included Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii and Zygosaccharomyces bailii. While these yeast species are known to occur in Kombucha, the enumeration of each species present throughout fermentation of each of the four Kombucha cultures demonstrated for the first time the dynamic nature of the yeast ecology. Kombucha fermentation is, in general, initiated by osmotolerant species, succeeded and ultimately dominated by acid-tolerant species.

Marine yeast isolation and industrial application

PubMed Central

Zaky, Abdelrahman Saleh; Tucker, Gregory A; Daw, Zakaria Yehia; Du, Chenyu

2014-01-01

Over the last century, terrestrial yeasts have been widely used in various industries, such as baking, brewing, wine, bioethanol and pharmaceutical protein production. However, only little attention has been given to marine yeasts. Recent research showed that marine yeasts have several unique and promising features over the terrestrial yeasts, for example higher osmosis tolerance, higher special chemical productivity and production of industrial enzymes. These indicate that marine yeasts have great potential to be applied in various industries. This review gathers the most recent techniques used for marine yeast isolation as well as the latest applications of marine yeast in bioethanol, pharmaceutical and enzyme production fields. PMID:24738708

Evidence that meiotic pairing starts at the telomeres: Molecular analysis of recombination in a family with a pericentric X chromosome inversion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shashi, V.; Allinson, P.S.; Golden, W.L.

1994-09-01

Recent studies in yeast have shown that telomeres rather than centromeres lead in chromosome movement just prior to meiosis and may have a role in recombination. Cytological studies of meiosis in Drosophila and mice have shown that in pericentric inversion heterozygotes there is lack of loop formation, with recobmination seen only outside the inversion. In a family with Duchenne muscular dystrophy (DMD) we recognized that only affected males and carrier females had a pericentric X chromosome inversion (inv X(p11.4;q26)). Since the short arm inversion breakpoint was proximal to the DMD locus, it could not be implicated in the mutational eventmore » causing DMD. There was no history of infertility, recurrent miscarriages or liveborn unbalanced females to suggest there was recombination within the inversion. We studied 22 members over three generations to understand the pattern of meiotic recombination between the normal and the inverted X chromosome. In total, 17 meioses involving the inverted X chromosome in females were studied by cytogenetic analysis and 16 CA repeat polymorphisms along the length of the X chromosome. Results: (a) There was complete concordance between the segregation of the DMD mutation and the inverted X chromosome. (b) On DNA analysis, there was complete absence of recombination within the inverted segment. We also found no recombination at the DMD locus. Recombination was seen only at Xp22 and Xq27-28. (c) Recombination was seen in the same individual at both Xp22 and Xq27-28 without recombination otherwise. Conclusions: (1) Pericentric X inversions reduce the genetic map length of the chromosome, with the physical map length being normal. (2) Meiotic X chromosome pairing in this family is initiated at the telomeres. (3) Following telomeric pairing in pericentric X chromosome inversions, there is inhibition of recombination within the inversion and adjacent regions.« less

APC/C-Cdc20 mediates deprotection of centromeric cohesin at meiosis II in yeast.

PubMed

Jonak, Katarzyna; Zagoriy, Ievgeniia; Oz, Tugce; Graf, Peter; Rojas, Julie; Mengoli, Valentina; Zachariae, Wolfgang

2017-06-18

Cells undergoing meiosis produce haploid gametes through one round of DNA replication followed by 2 rounds of chromosome segregation. This requires that cohesin complexes, which establish sister chromatid cohesion during S phase, are removed in a stepwise manner. At meiosis I, the separase protease triggers the segregation of homologous chromosomes by cleaving cohesin's Rec8 subunit on chromosome arms. Cohesin persists at centromeres because the PP2A phosphatase, recruited by the shugoshin protein, dephosphorylates Rec8 and thereby protects it from cleavage. While chromatids disjoin upon cleavage of centromeric Rec8 at meiosis II, it was unclear how and when centromeric Rec8 is liberated from its protector PP2A. One proposal is that bipolar spindle forces separate PP2A from Rec8 as cells enter metaphase II. We show here that sister centromere biorientation is not sufficient to "deprotect" Rec8 at meiosis II in yeast. Instead, our data suggest that the ubiquitin-ligase APC/C Cdc20 removes PP2A from centromeres by targeting for degradation the shugoshin Sgo1 and the kinase Mps1. This implies that Rec8 remains protected until entry into anaphase II when it is phosphorylated concurrently with the activation of separase. Here, we provide further support for this model and speculate on its relevance to mammalian oocytes.

APC/C-Cdc20 mediates deprotection of centromeric cohesin at meiosis II in yeast

PubMed Central

Jonak, Katarzyna; Oz, Tugce; Graf, Peter; Rojas, Julie; Mengoli, Valentina; Zachariae, Wolfgang

2017-01-01

ABSTRACT Cells undergoing meiosis produce haploid gametes through one round of DNA replication followed by 2 rounds of chromosome segregation. This requires that cohesin complexes, which establish sister chromatid cohesion during S phase, are removed in a stepwise manner. At meiosis I, the separase protease triggers the segregation of homologous chromosomes by cleaving cohesin's Rec8 subunit on chromosome arms. Cohesin persists at centromeres because the PP2A phosphatase, recruited by the shugoshin protein, dephosphorylates Rec8 and thereby protects it from cleavage. While chromatids disjoin upon cleavage of centromeric Rec8 at meiosis II, it was unclear how and when centromeric Rec8 is liberated from its protector PP2A. One proposal is that bipolar spindle forces separate PP2A from Rec8 as cells enter metaphase II. We show here that sister centromere biorientation is not sufficient to “deprotect” Rec8 at meiosis II in yeast. Instead, our data suggest that the ubiquitin-ligase APC/CCdc20 removes PP2A from centromeres by targeting for degradation the shugoshin Sgo1 and the kinase Mps1. This implies that Rec8 remains protected until entry into anaphase II when it is phosphorylated concurrently with the activation of separase. Here, we provide further support for this model and speculate on its relevance to mammalian oocytes. PMID:28514186

Construction of physical maps for the sex-specific regions of papaya sex chromosomes.

PubMed

Na, Jong-Kuk; Wang, Jianping; Murray, Jan E; Gschwend, Andrea R; Zhang, Wenli; Yu, Qingyi; Navajas-Pérez, Rafael; Feltus, F Alex; Chen, Cuixia; Kubat, Zdenek; Moore, Paul H; Jiang, Jiming; Paterson, Andrew H; Ming, Ray

2012-05-08

Papaya is a major fruit crop in tropical and subtropical regions worldwide. It is trioecious with three sex forms: male, female, and hermaphrodite. Sex determination is controlled by a pair of nascent sex chromosomes with two slightly different Y chromosomes, Y for male and Yh for hermaphrodite. The sex chromosome genotypes are XY (male), XYh (hermaphrodite), and XX (female). The papaya hermaphrodite-specific Yh chromosome region (HSY) is pericentromeric and heterochromatic. Physical mapping of HSY and its X counterpart is essential for sequencing these regions and uncovering the early events of sex chromosome evolution and to identify the sex determination genes for crop improvement. A reiterate chromosome walking strategy was applied to construct the two physical maps with three bacterial artificial chromosome (BAC) libraries. The HSY physical map consists of 68 overlapped BACs on the minimum tiling path, and covers all four HSY-specific Knobs. One gap remained in the region of Knob 1, the only knob structure shared between HSY and X, due to the lack of HSY-specific sequences. This gap was filled on the physical map of the HSY corresponding region in the X chromosome. The X physical map consists of 44 BACs on the minimum tiling path with one gap remaining in the middle, due to the nature of highly repetitive sequences. This gap was filled on the HSY physical map. The borders of the non-recombining HSY were defined genetically by fine mapping using 1460 F2 individuals. The genetically defined HSY spanned approximately 8.5 Mb, whereas its X counterpart extended about 5.4 Mb including a 900 Kb region containing the Knob 1 shared by the HSY and X. The 8.5 Mb HSY corresponds to 4.5 Mb of its X counterpart, showing 4 Mb (89%) DNA sequence expansion. The 89% increase of DNA sequence in HSY indicates rapid expansion of the Yh chromosome after genetic recombination was suppressed 2-3 million years ago. The genetically defined borders coincide with the common

Components of a Fanconi-like pathway control Pso2-independent DNA interstrand crosslink repair in yeast.

PubMed

Ward, Thomas A; Dudášová, Zuzana; Sarkar, Sovan; Bhide, Mangesh R; Vlasáková, Danuša; Chovanec, Miroslav; McHugh, Peter J

2012-01-01

Fanconi anemia (FA) is a devastating genetic disease, associated with genomic instability and defects in DNA interstrand cross-link (ICL) repair. The FA repair pathway is not thought to be conserved in budding yeast, and although the yeast Mph1 helicase is a putative homolog of human FANCM, yeast cells disrupted for MPH1 are not sensitive to ICLs. Here, we reveal a key role for Mph1 in ICL repair when the Pso2 exonuclease is inactivated. We find that the yeast FANCM ortholog Mph1 physically and functionally interacts with Mgm101, a protein previously implicated in mitochondrial DNA repair, and the MutSα mismatch repair factor (Msh2-Msh6). Co-disruption of MPH1, MGM101, MSH6, or MSH2 with PSO2 produces a lesion-specific increase in ICL sensitivity, the elevation of ICL-induced chromosomal rearrangements, and persistence of ICL-associated DNA double-strand breaks. We find that Mph1-Mgm101-MutSα directs the ICL-induced recruitment of Exo1 to chromatin, and we propose that Exo1 is an alternative 5'-3' exonuclease utilised for ICL repair in the absence of Pso2. Moreover, ICL-induced Rad51 chromatin loading is delayed when both Pso2 and components of the Mph1-Mgm101-MutSα and Exo1 pathway are inactivated, demonstrating that the homologous recombination stages of ICL repair are inhibited. Finally, the FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are also components of the genetic pathway controlled by Mph1-Mgm101-MutSα. Together this suggests that a prototypical FA-related ICL repair pathway operates in budding yeast, which acts redundantly with the pathway controlled by Pso2, and is required for the targeting of Exo1 to chromatin to execute ICL repair.

The X chromosome of monotremes shares a highly conserved region with the eutherian and marsupial X chromosomes despite the absence of X chromosome inactivation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watson, J.M.; Spencer, J.A.; Graves, J.A.M.

1990-09-01

Eight genes, located on the long arm of the human X chromosome and present on the marsupial X chromosome, were mapped by in situ hybridization to the chromosomes of the platypus Ornithorhynchus anatinus, one of the three species of monotreme mammals. All were located on the X chromosome. The authors conclude that the long arm of the human X chromosome represents a highly conserved region that formed part of the X chromosome in a mammalian ancestor at least 150 million years ago. Since three of these genes are located on the long arm of the platypus X chromosome, which ismore » G-band homologous to the Y chromosome and apparently exempt from X chromosome inactivation, the conservation of this region has evidently not depended on isolation by X-Y chromosome differentiation and X chromosome inactivation.« less

Chromosome aberration analysis in atomic bomb survivors and Thorotrast patients using two- and three-colour chromosome painting of chromosomal subsets.

PubMed

Tanaka, K; Popp, S; Fischer, C; Van Kaick, G; Kamada, N; Cremer, T; Cremer, C

1996-07-01

Chromosomal translocations in peripheral lymphocytes of three healthy Hiroshima atomic (A)-bomb survivors, as well as three Thorotrast patients and two non-irradiated age-matched control persons from the German Thorotrast study were studied by two- and three-colour fluorescence in situ hybridization (chromosome painting) with various combinations of whole chromosome composite probes, including chromosomes 1, 2, 3, 4, 6, 7, 8, 9 and 12. Translocation frequencies detected by chromosome painting in cells of the A-bomb survivors were compared with results obtained by G-banding. A direct comparison was made, i.e. only those cells with simple translocations or complex aberrations detected by G-banding were taken into consideration which in principle could be detected also with the respective painting combination. The statistical analysis revealed no significant differences from a 1:1 relationship between the frequencies of aberrant cells obtained by both methods. The use of genomic translocation frequencies estimated from subsets of chromosomes for biological dosimetry is discussed in the light of evidence that chromosomes occupy distinct territories and are variably arranged in human lymphocyte nuclei. This territorial organization of interphase chromosomes implies that translocations will be restricted to chromatin located at the periphery of adjacent chromosome territories.

Genomic reconstruction to improve bioethanol and ergosterol production of industrial yeast Saccharomyces cerevisiae.

PubMed

Zhang, Ke; Tong, Mengmeng; Gao, Kehui; Di, Yanan; Wang, Pinmei; Zhang, Chunfang; Wu, Xuechang; Zheng, Daoqiong

2015-02-01

Baker's yeast (Saccharomyces cerevisiae) is the common yeast used in the fields of bread making, brewing, and bioethanol production. Growth rate, stress tolerance, ethanol titer, and byproducts yields are some of the most important agronomic traits of S. cerevisiae for industrial applications. Here, we developed a novel method of constructing S. cerevisiae strains for co-producing bioethanol and ergosterol. The genome of an industrial S. cerevisiae strain, ZTW1, was first reconstructed through treatment with an antimitotic drug followed by sporulation and hybridization. A total of 140 mutants were selected for ethanol fermentation testing, and a significant positive correlation between ergosterol content and ethanol production was observed. The highest performing mutant, ZG27, produced 7.9 % more ethanol and 43.2 % more ergosterol than ZTW1 at the end of fermentation. Chromosomal karyotyping and proteome analysis of ZG27 and ZTW1 suggested that this breeding strategy caused large-scale genome structural variations and global gene expression diversities in the mutants. Genetic manipulation further demonstrated that the altered expression activity of some genes (such as ERG1, ERG9, and ERG11) involved in ergosterol synthesis partly explained the trait improvement in ZG27.

The CCR4-NOT Complex Is Implicated in the Viability of Aneuploid Yeasts

PubMed Central

Tange, Yoshie; Kurabayashi, Atsushi; Goto, Bunshiro; Hoe, Kwang-Lae; Kim, Dong-Uk; Park, Han-Oh; Hayles, Jacqueline; Chikashige, Yuji; Tsutumi, Chihiro; Hiraoka, Yasushi; Yamao, Fumiaki; Nurse, Paul; Niwa, Osami

2012-01-01

To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability. PMID:22737087

Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

PubMed Central

Vizeacoumar, Franco J.; van Dyk, Nydia; S.Vizeacoumar, Frederick; Cheung, Vincent; Li, Jingjing; Sydorskyy, Yaroslav; Case, Nicolle; Li, Zhijian; Datti, Alessandro; Nislow, Corey; Raught, Brian; Zhang, Zhaolei; Frey, Brendan; Bloom, Kerry

2010-01-01

We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions. PMID:20065090

Sex chromosome aneuploidies.

PubMed

Skuse, David; Printzlau, Frida; Wolstencroft, Jeanne

2018-01-01

Sex chromosome aneuploidies comprise a relatively common group of chromosome disorders characterized by the loss or gain of one or more sex chromosomes. We discuss five of the better-known sex aneuploidies: Turner syndrome (XO), Klinefelter syndrome (XXY), trisomy X (XXX), XYY, and XXYY. Despite their prevalence in the general population, these disorders are underdiagnosed and the specific genetic mechanisms underlying their phenotypes are poorly understood. Although there is considerable variation between them in terms of associated functional impairment, each disorder has a characteristic physical, cognitive, and neurologic profile. The most common cause of sex chromosome aneuploidies is nondisjunction, which can occur during meiosis or during the early stages of postzygotic development. The loss or gain of genetic material can affect all daughter cells or it may be partial, leading to tissue mosaicism. In both typical and atypical sex chromosome karyotypes, there is random inactivation of all but one X chromosome. The mechanisms by which a phenotype results from sex chromosome aneuploidies are twofold: dosage imbalance arising from a small number of genes that escape inactivation, and their endocrinologic consequences. Copyright © 2018 Elsevier B.V. All rights reserved.

Wine yeasts for the future.

PubMed

Fleet, Graham H

2008-11-01

International competition within the wine market, consumer demands for newer styles of wines and increasing concerns about the environmental sustainability of wine production are providing new challenges for innovation in wine fermentation. Within the total production chain, the alcoholic fermentation of grape juice by yeasts is a key process where winemakers can creatively engineer wine character and value through better yeast management and, thereby, strategically tailor wines to a changing market. This review considers the importance of yeast ecology and yeast metabolic reactions in determining wine quality, and then discusses new directions for exploiting yeasts in wine fermentation. It covers criteria for selecting and developing new commercial strains, the possibilities of using yeasts other than those in the genus of Saccharomyces, the prospects for mixed culture fermentations and explores the possibilities for high cell density, continuous fermentations.

Verification and characterization of chromosome duplication in haploid maize.

PubMed

de Oliveira Couto, E G; Resende Von Pinho, E V; Von Pinho, R G; Veiga, A D; de Carvalho, M R; de Oliveira Bustamante, F; Nascimento, M S

2015-06-26

Doubled haploid technology has been used by various private companies. However, information regarding chromosome duplication methodologies, particularly those concerning techniques used to identify duplication in cells, is limited. Thus, we analyzed and characterized artificially doubled haploids using microsatellites molecular markers, pollen viability, and flow cytometry techniques. Evaluated material was obtained using two different chromosome duplication protocols in maize seeds considered haploids, resulting from the cross between the haploid inducer line KEMS and 4 hybrids (GNS 3225, GNS 3032, GNS 3264, and DKB 393). Fourteen days after duplication, plant samples were collected and assessed by flow cytometry. Further, the plants were transplanted to a field, and samples were collected for DNA analyses using microsatellite markers. The tassels were collected during anthesis for pollen viability analyses. Haploid, diploid, and mixoploid individuals were detected using flow cytometry, demonstrating that this technique was efficient for identifying doubled haploids. The microsatellites markers were also efficient for confirming the ploidies preselected by flow cytometry and for identifying homozygous individuals. Pollen viability showed a significant difference between the evaluated ploidies when the Alexander and propionic-carmin stains were used. The viability rates between the plodies analyzed show potential for fertilization.

Chromosomal instability in Streptomyces avermitilis: major deletion in the central region and stable circularized chromosome

PubMed Central

2010-01-01

Background The chromosome of Streptomyces has been shown to be unstable, frequently undergoing gross chromosomal rearrangements. However, the mechanisms underlying this phenomenon remain unclear, with previous studies focused on two chromosomal ends as targets for rearrangements. Here we investigated chromosomal instability of Streptomyces avermitilis, an important producer of avermectins, and characterized four gross chromosomal rearrangement events, including a major deletion in the central region. The present findings provide a valuable contribution to the mechanistic study of genetic instability in Streptomyces. Results Thirty randomly-selected "bald" mutants derived from the wild-type strain all contained gross chromosomal rearrangements of various types. One of the bald mutants, SA1-8, had the same linear chromosomal structure as the high avermectin-producing mutant 76-9. Chromosomes of both strains displayed at least three independent chromosomal rearrangements, including chromosomal arm replacement to form new 88-kb terminal inverted repeats (TIRs), and two major deletions. One of the deletions eliminated the 36-kb central region of the chromosome, but surprisingly did not affect viability of the cells. The other deletion (74-kb) was internal to the right chromosomal arm. The chromosome of another bald mutant, SA1-6, was circularized with deletions at both ends. No obvious homology was found in all fusion sequences. Generational stability analysis showed that the chromosomal structure of SA1-8 and SA1-6 was stable. Conclusions Various chromosomal rearrangements, including chromosomal arm replacement, interstitial deletions and chromosomal circularization, occurred in S. avermitilis by non-homologous recombination. The finding of an inner deletion involving in the central region of S. avermitilis chromosome suggests that the entire Streptomyces chromosome may be the target for rearrangements, which are not limited, as previously reported, to the two

The Y chromosome of the Atelidae family (Platyrrhini): study by chromosome microdissection.

PubMed

Gifalli-Iughetti, C; Koiffmann, C P

2009-01-01

In order to study the intergeneric variability of the Y chromosome, we describe the hybridization of the Y chromosome of Brachytelesarachnoides, obtained by microdissection, to metaphases of Atelesbelzebuthmarginatus, Lagothrixlagothricha, and Alouatta male specimens. Brachytelesarachnoides (Atelinae) has 62 chromosomes and a very small Y chromosome. Our results showed that the Brachytelesarachnoides Y chromosome probe hybridized to Lagothrixlagothricha metaphases yielding one hybridization signal on only the tiny Y chromosome, and when hybridized with Atelesbelzebuthmarginatus metaphases it yielded one hybridization signal on two thirds of the small acrocentric Y chromosome. However, no hybridization signal was observed in Alouatta metaphases (subfamily Alouattinae), a closely related genus in the Atelidae family. Furthermore, our data support a close phylogenetic relationship among Brachyteles, Ateles, and Lagothrix and their placement in the Atelinae subfamily, but exclude Alouatta from this group indicating its placement as basal to this group. Copyright 2009 S. Karger AG, Basel.

Comparative Polygenic Analysis of Maximal Ethanol Accumulation Capacity and Tolerance to High Ethanol Levels of Cell Proliferation in Yeast

PubMed Central

Pais, Thiago M.; Foulquié-Moreno, María R.; Hubmann, Georg; Duitama, Jorge; Swinnen, Steve; Goovaerts, Annelies; Yang, Yudi; Dumortier, Françoise; Thevelein, Johan M.

2013-01-01

The yeast Saccharomyces cerevisiae is able to accumulate ≥17% ethanol (v/v) by fermentation in the absence of cell proliferation. The genetic basis of this unique capacity is unknown. Up to now, all research has focused on tolerance of yeast cell proliferation to high ethanol levels. Comparison of maximal ethanol accumulation capacity and ethanol tolerance of cell proliferation in 68 yeast strains showed a poor correlation, but higher ethanol tolerance of cell proliferation clearly increased the likelihood of superior maximal ethanol accumulation capacity. We have applied pooled-segregant whole-genome sequence analysis to identify the polygenic basis of these two complex traits using segregants from a cross of a haploid derivative of the sake strain CBS1585 and the lab strain BY. From a total of 301 segregants, 22 superior segregants accumulating ≥17% ethanol in small-scale fermentations and 32 superior segregants growing in the presence of 18% ethanol, were separately pooled and sequenced. Plotting SNP variant frequency against chromosomal position revealed eleven and eight Quantitative Trait Loci (QTLs) for the two traits, respectively, and showed that the genetic basis of the two traits is partially different. Fine-mapping and Reciprocal Hemizygosity Analysis identified ADE1, URA3, and KIN3, encoding a protein kinase involved in DNA damage repair, as specific causative genes for maximal ethanol accumulation capacity. These genes, as well as the previously identified MKT1 gene, were not linked in this genetic background to tolerance of cell proliferation to high ethanol levels. The superior KIN3 allele contained two SNPs, which are absent in all yeast strains sequenced up to now. This work provides the first insight in the genetic basis of maximal ethanol accumulation capacity in yeast and reveals for the first time the importance of DNA damage repair in yeast ethanol tolerance. PMID:23754966

The homologous recombination machinery modulates the formation of RNA–DNA hybrids and associated chromosome instability

PubMed Central

Wahba, Lamia; Gore, Steven K; Koshland, Douglas

2013-01-01

Genome instability in yeast and mammals is caused by RNA–DNA hybrids that form as a result of defects in different aspects of RNA biogenesis. We report that in yeast mutants defective for transcription repression and RNA degradation, hybrid formation requires Rad51p and Rad52p. These proteins normally promote DNA–DNA strand exchange in homologous recombination. We suggest they also directly promote the DNA–RNA strand exchange necessary for hybrid formation since we observed accumulation of Rad51p at a model hybrid-forming locus. Furthermore, we provide evidence that Rad51p mediates hybridization of transcripts to homologous chromosomal loci distinct from their site of synthesis. This hybrid formation in trans amplifies the genome-destabilizing potential of RNA and broadens the exclusive co-transcriptional models that pervade the field. The deleterious hybrid-forming activity of Rad51p is counteracted by Srs2p, a known Rad51p antagonist. Thus Srs2p serves as a novel anti-hybrid mechanism in vivo. DOI: http://dx.doi.org/10.7554/eLife.00505.001 PMID:23795288

Protein Arms in the Kinetochore-Microtubule Interface of the Yeast DASH Complex

PubMed Central

Miranda, JJ L.; King, David S.

2007-01-01

The yeast DASH complex is a heterodecameric component of the kinetochore necessary for accurate chromosome segregation. DASH forms closed rings around microtubules with a large gap between the DASH ring and the microtubule cylinder. We characterized the microtubule-binding properties of limited proteolysis products and subcomplexes of DASH, thus identifying candidate polypeptide extensions involved in establishing the DASH-microtubule interface. The acidic C-terminal extensions of tubulin subunits are not essential for DASH binding. We also measured the molecular mass of DASH rings on microtubules with scanning transmission electron microscopy and found that approximately 25 DASH heterodecamers assemble to form each ring. Dynamic association and relocation of multiple flexible appendages of DASH may allow the kinetochore to translate along the microtubule surface. PMID:17460120

Discussion of teleomorphic and anamorphic Ascomycetous yeasts and yeast-like taxa

USDA-ARS?s Scientific Manuscript database

The relationship of ascomycetous yeasts with other members of the ascomycete fungi (Ascomycota) has been controversial for over 100 years. Because yeasts are morphologically simple, it was proposed that they represent primitive forms of ascomycetes (e.g., Guilliermond 1912). Alternatively, the ide...

Physical Mapping and Refinement of the Painted Turtle Genome (Chrysemys picta) Inform Amniote Genome Evolution and Challenge Turtle-Bird Chromosomal Conservation

PubMed Central

Badenhorst, Daleen; Hillier, LaDeana W.; Literman, Robert; Montiel, Eugenia Elisabet; Radhakrishnan, Srihari; Shen, Yingjia; Minx, Patrick; Janes, Daniel E.; Warren, Wesley C.; Edwards, Scott V.; Valenzuela, Nicole

2015-01-01

Comparative genomics continues illuminating amniote genome evolution, but for many lineages our understanding remains incomplete. Here, we refine the assembly (CPI 3.0.3 NCBI AHGY00000000.2) and develop a cytogenetic map of the painted turtle (Chrysemys picta—CPI) genome, the first in turtles and in vertebrates with temperature-dependent sex determination. A comparison of turtle genomes with those of chicken, selected nonavian reptiles, and human revealed shared and novel genomic features, such as numerous chromosomal rearrangements. The largest conserved syntenic blocks between birds and turtles exist in four macrochromosomes, whereas rearrangements were evident in these and other chromosomes, disproving that turtles and birds retain fully conserved macrochromosomes for greater than 300 Myr. C-banding revealed large heterochromatic blocks in the centromeric region of only few chromosomes. The nucleolar-organizing region (NOR) mapped to a single CPI microchromosome, whereas in some turtles and lizards the NOR maps to nonhomologous sex-chromosomes, thus revealing independent translocations of the NOR in various reptilian lineages. There was no evidence for recent chromosomal fusions as interstitial telomeric-DNA was absent. Some repeat elements (CR1-like, Gypsy) were enriched in the centromeres of five chromosomes, whereas others were widespread in the CPI genome. Bacterial artificial chromosome (BAC) clones were hybridized to 18 of the 25 CPI chromosomes and anchored to a G-banded ideogram. Several CPI sex-determining genes mapped to five chromosomes, and homology was detected between yet other CPI autosomes and the globally nonhomologous sex chromosomes of chicken, other turtles, and squamates, underscoring the independent evolution of vertebrate sex-determining mechanisms. PMID:26108489

Complete denture biofilm after brushing with specific denture paste, neutral soap and artificial saliva.

PubMed

Paranhos, Helena de Freitas Oliveira; Salles, Antônio Eduardo Sparça; Macedo, Leandro Dorigan de; Silva-Lovato, Cláudia Helena da; Pagnano, Valéria Oliveira; Watanabe, Evandro

2013-01-01

This study compared the levels of biofilm in maxillary and mandibular complete dentures and evaluated the number of colony-forming units (cfu) of yeasts, after using auxiliary brushing agents and artificial saliva. Twenty-three denture wearers with hyposalivation and xerostomia were instructed to brush the dentures 3 times a day during 3 weeks with the following products: Corega Brite denture dentifrice, neutral liquid soap, Corega Brite combined with Oral Balance (artificial saliva) or tap water. For biofilm quantification, the internal surfaces of the dentures were disclosed, photographed and measured using a software. For microbiological analysis, the biofilm was scrapped off, and the harvested material was diluted, sown in CHROMagar™ Candida and incubated at 37°C for 48 h. Data were analyzed statistically by two-way ANOVA and Tukey's test (α=0.05). Mandibular dentures presented a mean biofilm percentage (µ=26.90 ± 21.10) significantly greater than the maxillary ones (µ=18.0 ± 15.0) (p<0.05). Brushing using Corega Brite combined with Oral Balance (µ=15.87 ± 18.47) was more effective (p<0.05) than using the denture dentifrice (µ=19.47 ± 17.24), neutral soap (µ=23.90 ± 18.63) or tap water (control; µ=32.50 ± 20.68). For the microbiological analysis, the chi-square test did not indicate significant difference between the hygiene products for either type of denture. The more frequently isolated species of yeasts were C. albicans, C. tropicalis and C. glabrata. In conclusion, mandibular dentures had more biofilm formation than maxillary ones. Denture brushing with Corega Brite dentifrice combined with the use of Oral Balance was the most effective method for reduction of biofilm levels, but the use of products did not show difference in yeast cfu counts.

Nuclear fusion and genome encounter during yeast zygote formation.

PubMed

Tartakoff, Alan Michael; Jaiswal, Purnima

2009-06-01

When haploid cells of Saccharomyces cerevisiae are crossed, parental nuclei congress and fuse with each other. To investigate underlying mechanisms, we have developed assays that evaluate the impact of drugs and mutations. Nuclear congression is inhibited by drugs that perturb the actin and tubulin cytoskeletons. Nuclear envelope (NE) fusion consists of at least five steps in which preliminary modifications are followed by controlled flux of first outer and then inner membrane proteins, all before visible dilation of the waist of the nucleus or coalescence of the parental spindle pole bodies. Flux of nuclear pore complexes occurs after dilation. Karyogamy requires both the Sec18p/NSF ATPase and ER/NE luminal homeostasis. After fusion, chromosome tethering keeps tagged parental genomes separate from each other. The process of NE fusion and evidence of genome independence in yeast provide a prototype for understanding related events in higher eukaryotes.

Micromechanics of human mitotic chromosomes

NASA Astrophysics Data System (ADS)

Sun, Mingxuan; Kawamura, Ryo; Marko, John F.

2011-02-01

Eukaryote cells dramatically reorganize their long chromosomal DNAs to facilitate their physical segregation during mitosis. The internal organization of folded mitotic chromosomes remains a basic mystery of cell biology; its understanding would likely shed light on how chromosomes are separated from one another as well as into chromosome structure between cell divisions. We report biophysical experiments on single mitotic chromosomes from human cells, where we combine micromanipulation, nano-Newton-scale force measurement and biochemical treatments to study chromosome connectivity and topology. Results are in accord with previous experiments on amphibian chromosomes and support the 'chromatin network' model of mitotic chromosome structure. Prospects for studies of chromosome-organizing proteins using siRNA expression knockdowns, as well as for differential studies of chromosomes with and without mutations associated with genetic diseases, are also discussed.

Yeasts and yeast-like organisms associated with fruits and blossoms of different fruit trees.

PubMed

Vadkertiová, Renáta; Molnárová, Jana; Vránová, Dana; Sláviková, Elena

2012-12-01

Yeasts are common inhabitants of the phyllosphere, but our knowledge of their diversity in various plant organs is still limited. This study focused on the diversity of yeasts and yeast-like organisms associated with matured fruits and fully open blossoms of apple, plum, and pear trees, during 2 consecutive years at 3 localities in southwest Slovakia. The occurrence of yeasts and yeast-like organisms in fruit samples was 2½ times higher and the yeast community more diverse than that in blossom samples. Only 2 species (Aureobasidium pullulans and Metschnikowia pulcherrima) occurred regularly in the blossom samples, whereas Galactomyces candidus, Hanseniaspora guilliermondii, Hanseniaspora uvarum, M. pulcherrima, Pichia kluyveri, Pichia kudriavzevii, and Saccharomyces cerevisiae were the most frequently isolated species from the fruit samples. The ratio of the number of samples where only individual species were present to the number of samples where 2 or more species were found (consortium) was counted. The occurrence of individual species in comparison with consortia was much higher in blossom samples than in fruit samples. In the latter, consortia predominated. Aureobasidium pullulans, M. pulcherrima, and S. cerevisiae, isolated from both the fruits and blossoms, can be considered as resident yeast species of various fruit tree species cultivated in southwest Slovakia localities.

Bird-like sex chromosomes of platypus imply recent origin of mammal sex chromosomes.

PubMed

Veyrunes, Frédéric; Waters, Paul D; Miethke, Pat; Rens, Willem; McMillan, Daniel; Alsop, Amber E; Grützner, Frank; Deakin, Janine E; Whittington, Camilla M; Schatzkamer, Kyriena; Kremitzki, Colin L; Graves, Tina; Ferguson-Smith, Malcolm A; Warren, Wes; Marshall Graves, Jennifer A

2008-06-01

In therian mammals (placentals and marsupials), sex is determined by an XX female: XY male system, in which a gene (SRY) on the Y affects male determination. There is no equivalent in other amniotes, although some taxa (notably birds and snakes) have differentiated sex chromosomes. Birds have a ZW female: ZZ male system with no homology with mammal sex chromosomes, in which dosage of a Z-borne gene (possibly DMRT1) affects male determination. As the most basal mammal group, the egg-laying monotremes are ideal for determining how the therian XY system evolved. The platypus has an extraordinary sex chromosome complex, in which five X and five Y chromosomes pair in a translocation chain of alternating X and Y chromosomes. We used physical mapping to identify genes on the pairing regions between adjacent X and Y chromosomes. Most significantly, comparative mapping shows that, contrary to earlier reports, there is no homology between the platypus and therian X chromosomes. Orthologs of genes in the conserved region of the human X (including SOX3, the gene from which SRY evolved) all map to platypus chromosome 6, which therefore represents the ancestral autosome from which the therian X and Y pair derived. Rather, the platypus X chromosomes have substantial homology with the bird Z chromosome (including DMRT1) and to segments syntenic with this region in the human genome. Thus, platypus sex chromosomes have strong homology with bird, but not to therian sex chromosomes, implying that the therian X and Y chromosomes (and the SRY gene) evolved from an autosomal pair after the divergence of monotremes only 166 million years ago. Therefore, the therian X and Y are more than 145 million years younger than previously thought.

DNA double strand break induction in yeast.

PubMed

Kiefer, J; Egenolf, R; Ikpeme, S E

2002-01-01

The induction of DNA double strand breaks (DSBs) by accelerated heavy ions was systematically measured in diploid yeast cells. Particles were provided by the accelerators at GSI, Darmstadt, and HMI, Berlin. DNA was separated using pulsed field gel electrophoresis and the intensity of the largest bands used to determine the loss of molecular weight. Since the DNA content of each chromosome is exactly known absolute values for DSB induction can be measured without calibration procedures. Ions used range from protons to uranium with LET values between 2 and about 15,000 keV.micron-1. Induction cross sections increase in the lower LET region approaching a plateau around 200 keV.micron-1. With higher LET values the dependence can no longer be described by a common curve with each ion showing a specific behaviour. With very heavy particles the influence of the penumbra becomes obvious: cross sections decrease with LET because of the reduced penumbra extensions. Classical target theory would predict cross sections to follow a simple saturation function which is not substantiated by the data. Track structure analysis as introduced by Butts and Katz in 1967 is also not able to predict the experimental results. A semi-empirical fit indicates a linear-quadratic dependence of induction cross sections on LET up to about 1000 keV.micron-1. RBE for DSB induction rises above unity reaching a maximum of about 2.5 around 200 keV.micron-1. This is different from many experiments in mammalian cells and is presumably due to differences in chromatin structure since yeast cells seem to lack a functional III histone.

Vibrio chromosomes share common history.

PubMed

Kirkup, Benjamin C; Chang, LeeAnn; Chang, Sarah; Gevers, Dirk; Polz, Martin F

2010-05-10

While most gamma proteobacteria have a single circular chromosome, Vibrionales have two circular chromosomes. Horizontal gene transfer is common among Vibrios, and in light of this genetic mobility, it is an open question to what extent the two chromosomes themselves share a common history since their formation. Single copy genes from each chromosome (142 genes from chromosome I and 42 genes from chromosome II) were identified from 19 sequenced Vibrionales genomes and their phylogenetic comparison suggests consistent phylogenies for each chromosome. Additionally, study of the gene organization and phylogeny of the respective origins of replication confirmed the shared history. Thus, while elements within the chromosomes may have experienced significant genetic mobility, the backbones share a common history. This allows conclusions based on multilocus sequence analysis (MLSA) for one chromosome to be applied equally to both chromosomes.

Forces in yeast flocculation

NASA Astrophysics Data System (ADS)

El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Vincent, Stéphane P.; Abellán Flos, Marta; Hols, Pascal; Lipke, Peter N.; Dufrêne, Yves F.

2015-01-01

In the baker's yeast Saccharomyces cerevisiae, cell-cell adhesion (``flocculation'') is conferred by a family of lectin-like proteins known as the flocculin (Flo) proteins. Knowledge of the adhesive and mechanical properties of flocculins is important for understanding the mechanisms of yeast adhesion, and may help controlling yeast behaviour in biotechnology. We use single-molecule and single-cell atomic force microscopy (AFM) to explore the nanoscale forces engaged in yeast flocculation, focusing on the role of Flo1 as a prototype of flocculins. Using AFM tips labelled with mannose, we detect single flocculins on Flo1-expressing cells, showing they are widely exposed on the cell surface. When subjected to force, individual Flo1 proteins display two distinct force responses, i.e. weak lectin binding forces and strong unfolding forces reflecting the force-induced extension of hydrophobic tandem repeats. We demonstrate that cell-cell adhesion bonds also involve multiple weak lectin interactions together with strong unfolding forces, both associated with Flo1 molecules. Single-molecule and single-cell data correlate with microscale cell adhesion behaviour, suggesting strongly that Flo1 mechanics is critical for yeast flocculation. These results favour a model in which not only weak lectin-sugar interactions are involved in yeast flocculation but also strong hydrophobic interactions resulting from protein unfolding.

The yeast actin cytoskeleton.

PubMed

Mishra, Mithilesh; Huang, Junqi; Balasubramanian, Mohan K

2014-03-01

The actin cytoskeleton is a complex network of dynamic polymers, which plays an important role in various fundamental cellular processes, including maintenance of cell shape, polarity, cell division, cell migration, endocytosis, vesicular trafficking, and mechanosensation. Precise spatiotemporal assembly and disassembly of actin structures is regulated by the coordinated activity of about 100 highly conserved accessory proteins, which nucleate, elongate, cross-link, and sever actin filaments. Both in vivo studies in a wide range of organisms from yeast to metazoans and in vitro studies of purified proteins have helped shape the current understanding of actin dynamics and function. Molecular genetics, genome-wide functional analysis, sophisticated real-time imaging, and ultrastructural studies in concert with biochemical analysis have made yeast an attractive model to understand the actin cytoskeleton, its molecular dynamics, and physiological function. Studies of the yeast actin cytoskeleton have contributed substantially in defining the universal mechanism regulating actin assembly and disassembly in eukaryotes. Here, we review some of the important insights generated by the study of actin cytoskeleton in two important yeast models the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Stable chromosome condensation revealed by chromosome conformation capture

PubMed Central

Eagen, Kyle P.; Hartl, Tom A.; Kornberg, Roger D.

2015-01-01

SUMMARY Chemical cross-linking and DNA sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed with regions of little or no interaction, in interphase nuclei. We find that TADs and the regions between them correspond with the bands and interbands of polytene chromosomes of Drosophila. We further establish the conservation of TADs between polytene and diploid cells of Drosophila. From direct measurements on light micrographs of polytene chromosomes, we then deduce the states of chromatin folding in the diploid cell nucleus. Two states of folding, fully extended fibers containing regulatory regions and promoters, and fibers condensed up to ten-fold containing coding regions of active genes, constitute the euchromatin of the nuclear interior. Chromatin fibers condensed up to 30-fold, containing coding regions of inactive genes, represent the heterochromatin of the nuclear periphery. A convergence of molecular analysis with direct observation thus reveals the architecture of interphase chromosomes. PMID:26544940

[Thermoresistance in Saccharomyces cerevisiae yeasts].

PubMed

Kaliuzhin, V A

2011-01-01

Under natural conditions, yeast Saccharomyces cerevisiae reproduce, as a rule, on the surface of solid or liquid medium. Thus, life cycle of yeast populations is substantially influenced by diurnal changes in ambient temperature. The pattern in the response of unrestricted yeast S. cerevisiae culture to changes in the temperature of cultivation is revealed experimentally. Yeast population, in the absence of environmental constraints on the functioning of cell chemosmotic bioenergetic system, demonstrates the ability of thermoresistance when the temperature of cultivation switches from the range of 12-36 degrees C to 37.5-40 degrees C. During the transient period that is associated with the temperature switching and lasts from 1 to 4 turnover cycles, yeast reproduction rate remains 1.5-2 times higher than under stationary conditions. This is due to evolutionary acquired adaptive activity of cell chemosmotic system. After the adaptive resources exhausting, yeast thermoresistance fully recovers at the temperature range of 12-36 degrees C within one generation time under conditions of both restricted and unrestricted nourishment. Adaptive significance of such thermoresistance seems obvious enough--it allows maintaining high reproduction rate in yeast when ambient temperature is reaching a brief maximum shortly after noon.

Interaction Between Yeasts and Zinc

NASA Astrophysics Data System (ADS)

Nicola, Raffaele De; Walker, Graeme

Zinc is an essential trace element in biological systems. For example, it acts as a cellular membrane stabiliser, plays a critical role in gene expression and genome modification and activates nearly 300 enzymes, including alcohol dehydrogenase. The present chapter will be focused on the influence of zinc on cell physiology of industrial yeast strains of Saccharomyces cerevisiae, with special regard to the uptake and subsequent utilisation of this metal. Zinc uptake by yeast is metabolism-dependent, with most of the available zinc translocated very quickly into the vacuole. At cell division, zinc is distributed from mother to daughter cells and this effectively lowers the individual cellular zinc concentration, which may become zinc depleted at the onset of the fermentation. Zinc influences yeast fermentative performance and examples will be provided relating to brewing and wine fermentations. Industrial yeasts are subjected to several stresses that may impair fermentation performance. Such stresses may also impact on yeast cell zinc homeostasis. This chapter will discuss the practical implications for the correct management of zinc bioavailability for yeast-based biotechnologies aimed at improving yeast growth, viability, fermentation performance and resistance to environmental stresses

21 CFR 172.896 - Dried yeasts.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Dried yeasts. 172.896 Section 172.896 Food and... PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis) and dried torula yeast (Candida utilis...

Decoding the principles underlying the frequency of association with nucleoli for RNA polymerase III–transcribed genes in budding yeast

PubMed Central

Belagal, Praveen; Normand, Christophe; Shukla, Ashutosh; Wang, Renjie; Léger-Silvestre, Isabelle; Dez, Christophe; Bhargava, Purnima; Gadal, Olivier

2016-01-01

The association of RNA polymerase III (Pol III)–transcribed genes with nucleoli seems to be an evolutionarily conserved property of the spatial organization of eukaryotic genomes. However, recent studies of global chromosome architecture in budding yeast have challenged this view. We used live-cell imaging to determine the intranuclear positions of 13 Pol III–transcribed genes. The frequency of association with nucleolus and nuclear periphery depends on linear genomic distance from the tethering elements—centromeres or telomeres. Releasing the hold of the tethering elements by inactivating centromere attachment to the spindle pole body or changing the position of ribosomal DNA arrays resulted in the association of Pol III–transcribed genes with nucleoli. Conversely, ectopic insertion of a Pol III–transcribed gene in the vicinity of a centromere prevented its association with nucleolus. Pol III–dependent transcription was independent of the intranuclear position of the gene, but the nucleolar recruitment of Pol III–transcribed genes required active transcription. We conclude that the association of Pol III–transcribed genes with the nucleolus, when permitted by global chromosome architecture, provides nucleolar and/or nuclear peripheral anchoring points contributing locally to intranuclear chromosome organization. PMID:27559135

Complete genome sequence and comparative genomics of the probiotic yeast Saccharomyces boulardii.

PubMed

Khatri, Indu; Tomar, Rajul; Ganesan, K; Prasad, G S; Subramanian, Srikrishna

2017-03-23

The probiotic yeast, Saccharomyces boulardii (Sb) is known to be effective against many gastrointestinal disorders and antibiotic-associated diarrhea. To understand molecular basis of probiotic-properties ascribed to Sb we determined the complete genomes of two strains of Sb i.e. Biocodex and unique28 and the draft genomes for three other Sb strains that are marketed as probiotics in India. We compared these genomes with 145 strains of S. cerevisiae (Sc) to understand genome-level similarities and differences between these yeasts. A distinctive feature of Sb from other Sc is absence of Ty elements Ty1, Ty3, Ty4 and associated LTR. However, we could identify complete Ty2 and Ty5 elements in Sb. The genes for hexose transporters HXT11 and HXT9, and asparagine-utilization are absent in all Sb strains. We find differences in repeat periods and copy numbers of repeats in flocculin genes that are likely related to the differential adhesion of Sb as compared to Sc. Core-proteome based taxonomy places Sb strains along with wine strains of Sc. We find the introgression of five genes from Z. bailii into the chromosome IV of Sb and wine strains of Sc. Intriguingly, genes involved in conferring known probiotic properties to Sb are conserved in most Sc strains.

Physical determinants of bipolar mitotic spindle assembly and stability in fission yeast

PubMed Central

Blackwell, Robert; Edelmaier, Christopher; Sweezy-Schindler, Oliver; Lamson, Adam; Gergely, Zachary R.; O’Toole, Eileen; Crapo, Ammon; Hough, Loren E.; McIntosh, J. Richard; Glaser, Matthew A.; Betterton, Meredith D.

2017-01-01

Mitotic spindles use an elegant bipolar architecture to segregate duplicated chromosomes with high fidelity. Bipolar spindles form from a monopolar initial condition; this is the most fundamental construction problem that the spindle must solve. Microtubules, motors, and cross-linkers are important for bipolarity, but the mechanisms necessary and sufficient for spindle assembly remain unknown. We describe a physical model that exhibits de novo bipolar spindle formation. We began with physical properties of fission-yeast spindle pole body size and microtubule number, kinesin-5 motors, kinesin-14 motors, and passive cross-linkers. Our model results agree quantitatively with our experiments in fission yeast, thereby establishing a minimal system with which to interrogate collective self-assembly. By varying the features of our model, we identify a set of functions essential for the generation and stability of spindle bipolarity. When kinesin-5 motors are present, their bidirectionality is essential, but spindles can form in the presence of passive cross-linkers alone. We also identify characteristic failed states of spindle assembly—the persistent monopole, X spindle, separated asters, and short spindle, which are avoided by the creation and maintenance of antiparallel microtubule overlaps. Our model can guide the identification of new, multifaceted strategies to induce mitotic catastrophes; these would constitute novel strategies for cancer chemotherapy. PMID:28116355

Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.

PubMed

Keniya, Mikhail V; Holmes, Ann R; Niimi, Masakazu; Lamping, Erwin; Gillet, Jean-Pierre; Gottesman, Michael M; Cannon, Richard D

2014-10-06

ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-β mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.

21 CFR 172.896 - Dried yeasts.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Dried yeasts. 172.896 Section 172.896 Food and... Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis) and dried torula yeast (Candida utilis) may be safely used in food provided the total folic acid...

21 CFR 172.896 - Dried yeasts.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Dried yeasts. 172.896 Section 172.896 Food and... Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis) and dried torula yeast (Candida utilis) may be safely used in food provided the total folic acid...

21 CFR 172.896 - Dried yeasts.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Dried yeasts. 172.896 Section 172.896 Food and... Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis) and dried torula yeast (Candida utilis) may be safely used in food provided the total folic acid...

21 CFR 172.896 - Dried yeasts.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Dried yeasts. 172.896 Section 172.896 Food and... Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis) and dried torula yeast (Candida utilis) may be safely used in food provided the total folic acid...