Forrest Spencer - Academia.edu (original) (raw)
Papers by Forrest Spencer
2004). A robust toolkit for functional profiling of the yeast genome. Mol Cell. 16(3):487-496. Mo... more 2004). A robust toolkit for functional profiling of the yeast genome. Mol Cell. 16(3):487-496. Modified for Production by PBM & CT For 20 + 2 control transformations (1 liter of culture) Day 1
Genetics
We have isolated 136 independent mutations in haploid yeast strains that exhibit decreased chromo... more We have isolated 136 independent mutations in haploid yeast strains that exhibit decreased chromosome transmission fidelity in mitosis. Eighty-five percent of the mutations are recessive and 15% are partially dominant. Complementation analysis between MATa and MAT alpha isolates identifies 11 chromosome transmission fidelity (CTF) complementation groups, the largest of which is identical to CHL1. For 49 independent mutations, no corresponding allele has been recovered in the opposite mating type. The initial screen monitored the stability of a centromere-linked color marker on a nonessential yeast chromosome fragment; the mitotic inheritance of natural yeast chromosome III is also affected by the ctf mutations. Of the 136 isolates identified, seven were inviable at 37 degrees and five were inviable at 11 degrees. In all cases tested, these temperature conditional lethalities cosegregated with the chromosome instability phenotype. Five additional complementation groups (ctf12 through...
The EMBO Journal
We have analyzed the CTF1 gene, identified in a screen for mutants with decreased chromosome tran... more We have analyzed the CTF1 gene, identified in a screen for mutants with decreased chromosome transmission fidelity and shown to correspond to the previously identified chl1 mutation. Chl1 null mutants exhibited a 200-fold increase in the rate of chromosome III missegregation per cell division, and near wild-type rates of marker homozygosis on this chromosome by mitotic recombination. Analysis of the segregation of a marker chromosome indicated that sister chromatid loss (1:0 segregation) and sister chromatid non-disjunction (2:0 segregation) contributed equally to chromosome missegregation. A genomic clone of CHL1 was isolated and used to map its physical position on chromosome XVI. Nucleotide sequence analysis of CHL1 revealed a 2.6 kb open reading frame with a 99 kd predicted protein sequence that contained two PEST sequences and was 23% identical to the coding region of a nucleotide excision repair gene, RAD3. Domains of homology between these two predicted protein sequences incl...
Progress in clinical and biological research
1. Prog Clin Biol Res. 1989;318:293-306. Chromosomal aneuploidy in Saccharomyces cerevisiae. Spen... more 1. Prog Clin Biol Res. 1989;318:293-306. Chromosomal aneuploidy in Saccharomyces cerevisiae. Spencer F, Connelly C, Gerring S, Shero J, Sikorski R, Hieter P. Department of Molecular Biology and Genetics, Johns Hopkins ...
Molecular and Cellular Biology
A chromosome transmission fidelity (ctf) mutant, s138, of Saccharomyces cerevisiae was identified... more A chromosome transmission fidelity (ctf) mutant, s138, of Saccharomyces cerevisiae was identified by its centromere (CEN) transcriptional readthrough phenotype, suggesting perturbed kinetochore integrity in vivo. The gene complementing the s138 mutation was found to be identical to the S. cerevisiae SPT4 gene. The s138 mutation is a missense mutation in the second of four conserved cysteine residues positioned similarly to those of zinc finger proteins, and we henceforth refer to the mutation of spt4-138. Both spt4-138 and spt4 delta strains missegregate a chromosome fragment at the permissive temperature, are temperature sensitive for growth at 37 degrees C, and upon a shift to the nonpermissive temperature show an accumulation of large budded cells, each with a nucleus. Previous studies suggest that Spt4p functions in a complex with Spt5p and Spt6p, and we determined that spt6-140 also causes missegregation of a chromosome fragment. Double mutants carrying spt4 delta 2::HIS3 and k...
Chromosoma
It has been more than two decades since the original chromosome transmission fidelity (Ctf) scree... more It has been more than two decades since the original chromosome transmission fidelity (Ctf) screen of Saccharomyces cerevisiae was published. Since that time the spectrum of mutations known to cause Ctf and, more generally, chromosome instability (CIN) has expanded dramatically as a result of systematic screens across yeast mutant arrays. Here we describe a comprehensive summary of the original Ctf genetic screen and the cloning of the remaining complementation groups as efforts to expand our knowledge of the CIN gene repertoire and its mutability in a model eukaryote. At the time of the original screen, it was impossible to predict either the genes and processes that would be overrepresented in a pool of random mutants displaying a Ctf phenotype or what the entire set of genes potentially mutable to Ctf would be. We show that in a collection of 136 randomly selected Ctf mutants, >65% of mutants map to 13 genes, 12 of which are involved in sister chromatid cohesion and/or kinetoc...
Current Protocols in Human Genetics, 2001
ABSTRACT This unit provides a protocol for moving yeast artificial chromosome (YAC) clones to new... more ABSTRACT This unit provides a protocol for moving yeast artificial chromosome (YAC) clones to new yeast hosts using basic microbial techniques and pulsed-field gel analysis. In contrast to other methods that can be used to transfer YAC clones, this requires neither optimization to achieve high-efficiency DNA-mediated transformation of chromosome-sized DNA nor specialized equipment for tetrad dissection and analysis. Instead, chromosome (YAC) transfer is selected in rare segregants ("YACductants") from a yeast mating that is rendered incomplete in most cell pairings by the presence of a kar1 (karyogamy-deficient) mutation in either parental strain. The Basic Protocol in this unit details the transfer of a YAC clone from yeast strain AB1380 (host to nearly all existing YAC libraries) to YPH925, a strain with nonreverting genetic markers compatible with existing plasmid constructs useful in YAC modification.
Methods in Molecular Biology™, 2008
TAG, or bar-code, microarrays allow measurement of the oligonucleotide sequences (TAGs) that mark... more TAG, or bar-code, microarrays allow measurement of the oligonucleotide sequences (TAGs) that mark each strain of deletion mutants in the Saccharomyces cerevisiae yeast knockout (YKO) collection. Comparison of genomic DNA from pooled YKO samples allows estimation of relative abundance of TAGs marking each deletion strain. Features of TAG hybridizations create unique challenges for analysis. Analysis is complicated by the presence of two TAGs in most YKO strains and the hybridization behavior of TAGs that may differ in sequence from array probes. The oligonucleotide size of labeled TAGs also results in difficulty with contaminating sequences that cause reduced specificity. We present methods for analysis that approach these unique features of TAG hybridizations.
Molecular biology of the cell, 2002
The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensurin... more The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensuring that anaphase is initiated only after kinetochore-microtubule associations of all sister chromatid pairs are complete. In this study, we find that known spindle checkpoint proteins do not contribute equally to chromosome segregation fidelity in Saccharomyces cerevisiae. Loss of Bub1 or Bub3 protein elicits the largest effect. Analysis of Bub1p reveals the presence of two molecular functions. An N-terminal 608-amino acid (nonkinase) portion of the protein supports robust checkpoint activity, and, as expected, contributes to chromosome segregation. A C-terminal kinase-encoding segment independently contributes to chromosome segregation through an unknown mechanism. Both molecular functions depend on association with Bub3p. A 156-amino acid fragment of Bub1p functions in Bub3p binding and in kinetochore localization by one-hybrid assay. An adjacent segment is required for Mad1p binding, d...
Methods in Enzymology, 1991
ABSTRACT The yeast Saccharomyces cerevisiae is an excellent organism for the study of mitotic and... more ABSTRACT The yeast Saccharomyces cerevisiae is an excellent organism for the study of mitotic and meiotic chromosome segregation because it is possible to isolate mutations that effect the fidelity of this process. This chapter presents two methods for analyzing chromosome segregation in these mutants. In the first method, digital imaging microscopy is used on individual live cells to analyze the segregation of fluorescently labeled chromosomes. In this method, changes in nuclear DNA staining pattern are observed using a microscope equipped with epifluorescence optics suitable for DAPI-stained sampies. A low-light level imaging device, such as a silicon-intensified tube camera or a multichannel plate intensifier, is coupled to the microscope and used to obtain video images. In the second method, the construction of artificial chromosomes is described that have been specifically designed for analysis of the fidelity of chromosome segregation in mitosis and meiosis.
Molecular Cell, 2004
three strain sets are subject to unavoidable selection pressure and accumulation of compensatory ... more three strain sets are subject to unavoidable selection pressure and accumulation of compensatory genetic 3 Department of Medicine 4 McKusick-Nathans Institute for Genetic Medicine changes. As a result, strain quality can deteriorate with time, possibly delivering misleading results. For exam-The Johns Hopkins University School of Medicine Baltimore, Maryland 21205 ple, DNA damage response pathway mutants (mec3⌬, ddc1⌬, rad17⌬, rad24⌬, and rad9⌬) behaved differently in screens with homozygous diploid YKOs and haploid MATa YKOs (see Supplemental Table S1 at http://www. ; Tong et al., 2001). However, beyond their potential genetic impurity, haploid mutants in different genes be-University of British Columbia Vancouver, British Columbia V5Z 4H4 have distinctly and thus are not well suited for manipulation as a population, the basis for microarray-based TAG Canada readouts (Ooi et al., 2003)
BMC Bioinformatics, 2005
Background: In a genetic interaction, the phenotype of a double mutant differs from the combined ... more Background: In a genetic interaction, the phenotype of a double mutant differs from the combined phenotypes of the underlying single mutants. When the single mutants have no growth defect, but the double mutant is lethal or exhibits slow growth, the interaction is termed synthetic lethality or synthetic fitness. These genetic interactions reveal gene redundancy and compensating pathways. Recently available large-scale data sets of genetic interactions and protein interactions in Saccharomyces cerevisiae provide a unique opportunity to elucidate the topological structure of biological pathways and how genes function in these pathways.
YAC Protocols, 1995
ABSTRACT Yeast artificial chromosome (YAC) clones are propagated in yeast, a host organism with a... more ABSTRACT Yeast artificial chromosome (YAC) clones are propagated in yeast, a host organism with a variety of established techniques for altering DNA sequences by homologous recombination in vivo. The modification of existing YAC clones allows the removal of undesired insert DNA (e.g., neighboring coding sequences or chimeric segments), the introduction of new selectable markers, or the replacement of wild-type DNA with defined mutant alleles. To use existing vector systems for YAC manipulation by homologous recombination, transfer to other yeast hosts is often necessary. The development of alternative host strains has been motivated in part by the paucity of nonreverting genetic markers in the genotype of the common library host AB1380 (1). In addition, clones with unstable inserts may be more faithfully propagated in recombination-deficient yeast strains (see, e.g., 2–4).
Trends in Genetics, 2006
The Saccharomyces genome-deletion project created &am... more The Saccharomyces genome-deletion project created >5900 'molecularly barcoded' yeast knockout mutants (YKO mutants). The YKO mutant collections have facilitated large-scale analyses of a multitude of mutant phenotypes. For example, both synthetic genetic array (SGA) and synthetic-lethality analysis by microarray (SLAM) methods have been used for synthetic-lethality screens. Global analysis of synthetic lethality promises to identify cellular pathways that 'buffer' each other biologically. The combination of global synthetic-lethality analysis, together with global protein-protein interaction analyses, mRNA expression profiling and functional profiling will, in principle, enable construction of a cellular 'wiring diagram' that will help frame a deeper understanding of human biology and disease.
Yeast, 1993
Eight independent chl (chromosome loss) mutants were isolated using yeast haploid strain disomic ... more Eight independent chl (chromosome loss) mutants were isolated using yeast haploid strain disomic for chromosome III. In these mutants, chromosome III is lost during mitosis 50-fold more frequently than in the wild-type strains. chl mutants are also incapable of stable maintenance of circular and linear artificial chromosomes. Seven of the eight mutations are recessive, and one is semidominant. Complementation tests placed these mutants into six complementation groups (chl11 through chl16). Based on tetrad analysis, chl12, chl14 and chl15 correspond to mutations in single nuclear genes. Tetrad analysis of the other mutants was not possible due to poor spore viability. Complementation analysis was also carried out between collection of chl mutants and ctf mutants (chromosome transmission fidelity) (Spencer et al., 1990). The chl3, chl4, chl8, chl12 and chl15 mutants were unable to complement ctf3, ctf17, ctf12, ctf18 and ctf4, respectively. Three CHL genes were mapped by tetrad analysis. The CHL3 gene is placed on the right arm of chromosome XII, between the ILV5 (33.3 cM) and URA4 (21.8 cM) loci. The CHL10 gene is located on the left arm of chromosome VI, 12.5 cM from the centromere. The CHL15 gene is tightly linked to the KAR3 marker of the right arm of chromosome XVI (8.8 cM). The mapping data indicate that these three genes differ from other genes known to affect chromosome stability in mitosis. Therefore, the total number of the CHL genes identified (including those described by us earlier) is 13 (CHL1-CHL10, CHL12, CHL14 and CHL15).
Proceedings of the National Academy of Sciences, 1992
Cytological observations of animal cell mitoses have shown that the onset of anaphase is delayed ... more Cytological observations of animal cell mitoses have shown that the onset of anaphase is delayed when chromosome attachment to the spindle is spontaneously retarded or experimentally interrupted. This report demonstrates that a centromere DNA (CEN) mutation carried on a single chromosome can induce a cell cycle delay observed as retarded mitosis in the yeast Saccharomyces cerevisiae. A 31-base-pair deletion within centromere DNA element II (CDEII delta 31) that causes chromosome missegregation in only 1% of cell division elicited a dramatic mitotic delay phenotype. Other CEN DNA mutations, including mutations in centromere DNA elements I and III, similarly delayed mitosis. Single division pedigree analysis of strains containing the CDEII delta 31 CEN mutation indicated that most (and possibly all) cells experienced delay in each cell cycle and that the delay was not due to increased chromosome copy number. Furthermore, a synchronous population of cells containing the CDEII delta 31 mutation underwent DNA synthesis on schedule with wild-type kinetics, but subsequently exhibited late chromosomal separation and concomitant late cell separation. We speculate that this delay in cell cycle progression before the onset of anaphase provides a mechanism for the stabilization of chromosomes with defective kinetochore structure. Further, we suggest that the delay may be mediated by surveillance at a cell cycle checkpoint that monitors the completion of chromosomal attachment to the spindle.
Proceedings of the National Academy of Sciences, 1994
A yeast artIal chromosome (YAC) containing a complete human adenovirus type 2 genome was construc... more A yeast artIal chromosome (YAC) containing a complete human adenovirus type 2 genome was constructed, and viral DNA derived from the YAC was shown to be infectious upon introduction into m cells. The adenovirus YAC could be mi
Proceedings of the National Academy of Sciences, 1996
All eukaryotes that have been studied to date possess the ability to detect and degrade transcrip... more All eukaryotes that have been studied to date possess the ability to detect and degrade transcripts that contain a premature signal for the termination of translation. This process of nonsense-mediated RNA decay has been most comprehensively studied in the yeast Saccharomyces cerevisiae where at least three trans-acting factors (Upf1p through Upf3P) are required. We have cloned cDNAs encoding human and murine homologues of Upf1p, termed rent1 (regulator of nonsense transcripts). Rent1 is the first identified mammalian protein that contains all of the putative functional elements in Upf1p including zinc finger-like and NTPase domains, as well as all motifs common to members of helicase superfamily I. Moreover, expression of a chimeric protein, N and C termini of Upf1p, complements the Upf1p-deficient phenotype in yeast. Thus, despite apparent differences between yeast and mammalian nonsense-mediated RNA decay, these data suggest that the two pathways use functionally related machinery.
Proceedings of the National Academy of Sciences, 2008
Although the majority of colorectal cancers exhibit chromosome instability (CIN), only a few gene... more Although the majority of colorectal cancers exhibit chromosome instability (CIN), only a few genes that might cause this phenotype have been identified and no general mechanism underlying their function has emerged. To systematically identify somatic mutations in potential CIN genes in colorectal cancers, we determined the sequence of 102 human homologues of 96 yeast CIN genes known to function in various aspects of chromosome transmission fidelity. We identified 11 somatic mutations distributed among five genes in a panel that included 132 colorectal cancers. Remarkably, all but one of these 11 mutations were in the homologs of yeast genes that regulate sister chromatid cohesion. We then demonstrated that down-regulation of such homologs resulted in chromosomal instability and chromatid cohesion defects in human cells. Finally, we showed that down-regulation or genetic disruption of the two major candidate CIN genes identified in previous studies (MRE11A and CDC4) also resulted in abnormal sister chromatid cohesion in human cells. These results suggest that defective sister chromatid cohesion as a result of somatic mutations may represent a major cause of chromosome instability in human cancers.
2004). A robust toolkit for functional profiling of the yeast genome. Mol Cell. 16(3):487-496. Mo... more 2004). A robust toolkit for functional profiling of the yeast genome. Mol Cell. 16(3):487-496. Modified for Production by PBM & CT For 20 + 2 control transformations (1 liter of culture) Day 1
Genetics
We have isolated 136 independent mutations in haploid yeast strains that exhibit decreased chromo... more We have isolated 136 independent mutations in haploid yeast strains that exhibit decreased chromosome transmission fidelity in mitosis. Eighty-five percent of the mutations are recessive and 15% are partially dominant. Complementation analysis between MATa and MAT alpha isolates identifies 11 chromosome transmission fidelity (CTF) complementation groups, the largest of which is identical to CHL1. For 49 independent mutations, no corresponding allele has been recovered in the opposite mating type. The initial screen monitored the stability of a centromere-linked color marker on a nonessential yeast chromosome fragment; the mitotic inheritance of natural yeast chromosome III is also affected by the ctf mutations. Of the 136 isolates identified, seven were inviable at 37 degrees and five were inviable at 11 degrees. In all cases tested, these temperature conditional lethalities cosegregated with the chromosome instability phenotype. Five additional complementation groups (ctf12 through...
The EMBO Journal
We have analyzed the CTF1 gene, identified in a screen for mutants with decreased chromosome tran... more We have analyzed the CTF1 gene, identified in a screen for mutants with decreased chromosome transmission fidelity and shown to correspond to the previously identified chl1 mutation. Chl1 null mutants exhibited a 200-fold increase in the rate of chromosome III missegregation per cell division, and near wild-type rates of marker homozygosis on this chromosome by mitotic recombination. Analysis of the segregation of a marker chromosome indicated that sister chromatid loss (1:0 segregation) and sister chromatid non-disjunction (2:0 segregation) contributed equally to chromosome missegregation. A genomic clone of CHL1 was isolated and used to map its physical position on chromosome XVI. Nucleotide sequence analysis of CHL1 revealed a 2.6 kb open reading frame with a 99 kd predicted protein sequence that contained two PEST sequences and was 23% identical to the coding region of a nucleotide excision repair gene, RAD3. Domains of homology between these two predicted protein sequences incl...
Progress in clinical and biological research
1. Prog Clin Biol Res. 1989;318:293-306. Chromosomal aneuploidy in Saccharomyces cerevisiae. Spen... more 1. Prog Clin Biol Res. 1989;318:293-306. Chromosomal aneuploidy in Saccharomyces cerevisiae. Spencer F, Connelly C, Gerring S, Shero J, Sikorski R, Hieter P. Department of Molecular Biology and Genetics, Johns Hopkins ...
Molecular and Cellular Biology
A chromosome transmission fidelity (ctf) mutant, s138, of Saccharomyces cerevisiae was identified... more A chromosome transmission fidelity (ctf) mutant, s138, of Saccharomyces cerevisiae was identified by its centromere (CEN) transcriptional readthrough phenotype, suggesting perturbed kinetochore integrity in vivo. The gene complementing the s138 mutation was found to be identical to the S. cerevisiae SPT4 gene. The s138 mutation is a missense mutation in the second of four conserved cysteine residues positioned similarly to those of zinc finger proteins, and we henceforth refer to the mutation of spt4-138. Both spt4-138 and spt4 delta strains missegregate a chromosome fragment at the permissive temperature, are temperature sensitive for growth at 37 degrees C, and upon a shift to the nonpermissive temperature show an accumulation of large budded cells, each with a nucleus. Previous studies suggest that Spt4p functions in a complex with Spt5p and Spt6p, and we determined that spt6-140 also causes missegregation of a chromosome fragment. Double mutants carrying spt4 delta 2::HIS3 and k...
Chromosoma
It has been more than two decades since the original chromosome transmission fidelity (Ctf) scree... more It has been more than two decades since the original chromosome transmission fidelity (Ctf) screen of Saccharomyces cerevisiae was published. Since that time the spectrum of mutations known to cause Ctf and, more generally, chromosome instability (CIN) has expanded dramatically as a result of systematic screens across yeast mutant arrays. Here we describe a comprehensive summary of the original Ctf genetic screen and the cloning of the remaining complementation groups as efforts to expand our knowledge of the CIN gene repertoire and its mutability in a model eukaryote. At the time of the original screen, it was impossible to predict either the genes and processes that would be overrepresented in a pool of random mutants displaying a Ctf phenotype or what the entire set of genes potentially mutable to Ctf would be. We show that in a collection of 136 randomly selected Ctf mutants, >65% of mutants map to 13 genes, 12 of which are involved in sister chromatid cohesion and/or kinetoc...
Current Protocols in Human Genetics, 2001
ABSTRACT This unit provides a protocol for moving yeast artificial chromosome (YAC) clones to new... more ABSTRACT This unit provides a protocol for moving yeast artificial chromosome (YAC) clones to new yeast hosts using basic microbial techniques and pulsed-field gel analysis. In contrast to other methods that can be used to transfer YAC clones, this requires neither optimization to achieve high-efficiency DNA-mediated transformation of chromosome-sized DNA nor specialized equipment for tetrad dissection and analysis. Instead, chromosome (YAC) transfer is selected in rare segregants ("YACductants") from a yeast mating that is rendered incomplete in most cell pairings by the presence of a kar1 (karyogamy-deficient) mutation in either parental strain. The Basic Protocol in this unit details the transfer of a YAC clone from yeast strain AB1380 (host to nearly all existing YAC libraries) to YPH925, a strain with nonreverting genetic markers compatible with existing plasmid constructs useful in YAC modification.
Methods in Molecular Biology™, 2008
TAG, or bar-code, microarrays allow measurement of the oligonucleotide sequences (TAGs) that mark... more TAG, or bar-code, microarrays allow measurement of the oligonucleotide sequences (TAGs) that mark each strain of deletion mutants in the Saccharomyces cerevisiae yeast knockout (YKO) collection. Comparison of genomic DNA from pooled YKO samples allows estimation of relative abundance of TAGs marking each deletion strain. Features of TAG hybridizations create unique challenges for analysis. Analysis is complicated by the presence of two TAGs in most YKO strains and the hybridization behavior of TAGs that may differ in sequence from array probes. The oligonucleotide size of labeled TAGs also results in difficulty with contaminating sequences that cause reduced specificity. We present methods for analysis that approach these unique features of TAG hybridizations.
Molecular biology of the cell, 2002
The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensurin... more The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensuring that anaphase is initiated only after kinetochore-microtubule associations of all sister chromatid pairs are complete. In this study, we find that known spindle checkpoint proteins do not contribute equally to chromosome segregation fidelity in Saccharomyces cerevisiae. Loss of Bub1 or Bub3 protein elicits the largest effect. Analysis of Bub1p reveals the presence of two molecular functions. An N-terminal 608-amino acid (nonkinase) portion of the protein supports robust checkpoint activity, and, as expected, contributes to chromosome segregation. A C-terminal kinase-encoding segment independently contributes to chromosome segregation through an unknown mechanism. Both molecular functions depend on association with Bub3p. A 156-amino acid fragment of Bub1p functions in Bub3p binding and in kinetochore localization by one-hybrid assay. An adjacent segment is required for Mad1p binding, d...
Methods in Enzymology, 1991
ABSTRACT The yeast Saccharomyces cerevisiae is an excellent organism for the study of mitotic and... more ABSTRACT The yeast Saccharomyces cerevisiae is an excellent organism for the study of mitotic and meiotic chromosome segregation because it is possible to isolate mutations that effect the fidelity of this process. This chapter presents two methods for analyzing chromosome segregation in these mutants. In the first method, digital imaging microscopy is used on individual live cells to analyze the segregation of fluorescently labeled chromosomes. In this method, changes in nuclear DNA staining pattern are observed using a microscope equipped with epifluorescence optics suitable for DAPI-stained sampies. A low-light level imaging device, such as a silicon-intensified tube camera or a multichannel plate intensifier, is coupled to the microscope and used to obtain video images. In the second method, the construction of artificial chromosomes is described that have been specifically designed for analysis of the fidelity of chromosome segregation in mitosis and meiosis.
Molecular Cell, 2004
three strain sets are subject to unavoidable selection pressure and accumulation of compensatory ... more three strain sets are subject to unavoidable selection pressure and accumulation of compensatory genetic 3 Department of Medicine 4 McKusick-Nathans Institute for Genetic Medicine changes. As a result, strain quality can deteriorate with time, possibly delivering misleading results. For exam-The Johns Hopkins University School of Medicine Baltimore, Maryland 21205 ple, DNA damage response pathway mutants (mec3⌬, ddc1⌬, rad17⌬, rad24⌬, and rad9⌬) behaved differently in screens with homozygous diploid YKOs and haploid MATa YKOs (see Supplemental Table S1 at http://www. ; Tong et al., 2001). However, beyond their potential genetic impurity, haploid mutants in different genes be-University of British Columbia Vancouver, British Columbia V5Z 4H4 have distinctly and thus are not well suited for manipulation as a population, the basis for microarray-based TAG Canada readouts (Ooi et al., 2003)
BMC Bioinformatics, 2005
Background: In a genetic interaction, the phenotype of a double mutant differs from the combined ... more Background: In a genetic interaction, the phenotype of a double mutant differs from the combined phenotypes of the underlying single mutants. When the single mutants have no growth defect, but the double mutant is lethal or exhibits slow growth, the interaction is termed synthetic lethality or synthetic fitness. These genetic interactions reveal gene redundancy and compensating pathways. Recently available large-scale data sets of genetic interactions and protein interactions in Saccharomyces cerevisiae provide a unique opportunity to elucidate the topological structure of biological pathways and how genes function in these pathways.
YAC Protocols, 1995
ABSTRACT Yeast artificial chromosome (YAC) clones are propagated in yeast, a host organism with a... more ABSTRACT Yeast artificial chromosome (YAC) clones are propagated in yeast, a host organism with a variety of established techniques for altering DNA sequences by homologous recombination in vivo. The modification of existing YAC clones allows the removal of undesired insert DNA (e.g., neighboring coding sequences or chimeric segments), the introduction of new selectable markers, or the replacement of wild-type DNA with defined mutant alleles. To use existing vector systems for YAC manipulation by homologous recombination, transfer to other yeast hosts is often necessary. The development of alternative host strains has been motivated in part by the paucity of nonreverting genetic markers in the genotype of the common library host AB1380 (1). In addition, clones with unstable inserts may be more faithfully propagated in recombination-deficient yeast strains (see, e.g., 2–4).
Trends in Genetics, 2006
The Saccharomyces genome-deletion project created &am... more The Saccharomyces genome-deletion project created >5900 'molecularly barcoded' yeast knockout mutants (YKO mutants). The YKO mutant collections have facilitated large-scale analyses of a multitude of mutant phenotypes. For example, both synthetic genetic array (SGA) and synthetic-lethality analysis by microarray (SLAM) methods have been used for synthetic-lethality screens. Global analysis of synthetic lethality promises to identify cellular pathways that 'buffer' each other biologically. The combination of global synthetic-lethality analysis, together with global protein-protein interaction analyses, mRNA expression profiling and functional profiling will, in principle, enable construction of a cellular 'wiring diagram' that will help frame a deeper understanding of human biology and disease.
Yeast, 1993
Eight independent chl (chromosome loss) mutants were isolated using yeast haploid strain disomic ... more Eight independent chl (chromosome loss) mutants were isolated using yeast haploid strain disomic for chromosome III. In these mutants, chromosome III is lost during mitosis 50-fold more frequently than in the wild-type strains. chl mutants are also incapable of stable maintenance of circular and linear artificial chromosomes. Seven of the eight mutations are recessive, and one is semidominant. Complementation tests placed these mutants into six complementation groups (chl11 through chl16). Based on tetrad analysis, chl12, chl14 and chl15 correspond to mutations in single nuclear genes. Tetrad analysis of the other mutants was not possible due to poor spore viability. Complementation analysis was also carried out between collection of chl mutants and ctf mutants (chromosome transmission fidelity) (Spencer et al., 1990). The chl3, chl4, chl8, chl12 and chl15 mutants were unable to complement ctf3, ctf17, ctf12, ctf18 and ctf4, respectively. Three CHL genes were mapped by tetrad analysis. The CHL3 gene is placed on the right arm of chromosome XII, between the ILV5 (33.3 cM) and URA4 (21.8 cM) loci. The CHL10 gene is located on the left arm of chromosome VI, 12.5 cM from the centromere. The CHL15 gene is tightly linked to the KAR3 marker of the right arm of chromosome XVI (8.8 cM). The mapping data indicate that these three genes differ from other genes known to affect chromosome stability in mitosis. Therefore, the total number of the CHL genes identified (including those described by us earlier) is 13 (CHL1-CHL10, CHL12, CHL14 and CHL15).
Proceedings of the National Academy of Sciences, 1992
Cytological observations of animal cell mitoses have shown that the onset of anaphase is delayed ... more Cytological observations of animal cell mitoses have shown that the onset of anaphase is delayed when chromosome attachment to the spindle is spontaneously retarded or experimentally interrupted. This report demonstrates that a centromere DNA (CEN) mutation carried on a single chromosome can induce a cell cycle delay observed as retarded mitosis in the yeast Saccharomyces cerevisiae. A 31-base-pair deletion within centromere DNA element II (CDEII delta 31) that causes chromosome missegregation in only 1% of cell division elicited a dramatic mitotic delay phenotype. Other CEN DNA mutations, including mutations in centromere DNA elements I and III, similarly delayed mitosis. Single division pedigree analysis of strains containing the CDEII delta 31 CEN mutation indicated that most (and possibly all) cells experienced delay in each cell cycle and that the delay was not due to increased chromosome copy number. Furthermore, a synchronous population of cells containing the CDEII delta 31 mutation underwent DNA synthesis on schedule with wild-type kinetics, but subsequently exhibited late chromosomal separation and concomitant late cell separation. We speculate that this delay in cell cycle progression before the onset of anaphase provides a mechanism for the stabilization of chromosomes with defective kinetochore structure. Further, we suggest that the delay may be mediated by surveillance at a cell cycle checkpoint that monitors the completion of chromosomal attachment to the spindle.
Proceedings of the National Academy of Sciences, 1994
A yeast artIal chromosome (YAC) containing a complete human adenovirus type 2 genome was construc... more A yeast artIal chromosome (YAC) containing a complete human adenovirus type 2 genome was constructed, and viral DNA derived from the YAC was shown to be infectious upon introduction into m cells. The adenovirus YAC could be mi
Proceedings of the National Academy of Sciences, 1996
All eukaryotes that have been studied to date possess the ability to detect and degrade transcrip... more All eukaryotes that have been studied to date possess the ability to detect and degrade transcripts that contain a premature signal for the termination of translation. This process of nonsense-mediated RNA decay has been most comprehensively studied in the yeast Saccharomyces cerevisiae where at least three trans-acting factors (Upf1p through Upf3P) are required. We have cloned cDNAs encoding human and murine homologues of Upf1p, termed rent1 (regulator of nonsense transcripts). Rent1 is the first identified mammalian protein that contains all of the putative functional elements in Upf1p including zinc finger-like and NTPase domains, as well as all motifs common to members of helicase superfamily I. Moreover, expression of a chimeric protein, N and C termini of Upf1p, complements the Upf1p-deficient phenotype in yeast. Thus, despite apparent differences between yeast and mammalian nonsense-mediated RNA decay, these data suggest that the two pathways use functionally related machinery.
Proceedings of the National Academy of Sciences, 2008
Although the majority of colorectal cancers exhibit chromosome instability (CIN), only a few gene... more Although the majority of colorectal cancers exhibit chromosome instability (CIN), only a few genes that might cause this phenotype have been identified and no general mechanism underlying their function has emerged. To systematically identify somatic mutations in potential CIN genes in colorectal cancers, we determined the sequence of 102 human homologues of 96 yeast CIN genes known to function in various aspects of chromosome transmission fidelity. We identified 11 somatic mutations distributed among five genes in a panel that included 132 colorectal cancers. Remarkably, all but one of these 11 mutations were in the homologs of yeast genes that regulate sister chromatid cohesion. We then demonstrated that down-regulation of such homologs resulted in chromosomal instability and chromatid cohesion defects in human cells. Finally, we showed that down-regulation or genetic disruption of the two major candidate CIN genes identified in previous studies (MRE11A and CDC4) also resulted in abnormal sister chromatid cohesion in human cells. These results suggest that defective sister chromatid cohesion as a result of somatic mutations may represent a major cause of chromosome instability in human cancers.