John Carbon

John Carbon
University of California, Santa Barbara | UCSB · Department of Molecular, Cellular, and Developmental Biology

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167
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Publications

Publications (167)
Article
DNA methylation is a common epigenetic signaling mechanism associated with silencing of repeated DNA and transcriptional regulation in eukaryotes. Here we report that DNA methylation in the human fungal pathogen Candida albicans is primarily localized within structural genes and modulates transcriptional activity. Major repeat sequences and multige...
Chapter
chromatography;ammonia;pteridines;anhydride converted;pyridine solution
Article
The centromere regions (CEN) of all eight chromosomes in Candida albicans have been characterized in terms of nucleotide sequence and size. The boundaries of each of the eight CEN DNA regions were mapped by chromatin immunoprecipitation-PCR using polyclonal rabbit antibodies generated against C. albicans centromere-specific protein CaCse4p (CENP-A...
Article
Full-text available
In the pathogenic yeast Candida albicans, the 3-kb centromeric DNA regions (CEN) of each of the eight chromosomes have different and unique DNA sequences. The centromeric histone CaCse4p (CENP-A homolog) occurs only within these 3-kb CEN regions to form specialized centromeric chromatin. Centromere activity was maintained on small chromosome fragme...
Article
The human pathogenic yeast Candida glabrata is the second most common Candida pathogen after Candida albicans, causing both bloodstream and mucosal infections. The centromere (CEN) DNA of C. glabrata (CgCEN), although structurally very similar to that of Saccharomyces cerevisiae, is not functional in S. cerevisiae. To further examine the structure...
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In an approach to clone and characterize centromeric DNA sequences of Candida albicans by chromatin immunoprecipitation, we have used antibodies directed against an evolutionarily conserved histone H3-like protein, CaCse4p (CENP-A homolog). Sequence analysis of clones obtained by this procedure reveals that only eight relatively small regions (appr...
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The gene encoding CaCse4p, a homolog of the evolutionarily conserved histone H3-like kinetochore protein CENP-A, has been cloned from the human pathogenic diploid yeast Candida albicans. To study the phenotype of C. albicans diploid cells depleted of CaCse4p, we deleted one copy of CaCSE4 and brought the other copy under control of a regulated PCK1...
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The CBF1 (centromere binding factor 1) gene ofCandida glabrata was cloned by functional complementation of the methionine biosynthesis defect of aSaccharomyces cerevisiae cbf1 deletion mutant. TheC. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 p...
Article
Yeast two-hybrid and genetic interaction screens indicate that Bir1p, a yeast protein containing phylogenetically conserved antiapoptotic repeat domains called baculovirus inhibitor of apoptosis repeats (BIRs), is involved in chromosome segregation events. In the two-hybrid screen, Bir1p specifically interacts with Ndc10p, an essential component of...
Article
In budding yeast (Saccharomyces cerevisiae), the majority of box H/ACA small nucleolar RNPs (snoRNPs) have been shown to direct site-specific pseudouridylation of rRNA. Among the known protein components of H/ACA snoRNPs, the essential nucleolar protein Cbf5p is the most likely pseudouridine (Psi) synthase. Cbf5p has considerable sequence similarit...
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We have identified a novel centromere-associated gene product from Saccharomyces cerevisiae that plays a role in spindle assembly and stability. Strains with a deletion of SLK19 (synthetic lethal Kar3p gene) exhibit abnormally short mitotic spindles, increased numbers of astral microtubules, and require the presence of the kinesin motor Kar3p for v...
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Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3-CEN DNA complex by atom...
Article
The Cbf5 protein of Saccharomyces cerevisiae was originally identified as a low-affinity centromeric DNA-binding protein, and chf5 mutants have a defect in rRNA synthesis. A closely related protein from mammals, NAP57, is a nucleolar protein that coimmunoprecipitates with the nucleolar phosphoprotein Nopp140. To study the function of this protein f...
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DNA from the centromere region of linkage group (LG) VII ofNeurospora crassa was cloned previously from a yeast artificial chromosome library and was found to be atypical ofNeurospora DNA in both composition (AT rich) and complexity (repetitive). We have determined the DNA sequence of a small portion (∼16.1 kb) of this region and have identified a...
Article
Yeast Cbf5p was originally isolated as a low-affinity centromeric DNA binding protein (W. Jiang, K. Middleton, H.-J. Yoon, C. Fouquet, and J. Carbon, Mol. Cell. Biol. 13:4884-4893, 1993). Cbf5p also binds microtubules in vitro and interacts genetically with two known centromere-related protein genes (NDC10/CBF2 and MCK1). However, Cbf5p was found t...
Article
Genetic and biochemical strategies have been used to identify Schizosaccharomyces pombe proteins with roles in centromere function. One protein, identified by both approaches, shows significant homology to the human centromere DNA-binding protein, CENP-B, and is identical to Abp1p (autonomously replicating sequence-binding protein 1) (Murakami, Y.,...
Article
CBF2/NDC10/CTF14 encodes the 110-kDa subunit of CBF3, a key component of the yeast centromere/kinetochore. Overexpression of yeast CDC34 specifically suppresses the temperature-sensitive growth phenotype of the ndc10-1 mutation. Mutations in CDC34, which specifies a ubiquitin-conjugating enzyme, arrest yeast cells in the G1 phase of the cell cycle,...
Article
We find that overexpression in yeast of the yeast MCK1 gene, which encodes a meiosis and centromere regulatory kinase, suppresses the temperature-sensitive phenotype of certain mutations in essential centromere binding protein genes CBF2 and CBF5. Since Mck1p is a known serine/threonine protein kinase, this suppression is postulated to be due to Mc...
Article
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We have used in vitro motility assays to investigate the mechanism of kinetochore function in the budding yeast Saccharomyces cerevisiae. Functional centromeric DNA plus a tripartite centromere binding protein complex, CBF3, was found to be necessary but not sufficient for in vitro kinetochore activity. A fourth required component was identified as...
Article
Full-text available
We have used in vitro motility assays to investigate the mechanism of kinetochore function in the budding yeast Saccharomyces cerevisiae. Functional centromeric DNA plus a tripartite centromere binding protein complex, CBF3, was found to be necessary but not sufficient for in vitro kinetochore activity. A fourth required component was identified as...
Article
Full-text available
The centromere locus from linkage group VII of Neurospora crassa has been cloned, characterized, and physically mapped. The centromeric DNA is contained within a 450-kb region that is recombination deficient, A+T-rich, and contains repetitive sequences. Repetitive sequences from within this region hybridize to a family of repeats located at or near...
Article
The centromere locus from linkage group VII of Neurospora crassa has been cloned, characterized, and physically mapped. The centromeric DNA is contained within a 450-kb region that is recombination deficient, A+T-rich, and contains repetitive sequences. Repetitive sequences from within this region hybridize to a family of repeats located at or near...
Article
Full-text available
Yeast centromere DNA (CEN) affinity column chromatography has been used to purify several putative centromere and kinetochore proteins from yeast chromatin extracts. The single yeast gene (CBF5) specifying one of the major low-affinity centromere-binding proteins (p64'/CBF5p) has been cloned and shown to be essential for viability of Saccharomyces...
Article
Yeast centromere DNA (CEN) affinity column chromatography has been used to purify several putative centromere and kinetochore proteins from yeast chromatin extracts. The single yeast gene (CBF5) specifying one of the major low-affinity centromere-binding proteins (p64'/CBF5p) has been cloned and shown to be essential for viability of Saccharomyces...
Article
Full-text available
We have cloned and determined the nucleotide sequence of the gene (CBF2) specifying the large (110 kD) subunit of the 240-kD multisubunit yeast centromere binding factor CBF3, which binds selectively in vitro to yeast centromere DNA and contains a minus end-directed microtubule motor activity. The deduced amino acid sequence of CBF2p shows no seque...
Article
Excerpt In budding yeast (Saccharomyces cerevisiae), the centromeric DNA sequences needed in cis to specify accurate mitotic and meiotic chromosome segregation are contained within a 125-bp locus known as CEN (for review, see Carbon and Clarke 1990; Clarke 1990). The presence of a CEN locus on an autonomously replicating yeast plasmid converts it i...
Article
During cell division, sister chromosomes segregate from each other on a microtubule-based structure called the mitotic spindle. Proteins bind to the centromere, a region of chromosomal DNA, to form the kinetochore, which mediates chromosome attachment to the mitotic spindle microtubules. In the budding yeast Saccharomyces cerevisiae, genetic analys...
Article
We have designed a colony color assay for monitoring centromere DNA-protein interactions in yeast (Saccharomyces cerevisiae). The assay is based on the ability of centromere DNA sequences to block (in cis) transcription initiated from a hybrid CEN-GAL1 promoter. Using a IacZ reporter gene under control of the CEN-GAL1 promoter, we screened colonies...
Article
We have determined the structural organization and functional roles of centromere-specific DNA sequence repeats in cen1, the centromere region from chromosome I of the fission yeast Schizosaccharomyces pombe. cen1 is composed of various classes of repeated sequences designated K', K"(dgl), L, and B', arranged in a 34-kb inverted repeat surrounding...
Article
We have determined the structural organization and functional roles of centromere-specific DNA sequence repeats in cen1, the centromere region from chromosome I of the fission yeast Schizosaccharomyces pombe. cen1 is composed of various classes of repeated sequences designated K', K"(dgl), L, and B', arranged in a 34-kb inverted repeat surrounding...
Article
A key protein component (CBF3) of the budding yeast (S. cerevisiae) centromere/kinetochore has been purified and characterized. CBF3 is a 240 kd multisubunit protein complex that binds specifically to the yeast wild-type centromere DNA (CEN), but not to nonfunctional CEN DNA containing a single base substitution in the critical CDEIII consensus seq...
Article
The centromere-associated B' and B DNA sequence repeats of Schizosaccharomyces pombe chromosomes I and II have been found to contain clusters of tRNA genes. The centromere II region (cen2) includes at least 22 tRNA genes distributed among five copies of the B sequence repeat containing genes specifying tRNA(Ile), tRNA(Ala), and tRNA(Val). Individua...
Article
Functional centromeric DNAs have now been isolated and characterized from both budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeasts. Artificial chromosomes containing these centromere DNA sequences segregate faithfully in both mitotic and meiotic cell divisions, but only in the parent organism. Structure-function analys...
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Full-text available
An electrophoretic karyotype of Aspergillus nidulans has been obtained using contour-clamped homogeneous electric field gel electrophoresis. Six chromosomal bands were separated, with two of the bands migrating as doublets. Using the Schizosaccharomyces pombe and Saccharomyces cerevisiae chromosomes as size standards, we estimate the sizes of the c...
Article
The centromere DNAs from chromosomes I and III of Schizosaccharomyces pombe have been cloned in an artificial chromosome vector in both budding and fission yeasts. In S. pombe, synthetic linear and circular minichromosomes containing an intact centromere are stable mitotically and behave as independent genetic linkage groups that segregate properly...
Article
Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 5...
Article
Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 5...
Article
Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletio...
Article
Full-text available
A 1.5-kilobase-pair SalI-HindIII (SH) restriction fragment from the region of Saccharomyces cerevisiae chromosome XIV immediately adjacent to the centromere appears to contain sequences that act as a hot spot for mitotic recombination. The presence of SH DNA on an autonomously replicating plasmid stimulates homologous genetic exchange between yeast...
Article
Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletio...
Article
A centromere (CEN) in Saccharomyces cerevisiae consists of approximately 150 bp of DNA and contains 3 conserved sequence elements: a high A + T region 78-86 bp in length (element II), flanked on the left by a conserved 8-bp element I sequence (PuTCACPuTG), and on the right by a conserved 25-bp element III sequence. We have carried out a structure-f...
Article
Two yeast (Saccharomyces cerevisiae) mutants that tolerate centromere (CEN) plasmids at high copy number have been isolated. The mutations relieve the restraint normally imposed on plasmid copy number by a cloned CEN sequence. Our CEN plasmids specify resistance to G418 and are high copy plasmids only when the mutant host cells are grown on medium...
Article
A 1.5-kilobase-pair SalI-HindIII (SH) restriction fragment from the region of Saccharomyces cerevisiae chromosome XIV immediately adjacent to the centromere appears to contain sequences that act as a hot spot for mitotic recombination. The presence of SH DNA on an autonomously replicating plasmid stimulates homologous genetic exchange between yeast...
Article
The Schizosaccharomyces pombe centromere-linked genes, LYS1 and CYH1 on chromosome I and TPS13 and RAN1 on chromosome II, have been isolated. The genetic order of these markers with respect to their centromeres was determined to establish relative directionality on the genetic and physical maps. Chromosome walking toward the centromeres reveals a g...
Article
Plasmids carrying a Saccharomyces cerevisiae centromere have a copy number of one or two, whereas other yeast plasmids have high copy numbers. The number of CEN plasmids per yeast cell was made artificially high by transforming cells simultaneously with several different CEN plasmids carrying different, independently selectable markers. Some host c...
Article
Plasmids carrying a Saccharomyces cerevisiae centromere have a copy number of one or two, whereas other yeast plasmids have high copy numbers. The number of CEN plasmids per yeast cell was made artificially high by transforming cells simultaneously with several different CEN plasmids carrying different, independently selectable markers. Some host c...
Article
Yeast (Saccharomyces cerevisiae) transposons (Ty elements) are excised from up to 20% of supercoiled plasmids during transformation of yeast cells. The excision occurs by homologous recombination across the direct terminal repeats (deltas) of the Ty element, leaving behind a single delta in the transforming plasmid. Only the initial transforming pl...
Article
Full-text available
The Schizosaccharomyces pombe acid phosphatase structural gene (PHO 1) was isolated by complementation of an S. pombe acid phosphatase mutant with a wild type S. pombe DNA recombinant plasmid library. Northern analysis indicates that acid phosphatase is encoded by a 1.4-kilobase mRNA of which approximately 100 bases are 3'-poly(A). The gene contain...
Article
Full-text available
We used DNA fragments from the centromere regions of yeast (Saccharomyces cerevisiae) chromosomes III and XI to examine the transcriptional activity within this chromosomal domain. DNA transcripts were found 200 to 300 base pairs from the 250-base-pair centromere core and lie within an ordered chromatin array. No transcripts were detected from the...
Article
In the yeast Saccharomyces cerevisiae, circular or linear plasmids containing a functional centromere (CEN) and a chromosomal replicator (ARS) are mitotically stable and segregate as ordinary yeast chromosomes in the first and second meiotic divisions. A centromere in S. cerevisiae consists of a region of DNA, approximately 150 bp in length, contai...
Article
We used DNA fragments from the centromere regions of yeast (Saccharomyces cerevisiae) chromosomes III and XI to examine the transcriptional activity within this chromosomal domain. DNA transcripts were found 200 to 300 base pairs from the 250-base-pair centromere core and lie within an ordered chromatin array. No transcripts were detected from the...
Article
Full-text available
A functional centromere located on a small DNA restriction fragment from Saccharomyces cerevisiae was identified as CEN14 by integrating centromere-adjacent DNA plus the URA3 gene by homologous recombination into the yeast genome and then by localizing the URA3 gene to chromosome XIV by standard tetrad analysis. DNA sequence analysis revealed that...
Article
A functional centromere located on a small DNA restriction fragment from Saccharomyces cerevisiae was identified as CEN14 by integrating centromere-adjacent DNA plus the URA3 gene by homologous recombination into the yeast genome and then by localizing the URA3 gene to chromosome XIV by standard tetrad analysis. DNA sequence analysis revealed that...
Article
Full-text available
The centromere region of Saccharomyces cerevisiae chromosome III has been replaced by various DNA fragments from the centromere regions of yeast chromosomes III and XI. A 289-base pair centromere (CEN3) sequence can stabilize yeast chromosome III through mitosis and meiosis. The orientation of the centromeric fragments within chromosome III has no...
Article
The complete nucleotide sequence of a 2296-bp DNA fragment containing the yeast (Saccharomyces cerevisiae) ARG4 gene has been determined. This gene specifies the synthesis of the arginine biosynthetic enzyme, argininosuccinate lyase (EC 4.3.2.1). The sequence contains one major open reading frame of 463 codons, giving a calculated Mr of 52 010 for...
Article
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Article
Structure-function analysis of a yeast (Saccharomyces cerevisiae) centromere (CEN3) has been carried out by altering the nucleotide sequence of the DNA within and surrounding the centromere of yeast chromosome III, and observing the behaviour of the resulting altered chromosomes during mitotic and meiotic cell divisions. A centromere substitution v...
Article
Plasmids containing a cloned yeast (Saccharomyces cerevisiae) centromere (CEN3) in combination with a suitable DNA replication system are maintained in yeast at the low copy number typical of a chromosome. In composite plasmids containing CEN3 plus the yeast 2 mu plasmid, the CEN3 copy number control is dominant over the amplification system that n...
Article
The centromere region of yeast chromosome III has been investigated by altering it in vivo. Deleting the functional centromere (CEN3) sequence leads to extreme instability of the resulting acentric chromosome. Inversion of CEN3, or its replacement by chromosome XI centromere DNA (CEN11) has no measurable effect on the mitotic and meiotic behaviour...
Article
This chapter focuses on the selection procedure for isolation of centromere DNAs from Saccharomyces cerevisiae. Hybrid plasmid derivatives of pBR322 that contain yeast centromere DNA in combination with a yeast DNA replicator and a suitable genetic marker are stably maintained in yeast through both mitotic and meiotic cell divisions and exhibit typ...
Article
Excerpt The centromere is a specific region of the eukaryotic chromosome that plays a fundamental role in chromosome movement throughout cell division. During the early stages of chromosome preparation for mitosis and meiosis, the centromere becomes visible as a distinct morphological entity along the chromatin fiber. The function of the centromere...
Article
We have examined the chromatin structure of the centromere regions of chromosomes III and XI in yeast by using cloned functional centromere DNAs (CEN3 and CEN11) as labeled probes. When chromatin from isolated nuclei is digested with micrococcal nuclease and the resulting DNA fragments separated electrophoretically and blotted to nitrocellulose fil...
Article
We determined the nucleotide sequence of DNA segments containing functional centromeres (CEN3 and CEN11) isolated from yeast chromosomes III and XI. The two centromere regions differ in primary nucleotide sequence, but contain structural features in common. Both centromere regions contain an extremely A + T-rich core segment 87-88 bp in length, fla...
Article
A specific autonomously replicating sequence (ars2) occurring near the ARG4 locus on chromosome VIII of yeast (Saccharomyces cerevisiae) has been localized on a 100 base-pair region of DNA. The nucleotide sequence of a 1517 bp† fragment spanning the ars2 region has been determined. The 100 bp ars2 region contains an 18 bp A + T sequence of perfect...
Article
We have cloned segments of yeast DNA containing the centromere XI-linked MET14 gene. This was done by selecting directly in Saccharomyces cerevisiae for complementation of a met14 mutation after transformation with a hybrid plasmid DNA genomic library. Genetic evidence indicates that functional centromere DNA (CEN11) from chromosome XI is also cont...
Article
We have cloned segments of yeast DNA containing the centromere XI-linked MET14 gene. This was done by selecting directly in Saccharomyces cerevisiae for complementation of a met14 mutation after transformation with a hybrid plasmid DNA genomic library. Genetic evidence indicates that functional centromere DNA (CEN11) from chromosome XI is also cont...