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J Carbon

Publications and source records attributed to J Carbon.

At least 19 recordsLinked to original sources
PubMed1995

Genetic and biochemical interactions between an essential kinetochore protein, Cbf2p/Ndc10p, and the CDC34 ubiquitin-conjugating enzyme.

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, with no intact spindles formed (M. G. Goebl, J. Yochem, S. Jentsch, J. P. McGrath, A. Varshavsky, and B. Byers, Science 241:1331-1335, 1988). The cdc34-2 mutation drastically alters the pattern of Cbf2p modification. Results of experiments using antibodies against Cbf2p and ubiquitin indicate that Cbf2p is ubiquitinated in vivo. Purified Cdc34p catalyzes the formation of Cbf2p-monoubiquitin conjugate in vitro. These data suggest that Cbf2p is an endogenous substrate of the CDC34 ubiquitin-conjugating enzyme and imply that ubiquitination of a kinetochore protein plays a regulatory role in kinetochore function.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1992

Microtubule-motor activity of a yeast centromere-binding protein complex.

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 analysis has shown that the 28-basepair (bp) CDEIII region of the 125-bp centromere DNA sequence (CEN sequence) is the main region controlling chromosome segregation in vivo. Therefore it is likely that proteins binding to the CDEIII region link the centromeres to the microtubules during mitosis. A complex of proteins (CBF3) that binds specifically to the CDEIII DNA sequence has been isolated by affinity chromatography. Here we describe kinetochore function in vitro. The CBF3 complex can link DNA to microtubules, and the complex contains a minus-end-directed microtubule-based motor. We suggest that microtubule-based motors form the fundamental link between microtubules and chromosomes at mitosis.

Adenosine Triphosphate↗
PubMed1992

A colony color assay for Saccharomyces cerevisiae mutants defective in kinetochore structure and function.

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 derived from mutagenized cells for a blue color phenotype indicative of derepression of the hybrid construct. A limited screen in which a 61-bp CEN11 DNA fragment containing an intact CDEIII subregion plus flanking sequences was used as the "pseudo-operator" led to the identification of mutations (blu) in three complementation groups. The blu1 mutants exhibited a decrease in activity of the major CEN DNA-binding proteins in vitro. The BLU1 gene was shown to be identical to the previously isolated SPT3 gene, known to be involved in the transcriptional regulation of a subset of yeast genes. Our results indicate that the BLU1/SPT3 gene product may also be required to maintain optimal levels of functional centromere DNA-binding proteins.

Acriflavine↗
PubMed1991

A 240 kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere.

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 sequence. When purified by affinity chromatography, CBF3 contains three protein components: CBF3A (110 kd), CBF3B (64 kd), and CBF3C (58 kd). Highly purified CBF3 requires the presence of a separate assembly factor or chaperone activity to bind to CEN DNA. Treatment with phosphatase inactivates CBF3, indicating that at least one of the CBF3 subunits must be phosphorylated for DNA binding to occur. A 56 bp region including the 26 bp CDEIII consensus is protected from DNAase I cleavage in the CBF3-CEN DNA complex.

Base Sequence↗
PubMed1991

Clustered tRNA genes in Schizosaccharomyces pombe centromeric DNA sequence repeats.

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). Individual B repeats are variously associated with other tRNA genes, including those specifying tRNA(Lys), tRNA(Arg), and tRNA(Glu2). The centromere I region (cen1) contains at least six tRNA genes in two copies of the B' repeated element, including genes specifying tRNA(Ile), tRNA(Ala), and tRNA(Glu3). Multiple tandemly arranged clusters of tRNA genes are presumably conserved due to restricted recombination frequencies in the centromere regions.

Base Sequence↗
PubMed1991

Identification of DNA regions required for mitotic and meiotic functions within the centromere of Schizosaccharomyces pombe chromosome I.

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 a 4- to 5-kb nonhomologous central core. Artificial chromosomes containing various portions of the cen1 region were constructed and assayed for mitotic and meiotic centromere function in S. pombe. Deleting K' and L from the distal portion of one arm of the inverted repeat had no effect on mitotic centromere function but resulted in greatly increased precocious sister chromatid separation in the first meiotic division. A centromere completely lacking K' and L, but containing the central core, one copy of B' and K" in one arm, and approximately 2.5 kb of the core-proximal portion of B' in the other arm, was also fully functional mitotically but again did not maintain sister chromatid attachment in meiosis I. However, deletion of K" from this minichromosome resulted in complete loss of centromere function. Thus, one copy of at least a portion of the K" (dgl) repeat is absolutely required but is not sufficient for S. pombe centromere function. The long centromeric inverted-repeat region must be relatively intact to maintain sister chromatid attachment in meiosis I.

Centromere↗
PubMed1990

Centromere structure and function in budding and fission yeasts.

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 analyses have revealed surprising fundamental differences between these two centromere classes. In the budding yeast centromeres, a 125-bp consensus DNA sequence contains all the information needed in cis to provide proper chromosome segregation. In contrast, the fission yeast centromeres each contain a long run (40 to 100 kb) of untranscribed repetitive DNA sequences arranged into a large inverted repeat, most of which is required for full centromere function. The fission yeast centromere-kinetochore appears to be a highly relevant experimental model for analysis of the mechanism of chromosome segregation in higher eukaryotes, in which the centromere regions often contain megabases of transcriptionally silent repetitive DNA sequences of unknown function.

Base Sequence↗
PubMed1989

Electrophoretic karyotype of Aspergillus nidulans.

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 chromosomes to be between 2.9 and 5.0 megabase pairs (mb) with a total genome size of approximately 31 mb. Four of the eight genetic linkage groups were assigned to chromosomal bands by hybridization of contour-clamped homogeneous electric field gel blots with various radiolabeled probes each specific to a particular linkage group. Contour-clamped homogeneous electric field gel analysis of reciprocal translocation strains gave chromosomal assignments for the four remaining linkage groups. In order of decreasing size, the A. nidulans chromosomes are: VIII (5.0 mb), VII (4.5 mb), II (4.2 mb), I and V (3.8 mb), III and VI (3.5 mb), and IV (2.9 mb).

Aspergillus nidulans↗
PubMed1989

Construction of functional artificial minichromosomes in the fission yeast Schizosaccharomyces pombe.

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 through meiosis. These experiments present a general strategy for the isolation of centromeres from other organisms.

Centromere↗
PubMed1988

Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

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 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.

Centromere↗
PubMed1987

Saccharomyces cerevisiae mutants that tolerate centromere plasmids at high copy number.

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 containing this antibiotic. The high copy number of the plasmids is independent of the specific cloned CEN sequence and recovered plasmids show no alteration in structure or function of the CEN DNA. The efficiency with which CEN plasmids go to high copy number is increased if the mutant cell is cotransformed by another CEN plasmid. The genomic mutation responsible for the high copy number (COP) is dominant and stable, and it segregates in a Mendelian manner. Homozygous COP/COP a/alpha diploids do not tolerate CEN plasmids at high copy number, suggesting that the mutation is regulated by mating type. The genomic DNA from both mutant cells contains an altered transposon (Ty) restriction fragment that cosegregates with the COP phenotype in crosses of mutant and wild-type strains. The mutations may be transposon-mediated events that identify a gene involved in centromere or mitotic spindle function.

Centromere↗
PubMed1987

Mutational analysis of meiotic and mitotic centromere function in Saccharomyces cerevisiae.

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-function analysis of the element I and II regions of CEN3 by constructing mutations in these sequences and subsequently determining their effect on mitotic and meiotic chromosome segregation. We have also examined the mitotic and meiotic segregation behavior of ARS plasmids containing the structurally altered CEN3 sequences. Replacing the periodic tracts of A residues within element II with random A + T sequences of equal length increases the frequency of mitotic chromosome nondisjunction only 4-fold; whereas, reducing the A + T content of element II while preserving the length results in a 40-fold increase in the frequence of chromosome nondisjunction. Structural alterations in the element II region that do not decrease the overall length have little effect on the meiotic segregation behavior of the altered chromosomes. Centromeres containing a deletion of element I or a portion of element II retain considerable mitotic activity, yet plasmids carrying these same mutations segregate randomly during meiosis I, indicating these sequences to be essential for maintaining attachment of the replicated sister chromatids during the first meiotic division. The presence of an intact element I sequence properly spaced from the element III region is absolutely essential for proper meiotic function of the centromere.

Base Sequence↗
PubMed1987

Characterization of a centromere-linked recombination hot spot in Saccharomyces cerevisiae.

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 genomic sequences and those present on the plasmid. In all recombinants characterized, exchange occurs in plasmid yeast sequences adjacent to rather than within the SH DNA. Hybridization analyses reveal that SH-containing plasmids are present in linear as well as circular form in S. cerevisiae and that linear forms are generated by cleavage at specific sites. Presumably, it is the linear form of the plasmid that is responsible for the stimulation of genetic exchange. Based on these observations, it is proposed that this DNA fragment contains a centromere-linked recombination hot spot and that SH-stimulated recombination occurs via a mechanism similar to double-strand-gap repair (J. W. Szostak, T. Orr-Weaver, J. Rothstein, and F. Stahl, Cell 33:25-35 1983).

Centromere↗
PubMed1987

Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae.

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 deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.

Base Sequence↗
PubMed1986

High frequency excision of Ty elements during transformation of yeast.

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 plasmid is susceptible to excision, and no high frequency excision is observed in plasmids that have become established in transformed cells or in plasmids that are resident in cells undergoing transformation. High frequency excision from plasmids during yeast transformation is not specific for Ty elements and can be observed with other segments of plasmid DNA bounded by direct repeats. The frequency of Ty excision from supercoiled plasmids is greatly reduced when the host yeast cells contain the rad52 mutation, a defect in double-strand DNA repair. When linear or ligated-linear plasmid DNAs containing a Ty element are used for transformation, few or no excision plasmids are found among the transformant colonies. These results suggest that when a yeast cell is transformed with a supercoiled plasmid, the plasmid DNA is highly susceptible to homologous recombination for a short period of time.

DNA Repair↗
PubMed1986

Isolation and characterization of the structural gene for secreted acid phosphatase from Schizosaccharomyces pombe.

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 contains no introns and the 3' and 5' untranslated regions are short. According to DNA and amino acid sequence data, the S. pombe acid phosphatase has a molecular weight of 50,600. An 18-amino acid sequence at the N terminus was found that is similar to previously identified signal peptides in other eukaryotic secretory proteins. This signal peptide is apparently removed during secretion, since it is absent in the mature secreted acid phosphatase. The gene can be induced 2--3-fold by starvation for phosphate. The signals required for this induction are contained on the isolated DNA clone. Although the gene can be expressed in Saccharomyces cerevisiae, secretion is abnormal.

Acid Phosphatase↗