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J Carbon

Publications and source records attributed to J Carbon.

At least 55 records · Page 3Linked to original sources
PubMed1986

Toxic effects of excess cloned centromeres.

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 cells carried five different CEN plasmids and an average total of 13 extra copies of CEN3. Several effects were noted. The copy number of each plasmid was unexpectedly high. The plasmids were mutually unstable. Cultures contained many dead cells. The viable host cells grew more slowly than control cells, even in nonselective medium. There was a pause in the cell cycle at or just before mitosis. We conclude that an excess of centromeres is toxic and that the copy number of centromere plasmids is low partly because of selection against cells carrying multiple centromere plasmids. The toxicity may be caused by competition between the centromeres for some factor present in limiting quantities, e.g., centromere-binding proteins, microtubules, or space on the spindle pole body.

Cell Cycle↗
PubMed1985

Identification and characterization of the centromere from chromosome XIV in Saccharomyces cerevisiae.

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 CEN14 possesses sequences (elements I, II, and III) that are characteristic of other yeast centromeres. Mitotic and meiotic analyses indicated that the CEN14 function resides on a 259-base-pair (bp) RsaI-EcoRV restriction fragment, containing sequences that extend only 27 bp to the right of the element I to III region. In conjunction with previous findings on CEN3 and CEN11, these results indicate that the specific DNA sequences required in cis for yeast centromere function are contained within a region about 150 bp in length.

Base Sequence↗
PubMed1984

The nucleotide sequence of the yeast ARG4 gene.

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 the protein, in good agreement with the experimentally determined value of 53 000. The sequence upstream from the ARG4 gene shares structural features in common with other yeast genes subject to general amino acid control.

Amino Acid Sequence↗
PubMed1984

Chromatin conformation of yeast centromeres.

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 effect on the normal mitotic or meiotic behavior of the chromosome. The structural integrity of the centromere region in these genomic substitution strains was examined by mapping nucleolytic cleavage sites within the chromatin DNA. A nuclease-protected centromere core of 220-250 base pairs was evident in all of the genomic substitution strains. The position of the protected region is determined strictly by the centromere DNA sequence. These results indicate that the functional centromere core is contained within 220-250 base pairs of the chromatin DNA that is structurally distinct from the flanking nucleosomal chromatin.

Base Sequence↗
PubMed1984

Structural and functional analysis of a yeast centromere (CEN3).

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 vector (pJC3-13) was constructed, which contains in the proper orientation: the DNA sequences that normally flank the chromosome III centromere, a wild-type URA3 gene for selection, and a unique BamHI restriction site for insertion of various DNA sequences to be assayed for centromere activity. Cleavage of the plasmid DNA with EcoRI generates a linear DNA fragment whose ends are homologous with the regions flanking the centromere. Transformation of the appropriate homozygous ura3 diploid yeast strain with this linear DNA results in URA3+ transformants in which the CEN3 region on one copy of chromosome III has been replaced by the URA3 gene and the DNA sequence previously inserted into the vector. These studies identify a 289 base-pair (bp) DNA fragment from the CEN3 region that retains full centromere function when used to replace the normal CEN3 sequence. Centromeres function equally well in either orientation, and the chromosome XI centromere (CEN11) can be used to replace CEN3, with no observable effect on mitotic or meiotic chromosome segregation. Various DNA restriction fragments occurring within the CEN3 region were used alone or in combinations to replace the normal CEN3 sequence. Yeast centromeres contain a high A + T region about 82-89 bp in length (element II) flanked by a highly conserved 11 bp sequence (III) and a less-conserved 14 bp sequence (I). The experiments demonstrate that both regions II and III are necessary for normal centromere function, although centromeres containing III plus truncated or rearranged portions of the high A + T region II retain partial activity. Chromosomes of the latter type often give abnormal segregation patterns through meiosis, including separation and random segregation of sister chromatids during the first meiotic division.

Base Sequence↗
PubMed1983

Copy number control by a yeast centromere.

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 normally drives the 2 mu plasmids to high copy number. The CEN3-2 mu composite plasmids are relatively stably maintained in yeast at a copy number of about one per haploid genome, and segregate through meiosis in a typical Mendelian pattern. Some of the CEN3-2 mu composite plasmids isolated from yeast contain deletions of variable size that remove the functional centromere, resulting in loss of the CEN3 control and reversion to high copy number. Formation of the CEN3 deletions requires the specialized recombination system (inverted repeat sequences and FLP gene) of the yeast 2 mu plasmid.

Centromere↗
PubMed1982

Nucleotide sequence comparisons and functional analysis of yeast centromere DNAs.

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, flanked by two short sequences (14 bp and 11 bp) that are identical in both DNAs. These elements plus one additional 10 bp region of perfect homology are positioned in an almost identical spatial arrangement within the two centromere regions. Significant homologies are also observed among the sequences flanking the high A + T region and various satellite DNA sequences from higher eucaryotes, although no repeated sequences occur near the yeast centromeres. Centromere activity in vivo is maintained on relatively small DNA fragments (627 bp for CEN3 and 858 bp for CEN11), as assayed by mitotic stabilization of autonomously replicating ars plasmids in yeast.

Base Sequence↗
PubMed1982

Yeast centromere DNA is in a unique and highly ordered structure in chromosomes and small circular minichromosomes.

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 filters, the centromeric nucleosomal subunits are resolved into significantly more distinct ladders than are those from the bulk of the chromatin. A discrete protected region of 220-250 bp of CEN sequence flanked by highly nuclease-sensitive sites was revealed by mapping the exact nuclease cleavage sites within the centromeric chromatin. On both sides of this protected region, highly phased and specific nuclease cutting sites exist at nucleosomal intervals (160 bp) for a total length of 12-15 nucleosomal subunits. The central protected region in the chromatin of both centromeres spans the 130 bp segment that exhibits the highest degree of sequence homology (71%) between functional CEN3 and CEN11 DNAs. This unique chromatin structure is maintained on CEN sequences introduced into yeast on autonomously replicating plasmids, but is not propagated through foreign DNA sequences flanking the inserted yeast DNA.

Base Sequence↗
PubMed1982

Isolation and subcloning analysis of functional centromere DNA (CEN11) from Saccharomyces cerevisiae chromosome XI.

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 contained on the segment of S. cerevisiae DNA cloned in pYe(MET14)2. This plasmid is maintained stably in budding S. cerevisiae cultures and segregates predominantly 2+:20- through meiosis. The CEN11 element has been subcloned in vector YRp7' on an S. cerevisiae DNA fragment 900 base pairs in length [pYe(CEN11)10]. The mitotic and meiotic behavior of plasmids containing CEN11 plus a DNA replicator (ars) indicates that the centromere DNA sequences enable these plasmids to function as true minichromosomes in S. cerevisiae.

Base Sequence↗
PubMed1981

Characterization of a yeast replication origin (ars2) and construction of stable minichromosomes containing cloned yeast centromere DNA (CEN3).

A yeast DNA sequence (ars2), capable of supporting autonomous replication of plasmids, in yeast, has been characterized. The ars2 replicator occurs about 7 kb from the ARG4 gene on yeast chromosome VIII. Plasmids containing ars2 and the ARG4 gene transform yeast arg4 mutants to ARG4+ with high frequency (about 103 transformants/micrograms DNA) and replicate autonomously in the transformed cells. The ars2 plasmids are mitotically unstable and are readily lost from yeast cultures when grown under nonselective conditions. The addition of a DNA segment containing functional yeast centromere (CEN3) and an ars2 plasmid effectively stabilizes the plasmid against both mitotic and meiotic loss. The ars2-CEN3 minichromosomes replicate autonomously in controlled copy number while segregating in a typical Mendelian pattern (2+ :2-) during meiosis. The requirement for a separate replicator sequence for stable mitotic and meiotic maintenance of centromere-containing minichromosomes is equally satisfied by the presence of either ars1 or ars2. The centromere controls plasmid copy number to a low value (usually one) regardless of the type of replicator used.

Base Sequence↗
PubMed1981

Direct selection procedure for the isolation of functional centromeric DNA.

Several plasmids, each containing a segment of DNA capable of conferring mitotic stability on the ars1 vector YRp7 replicating in yeast (Saccharomyces cerevisiae), have been isolated from a yeast genomic library by direct selection for stable plasmid maintenance. The mitotic-stabilizing sequences fall into two classes: (i) segments of the yeast "2-micrometers" plasmid and (ii) DNA segments containing yeast centromeric DNA. Plasmids of the first class segregate 4+:0- through meiosis whereas the centromere plasmids segregate predominantly 2+:2-, as typical chromosomes. Previously identified centromeres from chromosomes II (CEN3) and XI (CEN11), along with the CEN11-linked gene MET14, can be isolated by this direct selection procedure. In addition, several other unidentified centromere DNAs were obtained. There are no detectable sequence homologies between CEN3 and CEN11 DNAs nor do these DNAs hybridize to the other yeast centromere regions.

Aneuploidy↗
PubMed1980

Isolation and characterization of the yeast 3-phosphoglycerokinase gene (PGK) by an immunological screening technique.

An immunological screening technique has been used for the detection of a specific antigen-producing clone in a bank of bacterial colonies containing hybrid plasmids. This technique involves covalent attachment of antiserum to cyanogen bromide-activated paper discs, contact of this paper with lysed colonies on agar plates, and finally detection of the bound antigen with 125I-labeled antibody. Using this method, we have identified an Escherichia coli colony, containing a yeast DNA insert in plasmid ColE1, that produces antigen which combines with antibody directed against purified yeast 3-phosphoglycerate kinase. The hybrid plasmid (pYe57E2) obtained by this procedure has been shown by both biochemical and genetic methods to contain the structural gene PGK for yeast 3-phosphoglycerate kinase. The location of the PGK structural gene on pYe56E2 was determined by immunological screening of E. coli colonies bearing plasmids containing various reconstructions of the original yeast DNA insert. Examination of the expression of the cloned yeast PGK gene in both E. coli and yeast has shown that functional enzyme is synthesized from the cloned gene in yeast, but not in E. coli.

Cloning, Molecular↗