Nematode repetitive DNA with ARS and segregation function in Saccharomyces cerevisiae.

Several members of a repetitive DNA family in the nematode Caenorhabditis elegans have been shown to express ARS and centromeric function in Saccharomyces cerevisiae. The repetitive family, denoted CeRep3, consists of dispersed repeated elements about 1 kilobase in length, present 50 to 100 times in the nematode genome. Three elements were sequenced and found to contain DNA sequences homologous to yeast ARS and CEN consensus sequences. Nematode DNA segments containing these repeats were tested for ARS and CEN (or SEG) function after ligation to shuttle vectors and introduction into yeast cells. Such nematode segments conferred ARS function to the plasmid, as judged by an increased frequency of transformation compared with control plasmids without ARS function. Some, but not all, also conferred to the plasmid increased mitotic stability, increased frequency of 2+:2- segregation in meiosis, and decreased plasmid copy number. These effects are similar to those of yeast centromeric DNA. In view of these results, we suggest that the CeRep3 repetitive family may have replication and centromeric functions in C. elegans.

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Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation.

Molecular and Cellular Biology ◽

10.1128/mcb.9.4.1488 ◽

1989 ◽

Vol 9 (4) ◽

pp. 1488-1497 ◽

Cited By ~ 53

Author(s):

K W Runge ◽

V A Zakian

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Sequences ◽

Copy Number ◽

Plasmid Copy Number ◽

High Copy Number ◽

Limiting Factor ◽

Telomeric Dna ◽

Base Pairs ◽

Plasmid Copy ◽

Telomere Elongation

The termini of Saccharomyces cerevisiae chromosomes consist of tracts of C1-3A (one to three cytosine and one adenine residue) sequences of approximately 450 base pairs in length. To gain insights into trans-acting factors at telomeres, high-copy-number linear and circular plasmids containing tracts of C1-3A sequences were introduced into S. cerevisiae. We devised a novel system to distinguish by color colonies that maintained the vector at 1 to 5, 20 to 50, and 100 to 400 copies per cell and used it to change the amount of telomeric DNA sequences per cell. An increase in the number of C1-3A sequences caused an increase in the length of telomeric C1-3A repeats that was proportional to plasmid copy number. Our data suggest that telomere growth is inhibited by a limiting factor(s) that specifically recognizes C1-3A sequences and that this factor can be effectively competed for by long tracts of C1-3A sequences at telomeres or on circular plasmids. Telomeres without this factor are exposed to processes that serve to lengthen chromosome ends.

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Selection of yeast cells with a higher plasmid copy number in a Saccharomyces cerevisiae autoselection system

Yeast ◽

10.1002/(sici)1097-0061(19960315)12:3<199::aid-yea895>3.0.co;2-u ◽

1996 ◽

Vol 12 (3) ◽

pp. 199-205 ◽

Cited By ~ 6

Author(s):

Concetta Compagno ◽

Danilo Porro ◽

Stefania Radice ◽

Enzo Martegani ◽

Bianca Maria Ranzi

Keyword(s):

Saccharomyces Cerevisiae ◽

Copy Number ◽

Plasmid Copy Number ◽

Yeast Cells ◽

Plasmid Copy ◽

Selection Of

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Expression of plasmid R388-encoded type II dihydrofolate reductase as a dominant selective marker in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.4.3.407-414.1984 ◽

1984 ◽

Vol 4 (3) ◽

pp. 407-414

Author(s):

A Miyajima ◽

I Miyajima ◽

K Arai ◽

N Arai

Keyword(s):

Saccharomyces Cerevisiae ◽

Dihydrofolate Reductase ◽

Copy Number ◽

Enzyme Level ◽

Plasmid Copy Number ◽

Yeast Cells ◽

Type Ii ◽

Plasmid Copy ◽

Selective Media ◽

In Cells

The R388 plasmid-encoded drug-resistant type II dihydrofolate reductase gene (R . dhfr) was expressed in Saccharomyces cerevisiae by fusing the R . dhfr coding sequence to the yeast TRP5 promoter. Yeast cells harboring these recombinant plasmids grew in media with 10 micrograms of methotrexate per ml and 5 mg of sulfanilamide per ml, a condition which inhibits the growth of wild-type cells. Addition of a 390-base-pair fragment from the 3'-noncoding region of TRP5 downstream from R . dhfr increased expression. Presumably, the added segment promoted termination or polyadenylation or both of the R . dhfr transcript. The activity of the plasmid-encoded dihydrofolate reductase and the copy number of the R . dhfr plasmid in cells grown in drug-selective media were higher by one order of magnitude than those grown in nutrition-selective media. Plasmid copy number, as well as the plasmid-encoded enzyme level, decreased when cells were selected for prototrophy. In drug-selective media, the plasmid-encoded enzyme level and the content of R . dhfr transcripts were nearly constant in cells harboring R . dhfr plasmids containing different yeast promoters. In contrast, the plasmid copy number and beta-lactamase activity encoded in cis by plasmids were much higher when R . dhfr was associated with the weak TRP5 promoter than when it was fused to the strong ADC1 promoter. These results indicate that plasmid copy number, i.e., gene dosage of R . dhfr, correlates inversely with the strength of the promoter associated with R . dhfr, and cells with a higher plasmid copy number were enriched in drug-selective media. The transformation efficiency of R . dhfr fused to the ADC1 promoter was almost the same on drug-selective plates as on nutrition-selective plates, indicating that R . dhfr is suitable as a dominant selective transformation marker in S. cerevisiae.

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Expression of plasmid R388-encoded type II dihydrofolate reductase as a dominant selective marker in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.4.3.407 ◽

1984 ◽

Vol 4 (3) ◽

pp. 407-414 ◽

Cited By ~ 36

Author(s):

A Miyajima ◽

I Miyajima ◽

K Arai ◽

N Arai

Keyword(s):

Saccharomyces Cerevisiae ◽

Dihydrofolate Reductase ◽

Copy Number ◽

Enzyme Level ◽

Plasmid Copy Number ◽

Yeast Cells ◽

Type Ii ◽

Plasmid Copy ◽

Selective Media ◽

In Cells

The R388 plasmid-encoded drug-resistant type II dihydrofolate reductase gene (R . dhfr) was expressed in Saccharomyces cerevisiae by fusing the R . dhfr coding sequence to the yeast TRP5 promoter. Yeast cells harboring these recombinant plasmids grew in media with 10 micrograms of methotrexate per ml and 5 mg of sulfanilamide per ml, a condition which inhibits the growth of wild-type cells. Addition of a 390-base-pair fragment from the 3'-noncoding region of TRP5 downstream from R . dhfr increased expression. Presumably, the added segment promoted termination or polyadenylation or both of the R . dhfr transcript. The activity of the plasmid-encoded dihydrofolate reductase and the copy number of the R . dhfr plasmid in cells grown in drug-selective media were higher by one order of magnitude than those grown in nutrition-selective media. Plasmid copy number, as well as the plasmid-encoded enzyme level, decreased when cells were selected for prototrophy. In drug-selective media, the plasmid-encoded enzyme level and the content of R . dhfr transcripts were nearly constant in cells harboring R . dhfr plasmids containing different yeast promoters. In contrast, the plasmid copy number and beta-lactamase activity encoded in cis by plasmids were much higher when R . dhfr was associated with the weak TRP5 promoter than when it was fused to the strong ADC1 promoter. These results indicate that plasmid copy number, i.e., gene dosage of R . dhfr, correlates inversely with the strength of the promoter associated with R . dhfr, and cells with a higher plasmid copy number were enriched in drug-selective media. The transformation efficiency of R . dhfr fused to the ADC1 promoter was almost the same on drug-selective plates as on nutrition-selective plates, indicating that R . dhfr is suitable as a dominant selective transformation marker in S. cerevisiae.

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Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation

Molecular and Cellular Biology ◽

10.1128/mcb.9.4.1488-1497.1989 ◽

1989 ◽

Vol 9 (4) ◽

pp. 1488-1497

Author(s):

K W Runge ◽

V A Zakian

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Sequences ◽

Copy Number ◽

Plasmid Copy Number ◽

High Copy Number ◽

Limiting Factor ◽

Telomeric Dna ◽

Base Pairs ◽

Plasmid Copy ◽

Telomere Elongation

The termini of Saccharomyces cerevisiae chromosomes consist of tracts of C1-3A (one to three cytosine and one adenine residue) sequences of approximately 450 base pairs in length. To gain insights into trans-acting factors at telomeres, high-copy-number linear and circular plasmids containing tracts of C1-3A sequences were introduced into S. cerevisiae. We devised a novel system to distinguish by color colonies that maintained the vector at 1 to 5, 20 to 50, and 100 to 400 copies per cell and used it to change the amount of telomeric DNA sequences per cell. An increase in the number of C1-3A sequences caused an increase in the length of telomeric C1-3A repeats that was proportional to plasmid copy number. Our data suggest that telomere growth is inhibited by a limiting factor(s) that specifically recognizes C1-3A sequences and that this factor can be effectively competed for by long tracts of C1-3A sequences at telomeres or on circular plasmids. Telomeres without this factor are exposed to processes that serve to lengthen chromosome ends.

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Characterisation of a repetitive DNA family from Entamoeba histolytica containing Saccharomyces cerevisiae ARS consensus sequences

Gene ◽

10.1016/0378-1119(90)90253-n ◽

1990 ◽

Vol 96 (2) ◽

pp. 197-203 ◽

Cited By ~ 14

Author(s):

Anuradha Lohia ◽

N. Haider ◽

B.B. Biswas

Keyword(s):

Saccharomyces Cerevisiae ◽

Entamoeba Histolytica ◽

Repetitive Dna ◽

Consensus Sequences

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Identification of a mouse protein whose homolog in Saccharomyces cerevisiae is a component of the CCR4 transcriptional regulatory complex.

Molecular and Cellular Biology ◽

10.1128/mcb.15.7.3487 ◽

1995 ◽

Vol 15 (7) ◽

pp. 3487-3495 ◽

Cited By ~ 78

Author(s):

M P Draper ◽

C Salvadore ◽

C L Denis

Keyword(s):

Saccharomyces Cerevisiae ◽

Yeast Cells ◽

Significant Similarity ◽

Eukaryotic Transcription ◽

C Elegans ◽

Mouse Protein ◽

Transcriptional Regulatory ◽

Evolutionarily Conserved ◽

Regulatory Complex

The CCR4 protein from Saccharomyces cerevisiae is a component of a multisubunit complex that is required for the regulation of a number of genes in yeast cells. We report here the identification of a mouse protein (mCAF1 [mouse CCR4-associated factor 1]) which is capable of interacting with and binding to the yeast CCR4 protein. The mCAF1 protein was shown to have significant similarity to proteins from humans, Caenorhabditis elegans, Arabidopsis thaliana, and S. cerevisiae. The yeast gene (yCAF1) had been previously cloned as the POP2 gene, which is required for expression of several genes. Both yCAF1 (POP2) and the C. elegans homolog of CAF1 were shown to genetically interact with CCR4 in vivo, and yCAF1 (POP2) physically associated with CCR4. Disruption of the CAF1 (POP2) gene in yeast cells gave phenotypes and defects in transcription similar to those observed with disruptions of CCR4, including the ability to suppress spt10-enhanced ADH2 expression. In addition, yCAF1 (POP2) when fused to LexA was capable of activating transcription. mCAF1 could also activate transcription when fused to LexA and could functionally substitute for yCAF1 in allowing ADH2 expression in an spt10 mutant background. These data imply that CAF1 is a component of the CCR4 protein complex and that this complex has retained evolutionarily conserved functions important to eukaryotic transcription.

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Shuttle vectors for Candida albicans : control of plasmid copy number and elevated expression of cloned genes

Current Genetics ◽

10.1007/s00294-004-0499-3 ◽

2004 ◽

Vol 45 (6) ◽

pp. 390-398 ◽

Cited By ~ 19

Author(s):

Wenjin Du ◽

Melisa Coaker ◽

Jack D. Sobel ◽

Robert A. Akins

Keyword(s):

Candida Albicans ◽

Copy Number ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Shuttle Vectors ◽

Elevated Expression

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Enhancer and promoter elements from simian virus 40 and polyomavirus can substitute for an upstream activation sequence in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.10.3.947 ◽

1990 ◽

Vol 10 (3) ◽

pp. 947-957 ◽

Cited By ~ 4

Author(s):

N J Axelrod ◽

G G Carmichael ◽

P J Farabaugh

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Sequences ◽

Evolutionary Relationship ◽

Simian Virus 40 ◽

Regulatory Elements ◽

Simian Virus ◽

Yeast Cells ◽

Upstream Activation Sequence ◽

Beta Galactosidase ◽

Activation Sequence

Ten fragments of higher eucaryotic DNA were tested for upstream activation sequence activity in Saccharomyces cerevisiae by inserting them upstream of a CYC1::lacZ promoter lacking an upstream activation sequence. Fragments containing the 21-base-pair repeat region, the enhancer of simian virus 40 or both strongly stimulated beta-galactosidase synthesis, and three fragments from the polyomavirus enhancer region stimulated moderate levels. Three of the four controls of random DNA sequences failed to stimulate significant levels, and the fourth stimulated moderate levels. The stimulation in all cases was independent of the orientation of the inserted fragment. Two series of clones were examined in which between one and six tandemly arranged copies of a fragment were inserted into the XhoI site of the vector. Very interestingly, we detected an apparent exponential relationship between the number of copies of a fragment and the amount of beta-galactosidase produced. Southern analysis showed that increases in enzyme activity were not a result of increased plasmid copy number. Rather, quantitative S1 nuclease analysis demonstrated that the increases were correlated with steady-state levels of lacZ-specific mRNA. We suggest that there may be an evolutionary relationship between some transcriptional activation sequences in yeast cells and the higher eucaryotic regulatory elements that we tested.

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