Effect of limited homology on gene conversion in a Saccharomyces cerevisiae plasmid recombination system

1988 ◽  
Vol 8 (6) ◽  
pp. 2442-2448 ◽  
Author(s):  
B Y Ahn ◽  
K J Dornfeld ◽  
T J Fagrelius ◽  
D M Livingston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Conversion ◽  
Dna Sequences ◽  
Homologous Sequence ◽  
Insertion Mutation ◽  
Base Pairs ◽  
Recombination System ◽  
Plasmid Recombination ◽  
His3 Gene ◽  
Conversion Tract

Plasmids containing heteroallelic copies of the Saccharomyces cerevisiae HIS3 gene undergo intramolecular gene conversion in mitotically dividing S. cerevisiae cells. We have used this plasmid system to determine the minimum amount of homology required for gene conversion, to examine how conversion tract lengths are affected by limited homology, and to analyze the role of flanking DNA sequences on the pattern of exchange. Plasmids with homologous sequences greater than 2 kilobases have mitotic exchange rates as high as 2 x 10(-3) events per cell per generation. As the homology is reduced, the exchange rate decreases dramatically. A plasmid with 26 base pairs (bp) of homology undergoes gene conversion at a rate of approximately 1 x 10(-10) events per cell per generation. These studies have also shown that an 8-bp insertion mutation 13 bp from a border between homologous and nonhomologous sequences undergoes conversion, but that a similar 8-bp insertion 5 bp from a border does not. Examination of independent conversion events which occurred in plasmids with heteroallelic copies of the HIS3 gene shows that markers within 280 bp of a border between homologous and nonhomologous sequences undergo conversion less frequently than the same markers within a more extensive homologous sequence. Thus, proximity to a border between homologous and nonhomologous sequences shortens the conversion tract length.

scholarly journals Effect of limited homology on gene conversion in a Saccharomyces cerevisiae plasmid recombination system.

1988 ◽  
Vol 8 (6) ◽  
pp. 2442-2448 ◽  
Author(s):  
B Y Ahn ◽  
K J Dornfeld ◽  
T J Fagrelius ◽  
D M Livingston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Conversion ◽  
Dna Sequences ◽  
Homologous Sequence ◽  
Insertion Mutation ◽  
Base Pairs ◽  
Recombination System ◽  
Plasmid Recombination ◽  
His3 Gene ◽  
Conversion Tract

Plasmids containing heteroallelic copies of the Saccharomyces cerevisiae HIS3 gene undergo intramolecular gene conversion in mitotically dividing S. cerevisiae cells. We have used this plasmid system to determine the minimum amount of homology required for gene conversion, to examine how conversion tract lengths are affected by limited homology, and to analyze the role of flanking DNA sequences on the pattern of exchange. Plasmids with homologous sequences greater than 2 kilobases have mitotic exchange rates as high as 2 x 10(-3) events per cell per generation. As the homology is reduced, the exchange rate decreases dramatically. A plasmid with 26 base pairs (bp) of homology undergoes gene conversion at a rate of approximately 1 x 10(-10) events per cell per generation. These studies have also shown that an 8-bp insertion mutation 13 bp from a border between homologous and nonhomologous sequences undergoes conversion, but that a similar 8-bp insertion 5 bp from a border does not. Examination of independent conversion events which occurred in plasmids with heteroallelic copies of the HIS3 gene shows that markers within 280 bp of a border between homologous and nonhomologous sequences undergo conversion less frequently than the same markers within a more extensive homologous sequence. Thus, proximity to a border between homologous and nonhomologous sequences shortens the conversion tract length.

scholarly journals Mitotic gene conversion lengths, coconversion patterns, and the incidence of reciprocal recombination in a Saccharomyces cerevisiae plasmid system.

1986 ◽  
Vol 6 (11) ◽  
pp. 3685-3693 ◽  
Author(s):  
B Y Ahn ◽  
D M Livingston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Conversion ◽  
Average Length ◽  
Reciprocal Recombination ◽  
Allelic Sequence ◽  
Flanking Sequences ◽  
Mitotic Gene Conversion ◽  
His3 Gene ◽  
Conversion Tract

Plasmids capable of undergoing genetic exchange in mitotically dividing Saccharomyces cerevisiae cells were used to measure the length of gene conversion events, to determine patterns of coconversion when multiple markers were present, and to correlate the incidence of reciprocal recombination with the length of conversion tracts. To construct such plasmids, restriction site linkers were inserted both within the HIS3 gene and in the flanking sequences, and two different his3- alleles were placed in a vector. Characterization of the genetic exchanges in these plasmids showed that most occur with the conversion of one his3- allele. Many of these events included coconversions in which more than one marker along the allelic sequence was replaced. The frequency of coconversion decreased with the distance between two markers such that markers further than 1 kilobase apart were infrequently coconverted. From these results the average length of conversion was determined to be approximately 0.5 kilobase. Examination of coconversions involving three or more markers revealed an almost obligatory, simultaneous coconversion pattern of all markers. Thus, when two markers which flank an intervening marker are converted, the intervening marker is 20 times more likely to be converted than to remain unchanged. The results of these studies also showed that the incidence of reciprocal recombination, which accompanies more than 20% of the conversion events, is more frequent when the conversion tract is longer than average.

Mitotic gene conversion lengths, coconversion patterns, and the incidence of reciprocal recombination in a Saccharomyces cerevisiae plasmid system

1986 ◽  
Vol 6 (11) ◽  
pp. 3685-3693
Author(s):  
B Y Ahn ◽  
D M Livingston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Conversion ◽  
Average Length ◽  
Reciprocal Recombination ◽  
Allelic Sequence ◽  
Flanking Sequences ◽  
Mitotic Gene Conversion ◽  
His3 Gene ◽  
Conversion Tract

Plasmids capable of undergoing genetic exchange in mitotically dividing Saccharomyces cerevisiae cells were used to measure the length of gene conversion events, to determine patterns of coconversion when multiple markers were present, and to correlate the incidence of reciprocal recombination with the length of conversion tracts. To construct such plasmids, restriction site linkers were inserted both within the HIS3 gene and in the flanking sequences, and two different his3- alleles were placed in a vector. Characterization of the genetic exchanges in these plasmids showed that most occur with the conversion of one his3- allele. Many of these events included coconversions in which more than one marker along the allelic sequence was replaced. The frequency of coconversion decreased with the distance between two markers such that markers further than 1 kilobase apart were infrequently coconverted. From these results the average length of conversion was determined to be approximately 0.5 kilobase. Examination of coconversions involving three or more markers revealed an almost obligatory, simultaneous coconversion pattern of all markers. Thus, when two markers which flank an intervening marker are converted, the intervening marker is 20 times more likely to be converted than to remain unchanged. The results of these studies also showed that the incidence of reciprocal recombination, which accompanies more than 20% of the conversion events, is more frequent when the conversion tract is longer than average.

scholarly journals DNA SEQUENCES OF FRAMESHIFT AND OTHER MUTATIONS INDUCED BY ICR-170 IN YEAST

Genetics ◽  
1985 ◽  
Vol 111 (2) ◽  
pp. 233-241
Author(s):  
Joachim F Ernst ◽  
D Michael Hampsey ◽  
Fred Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Genetic Analysis ◽  
Dna Sequences ◽  
Induced Mutations ◽  
Sequence Analyses ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Frameshift Mutations

ABSTRACT ICR-170-induced mutations in the CYC1 gene of the yeast Saccharomyces cerevisiae were investigated by genetic and DNA sequence analyses. Genetic analysis of 33 cyc1 mutations induced by ICR-170 and sequence analysis of eight representatives demonstrated that over one-third were frameshift mutations that occurred at one site corresponding to amino acid positions 29-30, whereas the remaining mutations were distributed more-or-less randomly, and a few of these were not frameshift mutations. The sequence results indicate that ICR-170 primarily induces G·C additions at sites containing monotonous runs of three G·C base pairs. However, some (see PDF) sites within the CYC1 gene were not mutated by ICR-170. Thus, ICR-170 is a relatively specific mutagen that preferentially acts on certain sites with monotonous runs of G·C base pairs.

Meiotic recombination between repeated transposable elements in Saccharomyces cerevisiae

1988 ◽  
Vol 8 (7) ◽  
pp. 2942-2954
Author(s):  
M Kupiec ◽  
T D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transposable Elements ◽  
Gene Conversion ◽  
Meiotic Recombination ◽  
Small Region ◽  
Ty Elements ◽  
Ty Element ◽  
Reciprocal Exchange ◽  
Gene Conversions ◽  
Conversion Tract

We have measured the frequency of meiotic recombination between marked Ty elements in the Saccharomyces cerevisiae genome. These recombination events were usually nonreciprocal (gene conversions) and sometimes involved nonhomologous chromosomes. The frequency of ectopic gene conversion among Ty elements appeared lower than expected on the basis of previous studies of recombination between artificially constructed repeats. The conversion events involved either a subset of the total Ty elements in the genome or the conversion tract was restricted to a small region of the Ty element. In addition, the observed conversion events were very infrequently associated with reciprocal exchange.

scholarly journals Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (8) ◽  
pp. 1440-1448 ◽  
Author(s):  
M Johnston ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Base Pair ◽  
Transcription Initiation ◽  
Base Pairs ◽  
Regulation Of Expression ◽  
Notable Feature ◽  
His3 Gene

The GAL1 and GAL10 genes of Saccharomyces cerevisiae are divergently transcribed, with 606 base pairs of DNA separating their transcription initiation sites. These two genes are stringently coregulated: their expression is induced ca. 1,000-fold in cells growing on galactose and is repressed by growth on glucose. The nucleotide sequence of the region of DNA between these genes and the precise sites of transcription initiation are presented here. The most notable feature of the nucleotide sequence of this region is a 108-base-pair guanine-plus-cytosine-rich stretch of DNA located approximately in the middle of the region between GAL1 and GAL10. Analysis of the effects of mutations that alter the region between these two genes, constructed in vitro or selected in vivo, suggest that these guanine-plus-cytosine-rich sequences are required for the expression of both genes. The region of DNA between GAL1 and GAL10 is sufficient for regulation of expression of these genes: fusion of the region to the yeast HIS3 gene places HIS3 under GAL control.

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

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.

scholarly journals Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA.

1982 ◽  
Vol 2 (3) ◽  
pp. 221-232 ◽  
Author(s):  
V A Zakian ◽  
J F Scott
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Sequences ◽  
Recombinant Dna ◽  
Suitable Model ◽  
Chromosomal Dna ◽  
Base Pairs ◽  
Autonomously Replicating Sequence ◽  
Cell Cycles ◽  
Initiation Of Replication

Transformation studies with Saccharomyces cerevisiae (bakers' yeast) have identified DNA sequences which permit extrachromosomal maintenance of recombinant DNA plasmids in transformed cells. It has been hypothesized that such sequences (called ARS for autonomously replicating sequence) serve as initiation sites for DNA replication in recombinant DNA plasmids and that they represent the normal sites for initiation of replication in yeast chromosomal DNA. We have constructed a novel plasmid called TRP1 R1 Circle which consists solely of 1,453 base pairs of yeast chromosomal DNA. TRP1 RI Circle contains both the TRP1 gene and a sequence called ARS1. This plasmid is found in 100 to 200 copies per cell and is relatively stable during both mitotic and meiotic cell cycles. Replication of TRP1 RI Circle requires the products of the same genes (CDC28, CDC4, CDC7, and CDC8) required for replication of chromosomaL DNA. Like chromosomal DNA, its replication does not occur in cells arrested in the B1 phase of the cell cycle by incubation with the yeast pheromone alpha-factor. In addition, TRP1 RI Circle DNA is organized into nucleosomes whose size and spacing are indistinguishable from that of bulk yeast chromatin. These results indicate that TRP1 RI Circle has the replicative and structural properties expected for an origin of replication from yeast chromosomal DNA. Thus, this plasmid is a suitable model for further studies of yeast DNA replication in both cells and cell-free extracts.

scholarly journals Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences

1993 ◽  
Vol 13 (5) ◽  
pp. 2697-2705
Author(s):  
R H Schiestl ◽  
M Dominska ◽  
T D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Topoisomerase I ◽  
Target Sequence ◽  
Base Pairing ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Fragment

When the yeast Saccharomyces cerevisiae was transformed with DNA that shares no homology to the genome, three classes of transformants were obtained. In the most common class, the DNA was inserted as the result of a reaction that appears to require base pairing between the target sequence and the terminal few base pairs of the transforming DNA fragment. In the second class, no such homology was detected, and the transforming DNA was integrated next to a CTT or GTT in the target; it is likely that these integration events were mediated by topoisomerase I. The final class involved the in vivo ligation of transforming DNA with nucleus-localized linear fragments of mitochondrial DNA.

scholarly journals Yeast intrachromosomal recombination: long gene conversion tracts are preferentially associated with reciprocal exchange and require the RAD1 and RAD3 gene products.

Genetics ◽  
1989 ◽  
Vol 123 (4) ◽  
pp. 683-694 ◽  
Author(s):  
A Aguilera ◽  
H L Klein
Keyword(s):  
Gene Conversion ◽  
Excision Repair ◽  
Dna Helicase ◽  
Repair Gene ◽  
Tract Length ◽  
Intrachromosomal Recombination ◽  
Recombination System ◽  
Mitotic Gene Conversion ◽  
Gene Conversion Tract ◽  
Conversion Tract

Abstract A yeast intrachromosomal recombination system based on an inverted repeat has been designed to examine mitotic gene conversion tract length and the association of crossing over with gene conversion as a function of the conversion tract length. Short conversion tracts are found to be preferentially noncrossover while conversion tracts longer than 1.16 kb show a 50% association with crossover. Mutation in the excision repair gene RAD1 leads to a reduction in conversion tracts of at least 1.16 kb and a reduction in crossovers associated with conversion, regardless of the length of the conversion tract. Mutation in the excision repair gene RAD3, which encodes a DNA helicase, also leads to a reduction in conversion tracts of at least 1.16 kb, but has no effect on the frequency of associated crossovers. The roles of RAD1 and RAD3 in recombination are discussed.

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