Highly mutable sites for ICR-170-induced frameshift mutations are associated with potential DNA hairpin structures: studies with SUP4 and other Saccharomyces cerevisiae genes

1986 ◽  
Vol 6 (12) ◽  
pp. 4425-4432
Author(s):  
D M Hampsey ◽  
R A Koski ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Base Pair ◽  
Temporal Stability ◽  
Loop Formation ◽  
Induced Mutations ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mutagen Specificity ◽  
Hairpin Structures

The majority of the mutations induced by ICR-170 in both the CYC1 gene (J. F. Ernst et al. Genetics 111:233-241, 1985) and the HIS4 gene (L. Mathison and M. R. Culbertson, Mol. Cell. Biol. 5:2247-2256, 1985) of the yeast Saccharomyces cerevisiae were recently shown to be single G . C base-pair insertions at monotonous runs of two or more G . C base pairs. However, not all sites were equally mutable; in both the CYC1 and HIS4 genes there is a single highly mutable site where a G . C base pair is preferentially inserted at a [sequence in text]. Here we report the ICR-170 mutagen specificity at the SUP4-o tyrosine tRNA gene of yeast. Genetic fine structure analysis and representative DNA sequence determination of ICR-170-induced mutations revealed that there is also a single highly mutable site in SUP4-o and that the mutation is a G . C base-pair insertion at a monotonous run of G . C base pairs. Analysis of DNA sequences encompassing the regions of highly mutable sites for all three genes indicated that the mutable sites are at the bases of potential hairpin structures; this type of structure could not be found at any of the other, less mutable G . C runs in SUP4, CYC1, and HIS4. Based on these results and recent information regarding novel DNA structural conformations, we present a mechanism for ICR-170-induced mutagenesis. (i) ICR-170 preferentially binds to DNA in the beta conformation; factors that increase the temporal stability of this structure, such as adjacent stem-and-loop formation, increase the frequency of ICR-170 binding; (ii) the observed mutagen specificity reflects formation of a preferred ICR-170 intercalative geometry at [sequence in text] sites; (iii) during replication or repair, ICR-170 remains associated with the single-stranded template; (iv) stuttering or strand slippage by the polymerization complex as it encounters the mutagen results in nucleotide duplication; (v) subsequent replication or mismatch repair fixes the insertion into the genome. This mechanism accounts for both the IRC-170 mutagenic specificity and the molecular basis of the highly mutable sites in S. cerevisiae.

scholarly journals Highly mutable sites for ICR-170-induced frameshift mutations are associated with potential DNA hairpin structures: studies with SUP4 and other Saccharomyces cerevisiae genes.

1986 ◽  
Vol 6 (12) ◽  
pp. 4425-4432 ◽  
Author(s):  
D M Hampsey ◽  
R A Koski ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Base Pair ◽  
Temporal Stability ◽  
Loop Formation ◽  
Induced Mutations ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mutagen Specificity ◽  
Hairpin Structures

The majority of the mutations induced by ICR-170 in both the CYC1 gene (J. F. Ernst et al. Genetics 111:233-241, 1985) and the HIS4 gene (L. Mathison and M. R. Culbertson, Mol. Cell. Biol. 5:2247-2256, 1985) of the yeast Saccharomyces cerevisiae were recently shown to be single G . C base-pair insertions at monotonous runs of two or more G . C base pairs. However, not all sites were equally mutable; in both the CYC1 and HIS4 genes there is a single highly mutable site where a G . C base pair is preferentially inserted at a [sequence in text]. Here we report the ICR-170 mutagen specificity at the SUP4-o tyrosine tRNA gene of yeast. Genetic fine structure analysis and representative DNA sequence determination of ICR-170-induced mutations revealed that there is also a single highly mutable site in SUP4-o and that the mutation is a G . C base-pair insertion at a monotonous run of G . C base pairs. Analysis of DNA sequences encompassing the regions of highly mutable sites for all three genes indicated that the mutable sites are at the bases of potential hairpin structures; this type of structure could not be found at any of the other, less mutable G . C runs in SUP4, CYC1, and HIS4. Based on these results and recent information regarding novel DNA structural conformations, we present a mechanism for ICR-170-induced mutagenesis. (i) ICR-170 preferentially binds to DNA in the beta conformation; factors that increase the temporal stability of this structure, such as adjacent stem-and-loop formation, increase the frequency of ICR-170 binding; (ii) the observed mutagen specificity reflects formation of a preferred ICR-170 intercalative geometry at [sequence in text] sites; (iii) during replication or repair, ICR-170 remains associated with the single-stranded template; (iv) stuttering or strand slippage by the polymerization complex as it encounters the mutagen results in nucleotide duplication; (v) subsequent replication or mismatch repair fixes the insertion into the genome. This mechanism accounts for both the IRC-170 mutagenic specificity and the molecular basis of the highly mutable sites in S. cerevisiae.

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.

scholarly journals Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (2) ◽  
pp. 530-538 ◽  
Author(s):  
J McGrew ◽  
B Diehl ◽  
M Fitzgerald-Hayes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Dna Sequences ◽  
Base Pair ◽  
Site Directed Mutagenesis ◽  
Chromosome Stability ◽  
Functional Requirements ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Centromere Function

In this paper we show that a 211-base pair segment of CEN3 DNA is sufficient to confer wild-type centromere function in the yeast Saccharomyces cerevisiae. We used site-directed mutagenesis of the 211-base pair fragment to examine the sequence-specific functional requirements of a conserved 11-base pair segment of centromere DNA, element III (5'-TGATTTATCCGAA-3'). Element III is the most highly conserved of the centromeric DNA sequences, differing by only a single adenine X thymine base pair among the four centromere DNAs sequenced thus far. All of the element III sequences contain specific cytosine X guanine base pairs, including a 5'-CCG-3' arrangement, which we targeted for single cytosine-to-thymine mutations by using sodium bisulfite. The effects of element III mutations on plasmid and chromosome segregation were determined by mitotic stability assays. Conversion of CCG to CTG completely abolished centromere function both in plasmids and in chromosome III, whereas conversion of CCG to TCG decreased plasmid and chromosome stability moderately. The other two guanine X cytosine base pairs in element III could be independently converted to adenine X thymine base pairs without affecting plasmid or chromosome stability. We concluded that while some specific nucleotides within the conserved element III sequence are essential for proper centromere function, other conserved nucleotides can be changed.

Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (2) ◽  
pp. 530-538
Author(s):  
J McGrew ◽  
B Diehl ◽  
M Fitzgerald-Hayes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Dna Sequences ◽  
Base Pair ◽  
Site Directed Mutagenesis ◽  
Chromosome Stability ◽  
Functional Requirements ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Centromere Function

In this paper we show that a 211-base pair segment of CEN3 DNA is sufficient to confer wild-type centromere function in the yeast Saccharomyces cerevisiae. We used site-directed mutagenesis of the 211-base pair fragment to examine the sequence-specific functional requirements of a conserved 11-base pair segment of centromere DNA, element III (5'-TGATTTATCCGAA-3'). Element III is the most highly conserved of the centromeric DNA sequences, differing by only a single adenine X thymine base pair among the four centromere DNAs sequenced thus far. All of the element III sequences contain specific cytosine X guanine base pairs, including a 5'-CCG-3' arrangement, which we targeted for single cytosine-to-thymine mutations by using sodium bisulfite. The effects of element III mutations on plasmid and chromosome segregation were determined by mitotic stability assays. Conversion of CCG to CTG completely abolished centromere function both in plasmids and in chromosome III, whereas conversion of CCG to TCG decreased plasmid and chromosome stability moderately. The other two guanine X cytosine base pairs in element III could be independently converted to adenine X thymine base pairs without affecting plasmid or chromosome stability. We concluded that while some specific nucleotides within the conserved element III sequence are essential for proper centromere function, other conserved nucleotides can be changed.

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 Suppressible and nonsuppressible +1 G-C base pair insertions induced by ICR-170 at the his4 locus in Saccharomyces cerevisiae.

1985 ◽  
Vol 5 (9) ◽  
pp. 2247-2256 ◽  
Author(s):  
L Mathison ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Base Pair ◽  
Sequence Data ◽  
Induced Mutations ◽  
Coding Region ◽  
Base Pairs ◽  
Sequence Recognition ◽  
Frameshift Suppression ◽  
Target Sites

Fifteen independent ICR-170-induced his4 mutations in Saccharomyces cerevisiae were examined by DNA sequence analysis. All of the mutations contained a +1 G-C base pair addition in the HIS4 coding region. Eleven different sites of insertion were identified. Combined with previous DNA sequence data, 21 ICR-170-induced his4 mutations distributed at 16 different sites were analyzed. The insertions were always located in a consecutive run of two or more G-C base pairs, with all base pairs in each run having identical orientation. Long consecutive G-C runs were preferred target sites over short runs. Although some consecutive G-C runs appeared to be preferred target sites over others of identical length, such preference was not due to any particular type of nucleotide pair immediately adjacent to a given target site. In addition, DNA sequence analyses of the his4 mutations provided a basis for examining the mechanism of mRNA sequence recognition by extragenic suppressors of ICR-170-induced mutations. The implications of these results for mechanisms of frameshift suppression are discussed.

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 ROLE OF DNA SEQUENCES IN GENETIC RECOMBINATION IN THE ISO-1-CYTOCHROME c GENE OF YEAST. II. COMPARISON OF MUTANTS ALTERED AT THE SAME AND NEARBY BASE PAIRS

Genetics ◽  
1977 ◽  
Vol 85 (1) ◽  
pp. 1-22
Author(s):  
Carol W Moore ◽  
Fred Sherman
Keyword(s):  
Cytochrome C ◽  
Dna Sequences ◽  
Meiotic Recombination ◽  
Base Pair ◽  
Genetic Recombination ◽  
Initiation Codon ◽  
Recombination Rates ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
X Ray

ABSTRACT X-ray-induced mitotic recombination rates and spontaneous meiotic recombination rates have been determined in two-point crosses of various defined cyc1 mutants of the yeast Saccharomyces cerevisiae. All but one of the 17 cyc1 mutants chosen for this study contained either the addition, deletion or substitution of single base-pairs located within a defined segment of the gene that corresponds to the 11 amino acid residues at the amino terminus of iso-1-cytochrome c; approximately half of these mutants had alterations of the AUG initiation codon, some at the same base pair. Up to 66-fold differences in X-ray-induced recombination rates were observed when the same cyc1 mutant was crossed to cyc1 mutants having different alterations in the AUG initiation codon; over a ten-fold difference was observed in series of homologous crosses involving mutants with different changes at the same base-pair. Recombination rates that were associated with specific cyc1 mutants co-segregated with the particular alleles following meiosis, and comparable recombination patterns were also observed for independently isolated, identical mutations. With the mutants used in this study, the frequencies of meiotic recombination did not differ as markedly, suggesting a dissimilar dependence on specific DNA sequences for these two modes of recombination. These disproportionalities of recombination rates suggest that the nature of the mismatched bases influences the recombination process, but not in a way that can be simply interpreted.

Suppressible and nonsuppressible +1 G-C base pair insertions induced by ICR-170 at the his4 locus in Saccharomyces cerevisiae

1985 ◽  
Vol 5 (9) ◽  
pp. 2247-2256
Author(s):  
L Mathison ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Base Pair ◽  
Sequence Data ◽  
Induced Mutations ◽  
Coding Region ◽  
Base Pairs ◽  
Sequence Recognition ◽  
Frameshift Suppression ◽  
Target Sites

Fifteen independent ICR-170-induced his4 mutations in Saccharomyces cerevisiae were examined by DNA sequence analysis. All of the mutations contained a +1 G-C base pair addition in the HIS4 coding region. Eleven different sites of insertion were identified. Combined with previous DNA sequence data, 21 ICR-170-induced his4 mutations distributed at 16 different sites were analyzed. The insertions were always located in a consecutive run of two or more G-C base pairs, with all base pairs in each run having identical orientation. Long consecutive G-C runs were preferred target sites over short runs. Although some consecutive G-C runs appeared to be preferred target sites over others of identical length, such preference was not due to any particular type of nucleotide pair immediately adjacent to a given target site. In addition, DNA sequence analyses of the his4 mutations provided a basis for examining the mechanism of mRNA sequence recognition by extragenic suppressors of ICR-170-induced mutations. The implications of these results for mechanisms of frameshift suppression are discussed.

Micronuclear DNA sequences from Tetrahymena do not confer mitotic stability on ARS plasmids in Saccharomyces

Genome ◽  
1988 ◽  
Vol 30 (5) ◽  
pp. 690-696 ◽  
Author(s):  
Wendy H. Horsfall ◽  
Ronald E. Pearlman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Copy Number ◽  
Tetrahymena Thermophila ◽  
Assay System ◽  
Mitotic Stability ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genomic Libraries ◽  
Ade2 Gene

Genomic libraries containing micronuclear DNA sequences from Tetrahymena thermophila have been constructed in a vector containing ARS1, SUP11, and ura3 sequences from the yeast Saccharomyces cerevisiae. When transformed into a strain of S. cerevisiae carrying a suppressible ochre mutation in the ade2 gene, viable transformants are obtained only if the transforming plasmid is maintained at a copy number of one or two per cell. Mitotic segregation of the plasmid is easily assessed in a colour assay of transformants. Using this assay system, we showed that micronuclear DNA from Tetrahymena does not contain sequences that confer mitotic stability on yeast ARS-containing plasmids; i.e., sequences that function analogously to yeast centromere sequences. One transformant was analyzed that carries Tetrahymena sequences that maintain the copy number of the ARS plasmid at one or two per cell. However, these sequences do not confer mitotic stability on the transformants and they confer a phenotype in this assay similar to that of the REP3 gene of the yeast 2 μm plasmid.Key words: mitotic stability, centromere, Tetrahymena, Saccharomyces.

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