A plasmid model to study genetic recombination in yeast

Embretson, Janet E.; Livingston, Dennis

doi:10.1016/0378-1119(84)90058-1

Cited by 16 publications

(9 citation statements)

References 18 publications

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“…Relationship of reciprocal recombinations. As we reported previously (1,4), some of the plasmid conversion events are accompanied by reciprocal recombination. In addition, a few of the exchange events occur by crossovers between the heteroallelic markers.…”

Section: Discussionsupporting

confidence: 70%

“…Current models of exchange postulate that homology is required to synapse two homologous sequences by the formation of heteroduplex DNA in which a double helix is formed from complementary strands of the participating homologs (7,17,27,30). Examination of exchange between sequences of limited homology should define the absolute length of DNA needed to effect gene conversion and to find whether homology must exist on both sides of a marker for it to be included in the conversion event. Previously (1,4) we have constructed plasmids which undergo intramolecular gene conversion and crossing over in S. cerevisiae between heteroallelic copies of the HIS3 gene * Corresponding author.…”

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confidence: 99%

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

Ahn¹,

Dornfeld²,

Fagrelius³

et al. 1988

Mol. Cell. Biol.

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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-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.Numerous examples of genetic exchange between repeated sequences along individual chromosomes or between such elements present on different chromosomes have been observed in eucaryotic organisms. Many of the observed exchanges have been gene conversions in which a sequence of the donor replaces the homologous sequence of the recipient without concomitant change in the donor (12). In Saccharomyces cerevisiae, for which detailed studies have been possible, some of the events include crossing over concomitant with the conversion event (8,9,14), whereas others are mostly limited to conversion alone (5, 6,11,24). These exchanges are effected by the homology which exists between the repeated sequences. Although studies of homology requirements for exchange have been made with Escherichia coli (21,23,29) and mammalian cells (3,15,20), no systematic study has been made with S. cerevisiae to determine the requirements for homology in effecting exchange, particularly conversion, between elements of limited homology.Because exchange of this nature is thought to mimic the normal chromosomal exchange which occurs between DNA molecules of lengthy homology, exchange between elements of limited homology offers an opportunity to examine fundamental questions which relate to the basic mechanism of exchange. Current models of exchange postulate that homology is required to synapse two homologous sequences by the formation of heteroduplex DNA in which a double helix is formed from complementary strands of the participating homologs (7,17,27,30). Examination of exchange between sequences of limited homology should define the absolute length of DNA ne...

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Section: Discussionsupporting

confidence: 70%

mentioning

confidence: 99%

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

Ahn¹,

Dornfeld²,

Fagrelius³

et al. 1988

Mol. Cell. Biol.

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“…E. coli JM101 has the genotype D(lac-proAB) supE thi (F' traD36 proAB+ lacIqDM15) (36). E. coli RR1 has the genotype F-mcrB mrr hsdS20(rB-mB) leuB6 recA hisB ara-14proA2 lacYl galK2 xyl-S mtl-l rpsL20(Smr) supE44 (14). E. coli ECY5058 is strain HB101 (hsd20 recA13 proA2 leuB6 thi-1) (3) carrying pRK2013 (16 (18) or at 30°C in Luria broth (LB; 47).…”

Section: Materiails and Methodsmentioning

confidence: 99%

Cloning, sequencing, and expression of the Pseudomonas putida protocatechuate 3,4-dioxygenase genes

et al. 1993

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The genes that encode the a and 1a subunits of protocatechuate 3,4-dioxygenase (3,4-PCD [EC 1.13.11.31) were cloned from a Pseudomonas putida (formerly P. aenuginosa) (ATCC 23975) genomic library prepared in A phage. Plaques were screened by hybridization with degenerate oligonucleotides designed using known amino acid sequences. A 1.5-kb SnaI fragment from a 15-kb primary clone was subcloned, sequenced, and shown to contain two successive open reading frames, designatedpcaH andpcaG, corresponding to the c and a subunits, respectively, of 3,4-PCD. The amino acid sequences deduced from pcaHG matched the chemically determined sequence of 3,4-PCD in all except three positions. Cloning ofpcaHG into broad-host-range expression vector pKMY319 allowed high levels of expression in P. putida strains, as well as in Proteus mirabilis after specific induction of the plasmid-encoded nahG promoter with salicylate. The recombinant enzyme was purified and crystallized from P. mirabilis, which lacks an endogenous 3,4-PCD. The physical, spectrmscopic, and kinetic properties of the recombinant enzyme were indistinguishable from those of the wild-type enzyme. Moreover, the same transient enzyme intermediates were formed during the catalytic cycle. These studies establish the methodology which will allow mechanistic investigations to be pursued through site-directed mutagenesis of P. putida 3,4-PCD, the only aromatic ring-cleaving dioxygenase for which the three-dimensional stmcture is known.The catabolic pathways for bacterial degradation of aromatic compounds converge, in most instances, on a small group of single-ring aromatic compounds. This group includes protocatechuate, catechol, gentisate, and a few other, similar compounds (9,11,12,22,34). The aromatic ring of these compounds is opened during reactions catalyzed by dioxygenase enzymes in which both atoms of oxygen from 02 are incorporated into the substrate. These enzymes usually contain nonheme iron stabilized in either the Fe(II) or Fe(III) oxidation state (34,37,43,44 (Fig. 1). This pathway catalyzes the conversion of protocatechuate (PCA) to P-ketoadipyl coenzyme A and subsequently to citric acid cycle intermediates.3,4-PCD has been isolated from many widely divergent bacteria (6, 13, 18, 34, 56), and its elaboration has been used as a taxonomic characteristic in classifying bacterial strains (53). All of the well-characterized 3,4-PCDs are composed of equimolar amounts of two nonidentical subunits, termed a and 3, organized as a1 protomers. The number of a3 protomers present in the enzymes from different bacteria is quite variable, with a known range of 3 to 12 (34). Soon after this general quatemary structure was recognized (33, 60), each of the subunits of P. putida 3,4-PCD (classified as P. * Corresponding author. aeruginosa at the time) was chemically sequenced (26,27,29,30). Subsequently, we reported the crystal structure of this enzyme (38). This is the only structure of a mononuclear nonheme iron-containing dioxygenase known. On the basis of this crystal stru...

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“…The rad52 mutant SSL212A is the isogenic deletiondisruption of SSL204A in which RAD52 has been deleted from the HpaII site immediately upstream of the initiating codon to the SphI site 15 bp upstream of the stop codon (ADZUMA, OGAWA and OGAWA 1984) and replaced with LEU2. E. coli strain RR-1 hisB recA has been described previously (EMBRETSON and LIVINGSTON 1984).…”

Section: Methodsmentioning

confidence: 99%

“…If the order of mutations had been reversed, single strand annealing could result in a wild-type repeat copy but annealing would have to cover both mutated sites and mismatch correction would need to favor the wild-type sequence. verted repetitions (EMBRETSON and LIVINGSTON 1984;AHN and LIVINGSTON 1986). We have used these plasmids to examine conversion tract continuity, conversion tract length and associated crossovers which occur during mitotic recombination.…”

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confidence: 99%

Plasmid recombination in a rad52 mutant of Saccharomyces cerevisiae.

Dornfeld

Livingston

1992

Genetics

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Using plasmids capable of undergoing intramolecular recombination, we have compared the rates and the molecular outcomes of recombination events in a wild-type and a rad52 strain of Saccharomyces cerevisiae. The plasmids contain his3 heteroalleles oriented in either an inverted or a direct repeat. Inverted repeat plasmids recombine approximately 20-fold less frequently in the mutant than in the wild-type strain. Most events from both cell types have continuous coconversion tracts extending along one of the homologous segments. Reciprocal exchange occurs in fewer than 30% of events. Direct repeat plasmids recombine at rates comparable to those of inverted repeat plasmids in wild-type cells. Direct repeat conversion tracts are similar to inverted repeat conversion tracts in their continuity and length. Inverted and direct repeat plasmid recombination differ in two respects. First, rad52 does not affect the rate of direct repeat recombination as drastically as the rate of inverted repeat recombination. Second, direct repeat plasmids undergo crossing over more frequently than inverted repeat plasmids. In addition, crossovers constitute a larger fraction of mutant than wild-type direct repeat events. Many crossover events from both cell types are unusual in that the crossover HIS3 allele is within a plasmid containing the parental his3 heteroalleles.

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A plasmid model to study genetic recombination in yeast

Cited by 16 publications

References 18 publications

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

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

Cloning, sequencing, and expression of the Pseudomonas putida protocatechuate 3,4-dioxygenase genes

Plasmid recombination in a rad52 mutant of Saccharomyces cerevisiae.

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