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W P Wahls

Publications and source records attributed to W P Wahls.

17 recordsLinked to original sources
PubMed1999

High-efficiency gene targeting in Schizosaccharomyces pombe using a modular, PCR-based approach with long tracts of flanking homology.

Bähler et al.(1998) recently described a PCR-based system for the deletion, tagging and overexpression of endogenous genes in the fission yeast Schizosaccharomyces pombe. A small set of PCR primers can be used to generate gene-targeting substrates from each of several modules that differ in the selectable marker (ura4(+) or kanMX6), the presence or absence of specific epitope tags (HA, Myc, GST or GFP), the position in which the epitopes will be inserted (C- or N-terminal), and the presence or absence of a regulatable promoter (the nmt1 promoter). This is a straightforward and powerful system: nine different genes were C-terminal tagged at an average efficiency of 73%, using primers producing only 60-81 bp of homology. In contrast, when studying three transcriptionally-silent genes (rec8(+), rec10(+) and rec11(+)) we obtained an average homologous integration efficiency of 4% for 12 targeting constructs when using primers that contained 80 bp of homology. By using a PCR-based increase in the amount of flanking homology to >/=250 bp, we obtained homologous integration efficiencies of up to 100%. Thus, loci of S. pombe that are refractory to gene targeting when using short tracts of homology can be readily modified by increasing the extent of homology flanking the targeting modules. This straightforward and cost-effective approach might therefore be the one of choice for the modification of S. pombe loci in general and of targeting-refractory loci in particular.

Chromosome Mapping↗
PubMed1999

Centromere mapping functions for aneuploid meiotic products: Analysis of rec8, rec10 and rec11 mutants of the fission yeast Schizosaccharomyces pombe.

Recent evidence suggests that the position of reciprocal recombination events (crossovers) is important for the segregation of homologous chromosomes during meiosis I and sister chromatids during meiosis II. We developed genetic mapping functions that permit the simultaneous analysis of centromere-proximal crossover recombination and the type of segregation error leading to aneuploidy. The mapping functions were tested in a study of the rec8, rec10, and rec11 mutants of fission yeast. In each mutant we monitored each of the three chromosome pairs. Between 38 and 100% of the chromosome segregation errors in the rec8 mutants were due to meiosis I nondisjunction of homologous chromosomes. The remaining segregation errors were likely the result of precocious separation of sister chromatids, a previously described defect in the rec8 mutants. Between 47 and 100% of segregation errors in the rec10 and rec11 mutants were due to nondisjunction of sister chromatids during meiosis II. In addition, centromere-proximal recombination was reduced as much as 14-fold or more on chromosomes that had experienced nondisjunction. These results demonstrate the utility of the new mapping functions and support models in which sister chromatid cohesion and crossover position are important determinants for proper chromosome segregation in each meiotic division.

Aneuploidy↗
PubMed1999

Meiotic chromosome dynamics dependent upon the rec8(+), rec10(+) and rec11(+) genes of the fission yeast Schizosaccharomyces pombe.

During meiosis homologous chromosomes replicate once, pair, experience recombination, and undergo two rounds of segregation to produce haploid meiotic products. The rec8(+), rec10(+), and rec11(+) genes of the fission yeast Schizosaccharomyces pombe exhibit similar specificities for meiotic recombination and rec8(+) is required for sister chromatid cohesion and homolog pairing. We applied cytological and genetic approaches to identify potential genetic interactions and to gauge the fidelity of meiotic chromosome segregation in the mutants. The rec8(+) gene was epistatic to rec10(+) and to rec11(+), but there was no clear epistatic relationship between rec10(+) and rec11(+). Reciprocal (crossover) recombination in the central regions of all three chromosomes was compromised in the rec mutants, but recombination near the telomeres was nearly normal. Each of the mutants also exhibited a high rate of aberrant segregation for all three chromosomes. The rec8 mutations affected mainly meiosis I segregation. Remarkably, the rec10 and rec11 mutations, which compromised recombination during meiosis I, affected mainly meiosis II segregation. We propose that these genes encode regulators or components of a "meiotic chromatid cohesion" pathway involved in establishing, maintaining, and appropriately releasing meiotic interactions between chromosomes. A model of synergistic interactions between sister chromatid cohesion and crossover position suggests how crossovers and cohesion help ensure the proper segregation of chromosomes in each of the meiotic divisions.

Aneuploidy↗
PubMed1998

Recombination hotspot activity of hypervariable minisatellite DNA requires minisatellite DNA binding proteins.

Hypervariable minisatellite DNA repeats are found at tens of thousands of loci in the mammalian genome. These sequences stimulate homologous recombination in mammalian cells [Cell 60:95-103]. To test the hypothesis that protein-DNA interaction is required for hotspot function in vivo, we determined whether a second protein binding nearby could abolish hotspot activity. Intermolecular recombination between pairs of plasmid substrates was measured in the presence or absence of the cis-acting recombination hotspot and in the presence or absence of the second trans-acting DNA binding protein. Minisatellite DNA had hotspot activity in two cell lines, but lacked hotspot activity in two closely related cell lines expressing a site-specific helicase that bound to DNA adjacent to the hotspot. Suppression of hotspot function occurred for both replicating and non-replicating recombination substrates. These results indicate that hotspot activity in vivo requires site occupancy by minisatellite DNA binding proteins.

Animals↗
PubMed1998

Meiotic recombination hotspots: shaping the genome and insights into hypervariable minisatellite DNA change.

Meiotic homologous recombination serves three principal roles. First, recombination reassorts the linkages between newly-arising alleles to provide genetic diversity upon which natural selection can act. Second, recombination is used to repair certain types of DNA damage to provide a mechanism of genomic homeostasis. Third, with few exceptions homologous recombination is required for the appropriate segregation of homologous chromosomes during meiosis. Recombination rates are elevated near DNA sites called "recombination hotspots." These sites influence the distribution of recombination along chromosomes and the timing of recombination during the life cycle. Recent advances have revealed biochemical steps of hotspot activation and have suggested that hotspots may regulate when and where recombination occurs. Two models for hotspot activation, one in which hotspots act early in the recombination pathway and one in which hotspots act late in the recombination pathway, are presented. The latter model can account for changes at hypervariable minisatellite DNA in metazoan genomes by invoking resolution of Holliday junctions at minisatellite DNA repeats.

Animals↗
PubMed1998

Regulation of the Mts1-Mts2-dependent ade6-M26 meiotic recombination hot spot and developmental decisions by the Spc1 mitogen-activated protein kinase of fission yeast.

The M26 meiotic recombination hot spot in the ade6 gene of Schizosaccharomyces pombe is activated by the heterodimeric M26 binding protein Mts1-Mts2. The individual Mts1 (Atf1, Gad7) and Mts2 (Pcr1) proteins are also transcription factors involved in developmental decisions. We report that the Mts proteins are key effectors of at least two distinct classes of developmental decisions regulated by the mitogen-activated protein (MAP) kinase cascade. The first class (osmoregulation, spore viability, and spore quiescence) requires the Spc1 MAP kinase and the Mts1 protein but does not require the Mts2 protein. The second class (mating, meiosis, and recombination hot spot activation) requires the Spc1 kinase and the Mts1-Mts2 heterodimer. Northern and Western blotting eliminated any significant role for the Spc1 kinase in regulating the expression levels of the Mts proteins. Gel mobility shift experiments indicated that the Mts1-Mts2 heterodimer does not need to be phosphorylated to bind to ade6-M26 DNA in vitro. However, in vivo dimethyl sulfate footprinting demonstrated that protein-DNA interaction within cells is dependent upon the Spc1 MAP kinase, which phosphorylates the Mts1 protein. Thus, the Spc1 kinase helps regulate the effector activities of the Mts1-Mts2 heterodimer in part by modulating its ability to occupy the M26 DNA site in vivo. Meiotic recombination hot spot function is likely the result of DNA conformational changes imparted by binding of the Mts1-Mts2 meiotic transcription factor.

Activating Transcription Factor 1↗
PubMed1997

Transcription factor Mts1/Mts2 (Atf1/Pcr1, Gad7/Pcr1) activates the M26 meiotic recombination hotspot in Schizosaccharomyces pombe.

Homologous recombination hotspots increase the frequency of recombination in nearby DNA. The M26 hotspot in the ade6 gene of Schizosaccharomyces pombe is a meiotic hotspot with a discrete, cis-acting nucleotide sequence (5'-ATGACGT-3') defined by extensive mutagenesis. A heterodimeric M26 DNA binding protein, composed of subunits Mts1 and Mts2, has been identified and purified 40,000-fold. Cloning, disruption, and genetic analyses of the mts genes demonstrate that the Mts1/Mts2 heterodimer is essential for hotspot activity. This provides direct evidence that a specific trans-acting factor, binding to a cis-acting site with a unique nucleotide sequence, is required to activate this meiotic hotspot. Intriguingly, the Mts1/Mts2 protein subunits are identical to the recently described transcription factors Atf1 (Gad7) and Pcr1, which are required for a variety of stress responses. However, we report differential dependence on the Mts proteins for hotspot activation and stress response, suggesting that these proteins are multifunctional and have distinct activities. Furthermore, ade6 mRNA levels are equivalent in hotspot and nonhotspot meioses and do not change in mts mutants, indicating that hotspot activation is not a consequence of elevated transcription levels. These findings suggest an intimate but separable link between the regulation of transcription and meiotic recombination. Other studies have recently shown that the Mts1/Mts2 protein and M26 sites are involved in meiotic recombination elsewhere in the S. pombe genome, suggesting that these factors help regulate the timing and distribution of homologous recombination.

Activating Transcription Factor 1↗
PubMed1997

Region-specific meiotic recombination in Schizosaccharomyces pombe: the rec11 gene.

Mutations in the rec11 gene of Schizosaccharomyces pombe reduce meiotic recombinant frequencies by as much as a factor of 300 on chromosome III but less than a factor of 4 in the intervals tested on chromosomes I and II. To gain insight into the function of this region- (or chromosome-) specific activator of recombination, we have cloned and sequenced the rec11 gene. Meiotic crosses with rec11 disruption mutations placed the rec11 gene 6 cM from ade6 on chromosome III. Transcripts of rec11 accumulated transiently at 2-3 h after induction of melosis in a pat1-114 (Ts) mutant. Reverse transcriptase/polymerase chain reaction (RT-PCR) analysis of these transcripts revealed eight introns. The spliced RNA is predicted to encode a polypeptide of 923 amino acids with only very limited homology to reported proteins. The transient accumulation of rec11 transcripts and the phenotype of rec11 mutations suggest that the novel rec11 gene product acts early in meiosis to activate recombination preferentially on chromosome III.

Base Sequence↗
PubMed1994

A heteromeric protein that binds to a meiotic homologous recombination hot spot: correlation of binding and hot spot activity.

Homologous recombination hot spots are DNA sites that increase the frequency of recombination in their vicinity. The M26 allele of the ade6 gene in Schizosaccharomyces pombe is the first meiotic hot spot with an identified unique nucleotide sequence. We have purified 40,000-fold a heteromeric protein, containing polypeptides Mts1 (70 kD) and Mts2 (28 kD), that binds to the M26 site. Binding in vitro strictly correlates with hot spot activity in vivo for numerous single base pair substitutions in the vicinity of the M26 site, indicating that Mts1/Mts2 activates the M26 site and promotes a rate-limiting step of meiotic recombination. These and other data suggest that homologous recombination may be regulated primarily by discrete DNA sites and proteins that interact with those sites.

Activating Transcription Factor 1↗
PubMed1994

RNA associated with a heterodimeric protein that activates a meiotic homologous recombination hot spot: RL/RT/PCR strategy for cloning any unknown RNA or DNA.

The ade6-M26 mutation in the fission yeast Schizosaccharomyces pombe creates a meiotic homologous recombination hot spot. We have achieved 40,000-fold purification of a heterodimeric DNA-binding protein, Mts1/Mts2, that activates the recombination hot spot. Physical studies suggested the presence of a third subunit. It is demonstrated here that RNA molecules of approximately 210 nucleotides copurified with the heterodimer. To characterize the RNA component, it was necessary to develop a new strategy for cloning of the unknown, low-abundance, partially degraded RNAs that were present in purified Mts1/Mts2 protein preparations. The strategy uses RNA ligase to add DNA oligonucleotide priming sites to the RNA for subsequent reverse transcription and PCR (RNA ligase, reverse transcription-PCR, or RL/RT/PCR). This cloning procedure could be applied to the cloning of any unknown RNA or DNA molecules. Because the cDNA clones obtained from Mts1/Mts2 were largely heterogeneous, it seems likely that the RNAs copurified as a result of tight but nonspecific interactions with the heterodimeric protein.

Activating Transcription Factor 1↗
PubMed1993

Single-stranded DNA binding activity of C1-tetrahydrofolate synthase enzymes.

In eukaryotes C1-5,6,7,8-tetrahydrofolate (THF) synthase is a trifunctional enzyme that catalyzes the interconversion of reduced forms of folate to supply activated one-carbon units required for a variety of metabolic pathways. The enzymatic activities include 10-formyl-THF synthetase (EC 6.3.4.3), 5,10-methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5). In bacteria separate, monofunctional or bifunctional polypeptides catalyze the same reactions. We have purified C1-THF synthase from the fission yeast Schizosaccharomyces pombe and found its physical and enzymatic properties similar to those of other eukaryotic C1-THF synthase enzymes. Unexpectedly, the S. pombe enzyme bound strongly (Keq = 100 pM) to single-stranded DNA, but not to double-stranded DNA or to RNA. The binding was sequence-independent, apparently not cooperative, and not detectably inhibited by C1-THF synthase substrates or cofactors. Trifunctional cytoplasmic enzyme from Saccharomyces cerevisiae and monofunctional (synthetase) enzyme from Clostridium acidiurici also bound tightly to single-stranded DNA, while bifunctional (dehydrogenase and cyclohydrolase) enzyme from Escherichia coli did not, suggesting that single-stranded DNA binding is a conserved function of the synthetase domain of C1-THF synthase enzymes.

Aminohydrolases↗
PubMed1991

Two hypervariable minisatellite DNA binding proteins.

Hypervariable minisatellite DNA sequences are short, tandemly repeated sequences present at numerous loci in eukaryotes. They stimulate intermolecular homologous recombination up to 13-fold in human cells in culture and may be specific sites for the initiation of recombination in the eukaryotic genome (Wahls, W.P., Wallace, L.J., & Moore, P.D. (1990) Cell 60, 95-103). Reported here is the detection and partial purification of two hypervariable minisatellite DNA binding proteins, called Msbp-2 and Msbp-3, present in the nuclear extracts of human HeLa cells. The proteins elute from a gel filtration column with a native mass of 200-250 kDa and have sizes of 77 kDa and 115 kDa respectively.

Base Sequence↗
PubMed1990

Hypervariable minisatellite DNA is a hotspot for homologous recombination in human cells.

Hypervariable minisatellite DNA sequences are short tandemly repeated sequences that are present throughout the human genome and are implicated to enhance recombination. We have constructed a consensus hypervariable minisatellite sequence and analyzed its effect on homologous recombination in human cells in culture. The consensus sequence d(AGAGGTGGGCAGGTGG)6.5 is shown to stimulate homologous recombination up to 13.5-fold. The stimulation occurs at a distance and in both directions but does show a quantitative directionality. Stimulation occurs in a codominant manner, and the sequence is inherited equally in the products. Enhancement is maintained, but at a reduced level, when double-strand breaks are introduced into the substrates. Multiple unselected recombination events are promoted, and preferential stimulation of reciprocal exchange events is demonstrated.

Animals↗
PubMed1990

Relative frequencies of homologous recombination between plasmids introduced into DNA repair-deficient and other mammalian somatic cell lines.

Twelve mammalian somatic cell lines, some of them DNA damage-sensitive mutants paired with their respective wild-type parental lines, were assayed for their ability to catalyze extrachromosomal, intermolecular homologous recombination between pSV2neo plasmid recombination substrates. All of the somatic cell lines analyzed are capable of catalyzing homologous recombination; however, there is a wide range of efficiencies with which they do so. Five human cell lines display a fourfold range of recombination frequencies, and six hamster cell lines vary almost 20-fold. Linearizing one of the recombination substrates stimulates recombination in all but one of the cell lines. Two of the three paired mutant cell lines display a threefold reduction in their ability to catalyze homologous recombination when compared to their respective parental cell lines, indicating that the mutations that render them sensitive to DNA damaging agents might also play a role in homologous recombination.

Agammaglobulinemia↗
PubMed1990

The Z-DNA motif d(TG)30 promotes reception of information during gene conversion events while stimulating homologous recombination in human cells in culture.

Tracts of the alternating dinucleotide polydeoxythymidylic-guanylic [d(TG)].polydeoxyadenylic-cytidylic acid [d(AC)], present throughout the human genome, are capable of readily forming left-handed Z-DNA in vitro. We have analyzed the effects of the Z-DNA motif d(TG)30 upon homologous recombination between two nonreplicating plasmid substrates cotransfected into human cells in culture. In this study, the sequence d(TG)30 is shown to stimulate homologous recombination up to 20-fold. Enhancement is specific to the Z-DNA motif; a control DNA fragment of similar size does not alter the recombination frequency. The stimulation of recombination is observed at a distance (237 to 1,269 base pairs away from the Z-DNA motif) and involves both gene conversion and reciprocal exchange events. Maximum stimulation is observed when the sequence is present in both substrates, but it is capable of stimulating when present in only one substrate. Analysis of recombination products indicates that the Z-DNA motif increases the frequency and alters the distribution of multiple, unselected recombination events. Specifically designed crosses indicate that the substrate containing the Z-DNA motif preferentially acts as the recipient of genetic information during gene conversion events. Models describing how left-handed Z-DNA sequences might promote the initiation of homologous recombination are presented.

Animals↗
PubMed1990

Homologous recombination enhancement conferred by the Z-DNA motif d(TG)30 is abrogated by simian virus 40 T antigen binding to adjacent DNA sequences.

The Z-DNA motif polydeoxythymidylic-guanylic [d(TG)].polydeoxyadenylic-cytidylic acid [d(AC)], present throughout eucaryotic genomes, is capable of readily forming left-handed Z-DNA in vitro and has been shown to promote homologous recombination. The effects of simian virus 40 T-antigen-dependent substrate replication upon the stimulation of recombination conferred by the Z-DNA motif d(TG)30 were analyzed. Presence of d(TG)30 adjacent to a T-antigen-binding site I can stimulate homologous recombination between nonreplicating plasmids, providing that T antigen is absent, in both simian CV-1 cells and human EJ cells (W. P. Wahls, L. J. Wallace, and P. D. Moore, Mol. Cell. Biol. 10:785-793). It has also been shown elsewhere that the presence of d(TG)n not adjacent to the T-antigen-binding site can stimulate homologous recombination in simian virus 40 molecules replicating in the presence of T antigen (P. Bullock, J. Miller, and M. Botchan, Mol. Cell. Biol. 6:3948-3953, 1986). However, it is demonstrated here that d(TG)30 nine base pairs distant from a T-antigen-binding site bound with T antigen does not stimulate recombination between either replicating or nonreplicating substrates in somatic cells. The bound T antigen either prevents the d(TG)30 sequence from acquiring a recombinogenic configuration (such as left-handed Z-DNA), or it prevents the interaction of recombinase proteins with the sequence by stearic hindrance.

Animals↗