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Biomedical subjects

R E Malone

Publications and source records attributed to R E Malone.

At least 19 recordsLinked to original sources
PubMed1992

A meiotic gene conversion gradient opposite to the direction of transcription.

Genetic recombination involves classical crossing-over and gene conversion (aberrant segregation). In fungi that produce an ascus containing four spores, a gene conversion event is manifested as 3:1 or 1:3 (or more rarely 4:0 or 0:4) segregations, in contrast to the normal mendelian 2:2 segregation. Polarity is one of the properties of gene conversion; in almost all cases the frequency of conversion exhibits a gradient across the gene monitored. The frequency of conversion is usually independent of the specific allele used as a marker, but dependent on its location. An interpretation of conversion polarity is that it is caused by the existence of specific initiation sites for meiotic recombination, located at the high end of the polarity gradient. Here we show that the polarity gradient for the HIS2 gene of Saccharomyces cerevisiae is high at the 3' end of the gene, implying that the promoter of HIS2 is not the initiation site.

Alleles↗
PubMed1992

Construction of a human chromosome 4 YAC pool and analysis of artificial chromosome stability.

In order to construct a human chromosome 4-specific YAC library, we have utilized pYAC4 and a mouse/human hybrid cell line HA(4)A in which the only human chromosome present is chromosome 4. From this cell line, approximately 8Mb of chromosome 4 have been cloned. The library includes 65 human-specific clones that range in size from 30kb to 290kb, the average size being 108kb. In order to optimize the manipulation of YAC libraries, we have begun to investigate the stability of YACs containing human DNA in yeast cells; these studies will also determine if there are intrinsic differences in the properties of chromosomes containing higher eukaryotic DNAs. We are examining two kinds of stability: 1] mitotic stability, the ability of the YAC to replicate and segregate properly during mitosis, and 2] structural stability, the tendency of the YAC to rearrange. We have found that the majority of YACs examined are one to two orders of magnitude less stable than authentic yeast chromosomes. Interestingly, the largest YAC analyzed displayed a loss rate typical for natural yeast chromosomes. Our results also suggest that increasing the length of an artificial chromosome improves its mitotic stability. One YAC that showed a very high frequency of rearrangement by mitotic recombination proved to be a mouse/human chimera. In contrast to studies using total human DNA, the frequency of chimeras (i.e., mouse/human) in the YAC pool appeared to be low.

Blotting, Southern↗
PubMed1992

Characterization of REC104, a gene required for early meiotic recombination in the yeast Saccharomyces cerevisiae.

The REC104 gene was initially defined by mutations that rescued the inviability of a rad52 spo 13 haploid strain in meiosis. We have observed that rec104 mutant strains undergo essentially no induction of meiotic gene conversion, and we have not been able to detect any meiotic crossing over in such strains. The REC104 gene has no apparent role in mitosis, since mutations have no observable effect on growth, mitotic recombination, or DNA repair. The DNA sequence of REC104 reveals that it is a previously unknown gene with a coding region of 549-bp, and genetic mapping has localized the gene to chromosome VIII near FUR1. Expression of the REC104 gene is induced in meiosis, and it appears that the gene is not transcribed in mitotic cells. Possible roles for the REC104 gene product in meiosis are discussed.

Amino Acid Sequence↗
PubMed1992

Analysis of the spectrum of mutations induced by the rad3-102 mutator allele of yeast.

The product of the RAD3 gene of Saccharomyces cerevisiae is required for mitotic cell viability and excision repair of UV-induced pyrimidine dimers. Certain rad3 mutant alleles (originally called rem1) increase the rates of both spontaneous mitotic recombination and mutation. The increase in mutation rates is not dependent upon the presence of the RAD6 error-prone pathway. The mutator phenotype suggests that the wild-type RAD3 gene product may be involved in the maintenance of fidelity of DNA replication in addition to its known role in excision repair. To investigate the role that RAD3 might play in mutation avoidance, we have utilized a well-characterized shuttle vector system to study the mutational spectrum occurring in rad3-102 strains and compare it to that seen in RAD3 strains. The results put constraints on the role that the rad-102 mutant gene product must play if the RAD3 protein is a component of the replication complex. Alternatively, the mutational spectrum is consistent with the hypothesis that the rad3-102 mutant protein interferes with postreplication mismatch repair.

Adenosine Triphosphatases↗
PubMed1992

Molecular and genetic analysis of the yeast early meiotic recombination genes REC102 and REC107/MER2.

By selecting for mutations which could rescue the meiotic lethality of a rad52 spo13 strain, we isolated several new Rec genes required relatively early in the meiotic recombination process. This paper presents data to confirm that two of them, REC102 and REC107, are general, meiosis-specific recombination genes that have no detectable role during mitosis. Sequence analysis and genetic complementation indicate that REC107 is identical to the MER2 gene. No sequences related to REC102 have been found in the GenBank or EMBL collections. REC102 is expressed only in meiosis, prior to the reductional division, at about the time that genetic recombination occurs. Examination of the REC102 sequence indicates the presence of several sequences which may play a role in the regulation of its expression; however, the URS1 sequence commonly found in genes expressed early in meiosis is not present.

Amino Acid Sequence↗
PubMed1991

Isolation of mutants defective in early steps of meiotic recombination in the yeast Saccharomyces cerevisiae.

Using a selection based upon the ability of early Rec- mutations (e.g., rad50) to rescue the meiotic lethality of a rad52 spo13 strain, we have isolated 177 mutants. Analysis of 56 of these has generated alleles of the known Rec genes SPO11, ME14 and MER1, as well as defining five new genes: REC102, REC104, REC107, REC113 and REC114. Mutations in all of the new genes appear to specifically affect meiosis; they do not have any detectable mitotic phenotype. Mutations in REC102, REC104 and REC107 reduce meiotic recombination several hundred fold. No alleles of RED1 or HOP1 were isolated, consistent with the proposal that these genes may be primarily involved with chromosome pairing and not exchange.

Alleles↗
PubMed1990

Dual regulation of meiosis in yeast.

The two regulatory pathways appear to come together at the IME1 gene. It is clearly regulated by mating type and induced by starvation as well. Overexpression of IME1 completely overcomes MAT defects but may not circumvent all nutritional control. Kassir et al. (1988) found that overexpression of IME1 allowed sporulation in the presence of glucose and nitrogen. They also have found a meiotic level of message in temperature-sensitive cdc25 diploids shifted to high temperature in rich medium (Simchen and Kassir, 1989). Smith and Mitchell (1989) found that overexpression of IME1 induced an early meiotic event (recombination) in rich medium, but later meiotic events did not occur (i.e., they detected no spore formation). Mitchell (personal communication) has suggested that the difference may be due to differences in the amount of nitrogen present in the two experiments. Thus, while it is clear that IME1 is a necessary positive regulator of meiosis, responding both to mating type and nutritional conditions, it is not clear if it is sufficient. It is possible that other genes are involved in the response to starvation. One interpretation is that a separate nutritional control is exerted for events starting with meiosis I. Much of the regulatory pathway that allows yeast cells to enter meiosis has been determined. As in the case in many sensory transduction pathways, the initial signal for starvation is not yet known, nor is the nature of the proposed downstream phosphorylated effector. Given the power of yeast molecular genetics, answers to both these questions seem attainable. Another area that remains unclear is the difference between responses to nitrogen starvation versus carbon source. Many of the experiments discussed above do not address this question. The strategies used by yeast may be utilized in the developmental decisions used by other, more complex eukaryotes. Certainly several of the gene products involved in nutritional control in yeast have homologies in mammalian systems. For example, the human H-ras gene can substitute for yeast RAS; the relationship is sufficiently close that dominant Ha-ras mutations that inhibit CDC25 have been found (Powers et al., 1989). Furthermore, these dominant Ha-ras mutations have the appropriate phenotype in mammalian cells, suggesting the presence of a CDC25-like protein. Although the major components of mating type control appear to have been defined, the mechanism of the RME1-IME transcriptional control remains to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)

Crosses, Genetic↗
PubMed1990

The RAD50 gene, a member of the double strand break repair epistasis group, is not required for spontaneous mitotic recombination in yeast.

Mutations in the RAD50 gene of Saccharomyces cerevisiae have been shown to reduce double strand break repair, meiotic recombination, and radiation-inducible mitotic recombination. Several different point mutations (including ochre and amber alleles) have been previously examined for effects on spontaneous mitotic recombination and did not reduce the frequency of recombination. Instead, the rad50 mutations conferred a moderate hyper-rec phenotype. This paper examines a deletion/interruption allele of RAD50 that removes 998 of 1312 amino acids and adds 1.1 kb of foreign DNA. The results clearly indicate that spontaneous mitotic recombination can occur in the absence of RAD50; in fact, the frequency of recombination is elevated over the wild-type cell. One possible interpretation of these observations is that the initiating lesion in spontaneous recombination events in mitosis might not be a double strand break.

Alleles↗
PubMed1988

A reexamination of the role of the RAD52 gene in spontaneous mitotic recombination.

The RAD52 gene is required for much of the recombination that occurs in Saccharomyces cerevisiae. One of the two commonly utilized mutant alleles, rad52-2, increases rather than reduces mitotic recombination, yet in other respects appears to be a typical rad52 mutant allele. This raises the question as to whether RAD52 is really necessary for mitotic recombination. Analysis of a deletion/insertion allele created in vitro indicates that the null mutant phenotype is indeed a deficiency in mitotic recombination, especially in gene conversion. The data also indicate that RAD52 is required for crossing-over between at least some chromosomes. Finally, examination of the behavior of a replicating plasmid in rad52-1 strains indicates that the frequency of plasmid integration is substantially reduced from that in wild type, a conclusion consistent with a role for RAD52 in reciprocal crossing-over. Analysis of recombinants arising in rad52-2 strains suggests that this allele may result in the increased activity of a RAD52-independent recombinational pathway.

Alleles↗
PubMed1988

Spontaneous mitotic recombination in yeast: the hyper-recombinational rem1 mutations are alleles of the RAD3 gene.

The RAD3 gene of Saccharomyces cerevisiae is required for UV excision-repair and is essential for cell viability. We have identified the rem1 mutations (enhanced spontaneous mitotic recombination and mutation) of Saccharomyces cerevisiae as alleles of RAD3 by genetic mapping, complementation with the cloned wild-type gene, and DNA hybridization. The high levels of spontaneous mitotic gene conversion, crossing over, and mutation conferred upon cells by the rem1 mutations are distinct from the effects of all other alleles of RAD3. We present preliminary data on the localization of the rem1 mutations within the RAD3 gene. The interaction of the rem1 mutant alleles with a number of radiation-sensitive mutations is also different than the interactions reported for previously described (UV-sensitive) alleles of RAD3. Double mutants of rem1 and a defect in the recombination-repair pathway are inviable, while double mutants containing UV-sensitive alleles of RAD3 are viable. The data presented here demonstrate that: (1) rem1 strains containing additional mutations in other excision-repair genes do not exhibit elevated gene conversion; (2) triple mutants containing rem1 and mutations in both excision-repair and recombination-repair are viable; (3) such triple mutants containing rad52 have reduced levels of gene conversion but wild-type frequencies of crossing over. We have interpreted these observations in a model to explain the effects of rem1. Consistent with the predictions of the model, we find that the size of DNA from rem1 strains, as measured by neutral sucrose gradients, is smaller than wild type.

DNA Repair↗
PubMed1987

Hyper-mutation caused by the reml mutation in yeast is not dependent on error-prone or excision repair.

The reml mutations of Saccharomyces cerevisiae confer a semi-dominant hyper-recombination/hyper-mutation phenotype. Neither reml mutant allele has any apparent meiotic affect. We have examined spontaneous mutation in reml-2 strains and demonstrate that the reml-2 mutation, like reml-1, confers an average 10-fold increase in reversion and forward mutation rates. Unlike certain yeast rad mutations with phenotypes similar to reml, strains containing reml are resistant to MMS and only slightly UV sensitive at very high doses. To understand the mutator phenotype of reml, we have used a double-mutant approach, combining the reml mutation with radiation-sensitive mutations affecting DNA repair. Double mutants of reml-2 and a mutation in the yeast error-prone repair group (rad6-1) or a mutation in excision repair (rad1-2 or rad4) maintain the hyper-mutation phenotype. Since mutation rates remain elevated in these double-mutant strains, it appears as if the mutations which occur in the presence of reml resemble spontaneous mutation since they do not require the action of a repair system.

Alleles↗
PubMed1986

Meiosis can induce recombination in rad52 mutants of Saccharomyces cerevisiae.

The RAD52 and RAD50 genes have previously been shown to be required for normal meiotic recombination and for various types of recombination occurring in mitotic cells. Recent evidence suggests that rad52 mutants might be defective in an intermediate recombination step; we therefore examined recombination during meiosis in several rad52 mutants at several different loci and in genetic backgrounds that yield efficient sporulation and synchronous meiosis. Similar to previous reports, spores from rad52 diploids are inviable and meiotic recombination is greatly reduced by rad52 mutations. However, intragenic recombinants were detected when cells were plated on selective media during meiosis; rad52 mutants experience induction of recombination between homologues under these special conditions. The frequencies of recombination at four loci were considerably greater than the mitotic controls; however, they were still at least 20 times lower than corresponding Rad+ strains. The prototrophs induced by meiosis in rad52 mutants were not typical meiotic recombinants because incubation in nutrient-rich medium before plating to selective medium resulted in the complete loss of recombinants. We propose that previously observed single-strand breaks that accumulate in rad52 mutants may be associated with recombinational intermediates that are resolved when cells are returned to selective mitotic media and that the meiosis-induced recombination in rad52 cells does not involve double-strand breaks.

DNA Transposable Elements↗
PubMed1986

Excision repair functions in Saccharomyces cerevisiae recognize and repair methylation of adenine by the Escherichia coli dam gene.

Unlike the DNA of higher eucaryotes, the DNA of Saccharomyces cerevisiae (bakers' yeast) is not methylated. Introduction of the Escherichia coli dam gene into yeast cells results in methylation of the N-6 position of adenine. The UV excision repair system of yeast cells specifically responds to the methylation, suggesting that it is capable of recognizing modifications which do not lead to major helix distortion. The UV repair functions examined in this report are involved in the incision step of pyrimidine dimer repair. These observations may have relevance to the rearrangements and recombination events observed when yeast or higher eucaryotic cells are transformed or transfected with DNA grown in E. coli.

Adenine↗
PubMed1979

Chi.

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Alleles↗