The genetics of complex diseases.

Genetic factors influence virtually every human disorder, determining disease susceptibility or resistance and interactions with environmental factors. Our recent successes in the genetic mapping and identification of the molecular basis of mendelian traits have been remarkable. Now, attention is rapidly shifting to more-complex, and more-prevalent, genetic disorders and traits that involve multiple genes and environmental effects, such as cardiovascular disease, diabetes, rheumatoid arthritis and schizophrenia. Rather than being due to specific and relatively rare mutations, complex diseases and traits result principally from genetic variation that is relatively common in the general population. Unfortunately, despite extensive efforts by many groups, only a few genetic regions and genes involved in complex diseases have been identified. Completion of the human genome sequence will be a seminal accomplishment, but it will not provide an immediate solution to the genetics of complex traits.

Coincident gene conversion during mitosis in saccharomyces.

During mitosis, gene conversion events at the TRP5 locus on chromosome VII are coupled with conversion events at LEU1, a locus 18 cM away, 1200 times more frequently than would be expected for two independent acts of recombination. Such coincident conversion events that occur over relatively long distances could be due to several mechanisms. We discuss these possibilities and describe an experiment that indicates that a portion of coincident events is due to extensive heteroduplexes. The phenomenon of coincident gene conversion is discussed in relation to our earlier evidence that spontaneous recombination between homologues occurs prereplicationally in mitosis.

The effect of ochre suppression on meiosis and ascospore formation in Saccharomyces.

The effect of altered tyrosyl-tRNAs on the developmental process of sporulation was examined. Mutations in eight independent loci resulting in tyrosine-inserting nonsense suppressor were tested for their effects on sporulation. Different levels of inhibition were found ranging from SUP3-omicron, which caused the greatest reduction of sporulation (7-17% of wild type), to SUP11-omicron which caused no reduction in sporulation. Since the SUP3-omicron mutation exhibited the greatest effect, it was studied in detail. Although SUP3-omicron is a dominant nonsense suppressor, its effect on sporulation is recessive. Expression of the sporulation deficiency is dependent upon the stage of transfer from glucose growth medium (i.e., log, early stationary, etc.) to sporulation medium. SUP3-omicron/SUP3-omicron diploid cells transferred from log or early stationary phase are capable of sporulation, whereas cells transferred after early stationary phase (i.e., after adaptation to respiration) exhibit poor sporulative ability. Sporulation events were examined under restrictive conditions to observe those events completed by SUP3-omicron/SUP3-omicron diploids. The early events of sporulation occur in these cells. Later events are completed by progressively fewer cells. Premeiotic DNA synthesis occurred in approximately 40% of the cells, nuclear segregation occurred in 20%, and finally, only 2% formed asci. The fact that fewer late-sporulation events occur under restrictive conditions can be explained by increased efficiency of suppression.

Molecular mechanisms of recombination in Saccharomyces cerevisiae: testing mitotic and meiotic models by analysis of hypo-rec and hyper-rec mutations.

Recombination in the yeast Saccharomyces cerevisiae has been the subject of extensive genetic studies documenting the general properties of intragenic and intergenic recombination and the differences between mitotic and meiotic gene conversion and reciprocal exchange. Spontaneous mitotic and meiotic events differ in the time of onset of recombination relative to chromosomal replication, symmetry versus asymmetry of putative heteroduplex DNA regions, polarity of conversion of intragenic markers, and the lengths of DNA segments that undergo coincident conversion. The differences observed and the properties of yeast rec mutations provide evidence for multiple modes or pathways of mitotic and meiotic recombination. Several molecular models of recombination have been proposed to account for the basic parameters of genetic recombination and the differences between mitotic and meiotic recombination. Since the models differ with respect to the partial reactions comprising recombination they predict the isolation of different classes of hypo-recombination and hyper-recombination rec mutants. We have isolated a broad spectrum of yeast REC gene mutations that includes both hyper-rec and hypo-rec mutants. Five phenotypic classes of rec variants have been identified based upon their effects on spontaneous mitotic gene conversion and intergenic recombination. Their characteristics demonstrate that mitotic gene conversion and intergenic recombination are under independent as well as coordinate genetic control. Four gene mutations affecting recombination rad50, rad52, rem1 and spo11 have been extensively examined in several laboratories and illustrate the information that can be obtained by characterization of double mutant strains, detailed genotypic analysis of recombinants, and studies of meiotic recombination in cells in which the reductional division of meiosis has been bypassed by the spo13 mutation.

Evidence that spontaneous mitotic recombination occurs at the two-strand stage.

Spontaneous reciprocal mitotic recombination in the yeast Saccharomyces cerevisiae, associated with heteroallelic recombination, occurs almost exclusively at the two-strand stage and involves recombination of unduplicated chromosomes (i.e., during G1) or the unduplicated regions of chromosomes during the S phase of mitosis. The associated heteroallelic recombination frequently reflects the formation of symmetric Holliday structures, is not strongly polarized with respect to conversion at the heteroallelic trp5 sites studied, occasionally results in simultaneous conversion of widely separated genetic markers, and is positively correlated with recombination of flanking markers.

Conditional hyporecombination mutants of three REC genes of Saccharomyces cerevisiae.

We have isolated and characterized three conditional hyporecombination mutants, rec1-1, rec3-1 and rec4-1, that define three REC genes of Saccharomyces cerevisiae required for spontaneous general mitotic interchromosomal recombination. Each MATa/MAT alpha rec/rec diploid is deficient in mitotic single site gene conversion, intragenic recombination, intergenic recombination and sporulation at the restrictive temperature (36 degrees C). The rec1-1 mutation also confers conditional enhanced sensitivity to the killing effects of X-rays. The rec1-1 and rec3-1 mutations have been mapped to chromosome VII. The rec1-1, rec3-1 and rec4-1 mutations exhibit complementation at 36 degrees C for both mitotic recombination and sporulation.

Mitotic recombination: mismatch correction and replicational resolution of Holliday structures formed at the two strand stage in Saccharomyces.

In a preliminary report (Esposito 1978), evidence was presented which showed that heteroallelic recombination resulting in prototrophic colonies occurs at the 2-strand stage. A model utilizing replicative resolution of Holliday structures was proposed to explain how gene conversion at the 2-strand stage can result in exchange of outside markers. The object of the experiments reported herein was to present detailed genetic evidence for 2-strand recombination. In addition, we examined the features of mitotic recombination with respect to symmetry, length and polarity of heteroduplexes in wild type strains (REM1/REM1) and in strains bearing the hyper-recombination mutation rem1-1. To do this, we constructed strains so that prototrophs arising from heteroallelic recombination and recombinant for outside markers were detected by visual inspection. By analyzing these colonies genetically, we have inferred several features of mitotic recombination which distinguish it from its meiotic counterpart. Firstly, mitotic heteroduplexes are often symmetric while meiotic heteroduplexes are almost exclusively asymmetric. Secondly, heteroduplexes tend to be longer in mitosis that in meiosis. Thirdly, unlike meiotic conversion, mitotic conversion does not show strong polarity. Recombination in strains homozygous for the rem1-1 mutation also takes place at the 2-strand stage. The rem1-1 mutation, however, appears to alter the features of mismatch correction.

Enhancement of spontaneous mitotic recombination by the meiotic mutant spo11-1 in Saccharomyces cerevisiae.

Both nonreciprocal and reciprocal mitotic recombination are enhanced by the recessive mutant spo11-1, which was previously shown to affect meiosis by decreasing recombination and increasing nondisjunction. The mitotic effects are not distributed equally in all chromosomal regions. The genotypes of mitotic recombinants in spo11-1/spo11-1 diploid cells provide further evidence that widely spaced chromosomal markers undergo coincident conversion in mitosis.

Genetic recombination and commitment to meiosis in Saccharomyces.

Diploid cells of the yeast Saccharomyces cerevisiae become committed to recombination at meiotic levels without becoming committed to the meiotic disjunction of chromosomes. These two events of the meiotic process can be separated by removing cells from a meiosis-inducing medium and returning them to a medium that promotes vegetative cell division. Cells removed at an appropriate time remain diploid, revert to mitosis, and display recombination with meiotic-like frequencies. Those removed after this time are committed to the completion of meiosis. Diploids of three conditional sporulation-deficient mutants (spo1-1, spo2-1, and spo3-1) have been examined for recombination at restrictive temperatures. All exhibit commitment to recombination without commitment to meiotic disjunction as in the wild type. Cells of spo1-1/spo1-1 do not replicate the spindle pole body for meiosis I; thus, recombination ability can be acquired by cells that do not proceed beyond this cytological stage.

Diploid yeast cells yield homozygous spontaneous mutations.

A leucine-requiring hybrid of Saccharomyces cerevisiae, homoallelic at the LEU1 locus (leu1-12/leu1-12) and heterozygous for three chromosome-VII genetic markers distal to the LEU1 locus, was employed to inquire: (1) whether spontaneous gene mutation and mitotic segregation of heterozygous markers occur in positive nonrandom association and (2) whether homozygous LEU1/LEU1 mutant diploids are generated. The results demonstrate that gene mutation of leu1-12 to LEU1 and mitotic segregation of heterozygous chromosome-VII markers occur in strong positive nonrandom association, suggesting that the stimulatory DNA lesion is both mutagenic and recombinogenic. In addition, genetic analysis of diploid Leu+ revertants revealed that approximately 3% of mutations of leu1-12 to LEU1 result in LEU1/LEU1 homozygotes. Red-white sectored Leu+ colonies exhibit genotypes that implicate post-replicational chromatid breakage and exchange near the site of leu1-12 reversion, chromosome loss, and subsequent restitution of diploidy, in the sequence of events leading to mutational homozygosis. By analogy, diploid cell populations can yield variants homozygous for novel recessive gene mutations at biologically significant rates. Mutational homozygosis may be relevant to both carcinogenesis and the evolution of asexual diploid organisms.

Evidence for joint genic control of spontaneous mutation and genetic recombination during mitosis in Saccharomyces.

A procedure for detection of mutants exhibiting either enhanced or reduced spontaneous mutation during mitosis and/or meiosis has been developed to probe the joint genic control of spontaneous mutation and recombination in yeast. A semidominant mutator, rem1-1, recovered by this technique, exhibits enhanced spontaneous mutation,intragenic recombination, and intergenic recombination during mitosis. Diploids homozygous for rem1-1 exhibit normal levels of meiotic intragenic and intergenic recombination and diminished ascospore viability.

Mitotic versus meiotic recombination in Saccharomyces cerevisiae.

As part of a comparative analysis of spontaneous mitotic and meiotic recombination we have compared the mitotic and meiotic maps of the wild type and yeast hybrids homozygous for reml-l, a mitosis-specific hyper-rec mutation (Golin and Esposito, 1977; Golin, 1979). In wild type yeast strains recombination in centromere proximal intervals occurs relatively more frequently in mitosis than in meiosis. In reml-1/rem1-1 hybrids the distribution of mitotic exchange events is more similar to the distribution observed in meiosis.

Genetic analysis of two spored asci produced by the spo 3 mutant of Saccharomyces.

Ascospores present in two-spored asci formed by sop3-1 diploids at a semipermissive temperature (30 degrees C) represent random inclusion of haploid genomes into ascospores and exhibit normal viability, meiotic chromosome segregation and recombination. Genetic analysis and ultrastructural studies indicate that the function encoded by the sop3 locus is specifically required for the enclosure of the products of meiosis in prospore walls.

Nonrandomly-associated forward mutation and mitotic recombination yield yeast diploids homozygous for recessive mutations.

We have employed the analysis of spontaneous forward mutations that confer the ability to utilize L-alpha-aminoadipate as a nitrogen source (alpha-Aa+) to discern the events that contribute to mitotic segregation of spontaneous recessive mutations by diploid cells. alpha-Aa- diploid cells yield alpha-Aa+ mutants at a rate of 7.8 +/- 3.6 x 10(-9). As in haploid strains, approximately 97% (30/31) of alpha-Aa+ mutants are spontaneous lys2-x recessive mutations. alpha-Aa+ mutants of diploid cells reflect mostly the fate of LYS2/lys2-x heterozygotes that arise by mutation within LYS2/LYS2 populations at a rate of 1.2 +/- 0.4 x 10(-6). Mitotic recombination occurs in nonrandom association with forward mutation of LYS2 at a rate of 1.3 +/- 0.6 x 10(-3). This mitotic recombination rate is tenfold higher than that of a control LYS2/lys2-1 diploid. Mitotic segregation within LYS2/lys2-x subpopulations yields primarily lys2-x/lys2-x diploids and a minority of lys2-x aneuploids. Fifteen percent of lys2-x/lys2-x diploids appear to have arisen by gene conversion of LYS2 to lys2-x; 85% of lys2-x/lys2-x diploids appear to have arisen by mitotic recombination in the CENII-LYS2 interval. lys2-1/lys2-1 mitotic segregants of a control LYS2/lys2-1 diploid consist similarity of 18% of lys2-1/lys2-1 diploids that appear to have arisen by gene conversion of LYS2 to lys2-1 and 82% of lys2-1/lys2-1 diploids that appear to have arisen by mitotic recombination in the CENII-LYS2 interval.(ABSTRACT TRUNCATED AT 250 WORDS)

Antimutator activity during mitosis by a meiotic mutant of yeast.

Diploids homozygous for the mutation spo7-1 do not exhibit net premeiotic DNA synthesis at 34 degrees C and are defective in commitment to recombination following exposure to sporulation medium. The spo7-1 mutation confers antimutator activity during mitosis at 34 degrees C, indicating that the SPO7 gene product is involved in both mitotic and premeiotic DNA metabolism. Strains bearing spo7-1 are slightly more sensitive to killing by ultraviolet light than the wild type but are proficient in UV induced mutation and mitotic intragenic recombination. The mitotic antimutator activity of spo7-1 is directed against a class of forward mutations known to occur more frequently during mitosis than meiosis.

Simultaneous detection of changes in chromosome number, gene conversion and intergenic recombination during mitosis of Saccharomyces cerevisiae: spontaneous and ultraviolet light induced events.

We have employed a hyperhaploid strain of Saccharomyces cerevisiae disomic for chromosome VII to monitor spontaneous and ultraviolet light induced restitution of haploidy (chromosomal loss and/or nondisjunction), mitotic gene conversion and mitotic intergenic recombination. The disomic chromosomal pair incorporates six heterozygous markers, including cyh2 (r), distributed on both sides of the centromere. Cycloheximide resistant segregants of spontaneous origin were analyzed to calculate the spontaneous mitotic rates of restitution of haploidy, intergenic recombination and gene conversion that result in expression of the cyh2 (r) mutation. Restitution of haploidy was found to be the most common source of spontaneously arising cycloheximide resistant segregants. In contrast, those induced by ultraviolet light resulted most frequently from gene conversion of CYH2 (s) to cyh2 (r). The chromosome VII hyperhaploid system provides a sensitive method to detect the aneugenic and recombinagenic effects of suspect chemical and physical agents.

The REC46 gene of Saccharomyces cerevisiae controls mitotic chromosomal stability, recombination and sporulation: cell-type and life cycle stage-specific expression of the rec46-1 mutation.

The recessive hyperrecombination mutation rec46-1, isolated by ultraviolet light mutagenesis of the MAT alpha n+1 chromosome VII disomic strain LBL1 (Esposito et al. 1982), enhances the mitotic rates of spontaneous gene conversion, intergenic recombination and restitution of haploidy (due to chromosomal loss or mitotic nondisjunction) in MAT alpha n+1 chromosome VII disomic strains. The rec46-1 mutation does not prevent HO directed homothallic interconversion of mating types. MATa/MaT alpha rec46-1/rec46-1 diploids exhibit the same degree of hyperrecombinational activity as MAT alpha rec46-1 n+1 chromosome VII disomics with respect to gene conversion and intragenic recombination resulting in prototrophy. When compared to MAT alpha rec46-1 n+1 disomics however, MATa/MAT alpha rec46-1/rec46-1 diploids exhibit a ten fold reduced level of hyperrecombinational activity with respect to intergenic recombination and present no evidence of chromosomal loss or nondisjunction resulting in 2n-1 monosomic segregants. MATa/MAT alpha rec46-1/rec46-1 diploids are sporulation-deficient. The results obtained demonstrate that the REC46 gene product modulates mitotic chromosomal stability and recombination and is essential for sporulation (meiosis and ascospore formation).