Meiotic Recombination between Duplicated Genetic Elements in SACCHAROMYCES CEREVISIAE

We have studied the meiotic recombination behavior of strains carrying two types of duplications of an 18.6-kilobase HIS4 Bam HI fragment. The first type is a direct duplication of the HIS4 Bam HI fragment in which the repeated sequences are separated by Escherichia coli plasmid sequences. The second type, a tandem duplication, has no sequences intervening between the repeated yeast DNA. The HIS4 genes in each region were marked genetically so that recombination events between the duplicated segments could be identified. Meiotic progeny of the strains carrying the duplication were analyzed genetically and biochemically to determine the types of recombination events that had occurred. Analysis of the direct vs. tandem duplication suggests that the E. coli plasmid sequences are recombinogenic in yeast when homozygous. In both types of duplications recombination between the duplicated HIS4 regions occurs at high frequency and involves predominantly interchromosomal reciprocal exchanges (equal and unequal crossovers). The striking observation is that intrachromosomal reciprocal recombination is very rare in comparison with interchromosomal reciprocal recombination. However, intrachromosomal gene conversion occurs at about the same frequency as interchromosomal gene conversion. Reciprocal recombination events between regions on the same chromatid are the most infrequent exchanges. These data suggest that intrachromosomal reciprocal exchanges are suppressed.     

Meiotic exchange within and between chromosomes requires a common Rec function in Saccharomyces cerevisiae.

We used haploid yeast cells that express both the MATa and MAT alpha mating-type alleles and contain the spo13-1 mutation to characterize meiotic recombination within single, unpaired chromosomes in Rec+ and Rec- Saccharomyces cerevisiae. In Rec+ haploids, as in diploids, intrachromosomal recombination in the ribosomal DNA was detected in 2 to 6% of meiotic divisions, and most events were unequal reciprocal sister chromatid exchange (SCE). By contrast, intrachromosomal recombination between duplicated copies of the his4 locus occurred in approximately 30% of haploid meiotic divisions, a frequency much higher than that reported in diploids; only about one-half of the events were unequal reciprocal SCE. The spo11-1 mutation, which virtually eliminates meiotic exchange between homologs in diploid meiosis, reduced the frequency of intrachromosomal recombination in both the ribosomal DNA and the his4 duplication during meiosis by 10- to greater than 50-fold. This Rec- mutation affected all forms of recombination within chromosomes: unequal reciprocal SCE, reciprocal intrachromatid exchange, and gene conversion. Intrachromosomal recombination in spo11-1 haploids was restored by transformation with a plasmid containing the wild-type SPO11 gene. Mitotic intrachromosomal recombination frequencies were unaffected by spo11-1. This is the first demonstration of a gene product required for recombination between homologs as well as recombination within chromosomes during meiosis.     

Molecular analysis of gene conversion in spermatids from transgenic mice

Investigations into the mechanisms and properties of gene conversion in mammals are greatly restricted by the inability to recover all the products of a meiosis. Additionally, the study of this process has been hampered by the lack of visible markers to detect gene conversion, especially when the events are rare. In previous work, we developed a transgenic system for detection and quantitation of gene conversion events in the germline of mice (Murti, J.R., Bumbulis, M., Schimenti, J.C., 1992. High frequency germline gene conversion in transgenic mice. Mol. Cell. Biol. 12, 2545–2552) that could be exploited as an assay for recombinogenic chemicals (Murti, J.R, Schimenti, K.J., Schimenti, J.C., 1994. A recombination-based transgenic mouse system for genotoxicity testing. Mutat. Res. 307, 583–595). A specific intrachromosomal gene conversion event between two complementarily defective lacZ genes resulted in the production of β-galactosidase in spermatids, enabling a measurement of conversion frequency. Here, we report that the anticancer drug, cisplatin, increased gene conversion in meiotic stage cells in these transgenic mice. Furthermore, a method was developed for direct molecular analysis of transgene conversion events in single or pooled lacZ-positive spermatids. The ability to identify gametes that have undergone a rare gene conversion event, followed by molecular amplification of the recombinant gene, should make it possible to investigate the mechanisms of genetic recombination in mammals in greater detail than previously possible.     

Intrachromosomal Gene Conversion and the Maintenance of Sequence Homogeneity among Repeated Genes

Intrachromosomal gene conversion is the non-reciprocal transfer of information between a pair of repeated genes on a single chromosome. This process produces eventual sequence homogeneity within a family of repeated genes. An evolutionary model for a single chromosome lineage was formulated and analyzed. Expressions were derived for the fixation probability, mean time to fixation or loss, and mean conditional fixation time for a variant repeat with an arbitrary initial frequency. It was shown that a small conversional advantage or disadvantage for the variant repeat (higher or lower probability of producing two variant genes by conversion than two wild-type genes) can have a dramatic effect on the probability of fixation. The results imply that intrachromosomal gene conversion can act sufficiently rapidly to be an important mechanism for maintaining sequence homogeneity among repeated genes.     

Intrachromosomal gene conversion frequency in the H2 differs between haplotypes

 We examined two intrachromosomal gene conversion events with a polymerase chain reaction assay at the DNA level between the two major histocompatibility complex class II genes Eb and Ab in mice sperm before selection has occurred. The frequency of the intrachromosomal gene conversion event between Ebd and Abd was found to be at least one order of magnitude higher than between Ebk and Abk in the same mice. Parental imprinting of the genes appears not to have an effect on gene conversion, as both (d×k)F₁ and (k×d)F₁ mice have indistinguishable frequencies in both haplotypes. The number of DNA copies of the donor and acceptor genes present in the cell at the time of mutation does not seem to influence the frequency of the intrachromosomal gene conversion in the k haplotype, whereas the frequency in the d haplotype is increased when double the number of donor and acceptor genes is present. The DNA fragment transferred between Ebd and Abd is invariably short, and need not comprise more than six nucleotides. The fragment transferred within the k haplotype varies in length, and can attain at least 100 nucleotides. The difference between the haplotypes both in length and frequency might be attributed to a six-nucleotide deletion in the Abk gene, which might make base-pairing between the genes less efficient and less precise. Received: 24 October 1997 / Revised: 5 January 1998     

Conditional genomic rearrangement by designed meiotic recombination using VDE (PI-SceI) in yeast

Meiotic recombination plays critical roles in the acquisition of genetic diversity and has been utilized for conventional breeding of livestock and crops. The frequency of meiotic recombination is normally low, and is extremely low in regions called “recombination cold domains”. Here, we describe a new and highly efficient method to modulate yeast meiotic gene rearrangements using VDE (PI-SceI), an intein-encoded endonuclease that causes an efficient unidirectional meiotic gene conversion at its recognition sequence (VRS). We designed universal targeting vectors, by use of which the strain that inserts the VRS at a desired site is acquired. Meiotic induction of the strains provided unidirectional gene conversions and frequent genetic rearrangements of flanking genes with little impact on cell viability. This system thus opens the way for the designed modulation of meiotic gene rearrangements, regardless of recombinational activity of chromosomal domains. Finally, the VDE–VRS system enabled us to conduct meiosis-specific conditional knockout of genes where VDE-initiated gene conversion disrupts the target gene during meiosis, serving as a novel approach to examine the functions of genes during germination of resultant spores.     

Genetic and Molecular Analysis of Recombination Events in Saccharomyces Cerevisiae Occurring in the Presence of the Hyper-Recombination Mutation Hpr1

The hyper-recombination mutation hpr1 specifically increases mitotic intrachromatid crossovers, with no effect on other mitotic recombination events such as unequal sister chromatid exchange and plasmid-chromosome recombination, and no effect on meiotic recombination and a lesser effect on intrachromosomal gene conversion. The excision repair RAD1 gene is partially required for the expression on the hpr1 phenotype. The simplest hypothesis to account for some of the hpr1 stimulated recombination events is that a heteroduplex DNA intermediate and localized gene conversion are involved. hpr1 stimulated crossover events are independent of intrachromosomal gene conversion events stimulated by the hyper-gene conversion mutation hpr5. This result suggests that different intrachromosomal recombination processes are affected in each mutant strain. We propose that HPR1 may function to inhibit intrachromatid crossovers.     

A V region determinant (idiotope) expressed at high frequency in B lymphocytes is encoded by a large set of antibody structural genes.

Idiotope Ac38, a V region determinant of the lambda 1 chain-bearing, germ line encoded antibody B1-8, is expressed at high frequency (approximately 1/40) in lambda 1 chain-bearing B cells. Here, we describe the isolation of lambda-positive hybridomas from C57BL/6 mice which had been immunized with antibody Ac38, the antibody recognizing idiotope Ac38. In Northern blot analysis, mRNA isolated from 10 such hybridomas hybridizes with a cDNA probe from the VH gene expressed in the cell line B1-8. Amino acid sequence analysis of the VH regions of four of the hybridoma proteins reveals that they are all derived from related, though distinct, germ line VH genes. In one case the sequence data suggest that extensive somatic mutation has taken place. Only one of the four sequences derives from the same VH gene that is expressed in the cell line B1-8. Together with earlier evidence, the present data demonstrate that the Ac38 idiotope is a marker for at least five VH and three D region genes in the C57BL/6 germ line. This explains the high frequency at which this idiotope is expressed in the B cell population. In addition, our sequence determinations identify two VH genes in the C57BL/6 strain which are closely related (and possibly allelic) to two known BALB/c VH genes. One of these genes is the gene expressed in the BALB/c myeloma MOPC 104E.     

Concerted evolution of the immunoglobulin VH gene family

With the aim of understanding the concerted evolution of the immunoglobulin VH multigene family, a phylogenetic tree for the DNA sequences of 16 mouse and five human germ line genes was constructed. This tree indicates that all genes in this family have undergone substantial evolutionary divergence. The most closely related genes so far identified in the mouse genome seem to have diverged about 6 million years (MY) ago, whereas the most distantly related genes diverged about 300 MY ago. This suggests that gene duplication caused by unequal crossing-over or gene conversion occurs very slowly in this gene family. The rate of occurrence of gene duplication in the VH gene family has been estimated to be 5 x 10(-7) per gene per year, which seems to be at least about 100 times lower than that for the rRNA gene family. This low rate of concerted evolution in the VH gene family helps retain intergenic genetic variability that in turn contributes to antibody diversity. Because of accumulation of destructive mutations, however, about one-third of the mouse and human VH genes seem to have become nonfunctional. Many of these pseudogenes have apparently originated recently, but some of them seem to have existed in the genome for more than 10 MY. The rate of nucleotide substitution for the complementarity-determining regions (CDRs) is as high as that of pseudogenes. This suggests that there is virtually no purifying selection operating in the CDRs and that germ line mutations are effectively used for generating antibody diversity.      

The heavy chain genes of a lupus anti-DNA autoantibody are encoded in the germ line of a nonautoimmune strain of mouse and conserved in strains of mice polymorphic for this gene locus

The variable region of the heavy chain of a prototypic anti-DNA autoantibody from the lupus-prone mouse, MRL-lpr/lpr, was cloned and sequenced. The VH and JH genes expressed by this antoantibody were found to be identical to germ line genes from the nonautoimmune mouse strain, BALB/c. The D gene of this autoantibody differed by one nucleotide from several members of the germ line SP2 family, but has been found in expressed D genes from several strains of mice. These results show that a normal mouse strain contains all of the structural information necessary for the expression of the heavy chain variable region of a lupus autoantibody. A fragment that is present in both BALB/c and MRL mice is highly homologous in both coding and flanking sequences to the autoantibody VH gene (VH130) and is the same size as the BALB/c germ line gene. This suggests that these two strains may share the same allele of this VH gene, despite the fact that they are polymorphic for this VH gene family. Other mouse strains that are polymorphic for this locus contained one to three VH genes that were highly related to VH130 in both coding and flanking regions. Thus, VH genes that may be allelic to the antibody VH gene or that may have arisen by gene conversion, unequal crossing over or gene duplication, are conserved in many mouse strains.     

Base excision repair is limited by different proteins in male germ cell nuclear extracts prepared from young and old mice

The combined observations of elevated DNA repair gene expression, high uracil-DNA glycosylase-initiated base excision repair, and a low spontaneous mutant frequency for a lacI transgene in spermatogenic cells from young mice suggest that base excision repair activity is high in spermatogenic cell types. Notably, the spontaneous mutant frequency of the lacI transgene is greater in spermatogenic cells obtained from old mice, suggesting that germ line DNA repair activity may decline with age. A paternal age effect in spermatogenic cells is recognized for the human population as well. To determine if male germ cell base excision repair activity changes with age, uracil-DNA glycosylase-initiated base excision repair activity was measured in mixed germ cell (i.e., all spermatogenic cell types in adult testis) nuclear extracts prepared from young, middle-aged, and old mice. Base excision repair activity was also assessed in nuclear extracts from premeiotic, meiotic, and postmeiotic spermatogenic cell types obtained from young mice. Mixed germ cell nuclear extracts exhibited an age-related decrease in base excision repair activity that was restored by addition of apurinic/apyrimidinic (AP) endonuclease. Uracil-DNA glycosylase and DNA ligase were determined to be limiting in mixed germ cell nuclear extracts prepared from young animals. Base excision repair activity was only modestly elevated in pachytene spermatocytes and round spermatids relative to other spermatogenic cells. Thus, germ line short-patch base excision repair activity appears to be relatively constant throughout spermatogenesis in young animals, limited by uracil-DNA glycosylase and DNA ligase in young animals, and limited by AP endonuclease in old animals.     

Analysis of a Gene Conversion Gradient at the His4 Locus in Saccharomyces Cerevisiae

Heteroduplexes formed between genes on homologous chromosomes are intermediates in meiotic recombination. In the HIS4 gene of Saccharomyces cerevisiae, most mutant alleles at the 5' end of the gene have a higher rate of meiotic recombination (gene conversion) than mutant alleles at the 3' end of the gene. Such gradients are usually interpreted as indicating a higher frequency of heteroduplex formation at the high conversion end of the gene. We present evidence indicating that the gradient of conversion at HIS4 primarily reflects the direction of mismatch repair rather than the frequency of heteroduplex formation. We also identify a site located between the 5' end of HIS4 and the 3' end of BIK1 that stimulates heteroduplex formation at HIS4 and BIK1.     

Misexpression of cyclin D1 in embryonic germ cells promotes testicular teratoma initiation

Testicular teratomas result from anomalies in embryonic germ cell development. In the 129 family of inbred mouse strains, teratomas arise during the same developmental period that male germ cells normally enter G1/G0 mitotic arrest and female germ cells initiate meiosis (the mitotic:meiotic switch). Dysregulation of this switch associates with teratoma susceptibility and involves three germ cell developmental abnormalities seemingly critical for tumor initiation: delayed G1/G0 mitotic arrest, retention of pluripotency, and misexpression of genes normally restricted to embryonic female and adult male germ cells. One misexpressed gene, cyclin D1 (Ccnd1), is a known regulator of cell cycle progression and an oncogene in many tissues. Here, we investigated whether Ccnd1 misexpression in embryonic germ cells is a determinant of teratoma susceptibility in mice. We found that CCND1 localizes to teratoma-susceptible germ cells that fail to enter G1/G0 arrest during the mitotic:meiotic switch and is the only D-type cyclin misexpressed during this critical developmental time frame. We discovered that Ccnd1 deficiency in teratoma-susceptible mice significantly reduced teratoma incidence and suppressed the germ cell proliferation and pluripotency abnormalities associated with tumor initiation. Importantly, Ccnd1 expression was dispensable for somatic cell development and male germ cell specification and maturation in tumor-susceptible mice, implying that the mechanisms by which Ccnd1 deficiency reduced teratoma incidence were germ cell autonomous and specific to tumorigenesis. We conclude that misexpression of Ccnd1 in male germ cells is a key component of a larger pro-proliferative program that disrupts the mitotic:meiotic switch and predisposes 129 inbred mice to testicular teratocarcinogenesis.     

Me14, a Yeast Gene Required for Meiotic Recombination

Mutants at the MEI4 locus were detected in a search for mutants defective in meiotic gene conversion. mei4 mutants exhibit decreased sporulation and produce inviable spores. The spore inviability phenotype is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The MEI4 gene has been cloned from a yeast genomic library by complementation of the recombination defect and has been mapped to chromosome V near gln3. Strains carrying a deletion/insertion mutation of the MEI4 gene display no meiotically induced gene conversion but normal mitotic conversion frequencies. Both meiotic interchromosomal and intrachromosomal crossing over are completely abolished in mei4 strains. The mei4 mutation is able to rescue the spore-inviability phenotype of spo13 and 52 strains (i.e., mei4 spo13 rad52 mutants produce viable spores), indicating that MEI4 acts before RAD52 in the meiotic recombination pathway.     

Chromosomal Translocations Generated by High-Frequency Meiotic Recombination between Repeated Yeast Genes

We have examined meiotic and mitotic recombination between repeated genes on nonhomologous chromosomes in the yeast Saccharomyces cerevisiae. The results of these experiments can be summarized in three statements. First, gene conversion events between repeats on nonhomologous chromosomes occur frequently in meiosis. The frequency of such conversion events is only 17-fold less than the analogous frequency of conversion between genes at allelic positions on homologous chromosomes. Second, meiotic and mitotic conversion events between repeated genes on nonhomologous chromosomes are associated with reciprocal recombination to the same extent as conversion between allelic sequences. The reciprocal exchanges between the repeated genes result in chromosomal translocations. Finally, recombination between repeated genes on nonhomologous chromosomes occurs much more frequently in meiosis than in mitosis.     

Germ line maintenance of the pseudogene donor pool for somatic immunoglobulin gene conversion in chickens.

Somatic immunoglobulin diversity is generated in avian species by sequential gene conversion of variable (V) gene segments of the immunoglobulin heavy- and light-chain loci during B-cell development. The germ line pools of donor sequence information for somatic V-region gene conversion are found in families of V pseudogenes, located 5' of the single functional V gene of each locus. The sequence relationships among the pseudogenes (psi VL) and functional VL1 gene of the chicken light-chain alleles in three inbred strains were compared to determine the extent of diversity within the germ line pseudogene cluster. Numerous differences were observed. For example, compared with the previously reported CB allele and the G4 allele, the S3 allele contains two intact pseudogenes between psi VL16 and psi VL18. These two adjacent psi VL gene segments (psi VL17a and psi VL17b) could have given rise to the psi VL17 segment of the G4 and CB alleles by homologous recombination. The majority of other sequence polymorphisms among the psi VL alleles appear to be the result of meiotic gene conversion. The incidence of untemplated mutations within psi VL segments is significantly lower than the incidence of mutation within the pseudogene flanking regions. Together with the observations that most psi VL segments have open reading frames and lack stop codons, these data support the hypothesis that the psi VL cluster resembles a functional multigene family maintained by evolutionary selection for its functional role in generating somatic antibody diversity. Meiotic gene conversion events within the psi VL cluster serve both to introduce diversity by the exchange of short segments between family members and to prevent the accumulation of random mutations.