Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes.

Meiotic Recombination Between Paralogous RBCSB Genes on Sister Chromatids of Arabidopsis thaliana

Paralogous genes organized as a gene cluster can rapidly evolve by recombination between misaligned paralogs during meiosis, leading to duplications, deletions, and novel chimeric genes. To model unequal recombination within a specific gene cluster, we utilized a synthetic RBCSB gene cluster to isolate recombinant chimeric genes resulting from meiotic recombination between paralogous genes on sister chromatids. Several F1 populations hemizygous for the synthRBCSB1 gene cluster gave rise to Luc+ F2 plants at frequencies ranging from 1 to 3 × 10-6. A nonuniform distribution of recombination resolution sites resulted in the biased formation of recombinant RBCS3B/1B::LUC genes with nonchimeric exons. The positioning of approximately half of the mapped resolution sites was effectively modeled by the fractional length of identical DNA sequences. In contrast, the other mapped resolution sites fit an alternative model in which recombination resolution was stimulated by an abrupt transition from a region of relatively high sequence similarity to a region of low sequence similarity. Thus, unequal recombination between paralogous RBCSB genes on sister chromatids created an allelic series of novel chimeric genes that effectively resulted in the diversification rather than the homogenization of the synthRBCSB1 gene cluster.

Heterogeneity in the Sxr (sex-reversal) locus of the mouse as revealed by synthetic probes

SummaryThe sex-reversal mutation, Sxr and a variant form, Sxr′ have been established on the inbred C57BL/6Mcl background by repeated backcrossing to form the CB and CB′ strains, respectively. DNAs of normal XY, XX Sxr and XX Sxr′ as well as XY Sxr and XY Sxr′ carrier male mice have been digested with the restriction enzymes Hae III and Hinf I and electrophoresed. The DNAs show many common but also differing hybridization bands with synthetic oligonucleotide probes. In XY Sxr (and XY Sxr′) carrier males, the hybridization patterns of normal XY and those of XX Sxr (and XX Sxr′) males are simply superimposed. Individual differing bands can be categorized by their differential hybridization behaviour to the (GATA)4, (GACA)4, (GATA)2 GACA (GATA)2 and (GATA)3 (GACA)2 probes. In general, the hybridization patterns are regularly inherited. In addition to the predominant pattern in each strain, one additional XX Sxr and one additional XX Sxr′ hybridization pattern was observed: the additional pattern in the CB strain was transmitted (via variant XY Sxr carriers) while the secondary XX Sxr′ pattern in the CB′ strain could only be observed once. Thus ‘DNA finger printing’ with oligonucleotide probes can successfully be used to discriminate the DNAs of normal Y chromosomes, XX Sxr and XX Sxr′ variants as well as XY Sxr and XY Sxr′ carrier mice. Implications of the comparatively high unequal recombination rate involving the murine Y chromosome are discussed, as well as possible mechanisms.

Detection of new MHC mutations in mice by skin grafting, tumor transplantation and monoclonal antibodies: a comparison.

Two mechanisms of major histocompatibility complex (MHC) mutations have been described in mice; gene conversion and homologous but unequal recombination. However, our knowledge of mutations in MHC is incomplete because studies have been limited almost exclusively to two haplotypes, H-2b and H-2d, while hundreds of haplotypes exist in nature; it has been biased by the use of only one procedure of screening for mutation, skin grafting. We used three procedures to screen for MHC mutations: (1) conventional techniques of skin grafting, (2) syngeneic tumor transplantation and (3) typing with monoclonal anti-MHC antibodies (mAbs) and complement. The faster technique of tumor transplantation detected mutants similar to those discovered by skin grafting technique. Screening with mAbs allowed us to detect both mutants that are capable of rejecting standard skin grafts and those that are silent in skin grafting tests, and which therefore resulted in a higher apparent mutation frequency. Two mutants of the H-2a haplotype were found that carry concomitant class I and class II antigenic alterations. Both MHC mutants silent in skin grafting tests and mutants carrying concomitant class I and class II alterations have never been studied before and are expected to reveal new mechanisms of generating MHC mutations. 1-Ethyl-1-nitrosourea (ENU) failed to induce de novo MHC mutations in male mice in our skin grafting series.

MERODIPLOIDY IN ESCHERICHIA COLI-SALMONELLA TYPHIMURIUM CROSSES: THE ROLE OF UNEQUAL RECOMBINATION BETWEEN RIBOSOMAL RNA GENES

ABSTRACT Previous workers have shown that intergeneric crosses between Salmonella typhimurium and Escherichia coli produce a high proportion of merodiploid recombinants among the viable progeny. We have examined the unequal crossover event that was responsible for a number of intergeneric merodiploids. The merodiploids that we studied were all heterozygous for the metB–argH interval and were the products of intergeneric conjugal crosses. We found that when the S. typhimurium donor had its transfer origin closely linked to metB and argH, all recombinants examined were merodiploid, and they generally arose as F-prime factors. Many of these F-prime factors had been created by recombination between flanking rrn genes in the donor. When the S. typhimurium Hfr transfer origin was more distant from the selected markers, quite different results were obtained. (1) Depending on the donor, 19–47% of the recombinants that acquired the donor argH + or metB + genes were merodiploid for these loci, but none of the recombinants were F-prime. (2) A majority of the merodiploids had a novel (nonparental) rrn gene, indicating that unequal recombination between nonidentical rrn genes was a prevalent mechanism for establishing the merodiploidy. (3) Both tandem and nontandem duplications were found. (4) Some of the merodiploids duplicated E. coli genes in addition to acquiring S. typhimurium genes. (5) Some merodiploids contained the oriC region from each parent. Of a total of 118 intergeneric merodiploids characterized from all donors, 48 different genotypes were observed, and 38 of the 48 had one or more nonparental rrn operons.