A physical assay for detection of early meiotic recombination intermediates in Saccharomyces cerevisiae

Plasmodium gallinaceum ookinetes adhered to Aedes aegypti midgut epithelia when purified ookinetes and isolated midguts were combined in vitro. Ookinetes preferentially bound to the microvillated luminal surface of the midgut, and they seemed to interact with three types of structures on the midgut surface. First, they adhered to and migrated through a network-like matrix, which we have termed microvilli-associated network, that covers the surface of the microvilli. This network forms on the luminal midgut surface in response to blood or protein meals. Second, the ookinetes bound directly to the microvilli on the surface of the midgut and were occasionally found immersed in the thick microvillar layer. Third, the ookinetes associated with accumulations of vesicular structures found interspersed between the microvillated cells of the midgut. The origin of these vesicular structures is unknown, but they correlated with the surface of midgut cells invaded by ookinetes as observed by TEM. After binding to the midgut, ookinetes underwent extensive morphological changes: they frequently developed one or more annular constrictions, and their surface roughened considerably, suggesting that midgut components remain bound to the parasite surface. Our observations suggest that, in a natural infection, the ookinete interacts in a sequential manner with specific components of the midgut surface. Initial binding to the midgut surface may activate the ookinete and cause morphological changes in preparation for invasion of the midgut cells.     

Conversion-type and restoration-type repair of DNA mismatches formed during meiotic recombination in Saccharomyces cerevisiae

Meiotic recombination in yeast is associated with heteroduplex formation. Heteroduplexes formed between nonidentical DNA strands contain DNA mismatches, and most DNA mismatches in wild-type strains are efficiently corrected. Although some patterns of mismatch correction result in non-Mendelian segregation of the heterozygous marker (gene conversion), one predicted pattern of correction (restoration-type repair) results in normal Mendelian segregation. Using a yeast strain in which a marker leading to a well-repaired mismatch is flanked by markers that lead to poorly repaired mismatches, we present direct evidence for restoration-type repair in yeast. In addition, we find that the frequency of tetrads with conversion-type repair is higher for a marker at the 5' end of the HIS4 gene than for a marker in the middle of the gene. These results suggest that the ratio of conversion-type to restoration-type repair may be important in generating gradients of gene conversion (polarity gradients).     

Saccharomyces cerevisiae recA homologues RAD51 and DMC1 have both distinct and overlapping roles in meiotic recombination

BACKGROUND: Rad51 and Dmc1 are Saccharomyces cerevisiae homologues of the Escherichia coli recombination protein RecA. Mutant analysis has shown that both proteins are required for normal meiotic recombination, for timely and efficient formation of synaptonemal complex and for normal progression out from meiotic prophase. RESULTS: We have further characterized rad51 and dmc1 single mutants. A dmc1 mutation confers more severe defects in double strand break (DSB) resolution, crossover recombination and meiotic progression than does a rad51 mutant; in contrast, during return to growth, a rad51 mutation confers more severe defects in viability and intrachromosomal recombination than does a dmc1 mutation. Analysis of a rad51 dmc1 double mutant, in parallel with single mutants, shows that the double mutant is more defective with respect to the formation of crossovers during meiosis and, especially strikingly, with respect to interhomologue and intrachromosomal recombination during return to growth. Consistent with the observation of DMC1-dependent recombination in a rad51 mutant, subnuclear complexes of Dmc1 protein were detected for the first time in this mutant. In contrast to the effects on recombination, the effect of the double mutant on meiotic progression was similar to that of the rad51 single mutant. CONCLUSION: Rad51 and Dmc1 each make unique contributions to meiotic recombination. However, the two proteins are capable of substituting for one another under some circumstances, implying that they most likely share at least one recombination function. Recombination and cell cycle phenotypes are all consistent with the possibility that a dmc1 mutation causes an arrest of the post-DSB recombination complexes at a later, more stable stage than does a rad51 mutation.     

Isolation of COM1, a new gene required to complete meiotic double-strand break-induced recombination in Saccharomyces cerevisiae

We have designed a screen to isolate mutants defective during a specific part of meiotic prophase I of the yeast Saccharomyces cerevisiae. Genes required for the repair of meiotic double-strand breaks or for the separation of recombined chromosomes are targets of this mutant hunt. The specificity is achieved by selecting for mutants that produce viable spores when recombination and reductional segregation are prevented by mutations in SPO11 and SPO13 genes, but fail to yield viable spores during a normal Rec+ meiosis. We have identified and characterized a mutation com1-1, which blocks processing of meiotic double-strand breaks and which interferes with synaptonemal complex formation, homologous pairing and, as a consequence, spore viability after induction of meiotic recombination. The COM1/SAE2 gene was cloned by complementation, and the deletion mutant has a phenotype similar to com1-1, com1/sae2 mutants closely resemble the phenotype of rad50S, as assayed by phase-contrast microscopy for spore formation, physical and genetic analysis of recombination, fluorescence in situ hybridization to quantify homologous pairing and immunofluorescence and electron microscopy to determine the capability to synapse axial elements.     

Genome-wide expression monitoring in Saccharomyces cerevisiae

The genomic sequence of the budding yeast Saccharomyces cerevisiae has been used to design and synthesize high-density oligonucleotide arrays for monitoring the expression levels of nearly all yeast genes. This direct and highly parallel approach involves the hybridization of total mRNA populations to a set of four arrays that contain a total of more than 260,000 specifically chosen oligonucleotides synthesized in situ using light-directed combinatorial chemistry. The measurements are quantitative, sensitive, specific, and reproducible. Expression levels ranging from less than 0.1 copies to several hundred copies per cell have been measured for cells grown in rich and minimal media. Nearly 90% of all yeast mRNAs are observed to be present under both conditions, with approximately 50% present at levels between 0.1 and 1 copy per cell. Many of the genes observed to be differentially expressed under these conditions are expected, but large differences are also observed for many previously uncharacterized genes.     

Substrate length requirements for efficient mitotic recombination in Saccharomyces cerevisiae

An ectopic recombination system using ura3 heteroalleles varying in size from 80 to 960 bp has been used to examine the effect of substrate length on spontaneous mitotic recombination. The ura3 heteroalleles were positioned either on nonhomologous chromosomes (heterochromosomal repeats) or as direct or inverted repeats on the same chromosome (intrachromosomal repeats). While the intrachromosomal events occur at rates at least 2 orders of magnitude greater than the corresponding heterochromosomal events, the recombination rate for each type of repeat considered separately exhibits a linear dependence on substrate length. The linear relationships allow estimation of the corresponding minimal efficient processing segments, which are approximately 250 bp regardless of the relative positions of the repeats in the yeast genome. An examination of the distribution of recombination events into simple gene conversion versus crossover events indicates that reciprocal exchange is more sensitive to substrate size than is gene conversion.     

Requirement for end-joining and checkpoint functions, but not RAD52-mediated recombination, after EcoRI endonuclease cleavage of Saccharomyces cerevisiae DNA

RAD52 and RAD9 are required for the repair of double-strand breaks (DSBs) induced by physical and chemical DNA-damaging agents in Saccharomyces cerevisiae. Analysis of EcoRI endonuclease expression in vivo revealed that, in contrast to DSBs containing damaged or modified termini, chromosomal DSBs retaining complementary ends could be repaired in rad52 mutants and in G1-phase Rad+ cells. Continuous EcoRI-induced scission of chromosomal DNA blocked the growth of rad52 mutants, with most cells arrested in G2 phase. Surprisingly, rad52 mutants were not more sensitive to EcoRI-induced cell killing than wild-type strains. In contrast, endonuclease expression was lethal in cells deficient in Ku-mediated end joining. Checkpoint-defective rad9 mutants did not arrest cell cycling and lost viability rapidly when EcoRI was expressed. Synthesis of the endonuclease produced extensive breakage of nuclear DNA and stimulated interchromosomal recombination. These results and those of additional experiments indicate that cohesive ended DSBs in chromosomal DNA can be accurately repaired by RAD52-mediated recombination and by recombination-independent complementary end joining in yeast cells.     

Functional differences among the six Saccharomyces cerevisiae tRNATrp genes

The Saccharomyces cerevisiae haploid genome includes six copies of the gene encoding tRNATrp which are scattered on five chromosomes. Other, non-functional tDNATrp fragments also occur in the genome. The segments of all six genes which encode the 72-nucleotide mature tRNATrp, as well as a 34-nucleotide intervening sequence, are identical. However, the 5' and 3' flanking sequences diverge virtually at the boundaries of the coding region. We have used an assay based on suppression of UGA mutations by multi-copy clones of tDNATrp to search for functional differences among these genes. Previous studies with one tDNATrp had demonstrated that moderate suppression of a UGA mutation, leu2-2, resulted from introduction of a multi-copy clone of the gene. Attempts to use this assay to select tDNATrp clones from a yeast genomic library yielded only four of the six different clones. The other two genes were amplified by PCR and cloned in pRS202, a 2 mu vector also used for the genomic library. Plasmids bearing the six tRNA genes were transformed into S. cerevisiae strain JG369.3B and scored for their ability to suppress the leu2-2 mutation as well as his4-260, another UGA marker. Two of the six tRNATrp clones were unable to suppress either marker, two evidenced weak suppression of the Leu auxotrophy, and two were able to suppress both markers. Growth rates in liquid media requiring suppression were measured for cell lines carrying each of the clones. Differences greater than 50-fold were observed in media lacking histidine. An evolutionary tree based on 5'-flanking sequence corresponds reasonably well with suppressor activity, while a similar analysis of 3'-flanking sequence does not. This suggests that the functional differences are based on divergence in the 5'-flanking sequences of the tRNATrp genes.     

Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae

Female wild Japanese monkeys (Macaca fuscata), as with all male cercopithecoids, use the mesiobuccal surfaces or the elongated crests of the mandibular third premolars (P3s), as cutting blocks that wear against edges of maxillary canines during threat manifestation or food-eating. In other words, the crests of their P3s are honed with the maxillary canines. The crests become sloped during growth and more heavily striated with the advance of age. The number, directions, lengths, and widths of these striations have been analyzed quantitatively using scanning electron microscopy (SEM). Two samples showed two distinct types of parallel striations, one longer and thicker (171.5 microns long and 14.5 microns wide on average) than the other (114.8 microns long and 12.0 microns wide on average). These striations were caused by contact between the sharp edge of the upper canine and the P3 during honing (canine/premolar complex). The long and thick striations are asymmetrical with widened parts or pits on one end, and were easily distinguished from other thinner striations which may have been caused by fine particles. The third sample showed Hunter-Schreger bands with striae of Retzius on the sloping heavily worn mesiobuccal surface. The features of these thick parallel striations indicate that they result from closing movements of the jaw.     

The Saccharomyces cerevisiae RAD9 checkpoint reduces the DNA damage-associated stimulation of directed translocations

Genetic instability in the Saccharomyces cerevisiae rad9 mutant correlates with failure to arrest the cell cycle in response to DNA damage. We quantitated the DNA damage-associated stimulation of directed translocations in RAD9+ and rad9 mutants. Directed translocations were generated by selecting for His+ prototrophs that result from homologous, mitotic recombination between two truncated his3 genes, GAL1::his3-delta5' and trp1::his3-delta3'::HOcs. Compared to RAD9+ strains, the rad9 mutant exhibits a 5-fold higher rate of spontaneous, mitotic recombination and a greater than 10-fold increase in the number of UV- and X-ray-stimulated His+ recombinants that contain translocations. The higher level of recombination in rad9 mutants correlated with the appearance of nonreciprocal translocations and additional karyotypic changes, indicating that genomic instability also occurred among non-his3 sequences. Both enhanced spontaneous recombination and DNA damage-associated recombination are dependent on RAD1, a gene involved in DNA excision repair. The hyperrecombinational phenotype of the rad9 mutant was correlated with a deficiency in cell cycle arrest at the G2-M checkpoint by demonstrating that if rad9 mutants were arrested in G2 before irradiation, the numbers both of UV- and gamma-ray-stimulated recombinants were reduced. The importance of G2 arrest in DNA damage-induced sister chromatid exchange (SCE) was evident by a 10-fold reduction in HO endonuclease-induced SCE and no detectable X-ray stimulation of SCE in a rad9 mutant. We suggest that one mechanism by which the RAD9-mediated G2-M checkpoint may reduce the frequency of DNA damage-induced translocations is by channeling the repair of double-strand breaks into SCE.     

Gene conversions and crossing over during homologous and homeologous ectopic recombination in Saccharomyces cerevisiae

The pma1-105 mutation reduces the activity of the yeast plasma membrane H(+)-ATPase and causes cells to be both low pH and ammonium ion sensitive and resistant to the antibiotic hygromycin B. Revertants that can grow at pH 3.0 and on ammonium-containing plates frequently arise by ectopic recombination between pma1-105 and PMA2, a diverged gene that shares 85% DNA sequence identity with PMA1. The gene conversion tracts of revertants of pma1-105 were determined by DNA sequencing the hybrid PMA1::PMA2 genes. Gene conversion tracts ranged from 18-774 bp. The boundaries of these replacements were short (3-26 bp) regions of sequences that were identical between PMA1 and PMA2. These boundaries were not located at the regions of greatest shared identity between the two PMA genes. Similar results were obtained among low pH-resistant revertants of another mutation, pma1-147. One gene conversion was obtained in which the resulting PMA1::PMA2 hybrid was low pH-resistant but still hygromycin B-resistant. This partially active gene differs from a wild-type revertant only by the presence of two PMA2-encoded amino acid substitutions. Thus, some regions of PMA2 are not fully interchangeable with PMA1. We have also compared the efficiency of recombination between pma1-105 and either homeologous PMA2 sequence or homologous PMA1 donor sequences inserted at the same location. PMA2 x pma1-105 recombination occurred at a rate approximately 75-fold less than PMA1 x pma1-105 events. The difference in homology between the interacting sequences did not affect the proportion of gene conversion events associated with a cross-over, as in both cases approximately 5% of the Pma+ recombinants had undergone reciprocal translocations between the two chromosomes carrying pma1-105 and the donor PMA sequences. Reciprocal translocations were identified by a simple and generally useful nutritional test.     

Interaction between the MEC1-dependent DNA synthesis checkpoint and G1 cyclin function in Saccharomyces cerevisiae

The completion of DNA synthesis in yeast is monitored by a checkpoint that requires MEC1 and RAD53. Here we show that deletion of the Saccharomyces cerevisiae G1 cyclins CLN1 and CLN2 suppressed the essential requirement for MEC1 function. Wild-type levels of CLN1 and CLN2, or overexpression of CLN1, CLN2, or CLB5, but not CLN3, killed mec1 strains. We identified RNR1, which encodes a subunit of ribonucleotide reductase, as a high-copy suppressor of the lethality of mec1 GAL1-CLN1. Northern analysis demonstrated that RNR1 expression is reduced by CLN1 or CLN2 overexpression. Because limiting RNR1 expression would be expected to decrease dNTP pools, CLN1 and CLN2 may cause lethality in mec1 strains by causing initiation of DNA replication with inadequate dNTPs. In contrast to mec1 mutants, MEC1 strains with low dNTPs would be able to delay S phase and thereby remain viable. We propose that the essential function for MEC1 may be the same as its checkpoint function during hydroxyurea treatment, namely, to slow S phase when nucleotides are limiting. In a cln1 cln2 background, a prolonged period of expression of genes turned on at the G1-S border, such as RNR1, has been observed. Thus deletion of CLN1 and CLN2 could function similarly to overexpression of RNR1 in suppressing mec1 lethality.