Mutant Rec-1 Eliminates the Meiotic Pattern of Crossing over in Caenorhabditis Elegans

Meiotic crossovers are not randomly distributed along the chromosome. In Caenorhabditis elegans the central portions of the autosomes have relatively few crossovers compared to the flanking regions. We have measured the frequency of crossing over for several intervals across chromosome I in strains mutant for rec-1. The chromosome is ~50 map units in both wild-type and rec-1 homozygotes, however, the distribution of exchanges is very different in rec-1. Map distances expand across the gene cluster and contract near the right end of the chromosome, resulting in a genetic map more consistent with the physical map. Mutations in two other genes, him-6 and him-14, also disrupted the distribution of exchanges. Unlike rec-1, individuals homozygous for him-6 and him-14 had an overall reduction in the amount of crossing over accompanied by a high frequency of nondisjunction and reduced egg hatching. In rec-1; him-6 and rec-1; him-14 homozygotes the frequency of crossing over was characteristic of the Him mutant phenotype, whereas the distribution of the reduced number of exchanges was characteristic of the Rec-1 pattern. It appears that these gene products play a role in establishing the meiotic pattern of exchange events.     

The Rec102 Mutant of Yeast Is Defective in Meiotic Recombination and Chromosome Synapsis

A mutation at the REC102 locus was identified in a screen for yeast mutants that produce inviable spores. rec102 spore lethality is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The rec102 mutation completely eliminates meiotically induced gene conversion and crossing over but has no effect on mitotic recombination frequencies. Cytological studies indicate that the rec102 mutant makes axial elements (precursors to the synaptonemal complex), but homologous chromosomes fail to synapse. In addition, meiotic chromosome segregation is significantly delayed in rec102 strains. Studies of double and triple mutants indicate that the REC102 protein acts before the RAD52 gene product in the meiotic recombination pathway. The REC102 gene was cloned based on complementation of the mutant defect and the gene was mapped to chromosome XII between CDC25 and STE11.     

Hop1: A Yeast Meiotic Pairing Gene

The recessive mutation, hop1-1, was isolated by use of a screen designed to detect mutations defective in homologous chromosomal pairing during meiosis in Saccharomyces cerevisiae. Mutants in HOP1 displayed decreased levels of meiotic crossing over and intragenic recombination between markers on homologous chromosomes. In contrast, assays of the hop1-1 mutation in a spo13-1 haploid disomic for chromosome III demonstrated that intrachromosomal recombination between directly duplicated sequences was unaffected. The spores produced by SPO13 diploids homozygous for hop1 were largely inviable, as expected for a defect in interhomolog recombination that results in high levels of nondisjunction. HOP1 was cloned by complementation of the spore lethality phenotype and the cloned gene was used to map HOP1 to the LYS11-HIS6 interval on the left arm of chromosome IX. Electron microscopy revealed that diploids homozygous for hop1 fail to form synaptonemal complex, which normally provides the structural basis for homolog pairing. We propose that HOP1 acts in meiosis primarily to promote chromosomal pairing, perhaps by encoding a component of the synaptonemal complex.     

Meiotic Recombination and DNA Synthesis in a New Cell Cycle Mutant of SACCHAROMYCES CEREVISIAE

Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive temperature with a medial nuclear division phenotype. DNA replication is observed under these conditions, but most cells remain sensitive to hydroxyurea and do not complete the ongoing cell cycle if the drug is present during release from the temperature block. It is suggested that the cdc40 lesion affects an essential function in DNA synthesis. Normal meiosis is observed at the permissive temperature in cdc40 homozygotes. At the restrictive temperature, a full round of premeiotic DNA replication is observed, but neither commitment to recombination nor later meiotic events occur. Meiotic cells that are already committed to the recombination process at the permissive temperature do not complete it if transferred to the restrictive temperature before recombination is realized. These temperature shift-up experiments demonstrate that the CDC40 function is required for the completion of recombination events, as well as for the earlier stage of recombination commitment. Temperature shift-down experiments with cdc40 homozygotes suggest that meiotic segregation depends on the final events of recombination rather than on commitment to recombination.     

The Yeast Med1 Mutant Undergoes Both Meiotic Homolog Nondisjunction and Precocious Separation of Sister Chromatids

A mutant at the yeast MED1 locus was isolated in a screen for sporulation-proficient, meiotic-lethal mutants. Synaptonemal complex formation in the med1 mutant is apparently normal and med1 strains undergo meiotic crossing over at approximately 50% of the wild-type level. The med1 mutant undergoes homolog nondisjunction at meiosis I, presumably as a consequence of the decrease in crossing over. In addition, the mutant undergoes precocious separation of sister chromatids, resulting in chromosome missegregation at both meiotic divisions. We suggest that the med1 mutation perturbs chromosome structure, leading to a reduction in recombination and a defect in sister chromatid cohesion.     

Rapid Conformational Epitope Mapping of Anti-gp120 Antibodies with a Designed Mutant Panel Displayed on Yeast

gp120 is a substrate for protein engineering both for human immunodeficiency virus (HIV) immunogen design and as a bait for isolating anti-HIV antibodies from patient samples. In this work, we describe the display of a stripped core gp120 on the yeast cell surface. Validation against a panel of neutralizing antibodies confirms that yeast-displayed gp120 presents the CD4 binding site in the correct conformation. We map the epitope of the broadly neutralizing anti-gp120 antibody VRC01 using both a random mutagenesis library and a defined mutant panel and find that the resultant epitope maps are consistent with one another and with the crystallographically identified contact residues. Mapping the VRC01-competitive antibodies b12 and b13 reveals energetic differences in their epitopes that are not obvious from existing crystal structures. These data suggest mutation sets that abrogate binding to broadly neutralizing antibodies with greater specificity than the canonical mutation D368R, useful in rapidly assessing the nature of a vaccine response.     

Precocious Meiotic Centromere Separation of a Novel Yeast Chromosome

In Saccharomyces cerevisiae, a reciprocal translocation between chromosome II and a linear plasmid carrying a centromere (CEN6) has split chromosome II into two fragments: one, approximately 530 kilobase pairs (kbp) in size, has the left arm and part of the right arm of chromosome II; the other, a telocentric fragment approximately 350 kbp in size, has CEN6 and the rest of the right arm of chromosome II. A cross of this yeast strain with a strain containing a complete chromosome II exhibits a high frequency of precocious centromere separation (separation of sister chromatids during meiosis I) of the telocentric fragment. Precocious centromere separation is not due to the position of the centromere per se, since diploids that are homozygous for both fragments of chromosome II segregate the telocentric fragment with normal meiotic behavior. The precocious centromere separation described here differs from previously described examples in that pairing and synapsis of this telocentric chromosome seem to be normal. One model of how centromeres function in meiosis is that replication of the centromere is delayed until the second meiotic division. Data presented in this paper indicate that replication of the centromere is complete before the first meiotic division. The precocious separation of the centromere described here may be due to improper synapsis of sequences flanking the centromere.     

Morewright (Mwr), a New Meiotic Mutant of Drosophila Melanogaster Affecting Nonexchange Chromosome Segregation

A new meiotic mutation, morewright (mwr) was identified by screening for new mutations that act as dominant enhancers of the dosage-sensitive Drosophila melanogaster female meiotic mutant, nod(DTW). mwr is a recessive meiotic mutant, specifically impairing the segregation of nonexchange chromosomes. Cytological evidence suggests that the meitoic defect in mwr/mwr females is in homologue recognition because the chromosomes appear to be misaligned on an intact spindle. The mwr mutation was recovered during a screen of random P-element insertions on a chromosome with a single insertion located at 50C. The P-element insertion is a recessive female-sterile mutation. While excision of the P element from the mwr-bearing chromosome partially relieves the female sterility, the excisions retain the dominant nod(DTW)-enhancing activity. The mwr meiotic phenotype maps very close to the female-sterile P insertion. Thus the mwr locus appears to encode a function required for partner recognition in meiosis, although its relationship to the neighboring female-sterile mutation remains to be elucidated.     

Extensive Recombination of a Yeast Diploid Hybrid through Meiotic Reversion

In somatic cells, recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events (LOH), allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection. However, inter-homolog recombination in somatic cells is rare, thus reducing potential genetic variation. Here, we explored the property of S. cerevisiae to enter the meiotic developmental program, induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth, a process known as Return To Growth (RTG). Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome, derived from either parental origin. Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous (LOH) events of various lengths, varying between a few nucleotides up to several hundred kilobases. Furthermore, we show that reiteration of the RTG process shows incremental increases of homozygosity. Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci (QTLs) in diploid strains without undergoing sexual reproduction.     

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.     

Biosynthesis of Vitamin B6 by a Yeast Mutant

The gradient-plate technique was employed to isolate mutants of Saccharomyces marxianus (NRRL-Y-1550) which, when grown in a synthetic culture medium, excreted about 2 mug/ml of vitamin B(6) as ascertained by microbiological assay. The major component that possessed vitamin B(6) activity was isolated by ion-exchange column chromatography and identified as pyridoxol by ultraviolet and fluorescence spectroscopy, as well as by paper chromatography and various chemical tests. Pyridoxal was also identified as one of the excreted compounds. Two other compounds that possessed vitamin B(6) activity were excreted in smaller quantities in the growth medium and have not yet been identified; they are not phosphates of vitamin B(6). The amount of vitamin B(6) excreted was not increased when the mutant was grown in the presence of various oxidation products of this vitamin. The methods and results reported here may be helpful in future studies on the biosynthesis of vitamin B(6).     

一个新水稻温敏感叶色突变体的遗传分析及其基因分子定位

在粳稻品种嘉花1号（Oryza sativa L.ssp.japonica＇ Jiahua No.1＇）种子经60Coγ射线辐照处理的后代中,发现了1个低温敏感叶色突变体mr21。在较低温度（〈25.0°C）条件下,该突变体幼苗叶色呈黄色;随着温度逐渐升高,叶色由黄转绿,其临界温度约为27.5°C;在低温条件下,突变体幼苗总叶绿素含量以及叶绿素a、b的含量均较野生型嘉花1号明显下降,表明该突变体的叶色性状具有明显的温敏感性。遗传分析表明,该突变体叶色性状受1对隐性核基因控制,暂将该突变基因命名为thermo-sensitive leaf-color1（tsl-1）。以该突变体与籼稻9311（Oryza sativa L.ssp.indica＇ 9311＇）杂交的F2代分离群体作为定位群体,利用SSR分子标记将tsl-1基因初步定位在水稻（Oryza sativa）第1号染色体短臂上的MM1799与RM8132分子标记之间,其遗传距离分别为2.4cM和3.0cM;然后,进一步利用扩大F2代群体及新发展的分子标记将tsl-1基因定位在分子标记InDel2与InDel4之间的198kb内。研究结果为今后对该基因的克隆和功能分析奠定了基础。     

A Highly Revertible Cyc1 Mutant of Yeast Contains a Small Tandem Duplication

A mutant, cyc1-96, that reverts spontaneously at an extremely high rate, was uncovered after examining approximately 500 cyc1 mutants which lack or have defective iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. Cloning and DNA sequencing of appropriate fragments revealed that the cyc1-96 mutation contained a 19 bp duplication whereas the spontaneously arising revertants contained the normal wild-type sequence. Because the 19 bp segment in the wild-type sequence is flanked by a 5 bp repeat and because the cyc1-96 mutation arose spontaneously, the 19 bp duplication may have arisen by slippage and misalignment during DNA synthesis. The high reversion rate was not diminished in strains containing the rad52 mutation, which generally reduces mitotic recombination, including recombination associated with the elimination of a segment of a long direct repeat. Thus the loss of segments from short and long duplications occur by different mechanisms. We suggest that the high reversion rates of cyc1-96 and other short duplications are due to misalignment errors during replication.     

小麦吸收土壤磷转运子在酵母突变体中的功能互补分析

以小麦磷转运子全长编码cDMA(TaPT2)为探针与小麦基因组DNA进行Southern杂交,结果表明,在小麦基因组中存在该基因的不同家族成员,另外,将TaPT2基因转入酵母突变体MB192中,以野生型菌株YPH084为对照,分别检测YTaPT2,YPH084和MB192酸性磷酸酶分泌情况,生长情况以及对培养基的磷吸收情况,得到结论：TaPT2的功能与酵母磷转运子编码基因PHO84相似,具有增强酵母吸收磷的作用。     

Purity Control of the Production of Baker''s Yeast Employing a Fluorescent Antiserum

A simple staining procedure for the rapid detection of wild yeasts contaminating baker's yeast during the course of industrial production is described. Fluorescein-labeled, specific antiserum against Saccharomyces cerevisiae is applied to smears of baker's yeast which are then examined by ultraviolet microscopy. Optimal results are obtained with the combined phase contrast and fluorescence which makes the S. cerevisiae appear green, whereas cells of wild yeasts are visible in bright red counterstain. With this method, wild yeasts could be identified in baker's yeast at a dilution of 1:10,000.     

Dis1: A Yeast Gene Required for Proper Meiotic Chromosome Disjunction

Mutants at a newly identified locus, DIS1 (disjunction), were detected by screening for mutants that generate aneuploid spores (chromosome VIII disomes) at an increased frequency. Strains carrying the partially dominant alleles, DIS1-1 or DIS1-2, generate disomes at rates up to 100 times the background level. Mitotic nondisjunction is also increased 10- to 50-fold over background. Half-tetrad analysis of disomes for a marked interval on chromosome VIII yields wild-type map distances, indicating that a general recombination deficiency is not the cause of nondisjunction. Meiotic nondisjunction in DIS1 mutants is not chromosome specific; 5% of the spores disomic for chromosome VIII are also disomic for chromosome III. Although only one disomic spore is found per exceptional ascus most of the disomes appear to be generated in the first meiotic division because recovered chromosome VIII disomes contain mostly nonsister chromosomes. We propose that disome generation in the DIS1 mutants results from precocious separation of sister centromeres.     

A New Method to Distinguish between Meiotic and Premeiotic Recombinational Events in DROSOPHILA MELANOGASTER

A new method is proposed to distinguish between meiotic and premeiotic exchange events in Drosophila melanogaster males associated with male recombination activities. The method was applied to data that have accumulated in this laboratory during the past five years, and it was concluded that a large fraction, perhaps the overwhelming majority, of the male recombinants were due to exchange events that took place before meiosis.