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.     

A Yeast BiFC-seq Method for Genome-wide Interactome Mapping

Genome-wide physical protein±protein interaction(PPI) mapping remains a major challenge for current technologies. Here, we reported a high-efficiency BiFC-seq method, yeastenhanced green fluorescent protein-based bimolecular fluorescence complementation(y EGFPBiFC) coupled with next-generation DNA sequencing, for interactome mapping. We first applied y EGFP-BiFC method to systematically investigate an intraviral network of the Ebola virus.Two-thirds(9/14) of known interactions of EBOV were recap...     

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.     

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.     

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.     

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.     

A Meiotic Mutant Affecting Recombination in Female DROSOPHILA MELANOGASTER

mei-S282 is a female meiotic mutant isolated from a natural population of Drosophila melanogaster. It is a recessive mutation located at approximately map position 5 on the third chromosome which has two major effects. It causes a nonuniform decrease in recombination which is most drastic in distal chromosome regions and nondisjunction of all chromosome pairs is elevated at the first meiotic division. Nondisjunctional events are positively correlated; furthermore, nondisjoining chromosomes, themselves nonrecombinant, are preferentially recovered from cells in which nonhomologs are preferentially recovered from cells in which nonhomologs are also non-recombinant.-It is concluded that mei-S282 is a defect which occurs early in meiosis I prior to the time of exchange. In the mutant, the frequency of no-exchange tetrads for each of the major chromosomes is increased-and in cells which contain two or more no-exchange tetrads, an interaction between these chromosomes leads to correlated nondisjunction. mei-S282(+) then, is an exchange precondition necessary for the normal frequency and distribution of exchanges.     

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.     

Characterization and Molecular Mapping of a New Virescent Mutant in Rice

Plant leaves play a significant role in photosynthesis. Normal chloroplast development is critical for plant growth and yield performance. Defect of the chlorophyll in chloroplasts may cause abnormal leaf colors, such as yellow, white, or stripe. Chloroplasts have their own genomes encoding for about 100 genes that are essential for plastid protein synthesis and photosynthesis （Kanno and Hirai, 1993; Sato et al., 1999）. Moreover, over 3000 proteins encoded by plant nuclear genomes target to the chloroplasts and participate in the chloroplast development and/or photosynthesis. Hitherto, a number of plant genes, which encode for enzymes involved in chlorophyll biosynthetic pathways,     

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.     

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.     

利用酵母展示系统确定空间构象性抗原表位的方法研究

酵母展示系统是研究可溶性蛋白间相互作用的有效系统.利用酵母展示系统可以简单快速地研究抗原与抗体相互作用.充分利用此系统的优势,在确定空间构象性的抗原决定簇方面发展出新的应用.利用酵母同源重组把巨噬细胞迁移抑制因子(macrophage migration inhibitory factor,MIF)序列中的不同肽段以及不同点突变体展示在酵母表面,并用3个抗MIF单克隆抗体10C3,2A12和4E10分别标记,流式细胞仪检测抗体与抗原突变体的结合.用酵母展示方法确定了MIF上与3个单克隆抗体结合的关键氨基酸序列,从而建立起一个简便可靠的确定空间构象性抗原决定簇的方法.     

A Meiotic Mutant Defective in Distributive Disjunction in DROSOPHILA MELANOGASTER

The female meiotic mutant no distributive disjunction (symbol: nod) reduces the probability that a nonexchange chromosome will disjoin from either a nonexchange homolog or a nonhomolog; the mutant does not affect exchange or the disjunction of bivalents that have undergone exchange. Disjunction of nonexchange homologs was examined for all chromosome pairs; nonhomologous disjunction of the X chromosomes from the Y chromosome in XXY females, of compound chromosomes in females bearing attached-third chromosomes with and without a Y chromosome, and of the second chromosomes from the third chromosomes were also examined. The results suggest that the defect in nod is in the distributive pairing process. The frequencies and patterns of disjunction from a trivalent in nod females suggest that the distributive pairing process involves three separate events-pairing, orientation, and disjunction. The mutant nod appears to affect disjunction only.     

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.     

Homoaconitic Acid Accumulation by a Lysine-Requiring Yeast Mutant

Homoaconitic acid, the second intermediate of the proposed pathway for lysine biosynthesis in yeast, is accumulated in the growth medium of a lysine-requiring mutant. This acid has been identified on paper and column chromatography by comparing it with authentic cis-homoaconitic acid. The infrared spectrum of the isolated material was identical with that of synthetic cis-homoaconitic acid. In addition, the chemical structure of the enzymatic product has been verified by degradation to glyoxylic and α-ketoglutaric acids after treatment with KMnO₄ and HIO₄ and by catalytic reduction to the saturated acid 1,2,4-butanetricarboxylic acid. The isolated homoaconitic acid was also identified as a substrate for a purified enzyme preparation of homoaconitase.     

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).     

一个新的水稻白化转绿突变体的生理特性和基因定位

秋丰M来源于粳稻秋丰的自然白化转绿突变株。其主要特征为前三叶白化带绿,第四叶及以后叶片均为淡绿色,抽穗时,秋丰M的颖壳和前三叶一样仍出现带绿的白化现象。不同生长时期对野生型和突变型水稻叶片色素含量测定的结果与田间观察结果一致,秋丰M确实存在着一个叶色显著变化的过程。主要农艺性状的比较结果表明,秋丰与秋丰M除穗颈长和千粒重达到极显著差异外,其他农艺性状均无明显差异。遗传分析发现该突变性状受一对隐性核基因控制。以209株培矮64S×秋丰M F_2的隐性突变个体为定位群体,将突变基因定位在水稻第2染色体长臂上,位于 SSR 标记RM475和RM2-22之间,其遗传距离分别为17.3 cM和2.9 cM,并将该基因命名为gra_(t)。