用CHO细胞进行SCE检验方法探索

以姐妹染色单体交换(sister chromatid exchange,SCE)为指标,是继微核检测之后对各种化学物质的遗传毒性做进一步研究的有效方法。通过对化学物质进行SCE的数据统计,可以在一定程度上反映其致突变作用,是遗传毒性的敏感指标。试验中,应用CHO细胞株为主要材料,建立了以体外培养细胞SCE试验方法。在CHO细胞SCE试验中,在正式制片处理前3～4h加入秋水仙素;细胞通过65～70min低渗;第3次固定甲醇-冰乙酸比例为1：2;在加入5-溴脱氧尿嘧啶(BUda)后1～1．5个细胞周期后对细胞进行收获能取到最佳制片效果,得到良好的检测效果。     

Sister Chromatid Exchange Frequencies in Escherichia coli Analyzed by Recombination at the dif Resolvase Site

Sister chromatid exchange (SCE) in Escherichia coli results in the formation of circular dimer chromosomes, which are converted back to monomers by a compensating exchange at the dif resolvase site. Recombination at dif is site specific and can be monitored by utilizing a density label assay that we recently described. To characterize factors affecting SCE frequency, we analyzed dimer resolution at the dif site in a variety of genetic backgrounds and conditions. Recombination at dif was increased by known hyperrecombinogenic mutations such as polA, dut, and uvrD. It was also increased by a fur mutation, which increased oxidative DNA damage. Recombination at dif was eliminated by a recA mutation, reflecting the role of RecA in SCE and virtually all homologous recombination in E. coli. Interestingly, recombination at dif was reduced to approximately half of the wild-type levels by single mutations in either recB or recF, and it was virtually eliminated when both mutations were present. This result demonstrates the importance of both RecBCD and RecF to chromosomal recombination events in wild-type cells.     

Unequal Crossing over at the Rrna Tandon as a Source of Quantitative Genetic Variation in Drosophila

Abdominal bristle selection lines (three high and three low) and controls were founded from a marked homozygous line to measure the contribution of sex-linked "mutations" to selection response. Two of the low lines exhibited a period of rapid response to selection in females, but not in males. There were corresponding changes in female variance, in heritabilities in females, in the sex ratio (a deficiency of females) and in fitness, as well as the appearance of a mutant phenotype in females of one line. All of these changes were due to bb alleles (partial deficiencies for the rRNA tandon) in the X chromosomes of these lines, while the Y chromosomes remained wild-type bb⁺. We argue that the bb alleles arose by unequal crossing over in the rRNA tandon.—A prediction of this hypothesis is that further changes can occur in the rRNA tandon as selection is continued. This has now been shown to occur.—Our minimum estimate of the rate of occurrence of changes at the rRNA tandon is 3 x 10^-4. As this is substantially higher than conventional mutation rates, the questions of the mechanisms and rates of origin of new quantitative genetic variation require careful re-examination.     

Sequential and Combined G-Banding for Identifying Breakpoints in Sister Chromatid Exchanges

A simple new method is described for obtaining sequential and a combination of differential sister chromatid staining and G-banding in the same metaphase. Using this method the sister chromatid exchanges and chromosome lesion breakpoints can be precisely localized in particular bands of individual chromosomes.     

Sequential and Combined G-Banding for Identifying Breakpoints in Sister Chromatid Exchanges

A simple new method is described for obtaining sequential and a combination of differential sister chromatid staining and G-banding in the same metaphase. Using this method the sister chromatid exchanges and chromosome lesion breakpoints can be precisely localized in particular bands of individual chromosomes.     

The Relationship of Homologous Synapsis and Crossing over in a Maize Inversion

Frequency of homologous synapsis at pachytene for a relatively short heterozygous inversion was compared to the frequency of crossover occurrence within the inversion and to the frequency of the presence of a recombination nodule within the homologously synapsed inverted region. Crossover frequencies were estimated from bridge-fragment frequencies at anaphase I and anaphase II. Recombination nodules (RNs) were observed in electron micrographs. Results show very similar frequencies of homologous synapsis and the occurrence of reciprocal recombination within the inverted region, consistent with the interpretation that establishment of homologous synapsis in this case is related to at least commitment to the form of resolution of crossover intermediates which gives rise to reciprocal recombination, not conversion only, events. An RN was generally found at pachytene in homologously synapsed inverted regions.     

A Role for the RSC Chromatin Remodeler in Regulating Cohesion of Sister Chromatid Arms

The precise segregation of chromosomes is critical for the proliferation and development of living organisms. Defects in this process can result in tumorigenesis and hereditary diseases. The four-subunit cohesin complex plays an essential role in chromosome segregation and genome integrity. Recently, we reported that the association of cohesin with centromeres and chromosome arms is differentially regulated by the ATP-dependent RSC chromatin-remodeling complex. Here, we propose two models to explain why the cell should have evolved special mechanisms for centromeric and sister arm cohesion and why RSC differentially regulates these processes.     

The STRUCTURAL MAINTENANCE OF CHROMOSOMES 5/6 Complex Promotes Sister Chromatid Alignment and Homologous Recombination after DNA Damage in Arabidopsis thaliana

Sister chromatids are often arranged as incompletely aligned entities in interphase nuclei of Arabidopsis thaliana. The STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC) 5/6 complex, together with cohesin, is involved in double-strand break (DSB) repair by sister chromatid recombination in yeasts and mammals. Here, we analyzed the function of genes in Arabidopsis. The wild-type allele of SMC5 is essential for seed development. Each of the two SMC6 homologs of Arabidopsis is required for efficient repair of DNA breakage via intermolecular homologous recombination in somatic cells. Alignment of sister chromatids is enhanced transiently after X-irradiation (and mitomycin C treatment) in wild-type nuclei. In the smc5/6 mutants, the x-ray–mediated increase in sister chromatid alignment is much lower and delayed. The reduced S phase–established cohesion caused by a knockout mutation in one of the α-kleisin genes, SYN1, also perturbed enhancement of sister chromatid alignment after irradiation, suggesting that the S phase–established cohesion is a prerequisite for correct DSB-dependent cohesion. The radiation-sensitive51 mutant, deficient in heteroduplex formation during DSB repair, showed wild-type frequencies of sister chromatid alignment after X-irradiation, implying that the irradiation-mediated increase in sister chromatid alignment is a prerequisite for, rather than a consequence of, DNA strand exchange between sister chromatids. Our results suggest that the SMC5/6 complex promotes sister chromatid cohesion after DNA breakage and facilitates homologous recombination between sister chromatids.     

Loss of Rec8 from Chromosome Arm and Centromere Region is Required for Homologous Chromosome Separation and Sister Chromatid Separation,Respectively, in Mammalian Meiosis

Chromosome separation in meiosis I is different from those in mitosis and meiosis II inthat homologs separate from each other in the former while sisters do so in the latter. Weshow here that meiosis-specific cohesin subunit Rec8 in mouse oocytes showsessentially the same pattern of localization to those reported in yeasts1-3 and mammalianspermatocytes4,5; Rec8 along chromosome arm (armRec8) is lost at the metaphaseI-to-anaphase I transition, although centromeric Rec8 (cenRec8) is maintained until theonset of anaphase II. Suppression of the loss of armRec8 by microinjection of anti-Rec8antibody into the oocytes inhibits homolog separation but not the first polar bodyemission (cytokinesis). Similarly, the injection of anti-Rec8 antibody into metaphase IIoocytes prevents sister separation in anaphase II after oocyte activation. These datademonstrate that the loss of armRec8 and cenRec8 is required for separation ofhomologs and sisters, respectively, but both are not required for other late mitotic eventssuch as spindle elongation and cytokinesis in mouse oocytes. Further, we propose thatloss of armRec8 (homolog separation) and cytokinesis are suppressed until anaphase Iby Securin whose destruction is regulated by spindle checkpoint-proteasome pathway,and that Topoisomerase II is required for homolog separation independently from suchpathway.     

Nonrandom Sister Chromatid Segregation and Nuclear Migration in Hyphae of Aspergillus nidulans

Radioactive conidiospores of Aspergillus nidulans were prepared by growing a purine-requiring mutant with tritiated adenine. When these spores germinated in a nonradioactive medium, the dispersion of the original chromosome set could be followed by treating the hyphae with ribonuclease and preparing radioautograms. Germinating spores with four or eight nuclei contained two highly labeled nuclei and two or six nuclei with much less or no radioactivity. Successive mitotic divisions thus distributed the deoxyribonucleic acid (DNA) of the eight spore chromosomes among only two of the progeny nuclei. The two nuclei containing the original chromosome set were not dispersed at random along the linear hypha but were usually located near the growing tip. These results are compatible with the view that chromatids containing DNA strands of identical age segregate as a unit during mitosis. They further indicate that the mechanism which disperses newly formed nuclei in the growing hypha can distinguish between nuclei containing DNA strands of different ages.     

The PP2A Inhibitor I2PP2A Is Essential for Sister Chromatid Segregation in Oocyte Meiosis II

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Highlights? PP2A colocalizes with Rec8 in mouse oocyte meiosis II ? The PP2A inhibitor I2PP2A is expressed in ascidian and mouse oocyte meiosis ? I2PP2A colocalizes with PP2A in meiosis II, independently of bipolar attachment ? I2PP2A is required for sister separation in mouse oocyte meiosis II     

Meiotic Gene Conversion and Crossing over between Dispersed Homologous Sequences Occurs Frequently in Saccharomyces cerevisiae

We have examined meiotic recombination between two defined leu2 heteroalleles present at the normal LEU2 locus and in leu2-containing plasmids inserted at four other genomic locations. In diploids where the two leu2 markers were present at allelic locations on parental homologs, the frequency of Leu2+ spores varied 38-fold, in a location-dependent manner. These results indicate that recombination in a genetic interval can be modulated by sequences at least 2.7 kb outside that interval. Leu2+ meiotic segregants were also recovered from diploids where LEU2 was marked with one heteroallele, and the other leu2 heteroallele was inserted at another genomic location. These products of ectopic interactions, between dispersed copies of leu2 sharing only 2.2 kb of homology, were recovered at a frequency comparable to that observed in corresponding allelic crosses. This high frequency of ectopic meiotic recombination was observed in crosses where both recombining partners could potentially pair with sequences at an allelic position. In addition, a significant fraction (22-50%) of these ectopic recombinants were associated with crossing over of flanking sequences.     

Intersection Between the Regulators of Sister Chromatid Cohesion Establishment and Maintenance in Budding Yeast Indicates a Multi-Step Mechanism

Sister chromatid cohesion is established during S phase and maintained until anaphase. The cohesin complex (Mcd1p/Scc1p, Smc1p, Smc3p Irr1p/Scc3p in budding yeast) serves a structural role as it is required at all times when cohesion exists. Pds5p co-localizes temporally and spatially with cohesin on chromosomes but is thought to serve as a regulator of cohesion maintenance during mitosis. In contrast, Ctf7p/Eco1p is required during S phase for establishment but is not required during mitosis. Here we provide genetic and biochemical evidence that the pathways of cohesion establishment and maintenance are intimately linked. Our results show that mutants in ctf7 and pds5 are synthetically lethal. Moreover, over-expression of either CTF7 or PDS5 exhibits reciprocal suppression of the other mutant’s temperature sensitivity. The suppression by CTF7 is specific for pds5 mutants as CTF7 over-expression increases the temperature sensitivity of an mcd1 mutant but has no effect on smc1 or smc3 mutants. Three additional findings provide new insights into the process of cohesion establishment. First, over-expression of ctf7 alleles deficient in acetylase activity exhibit significantly reduced suppression of the pds5 mutant but exacerbated toxicity to the mcd1 mutant. Second, using chromosome spreads and chromatin immuno-precipitation, we find neither cohesin complex nor Pds5p chromosomal localization is altered in ctf7 mutants. Finally, biochemical analysis reveals that Ctf7p and Pds5p co-immunoprecipitate, which physically links these regulators of cohesion establishment and maintenance. We propose a model whereby Ctf7p and Pds5p co-operate to facilitate efficient establishment by mediating changes in cohesin complex on chromosomes after its deposition.     

Population Genetics of Tandem Repeats in Centromeric Heterochromatin: Unequal Crossing over and Chromosomal Divergence at the Responder Locus of Drosophila Melanogaster

The Responder (Rsp) locus in Drosophila melanogaster is the target locus of segregation distortion and is known to be comprised of a tandem array of 120-bp repetitive sequences. In this study, we first determined the large scale molecular structure of the Rsp locus, which extends over a region of 600 kb on the standard sensitive (cn bw) chromosome. Within the region, small Rsp repeat arrays are interspersed with non-Rsp sequences and account for 10-20% of the total sequences. We isolated and sequenced 32 Rsp clones from three different chromosomes. The main results are: (1) Rsp repeats isolated from the same chromosome are not more similar than those from different chromosomes. This implies either that there are more homologous exchanges at the Rsp locus than expected or, alternatively, that the second chromosomes of D. melanogaster have diverged from one another more recently at the centromeric heterochromatin than at the nearby euchromatin. (2) The repeats usually have a dimeric structure with an average difference of 16% between the left and right halves. The differences allow us to easily identify the products of unequal exchanges. Despite the large differences between the two halves, exchanges have occurred frequently and the majority of them fall within a 29-bp interval of identity between the two halves. Our data thus support the suggestion that recombination depends on short stretches of complete identity rather than long stretches of general homology. (3) Frequent unequal crossover events obscure the phylogenetic relationships between repeats; therefore, different parts of any single repeat could often have different phylogenetic histories. The high rate of unequal crossing over may also help explain the evolutionary dynamics of the Rsp locus.