Changes in the localization of the Saccharomyces cerevisiae anaphase-promoting complex upon microtubule depolymerization and spindle checkpoint activation

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase in the ubiquitin-mediated proteolysis pathway (UMP). To understand how the APC/C was targeted to its substrates, we performed a detailed analysis of one of the APC/C components, Cdc23p. In live cells, Cdc23-GFP localized to punctate nuclear spots surrounded by homogenous nuclear signal throughout the cell cycle. These punctate spots colocalized with two outer kinetochore proteins, Slk19p and Okp1p, but not with the spindle pole body protein, Spc42p. In late anaphase, the Cdc23-GFP was also visualized along the length of the mitotic spindle. We hypothesized that spindle checkpoint activation may affect the APC/C nuclear spot localization. Localization of Cdc23-GFP was disrupted upon nocodazole treatment in the kinetochore mutant okp1-5 and in the cdc20-1 mutant. Cdc23-GFP nuclear spot localization was not affected in the ndc10-1 mutant, which is defective in spindle checkpoint function. Additional studies using a mad2Delta strain revealed a microtubule dependency of Cdc23-GFP spot localization, whether or not the checkpoint response was activated. On the basis of these data, we conclude that Cdc23p localization was dependent on microtubules and was affected by specific types of kinetochore disruption.     

Microdissection and microcloning from the proximal region of mouse chromosome 7: isolation of clones genetically linked to the pudgy locus

Microdissection and microcloning have been utilized in order to create a bank of clones from the proximal region of mouse chromosome 7. Several important loci map to this area, including the albino locus (c), pink-eye dilution (p), and the developmental mutant, pudgy (pu). By use of interspecific crosses between Mus musculus domesticus and Mus spretus, we have generated backcross progeny segregating for the mutations chinchilla (cch) and pink-eye dilution (p). Exploiting the evolutionary divergence between the two species, we have analyzed the inheritance of restriction fragment length variants of three microclones and their linkage to the two markers cch and p, respectively. All three clones studied map to the dissected region, and as such also show genetic linkage to the pudgy locus. This bank of chromosome 7-derived microclones should provide molecular start points for the isolation of a variety of developmental loci of unknown gene product, including the pudgy locus.     

Investigation of the potential for mitotic recombination in the mouse

A variation of the mouse spot test is described that is designed to distinguish between spots of altered coat colour that arise by reciprocal mitotic recombination and those caused by somatic mutation or non-disjunction. Mouse fetuses that were heterozygous for two, linked coat colour genes were irradiated (1.5 Gy X-rays) in utero at 10.25-10.50 days post coitum (p.c.) or left untreated. Subsequently, the coats were classified for the presence of spots of altered colour. The irradiated embryos were heterozygous for the linked genes pink-eyed dilution (p) and albino (c) and were produced by both the repulsion and coupling crosses. Half of the reciprocal recombination events between the centromere and the proximal marker (p), in heterozygotes with p and c in repulsion, should produce twin spots. No such twin spots would be expected from a similar event in the coupling heterozygotes. The coats of 238 irradiated and 208 untreated repulsion heterozygotes plus 107 irradiated and 314 untreated coupling heterozygotes were classified for spots. One irradiated, repulsion heterozygote had a diffuse twin spot that was only recognisable by microscopic examination of the hairs. We conclude that if the treatment described induces mitotic recombination in the mouse, it does so with low efficiency.     

Separation of bovine spermatozoa proteins using 2D-PAGE revealed the relationship between tektin-4 expression patterns and spermatozoa motility

Poor semen quality has long been associated with bull infertility. However, the molecular basis in spermatozoa cells underlying the mechanisms of bull infertility remain unknown. The purpose of this study was to determine whether there is any protein in bovine spermatozoa related to semen quality. Semen samples from 18 Brahman bulls, 3 to 10 yrs of age, were assessed for semen quality in terms of spermatozoa motility and spermatozoa morphology. Spermatozoa extracts were separated using 2D-PAGE followed by staining with Coomassie blue. At least one duplicate gel was performed for each sample. Each gel was scanned with an ImageScanner System and analyzed for spots by ImageMaster 2D platinum software. The related protein spot(s) with semen quality was cut from the gel and identified by LC MS/MS. The results showed that at least 600 protein spots were detected in the spermatozoa extracts of the Brahman bulls. Of all these spots, there were 3 of 56 kDa at pI 6.4, 6.6 and 6.8 (Z₁, Z₂ and Z₃, respectively) that clearly showed different expression pattern among 18 Brahman bulls. Of 18 bulls (a) five showed the presence of spot Z₁ and Z₂ (pattern A) (b) one of spot Z₃ (pattern B) (c) five of spot Z₂ and Z₃ (pattern C) (d) one of spot Z₁ (pattern D) and (e) six of spot Z₂ (pattern E). Identification of spot Z₁, Z₂ and Z₃ by LC MS/MS had a similar result as matched to the tektin-4 protein of Bos taurus with a respective score of 171, 557 and 591. The statistical analysis of the 56 kDa protein patterns, tektin-4, indicated a significant effect on spermatozoa motility (P < 0.05) albeit non-significant on spermatozoa morphology. The bulls which showed pattern A had a higher percentage of spermatozoa motility than pattern E (P < 0.05) and not different from pattern C (P > 0.05). The statistical analysis also revealed that the presence of spot Z₁ had an effect on the percentage of spermatozoa motility (P < 0.01), whereas the presence of spot Z₂ and Z₃ had no effect (P > 0.05). The correlation coefficient between the relative protein content of spot Z₁ and the percentage of spermatozoa motility was 0.49. Our study demonstrates that the expression patterns of tektin-4 were a proxy for an effect on spermatozoa motility and consequently bull infertility. It may be that these protein patterns can be used as markers for improving bovine reproduction.     

Benzylideneacetone, an eicosanoid biosynthesis inhibitor enhances baculovirus pathogenicity in the diamondback moth, Plutella xylostella

Benzylideneacetone (BZA) is a monoterpenoid compound produced by an entomopathogenic bacterium, Xenorhabdus nematophila. BZA inhibits phospholipase A₂ to suppress biosynthesis of eicosanoids that mediate immune responses in insects. In response to per os infection of Autographa californica multiple nucleopolyhedrosis virus (AcMNPV), the diamondback moth, Plutella xylostella, developed red spots on the midgut epithelium. The midgut exhibiting red spot formation suffered abnormal cell integrity, such as genomic DNA fragmentation and condensed spots in the nucleoplasm. The number of red spots increased with viral dose and incubation time after the viral treatment. BZA inhibited the formation of the midgut red spots in a dose-dependent manner. However, the inhibitory effect of BZA on the red spot formation was reversed by addition of arachidonic acid, suggesting that the red spot response may be mediated by eicosanoids. BZA treatment resulted in significant enhancement of AcMNPV occlusion body (OB) pathogenicity to P. xylostella.     

碱性pH条件下白念珠菌钙离子通道CCH1和MID1基因对钙内流的影响及Crz1p转录因子对其的调控作用

白念珠菌Candida albicans对环境pH的适应能力与其致病性有密切关系,钙信号转导途径介导许多环境压力的应答并伴随胞内钙离子浓度的瞬间变化。通过构建钙通道基因CCH1和MID1的缺失突变株,在碱性pH条件下,研究其对胞内钙内流的影响以及转录因子Crz1p对CCH1和MID1基因的调控作用。使用二步法PCR介导的基因敲除技术构建cch1Δ/Δ和mid1Δ/Δ突变菌株,利用流式细胞术比较野生型和突变型菌株在碱性pH条件刺激下胞内钙的瞬间变化,进一步构建pPHO89-LacZ重组质粒并利用β-半乳糖苷     

Benzo(a)pyrene and X-rays induce reversions of the pink-eyed unstable mutation in the retinal pigment epithelium of mice

The pink-eyed unstable (p(un)) mutation is the result of a 70kb tandem duplication within the murine p gene. Homologous deletion/recombination of the locus to wild-type occurs spontaneously in embryos and results in pigmented spots in the fur and eye that persist for life. Such deletion events are also inducible by a variety of DNA damaging agents, as we have observed previously with the fur spot assay. Here, we describe the use of the retinal pigment epithelium (RPE) of the eye to detect reversion events induced with two differently acting agents. Benzo(a)pyrene (B(a)P) induces a high frequency, and X-ray exposure a more modest increase, of p(un) reversion in both the fur and the eye. The eye-spot assay requires fewer mice for significant results than the fur spot assay. Previous work had elucidated the cell proliferation pattern in the RPE and a position effect variegation phenotype in the pattern of p(un) reversions, which we have confirmed. Acute exposure to B(a)P or X-rays resulted in an increased frequency of reversion events. The majority of the spontaneous reversions lie toward the periphery of the RPE whereas induced events are found more centrally, closer to the optic nerve head. The induced distribution corresponds to the major sites of cell proliferation in the RPE at the time of exposure, and further advocates the proposal that dividing cells are at highest risk to develop deletions.     

微管解聚剂和光合作用抑制型除草剂诱导的黑麦和玉米中蛋白质组分变化的比较研究

研究了黑麦、玉米在经甲基胺草膦和Atrazine两种除草剂处理后叶绿素含量、叶绿体和根尖分生组织内的蛋白质组份变化,实验结果表明,0.1 mg·L^-1的Atrazine可使黑麦中的叶绿素含量下降（分别从对照的1.72±0.034 mg·g^-1 FW下降到1.62±0.05 mg·g^-1 FW、1.25±0.015 mg·g^-1 FW）。2种除草剂均可使黑麦、玉米中的蛋白质组份产生改变,如当分别用0.1 mg·L^-1的Atrazine处理时,黑麦分生组织中,有4个蛋白质斑点,斑点7、斑点18~20被诱导产生,12个蛋白质斑点,斑点6、斑点8~17、斑点21消失。玉米分生组织中,有4个蛋白质斑点,斑点5、斑点14~16被诱导产生,4个蛋白质斑点,斑点17~20消失。黑麦叶绿体中,有两个蛋白质斑点被诱导产生,13个蛋白质斑点,斑点1~13消失,但Atrazine处理不引起玉米叶绿素含量和叶绿体蛋白质组份的变化。4 mg·L^-1 APM可引起黑麦和玉米分生组织蛋白质组份变化,在黑麦中,1个蛋白质斑点被诱导产生,4个蛋白质斑点,斑点2~5消失。玉米分生组织中,15个蛋白质斑点,斑点4~5、斑点7~16、斑点21~23被诱导产生,4个蛋白质斑点,斑点1~3、斑点6消失。APM均不能引起2种作物中叶绿素含量和叶绿体蛋白质组份的变化。     

Chlorophyllide a Oxygenase mRNA and Protein Levels Correlate with the Chlorophyll a/b Ratio in Arabidopsis thaliana

Plants can change the size of their light harvesting complexes in response to growth at different light intensities. Although these changes are small compared to those observed in algae, their conservation in many plant species suggest they play an important role in photoacclimation. A polyclonal antibody to the C-terminus of the Arabidopsis thaliana chlorophyllide a oxygenase (CAO) protein was used to determine if CAO protein levels change under three conditions which perturb chlorophyll levels. These conditions were: (1) transfer to shaded light intensity; (2) limited chlorophyll synthesis, and (3) during photoinhibition. Transfer of wild-type plants from moderate to shaded light intensity resulted in a slight reduction in the Chl a/b ratio, and increases in both CAO and Lhcb1 mRNA levels as well as CAO protein levels. CAO protein levels were also measured in the cch1 mutant, a P642L missense mutation in the H subunit of Mg-chelatase. This mutant has reduced total Chl levels and an increased Chl a/b ratio when transferred to moderate light intensity. After transfer to moderate light intensity, CAO mRNA levels decreased in the cch1 mutant, and a concomitant decrease in CAO protein levels was also observed. Measurements of tetrapyrrole intermediates suggested that decreased Chl synthesis in the cch1 mutant was not a result of increased feedback inhibition at higher light intensity. When wild-type plants were exposed to photoinhibitory light intensity for 3 h, total Chl levels decreased and both CAO mRNA and CAO protein levels were also reduced. These results indicate that CAO protein levels correlate with CAO mRNA levels, and suggest that changes in Chl b levels in vascular plants, are regulated, in part, at the CAO mRNA level.     

由簇毛麦V染色体引起的小麦-簇毛麦染色体代换系、易位系中线粒体蛋白质组的变化(简报)

线粒体是需氧生物中的一种半自主性细胞器.在能量代谢中,它起了一个关键的作用,柠檬酸循环、电子传递和氧化磷酸化过程均在线粒体内完成.线粒体具有高度的生物化学和遗传上的独立性.含有DNA和核糖核蛋白体.负责合成生物体内2%-5%的蛋白质[1].线粒体DNA具有自身复制能力,控制着众多的遗传性状.在植物中广泛存在的细胞质雄性不育则被认为是由线粒体基因组控制的性状[2].     

Arabidopsis lyrata Small RNAs: Transient MIRNA and Small Interfering RNA Loci within the Arabidopsis Genus

Twenty-one-nucleotide microRNAs (miRNAs) and 24-nucleotide Pol IV-dependent small interfering RNAs (p4-siRNAs) are the most abundant types of small RNAs in angiosperms. Some miRNAs are well conserved among different plant lineages, whereas others are less conserved, and it is not clear whether less-conserved miRNAs have the same functionality as the well conserved ones. p4-siRNAs are broadly produced in the Arabidopsis genome, sometimes from active hot spot loci, but it is unknown whether individual p4-siRNA hot spots are retained as hot spots between plant species. In this study, we compare small RNAs in two closely related species (Arabidopsis thaliana and Arabidopsis lyrata) and find that less-conserved miRNAs have high rates of divergence in MIRNA hairpin structures, mature miRNA sequences, and target-complementary sites in the other species. The fidelity of miRNA biogenesis from many less-conserved MIRNA hairpins frequently deteriorates in the sister species relative to the species of first discovery. We also observe that p4-siRNA occupied loci have a slight tendency to be retained as p4-siRNA loci between species, but the most active A. lyrata p4-siRNA hot spots are generally not syntenic to the most active p4-siRNA hot spots of A. thaliana. Altogether, our findings indicate that many MIRNAs and most p4-siRNA hot spots are rapidly changing and evolutionarily transient within the Arabidopsis genus.     

Proximate basis of the covariation between a melanin-based female ornament and offspring quality

In contradiction to sexual selection theory, several studies showed that although the expression of melanin-based ornaments is usually under strong genetic control and weakly sensitive to the environment and body condition, they can signal individual quality. Covariation between a melanin-based ornament and phenotypic quality may result from pleiotropic effects of genes involved in the production of melanin pigments. Two categories of genes responsible for variation in melanin production may be relevant, namely those that trigger melanin production (yes or no response) and those that determine the amount of pigments produced. To investigate which of these two hypotheses is the most likely, I reanalysed data collected from barn owls (Tyto alba). The underparts of this bird vary from immaculate to heavily marked with black spots of varying size. Published cross-fostering experiments have shown that the proportion of the plumage surface covered with black spots, a eumelanin composite trait so-called plumage spottiness, in females positively covaries with offspring humoral immunocompetence, and negatively with offspring parasite resistance (i.e. the ability to reduce fecundity of ectoparasites) and fluctuating asymmetry of wing feathers. However, it is unclear which component of plumage spottiness causes these relationships, namely genes responsible for variation in number of spots or in spot diameter. Number of spots reflects variation in the expression of genes triggering the switch from no eumelanin production to production, whereas spot diameter reflects variation in the expression of genes determining the amount of eumelanin produced per spot. In the present study, multiple regression analyses, performed on the same data sets, showed that humoral immunocompetence, parasite resistance and wing fluctuating asymmetry of cross-fostered offspring covary with spot diameter measured in their genetic mother, but not with number of spots. This suggests that genes responsible for variation in the quantity of eumelanin produced per spot are responsible for covariation between a melanin ornament and individual attributes. In contrast, genes responsible for variation in number of black spots may not play a significant role. Covariation between a eumelanin female trait and offspring quality may therefore be due to an indirect effect of melanin production.     

基于ESDA的西北太平洋柔鱼资源空间热点区域及其变动研究

以西北太平洋(150°E—160°E、38°N—45°N)柔鱼Ommastrephes bartramii为研究对象,以2007和2010年鱿钓渔业的原始点位数据为基础,利用常规统计和探索性空间数据分析(ESDA)中的全局空间自相关分析方法,对西北太平洋柔鱼资源空间分布及其变动进行了研究。结果表明,西北太平洋柔鱼资源呈现较强的聚集分布特征,但不同空间位置差异较大。利用GIS和ESDA的局部自相关方法进行了柔鱼资源热冷点空间分布的制图,结果显示2007年研究区存在3个显著的热点和1个冷点,2010年与之存在较为明显的差异,具有1个热点和4个冷点。变化检测分析表明,两个年份间出现1个热点区域和1个冷点区域未发生变化,另有2个区域从2007年热点变成2010年冷点;此外,非热冷点之间的变动在研究区占据了主导地位。景观指数分析表明,研究区柔鱼资源热冷点格局的复杂性和自相似性并不高,但其异质性和聚集性非常强,且整体性和凝聚度均很高。分析认为,2007年西北太平洋柔鱼渔区的形成受温度和海流的影响,近一半作业渔区产量相对较高,但并未充分保证空间热点的大面积形成,空间热点和冷点分布面积大致相当;2010年整个西北太平洋柔鱼渔场受亲潮势力影响,空间热点较为集中、空间冷点较多且分散。     

Genotoxic activity in vivo of the naturally occurring glucoside,cycasin, in the Drosophila wing spot test

Cycasin, methylazoxymethanol-β-glucoside, is a naturally occurring carcinogenic compound. The genotoxicity of cycasin was assayed in the Drosophila wing spot test. Cycasin induced small single and large single spots on feeding at 10 μmol/g medium. The presence of these spots indicates that cycasin is genotoxic in Drosophila melanogaster. Microorganisms which showed β-glucosidase activity for cleaving cycasin to toxic aglycon were isolated from gut flora of the Drosophila larvae. Consequently, the Drosophila wing spot test would be useful for mutagenicity screening of other naturally occurring glucosides.     

The Saccharomyces cerevisiae Ca2+ channel Cch1pMid1p is essential for tolerance to cold stress and iron toxicity

Cch1p and Mid1p are components of a high-affinity Ca(2+)-permeable channel in the yeast plasma membrane. Here, we show that growth of mutants in the Cch1pMid1p channel is markedly hypersensitive to low temperature and to high iron concentration in the medium. Both phenotypes were suppressed by high Ca(2+) concentration. Iron stress elicited an increased Ca(2+) influx into both wild type and cch1Deltamid1Delta yeast. Inhibition of calcineurin strongly depressed growth of iron-stressed wild type yeast, indicating that calcineurin is a downstream element of the iron stress response. Iron hypersensitivity of the cch1Deltamid1Delta mutant was not associated with an increased iron uptake. An involvement of oxidative stress in the iron-hypersensitive phenotype was indicated by the findings that the antioxidants tocopheryl acetate and (ethyl)glutathione improved growth and viability of the iron-stressed mutant. Further, the degree of glutathione oxidation was increased in the presence of iron. The results indicate that iron stress leads to an increased oxidative poise and that Cch1pMid1p is essential to tolerate this condition.     

Characterization of X-ray induced increase of mitotic cross-overs in Glycine max

Summary Three types of spots can be identified on the leaves of heterozygous light green, Y/y, Glycine max (L.) Merrill: dark green (D) and aurea (A) single spots (resembling the phenotypes of the homozygotes) and double (Db) spots consisting of adjacent D and A tissue. X-irradiation increased the frequency of each type of spot on simple and first compound leaves. The Db spots, indicative of mitotic crossing-over (MCO), increase linearly with increasing dosage. Moisture content of the seeds was independent of the rate of spot increase. At high dosages morphological alterations were observed, including spots on homozygotes, leaf area reduction, smaller seedlings, and abnormal leaf shapes. The frequency of light green spots on normal dark green, Y/Y, seedlings was tabulated and, as with all other spot types, increased with increasing X-ray dosage. Dormant soybean seeds contain leaf primordia of both simple and first compound leaves. Mature simple leaves contained more spots, reflecting a larger primordial cell number, while first compound leaves had larger spots, since each affected cell underwent more mitoses prior to leaf maturation. Within first compound leaves, the terminal leaflets developed asynchronously in relation to the lateral leaflets. Terminal leaflets were shown to be initiated first, have a larger percentage of the leaflet area covered with spots, and have larger mature leaflet area. The spontaneous rate of MCO, 3.39×10^–5 MCO events per mitosis, was increased 282-fold by 1600 R. We also ascertained that Mitomycin C is more specific for Db spot induction than X-rays. These results are compared with our similar irradiation experiments on tobacco shoot apices.     

Involvement of eddy currents in the mutagenicity of ELF magnetic fields

Possible carcinogenic and/or mutagenic activity of extremely low frequency magnetic fields was examined using somatic mutation and recombination test system of Drosophila melanogaster. An X-linked semi-dominant DNA repair defective mutation mei-41(D5) was introduced into the conventional mwh/flr test system to enhance mutant spot frequency. Virgin females of w mei-41(D5)/FM6; flr/TM6 were crossed with w mei-41(D5)/Y; mwh jv; spa(pol) males. The F(1) third instar larvae were exposed to a 50Hz, 20mT sinusoidal AC magnetic field for 24h. After moulting from pupal cases, their wings were examined under a bright field microscope to detect hair spots with mwh or flr mutant morphology. The exposure caused a statistically significant enhancement in somatic recombination spot frequency. Mutant spots arising due to chromosomal non-disjunction or terminal deletion also increased but the frequency of spots resulting from point mutation was not altered. The enhancement in the recombination spot frequency was suppressed to the control level when a culture medium without electrolytes was used during exposure. When larvae were exposed to a magnetic field in an annular dish, flies from the outer ring showed more mutant spots compared to those from the inner ring. These results suggest that the detected mutagenic activity was that of the induced eddy current, rather than that of the magnetic field itself.     

Males with a Faster Courtship Display have More White Spots and Higher Pairing Success in the Diamond Firetail,Stagonopleura guttata

There is growing evidence that female mate choice could be based on a combination of multiple signals that often involve both ornamental colourful traits and behavioural displays. The Diamond Firetail is an Australian finch with a variable number of white spots on their black flank feathers. The number of white spots is a dimorphic characteristic: females have more spots than males, and males prefer females with many spots. Previously, we found assortative pairing for spot number despite the absence of experimental evidence for female preference for male spot number. Here, we test whether the male behavioural courtship display (bobbing while waving a grass stem) correlates with male spot number and pairing success. We also test whether male spot number predicts the outcome (winner or loser) of intrasexual competition over courtship materials (grass stem, perch, nest site). Males with many spots had higher pairing success, and male spot number correlated with the intensity of courtship display. In a multivariate statistical analysis, male courtship display was the stronger predictor of male pairing success. Finally, male spot number predicted the outcome of intrasexual interactions: males with many spots consistently won contests over grass stems, perches and nest sites. We suggest that intrasexual selection could favour male spot number, whereas courtship intensity appears to be under stronger intersexual selection.     

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their relation with semen freezability

The objective was to evaluate protein profiles of equine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine whether any of these proteins were related to semen freezability. Seminal plasma was collected from 10 stallions, of high and low semen freezability, housed at the State Stud of Lower Saxony, and routinely used in AI programs. Twenty-five protein spots were identified from the two-dimensional gel (12%), seven of which were present in all samples (all proteins were identified by MALDI-MS). Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used to generate ion images of samples in one or more mass-to-charge (m/z) values, providing the capability of mapping specific molecules to two-dimensional coordinates of the original sample. Of the 25 proteins identified, two spots had greater relative content (P < 0.05) in seminal plasma samples collected from stallions with high semen freezability: spot 5 (80-85 kDa, isoelectric point [pI] 7.54), identified as CRISP-3; and spot 45 (18.2 kDa, pI 5.0-5.2), identified as HSP-2. Conversely, protein content was greater (P < 0.05) in seminal plasma samples from stallions with low semen freezability: spot 7 (75.4 kDa, pI 6.9-7.4), identified as lactoferrin; spot 15 (26.7 kDa, pI 5.51), identified as kallikrein; spot 25 (25 kDa, pI 7.54), identified as CRISP-3; and spot 35 (13.9 kDa, pI 3.8-4.2), identified as HSP-1. In conclusion, there were differences in the seminal plasma protein profile from stallions with high and low semen freezability. Furthermore, CRISP-3 and HSP-2 were potential seminal plasma markers of high semen freezability.