A fragment of the yeast DNA repair protein Rad4 confers toxicity to E. coli and is required for its interaction with Rad7 protein

The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotide excision repair. Plasmids carrying the wild-type RAD4 gene cannot be propagated in Escherichia coli. In this study, a rad4 mutant that can be grown in E. coli was isolated. This rad4 allele is deleted of a large positively charged segment of the RAD4 coding region which is toxic to E. coli when expressed alone. The deletion mutant retains its ability to interact with Rad23 protein but not with Rad7 protein and is defective in nucleotide excision repair. The smallest Rad4 fragment that is toxic to E. coli consists of 336 amino acids with a calculated pI = 9.99.     

拟南芥组蛋白分子伴侣AtHIRA参与体细胞同源重组及盐胁迫响应

HIRA是组蛋白H3.3的特异分子伴侣, 在组蛋白H3.3掺入染色质的过程中发挥重要作用。研究表明, HIRA在哺乳动物胚胎发育和DNA损伤修复过程中不可或缺。而目前人们对于植物中HIRA同源基因功能的研究相对较少。该研究主要关注拟南芥(Arabidopsis thaliana) AtHIRA基因在植物体细胞同源重组以及减数分裂同源重组过程中的功能。将体细胞同源重组和减数分裂同源重组报告系统分别导入野生型和hira-1突变体后统计同源重组频率, 结果表明在正常生长条件下及在伯莱霉素(bleomycin)或UV-C处理条件下, hira-1突变体体细胞的分子内和分子间同源重组频率均低于野生型。而在正常生长条件下, 野生型与hira-1突变体花粉母细胞间的减数分裂同源重组频率没有明显差异, hira-1突变体的DNA损伤水平与野生型接近。qRT-PCR结果表明, DNA损伤修复相关基因RAD51和RAD54在hira-1突变体中的表达水平均高于野生型。此外, 盐胁迫处理实验表明, hira-1突变体对于高盐胁迫更加敏感。综上, AtHIRA在拟南芥体细胞同源重组及盐胁迫响应过程中发挥了一定作用。     

The Aspergillus niger carbon catabolite repressor encoding gene, creA

In order to undertake a comparative analysis of carbon catabolite repression in two Aspergillus species, the creA gene has been isolated from A. niger by cross hybridization, using the cloned A. nidulans gene. The A. niger gene has been shown to be functional in A. nidulans by heterologous complementation of the creA204 mutation of A. nidulans. Overall, the genes show 90% sequence similarity (82% identity) at the amino acid (aa) level. There were some striking similarities between the aa sequences encoded by the two fungal creA genes and two genes involved in carbon catabolite repression in Saccharomyces cerevisiae. The zinc-finger regions showed 96% similarity (84% identity) with the zinc-finger region of the MIG1 gene of S. cerevisiae. The CREA protein contains a stretch of 42 aa that is identical in A. niger and A. nidulans, and these show 81% similarity (33% identity) with a region of the S. cerevisiae RGR1 gene.     

夹竹桃灭钉螺效果初报

A laboratory experiment at 20±5℃ shows that the water extract of fresh Nerium indicum had an obvious effect on killing Oncomelania hupensis. Treated with 0.1% water extract for four days, the mortality of O. hupensis was up to 100%. The effect of different tissues of N. indicum on O. hupensis was in order of stem phloem>leaf>root phloem>flower. The effect of N. indicum on O. hupensis was about ten times higher than that of Pterocarya stenoptera and Rumex japonicus, and was equal to that of 1×10^-3mg·L^-1 niclosamidum.     

薏苡黑粉菌基因组测序揭示其物种分类及适应性进化

以薏苡黑粉菌病瘿为研究对象,采用固体和液体培养方法对薏苡黑粉菌进行分离纯化培养和形态观察;利用Illumina、Pacbio测序和Hi-C辅助基因组组装技术对黑粉菌进行测序、组装和注释,分析其基因组结构和组成特征。通过与其他6个黑粉菌物种基因组进行比较基因组学研究,分析薏苡黑粉菌的基因家族进化和系统发育关系。在pH 7的固体PDA和液体PDB培养基中实现了薏苡黑粉菌的纯化培养,固体培养菌落表现为白色、表面隆起、具褶皱,较湿润且不透明的形态特征。Illumina、Pacbio和Hi-C组装结果显示,薏苡黑粉菌具有20对染色体(2n=40),基因组大小20.093 417 Mb,GC含量53.81%;共预测和注释到7 476个蛋白编码基因和164个非编码基因,占整个基因组长度的61.02%和0.285 3%;查找到3 674个散在重复序列和8 139个串联重复序列,分别占整个基因组长度的2.754 4%和1.605 4%。同源基因家族聚类显示,7个黑粉菌物种Ustilago coicis、U. bromivora、U. maydis、U. hordei、Sporisorium reilianum、S. scitamineum和S. graminicola的编码基因被聚类为6 999个基因家族,其中共有基因家族5 379个。薏苡黑粉菌包含5 339个单拷贝基因、105个多拷贝基因、89个特异基因、752个其他基因和188个未分类基因。基因家族进化和系统发育聚类表明,薏苡黑粉菌U. coicis在~60.9 MYA分化出来,与雀麦黑粉菌U. bromivova、大麦坚黑粉菌U. hordei聚为一支,表现为更近的亲缘关系;而玉米黑粉菌U. maydis在~66.6 MYA分化出来与3个孢堆黑粉菌物种S. scitamineum、S. graminicola和S. reilianum聚为一支。     

Cloning and characterization of femA and femB from Staphylococcus epidermidis

A DNA fragment was identified and cloned from Staphylococcus epidermidis (Se) using femA from S. aureus (Sa) as a heterologous hybridization probe. DNA sequence analysis of a portion of this clone revealed two complete ORFs highly related to femA and femB of Sa. The genomic arrangement of the Se femA/B complex was nearly identical to that observed in Sa. Intra- and interspecies relatedness of these genes and conservation of genomic organization were consistent with gene duplication of one of these genes in an ancestral organism. Recombinant FEMA, produced in Escherichia coli (Ec), was purified to near homogeneity. Identity of the purified protein was verified by N-terminal amino acid (aa) sequence analysis.     

Schizosaccharomyces malidevorans and Sz. octosporus homologues of Sz. pombe rad9, a gene that mediates radioresistance and cell-cycle progression

The rad9 gene of Schizosaccharomyces pombe is involved in promoting resistance to ionizing radiation and UV light, as well as regulating cell cycle progression after irradiation. We have isolated functional rad9 cognates from two other fission yeasts, Sz. malidevorans and Sz. octosporus, that can restore radioresistance and the radiation-induced G2 delay response to Sz. pombe rad9::ura4 cells. The Sz. pombe and Sz. malidevorans genes are identical at the nucleotide sequence level, which reflects their close evolutionary relationship. Each bears three introns and codes for a 47464-Da protein that contains 426 amino acids (aa). In contrast, Sz. octosporus rad9 contains five introns and codes for a 48210-Da protein that is 432-aa long. The Sz. pombe rad9 product is only 65% identical and 80% similar to the corresponding Sz. octosporus gene product. All of the strains synthesize a rad9 RNA of approx. 1.6 kb. The presence of a rad9-like gene in these yeasts suggests that the cellular process(es) mediated by rad9, and used by these organisms to increase survival and transiently delay cycling in G2 after irradiation, are conserved. The isolation, analyses and comparison of rad9 genes from different organisms should aid in elucidating the specific biological role of the corresponding protein and especially help pinpoint regions important for function.     

Genomic organization of mouse Dishevelled genes

We report the isolation and characterization of two new genomic loci corresponding to the mouse Dishevelled (Dvl) genes Dvl2 and Dvl3. The Dvl genes are homologs of the Drosophila dsh segment polarity gene, and are involved in the Wnt/wingless signal transduction pathway. Dvl2 and Dvl3 genomic clones were isolated from a mouse 129 strain λFIXII genomic library and have identical exon/intron organization to Dvll. All three Dishevelled genes span 15 exons and 14 introns and have a number of conserved splice junction sites.     

Characterization of two highly similar Rad51 homologs of Physcomitrella patens

The moss Physcomitrella patens, which is a land plant with efficient homologous recombination, encodes two Rad51 proteins (PpaRad51.1 and PpaRad51.2). The PpaRad51.1 and PpaRad51.2 proteins, which share 94 % identity between them, interact with themselves and with each other. Both proteins bind ssDNA and dsDNA in a Mg(2+) and pH-dependent manner, with a stoichiometry of one PpaRad51.1 monomer per 3(+/-1) nt or bp and one PpaRad51.2 monomer per 1(+/-0.5) nt or bp, respectively. At neutral pH, a 1.6-fold excess of both proteins is required for ssDNA and dsDNA binding. PpaRad51.1 and PpaRad51.2 show ssDNA-dependent ATPase activity and efficiently promote strand annealing in a nucleotide-independent but in a Mg(2+)-dependent manner. Both proteins promote joint-molecule formation, DNA strand invasion and are able to catalyse strand exchange in the presence of Mg(2+) and ATP. No further increase in the activities is observed when both proteins are present in the same reaction. None of the PpaRad51 gene products complement the DNA repair and recombination phenotype of Saccharomyces cerevisiae rad51delta mutants. However, PpaRad51.1 confers a dominant-negative DNA repair phenotype, and both PpaRad51 proteins reduce the levels of double-strand break-induced recombination when overexpressed in S. cerevisiae wt cells. These results suggest that both PpaRad51 proteins are bona fide Rad51 proteins that may contribute, in a different manner, to homologous recombination, and that they might replace ScRad51 in a hypothetical yeast protein complex inactivating different functions required for recombinational repair.     

盐胁迫下大豆根组织定量PCR分析中内参基因的选择

实时荧光定量PCR已广泛用于基因表达的分析, 适当的内参基因选择是获得准确分析结果的关键。在大豆(Glycine max)分子生物学研究中, 逆境响应基因和microRNA (miRNA)表达的内参辅助检测基因均有哪些目前尚不清楚。该研究选用不同盐梯度和时间点组合处理的大豆根组织为材料, 对已报道的其它条件下表达相对稳定的内参基因(ACT、ACT2/7、CYP2、ELF1A、ELF1B、F-Box、TUA和UBC2)以及miRNA内参基因(U6、miR1515a、miR1520c、miR1520d、miR171a和miR171b)的表达情况进行了全面检测; 并采用Δ-Ct、Bestkeeper、NormFinder和Genorm四种方法对检测结果进行了综合分析, 发现ELF1B和CYP2适合作为大豆根系盐胁迫响应基因研究的内参基因, miR1515a和U6适合作为盐胁迫下大豆根组织miRNA研究的内参。上述研究结果为大豆盐胁迫响应基因和miRNA表达及其进一步的功能研究奠定了基础。     

基于Cfku70基因失活策略构建果生刺盘孢的高效基因敲除底盘菌株

果生刺盘孢侵染危害多种植物,是重要的植物病原真菌。在一些丝状真菌中,敲除非同源末端连接修复通路的关键基因ku70或ku80可显著提高同源重组效率,进而提高靶基因置换频率。本研究从果生刺盘孢基因组鉴定到Cfku70和Cfku80两个基因,并明确了基因失活对菌株生物学表型和基因敲除效率的影响。敲除Cfku70或Cfku80不影响菌株的菌落形态、营养生长、产孢、分生孢子萌发、侵染结构发育和致病;Cfku70基因敲除还大幅提升3个测试基因的敲除效率。本研究证实Cfku70基因失活能显著提高果生刺盘孢的基因敲除效率,适宜作为高效基因敲除的底盘菌株,研究结果为通过批量敲除策略筛选新型致病因子奠定重要基础。     

葡萄NCED基因家族进化及表达分析

9-顺式-环氧类胡萝卜素双加氧酶(NCED)是植物体内ABA生物合成的关键限速酶, 参与植物对干旱、外源ABA和高盐的响应过程, 降低环境胁迫对植株的危害。基于全基因组鉴定分析葡萄(Vitis vinifera) NCED基因家族成员, 探讨各成员的物种进化关系及各个基因成员在不同组织中的时空表达模式及对干旱、ABA和高盐(NaCl)胁迫的响应, 为进一步揭示该基因家族成员的生物学功能奠定基础。在葡萄基因组中共发现12个NCED基因。其推测的编码蛋白质长度在510 (VvNCED2)-625 aa (VvNCED10)之间。VvNCED蛋白的分子量最大值是70.53 kDa (VvNCED10), 最小值是57.85 kDa (VvNCED2)。在从祖先基因分化之后, 葡萄NCED基因发生了5次复制事件, 同时有2次丢失事件。NCED1/2、NCED3/4、NCED6/7和NCED9/10基因对被认为是通过片段复制产生。上述4对复制基因复制时间分布在3.08-120.0百万年前, 晚于单双子叶植物分化的时间。与对照相比, VvNCED1在ABA处理48小时后显著上调(72.1%), 而VvNCED2显著下调(84.0%)。VvNCED6只在干旱处理14、21和28天的根系中表达量高于对照, 分别为对照的2.49、1.05和1.09倍。VvNCED7只在干旱处理14天的根系中表达量高于对照, 为对照的1.07倍。在ABA处理72小时后, VvNCED3表达量较对照显著下调(59.5%), 而VvNCED4较对照显著上调(169.9%)。VvNCED3/VvNCED4分别在NaCl处理24和48小时出现显著性峰值, 较对照分别上调219.2%和114.4%。保守结构域不同组成和不同胁迫处理下差异表达模式是NCED蛋白发生功能分化的基础。推测NCED在进化过程中发生的功能分化有利于复制事件的发生。     

葡萄NCED基因家族进化及表达分析

9-顺式-环氧类胡萝卜素双加氧酶(NCED)是植物体内ABA生物合成的关键限速酶, 参与植物对干旱、外源ABA和高盐的响应过程, 降低环境胁迫对植株的危害。基于全基因组鉴定分析葡萄(Vitis vinifera) NCED基因家族成员, 探讨各成员的物种进化关系及各个基因成员在不同组织中的时空表达模式及对干旱、ABA和高盐(NaCl)胁迫的响应, 为进一步揭示该基因家族成员的生物学功能奠定基础。在葡萄基因组中共发现12个NCED基因。其推测的编码蛋白质长度在510 (VvNCED2)-625 aa (VvNCED10)之间。VvNCED蛋白的分子量最大值是70.53 kDa (VvNCED10), 最小值是57.85 kDa (VvNCED2)。在从祖先基因分化之后, 葡萄NCED基因发生了5次复制事件, 同时有2次丢失事件。NCED1/2、NCED3/4、NCED6/7和NCED9/10基因对被认为是通过片段复制产生。上述4对复制基因复制时间分布在3.08-120.0百万年前, 晚于单双子叶植物分化的时间。与对照相比, VvNCED1在ABA处理48小时后显著上调(72.1%), 而VvNCED2显著下调(84.0%)。VvNCED6只在干旱处理14、21和28天的根系中表达量高于对照, 分别为对照的2.49、1.05和1.09倍。VvNCED7只在干旱处理14天的根系中表达量高于对照, 为对照的1.07倍。在ABA处理72小时后, VvNCED3表达量较对照显著下调(59.5%), 而VvNCED4较对照显著上调(169.9%)。VvNCED3/VvNCED4分别在NaCl处理24和48小时出现显著性峰值, 较对照分别上调219.2%和114.4%。保守结构域不同组成和不同胁迫处理下差异表达模式是NCED蛋白发生功能分化的基础。推测NCED在进化过程中发生的功能分化有利于复制事件的发生。