One strand of ars189 from the maxicircle of Crithidia fasciculata, transforms Saccharomyces cerevisiae more efficiently than its complementary strand as a single stranded DNA

An autonomously replicating element (ars 189) has been isolated from the maxicircle DNA of an insect trypanosomatid Crithidia fasciculata. This 189-bp fragment contains two copies of the yeast consensus ARS sequence of (A/T)TTTATPuTTT(T/A), has an A + T composition of 79.4%, and shows a large asymmetry in the distribution of adenine and thymine residues between the two strands. The complementary strands of ars 189 have been cloned into an M 13 vector containing the URA3 gene of Saccharomyces cerevisiae. When these circular single-stranded (ss) DNAs were used to transform yeast spheroplasts, the M 13 chimeric DNA carrying the strand of ars189 rich in adenine generated approximately four times more yeast Ura⁺ transformants than the construct containing the thymine-rich strand. In contrast, both strands of yeast ARS1 cloned into an M 13 vector transformed yeast at an equivalent level. The conversion of ARS -containing ss DNAs to duplex forms in vivo and their subsequent autonomous replication have been verified by Southern hybridization analysis of extracts from yeast transformants.     

Two modules from the hypersuppressive rho mitochondrial DNA are required for plasmid replication in yeast

In the yeast hypersuppressive (HS) rho⁻ mutants most of the mitochondrial genome is deleted, but the remainder containing one of the three rep sequences is amplified. One of these sequences, rep2, and its flanking regions have been previously cloned and reported to promote autonomous plasmid replication in yeast. The present study suggests that the Ars activity associated with this HS rho⁻ mitochondrial DNA (mtDNA) fragment is due to the presence in cis of at least two modules: (i) the 11-bp consensus sequence 5′-^A_TAAA^C_TATAAA^A_T-3′, common to several ars sequences, and (ii) a palindromic sequence of the mitochondrial replicator. Proper spacing between the two modules, which varies from about 100 to 200 bp, is required for the Ars ⁺ activity.     

Unique features in the mitochondrial D-loop region of the European seabass Dicentrarchus labrax

We have cloned and sequenced the displacement-loop (D-loop) region of the mitochondrial DNA (mtDNA) from the European seabass Dicentrarchus labrax (Dl). This sequencing revealed the presence of four tandemly repeated elements (R1, R2, R3 and R4); the individual variation in mtDNA total length is entirely accounted for by their variable number. The individuals examined also possessed an imperfect copy of one of the tandem repeats (ΨR2). At least one termination-associated sequence (TAS) is present in each of the repeats and in two copies 5′ upstream from the tandem array as well. The alignment of the Dl D-loop region with D-loop sequences from four other Teleosts and one Chondrosteus showed the Dl sequence to be larger than that of other fish. The extraordinary length of the D1 D-loop sequence is also due to the 5′ and 3′ regions that are flanking the tandem array, the largest ones to date analyzed in fish. In this study, we also report the unique organization and localization of putative TAS and conserved-sequence block (CSB) elements, and the presence of a conserved 218-bp sequence in the D1 D-loop region.     

The chorion genes of the medfly. II. DNA sequence evolution of the autosomal chorion genes s18, s15, s19 and s16 in Diptera

We present a total of approximately 15 kb of DNA sequences, encompassing four chorion genes Ccs18, Ccs15, Ccs19, Cc16 and their flanking DNA in the medfly C. capitata. Comparison of coding regions, introns and intergenic sequences in five Dipteran species, D. melanogaster, D. subobscura, D. virilis, D. grimshawi and C. capitata documented an extensive divergence in introns and coding regions, but few well conserved elements in the proximal 5′ flanking regions in all species. These elements are related to conserved regulatory features of three of the genes, including tissue- and temporal regulation. In the fourth, gene s15, significant alterations in the 5′ flanking region may be responsible for its changed temporal regulation in C. capitata. One long intergenic sequence, located in the distal 5′ flanking region of gene s18, is homologous to ACE3, a major amplification control element and contains an 80-bp A/T-rich sequence, known to stimulate strong binding of the origin recognition complex (ORC) in D. melanogaster. Analysis of the nucleotide composition of all chorion genes in C. capitata and D. melanogaster showed that C. capitata exhibit less biased representation of synonymous codons than does D. melanogaster.     

Conservation of a 29-base-pair sequence within maxicircle ARS fragments from six species of trypanosomes

The maxicircles from Trypanosoma brucei, Herpetomonas samuelpessoai, Leptomonas seymouri, and Phytomonas davidi were examined for the presence of a 29-bp sequence termed CF29 that has been found in the ars 189 sequence from the Crithidia fasciculata maxicircle and in Lt-ars 189 from the maxicircle of Leishmania tarentolae. The CF29 sequence also contains a yeast consensus ARS of(T/A)TTTATPuTTT(T/A). All of the maxicircles examined contained specific fragments that hybridized to the CF29 probe. The non-replicating yeast plasmid vector YIp5 was used to clone these CF29-containing maxicircle fragments. High-frequency transformation was observed when these chimeric plasmids were used to transform Saccharomyces cerevisiae. Autonomous replication of these transforming plasmids was verified by Southern analysis of yeast-cell extracts using pBR322 as a hybridization probe. Therefore it appears that the CF29 sequence is widely conserved in kinetoplastid protozoa and is associated with ARS sequences in the maxicircles. Hybridization of the CF29 probe to a population of P. davidi minicircles was also observed. However, the YIp5 chimeric plasmid containing this CF29-hybridizing minicircle fragment failed to transform yeast.     

Sequence analysis of mouse cDNAs encoding ribosomal proteins L12 and L18

Mouse cDNAs encoding ribosomal proteins (r-proteins), L12 and L18, were isolated and their sequences determined. The L12 cDNA was found to contain 639 bp, including a coding sequence of 498 nucleotides (nt), 5' (78 nt) and 3' (45 nt) untranslated regions (UTRs), and a poly(A) tail of 18 nt. The L18 cDNA was shown to consist of 648 bp, including a coding sequence of 567 nt, 5' (26 nt) and 3' (39 nt) UTRs, and a poly(A) tail of 16 nt. The nt sequences of the protein-coding region from the mouse L12 and L18 cDNAs were found to exhibit 96% and 92% identity, respectively, with those of the rat. With the use of mouse L12 and L18 cDNA probes, multiple (at least 10) copies of the L12 and L18 gene families were shown to be present in the mouse and rat genomes. However, there was no sequence heterogeneity detected among seven L18 cDNA clones, indicating that only one copy of the L18 gene-related sequences is functional, and the other copies are presumably nonfunctional pseudogenes. The complete amino acid (aa) sequences of the mouse r-proteins, L12 and L18, were deduced from the nt sequences of their cDNA clones. L12 has 165 aa and a M_r, of 17 790, while L18 has 188 aa and a M_r of 21 570. The aa sequences of the mouse r-proteins, L12 and L18, exhibit 98% and 94% identity, respectively, to those of rat.     

稀有鮈鲫HSP70基因启动子的克隆及功能分析

热休克蛋白70(HSP70)作为一种分子伴侣,在环境毒理学中受到广泛研究。前期研究表明稀有鮈鲫HSP70基因(GrHSP70)表达量与五氯酚(pentachlorophenol, PCP)处理的浓度和时间在肝脏中呈现剂量/时间-依赖效应。为探究启动子在热休克蛋白70表达调控中的作用,根据已知的GrHSP70 cDNA序列,采用染色体步移技术克隆了GrHSP70的5'侧翼区的核苷酸序列。生物信息学分析从预测的转录起始位点(C)起的5'侧翼区域共1 487bp,潜在的转录因子结合位点包括雌激素响应元件(ERE)、Sp1结合位点(Sp1)、糖皮质激素响应元件(GRE)、TATA结合蛋白(TBP)、CCAAT/增强子蛋白结合位点(C/EBP)、Oct-1结合位点(Oct-1)、GATA转录因子结合位点(GATA-1)等。实验构建了含有启动子缺失片段的萤火虫萤光素酶(firefly luciferase)和海肾萤光素酶(renilla luciferase)报告基因表达载体,瞬时转染HeLa细胞后,利用双荧光活性检测确定获得的GrHSP70启动子具有启动活性,其核心启动位点位于转录起始点上游-1 487~-1 093bp。同时,用不同浓度PCP暴露成功转染了重组质粒(pGL-HSP70 promoter-Luc+)的HeLa细胞,培养24h后检测双荧光活性,与对照相比,随PCP浓度的增加,荧光活性均显著增加。说明在稀有鮈鲫肝脏中PCP会通过激活GrHSP70启动子来诱导GrHSP70表达,但PCP在稀有鮈鲫体内通过何种机制来调节HSP70的合成,仍然需要进一步研究。     

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.     

The nos (nitrous oxide reductase) gene cluster from the soil bacterium Achromobacter cycloclastes: Cloning, sequence analysis, and expression

The nitrous oxide (N₂O) reductase (nos) gene cluster from Achromobacter cycloclastes has been cloned and sequenced. Seven protein coding regions corresponding to nosR, nosZ (structural N₂O reductase gene), nosD, nosF, nosY, nosL, and nosX are detected, indicating a genetic organization similar to that of Rhizobium meliloti. To aid homology studies, nosR from R. meliloti has also been sequenced. Comparison of the deduced amino acid sequences with corresponding sequences from other organisms has also allowed structural and functional inferences to be made. The heterologous expression of NosD, NosZ (N₂O reductase), and NosL is also reported. A model of the Cu_A site in N₂O reductase, based on the crystal structure of this site in bovine heart cytochrome c oxidase, is presented. The model suggests that a His residue of the Cu_A domain may be a ligand to the catalytic Cu_Z site. In addition, the origin of the spectroscopically-observed Cys coordination to Cu_Z is discussed in terms of the sequence alignment of seven N₂O reductases.     

Stem Loop Sequences Specific to Transposable Element IS605 Are Found Linked to Lipoprotein Genes in Borrelia Plasmids

Background

Plasmids of Borrelia species are dynamic structures that contain a large number of repetitive genes, gene fragments, and gene fusions. In addition, the transposable element IS605/200 family, as well as degenerate forms of this IS element, are prevalent. In Helicobacter pylori, flanking regions of the IS605 transposase gene contain sequences that fold into identical small stem loops. These function in transposition at the single-stranded DNA level.

Methodology/Principal Findings

In work reported here, bioinformatics techniques were used to scan Borrelia plasmid genomes for IS605 transposable element specific stem loop sequences. Two variant stem loop motifs are found in the left and right flanking regions of the transposase gene. Both motifs appear to have dispersed in plasmid genomes and are found “free-standing” and phylogenetically conserved without the associated IS605 transposase gene or the adjacent flanking sequence. Importantly, IS605 specific stem loop sequences are also found at the 3′ ends of lipoprotein genes (PFam12 and PFam60), however the left and right sequences appear to develop their own evolutionary patterns. The lipoprotein gene-linked left stem loop sequences maintain the IS605 stem loop motif in orthologs but only at the RNA level. These show mutations whereby variants fold into phylogenetically conserved RNA-type stem loops that contain the wobble non-Watson-Crick G-U base-pairing. The right flanking sequence is associated with the family lipoprotein-1 genes. A comparison of homologs shows that the IS605 stem loop motif rapidly dissipates, but a more elaborate secondary structure appears to develop in its place.

Conclusions/Significance

Stem loop sequences specific to the transposable element IS605 are present in plasmid regions devoid of a transposase gene and significantly, are found linked to lipoprotein genes in Borrelia plasmids. These sequences are evolutionarily conserved and/or structurally developed in an RNA format. The findings show that IS605 stem loop sequences are multifaceted and are selectively conserved during evolution when the transposable element dissipates.     

过表达兴安落叶松TPP基因增强拟南芥对盐胁迫的耐受能力*

兴安落叶松(Larix gmelinii)是极为重要的造林针叶树种,具有早期速生、抗逆性强、生态效益好等特点。海藻糖参与调控干旱、寒冷、盐害等多种逆境胁迫,海藻糖-6-磷酸磷酸酶(TPP)是海藻糖合成通路的重要酶。从兴安落叶松逆境胁迫转录组中筛选到LgTPPI.1基因全长序列,克隆了其编码区(CDS),构建了重组载体并获得过表达LgTPPI.1拟南芥纯合株系。结果表明,LgTPPI.1 CDS全长1 236 bp,共编码411个氨基酸;LgTPPI.1基因的mRNA在根和茎中表达水平较低,在针叶中表达水平较高;过表达LgTPPI.1基因拟南芥在盐胁迫下海藻糖含量显著提高、脯氨酸和抗氧化酶类活性增加、胁迫响应标记基因表达上调、对盐胁迫的耐受性增强。这些结果表明,裸子植物利用与被子植物类似的海藻糖通路来耐受非生物胁迫。为后续进一步解析落叶松中海藻糖合成相关基因的功能,揭示裸子植物中针叶树对逆境的响应机制奠定基础。     

基于COI和ND5序列的中国南部唇鱼骨和间鱼骨群体遗传结构

唇鱼骨和间鱼骨均为分布较广的初级淡水鱼类,是理想的亲缘地理研究材料;且两者形态特征较为相似,不易鉴别,故两者的分布记述和物种有效性存在争议.为了解我国南部唇鱼骨和间鱼骨的群体遗传结构并探讨两者的物种有效性,本研究对8条水系的唇鱼骨和9条水系的间鱼骨共130尾个体的COI和ND5基因序列片段进行了测定,并对这两个基因的组合序列(2151 bp)进行了分析.结果表明: 在130尾个体的COI和ND5基因组合序列中,共有196个核苷酸变异位点,共检测出50个单倍型,单倍型多样性为0.964,核苷酸多样性为0.019,遗传多样性较高.基于COI和ND5基因组合序列构建的 NJ 树显示,所有种群可分为两支,支系Ⅰ包含了韩江和九龙江的全部单倍型以及瓯江的部分单倍型,余下的单倍型组成了支系Ⅱ.两支系间的遗传距离为0.036,而唇鱼骨与间鱼骨之间的遗传距离为0.027.单倍型网络图表明,韩江、九龙江种群和其他水系种群分化较大;漠阳江种群由海南岛种群扩散而来;海南岛各种群及漠阳江种群的单倍型分支与珠江水系单倍型的分支之间的亲缘关系较近,与长江水系单倍型分支之间的亲缘关系则较远;湘江、桂江和柳江之间的亲缘关系较近.AMOVA分析结果显示,地理区之间的变异约占71.2%,地理区内种群间变异约占16.6%,种群内的变异占12.2%,表明其遗传分化主要来自地理区之间.错配分析及中性检验结果显示,全部种群、唇鱼骨种群、间鱼骨种群、支系Ⅰ和支系Ⅱ在历史上均没有发生过明显的扩张.     

Two genes specifically expressed in fruiting dikaryons of Schizophyllum commune: homologies with a gene not regulated by mating-type genes

The nucleotide (nt) sequences of the Sc3 and Sc4 genes of the filamentous fungus Schizophyllum commune, and the deduced amino acid (aa) sequences, were determined; moreover, the previously published sequence for the ScI gene [Dons et al., EMBO J. 3 (1984) 2101–2106] was corrected. All three independently isolated genes were found to have similar structures and nt sequences of their coding regions. At the aa level the homology is 43–62% (63–69% in the C-terminal parts of the proteins), the hydrophobic aa predominate and the hydrophobicity patterns are similar. All three proteins contain leader sequences and eight cysteines among about 110 aa, conserved at the same positions. Yet these genes are differentially regulated: Sc1 and Sc4 are only expressed at high levels in fruiting dikaryons, whereas Sc3 is highly expressed in both monokaryons and dikaryons, independent from fruiting.