Eth-1, the Neurospora Crassa Locus Encoding S-Adenosylmethionine Synthetase: Molecular Cloning, Sequence Analysis and in Vivo Overexpression

Intense biochemical and genetic research on the eth-1(r) mutant of Neurospora crassa suggested that this locus might encode S-adenosylmethionine synthetase (S-Adomet synthetase). We have used protoplast transformation and phenotypic rescue of a thermosensitive phenotype associated with the eth-1(r) mutation to clone the locus. Nucleotide sequence analysis demonstrated that it encodes S-Adomet synthetase. Homology analyses of prokaryotic, fungal and higher eukaryotic S-Adomet synthetase polypeptide sequences show a remarkable evolutionary conservation of the enzyme. N. crassa strains carrying S-Adomet synthetase coding sequences fused to a strong heterologous promoter were constructed to assess the phenotypic consequences of in vivo S-Adomet synthetase overexpression. Studies of growth rates and microscopic examination of vegetative development revealed that normal growth and morphogenesis take place in N. crassa even at abnormally high levels of cellular S-Adomet. The degree of cytosine methylation of a naturally methylated genomic region was dependent on the cellular levels of S-Adomet. We conclude that variation in S-Adomet levels in N. crassa cells, which in addition to the status of genomic DNA methylation could modify the flux of other S-Adomet-dependent metabolic pathways, does not affect growth rate or morphogenesis.     

脱水应答转录因子CBF1的克隆与转基因小麦的分子检测

根据已发表的小麦(T.aestivum)转录因子CBF1基因序列(GenBank Accession No.AF376136),设计引物从小麦品种‘京花1号’叶片中克隆出该基因,用拟南芥RD29B基因为启动子构建含CBF1基因的逆境诱导表达载体pBAC127F(6 967 bp),以‘99-92’、‘5-98’、‘104’和‘轮选987’等冬小麦品种(系)的幼穗和幼胚为材料,基因枪转化该表达载体。经筛选与植株再生,共获得14株转基因植株及其后代株系。这14个株系经PCR分析和点杂交检测,最终确认了5-98-40、5-98-41这2个株系为转基因株系,结果表明拟南芥RD29B启动子调控下的转录因子CBF1基因已稳定整合到转基因植株中。     

Cloning of Flagellar Genes in Chlamydomonas Reinhardtii by DNA Insertional Mutagenesis

Chlamydomonas is a popular genetic model system for studying many cellular processes. In this report, we describe a new approach to isolate Chlamydomonas genes using the cloned nitrate reductase gene (NIT1) as an insertional mutagen. A linearized plasmid containing the NIT1 gene was introduced into nit1 mutant cells by glass-bead transformation. Of 3000 Nit(+) transformants examined, 74 showed motility defects of a wide range of phenotypes, suggesting that DNA transformation is an effective method for mutagenizing cells. For 13 of 15 such motility mutants backcrossed to nit(-) mutant strains, the motility phenotype cosegregated with the Nit(+) phenotype, indicating that the motility defects of these 13 mutants may be caused by integration of the plasmid. Further genetic analysis indicated that three of these mutants contained alleles of previously identified loci: mbo2 (move backward only), pf13 (paralyzed flagella) and vfl1 (variable flagellar number). Three other abnormal-flagellar-number mutants did not map to any previously described loci at which mutations produce similar phenotypes. Genomic sequences flanking the integrated plasmid in the mbo2 and vfl1 mutants were isolated and used as probes to obtain wild-type genomic clones, which complemented the motility defects upon transformation into cells. Our results demonstrate the potential of this new approach for cloning genes identified by mutation in Chlamydomonas.     

斑马鱼反转录病毒插入突变技术的发展及其在基因饱和突变与筛选中的应用

用于大规模基因突变与筛选的主要策略有化学诱变、插入突变、基因诱捕。插入突变是一种通过外源DNA整合的方式来获得突变体,并克隆得到对应突变基因的方法。运用反转录病毒介导的插入突变技术,在脊椎动物斑马鱼中已经获得了许多影响胚胎发育和细胞生长过程的突变体,并找到了对应的基因。基因诱捕技术也被运用于反转录病毒载体的构建。这套系统的建立使斑马鱼成为第一个有可能达到基因饱和突变和筛选的脊椎动物。     

Molecular Cloning of a Gene (Cfp) Encoding the Cytoplasmic Filament Protein P59nc and Its Genetic Relationship to the Snowflake Locus of Neurospora Crassa

P59Nc is a 59-kD polypeptide associated with 8-10-nm diameter cellular filaments in normal Neurospora crassa strains. Abnormally sized and shaped bundles of these structures are present in N. crassa strains carrying mutations at the locus sn (snowflake). By using molecular cloning and restriction fragment length polymorphism (RFLP) segregation analysis strategies we show here that sn is not the genetic locus of P59Nc. Several P59Nc cDNAs were cloned from a N. crassa lambda GT11 library after immunoscreening with specific polyclonal anti-P59Nc antibodies. Additional longer cDNAs were obtained from a N. crassa cDNA-lambda ZAP library. When used as probes in Southern blots of total DNA from wild-type strains, multicent-2 (a multiple mutant strain), and snowflake mutants, the P59Nc cDNAs revealed comparable patterns of hybridizing bands for all of the restriction enzymes tested. Analysis of segregation of BclI and ClaI RFLPs, detected in the genomic region of the P59Nc gene (locus cfp: cellular filament polypeptide), among a set of strains designed for RFLP mapping, or among selected progeny of crosses involving a snowflake parent, respectively, indicate that (i) there is in N. crassa a single cfp locus positioned on the right arm of linkage group VII between the locus for and the proximal breakpoint of the translocation T(VII----I)5936; (ii) the sn mutations in the centromere region of chromosome I do not represent translocations of cfp; and (iii) the snowflake mutants possesses a normal copy of the P59Nc gene on their chromosomes VII.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Vacuolar Atpase of Neurospora Crassa Is Indispensable: Inactivation of the Vma-1 Gene by Repeat-Induced Point Mutation

To analyze the phenotype of cells lacking the vacuolar ATPase, we inactivated the vma-1 gene, which encodes the catalytic subunit of the enzyme. Because preliminary experiments suggested the vma-1 gene was essential, we developed a method of simultaneously inactivating the gene and complementing it with a functional copy. We call this method repeat-induced point mutation (RIP) & Rescue. Two strains, both of which contained an extra copy of the vma-1 gene, were mated. Progeny that had inherited a functional copy of the gene at an ectopic site in the genome were selected. In some of these progeny the endogenous vma-1 gene had been altered by the RIP process. Sequencing showed the endogenous vma-1 gene had been inactivated by multiple point mutations. Progeny from strains with an inactive endogenous vma-1 gene were inviable unless a functional copy of the gene cosegregated, indicating that the vacuolar ATPase is essential in Neurospora crassa.     

羊草OEE1基因的克隆及盐胁迫下的表达

从羊草(Leymus chinensis )叶片cDNA文库中克隆得到可能编码33 kD的光系统Ⅱ(PSⅡ)外周蛋白(oxygen-evolving enhancer protein1,OEE1)全长cDNA(GenBank登录号为EF583851),命名为LcOEE1.序列分析结果表明,该cDNA全长1 107 bp,5′非编码区为32 bp,3′非编码区为71 bp,编码区长987 bp,编码328个氨基酸.BALSTp比对发现,该基因氨基酸序列与已报道的小麦和水稻中的OEE1序列具有95%和94%的相似性.聚类分析表明,该基因与小麦和水稻的亲缘关系较近,与拟南芥和菠菜OEE1基因的亲缘关系较远.Northern杂交结果表明,在200 mmol/L的NaCl处理7 d的幼叶中,OEE1 mRNA的表达量明显高于未处理的对照,说明羊草中OEEl基因受盐诱导.     

牦牛CAPN1基因的克隆与序列分析

CAPN1是影响肌肉嫩度的数量性状位点 (QTL)的候选基因。根据GenBank发表的普通牛CAPN1基因序列设计特异性引物,以天祝白牦牛cDNA为模板,分段进行PCR扩增,克隆,测序。应用生物软件BioEdit对各测序结果进行序列拼接共获得牦牛CAPN1 cDNA 片段2267bp,其中包含一个2151bp的完整的开放阅读框(ORF),以及3’和5’末端非编码区的部分序列(77bp和166bp) 。分析表明：牦牛CAPN1基因编码区全长2151bp,共编码716个氨基酸。与已报道的牛,猪,人小鼠的序列进行比较,核苷酸同源性分别为99.3%,93.9%,90.0% ,85.5% 。预测氨基酸的同源性分别为99.4%,96.1%,94.6%,89.0%,并且对牦牛CAPN1四个结构域分别进行NCBI BLAST发现四个结构域在以上四个物种中都显示出很好的保守性,最为保守的在结构域Ⅳ(>96%)。牦牛与牛产生的 14个核苷酸突变中,有3个产生了氨基酸突变,均发生在结构域Ⅲ。构建分子系统进化树表明：聚类结果与传统分类学相符。     

小麦细胞增殖核抗原基因启动子的克隆及活性分析

细胞增殖核抗原（proliferating cell nuclear antigen,PCNA）基因是DNA聚合酶δ的辅助因子,在真核细胞DNA复制及其损伤修复中发挥着重要的作用.采用高效热不对称交互PCR法（high-efficiency thermal asymmetric interlaced PCR,hiTAIL PCR）从小麦西农1 376基因组中扩增得到小麦PCNA基因启动子片段,并命名为TaPCNA启动子. PlantCARE启动子在线分析软件预测含有光应答调控元件（Box I）、脱落酸应答元件（ABRE）、花粉发育应答元件（GGTT motif,GTGA motif）及细胞周期转换结合位点（E2F-binding site）等.为了分析其启动子活性, 通过替换pBI121载体上的CaMV35S启动子,构建了TaPCNA启动子与β-葡糖醛酸酶（GUS）基因的融合表达载体,通过农杆菌介导法在烟草叶片中进行瞬时表达. GUS组织化学染色结果表明,TaPCNA基因启动子能够驱动GUS基因在烟草叶片中表达,证实了所获得的启动子序列具有启动活性.本研究通过hiTAIL-PCR法克隆得到TaPCNA基因的启动子,为深入研究该基因的功能奠定了基础.