耐辐射球菌转录因子DdrO的基因克隆与生物信息学分析

目的：克隆耐辐射球菌ddrO基因,并对其进行生物信息学分析,预测其功能。方法：根据耐辐射球菌ddrO基因序列,由Primer Premier 5设计一对引物,以提取的耐辐射球菌基因组为模板,PCR扩增获得耐辐射球菌ddrO基因,序列测定并利用生物信息学软件对ddrO基因的理化性质、高级结构及生物学功能等进行分析与预测。结果：成功获得了ddrO基因。生物信息学分析发现,ddrO基因核苷酸序列长度为396bp,编码一个131aa组成的相对分子质量为14.993kD的预测的DdrO转录因子。核酸同源性搜索及比较分析仅在与耐辐射球菌同属的Deinococcus geothermalis和Deinococcus deserti中发现高度相似的序列;蛋白同源性搜索发现一些与DdrO显著同源的蛋白,如Deide_20570（95%）,Dgeo_0336（90%）,Deide_3p02170（82%）等;结构域分析发现DdrO含有HTH（helix-turn-helix）DNA结合结构域。结论：根据生物信息学结果预测DdrO蛋白可能具有转录调控作用,参与DNA修复和复制,在耐辐射球菌的DNA损伤修复过程中发挥一定作用。     

耐辐射球菌转录因子DdrO 的基因克隆与生物信息学分析

目的:克隆耐辐射球菌ddrO基因,并对其进行生物信息学分析,预测其功能。方法:根据耐辐射球菌ddrO基因序列,由Primer Premier 5设计一对引物,以提取的耐辐射球菌基因组为模板,PCR扩增获得耐辐射球菌ddrO基因,序列测定并利用生物信息学软件对ddrO基因的理化性质、高级结构及生物学功能等进行分析与预测。结果:成功获得了ddrO基因。生物信息学分析发现,ddrO基因核苷酸序列长度为396bp,编码一个131aa组成的相对分子质量为14.993kD的预测的DdrO转录因子。核酸同源性搜索及比较分析仅在与耐辐射球菌同属的Deinococcus geothermalis和Deinococcus deserti中发现高度相似的序列;蛋白同源性搜索发现一些与DdrO显著同源的蛋白,如Deide₂0570（95%）,Dgeo₀336（90%）,Deide₃p02170（82%）等;结构域分析发现DdrO含有HTH（helix-turn-helix）DNA结合结构域。结论:根据生物信息学结果预测DdrO蛋白可能具有转录调控作用,参与DNA修复和复制,在耐辐射球菌的DNA损伤修复过程中发挥一定作用。     

家蝇变应原Tropomyosin基因的克隆、序列分析及诱导表达

对家蝇变应原原肌球蛋白(Tropomyosin)基因进行同源克隆,序列分析;构建原核表达载体并在大肠杆菌中表达。从家蝇幼虫cDNA文库中筛选获得Tropomyosin基因。以该基因的cDNA文库质粒为模板,进行PCR扩增,获得家蝇Tropomyosin完整编码序列。运用生物信息学方法对该基因及其编码蛋白的基本理化性质、信号肽、二级结构、三级结构、抗原表位和亚细胞定位等方面进行预测和分析。构建pEASY-E1-Tropomyosin重组质粒,转化到大肠杆菌OrigamiB(DE3)中进行诱导表达。Tropomyosin基因ORF全长828 bp,编码275个氨基酸,理论分子量31.6 kD;等电点为4.65,具有Tropomyosin家族的蛋白保守结构域。成功构建重组原核表达pEASY-E1-Tropomyosin并诱导表达重组蛋白。     

马铃薯StZnT11的电子克隆、表达及生物信息学分析

马铃薯锌转运蛋白(Solanum tuberosum zinc transporter 11,StZnT11)对于维持细胞中锌稳态至关重要。通过研究StZnT11在非生物胁迫和生物胁迫下的表达情况,为验证马铃薯StZnT11在青枯菌生物胁迫过程中的作用奠定了基础。从前期工作获得的表达文库中得到EST序列,利用NCBI中的Blast工具,对原始序列进行同源性分析,选择与原始序列的相似度、覆盖度、e期望值最高的一条同源对象序列。通过电子克隆,得到StZnT11基因。采用生物信息学方法对StZnT11基因的基因序列及编码的氨基酸组成、理化性质、分子进化、磷酸化位点、高级结构等多角度进行分析。结果表明,该基因cDNA全长1300bp,编码348个氨基酸,编码蛋白含23个磷酸化位点,有1个信号肽,有9个跨膜区域,是定位在质膜上的疏水性蛋白。通过氨基酸序列比对,StZnT11蛋白与烟草、番茄和辣椒等植物中的锌转运蛋白同源性较高。实时荧光定量聚合酶链反应检测结果表明,StZnT11在不同浓度的外源植物激素脱落酸(ABA)的作用下上调。组织定位检测提示StZnT11主要表达于特定组织(茎维管系统的韧皮部和叶片维管束)。这些结果为进一步进行该基因的实验克隆及功能验证研究提供了理论依据。     

油菜蔗糖转化酶基因的电子克隆和生物信息学分析

运用电子克隆技术获得油菜中一个蔗糖转化酶基因eDNA序列,同时根据此段序列设计引物以油菜eDNA为模板进行扩增。经测序得到证实。采用生物信息学方法,对该基因编码蛋白从氨基酸组成、基本理化性质、跨膜结构域、信号肽导肽、疏水性／亲水性、二级结构、亚细胞定位等方面进行了预测和分析。结果表明：该基因eDNA序列长度为2150bp,包含一个1779bp开放阅读框,编码592个氨基酸;该编码蛋白含有蔗糖转化酶的多个典型的保守结构域。同源比对分析显示,该基因编码的氨基酸序列与拟南芥等植物的蔗糖转化酶基因具有高度的相似性,进一步确定该蛋白为蔗糖蛋白酶。研究结果为该基因进一步的实验克隆,表达分析,功能鉴定奠定基础。     

植物内生枯草芽孢杆菌纳豆激酶同源基因的序列分析及其在大肠杆菌中的表达

利用基因组学和生物信息学的方法,从一种植物内生枯草芽孢杆菌菌株Bs0922的基因组DNA中克隆了纳豆激酶的同源基因NK-Bs0922,长度为1 149 bp。通过重组和转化在大肠杆菌BL21(DE3)中表达了一个大小为48.0 kD的重组蛋白。     

珍珠黄杨矮化基因BsGAI2的克隆及功能分析

GA信号转导途径或生物合成受阻是导致植物矮化的重要因素,而DELLA蛋白是GA信号传导通路中一类重要的负调节因子。本研究利用同源克隆和RACE技术,从珍珠黄杨中克隆得到BsGAI2基因的c DNA序列,全长为2305 bp,包括1836 bp完整的ORF。实时荧光定量表达分析表明,BsGAI2基因在珍珠黄杨茎中表达量最高,在根中表达量最低;BsGAI2转基因烟草呈明显矮化,节间变短,长势较慢,延迟开花等特征。利用GFP荧光检测方法鉴定转化型烟草,发现转化苗的叶、茎中均有荧光信号,并且茎木质部有明亮富集的荧光信号。这些结果均表明BsGAI2基因可能在珍珠黄杨茎节间缩短导致矮化的过程中扮演重要角色。本研究分析了BsGAI2基因编码蛋白的理化性质和功能鉴定,为今后进一步研究BsGAI2基因及DELLA蛋白家族的功能提供了理论依据。     

甘蔗MYB2转录因子的电子克隆和生物信息学分析

用电子克隆方法获得甘蔗MYB2基因,采用生物信息学方法,对该基因编码蛋白从氨基酸组成、理化性质、跨膜结构域、疏水性/亲水性、亚细胞定位、高级结构及功能域等方面进行了预测和分析。结果表明:甘蔗MYB2基因全长991bp,包含570bp的ORF,编码189个氨基酸。甘蔗MYB2基因包含有MYB功能域,在序列组成、高级结构及活性位点等方面,与玉米等其它植物的MYB2基因具有高度的相似性。研究结果为该基因的实验克隆奠定基础。     

辣椒CaPI的克隆与功能分析

【目的】PISTILLATA(PI)基因属于典型的Type II型MADS-box基因家族成员,是ABC(D)E模型中的B类基因,在植物发育过程中起着重要作用,但辣椒PI同源基因功能研究未见报道。探索辣椒PI基因功能,为深入研究植物PI同源基因的功能机制奠定基础。【方法】利用RT-PCR的方法从一年生辣椒（Capsicum annuum L.）花器官的cDNA中克隆PI同源基因CaPI,并通过生物信息学方法分析其理化性质、亚细胞定位、蛋白结构和系统进化关系;利用实时荧光定量PCR(RT-qPCR)技术分析基因在辣椒不同组织中的表达特征;构建植物过表达载体35S:CaPI,通过floral-dipping法转化拟南芥。【结果】该基因开放阅读框648 bp,编码215个氨基酸,相对分子质量为25.13 kD。氨基酸多重序列比对表明,CaPI蛋白N端含有“MGRGKIEIKRIEN”保守基序。系统进化树分析证实,CaPI与马铃薯、番茄和矮牵牛的PI同源基因亲缘关系较近。RT-qPCR证实CaPI主要在花中表达,在花萼中表达量最高,其次是花瓣,在雄蕊中低表达,而在雌蕊中几乎不表达。在拟南芥中过...     

新基因Nischarin生物信息学分析及初步验证

目的:对新基因Nischarin进行生物信息学分析,探索其新功能特征,并通过实验进行初步验证。方法:用生物信息学方法对Nischarin进行初步分析,阐明了它的基因结构、染色体定位、编码蛋白质的理化性质、相互作用基因、相互作用蛋白、亚细胞定位、蛋白质功能域等信息。最后采用细胞免疫荧光对其DNA结合位点进行初步验证。结果:对新基因Nischarin的上述性质进行了有效的预测,分析表明该基因结构复杂,相互作用基因或蛋白多,亚细胞分布预测复杂。验证了Nishcarin存在的DNA结合位点。结论:通过生物信息学分析,表明新基因Nischarin是一个复杂的基因,可能存在的多种蛋白表达形式、这些不同的蛋白可能存在不同的亚细胞分布,且该蛋白可能与多种蛋白存在相互作用,上述基因和蛋白特性可能是Ⅰ型咪唑啉受体(Imidazoline-1 receptor,I1R)复杂药理学作用的分子基础。     

A RAD52‐like single‐stranded DNA binding protein affects mitochondrial DNA repair by recombination

The plant mitochondrial DNA‐binding protein ODB1 was identified from a mitochondrial extract after DNA‐affinity purification. ODB1 (organellar DNA‐binding protein 1) co‐purified with WHY2, a mitochondrial member of the WHIRLY family of plant‐specific proteins involved in the repair of organellar DNA. The Arabidopsis thaliana ODB1 gene is identical to RAD52‐1, which encodes a protein functioning in homologous recombination in the nucleus but additionally localizing to mitochondria. We confirmed the mitochondrial localization of ODB1 by in vitro and in vivo import assays, as well as by immunodetection on Arabidopsis subcellular fractions. In mitochondria, WHY2 and ODB1 were found in large nucleo‐protein complexes. Both proteins co‐immunoprecipitated in a DNA‐dependent manner. In vitro assays confirmed DNA binding by ODB1 and showed that the protein has higher affinity for single‐stranded than for double‐stranded DNA. ODB1 showed no sequence specificity in vitro. In vivo, DNA co‐immunoprecipitation indicated that ODB1 binds sequences throughout the mitochondrial genome. ODB1 promoted annealing of complementary DNA sequences, suggesting a RAD52‐like function as a recombination mediator. Arabidopsis odb1 mutants were morphologically indistinguishable from the wild‐type, but following DNA damage by genotoxic stress, they showed reduced mitochondrial homologous recombination activity. Under the same conditions, the odb1 mutants showed an increase in illegitimate repair bypasses generated by microhomology‐mediated recombination. These observations identify ODB1 as a further component of homologous recombination‐dependent DNA repair in plant mitochondria.     

Identification and characterization of uvrA, a DNA repair gene of Deinococcus radiodurans.

Deinococcus radiodurans is extraordinarily resistant to DNA damage, because of its unusually efficient DNA repair processes. The mtcA+ and mtcB+ genes of D. radiodurans, both implicated in excision repair, have been cloned and sequenced, showing that they are a single gene, highly homologous to the uvrA+ genes of other bacteria. The Escherichia coli uvrA+ gene was expressed in mtcA and mtcB strains, and it produced a high degree of complementation of the repair defect in these strains, suggesting that the UvrA protein of D. radiodurans is necessary but not sufficient to produce extreme DNA damage resistance. Upstream of the uvrA+ gene are two large open reading frames, both of which are directionally divergent from the uvrA+ gene. Evidence is presented that the proximal of these open reading frames may be irrB+.     

Properties and applications of human DNA repair genes

The importance of understanding DNA repair processes is discussed in terms of the origins of human cancer. Several human repair genes have been mapped to specific human chromosomes using somatic cell hybrids. It is noteworthy that 3 of these genes lie in the same region of chromosome 19: genes ERCC1 and ERCC2, which are involved in nucleotide excision repair, and XRCC1, which is involved in the repair of strand breaks. The genes XRCC1 and ERCC2 were cloned from cosmid libraries prepared from DNA transformants of the CHO mutants EM9 and UV5, respectively. Analysis of the cDNA sequence of ERCC2 showed that the protein encoded by this gene is highly homologous (73%) to the RAD3 repair protein in the yeast Saccharomyces cerevisiae. Thus, the known properties of RAD3 combined with the high homology provide the first insight about the biochemical role of a human repair protein involved in the incision step of nucleotide excision repair. So far XRCC1 is the only cloned mammalian gene involved in repairing damage from ionizing radiation. The UV5 mutant line was also applied to problems in environmental mutagenesis by introducing the mouse cytochrome P(3)450 (P450IA2 subfamily) gene for metabolic activation of aromatic amines. We show in a rapid differential cytotoxicity assay with 2 compounds found in cooked beef (IQ, 2-amino-3-methylimidazo[4,5-f]quinoline and PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) that this gene is efficiently expressed in the transformed UV5P3 cells. Reversion of the repair deficiency in these cells will give a matched pair of cell lines that are metabolically proficient and repair deficient. Such lines will provide a rapid assay for genotoxic heterocyclic amines requiring activation.     

Saccharomyces cerevisiae MPH1 gene, required for homologous recombination-mediated mutation avoidance, encodes a 3' to 5' DNA helicase

The MPH1 (mutator pHenotype 1) gene of Saccharomyces cerevisiae was identified on the basis of elevated spontaneous mutation rates of haploid cells deleted for this gene. Further studies showed that MPH1 functions to channel DNA lesions into an error-free DNA repair pathway. The Mph1 protein contains the seven conserved motifs of the superfamily 2 (SF2) family of nucleic acid unwinding enzymes. Genetic analyses have found epistasis of the mph1 deletion with mutations in the RAD52 gene group that mediates homologous recombination and DNA repair by homologous recombination. To begin dissecting the biochemical functions of the MPH1-encoded product, we have expressed it in yeast cells and purified it to near homogeneity. We show that Mph1 has a robust ATPase function that requires single-stranded DNA for activation. Consistent with its homology to members of the SF2 helicase family, we find a DNA helicase activity in Mph1. We present data to demonstrate that the Mph1 DNA helicase activity is fueled by ATP hydrolysis and has a 3' to 5' polarity with respect to the DNA strand on which this protein translocates. The DNA helicase activity of Mph1 is enhanced by the heterotrimeric single-stranded DNA binding protein replication protein A. These results, thus, establish Mph1 as an ATP-dependent DNA helicase, and the availability of purified Mph1 should facilitate efforts at deciphering the role of this protein in homologous recombination and mutation avoidance.     

araB Gene and nucleotide sequence of the araC gene of Erwinia carotovora.

The araB and araC genes of Erwinia carotovora were expressed in Escherichia coli and Salmonella typhimurium. The araB and araC genes in E. coli, E. carotovora, and S. typhimurium were transcribed in divergent directions. In E. carotovora, the araB and araC genes were separated by 3.5 kilobase pairs, whereas in E. coli and S. typhimurium they were separated by 147 base pairs. The nucleotide sequence of the E. carotovora araC gene was determined. The predicted sequence of AraC protein of E. carotovora was 18 and 29 amino acids longer than that of AraC protein of E. coli and S. typhimurium, respectively. The DNA sequence of the araC gene of E. carotovora was 58% homologous to that of E. coli and 59% homologous to that of S. typhimurium, with respect to the common region they share. The predicted amino acid sequence of AraC protein was 57% homologous to that of E. coli and 58% homologous to that of S. typhimurium. The 5' noncoding regions of the araB and araC genes of E. carotovora had little homology to either of the other two species.     

repE--the Dictyostelium homolog of the human xeroderma pigmentosum group E gene is developmentally regulated and contains a leucine zipper motif.

We have cloned and characterized the Dictyostelium discoideum repE gene, a homolog of the human xeroderma pigmentosum (XP) group E gene which encodes a UV-damaged DNA binding protein. The repE gene maps to chromosome 4 and it is the first gene identified in Dictyostelium that is homologous to those involved in nucleotide excision repair and their related XP diseases in humans. The predicted protein encodes a leucine zipper motif. The repE gene is not expressed by mitotically dividing cells, and repE mRNA is first detected during the aggregation phase of development when the cells have ceased dividing and replicating genomic DNA. The mRNA level plateaus by the time the developing cells have entered multicellular aggregates and remains at the same steady-state level for the remainder of development. In addition, we have demonstrated that the level of mRNA is very low in developing cells. These observations suggest that repE may play a regulatory role in development. The data indicate that potential developmental roles for XP-related genes can be profitably studied in this system.     

Genetic analysis of DNA repair in the hyperthermophilic archaeon, Thermococcus kodakaraensis

Extensive biochemical and structural analyses have been performed on the putative DNA repair proteins of hyperthermophilic archaea, in contrast to the few genetic analyses of the genes encoding these proteins. Accordingly, little is known about the repair pathways used by archaeal cells at high temperature. Here, we attempted to disrupt the genes encoding the potential repair proteins in the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis. We succeeded in isolating null mutants of the hjc, hef, hjm, xpb, and xpd genes, but not the radA, rad50, mre11, herA, nurA, and xpg/fen1 genes. Phenotypic analyses of the gene-disrupted strains showed that the xpb and xpd null mutants are only slightly sensitive to ultraviolet (UV) irradiation, methyl methanesulfonate (MMS) and mitomycin C (MMC), as compared with the wild-type strain. The hjm null mutant showed sensitivity specifically to mitomycin C. On the other hand, the null mutants of the hjc gene lacked increasing sensitivity to any type of DNA damage. The Hef protein is particularly important for maintaining genome homeostasis, by functioning in the repair of a wide variety of DNA damage in T. kodakaraensis cells. Deletion of the entire hef gene or of the segments encoding either its nuclease or helicase domain produced similar phenotypes. The high sensitivity of the Δhef mutants to MMC suggests that Hef performs a critical function in the repair process of DNA interstrand cross-links. These damage-sensitivity profiles suggest that the archaeal DNA repair system has processes depending on repair-related proteins different from those of eukaryotic and bacterial DNA repair systems using homologous repair proteins analyzed here.