新型抗肿瘤力达霉素合成酶基因sgcD的克隆、表达和性质研究

力达霉素是由球孢链霉菌(Streptomyces globisporus)C1027产生的一种新型烯二炔类抗肿瘤抗生素, 具有很强的抗肿瘤活性. 从S. globisporus C1027中克隆获得长度为75 kb的力达霉素生物合成基因簇, 包含33个开放阅读框架(ORF). 以sgcD(ORF24)为研究对象, 对其功能进行了研究. 基因中断实验证明, sgcD与力达霉素生物合成相关. 经同源性分析, 推测sgcD编码氨基变位酶, 催化a-酪氨酸转化为b-酪氨酸, 参与力达霉素生物合成中β-酪氨酸的合成. 为了确定sgcD编码的酶的性质, 将sgcD克隆至大肠杆菌表达载体pET30a中进行诱导表达, 然后对表达产物进行酶学分析. 实验结果表明, sgcD的表达产物具有氨基变位酶的活性. sgcD编码的氨基变位酶是第一个被定性的烯二炔类抗肿瘤抗生素生物合成酶. 本研究将有助于这类抗生素生物合成机制的阐明, 对改造和研发新型抗肿瘤药物具有显著意义.     

链霉菌大肠杆菌穿梭质粒载体pSGLgpp的构建及应用

质粒pSGL1(74kb)是从球孢链霉菌(\%Streptomyces geobisporus)\%中分离得到的一个高拷贝质粒,已测定其最小复制子序列。从球孢链霉菌总DNA中采用PCR方法扩增获得编码C1027前蛋白信号肽的DNA片断gpp。将gpp克隆至pSGL1的衍生质粒pSGLN中,获得新的链霉菌表达型质粒载体pSGLgpp。应用该质粒进行了人的河溶性白细胞介素1受体I型的表达。     

日本血吸虫琥珀酸脱氢酶铁硫蛋白全长cDNA的克隆、表达及保护性免疫评价

根据GenBank日本血吸虫(Schistosoma japonicum)琥珀酸脱氢酶铁硫蛋白(SjSDISP)不完整的表达序列标签(BU804141)以及日本血吸虫成虫cDNA文库载体多克隆位点邻近核苷酸序列设计引物, 以日本血吸虫成虫cDNA文库为模板, 采用锚式PCR策略, 对SjSDISP cDNA不完整的3′端和5′端进行扩增、测序, 用序列比对拼接电子软件比对, 基于重叠区进序列合并, 获得1071 bp的SjSDISP全长cDNA. 序列分析推断该片段含有编码SjSDISP基因的完整阅读框, 编码278个氨基酸残基. 将其编码基因克隆到原核表达载体pQE30上, 在大肠杆菌M15中获得准确、高效表达, 表达产物分子量约为32 kD. 用日本血吸虫成虫抗原免疫血清对表达产物进行Western blot检测, 在预测位置上出现明显的识别条带. 用纯化的重组蛋白rSjSDISP免疫小鼠, 进行动物免疫保护性评价, 在虫荷、每克肝卵、每克粪卵和每雌子宫内卵数方面, 与佐剂对照组比较差异均具有显著性(P<0.05, P<0.01). 结果表明, 日本血吸虫琥珀酸脱氢酶铁硫蛋白酶(SjSDISP)全长cDNA成功克隆并在大肠菌中得到表达; 表达产物具有良好的抗原性和动物免疫保护效果, 是一种潜在的具有部分免疫保护性的抗日本血吸虫病疫苗候选分子.     

L-乳酸脱氢酶基因克隆及功能分析

构建了一株产D ,L_乳酸的乳杆菌 (Lactobacillussp .)MD_1的基因文库。利用乳酸脱氢酶和丙酮酸裂解酶缺陷的EscherichiacoliFMJ14 4作为宿主 ,通过互补筛选分离克隆到乳酸脱氢酶基因 (ldhL)。核酸序列分析表明 ,该基因以ATG为起始密码子编码 316个氨基酸残基组成的蛋白质 ,预测的分子量为 33 84kD ;5′端存在典型的启动子结构 ,3′端的终止子是不依赖于 ρ因子的转录终止子。ldhL编码的蛋白质有 3个保守区域 ,其中Gly13～Asp50保守区域是NADH的结合位点 ,Asp73～Ile10 0和Asn12.3～Arg15.4保守区是酶的活性部位。该ldhL和其他乳杆菌的ldhL基因和编码的氨基酸序列相似性较低 ,核苷酸序列相似性最高仅为 64.1% ,氨基酸序列相似性最高仅为 68.9% ,是新的L_乳酸脱氢酶基因     

弗氏链霉菌丝氨酸蛋白酶基因的克隆及表达

从一株具有极强的降解羽毛能力的弗氏链霉菌菌株（Streptomyces fradiae var.k11）中纯化得到了一种丝氨酸蛋白酶SFP2。经蛋白测序,得到部分氨基酸序列,设计简并引物,PCR扩增得到部分基因序列,通过构建基因文库,获得了包括信号肽序列在内的完整的基因sfp2(EMBL收录号AJ784940),开放阅读框全长924bp,包括114bp的信号肽编码序列和810bp的酶原编码序列, 其中成熟蛋白编码基因长576bp,编码191个氨基酸,理论分子量为19.112kD。酶原编码基因和成熟蛋白编码基因均在大肠杆菌和枯草芽孢杆菌中得到了表达,酶原编码基因表达产物具有正常的生物学活性,证明了克隆基因的生物学功能。     

类大麦属高分子量谷蛋白基因Kx的序列测定及表达

利用SDS_PAGE检测了2份类大麦属(Crithopsisdelileana)材料的高分子量谷蛋白亚基组成,并对其中1份材料的x型亚基进行了克隆和测序。结果表明,2份材料具有完全相同的蛋白电泳图谱。在小麦的高分子量区域仅检测到一条蛋白质带,与小麦y型亚基的迁移率接近,但克隆测序表明其为x型高分子量谷蛋白亚基,其编码基因命名为Kx。Kx基因编码区序列长度为2 0 5 2bp ,编码长度为6 6 1个氨基酸残基的蛋白质,其序列具有典型的x型高分子量谷蛋白亚基的特征。Kx基因能在原核表达系统内正确表达,其表达蛋白与来源于种子中的Kx亚基的迁移率完全一致。Kx亚基与小麦属A、B和D ,山羊草属C和U以及黑麦属R染色体组编码的高分子量谷蛋白亚基氨基酸序列非常相似,但在N和C保守区的氨基酸组成以及重复区长度上与它们存在明显差异。聚类分析可将Kx与Ax1聚类为平行的分支。由此可见,来源于C .delileana的Kx基因为一新的x型高分子量谷蛋白亚基基因。     

绵羊tnp2基因的克隆及其在体外共培养系统中圆形精子细胞的转录分析

过渡蛋白2基因（tnp2）是圆形精子细胞特异表达的基因。为了开展绵羊圆形精子细胞标记基因的研究,根据GenBank上已公布的牛的cDNA序列设计引物,采用RT-PCR和分子克隆方法,克隆了蒙古绵羊tnp2基因cDNA部分编码区序列。DNA 序列测定结果与牛的核苷酸序列比对,同源性为95.3%。根据绵羊tnp2基因的cDNA序列设计引物,对共培养的四月龄绵羊睾丸生殖细胞进行RT-PCR鉴定。结果显示体外共培养的绵羊睾丸生殖细胞一直到第十周后仍有圆形精子细胞产生。绵羊tnp2基因的cDNA克隆和序列测定为进一步研究绵羊精子发生过程奠定了基础。     

来源于Bacillus subtilis的中性植酸酶基因的克隆及在大肠杆菌中的表达

通过PCR的方法从Bacillus subtilis基因组中克隆了中性植酸酶基因nphy,DNA全序列分析表明其结构基因全长1152个核苷酸(编码383个氨基酸),5′端有一编码26个氨基酸的信号肽序列。去除信号肽编码序列的nphy克隆到大肠杆菌IPTG诱导表达载体pTYB40上,在大肠杆菌中得到了高效表达,表达量达到大肠杆菌可溶性蛋白的40%以上,表达产物具有生物学活性,证实了克隆到的中性植酸酶的编码基因有正常的生物学功能。     

水稻稻米糊化温度控制基因ALK的图位克隆及其序列分析

糊化温度是仅次于直链淀粉含量的评价水稻稻米蒸煮品质的重要指标. 许多文献报道, 糊化温度受一主效基因控制. 采用图位克隆法分离克隆了水稻糊化温度基因(ALK), 序列分析表明其编码可溶性淀粉合酶Ⅱ. 进一步比较不同品种间该基因DNA序列与碱消法分析结果, 推测出ALK基因编码区内的碱基替换可能引起了支链淀粉晶体层结构的改变, 从而导致糊化温度的变化.     

青花菜雄性不育相关基因BoDHAR的克隆与表达分析

以一个与甘蓝显性核不育相关的差异表达片段的序列为信息探针,通过在NCBI与TAIR网站数据库中进行同源EST序列搜索,经人工拼接、RT-PCR、PCR 克隆与序列分析,获得了青花菜脱氢抗坏血酸还原酶DHAR dehydroascorbate reductase 基因的 cDNA 与 DNA 全长序列,命名为BoDHAR。并利用双链接头介导 PCR 的染色体步行技术（genome walking）克隆了其上游 644bp 的5′端序列。所获的BoDHAR基因全长 1486bp,存在两个内含子,DNA 编码区序列633bp,编码210个氨基酸;序列分析表明：BoDHAR与同源基因AT1G195701cDNA 序列有 82.3% 的一致性,推导的氨基酸序列有 79.6% 的一致性;编码的水溶性蛋白存在多个磷酸化位点;5′端上游区存在明显的转录调控序列。半定量RT-PCR结果表明：BoDHAR 在可育系花蕾中的表达量明显高于不育系花蕾,在花药中的表达明显高于其它部位。     

C-1027-induced alterations in Epstein-Barr viral DNA replication in latently infected cultured human Raji cells: relationship to DNA damage.

This study is the first detailing drug-induced changes in EBV DNA replication intermediates (RIs). Both EBV replication inhibition and damage induction were studied in latently infected human Raji cells treated with the enediyne DNA strand-scission agent C-1027. Analysis of RIs on two-dimensional agarose gels revealed a rapid loss in the EBV bubble arc. When elongation of nascent chains was blocked by aphidicolin, this loss was inhibited, suggesting that C-1027-induced disappearance of RIs was related to maturation of preformed replication molecules in the absence of initiation of new RIs. C-1027 damage to EBV DNA was limited at concentrations where loss of the bubble arc was nearly complete, and none was detected within the replicating origin (ori P)-containing fragment, indicating that replication inhibition occurred in trans. By contrast, the non-nuclear mitochondrial genome was insensitive to replication inhibition but highly sensitive to damage induced by C-1027. C-1027-induced trans inhibition of nuclear but not mitochondrial DNA replication is consistent with a cell cycle checkpoint response to a DNA-damaging agent. EBV replication and Raji cell growth were inhibited at equivalent C-1027 doses.     

DNA damage by the enediyne C-1027 results in the inhibition of DNA replication by loss of replication protein A function and activation of DNA-dependent protein kinase.

Treatment of cells with the enediyne C-1027 is highly efficient at inducing single- and double-strand DNA breaks. This agent is highly cytotoxic when used at picomolar levels over a period of days. For this study, C-1027 has been used at higher levels for a much shorter time period to look at early cellular responses to DNA strand breaks. Extracts from cells treated with C-1027 for as little as 2 h are deficient in SV40 DNA replication activity. Treatment with low levels of C-1027 (1-3 nM) does not result in the presence of a replication inhibitor in cell extracts, but they are deficient in replication protein A (RPA) function. Extracts from cells treated with high levels of C-1027 (10 nM) do show the presence of a trans-acting inhibitor of DNA replication. The deficiency in RPA in extracts from cells treated with low levels of C-1027 can be fully complemented by the addition of exogenous RPA, and may be due to a C-1027-induced decrease in the extractability of RPA. This decrease in the extractability of RPA correlates with the appearance of many extraction-resistant intranuclear RPA foci. The trans-acting inhibitor of DNA replication induced by treatment of cells with high levels of C-1027 (10 nM) is DNA-dependent protein kinase (DNA-PK). DNA-PK is activated by the presence of DNA fragments induced by C-1027 treatment, and can be abrogated by removal of the DNA fragments. Although it is activated by DNA damage and phosphorylates RPA, DNA-PK is not required for either RPA focalization or loss of RPA replication activity.     

The radiomimetic enediyne C-1027 induces unusual DNA damage responses to double-strand breaks

Cells lacking the protein kinase ataxia telangiectasia mutated (ATM) have defective responses to DNA double-strand breaks (DSBs), including an inability to activate damage response proteins such as p53. However, we previously showed that cells lacking ATM robustly activate p53 in response to DNA strand breaks induced by the radiomimetic enediyne C-1027. To gain insight into the nature of C-1027-induced ATM-independent damage responses to DNA DSBs, we further examined the molecular mechanisms underlying the cellular response to this unique radiomimetic agent. Like ionizing radiation (IR) and other radiomimetics, breaks induced by C-1027 efficiently activate ATM by phosphorylation at Ser1981, yet unlike other radiomimetics and IR, DNA breaks induced by C-1027 result in normal phosphorylation of p53 and the cell cycle checkpoint kinases (Chk1 and Chk2) in the absence of ATM. In the presence of ATM, but under ATM and Rad3-related kinase (ATR) deficient conditions, C-1027 treatment resulted in a decrease in the level of Chk1 phosphorylation but not in the level of p53 and Chk2 phosphorylation. Only when cells were deficient in both ATM and ATR was there a reduction in the level of phosphorylation of each of these DNA damage response proteins. This reduction was also accompanied by an increased level of cell death in comparison to that of wild-type cells or cells lacking either ATM or ATR. Our findings demonstrate a unique cellular response to C-1027-induced DNA DSBs in that DNA damage response proteins are unaffected by the absence of ATM, as long as ATR is present.     

球孢链霉菌质粒pSGL1的性质研究

链霉菌质粒pSGL1是本实验室在球孢链霉菌（Streptomycelglobisporus）中发现的新的质粒,它可以在变铅青链霉菌中复制。用缺失分析确定了pSGL1的基本复制区位于一个2．0kb的片段上。pSGL1能够与pIJ101相容。pSGL1是一个高拷贝质粒,拷贝数约为70～250。在对质粒进行分析的过程中得到的一些衍生质粒如pSGLN和pSGLS3等可以作为新的链霉菌基因克隆载体,并可用于构建新的高效分泌表达的链霉菌载体。     

Cellular responses to the DNA strand-scission enediyne C-1027 can be independent of ATM, ATR, and DNA-PK kinases

The current paradigm based upon ionizing radiation (IR) studies states that cells deficient in either ataxia-telangiectasia-mutated kinase (ATM) or related phosphatidylinositol 3 (PI 3) -kinases (ATR and DNA-PK) are hypersensitive to DNA strand breaks because they are unable to rapidly activate downstream effectors such as p53. Here we have contrasted cell responses to IR and C-1027, a radiomimetic antibiotic that induces DNA strand breaks. At equal levels of DNA double strand breaks, cell lines with inactive ATM or other phosphatidylinositol 3-kinases displayed classical hypersensitivity to IR but not to C-1027. Moreover, phosphorylation of p53 Ser-15 induced by C-1027 was independent of ATM, ATR, or DNA-PK function. We have concluded that the model based on IR studies cannot always be directly applied to DNA damage induced by other strand-scission agents.     

ESR studies on DNA cleavage induced by enediyne C-1027 chromophore

C-1027 belongs to the family of chromoprotein antitumor antibiotics, which contain a carrier apoprotein and a highly unstable enediyne chromophore. The enediyne spontaneously aromatizes to generate p-benzyne biradical, and subsequently abstracts hydrogens from the DNA sugar backbone, resulting in cleavage of the double strand. Using spin-trapping methods, we obtained direct proof of radical intermediates during an DNA cleavage, and found intriguing difference in behavior between the trapping agents 2-methyl-2-nitrosopropane (MNP) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO): MNP added to the sugar radicals of the DNA, whereas DMPO directly trapped a phenyl radical or p-benzyne biradical derived from the C-1027 chromophore.     

Role of sgcR3 in positive regulation of enediyne antibiotic C-1027 production of Streptomyces globisporus C-1027

Background  

C-1027, produced by Streptomyces globisporus C-1027, is one of the most potent antitumoral agents. The biosynthetic gene cluster of C-1027, previously cloned and sequenced, contains at least three putative regulatory genes, i.e. sgcR1, sgcR2 and sgcR3. The predicted gene products of these genes share sequence similarities to StrR, regulators of AraC/XylS family and TylR. The purpose of this study was to investigate the role of sgcR3 in C-1027 biosynthesis.     

The cellular response to DNA damage induced by the enediynes C-1027 and neocarzinostatin includes hyperphosphorylation and increased nuclear retention of replication protein a (RPA) and trans inhibition of DNA replication

This study examined the cellular response to DNA damage induced by antitumor enediynes C-1027 and neocarzinostatin. Treatment of cells with either agent induced hyperphosphorylation of RPA32, the middle subunit of replication protein A, and increased nuclear retention of RPA. Nearly all of the RPA32 that was not readily extractable from the nucleus was hyperphosphorylated, compared to < or =50% of the soluble RPA. Enediyne concentrations that induced RPA32 hyperphosphorylation also decreased cell-free SV40 DNA replication competence in extracts of treated cells. This decrease did not result from damage to the DNA template, indicating trans-acting inhibition of DNA replication. Enediyne-induced RPA hyperphosphorylation was unaffected by the replication elongation inhibitor aphidicolin, suggesting that the cellular response to enediyne DNA damage was not dependent on elongation of replicating DNA. Neither recovery of replication competence nor reversal of RPA effects occurred when treated cells were further incubated in the absence of drug. C-1027 and neocarzinostatin doses that caused similar levels of DNA damage resulted in equivalent increases in RPA32 hyperphosphorylation and RPA nuclear retention and decreases in replication activity, suggesting a common response to enediyne-induced DNA damage. By contrast, DNA damage induced by C-1027 was at least 5-fold more cytotoxic than that induced by neocarzinostatin.     

麦迪霉素产生菌具有启动功能的DNA片段的克隆和分析

利用启动子探针质粒载体pIJ486从麦迪霉素产生菌总DNA中克隆得到了一段具有启动功能的DNA片段.通过限制性酶酶切分析,测定插入DNA片段大小为2.3kb.又利用载体pIJ486和pIJ487的新霉素抗性结构基因上游有多酶切点方向相反的性质,分析了插入片段在两个不同方向上的启动能力.结果表明,在两个方向上均有启动功能,但强弱相差六倍.其中在XbaI-HindIII方向上具有较强的启动能力,在变铅青链霉菌中新霉素抗性水平可达20mg/ml以上.进一步对插入片段的三个BamHI小片段进行分析的结果表明,较强启动子区域集中在BamHI-BamHI 0.79kb DNA片段上.