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CONTROL OF KLEBSIELLA PNEUMONIAE NITROGEN FIXATION (NIF) GENES BY THE NIFA PRODUCT.

机译:通过NIFA产品控制肺炎克雷伯菌肺炎固氮(NIF)基因。

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摘要

The products of the nifLA operon regulate the expression of nitrogen fixation (nif) genes in Klebsiella pneumoniae. The first part of this thesis describes regulatory factors that control the activity of the nifLA promoter. Specifically, the products of glnG (ntrC), glnF (ntrA), glnL (ntrB) and possibly glnB are shown or implicated to exert regulatory effects on nifLA expression. This thesis also describes the discovery that the nifA product can substitute for the glnG product in activating a number of genes involved in nitrogen metabolism (eg. glnALG, hut, aut), including its own nifLA promoter. As with the case of the glnG product, activation mediated by the nifA product appears to require the glnF product.; The second section of this thesis describes the structure of the nifLA promoter and a consensus heptameric sequence (TTTTGCA) found among five glnG and nifA regulated promoters examined. For two of these promoters, where the transcriptional start points have been determined, the consensus sequence is located at the -15 region, suggesting that it may be involved in glnG and nifA mediated transcription. In contrast, a promoter that responds to nifA, but not glnG mediated activation, does not contain this heptameric sequence, but instead the sequence CCCTGCA in its -15 region.; The last section of this thesis centers on the development of the P2 helper-dependent phage P4 as a specialized cloning vector for K. pneumoniae. This thesis describes that P4virl, an immunity-insensitive mutant, can replicate autonomously as a multicopy plasmid in E. coli and K. pneumoniae (in the absence of a P2 helper). It also describes experiments that show P4 can integrate site specifically into the K. pneumoniae chromosome, in which the event occurs at a low frequency. In order to convert cloned DNA into single copy states, vectors were constructed derived from P4virl. In addition to antibiotic resistance genes, these derivatives carry a nonsense mutation in its replication gene, and hence cannot replicate in a wild type host. Consequently, this allows direct selection of integrated lysogens. This approach, which involves cloning DNA as recombinant P4 plasmids in host strains that carry nonsense suppressors, and the subsequent integration of a single copy into a wild type host, was demonstrated for certain nif genes which would otherwise exhibit multicopy-associated inhibitory effects on nif expression.
机译:nifLA操纵子的产物调节肺炎克雷伯菌中固氮(nif)基因的表达。本论文的第一部分描述了控制nifLA启动子活性的调节因子。具体而言,显示或暗示了glnG(ntrC),glnF(ntrA),glnL(ntrB)和可能的glnB的产物对nifLA表达具有调节作用。本论文还描述了发现,nifA产物可以替代glnG产物,从而激活许多参与氮代谢的基因(例如glnALG,小屋,aut),包括其自身的nifLA启动子。与glnG产物的情况一样,由nifA产物介导的活化似乎需要glnF产物。本论文的第二部分描述了nifLA启动子的结构以及在所检测的5个glnG和nifA调控的启动子中发现的共有七聚体序列(TTTTGCA)。对于其中两个启动子已确定转录起始点的启动子,共有序列位于-15区,这表明它可能参与glnG和nifA介导的转录。相反,对nifA有反应但对glnG介导的激活没有反应的启动子在其-15区不包含该七聚体序列,而是序列CCTCGCA。本论文的最后一部分集中于作为肺炎克雷伯菌的专门克隆载体的P2辅助依赖性噬菌体P4的开发。本论文描述了一种对免疫不敏感的突变体P4vir1,可以在大肠杆菌和肺炎克雷伯氏菌中(无P2辅助物时)作为多拷贝质粒自主复制。它还描述了表明P4可以将位点特异性整合到肺炎克雷伯菌染色体中的实验,其中该事件以较低频率发生。为了将克隆的DNA转化为单拷贝状态,构建了衍生自P4vir1的载体。除了抗生素抗性基因外,这些衍生物还在其复制基因中带有无意义的突变,因此不能在野生型宿主中复制。因此,这允许直接选择整合的溶原。对于某些nif基因,这种方法涉及将DNA作为重组P4质粒克隆到携带无义抑制因子的宿主菌株中,然后将单拷贝整合到野生型宿主中,而对于某些nif基因,该方法将对nif表现出多拷贝相关的抑制作用表达。

著录项

  • 作者

    OW, DAVID WING.;

  • 作者单位

    Harvard University.;

  • 授予单位 Harvard University.;
  • 学科 Biology Genetics.
  • 学位 Ph.D.
  • 年度 1983
  • 页码 234 p.
  • 总页数 234
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 遗传学;
  • 关键词

  • 入库时间 2022-08-17 11:51:14

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