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首页> 外文学位 >Studies of schistosome DNA: Species and strain differences from melting and drug resistance DNA analysis and cloning, sequencing and expression analysis of a putative praziquantel binding Ca(++)-ATPase gene.
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Studies of schistosome DNA: Species and strain differences from melting and drug resistance DNA analysis and cloning, sequencing and expression analysis of a putative praziquantel binding Ca(++)-ATPase gene.

机译：血吸虫DNA的研究：物种和菌株差异从融化和耐药性DNA分析以及推定的吡喹酮结合Ca（++）-ATPase基因的克隆，测序和表达分析。

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摘要

This thesis addresses the investigation of schistosome DNA with melting, drug-resistance and cloning, sequencing and characterization of a putative Ca++-ATPase gene. The study of experimental and computed high resolution melting curves for S. mansoni, S. japonicum and S. haematobium presented in Chapter 2 show the %(G+C) in S. mansoni, S. japonicum and S. haematobium genomes are 29.4, 26.1 and 31.5, respectively. Moreover, they have their own distribution of repetitive DNAs in their experimental melts. Despite the limited size of the datasets available in GENBANK, those tables presented in Chapter 2 show that coding sequences of S. mansoni, S. japonicum and S. haematobium melt over a higher temperature range than flanking and intron sequences. So MELTSIM could potentially be used as a useful tool to distinguish these DNA functional classes in the genome.;The genetic differences between Schistosoma mansoni strains from different geographic areas, which were reportedly resistant or sensitive to antischistosomal drugs were studied with randomly amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) PCR and representational difference analysis (RDA) assays in Chapter 3. Out of the 20 RAPD primers we chose, 19 of them showed the capacity to produce a medium to high level of amplification and 6 revealed difference PCR bands between drug-resistant and drug-sensitive strains. One particular primer, 5'-CAGCGACAAG-3 ', showed 2 major difference bands between Praziquantel (PZQ)-resistant and PZQ-sensitive strains from one endemic area of Egypt. Those RAPD and RDA results demonstrate that defined sequence primers could be applied as a useful tool for differentiating drug-resistant and sensitive schistosome parasites in the field; genomic differences between drug-resistant and drug-sensitive strains could also be isolated by representational difference analysis.;Chapter 4 has been cloned, isolated, sequenced and characterized a novel Ca++-ATPase homologue, SM3, from S. mansoni. This clone has 3900 bp in length, including a 3030 bp open reading frame (ORF) and a 870 bp 3'-untranslated sequence. The %(G+C) in ORF is 37%, while the 3'-untranslated sequence is 27% in agreement with our MELTSIM results. An initiator methionine has not yet been unambiguously identified. Therefore, all of the sequence analysis is currently based on the near-complete sequence. From the analysis of the sequence and amino acid alignment compared to those Ca++-ATPases from other organisms in the public database, SM3 has approximately 51% identity and 64% similarity to the third class of Ca++-ATPase from rat (RS10-31) and 40% identity and 53% similarity to yeast (PMR1). SM3 only has 24-27% identity and 41--43% similarity to the PMCAs and SERCAs from rat, human and C. elegans. SM3 is predicted to have a Mr 110,794.5, pI of 6.48 and 10 hydrophobic, potentially transmembrane domains. The deduced amino acid sequence shows the presence in SM3 of all the conserved domains commonly found in P-type ATPases.
机译：本论文致力于对血吸虫DNA的研究，包括融合，耐药性以及推测的Ca ++-ATPase基因的克隆，测序和鉴定。第2章介绍的曼氏沙门氏菌，日本血吸虫和沙门氏菌的实验和计算的高分辨率解链曲线研究表明，曼氏沙门氏菌，日本血吸虫和沙门氏菌基因组的％（G + C）为29.4， 26.1和31.5。而且，它们在实验熔体中具有自己的重复DNA分布。尽管GENBANK中可用的数据集的大小有限，但第2章中提供的那些表显示，曼索氏链球菌，日本血吸虫和血红球菌的编码序列在比侧翼和内含子序列更高的温度范围内融化。因此，MELTSIM有望成为区分基因组中这些DNA功能类别的有用工具。;使用随机扩增的多态性DNA研究了来自不同地理区域的曼氏血吸虫菌株之间的遗传差异，据报道它们对抗血吸虫病药物具有抗药性或敏感性。 RAPD），简单重复序列（SSR）PCR和代表性差异分析（RDA）分析在第3章中进行了选择。在我们选择的20种RAPD引物中，其中19种具有产生中等至高水平扩增能力的能力，而6种具有差异性耐药菌株和耐药菌株之间的PCR条带。一种特殊的引物5'-CAGCGACAAG-3'在来自埃及一个流行地区的对吡喹酮（PZQ）耐药和对PZQ敏感的菌株之间显示了2条主要差异带。 RAPD和RDA的结果表明，确定的序列引物可用作区分本领域耐药性和敏感血吸虫寄生虫的有用工具。还可以通过代表性差异分析来分离耐药菌株和耐药菌株之间的基因组差异。第四章已从曼氏酵母中克隆，分离，测序并鉴定了一种新型的Ca ++-ATPase同源物SM3。该克隆的长度为3900 bp，包括一个3030 bp的开放阅读框（ORF）和一个870 bp的3'非翻译序列。与我们的MELTSIM结果一致，ORF中的％（G + C）为37％，而3'-非翻译序列为27％。尚未明确鉴定引发剂蛋氨酸。因此，所有序列分析当前都基于接近完整的序列。与公共数据库中其他生物的Ca ++-ATPase相比，从序列和氨基酸比对分析来看，SM3与来自大鼠（RS10-31）的第三类Ca ++-ATPase具有51％的同源性和64％的相似性。与酵母（PMR1）的同一性为40％，相似性为53％。 SM3与大鼠，人和秀丽隐杆线虫的PMCA和SERCA仅具有24-27％的同一性和41--43％的相似性。预测SM3具有110.794.5的Mr，pI为6.48和10个疏水性，潜在跨膜结构域。推导的氨基酸序列显示出SM3中通常存在于P型ATPase中的所有保守结构域。

著录项

  • 作者

    Tsai, Meng-Hsiun.;

  • 作者单位

    University of Massachusetts Lowell.;

  • 授予单位 University of Massachusetts Lowell.;
  • 学科 Biology Molecular.
  • 学位 Ph.D.
  • 年度 1998
  • 页码 120 p.
  • 总页数 120
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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