NOVEL GENE DISCOVERY FROM NUCLEOCYTOPLASMIC LARGE DNA VIRUSES (NCLDV)

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 本研究的主要目的是寻找核质大DNA病毒(NCLDV)的新基因。这一组的模型病毒--草履虫Bursaria小球藻病毒-1(PBVC-1)，与该组的其他7种病毒一样，已经被测序。这些病毒包含许多新的基因/蛋白质，其中一些是已知最小的，其中一些具有商业应用(下一段和第B.3节)。PBCV-1属于一个古老的真核病毒家族，它感染一种类似小球藻的绿藻，该绿藻是草履虫的共生体。PBVC-1是一种斑块形成病毒，已被证明非常适合实验室研究。詹姆斯·范·埃滕(JVE)是这个项目的导师/合作者(见下文E节)，他在这种病毒上工作了25年以上，目前拥有NIH的资金来研究这种病毒的DNA复制和基因表达。此外，JVE拥有一个合作者网络，他们正在研究PBVC-1的各种性质。该病毒将被用作对抗所有新测序病毒的参考。 关注NCLDV病毒组的原因是，根据定义，它们拥有巨大的基因组(拟态病毒编码1000多种蛋白质)，而且它们是古老的。它们庞大的基因组是由来自各种来源的高水平基因和基因复制而来的。它们古老的历史和短暂的生命周期使它们能够进化出一些最小的功能蛋白质，而其他蛋白质已被证明具有医学重要性(见第B.3节)。例如，PBCV-1具有编码蛋白质的基因，这些蛋白质涉及DNA复制、重组和修复、细胞信号、DNA限制/修饰等。PBCV-1II型DNA拓扑异构酶是迄今为止被描述的最小的一种，但具有非常高的活性水平(Lavrukhin等人，2000年)。PBCV-1的拓扑异构酶II在癌细胞的研究中引起了人们的兴趣。它的限制性内切酶和甲基化酶由新英格兰生物实验室商业化销售。这种病毒还编码最小的K+通道蛋白。Plugge等人。(2000)广泛地表征了这种来自PBCV-1的蛋白质。离子通道蛋白影响离子浓度，是包括心脏在内的肌肉通道阻滞剂的靶标。此外，钾离子(K+)通道在乳腺癌的增殖中起着关键作用(Abdul，Santo和Hoosein，2003)。 这项建议的主要目标是：(I)对这一组中的其他病毒进行测序；(Ii)对这些新测序的基因组进行基因组、生物信息学和系统发育分析。随着新的数据库和方法的出现，分析将继续进行。(三)一旦识别出新的或不寻常的基因，就把它们交给Van Etten实验室的其他人和他们的同事，他们将克隆、表达和鉴定这些基因。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The major goal of this research endeavor is to search for novel genes among the Nucleocytoplasmic Large DNA Viruses (NCLDV). The model virus of this group, Paramecium bursaria chlorella virus-1 (PBVC-1), which infect a green alga, has been sequenced, as have 7 other viruses of this group. These viruses contain many novel genes/proteins, some of which are the smallest known, several of which have commercial applications (next paragraph and section B.3.). PBCV-1 belongs to an ancient family of eukaryotic viruses that infect a chlorella-like green alga, a symbiont of Paramecium bursaria. PBVC-1 is a plaque-forming virus that has proven to be very amenable to laboratory study. James Van Etten, (JVE) the mentor/collaborator for this project (see section E below), has 25+ years working on this virus and has current NIH funding to investigate the DNA replication and gene expression of this virus. Furthermore, JVE has a network of collaborators who are studying a wide variety of PBVC-1 properties. This virus will be used as a reference against all newly sequenced viruses. The reasons for focusing on the NCLDV group of viruses are that they have, by definition, large genomes (the mimivirus encodes over 1000 proteins) and they are ancient. Their large genomes have come about by high jacking genes from a variety of sources and by gene duplication. Their ancient history and short life cycles have enabled them to evolve some of the smallest functional proteins, while other proteins have been shown to have medical importance (see section B.3.). For example, PBCV-1 has genes that encode proteins involved in DNA replication, recombination and repair, cell signaling, DNA restriction/modification, to name a few. The PBCV-1 type II DNA topoisomerase is the smallest one to be described to date and yet has very high activity levels (Lavrukhin et al., 2000). Topoisomerase II from PBCV-1 has been of interest in the study of cancer cells. Its restriction and methylation enzymes are commercially sold by New England Biolabs. This virus also encodes the smallest K+ channel protein. Plugge et al. (2000) have extensively characterized this protein from PBCV-1. Ion channel proteins affect ion concentrations and are the target of channel blockers for muscles, including the heart. As well, potassium ion (K+) channels play a key role in the proliferations of breast cancer (Abdul, Santo, and Hoosein, 2003). The primary objectives towards this proposal are: (i.) sequence additional viruses from this group; (ii.) conduct genomic, bioinformatic and phylogenetic analyses of these newly sequenced genomes. The analyses will be ongoing as new databanks and methods emerge. (iii.) Upon identification of novel or unusual genes, hand them off to others in the Van Etten lab and their colleagues who will clone, express and characterize them.