NOVEL GENE DISCOVERY FROM NUCLEOCYTOPLASMIC LARGE DNA VIRUSES (NCLDV)

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 本奋进的主要目的是在核质大DNA病毒（NCLDV）中寻找新基因。 该组的模式病毒，草履虫bursaria绿草履虫病毒-1（PBVC-1），感染一个绿色昆虫，已被测序，因为有7个其他病毒的该组。 这些病毒含有许多新的基因/蛋白质，其中一些是已知的最小的，其中一些具有商业用途（下一段和第B.3节）。 PBCV-1属于一个古老的真核病毒家族，感染草履虫共生体草履虫样绿色虫。 PBVC-1是一种噬斑形成病毒，已被证明非常适合实验室研究。 James货车Etten（JVE）是该项目的导师/合作者（见下文E节），他在该病毒方面有25年以上的工作经验，目前有NIH的资金来研究该病毒的DNA复制和基因表达。 此外，JVE拥有一个合作者网络，他们正在研究各种PBVC-1特性。 该病毒将用作所有新测序病毒的参考。 关注NCLDV组病毒的原因是，根据定义，它们具有大的基因组（拟病毒编码超过1000种蛋白质），并且它们是古老的。 它们庞大的基因组是通过从各种来源的基因中劫持基因和基因复制而形成的。 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具有编码参与DNA复制、重组和修复、细胞信号传导、DNA限制/修饰等的蛋白质的基因。 PBCV-1 II型DNA拓扑异构酶是迄今为止描述的最小的拓扑异构酶，但具有非常高的活性水平（Lavrukhin等人，2000年）。 来自PBCV-1的拓扑异构酶II在癌细胞的研究中引起了兴趣。 其限制性酶和甲基化酶由新英格兰生物实验室商业销售。 该病毒还编码最小的K+通道蛋白。 Plugge等人（2000）已经广泛表征了来自PBCV-1的这种蛋白质。 离子通道蛋白影响离子浓度，并且是肌肉（包括心脏）通道阻滞剂的目标。 同样，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）。 本建议的主要目标是：对来自该组的另外的病毒进行测序;（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，这些分析将继续进行。 （三）一旦发现新的或不寻常的基因，就把它们交给货车埃滕实验室的其他人和他们的同事，他们将克隆、表达和鉴定它们。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.