CHLORELLA VIRUS-1 (PBCV-1)

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 草履虫小球藻病毒-1(PBCV-1)基因组全长330 kb，已完成全序列测定。它被发现含有373个大的开放阅读框架(ORF)，推测这些ORF编码蛋白质。这些开放源码框架中约有一半在任何数据库中都没有对应的数据库。在过去的一年里，我为371个非冗余ORF中的每一个安排了50聚体单链DNA探针的设计和合成。这些PBCV-1 DNA探针已经被内布拉斯加大学医学中心的基因组核心设施发现在微阵列平板上。去年夏天，我一直在学习如何从感染了PBCV-1病毒的小球藻细胞中提取总RNA。我现在已经开发了一种产生高质量总RNA的方法(A260/2801.9，浓度2g/？L，甲醛RNA凝胶)。我收集了未感染的小球藻细胞的总RNA(对照)，以及从感染PBCV-1 30、75、120和240分钟的细胞中收集的总RNA。所有5个组的总RNA都被送到基因组学核心设施，在那里它们被逆转录，用Cy3和Cy5染料标记，并与微阵列平板上的探针竞争性杂交。例如，从感染0分钟的细胞(即只含有宿主RNA的未感染细胞)中分离的总RNA反转录的cDNA被标记为Cy3(绿色)，而从感染30分钟的细胞中分离的总RNA反转录的cDNA被标记为Cy5(红色)；两组cDNA混合并与微阵列上的PBCV-1探针杂交。每种基因的表达谱正在开发中。虽然分析还没有完成，但很明显，一些PBCV-1的mRNAs在早期表达，而另一些表达的时间较晚。也许最令人惊讶的是，病毒基因组的大约10%似乎与其小球藻宿主共享，这一点从未感染(0分钟)的小球藻细胞对PBCV-1微阵列探针的热处理中得到了证明。这些共享的ORF(基因)现在正在被描述。在病毒及其宿主共有的基因中，主要的病毒外壳蛋白。

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 330 kb genome of Paramecium bursaria Chlorella Virus-1 (PBCV-1) has been completely sequenced. It was found to contain 373 large open reading frames (ORFs) that putatively code for proteins. About half of these ORFs have no counterparts in any of the databases. During this past year I arranged for the design and synthesis of 50-mer ssDNA probes for each of the 371 non-redundant ORFs. These PBCV-1 DNA probes have been spotted onto microarray plates by the Genomics Core Facility at the University of Nebraska Medical Center. I spent the past summer learning how to isolate total RNA from Chlorella cells infected with PBCV-1. I have now developed a protocol that yields high quality total RNA (A260/280 1.9, concentration 2 ¿g/¿L, formaldehyde RNA gels). I have collected total RNA from uninfected Chlorella cells (control), and from cells that have been infected with PBCV-1 for 30, 75, 120 and 240 minutes. Total RNA from all 5 groups have been sent to the Genomics Core Facility where they have been reverse transcribed, labeled with Cy3 and Cy5 dyes and competitively hybridized to the probes on the microarray plate. For example, the cDNA reverse transcribed from total RNA isolated from cells infected for 0 minutes (i.e., uninfected cells that contains only host RNA) were labeled with Cy3 (green), while cDNA reverse transcribed from total RNA isolated from cells infected for 30 minutes were labeled with Cy5 (red); the two groups of cDNAs were mixed and hybridized to the PBCV-1 probes on a microarray. Expression profiles for each gene are being developed. While the analysis is not yet complete, it is clear that some PBCV-1 mRNAs are expressed early, while others are expressed late. Perhaps the biggest surprise is the approximately 10% of the virus genome appears to be shared with its Chlorella host as evidenced by annealing of the uninfected (0 minutes) Chlorella cells to the PBCV-1 microarray probes. Those shared ORFs (genes) are now being characterized. Among the genes common to the virus and its host is the major viral coat protein.