CHLORELLA VIRUS-1 (PBCV-1)

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。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。所列机构为中心机构，不一定为研究者机构。测定了绿草履虫小球藻病毒1型（ParameciumbursariaChlorellaVirus-1，PBCV-1）330 kb的全基因组序列。发现它含有373个大的开放阅读框（ORF），这些ORF编码蛋白质。这些ORF中大约有一半在任何数据库中都没有对应的ORF。在过去的一年里，我为371个非冗余ORF中的每一个设计和合成了50聚体ssDNA探针。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学核心设施点到微阵列板上。在过去的一个夏天里，我一直在学习如何从感染了PBCV-1的小球藻细胞中分离总RNA。我现在已经开发了一种产生高质量总RNA的方案（A260/280 1.9，浓度2 μ g/μ L，甲醛RNA凝胶）。我已经从未感染的小球藻细胞（对照）和已经用PBCV-1感染30、75、120和240分钟的细胞中收集了总RNA。将来自所有5组的总RNA送至Genomics Core Facility，在那里它们被逆转录，用Cy 3和Cy 5染料标记，并与微阵列板上的探针竞争性杂交。例如，从感染0分钟的细胞分离的总RNA逆转录的cDNA（即，仅含有宿主RNA的未感染细胞）用Cy 3（绿色）标记，而从感染30分钟的细胞分离的总RNA逆转录的cDNA用Cy 5（红色）标记;将两组cDNA混合并与微阵列上的PBCV-1探针杂交。每个基因的表达谱正在开发中。虽然分析尚未完成，但很明显，一些PBCV-1 mRNA表达较早，而其他mRNA表达较晚。也许最大的惊喜是大约10%的病毒基因组似乎与其小球藻宿主共享，如通过未感染的（0分钟）小球藻细胞与PBCV-1微阵列探针退火所证明的。这些共享的ORF（基因）现在正在被表征。在病毒及其宿主共有的基因中，有一种是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.