CHLORELLA VIRUS-1 (PBCV-1)

小球藻病毒-1 (PBCV-1)

• 批准号: 7381537
• 负责人: Gary E Duncan
• 金额: $ 4.99万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381537
• 关键词: CHLORELLA; VIRUS; PBCV

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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。草履虫小球藻病毒 1 (PBCV-1) 的 330 kb 基因组已完成测序。研究发现它包含 373 个大型开放阅读框 (ORF)，推测这些开放阅读框编码蛋白质。这些 ORF 中约有一半在任何数据库中都没有对应项。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 ssDNA 探针的设计和合成。内布拉斯加大学医学中心基因组学核心设施已将这些 PBCV-1 DNA 探针点样到微阵列板上。去年夏天我学习了如何从感染 PBCV-1 的小球藻细胞中分离总 RNA。我现在开发了一种可产生高质量总 RNA 的方案（A260/280 1.9，浓度 2 µg/L，甲醛 RNA 凝胶）。我从未感染的小球藻细胞（对照）以及已感染 PBCV-1 30、75、120 和 240 分钟的细胞中收集了总 RNA。所有 5 组的总 RNA 均已发送至基因组学核心设施，在那里进行逆转录，用 Cy3 和 Cy5 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（即仅含有宿主RNA的未感染细胞）中分离的总RNA反转录的cDNA用Cy3（绿色）标记，而从感染30分钟的细胞中分离的总RNA反转录的cDNA用Cy5（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 早期表达，而另一些则晚期表达。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，未感染的小球藻细胞（0 分钟）与 PBCV-1 微阵列探针的退火证明了这一点。这些共享的 ORF（基因）现在正在被表征。病毒及其宿主共有的基因之一是主要的病毒外壳蛋白。