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.
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