1987 Fiscal Year Final Research Report Summary

Studies on transcription and replication of viral genomes in infected plant cells by situ hybridization

Research Project

Project/Area Number	61560049
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	植物保護
Research Institution	Tokyo University of Agriculture and Technology
Principal Investigator	HOSOKAWA Daijiro Faculty of Agriculture,Tokyo University of Agriculture and Technology (associate professor), 農学部, 助教授 (50014957)
Project Period (FY)	1986 – 1987
Keywords	In situ hybridization / タバコモザイクウイルス / ビオチン標識核酸プローブ
Research Abstract	In situ hybridization is potentially a very powerful technique for detecting the localization of viral gemones in infected cells. Unfortunatelly until now, it have no wide application to plant virus-infected cells. We have developed convenient method of in situ hybridization for tobacco mosaic virus (TMV)-infected cells using biotinylated RNA probes. To prepare sense-strand RNA probes, TMV-RNA were fragmented (200-600 nucleoties long) by alkali treatment in the presence of Mg^<2+>. The RNA fragments were labeled with photobiotin by mercury vapour lamp irradiation. To prepare antisense-strand RNA probes, the cloned cDNA of TMV coat protein gene was inserted into a transcription vector, pGITC, that carries the SP 6 promoter at the transcription initiation site. The transcription vector was linearized just downstream of the cDNA insert with Bam H I, and was transcribed to obtain biotin labeled antisense RNA probe, using the SP 6 polymerase in the presence of biotin-11-UTP. When these probes were hybridized with TMV-RNA isolated from virions or doublestrand TMV-RNA extracted from TMV-infected tobacco plant on nylon membrane, hybridization reactions were detected by tagging avidin-al^kaline phosphatase followed by incubation in nitro blue tetrazolium. By in situ hybridization with these probes, viral nucleic acids were detected in the TMV-infected tobacco protoplasts spread on slides. In situ hybridization at electron microscope levels are in progress.