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Mapping of Late Transcripts of Autographa californica Nuclear Polyhedrosis Virus by ‘Criss-Cross’ DNA-RNA Hybridization

Mark A. Cochran

Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada, K7L 3N6

A ‘criss-cross’ DNA-RNA hybridization technique and Northern blot analysis were used to analyse late RNA transcripts in Autographa californica nuclear polyhedrosis virus (AcNPV)-infected Spodoptera frugiperda cells. Infected cells were pulse-labelled with [³²P]orthophosphate at 24 to 30 h post-infection and cytoplasmic poly(A)⁺ RNA was isolated at 30 h post-infection. Labelled RNA was fractionated on preparative methylmercury gels and blotted under annealing conditions onto Southern blots of preparative DNA gels containing the AcNPV DNA digested with EcoRI, BamHI or HindIII restriction endonucleases. During hybridization the axis of electrophoresis of the DNA fragments was at right angles to the axis of electrophoresis of the RNA. The ‘criss-cross’ hybridization pattern was used to identify viral transcripts, to estimate their relative sizes and to map them on the genome. A transcription map for at least 12 late RNA species was drawn based on the consensus data obtained from several ‘criss-cross’ blot hybridizations and concurrent Northern blot analysis.

Keywords: AcNPV, baculovirus, transcription mapping, ‘criss-cross’ hybridization

^Dg Present address: Laboratory of Biology of Viruses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20205, U.S.A.

Received 1 August 1983; accepted 12 December 1983.