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Molecular Cloning and Restriction Endonuclease Mapping of the Rat Cytomegalovirus Genome

Department of Medical Microbiology, State University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands

Rat cytomegalovirus (RCMV) DNA was cleaved by restriction endonuclease EcoRI into 24 fragments ranging in mol. wt. from 34 x 10⁶ to 0.20 x 10⁶, of which 18 fragments could be cloned in plasmid pACYC 184. Restriction endonuclease XbaI cleaved the RCMV genome into 28 fragments, ranging in size from 44 x 10⁶ to 0·81 x 10⁶, of which 24 fragments were cloned in plasmid pSP62-PL. Among the restriction fragments that could not be cloned were two major terminal colinear fragments, EcoRI-A (34 x 10⁶) and XbaI-A (44 x 10⁶). Thus, the complete sets of recombinant plasmids spanned about 70% of the RCMV genome. Our mapping results including determination of the termini of the genome, characterization of double digestion products of restriction fragments and cross-hybridization of ³⁵S-labelled (cloned) EcoRI and XbaI fragments to Southern blots of EcoRI-, XbaI- or BglII-cleaved RCMV DNA, allowed us to construct the EcoRI and XbaI restriction maps of RCMV DNA. Since no cross-hybridization between internal fragments was seen, it is concluded that the RCMV genome consists of a long unique sequence of 224 kilobases without internal inverted repeat sequences, which is similar to the structures of murine and guinea-pig CMV DNA but unlike that of human CMV DNA. In a minor population (approx. 20%) of the RCMV DNA, one terminus was found to be larger by 0.35 x 10⁶ mol. wt. The nature of this fragment is unclear at the moment.

Keywords: RCMV, genome, cytomegalovirus

Received 31 December 1985; accepted 26 March 1986.

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