Molecular cloning and physical mapping of the tupaia herpesvirus genome

Abstract

Purified virion DNA of about 200 kilobase pairs of tupaia herpesvirus strain 2 was cleaved with EcoRI or HindlIl restriction endonuclease. Restriction fragments representing the complete viral genome including both termini were inserted into the EcoRI, HindIII, and EcoRI-HindIII sites of the bacterial plasmid pAT153. Restriction maps for the restriction endonucleases EcoRI and Hindlll were constructed with data derived from Southern blot hybridizations of individual viral DNA fragments or cloned DNA fragments which were hybridized to either viral genome fragments or recombinant plasmids. The analysis revealed that the tupaia herpesvirus genome consists of a long unique sequence of 200 kilobase pairs and that inverted repeat DNA sequences of greater than 40 base pairs do not occur, in agreement with previous electron microscopic data. No DNA sequence homology was detectable between the tupaia herpesvirus DNA and the genome of murine cytomegalovirus, which was reported to have a similar structure. In addition, seven individual isolates of tupaia herpesvirus were characterized. The isolates can be grouped into five strains by their DNA cleavage patterns.

Analysis of the genomic termini of tupaia herpesvirus DNA by restriction mapping and nucleotide sequencing

“…Taken together with the results discussed above, the sequence of the cloned terminal joint of THV DNA must be considered to be more representative of the true ends of the viral genome than the heterogeneous THV DNA populations with variable termini that are also present in mature virions. This result is also consistent with the fact that the terminal HindIII B and J DNA fragments of THV DNA do not hybridize to each other as reported previously (8).…”

“…This heterogeneity raises the interesting question to what extent the terminal junction of THV DNA represents the true ends of the viral genome. Since extensive restriction analysis and contour length measurements of intact THV DNA molecules clearly indicate that the majority of mature THV DNA is of a unit length of approximately 200 kbp (3,8), those THV DNA molecules that are greater in size than unit length and vary from it by about 10 to 3,800 bp must be extremely small. In agreement with this result, staining of mature THV DNA with ethidium bromide in agarose gels does not reveal any of the heterogeneous DNA populations.…”

“…Construction of plasmids. The recombinant plasmids containing the HindIII J and B DNA fragments of THV (strain 2) and plasmid vector pAT153 were constructed and isolated as previously described (8). Subcloning of the terminal junction of fragments was done with the BamHI and SalI subfragments of the HindlIl B junction fragment.…”

“…There are some herpesviruses that seem to have a less complex structure than HSV, but they retain the feature of having more or less long terminal repeats, e.g., Epstein-Barr virus (EBV) (1, 7), herpesvirus saimiri (6), and channel catfish virus (2). At the other extreme, there are at least two herpesviruses that were reported to lack any conventionally detectable direct or inverted terminal repeats, namely tupaia herpesvirus (THV) (3,8) and murine cytomegalovirus (5,11). The terminal repeat element a of HSV has been shown to encode recognition sequences for encapsidation of viral DNA and for inversions of HSV DNA (12).…”

Nucleotide sequence analysis of a cloned DNA fragment from human cells reveals homology to retrotransposons.

“…Above a broad background, discrete and intense DNA bands became visible, particularly in fraction B, which moved at the approximate position of supercoiled human mitochondrial DNA or were of higher mobility. The HindIII DNA fragments of fraction B that corresponded to 4 to 6 kilobase pairs were isolated from the low-melting-point agarose gel, purified, and inserted into the HindIII sites of the pAT153 vector (13,25). A total of 314 recombinant clones were analyzed.…”