Lymantria dispar nuclear polyhedrosis virus homologous regions: characterization of their ability to function as replication origins.
Homologous regions (hrs) were identified in the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) genome. A 1.58-kb region surrounding hr4 was sequenced and found to have two distinct domains. Domain I (about 600 bp) is composed of seven repeats of about 80 bp including a series of palindromes containing MluI sites and overlapping XhoI and SacI sites. Domain II (about 700 bp) is composed of eight partially repeated sequences of 60 to 100 bp containing a 15- to 25-bp sequence that is 80 to 100% A+T in addition to a 6- to 10-bp palindrome containing an NruI site. Hybridization of a domain I sequence to cosmids containing the LdMNPV genome indicated its presence at eight positions (hr1 to -8) on the genome. In contrast, hybridization of domain II indicated that it was present only at the hr4 locus. A DpnI-based transient-replication assay was used to determine if subclones of hr4 transfected into LdMNPV-infected L. dispar cells functioned as replication origins. Subclones of hr4 containing either domain I or domain II replicated at very low or moderate levels, respectively. However, when domain I and domain II were linked on the same plasmid, high levels of replication were observed. A 1.4-kb region containing hr1 was also sequenced. It lies immediately upstream of the polyhedrin gene and contains six domain I-type repeats. Four-hundred-base-pair regions of domain I repeats from hr1 and hr4 showed 89% sequence identity. Plasmids containing the hr1 domain I replicated at low levels. However, hybrid plasmids in which the AT-rich hr4 domain II was inserted adjacent to hr1 domain I replicated to high levels, indicating that the AT-rich domain II greatly enhances replication. The orientation and position of domains I and II relative to each other did not have major effects on the levels of replication.
Pearson
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MN
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331-7301.
Rohrmann
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GF
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D016679:Genome, Viral; D008969:Molecular Sequence Data; D009036:Moths / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000502:physiology; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid; D018741:Replication Origin; D015183:Restriction Mapping; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014779:Virus Replication / Q000235:genetics
D009838:Oligodeoxyribonucleotides