Analysis of unselected HSV-1 McKrae/HSV-2 HG 52 recombinants demonstrates preferential recombination between intact genomes and restriction endonuclease fragments containing an origin of replication.
To identify viral genes involved in reactivation of herpes simplex virus from latency, intertypic HSV-1 strain McKrae/HSV-2 strain HG 52 recombinants were selected following cotransfection of intact McKrae DNA and XbaI or HpaI cleaved HG 52 DNA. Eleven separately obtained recombinants containing HG 52 inserts between 0.35-0.56 and/or 0.82-1.0 map units (mu) were isolated. It was noted that with HpaI digested HG 52 DNA, only recombinants containing type 2 inserts from HpaI d (0.35-0.57) and/or containing an intact type 2 [S] region were isolated. Similarly with XbaI cleaved HG 52 DNA only recombinants containing type 2 sequences from XbaI c (0-0.45) were isolated. In effect, the type 2 insert always contained one or both origins of replication (ORIL/ORIS). In reciprocal experiments isolation of two recombinants from cotransfection of HpaI cleaved McKrae DNA with intact HG 52 DNA confirmed this finding; one contained both copies of ORIS and the intervening short region sequences of McKrae, the other contained approximately 3 kb of McKrae in which ORIL is located. These results indicate that either (a) the presence of an origin of replication in a RE fragment amplifies the fragment thereby increasing its concentration and hence recombination potential with intact genomes; and/or (b) recombination and replication may be correlated. In either case isolation of recombinants containing ORIL and ORIS from the restricted DNA parent strongly suggests that both origins are functional in vitro.
Batra
S K
SK
MRC Virology Unit, Institute of Virology, Glasgow, Scotland.
Brown
S M
SM
D003434:Crossing Over, Genetic; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics*; D011995:Recombination, Genetic; D015183:Restriction Mapping; D018139:Simplexvirus / Q000235:genetics*; D014162:Transfection; D014779:Virus Replication
D004279:DNA, Viral; D004262:DNA Restriction Enzymes