CHANGES IN LOCATIONS OF CROSSOVER SITES OVER TIME IN DE-NOVO GENERATED RNA RECOMBINANTS

Recombinant RNAs generated in plants 3 weeks postinoculation with turn ip crinkle virus (TCV) genomic RNA and an associated satellite RNA, sa t-RNA D, have a majority of TCV crossover sites in a 24-nucleotide rep eat (motif IIIA/IIIB) that forms part of a stable hairpin (Carpenter e t al., 1995, J. Mol. Biol. 245, 608-622). To determine ii parameters o ther than nucleotide sequence in the crossover region affect junction site selection, recombinants were assayed at various times postinocula tion of plants and protoplasts. Populations of recombinants became pro gressively shorter in plants and larger in protoplasts. Levels of inoc ulated transcript and age of the plant were not substantial factors in the shifts in crossover site locations. The two most commonly cloned recombinant species were not amplified to detectable levels in protopl asts, suggesting that these molecules are not viable templates for rep lication. These results suggest that recombination between sat-RNA D a nd TCV is a very frequent event, and populations of recombinants are l ikely generated de novo in each infected cell and represent the origin al recombinant molecules rather than progeny of such molecules. Theref ore, factors other than simple selection for recombinants that are mor e fit to replicate are probably responsible for the differences in jun ction sites in populations of sat-RNA D/TCV recombinants. (C) 1996 Aca demic Press, Inc.