RNA recombination has been described for a number of viruses in the plant and animal kingdoms, but the mechanisms of selection of recombination sites are poorly understood. The nonrandom recombination between two subviral RNAs associated with turnip crinkle virus was used to study the requirement for specific sequences and structures in the generation of recombinant molecules. Single-base mutations that disrupted either the stem or the loop of one of the two computer-predicted stem-loop structures eliminated detectable recombinant molecules. However, recombinants were detected if compensatory mutations were generated that re-formed a stable hairpin structure. These results provide evidence for the necessity of specific structures in the formation of recombinant molecules in this system.

Turnip crinkle virus (TCV) is associated with satellite (sat) RNAs (sat‐RNA D, sat‐RNA F), defective interfering (DI) RNAs (DI RNA G, DI1 RNA), and one RNA with properties of both sat‐RNAs and DI RNAs (sat‐RNA C). When plants were inoculated with TCV, sat‐RNA D and in vitro sat‐RNA C transcripts containing non‐viable mutations in the 5′ domain, recombinant sat‐RNAs were recovered. These recombinants were composed of sat‐RNA D at the 5′ end and sat‐RNA C sequences at the 3′ end. Analysis of 20 independent recombination junctions revealed that unequal crossing‐over had occurred in planta in a region of sequence similarity between the two sat‐RNAs which resulted in the duplication of 3‐16 nucleotides. Thirty percent of the sat‐RNA recombinants also had one to three additional nucleotides inserted at the crossover junctions which did not correspond to either sat‐RNA C or sat‐RNA D sequence. The right side of the recombination junctions always began with one of three consecutive nucleotides of sat‐RNA C. Based on the similarity between this sequence of sat‐RNA C, the right side junction of DI RNA G and the 5′ end of TCV, as well as the sequence similarity between right side junctions of DI1 RNA and sat‐RNA C and the 5′ end of the sat‐RNAs, a replicase‐driven copy choice mechanism is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

During myogenesis, reductions in trophic factor availability signal most myoblasts to fuse, up-regulate the expression of muscle-specific genes, and form myotubes. Those cells failing to differentiate into myotubes initiate apoptosis and rapidly die. At present, the signal-transduction molecules that determine whether myoblasts should differentiate or die are largely unknown. In this report, we describe the cloning and characterization of DALP, a small LIM-only type zinc-finger protein that is induced when the intersegmental muscles (ISMs) of the moth Manduca sexta become committed to die at the end of metamorphosis. Forced expression of death-associated LIM-only protein (DALP) in Drosophila results in skeletal muscle atrophy. Ectopic expression of DALP, or its mammalian ortholog Hic-5, blocks differentiation and induces apoptosis in mouse C 2 C 12 myoblasts. Both of these effects can be overcome by contact with normal myoblasts or by ectopic expression of the muscle-specific transcription factor MyoD. Hic-5 expression is specifically and dramatically induced in normal myoblasts that die after removal of trophic support. Taken together, these data suggest that DALP and Hic-5 act upstream of MyoD and function as phylogenetically conserved ''switches'' to block muscle differentiation and induce death.

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While considerable attention has focused on the role of specific proteins in mediating programmed cell death, few studies have examined the possible involvement of post-transcriptional regulation of mRNAs associated with this developmental process. We have examined developmental changes in transcript stability and translatability using protein extracts generated from the intersegmental muscles (ISM) of the moth Manduca sexta as a cell-free model system to examine three genes that are representative of the patterns of expression observed in condemned ISMs: repressed (actin), induced (polyubiquitin) and constitutively expressed (ubiquitin-fusion 80; ubf80). In addition, we have used luciferase mRNA as a generic reporter transcript to determine if there are sequence-specific controls of mRNA function related to programmed cell death. Among the three Manduca transcripts, polyubiquitin displayed the shortest half-life (t1/2) in all ISM extracts tested. The stability and translatability of all mRNAs were most affected in extracts from muscle cells from day 17 animals, just prior to the commitment of the muscles to die. Transfer of the 3' untranslated regions from the Manduca transcripts to luciferase mRNA did not appreciably change the stability or translatability of this test transcript. These data suggest that there may be global removal of cellular transcripts just prior to death to allow newly expressed mRNAs to rapidly accumulate to high levels. Such changes in message abundance, translatability and stability may facilitate the efficient activation of death (and perhaps other differentiation programs) in some developmental systems.

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