Bujarski, Jozef J.
M.S. (Master of Science)
Department of Biological Sciences
Fava bean--Genetics; Plant viruses--Genetics; RNA viruses--Genetics
Full-length cDNAs of broad bean mottle bromovirus (BBMV) were synthesized by employing a novel, reverse transcriptasepolymerase chain reaction (RT-PCR) that utilized Vent thermostable DNA polymerase. In vitro transcribed RNAs synthesized from the full-length cDNA clones of either Bawden (Ba) or Morocco (Mo) strains of BBMV have been shown to be infectious when inoculated onto leaves of Vicia faba. The modified 5' termini of the transcribed BBMV RNAs were repaired during the course of infection in planta suggesting the importance of a correct 5' terminus in the life cycle of BBMV RNAs. One application of the in vitro transcription system was to examine defective interfering (Dl) RNAs of BBMV strains. In vitro transcribed Dl RNA of the Mo strain was demonstrated to be biologically active in Vicia faba upon co-inoculation with Dl RNAfree virion RNA preparations of either homologous Mo or heterologous Ba BBMV strains. The elimination of the translation initiation codon in Mo Dl transcripts by in vitro mutagenesis resulted in the reduction of the accumulation of mutant Dl RNAs under the detectable level in planta suggesting the role of coding capacity in Dl RNA accumulation. Sequence heterogeneity at the 5' end of BBMV Ba RNA3 was observed during the experiments. The extra nucleotides resulted from the duplication of two short, overlapping regions of the leader sequence. The novel RNA3 molecule was found to be very competitive in some plant hosts and less competitive in others. The developed in vitro transcription system should facilitate future studies aimed at understanding the molecular biology of BBMV and its Dl RNAs.
Pogany, Judit, "Molecular studies on broad bean mottle virus (BBMV) using in vitro transcribed viral RNAs" (1993). Graduate Research Theses & Dissertations. 4020.
ix, 82 pages
Northern Illinois University
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