Synthesis of figwort mosaic virus (FMV) coat protein - reverse transcriptase fusion protein in reticulocyte lysate from a full length transcript
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abstract
The genetic organization of FMV genome (cirucular, double trended DMA of about 8Kbp) from sequence analysis reveals six major (I-VI) and a small (VII) ORFs. IQ vivo two transcripts are synthesized. One transcript spans the gene VI; the other spans the entire length of the viral genome and may function as a messenger RNA for the translation of 5-6 closely spaced genes appearing on this RNA. In a previous investigation, we showed that the CAT reporter gene placed in proximal, middle or distal ctetron was translated in vitro from a full length transcript prepared from the reconstructed viral genome In pGEM4Z expression vector. Constructs pH52 and pHSS were prepared from a naturally occurring deletion mutant of FMV which results in fusion of coat protein (TV) and reverse transcriptase (V) dstrons. CAT gene in these constructs is positioned in cfetron II and VI, respectively. In vitro translation of transcripts prepared from these constructs results in simultaneous synthesis of CAT polypeptide and predicted size of fusion (TV/V) porypepode of 86,000 dallons. These polypeptides are Immuno selected using anflCAT and antiviral coat protein antibodies. When IV/V ORF Is deleted from these DNA constructs by digestion with the Sac 1 before transcript synthesis, the expression of fusion polypeptide is abolished. In pH52 contracts synthesis of CAT polypeptide is still observed. These results provide further support to the translational coupling/relay race model hi the translation of full length transcript of FMV. (CSU Biotech. and UK USDA grants).
