Abstract
Movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV) belongs to “30 K” superfamily of proteins and members of this family are known to show a wide array of functions. In the present study this gene was found to be genetically unstable in E. coli when transformed DH5α cells were grown at 28 °C and 37 °C. However, genetic instability was not encountered at 20 °C. Heterologous over expression failed despite the use of different transcriptional promoters and translational fusion constructs. Total cell lysate when subjected to western blotting using anti-ACLSV MP antibodies, showed degradation/cleavage of the expressed full-length protein. This degradation pointed at severe proteolysis or instability of the corresponding mRNA. Predicted secondary structure analysis of the transcript revealed a potential cleavage site for an endoribonuclease (RNase E) of E. coli. The negating effect of RNase E on transcript stability and expression was confirmed by northern blotting and quantitative RT-PCR of the RNA extracted from RNase E temperature sensitive mutant (strain N3431). The five fold accumulation of transcripts at non-permissive temperature (43 °C) suggests the direct role of RNase E in regulating the expression of ACLSV MP in E. coli.
Highlights
Apple chlorotic leaf spot virus (ACLSV), a member of genus Trichovirus, family Betaflexiviridae, is a positive sense, single stranded RNA virus having particle size of about 600–700 nm
Studies on Movement protein (MP) sequence from ACLSV-P isolate which shares only 79.9% homology to ACLSV-A MP sequence have not been undertaken to date
There is a lot of ambiguity related to the initiation codon marked in gene sequences of ACLSV MP submitted in nucleotide database maintained by NIH, Bethesda, Maryland, USA
Summary
Apple chlorotic leaf spot virus (ACLSV), a member of genus Trichovirus, family Betaflexiviridae, is a positive sense, single stranded RNA virus having particle size of about 600–700 nm. Irrespective of the various modes of cell to cell movement, based on structural conservation, many viral movement proteins including the ACLSV MP are assigned to “30 K” superfamily (members contain a conserved D motif) of proteins[15, 16]. Plasmid instability may be caused by the following factors (i) copy number of plasmid, (ii) toxic gene products, (iii) metabolic burden on host cells to maintain the plasmids, (iv) recombination potential of certain inserts, (v) presence of cryptic bacterial promoters in eukaryotic sequences may produce spurious/toxic peptides which create problems in stabilization of plasmids or leads to promoter occlusion[23,24,25], (vi) secondary structures of insert- DNA or presence of AT rich regions[26], poly d(T) regions[27] and presence of multiple repeats[28, 29].
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