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• W3150210487 abstract "Creation of an artificial mRNA-interfering complementary RNA (micRNA) immune system, utilizing anti-sense RNAs to inhibit viral gene expression, has been shown to be an effective way to prevent viral infection. In the RNA coliphage SP, the gene for the maturation protein was found to be the best target for this type of immune system; mRNA-interfering complementary RNAs specific to the genes for coat protein and replicase were less effective in preventing infection. The greatest inhibitory effect was observed with a 240-base sequence encompassing the 24-base noncoding region of the maturation gene plus the 216-base coding sequence. Significantly, even a 19-base sequence covering only the Shine-Dalgarno sequence (ribosome-binding region) without the coding region exerted a strong inhibitory effect on phage proliferation. In contrast to the highly specific action against phage SP exhibited by the longer mRNA-interfering complementary RNA, the specificity with the shorter mRNA-interfering complementary RNA was broadened to phages Q,f and GA as well as SP, all of mhich are classified in the different groups of RNA coliphages. Therefore, this type of anti-viral reagent may be designed to have a particular breadth of specificity, thus increasing its value in various research and possibly clinical applications. An anti-sense RNA to a specific mRNA has been shown to function as a repressor for gene expression in Escherichia coli (1, 2). The micF gene maps at 21 min on the E. coli chromosome and encodes a small RNA, micF RNA, which is complementary to the mRNA for the major outer membrane protein, OmpF. Production of the micF RNA has been shown to inhibit OmpF production. Since the artificial production of an anti-sense RNA against a specific gene can be easily achieved in both prokaryotes and eukaryotes, several attempts have been made to artificially regulate specific genes in a manner analogous to the micF system (refs. 3-12; for review see also ref. 13). We have termed these regulators micRNAs for mRNA-interfering complementary RNA. In addition to regulating cellular genes by artificial micRNA, we have designed a specific E. coli immune system against viral infection (micRNA immune system) using micRNAs against viral genes (14). E. coli cells containing a plasmid carrying an inducible gene for micRNAs against coat protein and replicase of the positive, single-stranded RNA coliphage SP became resistant or immune to the phage upon induction of the micRNA (14). In the present paper we explore the design of a construct that will more effectively confer immunity against phage SP and discuss how to broaden the specificity of the micRNA immune system to include other related RNA phages. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. ?1734 solely to indicate this fact. MATERIALS AND METHODS Bacterial Strains and Plasmids. E. coli K12 strain JA221 (lacYhsdR trpES leuB6 recA/F' laCq lac+ pro-) (15) and E. coli A/X (F+, Su', pro-) were used. Cell culture and phage infection were carried out as described (14). Plasmid pJDC406 (14) was used for construction of various micRNA immune systems. DNA Manipulation. DNA manipulation was carried out according to Maniatis et al. (16). Phage Titers. Phage titers in Fig. 3 were estimated using E. coli A/X as indicator. E. coli cells carrying pMIC-Dl and/or pJDC406 were grown at 37?C in L broth containing 5 mM CaCl2, and isopropyl /3-D-thiogalactoside (IPTG) was added at a final concentration of 2 mM at a Klett-Summerson reading of 10 to induce the micRNA gene. At a Klett unit of 55, phage SP was added at a multiplicity of 1. After 6 min, 0.1 ml of the culture was mixed with 0.9 ml of L broth containing an excess amount of anti-SP serum (K = 10) and 2 mM IPTG. The mixture was incubated at 37?C to remove unabsorbed phage particles. At 12 min after infection, the mixture was further diluted 1:10,000. At each point for the first 29 min, an aliquot, after an appropriate dilution, was immediately mixed with a large excess of stationary culture of E. coli A/X and plated onto L-broth agar plates. After 35 min of infection, samples were treated with chloroform before an appropriate dilution. Numbers of plaques were measured after incubation at 37?C overnight." @default.
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• W3150210487 date "2016-01-01" @default.
• W3150210487 modified "2023-09-26" @default.
• W3150210487 title "Engineering of the mRNA-interfering complementary RNA immune system against viral infection" @default.
• W3150210487 hasPublicationYear "2016" @default.
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