Inversing the natural hydrogen bonding rule to selectively amplify GC-rich ADAR-edited RNAs

DNA complementarity is expressed by way of three hydrogen bonds for a G:C base pair and two for A:T. As a result, careful control of the denaturation temperature of PCR allows selective amplification of AT-rich alleles. Yet for the same reason, the converse is not possible, selective amplification of GC-rich alleles. Inosine (I) hydrogen bonds to cytosine by two hydrogen bonds while diaminopurine (D) forms three hydrogen bonds with thymine. By substituting dATP by dDTP and dGTP by dITP in a PCR reaction, DNA is obtained in which the natural hydrogen bonding rule is inversed. When PCR is performed at limiting denaturation temperatures, it is possible to recover GC-rich viral genomes and inverted Alu elements embedded in cellular mRNAs resulting from editing by dsRNA dependent host cell adenosine deaminases. The editing of Alu elements in cellular mRNAs was strongly enhanced by type I interferon induction indicating a novel link mRNA metabolism and innate immunity

The regulation of interferon synthesis in animal cells.

The inability of the VERO cell line to produce interferon suggested that it might represent a regulatory variant which would be useful for somatic cell genetic studies of the regulation of interferon production. It was confirmed that VERO cells are unable to produce interferon in response to virus infection whereas BSC.B cells (similarly derived from the African Green monkey) were able to do so. Treatment of a number of cell lines and embryonic cells with polyriboinosinic. polyribocytidylic acid (poly(rI). poly(rC)) (usually in the presence of diethylaminoethyl (DEAE)-dextran) resulted in the development of an anti-viral state but VERO cells were found to be only weakly responsive. No effective method for significantly enhancing the anti-viral action of poly(rI). poly(rC) or interferon production was found. The cytotoxic effect of interferon and poly(rI). poly(rC) treatment was found to be limited to L929 cells and thus could give no information on the non-inducibility of VERO cells. Mutagenized VERO cells surviving selection by infection with temperature-sensitive Sindbis virus after treatment with poly(rI). poly(rC) were similar to VERO cells in their ability to support the growth of wild-type or temperature-sensitive Sindbis virus or to respond to poly(rI). poly(rC) treatment and they did not release virus or interferon into the medium, VERO cells cannot therefore give rise to inducible revertants. HAT medium containing ouabain was found to be suitable for the selection of hybrids of VERO cells with mouse D1 cells lacking thymidine kinase. Mouse D1 cells are able to produce interferon. Polyethylene glycol treatment effectively replaced treatment with inactivated Sendai virus for the promotion of cell fusion. All VERO-mouse hybrid clones examined were sensitive to the anti-viral action of poly(rI). poly(rC) treatment and many were shown to produce mouse interferon, however, none were able to produce monkey interferon. Certain clones were less sensitive than mouse D1 cells to the anti-viral action of poly(rI). poly(rC) and this may have been an indication of the activity of a VERO cell-coded repressor of interferon production. Thus there is no evidence that VERO cells are able to produce interferon and only slight evidence for an altered regulatory function in these cells