Short nucleotide sequence analysis of seven restriction fragments of murine herpesvirus 68 (MHV-68) DNA has been undertaken and used to determine the overall genome organization and relatedness of this virus to other well characterized representatives of the alpha-, beta-and gammaherpesvirus subgroups. Nine genes have been identified which encode amino acid sequences with greater similarity to proteins of the gammaherpesvirus Epstein-Barr virus (EBV) than to the homologous products of the alphaherpesviruses varicella-zoster virus and herpes simples virus type 1 or the betaherpesvirus human cytomegalovirus. In addition, the genome organization of MHV-68 is shown to have an overall collinearity with that of the gammaherpesviruses EBV and herpesvirus saimiri. In common with these viruses, dinucleotide frequency analysis of MHV-68 coding sequences reveals a marked reduction in CpG dinucleotide frequency thus implicating a dividing cell population as the site of latency in vivo.
Guanylyl cyclase-activating proteins (GCAPs) are Ca 2ϩ -binding proteins that activate guanylyl cyclase when free Ca 2ϩ concentrations in retinal rods and cones fall after illumination and inhibit the cyclase when free Ca 2ϩ reaches its resting level in the dark. Several forms of retinal dystrophy are caused by mutations in GUCA1A, the gene coding for GCAP1. To investigate the cellular mechanisms affected by the diseased state, we created transgenic mice that express GCAP1 with a Tyr99Cys substitution (Y99C GCAP1) found in human patients with a late-onset retinal dystrophy (Payne et al., 1998). Y99C GCAP1 shifted the Ca 2ϩ sensitivity of the guanylyl cyclase in photoreceptors, keeping it partially active at 250 nM free Ca 2ϩ , the normal resting Ca 2ϩ concentration in darkness. The enhanced activity of the cyclase in the dark increased cyclic nucleotide-gated channel activity and elevated the rod outer segment Ca 2ϩ concentration in darkness, measured by using fluo-5F and laser spot microscopy. In different lines of transgenic mice the magnitude of this effect rose with the Y99C GCAP1 expression. Surprisingly, there was little change in the rod photoresponse, indicating that dynamic Ca 2ϩ -dependent regulation of cGMP synthesis was preserved. However, the photoreceptors in these mice degenerated, and the rate of the cell loss increased with the level of the transgene expression, unlike in transgenic mice that overexpressed normal GCAP1. These results provide the first direct evidence that a mutation linked to congenital blindness increases Ca 2ϩ in the outer segment, which may trigger the apoptotic process.
Murine herpesvirus 68 (MHV-68) is a naturally occurring herpesvirus of small free-living rodents. In order to facilitate the molecular characterization of the virus genome, a library of cloned restriction fragments has been produced and restriction enzyme cleavage maps deduced for the enzymes BamHI, EcoRI and HindIII. The MHV-68 genome comprises a region of unique DNA of approximately 118 kbp which is flanked by variable numbers of a 1.23 kb repeat unit. The organization of the MHV-68 genome is, therefore, most similar to that of the lymphotropic Y2 group of herpesviruses which include herpesvirus saimiri and herpesvirus ateles.
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