Molecular localisation of a G-protein mRNA using differential display and in situ hybridization.
Defining molecular repertoires within virally infected tissues of the nervous system may provide insight into the pathogenesis of, and immunity to, neurotropic viruses. Here we report the application of such a method, namely mRNA differential display (DD), to the identification of mRNAs that are expressed at different levels in herpes simplex virus (HSV) infected nervous tissue from immunocompetent and CD8(+) lymphocyte depleted mice. Small amounts of input RNA can be used by DD, making the method ideal for experiments based on murine sensory ganglia (DRG), which on average yield less than 0.5 microg of total RNA. In the current work, DD facilitated the identification of a mRNA whose abundance in HSV-infected ganglia, based on Northern blot analysis, was reduced in mice depleted of CD8(+) cells. The cloned product of this mRNA was of particular interest to our research as sequence data strongly suggested that it represented the murine homologue of the alpha chain of a G protein termed Golf. This G protein had not previously been reported from dorsal root ganglial tissue. RT-PCR confirmed the presence of Golf in DRG and in situ hybridization studies localised this molecule to primary sensory neurons. These data indicate that DD is sufficiently robust to be applied to the study of virus pathogenesis within the nervous system.
Wilkinson
R
R
Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, Frome Road, South Australia 5000, Adelaide, Australia. ray.wilkinson@imvs.sa.gov.au
Tscharke
D
D
Simmons
A
A
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015152:Blotting, Northern; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D005260:Female; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D005727:Ganglia, Spinal / Q000166:cytology / Q000502:physiology / Q000821:virology; D015967:Gene Expression Regulation, Viral; D006561:Herpes Simplex / Q000175:diagnosis; D018259:Herpesvirus 1, Human / Q000235:genetics; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics*; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D009475:Neurons, Afferent / Q000502:physiology / Q000821:virology; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA
D000906:Antibodies; D012333:RNA, Messenger; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins