Analysis of Ross River virus genomic RNA using HaeIII digests of single-stranded cDNA to infected-cell RNA and virion RNA.
To study genetic relationships between isolates of Ross River virus (RRV), an alphavirus with a chromosome of approximately 12,000 nucleotides, total high-molecular-weight RNA from RRV-infected baby hamster kidney (BHK) cells was transcribed into 32P-labeled, complementary DNA using reverse transcriptase and random calf-thymus DNA primers. The cDNA was digested with HaeIII or TaqI (restriction nucleases which cleave single-stranded DNA), and the restriction fragments separated on a standard DNA sequencing gel. The resulting HaeIII or TaqI restriction digest profiles mainly comprised virus-specific bands; cell RNAs were transcribed poorly. In reconstruction experiments, purified 49 S RRV genomic RNA and a 10-fold mass excess of mock-infected-cell RNA were reverse transcribed in the same reaction mix. Under these conditions there was no interference with the transcription of viral RNA sequences. When the level of viral RNA was lowered to one-hundredth that of cell RNA in the reaction mix, there was no qualitative change in restriction digest profiles. The procedure is rapid, simple, uses small amounts of 32P, does not require purification of virus or viral RNA, and permits cross-comparison between several virus strains on a single one-dimensional gel. The method should be applicable to other single-stranded RNA viruses of moderate genome complexity.
Faragher
S G
SG
Hutchison
C A
CA
Dalgarno
L
L
D000524:Alphavirus / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D004247:DNA; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded; D015252:Deoxyribonucleases, Type II Site-Specific; D004591:Electrophoresis, Polyacrylamide Gel; D005814:Genes, Viral; D007668:Kidney; D012313:RNA / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics*; D012398:Ross River virus / Q000235:genetics*
D004277:DNA, Single-Stranded; D012367:RNA, Viral; D012313:RNA; D004247:DNA; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases; C027046:TCGA-specific type II deoxyribonucleases