Mapping Temperature-sensitive Mutants of Vesicular Stomatitis Virus by RNA Heteroduplex Formation

Abstract

SUMMARYDuplex RNA molecules made by hybridization of virion and mRNA of vesicular stomatitis virus (VSV) were digested with ribonuclease and separated into five size classes, each containing the gene and the mRNA for one of the VSV proteins. Denaturation of the duplexes yielded full size mRNA lacking poly(A) tails. Utilizing duplex formation between the RNAs from VSV temperature-sensitive (ts) mutants and their revertants and subsequent RNase digestion under varying salt conditions, specific cleavages within a certain duplex were seen for representative mutants from complementation groups III, IV and V. Specific cleavages were not seen for a group II mutant. From these results gene assignments cannot be made for group II; equivocal assignments are made for group III and clear assignments made for group IV and V. The assignment for the group V mutants, however, does not conform to expectations. Nevertheless, from these studies and other published ones, there is the suggestion that interactions may exist between the gene products of complementation groups II and V during VSV transcription and morphogenesis. These results also support the lack of transcriptional splicing for VSV mRNAs.

Sensitive and fast mutation detection by solid phase chemical cleavage

“…Several methods have been developed for analyzing point mutations, small deletions, and insertions. The methods include DNA sequencing, denaturing gradient gel electrophoresis (DGGE) (Myers et al, 1985;Abrams et al, 1990) heteroduplex method (White et al, 1992), single strand conformation polymorphism (SSCP) (Orita et al, 1989;Danenberg et al, 1992), ribonuclease A cleavage (Freeman and Huang, 1981;Gibbs and Caskey, 1987), carbodiimide modification (Novack et al, 1986;Ganguly and Prockop, 1990), MutS binding (Ellis et al, 1994), enzyme mismatch cleavage (Youil et al, 1995), and the chemical cleavage of mismatches (CCM) (Cotton et al, 1988). The chemical cleavage method has great advantages compared to the other methods: large fragments (up to 2kb) can be analyzed in one operation, the nature and position (within 10-15bp) of the mutation are revealed, and close to 100% of all mutations are found (Cotton, 1989).…”

Somatic Mutation Detection in Human Biomonitoring

“…It utilizes the enzymatic ability to cleave RNNRNA or RNAIDNA heteroduplexes (Freeman & Huang 1981; Myers e f al. Different kinds of enzymes can detect non-paired bases and restrict DNA at these sites.…”

“…One assay used, is the Ribonuclease A mismatch assay. It utilizes the enzymatic ability to cleave RNNRNA or RNAIDNA heteroduplexes (Freeman & Huang 1981; Myers e f al. 1985a).…”

Ανάπτυξη Μεθόδου Δεσμευτικής Αναλύσεως Για Την Ανίχνευση-Προσδιορισμό Βιομορίων Σε Βιολογικά Δείγματα

“…Πολλές ριβονουκλεάσες διασπούν μονόκλωνες αλυσίδες του RNA στα κατάλοιπα της πυριμιδίνης. Η παραπάνω ιδιότητα αξιοποιήθηκε στη μέθοδο RPA όταν βρέθηκε ότι ορισμένες ριβονουκλεάσες μπορούσαν να διασπάσουν ετερόδιπλα μόρια RNA-RNA [16], καθώς και RNA-DNA [17] που διέφεραν έστω και σε ένα ζεύγος βάσεων. Αρχικά RNA συμπληρωματικό με την αλληλουχία ενδιαφέροντος μεταγράφεται in vitro και στη συνέχεια επισημαίνεται με ραδιοϊσότοπο ή βιοτίνη.…”