ABSTRACT
A method for the amplification of viral signal present in infected samples is described. Viral suspensions are spotted on nitrocellulose paper, and immediately afterwards an adequate amount of a permissive cell line is added. The nitrocellulose filter is then incubated overnight, fixed and hybridized to labeled viral probes. The technique is extremely fast, reproducible and inexpensive, and may be readily applicable to the clinical diagnosis of many viral diseases.
Subject(s)
Virology/methods , Viruses/isolation & purification , Biotechnology , Collodion , DNA, Viral/genetics , DNA, Viral/isolation & purification , Filtration/instrumentation , Molecular Probes , Nucleic Acid Hybridization , RNA, Viral/genetics , RNA, Viral/isolation & purification , Viruses/geneticsABSTRACT
A quick and sensitive method to quantitate viral RNA synthesis has been developed. Utilizing glutaraldehyde to fix infected cells onto nitrocellulose paper, viral RNA can be probed directly in situ. Viral message can be detected from as few as 10(4) infected cells. This technique can be used to study viral gene expression and can be adapted to screen the activity of antiviral agents such as interferon.
Subject(s)
Encephalomyocarditis virus/genetics , RNA, Viral/analysis , Simplexvirus/genetics , Virology/methods , Animals , Cell Line , DNA, Viral/genetics , Gene Expression Regulation , Glutaral , Humans , Interferons/pharmacology , Nucleic Acid Hybridization , RNA, Viral/biosynthesis , Transcription, Genetic , Vero CellsABSTRACT
2,6-Diaminopurine(DAP)-resistant mutants have been isolated from mouse lymphoma 5178Y TK+/TK- heterozygotes. In the presence of 50 microM DAP, two colony types were isolated. Small colonies contained 50% wild-type adenine phosphoribosyl transferase (APRT) activity (partial mutants), whereas large colonies have undetectable levels of APRT (aprt- mutants). aprt- mutants could be isolated following mutagenesis with ICR-191 or EMS from the partial mutants. Southern blot analysis of EcoRI digested wild-type DNA using a 3.1 kb mouse aprt genomic probe indicated sequence polymorphism at one or both EcoRI sites flanking the allele. Southern blot analysis of one of the partial mutants and one ICR-induced aprt- mutant (single step) indicated that both strains were hemizygous at the APRT locus. Such stable hemizygous strains would be useful in short-term mutagen tests.
