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Cloning and gene assignment of mRNAs of human parainfluenza virus 3
Abstract:
Cytoplasmic poly(A)-containing RNA from parainfluenza virus 3-infected cells was used as template to construct a cDNA library that was cloned into the EcoRV site of the plasmid pMG5. The resulting clones were screened with [32P]-labeled cDNA probes made from infected and mock-infected cell mRNAs. The virus specificity of the clones was confirmed by Northern blot hybridization. The viral clones were grouped into five different families by hybridization with individual size-selected reverse transcripts representing the major classes of poly(A)+-RNA from virus-infected cells. The five groups were shown to be unrelated on the basis of cross-colony hybridization and corresponded to five unique classes of intracellular viral poly(A)+-RNAs. Clones representing the NP and P genes of PIV-3 were identified by both hybrid-selected and hybrid-arrested translation. Clones specific for the P gene selected mRNA that directed the synthesis of P protein and another polypeptide of 21 kDa. This additional polypeptide comigrated with protein VP8 previously identified in virions and in infected cell lysates.
Insights
Researchers created a cDNA library from parainfluenza virus 3-infected cells to identify viral genes. This study successfully identified clones for the NP and P genes, aiding in understanding viral gene expression.
Area of Science:
- Molecular Virology
- Gene Expression Analysis
Background:
- Parainfluenza virus 3 (PIV-3) is a significant human respiratory pathogen.
- Understanding PIV-3 gene expression is crucial for developing antiviral strategies.
Purpose of the Study:
- To construct a complementary DNA (cDNA) library from PIV-3 infected cells.
- To identify and characterize clones representing specific PIV-3 genes.
Main Methods:
- Construction of a cDNA library using cytoplasmic poly(A)-containing RNA from PIV-3 infected cells.
- Screening of clones using radiolabeled cDNA probes and Northern blot hybridization.
- Identification of specific gene clones via hybrid-selected and hybrid-arrested translation.
Main Results:
- A PIV-3 cDNA library was successfully constructed and screened.
- Viral clones were categorized into five distinct, unrelated families based on RNA hybridization.
- Clones corresponding to the nucleoprotein (NP) and phosphoprotein (P) genes of PIV-3 were identified.
- P gene clones selected mRNA encoding the P protein and a 21 kDa polypeptide, potentially VP8.
Conclusions:
- The study established a method for isolating and characterizing PIV-3 specific cDNA clones.
- Identification of NP and P gene clones provides tools for further PIV-3 research.
- The 21 kDa polypeptide associated with the P gene warrants further investigation regarding its role and identity.