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Expression of enzymatically active adenovirus DNA polymerase from cloned DNA requires sequences upstream of the main
L M Shu1, M S Horwitz, J A Engler
1Department of Biochemistry, University of Alabama at Birmingham 35294.
Abstract:
Replication of human adenovirus (Ad) DNA requires three virus-encoded proteins that are coordinately transcribed from a single promoter at early times after infection. The mRNAs for two of these proteins, the preterminal protein (pTP) and the Ad DNA polymerase (Ad Pol), share several exons, including one encoded near Ad genome coordinate 39. Plasmids containing the putative exons that encode Ad Pol mRNA were constructed to determine if enzymatically active Ad Pol protein could be synthesized. An Ad Pol of 140 kDa was detected by immunoprecipitation with specific antibody and its enzymatic activity was confirmed by complementation of Ad DNA replication in vitro. In addition to an Ad2 DNA fragment from 24.7 to 9.2 map units which contains an open reading frame for a protein of 120 kDa, the HindIII-J fragment that encodes the exon at genome coordinate 39 can be shown to be essential for production of full-length (140 kDa), enzymatically active Ad Pol.
Insights
Human adenovirus DNA replication needs specific viral proteins. Researchers confirmed the essential role of the HindIII-J fragment in producing active adenovirus DNA polymerase (Ad Pol).
Area of Science:
- Molecular Biology
- Virology
Background:
- Human adenovirus (Ad) DNA replication relies on three virus-encoded proteins.
- These proteins are transcribed from a single promoter early in infection.
- The mRNAs for preterminal protein (pTP) and Ad DNA polymerase (Ad Pol) share exons, including one at genome coordinate 39.
Purpose of the Study:
- To determine if enzymatically active Ad Pol protein could be synthesized.
- To investigate the role of specific DNA fragments in Ad Pol production.
Main Methods:
- Construction of plasmids containing putative Ad Pol mRNA exons.
- Detection of Ad Pol protein using immunoprecipitation with a specific antibody.
- Confirmation of enzymatic activity through complementation of Ad DNA replication in vitro.
Main Results:
- A 140 kDa Ad Pol protein was detected and confirmed to be enzymatically active.
- The HindIII-J fragment, encoding an exon at genome coordinate 39, was found essential for producing full-length, active Ad Pol.
- An Ad2 DNA fragment (24.7 to 9.2 map units) containing a 120 kDa open reading frame was also studied.
Conclusions:
- The HindIII-J fragment is crucial for the synthesis of enzymatically active, full-length adenovirus DNA polymerase.
- Understanding Ad Pol synthesis is key to deciphering adenovirus DNA replication mechanisms.