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In Vitro Transcription: Probing the Molecular Functions of Adenovirus Regulatory Proteins

Adenoviruses (Ads), like other DNA tumor viruses, have evolved specific regulatory genes that facilitate virus replication by controlling the transcription of other viral genes as well as that of key cellular genes that regulate cell-cycle progression and cellular DNA synthesis. Of p ...

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Cell Microinjection: In Vivo Analysis of the Functional Domains of Viral Regulatory Proteins

Mammalian-cell microinjection is a powerful method for analyzing the in vivo functions of viral genes and viral gene products. By microinjection, a controlled amount (ranging from 1 to many thousands of copies) of a viral or cellular gene, a protein product of a gene, a polypeptide fragment encod ...

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Immunoprecipitation of E1 A-Containing Protein Complexes

Important insights into cell growth and transcriptional regulation have been realized by correlating adenovirus ElA-induced cell-growth-control mechanisms with ElA’s specific interactions with host-cell proteins (see ref. 4). A key method for directly demonstrating these ...

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Preparation of Splicing-Competent Nuclear Extracts from Adenovirus-lnfected Cells

Adenovirus has contributed significantly to our current understanding of the organization and expression of genes in eukaryotic cells. The most startling discovery was probably the observation that most adenovirus mRNAs are encoded from discontinuous DNA segments. The split- ...

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Adenovirus Entry into Cells: A Quantitative Fluorescence Microscopy Approach

Adenoviruses carry their DNA genome into postmitotic nuclei of a variety of human cells, either within an organism (in vivo) or outside an organism in culture (ex vivo) (1). Recombinant adenoviruses are developed m many laboratories as gene-delivery vehicles to treat hereditary and acquir ...

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Simultaneous Detection of RNA and Proteins in Adenovirus-Infected Cells by Fluorescence In Situ Hybridization (FISH) and lmmunostaining

Adenovirus (Ad)-infected cells have been used extensively as a model system for studying the expression of eukaryotic genes and were instrumental in elucidating steps m the production of RNA. Much of this work involved genetic and molecular analyses of viral gene expression. Recent advan ...

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Preparation and Titration of CsCl-Banded Adenovirus Stock

An important step m the development of modern experimental virology was the development of the plaque assay, first with bacteriophage, then with eukaryotic viruses. In order to obtain quantitative, interpretable, and reproducible results, it is necessary to know how much virus is being us ...

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Adenovirus Protease

Adenoviruses encode a cysteme endopeptidase synthesized late in virus infection which is essential for virlon maturation and infectivity (1,2). The enzyme 1s encapsidated (approx 20 molecules per vinon) and may also have a role during decapsidation (3–5). Although there are approx 100 a ...

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Transfection Complexes Generated with Adenovirus and Polyethylenimine-Condensed DNA

Many types of viral infection produce a transient permeabilization of the Infected cell (see refs. 1 and 2; reviewed in refs. 3 and 4). A large improvement in transfection efficiency was obtained when this phenomenon was applied to receptor-mediated gene delivery (5). A number of permeabilizing ...

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Methods for Growth and Purification of Enteric Adenovirus Type 40

The enteric adenoviruses of subgroup F (Ad40 and Ad41) pose some special problems of cultivation, as they cannot be readily passaged in many of the cell types used to propagate the more commonly used subgroup C serotypes (Ad2 and Ad5), and there is no standard plaque assay: A full discussion of the observ ...

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Phylogenetic Analysis of Adenovirus Sequences: Proof of the Necessity of Establishing a Third Genus in the Adenoviridae Family

Bovine adenovirus (BAV) serotypes 4 through 8 have been found clearly distingushable from BAVs 1,2,3, and 9 and from all other mastadenoviruses, and were therefore classified as subgroup 2 BAVs (1) and were considered as candidate members of a new taxon (2). The distinction was originally based on b ...

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Construction of Mutations in the Adenovirus Early Region 3 (E3) Transcription Units

The E3 transcription unit of the well-studied subgroup C adenoviruses (Ad) (prototypic serotypes 2 and 5 ) is located between map units 76–86 (see Fig. 1). The E3 region is surrounded by the genes for virion protein VIII and fiber, and is transcribed off the r-strand. The E3-region transcription unit is co ...

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Isolation, Growth, and Purification of Defective Adenovirus Deletion Mutants

Adenovirus mutants that lack essential genes must be grown by complementation, the products of the missing genes supplied by a source other than the viral genome. Two methods are available for the growth of defective adenovirus mutants by complementation. For mutations confined to E1, E4, or p ...

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Manipulation of Early Region 4

Early region 4 (E4; Fig. 1A) occupies the right-hand 3000 bp of the human adenovirus genome. The sequences of E4 and E4 cDNAs indicate that E4 encodes seven polypeptides (1–4), most of which have been detected in infected cells. Analysis of E4 mutants has implicated E4 products in a variety of processes that ...

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Adenovirus DNA Packaging: Construction and Analysis of Viral Mutants

The selective packaging of adenovirus DNA into a capsid at late times after viral infection raises a number of interesting questions. How is the viral DNA specifically selected from the pool of viral and cellular DNA for encapsidation? Is the packaging process coordinated with viral DNA repli ...

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Methods for Creating and Analyzing Adenovirus Vectors that Express Proteins that Act on the Viral Genome

Adenovirus genetic research has entered a new phase with the recent upsurge in interest in using the virus as a vector for gene transfer, gene therapy, and as a vaccine (reviewed in refs. 1–3). Although the idea of using adenovirus as a vector is not new (4), recent advances both in understanding the functions ...

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Construction of Mouse Adenovirus Type 1 Mutants

Construction of mouse adenovirus type 1 (MAV-1) mutants is facilitating studies of adenoviral pathogenesis in the natural host. We have isolated the first site-directed viral mutants of MAV-1, and we are comparing effects of wild-type (wt) and these mutant viruses in infections of mice (Smith ...

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Generation of Adenovirus-Specific Cytotoxic T Lymphocytes

When using adenovirus as a vector for the delivery of transgenes, the induction of a cytotoxic T lymphocytes (CTL) can limit the longevity of the transgene expression (1,2). To detect the CTL response to adenovirus proteins or inserted transgenes in a mouse model, even with multiple in vivo immuniz ...

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Measurement of Macrophage-Mediated Cytolysis of Adenovirus-Infected Cells

Macrophages are well-known for their ability to serve as phagocytes, ingesting and destroying microorganisms such as bacteria, and for their function in antigen presentation. Less appreciated, perhaps, is the fact that macrophages are also capable of recognizing and destroying cer ...

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Interaction of Cationic Peptides with Bacterial Membranes

A common feature of cationic peptides is that their site of action is at the membrane due to channel formation, and that they tend to possess strong selectivity towards then target membrane. For example, although moth cecropin and bee melittin are members of the same family of peptides that adopt amp ...

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