Biomaterial Database

Isolation and analysis of adenovirus type 5 mutants containing deletions in the gene encoding the DNA-binding protein. 1985

S A Rice, and D F Klessig

A genetic system is described which allows the isolation and propagation of adenovirus mutants containing lesions in early region 2A (E2A), the gene encoding the multifunctional adenovirus DNA-binding protein (DBP). A cloned E2A gene was first mutagenized in vitro and then was introduced into the viral genome by in vivo recombination. The E2A mutants were propagated by growth in human cell lines which express an integrated copy of the DBP gene under the control of a dexamethasone-inducible promoter (D. F. Klessig, D. E. Brough, and V. Cleghon, Mol. Cell. Biol. 4:1354-1362, 1984). The protocol was used to construct five adenovirus mutants, Ad5d1801 through Ad5d1805, which contained deletions in E2A. One of the mutants, Ad5d1802, made no detectable DBP and thus represents the first DBP-negative adenovirus mutant, while the four other mutants made truncated DBP-related polypeptides. All five mutants were completely defective for growth and plaque formation on HeLa cell monolayers. Furthermore, the two mutants which were tested, Ad5d1801 and Ad5d1802, did not replicate their DNA in HeLa cells. The mutant Ad5d1804 encoded a truncated DBP-related protein which contained an entire amino-terminal domain derived from the host range mutant Ad5hr404, a variant of Ad5 which multiplies efficiently in monkey cells. While results of a previous study suggest that the amino-terminal domain of DBP could act independently of the carboxyl-terminal domain to enhance late gene expression in monkey cells, the Ad5d1804 polypeptide failed to relieve the block to late viral protein synthesis in monkey cells. The mutant Ad5d1802 was used to study the role of DBP in the regulation of early adenovirus gene expression in infected HeLa cells. These experiments show that E2A mRNA levels are consistently reduced approximately fivefold in Ad5d1802-infected cells, suggesting either a role for DBP in the expression of its own gene or a cis-acting defect caused by the E2A deletion. DBP does not appear to play a significant role in the regulation of adenovirus early regions 1A, 1B, 3, or 4 mRNA levels in infected HeLa cell monolayers since wild-type Ad5- and Ad5d1802-infected cells showed very little difference in the patterns of expression of these genes.

UI	MeSH Term	Description	Entries
D009154	Mutation	Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.	Mutations
D002872	Chromosome Deletion	Actual loss of portion of a chromosome.	Monosomy, Partial,Partial Monosomy,Deletion, Chromosome,Deletions, Chromosome,Monosomies, Partial,Partial Monosomies
D004261	DNA Replication	The process by which a DNA molecule is duplicated.	Autonomous Replication,Replication, Autonomous,Autonomous Replications,DNA Replications,Replication, DNA,Replications, Autonomous,Replications, DNA
D004268	DNA-Binding Proteins	Proteins which bind to DNA. The family includes proteins which bind to both double- and single-stranded DNA and also includes specific DNA binding proteins in serum which can be used as markers for malignant diseases.	DNA Helix Destabilizing Proteins,DNA-Binding Protein,Single-Stranded DNA Binding Proteins,DNA Binding Protein,DNA Single-Stranded Binding Protein,SS DNA BP,Single-Stranded DNA-Binding Protein,Binding Protein, DNA,DNA Binding Proteins,DNA Single Stranded Binding Protein,DNA-Binding Protein, Single-Stranded,Protein, DNA-Binding,Single Stranded DNA Binding Protein,Single Stranded DNA Binding Proteins
D005786	Gene Expression Regulation	Any of the processes by which nuclear, cytoplasmic, or intercellular factors influence the differential control (induction or repression) of gene action at the level of transcription or translation.	Gene Action Regulation,Regulation of Gene Expression,Expression Regulation, Gene,Regulation, Gene Action,Regulation, Gene Expression
D005814	Genes, Viral	The functional hereditary units of VIRUSES.	Viral Genes,Gene, Viral,Viral Gene
D005818	Genetic Engineering	Directed modification of the gene complement of a living organism by such techniques as altering the DNA, substituting genetic material by means of a virus, transplanting whole nuclei, transplanting cell hybrids, etc.	Genetic Intervention,Engineering, Genetic,Intervention, Genetic,Genetic Interventions,Interventions, Genetic
D000260	Adenoviruses, Human	Species of the genus MASTADENOVIRUS, causing a wide range of diseases in humans. Infections are mostly asymptomatic, but can be associated with diseases of the respiratory, ocular, and gastrointestinal systems. Serotypes (named with Arabic numbers) have been grouped into species designated Human adenovirus A-G.	APC Viruses,APC Virus,Adenovirus, Human,Human Adenovirus,Human Adenoviruses
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D013329	Structure-Activity Relationship	The relationship between the chemical structure of a compound and its biological or pharmacological activity. Compounds are often classed together because they have structural characteristics in common including shape, size, stereochemical arrangement, and distribution of functional groups.	Relationship, Structure-Activity,Relationships, Structure-Activity,Structure Activity Relationship,Structure-Activity Relationships