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M L Harter

Publications and source records attributed to M L Harter.

34 records · Page 2Linked to original sources
PubMed1988

DNA-binding properties of an adenovirus 289R E1A protein.

An adenovirus 2 289 amino acid (289R) E1A protein purified from Escherichia coli has been shown to interact with DNA by two independent methods. UV-crosslinking of complexes containing unmodified, uniformly 32P-labelled DNA and purified E1A protein induced efficient labelling of the protein with covalently attached oligonucleotides, indicating that the E1A protein itself contacts DNA. Discrete nucleoprotein species were also observed when E1A protein--DNA complexes were analysed by gel electrophoresis. Although the 289R E1A protein exhibited no significant binding to single-stranded DNA or to RNA, no evidence for its sequence-specific binding to double-stranded DNA was obtained with either assay. Identification of the sites of covalent attachment of 32P-labelled oligonucleotides by partial proteolysis of the crosslinked E1A protein indicated that the interaction of this protein with DNA is mediated via domain(s) in the C-terminal half of the protein. Such previously unrecognized DNA-binding activity is likely to contribute to the regulatory activities of this important adenoviral protein.

Adenovirus Early Proteins↗
PubMed1987

Activation of adenovirus promoters by the adenovirus E1A protein in cell-free extracts.

The primary product of the adenovirus E1A gene is a protein that is sufficient for controlling host-cell proliferation and immortalizing primary rodent cells. The mechanism by which the protein induces these cellular effects is poorly understood, but might be linked to its ability to regulate RNA transcription from a number of viral and cellular genes. The mechanism of E1A's transcriptional-activation (trans-activation) was studied here by monitoring the protein's effect on specific adenovirus promoters in two types of transcriptional systems in vitro. One of these systems consisted of extracts from transformed cells constitutively expressing E1A, and the other consisted of extracts of HeLa cells supplemented with a plasmid-encoded E1A protein purified from Escherichia coli. The results show that the E1A protein specifically stimulates transcription from adenovirus promoters; thus, the induction of cellular transcription factors is not necessary to explain the stimulation of transcription by E1A.

Adenoviridae↗
PubMed1986

Adenovirus-2 early region IA protein synthesized in Escherichia coli extracts indirectly associates with DNA.

The interaction of adenovirus-2 (Ad2) early region IA (EIA) protein (encoded by the 13S mRNA) with DNA was examined using EIA protein synthesized in Escherichia coli extracts directed by a plasmid containing the cloned EIA gene. Without any purification, this protein when chromatographed over calf thymus DNA immobilized on cellulose, showed at least two types of salt-sensitive activities after associating with equal efficiency to both single- and double-stranded DNA; however, a putative C-terminal proteolytic fragment of the EIA protein (identified by immunoprecipitation with anti-serum specific to the EIA carboxy-terminus) showed 10-fold greater affinity to double- versus single-stranded DNA. When examined with Ad2 DNA, the EIA protein had a retention that was at least 2-fold higher compared to calf thymus DNA, suggesting some substrate specificity. It was also found that a 1.0 M salt concentration was required for the elution of the EIA protein from pBR322 DNA containing cloned regulatory sequences of adenovirus early regions II and III. This suggests that the strength of the protein interaction depends on the target DNA sequence. Finally, addition of uninfected HeLa cell extract to bacterial extracts containing EIA-like protein potentiated the association of the protein to double-stranded calf thymus DNA up to 7-fold. These data support the hypothesis that the EIA protein interacts with target DNA, presumably mediated by co-factor(s) in an indirect fashion.

Adenovirus Early Proteins↗
PubMed1984

Plasmid-directed synthesis of genuine adenovirus 2 early-region 1A and 1B proteins in Escherichia coli.

The transforming region of human adenovirus 2 is located in the left 11.2% of the viral genome and is comprised of two distinct genetic units termed E1A and E1B. cDNAs containing the entire nucleotide sequence of the mature E1A 13S and E1B 22S mRNAs that are complementary to these genetic units have been introduced into bacterial plasmids a short distance downstream from the Escherichia coli lac promoter. Upon transformation into appropriate E. coli hosts, one of these plasmids, pKHAO, directed the synthesis of a 45-kilodalton (kd) protein, and the other, pKHBO, synthesized a protein of 54.9 kd. Both of these plasmid-encoded proteins constituted 0.1 to 0.3% of the total cellular protein and were virtually identical to the authentic adenovirus 2 E1A 42- to 50-kd and E1B 53- to 58-kd tumor antigens (T antigen) as determined by gel electrophoresis, immunoprecipitation, and tryptic fingerprint analysis. With the use of our pKHBO expression plasmid we were also able to demonstrate that the second AUG sequence appearing in the E1B 22S mRNA corresponded to the start of the gene encoding the large adenovirus 2 T antigen. This confirms theoretical deductions based on DNA sequencing analysis that translation of the large T antigen initiates translation at an internal ATG rather than at the 5'-proximal AUG.

Adenoviruses, Human↗
PubMed1983

The photobinding of 5,7-dimethoxycoumarin to adenovirus type-2 DNA. A method for in vitro mutagenesis.

The photobinding of 5,7-dimethoxycoumarin to isolated adenovirus-type 2 DNA has been investigated with respect to the influence of the ionic environment, and varying molar ratios of DNA(p): 5,7-dimethoxycoumarin. In particular, the ultraviolet radiation-induced covalent addition of 5,7-dimethoxycoumarin to adenovirus DNA was increased by reducing the concentration of Na+. The maximum photobinding of 5,7-[3H]dimethoxycoumarin to adenovirus DNA under the given ionic condition was one 5,7-dimethoxycoumarin per 101 nucleotides. Moreover, restriction enzyme analysis of the 5,7-dimethoxycoumarin-DNA photoadduct versus unmodified viral DNA, suggested that the sequence d(A-T) is the preferential site for intercalation and subsequent photobinding of 5,7-dimethoxycoumarin. This susceptibility of d(A-T) sequences to 5,7-dimethoxycoumarin interaction has a corresponding influence on the survival of adenovirus because of the A-T-rich sequences that occur in some of the early gene regions of the adenovirus genome. Specifically, 5,7-dimethoxycoumarin per 800 nucleotides in adenovirus DNA reduced the surviving fraction of adenovirus to a value of 0.1 after DNA infectivity (transfection) into human 293 cells. Results suggest that 5,7-dimethoxycoumarin may be used for generating a limited 'library' of mutations in each of the five early gene regions of the adenovirus genome.

Adenoviridae↗
PubMed1979

Adenovirus type 2 terminal protein: purification and comparison of tryptic peptides with known adenovirus-coded proteins.

The protein covalently bound to the 5' termini of adenovirus type 2 DNA has been purified from virus labeled with [35S]methionine, using exclusion chromatography of disrupted virions to isolate the DNA-protein complex, which is then digested with DNase. The terminal protein isolated from mature virus is most effectively labeled if the cells are exposed to [35S]methionine during the "intermediate" period of 13 to 21 h postinfection, suggesting that the protein is synthesized during this interval. The tryptic peptides of the terminal protein were compared with those of several known adenovirus-coded proteins and found to be unrelated. In particular, the terminal protein is not related to the 38-50K early proteins encoded by the leftmost 4.4% of the adenovirus genome, one region essential for the transforming activity of the virus. Neither is it related to the 72K single-strand-specific DNA binding protein, the minor virion component IVa2, or the major capsid component hexon.

Adenoviruses, Human↗
PubMed1978

Adenovirus type 2 early proteins synthesized in vitro and in vivo: identification in infected cells of the 38,000- to 50,000- molecular-weight protein encoded by the left end of the adenovirus type 2 genome.

Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein synthesized early after injection of human cells with adenovirus type 2 (Ad2) showed that polypeptides of 11,000 (11K), 12K, 14K, 15K, 19K, 21K, 24K, and 72k molecular weight were present in infected but not in mock-infected cells. These polypeptides corresponded in electrophoretic mobility to the following polypeptides synthesized in vitro by using mRNA complementary to specific regions of the Ad2 genome: 11K, 19K, 21K (91.5 to 96.8 map units), 14K (78.2 to 83.4 map units), 72K (62.4 to 67.9 map units), and 15K (4.9 to 11.0 map units). Polypeptides of 25K, 17K, 15.5K, and 13K were also synthesized in vitro, but have not yet been detected in infected cells. In addition, six adeno-specific polypeptides of 38 to 50K molecular weight could be discerned in infected cells if two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis was used to compare extracts from infected and mock-infected cells. Partial protease digestion showed these 38 to 50K polypeptides to be related in sequence to each other and to the 40 to 50K polypeptide made in vitro (1.3 to 4.0 map units).

Adenoviruses, Human↗
PubMed1976

Detection of adenovirus type 2-induced early polypeptides using cycloheximide pretreatment to enhance viral protein synthesis.

(35S) methionine-labeled polypeptides synthesized by adenovirus type 2-infected cells have been analyzed by polyacrylamide gradient gel electrophoresis and autoradiography. Cycloheximide (CH) was added to infected cultures to accumulate early viral mRNA relative to host cell mRNA. This allowed viral proteins to be synthesized in increased amounts relative to host proteins after removal of CH and pulse-labeling with (35S)methionine. During the labeling period arabinosyl cytosine was added to prevent the synthesis of late viral proteins. This procedure facilitated the detection of six early viral-induced polypeptides, designated EP1 through EP6 (early protein), with apparent molecular weights of 75,000 (75K), 42K, 21K, 18K, 15K, and 11K. Supportive data were obtained by coelectrophoresis of (35S)- and (3H)methionine-labeled polypeptides from infected and uninfected cells, respectively. Three of these early polypeptides have not been previously reported. CH pretreatment enhanced the rates of synthesis of EP4 and EP6 20- to 30-fold and enhanced that of the others approximately twofold. The maximal rates of synthesis of the virus-induced proteins varied, in a different manner, with time postinfection and CH pretreatment. Since CH pretreatment appears to increase the levels of early viral proteins, it may be a useful procedure to assist their isolation and functional characterization.

Adenoviridae↗