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D J Spector

Publications and source records attributed to D J Spector.

At least 19 recordsLinked to original sources
PubMed1998

Ex vivo gene transfer of endothelial nitric oxide synthase to atherosclerotic rabbit aortic rings improves relaxations to acetylcholine.

Cholesterol feeding results in impaired endothelium dependent vasorelaxation. The role of nitric oxide in this process is unclear. The aim of this study was to evaluate the role of nitric oxide in cholesterol-induced vasomotor dysfunction by examining the effect of overexpression of eNOS in the hypercholesterolemic rabbit aorta on vascular reactivity. Vascular rings from the thoracic aorta of hypercholesterolemic rabbits were exposed ex vivo either to an adenoviral vector encoding endothelial nitric oxide synthase (AdeNOS) or Escherichia coli beta Galactosidase (AdbetaGal). Transgene expression was examined by histochemistry for beta galactosidase, immunohistochemistry for eNOS and cyclic GMP measurements and vasomotor studies were performed. Transgene expression was found to localize to the endothelium and adventitia. cGMP levels were significantly greater in AdeNOS compared to AdbetaGal transduced rings. Acetylcholine mediated relaxation was significantly impaired in cholesterol fed rabbits and was markedly improved by overexpression of eNOS. These results suggest that reduced NO bioavailability observed in cholesterol-induced vascular dysfunction can be partially overcome by eNOS gene transfer.

Acetylcholine↗
PubMed1997

Readthrough activation of early adenovirus E1b gene transcription.

In cells productively infected with adenovirus type 5, transcription is not terminated between the E1a gene and the adjacent downstream E1b gene. Insertion of the mouse beta(maj)-globin transcription termination sequence (GGT) into the E1a coding region dramatically reduces early, but not late, E1b expression (E. Falck-Pedersen, J. Logan, T. Shenk, and J. E. Darnell, Jr., Cell 40:897-905, 1985). In the study described herein, we showed that base substitution mutations in the globin DNA that specifically relieved transcription termination also restored early E1b promoter activity in cis, establishing that maximal early E1b expression requires readthrough transcription originating from the adjacent upstream gene. To identify potential targets of readthrough activation, a series of recombinant viruses with double mutations was constructed. Each double-mutant virus strain had the transcription termination sequences in the first exon of E1a and a deletion within the transcription control region of E1b. Early E1b expression from the double-mutant strains was more defective than that from strains containing either mutation alone, indicating that the deleted regions (positions -362 to -35) are not the target for readthrough activation. Two findings suggested that a cis-dominant property of early viral templates is important for readthrough activation. First, the early E1b defect caused by the GGT insertion was not complemented in trans by factors present in late-infected cells. Second, restoration of E1b transcription at late times occurred concurrently with viral DNA replication. Readthrough activation may help convert virion DNA into a transcriptionally competent template prior to DNA replication and late transcription.

Adenovirus E1A Proteins↗
PubMed1997

Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery.

Endothelial NO synthase (eNOS) is an enzyme responsible for the production of a potent vasodilator and a key regulator of vascular tone, NO. In peripheral arteries, expression of a recombinant eNOS gene increases production of NO in the blood vessel wall. This approach appears to be a promising strategy for gene therapy of cerebrovascular disease. The major objective of the present study was to determine whether a recombinant eNOS gene (AdCMVNOS) can be functionally expressed in cerebral arteries. Replication-defective recombinant adenovirus vectors encoding bovine eNOS and Escherichia coli beta-galactosidase (AdCMVLacZ) genes, driven by the cytomegalovirus promoter, were used for ex vivo gene transfer. Rings of canine basilar artery were incubated with increasing titers of the vectors in MEM. Twenty-four or forty-eight hours after gene transfer, expression and function of AdCMVNOS were evaluated by (1) immunohistochemical staining, (2) isometric tension recording, and (3) cGMP radioimmunoassay. Transfection with AdCMVNOS resulted in the expression of recombinant eNOS protein in the vascular adventitia and endothelium, associated with significantly reduced contractile responses to UTP and enhanced endothelium-dependent relaxation to calcium ionophore A23187. Basal production of cGMP was significantly increased in the transfected vessels. The reduced contractions to UTP with increased cGMP production were reversed by a NOS inhibitor, N(G)-monomethyl-L-arginine. Contractions to UTP or production of cGMP were not affected in arteries transfected with AdCMVLacZ reporter gene. The results of the present study represent the first successful transfer and functional expression of recombinant eNOS gene in cerebral arteries. Our findings suggest that cerebral arterial tone can be modulated by recombinant eNOS expression in the vessel wall.

Animals↗
PubMed1994

Protein-protein interactions between human cytomegalovirus IE2-580aa and pUL84 in lytically infected cells.

The human cytomegalovirus immediate-early protein IE2-580aa (ppUL122a) activates transcription of viral and cellular genes and represses its own transcription through sequence-specific binding to the major immediate-early promoter. In lytically infected cells, IE2-580aa interacts with a 75-kDa viral protein (p75), an early protein that is also synthesized at late times after infection. Here we show that p75 is the product of the UL84 gene. Its association with IE2-580aa in infected cells suggests that pUL84 is involved in transcription control.

Cells, Cultured↗
PubMed1994

The human cytomegalovirus 86-kilodalton immediate-early 2 protein: synthesis as a precursor polypeptide and interaction with a 75-kilodalton protein of probable viral origin.

The immediate-early 2 (IE2) 86-kDa polypeptide, a major immediate-early gene product of human cytomegalovirus, regulates transcription both positively and negatively. We report two new properties of the IE2 86-kDa polypeptide in infected cells. Immunoprecipitation of infected cell proteins from human embryonic lung cells by antipeptide or monoclonal antibodies specific for IE2 epitopes revealed three closely migrating polypeptide species. The slowest, p86, behaved as expected for the mature 86-kDa IE2 polypeptide. The middle species, p80, was immunoprecipitated from denatured as well as native samples and labeled to steady state rapidly. Pulse-chase analysis demonstrated directly that p80 was a metabolic precursor to p86. The fastest-migrating species, p75, was not detected by probing blots of the immunoprecipitated proteins with IE2-specific antisera; p75 was not precipitated from denatured protein samples; and the products of partial proteolysis of p75 were distinct from those of p86. These properties established p75 as an unrelated coprecipitated polypeptide complexed with p86. The p75 proteins coprecipitated from cells infected with two different strains of human cytomegalovirus, AD169 and Towne, had different mobilities. p75 was detected as early as 6 h and as late as 72 h after infection, but it was not synthesized in cells released from a cycloheximide block. Thus, it is likely that p75 is an early viral protein.

Antibodies, Viral↗
PubMed1993

A multicomponent cis-activator of transcription of the E1b gene of adenovirus type 5.

We report that transcription of the adenovirus type 5 E1b gene is activated substantially in cis by sequences located between positions -362 and -49 with respect to the RNA start site. DNA fragments consisting of the -362 to -49 sequences, or subsets thereof, were inserted into a reporter plasmid containing a minimal E1b promoter (positions -48 to +14) joined to the Escherichia coli cat gene. In the presence of cotransfected E1a and E1b genes in trans, CAT enzyme synthesis in transfected KB cells was stimulated about 20-fold by sequences from -362 to -49 (XY) in cis and to a lesser extent by sequences from either -362 to -128 (X) or -127 to -49 (Y). Adenoviruses were isolated lacking the X, Y, or XY sequences and KB cells were infected with one of the mutants, as well as wild-type virus to provide E1a and E1b in trans. Deletion of both X and Y resulted in a 20-fold reduction in early E1b RNA and a 12-fold reduction in late RNA. Deletion of X or Y alone produced up to 5-fold reductions in early or late E1b RNA accumulation. In vitro DNA-protein interactions in the Y sequence were revealed by modification of the procedure used for previous detection of X region footprints. These data indicate that X and Y sequences, which include E1a protein coding and 3' untranslated DNA, also participate in DNA-protein interactions necessary for high levels of E1b promoter activity. The presence of such overlapping genetic elements raises the interesting possibility that functional E1a and E1b mRNAs must be synthesized from separate templates.

Adenoviridae↗
PubMed1992

Differential effect of DNA supercoiling on transcription of adenovirus genes in vitro.

We examined the effect of DNA template topology on the transcription of immediate early (E1a), early (E1b) and late (pIX) adenovirus genes in vitro. Transcription in whole cell extracts was measured by quantitative hybridization to end-labelled DNA and protection of hybrids from S1 nuclease digestion. Two- to fourfold more E1a RNA was synthesized from supercoiled, compared to linear, DNA templates. Similarly, transcription of the E1b gene was stimulated three- to sevenfold when the template was supercoiled. In contrast, RNA synthesis from the late pIX gene was found to be independent of DNA topology. These results show that DNA topology affects transcription in a promoter-specific manner.

Adenoviridae↗
PubMed1990

cis-dominant defect in activation of adenovirus type 5 E1b early RNA synthesis.

A cis-dominant mutation in the adjacent E1a gene disrupted the accumulation of adenovirus type 5 E1b mRNA during the early phase of infection. Steady-state levels of cytoplasmic and nuclear E1b RNAs in cells infected with dl312, a strain that lacks the E1a TATA box, cap site, and much of the coding sequence, were reduced 5- to 10-fold even when the E1a activator was provided in trans. The strain was defective for early E1b RNA synthesis but not for E1b RNA made late or during prolonged incubation in the presence of an inhibitor of DNA replication. The defect in E1b RNA synthesis could not be attributed to the E1a promoter sequences missing in dl312 DNA. If the E1a protein-coding region contains cis-acting regulatory sequences, they are not part of the previously mapped E1b transcriptional control region and may represent additional regulatory elements that ensure prompt and efficient E1b expression during the early phase of infection.

Adenovirus Early Proteins↗
PubMed1988

Organization of the transcriptional control region of the E1b gene of adenovirus type 5.

Genetic analysis of the transcriptional control sequences of the E1b gene of adenovirus type 5 identified two regions that stimulated specific transcription by whole cell extracts from uninfected cells. The first region, located within 50 nucleotides (position -50) 5' to the transcription initiation (cap) site, contains a G+C-rich consensus-binding site (GC box) for the transcription factor Sp1 and a TATA box. Unambiguous stimulatory activity of the second region, between positions -358 and -127, was observed only in the absence of the GC box. DNase I protection experiments (footprinting) with crude nuclear extracts from uninfected cells revealed multiple DNA-protein interactions at the control region. Proximal to the initiation site, both the GC box and the cap site were protected; however, protection of the TATA box was not observed. In the distal region, four protein-binding sites, designated I through IV, were located between positions -250 and -120. Three of the four mapped in protein-coding sequences of the adjacent E1a gene. Sites I and II were 5' to position -218 whereas sites III and IV were 3' to position -218. This finding was consistent with results of the transcriptional analysis indicating that subsets of the distal region were sufficient for stimulation of transcription in vitro in the absence of the GC box. Within the boundaries of site I, a 10-base-pair protected sequence was similar to one located 5' to the adenovirus E1a, E2a, E3, E4, E2 late, and polypeptide IX transcription initiation sites. Sequences within the boundaries of the other three sites were similar to those within other viral and cellular enhancers.

Adenoviruses, Human↗
PubMed1987

Participation of two human cytomegalovirus immediate early gene regions in transcriptional activation of adenovirus promoters.

The participation of human cytomegalovirus (HCMV) immediate early genes in the activation of the expression of adenovirus genes in trans (trans-activation) was examined. The initial strategy used was to determine the ability of HCMV genes to complement mutants of adenovirus E1a, an immediate early gene which encodes a trans-activator. The HCMV immediate early gene regions IE1 and IE2 complemented E1a-deficient mutants in three separate assays. IE1 and IE2 substituted for E1a in the synthesis of infectious adenovirus, late adenovirus RNA, and adenovirus DNA. Complementation by the IE2 gene region alone, but not by IE1 alone, was observed using the most discriminating assay, that for late adenovirus RNA synthesis. A role for both HCMV gene regions in positive transcriptional control was indicated by their ability to increase expression of chloramphenicol acetyltransferase (CAT) mediated by the adenovirus E2a promoter. The IE2 region alone activated CAT synthesis but IE1 alone had no detectable activity. Moreover, the activity of both gene regions was about 10-fold higher than that of IE2 alone. These data indicate that efficient complementation of E1a-deficient mutants and trans-activation of adenovirus early promoters involved the participation of both HCMV immediate early gene regions.

Adenoviruses, Human↗
PubMed1987

Hybridization between a repeated region of herpes simplex virus type 1 DNA containing the sequence [GGC]n and heterodisperse cellular DNA and RNA.

A small DNA fragment containing the simple sequence [GGC]10 from the long repeat of herpes simplex virus type 1 (HSV-1) DNA hybridized to cellular DNA and polyadenylated RNA from different mammalian species. The number and intensity of blot hybridization signals were increased in human compared with rodent and simian nucleic acids. The hybridization was blocked specifically by human 28S ribosomal DNA, which shares only the GGC repeats with the herpes simplex virus DNA. These data indicate that GGC repeats were common components of cellular DNA and were expressed in mRNA. Blot hybridization analysis of viral RNA from the HSV-1 gene regions encompassing the GGC repeats revealed abundant stable mRNAs from portions of the virus genome not previously analyzed in detail and indicated that the viral GGC sequence was not expressed in stable cytoplasmic mRNA.

Animals↗
PubMed1986

Identification of a human cytomegalovirus virus DNA segment that complements an adenovirus 5 immediate early mutant.

Human cytomegalovirus (HCMV) complements adenovirus mutant dl312, which is completely defective for expression of the adenovirus immediate early E1a gene region, for lytic growth (Tevethia and Spector, 1984). This assay defines at least one HCMV function, activation of the transcription of adenovirus early genes in trans, that must be provided for dl312 replication in coinfected cells. We show here that trans-activation depended on the expression of one or more HCMV gene. Human embryonic lung cells were transfected with dl312 DNA-protein complex and either HCMV DNA or recombinant plasmids containing the adenovirus E1a gene region or HCMV (Towne strain) DNA fragments. Replication of dl312 occurred only in cells that received both DPC and either the E1a gene, HCMV DNA, or the XbaI-E HCMV DNA fragment (0.68 to 0.77 map units). In addition, we show that HCMV also complemented adenovirus mutant pm975 for growth in 0.2% serum. Since pm975 grows poorly in low concentrations of serum due to a defect in an E1a gene product, this assay identified a second HCMV E1a-like function.

Adenoviruses, Human↗
PubMed1985

Hybridization of herpes simplex virus DNA and human ribosomal DNA and RNA.

A small DNA segment from the inverted repeats at the termini of the unique long sequence region of herpes simplex virus DNA was found to hybridize with human 28 S ribosomal DNA and RNA but not 18 S ribosomal DNA and RNA. The hybridization occurred under stringent conditions and was not blocked by nucleic acids high in guanine plus cytosine content. These data strongly suggest that the hybridization represented authentic base sequence homology.

Chromosome Mapping↗
PubMed1984

Complementation of an adenovirus 5 immediate early mutant by human cytomegalovirus.

The initiation of the lytic cycle of adenovirus 5 requires synthesis of a transcriptional activator encoded by the viral early genetic unit, E1a. Mutant viruses lacking E1a are defective. Human cytomegalovirus activated transcription of early adenovirus genes and complemented an E1a- adenovirus mutant in cells permissive for both viruses.

Adenoviruses, Human↗
PubMed1983

The pattern of integration of viral DNA sequences in the adenovirus 5-transformed human cell line 293.

The pattern of integrated adenovirus 5 DNA in the adenovirus 5-transformed human cell line 293 was analyzed by DNA blot hybridization. In contrast to a previous report, only sequences from the left end of the genome were detected. The viral DNA was contained in a unique DNA fragment generated by cleavage of 293 DNA with either EcoRI or BamHI, two enzymes which do not cut the integrated viral DNA. Further mapping studies indicated that the integrated sequence was probably colinear with viral DNA. The joining to host cell sequences occurred between viral nucleotide base pairs 1 and 270 (a site for BalI) on one end and 4123 (SmaI site) and 5372 (BalI site) at the other end. The viral DNA was not tandemly repeated. These results suggest that integration of viral sequences into the host genome probably occurred at a single site. If any subsequent duplications of viral DNA took place, then duplication of extensive cellular sequences also must have occurred.

Adenoviruses, Human↗
PubMed1982

Transcription of adenovirus 5 early region 1b is elevated in permissive cells infected by a mutant with an upstream deletion.

Early region 1b (E1b) of adenovirus 5 consists of a single transcription unit that lies from 1,702 to 4,070 nucleotides from the conventional left end of the genome. The effect of mutations that map upstream of E1b on the production of E1b mRNA was examined in vivo with mutants defective in gene functions from the neighboring early region 1a (E1a) transcription unit (499 to 1,632 nucleotides from the left end). These host range mutants replicate in the adenovirus 5-transformed human cell line 293. E1b mRNA accumulation was assayed by DNA-RNA hybridization late after productive infection when the E1b transcripts are abundant in the cytoplasm. Cells infected by wild-type virus, mutant dl311, or mutant hr1. The elevated levels of E1b mRNA were also detected in steady-state nuclear RNA, pulse-labeled polyadenylated nuclear RNA, and pulse-labeled total nuclear RNA. These data indicate that E1b transcription was elevated in human 293 cells infected with dl312. There was no evidence of increases in genomic DNA in dl312-infected cells, suggesting that the rate of transcription may be elevated. When mixed infections with a 10-fold excess of either dl312 or wild-type virus were performed, the phenotype was that of the more abundant genome. This result suggests that the respective phenotypes were cis dominant. The increased rate of transcription can be attributed to cis-active regulatory effects of the deletion of nucleotides 448 to 1,349 in mutant dl312 DNA.

Adenoviruses, Human↗