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J Brady

Publications and source records attributed to J Brady.

At least 73 records · Page 4Linked to original sources
PubMed1989

The adenovirus EIIA enhancer. Analysis of regulatory sequences and changes in binding activity of ATF and EIIF following adenovirus infection.

Adenovirus EIIA upstream sequences which contain the binding sites for proteins ATF and EIIF act as an enhancer and can be trans-activated by both E1A and SV40 T/t-antigens. Specific mutation of either the ATF or EIIF binding site demonstrates that both act as positive regulators, decreasing transcription greater than 10-fold. Mutation of both the ATF and EIIF binding sites inhibited the EIIA enhancer 200-fold. Analysis of insertion mutations suggests that the spatial alignment of the upstream ATF and EIIF binding sites with respect to the downstream EIIF binding site on the DNA helix is important. Consistent with previous findings, using gel shift analysis we demonstrate that the binding activity of EIIF is increased following wild-type adenovirus infection. In contrast, using identical gel shift conditions, the binding activity of ATF is decreased by viral infection.

Adenovirus Early Proteins↗
PubMed1989

The human cytomegalovirus mtrII colinear region in strain Tanaka is transformation defective.

The morphological transforming region II (mtrII) of human cytomegalovirus (HCMV) strain Towne has been localized to a 980-base-pair fragment containing three putative open reading frames (ORFs) of 79, 83, and 34 amino acids (aa). In addition, noncoding DNA sequence elements which have the potential to form stem-loop structures were also observed within mtrII. To determine what elements within HCMV Towne mtrII are important in transformation, colinear regions in other HCMV strains (AD169 and Tanaka) were isolated and a comparison of transforming potential was performed. The results indicated that the 2.2-kilobase colinear region in strain AD169 was transforming, whereas the colinear mtrII region in strain Tanaka showed significantly reduced transforming potential. Analysis of the nucleotide sequence data of these colinear regions revealed the presence of the 79-aa ORF in strains Towne and AD169 and its absence in strain Tanaka. In addition, BglII-digested Towne mtrII, which was cleaved within the 79-aa ORF, was shown to display significantly reduced transforming potential. Since the 83- and 34-aa coding sequences were interrupted in both the transforming AD169 colinear region and the nontransforming Tanaka strains, these ORFs were thought not to be important in transformation. Analysis of the stem-loop structures within each of the mtrII colinear regions did not reveal significant changes among the transforming and nontransforming colinear fragments. Thus, the comparative data indicate an important role for the 79-aa ORF in transformation.

Base Sequence↗
PubMed1988

trans-activation of RNA polymerase II and III promoters by SV40 small t antigen.

A biochemical role for SV40 small t antigen (t) in the viral infectious cycle that would explain the strong conservation of t structure among papovaviruses and its role as a helper of SV40 large T antigen function in the viral transforming process is not understood. Here, we report an intracellular biochemical function of the protein--the capacity to trans-activate selected RNA polymerase II and III-requiring promoters. Since t has failed in the past to bind to DNA and did not stimulate all polymerase II-requiring promoters tested, it likely trans-activates, at least in part, by modifying the activity of selected transcription factors.

Antigens, Polyomavirus Transforming↗
PubMed1988

Localization and DNA sequence analysis of the transforming domain (mtrII) of human cytomegalovirus.

To define the morphological transforming region II (mtrII) of human cytomegalovirus (HCMV), a series of subclones of the Xba I/BamHI fragment EM was constructed in vitro and tested for focus-forming activity and tumorigenicity. A 980-base-pair subclone of fragment EM was identified, and its nucleotide sequence revealed three small open reading frames (ORFs), encoding 79, 83, and 34 amino acid residues. S1 nuclease analysis of HCMV-infected cells identified several distinct early RNA species within mtrII, two of which (P1 and P2) were of particular interest, since the length of the protected DNA fragments would position the 5' end of the RNAs upstream of the open reading frames. In addition, the 980-base-pair transforming sequence revealed DNA elements capable of forming stem-loop structures. Thus the transforming mtrII domain of HCMV strain Towne contains both small open reading frames that are expressed in lytically infected cells and sequences resembling insertion-like structures that may be involved in transformation.

Animals↗
PubMed1988

Carboxyl-terminal mutants of the large tumor antigen of simian virus 40: a role for the early protein late in the lytic cycle.

Simian virus 40 (SV40) mutants dl1066 and dl1140 contain deletions within the region encoding the carboxyl terminus of the large tumor (T) antigen. Although these mutations have little effect on the efficiency of viral DNA replication, they decrease the yield of infectious virus particles by 3-4 orders of magnitude [Pipas, J. (1985) J. Virol. 54, 569-575]. Here we show that the level of late RNA is lower by a factor of 5-15 in CV-1P monkey cells infected with these mutants compared to cells infected with wild-type SV40. Consistent with this decrease in RNA, synthesis of late viral structural proteins VP1 and VP3 decreases by a factor of 5-15. In contrast, the synthesis of SV40 agnoprotein decreases by a factor greater than 100. Intercistronic complementation of these mutants with pm1493 and dl121, two SV40 mutants that are defective in agnoprotein but encode wild-type T antigen, results in an increased synthesis of agnoprotein in the infected cells. These results suggest that the carboxyl-terminal portion of T antigen participates in the posttranscriptional regulation of agnoprotein.

Animals↗
PubMed1988

Characterization of cellular factors that interact with the human T-cell leukemia virus type I p40x-responsive 21-base-pair sequence.

Transcriptional activation of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) by viral protein p40x requires a 21-base-pair (bp) sequence which is repeated three times within the LTR. This sequence contains a core octanucleotide (TGACGTCT) which has been attributed to be a cyclic-AMP (cAMP)-responsive element. We demonstrate here that the HTLV-I LTR can be specifically stimulated by cAMP regulators and have identified four proteins in HeLa cells that bind to the HTLV-I 21-bp sequence. We correlated the in vitro binding and transcriptional activity of one of these cellular factors (Mr, 180,000) to the trans-activation of the HTLV-I LTR by p40x. Point mutations were generated within the cAMP octanucleotide of the HTLV-I 21-bp sequence that simultaneously abolished biological responsiveness to trans-activation by p40x and to stimulation by cAMP. We found that these mutations also eliminated the binding of the 180-kilodalton HeLa factor to the HTLV-I 21-bp element. In the absence of a demonstrable DNA-binding property for p40x, we hypothesize that cellular proteins are involved, possibly through signal transduction pathways, in its trans-activation of responsive promoters.

Base Sequence↗
PubMed1987

Translational regulation of SV40 early mRNA defines a new viral protein.

SP6-initiated in vitro transcripts, representing the three major classes of early SV40 mRNAs, early-early (EE) and two late-early (LE) transcripts, were assayed by in vitro translation to compare their relative efficiencies for synthesis of the SV40 T antigens. The presence of one or two potential AUG initiator codons in the leader sequences of the LE RNAs inhibits efficient translation from the downstream T-antigen initiator AUG. In vitro translation of the capped form of the shorter SV40 LE RNA resulted in the synthesis of a 2.7-kd protein. In vivo pulse labeling of SV40-infected CV-1 cells demonstrated the accumulation of a peptide of similar size at late times after lytic infection, indicating that it is an authentic viral protein encoded by the early leader sequence of SV40.

Animals↗
PubMed1987

72-bp element contains a critical control region for SV40 late expression in Xenopus laevis oocytes.

The SV40 late promoter is transcribed at least 10-fold more efficiently than the SV40 early promoter when SV40 DNA is injected into the germinal vesicle of Xenopus laevis oocytes. Late expression in the oocyte is independent of T antigen and does not require DNA replication. To identify DNA sequences required for SV40 late gene expression, 12 mutants spanning nucleotide position (np) 5187 to np 304 were injected into the germinal vesicles of X. laevis oocytes, and RNA was extracted 18 to 24 hr later. S1 nuclease analysis of the 5' ends of SV40 late mRNA revealed that mutations in the origin of replication had no quantitative or qualitative effect on the 5' late start sites. Mutants which deleted the 21-bp repeats did not reduce or alter use of the major RNA initiation site (np 295), but did reduce use of a minor initiation site within the 72-bp repeats. In contrast, deletion of or certain point mutations in the 72-bp repeat decreased initiation from the major late start site. An 85-bp insertion containing a complete set of the 21-bp repeats positioned to the late side of the enhancer elements also decreased initiation from the major late start site. Thus, an element in the 72-bp repeat appears to be the major promoter element for late SV40 transcription in the oocyte.

Animals↗
PubMed1987

Identification of p40x-responsive regulatory sequences within the human T-cell leukemia virus type I long terminal repeat.

Distinct transcriptional regulatory sequences located within the upstream sequences required for p40x trans-activation of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) were chemically synthesized and cloned upstream of the basal HTLV-I LTR promoter. Plasmids containing a single 21-base-pair (bp) repeat were weakly inducible by p40x. The level of trans-activation by p40x was increased when two (30-fold) or three (40-fold) 21-bp repeats were present in the upstream control region. In the mutant containing two 21-bp repeats, the upstream 21-bp repeat could be positioned in either the sense (30-fold) or the antisense (16-fold) orientation. Plasmids containing a 51-bp repeat element, which included a single 21-bp repeat, were induced to levels similar to that obtained with the 21-bp repeat sequence alone. Template DNAs containing a single copy of the HTLV-I sequences between -117 and -160 were stimulated approximately 10-fold by p40x when one copy of the 21-bp element was located downstream.

Base Sequence↗
PubMed1986

Spacing between simian virus 40 early transcriptional control sequences is important for regulation of early RNA synthesis and gene expression.

We have analyzed the effect of insertion mutants between the simian virus 40 (SV40) 21-base pair (bp) repeats and the early-early (EE) TATA sequence. Insertion of 4, 42, or 90 bp of DNA at the SV40 NcoI site (map position 37) has been analyzed for its effect on expression of the SV40 early gene and positioning of the RNA 5' ends. Insertion of 4 bp reduced SV40 early promoter-dependent chloramphenicol acetyltransferase (CAT) expression by six- to eightfold. Increasing the size of the insertion to 42 or 90 bp resulted in a further drop in early gene expression to basal levels. At the RNA level, the 4-bp insertion reduced EE RNA synthesis approximately 10-fold. No concomitant increase in late-early (LE) RNA synthesis was observed. Insertion of 42 or 90 bp of DNA resulted in a decrease of EE RNA synthesis and a stimulation of LE RNA synthesis. Deletion of the SV40 72-bp repeats from the insertion mutants demonstrated that some, but not all, of the LE RNA depends upon the presence of these sequences. These studies suggest that the ability of RNA polymerase II to utilize the EE (TATA-directed) transcriptional control sequence requires an interaction with the upstream 21-bp repeats or the 72-bp repeats or both. That LE RNA levels in pJI1-in42 CAT and pJI1-in90 CAT were equivalent to the level of EE RNA in pJI1-CAT, yet the level of CAT gene expression was decreased greater than 10-fold, suggests that LE mRNA is under translational control and probably prefers a 5' initiation codon proximal to that of the CAT gene.

Base Sequence↗
PubMed1986

Stimulation of the adenovirus E2 promoter by simian virus 40 T antigen or E1A occurs by different mechanisms.

We have examined the ability of simian virus 40 T antigen to stimulate transcription from the adenovirus E2 promoter. T antigen, produced from a cotransfected plasmid, stimulated chloramphenicol acetyltransferase enzyme and mRNA production from an E2 promoter-chloramphenicol acetyltransferase fusion plasmid (pEC113) in monkey kidney CV-1 cells. The level of stimulation of E2 transcription by simian virus 40 T antigen was equal to that observed in cotransfections of pEC113 and the adenovirus E1A gene product. Deletion mutations from the 5' end of the E2 promoter were examined for their ability to express basal, T-antigen, or E1A trans-activated promoter activity. In each case, deletion of upstream promoter sequences to -70 base pairs reduced chloramphenicol acetyltransferase expression to approximately 30% of the level observed with the intact E2 promoter. Deletion to -59 base pairs resulted in chloramphenicol acetyltransferase expression that was 3 to 5% of that observed with the intact E2 promoter. At saturating levels of the stimulatory proteins, the chloramphenicol acetyltransferase levels obtained in response to T antigen and adenovirus E1A were additive. COS-1 cells, which are derived from CV-1 cells and constitutively express simian virus 40 T antigen, do not support E2 promoter trans activation by T antigen. E1A trans activation of the E2 promoter is efficient in COS-1 cells. These results suggest that although promoter sequence requirements are similar, T antigen and E1A trans activate the E2 promoter by different mechanisms.

Adenovirus Early Proteins↗
PubMed1986

Trends in private insurance coverage for mental illness.

The authors analyzed data from the U.S. Bureau of Labor Statistics Level of Benefits Surveys for the period 1979-1984 to examine coverage for psychiatric disorders in the private sector. While the overall number of employees with psychiatric benefits has increased, a greater percentage have more restrictions on those benefits. The extent of coverage is wide ranging, but there is little depth and the majority of psychiatric care expenses are not insured. Although the figures do not suggest a dramatic cutback in insurance coverage, the trends toward increased reductions in benefits for all health care threaten the more vulnerable psychiatric benefits.

Alcoholism↗