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J N Miner

Publications and source records attributed to J N Miner.

9 recordsLinked to original sources
PubMed1992

The basic region of AP-1 specifies glucocorticoid receptor activity at a composite response element.

Unrelated factors collaborate at composite response elements to confer novel patterns of transcriptional regulation. For example, AP-1 and glucocorticoid receptor bind and mutually affect their activities at a 25-bp composite element, plfG. We found that different members of the AP-1 factor family that behave similarly in the absence of receptor are strikingly distinct in its presence: They specify opposite (enhancement vs. repression) regulatory actions by the receptor. Four amino acids within the AP-1 DNA-binding domain were identified as crucial determinants of receptor regulatory activity at plfG. We conclude that interactions of factors from separate transcription factor families at composite response elements provide a mechanism by which a single factor can regulate both positively or negatively, and a potential resolution of the apparent functional redundancy within regulatory factor families.

Amino Acid Sequence↗
PubMed1991

Regulatory crosstalk at composite response elements.

Transcriptional regulatory factors from different families interact with each other when bound to DNA at composite response elements. This level of communication has two striking consequences: ubiquitous factors can effect cell specificity, and closely related factors from a given family can produce very different regulatory patterns.

Animals↗
PubMed1990

Transcription factor interactions: selectors of positive or negative regulation from a single DNA element.

The mechanism by which a single factor evokes opposite regulatory effects from a specific DNA sequence is not well understood. In this study, a 25-base pair element that resides upstream of the mouse proliferin gene was examined; it conferred on linked promoters either positive or negative glucocorticoid regulation, depending upon physiological context. This sequence, denoted a "composite" glucocorticoid response element (GRE), was bound selectively in vitro both by the glucocorticoid receptor and by c-Jun and c-Fos, components of the phorbol ester-activated AP-1 transcription factor. Indeed, c-Jun and c-Fos served as selectors of hormone responsiveness: the composite GRE was inactive in the absence of c-Jun, whereas it conferred a positive glucocorticoid effect in the presence of c-Jun, and a negative glucocorticoid effect in the presence of c-Jun and relatively high levels of c-Fos. The receptor also interacted selectively with c-Jun in vitro. A general model for composite GRE action is proposed that invokes both DNA binding and protein-protein interactions by receptor and nonreceptor factors.

Animals↗
PubMed1990

Anchoring a vaccinia virus promoter in the nucleus prevents its trans-activation by viral infection.

The vaccinia virus 7.5 kDa constitutive promoter, when fused to a reporter gene and recombined into the genome of L cells, is not activatable upon subsequent infection with vaccinia virus. However, the same promoter is actively transcribed during transient cytoplasmic transfection procedures or within the context of the viral genome. This suggests that the intact vaccinia transcriptional machinery either does not enter the nucleus or, if it does, is unable to interact with cellular chromatin.

Cell Nucleus↗
PubMed1990

Vaccinia virus: a versatile tool for molecular biologists.

Continued advances in genetic engineering have made possible the high-level expression of correctly processed cellular, viral and bacterial polypeptides. This article focuses on viral expression vectors and, more specifically, the vaccinia virus expression system. Vaccinia virus has been used to express a variety of proteins with useful immunogenic, catalytic or pharmaceutical properties. We discuss briefly the biology of vaccinia and its significance in the use of vaccinia as an expression vector, the variety of vaccinia systems currently in use and, finally, we summarize some recent developments which bode well for future applications of vaccinia virus technology.

Animals↗
PubMed1989

Rifampicin prevents virosome localization of L65, an essential vaccinia virus polypeptide.

In contrast to its irreversible effect on the Escherichia coliRNA polymerase beta-subunit, the antibiotic rifampicin reversibly inhibits vaccinia virus morphogenesis at a step during the formation of immature viral particles. The protein affected by the presence of rifampicin is L65, a major late vaccinia polypeptide to which mutations that confer rifampicin resistance have been mapped. We now provide evidence using a monospecific anti-L65 serum in concert with immunofluorescence and sucrose gradient analysis that the mechanism of action of rifampicin on vaccinia virus replication involves the inhibition of localization of L65 to the viral factories (virosomes) thereby blocking further development. Studies on the expression and distribution of L65 during the infection cycle reveal that L65 is a stable, nonglycosylated late protein associated with virions. These results are discussed in relationship to the possible in vivo functions of the L65 protein.

Blotting, Western↗
PubMed1989

DNA sequences that regulate expression of a vaccinia virus late gene (L65) and interact with a DNA-binding protein from infected cells.

To be efficiently expressed in vivo, the vaccinia virus late gene, L65, requires 5'-proximal cis-acting elements which bind a factor from infected cells. Deletion mutagenesis and vaccinia virus helper-dependent transient expression procedures were used to demonstrate that two distinct late promoter elements direct transcription from two different start sites (proximal [+1] and distal [-92]). The -128 to -112 region was essential for L65 distal promoter function, while sequences between -59 and +50 were sufficient for L65 proximal promoter function. The proximal DNA sequences interact with a protein, binding factor I (BF-I), which was isolated and partially purified from vaccinia virus-infected cells at late times postinfection. This activity is not detectable in uninfected cells or in purified virions. This factor binds specifically to two different sites within the proximal promoter, one 5' and one 3' to the transcription start site, but does not bind to the distal promoter element.

Animals↗
PubMed1988

Molecular dissection of cis-acting regulatory elements from 5'-proximal regions of a vaccinia virus late gene cluster.

Promoter elements responsible for directing the transcription of six tightly clustered vaccinia virus (VV) late genes (open reading frames [ORFs] D11, D12, D13, A1, A2, and A3) from the HindIII D/A region of the viral genome were identified within the upstream sequences proximal to each individual locus. These regions were identified as promoters by excising them from the VV genome, abutting them to the bacterial chloramphenicol acetyl transferase gene, and demonstrating their ability to drive expression of the reporter gene in transient-expression assays in an orientation-specific manner. To delineate the 5' boundary of the upstream elements, two of the VV late gene (A1 and D13) promoter: CAT constructs were subjected to deletion mutagenesis procedures. A series of 5' deletions of the ORF A1 promoter from -114 to -24 showed no reduction in promoter activity, whereas additional deletion of the sequences from -24 to +2 resulted in the complete loss of activity. Deletion of the ORF A1 fragment from -114 to -104 resulted in a 24% increase in activity, suggesting the presence of a negative regulatory region. In marked contrast to previous 5' deletion analyses which have identified VV late promoters as 20- to 30-base-pair cap-proximal sequences, 5' deletions to define the upstream boundary of the ORF D13 promoter identified two positive regulatory regions, the first between -235 and -170 and the second between -123 and -106. Background levels of chloramphenicol acetyltransferase expression were obtained with deletions past -88. Significantly, this places the ORF D13 regulatory regions within the upstream coding sequences of the ORF A1. A high-stringency computer search for homologies between VV late promoters that have been thus far characterized was carried out. Several potential consensus sequences were found just upstream from RNA start sites of temporally related promoter elements. Three major conclusions are drawn from these experiments. (i) The presence of promoters preceding each late ORF supports the hypothesis that each is expressed as an individual transcriptional unit. (ii) Promoter elements can be located within the coding portion of the upstream gene. (iii) Sequence homologies between temporally related promoter elements support the notion of kinetic subclasses of late genes.

Base Sequence↗