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W M Flanagan

Publications and source records attributed to W M Flanagan.

28 records · Page 2Linked to original sources
PubMed1992

In vitro transcription faithfully reflecting T-cell activation requirements.

T-cell activation is a complex process mediated by cell membrane molecules including the T-cell antigen receptor (TCR), adhesive molecules, and cytokine receptors that collectively produce an increase in intracellular Ca2+, and activation of protein kinase C that initiate a genetic program resulting in immunologic function and irreversible differentiation. To understand how these cell membrane events are translated into a genetic regulatory cascade resulting in T-cell function, we have developed an in vitro transcription system, derived from Jurkat T-cells, which demonstrates inducible, cell-type-specific transcription following T-cell stimulation. Nuclear extracts from cells stimulated with phorbol 12-myristate 13-acetate and ionomycin, which activate protein kinase C and mimic physiological activation through the T-cell antigen receptor, transcribe an interleukin-2 (IL-2) enhancer (-326 to +24) template 5-fold more efficient than nuclear extracts from resting T-cells and severalfold more efficient than extracts from Jurkat cells treated with phorbol 12-myristate 13-acetate or ionomycin alone. Further results demonstrate that in vitro transcription of the IL-2 enhancer is T-cell specific since nuclear extracts from rat liver and stimulated HeLa cells are unable to induce IL-2 transcription. The activation-dependent, T-cell-specific in vitro transcription system described here should facilitate the dissection of signals that emanate from the T-cell surface resulting in IL-2 transcription and T-cell activation.

Cells, Cultured↗
PubMed1991

Activation of early gene expression in T lymphocytes by Oct-1 and an inducible protein, OAP40.

After antigenic stimulation of T lymphocytes, genes essential for proliferation and immune function, such as the interleukin-2 (IL-2) gene, are transcriptionally activated. In both transient transfections and T lymphocyte-specific in vitro transcription, the homeodomain-containing protein Oct-1 participated in the inducible regulation of transcription of the IL-2 gene. Oct-1 functioned in this context with a 40-kilodalton protein called Oct-1-associated protein (OAP40). In addition to interacting specifically with DNA, OAP40 reduced the rate of dissociation of Oct-1 from its cognate DNA-binding site, suggesting that a direct interaction exists between Oct-1 and OAP40.

Amino Acid Sequence↗
PubMed1991

Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A.

Cyclosporin A and FK506 inhibit T- and B-cell activation and other processes essential to an effective immune response. In T lymphocytes these drugs disrupt an unknown step in the transmission of signals from the T-cell antigen receptor to cytokine genes that coordinate the immune response. The putative intracellular receptors for FK506 and cyclosporin are cis-trans prolyl isomerases. Binding of the drug inhibits isomerase activity, but studies with other prolyl isomerase inhibitors and analysis of cyclosporin-resistant mutants in yeast suggest that the effects of the drug result from the formation of an inhibitory complex between the drug and isomerase, and not from inhibition of isomerase activity. A transcription factor, NF-AT, which is essential for early T-cell gene activation, seems to be a specific target of cyclosporin A and FK506 action because transcription directed by this protein is blocked in T cells treated with these drugs, with little or no effect on other transcription factors such as AP-1 and NF-kappa B. Here we demonstrate that NF-AT is formed when a signal from the antigen receptor induces a pre-existing cytoplasmic subunit to translocate to the nucleus and combine with a newly synthesized nuclear subunit of NF-AT. FK506 and cyclosporin A block translocation of the cytoplasmic component without affecting synthesis of the nuclear subunit.

Amino Acid Isomerases↗
PubMed1991

Analysis of the herpes simplex virus type 1 promoter controlling the expression of UL38, a true late gene involved in capsid assembly.

The cistrons encoding the herpes simplex virus type 1 (HSV-1) UL37 and UL38 genes are adjacent to one another but are transcribed from opposite strands of the viral DNA. The UL37 gene encodes a 1,123-amino-acid protein of unknown function, while the 465-amino-acid UL38 protein is involved in capsid assembly. Previous work from our laboratory indicated that the transcripts encoding these proteins are expressed with significantly different kinetics in productive infection. In the present communication we confirm the kinetic classes and precisely map the cap sites of the UL37 and UL38 mRNAs. A bifunctional reporter gene vector was used to demonstrate that divergent promoters control the expression of these reporter genes in trans-activation assays. The UL38 promoter is functionally separable from that controlling UL37 in a recombinant virus. We used deletion analysis to demonstrate that as few as 29 bases 5' of the mRNA cap site are adequate for full activity of the UL38 promoter in trans-activation assays. Finally, we analyzed the protein-binding properties of the UL38 promoter; several sites that form complexes containing ICP4, with clear homology to those identified in the HSV-1 gamma 42 promoter, are present. Thus, in general, the properties of this promoter are quite similar to those of other gamma promoters.

Animals↗
PubMed1989

Identification of the latency-associated transcript promoter by expression of rabbit beta-globin mRNA in mouse sensory nerve ganglia latently infected with a recombinant herpes simplex virus.

The herpes simplex virus type 1 latency-associated transcript (LAT) is expressed as a major species in latently infected mouse neurons. Previous sequence analysis revealed no obvious promoter elements near the 5' end of the LAT, but a TATA box and other potential promoter elements were found 700 base pairs upstream. A recombinant virus in which the rabbit beta-globin gene was inserted immediately downstream of the TATA box expressed globin mRNA and did not express the LAT. A second recombinant virus, in which this TATA box was removed, was negative for LAT expression in a latent infection. The location of the LAT promoter suggested that RNA upstream of the LAT was synthesized and degraded during latent-phase transcription. Low levels of this RNA were observed by in situ hybridization. In other experiments, RNA from a productive infection was used to detect a transcript extending from the LAT promoter to a polyadenylation signal approximately 8.5 kilobase downstream. These data suggest that the LAT may be processed from a larger transcription unit which begins distal to the TATA box 700 base pairs upstream of the LAT and extends to a polyadenylation signal almost 5 kilobases downstream of the 3' end of the LAT.

Animals↗
PubMed1988

The herpes simplex virus latency-associated transcript is spliced during the latent phase of infection.

The herpes simplex virus type 1 latency-associated transcript (LAT) is expressed as a major species 2,100 to 2,200 bases in length and a less abundant one ca. 730 bases shorter in latently infected mouse and rabbit neurons. RNA blot hybridization experiments using 20- to 22-base synthetic oligonucleotides and mung bean nuclease protection assays have demonstrated that the smaller LAT species is colinear with the larger one, except for a 730-base intron. On the basis of Northern blot analysis, the spliced species which comprises as much as 50% of the total LAT in latent infections of mice with several strains of herpes simplex virus type 1 and latent infections of rabbits with either the McKrae or the KOS(M) strains of virus is not present in the acute phase of infection. Further and rather surprisingly, in mice latently infected with the KOS(M) strain of virus, the spliced LAT species is considerably less abundant. This suggests that both the strain of virus and the animal in which the latent infection occurs are important in either the processing or stability of spliced LAT. Finally, an exhaustive series of experiments failed to provide convincing evidence that a unique, poly(A)+ species of LAT exists in the latent phase of infection.

Acute Disease↗
PubMed1988

Physical characterization of the herpes simplex virus latency-associated transcript in neurons.

RNA transfer (Northern) blot analysis was used to perform the physical characterization of the transcript expressed in murine sensory nerve ganglia latently infected with herpes simplex virus type 1. Most of this latency-associated transcript (LAT) was isolated in the poly(A)- fraction from ganglia. A smaller RNA species was also detected at less than 10% the abundance of the major one. LAT was not detected with probes from DNA outside the limits of the larger species. In situ hybridization data correlated well with Northern blot analysis; however, low levels of hybridization were seen with probes immediately outside the region of viral DNA giving positive Northern blot signals. S1 nuclease and primer extension mapping were used to locate the 5' end of the LAT 510 bases to the left of a KpnI site at 0.783 map units. The 3' end of the major latency-associated species was mapped to just within a 310-base-pair SmaI fragment located 660 to 970 base pairs to the right of the SalI site at 0.790 map units. These data were correlated with an analysis of the sequence of the DNA encoding this transcript and its possible function in the latent phase of infection.

Amino Acid Sequence↗
PubMed1987

A bi-functional reporter plasmid for the simultaneous transient expression assay of two herpes simplex virus promoters.

We have constructed a transient expression vector (pCAL) containing two reporter genes, chloramphenicol acetyltransferase (CAT) and beta-galactosidase (beta-gal) for use in studying herpes simplex virus type 1 (HSV-1) promoter activity in mammalian cells. The construct was designed to be useful in analyzing the simultaneous expression from two different promoters. To test the utility of the vector, we used three HSV-1 promoters that had been characterized previously by workers in this laboratory. Two are early (beta) promoters, for alkaline exonuclease and deoxyuridine triphosphate nucleotidohydrolase; the third promoter controls the major capsid protein transcript and is late (beta gamma). The two different kinetic classes of promoters were ligated in a divergent orientation into pCAL and transfected into rabbit skin fibroblast. Transfected cells were then superinfected with low multiplicities of HSV-1; 18 hr later, we observed the simultaneous expression of both marker genes under control of the respective promoters. The usefulness of such a transient expression reporter vector is discussed.

Animals↗