Biomedical subjects

L Buckbinder

Publications and source records attributed to L Buckbinder.

6 recordsLinked to original sources

PubMed1992

Thyroid hormone-induced gene expression changes in the developing frog limb.

Greater than 120 genes are up-regulated in Xenopus laevis limb buds within the first 24 h after induction of metamorphosis by thyroid hormone. Fourteen of these have been isolated and characterized. Four encode heat shock proteins. The identified regulated genes have in common a relatedness with cell growth as exemplified by the serum response of quiescent fibroblasts. Some of the genes respond directly to hormone. However, the majority appear to be secondary response genes judging from their delayed kinetics and cycloheximide sensitivity. This indicates that there are at least two periods of gene expression change in the first 24 h. DNA replication increases in the second 24 h. Growth of the limb bud occurs for several days before the genes that characterize terminal differentiation of its cell types are up-regulated.

Animals↗

PubMed1991

The initiator directs the assembly of a transcription factor IID-dependent transcription complex.

Highly purified RNA polymerase II was found to be able to weakly recognize the initiator (Inr) present in the adenovirus IVa2 and major late promoters. The association of RNA polymerase II with the Inr was enhanced by the general transcription factors. The Inr was capable of directing the formation of a DNA-protein complex. Transcription competent complexes on the adenovirus major late and IVa2 promoters appear to be formed by alternative pathways mediated through the Inr and/or "TATA" motif. The presence of both motifs, however, is required for efficient transcription utilizing a discrete start site. Complexes formed at either site required transcription factor TFIID, the TATA binding protein. Consistent with this observation, a TFIID requirement was demonstrated for transcription from a mutant adenovirus major late promoter construct lacking a functional TATA motif.

Adenoviridae↗

PubMed1989

TPA can overcome the requirement for EIa and together act synergistically in stimulating expression of the adenovirus EIII promoter.

We have examined the control of gene expression from the adenovirus early region III (Ad-EIII) promoter, which contains two previously defined elements, the AP1 and ATF sites. We found that the AP1 element is capable of mediating activation by the adenovirus immediate early (EIa) gene products. Consistent with studies demonstrating that the AP1 site mediates signal transduction in response to 12-O-tetradecanoylphorbol 13-acetate (TPA) we have shown that TPA can activate Ad-EIII expression and overcome the requirement for EIa. Together TPA and EIa elicited a synergistic response in expression from the Ad-EIII promoter during both transient expression assays and viral infections. This synergistic effect required the AP1 element. An EIII promoter construct, in which sequences upstream of the TATA box had been replaced with four AP1 sites, was responsive to TPA and EIa and in combination promoted the synergistic effect. The analysis of specific factors involved in transcription from the Ad-EIII indicated that proteins recognizing the ATF and AP1 sites were important in expression from this promoter in vitro. Purification of protein factors that specifically stimulated EIII expression resulted in the isolation of a set of factors of the AP1 family. Affinity purified AP1 recognized and activated transcription through both the AP1 and ATF elements. In addition, a protein fraction was identified with DNA binding activity specific for the ATF element. This fraction was dependent on the ATF site for transcriptional activity.

Adenovirus Early Proteins↗

PubMed1989

Phosphorylation of cellular proteins regulates their binding to the cAMP response element.

We have studied the protein factors that promote transcription via binding to the cAMP response element (CRE) present in the adenovirus early region III (EIII) and early region IV (EIV) promoters. Three sets of CRE-binding phosphoproteins, ranging in molecular mass from 65-72, 38-43, and 31-37 kDa, were identified in vivo from HeLa cells. Western blot analysis revealed that all three sets of proteins identified were immunologically related to the transcription factor AP1. We found that binding of these proteins to the CRE could be regulated by phosphorylation in vitro. EivF, a 65-72-kDa protein was found to bind specifically to the adenovirus EIV promoter. We have also shown that the smaller molecular mass proteins of 31-37 and 38-43 kDa were able to bind to the CRE present in the adenovirus EIV promoter, as well as to two related DNA elements present in the adenovirus EIII promoter, the ATF and AP1 sites. Phosphorylation of these proteins with the cAMP-dependent protein kinase, affected their transcriptional activity and binding affinity to the three sites. Furthermore, the binding specificity of the 31-37-kDa polypeptides was mediated by cAMP-dependent protein kinase in vitro. Our data suggests that phosphorylation of factors that bind to the CRE may, in part, underlie the cellular response to the adenovirus-encoded Ela protein.

Adenoviridae↗

PubMed1989

Factors involved in specific transcription by mammalian RNA polymerase II. Role of factors IID and MLTF in transcription from the adenovirus major late and IVa2 promoters.

The role of the adenovirus major late upstream transcription factor (MLTF) in transcription from the adenovirus major late and the IVa2 promoters was studied. The transcription initiation site of the IVa2 promoter is located 210 nucleotides upstream from the CAP site of the major late promoter. Transcription from these two promoters occurs on different DNA strands. Thus, this divergent transcription suggests that the same factor could simultaneously regulate the expression of two different genes. This was investigated utilizing a reconstituted transcription system in vitro. The addition of MLTF to reaction mixtures containing the purified general transcription factors and the major late promoter resulted in a 10-12-fold stimulation of transcription. This stimulation was because of an increase of the stability of the preinitiation complex. MLTF allowed DNA template molecules to undergo multiple rounds of transcription. MLTF also stimulated transcription from the adenovirus-encoded IVa2 promoter. Surprisingly, reconstitution experiments indicated that transcription from the IVa2 promoter which does not have a TATA sequence required all the previously described general transcription factors, including TFIID, the TATA binding protein. The requirement for TFIID was demonstrated by reconstitution experiments as well as by oligonucleotide competition experiments. The implications of this observation are discussed.

Adenoviruses, Human↗

PubMed1988

EivF, a factor required for transcription of the adenovirus EIV promoter, binds to an element involved in EIa-dependent activation and cAMP induction.

The isolation of a cellular factor that was specifically required for transcription from the promoter of the adenovirus early gene iv, an EIa-activated promoter, is described. This factor (EivF) was purified from HeLa cells using a functional transcription assay and identified as a 72,000- to 65,000-dalton protein. DNase I footprinting experiments demonstrated that purified EivF bound to the sequence 5'-GT(G/T)ACGT-3' present two times upstream of the Eiv TATA box. Nuclear extracts prepared from HeLa cells contained more than one factor capable of binding to the EivF recognition site. Previous studies have indicated that a sequence similar to the EivF-binding site was recognized by a 43,000-dalton protein and participated in the cAMP response of the somatostatin promoter. The purified and transcriptionally active EivF also bound to DNA sequence elements present in the somatostatin and alpha-gonadotropin promoters shown previously to be responsive to cAMP.

Adenoviridae↗