Search PubMed⌕ Search

Biomedical subjects

L Sealy

Publications and source records attributed to L Sealy.

At least 37 records · Page 2Linked to original sources
PubMed1990

Rous sarcoma virus enhancer factor I is a ubiquitous CCAAT transcription factor highly related to CBF and NF-Y.

We have characterized enhancer factor I (EFI), a trans-acting factor present in avian nuclear extracts which binds to the Rous sarcoma virus long terminal repeat enhancer and promoter. Through deletion and point mutagenesis, we show that EFI is a member of the CCAAT family of transcription factors. Although the CCAAT motif is essential for protein-DNA recognition, EFI shows surprising latitude in the nucleotide sequences flanking the CCAAT motif to which it will bind with high affinity. EFI will cross-bind to the binding sites of a number of previously described CCAAT factors, including CBF, NF-Y, CP2, CP1, CTF/NF-1, and c/EBP, with a range of affinities that is at most 10-fold lower than the high affinity binding site for EFI in the Rous sarcoma virus long terminal repeat. We present evidence, however, that EFI is probably identical or very closely related to CBF and NF-Y. This is based on the fact that EFI in avian nuclear extracts binds with equal or 2-fold greater affinity to the binding sites of NF-Y and CBF, despite less than 50% homology (outside the CCAAT motif) between the EFI, NF-Y, and CBF recognition sequences. Moreover, radiolabeled EFI, NF-Y, or CBF DNAs give rise to identical gel retardation patterns in extracts from a variety of different cell types. EFI, CBF, and NF-Y appear to fractionate identically upon ion exchange chromatography, separating into two heterologous components (A and B) which must be recombined to recover substantial DNA binding activity. Molecular weight estimates for the two heterologous components of EFI, CBF, and a Y-box binding protein (Celada, A., and Maki, R.A. (1989) Mol. Cell. Biol. 9, 3097-3100) are very similar. EFI DNA binding activity has recently been shown to be induced by serum and the oncogene v-src (Dutta, A., Stoeckle, M.Y., and Hanafusa, H. (1990) Genes & Dev. 4, 243-254). The close relationship or identity between EFI, CBF, and NF-Y, thus has important implications regarding the mechanisms by which serum or the oncogene v-src may affect changes in gene expression.

Animals↗
PubMed1990

Identification of a third protein factor which binds to the Rous sarcoma virus LTR enhancer: possible homology with the serum response factor.

We have identified a new protein factor (EFIII) in nuclear extracts of quail fibroblasts and chick embryos which binds specifically in vitro to a 26-bp region of the Rous sarcoma virus (RSV) long terminal repeat (LTR) enhancer. The EFIII binding site in the RSV LTR exhibits a strong sequence homology to the serum response element (SRE). The SRE is a 22-bp cis-acting DNA sequence element, first identified upstream of the human c-fos gene, which can confer serum inducibility to heterologous promotors. The binding site for EFIII in the RSV LTR enhancer is also of interest because this region has been implicated in mediating trans-activation of the RSV LTR enhancer by the protein product of the v-fos gene. We show that avian EFIII binds with equal efficiency to both its binding sites in the RSV LTR and the human c-fos SRE. A dyad symmetry element in the c-fos SRE, previously shown to be critical for binding of the cognate human serum response factor (SRF), is also critical for EFIII binding to the LTR SRE-homologous sequences; similarly, EFIII and the human SRF exhibit identical protein-DNA contacts with their corresponding recognition sequences. We suggest that EFIII may be the avian homolog of the mammalian SRF and, in fact, have evidence to indicate that the RSV LTR is serum responsive.

Animals↗
PubMed1990

Transcriptional activity of the Rous sarcoma virus long terminal repeat correlates with binding of a factor to an upstream CCAAT box in vitro.

The avian nuclear protein, enhancer factor 1 (EF1), binds specifically to the long terminal repeat (LTR) of Rous sarcoma virus (RSV) in a region that has been implicated in enhancer/promoter function. We have characterized the in vitro binding properties of this factor from chick embryo nuclear extracts by methylation interference/protection foot-printing of the wild-type LTR and also by gel electrophoretic mobility shift assays performed on a series of LTR mutants. We find that the inverted CCAAT pentanucleotide located at position -129 is essential for EF1 binding in vitro. Nucleotides flanking this element exert a smaller effect on binding. Linker-substitution and point mutations which reduce EF1 binding to this site in vitro also reduce promoter activity in transiently transfected cells. EF1 also binds with lower affinity to another inverted CCAAT box at position -65, an element which we show is also essential for transcriptional activity of the RSV LTR. We conclude, therefore, that EF1 is a CCAAT box-binding factor which is involved in the activation of RSV transcription in avian cells. Furthermore, we show that EF1 can recognize the CCAAT boxes of several other promoters in which the functional importance of this pentanucleotide has been established.

Animals↗
PubMed1986

Novobiocin inhibits passive chromatin assembly in vitro.

Novobiocin, an inhibitor of prokaryotic DNA gyrase and eukaryotic type II topoisomerase enzymes, interferes with in vitro chromatin assembly using purified histones, DNA and nucleoplasmin. The target of inhibition is not topoisomerase II; this energy-independent assembly system lacks any ATP and Mg2+-dependent type II topoisomerase or gyrase activities. Rather, novobiocin interacts with histones, disrupting histone-histone associations required for octamer formation, and causing histones to precipitate from both nucleoplasmin-histone and histone-DNA complexes. Thus, novobiocin is able to generate 'dynamic' chromatin in vitro in the absence of ATP and Mg2+ by removing histones from previously assembled static chromatin, so that the DNA supercoils, previously constrained by conventional nucleosomes, become susceptible to removal by topoisomerase I.

Animals↗
PubMed1986

Massive phosphorylation distinguishes Xenopus laevis nucleoplasmin isolated from oocytes or unfertilized eggs.

Nucleoplasmin isolated from unfertilized Xenopus laevis eggs possesses an in vitro chromatin assembly activity which is superior to nucleoplasmin isolated from oocytes. It is demonstrated here that the two forms of the protein differ in the amount of attached phosphate, with the egg protein possessing nearly 20 phosphate groups per protein monomer and the oocyte protein possessing less than 10 phosphate groups per monomer. A kinase preparation from unfertilized eggs is shown to be capable of modifying oocyte nucleoplasmin so that it displays the electrophoretic heterogeneity of egg nucleoplasmin. Furthermore, when the egg protein is treated with phosphatase and repurified, the chromatin assembly activity deteriorates to the level of the oocyte protein.

Animals↗
PubMed1986

Xenopus nucleoplasmin: egg vs. oocyte.

Nucleoplasmin has been purified from either oocytes or unfertilized eggs of the frog, Xenopus laevis. We find that the pentameric form of egg nucleoplasmin exhibits an apparent molecular mass approximately 15 000 daltons larger than its oocyte counterpart upon sodium dodecyl sulfate (SDS)-acrylamide gel electrophoresis. Egg nucleoplasmin monomers are more heterogeneous, substantially more acidic, and overall larger in apparent molecular weight than oocyte nucleoplasmin monomers when analyzed by isoelectric focusing or SDS gel electrophoresis. Protease digestions indicate that the structural differences between egg and oocyte nucleoplasmin are primarily confined to the N-terminal halves of the proteins. The structural diversity observed is accompanied by a difference in the ability of nucleoplasmin from the two sources to act as a nucleosome assembly agent in vitro. Egg nucleoplasmin efficiently promotes the formation of nucleosomes onto circular pBR322 DNA in vitro at physiological ionic strength and at physiological histone:DNA ratios, while oocyte nucleoplasmin is markedly deficient in serving as an in vitro chromatin assembly agent under all conditions which we have tested. Treatment of egg nucleoplasmin in vitro with alkaline phosphatase demonstrates that the structural diversity between egg and oocyte nucleoplasmin results primarily from extensive additional phosphorylation of the egg protein. The relevance of nucleoplasmin phosphorylation in leading to differences in the chromatin assembly activity of this protein both in vitro and in vivo is considered.

Animals↗
PubMed1986

Novobiocin precipitates histones at concentrations normally used to inhibit eukaryotic type II topoisomerase.

At concentrations normally used to inhibit eukaryotic type II topoisomerase activity (100-1000 micrograms/ml) novobiocin binds core histones. Approximately 15 moles of novobiocin bind per mole of histone resulting in histone precipitation from solution in either 0.15 M or 2 M NaCl. The interaction between novobiocin and proteins appears to involve arginine residues: histones H3 and H4 (13.5 and 14 mole percent arginine) are precipitated at lower novobiocin concentrations than histones H2A and H2B (9.5 and 6.5 mole percent arginine). Furthermore, polyarginine but not polyornithine competes for novobiocin in histone precipitation. Moreover, histones with arginine residues modified with 1,2-cyclohexanedione are soluble in 1000 micrograms/ml novobiocin. Because novobiocin can remove histones from solution as well as inhibit topoisomerase activity, and because both of these events can alter DNA topology, novobiocin should be used with caution in experiments designed to implicate topoisomerase activity in chromatin dynamics.

Animals↗
PubMed1983

Site-specific mutagenesis of avian erythroblastosis virus: erb-B is required for oncogenicity.

Avian erythroblastosis virus (AEV) induces both erythroblastosis and fibrosarcomas in susceptible birds. Two domains within its replication-defective genome, erb-A and erb-B, have been implicated in AEV-mediated oncogenesis. An efficient transfection system for generating infectious, transforming virus from molecular clones of AEV and RAV-1 (helper virus) was combined with the techniques of site-specific mutagenesis to investigate the contribution of erb-B to the two forms of oncogenesis induced by AEV. Deletion and frameshift mutations were constructed in the erb-B locus of cloned AEV DNA in vitro. Infectious retroviruses harboring these mutations were recovered and their ability to transform fibroblasts in vitro or induce erythroleukemia in vivo was assessed. The presence of mutant viral genomes in chick embryo fibroblasts or erythroblasts of infected birds was confirmed by suitable biochemical analyses. Expression of viral genes in cells infected with AEV mutants was examined by immunoprecipitation with antisera to erb-A and erb-B proteins. It was found that the product of erb-B is necessary for transformation of fibroblasts and induction of erythroblastosis by AEV, although a small portion of this protein at the carboxy terminus is dispensable.

Alpharetrovirus↗
PubMed1983

Site-specific mutagenesis of avian erythroblastosis virus: v-erb-A is not required for transformation of fibroblasts.

Avian erythroblastosis virus (AEV) is an acutely transforming retrovirus whose putative oncogenes (v-erb-A and v-erb-B) encode the proteins P74gag-erb-A and P61-68erb-B. The existence of these two gene products has prompted the question of whether one or both proteins are required in the transformation of erythroblasts and fibroblasts by AEV. In the accompanying manuscript, we describe the use of site-specific mutagenesis to generate mutants of AEV unable to synthesize P61-68erb-B. Here we present our analysis of the oncogenic potential of an AEV mutant unable to synthesize P74gag-erb-A due to a large deletion encompassing both gag and v-erb-A sequences. The erb-A-mutant retrovirus propagated quite poorly on fibroblasts in culture; however, fibroblasts harboring the erb-A mutant genome were transformed in the absence of P74gag-erb-A expression. The mutant virus failed to induce erythroleukemias in chickens, but the validity of this finding is compromised by the poor replicative capacity of the mutant. The results presented in this and the preceding manuscript indicate that P61-68erb-B is both necessary and sufficient for neoplastic transformation of fibroblasts by AEV; by contrast, a role for p74gag-erb-A in leukemogenesis by AEV has not yet been rigorously excluded.

Alpharetrovirus↗
PubMed1983

The product of the avian erythroblastosis virus erbB locus is a glycoprotein.

Avian erythroblastosis virus (AEV) induces both erythroblastosis and fibrosarcomas in susceptible birds. A locus, v-erbB, within the viral genome has been implicated in AEV-mediated oncogenesis. We report here the detection and partial characterization of the protein product of the v-erbB oncogene in AEV-transformed cells. We obtained the antisera necessary for our analysis by expressing a portion of the molecularly cloned v-erbB locus in Escherichia coli and immunizing rabbits with the resulting bacterial erbB polypeptide. Antisera directed against the bacterial polypeptide reacted with v-erbB proteins obtained from virus-infected avian cells. By three criteria--tunicamycin inhibition, lectin binding and metabolic labeling with radioactive sugar precursors--the product of the v-erbB gene appears to be a glycoprotein.

Alpharetrovirus↗
PubMed1982

Histone hypoacetylation following release of HTC cells from butyrate.

Exposure of HTC cells to 6 mM sodium butyrate for 20 h results in significantly reduced amounts of nuclear histone acetyltransferase. While the direct butyrate inhibition of histone deacetylases is rapidly reversed upon removal of cells from butyrate, histone acetyltransferase activity increases only gradually following release of cells from butyrate, requiring several hours to regain control cell levels. As a result, HTC cells released from a 20-h exposure to butyrate display normal kinetics of histone acetate turnover but markedly reduced rates of histone acetylation, the imbalance in these two processes produces histone hypoacetylation.

Acetylation↗
PubMed1979

Comparative studies on highly metabolically active histone acetylation.

Histone acetate is hydrolyzed rapidly in logarithmically dividing hepatoma tissue culture cells (Jackson, V., Shires, A., Chalkley, R. and Granner, D.K. (1975) J. Biol. Chem. 250, 4856--4863). The phenomenon has been analyzed further in hepatoma tissue culture cells at various stages of the cell cycle, in stationary phase, and in the presence of actinomycin D. We also investigated the phenomenon in Tetrahymena pyriformis macronuclei, bovine thymocytes, and human foreskin fibroblasts. The data suggest that this highly metabolically active histone acetylation while altered in mitotic cells, is independent of the overall rate of cell division, and is only slightly sensitive to actinomycin D. Finally, we conclude that the same general phenomenon is found in both cancerous and normal cells and is apparently common to cells from various stages of the evolutionary scale.

Acetylation↗
PubMed1978

The effect of sodium butyrate on histone modification.

The hyperacetylation of histones due to treatment of cultured cells with sodium butyrate has been studied. The hyperacetylation is due to inhibition of histone deacetylase. Other short chain fatty acids including acetic, isobutyric and propionic acid also produce increased modification. Histone H4 already deposited on the chromosome can be rapidly acetylated to the extent of about 70%. That 80% of histone H4 is acetylated after a 24 hr exposure to butyrate is due to the fact that incoming H4 histone is 100% acetylated and does not return to the parental unmodified form in the presence of butyrate.

Acetates↗