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Biomedical subjects

B H Sells

Publications and source records attributed to B H Sells.

At least 19 recordsLinked to original sources
PubMed1997

GA-binding protein is involved in altered expression of ribosomal protein L32 gene.

Differentiation of BC3H1 myoblasts to myocytes is accompanied by a 67% drop in the rate of rpL32 gene transcription. Addition of high concentrations of serum to resting myocyte populations stimulates cell growth and subsequent dedifferentiation to proliferating myoblasts with a return to the normal rate of rpL32 gene transcription. During these growth rate changes the binding activities of previously identified factors (beta, gamma, delta) which interact with the rpL32 gene promoter were examined by mobility shift assays. Binding of the beta factor (an Ets related protein) to an oligonucleotide containing the beta element was reduced significantly in myocyte nuclear extracts, but subsequent dedifferentiation increased binding within 30 min in either the presence or absence of the cycloheximide. Binding of the gamma and delta factors to their respective elements changed only slightly during these processes. Dephosphorylation of either myoblast or myocyte extracts resulted in increased binding of the beta factor suggesting that binding activity of the beta factor is modulated by phosphorylation during the changes in BC3H1 myoblasts growth rate. In addition, mobility shift assays with recombinant GABP alpha and beta proteins and their specific antibodies revealed that GABP proteins bind to the rpL32 gene promoter in a sequence dependent manner, and that similar proteins are present in BC3H1 myoblast/myocyte extracts. These results support the premise that the GABP heterodimer is the rpL32 beta factor. Furthermore, during BC3H1 myoblast differentiation and dedifferentiation neither the levels of the GABP alpha and beta proteins nor their respective mRNAs change. These results suggest that GABP is a constitutively expressed protein and is involved in regulating rpL32 gene by post-transcriptional modifications.

Animals↗
PubMed1996

Altered subcellular distribution of U3 snRNA in response to serum in mouse fibroblasts.

To extend our understanding of the mechanisms regulating ribosome biosynthesis during changes in cellular growth rate, the expression and subcellular distribution of U3 snRNA and one of its associated proteins, fibrillarin, were examined in mouse 3T6 fibroblasts. Altering serum concentrations produces changes in the ribosome content of the cell as reflected by total RNA levels. When exponentially growing 3T6 cells are induced to become quiescent by serum starvation, a significant downshift in U3 snRNA gene transcription occurs in parallel to a decrease in pre-rRNA synthesis. Serum stimulation results in an increase in the rate of synthesis of both U3 snRNA and pre-rRNA. However, U3 snRNA synthesis lags behind that of pre-rRNA. Furthermore, in serum-starved fibroblasts, a significant portion of the total cellular U3 snRNA appears in the cytoplasm. Following serum stimulation, a redistribution occurs and U3 snRNA is localized predominantly in the nucleolus at a level similar to that observed in exponentially growing cells. This redistribution is inhibited when RNA or protein synthesis is repressed in serum-stimulated fibroblasts by actinomycin D or cycloheximide. In contrast, the level and subcellular distribution of fibrillarin remain unchanged during serum starvation. These results suggest that during changes in ribosome production, distinct pools of U3 snRNPs exist within the cell.

Animals↗
PubMed1993

Coordinated decreases in rRNA gene transcription factors and rRNA synthesis during muscle cell differentiation.

rRNA synthesis decreases significantly during the differentiation of rat L6 myoblasts to myotubes. Nuclear run-on assays demonstrated that the decrease was attributable to decreased rates of rRNA gene transcription. Immunoblot analysis indicated a marked reduction in amounts of the RNA polymerase I transcription factors UBF1 and UBF2 (upstream binding factors 1 and 2, respectively). The levels of these factors dropped in parallel with the down-shift in rRNA gene transcription. The amount of UBF does not fall due to a general decrease in cellular protein, as myosin heavy-chain protein accumulates markedly during this same time. RNA blots of total RNA isolated from myoblasts and differentiating myotubes showed a decrease in the mRNA for UBF, at the same time the mRNA for myogenin was accumulating. The down-shift in UBF mRNA levels preceded the decrease in the protein levels for UBF. There have been reports that the acute response of the rRNA gene transcription system to physiological signals in many systems involves an RNA polymerase I-associated factor. However, our results imply that the regulation of rRNA gene DNA transcription in response to physiological processes, such as differentiation, may involve multiple regulatory pathways.

Adenosine Triphosphate↗
PubMed1993

Direct binding of yeast transcription factor (TFIID) to the ribosomal protein L32 (rpL32) TATA-less promoter sequence.

The ribosomal protein L32 (rpL32) gene transcribed by RNA polymerase II lacks a canonical TATA element, that binds the transcription factor TFIID tau or TBP (TATA binding protein). Instead this promoter contains an element, termed gamma, located at -30 relative to the transcription initiation site. We previously reported that, despite the lack of a canonical TATA element the rpL32 gene utilizes yeast TFIID tau for its transcriptional initiation. Whether TFIID tau participates in rpL32 gene transcription by binding directly to a promoter element or through another protein has not been resolved. These studies reveal that proteins ranging in size from 20-40 kDa binds to the gamma-element. The 40 kDa protein(s) displays strong affinity for the canonical TATA element and may be related or equivalent to TFIID tau. Furthermore, cloned and purified yeast TFIID (TBP) binds directly to the gamma-element implying that the gamma-element directs RNA polymerase II-dependent transcription of the rpL32 gene.

Base Sequence↗
PubMed1993

Dexamethasone stimulates rRNA gene transcription in rat myoblasts.

The glucocorticoid analogue, dexamethasone, stimulated RNA synthesis more than two-fold in rat L6 myoblasts, without affecting the rate of cell proliferation. Treatment of myoblasts for 24 h with 10(-7) M dexamethasone resulted in a 30% increase in the cellular RNA level. More than a two-fold stimulation of pre-rRNA gene transcription by dexamethasone, as measured in isolated nuclei and by cell-free transcription, was accompanied by a corresponding increase in pre-rRNA levels. Co-incubation of myoblasts with cycloheximide and dexamethasone did not affect the enhanced pre-rRNA gene transcription demonstrating that de novo protein synthesis was unnecessary to manifest the dexamethasone effect on rDNA transcription. Support for this conclusion is provided by the finding that the levels of UBF1 and UBF2, rDNA upstream binding transcription factors, remain unchanged. The glucocorticoid antagonist RU38486 [11 beta-(4-dimethylaminophenyl)17 beta-hydroxy-17 alpha-(prop-1-ynyl)estra- 4,9-dien-3-one] inhibited the dexamethasone-stimulated rRNA gene transcription suggesting that the glucocorticoid receptor is involved in the response mechanism.

Animals↗
PubMed1992

A positive regulator of the ribosomal protein gene, beta factor, belongs to the ETS oncoprotein family.

The beta factor, which interacts with the rpL32 promoter, binds to the sequence 5'-GAGCCGGAAGTG and trans-activates this gene. Comparison of the DNA sequences bound by the beta factor with those bound by other known DNA-binding proteins revealed that the ETS proteins interact with similar DNA sequences. Consequently we have examined the relationship of the beta factor to the several ETS proteins so far reported. Antibody and oligonucleotide competition experiments, performed by using electrophoretic shift analysis, revealed that the beta factor contains ETS epitopes and that it is immunologically related to both of the GA-binding proteins (GABPs), implying that the beta factor may consist of two separate protein subunits.

Amino Acid Sequence↗
PubMed1992

Yeast transcription factor IID participates in cell-free transcription of a mammalian ribosomal protein TATA-less promoter.

We analysed transcription of the gene for the ribosomal protein (rp) L32 of the mouse, which is transcribed in mouse L1210 nuclear extracts in vitro. The rpL32 gene lacks a canonical TATA box. Hence it has been suggested that this gene has an alternative transcription pathway not requiring transcription factor IID (TFIID). Selective inactivation of TFIID in nuclear extract completely abolished the transcription of rpL32 in vitro. Selective inactivation was restored by the addition of cloned and purified yeast TFIID (yTFIID), indicating that this TATA-less rpL32 promoter utilizes TFIID for its transcription initiation. Furthermore, addition of an oligonucleotide-containing TATA sequence interfered with the rpL32 transcription and this was overcome by the addition of yTFIID. To further examine the stage of involvement of TFIID in rpL32 transcription, TATA oligonucleotide was added to nuclear extract before and after the formation of the transcription complex. The results reveal that TFIID associates with the pre-initiation complex and that this complex is largely resistant to added TATA oligonucleotide. Our results show, for the first time, that the TATA-less rpL32 gene utilizes TFIID for transcription initiation.

Animals↗
PubMed1992

Enhanced cell-free transcription of the ribosomal protein L32 gene by the polyoma virus enhancer PEA3 DNA-binding protein.

The mouse-ribosomal-protein-L32-gene promoter contains a 12-bp sequence motif within the 5'-upstream region termed the beta element which shows significant similarity with the consensus sequence of the polyoma-virus-enhancer PEA3. A cloned PEA3 DNA-binding protein, expressed in Escherichia coli and purified, activates the expression of the ribosomal-protein-L32 gene in a cell-free system. Moreover, the PEA3 protein participates in the formation of the ribosomal-protein-L32-promoter-preinitiation-transcription complex. The preinitiation complex formed with PEA3 is resistant to competition by oligonucleotides containing the beta element. In addition anti-PEA3 serum interacts with a factor in mouse L1210 nuclear extract that binds to the beta element, causing a supershift in a mobility-shift assay. Our study demonstrates for the first time that the PEA3 protein can transactivate a cellular gene in a cell-free transcription system.

Animals↗
PubMed1992

Histone H4 mRNA levels are down-regulated by 3' RNA processing during terminal differentiation of myoblasts.

The capacity for 3' processing of the histone H4 pre-mRNA is lost following differentiation of rat L6 myoblasts to myotubes. Nuclear extracts prepared from proliferating myoblasts, but not differentiated myotubes, actively process histone H4 pre-mRNA in vitro. The activity of two factors required for 3' processing, the heat-labile factor and U7 snRNP, also changes during the differentiation period, concurrent with the loss of 3' processing activity. During myotube formation, the activity of the heat-labile factor decreases significantly while the 5' sequences of the U7 snRNA become progressively resistant to micrococcal nuclease digestion. Thus, the dramatic down-shift in histone H4 mRNA levels which occurs during myoblast differentiation is controlled at both the transcriptional and posttranscriptional level.

Animals↗
PubMed1992

Identification of proteins associating with poly(A)-binding-protein mRNA.

Synthesis of poly(A)-binding protein is regulated at the translational level. We have investigated the binding of proteins to this mRNA on the premise that the protein(s) of the mRNP complex may be involved in regulating the expression of the mRNA. We found the first 243 nucleotides of the 5' untranslated region to contain sequences essential for RNP formation. A large, single-stranded bulge structure encompassing stretches rich in adenine nucleotides and a potential stem-loop domain appear to be the primary sites for protein binding. Removal of the 243-nucleotide segment results in a drastic reduction in protein binding and a concomitant increase in translational efficiency in vitro. We suggest that proteins binding to this region, including poly(A)-binding protein itself, may be essential for regulating translation of this mRNA.

Base Sequence↗
PubMed1992

Regulation of U3 snRNA expression during myoblast differentiation.

Differentiation of proliferating rat L6 myoblasts to syncytial multinucleated myotubes results in a significant downshift in the rate of U3 snRNA gene transcription, paralleling the decrease in rRNA synthesis previously documented. Coordinate production of U3 snRNA and rRNA during the differentiation process adds further support for a role of U3 snRNA in ribosome biogenesis. Despite the dramatic decrease in U3 snRNA transcription during differentiation, a corresponding drop in the cellular level of U3 snRNA does not occur. In myotubes, the amount of U3 snRNA is regulated at the post-transcriptional level in which there is a significant accumulation of U3 snRNA in the cytoplasm of myotubes. This intracellular redistribution of U3 snRNA may significantly affect the entire process of rRNA maturation or result from the decrease in ribosome production accompanying terminal differentiation of myoblasts.

Animals↗
PubMed1992

Visualization of a mammalian transcription initiation complex.

Various proteins required for the initiation of eukaryotic gene transcription by RNA polymerase II have been identified and characterized, but little is known about their organization into a functional unit. Here, we describe the appearance of the murine ribosomal protein (rp) L32 gene transcription initiation complex as determined by transmission electron microscopy. Using a fractionated nuclear extract enriched for transcription factors necessary for rpL32 gene transcription in vitro and a DNA fragment containing the rpL32 gene promoter, the transcription initiation complex was imaged by standard transmission electron microscopy. Quantitative image analysis demonstrated that the complex is a multilobed structure whose two-dimensional projections are approximately 24 x 34 nm in size. Looping of the DNA seen in these images suggests that the proteins residing at the promoter region associate with proteins several hundred base pairs distant to the RNA start site, with bending of the DNA allowing these interactions to occur.

Animals↗
PubMed1992

Modification of mRNA-associated proteins during changes in growth conditions.

Following serum stimulation of quiescent 3T6 cells, an elevated in vivo rate of translation was observed. These studies were designed to identify the proteins associated with polysomal mRNA under different growth conditions in an attempt to establish a relationship between translational rate and the mRNA-associated proteins. Ultraviolet cross-linking of proteins to mRNA was employed to ensure that only genuine mRNA-associated proteins were investigated. Our results revealed little change in the population of mRNA-binding proteins, although minor variations in the synthesis of several proteins, most notably a 32 kilodalton species, were observed during growth transitions. These investigations demonstrate further that most of the mRNA-binding proteins were phosphorylated with the degree of phosphorylation of several proteins influenced by growth conditions.

Animals↗
PubMed1992

Translation of poly(A)-binding protein mRNA is regulated by growth conditions.

Translational efficiency of a minor group of mRNAs is regulated by serum levels in 3T6 fibroblasts. Included within this group is the poly(A)-binding protein (PABP) mRNA. We analyzed the distribution of PABP mRNA in polysome profiles and found a large percentage of this mRNA to be translationally repressed in both actively growing (approximately 60%) and resting cells (approximately 70%). Elevated serum levels induced a distinct bimodal distribution of this mRNA between actively translated and repressed fractions. Similarly, treatment of cells with low doses of cycloheximide also generated a partial shift of repressed PABP mRNA into the actively translated fraction. In an attempt to characterize the factors which regulate PABP mRNA translation we have identified the proteins which bind to this mRNA in vitro. Sequences within the 5' untranslated region were found to be sufficient for binding of all proteins to this mRNA. We suggest that this region and the proteins associated with it may be essential for translation control of PABP mRNA.

Blood Physiological Phenomena↗
PubMed1992

A downstream sequence of the rpL32 promoter competes with the glucocorticoid responsive element for a protein factor.

The murine ribosomal protein (rp) L32 gene contains essential promoter sequences located both upstream and downstream of the cap site. A combination of gel mobility shift, UV cross-linking, and cell-free transcription assays were used to analyze the interaction of factors binding to a downstream element (located at position +25 to +37). The rpL32 downstream element identified polypeptides (transcription factors) ranging in size from 45 to 25 kilodaltons (kDa). Four base pair changes in the wild-type sequence of the downstream element eliminated binding. An oligonucleotide containing the glucocorticoid responsive element sequence competed specifically for the 45-kDa protein in both the gel mobility shift assay and in the UV cross-linking studies. Our data also indicate that the downstream binding factors contribute to cell-free transcription of the rpL32 gene.

Animals↗
PubMed1991

Identification of a polypeptide bound to the beta region of the mouse r protein L32 promoter.

Studies have been initiated to identify the protein component(s) which interact with the beta regulatory region of the mouse ribosomal protein L32 gene promoter. By the combined use of the mobility shift assay and UV cross-linking, a factor specific for the upstream transcriptional control sequence of the beta region of the ribosomal protein L32 promoter has been detected in mouse L1210 nuclear extracts. A mutation (GT----TC at -71 to -70) in this sequence eliminates the binding. Beta factor is identified as a 55 kDa polypeptide by UV cross-linking. Addition of excess beta element (double-stranded oligonucleotide) to a cell-free transcription system reduces transcription of the ribosomal protein L32 gene. Our results provide evidence that the interaction between the beta element and the beta factor is involved in ribosomal protein L32 transcription.

Animals↗
PubMed1991

Control points in eucaryotic ribosome biogenesis.

Ribosome biogenesis in eucaryotic cells involves the coordinated synthesis of four rRNA species, transcribed by RNA polymerase I (18S, 28S, 5.8S) and RNA polymerase III (5S), and approximately 80 ribosomal proteins translated from mRNAs synthesized by RNA polymerase II. Assembly of the ribosomal subunits in the nucleolus, the site of 45S rRNA precursor gene transcription, requires the movement of 5S rRNA and ribosomal proteins from the nucleoplasm and cytoplasm, respectively, to this structure. To integrate these events and ensure the balanced production of individual ribosomal components, different strategies have been developed by eucaryotic organisms in response to a variety of physiological changes. This review presents an overview of the mechanisms modulating the production of ribosomal precursor molecules and the rate of ribosome biogenesis in various biological systems.

Cell Nucleolus↗