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S G Roberts

Publications and source records attributed to S G Roberts.

At least 37 records · Page 2Linked to original sources
PubMed1995

A role for activator-mediated TFIIB recruitment in diverse aspects of transcriptional regulation.

BACKGROUND: Transcription by RNA polymerase II in eukaryotic cells requires the ordered assembly of general transcription factors on the promoter to form a preinitiation complex. Transcriptional activator proteins (activators) stimulate transcription by increasing the rate and/or extent of preinitiation complex assembly. We have shown previously that acidic activators increase the stable association of TFIIB on the promoter, a process we refer to as 'recruitment'. In this study, we provide evidence that diverse activators facilitate TFIIB assembly by a related mechanism. We then investigate the activator-mediated assembly of TFIIB with regard to two aspects of transcription: the distance-dependence of activator function, and reinitiation. RESULTS: We have previously described amino-acid-substitution mutants of TFIIB that are able to support an activator-independent basal level of transcription but do not respond to acidic activators. We now show that these mutants also do not respond to other classes of activators. We demonstrate that this defect is due to a failure of the activators to recruit the mutant TFIIB to the promoter. Activators often lose activity as their distance from the initiation site is increased. We show that this impaired transcriptional activity correlates with a decrease in TFIIB recruitment. Finally, we find that following the initiation of transcription, TFIIB dissociates from the promoter, requiring the activator-mediated reassembly of TFIIB in the preinitiation complex for each new round of transcription. CONCLUSION: We have provided evidence that diverse activators recruit TFIIB to the promoter by a related mechanism. This central step in transcriptional activation is sensitive to promoter architecture, and is required for each new round of transcription.

Gene Expression Regulation↗
PubMed1994

Activator-induced conformational change in general transcription factor TFIIB.

Transcriptional activator proteins (activators) function, at least in part, by increasing preinitiation complex assembly (for reviews see refs 1-4). Previous studies have shown that an acidic activator forms a contact with the general transcription factor TFIIB (refs 5-7) and recruits it into the preinitiation complex. Mutational studies indicate that this interaction between the acidic activator and TFIIB is required for transcriptional stimulation. We show here that the acidic activator-TFIIB interaction has an additional function in preinitiation complex assembly. We provide evidence that in native TFIIB the amino- and carboxy-terminal domains are engaged in an intramolecular interaction. The acidic activator disrupts this intramolecular interaction to expose binding sites for general transcription factors that enter the preinitiation complex through association with TFIIB. Thus, the acidic activator induces a conformational change in TFIIB that drives preinitiation complex assembly forward.

Glutathione Transferase↗
PubMed1994

Nuclear protein CBP is a coactivator for the transcription factor CREB.

The transcription factor CREB binds to a DNA element known as the cAMP-regulated enhancer (CRE). CREB is activated through phosphorylation by protein kinase A (PKA), but precisely how phosphorylation stimulates CREB function is unknown. One model is that phosphorylation may allow the recruitment of coactivators which then interact with basal transcription factors. We have previously identified a nuclear protein of M(r)265K, CBP, that binds specifically to the PKA-phosphorylated form of CREB. We have used fluorescence anisotropy measurements to define the equilibrium binding parameters of the phosphoCREB:CBP interaction and report here that CBP can activate transcription through a region in its carboxy terminus. The activation domain of CBP interacts with the basal transcription factor TFIIB through a domain that is conserved in the yeast coactivator ADA-1 (ref. 8). Consistent with its role as a coactivator, CBP augments the activity of phosphorylated CREB to activate transcription of cAMP-responsive genes.

Animals↗
PubMed1993

Interaction between an acidic activator and transcription factor TFIIB is required for transcriptional activation.

How eukaryotic promoter-specific activator proteins (activators) stimulate transcription is a central question. We have previously shown that an acidic activator can directly interact with the general transcription factor TFIIB and increase its stable assembly into a preinitiation complex. We have proposed that this increase in TFIIB assembly is at least part of the mechanism by which an acidic activator functions. A prediction of this hypothesis is that a TFIIB mutant unable to interact with an acidic activator could not support activated transcription, and here we present experiments that verify this prediction. In conjunction with previous studies, our results argue that interaction between an acidic activator and TFIIB is necessary for transcriptional activation.

Binding Sites↗
PubMed1993

Evaluation of the chick wing territory as an equipotential self-differentiating system.

Harrison (1918: J. Exp. Zool. 25: 413-461) described a developmental field as an "equipotential self-differentiating system." The present study was undertaken to address the question: To what extent can be pre-limb territory of a chick embryo be considered a developmental field? To what extent is the chick pre-limb territory an equipotential self-differentiating system? Two sets of experiments were undertaken to address these questions: (1) Whole and half limb territories were explanted to the celoma of host embryos, and (2) portions of the wing territories were extirpated. The wing exhibited the quality of self-differentiation after stage 12, in that the isolated wing territory, grafted to a host celom, could form limbs beginning at stage 12 (however, complete wings formed only from wing territories of stage 16 and older). On the other hand, the chick wing territory did not appear to exhibit equipotentiality. No posterior half limb graft formed normal limbs, and only in two exceptional cases did anterior half limb grafts form limbs. If part or all of the wing territory was removed from chick embryos, normal limbs formed in less than 15% of the cases after stage 15, in about 30% of the cases at stages 13 and 14, but in over half the cases at stages 10-12. Wound healing and reinitiation of limb potential may be responsible for the higher incidence of limb formation at the younger ages.

Animals↗
PubMed1993

Cold shock induces the synthesis of stress proteins in human keratinocytes.

Heat shock proteins or stress proteins are synthesized when cells are exposed to a wide variety of physiologic stresses. The stress response is evolutionarily highly conserved, suggestive of an essential function(s) for the survival of organisms, protecting them from harmful trauma. Exposure to cold induces a stress response in organisms such as Drosophila melanogaster and Sarcophaga crassipalpis and this led us to determine whether or not cold shock responses occur in human skin after exposure to cold such as might occur during cryopreservation of tissues or cryosurgery. Biopsies taken from fresh human skin at chest surgery were exposed to 4, 15, 20, and 37 degrees C (control) for 60 min and then allowed to incorporate 35S-methionine at 37 degrees C for up to 3 h. Proteins from the epidermis were extracted and analyzed by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis. At 15 degrees C and below there was increased synthesis of 90 and 72 kD proteins 2 h after shocking. The 72-kD protein was identified as a heat shock protein using a monoclonal antibody to HSP72 and it is proposed from electrophoretic evidence that the 90-kD protein is also a heat shock protein. Clearly, cold shock stimulates a stress response in human epidermis altering the spectrum of proteins expressed and inducing the synthesis of heat shock proteins.

Cold Temperature↗
PubMed1991

Gene sequence of mouse B-type proline-rich protein MP4. Transcriptional start point and an upstream phylogenetic footprint with ets-like and rel/NFkB-like elements.

A mouse genomic B-type proline-rich protein (PRP) cosmid clone was isolated by cDNA hybridisation and mapped, the gene region was subcloned and 3770 bp were sequenced. This gene (MP4) contained three introns and encoded a 1020-nt (nt, nucleotide) mRNA for a PRP precursor 300 amino acids long arranged with 11 imperfect 18-residue proline-rich repeats. The transcriptional start point was determined by S1 nuclease mapping and primer extension to be 26 bp downstream of a TATAA sequence. Sequence comparisons revealed that only two regions from positions -650 bp - -30 bp were highly conserved in all other PRP genes, PRP boxes 1 and 2. Box 1 at positions -112 to -135 contained ets-like and rel/NFkB-like elements and was 74% conserved over 23 bp. Box 2 at positions -33 - -51 was 53% conserved over 19 bp. A search of the EMBL and GenBank sequence libraries indicated that PRP box 1 was only present upstream of the known mammalian PRP gene sequences and was absent from other genes. These conserved sequences may thus be relevant to the tissue-specific and beta-adrenergic regulation of PRP gene transcription.

Amino Acid Sequence↗
PubMed1991

The mouse proline-rich protein MP6 promoter binds isoprenaline-inducible parotid nuclear proteins via a highly conserved NFkB/rel-like site.

Proline-rich protein (PRP) gene MP6 was isolated from a mouse BALB/c genomic DNA library in lambda EMBL3, characterised by hybridisation and restriction mapping and the promoter region, from -162 to +72 around the PRP consensus cap-site, was sequenced. In gel shift assays this region formed complexes C1 and C2 with parotid nuclear proteins which were induced by the beta-adrenergic agonist isoprenaline. DNA competition studies and direct binding assays of promoter subfragments showed that it was the sequence from -157 to -91 that was forming the isoprenaline-dependent complexes. All PRP genes conserve a 23bp. sequence, termed PRP Box1, with ets and NFkB/rel binding site-like elements, upstream of their promoters. In the MP6 promoter, PRP Box1 was within the region forming the complexes. Further gel shift assays using PRP Box1 oligonucleotides as competitors and targets indicated that the NFkB/rel binding site-like element was important in formation of the isoprenaline-inducible complexes. HeLa nuclear extracts also formed complexes with PRP Box1 similar to C1 and C2 but nuclear extracts from spleen, submandibular gland and liver did not. These complexes are thus candidate regulators for the isoprenaline-dependent and tissue-specific transcription of PRP genes.

Amino Acid Sequence↗
PubMed1991

Beta-adrenergic regulation of beta-actin mRNA abundance in mouse parotid glands by a post-transcriptional mechanism.

In the first 24 h after a single injection of the beta-adrenergic agonist isoprenaline to mice, the level of beta-actin mRNA in the parotid glands increased significantly above that observed in untreated mice. The increase was transient, reaching 11 times the normal level 18 h after treatment and declining thereafter. Repeated daily doses of isoprenaline did not result in any further increase in beta-actin mRNA. Nuclear transcription experiments showed that there was no increase in the transcription rate of the beta-actin gene 8 h after an injection of isoprenaline, although beta-actin mRNA levels were increasing at this time. Immunoblotting revealed an increase in beta-actin protein in parotid gland samples after isoprenaline treatment, although the increase was not to the same extent as the mRNA, perhaps indicating that degradation of beta-actin had also increased. Using immunocytochemistry it was found that beta-actin was located mainly in the apical cortex of the normal acinar cell. There was a significant decrease in cortical beta-actin 24 h after isoprenaline treatment, suggesting that the beta-actin was under the process of redistribution. From these data we propose that isoprenaline caused an increase in beta-actin synthesis by a posttranscriptional mechanism and a redistribution of beta-actin in preparation for the well-known subsequent change in morphology and function of the parotid glands.

Actins↗
PubMed1989

Epidermal keratin levels during oral 1-alpha-hydroxyvitamin D3 treatment for psoriasis.

Recently the treatment of psoriasis with vitamin D3, its metabolites or analogues, has been reported to be clinically effective and free from side effects. We have quantified the changes in the levels of epidermal lesional keratins in 15 psoriatic patients receiving oral 1 alpha(OH)D3 treatment. Lesions were sampled before treatment commenced and at monthly intervals for 4-6 months. Clinical resolution occurred in 7 patients within this time; 3 other patients showed incomplete lesion resolution and the remaining 5 patients showed a complete lack of response within the treatment period. Lesion resolution, as judged by clinical criteria, was accompanied by significant changes in the levels of three of the keratin polypeptides and smaller changes in others. Keratin 2 increased to levels greater than those in normal epidermis, while keratins 16 and 18 decreased to normal levels. Changes in the levels of keratins 1 and 5 were small and those of keratins 7, 10 and 14 minimal. These changes were compared with values found during lesion resolution with other therapies used in psoriasis, i.e. topical dithranol, PUVA, oral etretinate and hydroxyurea and were highly reminiscent of those observed during PUVA therapy but contrasted with those during etretinate treatment. The decrease in level of keratin 16, a hyperproliferation marker, suggests that 1 alpha(OH)D3 inhibits keratinocyte proliferation, but at the same time the overproduction of keratin 2, a major keratin of the granular cells, indicates that there is an increase in the number of cells in the later stages of differentiation.

Anthralin↗
PubMed1984

Amitriptyline and its basic metabolites determined in plasma by gas chromatography.

Gas chromatography was used to determine plasma levels of amitriptyline, nortriptyline and their 10-hydroxy derivatives after conversion to the dehydro compounds by heating with acid. The primary amine 10-hydroxydesmethylnortriptyline is also dehydrated and the dehydro compound coincides on the chromatogram with dehydronortriptyline. Treatment of the extract with salicylaldehyde selectively removed the primary amine, which was determined by difference. Cis- and trans-hydroxydesmethylnortriptyline were isolated from urine by thin-layer chromatography and used to standardize the estimation. The stability of all the metabolites in plasma was investigated. Results are given for hydroxydesmethylnortriptyline levels in the plasma of 41 patients treated with amitriptyline.

Amines↗
PubMed1982

Time course of plasma drug levels during once-daily oral administration of clomipramine.

Fourteen depressed in-patients were treated with 150 mg clomipramine (CLO) daily, given as one oral dose. Using a gas-chromatographic method, concentrations of CLO and desmethyl clomipramine (DMCLO) were determined in plasma samples taken at frequent intervals during 24 h. The plasma level of each compound 12 h after the dose correlated well with the average value in the same patient, calculated over the whole 24-h period. Levels at other times gave poorer correlations, and at 24 h it was particularly poor. Plasma DMCLO concentrations were usually maximum 4-6 h after the dose. The ratios of maximum to minimum levels averaged only 1.31 +/- 0.15 SD. Peak CLO levels occurred 3 or 4 h after the dose. Maximum; minimum ratios averaged 2.72 +/- 0.73 SD, contrasting with the much smaller fluctuations of plasma nortriptyline (NT) levels observed in patients given this drug once daily. The difference is not due to a shorter half-life of CLO, but to the absorption and/or distribution behaviour of the two drugs. Although not fully understood, this difference between tertiary and secondary amines appears to hold generally among the tricyclic antidepressants.

Administration, Oral↗
PubMed1981

Binding of amitriptyline and nortriptyline in plasma determined from their equilibrium distributions between red cells and plasma, and between red cells and buffer solution.

In a new method for the measurement of plasma binding, amitriptyline (AT) and nortriptyline (NT) were allowed to reach equilibrium distribution between the cells and plasma of whole blood. A separate sample of the cells was equilibrated with a buffer solution containing the drugs. From the two distribution ratios for each drug, the fraction free in the plasma was calculated. Equilibria were achieved rapidly, avoiding denaturation of binding proteins. The pH was adequately controlled and the composition of the plasma was not altered by the experimental procedures. Large volumes of buffer solution gave amounts of free drug readily measurable by gas chromatography. Duplicate determinations showed coefficients of variation of 6% and 4.8% respectively for the free fractions of AT and NT in a given plasma sample. In 51 subjects the mean percentage of AT free in plasma was 4.31 +/- 0.59 SD and, of NT, 8.59 +/- 0.86. Binding was independent of drug concentration in the therapeutic range and did not differ between males and females nor between patients and normal subjects. It increased slightly with age. It was not affected by chylomicrons in the blood.

Amitriptyline↗