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D A Gillespie

Publications and source records attributed to D A Gillespie.

34 records · Page 2Linked to original sources
PubMed1993

Transformation by the fos or jun oncogene does not increase AP-1 DNA-binding activity.

The c-fos and c-jun proto-oncogenes encode components of the transcription factor AP-1. To determine whether transformation by the v-fos or v-jun oncogene results in alterations in the level or regulation of this factor, we have characterized AP-1 DNA-binding activity in nuclear extracts prepared from v-fos- and c-fos-transformed rat fibroblast cell lines and v-jun-transformed chicken embryo fibroblasts under various growth conditions. During proliferation, the level of AP-1 DNA-binding activity does not differ among the v-fos, c-fos, or v-jun-transformed cells and their normal progenitors, despite constitutive overexpression of the corresponding oncoproteins. Therefore, although necessary, it is not likely that an increase in DNA binding is sufficient for fos or jun transformation. Normal rat and chicken fibroblasts demonstrate very low levels of AP-1 DNA-binding activity when quiescent, and upon serum stimulation a biphasic increase is observed. A similar cyclical pattern is seen in v-fos-transformed cells, but in v-jun-transformed cells AP-1 DNA-binding activity does not fluctuate in response to serum stimulation, which suggests that this level of control may be exerted through the Jun component of the AP-1 complex.

Animals↗
PubMed1993

Gene-regulatory properties of Myc helix-loop-helix/leucine zipper mutants: Max-dependent DNA binding and transcriptional activation in yeast correlates with transforming capacity.

Max is a basic helix-loop-helix/leucine zipper (bHLH/LZ) protein that forms sequence-specific DNA-binding complexes with the c-Myc oncoprotein (Myc). Using Saccharomyces cerevisiae, we have shown that the Max bHLH/LZ domain enables Myc to activate transcription through CACGTG and CACATG sequences in vivo, and that the number and context of such sites determines the level of activation. In addition, we have used yeast to investigate the role of the Myc helix-loop-helix (HLH) and leucine zipper (LZ) motifs in mediating Max-dependent DNA-binding and transcriptional activation in vivo using HLH/LZ mutants generated by site-directed mutagenesis. The results show that, while both motifs are essential for Myc to activate transcription, helix 2 of the HLH together with the contiguous LZ suffice to mediate complex formation with Max, whilst helix 1 is essential for sequence-specific DNA binding of Myc-Max complexes. Furthermore, the ability of Myc HLH/LZ mutants to bind DNA and activate transcription in collaboration with Max correlates closely with their neoplastic transforming activity in higher eukaryotic cells.

Base Sequence↗
PubMed1991

Protein phosphatase 2A reverses phosphorylation of c-Jun specified by the delta domain in vitro: correlation with oncogenic activation and deregulated transactivation activity of v-Jun.

Chicken c-Jun proteins synthesized in vitro in reticulocyte extract consist of several electrophoretic isoforms resulting from phosphorylation which can be specifically reversed by purified protein phosphatase 2A (PP2A). Using the phosphatase inhibitors okadaic acid and microcystin-LR, we conclude that the isoforms seen in vitro represent a balance between the action of an unidentified kinase(s) which phosphorylates c-Jun and dephosphorylation by an endogenous PP2A-like phosphatase. c-Jun proteins are also subject to phosphorylation in vivo in chick embryo fibroblasts (CEF), which can be reversed by PP2A. In contrast, the viral Jun oncoprotein encoded by ASV17 is not subject to PP2A-sensitive phosphorylation in vitro and is hypophosphorylated in comparison with c-Jun in ASV17-transformed CEF. Hybrids between c-Jun and v-Jun demonstrate that differential phosphorylation in vitro is a consequence of deletion of 27 amino acids in the N-terminal third of v-Jun. The deletion is important for oncogenic activation and lies in a domain, termed delta, which regulates c-Jun transactivation function. PP2A-sensitive phosphorylation in vitro correlates with the differential responsiveness of c-Jun and v-Jun to a recently identified cell type-specific inhibitor of transactivation function.

Amino Acid Sequence↗
PubMed1991

Regulation of AP-1/DNA complex formation in vitro.

The level of AP-1 DNA-binding activity exhibited in vitro by unfractionated extracts of Hela nuclei can be stimulated by a low molecular weight fraction from rabbit reticulocyte lysate. Stimulation also requires a heat labile component of the nuclear extract, probably a protein. Stimulated and unstimulated extracts with high and low AP-1 DNA-binding activities contain the same levels of proteins reactive with antisera against Jun and Fos, proteins which are shown to be involved in the AP-1/DNA complexes detected in vitro. The low molecular weight fraction from reticulocyte lysate can be substituted by the reducing agent dithiothreitol (DTT) in the stimulation reaction and conversely oxidised glutathione greatly reduces formation of AP-1/DNA complexes. The binding activities of transcription factors SP-1, NF-1 and CBP are unaffected by DTT or oxidised glutathione. These observations, taken together, suggest that the efficiency with which pre-existing Fos and Jun proteins can bind an AP-1 target sequence in vitro can be controlled by a nuclear activity which is sensitive to oxidation/reduction and that this control mechanism is specific for AP-1.

Animals↗
PubMed1990

The leucine zipper domain of avian cMyc is required for transformation and autoregulation.

Small deletions of 7 to 48 amino acids have been generated in the leucine zipper domain of the avian cMyc protein and the mutant cMyc proteins expressed using an avian retroviral vector. Retrovirally encoded cMyc protein transforms primary chick embryo fibroblasts and leads to abnormal regulation of the endogenous c-myc gene. Deletion of the most C-terminal leucine of the zipper motif confers a partial phenotype affecting some but not all parameters of transformation. Complete loss of transforming activity results from deletion of further leucine residues, including one which is not part of the heptad repeat. In cMyc transformed cells endogenous c-myc mRNA is expressed at a low level and is abnormally refractory to induction by serum stimulation. In contrast, a non-transforming cMyc protein which lacks the zipper does not affect normal c-myc expression. These results demonstrate that the leucine zipper domain of avian cMyc is required for both transformation and autoregulation, and suggests that essential leucine residues within the motif may be spaced differently from those in the zippers of Fos and Jun.

Amino Acid Sequence↗
PubMed1989

Proviral position effects: possible probes for genes that suppress transcription.

Rous sarcoma virus, an oncogenic avian retrovirus, readily causes morphological transformation of chick cells, but in infected rat cells transformation is rare because proviral transcription is inefficient. This constraint is not due to a lack of positive transcriptional factors, or an excess of negative ones, but reflects the site of proviral integration in rat cell DNA. In most sites the provirus is almost invariably silent, in others it is correspondingly active, whilst in a third category expression fluctuates in concert with transitions in chromatin structure. Transcriptional fluctuations are mediated both by flanking cell DNA in cis and by trans-acting cell genes, suggesting that proviral position effects are sensors for genes that down-regulate transcription, perhaps by determining chromatin configuration. We have tried to identify such genes by gene transfer, karyology and insertional mutagenesis. The variable success of these three approaches indicates that the transcriptional down-regulator(s) need act only transiently. This is consistent with a function that operates in ontogeny or differentiation to down-regulate genes whose silence is then perpetuated by other means. The loss of such functions may predispose to neoplasia.

Animals↗
PubMed1989

Detection of a Myc-associated protein by chemical cross-linking.

A single nuclear protein (Myc-associated protein) can be specifically cross-linked to avian Myc proteins by treatment of nuclei or cells with the reversible cross-linker dimethyl 3,3'-dithiobis-propionimidate. Myc-associated protein has a molecular weight of approximately 500,000, is not detectably phosphorylated and, in contrast to Myc, has a long apparent half-life of greater than 3 h.

Animals↗
PubMed1986

Expression of integrated Rous sarcoma viruses: DNA rearrangements 5' to the provirus are common in transformed rat cells but not seen in infected but untransformed cells.

The study of Rous sarcoma virus (RSV)-infected rat cell clones offers a novel approach to unravelling the mechanisms controlling eukaryotic gene expression. RSV-transformed rat cell clones frequently contain duplicated proviral sequences immediately upstream of an intact provirus. This category of proviral rearrangement is not seen in cells that remain untransformed after RSV infection nor in subsequently segregating transformants. These results suggest that such rearrangements occur during or soon after proviral integration, and that they may favour early proviral expression.

Animals↗
PubMed1986

Control of expression of an integrated Rous sarcoma provirus in rat cells: role of 5' genomic duplications reveals unexpected patterns of gene transcription and its regulation.

Rat cells transformed by Rous sarcoma virus frequently contain duplications of viral (and sometimes cellular) DNA 5' to the integrated provirus, suggesting that such rearrangements favor provirus expression. In one cell line, A11, the duplication includes the viral src gene and proviral sequences that flank it. We examined three possible roles for this structure. Since the proviral v-src gene transformed recipient cells upon DNA transfer and was the major template for v-src transcription in A11 cells, the presence of v-src in the duplication is presumably not necessary for transformation. Since the size and structure of transcripts from the proviral v-src gene in A11 cells were conventional, the duplication does not facilitate transformation by providing a novel transcriptional strategy. Thus, we favor the concept that the duplication either attenuates a negative effect of flanking elements at the host chromosome integration site or augments the positive regulation of conventional provirus expression or both. Gene transfer and transcription analyses with both genomic and cloned DNA showed that the mechanisms of such regulatory phenomena are complex. Identical sequences in the provirus and the 5' duplication displayed different patterns of expression in A11 cells that could be disrupted in segments of cloned DNA. Among the elements that influenced such expression were sequences from the gag-pol region of the provirus.

Animals↗
PubMed1985

Rearrangements of viral and cellular DNA are often associated with expression of Rous sarcoma virus in rat cells.

Rous sarcoma virus (RSV) proviruses integrated within the DNA of transformed rat cells frequently display duplications of variable segments of proviral DNA upstream of an intact provirus. The rearrangement in the A11 clone of transformed rat cells consists of a partial duplication of both viral and cellular DNA segments whose origin is a region of approximately 4 kb encompassing the 3' virus-cell junction. Transposition of this DNA appears to have occurred at or after virus integration by a mechanism involving at least two recombination events. In every case examined, including A11, the transcriptional organization of the original provirus has been conserved and viral RNA expression appears to occur normally. The frequency of such rearranged proviruses in the DNA of transformed rat cells suggests that upstream rearrangements may influence provirus expression.

Animals↗
PubMed1984

Analysis of the variations in proviral cytosine methylation that accompany transformation and morphological reversion in a line of Rous sarcoma virus-infected Rat-1 cells.

Cells of the A11 lineage of Rat-1 contain a single complete Rous sarcoma provirus. Variation in the activity of this provirus accompanies fluctuations in the lineage between normal and transformed phenotypes. Increased proviral cytosine methylation of the doublet CpG in the tetranucleotide CCGG correlates with transcriptional inactivity and this pattern of cytosine hypermethylation is stable, even when the cells are transformed by another virus. However, transformation can also be induced by 5-azacytidine (but not by other mutagens) and in these transformants reduced proviral cytosine methylation is accompanied by increased proviral transcription. Differences in CCGG methylation between normal and transformed cells are found mainly in the 3' half of the provirus; sites near and within the src gene are heavily methylated only when the provirus is transcriptionally inactive. On the other hand, both transformed and normal A11 derivatives show little, if any, cytosine methylation of CCGG sequences in and flanking the 5' portion of the provirus.

5-Methylcytosine↗
PubMed1982

The changes in proviral chromatin that accompany morphological variation in avian sarcoma virus-infected rat cells.

The clone All of avian sarcoma virus B77-infected Rat-1 cells comprises both morphologically normal and morphologically transformed derivatives. Transformed subclones, in which virus-specific RNA is readily detectable, contain a provirus that is very sensitive to DNase 1 digestion of chromatin, and show DNase 1 hypersensitive sites at the 5' end of the provirus and in 5' flanking cell DNA. Normal subclones with no detectable virus-specific RNA, whether infected cells that have never been transformed or revertants derived from transformed cells, contain a provirus that is far more resistant to DNase 1 digestion. Moreover this provirus lacks hypersensitive sites at its 5' end, although DNase 1 hypersensitive sites were detected at the 3' end of the provirus in either normal or transformed clones. The pattern of cytosine methylation in the proviral restriction sites of the isoschizomers Msp I and Hpa II differed between transformed and revertant clones; the revertants show additional methylation at some CpG doublets.

Alpharetrovirus↗
PubMed1980

DNA replication in Physarum polycephalum. Analysis of replicating nuclear DNA using the electron microscope.

DNA has been isolated from Physarum polycephalum nuclei obtained from macroplasmodia at different stages in the mitotic cycle, and examined using the electron microscope. Putative replicating structures were identified, the majority of which contained clusters of 2--37 'microbubbles', each microbubble corresponding to a segment of DNA 100--5000 nucleotides long. The microbubble-containing structures are unstable in the formamide hyperphase used to prepare specimens for electron microscopy, possibly due to dissociation of newly replicated nascent DNA fragments from the parental DNA template during manipulation. The microbubble clusters present in early S-phase DNA extent over segments averaging 16400 nucleotide residues, and are separated by non-replicated regions of DNA varying in length from 10000 to 50000 nucleotides. It is suggested that each microbubble cluster may represent a 'replicon', and that many 'replicons' in Physarum DNA may contain several sites for the initiation of DNA synthesis that are active during S-phase.

Cell Nucleus↗
PubMed1979

Microbubbles in replicating nuclear deoxyribonucleic acid from Physarum polycephalum.

Clusters of microbubbles, represent probable sites of newly initiated DNA synthesis, were identified in nuclear DNA from Physarum polycephalum by using the electron microscope. Their presence is associated specifically with S-phase. Each microbubble corresponds in size to a replicating segment of DNA about 100-5000 nucleotide residues in length. The DNA structures containing microbubbles are metastable, and revert to native DNA in the presence of moderate concentrations of formamide used to prepare samples for electron microscopy. It is suggested that each cluster of microbubbles may correspond to a unit of replication (a replicon) in Physarum DNA.

Cell Nucleus↗
PubMedDate not supplied

Pruritus and cholestasis: therapeutic options.

The pathogenesis of pruritus of cholestasis remains unclear. Bile salts do not appear to be the sole prurogens in cholestasis. Histaminergic pathways may be involved, and central opiate receptor processes seem much more important than has previously been recognized. The therapeutic options for relief of cholestatic pruritus are summarized in Table 2. Resins such as cholestyramine are the first line of therapy. In cases where cholestyramine has failed, rifampicin and antihistamines may be beneficial. Opiate antagonists hold great potential if opioid withdrawal-like syndromes can be avoided. Ursodeoxycholic acid and methotrexate have an advantage in not only relieving pruritus but also potentially retarding disease progression in PBC and PSC, respectively, although this remains to be proved. Other agents such as EPO and SAMe remain experimental and require further study to clarify their effectiveness before they can be recommended.

Cholestasis↗