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Biomedical subjects

R J Watson

Publications and source records attributed to R J Watson.

At least 37 records · Page 2Linked to original sources
PubMed1996

Interference of Myb transactivation activity by a conditional dominant negative protein: functional interference in a cytotoxic T-cell line results in G1 arrest.

The ability to ablate the activity of specific transcription factors in vivo is a potentially important tool to study their roles in cellular processes such as the cell cycle. Previously, production of a dominant interfering c-Myb protein (comprising a fusion of the c-Myb DNA binding domain with the Drosophila Engrailed transrepressor) was found to inhibit the proliferation of immature thymocytes in the developing thymus of transgenic mice. We report here the further development of this stratagem by rendering the c-Myb/Engrailed protein conditionally active by fusion to a modified estrogen receptor hormone binding domain, ER. Co-transfection experiments in NIH 3T3 fibroblasts showed that the resulting chimeric protein, Myb/En/ER, repressed transactivation of a c-Myb-responsive reporter only in the presence of the synthetic steroid, 4-hydroxytamoxifen (OHT). Additionally, we found that Myb/En/ER could counteract transactivation by C/EBP-beta of the mim-1 promoter, which contains juxtaposed Myb and C/EBP binding sites. Cytotoxic T-cells stably producing the inactive Myb/En/ER protein were readily obtained by gene transfection. The addition of OHT to these cells resulted in inhibition of proliferation and arrest in G1. The utility of this experimental system to study Myb and other transcription factors is discussed.

Animals↗
PubMed1996

E2F binding is required but not sufficient for repression of B-myb transcription in quiescent fibroblasts.

We have previously shown in mouse NIH3T3 fibroblasts that transcription of the B-myb gene, which encodes a transcription factor required for S phase entry, is repressed through a promoter E2F site in G0/early G1. Transcription repression at this stage of the cell cycle was correlated with binding of a specific p107/E2F complex to this site. We report here, however, that transfection of cells with the known components of this complex, p107, E2F-4 and DP-1, did not repress the B-myb promoter in cycling NIH3T3 cells, although p107 inhibited transcription transactivation by E2F-4/DP-1. To establish definitively the contribution of E2F to repression, the effects of further mutations within and surrounding the E2F site were examined. It was evident that E2F binding and repression were closely correlated, lending greater weight to the contention that E2F itself is implicated in this activity. These studies also identified a closely linked site, designated the downstream repression site (DRS), which was not required for E2F binding or transactivation but which was necessary for repression. These findings indicated that E2F-dependent repression and activation are independently regulated phenomena and suggest that repression involves additional interactions determined by the promoter context.

3T3 Cells↗
PubMed1996

B-myb promotes S phase and is a downstream target of the negative regulator p107 in human cells.

The retinoblastoma protein family has been implicated in growth control and modulation of the activity of genes involved in cell proliferation, such as B-myb. Recent evidence indicates that the product of the B-myb gene is necessary for the growth and survival of several human and murine cell lines. Upon overexpression, B-myb induces deregulated cell growth of certain cell lines. Here we show that B-myb overexpression is able to induce DNA synthesis in p107 growth-arrested human osteosarcoma cells (SAOS2). p107 might exert its growth-suppressive activity by regulating B-myb gene transcription. Indeed, p107 down-modulated B-myb promoter activity and drastically decreased E2F-mediated transactivation. Finally, B-myb was able to stimulate DNA synthesis of both stably and transiently transfected human glioblastoma cells (T98G). Altogether, these data provide definitive evidence that the human B-myb protein is involved in growth control of human cells, and that p107 has a significant role in regulating B-myb gene activity.

Adenovirus E2 Proteins↗
PubMed1996

Identification and analysis of the dissimilatory nitrous oxide reduction genes, nosRZDFY, of Rhizobium meliloti.

The complete nos region essential for dissimilatory nitrous oxide reduction by the endosymbiotic diazotroph Rhizobium meliloti was identified in a cosmid (pYC7) carrying a 10.1-kb EcoRI fragment of the nod megaplasmid. This gene region was localized by Southern hybridization and Tn5 mutagenesis to within 8 kb downstream from the fixGHIS cluster. Nucleotide sequence determination of a 4.6-kb DNA segment including the structural gene nosZ and its flanking regions showed sequence homology and similarity in genetic organization with the nosRZDFY genes of Pseudomonas stutzeri Zobell. The genes were arranged in three complementation groups, comprising the nosZ structural gene, the nosR regulatory gene, and the nosDFY copper-processing genes. The derived amino acid sequence of the R. meliloti nosZ product (a multi-copper nitrous oxide reductase) was more similar to those of the analogous gene products of Paracoccus and Pseudomonas species than to that of Alcaligenes eutrophus. The nosZ gene was preceded by nosR, which encodes a regulatory protein containing C-terminal cysteine clusters similar to those present in the 4Fe-4S binding region of bacterial ferredoxins, The nosDFY genes, located downstream from nosZ, were identified as copper-processing genes encoding a periplasmic protein, an ATP/GTP-binding protein, and a membrane protein presumably forming a copper-processing system. A consensus sequence for an Anr- or Fnr-binding site similar to that in the upstream sequence of nosZ in Paracoccus denitrificans or P. stutzeri was absent in R. meliloti. No rpoN-binding site preceding the nos genes was detected, and none of the Tn5 insertions in the nos gene region affected symbiotic N2-fixing ability.

Amino Acid Sequence↗
PubMed1996

Epstein-Barr virus nuclear antigen 3C is a powerful repressor of transcription when tethered to DNA.

The expression of Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for the activation and immortalization of human B lymphocytes by EBV. EBNA3C consists of 992 amino acids and includes a potential bZIP motif and regions rich in acidic, proline, and glutamine residues. Thus, EBNA3C resembles several trans regulators of gene expression. It has recently been shown that a fragment of EBNA3C can activate reporter gene expression when fused to the DNA-binding domain of GAL4 (D. Marshall and C. Sample, J. Virol. 69:3624-3630,1995). Although EBNA3C binds DNA, a specific site for EBNA3C binding has not been identified; to test the ability of full-length EBNA3C to regulate transcription, EBNA3C (amino acids 11 to 992) was fused to the DNA-binding domain of GAL4. We show that this fusion protein does not transactivate but rather is a potent repressor of reporter gene expression. Repression is dependent on the dose of GAL4-EBNA3C and on the presence of GAL4-binding sites within reporter plasmids. Repression is not restricted to B cells nor is it species or promoter specific. Repression is independent of the location of the GAL4-binding sites relative to the transcription start site. A fragment of EBNA3C (amino acids 280 to 525) which represses expression in a manner which is nearly identical to that of the full-length protein has been identified; this fragment is rich in acidic and proline residues. A second, less potent repressor region located C terminal to amino acids 280 to 525 has also been identified; this domain is rich in proline and glutamine residues. We also show binding of EBNA3C, in vitro, to the TATA-binding protein component of TFIID, and this suggests a mechanism by which EBNA3C may communicate with the basal transcription complex.

3T3 Cells↗
PubMed1995

Cell-cycle regulation of human B-myb transcription.

We demonstrate here that activity of the human B-myb promoter is regulated during the cell cycle by the E2 transcription factor (E2F). Comparison of the human B-myb promoter sequence with that of its murine counterpart revealed an evolutionally conserved sequence that contains an E2F-binding site. In transiently transfected murine NIH3T3 and human HaCaT cells, luciferase (Luc) reporter activity directed by the human B-myb promoter was found to increase significantly in late G1/S phase of the cell cycle. Mutation of the promoter E2F site resulted in significantly greater Luc activity in NIH3T3 and HaCaT cells made quiescent by serum deprivation, indicating that E2F repressed transcription of this gene during G0. Analysis of E2F DNA-binding activity in G0 HaCaT cells revealed a distinct complex that apparently contained neither the retinoblastoma gene protein, pRb, nor the related p107 protein. De-repression of transcription in S phase was accompanied by the disappearance of this G0 E2F complex and the appearance of a distinct complex containing p107. In addition, complexes containing pRb were detected at both stages of the cell cycle.

3T3 Cells↗
PubMed1995

Conditional inhibition of erythroid differentiation by c-Myb/oestrogen receptor fusion proteins.

The c-myb proto-oncogene encodes a transcription factor that has been implicated in the regulation of haemopoietic cell differentiation and appears also to be required for cell proliferation in a number of different lineages. Typically, transcription of c-myb is down-regulated during haemopoietic cell differentiation, and it has been found in several erythroid and myeloid cell lines that constitutive c-myb expression, from a transfected plasmid, blocks this differentiation process. To investigate further the activity of c-myb in haemopoietic cell differentiation, we have transfected Friend murine erythroleukaemia (F-MEL) cells with plasmids encoding conditionally active c-Myb/oestrogen receptor (Myb/ER) fusion proteins. Transcriptional activity of the Myb/ER fusion proteins was found to be strictly hormone-dependent, and this property was correlated with the ability of these proteins to inhibit erythroid differentiation. From analysis of a Myb/ER protein that lacks the c-Myb transactivation domain, it was apparent that the C-terminal ER transactivation domain could substitute for that of c-Myb in inhibition of differentiation. Activation of Myb/ER in F-MEL cells had no effect upon the early and transient inhibition of entry into S phase associated with dimethyl sulfoxide (DMSO) induction. Further analyses of alpha-globin and PU.1 gene transcription suggested that c-Myb is unable to influence gene expression immediately following DMSO-induction and that inhibition of F-MEL cell differentiation must therefore result from the function of c-Myb in the post-commitment period. Nonetheless, c-Myb had effects on the erythroid differentiation programme that were clearly dissociated from its role in cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1995

Detection of Rhizobium meliloti cells in field soil and nodules by polymerase chain reaction.

A genetically marked Rhizobium meliloti strain, R692, was prepared by insertion of a 1.7-kb DNA segment from Tn903 between the nifHDK and fixABC genes in the nod megaplasmid. This DNA was used as a marker, detectable by polymerase chain reaction (PCR), for the specific identification of bacteria in soil samples and alfalfa nodules. This detection technique was tested by applying different titres of the marked strain to field plots seeded with alfalfa. Samples of soil and nodules were assayed for the presence of the marker DNA fragment by PCR using primers specific to the marker sequence. The experiments revealed that the bacteria could be detected directly in soil containing about 10(3)-10(4) bacteria/g, but greater sensitivity was prevented by potent PCR inhibitors present in the samples. The titre of the bacteria in the soil decreased rapidly after inoculation, dropping about 10-fold per week. Tests of vertical location of the bacteria in soil cores showed that the bacteria were initially dispersed to a depth of 18 cm, and subsequently retained viability in the top 2-8 cm. As few as 10 marked R. meliloti per gram of soil resulted in its establishment at detectable levels in nodules. Application of about 10(4)-10(5) bacteria/g soil was sufficient to give the maximum number of nodules per plant and resulted in 70-90% occupancy by the marked strain. Limited movement of the inoculant was detected by analysis of nodules from plants adjacent to the sites where the bacteria were applied, probably by movement in water. The experiments demonstrated the advantages of PCR for the monitoring of marked microorganisms in the environment.

Base Sequence↗
PubMed1994

HPV16 E7 oncoprotein deregulates B-myb expression: correlation with targeting of p107/E2F complexes.

HPV16 is a human tumour virus encoding two principal oncoproteins, E6 and E7. Expression of E7 can induce DNA synthesis in quiescent cells and this property coincides with its ability to bind to the cell proteins pRb and p107. As these cell proteins are regulators of the transcription factor E2F, we have investigated whether the interaction with E7 could result in induction of cell cycle regulated genes. We show that B-myb, whose induction at the G1/S boundary is regulated by release from E2F mediated transcriptional repression, is a target for transcriptional activation by E7 and is the first E7 responsive cell gene to be identified. E7 transactivation leads to both inappropriate transcription of B-myb during G1 and constitutive over-expression in cycling cells. B-Myb plays an essential role in cell cycle progression, and activation by E7 is likely to contribute to the mitogenic activity of the viral oncoprotein. Regulation of the B-myb promoter in NIH3T3 cells correlates with binding of distinct p107-containing complexes at the E2F binding site, and analysis of E7 mutants confirms that B-myb transcription in these cells is regulated through interactions with p107 rather than pRb. These results provide the first example of a potentially specific role for p107 in the regulation of the cell cycle.

3T3 Cells↗
PubMed1994

Lung and chest wall mechanical properties before and after cardiac surgery with cardiopulmonary bypass.

From measurements of airway and esophageal pressures and flow, we calculated the elastance and resistance of the total respiratory system (Ers and Rrs), chest wall (Ecw and Rcw), and lungs (EL and RL) in 11 anesthetized-paralyzed patients immediately before cardiac surgery with cardiopulmonary bypass and immediately after chest closure at the end of surgery. Measurements were made during mechanical ventilation in the frequency and tidal volume ranges of normal breathing. Before surgery, frequency and tidal volume dependences of the elastances and resistances were similar to those previously measured in awake seated subjects (Am. Rev. Respir. Dis. 145: 110-113, 1992). After surgery, Ers and Rrs increased as a result of increases in EL and RL (P < 0.05), whereas Ecw and Rcw did not change (P > 0.05). EL and RL exhibited nonlinearities (i.e., decreases with increasing tidal volume) that were not seen before surgery, and RL showed a greater dependence on frequency than before surgery. The changes in RL or EL after surgery were not correlated with the duration of surgery or cardiopulmonary bypass time (P > 0.05). We conclude that 1) frequency and tidal volume dependences of respiratory system properties are not affected by anesthesia, paralysis, and the supine posture, 2) open-chest surgery with cardiopulmonary bypass does not affect the mechanical properties of the chest, and 3) cardiac surgery involving cardiopulmonary bypass causes changes in the mechanical behavior of the lung that are generally consistent with those caused by pulmonary edema induced by oleic acid (J. Appl. Physiol. 73: 1040-1046, 1992) and decreases in lung volume.

Adult↗
PubMed1993

Transcription regulation by murine B-myb is distinct from that by c-myb.

The transcription regulatory properties of murine B-myb protein were compared to those of c-myb. Whereas c-Myb trans-activated an SV40 early promoter containing multiple copies of an upstream c-Myb DNA-binding site (MBS-1), and similarly the human c-myc promoter, B-Myb was unable to do so. Full-length B-Myb translated in vitro did not bind MBS-1; however, truncation of the B-Myb C-terminus or fusion of the B-Myb DNA-binding domain to the c-Myb C-terminus showed that it was inherently competent to interact with this motif. Further evidence from co-transfection experiments, demonstrating that B-Myb inhibited trans-activation by c-Myb, suggested that failure of B-Myb to trans-activate these promoters did not simply occur through lack of binding to MBS-1. Moreover, using GAL4/B-Myb fusions, it was found that an acidic region of B-Myb, which by comparison to c-Myb was expected to contain a transcription activation domain, actually had no inherent trans-activation activity and indeed appeared to trans-inhibit c-Myb. In contrast to the above findings, both B-Myb and c-Myb were able to weakly trans-activate the DNA polymerase alpha promoter. Results obtained here demonstrate that the activities of B-Myb and c-Myb are clearly distinct and suggest that these related proteins may have different functions in regulation of target gene expression.

3T3 Cells↗
PubMed1993

An E2F-binding site mediates cell-cycle regulated repression of mouse B-myb transcription.

Transcription of the B-myb gene is regulated at the G1/S boundary of the cell cycle. To begin to examine the mechanism controlling expression of this gene during the cell-cycle, a mouse B-myb 5' flanking sequence was isolated from a cosmid library and shown to promote efficiently the transcription of a luciferase reporter gene when transfected into NIH3T3 fibroblasts. It was further shown that in transfected cells released from G0 by readdition of serum, luciferase activity directed by the B-myb promoter was induced substantially as cells entered S phase, thus paralleling the regulation of endogenous B-myb. Analysis of the B-myb promoter identified a region that appeared to have no intrinsic promoter activity yet which acted to regulate transcription negatively in G0. Mutagenesis of an E2F consensus binding site within this region was sufficient to relieve transcription repression in G0, resulting in a promoter with constitutively high activity. Specific G0 and S phase E2F complexes binding to this B-myb element were detected in NIH3T3 cell extracts by mobility shift assays. These studies demonstrate for the first time a direct role for E2F in regulation of cell cycle gene expression by repression of transcription in G0/early G1.

3T3 Cells↗
PubMed1993

Characterization of [3H]vesamicol binding in rat brain preparations.

The binding of (1)-[3H]vesamicol was characterized in several subcellular fractions and brain regions of the rat. Binding to a lysed P2 fraction from the rat cerebral cortex reached equilibrium within 4 min at 37 degrees C and was reversible (dissociation half-time 4.9 min). At least two binding affinities were found in P2 fractions from the cerebral cortex (Kd: 21 nM and 980 nM), striatum (Kd: 28 nM and 690 nM), and cerebellum (Kd: 22 nM and 833 nM). High affinity Bmax values were highest in striatum (1.17 pmol/mg protein), followed by cerebellum (0.67 pmol/mg protein), and cerebral cortex (0.38 pmol/mg protein). Low affinity Bmax values were highest in cerebellum (5.2 pmol/mg protein), with similar values for cerebral cortex (3.7 pmol/mg protein) and striatum (3.8 pmol/mg protein). High affinity but not low affinity binding in each brain region was stereospecific. Another inhibitor of vesicular ACh-transport also displaced 1-vesamicol binding potently (IC50: 17 nM) and efficaciously (over 90%). Both high affinity and low affinity Bmax values for [3H]vesamicol-binding were highest in a partially purified synaptic vesicle fraction, followed by purified synaptosomes, crude membranes and P2 fractions. Specific binding was not observed in a mitochondria-enriched fraction. Crude membrane preparations of primary, neuron-enriched whole brain cultures also exhibited high (64 nM) and low affinity (1062 nM) [3H]vesamicol binding. Isoosmotic replacement of 0.18 M KCl in the binding-buffer with NaCl had no effect on binding. These results suggest that at least some high affinity [3H]vesamicol binding in rat brain preparations may be associated with synaptic vesicles, some of which may not be cholinergic in origin.

Animals↗
PubMed1993

Gender identity disorder and courtship disorder.

A hypothetical connection between gender identity disorder and courtship disorder was investigated in 274 heterosexual male patients of the following types: nontransvestic fetishists for female attire; transvestic fetishists; patients with gender identity disorder of adolescence and adulthood, nontranssexual type; and transsexuals. Of these patients, 53 had also demonstrated one or more of the putative expressions of courtship disorder. The proportion each of these types contributed to this group of 53 patients with a courtship disorder was compared with the same type's proportional contribution to the group of 221 gender identity patients without a courtship disorder. The transvestic fetishists contributed a significantly larger percentage and the transsexuals a significantly smaller percentage of individuals to the group with a courtship disorder than to that without a courtship disorder. Theoretical implications of this asymmetry are discussed.

Adult↗
PubMed1993

Cloning and nucleotide sequencing of Rhizobium meliloti aminotransferase genes: an aspartate aminotransferase required for symbiotic nitrogen fixation is atypical.

In Rhizobium meliloti, an aspartate aminotransferase (AspAT) encoded within a 7.3-kb HindIII fragment was previously shown to be required for symbiotic nitrogen fixation and aspartate catabolism (V. K. Rastogi and R.J. Watson, J. Bacteriol. 173:2879-2887, 1991). A gene coding for an aromatic aminotransferase located within an 11-kb HindIII fragment was found to complement the AspAT deficiency when overexpressed. The genes encoding these two aminotransferases, designated aatA and tatA, respectively, have been localized by subcloning and transposon Tn5 mutagenesis. Sequencing of the tatA gene revealed that it encodes a protein homologous to an Escherichia coli aromatic aminotransferase and most of the known AspAT enzymes. However, sequencing of the aatA gene region revealed two overlapping open reading frames, neither of which encoded an enzyme with homology to the typical AspATs. Polymerase chain reaction was used to selectively generate one of the candidate sequences for subcloning. The cloned fragment complemented the original nitrogen fixation and aspartate catabolism defects and was shown to encode an AspAT with the expected properties. Sequence analysis showed that the aatA protein has homology to AspATs from two thermophilic bacteria and the eukaryotic tyrosine aminotransferases. These aminotransferases form a distinct class in which only 13 amino acids are conserved in comparison with the well-known AspAT family. DNA homologous to the aatA gene was found to be present in Agrobacterium tumefaciens and other rhizobia but not in Klebsiella pneumoniae or E. coli.

Amino Acid Sequence↗
PubMed1992

Reduction in apoptosis relative to mitosis in histologically normal epithelium accompanies fibrocystic change and carcinoma of the premenopausal human breast.

The aims of this study in 227 premenopausal women were (a) to determine the mitotic index (MI), the thymidine labelling index (LI), and the apoptotic index (AI) within the epithelial cells of histologically 'normal' human breast biopsy material removed away from the site of either a fibroadenoma or a carcinoma; and (b) to relate differences in the kinetic indices of the 'normal' epithelium to the pathology in the same breast diagnosed as fibroadenoma alone (125 patients), fibroadenoma with accompanying mild fibrocystic change (79 patients), or carcinoma (23 patients). Ratios of the average indices (AI/MI, AI/LI, MI/LI) were also calculated to minimize uncertainties related to the total cell population counted, the denominator in the LI, MI, and AI. All indices and ratios of indices were corrected for age, averaged over the cycle, and expressed as log-transformed values for analysis. Significant (P less than 0.001) reductions in AI and in apoptosis relative to mitosis (reduced AI/MI) were found in 'normal' epithelium from breasts having fibrocystic change (AI = 0.17 +/- 0.02; AI/MI = 1.01 +/- 0.18) and carcinoma (AI = 0.19 +/- 0.04; AI/MI = 0.88 +/- 0.29), compared with breast with fibroadenoma alone (AI = 0.27 +/- 0.03; AI/MI = 1.29 +/- 0.39). In the absence of significant differences in MI and LI between the 'normal' tissue groups, this finding raises the possibility that reduced epithelial cell apoptosis might be causally associated with the development of fibrocystic change and with an increased risk of development of carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenofibroma↗