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J B Bell

Publications and source records attributed to J B Bell.

At least 19 recordsLinked to original sources
PubMed1995

Distinguishable functions for engrailed and invected in anterior-posterior patterning in the Drosophila wing.

Subdivision of the limb primordia into compartments initiates pattern formation in the developing limbs. Interaction between distinctly specific cells in adjacent compartments leads to localized expression of the secreted signalling molecules Wingless (Wg) or Decapentaplegic (Dpp), which in turn organize pattern and control growth of the limbs. The homeobox gene engrailed has been implicated in specification of posterior cell fate, whereas the LIM/homeobox gene, apterous, specifies dorsal fate. Removing apterous activity causes a complete transformation from dorsal to ventral fate and leads to the formation of an ectopic dorsal-ventral boundary organizer. By contrast, removing engrailed activity causes incomplete morphological transformation from posterior to anterior fate in the wing, and fails to produce an ectopic anterior-posterior organizer (reviewed in ref.2). Complete transformation can only be effected by simultaneously eliminating activity of engrailed and its homologue invected. Here we show that invected functions principally to specify posterior cell fate. Thus establishment of the anterior-posterior organizer and control of compartment identity are genetically distinguishable, and invected may perform a discrete subset of functions previously ascribed to engrailed.

Animals↗
PubMed1995

The proliferation of multiple myeloma colonies (MY-CFUc) in vitro is independent of prognosis and is not associated with mutated N- or K-ras alleles in human bone marrow aspirates.

During the period September 1987 to March 1993 the proliferation of myeloma cells as colonies (MY-CFUc) in vitro was examined in bone marrow aspirates from 43 patients with multiple myeloma and two patients with Waldenström's macroglobulinaemia. Twenty-four samples from 45 patients, of whom three were at presentation, four were in complete remission (CR), six had achieved a partial response (PR) and 11 had progressive disease (PD), produced MY-CFUc in vitro. The same bone marrow aspirates or one taken within 2 months of that assessed for MY-CFUc were used in the polymerase chain reaction (PCR). Genomic DNA was analysed for mutations in N- and K-ras by slot blotting of the amplified products from the PCR with 32P-labelled probes and by direct sequencing. No mutations were detected in N- or K-ras proto-oncogenes at codons 12, 13 or 61 in any sample. Eleven of the patients from whom MY-CFUc were produced remain alive with a median survival of 73 months (range 15-75 months). MY-CFUc have been cultured from 19 of these 24 patients on subsequent occasions, of whom nine remain alive. Among patients whose cells did not produce MY-CFUc in vitro at the time of sampling for mutated ras alleles, biopsy samples from four patients have produced MY-CFUc in vitro on subsequent occasions, of whom one patient remains alive. The data show that the proliferation of MY-CFUc in vitro occurred independently of disease status and was not indicative of prognosis. The failure to detect mutated N- or K-ras alleles in any sample suggests that if such mutations were present in the cells which form colonies in vitro they represented less than 0.1% of the tumour burden and did not affect the survival of this group of patients.

Alleles↗
PubMed1995

Protected P-element termini suggest a role for inverted-repeat-binding protein in transposase-induced gap repair in Drosophila melanogaster.

P-element transposition is thought to occur by a cut-and paste mechanism that generates a double-strand break at the donor site, the repair of which can lead to internally deleted elements. We have generated a series of both phenotypically stronger and weaker allelic derivatives of vg21, a vestigial mutant caused by a P-element insertion in the 5' region of the gene. Virtually all of the new alleles arose by internal deletion of the parental element in vg21, and we have characterized a number of these internally deleted P elements. Depending upon the selection scheme used, we see a very different spectrum of amount and source of P-element sequences in the resultant derivatives. Strikingly, most of the breakpoints occur within the inverted-repeats such that the last 15-17 bp of the termini are retained. This sequence is known to bind the inverted-repeat-binding protein (IRBP). We propose that the IRBP may act to preserve the P-element ends when transposition produces a double-strand gap. This allows the terminus to serve as a template upon which DNA synthesis can act to repair the gap. Filler sequences found at the breakpoints of the internally deleted P elements resemble short stretches, often in tandem arrays, of these terminal sequences. The structure of the filler sequences suggests replication slippage may occur during the process of gap repair.

Alleles↗
PubMed1995

G-CSF is a major component of colony-stimulating activity (CSA) in the plasma of patients with multiple myeloma after treatment with high-dose melphalan (HDM).

Colony-stimulating activity (CSA) was measured by the production of granulocyte-macrophage colony-forming units (GM-CFU) from normal donor bone marrow in the plasma of 29 patients with multiple myeloma (MM) after intensive treatment with high-dose melphalan (HDM) with or without autologous bone marrow rescue (ABMR). Although patients who received ABMR had an earlier recovery of circulating neutrophils compared with those who received HDM alone, the time at which CSA reached a maximum was similar in both groups (10 to 11 days) after therapy. The decline in CSA correlated with the recovery of the neutrophil count. In plasma from patients who received recombinant human granulocyte colony-stimulating factor (rhG-CSF), in addition to an autograft, CSA reached a maximum earlier (7 days). Furthermore, neutrophil recovery was earlier in these patients. Platelet recovery was not increased by rhG-CSF. The time at which CSA was maximum in four patients who were undergoing intensive therapy for the second time occurred 9 days after treatment with HDM. Although the period without neutrophils was longer in three (of four) patients who survived long term, one patient who received rhG-CSF had a shorter period of neutropenia than the two who had not had the cytokine. G-CSF was detected in plasma from seven of seven patients but not at all times after treatment. In plasma samples that contained G-CSF, colony numbers were increased by recombinant interleukin-4 (rIL-4) in vitro. Neither IL-3 nor GM-CSF was detected in plasma; however, antibody to GM-CSF reduced CSA in all samples after intensive therapy. The data suggest that CSA is a consistent physiologic response to intensive therapy, even in previously treated patients, but that hematologic recovery is dependent on the availability of viable progenitor cells.

Bone Marrow Transplantation↗
PubMed1994

Targeting of an enhancer trap to vestigial.

The vestigial gene (vg+) is required for normal wing development and is expressed in a spatially distinct pattern in imaginal discs. We have exploited a general property of P element alleles to target an enhancer trap to the 5' region of the gene. By replacing the P element resident at this site in vg21 with a P element carrying a lacZ reporter gene, the vglacZ1 allele was selected on the basis of its increased mutant phenotype. In contrast to vg+ expression, which occurs primarily in the presumptive wing margin and hinge, beta-galactosidase expression in vglacZ1 wing discs is localized to the dorsal wing surface and displays homologous haltere expression. The targeting of P element enhancer traps could be readily extended to other genes with low rates of primary P element insertion.

Animals↗
PubMed1994

Neutralising antibodies in patients with multiple myeloma receiving maintenance therapy with interferon alpha 2b.

In a study of 29 patients who were receiving or had received interferon alpha 2b (IFN-alpha 2b) as maintenance therapy for multiple myeloma, antibodies were detected in 58% (17/29) of patients measured by a solid-phase enzyme-linked immunosorbent assay (ELISA). Only 7/17 patients who were positive for antibody in the ELISA had neutralising antibody to IFN-alpha 2b, measured by virus growth inhibition. These patients comprised six who were receiving IFN-alpha 2b at the time of assessment and one who had finished treatment. Among patients who were receiving the cytokine, four had progressive disease, one was in complete remission and one in partial remission. Neutralising activity was also detected to natural human leucocyte IFN-alpha in the same patients. Two patients who were positive for neutralising antibody remain in remission and are continuing to receive IFN-alpha 2b. These two patients have since lost their neutralising titre. No neutralising antibody to IFN-alpha 2b or natural human leucocyte IFN-alpha was detected in serum from six normal donors. The data suggest that neutralising antibody formation in patients with multiple myeloma is not responsible for relapse in patients receiving IFN-alpha 2b. The transient nature of neutralising antibody production in patients who remain in remission suggests that this response to IFN-alpha 2b is not associated with memory B cells.

Antibodies, Neoplasm↗
PubMed1994

TGF beta 1 and IL-4 have opposing effects on the proliferation of chronic phase chronic myeloid leukaemic cells stimulated by G-CSF in vitro.

The effects of transforming growth factor beta 1 (TGF beta 1) have been studied in vitro on the clonogenicity of haemopoietic progenitor cells (CFU-CML) from 14 patients with chronic myeloid leukaemia (CML) in chronic phase and 13 normal donors. In 14/14 patients with CML, 5 ng of TGF beta 1/dish decreased CFU-CML-formation in cultures stimulated with 15 ng of granulocyte colony-stimulating factor (G-CSF)/dish (p < 0.0005) and in 13/14 patients TGF beta 1 reduced CFU-CML-formation induced by G-CSF in combination with 5 ng of recombinant human interleukin-4 (rhIL-4)/dish (p < 0.005). In 10/14 samples the number of CFU-CML were reduced to levels lower than in cultures containing G-CSF alone (p < 0.01). In contrast, TGF beta 1 had no significant inhibitory effect on the G-CSF-directed proliferation of normal donor mononuclear cells (MNC) either alone or in combination with rhIL-4. RhIL-4 increased G-CSF-induced colony formation in 13/14 CML samples (p < 0.001), but did not have the same effect in the normal donor samples. The in vitro clonogenicity of CML peripheral blood MNC stimulated with 15 ng of G-CSF could not be correlated with the white cell count or the percentage of CD34+ cells at diagnosis.

Antigens, CD↗
PubMed1993

The functional analysis of nonsense suppressors derived from in vitro engineered Saccharomyces cerevisiae tRNA(Trp) genes.

Nonsense suppressors derived from Saccharomyces cerevisiae tRNA(Trp) genes have not been identified by classical genetic screens, although one can construct efficient amber (am) suppressors from them by making the appropriate anticodon mutation in vitro. Herein, a series of in vitro constructed putative suppressor genes was produced to test if pre-tRNA(Trp) processing difficulties could help to explain the lack of classical tRNA(Trp)-based suppressors. It is clear that inefficient processing of introns from precursor tRNA(Trp), or inaccurate overall processing, may explain why some of these constructs fail to promote nonsense suppression in vivo. However, deficient processing must be only one of the reasons why classical tRNA(Trp)-based suppressors have not been characterized, as suppression may still be extremely weak or absent in instances where the in vitro construct can lead to an accumulation of mature tRNA(Trp). Furthermore, suppression is also very weak in strains transformed with an intronless derivative of a putative tRNA(Trp) ochre (oc) suppressor gene, wherein intron removal cannot pose a problem.

Base Sequence↗
PubMed1993

Expression of a Drosophila melanogaster amber suppressor tRNA(Ser) in Caenorhabditis elegans.

The purpose of this study was to test a cloned amber-suppressing tRNA(Ser) gene derived from Drosophila melanogaster for its ability to produce amber suppression in the nematode Caenorhabditis elegans. To date, all characterized nonsense suppressors in C. elegans have been derived from tRNA(Trp) genes. Suppression was assayed by monitoring the reversal of a mutant tra-3 phenotype among individuals transformed with the cloned Drosophila suppressor gene. An amber allele of tra-3 results in masculinization of XX animals with accompanying sterility. Complete suppression was observed among the transformants. The presence of the heterologous transgene, in both suppressed experimental animals and controls injected with a non-suppressing wild-type Drosophila tRNA(Ser) gene, was verified by PCR amplification of DNA from single worms using primers flanking the tRNA(Ser) gene. Suppression by the heterologous transgene was comparable in quality to that produced by endogenous C. elegans suppressors, and, in frequency as well as quality, to that produced by a transgenic C. elegans tRNA(Trp)-derived suppressors. Thus, a heterologous suppressor gene will function in C. elegans, and it need not be based on tRNA(Trp).

Animals↗
PubMed1992

Characterization of the tRNA(Trp) genes of Saccharomyces cerevisiae.

The purpose of this work was to examine the tRNA(Trp)-encoding genes (tRNA(Trp)) of Saccharomyces cerevisiae to gain insight as to why tRNA(Trp) amber suppressors, isolated by conventional genetic techniques, have not been reported. The results herein indicate that the haploid yeast genome contains six tRNA(Trp) genes which map to five or six chromosomes. Not only do the six genes have identical coding sequences but their introns are also identical. Gene replacement experiments indicate that five copies of tRNA(Trp) are sufficient for cell viability. Thus, mutation of one tRNA(Trp) gene to a suppressor in vivo, lowering the functional number of tRNA(Trp) genes, would not be expected to be lethal.

Base Sequence↗
PubMed1992

A P element chimera containing captured genomic sequences was recovered at the vestigial locus in Drosophila following targeted transposition.

A P element carrying the Dopa decarboxylase gene, P[Ddc], was targeted into vg21, a cryptic P element induced mutant allele of the vestigial (vg) locus. The resulting allele, vg28w, contained the expected P[Ddc] plus an additional 9.5 kb of DNA, captured from elsewhere on chromosome II. Reversion of the vg28w mutant allele demonstrated that the entire insert can excise but cannot reinsert at an appreciable frequency. We explain the targeted transposition as the repair of a double stranded gap, created by the excision of the P element at vg21, and suggest that the formation of chimeric elements may be an important component of P element dependent genomic instability.

Alleles↗
PubMed1992

Colony-stimulating activity in the serum of patients with hemopoietic malignancies after intensive chemotherapy/radiotherapy: its augmentation by GM-CSF in vivo and interleukin 4 in vitro.

Colony-stimulating activity (CSA) in the serum of patients with hematological malignancies increased substantially after intensive therapy with cyclophosphamide/busulfan, cyclophosphamide/total body irradiation, or melphalan/total body irradiation. This was not dependent on patients receiving allogeneic bone marrow transplantation (ABMT) or autologous bone marrow rescue (ABMR). In 44 of 62 patients CSA was maximum approximately 7 days after chemotherapy/radiotherapy, whereas in 18 of 62 patients CSA was maximum between 9 and 20 days after therapy and decreased thereafter. The time course of CSA was not dependent on disease and was not affected by recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) given as a continuous infusion for 14 days after therapy; however, serum from patients receiving rhGM-CSF produced significantly more colonies from donor bone marrow than serum from patients who did not receive the cytokine (p = 0.013). Despite the early peak in CSA in the majority of patients, there was no correlation between the time at which CSA was maximum and the return of patients' neutrophils to 500/microliters. Recombinant human interleukin 4 (IL-4) increased the number of granulocyte-macrophage colony-forming unit colonies, principally granulocyte colony-forming unit colonies, from normal bone marrow exposed to patients' serum after intensive therapy and antibody to GM-CSF reduced colony numbers. The results suggest that after intensive therapy granulocyte colony-stimulating factor (G-CSF) as well as GM-CSF is released into the serum and, in addition to acting directly with G-CSF, IL-4 may stimulate mononuclear cells to produce and/or release G-CSF.

Bone Marrow Cells↗
PubMed1991

Eukaryotic DNA polymerase amino acid sequence required for 3'----5' exonuclease activity.

We have identified an amino-proximal sequence motif, Phe-Asp-Ile-Glu-Thr, in Saccharomyces cerevisiae DNA polymerase II that is almost identical to a sequence comprising part of the 3'----5' exonuclease active site of Escherichia coli DNA polymerase I. Similar motifs were identified by amino acid sequence alignment in related, aphidicolin-sensitive DNA polymerases possessing 3'----5' proofreading exonuclease activity. Substitution of Ala for the Asp and Glu residues in the motif reduced the exonuclease activity of partially purified DNA polymerase II at least 100-fold while preserving the polymerase activity. Yeast strains expressing the exonuclease-deficient DNA polymerase II had on average about a 22-fold increase in spontaneous mutation rate, consistent with a presumed proofreading role in vivo. In multiple amino acid sequence alignments of this and two other conserved motifs described previously, five residues of the 3'----5' exonuclease active site of E. coli DNA polymerase I appeared to be invariant in aphidicolin-sensitive DNA polymerases known to possess 3'----5' proofreading exonuclease activity. None of these residues, however, appeared to be identifiable in the catalytic subunits of human, yeast, or Drosophila alpha DNA polymerases.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of yeast orotidine 5'-monophosphate decarboxylase overexpressed from plasmid PGU2.

Orotidine 5'-monophosphate decarboxylase (ODCase) has been overexpressed in yeast 15C cells transformed with a plasmid carrying the URA3 gene that encodes ODCase. Twenty g of cells having ODCase activity equal to 30 mg of pure enzyme per liter of cell culture were obtained after 9 h of galactose induction. To remove yeast proteases, a 60-90% ammonium sulfate fractionation step plus the addition of EDTA as an inhibitor of metallopeptidases was necessary. The purification protocol yielded ODCase that was protease-free and stable to storage at 4 degrees C for 16 months. The pure enzyme had a specific activity of 40 units/mg in 50 mM phosphate buffer, pH 6, and could be stored at -20 degrees C in 20% glycerol with retention of full activity for more than 2 years. The enzyme had a Km for orotidine 5'-monophosphate of 0.7 microM at pH 6 and 25 degrees C. The molecular weight of the plasmid-derived ODCase monomer determined by electrophoresis on denaturing polyacrylamide gels was 29,500. ODCase sedimented through sucrose density gradients as a monomer of about 30 kDa at low protein concentration and in the absence of ligands that bind at the catalytic site. An increase in the sedimentation rate could be induced by increasing the ODCase concentration or by adding ligands that are competitive inhibitors. ODCase sedimented in a single band typical of a protein of 46 kDa at the highest protein concentration studied or in the presence of 50 mM phosphate or 933 microM substrate (orotidine 5'-monophosphate) or product (UMP). A dimer sedimenting as a protein of about 64 kDa occurred in the presence of 50 microM 6-azauridine 5'-monophosphate or 2 microM 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid, competitive inhibitors of ODCase. These results resemble the ligand-induced subunit association of the ODCase domain of bifunctional UMP synthase and support the use of yeast ODCase as a model for ODCases from other species.

Binding, Competitive↗
PubMed1991

Investigation of the enzymatic mechanism of yeast orotidine-5'-monophosphate decarboxylase using 13C kinetic isotope effects.

Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed 13C kinetic isotope effect of 1.0247 +/- 0.0008 at 25 degrees C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494 +/- 0.0006 measured at pH 4.0, 25 degrees C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently being observed under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel [Beak, P. & Siegel, B. (1976) J. Am. Chem. Soc. 98, 3601-3606], which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25 degrees C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. The observed isotope effect does not rise to the intrinsic value above pH 8.5; instead, the observed isotope effects at 25 degrees C plotted against pH yield an asymmetric curve that at high pH plateaus at about 1.035. These data, in conjunction with the pH profile of Vmax/km, fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at high pH, thus providing evidence for the catalytic mechanism of Beak and Siegel (1976).

Carbon Isotopes↗
PubMed1991

Crystallization of yeast orotidine 5'-monophosphate decarboxylase complexed with 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid.

Using an incomplete factorial experimental design, we have identified conditions for crystallization of yeast orotidine 5'-monophosphate decarboxylase (ODCase) in an unliganded state and complexed separately to two inhibitors: 6-azauridine 5'-monophosphate (aza-UMP) and 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid (BMP). Crystals of X-ray diffraction quality have been obtained of yeast ODCase complexed with BMP, a putative transition state analog inhibitor (Ki = 8.8 x 10(-12) M). ODCase:BMP complex crystals with a hexagonal rod habit were grown from a solution initially containing 12 mg/ml ODCase (205 microM dimer) plus 450 microM BMP by microdialysis at 4 degrees C against a mother liquor which consisted of 0.1 M Na-PIPES-acetate (pH 6.4), 37.5 microM BMP, 5 mM mercaptoethanol, 1% polyethylene glycol 400, and 2.3 M ammonium sulfate. Crystals were analyzed using precession photography and were assigned to trigonal space group R32 with unit cell dimensions a = b = 115 A, c = 385 A. The crystal density is 1.245 g/cm3 indicating the presence of two ODCase: BMP complex dimers (118 kDa each) per asymmetric unit with a packing density of 2.08 A3/Da and 41% solvent content. The morphological habit of crystals of the ODCase:BMP complex changed when the initial ammonium sulfate concentration was increased in 0.05 M steps from 2.3 to 2.45 M. All of these crystals diffracted to at least 3.0 A resolution over a period of several weeks at room temperature and are isomorphous.

Ammonium Sulfate↗
PubMed1991

Control of Drosophila wing and haltere development by the nuclear vestigial gene product.

The Dipteran flight appendages, the wings and halteres, develop from larval imaginal discs that also produce other sections of the second and third thoracic adult body segments. Loss of vestigial (vg) function in Drosophila selectively eliminates wing and haltere formation. Here, we show that vg expression is spatially restricted to the presumptive wing and haltere regions of these imaginal discs. An intronic regulatory element mediates this restriction and may elaborate upon cues that activate vg expression in the embryonic disc primordia. The nuclear vg protein lacks any recognized nucleic acid-binding motif but is comprised of two putative functional domains, one of which bears similarity to part of the Deformed homeotic protein and may mediate protein-protein interactions. These results suggest that vg is directly involved in determining which thoracic imaginal disc cells will form wings and halteres, perhaps by interacting with other nuclear regulatory proteins.

Alleles↗