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N Davidson

Publications and source records attributed to N Davidson.

At least 145 records · Page 8Linked to original sources
PubMed1990

A combination of derepression of the lac operator-repressor system with positive induction by glucocorticoid and metal ions provides a high-level-inducible gene expression system based on the human metallothionein-IIA promoter.

We and others have introduced the use of the lac operator-repressor system as a method for providing inducible gene expression for gene transfer experiments in animal cells (M. C.-T. Hu, and N. Davidson, Cell 48:555-566, 1987; J. Figge, C. Wright, C. J. Collins, T. M. Roberts, and D. M. Livingston, Cell 52:713-722, 1988). To improve the dynamic range of such an inducible system, we have investigated the effects of combining the relief by isopropyl-beta-D-thiogalactoside (IPTG) of negative control by the lac system with positive induction by the natural inducers glucocorticoids and cadmium ion for a system based on the human metallothionein-IIA gene promoter. We used the chloramphenicol acetyltransferase gene as a reporter gene and inserted a lacO sequence into the promoter between the GC box and metal-responsive element 1, between metal-responsive element 1 and the TATA box, or between the TATA box and the transcription start site. Surprisingly, all of these insertions had a significant inhibitory effect on promoter activity even in the absence of repressor. However, with these lacO-containing constructs, the levels of gene expression after induction by glucocorticoid, Cd2+, or both were considerably reduced in cells engineered to express the lac repressor. Derepression by IPTG, coupled with induction by both dexamethasone and Cd2+ ion, then provided a high level of induced expression, i.e., by a factor of approximately 100 over the basal level of expression. However, inserting the lacO sequence well upstream just before the glucocorticoid-responsive element had much smaller effects on expression levels in both repressor-negative and repressor-positive cells. This study describes a new, high-level-inducible promoter system for gene transfer experiments. The observed effects are discussed in terms of current models of the mechanisms by which transcription factors control gene expression.

Animals↗
PubMed1989

Functional expression of the yeast alpha-factor receptor in Xenopus oocytes.

The STE2 gene of the yeast Saccharomyces cerevisiae encodes a 431-residue polypeptide that has been shown by chemical cross-linking and genetic studies to be a component of the receptor for the peptide mating pheromone, alpha-factor. To demonstrate directly that the ligand binding site of the alpha-factor receptor is comprised solely of the STE2 gene product, the STE2 protein was expressed in Xenopus oocytes. Oocytes microinjected with synthetic STE2 mRNA displayed specific surface binding for 35S-labeled alpha-factor (up to 40 sites/micron2/ng RNA). Oocytes injected with either STE2 antisense RNA or heterologous receptor mRNA (nicotinic acetylcholine receptor alpha, beta, gamma, and delta subunit mRNAs) showed no binding activity (indistinguishable from uninjected control oocytes). The apparent KD (7 nM) of the alpha-factor binding sites expressed on the oocyte surface, determined by competition binding studies, agreed with the values reported for intact yeast cells and yeast plasma membrane fractions. These findings demonstrate that the STE2 gene product is the only yeast polypeptide required for biogenesis of a functional alpha-factor receptor. Electrophysiological measurements indicated that the membrane conductance of oocytes injected with STE2 mRNA, or with both STE2 and GPA1 (encoding a yeast G protein alpha-subunit) mRNAs, did not change and was not affected by pheromone binding. Thus, the alpha-factor receptor, like mammalian G protein-coupled receptors, apparently lacks activity as an intrinsic or ligand-gated ion channel. This report is the first instance in which a membrane-bound receptor from a unicellular eukaryote has been expressed in a vertebrate cell.

Animals↗
PubMed1989

Mutations in the p53 gene occur in diverse human tumour types.

The p53 gene has been a constant source of fascination since its discovery nearly a decade ago. Originally considered to be an oncogene, several convergent lines of research have indicated that the wild-type gene product actually functions as a tumour suppressor gene. For example, expression of the neoplastic phenotype is inhibited, rather than promoted, when rat cells are transfected with the murine wild-type p53 gene together with mutant p53 genes and/or other oncogenes. Moreover, in human tumours, the short arm of chromosome 17 is often deleted. In colorectal cancers, the smallest common region of deletion is centred at 17p13.1; this region harbours the p53 gene, and in two tumours examined in detail, the remaining (non-deleted) p53 alleles were found to contain mutations. This result was provocative because allelic deletion coupled with mutation of the remaining allele is a theoretical hallmark of tumour-suppressor genes. In the present report, we have attempted to determine the generality of this observation; that is, whether tumours with allelic deletions of chromosome 17p contain mutant p53 genes in the allele that is retained. Our results suggest that (1) most tumours with such allelic deletions contain p53 point mutations resulting in amino-acid substitutions, (2) such mutations are not confined to tumours with allelic deletion, but also occur in at least some tumours that have retained both parental 17p alleles, and (3) p53 gene mutations are clustered in four 'hot-spots' which exactly coincide with the four most highly conserved regions of the gene. These results suggest that p53 mutations play a role in the development of many common human malignancies.

Alleles↗
PubMed1989

Regulation of chicken alpha and beta actin genes and their hybrids inserted into myogenic mouse cells.

We have investigated the regulation of intact non-muscle (beta) and muscle-specific (skeletal alpha) chicken actin genes and of hybrids of these two genes (alpha 5'-beta 3' and beta 5'-alpha 3') transferred into the mouse myogenic non-fusing cell line BC3H1. BC3H1 cells express members of the actin multigene family in a differentiation-dependent manner. When proliferating, the cells accumulate large amounts of non-muscle actin mRNA; when the cells are induced to differentiate, the amount of non-muscle actin mRNA decreases and the amount of muscle-specific actin mRNA increases. The transferred beta-actin gene is efficiently expressed in undifferentiated cells and appropriately down-regulated upon differentiation. In contrast, the transferred alpha-actin gene is inefficiently expressed and not consistently up-regulated. Results with the intact and hybrid genes, taken together, are consistent with the hypothesis that both 5' and 3' halves of these genes contain sequences important in regulating the efficiency and/or developmental timing of their expression in BC3H1 cells. By nuclear run-on experiments we found no evidence for gene-specific changes in the rate of transcription of the transferred actin genes during myogenesis. We conclude that the differentiation-dependent changes in expression of the intact beta-actin gene in BC3H1 cells must be regulated at the post-transcriptional level.

Actins↗
PubMed1989

Secondary acute myelocytic leukemia after adjuvant therapy for early-stage breast carcinoma. A new complication of cyclophosphamide, methotrexate, and 5-fluorouracil therapy.

The occurrence of treatment-related hematologic malignancies after adjuvant therapy with alkylating agents for gastrointestinal cancers, ovarian carcinoma, and breast cancer and after treatment for Hodgkin's disease, non-Hodgkin's lymphoma, germ-cell tumors, and multiple myeloma has been well documented. Adjuvant chemotherapy is frequently used for the treatment of early stage breast cancer, and to date there has been no increase in the incidence of secondary myelodysplastic syndromes or acute leukemia after cyclophosphamide-based regimens when compared with surgical controls. This report describes two patients who developed acute myelocytic leukemia only after exposure to cyclophosphamide, methotrexate, and 5-fluorouracil adjuvant therapy. These two cases of acute leukemia, which developed 3 years after diagnosis of breast cancer and initiation of chemotherapy, were characterized by trilineage dysplasia and pancytopenia, and had abnormalities of chromosomes 5 and 7: characteristics consistent with treatment-related leukemia. Many women are diagnosed with early stage breast cancer each year who are potential candidates for adjuvant therapy. Although certain subgroups of patients have been shown to benefit from adjuvant therapy, continued efforts must be directed at identifying responders so that others will not be exposed to the additional risks of chemotherapy.

Aged↗
PubMed1989

Developmental regulation of sodium channel expression in the rat forebrain.

Na+ channels in adult rat brain are heterotrimeric complexes consisting of alpha subunits (260 kDa) noncovalently associated with a beta 1 subunit (36 kDa) and disulfide-linked to a beta 2 subunit (33 kDa). The time course of developmental accumulation of the 9-kilobase mRNA encoding sodium channel alpha subunits in the rat forebrain was measured by RNA blotting. These transcripts were present at low levels until birth, increased rapidly in abundance to peak by postnatal day 7, and subsequently declined to 50% of this maximum value in adult animals. Sodium channel gene transcription measured by a nuclear run-on assay was first detectable on embryonic day 16, increased to a maximum on postnatal days 1 through 7, and declined in adulthood. The level of gene transcription was highest during the period of rapid rise of Na+ channel alpha subunit mRNA levels and decreased during the period of Na+ channel mRNA decline. The levels of Na+ channel alpha subunit protein measured by immunoblotting increased from postnatal day 1 to postnatal day 21, with the greatest rate of increase falling between days 7 and 21. The number of high affinity saxitoxin binding sites increased in parallel to the increase in alpha subunit protein. The period of most rapid rise in Na+ channel alpha subunit levels corresponded to the period of greatest Na+ channel mRNA abundance. Na+ channel alpha subunits were resolved into free alpha subunits and alpha subunits disulfide-linked to beta 2 subunits. On postnatal day 1, virtually all Na+ channel alpha subunits were in the free alpha form. The fraction of disulfide-linked alpha subunits increased to 60% by postnatal day 21 and 90% by postnatal day 90. The concentration of free alpha subunits was maximum on postnatal days 7 to 14 and declined to less than 10% in adulthood. We conclude from these data that the formation of mature heterotrimeric sodium channel complexes is regulated by at least two processes in developing rat forebrain. Activation of Na+ channel alpha subunit gene transcription and the subsequent increase in Na+ channel mRNA are responsible for the major increases in alpha subunit protein and functional Na+ channels in the neonatal brain. However, changes in alpha subunit mRNA abundance alone are not sufficient to explain the kinetics of alpha subunit protein accumulation. Kinetic analysis suggests a requirement for a developmentally regulated translational or post-translational step in brain sodium channel expression.

Aging↗
PubMed1989

Further evidence demonstrating that N-methyl-D-aspartate and kainate activate distinct ion channels.

Several excitatory amino acid receptors encoded by rat brain mRNA were expressed in Xenopus oocytes. Experimental protocols using an open channel blocker (MK-801) were designed to test the common receptor-channel hypothesis in which N-methyl-D-aspartate (NMDA) and kainate activate the same ion channel but induce different open channel conformations with different ionic permeabilities. The present data demonstrate that NMDA exposes previously trapped MK-801 molecules to the transmembrane field and accelerates their dissociation from the channel at positive potentials, while kainate lacks this effect. Therefore, kainate does not activate the same ion channel as NMDA does. Furthermore, differential inhibition of the NMDA response or the kainate response by the competitive antagonists D-2-amino-5-phosphonopentanoic acid (D-AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) indicates that NMDA and kainate do not share the same binding site. Thus, these several lines of evidence demonstrate that two distinct receptor-channels are activated by NMDA and kainate, respectively.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1989

Expression of Drosophila Shaker potassium channels in mammalian cells infected with recombinant vaccinia virus.

A recombinant vaccinia virus containing a Drosophila potassium channel (Shaker H4) cDNA was constructed by homologous recombination between wild-type vaccinia virus DNA and a transfer plasmid. The new virus was used to infect four types of mammalian cells in culture. Electrophysiological recording 24-72 hr after infection revealed the expression of voltage-gated transient potassium channels in all four cell types. The properties of the induced currents were identical to those previously observed following injection of the Shaker H4 transcript into oocytes. Vaccinia promises to be an effective vehicle for the heterologous expression of transmembrane ion channels in a variety of cell types.

4-Aminopyridine↗
PubMed1988

Evidence that the M2 membrane-spanning region lines the ion channel pore of the nicotinic receptor.

Site-directed mutagenesis and expression in Xenopus oocytes were used to study acetylcholine receptors in which serine residues (i) were replaced by alanines (alpha, delta subunits) or (ii) replaced a phenylalanine (beta subunit) at a postulated polar site within the M2 transmembrane helix. As the number of serines decreased, there were decreases in the residence time and consequently the equilibrium binding affinity of QX-222, a quaternary ammonium anesthetic derivative thought to bind within the open channel. Receptors with three serine-to-alanine mutations also displayed a selective decrease in outward single-channel currents. Both the direction of this rectification and the voltage dependence of QX-222 blockade suggest that the residues mutated are within the aqueous pore of the receptor and near its cytoplasmic (inner) surface.

Animals↗
PubMed1988

Properties of two classes of rat brain acidic amino acid receptors induced by distinct mRNA populations in Xenopus oocytes.

The Xenopus laevis oocyte expression system was used to study the molecular composition of mRNAs encoding acidic amino acid (AA) receptors from rat brain. Xenopus oocytes injected with poly(A) mRNA express two general classes of AA receptors. One class consists of AA-gated cation channels. Responses are evoked by N-methyl-D-aspartate (NMDA), by kainate, and to a lesser extent by L-glutamate or quisqualate. The second class of receptor is coupled to an intracellular second messenger pathway activating an oocyte-encoded Ca2+-activated Cl- conductance. This second messenger-coupled AA receptor can be activated by L-glutamate or quisqualate. DL-2-amino-5-phosphonopentanoic acid and D-alpha-aminohexanedioic acid inhibit the AA-gated cation conductances activated by NMDA or kainate with different potencies but do not inhibit the second messenger-coupled AA receptor. Responses to NMDA are enhanced by micromolar level of glycine and are inhibited by Mg2+, Zn2+, or MK-801. Dose-response analysis reveals that the AA-gated cation conductance activated by kainate requires the binding of two agonist molecules. To study the molecular composition, the mRNAs were size-fractionated by denaturing agarose gel electrophoresis. About 20-fold purification in specific activity (nA/ng of mRNA injected) of mRNAs encoding the second messenger-coupled AA receptor was achieved. In contrast, only a slight enrichment of the mRNAs encoding the AA-gated channel was observed. This suggests that the second messenger-coupled AA receptor is encoded by a single size class of mRNA, whereas the AA-gated cation channel(s) is encoded by multiple species of mRNAs or by mRNAs whose size distribution is heterogeneous.

Amino Acids↗
PubMed1988

Tetrodotoxin-sensitive voltage-dependent Na currents recorded from Xenopus oocytes injected with mammalian cardiac muscle RNA.

Voltage-sensitive sodium (Na) channel currents recorded from mammalian cardiac muscle are blocked by tetrodotoxin (TTX) with a Kd of 1-3 microM. We have observed a Kd for TTX of 4-10 nM for Na currents recorded from Xenopus oocytes injected with RNA extracted from rabbit cardiac muscle. This result suggests that the degree of TTX sensitivity of Na channels encoded by cardiac muscle mRNA is in part determined by post-translational modification(s) or of associations with accessory proteins in the membrane.

Animals↗
PubMed1988

Transcription from each of the Drosophila act5C leader exons is driven by a separate functional promoter.

The Drosophila act5C gene has two leader exons at which transcription initiation occurs. In this way two classes of transcripts that are different with respect to the 5'-untranslated sequences are synthesized. Both are present in Drosophila Kc cell mRNA. To define the sequences necessary for transcription from each start point and to determine if each is driven by a separate promoter, 5'-flanking regions from the act5C gene were inserted upstream from the bacterial chloramphenicol acetyltransferase gene and tested for promoter activity by transient assays in Drosophila Kc cells. We show that both leader exons are preceded by separate, functional, promoters. The exon 1 proximal promoter contains at least two regions important for optimal expression. One is at more than 1.9 kb upstream from the exon 1 cap site while the other lies between 1.2 and 0.09 kb of the cap site. The promoter elements necessary for transcription from exon 2 are within 450 bp upstream from its cap site. The data suggest that, in some constructions, transcription initiation at exon 1 inhibits transcription initiation at exon 2. There is a sequence of dyad symmetry which is present upstream from both exon 1 and exon 2 of the form CC(A-rich)6GG. The same sequences are found upstream from many mammalian and chicken actin genes and of the human and mouse c-fos genes, where they are believed to be transcription regulatory sequences.

Actins↗
PubMed1988

The inducible lac operator-repressor system is functional for control of expression of injected DNA in Xenopus oocytes.

We have investigated the use of the Escherichia coli lac operator-repressor system to regulate the expression of genes introduced by microinjection into Xenopus laevis oocytes. We observe that expression of an MSV-cat fusion gene, in which the lac operator was inserted between the TATA box and the transcription start point (tsp), or between the tsp and the start codon (ATG), is completely repressed when the lac repressor protein is added to the plasmid suspension prior to injection. The lac repressor had no detectable effect on the expression of a coinjected HSV-1 tk gene that had no operator insertion (or on an MSV-cat gene without an operator), indicating that the nonspecific DNA-binding properties of the repressor do not inhibit transcription. CAT activity expressed from the operator-containing MSV-cat genes transcribed in the oocyte nucleus was also inhibited by repressor injected into the oocyte cytoplasm, showing that biologically active repressor proteins can enter the nucleus from the cytoplasm. Injection of the inducer IPTG into the oocyte cytoplasm markedly derepressed the repressed cat genes but not the HSV-1 tk gene coinjected as an internal control. Overall, our results show that the lac operator-repressor system can be useful as a genetic switch in the regulation of gene expression of injected DNA in frog oocytes. Finally, our observations on the vectors used in this work show that the MSV enhancer significantly activates transcription from the SV40 early promoter in frog oocytes, although previous studies have indicated that the MSV enhancer is not necessary for the activity of the MSV promoter in oocytes [Graves et al., Mol. Cell. Biol. 5 (1985) 1945-1958].

Animals↗
PubMed1988

At least two mRNA species contribute to the properties of rat brain A-type potassium channels expressed in Xenopus oocytes.

Fast transient K+ channels (A channels) of the type operating in the subthreshold region for Na+ action potential generation were expressed in Xenopus oocytes injected with rat brain poly(A) RNA. Sucrose gradient fractionation of the RNA separates mRNAs encoding A-currents (6-7 kb) from mRNAs encoding other voltage-dependent K+ channels. A-currents expressed with fractionated mRNA differ in kinetics and pharmacology from A-currents expressed with total mRNA. The original properties of the A-currents can be reconstituted when small mRNAs (2-4 kb) are added to the large mRNA fraction. Thus the properties of the A-currents expressed with total poly(A) RNA depend on the presence of more than one mRNA species. mRNA(s) present in the large RNA fraction must encode channel subunits since they express an A-current by themselves. The small mRNA(s) may encode a second subunit(s) or a factor, such as an enzymatic activity that modulates the properties of the channels, which could play a role in generating A-channel functional diversity.

Animals↗
PubMed1988

A rat brain Na+ channel alpha subunit with novel gating properties.

We have constructed a full-length rat brain Na+ channel alpha subunit cDNA that differs from the previously reported alpha subunit of Noda et al. at 6 amino acid positions. Transcription of the cDNA in vitro and injection into Xenopus oocytes resulted in the synthesis of functional Na+ channels. Although the single-channel conductance of the channels resulting from cloned cDNA was the same as that of channels resulting from injection of rat brain RNA, we observed two significant differences in the gating properties of the channels. The Na+ currents from cloned cDNA displayed much slower macroscopic inactivation compared with those from rat brain mRNA. In addition, the current-voltage relationship for currents from cloned cDNA was shifted 20-25 mV in the depolarizing direction compared with currents from rat brain RNA. Coinjection of low MW rat brain RNA restored normal inactivation of the channels indicating the presence of a component, either a structural subunit of the channel complex or a modifying enzyme, necessary for normal gating of the channel.

Amino Acid Sequence↗
PubMed1988

A-type potassium channels expressed from Shaker locus cDNA.

A-type K+ currents are expressed in Xenopus oocytes injected with in vitro-synthesized transcripts from cDNAs for the Drosophila Shaker (Sh) locus. A single Sh gene product, possibly as a multimer, is sufficient for formation of functional A channels. Various Sh RNAs express A currents with distinct kinetic properties. An analysis of structure-function relationships shows that the conserved central region of Sh polypeptides determines ionic selectivity and overall channel behavior, whereas the divergent amino and carboxyl termini can modify channel kinetics. Alternative splicing of Sh gene transcripts may provide one mechanism for the generation of K+ channel diversity.

Animals↗