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M Vidal

Publications and source records attributed to M Vidal.

At least 19 recordsLinked to original sources
PubMed1992

A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F.

The retinoblastoma protein (pRB) plays an important role in the control of cell proliferation, apparently by binding to and regulating cellular transcription factors such as E2F. Here we describe the characterization of a cDNA clone that encodes a protein with properties of E2F. This clone, RBP3, was identified by the ability of its gene product to interact with pRB. RBP3 bound to pRB both in vitro and in vivo, and this binding was competed by viral proteins known to disrupt pRB-E2F association. RBP3 bound to E2F recognition sequences in a sequence-specific manner. Furthermore, transient expression of RBP3 caused a 10-fold transactivation of the adenovirus E2 promoter, and this transactivation was dependent on the E2F recognition sequences. These properties suggest that RBP3 encodes E2F, or an E2F-like protein.

Amino Acid Sequence↗
PubMed1991

The influence of transferrin binding to L2C guinea pig leukemic lymphocytes on the endocytosis cycle kinetics of its receptor.

The parameters regulating the internalization and recycling of transferrin-specific receptors were determined in guinea pig leukemic B lymphocytes, in the absence or presence of ligand. We show that after the cells were purified, 45-56% of the total receptors were on the cell surface. In the absence of transferrin, unoccupied receptors are quickly internalized (rate constant, 0.12 min-1) whereas their recycling is much slower (rate constant, 0.026 min-1). This difference between endocytosis and recycling rates leads to a balanced receptor distribution with only 22% of the total receptors outside after incubation of the cells for 20-30 min at 37 degrees C. The internalization rate of occupied receptors, measured in the presence of transferrin is faster (rate constant, 0.21 min-1) than that of unoccupied receptors calculated in the absence of transferrin (0.12 min-1; see above). On the other hand, mere binding of transferrin to its receptor, without internalization, arrested by cytoplasm acidification, is sufficient to induce a large increase (by a factor of seven) in the recycling rate of unoccupied internal receptors from 0.026 min-1 to 0.17 min-1. Thus, in these lymphocytes, transferrin mobilizes internal receptors by modifying the kinetic rates of internalization and recycling, leading to a new equilibrium between external and internal receptors.

Animals↗
PubMed1991

Molecular cloning and expression of the VP1 gene of foot-and-mouth disease virus C1 in E. coli: effect on bacterial cell viability.

The VP1 gene of foot-and-mouth disease virus (serotype C1) has been cloned in Escherichia coli Clts cells, under the control of the bacteriophage lambda pL promoter. The expressed VP1 protein was complete and non-fused, and its molecular weight was indistinguishable from that of the VP1 obtained from virions. Cells harbouring the recombinant vectors exhibited symptoms of plasmid instability and toxicity and died in a few weeks even when never exposed to inducing conditions. A new plasmid clone in which a segment of the VP1 gene was fused with contiguous genes of the viral genome was very stable. The expressed partial VP1 protein contains the two major immunogenic domains of the virion. This system can be used as a tool to design an immunogenic VP1, and to explore possible synthetic vaccines against foot-and-mouth disease.

Animals↗
PubMed1991

RPD1 (SIN3/UME4) is required for maximal activation and repression of diverse yeast genes.

We show that the extent of transcriptional regulation of many, apparently unrelated, genes in Saccharomyces cerevisiae is dependent on RPD1 (and RPD3 [M. Vidal and R. F. Gaber, Mol. Cell. Biol. 11:6317-6327, 1991]). Genes regulated by stimuli as diverse as external signals (PHO5), cell differentiation processes (SPO11 and SPO13), cell type (RME1, FUS1, HO, TY2, STE6, STE3, and BAR1), and genes whose regulatory signals remain unknown (TRK2) depend on RPD1 to achieve maximal states of transcriptional regulation. RPD1 enhances both positive and negative regulation of these genes: in rpd1 delta mutants, higher levels of expression are observed under repression conditions and lower levels are observed under activation conditions. We show that several independent genetic screens, designed to identify yeast transcriptional regulators, have detected the RPD1 locus (also known as SIN3, SD11, and UME4). The inferred RPD1 protein contains four regions predicted to take on helix-loop-helix-like secondary structures and three regions (acidic, glutamine rich, and proline rich) reminiscent of the activating domains of transcriptional activators.

Alleles↗
PubMed1991

RPD3 encodes a second factor required to achieve maximum positive and negative transcriptional states in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, TRK1 and TRK2 encode the high- and low-affinity K+ transporters, respectively. In cells containing a deletion of TRK1, transcription levels of TRK2 are extremely low and are limiting for growth in media containing low levels of K+ (Trk- phenotype). Recessive mutations in RPD1 and RPD3 suppress the TRK2, conferring an approximately fourfold increase in transcription. rpd3 mutations confer pleiotropic phenotypes, including (i) mating defects, (ii) hypersensitivity to cycloheximide, (iii) inability to sporulate as homozygous diploids, and (iv) constitutive derepression of acid phosphatase. RPD3 was cloned and is predicted to encode a 48-kDa protein with no extensive similarity to proteins contained in current data bases. Deletion of RPD3 is not lethal but confers phenotypes identical to those caused by spontaneous mutations. RPD3 is required for both full repression and full activation of transcription of target genes including PHO5, STE6, and TY2. RPD3 is the second gene required for this function, since RPD1 is also required. The effects of mutations in RPD1 and RPD3 are not additive, suggesting that these genes are involved in the same transcriptional regulatory function or pathway.

Amino Acid Sequence↗
PubMed1991

Dissociation between endothelium-dependent relaxations and increases in cGMP in systemic veins.

In canine systemic veins, in contrast to what is observed in mammalian systemic arteries, endothelium-dependent relaxations to the calcium ionophore A23187 are not diminished by the inhibitor of soluble guanylate cyclase, methylene blue. Therefore, experiments were designed to determine whether these relaxations in the veins are associated with the accumulation of guanosine 3',5'-cyclic monophosphate (cGMP). Rings of canine femoral arteries and veins with and without endothelium were suspended for the measurement of isometric force in organ chambers; cGMP was measured by radioimmunoassay. In arteries and veins contracted with norepinephrine, the tissue content of cGMP was greater in rings with than without endothelium. This difference was decreased by methylene blue (10(-5) M). A23187 (3 X 10(-7) M, for 1 min) increased the accumulation of cGMP, which was temporally related with the onset of relaxation in tissues with endothelium. Methylene blue inhibited the accumulation of cGMP in both blood vessels but inhibited the relaxations only in the arteries. In rings without endothelium, sodium nitroprusside (3 X 10(-7) and 10(-5) M) initiated increases in cGMP, which followed the onset of relaxation. Neither response to sodium nitroprusside was reduced by methylene blue. These results suggest that in canine femoral arteries and veins, relaxation of the smooth muscle to sodium nitroprusside are mediated by a mechanism distinct from changes in cGMP. Likewise, in canine systemic veins, endothelium-derived factor(s) released in response to A23187 also can initiate relaxation of the smooth muscle by a mechanism distinct from changes in cGMP.

Animals↗
PubMed1991

Activation of T lymphocytes by cross-linking of glycophospholipid-anchored Thy-1 mobilizes separate pools of intracellular second messengers to those induced by the antigen-receptor/CD3 complex.

T cells can be activated, not only by the conventional (antigen-receptor/CD3 complex) route, but also by cross-linking any one of their lipid-anchored surface glycoproteins. We have compared early transmembrane signalling events mediated through CD3 with those mediated through Thy-1, a lipid-linked surface glycoprotein, on the human lymphoid cell line Jurkat and transfectants expressing higher levels of Thy-1. Cross-linking of Thy-1 causes immediate phosphatidylinositol (PI) turnover and an influx of extracellular Ca2+, while releasing very little Ca2+ from intracellular stores. CD3 activation, on the other hand, causes PI turnover which releases intracellular Ca2+, and only secondarily induces an influx of extracellular ions. The Thy-1 response is detectable at very low levels of surface Thy-1, and is not mimicked by enzymatic removal of lipid-linked proteins from the cell surface. The Thy-1-induced Ca2+ influx is more sensitive to L channel blockers than the CD3-mediated flux. These results indicate that the initial stages of Thy-1-mediated activation involve the rapid and extensive mobilization of the intracellular second messengers, PI and Ca2+, by mechanisms separate to those activated by the antigen-receptor/CD3 complex.

Antigens, CD↗
PubMed1991

Fatty acylation of human platelet proteins: evidence for myristoylation of a 50 kDa peptide.

Fatty acid acylation of platelet proteins was studied by measuring incorporation of [3H]palmitate and [3H]myristate after incubation at 37 degrees C for 4 h. About ten major radiolabeled proteins were detected after SDS-polyacrylamide gel electrophoresis and fluorography, for both fatty acids. Cleavage by hydroxylamine treatment indicated an ester bond of either palmitate or myristate to these proteins. Nevertheless, a single 50 kDa peptide was specifically modified by an amide-linked myristate. The functions of acylated proteins in platelets are still unknown, but their relation with DLPC-induced shape changes and vesicle shedding is excluded.

Acylation↗
PubMed1990

Differences in human cell lines to support stable replication of Epstein-Barr virus-based vectors.

Vectors carrying the origin of replication, ori-P, of the Epstein-Barr virus (EBV) are maintained extrachromosomally in human cells expressing the EBV nuclear antigen 1 (EBNA-1). We have studied the EBV vectors p201 and p292 in which both ori-P and EBNA-1 functions are present using the human cell lines A431 and HeLa. The two lines showed differences in their transfectability by the EBV vectors. Thousands of HeLa transfectants were obtained with either vector and these remained intact as episomes. A431 could only be efficiently transfected with p292 and a high ratio of chromosomal integrations and rearrangements were observed. The vector p292 expressed the EBNA-1 gene more efficiently than p201 and this was found to be associated with a harmful effect on the grown of both HeLa and A431 lines. These results indicate that EBV vectors behave differently, depending on the cell line and that over-expression of EBNA 1 from these vectors may be detrimental to the cells.

Antigens, Viral↗
PubMed1990

Effects of benzyl alcohol on transferrin and low density lipoprotein receptor mediated endocytosis in leukemic guinea pig B lymphocytes.

We demonstrated that benzyl alcohol, a neutral local anesthetic drug, inhibits the uptake and degradation of lowdensity lipoprotein and endocytosis of transferrin receptors of guinea pig leukemic B lymphocytes (L2C). This inhibition is very rapid, concentration dependant and reversible by simple washing. Membrane fluidity of the living cells is also modified.

Animals↗
PubMed1990

Tissue specific and position independent expression of the complete gene domain for chicken lysozyme in transgenic mice.

A 21.5 kb DNA fragment carrying the entire chicken lysozyme gene locus was introduced into the germ line of mice. The fragment contains the transcribed region plus 11.5 kb 5'-flanking and 5.5 kb 3'-flanking sequences including all known cis-regulatory elements and the 5' and 3' attachment elements (A-elements) which define the borders of the DNase I sensitive chromatin domain. All sequences which adopt a DNase I hypersensitive chromatin conformation in vivo are present on the construct. Seven founder mice were analysed. All of these expressed chicken lysozyme RNA at high levels specifically in macrophages, as is the case in the donor species. Expression levels are dependent on the copy number of integrated genes indicating that a complete gene locus, as defined by its chromatin structure, functions as an independent regulatory unit when introduced into a heterologous genome.

Animals↗
PubMed1990

Tissue-specific control elements of the Thy-1 gene.

We have exploited the structural homology, but different patterns of expression of the murine and human Thy-1 genes to map a number of tissue-specific enhancer elements in the genes. All of these are located downstream from the site of transcriptional initiation. The human gene contains separate elements which direct expression to the kidney or spleen epithelium. The murine gene lacks these elements but instead contains a thymocyte specific enhancer in the third intron. Developmentally-regulated expression in nerve cells is directed (at least in part) by an atypical element in the first intron. The latter is active on heterologous promoters, but is position and distance dependent.

Animals↗
PubMed1990

Direct selection for mutants with increased K+ transport in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells containing a deletion of TRK1, the gene encoding the high affinity potassium transporter, retain only low affinity uptake of this ion and consequently lose the ability to grow in media containing low levels (0.2 mM) of potassium. Using a trk1 delta strain, we selected spontaneous Trk+ pseudorevertants that regained the ability to grow on low concentrations of potassium. The revertants define three unlinked extragenic suppressors of trk1 delta. Dominant RPD2 mutations and recessive rpd1 and rpd3 mutations confer increased potassium uptake in trk1 delta cells. Genetic evidence suggests that RPD2 mutations are alleles of TRK2, the putative low affinity transporter gene, whereas rpd1 and rpd3 mutations increase TRK2 activity: (1) RPD2 mutations are closely linked to trk2, and (2) trk2 mutations are epistatic to both rpd1 and rpd3. rpd1 maps near pho80 on chromosome XV and rpd3 maps on the left arm of chromosome XIV, closely linked to kre1.

Alleles↗
PubMed1990

Enhancer sequences located 3' of the mouse immunoglobulin lambda locus specify high-level expression of an immunoglobulin lambda gene in B cells of transgenic mice.

In contrast to the mouse immunoglobulin heavy chain and kappa light chain genes, very little is known about the regulation of expression of the immunoglobulin lambda light chain locus. To identify elements responsible for lambda gene regulation we mapped DNaseI hypersensitive sites associated with a functionally rearranged lambda 1 gene in nuclei from the myeloma cell line J558L. Tissue-specific hypersensitive sites were identified 2.3 to 2.5 kb upstream of the CAP site of both the lambda 1 gene and the unrearranged variable (V) lambda 2 gene segments. DNA sequences flanking the lambda 1 gene were isolated and tested for their influence on expression of the lambda 1 gene after transfection into myeloma cells and after injection into fertilized mouse eggs. Two enhancer elements were identified downstream of the lambda 1 gene. A proximal element (located 4 to 10 kb 3' of the gene) enhanced expression of a lambda 1 gene in stable myeloma cell transfectants but had no effect on the expression of a heterologous reporter gene in transient assays. A second, distal element, located approximately 30 kb 3' of the gene, enhanced heterologous expression in J558L cells expressing a lambda gene but not in a non-lambda myeloma cell line (SP2/0-Ag14). Co-injection of cosmids containing the lambda 1 gene and both the proximal and distal downstream elements into fertilized mouse eggs resulted in high-level expression of the lambda 1 transgene in B cells of transgenic mice. The identification of these lambda regulatory elements, in addition to contributing to an understanding of lambda gene regulation per se, will facilitate the study of the regulation of differential expression of kappa and lambda light chain genes in the immune system.

Animals↗
PubMed1989

Modifications of LDL-receptor-mediated endocytosis rates in CEM lymphoblastic cells grown in lipoprotein-depleted fetal calf serum.

The efficiency of supplying cholesterol by the LDL endocytic pathway of lymphoblastic T CEM cells was compared when incubated in the presence of either fetal calf serum (FCS) or lipoprotein-depleted fetal calf serum (LDFCS). In the presence of FCS, there were 8600 +/- 2000 LDL receptors/cell with a Kd of (2.2 +/- 0.8).10(-8) M and a receptor cycling time of about 7 min; about 90% of the internalized LDL was degraded. LDL degradation produced 98% of total cellular cholesterol and only 2% came from endogenous synthesis. The absence of LDL in the culture medium of lymphoblastic CEM cells deeply modified certain metabolic and structural characteristics of the cells. Their cholesterol content decreased; the total number of LDL receptors increased 6-fold, whereas their affinity for the ligand decreased by the same factor (Kd = (1.2 +/- 0.2).10(-7) M); the receptor cycling time increased 3-fold. Finally, LDL degraded by cholesterol-depleted CEM cells amounted to about 40% of that degraded by untreated CEM cells.

Cholesterol↗
PubMed1989

A dominant control region from the human beta-globin locus conferring integration site-independent gene expression.

The regulatory elements that determine the expression pattern of a number of eukaryotic genes expressed specifically in certain tissues have been defined and studied in detail. In general, however, the expression conferred by these elements on genes reintroduced into the genomes of cell lines and transgenic animals has turned out to be at a low level relative to that of endogenous genes, and influenced by the chromosomal site of insertion of the exogenous construct. We have previously shown that if regions flanking the human beta-globin locus are introduced into the mouse genome along with the human beta-globin gene, a level of expression comparable to that of endogenous genes can be achieved that is also independent of integration site. We have now defined a dominant control region with these properties consisting of 6.5 kilobases of DNA encompassing erythroid cell-specific DNase I hypersensitive sites. The identification of such dominant control regions could have important applications in somatic gene therapy.

Animals↗
PubMed1989

Asymmetric distribution of phospholipids in the membrane of vesicles released during in vitro maturation of guinea pig reticulocytes: evidence precluding a role for "aminophospholipid translocase".

Guinea pig reticulocytes lose their transferrin (Tf) binding activity during maturation, in the form of vesicles (exosomes) released into the extracellular medium. Vesicles were prepared from cultures of reticulocytes to study the possible externalization of a particular membrane-associated activity, i.e., that of "aminophospholipid translocase." Analysis of the peptide composition of these vesicles revealed that the major proteins are the Tf receptor and another peptide (70kDa), which is probably the "clathrin-uncoating ATPase" described by Johnstone et al. (1987). The exosome had a lipid composition similar to erythrocyte membrane, although with a lightly but significantly lower phosphatidylethanolamine content. The aminophospholipid distribution in the vesicle membrane was determined by fluorescamine labeling. The exosomes showed an asymmetric aminophospholipid distribution similar to that of erythrocytes. "Aminophospholipid translocase" activity was absent, as no transverse diffusion of spin-labeled phospholipids occurred over more than 2 hours at 37 degrees C.

Animals↗