PubMed Health⌕ Search

Biomedical subjects

S Rusconi

Publications and source records attributed to S Rusconi.

At least 91 records · Page 5Linked to original sources
PubMed1991

Transgenic regulation in laboratory animals.

This chapter is an attempt to summarize some commonly accepted and some more subjective opinions about the regulation of transgene expression in laboratory animals. After a short historical introduction, I present some general notions regarding gene structure/function. The spotlight shifts then to the description of the most popular techniques for gene transfer, including the targeted gene replacement. The different approaches are briefly discussed in terms of intrinsic advantages and limitations regarding gene expression patterns. Furthermore, the role of enhancers, promoters and other cis-acting elements such as silencers and dominant control regions as well as their involvement in the chromatin on-off state are discussed on the basis of a specific example studied in our laboratory. The review concludes by presenting recent results and the new perspectives opening in the field of 'surrogate' (also called 'reversed') genetics. Some problems which remain to be solved both at the technical as well as at the social-ethical level are also briefly presented.

Animals↗
PubMed1991

Interference and synergism of glucocorticoid receptor and octamer factors.

We have analysed the interplay of glucocorticoid receptor (GR) and the lymphocyte-specific factor Oct-2A with transient co-transfection assays. Our data confirm our previously described observation that GR and the apparently unrelated factors belonging to the Octamer family can synergize when permitted to bind in cis. However, when GR binding sites are not present in the reporter genes, we observe that the action of the cloned factor Oct-2A expressed in HeLa cells is strongly inhibited in the presence of active GR molecules. We can demonstrate that this GR-mediated inhibition of Oct-2A action is neither due to competitive binding to DNA target sites nor to a reduction of DNA binding competent Oct-2A in the transfected cells. We observe that the phenomenon is not reciprocal, since co-expression of Oct-2A does not inhibit GR-dependent transcription activation. Furthermore, we provide evidence that the observed GR-Oct-2A interference may be dependent on the type of cell line hosting the co-transfected molecules. We consider it likely that the GR-mediated inhibition is due to the exhaustion of some rate-limiting co-activators.

Base Sequence↗
PubMed1990

Transactivation of the HIV promoter by Tat can be estimated by a bacterial blue-white color system.

We describe an assay system which allows easy quantitation of transactivation of the human immunodeficiency virus (HIV) promoter by the viral Tat protein. We make use of the novel expression assay for the study of transcriptional activators [Rusconi et al., Gene 89 (1990) 211-221]. After transfection of a reference plasmid, a Tat expression plasmid, a plasmid in which the expression of simian virus 40 (SV40) large T antigen is driven by the HIV promoter and a replicator plasmid containing an SV40 ori into mammalian cells, low Mr DNA is shuttled back into Escherichia coli. Transactivation is quantitated by comparing the number of white colonies (due to the replicator plasmid) in presence and absence of Tat to the number of blue colonies (due to the reference plasmid). At high copy numbers of transfected reporter plasmid the system was saturated with respect to large T antigen and not accessible to transactivation by the viral Tat protein. Gradual decrease of the concentration of the HIV-promoter-containing plasmid resulted in continuous improvement of transactivation of this promoter. The demonstration of a 200-fold stimulation of the HIV-1 promoter indicates the sensitivity of the assay and its general applicability to analyse the interplay between a transacting factor and the responsive DNA/RNA sequences.

Animals↗
PubMed1990

Immediate early protein of pseudorabies virus is a general transactivator but stimulates only suboptimally utilized promoters. A clue to specificity?

Pseudorabies virus, a herpesvirus, encodes an immediate early (IE) protein that is known to be a general and strong transactivator of transcription. We have tested the activity of this IE protein with a set of well-defined promoters containing a TATA box and one type of upstream factor binding site (for Sp1, NF-kappa B, heavy metal responsive factors, octamer factors or glucocorticoid receptor). All promoters were strongly activated by IE protein, i.e. the IE protein did not preferentially activate transcription via a particular type of upstream element. Activation did not require a bona fide TATA box, since a promoter construct with three Sp1 sites but no TATA box was also activated. Our data are not compatible with a model in which IE protein would bypass the need for upstream factors. Rather, the properties of IE protein, especially a failure to induce strong transcription from a promoter with only a TATA box but no upstream sequences, mimic the action of a remotely placed, cis-active, enhancer DNA. The IE protein was found to have no effect on transcription units that are expressed to their maximal potential, irrespective of whether this was high or low. Such optimal transcription conditions are observed in the presence of a strong enhancer, or with multiple tandem copies of an upstream binding site and/or a high concentration of the corresponding factor. The property of stimulating only "suboptimally" utilized promoters may be exploited by pseudorabies virus to restrict the specificity of the IE protein to the viral early promoters and a subset of cellular promoters.

Base Sequence↗
PubMed1990

Role of TATA-element in transcription from glucocorticoid receptor-responsive model promoters.

Transcription activation properties of the rat glucocorticoid receptor (GR) on minimal, TATA-box containing or depleted promoters have been tested. We show that a cluster of Glucocorticoid Responsive Elements (GRE), upon activation by the GR, is sufficient to mediate abundant RNA-polymerase II transcription. We find that in absence of a bona fide TATA-element transcription initiates at a distance of 45-55bp from the activated GRE cluster with a marked preference for sequences homologous to the initiator element (Inr). Analyzing defined, bi-directional transcription units we demonstrate that the apparent reduction of specific transcription in strong, TATA-depleted promoters, is mainly due to loss of short-range promoter polarization. The implications for long-range promoter/enhancer communication mechanisms are also discussed.

Base Sequence↗
PubMed1990

A novel expression assay to study transcriptional activators.

A novel assay to study transcriptional regulation in vivo designated trans-activation-dependent replication (TDR) assay is based on the modulation of a simian virus 40 (SV40)-derived replication system. A mixture of four plasmids (pPARA + pCIS + pTRANS + pREF) is co-transfected into vertebrate cells. After appropriate incubation, the replication of the pPARA plasmid (containing an SV40 origin of replication) is measured with a simple enzymatic test. We demonstrate that the level of replication is dependent on the differential trans-activation of the reporter pCIS (in which SV40 T-antigen is brought under control of the desired promoter) by the specific regulator protein encoded by the pTRANS plasmid. Three advantages make this assay a convenient tool for the systematic analysis of trans-activation in vivo: (1) remarkable sensitivity (higher than conventional assays); (2) rapid sample processing combined with a built-in standard (pREF-plasmid); (3) avoidance of expensive reagents such as freshly radiolabelled probes. We present the application of the TDR assay to the analysis of deletion mutants of the glucocorticoid receptor (GR) and other, GR-based chimeric trans-activators. The results demonstrate that the properties of protein domains are not always additive in a particular chimaera. Further application possibilities of the TDR assay are also discussed.

Animals↗
PubMed1990

A single DNA-binding transcription factor is sufficient for activation from a distant enhancer and/or from a promoter position.

Typical cell type-specific or inducible mammalian genes are under the control of one or more remote enhancers which transmit their effect to the promoter region located at the initiation site of transcription. Both enhancers and promoters are composed of multiple binding sites for transcription factors. To study the requirements for promoter and enhancer function, we have used a reporter gene that is completely dependent on a single DNA-binding transcription factor in vivo. This factor is a truncated, hormone-independent form of the glucocorticoid receptor which interacts strongly with a palindromic binding site. After transfection into HeLa cells, transcription of a reporter gene with one, two or four copies of the binding site upstream of the TATA box is enhanced less than 10, at least 100 and greater than 1000-fold respectively, in the presence of the receptor. Even when the TATA box is deleted, the four upstream binding sites confer receptor-dependent transcription, though from scattered initiation sites. When four copies of the palindromic binding site are placed downstream of the transcription unit, they form a very strong receptor-dependent enhancer. This enhancer can activate comparably well promoters containing binding sites for either glucocorticoid receptor, Sp1 factor, or octamer factor. Our data show that a single defined DNA-binding factor can mediate both promoter and enhancer activity, and that it can co-operate functionally both with itself and with seemingly unrelated transcription factors.

Animals↗
PubMed1989

Down-regulation and phosphorylation of glucocorticoid receptors in cultured cells. Investigations with a monospecific antiserum against a bacterially expressed receptor fragment.

The expression of the glucocorticoid receptor (GR) gene and the phosphorylation of the GR protein has been studied as a function of time after hormone addition to NIH 3T3 cells. We detected a ligand-induced decrease of GR gene expression at both the level of RNA and protein. GR mRNA declined to 25% of the control within 3 h of dexamethasone treatment and remained at this level for at least 24 h. GR protein was analyzed with a monospecific antiserum directed against the DNA- and hormone-binding domains of the rat GR synthesized in Escherichia coli. Pulse-chase experiments revealed a decrease in GR half-life from 8 h in the absence of hormone to 3 h following hormone treatment. Both effects resulted in a reduction of total GR protein to 20% as determined by quantitative immunoblotting. The level of the GR protein returned to that of untreated cells within 24 h after withdrawal of hormone. Furthermore, dexamethasone treatment led to a 3-4-fold increase in GR phosphorylation within 60 min. The glucocorticoid antagonist 17 beta-hydroxy-11 beta-(4-dimethylamino-phenyl)-17 alpha-(1-propynyl)-oestra-4,9-dien-3-one (RU 486) did not change the phosphorylation state of the receptor, but a down-regulation of the GR was still observed. These results suggest that ligand-dependent receptor phosphorylation is not involved in down-regulation of the GR but may be important in the transcriptional activation of hormone responsive genes.

Animals↗
PubMed1988

Metal binding 'finger' structures in the glucocorticoid receptor defined by site-directed mutagenesis.

The glucocorticoid receptor and the other members of the steroid receptor super-family share a highly conserved, cysteine-rich region which coincides with the DNA binding/transactivating domain. It has been postulated that this region is folded into two 'zinc finger' structures, similar to those originally reported for the transcription factor TFIIIA. The first potential finger domain contains four conserved cysteines and one conserved histidine, while the second contains five conserved cysteines. Using site-directed mutagenesis, we have analysed the consequences of altering the proposed finger-like structures. Our results show that most of the mutations affecting the conserved cysteines result in a total loss of glucocorticoid receptor function. In one important exception, however, a conserved cysteine (Cys500) is dispensable for glucocorticoid receptor activity and therefore cannot be involved in complexing a metal ion to form a finger structure. Moreover, the replacement of either Cys476 or Cys482 by His residues maintains partial in vivo activity of the glucocorticoid receptor, while their exchange for an alanine or serine residue, respectively, eliminates receptor function. These results support, at a genetic level, the involvement of cysteines of the glucocorticoid receptor DNA binding domain in metal ion complexation and define the candidate residues involved in such coordination.

Amino Acid Sequence↗
PubMed1987

Functional dissection of the hormone and DNA binding activities of the glucocorticoid receptor.

We have identified two separate regions of the 795 amino acid rat glucocorticoid receptor that interact with hormonal ligands and DNA respectively. The functional regions were defined by direct assays of segments of the receptor coding sequence translated in vitro. Hormone affinity measurements suggested that residues near the receptor C-terminus are the primary determinants of ligand binding, whereas sequence-specific DNA binding activity resides between amino acids 440 and 546. DNA binding efficiency was stimulated only modestly by prior hormone binding. The receptor regions identified in these in vitro studies correspond to those that mediate ligand-dependent transcriptional enhancement in vivo.

Affinity Labels↗
PubMed1986

Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA.

We isolated and sequenced 6.3 kb of cDNA encoding that rat glucocorticoid receptor, a protein that binds and activates a class of hormone-dependent transcriptional enhancers. Receptor-containing cells produce receptor mRNAs of approximately equal to 6.5 kb and approximately equal to 4.8 kb that differ only in their 3' nontranslated regions; an open reading frame of 795 amino acids resides within the 5' portion of the transcripts. The coding region was expressed in vitro, in transient transfections, and in stable transfectants of a receptor-deficient cell line. The protein products are indistinguishable from bona fide receptor with respect to sedimentation and electrophoretic mobility, antibody reactivity, and hormone and DNA binding. Moreover, the cloned receptor protein activates its corresponding enhancers, restoring to the receptor-deficient cells the full capacity for regulated enhancement.

Amino Acid Sequence↗
PubMed1982

An unusual evolutionary behaviour of a sea urchin histone gene cluster.

DNA sequences of cloned histone coding sequences and spacers of sea urchin species that diverged long ago in evolution were compared. The highly repeated H4 and H3 genes active during early embryogenesis had evolved (in their silent sites) at a rate (0.5-0.6% base changes/Myr) similar to single-copy protein-coding genes and nearly as fast as spacer DNA (0.7% base changes/Myr) and unique DNA. Thus, evolution in the major histone genes conforms to a universal evolutionary clock based on the rate of base sequence change. By contrast, the H4 and H3 coding sequences and a non-transcribed spacer of the DNA clone h19 of Psammechinus miliaris show an exceptionally low rate of sequence evolution only 1/100 to 1/200 that predicted from the clock hypothesis. According to the classical model of gene inheritance, the h19 DNA sequences in the Psammechinus genome require unusual conservation mechanisms by selection at the level of the gene and spacer sequences. An alternative explanation could be recent horizontal gene transfer of a histone gene cluster from the very distantly related Strongylocentrotus dröbachiensis to the P. miliaris genome.

Journal Article↗
PubMed1981

Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences.

We have studied the transient expression of a cloned rabbit hemoglobin beta 1 gene after its introduction into HeLa cells. Two and one-half days after transfection using the calcium phosphate technique we extracted RNA from the entire cell population and analyzed it by the S1 nuclease hybridization assay. Transcripts were barely detectable when beta-globin gene-plasmid recombinants were used. However, 200 times more beta-globin gene transcripts were found when the beta-globin gene recombinants also contained SV40 DNA, and 90% of these transcripts (about 1000 per cell) had the same 5' end as authentic rabbit globin mRNA. In the latter case, abundant production of beta-globin protein was readily detected in a fraction of transfected cells by immunofluorescent staining. Enhancement of globin gene expression was dependent on SV40 sequences acting in cis, but independent of the viral origin of DNA replication. The enhancing activity was associated with the 72 bp repeated sequence element located at the beginning of the viral late gene region. Viral DNA fragments containing the transcriptional enhancer element could act in either orientation at many positions, including 1400 bp upstream or 3300 bp downstream from the transcription initiation site of the rabbit beta-globin gene. These studies define a class of DNA elements with a mode of action that has not been heretofore described. The activation of genes by specific enhancer elements seems to be a widespread mechanism that may be used for the regulation of gene expression.

Animals↗
PubMed1981

Transformation of frog embryos with a rabbit beta-globin gene.

In order to study the fate and possible expression of foreign DNA during embryogenesis of the frog Xenopus laevis, we have injected a rabbit beta-globin gene into fertilized Xenopus eggs. Frog embryo DNA was extracted at various stages of development, fractionated by agarose gel electrophoresis, transferred to nitrocellulose filters, and hybridized to labeled beta-globin recombinant plasmid DNA. It was found that the injected DNA replicated extrachromosomally, reaching, at gastrula stage, a level equivalent to a 10- to 200-fold amplification of input DNA. At later stages, a majority of the foreign DNA was degraded, but a small fraction was maintained. Six-week-old tadpoles as well as six-month-old frogs contained an average of 3-10 copies of the rabbit globin gene per cell. Most of these persisting globin genes were present as long tandem repeats and comigrated in agarose gel electrophoresis with high molecular weight Xenopus DNA. Analysis of globin gene expression by S1 nuclease mapping showed that the rabbit beta-globin promoter was recognized in the frog embryo and that the transcripts were correctly spliced.

Animals↗
PubMedDate not supplied

Transmission and expression of a specific pair of rearranged immunoglobulin mu and kappa genes in a transgenic mouse line.

Two genes that code for a hapten-specific immunoglobulin M (IgM) have been introduced into the mouse germ line. The transgenic antibody represents 10-50% of the serum IgM and is expressed on the membrane of B cells. B-cell hybridoma lines show that a negative feedback inhibition of mu and kappa transgenic products on the immunoglobulin heavy-chain rearrangement is possible.

Alleles↗
PubMedDate not supplied

Glucocorticoid receptor mutants that are constitutive activators of transcriptional enhancement.

Glucocorticoids, a class of steroid hormones, associate specifically with intracellular receptors, facilitating a conformational change that converts the receptor in vitro to a DNA-binding protein and in vivo to a nuclear species that activates a class of transcriptional enhancers termed glucocorticoid response elements (GREs). The DNA sequences recognized specifically by the hormone-receptor complex correspond directly to those required for GRE enhancement. The structural transition that accompanies steroid binding, 'receptor transformation', has been monitored by changes in receptor chromatographic properties, accessibility to monoclonal antibodies, association with other receptor subunits or with heterologous proteins, and aqueous two-phase partition coefficient. However, the significance of the structural change for the biological activity of the receptor is not understood. We have used cloned rat glucocorticoid-receptor coding sequences to produce and characterize a novel class of receptor mutants that elicit GRE enhancer function in transfected cells even in the absence of hormone. The constitutive activity of those receptor derivatives, together with mapping studies that distinguish between the DNA- and hormone-binding domains of the receptor, imply that the conformational change corresponding to receptor transformation may simply unmask pre-existing functional domains for DNA binding, enhancer activation, or both.

Amino Acid Sequence↗
PubMedDate not supplied

Prevalence of HIV-1 resistant strains in recent seroconverters.

Twenty-nine HIV-1 recently infected subjects were retrospectively studied to investigate both the prevalence of nucleoside reverse transcriptase inhibitors (NRTI)-related mutations at primary infection and the proportion of naturally occurring mutations in protease inhibitor (PI)-naive patients. Neither HIV-1 plasma viremia nor CD4 absolute count at baseline could distinguish patients with NRTI pre-existing mutations from those with wild-type virus. An increasing proportion of ZDV-related mutations was observed over time with an overall frequency of 20.7% in the study period. Only 1 out of 6 patients (16.7%) with ZDV-related mutations showed a phenotypically ZDV resistant isolate. A striking proportion of polymorphic changes was present in the protease region of pol gene in newly infected individuals. As many as 80% of seroconverters presented at least one naturally occurring substitution. Some PI-associated substitutions, thought to be compensatory in protease enzymatic function, could confer intermediate to high PI-resistance. Their role following PI administration remains to be elucidated. Our data suggest that the choice of drugs should be oriented by both genotypic and phenotypic evaluations to tailor individual regimens in seroconverters.

Acquired Immunodeficiency Syndrome↗