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G Ciliberto

Publications and source records attributed to G Ciliberto.

At least 127 records · Page 7Linked to original sources
PubMed1987

Inducible and tissue-specific expression of human C-reactive protein in transgenic mice.

C-reactive protein (CRP) is a major acute phase reactant in man but not in mouse. It is synthesized in abundant quantities by human hepatocytes during the course of several diseases, mainly acute inflammations. To investigate the regulation of CRP expression, the human CRP gene was introduced into fertilized eggs by microinjection and transgenic mouse lines were derived. The CRP gene is exclusively transcribed in the liver and expression is strictly dependent on experimental inflammation. The kinetics of induction both for RNA and protein synthesis is very fast; RNA is first detectable after 2 h in the liver, the protein after 6 h in the serum. Human CRP levels in the sera of transgenic mice are comparable to those observed in human diseases. Nuclear run-on experiments indicate that regulation is primarily at the transcriptional level.

Animals↗
PubMed1987

Properties of a U1 RNA enhancer-like sequence.

The properties of a X.laevis U1B snRNA gene enhancer have been studied by microinjection in Xenopus oocytes. The enhancer-like sequence, defined as a short DNA stretch that is able to activate transcription in an orientation independent manner, is interchangeable between different U snRNA genes. The enhancer sequence alone does not, however, efficiently activate transcription from an SV40 pol II promoter but regains its activity when combined with the U-gene specific proximal sequence element. DNase I protection experiments show that the X.laevis U1B enhancer can interact specifically with a nuclear factor present in mammalian cells.

Animals↗
PubMed1987

Cis- and trans-acting elements responsible for the cell-specific expression of the human alpha 1-antitrypsin gene.

The 5' flanking region of the human alpha 1-antitrypsin (alpha 1-AT) gene contains cis-acting signals for liver-specific expression and, when fused to a reporter gene, is able to drive the expression of this gene specifically in liver cells. Here we report the results of a functional dissection of the alpha 1-AT regulatory region. The expression of the bacterial chloramphenicol-transacetylase (CAT) gene, fused to a set of alpha 1-AT 5' flanking regions shortened by progressive deletions or mutated by base pair substitutions, has been compared by transfection in HepG2 (hepatocyte) and HeLa (non-hepatocyte) human cell lines. A minimal tissue-specific element has been identified between the nucleotides -137 and -37 (from the transcriptional start site). This DNA segment activates the heterologous SV40 promoter in hepatoma cell lines but not in HeLa cells. This element contains at least two regions referred to as the A (-125/-100) and B (-84/-70) domains, both essential for transcription. There are at least two other regulatory domains located upstream of the 'minimal element'; the most active of these is located between positions -261 and -210 from the cap site. These upstream elements activate the heterologous SV40 early promoter both in hepatoma cell lines and in HeLa cells. Upon fractionation of rat liver nuclear extracts two proteins have been identified, alpha 1TF-A and alpha 1TF-B, which bind specifically to the A and B domains respectively. Transcriptionally inactive A and B domain mutants are not able to bind these proteins.

Base Sequence↗
PubMed1987

The human alpha 1-antitrypsin gene is transcribed from two different promoters in macrophages and hepatocytes.

In order to investigate the mechanism of expression of the human alpha 1-antitrypsin (alpha 1-AT) gene in macrophages, we have characterized the alpha 1-AT transcriptional units in these cells and discovered that there is a macrophage-specific promoter located approximately 2000 bp upstream of the hepatocyte-specific promoter. Transcription from the two alpha 1-AT promoters is mutually exclusive: the macrophage promoter is silent in hepatocytes and the hepatocyte promoter is silent in macrophages. In addition, in macrophages two distinct mRNAs are generated transcript by alternative splicing. These results suggest that alpha 1-AT gene transcription responds to two different cell-specific regulatory mechanisms.

Base Sequence↗
PubMed1986

Formation of the 3' end on U snRNAs requires at least three sequence elements.

The structural requirements for 3' end formation on the Xenopus laevis U1B snRNA gene have been studied. Three sequence elements are shown to be required. The first is a conserved sequence element found immediately 3' of all vertebrate U snRNA genes studied so far. The second is a gene internal sequence potentially capable of forming a stem-loop structure close to the 3' end of the RNA. The third element lies upstream of these, and may be part of the gene promoter. Experiments designed to investigate the mechanism of 3' end formation on primary U1B snRNA transcripts failed to find evidence for a processing event.

Acetyltransferases↗
PubMed1985

Alu sequences transcription in X. laevis oocytes: nuclear-cytoplasmic partitioning and evidence for 3' end processing reactions.

A large Alu-family cluster in the 5' flanking region of a human alpha 1-acid glycoprotein gene has been identified and sequenced. Individual members microinjected into X. laevis oocytes are transcribed only when canonical box A and B components of the split pol III promoter are present. Alu transcripts accumulate in the nucleus. An unusually short Alu transcript, able to assume a stable secondary structure, undergoes a 3' end processing reaction similar to the one required for tRNA 3' end maturation.

Animals↗
PubMed1985

Functional activity and chromatin configuration of SV40 enhancer injected in Xenopus laevis oocytes.

The SV40 enhancer microinjected into the nuclei of X. laevis oocytes is able to activate transcription about 100-fold when cloned upstream of the SV40 early promoter region or about 10-fold regardless of its orientation when located at a long distance from a test gene (bacterial chloramphenicol acetyl transferase). This effect is qualitatively and quantitatively similar to that observed when analogous constructions were transfected into mammalian cells. Making use of a direct labelling technique for the analysis of the chromatin structure we could show that the SV40 enhancer region in the microinjected plasmids is particularly accessible to cleavage with MNase or DNAse I. Single sites in the 72 bp repeats have been mapped at the nucleotide level.

Animals↗
PubMed1985

Structure of the human alpha 1-acid glycoprotein gene: sequence homology with other human acute phase protein genes.

We have determined the sequence coding for human alpha 1-acid glycoprotein from two independently isolated cDNA clones and a genomic clone. The aminoacid sequences deduced from the three clones, deriving from three different individuals, are identical. Southern blot analysis on human DNA indicates that there are at least two genes coding for alpha 1-AGP. We propose that alpha 1-AGP found in plasma is a mixture of the products of these two different genes. This is the simpler explanation for the heterogeneity in the aminoacid composition in purified alpha 1-AGP observed by Schmid et al. (1). DNA sequence comparison with cDNA clones coding for human alpha 1-antitrypsin and haptoglobin shows a conserved sequence within the 5' untranslated region which may play a role in the acute phase response.

Acute-Phase Proteins↗
PubMed1985

Transcription signals in embryonic Xenopus laevis U1 RNA genes.

A genomic clone of the most abundant U1 RNA genes from Xenopus laevis was isolated from erythrocyte DNA and sequenced. Two different U1 RNA genes, U1A and U1B, are encoded in an HindIII 1.5-kb fragment and both are expressed after microinjection in Xenopus oocytes. Deletions and site-directed mutagenesis of the clones revealed two promoter elements in the U1B gene; one, located 250-220 nucleotides upstream from the 5' terminus of mature U1 RNA, functions as an activator, yielding a 10-fold promotion of transcription; the other, located 60-50 nucleotides upstream of the cap site, functions as an essential element for promotion of transcription. The U1A gene contained only the latter element in the cloned fragment. Homologous sequences can be identified in several U RNA genes of X. laevis.

Animals↗
PubMed1985

Cell-specific expression of a transfected human alpha 1-antitrypsin gene.

We have cloned the human alpha 1-antitrypsin (alpha 1-AT) gene and identified the promoter and the transcription initiation point. The cloned gene, following transfection, is expressed in a cell-specific manner, being transcribed in a human hepatoma cell line (Hep3B) but not in HeLa cells. We show that the 5' flanking region of the alpha 1-AT gene contains DNA sequences sufficient for efficient transcription in Hep3B but not in HeLa cells. This DNA sequence also activates, in a cell-specific manner, heterologous promoters such as that of SV40; however, the effect is only obtained in one orientation, suggesting that this cis-acting cell-specific element does not share all the features generally associated with enhancers. By cotransfection-competition experiments we also show the existence of a limiting trans-acting factor, essential for the expression of the alpha 1-AT gene in Hep3B cells.

Base Sequence↗
PubMed1984

Transcription of multimeric tRNA genes.

We have constructed a set of plasmids carrying a tRNAPro gene from C. elegans in a head to tail dimeric and trimeric arrangement with virtually no spacer sequence. We show that in two different transcriptional systems, each coding region functions as an internal promoter directing the synthesis of independent transcriptional products. This is in contrast with the property of natural head to tail dimeric arrangements found in yeast, where only one coding region functions as promoter (Mao et al., 1980 Cell 20, 589; Schmidt et al., 1980 Nature 287, 750). The evolutionary significance of our finding is discussed.

Animals↗
PubMed1984

Inhibition of eukaryotic tRNA transcription by potential Z-DNA sequences.

The effect of d(CA/TG)n DNA segments on tRNA transcription has been examined. Alternating purine-pyrimidine tracts were cloned at a long distance from, adjacent to, or within the coding sequence of a tRNAPro gene from Caenorhabditis elegans and shown to be able to assume the A-DNA conformation in vitro in physiological salt concentrations. The transcriptional level of these constructs was compared to that of normal tDNAPro by micro-injection into Xenopus laevis oocytes. Our results show a strong inhibitory effect by potential Z-DNA sequences only when these are placed in the flanking regions of the gene or when they are located between the elements (Box A and Box B) of the split promoter. Transcription was studied in parallel with supercoiled and linear DNA molecules carrying a d(CA/TG) stretch 124-bp long in front of the tRNAPro gene. The results show the same level of inhibition of Po/III transcription regardless of the topological status of the injected DNA.

Animals↗
PubMed1984

Mutations in Box B of the promoter of a eucaryotic tRNAPro gene affect rate of transcription, processing, and stability of the transcripts.

We have constructed a series of single and double base-pair-substitution mutants in and around the second component (Box B) of the promoter of a tRNAPro gene from C. elegans. Their analysis in in vivo and in vitro transcriptional systems establishes the importance of single nucleotides in the promotion of transcription. Most mutants in the region coding for the TpsiCG stem-loop show a reduced gene expression associated with lack of processing of the primary transcriptional products; in the oocytes these are rapidly degraded, with a half-life considerably shorter than that of wild-type tRNA molecules. In contrast, mutations in the DNA region coding for the anticodon stem-loop do not alter the efficiency of transcription or the processing of the transcripts.

Animals↗
PubMed1983

A general method to select for M13 clones carrying base pair substitution mutants constructed in vitro.

In this paper we describe a method to select base pair substitution mutants constructed in vitro. The mutagenesis is performed by forcing mistakes during in vitro synthesis from a primer annealed to a single stranded DNA template. The selection is based on the fact that, following transformation, the progeny of the strand elongated in vitro and the template strand have different phenotypes. The method is general and applicable to any DNA segment; the type of base pair substitution and its position can be chosen at will. The combined efficiency of mutagenesis and selection allows for 85% frequency of mutants in all analyzed clones.

Animals↗
PubMed1983

Isolation and characterization of a tRNA(guanine-7-)-methyltransferase from Salmonella typhimurium.

The tRNA modifying enzyme, S-adenosylmethionine:tRNA(guanine-7-)-methyltransferase, has been extensively purified from Salmonella typhimurium. A rapid and efficient purification method using phosphocellulose chromatography followed by ammonium sulfate precipitation and Sephadex G-100 gel filtration is described. The enzyme appears to be a single polypeptide chain with a molecular weight of approximately 25 000--30 000 daltons. The Km for S-adenosylmethionine and for undermethylated tRNA is 53 microM and 3.4 microM, respectively. The methylation reaction is dependent on added monovalent or divalent cations; 5 mM spermidine, 3 mM MgCl2 and 1 mM spermine are the most effective. The enzyme, though not homogeneous, is free from contaminating ribonucleases and other tRNA methyltransferases.

Chromatography, Gel↗
PubMed1983

Common and interchangeable elements in the promoters of genes transcribed by RNA polymerase iii.

We have shown that the 34 bp internal control region of the somatic 5S RNA gene from Xenopus borealis can be split into two separable components. A maxigene carrying an insertion between nucleotide 71 and nucleotide 74 of the coding region is actively transcribed in the nucleus of X. laevis oocytes, giving rise to a maxitranscript with initiation and termination points identical with those of the wild-type transcript. The first 11 bases of the 5S RNA gene promoter are shown to be structurally and functionally homologous with the first component (box A) of the promoter for tRNA genes. This was shown by constructing hybrid 5S RNA-tRNAPro and tRNAPro-5S RNA genes that were efficiently transcribed in the X. laevis oocytes. Initiation of transcription appears to be a complex phenomenon in which both components of the internal promoter play a role.

Animals↗