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R Cortese

Publications and source records attributed to R Cortese.

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

The human alpha-1-antitrypsin gene is efficiently expressed from two tissue-specific promotors in transgenic mice.

Alpha 1-antitrypsin (alpha 1AT) present in large amounts in human serum and synthesized predominantly in hepatocytes, is the most abundant protease inhibitor and alpha 1AT mutant proteins are associated with hereditary disorders. To investigate the regulation of the normal human alpha 1AT gene, we have microinjected fertilized mouse eggs with a 17.5 kb DNA fragment containing the entire gene with 7 kb 5' and 0.3 kb 3' flanking sequences. We show that this DNA fragment contains all the information for efficient, accurate and tissue-specific expression. High serum concentration of the human protein was found in three independent transgenic mouse lines. The human alpha 1AT RNA is transcribed efficiently in liver, kidney and macrophages and we demonstrate that two different promoters are used for the expression in liver and macrophages of the transgenic mice.

Animals↗
PubMed1987

The human haptoglobin gene: transcriptional regulation during development and acute phase induction.

Haptoglobin is a plasma protein scarcely present in fetal but abundant in adult serum, where it is present at a concentration of approximately 150 mg/100 ml. In this paper we show by run-on experiments that the haptoglobin (Hp) gene is actively transcribed in adult but not in fetal liver nuclei. Studies with established cell lines indicate that the Hp gene is expressed in the hepatoma cells HepG2 but not in the hepatoma cell line Hep3B nor in HeLa cells. Plasmids carrying various segments of the 5' flanking region of the Hp gene fused to the chloramphenicol acetyl transferase (CAT) gene direct CAT transcription when introduced into HepG2 but are inactive in Hep3B and in HeLa cells, thus behaving like the resident chromosomal Hp gene. Deletion analysis defines a region, upstream to the transcription initiation site, essential for cell-specific expression. The Hp gene is induced in Hep3B cells by treatment with supernatant from LPS-stimulated monocytes (SMS), in a manner mimicking the acute phase reaction. We characterize the DNA segment necessary and sufficient for cell-specific expression of the Hp-CAT constructions in HepG2 and show that the same segment is also sufficient for acute phase induction in Hep3B.

Adult↗
PubMed1987

Structure and expression of the genes coding for human alpha 1-acid glycoprotein.

alpha 1-acid glycoprotein (alpha AGP) is a well-characterized human plasma protein. Its structural properties have been studied for many years but little is known about its function. Amino acid sequence analysis of purified human alpha AGP from plasma pooled from several individuals showed considerable heterogeneity. We have cloned the genomic DNA segment encoding alpha AGP and we show that it contains three adjacent alpha AGP coding regions, AGP-A, B and B', identical in exon--intron organization but with slightly different coding potential. These results account for the heterogeneity observed by protein sequencing. Southern blot analysis indicates that the cloned cluster contains all the alpha AGP coding sequences present in the human genome. The larger majority of alpha AGP mRNA in human liver is transcribed from AGP-A, whose promoter and cap site have been determined while the level of AGP-B and B' mRNA in human liver is very low. Using Hep3B hepatoma cells as a model system for the in vitro study of the acute phase reaction, we show that only AGP-A is strongly induced by treatment with culture medium of LPS stimulated monocytes.

Amino Acid Sequence↗
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↗
PubMed1987

A recombinant apoA-1--protein A hybrid reproduces the binding parameters of HDL to its receptor.

We have constructed a plasmid, pLM8, containing the coding sequence of the mature human apoA-1 fused to the coding sequence of the IgG-binding domains of protein A (PA) from Staphylococcus aureus. The hybrid gene is transcribed in Escherichia coli under the control of a heat-sensitive repressor, leading to the synthesis of large amounts of hybrid protein (apoA-1--PA). The hybrid protein was purified by denaturation with urea and alkali, renaturation and affinity chromatography on an IgG Sepharose column. ApoA-1--PA is soluble and has an Mr of 316 kd, as determined by gel filtration. This is five times the monomer size of 62 kd, predicted from the sequence and found by SDS-PAGE analysis. Cell surface binding activity of the hybrid protein was tested using two different cell types (J774 macrophages and Fao hepatocytes) and compared to human high density lipoprotein (HDL). High-affinity binding was found for both ligands in both cell lines (Kd = 3.4 X 10(-8)M in Fao cells, 4.9 X 10(-8) M in J774 cells for apoA-1--PA and 3.0 X 10(-8) M in Fao cells, 2.8 X 10(-8) M in J774 cells for HDL), with approximately 2 X 10(5) high-affinity binding sites per cell. ApoA-1--PA and HDL effectively competed with each other for binding to the cell surface. Additionally, they both bound to a 110-kd polypeptide on a ligand blot, identifying an HDL receptor. The binding parameters of HDL were very similar to those of apoA-1--PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Negative control of liver-specific gene expression: cloned human retinol-binding protein gene is repressed in HeLa cells.

It has been found that 334 bases of the 5' flanking region of the human retinol-binding protein (RBP) gene contain sufficient information to direct accurate and specific transcription in human hepatoma but not in HeLa cells. Dissection of this region reveals the existence of at least three distinct controlling elements: an enhancer, which can activate promoters in a variety of cell lines and is therefore non-tissue specific; a negative cis-acting element, which apparently binds a repressor molecule not present or non-functional in hepatoma cell lines; a promoter element. Transcription is confined to hepatoma cells only when both the negative element and the promoter element are present.

Carcinoma, Hepatocellular↗
PubMed1987

Characterization of human ferritin H chain synthetized in Escherichia coli.

We have inserted the coding region of the cDNA for human ferritin H chain into the expression vector pEMBLex2. The plasmid obtained is able to direct the synthesis of the ferritin H chain in Escherichia coli up to a concentration of 15% of total soluble proteins. All expressed subunits are found correctly assembled in the complete ferritin molecule, which can be easily purified. We have shown that the ferritin synthesized in E. coli has an Mr, electrophoretic mobility, and thermal stability similar to natural human isoferritins and is recognized by monoclonal antibodies specific for the H, but not by those for the L human ferritin chains.

Cloning, Molecular↗
PubMed1987

Properties of a genetically engineered G domain of elongation factor Tu.

The G domain of elongation factor Tu (EF-Tu), representing the N-terminal half of the factor according to its three-dimensional model traced at high resolution, has been isolated by genetic manipulation of tufA and purified to homogeneity. The G domain, whose primary structure shares homology with the eukaryotic protein p21, is capable of supporting the basic activities of the intact molecule (guanine nucleotide binding in 1:1 molar ratio and GTPase activity). However, it is no longer exposed to the allosteric mechanisms regulating EF-Tu. The G-domain complexes with GTP and GDP display similar K'd values in the microM range, in contrast to EF-Tu that binds GDP much more tightly than GTP. Its GTPase shows the characteristics of a slow turnover reaction (0.1 mmol X sec-1 X mol-1 of G domain), whose rate closely corresponds to the initial hydrolysis rate of EF-Tu X GTP in the absence of effectors and lies in the typical range of GTPase of the p21 protein. Of the EF-Tu ligands only the ribosome displays a clear effect enhancing the G-domain GTPase. Our results suggest that the middle and C-terminal domain play an essential role in regulating the activity of the N-terminal domain of the intact molecule as well as in the interactions of EF-Tu with aminoacylated tRNA, elongation factor Ts, and kirromycin. With the isolation of the G domain of EF-Tu, a model protein has been constructed for studying and comparing common characteristics of the guanine nucleotide-binding proteins.

Binding Sites↗
PubMed1986

Cloning of the gene coding for human L apoferritin.

A recently reported cDNA clone coding for human promyelocytic L apoferritin, shows some differences with a liver L apoferritin cDNA. We have investigated if these differences are due to the expression of different genes or to an alternative transcription of an unique gene. In this paper we report data suggesting that a single gene is mainly expressed in several tissues examined. This gene has been cloned and characterized. Its sequence shows three introns: the exon sequence is identical to that of cDNA clone isolated from human liver. A minimum of five related pseudogenes have been also analysed. One of them is a processed pseudogene interrupted by an intron-like fragment.

Amino Acid Sequence↗
PubMed1986

Structure of gene and pseudogenes of human apoferritin H.

Ferritin is composed of two subunits, H and L. cDNA's coding for these proteins from human liver (1,2,3), lymphocytes (4) and from the monocyte-like cell line U937 (5) have been cloned and sequenced. Southern blot analysis on total human DNA reveals that there are many DNA segments hybridizing to the apoferritin H and L cDNA probes (1,2,4,6). In view of the tissue heterogeneity of ferritin molecules (7,8), it appeared possible that apoferritin molecules could be coded by a family of genes differentially expressed in various tissues (1,2). In this paper we describe the cloning and sequencing of the gene coding for human apoferritin H. This gene has three introns; the exon sequence is identical to that of cDNA's isolated from human liver, lymphocytes, HeLa cells and endothelial cells. In addition we show that at least 15 intronless pseudogenes exist, with features suggesting that they were originated by reverse transcription and insertion. On the basis of these results we conclude that only one gene is responsible for the synthesis of the majority of apoferritin H mRNA in various tissues examined, and that probably all the other DNA segments hybridizing with apoferritin cDNA are pseudogenes.

Apoferritins↗
PubMed1985

Cloning and sequencing of a full length cDNA corresponding to human cellular retinol-binding protein.

We have isolated and sequenced a cDNA clone corresponding to the human cellular retinol-binding protein (CRBP). The deduced amino acid sequence, which encompasses 134 amino acid residues, shows significant homology with several low molecular weight proteins which bind hydrophobic ligands. No homology to the plasma retinol-binding protein was observed. Southern and Northern blot analyses suggest that the CRBP gene is present in a single copy in the haploid genome and that it is transcribed in a single mRNA species.

Amino Acid Sequence↗
PubMed1985

The primary structure of human hemopexin deduced from cDNA sequence: evidence for internal, repeating homology.

We have cloned and analyzed a cDNA containing the coding sequence for human hemopexin. We have first identified, by immunological screening of 30.000 colonies of a liver cDNA library in the expression vector pEX1, a clone carrying an insert 1170 base pairs long that shows 100% homology with a known human hemopexin peptide. The complete sequence coding for hemopexin was isolated from a liver cDNA library in the vector pAT218. The DNA insert of 1523 base pairs shows an open reading frame coding for 439 amino acids, a 3' noncoding region of 159 nucleotides long, followed by a poly(A) tail. The insert spans the entire coding region and from which the primary structure of the protein was deduced. By computer assisted analysis of the amino acid sequence, it was possible to recognize a core unit, of about 45 amino acids, which is repeated 8 or possibly even 10 fold along the polypeptide chain. This feature suggests that the gene might have evolved through a series of duplications. This characteristic, together with prediction of secondary structure, suggest a rough model for the tridimensional folding that allows some speculations on the function of hemopexin. Blot hybridization of total RNA from human liver with nick translated hemopexin cDNA detected a message of about 1600 nucleotides. Southern blot experiments to identify the hemopexin gene (s) suggest that it is not a large multi-gene family, but that there is only one or at most a few genes in the human genome.

Amino Acid Sequence↗
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

Structure and expression of the human haptoglobin locus.

Human genomic clones of the haptoglobin Hp1F and the "haptoglobin related' gene (Hpr) have been isolated. The two genes are adjacent, spanning a region of approximately 21 kb. A comparison of their coding sequences shows that Hpr differs from Hp1F at 28 codons. Northern blot and primer elongation analyses with human liver RNA show that the haptoglobin gene Hp1F appears to be transcribed some 1000-fold less in fetal than in adult liver. In adult liver the amount of Hpr mRNA is at the lower limit of detection, therefore the extent of its expression is at most less than 1000-fold that of the Hp1F gene. No Hpr mRNA can be detected in fetal liver.

Base Sequence↗