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L Dente

Publications and source records attributed to L Dente.

30 records · Page 2Linked to original sources
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

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

Mapping through somatic cell hybrids and cDNA probes of protein C to chromosome 2, factor X to chromosome 13, and alpha 1-acid glycoprotein to chromosome 9.

The previously unassigned gene coding for the anti-coagulatory protein C has been mapped on chromosome 2 using a cDNA probe and genomic blots from a human-hamster somatic cell hybrid panel. The assignments of the genes coding for the coagulation factor X to chromosome 13, and for alpha 1-acid glycoprotein to chromosome 9 have been confirmed using a similar direct approach.

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

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↗
PubMed1983

pEMBL: a new family of single stranded plasmids.

We have constructed a series of plasmids, the pEMBL family, characterized by the presence of 1) the bla gene as selectable marker, 2) a short segment coding for the alpha-peptide of beta-galactosidase and containing a multiple cloning sites polylinker, 3) the intragenic region of phage F1. pEMBL plasmids have the property of being encapsidated as single stranded DNA, upon superinfection with phage F1. These vectors have been used successfully for DNA sequencing with the dideoxy-method, and can be used for any other purpose for which M13 derivatives are used. However, the pEMBL plasmids have the advantage of being smaller than M13 vectors, and the purification of the DNA is simpler. In addition, and most importantly, long inserts have a higher stability in pEMBL plasmids than M13 vectors.

Base Sequence↗
PubMed1983

Cloning of several cDNA segments coding for human liver proteins.

A human cDNA library was constructed using M13 derivative vectors. The simple and rapid procedures for sequencing single-stranded DNA by the dideoxy chain termination method allowed a screening of individual clones directly by DNA sequence analysis. Some of these clones were identified as coding for: serum albumin, alpha1-antitrypsin, retinol-binding protein, prothrombin, haptoglobin, and metallothionein. Furthermore, a clone coding for aldolase B was tentatively identified on the basis of high sequence homology with rabbit muscle aldolase.

Amino Acid Sequence↗
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↗
PubMed1982

Pyruvate kinase deficiency: characterization of two new genetic variants.

Pyruvate kinase (PK) has been purified from the red blood cells of two sisters who had suffered severe chronic non-spherocytic haemolytic anaemia since infancy, and of one patient who had haemolytic anaemia during pregnancy. The two sisters showed remarkable clinical improvement following splenectomy. The enzyme from their red cells was found to exhibit low activity (about 25% of normal) in crude haemolysates, low affinity for the substrate, phosphoenol pyruvate (PEP), and high sensitivity to fructose-1,6-diphosphate (FDP) activation. This PK differs from previously reported variants and it is provisionally designated PK 'Torre Annunziata'. The enzyme from the other patient had near-normal activity in crude haemolysates, slight changes in kinetics with respect to the substrate, PEP, and with respect to the effects of FDP, ATP and pH, and a markedly reduced thermostability. This PK also differs from previously reported variants and it is provisionally designated PK 'Torre del Greco'. During the course of this study an improved method for purification of PK using Cibacron blue sepharose has been developed.

Adult↗
PubMed1982

A prokaryotic tRNATyr gene, inactive in Xenopus laevis oocytes, is activated by recombination with an eukaryotic tRNAPro gene.

Eukaryotic tDNA promoters are composed of two essential regions contained within the coding sequence (Box A and Box B). Due to the highly conserved structure of prokaryotic and eukaryotic tRNA, most prokaryotic tRNA genes are expected to be active templates in eukaryotic transcriptional systems. In this paper we show that Escherichia coli tDNATyr is not transcribed in the nucleus of Xenopus laevis oocytes. By in vitro construction of hybrid molecules between inactive prokaryotic tDNATyr from E. coli, and active eukaryotic tDNAPro from Caenorhabditis elegans, we show that tDNATyr can be made into an active gene if its first third, including the Box A region, is replaced by that of the eukaryotic tDNA . These results suggest that an improper Box A sequence is responsible for the inactivity of the E. coli tRNATyr gene, and argue against the role of secondary and tertiary DNA conformations in RNA polymerase III transcription.

Animals↗