Search PubMed⌕ Search

Biomedical subjects

V Sgaramella

Publications and source records attributed to V Sgaramella.

At least 19 recordsLinked to original sources
PubMed2001

A model for the involvement of Okazaki fragments maturation in the expansion of short tandem repeats.

We propose a model for the expansion of short tandem repeats (ESTR), a phenomenon which has been found to occur in human DNA and is associated with a dozen of neuromuscular diseases. The model is based mainly on theoretical considerations and recovers experimental data from the literature; it also finds support in preliminary results obtained by us in multiprimed polymerase chain reactions designed to assess the effects of a downstream primer on the fidelity of the elongation of an upstream one. The model links the occurrence of the ESTR to a defective maturation of the Okazaki fragments (OF), and in particular to an improper processing of their 3' termini. This may occur when the last OF approaches the 5' terminus of the previous one in a susceptible region of the template. It is postulated here that when a growing OF has progressed past the priming region and its main portion has been synthesized, upon approaching its conclusion, the final elongation may take place in a region of the template where certain triplets are repeated: in that case a series of aberrations on the elongation mechanism may occur. These aberrations could involve (a) the displacement of the 5' terminus of the penultimate, properly matured OF, enacted by the incoming 3' terminus of the last OF, (b) the switch of the latter to the displaced strand of the former as template, (c) the fold-back on itself of the growing 3' terminus of the last OF, (d) its assumption of an unusual structure because of the repetition, and (e) some impairment of its removal by structure-specific exo-endonuclease(s). Derangements of this last part of the process may trigger the ESTR.

Base Sequence↗
PubMed1998

A procedure for cloning genomic DNA fragments with increasing thermoresistance.

Genomic DNAs have been cleaved by restriction or sonication, and the resulting double-stranded fragments have been exposed to increasing temperatures. This treatment may induce the helix-coil transition either in a single or in several steps, depending on the size and composition of the duplexes. Eventually, a critical temperature is reached at which each duplex melts completely and the two constitutive single strands separate. A transition interval can thus be defined for each duplex by the temperature at which the earliest strand separation takes place and that at which the most resistant double-stranded core collapses. If solutions containing a mixture of DNA duplexes are exposed to temperatures within their transition intervals, three kinds of molecules should originate: (1) duplexes that have not yet initiated the melting phase; (2) duplexes that have undergone only partial melting; and (3) single strands that derive from fully melted duplexes. If the heated solutions are quickly cooled to 0 degreesC, only the molecules from the first two classes can be ligated to a compatibly ended vector and cloned: class (1) are intact duplexes, and class (2) are molecules that snap immediately back to fully duplex structures: both are double-stranded. Conversely, the single strands of class (3) may not reanneal and thus be neither ligated nor cloned. We have tested the procedure on restricted coliphage lambda DNA, in view of its compartmentalized organization and known sequence. Then, we have applied it to human genomic DNA fragmented by sonication. After cloning of the available duplexes in a bacterial plasmid, libraries of molecules endowed with a progressively higher thermoresistance can be prepared for thermodynamic and genomic studies.

Bacteriophage lambda↗
PubMed1997

Isolation of a pancreas-specific gene located on human chromosome 14q31: expression analysis in human pancreatic ductal carcinomas.

We have isolated a novel cDNA (SEL1L) that shows sequence similarities to SEL-1, a gene identified as an extragenic suppressor of the lin-12 hypomorphic mutant from Caenorhabditis elegans (7, 8). SEL1L exhibits a tissue-specific pattern of expression: a single poly(A)+ RNA species of 7.5 kb is abundantly expressed only in the pancreas of healthy individuals, whereas low to undetectable levels are observed in other adult and in some fetal tissues. Somatic hybrid panel and fluorescence in situ hybridization positioned this gene in the q31 band of human chromosome 14. The tissue-specific expression of this gene induced us to study its role in human pancreatic carcinomas. Our analysis revealed that 17% of adenocarcinomas of the pancreas did not express SEL1L to a detectable level; however, no gross genomic alterations were apparent in the few hundred kilobases of the relevant region.

Adult↗
PubMed1997

Functional characterization of a human DNase-like protein encoded by a gene positioned in Xq28.

Xib, a gene recently reported to reside on the q28 region of the human X chromosome [Pergolizzi et al. (1996) Gene 168, 267-270], contains an open reading frame homologous to those of the DNase I family enzymes. The full open reading frame of this gene has been fused to the E. coli gene of the maltose binding protein and expressed in bacteria as a chimeric protein. The partially purified chimeric protein is enzymatically active. It introduces single and double stranded breaks into supercoiled DNA, at 30 degrees C in the absence of divalent cations and at a pH optimum of 5.2. To our knowledge this enzyme represents the first cloned human endonuclease with characteristics similar to those of acidic DNase II.

Cloning, Molecular↗
PubMed1996

Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops.

In PCR, DNA polymerases from thermophilic bacteria catalyze the extension of primers annealed to templates as well as the structure-specific cleavage of the products of primer extension. Here we show that cleavage by Thermus aquaticus and Thermus thermophilus DNA polymerases can be precise and substantial: it occurs at the base of the stem-loop structure assumed by the single strand products of primer extension using as template a common genetic element, the promoter-operator of the Escherichia coli lactose operon, and may involve up to 30% of the products. The cleavage is independent of primer, template, and triphosphates, is dependent on substrate length and temperature, requires free ends and Mg2+, and is absent in DNA polymerases lacking the 5'-->3' exonuclease, such as the Stoffel fragment and the T7 DNA polymerase. Heterogeneity of the extension products results also from premature detachment of the enzyme approaching the 5' end of the template.

Base Sequence↗
PubMed1994

Italy's role in EMBL.

Explore the source record for details and available documents.

Academies and Institutes↗
PubMed1993

Lawyers' delights and geneticists' nightmares: at forty, the double helix shows some wrinkles.

The National Institutes of Health (NIH) request to patent the base sequences of incomplete and uncharacterized fragments of DNA copied on messenger RNAs (cDNAs) extracted from human tissues, the refusal by the patent office, and the appeal placed by NIH, have incited a violent controversy, fueled by rational, as well as emotional elements. In a compromising mode between liberalism and protectionism, I propose that legal protection be considered only for those RNA/DNA sequences, either natural or artificial, which can generate practical applications per se, and not through their expression products. Another controversy is developing around a popular tool for genomic research: the fidelity of yeast artificial chromosome (YAC) libraries being distributed worldwide for physical mapping is being questioned. Some of these libraries have been shown to be affected by surprisingly high levels of co-cloning, in addition to more common gene reshuffling instances. Also in this case, scientific as well as non-scientific components have to be considered. Possible remedies for the underlying problems may be found in the proper use of kinetic, enzymatic and microbiological variables in the production of YACs. Here too, a sharper distinction between the secular and scientific gratifications of research could help.

Chromosomes, Artificial, Yeast↗
PubMed1993

An ordered collection of Bacillus subtilis DNA segments cloned in yeast artificial chromosomes.

A collection of 772 Bacillus subtilis DNA segments was obtained by cloning in yeast artificial chromosomes. The B. subtilis inserts of 288 clones were mapped by hybridization using as probes 65 cloned genes and 188 isolated insert ends. In this way, 59 inserts were ordered in four contigs that cover > 98% of the B. subtilis chromosome. This ordered collection is now available for further genetic and physical analysis of the B. subtilis genome.

Bacillus subtilis↗
PubMed1991

Molecular cloning of DNA from a sorted human minichromosome.

A human supernumerary minichromosome (MC), found in a newborn baby and sorted on a fluorescence-activated cell sorter (FACS-440) has been previously described [Ferretti et al., Cytotechnology 1 (1987) 7-12]. We report here on the construction of a library of EcoRI fragments in the phage lambda gtWES.lambda B', starting from 7.5 ng of MC DNA, and describe the isolation of single-copy DNA clones from the library in a two-step procedure. We employed in situ hybridization to unambiguously select the clones specific for the MC, and used three of these clones to demonstrate that it originated from chromosome 9.

Chromosomes, Human, Pair 9↗
PubMed1991

The origin of a morphologically unidentifiable human supernumerary minichromosome traced through sorting, molecular cloning, and in situ hybridisation.

A supernumerary minichromosome has been detected in a severely malformed patient. Attempts at identifying the marker by conventional approaches were unsuccessful. The physical isolation of the minichromosome by fluorescence activated sorting, molecular cloning of its DNA, and in situ hybridisation experiments performed with single copy DNA probes allowed us to show that it was derived from a rearrangement involving the centromere and the proximal region of the short arm of chromosome 9.

Abnormalities, Multiple↗
PubMed1990

Long range restriction analysis of the bovine casein genes.

Pulsed field gel electrophoresis (PFGE) was used to analyse the organization of the bovine alpha s1, alpha s2, beta and kappa casein genes. High molecular weight DNA was prepared from fibroblasts and lymphocytes embedded in agarose and was digested with the restriction endonucleases Clal, Sall, Smal, Xhol. The digestion products were separated by PFGE, transfered to nitrocellulose filters and hybridized to probes corresponding to the cDNAs of the four bovine casein genes. The casein genes were demonstrated to be physically linked within a region of 300 kb, represented by two adjacent Xhol fragments in fibroblasts and by a single fragment in lymphocytes. A restriction map of the casein locus was derived and the order of the genes was shown to be kappa, alpha s2, beta, alpha s1.

Animals↗
PubMed1990

Restriction fragment length polymorphism analysis of the kappa-casein locus in cattle.

The two common genetic variants (A and B) of bovine kappa-casein originate from two point mutations in the codons for the aminoacids in position 136 and 148. These mutations give rise to polymorphic sites for the restriction endonucleases Hin dIII, AluI, HinfI, Mbo II and TaqI. We have examined DNAs of several Italian Friesian cows and bulls of known and unknown genotype by Southern analyses using kappa-casein cDNA probes. Restriction fragment length polymorphisms (RFLPs) specific for the A and B alleles were identified for each of the above enzymes, except for AluI, which has a non-polymorphic site 12bp away from the polymorphic one. We have also found two new polymorphic sites for MboII and TaqI in the non-coding regions. These sites differentiate the A allele into two new variants, named A1 and A2. The RFLP analysis permits the characterization of kappa-casein alleles even in the absence of their expression. This should facilitate selective breeding programmes aimed at increasing the frequency of the kappa-casein B allele whose product improves the cheesemaking properties of milk.

Amino Acid Sequence↗
PubMed1990

A procedure for cloning restriction fragments of DNA as single inserts in yeast artificial chromosomes.

A novel procedure is described for the cloning of partial EcoRI fragments of bovine DNA: it reduces the chance of sequence rearrangements due to multiple insertions (co-cloning) of restriction fragments in the resulting YAC. The DNA to be inserted has been dephosphorylated, whereas the matching ends of the vector, pYAC4, have not. The ligation was essentially complete, the transformation efficiency was close to 19 transformants per ng of vector and the frequency of clones carrying YAC, 60-100 kb in size, was close to 70%. The YACs show segregative and replicative stability.

Animals↗
PubMed1989

Cloning of histidine genes of Azospirillum brasilense: organization of the ABFH gene cluster and nucleotide sequence of the hisB gene.

A cluster of four Azospirillum brasilense histidine biosynthetic genes, hisA, hisB, hisF and hisH, was identified on a 4.5 kb DNA fragment and its organization studied by complementation analysis of Escherichia coli mutations and nucleotide sequence. The nucleotide sequence of a 1.3 kb fragment that complemented the E. coli hisB mutation was determined and an ORF of 624 nucleotides which can code for a protein of 207 amino acids was identified. A significant base sequence homology with the carboxy-terminal moiety of the E. coli hisB gene (0.53) and the Saccharomyces cerevisiae HIS3 gene (0.44), coding for an imidazole glycerolphosphate dehydratase activity was found. The amino acid sequence and composition, the hydropathic profile and the predicted secondary structures of the yeast, E. coli and A. brasilense proteins were compared. The significance of the data presented is discussed.

Amino Acid Sequence↗
PubMed1987

Structure-function studies on bacteriorhodopsin. II. Improved expression of the bacterio-opsin gene in Escherichia coli.

The aims of this work have been to express bacterio-opsin with minimal variation from the native primary structure and to improve the level of expression in Escherichia coli. We describe the construction of plasmids in which the bacterio-opsin gene contains only an additional methionine residue at the N terminus and in which the C-terminal aspartic acid encoded in the gene has been deleted to conform to the mature protein. In attempts to improve bacterio-opsin expression, a variety of expression plasmids were constructed in which the promoters and the ribosome-binding sequences were varied. Invariably, in these plasmids, translation but not transcription of the bacterio-opsin gene was limiting. A striking increase in expression of the gene occurred when the codons for several of the N-terminal amino acids were changed to increase the A = T content. Bacterio-opsin expressed in E. coli was degraded with a half-life of 8-10 min. The addition of hydrophobic signal sequences at the N terminus increased the half-life and overall yield of the protein. Bacterio-opsin thus produced regenerated the native bacteriorhodopsin-like chromophore and carried out light-dependent proton translocation at a rate comparable to that of the native bacterio-opsin prepared from the purple membrane.

Amino Acid Sequence↗