Biomedical subjects

S Volinia

Publications and source records attributed to S Volinia.

At least 37 records · Page 2Linked to original sources

PubMed1996

YAC clone H10 discriminates between 3q26.2 and 3q27 chromosome rearrangements in hematological disorders.

To discriminate with molecular-cytogenetic resolution between 3q26.2 breakpoint, associated to various myeloproliferative disorders, and 3q27 breakpoint, recurrent in several types of non-Hodgkin lymphoma, we tested the feasibility of using a yeast artificial chromosome, YAC clone H10, mapped on 3q26.3. Fluorescent in situ hybridization of the biotinylated polymerase chain reaction product of the YAC H10 was performed in three myeloproliferative diseases and one follicular non-Hodgkin lymphoma carrying different rearrangements of chromosome 3 involving region q26-q27. Our study shows that YAC H10 signal was telomeric to all three myeloid breakpoints, while it was centromeric in the lymphoid one thus showing that this probe can discriminate between these two subsets of chromosome 3 rearrangements. These results point out the opportunity of using additional YACs in the characterization of polymorphic chromosome alterations acquired in neoplastic cells.

Aged↗

PubMed1995

Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

Amino Acid Sequence↗

PubMed1995

A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system.

Phosphoinositide (PI) 3-kinases have been characterized as enzymes involved in receptor signal transduction in mammalian cells and in a complex which mediates protein trafficking in yeast. PI 3-kinases linked to receptors with intrinsic or associated tyrosine kinase activity are heterodimeric proteins, consisting of p85 adaptor and p110 catalytic subunits, which can generate the 3-phosphorylated forms of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 as potential second messengers. Yeast Vps34p kinase, however, has a substrate specificity restricted to PtdIns and is a PtdIns 3-kinase. Here the molecular characterization of a new human PtdIns 3-kinase with extensive sequence homology to Vps34p is described. PtdIns 3-kinase does not associate with p85 and phosphorylates PtdIns, but not PtdIns4P or PtdIns(4,5)P2. In vivo PtdIns 3-kinase is in a complex with a cellular protein of 150 kDa, as detected by immunoprecipitation from human cells. Protein sequence analysis and cDNA cloning show that this 150 kDa protein is highly homologous to Vps15p, a 160 kDa protein serine/threonine kinase associated with yeast Vps34p. These results suggest that the major components of the yeast Vps intracellular trafficking complex are conserved in humans.

Amino Acid Sequence↗

PubMed1995

The usage of oligopeptides in proteins correlates negatively with molecular weight.

We scanned the Swissprot databank to study the distribution of oligopeptides in sequenced proteins. We observed that in 7839,000 amino acids in the bank, the frequency is strongly and negatively correlated with molecular weight. The correlation coefficient is -0.63 for single amino acids, -0.58 for dipeptides, -0.54 for tri- and -0.46 for tetrapeptides. In subsets of proteins of man, mouse, rat drosophila, yeast and E. coli, the correlations were very similar to those for the total database. The intensity of the correlation diminishes linearly with the length of the peptide. We attributed the loss of dominance of molecular weight in determining the frequency of oligopeptides of higher order, to the emergence of function in longer oligopeptides. In the case of dipeptides, we observed that those composed of the same amino acid are in great excess in comparison with their expectation. The WW dipeptide is singular, in the sense that is observed/expected ratio is 13.5 standard deviations above the average ratio.

Amino Acids↗

PubMed1994

Characterization of a phosphatidylinositol-specific phosphoinositide 3-kinase from mammalian cells.

BACKGROUND: As phosphoinositides can serve as signalling molecules within cells, the enzymes responsible for their synthesis and cleavage are likely to be involved in the transduction of signals from the cell surface through the cytoplasm. The precise role of the phosphoinositide 3-kinase that has been cloned from mammalian cells is not known, but it has been implicated in receptor-stimulated mitogenesis, glucose uptake and membrane ruffling. The enzyme can use phosphatidylinositol (PtdIns), PtdIns 4-phosphate and PtdIns (4,5)-bisphosphate as substrates in vitro, but it seems to phosphorylate PtdIns (4,5)-bisphosphate preferentially in vivo. The VPS34 gene product of yeast, by contrast, is a phosphoinositide 3-kinase homologue implicated in vacuolar protein sorting that apparently utilizes only PtdIns as a substrate. The significance of this difference in lipid-substrate preference and its relationship to the functions of the two phosphoinositide kinases is unknown. RESULTS: We have characterized a distinct PtdIns-specific phosphoinositide 3-kinase activity in mammalian cells. Unlike the previously identified, broad-specificity mammalian phosphoinositide kinase, this enzyme is resistant to the drug wortmannin and uses only PtdIns as a substrate in vitro; it therefore has the capacity to generate PtdIns 3-phosphate specifically. The newly characterized enzyme, which was purified by chromatography from cytosol, has biochemical and pharmacological characteristics distinct from those of the broad-specificity enzyme. CONCLUSIONS: The enzyme we have characterized may serve to generate PtdIns 3-phosphate for fundamentally different roles in the cell from those of PtdIns (3,4)-bisphosphate and/or PtdIns (3,4,5)-trisphosphate. Furthermore, the functions of the VSP34 gene product, which may not be relevant to the broad-specificity mammalian phosphoinositide 3-kinase, may be related to those of the enzyme we describe.

Amino Acid Sequence↗

PubMed1994

Molecular cloning, cDNA sequence, and chromosomal localization of the human phosphatidylinositol 3-kinase p110 alpha (PIK3CA) gene.

Phosphatidylinositol (PI) 3-kinase is a heterodimeric enzyme comprising a 110-kDa catalytic subunit and an 85-kDa regulatory subunit that binds to tyrosine phosphopeptide sites linked directly or indirectly to receptors serving diverse signal functions. Knowledge of the structure and function of PI 3-kinase was greatly advanced by the purification, cDNA cloning, and subsequent expression of the bovine enzyme. Here the cloning of the cDNA for the human p110 alpha subunit of PI 3-kinase (PIK3CA), encoding a protein 99% identical to the bovine p110, and of its gene in YAC is described. The chromosomal localization of the gene for PIK3CA is shown to be at 3q21-qter as determined using somatic cell hybrids. In situ hybridization performed using Alu-PCR from the YAC DNA located the gene in 3q26.3.

Animals↗

PubMed1994

Identification of a set of frequent decanucleotides in plants and in animals.

We studied the frequency distribution of 1,048,576 oligonucleotides 10 bp long in a sample of 1.961 Mbase of genes from plants, made of 635 sequences extracted from GenBank 71.0, with the aim of detecting transcription control signals. Among all decamers, 3255, or 0.3%, had a frequency 10 times higher than the mean and were subjected to further statistical analysis. For each of the 3255 decamers (parents), we counted the individual frequencies of the 30 decamers (progeny) differing from the parent by one base mutation, and calculated two variance/mean chi-squares for the progeny, with and without the parent decamer. By studying the distribution of the ratio between the two chi-squares we observed that out of 3255 decamers > 10 times frequent than average, 432 had a chi-square ratio > 1.9. In this residual set, which corresponds to < 0.04 per cent of all possible decamers, only 15 known eukaryotic transcription control elements were found; on the other hand, it included 29 decanucleotides that matched with decanucleotides of a set of Drosophila, 24 with a set from mammals, 13 with a set from yeast and four with a set of viruses--all sets identified with the statistical procedures here described. These decanucloetides are highly repetitive and seem to be present throughout all higher organisms, whereas they are uncommon in mammalian viruses.

Animals↗

PubMed1994

A set of Alu-free frequent decamers from mammalian genomes enriched in transcription factor signals.

We have recently reported that the statistical analysis of the frequency distribution of short oligonucleotides within mammalian and viral genomes allows the production of sets of DNA sequences enriched in signals for transcription factors. Such statistical approaches could facilitate the identification of new promoter regions playing a role in the transcriptional regulation of gene expression. In the case of mammalian oligonucleotides, we found that the published set of frequent decamers enriched in transcriptional motifs is not suitable for studies on genes of Homo sapiens and evolutionarily related genomes, because it contains decameric sequences belonging to genomic repeats. We report here that most of the decameric sequences of DNA repeats belong to Alu repeats. Accordingly, we produced a subset of Alu-free frequent decamers. In addition, we eliminated from the subset of Alu-free frequent decamers those that are frequently present within other common human repeats, including (GT)n, (AT)n, (CA)n, (ATT)n, (CAA)n and (GTT)n. The Alu-free (repeats-free) subset of frequent mammalian decamers is enriched in signals for transcription factors and allows the identification of putative signals in genes, such as those coding for plasminogen activator, adenosine deaminase and p53, that contain a large number of Alu-like repeats interspersed within our genomic sequences. The newly generated compilation of frequent decamers described here might be used to locate genomic regions playing functional roles in the expression of genes of Homo sapiens and related primates.

Animals↗

PubMed1993

Enrichment of oligonucleotide sets with transcription control signals. III: DNA from non-mammalian vertebrates.

We studied the frequency distribution of 1,048,576 oligonucleotides 10 bp long in a sample of 1.072 x 10(6) bases of genes from non-mammalian vertebrates, made of 322 sequences extracted from EMBL(R) 29.0, with the aim of detecting transcription control signals. Among all decamers, 2097 (0.2%) had a frequency 10 times higher than the mean and were subjected to further statistical analysis. For each of the 2097 decamers (parents), we counted the individual frequencies of the 30 decamers differing from the parent by one base mutation (progeny) and we calculated two variance/mean chi squares for the progeny, with and without the parent decamer. By studying the distribution of the ratio between the two chi squares we observed that out of 2097 decamers that occurred > 10 times more frequently than average, 1017 had a chi square ratio of between 1 and 1.5; in this final set, which corresponds to < 0.097% of all possible decamers, 75 decamers were found to contain 100 transcription control elements, like CCAAT and others. The final set contains a high excess of signals when compared to 100 random sets of 1017 decamers. Some of the decamers selected with the procedure are members of consensus sequences rather than unique sequences.

Algorithms↗

PubMed1992

Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit.

Purified bovine brain phosphatidylinositol 3-kinase (Pl3-kinase) is composed of 85 kd and 110 kd subunits. The 85 kd subunit (p85 alpha) lacks Pl3-kinase activity and acts as an adaptor, coupling the 110 kd subunit (p110) to activated protein tyrosine kinases. Here the characterization of the p110 subunit is presented. cDNA cloning reveals p110 to be a 1068 aa protein related to Vps34p, a S. cerevisiae protein involved in the sorting of proteins to the vacuole. p110 expressed in insect cells possesses Pl3-kinase activity and associates with p85 alpha into an active p85 alpha-p110 complex that binds the activated colony-stimulating factor 1 receptor. p110 expressed in COS-1 cells is catalytically active only when complexed with p85 alpha.

Amino Acid Sequence↗

PubMed1992

Enrichment of oligonucleotide sets with transcription control signals. II: Mammalian DNA.

We studied the frequency distribution of oligonucleotides 10 bp long in a sample of 1.6 Mb of mammalian genes, containing 579 sequences from GenBank(R) 55.0, with the aim of detecting transcription control signals. 2216 decamers had a frequency higher than 10 times the mean and were subjected to further statistical analysis. For each of the 2216 decamers (parents), we counted the individual frequencies of the 30 decamers differing from the parent by one base mutation (progeny) and then calculated two variance/mean chi squares for the progeny, with and without the parent. We then studied the distribution of the ratio between the two chi squares. Out of 2216 decamers, 346 had a chi square ratio of 1.9 or larger. In this final set, which corresponds to less than 0.033 per cent of all possible decamers, 18 were found to contain 23 eukaryotic transcription control elements 5-10 bp of length, such as Sp1 and others. Furthermore, when compared to 210 random sets containing 346 decamers, this set contains a highly significant excess of the longer signals.

Algorithms↗

PubMed1992

Chromosomal localization of human p85 alpha, a subunit of phosphatidylinositol 3-kinase, and its homologue p85 beta.

The human phosphatidylinositol (PI) 3-kinase p85 alpha subunit gene and its homologue p85 beta were assigned to human chromosomes by analysis of their segregation in a panel of somatic cell hybrids using human-specific polymerase chain reaction primers. The p85 alpha locus was only present in hybrids retaining the human chromosome 5q. The presence of the p85 beta locus coincided with the presence of chromosome 19. The precise chromosomal sublocalization of these two genes was then determined by in situ hybridization. We confirmed the localization of the p85 alpha gene at 5q12-q13, as recently described (Cannizzaro, L.A., Skolnik, E.Y., Margolis, B., Croce, C.M., Schlesinger, J. & Huebner, K. (1991). Cancer Res., 51, 3818-3820) and positioned the p85 beta locus at 19q13.2-q13.4.

Base Sequence↗

PubMed1992

Simplified construction and characterization of yeast artificial chromosome libraries.

Three yeast artificial chromosome (YAC) libraries were constructed using two human cell lines and the pYAC-RC vector. The main differences from the previously described methods were: i) genomic DNA was digested in low melting point (LMP) agarose blocks with the rare cutting enzyme ClaI; ii) DNA was ligated in melted LMP agarose after agarase treatment; iii) spheroplast regeneration plating was done in calcium alginate thin layer. In addition, a panel of PCR primers was used to identify quickly the presence in the libraries of repetitive and single copy human DNA sequences.

Base Sequence↗

PubMed1991

A set of viral DNA decamers enriched in transcription control signals.

We studied the frequency distribution of oligonucleotides 10 bp long in a sample of 620 Kb of viral genomes, containing 102 sequences from GenBank, with the aim of detecting transcription control signals. Two thousand three hundred decamers had a frequency 10 times higher than the mean and were subjected to further statistical analysis. For each of the 2300 decamers (parents), we counted the individual frequencies of the 30 decamers differing from the parent by one base mutation (progeny) and then calculated two variance/mean chi squares for the progeny, with and without the parent. We then studied the distribution of the ratio between the two chi squares. Out of 2300 decamers, 10 times more frequent than average, 479 decamers had a chi square ratio of 1.9 or larger. In this final set, which corresponds to less than 0.05% of all possible decamers, 58 decamers were found to contain viral and eukaryotic transcription control elements, like NF-kB, Sp1 and others. Furthermore, this set contains an excess of signals of length 5, 6, 7, 8, 9 and 10, when compared to 150 random sets, bootstrapped from the same viral genomes.

Algorithms↗

PubMed1991

Characterization of the pseudogenic and genic homologous regions of von Willebrand factor.

The homologous pseudogenic and genic regions of von Willebrand factor (vWF) were studied in DNA from a patient with homozygous deletion of vWF genes and compared with a normal control. This analysis indicates informative restriction patterns for the investigation of restriction fragment length polymorphisms (RFLPs) and gene lesions, and for molecular cloning. A useful new genic XbaI RFLP was found and characterized. A large BgIII fragment of the pseudogenic region was cloned and mapped, and single sequences (9 kb) were used as probes. Corresponding genic and pseudogenic fragments, which contain exons 23-28, and specific restriction patterns were identified, including a new polymorphic TaqI site that was mapped in the gene. A cloned fragment contains the 5' boundary of the pseudogene and recognizes an additional and unknown homologous sequence in the genome. The chromosomal localization of the vWF pseudogene and of the breakpoint cluster region (BCR) gene were compared by 'in situ' hybridization: overlapping patterns were detected. The cloning, characterization and mapping of the pseudogenic region improves the analysis of this portion of chromosome 22 affected by several somatic and constitutional alterations, and also of the corresponding genic region on chromosome 12.

Base Sequence↗

PubMed1990

Human HLA-DR alpha gene: a rare oligonucleotide (GTATA) identifies an upstream sequence required for nuclear protein binding.

Synthetic oligonucleotides containing putative regulatory sequences are currently employed to identify and isolate genes coding for nuclear binding factors. Upstream DNA sequences of eukaryotic genes required for transcriptional activity and tissue specificity can be identified by means of biochemical techniques as well as computer analysis using homology searching. An alternative approach has been recently proposed by our research group. Scanning DNA sequences 1.8 megabases in length from a Genetic Sequence Data Bank, we have identified rare oligonucleotides 5 base pairs (bp) long, which are localized within or close to regulatory segments in mammalian promoters. In this paper we demonstrate that the rare GTATA sequence identifies an upstream region of the HLA-DR alpha gene which operates in conjunction with the sequence AGAAGTCAG, homologous to a box found in many interferon-inducible genes, in binding nuclear proteins.

Base Sequence↗

PubMed1990

Oligonucleotide correlations between infector and host genomes hint at evolutionary relationships.

The frequencies of oligonucleotides of length 3-6 were studied in 211 sequences of human DNA (659 kilobases), 22 sequences of DNA of human viruses (120 kbs), in 181 sequences of E. coli (442 kbs), and in 42 sequences of phages of E. coli (137 kbs). The sequences were obtained from Genbank(R) 48. The observed frequencies (O) were compared to the expected frequencies (E) obtained in two ways: 1) according to nucleotide composition for each series, and 2) according to first order Markow chains for triplets, second order for quadruplets, and third order for quintuplets and sextuplets. The ratio O/E was obtained for each oligonucleotide. Then, the correlation between the ratio O/E in a pair of series was calculated. Strong correlations were observed for sequences of man and human viruses, and for E. coli and its phages. Other correlations were small. For higher order Markov chains, there is indication of some correlation also between viruses and phages. It was concluded that through analysis of parallel oligonucleotide series it may be possible to infer some of the complex evolutionary relationships existing between cells and their infectors beyond the level of codon usage.

Base Composition↗

PubMed1990

Direct detection of a missense mutation causing severe hemophilia A by PCR amplification and fluorescence scanning.

The amplification of Factor VIII gene-specific sequences, obtained by polymerase chain reaction, was used for hemophilia A carrier detection. Exon 24 sequences were employed in the carrier status determination of a missense mutation causing severe hemophilia A in two unrelated patients. After agarose gel electrophoresis, the digested DNA was subjected to quantitative determination of fluorescence. This technique significantly improves the digest analysis.

Base Sequence↗