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M Savino

Publications and source records attributed to M Savino.

At least 37 records · Page 2Linked to original sources
PubMed1998

DNA superstructural features and nucleosomal organization of the two centromeres of Kluyveromyces lactis chromosome 1 and Saccharomyces cerevisiae chromosome 6.

Superstructural features of the Kluyveromyces lactis chromosome 1 (KlCEN1) and of the Saccharomyces cerevisiae chromosome 6 (SCEN6) centromeric DNAs were evaluated using a theoretical method, developed by our group, and experimentally measured by gel electrophoretic retardation. Both methods show that, in spite of the remarkable AT richness of the two centromeric sequences, their curvature is not very high. However the peculiar sequence features of the two centromeres allow to organize highly stable nucleosomes, with a free energy about that of the nucleosome formed on the 5S RNA gene. The good agreement between experimental and theoretical evaluation of nucleosome free energies as well as of their multiple positioning shows that in centromeres both DNA curvature and flexibility are relevant in determining nucleosomal features.

Centromere↗
PubMed1998

Nucleosome assembly on telomeric sequences.

The organization of telomeric chromatin differs from that of bulk chromatin in some peculiar features, such as the unusually short nucleosomal spacing found in vertebrates. Telomeric DNAs are straight, since they consist mostly of 6-8-bp repeated sequences, therefore out of phase with the B DNA period. This feature should be of relevance in nucleosome formation, suggesting the usefulness of studying simple model systems of nucleosome assembly. We reconstituted nucleosomes in vitro, by using purified histone octamers and/or by octamer transfer from chicken erythrocyte nucleosomes, onto telomeric sequences from human, Arabidopsis thaliana, and Saccharomyces cerevisiae. All of these telomeres contain GGG and GGT triplets but are characterized by different repeat lengths (6, 7, and 8 bp). The free energies involved in the association process are the highest among the biological sequences so far assayed, suggesting a main role of DNA flexibility in the assembly of telomeric chromatin. Digestion studies with DNase I, hydroxyl radicals, exonuclease III, and lambda exonuclease indicate that telomeric nucleosomes are characterized by multiple translational positioning without rotational phasing, whereas the telomeric DNA folding around the histone octamer shows the canonical periodicity of about 10.2 bp. The experimental results and a theoretical simulation of DNase I digestion indicate a multiple nucleosome positioning with the periodicity of telomeric DNA. This suggests a main role of local chemical recognition between telomeric sequences and the histone octamer in nucleosome assembly.

Animals↗
PubMed1998

Mutational screening of thrombopoietin receptor gene (c-mpl) in patients with congenital thrombocytopenia and absent radii (TAR).

Thrombocytopenia with absent radii (TAR) is a rare autosomal recessive disease characterized by hypomegakaryocytic thrombocytopenia and bilateral radial aplasia. We performed mutational screening of coding and promoter regions of the c-mpl gene, encoding thrombopoietin (TPO) receptor, by sequence analysis in four unrelated patients affected by TAR syndrome. Our results indicate that c-mpl gene mutations are not a common cause of thrombocytopenia in TAR syndrome.

Adolescent↗
PubMed1997

Multiple nucleosome positioning with unique rotational phasing on multimers of the light-responsive elements of pea rbcS-3A and rbcS-3.6 genes: comparison between experimental and theoretical mapping.

Nucleosome positioning along two DNA tracts, corresponding to tetramers of the light-responsive elements of pea rbcS-3A and rbcS-3.6 genes, were studied by experimental (exonuclease III mapping and band shift electrophoresis) as well as theoretical methods. Multiple nucleosome positioning with unique rotational phase was derived from both methods in satisfactorily good agreement, if nucleosome dyad axis positions are considered. Theoretical and experimental distributions of nucleosome frequencies appear different, probably on account of DNA sequence dependent digestion kinetics of exonuclease III.

Base Sequence↗
PubMed1997

Different bindings of the minor groove ligands DAPI and Hoechst 33258 to multimers of the curved (CA4T4G) and noncurved (CT4A4G) DNA sequences.

Equilibrium binding properties of DAPI and Hoechst for prototype curved (CA4T4G)n and straight (CT4A4G)n DNAs were derived from fluorescence intensity. Both decamers display a single strong binding site for each ligand. Hoechst binds seven times stronger to curved than to straight DNA. An additional cooperative, much weaker binding site is found for DAPI.

Base Sequence↗
PubMed1997

The genomic organization of the Fanconi anemia group A (FAA) gene.

Fanconi anemia (FA) is a genetically heterogenous disease involving at least five genes on the basis of complementation analysis (FAA to FAE). The FAA gene has been recently isolated by two independent approaches, positional and functional cloning. In the present study we describe the genomic structure of the FAA gene. The gene contains 43 exons spanning approximately 80 kb as determined by the alignment of four cosmids and the fine localization of the first and the last exons in restriction fragments of these clones. Exons range from 34 to 188 bp. All but three of the splice sites were consistent with the ag-gt rule. We also describe three alternative splicing events in cDNA clones that result in the loss of exon 37, a 23-bp deletion at the 5' end of exon 41, and a GCAG insertion at the 3' portion also in exon 41. Sequence analysis of the 5' region upstream of the putative transcription start site showed no obvious TATA and CAAT boxes, but did show a GC-rich region, typical of housekeeping genes. Knowledge of the structure of the FAA gene will provide an invaluable resource for the discovery of mutations in the gene that accounts for about 60-66% of FA patients.

Alternative Splicing↗
PubMed1997

In vitro low propensity to form nucleosomes of four telomeric sequences.

The structural aspects of nucleosome assembly on telomeres are largely unknown. We analyzed by competitive reconstitution the affinities for the histone octamer of telomeric sequences from four different eukaryotic groups, Arabidopsis thaliana, mammals, Tetrahymena, and Saccharomyces cerevisiae. All telomeres reconstitute in nucleosomes with lower association constants than average nucleosomal DNA. DNase I digestion analysis suggests a multiple translational positioning and the lack of rotational positioning, probably due to telomeric repeats length (in most cases 6-8 bp), out of phase with the DNA helical repeat on the nucleosome (10.2 bp). These results could partly explain the lack of nucleosomes on lower eukaryote telomeres, and suggest a high in vivo mobility of telomeric nucleosomes.

Animals↗
PubMed1997

Linkage analysis of Fanconi anaemia in Italy and mapping of the complementation group A gene.

Fanconi anaemia (FA) is an autosomal recessive disease characterised by genetic heterogeneity, with at least five complementation groups (FA-A to FA-E). The FAC gene has been cloned and localised to 9q22.3. The most frequent defective gene, FAA, was recently mapped to chromosome 16q24.3, in a region of 10 cM between D16S498 and the telomere. Eleven FA-A and 16 unclassified Italian families were analysed by microsatellite markers. To define the localisation of the FAA locus further, microsatellites were analysed at 16q24. All the families were consistent with linkage, the highest lod score being observed with D16S1320. Evidence for common haplotypes was obtained in two genetic isolates from the Brenta basin and the Naples region. Autozygosity mapping and haplotype analysis suggest that the FAA locus is distal to D16S305.

Cell Cycle Proteins↗
PubMed1997

Prevalence of anxiety disorders comorbidity in bipolar depression, unipolar depression and dysthymia.

Eighty-seven patients with current episode of depression were assessed by the SCID-P and subdivided in bipolar depressives (N = 24), unipolar depressives (n = 38) and dysthymics (n = 25). Anxiety disorders comorbidity in these three groups was investigated by means of the SCID-P. Panic disorder comorbidity was found in 36.8% of bipolar depressives, 31.4% of unipolar depressives and 13% of dysthymics. Prevalence of obsessive-compulsive disorder was 21.1% in bipolars, 14.3% in unipolars and 8.7% in dysthymics. Generalized anxiety disorder resulted in being much more associated with dysthymia (65.2%) than with bipolar (31.6%) or unipolar depression (37.1%). Social phobia comorbidity was exhibited mainly by unipolars (11.4%), while no cases were detected in the bipolar group. Odds ratios revealed that generalized anxiety disorder is significantly more likely to co-occur with dysthymia. Panic disorder showed a higher trend to be associated with bipolar and unipolar depression. Social phobia was more frequent among unipolar depression.

Adult↗
PubMed1997

Molecular approach to the nucleo-melanosomal interaction in human melanoma cells.

This paper presents evidence that L-tyrosine oxidation products and 5,6-dihydroxyindole, an intermediate of melanin synthesis bind to and modify DNA structure, as tested by extracting cell DNA, using topoisomerase I and denaturation assays. When supercoiled plasmid pCU18 or pBR322 DNAs are treated with 5,6-dihydroxyindole the supercoiled species disappear and are converted to species less mobile in a gel retardation test with respect to relaxed DNA, 5,6-Dihydroxyindole causes an easier acid denaturation of the double helix. The results, that are dose dependent, would point to both intercalation and cross-linking of DNA by 5,6-dihydroxyindole and its oxidation product(s). 3H-L-tyrosine deriving radioactivity, bound to nuclear DNA, is higher at low pH, (5.6) if compared to pH 6.8. The highest radioactivity bound to cell DNA is found during the transition from the amelanotic to the melanotic phenotype in human melanoma cell lines. As a control, the binding of 3H-L-tyrosine radioactivity to human prostate fibroblast DNA was investigated.

Animals↗
PubMed1997

Mutations of the Fanconi anemia group A gene (FAA) in Italian patients.

Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive pancytopenia, congenital malformations, and predisposition to acute myeloid leukemia. At least five complementation groups (FA-A-FA-E) have been identified. The relative prevalence of FA-A has been estimated at an average of approximately 65% but may widely vary according to ethnic background. In Italy, 11 of 12 patients analyzed by cell-fusion studies were assigned to group FA-A, suggesting an unusually high relative prevalence of this FA subtype in patients of Italian ancestry. We have screened the 43 exons of the FAA gene and their flanking intronic sequences in 38 Italian FA patients, using RNA-SSCP. Ten different mutations were detected: three nonsense and one missense substitutions, four putative splice mutations, an insertion, and a duplication. Most of the mutations are expected to cause a premature termination of the FAA protein at various sites throughout the molecule. Four protein variants were also found, three of which were polymorphisms. The missense mutation D1359Y, not found in chromosomes from healthy unrelated individuals, was responsible for a local alteration of hydrophobicity in the FAA protein, and it was likely to be pathogenic. Thus, the mutations so far encountered in the FAA gene are essentially all different. Since screening based on the analysis of single exons by genomic DNA amplification apparently detects only a minority of the mutations, methods designed to detect alterations in the genomic structure of the gene or in the FAA polypeptide may be helpful in the identification of FAA mutations.

Cell Cycle Proteins↗
PubMed1996

Influence of DNA superstructural features and histones aminoterminal domains on mononucleosome and dinucleosome positioning.

Mononucleosome and dinucleosome positioning was studied in the complexes between two DNA fragments of different lengths, both containing a strongly curved sequence from Crithidia fasciculata kinetoplast, and histone octamers either normal or lacking aminoterminal domains. The results obtained by Exo III and DNase I selective digestion were: (a) The first and most stable nucleosome, formed with both types of histone octamers, is positioned on the curved sequence, showing a multiple dyad axis translational positioning with the same rotational phasing. This result is in very good agreement with the theoretical prediction, obtained by adopting a method developed by us, based on the evaluation of DNA distortion energy from the nucleotide sequence. (b) The second nucleosome has two main different positions. The first one, near the extremity of the DNA fragment opposite to the curved sequence, presents a higher frequency in the case of normal nucleosome, whereas an intermediate position appears populated with a higher frequency in the case of the "tailless' nucleosome.

Animals↗
PubMed1995

Influence of DNA superstructural features and histone amino-terminal domains on nucleosome positioning.

Nucleosome positioning has been studied on a strongly curved 268 bp DNA fragment from a Crithidia fasciculata kinetoplast, complexed with a histone octamer either normal or lacking amino-terminal domains. A very similar nucleosome multiple positioning, with the same rotational phasing, has been found, by Exo III mapping, in both cases. The experimental positioning is in fairly good agreement with that predicted using a theoretical method based on DNA distortion energy, derived from the nucleotide sequence. Taking into account that nucleosomes, without histone amino-terminal domains, lack thirty percent of electrostatic interactions, these results suggest a dominant role on nucleosome positioning of DNA distortion energy with respect to modifications in histone domains.

Animals↗
PubMed1995

Superstructural features of the upstream regulatory regions of two pea rbcS genes and nucleosomes positioning: theoretical prediction and experimental evaluation.

Nucleosome positioning on two 384 bp DNA fragments, obtained from the upstream regulatory region of two pea rbcS genes, relevant in photoregulated transcription, was predicted using our theoretical method, based on the evaluation of the sequence dependent DNA bending energy. The theoretical prediction was checked by experimental evaluation of nucleosome positions after in vitro reconstitution, by mapping Exonuclease III-resistant borders and by digesting monomeric sequences with various restriction enzymes. Both approaches satisfactorily confirmed the theoretical predictions, showing that the nucleotide sequence intrinsic bendability has a dominant role in nucleosome positioning.

Base Sequence↗
PubMed1995

Influence of dynamic fluctuations on DNA curvature.

The effect of dynamic fluctuations on physical manifestations of DNA curvature such as electrophoretic retardation, circularization of DNA tracts and nucleosomes positioning is examined. It is shown that in all cases the main features of the processes can be satisfactorily explained by a static curvature model, which appears to be a good representation of time and ensemble averaged superstructures of DNA chains. The dynamic fluctuations around the average curvature appear to influence only the kinetics of these processes. In the case of polyacrylamide gel electrophoretic retardation it is demonstrated that the approximation of the static model holds on the assumption that dynamic fluctuations are independent from intrinsic curvature. The actual validity of the static model we proposed several years ago is satisfactorily demonstrated by the explanation and prediction of different experiments, such as cyclic permutation gel electrophoresis, differential DNAase I cleavage of cyclic versus linear DNA tracts and nucleosome positioning.

Animals↗
PubMed1994

Modifying quiet room design enhances calming of children and adolescents.

OBJECTIVE: To determine whether altering design of a quiet room (QR) produced more rapid calming of agitated or aggressive hospitalized children. METHOD: One of five similar QRs was modified by painting the white walls tea rose, carpeting the vinyl floor, and painting a picturesque mural on one wall. The effects of these modifications were assessed in 19 patients (mean age = 9.6 years), using a within-subjects, repeated-measures design. Overt Aggression ratings were made at the time of placement, and at 5-minute intervals thereafter, until the child was dismissed. Children were blind to the fact that a study was being conducted; raters and staff were not. RESULTS: Total aggression ratings were 45% lower in the modified QR than in the standard QR (p < .03), and initial aggression scores fell by 50% during 5 minutes of placement in the modified QR, but only after 20 minutes of placement in the standard QR (p < .0001). Motor excitement and verbal aggression were the two component factors most strongly influenced by QR design. CONCLUSION: This preliminary report suggests that it may be possible to modify QRs to facilitate calming of aggressive, agitated children and provides preliminary support for redesign of QRs.

Acting Out↗