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Biomedical subjects

L De Carli

Publications and source records attributed to L De Carli.

At least 19 recordsLinked to original sources
PubMed2001

Insertion of a loxP site in a size-reduced human accessory chromosome.

The generation in vitro of mammalian artificial chromosomes, in view of the possibility of developing new technologies for gene therapy, is still an ambitious goal. Mammalian artificial chromosomes, to be used as cloning and expression vectors, have been constructed either by de novo synthesis or by reduction of pre-existing chromosomes. In the work here reported, we introduced a loxP sequence into the pericentromeric region of a chromosome 9-derived X-ray-reduced minichromosome, with the purpose of generating a human chromosome vector (HCV). The modified accessory chromosome is linear and mitotically stable, has lost at least 1400 kb of alpha satellite DNA and normally binds CENP-B, CENP-C and CENP-E. The efficiency of gene targeting via loxP mediated homologous recombination was tested using the histone H2B-Green Fluorescent Protein chimaeric gene as a reporter. The frequency of site-specific insertion of the exogenous sequence was found to be about 50% and to occur in a controlled way with regard to the number of copies. The expression level of the fusion protein was stable over prolonged time in culture.

Animals↗
PubMed1996

Gene targeting to the centromeric DNA of a human minichromosome.

A human supernumerary minichromosome (MC), previously identified as a derivative of chromosome 9, has been introduced into Chinese hamster ovary (CHO) cells by means of cell fusion. A hybrid clone containing the MC as the only free human chromosome was isolated. A selectable marker gene (neo) inserted into a yeast artificial chromosome (YAC) has been successfully targeted to the MC centromeric DNA via co-transfection with chromosome-9-specific alpha satellite DNA. In situ hybridization and Southern blotting experiments demonstrated that the intact neo gene was integrated into the MC centromeric DNA. Studies on the clonal distribution and on the stability of the MC either in the presence or in the absence of the selective agent have been carried out. The MC is susceptible to further manipulations and may thus represent a model for the construction of a large-capacity vector for somatic gene therapy.

Animals↗
PubMed1994

Assignment of the human cytokeratin 3 gene (KRT3) to 12q12-->q13 by FISH.

We used fluorescence in situ hybridization to localize the human gene for cytokeratin 3 (KRT3), a member of the type II subfamily of cytokeratins, within the human genome. The results show that KRT3 is located within chromosome region 12q12-->q13. All human type II keratin genes mapped to date have been assigned to chromosome 12, where they are likely to be organized into one homotypic cluster.

Chromosome Mapping↗
PubMed1993

Variation of minisatellites in chemically induced mutagenesis and in gene amplification.

A mutation assay in cultured mammalian cells was developed based on direct analysis of minisatellite DNA. Chinese hamster cells (V79) were mutagenized with nitrosoguanidine and independent colonies were isolated and expanded. DNA fingerprints were then obtained after digestion with HinfI or HaeIII and hybridization with 33.15 and 33.6 probes (Jeffreys et al., 1985). 12 colonies from untreated cells were also analyzed. Digestion with HaeIII and hybridization with 33.15 probe detected the highest frequency of induced variants. The results suggest that minisatellite sequences are hypermutable sites that can be used to monitor the mutagenic effect of chemical agents. We have also analyzed the DNA fingerprints of 17 independent Chinese hamster (CHO) cell lines carrying amplification of the CAD gene. The DNA fingerprint analysis showed a variation in minisatellite regions in 3 lines while no variation was observed in independent colonies from the CHO parental cell line. The results suggest that these sequences may be hot spots for recombination during gene amplification.

Animals↗
PubMed1992

Assignment of the human connexin 32 gene (GJB1) to band Xq13.

The chromosomal localization of the human gene coding for connexin 32 (GJB1) was determined by in situ suppression hybridization (ISSH). The results allowed assignment of the gene to band Xq13, thus refining previous localization data obtained by means of somatic cell hybrid analysis.

Blotting, Southern↗
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

An improved method for chromosome banding after fluorescence in situ hybridization: use of a tetramethylrhodhamine-conjugated BrdU antibody.

A method for high quality chromosome banding after in situ hybridization with biotinylated probes has been developed. Fluoresceine-conjugated avidin is used for probe detection, while chromosome banding is performed with a tetramethylrodhamine-conjugated anti-BrdU antibody. In this way probe localization and chromosome identification can be performed simultaneously simply by changing the incidental light wavelength.

Adult↗
PubMed1991

Localization of DNA probes tightly linked to the Friedreich's ataxia locus by in situ hybridization in a case of pericentric inversion of chromosome 9.

The gene for Friedreich's ataxia (FA), an autosomal recessive neurodegenerative disorder, has been recently assigned to the long arm of chromosome 9. Linkage disequilibrium between FA and two diverse chromosome 9 markers, D9S5 and D9S15, has been detected in French, French-Canadian and Italian populations. Here, we report the physical localization of these loci by in situ hybridization of probes 26P and MCT112S identifying the D9S5 and D9S15 loci, respectively. Experiments performed on lymphocytes carrying a chromosome 9 pericentric inversion have allowed us to assign both the loci to band 9q21. Furthermore, in situ hybridization data and partial sequencing of the probe MCT112S indicate the presence of alphoid satellite DNA within this region. This suggests that MCT112S is more proximal to the centromere than 26P.

Base Sequence↗
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

Aneuploidy assay on diethylstilbestrol by means of in situ hybridization of radioactive and biotinylated DNA probes on interphase nuclei.

Aneuploidy tests by means of in situ hybridization with chromosome-specific DNA probes on interphase nuclei have been carried out on human lymphocytes treated with diethylstilbestrol (DES). A DNA probe specific for chromosome Y (Y97), either radioactive or biotinylated, was used for the assays. Autoradiography or FITC-conjugated antibiotin antibodies were employed to visualize the hybridization sites. A significant increase of hyperdiploid nuclei was obtained with both procedures and a dose-related effect was revealed using the biotinylated probe. The results obtained, while giving further support to the evidence that DES is able to induce aneuploidy in cultured human cells, also indicate that the sensitivity of the assay can be improved by using biotinylated probes coupled with fluorescent antibodies.

Adult↗
PubMed1990

Aneuploidy induced by chloral hydrate detected in human lymphocytes with the Y97 probe.

Chloral hydrate (CH) has been assayed for its ability to induce chromosome number variation in human lymphocytes in culture. Aneuploidy induction has been detected by means of in situ hybridization on interphase nuclei with a chromosome Y-specific DNA probe. A dose-dependent increase in the number of hyperdiploid nuclei was found at CH concentrations ranging from 250 to 750 micrograms/ml. These results represent a further contribution to the validation of the method which gave a positive response with drugs characterized by different mechanisms of action and therefore may be of general use in detecting aneuploidy in mammalian cells.

Aneuploidy↗
PubMed1990

Fragile-X mental retardation in a large five-generation family. A clinical and cytogenetic study.

A number of problems concerning both clinical and genetic or cytogenetic aspects of the fragile-X syndrome remain unsolved. In the present work, a large 5-generation fragile-X family has been clinically and cytogenetically investigated. The results of our study indicated that an unusually high proportion of affected males was present in the family examined; all fragile-X-positive males were profoundly retarded and showed the phenotype typically described for this syndrome; moreover, we observed a variability of penetrance within the pedigree and all fragile-X-positive females in the 4th and 5th generation had some degree of mental impairment. A multistep mutation model has been proposed in order to explain some of these findings.

Adolescent↗
PubMed1989

Overexpression of N-ras oncogene and epidermal growth factor receptor gene in human glioblastomas.

Five human glioblastoma cell lines were analyzed for oncogene activation with a panel of probes. Abnormal expression of the epidermal growth factor receptor (EGFr) gene was detected in four of five lines; N-ras oncogene overexpression was found in all five cell lines. These results were subsequently confirmed with fresh brain tumor and nonneoplastic brain tissue biopsy samples; increased expression of the N-ras proto-oncogene was observed in five of five glioblastomas, all of which also showed EGFr gene overexpression, but not in well-differentiated gliomas or in nonneoplastic brain tissue specimens. No significant differences in Ha-ras and Ki-ras expression were observed. Preliminary histochemical observations showed that intracellular levels of transforming growth factor alpha, a putative biochemical link between these two oncogenes, were significantly higher in glioblastoma cells than in controls.

Blotting, Northern↗
PubMed1989

Aneuploidy assays on interphase nuclei by means of in situ hybridization with DNA probes.

A method has been developed to detect chemically induced aneuploidy in interphase nuclei by means of in situ hybridization with chromosome-specific DNA probes. Lymphocyte cultures were treated with two known aneuploidy inducers, Benomyl and Griseofulvin. Two DNA fragments, QP23 and Y97, homologous to repetitive sequences, localized in the pericentromeric region of chromosome 9 and in the centromeric region of Y chromosome respectively, were used as probes. Following autoradiography, grain clusters, revealing the presence of the target chromosomes, were scored in resting nuclei. A marked increase in the frequency of cells with supernumerary autoradiographic signals was observed with both probes at all concentrations of the test compounds. The assay procedure appeared to be reproducible, sensitive and efficient in scoring large cell samples. It may therefore provide a useful tool for preliminary screening of potential aneuploidy inducers.

Adult↗
PubMed1989

Characterization of an X;X translocation by cytological analysis and in situ hybridization.

An X;X chromosomal translocation was ascertained by conventional cytogenetic analysis in a phenotypically normal woman with secondary amenorrhea. In situ hybridization was performed with previously mapped X-specific DNA sequences to study the rearrangement at the molecular level. The results allowed us to demonstrate that the subject is monosomic for the distal region of the short arm of the X and trisomic for the distal region of the long arm.

Adult↗