L. De Carli

Frequencies of sister-chromatid exchanges in relation to cell kinetics in lymphocyte cultures

The frequency of sister-chromatid exchanges (SCE) was determined on second-division metaphases of lymphocytes stimulated by phytohaemagglutinin (PHA) during 9 days of culture. By using either a continuous or a pulsed bromodeoxyuridine (BUdR) treatment, cells were selected that had divided only twice, or at least twice, after different culture periods. No significant differences were observed in the SCE frequencies among the various samples. The incidence of SCE appears to be independent of the proliferation properties of cultured lymphocytes, such as length of cell cycle, fast or delayed response to PHA and number of divisions performed in vitro.

Double Y as an indicator in a test of mitotic non-disjunction in cultured human lymphocytes

A cytological test for the detection of non-disjunction in human cultured lymphocytes has been developed. The Y chromosome was used as a marker, because this chromosome is easily identifiable in mitosis after staining with quinacrine dihydrochloride without karyotyping. Non-disjunction can be directly revealed by scoring mitoses with two Ys. The test was applied to agents known for their ability to induce numerical and/or structural changes in chromosomes, namely benomyl, colcemid, distamycin A, mitomycin C and X-rays. In treated lymphocytes, increased frequencies of YY mitoses were found, ranging from 2.5 X 10(-4) to 61.2 X 10(-4) (control value less than or equal to 1.1 X 10(-4). The results suggest that the detection of fluorescent Y chromosomes on metaphases is suitable for determining induced non-disjunction in human cultured cells.

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.

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.

Regional mapping of the human placental alkaline phosphatase gene (Alpp) to 2q37 by in situ hybridization

In order to determine the subchromosomal location of the gene for human placental alkaline phosphatase (ALPP; EC 3.1.3.1.), a cDNA probe encompassing most of the ALPP translated sequences was hybridized in situ to metaphase chromosomes. Our results confirm previous assignment of the gene to chromosome 2 and allow its regional mapping to band q37.

Incorporation of amino acids by a cell-free system prepared from human cells cultured in vitro

Abstract 1. A cell-free amino acid-polymerizing system has been prepared from human cells (EUE) cultured in vitro . The requirements and properties of the system are those expected for protein-synthesizing activity in vitro . 2. The system responds to endogenous mRNA, tobacco mosaic virus RNA and synthetic polynucleotides. 3. Of the compounds known to interfere with protein synthesis, pederin appears to be the most active, being inhibitory at concentrations as low as 0.01 μg/ml. 4. Ribosomes from EUE cells are active in the presence of polymerizing enzymes prepared from other organisms containing 80-S ribosomes but not from those endowed with 70-S ribosomes. Likewise, polymerizing enzymes from EUE cells are active only when added to 80-S ribosomes.

Liposomes induce chromosome aberrations in human cultured cells

The genotoxic effect of multilamellar lipid vesicles (MLV) was analysed on cultured heteroploid and diploid human cells. Dose-dependent reduction of cell survival and mitotic rate as well as induction of chromosome aberrations were observed. Chromatid and chromosome breaks and chromatid exchanges were found in 24-h culture after liposome treatment, whereas chromosome rearrangements were prevalent at 48 h. Neutral (PC/Chol) and positive (PC/SA) MLV showed a greater damage than negative (PS/PC; PS) MLV. Fibroblasts were the most sensitive cell type. In the case of PC/Chol MLV vesicles, control experiments with PC and Chol of controlled purity ruled out the possibility that the observed chromosome aberrations were caused by toxic oxidation products present in commercial preparations.

Localization of ribosomal RNA genes on human acrocentric chromosomes

SummaryClonal derivatives of a human heteroploid cell line, with different numbers of acrocentric chromosomes, show different rDNA contents. A linear relationship has been found between the rDNA content and the relative mass of the acrocentric chromosomes (D+G) expressed as the ratio between the mass of their DNA and the mass of the DNA of the whole chromosomal complement. The results suggest that human rRNA genes are located exclusively on the chromosomes of the groups D and G and that all these chromosomes contain rRNA genes.

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.

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.