G. Della Valle

Differences in the organization and chromosomal allocation of satellite DNA between the European long tailed house mice Mus domesticus and Mus musculus

We compared the organization of satellite DNA (stDNA) and its chromosomal allocation inMus domesticus and inMus musculus. The two stDNAs show similar restriction fragment profiles after digestion (probed withM. domesticus stDNA) with some endonucleases of which restriction sequences are present in the 230–240 bp repetitive unit of theM. domesticus stDNA. In contrast, EcoRI digestion reveals thatM. musculus stDNA lacks most of the GAATTC restriction sites, particularly at the level of the half-monomer. The chromosome distribution of stDNA (revealed by anM. domesticus stDNA probe) shows different patterns in theM. domesticus andM. musculus karyotypes, with about 60% ofM. domesticus stDNA retained in theM. musculus genome. It is particularly noteworthy that the pericentromeric regions ofM. musculus chromosomes 1 and X are totally devoid ofM. domesticus stDNA sequences. In both groups, the differences in energy transfer between the stDNA-bound fluorochromes Hoechst 33258 and propidium iodide suggest that AT-rich repeated sequences have a much more clustered array in theM. domesticus stDNA, as if they are organized in tandem repeats longer than those ofM. musculus. Considering the data as a whole, it seems likely that the evolutionary paths of the two stDNAs diverged after the generation of the ancestral 230–240 bp stDNA repetitive unit through the amplification, in theM. domesticus genome, of a family repeat which included the EcoRI GAATTC restriction sequence.

Identification of a human clustered G + C-rich DNA family of repeats (Sau3A family)

Sau3A digestion of human G + C-rich DNA molecules yields discrete bands of approximately 70 and 140 base-pairs, under-represented in A + T-rich DNA molecules and in total DNA. We have cloned the 70 base-pair band in a plasmid vector and isolated a representative recombinant clone that identifies a new human family of repeats, the Sau3A family. The new family has been characterized for a number of parameters: genomic organization; reiteration frequency; sequence analysis; and distribution in a human genomic library. The Sau3A sequence (68 base-pairs in length, 53% G + C) is present in approximately 4 X 10(4) copies/haploid genome; the family is characterized by a cluster organization and is confined to a limited fraction (0.5%) of phages of a human genomic library. Southern blot hybridizations of the cloned sequence to restriction digests of total human DNA and of isolated genomic clones does not show the involvement of Sau3A blocks in long-range periodicities for any of the enzymes tested. The data suggest either a high sequence variability in the family or a complex organization of Sau3A sequence domains.

Expression of β2m-Free HLA Class I Heavy Chains in Neuroblastoma Cell Lines

Flow cytometry with the specific monoclonal antibody (MoAb) L31 was used to analyse the expression of HLA class I heavy chains not bound with β2‐microglobulin (β2m) by neuroblastoma (NB) cell lines IMR‐32 and LA‐N‐1. The cells, which express barely detectable amounts of β2m‐free (L31‐positive molecules) and β2m‐complexed HLA class I antigens (W6.32‐ and BBM. I‐reactive molecules), expressed MHC class I molecules not bound to light chains upon differentiation with either retinoic acid or serum starvation. The expression was not accompanied by an increase of surface heterodimers. Conversely, recombinant interferon‐γ (rIFN‐γ) treatment led IMR‐32 and LA‐N‐1 cells to almost exclusively express β2m‐complexed HLA class I heavy chains. Surface β2m‐free MHC class I molecules displayed a molecular mass of ~45 kDa and did not bind exogenously added β2m. No changes in the synthesis of either HLA class I and β2m mRNAs or of L31 proteins were observed in differentiated NB cells, thus suggesting that the surface exposure of unusual HLA class I antigens is regulated post‐translationally. These findings indicate that, in addition to activated lymphocytes, the surface expression of β2m‐free class I heavy chains is a feature of other cell types, such as NB cells.

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.

Satellite DNA induces unstable expression of the adjacent herpes simplex virus tk gene cotransfected in mouse cells.

To study the influence of clustered highly repetitive DNA sequences on the expression of adjacent genes, LTK- cells were cotransfected with the herpes simplex virus thymidine kinase (tk) gene and mouse satellite DNA. TK+ transformants containing a few copies of the tk genes flanked by satellite DNA were isolated. In situ hybridization on the metaphase chromosomes indicated that in each cell line the TK sequences resided at a single chromosomal site and that integration occurred preferentially into regions of the cellular DNA rich in highly repetitive sequences. The prominent feature of these cell lines was their phenotypic instability. Suppression and reexpression of the tk gene occurred at high frequency (greater than 3%) and did not correlate with any significant change in the organization of foreign DNA or with the presence of selective agents. These results indicate that satellite DNA, the major component of constitutive heterochromatin, may influence the expression of adjacent genes by affecting the chromatin structure.

Chromosomal aberrations induced in human cultured cells by liposome-encapsulated deoxyribonuclease I

Experiments of incorporation of a nucleolytic enzyme into human cells cultured in vitro have been carried out with the aim of inducing structural chromosome variations. Human heteroploid cells, either as asynchronous populations or enriched in mitoses, and PHA-stimulated lymphocytes were used as recipients. We found that all these cells when exposed to pancreatic DNAase I encapsulated in liposomes, either of multilamellar (MLV) or of small unilamellar (SUV) type, show an incidence of chromosome damage higher than that induced by the enzyme free in the incubation buffer. Our results indicate that liposomes are suitable vehicles for the transfer of an exogenous nuclease into human cultured cells. The enzyme remains functionally active and interacts with nuclear DNA, giving rise to chromosome lesions.

A human DNA telomeric fragment contains yeast ars and mitotic stabilizing sequences

A minilibrary of human DNA fragments was prepared in the vector YIP5 from a DNA preparation enriched for telomeric sequences. Screening of the library produced one clone that hybridized to the TTAGGG sequence. The cloned DNA fragment was shown to be telomeric by a number of criteria. In situ hybridization to metaphase human chromosomes showed that the fragment hybridized to the tips of all human chromosomes. The fragment contained at least two yeast autonomously replicating sequences (ARS) and stabilizing sequences, since it transformed Saccharomyces cerevisiae with high efficiency, giving rise to clones which were mitotically stable under non-selective growth.

Assignment of the E1A-regulated transcription factor E4F gene (E4F1) to human chromosome band 16p13.3 by in situ hybridization and somatic cell hybrids.

a Dipartimento di Biologia Animale Università degli Studi di Catania, Catania; b Laboratorio Nazionale Consorzio Interuniversitario per le Biotecnologie, Trieste; c Telethon Institute of Genetics and Medicine (TIGEM), Milano; d Dipartimento di Biologia Evoluzionistica Sperimentale, Università degli Studi di Bologna, Bologna; e Dipartimento di Biochimica Biofisica e Chimica delle Macromolecole, Università degli Studi di Trieste, Trieste (Italy)

Expression of Multiple Activated Cellular Oncogenes in Human Brain Tumors

Activated cellular oncogenes have frequently been reported in primitive neuroectodermal tumors. As regards malignant gliomas, either primary tumors or cell lines, gene amplifications, rearrangements, overexpressions of a spectrum of oncogenes have been described.

Searching for Proteins and Sequences of DNA Replication in Mammalian Cells

We have approached the problem of describing the process of DNA replication of mammalian cells at the molecular level from two sides: 1) Purification and description of the properties of proteins (presumably) involved in the advancement of the growing fork. 2) Attempts at cloning sequences of human DNA allowing autonomous replication in mammalian cells of plasmids bearing them.