To improve DNA resolution of fluorescence in situ hybridization we have adapted a nuclear extraction technique, resulting in highly extended DNA loops arranged around the nuclear matrix in a halo-like structure. In situ hybridization signals from alphoid and cosmid DNAs appear as beads-on-a-string, which, according to preliminary experiments, results from the association of individual probe fragments. By multicolor hybridizations we have been able to determine relative map position and to easily detect 10 kb overlap between individual cosmid clones, each of which shows linear beaded signals of ca. 10 microns, suggesting that the DNA is essentially linearized in our protocol. The map configuration can be typically derived from analysis of 5-10 cells only. The resolution range of the technique is at least 10-200 kb, and probably as little as a few kb, thus greatly extending the abilities of the existing FISH methodologies. This novel technique is much more efficient and practicable than pronuclei hybridizations, another method for high resolution FISH, and readily produces results with probes of a variety of genomic origin. In conclusion the DNA halo technique should be able to contribute significantly to the assessment of cosmid and YAC overlaps as well as to the sizing of gaps between adjacent contigs generated in genome projects.

SummaryRat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparation techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference.AFM images of LEC show structures that correlate well with SEM images. Dried-coated, dried-uncoated and wetfixed LEC show a central bulging nucleus and flat fenestrated cellular processes. It was also possible to obtain height information which is not available in SEM. After treatment with ethanol or serotonin the diameters of fenestrae increased (6%) and decreased (ÿ15%), respectively. The same alterations of fenestrae could be distinguished by measuring AFM images of dried-coated, dried-uncoated and wet-fixed LEC. Comparison of dried-coated (SEM) and wetfixed (AFM) fenestrae indicated a mean shrinkage of 20% in SEM preparations. In conclusion, high-resolution imaging with AFM of the cell surface of cultured LEC can be performed on dried-coated, dried-uncoated and wet-fixed LEC, which was hitherto only possible with fixed, dried and coated preparations in SEM and transmission electron microscopy (TEM).

Xeroderma pigmentosum (XP) is a rare hereditary human disorder clinically associated with severe sun sensitivity and predisposition to skin cancer. Some XP patients also show clinical characteristics of Cockayne syndrome (CS), a disorder associated with defective preferential repair of DNA lesions in transcriptionally active genes. Cells from the two XP-patients who belong to complementation group D and exhibit additional clinical symptoms of CS are strikingly more sensitive to the cytotoxic effects of UV-light than cells from classical XP-D patients. To explain the severe UV-sensitivity it was suggested that XP-D-CS cells have a defect in preferential repair of UV-induced 6-4 photoproducts (6-4PP) in active genes. We investigated the capacity of XP-D and XP-D-CS cells to repair UV-induced DNA lesions in the active adenosine deaminase gene (ADA) and in the inactive 754 gene by determining (i) the removal of specific lesions, i.e. cyclobutane pyrimidine dimers (CPD) and 6-4PP, or (ii) the formation of BrdUrd-labeled repair patches. No differences in repair capacity were observed between XP-D and XP-D-CS cells. In both cell types repair of CPD was completely absent whereas 6-4PP were inefficiently removed from the ADA gene and the 754 gene with similar kinetics. However, whereas XP-D cells were able to restore UV-inhibited RNA synthesis after a UV-dose of 2 J/m2, RNA synthesis in XP-D-CS cells remained repressed up to 24 h after irradiation. Our results are inconsistent with the hypothesis that differences in the capacity to perform preferential repair of UV-induced photolesions in active genes between XP-D and XP-D-CS cells are the cause of the extreme UV-sensitivity of XP-D-CS cells. Rather, the enhanced sensitivity of XP-D-CS cells may be associated with a defect in transcription regulation superimposed on the repair defect.

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Cationic liposomes are commonly used for transfection of plasmids into mammalian cells, while microspheres have been traditionally used for selective delivery of anticancer agents into tumor vasculature. We have developed a novel vector, comprised of cationic liposomes electrostatically bound to ion-exchange microspheres (termed 'microplex') for targeted gene therapy of solid tumors. The delivery modes tested in a rat solid tumor model were free plasmids, plasmids bound to microspheres, to liposomes, or to the combination vector. The greatest amount of chloramphenicol acetyltransferase (CAT) reporter gene expression in tumors was achieved using the microplex vector; 3.4-fold compared with free, and 1.8-fold compared with both microspherical and liposomal deliveries (p < 0.01). Tumor-to-normal kidney tissue CAT expression ratios were as follows: free 1.9:1; microspherical 3.7:1; liposomal 1.4:1 and microplexical 2.7:1. Expression between the two types of tissues was significantly different (p < 0.01) for all delivery modes. Microspheres targeted the plasmids to the tumors, while the action of cationic liposomes on cellular membranes allowed more plasmids to breach the cell membrane. This study has proven that the novel microplex vector is capable of selective delivery of genes to tumors and has the potential to target genes in clinical trials.