Fluorescence in situ hybridization to metaphase chromosomes or chromatin fibers In Interphase nuclei is a powerful technique in mapping genes and DNA segments to specific chromosome region. We have been able to release the chromatin fibers from cells arrested at GI and G2 phases using different drugs and a simple alkailne lysis procedure. We have also demonstrated specific hybridization of fluorescencelabeled probes to single-copy genoic DNA sequences on the free chromatins. Fluorescence in situ hybridztion signals have been detected for sequences separated as close as 21 klobase pairs and as far as 350 kilobase pairs, with excellent correspondence between the observed and expetd distances. The resolution of this technique should approach 10 kilobase pairs and its coverage should span millions of base pairs. Therefore, free chromatin mapping can be generally used to study the structure and organization of mammalian genomes.

DEAD box proteins, characterized by the conserved motif Asp-Glu-Ala-Asp, are putative RNA helicases implicated in a number of cellular processes involving alteration ofRNA secondary structure such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. Based on their distribution patterns, some members of this family are believed to be involved in embryogenesis, spermatogenesis, and cellular growth and division. Here, we report that the mRNA encoding a DEAD box protein, designated HuDBP-RB, is present at elevated levels in two ofsix retinoblastoma (RB) cell lines tested and is preferentially expressed in fetal tissues of neuroectodernal origin. It is not possible to dassify HuDBP-RB as a member of any of the DEAD box protein subgroups identified to date since the regions of amino acid similarity between HuDBP-RB and other

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Life span determination in normal human cells may be regulated by nucleoprotein structures called telomeres, the physical ends of eukaryotic chromosomes. Telomeres have been shown to be essential for chromosome stability and function and to shorten with each cell division in normal human cells in culture and with age in vivo. Reversal of telomere shortening by the forced expression of telomerase in normal cells has been shown to elongate telomeres and extend the replicative life span (H. Vaziri and S. Benchimol, Curr. Biol. 8:279-282, 1998; A. G. Bodnar et al., Science 279:349-352, 1998). Extension of the life span as a consequence of the functional inactivation of p53 is frequently associated with loss of genomic stability. Analysis of telomerase-induced extended-life-span fibroblast (TIELF) cells by G banding and spectral karyotyping indicated that forced extension of the life span by telomerase led to the transient formation of aberrant structures, which were subsequently resolved in higher passages. However, the p53-dependent G1 checkpoint was intact as assessed by functional activation of p53 protein in response to ionizing radiation and subsequent p53-mediated induction of p21(Waf1/Cip1/Sdi1). TIELF cells were not tumorigenic and had a normal DNA strand break rejoining activity and normal radiosensitivity in response to ionizing radiation.