A highly conserved repetitive DNA sequence, (TTAGGG)., has been isolated from a human recombinant repetitive DNA library. Quantitative hybridization to chromosomes sorted by flow cytometry indicates that comparable amounts of this sequence are present on each human chromosome. Both fluorescent in situ hybridization and BAL-31 nuclease digestion experiments reveal major clusters of this sequence at the telomeres of all human chromosomes. The evolutionary conservation of this DNA sequence, its terminal chromosomal location in a variety of higher eukaryotes (regardless of chromosome number or chromosome length), and its similarity to functional telomeres isolated from lower eukaryotes suggest that this sequence is a functional human telomere.The human genome contains a variety of DNA sequences present in multiple copies (1). These repetitive DNA sequences are thought to arise by many mechanisms, from direct sequence amplification by the unequal recombination of homologous DNA regions to the reverse flow of genetic information (2). While it is likely that some ofthese repetitive DNA sequences influence the structure and function of the human genome, little experimental evidence supports this idea at present. We reasoned, however, that evolutionary conservation of a particular repetitive DNA sequence family might imply that the sequence is essential to cellular function. To isolate highly conserved repetitive DNA sequences, we constructed a recombinant human repetitive DNA library (pHuR library, for plasmid human repeat) and isolated clones that shared a high degree of sequence identity with rodent repetitive DNA. Four of the six most conserved cloned sequences isolated in this manner consisted of tandem arrays of the alternating (dG-dT)-(dA-dC) sequence, known to be ubiquitously interspersed in eukaryotic genomes and capable of forming the alternative Z-DNA conformation (3).The remaining two highly conserved cloned DNA sequences consisted of tandem arrays of the hexanucleotide sequence (TTAGGG), ¶ identical to the hexanucleotide sequence known to be at the telomeres of trypanosome chromosomes (4, 5). A telomere is functionally defined as a region of DNA at the molecular end of a linear chromosome that is required for replication and stability of the chromosome (6). Replicating a linear DNA molecule presents unique challenges, since all known DNA polymerases require a polynucleotide primer bearing a 3'-hydroxyl group. A variety of mechanisms are used to circumvent this replication problem, from the production of concatemeric genomes (7) to the evolution of specific telomere terminal transferase enzymes (8). In addition to their role in chromosome replication, functional telomeric DNA sequences are believed to confer stability to chromosomes, preventing the end-to-end fusions and DNA degradation normally observed after breakage of chromosomes by x-irradiation or physical rupture (6).In this paper, we present the results of fluorescent in situ hybridization (9) and BAL-31 nuclease digestion experiments (4, 5), ...

Two recombinant DNA clones that are localized to single human chromosomes were isolated from a human repetitive DNA library. Clone pHuR 98, a variant satellite 3 sequence, specifically hybridizes to chromosome position 9qh. Clone pHuR 195, a variant satellite 2 sequence, specifically hybridizes to chromosome position 16qh. These locations were determined by fluorescent in situ hybridization to metaphase chromosomes, and confirmed by DNA hybridizations to human chromosomes sorted by flow cytometry. Pulsed field gel electrophoresis analysis indicated that both sequences exist in the genome as large DNA blocks. In situ hybridization to intact interphase nuclei showed a well-defined, localized organization for both DNA sequences. The ability to tag specific human autosomal chromosomes, both at metaphase and in interphase nuclei, allows novel molecular cytogenetic analyses in numerous basic research and clinical studies.

To define the role of SV40 large T antigen in the transformation and immortalization of human cells, we have constructed a plasmid lacking most of the unique coding sequences of small t antigen as well as the SV40 origin of replication. The promoter for T antigen, which lies within the origin of replication, was deleted and replaced by the Rous sarcoma virus promoter. This minimal construct was co-electroporated into normal human fibroblasts of neonatal origin along with a plasmid containing the neomycin resistance gene (neo). Three G418-resistant, T antigen-positive clones were expanded and compared to three T antigen-positive clones that received the pSV3neo plasmid (capable of expressing large and small T proteins and having two origins of replication). Autonomous replication of plasmid DNA was observed in all three clones that received pSV3neo but not in any of the three origin minus clones. Immediately after clonal expansion, several parameters of neoplastic transformation were assayed. Low percentages of cells in T antigen-positive populations were anchorage independent or capable of forming colonies in 1% fetal bovine serum. The T antigen-positive clones generally exhibited an extended lifespan in culture but rarely became immortalized. Large numbers of dead cells were continually generated in all T antigen-positive, pre-crisis populations. Ninety-nine percent of all T antigen-positive cells had numerical or structural chromosome aberrations. Control cells that received the neo gene did not have an extended life span, did not have noticeable numbers of dead cells, and did not exhibit karyotype instability. We suggest that the role of T antigen protein in the transformation process is to generate genetic hypervariability, leading to various consequences including neoplastic transformation and cell death.

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A modification of the propidium-iodide hypotonic sodium citrate method has been developed specifically for high-resolution staining of mouse 3T3 cell nuclei for analysis by flow cytometry. The method employs a brief treatment of cells at 37°C with Triton X-100 and RNAse in the presence of propidium iodide in hypotonic sodium citrate, followed by restoration to isotonicity with NaCI. The average CV obtained for the GI peak was 3.5%, and the samples were stable for 1-2 weeks at 4°C. Compared to this technique, previously described propidium iodide-staining methods gave poor resolution with 3T3 cells.Key terms: Flow cytometry, DNA staining method, propidium iodide, 3T3 cells, hypotonic staining conditions Several methods for staining the DNA of mammalian cells for flow cytometric analysis have been reported. These include the mithramycin procedure of Crissman and Tobey (2) developed using ethanol-fixed Chinese hamster ovary cells and the propidium iodide technique of Krishan (5) developed using human lymphocytes and a human leukemic lymphoblast line. The latter technique utilizes hypotonic staining conditions to lyse the cells but leaves the nucleus intact. Because lymphocytes have very little cytoplasm, clean preparations can be obtained by this procedure without further treatment. In contrast, it is more difficult to obtain clean preparations from cells that have a large amount of cytoplasm and double-stranded RNA in their nuclei. For these types of cells, staining is routinely carried out in the presence of ribonuclease (RNAse) and a nonionic detergent (4,lO).When we began growth control studies utilizing mouse 3T3 cells, we found that previously described methods for staining DNA with propidium iodide (5,lO) gave poorly resolved DNA distributions. In this report, we present a propidium iodide staining procedure that was developed specifically for 3T3 cells and offers highly improved resolution over staining techniques developed for other cell types. MATERIALS ANI) METHODS MaterialsPropidium iodide was obtained from Calbiochem, Dulbecco's Modified Eagle Medium (DMEM) from Grand Island Biological Company, calf serum from Colorado Serum Company, RNAse (3433 R 373832) and trypsin (crystalline) from Worthington, fluorescent micropheres (Lot #3065) from Coulter Electronics, and Triton X-100 (TX-100) from Sigma. All other chemicals were reagent grade. Cell CultureBALBIC-3T3 (clone A31) cells, obtained from Dr. W.J. Pledger, were grown in T-75 flasks in DMEM supplemented with 10% calf serum, 2 mM L-glutamine, 100 unitsiml penicillin G, and 0.2 mg/ml streptomycin sulfate. The cells were routinely subcultured a t a 1:lO dilution every 3 days. Culture were mycoplasma-free. Harvesting and DNA StainingA confluent T-75 flask was rinsed once with 0.1% trypsin, 80 pM ethylenediamine tetraacetic acid (EDTA) in phosphate buffered saline (PBS), pH 7.4, and then treated briefly with 1 ml of the trypsin-EDTA solution to remove the cells from the flask. We then added 10 ml of medium containing 20% calf serum and the cells were harve...

Chinese hamster cell cultures derived from either fetal cell suspensions or adult ear clippings invariably became permanent cell lines during conventional subcultivation. The immortal cell cultures arose from rare spontaneous cellular events during the in vitro cultivation of cells with limited proliferative capacity. Immortality was not related to rare, precommitted cells from the animals. The expansion of clones of cells with limited life-span to form permanent cell lines was routinely successful only when the initial, unsubdivided culture achieved a total number in excess of 10(6) cells. On the basis of this observation, a serial clonogenicity assay was developed for determining the life-span of the cells with limited proliferative capacity and for determining whether a cell population is immortal. In addition, the technique of clonal expansion was used for a fluctuation analysis to determine the rate of immortalization. This analysis yielded a rate of 1.9 X 10(6) per cell per generation.

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