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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