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), ...

The rat serum albumin gene has been isolated from a recombinant library containing the entire rat genome cloned in the X phage Charon 4A. Preliminary R-loop and restriction analysis has revealed that this gene is split into at least 14 fragments (exons) by 13 intervening sequences (introns), and that it occupies a minimum of 14.5 kilobases of genomic DNA.Recent advances in recombinant DNA technology have made it possible to obtain virtually any desired single-copy genomic sequence in cloned form, provided an appropriate probe is available. We have used these techniques to isolate the rat serum albumin gene. Serum albumin synthesis is one of the major characteristics of vertebrate liver. Observation of the activity and state of this gene during development and in adult tissues should be informative as to the process of terminal differentiation. Albumin synthesis is essentially constitutive, but does respond significantly to a variety of stimuli (1). It is also expressed to variable extents in different hepatoma cell lines (2). The availability of cloned albumin genomic DNA will greatly facilitate the study of this variable expression, particularly at the level of transcript processing.Determination of the sequence organization of the albumin gene is also of interest, especially with regard to the disposition of repetitive elements and intervening sequences. Although regulatory (3) and evolutionary (4) significance has been postulated, the functional role, if any, of these striking features of eukaryotic genomes remains unknown. The comparative studies that will be possible as other genes are extracted from the rat and related species can be expected to provide considerable insight into this fascinating problem. MATERIALS AND METHODSRat Genome Library. High molecular weight liver DNA was extracted from an adult male Sprague-Dawley rat (Simonsen Labs, Gilroy, CA) by the method of Blin and Stafford (5) and aliquots were digested with EcoRI (Boehringer Mannheim) under conditions adjusted to cleave either one-third or one-fifth of the EcoRI sites in an equivalent amount of bacteriophage X DNA. The fragments resulting from this partial digestion were sedimented through a 10-30% sucrose gradient; the material between 10 and 20 kilobases (kb) was recovered by ethanol precipitation. A sample of this rat DNA (2.5 ,g) was ligated with 8.5 ,ig of a preparation of Charon 4A "cloning fragments" (6, 7). This recombinant DNA was packaged in vitro by using extracts from defective X lysogens provided by N. Sternberg (6). The method used was that of Hohn and Murray (8). Approximately 2,000,000 independent clones were obtained. The library was amplified 100,000-fold by subconfluent plating on Escherichia coli strain DP5OSupF (9).cDNA Clones. cDNA was synthesized from purified albumin mRNA as described (10). This cDNA contained a small amount of full-length material and had a number average size of approximately 1000 nucleotides. It was rendered double-stranded by sequential treatment with E. coli DNA polymerase I and S1 nuc...

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Parameters of repetitive sequence organization have been measured in the rat genome. Experiments using melting, hydroxylapatite binding, and single strand specific nuclease digestion have been used to measure the number, length, and arrangement of repeated DNA sequences. Renaturation and melting or S1 nuclease digestion of 1.0 kbp DNA fragment show about 20% of rat DNA sequences are 3000-fold repeated. Renatured duplexes from 4.0 kbp DNA fragments display two repetitive size fractions after nuclease digestion. About 60% of the repeated sequences are 0.2-0.4 kbp long while the remainder are longer than 1.5 kbp. The arrangement of the repeated sequences has been measured by hydroxylapatite fractionation of DNA fragments of varying lengths bearing a repeated sequence. Repeated DNA sequences are interspersed among 2.5 kbp long nonrepeated sequences throughout more than 70% of the rat genome. There are approximately 350 different 3000-fold short repeated sequences in the rat interspersed among 600,000 nonrepeated DNA sequences.