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), ...

Histone phosphorylation and chromatin structure were examined in synchronized CHO Chinese hamster cells during progression through mitosis. Cell population distribution in various phases of mitosis was determined by electron microscopy. Entry into mitosis was seen to occur in two stages: (1) the gathering of chromatin into aggregates of dense chromatin clumps during preprophase, followed by (2) the condensation of these aggregates into chromosome structures during prophase. Exit from mitosis was observed essentially as the reverse process, chromosomes first being disorganized into dense chromatin clumps during telophase, followed by dispersion of these aggregates in early G1. Correlating these structural changes with histone phosphorylation revealed that interphase-type histone H1 phosphorylation (H1 I) involving 1 -3 phosphates per molecule existed in interphase and during the chromatin aggregation stages of mitosis (preprophase and telophase). Also, no histone H3 phosphorylation occurred during these periods of the cell cycle. It is proposed that HIl phosphorylation may be involved with the submicroscopic changes in chromatin organization observed during interphase using molecular probes of chromatin structure. However, during mitosis, histone phosphorylation was correlated with microscopic chromatin structural changes. During the second stage of mitosis (prophase, metaphase, and anaphase), when chromosome structures were fully condensed, virtually all histone H1 existed as superphosphorylated molecules (H 1M) containing 3-6 phosphates, and all histone H 3 molecules were phosphorylated. Exit of cells from anaphase correlated closely with the dephosphorylation of H3 to unphosphorylated H3 and with the dephosphorylation of H~M to subphosphorylated H1 containing 0-3 phosphates. Further dephosphorylation of subphosphorylated HI to unphosphorylated H1 occurred as these cells left telophase and entered G1. These experiments demonstrated that H1M superphosphorylation and H3 phosphorylation are strictly mitotic events which occur only when chromosomes are fully condensed. The absence of Colcemid in some of these experiments eliminates the possibility that H I M and H3 phosphorylations are artifacts of the Colcemid treatment. It is proposed that histones H1 and H3 may impose a restriction on chromatin structure which prevents chromosome condensation during interphase and that the HlM and H3 phosphorylations remove this restriction during mitosis.Many investigators have proposed that DNA activities might be controlled by modulating the structure of chromatin through reversible modifications of the histone proteins (for recent review see [l I). Experimental results from our laboratory have supported this concept by demonstrating that the modification of histones by phosphorylation is associated with changes in chromatin structure [2-101. The strongest evidence for a correlation between histone phosphorylation and chromatin structural change has been found at mitosis. For example, in synchronized cultured mammalian cells,...

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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.

A 9.7 kb segment encompassing exons 7-10 of the adrenoleukodystrophy (ALD) locus of the X chromosome has duplicated to specific locations near the pericentromeric regions of human chromosomes 2p11,10p11, 16p11 and 22q11. Comparative sequence analysis reveals 92-96% nucleotide identity, indicating that the autosomal ALD paralogs arose relatively recently during the course of higher primate evolution (5-10 million years ago). Analysis of sequences flanking the duplication region identifies the presence of an unusual GCTTTTTGC repeat which may be a sequence-specific integration site for the process of pericentromeric-directed transposition. The breakpoint sequence and phylogenetic analysis predict a two-step transposition model, in which a duplication from Xq28 to pericentromeric 2p11 occurred once, followed by a rapid distribution of a larger duplicon cassette among the pericentromeric regions. In addition to facilitating more effective mutation detection among ALD patients, these findings provide further insight into the molecular basis underlying a pericentromeric-directed mechanism for non-homologous interchromosomal exchange.

A human umbilical vein endothelial cell cDNA library in lambda gt11 was screened for expression of thrombomodulin antigens with affinity-purified rabbit polyclonal anti-thrombomodulin immunoglobulin G (IgG) and mouse monoclonal anti-human thrombomodulin IgG. Among 7 million recombinant clones screened, 12 were recognized by both antibodies. Two of these, lambda HTm10 and lambda HTm12, were shown to encode thrombomodulin by comparison of the amino acid sequence deduced from the nucleotide sequence to the amino acid sequence determined directly from tryptic peptides of thrombomodulin. Thrombomodulin mRNA was estimated to be 3.7 kilobases in length by Northern blot analysis of endothelial cell and placental poly(A)+ RNA. Thrombomodulin mRNA was not detected in human brain, HepG2 hepatoma cells, or the monocytic U937 cell line. Additional cDNA clones were selected by hybridization with the 1.2-kilobase insert of lambda HTm10. One isolate, lambda HTm15, contained a 3693 base pair cDNA insert with an apparent 5'-noncoding region of 146 base pairs, an open reading frame of 1725 base pairs, a stop codon, a 3'-noncoding region of 1779 base pairs, and a poly(A) tail of 40 base pairs. The cDNA sequence encodes a 60.3-kDa protein of 575 amino acids. The predicted protein sequence includes a signal peptide of approximately 21 amino acids, an amino-terminal ligand-binding domain of approximately 223 amino acids, an epidermal growth factor (EGF) homology region of 236 amino acids, a serine/threonine-rich segment of 34 amino acids, a membrane-spanning domain of 23 amino acids, and a cytoplasmic tail of 38 amino acids. The EGF-homology region consists of six tandemly repeated EGF-like domains.(ABSTRACT TRUNCATED AT 250 WORDS)

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The number of pore complexes per nucleus was determined for a wide variety of cultured cells selected for their variable DNA content over a range of 1-5,600. The pore number was compared to DNA content, nuclear surface area, and nuclear volume. Values for pore frequency (pores/square micrometer) were relatively constant in the species studied. When the pore to DNA ratio was plotted against the DNA content, there was a remarkable correlation which decreased exponentially for the cells of vertebrate origin. Exceptions were the heteroploid mammalian ceils which had the same ratio as the diploid mammalian cells despite higher DNA content.The results are interpreted to mean that neither the nuclear surface, the nuclear volume, nor the DNA content alone determines the pore number of the nucleus, but rather an as yet undetermined combination of different factors. The surface and the volume of vertebrate nuclei do not decrease with decreasing DNA content below a given value. The following speculation is suggested to account for the anomalous size changes of the nucleus relative to DNA content in vertebrates. Species with small DNA complements have a relatively large proportion of active chromatin which determines the limits of the physical parameters of the nucleus. The amount of active chromatin may be the same for at least the vertebrates with low DNA content. At high DNA content, the nuclear parameters may be determined by the relatively high proportion of inactive condensed chromatin which increases the nuclear surface and volume.The two membranes of the nuclear envelope are interrupted by nuclear pore complexes, the structure and possible function of which have been the subject of several reviews (9-13, 19, 20, 32, 35). These pore complexes could control the exchange of macromolecules between the two major compartments of the cell either by a change in total number of pores or by a selective filter mechanism. Feldherr (8) determined that gold particle size rather than pore frequency seemed to influence the exchange rate. Paine et al. (29) found that the pore complex has a patent radius of 45 and, therefore, allows free exchange of small molecules. No selective filter function other than pore size has been demonstrated up to now. An increase in the nuclear pore number, however, could be brought about experimentally in lymphocytes that had been stimulated with phytohemag- 748THE JOURNAL OF CELL BIOLOGY-VOLUME 73, 1977" pages 748-760 on August 29, 2018 jcb.rupress.org Downloaded from http://doi.org/10. 1083/jcb.73.3.748 Published Online: 1 June, 1977 glutinin (PHA) (23,24), indicating a change in nuclear pore number during a change in metabolic activity. Also, the total pore number per nucleus doubled from a plateau in the G1 phase to the end of the cell cycle in HeLa cells, and the porevolume ratio stayed equal during this time (24). The question which arose from these observations is whether there is any relationship between the amount of DNA and the nuclear pore number and whether the volume or the sur...