Carl E. Hildebrand

Evolution and distribution of (GT)n repetitive sequences in mammalian genomes

The dinucleotide repetitive sequence, (GT)n, is highly interspersed in eukaryotic genomes and may have functional roles in genetic recombination or the modulation of transcriptional activity. We have examined the distribution and conservation of position of GT repetitive sequences in several mammalian genomes. The distribution of GT repetitive sequences in the human genome was determined by the analysis of over 3700 cosmid clones containing human insert DNA. On average, a GT repetitive sequence occurs every 30 kb in DNA from euchromatic regions. GT repetitive sequences are significantly underrepresented in centric heterochromatin. The density of GT repetitive sequences in the human genome could also be estimated by analyzing GenBank genomic sequences that include introns and flanking sequences. The frequency of GT repetitive sequences found in GenBank human DNA sequences was in close agreement with that obtained by experimental methods. GenBank genomic sequences also revealed that (GT)n repetitive sequences (n greater than 6) occur every 18 and 21 kb, on average, in mouse and rat genomes. Comparative analysis of 31 homologous sequences containing (GT)n repetitive sequences from several mammals representing four orders revealed that the positions of these repeats have been conserved between closely related species, such as humans and other primates. To a lesser extent, positions of GT repetitive sequences have been conserved between species in distantly related groups such as primates and rodents. The distribution and conservation of GT repetitive sequences is discussed with respect to possible functional roles of the repetitive sequence.

Human chromosome-specific repetitive DNA sequences: novel markers for genetic analysis

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.

Human metallothionein genes: Structure of the functional locus at 16q13

The functional human metallothionein (MT) genes are located on chromosome 16q13. We have physically mapped the functional human MT locus by isolation and restriction digest mapping of cloned DNA. The mapped region contains all sequences on chromosome 16 that hybridize to metallothionein gene probes and comprises 14 tightly linked MT genes, 6 of which have not been previously described. This analysis defines the genetic limits of metallothionein functional diversity in the human genome.

A cadmium-resistant variant of the Chinese hamster (CHO) cell with increased metallothionein induction capacity.

Abstract The toxic trace metal Cd 2+ has been used to select a variant (designated Cd r ) of the Chinese hamster cell (line CHO) resistant to the growth-inhibitory and cytotoxic effects of Cd 2+ . Resistance of the Cd r cell to Cd 2+ -mediated cytotoxicity is not due to a decreased capability of the Cd r cell to accumulate Cd 2+ since Cd 2+ uptake in the Cd r cell is indistinguishable from that in the CHO cell at both toxic and subtoxic Cd 2+ exposures. Comparison of the relative capacities of these two cell types to induce specific low molecular weight Cd 2+ -binding proteins (metallothioneins) reveals that the Cd r cell has an increased capacity to induce metallothionein and to sequester intracellular Cd 2+ in metallothioneins. These results suggest that the greater competence of the Cd r cell to induce metallothionein is a major factor in the Cd 2+ -resistant phenotype of the variant.

Hydroxyurea does not prevent synchronized G1 chinese hamster cells from entering the DNA synthetic period

Using very high concentrations of radioactively labeled thymidine, we show that synchronized G/sub 1/ cells treated with hydroxyurea entered the DNA synthetic period at a time and rate indistinguishable from that of untreated cells, although the rate of DNA synthesis was greatly reduced in the drug-treated cultures. The DNA synthesized in the presence of hydroxyurea was less than or equal to 1 x 10/sup 7/ daltons, all of which could be chased into bulk DNA of approximately 3.5 x 10/sup 8/ daltons within 3 hr after removal of hydroxyurea. Hydroxyurea synchronized cells are apparently not blocked at the G/sub 1//S boundary but in the S phase itself.

Cd2+ responses of cultured human blood cells

Cd2+ cytotoxicity, uptake, and partitioning, and Cd2+-induced metallothioneine synthesis were studied in cultured peripheral human blood cells. Mononuclear cells were found to resist relatively high levels of Cd2+. Few cells were killed below 50 microM Cd2+. Above this value, survival decreased exponentially with dose. The mean LD50 for mononuclear cells cultured in Cd2+ for 40 hr was 100 microM. Polymorphonuclear cells (granulocytes) were found to be more resistant, with a significantly higher threshold and LD50, and a more complex dose response. Most of the Cd2+ incorporated by blood cells was taken up by nucleated cells. Despite their greater resistance, polymorphonuclear cells incorporated more Cd2+ at higher doses (50 to 150 microM) than did mononuclear cells. No Cd2+ was bound to metallothioneine in polymorphonuclear cells following exposure to Cd2+ for even extended periods of time (18 hr) at high doses of 109Cd2+ (25 microM). Instead Cd2+ appeared in a Sephadex G-75 peak of approximately 60,000 Da, as well as in the void peak. No significant amount of preexisting metallothioneine (MT) or metallothioneine mRNA was found in the mononuclear cells. However, MT synthesis was induced rapidly following exposure to Cd2+. [109Cd2+]MT appeared within 1 hr following exposure to 50 microM 109Cd2+, and MT synthesis rates measured from [35S]cysteine incorporation were found to be maximal within 4 hr.

Evaluation of a cosmid contig physical map of human chromosome 16

A cosmid contig physical map of human chromosome 16 has been developed by repetitive sequence finger-printing of approximately 4000 cosmid clones obtained from a chromosome 16-specific cosmid library. The arrangement of clones in contigs is determined by (1) estimating cosmid length and determining the likelihoods for all possible pairwise clone overlaps, using the fingerprint data, and (2) using an optimization technique to fit contig maps to these estimates. Two important questions concerning this contig map are how much of chromosome 16 is covered and how accurate are the assembled contigs. Both questions can be addressed by hybridization of single-copy sequence probes to gridded arrays of the cosmids. All of the fingerprinted clones have been arrayed on nylon membranes so that any region of interest can be identified by hybridization. The hybridization experiments indicate that approximately 84% of the euchromatic arms of chromosome 16 are covered by contigs and singleton cosmids. Both grid hybridization (26 contigs) and pulsed-field gel electrophoresis experiments (11 contigs) confirmed the assembled contigs, indicating that false positive overlaps occur infrequently in the present map. Furthermore, regional localization of 93 contigs and singleton cosmids to a somatic cell hybrid mapping panel indicates that there is no bias in the coverage of the euchromatic arms.

Rapid assembly of newly synthesized DNA into chromatin subunits prior to joining of small DNA replication intermediates

Abstract Nuclei from cells having the replicating DNA pulse-labeled with [3H]thymidine and the nonreplicating DNA uniformly labeled with [14C]thymidine were treated with micrococcal nuclease according to procedures which have been used to study the subunit structure of chromatin. Sedimentation analyses of chromatin from nuclease-treated nuclei, together with measurements of the size of newly synthesized DNA, indicate that (1) chromatin subunits near the replication fork are more susceptible to nuclease attack than subunits in non-replicating chromatin; (2) newly synthesized DNA is rapidly assembled into chromatin subunits prior to joining of small DNA replication intermediates; and (3) within 10 min after synthesis, DNA in newly replicated chromatin acquires a susceptibility to nuclease treatment similar to that of non-replicating chromatin.

Construction and characterization of partial digest DNA libraries made from flow-sorted human chromosome 16☆

In this report, we present the techniques used for the construction of chromosome-specific partial digest libraries from flow-sorted chromosomes and the characterization of two such libraries from human chromosome 16. These libraries were constructed to provide materials for use in the development of a high-resolution physical map of human chromosome 16, and as part of a distributive effort on the National Laboratory Gene Library Project. Libraries with 20-fold coverage were made in Charon-40 (LA16NL03) and in sCos-1 (LA16NC02) after chromosome 16 was sorted from a mouse-human monochromosomal hybrid cell line containing a single homologue of human chromosome 16. Both libraries are approximately 90% enriched for human chromosome 16, have low nonrecombinant backgrounds, and are highly representative for human chromosome-16 sequences. The cosmid library in particular has provided a valuable resource for the isolation of coding sequences, and in the ongoing development of a physical map of human chromosome 16.

Action of heparin on mammalian nuclei: I. Differential extraction of histone H1 and cooperative removal of histones from chromatin

Heparin interacts strongly with the histone component of chromatin, forming heparin-histone complexes which resist dissociation by 0.2 M H2SO4. Heparin treatment of unfractionated histones isolated from nuclei of Chinese hamster cells indicates that the affinities of the histone classes for heparin appear in the order from greatest to least: (H3, H4) greater than (H2A, H2B) greater than H1. However, when isolated nuclei are treated with heparin, H1 is released from the chromatin more readily than the other four histone classes. The release of these four histones (H2A, H2B, H3, and H4) is coordinate and occurs in a highly cooperative manner, as indicated by (1) dependence of the initial kinetics of histone removal upon heparin concentration, (2) analysis of DNA and histones in the fractions obtained from differential sedimentation of heparin-treated nuclei, and (3) analysis of the products from heparin-treated nuclei by equilibrium centrifugation in metrizamide density gradients. The results suggest rapid procedures for using heparin as an agent for studying the accessibility of histones in chromatin of intact nuclei. The relationship of these results to current models of chromatin structure is discussed.