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.

Cytological and molecular characterization of centromeres in Mus domesticus and Mus spretus.

We have applied EM in situ hybridization (EMISH) and pulsed field gel electrophoresis (PFGE) to samples from diploid primary cell cultures and an established cell line to examine in detail the relative organization of the major and minor satellite DNAs and telomere sequences in the genomes of Mus domesticus and Mus spretus. EMISH localizes the Mus domesticus minor satellite to a single site at the centromere-proximal end of each chromosome. Double label hybridizations with both minor satellite and telomere probes show that they are in close proximity and possibly are linked. In fact, PFGE of M. domesticus DNA digested with Sal I and Sfi I reveals the presence of fragments which hybridize to both probes and is consistent with the physical linkage of these two sequences. The M. domesticus minor satellite is the more abundant satellite in Mus spretus. Its distribution in M. spretus is characterized by diffuse labeling with no obvious concentration near chromosome ends. In addition to this repeat the M. spretus genome contains a small amount of DNA that hybridizes to a M. domesticus major satellite probe. Unlike the M. domesticus minor satellite, it is not telomere proximal but is confined to a domain at the border of the centromere and the long arm. Thus, although both species possess all three sequences, except for the telomeres, their distribution relative to one another is not conserved. Based on the results presented, we propose preliminary molecular maps of the centromere regions of Mus domesticus and Mus spretus.

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.

A refined physical map of the long arm of human chromosome 16.

Mapping of 33 anonymous DNA probes and 12 genes to the long arm of chromosome 16 was achieved by the use of 14 mouse/human hybrid cell lines and the fragile site FRA16B. Two of the hybrid cell lines contained overlapping interstitial deletions in bands q21 and q22.1. The localization of the 12 genes has been refined. The breakpoints present in the hybrids, in conjunction with the fragile site, can potentially divide the long arm of chromosome 16 into 16 regions. However, this was reduced to 14 regions because in two instances there were no probes or genes that mapped between pairs of breakpoints.

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.

Characterization of polymorphic loci on a telomeric fragment of DNA from the long arm of human chromosome 7.

The 240-kb yeast artificial chromosome (YAC) HTY146 (D7S427) containing the telomere from the q arm of human chromosome 7 was subcloned into the cosmid vector sCOS-1. Cosmid subclones were screened for DNA polymorphisms by Southern blot analysis of restriction digests of DNA from random individuals. Four distinct polymorphisms were characterized. These markers provide a resource for defining the end of the genetic map for the long arm of human chromosome 7.

Physical mapping of human chromosomes by repetitive sequence fingerprinting.

We have developed an approach for identifying overlapping cosmid clones by exploiting the high density of repetitive sequences in complex genomes. Individual clones are fingerprinted, using a combination of restriction enzyme digestions followed by hybridization with selected classes of repetitive sequences. This "repeat fingerprinting" technique allows small regions of clone overlap (10-20%) to be unambiguously assigned. We demonstrate the utility of this approach, using the fingerprinting of 3145 cosmid clones (1.25 x coverage), containing one or more (GT)n repeats, from human chromosome 16. A statistical analysis was used to link these clones into 460 contiguous sequences (contigs), averaging 106 kilobases (kb) in length and representing approximately 54% (48.7 Mb) of the euchromatic arms of this chromosome. These values are consistent with theoretical calculations and indicate that 150- to 200-kb contigs can be generated with 1.5 x coverage. This strategy requires the fingerprinting of approximately one-fourth as many cosmids as random strategies requiring 50% minimum overlap for overlap detection. By "nucleating" at specific regions in the human genome, and exploiting the high density of interspersed sequences, this approach allows (i) the rapid generation of large (greater than 100-kb) contigs in the early stages of contig mapping and (ii) the production of a contig map with useful landmarks for rapid integration of the genetic and physical maps.

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.

Molecular and cellular mechanisms of cadmium resistance in cultured cells.

Heavy metal induction of the synthesis of metallothioneins (MTs) provides an ideal model system for basic mechanistic studies of gene expression. Cell lines varying in their resistance to heavy metals have been isolated through a regime of exposure to serially increasing levels of Cd followed by clonal isolation. These cell lines have been used to examine the role of methylation and amplification in the Cd-resistant (Cdr) phenotype. It is suggested that regulation of expression of the MT genes in Cdr Chinese hamster cells is modulated at both the transcriptional and translational levels. An analysis of the MT2 gene sequence has uncovered a potential alternative splice site in the first intron. Usage of this site would insert 3 or 12 additional amino acids between amino acids 9 and 10. Analysis of the splicing pattern of the MT2 gene transcript in cultured cells has indicated that the second intron is preferentially removed prior to first intron excision.

5-Azacytidine-induced conversion to cadmium resistance correlates with early S phase replication of inactive metallothionein genes in synchronized CHO cells.

Previous studies have shown both hypermethylation and late replication of DNA sequences to be associated with gene inactivity. To determine whether there is a causal relationship between patterns of DNA methylation and replication timing during S phase, we have examined the timing of replication of the inactive, hypermethylated metallothionein (MT) I and II genes in synchronized, cadmium-sensitive (Cds) CHO cells. The time of S-phase replication of the MT genes was ascertained by determining the period of S phase wherein cadmium-resistant (Cdr) cells could be induced with highest frequency by pulse treatment of synchronized Cds cells with the hypomethylating drug 5-azacytidine (5-aza-CR), and by analyzing Southern blots of density fractionated DNAs isolated from synchronized cells pulse-labeled with BrdU during different intervals after release from hydroxyurea blockade. Southern filter hybridization analyses demonstrated replication of both MTI and II gene sequences within the first half of S phase. Consistent with this result, phenotypic conversion of Cds to Cdr was maximal immediately after hydroxyurea release and decreased abruptly within three hours. The replication of inactive hypermethylated MT genes in early S phase argues that transcriptional inactivity and gene-specific hypermethylation are not sufficient conditions for late DNA replication.

Overview of VEGA Venus Balloon in Situ Meteorological Measurements.

The VEGA balloons made in situ measurements of pressure, temperature, vertical wind velocity, ambient light, frequency of lightning, and cloud particle backscatter. Both balloons encountered highly variable atmospheric conditions, with periods of intense vertical winds occurring sporadically throughout their flights. Downward winds as large as 3.5 meters per second occasionally forced the balloons to descend as much as 2.5 kilometers below their equilibrium float altitudes. Large variations, in pressure, temperature, ambient light level, and cloud particle backscatter (VEGA-1 only) correlated well during these excursions, indicating that these properties were strong functions of altitude in those parts of the middle cloud layer sampled by the balloons.

Determination of Venus Winds by Ground-Based Radio Tracking of the VEGA Balloons.

A global array of 20 radio observatories was used to measure the three-dimensional position and velocity of the two meteorological balloons that were injected into the equatorial region of the Venus atmosphere near Venus midnight by the VEGA spacecraft on 11 and 15 June 1985. Initial analysis of only radial velocities indicates that each balloon was blown westward about 11,500 kilometers (8,000 kilometers on the night side) by zonal winds with a mean speed of about 70 meters per second. Excursions of the data from a model of constant zonal velocity were generally less than 3 meters per second; however, a much larger variation was evident near the end of the flight of the second balloon. Consistent systematic trends in the residuals for both balloons indicate the possibility of a solar-fixed atmospheric feature. Rapid variations in balloon velocity were often detected within a single transmission (330 seconds); however, they may represent not only atmospheric motions but also self-induced aerodynamic motions of the balloon.