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J L Longmire

Publications and source records attributed to J L Longmire.

18 recordsLinked to original sources
PubMed1996

A high-resolution annotated physical map of the human chromosome 13q12-13 region containing the breast cancer susceptibility locus BRCA2.

Various types of physical mapping data were assembled by developing a set of computer programs (Integrated Mapping Package) to derive a detailed, annotated map of a 4-Mb region of human chromosome 13 that includes the BRCA2 locus. The final assembly consists of a yeast artificial chromosome (YAC) contig with 42 members spanning the 13q12-13 region and aligned contigs of 399 cosmids established by cross-hybridization between the cosmids, which were selected from a chromosome 13-specific cosmid library using inter-Alu PCR probes from the YACs. The end sequences of 60 cosmids spaced nearly evenly across the map were used to generate sequence-tagged sites (STSs), which were mapped to the YACs by PCR. A contig framework was generated by STS content mapping, and the map was assembled on this scaffold. Additional annotation was provided by 72 expressed sequences and 10 genetic markers that were positioned on the map by hybridization to cosmids.

BRCA2 Protein↗
PubMed1995

How bats achieve a small C-value: frequency of repetitive DNA in Macrotus.

Bats possess a genome approximately 50-87% the size of other eutherian mammals. We document that the events that have achieved or maintained a small genome size in the Mexican leaf-nosed bat Macrotus waterhousii have resulted in a lower copy number of interspersed and tandemly repetitive elements. These conclusions are based on examination of 1726 randomly chosen recombinant cosmids, with an average insert size of 35.7 kb and representing 2.6% of the haploid genome of M. waterhousii. Probes representative of microsatellites [(GT)n, (CT)n, (AT)n, (GC)n] and a tandem repeat (rDNA) were used to estimate frequency of repetitive elements in the M. waterhousii genome. Of the four dinucleotides, (GT)n was present in 33.5% of the clones, (CT)n was present in 31.0% of the clones, and (AT)n and (GC)n were not represented in any of the clones examined. The 28S rDNA and a repetitive element from M. californicus were found in three and four clones, respectively. The dinucleotides (GT)n and (CT)n occurred together in the same clone more frequently than expected from chance. Although our data do not allow us to empirically test which mechanisms are maintaining copy number of repetitive DNA in the bat genome, the nonrandom association of these different families of repetitive DNA may provide insight into a mechanism that proportionately reduces diverse families of repetitive DNA that are known to be amplified by very different mechanisms.

Animals↗
PubMed1994

Characterization of a cosmid library from flow-sorted chromosomes 11.

A cosmid library specific for human chromosome 11 has been constructed from flow-sorted chromosomes. The flow-purified chromosomes were prepared from the hamster/human hybrid line J1 which contains chromosome 11 as the only human chromosome. Individual clones were sampled in 187 microtitre plates, resulting in a total of 17,952 colonies. Hybridization analysis revealed that 83.7% of these clones were of human and 10.4% of hamster origin. The average insert size was estimated at 33.6 kb, and only 2.4% of insert fragments appear to be rearranged. This should result in 494,487 kb of cloned human DNA representing 3.5 chromosome 11 equivalents. We have prepared high-density nylon membranes of the arrayed library containing 1,536 single colonies per filter. We have demonstrated the usefulness of the library in the molecular genetic analysis of human chromosome 11 by testing for the presence of possibly polymorphic simple repeat motifs, by identifying cosmids that contain inserts from the telomeric ends of chromosome 11 and by assessing the potential of the library for rapid chromosome walking.

Animals↗
PubMed1994

Enzymatic elongation of microsatellite oligomers for use in direct-label chemiluminescent hybridizations.

Short, synthetic oligonucleotide sequences representing microsatellites and other short tandem repeats can be elongated (concatamerized) using a simple method in which complementary strands are annealed, phosphorylated, primer extended and ligated. When used in direct-label chemiluminescent hybridizations, the elongated microsatellite sequences provide an approximately 30-fold increase in signal strength compared with microsatellite oligomers that have not been concatamerized. Concatamerization of simple repeat oligomers further enables the use of relatively short oligonucleotide sequences in direct-label chemiluminescent hybridization experiments, thereby reducing the overall need for radioisotopes in certain commonly performed laboratory procedures such as DNA fingerprinting and selection of clones containing microsatellite sequences.

Animals↗
PubMed1993

Genome organization of repetitive elements in the rodent, Peromyscus leucopus.

To document the frequency and distribution of repetitive elements in Peromyscus leucopus, the white-footed mouse, a cosmid genomic library was examined. Two thousand thirteen randomly chosen recombinants, with an average insert size of 35 kb and representing 2.35% of the haploid genome of P. leucopus, were screened with probes representing microsatellites, tandem repeats, and transposable elements. Of the four dinucleotides, (GT)n was present in 87% of the clones, (CT)n was present in 59% of the clones, and (AT)n and (GC)n each was represented in our sample by a single clone (0.05%). (TCC)n was present in 8% of the clones. Of the tandem repeats, the 28S ribosomal probe and the (TTAGGG)n telomere probe were not represented in the library, whereas a heterochromatic fragment was present in 9% of the clones. A transposable element, mys, was estimated to occur in 4700 copies, whereas a long interspersed element (LINE) was estimated to occur in about 41,000 copies per haploid genome. LINE and mys occurred together in the same clones more frequently than expected on the basis of chance. Hybridizing the library to genomic DNA from P. leucopus, Reithrodontomys fulvescens, Mus musculus, and human produced general agreement between phylogenetic relatedness and intensity of hybridization. However, dinucleotide repeats appeared to account for a disproportionately high number of positive clones in the more distantly related taxa.

Animals↗
PubMed1991

Use of sex-linked minisatellite fragments to investigate genetic differentiation and migration of North American populations of the peregrine falcon (Falco peregrinus).

The M13 repeat detects different levels of genetic variation in falcons. First, this minisatellite probe reveals typically highly variant restriction fragments that show no apparent unequal distribution between the sexes. Secondly, the M13 repeat detects sets of fragments that are only present in DNAs from female falcons. The level of polymorphism displayed by the sex-linked fragments is greatly reduced relative to most autosomal minisatellites. In addition, the size of these fragments (in kilobase pairs) is species-specific among Mauritius kestrels (Falco punctatus) and peregrines (Falco peregrinus). Variation observed at one o of the sex-linked fragments in peregrines has proven to be useful in distinguishing a subset of the tundrius subspecies of this endangered raptor. This correlation has enabled a genetic test to be used to examine the representation of tundrius peregrines during mass migration.

Animals↗
PubMed1990

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.

Chromosome Mapping↗
PubMed1988

Isolation and molecular characterization of a highly polymorphic centromeric tandem repeat in the family Falconidae.

An abundant tandem repeat has been cloned from genomic DNA of the merlin (Falco columbarius). The cloned sequence is 174 bp in length, and maps by in situ hybridization to the centromeric regions of several of the large chromosomes within the merlin karyotype. Complementary sequences have been identified within a variety of falcon species; these sequences are either absent or in very low copy number in the family Accipitridae. The cloned merlin repeat reveals highly polymorphic restriction patterns in the peregrine falcon (Falco peregrinus). These polymorphisms, which have been shown to be stably inherited within a family of captive peregrines, can be used to differentiate the Greenland and Argentina populations of this endangered raptor species.

Animals↗
PubMed1987

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.

Base Sequence↗
PubMed1985

Oncogenes and linkage groups: conservation during mammalian chromosome evolution.

Proto-oncogenes, which represent the cellular progenitors of the transforming genes harbored by acute transforming oncogenic retroviruses, have been highly conserved during vertebrate evolution. In this report, we have assigned experimentally a subset of proto-oncogenes (SRC, ABL, FES, and FMS-all related to the SRC family) to Chinese hamster chromosomes by Southern filter hybridization analyses of DNAs isolated from both somatic cell hybrids and flow-sorted hamster chromosomes. These results demonstrate that several autosomal linkage groups containing proto-oncogenes originated prior to the radiation and speciation of mammals and have remained remarkably stable for nearly 80 million years.

Animals↗
PubMed1985

Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulating metallothionein gene expression.

We describe here the derivation, characterization, and use of clonal cadmium-resistant (Cdr) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylamide gel electrophoresis, we showed that the stable Cdr phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 microM, coordinate amplification of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which we have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cdr variants expressing abundant MT and their corresponding Cds parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expression of the isometallothioneins.

Animals↗
PubMed1984

Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosome 3: evidence for the role of chromosome rearrangement in gene amplification.

Cadmium resistant (Cdr) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cdr variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with a Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster X mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. We speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cdr hamster cell lines.

Animals↗
PubMedDate not supplied

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.

Animals↗