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J J Fawcett

Publications and source records attributed to J J Fawcett.

9 recordsLinked to original sources
PubMed2000

Construction of a BAC contig map of chromosome 16q by two-dimensional overgo hybridization.

We have used sequence-based markers from an integrated YAC STS-content/somatic cell hybrid breakpoint physical map and radiation hybrid maps of human chromosome 16 to construct a new sequence-ready BAC map of the long arm of this chromosome. The integrated physical map was generated previously in our laboratory and contains 1150 STSs, providing a marker on average every 78 kb on the euchromatic arms of chromosome 16. The other two maps used for this effort were the radiation hybrid maps of chromosome 16 from Whitehead Institute and Stanford University. To create large sequenceable targets of this chromosome, we used a systematic approach to screen high-density BAC filters with probes generated from overlapping oligonucleotides (overgos). We first identified all available sequences in the three maps. These include sequences from genes, ESTs, STSs, and cosmid end sequences. We then used BLASTto identify 36-bp unique fragments of DNA for overgo probes. A total of 906 overgos were selected from the long arm of chromosome 16. Hybridizations occurred in three stages: (1) superpool hybridizations against the 12x coverage human BAC library (RPCI-11); (2) two-dimensional hybridizations against rearrayed positive BACs identified in the superpool hybridizations; and (3) pooled tertiary hybridizations for those overgos that had ambiguous positives remaining after the two-dimensional hybridization. For the superpool hybridizations, up to 236 overgos have been pooled in a single hybridization against the 12x BAC library. A total of 5187 positive BACs from chromosome 16q were identified as a result of five superpool hybridizations. These positive clones were rearrayed on membranes and hybridized with 161 two-dimensional subpools of overgos to determine which BAC clones were positive for individual overgos. An additional 46 tertiary hybridizations were required to resolve ambiguous overgo-BAC relationships. Thus, after a total of 212 hybridizations, we have constructed an initial probe-content BAC map of chromosome 16q consisting of 828 overgo markers and 3363 BACs providing >85% coverage of the long arm of this chromosome. The map has been confirmed by the fingerprinting data and BAC end PCR screening.

Chromosomes, Bacterial↗
PubMed1998

The mouse Y chromosome: enrichment, sizing, and cloning by bivariate flow cytometry.

In this report, we demonstrate the utility of interleukin-2 (IL-2) stimulation of spleen cell cultures and bivariate flow cytometry in the analysis and purification of the C57BL/6J mouse Y Chromosome. We determined that the DNA content of the C57BL/6J Y Chromosome is approximately 94.7 Mb, making it similar in size to human Chromosome 16 and significantly larger than previous estimates. In addition, we describe the bulk isolation of mouse Y Chromosomes and demonstrate enrichment of the isolated material using a fluorescence in situ hybridization strategy. We detail the construction of two small insert Y Chromosome-specific libraries, ideal for sampling Y Chromosome sequences. From these libraries 1566 clones were analyzed. We provide a detailed characterization of 103 clones, generating nearly 50 kb of sequence. For 30 of these clones, we identify regions of homology to known Y chromosomal sequences, confirming the enrichment of the sorted DNA. From the remaining characterized clones, we describe the development of 15 male-specific PCR assays and 19 male-female PCR assays potentially originating from the pseudoautosomal region or other areas of X-Y or autosome-Y homology.

Animals↗
PubMed1997

New flow cytometric technologies for the 21st century.

The envelope that defines the limits within which flow cytometry was developed is being rapidly expanded. For example: detection sensitivity has been extended to single molecules, the size range of "particle" analysis now extends from DNA fragments to plankton (1,000.+ microns), cell and chromosome sorting rates are being increased dramatically by using inactivation procedures (50,000 per second versus 2,000 per second), rapid kinetic flow cytometry enables real-time analysis of molecular assembly and cell function in the sub-second time domain, the lifetime of a fluorochrome bound to a single cell can be measured with nsec precision, and classical karyotype information (cell to cell heterogeneity) can be determined in a flow based system. These frontiers have greatly expanded the range of new and exciting flow cytometric based biomedical applications. New enabling technologies have provided the means to measure DNA cleavage by the structure-specific nuclease, human Flap Endonuclease (FEN-1), in the 300 msec time frame. Phase sensitive measurements and fluorescence lifetime are proving to be major advances for understanding molecular environments that change with, for example, the process of apoptosis. The ability to detect single fluorescent molecules has been applied to the analysis of DNA fragments obtained from enzymatic digestion of lambda DNA. This technology is being used to rapidly and very accurately size DNA fragments for the human genome project. Optical chromosome selection is a faster, better, less complex approach to chromosome sorting. This method is based on the induction of specific damage to the DNA of selected chromosomes. Lastly, the miniaturization of a single cell fractionator has made it possible to perform single cell flow cytogenetics.

Chromosomes, Human↗
PubMed1994

TOTO and YOYO: new very bright fluorochromes for DNA content analyses by flow cytometry.

Flow cytometric (FCM) studies were performed on nuclei, ethanol-fixed CHO cells, and isolated human GM130 chromosomes stained with two new cyanine dyes, TOTO and YOYO. These fluorochromes, which are dimers of thiazole orange and oxazole yellow, respectively, have high quantum efficiencies and exhibit specificities for both DNA and RNA. Bound to dsDNA in solution, TOTO and YOYO emit at 530 and 510 nm, respectively, when excited at 488 nm and 457 nm, wavelengths available from most lasers employed in FCM. RNase-treated CHO nuclei, stained with either TOTO or YOYO, provided DNA histograms, with low coefficients of variation, that were as good as or better than those obtained with nuclei stained with propidium iodide (PI) or mithramycin (MI). In addition, by comparison on an equimolar basis, nuclei stained with YOYO fluoresced over 1,000 times more intensely than nuclei stained with MI. Fluorescence ratio analyses of nuclei stained with both YOYO and Hoechst 33258 showed that the ratio of YOYO to Hoechst fluorescence remained relatively constant for G1 and S phase cells, but decreased significantly for cells in G2/M. These results indicate that the cyanine dyes may be useful in examining specific changes in chromatin structure during G2/M phases of the cell cycle. Ethanol-fixed CHO cells stained with TOTO or YOYO did not yield reproducible DNA histograms of good quality, presumably because of the poor accessibility of DNA to these large fluorochromes. However, bivariate analyses of human GM130 chromosomes stained with TOTO or YOYO alone and excited sequentially with uv and visible wave-lengths showed resolution of many individual chromosome peaks similar to results obtained for chromosomes stained with HO and chromomycin A3. Collectively, these studies show potential advantages for the use of these new cyanine dyes in FCM studies that require the sensitive detection of DNA.

Animals↗
PubMed1994

Generation of five high-complexity painting probe libraries from flow-sorted mouse chromosomes.

Mouse metaphase chromosomes were purified by flow sorting from the murine fibroblast cell line Mus spretus clone 5A. We sorted chromosomes that fell into five individual peaks based on the Hoechst 33258/chromomycin A3 DNA histogram: three peaks corresponding to the least amount of DNA and two peaks representing chromosomes with the most DNA content. This is the first example of the successful application of bivariate flow karyotyping to murine chromosome sorting. We then applied primer-directed in vitro DNA amplification using the polymerase chain reaction (PCR) to generate and label larger amounts of chromosome-specific DNA. In situ hybridization showed specific binding of the PCR products to mouse chromosomes Y, 19, 18, 3, and X as well as chromosomes 1 and 2. The combination of chromosome sorting from the M. spretus cell line and PCR proved to be highly valuable for generation of pools of DNA fragments that exhibit specific binding to mouse chromosomes and can be used to identify and delineate mouse metaphase chromosomes.

Animals↗
PubMed1989

The clinical usefulness of chromosome analysis by flow cytometry.

Historical and clinical aspects of chromosome analysis by flow cytometric methods are reviewed. A new method of preparing small samples (1.0 mL of blood) of peripheral lymphocytes for flow karyotype analysis using phytohemagglutinin and interleukin-2 is presented in detail. Figures of flow karyotypes, partial banded karyotypes, and idiograms of patients with inv(8), rec dup(8), rob t(14;21), and t(1;22) are presented, as well as examples of univariate and bivariate flow histograms from other researchers' published works. The clinical utility of these techniques is explored, with specific reference to recent work in chromosome polymorphisms and cultured amniocyte lines. We conclude that, although flow karyotyping is not a replacement for classical banded chromosome analysis, used as an adjunct, this new technique has some clinical usefulness relating to its capability to resolve small differences in chromosomal DNA content.

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↗