PubMed Health⌕ Search

Biomedical subjects

J H Jett

Publications and source records attributed to J H Jett.

At least 19 recordsLinked to original sources
PubMed1991

Rapid DNA sequencing based upon single molecule detection.

We are developing a laser-based technique for the rapid sequencing of 40-kb or larger fragments of DNA at a rate of 100 to 1000 bases per second. The approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA fragment into a flowing sample stream, and detection of individual fluorescently labeled bases as they are cleaved from the DNA fragment by an exonuclease. The ability to sequence large fragments of DNA will significantly reduce the amount of subcloning and the number of overlapping sequences required to assemble megabase segments of sequence information.

Base Sequence↗
PubMed1990

Flow cytometric competitive binding assay for determination of actinomycin-D concentrations.

A single step, separation free competitive binding reaction between the fluorescent antibiotic mithramycin and actinomycin-D for common binding sites on DNA coated 10 microns diameter microspheres is described. The fluorescence of the microspheres is measured with a flowcytometer. In the presence of a constant amount of mithramycin, the microsphere fluorescence is inversely proportional to actinomycin-D concentration.

Binding, Competitive↗
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↗
PubMed1989

Use of xantham gum to suspend large particles during flow cytometric analysis and sorting.

In this report we describe the use of xantham gum as a biologically inert material for increasing the viscosity of a suspension of cells or particles during flow cytometric analysis and sorting. A 0.1% concentration of xantham gum in culture medium or saline will increase the viscosity approximately 9-fold. For suspensions of multicellular spheroids 100-400 microns in diameter the measured sedimentation velocity was approximately 9 times slower than that in medium alone. Thus, spheroids of 100 microns diameter remain in suspension in 0.1% xantham gum for 66 min, compared to 7.5 min in culture medium. This allows extended periods of sorting without stirring or agitating the sample suspension. The xantham gum solution is noncytotoxic for periods up to 8 h as measured by clonogenicity assay. Xantham gum has the added advantage that the viscosity is significantly reduced when the solution is subjected to shear stress, such as during flow. This technique should be applicable to extended sorting of suspensions of spheroids, plant cells, and other large particles, as well as for analyzing and sorting single cells for extended periods.

Animals↗
PubMed1989

High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules.

We are developing a laser based technique for the rapid sequencing of large fragments (approximately 40 kb) of DNA based upon the detection of single, fluorescently tagged nucleotides cleaved from a single DNA fragment. We have demonstrated significant progress on several of the important steps of this technique. The projected rate of sequencing is several hundred bases per second which is orders of magnitude faster than existing methods. Once developed, this technology could be utilized by investigators for rapid sequencing of genetic material from virtually any source.

Base Sequence↗
PubMed1987

Viable sorting of intact multicellular spheroids by flow cytometry.

A flow cytometric method has been developed for sorting viable, intact multicellular spheroids in order to obtain uniformly-sized populations with diameters in the range of 50-100 microns. A FACS II instrument was modified for this purpose by installing a 200-microns-diameter exit orifice and by making adjustments in the sheath flow, oscillator frequency, and number of droplets sorted. Polystyrene microspheres (44 and 88 microns diameter) and 41-96-microns-diameter spheroids could be sorted and recovered with 70-100% efficiency, an improvement over previous reports. Unstained, viable spheroids were simultaneously analyzed for small-angle forward light scatter, 90 degree light scatter, and autofluorescence using a 488-nm laser operating at 100 mW. Analysis of the data demonstrated a considerable variation in both the 90 degrees light scatter and the autofluorescence signals for a given forward angle light scattering signal. By setting narrow sort windows on the forward angle light scattering signal and either the 90 degree light scatter or autofluorescence signals, uniformly spherical spheroid populations could be recovered. These sorted populations had coefficients of variation of the mean diameter in the range of 5-9%. This represents a variation of less than one cell diameter, and is a major improvement over any other technique. There was no significant difference in the subsequent growth rates of sorted spheroids compared to the unsorted spheroids. This technique will apply when uniform populations of small spheroids are required, such as investigations of the contact effect or in the initiation of growth curve studies.

Animals↗
PubMed1987

Evolutionary relationships of the Chinese hamster X chromosome and autosomes: a comparison using solution hybridization techniques.

The evolutionary relationships of Chinese hamster X chromosome and autosome DNA sequences were compared by solution hybridization techniques. Chinese hamster X chromosome tracer was prepared by radiolabeling DNA from chromosomes isolated by fluorescence-activated sorting. Radiolabeled Chinese hamster total genomic DNA, approximately 90% of which is of autosome origin, was used as autosome tracer. Each tracer was mixed with excess driver DNA of Chinese hamster, Syrian hamster, rat, rabbit, cat, cow, or human origin. Reaction mixtures were melted and allowed to reassociate to an equivalent CoT of 12,000, under conditions which permitted 35% mismatch in DNA duplexes. Both the extent of duplex formation (the normalized percentage hybridization or NPH) and the average thermal stability of the duplexes formed (melting temperature or Tm) were measured; these values were used to compare the evolutionary relatedness of tracer and driver DNAs. The pattern of evolutionary relatedness revealed by comparing either the Tm or NPH values obtained with different drivers was the same for X chromosome and autosome DNA and was consistent with the phylogeny of the species examined. Although NPH and Tm values for X chromosome and autosome tracers differed, differences fell within the range of experimental error. The results of these studies provide no evidence for differential conservation of Chinese hamster X chromosome sequences, suggesting that the constraints on the mammalian X chromosome which act to maintain its gene linkage group intact do not markedly reduce the extent to which its sequences diverge during evolution.

Animals↗
PubMed1985

Droplet sorting of large particles.

The systematics of droplet formation conditions for orifices with diameters up to 200 micron are described. Sorting recovery experiments indicate that particles up to 44 micron in diameter can be recovered by charged droplet deflection of two drops with at least 75% recovery. By reducing the jet velocity, a deflection of greater than 1 cm was obtained for all droplet sizes.

Cell Separation↗
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

Amplified flow-cytometric separation-free fluorescence immunoassays.

An equilibrium-type competitive-binding fluorescence immunoassay with high sensitivity and excellent precision is described that obviates separation of free from bound label. In the assay relatively large (10 microns diameter) antibody-coated non-fluorescent particles and very small (0.10 micron diameter) antigen-coated fluorescent latex particles are used. Soluble nonlabeled antigen competes with antigen on the microspheres for antibody binding sites on the larger spheres. After equilibrium is attained, the fluorescence distribution of 5000 of the large spheres is measured in a flow cytometer. The mean values for the fluorescence distribution obtained from samples containing known concentrations of soluble antigen are used to construct a standard displacement curve. In a prototype assay for the antigen horseradish peroxidase, a sensitivity of 10(-12) mol/L has been achieved. Undiluted serum can be assayed without loss of sensitivity. Preliminary experiments also indicate that double-antibody "sandwich"-type assays of very high sensitivity (10(-14) mol/L) are also possible when this dual-sphere concept is exploited.

Binding, Competitive↗
PubMed1979

Light scattering with stream-in-air flow systems.

Both forward angle and 90 degrees light-scattering measurements have been used for cell sizing with stream-in-air flow systems with very little theoretical base for the measurements. Mie theory calculations are compared with measurements on plastic microspheres. Detector response for homogeneous spheres is shown to be sensitive to refractive index.

Cell Count↗
PubMed1979

Laser flow cytometric light scatter and fluorescence pulse width and pulse rise-time sizing of mammalian cells.

In laser flow cytometry, an increasingly popular technique of analytical cytology, quantitative measurements of interest include cell and nuclear diameters. Electronic circuitry for a new cell sizing technique has been developed which measured the time that signal pulses from either fluorescence or light scatter sensors exceed a preset constant fraction of the peak signal amplitude (pulse width) or the time that it takes a signal to rise between constant fractions of the peak signal amplitude on the rising side of the pulse (pulse rise-time). These pulse width or pulse rise-time measurements were related to cell or nuclear diameters and were used in combination to determine nuclear size to cell size ratios. This method of sizing was found to be independent of fluorescent or light-absorbing stain intensity, linearly related to cell or nuclear diameter, and capable of resolving small diameter differences.

Animals↗
PubMed1978

Quantification of cell fusion by twenty-one strains of Newcastle disease virus using flow microfluorometry.

The cell-fusing ability of Newcastle disease virus (NDV) was quantified using flow microfluorometry (FMF). The rate of polykaryocyte formation, fusion dependence on multiplicity of infection, and cell fusion differences for 21 NDV strains were measured using this technique. No correlation was found between the virulence of a virus strain and its cell fusion index calculated from the FMF data.

Cell Fusion↗
PubMed1977

Chromosomes of Peromyscus (rodentia, cricetidae). VI. The genomic size.

In the genus Peromyscus cells of all species contain 48 chromosomes; however, the fundamental number varies from 56 (P. Crinitus, P. boylei) to 96 (P. eremicus). In some cases biarmed chromosomes are the result of pericentric inversions, while in others they are the result of addition of large amounts of constitutive heterochromatin. Flow microfluorometric DNA-per-cell determinations demonstrated that in some species (P. eremicus) the genome is increased by 36% over the amount of DNA found in most mammalian species. Studies of unique karyotypes with increased amounts of DNA added as constitutive heterochromatin may ultimately help in the elucidation of the mechanisms involved in karyotype evolution and speciation.

Animals↗
PubMed1976

Prophage induction and inactivation by UV light.

Analysis of the induction curves for UV light-irradiated Haemophilus influenzae lysogens and the distribution of pyrimidine dimers in a repair-deficient lysogen suggests that one dimer per prophage-size segment of the host bacterial chromosome is necessary as a preinduction event. The close correlations obtained prompted a renewed consideration of the possibility that direct prophage induction occurs when one dimer is stabilized within the prophage genome. The host excision-repair system apparently functions to reduce the probability of "stabilizing" within the prophage those dimers that are necessary for induction and inactivation. The presence of the inducible defective prophage in strain Rd depresses the inducibility of prophage HP1c1.

Bacteriophages↗
PubMed1976

Flow microfluorometric quantitation of the blastogenic response of lymphocytes.

A method for quantitating the specific stimulation of peripheral lymphocytes has been developed using the techniques of flow microfluorometry. Peripheral bovine lymphocytes were collected and specifically stained for deoxyribonucleic acid (DNA) content using a low-salt propidium iodide procedure. Flow microfluorometry was used to determine, on the basis of DNA content, the percentage of cells in a population that was stimulated. Extremely uniform staining of the lymphocytes (coefficient of variation of less than 2%) provides a high resolution between proliferating and nonproliferating cells. The method provides a rapid, highly repoducible technique for determing the fraction of lymphocytes stimulated in response to tuberculin antigens based on an increase in cellular DNA content. Specific and nonspecific stimulation by a defined antigen can be measured and resolved.

Animals↗