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B Trask

Publications and source records attributed to B Trask.

At least 19 recordsLinked to original sources
PubMed1992

Assembly and analysis of cosmid contigs in the CEA-gene family region of human chromosome 19.

The carcinoembryonic antigen (CEA)-like genes are members of a large gene family which is part of the immunoglobulin superfamily. The CEA family is divided into two major subgroups, the CEA-subgroup and the pregnancy-specific glycoprotein (PSG)-subgroup. In the course of an effort to develop a set of overlapping cosmids spanning human chromosome 19, we identified 245 cosmids in a human chromosome 19 cosmid library (6-7X redundant) by hybridization with an IgC-like domain fragment of the CEA gene. A fluorescence-based restriction enzyme digest fingerprinting strategy was used to assemble 212 probe-positive cosmids, along with 115 additional cosmids from a collection of approximately 8,000 randomly selected cosmids, into five contigs. Two of the contigs contain CEA-subgroup genes while the remaining three contigs contain PSG-subgroup genes. These five contigs range in size from 100 kb to over 300 kb and span an estimated 1 Mb. The CEA-like gene family was determined by fluorescence in situ hybridization to map in the q13.1-q13.2 region of human chromosome 19. Analysis of the two CEA-subgroup contigs provided verification of the contig assembly strategy and insight into the organization of 9 CEA-subgroup genes.

Carcinoembryonic Antigen↗
PubMed1992

Immunofluorescent labeling of centromeres for flow cytometric analysis.

A procedure to stain the centromeric region of chromosomes for dual beam flow cytometric analysis is described. Serum from a CREST (Scleroderma syndrome) patient presenting a high titer of anticentromeric antibodies was chosen on the basis of specificity of labeling of cells on slides. The high affinity of the antibodies to centromeres and low binding to chromosomal arms allowed the development of an indirect immunofluorescent labeling procedure using isolated and unfixed chromosomes stabilized by Mg++ ions. Discontinuous Ficoll gradients were used to separate chromosomes from unbound antibodies. With this procedure, chromosome clumping and degradation were minimal. The chromosomes were then stained with the DNA dyes Hoechst 33258 and chromomycin A3, before dual beam flow cytometric analysis. Flow karyotypes, with good chromosome peak resolution, were obtained for both human and hamster chromosomes subjected to the immunolabeling procedure. For quantification of FITC fluorescence due to bound antibody, chromosomes were counterstained with Hoechst only. The FITC intensity of antibody-labeled human and hamster chromosomes were 4-10 and 20 times greater than control chromosomes, respectively. These results suggest that the staining procedure may be suitable for immunolabeling of chromosomes with antibodies recognizing other nuclear proteins and their subsequent quantification by flow cytometry.

Animals↗
PubMed1992

The CCAAT/enhancer binding protein (C/EBP alpha) gene (CEBPA) maps to human chromosome 19q13.1 and the related nuclear factor NF-IL6 (C/EBP beta) gene (CEBPB) maps to human chromosome 20q13.1.

The CEBPA gene encoding CCAAT/enhancer binding protein (C/EBP alpha) has been mapped to human chromosome 19 and the CEBPB (formerly TCF5) gene encoding NF-IL6 (C/EBP beta) to human chromosome 20 by Southern blot analysis of Chinese hamster x human and mouse x human somatic cell hybrids. CEBPA has been further mapped to 19q13.1 between the loci GPI and TGFB using human x hamster somatic cell hybrids containing restricted fragments of human chromosome 19. This position was confirmed by fluorescence in situ hybridization. Furthermore, CEBPB has been mapped to 20q13.1 by fluorescence in situ hybridization.

Animals↗
PubMed1992

Fluorescence in situ hybridization mapping of human chromosome 19: mapping and verification of cosmid contigs formed by random restriction enzyme fingerprinting.

Automated restriction enzyme fingerprinting of 7900 cosmids from chromosome 19 and calculation of the likelihood of their overlap based on shared fragments have resulted in the assembly of 743 sets of overlapping cosmids (contigs). We have mapped 22% of the formed contigs (n = 165) and all of the contigs with minimal tiling paths exceeding 6 members (n = 50) to chromosomal bands by fluorescence in situ hybridization using DNA from at least one member cosmid. The estimated average size of the formed contigs is 60-70 kb. Thus, members of a correctly formed contig are expected to lie close to each other in metaphase and interphase chromatin. Therefore, we tested the contig assembly process by comparing the band assignment of two or more members selected from each of 97 contigs. Forty-two of these contigs were further characterized for valid assembly by determining the proximity of members in interphase chromatin. Using these tests, we surveyed a total of 431 joins counted along the minimal tiling path (280 in interphase as well as metaphase) and found 6 erroneous joins, one in each of 6 contigs (6% of tested).

Chromosomes, Human, Pair 19↗
PubMed1991

A new system for high-resolution DNA sequence mapping interphase pronuclei.

Cosmid clones containing human or hamster inserts have been hybridized in situ and localized with fluorescent reporter molecules in interphase nuclei (pronuclei) obtained after fusion of hamster eggs with either human or hamster sperm. Hamster egg cytoplasm processes the tightly packaged sperm DNA into large diffuse networks of chromatin fiber bundles, providing hybridization targets more extended than those available in somatic interphase cell nuclei. Pronuclear physical distances between hybridization signals were measured in micrometers and correlated to genomic distances determined by restriction fragment analyses, using cosmids from the Chinese hamster DHFR region and from the human Factor VIII/color vision pigment gene region (Xq28). The mean pronuclear distances between hybridization sites were about three times as large as those measured in somatic interphase cells for equivalent genomic distances. The relationship between physical and genomic distances was linear from less than 50 kb to at least 800 kb. The results show that physical distance in the sperm-egg system promises to extend the mapping range obtainable in somatic interphase nuclei below 50 kb and up to at least 800 kb.

Animals↗
PubMed1990

A computer program for analyzing bivariate flow karyotypes.

This article describes a computer program for analyzing bivariate flow karyotypes of human chromosomes stained with Hoechst 33258 (HO) and chromomycin A3 (CA). The karyotype first is divided into regions that contain chromosome peaks. The chromosomes that are associated with those areas are identified. The distributions in these areas then are fitted with mathematical functions of increasing complexity. The process starts by fitting a specified number of univariate Gauss functions to projections of the HO and CA distributions of each area. The final fit can include multiple bivariate Gauss functions, including a background function for debris subtraction. The results of one stage in the fitting process serve as seed values for the next, more complex step. Since the program autonomously estimates the starting values for the iterative fitting procedures, the fit results are insensitive to operator bias and the program will consistently converge to the same solutions. The resulting table of parameter values can be used to compare flow karyotypes to a reference data set.

Chromomycin A3↗
PubMed1990

Quantification of the DNA content of structurally abnormal X chromosomes and X chromosome aneuploidy using high resolution bivariate flow karyotyping.

Quantification of the Hoechst and chromomycin A3 fluorescence intensities of mitotic human chromosomes isolated from karyotypically normal and abnormal cells was performed with a dual beam flow cytometer. The resultant flow karyotypes contain information about the relative DNA content and base composition of chromosomes and their relative frequencies in the mitotic cell sample. The relative copy number of X and Y chromosomes was determined for 38 normal males and females and 6 cell lines with X or Y chromosome aneuploidy. Flow karyotype diagnoses corresponded with conventional cytogenetic results in all cases. We show that chromosome DNA content can be derived from peak position in Hoechst vs. chromomycin flow karyotypes. These values are linearly related to propidium iodide staining intensity as measured with flow cytometry and to the binding of gallocyanin chrome alum to phosphate groups as measured with slide-based scanning photometry. Cell lines with deleted or dicentric X chromosomes ranging in length from 0.53 to 1.95 times normal were analyzed by using flow cytometry. The measured difference in DNA content between a normal X and each of the structurally abnormal chromosomes was linearly correlated to the difference predicted from cytogenetics and/or probe analyses. Deletions of 3-5 Mb, which were at and below the detection limits of conventional cytogenetics, could be quantified by flow karyotyping in individuals with X-linked diseases such as Duchenne muscular dystrophy, choroideremia, and ocular albinism/ichthyosis. The results show that the use of flow karyotyping to quantify the size of restricted regions of the genome can complement conventional cytogenetics and other physical mapping techniques in the study of genetic disorders.

Aneuploidy↗
PubMed1990

Analytical approaches to detection and characterization of disease-linked chromosome aberrations.

Flow karyotyping and FISH with chromosome specific or disease-locus-specific probes are powerful adjuncts to conventional cytogenetic analysis. Flow karyotyping is well suited to quantitative analysis of DNA content changes that occur during structural rearrangement. FISH with probes for repeated sequences allows ready detection of aneuploidy in interphase cells. FISH with whole chromosome composite probes to metaphase spreads facilitates detection of subtle structural changes and allows detection of structural aberrations that occur at frequencies as low as 10(-3). FISH with locus-specific probes facilitates diagnosis of specific genetic diseases, allows phenotype-genotype correlation on a cell-by-cell basis and may be developed into a sensitive method for detection of residual disease.

Chromosome Aberrations↗
PubMed1989

The proximity of DNA sequences in interphase cell nuclei is correlated to genomic distance and permits ordering of cosmids spanning 250 kilobase pairs.

The physical distance between DNA sequences in interphase nuclei was determined using eight cosmids containing fragments of the Chinese hamster genome that span 273 kb surrounding the dihydrofolate reductase (DHFR) gene. The distance between these sequences at the molecular level has been determined previously by restriction enzyme mapping (J.E. Looney and J.L. Hamlin, 1987, Mol. Cell Biol. 7: 569-577; C. Ma et al., 1988, Mol. Cell Biol. 8: 2316-2327). Fluorescence in situ hybridization was used to localize the DNA sequences in interphase nuclei of cells bearing only one copy of this genomic region. The distance between DNA sequences in interphase nuclei was correlated to molecular distance over a range of 25 to at least 250 kb. The observed relationship was such that genomic distance could be predicted to within 40 kb from interphase distance. The correct order of seven probes was derived from interphase distances measured for 19 pair-wise combinations of the probes. Measured distances between sequences approximately 200 kb apart indicate that the DNA is condensed 70- to 100-fold in hybridized nuclei relative to a linear DNA helix molecule. Cell lines with chromosome inversions were used to show that interphase distance increases with genomic distance in the 50-90 Mb range, but less steeply than in the 25-250 kb range.

Animals↗
PubMed1989

Three-dimensional analysis of the organization of human chromosome domains in human and human-hamster hybrid interphase nuclei.

This report describes the intranuclear organization of chromosomes in human-hamster hybrid nuclei and in human cell nuclei. The target chromosomes were stained using in situ hybridization with biotinylated, chromosome-specific DNA probes. Bound probe was detected with fluorescein-avidin. Hybridizations were performed to fixed nuclei in aqueous suspension in order to preserve their three-dimensional morphology. Total nuclear DNA was stained with DAPI. Three-dimensional information about the organization of DNA and probe within the nucleus was obtained by optical sectioning. The human chromosomes in human-hamster hybrid nuclei were found to be confined to 'domains' that were maintained during the cell cycle. Different spatial localization patterns of the human chromosomes were seen in interphase nuclei of two different hybrid cell lines. The positions of chromosome-specific repetitive sequences in human fibroblast interphase nuclei were also studied using probes for the telomeric region of chromosome 1p (1p36), the centromeric region of chromosome 9 (9q12) and the long arm of the Y chromosome (Yq12). These studies showed that the two 1p telomeric loci are located near the nuclear surface. The chromosome 9 centromeric loci are similarly located. Simultaneous hybridization of the chromosome 1 telomeric probe (target size approximately 200 kb; b, base) and the Y-specific probe (target size greater than 2Mb), demonstrate that the binding sites of the two probes can be distinguished in the same nucleus on the basis of domain size.

Animals↗
PubMed1989

Chromosome heteromorphism quantified by high-resolution bivariate flow karyotyping.

Maternal and paternal homologues of many chromosome types can be differentiated on the basis of their peak position in Hoechst 33258 versus chromomycin A3 bivariate flow karyotypes. We demonstrate here the magnitude of DNA content differences among normal chromosomes of the same type. Significant peak-position differences between homologues were observed for an average of four chromosome types in each of the karyotypes of 98 different individuals. The frequency of individuals with differences in homologue peak positions varied among chromosome types: e.g., chromosome 15, 61%; chromosome 3, 4%. Flow karyotypes of 33 unrelated individuals were compared to determine the range of peak position among normal chromosomes. Chromosomes Y, 21, 22, 15, 16, 13, 14, and 19 were most heteromorphic, and chromosomes 2-8 and X were least heteromorphic. The largest chromosome 21 was 45% larger than the smallest 21 chromosome observed. The base composition of the variable regions differed among chromosome types. DNA contents of chromosome variants determined from flow karyotypes were closely correlated to measurements of DNA content made of gallocyanin chrome alum-stained metaphase chromosomes on slides. Fluorescence in situ hybridization with chromosome-specific repetitive sequences indicated that variability in their copy number is partly responsible for peak-position variability in some chromosomes. Heteromorphic chromosomes are identified for which parental flow karyotype information will be essential if de novo rearrangements resulting in small DNA content changes are to be detected with flow karyotyping.

Bisbenzimidazole↗
PubMed1989

Choroideremia and deafness with stapes fixation: a contiguous gene deletion syndrome in Xq21.

The study of contiguous gene deletion syndromes by using reverse genetic techniques provides a powerful tool for precisely defining the map location of the genes involved. We have made use of individuals with overlapping deletions producing choroideremia as part of a complex phenotype, to define the boundaries on the X chromosome for this gene, as well as for X-linked mixed deafness with perilymphatic gusher (DFN3). Two patients with deletions and choroideremia are affected by an X-linked mixed conductive/sensorineural deafness; one patient, XL-62, was confirmed at surgery to have DFN3, while the other patient, XL-45, is suspected clinically to have the same disorder. A third choroideremia deletion patient, MBU, has normal hearing. Patient XL-62 has a cytogenetically detectable deletion that was measured to be 7.7% of the X chromosome by dual laser flow cytometry; the other patient, XL-45, has a cytogenetically undetectable deletion that measures only 3.3% of the X chromosome. We have produced a physical map of the X-chromosome region containing choroideremia and DFN3 by using routine Southern blotting, chromosome walking and jumping techniques, and long-range restriction mapping to generate and link anonymous DNA sequences in this region. DXS232 and DXS233 are located within 450 kb of each other on the same SfiI and MluI fragments and share partial SalI fragments of 750 and greater than 1,000 kb but are separated by at least one SalI site. In addition, DXS232, which lies outside the MBU deletion, detects the proximal breakpoint of this deletion. We have isolated two new anonymous DNA sequences by chromosome jumping from DXS233; one of these detects a new SfiI fragment distal to DXS233 in the direction of the choroideremia gene, while the other jump clone is proximal to DXS233 and detects a new polymorphism. These data refine the map around the loci for choroideremia and for mixed deafness with stapes fixation and will provide points from which to isolate candidate gene sequences for these disorders.

Adolescent↗
PubMed1989

Inheritance of chromosome heteromorphisms analyzed by high-resolution bivariate flow karyotyping.

The DNA content of the mitotic chromosomes from 10 children and their parents in four families were quantified by bivariate flow karyotyping. In all cases, each chromosome peak in the flow karyotype of the child could be traced to one of the two parents. The measured absolute difference in homologue DNA content between children and their parents averaged 0.8%, or approximately 1 Mbp over all chromosome types. This study demonstrates that flow karyotypes of a proband's parents can be an aid in detecting and quantifying the size of de novo deletions that involve heteromorphic chromosome types.

Chromosomes, Human↗
PubMed1988

Prenatal detection of 46,XY,rec(5),dup q, inv(5)(p13q33) using DNA analysis, flow cytometry, and in situ hybridization to supplement classical cytogenetic analysis.

Distinguishing between balanced and unbalanced chromosome complements segregating from parental rearrangements may be difficult using only classical cytogenetic techniques if banding morphology is similar under both expectations. In these situations, supplementing cytogenetic analysis with molecular genetic techniques and flow cytometry may provide increased diagnostic accuracy. To illustrate this, we present a case in which similar band pattern morphology would be expected for both the balanced carrier (heterozygote) and the recombinant dup q chromosome complements segregating from a mother with a balanced inversion [46,XX,inv(5)(p13q33)]. The parents came to Northwestern for consultation after receiving conflicting interpretations of their first amniotic fluid cultures. An ultrasound examination was said to be normal. They inquired whether there were ways to increase their confidence that the complement was unbalanced. Their reluctance to terminate the pregnancy was due to a 6-year history of infertility. After extensive counselling, the couple elected repeat amniocentesis. Further cytogenetic analysis of repeat amniotic fluid cultures by G-banding and R-banding, molecular genetic analysis with highly polymorphic DNA probes, and quantitative flow cytometry were performed. Results agreed that an unbalanced fetal complement was present. Southern blot analysis with a 5p marker definitively demonstrated a lack of maternal 5p material in the fetus, and in situ hybridization showed a 5q marker at either end of the recombinant chromosome. Flow cytometry was consistent with this interpretation. Because of the advanced gestational age, the parents elected to terminate based on cytogenic results of the second amniocentesis, rather than to wait another 1-2 weeks for results of other methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1988

Improved resolution of flow cytometric measurements of Hoechst- and chromomycin-A3-stained human chromosomes after addition of citrate and sulfite.

The resolution of bivariate flow karyotypes of human chromosomes stained with Hoechst 33258 and chromomycin A3 can be increased by adding sodium citrate and sodium sulfite to the chromosomes shortly before measurement. A flow karyotype of a patient with chronic myelocytic leukemia is shown to illustrate that the addition of these compounds allows high-resolution measurements to be made and evaluated reliably from clinical samples.

Benzimidazoles↗