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Biomedical subjects

B J Trask

Publications and source records attributed to B J Trask.

At least 19 recordsLinked to original sources
PubMed1995

Direct selection of cDNAs using whole chromosomes.

We have developed a method for direct selection of cDNAs using whole chromosomes as target DNA. Double-strand cDNAs were synthesized from human fetal brain polyadenylated mRNAs. Flow-sorted chromosomes 17 and 19 were amplified by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and used to capture ds cDNAs by an improved magnetic bead capture protocol. To demonstrate the capabilities of this method, the selected cDNAs were used as probes in FISH experiments. The selected cDNA populations specifically painted chromosomes 17 or 19 on metaphase spreads. These results demonstrate that it is possible to do chromosome painting using cDNA probes and that this method is a means to rapidly select expressed sequences encoded by any portion of the genome.

Brain↗
PubMed1995

Assignment of the human p27Kip1 gene to 12p13 and its analysis in leukemias.

The p27Kip1 (p27) gene encodes an inducible inhibitor of cyclin-dependent kinase activity. Using a murine p27 cDNA as probe, we obtained a human cDNA clone and subsequently used it to isolate a genomic clone of this gene. The coding region of the human p27 gene was contained in two exons. Both the amino acid sequence and intron-exon organization of p27 were similar to those previously found for the related cyclin-dependent kinase inhibitor p21Waf1 (p21). The p27 gene was localized to chromosome band 12p13 by a combination of somatic cell hybrid and fluorescence in situ hybridization analyses. The p27 gene product is thought to control the leukocyte cell cycle and the 12p13 chromosomal band is known to be deleted in leukemias, suggesting that the p27 gene may act as a tumor suppressor gene in leukemias. Although p27 was found to reside in the minimal region of chromosomal loss in hematological malignancies, no mutations of p27 were observed in leukemia samples. Haploinsufficiency of p27 may confer a growth advantage to leukemia cells.

Base Sequence↗
PubMed1995

The gene encoding the VP16-accessory protein HCF (HCFC1) resides in human Xq28 and is highly expressed in fetal tissues and the adult kidney.

After herpes simplex virus (HSV) infection, the viral regulatory protein VP16 activates transcription of the HSV immediate-early promoters by directing complex formation with two cellular proteins, the POU-homeodomain transcription factor Oct-1 and the host cell factor HCF. The function of HCF in uninfected cells is unknown. Here we show by fluorescence in situ hybridization and somatic cell hybrid analysis that the gene encoding human HCF, HCFC1, maps to the q28 region of the X chromosome. Yeast artificial chromosome and cosmid mapping localizes the HCFC1 gene within 100 kb distal of the renal vasopressin type-2 receptor (V2R) gene and adjacent to the renin-binding protein gene (RENBP). The HCFC1 gene is apparently unique. HCF transcripts and protein are most abundant in fetal and placental tissues and cell lines, suggesting a role in cell proliferation. In adults, HCF protein is abundant in the kidney, but not in the brain, a site of latent HSV infection and where HCF levels may influence progression of HSV infection.

Animals↗
PubMed1995

Treatment of cells with alkaline borate buffer extends the capability of interphase FISH mapping.

Interphase fluorescence in situ hybridization (FISH) has been shown to be a means to map DNA sequences relative to each other in the 100 kb to 1-2 Mb genomic-separation range. At distances below 0.1 Mb, probe sites are infrequently resolved in interphase chromatin. In the 0.1- to 1-Mb range, interphase chromatin can be modeled as a freely flexible chain. The mean square interphase distance between two probes is proportional to the genomic separation between the probes on the linear DNA molecule. Above 1-2 Mb, the relationship between interphase distance and genomic separation changes abruptly and appears to level off. We have used alkaline-borate treatment to expand the capability of interphase FISH mapping. We show here that alkaline-borate treatment increases nuclear diameter, the interphase distance between probes on homologous chromosomes, and the distance between probes on the same chromosome. We also show that the mean square distance between hybridization sites in borate-treated nuclei is proportional to genomic separation up to 4 Mb. Thus, alkaline-borate treatment enhances the capability of interphase FISH mapping by increasing the absolute distance between probes and extending the range of the simple relationship between interphase distance and genomic separation.

Borates↗
PubMed1995

Evidence for the organization of chromatin in megabase pair-sized loops arranged along a random walk path in the human G0/G1 interphase nucleus.

We determined the folding of chromosomes in interphase nuclei by measuring the distance between points on the same chromosome. Over 25,000 measurements were made in G0/G1 nuclei between DNA sequences separated by 0.15-190 megabase pairs (Mbp) on three human chromosomes. The DNA sequences were specifically labeled by fluorescence in situ hybridization. The relationship between mean-square interphase distance and genomic separation has two linear phases, with a transition at approximately 2 Mbp. This biphasic relationship indicates the existence of two organizational levels at scales > 100 kbp. On one level, chromatin appears to be arranged in large loops several Mbp in size. Within each loop, chromatin is randomly folded. On the second level, specific loop-attachment sites are arranged to form a supple, backbonelike structure, which also shows characteristic random walk behavior. This random walk/giant loop model is the simplest model that fully describes the observed large-scale spatial relationships. Additional evidence for large loops comes from measurements among probes in Xq28, where interphase distance increases and then locally decreases with increasing genomic separation.

Cell Cycle↗
PubMed1995

Molecular analysis of a novel subtelomeric repeat with polymorphic chromosomal distribution.

DNA from a 50-kb yeast artificial chromosome (YAC) containing one human telomere was characterized. Cloned sequences from the centromeric end of this YAC (designated yRM2001) localized to several human chromosomes by somatic hybrid panel mapping. The telomeric end of the YAC contained both (TTAGGG)n sequences and the previously characterized TelBam3.4 subterminal repeat element. A novel low-copy repeat element (designated HC1103) mapped 19 kb from the telomeric end of the YAC. This repeat was shown by fluorescence in situ hybridization to be present in several subtelomeric regions (3q, 12p, 15q, 19p, and 20p) and at an interstitial site (2q13-->q14) in all individuals studied, but to be polymorphically distributed at several other telomeres. The YAC vector-insert EcoRI cloning site was positioned 50 kb to 70 kb from chromosome termini in human genomic DNA using RecA-assisted restriction endonuclease (RARE) cleavage analysis. Our results suggest that the DNA segment cloned in yRM2001 contains a novel block of low-copy DNA consistently present at some human telomeres, but polymorphically distributed at others.

Base Sequence↗
PubMed1995

The 18q- syndrome: analysis of chromosomes by bivariate flow karyotyping and the PCR reveals a successive set of deletion breakpoints within 18q21.2-q22.2.

The 18q- syndrome is one of several terminal deletion disorders that occur in humans. Previous G-banding studies suggest that the loss of a critical band, 18q21.3, results in mental retardation, craniofacial anomalies, and metabolic defects. However, it is difficult to reconcile the consistent loss of a single region with the large variability in clinical phenotype. The purpose of this study was to reassess the extent of chromosomal loss in a cohort of 17 18q- syndrome patients by using fluorescent-activated chromosome sorting, PCR, and FISH. Bivariate flow karyotypes revealed heterogeneity among the deletions; they ranged in size from 9 to 26 Mb. To confirm this heterogeneity at a molecular level, deleted and normal chromosomes 18 of six patients were collected by flow sorting, preamplified by random priming, and assayed for marker content by the PCR. This analysis defined five unique breakpoints among the six patients. We conclude that the terminal deletions in the 18q- syndrome occur over a broad region spanning the interval from 18q21.2 to 18q22.2. Our results suggest that the variability in clinical phenotype may be more representative of a contiguous-gene syndrome with a baseline deficit of 18q22.2-qter than of the loss of a single critical region within 18q21.3.

Chromosome Deletion↗
PubMed1994

Cloning, sequencing, and mapping of the human chromosome 14 heat shock protein gene (HSPA2).

A genomic clone for the human heat shock protein (HSP) 70 gene located on chromosome 14 was isolated and sequenced. The gene, designated HSPA2, has a single open reading frame of 1917 bp that encodes a 639-amino acid protein with a predicted molecular weight of 70,030 Da. Analysis of the sequence indicates that HSPA2 is the human homologue of the murine Hsp70-2 gene with 91.7% identity in the nucleotide coding sequence and 98.2% in the corresponding amino acid sequence. HSPA2 has less amino acid homology to other members of the human HSP70 gene family, 83.3% to the heat-inducible HSP70-1 gene and 86.1% with the human heat shock cognate gene HSC70. HSPA2 is constitutively expressed in most tissues, with very high levels in testis and skeletal muscle. Significant but lower levels are also expressed in ovary, small intestine, colon, brain, placenta, and kidney. A yeast artificial chromosome (YAC) clone containing HSPA2 (YAC741H4) that also contained the polymorphic marker D14S63 was identified. This 670-kb YAC was mapped to 14q24.1 by fluorescence in situ hybridization (FISH). Subsequent two-color FISH and genetic mapping placed HSPA2/D14S63 proximal to the markers D14S57 and D14S77.

Amino Acid Sequence↗
PubMed1993

Discordant phenotype of two overlapping deletions involving the PAX3 gene in chromosome 2q35.

Waardenburg syndrome (WS), the most common form of inherited congenital deafness, is a pleiotropic, autosomal dominant condition with variable penetrance and expressivity. WS is clinically and genetically heterogeneous. The basis for the phenotypic variability observed among and between WS families is unknown. However, mutations within the paired-box gene, PAX3, have been associated with a subset of WS patients. In this report we use cytogenetic and molecular genetic techniques to study a patient with WS type 3, a form of WS consisting of typical WS type 1 features plus mental retardation, microcephaly, and severe skeletal anomalies. Our results show that the WS3 patient has a de novo paternally derived deletion, del (2)(q35q36), that spans the genetic loci PAX3 and COL4A3. A molecular analysis of a chromosome 2 deletional mapping panel maps the PAX3 locus to 2q35 and suggests the locus order: centromere-(INHA, DES)-PAX3-COL4A3-(ALPI, CHRND)-telomere. Our analyses also show that a patient with a cleft palate and lip pits, but lacking diagnostic WS features, has a deletion, del (2)(q33q35), involving the PAX3 locus. This result suggests that not all PAX3 mutations are associated with a WS phenotype and that additional regional loci may modify or regulate the PAX3 locus and/or the development of a WS phenotype.

Bone and Bones↗
PubMed1993

Studies of metaphase and interphase chromosomes using fluorescence in situ hybridization.

Our measurements have bearing on the resolution with which maps can be constructed and abnormalities can be detected by studying the proximity of DNA sequences in metaphase and interphase chromosomes. The results of our analyses are summarized in Figure 8. Metaphase chromosomes are compacted sufficiently that it is impractical to order sequences separated by less than approximately 1 Mbp. In contrast, 100-kbp resolution can be obtained in interphase chromosomes. Distance measurements reveal that interphase chromatin behaves as a random polymer over distances up to 1-2 Mbp. At greater distances, higher order constraints, perhaps the dimensions of the individual chromosome domains, come into play. A caveat remains: Because the effect of the FISH procedure on native chromosome organization is not well understood, these conclusions may not be applicable to native chromatin. We have illustrated that FISH, with appropriately chosen probes, can supplement conventional cytogenetics in the study of chromosome abnormalities. The technique is increasingly being applied in research laboratories to detect and characterize chromosome abnormalities and point the way to the location of genes involved in human disease.

Chromosome Aberrations↗
PubMed1992

Estimating genomic distance from DNA sequence location in cell nuclei by a random walk model.

The folding of chromatin in interphase cell nuclei was studied by fluorescent in situ sequences chromatin according to a random walk model. This model provides the basis for calculating the spacing of sequences along the linear DNA molecule from interphase distance measurements. An interphase mapping strategy based on this model was tested with 13 probes from a 4-megabase pair (Mbp) region of chromosome 4 containing the Huntington disease locus. The results confirmed the locations of the probes and showed that the remaining gap in the published maps of this region is negligible in size. Interphase distance measurements should facilitate construction of chromosome maps with an average marker density of one per 100 kbp, approximately ten times greater than that achieved by hybridization to metaphase chromosome. achieved by hybridization to metaphase chromosomes.

Cell Nucleus↗
PubMed1992

Isolation and characterization of the cDNA encoding human DNA methyltransferase.

We have cloned a series of overlapping cDNA clones encoding a 5194 bp transcript for human DNA methyltransferase (DNA MTase). This sequence potentially codes for a protein of 1495 amino acids with a predicted molecular weight of 169 kDa. The human DNA MTase cDNA has eighty percent homology at the nucleotide level, and the predicted protein has seventy-four percent identity at the amino acid level, to the DNA MTase cDNA cloned from mouse cells. Like the murine DNA MTase, the amino terminal two-thirds of the human protein contains a cysteine-rich region suggestive of a metal-binding domain. The carboxy terminal one-third of the protein shows considerable similarity to prokaryotic (cytosine-5)-methyltransferases. The arrangement of multiple motifs conserved in the prokaryotic genes is preserved in the human DNA MTase, including the relative position of a proline-cysteine dipeptide thought to be an essential catalytic site in all (cytosine-5)-methyltransferases. A single 5.2 kb transcript was detected in all human tissues tested, with the highest levels of expression observed in RNA from placenta, brain, heart and lung. DNA MTase cDNA clones were used to screen a chromosome 19 genomic cosmid library. The DNA MTase-positive cosmids which are estimated to span a genomic distance of 93 kb have been localized to 19p13.2-p13.3 by fluorescence in situ hybridization. Isolation of the cDNA for human DNA MTase will allow further study of the regulation of DNA MTase expression, and of the role of this enzyme in establishing DNA methylation patterns in both normal and neoplastic cells.

Amino Acid Sequence↗
PubMed1992

Somatic cell hybrids, sequence-tagged sites, simple repeat polymorphisms, and yeast artificial chromosomes for physical and genetic mapping of proximal 17p.

Somatic cell hybrids retaining the deleted chromosome 17 from 15 unrelated Smith-Magenis syndrome (SMS) [del(17)(p11.2p11.2)] patients were obtained by fusion of patient lymphoblasts with thymidine kinase-deficient rodent cell lines. Seventeen sequence-tagged sites (STSs) were developed from anonymous markers and cloned genes mapping to the short arm of chromosome 17. The STSs were used to determine the deletion status of these loci in these and four previously described human chromosome 17-retaining hybrids. Ten STSs were used to identify 28 yeast artificial chromosomes (YACs) from the St. Louis human genomic YAC library. Four of the 17 STSs identified simple repeat polymorphisms. The order and location of deletion breakpoints were confirmed and refined, and the regional assignment of several probes and cloned genes were determined. The cytogenetic band locations and relative order of six markers on 17p were established by fluorescence in situ hybridization mapping to metaphase chromosomes. The latter data confirmed and supplemented the somatic cell hybrid results. Most of the hybrids derived from [del(17)(p11.2p11.2)] patients demonstrated a similar pattern of deletion for the marker loci and were deleted for D17S446, D17S258, D17S29, D17S71, and D17S445. However, one of them demonstrated a unique pattern of deletion. This patient is deleted for several markers known to recognize a large DNA duplication associated with Charcot-Marie-Tooth (CMT) disease type 1A. These data suggest that the proximal junction of the CMT1A duplication is close to the distal breakpoint in [del(17)(p-11.2p11.2)] patients.

Abnormalities, Multiple↗
PubMed1992

Fluorescence in situ hybridization establishes the order cen-DXS28(C7)-DXS67(B24)-DXS68(L1)-tel in human chromosome Xp21.3.

We report here on the order of three DNA markers, C7, B24, and L1, based on the arrangement of their fluorescently labeled hybridization sites in interphase cell nuclei. The three markers map distal to the Duchenne muscular dystrophy (DMD), glycerol kinase deficiency (GKD), and adrenal hypoplasia (AHC) loci on human chromosome Xp21.3. Their order has been a matter of controversy. In interphase chromatin, B24 maps between C7 and L1. We estimate from interphase distance that C7 and L1 are 300-500 kb apart. When the three markers are hybridized to interphase cells of Nijmegen1, a patient with DMD, GKD, and AHC, only C7 appears to be deleted, rather than both C7 and L1, as had been reported elsewhere. C7 is also the only one of the three markers deleted in several other DMD patients studied by others. The deletion results indicate that C7 is the most proximal of the three markers and allow the trio of ordered probes to be oriented on the chromosome: cen-C7(DXS28)-B24(DXS67)-L1(DXS68)-tel.

DNA Probes↗
PubMed1992

Order and genomic distances among members of the carcinoembryonic antigen (CEA) gene family determined by fluorescence in situ hybridization.

Fluorescence in situ hybridization was used to establish the order of, and to estimate genomic distances among, members of the carcinoembryonic antigen (CEA) and pregnancy-specific glycoprotein (PSG) subgroups on chromosome 19. Fluorescence in situ hybridization to metaphase chromosomes localized the PSG subgroup telomeric to the CEA subgroup. Cosmid clones containing sequences for individual genes in the CEA and PSG subgroups were also hybridized to human sperm pronuclear and somatic interphase nuclear chromatin targets. The mapping results lead to the gene order cen-CGM7-CEA-NCA-CGM1-BGP-CGM9-CGM8-PSG-te l. The genomic distances between selected pairs of gene family members were estimated from the physical distances between hybridization sites measured in pronuclei. The CEA-PSG gene family region is estimated to span 1.1 to 1.2 Mb.

Carcinoembryonic Antigen↗
PubMed1992

Assignment of the gene encoding DNA ligase I to human chromosome 19q13.2-13.3.

The gene encoding DNA ligase I has been mapped on human chromosome 19 by analysis of rodent-human somatic cell hybrids informative for this chromosome and by two-color fluorescence in situ hybridization. The DNA ligase I gene (LIG1) is localized to 19q13.2-13.3 and is distal to ERCC1, the most telomeric of three DNA repair genes on this chromosome.

Blotting, Southern↗
PubMed1992

Evolution of the mammalian G protein alpha subunit multigene family.

Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce extracellular signals received by transmembrane receptors to effector proteins. The multigene family of G protein alpha subunits, which interact with receptors and effectors, exhibit a high level of sequence diversity. In mammals, 15 G alpha subunit genes can be grouped by sequence and functional similarities into four classes. We have determined the murine chromosomal locations of all 15 G alpha subunit genes using an interspecific backcross derived from crosses of C57BL/6J and Mus spretus mice. These data, in combination with mapping studies in humans, have provided insight into the events responsible for generating the genetic diversity found in the mammalian alpha subunit genes and a framework for elucidating the role of the G alpha subunits in disease.

Animals↗