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D Housman

Publications and source records attributed to D Housman.

At least 73 records · Page 4Linked to original sources
PubMed1990

The candidate Wilms' tumour gene is involved in genitourinary development.

Wilms' tumour is an embryonic kidney tumour thought to arise through aberrant mesenchymal stem cell differentiation and to result from loss of function of a 'tumour suppressor' gene(s). Both sporadic and syndrome-associated Wilms' tumours are accompanied by an increased frequency of abnormalities of the urinary tract and genitalia. Deletional analysis of individuals with the WAGR syndrome (for, Wilms' tumour, aniridia, genitourinary abnormalities and mental retardation) showed that a Wilms' tumour gene lies at chromosomal position 11p13. This led to the isolation of a candidate Wilms' tumour gene, encoding a zinc-finger protein which is likely to be a transcription factor. To gain insight into the role of this candidate gene in normal development and tumorigenesis, we have now performed in situ messenger RNA hybridization on sections of human embryos and Wilms' tumours. The candidate Wilms' tumour gene is expressed specifically in the condensed mesenchyme, renal vesicle and glomerular epithelium of the developing kidney, in the related mesonephric glomeruli and in cells approximating these structures in tumours. The other main sites of expression are the genital ridge, fetal gonad and mesothelium. These data suggest that (1) this candidate is indeed a Wilms' tumour gene, (2) the associated genital abnormalities are pleiotropic effects of mutation in the Wilms' tumour gene itself, in support of recent genetic analysis, and (3) this gene has a specific role in kidney development and a wider role in mesenchymal-epithelial transitions.

Blotting, Northern↗
PubMed1990

High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones.

Cosmid clones containing human DNA inserts have been mapped on chromosome 11 by fluorescence in situ hybridization under conditions that suppress signal from repetitive DNA sequences. Thirteen known genes, one chromosome 11-specific DNA repeat, and 36 random clones were analyzed. High-resolution mapping was facilitated by using digital imaging microscopy and by analyzing extended (prometaphase) chromosomes. The map coordinates established by in situ hybridization showed a one to one correspondence with those determined by Southern (DNA) blot analysis of hybrid cell lines containing fragments of chromosome 11. Furthermore, by hybridizing three or more cosmids simultaneously, gene order on the chromosome could be established unequivocally. These results demonstrate the feasibility of rapidly producing high-resolution maps of human chromosomes by in situ hybridization.

Blotting, Southern↗
PubMed1990

Multidrug resistance gene expression is controlled by steroid hormones in the secretory epithelium of the uterus.

The multidrug resistance (mdr) gene family has been shown to encode a membrane glycoprotein, termed the P-glycoprotein, which functions as a drug efflux pump with broad substrate specificity. This multigene family is expressed in a tissue-specific fashion in a wide variety of normal and neoplastic tissues. The regulation of mdr gene expression in normal tissues is not understood. We have recently shown that mdr mRNA and the P-glycoprotein increases dramatically in the secretory luminal and glandular epithelium of the gravid murine uterus. This observation has suggested that mdr gene expression in the uterus is controlled by the physiologic changes associated with pregnancy. This report now demonstrates that mdr mRNA and P-glycoprotein are induced at high levels in the uterine secretory epithelium by the combination of estrogen and progesterone, the major steroid hormones of pregnancy. This regulation of mdr gene expression in the uterus does not require any other contribution from the fetus or placenta. The data indicate that this gene locus is hormonally responsive to estrogen and progesterone in the uterine secretory epithelium, suggesting an important and physiologically regulated role during pregnancy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

A panel of irradiation-reduced hybrids selectively retaining human chromosome 11p13: their structure and use to purify the WAGR gene complex.

The irradiation-fusion technique offers a means to isolate intact subchromosomal fragments of one mammalian species in the genetic background of another. Irradiation-reduced somatic cell hybrids can be used to construct detailed genetic and physical maps of individual chromosome bands and to systematically clone genes responsible for hereditary diseases on the basis of their chromosomal position. To assess this strategy, we constructed a panel of hybrids that selectively retain the portion of human chromosome band 11p13 that includes genes responsible for Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (constituting the WAGR syndrome). A hamster-human hybrid containing the short arm of chromosome 11 as its only human DNA (J1-11) was gamma-irradiated and fused to a Chinese hamster cell line (CHO-K1). We selected secondary hybrid clones that express MIC1 but not MER2, cell-surface antigens encoded by bands 11p13 and 11p15, respectively. These clones were characterized cytogenetically by in situ hybridization with human repetitive DNA and were tested for their retention of 56 DNA, isozyme, and antigen markers whose order on chromosome 11p is known. These cell lines appear to carry single, coherent segments of 11p spanning MIC1, which range in size from 3000 kb to more than 50,000 kb and which are generally stable in the absence of selection. In addition to the selected region of 11p13, two cell lines carry extra fragments of the human centromere and two harbor small, unstable segments of 11p15. As a first step to determine the size and molecular organization of the WAGR gene complex, we analyzed a subset of reduced hybrids by pulsed-field gel electrophoresis. A small group of NotI restriction fragments comprising the WAGR complex was detected in Southern blots with a cloned Alu repetitive probe. One of the cell lines (GH3A) was found to carry a stable approximately 3000-kb segment of 11p13 as its only human DNA. The segment encompasses MIC1, a recurrent translocation breakpoint in acute T-cell leukemia (TCL2), and most or all of the WAGR gene complex, but does not include the close flanking markers D11S16 and delta J. This hybrid forms an ideal source of molecular clones for the developmentally fascinating genes underlying the WAGR syndrome.

Animals↗
PubMed1990

Monochromosomal rodent-human hybrids from microcell fusion of human lymphoblastoid cells containing an inserted dominant selectable marker.

An improved system for the production of a series of rodent-human hybrids selectively retaining single human chromosomes marked in known locations is described. Such hybrids have significant applications in gene mapping and other genetic studies. Human lymphoblastoid lines were infected with the retroviral vector SP-1, which contains the bacterial his-D gene allowing mammalian cells to grow in the presence of histidinol. Microcell fusion of the infected lymphoblastoid cells with CHO cells was used to produce hybrids containing single human chromosomes retained by histidinol selection. Hybrids containing a single human chromosome 9 and a single human chromosome 19 are described. These have been characterized cytogenetically by G-banding, in situ hybridization, and Southern blot analysis.

Animals↗
PubMed1989

A fine-structure deletion map of human chromosome 11p: analysis of J1 series hybrids.

Deletion analysis offers a powerful alternative to linkage and karyotypic approaches for human chromosome mapping. A panel of deletion hybrids has been derived by mutagenizing J1, a hamster cell line that stably retains chromosome 11 as its only human DNA, and selecting for loss of MIC1, a surface antigen encoded by a gene in band 11p13. A unique, self-consistent map was constructed by analyzing the pattern of marker segregation in 22 derivative cells lines; these carry overlapping deletions of 11p13, but selectively retain a segment near the 11p telomere. The map orders 35 breakpoints and 36 genetic markers, including 3 antigens, 2 isozymes, 12 cloned genes, and 19 anonymous DNA probes. The deletions span the entire short arm, dividing it into more than 20 segments and define a set of reagents that can be used to rapidly locate any newly identified marker on 11p, with greatest resolution in the region surrounding MIC1. The approach we demonstrate can be applied to map any mammalian chromosome. To test the gene order, we examined somatic cell hybrids from five patients, whose reciprocal translocations bisect band 11p13; these include two translocations associated with familial aniridia and two with acute T-cell leukemia. In each patient, the markers segregate in telomeric and centromeric groups as predicted by the deletion map. These data locate the aniridia gene (AN2) and a recurrent T-cell leukemia breakpoint (TCL2) in the marker sequence, on opposite sides of MIC1. To provide additional support, we have characterized the dosage of DNA markers in a patient with Beckwith-Wiedemann syndrome and an 11p15-11pter duplication. Our findings suggest the following gene order: TEL - (HRAS1, MER2, CTSD, TH/INS/IGF2, H19, D11S32) - (RRM1, D11S1, D11S25, D11S26) - D11S12 - (HBBC, D11S30) - D11S20 - (PTH, CALC) - (LDHA, SAA, TRPH, D11S18, D11S21) - D11S31 - D11S17 - HBVS1 - (FSHB, D11S16) - AN2 - MIC1 - TCL2 - delta J - CAT - MIC4 - D11S9 - D11S14 - ACP2 - (D11S33, 14L) - CEN. We have used the deletion map to show the distribution on 11p of two centromeric repetitive elements and the low-order interspersed repeat A36Fc. Finally, we provide evidence for an allelic segregation event in the hamster genome that underlies the stability of chromosome 11 in J1. The deletion map provides a basis to position hereditary disease loci on 11p, to distinguish the pattern of recessive mutations in different forms of cancer and, since many of these genes have been mapped in other mammalian species, to study the evolution of a conserved syntenic group.

Animals↗
PubMed1989

A highly polymorphic locus cloned from the breakpoint of a chromosome 11p13 deletion associated with the WAGR syndrome.

Children with constitutional deletions of chromosome 11p13 suffer from aniridia, genitourinary malformations, and mental retardation and are predisposed to develop bilateral Wilms tumor (the WAGR syndrome). The critical region for these defects has been narrowed to a segment of band 11p13 between the catalase and the beta-follicle-stimulating hormone genes. In this report, we have cloned the endpoints from a WAGR patient whose large cytogenetic deletion, del(11)(p14.3::p13), does not include the catalase gene. The deletion was characterized using DNA polymorphisms and found to originate in the paternally derived chromosome 11. The distal endpoint was identified as a rearrangement of locus D11S21 in conventional Southern blots of the patient's genomic DNA, but was not detected in leukocyte DNA from either parent or in sperm DNA from the father. The proximal endpoint was isolated by cloning the junction fragment and was mapped in relation to other markers and breakpoints. It defines a new locus in 11p13-delta J, which is close to the Wilms tumor gene and the breakpoint cluster region (TCL2) of the frequent t(11;14)(p13;q11) translocation in acute T-cell leukemia. An unusual concentration of base pair substitutions was discovered at delta J, in which 9 of 44 restriction sites tested (greater than 20%) vary in the population. This property makes delta J one of the most polymorphic loci on chromosome 11 and may reflect an underlying instability that contributed to the original mutation. The breakpoint extends the genetic map of this region and provides a useful marker for linkage studies and the analysis of allelic segregation in tumor cells.

Adult↗
PubMed1989

Suicidal behavior in "normal" adolescents: risk and protective factors.

Risk and protective factors were examined in suicidal and nonsuicidal public high school students. With life stress and depression as independent risk factors, family cohesion was found to offset the effect of stress, and friendships to have a more indirect effect. Differential effects of ten sources of stress were analyzed from a developmental perspective, and the probability of suicidal behavior associated with clusters of factors was estimated for the general population.

Achievement↗
PubMed1989

Identification of a region within the Na,K-ATPase alpha subunit that contributes to differential ouabain sensitivity.

To analyze determinants within the Na,K-ATPase alpha subunit that contribute to differential ouabain sensitivity, we constructed and expressed a panel of chimeric cDNA molecules between ouabain-resistant and ouabain-sensitive alpha subunit cDNAs. When introduced into ouabain-sensitive monkey CV-1 cells, ouabain-resistant rat alpha 1 subunit cDNA and chimeras in which the 5' end of ouabain-sensitive human alpha 1 or rat alpha 2 subunit cDNA was replaced by the 5' end of rat alpha 1 subunit cDNA conferred resistance to 100 microM ouabain. Monkey cells transfected with the reciprocal chimeras were unable to survive selection in 1 microM ouabain. Rat alpha 2 subunit cDNA and a chimera in which the 5' end of rat alpha 1 subunit cDNA was replaced by the 5' end of rat alpha 2 subunit cDNA conferred resistance to 0.5 microM ouabain. These results suggest that determinants of ouabain resistance reside within the amino-terminal portions of the rat alpha 1 and alpha 2 subunits. Expression of chimeric alpha subunit cDNAs should prove useful for elucidating the structural basis of Na,K-ATPase function.

Animals↗
PubMed1988

Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit.

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.

Algorithms↗
PubMed1988

The gene encoding multidrug resistance is induced and expressed at high levels during pregnancy in the secretory epithelium of the uterus.

A survey of the expression of the multidrug-resistance gene (mdr) in mouse tissues revealed that a mdr mRNA species is expressed at extremely high levels in the gravid uterus. mdr mRNA expression levels increase dramatically during pregnancy compared to the relatively low levels of expression observed in the nongravid uterus. In situ hybridization experiments revealed that the increased expression of the mdr mRNA is specifically localized to the secretory epithelial cells of the endometrium. Immunocytochemistry studies with a mdr glycoprotein-specific antiserum demonstrate that the mdr glycoprotein is predominantly localized to the luminal surface of the secretory epithelial cells. These results indicate that the mdr gene expression in the uterus is controlled by the physiologic changes associated with pregnancy. Our data are consistent with a potential role for the mdr glycoprotein in the transport of substrate across the secretory epithelium of the gravid uterus.

Animals↗
PubMed1988

Cloning and characterization of a second member of the mouse mdr gene family.

The mammalian mdr gene family comprises a small number of closely related genes. Previously, we have shown that one member, mdr1, has the capacity to convey multidrug resistance to drug-sensitive recipient cells in a gene transfer protocol. However, the functional characteristics of other members of this gene family have not been examined. In this report, we characterize a second member of the mdr gene family which we designated mdr2. We determined the nucleotide sequence corresponding to the complete coding region of this mdr2 transcript. The predicted amino acid sequence of this protein (1,276 amino acids) showed that it is a membrane glycoprotein highly homologous to mdr1 (85%), strongly suggesting that both genes originate from a common ancestor. Regions of divergence between mdr1 and mdr2 proteins are concentrated in two discrete segments of the predicted polypeptides, each approximately 100 residues in length. The mdr2 protein appears to be formed by the duplication of a structural unit which encodes three putative transmembrane loops and a predicted nucleotide-binding fold and is highly homologous to bacterial transport proteins such as hlyB. This strong homology suggests that mdr2 also participates in an energy-dependent membrane transport process. However, the direct relationship, if any, of this new member of the mdr family to multidrug resistance remains to be established. Knowledge of the complete nucleotide sequence and predicted amino acid sequence of the mdr2 gene product will enable the preparation of gene-specific probes and antibodies necessary to study the functional role of this gene in multidrug resistance and normal physiological processes.

Amino Acid Sequence↗
PubMed1988

Restriction analysis of the structural alpha-L-fucosidase gene and its linkage to fucosidosis.

Human alpha-L-fucosidase is a lysosomal enzyme responsible for hydrolysis of alpha-L-fucoside linkages in fucoglycoconjugates. A single gene, FUCA 1, located on chromosome 1p34.1-1p36.1 encodes for alpha-L-fucosidase activity. To gain insight into the nature of the molecular defects leading to fucosidosis, we have characterized the genomic structure of FUCA 1. Restriction-endonuclease analysis suggests that at least seven exons dispersed over 22 kb are present in genomic FUCA 1. Two restriction-fragment-length polymorphisms (RFLPs) have been identified in the Caucasian population. The PvuII and BglI RFLPs each have two codominant alleles in Hardy-Weinberg equilibrium. Allele frequencies for the PvuII RFLP are .70/.30, and those for the BglI RFLP .63/.37. Both RFLPs are in strong linkage disequilibrium with each other, with a correlation coefficient of .94. The polymorphism information content (PIC) of the combined DNA markers is .38, high enough to be useful in the prenatal diagnosis of fucosidosis. The combined lod score for linkage between the fucosidosis mutation and FUCA 1 markers in two families was significant at a recombination fraction of 0. This suggests that the fucosidosis mutation resides in FUCA 1.

Blotting, Southern↗
PubMed1987

Analysis of human chromosome 11 by somatic cell genetics: reexamination of derivatives of human-hamster cell line J1.

Through the fusion of a CHO cell population to a human cell population, a hybrid cell line which has lost all human chromosomes except chromosome 11 was derived. This cell line, J1, does not appear to segregate human chromosome 11 during growth. A series of deletion segregants were isolated from J1 which had lost a portion of either the long, short, or both arms of chromosome 11. This panel of deletion segregants was used for mapping a number of genetic markers on the short arm of chromosome 11. Karyotypic analysis led to the interpretation that derivatives of J1 selected for the loss of cell surface antigens encoded by genes on the short arm of the chromosome had simple terminal deletions of this chromosome arm. More recently, we have applied recombinant DNA and in situ hybridization techniques to the analysis of the structure of chromosome 11. In the course of this analysis, we have obtained data that indicate that all J1 deletion segregants retain a small chromosomal segment containing the structural genes for insulin and HRAS1. Analysis of in situ hybridization data indicates that in cell lines in which a chromosome 11 fragment cannot be identified by karyotype analysis, human DNA has been translocated to a Chinese Hamster chromosome. These results suggest that the original interpretation of the karyotypes of deletion segregants derived from J1 as simple terminal deletions is not correct. A reanalysis of gene localization studies based on these deletion segregants suggests that some assignments of genes to specific bands on chromosome 11 should be reconsidered. In particular, data on additional deletion segregants are consistent with localization of the beta-globin gene complex to band 11p15. The data presented here suggest that in several hybrid derivatives of J1, a continuous DNA segment of approximately 10(7) base pairs in length which includes the insulin and HRAS1 (cellular homolog of retroviral oncogene Harvey ras) genes has been isolated from the remainder of the human genome. We propose that the stability of chromosome 11 in the original hybrid was due to complementation of a genetic defect in the original CHO cell parent by a gene located in close physical proximity to the insulin and HRAS1 genes on chromosome 11. Data are presented which test and support this hypothesis.

Animals↗
PubMed1987

Insertion of dominant selectable markers into the human genome.

In the above discussion, I have outlined the current status of our efforts to use retroviral vectors to introduce selectable markers into the human genome and to use these markers for mapping specific chromosomal regions. I have not reviewed in detail the extensive characterization of the mouse H-2 region carried out by David Nelson and John Weis based on the insertion of a single retroviral element. This analysis has provided a model for the application of retroviral elements into various regions of the human genome. The prospects for increasing the resolution of the human genetic map and identifying many genes relevant to human health and development are likely to be enhanced by increasing the precision of this methodology.

Chromosome Mapping↗
PubMed1986

The inhibition of commitment of mouse erythroleukemia cells by steroids involves a glucocorticoid-receptor mediated process(es) acting at the nuclear level.

Dexamethasone has been shown to inhibit dimethylsulfoxide (DMSO)-induced differentiation of mouse erythroleukemia (or Friend) cells by blocking commitment to terminal erythroid maturation. In this study, we confirmed previous reports indicating the presence of glucocorticoid receptors in murine erythroleukemia cells and examined the mechanism(s) by which steroids block commitment. Untreated murine erythroleukemia cells contain dexamethasone receptors which decrease in number during DMSO-induced cell differentiation. When steroids of different classes (estrogenic, androgenic, glucocorticoid) were tested for inhibition of commitment and for displacement of [3H]dexamethasone from its receptors in DMSO-treated cells, we observed that the glucocorticoids dexamethasone, prednisolone and hydrocortisone, all blocked commitment and substantially displaced [3H]dexamethasone. In contrast, steroids other than glucocorticoids failed to inhibit commitment or displace [3H]dexamethasone. Analysis of kinetics of dexamethasone binding to chromatin revealed that dexamethasone binds to the nucleus via the receptor and preferentially interacts with active chromatin. Inhibition of commitment by dexamethasone persisted in cells released from this agent and reincubated with DMSO in the presence of another glucocorticoid of similar affinity to steroid receptors; inhibition of commitment, however, was not obtained when cells removed from dexamethasone were incubated in the presence of beta-estradiol, progesterone and testosterone. These data indicate that inhibition of commitment of mouse erythroleukemia cells by steroids is associated with binding to glucocorticoid receptors and may involve interactions of steroids and their receptors with regions of chromatin.

Animals↗
PubMed1986

Mammalian multidrug resistance gene: complete cDNA sequence indicates strong homology to bacterial transport proteins.

The complete nucleotide and primary structure (1276 amino acids) of a full length mdr cDNA capable of conferring a complete multidrug-resistant phenotype is presented. The deduced amino acid sequence suggests that mdr is a membrane glycoprotein which includes six pairs of transmembrane domains and a cluster of potentially N-linked glycosylation sites near the amino terminus. A striking feature of the protein is an internal duplication that includes approximately 500 amino acids. Each duplicated segment includes a consensus ATP-binding site. Amino acid homology is observed between the mdr gene and a series of bacterial transport genes. This strong homology suggests that a highly conserved functional unit involved in membrane transport is present in the mdr polypeptide. We propose that an energy-dependent transport mechanism is responsible for the multidrug-resistant phenotype.

Amino Acid Sequence↗