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Biomedical subjects

D E Housman

Publications and source records attributed to D E Housman.

At least 73 records · Page 4Linked to original sources
PubMed1993

Single-strand conformational polymorphism (SSCP) mapping of the mouse genome: integration of the SSCP, microsatellite, and gene maps of mouse chromosome 1.

Interspersed repetitive sequence (IRS) PCR and repetitive element-to-bubble (IRS-bubble) PCR have been utilized to rapidly generate large numbers of mouse-specific, chromosome 1-enriched STSs from mouse-hamster somatic cell hybrids. Single-strand conformational polymorphism (SSCP) has been used to localize 39 new repetitive element-linked STSs to the mouse map: 22 to Chr 1, 10 to Chr. 15, 2 each to Chrs 12 and 14, and three to Chr 7. In addition, we have integrated the SSCP, single-strand length polymorphism, and restriction fragment length polymorphism maps of mouse Chr. 1, resulting in a high-density map of the chromosome, containing over 100 loci, all typed on a single interspecific backcross.

Animals↗
PubMed1993

Tissue specific expression of FMR-1 provides evidence for a functional role in fragile X syndrome.

We have performed mRNA in situ hybridization studies and northern blot analysis in the mouse and human, respectively, to determine the normal gene expression patterns of FMR-1. Expression in the adult mouse was localized to several regions of the brain and the tubules of the testes, which are two of the major organs affected in fragile X syndrome. Universal and very strong expression was observed in early mouse embryos, with differentially decreasing expression during subsequent stages of embryonic development. The early embryonic onset and tissue specificity of FMR-1 gene expression is consistent with involvement in the fragile X phenotype, and also suggests additional organ systems in which clinical manifestations of reduced FMR-1 gene expression may occur.

Adult↗
PubMed1993

Mutations in the neurofibromatosis 1 gene in sporadic malignant melanoma cell lines.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder characterized by progressive and variable involvement of tissues predominantly derived from the neural crest and a predisposition toward malignancies. The NF1 gene encodes neurofibromin, a GTPase-activating protein containing a GAP-related domain (NF1-GRD) that is capable of down-regulating ras by stimulating its intrinsic GTPase activity. We report a homozygous deletion of most of NF1 in one of eight malignant melanoma cell lines leading to loss of detectable mRNA and protein, as well as the apparent absence of protein and mRNA in another melanoma. This data suggests that NF1 can function as a tumour suppressor gene in the development or progression of malignant melanoma.

Chromosome Mapping↗
PubMed1993

Direct detection of novel expanded trinucleotide repeats in the human genome.

Expansion of trinucleotide repeats can give rise to genetic disease. We have developed a technique, repeat expansion detection (RED), that can identify potentially pathological repeat expansion without prior knowledge of chromosomal location. Human genomic DNA is used as a template for a two-step cycling process that generates oligonucleotide multimers when expanded trinucleotide sequences are present at the level found in myotonic dystrophy and fragile-X patients. We have identified at least one new locus exhibiting trinucleotide expansion. Analysis of three families transmitting a long CTG repeat shows that the allele in these families corresponds to a locus on chromosome 18. RED constitutes a powerful tool to identify other diseases caused by this mechanism, particularly diseases associated with anticipation.

Chromosomes, Human, Pair 18↗
PubMed1993

Identification of human chromosome 9 specific genes using exon amplification.

We have recently developed a method, exon amplification, that is designed for isolation of exon sequences from genomic DNA. To assess the efficacy of this method we have analyzed cosmid genomic clones derived from human chromosome 9, and have cloned several products from this analysis. Approximately 63% of cosmids produced at least one product derived from functioning splice sites within the target genomic fragment, and in many cases multiple products were isolated. In addition, an easily identifiable class of false positives was produced from 56% of cosmids analyzed; these are readily eliminated from subsequent study. Sequence analysis and database searches revealed that the majority (87%) of the putative exon clones were unique, the remainder being derived from repetitive sequences. Analysis of sequence conservation by Southern blotting in addition to cDNA screening experiments suggested that most, if not all, of these unique sequences represent true exons. The results of these studies indicate that exon amplification is a rapid and reliable approach for isolation of exon sequences from mammalian genomic DNA.

Animals↗
PubMed1993

A gene from chromosome 4p16.3 with similarity to a superfamily of transporter proteins.

We have previously used exon amplification to identify the ADD1 gene in cosmid Y24 from the Huntington's disease (HD) region of 4p16.3. The same technique has now yielded a second gene from this cosmid. This gene appears to encode a novel member of a superfamily of transporter proteins that includes active and passive transporters in a number of species. The predicted protein of 455 amino acids displays sequence similarity with the E. coli tetracycline resistance efflux protein encoded by cloning vector pBR322, and with a number of related transporters. This gene should open a route to isolating additional mammalian members of this growing superfamily.

Amino Acid Sequence↗
PubMed1993

Mouse beta-globin DNA-binding protein B1 is identical to a proto-oncogene, the transcription factor Spi-1/PU.1, and is restricted in expression to hematopoietic cells and the testis.

The hematopoietic-specific DNA-binding protein B1 binds to the DNA consensus sequence AAAGRGGAARYG located twice in intervening sequence 2 of both of the mouse beta-globin genes (D. L. Galson and D.E. Housman, Mol. Cell. Biol. 8:381-392, 1988). B1 was cloned by expression of a murine erythroleukemia (MEL) cell cDNA library in transfected COS cells and screening by electrophoretic mobility shift analysis. B1 is identical to the proto-oncogene Spi-1/PU.1 (Spi-1), an ets family member. Protein-DNA contacts are shown to resemble those of the helix-turn-helix homeodomain proteins. By Northern (RNA) analysis, we found that Spi-1 mRNA is present at low levels during murine CFU-E maturation and is at least 20-fold higher in uninduced MEL, a transformed proerythroblast-like cell line which contains an activating/transforming insertion of spleen focus-forming virus at the Spi-1 locus. Dimethyl sulfoxide-induced MEL cell differentiation decreases Spi-1 mRNA to approximately 20% of the uninduced level before commitment occurs. In addition to erythroid cells, Spi-1 mRNA is present in B cells, myelomonocytes, and mast cells but not in T cells and nonhematopoietic cell types. In situ hybridization demonstrated Spi-1 mRNA expression in bone marrow, spleen, interstitial nonhepatocytes of the liver, and interstitial nontubular cells of the testis. The Spi-1 locus was mapped on human chromosome 11 to the same interval as ACP2 (lysosomal acid phosphatase), between the anonymous DNA markers D11S33 and D11S14. This region has not yet been found to be associated with a human malignancy.

Amino Acid Sequence↗
PubMed1993

Molecular definition of a chromosome 9p21 germ-line deletion in a woman with multiple melanomas and a plexiform neurofibroma: implications for 9p tumor-suppressor gene(s).

Cutaneous malignant melanoma (CMM) is often familial, but the mode of inheritance and the chromosomal location of melanoma susceptibility locus are controversial. Identification of a 34-year-old woman with eight primary malignant melanomas, multiple atypical moles, and a de novo constitutional cytogenetic rearrangement involving chromosomes 5p and 9p suggested the presence of a melanoma predisposition gene at one of these locations. A high-resolution karyotype showed a partial deletion of a dark-staining Giemsa band, either 5p14 or 9p21. The patient was heterozygous for five 5p14 RFLPs. In situ hybridization with D9S3 indicated that this 9p21 marker was deleted. Gene dosage studies demonstrated the deletion of two more distal 9p21 markers, D9S126 and IFNA. In addition, she was hemizygous for the more proximal 9p21 short tandem-repeat polymorphism at D9S104. D9S18, D9S19, and D9S33 were retained, localizing the deletion to 9p21 between D9S19 on the proximal side and D9S33 on the distal side. Pulsed-field gel electrophoresis with D9S19 and D9S33 did not reveal any junction fragments in the patient's DNA. This germ-line deletion suggests that mutations in a 9p21 gene may initiate melanoma tumorigenesis.

Adult↗
PubMed1992

mdr2 encodes P-glycoprotein expressed in the bile canalicular membrane as determined by isoform-specific antibodies.

We have produced antibodies specific for the three P-glycoprotein (P-gp) isoforms encoded by the mouse mdr1, mdr2, and mdr3 genes. The anti-Mdr2 and anti-Mdr3 antibodies were generated against synthetic peptides derived from the "linker" region, whereas the anti-Mdr1 antibody was raised against a fusion protein containing the amino terminus of Mdr1. Western blot analysis showed that the three antibodies could discriminate between the three isoforms in membrane fractions from Hamster cells transfected with the corresponding full-length or chimeric mdr cDNAs. Immunocytochemistry studies of mdr-transfected cells showed that the three antibodies specifically recognized each P-gp isoform expressed in whole cells. Immunoblotting of normal mouse tissues revealed that the Mdr2 isoform was expressed at very high levels in liver canalicular membrane vesicles (CMV) but not in membrane vesicles prepared from the basolateral (sinusoidal) domain (SMV). Mdr3 was detected in intestinal brush border membrane vesicles and also in CMV, although at levels much lower than Mdr2. Mdr1 was not detected in CMV or SMV but was detected in endometrial tissue from the gravid uterus. Photolabeling experiments with [125I]iodoarylazidoprazosin followed by immunoprecipitation with isoform-specific antibodies indicated that, in CMV, Mdr3 but not Mdr2 could bind the drug analogue.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

A dominant mutation in the Wilms tumor gene WT1 cooperates with the viral oncogene E1A in transformation of primary kidney cells.

Wilms tumor is a pediatric kidney cancer that has been linked to the inactivation of a tumor-suppressor gene at chromosome locus 11p13. The WT1 gene, mapping to this locus, is developmentally regulated in the kidney and encodes a putative transcription factor that has been shown to be mutated in Wilms tumor specimens. We have suggested that one such altered product of the WT1 gene may be capable of trans-dominant suppression, since the mutated allele was found to be coexpressed with the wild-type allele in a sporadic Wilms tumor. We therefore tested the ability of this mutant WT1 allele, containing an in-frame deletion within the DNA-binding domain, to transform primary baby rat kidney cells. The mutant WT1 gene was found to cooperate with the adenoviral E1A gene in transforming baby rat kidney cells, as demonstrated by growth in soft agar and tumorigenicity in nude mice. The wild-type WT1 gene in all of its alternatively spliced forms neither suppressed E1A-induced focus formation nor cooperated with E1A. Our results indicate that impairment of DNA binding of the WT1 tumor-suppressor gene product can result in a dominant negative mutation.

Adenovirus Early Proteins↗
PubMed1992

Isolation and characterization of human cDNA clones encoding a high mobility group box protein that recognizes structural distortions to DNA caused by binding of the anticancer agent cisplatin.

Human cDNA clones encoding a structure-specific recognition protein, SSRP1, that binds specifically to DNA modified with cisplatin have been isolated and characterized. The SSRP1 gene maps to human chromosome 11q12. The cDNA clones, obtained by using partial-length cDNAs described previously, predict an 81-kDa protein containing several highly charged domains and a stretch of 75 amino acids 47% identical to a portion of the high mobility group (HMG) protein HMG1. This HMG box most likely constitutes the structure recognition element for cisplatin-modified DNA, with the probable recognition motif being the local duplex unwinding and bending toward the major groove that occurs upon formation of intrastrand cis-[Pt(NH3)2]2+ d(GpG) and d(ApG) cross-links. Although the DNA recognition properties of members of the HMG-box family of proteins have been characterized with respect to their sequence specificity, the present work demonstrates that proteins with this domain can recognize particular DNA structures as well. The Pt-DNA SSRP described here is the human homolog of a recently identified mouse protein that binds to recombination signal sequences [Shirakata, M., Hüppi, K., Usuda, S., Okazaki, K., Yoshida, K. & Sakano, H. (1991) Mol. Cell. Biol. 11, 4528-4536]. These sequences have been postulated to form stem-loop structures, further implicating local bends and unwinding in DNA as a recognition target for HMG-box proteins. Expression analysis in a variety of tissues and cisplatin-resistant cell lines and the inability of cisplatin to induce the message in HeLa cells argue against a direct link between SSRP1 mRNA levels and the response of cells to the drug.

Amino Acid Sequence↗
PubMed1992

Expansion of an unstable DNA region and phenotypic variation in myotonic dystrophy.

Myotonic dystrophy is the commonest adult form of muscular dystrophy, with an estimated incidence of 1 per 7,500, although this is likely to be an underestimate because of the difficulty of detecting minimally affected individuals. It is a multisystem autosomal dominant disorder of unknown biochemical basis. No case of new mutation has been proven. We have isolated a human genomic clone that detects novel restriction fragments specific to individuals with myotonic dystrophy. A two-allele EcoRI polymorphism is seen in normal individuals, but in most affected individuals one of the normal alleles is replaced by a larger fragment, which varies in length both between unrelated affected individuals and within families. The unstable nature of this region may explain the characteristic variation in severity and age at onset of the disease. A second polymorphism at this locus is in almost complete linkage disequilibrium with myotonic dystrophy, strongly supporting our earlier results which indicated that most cases are descended from one original mutation.

Blotting, Southern↗
PubMed1992

Mapping and characterization of 129 cosmids on human chromosome 11p.

We constructed cosmid libraries from human-hamster somatic cell hybrids that possess all or part of the short arm of chromosome 11 as their only human complement and isolated 129 human 11p clones. These cosmids map to 22 of 25 intervals distinguished by a hybrid panel for chromosome 11p. Forty-eight single-copy sequences were subcloned from 25 cosmids. Six of 17 (35%) single-copy sequences tested identify 11 new polymorphisms. Restriction endonuclease analysis identified CpG islands in 16 of 68 cosmids (23.5%). Analysis of the distribution of restriction endonuclease sites recognizing CpG dinucleotides showed that clusters of these sites, including those associated with the 5' region of an 11p13 Wilms' tumor gene, WT1, can span greater distances than generally recognized. The cosmids reported here should contribute to the construction of long-range physical maps and the isolation of additional genes on the short arm of chromosome 11.

Blotting, Southern↗
PubMed1992

Generation and characterization of a human chromosome 9 cosmid library.

A cosmid library has been constructed from the hamster-human hybrid cell line PK-87-9, which contains chromosome 9 as its sole known human component. Ten thousand colonies were produced, of which approximately 200, or 2%, contain human material. Fifty of these 200 were regionally mapped by an Alu-primed PCR product hybridization procedure. These cosmids were localized to all regions of chromosome 9, but were especially concentrated in the distal portion of 9q. The map location derived by the Alu-primed PCR product hybridization procedure was compared to the map location derived by fluorescent in situ hybridization. Assignment of chromosomal location by the two methods was correspondent in all but a few cases. The presumptive presence of HTF islands was investigated for 130 cosmids by digestion with the restriction enzyme NotI. Twenty percent of cosmids contained at least one NotI site. A number of simple sequence repeat polymorphisms identified from the cosmid set were characterized and will provide a link between the genetic and physical maps for this chromosome.

Animals↗
PubMed1992

Construction and characterization of radiation hybrids for chromosome 9, and their use in mapping cosmid probes on the chromosome.

Radiation hybrids were produced from a monochromosomal microcell hybrid (PK87-9) which contains only human chromosome 9 with an inserted marker on 9p. Doses of radiation ranging from 1000 to 8000 rads were used to produce a series of hybrids with different size fragments of human chromosome 9. The inserted dominant selectable marker was used to select for hybrids that preferentially maintain fragments of 9p. A panel of 53 radiation hybrids were characterized for 17 chromosome 9 markers. In addition, 17 hybrids were analyzed by fluorescent in situ hybridization (FISH). Hybrids were produced with breaks on both 9p and 9q, many of which appear to contain a single fragment of human chromosome 9. These hybrid cell lines were used to regionally localize 31 cosmids isolated from a chromosome 9 cosmid library. Six cosmids were mapped to intervals on 9p, six cosmids mapped to the centromeric region of the chromosome, and 19 mapped to 9q.

Animals↗
PubMed1992

Isolation and chromosomal assignment of 100 highly informative human simple sequence repeat polymorphisms.

One hundred highly informative simple sequence repeat (SSR) polymorphisms have been isolated and mapped to specific human chromosomes by somatic cell hybrid analysis. These markers include 97 (CA)n, 2 (AGAT)n, and a single (AACT)n repeat. All the SSRs have heterozygosities greater than 0.50 and can be amplified using identical PCR conditions. At least one SSR was detected on every chromosome, except for chromosomes 22 and Y. The frequency of (CA)n repeats on each chromosome was proportional to the relative chromosomal length, except for chromosome 15, on which a substantial excess of markers was identified.

Base Sequence↗
PubMed1992

Sperm typing allows accurate measurement of the recombination fraction between D3S2 and D3S3 on the short arm of human chromosome 3.

The interval between the D3S2 and D3S3 loci on human chromosome 3p is a frequent site of deletions in a number of different cancers and contains the most common fragile site in man. Both loci have been physically mapped to 3p but because heterozygosity for D3S3 is so infrequent, recombination between them could not be determined accurately by using family studies. Sperm typing can measure recombination between DNA polymorphisms even in a single individual and thus can make use of polymorphisms with a low PIC. The recombination fraction between D3S2 and D3S3 was estimated to be 0.28 based on analyzing 189 and 77 sperm from two doubly heterozygous donors, respectively. These results demonstrate one of the ways in which sperm typing can complement pedigree analysis in constructing genetic maps.

Base Sequence↗