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Biomedical subjects

D I Smith

Publications and source records attributed to D I Smith.

At least 91 records · Page 5Linked to original sources
PubMed1994

Characterization of a microdissection library from human chromosome region 3p14.

Structural alterations in human chromosome region 3p14-p23 resulting in the inactivation of one or more tumor suppressor genes are thought to play a pathogenic role in small cell lung cancer, renal cell carcinoma, and other human neoplasms. To identify putative tumor suppressor genes, 428 recombinant clones from a microdissection library specific for human chromosome region 3p14 were isolated and characterized. Ninety-six of these (22.5%) were human single-copy DNA sequences, 57 of which were unique sequence clones. Forty-four of these were mapped to the microdissected region using a cell hybrid mapping panel. Within this mapping panel, four probes detected two new chromosome breakpoints that were previously indistinguishable from the translocation breakpoint t(3;8) in 3p14.2 in hereditary renal cell carcinoma. One probe maps to the homozygously deleted region of the small cell lung cancer cell line U2020. In addition, microdissection clones have been shown to be suitable for isolation of yeast artificial chromosomes.

Animals↗
PubMed1994

Isolation of two contigs of overlapping cosmids derived from human chromosomal band 3p21.1 and identification of 5 new 3p21.1 genes.

Consistent loss of DNA sequences from several regions on the short arm of human chromosome 3 has suggested that multiple tumor suppressor genes reside on chromosome 3p in various types of cancer cells. We have focused our efforts on an analysis of chromosomal band 3p21.1 since aminoacylase-1 (ACY1), which is localized to this band, has been shown to have lower levels of expression in several small cell and non-small cell lung cancer cell lines. Starting with two cosmids within 3p21.1, D3S92 and D3S93, we have isolated two separate contigs of overlapping cosmids within 3p21.1, by screening a library of 5700 chromosome 3-specific cosmid clones. Detailed restriction maps for these two contigs show that they contain multiple clusters of rare cutting restriction endonuclease sites. One contig extends for 100 kb and encompassed both ACY1 and D3S92, and the other extends about 80 kb around the D3S93 locus. Many different restriction fragments derived from these two contigs were found to be evolutionarily conserved and hybridized to distinct message transcripts. These fragments were used to identify homologous cDNAs from an adenogastric cDNA library, and several of these cDNAs were partially sequenced. We have identified five new genes from these two contigs and there is evidence to suggest that several additional genes reside within these cosmid contigs. The genes identified from 3p21.1 were then hybridized to DNA, isolated from a series of lung cancer cell lines and matched normal and tumor DNA from lung cancer patients. No alterations were detected with any of these probes, both at the DNA or RNA levels. A similar analysis with DNA fragments derived from these two genomic regions also failed to detect any alterations.

Animals↗
PubMed1994

Physical map of small cell lung cancer deletion region on short arm of human chromosome 3 (3p13-22) based on radiation fusion hybrid analysis.

Deletion of DNA sequences from various regions of the short arm of human chromosome 3 (3p13-14, 3p21, and 3p25) has been observed during the development of a variety of solid tumors, including lung and renal cell carcinomas. In this study we have used a set of radiation fusion hybrids to generate a physical map of chromosome 3p to orient the search for putative tumor suppressor genes. Eighty-six human-hamster radiation fusion hybrids were screened on Southern blots for the retention of 55 human chromosome 3p DNA markers. The high marker density enabled us to identify a set of successively overlapping chromosome fragments in the 3p13-22 area guided by eight markers with previously known order. Twenty-four map intervals were suggested using breakpoints determined by partial fragment overlaps. The final order between the markers derived is consistent with previous information about localizations for 26 of the markers to three larger cytogenetic intervals.

Animals↗
PubMed1994

Identification of human chromosome region 3p14.2-21.3-specific YAC clones using Alu-PCR products from a radiation hybrid.

Deletion of DNA sequences from at least three different regions on the short arm of human chromosome 3 (3p13-14, 3p21 and 3p25) are frequently observed during the development of many solid tumors, including lung cancers and renal cell carcinomas. In order to physically characterize the 3p21 region, we previously identified a radiation fusion hybrid that contained about 20 megabases of DNA from chromosome region 3p14.2-p21.3. In this study total Alu-PCR products from this hybrid were used as a probe to isolate 86 yeast artificial chromosomes (YAC) clones from a 620-kb average insert YAC library (ICRF). Sixty-nine Alu-PCR markers, generated from the YACs, and seven PCR primers were used to screen for overlaps between individual clones. Seven contigs were identified encompassing 32 YAC clones. Based on previous information about localization of the PCR primers, the three largest contigs could be assigned to smaller subregions between 3p14.2 and 3p21.3. By this work a large proportion of the 3p14.2-21.3 region is covered with large-insert YAC clones.

Base Sequence↗
PubMed1994

Molecular characterization of a novel human gene, SEC13R, related to the yeast secretory pathway gene SEC13, and mapping to a conserved linkage group on human chromosome 3p24-p25 and mouse chromosome 6.

We previously described sequence tags from 58 novel directionally cloned human cDNAs from an enriched retinal pigment epithelial cell line library (Gieser and Swaroop, 1992). The nucleotide sequence of one of the cDNA clones, AA35 (D3S1231E), showed strong homology to the yeast SEC13 gene, required for vesicle biogenesis from endoplasmic reticulum during the transport of proteins. We have designated the human gene SEC13R (SEC13-Related). The amino acid sequence of the SEC13R gene product shows 70% similarity to yeast Sec13p, suggesting that SEC13R may be the human homolog of SEC13. The deduced polypeptide sequence contains several beta-transducin like 'WD40' repeats, and is rich in serine and threonine residues. The 1.4 kb transcript of SEC13R is detected by Northern analysis in many human tissues. However, RT-PCR analysis using two primer sets from different regions of the gene suggests differential expression of alternately spliced transcripts in various tissues. Somatic cell hybrid and in situ hybridization studies localized the SEC13R gene to human chromosome 3p24-p25. A related sequence was mapped to chromosome 18q11.2-q12. SEC13R was physically mapped to a yeast artificial chromosome (YAC) clone spanning the D3S720 marker from the region of the Von Hippel-Lindau disease locus. The mouse Sec13r gene was mapped to the conserved linkage group on chromosome 6 that corresponds to human chromosome 3p24-p25.

Amino Acid Sequence↗
PubMed1993

Identification of a G protein coupled receptor induced in activated T cells.

Many genes are induced after T cell activation to make a cell competent for proliferation and ultimately, function. Many of these genes encode surface receptors for growth factors that signal a cell to proliferate. We have cloned a novel gene (clone 6H1) that codes for a member of the G protein-coupled receptor superfamily. This gene was isolated from a chicken activated T cell cDNA library by low level hybridization to mammalian IL-2 cDNA probes. The 308 amino acid open reading frame has seven hydrophobic, presumably transmembrane domains and a consensus site for interaction with G proteins. Tissue distribution studies suggest that gene expression is restricted to activated T cells. The message appears by 1 h after activation and is maintained for at least 45 h. Transcription of 6H1 is induced by a number of T cell stimuli and is inhibited by cyclosporin A, but not by cycloheximide. This is the first description of a member of this superfamily expressed specifically in activated T cells. The gene product may provide a link between T cell growth factors and G protein activation.

Amino Acid Sequence↗
PubMed1993

Cloning of tumor suppressor genes involved in solid tumor development.

OBJECTIVE: The homozygous inactivation of tumor suppressor genes is a common event in the multistep process of tumorigenesis. The identification of most tumor suppressor genes known to date has come from the study of individuals with a defective copy of such a gene that increases their predisposition to malignancy. Although much is still to be learned about the function of these genes, they all seem to play a role in cell growth regulation. Karyotypic and molecular studies of several solid tumors suggest the presence of at least three tumor suppressor genes on the short arm of human chromosome 3. We present an overview on the progress made in the identification of tumor suppressor genes involved in solid tumor development. CONCLUSION: The explosive effort to map the human genome fueled by the Human Genome Project will facilitate the identification of tumor suppressor genes on chromosome 3 and on other chromosomes within the next 2 to 3 years. This information will not only provide a better understanding in cell cycle regulation and differentiation but also new insights in the design of new therapies for the treatment of human cancer.

Chromosome Deletion↗
PubMed1993

Determination of the specificity of aphidicolin-induced breakage of the human 3p14.2 fragile site.

The constitutive 3p14.2 fragile site is the most highly inducible fragile site in the human genome. This locus may predispose chromosome 3 to specific losses due to deletions and translocations that have been associated with several malignancies, including hereditary renal cell carcinoma. Using aphidicolin concentrations of 0.4 and 4.0 microM, we have generated and isolated 23 and 22 respective somatic cell hybrids that contain chromosome 3 short-arm breaks. The breakpoints in these hybrids have been localized with numerous chromosome 3 markers. We have observed that at the low aphidicolin dose, chromosome-3 breaks cluster within the 3p14.2 region, whereas at the high aphidicolin dose, two new loci, one within 3p14.1 and the other near the centromere, become predominantly affected. Our studies have failed to differentiate any of the 3p14.2 breakpoints from each other or from the t(3;8)(p14.2;q24.13) familial renal cell carcinoma translocation breakpoint, suggesting that the fragile site may have played a role in the generation of this balanced translocation. The resulting somatic cell hybrids generated from this work have refined the marker localizations within 3p13-p21.1 and should facilitate the physical characterization of this interesting region.

Animals↗
PubMed1993

Lamin A/C gene and a related sequence map to human chromosomes 1q12.1-q23 and 10.

Lamins A and C are products of alternate splicing of one transcript from a single gene. We have isolated a partial cDNA, cE1-2, whose 800-bp sequence is 99% identical to the 3' untranslated region of the lamin A/C gene. We report here the mapping of this gene and a closely related sequence to human chromosomes 1 and 10, more specifically, to 1q12.1-q23. The localization of cE1-2 hybridizing sequences to two different chromosomes suggests that one of these loci represents an as yet unknown member of the lamin gene family, either a pseudogene or an expressed gene.

Animals↗
PubMed1993

Cloning and characterization of the human t(3;6)(p14;p11) translocation breakpoint associated with hematologic malignancies.

The t(3;6)(p14;p11) chromosome translocation was identified in a family in which three members developed hematologic malignancies. To help characterize the region on chromosome 3 surrounding this translocation breakpoint, two flanking lambda clones, MS156 and MJ1525, were linked by pulsed-field gel electrophoresis to the same 510-kb NotI fragment on chromosome 3. MS156 was localized to a region proximal to the breakpoint of a der(3) chromosome somatic cell hybrid (derived from the t(3;6) cell line), and MJ1525 localized distal to the breakpoint. MJ1525 was used to screen the CEPH yeast artificial chromosome (YAC) library, which revealed a YAC, 195F3, that spanned the breakpoint. Subcloning into Lambda DASH II and production of a contiguous array of overlapping lambda clones revealed a clone, L17, that spanned the breakpoint. A rare restriction endonuclease map for the YAC 195F3 was constructed, and multiple clusters of rare restriction sites within the YAC were identified, possibly indicating the disruption of a gene by the t(3;6) translocation breakpoint.

Animals↗
PubMed1993

The isolation of a yeast artificial chromosome (YAC) contig extending for 2 megabases in the vicinity of the von Hippel Lindau disease gene.

Von Hippel Lindau disease (VHL) is a rare autosomal dominant disease associated with tumors and cysts in multiple organ systems. The VHL disease gene is tightly linked to the polymorphic DNA marker 233E2 (D3S720) and flanked by 479H4 (D3S719) on its telomeric and RAF1 on its centromeric side. Two additional markers, D3S1038 and D3S601, have also been identified, and these markers, like D3S720, are very tightly linked to VHL. Previously 93 cosmid clones were mapped to the larger region, 3p24.2-pter, surrounding the VHL disease gene. Using a Southern-based screening strategy on pools of YAC clones we have isolated a contig of overlapping YAC clones that extends about 0.7 megabase centromeric, and about 1.3 megabases telomeric of D3S720 and contains all three tightly linked VHL markers. Individual YACs in this contig were hybridized to grids containing cosmids localized between 3p24.2-pter and to several cosmids localized by fluorescent in situ hybridization (FISH) to 3p25. A total of 28 cosmids were positioned on this contig of overlapping YAC clones. We have also identified homologous YAC clones to many additional cosmid clones localized between 3p24.2-p25, although these have not yet been precisely localized relative to the contig of YAC clones. This contig of YAC clones probably contains the VHL disease gene and should facilitate the isolation and characterization of this gene.

Base Sequence↗
PubMed1993

Genetic mapping of dinucleotide repeat polymorphisms and von Hippel-Lindau disease on chromosome 3p25-26.

A genetic map of highly polymorphic microsatellite markers spanning the von Hippel-Lindau region (VHL) of 3p25 was constructed using the CEPH reference pedigrees. A greater than 1000:1 odds map of pter-D3S1038-RAF1-D3S651-D3S656-D3S110- D3S1255-cen was found. Genotyping of six multigenerational VHL families showed the region surrounding the D3S1038 marker to be the most likely location for the VHL gene with a peak location score of 10.04 with VHL completely linked to D3S1038. These data provide an initial high resolution genetic map of this region; D3S1038 appears to be a highly polymorphic marker that should prove useful in the future for presymptomatic diagnosis.

Adult↗
PubMed1993

Identification of a specific radioligand for the cardiac rapidly activating delayed rectifier K+ channel.

Class III antiarrhythmic drugs show promise as effective treatments for the suppression of potentially lethal cardiac arrhythmias. Dofetilide (UK-68,798), is a potent class III antiarrhythmic agent that is presently under clinical investigation. The objective of this study was to determine whether [3H]dofetilide could be used as a specific radioligand for the rapidly activating delayed rectifier K+ channel of the heart. We find that [3H]dofetilide binds to high-affinity sites on guinea pig cardiac myocytes. Competition studies using unlabeled dofetilide indicate that binding is characterized by an IC50 of 100 +/- 30 nM (mean +/- SD, n = 13). Scatchard analyses of binding indicate a Kd of 70 +/- 6 nM and a maximal binding capacity of 0.30 +/- 0.02 pmol/mg protein. [3H]Dofetilide is displaced from guinea pig myocytes by dofetilide, clofilium, quinidine, sotalol, and sematilide with a rank order of potency that correlates with functional blockade of the rapidly activating delayed rectifier K+ current (correlation coefficient, 0.951; slope, 0.99 +/- 0.19; p = 0.014). High-affinity [3H]dofetilide binding is not detected in rat myocytes, which are devoid of delayed rectifier K+ current. We conclude that [3H]dofetilide specifically binds to sites associated with the rapidly activating delayed rectifier K+ channel of guinea pig myocardium.

Animals↗
PubMed1992

Localization of three novel hybrid breakpoints and refinement of 18 marker assignments in the human 3cen-p21.1 region.

Using the human/hamster cell line UCTP2A-3, we have generated and isolated three hybrids, each containing a novel human chromosome 3p break. All chromosome 3 materials distal to the breaks were lost. Two of the breakpoints were produced using aphidicolin induction; the third breakpoint occurred spontaneously. The aphidicolin-induced breaks were localized to 3p21.1 in hybrid AR1 and to p14.1 in hybrid AR2. The spontaneous break was localized to 3p11 in hybrid 2A-3-1. These hybrids were used to sublocalize 18 chromosome 3 probes further to five regions within 3cen-p21.1. The new hybrids and sublocalized markers will be useful in the study and characterization of the 3p11, 3p14.1, and 3p21.1 segments of chromosome 3.

Animals↗
PubMed1992

Relationship between the number and type of alcohol outlets and mortality due to liver cirrhosis and traffic accidents.

The aim of the study was to determine the effect on liver cirrhosis and driver and motorcyclist mortality of changing the alcohol outlet rates in New South Wales relative to the adjacent control State of Victoria. A longitudinal study design with a six year before period (1968-1973) and a nine year after period (1974-1982) was used. A 10.5% relative increase in the total outlet rate for New South Wales during the after period was associated with significant increases in liver cirrhosis mortality (males + 22.1%, females + 17.7%) and driver and motorcyclist mortality (males + 19.8%, females + 18.9%) in comparison to the control state. The analyses also indicated that the increases in the dependent variables were apparently due to the higher outlet rates for restaurants and stores rather than for hotels and taverns. Attention is drawn to a number of limitations of the study design.

Journal Article↗