[Show abstract][Hide abstract] ABSTRACT: Mutations of the PPP2R1B gene, which encodes the Abeta scaffolding subunit of serine/threonine protein phosphatase 2A (PP2A), have been identified in several types of cancer including lung and breast carcinoma. One of these mutations results in an alteration of glycine 90 to aspartic acid (G90D), which has been found in both tumor and genomic DNA, raising the possibility that it is associated with an increased risk for cancer. A novel microarray-based technology was used to screen for this single-nucleotide polymorphism in 387 cancer patients and 329 control individuals. These data were used for case-control and family-based comparisons in order to study the association of this polymorphism with susceptibility to lung carcinoma, breast carcinoma, and acute lymphoblastic leukemia. The frequency of the G90D polymorphism in breast cancer patients was significantly higher in cases (3%) than in controls (0.3%). The wild-type Abeta subunit interacted with the B56gamma (PPP2R5C), PR72 (PPP2R3A), and PR48 subunits of PP2A but did not interact with the B55alpha (PPP2R2A), B56alpha (PPP2R5A), or B56beta (PPP2R5B) regulatory subunits in an in vitro binding assay. The G90D alteration inhibited the interaction of Abeta with the B56gamma subunit but had no effect on binding to the PR72 subunit. These results provide evidence that the G90D alteration of the Abeta subunit of PP2A is associated with a low frequency of breast carcinoma and that the role of this alteration in transformation is likely to involve decreased interaction with the B56gamma regulatory subunit.
Genes Chromosomes and Cancer 03/2006; 45(2):182-90. · 3.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have determined the complete sequence of 951,695 bp from the class I region of H2, the mouse major histocompatibility complex (Mhc) from strain 129/Sv (haplotype bc). The sequence contains 26 genes. The sequence spans from the last 50 kb of the H2-T region, including 2 class I genes and 3 class I pseudogenes, and includes the H2-M region up to Gabbr1. A 500-kb stretch of the H2-M region contains 9 class I genes and 4 pseudogenes, which fall into two subfamilies, M1 and M10, distinct from other mouse class I genes. This M1/M10 class I gene-cluster is separated from the centromeric H2-T and the telomeric H2-M4, -5 and -6 class I genes by "nonclass I genes". Comparison with the corresponding 853-kb region of the human Mhc, which includes the HLA-A region, shows a mosaic of conserved regions of orthologous nonclass I genes separated by regions of species-specific expansion of paralogous Mhc class I genes. The analysis of this mosaic structure illuminates the dynamic evolution of the Mhc class I region among mammals and provides evidence for the framework hypothesis.
Genome Research 05/2003; 13(4):589-600. · 13.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
[Show abstract][Hide abstract] ABSTRACT: Hereditary multiple exostoses (HME) is a genetically heterogeneous disease characterized by the development of bony protuberances at the ends of all long bones. Genetic analyses have revealed HME to be a multigenic disorder linked to three loci on chromosomes 8q24 (EXT1), 11p11-13 (EXT2), and 19p (EXT3). The EXT1 and EXT2 genes have been cloned and defined as glycosyltransferases involved in the synthesis of heparan sulfate. EST database analysis has demonstrated additional gene family members, EXT-like genes (EXTL1, EXTL2, and EXTL3), not associated with a HME locus. The mouse homologs of EXT1 and EXT2 have also been cloned and shown to be 99% and 95% identical to their human counterparts, respectively. Here, we report the identification of the mouse EXTL1 gene and show it is 74% identical to the human EXTL1 gene. Expression studies of all three mouse EXT genes throughout various stages of embryonic development were carried out and whole-mount in situ hybridization in the developing limb buds showed high levels of expression of all three EXT genes. However, in situ hybridization of sectioned embryos revealed remarkable differences in expression profiles of EXT1, EXT2, and EXTL1. The identical expression patterns found for the EXT1 and EXT2 genes support the recent observation that both proteins form a glycosyltransferase complex. We suggest a model for exostoses formation based on the involvement of EXT1 and EXT2 in the Indian hedgehog/parathyroid hormone-related peptide (PTHrP) signaling pathway, an important regulator of the chondrocyte maturation process.
[Show abstract][Hide abstract] ABSTRACT: 11q23-24 chromosome is a region containing frequent allelic loss (loss of heterozygosity; LOH) in human cancers. To examine cancer-related allelic loss in the region between D11S940 and APOC3, we used 17 polymorphic markers and allotyped 28 lung cancer-derived cell lines and their corresponding matched lymphoblastoid cell lines. LOH was found in 71.4% (20/28) of the lung cancer cell lines and was localized to two distinct minimal regions of loss. One region is bracketed by markers D11S1647 and NCAM2 and contains the gene encoding the beta isoform of the A subunit of the human protein phosphatase 2A (PPP2R1B). Recently, mutations in this gene were described in lung and colon cancers, suggesting that PPP2R1B functions as a tumor-suppressor gene. A second minimal region of loss was defined between markers D11S1792 and D11S1885, a region estimated to be less than I Mb. Thus, chromosome 11 likely harbors two sites of suppressor oncogene activity in lung cancer, one defined by the PPP2R1B gene and the second located telomeric to PPP2R1B. This study facilitates the identification and cloning of a second critical tumor-suppressor gene involved in lung cancer, and possibly a variety of other cancers, on human chromosome band 11q23.
Genes Chromosomes and Cancer 07/1999; 25(2):154-9. · 3.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Olfactory receptors (OR) are encoded by a large multigene family including hundreds of members dispersed throughout the human genome. Cloning and mapping studies have determined that a large proportion of the olfactory receptor genes are located on human chromosomes 6, 11, and 17, as well as distributed on other chromosomes. In this paper, we describe and characterize the organization of olfactory receptor genes on human chromosome 11 by using degenerate PCR-based probes to screen chromosome 11-specific and whole genome clone libraries for members of the OR gene family. OR genes were identified by DNA sequencing and then localized to regions of chromosome 11. Physical maps of several gene clusters were constructed to determine the chromosomal relationships between various members of the family. This work identified 25 new OR genes located on chromosome 11 in at least seven distinct regions. Three of these regions contain gene clusters that include additional members of this gene family not yet identified by sequencing. Phylogenetic analysis of the newly described OR genes suggests a mechanism for the generation of genetic diversity.
[Show abstract][Hide abstract] ABSTRACT: The PPP2R1B gene, which encodes the β isoform of the A subunit of the serine/threonine protein phosphatase 2A (PP2A), was identified
as a putative human tumor suppressor gene. Sequencing of thePPP2R1B gene, located on human chromosome 11q22-24, revealed somatic alterations in 15% (5 out of 33) of primary lung tumors, 6%
(4 out of 70) of lung tumor–derived cell lines, and 15% (2 out of 13) of primary colon tumors. One deletion mutation generated
a truncated PP2A-Aβ protein that was unable to bind to the catalytic subunit of the PP2A holoenzyme. The PP2R1B gene product may suppress tumor development through its role in cell cycle regulation and cellular growth control.
[Show abstract][Hide abstract] ABSTRACT: SM22 is a 22-kDa protein identified variously as SM22, transgelin, WS3-10, or mouse p27. Though its precise function is unknown, it is abundant in smooth muscle and so may contribute to the physiology of this widespread tissue. We found that cosmid 16b6 contains the entire 5.4-kb, five-exon human SM22 gene (HGMW-approved symbol, TAGLN), and we cytogenetically localized the gene to chromosome 11q23.2. Northern analysis of human adult tissues showed that SM22 mRNA is most prevalent in smooth muscle-containing tissues, but is also found at lower levels in heart. The human SM22 promoter contains nuclear factor-binding motifs known to regulate transcription in smooth muscle, and human SM22 promoter-luciferase reporter constructs exhibited high transcriptional activity in A7r5 or primary canine aortic smooth muscle cells, but show little activity in nonmuscle COS7 cells. In addition, human SM22 promoter activity increased by two- to threefold upon serum stimulation of nonmuscle cells.
[Show abstract][Hide abstract] ABSTRACT: Analysis of an extended pedigree in which a balanced t(9;11)(p24;q23.1) translocation was found to cosegregate with bipolar affective disorder revealed that five of 11 translocation carriers had bipolar affective disorder and one carrier had unipolar depression. There were no affected individuals in the pedigree without the balanced translocation. We hypothesized that gene(s) or gene regulatory regions disrupted by the translocation might be contributing to the bipolar affective disorder in a dominant fashion. To test this hypothesis, we isolated the derivative chromosome 9 and derivative chromosome 11 in somatic cell hybrids and identified the nearest flanking markers on chromosome 9 (D9S230 and D9S2011E/HRFX3) and chromosome 11 (EST00652 and CRYA2). YAC contigs were constructed in the region of flanking markers for both chromosomes 9 and 11. Chromosome 11 breakpoint was localized within an 8-kb region in a small insert (100 kb) YAC. Chromosome 9 breakpoint was localized within approximately 2 Mb region. Several genes and ESTs including EST00652, CRYA2, DRD2, 5HTR3 on chromosome 11 and VLDLR and SLC1A1 on chromosome 9 were mapped within the vicinity of the breakpoint but were shown not to be disrupted by the translocation breakpoint. Although several possibilities exist regarding the role of the balanced translocation in developing bipolar affective disorder in this pedigree, including a chance cosegregation, identification of a disrupted gene or gene regulatory region with the help of physical mapping resources described in this study may help to identify the presence of a susceptibility gene for this disorder.
American Journal of Medical Genetics 02/1998; 81(1):81-91. · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Usher syndrome 1C (USH1C) is a congenital condition manifesting profound hearing loss, the absence of vestibular function, and eventual retinal degeneration. The USH1C locus has been mapped genetically to a 2- to 3-cM interval in 11p14–15.1 between D11S899 and D11S861. In an effort to identify the USH1C disease gene we have isolated the region between these markers in yeast artificial chromosomes (YACs) using a combination of STS content mapping and Alu–PCR hybridization. The YAC contig is ∼3.5 Mb and has located several other loci within this interval, resulting in the order CEN-LDHA-SAA1-TPH-D11S1310-(D11S1888/KCNC1)-MYOD1-D11S902D11S921-D11S1890-TEL. Subsequent haplotyping and homozygosity analysis refined the location of the disease gene to a 400-kb interval between D11S902 and D11S1890 with all affected individuals being homozygous for the internal marker D11S921. To facilitate gene identification, the critical region has been converted into P1 artificial chromosome (PAC) clones using sequence-tagged sites (STSs) mapped to the YAC contig, Alu–PCR products generated from the YACs, and PAC end probes. A contig of >50 PAC clones has been assembled between D11S1310 and D11S1890, confirming the order of markers used in haplotyping. Three PAC clones representing nearly two-thirds of the USH1C critical region have been sequenced. PowerBLAST analysis identified six clusters of expressed sequence tags (ESTs), two known genes (BIR,SUR1) mapped previously to this region, and a previously characterized but unmapped gene NEFA (DNA binding/EF hand/acidic amino-acid-rich). GRAIL analysis identified 11 CpG islands and 73 exons of excellent quality. These data allowed the construction of a transcription map for the USH1C critical region, consisting of three known genes and six or more novel transcripts. Based on their map location, these loci represent candidate disease loci for USH1C. The NEFA gene was assessed as the USH1C locus by the sequencing of an amplified NEFA cDNA from an USH1C patient; however, no mutations were detected.
[Show abstract][Hide abstract] ABSTRACT: The parasitic protozoan Giardia lamblia represents one of the earliest diverging lineages in the evolutionary history of eukaryotic organisms as well as an important human pathogen. A representative sampling of gene sequences from this early diverging protozoan could provide insights into genotypic and phenotypic innovations associated with the origin of eukaryotes. Currently, known giardial gene sequences are heavily biased toward a few gene families, including variant surface proteins (VSPs), structural proteins, and ribosomal RNA genes. One-pass sequences of Giardia genomic DNA were obtained using vector flanking priming sequences on the ends of cosmids in two independent libraries. Comparisons of 2304 of these sequences against the GenBank™ database identified 205 potential giardial genes with BLAST scores P(n)
Molecular and Biochemical Parasitology 01/1998; 95(2):267-280. · 2.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multiple exostoses is a polygenic disease of bone formation and development characterized by the presence of cartilage-capped osseous projections emanating from the end of the long bones. Two members of a recently defined multigene family of proteins (EXT1 and 2) were shown to be involved in this disease. To investigate the evolutionary relatedness of EXT genes across species we isolated the mouse EXT2 cDNA. As in the human counterpart, the mouse EXT2 cDNA contains an open reading frame of 2154 bp encoding a predicted protein of 718 amino acids. The nucleic acid sequence is 87% identical to the human EXT2 transcript, resulting in an amino acid sequence which is 95% identical to the human protein. The mouse EXT2 gene also shows significant sequence similarity to the mouse and human EXT1 gene. Northern blot analysis shows that this gene is expressed in early stages of embryonic development, and in situ hybridizations suggest that EXT2 plays a role in limb development. The identification of the mouse EXT2 gene will allow functional analysis through insertional inactivation and reverse genetics in mice in order to better understand the formation of exostoses during bone formation.
Biochemical and Molecular Medicine 07/1997; 61(1):16-21.
[Show abstract][Hide abstract] ABSTRACT: PRIMO is a computer program that designs walking primers for large-scale DNA sequencing projects. Oligonucleotide primers are predicted automatically, using quality information associated with each base call, eliminating the need for manually viewing the sequence traces or inspecting contig assemblies to determine appropriate locations for primer design. This allows PRIMO to run in batch mode on an arbitrarily large number of templates. For shotgun sequencing, PRIMO reads assembled sequence contigs with corresponding base quality statistics and automatically designs walking primers as needed to extend and join contigs, or improve their overall quality. In the opposite extreme of single-pass or completely directed sequencing, PRIMO reads the unassembled sequence for each template and designs walking primers for extending each read. If the base-calling software does not provide base quality statistics, PRIMO assigns its own measure of base quality determined by the shapes of individual peaks in the trace data for each template. In this way, PRIMO can be used in the finishing stages of a shotgun sequencing project, in sequencing by directed primer walking, or in some intermediate strategy. The code is written in ANSI C and maintained in two versions: one for the Macintosh and the other for UNIX.
[Show abstract][Hide abstract] ABSTRACT: Hereditary multiple exostoses (EXT) is an autosomal dominant condition characterized by short stature and the development of bony protuberances at the ends of all the long bones. Three genetic locl have been identified by genetic linkage analysis at chromosomes 8q24.1, 11p11-13 and 19p. The EXT1 gene on chromosome 8 was recently identified and characterized. Here, we report the isolation and characterization of the EXT2 gene. This gene shows striking sequence similarity to the EXT1 gene, and we have identified a four base deletion segregating with the phenotype. Both EXT1 and EXT2 show significant homology with one additional expressed sequence tag, defining a new multigene family of proteins with potential tumour suppressor activity.
[Show abstract][Hide abstract] ABSTRACT: Physical mapping of human chromosomes at a resolution of 100 kb to 1 Mb will provide important reagents for gene identification and framework templates for ultimately determining the complete DNA sequence. Sequence-tagged site (STS) content mapping, coupled with large fragment cloning in yeast artificial chromosomes, provides an efficient mechanism for producing first-generation, low-resolution maps of human chromosomes. Previously, we produced a set of standardized STSs for human chromosome 11 regionally localized by fluorescencein situhybridization or somatic cell hybrid analysis. In this paper, we used these as well as other STSs to map over 900 YAC clones to chromosome 11, organize yeast artificial chromosome (YAC) clones into contigs by STS content, and identify 109 islands spanning an estimated 218 Mb on the 126-Mb chromosome. Since about 62% of the islands contain markers ordered on chromosome 11 by genetic or radiation hybrid analysis, this data set represents a first-order approximation of a physical map of human chromosome 11. This set of clones, contigs, and associated STSs will provide the material for the production of a continuous overlapping set of YACs as well for high-resolution physical mapping based upon sampled and complete DNA sequencing.
[Show abstract][Hide abstract] ABSTRACT: Inner product mapping (IPM) has been proposed as a hybridization-based method for achieving low-cost, high-throughput, high-resolution radiation hybrid (RH) mapping of clones. Using Alu-PCR products of chromosome 11-specific clones, we serially hybridized a set of RHs against gridded filters of YACs having an average size of 350 kb. We then combined these hybridization data with preexisting RH map data to build an inner product map. This binning of 865 YACs provides the first high-resolution large-scale (> twofold redundancy) clonal coverage of human chromosome 11 and is the first inner product map ever constructed. We verified the accuracy and precision of this chromosome 11 map by performing a novel likelihood analysis on independent YAC hybridization data. These results establish that IPM is a highly rapid, inexpensive, accurate, and precise large-scale long-range mapping method, particularly when preexisting RH maps are available, and that IPM can replace or complement more conventional short-range mapping methods. IPM may enable the rapid construction of sequence-ready maps and the binning of expressed sequences.
[Show abstract][Hide abstract] ABSTRACT: A method for high-throughput, high-resolution physical mapping of complex genomes and human chromosomes called Genomic Sequence Sampling (GSS) has recently been proposed (Smith et al., 1994, Nature Genet. 7: 40-47). This mapping strategy employs high-density cosmid contig assembly over 200-kb to 1-Mb regions of the target genome coupled with DNA sequencing of the cosmid ends. The relative order and spacing of the sequence fragments is determined from the template contig, resulting in a physical map of 1- to 5-kb resolution that contains a substantial portion of the entire sequence at one-pass accuracy. The purpose of this paper is to determine the theoretical parameters for GSS mapping, to evaluate the effectiveness of the contig-building strategy, and to calculate the expected fraction of the target genome that can be recovered as mapped sequence. A novel aspect of the cosmid fingerprinting and contig-building strategy involves determining the orientation of the genomic inserts relative to the cloning vectors, so that the sampled sequence fragments can be mapped with high resolution. The algorithm is based upon complete restriction enzyme digestion, contig assembly by matching fragments, and end-orientation of individual cosmids by determining the best consistent fit of the labeled cosmid end fragments in the consensus restriction map.