Biomedical subjects

R Kucherlapati

Publications and source records attributed to R Kucherlapati.

At least 37 records · Page 2Linked to original sources

PubMed2000

Human KLRF1, a novel member of the killer cell lectin-like receptor gene family: molecular characterization, genomic structure, physical mapping to the NK gene complex and expression analysis.

The human NK gene complex localized on chromosome 12p12.3 - p13.2 codes for several lectin-like receptor genes expressed by NK cells as well as by other hematopoietic cells. In this study, by using the expressed sequence tag database we identified a novel receptor gene, designated as killer cell lectin-like receptor, subfamily F, member 1 (KLRF1), encoding a putative type II transmembrane glycoprotein. The KLRF1 gene has been localized on the high-resolution physical map of chromosome 12p. The genomic structure of the KLRF1 gene and the existence of one spliced variant are also described. KLRF1 was expressed at the mRNA level in peripheral blood leukocytes, activated NK cells, monocytes and NK and myeloid cell lines. The presence of two immunoreceptor tyrosine-based inhibitory-like motifs within the cytoplasmic tail of KLRF1 suggests an inhibitory role in NK cell and monocyte activity.

Amino Acid Sequence↗

PubMed2000

Cardiac defects and renal failure in mice with targeted mutations in Pkd2.

PKD2, mutations in which cause autosomal dominant polycystic kidney disease (ADPKD), encodes an integral membrane glycoprotein with similarity to calcium channel subunits. We induced two mutations in the mouse homologue Pkd2 (ref.4): an unstable allele (WS25; hereafter denoted Pkd2WS25) that can undergo homologous-recombination-based somatic rearrangement to form a null allele; and a true null mutation (WS183; hereafter denoted Pkd2-). We examined these mutations to understand the function of polycystin-2, the protein product of Pkd2, and to provide evidence that kidney and liver cyst formation associated with Pkd2 deficiency occurs by a two-hit mechanism. Pkd2-/- mice die in utero between embryonic day (E) 13.5 and parturition. They have structural defects in cardiac septation and cyst formation in maturing nephrons and pancreatic ducts. Pancreatic ductal cysts also occur in adult Pkd2WS25/- mice, suggesting that this clinical manifestation of ADPKD also occurs by a two-hit mechanism. As in human ADPKD, formation of kidney cysts in adult Pkd2WS25/- mice is associated with renal failure and early death (median survival, 65 weeks versus 94 weeks for controls). Adult Pkd2+/- mice have intermediate survival in the absence of cystic disease or renal failure, providing the first indication of a deleterious effect of haploinsufficiency at Pkd2on long-term survival. Our studies advance our understanding of the function of polycystin-2 in development and our mouse models recapitulate the complex human ADPKD phenotype.

Animals↗

PubMed2000

Mutations in KERA, encoding keratocan, cause cornea plana.

Specialized collagens and small leucine-rich proteoglycans (SLRPs) interact to produce the transparent corneal structure. In cornea plana, the forward convex curvature is flattened, leading to a decrease in refraction. A more severe, recessively inherited form (CNA2; MIM 217300) and a milder, dominantly inherited form (CNA1; MIM 121400) exist. CNA2 is a rare disorder with a worldwide distribution, but a high prevalence in the Finnish population. The gene mutated in CNA2 was assigned by linkage analysis to 12q (refs 4, 5), where there is a cluster of several SLRP genes. We cloned two additional SLRP genes highly expressed in cornea: KERA (encoding keratocan) in 12q and OGN (encoding osteoglycin) in 9q. Here we report mutations in KERA in 47 CNA2 patients: 46 Finnish patients are homozygous for a founder missense mutation, leading to the substitution of a highly conserved amino acid; and one American patient is homozygous for a mutation leading to a premature stop codon that truncates the KERA protein. Our data establish that mutations in KERA cause CNA2. CNA1 patients had no mutations in these proteoglycan genes.

Amino Acid Sequence↗

PubMed2000

A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains.

We explored the role of hypocretins in human narcolepsy through histopathology of six narcolepsy brains and mutation screening of Hcrt, Hcrtr1 and Hcrtr2 in 74 patients of various human leukocyte antigen and family history status. One Hcrt mutation, impairing peptide trafficking and processing, was found in a single case with early onset narcolepsy. In situ hybridization of the perifornical area and peptide radioimmunoassays indicated global loss of hypocretins, without gliosis or signs of inflammation in all human cases examined. Although hypocretin loci do not contribute significantly to genetic predisposition, most cases of human narcolepsy are associated with a deficient hypocretin system.

Adult↗

PubMed2000

High-resolution transcript map of the region spanning D12S1629 and D12S312 at chromosome 12q13: triple A syndrome-linked region.

For those searching for human disease-causing genes, information on the position of genes with respect to genetic markers is essential. The physical map composed of ESTs and genetic markers provides the positional information of these markers as well as the starting point of gene identification in the form of genomic clones containing exons. To facilitate the effort of identification of genes in the region spanning D12S1629 and D12S312, we constructed a high-resolution transcript map with PAC/BAC/cosmid clones. The strategy for the construction of such a map involved utilization of STSs for the screening of the large insert bacterial chromosome libraries and a chromosome 12-specific cosmid library by hybridization. The contig was constructed based on the STS contents of the clones. The resulting high-resolution transcript map of the region between P273P14/SP6 and D12S312 spans 4.4 cM from 66.8 to 71.2 cM of the Généthon genetic map and represents approximately 2.4 Mb. It was composed of 81 BAC, 45 PAC, and 91 cosmid clones with a minimal tiling path consisting of 16 BAC and 4 PAC clones. These clones are being used to sequence this part of chromosome 12. We determined the order of 135 STSs including 74 genes and ESTs in the map. Among these, 115 STSs were unambiguously ordered, resulting in one ordered marker per 21 kb. The order of keratin type II locus genes was determined. This map would greatly enhance the positional cloning effort of the responsible genes for those diseases that are linked to this region, including male germ cell tumor as well as palmoplantar keratoderma, Bothnian-type, and triple A syndrome. This transcript map was localized at human chromosome 12q13.

Addison Disease↗

PubMed2000

Mutations in the protein kinase A R1alpha regulatory subunit cause familial cardiac myxomas and Carney complex.

Cardiac myxomas are benign mesenchymal tumors that can present as components of the human autosomal dominant disorder Carney complex. Syndromic cardiac myxomas are associated with spotty pigmentation of the skin and endocrinopathy. Our linkage analysis mapped a Carney complex gene defect to chromosome 17q24. We now demonstrate that the PRKAR1alpha gene encoding the R1alpha regulatory subunit of cAMP-dependent protein kinase A (PKA) maps to this chromosome 17q24 locus. Furthermore, we show that PRKAR1alpha frameshift mutations in three unrelated families result in haploinsufficiency of R1alpha and cause Carney complex. We did not detect any truncated R1alpha protein encoded by mutant PRKAR1alpha. Although cardiac tumorigenesis may require a second somatic mutation, DNA and protein analyses of an atrial myxoma resected from a Carney complex patient with a PRKAR1alpha deletion revealed that the myxoma cells retain both the wild-type and the mutant PRKAR1alpha alleles and that wild-type R1alpha protein is stably expressed. However, in this atrial myxoma, we did observe a reversal of the ratio of R1alpha to R2beta regulatory subunit protein, which may contribute to tumorigenesis. Further investigation will elucidate the cell-specific effects of PRKAR1alpha haploinsufficiency on PKA activity and the role of PKA in cardiac growth and differentiation.

Abnormalities, Multiple↗

PubMed2000

A sequence-ready map for human chromosome 12q15-21.

Construction of sequence-ready clone map is an essential step toward sequencing the human genome. We chose a region that is frequently amplified in liposarcoma between D12S350 and D12S106 in chromosome 12q15-21 to build a PAC/BAC clone contig map. This region was spanned by 4 YACs and contained 30 STS on the YAC and radiation hybrid (RH) framework maps, providing an average STS spacing of 160 kb if each YAC is approximately 1.2 Mb in size. To convert a STS-based YAC map to a STS-based contig map of bacterial clones, 22 non-polymorphic STS markers were used as probes to screen the high density gridded arrays of PAC and BAC clones by filter hybridizations, followed by assembly of clones into contigs by marker content. Contigs have been extended and joined by direct end sequencing of appropriate clones, generating new STSs and rescreening the library as necessary. Using these approaches, we have constructed 5 contigs covering the region with the largest single contig being 1.4 Mb and a final size estimation of 3.6 Mb. The map is comprised of 17 YACs, 187 PACs, 160 BACs, and 17 cosmids; onto this, 6 polymorphic, 97 non-polymorphic, 24 ESTs, and 4 gene-based markers are now placed in a unique order, providing an average resolution of approximately 28 kb. Of a total of 131 markers, 97 were developed in the present study. The sequence-ready map should provide a framework to generate complete DNA sequence and ultimately gene map of this segment of chromosome 12.

Base Sequence↗

PubMed1999

Short-chain fatty acid metabolism, apoptosis, and Apc-initiated tumorigenesis in the mouse gastrointestinal mucosa.

Short-chain fatty acids (SCFAs) are physiological regulators of growth and differentiation in the gastrointestinal tract, and we have previously shown that apoptosis induced in colonic cell lines by these compounds is dependent on their metabolism by B-oxidation in the mitochondria (B. G. Heerdt et al., J. Biol. Chem., 266: 19120-19126, 1991; Cancer Res., 54: 3288-3293, 1994). Because tumors initiated by an inherited Apc mutation have been reported to be linked to decreases in apoptosis in the flat mucosa of the gastrointestinal tract, the aims were to determine whether elimination of efficient metabolism of SCFAs affected apoptosis in the gastrointestinal mucosa of the mouse, and whether this altered tumorigenesis initiated by an inherited Apc mutation. We, therefore, generated mice that have a chain-terminating mutation in the Apc gene and that were either wild-type for SCFA metabolism, or deficient, due to homozygous deletion of the gene (Scad) that encodes the enzyme short-chain acyl dehydrogenase, which catalyzes the first step in SCFA B-oxidation. Scad+/+ mice maintained on a wheat bran-fiber-supplemented diet gained significantly more weight than mice maintained on AIN76A, but this was eliminated by the Scad mutation, demonstrating that uptake and metabolism of SCFAs in the gastrointestinal tract can be a significant energy source. As predicted, on either AIN76A or wheat bran diet, the Scad mutation almost completely eliminated apoptosis in the flat mucosa of the proximal colon and reduced apoptosis by 50% in the distal colon compared with littermates that were wild-type for Scad. The mutation also reduced apoptosis by approximately 50% in the duodenum in AIN76A-fed mice. These reductions in apoptosis had no effect on incidence, frequency, or site specificity of tumors initiated by the Apc mutation. Therefore, the metabolism of SCFAs by the gastrointestinal mucosa plays a role in modulating apoptosis, but a general decrease in apoptosis in the mucosa of the gastrointestinal tract is not linked to gastrointestinal tumorigenesis initiated by an inherited Apc mutation.

Animals↗

PubMed1999

Postgastrulation Smad2-deficient embryos show defects in embryo turning and anterior morphogenesis.

SMAD2 is a member of the transforming growth factor beta and activin-signaling pathway. To examine the role of Smad2 in postgastrulation development, we independently generated mice with a null mutation in this gene. Smad2-deficient embryos die around day 7.5 of gestation because of failure of gastrulation and failure to establish an anterior-posterior (A-P) axis. Expression of the homeobox gene Hex (the earliest known marker of the A-P polarity and the prospective head organizer) was found to be missing in Smad2-deficient embryos. Homozygous mutant embryos and embryonic stem cells formed mesoderm derivatives revealing that mesoderm induction is SMAD2 independent. In the presence of wild-type extraembryonic tissues, Smad2-deficient embryos developed beyond 7.5 and up to 10.5 days postcoitum, demonstrating a requirement for SMAD2 in extraembryonic tissues for the generation of an A-P axis and gastrulation. The rescued postgastrulation embryos showed malformation of head structures, abnormal embryo turning, and cyclopia. Our results show that Smad2 expression is required at several stages during embryogenesis.

Alleles↗

PubMed1999

Mouse models for colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers in the Western world. Much has been learned about colorectal cancer from human inherited syndromes, such as familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC). Mouse models for CRC were generated by introducing mutations into the mouse genes, whose human counterparts were implicated in the onset and progression of CRC. Central among these are mice carrying mutations in the Adenomatous polyposis coli (Apc) gene. Although most of these Apc mutations share some common phenotypes as homozygous embryonic lethality and tumor predisposition, the severity of the tumor predisposition is variable. Mice with mutations in the mismatch repair genes, Msh2 and Mlh1, exhibit a mismatch repair defect and are predisposed to developing gastrointestinal cancer, lymphomas and tumors of other organ systems. Mice carrying a mutation in the Pms2 gene are predisposed to lymphomas and other tumors. Mice with a mutation in the Msh6 gene have a defect in base mismatch repair and show a tumor predisposition phenotype. Mice with mutations in Mlh1, Pms2 and Msh5 have defects in meiosis suggesting unique roles for these genes in gametogenesis.

Adenomatous Polyposis Coli↗

PubMed1999

Reduced isotype switching in splenic B cells from mice deficient in mismatch repair enzymes.

Mice deficient in various mismatch repair (MMR) enzymes were examined to determine whether this repair pathway is involved in antibody class switch recombination. Splenic B cells from mice deficient in Msh2, Mlh1, Pms2, or Mlh1 and Pms2 were stimulated in culture with lipopolysaccharide (LPS) to induce immunoglobulin (Ig)G2b and IgG3, LPS and interleukin (IL)-4 to induce IgG1, or LPS, anti-delta-dextran, IL-4, IL-5, and transforming growth factor (TGF)-beta1 to induce IgA. After 4 d in culture, cells were surface stained for IgM and non-IgM isotypes and analyzed by FACS((R)). B cells from MMR-deficient mice show a 35-75% reduction in isotype switching, depending on the isotype and on the particular MMR enzyme missing. IgG2b is the most affected, reduced by 75% in Mlh1-deficient animals. The switching defect is not due to a lack of maturation of the B cells, as purified IgM(+)IgD(+) B cells show the same reduction. MMR deficiency had no effect on cell proliferation, viability, or apoptosis, as detected by [(3)H]thymidine incorporation and by propidium iodide staining. The reduction in isotype switching was demonstrated to be at the level of DNA recombination by digestion-circularization polymerase chain reaction (DC-PCR). A model of the potential role for MMR enzymes in class switch recombination is presented.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Apc1638T: a mouse model delineating critical domains of the adenomatous polyposis coli protein involved in tumorigenesis and development.

The adenomatous polyposis coli (APC) gene is considered as the true gatekeeper of colonic epithelial proliferation: It is mutated in the majority of colorectal tumors, and mutations occur at early stages of tumor development in mouse and man. These mutant proteins lack most of the seven 20-amino-acid repeats and all SAMP motifs that have been associated with down-regulation of intracellular beta-catenin levels. In addition, they lack the carboxy-terminal domains that bind to DLG, EB1, and microtubulin. APC also appears to be essential in development because homozygosity for mouse Apc mutations invariably results in early embryonic lethality. Here, we describe the generation of a mouse model carrying a targeted mutation at codon 1638 of the mouse Apc gene, Apc1638T, resulting in a truncated Apc protein encompassing three of the seven 20 amino acid repeats and one SAMP motif, but missing all of the carboxy-terminal domains thought to be associated with tumorigenesis. Surprisingly, homozygosity for the Apc1638T mutation is compatible with postnatal life. However, homozygous mutant animals are characterized by growth retardation, a reduced postnatal viability on the B6 genetic background, the absence of preputial glands, and the formation of nipple-associated cysts. Most importantly, Apc1638T/1638T animals that survive to adulthood are tumor free. Although the full complement of Apc1638T is sufficient for proper beta-catenin signaling, dosage reductions of the truncated protein result in increasingly severe defects in beta-catenin regulation. The SAMP motif retained in Apc1638T also appears to be important for this function as shown by analysis of the Apc1572T protein in which its targeted deletion results in a further reduction in the ability of properly controlling beta-catenin/Tcf signaling. These results indicate that the association with DLG, EB1, and microtubulin is less critical for the maintenance of homeostasis by APC than has been suggested previously, and that proper beta-catenin regulation by APC appears to be required for normal embryonic development and tumor suppression.

Adenomatous Polyposis Coli Protein↗

PubMed1999

Organization of human and mouse skeletal myosin heavy chain gene clusters is highly conserved.

Myosin heavy chains (MyHCs) are highly conserved ubiquitous actin-based motor proteins that drive a wide range of motile processes in eukaryotic cells. MyHC isoforms expressed in skeletal muscles are encoded by a multigene family that is clustered on syntenic regions of human and mouse chromosomes 17 and 11, respectively. In an effort to gain a better understanding of the genomic organization of the skeletal MyHC genes and its effects on the regulation, function, and molecular genetics of this multigene family, we have constructed high-resolution physical maps of both human and mouse loci using PCR-based marker content mapping of P1-artificial chromosome clones. Genes encoding six MyHC isoforms have been mapped with respect to their linear order and transcriptional orientations within a 350-kb region in both human and mouse. These maps reveal that the order, transcriptional orientation, and relative intergenic distances of these genes are remarkably conserved between these species. Unlike many clustered gene families, this order does not reflect the known temporal expression patterns of these genes. However, the conservation of gene organization since the estimated divergence of these species (approximately 75-110 million years ago) suggests that the physical organization of these genes may be significant for their regulation and function.

Animals↗

PubMed1999

Tumorigenesis in Mlh1 and Mlh1/Apc1638N mutant mice.

An3 1 KAL I MutL homologue 1 (MLH1) is a member of the family of proteins required for DNA mismatch repair. Germ-line mutations in MLH1 lead to the cancer susceptibility syndrome hereditary nonpolyposis colorectal cancer (HNPCC). We generated mice carrying a null mutation in the Mlh1 gene. We showed that mice heterozygous and homozygous for the Mlh1 gene are predisposed to developing tumors of the gastrointestinal (GI) tract, lymphomas, and a number of other tumor types. We also examined the role of adenomatous polyposis coli gene (Apc) gene mutations in the GI tumors of Mlh1 mutant mice by different methods and showed that the GI tumors in Mlh1 mice express little or no adenomatous polyposis coli protein. When an Apc gene mutation was bred into the Mlh1 mutant mice, the GI tumor incidence increased 40-100-fold. The wild-type Apc allele in these tumors was found to contain mutations. Together, these results show that we have developed two mouse models for human HNPCC and that the mechanisms of tumor development in the GI tract of these mice involve loss of Apc gene function in a manner very similar to that seen in the GI tumors of HNPCC.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Genotype-phenotype correlation in murine Apc mutation: differences in enterocyte migration and response to sulindac.

The adenomatous polyposis coli (APC) gene product mediates coordinated cell growth in the intestinal mucosa. In humans, germ-line mutations of APC are associated with colorectal carcinogenesis, a process that varies in severity depending on the length of the protein resulting from the mutant allele. In a previous study of the C57BL/6J-Min/+ (Min/+) mouse, we found that the protein fragment resulting from truncation at codon 850 of murine Apc was associated with changes in enterocyte migration, proliferation, apoptosis, and beta-catenin expression. This effect was reversed upon treatment of Min/+ mice with the chemopreventive drug sulindac sulfide. In this study, we measured enterocyte migration in the Apc1638N mouse, an animal with an Apc mutation that yields no detectable APC protein. We found no difference in enterocyte migration, proliferation, apoptosis, or beta-catenin levels in the Apc1638N mouse when compared to wild-type littermates bearing two normal Apc alleles. Furthermore, administration of sulindac sulfide to Apc1638N mice did not alter enterocyte migration. These observations suggest that a dominant negative effect altering cell migration is exerted by the truncated APC protein present in the Min/+ mouse. These data also suggest that the effectiveness of chemopreventive agents in preventing Apc-related tumor formation may depend on which type of mutation is present.

Adenomatous Polyposis Coli↗

PubMed1999

Making and reading microarrays.

There are a variety of options for making microarrays and obtaining microarray data. Here, we describe the building and use of two microarray facilities in academic settings. In addition to specifying technical detail, we comment on the advantages and disadvantages of components and approaches, and provide a protocol for hybridization. The fact that we are now making and using microarrays to answer biological questions demonstrates that the technology can be implemented in a university environment.

DNA↗

PubMed1999

Mammalian MutS homologue 5 is required for chromosome pairing in meiosis.

MSH5 (MutS homologue 5) is a member of a family of proteins known to be involved in DNA mismatch repair. Germline mutations in MSH2, MLH1 and GTBP (also known as MSH6) cause hereditary non-polyposis colon cancer (HNPCC) or Lynch syndrome. Inactivation of Msh2, Mlh1, Gtmbp (also known as Msh6) or Pms2 in mice leads to hereditary predisposition to intestinal and other cancers. Early studies in yeast revealed a role for some of these proteins, including Msh5, in meiosis. Gene targeting studies in mice confirmed roles for Mlh1 and Pms2 in mammalian meiosis. To assess the role of Msh5 in mammals, we generated and characterized mice with a null mutation in Msh5. Msh5-/- mice are viable but sterile. Meiosis in these mice is affected due to the disruption of chromosome pairing in prophase I. We found that this meiotic failure leads to a diminution in testicular size and a complete loss of ovarian structures. Our results show that normal Msh5 function is essential for meiotic progression and, in females, gonadal maintenance.

Animals↗

PubMed1999

Mutations in ATP2A2, encoding a Ca2+ pump, cause Darier disease.

Darier disease (DD) is an autosomal-dominant skin disorder characterized by loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization. Recently we constructed a 2.4-Mb, P1-derived artificial chromosome contig spanning the DD candidate region on chromosome 12q23-24.1. After screening several genes that mapped to this region, we identified mutations in the ATP2A2 gene, which encodes the sarco/endoplasmic reticulum Ca2(+)-ATPase type 2 isoform (SERCA2) and is highly expressed in keratinocytes. Thirteen mutations were identified, including frameshift deletions, in-frame deletions or insertions, splice-site mutations and non-conservative missense mutations in functional domains. Our results demonstrate that mutations in ATP2A2 cause DD and disclose a role for this pump in a Ca(2+)-signalling pathway regulating cell-to-cell adhesion and differentiation of the epidermis.

Calcium-Transporting ATPases↗