RESUMO
Hirschsprung disease (HSCR) is a complex genetic disorder characterized by the absence of enteric nervous system (ENS) in the distal region of the intestine. Down Syndrome (DS) patients have a >50-fold higher risk of developing HSCR than the general population, suggesting that overexpression of human chromosome 21 (Hsa21) genes contribute to HSCR etiology. However, identification of responsible genes remains challenging. Here, we describe a genetic screening of potential candidate genes located on Hsa21, using the zebrafish. Candidate genes were located in the DS-HSCR susceptibility region, expressed in the human intestine, were known potential biomarkers for DS prenatal diagnosis, and were present in the zebrafish genome. With this approach, four genes were selected: RCAN1, ITSN1, ATP5PO and SUMO3. However, only overexpression of ATP5PO, coding for a component of the mitochondrial ATPase, led to significant reduction of ENS cells. Paradoxically, in vitro studies showed that overexpression of ATP5PO led to a reduction of ATP5PO protein levels. Impaired neuronal differentiation and reduced mitochondrial ATP production, were also detected in vitro, after overexpression of ATP5PO in a neuroblastoma cell line. Finally, epistasis was observed between ATP5PO and ret, the most important HSCR gene. Taken together, our results identify ATP5PO as the gene responsible for the increased risk of HSCR in DS patients in particular if RET variants are also present, and show that a balanced expression of ATP5PO is required for normal ENS development.
Assuntos
Síndrome de Down , Sistema Nervoso Entérico , Doença de Hirschsprung , Animais , Humanos , Doença de Hirschsprung/genética , Doença de Hirschsprung/metabolismo , Síndrome de Down/genética , Síndrome de Down/metabolismo , Peixe-Zebra/genética , Sistema Nervoso Entérico/metabolismo , Biomarcadores/metabolismoRESUMO
Trisomy 21 or Down syndrome (DS) is the most frequent genetic cause of mental retardation, affecting one in 800 live born human beings. Mice with segmental trisomy 16 (Ts65Dn mice) are at dosage imbalance for genes corresponding to those on human chromosome 21q21-22.3--which includes the so-called DS 'critical region'. They do not show early-onset of Alzheimer disease pathology; however, Ts65Dn mice do demonstrate impaired performance in a complex learning task requiring the integration of visual and spatial information. The reproducibility of this phenotype among Ts65Dn mice indicates that dosage imbalance for a gene or genes in this region contributes to this impairment. The corresponding dosage imbalance for the human homologues of these genes may contribute to cognitive deficits in DS.
Assuntos
Mapeamento Cromossômico , Síndrome de Down/genética , Síndrome de Down/fisiopatologia , Aprendizagem , Atividade Motora , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/genética , Análise de Variância , Animais , Cromossomos Humanos Par 21 , Modelos Animais de Doenças , Síndrome de Down/psicologia , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Caracteres SexuaisRESUMO
The existence of tumor-suppressor genes was originally demonstrated by functional complementation through whole-cell and microcell fusion. Transfer of chromosome 11 into a human non-small-cell lung cancer (NSCLC) cell line, A549, suppresses tumorigenicity. Loss of heterozygosity (LOH) on the long arm of chromosome 11 has been reported in NSCLC and other cancers. Several independent studies indicate that multiple tumor-suppressor genes are found in this region, including the gene PPP2R1B at 11q23-24 (ref. 7). Linkage studies of NSCLC are precluded because no hereditary forms are known. We previously identified a region of 700 kb on 11q23.2 that completely suppresses tumorigenicity of A549 human NSCLC cells. Most of this tumor-suppressor activity localizes to a 100-kb segment by functional complementation. Here we report that this region contains a single confirmed gene, TSLC1, whose expression is reduced or absent in A549 and several other NSCLC, hepatocellular carcinoma (HCC) and pancreatic cancer (PaC) cell lines. TSLC1 expression or suppression is correlated with promoter methylation state in these cell lines. Restoration of TSLC1 expression to normal or higher levels suppresses tumor formation by A549 cells in nude mice. Only 2 inactivating mutations of TSLC1 were discovered in 161 tumors and tumor cell lines, both among the 20 primary tumors with LOH for 11q23.2. Promoter methylation was observed in 15 of the other 18 primary NSCLC, HCC and PaC tumors with LOH for 11q23.2. Thus, attenuation of TSLC1 expression occurred in 85% of primary tumors with LOH. Hypermethylation of the TSLC1 promoter would seem to represent the 'second hit' in NSCLC with LOH.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Genes Supressores de Tumor , Imunoglobulinas , Neoplasias Pulmonares/genética , Proteínas de Membrana , Proteínas/genética , Animais , Sequência de Bases , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Primers do DNA , DNA Complementar , Ligação Genética , Humanos , Perda de Heterozigosidade , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Proteínas Supressoras de TumorRESUMO
Down syndrome (DS), trisomy of human chromosome 21, is the most common genetic cause of intellectual disability. With an incidence in some countries as high as one in approximately 700 live births, and a complex, extensive and variably severe phenotype, Down syndrome is a significant medical and social challenge. In recent years, there has been a rapid increase in information on the functions of the genes of human chromosome 21, as well as in techniques and resources for their analysis. A recent workshop brought together experts on the molecular biology of Down syndrome and chromosome 21 with interested researchers in other fields to discuss advances and potentials for generating gene-phenotype correlations. An additional goal of the workshop was to work towards identification of targets for therapeutics that will correct features of DS. A knowledge-based approach to therapeutics also requires the correlation of chromosome 21 gene function with phenotypic features.
Assuntos
Cromossomos Humanos Par 21/genética , Síndrome de Down/genética , Animais , Citogenética , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Síndrome de Down/terapia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , MicroRNAs/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Musculares/genética , Sistema Nervoso/crescimento & desenvolvimento , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteína cdc42 de Ligação ao GTP/genética , Quinases DyrkRESUMO
The molecular mechanisms underlying the specific traits in individuals with Down syndrome (DS) have been postulated to derive either from nonspecific perturbation of balanced genetic programs, or from the simple, mendelian-like influence of a small subset of genes on chromosome 21. However, these models do not provide a comprehensive explanation for experimental or clinical observations of the effects of trisomy 21. DS is best viewed as a complex genetic disorder, where the specific phenotypic manifestations in a given individual are products of genetic, environmental and stochastic influences. Mouse models that recapitulate both the genetic basis for and the phenotypic consequences of trisomy provide an experimental system to define these contributions.
Assuntos
Síndrome de Down/genética , Aneuploidia , Animais , Modelos Animais de Doenças , Síndrome de Down/patologia , Humanos , Camundongos , Camundongos Transgênicos , Modelos Genéticos , FenótipoRESUMO
A neomycin resistance cassette was integrated into the human-derived insert of a 360-kilobase yeast artificial chromosome (YAC) by targeting homologous recombination to Alu repeat sequences. The modified YAC was transferred into an embryonal carcinoma cell line by using polyethylene glycol-mediated spheroplast fusion. A single copy of the human sequence was introduced intact and stably maintained in the absence of selection for over 40 generations. A substantial portion of the yeast genome was retained in hybrids in addition to the YAC. Hybrid cells containing the YAC retained the ability to differentiate when treated with retinoic acid. This approach provides a powerful tool for in vitro analysis because it can be used to modify any human DNA cloned as a YAC and to transfer large fragments of DNA intact into cultured mammalian cells, thereby facilitating functional studies of genes in the context of extensive flanking DNA sequences.
Assuntos
Cromossomos Fúngicos , Biblioteca Gênica , Genoma Humano , Saccharomyces cerevisiae/genética , Transfecção , Animais , Linhagem Celular , Clonagem Molecular , Vetores Genéticos , Humanos , Hibridização de Ácido Nucleico , Plasmídeos , Mapeamento por Restrição , TeratomaRESUMO
The Subsurface Microbial Culture Collection (SMCC) was established by the U.S. Dept. of Energy (DOE) and contains nearly 10,000 strains of microorganisms (mostly bacteria) isolated from terrestrial subsurface environments. Selected groups of bacterial isolates from three sample sites situated above geochemically and hydrologically different subsurface environments have been characterized by phylogenetic analysis of 16S ribosomal RNA (rRNA) gene nucleotide sequences. Among these isolates were members of six major phylogenetic groups of bacteria: the high-G+C and low-G+C Gram-positive bacteria; the alpha-, beta-, and gamma-subdivisions of the Proteobacteria; and the Flexibacter/Cytophaga/Bacteroides group. A small number of the SMCC strains may be members of new bacterial genera, but most of them could be placed with reasonable confidence into more than 35 previously described genera. The majority of the Gram-positive isolates were species of Arthrobacter, Bacillus, or Streptococcus, whereas Acinetobacter, Comamonas, Pseudomonas, Sphingomonas, and Variovorax were among the most frequently encountered Gram-negative genera. A high proportion of the strains were placed in fewer than 10 genera, implying that there is substantial duplication within the SMCC at the genus level. When groups of isolates assigned to Acinetobacter, Arthrobacter, or Sphingomonas were analyzed in more detail, however, it was found that each group consisted of subgroups of strains that probably differed at the species level. Restriction endonuclease analysis (applied to the strains from one sample site) indicated that additional diversity was present at the strain level. Most of the SMCC isolates assigned to some genera (e.g., Acinetobacter) were very closely related to previously described species in those genera, but most of the isolates assigned to other genera (e.g., Arthrobacter and Sphingomonas) appeared (or were shown) to be new species, thereby indicating that a reasonable amount of novelty is present within the SMCC at the species level.
Assuntos
Bactérias/classificação , Microbiologia Ambiental , Filogenia , RNA Ribossômico 16S/genética , Bancos de Espécimes Biológicos , DNA Ribossômico/genética , Fenômenos Geológicos , Geologia , Órgãos Governamentais , Dados de Sequência Molecular , RNA Bacteriano/genética , Estados UnidosRESUMO
Chromosome fragmentation vectors (CFVs) are used to create deletion derivatives of large fragments of human DNA cloned as yeast artificial chromosomes (YACs). CFVs target insertion of a telomere sequence into the YAC via homologous recombination with Alu repetitive elements. This event results in the loss of all YAC sequences distal to the site of integration. A new series of CFVs has been developed. These vectors target fragmentation to both Alu and LINE human repetitive DNA elements. Recovery of deletion derivatives is ten- to 20-fold more efficient with the new vectors than with those described previously.
Assuntos
Cromossomos Fúngicos , Vetores Genéticos , Biblioteca Gênica , Genoma Humano , Humanos , Mapeamento por Restrição , Transformação GenéticaRESUMO
Loss of heterozygosity for a locus on human chromosome 11q22-23 is observed at high frequency in non-small cell lung carcinoma (NSCLC). Introduction of a 1.1 Mb fragmented yeast artificial chromosome (YAC) mapping to this region completely suppresses the tumorigenic properties of a human NSCLC cell line, A549. Smaller fragmented YACs give partial but not complete suppression. To further localize the gene(s) responsible for this partial suppression, a bacterial artificial chromosome (BAC) and P1-based artificial chromosome (PAC) contig was constructed, completely spanning the candidate region. End sequence generated in the construction of the BAC/PAC contig identified a previously unmapped EST and served to order genomic sequence contigs from the publicly available Celera Genomics (CG) and Human Genome Project (HGP) efforts. Comparison showed that CG provided larger contigs, while HGP provided more coverage. Neither CG nor HGP provided complete sequence coverage, alone or in combination. The sequence was used to map 110 ESTs and to predict new genes, including two GenScan gene predictions that overlapped ESTs and were shown to be differentially expressed in tumorigenic and suppressed A549 cell lines.
Assuntos
Cromossomos Humanos Par 11/genética , Genes Supressores de Tumor/genética , Predisposição Genética para Doença/genética , Imunoglobulinas , Proteínas de Membrana , Proteínas/genética , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular , Mapeamento de Sequências Contíguas , DNA de Neoplasias/química , DNA de Neoplasias/genética , Humanos , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , Análise de Sequência de DNA , Células Tumorais Cultivadas , Proteínas Supressoras de TumorRESUMO
Multiple organ dysfunction syndrome (MODS) appears to be the result of a complex program influenced by multiple factors, including environmental, physiological, and immunological conditions. Thus, an uncontrolled inflammatory response following a stochastic event, the initial injury, is believed to be the cause for the development of this syndrome. Several lines of evidence suggest that a genetic component could contribute to the regulation of the inflammatory response, as well, but no direct evidence demonstrates a heritable predisposition to MODS. In the present study, a genetic contribution was demonstrated for the inflammatory response induced by the administration of bacterial lipopolysaccharide (LPS) in different, genetically distinct strains of inbred mice. A survey of five inbred strains showed that mortality following administration of Escherichia coli LPS (20 mg/kg) was highest in C57BL/6J (B6) mice, while A/J mice were the most resistant. Accordingly, B6 and A/J mice were examined further for differences in the inflammatory response elicited by LPS. B6 mice showed higher levels of circulating interleukin-1beta and interleukin-6, as well as higher mRNA levels of hepatic beta-fibrinogen (an acute-phase gene) and metallothionein. Surprisingly, the circulating levels of tumor necrosis factor-alpha were significantly higher in A/J than in B6 mice after LPS administration. Since B6 and A/J mice were bred and raised in identical environments and received the same LPS challenge, the contrasting inflammatory response that was observed is largely attributable to genetic differences between these two strains. These data illustrate that the response to injury could be modulated by the genetic background of the individual. This information may be pertinent for the care of critically ill patients.
Assuntos
Citocinas/sangue , Inflamação/genética , Inflamação/mortalidade , Lipopolissacarídeos/toxicidade , Camundongos Endogâmicos/genética , Animais , Fibrinogênio/metabolismo , Inflamação/metabolismo , Fígado/metabolismo , Fígado/microbiologia , Masculino , Metalotioneína/metabolismo , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBARESUMO
Chromosomal protein HMG-14 is a ubiquitous nuclear protein that may modulate the chromatin structure of transcriptionally active genes. To gain insights into the cellular function of the HMG-14 protein, we generated two transgenic mouse lines carrying either two or six copies of the human HMG-14 gene. The transgenic mice express human HMG-14 mRNA and protein in all tissues examined at a level reflecting the increased gene dosage, suggesting that the HMG14 transgene contains all the control regions necessary for regulated gene expression. Expression of the human HMG-14 protein does not alter the expression of the endogenous mouse HMG-14 protein or its close homolog, protein HMG-17. The intracellular distribution of the exogenous human protein is indistinguishable from that of the endogenous mouse protein, resulting in a three-fold increase in the level of the chromatin-bound HMG-14. The transgenic mice had a higher incidence of epithelial cysts in their thymus than did control animals. We conclude that the cellular levels of HMG-14/-17 are determined by gene copy number, that the DNA fragment containing the gene and about 1,000 bp flanking its 5' and 3' ends contain most of the elements necessary for gene expression, that the upper limits of HMG-14 in chromatin are not stringently regulated, and that a three-fold increase in chromatin-bound protein cause only mild phenotypic changes in the transgenic mice.
Assuntos
Cromatina/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Animais , Feminino , Expressão Gênica , Heterozigoto , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/genética , TransgenesRESUMO
S100 beta protein, a member of a Ca(2+)-binding protein family present primarily in the nervous system, affects the survival and differentiation of both neurons and glia cells. Elevated levels of S100 beta protein have been observed in the brains of individuals with Alzheimer Disease (AD), as well as in those with Down Syndrome (DS). We have examined transcript levels from the gene encoding the amyloid precursor protein (APP) in four brain regions of mice from 1 to 24 months of age. After stable adult levels of expression are reached, APP mRNA levels do not change with aging. APP mRNA levels are independent of normal regional variation in S100 beta mRNA and protein. Further, chronic exposure to S100 beta elevated 2- or 7-fold above normal did not alter the transcript levels of APP in transgenic mice. These results leave open the possibility of focal changes in APP transcription and do not address possible effects of S100 beta on the complex processing known to occur with APP protein. However, neither control nor transgenic aged mice showed any evidence of abnormal deposition of amyloid in neuritic plaques. These results are discussed in the context of hypotheses about the role of elevated S100 beta in DS and AD.
Assuntos
Envelhecimento/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , RNA Mensageiro/metabolismo , Proteínas S100/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Fatores de TempoRESUMO
Mice homozygous for the gene dwarf (dw) have elevated levels of somatostatin (SS) in extra-hypothalamic brain regions. By in situ hybridization, increased levels of SS mRNA were observed in regions shown previously to contain higher levels of the SS peptide. Thus, the rate of transcription and/or the stability of SS mRNA are affected by the dw mutation. Since both dw and the gene encoding SS, Smst, are located on mouse chromosome 16, two backcrosses segregating dw and Smst were used to determine whether dw is an allele of Smst. In one backcross, an inbred strain derived from the subspecies Mus musculus molossinus was used to provide a high degree of DNA sequence polymorphism. The gene order and map distances determined on this backcross were: (centromere) - Prm-1 - 7 - Igl-1 - 3 - Smst - 29 - dw - 15 - Sod-1 - 4 - Ets-2, demonstrating clearly that Smst and dw are distinct genes. Additional evidence against a primary role for SS excess in the pathogenesis of dw/dw mice was obtained by injecting normal newborn mice with a potent SS analog (cyclo II). In contrast to the pattern of cell loss observed in the dwarf anterior pituitary, the pituitaries of injected mice were indistinguishable from normal controls, further suggesting that the Smst locus is not the primary site of dw gene action.
Assuntos
Encéfalo/metabolismo , Mapeamento Cromossômico , Genes , Camundongos Mutantes/genética , Somatostatina/genética , Alelos , Animais , Clonagem Molecular , Cruzamentos Genéticos , DNA/genética , DNA/isolamento & purificação , Nanismo/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL/genética , Camundongos Endogâmicos/genética , Hibridização de Ácido Nucleico , Mapeamento por RestriçãoRESUMO
Recently, the gene encoding the cerebrovascular and neuritic plaque amyloid, a pathologic stigma of Alzheimer's disease (AD), has been molecularly cloned and mapped to human chromosome 21, band q21. Changes in the brains of individuals with trisomy 21 (Down syndrome, DS) over 35 years of age closely resemble AD neuropathology. Genetic homology which exists between human chromosome 21 (HSA 21) and mouse chromosome 16 (MMU 16) has led to the use of mice with trisomy 16 as a model system for studies relevant to DS. Mice with Ts16 exhibit numerous developmental abnormalities that can be correlated with features observed in DS, including neurochemical and neuroanatomic alterations. In this study, we show that the genetic homology between HSA 21 and MMU 16 extends to the gene encoding the amyloid peptide. The homologous mouse gene, designated Cvap, for cerebrovascular amyloid peptide, is localized on MMU 16 band C3----ter, and is in close proximity to both superoxide dismutase-1 (Sod-1), and the protooncogene, Ets-2, two of the genes known to localize to the DS region of HSA 21. Linkage of these genes has been maintained since the divergence of the common ancestor of mouse and man, despite a chromosomal rearrangement which has changed the gene order between the two species. These findings expand the region of HSA 21 with known homology to MMU 16, and provide a genetic basis to suggest that studies of the trisomy 16 mouse, in addition to being relevant to DS, may also clarify the role of abnormal gene expression in AD.
Assuntos
Doença de Alzheimer/genética , Amiloide/genética , Mapeamento Cromossômico , Síndrome de Down/genética , Ligação Genética , Doença de Alzheimer/metabolismo , Animais , Cromossomos Humanos Par 21/análise , Cricetinae , Modelos Animais de Doenças , Síndrome de Down/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The expression during development of 3 genes located on mouse chromosome 16 (MMU 16) which are implicated in neurobiological processes was examined by blot hybridization beginning at early gestational ages in the mouse. The 3 genes, amyloid precursor protein (App), preprosomatostatin (Smst), and growth-associated protein 43 (Gap43), exhibited distinct profiles of expression. App expression increased steadily throughout fetal and postnatal development. Smst expression peaked during the third postnatal week, then reached a plateau at a slightly lower level in adults, and Gap43 expression was highest in the early postnatal period, declining in adults to levels below those seen at the earliest timepoints examined. Smst message levels exhibited a 1.5-fold increase in the brains of trisomy 16 (Ts16) mice as compared to normal littermates on day 15 of gestation, while Gap43 and App message levels were elevated approximately 2-fold.
Assuntos
Amiloide/metabolismo , Encéfalo/metabolismo , Síndrome de Down/metabolismo , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Precursores de Proteínas/metabolismo , Somatostatina/metabolismo , Amiloide/genética , Precursor de Proteína beta-Amiloide , Animais , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Modelos Animais de Doenças , Proteína GAP-43 , Proteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Hibridização de Ácido Nucleico , Precursores de Proteínas/genética , Somatostatina/genéticaRESUMO
The Trisomy 16 (Ts16) mouse has been proposed as a model for Down Syndrome (DS) in humans, based on genetic homology between mouse chromosome 16 (MMU 16) and human chromosome 21 (HSA 21). Translocations of HSA 21 resulting in trisomy for only a portion of the genetic information contained on this chromosome can result in a DS phenotype. Thus, these translocations help to define a "DS region" of the chromosome. A number of genes from this DS region of HSA 21 have been mapped to MMU 16. Techniques for localizing genes on chromosomes have been used to identify the portion of MMU 16 which corresponds to the DS region of HSA 21. This region appears to be highly conserved between mouse and human, providing further support for a mouse model of DS.
Assuntos
Cromossomos Humanos Par 21 , Síndrome de Down/genética , Camundongos Mutantes Neurológicos/genética , Animais , Mapeamento Cromossômico , Modelos Animais de Doenças , Ligação Genética , Humanos , Camundongos , Hibridização de Ácido Nucleico , Translocação GenéticaRESUMO
Current techniques used to measure lengths of DNA fragments in atomic force microscope (AFM) images require a user to operate interactive software and execute tedious error-prone cursor selections. An algorithm is proposed which provides an automated method for determining DNA fragment lengths from AFM images without interaction from the computer operator (e.g. cursor selections or mouse clicks). The approach utilises image processing techniques tailored to characteristics of AFM images of DNA fragments. The automated measurements have a mean absolute deviation of less than 1 pixel when compared to manual image-based measurements. The DNA length determined from the histogram of calculated lengths is accurate to within 3% of the actual DNA length in solution. For fragments that are 250 base-pairs long, the precision is estimated to be within 17 nm, which is about 20% of the total length. This precision was confirmed when the algorithm easily resolved fragments in one image that differed by only 17 nm. Fragment sizes up to 2000 base-pairs have been tested and successfully sized. This algorithm is being developed as part of a new solid-state DNA sizing technique for applications such as genotyping and construction of physical genome maps.