RESUMO
Accumulation of amyloid-beta (Aß) is one of the hallmarks of Alzheimer's disease (AD), and efficient clearance of Aß by cells of the innate immune system may be an important mechanism for controlling or preventing disease onset. It was reported that peripheral blood mononuclear cells (PBMCs) of most AD patients are defective in the phagocytosis of soluble Aß. Natural curcumins were shown to restore Aß phagocytosis by AD PBMCs and to up-regulate the expression of key genes including MGAT3 and those encoding Toll-like receptors (TLRs). Bisdemethoxycurcumin (BDC), a minor component of natural curcumin, was shown to have the greatest potency for stimulating AD PBMCs. Because natural curcumins have inherent limitations with regard to physicochemical properties, synthetic curcumin analogues were developed that showed improved solubility, stability, and bioavailability. An in vitro system using human monocytic cell lines (U-937, THP-1) was used to evaluate analogues for the potency of innate immune cell stimulation. These cell lines showed responses to curcuminoids and to 1α,25-dihydroxyvitamin D3 (VD3) resembling those seen in human PBMCs. From more than 45 curcuminoids analyzed, the most potent compounds possessing enhanced pharmaceutical properties were identified. The most promising candidates included prodrug versions containing water solubility-enhancing amino acids and stability-increasing modifications near the central diketone. In vivo studies showed compound (5) substantially increased bioavailability by combining several promising structural modifications. Studies examining ex vivo phagocytosis of Aß and bead particles in mouse microglia showed that BDC and several water-soluble analogues were quite effective compared to curcumin or an unnatural analogue. In vitro studies using monocytic cell lines reported herein complement those using human PBMCs and represent a routinely accessible and uniform cellular resource allowing direct comparisons between compounds.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Curcumina/análogos & derivados , Curcumina/farmacologia , Expressão Gênica/efeitos dos fármacos , Microglia/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Curcumina/farmacocinética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Microglia/metabolismo , Monócitos/metabolismo , Fagocitose/efeitos dos fármacos , RNA Mensageiro/metabolismo , Relação Estrutura-AtividadeRESUMO
Neurodegenerative diseases are associated with accumulation of modified proteins or peptides including amyloid-ß (Aß) in Alzheimer's disease (AD), and misfolded superoxide dismutase-1 (SOD-1) in amyotrophic lateral sclerosis (ALS). Clearance of Aß or SOD-1 by the innate immune system may be important for controlling or preventing disease onset. Curcumins restore Aß phagocytosis by peripheral blood mononuclear cells (PBMCs) from AD patients and Aß clearance with upregulation of key genes including MGAT3, vitamin D receptor (VDR) and Toll-like receptors (TLRs). Certain curcumins inhibit inflammatory processes of PBMCs from ALS patients. We developed an in vitro system using human monocytes from patients and monocytic cell lines (i.e. U-937, THP-1) for evaluating curcuminoid potency of innate immune cell stimulation. Bisdemethoxycurcumin and certain analogs potentiated MGAT3,VDR and TLR gene expression 3- to 300-fold in U-937 cells. The effect of curcumins on inflammation in monocytes from patients with ALS was examined. Recursive medicinal chemistry was applied to identify compounds that stimulate the innate immune system for use in the clearance of Aß in AD and the reversal of neuroinflammation and defective SOD-1 accumulation in ALS.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Esclerose Lateral Amiotrófica/patologia , Anti-Inflamatórios não Esteroides/farmacologia , Curcumina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Doença de Alzheimer/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Linhagem Celular Transformada , Células Cultivadas , Curcumina/análogos & derivados , Citocinas/genética , Citocinas/metabolismo , Diarileptanoides , Humanos , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , RNA Mensageiro/metabolismo , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
OBJECTIVE: To determine the safety of polygeline, a gelatine-derived plasma substitute produced from bovine bones, in terms of safety for bovine spongiform encephalopathy (BSE) by evaluating the ability of the manufacturing process of polygeline to eliminate agents related to transmissible spongiform encephalopathy (TSE) through the validation of three main production steps. DESIGN: Laboratory scale experimental process (in duplicate) using 20% hamster-adapted 263K scrapie-infected brain homogenate as infective titrated source (10(9) LD50/2 ml), added to each material before being processed and titrated in hamsters. Experiment 1: time/temperature dependency of gelatine autoclaving. Experiment 2: cross-linking and distillation. Experiment 3: final sterilization. Monitoring period: 1 year with daily animal clinical observation. Histology of all brains. SETTING: LCG-RBM laboratories, Italy; strict GLP compliance. MEASUREMENTS AND RESULTS: Heating the gelatine (at conditions lower than those used in production process) was very effective in inactivating the infectivity of TSE agents. Clearance factors were reproducible, dependent upon time and temperature, reaching a total theoretical process clearance in the range of 9.2-13.8 [6.9 + 2.3 (+ 4.6)] log10 LD50. CONCLUSIONS: These experimental results provide further important data confirming the safety of the procedural steps; this complements the safety due to the careful sourcing of the raw material. There is high assurance that there is no significant risk of TSE transmission to humans by the therapeutic administration of polygeline.
Assuntos
Encefalopatia Espongiforme Bovina/transmissão , Substitutos do Plasma , Poligelina , Scrapie/transmissão , Animais , Encéfalo/patologia , Encéfalo/virologia , Bovinos , Cricetinae , Composição de Medicamentos/métodos , Contaminação de Medicamentos , Feminino , Dose Letal Mediana , Mesocricetus , EsterilizaçãoRESUMO
Inherited mutations in the BRCA2 gene predispose women to breast and ovarian cancer. We created a mutation in the mouse Brca2 gene that terminates translation in exon 11 at 45% of the normal transcript length. Ninety % of Brca2(tm1Cam) homozygous mutant mice die prenatally or perinatally. The location of the Brca2(tm1Cam) mutation differs from those reported previously, and this phenotype suggests a correlation with genotype analogous to that previously reported in humans. Although heterozygote mice have remained free of tumors for 10 months, Brca2(tm1Cam) homozygous mutants that survived to adulthood died with thymic lymphomas between 12 and 14 weeks of age.
Assuntos
Linfoma/genética , Mutação , Proteínas de Neoplasias/genética , Timo/fisiologia , Fatores de Transcrição/genética , Alelos , Animais , Proteína BRCA2 , Éxons , Genótipo , Homozigoto , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/fisiologia , Fenótipo , Fatores de Transcrição/fisiologiaRESUMO
Recent transcription mapping efforts within chromosome 17q21 have led to the identification of a human homolog of the Drosophila gene Enhancer of zeste, E(z). A member of the Polycomb group (Pc-G) of proteins, Drosophila E(z) acts as a negative regulator of the segment identity genes of the Antennapedia and Bithorax complexes. Here we report the full-length protein coding sequence of human EZH1 (Enhancer of zeste homolog 1) and compare the respective protein sequences in both species. EZH1 encodes a protein of 747 amino acids that displays 55% amino acid identity overall (70% similarity) with Drosophila E(z). The strongest homology was noted (79% identity, 89% similarity) within the carboxy-terminal 245 amino acids, including the SET domain, a region of E(z) also conserved in other Drosophila proteins with roles in development and/or chromatin structure. A large Cysrich region with a novel spatial pattern of cysteine residues was also conserved in both EZH1 and E(z). The strong sequence conservation suggest potential roles for EZH1 in human development as a transcriptional regulator and as a component of protein complexes that stably maintain heterochromatin. EZH1 is expressed as two major transcripts in all adult and fetal human tissues surveyed; comparison of cloned cDNAs suggests that alternative splicing may account for at least part of the transcript size difference. Analysis of one cDNA revealed an unusual splicing event involving EZH1 and a tandemly linked gene GPR2 and suggests a potential mechanism for modifying the EZH1 protein in the conserved C-terminal domain. The sequence and isolated cDNAs will provide useful reagents for determining the function of EZH1 and the importance of the evolutionarily conserved domains.
Assuntos
Proteína BRCA1/genética , Proteínas de Drosophila , Proteínas de Insetos/genética , Proteínas Nucleares , Proteínas/genética , Proteínas Repressoras , Adulto , Animais , Mapeamento Cromossômico , Cromossomos Humanos Par 17 , Clonagem Molecular , DNA Complementar , Proteínas de Ligação a DNA , Drosophila , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Complexo Repressor Polycomb 2 , Polimorfismo Genético , Homologia de Sequência de Aminoácidos , Fatores de TranscriçãoRESUMO
Large pools of cosmids from the BRCA1 region of human chromosome 17 were screened for tetranucleotide repeat polymorphisms by hybridizing shotgun subcloned pools with a mixture of 25 oligonucleotides. Identified subclones were PCR amplified and directly sequenced to design PCR primers for short tandem repeat polymorphism (STRP) analysis of family DNAs. With the identification of the BRCA1 gene and the observation that most mutations in this > 100-kb gene are unique, haplotyping and linkage analysis may play a significant role in diagnosis and carrier detection of BRCA1-associated breast and ovarian cancers. We report the characterization of 15 new STRPs flanking the BRCA1 locus.
Assuntos
Genes Supressores de Tumor , Repetições de Microssatélites , Proteínas de Neoplasias/genética , Polimorfismo Genético , Fatores de Transcrição/genética , Proteína BRCA1 , Sequência de Bases , Neoplasias da Mama/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 17/genética , Clonagem Molecular , Cosmídeos , Primers do DNA/genética , Feminino , Humanos , Dados de Sequência Molecular , Neoplasias Ovarianas/genética , Reação em Cadeia da PolimeraseRESUMO
The human genes BRCA1, conferring susceptibility to early-onset breast and ovarian cancer, has recently been isolated. Here we describe isolation of cDNAs, sequence analysis, and genomic localization of the murine homolog, Brac1. The mouse cDNA sequence predicts a protein of 1812 amino acids; a number of small gaps account for the 51 fewer residues in the mouse protein relative to human BRCA1. While the predicted mouse and human proteins display on the whole a high level of homology (58% identity, 73% similarity), the regions of greatest homology are at the respective amino and carboxyl termini. Most reported disease-associated missense mutations in human BCRA1 occurred within these more highly conserved terminal regions. A predicted zinc-building RING finger domain near the amino terminus lies within a 50 amino acid stretch that is perfectly conserved in both species. The strong conservation during mammalian evolution argues for the importance of this domain, perhaps mediating a role for BRCA1 in DNA and/or protein binding. We have also identified a conserved highly acidic domain in the carboxyl terminal half of the BCRA1 protein resembling acidic transactivation domains of certain transcription factors. Using an interspecific backcross panel, Brca1 was mapped to a region of mouse chromosome 11 that exhibits conserved linkage with 17q21. The sequence and isolated cDNAs will provide useful reagents for studying the expression of Brca1 in the mouse, and for testing the importance of the evolutionarily conserved domains.
Assuntos
Sequência Conservada , Proteínas de Neoplasias/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Proteína BRCA1 , Sequência de Bases , Evolução Biológica , DNA Complementar/isolamento & purificação , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
The human homolog of the Saccharomyces cerevisiae cell division control 27 gene (CDC27) was mapped to human chromosome 17q12-q21 using a panel of human/rodent somatic cell hybrids and localized distal to the breast cancer susceptibility gene, BRCA1, using a panel of radiation hybrids. The radiation hybrid panel indicates that the most likely position of human CDC27 on human chromosomes 17 is between the marker D17S409 and the beta 3 subunit of integrin (ITGB3). Further confirmation of this localization comes from the sequence tagged site (STS) mapping of human CDC27 to the same yeast artificial chromosomes (YACs) positive for ITGB3. The estimated distance between ITGB3 and human CDC27 is less than 600 kb.
Assuntos
Antígenos CD/genética , Proteínas de Ciclo Celular/genética , Cromossomos Humanos Par 17 , Proteínas de Neoplasias/genética , Glicoproteínas da Membrana de Plaquetas/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Animais , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase , Proteína BRCA1 , Sequência de Bases , Neoplasias da Mama/genética , Divisão Celular/genética , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Cricetinae , DNA Polimerase III , Primers do DNA , Feminino , Marcadores Genéticos , Humanos , Células Híbridas , Integrina beta3 , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Ratos , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe , Sitios de Sequências Rotuladas , Ubiquitina-Proteína LigasesRESUMO
We have examined the developmental expression of the murine breast and ovarian cancer susceptibility gene, Brca1, to investigate its role in the control of cell growth and differentiation. Specifically, we have analysed Brca1 expression during embryonic development, in adult tissues, and during postnatal mammary gland development, particularly in response to ovarian hormones. Our results suggest that Brca1 is expressed in rapidly proliferating cell types undergoing differentiation. In the mammary gland, Brca1 expression is induced during puberty, pregnancy, and following treatment of ovariectomized animals with 17 beta-estradiol and progesterone. These observations imply that Brca1 is involved in the processes of proliferation and differentiation in multiple tissues, notably in the mammary gland in response to ovarian hormones.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Glândulas Mamárias Animais/metabolismo , Proteínas de Neoplasias/biossíntese , Fatores de Transcrição/biossíntese , Animais , Proteína BRCA1 , Sequência de Bases , Divisão Celular/genética , Células Cultivadas , Desenvolvimento Embrionário e Fetal , Epitélio/metabolismo , Estradiol/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Lactação , Masculino , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/embriologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Oligonucleotídeos Antissenso , Especificidade de Órgãos , Ovariectomia , Paridade , Gravidez , Progesterona/farmacologia , Maturidade Sexual , Fatores de Transcrição/genéticaRESUMO
We recently cloned human cDNA representing several mRNA isoforms of human TCF11, a transcription factor of the basic-region, leucine-zipper (bZIP) family located on chromosome 17q22 as well as a genomic clone of this gene. We have now determined the complete genomic organization of the TCF11 gene, which consists of 9 exons distributed over 15 kb of genomic DNA. Pulsed-field gel electrophoresis was used to construct a physical map around TCF11, to characterize a 450-kb YAC clone that contains the gene, and to link TCF11 physically to BTR, a marker on chromosome 17.
Assuntos
Proteínas de Caenorhabditis elegans , Proteínas de Ligação a DNA , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Caenorhabditis/genética , Mapeamento Cromossômico , Sondas de DNA , DNA Complementar/química , Éxons , Proteínas de Helminto/genética , Humanos , Camundongos , Dados de Sequência Molecular , Fator 1 Relacionado a NF-E2 , Homologia de SequênciaRESUMO
We have used a combination of methods (exon amplification, direct selection, direct screening, evolutionary conservation, island rescue-PCR, and direct sequence analysis) to survey approximately 600 kb of genomic DNA surrounding the BRCA1 gene for transcribed sequences. We have cloned a set of fragments representing at least 26 genes. The DNA sequence of these clones reveals that 5 are previously cloned genes; the precise chromosomal location of 2 was previously unknown, and 3 have been cloned and mapped by others to this interval. Three other genes, including BRCA1 itself, have recently been mapped independently to this region. Sequences from 11 genes are similar but not identical matches to known genes; 5 of these appear to be the human homologues of genes cloned from other species. Another 7 genes have no similarity with known genes. In addition, 39 putative exons and 14 expressed sequence tags have been identified and mapped to individual cosmids. This transcript map provides a detailed description of gene organization for this region of the genome.
Assuntos
Cromossomos Humanos Par 17 , Proteínas de Neoplasias/genética , Fatores de Transcrição/genética , Animais , Proteína BRCA1 , Sequência de Bases , Evolução Biológica , Mama/metabolismo , Neoplasias da Mama/genética , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Clonagem Molecular , Sequência Conservada , Cosmídeos , DNA/genética , DNA/isolamento & purificação , Éxons , Feminino , Expressão Gênica , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido Nucleico , Sitios de Sequências Rotuladas , Transcrição GênicaRESUMO
A familial early-onset breast cancer gene (BRCA1) has been localized to chromosome 17q21. To characterize this region and to aid in the identification of the BRCA1 gene, a physical map of a region of 1.0-1.5 Mb between the EDH17B1 and the PPY loci on chromosome 17q21 was generated. The physical map is composed of a yeast artificial chromosome (YAC) and P1 phage contig with one gap. The majority of the interval has also been converted to a cosmid contig. Twenty-three PCR-based sequence-tagged sites (STSs) were mapped to these contigs, thereby confirming the order and overlap of individual clones. This complex physical map of the BRCA1 region was used to isolate genes by a number of gene identification techniques and to generate transcript maps of the region, as presented in the three accompanying manuscripts of Brody et al. (1995), Osborne-Lawrence et al. (1995), and Friedman et al. (1995).
Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos Par 17 , Proteínas de Neoplasias/genética , Fatores de Transcrição/genética , Proteína BRCA1 , Sequência de Bases , Mapeamento Cromossômico , Passeio de Cromossomo , Cromossomos Artificiais de Levedura , Clonagem Molecular , Cosmídeos , Primers do DNA , Biblioteca Gênica , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Chromosome 17q12-21 is known to contain a gene (or genes) which confers susceptibility to early-onset breast cancer and ovarian cancer (BRCA1). Identification and isolation of BRCA1 will likely provide the basis for increased understanding of the pathogenesis of breast and ovarian cancer, the development of targeted diagnostic and therapeutic approaches, and a means of screening women at risk of being BRCA1 mutation carriers. Genetic and physical maps of the BRCA1 candidate region have been largely completed and efforts are being directed at identification of candidate genes from within this region. We have begun the task of identifying transcripts from this region employing three complementary strategies. These include: 1) direct cDNA screening with cosmids derived from the BRCA1 region; 2) exon amplification; and 3) magnetic bead capture. Transcripts identified using these approaches are being characterized for: 1) tissue expression pattern; 2) the presence of genomic rearrangement in DNA derived from affected members of families believed to show linkage between breast cancer and genetic markers in the BRCA1 candidate interval; 3) altered size and/or expression pattern in RNA prepared from such individuals; and 4) homology to known genes or functional motifs. Germline mutations in affected individuals from these families will serve as presumptive evidence of BRCA1 identity.
Assuntos
Neoplasias da Mama/genética , Genes Supressores de Tumor/genética , Neoplasias Ovarianas/genética , Transcrição Gênica/genética , DNA de Neoplasias/genética , Éxons , Feminino , Amplificação de Genes , Biblioteca Gênica , Ligação Genética , HumanosRESUMO
Family history is recognized widely as a significant risk factor for the development of breast cancer. A gene (BRCA1), mutations in which confer susceptibility to early-onset breast and ovarian cancer, has been mapped to chromosome 17q12-21. An intensive search for this gene is currently underway in a number of laboratories. Recent data support the hypothesis that BRCA1 is a tumor suppressor gene that may be important in the development of both inherited and sporadic breast and ovarian cancers. Genetic and physical maps of the BRCA1 candidate region largely have been completed and efforts are being directed at identification of candidate genes from within this region. A small number of families recently have received results of genetic-linkage testing, indicating which family members likely are to be carriers of a germline BRCA1 mutation, and, therefore, have a lifetime risk of developing breast cancer of approximately 85%. The imminent isolation of BRCA1 will make predictive testing for breast cancer a reality for many women and likely will pave the way for novel diagnostic and therapeutic strategies in the future.
Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos Par 17 , Mapeamento Cromossômico , Feminino , Genes Supressores de Tumor , Predisposição Genética para Doença , Humanos , MutaçãoRESUMO
The gene encoding ATP-citrate lyase, designated ACLY, was mapped to human chromosome 17q12-q21 by PCR on a panel of human/rodent somatic cell hybrids and localized to 17q21.1 by PCR on a panel of radiation hybrids. The radiation hybrid panel indicates that the most likely position of ACLY on 17q21.1 is between gastrin (GAS) and D17S856 at a distance of 170-290 kb from the GAS locus.
Assuntos
ATP Citrato (pro-S)-Liase/genética , Cromossomos Humanos Par 17 , Gastrinas/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA , Marcadores Genéticos , Humanos , Células Híbridas , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , RatosRESUMO
The high incidence of breast cancer and/or ovarian cancer in some families appears to be due to germ-line mutations in BRCA1. Genetic analysis of such families suggests that the BRCA1 candidate region lies between D17S857 and D17S78 on chromosome 17q21 (Kelsell et al. 1993; Simard et al. 1993). To identify and isolate BRCA1, we have used linkage and meiotic recombination analysis, characterized regions displaying LOH in tumor DNA from BRCA1-linked families, performed YAC and cosmid clone isolation and ordering, and used three complementary transcript-searching strategies. We have identified as many as 28 genes from the BRCA1 candidate region, and we are searching for constitutive mutations in these candidate genes by several methods in an attempt to identify BRCA1.
Assuntos
Neoplasias da Mama/genética , Genes Supressores de Tumor , Mutação em Linhagem Germinativa , Proteínas de Neoplasias/genética , Fatores de Transcrição/genética , Proteína BRCA1 , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/genética , Feminino , Marcadores Genéticos , Humanos , Neoplasias Ovarianas/genéticaRESUMO
The chromosomal region 17q12-q21 contains a gene (BRCA1) conferring susceptibility to early-onset familial breast and ovarian cancer. An 8000-rad radiation-reduced hybrid (RH) panel was constructed to provide a resource for long-range mapping of this region. A large fraction of the hybrids (approximately 90%) retained detectable human chromosome 17 sequences. The complete panel of 76 hybrids was scored for the presence or absence of 22 markers from this chromosomal region, including 14 cloned genes, seven microsatellite repeats, and one anonymous DNA segment. Statistical analysis of the marker retention data employing multipoint methods provided both comprehensive and framework maps of this chromosomal region, including distance estimates between adjacent markers. The comprehensive RH map includes 17 loci and spans 179 cRays(8000). Likelihood ratios of at least 1000:1 support the 10-locus framework order: cen-D17S250-ERBB2-(THRA1, TOP2A)-D17S855-PPY-D17S190-MTBT1-GP3A++ +-BTR-D17S588-tel. The order obtained from RH mapping, when used in conjunction with other methods, will be useful in linkage analysis of breast cancer families and will facilitate the development of a physical map of this region.
Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos Par 17 , Oncogenes , Neoplasias Ovarianas/genética , Sequência de Bases , Mapeamento Cromossômico/métodos , DNA/genética , Primers do DNA , DNA Satélite/genética , Feminino , Marcadores Genéticos , Humanos , Células Híbridas/efeitos da radiação , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Sequências Repetitivas de Ácido NucleicoRESUMO
The components of the renin-angiotensin system have been detected in various reproductive tissues of mammals including the testis, epididymis, ovary, and placenta. Using Northern blot and in situ hybridization analyses, we detected abundant levels of renin mRNA in the coagulating gland (anterior prostate) of mice with one (Ren-1) but not two (Ren-1/Ren-2) copies of the renin gene. In contrast to mice, the single renin gene of the rat (R. norvegicus) was silent in the coagulating gland. The results of this survey suggest that the ability of the renin gene to express in the coagulating gland was acquired during the speciation of mice and subsequently lost as a result of the duplication event at the renin locus. In the coagulating gland, we found that renin mRNA transcripts initiated at a series of upstream start sites, some of which map within a 0.5-kb transposable-like element previously identified in the promoter region of the mouse, but not the rat, renin gene. Furthermore, renin gene expression in the coagulating gland was positively regulated by testosterone. The coagulating gland thus represents a male reproductive tissue that demonstrates high-level, species-specific, and differential expression of renin mRNA.
Assuntos
Regulação da Expressão Gênica , Próstata/metabolismo , Renina/genética , Animais , Northern Blotting , Mapeamento Cromossômico , Di-Hidrotestosterona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Próstata/química , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Transcrição GênicaRESUMO
A targeted yeast artificial chromosome (YAC) library for chromosome 11 has been constructed from the J1 cell line that carries a single human chromosome 11 within a hamster DNA background. Interspecies chimeric clones generated during construction of the library were detected during the screening process and eliminated from the library. Contig assembly becomes much less difficult using such a library as the complexity is decreased and the ends of the clone inserts can be rescued for walking to neighboring clones. The library contains > 1824 clones with an average insert length of 337 kb. This represents a fourfold coverage of chromosome 11 or a > 95% chance of recovering a unique single-copy sequence from the library. Two hundred YAC clones were localized by fluorescence in situ hybridization and found to be randomly distributed along the chromosome. The library has been screened with probes for the chromosome 11 markers HBB, GLUR4, H19, and D11S193. Corresponding YAC clones have been isolated for each locus. This analysis has indicated that the library is unbiased, that cognate YAC clones can be recovered with chromosome 11 markers, and that extensive contig assembly should be feasible.
Assuntos
Cromossomos Fúngicos , Cromossomos Humanos Par 11 , Biblioteca Gênica , Genoma Humano , Animais , Southern Blotting , Linhagem Celular , Bandeamento Cromossômico , Clonagem Molecular , Cricetinae , Humanos , Hibridização In SituRESUMO
Somatic cell hybrid mapping panels have previously been constructed to assist in the regional assignment of anonymous DNA probes and cloned genes to human chromosome 17. While a substantial number of hybrids are available that subdivide the short arm of this chromosome and the proximal portion of its long arm into specific regions, relatively few exist with breakpoints in the distal portion of the long arm. To increase the resolution of this region, four additional human x rodent somatic cell hybrids have been constructed that include breakpoints spanning the region 17q22-->q24. Hybrid clones carrying the long-arm derivative of chromosome 17 were initially identified by fluorescence in situ hybridization. Hybrids were subsequently screened using the polymerase chain reaction with primer sets representing DNA markers previously mapped to chromosome 17. These hybrids expand the existing somatic cell hybrid panel for the distal portion of the long arm of chromosome 17.