RESUMO
The Ras signal transduction pathway is often deregulated in human myeloid leukaemia. For example, activating point mutations in RAS genes are found in some patients with juvenile chronic myelogenous leukaemia (JCML), while other patients with JCML show loss of the neurofibromatosis type 1 (NF1) gene, a Ras GTPase activating protein. By generating mice whose haematopoietic system is reconsituted with Nf1 deficient haematopoietic stem cells we show that Nf1 gene loss, by itself, is sufficient to produce the myeloproliferative symptoms associated with human JCML. We also provide evidence to indicate that Nf1 gene loss induces myeloproliferative disease through a Ras-mediated hypersensitivity to granulocyte/macrophage-colony stimulating factor (GM-CSF). Finally, we describe a genetic screen for identifying genes that cooperate with Nf1 gene loss during progression to acute myeloid leukaemia.
Assuntos
Genes da Neurofibromatose 1 , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas ras/fisiologia , Doença Aguda , Animais , Medula Óssea/imunologia , Diferenciação Celular , Linhagem Celular Transformada , Cruzamentos Genéticos , Modelos Animais de Doenças , Progressão da Doença , Feminino , Guanosina Trifosfato/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Leucemia Mieloide , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Proteínas ras/metabolismoRESUMO
Retroviruses induce myeloid leukaemia in BXH-2 mice by the insertional mutation of cellular proto-oncogenes or tumour suppressor genes. Disease genes can thus be identified by proviral tagging through the identification of common viral integration sites in BXH-2 leukaemia. Here, we describe a new approach for proviral tagging that greatly facilitates the identification of BXH-2 leukaemia genes. Using this approach, we identify three genes whose expression is activated by proviral integration in BXH-2 leukaemias; Hoxa7, Hoxa9, and a Pbx1-related homeobox gene, Meis1. Proviral activation of Hoxa7 or Hoxa9 is strongly correlated with proviral activation of Meis1 implying that Hoxa7 and Hoxa9 cooperate with Meis1 in leukaemia formation. These studies provide the first genetic evidence that Pbx1-related genes cooperate with Hox genes in leukaemia formation and identify a number of new murine myeloid leukaemia genes.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Homeodomínio/genética , Leucemia Mieloide/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Animais , Mapeamento Cromossômico , Clonagem Molecular/métodos , Ilhas de CpG , Desoxirribonucleases de Sítio Específico do Tipo II , Modelos Animais de Doenças , Feminino , Genes Homeobox/genética , Genes Homeobox/fisiologia , Genes Neoplásicos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Proteína Meis1 , Fator de Transcrição 1 de Leucemia de Células Pré-B , Provírus/genética , Ativação Viral , Integração Viral/genéticaRESUMO
Prader-Willi syndrome (PWS) is associated with paternal gene deficiencies in human chromosome 15q11-13, suggesting that PWS is caused by a deficiency in one or more maternally imprinted genes. We have now mapped a gene, Snrpn, encoding a brain-enriched small nuclear ribonucleoprotein (snRNP)-associated polypeptide SmN, to mouse chromosome 7 in a region of homology with human chromosome 15q11-13 and demonstrated that Snrpn is a maternally imprinted gene in mouse. These studies, in combination with the accompanying human mapping studies showing that SNRPN maps in the Prader-Willi critical region, identify SNRPN as a candidate gene involved in PWS and suggest that PWS may be caused, in part, by defects in mRNA processing.
Assuntos
Autoantígenos/genética , Síndrome de Prader-Willi/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 15 , Cruzamentos Genéticos , DNA/genética , Feminino , Ligação Genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Dados de Sequência Molecular , Muridae , Processamento Pós-Transcricional do RNA/genética , Proteínas Centrais de snRNPRESUMO
Recent advances in gene mapping technologies have led to increased emphasis in developing representative genetic maps for several species, particularly domestic plants and animals. These maps are being compiled with two distinct goals: to provide a resource for genetic analysis, and to help dissect the evolution of genome organization by comparing linkage relationships of homologous genes. We propose here a list of 321 reference anchor loci suitable for comparative gene mapping in mammals and other vertebrate classes. We selected cloned mouse and human functional genes spaced an average of 5-10 centiMorgans throughout their respective genomes. We also attempted to include loci that are evolutionarily conserved and represented in comparative gene maps in other mammalian orders, particularly cattle and the domestic cat. We believe that the map may provide the basis for a unified approach to comparative analysis of mammalian species genomes.
Assuntos
Mapeamento Cromossômico , Mamíferos/genética , Animais , Gatos , Bovinos , Feminino , Marcadores Genéticos , Genoma , Genoma Humano , Humanos , Masculino , Camundongos , Especificidade da EspécieRESUMO
Precise comparisons of mammalian gene maps require common anchor loci as landmarks for conserved chromosomal segments. Using a computer script that automates DNA sequence database alignments, we designed 410 evolutionarily conserved primer pair sequences which are specific for anchor locus gene amplification from any mammalian species' DNA. Primer pairs were designed to span introns for polymorphism ascertainment, and to include sufficient exonic sequence (25-400 bp) to allow for gene identification. A total of 318 primer pairs were optimized for domestic cats, and 86% of the sequenced feline PCR products showed homology to the gene of primer origin. A screen of 20 mammals from 11 orders revealed that 35-52% of the 318 primers yielded a single PCR product without further optimization suggesting that nearly 75% can be optimized for any eutherian mammal.
Assuntos
Mapeamento Cromossômico , Animais , Mapeamento Cromossômico/métodos , Primers do DNA , Bases de Dados Factuais , Marcadores Genéticos , Genoma , Humanos , Mamíferos , Dados de Sequência Molecular , Polimorfismo Genético , Alinhamento de SequênciaRESUMO
Imprinting in the 15q11-q13 region involves an 'imprinting centre' (IC), mapping in part to the promoter and first exon of SNRPN. Deletion of this IC abolishes local paternally derived gene expression and results in Prader-Willi syndrome (PWS). We have created two deletion mutations in mice to understand PWS and the mechanism of this IC. Mice harbouring an intragenic deletion in Snrpn are phenotypically normal, suggesting that mutations of SNRPN are not sufficient to induce PWS. Mice with a larger deletion involving both Snrpn and the putative PWS-IC lack expression of the imprinted genes Zfp127 (mouse homologue of ZNF127), Ndn and Ipw, and manifest several phenotypes common to PWS infants. These data demonstrate that both the position of the IC and its role in the coordinate expression of genes is conserved between mouse and human, and indicate that the mouse is a suitable model system in which to investigate the molecular mechanisms of imprinting in this region of the genome.
Assuntos
Impressão Genômica , Mutação , Síndrome de Prader-Willi/genética , Ribonucleoproteínas Nucleares Pequenas , Animais , Autoantígenos/genética , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Mutantes , Fenótipo , Deleção de Sequência , Proteínas Centrais de snRNPRESUMO
The growth hormone-releasing hormone receptor (GHRHR) is a member of the family of G protein-coupled receptors that is expressed on pituitary somatotrope cells and mediates the actions of GHRH in stimulating growth hormone (GH) synthesis and secretion. We report that the Ghrhr gene is located in the middle of mouse chromosome 6 in the same region as the little mutation. Mice homozygous for this mutation have reduced GH secretion and a dwarf phenotype. A missense mutation was identified in the extracellular domain of the little GHRHR that disrupts receptor function, suggesting that the growth deficit in these mice results from a defect in the GHRHR. Similar alterations in GHRHR might explain some isolated GH deficiencies in humans.
Assuntos
Mutação , Receptores de Neuropeptídeos , Receptores de Neurotransmissores/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , DNA/genética , Nanismo/genética , Feminino , Hormônio do Crescimento/deficiência , Hormônio do Crescimento/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , FenótipoRESUMO
The mouse represents an excellent model system for the study of genetic deafness in humans. Many mouse deafness mutants have been identified and the anatomy of the mouse and human ear is similar. Here we report the use of a positional cloning approach to identify the gene encoded by the mouse recessive deafness mutation, Snell's waltzer (sv). We show that sv encodes an unconventional myosin heavy chain, myosin VI, which is expressed within the sensory hair cells of the inner ear, and appears to be required for maintaining their structural integrity. The requirement for myosin VI in hearing makes this gene an excellent candidate for a human deafness disorder.
Assuntos
Surdez/genética , Células Ciliadas Auditivas Internas/química , Cadeias Pesadas de Miosina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Inversão Cromossômica , Clonagem Molecular , Análise Mutacional de DNA , Surdez/patologia , Genes Recessivos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/análise , Cadeias Pesadas de Miosina/fisiologia , Órgão Espiral/química , RNA Mensageiro/análise , Mapeamento por Restrição , Deleção de Sequência/genéticaRESUMO
Non-agouti-lethal 18H (a18H) mice are dark agouti with black pinna hairs. What makes these mice unique is that they develop a spectrum of immunological diseases not seen in other agouti mutant mice. On the JU/Ct background, a18H mice develop an inflammatory disease of the large intestine. On the C57BL/6J background, they develop a fatal disease characterized by pulmonary chronic interstitial inflammation and alveolar proteinosis, inflammation of the glandular stomach and skin resulting in scarring due to constant itching, and hyperplasia of lymphoid cells, haematopoietic cells and the forestomach epithelium. Previous studies suggested that the a18H mutation results from a paracentric inversion that affects two loci: agouti and another, as yet unidentified locus designated itchy (the provisional gene symbol is Itch), that is responsible for the immunological phenotype of a18H mice. Here we confirm that a18H results from an inversion and show that Itch encodes a novel E3 ubiquitin protein ligase, a protein involved in ubiquitin-mediated protein degradation. Our results indicate that ubiquitin-dependent proteolysis is an important mediator of the immune response in vivo and provide evidence for Itch's role in inflammation and the regulation of epithelial and haematopoietic cell growth.
Assuntos
Inversão Cromossômica , Inflamação/genética , Ligases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , Humanos , Inflamação/enzimologia , Ligases/biossíntese , Ligases/química , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Ubiquitina-Proteína LigasesRESUMO
Retroviral insertional mutagenesis in BXH2 and AKXD recombinant inbred mice induces a high incidence of myeloid or B- and T-cell leukaemia and the proviral integration sites in the leukaemias provide powerful genetic tags for disease gene identification. Some of the disease genes identified by proviral tagging are also associated with human disease, validating this approach for human disease gene identification. Although many leukaemia disease genes have been identified over the years, many more remain to be cloned. Here we describe an inverse PCR (IPCR) method for proviral tagging that makes use of automated DNA sequencing and the genetic tools provided by the Mouse Genome Project, which increases the throughput for disease gene identification. We also use this IPCR method to clone and analyse more than 400 proviral integration sites from AKXD and BXH2 leukaemias and, in the process, identify more than 90 candidate disease genes. Some of these genes function in pathways already implicated in leukaemia, whereas others are likely to define new disease pathways. Our studies underscore the power of the mouse as a tool for gene discovery and functional genomics.
Assuntos
Clonagem Molecular/métodos , Leucemia Experimental/genética , Oncogenes/genética , Transdução de Sinais/genética , Animais , Cromossomos/genética , Etiquetas de Sequências Expressas , Genoma , Humanos , Linfoma/genética , Camundongos , Camundongos Endogâmicos , Mutagênese Insercional , Mapeamento Físico do Cromossomo , Reação em Cadeia da Polimerase/métodos , Provírus/genética , Reprodutibilidade dos Testes , Retroviridae/genética , Sensibilidade e Especificidade , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
The best examples of imprinting in humans are provided by the Angelman and Prader-Willi syndromes (AS and PWS) which are associated with maternal and paternal 15q11-13 deletions, respectively, and also with paternal and maternal disomy 15. The region of the deletions has homology with a central part of mouse chromosome 7, incompletely tested for imprinting effects. Here, we report that maternal duplication for this region causes a murine imprinting effect which may correspond to PWS. Paternal duplication was not associated with any detectable effect that might correspond with AS. Gene expression studies established that Snrpn is not expressed in mice with the maternal duplication and suggest that the closely-linked Gabrb-3 locus is not subject to imprinting. Finally, an additional new imprinting effect is described.
Assuntos
Autoantígenos/genética , Modelos Genéticos , Síndrome de Prader-Willi/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Animais , Mapeamento Cromossômico , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , Família Multigênica , Translocação Genética , Proteínas Centrais de snRNPRESUMO
The gene encoding the granulocyte macrophage colony stimulating factor receptor alpha subunit (CSF2RA) has previously been mapped to the pseudoautosomal region of the human sex chromosomes. In contrast, we report that the murine locus, Csf2ra, maps to an autosome in the laboratory mouse. By in situ hybridization and genetic mapping, Csf2ra maps at telomeric band D2 of mouse chromosome 19. This first instance of a pseudoautosomal locus in human being autosomal in mouse, indicates incomplete conservation between the human and mouse X chromosomes and suggests that the genetic content of the pseudoautosomal region may differ between species of eutherian mammals due to chromosomal rearrangements.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Cromossomo X , Animais , Bandeamento Cromossômico , Mapeamento Cromossômico , Cruzamentos Genéticos , Feminino , Humanos , Hibridização In Situ , Substâncias Macromoleculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Muridae , Polimorfismo de Fragmento de Restrição , TelômeroRESUMO
The mouse gene Mash2 encodes a transcription factor required for development of trophoblast progenitors. Mash2-homozygous mutant embryos die at 10 days postcoitum from placental failure. Here we show that Mash2 is genomically imprinted. First, Mash2+/- embryos inheriting a wild-type allele from their father die at the same stage as -/- embryos, with a similar placental phenotype. Second, the Mash2 paternal allele is initially expressed by groups of trophoblast cells at 6.5 and 7.5 days post-coitum, but appears almost completely repressed by 8.5 days post-coitum. Finally, we have genetically and physically mapped Mash2 to the distal region of chromosome 7, within a cluster of imprinted genes, including insulin-2, insulin-like growth factor-2 and H19.
Assuntos
Proteínas de Ligação a DNA/genética , Impressão Genômica , Fatores de Transcrição , Trofoblastos/metabolismo , Alelos , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Mapeamento Cromossômico , Cruzamentos Genéticos , Primers do DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Ligação Genética , Idade Gestacional , Heterozigoto , Homozigoto , Masculino , Camundongos , Dados de Sequência Molecular , Mutação , GravidezRESUMO
Expression of Hoxa7 and Hoxa9 is activated by proviral integration in BXH2 murine myeloid leukaemias. This result, combined with the mapping of the HOXA locus to human chromosome 7p15, suggested that one of the HOXA genes might be involved in the t(7;11)(p15;p15) translocation found in some human myeloid leukaemia patients. Here we show that in three patients with t(7;11), the chromosome rearrangement creates a genomic fusion between the HOXA9 gene and the nucleoporin gene NUP98 on chromosome 11p15. The translocation produces an invariant chimaeric NUP98/HOXA9 transcript containing the amino terminal half of NUP98 fused in frame to HOXA9. These studies identify HOXA9 as an important human myeloid leukaemia gene and suggest an important role for nucleoporins in human myeloid leukaemia given that a second nucleoporin, NUP214, has also been implicated in human myeloid leukaemia.
Assuntos
Cromossomos Humanos Par 11 , Cromossomos Humanos Par 7 , Proteínas de Homeodomínio/genética , Leucemia Mieloide/genética , Proteínas de Membrana/genética , Complexo de Proteínas Formadoras de Poros Nucleares , Proteínas Nucleares/genética , Translocação Genética , Doença Aguda , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Genes Homeobox/genética , Humanos , Íntrons/genética , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Mapeamento por Restrição , Análise de Sequência de DNARESUMO
Using a bioassay consisting of the proliferation of a murine B cell line, a cDNA of a gene whose product supports the growth of that cell line was isolated from a thymic stromal cell line. This factor, termed thymic stromal lymphopoietin (TSLP), is a protein of 140 amino acids. The gene encoding TSLP was mapped to murine chromosome 18. Purified recombinant TSLP supported the growth of pre-B cell colonies in vitro, but had no myelopoietic activity. TSLP had comitogenic activity for fetal thymocytes, but was not as potent as interleukin 7 in lobe submersion cultures. Injection of TSLP into neonatal mice induced the expansion of B220(+)BP-1(+) pre-B cells.
Assuntos
Linfócitos B/efeitos dos fármacos , Citocinas/farmacologia , Hematopoese/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Citocinas/química , Citocinas/genética , DNA Complementar/isolamento & purificação , Feminino , Interleucina-7/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA Mensageiro/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The cellular receptor for murine thymic stromal lymphopoietin (TSLP) was detected in a variety of murine, but not human myelomonocytic cell lines by radioligand binding. cDNA clones encoding the receptor were isolated from a murine T helper cell cDNA library. TSLP receptor (TSLPR) is a member of the hematopoietin receptor family. Transfection of TSLPR cDNA resulted in only low affinity binding. Cotransfection of the interleukin 7 (IL-7)Ralpha chain cDNA resulted in conversion to high affinity binding. TSLP did not activate cells from IL-7Ralpha(-/)- mice, but did activate cells from gammac(-/)- mice. Thus, the functional TSLPR requires the IL-7Ralpha chain, but not the gammac chain for signaling.
Assuntos
Hematopoese/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Receptores de Citocinas/fisiologia , Receptores de Interleucina-7/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , Citocinas/farmacologia , Humanos , Interleucina-7/farmacologia , Linfócitos/fisiologia , Masculino , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/análise , Receptores de Citocinas/química , Receptores de Citocinas/genética , Receptores de Interleucina-7/química , Proteínas Recombinantes/química , Transdução de SinaisRESUMO
Itchy mice possess a loss-of-function mutation in a HECT-domain-containing ubiquitin ligase (E3), Itch. Homozygous itchy mice develop a systemic and progressive autoimmune disease that proves lethal beginning at 6 months of age. Numerous targets of Itch-mediated ubiquitination have been identified, and some of these have defined physiological roles for Itch signaling in Tcell anergy and T cell differentiation. Studies of itchy mice have also allowed for the identification of a novel pathway involved in autoimmunity: noncanonical Notch signaling. In itchy mice carrying an activated Notch1 transgene, there are increased amounts of full-length Notch1, which can complex with p56(lck) and PI3K to activate a cell survival signal that is mediated by phospho-AKT. This, in turn, leads to a reduction in apoptosis in the thymus and may have consequences in T cell tolerance. A role for noncanonical Notch signaling in autoimmune disease is also supported by numerous mouse knockout studies, and suggests possible new therapeutic approaches for the treatment of autoimmune disease.
Assuntos
Doenças Autoimunes/etiologia , Transdução de Sinais , Ubiquitina-Proteína Ligases/fisiologia , Animais , Doenças Autoimunes/terapia , Camundongos , Mutação , Receptores Notch/fisiologia , Linfócitos T/imunologia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
We have isolated a murine cDNA encoding a 9-kD protein, Chisel (Csl), in a screen for transcriptional targets of the cardiac homeodomain factor Nkx2-5. Csl transcripts were detected in atria and ventricles of the heart and in all skeletal muscles and smooth muscles of the stomach and pulmonary veins. Csl protein was distributed throughout the cytoplasm in fetal muscles, although costameric and M-line localization to the muscle cytoskeleton became obvious after further maturation. Targeted disruption of Csl showed no overt muscle phenotype. However, ectopic expression in C2C12 myoblasts induced formation of lamellipodia in which Csl protein became tethered to membrane ruffles. Migration of these cells was retarded in a monolayer wound repair assay. Csl-expressing myoblasts differentiated and fused normally, although in the presence of insulin-like growth factor (IGF)-1 they showed dramatically enhanced fusion, leading to formation of large dysmorphogenic "myosacs." The activities of transcription factors nuclear factor of activated T cells (NFAT) and myocyte enhancer-binding factor (MEF)2, were also enhanced in an IGF-1 signaling-dependent manner. The dynamic cytoskeletal localization of Csl and its dominant effects on cell shape and behavior and transcription factor activity suggest that Csl plays a role in the regulatory network through which muscle cells coordinate their structural and functional states during growth, adaptation, and repair.
Assuntos
Fator de Crescimento Insulin-Like I/farmacologia , Proteínas Musculares/metabolismo , Músculos/citologia , Músculos/efeitos dos fármacos , Proteínas Nucleares , Proteínas de Xenopus , Envelhecimento/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Calcineurina/metabolismo , Diferenciação Celular , Fusão Celular , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição MEF2 , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Músculos/embriologia , Músculos/metabolismo , Fatores de Regulação Miogênica , Fatores de Transcrição NFATC , Especificidade de Órgãos , Mapeamento Físico do Cromossomo , Transporte Proteico , RNA Mensageiro/análise , RNA Mensageiro/genética , Fatores de Transcrição/metabolismo , CicatrizaçãoRESUMO
Laminin trimers composed of alpha, beta, and gamma chains are major components of basal laminae (BLs) throughout the body. To date, three alpha chains (alpha1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional alpha chains (alpha4 and alpha5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant alpha4 and alpha5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that alpha4 and alpha5 assemble into four novel laminin heterotrimers (laminins 8-11: alpha4beta1gamma1, alpha4beta2gamma1, alpha5beta1gamma1, and alpha5beta2gamma1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of alpha1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, alpha4 and alpha5 exhibited the broadest patterns of expression, while expression of alpha1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the alpha chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one alpha chain, all alpha chains were present in multiple BLs, and some BLs contained two or three alpha chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different alpha chains and two different beta chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.
Assuntos
Laminina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Membrana Basal/metabolismo , Mapeamento Cromossômico , DNA Complementar/genética , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Rim/metabolismo , Laminina/química , Laminina/metabolismo , Pulmão/metabolismo , Camundongos , Dados de Sequência Molecular , Família Multigênica , Miocárdio/metabolismoRESUMO
A recessive mutation was identified in a family of transgenic mice that resulted in a reversal of left-right polarity (situs inversus) in 100 percent of the homozygous transgenic mice tested. Sequences that flanked the transgenic integration site were cloned and mapped to mouse chromosome 4, between the Tsha and Hxb loci. During early embryonic development, the direction of postimplantation turning, one of the earliest manifestations of left-right asymmetry, was reversed in homozygous transgenic embryos. This insertional mutation identifies a gene that controls embryonic turning and visceral left-right polarity.