RESUMO
Using homology-based database screening, we have identified the mouse homologue for the recently described matrix metalloproteinase-19 (MMP-19). Sequencing of mouse MMP-19 resulted in a putative open reading frame (ORF) of 527 amino acids showing 84% identity to the human homologue. In mouse, MMP-19 appears to be most highly expressed in the liver; however, there is a detectable level of expression in all tissues analyzed. The major mouse MMP-19 transcript is almost twice as long as that of human. The COOH-terminal serine and threonine-rich domain is considerably longer in the mouse homologue. The mouse MMP-19 gene maps to very distal end of mouse chromosome 10.
Assuntos
Regulação Enzimológica da Expressão Gênica , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Domínio Catalítico , Mapeamento Cromossômico , Clonagem Molecular , Humanos , Fígado/fisiologia , Metaloproteinases da Matriz Secretadas , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Análise de Sequência , Homologia de Sequência de AminoácidosRESUMO
To understand the biologic function of TIMP-2, a member of the tissue inhibitors of metalloproteinases family, an inactivating mutation was introduced in the mouse Timp-2 gene by homologous recombination. Outbred homozygous mutants developed and procreated indistinguishably from wild type littermates, suggesting that fertility, development, and growth are not critically dependent on TIMP-2. Lack of functional TIMP-2, however, dramatically altered the activation of proMMP-2 both in vivo and in vitro. Fully functional TIMP-2 is essential for efficient activation of proMMP-2 in vivo. No evidence of successful functional compensation was observed. The results illustrate the duality of TIMP-2 function, i.e. at low concentrations, TIMP-2 exerts a "catalytic" or enhancing effect on cell-mediated proMMP-2 activation, whereas at higher concentrations, TIMP-2 inhibits the activation and/or activity of MMP-2.
Assuntos
Precursores Enzimáticos/metabolismo , Gelatinases/metabolismo , Metaloendopeptidases/metabolismo , Inibidor Tecidual de Metaloproteinase-2/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Ativação Enzimática , Feminino , Masculino , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Recombinação Genética , Inibidor Tecidual de Metaloproteinase-2/metabolismoRESUMO
DNase I hypersensitive site 2 (HS 2) of the human beta-globin Locus Control Region (LCR) directs high level expression of the beta-globin gene located 50 kilobases downstream. Experiments in cultured cells and in transgenic mice demonstrate that duplicated AP1-like sites in HS 2 are required for this powerful enhancer activity. A cDNA clone encoding a basic, leucine-zipper protein that binds to these sites was isolated and designated Locus Control Region-Factor 1 (LCR-F1). This protein is a member of a new family of regulatory factors that contain a 63 amino acid 'CNC domain' overlapping the basic region. This domain is approximately 70% identical in the Drosophila Cap N Collar (CNC) protein, NF-E2 and LCR-F1. LCR-F1 transactivates an HS 2/gamma-globin reporter gene over 170-fold in transient transfection experiments specifically in erythroid cells. These results suggest that LCR-F1 may be a critical factor involved in LCR-mediated, human globin gene expression.
Assuntos
Eritrócitos/metabolismo , Regulação da Expressão Gênica , Globinas/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Globinas/biossíntese , Células HeLa , Humanos , Leucemia Eritroblástica Aguda , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Fator 1 Relacionado a NF-E2 , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
The human beta-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human epsilon-, gamma- and beta-globin genes and, secondly, these sequences function as a powerful enhancer of epsilon-, gamma- and beta-globin gene expression. Erythroid-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5' HS 2 site confers position-independent expression in transgenic mice and enhances human beta-globin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease beta-globin gene expression. In contrast, no single site is required for position-independent transgene expression; all mice with site-specific mutations in 5' HS 2 express human beta-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5' HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression.
Assuntos
Desoxirribonuclease I/metabolismo , Elementos Facilitadores Genéticos , Expressão Gênica , Globinas/genética , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Deleção de Genes , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Sequências Reguladoras de Ácido Nucleico , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Fatores Estimuladores UpstreamRESUMO
The human beta-globin locus control region (LCR) is essential for high-level expression of human epsilon-, gamma-, and beta-globin genes. Developmentally stable DNase I hypersensitive sites (designated HS) mark sequences within this region that are important for LCR activity. A 1.9-kilobase (kb) fragment containing the 5' HS 2 site enhances human beta-globin gene expression 100-fold in transgenic mice and also confers position-independent expression. To further define important sequences within this region, deletion mutations of the 1.9-kb fragment were introduced upstream of the human beta-globin gene, and the constructs were tested for activity in transgenic mice. Although enhancer activity was gradually lost with deletions of both 5' and 3' sequences, a 373-base-pair (bp) fragment retained the ability to confer relative position-independent expression. Three prominent DNase I footprints were observed in this region with extracts from the human erythroleukemia cell line K-562, one of which contained duplicated binding sites for transcription factor AP-1 (activator protein 1). When the 1.9-kb fragment containing an 18-bp deletion of the AP-1 binding sites was tested in transgenic mice, enhancer activity decreased 20-fold but position-independent expression was retained.