RESUMO
MUNC18-1 (n-Sec1) is a brain-specific protein and is known to play a role in neurotransmitter release by mediating docking and fusion of synaptic vesicles to presynaptic membranes. The protein is also implicated in the cellular excretion process of hormones and other biological substances in other mammalian tissues and yeasts. We have studied the structure of mouse munc18-1 gene by sequencing the genomic munc18-1 gene and its 5'-flanking region. munc18-1 gene comprises 19 exons whose size ranges from 50 base pairs (2nd exon) to 1676 base pairs (19th exon) with a total gene size of approximately 56 kilobases. In the 5'-flanking region, there are several transcription factor binding sites such as for HSF2, Lyf-1, and Sp1 but no TATA or CAAT sequences. munc18-1 gene was mapped on mouse chromosome 2 between two anchor markers D2Mit152 and D2Mit242. Transfection experiments employing these and upstream sequences suggest the presence of a sequence(s) that negatively regulates the expression of munc18-1 gene.
Assuntos
Encéfalo/citologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Vesículas Sinápticas/fisiologia , Proteínas de Transporte Vesicular , Animais , Sequência de Bases , Fusão Celular , Mapeamento Cromossômico , Camundongos , Dados de Sequência Molecular , Proteínas Munc18 , Células PC12 , Poli A/metabolismo , Regiões Promotoras Genéticas , Ratos , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismoRESUMO
We describe the principle and actual processes of a differential cloning procedure designed for cloning of anonymous restriction DNA fragments whose molecular sizes differ between two genomic DNA preparations from higher organisms as a result of DNA rearrangement, polymorphism, etc. The procedure, which was extensively modified from the original one and still employs in-gel competitive reassociation (IGCR) as the basic principle, aims for cloning of DNA fragments which exist in one copy or less per mammalian genome. The modified procedure consists of dissociation and reassociation of biotinylated restriction digests of target DNA fragments (from which clones are to be isolated) in the presence of a large excess of reference (competitor) DNA in gel after electrophoresis, which is followed by absorption of the target DNA fragments to streptavidin-coated tubes and solid-phase polymerase chain reaction. After repeating these steps we attained substantial enrichment of altered DNA fragments which were originally present in one copy or less per complex mammalian genome.
Assuntos
Clonagem Molecular/métodos , DNA/genética , DNA/isolamento & purificação , Eletroforese/métodos , Animais , Sequência de Bases , DNA/normas , Primers do DNA/genética , Feminino , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos , Ratos Wistar , Deleção de SequênciaRESUMO
We have developed a substantially improved differential cloning procedure designed for cloning anonymous altered restriction DNA fragments from higher organisms. The improvements include (i) in-gel dissociation and reassociation of biotinylated restriction digests of target DNA fragments, (ii) replacement of agarose gel by a synthetic gel material for electrophoresis, (iii) use of a reassociation enhancing reagent (CTAB) for in-gel reassociation, and (iv) introduction of PCR. After several cycles of IGCR, we attained considerable enrichment of altered or rearranged DNA fragments which were originally present at one copy or less per complex eukaryotic genome. Examples of enrichment include those of an exogenously added DNA fragment, a chromosomal DNA sequence that has undergone a deletion, and DNA fragments containing a recombination junction.
Assuntos
Clonagem Molecular/métodos , DNA/genética , Animais , Sequência de Bases , Fenômenos Biofísicos , Biofísica , DNA/isolamento & purificação , Rearranjo Gênico , Marcadores Genéticos , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Recombinação GenéticaRESUMO
We have developed a differential cloning procedure designed for cloning of anonymous restriction DNA fragments whose molecular sizes differ between two genomic DNA preparations from higher organisms. The procedure, which was extensively revised from the original one, consists of several steps as summarized below. (i) Digestion of two DNA preparations (target and reference DNA) with the same restriction enzyme (4 base cutter). (ii) Biotinylation of target DNA and conversion of reference DNA to nonamplifiable form by terminal dephosphorylation. (iii) Electrophoresis of the two DNA preparations through a synthetic gel with a large excess of reference DNA as a competitor. (iv) In-gel alkaline dissociation of DNA, followed by reassociation (in-gel competitive reassociation). (v) Elution of DNA from the gel and PCR after adapter ligation and adsorption of DNA onto streptavidin-coated matrix. By repeating these steps, we attained substantial enrichment (approximately 10,000-fold) of DNA fragments which were originally present at one copy or less per complex mammalian genome. The details of the procedure and its unique characteristics in cloning of altered genomic DNA fragments, particularly from mammalian genome, are discussed.