RESUMO
BamHI, a Type II restriction modification system from Bacillus amyloliquefaciensH recognizes the sequence GGATCC. The methylase and endonuclease genes have been cloned into E. coli in separate steps; the clone is able to restrict unmodified phage. Although within the clone the methylase and endonuclease genes are present on the same pACYC184 vector, the system can be maintained in E. coli only with an additional copy of the methylase gene present on a separate vector. The initial selection for BamHI methylase activity also yielded a second BamHI methylase gene which is not homologous in DNA sequence and hybridizes to different genomic restriction fragments than does the endonuclease-linked methylase gene. Finally, the interaction of the BamHI system with the E. coli Dam and the Mcr A and B functions, have been studied and are reported here.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/genética , Clonagem Molecular , Desoxirribonuclease BamHI/genética , Sequência de Aminoácidos , Bacillus/genética , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Clonagem Molecular/métodos , Metilases de Modificação do DNA/genética , Sondas de DNA/genética , Sondas de DNA/isolamento & purificação , Elementos de DNA Transponíveis , DNA Bacteriano/isolamento & purificação , Desoxirribonuclease BamHI/isolamento & purificação , Desoxirribonuclease BamHI/fisiologia , Escherichia coli/genética , Rearranjo Gênico , Dados de Sequência Molecular , Plasmídeos , Mapeamento por Restrição/métodos , Transformação GenéticaRESUMO
The Taq I modification and restriction genes (recognition sequence TCGA) have been cloned in E. coli and their DNA sequences have been determined. Both proteins were characterized and the N-terminal sequence of the endonuclease was determined. The genes have the same transcriptional orientation with the methylase gene 5' to the endonuclease gene. The methylase gene is 1089 bp in length (363 amino acids, 40,576 daltons); the endonuclease gene is 702 bp in length (234 amino acids, 27,523 daltons); they are separated by 132 bp. Both methylase and endonuclease activity can be detected in cell extracts. The clones fully modify the vector and chromosomal DNA but they fail to restrict infecting phage. Clones carrying only the restriction gene are viable even in the absence of modification. The restriction gene contains 7 Taq I sites; the modification gene contains none. This asymmetric distribution of sites could be important in the regulation of the expression of the endonuclease gene.
