Morrison M, Mackie R I, White B A
Department of Animal Sciences, University of Illinois, Urbana 61801.
Appl Environ Microbiol. 1992 Jan;58(1):66-9. doi: 10.1128/aem.58.1.66-69.1992.
The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.
本文描述了纤维素分解瘤胃细菌黄化瘤胃球菌FD-1的主要DNA限制修饰系统。限制性内切酶RflFI可通过磷酸纤维素和肝素琼脂糖层析从细胞提取物中分离出来。单独使用RflFI或与从白色链霉菌G中分离出的限制性内切酶SalI联合进行的限制性酶切消化表明，RflFI识别的DNA序列与SalI识别的序列重叠或相同。DNA序列分析证实，RflFI的活性与SalI相同，识别序列为5'-GTCGAC-3'，切割发生在G和T之间。该序列内的腺嘌呤甲基化可在体外由TaqI甲基化酶催化，这抑制了RflFI和SalI对质粒DNA分子的切割。来自黄化瘤胃球菌FD-1的染色体DNA在该DNA序列内也被甲基化，因为两种限制性内切酶都不能降解该DNA底物。这些发现为在对黄化瘤胃球菌FD-1进行基因转移实验之前保护质粒分子不被降解提供了一种方法。
