Stein D C, Gunn J S, Piekarowicz A
Department of Microbiology, University of Maryland, College Park 20742, USA.
Biol Chem. 1998 Apr-May;379(4-5):575-8.
The DNA sequence encoding the S.NgoI restriction/modification (R/M) system was identified from a gene bank made from Neisseria gonorrhoeae strain WR302 by identifying recombinant plasmids that induced the reporter system in a methylase detection strain AP1-200-9 (Piekarowicz et al., 1991) and were resistant to digestion with NgoI. The DNA sequence was determined from one of these (pUCP30). M.NgoI is a protein of 315 aa with a predicted MW of 35296 Da and R.NgoI is a protein of 350 aa with a predicted MW of 40650 Da. The termination codon of M.NgoI overlapped the start codon of R.NgoI. The same strategy was used to clone the R/M system encoding HaeII from Haemophilus aegyptius strain ATCC 11116. The DNA sequence from one clone representing this class (pAP704) was determined. HaeII methylase is a protein of 318 aa with a predicted MW of 35669 Da and R.HaeII contains 352 aa with a predicted MW of 40800 Da. aa alignments between the two methylases indicated that they were 74.3% identical and 79% similar. DNA sequence alignments revealed 68% identity. An aa alignment between the two restriction enzymes indicated that they were 60% identical and 68% similar. DNA sequence alignments revealed 61% identity. The DNA sequences flanking these two systems were identified and used to determine the genomic organization of the two systems. The S.NgoI genes were found between two genes, one with high homology to GTP binding proteins of unknown function and one with homology to genes involved in tRNA synthetase synthesis. The HaeII R/M genes were located between two genes, mucF and mucE. The DNA sequence of the HaeII R/M system was compared to the genomic DNA sequence of H. influenzae Rd. Although the DNA sequences flanking the HaeII system were > 99% identical to contiguous DNA fragments found in the genome of H. influenzae Rd, no homology was seen with the DNA sequences encoding the HaeII R/M system, indicating that it is not found in this strain. Given the vast difference in the GC content of S.NgoI and HaeII, their apparent insertion into polycistronic operons, and their difference in codon usage when compared to the species from which they were isolated, the data suggest that these R/M systems originated in an organism other than Neisseria or Haemophilus.
通过在甲基化酶检测菌株AP1-200-9（Piekarowicz等人，1991年）中鉴定能诱导报告系统且对NgoI消化具有抗性的重组质粒，从淋病奈瑟菌菌株WR302构建的基因文库中鉴定出编码S.NgoI限制/修饰（R/M）系统的DNA序列。从其中一个（pUCP30）确定了DNA序列。M.NgoI是一种315个氨基酸的蛋白质，预测分子量为35296 Da，R.NgoI是一种350个氨基酸的蛋白质，预测分子量为40650 Da。M.NgoI的终止密码子与R.NgoI的起始密码子重叠。采用相同策略从埃及嗜血杆菌菌株ATCC 11116中克隆编码HaeII的R/M系统。确定了代表此类的一个克隆（pAP704）的DNA序列。HaeII甲基化酶是一种318个氨基酸的蛋白质，预测分子量为35669 Da，R.HaeII包含352个氨基酸，预测分子量为40800 Da。两种甲基化酶之间的氨基酸比对表明它们的同一性为74.3%，相似性为79%。DNA序列比对显示同一性为68%。两种限制酶之间的氨基酸比对表明它们的同一性为60%，相似性为68%。DNA序列比对显示同一性为61%。确定了这两个系统侧翼的DNA序列，并用于确定这两个系统的基因组组织。发现S.NgoI基因位于两个基因之间，一个与功能未知的GTP结合蛋白具有高度同源性，另一个与参与tRNA合成酶合成的基因具有同源性。HaeII R/M基因位于mucF和mucE这两个基因之间。将HaeII R/M系统的DNA序列与流感嗜血杆菌Rd的基因组DNA序列进行比较。尽管HaeII系统侧翼的DNA序列与流感嗜血杆菌Rd基因组中相邻的DNA片段>99%相同，但未发现与编码HaeII R/M系统的DNA序列具有同源性，表明该菌株中不存在该系统。鉴于S.NgoI和HaeII的GC含量差异巨大、它们明显插入多顺反子操纵子以及与它们所分离物种相比密码子使用的差异，数据表明这些R/M系统起源于奈瑟菌或嗜血杆菌以外的生物体。
