Nwankwo D, Wilson G
New England Biolaboratories Inc, Beverly, MA 01915.
Mol Gen Genet. 1987 Oct;209(3):570-4. doi: 10.1007/BF00331164.
The modification genes of Flavobacterium okeanokoites and Haemophilus galinarum have been cloned into the vector pBR322 and expressed in Escherichia coli cells. FokI methylase gene is contained on a 3.80 kb piece of F. okeanokoites DNA. Plasmid constructs carrying this fragment of DNA are resistant to digestion by FokI restriction endonuclease but are sensitive to cleavage by HindIII, EcoRI and PstI. Unmodified lambda DNA molecules, exposed in vitro to cell extracts prepared from cells habouring this plasmid, became resistant to digestion by FokI. The smallest HgaI methylase clone carries the pBR322 plasmid containing a 3.50 kb piece of H. galinarum DNA. This plasmid is resistant to digestion by HgaI. Neither the FokI nor the HgaI restriction endonuclease was detected in either clone. This is the first report of cloning modification genes whose protein products recognise asymmetric nucleotide sequences.
食海洋黄杆菌(Flavobacterium okeanokoites)和鸡嗜血杆菌(Haemophilus galinarum)的修饰基因已被克隆到载体pBR322中,并在大肠杆菌细胞中表达。食海洋黄杆菌的一段3.80 kb的DNA片段上含有FokI甲基化酶基因。携带该DNA片段的质粒构建体对FokI限制性内切酶的消化具有抗性,但对HindIII、EcoRI和PstI的切割敏感。在体外将未修饰的λDNA分子暴露于含有该质粒的细胞制备的细胞提取物中后,其对FokI的消化具有抗性。最小的HgaI甲基化酶克隆携带含有一段3.50 kb鸡嗜血杆菌DNA的pBR322质粒。该质粒对HgaI的消化具有抗性。在这两个克隆中均未检测到FokI或HgaI限制性内切酶。这是关于克隆其蛋白质产物识别不对称核苷酸序列的修饰基因的首次报道。