Soper B W, Reddy K J
Department of Biological Sciences, State University of New York-Binghamton 13902.
J Bacteriol. 1994 Sep;176(17):5565-70. doi: 10.1128/jb.176.17.5565-5570.1994.
In the process of developing a gene transfer system for the marine, unicellular, nitrogen-fixing cyanobacterium Cyanothece sp. strain BH68K, two major restriction barriers have been identified. A cell wall-associated nuclease exhibited non-site-specific degradation of covalently closed circular and linear double-stranded DNA molecules, including Cyanothece sp. strain BH68K chromosomal DNA. The nuclease is easily released from intact cells by using water or buffer containing Triton X-100. Nuclease activity was undetectable in cell extracts prepared from water-washed cells. Comparison of the restriction endonuclease susceptibility of Cyanothece sp. strain BH68K DNA to that of Anabaena sp. strain PCC 7120 revealed that these organisms have a nearly identical pattern of restriction and therefore may contain similar systems for DNA methylation. Restriction by DpnI, MboI, and Sau3AI indicated the presence of adenine methylation. Cyanothece sp. strain BH68K cell extracts contain a type II restriction endonuclease, Csp68KI. The activity of Csp68KI was easily detected in cell extracts without extensive purification. Csp68KI is an isoschizomer of AvaII and recognizes the nucleotide sequence 5'-GG(A/T)CC-3'. Cleavage occurs between the guanosine nucleotides producing 3-bp 5' overhang ends.
在为海洋单细胞固氮蓝细菌蓝藻菌属BH68K菌株开发基因转移系统的过程中，已确定了两个主要的限制屏障。一种与细胞壁相关的核酸酶对共价闭合环状和线性双链DNA分子表现出非位点特异性降解，包括蓝藻菌属BH68K菌株的染色体DNA。通过使用含有 Triton X-100的水或缓冲液，该核酸酶很容易从完整细胞中释放出来。在用水洗涤的细胞制备物的细胞提取物中未检测到核酸酶活性。比较蓝藻菌属BH68K菌株DNA与鱼腥藻属PCC 7120菌株DNA对限制性内切酶的敏感性，发现这些生物体具有几乎相同的限制模式，因此可能含有类似的DNA甲基化系统。DpnI、MboI和Sau3AI的限制表明存在腺嘌呤甲基化。蓝藻菌属BH68K菌株的细胞提取物含有一种II型限制性内切酶Csp68KI。在未经广泛纯化的细胞提取物中很容易检测到Csp68KI的活性。Csp68KI是AvaII的同裂酶，识别核苷酸序列5'-GG(A/T)CC-3'。切割发生在鸟苷核苷酸之间，产生3个碱基对的5'突出末端。
