Pomati Francesco, Neilan Brett A
Cyanobacteria and Astrobiology Research Laboratory, School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney 2052, NSW, Australia.
Nucleic Acids Res. 2004 Jan 12;32(1):e7. doi: 10.1093/nar/gnh012.
A PCR-based positive hybridization (PPH) method was developed to explore toxic-specific genes in common between toxigenic strains of Anabaena circinalis, a cyanobacterium able to produce saxitoxin (STX). The PPH technique is based on the same principles of suppression subtractive hybridization (SSH), although with the former no driver DNA is required and two tester genomic DNAs are hybridized at high stringency. The aim was to obtain genes associated with cyanobacterial STX production. The genetic diversity within phylogenetically similar strains of A.circinalis was investigated by comparing the results of the standard SSH protocol to the PPH approach by DNA-microarray analysis. SSH allowed the recovery of DNA libraries that were mainly specific for each of the two STX-producing strains used. Several candidate sequences were found by PPH to be in common between both the STX-producing testers. The PPH technique performed using unsubtracted genomic libraries proved to be a powerful tool to identify DNA sequences possibly transferred laterally between two cyanobacterial strains that may be candidate(s) in STX biosynthesis. The approach presented in this study represents a novel and valid tool to study the genetic basis for secondary metabolite production in microorganisms.
开发了一种基于聚合酶链反应的阳性杂交（PPH）方法，以探索能产生石房蛤毒素（STX）的蓝藻——环状鱼腥藻产毒菌株之间共有的毒素特异性基因。PPH技术基于与抑制性消减杂交（SSH）相同的原理，不过前者不需要驱动DNA，且两个测试基因组DNA在高严格度下杂交。目的是获得与蓝藻STX产生相关的基因。通过DNA微阵列分析，将标准SSH方案的结果与PPH方法的结果进行比较，研究了系统发育相似的环状鱼腥藻菌株内的遗传多样性。SSH能够获得主要针对所使用的两种产STX菌株中每一种的DNA文库。通过PPH发现，几个候选序列在两个产STX测试菌株中是共有的。使用未消减基因组文库进行的PPH技术被证明是一种强大的工具，可用于鉴定可能在两个蓝藻菌株之间横向转移的DNA序列，这些序列可能是STX生物合成中的候选序列。本研究中提出的方法是研究微生物次级代谢产物产生的遗传基础的一种新颖且有效的工具。
