Akopyants N S, Fradkov A, Diatchenko L, Hill J E, Siebert P D, Lukyanov S A, Sverdlov E D, Berg D E
Departments of Molecular Microbiology and Genetics, Washington University Medical School, St. Louis, MO 63110, USA.
Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):13108-13. doi: 10.1073/pnas.95.22.13108.
Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based subtractive hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by subtractive hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot hybridizations indicated that these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.
仅在细菌物种的某些菌株中具有特征的基因可能具有重大的生物学意义。在此，我们描述了一种基于聚合酶链反应（PCR）的消减杂交方法，用于高效检测此类DNA，并将其应用于胃病原体幽门螺杆菌。通过与基因组已完全测序的无关菌株（26695）进行消减杂交，获得了18个对猴定植菌株（J166）特异的DNA。7个J166特异的克隆与26695基因组没有DNA序列匹配，另外11个克隆是混合的，相邻片段与26695中的任何序列匹配或不匹配。在蛋白质水平上，7个克隆与推定的DNA限制修饰酶具有同源性，2个与推定的代谢酶具有同源性。另外9个与已指定功能的蛋白质没有数据库匹配。使用针对12个消减克隆特异的引物对13个无关幽门螺杆菌菌株进行PCR检测，并进行互补Southern印迹杂交，结果表明这些DNA在幽门螺杆菌群体中高度多态，每个菌株产生不同的基因特异性PCR扩增模式。如本文所述，寻找多态性DNA应有助于鉴定病原体中以前未知的毒力基因，并为微生物遗传多样性和进化提供新的见解。
