Sanson G F, Briones M R
Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo S.P. 04023-062, Brazil.
J Clin Microbiol. 2000 Jan;38(1):227-35. doi: 10.1128/JCM.38.1.227-235.2000.
We tested whether comparative sequence analysis of the mitochondrion-encoded cytochrome c oxidase subunit 2 gene (COX2) could be used to distinguish intraspecific variants of Candida glabrata. Mitochondrial genes are suitable for investigation of close phylogenetic relationships because they evolve much faster than nuclear genes, which in general exhibit very limited intraspecific variation. For this survey we used 11 clinical isolates of C. glabrata from three different geographical locations in Brazil, 10 isolates from one location in the United States, 1 American Type Culture Collection strain as an internal control, and the published sequence of strain CBS 138. The complete coding region of COX2 was amplified from total cellular DNA, and both strands were sequenced twice for each strain. These sequences were aligned with published sequences from other fungi, and the numbers of substitutions and phylogenetic relationships were determined. Typing of these strains was done by using 17 substitutions, with 8 being nonsynonymous and 9 being synonymous. Also, cDNAs made from purified mitochondrial polyadenylated RNA were sequenced to confirm that our sequences correspond to the expressed copies and not nuclear pseudogenes and that a frameshift mutation exists in the 3' end of the coding region (position 673) relative to the Saccharomyces cerevisiae sequence and the previously published C. glabrata sequence. We estimated the average evolutionary rate of COX2 to be 11.4% sequence divergence/10(8) years and that phylogenetic relationships of yeasts based on these sequences are consistent with rRNA sequence data. Our analysis of COX2 sequences enables typing of C. glabrata strains based on 13 haplotypes and suggests that positions 51 and 519 indicate a geographical polymorphism that discriminates strains isolated in the United States and strains isolated in Brazil. This provides for the first time a means of typing of Candida strains that cause infections by use of direct sequence comparisons and the associated divergence estimates.
我们测试了线粒体编码的细胞色素c氧化酶亚基2基因（COX2）的比较序列分析是否可用于区分光滑念珠菌的种内变体。线粒体基因适合用于研究密切的系统发育关系，因为它们的进化速度比核基因快得多，而核基因通常种内变异非常有限。在本次调查中，我们使用了来自巴西三个不同地理位置的11株光滑念珠菌临床分离株、来自美国一个地点的10株分离株、1株美国模式培养物保藏中心菌株作为内部对照，以及菌株CBS 138的已发表序列。从总细胞DNA中扩增出COX2的完整编码区，每个菌株的两条链都进行了两次测序。这些序列与其他真菌的已发表序列进行比对，并确定替换数和系统发育关系。通过使用17个替换对这些菌株进行分型，其中8个为非同义替换，9个为同义替换。此外，对从纯化的线粒体聚腺苷酸化RNA制备的cDNA进行测序，以确认我们的序列对应于表达拷贝而非核假基因，并且相对于酿酒酵母序列和先前发表的光滑念珠菌序列，在编码区3'端（位置673）存在移码突变。我们估计COX2的平均进化速率为11.4%序列分歧/10⁸年，并且基于这些序列的酵母系统发育关系与rRNA序列数据一致。我们对COX2序列的分析能够基于13种单倍型对光滑念珠菌菌株进行分型，并表明位置51和519显示出一种地理多态性，可区分在美国分离的菌株和在巴西分离的菌株。这首次提供了一种通过直接序列比较和相关分歧估计对引起感染的念珠菌菌株进行分型的方法。
