PHASE AND ANTIGENIC VARIATION OF NEISSERIA GONORRHOEAE OPA GENES

淋病奈瑟菌（Neisseria gonorrhoeae，Ng）是淋病的病原体，在其发病过程中具有较高的频率和抗原变异。负责表面成分（即应变基因）的可变表达的主要遗传机制涉及串联DNA重复序列的缺失或插入。opa基因家族是N.淋病是结构上相关的一组偶然性基因的最著名的例子。奈瑟球菌opa基因编码一个外膜蛋白家族，其在实验室和体内显示出时相可变表达（如在自然和男性志愿者感染研究中所示）。相位变化是由编码加工信号肽的区域中每个opa基因内的五聚体（[CTTCT]）重复元件数目的变化引起的。给定基因内的重复数目用于使起始密码子与编码成熟Opa蛋白的序列对齐（框内）或错配（框外）。我们的目标是通过确定遗传和环境对相位变化的影响来了解非法重组的机制。报告系统已被用于测量相位变化频率。遗传对相位变异的影响：以前的研究表明，细菌细胞的DNA修复途径在利用串联DNA重复序列的超变基因中发生的遗传转换中很重要。我们在N中有突变的基因。淋病涉及主要的DNA修复途径。此时鉴定和突变的基因涉及错配修复（mutS/hexA，mutL/hexB），转录偶联修复（tcrF），核苷酸切除修复（uvrA，uvrB和uvrC）和氧化损伤控制和修复（fpg，oxyR和katF）。将这些突变引入Ng中应增加opa基因报告基因的相位变化频率，但同时增加利用非法重组的所有基因的相位变化速率，导致同时表达所有可变组分的菌株。这些菌株将帮助我们了解变异在细菌发病机制中的作用，也可能有助于产生具有广泛免疫反应性的减毒菌株。环境对相位变化的影响使用Ng opa：：phoA报道菌株测量在许多环境条件下的相位变化频率。我们通过制造opa：：bla报告菌株，扩大了我们的报告基因结构库，使我们能够直接选择特定的基因构型。在生长阶段的过程中，通过简单地比较相位变体的数量（或者？在关闭？还是？关上吗？）存在于人口中。增加时相变化频率的环境因素包括i）生长期和饥饿，ii）真核细胞接触，iii）DNA转化和iv）DNA损伤。- 淋病奈瑟菌，淋病，性传播疾病，时相变异，抗原变异，Opa，非法生殖

Neisseria gonorrhoeae (Ng), the causative agent of gonorrhea, exhibits high frequency phase and antigenic variation during the course of disease. The predominant genetic mechanism responsible for variable expression of surface constituents (i.e. contingency genes) involves the deletion or insertion of tandem DNA repeats. The opa gene family of N. gonorrhoeae is the best known example of a structurally related group of contingency genes. Neisserial opa genes encode a family of outer-membrane proteins that show phase variable expression in the laboratory and in vivo (as shown in natural and male-volunteer infection studies). Phase variation results from changes in the number of pentameric ([CTTCT]) repeat elements within each opa gene in the region encoding the processed signal-peptide. The number of repeats within a given gene serves to either align (in frame) or misalign (out- of-frame) the start codon with the sequence encoding the mature Opa protein. Our goal is to understand the mechanisms for illegitimate recombination by determining the genetic and environmental influences on phase variation. Reporter systems have been used to measure phase variation frequencies.A. Genetic Influences on Phase Variation.Previous studies have shown that the DNA repair pathways of the bacterial cell are important in the genetic switching that occurs in hypermutable genes that utilize tandem DNA repeats. We have mutated genes in N. gonorrhoeae involved in the major DNA repair pathways. The genes identified and mutated at this time are involved in Mismatch Repair (mutS/hexA, mutL/hexB), Transcription Coupled Repair (tcrF), Nucleotide Excision Repair (uvrA, uvrB and uvrC) and Oxidative Damage Control and Repair (fpg, oxyR and katF). Introduction of these mutations into Ng should increase the phase variation frequency of the opa gene reporter but, at the same time, increase the phase change rates of all of the genes that utilize illegitimate recombination, resulting in strains that express all of the variable components simultaneously. These strains will help us understand the role of variation in bacterial pathogenesis and may also be useful in the generation of attenuated strains with broad immunological reactivity.B. Environmental Influences on Phase Variation.The Ng opa::phoA reporter strain was used to measure phase variation frequencies under a number of environmental conditions. We have expanded our repertoire of reporter costructions by making opa::bla reporter strains, allowing us to directly select for specific gene configurations. Frequencies were measured during the course of the growth phase by simply comparing the number of phase variants (either ?on to off? or ?off to on?) present in the population. Environmental factors which increased the frequency of phase variation included i) growth phase and starvation, ii) eukaryotic cell contact, iii) DNA transformation and iv) DNA damage. - Neisseria gonorrhoeae, gonorrhea, Sexually Transmitted Diseases, Phase Variation, Antigenic Variation, Opa, Illegitimate Recombination