GENETIC RECOMBINATION IN MYCOPLASMAS

支原体是最小的自由生活的微生物，广泛存在于 分布于自然界，通常在植物，昆虫， 和动物，包括人类。虽然这些对经济的影响 疾病是巨大的，关于机制的信息很少 发病机理和有效的防治方法尚不清楚。的 支原体的表面性质以约103 每代每细胞。高频表型变异影响 表面抗原的结构，集落形态， 微生物对支原体病毒的吸附，以及支原体对 红细胞表面特性的高频变化也 已被证明是重要的疾病发病机制。一些高频 肺支原体的表型变异与 位点特异性DNA重排是本提案的重点。两 同时发生的DNA重排特别令人感兴趣。 是编码限制的新DNA元件的倒位， 修改（R-M）系统。另一个涉及DNA序列编码 存在于可变表面抗原中的表位。长期目标是 了解支原体染色体的分子基础 表型变异，这些现象在疾病中的作用 致病机制，以及它们在支原体进化中所起的作用。一 初步假设是R-M系统调节染色体DNA 通过双链断裂修复途径在支原体中的重排。 这样的重组机制与已知的重组机制根本不同。 原核生物中的重组途径，这些研究可能有 对目前关于遗传变异的思想产生了相当大的影响， 致病菌目前这项提案的目标是 刻画了M.肺，以表征基因 编码可变表面抗原，并确定DNA如何 重排调节这些系统的表达。第一个目标是 在核苷酸水平克隆和表征遗传基因座， 与可逆元素高度同源。该基因座可以编码一种 第二个R-M系统。第二个目标是确定DNA倒位 影响R-M通过检查相关的转录调节 的位点第三个目标是在M.肺内 为了确定R-M酶是否在高- 频率DNA重排和表型转换的支原体。的 第四个目标是确定DNA重排如何影响 可变表面抗原这将通过表征 编码可变表面抗原的基因和DNA 在其附近发生的重排。第五个也是最后一个目标是 为了扩大我们的亚克隆库，含有DNA倒位和其他 重新安排，以便更充分地表征 遗传变异在这个系统中。

Mycoplasmas, the smallest free-living microorganisms, arc widely distributed in nature and commonly produce disease in plants, insects, and animals, including man. Although the economic impact of these diseases is great, little information is available concerning mechanisms of pathogenesis and effective methods of control are unavailable. The surface properties of mycoplasmas vary at a high frequency of about 103 per cell per generation. High-frequency phenotypic variations affect the structure of surface antigens, colony morphology, the susceptibility of the organism to mycoplasma viruses, and the adsorption of mycoplasmas to red blood cells. High-frequency changes in surface properties have also been shown to be important to disease pathogenesis. Some high-frequency phenotypic variations in Mycoplasma pulmonis have been correlated with site-specific DNA rearrangements that are the focus of this proposal. Two DNA rearrangements that occur concurrently are of particular interest One is the inversion of a novel DNA element encoding a restriction and modification (R-M) system. The other involves DNA sequences encoding epitopes present in variable surface antigens. The long-range goals are to understand the molecular basis of mycoplasmal chromosomal and phenotypic variation, the role these phenomena play in disease pathogenesis, and the role they have played in mycoplasmal evolution. A tentative hypothesis is that R-M systems regulate chromosomal DNA rearrangements in mycoplasmas via double-strand break repair pathways. Such a recombination mechanism is fundamentally different from known pathways of recombination in prokaryotes, and these studies may have considerable impact on current thinking regarding genetic variation in pathogenic bacteria The goals of this current proposal are to characterize the R-M systems of M. pulmonis, to characterize the genes encoding the variable surface antigens, and to determine how DNA rearrangements regulate expression of these systems. The first aim is to clone and characterize at the nucleotide level a genetic locus that is highly homologous to the invertible element. This locus may encode a second R-M system. The second aim is to determine how DNA inversion affects R-M by examining the transcriptional regulation of the pertinent loci. The third aim is to inactivate the R-M systems in M. pulmonis in order to determine whether R-M enzymes play a causal role in high- frequency DNA rearrangements and phenotypic switching in mycoplasmas. The fourth aim is to determine how DNA rearrangements affect expression of variable surface antigens. This will be accomplished by characterizing both the genes encoding variable surface antigens and the DNA rearrangements that occur in their vicinity. The fifth and final aim is to expand our repertoire of subclones containing DNA inversions and other rearrangements in order to more fully characterize the potential for genetic variation in this system.