Expansion of an unstable GAA·TTC repeat in the first intron of the FXN gene causes Friedreich ataxia by reducing frataxin expression. Deficiency of frataxin, an essential mitochondrial protein, leads to progressive neurodegeneration and cardiomyopathy. The degree of frataxin reduction correlates with GAA·TTC tract length, but the mechanism of reduction remains controversial. Here we show that transcription causes extensive RNA·DNA hybrid formation on GAA·TTC templates in bacteria as well as in defined transcription reactions using T7 RNA polymerase in vitro. RNA·DNA hybrids can also form to a lesser extent on smaller, so-called ‘pre-mutation’ size GAA·TTC repeats, that do not cause disease, but are prone to expansion. During in vitro transcription of longer repeats, T7 RNA polymerase arrests in the promoter distal end of the GAA·TTC tract and an extensive RNA·DNA hybrid is tightly linked to this arrest. RNA·DNA hybrid formation appears to be an intrinsic property of transcription through long GAA·TTC tracts. RNA·DNA hybrids have a potential role in GAA·TTC tract instability and in the mechanism underlying reduced frataxin mRNA levels in Friedreich Ataxia.

Mycoplasma genitalium has been increasingly recognized as an important microbe not only because of its significant association with human genital tract diseases but also because of its utility as a model for studying the minimum set of genes necessary to sustain life. Despite its small genome, 4.7% of the total genome sequence is devoted to making the MgPa adhesin operon and its nine chromosomal repetitive elements (termed MgPars). The MgPa operon, along with 9 MgPars, is believed to play an important role in pathogenesis of M. genitalium infection and has also served as the main target for development of diagnostic tools. However, genetic variation in the complete MgPa operon and MgPars among clinical strains of M. genitalium has not been addressed. In this study we examined the genetic variation in the complete MgPa operon (approximately 8.5 kb) and full or partial MgPar sequences (0.4–2.6 kb) in 15 geographically diverse strains of M. genitalium. Extensive variation was present in four repeat regions of the MgPa operon (with homology to MgPars) among and within strains while the non-repeat regions (without homology to MgPars) showed low-level variation among strains and no variation within strains. MgPars showed significant variation among strains but were highly homogeneous within strains, supporting gene conversion as the likely recombination mechanism. When applying our sequence data to evaluate published MgPa operon-based diagnostic PCR assays and genotyping systems, we found that 11 of 19 primers contain up to 19 variable nucleotides and that the target for one of two typing systems is located in a hypervariable repeat region, suggesting the likelihood of false results with some of these assays. This study not only provides new insights into the role of the MgPa operon in the pathogenesis of M. genitalium infection but has important implications for the development of diagnostic tools.

genitalium has yet to be defined, significant associations exist between M. genitalium and cervicitis, pelvic inflammatory disease (PID), and tubal infertility (23,25,30). Among female reproductive tract tissues, M. genitalium DNA has been detected in specimens from the vagina, endocervix, endometrium, and the fallopian tubes, suggesting that, after sexual transmission, the organism can disseminate from the lower to the upper tract. Similarly, outbred mice inoculated vaginally showed time-dependent ascension of M. genitalium from the vagina to the cervix, followed by detection in the uterus, fallopian tubes, and ovaries (27). Therefore, the cervix appears to be either a primary or transient target of infection. Considering the clinical associations of M. genitalium with urogenital inflammation, it remains imperative to investigate the basic mechanisms of mucosal infection and disease induced by this organism.Among male and female reproductive tract syndromes for which M. genitalium has been implicated, all could be attributed to long-term colonization of the urogenital tract, which we refer to here as "persistence." Consistent with the ability to survive longterm in urogenital tissues and similar to other sexually acquired urogenital pathogens, it is hypothesized that M. genitalium has evolved specific mechanisms to evade the host immune system. Indeed, persistent cervical infection has been observed in several clinical studies (4,6,10,13) and has been associated with chronic symptoms (4). Persistent infection by M. genitalium is likely mediated, at least in part, by recombinational variation of genes encoding surface-exposed antigens (13,14,22) and through intracellular localization (2,7,17,26,31). Despite these mechanisms for avoiding the host's immune system, M. genitalium appears to induce a significant inflammatory response, as demonstrated with epidemiologic associations with urogenital disease (25, 30) and the observation that human ecto-and endocervical epithelial cells respond to acute infection with proinflammatory cytokine secretion (26). A link to human immunodeficiency virus (HIV) infection has been observed (29) since cervicitis caused by M. genitalium occurs more often in HIV-positive subjects (20) and, importantly, since M. genitalium persists longer in these women (6). In addition, high M. genitalium burden is correlated with increased HIV shedding from the cervix (24). Collectively, under-

e Mycoplasma genitalium causes persistent urogenital tract infection in humans. Antigenic variation of the protein encoded by the MG192 gene has been proposed as one of the mechanisms for persistence. The aims of this study were to determine MG192 sequence variation in patients with chronic M. genitalium infection and to analyze the sequence structural features of the MG192 gene and its encoded protein. Urogenital specimens were obtained from 13 patients who were followed for 10 days to 14 months. The variable region of the MG192 gene was PCR amplified, subcloned into plasmids, and sequenced. Sequence analysis of 220 plasmid clones yielded 97 unique MG192 variant sequences. MG192 sequence shift was identified between sequential specimens from all but one patient. Despite great variation of the MG192 gene among and within clinical specimens from different patients, MG192 sequences were more related within M. genitalium specimens from an individual patient than between patients. The MG192 variable region consisted of 11 discrete subvariable regions with different degrees of variability. Analysis of the two most variable regions (V4 and V6) in five sequential specimens from one patient showed that sequence changes increased over time and that most sequences were present at only one time point, suggesting immune selection. Topology analysis of the deduced MG192 protein predicted a surface-exposed membrane protein. Extensive variation of the MG192 sequence may not only change the antigenicity of the protein to allow immune evasion but also alter the mobility and adhesion ability of the organism to adapt to diverse host microenvironments, thus facilitating persistent infection.A s a sexually transmitted human pathogen, Mycoplasma genitalium causes nongonococcal urethritis (NGU) in men and is associated with genital tract inflammatory diseases in women, including endometritis, cervicitis, pelvic inflammatory disease, and tubal factor infertility (reviewed in reference 1). Additionally, there are increasing numbers of studies suggesting that M. genitalium increases the risk of HIV-1 acquisition and/or transmission (2-4). Like other pathogenic mycoplasmas, M. genitalium is capable of causing chronic infections, as has been documented in cultured human endocervical epithelial cells (5), in animal models (6, 7), in men with NGU (8, 9), and in women with cervicitis (10). The mechanisms for persistence remain poorly understood.The MgPa operon in the M. genitalium genome encodes three proteins, MG190 (mgpA), MG191 (mgpB or P140), and MG192 (mgpC or P110). The latter two are the known major adhesion proteins located on the surface of the terminal structure of M. genitalium, which plays a major role in the attachment of the organism to host epithelial cells (11,12). Both proteins are highly antigenic and capable of eliciting strong antibody responses in M. genitalium-infected patients and experimentally infected animals (6,(12)(13)(14), suggesting an important role in pathogenesis. According to the genome sequence of M. genital...

Mycoplasma genitalium, a human pathogen associated with sexually transmitted diseases, is unique in that it has the smallest genome of any known free-living organism. Despite its small genome, 4.7 % of the total genomic sequence is devoted to making the MgPa adhesin operon (containing the MG190, MG191 and MG192 genes) and its repetitive chromosomal sequences (known as MgPars). The goals of this study were to investigate the location, organization and variability of trinucleotide tandem repeats (TTRs) in the MgPa operon and MgPars and to explore the possible mechanisms and role of TTR variations. By analysing the complete MgPa operon and complete or partial MgPar sequences in a collection of 15 geographically diverse clinical strains of M. genitalium, TTR sequences were identified in four regions in MG191, one region in MG192, and two or three regions in each of all nine MgPars except for MgPar 3. These TTRs were variable not only in the repeat copy number but also in the repeat unit sequence among or within strains. The key mechanisms for the TTR variations likely include recombination between MgPa and MgPars, and slipped-strand mispairing. TTR variation may represent a mechanism to maximize the variation of the MgPa operon, which is complementary to genetic variation involving segmental recombination between MgPa and MgPars, thus enhancing the organism's ability to adhere to and colonize host cells as well as evasion of the host immune system.