DNA polymerase beta is critical for mouse meiotic synapsis.

We have shown earlier that DNA polymerase beta (Pol beta) localizes to the synaptonemal complex (SC) during Prophase I of meiosis in mice. Pol beta localizes to synapsed axes during zygonema and pachynema, and it associates with the ends of bivalents during late pachynema and diplonema. To test whether these localization patterns reflect a function for Pol beta in recombination and/or synapsis, we used conditional gene targeting to delete the PolB gene from germ cells. We find that Pol beta-deficient spermatocytes are defective in meiotic chromosome synapsis and undergo apoptosis during Prophase I. We also find that SPO11-dependent gammaH2AX persists on meiotic chromatin, indicating that Pol beta is critical for the repair of SPO11-induced double-strand breaks (DSBs). Pol beta-deficient spermatocytes yielded reduced steady-state levels of the SPO11-oligonucleotide complexes that are formed when SPO11 is removed from the ends of DSBs, and cytological experiments revealed that chromosome-associated foci of replication protein A (RPA), RAD51 and DMC1 are less abundant in Pol beta-deficient spermatocyte nuclei. Localization of Pol beta to meiotic chromosomes requires the formation of SPO11-dependent DSBs. Taken together, these findings strongly indicate that Pol beta is required at a very early step in the processing of meiotic DSBs, at or before the removal of SPO11 from DSB ends and the generation of the 3' single-stranded tails necessary for subsequent strand exchange. The chromosome synapsis defects and Prophase I apoptosis of Pol beta-deficient spermatocytes are likely a direct consequence of these recombination defects.

Effects of organochlorines, individually and in mixtures, on B-cell proliferation in marine mammals and mice.

Organochlorines (OC) are lipophilic and stable, and therefore accumulate in tissues of top predators, such as marine mammals. While the immunomodulatory effects of individual OC have been studied in lab animals, their effects in other species (such as marine mammals) and the possible interactions between chemicals in mixtures are not well understood. This study investigated the immunomodulatory effects of four polychlorinated biphenyls (PCB, IUPAC numbers 138, 153, 169, and 180), as well as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), individually and in mixtures, in marine mammals and mice. Mitogen-induced B lymphocyte proliferation was mostly modulated by non-coplanar PCBs, for which general mechanisms underlying toxicity are poorly understood. Simple additive effects of OC in mixtures were found only in mice, while both synergistic and antagonistic interactions between OC were found in marine mammals. The toxic equivalency (TEQ) approach, which is currently used to assess the dioxin-like toxicity of OC mixtures, failed to predict immunotoxicity in mice and marine mammals, likely due to the complexity of interactions between OC and effects via dioxin-independent pathways. The commonly used mouse model failed to predict the immunotoxicity due to OC in the marine mammals tested. In addition, clustering data suggested that phylogeny might not help predict the toxicity of OC. Lymphoproliferative response was modulated in most species tested suggesting the possibility of increased susceptibility to infectious diseases in these animals. These findings may be helpful in more accurately characterizing the immunotoxic potential of OC in different target species and help in more relevant risk assessment.

Development of real-time diagnostic assays specific for Mycoplasma mycoides subspecies mycoides Small Colony.

Rapid and specific detection of Mycoplasma mycoides subsp. mycoides Small Colony (M. mycoides SC) is important for the effective control of contagious bovine pleuropneumonia. Although the United States has been free of this disease for over 100 years, it is necessary to develop modern diagnostic assays that are sensitive and specific for biological agents that would affect the US agricultural industry following accidental or intentional introduction into the US agricultural population. With this aim in mind, we have identified M. mycoides SC-specific genetic loci and developed TaqMan-based PCR assays for the detection of M. mycoides SC. The TaqMan assay allows for real-time detection of specific, amplified PCR products using portable equipment, enabling testing to be performed in the field. These assays are specific for M. mycoides SC, failing to amplify DNA from other organisms belonging to the M. mycoides cluster or two phylogenetically unrelated bovine mycoplasma species. Standard curves were drawn based on the linear relationships measured between the threshold fluorescence (C(T)) values and a measured quantity of genomic DNA. M. mycoides SC was successfully detected in bronchoalveolar lavage samples obtained from experimentally infected cattle. These TaqMan-based real-time PCR assays will allow for the rapid and specific detection of M. mycoides SC.

Transcriptional responses of Mycoplasma gallisepticum strain R in association with eukaryotic cells.

Mycoplasma gallisepticum is an etiologic agent of chronic respiratory disease in chickens and infectious sinusitis in turkeys. Other than proteins important for cytadherence, few M. gallisepticum factors or pathways contributing to host cell interactions have been identified. In this study, an oligonucleotide-based microarray was utilized to investigate transcriptional changes in M. gallisepticum strain R(low) upon exposure to eukaryotic cells. Fifty-eight genes were either up- or downregulated upon exposure to MRC-5 lung fibroblasts grown in vitro, including genes encoding transport-, metabolism-, and translation-associated proteins. Twenty of the 58 regulated genes have no assigned function. These results indicate that M. gallisepticum regulates gene expression upon exposure to eukaryotic cells, revealing genes and pathways likely to be important for host-bacterium interaction.

Identification of fibronectin-binding proteins in Mycoplasma gallisepticum strain R.

We have determined that virulent Mycoplasma gallisepticum strain Rlow is capable of binding the extracellular matrix protein fibronectin. Fibronectin was found to be present in M. gallisepticum Rlow protein extracts by Western blotting and peptide sequencing. Mycoplasma gallisepticum Rhigh, the attenuated, high-passage derivative of Rlow, is deficient in this ability. MGA_1199, the M. gallisepticum homologue of the cytadherence-associated protein P65 from Mycoplasma pneumoniae, and MGA_0928, the M. gallisepticum homologue of the M. pneumoniae cytoskeletal protein HMW3, were identified as fibronectin-binding proteins. Peptides from the regions of MGA_1199 and MGA_0928 exhibiting the highest degree of homology with known fibronectin-binding proteins were shown to bind the gelatin/heparin-binding domain of fibronectin. MGA_1199 and MGA_0928 were shown to be absent and aberrant, respectively, in Rhigh, explaining its lack of fibronectin-binding capability. Consistent with its M. pneumoniae counterpart, MGA_1199 (renamed PlpA) was demonstrated to be surface exposed, despite a lack of classical membrane-spanning domains. Due to its demonstrated topology and the strength of interaction between its binding peptide and fibronectin, we propose that PlpA functions as a fibronectin-binding protein in vivo and may possess atypical transmembrane domains.

Isolation and characterization of Mycoplasma sphenisci sp. nov. from the choana of an aquarium-reared jackass penguin (Spheniscus demersus).

Strain UCMJ was isolated from the choana of a jackass penguin (Spheniscus demersus) with recurrent mucocaseous choanal discharge. Isolation of this mycoplasma expands the known range of species hosting mycoplasmas. The name Mycoplasma sphenisci sp. nov. is proposed for this new species, for which strain UCMJ is the type strain.

The complete genome sequence of the avian pathogen Mycoplasma gallisepticum strain R(low).

The complete genome of Mycoplasma gallisepticum strain R(low) has been sequenced. The genome is composed of 996,422 bp with an overall G+C content of 31 mol%. It contains 742 putative coding DNA sequences (CDSs), representing a 91 % coding density. Function has been assigned to 469 of the CDSs, while 150 encode conserved hypothetical proteins and 123 remain as unique hypothetical proteins. The genome contains two copies of the rRNA genes and 33 tRNA genes. The origin of replication has been localized based on sequence analysis in the region of the dnaA gene. The vlhA family (previously termed pMGA) contains 43 genes distributed among five loci containing 8, 2, 9, 12 and 12 genes. This family of genes constitutes 10.4% (103 kb) of the total genome. Two CDSs were identified immediately downstream of gapA and crmA encoding proteins that share homology to cytadhesins GapA and CrmA. Based on motif analysis it is predicted that 80 genes encode lipoproteins and 149 proteins contain multiple transmembrane domains. The authors have identified 75 proteins putatively involved in transport of biomolecules, 12 transposases, and a number of potential virulence factors. The completion of this sequence has spawned multiple projects directed at defining the biological basis of M. gallisepticum.