Interaction between the Msh2 and Msh6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-Msh6 Complex

Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes requires ATP binding to only the Msh6 nucleotide-binding site, whereas the formation of Msh2-Msh6 sliding clamps requires ATP binding to both the Msh2 and Msh6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and Msh6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps.     

Sex ratios under asymmetrical local mate competition in the parasitoid wasp Nasonia vitripennis

Sex ratio theory has proved remarkably useful in testing theadaptive nature of animal behavior. A particularly productivearea in this respect is Hamilton's theory of local mate competition(LMC), which has been extended in numerous directions to includegreater biological realism, allowing more detailed tests inspecific organisms. We have presented one such extension, termedasymmetrical LMC, which occurs when egg laying by females ona patch is asynchronous, and emerging males do not disperse,resulting in the extent of LMC on a patch varying over time.Our aim here is to test whether the parasitoid wasp Nasoniavitripennis responds to variation in the degree of asymmetricalLMC. Specifically, we show that females adjust their offspringsex ratios in response to (1) variation in the amount of asynchronyin emergence between broods on a patch and (2) the number andproportion of previously parasitized hosts on the patch. Ourresults provide qualitative support for the predictions of theory,suggesting new levels of complexity in the sex ratio behaviorof this much-studied organism. However, our results do not alwaysprovide quantitative support for theory, suggesting furthercomplexities that must be clarified.     

Using FTA® cards to store avian blood samples for genetic studies. Their application in sex determination

FTA® cards were used for long‐term storage of avian blood samples. Blood DNA was extracted by a simple method and used in PCR for sex identification of adult and nestling Great Grey Shrikes Lanius excubitor.     

Typing of Genetic Markers Involved in Stress Response by Fluorescent cDNA-Amplified Fragment Length Polymorphism Technique

Identification of genetic markers involved in stress response to physical factors or chemical substances in organisms is a challenging task. Typing of upregulated gene expression due to selective antibacterial pressure is a promising approach in the search of molecular mechanisms responsible for development of resistance. cDNA-Fluorescent Amplified Fragment Length Polymorphism (cDNA-FAFLP) strategy was developed and applied in the search of antimycotic drug resistance marker(s) in medically important fungi as an alternative method to microarray analysis. We compared differential gene expression of two sensitive Candida albicans reference strains (ATCC 10231 and ATCC 60133) and two of their paired resistant to fluconazole and itraconazole mutants. Resistant mutants Candida albicans FLC-R, resistant to fluconazole (MIC > 128 μg/ml) and Candida albicans ICZ-R, resistant to itraconazole (MIC > 4 μg/ml) were obtained in subcultures with gradual increase of the antifungal in the culture medium. cDNA-AFLP profile in both itraconazole resistant mutants showed specific spectrophotometric peaks with 5–6-fold RNA overexpression product of 500 bp length compared to the sensitive strains. Fluconazole mutants do not reveal RNA level changes under tested by us typing conditions. These results indicate that the cDNA-FAFLP strategy is a relatively rapid, simple, and reliable method for simultaneous typing of both constitutive and induced differences in expression of host genes providing insight into the biological processes involved in response to drugs in bacteria and fungi. Moreover, this methodology could be tested for typing of the genome response of any organism to physical or chemical stress factors.