T-track PCR fingerprinting for the rapid detection of genetic polymorphism

The diversity of DNA sequences can be analyzed by comparing randomly amplified polymorphic DNA, or restriction fragment length polymorphism fragments of DNA. Such analyses are dependent on the selection of appropriate restriction enzyme(s) and/or primers. We have investigated a simpler approach to providing sensitive and specific genotyping. Cyclic extension of target sequences with dideoxythymidine generates PCR products with variable lengths. We analyzed these variable PCR products by scoring the number of variable bands and comparing the scores (numerical profiles) to establish similarities. We found that the polymorphic lengths of the PCR products were comparable among serologically defined strains. It suggests that this single PCR reaction followed by a one-step electrophoresis yields easily analyzable data that can be compared with data from other gels.     

Protective effects of P2X7 receptor deletion on aging-related memory alterations

Neuroinflammation is one of the factors implicated in cognitive aging. Recent studies have shown that the P2X7 receptor (P2X7R) is a major inflammatory regulator in microglia. The present study examined the effects of deletion of P2X7R on cognitive aging. Cognitive status was examined in 5-month-old (young) and 11- to 14-month-old (middle-aged) mice with P2X7R deletion (P2X7R knockout (KO)), and in age-matched control wild type mice, using the Morris water maze task. No differences were observed between the performances of young control mice and young P2X7R KO mice in the Morris water maze task. However, middle-aged P2X7R KO mice demonstrated better performance than middle-aged control mice. No differences were observed in hippocampal interleukin-1β levels and the number of microglial cells in the cornus ammonis (CA1) region of the hippocampus during microglial activation in young and middle-aged P2X7R KO mice, and control mice. Hippocampal levels of the inhibitory form of glycogen synthase kinase-3β were lower in young control mice than in mice in the other groups. These results suggest that the inhibition of P2X7R might have beneficial effects on aging-related cognitive impairments.     

Localization and function of three monothiol glutaredoxins in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe contains two dithiol glutaredoxins (Grx1 and Grx2) and genes for three putative monothiol glutaredoxins (grx3, 4, and 5). We investigated the expression, sub-cellular localization, and functions of the three monothiol glutaredoxins. Fluorescence microscopy revealed that Grx3 is targeted to nuclear rim and endoplasmic reticulum, Grx4 primarily to the nucleus, and Grx5 to mitochondria. Null mutation of grx3 did not significantly affect growth and resistance against various oxidants, whereas grx5 mutation caused slow growth and sensitivity toward oxidants such as hydrogen peroxide, paraquat, and diamide. The grx2grx5 double mutation, deficient in all mitochondrial glutaredoxins, caused further retardation in growth and severe sensitivity toward all the oxidants tested. The grx4 mutation was not viable, suggesting a critical role of Grx4 for the physiology of S. pombe. Overproduction of Grx3 and Grx5, but not the truncated form of Grx5 without mitochondrial target sequence, severely retarded growth as Grx2 did, supporting the idea that Grx2, 3, and 5 are targeted to organellar compartments. Our results propose a distinct role for each glutaredoxin to maintain thiol redox balance, and hence the growth and stress resistance, of the fission yeast.     

Genetic Analysis of Borrelia burgdorferi Sensu Lato in Korea Using Genomic Hybridization and 16S rRNA Gene Sequence Determination

Nine Borrelia burgdorferi sensu lato isolated in Korea were subjected to genomic hybridization using 16S rRNA gene probe and specific restriction patterns (HindIII and EcoRV) led these nine Borrelia into five subtypes. The evolutionary relationships of the five isolates corresponding to five RFLP groups were measured through the sequence determination of 16S rRNA gene and phylogenetic analysis. The isolates 935T (group I), 934U and 17Y (Group IIa, IIb) were well clustered with B. garinii and B. afzelii. 5MT and 9MT strains (Group IIIa and Group IIIb) formed a common branch shared with B. afzelii cluster although the evolutionary distance was rather long. So, most of B. burgdorferi sensu lato in Korea was B. afzelii or B. afzelii-related group and some minor group such as B. garinii also existed.     

Functional interplay between the oxidative stress response and DNA damage checkpoint signaling for genome maintenance in aerobic organisms

The DNA damage checkpoint signaling pathway is a highly conserved surveillance mechanism that ensures genome integrity by sequential activation of protein kinase cascades. In mammals, the main pathway is orchestrated by two central sensor kinases, ATM and ATR, that are activated in response to DNA damage and DNA replication stress. Patients lacking functional ATM or ATR suffer from ataxia-telangiectasia (A-T) or Seckel syndrome, respectively, with pleiotropic degenerative phenotypes. In addition to DNA strand breaks, ATM and ATR also respond to oxidative DNA damage and reactive oxygen species (ROS), suggesting an unconventional function as regulators of intracellular redox status. Here, we summarize the multiple roles of ATM and ATR, and of their orthologs in Saccharomyces cerevisiae, Tel1 and Mec1, in DNA damage checkpoint signaling and the oxidative stress response, and discuss emerging ideas regarding the possible mechanisms underlying the elaborate crosstalk between those pathways. This review may provide new insights into the integrated cellular strategies responsible for maintaining genome stability in eukaryotes with a focus on the yeast model organism.     

Direct DNA binding activity of the Fanconi anemia D2 protein

It is known that the Fanconi anemia D2 protein is vital for protecting the genome from DNA damage, but what activities this protein has are unknown. In these experiments we purified full-length Fanconi anemia protein D2 (FANCD2), and we found that FANCD2 bound to DNA with specificity for certain structures: double strand DNA ends and Holliday junctions. Proteins containing patient-derived mutations or artificial variants of the FANCD2 protein were similarly expressed and purified, and each variant bound to the Holliday junction DNA with similar affinity as did the wild-type protein. There was no single discrete domain of FANCD2 protein that bound to DNA, but rather the full-length protein was required for structure-specific DNA binding. This finding of DNA binding is the first biochemical activity identified for this key protein in the Fanconi anemia pathway.