Targeted DNA damage at individual telomeres disrupts their integrity and triggers cell death

Cellular DNA is organized into chromosomes and capped by a unique nucleoprotein structure, the telomere. Both oxidative stress and telomere shortening/dysfunction cause aging-related degenerative pathologies and increase cancer risk. However, a direct connection between oxidative damage to telomeric DNA, comprising <1% of the genome, and telomere dysfunction has not been established. By fusing the KillerRed chromophore with the telomere repeat binding factor 1, TRF1, we developed a novel approach to generate localized damage to telomere DNA and to monitor the real time damage response at the single telomere level. We found that DNA damage at long telomeres in U2OS cells is not repaired efficiently compared to DNA damage in non-telomeric regions of the same length in heterochromatin. Telomeric DNA damage shortens the average length of telomeres and leads to cell senescence in HeLa cells and cell death in HeLa, U2OS and IMR90 cells, when DNA damage at non-telomeric regions is undetectable. Telomere-specific damage induces chromosomal aberrations, including chromatid telomere loss and telomere associations, distinct from the damage induced by ionizing irradiation. Taken together, our results demonstrate that oxidative damage induces telomere dysfunction and underline the importance of maintaining telomere integrity upon oxidative damage.     

Genetic and thermal effects on the latitudinal variation in the timing of fin development of a fish Oryzias latipes

Heterochrony, an evolutionary change in developmental processes, is one of the major proximate causes of morphological diversity of organisms. It has been reported in the medaka Oryzias latipes that higher-latitude larvae have a genetic tendency to complete fin ray formation at larger body sizes, which results in relatively shorter anal and dorsal fins in adults. However, this latitudinal, heterochronic variation in fin length in the wild may be partially explained by latitudinal differences in thermal environments, if temperatures affect the timing of fin ray formation. Common-environment experiments revealed that the body size at which fin pterygiophore (a basal skeleton of fin rays) formation was completed was larger in higher-latitude larvae than in lower-latitude larvae at all temperatures examined, supporting the proposal that fin ray formation of the former is genetically delayed. However, phenotypic plasticity in response to temperature was also evident; lower temperatures caused delayed fin ray formation until a larger body size had been achieved in both high- and low-latitude larvae. These observations suggest that habitat temperatures also contribute to the latitudinal difference in the timing of fin development, magnifying phenotypic variation in fin length across latitudes. We discuss reasons for this positive covariance between genetic and environmental effects on the latitudinal, heterochronic variation, from the viewpoint of local adaptation and evolution of phenotypic plasticity.     

Preparation of recombinant rat eosinophil-associated ribonuclease-1 and -2 and analysis of their biological activities

Rat eosinophils contain eosinophil-associated ribonucleases (Ears) in their granules. Ears are thought to be synthesized as pre-forms and stored in the granules as mature forms. However, the N-terminal amino acid of mature Ear-1 and Ear-2 is still controversial. Therefore, we prepared two recombinant mature forms of Ear-1 and Ear-2 in which the N-terminal amino acids are Ser24 (S) [Ear-1 (S) and Ear-2 (S)] and Gln26 (Q) [Ear-1 (Q) and Ear-2 (Q)], and analyzed their biological activities by comparing them with those of pre-form Ear-1 and pre-form Ear-2. The four mature Ears showed RNase A activity as well as bovine pancreatic RNase A activity, but pre-Ear-1 and pre-Ear-2 showed no RNase A activity. Mature Ear-1 (Q) and mature Ear-2 (Q) showed more potent RNase A activity than mature Ear-1 (S) and mature Ear-2 (S), respectively. The RNase A activities of mature Ear-1 (Q) and mature Ear-2 (Q) were reduced by treatment at 96 degrees C for 20 min or with RNase inhibitor. The growth of Escherichia coli was inhibited by both pre-Ears and mature Ears in a concentration-dependent manner, and was almost completely suppressed at 1.0 microM. The bactericidal activities of mature Ear-1 (Q) and mature Ear-2 (Q) were not inhibited by RNase inhibitor, but was increased by treatment at 96 degrees C for 20 min.     

RBM10 regulates alternative splicing

RBM10, originally called S1-1, is a nuclear RNA-binding protein with domains characteristic of RNA processing proteins. It has been reported that RBM10 constitutes spliceosome complexes and that RBM5, a close homologue of RBM10, regulates alternative splicing of apoptosis-related genes, Fas and cFLIP. In this study, we examined whether RBM10 has a regulatory function in splicing similar to RBM5, and determined that it indeed regulates alternative splicing of Fas and Bcl-x genes. RBM10 promotes exon skipping of Fas pre-mRNA as well as selection of an internal 5′-splice site in Bcl-x pre-mRNA. We propose a consensus RBM10-binding sequence at 5′-splice sites of target exons and a mechanistic model of RBM10 action in the alternative splicing.     

The molecular basis for genetic polymorphism of human deoxyribonuclease II (DNase II): a single nucleotide substitution in the promoter region of human DNase II changes the promoter activity

We report that, contrary to common belief, polypeptides fused to the carboxy-terminus of the M13 gene-3 minor coat protein are functionally displayed on the phage surface. In a phagemid display system, carboxy-terminal fusion through optimized linker sequences resulted in display levels comparable to those achieved with conventional amino-terminal fusions. These findings are of considerable importance to phage display technology because they enable investigations not suited to amino-terminal display, including the study of protein–protein interactions requiring free carboxy-termini, functional cDNA cloning efforts, and the display of intracellular proteins.