Creation of a novel telomere-cutting endonuclease based on the EN domain of telomere-specific non-long terminal repeat retrotransposon, TRAS1

<p>Abstract</p> <p>Background</p> <p>The ends of chromosomes, termed telomeres consist of repetitive DNA. The telomeric sequences shorten with cell division and, when telomeres are critically abbreviated, cells stop proliferating. However, in cancer cells, by the expression of telomerase which elongates telomeres, the cells can continue proliferating. Many approaches for telomere shortening have been pursued in the past, but to our knowledge, cutting telomeres <it>in vivo </it>has not so far been demonstrated. In addition, there is lack of information on the cellular effects of telomere shortening in human cells.</p> <p>Results</p> <p>Here, we created novel chimeric endonucleases to cut telomeres by fusing the endonuclease domain (TRAS1EN) of the silkworm's telomere specific non-long terminal repeat retrotransposon TRAS1 to the human telomere-binding protein, TRF1. An <it>in vitro </it>assay demonstrated that the TRAS1EN-TRF1 chimeric endonucleases (T-EN and EN-T) cut the human (TTAGGG)_nrepeats specifically. The concentration of TRAS1EN-TRF1 chimeric endonucleases necessary for the cleavage of (TTAGGG)_nrepeats was about 40-fold lower than that of TRAS1EN alone. When TRAS1EN-TRF1 endonucleases were introduced into human U2OS cancer cells using adenovirus vectors, the enzymes localized at telomeres of nuclei, cleaved and shortened the telomeric DNA by double-strand breaks. When human U2OS and HFL-1 fibroblast cells were infected with EN-T recombinant adenovirus, their cellular proliferation was suppressed for about 2 weeks after infection. In contrast, the TRAS1EN mutant (H258A) chimeric endonuclease fused with TRF1 (ENmut-T) did not show the suppression effect. The EN-T recombinant adenovirus induced telomere shortening in U2OS cells, activated the p53-dependent pathway and caused the senescence associated cellular responses, while the ENmut-T construct did not show such effects.</p> <p>Conclusions</p> <p>A novel TRAS1EN-TRF1 chimeric endonuclease (EN-T) cuts the human telomeric repeats (TTAGGG)_nspecifically <it>in vitro </it>and <it>in vivo</it>. Thus, the chimeric endonuclease which is expressed from an adenoviral vector can suppress cell proliferation of cancer cells.</p

Molecular cloning of haploid germ cell-specific tektin cDNA and analysis of the protein in mouse testis

AbstractTektins are a class of proteins that form filamentous polymers in the walls of ciliary and flagellar microtubules. We report here the molecular cloning of a new member of the tektin family, tektin-t, identified from a mouse haploid germ cell-specific cDNA library. Tektin-t mRNA encodes a protein of 430 deduced amino acids possessing RSNVELCRD, the conserved sequence of tektin family proteins. Western blotting showed a single band having a molecular weight of 86 kDa in the mouse testis. Immunohistochemistry of the testis showed that tektin-t is localized in the flagella of elongating spermatids from developmental step 15 to maturity

Temperature-dependent magnetoresistance effects in FeSi/FeSi/FeSi trilayered spin valve junctions

Fe3Si/FeSi2/Fe3Si trilayered junctions were fabricated by facing targets direct-current sputtering combined with a mask method, and the spin valve signals of the junctions were studied in the temperature range from 50 to 300 K. Whereas the magnetoresistance ratio of giant magnetoresistance and tunnel magnetoresistance junctions monotonically increases with decreasing temperature, that of our samples has the maximum value around 80 K and decreases with decreasing temperature at lower than 80 K, which might be due to an increase in the electrical conductivity mismatch between the metallic Fe3Si layers and semiconducting FeSi2 interlayer in the low temperature range.Asia-Pacific Conference on Semiconducting Silicides and Related Materials — Science and Technology Towards Sustainable Electronics (APAC Silicide 2016), July 16-18, 2016, Fukuoka, Japa

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

Transcriptional landscape of small non-coding RNAs reveals diversity of categories and functions in molluscs

ABSTRACTSmall non-coding RNAs (sncRNAs) are non-coding RNA molecules that play various roles in metazoans. Among the sncRNAs, microRNAs (miRNAs) guide post-translational gene regulation during cellular development, proliferation, apoptosis, and differentiation, while PIWI-interacting RNAs (piRNAs) suppress transposon activity to safeguard the genome from detrimental insertion mutagenesis. While an increasing number of piRNAs are being identified in the soma and germlines of various organisms, they are scarcely reported in molluscs. To unravel the small RNA (sRNA) expression patterns and genomic function in molluscs, we generated a comprehensive sRNA dataset by sRNA sequencing (sRNA-seq) of eight mollusc species. Abundant miRNAs were identified and characterized in all investigated molluscs, and ubiquitous piRNAs were discovered in both somatic and gonadal tissues in six of the investigated molluscs, which are more closely associated with transposon silencing. Tens of piRNA clusters were also identified based on the genomic mapping results, which varied among different tissues and species. Our dataset serves as important reference data for future genomic and genetic studies on sRNAs in these molluscs and related species, especially in elucidating the ancestral state of piRNAs in bilaterians

Use of stepwise subtraction to comprehensively isolate mouse genes whose transcription is up-regulated during spermiogenesis

We report the isolation of 153 mouse genes whose expression is dramatically up-regulated during spermiogenesis. We used a novel variation of the subtractive hybridization technique called stepwise subtraction, wherein the subtraction process is systematically repeated in a stepwise manner. We named the genes thus identified as TISP genes (transcript induced in spermiogenesis). The transcription of 80 of these TISP genes is almost completely specific to the testis. This transcription is abruptly turned on after 17 days of age, when the mice enter puberty and spermiogenesis is initiated. Considering that the most advanced cells present at these stages of spermatogenesis are the spermatids, it is likely that we could isolate most of the spermatid-specific genes. DNA sequencing revealed that about half the TISP genes are novel and uncharacterized genes, confirming the utility of the stepwise subtraction approach for gene discovery

Transcriptional landscape of small non-coding RNAs reveals diversity of categories and functions in molluscs

Small non-coding RNAs (sncRNAs) are non-coding RNA molecules that play various roles in metazoans. Among the sncRNAs, microRNAs (miRNAs) guide post-translational gene regulation during cellular development, proliferation, apoptosis, and differentiation, while PIWI-interacting RNAs (piRNAs) suppress transposon activity to safeguard the genome from detrimental insertion mutagenesis. While an increasing number of piRNAs are being identified in the soma and germlines of various organisms, they are scarcely reported in molluscs. To unravel the small RNA (sRNA) expression patterns and genomic function in molluscs, we generated a comprehensive sRNA dataset by sRNA sequencing (sRNA-seq) of eight mollusc species. Abundant miRNAs were identified and characterized in all investigated molluscs, and ubiquitous piRNAs were discovered in both somatic and gonadal tissues in six of the investigated molluscs, which are more closely associated with transposon silencing. Tens of piRNA clusters were also identified based on the genomic mapping results, which varied among different tissues and species. Our dataset serves as important reference data for future genomic and genetic studies on sRNAs in these molluscs and related species, especially in elucidating the ancestral state of piRNAs in bilaterians.</p

Transcriptome analysis of Edwardsiella piscicida during intracellular infection reveals excludons are involved with the activation of a mitochondrion-like energy generation program

ABSTRACTPhylogenetic evidence suggests a shared ancestry between mitochondria and modern Proteobacteria, a phylum including several genera of intracellular pathogens. Studying these diverse pathogens, particularly during intracellular infection of their hosts, can reveal characteristics potentially representative of the mitochondrial-Proteobacterial ancestor by identifying traits shared with mitochondria. While transcriptomic approaches can provide global insights into intracellular acclimatization by pathogens, they are often limited by excess host RNAs in extracts. Here, we developed a method employing magnetic nanoparticles to enrich RNA from an intracellular Gammaproteobacterium, Edwardsiella piscicida, within zebrafish, Danio rerio, fin fibroblasts, enabling comprehensive exploration of the bacterial transcriptome. Our findings revealed that the intracellular E. piscicida transcriptome reflects a mitochondrion-like energy generation program characterized by the suppression of glycolysis and sugar transport, coupled with upregulation of the tricarboxylic acid (TCA) cycle and alternative import of simple organic acids that directly flux into TCA cycle intermediates or electron transport chain donors. Additionally, genes predicted to be members of excludons, loci of gene pairs antagonistically co-regulated by overlapping antisense transcription, are significantly enriched in the set of all genes with perturbed sense and antisense transcription, suggesting a general but important involvement of excludons with intracellular acclimatization. Notably, genes involved with the activation of the mitochondrion-like energy generation program, specifically with metabolite import and glycolysis, are also members of predicted excludons. Other intracellular Proteobacterial pathogens appear to employ a similar mitochondrion-like energy generation program, suggesting a potentially conserved mechanism for optimized energy acquisition from hosts centered around the TCA cycle.IMPORTANCEPhylogenetic evidence suggests that mitochondria and Proteobacteria, a phylum encompassing various intracellular pathogens, share a common ancestral lineage. In this study, we developed a novel method employing magnetic nanoparticles to explore the transcriptome of an aquatic Gammaproteobacterium, Edwardsiella piscicida, during intracellular infection of host cells. We show that the strategy E. piscicida uses to generate energy strikingly mirrors the function of mitochondria—energy generators devoid of glycolytic processes. Notably, several implicated genes are members of excludons—gene pairs antagonistically co-regulated by overlapping antisense transcription. Other intracellular Proteobacterial pathogens appear to adopt a similar mitochondrion-like energy generation program, indicating a possibly conserved strategy for optimized energy acquisition from hosts centered around the tricarboxylic acid cycle