SummarySmall RNAs are used to silence transposable elements (TEs) in many eukaryotes, which use diverse evolutionary solutions to identify TEs. In ciliated protozoans, small-RNA-mediated comparison of the germline and somatic genomes underlies identification of TE-related sequences, which are then eliminated from the soma. Here, we describe an additional mechanism of small-RNA-mediated identification of TE-related sequences in the ciliate Tetrahymena. We show that a limited set of internal eliminated sequences (IESs) containing potentially active TEs produces a class of small RNAs that recognize not only the IESs from which they are derived, but also other IESs in trans. This trans recognition triggers the expression of yet another class of small RNAs that identify other IESs. Therefore, TE-related sequences in Tetrahymena are robustly targeted for elimination by a genome-wide trans-recognition network accompanied by a chain reaction of small RNA production.

Tetrahymena is a useful eukaryotic model for biochemistry and molecular cell biology studies. We previously demonstrated that targeted ectopic DNA elimination, also called co-Deletion (coDel), can be induced by the introduction of an internal eliminated sequence (IES)-target DNA chimeric construct. In this study, we demonstrate that coDel occurs at most of the loci tested and can be used for the production of somatic gene KO strains. We also showed that coDel at two loci can be simultaneously induced by a single transformation; thus, coDel can be used to disrupt multiple gene loci in a single cell. Therefore, coDel is a useful tool for functional genetics in Tetrahymena and further extends the usefulness of this model organism.KEYWORDS Tetrahymena; DNA elimination; RNAi; gene knockout T HE ciliated protozoan Tetrahymena thermophila can grow at an exceptionally high rate (its doubling time is approximately 2 hr) and can reach a high density (a few million cells per milliliter) under simple and inexpensive culture conditions (reviewed in Orias et al. 2000). In combination with robust genetic manipulation methods (reviewed in Chalker 2012), Tetrahymena is a useful model eukaryote for biochemistry and molecular cell biology studies (reviewed in Collins and Gorovsky 2005).Three strategies for loss-of-function genetic studies have been established for this organism. The first strategy is a germline knockout (KO), in which one of the two gene copies in the diploid micronucleus (MIC) is replaced with a drug-resistance gene by homologous recombination, and two heterozygous strains are then sexually crossed to obtain a homozygous germline KO strain (Cassidy-Hanley et al. 1997). The second strategy is somatic KO, in which one of the 45 copies of a gene in the polyploid macronucleus (MAC) is replaced with a drugresistance gene by homologous recombination, and "phenotypic assortment" is used to obtain cells with the drug-resistance gene at all loci (Merriam and Bruns 1988). Phenotypic assortment is possible because MAC chromosomes are randomly segregated into daughter cells in vegetative cell division and a stepwise increase of the drug in culture selects for cells that have more drug-resistance genes. The final strategy is gene knockdown (KD) by RNA interference (RNAi), in which a construct expressing long hairpin RNA that is complementary to the gene of interest is introduced into a nonessential locus in the MAC and small RNAs produced from the hairpin RNA posttranscriptionally silence the expression of the gene (HowardTill and Yao 2006).Although the first two gene KO strategies have been reliably used to study individual gene functions, they are not suitable for high-throughput genetic screening because the integration of a KO construct into the MIC germline occurs at very low efficiency and the phenotypic assortment process used in somatic KOs require the careful control of drug concentrations in each strain and culture step. Moreover, it takes 1 month to obtain KO strains using these methods. For germline KO, t...

We examined the relationship between the amino acid sequences of the V2 and V3 regions of the envelope protein and the biological properties of ten human immunodeficiency virus type 1 (HIV-1) primary isolates. The infectivity, cytopathic effect (CPE), and syncytium forming activity of these primary isolates were tested against three T cell lines (CEM, MT2, and MOLT4/CL.8 cells), CD8-depleted peripheral blood mononuclear cells (PBMC), and primary monocyte-derived macrophages (MDM) from seronegative donors. In addition to the viral groups which had the syncytium inducing/T-cell line tropic (SI/TT) phenotype or non-syncytium inducing/non-T cell line tropic (NSI/NT) phenotype (including the NSI/macrophage tropic (NSI/MT) phenotype), there was a group of viruses that infected one or two T cell lines and PBMC but could not mediate syncytium formation. We therefore classified this group of viruses as a non-syncytium inducing/partial T-cell line tropic (NSI/pTT) virus. To investigate the relationship between these viral phenotypes and the sequence variability of the V2 and V3 regions of the envelope, we cloned the viral gene segment and sequenced the individual isolates. The sequence data suggested that the SI/TT type changes in the V3 sequence alone mediate a partial T cell line tropism and mild cytopathic effect and that an isolate became more virulent (SI/TT phenotype) if there were additional changes in the V2 or other regions. On the other hand, sequence changes in the V2 region alone could not mediate phenotypic changes but some additional changes in the other variable regions (for example, V3) might be required for the phenotypic changes in combination with changes in V2. These findings also suggested that amino acid changes in both the V2 and V3 region are required for the development of virulent variants of HIV-1 that outgrow during advanced stages of the disease.Key words: Human immunodeficiency virus type 1 (HIV-1), Primary isolates, V2 and V3 sequences, Viral phenotype Genetic variability within the human immunodeficiency virus type 1 (HIV-1) genome is most pronounced in the env gene, in which nucleotide substitutions, duplications, and deletions produce extensive amino acid diversity within five hypervariable domains in the envelope glycoprotein. The hypervariable domains, designated V1 through V5, are interspersed with conserved regions along the gp120 molecule (20,22,33). Sequence variation in the env gene can affect the biological phenotype with respect to the replication rate in different cells, as well as cytotropism and the capacity to induce syncytia in vitro (4,5,8,13,15,23,24,26,35).A comparative analysis of a large set of V3 sequences derived from primary or culture-adapted isolates revealed that an acidic amino acid or alanine predominates at position 25 among the macrophage tropic isolates, whereas a basic or neutral amino acid at this site is associated with non-conservative basic amino acid substitutions mainly at positions 11, 24, and 32 and that both features correlate with the T c...

A pilot-scale sustainable hydrogen production system using reverse electrodialysis (RED) technology was launched. The system is based on direct conversion of salinity gradient energy (SGE) between seawater (SW) and sewage treated water (STW) to hydrogen production by water electrolysis. The hydrogen production rate was almost the same as the theoretical value. This indicates that the RED hydrogen production system can convert SGE between SW and STW to hydrogen energy at high current efficiency.