Diverged and Active Partitiviruses in Lichen.

The lichen is a microbial consortium that mainly consists of fungi and either algae (Viridiplantae) or cyanobacteria. This structure also contains other bacteria, fungi, and viruses. However, RNA virus diversity associated with lichens is still unknown. Here, we analyzed RNA virus diversity in a lichen dominated by fungi and algae using dsRNA-seq technology and revealed that partitiviruses were dominant and active in the microbial consortium. The Partitiviridae sequences found in this study were classified into two genera, which have both plant- and fungi-infecting partitiviruses. This observation suggests that the lichen provides an opportunity for horizontal transfer of these partitiviruses among microbes that form the lichen consortium.

The acquired radioresistance in HeLa cells under conditions mimicking hypoxia was attenuated by a decreased expression of HIF subunit genes induced by RNA interference.

The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment.

Utilization of microRNAs with decreased expression levels in response to X-ray irradiation for fine-tuning radiation-controlled gene regulation.

We previously developed a promoter that was responsive to radiation by randomly combining cis-elements of transcription factors that are activated in response to radiation in prostate cancer cells. The promoter enhanced the expression of the luciferase gene linked downstream by more than 10-fold 12 h after X-ray irradiation at 10 Gy. However, without radiation, it still significantly drove its expression. To suppress expression while retaining its enhancement in response to radiation, we focused our attention on microRNAs (miRNAs). miRNAs are a group of non-coding RNAs approximately 22 nucleotides long that control gene expression by binding to a target sequence residing on the 3'-untranslated region (3'UTR) of a target gene. We identified 8 miRNAs that were downregulated in response to X-ray irradiation, and inserted artificial target sequences composed of randomly combined complementary sequences into 3 representative miRNAs into the 3'UTR of the luciferase gene. The target sequences suppressed the expression, and released the expression, after X-ray irradiation, as expected. When we combined an artificial target sequence with the radiation-responsive promoter, it resulted in a clear-cut gene regulation of expression that was greater than that induced by the promoter alone.

Regulation of gene expression in human prostate cancer cells with artificially constructed promoters that are activated in response to ultrasound stimulation.

We chose promoters responsive to sonication in LNCap cells, a prostate cancer cell line, out of a library composed of DNA fragments constructed by linking the TATA box sequence to randomly combined cis-acting elements of transcription factors activated in response to radiation in prostate cancer cells. When a plasmid containing the luciferase gene under control of a promoter was transfected into LNCap cells and sonicated with 1 MHz ultrasound at 0.5 W/cm(2), 10% DF for 60s, 13 promoters showed more than 10-fold enhancement compared with their counterparts without sonication 12h after sonication. As to their responsiveness to sonication, the best two promoters were then compared to clone 880-8, a derivative from clone 880 that was created by random introduction of point mutations and was shown to have an improved response to X-ray irradiation. We then took clone 880-8 for further analyses since it showed the highest enhancement to sonication, though not statistically significant from the others. Next, we employed a retrovirus vector and stably introduced the luciferase gene under control of clone 880-8 into LNCap cells to establish a cell line. When the cell line was sonicated with 1 MHz ultrasound at 0.5 W/cm(2), 10% DF for 60s, luciferase expression was enhanced up to 14.8-fold 12h after sonication. We then established another cell line by replacing the luciferase gene with the fcy::fur gene, a suicide gene, and when the cell line was sonicated with 1 MHz ultrasound at 0.5 W/cm(2), 10% DF for 60s, expression of the gene was enhanced, showing the maximum expression 12-24h after sonication. When the cells were incubated in medium containing 5-fluorocytosine, cell survival ratio decreased dose dependently with 5-fluorocytosine only after sonication treatment, suggesting this promoter could be utilized for gene expression control with ultrasound.

Functional expression of a plant hydroxynitrile lyase in Escherichia coli by directed evolution: creation and characterization of highly in vivo soluble mutants.

Low protein solubility of recombinantly expressed proteins in Escherichia coli is a major factor hindering their application and analysis. We generated highly in vivo soluble mutants of a hydroxynitrile lyase in E.coli using protein engineering. Structure-guided saturation mutagenesis caused high solubility of single Lys-Pro mutations at positions 176, 199 and 224 of this low soluble wild-type enzyme. The triple Lys-Pro mutant generated at these surface conserved residues showed up to 8-fold increase in specific activity in the cell-free extract. Random mutagenesis also created a mutant of His103Met with 18.5-fold increase. The main expression form was reversed from insoluble to the soluble fraction following both types of above-mentioned mutations in E.coli at 37°C. The findings challenge the rationale of producing recombinant proteins in this host at 37°C. Formerly wild type low soluble protein was then present as soluble protein by these mutations, which also elevated the total soluble protein fraction in E.coli. Saturation mutagenesis of His103 provided other highly soluble mutants with hydrophobic substitutions. These mutations caused only minor secondary structural changes as determined by circular dichroism and Fourier-transform infrared spectroscopy and affected catalytic efficiency slightly for the purified mutants (0.82-1.6-fold for benzaldehyde and 0.9-1.9-fold for mandelonitrile). The stability of the mutants was differed from that of the wild type at high temperatures and at pH >8. Exchanging the buried basic-polar residue His103 with hydrophobic amino acids is in line with the overall structure of the enzyme, i.e. having hydrophilic residues in solvent-exposed areas and hydrophobic residues in the core.

Screening for new hydroxynitrilases from plants.

We established a simple HPLC method to determine the activity and stereochemistry of the chiral mandelonitrile synthesized from benzaldehyde and cyanide, and applied it to screen for hydroxynitrile lyase (HNL) activity of plant origin. A total of 163 species of plants among 74 families were examined for (R)- and (S)-HNL activities using the method. We discovered that homogenate of leaves of Baliospermum montanum shows (S)-HNL activity, while leaves and seeds from Passiflora edulis, and seeds from Eriobotrya japonica, Chaenomles sinensis, Sorbus aucuparia, Prunus mume, and Prunus persica show (R)-HNL activity. Partially purified (R)-HNLs from Passiflora edulis and Eriobotrya japonica acted not only on benzaldehyde but also on aliphatic ketone. The enantiomeric excess of (R)-methylpropylketone cyanohydrin synthesized from 2-pentanone using homogenate from leaves of Passiflora edulis was 87.0%, and that of (R)-mandelonitrile synthesized by homogenate from seeds of Eriobotrya japonica was 85.0%.