Development of a 60-mer oligonucleotide microarray on the basis of benzene monooxygenase gene diversity

We constructed a 60-mer oligonucleotide microarray on the basis of benzene monooxygenase gene diversity to develop a new technology for simultaneous detection of the functional gene diversity in environmental samples. The diversity of the monooxygenase genes associated with benzene degradation was characterized. A new polymerase chain reaction (PCR) primer set was designed using conserved regions of benzene monooxygenase gene (BO12 primer) and used for PCR-clone library analysis along with a previously designed RDEG primer which targeted the different types of benzene monooxygenase gene. We obtained 20 types of amino acid sequences with the BO12 primer and 40 with the RDEG primer. Phylogenetic analysis of the sequences obtained suggested the large diversity of the benzene monooxygenase genes. A total of 87 60-mer probes specific for each operational taxonomical unit were designed and spotted on a microarray. When genomic DNAs of single strains were used in microarray hybridization assays, corresponding sequences were successfully detected by the microarray without any false-negative signals. Hybridization with soil DNA samples showed that the microarray was able to detect sequences that were not detected in clone libraries. Constructed microarray can be a useful tool for characterizing monooxygenase gene diversity in benzene degradation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Orchidantha inouei (Lowiaceae), a new species from Borneo

A new species, Orchidantha inouei , is described from Sarawak. Two types of paracladium in the inflorescence are reported with a discussion on the inflorescence morphology of Lowiaceae.     

Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device

The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.     

Ecological response of nitrification to oil spills and its impact on the nitrogen cycle

Marine oil spills are catastrophic events that cause massive damage to ecosystems at all trophic levels. While most of the research has focused on carbon-degrading microorganisms, the potential impacts of hydrocarbons on microbes responsible for nitrification have received far less attention. Nitrifiers are sensitive to hydrocarbon toxicity: ammonia-oxidizing bacteria and archaea being 100 and 1000 times more sensitive than typical heterotrophs respectively. Field studies have demonstrated the response of nitrifiers to hydrocarbons is highly variable and the loss of nitrification activity in coastal ecosystems can be restored within 1–2 years, which is much shorter than the typical recovery time of whole ecosystems (e.g., up to 20 years). Since the denitrification process is mainly driven by heterotrophs, which are more resistant to hydrocarbon toxicity than nitrifiers, the inhibition of nitrification may slow down the nitrogen turnover and increase ammonia availability, which supports the growth of oil-degrading heterotrophs and possibly various phototrophs. A better understanding of the ecological response of nitrification is paramount in predicting impacts of oil spills on the nitrogen cycle under oil spill conditions, and in improving current bioremediation practices.     

Detection of micro-RNA by a combination of nucleic acid sequence-based amplification and a novel chemiluminescent pyrophosphate assay

Micro-RNA has attracted much attention as a biomarker for disease progression and malignancy. A compact, simple, rapid, and highly sensitive method is required to perform simple genetic analyses, such as point-of-care testing (POCT), at the clinic or bedside. Nucleic acid sequence-based amplification (NASBA) is a specific amplification method for a single-stranded RNA fragment that is useful for the highly sensitive detection of miRNAs. In this work, we developed a novel miRNA analytical system for POCT by combining the NASBA and chemiluminescence methods. Because the NASBA reaction is conducted at a constant temperature (41°C) and detection by chemiluminescence reaction does not require a light source, these methods could be combined to amplify 100 ng/assay miRNA. This combined miRNA detection method could be useful for the future development of compact POCT systems.     

Balance between DNA‐binding affinity and specificity enables selective recognition of longer target sequences in vivo

Although genome‐editing enzymes such as TALEN and CRISPR/Cas9 are being widely used, they have an essential limitation in that their relatively high‐molecular weight makes them difficult to be delivered to cells. To develop a novel genome‐editing enzyme with a smaller molecular weight, we focused on the engrailed homeodomain (EHD). We designed and constructed proteins composed of two EHDs connected by a linker to increase sequence specificity. In bacterial one‐hybrid assays and electrophoresis mobility shift assay analyses, the created proteins exhibited good affinity for DNA sequences consisting of two tandemly aligned EHD target sequences. However, they also bound to individual EHD targets. To avoid binding to single target sites, we introduced amino acid mutations to reduce the protein–DNA affinity of each EHD monomer and successfully created a small protein with high specificity for tandem EHD target sequences.     

Erratum to: De novo sequencing and comparative analysis of expressed sequence tags from gynodioecious fig (Ficus carica L.) fruits: caprifig and common fig

Tree Genetics & Genomes - Negative correlation caused by competition among individuals and positive spatial correlation due to environmental heterogeneity may lead to biases in estimating...