No abstract

The strain-promoted "double-click" (SPDC) reaction using Sondheimer diyne, a novel convergent method conjugating three molecules spontaneously, has enabled us to readily modify an azido-biomolecule with a small reporter azido-molecule.

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The female flower of hop (Humulus lupulus var. lupulus) is an essential ingredient that gives characteristic aroma, bitterness and durability/stability to beer. However, the molecular genetic basis for identifying DNA markers in hop for breeding and to study its domestication has been poorly established. Here, we provide draft genomes for two hop cultivars [cv. Saazer (SZ) and cv. Shinshu Wase (SW)] and a Japanese wild hop [H. lupulus var. cordifolius; also known as Karahanasou (KR)]. Sequencing and de novo assembly of genomic DNA from heterozygous SW plants generated scaffolds with a total size of 2.05 Gb, corresponding to approximately 80% of the estimated genome size of hop (2.57 Gb). The scaffolds contained 41,228 putative protein-encoding genes. The genome sequences for SZ and KR were constructed by aligning their short sequence reads to the SW reference genome and then replacing the nucleotides at single nucleotide polymorphism (SNP) sites. De novo RNA sequencing (RNA-Seq) analysis of SW revealed the developmental regulation of genes involved in specialized metabolic processes that impact taste and flavor in beer. Application of a novel bioinformatics tool, phylogenetic comparative RNA-Seq (PCP-Seq), which is based on read depth of genomic DNAs and RNAs, enabled the identification of genes related to the biosynthesis of aromas and flavors that are enriched in SW compared to KR. Our results not only suggest the significance of historical human selection process for enhancing aroma and bitterness biosyntheses in hop cultivars, but also serve as crucial information for breeding varieties with high quality and yield.

Tissue-specific gene expression is tightly regulated by various elements such as promoters, enhancers, and long noncoding RNAs (lncRNAs). In the present study, we identified a conserved noncoding sequence (CNS1) as a novel enhancer for the spermatocyte-specific mouse testicular cell adhesion molecule 1 (Tcam1) gene. CNS1 was located 3.4 kb upstream of the Tcam1 gene and associated with histone H3K4 mono-methylation in testicular germ cells. By the in vitro reporter gene assay, CNS1could enhance Tcam1 promoter activity only in GC-2spd(ts) cells, which were derived from mouse spermatocytes. When we integrated the 6.9-kb 5'-flanking sequence of Tcam1 with or without a deletion of CNS1 linked to the enhanced green fluorescent protein gene into the chromatin of GC-2spd(ts) cells, CNS1 significantly enhanced Tcam1 promoter activity. These results indicate that CNS1 could function as a spermatocyte-specific enhancer. Interestingly, CNS1 also showed high bidirectional promoter activity in the reporter assay, and consistent with this, the Smarcd2 gene and lncRNA, designated lncRNA-Tcam1, were transcribed from adjacent regions of CNS1. While Smarcd2 was ubiquitously expressed, lncRNA-Tcam1 expression was restricted to testicular germ cells, although this lncRNA did not participate in Tcam1 activation. Ubiquitous Smarcd2 expression was correlated to CpG hypo-methylation of CNS1 and partially controlled by Sp1. However, for lncRNA-Tcam1 transcription, the strong association with histone acetylation and histone H3K4 tri-methylation also appeared to be required. The present data suggest that CNS1 is a spermatocyte-specific enhancer for the Tcam1 gene and a bidirectional promoter of Smarcd2 and lncRNA-Tcam1.

Steric character is one of the most fundamental factors to determine the reactivity of the substrate in organic synthesis. In bimolecular reaction, the sterically-bulky group situated close to the reactive center generally prevents the approach of the reaction partner retarding the bond formation. This report describes, to the contrary, significantly enhanced reactivity of 2,6-disubstituted phenyl azides observed in catalyst-free 1,3-dipolar cycloaddition with alkynes, unexpectedly reacting faster than unsubstituted phenyl azide and even more faster than unhindered alkyl azide, despite the steric hindrance adjacent to the reactive azido group. Experimental and computational studies have indicated that the steric hindrance eliciting the inhibition of resonance between azido group and the aromatic ring is the primary cause of this apparently-paradoxical phenomenon. This is the first type of steric acceleration, indicating a possibility of designing a highly reactive functional group by strategically locating it in the sterically-congested environment.

(+)-Sesamin, (+)-sesamolin, and (+)-sesaminol glucosides are phenylpropanoid-derived specialized metabolites called lignans, and are rich in sesame (Sesamum indicum) seed. Despite their renowned anti-oxidative and health-promoting properties, the biosynthesis of (+)-sesamolin and (+)-sesaminol remained largely elusive. Here we show that (+)-sesamolin deficiency in sesame is genetically associated with the deletion of four C-terminal amino acids (Del4C) in a P450 enzyme CYP92B14 that constitutes a novel clade separate from sesamin synthase CYP81Q1. Recombinant CYP92B14 converts (+)-sesamin to (+)-sesamolin and, unexpectedly, (+)-sesaminol through an oxygenation scheme designated as oxidative rearrangement of α-oxy-substituted aryl groups (ORA). Intriguingly, CYP92B14 also generates (+)-sesaminol through direct oxygenation of the aromatic ring. The activity of CYP92B14 is enhanced when co-expressed with CYP81Q1, implying functional coordination of CYP81Q1 with CYP92B14. The discovery of CYP92B14 not only uncovers the last steps in sesame lignan biosynthesis but highlights the remarkable catalytic plasticity of P450s that contributes to metabolic diversity in nature.

The development of selective and multitargeted kinase inhibitors has received much attention, because cross-reactivity with unintended targets may cause toxic side effects, while it can also give rise to efficacious multitargeted drugs. Here we describe a deconvolution approach to dissecting kinase profiling data in order to gain knowledge about cross-reactivity of inhibitors from large-scale profiling data. This approach not only enables activity predictions of given compounds on a kinome-wide scale, but also allows to extract residue-fragment pairs that are associated with activity. We demonstrate its effectiveness using a large-scale public chemogenomics data set and also apply our proposed model to a recently published bioactivity data set. We further illustrate that the preference of given compounds for kinases of interest is better understood by residue-fragment pairs, which could provide both biological and chemical insights into cross-reactivity.