Explore the vast range of titles available.

The majority of base pairs in double-stranded DNA exist in the canonical Watson-Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various complexes of DNA with proteins and small molecules, which are key for biological function and mechanism. While detection of Hoogsteen base pairs in large DNA complexes and assemblies poses considerable challenges for traditional structural biology techniques, we show here that multidimensional dynamic nuclear polarization–enhanced solid-state NMR can serve as a unique spectroscopic tool for observing and distinguishing Watson-Crick and Hoogsteen base pairs in a broad range of DNA systems based on characteristic NMR chemical shifts and internuclear dipolar couplings. We illustrate this approach using a model 12-mer DNA duplex, free and in complex with the antibiotic echinomycin, which features two central adenine-thymine base pairs with Watson-Crick and Hoogsteen geometry, respectively, and subsequently extend it to the ∼200 kDa Widom 601 DNA nucleosome core particle.

Hfq‐binding small RNAs (sRNAs) in bacteria modulate the stability and translational efficiency of target mRNAs through limited base‐pairing interactions. While these sRNAs are known to regulate numerous mRNAs as part of stress responses, what distinguishes targets and non‐targets among the mRNAs predicted to base pair with Hfq‐binding sRNAs is poorly understood. Using the Hfq‐binding sRNA Spot 42 of Escherichia coli as a model, we found that predictions using only the three unstructured regions of Spot 42 substantially improved the identification of previously known and novel Spot 42 targets. Furthermore, increasing the extent of base‐pairing in single or multiple base‐pairing regions improved the strength of regulation, but only for the unstructured regions of Spot 42. We also found that non‐targets predicted to base pair with Spot 42 lacked an Hfq‐binding site, folded into a secondary structure that occluded the Spot 42 targeting site, or had overlapping Hfq‐binding and targeting sites. By modifying these features, we could impart Spot 42 regulation on non‐target mRNAs. Our results thus provide valuable insights into the requirements for target selection by sRNAs. Target recognition by bacterial small RNAs is not well understood. The accessibility of the targeted sequences, stability of the duplex, and the binding and positioning of the RNA chaperone Hfq on the mRNA are critical.

Gibberellins (GAs) are phytohormones that regulate many aspects of plant growth and development, including germination, elongation, flowering, and floral development. Negative feedback regulation contributes to homeostasis of the GA level. DELLAs are negative regulators of GA signaling and are rapidly degraded in the presence of GAs. DELLAs regulate many target genes, including AtGA20ox2 in Arabidopsis (Arabidopsis thaliana), encoding the GA-biosynthetic enzyme GA 20-oxidase. As DELLAs do not have an apparent DNA-binding motif, transcription factors that act in association with DELLA are necessary for regulating the target genes. Previous studies have identified GAI-ASSOCIATED FACTOR1 (GAF1) as such a DELLA interactor, with which DELLAs act as coactivators, and AtGA20ox2 was identified as a target gene of the DELLA-GAF1 complex. In this study, electrophoretic mobility shift and chromatin immunoprecipitation assays showed that four GAF1-binding sites exist in the AtGA20ox2 promoter. Using transgenic plants, we further evaluated the contribution of the DELLA-GAF1 complex to GA feedback regulation. Mutations in four GAF1-binding sites abolished the negative feedback of AtGA20ox2 in transgenic plants. Our results showed that GAF1-binding sites are necessary for GA feedback regulation of AtGA20ox2, suggesting that the DELLA-GAF1 complex is a main component of the GA feedback regulation of AtGA20ox2.

High aluminum (Al) tolerance of rice (Oryza sativa) is controlled by multiple tolerance genes, but the regulatory mechanisms underlying the differential expression of these genes are poorly understood. Here, we investigated the factors regulating the expression of OsFRDL4, a gene encoding a citrate efflux transporter involved in Al-induced citrate secretion from the roots. Analysis with chromosome segment substitution lines derived from cv Nipponbare (high OsFRDL4 expression) and cv Kasalath (low OsFRDL4 expression) revealed that the differential expression of OsFRDL4 is responsible for the quantitative trait locus for Al tolerance detected previously on chromosome 1. Comparison of the OsFRDL4 gene structure in cv Nipponbare and cv Kasalath showed that there was no difference in the position of the transcriptional start site, but a 1.2-kb insertion showing high similarity to the solo long terminal repeat of the retrotransposon was found in the promoter region of OsFRDL4 in cv Nipponbare. This insertion showed higher promoter activity and contained nine cis-acting elements for ALUMINUM RESISTANCE TRANSCRIPTION FACTOR1 (ART1). However, this insertion did not alter the spatial expression or cellular localization of OsFRDL4. Furthermore, this insertion was found in most japonica varieties but was largely absent from indica varieties or wild rice species. These results indicate that the 1.2-kb insertion in the OsFRDL4 promoter region in japonica subspecies is responsible for their higher expression level of OsFRDL4 due to the increased number of cis-acting elements of ART1. Our results also suggest that this insertion event happened at the initial stage of domestication of japonica subspecies.

Deep sequencing has enabled the investigation of a wide range of environmental microbial ecosystems, but the high memory requirements for de novo assembly of short-read shotgun sequencing data from these complex populations are an increasingly large practical barrier. Here we introduce a memory-efficient graph representation with which we can analyze the k -mer connectivity of metagenomic samples. The graph representation is based on a probabilistic data structure, a Bloom filter, that allows us to efficiently store assembly graphs in as little as 4 bits per k -mer, albeit inexactly. We show that this data structure accurately represents DNA assembly graphs in low memory. We apply this data structure to the problem of partitioning assembly graphs into components as a prelude to assembly, and show that this reduces the overall memory requirements for de novo assembly of metagenomes. On one soil metagenome assembly, this approach achieves a nearly 40-fold decrease in the maximum memory requirements for assembly. This probabilistic graph representation is a significant theoretical advance in storing assembly graphs and also yields immediate leverage on metagenomic assembly.

Flowering time of summer annual Arabidopsis thaliana accessions is largely determined by the timing of FLOWERING LOCUS T (FT) expression in the leaf vasculature. To understand the complex interplay between activating and repressive inputs controlling flowering through FT, cis-regulatory sequences of FT were identified in this study. A proximal and an ~5-kb upstream promoter region containing highly conserved sequence blocks were found to be essential for FT activation by CONSTANS (CO). Chromatin-associated protein complexes add another layer to FT regulation. In plants constitutively overexpressing CO, changes in chromatin status, such as a decrease in binding of LIKE HETEROCHROMATIN PROTEIN1 (LHP1) and increased acetylation of H3K9 and K14, were observed throughout the FT locus, although these changes appear to be a consequence of FT upregulation and not a prerequisite for activation. Binding of LHP1 was required to repress enhancer elements located between the CO-controlled regions. By contrast, the distal and proximal promoter sequences required for FT activation coincide with locally LHP1 and H3K27me3 depleted chromatin, indicating that chromatin status facilitates the accessibility of transcription factors to FT. Therefore, distant regulatory regions are required for FT transcription, reflecting the complexity of its control and differences in chromatin status delimit functionally important cis-regulatory regions.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that play vital regulatory roles in plant growth, development, and environmental stress responses. Cadmium (Cd) is a non-essential heavy metal that is highly toxic to living organisms. To date, a number of conserved and non-conserved miRNAs have been identified to be involved in response to Cd stress in some plant species. However, the miRNA-mediated gene regulatory networks responsive to Cd stress in radish (Raphanus sativus L.) remain largely unexplored. To dissect Cd-responsive miRNAs and their targets systematically at the global level, two small RNA libraries were constructed from Cd-treated and Cd-free roots of radish seedlings. Using Solexa sequencing technology, 93 conserved and 16 non-conserved miRNAs (representing 26 miRNA families) and 28 novel miRNAs (representing 22 miRNA families) were identified. In all, 15 known and eight novel miRNA families were significantly differently regulated under Cd stress. The expression patterns of a set of Cd-responsive miRNAs were validated by quantitative real-time PCR. Based on the radish mRNA transcriptome, 18 and 71 targets for novel and known miRNA families, respectively, were identified by the degradome sequencing approach. Furthermore, a few target transcripts including phytochelatin synthase 1 (PCS1), iron transporter protein, and ABC transporter protein were involved in plant response to Cd stress. This study represents the first transcriptome-based analysis of miRNAs and their targets responsive to Cd stress in radish roots. These findings could provide valuable information for functional characterization of miRNAs and their targets in regulatory networks responsive to Cd stress in radish.

Although mutation rates are a key determinant of the rate of evolution they are difficult to measure precisely and global mutations rates (mutations per genome per generation) are often extrapolated from the per-base-pair mutation rate assuming that mutation rate is uniform across the genome. Using budding yeast, we describe an improved method for the accurate calculation of mutation rates based on the fluctuation assay. Our analysis suggests that the per-base-pair mutation rates at two genes differ significantly (3.80 × 10−10 at URA3 and 6.44 × 10−10 at CAN1) and we propose a definition for the effective target size of genes (the probability that a mutation inactivates the gene) that acknowledges that the mutation rate is nonuniform across the genome.

5-Amino-1-β-D-ribofuranosylimidazole-4-carboxamide 5′-monophosphate (ZMP) is a central intermediate in de novo purine nucleotide biosynthesis. Its nucleobase moiety, 5-aminoimidazole-4-carboxamide (Z-base), is considered an ambiguous base that can pair with any canonical base owing to the rotatable nature of its 5-carboxamide group. This idea of ambiguous base pairing due to free rotation of the carboxamide has been applied to designing mutagenic antiviral nucleosides, such as ribavirin and T-705. However, the ambiguous base-pairing ability of Z-base has not been elucidated, because the synthesis of Z-base-containing oligomers is problematic. Herein, we propose a practical method for the synthesis of Z-base-containing DNA oligomers based on the ring-opening reaction of an N1-dinitrophenylhypoxanthine (HxaDNP) base. Thermal denaturation studies of the resulting oligomers revealed that the Z-base behaves physiologically as an A-like nucleobase, preferentially forming pairs with T. We tested the behavior of Z-base-containing DNA oligomers in enzyme-catalyzed reactions: in single nucleotide insertion, Klenow fragment DNA polymerase recognized Z-base as an A-like analog and incorporated dTTP as a complementary nucleotide to Z-base in the DNA template; in PCR amplification, Taq DNA polymerase similarly incorporated dTTP as a complementary nucleotide to Z-base. Our findings will contribute to the development of new mutagenic antiviral nucleoside analogs.

Inflorescence architecture in small-grain cereals has a direct effect on yield and is an important selection target in breeding for yield improvement.We analyzed the recessivemutation laxatum-a (lax-a) in barley (Hordeum vulgare), which causes pleiotropic changes in spike development, resulting in (1) extended rachis internodes conferring a more relaxed inflorescence, (2) broadened base of the lemma awns, (3) thinner grains that are largely exposed due to reduced marginal growth of the palea and lemma, and (4) and homeotic conversion of lodicules into two stamenoid structures. Map-based cloning enforced by mapping-by-sequencing of the mutant lax-a locus enabled the identification of a homolog of BLADE-ON-PETIOLE1 (BOP1) and BOP2 as the causal gene. Interestingly, the recently identified barley uniculme4 gene also is a BOP1/2 homolog and has been shown to regulate tillering and leaf sheath development. While the Arabidopsis (Arabidopsis thaliana) BOP1 and BOP2 genes act redundantly, the barley genes contribute independent effects in specifying the developmental growth of vegetative and reproductive organs, respectively. Analysis of natural genetic diversity revealed strikingly different haplotype diversity for the two paralogous barley genes, likely affected by the respective genomic environments, since no indication for an active selection process was detected