Group II intron mobility occurs by target DNA-primed reverse transcription

AbstractMobile group II introns encode reverse transcriptases and insert site specifically into intronless alleles (homing). Here, in vitro experiments show that homing of the yeast mtDNA group II intron a12 occurs by reverse transcription at a double-strand break in the recipient DNA. A site-specific endonuclease cleaves the antisense strand of recipient DNA at position +10 of exon 3 and the sense strand at the intron insertion site. Reverse transcription of al2-containing pre-mRNA is primed by the antisense strand cleaved in exon 3 and results in cotransfer of the intron and flanking exon sequences. Remarkably, the DNA endonuclease that initiates homing requires both the a12 reverse transcriptase protein and a12 RNA. Parallels in their reverse transcription mechanisms raise the possibility that mobile group II introns were ancestors of nuclear non-long terminal repeat retrotransposons and telomerases

DGR mutagenic transposition occurs via hypermutagenic reverse transcription primed by nicked template RNA.

Diversity-generating retroelements (DGRs) are molecular evolution machines that facilitate microbial adaptation to environmental changes. Hypervariation occurs via a mutagenic retrotransposition process from a template repeat (TR) to a variable repeat (VR) that results in adenine-to-random nucleotide conversions. Here we show that reverse transcription of the Bordetella phage DGR is primed by an adenine residue in TR RNA and is dependent on the DGR-encoded reverse transcriptase (bRT) and accessory variability determinant (Avd ), but is VR-independent. We also find that the catalytic center of bRT plays an essential role in site-specific cleavage of TR RNA for cDNA priming. Adenine-specific mutagenesis occurs during reverse transcription and does not involve dUTP incorporation, indicating it results from bRT-catalyzed misincorporation of standard deoxyribonucleotides. In vivo assays show that this hybrid RNA-cDNA molecule is required for mutagenic transposition, revealing a unique mechanism of DNA hypervariation for microbial adaptation

A new topology of the HK97-like fold revealed in Bordetella bacteriophage by cryoEM at 3.5 A resolution.

Bacteriophage BPP-1 infects and kills Bordetella species that cause whooping cough. Its diversity-generating retroelement (DGR) provides a naturally occurring phage-display system, but engineering efforts are hampered without atomic structures. Here, we report a cryo electron microscopy structure of the BPP-1 head at 3.5 Å resolution. Our atomic model shows two of the three protein folds representing major viral lineages: jellyroll for its cement protein (CP) and HK97-like ('Johnson') for its major capsid protein (MCP). Strikingly, the fold topology of MCP is permuted non-circularly from the Johnson fold topology previously seen in viral and cellular proteins. We illustrate that the new topology is likely the only feasible alternative of the old topology. β-sheet augmentation and electrostatic interactions contribute to the formation of non-covalent chainmail in BPP-1, unlike covalent inter-protein linkages of the HK97 chainmail. Despite these complex interactions, the termini of both CP and MCP are ideally positioned for DGR-based phage-display engineering. DOI: http://dx.doi.org/10.7554/eLife.01299.001

Online sensor information and redundancy resolution based obstacle avoidance for high DOF mobile manipulator teleoperation

High degrees of freedom (DOF) mobile manipulators provide more flexibility than conventional manipulators. They also provide manipulation operations with a mobility capacity and have potential in many applications. However, due to high redundancy, planning and control become more complicated and difficult, especially when obstacles occur. Most existing obstacle avoidance methods are based on off-line algorithms and most of them mainly focus on planning a new collision-free path, which is not appropriate for some applications, such as teleoperation and uses many system resources as well. Therefore, this paper presents an online planning and control method for obstacle avoidance in mobile manipulators using online sensor information and redundancy resolution. An obstacle contour reconstruction approach employing a mobile manipulator equipped with an active laser scanner system is also introduced in this paper. This method is implemented using a mobile manipulator with a seven-DOF manipulator and a four-wheel drive mobile base. The experimental results demonstrate the effectiveness of this method. © 2013 Zhang et al.; licensee InTech.Link_to_subscribed_fulltex

Genome-wide analysis of soybean hypoxia inducible gene domain containing genes: a functional investigation of GmHIGD3

The response of Hypoxia Inducible Gene Domain (HIGD) proteins to hypoxia plays a crucial role in plant development. However, the research on this gene family in soybean has been lacking. In this study, we aimed to identify and comprehensively analyze soybean HIGD genes using the Glycine max genome database. As a result, six GmHIGD genes were successfully identified, and their phylogeny, gene structures, and putative conserved motifs were analyzed in comparison to Arabidopsis and rice. Collinearity analysis indicated that the HIGD gene family in soybean has expanded to some extent when compared to Arabidopsis. Additionally, the cis-elements in the promoter regions of GmHIGD and the transcription factors potentially binding to these regions were identified. All GmHIGD genes showed specific responsiveness to submergence and hypoxic stresses. Expression profiling through quantitative real-time PCR revealed that these genes were significantly induced by PEG treatment in root tissue. Co-expressed genes of GmHIGD were primarily associated with oxidoreductase and dioxygenase activities, as well as peroxisome function. Notably, one of GmHIGD genes, GmHIGD3 was found to be predominantly localized in mitochondria, and its overexpression in Arabidopsis led to a significantly reduction in catalase activity compared to wild-type plants. These results bring new insights into the functional role of GmHIGD in terms of subcellular localization and the regulation of oxidoreductase activity

Target Site Recognition by a Diversity-Generating Retroelement

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification

Methods for analyzing the insertion capabilities of modified group II introns

The present invention provides a system and methods for analyzing the function of nucleotide integrases and modified group II introns. The system comprises a donor plasmid comprising a wild-type or modified group II intron, a recipient plasmid comprising a DNA recognition site and a promoterless reporter gene downstream of the DNA target site, and a host cell. The method comprises the steps of transforming a host cell with the donor and recipient plasmids, assaying for expression of the reporter gene, isolating plasmid DNA from the cotransformed cells, and analyzing the plasmid DNA to confirm that the group II intron has been inserted into the target sequence. The present invention also provides a method for simultaneously analyzing the activity of two or more modified nucleotide integrases. The present invention also relates to methods of preparing a library of donor plasmids containing a plurality of diverse modified group II intron DNA sequences.Board of Regents, University of Texas Syste

Diversity-generating Retroelements in Phage and Bacterial Genomes.

Diversity-generating retroelements (DGRs) are DNA diversification machines found in diverse bacterial and bacteriophage genomes that accelerate the evolution of ligand-receptor interactions. Diversification results from a unidirectional transfer of sequence information from an invariant template repeat (TR) to a variable repeat (VR) located in a protein-encoding gene. Information transfer is coupled to site-specific mutagenesis in a process called mutagenic homing, which occurs through an RNA intermediate and is catalyzed by a unique, DGR-encoded reverse transcriptase that converts adenine residues in the TR into random nucleotides in the VR. In the prototype DGR found in the Bordetella bacteriophage BPP-1, the variable protein Mtd is responsible for phage receptor recognition. VR diversification enables progeny phage to switch tropism, accelerating their adaptation to changes in sequence or availability of host cell-surface molecules for infection. Since their discovery, hundreds of DGRs have been identified, and their functions are just beginning to be understood. VR-encoded residues of many DGR-diversified proteins are displayed in the context of a C-type lectin fold, although other scaffolds, including the immunoglobulin fold, may also be used. DGR homing is postulated to occur through a specialized target DNA-primed reverse transcription mechanism that allows repeated rounds of diversification and selection, and the ability to engineer DGRs to target heterologous genes suggests applications for bioengineering. This chapter provides a comprehensive review of our current understanding of this newly discovered family of beneficial retroelements

Research on Centroid Position for Stairs Climbing Stability of Search and Rescue Robot

This paper represents the relationship between the stability of stairs climbing and the centroid position of the search and rescue robot. The robot system is considered as a mass point-plane model and the kinematics features are analyzed to find the relationship between centroid position and the maximal pitch angle of stairs the robot could climb up. A computable function about this relationship is given in this paper. During the stairs climbing, there is a maximal stability-keeping angle depends on the centroid position and the pitch angle of stairs, and the numerical formula is developed about the relationship between the maximal stability-keeping angle and the centroid position and pitch angle of stairs. The experiment demonstrates the trustworthy and correction of the method in the paper