Circular Template Research Articles

How Can a Patient Blind to Radial Motion Discriminate Shifts in the Center-of-Motion?

Within biologically constrained models of heading and complex motion processing, localization of the center-of-motion (COM) is typically an implicit property arising from the precise computation of radial motion direction associated with an observer's forward self-motion. In the work presented here we report psychophysical data from a motion-impaired stroke patient, GZ, whose pattern of visual motion deficits is inconsistent with this view. We show that while GZ is able to discriminate direction in circular motions she is unable to discriminate direction in radial motion patterns. GZ's inability to discriminate radial motion is in stark contrast with her ability to localize the COM in such stimuli and suggests that recovery of the COM does not necessarily require an explicit representation of radial motion direction. We propose that this dichotomy can be explained by a circular template mechanism that minimizes a global motion error relative to the visual motion input, and we demonstrate that a sparse population of such templates is computationally sufficient to account for human psychophysical performance in general and in particular, explains GZ's performance. Recent re-analysis of the predicted receptive field structures in several existing heading models provides additional support for this type of circular template mechanism and suggests the human visual system may have available circular motion mechanisms for heading estimation.

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The post-genomic era brings with it many challenges to the understanding of protein structure, function and interaction. The increasing number of newly identified proteins that need to be expressed, problems associated with cell cultivation, and traditional sub-cloning procedures are some of the key bottlenecks.Rapid Translation System “RTS” is a scalable in vitro transcription/translation protein expression platform, that produces sufficient amounts of protein for characterization studies, functional assays, or structural analysis. RTS offers the advantages of a cell-free protein expression system, eliminating the need for laborious up- and down-stream steps (e.g. host cell transformation, culturing, or lysis) typically associated with cell-based expression systems.Generally, to further fasten the protein expression screening, we recommend to use linear (PCR-generated) templates with the RTS screening kits. These templates are produced via the RTS Linear Template Generation Sets. For scale-up with RTS500 (1ml reaction) and RTS9000 (10, 30ml reaction), the use of circular templates is recommended, which are generated with the RTS pIVEX Vector Sets. In order to yield mg quantities, expressions should be performed using the RTS ProteoMaster instrument.For optimization of the prokaryotic Rapid Translation, we offer a special software, the ProteoExpert RTS E. coli HY. The software delivers high-yield variants of an input ORF sequence.

Transcription arrest caused by long nascent RNA chains

The transcription process is highly processive. However, specific sequence elements encoded in the nascent RNA may signal transcription pausing and/or termination. We find that under certain conditions nascent RNA chains can have a strong and apparently sequence-independent inhibitory effect on transcription. Using phage T3 RNA polymerase (T3 RNAP) and covalently closed circular (cccDNA) DNA templates that did not contain any strong termination signal, transcription was severely inhibited after a short period of time. Less than ∼10% residual transcriptional activity remained after 10 min of incubation. The addition of RNase A almost fully restored transcription in a dose dependent manner. Throughout RNase A rescue, an elongation rate of ∼170 nt/s was maintained and this velocity was independent of RNA transcript length, at least up to 6 kb. Instead, RNase A rescue increased the number of active elongation complexes. Thus transcription behaved as an all-or-none process. The mechanism of transcription inhibition was explored using electron microscopy and further biochemical experiments. The data suggest that multiple mechanisms may contribute to the observed effects. Part of the inhibition can be ascribed to the formation of R-loops between the nascent RNA and the DNA template, which provides “roadblocks” to trailing T3 RNAPs. Based on available literature we discuss possible in vivo implications of the results.

Vaccinia Virus Intermediate Stage Transcription Is Complemented by Ras-GTPase-activating Protein SH3 Domain-binding Protein (G3BP) and Cytoplasmic Activation/Proliferation-associated Protein (p137) Individually or as a Heterodimer

Transcription of the DNA genome of vaccinia virus occurs in the cytoplasm and is temporally programmed by early, intermediate, and late stage-specific transcription factors in conjunction with a viral multisubunit RNA polymerase. The RNA polymerase, capping enzyme, and three factors (VITF-1, VITF-2, and VITF-3) are sufficient for in vitro transcription of a DNA template containing an intermediate stage promoter. Vaccinia virus intermediate transcription factor (VITF)-1 and -3 are virus-encoded, whereas VITF-2 was partially purified from extracts of uninfected HeLa cells. Using purified and recombinant viral proteins, we showed that the HeLa cell factor was required for transcription of linear or nicked circular templates but not of super coiled DNA. HeLa cell polypeptides of approximately 110 and 66 kDa copurified with VITF-2 activity through multiple chromatographic steps. The polypeptides were separated by SDS-polyacrylamide gel electrophoresis and identified by mass spectrometry as Ras-GTPase-activating protein SH3 domain-binding protein (G3BP) and p137, recently named cytoplasmic activation/proliferation-associated protein-1. The co-purification of the two polypeptides with transcription-complementing activity was confirmed with specific antibodies, and their association with each other was demonstrated by affinity chromatography of tagged recombinant forms. Furthermore, recombinant G3BP and p137 expressed individually or together in mammalian or bacterial cells complemented the activity of the viral RNA polymerase and transcription factors. The involvement of cellular proteins in transcription of intermediate stage genes may regulate the transition between early and late phases of vaccinia virus replication.

Replicative intermediates of Tomato leaf curl virus and its satellite DNAs

Several plant geminiviruses have been shown recently to utilize both rolling-circle replication (RCR) and recombination-dependent replication (RDR) strategies. A highly specific binding of the viral replication-associated protein (Rep) to its cognate DNA is essential for initiation of viral DNA replication and for the recognition of DNA components of the bipartite geminiviruses of the Begomovirus genus. We have extended the replication analysis to the monopartite Australian Tomato leaf curl virus (ToLCV), its Rep binding deficient mutants, and the satellite DNAs it supports. Analyses of viral DNA by two-dimensional agarose gel electrophoresis after fractionation by single-stranded (ss) DNA-selective cellulose chromatography revealed that DNA intermediates of ToLCV and its mutant were identical. Both RCR and RDR intermediates were identified. New ToLCV DNA forms were observed and characterized as subgenomic topoisomers, heterogeneous open circular double-stranded (ds) DNA, and degradation products. A 1350-nt DNA β satellite associated with the unrelated Cotton leaf curl Multan virus (CLCuMV) was supported by ToLCV and produced intermediates of both RCR and RDR, suggesting that replication strategies of satellites are determined by the helper virus. Replicative intermediates of the 682 nt ToLCV satellite DNA could not be resolved; however, concatemers of up to octamer were detected, together with a field of hybridizing material suggestive of complementary strand replication on heterogeneous circular ssDNA templates.

Coupling DNA Supercoiling to Transcription in Defined Protein Systems

Transcription of closed circular DNA templates in the presence of DNA gyrase is known to stimulate negative DNA supercoiling both in vivo and in vitro. It has proven elusive, however, to establish a general system in vitro that supports transcription-coupled DNA supercoiling (TCDS) by the "twin-domain" mechanism (Liu, L. F. and Wang, J. C. (1987) Proc. Natl. Acad. Sci. USA 84, 7024-7027) that operates in bacteria. In this report, we examine the properties of TCDS in defined protein systems that minimally contained T7 RNA polymerase and DNA gyrase. Specifically designed plasmid DNA templates permitted us to control the location and length of RNA transcripts. We demonstrate that TCDS takes place by two separate, and apparently independent, mechanistic pathways in vitro. The first supercoiling pathway, which is not likely to be significant in vivo, was found to be dependent on R-loop formation and could be suppressed by the presence of RNase H or bacterial HU protein. The second pathway for TCDS was much more potent, but became predominant in vitro only when sequence-specific DNA-bending proteins were present during transcription, and RNA transcript lengths exceeded 3 kb. This major supercoiling route was shown to be resistant to RNase H and had functional properties consistent with those predicted for the twin-domain mechanism. For example, DNA supercoiling activity was proportional to RNA transcript length and was greatly stimulated by macromolecular crowding agents. Under optimal conditions, the twin domain pathway of TCDS rapidly and efficiently generated superhelicity levels more than twice that typically found in vivo.

Observation of internal cleavage and ligation reactions of a ribozyme.

We have used single-molecule spectroscopy to untangle conformational dynamics and internal chemistry in the hairpin ribozyme. The active site of the ribozyme is stably formed by docking two internal loops, but upon cleavage undocking is accelerated by two orders of magnitude. The markedly different kinetic properties allow us to differentiate cleaved and ligated forms, and thereby observe multiple cycles of internal cleavage and ligation of a ribozyme in a uniquely direct way. The position of the internal equilibrium is biased toward ligation, but the cleaved ribozyme undergoes several undocking events before ligation, during which products may dissociate. Formation of the stably docked active site, rapid undocking after cleavage, and a strong bias toward ligation should combine to generate a stable circular template for the synthesis of the viral (+) strand and thus ensure a productive replication cycle.

Synthesis and characterization of topologically linked single-stranded DNA rings

We investigated the synthesis of linked-ring DNAs by two DNA-ligation-based methods. In the first method, two DNA oligonucleotides were associated through a duplex segment of more than a full helical turn. Circularization of the entwined oligonucleotides by T4 DNA ligase resulted in two linked-ring DNAs with a total yield of ∼40%. In the second method, a DNA oligonucleotide was circularized over a circular DNA template, resulting in the formation of ∼10% linked-ring product. The circular nature of linked-ring DNAs was verified with exonuclease digestion and the existence of topological linkages was demonstrated by analyzing the electrophoretic mobility pattern of DNA products obtained from the digestion of each linked-ring DNA using specific restriction endonucleases. A linked-ring DNA library in which one of the two rings contained random-sequence nucleotides was also constructed and tested for compatibility with in vitro selection.

The Biochemical Requirements of DNA Polymerase V-mediated Translesion Synthesis Revisited

In addition to replicative DNA polymerases, cells contain specialized DNA polymerases involved in processes such as lesion tolerance, mutagenesis and immunoglobulin diversity. In Escherichia coli, DNA polymerase V (Pol V), encoded by the umuDC locus, is involved in translesion synthesis (TLS) and mutagenesis. Genetic studies have established that mutagenesis requires both UmuC and a proteolytic product of UmuD (UmuD′). In addition, RecA protein and the replication processivity factor, the β-clamp, were genetically found to be essential co-factors for mutagenesis. Here, we have reconstituted Pol V-mediated bypass of three common replication-blocking lesions, namely the two major UV-induced lesions and a guanine adduct formed by a chemical carcinogen (G-AAF) under conditions that fulfil these in vivo requirements. Two co-factors are essential for efficient Pol V-mediated lesion bypass: (i) a DNA substrate onto which the β-clamp is stably loaded; and (ii) an extended single-stranded RecA/ATP filament assembled downstream from the lesion site. For efficient bypass, Pol V needs to interact simultaneously with the β-clamp and the 3′ tip of the RecA filament. Formation of an extended RecA/ATP filament and stable loading of the β-clamp are best achieved on long single-stranded circular DNA templates. In contrast to previously published data, the single-stranded DNA-binding protein (SSB) is not absolutely required for Pol V-mediated lesion bypass provided ATP, instead of ATPγS, activates the RecA filament. Further discrepancies with the existing literature are explainable by the use of either inadequate DNA substrates or a UmuC fusion protein instead of native Pol V.

Features affecting the ability of hepatitis delta virus RNAs to initiate RNA-directed RNA synthesis.

In models of the replication of human hepatitis delta virus (HDV) RNA, it is generally assumed that circular RNAs are the only templates. However, noncircular HDV RNAs are also produced during replication, and it is known that replication can be initiated by transfection with noncircular RNAs. Therefore, strategies were devised to determine the relative ability of different HDV RNA species to initiate RNA replication. One strategy used in vivo intermolecular competition following cotransfection into cells, between two sequence-marked HDV RNA species. Circular RNA templates were found to be at least severalfold more efficient than a dimeric linear template. Unit-length linear species, that is, equivalent to circles opened at different sites, were in most cases but not always of efficiency comparable to that of each other. Greater-than-unit-length linear species were more efficient than unit-length species, presumably because of the increased opportunities for template switching. Genomic linear RNAs were generally of initiation ability comparable to that of antigenomic RNAs. A second strategy measured the ability of initiation to occur on different regions of HDV RNAs that were twice the unit length. In summary, results from these two experimental strategies make clear that linear HDV RNA species, as well as circles, can contribute to the overall process of HDV genome replication. In addition, the results from the two experimental strategies provided information on the impact of template switching during RNA-directed transcription.

Circular rapid amplification of cDNA ends for high-throughput extension cloning of partial genes

The rapid amplification of cDNA ends (RACE) procedure is a widely used PCR-based method to clone the cDNA ends of mRNA transcripts. Current RACE methods often produce a high background of nonspecific PCR products, which can exclude the identification of the target cDNA of interest. We describe here an improved RACE procedure using circular cDNA templates and demonstrate the successful extension cloning of 4406 cDNAs.

Rotation Invariant Image Matching by Using Adaptive Concentric Ring Template

This paper proposed a method to search a target image area that has a free location and inclination in an objective image. The gray-level histogram and the gray-level ratio of pixels pair histogram are used as the invariant feature to change of inclination in this method. The proposed method separates a circular template to ring regions with the common center and generates histograms about each separated ring region. These generated histograms are used for matching. Moreover, in order to reduce the computation cost with maintaining the reliability, effective ring regions for matching are selected by the genetic algorithm. The experimental results show that the target image areas were searched successfully in spite of some ring regions were not used and the high-speed matching was realized by using the generated concentric ring template image in comparison with by using the whole template image.

Rotation invariant image template matching based on correlation of curvature distribution

AbstractThis paper proposes a method of searching for a target image area that has an arbitrary location and inclination in an objective image. The method uses the curvature of the edge pixels, which is invariant to the location and inclination of a target image area. The curvature of an edge pixel and the distance between the central pixel and the edge pixel are measured in a circular image area. The stored value in the two‐dimensional space that is spread by the curvature and the distance is incremented. This process is repeated about all edge pixels in the circular image area. The matching is executed between the two‐dimensional spaces that are generated by the circular template image and generated by a circular image area in an objective image. The experimental results show that the recognition rate of the target image areas recorded 97.8% of four kinds of images. The averaged processing time to search the target image areas was 20.4 seconds. © 2003 Wiley Periodicals, Inc. Electr Eng Jpn, 145(4): 56–3, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/eej.10235

Architecture of the Replication Complex and DNA Loops at the Fork Generated by the Bacteriophage T4 Proteins

Rolling circle replication has previously been reconstituted in vitro using M13 duplex circles containing preformed forks and the 10 purified T4 bacteriophage replication proteins. Leading and lagging strand synthesis in these reactions is coupled and the size of the Okazaki fragments produced is typical of those generated in T4 infections. In this study the structure of the DNAs and DNA-protein complexes engaged in these in vitro reactions has been examined by electron microscopy. Following deproteinization, circular duplex templates with linear tails as great as 100 kb are observed. The tails are fully duplex except for one to three single-stranded DNA segments close to the fork. This pattern reflects Okazaki fragments stopped at different stages in their synthesis. Examination of the DNA-protein complexes in these reactions reveals M13 duplex circles in which 64% contain a single large protein mass (replication complex) and a linear duplex tail. In 56% of the replicating molecules with a tail there is at least one fully duplex loop at the replication complex resulting from the portion of the lagging strand engaged in Okazaki fragment synthesis folding back to the replisome. The single-stranded DNA segments at the fork bound by gene 32 and 59 proteins are not extended but rather appear organized into highly compact structures ("bobbins"). These bobbins constitute a major portion of the mass of the full replication complex.

PriA Mediates DNA Replication Pathway Choice at Recombination Intermediates

We report the reconstitution of the initial steps of the double-strand break-repair pathway where joint molecule formation between a duplex DNA fragment and a circular template by the combined action of RecA, RecBCD, and the single-stranded DNA binding protein provides the substrate for replication fork formation by the restart primosome and the DNA polymerase III holoenzyme. We show that PriA dictates the pathway of replication from the recombination intermediate by inhibiting a nonspecific, strand displacement DNA synthesis reaction and favoring the formation of a bona fide replication fork. Furthermore, we find that RecO and RecR significantly stimulate this recombination-directed DNA replication reaction, and that this stimulation is modulated by the presence of RecF, suggesting that the latter protein may also act as a regulator of the pathway of resolution of the recombination intermediate.

207 Precision Profile Grinding by Depth Control with PZT-Actuating Table and Templates

Grinding method of correct plane shape and shallow pattern shape is investigated. Conventional surface grinder is used. The table that has permanent magnetic chuck on six piezo-electric actuators is used. Firstly two pairs of sensor and circular templates are used for compensating grinding in x and y feed direction. Ground surface by this method was compensated to the correct plane surface. For nearer compensating grinding performance, 3D contour template is used. Contour of any flexible shape is obtained with 9 adjustable screws. From the result the maximum error from correct plane is less than 0.4μm on the work of 100mm width and 130mm length. Then shallow square pads such as oil pad was able to be ground by using the contour template and usefulness of proposed method is examined.

Dominant Effect of Protein Charge Rather Than Protein Shape in Apparent DNA Bending by Engineered bZIP Domains

We are interested in how asymmetric charge neutralization of DNA by proteins results in DNA bending. We have previously reported electrophoretic phasing experiments utilizing homodimer peptides derived from GCN4, a yeast basic zipper (bZIP) transcription factor. Here we report the results of experiments that examine the importance of peptide sequence context in DNA bending and test the hypothesis that peptide structural asymmetry causes electrophoretic anomalies in the absence of DNA bending. We prepared two new series of bZIP peptides that differed dramatically in overall size, structure, and peptide sequence near the DNA. The magnitude of apparent DNA bending is independent of the structure of the protein. This result reduces the concern that bZIP protein structure causes electrophoretic anomalies in the absence of DNA bending. In all cases, both the magnitude and direction of the apparent DNA bend angle are strongly dependent on the local peptide charge. We attempted to validate independently our results with a minicircle competition binding assay. Binding preferences of severalfold for properly phased circular versus linear DNA templates were predicted. However, no binding preferences were observed. We propose that the minicircle binding assay may be intrinsically insensitive to DNA bending or flexibility induced by the bZIP peptides studied, and we provide a unifying explanation for the discrepancies between the cyclization and electrophoretic experiments.

First Report of Rhynchosia golden mosaic virus (RhGMV) Infecting Tobacco in Chiapas, Mexico.

After a tobacco virus outbreak associated with whiteflies in Chiapas, Mexico, we conducted a survey to detect the presence of begomoviruses. Previously, two tobacco-infecting geminiviruses were reported in the same geographical area: Texas pepper virus-Chiapas and Tobacco apical stunt virus (TPV-CPS and TbASV, respectively) (2). DNA extracts from symptomatic tobacco plants (yellow mosaic, severe foliar distortion, and dwarfing) were used to biolistically inoculate tobacco plants (1). After symptom expression, the viruses were analyzed by polymerase chain reaction (PCR) and sequencing. For the first PCR procedure, the primers used (RepMot: 5'GAGTCTAGAGGATANGTRAGGAAATARTTCTT GGC3' and CPMot: 5'CGCGAATTCGACTGGACCTTACATGGNCCTT CAC3') were designed from conserved regions of the Rep and CP genes, and directed the amplification of a fragment that includes the intergenic region and varies in size from 600 (for New World begomoviruses) to 750 bp (Old World begomoviruses). Cloning of the PCR products (approximately 600 bp) was performed in the pCRII vector (Invitrogene, San Diego, CA), and viral inserts derived from different symptomatic plants were sequenced. Nucleotide sequence comparisons were performed using the Clustal method (MegAlign, DNAStar software, Madison, WI) with GenBank databases. Analysis of the PCR products allowed the identification of two types of viral sequences. The first virus identified was 98% identical to TPV-CPS, whereas the second virus was clearly related to Rhynchosia golden mosaic virus (RhGMV; 91% identity in the amplified region), and 65% identical to Pepper Huasteco virus (PHV). To disclose the identity of the second virus, another set of primers was used, p260 and p261 (4). These primers are located back-to-back in a conserved region of the CP gene, and direct the amplification of a full-length DNA-A from circular templates. The resulting PCR fragment (2.6 kb) was cloned in pCRII and fully sequenced (GenBank Accession No. AF408199). Analysis showed that this tobacco-infecting geminivirus is a strain of the recently described RhGMV from Honduras (3) (overall DNA A sequence identity, 94%; protein similarities: CP, 98.4%; AL1, 93.6%; AL2, 92.8%; and AL3, 91.7%). Comparative analysis of the intergenic regions of RhGMV-Tob, TPV-CPS, and TbASV showed that these viruses display different Ori-associated iterative motifs (iterons): RhGMV-Tob (GGTRT/G), TPV-CPS (GGAGTC), and TbASV (GGTAT). Since iterons are critical determinants of replication specificity, this observation indicates that those viruses are probably unable to form infectious pseudorecombinants in nature. To date, at least three different geminiviruses have been identified from symptomatic tobacco samples in Chiapas (2), showing how complex a geminiviral outbreak can be in a permissive environment. To our knowledge, this is the first time that the presence of RhGMV has been reported in Mexico and also the first time that this virus has been associated with an economically important crop.

Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro.

The ability of cell-free extracts to correct DNA mismatches has been demonstrated in both prokaryotes and eukaryotes. Such an assay requires a template containing both a mismatch and a strand discrimination signal, and the multi-step construction process can be technically difficult. We have developed a three-step procedure for preparing DNA heteroduplexes containing a site-specific nick. The mismatch composition, sequence context, distance to the strand signal, and the means for assessing repair in each strand are adjustable features built into a synthetic oligonucleotide. Controlled ligation events involving three of the four DNA strands incorporate the oligonucleotide into a circular template and generate the repair-directing nick. Mismatch correction in either strand of a prototype G.T mismatch was achieved by placing a nick 10-40 bp away from the targeted base. This proximity of nick and mismatch represents a setting where repair has not been well characterized, but the presence of a nick was shown to be essential, as was the MSH2/MSH6 heterodimer, although low levels of repair occurred in extract defective in each protein. All repair events were inhibited by a peptide that interacts with proliferating cell nuclear antigen and inhibits both mismatch repair and long-patch replication.

Circular mRNA encoding for monomeric and polymeric green fluorescent protein.

Many proteins with unusual structural properties are comprised of multiple repeating amino acid sequences, and are often fractious to expression in recombinant systems. To facilitate recombinant production of such proteins for structural and engineering studies, the author has developed a method for producing messenger RNAs on circular RNA templates. This circularization process is derived from a rearranged group I intron, from which circular RNA is produced through the splicing activity of autocatalytic group I RNA elements (Fig. 1; 1,2). Because the only cofactors required for splicing of the group I intron are magnesium and guanosine, the process can take place in a variety of organisms, making it amenable to a wide variety of protein expression systems (1–4).