Cloning and sequence analysis of linear plasmid telomeres of the bacterium Borrelia burgdorferi

Borrelia burgdorferi, the Lyme disease agent, has double-stranded linear plasmids with covalently closed ends. DNA at the ends, or telomeres, of two linear plasmids of B. burgdorferi strain B31 was examined. Telomeric sequences from both ends of a 16 kb linear plasmid and from one end of a 49 kb linear plasmid were cloned and sequenced. An 18 bp AT-rich inverted repeat was found at each end of the 16 kb linear plasmid. The sequences of the two ends of this plasmid were different beyond these short inverted terminal repeats. The cloned end of the 49 kb linear plasmid had sequence identity with one end of the 16 kb linear plasmid. The end sequence common to both plasmids contained a series of phased, short direct repeats and a 52 bp palindrome adjacent to a highly AT-rich region. These findings indicate that Borrelia linear plasmid telomeres have structural features different from those of other known replicons.     

DNA substrate requirements for stable joint molecule formation by the RecA and single-stranded DNA-binding proteins of Escherichia coli

In reactions between linear single-stranded DNAs (ssDNAs) and circular double-stranded DNAs (dsDNAs), stable joint molecule formation promoted by the recA protein (RecA) requires negative superhelicity, a homologous end, and an RecA-ssDNA complex. Linear ssDNAs with 3'-end homology react more efficiently than linear ssDNAs with 5'-end homology. This 3'-end preference is explained by the finding that 3'-ends are more effectively coated by RecA than 5'-ends, as judged by exonuclease VII protection, and are thus more reactive. The ability of linear ssDNAs with 5'-end homology to react is improved by the presence of low concentrations of exonuclease VII. In reactions between ssDNAs and linear dsDNAs with end homology, stable joint molecule formation occurs more efficiently when the homology is at the 3'-end rather than at the 5'-end of the complementary strand. In addition, linear dsDNAs with homology at the 3'-end of the complementary strand react more efficiently with linear ssDNAs with 3'-end homology than with linear ssDNAs with 5'-end homology. The ability of linear ssDNAs with 5'-end homology to react, in the absence of single-stranded DNA-binding protein, is improved by adding 33-46 nucleotides of heterologous sequence to the 5'-end of the linear ssDNA. The poor reactivity of linear ssDNAs with 5'-end homology is explained by a lack of RecA at the 5'-ends of linear ssDNAs, which is a consequence of the polar association and dissociation of RecA.     

Recombination between linear double-stranded DNA substrates in vivo

Recombineering technology in Escherichia coli enables targeting of linear donor DNA to circular recipient DNA using short shared homology sequences. In this work, we demonstrate that recombineering is also able to support recombination between a pair of linear DNA substrates (linear/linear recombineering) in vivo in E. coli. Linear DNA up to 100 kb is accurately modified and remains intact without undergoing rearrangements after recombination. This system will be valuable for direct in vivo manipulation of large linear DNA including the N15 and PY54 prophages and linear animal viruses, and for assembly of linear constructs as artificial chromosome vectors.     

Covalently closed circular viral DNA formed from two types of linear DNA in woodchuck hepatitis virus-infected liver.

We found that livers from woodchucks chronically infected with woodchuck hepatitis virus (WHV) contained covalently closed circular DNA (cccDNA) molecules with deletions and insertions indicative of their formation from linear viral DNA by nonhomologous recombination, as we previously described for the duck hepatitis B virus (W. Yang and J. Summers, J. Virol. 69:4029-4036, 1995). However, evidence for two different types of linear precursors was obtained by analysis of the recombination joints in WHV cccDNA. Type 1 linear precursors possessed the structural properties that correspond to those of in situ-primed linear DNA molecules, which constitute between 7 and 20% of all viral DNA replicative intermediates synthesized in the liver. Type 2 linear precursors are hypothetical species of linear DNAs with a terminal duplication of the cohesive-end region, between DR1 and DR2. This type of linear DNA has not been previously described and was not detected among the DNA species present in nucleocapsids. A fraction of cccDNAs formed from both type 1 and type 2 linear DNAs are predicted to be functional for further DNA synthesis, and some evidence for the formation of two or more generations of cccDNA from linear DNA was observed.     

Enhancement of linear gramicidin expression from Bacillus brevis ATCC 8185 by casein peptide

Bacillus brevis (Brevibacillus parabrevis) ATCC 8185 synthesizes two kinds of antibiotic peptides, cyclopeptide tyrocidine and linear gramicidin. The production of linear gramicidin can be induced by the standard method (using a skim milk medium for pre-culture and beef broth for the main culture) employed for the induction of tyrocidine. In this study, we tried to determine the optimal growth medium for B. brevis ATCC 8185 for synthesizing linear gramicidin. The yield of linear gramicidin produced by the standard method was 3.11 microg/ml. When beef broth was used both as the pre-medium and the main medium, the yield of the antibiotic was only 0.59 microg/ml. To confirm the influence of skim milk, the strain was grown in a 1% skim milk medium. As a result, the amount of linear gramicidin produced reached 20.3 microg/ml. These findings show the importance of skim milk in the production of linear gramicidin. In the skim milk medium, the cells produced an extracellular protease 2 h before the linear gramicidin was expressed. The 1% skim milk medium pretreated by this protease did not allow the induction of linear gramicidin into the cells, and protease activity was not detected in the supernatant of the culture. When the cells were cultivated in a 1% egg albumin medium, protease activity from the supernatant of the culture was detected, but production of linear gramicidin was not observed. Therefore, a 1% casein medium was used for production of linear gramicidin. As a result, the yield of linear gramicidin produced in the medium reached 6.69 microg/ml. We concluded that a digested product of the extracellular protease from casein enhances linear gramicidin production.     

Linear approximations for swing leg motion during gait

The applicability of a linear systems analysis of two-dimensional swing leg motion was investigated. Two different linear systems were developed. A linear time-varying system was developed by linearizing the nonlinear equations describing swing leg motion about a set of nominal system and control trajectories. Linear time invariant systems were developed by linearizing about three different fixed limb positions. Simulations of swing leg motion were performed with each of these linear systems. These simulations were compared to previously performed nonlinear simulations of two-dimensional swing leg motion and the actual subject motion. Additionally, a linear system analysis was used to gain some insight into the interdependency of the state variables and controls. It was shown that the linear time varying approximation yielded an accurate representation of limb motion for the thigh and shank but with diminished accuracy for the foot. In contrast, all the linear time invariant systems, if used to simulate more than a quarter of the swing phase, yielded generally inaccurate results for thigh shank and foot motion.     

Cruciform transitions in DNA

The rates of transition between the cruciform and linear conformations of a perfectly inverted repeated lac operator DNA sequence have been measured using a trimethylpsoralen intrastrand cross-linking assay. The rate and extent of the linear to cruciform transition were dependent on temperature and on the superhelical density of the DNA. Apparent half-lives for this transition were between 4-9 min at 37 degrees C for supercoiled DNAs as isolated from cells. The half-life for the cruciform to linear transition in relaxed DNA was about 30 s at 37 degrees C. Mg2+ stabilized both conformations but stabilized the linear form to a greater degree than the cruciform. The rates of transition were temperature dependent suggesting enthalpies of activation of 26.3 kcal mol-1 for the cruciform to linear transition and 33.4 kcal mol-1 for the linear to cruciform transition. The rate of the linear to cruciform transition was slower at 50 than 37 degrees C. Heating above 70 degrees C resulted in the loss of the cruciform structure.     

Formation of a linear [3Fe-4S] cluster in a seven-iron ferredoxin triggered by polypeptide unfolding

Cubic iron-sulfur ([Fe-S]) clusters are common inorganic cofactors in proteins. The presence of a linear [3Fe-4S] cluster in a protein was first observed in beef-heart aconitase at high pH, where the protein structure was perturbed. Not long ago, the same linear cluster was discovered upon unfolding of a thermophilic di-cluster seven-iron ferredoxin, suggesting a more general relevance for this type of linear clusters in Nature. Since structure-induced cluster rearrangements may be important regulatory, on-going processes in living systems, we decided to further characterize the formation of the linear iron-sulfur cluster observed upon ferredoxin unfolding. Here we present a kinetic investigation of parameters that affect the linear-cluster formation and disassembly in the Sulfolobus acidocaldarius seven-iron ferredoxin. We find the linear cluster to be an intermediate on the protein-mediated cluster-degradation pathway under a wide range of pH and denaturant conditions. The linear species forms in parallel with secondary-structure disappearance. In contrast, the disassembly rate constant for the linear cluster is independent of denaturant concentration but depends strongly on solution pH. At high pH, the disassembly rate is slower and the linear iron-sulfur species has a longer lifetime, than at low pH.     

Low transfecting efficiency of phage lambda ring chromosomes and their fragments formed by membrane nucleases

Transfection efficiency of a number of lambda DNA samples differing in ring to linear molecules ratio was determined. Graphic extrapolation to the zero content of linear molecules showed that efficiency of ring molecules did not exceed 5% of that of linear molecules. Probably, this difference is caused by more fast penetration of linear molecules into the cell and, therefore, by lower probability of their degradation by cell wall nucleases. Fragments of both ring and linear molecules formed by cell wall nucleases proved to be inactive in marker rescue experiments.     

The differential aspects of the linear isobole in the study of combined action of agents

Although the isobologram is presently the most widely used method of analysis for combined effects of agents, there are several different interpretations of the linear isobole isobole in regard to its use as a criterion of interaction. An investigation of the differential aspects of the linear isobole relation may cast some light in this regard. By conceptual extension of the present single effect level (i.e. effect-point) relation of the linear isobole to an effect-neighbourhood relation in which the linear isobole holds over a small continuous range of effect levels, the mathematical differential of the linear isobole can be developed and investigated. This differential aspect provides some useful insights into the implication and interpretation of the linear isobole relation when used as a general criterion in agent interaction studies. it can also serve as the mathematical basis for the formulation of analytic schemes in which the linear isobole relation is applicable over a continuous range of effect levels.     

Giant linear plasmids of β-lactam antibiotic producing Streptomyces

Abstract A survey of the total cellular DNA from five β-lactam antibiotic-producing Streptomyces spp. by pulsed field gel electrophoresis was conducted to investigate the presence of linear plasmids. Streptomyces clavuligerus NRRL 3585 contained two giant linear plasmids of 120 and 430 kb, in addition to the well-characterized 11.7 kb linear plasmid. Streptomyces griseus NRRL 3851 contained a single giant linear plasmid of 120 kb, and Streptomyces jumonjinensis NRRL 5741 contained two giant linear plasmids (220 and 280 kb), and two smaller linear plasmids. No plasmids were identified in Streptomyces cattleya NRRL 3841 or Streptomyces lipmannii NRRL 3584. Southern hybridization did not reveal any homology shared by these plasmids, and β-lactam antibiotic synthesis gene clusters were located on the chromosome.     

Should artificial neural networks replace linear models in tree ring based climate reconstructions?

Studies focused on tree ring—climate relationships usually use linear methods to find the optimal transfer function. In our study, three sites with three different tree species from the Western Balkan region were selected to compare linear and artificial neural network (ANN) nonlinear models and to see whether linear models can be potentially replaced with ANN in climate reconstruction. For each site, one linear and two different ANN models were calculated. For all analysed sites, we were able to find a better fit using the advanced technique of ANN. All calibration and verification statistics were in favour of ANN models. A climate variable was reconstructed for a selected site using linear and nonlinear ANN methods. We demonstrated that ANN is always a more effective method, which always produce better results than linear models. The key to success is a properly selected training algorithm, which prevents overfitting and is able to find the optimal transfer function, also linear, if that is the case.     

Conversion of a linear to a circular plasmid in the relapsing fever agent Borrelia hermsii.

Spirochetes of the genus Borrelia have genomes composed of both linear and circular replicons. We characterized the genomic organization of B. burgdorferi, B. hermsii, B. turicatae, and B. anserina with pulsed-field gel electrophoresis. All four species contained a linear chromosome approximately 1 Mb in size and multiple linear plasmids in the 16- to 200-kb size range. Plasmids 180 and 170 kb in size, present in the relapsing fever agents B. hermsii and B. turicatae but not in the other two species, behaved as linear duplex DNA molecules under different electrophoretic conditions. A variant of strain HSI of B. hermsii had a 180-kb circular instead of linear plasmid. There were no detectable differences in the growth rates or in the expression of cellular proteins between cells bearing linear forms and those bearing circular forms of the plasmid. The conversion to a circular conformation of monomeric length was demonstrated by the introduction of strand breaks with irradiation, restriction endonuclease analysis, and direct observation of the DNA molecules by fluorescent microscopy. Consideration of different models for the replication of linear DNA suggests that circular intermediates may be involved in the replication of linear replicons in Borrelia spp.     

Mechanism Underlying IκB Kinase Activation Mediated by the Linear Ubiquitin Chain Assembly Complex

The linear ubiquitin chain assembly complex (LUBAC) ligase, consisting of HOIL-1L, HOIP, and SHARPIN, specifically generates linear polyubiquitin chains. LUBAC-mediated linear polyubiquitination has been implicated in NF-κB activation. NEMO, a component of the IκB kinase (IKK) complex, is a substrate of LUBAC, but the precise molecular mechanism underlying linear chain-mediated NF-κB activation has not been fully elucidated. Here, we demonstrate that linearly polyubiquitinated NEMO activates IKK more potently than unanchored linear chains. In mutational analyses based on the crystal structure of the complex between the HOIP NZF1 and NEMO CC2-LZ domains, which are involved in the HOIP-NEMO interaction, NEMO mutations that impaired linear ubiquitin recognition activity and prevented recognition by LUBAC synergistically suppressed signal-induced NF-κB activation. HOIP NZF1 bound to NEMO and ubiquitin simultaneously, and HOIP NZF1 mutants defective in interaction with either NEMO or ubiquitin could not restore signal-induced NF-κB activation. Furthermore, linear chain-mediated activation of IKK2 involved homotypic interaction of the IKK2 kinase domain. Collectively, these results demonstrate that linear polyubiquitination of NEMO plays crucial roles in IKK activation and that this modification involves the HOIP NZF1 domain and recognition of NEMO-conjugated linear ubiquitin chains by NEMO on another IKK complex.     

Construction of linear invariants in phylogenetic inference.

An analytical method is presented for constructing linear invariants. All linear invariants of a k-species tree can be derived from those of (k-1)-species trees using this method. The new method is simpler than that of Cavender, which relies on numerical computations. Moreover, the new method provides a convenient tool to study the relationships between linear invariants of the same tree or of different trees. All linear invariants of trees of up to five species are derived in this study. For four species, there are 16 independent linear invariants for each of the three possible unrooted trees, 14 of which are shared by two unrooted trees and 12 of these are shared by all three unrooted trees; the last types of linear invariants can be used to construct tests on the assumptions about nucleotide substitutions. The number of linear invariants for a tree is found to increase rapidly with the number of species.     

Efficacy of linear and convex transducers for ultrasound-guided transvaginal follicle aspiration

A new device was developed to hold linear transducers for transvaginal follicle aspiration. Efficacy of follicle aspiration was compared using a linear 6 MHz and a convex 5 MHz transducer. Fifty-five cows were submitted to follicle aspiration at random days of the estrous cycle. Aspirations were conducted with linear (n=28) and convex (n=38) transducers with 18 G needles at a negative pressure corresponding to 13 ml H(2)O/min. A greater number of follicles were aspirated using convex than to linear probe (12.4 versus 7.8, respectively, P<0.05). Mean number of oocytes and recovery rates were similar for convex (5.4 and 48.6%) and linear (4.6 and 59.3%) transducers. Limited space between the linear transducer and needle guide restricted access to some portions of the ovary, reducing the number of follicles aspirated using a linear transducer. The newly developed adaptor allowed greater stability, holding the ovaries firmly against the linear transducer. This diminished mobility permitted a similar number of oocytes to be recovered with both transducers. In conclusion, this new adaptor provided a low cost alternative for routine follicle aspiration and oocyte recovery in cattle.     

CIRCexplorer3:A CLEAR Pipeline for Direct Comparison of Circular and Linear RNA Expression

Sequences of circular RNAs(circ RNAs) produced from back-splicing of exon(s) completely overlap with those from cognate linear RNAs transcribed from the same gene loci with the exception of their back-splicing junction(BSJ) sites.Therefore,examination of global circ RNA expression from RNA-seq datasets generally relies on the detection of RNA-seq fragments spanning BSJ sites,which is different from the quantification of linear RNA expression by normalized RNA-seq fragments mapped to whole gene bodies.Thus,direct comparison of circular and linear RNA expression from the same gene loci in a genome-wide manner has remained challenging.Here,we update the previously-reported CIRCexplorer pipeline to version 3 for circular and linear RNA expression analysis from ribosomal-RNA depleted RNA-seq(CIRCexplorer3-CLEAR).A new quantitation parameter,fragments per billion mapped bases(FPB),is applied to evaluate circular and linear RNA expression individually by fragments mapped to circ RNA-specific BSJ sites or to linear RNA-specific splicing junction(SJ) sites.Comparison of circular and linear RNA expression levels is directly achieved by dividing FPBcircby FPBlinearto generate a CIRCscore,which indicates the relative circ RNA expression level using linear RNA expression level as the background.Highlyexpressed circ RNAs with low cognate linear RNA expression background can be readily identified by CIRCexplorer3-CLEAR for further investigation.CIRCexplorer3-CLEAR is publically available at https://github.com/Yang Lab/CLEAR.     

Conservation of plasmid maintenance functions between linear and circular plasmids in Borrelia burgdorferi

The Lyme disease agent Borrelia burgdorferi maintains both linear and circular plasmids that appear to be essential for mammalian infection. Recent studies have characterized the circular plasmid regions that confer autonomous replication, but the genetic elements necessary for linear plasmid maintenance have not been experimentally identified. Two vectors derived from linear plasmids lp25 and lp28-1 were constructed and shown to replicate autonomously in B. burgdorferi. These vectors identify internal regions of linear plasmids necessary for autonomous replication in B. burgdorferi. Although derived from linear plasmids, the vectors are maintained in circular form in B. burgdorferi, indicating that plasmid maintenance functions are conserved, regardless of DNA form. Finally, derivatives of these vectors indicate that paralogous gene family 49 is apparently not required for either circular or linear plasmid replication.     

Nature of φX174 Linear DNA from a DNA Ligase-Defective Host

Linear DNAs have been prepared from phiX phage and from phiX RF II (double-stranded circular form of phiX DNA, formed during infection and nicked in one or both strands) molecules derived from infection at the restrictive temperature of Escherichia coli ts7, a host mutant with a temperature-sensitive DNA ligase activity. The linear DNA from these phages can be circularized by annealing with fragments of phiX RF DNA produced by the Haemophilus influenzae restriction nuclease. The circularization experiment indicated that the site of breakage of the linear phage DNAs is not unique nor confined to a particular region of the genome. These linear DNAs were less than 0.1% as infective as circular phage DNA. The linear, positive strand of late RF II DNA, however, is uniquely nicked in the region of the phiX genome corresponding to cistron A. Although a low level of infectivity is associated with the linear DNA derived from late RF II, this infectivity appears to be a result of the association of linear positive and linear negative strands during the infectivity assay.