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At least 109 records · Page 6Linked to original sources
PubMed1990

Gene replacement in Bordetella pertussis by transformation with linear DNA.

We replaced the wild-type TOX operon of Bordetella pertussis with in vitro mutated, detoxified alleles by electroporetic transformation using unmarked linear DNA. Uptake of DNA was selected by transient ampicillin resistance and two simultaneous recombination events resulted in gene-replacement at the natural locus with no integration of heterologous DNA. TOX alleles were stable without selection and recombinant strains secreted non-toxic, fully assembled, protective pertussis toxin (PT) analogues with kinetics similar to the parental vaccine strain under production-scale fermentation conditions. Strains generated in this way are suitable for the production of recombinant whole-cell or component whooping cough vaccines that require no chemical modification of PT.

Alleles↗
PubMed1997

Thermodynamics of dT--dT base pair mismatching in linear DNA duplexes and three-arm DNA junctions.

We have used a combination of magnetic-suspension densimetry and calorimetry to derive complete thermodynamic profiles, including volume changes, for the formation of linear DNA duplexes and three-arm branched DNA junctions, from their component strands, with and without dT-dT mismatches. The formation of each type of complex at 20 degrees C is accompanied by a favorable free energy, with a favorable enthalpy term partially compensated by an unfavorable entropy. Formation is associated also with net uptake of water molecules. Using the formation of the fully-paired linear duplex or three-arm junction as reference states, we can establish a thermodynamic cycle in which the contribution of the single-strand species cancels. From this cycle, we determine that substitution of dA for dT has a differential free energy of deltadeltaG degrees of +2.4 kcal mol(-1) for mismatched duplex and +2.0 kcal mol(-1) (on the average) for the mismatched junction. These unfavorable differential free energies result from an unfavorable enthalpy, partially compensated by a favorable entropy, and a negative deltadeltaV. The free energies in the two cases have signs opposed to those of deltadeltaV, a situation that implicates hydration changes in creating the mismatch. When the deltadeltaV terms are normalized by the total number of base pairs involved, the immobilization of structural water molecules (and/or substitution of electrostricted for hydrophobic water molecules) is about 7 times greater for junctions than duplexes. This is consistent with more extensive hydrophobic hydration of branched DNA structures than of duplexes.

Base Composition↗
PubMed1994

Expression of the linear DNA plasmid pRS64 in the plant pathogenic fungus Rhizoctonia solani.

The plant pathogenic isolate RI-64 of anastomosis group 4 of Rhizoctonia solani possesses three linear DNA plasmids (pRS64-1, -2, and -3). Unique poly(A)- RNA, 0.5 kb in length and hybridizable with the pRS64 DNAs was found in mycelial cells of the isolate RI-64. The overall homology at the nucleotide level between pRS64-1, -2, and -3, and the cDNA prepared from the poly(A)- RNA was 100%, 73%, and 84%, respectively. The open reading frames found in pRS64-1, -2, and -3 (ORF1-1, ORF2-1, and ORF3-1) are 68 amino acids long. The amino acids sequence showed no significant homology with known proteins. Extracts from Escherichia coli cells expressing ORF1-1 contain a specific protein of 7 kDa. Antisera raised against the ORF1-1 product obtained from E. coli cells cross-reacted with the specific proteins found in the mycelia. The results indicate that the DNA plasmids found in R. solani contain a sequence that encodes a specific protein which may be involved in determination of plant pathogenicity.

Amino Acid Sequence↗
PubMed2004

Binding of phage Phi29 architectural protein p6 to the viral genome: evidence for topological restriction of the phage linear DNA.

Bacillus subtilis phage Phi29 protein p6 is required for DNA replication and promotes the switch from early to late transcription. In vivo it binds all along the viral linear DNA, which suggests a global role as an architectural protein; in contrast, binding to bacterial DNA is negligible. This specificity could be due to the p6 binding preference for less negatively supercoiled DNA, as is presumably the case with viral (with respect to bacterial) DNA. Here we demonstrate that p6 binding to Phi29 DNA is greatly increased when negative supercoiling is decreased by novobiocin; in addition, gyrase is required for DNA replication. This indicates that, although non-covalently closed, the viral genome is topologically constrained in vivo. We also show that the p6 binding to different Phi29 DNA regions is modulated by the structural properties of their nucleotide sequences. The higher affinity for DNA ends is possibly related to the presence of sequences in which their bendability properties favor the formation of the p6-DNA complex, whereas the lower affinity for the transcription control region is most probably due to the presence of a rigid intrinsic DNA curvature.

Bacillus Phages↗
PubMed2006

Mitochondrial genome of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa): A linear DNA molecule encoding a putative DNA-dependent DNA polymerase.

The 16,937-nuceotide sequence of the linear mitochondrial DNA (mt-DNA) molecule of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa) - the first mtDNA sequence from the class Scypozoa and the first sequence of a linear mtDNA from Metazoa - has been determined. This sequence contains genes for 13 energy pathway proteins, small and large subunit rRNAs, and methionine and tryptophan tRNAs. In addition, two open reading frames of 324 and 969 base pairs in length have been found. The deduced amino-acid sequence of one of them, ORF969, displays extensive sequence similarity with the polymerase [but not the exonuclease] domain of family B DNA polymerases, and this ORF has been tentatively identified as dnab. This is the first report of dnab in animal mtDNA. The genes in A. aurita mtDNA are arranged in two clusters with opposite transcriptional polarities; transcription proceeding toward the ends of the molecule. The determined sequences at the ends of the molecule are nearly identical but inverted and lack any obvious potential secondary structures or telomere-like repeat elements. The acquisition of mitochondrial genomic data for the second class of Cnidaria allows us to reconstruct characteristic features of mitochondrial evolution in this animal phylum.

Amino Acid Sequence↗
PubMed1989

Physical and biological characterization of linear DNA plasmids of the yeast Pichia inositovora.

Three cryptic DNA plasmids have been identified in a strain of the yeast Pichia inositovora that are 18, 13, and 10 kbp in size. All are sensitive to digestion by DNase I, restriction endonucleases, and exonuclease III, but are resistant to the activities of RNase A and lambda exonuclease. These results indicate that each plasmid is a linear DNA molecule whose 5' ends are protected. A restriction map has been developed for each of the plasmids, demonstrating that each is unique and confirming their linear nature. The plasmids are a major constituent of DNA prepared from whole cells, but are absent from DNA preparations of purified mitochondria and nuclei, indicating that the plasmids are located in the cytoplasm. These plasmids share many of the physical characteristics described for the linear plasmids of the yeasts Kluyveromyces lactis and Saccharomycopsis crataegensis. Unlike the linear plasmids of K. lactis, however, they appear not to be capable of killer toxin production.

Autoradiography↗
PubMed2000

Binding affinity of T7 RNA polymerase to its promoter in the supercoiled and linearized DNA templates.

A promoter competition assay was used to measure the stability of T7 RNA polymerase with its promoter. When T7 RNA polymerase was incubated with GTP for 5 minutes before the elongation of transcription in either the supercoiled or linearized template, the half-lives of T7 RNA polymerase-DNA complexes were reduced. The transcription product increased when T7 RNA polymerase preincubated with GTP in the supercoiled DNA but not in the linearized DNA template. On the other hand, preincubation of ATP with T7 RNA polymerase decreased the stability of T7 RNA polymerase with the supercoiled DNA, but did not affect the stability of T7 RNA polymerase with the linearized DNA. Furthermore, the production of RNA transcript was increased when T7 RNA polymerase was incubated with ATP in either supercoiled or linearized template before transcription elongation. This study is important to understand the relationship between the transcription initiation and the stability of the ternary complex, and to produce large quantities of RNA transcript in vitro.

Adenosine Triphosphate↗
PubMed1984

Incompatibility of linear DNA killer plasmids pGKL1 and pGKL2 from Kluyveromyces lactis with mitochondrial DNA from Saccharomyces cerevisiae.

Two linear killer plasmids (pGKL1 and pGKL2) from Kluyveromyces lactis stably replicated and expressed the killer phenotype in a neutral petite mutant [( rho0]) of Saccharomyces cerevisiae. However, when cytoplasmic components were introduced by cytoduction from a wild-type [( rho+]) strain of S. cerevisiae, the linear plasmids became unstable and were frequently lost from the cytoductant cells during mitosis, giving rise to nonkiller clones. The phenomenon was ascribed to the incompatibility with the introduced S. cerevisiae mitochondrial DNA (mtDNA), because the plasmid stability was restored by [rho0] mutations in the cytoductant cells. Incompatibility with mtDNA was also apparent for the transmission of plasmids into diploid progeny in crosses between killer cells carrying the pGKL plasmids and [rho+] nonkiller cells lacking the plasmids. High-frequency transmission of the plasmids was observed in crosses lacking mtDNA [( rho0] by [rho0] crosses) and in crosses involving mutated mtDNA with large deletions of various regions of mitochondrial genome. In contrast, mutated mtDNA from various mit- mutations also exerted the incompatibility effect on the transmission of plasmids. Double-stranded RNA killer plasmids were stably maintained and transmitted in the presence of wild-type mtDNA and stably coexisted with pGKL killer plasmids in [rho0] cells of S. cerevisiae.

Ascomycota↗
PubMed1990

Invertrons, a class of structurally and functionally related genetic elements that includes linear DNA plasmids, transposable elements, and genomes of adeno-type viruses.

Invertrons are genetic elements composed of DNA with inverted terminal repeats at both ends, covalently bonded to terminal proteins involved in the initiation of DNA replication at both their 5' termini when they exist in the cytoplasm of their host in free form. They function as viruses, linear DNA plasmids, transposable elements, and sometimes combinations of two of these properties. They differ from retroviruses and related retro-type transposons which have direct repeats on both their genomic ends and exploit RNA intermediates for replication of their DNA. A model for replication and integration of invertrons is presented, as well as a model for transposition of transposable elements.

Adenoviridae↗
PubMed1986

Efficient isolation of the linear DNA killer plasmid of Kluyveromyces lactis: evidence for location and expression in the cytoplasm and characterization of their terminally bound proteins.

Differential centrifugation of an osmotic lysate of K. lactis protoplasts showed that the linear DNA killer plasmids of K. lactis, pGKL1 and pGKL2, are almost exclusively present in the cytoplasmic fraction. This fractionation procedure allows the rapid isolation of large amounts of plasmid DNA without contamination by chromosomal and mitochondrial DNA. With these DNA preparations the size of the terminally bound proteins was estimated to be 28 and 36 kDal for pGKL1 and pGKL2, respectively. The entire pGKL1 sequence (except for 21 base pairs at the right terminus) was cloned in a shuttle vector that permits autonomous replication in the nucleus of K. lactis. However, killer gene expression could not be established in transformants. In connection with the observed cytoplasmic localization, this result suggests that gene expression of the killer DNA plasmids is entirely cytoplasmic.

Cloning, Molecular↗
PubMed1991

A single-stranded gap in human immunodeficiency virus unintegrated linear DNA defined by a central copy of the polypurine tract.

The structure of unintegrated human immunodeficiency virus type 1 (HIV-1) DNA from acutely infected human lymphoid cells was analyzed by nuclease S1 cleavage. We observed a unique, discrete single-stranded gap in unintegrated linear DNA molecules, located near the center of the genome. Oligonucleotide primer extension experiments determined that the downstream limit of this gap coincides with the last nucleotide of a central copy of the polypurine tract found in all sequenced lentivirus genomes. Other retroviruses have only one copy of the polypurine tract at the 5' boundary of the 3' long terminal repeat, which has been shown to determine initiation of retroviral DNA plus-strand synthesis. We conclude from our observations that the central repeat of the polypurine tract can create an additional site for plus-strand synthesis initiation in lentiviruses. The central single-stranded gap was not found in circular DNA molecules, the vast majority of them carrying only one long terminal repeat. This finding suggests that the generation of such circular molecules is associated with early DNA ligation events.

Base Sequence↗
PubMed1984

Nucleotide sequence and transcription analysis of a linear DNA plasmid associated with the killer character of the yeast Kluyveromyces lactis.

In killer strains of the yeast Kluyveromyces lactis, production of a protein toxin which inhibits the growth of sensitive yeast cells is associated with the presence of two linear DNA plasmids, k1 and k2. We have determined the nucleotide sequence of the smaller plasmid k1 (8.9kb) which is thought to carry the structural gene(s) encoding the toxin. The plasmid has a low G + C content (26.8%) and contains four long open reading frames which account for over 95% of the total sequence. The longest open reading frame (1146 amino acids) probably corresponds to a structural gene for the killer toxin. Transcripts from three of the putative genes have been detected in K.lactis by Northern hybridisation.

Amino Acid Sequence↗
PubMed1993

Atomic force microscopy of oriented linear DNA molecules labeled with 5nm gold spheres.

The atomic force microscope (AFM;1) can image DNA and RNA in air and under solutions at resolution comparable to that obtained by electron microscopy (EM) (2-7). We have developed a method for depositing and imaging linear DNA molecules to which 5nm gold spheres have been attached. The gold spheres facilitate orientation of the DNA molecules on the mica surface to which they are absorbed and are potentially useful as internal height standards and as high resolution gene or sequence specific tags. We show that by modulating their adhesion to the mica surface, the gold spheres can be moved with some degree of control with the scanning tip.

DNA↗
PubMed1998

Ku70/Ku80 protein complex inhibits the binding of nucleotide excision repair proteins on linear DNA in vitro.

We have previously reported that the incision efficiency of the nucleotide excision repair (NER) reaction measured in vitro with cell-free human protein extracts was reduced by up to 80% on a linearized damaged plasmid DNA substrate when compared to supercoiled damaged DNA. The inhibition stemed from the presence of the DNA-end binding Ku70/Ku80 heterodimer which is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). Here, the origin of the repair inhibition was assessed by a new in vitro assay in which circular or linear plasmid DNA, damaged or undamaged, was quantitatively adsorbed on sensitized microplate wells. The binding of two NER proteins, XPA and p62-TFIIH, indispensable for the incision step of the reaction, was quantified either directly in an ELISA-like reaction in the wells with specific antibodies or in Western blotting experiments on the DNA-bound fraction. We report a dramatic inhibition of XPA and p62-TFIIH association with UVC photoproducts on linear DNA. XPA and p62-TFIIH binding to DNA damage was regained when the reaction was performed with extracts lacking Ku activity (extracts from xrs6 rodent cells) whereas addition of purified human Ku complex to these extracts restored the inhibition. Despite the fact that DNA-PK was active during the NER reaction, the mechanism of inhibition relied on the sole Ku complex, since mutant protein extracts lacking the catalytic DNA-PK subunit (extracts from the human M059J glioma cells) exhibited a strong binding inhibition of XPA and p62-TFIIH proteins on linear damaged DNA, identical to the inhibition observed with the DNA-PK+ control extracts (from M059K cells).

Animals↗
PubMed1984

Homologous in vitro transcription of linear DNA fragments containing the tRNAArg-tRNAAsp gene pair from Saccharomyces cerevisiae.

Transcription of a tRNAArg-tRNAAsp gene pair from Saccharomyces cerevisiae by an homologous yeast extract results in a dimeric percursor molecule which is processed to mature-sized tRNAArg and tRNAAsp molecules. We have transcribed linear DNA fragments cleaved within the gene sequences to show that precursor synthesis is not dependent on the internal promoter of the second gene (tRNAAsp). Furthermore, the second gene does not support independent transcription when the normal upstream initiation site is removed.

DNA, Fungal↗
PubMed2002

Nucleosome-like complex of the histone from the hyperthermophile Methanopyrus kandleri (MkaH) with linear DNA.

The MkaH protein from the archaeon Methanopyrus kandleri, an unusual assembly of two histone-fold domains in a single polypeptide chain, demonstrates high structural similarity to eukaryal histones. We studied the DNA binding and self-association properties of MkaH by means of the electrophoretic mobility shift assay (EMSA), electron microscopy (EM), chemical cross-linking, and analytical gel filtration. EMSA showed an increased mobility of linear DNA complexed with MkaH protein with a maximum at a protein-DNA weight ratio (R(w)) of approximately 3; the mobility decreased at higher protein concentration. EM of the complexes formed at Rw or=9) thickened compact nucleoprotein structures were observed; no individual loops were seen within the complexes. Gel filtration chromatography and chemical fixation indicated that in the absence of DNA the dominant form of the MkaH in solution, unlike other archaeal histones, is a stable dimer (pseudo-tetramer of the histone-fold domain) apparently resembling the eukaryal (H3-H4)(2) tetramer. Similarly, dimers are the dominant form of the protein interacting with DNA. The properties of MkaH supporting the assignment of its intermediate position between other archaeal and eukaryal histones are discussed.

Archaeal Proteins↗
PubMed2006

Complete DNA sequences of the mitochondrial genomes of the pathogenic yeasts Candida orthopsilosis and Candida metapsilosis: insight into the evolution of linear DNA genomes from mitochondrial telomere mutants.

We determined complete mitochondrial DNA sequences of the two yeast species, Candida orthopsilosis and Candida metapsilosis, and compared them with the linear mitochondrial genome of their close relative, C.parapsilosis. Mitochondria of all the three species harbor compact genomes encoding the same set of genes arranged in the identical order. Differences in the length of these genomes result mainly from the presence/absence of introns. Multiple alterations were identified also in the sequences of the ribosomal and transfer RNAs, and proteins. However, the most striking feature of C.orthopsilosis and C.metapsilosis is the existence of strains differing in the molecular form of the mitochondrial genome (circular-mapping versus linear). Their analysis opens a unique window for understanding the role of mitochondrial telomeres in the stability and evolution of molecular architecture of the genome. Our results indicate that the circular-mapping mitochondrial genome derived from the linear form by intramolecular end-to-end fusions. Moreover, we suggest that the linear mitochondrial genome evolved from a circular-mapping form present in a common ancestor of the three species and, at the same time, the emergence of mitochondrial telomeres enabled the formation of linear monomeric DNA forms. In addition, comparison of isogenic C.metapsilosis strains differing in the form of the organellar genome suggests a possibility that, under some circumstances, the linearity and/or the presence of telomeres provide a competitive advantage over a circular-mapping mitochondrial genome.

Base Sequence↗