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At least 91 records · Page 5Linked to original sources
PubMed1988

Transfection of the hypotrichous ciliate Stylonychia lemnae with linear DNA vectors.

We present a new protocol for the transfection of Stylonychia lemnae with linear DNA vectors containing the neomycin-resistance gene from Escherichia coli transposon Tn5. The taking up of heterologous DNA is achieved by damaging the cells' protein coat with urea prior to transfection according to the calcium phosphate co-precipitation procedure. After transfection, transformed cells can be enriched by selection with the antibiotic drug G418. Hybridization experiments show that macronuclear DNA of these G418-selected cells contains molecules homologous to the transfected vector DNA, which are altered by some recombination process. Transfected cells, which have grown for more than 100 cell cycles in antibiotic-free medium, still contain vector-homologous DNA, but recombination continued during this time. We are able to transfect Stylonychia early after conjugation, a process which is followed by complex genome rearrangements and amplification. In these experiments we observed considerable amplification of vector-homologous DNA molecules as compared to transfected vegetative cells. Again these molecules are altered by recombination in respect to the original vector DNA. As soon as suitable vectors are available, the transfection protocol presented here can be a basic tool for the study of DNA replication, transcription and macronuclear development in Stylonychia.

Animals↗
PubMed2003

Expression and localization of the linear DNA plasmid-encoded protein (RS224) in Rhizoctonia solani AG2-2.

Expression of the linear DNA plasmid-encoded protein (RS224) from the plant-pathogenic fungus Rhizoctonia solani isolate H-16, anastomosis group 2-2, and its localization were studied. Extracts from Escherichia coli cells expressing the open reading frame (ORF) of RS224 (RS224ORF in pRS224) contain a 92-kDa T7.Tag-RS224orf fusion protein. Antisera raised against the fusion protein obtained from E. coli cells cross-reacted with a 90-kDa protein in the mycelia. To analyze the subcellular localization of the 92-kDa protein, mycelia of R. solani were disrupted and fractionated. Antibodies against RS224 proteins specifically reacted to the mitochondrial fraction, suggesting that RS224 is localized in mitochondria.

Base Sequence↗
PubMed1983

In vitro coupled transcription-translation of linear DNA fragments in a lysate derived from a recB rna pnp strain of Escherichia coli.

A recB21 derivative (CLB7) of an Escherichia coli rna-19 pnp-7 strain (PR7) was constructed for use in examining the in vitro coupled transcription-translation of linear DNA. The expression of linearized DNAs in CLB7 (recB21 rna-19 pnp-7) lysates was enhanced significantly when compared with expression of the same DNAs in lysates prepared from the PR7 or the original recB21 (CF300) strains. In addition, the endogenous incorporation of [35S]methionine into protein was considerably reduced in CLB7 lysates relative to lysates derived from the original recB21 strain.

Bacterial Proteins↗
PubMed1987

Terminal segment of Kluyveromyces lactis linear DNA plasmid pGKL2 supports autonomous replication of hybrid plasmids in Saccharomyces cerevisiae.

By use of linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis we have constructed hybrid plasmids carrying a LEU2 gene of Saccharomyces cerevisiae as a selectable marker. The replication properties of hybrid plasmids in yeasts were investigated. We demonstrated that the insertion of a LEU2 gene into pGKL2 resulted in circularization of the hybrid plasmids and pGKL2 segment supported autonomous replication of the plasmids. Moreover, the hybrid plasmids propagated autonomously, independently of the presence of the natural pGKL2 plasmid.

Base Sequence↗
PubMed1984

Characterization of a novel killer toxin encoded by a double-stranded linear DNA plasmid of Kluyveromyces lactis.

A novel killer toxin, encoded by a double-stranded linear DNA plasmid pGK l-1 (5.4 MDa) in Kluyveromyces lactis IFO 1267 was purified 320 000-fold from the culture broth of yeast. The toxin was obtained in an electrophoretically homogeneous state with a yield of 24% by hydroxyapatite column chromatography, chromatofocusing and polyacrylamide gel electrophoresis. The purified toxin was dissociated into two subunits with molecular masses of 27 kDa and above 80 kDa, as estimated by Laemmli's sodium dodecylsulfate gel electrophoresis; the exact composition ratio of the two subunits remains unestablished. The isoelectric point was between 4.4 and 4.8. As compared with the reported narrow pH range of action and instability of k1 killer toxin encoded by a double-stranded RNA plasmid of Saccharomyces cerevisiae, the K. Lactis toxin was effective with sensitive strains of S. cerevisiae in a relatively wider pH range between 4 and 8; it was stable for several months at pH 6.0 when stored below -20 degrees C. In contrast to the simple protein nature of the k1 killer toxin with a molecular mass of 11.47 kDa, the K. lactis toxin maintained a mannoprotein nature, as it was absorbed by a ConA-Sepharose column and eluted by methyl alpha-D-mannoside. The growth inhibitory activity of K. lactis toxin was enhanced 2-35-fold by the presence of 4-60% glycerol.

Ascomycota↗
PubMed1984

Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments.

Deletion of an essential gene in Escherichia coli was accomplished by transformation of linear DNA fragments that have a Kanr gene segment flanked by sequences homologous to closely spaced regions on the E. coli chromosome. Selection for a double crossover within homologous sequences can effectively delete an entire gene. Cell viability is maintained by provision of the essential gene on a plasmid with a temperature-sensitive replicon, resulting in cells which have a temperature-sensitive phenotype.

Base Sequence↗
PubMed1978

Studies on the replication of KRV single-stranded linear DNA.

The autonomous parvovirus, Kilham rat virus (KRV), is composed of three structural proteins (Salzman & White, 1970) and a single molecule of DNA. The DNA molecule is linear and single-stranded (ss). It is believed to have both 3' and 5'-terminal palindromic sequences (Salzman, 1977). Replication of the ss DNA may involve double-stranded (ds) DNA intermediates of one unit length as found in the virion or multiple length concatamers (Salzman & White, 1973; Gunther & May, 1976; Lavelle & Li, 1977). We have attempted to determine when the synthesis of KRV ds DNA can be determined in synchronized infected cells and the structure of the ds DNA molecules.

Culture Techniques↗
PubMed1991

Telomeres inhibit end to end fusion and enhance maintenance of linear DNA molecules injected into the Paramecium primaurelia macronucleus.

Direct injection into the macronucleus of Paramecium tetraurelia of DNA molecules coding for the A-antigen leads to expression of the gene and autonomous replication. When injected into Paramecium primaurelia DNA from probably any origin, procaryote or eucaryote, can replicate as linear telomerized molecules and the number of copies maintained can be very high (up to 20000 copies). We present here evidence that if the injected linear DNA molecules harbour preexisting telomeres at both extremities they are protected from degradation, the number of DNA molecules maintained being 15- to 30-fold higher than if the molecules are injected without telomeres. Some of the injected molecules replicate as multimers, but, only when the fused ends are devoid of preexisting telomeric repeats.

Animals↗
PubMed1989

Identification of temperature-sensitive mutants of vaccinia virus that are defective in conversion of concatemeric replicative intermediates to the mature linear DNA genome.

Pulsed-field gel electrophoresis was used to screen temperature-sensitive mutants of vaccinia virus for the ability to convert replicated viral DNA into mature linear 185-kilobase hairpin-terminated genomes. Of 30 mutually noncomplementing mutants tested, 5 displayed a temperature-sensitive defect in the resolution of the telomere fusion configuration within concatemeric replicative intermediates, resulting in a failure to convert such intermediates to the linear monomeric genome. Adjacent genomic units in the concatemeric arrays generated in these mutants were arranged in both tandem and inverted orientations. The observation that four of the five mutants had a severe general defect in the synthesis of the late class of viral proteins suggests that at least one late protein is directly required to resolve the telomere fusion intermediate to hairpin termini. The identification of such telomere resolution proteins should be facilitated by genetic and molecular characterization of resolution-defective mutants, such as C63, in which late protein synthesis is not severely affected.

Animals↗
PubMed1993

Sequence-dependent reactivity of linear DNA to chemical cleavage by Cu(II):thiol combinations including cysteine or glutathione.

The chemical cleavage of linear DNA by Cu(II):thiol combinations was investigated using end-labelled double-stranded restriction fragments. Single-strand cleavage of the target DNA occurred with apparent sequence preference, but there appeared to be no apparent consensus sequence for such preferentially cleaved sites based on studies of three different restriction fragments. For any given restriction fragment, the observed Cu(II):thiol DNA-cleavage sequence preference was found to be independent of the nature of the thiol used. Cleavage of 3'-end-labelled DNA generated fragments bearing 5'-phosphoryl termini, and cleavage of 5'-end-labelled DNA gave rise to fragments bearing 3'-phosphoryl and 3'-'phosphatase-inert' termini in equal proportions. No appreciable amounts of fragments bearing 3'- or 5'-hydroxy termini were detected. This pattern of cleavage products is similar to that observed using the hydroxyl-radical-generating systems, 60Co gamma-irradiation or methidium propyl EDTA:Fe(II), but is dissimilar to that found with the Cu(II):phenanthroline or bleomycin systems. Cu(II):thiol cleavage of DNA fragments containing inserts of repeated dinucleotide sequence showed alternating patterns of relative cleavage intensities within the insert-sequence regions which were not seen in the sequences flanking the insert sequence. The assembled experimental data indicate that a reaction product of the Cu(II):thiol interaction, probably the hydroxyl radical, causes DNA cleavage. The Cu(II):thiol interaction may occur free in solution or via a copper species which is bound non-specifically to DNA. The observed low sequence-dependence of the cleavage reaction probably reflects the nature of the DNA structure itself, the Cu(II):thiol system serving as a structural probe of sequence-dependent structural variations in the DNA structure.

Base Sequence↗
PubMed1997

The complete nucleotide sequence and characterization of the linear DNA plasmid pRS64-2 from the plant pathogenic fungus Rhizoctonia solani.

The complete nucleotide sequence of one of three linear DNA plasmids (pRS64-2) from the plant pathogenic fungus Rhizoctonia solani was determined. The pRS64-2 DNA consisted of 2877 nucleotides. The nucleotide sequences of the middle 2.2-kb regions of the other two plasmids (pRS64-1 and pRS64-3) were also determined. Comparison of the nucleotide sequences among the three plasmid DNAs indicated the presence of four regions with more than 86% sequence homology, suggesting the development of three plasmid DNAs from a common ancestor. A computer-based study of the pRS64-2 DNA-folding at both termini predicted hairpin loop structures. The hairpin loops consisted of the left- and right-hand terminal 113 and 105 nucleotides, respectively, and had no sequence homology. They had not undergone flip-flop inversion. The hairpins form cruciform base-paired structures.

Base Sequence↗
PubMed1988

The cyclization of linear DNA in Escherichia coli by site-specific recombination.

The efficiency with which linearized plasmid DNA can transform competent Escherichia coli can be significantly increased by use of the Cre-lox site-specific recombination system of phage P1. Linear plasmid molecules containing directly repeated loxP sites (lox2 plasmids) are cyclized in Cre+ E. coli strains after introduction either by transformation or by mini-Mu transduction. Exonuclease V activity of the RecBC enzyme inhibits efficient cyclization of linearized lox2 plasmids after transformation. By use of E. coli mutants which lack exonuclease V activity, Cre-mediated cyclization results in transformation efficiencies for linearized lox2 plasmids identical to those obtained with covalently closed circular plasmid DNA. Moreover, Cre+ E. coli recBC strains allow the efficient recovery of lox2 plasmids integrated within large linear DNA molecules such as the 150-kb genome of pseudorabies virus.

Bacteriophages↗
PubMed2001

Complete nucleotide sequence of the linear DNA plasmid pRS224 with hairpin loops from Rhizoctonia solani and its unique transcriptional form.

The complete nucleotide sequence of the linear DNA plasmid (pRS224-1) from the plant-pathogenic fungus Rhizoctonia solani isolate H-16 was determined; and its unique RNA transcripts were characterized. The pRS224-1 DNA consists of 4,986 nucleotides. A computer-based study of the folding of pRS224-1 at both termini predicted hairpin-loop structures. The hairpin loops consisted of the left and right termini of 236 and 264 nucleotides, respectively, and share no sequence homology. Unique poly(A) RNAs, 4.7 kb and 7.4 kb in length and hybridizing with the pRS224 DNA, were found in mycelial cells of R. solani H-16. Transcript product-mapping allowed the prediction of the locations of different expression signals. The 7.4-kb transcript is generated from the left terminal region of the complementary strand, via the full-length sense-strand, to the right terminal region of the complementary strand. The 4.7-kb transcript is generated from the center region of the sense strand to the right terminal region of the complementary strand. One open reading frame (ORF) found in pRS224-1 is 887 amino acids long and has a potential coding capacity of 102 kDa. The ORF contains the highly conserved domains characteristic of reverse transcriptase sequences, including the highly conserved YXDD sequence.

Amino Acid Sequence↗
PubMedDate not supplied

[Entry of double-and single-stranded linear DNA in Ca2+-treated Escherichia coli cells].

Hydroxylapatite chromatography technique was used to investigate the conformational changes undergone by exogenous linear DNA during the uptake by Ca2+-treated EScherichia coli cells. Both single- (heat denatured) and double-stranded DNAs were examined. While native DNA preserved its double-strandedness during the uptake, the heat denatured (single-stranded) DNA was rapidly converted to the double-stranded form. This process was energy-dependent and is supposed to be dependent on gene recA product.

Biological Transport↗
PubMed1983

Enhanced polypeptide synthesis programmed by linear DNA fragments in cell-free extracts lacking exonuclease V.

Preparation of an in vitro coupled transcription-translation system from E. coli strains lacking exonuclease V has greatly improved the system for use with added linear DNA fragments. In fact, in extracts of these mutants linear fragments are stable for several hours. However the cell extracts show a high level of endogenous background. To avoid this complication extracts were prepared at 30 degrees C from a mutant carrying a temperature-sensitive exonuclease V. Polypeptides coded by a specific DNA region, e.g., delineated by restriction endonuclease sites, can now be easily identified.

DNA, Bacterial↗
PubMed2001

Cloning and nucleotide sequence of a linear DNA plasmid from Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

Extrachromosomal elements were found in a strain of X. dendrorhous, and were characterized as linear DNA forming two well defined groups, pPh1 with 3 high-copy-number molecules, pPh11 (6.9 kb), pPh12 (5.7), pPh13 (4.7), and pPh2 with 2 low-copy-number molecules, pPh21 (3.6 kb), pPh22 (3.0). A 4077 bp fragment from pPh13 was cloned in pUC18 (pDK1) and sequenced (accession no. AJ 278,424). Seven putative ORF and some possible regulator sequences were defined.

Amino Acid Sequence↗
PubMed1994

scid cells efficiently integrate hairpin and linear DNA substrates.

The scid mouse mutation affects V(D)J rearrangement and double-strand break repair. scid V(D)J rearrangement is characterized by defective coding joint formation which prevents the development of mature B and T cells. Hairpin DNA has been implicated in the formation of V(D)J coding joints. We found scid cells to be proficient in hairpin processing in the context of DNA integration. In addition, we found that the scid defect did not impair integration of linear DNA via nonhomologous recombination. Therefore, hairpin processing and integration of DNA into the genome are distinct from hypersensitivity to ionizing radiation and the defect in V(D)J recombination.

3T3 Cells↗