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PubMedDate not supplied

[Kinetics of the reactions joining DNA fragments, catalyzed by DNA-ligase. II. Heterogeneous mixture of fragments. Optimization of the reaction conditions].

The system of kinetic equations was proposed describing the joining of heterogeneous DNA fragments by DNA-ligase. The DNA fragments were considered as flexible rods and characterized only by molecular weights and concentrations. The program for calculating the concentrations of all circular and linear DNA forms is described. The program has been written in FORTRAN IV and calculations were made with EC 10--10. For the maximal yield of the multimeric circular DNA molecules it is recommended to begin the ligation at a high DNA concentration and then after a certain fixed time to dilute the reaction mixture. The optimum time intervals and ratios of fragment concentrations were calculated. The yield of different ligation products has been determined. The calculations of optimum reaction conditions were carried out for the construction of hybrid molecules containing DNA fragments with or without selective genetic markers.

DNA↗
PubMed1988

N4 virion DNA dependent-RNA polymerase: initiation sequences utilized by the enzyme on heterologous templates.

Bacteriophage N4 virion-encapsulated RNA polymerase, the enzyme responsible for transcription of the phage early RNAs, is unable to use duplex linear DNA as a template. In contrast to other RNA polymerases, the enzyme transcribes denatured N4 DNA with in vivo specificity. The promoter sequences for three sites of transcription initiation on the N4 genome have been determined and found to contain conserved sequences and two sets of inverted repeats. In order to define the minimal sequence requirements for N4 virion RNA polymerase activity, we have screened several heterologous DNAs, amounting to 64,328 bases, for their ability to support transcription. Several sequences allowing specific initiation were found. Their location, properties and the relation to N4 virion RNA polymerase promoters are discussed.

Coliphages↗
PubMed1980

Localization of the binding sites of prokaryotic and eukaryotic RNA polymerases on simian virus 40 DNA.

The binding sites of calf thymus RNA polymerase (B) II, wheat germ RNA polymerase B and of the Escherichia coli RNA polymerase were mapped on the simian virus 40 genome by observation of enzyme-linear DNA complexes by electron microscopy. Three to four major sites and several minor sites are observed for each enzyme; common binding sites for the three enzymes are found in positions 0.17, 0.53 and 0.90 of the viral physical map. Initiation complexes with these enzymes can be stabilized with specific ribodinucleotides and a single ribonucleoside triphosphate. Whereas ApA and ATP greatly enhances the binding of the E. coli enzyme at position 0.17, they stabilize the binding of the eukaryotic enzyme at many sites, some of them located in close proximity of the origin of replication.

Adenosine Monophosphate↗
PubMed1986

Gene-sized macronuclear DNA molecules are clustered in micronuclear chromosomes of the ciliate Oxytricha nova.

Following the sexual phase of its life cycle, the hypotrichous ciliate Oxytricha nova transforms a copy of its chromosomal micronucleus into a macronucleus containing short, linear DNA molecules with an average size of 2.2 kilobase pairs. In addition, more than 90% of the DNA sequences in the micronuclear genome are eliminated during this process. We have examined the organization of macronuclear DNA molecules in the micronuclear chromosomes. Macronuclear DNA molecules were found to be clustered and separated by less than 550 base pairs in two cloned segments of micronuclear DNA. Recombinant clones of two macronuclear DNA molecules that are adjacent in the micronucleus were also isolated and examined by DNA sequencing. The two macronuclear DNA molecules were found to be separated by only 90 base pairs in the micronuclear genome.

Animals↗
PubMed1995

Tandem integration of multiple ILV5 copies and elevated transcription in polyploid yeast.

An industrial yeast strain was modified by introducing DNA into brewing yeast such that the derived cells contain only yeast DNA. Thus selectable markers and bacterial sequences are not present in the final strain, making this procedure attractive for the development of generally acceptable brewing yeast. Linear DNA containing the cloned ILV5 gene was introduced into lager yeast along with an unlinked circular bifunctional plasmid containing a dominant resistance marker. Resistant colonies were screened for site-directed integration of the ILV5 DNA. Candidates were examined by several methods including Southern transfer and polymerase chain reaction. In this way, a strain WM56 was identified containing three tandem copies of ILV5. The amplified ILV5 region is stable during repeated subculturing in the absence of selective pressure. Correspondingly elevated levels of ILV5 transcript in strain WM56 compared to the control (i.e. non-tandem) parental strain led to increased amounts of encoded acetohydroxyacid reductoisomerase as evidenced by significantly lower diacetyl production. WM56 appears to be identical to the parental strain judged by CHEF, total restriction digestion patterns, and probing, but differs in the ILV5 region of the chromosome. The method is generally applicable to other yeast strains, and if desired, is amenable to iterated cycles of integration to increase the number of copies.

Genes, Fungal↗
PubMed1989

Estimation of circular DNA size using gamma-irradiation and pulsed-field gel electrophoresis.

A method is described for estimating the size of large circular DNAs found within complex chromosomal DNA preparations. DNAs are treated with low levels of gamma-irradiation, sufficient to introduce a single double-stranded break per circle, and the resulting linear DNA is sized by pulsed-field electrophoresis and blot hybridization. The method is fast, reproducible, and very conveniently applied to the agarose-enclosed chromosomal DNA preparations commonly used in pulsed field electrophoresis.

Animals↗
PubMed1994

The actual incision determines the efficiency of repair of cisplatin-damaged DNA by the Escherichia coli UvrABC endonuclease.

The UvrABC endonuclease from Escherichia coli repairs a broad spectrum of DNA lesions with variable efficiencies. The effectiveness of repair is influenced by the nature of the lesion, the local DNA sequence, and/or the topology of the DNA. To get a better understanding of the aspects of this multistep repair reaction that determine the effectiveness of repair, we compared the incision efficiencies of linear DNA fragments containing either a site-specific cis-[Pt(NH3)2(d(GpG)-N7(1),-N7(2)]] or a cis- Pt(NH3)2[d(GpCpG)-N7(1),-N7(3)]] adduct. Overall the DNA with the cis-PtGG adduct was incised about 3.5 times more efficiently than the cis-Pt.GCG-containing DNA. The rate of UvrB-DNA preincision complex formation for both lesions was similar and high in relation to the incision. DNase I footprints, however, showed that the local structure of the two preincision complexes is different. An assay was developed to measure the binding of UvrC to the preincision complexes and it was found that the binding rate of UvrC to the more slowly incised cis-Pt.GCG preincision complex was higher than to the cis-Pt.GG preincision complex. This most likely reflects a qualitative difference in preincision complex structures. For both lesions the binding of UvrC to the preincision complex was fast compared to the kinetics of actual incision. Apparently, direct incision of cisplatin damage requires an additional conformational change after the binding of UvrC.

Adenosine Triphosphatases↗
PubMed2006

Polychlorinated biphenyl quinone metabolites poison human topoisomerase IIalpha: altering enzyme function by blocking the N-terminal protein gate.

Polychlorinated biphenyls (PCBs) are associated with a broad spectrum of human health problems and cause cancer in rodents. In addition, these compounds cause chromosomal aberrations in humans and treated human cells. Although the underlying basis for the chromosomal damage induced by PCBs is not understood, it is believed that these compounds act through a series of phenolic and quinone-based metabolites. Recent studies indicate that several quinones that promote chromosomal damage also act as topoisomerase II poisons. Therefore, the effects of PCB quinone metabolites (including mono and dichlorinated compounds and p- and o-quinones) on the activity of human topoisomerase IIalpha were examined. Results indicate that these compounds are potent topoisomerase IIalpha poisons in vitro and act by adducting the enzyme. They also increase DNA cleavage by topoisomerase IIalpha in cultured human cells. In contrast, incubation of topoisomerase IIalpha with PCB metabolites in the absence of DNA leads to a rapid loss of enzyme activity. On the basis of (1) the differential ability of quinone-treated enzyme to bind circular and linear DNA molecules and (2) the generation of salt-stable noncovalent complexes between topoisomerase IIalpha and circular plasmids in the presence of PCB quinones, it appears that these compounds alter enzyme function (at least in part) by blocking the N-terminal gate of the protein. Finally, exposure to quinones generates a protein species with a molecular mass approximately twice that of a monomeric topoisomerase IIalpha protomer. This finding suggests that PCB quinones block the N-terminal gate by cross-linking the protomer subunits of topoisomerase IIalpha.

Antigens, Neoplasm↗
PubMed1991

Melting of oligodeoxynucleotides with various structures.

Effects of DNA fragments end structures on their melting profiles were studied experimentally and theoretically. We examined melting of hairpins and dumbbells obtained from 62-bp-long linear DNA duplex which is a perfect palindromic sequence. To fit theoretical melting profile to experimental ones additional theoretical parameters were incorporated into the standard statistical mechanical helix-coil transition theory. From comparison theoretical and experimental melting profiles theoretical parameters connected with end-structure effects were evaluated. Analysis revealed the stabilization effect of the hairpin loops and helix ends with respect to DNA duplex melting. Both type of ends make melting these oligodeoxynucleotides more cooperative than predicted by the standard helix-coil transition theory. At low ionic strength ([Na+] less than 0.04 M) this effect becomes so pronounced that melting of the DNA duplexes 30-40 bp-long conforms to the two state model. From the analysis experimental data obtained for dumbbell structures loop-weighting factor for single-stranded loop consisting of 132 nucleotides was determined. This parameter decreases 10 times with the ionic strength decreasing by an order of magnitude from 0.2 to 0.02 M Na+.

Base Sequence↗
PubMed1994

Distribution of exchanges upon homologous recombination of exogenous DNA in Xenopus laevis oocytes.

Homologous recombination between DNA molecules injected into Xenopus oocyte nuclei was investigated by examining the recovery of information from differentially marked parental sequences. The injected recombination substrate was a linear DNA with terminal direct repeats of 1246 bp; one repeat differed from the other by eight single base-pair substitutions, distributed throughout the region of homology, each of which created or destroyed a restriction enzyme site. Recombination products were recovered and analyzed for their content of the diagnostic sites, either directly by Southern blot-hybridization or after cloning in bacteria. The majority (76%) of the cloned products appeared to be the result of simple exchanges-i.e., there was one sharp transition from sequences derived from one parent to sequences derived from the other. These simple exchanges were concentrated near the ends of the homologous interval and, thus, near the sites of the original molecular ends. Placing marked sites on only one side of the homologous overlap showed that marker recovery was governed largely by the positions of the molecular ends and not by the markers themselves. When a terminal nonhomology was present at one end of the substrate, the yield of recombinants was sharply decreased, but the pattern of exchanges was not affected, suggesting that products from end-blocked substrates arise by the same recombination pathway. Because of considerable evidence supporting a nonconservative, resection-annealing mechanism for recombination in oocytes, we interpret the distribution of exchanges as resulting from long-patch repair of extensive heteroduplex intermediates.

Animals↗
PubMedDate not supplied

DNA-binding properties of the recombinant high-mobility-group-like AT-hook-containing region from human BRG1 protein.

The hBRG1 protein, a central ATPase of the human switching/sucrose non-fermenting (SWI/SNF) remodeling complex, has a catalytic ATPase domain, an AT-hook motif and a bromodomain. Bromodomains, found in many chromatin-associated proteins, recognize N-acetyl-lysine in histones and other proteins. The AT-hook motif, first described in the high-mobility group of non-histone chromosomal proteins HMGA1/2, is a DNA-binding motif. The AT-hook binds to the AT-rich DNA sequences in the minor groove of B-DNA in a non-sequence specific manner. AT-hook motifs have been identified in many other DNA-binding proteins. In this study we cloned and purified a fragment of hBRG1 encompassing the AT-hook region and the bromodomain. Nuclear magnetic resonance (NMR) and circular dichroism (CD) analyses show that the recombinant domains are structured. The functionality of subdomains was checked by assessing their interactions with N-acetylated peptides from histones and with DNA. Isothermal titration calorimetric (ITC) analysis demonstrates that the primary micromolar interaction is through the AT-hook motif. The AT-hook region binds to linear DNA by unwinding it. These properties resemble the characteristics of the HMGA1/2 proteins and their interaction with DNA.

Acetylation↗
PubMed1997

[Inhibitory effect of trisodium phosphonoformate (PFA) on duck hepatitis B virus (DHBV) DNA in vivo].

This paper described that DHBV infected ducklings were given with PFA 250 mg, 125 mg, 62.5 mg/kg b. i. d for 21 days intraperitioneally (i.p.) and dynamics of duck hepatitis B virus DNA (DHBV DNA) and duck hepatitis B surface antigen (DHBsAg) were detected in serum and distribution of DHBV DNA was tested in liver, kidney, pancreas and spleen. Supercoiled DNA (SC DNA) extracted from liver was analysed with Southern blot. The results showed that PFA in a dose of 125 mg and 250 mg/kg inhibited significantly the serum DHBsAg; doses of 125 mg and 250 mg/kg inhibited the serum DHBV DNA from 14 to 21 days, and dose of 250 mg/kg inhibited the serum DHBV DNA earlier even at 7 days after being given with PFA. Doses of 125 mg and 250 mg/kg inhibited obviously DHBV DNA in liver and kidney, but no inhibition was found in spleen and pancreas. The replication of DHBV DNA in liver of ducklings treated with PFA in a dose of 250 mg/kg for 21 days showed that the PFA inhibited significantly the synthesis of linear DNA (L DNA), relaxed circular DNA (RC DNA) and SC DNA. But DHBsAg and DHBV DNA in serum and DHBV DNA in liver, kidney, pancreas and spleen after treated with PFA for 21 days were still present, this meant that PFA treatment didn't clear off the virus genome in infected ducklings, the "rebounding" of DHBV appeared after cessation of the PFA treatment.

Animals↗
PubMed1985

Dynamics of DNA polymerase III holoenzyme of Escherichia coli in replication of a multiprimed template.

Movements of DNA polymerase III holoenzyme (holoenzyme) in replicating a template multiprimed with synthetic pentadecadeoxynucleotides (15-mers) annealed at known positions on a single-stranded circular or linear DNA have been analyzed. After extension of one 15-mer on a multiprimed template, holoenzyme moves downstream in the direction of chain elongation to the next primer. Holoenzyme readily traverses a duplex, even 400 base pairs long, to exploit its 3'-hydroxyl end as the next available primer. This downstream polarity likely results from an inability to diffuse upstream along single-stranded DNA. These holoenzyme movements, unlike formation of the initial complex with a primer, do not require ATP. Time elapsed between completion of a chain and initiation on the next downstream primer is rapid (1 s or less); dissociation of holoenzyme to form a complex with another primed template is slow (1-2 min). Thus, holoenzyme diffuses rapidly only on duplex DNA, probably in both directions, and forms an initiation complex with the first primer encountered. Based on these findings, schemes can be considered for holoenzyme action at the replication fork of a duplex chromosome.

Adenosine Triphosphate↗
PubMed1977

A restriction endonuclease cleavage map of mouse mitochondrial DNA.

A restriction endonuclease cleavage map is presented for mouse mitochondrial DNA. This map was constructed by electron microscopic measurements on partial digests containing fixed D-loops, and by electrophoretic analysis of partial and complete single enzyme digests, and of double digests. No map differences were detected between mitochondrial DNA from cultured LA9 cells and an inbred mouse line for the six endonucleases used. Three cleavage sites recognized by HpaI, five sites recognized by HincII, two sites recognized PstI and four sites recognized by BamI were located with respect to the origin of replication and the EcoRI and HinIII sites previously determined by others. No cleavages were produced by KpnI or SalI. The migration of linear DNA with a molecular weight greater than 1 X 10(6) was not a linear function of log molecular weight in 1% agarose gels run at 6.6 volts/cm.

Animals↗
PubMed1993

DNA sequence analysis of conserved and unique regions of swinepox virus: identification of genetic elements supporting phenotypic observations including a novel G protein-coupled receptor homologue.

Swinepox virus (SPV) contains a double-stranded cross-linked linear DNA genome of approximately 175 kilobase pairs with terminal inverted repetitions (TIRs) of 4.3 kb. The nucleotide sequence was determined for fragments from several regions of the genome including a 2.85-kb fragment from the central potentially conserved portion and two fragments within the presumed variable near-terminal regions which tend to be unique to a given poxvirus. The core sequence contains one partial and two complete open reading frames that are highly conserved and colinear with three contiguous ORFs within the HindIII D fragment of vaccinia virus (VV). The two near-terminal fragments, encompassing 14.2 and 3.6 kb, are respectively located 2.1 kb internal to the left and right cross-linked termini of the DNA and span the TIR junctions. The sequences encode 25 open reading frames including numerous proteins predicted to be membrane-bound or secreted in infected cells. Several ORFs unique to SPV were identified that may be involved in cell attachment, immune modulation, and pathogenesis including a novel poxvirus G protein-coupled receptor. In addition, several polypeptides encoded within the near-terminal regions of vaccinia virus DNA that function as host range or virulence factors are lacking within this region of swinepox virus including the VV growth factor, complement-binding protein, and ORFs C7L and K1L, associated with host range. The lack of these functional homologues could explain the characteristic attenuated phenotype and limited host range of SPV.

Amino Acid Sequence↗
PubMed1999

Streptomyces genomes: circular genetic maps from the linear chromosomes.

Streptomyces chromosomes are linear DNA molecules and yet their genetic maps based on linkage analysis are circular. The only other known examples of this phenomenon are in the bacteriophages T2 and T4, the linear genomic sequences of which are circularly permuted and terminally redundant, and in which replication intermediates include long concatemers. These structural and functional features are not found in Streptomyces. Instead, the circularity of Streptomyces genetic maps appears to be caused by a completely different mechanism postulated by Stahl & Steinberg (1964, Genetics 50, 531-538)--a strong bias toward even numbers of crossovers during recombination creates misleading genetic linkages between markers on the opposite arms of the chromosome. This was demonstrated by physical inspection of the telomeres in recombinant chromosomes after interspecies conjugation promoted by a linear or circular plasmid. The preference for even numbers of crossovers is probably demanded by the merozygosity of the recombining chromosomes, and by the association between the telomeres mediated by interactions of covalently bound terminal proteins.

Chromosome Mapping↗
PubMed1985

SV40 chromatin structure is not essential for viral gene expression.

The biological activity and the fate of SV40 DNA (minichromosomes, DNA I, DNA II, DNA III) were tested in culture cells by immunofluorescence staining and blot analysis. Following microinjection of 2-4 circular SV40 molecules (minichromosomes, DNA I, DNA II) into the cytoplasm or the nuclei of monkey and rat cells, T- and V-antigen synthesis was demonstrable in nearly every recipient cell. Only linear DNA induced T-antigen synthesis with a very low efficiency after cytoplasmic injection. This low activity correlates with a rapid degradation of DNA III in the recipient cells. Further modifications observed immediately after injection are relaxation of superhelical molecules and formation of high-Mr DNA. Assembly of the injected DNA into SV40 chromatin-like structure, however, occurred only late after early viral gene expression.

Animals↗
PubMed1982

Purification and properties of a neutral endodeoxyribonuclease from rat small intestinal mucosa.

An endodeoxyribonuclease has been purified to near homogeneity from rat small intestinal mucosa by a procedure involving Con A-Sepharose affinity chromatography. During the initial steps of purification, the presence of 5 mM CaCl2 was essential for stability of the enzyme activity. The enzyme has a molecular weight of 32 000 and an isoelectric point of 4.7. NaCl, sulfhydryl reagents, and iodoacetate strongly inhibited the reaction, but tRNA did not. The enzyme required divalent cations for activity and had a pH optimum of pH 6.2 with Co2+ and pH 7.7 with Mn2+. In both optimum conditions, the enzyme hydrolyzed native DNA more rapidly than denatured DNA, and the average chain lengths of limit digestion products of native and denatured DNA were 8 and 10, respectively, at pH 6.2 and 9 and 11, respectively, at pH 7.7. The enzyme activity to produce acid-soluble fractions from linear DNA substrate was similar in the two optimum conditions, but the activity to nick double-stranded, superhelical circular DNA substrate was significantly higher at pH 6.2 than at pH 7.7. The endonuclease formed single-strand breaks making 5'-phosphoryl and 3'-hydroxyl termini, and deoxythymidine was present at the 5' termini with a frequency of about 50% in both optimum conditions. Bovine pancreatic DNase I antibody and G-action inhibited the enzyme activity. Thus this endonuclease is classified as a DNase I.

Animals↗