Circular Deoxyribonucleic Acid Molecules Research Articles

Ultrasonic atomization of the DNA solution for atomic force microscopy

We have investigated the ultrasonic atomization of the deoxyribonucleic acid (DNA) solution as a simple preparation technique to image circular DNA molecules with the atomic force microscope. The DNA solution with a relatively high concentration (∼25 μg/ml) is ultrasonically atomized at the exciting frequency of 2.4 MHz. Preliminary results show that this technique allows an even distribution of unaggregated strands.

Use of plasmid profiles in epidemiologic surveillance of disease outbreaks and in tracing the transmission of antibiotic resistance.

Plasmids are circular deoxyribonucleic acid molecules that exist in bacteria, usually independent of the chromosome. The study of plasmids is important to medical microbiology because plasmids can encode genes for antibiotic resistance or virulence factors. Plasmids can also serve as markers of various bacterial strains when a typing system referred to as plasmid profiling, or plasmid fingerprinting is used. In these methods partially purified plasma deoxyribonucleic acid species are separated according to molecular size by agarose gel electrophoresis. In a second procedure, plasmid deoxyribonucleic acid which has been cleaved by restriction endonucleases can be separated by agarose gel electrophoresis and the resulting pattern of fragments can be used to verify the identity of bacterial isolates. Because many species of bacteria contain plasmids, plasmid profile typing has been used to investigate outbreaks of many bacterial diseases and to trace inter- and intra-species spread of antibiotic resistance.

21] Site-specific mutagenesis to modify the human tumor necrosis factor gene

Publisher Summary The development of oligonucleotide-directed site-specific mutagenesis on single-stranded phage template has provided a powerful new technique in the study of structure-function relationships of proteins. This chapter describes a simplified procedure for oligonucleotide-directed site-specific mutagenesis on M13 single-stranded phage deoxyribo nucleic acid (DNA) templates. The rationale behind this approach is to make use of the DNA repair mechanism present in the E. coli host to repair the gapped M13 phage DNA into the covalently closed circular form before replication of the DNA molecules. The mutagenesis procedure described in this chapter provides a rapid method for the modification of cloned genes. It takes advantage of the ability of E. coli to efficiently repair gapped circular DNA molecules, to shorten the overall time of the mutagenesis, from initiation of the mutagenesis reaction to the identification of the mutant phage plaque. Although this faster method is less efficient in the conversion of parent DNA to mutant DNA molecules, the ability to use the same oligonucleotide primer as a probe to identify the mutant phage plaques effectively compensates for this inefficiency.

A multiple plasmid-containing Escherichia coli strain: Convenient source of size reference plasmid molecules

A strain of Escherichia coli is described that contains eight plasmid species ranging in size from 1.36 × 10 6 to 35.8 × 10 6 daltons. This strain can be employed as a single source of covalently closed circular deoxyribonucleic acid molecules of different sizes for use as references in agarose gel electrophoretic analysis.

Isolation of extrachromosomal deoxyribonucleic acids from extremely thermophilic bacteria.

Eight strains of thermophilic bacteria were examined for the presence of covalently closed circular deoxyribonucleic acid molecules by caesium chloride-ethidium bromide density gradient centrifugation. Four of the eight strains tested, Thermus flavus BS1, AT61, AT62 and Thermus thermophilus HB8 carried covalently closed circular DNA molecules. Thermus flavus BS1 haboured two species of plasmids with molecular weights of 6.1 X 10(6) and 17.0 X 10(6) as determined by electron microscopy. Thermus thermophilus HB8, T. flavus AT61 and T. flavus AT62 carried plasmids with molecular weights of 6.2 X 10(6), 6.6 X 10(6) and 6.6 X 10(6), respectively. Plasmids from T. flavus AT61 and AT62 were indistinguishable in their electrophoretic patterns in agarose or acrylamide gel after digestion with various restriction endonucleases. This is the first evidence for the presence of plasmids in extremely thermophilic bacteria, though their functions are unknown.

Generation of miniplasmids from copy number mutants of the R plasmid NR1.

Small, closed circular deoxyribonucleic acid molecules, called miniplasmids, were observed in Escherichia coli harboring copy number mutants of the R plasmid NR1 after growth in medium containing tetracycline. The level of tetracycline resistance conferred by the copy mutant plasmids was lower (3 to 6 microgram/ml) than that conferred by NR1 (100 MICROGRAM/ML). The presence of the miniplasmid enhanced the level of tetracycline resistance conferred by the copy mutant. Miniplasmids of molecular weights 4 X 10(6) to 13 X 10(6) were found. They carried no antibiotic resistance markers and could be eliminated by growth in the presence of chloramphenicol and/or streptomycin-spectinomycin. Studies with the restriction endonucleases EcoRI and Sal I indicated that the miniplasmids are derived from the region of the copy mutant plasmids that contains the origin for replication of the resistance transfer factor. There were approximately 12 copies of the miniplasmid per chromosome, compared with 3 and 6 copies of the copy mutants of NR1. The miniplasmids appeared to be incompatible with the copy mutant plasmids.

Extrachromosomal deoxyribonucleic acid in R factor-harboring Enterobacteriaceae.

Extrachromosomal deoxyribonucleic acid (DNA) from 24 different R factor-harboring Enterobacteriaceae was isolated and characterized by analytical ultracentrifugation and electron microscopy. The R factors represented 15 different patterns of transferable drug resistance found in enterobacteria from an enclosed geographic area. All of the strains contained extrachromosomal, circular DNA molecules within the range of 0.4 to 52 mum. More than one size class of circular DNA molecules was observed in the majority of the extrachromosomal DNA preparations. The buoyant density of the extrachromosomal DNA ranged from 1.700 to 1.720 g/cm3. The majority of the bacteria contained extrachromosomal DNAs of various densities. Three-fourths of the R factors were classified as fi+. The investigation illustrates the extensive variability in the physical characteristics of plasmid DNA from R factor-harboring strains.

Characterization of transmissible genetic elements from sucrose-fermenting Salmonella strains.

Two of seven sucrose-fermenting Salmonella strains obtained from clinical sources were found capable of conjugal transfer of the sucrose fermentation (Scr+) property to the Escherichia coli K-12 strain WR3026. The genetic elements conferring this Scr+ property, designated scr-53 and scr-94, were then conjugally transmissible from Escherichia coli WR3026 Scr+ exconjugants to other strains of Escherichia coli at frequences of 5 times 10- minus 6 to 5 times 10- minus 3 for the scr-53 element and 10- minus 6 to 10- minus 5 for the scr-94 element. In Escherichia coli hosts, both of these elements were compatible with F-lac and with each of six previously characterized transmissible lac elements. No antibiotic resistance characteristics or colicin production were discovered to be associated with either scr-53 or scr-94. Neither scr element generated a male host response to the female-specific phage phiII, but the scr-53 element rendered its Escherichia coli host sensitive to the male-specific phage R-17. Escherichia coli hosts containing scr-53 were susceptible to lysis by P1vir, and transduction of the scr-53 element was accomplished with this phage. The scr-53 element was isolated from Escherichia coli WR3026, Scr+ transductants, and Escherichia coli WR2036 Scr+ exconjugants as a covalently closed circular deoxyribonucleic acid molecule with a molecular weight (determined by electron microscopy) of approximately 52 times 10-6. Receipt of the scr-94 element rendered Escherichia coli hosts of this element unsusceptible to lysis by P1vir, although adsorption of the phage by an Escherichia coli WR3026 exconjugant containing scr-94 occurred as efficiently as it did on WR3026 itself. Repeated examination of Escherichia coli strains harboring scr-94, as well as of the Salmonella strain which initially contained it, did not reveal the presence of circular deoxyribonucleic acid. The synthesis of the sucrose cleaving enzyme was inducible in Escherichia coli exconjugants containing either scr-53 or scr-94.

Nature of R-factor replication in the presence of chloramphenicol.

Covalently closed circular deoxyribonucleic acid molecules of RSF1030, a nonconjugative R-factor, initiate and complete rounds of semiconservative replication in the absence of protein synthesis long after the bacterial chromosome has ceased its replication. RSF1030 replication under these conditions is sensitive to the inhibitor of ribonucleic acid synthesis, rifampicin. The product of this replication, a covalently closed DNA molecule, shows, in contrast to those molecules produced during the replication in a logarithmically growing culture of Escherichia coli, a transition to the open circular form upon treatment with ribonucleases of alkali. Analysis of the product resulting from alkali treatment indicates that there is a single break in one strand of the original circular duplex. This alkali-sensitive site occurs with equal probability in either of the complementary strands. These results are interpreted as a requirement for an RNA primer for the initiation of RSF1030 DNA synthesis and as showing that its removal from the covalently closed molecule is inhibited in the absence of protein synthesis.

Isolation and Characterization of Circular Deoxyribonucleic Acid Obtained from Lactose-Fermenting Salmonella Strains

Six lac elements originally contained in Salmonella strains were transferred to Escherichia coli WR3026. All of the six E. coli strains that received one of the lac elements were observed to contain supercoiled, circular deoxyribonucleic acid (DNA) when examined by the dye-buoyant density method. Segregants of each of these E. coli WR3026 strains that had lost the ability to utilize lactose, when examined in the same manner as the lactose-fermenting strains, were not observed to contain these supercoiled, circular DNA molecules. Thus the DNA of the lac elements is maintained in E. coli WR3026 in the supercoiled, circular form. Molecular weights of the supercoiled, circular molecules isolated from strains carrying the lac elements were determined by sucrose density gradient centrifugation to be 30 million to 56 million. The calculated number of copies per chromosome of the lac elements varied from 1.4 to 3.7, depending upon the particular lac element examined. Each of the elements was determined to have a guanine plus cytosine composition of 50%. All six of the E. coli WR3026 strains containing a transmissible lac element were tested with the E. coli male-specific phage, R-17, and the E. coli female-specific phage, phiII, and did not respond to either of these phages as do F-containing derivatives of E. coli K-12.

Characterization of the polydisperse closed circular deoxyribonucleic acid molecules of Bacillus megaterium.

The polydisperse circular deoxyribonucleic acid (DNA) molecules which comprise up to 30% of the total extractable DNA of Bacillus megaterium strain 216 have been purified and partially characterized. Banding in cesium chlorideethidium bromide by "gradient relaxation" in a fixed-angle rotor provided good resolution of circular and chromosomal DNAs for preparative separations. Renaturation studies on purified circular DNA failed to reveal a rapidly renaturing fraction, and DNA-DNA hybridization studies indicated that the majority of the chromosomal nucleotide sequences are represented in the heterogeneous-size population of circular molecules. It is concluded that the circular DNA of B. megaterium does not represent typical bacterial plasmid DNA. The possibility that the circular DNA molecules are the result of the expression of a defective bacteriophage is discussed.

Extrachromosomal deoxyribonucleic acid in different enterobacteria.

Eighty-seven different enterobacteria and pseudomonas strains were examined for the presence of extrachromosomal deoxyribonucleic acid (DNA). Thirty-four strains contained closed circular DNA by the ethidium bromide CsCl density technique. Extrachromosomal DNA was most frequent in Escherichia and Klebsiella strains. The extrachromosomal DNA was isolated and characterized by analytical ultracentrifugation and electron microscopy. All the extrachromosomal DNA-containing bacteria contained circular DNA molecules of small size (0.5-4 mum). Most of these bacteria also contained larger circles (20-40 mum). The number of different size classes of circular DNA in each strain varied from one to five. The buoyant density of the extrachromosomal DNA ranged from 1.692 to 1.721 g/cm(3). Many bacteria contained extrachromosomal DNA of more than one density.

Isolation and characterization of the fertility factor P of Vibrio cholerae.

Vibrio cholerae strains with the transmissible fertility factor P contained a supercoiled circular deoxyribonucleic acid (DNA) component amounting to between 2 and 6% of the total DNA obtained from the cells. Such a component was not observed in V. cholerae strains lacking the fertility factor. This supercoiled circular DNA was isolated from P(+) cells, and the molecular weight was determined by sedimentation velocity experiments and electron microscopy to be approximately 80 million daltons. These supercoiled circular DNA molecules, which have a guanine plus cytosine (G + C) composition of 42%, were concluded to be the extrachromosomal P factor. It was calculated that there is approximately one copy of the P factor per chromosome. A small amount of supercoiled circular DNA was occasionally isolated from the P(-) strains of V. cholerae. The function of this component, which has a molecular weight of 40 million daltons, is not known. The molecules found in the P(-) strains were readily distinguished from the P(+) circular molecules by their smaller molecular weight and different G + C composition.

Molecular nature of R-factor deoxyribonucleic acid isolated from Salmonella typhimurium minicells.

In earlier reports it was shown that a variety of extrachromosomal elements harbored in the Escherichia coli minicell producer segregate into the minicells. We show in this report that the fi(+) R factor R-100-1 (derepressed derivative of R-100) similarly segregates into minicells produced by a Salmonella typhimurium strain. Four distinct classes of covalently closed circular deoxyribonucleic acid molecules are found in minicells derived from the R(+)Salmonella minicell producer. The sum of the average molecular weights or contour lengths of the circular molecules in two of the classes is equal to the average molecular weight or contour length of those in a third class. The data suggest that R-100-1 dissociates into resistance determinants (i.e., genes that specify the molecules that confer resistance) and the resistance transfer factor (i.e., genes responsible for the transferability of the R factor to a recipient). In contrast, only one molecular species is found in minicells derived from the R(+) (R-100-1) Escherichia coli minicell producer. The fourth size class consists of small covalently closed circles (minicircles), which were originally found in the R(-)Salmonella minicell producer and are shown in this report to be enhanced in number in R(+)Salmonella minicells.

Light-scattering studies on deoxyribonucleic acid flexibility. The solution properties of a small circular deoxyribonucleic acid molecule.

Previous investigations on the persistence length of DNA in solution have revealed large discrepancies between hydrodynamic results and those from light-scattering techniques which have potentially a greater resolving power. The information obtained from experiments on a small circular DNA molecule has resolved these discrepancies. The non-superhelical circular double-stranded DNA molecule from bacteriophage [unk]X174-infected cells is small enough to permit accurate light-scattering extrapolations, and its solutions have negligible anisotropy. The persistence length obtained from experimental investigations on this molecule is comparable with that obtained by hydrodynamic techniques, even with variation of the excluded-volume factor.

Characterization of deoxyribonucleic acid isolated from the granulosis viruses of the cabbage looper, Trichoplusia ni and the fall armyworm, Spodoptera frugiperda

Large, covalently closed circular deoxyribonucleic acid molecules have been isolated from the granulosis viruses that infect the cabbage looper, Trichoplusia ni, and the fall armyworm, Spodoptera frugiperda. As extracted from either virus, the nucleic acid is a mixture of double-stranded covalently closed circular molecules (about 30%), circular molecules (about 60%) with one or few nicks, and linear molecules (less than 10%). Centrifugation to equilibrium in cesium chloride gradients containing ethidium bromide shows the presence of three bands of DNA: a light band of DNA containing linear and relaxed circular DNAs, a heavy band of covalently closed DNA, and a third band of DNA intermediate to the light and heavy bands of DNA. Significantly, lyophilization of the occluded virus preparation before purification of the DNA does not allow the recovery of covalently closed DNA as compared to that recovered from nonlyophilized virus. Data herein suggest that lyophilization introduces, for the most part, a single-strand nick in the double-stranded molecule. A comparison of the sedimentation rates of the intact linear forms of the insect virus DNAs with those of T4 and T7 phage DNA standards on both neutral and alkaline sucrose gradients was used to estimate the molecular weight of the insect virus genomes. The genome from Spodoptera frugiperda granulosis virus is about 95 × 10 6 daltons and that from Trichoplusia ni granulosis virus is about 100 × 10 6 daltons. The base compositions of the virus and host DNAs were determined by thermal denaturation measurements.

Intracellular forms of adenovirus deoxyribonucleic acid. I. Evidence for a deoxyribonucleic acid-protein complex in baby hamster kidney cells infected with adenovirus type 12.

The total intracellular deoxyribonucleic acid (DNA) from baby hamster kidney cells abortively infected with (3)H-adenovirus type 12 was analyzed in dye-buoyant density gradients. Between 10 and 20% of the cell-associated radioactivity derived from viral DNA bands in a density position which is 0.043 to 0.085 g/cm(3) higher than that of viral DNA extracted from purified virions. The DNA in the high-density region (HP-fraction) is almost completely absent when DNA, ribonucleic acid (RNA) or protein synthesis is chemically inhibited in separate experiments. The HP-fraction is not found when the virus does not adsorb to and enter the cell. The DNA in the HP-fraction appears as early as 2 hr after inoculation. At 2 hr after infection, the HP-fraction is present both in the nucleus and the cytoplasm. This DNA hybridizes exclusively with viral DNA and sediments at approximately the same rate in both neutral and alkaline sucrose density gradients. Electron microscopy has revealed no circular DNA molecules in this fraction. Evidence indicates that the viral DNA in the HP-fraction exists in a complex with protein and possibly RNA. The protein component of the complex is resistant to enzymatic digestion, whereas the complex is susceptible to ribonuclease treatment. Digestion with deoxyribonuclease reduces the amount of DNA found in the HP-fraction. The structure and biological function of this complex are currently being investigated.

Isolation of circular deoxyribonucleic acid from Salmonella typhosa hybrids obtained from matings with Escherichia coli Hfr donors.

Heterozygous, partial diploid Salmonella typhosa hybrids obtained from matings with Escherichia coli K-12 Hfr strains were observed to contain supercoiled, circular deoxyribonucleic acid (DNA) when examined by the dye-buoyant density method. Examination of one such S. typhosa hybrid after its loss, by segregation, of the inherited E. coli genetic markers revealed a concurrent loss of its supercoiled circular DNA. Subsequent remating of this segregant with various E. coli Hfr strains resulted in the reappearance of the circular DNA. Molecular weight determinations of circular DNA molecules isolated from a number of S. typhosa partial diploid hybrids were made by sucrose density gradient ultracentrifugation and electron microscopy. These studies revealed a range of molecular sizes among the various hybrids examined, but each hybrid exhibited only a single characteristic size for its contained circular DNA. The range of size is consistent with the presence in each hybrid of a different length of E. coli chromosome. It was concluded that the E. coli Hfr genetic segments transferred to these S. typhosa hybrids were conserved, in the diploid state, in the form of supercoiled, circular DNA molecules.

Isolation and characterization of supercoiled circular deoxyribonucleic acid from beta-hemolytic strains of Escherichia coli.

Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated by cesium chloride centrifugation in the presence of ethidium bromide from a naturally occurring beta-hemolytic Escherichia coli strain (SC52). The open circular forms have contour lengths of 2.25 +/- 0.1 mum, 24.0 +/- 0.3 mum, and 29.5 +/- 0.5 mum. The beta-hemolytic character of E. coli SC52 can be transferred by conjugation to a nonhemolytic recipient strain. Analysis of the supercoiled DNA of the hemolytic recipient demonstrated that the two large supercoiled DNA molecules of E. coli SC52 are transferred during this event, too. A beta-hemolytic laboratory E. coli strain and several of its derivatives have been shown to contain at least one circular DNA molecule, slightly larger in size than those isolated from E. coli SC52 and its conjugant. The possible significance of these DNA molecules for hemolysin production and transfer is discussed.

Genetic Map of Bacteriophage φX174

Bacteriophage phiX174 temperature-sensitive and nonsense mutations in eight cistrons were mapped by using two-, three-, and four-factor genetic crosses. The genetic map is circular with a total length of 24 x 10(-4)wt recombinants per progeny phage. The cistron order is D-E-F-G-H-A-B-C. High negative interference is seen, consistent with a small closed circular deoxyribonucleic acid molecule as a genome.