Dye-buoyant Density Centrifugation Research Articles

Comparison of plasmids in strains of Zymomonas mobilis

Four strains of Zymomonas mobilis were examined for their resistance to antimicrobial agents and found to have similar resistance profiles. Plasmid DNA was extracted and purified by CsCl dye-buoyant density centrifugation; molecular weights were determined by agarose gel electrophoresis and electron microscopy. All four strains harbored a large plasmid (46 × 10 6 Da) and a smaller plasmid (16–21 × 10 6 Da) whose molecular weight was strain dependent. Two strains, Ag11 and ATCC 10988, had smaller plasmids of unique molecular weight. Homology existed between the plasmids in the four strains as shown by cross-reaction in DNA-DNA blot hybridizations. Only one plasmid appeared unique to the host from which it was isolated.

Electron Microscopic Evidence for a Multimeric System of Plasmids in Fast-Growing Rhizobium Spp.

Covalently closed circular (CCC)-DNA could be isolated, by dye-buoyant density centrifugation, from l3 out of 15 strains from three species of fast-growing rhizobia. The plasmid band was also present in ineffective mutants and non-infective mutants derived from symbiotically effective, plasmid-carrying parent strains. The buoyant density in caesium chloride was 1� 719 g/cm3 for chromosomal DNA and 1� 715-1� 716 g/cm3 for plasmid DNA in the four strains examined. Electron microscopy of the CCC-DNA from five strains revealed a wide size range of large circular molecules, with the contour length ranging from l3. 5 to c. 170 jim (mol. wt range 28 x 106-352 x 106 ). Analysis of the size distribution of open circular molecules from the five strains indicated that a multimeric series of plasmid molecules may occur in these bacteria, with an estimated mean monomer length of l3�5 jlin.

Origin and direction of replication of the bacteriocinogenic plasmid Clo DF13

Cairn's type replicative intermediates of both the wildtype Clo DF13 plasmid and the copy mutant CLO DF13 cop3 were isolated by dye-buoyant density centrifugation. Replicative intermediates were linearized at the HpaI or Sa1I cleavage site, and examined with the electron-microscope. The data show that replication of both the Clo DF13 wild type plasmid and the Clo DF13 cop3 plasmid, initiates at about 2.8% on the physical map. Replication proceeds unindirectionally and counterclockwise on this map.

Large Plasmids in Different Rhizobium Species

Summary Large plasmids were detected in Rhizobium species by sedimentation analysis of lysates on alkaline sucrose gradients and by dye buoyant density centrifugation. Mitomycin C treatment did not increase the amount of plasmid DNA in the bacteria. Renaturation kinetics were used to confirm that these large DNA molecules had the molecular complexity of plasmids and to estimate their molecular weights. These ranged from 0·7 × 108 to 4·0 × 108 daltons. The maximum yield of isolated plasmid DNA relative to chromosomal DNA was 3·8%.

Method for obtaining more-accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation

A method that gives high recovery of deoxyribonucleic acid (DNA) from crude bacterial lysates using ethidium bromide-cesium chloride density gradient centrifugation is presented. After Pronase digestion and shearing of the lysate, essentially 100% recovery of chromosomal DNA and a reproducible recovery of covalently closed circular (CCC) plasmid DNA is obtained for a specific plasmid in a given strain. This method should be useful for comparing the CCC plasmid/chromosome ratio of various plasmid-host combinations.

Characterization of the antibiotic resistance plasmid ERL1 from Streptococcus pyogenes.

The streptococcal plasmid ERL1 determining inducible resistance to erythromycin, lincomycin, and staphylomycin S was isolated by dye-buoyant density centrifugation and shown to have a molecular weight of about 17.5 Mdal, as revealed by sedimentation through neutral sucrose gradients. In SM60 cells entering the stationary phase its covalently closed circular form was present to the extent of 5 copies per chromosomal genome equivalent, ERL1 was subject to the DNA restriction and modification mechanism discovered in strain 56188. It did not appear to exercise restriction of phage DNA but mediated a partial release of the restricted growth of A25.

Characterization of a Nosocomially Significant, Multiple Drug-Resistant Strain of <i>Serratia marcescen</i><i>s</i>

A multiple drug-resistant strain of Serratia marcescens (bacteriocin type 18) was isolated from three clinical patients. The isolates were found to carry a conjugally nontransferable, nonmobilizeable resistance plasmid (R-plasmid) with resistance-(r)-determinants against ten antimicrobial drugs: ampicillin, carbenicillin, chloramphenicol, gentamicin, kanamycin, neomycin, streptomycin, tobramycin, triple sulfonamides, cotrimoxazole, and--possibly--nalidixic acid, as determined with exposure to 'curing' agents (ethidium bromide, acridine orange, and sodium dodecyl sulfate) and by the high rate of spontaneous loss of r-determinants. Dyebuoyant density centrifugation allowed recovery of R-plasmid DNA that measured roughly 24 mum in contour length; after 'curing' with concomitant loss of 9 r-determinants, the contour length of the R-plasmid DNA of one isolate (No. SE 154) had decreased to roughly 15 mum, and none was detected in the sole variant of the isolate that spontaneously had lost 11 r-determinants.

Characterization of defective SV40 isolated from SV40-transformed cells

Defective SV40 viruses were isolated from SV40-transformed monkey, human and hamster cells after Sendai virus-mediated fusion of the transformed cells with TC7 cells, a stable line of African green monkey kidney cells. Viral isolates were concentrated and purified and the defective viruses examined by electron microscopy. The buoyant densities in CsCl of the defective viruses ranged between 1.32 and 1.33 g/cc. DNA isolated from defective viruses was characterized by dye-buoyant density centrifugation and by velocity sedimentation in neutral CsCl. The DNA was heterogeneous in size and contained some covalently closed double-stranded circular molecules.

Studies on the structure of replicative intermediates in bacteriophage M13 single stranded DNA synthesis.

Pulse-labeled replicative intermediates in M 13 single stranded DNA synthesis can be separated by dye-buoyant density centrifugation into two major fractions: Supercoiled molecules (RI I) containing viral strands of more than one genome length, and "relaxed" molecules (RI II) with labeled DNA chains shorter than unit length. It is postulated that RI II molecules might be formed in vivo by site-specific nicking of RF I molecules.

Supercoiled circular DNA in crown-gall inducing Agrobacterium strains

By alkaline sucrose gradient, neutral sucrose gradient and dye-buoyant density centrifugation a large plasmid was shown to be present in the crown-gall inducing Agrobacterium tumefaciens strain B6-S3. Measurements of contour lengths carried out on electron micrographs resulted in a mean length of 54·1 μ m, corresponding to a molecular weight of 112 × 10 6 . Only one or a few copies of this plasmid are present per bacterial chromosome. Mitomycin C induction has no influence on the amount of plasmid DNA in the cell. At present this plasmid must be considered as cryptic, for no genetic markers on it are known. Furthermore, large plasmids were isolated from crown-gall inducing strains belonging to seven Agrobacterium groups described by Kersters et al. (1973). Contour length measurements carried out on the plasmids isolated from the various crown-gall inducing strains fell in the range from 54·1 μ m to 75·4 μ m, depending on the strain examined. We were not able to find such plasmids in eight non-pathogenic strains belonging to four of the same groups. The hypothesis is formulated that the large plasmid present in crown-gall inducing bacteria could be the “tumor-inducing principle”.

Plasmid-Specific Transformation in Staphylococcus aureus

Transformation of Staphylococcus aureus cells with circular duplex deoxyribonucleic acid prepared from plasmid-carrying strains by alkali denaturation and selective renaturation or by dye-buoyant density centrifugation is reported. In all of the transformants tested, the transformed markers became established as autonomous plasmids that were biologically and physically indistinguishable from those carried by the donor strains. Transformation with bulk deoxyribonucleic acid from a strain carrying the penicillinase plasmid, PI(258), gave rise to transformants in which the erythromycin locus, the only plasmid marker transformed, was shown to be integrated into the host chromosome.

The replication of the ring-shaped DNA of polyoma virus: II. Identification of molecules at various stages of replication

Viral DNA was selectively extracted from mouse embryo cells which had been subjected to short pulses of radioactive thymidine during productive infection with polyoma virus. The replieative intermediate of the viral DNA, component II ∗, was then isolated and characterized using dye buoyant density centrifugation, chromatography on benzoylated-naphthoylated DEAE cellulose and velocity sedimentation in sucrose solutions. The results of the sedimentations performed at both neutral and alkaline pH values suggested that component II ∗ represented a collection of molecules at all stages of replication. This was further supported by the electron microscopic observation of purified preparations of component II ∗, which revealed circular molecules with two branch points.

Characterization of two circular satellite species of deoxyribonucleic acid in Rhodopseudomonas spheroides

DNA, isolated from a strain of Rhodopseudomonas spheroides, showed two components when banded in CsCl, with buoyant densities of 1.719 and 1.724 g/ml. The lighter band comprised about 8% of the total. Using a mild lysis procedure, it was possible to extract DNA of which 15–20% was circular. Circular DNA, isolated by dye-buoyant density centrifugation, contained two components with buoyant densities 1.718 and 1.724 g/ml. Sedimentation of the circular DNA gave two peaks, with S 20,w values of 78.2 S and 52.7 S; sedimentation in alkali, and in neutral solution after denaturation, showed that the first peak was closed circular and the second peak open circular DNA. Band-buoyant density sedimentation of the circular DNA in alkaline CsCl initially showed two peaks; after a time, each peak split into two. The alkaline buoyant densities of the four peaks were consistent with open and closed circles with neutral buoyant densities 1.718 and 1.724 g/ml. It is concluded that Rps. spheroides contains two circular satellite DNA species, with the same molecular weight of 70–75 × 10 6 daltons, but with slightly different base compositions.

Heterogeneous circular DNA elements in vegetative cultures of Bacillus megaterium.

DNA preparations from two strains of Bacillus megaterium, one which produces megacin and another that is megacin sensitive, were analyzed by dye-buoyant density centrifugation, cesium chloride gradient equilibrium centrifugation in the absence of dye, zone sedimentation in sucrose, and electron microscopy. Both strains were found to contain a substantial proportion of their total DNA as circular, covalently closed, double helical molecules. These DNA elements were heterogeneous in size and appeared slightly less dense than chromosomal DNA. The physiological role and origin of these elements is unknown at present.