Characterization of Inducible Bacteriophages in Bacillus licheniformis

Two morphologically distinct and physically separable defective phages have been found in Bacillus licheniformis NRS 243 after induction by mitomycin C. One of them (PBLB) is similar to the defective phage PBSX of B. subtilis, which has a density of 1.373 g/cm(3) in CsCl and a sedimentation coefficient of 160S. PBLB incorporates into its head mainly bacterial deoxyribonucleic acid (DNA) which has a sedimentation coefficient of 22S and a buoyant density in CsCl of 1.706 g/cm(3). The other phage (PBLA) has a morphology similar to the temperate phage phi105 of B. subtilis; the head diameter is about 66 nm, and it possesses a long and noncontractile tail. PBLA has a density of 1.484 g/cm(3) in CsCl and the phage-specific DNA, which is exclusively synthesized after induction by mitomycin C, has a density of 1.701 g/cm(3). PBLA DNA is double-stranded and has a sedimentation coefficient of 36S, corresponding to a molecular weight of 34 x 10(6) to 35 x 10(6) daltons. The phage DNA has one interruption per single strand, giving single-stranded segments with molecular weights of 13 x 10(6) and 4 x 10(6) daltons. Common sequences between the two phage DNA species and with their host DNA have been demonstrated by DNA-DNA hybridization studies. Both phage particles kill sensitive bacteria. However, all attempts thus far to find an indicator strain to support plaque formation have been unsuccessful.     

The binding of poly(rA) and poly(rU) to denatured DNA. I. Model studies with homopolymers.

We have compared the properties of the poly(rA).oligo(dT) complex with those of the poly(rU).oligo(dA)n complex. Three main differences were found. First, poly(rA) and oligo(dT)n do not form a complex in concentrations of CsCl exceeding 2 M because the poly(rA) is insoluble in high salt. If the complex is made in low salt, it is destabilized if the CsCl concentration is raised. Complexes between poly(rU) and oligo(dA)n, on the other hand, can be formed in CsCl concentrations up to 6.6 M. Second, complexes between poly(rA) and oligo(dT)n are more rapidly destabilized with decreasing chain length than complexes between poly(rU) and oligo(dA)n. Third, the density of the complex between poly(rA) and poly(dT) in CsCl is slightly lower than that of poly(dT), whereas the density of the complex between poly(rU) and poly(dA) in CsCl is at least 300 g/cm3 higher than that of poly(dA). These results explain why denatured natural DNAs that bind poly(rU) in a CsCl gradient usually do not bind poly(rA).     

Density-gradient centrifugation in lithium metatungstate and tris-(hydroxymethyl)aminomethane phosphotungstate

New substances--lithium metatungstate (MTL) and tris-(hydroxymethyl)aminomethane phosphotungstate (PTT)--have been presented for density-gradient preparation. The buoyant densities of protein, RNA, DNA and some nucleoproteins were determined in solutions of these salts. Nucleic acids have been smaller buoyant density (1.1 g/cm3) than the proteins in contrast to CsCl-gradients. The protein in PTT solution have buoyant density 1.5 g/cm3 and in MTL solution 2.0-2,3 g/cm3. It was shown that MTL gradients allow to reach better resolution in nucleoprotein analysis than CsCl gradients.     

Mitochondrial DNA of Yoshida ascites tumour cells. Physicochemical properties and biological function.

Mitochondrial DNA from Yoshida A.H. 130 cells, has been characterized by determination of the buoyant density by CsCl equilibrium density gradient centrifugation and the thermal denaturation and renaturation behaviour. These studies have been carried out parallelly on nuclear DNA from the same cells in order to search for possible differences between both DNAs. Mitochondrial DNA of Yoshida cells presents an equilbrium in CsCl of 1.7154 g/cm3 and a sharp melting with a Tm of 92 degrees C. Nuclear DNA presents an equilibrium of 1.7030 g/cm3 and a Tm of 88 degrees C. The guanine plus cytosine content in both DNAs has been calculated from tumour results and compared with the content in normal rat liver cells M-DNA of tumour cells presents a higher guanine plus cytosine content than N-DNA, whereas in normal liver cells is higher in N-DNA. N-DNAs of both normal and tumour cells have the same guanine plus cytosine content, whereas M-DNA from tumour cells presents a significant increase (about 35%) with regard to this from normal liver cells.     

CsCl Density Gradient Centrifugation Studies of Intact Bacterial Cells

Cells of Escherichia coli have been successfully banded in CsCl density gradients and a portion of the population reclaimed in a viable state. Differentiation between two strains of this organism in a CsCl density gradient has been demonstrated also. Several studies were undertaken to see whether differences could be detected between two samples of cells of the same strain which had been subjected to different conditions. The results were as follows: (a) Introduction of a heavy label (5-bromouracil) into the DNA during a 90 minute period did not produce an observable change in cell density. (b) Removal of a required amino acid from the growth medium of an E. coli auxotroph resulted in an increase in both the density and heterogeneity of the cells. (c) Exposure of cells to 27 kr of gamma radiation, followed by a period during which portions of both DNA and RNA were lost, yielded two distinct bands, one at the normal position in the gradient and the other shifted to a lighter region.     

An analysis of the bovine genome by Cs2SO4-Ag density gradient centrifugation

Calf DNA preparations having molecular weights of 5 to 7 × 10⁶ have been fractionated by preparative Cs₂SO₄—Ag⁺ density gradient centrifugation into a number of components. These may be divided into three groups: (1) the main DNA component (1.697 g/cm³; all densities quoted are those determined in CsCl density gradients), the 1.704 and 1.709 g/cm³ components form about 50, 25 and 10% of the genome, respectively; they are characterized by having symmetrical CsCl bands and melting curves, both of which have standard deviations close to those of bacterial DNAs of comparable molecular weight, and by their G + C contents being equal to 39, 48 and 54%, respectively; after heat-denaturation and reannealing, their buoyant densities in CsCl are greater than native DNA by 12, 10 and 3 mg/cm³, respectively. (2) The 1.705, 1.710, 1.714 and 1.723 g/cm³ components represent 4, 1.5, 7 and 1.5% of the DNA, respectively, and exhibit the properties of “satellite” DNAs; their CsCl bands and melting curves have standard deviations lower than those of bacterial DNAs; after heat-denaturation and reannealing, their buoyant densities are identical to native DNA, except for the 1.705 g/cm³ component, which remains heavier by 5 mg/cm³; in alkaline CsCl, only the 1.714 g/cm³ component shows a strand separation. (3) A number of minor components, forming 1% of the DNA, have been recognized, but they have not been investigated in detail; two of them (1.719 and 1.699 g/cm³) might correspond to ribosomal cistrons and mitochondrial DNA, respectively.     

Stable isotope probing with 15N achieved by disentangling the effects of genome G+C content and isotope enrichment on DNA density

Stable isotope probing (SIP) of nucleic acids is a powerful tool that can identify the functional capabilities of noncultivated microorganisms as they occur in microbial communities. While it has been suggested previously that nucleic acid SIP can be performed with 15N, nearly all applications of this technique to date have used 13C. Successful application of SIP using 15N-DNA (15N-DNA-SIP) has been limited, because the maximum shift in buoyant density that can be achieved in CsCl gradients is approximately 0.016 g ml-1 for 15N-labeled DNA, relative to 0.036 g ml-1 for 13C-labeled DNA. In contrast, variation in genome G+C content between microorganisms can result in DNA samples that vary in buoyant density by as much as 0.05 g ml-1. Thus, natural variation in genome G+C content in complex communities prevents the effective separation of 15N-labeled DNA from unlabeled DNA. We describe a method which disentangles the effects of isotope incorporation and genome G+C content on DNA buoyant density and makes it possible to isolate 15N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of "heavy" DNA from primary CsCl density gradients followed by purification of 15N-labeled DNA from unlabeled high-G+C-content DNA in secondary CsCl density gradients containing bis-benzimide. This technique, by providing a means to enhance separation of isotopically labeled DNA from unlabeled DNA, makes it possible to use 15N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in 13C-DNA-SIP applications.     

Cucumis sativus Cryptic Virus, a New Virus in Cucumber

Isometric virus-like particles (VLPs) have been purified from cucumber leaf tissue. Three dsRNA segments with estimated molecular weights of 1.8, 1.1 and 1.0 × 10⁶d have been isolated from VLPs occurring in CsCl density gradient fractions but were also readily detected in preparations from as little as 1 g of fresh leaf tissue. The VLPs resemble dsRNA containing cryptic viruses and have been named Cucumis sativus cryptic virus (CsCV).     

Analysis of the genome of plants. II. Characterization of repetitive DNA in barley (Hordeum vulgare) and wheat (Triticum aestivum).

Barley and wheat DNAs have been characterized by studying their kinetics of reassociation, melting properties and sedimentation behaviour in neutral CsCl gradients as well as in Cs2SO4 gradients containing Ag+ or Hg2+. In both species, reassociation kinetics have revealed the presence of approx. 76% redundant nucleotide sequences which have been grouped into very rapidly reassociating (Cot 0-0.01), rapidly reassociating (Cot 0.01-1.0) and slowly reassociating (Cot 1-100) fractions. The barley Cot 0-0.01 and Cot 0.01-1.0 fractions as well as the wheat Cot 0.01-1.0 fraction form narrow bands upon centrifugation in CsCl gradients. Under similar experimental conditions both Cot 0.01 and Cot 1.0-100 wheat fractions and the barley Cot 1.0-100 fraction form broad bands each having several shoulders. Thermal denaturation studies of most of the above reassociated fractions have shown a considerable degree of order in their duplexes with an average hyperchromicity of 21.5%. When native, high molecular weight barley DNA is centrifuged in Ag+/CS2SO4 density gradients (RF = 0.2), two satellites appear on the heavier side of the main band, as against one in the case of wheat. The two minor peaks, designated as satellites I and II, have buoyant densities of 1.702 and 1.698 g/cm3, respectively, in neutral CsCl gradients and together represent about 8-9% of total barley DNA. Upon centrifugation in Hg2+/CS2SO4 density gradients, one satellite is observed in both barley and wheat and it accounts for 1-2% of their genomes.     

Diversity and phylogenetic implications of CsCl profiles from rodent DNAs

Buoyant density profiles of high-molecular-weight DNAs sedimented in CsCl gradients, i.e., compositional distributions of 50- to 100-kb genomic fragments, have revealed a clear difference between the murids so far studied and most other mammals, including other rodents. Sequence analyses have revealed other, related, compositional differences between murids and nonmurids. In the present study, we obtained CsCl profiles of 17 rodent species representing 13 families. The modal buoyant densities obtained for rodents span the full range of values observed in other eutherians. More remarkably, the skewness (asymmetry, mean - modal buoyant density) of the rodent profiles extends to values well below those of other eutherians. Scatterplots of these and related CsCl profile parameters show groups of rodent families that agree largely with established rodent taxonomy, in particular with the monophyly of the Geomyoidea superfamily and the position of the Dipodidae family within the Myomorpha. In contrast, while confirming and extending previously reported differences between the profiles of Myomorpha and those of other rodents, the CsCl data question a traditional hypothesis positing Gliridae within Myomorpha, as does the recently sequenced mitochondrial genome of dormouse. Analysis of CsCl profiles is presented here as a rapid, robust method for exploring rodent and other vertebrate systematics.     

Buoyant and potentiometric titrations of synthetic polpeptides. II. Five copolypeptides and two nonionizable homopolypeptides in CsCl solutions.

The buoyant density titrations of five ionizable copolypeptides in concentrated CsCl solutions have been determined. The results are used to formulate models for predicting the buoyant density titration behavior of copolypeptides and proteins using the previously reported homopolypeptide buoyant density titration curves. It was determined for these copolypeptides that the best predictive model must include not only the buoyant densities of the constituent amino acid residues and the relative composition, but also hydration and salt binding. Hydrations determined for the homopolypeptides are used in the copolypeptide predictive model. The hydrations of the neutral homopolypeptides were readily calculable since their buoyant densities are thermodynamically defined in terms of their partial specific volumes and hydrations. For the case of a charged macromolecule, an expression for the buoyant density as a function of the number and nature of the bound ions, its partial specific volume, and its relative hydration has also been available for some time. This heretofore intuitive relationship is now derived from thermodynamic principles and allows calculations of hydrations to charged macromolecules which bind either cations, anions, or both. The potentiometric titrations of three of the five copolypeptides in concentrated CsCl solutions were determined in order to study the effect of residue interaction and solvation effects on their ionization behavior. The potentiometric results are also combined directly with the buoyant density titration results to determine the correlation of the buoyant density with the degree of ionization. As in the cases of poly(Glu) and poly(His), the buoyant density of the copolypeptides changed linearily with the degree of ionization. The buoyant density titrations of two nonionizable homopolypeptides, poly(Gly) and poly(Ala), were determined in concentrated CsCl solutions. The buoyant density was found to increase with increasing pH, despite the fact that side chains do not contain ionizable groups. This is the first evidence from homopolypeptide or copolypeptide data that buoyant density changes can be observed from effects other than side-chain ionizations.     

Occurrence of a kinetoplast DNA-protein complex in Trypanosoma cruzi

The kinetoplast DNA has been purified in high yield from $\text{Trypanosoma cruzi}$ by centrifugation on 3 M CsCl after lysis with a detergent. In exponentially growing trypanosomes, a light component causing a density shift of the kinetoplast DNA in CsCl density gradients is observed. This component is presumably a protein since it is removed by digestion with proteolytic enzymes. The DNA-protein complex is resistant to phenol extraction and 4 M guanidinium chloride, which indicates the possibility of a covalent bond between the protein and the DNA. The Kinetoplast DNA isolated from trypanosomes at the stationary phase by centrifugation on 3 M CsCl is free of proteins.     

Cat satellite DNA. Isolation using netropsin with CsCl gradients

The absence of centromeric bands in the karyotype of Felis catus is confirmed. It is also confirmed that no satellite band is visible in CsCl density gradients. However, a satellite is observed both by recentrifuging the fraction of the DNA that bands at high density in CsCl and by using netropsin to enhance the resolution of a CsCl gradient containing total F. catus DNA. The satellite, about 0.5% of total DNA, was isolated by repeated centrifugation in CsCl alone and in CsCl with netropsin. Netropsin was removed and a pure satellite DNA obtained. The reassociation kinetics (C0t1/2 less than 10(-3) M . s) show that the satellite is of the simple sequence type and hence a candidate for centromeric heterochromatin. Its cytological localisation awaits in situ hybridisation experiments.     

Biochemistry and ultrastructure of iridescent virus type 29

Physical and chemical parameters of iridescent virus type 29, isolated from the mealworm, Tenebrio molitor, have been analyzed. The icosahedral capsid is 130–135 nm in diameter and is surrounded by a fringe of coarse filaments. The virus has a buoyant density in CsCl of 1.31 g cm^?3 and contains 20 to 25 structural proteins as analyzed by isoelectric focusing and SDS-polyacrylamide gel electrophoresis. The DNA has a buoyant density in CsCl of 1.6874 g cm^?3 indicating a G + C content of approximately 28%. The lipid components of this virus differ from those of the host cell; the virus contains about 80% cardiolipin and 20% phosphatidyl choline.     

Isolation of coliphage lambda ghosts able to adsorb onto bacterial cells

We have examined three methods of γ ghost production, starting with the [³H]eucine-labelled phage, purified by CsCl density gradient sedimentation. Ghosts obtained by the osmotic shock or by incubation in 5 M LiCl do not adsorb on bacteria. Ghosts obtained by the treatment with the chelating agent EDTA and purified by CsCl density gradient sedimentation posses well preserved adsorption properties and are virtually free of DNA and infectious phage particles.     

Ribosomal RNA cistrons in Allomyces arbuscula

Bulk and nuclear DNA have been fractionated by preparative neutral CsCl equilibrium density gradient centrifugation and each fraction hybridized to labeled rRNA (25 + 18 S). The cistrons coding for rRNA appeared on the light side of the main peak. Hybridization of the nuclear DNA fractionated by preparative Ag+-Cs2SO4 gradients at different pHs showed that the banding profile did not change as compared to the CsCl pattern. In Hg2+-Cs2SO4 gradients, however, the peak of the fRNA-DNA hybrids shifted on the heavier side of the profile. This indicates that the ribosomal RNA cistrons in Allomyces are A-T-rich. Hybridization with homologous rRNA showed that, at saturation, 3.25% of the DNA is complementary to rRNA. With the genome size of 1.7-10(10) daltons, the multiplicity of rRNA cistrons has been found to be close to 270.     

Partial denaturation of mouse DNA in preparative CsCl density gradients at alkaline pH.

A new technique--partial denaturation of DNA in equilibrium CsCl density gradients at pH 11.4--is used to determine the distribution of intermediate states in the melting of mouse DNA. When the technique is applied in the preparative ultracentrifuge, the DNA is fractionated according to stability. Neutralization of the partially denatured fractions results in the recovery of most of the DNA in its native form. The individual fractions are more homogeneous than the total DNA: they have decreased density heterogeneity (smaller band widths), neutral CsCl buoyant densities that differ from the average, and more homogeneous melting profiles with melting temperatures that differ from the average.     

Prediction of centrifugation times for equilibrium and velocity sedimentation on various gradients

Computer and calculator programs have been prepared which can predict sedimentation times for a significant fraction of the kinds of density gradient centrifugation experiments currently being carried out in biochemistry. Gradients of sucrose, glycerol, or CsCl were accommodated for linear or several forms of convex or concave gradient profiles. Times of sedimentation to various uniformly spaced points between the meniscus and the bottom of the tube, or to a point near the isopycnic density, are predicted.