Correlation of Melting Temperature and Cesium Chloride Buoyant Density of Bacterial Deoxyribonucleic Acid

Samples of bacterial deoxyribonucleic acid from bacteria having guanine plus cytosine (GC) contents in the range of 27 to 72 moles per cent GC were analyzed by optical melting (T(m)) and equilibrium buoyant density methods. The relation between these properties is shown to be linear. The relative value of 1.99 moles per cent GC per degree C change in T(m) is calculated, and a reference method for the calculation of GC contents relative to a standard is derived.     

Deoxyribonucleic acid homology in bacterial taxonomy: effect of incubation temperature on reaction specificity

Parameters affecting deoxyribonucleic acid duplex (DNA-DNA) formation on membrane filters were evaluated. The reference strains used were Cytophaga succinicans strain 8, which has a guanine plus cytosine (GC) content of 38%, and Myxococcus xanthus strain FB, which has a GC content of 70%. Both organisms are gliding bacteria classified among the myxobacteria. Among the parameters evaluated, the incubation temperature used during duplex formation was found to be the most important in terms of the physical nature of the reaction product. When an incubation temperature 25 C below the melting point (T(m)) of the native DNA was used, homologous duplexes exhibited a thermal stability similar to that of native DNA. At 35 C below the T(m), a considerable proportion of the duplexes were of much lower stability; at 40 C below the T(m), most of the duplexes were of much lower stability. Similar duplexes of low stability were also formed between DNA molecules from morphologically and nutritionally diverse organisms, provided the GC percentages of the DNA preparations were similar. Competition between unlabeled and labeled DNA fragments for binding sites on immobilized DNA was also greatly influenced by the incubation temperature. Heterologous DNA-DNA complexes exhibited thermal stabilities which correlated with measurements of DNA homology in experiments involving competition. In addition, the difference in thermal stabilities of heterologous and homologous DNA complexes (DeltaT'(m)) may provide a measure of divergence in nucleotide sequences.     

Coding and non-coding DNA thermal stability differences in eukaryotes studied by melting simulation, base shuffling and DNA nearest neighbor frequency analysis

The melting of the coding and non-coding classes of natural DNA sequences was investigated using a program, MELTSIM, which simulates DNA melting based upon an empirically parameterized nearest neighbor thermodynamic model. We calculated T(m) results of 8144 natural sequences from 28 eukaryotic organisms of varying F(GC) (mole fraction of G and C) and of 3775 coding and 3297 non-coding sequences derived from those natural sequences. These data demonstrated that the T(m) vs. F(GC) relationships in coding and non-coding DNAs are both linear but have a statistically significant difference (6.6%) in their slopes. These relationships are significantly different from the T(m) vs. F(GC) relationship embodied in the classical Marmur-Schildkraut-Doty (MSD) equation for the intact long natural sequences. By analyzing the simulation results from various base shufflings of the original DNAs and the average nearest neighbor frequencies of those natural sequences across the F(GC) range, we showed that these differences in the T(m) vs. F(GC) relationships are largely a direct result of systematic F(GC)-dependent biases in nearest neighbor frequencies for those two different DNA classes. Those differences in the T(m) vs. F(GC) relationships and biases in nearest neighbor frequencies also appear between the sequences from multicellular and unicellular organisms in the same coding or non-coding classes, albeit of smaller but significant magnitudes.     

Deoxyribonucleic acid base composition of simonsiellaceae

The molar percentages of guanine plus cytosine in the DNA of 51 strains of Simonsiellaceae were determined by buoyant density ultracentrifugation of cell lysates in CsCl. The DNA base ratios ranged from 41–55 mole-% guanine plus cytosine. These values fall within the range known for the Order Cytophagales, the non-fruiting gliding bacteria, and are out-side the range of the Order Myxobacterales, the fruiting myxobacteria. Among the strains of the genus Simonsiella, four distinct groups can be delineated on the basis of source of origin (sheep, dog, cat, human) and GC content. The neotype of Alysiella filiformis has a GC content of 45.4 mole-%.     

Development and experimental validation of a predictive threshold cycle equation for quantification of virulence and marker genes by high-throughput nanoliter-volume PCR on the OpenArray platform

Development of quantitative PCR (QPCR) assays typically requires extensive screening within and across a given species to ensure specific detection and lucid identification among various pathogenic and nonpathogenic strains and to generate standard curves. To minimize screening requirements, multiple virulence and marker genes (VMGs) were targeted simultaneously to enhance reliability, and a predictive threshold cycle (C(T)) equation was developed to calculate the number of starting copies based on an experimental C(T). The empirical equation was developed with Sybr green detection in nanoliter-volume QPCR chambers (OpenArray) and tested with 220 previously unvalidated primer pairs targeting 200 VMGs from 30 pathogens. A high correlation (R(2) = 0.816) was observed between the predicted and experimental C(T)s based on the organism's genome size, guanine and cytosine (GC) content, amplicon length, and stability of the primer's 3' end. The performance of the predictive C(T) equation was tested using 36 validation samples consisting of pathogenic organisms spiked into genomic DNA extracted from three environmental waters. In addition, the primer success rate was dependent on the GC content of the target organisms and primer sequences. Targeting multiple assays per organism and using the predictive C(T) equation are expected to reduce the extent of the validation necessary when developing QPCR arrays for a large number of pathogens or other targets.     

Effect of the GC content of DNA on the distribution of UVB-induced bipyrimidine photoproducts.

Solar UV radiation is a major mutagen that damages DNA through the formation of dimeric photoproducts between adjacent thymine and cytosine bases. A major effect of the GC content of the genome is thus anticipated, in particular in prokaryotes where this parameter significantly varies among species. We quantified the formation of UV-induced photolesions within both isolated and cellular DNA of bacteria of different GC content. First, we could unambiguously show the favored formation of cytosine-containing photoproducts with increasing GC content (from 28 to 72%) in isolated DNA. Thymine-thymine cyclobutane dimer was a minor lesion at high GC content. This trend was confirmed by an accurate and quantitative analysis of the photochemical data based on the exact dinucleotide frequencies of the studied genomes. The observation of the effect of the genome composition on the distribution of photoproducts was then confirmed in living cells, using two marine bacteria exhibiting different GC content. Because cytosine-containing photoproducts are highly mutagenic, it may be predicted that species with genomes exhibiting a high GC content are more susceptible to UV-induced mutagenesis.     

Nucleotide Composition of Deoxyribonucleic Acid of Some Species of Cryptococcus, Rhodotorula, and Sporobolomyces

The buoyant density of deoxyribonucleic acid (DNA) from nine species and two varieties of Cryptococcus, three species and two varieties of Rhodotorula, and six species of Sporobolomyces was determined by CsCl density gradient equilibrium centrifugation. Several species were represented by two to four different strains. Expressed in moles per cent of guanine plus cytosine (GC content) the ranges were 49 to 65%, 52 to 70%, and 51 to 65% for Cryptococcus, Rhodotorula, and Sporobolomyces, respectively. For each genus, the GC content was distributed into two discrete groups with averages ranging from 52 to 54 and 60 to 66, respectively. An analysis of these results suggested that the determination of GC content of DNA had a taxonomic value for these yeast genera.     

Deoxyribonucleic Acid Base Composition of Some Members of the Subgenera Betabacterium and Streptobacterium

The base composition of deoxyribonucleic acid (DNA) prepared from four Betabacterium strains and four Streptobacterium strains was determined. Per cent GC values (guanine + cytosine/total bases) of the DNA were evaluated from the “melting-temperatures” (Tm) of the nucleic acids. For the Betabacterium strains, these values ranged from 44 to 51.5% GC, and those for the Streptobacterium strains ranged from 43 to 47.5% GC. The taxonomic division into these two subgenera is not, therefore, supported by these findings.     

Deoxyribonucleic acid base composition of pediococci and aerococci

Summary The guanine+cytosine content (GC) of DNAs of 9 strains of pediococci and 6 strains of aerococci was determined using different physicochemical methods. It was found that the GC content of DNA of the pediococci ranged from 37.8 to 41.2%, while that of the aerococci varied from 42.0 to 43.2%. The results obtained show that genetically related species are involved whose mutual position in the system is determined by only slight differences in the phenotype.     

Cytosine deamination plays a primary role in the evolution of mammalian isochores

DNA melting is rate-limiting for cytosine deamination, from which we infer that the rate of cytosine deamination should decline twofold for each 10% increase in GC content. Analysis of human DNA sequence data confirms that this is the case for 5-methylcytosine. Several lines of evidence further confirm that it is also the case for unmethylated cytosine and that cytosine deamination causes the majority of all C-->T and G-->A transitions in mammals. Thus, cytosine deamination and DNA base composition each affect the other, forming a positive feedback loop that facilitates divergent genetic drift to high or low GC content. Because a 10 degrees C increase in temperature in vitro increases the rate of cytosine deamination 5. 7-fold, cytosine deamination must be highly dependent on body temperature, which is consistent with the dramatic differences between the isochores of warm-blooded versus cold-blooded vertebrates. Because this process involves both DNA melting and positive feedback, it would be expected to spread progressively (in evolutionary time) down the length of the chromosome, which is consistent with the large size of isochores in modern mammals.     

Deoxyribonucleic Acid Base Composition of the Genus Lactobacillus

Deoxyribonucleic acids of 45 strains of Lactobacillus and 5 strains of Bifidobacterium which had been analyzed for base composition by chromatographic means were examined at equilibrium in a CsCl density gradient. Regression analysis showed that there have been systematic errors involved in the estimation of guanine plus cytosine (GC) content by the chemical method, and that the relation between buoyant density and base composition is indeed linear and best fitted by the equation GC = 10.309 (rho-1.662), which compares well in slope with the equation of Schildkraut, Marmur, and Doty. With the improved data obtained in this study, the specific groupings of the species of both genera were reevaluated.     

The negative ion states of molecules: adenine and guanine.

Anions of cytosine and thymine predominate in radiation-damaged DNA. This is in contrast to the experimental order of adiabatic electron affinities: A, 0.95; G, 1.51; >T, 0.79, U, 0.80; C, 0.56 (+/-0.05 eV). Excited negative-ion states of adenine (A) and guanine (G) are identified using semiempirical AM1-MCCI quantum mechanical calculations. A planar G(-) has an excited state adiabatic electron affinity, AEA*, of 0.3 +/- 0.05 eV. This state and the unique Watson-Crick structure are responsible for the preponderance of charge on C(-) in radiation-damaged DNA. By analogy to the value for cytosine, the dipole-bound EA of G is estimated as 0.25 eV. New AEA values from literature reduction potentials for the ribose nucleotides are rC, 0.6; rU, 0.8; and rT, 0.8 (+/-0.1 eV). From literature photoelectron spectroscopy, AEA* vales for U are 0.15, 0.3, 0.5, and 0.6 eV. In GC(-2), stacked [GC:GC](-3), and [GC:GC:GC](-4), the charge moves to G. In [GC:GC:GC](-2 to -4), the charge moves from GC(1) to GC(3) through space without a bridge or bond. This is important to electron conduction, radiation damage and repair, and nanoscale devices.     

Use of Lysostaphin in the Isolation of Highly Polymerized Deoxyribonucleic Acid and in the Taxonomy of Aerobic Micrococcaceae

By use of the staphylolytic enzyme lysostaphin, a method was devised for isolating and purifying highly polymerized deoxyribonucleic acid (DNA) from lysostaphinsusceptible Micrococcaceae. Staphylococcus aureus DNA isolated by this procedure gave an estimated molecular weight of ca. 2 x 10(8) and a residual protein content of 2.3%. The mole percentage of guanine + cytosine (GC) present in the DNA from 21 strains of aerobic Micrococceae was determined by buoyant density in cesium chloride. DNA from 12 biochemically typical members of the genus Staphylococcus gave a mean GC composition of 35.2 +/- 0.5 mole per cent. Four biochemically atypical Staphylococcus strains and one biochemically typical strain of the genus Micrococcus (M. candicans) were found to be susceptible to lysostaphin and gave typical Staphylococcus spp. GC base ratios. One biochemically atypical member of the genus Micrococcus (M. varians) was not susceptible to lysostaphin and gave a typical Micrococcus spp. GC base ratio. Lysostaphin susceptibility is an easy test to perform, and the results of this test appear to correlate with GC base ratio studies of the genera of Micrococcaceae.     

Quantitative Aspects of Deoxyribonucleic Acid Renaturation: Base Composition, State of Chromosome Replication, and Polynucleotide Homologies

The base composition of a deoxyribonucleic acid (DNA) sample affects its intrinsic rate of renaturation. In agreement with the information of Wetmur and Davidson, it was established that high guanosine plus cytosine (GC) DNA renatures faster than expected from analytical measurement of its molecular weight. A calculated correction factor of 1.8% of the observed C(0)t(.5) is required for every mole per cent GC difference from 51% GC. The correction factor is now established in the range of 32 to 65% GC. Renaturation of DNA mixtures prepared from pairs of organisms has been studied. When no similarity existed between the two organisms, the observed C(0)t(.5) of the mixture was the sum of the independently determined C(0)t(.5) values. Lack of additivity was correlated with similarities in polynucleotide sequence of the reassociating DNA molecules. A quantitative relationship was formulated to relate C(0)t(.5) values of renatured DNA mixtures to per cent binding ("homology"). Finally, it was demonstrated that DNA prepared from log-phase cells renatures faster than stationary-phase DNA and also departs from theoretical second-order kinetics.     

Use of Shake Cultures in a Semisolid Thioglycolate Medium for Differentiating Staphylococci from Micrococci

The standard diagnostic test for differentiating staphylococci from micrococci is based on the ability of the former to produce acid anaerobically in a glucose-containing growth medium. This test has been modified to provide greater convenience, easier interpretation of results, and better correlation with deoxyribonucleic acid (DNA) base composition. In the modified test, shake cultures in Brewer's fluid thioglycolate medium with 0.3% agar added are observed for growth in the anaerobic zone of the tubes. This test was applied to 125 strains of staphylococci and micrococci, and all except two strains gave results that were consistent with other criteria. Of particular interest were eight strains of Micrococcus saprophyticus and three strains of M. lactis that have a DNA composition of 30 to 37% guanine plus cytosine (GC). All 11 of these cultures produced anaerobic growth and thus would be classified as staphylococci. Strains of M. lactis that have a high GC content in their DNA grew only aerobically. Some cultures of staphylococci produced characteristic band patterns of anaerobic growth and other cultures produced only a few anaerobic colonies from an inoculum of 10(6) to 10(7) cells. These observations suggest some interesting genetic and metabolic capabilities in such cultures.     

Bacterial DNA methylation and gene transfer efficiency

The necessary amplification step in bacteria of any plasmid currently used in DNA immunization or gene therapy introduces modification in the nucleotide sequence of plasmid DNA used in gene transfer. These changes affect the adenine and the internal cytosine in respectively all of the GATC and CC(A/T)GG sequences. These modifications which introduce 6-methyladenine and 5-methylcytosine in plasmidic DNA are the consequence of the existence of the bacterial modification systems Dam and Dcm. In eucaryotes, the presence of 5-methylcytosine at dinucleotides -CG- is involved in silencing gene expression, but the possible consequences of the presence of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in the plasmids used in gene transfer experiments are presently unknown. Since the possibility exists to obtain plasmid DNA lacking this specific bacterial pattern of methylation by using (dam(-), dcm(-)) bacteria we performed experiments to compare in vitro and in vivo gene transfer efficiency of a pCMV-luc reporter plasmid amplified either in the JM109 (dam(+), dcm(+)) or JM110 (dam(-), dcm(-)) bacteria. Data obtained demonstrated that the presence of 6-methyladenine in GATC sequences and 5-methylcytosine in the second C of CC(A/T)GG motifs does not reduce the levels of luciferase activity detected following in vitro or in vivo gene transfer. On the contrary, gene transfer with a pCMV-luc amplified in JM109 (dam(+), dcm(+)) bacteria gives greater amounts of luciferase than the same transfection performed with a plasmid amplified in the mutated JM110 (dam(-), dcm(-)) counterpart. Therefore, these data do not suggest that the use of (dam(-), dcm(-)) bacteria to amplify plasmid DNA may increase gene transfer efficiency. However, the persistence of the use of (dam(+), dcm(+)) bacteria in order to amplify plasmid DNA raises the question of the possible biological consequences of the introduction of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in eukaryotic cells or organisms.     

Comparison of the Morphology and Deoxyribonucleic Acid Composition of 27 Strains of Nitrifying Bacteria

The gross morphology, fine structure, and per cent guanine plus cytosine (GC) composition of deoxyribonucleic acid of 27 strains of nitrifying bacteria were compared. Based on morphological differences, the ammonia-oxidizing bacteria were separated into four genera. Nitrosomonas species and Nitrosocystis species formed one homogenous group, and Nitrosolobus species and Nitrosospira species formed a second homogenous group in respect to their deoxyribonucleic acid GC compositions. Similarly, the nitrite-oxidizing bacteria were separated into three genera based on their morphology. The members of two of these nitrite-oxidizing genera, Nitrobacter and Nitrococcus, had similar GC compositions, but Nitrospina gracilis had a significantly lower GC composition than the members of the other two genera.