Comparison of different methods of introducing a label into the DNA of microorganisms

Different methods of isotope introduction into plasmid and chromosomal DNA have been compared. The efficiency of label introduction into DNA was estimated by the results of DNA--DNA hybridisation and by the thermostability of hybrid duplexes. Using the enzymatic methods of label introduction levels of DNA specific radioactivity and label binding in homologous and heterologous reactions were close. DNA labeled by the method of enzymatic methylation forms more thermostable hybrid duplexes than "nick-translation" DNA. The advantages of enzymatic methods of label introduction for creating a microorganism reference DNA bank are discussed.     

Repeating DNA sequences of closely related species of mosquitoes]

Repeated DNA sequences of mosquitoes were studied by using of reassociation kinetics, molecular hybridization, restriction analysis and Southern blot-hybridization. Mosquitoes of two genera, the species of one of them being sibling species, were investigated. The content of all repeated families is the same both in sibling species and in species of different genera DNA. The percent of homologous sequences is low as compared to the high thermal stability of heterologous duplexes both in sibling species DNA and in different genera DNA. Restriction analysis of DNA and blot-hybridization with 35S repeated fraction revealed certain specific families of repeated sequences in the DNA of sibling species and of different genera of mosquitoes.     

Intra- and interpopulation differentiation of human genomes by molecular DNA hybridization

The DNA-DNA hybridization method was used to compare the repetitive sequences with a low degree of intragenomic divergence in various etno-territorial groups (Russians, Bouriats and Paleoasiats). Values of intergenomic divergence within groups and between them were estimated by a decrease in melting temperature of hybrid duplexes in relation to homologous 3H-labeled thermostable fraction reassociates of DNA of a Russian. Statistically valid differences in melting temperature were revealed when Russian, Bouriat and Paleoasiatic groups were compared. No such differences were found within each of the groups. Though the thermostability profiles had much in common in each case, some quantitative differences in melting temperature allowed to differentiate local groups in humans.     

Serologic and immunochromatographic detection of oxygenated polyenoic acids in Euglena gracilis var. bacillaris

The extent of evolutionary conservation of DNA complimentary to RNA stored in the mature oocyte of the sea urchin S. purpuratus has been assessed. To do this, such DNA was hybridized with total genomic DNA of S. purpuratus and S. franciscanus and the thermal stability of the resultant duplexes was measured by two methods. In the first method, the duplexes were bound to hydroxylapatite and thermally eluted; the difference in thermal stability between homologous and heterologous duplexes averaged 6.9 degrees C in duplicate determinations. In the second experiment, the same hybrids were thermally melted in 2.4M tetraethylammonium chloride, then assayed with S1 nuclease; the difference in thermal stability of homologous and heterologous duplexes was 4.8 degrees C. Either value is significantly lower than the divergence of total single-copy DNA among these species as measured by the same techniques. This demonstrates that DNA sequences complimentary to maternal RNA are conserved during evolution, and thus that a high fraction of them are likely to be physiologically functional.     

A quantitative bacterial dot method for DNA-DNA hybridization and its correlation to the hydroxyapatite method

A filter hybridization method employing bacterial samples and [¹²⁵I]labeled chromosomal DNA as a probe was used for DNA-DNA hybridization. It was found that the hybrids had a thermal melting temperature very similar to that of duplexes formed by purified filterbound DNA. The difference in thermal denaturation midpoint between homologous and heterologous duplexes was determined for a number of strains ofAcinetobacter spp. andEnterobacter agglomerans. A comparison with the corresponding data obtained by the hydroxyapatite method showed good correlation between the two methods. The use of bacterial samples in filter hybridization omits the time-consuming DNA preparation procedure necessary for traditional DNA-DNA hybridization procedures. A simplified, two-step elution procedure is suggested for processing large numbers of strains.     

Tobacco single-copy DNA is highly homologous to sequences present in the genomes of its diploid progenitors

Summary We compared the single-copy DNA sequences of the tetraploid tobacco plant, Nicotiana tabacum, with those of its diploid progenitors N. sylvestris and N. tomentosiformis. We observed that 65% of N. sylvestris and N. tomentosiformis single-copy DNA fragments reacted with each other using moderately stringent hybridization conditions (60° C, 0.18 M Na⁺). An additional 10% sequence homology was detected when the hybridization temperature was reduced by 10° C. The thermal stability of interspecific single-copy DNA duplexes indicated that they were approximately 6% more mispaired than homologous single-copy DNA duplexes. In contrast, we observed almost no single-copy DNA divergence between N. tabacum and its diploid progenitors. Greater than 99% of N. sylvestris and N. tomentosiformis single-copy DNAs reacted with N. tabacum DNA using moderately stringent hybridization conditions. The thermal stability of these duplexes indicated that they contained no more sequence mismatch than homologous single-copy duplexes. Together, our results show that significant single-copy DNA sequence divergence has occurred between the diploid N. sylvestris and N. tomentosiformis genomes. However, by applying our experimental criteria these single-copy DNAs are indistinguishable from their counterparts in the hybrid N. tabacum nucleus.     

Thermal stability of homologous and heterologous bacterial DNA duplexes

Thermal stability of homologous and heterologous DNA duplexes renatured according to the renaturation-rate method of De Ley et al. (1970) for 35 min or 17 hr, was estimated from the melting profiles of the duplexes. Comparison of the melting points of native and renatured DNA revealed that in the first 35 min of renaturation highly stable homologous duplexes were mainly formed, whereas up to 7% mismatching occurred in duplexes renatured for 17 hr. Up to 8% more mismatching was found in heterologous DNA duplexes of moderately related coryneform bacteria than in homologous ones after 35 min renaturation. It can be concluded that mismatching in heterologous hybrids of closely related DNAs had been restricted to a few % and of moderately related DNAs to approximately 10% in the initial renaturation phase.     

Use of hybridization melting kinetics for detecting Phytophthora species using three-dimensional microarrays: demonstration of a novel concept for the differentiation of detection targets

Microarray-based detection is limited by variable and inconsistent hybridization intensities across the diversity of probes used in each array. In this paper, we introduce a novel concept for the differentiation of detection targets using duplex melting kinetics. A microarray assay was developed on a PamChip microarray enabling the differentiation of target Phytophthora species using the melting kinetics of probe-target duplexes. In the majority of cases the hybridization kinetics of target and non-target duplexes differed significantly. Analysis of the melting kinetics of duplexes formed by probes with target and non-target DNA was found to be an effective method for determining specific hybridization and was independent of fluctuations in hybridization signal intensity. This form of analysis was more robust than the traditional approach based on hybridization intensity, and enabled the detection of individual Phytophthora species and mixtures thereof.     

Rapid evolution in a fraction of theDrosophila nuclear genome

Summary Previous observations have indicated thatDrosophila DNA contains a component that evolves so rapidly that it fails to hybridize between the DNAs of sibling species. To establish the reality of this component and study its properties, the fraction (about 20%) ofDrosophila simulans (Dsim) DNA that fails to hybridize toDrosophila melanogaster (Dmel) DNA has been isolated. The majority of the hybridizable part of this isolated fraction (based on control tests on Dsim DNA) fails to hybridize with Dmel DNA under the conditions used for the initial fractionation. Clones of this fraction do hybridize with Dmel DNA at open criterion producing duplexes with greatly reduced thermal stability, indicating that the underlying process is rapid sequence divergence rather than loss of the homologous sequences by relatively large deletions.Cloned fragments from the nonhybridizing fraction from Dsim are more than 15% divergent from the Dmel homologues, whereas the fraction that does hybridize is only 3–5% divergent. In comparison, synonymous substitutions in the coding regions of five genes show a 9% average divergence between Dsim and Dmel. They appear to be intermediate in their degree of divergence between the hybridizing and nonhybridizing components.     

Discrimination between perfect and mismatched duplexes with oligonucleotide gel microchips: role of thermodynamic and kinetic effects during hybridization

The efficiency of discrimination between perfect and mismatched duplexes during hybridization on microchips depends on the concentrations of target DNA in solution and immobilized probes, buffer composition, and temperature of hybridization and is determined by both thermodynamic relationships and hybridization kinetics. In this work, optimal conditions of discrimination were studied using hybridization of fluorescently labeled target DNA with custom-made gel-based oligonucleotide microchips. The higher the concentration of immobilized probes and the higher the association constant, the higher the concentration of the formed duplexes and the stronger the corresponding fluorescence signal, but, simultaneously, the longer the time needed to reach equilibrium. Since mismatched duplexes hybridize faster than their perfect counterparts, perfect-to-mismatch signal ratio is lower in transient regime, and short hybridization times may hamper the detection of mutations. The saturation time can be shortened by decreasing the probe concentration or augmenting the gel porosity. This improves the detection of mutations in transient regime. It is shown that the decrease in the initial concentration of oligonucleotide probes by an order of magnitude causes only 1.5-2.5-fold decrease of fluorescence signals after hybridization of perfect duplexes for 3-12 h. At the same time, these conditions improve the discrimination between perfect and mismatched duplexes more than two-fold. A similar improvement may be obtained using an optimized dissociation procedure.     

Functional polarity is introduced by Dicer processing of short substrate RNAs

Synthetic RNA duplexes that are substrates for Dicer are potent triggers of RNA interference (RNAi). Blunt 27mer duplexes can be up to 100-fold more potent than traditional 21mer duplexes. Not all 27mer duplexes show increased potency. Evaluation of the products of in vitro dicing reactions using electrospray ionization mass spectrometry reveals that a variety of products can be produced by Dicer cleavage. Use of asymmetric duplexes having a single 2-base 3'-overhang restricts the heterogeneity that results from dicing. Inclusion of DNA residues at the ends of blunt duplexes also limits heterogeneity. Combination of asymmetric 2-base 3'-overhang with 3'-DNA residues on the blunt end result in a duplex form which directs dicing to predictably yield a single primary cleavage product. It is therefore possible to design a 27mer duplex which is processed by Dicer to yield a specific, desired 21mer species. Using this strategy, two different 27mers can be designed that result in the same 21mer after dicing, one where the 3'-overhang resides on the antisense (AS) strand and dicing proceeds to the 'right' ('R') and one where the 3'-overhang resides on the sense (S) strand and dicing proceeds to the 'left' ('L'). Interestingly, the 'R' version of the asymmetric 27mer is generally more potent in reducing target gene levels than the 'L' version 27mer. Strand targeting experiments show asymmetric strand utilization between the two different 27mer forms, with the 'R' form favoring S strand and the 'L' form favoring AS strand silencing. Thus, Dicer processing confers functional polarity within the RNAi pathway.     

Optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene DNA probes

We present a study on the refinement of filter-hybridization conditions for a series of synthetic oligonucleotides in the range from 17 to 50 base residues in length. Experimental conditions for hybridization and the subsequent washing steps of the filter were optimized for different lengths of the synthetic oligonucleotides by varying the formamide concentration and washing conditions (temperature and monovalent cation concentration). Target DNA was immobilized to the nitrocellulose filter with the slot blot technique. The sequences of the synthetic oligonucleotides are derived from the third exon of the human oncogene c-myc and the corresponding viral gene v-myc and the G + C content was between 43 and 47%. Optimal conditions for hybridization with a 82% homologous 30-mer and 100% homologous 17-, 20-, 25-, 30-, and 50-mers were found to be a concentration of formamide of 15, 15, 30, 30, 40, and 50%, respectively. Optimal conditions for washing were 0.5X standard sodium citrate (SSC) at 42 degrees C for 2 X 15 min. The melting temperature for these optimal hybridization and washing conditions was calculated to be up to 11 degrees C below the hybridization temperature actually used. This confirms that the duplexes are more stable than expected. The melting points for 17-, 20-, and 30-mers were measured in the presence of 5X SSC and found to be 43, 58, and 60 degrees C, respectively. Competition between double- and single-stranded DNA probes to the target DNA was investigated. The single-stranded DNA probes were about 30- to 40-fold more sensitive than the double-stranded DNA probes.     

Genetic mobility of plasmids of Streptomyces erythraeus

Grossing of S. erythraeus 4 with S. erythraeus 1 resulted in transfer of genetic elements from strain 4 to strain 1 as evidence by the 20 and 18 kb fragments in the experiments on DNA-DNA hybridization. The presence of the genetic elements in strain 1 was the cause of plasmid pSE 21 mobility. In strain 6, a derivative of S. erythraeus 1 plasmid pSE 21 was accompanied by other extrachromosomal DNAs characterized by high instability. During storage of the strain at a temperature of 4 degrees C for more than 1 or 2 months the number of the plasmid pSE 21 copies decreased. When the strain was stored for longer periods (6 months or more) the plasmid DNA was not detectable even with the DNA-DNA hybridization procedure. The results of hybridization of a fraction of the extrachromosomal DNA of S. erythraeus 6, the Bam HIB fragment of plasmid pSE 21 with the total DNA of strains 1, 4, 5, 6 and BTCC 2 of S. erythraeus and hybridization of DNA of plasmid pSE 21 with the total DNA of S. erythraeus 6 and 1 showed that (1) strains 1, 5 and BTCC 2 had the same hybridization patterns, (2) the other extrachromosomal DNAs present in the fraction were homologous with the Bam HIA fragment of plasmid pSE 21, (3) chromosomes of strains 1, 4, 5, 6 and BTCC 2 of S. erythraeus also contained DNA homologous to the plasmid Bam HIA fragment. It was suggested that plasmid pSE 21 could be used as a basis for constructing the integrative vector for S. erythraeus.     

DNA composition in South American camelids I. Characterization and in situ hybridization of satellite DNA fractions

The DNA composition and the in situ hybridization of satellite fractions were analysed in the New World camelids llama, alpaca, guanaco and vicuña. In the four camelid forms, it was possible to identify a similar main band DNA and five satellite fractions (I–V) with G+C base contents ranging from 32% to 66%. Satellites II–V from llama were in situ reannealed on chromosomes from the four camelid forms. The results obtained were: (a) the four satellites hybridized with regions of C-banding (centromeric regions of all chromosomes and short arms of some autosomes); (b) in general, homologous hybridizations (llama DNA versus llama chromosomes) were more efficient than heterologous reassociations; there were however three exceptions to this rule (vicuña and alpaca satellite fraction II, chromosome group B; vicuña fraction V, chromosome groups A and B); (c) X chromosomes from the four camelids had satellites III–V but lacked satellite II, (d) no satellite fraction was detected on chromosome Y. The analysis of the in situ hybridization patterns allowed to conclude that most or all C-banded chromosome regions comprise several satellite DNA fractions. It is, moreover, proposed that there is an ample interspecies variation in the number of chromosomes that cross-react with a given satellite fraction. Our data give further support to the close genomic kinship of New World camelids.     

Titration of variant DNA sequences differing by a single point-mutation by selective dot-blot hybridization with synthetic oligonucleotides

A dot-blot hybridization procedure with synthetic oligonucleotide probes is reported, which allows the quantitative titration in genomic DNA of variant forms of repeated genes differing by a single nucleotide change. It involves the utilization of a pair of 22-base long oligonucleotides matching the two variant sequences and the choice of an hybridization temperature very close to the Td of the oligonucleotide/DNA duplexes. The selectivity is achieved through a competition between the cognate labeled and the non-cognate unlabeled probes in the hybridization mixture.     

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.     

Clostridium sporogenes isolates and their relationship to C. botulinum based on deoxyribonucleic acid reassociation.

Sixty-two isolates of Clostridium sporogenes from canned foods were examined for cultural properties, heat resistance and DNA-DNA homology to Clostridium botulinum type A190. Sporulation was observed in most of 21 umbonate and rhizoidal colony-forming strains (colony-type I strains), but not in most of the 41 strains with convex and circular or crenate colonies with a mat to semi-glossy surface (colony-type II strains). More than half of the latter strains showed much higher heat resistance than the rhizoidal colony-forming strains. The DNA isolated from colony-type II strains was 81% or more homologous to C. botulinum A190 DNA, forming duplexes which had thermostabilities similar to homologous duplexes of strain A190 DNA. Colony-type I strains differed from C. botulinum by 30 to 40% DNA homology and the DNA duplexes formed between these strains and strain A190 showed deltaT m(e) values of 7-0 degrees C when compared with the T m(e) of homologous DNA duplexes of strain A190.     

Analysis of genomic similarity in Streptomycetes belonging to the fluorescent subgroup as a confirmation of their taxonomic revision using a population method

Genomic similarity was analysed in streptomycetes belonging to the fluorescent subgroup: Streptomyces chrysomallus, S. fluorescens, S. galbofluorescens and S. citreofluorescens. The degree of reference S. chrysomalius DNA hybridization with S. fluorescens and S. galbofluorescens DNAs was 75 and 82%, respectively, thus being within the limits of the intraspecial hybridization level. S. citreofluorescens DNA showed a 55% homology with reference S. chrysomallus DNA, which corresponded to the range of interspecies hybridization. These conclusions were confirmed by the results obtained in analysing the thermostability of hybrid duplexes. Therefore, these findings are consistent with the data of revising the species taxonomy of this streptomycetes subgroup which was done using the method of comparative population analysis. The population model proposed by one of the authors can be used to assess the intraspecies level of DNA-DNA hybridization.     

DNA hydration studied by pressure perturbation scanning microcalorimetry

Pressure perturbation differential scanning calorimetry was used to determine thermal expansion coefficients and thus temperature-induced volume changes of DNA duplexes differing in their GC/AT content. It was shown that the temperature-induced unfolding of the DNA duplexes proceeds with a significant increase of the thermal expansion coefficient and the partial volume of the DNA. Unusually, large temperature-induced changes in the partial volume were observed for an AT-rich dodecamer, a finding consistent with previous crystallographic studies showing the presence of highly ordered water molecules hydrating the minor groove of such duplexes. The data show that the density of this ordered water is substantially higher than that of the bulk water. This ordered water cannot, therefore, be equated to ice at normal pressures but it thermodynamically resembles ice formed at high pressures.     

Pairing statistics and melting of random DNA oligomers: Finding your partner in superdiverse environments

Understanding of the pairing statistics in solutions populated by a large number of distinct solute species with mutual interactions is a challenging topic, relevant in modeling the complexity of real biological systems. Here we describe, both experimentally and theoretically, the formation of duplexes in a solution of random-sequence DNA (rsDNA) oligomers of length L = 8, 12, 20 nucleotides. rsDNA solutions are formed by 4^L distinct molecular species, leading to a variety of pairing motifs that depend on sequence complementarity and range from strongly bound, fully paired defectless helices to weakly interacting mismatched duplexes. Experiments and theory coherently combine revealing a hybridization statistics characterized by a prevalence of partially defected duplexes, with a distribution of type and number of pairing errors that depends on temperature. We find that despite the enormous multitude of inter-strand interactions, defectless duplexes are formed, involving a fraction up to 15% of the rsDNA chains at the lowest temperatures. Experiments and theory are limited here to equilibrium conditions.