Indexes to the reassociation and stability of solution DNA hybrids

Summary The computation, assumptions, and properties of DNA-hybrid stability and reassociation indexes were reviewed. Different methods of computing the same index typically yielded similar values. However, because dissociation curves change from asymmetric to symmetric as increasingly divergent DNAs are compared, adequate determination of mode required fitting a complex function. Delta Tm, delta mode, and delta T50H correlated well up to ca. 12, and all were found to be useful indexes of genomic similarity in that range. They also exhibited similar levels of error, even though T50H comprises a percent reassociation component with relatively large variance. At greater distances, the delta Tm scale became markedly compressed because of the boundary imposed by the temperature of hybrid formation (incubation temperature). Though not compressed or technically limited by it, delta mode and delta T50H could not be extrapolated with certainty below the incubation temperature. Among theoretical problems discussed: Tm and mode index an increasingly small percentage of the genome as the extent of reassociation decreases, and they may compare different genomic segments as DNAs become highly diverged. T50H relies upon the assumptions that all sequences evolve at a constant rate and that reassociation behavior is the same among all sequences regardless of their extent of divergence. Tm and T50H may be biased by selfhybridization of repetitive elements or cross-hybridization of paralogous sequences. Delta mode is free of such biases as long as the genomes under comparison are not too diverged. No index was found to be best in all circumstances.     

A method of equalizing the level of DNA sequences of varying amount in a heterogeneous DNA mixture]

A method for the equalization of double-stranded DNA concentrations in the mixture which may be used for equalizing double-stranded cDNA concentrations involves thermal denaturation of the double-stranded DNA mixture followed by reassociation. The initial reassociation rate is Vi = Ki.(single-stranded DNA)2, and by the end of the process the concentrations of the unreassociated molecules for different DNAs should be approximately equal. Using hydroxylapatite chromatography one can separate single-stranded DNAs from double-stranded DNAs and carry out complete single-stranded DNAs reassociation. The new ratio of different double-stranded DNA concentrations would be almost 1.     

Nucleotide sequence divergence among DNA fractions of different syngens of Tetrahymena pyriformis

The magnitude of the differences in base sequence of DNA fractions derived from different syngens of the ciliated protozoan Tetrahymena pyriformis was investigated. Each DNA was fractionated into unique and repeated sequences by hydroxylapatite chromatography, and the fractions were tested by in vitro molecular hybridization techniques. The amount of hybrid formed and the thermal stability of the hybrid molecules were examined at different incubation temperatures (50 and 65 C) for unique sequences and at 50 C for repeated sequences. The extent of the reactions involving either unique or repeated sequences was nearly complete when the two DNAs compared were derived from the same syngen. Moreover, intrasyngenic hybrids formed at 50 C (and 65 C for unique sequences) exhibit a high degree of thermal stability. In contrast, the extent of the reactions involving sequences derived from different syngens was low, as expected from the effect of mismatching on rate of reassociation, and intersyngenic hybrids formed at 50 C have low thermal stability. The reaction of unique sequences is further reduced at 65 C and the intersyngenic hybrids formed have a higher thermal stability than those formed at 50 C. The degree to which thermal stability is lowered was then used to estimate the percentage of mispaired bases. The average divergence of unique sequences between syngens is large and of the magnitude found for rodent DNAs from different genera or for Drosophila DNAs from nonsibling species. The repeated sequence fraction may contain more than one component and may be more conserved than the unique sequence fraction.     

Detection and quantitation of Staphylococcus aureus penicillinase plasmid deoxyribonucleic acid by reassociation kinetics.

The quantity of penicillinase plasmid deoxyribonucleic acid (DNA) in various strains of Staphylococcus aureus has been determined by DNA-DNA reassociation kinetics. Specifically, 32P- or 125I-labeled denatured probes of purified plasmid DNA were reassociated in the presence of denatured DNAs isolated from the bacterial strains in question. The number of plasmid copies per cell was calculated from the effect of the latter nucleic acid samples on the reassociation rate of the radiolabeled probe. Among the S. aureus strains examined were monoplasmid, diplasmid and replication-defective representatives, and the effect of temperature on wild-type plasmid content was also investigated.     

A simple linear transform for the Folin-Lowry protein calibration curve to 1.0 mg/ml

The stability of covalently mercurated DNAs during DNA:DNA reassociation, heteroduplex recovery on sulfhydryl-Sepharose, and S₁ nuclease digestion under a variety of solvent and temperature conditions is described. The nonspecific loss of²⁰³Hg from mercurated DNA can be minimized by use of aqueous formamide solvents in reassociation experiments and by minimizing exposure to sulfhydryl reagents and temperatures above 35°C. Single-stranded DNA is shown to be more sensitive to demercuration than is native, duplex DNA.     

Effects of Sequence Divergence on the Reassociation Properties of Repetitive DNAs

PYRIMIDINE tract analysis of two satellite DNAs suggests that their basic sequences are simpler than those calculated from their rates of reassociation (réf. 1 and unpublished results of A. Carr-Brown, E. M. S. and P. M. B. Walker). One possible explanation for this discrepancy would be that mismatched base pairs, which are known to affect the stability of duplexes², might have a serious effect on the rate of reassociation of DNA. The work of Sutton and McCallum³ (see following article³) shows that this is indeed the case for mouse satellite DNA. If this effect were general it would have serious consequences for the interpretation of the reassociation kinetics of the repeated sequences in higherorganism DNA, because the bulk of these sequences reassociate to give badly matched duplexes. I now discuss a simple modification of the mechanism for DNA reassociation put forward by Wetmur and Davidson⁵, which may explain why the effect of mismatching is so large and which suggests a relationship which may eventually be used to correct for the effect.     

Capillary electrophoresis as a method to study DNA reassociation

To develop analytical methodology to assess the genetic complexity of a DNA sample, capillary electrophoresis with laser-induced fluorescence detection is used to monitor the annealing process of DNA samples. Coated columns are filled with an entangled polymer solution shown to optimally separate DNA through size-selective capillary electrophoresis. DNA samples are denatured by heating in a boiling water bath for approximately 10 min and then cooled to approximately 25 degrees C below the melting point of the DNA sample to initiate the reassociation process. The DNA is detected by means of the laser-induced fluorescence of intercalated ethidium bromide, which produces a substantially greater signal for double- versus single-stranded DNA. The rate of reassociation is dependent upon the rate at which complimentary strands of DNA encounter each other and the degree of repeating base sequences in the sample (hence, the diversity of the DNA). Experimental parameters also influence the reassociation rate. The effects of salt concentration and incubation temperature are presented. Traditional plots of C(o)t (C(o) = DNA concentration and t = reassociation time) versus % recovery of double-stranded DNA signal are generated for PhiX 174 Hae III digest and 50 bp stepladder DNA, individually and combined, to calculate the reassociation rate constants for these samples. Because reassociation of individual fragments is observed by the CE-LIF method, more information about the samples is available than with less specific and time-consuming traditional methods of investigating DNA reassociation.     

Does the concentration of DNA (Co) and the time of incubation (t) as parameters of Cot influence the thermal stability of the DNA duplexes?

It has been shown in a previous paper (8) that the prime product of reassociation of related DNA sequences under open experimental conditions are mismatched duplexes which undergo maturation upon further incubation. Due to this feature, the Tm value of the duplexes of a large number of DNAs is strongly dependent on the Cot value. Here we present data showing that the Tm of the duplexes of such type of DNAs depends also on the concentration of DNA in the range of one and the same Cot value. The significance of this finding in studying the taxonomic relationship by DNA-DNA hybridisation is discussed.Abbreviations Co = initial concentration of single-stranded DNA in moles of nucleotides per liter - t = time of incubation in seconds - Cot = the product of Co and t (mol. sec. 1¹) - PB = an equimolar mixture of NaH₂PO₄ and Na₂HPO₄, pH 6.8 - HAP = hydroxyapatite - Ti = incubation temperature - Tm = melting temperature - Te = elution temperature, i.e. the temperature at which one half of the DNA is eluted as single strands by HAP-thermal chromatography     

An alternative view of mammalian DNA sequence organization: I. Repetitive sequence interspersion in Syrian hamster DNA: A model system

The biochemical and biophysical techniques originally introduced by Davidson et al. (1973) and Graham et al. (1974) for the determination of the general organization and length of repetitive and non-repetitive sequences in eukaryotic DNA have been extended and modified. Improvements in the experimental methods employed in these pioneering works have led to novel interpretations and conclusions about mammalian DNA sequence organization. In what is commonly referred to as an interspersion experiment, the average spacing of repetitive DNA regions is inferred from the length dependence of hydroxyapatite binding of radio-labeled tracer DNAs reassociated with an excess of short 200 nucleotide repetitive sequence driver DNA. Studies on Syrian hamster DNA, using an improved procedure for conducting interspersion experiments, suggest that either a frequent cluster in the distribution of non-repetitive DNA sequence lengths occurs at 7200 (±2000) nucleotides or that repetitive sequences are randomly spaced on a number average basis. In contrast, measurements obtained using the traditional methods suggest that a frequent cluster in the distribution of non-repetitive DNA sequence lengths occurs at approximately 1000 nucleotides. When reassociations were conducted at elevated temperatures, to allow only well-matched repetitive sequences to hybridize, the amount of DNA operationally observed as “repetitive” was reduced. Interspersion experiments conducted with Syrian hamster DNA at a reassociation temperature of 75 °C yielded data similar to those obtained by Manning et al. (1975) for Drosophila melanogaster DNA reassociated at 60 °C.     

Single copy DNA homology in sea stars

Summary The sequence homology in the single copy DNA of sea stars has been measured. Labeled single copy DNA fromPisaster ochraceus was reannealed with excess genomic DNA fromP. brevispinus, Evasterias troschelii, Pycnopodia helianthoides, Solaster stimpsoni, andDermasterias imbricata. Reassociation reactions were performed under two criteria of salt and temperature. The extent of reassociation and thermal denaturation characteristics of hybrid single copy DNA molecules follow classical taxonomic lines.P. brevispinus DNA contains essentially all of the sequences present inP. ochraceus single copy tracer whileEvasterias andPycnopodia DNAs contain 52% and 46% of such sequences respectively. Reciprocal reassociation reactions with labeledEvasterias single copy DNA confirm the amount and fidelity of the sequence homology. There is a small definite reaction of uncertain homology betweenP. ochraceus single copy DNA andSolaster orDermasterias DNA. SimilarlySolaster DNA contains sequences homologous to approximately 18% ofDermasterias unique DNA. The thermal denaturation temperatures of heteroduplexes indicate that the generaPisaster andEvasterias diverged shortly after the divergence of the subfamilies Pycnopodiinae and Asteriinae. The twoPisaster species diverged more recently, probably in the most recent quarter of the interval since the separation of the generaPisaster andEvasterias.     

DNA reassociation using oscillating phenol emulsions

Reassociating double-stranded DNA from single-stranded components is necessary for many molecular genetics experiments. The choice of a DNA reassociation method is dictated by the complexity of the starting material. Reassociation of simple oligomers needs only slow cooling in an aqueous environment, whereas reannealing the many single-stranded DNAs of complex genomic mixtures requires both a phenol emulsion to accelerate DNA reassociation and dedicated equipment to maintain the emulsion. We present a method that is equally suitable for reassociating either simple or complex DNA mixtures. The Oscillating Phenol Emulsion Reassociation Technique (OsPERT) was primarily developed to prepare heteroduplex DNA from alkali-denatured high molecular weight human genomic DNA samples in which hundreds of thousands of fragments need to be reannealed, but the simplicity of the technique makes it practical for less demanding DNA reassociation applications.     

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.     

Single copy DNA and structural gene sequence relationships among four sea urchin species

Measurements of the divergence of single copy DNA sequences among four sea urchin species are presented. At a standard criterion for reassociation (0.12 M phosphate buffer, 60° C, hydroxyapatite binding) we observe the following extents of reaction and reductions in thermal stability for single copy DNA reassociation between Strongylocentrotus purpuratus tracer and heterologous driver DNA: S. dröbachiensis 68% and 2.5°C; S. franciscanus 51% and 3.5° C; Lytechinus pictus 12% and 7.5° C. The implied extents of sequence relatedness are consistent with the phylogenetic relationships of these species. The rate of single copy sequence divergence in the evolutionary lines leading to the Strongylocentrotus species is estimated to be 0.06–0.35% per million years. The rate of divergence of total single copy sequence has been compared to that of structural gene sequences represented in S. purpuratus gastrula polysomal messenger RNA. When closely related species, S. purpuratus and S. franciscanus, are compared, these polysomal sequences are found to diverge at a lower rate than does the total single copy sequence. For two very distantly related species, S. purpuratus and L. pictus, a small fraction of the single copy DNA sequence is probably conserved. These conserved sequences are not enriched in their content of structural gene sequences.Also staff member, Carnegie Institution of Washington, Washington, D.C. 20015     

Structural genes adjacent to interspersed repetitive DNA sequences

The observation that repetitive and single copy sequences are interspersed in animal DNAs has suggested that repetitive sequences are adjacent to single copy structural gene sequences. To test this concept, single copy DNA sequences contiguous to interspersed repetitive sequences were prepared from sea urchin DNA by hydroxyapatite fractionation (repeat-contiguous DNA fraction). These single copy sequences included about one third of the total nonrepetitive sequence in the genome as determined by the amounts recovered during the hydroxyapatite fractionation and by reassociation kinetics. ³H-labeled mRNA from sea urchin gastrula was prepared by puromycin release from polysomes and used in DNA-driven hybridization reactions. The kinetics of mRNA hybridization reactions with excess whole DNA were carefully measured, and the rate of hybridization was found to be 3–5 times slower than the corresponding single copy DNA driver reassociation rate. The mRNA hybridized with excess repeat-contiguous DNA with similar kinetics relative to the driver DNA. At completion 80% of that mRNA hybridizable with whole DNA (approximately 65%) had reacted with the repeat-contiguous DNA fraction (50%). This result shows that 80–100% of the mRNA molecules present in sea urchin embryos are transcribed from single copy DNA sequences adjacent to interspersed repetitive sequences in the genome.     

Measurement of the Sequence Complexity of Cloned Moloney Murine Leukemia Virus 60 to 70S RNA: Evidence for a Haploid Genome

The sequence complexity of the 60-70S RNA complex from Moloney murine leukemia virus (M-MuLV) was determined by measuring the annealing rate of radioactively labeled virus-specific DNA with M-MuLV 60-70S RNA in conditions of vast RNA excess. The M-MuLV RNA annealing rate, characterized by the quantity C(r)t((1/2)), was compared with the C(r)t((1/2)) values for annealing of poliovirus 35S RNA (2.6 x 10(6) molecular weight) with poliovirus-specific DNA and Sindbis virus 42S RNA (4.3 x 10(6) molecular weight) with Sindbis-specific DNA. M-MuLV-specific DNA was prepared in vitro by the endogenous DNA polymerase reaction of M-MuLV virions, and poliovirus and Sindbis virus DNAs were prepared by incubation of viral RNA and DNA polymerase purified from avian myeloblastosis virus and an oligo deoxynucleotide primer. The poliovirus and Sindbis virus DNAs were sedimented through alkaline sucrose gradients, and those portions of the DNA with sizes similar to the M-MuLV DNA were selected out for the annealing measurements. M-MuLV was cloned on NIH-3T3 cells because it appeared possible that the standard source of M-MuLV for these experiments was a mixture of viruses. The annealing measurements indicated a sequence complexity of approximately 9 x 10(6) daltons for the cloned M-MuLV 60-70S RNA when standardized to poliovirus and Sindbis virus RNAs. This value supports the hypothesis that each of the 35S RNA subunits of M-MuLV 60-70S RNA has a different base sequence.     

Related base sequences in the DNA of simple and complex organisms. V. The specificity of interactions between oligonucleotides and denatured DNA

A mixture of differentially labeled mouse and Bacillus subtilis DNA was used as a source of oligodeoxynucleotides of chain lengths from 15 to 40 nucleotides. The extent of interaction of these oligonucleotides with homologous or heterologous DNA bound to membrane filters was measured. The specificity of such interactions increases with chain length and with the incubation temperature. The thermal stability of the complexes is a function of chain length. Homologous oligonucleotide/DNA duplexes of B. subtilis are more stable than those of mouse of corresponding size, consistent with the incidence of partially related base sequences in mouse DNA. Oligonucleotides in this size range are also able to recognize partially complementary base sequences in the DNA of closely related organisms. This approach shows promise as a means of obtaining quantitative estimates of base sequence divergence between the DNAs of related organisms.This research was supported by a grant from the National Science Foundation (GB 6099).     

Reassociation kinetics of three satellite components of calf thymus DNA.

Using absorption measurements the reassociation kinetics of three satellite DNA components isolated from calf thymus was studied under various conditions. A different method using CsC1 density gradient determinations particularly suited for kinetic analysis of mixtures was also used and shown to give similar results. Reassociation rate constants were corrected for mismatching during strand reassociation using data obtained by kinetic analysis of fractions of the 1.714 g/cm-3 satellite component. The values of corrected as well as uncorrected complexities were calculated and compared with results of other methods. They were shown to be compatible with the concept of sequence repetition at various levels.     

Ethidium bromide-mediated renaturation of denatured closed circular DNAs: mechanistic aspects and fractionation of DNA on a molecular weight basis

When closed circular duplex DNAs are exposed to alkali in the presence of ethidium bromide, from 0 to 100% of the DNA can be recovered as the fully base-paired duplex (native) form upon neutralization of the solutions. The fraction of native DNA depends on the concentration of ethidium bromide, time of incubation, ionic strength and temperature of the solutions before neutralization as well as the molecular weight and superhelix density of the DNA. Limiting ethidium concentrations exist below and above which 0 and 100% of the DNA, respectively, is recovered as native material under a given set of incubation conditions regardless of the length of time of incubation before neutralization. The strong molecular weight dependence of the fraction of DNA recovered in the native form after a given time of pre-neutralization incubation at ethidium concentrations between the limiting values noted above allows larger DNAs to remain fully denatured upon neutralization while smaller DNAs in the same mixture are fully renatured. This permits the rapid fractionation of mixtures of closed duplex DNAs on the basis of molecular weight when a technique for the separation of denatured from fully base-paired DNA is applied to such mixtures. Such a separation has been demonstrated through the marked enrichment of plasmid cloning vector DNA containing cloned inserts in the fractions that remain denatured after neutralization of alkaline solutions of these DNAs containing ethidium bromide.     

A study of the evolution of repeated DNA sequences in primates and the existence of a new class of repetitive sequences in primates.

A new class of lowly repetitive DNA sequences has been detected in the primate genome. The renaturation rate of this sequence class is practically indistinguishable from the renaturation rate of single-copy sequences. Consequently, this lowly repetitive sequence class has not been previously observed in DNA renaturation rate studies. This new sequence class is significant in that it might occupy a major fraction of the primate genome.Based on a study of the thermal stabilities of DNA heteroduplexes constructed from human DNA and either bonnet monkey or galago DNAs, we are able to compare the relative mutation rates of repetitive and single-copy sequences in the primate genome. We find that the mutation rate of short, interspersed repetitive sequences is either less than or approximately equal to the mutation rate of single-copy sequences. This implies that the base sequence of these repetitive sequences is important to their biological function.We also find that numerous mutations have accumulated in interspersed repeated sequences since the divergence of galago and human. These mutations are only recognizable because they occur at specific sites in the repeated sequence rather than at random sites in the sequence. Although interspersed repetitive sequences from human and galago can readily cross-hybridize, these site-specific mutations identify them as being two distinct classes. In contrast, far fewer site-specific mutations have occurred since the divergence of human and monkey.     

DNA sequence organization in the genome of Petroselinum sativum (Umbelliferae)

The genome of parsley was studied by DNA/DNA reassociation to reveal its spectrum of DNA reiteration frequencies and sequence organization. The reassociation of 300 nucleotide DNA fragments indicates the presence of four classes of DNA differing in repetition frequency. These classes are: highly repetitive sequences, fast intermediate repetitive sequences, slow intermediate repetitive sequences, and unique sequences. The repeated classes are reiterated on average 136,000, 3000, and 42 times respectively. A minor part of the genome is made up of palindromes. — The organization of DNA sequences in the P. sativum genome was determined by the reassociation kinetics of DNA fragments of varying length. Further information was derived from S1 nuclease resistance and from hyperchromicity measurements on DNA fragments reassociated to defined C₀t values. — The portion of the genome organized in a short period interspersion pattern amounts to 47%, with the unique sequences on an average 1000 nucleotides long, and most of the repetitive sequences about 300 nucleotides in length, whereas the weight average length may be up to 600 nucleotides. — About 5% unique DNA and 11% slow intermediate repetitive DNA consist of sequences from 10³ up to 10⁴ nucleotides long; these are interspersed with repetitive sequences of unknown length. Long repetitive sequences constitute 33% of the genome, 13% are satellite-like organized, and 20% in long stretches of intermediate repetitive DNA in which highly divergent sequences alternate with sequences that show only minimal divergence. — The results presented indicate remarkable similarities with the genomes of most animal species on which information is available. The most intriguing pecularity of the plant genome derives from its high content of repetitive DNA and the presumed organization of the latter.