Reduction in the rate of DNA reassociation by sequence divergence

An estimate is made of the effect of imperfectly complementary sequences on the rate of reassociation of DNA. Rate measurements are reported for the reassociation of deaminated DNA and for the pairing of DNAs from related bacteria. A method is presented for separating the effect of the incubation temperature on the rate from the effect of sequence divergence. After correction to the optimum incubation temperature, the rate of DNA reassociation appears to be reduced by a factor of two for each 10 deg. C reduction in melting temperature due to sequence divergence. For most typical cases this effect is modest. However it can be quite important for measurements of the relation between the DNAs of different species.     

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     

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.     

Reduced-stringency DNA reassociation: sequence specific duplex formation.

Reduced-stringency DNA reassociation conditions allow low stability duplexes to be detected in prokaryotic, plant, fish, avian, mammalian, and primate genomes. Highly diverged families of sequences can be detected in avian, mouse, and human unique sequence dNAs. Such a family has been described among twelve species of birds; based on species specific melting profiles and fractionation of sequences belonging to this family, it was concluded that permissive reassociation conditions did not artifactually produce low stability structures (1). We report S1 nuclease and optical melting experiments, and further fractionation of the diverged family to confirm sequence specific DNA reassociation at 50 degrees in 0.5 M phosphate buffer.     

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.     

Stacking energies in DNA

Variations in base mono- and dipoles result in variations in stacking energies for the 10 unique neighbor pairs in DNA. Stacking energies for pair M on N, expressed as TMN, were derived by matrix decomposition of a large set of linear algebraic expressions relating the measured Tm for subtransitions emanating from large polymeric DNAs, and the fractional neighbor frequencies, fMN, for the domains responsible for the transitions, Tm = sigma fMNTMN. Tm were determined for subtransitions that dissociate in approximately all-or-none fashion in high resolution melting profiles of partially deleted and recombinant forms of pBR322 DNA. Three different analytical maneuvers were undertaken to resolve subtransitions: site-specific cleavage of domains; deletion of domains; and addition of domains. Three dozen domains of widely divergent, quasi-random neighbor frequencies were identified and assigned, resulting in a unique set of values for TMN with standard deviation, sigma = +/- 0.23 degree C. The average difference between calculated and experimental Tm for domains is only +/- 0.17 degree C, indicating that the thermodynamic properties of these domains are not in any way unusual. Assuming delta S to be constant for all pairs, the corresponding delta HMN are found to have a precision of +/- 10 calories.mol-1 and an accuracy of +/- 606 calories.mol-1. TMN used to calculate melting curves by statistical mechanical analysis of sequences of the different plasmid specimens in this study were in quantitative agreement with observed curves for most sequences. These TMN differ significantly from those determined previously and also correlate poorly with values determined by quantum chemical analysis. Stabilities of neighbor pairs, expressed as the difference in free energy between that for a given pair (MN) and that for the average of like pairs (M, N), depend on the relationship of stacked purines and pyrimidines as follows. delta delta Gpu-py(-466 cal) greater than delta delta Gpu-pu(+52 cal) greater than delta delta Gpy-pu(+335 cal) Differences between experimental Tm and Tm calculated with TMN for the isolated neighbor pairs in the B-conformation are useful in the identification of altered structures and unusual modes of dissociation of helixes. A significantly higher Tm is observed for the highly biased repeated sequence synthetic helixes dA.dT, d(AGC).d(GCT), and d(GAT).d(ATC), reflecting auxiliary sources of stability such as bifurcated hydrogen bonds and/or altered structures for these helixes.     

Thermotropic and mixing behavior of mixed-chain phosphatidylcholines with molecular weights identical with that of L-alpha-dipalmitoylphosphatidylcholine.

The thermotropic phase behavior of 10 mixed-chain phosphatidylcholines, in excess water, has been examined and compared with that of identical-chain C(16):C(16)PC by using high-resolution differential scanning calorimetry (DSC). The molecular weights (MW) of these 11 molecular species are the same, but their delta C/CL values, or the normalized chain length differences, vary considerably, ranging from 0.035 to 0.540. The thermodynamic parameters (Tm, delta H, and delta S) associated with the main phase transitions for these lipid dispersions exhibit biphasic V-shaped curves, when plotted against delta C/CL. Similar characteristic curves have been reported previously for aqueous dispersions of mixed-chain phosphatidylcholines with MW identical with that of C(17):C(17)PC [Lin et al. (1990) Biochemistry 29, 7063-7072]. The initial decrease in Tm (delta H or delta S) with increasing values of delta C/CL is attributed to the progressive increase in the magnitude of the chain-terminal perturbations on the conformational statistics of the adjacent hydrocarbon chains and hence the lateral chain-chain interactions of these mixed-chain phosphatidylcholines in the gel-state bilayer. At delta C/CL approximately equal to 0.42, the chain-end perturbation is presumably at its maximum; beyond this point, the highly asymmetric phosphatidylcholines are proposed to pack, at T less than Tm, into the mixed interdigitated bilayer. In this new packing mode, the methyl ends of the longer acyl chains are relocated at the interfaces between the hydrocarbon core of the bilayer and the aqueous medium. This disposition of the bulky chain ends releases a certain degree of chain-chain packing disorders, leading to an increase in Tm (delta H or delta S) with increasing delta C/CL.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Melting and reassociation of double-stranded RNA of the encephalomyocarditis virus]

The processes of melting and reassociation of double-stranded RNA in dimethylsulfoxide were studied. The addition of a small amount of LiCl results in great results in great reduction of Tm (temperature of melting), whereas the NaCl produces the opposite effect. It is suggested, that LiCl coordinates the molecules of H2O, reducing their activity, and consequently destabilises dsRNA. Mild conditions for melting and reassociation of RNA can be created. It was found that under optimal conditions for dsRNA melting, the degree of strand separation depends on the overall concentration of RNA, irrespective of the type of RNA added to the dsRNA preparation. Reassociation of dsRNA of EMC virus proceeds much faster than that of dsRNA of a related poliovirus. Addition of poly(C) to an annealing mixture slows down the rate of reassociation of EMC dsRNA, producing no effect on the poliovirus dsRNA reassociation. It is suggested that the presence of large poly(C) and poly(G) tracts in the complementary strands of the RNA determines its anomalous fast reassociation. Upon incubation of completely separated strands of EMC dsRNA in a water solution with high ionic strength partially double-stranded aggregates are formed. The formation of aggregates is prevented by addition of poly(A), which indicates that they are produced by "zippening" of a molecule starting with poly(A):poly(U) region. The significance of homopolymeric regions for stability of dsRNA of the EMC virus as well as their role in viral multiplication are discussed.     

Characteristic of segments of transgenic animal genomes, adjacent to integrated sequences of foreign DNA

DNAs of seven transgenic mice and one transgenic rabbit was divided into fractions according to reassociation kinetics and GC-content. Moderate and/or frequent (reverse) repeated sequences of the genome were revealed in all cases next to different transgenes. DNA fractions containing foreign sequences differed by the GC-content in different transgenic animals.     

Genetic relatedness of the genomes of equine herpesvirus types 1, 2, and 3.

Genomic DNAs of equine herpesvirus type 1 (EHV-1), EHV-2 (equine cytomegalovirus), and EHV-3 were examined by reassociation kinetic and thermal denaturation analyses to determine the extent and degree of homology among the three viral DNAs. Results of reassociation analyses indicated a limited homology among the three EHV genomes. Homologous DNA sequences equivalent to 1.8 to 3.7 megadaltons between EHV-1 and equine cytomegalovirus, 7.6 to 8.2 megadaltons between EHV-1 and EHV-3, and 1.3 to 1.9 megadaltons between equine cytomegalovirus and EHV-3 were detected. Examination by thermal denaturation of the DNA homoduplexes and heteroduplexes formed during reassociation revealed a high degree of base pairing within the duplexes, suggesting that closely related sequences may be conserved among the genomes of EHV.     

Genomic methylation: a tool for typing Helicobacter pylori isolates

The genome sequences of three Helicobacter pylori strains revealed an abundant number of putative restriction and modification (R-M) systems within a small genome (1.60 to 1.67 Mb). Each R-M system includes an endonuclease that cleaves a specific DNA sequence and a DNA methyltransferase that methylates either adenosine or cytosine within the same DNA sequence. These are believed to be a defense mechanism, protecting bacteria from foreign DNA. They have been classified as selfish genetic elements; in some instances it has been shown that they are not easily lost from their host cell. Possibly because of this phenomenon, the H. pylori genome is very rich in R-M systems, with considerable variation in potential recognition sequences. For this reason the protective aspect of the methyltransferase gene has been proposed as a tool for typing H. pylori isolates. We studied the expression of H. pylori methyltransferases by digesting the genomic DNAs of 50 strains with 31 restriction endonucleases. We conclude that methyltransferase diversity is sufficiently high to enable the use of the genomic methylation status as a typing tool. The stability of methyltransferase expression was assessed by comparing the methylation status of genomic DNAs from strains that were isolated either from the same patient at different times or from different stomach locations (antrum and corpus). We found a group of five methyltransferases common to all tested strains. These five may be characteristic of the genetic pool analyzed, and their biological role may be important in the host/bacterium interaction.     

Human-specific sequences: isolation of species-specific DNA regions by genome subtraction

Uniqueness is fundamental to the individuality of species, and this in turn is based on the uniqueness of their genomes. For the purpose of resolving the genetic basis of human uniqueness, we describe here the isolation of human-specific sequences using the technique of genome subtraction, i.e., competitive reassociation of genomic DNAs between two very closely related species. One such sequence, HS5, was found to be present only in the human genome and absent in the genomes of non-human primates including chimpanzees, the species most closely related to humans.     

Characterization of the nuclear genome of pearl millet.

The nuclear genome of pearl millet has been characterized with respect to its size, buoyant density in CsCl equilibrium density gradients, melting temperature, reassociation kinetics and sequence organization. The genome size is 0.22 pg. The mol percent G + C of the DNA is calculated from the buoyant density and the melting temperature to be 44.9 and 49.7%, respectively. The reassociation kinetics of fragments of DNA 300 nucleotides long reveals three components: a rapidly renaturing fraction composed of highly repeated and/or foldback DNA, middle repetitive DNA and single copy DNA. The single copy DNA consists of 17% of the genome. 80% of the repetitive sequences are at least 5000 nucleotide pairs in length. Thermal denaturation profiles of the repetitive DNA sequences show high Tm values implying a high degree of sequence homogeneity. About half of the single copy DNA is short (750--1400 nucleotide paris) and interspersed with long repetitive DNA sequences. The remainder of the single copy sequences vary in size from 1400 to 8600 nucleotide pairs.     

Calorimetry of tetraether lipids from Thermoplasma acidophilum: incorporation of alamethicin, melittin, valinomycin, and nonactin.

The development and application of model membrane systems on the basis of tetraether lipids from Thermoplasma acidophilum has been proposed. In this respect incorporation of membrane proteins and ionophores is indispensable and is demonstrated in the case of alamethicin, melittin, nonactin, and valinomycin by calorimetry. Dipalmitoylphosphatidylcholine (DPPC) and dihexadecylmaltosylglycerol (DHMG) were chosen for comparison. Melittin and alamethicin prove to broaden the lipid phase transition and to reduce the melting temperature Tm and enthalpy change (delta H) of the main phospholipid from T. acidophilum (MPL) and DPPC. The decrease in Tm, however, is more pronounced in DPPC than in MPL. Valinomycin shows only a marginal effect on the temperature and width of the transition; delta H is reduced in MPL and remains constant in DPPC and DHMG. With nonactin the phase transition of DPPC is quenched, and delta H and the half-height width are increased. DHMG is affected to a lesser extent and MPL only marginally. The four ionophores exhibit different modulation of the phase transition behavior of the various lipids as expected from their varying molecular structures. Thus, the integral membrane protein alamethicin, the peripheral protein melittin, valinomycin, and nonactin interact primarily with lipid head groups and are readily incorporated into the tetraether lipid structures.     

Thermal stability of proteins in the presence of poly(ethylene glycols)

Thermal unfolding of ribonuclease, lysozyme, chymotrypsinogen, and beta-lactoglobulin was studied in the absence or presence of poly(ethylene glycols). The unfolding curves were fitted to a two-state model by a nonlinear least-squares program to obtain values of delta H, delta S, and the melting temperature Tm. A decrease in thermal transition temperature was observed in the presence of poly(ethylene glycol) for all of the protein systems studied. The magnitude of such a decrease depends on the particular protein and the molecular size of poly(ethylene glycol) employed. A linear relation can be established between the magnitude of the decrease in transition temperature and the average hydrophobicity of these proteins; namely, the largest observable decrease is associated with the protein of the highest hydrophobicity. Further analysis of the thermal unfolding data reveals that poly(ethylene glycols) significantly effect the relation between delta H degrees of unfolding and temperature for all the proteins studied. For beta-lactoglobulin, a plot of delta H versus Tm indicates a change in slope from a negative to a positive value, thus implying a change in delta Cp in thermal unfolding caused by the presence of poly(ethylene glycols). Results from solvent-protein interaction studies indicate that at high temperature poly(ethylene glycol) 1000 preferentially interacts with the denatured state of protein but is excluded from the native state at low temperature. These observations are consistent with the fact that poly(ethylene glycols) are hydrophobic in nature and will interact favorably with the hydrophobic side chains exposed upon unfolding; thus, it leads to a lowering of thermal transition temperature.     

Calorimetric and spectroscopic investigation of the unfolding of human apolipoprotein B

The unfolding of human apolipoprotein B-100 in its native lipid environment, low density lipoprotein (LDL), and in a soluble, lipid-free complex with sodium deoxycholate (NaDC) has been examined using differential scanning calorimetry (DSC) and near UV circular dichroic (CD) spectroscopy. High resolution DSC shows that LDL undergoes three thermal transitions. The first is reversible and corresponds to the order-disorder transition of the core-located cholesteryl esters (CE) (Tm = 31.1 degrees C, delta H = 0.75 cal/g CE). The second, previously unreported, is reversible with heating up to 65 degrees C (Tm = 57.1 degrees C, delta H = 0.20 cal/g apoB) and coincides with a reversible change in the tertiary structure of apoB as shown by near UV-CD. No alteration in the secondary structure of apoB is observed over this temperature range. The third transition is irreversible (Tm = 73.5 degrees C, delta H = 0.99 cal/g apoB) and coincides with disruption of the LDL particle and denaturation of apoB. The ratio of delta H/delta HvH for the reversible protein-related transition suggests that this is a two-state event that correlates with a change in the overall tertiary structure of the entire apoB molecule. The second protein-related transition is complex and coincides with irreversible denaturation. ApoB solubilized in NaDC undergoes three thermal transitions. The first two are reversible (Tm = 49.7 degrees C, delta H = 1.13 cal/g apoB; Tm = 56.4 degrees C, delta H = 2.55 cal/g apoB, respectively) and coincide with alterations in both secondary and tertiary structure of apoB. The changes in secondary structure reflect an increase in random coil conformation with a concomitant decrease in beta-structure, while the change in tertiary structure suggests that the conformation of the disulfide bonds is altered. The third transition is irreversible (Tm = 66.6 degrees C, delta H = 0.54 cal/g apoB) and coincides with complete denaturation of apoB and disruption of the NaDC micelle. The ratio of delta H/delta HvH for the two reversible transitions indicates that each of these transitions is complex which may suggest that several regions or domains of apoB are involved in each thermal event.     

Thermal denaturation of DNA from bromodeoxyuridine substituted cells.

The thermal denaturation of DNA from cell lines extensively substituted with bromodeoxyuridine has been examined spectrophotometrically over a wide range in ionic strength and by thermal elution from hydroxyapatite columns. BrdU substitution stabliizes DNA at all ionic strengths between 7.5 mM and 1350 mM potassium ion concentration, although a plot of log ionic strength vs Tm deviates from linearity above 150 mM. This nonlinearity is most pronounced with BrdU-substituted DNAs, resulting in a lowered delta Tm between unsubstituted and substituted DNA with increasing ionic strength. DMSO is shown to decrease the Tm of both unsubstituted and BrdU-substituted DNA equally, at a rate of .5 degrees C per 1% DMSO.     

Isolation and characterization of Methanothermobacter crinale sp. nov., a novel hydrogenotrophic methanogen from the Shengli oil field

Syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis is an alternative methanogenic pathway in certain thermophilic anaerobic environments such as high-temperature oil reservoirs and thermophilic biogas reactors. In these environments, the dominant thermophilic methanogens were generally related to uncultured organisms of the genus Methanothermobacter. Here we isolated two representative strains, Tm2(T) and HMD, from the oil sands and oil production water in the Shengli oil field in the People's Republic of China. The type strain, Tm2(T), was nonmotile and stained Gram positive. The cells were straight to slightly curved rods (0.3 μm in width and 2.2 to 5.9 μm in length), but some of them possessed a coccal shape connecting with the rods at the ends. Strain Tm2(T) grew with H(2)-CO(2), but acetate is required. Optimum growth of strain Tm2(T) occurred in the presence of 0.025 g/liter NaCl at pH 6.9 and a temperature of 65°C. The G+C content of the genomic DNA was 40.1 mol% ± 1.3 mol% (by the thermal denaturation method) or 41.1 mol% (by high-performance liquid chromatography). Analysis of the 16S rRNA gene sequence indicated that Tm2(T) was most closely related to Methanothermobacter thermautotrophicus ΔH(T) and Methanothermobacter wolfeii VKM B-1829(T) (both with a sequence similarity of 96.4%). Based on these phenotypic and phylogenic characteristics, a novel species was proposed and named Methanothermobacter crinale sp. nov. The type strain is Tm2(T) (ACCC 00699(T) = JCM 17393(T)).     

Evolutionary relationships of the Chinese hamster X chromosome and autosomes: a comparison using solution hybridization techniques

The evolutionary relationships of Chinese hamster X chromosome and autosome DNA sequences were compared by solution hybridization techniques. Chinese hamster X chromosome tracer was prepared by radiolabeling DNA from chromosomes isolated by fluorescence-activated sorting. Radiolabeled Chinese hamster total genomic DNA, approximately 90% of which is of autosome origin, was used as autosome tracer. Each tracer was mixed with excess driver DNA of Chinese hamster, Syrian hamster, rat, rabbit, cat, cow, or human origin. Reaction mixtures were melted and allowed to reassociate to an equivalent CoT of 12000, under conditions which permitted 35% mismatch in DNA duplexes. Both the extent of duplex formation (the normalized percentage hybridization or NPH) and the average thermal stability of the duplexes formed (melting temperature or Tm) were measured; these values were used to compare the evolutionary relatedness of tracer and driver DNAs. The pattern of evolutionary relatedness revealed by comparing either the Tm or NPH values obtained with different drivers was the same for X chromosome and autosome DNA and was consistent with the phylogeny of the species examined. Although NPH and Tm values for X chromosome and autosome tracers differed, differences fell within the range of experimental error. The results of these studies provide no evidence for differential conservation of Chinese hamster X chromosome sequences, suggesting that the constraints on the mammalian X chromosome which act to maintain its gene linkage group intact do not markedly reduce the extent to which its sequences diverge during evolution.