The single-copy DNA sequence polymorphism of the sea urchin strongylocentrotus purpuratus

The single-copy DNA sequence difference between individual sea urchins of the species Strongylocentrotus purpuratus has been estimated by comparing the thermal stability of reassociated DNA duplexes from two individuals with that for DNA from an individual. Thermal stability was measured by hydroxyapatite thermal chromatography, S1 nuclease resistance after heating in a solvent which neutralizes the effect of DNA base composition, and spectrophotometric melting. One pair of individuals appear to differ from each other in about 4% of the nucleotide pairs of their single-copy DNA sequence. The differences in DNA sequence among individuals in local populations are not distinguishably smaller than those among populations as far apart as 2000 kilometers along the Pacific coast of North America.     

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.     

Evolution of sea urchin non-repetitive DNA

Summary New methods have been applied to the determination of single copy DNA sequence differences between the sea urchin speciesStrongylocentrotus purpuratus, S. franciscanus, S. drobachiensis, andLytechinus pictus. The thermal stability of interspecies DNA duplexes was measured in a solvent (2.4 M tetraethylammonium chloride) that suppresses the effect of base composition on melting temperature. The lengths of duplexes were measured after digestion with S1 nuclease and correction made for the effect of length on thermal stability. The degree of base substitution that has occurred in the single copy DNA during sea urchin evolution is significantly larger than indicated by earlier measurements. We estimate that 19% of the nucleotides of the single copy DNA are different in the genomes of the two sea urchin congeners,S. purpuratus, andS. franciscanus, which apparently diverged only 15 to 20 million years ago.     

The divergence between human and baboon globin genes.

Complementary DNA (cDNA) was prepared with viral RNA-directed DNA polymerase from purified baboon globin messenger RNA (mRNA). Homologous and heterologous hybrids between human and baboon mRNAs and cDNAs were compared for extent of hybridisation and thermal stability. Higher mRNA inputs to the hybridizations were required to reach saturation in the heterologous cases. The melting temperature of the heterologous hybrid was 5 degrees C lower than the homologous hybrid. Between these two primates, divergence has occurred in the globin gene to a smaller extent than that possible from third position changes in the coding sequences of the divergence of total DNA. Globin cDNA prepared from baboon will not in general be useful as a probe for human globin mRNA or human globin gene sequences.     

CD, absorption and thermodynamic analysis of repeating dinucleotide DNA, RNA and hybrid duplexes [d/r(AC)]12.[d/r(GT/U)]12 and the influence of phosphorothioate substitution.

Circular dichroism (CD) spectra and melting temperature (Tm) data for five duplexes containing phosphorothioate linkages were compared with data for four unmodified duplexes to assess the effect of phosphorothioate modification on the structure and stability of DNA. DNA and DNA.RNA duplexes. Nine duplexes were formed by mixing oligomers 24 nt long in 0.15 M K+(phosphate buffer), pH 7.0. Unmodified DNA.DNA and RNA.RNA duplexes were used as reference B-form and A-form structures. The CD spectra of the modified hybrids S-d(AC)12.r(GU)12 and r(AC)12.S-d(GT)12 differed from each other but were essentially the same as the spectra of the respective unmodified hybrids. They were more A-form than B-form in character. CD spectra of duplexes S-d(AC)12.d(GT)12 and d(AC)12.S-d(GT)12 were similar to that of d(AC)12.d(GT)12, except for a reduced long wavelength CD band. Sulfur modifications on both strands of the DNA duplex caused a pronounced effect on its CD spectrum. The order of thermal stability was: RNA.RNA > DNA.DNA > DNA.RNA > S-DNA.DNA > S-DNA. RNA > S-DNA.S-DNA. Phosphorothioation of one strand decreased the melting temperature by 7.8+/-0.6 degrees C, regardless of whether the substitution was in a hybrid or DNA duplex. Thermodynamic parameters were obtained from a multistate analysis of the thermal melting profiles. Interestingly, the destabilizing effect of the phosphorothioate substitution appears to arise from a difference in the entropy upon forming the DNA.DNA duplexes, while the destabilizing effect in the DNA.RNA hybrids appears to come from a difference in enthalpy.     

Polynucleotide Sequence Relationships among Members of Enterobacteriaceae

Polynucleotide relationships were examined among many representatives of the Enterobacteriaceae by means of agar, membrane filter, and hydroxyapatite procedures. The amount of deoxyribonucleic acid (DNA) that reassociated was dependent, especially in interspecific reactions, on the annealing temperature. In only three cases: Escherichia coli-Shigella flexneri, Salmonella typhimurium-S. typhi, and Proteus mirabilis-P. vulgaris, was relative interspecific duplex formation 80% or higher. In most cases interspecies DNA duplex formation was 40% or less of that obtained from intraspecies DNA reassociation reactions. The stability of E. coli-S. flexneri DNA duplexes formed at either 60 or 75 C was virtually identical to that of homologous E. coli DNA duplexes, and the degree of interspecies duplex formation was minimally affected by the temperature increase (86% at 60 C; 77% at 75 C). The thermal stability of DNA duplexes formed at 60 C between DNA from E. coli and DNA from strains of Aerobacter aerogenes, S. typhimurium, S. typhi, and P. mirabilis was about 12 to 14 C below that of reassociated E. coli DNA. At 75 C, the formation of the interspecific DNA duplexes was markedly decreased, but the stability of the DNA able to reassociate at this temperature approximated that of reassociated E. coli DNA. The degree of reassociation and the thermal stability of E. coli-S. flexneri DNA duplexes suggests relatively little evolutionary divergence in these organisms. The other enterobacteria tested, however, have diverged to a point where less than one-half of their DNA can reanneal with E. coli DNA at 60 C and less than 10% reacts at 75 C. The degree of divergence between various enterobacteria does not appear to be uniform along the DNA molecule. Ribosomal ribonucleic acid (RNA)-specific sequences are conserved among most enterobacteria. An examination of messenger RNA relatively specific for the lactose operon suggests that specific chromosomal genes may diverge more or less than the genome as a whole.     

Investigation of some properties of oligodeoxynucleotides containing 4'-thio-2'-deoxynucleotides: duplex hybridization and nuclease sensitivity.

The thermal stabilities of the duplexes formed between 4'-thio-modified oligodeoxynucleotides and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligodeoxynucleotides. A 16mer oligodeoxynucleotide containing 10 contiguous 4'-thiothymidylate modifications formed a less stable duplex with the DNA target (deltaTm/modification -1.0 degrees C) than the corresponding unmodified oligodeoxynucleotide. However, when the same oligodeoxynucleotide was bound to the corresponding RNA target, a small increase in Tm was observed (deltaTm/modification +0.16 degrees C) when compared with the unmodified duplex. A study to identify the specificity of an oligodeoxynucleotide containing a 4'-thiothymidylate modification when forming a duplex with DNA or RNA containing a single mismatch opposite the modification found the resulting Tms to be almost identical to the wild-type duplexes, demonstrating that the 4'-thio-modification in oligodeoxynucleotides has no deleterious effect on specificity. The nuclease stability of 4'-thio-modified oligodeoxynucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. No significant resistance to degradation by the exonuclease SVPD was observed when compared with the corresponding unmodified oligodeoxynucleotide. However, 4'-thio-modified oligodeoxynucleotides were found to be highly resistant to degradation by the endonuclease S1. It was also demonstrated that 4'-thio-modified oligodeoxynucleotides elicit Escherichia coli RNase H hydrolysis of the RNA target only at high enzyme concentration.     

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.     

Interactions of oligonucleotide analogs containing methylphosphonate internucleotide linkages and 2'-O-methylribonucleosides.

The interactions of oligonucleotide analogs, 12-mers, which contain deoxyribo- or 2'-O-methylribose sugars and methylphosphonate internucleotide linkages with complementary 12-mer DNA and RNA targets and the effect of chirality of the methylphosphonate linkage on oligomer-target interactions was studied. Oligomers containing a single Rp or Sp methylphosphonate linkage (type 1) or oligomers containing a single phosphodiester linkage at the 5'-end followed by 10 contiguous methylphosphonate linkages of random chirality (type 2) were prepared. The deoxyribo- and 2'-O-methylribo- type 1 12-mers formed stable duplexes with both the RNA and DNA as determined by UV melting experiments. The melting temperatures, Tms, of the 2'-O-methylribo-12-mer/RNA duplexes (49-53 degrees C) were higher than those of the deoxyribo-12mer/RNA duplexes (31-36 degrees C). The Tms of the duplexes formed by the Rp isomers of these oligomers were approximately 3-5 degrees C higher than those formed by the corresponding Sp isomers. The deoxyribo type 2 12-mer formed a stable duplex, Tm 34 degrees C, with the DNA target and a much less stable duplex with the RNA target, Tm < 5 degrees C. In contrast, the 2'-O-methylribo type 2 12-mer formed a stable duplex with the RNA target, Tm 20 degrees C, and a duplex of lower stability with the DNA target, Tm < 5 degrees C. These results show that the previously observed greater stability of oligo-2'-O-methylribonucleotide/RNA duplexes versus oligodeoxyribonucleotide/RNA duplexes extends to oligomers containing methylphosphonate linkages and that the configuration of the methylphosphonate linkage strongly influences the stability of the duplexes.     

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.     

Synthesis and hybridization studies of oligonucleotides containing 1-(2-deoxy-2-alpha-C-hydroxymethyl-beta-D-ribofuranosyl)thymine (2'-alpha-hm-dT)

We report the first investigation of oligoribonucleotides containing a few 1-(2-deoxy-2-alpha-C-hydroxymethyl-beta-D-ribofuranosyl)thymine units (or 2'-hm-dT, abbreviated in this work as 'H'). Both the 2'-CH2O-phosphoramidite and 3'-O-phosphoramidite derivatives of H were synthesized and incorporated into both 2',5'-RNA and RNA chains. The hybridization properties of the modified oligonucleotides have been studied via thermal denaturation and circular dichroism studies. While 3',5'-linked H was shown previously to significantly destabilize DNA:RNA hybrids and DNA:DNA duplexes (modification in the DNA strand; DeltaT(m) approximately -3 degrees C/insert), we find that 2',5'-linked H have a smaller effect on 2',5'-RNA:RNA and RNA:RNA duplexes (DeltaT(m) = -0.3 degrees C and -1.2 degrees C, respectively). The incorporation of 3',5'-linked H into 2',5'-RNA:RNA and RNA:RNA duplexes was found to be more destabilizing (-0.7 degrees C and -3.6 degrees C, respectively). Significantly, however, the 2',5'-linked H units confer marked stability to RNA hairpins when they are incorporated into a 2',5'-linked tetraloop structure (DeltaT(m) = +1.5 degrees C/insert). These results are rationalized in terms of the compact and extended conformations of nucleotides.     

Characteristics of individual repetitive sequence families in the sea urchin genome studied with cloned repeats

Cloned repetitive sequences from the S. purpuratus genome a few hundred to approximately 1000 nucleotides long were used to investigate the characteristics of individual repetitive sequence families. They were terminally labeled by the kinase procedure and reacted with sheared S. purpuratus DNA. Repetition frequencies were measured for 26 individual families and were found to vary from a few to several thousand copies per genome. Estimates of sequence divergence were made for 18 cloned repeat families by measuring thermal stability of the heteroduplexes formed between the genomic DNA and the cloned fragments, compared with that of the renatured cloned fragments. The difference was <4°C for three of the 18 families, and <10°C for 13 of the 18 families. These 13 repetitive sequence families lack any detectable highly divergent sequence relatives, and the results reported are shown not to change when the renaturation criterion is lowered below 55°C in 0.18 M Na⁺. Five of the 18 cloned families displayed greater sequence divergence. The average sequence divergence of the total short repetitive sequence fraction of S. purpuratus DNA was found to match closely the average of the divergences of the cloned repeat sequences.     

Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes.

To improve the previous DNA/DNA nearest-neighbor parameters, thermodynamic parameters (deltaH degrees, deltaS degrees and deltaG degrees) of 50 DNA/DNA duplexes were measured. Enthalpy change of a helix initiation factor is also considered though the parameters reported recently did not contain the factor. A helix initiation factor for DNA/DNA duplex determined here was the same as that of RNA/RNA duplex (deltaG degrees(37) = 3.4 kcal/mol). The improved nearest-neighbor parameters reproduced not only these 50 experimental values used here but also 15 other experimental values obtained in different studies. Comparing deltaG degrees(37) values of DNA/DNA nearest-neighbor parameters obtained here with those of RNA/RNA and RNA/DNA, RNA/RNA duplex was generally the most stable of the three kinds of duplexes with the same nearest-neighbor sequences. Which is more stable between DNA/DNA and RNA/DNA duplexes is sequence dependent.     

Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch.

Oligodeoxyribonucleotides complementary to the DNA of the wild type (wt) bacteriophage phi chi 174 have been synthesized by the phosphotriester method. The oligomers, 11, 14, and 17 bases long, are complementary to the region of the DNA which accounts for the am-3 point mutation. When hybridized to am-3 DNA, the oligonucleotides form duplexes with a single base pair mismatch. The thermal stability of the duplexes formed between wt and am-3 DNAs has been measured. The am-3 DNA:oligomer duplexes dissociate at a temperature about 10 degrees C lower than the corresponding wt DNA:oligomer duplexes. This dramatic decrease in thermal stability due to a single mismatch makes it possible to eliminate the formation of the mismatched duplexes by the appropriate choice of hybridization temperature. These results are discussed with respect to the use of oligonucleotides as probes for the isolation of specific cloned DNA sequences.     

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.     

Studies on nucleic acid interactions. I. Stabilities of mini-duplexes (dG2A4XA4G2-dC2T4YT4C2) and self-complementary d(GGGAAXYTTCCC) containing deoxyinosine and other mismatched bases.

The thermal stability of DNA duplexes containing deoxyinosine in a pairing position in turn with each of the four major deoxynucleotides has been investigated by measuring ultraviolet-absorbance at different temperatures. d(G2A4 X A4G2) and d(C2T4YT4C2) were prepared by the solid-phase phosphotriester method. When X is deoxyinosine, the Tm values of the duplexes are in the order Y = dC greater than dA greater than dG greater than dT greater than dU. The Tm of other duplexes containing dG, dA and dT at X were also measured. Self-complementary duplexes d(GGGAAINTTCCC) showed the same order of stability with N being dC, dA, dG and dT. Thermal stabilities of duplexes containing dG instead of dI were compared with other matched and mismatched duplexes. The Tm values of sequence isomers containing purine-pyrimidine combinations were compared. Self-complementary duplexes containing G-C and A-T in the central positions showed Tm values ca. 10 degrees higher than those containing C-G and T-A in the same positions. Thermodynamic parameters and circular dichroism spectra of these oligonucleotides were compared.     

Structure and stability of the consecutive stereoregulated chiral phosphorothioate DNA duplex

The duplex structures of the stereoregulated phosphorothioate DNAs, [R(p),R(p)]- and [S(p),S(p)]-[d(GC(ps)T(ps)ACG)] (ps, phosphorothioate; PS-DNA), with their complementary RNA have been investigated by combined use of (1)H NMR and restrained molecular dynamics calculation. Compared to those obtained for the unmodified duplex structures (PO-DNA.RNA), the NOE cross-peak intensities are virtually identical for the PS-DNA.RNA hybrid duplexes. The structural analysis on the basis of the NOE restraints reveals that all of the three DNA.RNA duplexes take a A-form conformation and that there is no significant difference in the base stacking for the DNA.RNA hybrid duplexes. On the other hand, the NOE cross-peak intensities of the protons around the central T(ps)A step of the PS-DNA.DNA duplexes are apparently different from those of PO-DNA. DNA. The chemical shifts of H8/6 and H1' at the T(ps)A step are also largely different among PS-DNA.DNAs and PO-DNA.DNA, suggesting that the DNA.DNA structure is readily changed by the introduction of the phosphorothioate groups to the central T(p)A step. The structure calculations indicate that all of these DNA.DNA duplexes are B-form although there exist some small differences in helical parameters between the [R(p),R(p)]- and [S(p),S(p)]PS-DNA.DNA duplexes. The melting temperatures (T(m)) were determined for all of the duplexes by plotting the chemical shift change of isolated peaks as a function of temperature. For the PS-DNA.RNA hybrid duplexes, the [S(p),S(p)] isomer is less stable than the [R(p),R(p)] isomer while this trend is reversed for the PS-DNA.DNA duplexes. Consequently, although the PS-DNA.RNA duplexes take the similar A-form structure, the duplex stability is different between PS-DNA.RNA duplexes. The stability of the DNA.RNA duplexes may not be governed by the A-form structure itself but by some other factors such as the hydration around the phosphorothioate backbone, although the T(m) difference of the DNA.DNA duplexes could be explained by the structural factor.     

Synthesis and properties of cationic oligopeptides with different side chain lengths that bind to RNA duplexes

A series of artificial peptides bearing cationic functional groups with different side chain lengths were designed, and their ability to increase the thermal stability of nucleic acid duplexes was investigated. The peptides with amino groups selectively increased the stability of RNA/RNA duplexes, and a relationship between the side chain length and the melting temperature (T_m) of the peptide–RNA complexes was observed. On the other hand, while peptides with guanidino groups exhibited a similar tendency with respect to the peptide structure and thermal stability of RNA/RNA duplexes, those with longer side chain lengths, such as l-2-amino-4-guanidinobutyric acid (Agb) or l-arginine (Arg) oligomers, stabilized both RNA/RNA and DNA/DNA duplexes, and those with shorter side chain lengths exhibited a higher ability to selectively stabilize RNA/RNA duplexes. In addition, peptides were designed with different levels of flexibility by introducing glycine (Gly) residues into the l-2-amino-3-guanidinopropionic acid (Agp) oligomers. It was found that insertion of Gly did not affect the thermal stability of the peptide–RNA complexes, but an alternate arrangement of Gly and Agp apparently decreased the thermal stability. Therefore, in the Agp oligomer, consecutive Agp sequences are essential for increasing the stability of RNA/RNA duplexes.