The analysis of circular dichroism spectra of natural DNAs using spectral components from synthetic DNAs

We have tested 21 different basis sets of synthetic DNA circular dichroism spectra and have slected one for use in spectral analyses of natural DNAs. This “standard” set consists of spectra of eight polymers: poly[d(A-A-T)·d(A-T-T)], poly[d(A-G-G)·d(C-C-T)], poly[d(A-T)·d(A-T)], poly[d(G-C)·d(G-C)], poly[d(A-G)·d(C-T)], poly[d(A-C)·d(G-T)], poly[d(A-T-C)·d(G-A-T)], and poly[d(A-G-C)·d(G-C-T)]. This basis set, applied according to the first-neighbor polymer procedure of Gray and Tinoco, allows a more uniformly accurate spectral analysis of six natural complex DNAs and eight (A+T)-rich satellite DNAs for base composition and first-neighbor frequencies than was previously possible. We find that spectra of poly[d(A)·d(T)] and/or poly[d(A-C-T-)·d(A-G-T)] are not generally required for good analysis results but we show in this and the following paper that these spectra are needed for the most accurate analyses of some satellite DNAs.     

Sequence dependence of the circular dichroism of synthetic double-stranded RNAs

We have synthesized and studied the CD spectra of five new double-stranded RNA polymers: poly[r(A-G)·r(C-U)], poly[r(A-U-C)·r(G-A-U)], poly[r(A-C-U)·r(A-G-U)], poly[r(A-A-C)·r(G-U-U)], and poly[r(A-C-C)·r(G-G-U)]. Together with previously published spectra of seven other RNA sequences, the spectra of these new sequences provide a library sufficient to approximate the spectra of all other RNA sequences by first-neighbor formulas and, in addition, give four spectra with which we may test the validity of first-neighbor approximations. (1) We find that the spectra of RNA sequence isomers are very different, but that the spectra essentially do obey first-neighbor relationships. (2) We have derived tentative first-neighbor assignments of negative bands at about 295 and 210 nm in the CD spectra. (3) A test of spectral independence shows that among the 12 polymer spectra there are at least seven significant independent spectral shapes, one less than the eight needed to give the most accurate spectral analysis of an unknown RNA sequence for its first-neighbor frequencies. (4) Spectra are calculated for RNAs of random base composition, approximating natural RNAs having complex sequences. (5) A T-matrix of spectral components assigned to the first-neighbor base pairs is derived from 10 of the spectra. This matrix allows an estimation of the CD spectrum of any other known RNA sequence or an analysis of the spectrum of an unknown sequence for its distribution of first-neighbor base-pair frequencies. (6) Test analyses of two of the synthetic polymers and of two natural RNAs set a probable limit on the accuracy of first-neighbor frequency determinations using this T-matrix. (7) Finally, we summarize in an appendix the melting temperatures for all the RNA and corresponding DNA sequences; it appears that the T_m values of both DNAs and RNAs approximately obey first-neighbor relationships.     

An ochre suppressor of bacteriophage T4 that is associated with a transfer RNA

Ultraviolet circular dichroism spectra have been obtained for native and heat-denatured Drosophila virilis satellite DNAs I, II and III. Gall &; Atherton (1974) have found that these DNAs have simple, unique sequences. We compare here the circular dichroism spectra of these satellite sequences with the circular dichroism spectra of synthetic DNAs of simple sequences which are combined in first-neighbor calculations. We also apply an analytical procedure for determining nearest-neighbor frequencies from the DNA spectra (Allen et al., 1972). The results are an indication of the potential usefulness and present limitations of circular dichroism measurements in confirming or determining the nearestneighbor frequencies of satellite DNAs of simple sequences.     

A comparison of the circular dichroism spectra of synthetic DNA sequences of the homopurine . homopyrimidine and mixed purine- pyrimidine types.

We have obtained the ultraviolet circular dichroism spectra of two repeating trinucleotide DNAs, poly [d(A-G-G).d(C-C-T)] and poly[d(A-A-G).d(C-T-T)], that have all purines on one strand and all pyrimidines on the other. These spectra, together with spectra of other synthetic polymers, can be combined to give 3 first-neighbor calculations of the spectrum of poly[d(A).d(T)] and 2 first-neighbor calculations of the spectrum of poly [d(G).d(C)]. The results show (1) that first-neighbor calculations utilizing only spectra of homopurine.homopyrimidine DNA sequences are no more accurate than are similar calculations that involve spectra of mixed purine-pyrimidine sequences, demonstrating that double-stranded homopurine.homopyrimidine sequences do not obviously belong to a special class of secondary conformations, and (2) that the wavelength region above 250 nm in the CD spectra of synthetic DNAs is least predictable from first-neighbor equations, probably because this region is especially sensitive to sequence-dependent conformational differences.     

The effect of silver ion binding and pH on the buoyant density of DNA and its use in fractionating heterogeneous DNA.

1. The effect of pH on the buoyant density of the complexes of Ag+ with DNA has been studied using 3H-labeled human DNA and several bacterial DNAs to determine the conditions necessary for the maximum resolution of compositional heterogeneity. In neutral CS2SO4 density gradients, Ag+ complexes with (G - C)-rich components are always denser than those with (A - T)-rich components, since (G - C)rich DNAs have a larger affinity for Ag+ than (A - T)-rich DNAs and their complexes are denser than (A - T)-rich complexes. In alkaline (pH greater than 9) CS2SO4 gradients, the buoyant density of the Ag+ - DNA complex is not a simple function of base composition. The Ag+ affinity of (A - T)-rich DNA is larger than that of (G - C)-rich DNA but the density of a (G - C)-rich complex is larger. Thus the ordering of the buoyant density changes depends on the amount of added Ag+. 2. The problem of resolving the density heterogeneity within a tracer DNA, and minor components of DNA, is explored and useful fractionation techniques are developed.     

Isolation and comparison of tribe-specific centromeric repeats within Bovidae

A taxonomic division of the family Bovidae (Artiodactyla) is difficult and the evolutionary relationships among most bovid subfamilies remain uncertain. In this study, we isolated the cattle satellite I clone BTREP15 (1.715 satellite DNA family) and autosomal centromeric DNAs of members of ten bovid tribes. We wished to determine whether the analysis of fluorescence in situ hybridization patterns of the cattle satellite I clone (BTREP15) and tribe-specific centromeric repeats isolated by laser microdissection would help to reveal some of the ambiguities occurring in the systematic classification of the family Bovidae. The FISH study of the presence and distribution of the cattle satellite I clone BTREP15 (1.715 satellite DNA family) within members of ten bovid tribes was not informative. FISH analysis of autosomal centromeric DNA probes in several species within one tribe revealed similar hybridization patterns in autosomes confirming tribal homogeneity of these probes. Sex chromosomes showed considerable variation in sequence composition and arrangement not only between tribes but also between species of one tribe. According to our findings it seems that Oreotragus oreotragus developed its own specific satellite DNA which does not hybridize to any other bovid species analysed. Our results suggest O. oreotragus as well as Aepyceros melampus may be unique species not particularly closely related to any of the recognized bovid tribes. This study indicates the isolation of tribe-specific centromeric DNAs by laser microdissection and cloning the sequence representing the main motif of these repetitive DNAs could offer the perspectives for comparative phylogenetic studies.     

Analysis of DNA from related and diverse species of Barley

DNAs of three closely related diploid species Hordeum vulgare, H. agriocrithon and H. spontaneum were compared among themselves and with a group of three species related to each other H. californicum (2x), H. jubatum (4x), and H. arizonicum (6x) having one genome in common.Buoyant densities of the DNAs from these species fall within a narrow range (1.700–1.701 g cm⁻³). Melting temperatures (T_m) of the DNAs are also similar (85.2–86.2°C). None of the DNAs showed any satellite band in neutral Cesium chloride gradients. In silver-cesium sulphate gradients, however, DNAs of all the species formed satellites on both the light and heavy sides of the main. band The diploid series have similar but not identical satellite patterns. The members of the polyploid series showed similar satellite patterns among themselves but distinct from those of the diploid series. In the polyploid group the amount of satellite showed a progressive decrease with the increase in the ploidy.The amount of repeated DNA (reassociating up to Cot 100) in H. vulgare was 69% and in H. arizonicum 65% of the total. The two species differed in their Cot curves. The low Cot fractions of H. arizonicum contained components of low buoyant densities which were absent in the corresponding Cot fractions of H. vulgare DNA.     

Effects of shell stress on the growth of hermit crabs

Growth rates of the hermit crabs Pagurus longicarpus Say and P. pollicaris Say maintained in preferred shells and in shells smaller than the preferred size have been compared. Changes in wet weight, shield length, and duration of the molt cycle were determined. Crabs in preferred shells grew significantly faster than those in small shells. P. pollicaris molted at approximately the same rate in both cases but grew more each molt in preferred shells. Von Bertalanffy growth curves were fitted to changes in shield length among crabs in preferred shells. These curves indicate that P. longicarpus may mature four months after settling from the plankton and reach its asymptotic size within the next eight months while P. pollicaris also matures four months after leaving the plankton but does not reach its asymptotic size for approximately three years. The rapid growth of P. longicarpus may enable it to preempt shells which are required for the successful brooding of a large clutch before these shells are required by more aggressive competitors, such as P. pollicaris and Clibanarius vittatus.     

Cooperative lengths of DNA during melting

The mean cooperative length of domains of DNA, determined from the variance in (G + C) content in derivative melting curves of large bacterial DNAs, varies from 230 base pairs (bp) for (A ? T)-rich domains to 580 bp for (G ? C) domains. These values correspond to values for the cooperativity parameter of 2(±2) × 10^?5 and 3(±2) × 10^?6, respectively, and to +7.2 and +9.6 kcal for the free energy of helix interruption in those regions.     

Detection and location of three simple sequence DNAs in polytene chromosomes from virilis group species of Drosophila

In vitro synthesized RNAs complementary to the three satellite DNAs of Drosophila virilis have been used in a series of in situ hybridization experiments with polytene chromosomes from virilis group species. Gall and Atherton (1974) demonstrated that each of the satellites of D. virilis is comprised of many repeats of a distinct, seven base pair long, simple sequence. With few exceptions, copies of each of these simple sequences are detected in the chromocenters of all virilis group species. This is true even in species which do not possess satellite DNAs at buoyant densities corresponding to those of the satellite DNAs of D. virilis. Small quantities of the three simple sequences are also detected in euchromatic arms of several different species. The same euchromatic location may contain detectable copies of one, two, or all three simple sequence DNAs. The amounts of simple sequences at each location in the euchromatin may vary between species, between different stocks of the same species, and even between individuals of the same stock. The simple sequences located in the euchromatin appear to undergo DNA replication during formation of polytene chromosomes unlike those in heterochromatin. The locations of the euchromatic sequences are not the results of single chromosomal inversion events involving heterochromatic and euchromatic breakpoints.     

Detection of satellite DNA in Palorus ratzeburgii: Analysis of curvature profiles and comparison with Tenebrio molitor satellite DNA

Very abundant and homogenous satellite DNA has been found in the flour beetle Palorus ratzeburgii, representing 40% of its genome. Sequencing of 14 randomly cloned satelite monomers revealed a conserved monomer length of 142 bp and an average A+T content of 68%. Sequence variation analysis showed that base substitutions, appearing with a frequency of 2.3%, are predominant differences among satellite monomers. The satellite sequence is unique without significant direct repeats and with only two potentially stable inverted repeats. After electrophoresis of satellite monomers on native polyacrylamide gel retarded mobilities characteristic for curved DNA molecules are observed. The curvature profiles and DNA helix axis trajectory are calculated on the basis of three different algorithms. These calculations predict that P ratzeburgii satellite DNA forms a left-handed solenoid superstructure. Comparison of described features with other satellite DNAs reveals some striking similarities with satellite DNA from related species Tenebrio molitor, which belongs to the same family of Tenebrionidae. Both satellites are very abundant and homogenous with the same, highly conserved monomer length, although there is no homology at the nucleotide level. Their monomers, as well as multimers, exhibit very similar retarded electrophoretic mobilities. The calculated curvature profiles predict two bend centers in monomers of each satellite, resulting in a model of left-handed solenoid superstructures of similar appearance.     

REVEALING GENETIC MARKERS IN GELIDIUM VAGUM (RHODOPHYTA) THROUGH THE RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) TECHNIQUE1

The recently developed random amplified polymorphic DNA technique was evaluated as a method for characterizing isolates of the agarophyte Gelidium vagum Okamura. Reaction conditions for single primer polymerase chain reaction were optimized to obtain a high degree of reproducibility of the amplified bands generated from purified G. vagum DNA. A total of 165 primers, including both (A + T)- and (G + C)-rich sequences, was screened for DNA amplification using template DNA from a single Gelidium isolate. None of the 45 (A + T)-rich primers was positive (i.e. band-producing). Of the (G + C)-rich primers, 47 were positive, generating a total of 322 prominent amplification products for DNA from 13 different G. vagum isolates. Polymorphic DNA loci were detected by 37 of the primers. Unweighted pair-group arithmetic average cluster analysis (UPGMA) of these loci was used to group the G. vagum isolates and thereby determine which were most similar. G. latifolium, used as an out-group for the UPGMA analysis, showed a high degree of dissimilarity.     

Reduced Rates of Sequence Evolution of Y-Linked Satellite DNA in Rumex (Polygonaceae)

One characteristic of sex chromosomes is the accumulation of a set of different types of repetitive DNA sequences in the Y chromosomes. However, little is known about how this occurs or about how the absence of recombination affects the subsequent evolutionary fate of the repetitive sequences in the Y chromosome. Here we compare the evolutionary pathways leading to the appearance of three different families of satellite-DNA sequences within the genomes of Rumex acetosa and R. papillaris, two dioecious plant species with a complex XX/XY₁Y₂ sex-chromosome system. We have found that two of these families, one autosomic (the RAE730 family) and one Y-linked (the RAYSI family), arose independently from the ancestral duplication of the same 120-bp repeat unit. Conversely, a comparative analysis of the three satellite-DNA families reveals no evolutionary relationships between these two and the third, RAE180, also located in the Y chromosomes. However, we have demonstrated that, regardless of the mechanisms that gave rise to these families, satellite-DNA sequences have different evolutionary fates according to their location in different types of chromosomes. Specifically, those in the Y chromosomes have evolved at half the rate of those in the autosomes, our results supporting the hypothesis that satellite DNAs in nonrecombining Y chromosomes undergo lower rates of sequence evolution and homogenization than do satellite DNAs in autosomes.[Reviewing Editor: DR. Jerzy Jurka]     

Analysis of DNAs from two species of the virilis group of Drosophila and implications for satellite DNA evolution

The DNAs from two virilis group species of Drosophila, D. lummei and D. kanekoi, have been analyzed. D. lummei DNA has a major satellite which, on the basis of CsCl equilibrium centrifugation, thermal denaturation, renaturation and in situ hybridization is identical to D. virilis satellite I. D. kanekoi DNA has a major satellite at the same buoyant density in neutral CsCl gradients as satellite III of D. virilis. However, on the basis of alkaline CsCl gradients, the satellite contains a major and a minor component, neither one of which is identical to D. virilis satellite III. By in situ hybridization experiments, sequences complementary to the major component of the D. kanekoi satellite are detected in only some species and in a way not consistent with the phylogeny of the group. However, by filter hybridization experiments using nick-translated D. kanekoi satellite as well as D. lummei satellite I and D. virilis satellite III DNAs as probes, homologous sequences are detected in the DNAs of all virilis group species. Surprisingly, sequences homologous to these satellite DNAs are detected in DNAs from non-virilis group Drosophila species as well as from yeast, sea urchin, Xenopus and mouse.     

Hysteresis and partial irreversibility of denaturation of DNA as a means of investigating the topology of base distribution constraints: application to a yeast rho- (petite) mitochondrial DNA

Low salt concentrations prevent reassociation of separated single strands of DNA, but not the renaturation of partially melted molecules. Rewinding, however, may be delayed (hysteresis) and/or incomplete (partial irreversibility). Long-range fluctuations in base compositioncould account for these observations: (a) the “zippering-up” of a denatured (G + C)-rich section may have to await that of one of its neighbouring (A + T)-rich sections, hence a temperature lag in rewinding; (b) the removal of intramolecular heterogeneities in base composition by fragmentation will give rise to a dispersal of strand-separation temperatures. Conversely, it is shown how a considerable amount of information about the topology of base distribution constraints could be derived from these phenomena.Some yeast ρ^? (petite) mitochondrial DNAs, the melting of which is quasidiscontinuous, provide an excellent opportunity for testing the applicability of this new approach to denaturation mapping. Alternating partial denaturation and renaturation with a low rate of temperature change were followed by high-frequency recording of absorbance at 260 nm. A typical experiment (counterion concentration 0.015 m-Na⁺) carried out on a low-complexity (length of repetitive unit about 3000 base-pairs) ρ^? DNA is reported in full detail. Analysis of the data disclosed the existence of two relatively (G + C)-rich clusters separated by long homogeneous stretches of high (A + T) content.The rewinding of ρ^? DNAs is a discontinuous process. Both equilibrium and non-equilibrium melting processes were observed. Hysteresis in rewinding, which is restricted to the melting range, increases discontinuously with the extent of unwinding reached prior to cooling. Results are shown to be fully consistent with a model that presupposes that nucleation does not play any part in the renaturation process. They are briefly discussed further in the light of current concepts in the theory of helix-coil transitions of DNA.     

Studies on maternal inheritance in polyploid wheats with cytoplasmic DNAs as genetic markers

Summary Restriction fragment patterns of DNA fragments obtained after EcoRI cleavage of chloroplastic (cp) and mitochondrial (mt) DNAs isolated from different wheat species were compared. T. aestivum, T. timopheevi, Ae. speltoides, Ae. sharonensis and T. urartu gave species specific mt DNA patterns. Consequently, the cytoplasmic genomes of wheat cannot have originated from contemporary Ae. speltoides, Ae. sharonensis and T. urartu species. It is shown that cp and mt DNAs of Ae. ventricosa, a tetraploid used to transfer eyespot resistance into T. aestivum, contains cp and mt DNAs differing from DNAs isolated from T. aestivum and other wheats. In contrast, the cytoplasmic DNAs of Ae. ventricosa and Ae. squarrosa reveal an important homology, suggesting that Ae. squarrosa was the female parent of Ae. ventricosa. Disomic addition lines (T. aestivum — Ae. ventricosa) in both Ae. ventricosa cytoplasm and T. aestivum cytoplasm contained cytoplasmic DNAs identical to those of the maternal parent. Restriction patterns of the cp and mt DNAs isolated from eight lines of Triticale differing in their cytoplasm have been compared to those of the maternal parent. A strict maternal inheritance has been observed in each case.     

Characterization of Satellite DNA from Three Marine Dinoflagellates (Dinophyceae): Glenodinium sp. and Two Members of the Toxic Genus, Protogonyaulax

Using CsCl-Hoechst dye or CsCl-ethidium bromide gradients, satellite and nuclear DNAs were separated and characterized in three marine dinoflagellates: Glenodinium sp., and two toxic dinoflagellates, Protogonyaulax tamarensis and Protogonyaulax catenella. In all three dinoflagellates, the lowest density fraction, satellite DNA₁, hybridized to chloroplast genes derived from terrestrial plants and/or other algae. Dinoflagellate chloroplast DNAs exhibited molecular sizes of 114 to 125 kilobase pairs, which is consistent with plastid sizes determined for other chromophytic algae (120-150 kilobase pairs). Mitochondrial DNA was not resolved from nuclear DNA in this system. Two additional satellite DNAs, satellite DNA₂ and satellite DNA₃, recovered from P. tamarensis and P. catenella were similar to one another, both within and between species, when characterized by restriction enzyme analysis. These satellites were 85 to 95 kilobase pairs in size, and exhibited restriction fragments that hybridized to yeast nuclear ribosomal RNA genes. Restriction enzyme analyses and DNA hybridization studies of cpDNA document that the two Protogonyaulax isolates are not evolutionarily identical.     

Satellite DNA sequences in the neotropical marmoset Callimico goeldii (Primates,Platyrrhini)

Two families of tandemly repeated satellite DNAs were isolated from the neotropical primate Callimico goeldii (Goeldi's marmoset). One satellite, CgoA, is over 70% A+T and has a monomer length of 338 bp. The other satellite, CgoB, is 50% A+T and has a monomer length of 916 bp. Both CgoA and CgoB hybridize strongly with Callimico DNA, but not with the DNA of other new and old world primates. Based upon a neutral substitution rate of 1.5×10^–9/site per year for primates, sequence data from 15 CgoA monomers indicate that the tandem array is at least 30 million years old. Since no other neotropical primate has amplified CgoA sequences, the data suggest that the ancestor of Callimico separated from the other neotropical primates at least 30 million years ago. This value is about fourfold larger than the value of 7–9 million years derived from immunological data by Sarich and Cronin (1980). Possible reasons for this discrepancy are discussed. On leave from: Genetics Section, Instituto Nacional do Cancer, Rio de Janeiro/Department of Genetics, Universidade Federal do Rio de Janeiro, Brazil     

Salt-concentration dependence of melting profiles of lambda phage DNAs: evidence for long-range interactions and pronounced end effects.

Melting profiles of DNAs from wild-type λ phage and a deletion mutant phage λb2 were examined in a wide range of salt concentration. The fine structure of the melting profiles changed sharply with salt concentration, especially in the range [Na⁺] ? 10 mM. A comparison of the melting profiles between the wild-type and the deletion mutant DNAs provided good evidence for extremely high melting cooperativity under low salt conditions, which is clearly manifested as the long-range interactions and the pronounced end effects; a large melting peak appeared as a result of the b2 deletion without any inserted sequence in the salt range [Na⁺] ? 2.8 mM. It was also suggested that in the further reduced salt range [Na⁺] ? 2.0 mM, melting of a λ DNA molecule starts from its right end rather than the most (A + T)-rich central region. The molecular basis of the high melting cooperativity at low salt concentrations can be explained in terms of the increased free energy associated with loop formation in the double-helical structure of DNA.     

The ultraviolet circular dichroism of some natural DNAs and an analysis of the spectra for sequence information

The circular dichroism spectra of many natural DNAs and double-stranded synthetic polynucleotides were obtained. The eight first-neighbor contributions to the CD spectra of a DNA have been extracted from these data. Therefore, the CD spectrum for any DNA with known first-neighbor frequencies may be easily calculated. For a natural DNA the CD spectrum may be approximated by assuming the first-neighbor frequencies have the most probable values consistent with the base composition. Under favorable conditions, the measured CD spectrum can be used to determine thirteen of the sixteen first-neighbor frequencies of a DNA to ± 0.02 mole percent. The TG, CA, and TA first-neighbor cannot be unambiguously resolved by our method. The accuracy of the first-neighbor frequency analysis depends on the number of different first-neighbors present in the DNA and the extent to which they differ from the most probable value. The extinction coefficient at 260 nm and the base composition can also be calculated from the CD spectrum.