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In this paper, we present an electrochemically driven large amplitude pH alteration method based on a serial electrolytic cell involving a hydrogen permeable bifacial working electrode such as Pd thin foil. The method allows solution pH to be changed periodically up to ±4~5 units without additional alteration of concentration and/or composition of the system. Application to the acid-base driven cyclic denaturation and renaturation of 290 bp DNA fragments is successfully demonstrated with in situ real-time UV spectroscopic characterization. Electrophoretic analysis confirms that the denaturation and renaturation processes are reversible without degradation of the DNA. The serial electrolytic cell based electrochemical pH alteration method presented in this work would promote investigations of a wide variety of potential-dependent processes and techniques.

We employed salt-dependent differential scanning calorimetric measurements to characterize the stability of six oligomeric DNA duplexes (5'-GCCGGAXTGCCGG-3'/5'-CCGGCAYTCCGGC-3') that contain in the central XY position the GC, AT, GG, CC, AA, or TT base pair. The heat-induced helix-to-coil transitions of all the duplexes are associated with positive changes in heat capacity, DeltaC(p), ranging from 0.43 to 0.53 kcal/mol. Positive values of DeltaC(p) result in strong temperature dependences of changes in enthalpy, DeltaH degrees, and entropy, DeltaS degrees , accompanying duplex melting and cause melting free energies, DeltaG degrees, to exhibit characteristically curved shapes. These observations suggest that DeltaC(p) needs to be carefully taken into account when the parameters of duplex stability are extrapolated to temperatures distant from the transition temperature, T(M). Comparison of the calorimetric and van't Hoff enthalpies revealed that none of the duplexes studied in this work exhibits two-state melting. Within the context of the central AXT/TYA triplet, the thermal and thermodynamic stabilities of the duplexes in question change in the following order: GC > AT > GG > AA approximately TT > CC. Our estimates revealed that the thermodynamic impact of the GG, AA, and TT mismatches is confined within the central triplet. In contrast, the thermodynamic impact of the CC mismatch propagates into the adjacent helix domains and may involve 7-9 bp. We discuss implications of our results for understanding the origins of initial recognition of mismatched DNA sites by enzymes of the DNA repair machinery.

The thermodynamics of nine hairpin DNAs were evaluated using UV-monitored melting curves and differential scanning calorimetry (DSC). Each DNA has the same five-base loop and a stem with 8-10 base pairs. Five of the DNAs have a tandem mismatch in the stem, while four have all base pairs. The tandem mismatches examined (ga/ga, aa/gc, ca/gc, ta/ac, and tc/tc) spanned the range of stability observed for this motif in a previous study of 28 tandem mismatches. UV-monitored melting curves were obtained in 1.0 M Na(+), 0.1 M Na(+), and 0.1 M Na(+) with 5 mM Mg(2+). DSC studies were conducted in 0.1 M Na(+). Transition T(m) values were unchanged over a 50-fold range of strand concentration. Model-independent enthalpy changes (DeltaH degrees ) evaluated by DSC were in good agreement (+/-8%) with enthalpy values determined by van't Hoff analyses of the melting curves in 0.1 M Na(+). The average heat capacity change (DeltaC(p)) associated with the hairpin to single strands transitions was estimated from plots of DeltaH degrees and DeltaS degrees with T(m) and ln T(m), respectively, and from profiles of DSC curves. The average DeltaC(p) values (113 +/- 9 and 42 +/- 27 cal x K(-1) x mol(-1) of bp), were in the range of values reported in previous studies. Consideration of DeltaC(p) produced large changes in DeltaH degrees and DeltaS degrees extrapolated from the transition region to 37 degrees C and smaller but significant changes to free energies. The loop free energy of the five tandem mismatches at 37 degrees C varied over a range of approximately 4 kcal x mol(-1) for each solvent.

Mg(2+)-induced folding of yeast tRNA(Phe) was examined at low ionic strength in steady-state and kinetic experiments. By using fluorescent labels attached to tRNA, four conformational transitions were revealed when the Mg(2+) concentration was gradually increased. The last two transitions were not accompanied by changes in the number of base pairs. The observed transitions were attributed to Mg(2+) binding to four distinct types of sites. The first two types are strong sites with K(diss) of 4 and 16 microM. The sites of the third and fourth types are weak with a K(diss) of 2 and 20 mM. Accordingly, the Mg(2+)-binding sites previously classified as "strong" and "weak" can be further subdivided into two subtypes each. Fluorescent transition I is likely to correspond to Mg(2+) binding to a unique strong site selective for Mg(2+); binding to this site causes only minor A(260) change. The transition at 2 mM Mg(2+) is accompanied by substantial conformational changes revealed by probing with ribonucleases T1 and V1 and likely enhances stacking of the tRNA bases. Fast and slow kinetic phases of tRNA refolding were observed. Time-resolved monitoring of Mg(2+) binding to tRNA suggested that the slow kinetic phase was caused by a misfolded tRNA structure formed in the absence of Mg(2+). Our results suggest that, similarly to large RNAs, Mg(2+)-induced tRNA folding exhibits parallel folding pathways and the existence of kinetically trapped intermediates stabilized by Mg(2+). A multistep scheme for Mg(2+)-induced tRNA folding is discussed.

Under nonenzymatic conditions in vitro, Adriamycin appears to form interstrand cross-links with DNA over 1-2 days. This is the first report of such Adriamycin-induced interstrand cross-links in vitro. Cross-links were measured by a fluorescence based renaturation assay and also by gel electrophoresis. Both procedures revealed an increase of cross-linking with reaction time and with increasing Adriamycin concentration and a 5-6-fold enhancement in the presence of Fe3+ ions. The cross-link contains the Adriamycin chromophore, with a lambda max of 508 nm, intercalated at the GpC site of cross-linking. Maximal stoichiometry of the cross-link was one per 11-20 bp. The cross-link appears to involve adducts of the Adriamycin chromophore linked to the N2 of guanine, with no indication that N7 of guanine is involved. Given that the mode of action of Adriamycin still remains obscure, even after 20 years of clinical use, the possibility that interstrand DNA cross-links may be associated with the clinical mechanism of action of this drug should now be fully addressed.

Thermodynamic and kinetic parameters for the triplex-forming reactions between a homopurine-homopyrimidine 22-base-pair duplex (sequence of the purine strand: 5'd[AAAGGAGGAGAAGAAGAAAAAA]3') and the four 22-dN third strands (22 dN: 5'd[TTTCCTCCTCTNCTTCTTTTTT]3', where N = A, C, T, or G) were determined from thermal denaturation and renaturation UV absorbance profiles. Cooling and heating curves were not superimposable and thus allowed us to determine the rate constants of association (k(on)) and dissociation (k(off)) as a function of temperature, assuming a two-state model analogous to that developed for duplex-forming reactions. Experiments were performed in 10 mM cacodylate buffer (pH 6.8) in the presence of NaCl concentrations ranging from 20 to 300 mM. Within experimental accuracy, the main results are the following: (i) The rate constants k(on) and k(off) result in linear Arrhenius plots, consistent with the prediction of two-state association and dissociation (ii) k(on) is independent of the nature of the base N located in the center of the third strand. (iii) k(on) strongly decreases when the NaCl concentration is decreased. (iv) The activation energy, E(on), is always negative and becomes more negative when the NaCl concentration is decreased. (v) k(off) is independent of NaCl concentration but depends on the base N, with its magnitude following the order C greater than G greater than A much greater than T. (vi) The activation energy, E(off), is independent of the base N. All these results are discussed in the light of a nucleation-zipping model similar to that developed for the duplex-coil transitions [Craig, M. E., Crothers, D. M., & Doty, P. (1971) J. Mol. Biol. 62, 383-401; Pörschke, D., Eigen, M. (1971) J. Mol. Biol. 62, 361-381].

In the preceding paper (Norris & Kolodner, 1990), we described the purification of a Mr 33,000 polypeptide which dramatically stimulated the activity of SEP1, the yeast mitotic strand exchange protein. In this paper, we characterized this new protein, which was designated SF1, in the absence of SEP1. SF1 had a sedimentation coefficient of 1.7 S and a Stokes radius of 30 A, which was consistent with a calculated native molecular weight of 31,000, indicating that SF1 existed in solution as a monomer. Filter binding assays showed that SF1 bound preferentially to single-stranded rather than double-stranded DNA. Fluorescence spectroscopy analysis indicated that SF1 occluded approximately eight nucleotides when bound to single-stranded DNA and exhibited a dissociation constant, KD, of 2.83 x 10(-6) M. The binding of SF1 to single-stranded DNA was noncooperative and appeared to involve at least one tyrosine residue. SF1, in the absence of SEP1, stimulated the renaturation of homologous single-stranded DNA, suggesting that it might act directly in some phase of the strand exchange reaction.

Kinetoplast DNA (kDNA) of the culture form of Trypanosoma cruzi is cleaved by restriction endonucleases (HpaII, HindII, EcoRI, and HaeIII). The analysis of the cleavage patterns proves that the minicircles (free circulargenome units) are heterogeneous in base sequences. The same results are obtained with the complex kDNA network which is composed of the association of minicircles and linear molecules. Kinetic studies of the renaturation of kDNA previously cleaved by HpaII into fragments of the genome unit size show at least two populations of molecules. About 75% of these molecules correspond to the fast renaturing population having the molecular complexity of the minicircles. The molecules of the slow renaturing population have a much higher molecular complexity than the minicircles and do not seem to be related to the majority of the long linear molecules.

Evidence from absorbance, fluorescence, and circular dichroism (CD) measurements strongly suggests that adduct conformations at the binding sites are grossly different before and after thermal denaturation of (+)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]py ren e [(+)-anti-BPDE] modified DNAs. This conclusion is reached through the following observations: (1) upon melting and cooling, the (+)-anti-BPDE-modified DNA exhibits pronounced hypochromic effects with concomitant spectral red shifts for the pyrenyl absorbance; (2) the pyrenyl CD spectrum reverses sign upon thermal denaturation-renaturation; (3) the fluorescence emission spectra resulting from excitations at 353 nm (10 nm to the red of hydrolyzed and unbound anti-BPDE) exhibit enhanced intensities and spectral red shifts for the thermally denatured and cooled adducts; and (4) in contrast to the absence of a shoulder prior to melting, the postmelt adducts exhibit a prominent 355-nm maximum (evidence of stacking interactions) in the excitation spectrum when 384-387-nm emission is monitored. Studies with synthetic polynucleotides further reveal that (+)-anti-BPDE-modified poly(dG).poly(dC) exhibits the greatest nonreversible renaturation at the binding sites, possibly as a consequence of pyrenyl self-stacking. This, coupled with the previous findings that this polymer suffers the most extensive (+)-anti-BPDE modification, appears to suggest that (dG)n . (dC)n regions may be responsible for such observed effects in native DNA.

The 5S RNA complexed in the 7S particle of immature Xenopus laevis oocytes was 32P labeled at its 3' end and then subjected in situ to partial digestion using ribonucleases T1, T2, A, and V1 in order to study the conformation of the complexed RNA and its interaction with the transcription factor A (TFIIIA). Digested samples were gel purified to retrieve 5S RNA that was still complexed with the transcription factor protein, and the cleavages in these RNAs were analyzed on sequencing gels. The RNA associated with the 7S particle is very susceptible to ribonuclease activity despite the presence of the protein. Also, the 5S RNA in the 7S particle is in a different conformation from renatured Xenopus laevis (Xlo) 5S RNA and appears to have less secondary structure predominantly in the stem that includes helices IV and V. A species of native Xlo 5S RNA which was isolated from 7S particle preparations under nondenaturing conditions revealed a conformation that was more similar to the 5S RNA in the 7S particle than to renatured 5S RNA. Comparison of data from partial ribonuclease digestions performed on renatured 5S RNA, on the native 5S RNA, and on the complexed 5S RNA allowed us to approximate sites of protein-induced structural change in the complexed 5S RNA, which may signal protein interaction domains. These sites include the 5' side of helices III and V. In another approach to the study of 5S RNA-TFIIIA interactions, we have observed that incubation of 32P-labeled Xlo 5S RNA with 7S particles results in the incorporation of labeled RNA into 7S particles.(ABSTRACT TRUNCATED AT 250 WORDS)

The fraction of chloroplast DNA transcribed at different stages of chloroplast development in Euglena gracilis was measured by RNA-DNA hybridization. Euglena cells were grown in the dark in a heterotrophic medium to stationary phase and then transferred to the light. Chloroplast development was monitored by the increase in the cellular chlorophyll content in the absence of cell division. Total cell RNA was isolated at various stages of chloroplast development, and hybridized in a vast excess to [125I]chloroplast DNA. The fraction of [125I]chloroplast DNA in the form of a duplex was monitored by chromatography on hydroxylapatite columns. The amount of RNA-DNA hybrid in the duplex mixture was determined by correcting for the contribution of DNA-DNA renaturation under the same conditions. The fraction of chloroplast DNA transcribed was calculated by multiplying by two the amount of single-stranded DNA in the form of an RNA-DNA hybrid. Prior to the initiation of chloroplast development (i.e., in dark grown cells) the fraction of chloroplast DNA represented as RNA transcripts in the cell is 0.53. As chloroplast development proceeds, the fraction of the chloroplast DNA transcribed decreases to 0.47. Experiments in which mixtures of various RNA samples were hybridized to the chloroplast DNA indicate that there is a small portion of chloroplast DNA transcribed at later stages of chloroplast development which is not represented as transcripts at the onset of chloroplast development. Melting properties of the RNA-DNA hybrids show that the RNA-DNA duplexes are slightly less stable than renatured [125I]chloroplast DNA.

Nuclear DNA of the marine chromophytic alga Olisthodiscus luteus was analyzed in this study. Reassociation kinetics analysis has shown that 440-nucleotide DNA fragments from the genome of this alga contain 4% foldback, 58% repetitive, and 34% single-copy sequences. Precise analysis using isolated single-copy DNA revealed that Olisthodiscus has a large haploid DNA content of 1.66 x 10(-12) g/cell. For determination of the organization of single-copy and repetitive sequences within this genome, DNA fragments 3000 nucleotides in length were reassociated to C0t= 100 M . s. At this low C0t value 89% of the DNA bound to hydroxylapatite, suggesting that single-copy and repetitive elements are interspersed. The lengths of the duplexed repetitive DNA on these 3000-nucleotide fragments were measured by electron microscopy after digestion with S1 nuclease which removed the unreassociated single-copy DNA regions. Of these repetitive sequences, 68% were shorter than 1200 nucleotide pairs in length and had a modal length of 350 nucleotide pairs. A minor class of longer (to 4000 nucleotide pairs) repetitive sequences was also observed.

We have investigated the renaturation kinetics of a single family of cloned interspersed repeated sequences isolated from human DNA. Cross-renaturation studies of individual cloned sequences reveal heterogeneity in both the renaturation rate and the thermal stability of heteroduplexes formed from members of this family of sequences. However, cloned members of this family all renature with approximately the same number of copies in the human genome, demonstrating that they are a single family of sequences by the criterion of DNA renaturation kinetics. When a single cloned member of the family is renatured with total human DNA as a function of temperature, the thermal stability of the renatured heteroduplexes is found to be independent of the renaturation temperature over the range 25 degrees C below to 4 degrees C above the melting temperature. Further, the number of genomic copies with which this cloned family member reacts is also independent of the renaturation temperature from 25 degrees C below up to the melting temperatures. These observations demonstrate a remarkable degree of homogeneity in the evolutionary sequence divergence of members of this family. These results also demonstrate that renaturation kinetics can accurately measure the number of genomic copies of interspersed repeated DNA sequences.

The recA enzyme of Escherichia coli catalyzes renaturation of DNA coupled to hydrolysis of ATP. The rate of enzymatic renaturation is linearly dependent on recA protein concentration and shows saturation kinetics with respect to DNA concentration. The kinetic analysis of the reaction indicates that the Km for DNA is 65 microM while the kcat is approximately 48 pmol of duplex formed (pmol of recA)-1 (20 min)-1. RecA protein catalyzed renaturation has been characterized with respect to salt sensitivity, Mg2+ ion and pH optima, requirements for nucleoside triphosphates, and inhibition by nonhydrolyzable nucleoside triphosphates and analogues. These results are consistent with a Michaelis-Menten mechanism for DNA renaturation catalyzed by recA protein. A model is described in which oligomers of recA protein bind rapidly to single-stranded DNA, and in the presence of ATP, these nucleoprotein intermediates aggregate to bring complementary sequences into close proximity for homologous pairing. As with other DNA pairing reactions catalyzed by recA protein, ongoing DNA hydrolysis is required for renaturation. However, unlike the strand assimilation or transfer reaction, renaturation is inhibited by E. coli helix-destabilizing protein.

A DNA fraction which is highly enriched in active gene sequences and tightly associated with a subset of nonhistone chromosomal proteins has been isolated from human placenta. After extraction with 2 M NaCl, placental chromatin was separated into two distinct components by centrifugation. Of the total DNA, approximately 96% (DNA-S) is protein free, while the remaining 4% (DNA-P) is tightly complexed with nonhistone chromosomal proteins. Reassociation studies revealed that the DNA-P fraction was enriched 22-fold in actively transcribed human placental lactogen gene sequences, while the DNA-S fraction was correspondingly depleted 22-fold in these sequences. Approximately 45% of the sequences present in DNA-P (equivalent to 1.8% of the genome) were not present in the DNA-S fraction. Reassociation of nick-translated DNA-P to DNA from a partial digest of DNase I treated nuclei indicated that 27% of the DNA-P sequences were DNAase I sensitive, suggesting they may represent actively transcribed gene sequences. Analysis of the overall sequence organization of DNA-P showed that relative to unfractionated DNA and DNA-S, DNA-P was enriched in single-copy sequences, slightly enriched in the class of middle repetitive sequences from C0t 0.01 to 100 M.s, devoid of the more highly repetitive sequences (C0t less than or equal to 0.01). The distribution of total active placental genes between DNA-P and DNA-S was measured by hybridization with a complementary DNA probe transcribed from total polysomal poly(A+) messenger RNA. We found that 57% of this cDNA probe reassociated to DNA-P and 58% to DNA-S, while 95% reassociated to DNA-P mixed with DNA-S at the observed ratio of 4 to 96, suggesting that the DNA-P fraction contained a different population of active gene sequences than DNA-S. From these results we estimate that approximately 85% of the transcribed sequences appear to be distinctly distributed and equally proportioned between DNA-P and DNA-S, while approximately 15% of the transcribed sequences are common to both fractions. We suggest that the strong affinity of the tightly bound nonhistone chromosomal proteins for the DNA-P fraction indicates a likely role for these proteins in the regulation of gene expression.

Details of the general DNA sequence organization in the dinoflagellate Crypthecodinium cohnii have been obtained by using hydroxylapatite binding experiments, S1 nuclease digestion .and electron microscopy of reassociated DNA. It has been found that roughly half of the genome is made up of unique sequences interspersed with repeated sequence elements with a period of approximately 600 nucleotides. This class represents roughly 95% of the total number of interspersed unique elements in the genome. The remaining 5% are uninterrupted by repeated sequences for at least 4000 nucleotide pairs. The interspersed repeated elements are narrowly distributed in length with 80% under 300 nucleotide pairs in length. About half of the repeated DNA (20-30% of the genome) is not interspersed among unique sequences. The close spacing of the short repeats interspersed throughout much of the genome is consistent with the occurrence of the huge network structures observed in the electron microscope for low Cot reassociation of moderately long fragments. An unusual class of heteroduplexes was detected in the electron microscope which is believed to derive from the reassociation of repeated sequences from different families which are frequently found adjacent to one another in different locations in the genome. The occurrence of this novel arrangement of repeated sequences may reflect the unusual organization of the dinoflagellate nucleus. However, in most respects the sequence arrangement in this unicellular alga is very typical of higher plants and animals.

We have analyzed the effect of estrogen on the kinds of unique DNA sequences which are transcriptionally expressed in chick oviduct with an "expressed" DNA probe. Steady-state nRNA in estrogen-stimulated chick oviduct represents about 25% of the complexity of total chick unique DNA. To purify this expressed DNA fraction, chick unique DNA was isolated, nick-translated, and hybridized to chemically mercurated oviduct nRNA (Hg-nRNA); the resulting hybrids were bound to sulfhydryl-Sepharose, and DNA was selectively recovered by thermal elution in formamide buffer. To compare the sequence homology between nRNAs isolated from oviduct before or up to 6 days after estrogen withdrawal, trace amounts of expressed DNA derived from estrogen-stimulated oviduct were hybridized in RNA-excess reactions. All nRNAs hybridized with equal efficiency. Furthermore, hybridization of expressed DNA to nRNA mixtures showed that nRNA from nonwithdrawn and withdrawn oviduct contained a similar set of unique sequences. The data indicate that, at most, only a small percentage (0--5%) of transcriptionally active unique DNA sequences are shut down when estrogen is removed from the circulation.

The electron microscope was used to count the number of double-stranded linear and circular renaturation products of a 2:1 by weight mixture of restricted double-stranded linear phi X174 RFI and single-stranded circular phi X174 viral deoxyribonucleic acid (DNA). More linear molecules than circular molecules are observed. However, when a 1:1 mixture of aliquots of either phi X174 RFI or SV40 DNA, each previously cleaved with a different single-site restriction enzyme, is denatured and renatured under conditions which assure circularization with out of phase molecules, an equal number of linear and circular molecules is observed. These experiments indicate that the nucleation rate is not affected by circular permutation of linear strands but is decreased approximately 3-fold when one of the reacting strands is circular. An excluded volume theory is developed which is consistent with these as well as previous results concerning the effects of DNA strand lengths on renaturation rates.

Parameters of repetitive sequence organization have been measured in the rat genome. Experiments using melting, hydroxylapatite binding, and single strand specific nuclease digestion have been used to measure the number, length, and arrangement of repeated DNA sequences. Renaturation and melting or S1 nuclease digestion of 1.0 kbp DNA fragment show about 20% of rat DNA sequences are 3000-fold repeated. Renatured duplexes from 4.0 kbp DNA fragments display two repetitive size fractions after nuclease digestion. About 60% of the repeated sequences are 0.2-0.4 kbp long while the remainder are longer than 1.5 kbp. The arrangement of the repeated sequences has been measured by hydroxylapatite fractionation of DNA fragments of varying lengths bearing a repeated sequence. Repeated DNA sequences are interspersed among 2.5 kbp long nonrepeated sequences throughout more than 70% of the rat genome. There are approximately 350 different 3000-fold short repeated sequences in the rat interspersed among 600,000 nonrepeated DNA sequences.

The structure of Xenopus laevis oocyte (Xlo) 5S ribosomal RNA has been probed with single-strand-specific ribonucleases T1, T2, and A with double-strand-specific ribonuclease V1 from cobra venom. The digestion of 5'- or 3'-labeled renatured 5S RNA samples followed by gel purification of the digested samples allowed the determination of primary cleavage sites. Results of these ribonuclease digestions provide support for the generalized 5S RNA secondary structural model derived from comparative sequence analysis. However, three putative single-stranded regions of the molecule exhibited unexpected V1 cuts, found at C36, U73, U76, and U102. These V1 cuts reflect additional secondary structural features of the RNA including A.G base pairs and support the extended base pairing in the stem containing helices IV and V which was proposed by Stahl et al. [Stahl, D. A., Luehrsen, K. R., Woese, C. R., & Pace, N. R. (1981) Nucleic Acids Res. 9, 6129-6137]. A conserved structure for helix V having a common unpaired uracil residue at Xlo position 84 is proposed for all eukaryotic 5S RNAs. Our results are compared with nuclease probes of other 5S RNAs.

The heptaethyl ester of heptadeoxythymidylyl-(3'-5')-deoxythymidine (d-[Tp(Et)]7T or d-T8-Et) has been prepared by chemical methods. The material, consisting of a mixture of diastereoisomers, forms a 1:1 complex with (dA)n in neutral aqueous buffer; this interaction is virtually independent of ionic strength. The octamer triester does not bind to (dA)n-(dT)n, and it interacts with (rA)n only at low temperatures. By cochromatography with (dA)n on Sephadex G-50, d-T8-Et fractions with different binding affinities for the polyadenylates were obtained. This heterogeneity in binding affinity is ascribed to the diastereoisomerism of d-T8-Et. Enthalpies of dupoex formation were determined by the concentration variation method. At 0.1 M sodium ion concentration, the enthalpy of binding of the various d-T8-Et fractions to (dA)n is essentially invariant (-8.1 kcal/mol of base pairs at 0 degrees C to -8.6 kcal at 25 degrees C) and 1.6 kcal/mol of base pairs more negative than the enthalpy of binding of the phosphodiester analogue, d-(Tp)7T, to (dA)n (-6.8 kcal/mol of base pairs at 11 degrees C). This difference is the electrostatic contribution to the enthalpy of duplex formation, arising from the interstrand electrostatic repulsion and the intrastrand repulsion in d-(Tp)7T. The entropy of binding to (dA)n is more negative for the octamer triesters than for the diester analogue, and is different for the various d-T8-Et fractions. This is interpreted in terms of varying degrees of restriction of rotational freedom for the ethyl substituents upon double helix formation.

The complexity of the DNA of the free-living nematode Panagrellus silusiae has been examined. Reassociation kinetics of pressure-sheared fragments (approximately 290 nucleotides) in 0.18 M Na+ at 60 degrees C showed the presence of foldback, repetitive, and unique DNA sequence elements. The three classes comprise 9.3%, 26.1%, and 61.3% of the total DNA, respectively. The mean length of the foldback duplex DNA after digestion with S1 nuclease is about 185 nucleotides. There are about 1.8 x10(4) inverted repeats per genome. Sequence arrangement was deduced from (1) renaturation kinetic profiles of long and short fragments on hydroxylapatite; (2) the pattern of renaturation of tracer DNA, labeled in vitro with 125I, of various sizes after incubation with excess short fragments; and (3) thermal denaturation behavior of DNA that had been reassociated to various C0t values. It was found that DNA fragments of the repetitive fraction that are, at least, 2000 nucleotides in length are virtually free of unique sequences. Moreover, it is estimated that the repeated segments in this species could extend for 10,000 nucleotide pairs. Thus, Panagrellus DNA lacks the pattern of extensive short period interspersion that is typified by the DNA of Xenopus.

Labeled unique sequence human X chromosomal DNA has been isolated by hybridization of labeled human DNA with DNA from a human-mouse hybrid cell line, A9/HRBC2-A, which contains a single human chromosome, the X. Homopolymer tails of poly(dA) were added to nick-translated unique sequence human DNA to permit separation of the labeled DNA from unlabeled driver DNA by binding to oligo(dT)-cellulose. Human DNA sequences homologous with mouse DNA were removed from this labeled poly-(dA)-tailed human probe by hybridization with excess unlabeled mouse DNA. The labeled human DNA which did not hybridize with mouse DNA was then hybridized with excess unlabeled A9/HRBC2-A DNA, with which only X chromosomal human DNA will hybridize. After hybridization, the labeled human X chromosomal DNA was separated from the unlabeled A9/HRBC2-A DNA by binding to oligo(dT)-cellulose. The purity of the final X chromosomal DNA preparation was greater than 90%, and the hybridization with mouse DNA was less than 2%. When carried out under standard conditions for DNA reassociation, this procedure is complicated by the formation of hybrids between the poly(dA) tails of the probe and T-rich sequences in mouse and human DNA. However, these imperfectly paired hybrids are less stable than those of unique sequence DNA and can be eliminated by carrying out the hydroxylapatite chromatography at an elevated temperature of 71 degrees C.

Quantitative agarose gel assays reveal that the recA protein promoted renaturation of complementary DNA strands (phi X DNA) proceeds in two stages. The first stage results in the formation of unit-length duplex DNA as well as a distribution of other products ("initial products"). In the second stage, the initial products are converted to complex multipaired DNA structures ("network DNA"). In the presence of ATP, the initial products are formed within 2 min and are then rapidly converted to network DNA. In the absence of ATP, the initial products are formed nearly as fast as with ATP present, but they are converted to network DNA at a much lower rate. The time-dependent formation of initial products and network DNA from complementary single strands for both the ATP-stimulated and ATP-independent reactions can be modeled by using a simple two-step sequential kinetic scheme. This model indicates that the primary effect of ATP in the recA protein promoted renaturation reaction is not on the initial pairing step (which leads to the formation of initial products) but rather is to increase the rate at which subsequent pairing events can occur.

The base composition dependence of double-stranded ribonucleic acid (RNA) melting was studied by observing the structure and widths of melting transitions for Penicillium chrysogenum mycophage RNA as well as differences in melting temperatures of two RNAs of different base composition. Double-stranded RNA melting is independent of base compositions in 3.5 M Et4NCl and 4.6 M Me4NCl, where the melting temperatures are 25 and 92 degrees C, respectively. Double-stranded RNA renaturation rate constants are reported in Et4NCl solutions. The nucleation rate constant is about 10 times lower than that for double-stranded deoxyribonucleic acid. Analyses of renaturation kinetics results lead to the conclusion that each of the three similar but separable RNA segments of Penicillium chrysogenum mycophage is unique.

The diversity, abundance, and DNA sequence representation of poly(adenylic acid) containing RNA derived from cells of Achlya ambisexualis cultured in defined and undefined media have been determined. The kinetics of hybridization of polyadenylated RNA with complementary DNA were the same for both culture conditions and revealed the presence of three frequency classes containing 29, 220, and 3000 different sequences of an average length of 1150 nucleotides. Complexity estimates derived from experiments in which polyadenylated RNA was hybridized to unique sequence DNA were in good agreement with these results. The kinetics of hybridization of complementary DNA with an excess of nuclear DNA indicate that approximately 10% of the RNA is transcribed from reiterated DNA sequences while the remainder is transcribed from single copy sequences.

A method of enriching, from the total DNA of an organism, for long DNA strands carrying a particular gene is described. The purified RNA corresponding to the gene is covalently attached to biotin via a cytochrome c bridge. This modified RNA is hybridized to the total DNA. Those DNA strands which hybridize are separated from all the other DNA, using the avidin-biotin interaction, by one of two methods. Avidin is covalently attached to submicroscopic polymer spheres; the complexes of avidin spheres with the DNA: RNA-biotin hybrids band in CsCl at a much lower buoyant density than does free DNA. Alternatively, the DNA:RNA-biotin hybrids are isolated by affinity chromatography on an avidin-solid support column. These methods have been used to prepare long single strands of Drosophila ribosomal DNA (rDNA) in high yield and 42 to 80% pure.

Fragments of 3.4 kilobases (kb) are released from DNA of human males, but not DNA of human females, by cleavage with restriction endonucleases HaeIII, EcoRI, or EcoRII. Most, if not all, reiterated DNA which is specific for the Y chromosome (it-Y DNA) is present within these male-specific 3.4-kb molecules. Although such 3.4-kb molecules are themselves localized to the Y chromosome, this is not true for all sequences within them. At least two distinguishable types of reiterated sequences are found within each 3.4-kb molecule. One type consists of at least two families which are highly reiterated and are not confined to the Y chromosome. The other type is composed of an estimated minimum of 39 families, each moderately reiterated and localized to the Y chromosome. Y-specific and non-Y-specific sequences are interspersed with one another in the same 3.4-kb molecule. In the average 3.4-kb molecule, three 800 nucleotide lengths of Y-specific sequences alternate with four 250 nucleotide lengths of non-Y-specific sequences. Since the total number of families of Y-specific sequences, calculated on the basis of reiteration frequency and total abundance in a male genome, greatly exceeds the number of Y -specific sequences present in a single 3.4-kb molecule, it necessarily follows that the population of these 3.4-kb molecules is heterogeneous.

Transcription by purified mammalian RNA polymerase II in vitro leads to extensive formation of DNA-RNA hybrids between nascent RNA and the template DNA strand. This is especially clear during transcription of 3'-extended (dC-tailed) DNA templates where the nontranscribed DNA strand is progressively displaced as transcription proceeds [Kadesch, T. R., & Chamberlin, M. J. (1982) J. Biol. Chem. 257, 5286-5295]. Addition of small amounts of a HeLa cell extract to such a transcription system enhances renaturation of the template DNA and displacement of the nascent RNA, as measured by the sensitivity of the RNA to pancreatic ribonuclease. Using this latter assay, we have purified a protein factor (renaturase) 250-fold from HeLa cell extracts using chromatography on DEAE-cellulose, DNA-cellulose, and hydroxylapatite. Renaturase preparations facilitate complete renaturation of the template DNA duplex during transcription by RNA polymerase II and lead to concurrent displacement of the nascent RNA. Current preparations are free from all but traces of deoxyribonuclease or ribonuclease. The active component has a molecular weight of about 30000 as estimated by preparative density gradient sedimentation. We have examined the structure of transcribing RNA polymerase II complexes in the presence and absence of renaturase, using the electron microscope and the Williams polylysine technique [Williams, R. C. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2311-2315]. In the presence of renaturase, the DNA template is fully renatured, and a ternary complex in which the nascent RNA is displaced during transcription is seen.(ABSTRACT TRUNCATED AT 250 WORDS)

The size and arrangement of repetitive and inverted repeat (foldback) sequences in rat DNA were studied by visualization of hybrid and heteroduplex structures in the electron microscope. The self-reassociation of repetitive sequence-bearing DNA strands often results in the formation of four-ended "H" structures, whose duplex regions equal the repetitive sequence length and can be measured in the electron microscope. In this way, it was determined that the average size of the class of numerous short repetitive sequences is 0.40 +/- 0.15 kbp. Heteroduplex structures were prepared between long whole DNA single strands and short repeat-sequence-bearing strands. The analysis of these structures confirms that the size of the repetitive sequences in 0.4 kbp on average. Length measurements between adjacent duplexes show that the average spacing between two interspersed repeats is at least 1.5-1.8 kbp. By examining 29.4-kbp single strands after brief renaturation, the size and distribution of foldback sequences were determined. There are 1.9 X 10(5) foldback apirs per rat genome, spaced an average of 9.7 kbp apart according to our measurement. Repetitive, inverted repeat and unique sequences are interspersed with each other in at least half the genome.

The reassociation kinetics of pea (Pisum sativum L.) DNA fragments (300 nucleotides) were measured with hydroxylapatite. The most slowly reassociating fragments do so with a rate constant of 2 X 10(-4) L mol-1s-1, as determined from experiments with total DNA as well as with a tracer enriched for slowly renaturing sequences. This rate is about 1000 times slower than that observed for Escherichia coli DNA included as an internal kinetic standard, indicating a kinetic complexity of 4.5 X 10(9) nucleotide pairs or 4.6 pg of DNA per haploid nucleus. This estimate is in good agreement with previous chemical and cytophotometric measurements. The majority (85%) of the 300 nucleotide fragments contain repetitive sequences. While the reassociation of repetitive DNA could be modeled with two theoretical second-order components, the data did not specify a unique solution. The reassociation kinetics of isolated high- and low-frequency fractions indicate that repetitive sequence families in pea DNA probably cover a broad range of frequencies ranging from 100 to 10 000 or more copies per haploid genome. Single-copy sequences account for about 30% of the DNA, but because of extensive interpersion of repetitive sequences only about 15% of 300 nucleotide fragments reassociate with single-copy kinetics. From studies of hydroxylapatite binding as a function of fragment length, we conclude that the major class of single-copy sequences has a modal length of about 300 nucleotides. Long tracer reassociation kinetics indicate that sequences with an apparent repetition frequency of about 10 000 copies are interspersed at intervals of less than 1300 nucleotides throughout 75% of the genome. At a detection limit of about 3%, we find no single-copy sequences longer than 1000 nucleotides.

Complementary DNA, synthesized with L-cell polyadenylated RNA as template, renatured with total L-cell DNA to about 70%. About 30% complementary to unique sequence DNA and another 10 and 30% corresponded to sequences about 20- and 500-fold repetitive. Complementary DNA was fractionated after partial hybridization with total polyadenylated RNA to obtain preparations enriched or impoverished in complements of the most frequent polyadenylated RNA. Renaturation of these complementary DNA fractions with L-cell DNA revealed that most frequent RNAs are transcribed from repetitive DNA sequences, Complementary DNA, density labeled with bromodeoxyuridine, was fractionated by renaturation with L-cell DNA to yield fractions enriched in repetitive and unique sequence DNA. The denisty labeled complementary DNA was purified by equilibrium centrifiguation in an alkaline Cs2SO4 gradient. The complementary DNA representing mainly repetitive DNA sequences hybridized preferentially to frequent polyadenylated RNA.

A simple method for the isolation and characterization of DNA-DNA and DNA-RNA hybrid molecules formed in solution was developed. It was based on the fact that, in appropriate salt concentration, such as 5% Na2HPO4, DNA in either double-stranded (DNA-DNA or DNA-RNA) or single-stranded forms, but not free nucleotides, can bind to diethylaminoethylcellulose disc filters (DE81). Thus tested samples were treated with the single-strand-specific nuclease S1 and then applied to DE81 filters. The free nucleotides, resulting from degrading the single-stranded molecules, were removed by intensive washing with 5% Na2HPO4, leaving only the hybrid molecules on the filters. The usefulness of this method was illustrated in dissociation and reassociation studies of viral (SV40) or cellular (NIH/3T3) DNAs and DNA-RNA hybrid molecules. Using this technique the reassociation of denatured SV40 DNA was found to be a very rapid process. Dissociation studies revealed that the melting curves of tested DNAs were dependent on salt concentration. Thus the melting temperatures (tm) obtained for SV40 DNA were 76 degrees C at 1 X SSC (0.15 M NaCl-0.015 M sodium citrate) and 65 degrees C at 0.1 X SSC, and for NIH/3T3 DNA 82 degrees C at 1 X SSC and 68 degrees C at 0.1 X SSC. MuLV DNA-RNA hybrid molecules were formed by annealing in vitro synthesized MuLV DNA with 70S MuLV RNA at 68 degrees C. The melting temperature of this hybrid in the annealing solution was 87 degrees C. Another important feature of this procedure was that, after being selectively bound to the filters, the hybrid molecules could efficiently be recovered by heating the filters for 5 min at 60 degrees C in 1.5-1.7 M KCl. The recovered molecules were intact hybrids as they were found to be completely resistant to S1 nuclease.

The base-sequence complexities and relative abundance of polysomal and nuclear polyadenylated [poly(A+)] RNA sequences have been analyzed in a pluripotent embryonal carcinoma cell line. Polysomal RNA and nuclear poly(A+) RNA have a complexity representing respectively 0.5% and 2.5% of the single copy component of haploid mouse DNA (1.8 X 10(6) K base pairs). By hybridization with specific cDNAs, three abundance classes were found in polysomal poly(A+) RNA, representing respectively 31%, 33%, and 36% of the RNA, with base sequence complexities of 0.1 X 10(3), 0.9 X 10(3), and 14.5 X 10(3) kilobases. This corresponds to 7000-8000 different mRNA species of an average length of 2000 nucleotides, present on an average of 5 to 600 copies per cell. In nuclear RNA, a major class of abundance was found with a complexity of 100 X 10(3) kilobases, each sequence being present in 1 copy per nucleus. The majority of the polysomal poly(A+) RNA sequences are represented in the nuclear poly(A+) RNA but are present in a more restricted range of relative abundance implying posttranscriptional mechanisms of quantitative modulation: polysomal RNA sequences appear to be preferentially transcribed into nuclear cDNA suggesting a preferential location of these sequences close to poly(A) sequences. The presence of a specialized gene product, globin specific RNA, could not be detected either in the nuclear or polysomal compartments of embryonal carcinoma cells, even at levels that would have detected one sequence per 50 cells.

Repeatable fractionation of sheared chromatin from purified populations of chicken erythroid cells has been achieved, based on the Bio-Gel procedure of Janowski et al. ((1972) Karolinska Symp. 5, 112). For reticulocytes, 3-5% of chromatin DNA is excluded from Bio-Gel A-50 m (peak I) and over 90% elutes in the included volume of the column(peak II). Peak I material has a higher protein/DNA ratio than peak II chromatin and the two fractions have melting profiles characteristic of "active" and "inactive" chromatin, respectively. In cells prelabeled with [3H]uridine or [3H]leucine there was very pronounced preferential association of radioactivity with the "active" peak I chromatin. The distribution of "active" (globin) and "inactive" (keratin) gene sequences in the DNA of fractions from peak I and peak II chromatin was determined with complimentary DNA (cDNA) probes to chicken globin mRNA and chicken feather keratin mRNA. While slight enrichment for globin gene sequences was found in peak I (relative to DNA in these fractions), some 80% of the total globin hybrid formed was found in peak II fractions. Experiments with the keratin cDNA probe showed that these genes were equally distributed in both chromatin fractions rather than being confined to the "inactive" peak II material. The hybridization data in particular question the validity of claims for fractionation of chromatin into "active" and "inactive" material.