Protonated polypurine/polypyrimidine DNA tracts that appear to lack the single-stranded pyrimidine loop predicted by the "H" model.

Three synthetic oligomers: 5'd(AG)8.dA.d(CT)(8)3'(A), 5'd(TC)7.d(TTA).d(GA)(8)3'(B) and d(GA)17(C) were cloned into the plasmid vector p915 in order to study the effects of sequence symmetry on pH-dependent structural transitions in polypurine/polypyrimidine DNA. When present in linear molecules all three sequences undergo transitions to protonated states. These are kinked to different degrees as determined by a non-denaturing gel mobility shift assay. Chemical probe analysis shows that the protonated states adopted by the linear forms of A and C exhibit certain features which have been regarded as indicating partially triple stranded "H" transition structures. The chemical reactivities of the transition structure adopted by linear molecule B and certain features of those exhibited by the transition structures of linear molecules A and C do not conform to the predictions of the "H" model.

Triplex DNA in plasmids and chromosomes.

Circular plasmids containing pyrimidine purine tracts can form both inter-and intramolecular triplexes. Addition of poly(dTC) to plasmid pTC45, which contains a (TC)45.(GA)45 insert, results in intermolecular triplex formation. Agarose-gel electrophoresis gives rise to many well-resolved bands, which correspond to 1, 2, 3, 4... plasmid molecules attached to the added pyrimidine strand. In the electron microscope these complexes appear as a rosette of petals. The mobility of these triplex-containing complexes can be retarded by the addition of a triplex-specific monoclonal antibody, Jel318. Intramolecular triplex formation can be demonstrated at pH 5 in pTC45 and also in pT463-I, a plasmid containing a segment of a crab satellite DNA with both (G)n.(C)n and (TCC)n.(GGA)n inserts. However, although the intermolecular triplex remains stable for some time at pH 8, intramolecular triplex formation only occurs at low pH. Triplexes can also be detected by an immunoblotting procedure with Jel318. This unfamiliar structure is readily demonstrated in eukaryotic extracts, but not in cell extracts from Escherichia coli. Triplexes may thus be an inherent feature of eukaryotic chromosome structure.

The in-vivo occurrence of Z DNA.

The energetics of the B-Z transition of two different types of cloned alternating purine/pyrimidine DNA sequences have been analysed by a two dimensional electrophoretic technique. Since the transition between right handed and left handed forms of these polymers is detected by alterations of electrophoretic mobilities of topoisomers of the plasmid DNA molecules, the method is not dependent on Z-DNA binding ligands. The measurements reflect intrinsic properties of the DNA unperturbed by the free energy of binding such a ligand. Direct evidence from the analysis of topoisomer distributions is presented which shows that d(GC)n.d(GC)n sequence elements within an E. coli plasmid will adopt a Z conformation in-vivo under conditions of blocked protein synthesis. Evidence for the in-vivo occurrence of Z-DNA was not detected in plasmid DNA isolated from bacterial cells growing in the absence of protein synthesis inhibitors. A model is proposed for a function for the B-Z transition in ensuring the correct pairing of homologous chromosomes during meiosis.

Bovine elastin cDNA clones: evidence for the occurrence of a new elastin-related protein in fetal calf ligamentum nuchae.

Poly (A+) mRNA was isolated from fetal calf ligamentum nuchae and used for the construction of cDNA libraries. A fraction highly enriched in elastin mRNA was used to prepare the cDNA probes for screening the libraries. A 2 kb clone, pRE1, gave the most positive signal in colony hybridization. It hybridized to a mRNA of the same size as reported for elastin mRNAs from chick and sheep. Hybrid-arrested translation showed that translation of mRNAs for proteins other than elastin doublet was not inhibited by pRE1. Southern blot analysis showed that pRE1 has sequence homology with pVE6 and pVE10, which were tentatively identified as elastin-related cDNA clones representing two distinct mRNAs. DNA sequence data from the 5' end of pRE1 show that the translated amino acid sequence is not typical of known elastin sequences but contains some elastin-like sequences. All of this evidence strongly suggests the occurrence in fetal calf nuchal ligament of a mRNA which codes for a previously unknown elastin-related protein.

Improved methods for the fluorographic detection of weak beta-emitting radioisotopes in Agarose and acrylamide gel electrophoresis media.

The use of acetic acid as a solvent for diphenyloxazole (PPO) in fluorographic procedures has been investigated. It is demonstrated to be superior to both dimethyl sulfoxide and methanol with respect to its suitability in both agarose and acrylamide gel electrophoresis systems. In addition, a method has been developed for impregnating fragile gels such as those used for immunodiffusion with PPO in preparation for fluorography.

Yeast plasmids have fewer superhelical turns than predicted by the nucleosome repeat of yeast DNA.

The superhelical density of three Saccharomyces cerevisiae plasmids was determined with respect to a defined reference state during vegetative growth and stationary phase. The levels of supercoiling determined were approximately 20% lower than predicted by comparisons with SV40 DNA and reconstituted minichromosomes using histones from higher eukaryotes. In two different plasmids with the ARS1 origin of replication, the level of supercoiling changed substantially as the host cells entered stationary phase. Supercoiling of the endogenous 2-microns plasmid during vegetative growth was lower than in the ARS1-containing plasmids but did not change significantly upon entry of the cells into stationary phase.

Partial purification of "omega" protein from calf thymus.

Proteins which relax supercoiled DNA, called "omega" proteins, are thought to be involved in DNA replication. Calf thymus is a plentiful source of "omega" protein activity. It has been extensively purified and has been characterized as behaving similarly to other eukaryotic "omega" proteins in completely relaxing either positively or negatively supercoiled DNA, requiring a salt concentration of about 0.2 M NaCl or KCl, and not requiring Mg2+. A transient nick must occur but could not be detected. A new assay for "omega" activity is described which is rapid and sensitive, and depends on the fluorescence enhancement of ethidium intercalating duplex DNA.

Facile transition of poly[d(TG) x d(CA)] into a left-handed helix in physiological conditions.

The DNA polymer d(GC)n . d(GC)n can undergo a transition from the usual right-handed 10.4 base pairs (bp) per turn B form to a novel left-handed 12 bp per turn Z form in response to altered environmental conditions. Several other alternating purine-pyrimidine DNA polymers with modified bases have been shown to undergo transitions from B to Z conformations, with varying degrees of difficulty. We report here that the unmodified DNA polymer d(TG)n . d(CA)n readily undergoes a transition to a Z conformation when subjected to unwinding torsional stress in ionic conditions that are close to physiological. By using a two-dimensional gel electrophoresis system, we have determined both the critical free energy of supercoiling that is required to initiate the transition and the free energy of supercoiling that is required to maintain this polymer in the Z form.

Transition of a cloned d(AT)n-d(AT)n tract to a cruciform in vivo.

A 34 base pair tract of the simple repeating dinucleotide d(AT)n-d(AT)n cloned into a 2.4 kb polylinker plasmid vector undergoes a structural transition in response to negative superhelical coiling. The transition has been characterized by 2 dimensional gel electrophoresis, mapping of S1, P1 and T7 endonuclease 1 sensitive sites, and mapping of sites that are sensitive to modification by bromoacetaldehyde. After S1 nuclease treatment it is possible to trap supercoiled species that are nicked on one or both strands near the center of the palindrome. These data show that the alternate state adopted by the d(AT)n-dAT)n insert is a cruciform rather than a Z conformation. Unlike other B-cruciform transitions the transition in d(AT)n-d(AT)n has a low activation energy and the transition is facilitated by the presence of magnesium ions. Evidence from in-vivo topoisomer distributions is presented which shows that under conditions of blocked protein synthesis the d(AT)n-d(AT)n insert will spontaneously adopt the cruciform state in-vivo in E. coli.

Purification and properties of type 1 topoisomerase from chicken erythrocytes: mechanism of eukaryotic topoisomerase action.

A simple method for the purification of the major topoisomerase (topoisomerase 1) from chicken erythrocytes is described. Because of the generally repressed state of the chromatin from these nuclei, the heterogeneity of the non-histone proteins is reduced, and it is possible to purify this enzyme from a nuclear extract by a single chromatographic step. The chicken erythrocyte topoisomerase appears to be similar to previously described eukaryotic type I topoisomerases with respect to its physical and enzymological properties. The pattern of intermediate products generated during the action of chicken erythrocyte topoisomerase on a supercoiled closed circular DNA substrate has been examined quantitatively and has been shown to be consistent with a mechanism in which the enzyme closes its substrate DNA molecular after the removal of each superhelical turn and in which dissociation of the enzyme substrate complex may, but does not necessarily, occur after each cycle of the reaction.

A topoisomerase from chicken erythrocyte nuclei which does not assemble nucleosome core particles in vitro.

We have examined the ability of a topoisomerase purified from chicken erythrocyte nuclei to mediate nucleosome core assembly in vitro at physiological ionic strength (0.15 M NaCl). Although we have detected limited amounts of spontaneously assembled nucleosome cores at this salt concentration, the addition of this topoisomerase does not increase the amount of assembly observed. Nucleosome assembly was assayed by quantitating the amount of core particle length DNA accumulated with time upon the nuclease digestion of histone-DNA complexes. In addition, the amount of negative supercoils introduced into relaxed closed circular DNA upon nucleosome core particle assembly was determined. Correctly assembled complexes do not protect more DNA from nuclease digestion than random histone-DNA complexes but shift the heterogeneous size distribution of protected fragments to a more homogeneous distribution centred around 145 base pairs. Under our conditions of nucleosome assembly, a second histone-DNA complex which is distinct from the nucleosome core can be detected under physiological ionic strength conditions. This particle does not form in high salt assembly experiments. Similarly, the assembly of this particle is unaffected by the presence or absence of topoisomerase.

Internal structure of discrete nucleohistone complexes which form in vitro under conditions of physiological ionic strength.

Three discrete histone-DNA complexes assemble spontaneously when the four core histones are mixed with DNA under conditions which are close to physiological (0.15 M NaCl, pH 8). These species include the (H2A,H2B) dimeric complex (P1), the (H2A2,H2B2,H3,H4) hexameric complex (P2) and the nucleosome core complex (P3). This report compares several properties of these complexes with the properties of nucleosome cores assembled at high ionic strength (0.6 M NaCl). Based on histone-histone cross-linking studies, CD spectra, and thermal denaturation experiments, P1 is structurally similar to the subnucleosomal (H2A,H2B) fragment isolated from nuclease-digested chromatin. P1, P2, P3, and high salt-assembled nucleosome cores can all incorporate (H2A,H2B) pairs which have been previously cross-linked. Although the CD spectra and thermal denaturation profiles of P2 and P3 are closely related to those of nucleosome cores assembled in 0.6 M NaCl, cross-linking studies indicate that the arrangement of the histones in P2, and in a proportion of the P3 particles assembled in 0.15 M NaCl, are significantly different from their arrangement in nucleosome cores assembled in 0.6 M NaCl. The single cysteine residue on the H3 of P2 is accessible to the solvent. The two fluorescently labeled cysteine residues in a large proportion of the P3 particles assembled in 0.15 M NaCl are in a different orientation with respect to each other than the same residues in nucleosome cores assembled at high ionic strength.

Pathways of assembly of nucleohistone complexes formed in vitro under physiological conditions. Implications for the structure of the nucleosome.

The composition and structure of three discrete nucleohistone particles produced by micrococcal nuclease digestion of the complex formed when the four core histones are mixed directly with DNA under conditions that are close to physiological have been reported in accompanying articles (Ellison, M. J., and Pulleyblank, D. E. (1983) J. Biol. Chem. 258, 13307-13313; 13314-13320). These include a peak containing a compact hexameric complex composed of one pair of (H3,H4) and two pairs of (H2A,H2B) (P2) and a peak containing complexes with the composition and sedimentation properties of nucleosome cores (P3). We have examined the pathways of assembly of P2 and P3 by determining the effect of order of histone addition on the yields of these species. The assembly of P2 is initiated by the binding of an (H2A,H2B) pair to the DNA which then directs the placement of an (H3,H4) pair. The incorporation of the final (H2A,H2B) pair to complete the particle is probably cooperative. The assembly of P3 is less dependent than the assembly of P2 upon the order in which the histone pairs are added to the DNA. More than one pathway has been implicated in the formation of these particles. P3 contains a component which has many but not all of the properties of nucleosome cores assembled at elevated ionic strength. Alternative pathways of assembly of histone pairs onto DNA are discussed which could lead to the formation of the discrete histone DNA structures that have been isolated here. A model is proposed in which the nucleosome unfolds and refolds into an alternative configuration.

The assembly of an H2A2,H2B2,H3,H4 hexamer onto DNA under conditions of physiological ionic strength.

A novel nucleohistone particle is generated in high yield when a complex of DNA with the four core histones formed under conditions that are close to physiological (0.15 M NaCl, pH 8) is treated with micrococcal nuclease. The particle was found to contain 102 base pairs of DNA in association with six molecules of histones in the ratio 2H2A:2H2B:1H3:1H4 after relatively brief nuclease treatment. Prolonged nuclease digestion resulted in a reduction in the DNA length to a sharply defined 92-base pair fragment that was resistant to further degradation. Apparently normal nucleosome core particles containing two molecules each of the four core histones in association with 145 base pairs of DNA and a particle containing one molecule each of histones H2A and H2B in association with approximately 40 base pairs of DNA were also generated during nuclease treatment of the histone-DNA complexes formed under physiological ionic strength conditions. Kinetic studies have shown that the hexamer particle is not a subnucleosomal fragment produced by the degradation of nucleosome core particles. Furthermore, the hexamer particle was not found among the products of nuclease digestion when histones and DNA were previously assembled in 0.6 M NaCl. The high sedimentation coefficient of the hexameric complex (8 S) suggests that the DNA component of the particle has a folded conformation.

H3 Cys-110 is in close proximity to the C-terminal regions of H2B and H4 in a nucleosome core with an altered internal arrangement of histones.

A particle obtained by nuclease digestion of nucleohistone complexes prepared by direct mixing of histones with DNA in 0.15 M NaCl was indistinguishable by composition and physical properties from nucleosome cores prepared under the same conditions from nucleohistone preannealed in 0.6 M NaCl. We show here that different photo-cross-links form when these particles are prepared from H3 labeled with photoaffinity reagents on the unique histone H3 cysteine. H3-H3 histone dimers were dominant when the particles were prepared by dilution of the nucleohistone from 0.6 M NaCl while H3-H2B and H3-H4 histone dimers were prominent if the nucleohistone complex was prepared directly in 0.15 M NaCl. Peptide mapping of the novel H3-H4 and H3-H2B dimers showed that Cys-110 of histone H3 is cross-linked to the 18 amino acid C-terminal end of H4 or to the 66 amino acid C-terminal half of H2B.

A structural basis for S1 nuclease sensitivity of double-stranded DNA.

A protonated form of a cloned simple repeating DNA sequence d(TC)n X d(GA) is detectable in equilibrium with the usual Watson-Crick base-paired form at pHs up to 7. This form is anomalously sensitive to a variety of single-strand-specific endonucleases. The observed pH dependent protection of N-7 of dG residues within the insert suggests that these residues are either Hoogsteen or reverse Hoogsteen base-paired to protonated dC residues of the polypyrimidine strand. A structure in which dA:dT Watson-Crick base pairs alternate with Hoogsteen syndG:dCH+ pairs appears to be the most stereochemically acceptable structure consistent with the chemical properties of this protonated DNA. Protonated d(TC)n X d(GA)n interacts with an anti-Z DNA antibody raised against brominated d(GC)n X d(GC)n.