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The effect of histones on the reaction of reassociation of the two complementary strands of DNA from different sources has been investigated. The reassociation rate of denatured linear DNA from bacteriophage M13 monitored spectrophotometrically and using nuclease S1 is roughly the same in the presence and absence of core histones at physiological ionic strength. Electron microscopy reveals that in the samples containing histones a large network of duplex DNA is produced. Nevertheless, closed circular M13 DNA and a cloned DNA fragment (158 bp) from nucleosomal origin are entirely renatured in the presence of histones as demonstrated by the well-defined double-stranded DNA bands seen in electrophoretic gels. Various experiments performed using the purified (+) and (-) strands of the cloned nucleosome DNA fragment at low ionic strength indicate that core histones initially bound to one or even to the two strands allow the formation of duplex DNA. These findings and the results obtained with partially denatured closed circular M13 DNA allow us to conclude that core histones neither prevent the nucleation nor inhibit the rapid zippering reactions leading to the formation of double-stranded DNA. The mechanism that allows the renaturation of DNA in the presence of histones may also participate in biological processes involving the pairing of complementary nucleotides.

During the initial stages of human immunodeficiency virus (HIV) replication, 5'-terminally redundant (R') DNA, the minus strand synthesized as the complement of the 5'-long terminal repeat (LTR) terminal redundancy, must anneal to the 3'-LTR RNA to enable template transfer. The (R')DNA sequences contain the site involved in the tat-TAR interaction and extensive secondary structures that strongly interfere with annealing. The novel annealing reaction between (R')DNA and 3'-LTR RNA follows first-order kinetics, consistent with an unusually slow unfolding of the secondary structure as the rate-limiting step followed by a more rapid nucleation step. The HIV nucleocapsid protein accelerates the annealing reaction 3000-fold under optimal conditions. This acceleration may be necessary for strand transfer to efficiently occur in vivo and may provide a target for anti-HIV chemotherapeutic agents.

In contrast to the horse heart apocytochrome c, the chicken heart apocytochrome c underwent a conformational change from random coil to partial folding during a renaturation process. When the apocytochrome horse heart and that of chicken heart c were subjected to a translocation assay in vitro using large trypsin-enclosed unilamellar vesicles from soybean phospholipids, the ability of the chicken heart apocytochrome c to penetrate into the liposomes was found to decrease markedly with the renaturation procedure, while that of horse heart apocytochrome c remained relatively constant. Observations from circular dichroism measurement on the induction of secondary folding of these two species of apocytochrome c upon interaction with soybean phospholipid vesicles suggested that a more flexible structure of apocytochrome c embedded in the lipid matrix be required for its efficient translocation across the bilayer.

To obtain mechanistic insights about RecA-promoted base pairing between complementary polynucleotides, the complex formation of RecA with poly(dA) and poly(dT) in the presence of ATP (and ATP-regenerating system) has been studied. The reaction was followed using a fluorescent probe, benzopyrenediolepoxide (BPDE), covalently attached to less than 1% of the adenine bases of poly(dA). BPDE is sensitive to its environment and has been found useful for detection of interactions between DNA strands, in the three binding positions of the RecA filament, in the presence of adenosine 5'-O-3-thiotriphosphate (ATP[S]) [Wittung, P., Norden, B. and Takahashi, M. (1994) J. Biol. Chern. 269, 5799-5803]. The emission spectrum of RecA:BPDE-poly(dA) formed in the presence of ATP is similar to that observed with ATP[S] supporting similar structures of the complexes. However, the fluorescence anisotropy is considerably reduced, suggesting a higher degree of freedom of DNA in the presence of ATP hydrolysis. Upon addition of a complementary strand, poly(dT), to a preformed filament of RecA:BPDE-poly(dA) in the presence of ATP, the fluorescence intensity slowly decreases and a change of emission profile consistent with Watson-Crick base pairing is observed. This contrasts with the case of ATP[S] in which normal base pairing is never observed. Hence, ATP hydrolysis appears necessary for the RecA filament to be able to promote true renaturation. The renaturation reaction is found more effective when one of the complementary DNA strands is bound in the primary RecA DNA-binding position and the other is added as the third strand, but the reaction can also occur between DNA strands in any combination of binding positions in the RecA filament. This observation suggests the importance of the third DNA-binding position of the RecA filament. Renaturation between DNA strands in the other two combinations of binding positions is speculated to have a role in aborting the strand-exchange reaction when the strands are insufficiently complementary.

The Polymerase Chain Reaction is a technique in molecular biology, that allows million-fold amplification of DNA-fragments. It is based on specific oligonucleotides used as starting segments (primers) of both sides of the genomic region to be studied. This chain reaction is characterized by three steps: denaturation of DNA by increasing the temperature; renaturation of the DNA allowing for competition between the two original DNA segments and the numerous primers, and finally synthesis of DNA by the polymerase. The cycle is repeated from 25 to 35 times in order to exponentially duplicate the DNA-fragments between the two primers. Usage of a thermoresistant polymerase permitted automatization of the procedure. This technique represents a revolution for practical molecular biology and medicine. Its very high sensitivity, however, may cause errors that have to be recognized. Several practical examples are described and analyzed in order to illustrate the PCR-concept to physicians in practise.

A protein that catalyzes the renaturation of complementary strands of DNA has been purified from mitotic cells of the lower eukaryote Ustilago maydis. The most highly purified fraction contains a polypeptide with a molecular mass of 20 kDa as determined by SDS/PAGE and glycerol gradient sedimentation. DNA reannealing is enhanced by the presence of a divalent cation but does not require ATP nor any other nucleotide triphosphate. Reassociation proceeds with fast kinetics as more than 60% of the DNA is reannealed within 4 min at a 30:1 nucleotide/protein monomer ratio, results which suggest that the protein acts in a stoichiometric fashion. Amino acid analysis revealed that the protein contained an elevated level of basic residues and low levels of tryptophan and tyrosine. The protein binds to an oligonucleotide of ten residues but not to one having only five. As judged by agarose gel assays, the protein does not catalyze strand-transfer reactions but does promote the annealing of a 58-residue polynucleotide onto single-stranded circles and gapped linear duplexes. These latter reactions are dependent on the presence of DNA sequence similarity between the pairing partners.

The recO gene product is required for RecF pathway-mediated recombination and the repair of DNA damage after UV irradiation or mitomycin C exposure in Escherichia coli. In this study, the E. coli recO gene product was overexpressed and purified to at least 99% homogeneity. N-Terminal protein sequence analysis of the overexpressed 31 kDa polypeptide confirmed that this polypeptide was encoded by the recO gene. The N-terminal protein sequence of RecO also confirmed that the first 12 amino acids of functional RecO protein are encoded within the upstream era gene. The purified protein chromatographs with the same Stokes radius (25 A) as a globular protein having a molecular mass of 28 kDa, indicating that RecO is a monomer in solution. The purified RecO protein binds to both single-stranded and double-stranded DNA, and promotes renaturation of complementary single-stranded DNA molecules in the absence of any high energy cofactor. The rate constant for this reaction is independent of the concentration of DNA, suggesting that the reaction follows first-order reaction kinetics. In addition, this reaction is inhibited by 160 mM NaCl, requires Mg2+, and is not stimulated by ATP. These biochemical characteristics support a role for RecO protein in an early phase of homologous recombination.

Purification of the ATP-dependent homologous pairing activity from Ustilago maydis yields a protein preparation that is enriched for a 70-kDa polypeptide as determined by SDS-gel electrophoresis. The protein responsible for the ATP-dependent pairing activity, using renaturation of complementary single strands of DNA as an assay, has a Stokes radius of 3.6 nm and a sedimentation coefficient of 4.3 S consistent with the interpretation that the activity arises from a monomeric globular protein of 70 kDa. Including heparin-agarose and FPLC gel filtration chromatography steps in the previously published protocol improves the purification of the protein. ATP and Mg2+ are necessary cofactors for optimal DNA renaturation activity. ADP inhibits the reaction. Analysis of the ATP-dependent renaturation kinetics indicates the reaction proceeds through a first-order mechanism. The protein has an associated DNA-dependent ATPase as indicated by co-chromatography with the purified ATP-dependent renaturation activity through an FPLC gel-filtration column. Single-stranded DNA and Mg2+ are required for optimal ATP hydrolytic activity, although a number of other polynucleotides and divalent cations can substitute to varying degrees. Hydrolysis of ATP is activated in a sigmoidal manner with increasing amounts of the protein. At ATP concentrations below 0.1 mM the ATPase activity exhibits positive cooperativity as indicated from the Hill coefficient of 1.8 determined by steady-state kinetic analysis of the reaction. ADP and adenosine 5'-[beta,gamma-imido]triphosphate are inhibitors of the ATPase activity although they appear to exert their inhibitory effects through different modes. These results are interpreted as evidence for protein-protein interactions.

The p53 tumor-suppressor protein has previously been shown to bind double-stranded and single-stranded DNA. We report that the p53 protein can bind single-stranded DNA ends and catalyze DNA renaturation and DNA strand transfer. Both a bacterially expressed wild-type p53 protein and a glutathione S-transferase-wild-type p53 fusion protein catalyzed renaturation of different short (25- to 76-nt) complementary single-stranded DNA fragments and promoted strand transfer between short (36-bp) duplex DNA and complementary single-stranded DNA. Mutant p53 fusion proteins carrying amino acid substitutions Glu-213, Ile-237, or Tyr-238, derived from mutant p53 genes of Burkitt lymphomas, failed to catalyze these reactions. Wild-type p53 had significantly higher binding affinity for short (36- to 76-nt) than for longer (> or = 462-nt) single-stranded DNA fragments in an electrophoretic mobility-shift assay. Moreover, electron microscopy showed that p53 preferentially binds single-stranded DNA ends. Binding of DNA ends to p53 oligomers may allow alignment of complementary strands. These findings suggest that p53 may play a direct role in the repair of DNA breaks, including the joining of complementary single-stranded DNA ends.

The human genome contains about 3 x 10(9) base pairs which, based on average estimates for the size of coding regions, might suggest an upper limit of about 3 million genes. Evidence from a variety of approaches indicates a much lower figure--in the range 40,000-100,000 genes. Renaturation kinetic analysis reveals the presence of many repetitive elements in the human genome. Some of these are quite simple and highly reiterated, others are more complex and exist in fewer copies. The distribution and function of some of these families and the evolution of complex genomes from simpler ones are discussed.

ICP8, the major single-stranded DNA-binding protein of herpes simplex virus type 1, promotes renaturation of complementary single strands of DNA. This reaction is ATP independent but requires Mg2+. The activity is maximal at pH 7.6 and 80 mM NaCl. The major product of the reaction is double-stranded DNA, and no evidence of large DNA networks is seen. The reaction occurs at subsaturating concentrations of ICP8 but reaches maximal levels with saturating concentrations of ICP8. Finally, the renaturation reaction is second order with respect to DNA concentration. The ability of ICP8 to promote the renaturation of complementary single strands suggests a role for ICP8 in the high level of recombination seen in cells infected with herpes simplex virus type 1.

An extensive compilation of renaturation rate data for human DNA is presented as an empirical guide for genomic hybridization studies and to document as accurately as possible the repetitive sequence content of the human genome. Approximately 15% of the genome renatures with heterogeneous rates characteristic of moderately repetitive sequences. Cross hybridization studies of this fraction with known repetitive sequences show that it is largely composed of known moderately repetitive sequences and contaminating highly repetitive sequences. These findings limit the maximum possible abundance of unidentified families of short interspersed elements (SINEs) in human DNA. Exhaustive analysis by subtractive hybridization of a small library constructed from short fragments of repetitive sequences yields few additional new SINEs, again suggesting the limited abundance of such sequences.

We have purified phage lambda beta protein produced by a recombinant plasmid carrying bet gene and confirm that it forms a complex with a protein of relative molecular mass 70 kDa. Therefore, beta protein, a component of general genetic recombination, is associated with two functionally diverse complexes; one containing exonuclease and the other 70 kDa protein. Using a number of independent methods, we show that 70 kDa protein is the ribosomal S1 protein of E. coli. Further, the association of 70 kDa protein with beta protein is biologically significant, as the former inhibits joining of the terminal ends of lambda chromosome and renaturation of complementary single stranded DNA promoted by the latter. More importantly, these findings initiate an understanding of an important mode of host- virus interaction in general with specific implication(s) in homologous genetic recombination.

The Adenovirus DNA binding protein (DBP) imposes a regular, rigid and extended conformation on single stranded DNA (ssDNA) and removes secondary structure. Here we show that DBP promotes renaturation of complementary single DNA strands. Enhancement of intermolecular renaturation is sequence independent, can be observed over a broad range of ionic conditions and occurs only when the DNA strands are completely covered with DBP. When one strand of DNA is covered with DBP and its complementary strand with T4 gene 32 protein, renaturation is still enhanced compared to protein-free DNA, indicating that the structures of both protein-DNA complexes are compatible for renaturation. In contrast to promoting intermolecular renaturation, DBP strongly inhibits intramolecular renaturation required for the formation of a panhandle from an ssDNA molecule with an inverted terminal repeat. We explain this by the rigidity of an ssDNA-DBP complex. These results will be discussed in view of the crystal structure of DBP that has recently been determined.

We have used site-directed mutagenesis to prepare two new mutant recA proteins, one in which histidine 97 has been replaced by alanine, and another in which lysine 248 has been replaced by alanine. Although these mutant proteins were originally designed from different considerations, they turned out to have remarkably similar properties. Both the [H97A]recA protein and the [K248A]recA protein bind poorly to single-stranded DNA, have no single-stranded DNA-dependent ATP hydrolysis activity, and do not promote renaturation of complementary single-stranded DNA molecules or the ATP-dependent three-strand exchange reaction. Furthermore, both mutant proteins are defective in Mg(2+)-induced helical filament formation. To account for these results, we propose that the mutation of either histidine 97 or lysine 248 alters subunit interactions between recA monomers and that this leads to the loss of cooperative single-stranded DNA binding and DNA pairing activities. This proposal is consistent with the recently determined x-ray structure of the recA protein, which shows that although histidine 97 and lysine 248 are distant from one another in the monomer structure, these two residues are on the opposing complementary faces of the recA subunit and pack against each other at the interface between adjacent recA monomers in the helical filament (Story, R. M., Weber, I. T., and Steitz, T. A. (1992) Nature 355, 318-325).

Drosophila Rrp1 protein purified from embryos has four tightly associated enzymatic activities: DNA strand transfer, single-strand DNA renaturation, 3'-exonuclease, and apurinic endonuclease. Copurifying with these activities is a single polypeptide that has an apparent M(r) of 105,000 when estimated by SDS-polyacrylamide gel electrophoresis. To determine if this polypeptide is sufficient for these activities, it has been overexpressed in Escherichia coli. In crude extracts of E. coli cells, an ATP-independent Mg(2+)-dependent strand transfer activity is observed upon activation of the promoter that drives expression of Rrp1. Rrp1 protein purified from induced E. coli cells has electrophoretic, chromatographic, and enzymatic properties similar to those of Drosophila Rrp1 protein. The carboxyl-terminal region of Rrp1 (amino acids 428-679) is homologous to E. coli exonuclease III. Rrp1 deleted for this region cannot carry out DNA strand transfer, but can renature complementary single-strand DNA. The strand transfer activity of this truncated protein can be restored if DNA 3'-exonuclease is provided in trans by pretreating the double-strand DNA substrate with E. coli exonuclease III. This demonstrates a likely role of the exonuclease in the in vitro DNA strand transfer reaction carried out by Rrp1 protein. Such a role is also suggested by an analysis of the polarity of the strand transfer reaction.

DNA strandness changes occurring during in situ hybridization were monitored using monoclonal antibodies. Our results show that: 1) RNase induces formation of single stranded DNA, 2) after denaturation-renaturation, single stranded DNA is found principally in centromeric areas, and 3) double stranded DNA is still observed after each step.

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; Porschke, D., Eigen, M. (1971) J. Mol. Biol. 62, 361-381].

Single-stranded DNA binding proteins (SSBs) have been isolated from many organisms, including Escherichia coli, Saccharomyces cerevisiae and humans. Characterization of these proteins suggests they are required for DNA replication and are active in homologous recombination. As an initial step towards understanding the role of the eukaryotic SSBs in DNA replication and recombination, we examined the DNA binding and strand exchange stimulation properties of the S. cerevisiae single-strand binding protein y-RPA (yeast replication protein A). y-RPA was found to bind to single-stranded DNA (ssDNA) as a 115,000 M(r) heterotrimer containing 70,000, 36,000 and 14,000 M(r) subunits. It saturated ssDNA at a stoichiometry of one heterotrimer per 90 to 100 nucleotides and binding occurred with high affinity (K omega greater than 10(9) M-1) and co-operativity (omega = 10,000 to 100,000). Electron microscopic analysis revealed that y-RPA binding was highly co-operative and that the ssDNA present in y-RPA-ssDNA complexes was compacted fourfold, arranged into nucleosome-like structures, and was free of secondary structure. y-RPA was also tested for its ability to stimulate the yeast Sepl and E. coli RecA strand-exchange proteins. In an assay that measures the pairing of circular ssDNA with homologous linear duplex DNA, y-RPA stimulated the strand-exchange activity of Sepl approximately threefold and the activity of RecA protein to the same extent as did E. coli SSB. Maximal stimulation of Sepl occurred at a stoichiometry of one y-RPA heterotrimer per 95 nucleotides of ssDNA. y-RPA stimulated RecA and Sepl mediated strand exchange reactions in a manner similar to that observed for the stimulation of RecA by E. coli SSB; in both of these reactions, y-RPA inhibited the aggregation of ssDNA and promoted the co-aggregation of single-stranded and double-stranded linear DNA. These results demonstrate that the E. coli and yeast SSBs display similar DNA-binding properties and support a model in which y-RPA functions as an E. coli SSB-like protein in yeast.

A1 hnRNP protein, which rapidly renatures complementary strands of nucleic acids in vitro, affects both the equilibrium and kinetic properties of the reaction (single-stranded DNA in equilibrium with double-stranded DNA). A1 lowers the melting transition of duplex DNA. However, at temperatures above this new Tm, both single- and double-stranded DNAs are present at equilibrium and are rapidly interconverting. Although the ratio of single and double strands under these conditions is a function of both the A1 protein and complementary DNA strand concentrations, it is not strongly affected by further increases in temperature. These surprising results demonstrate that A1 does not act as a simple catalyst in promoting renaturation and indicate how A1 and other proteins could act to speed the turnover of intermediate complexes in important biological processes.

The effects of serine phosphorylation on the DNA cleavage/religation equilibrium of topoisomerase II and the sensitivity of the enzyme to antineoplastic drugs were characterized. Both casein kinase II and protein kinase C were used for these studies. Each kinase incorporated a maximum of approximately 1.4 phosphate molecules per homodimer of topoisomerase II. When the enzyme was incubated with both kinases simultaneously, phosphate incorporation increased to approximately 2.6 molecules/homodimer. In the absence of antineoplastic drugs, phosphorylation had only a slight effect on the DNA cleavage/religation equilibrium of topoisomerase II. However, in the presence of etoposide or 4'-(9-acridinylamino)methane-sulfon-m-anisidide, phosphorylation attenuated the ability of drugs to stabilize enzyme-DNA cleavage complexes. Levels of drug-induced DNA cleavage products decreased approximately 33% following phosphorylation of topoisomerase II by casein kinase II, approximately 17% following modification by protein kinase C, and approximately 50% following simultaneous phosphorylation of the enzyme by both kinases. This latter 50% reduction in DNA cleavage products correlated with an approximately 2-fold increase in the apparent first order rate constant for DNA religation mediated by simultaneously modified topoisomerase II. These results strongly suggest that the sensitivity of topoisomerase II toward antineoplastic drugs can be modulated by altering the phosphorylation state of the enzyme.

The RAD10 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of ultraviolet-damaged DNA, and it functions in mitotic recombination. RAD10 has homology to the human excision repair gene ERCC-1. Here we describe the purification of the protein encoded by RAD10 and show that it is a DNA-binding protein with a strong preference for single-stranded DNA. We also show that RAD10 promotes the renaturation of complementary DNA strands.

The rate of renaturation for complementary DNA strands can be enhanced greater than 10(4)-fold by the addition of simple cationic detergents, and the reaction is qualitatively and quantitatively very similar to that found with purified heterogeneous nuclear ribonucleoprotein A1 protein. Under optimal conditions, renaturation rates are greater than 2000-fold faster than reactions run in 1 M NaCl at 68 degrees C. The reaction is second-order with respect to DNA concentration, and reaction rates approach or equal the rate with which complementary strands are expected to encounter each other in solution. Renaturation can even be observed well above the expected melting temperature of the duplex DNA, demonstrating that some cationic detergents have DNA double-helix-stabilizing properties. The reaction is also extremely rapid in the presence of up to a 10(6)-fold excess of noncomplementary sequences, establishing that renaturation is specific and relatively independent of heterologous DNA. This finding also implies that up to several thousand potential target sequences can be sampled per strand per second. Such reagents may be useful for procedures that require rapid nucleic acid renaturation, and these results suggest ways to identify and design other compounds that increase the kinetics of association reactions. Moreover, this work provides further support for a model relating the existence of flexible, weakly interacting, repeating domains to their function in rapid molecular assembly processes in vitro and in vivo.

The uvsX protein of bacteriophage T4 is a recA-type recombinase. This protein has previously been shown to help initiate DNA replication on a double-stranded DNA template by catalyzing synapsis between the template and a homologous DNA single strand that serves as primer. Here, we demonstrate that this replication-initiating activity of the uvsX protein greatly amplifies the snap-back (hairpin-primed) DNA synthesis that is catalyzed by the T4 DNA polymerase holoenzyme on linear, single-stranded DNA templates. Amplification requires the presence of uvsX protein, the DNA polymerase holoenzyme, T4 gene 32 protein, and a T4 DNA helicase, in a reaction that is modulated by the T4 uvsY protein (an accessory protein to the uvsX recombinase). The reaction products consist primarily of large networks of double-stranded and single-stranded DNA. With alkali or heat treatment, these networks resolve into dimer-length single-stranded DNA chains that renature instantaneously to reform a monomer-length double helix. A simple model can explain this uvsX protein-dependent amplification of snap-back DNA synthesis; the mechanism proposed makes several predictions that are confirmed by our experiments.

Summary Twelve human isolates exhibiting the properties of the family Pasteurellaceae but phenotypically deviating from established species, or resembling species of animal origin that are only rarely reported to occur in human materials, were checked for their identities by DNA-DNA hybridization. The collection consisted of one strain of Actinobacillus lignieresii , two strains of Actinobacillus hominis (mannose-positive), two hitherto undescribed Actinobacillus or Actinobacillus -like species, [ Pasteurella ] haemolytica biovar T, CDC group HB-5 (initially oxidase and indole-negative), a new species in the [ Haemophilus ] aphrophilus /[ Haemophilus ] segnis group, a new sucrose-negative and gas-producing Pasteurella -like species, and three strains of Bisgaard's Pasteurella -like taxon 16. Some diagnostically useful features of these unusual human Pasteurellaceae are described.

Hydroxyapatite column chromatography has been used to study some properties of the extensively sheared DNA of the Rainbow lizard, Agama agama agama. Reassociation studies show that the genome has a Cot1/2 of 370. Approximately 15% of the genome is highly repetitive in nature. This repetitive fraction is resolved into thermally stable and less stable fractions. The stable fraction has a base composition of 47% GC, higher than the 40.2% GC for the native DNA. This stable fraction is believed to be of recent origin. Chromatography of the total DNA of the lizard with linear gradients of phosphate buffer containing 1 M urea resolves it into two components which were shown by thermal fractionation, also in the presence of 1 M urea, to vary in base composition. This behaviour may be characteristic of reptilian genomes and may be used as a basis for studying the structural organisation of the reptilian genome.

Hepatitis delta virus (HDV) RNA subfragments undergo self-cleavage at varying efficiencies. We have developed a procedure of using repeated cycles of heat denaturation and renaturation of RNA to achieve a high efficiency of cleavage. This effect can also be achieved by gradual denaturation of RNA with heat or formamide. These results suggest that only a subpopulation of the catalytic RNA molecules assumes the active conformation required for self-cleavage. This procedure could be of general use for detecting catalytic RNA activities.

Simian virus 40 large tumour antigen (T antigen) is shown to catalyse the formation of duplex DNA from complementary strands in specific conditions. The activity is dependent on an excess of unspecific double-stranded DNA and seems not to function by T antigen mediated destabilization of secondary structure. Rather, protein-protein contacts between T antigen molecules appear to be involved. Protein-protein interactions between T antigen molecules bound to physically separated DNA sites are also demonstrated by the formation of specific DNA loops and by cyclization of DNA molecules with 3'-extended single-stranded ends where T antigen specifically binds to the single-stranded/double-stranded junctions. The relevance of these properties for T antigen functions in DNA replication, transcription and(or) recombination is discussed.

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.

The technique of forming interspecific DNA heteroduplexes and estimating phylogenetic distances from the depression in their duplex melting temperature has several physical and chemical constraints. These constraints determine the maximum phylogenetic distance that may be estimated by this technique and the most appropriate method of analyzing that distance. Melting curves of self-renatured single copy primate DNAs reveal the presence of components absent from the renaturation products of exactly paired sequences. This observation, which confirms existing literature, challenges a fundamental assumption: that orthologous (i.e., corresponding) DNA sequences in the divergent species are being compared in DNA heteroduplex melting experiments. As a model system, the thermal stabilities of heteroduplexes formed between a human alpha-globin cDNA and four alpha-like globin genes isolated from chimpanzee are qualitatively compared. The results of this comparison show that the cross-hybrids of imperfectly matched gene duplicates from divergent species can contribute to the additional components that are present in renatured single copy DNAs. Single copy DNA, as usually defined, includes sequence duplicates that will obscure phylogenetic comparisons in a mass hybridization of genomes.

AIM: To express chimeric Fd (cFd) and chimeric light chain (cL) in E. coli respectively and refold them into chimeric Fab (cFab) antibody. METHODS: cFd and cL genes were respectively inserted into the prokaryotic expression vector pET32a to construct recombinant vectors pET32a/cFd and pET32a/cL. Then, the competent E. coli cells were transformed by the recombinant vectors and induced by IPTG. Moreover, a large quantity of cFd and cL expression products were prepared and mixed with equal molar to refold into cFab by gradient dialysis. The refolded products were identified and analyzed by sodium SDS-PAGE, Western blotting, ELISA and HPLC. RESULTS: High efficient prokaryotic expressions of both cFd and cL in the form of non-fusion protein were obtained with the expression levels of 28.3% and 32.3% of total bacteria proteins, respectively. Their relative molecular masses were all 24 ku or so, and both of them mainly existed in the form of inclusion bodies. In addition, cFd and cL were successfully refolded into cFab by gradient dialysis, with about 59.45% of recovery when the starting total protein concentration was 100 μg/mL. The renatured cFab could specifically bind to related antigen with high affinity. CONCLUSION: The cFab antibody against human hepatoma was highly and efficiently expressed and refolded, which laid a solid foundation for studying its application in the treatment of hepatoma.

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

Ciliated protozoa are characterized by generative micronuclei and vegetative polyploid macronuclei. Micronuclei of Stylonychia mytilus contain 1 600 times as much DNA per haploid genome as E. coli. Most of this DNA is shown to be repetitive. The development of the macronucleus involves, as demonstrated by cytology, only 1/3 of the chromosomes which in a first replication phase are polytenized in probably 5 replication steps and appear as giant chromosomes. At this developmental stage considerable amounts of repetitive DNA are still present in the chromosomes. During the subsequent disintegration phase more than 90% of the DNA are eliminated from the macronucleus anlage. The remainder is further replicated five times and composes the final macronucleus. Since this DNA reassociates with a reaction rate almost identical to an ideal second order reaction its kinetic complexity can be determined by comparison with the kinetic complexity of E. coli DNA. Macronuclear DNA reassociates with a kinetic complexity of 26 times the kinetic complexity of E. coli DNA (corrected for GC content) which indicates that macronuclear DNA sequences exist at a ploidy level of 4 096 C. We assume that macronuclear DNA may be present only once per haploid genome. In this case it represents only 1.6% of the DNA in micronuclei or 10% of the DNA in the giant chromosome stage.

Differences in the RNA-driven hybridization kinetics of genomic DNA and cDNA probes led us to examine physical parameters affecting these reactions. Cloned cDNA complementary to serum albumin (SA) mRNA hybridized in accordance with single component kinetics, whereas cloned SA genomic DNA hybridized more slowly and with multiple component kinetics. This difference is largely attributable to the relatively short and variable lengths of the mRNA complementary regions in the cloned genomic DNA. The rate of mRNA driven hybridization is affected to about half the extent observed for DNA renaturation as Na+ is increased or decreased from 0.18M. In the annealing of nucleic acids of high sequence complexity, after approximately 70% of reaction has been reached, the rate of the reaction is slowed and completion is not reached under "static" conditions. In practical terms, this is not the case for systems of low sequence complexity. This problem can be largely overcome by continuous or frequent mixing of the reactants, so that complex cDNA probes are hybridized essentially to completion, and kinetics can therefore be more readily compared to simple complexity standards.

Approximately 75% of the wheat and rye genomes consist of repeated sequence DNA. Three-quarters of the non-repeated or few copy sequences in wheat are less than 1000 base-pairs long, whilst in rye approximately half of the non-repeated or few copy sequences are in this size class. Most of the remaining non-repeated or few copy sequences appear to be a few thousand base-pairs long. In this paper a somewhat novel approach has been used to quantitatively analyse the linear organisation of the large proportion of repeated sequence DNA as well as the non-repeated DNA in the wheat and rye genomes. Repeated sequences in the genomes of oats, barley, wheat and rye have been used as probes to distinguish and isolate four different groups of repeated sequences and their neighbouring sequences from the wheat and rye genomes. Radioactively labelled wheat or rye DNA fragments ranging from 200 to over 9000 nucleotides long were incubated separately with large excesses of denatured unlabelled oats, barley, wheat and rye DNAs to Cot values which enable all the repeated sequences of the unlabelled DNA to renature. The following parameters were then determined from the proportions of total labelled DNA in fragments which had at least partially renatured. (1) The proportions of the repeated sequences in the labelled DNAs that were able to hybridise to each unlabelled DNA; (2) the mean distance apart of the hybridising sequences on the longer labelled fragments; and (3) the proportion of the genome in which the hybridising sequences were concentrated. Analysis of these results, together with those of separate experiments designed to quantitatively estimate the nature of sequences unable to reanneal with the repeated sequences of each of the probe DNAs, have enabled schematic maps to be drawn which show how the repeated and non-repeated sequences are arranged in the wheat and rye genomes. Both genomes are constructed from millions of relatively short sequences, most of them considerably shorter than 3000 base-pairs. This structure was recognised because adjacent sequences can be distinguished by their frequency of repetition (i.e. repeated or non-repeated) or by their evolutionary origin. Approximately 40 to 45% of the wheat genome and 30 to 35% of the rye genome consists of short non-repeated sequences interspersed between short repeated sequences. Approximately 50% of the wheat genome and 60% of the rye genome consists of tandemly arranged repeated sequences of different evolutionary origins. It is postulated that much of this complex repeated sequence DNA could have arisen from amplification of compound sequences, each containing repeated and non-repeated sequence DNA. Short repeated sequences with a number average length of around 200 base-pairs and which occupy about 20% of the wheat and rye genomes are related to repeated sequences also found in oats and barley. They are concentrated in 60 to 70% of the wheat and rye genomes, being interspersed with different short repeated sequences and a significant proportion of the short non-repeated sequences. Rye chromosomes contain more DNA than wheat chromosomes. This is principally, but not entirely, due to additional repeated sequence DNA. Many quantitative changes appear to have occurred in both genomes, possibly affecting most families of repeated sequences, since wheat and rye diverged from a common ancestor. Both species contain species-specific repeated sequences (24% of rye genome; 16% of wheat genome) but a large proportion of these are closely interspersed with repeated sequences found in both genomes.

A rapidly sedimenting DNA-protein complex was isolated from nuclear lysates in 2 M NaCl and characterized with regard to its polypeptide composition and the DNA-binding properties of the purified proteins. The complex consists of the nuclear matrix with attached DNA. Electrophoresis in SDS-polyacrylamide gels revealed two major and five minor polypeptide bands, mainly in the 60 to 75 kDa molecular weight region. The DNA-matrix complex dissociated into free DNA and proteins in the presence of 2 M NaCl and 5 M urea. The proteins could be purified by chromatography on hydroxyapatite and showed a strong tendency to reassociate at 0.15 M NaCl concentration in the absence of urea. DNA was bound to the reassociated proteins at 0.15 M NaCl concentration. Part of the DNA-protein complex was stable at 1 M NaCl concentration. The binding appeared to be random with regard to the DNA sequence.

The sequence complexity of several plasmids of Bacillus megaterium has been determined by reassociation kinetics, both optically and by the S1 nuclease method. G + C values calculated from T m 's obtained at several salt concentrations were compared to G + C values calculated from buoyant densities determined by isopycnic centrifugation. The G + C values for each plasmid are not substantially different from each other or from the chromosome, and the comparison of values calculated by the two methods shows that there are few, if any, modified or unusual bases present. Reassociation kinetics imply that each plasmid size class is unique in nucleotide sequence and that they contain no detectable high-complexity chromosomal DNA. Two plasmid species, the 4.0- and 6.2-md species, contain internal AT-rich regions. The role of dioxane in revealing such intramolecular heterogeneity is discussed.

Genetic recombination is the set of processes that results in new linkage relationships of genes or parts of genes (l), Three subclasses of recombination are currently recognized: (a) General recombination describes exchanges that occur between homologous genophores, more or less anywhere along their length. (b) Site-specific recombination describes exchanges that occur at a limited number of specific sites, as seen for example in the integration of some viral genomes into the host genome (2, 3). (c) Illegitimate or nonhomologous recombi­ nation are terms used for exchanges between genophores that are largely nonhomologous. Examples include the generation of specialized transducing particles (4) and the random chromosomal insertion of the bacteriophage Mu genome (5, 6). Although most of this review will deal with general recombina­ tion, the other classes of recombination are no less important (7). The macromolecular metabolism of DNA includes the partially overlapping areas of replication, repair, and recombination. The recent recognition of the role of transcription in replication and the possible implication of transcription in recombination indicate even greater complexity in these overlaps. Moreover, the degree of overlapping varies from one organism to another (8). As examples, the replication and recombination of phage T4 are extensively overlapping (9-11). The genes required for recombination are those required for replication. By contrast, the genes that govern recombination in bacteriophage A can be deleted without eliminating replication (12), although there are important relationships between replication and recombination in A as well (see below). Figure I illustrates the probable role of the heteroduplex joint in producing variations in the genetic output from an exchange. New DNA synthesis in the joint or asymmetry in the formation of joints may be the source of unequal recoveries of genetic information from the respective parents. In organisms like

It has been shown previously that after self-annealing of the nuclear precursor of messenger RNA (premRNA) at high values of Cot about 20% of the material becomes resistant to ribonuclease digestion [ 1 ] . This was explained by the existence of self-complementary regions in the molecules of pre-mRNA and by the ability of these sequences to form doublestranded complexes after annealing. In this paper we report on the isolation and the most important properties of this fraction of RNA (self-complementary RNA or scRNA). Its possible origin is discussed.

Complementary DNA was synthesized using polyadenylated nuclear RNA of cultured Drosophila cells as template. The kinetics of hybridization of this cDNA with nuclear RNA indicated that the complexity of this RNA population is five to ten times greater than that of cytoplasmic mRNA. The same difference in the fraction of DNA represented was obtained when nuclear and cytoplasmic RNA were hybridized with labeled unique sequence DNA. The fraction of the DNA sequences represented in total number of polyadenylated nuclear RNA is much higher than that represented in cytoplasmic RNA.

Sindbis virus 42 S RNA was efficiently transcribed into complementary DNA (cDNA) by avian myeloblastosis virus αβ DNA polymerase using oligo(dT) or single-stranded calf thymus DNA as primers. Both of the Sindbis virus cDNA products were able to protect 60% of 125I-labeled Sindbis virus RNA, at near equal weight ratios, from RNAase A and T1 digestion. Using hybridization kinetics, the C r t 1 2 value for hybridization of the calf thymus-primed cDNA product with excess Sindbis RNA was determined to be 1.8 · 10−2 mol · s · l−1. These data demonstrate that the Sindbis virus cDNA products are relatively uniform representations of Sindbis virus RNA sequences.

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)