Aggregation and adsorption at the air-water interface of bacteriophage phiX174 single-stranded DNA.

We study the phase behavior of phage phiX174 single-stranded DNA in very dilute solutions in the presence of monovalent and multivalent salts, in both water (H(2)O) and heavy water (D(2)O). DNA solubility depends on the nature of the salts, their concentrations, and the nature of the solvent. The appearance of attractive interactions between the monomers of the DNA chains in the bulk of the solution is correlated with an adsorption of the chains at the air-water interface. We characterize this correlation in two types of aggregation processes: the condensation of DNA induced by the trivalent cation spermidine and its salting out in the presence of high concentrations (molar and above) of monovalent (sodium) cations, both in water and in heavy water. The overall solubility of single-stranded DNA is decreased in D(2)O compared to H(2)O, pointing to a role of DNA hydration in addition to electrostatic factors in the observed phase separations. DNA adsorption involves attractive van der Waals forces, and these forces are also operating in the bulk aggregation process.

Complementary recognition in condensed DNA: accelerated DNA renaturation.

The functional consequences of DNA condensation are investigated. The recognition of complementary strands is profoundly modified by this critical phenomenon. (1) Condensation of denatured DNA greatly accelerates the kinetics of DNA renaturation. We propose a unifying explanation for the effects of several accelerating solvents studied here including polymers, di- and multivalent cations, as well as effects seen with the phenol emulsions and single-stranded nucleic acid binding proteins. Optimal conditions for renaturation at or above the calculated three dimensional diffusion limit are theoretically consistent with a limited search space in the condensed phases. (2) In addition to these effects on association of two single strands, similar condensation acceleration effects can be seen in strand exchange experiments with double stranded DNA without proteins. These may model a mechanism of recombinational protein function.

DNA crossovers and type II DNA topoisomerases: A thermodynamical study.

We present a theoretical study of the interaction of tight DNA crossovers with eukaryotic type II DNA topoisomerases. A quantitative analysis of the role of the enzyme during anaphase first shows that a tight DNA crossover should be an intermediate of the strand-passage reaction. We then focus on the initial steps of the strand-passage reaction in vitro which lead to the formation of a ternary complex ES1S2 between the enzyme and a tight DNA crossover (where E is the enzyme, S1 (respectively S2) is the first (respectively the second) DNA segment bound by the enzyme, and S1S2 is a tight crossover). This formation can be described by three equilibrium association constants: KS1 (for the reaction E+S1left arrow over right arrow ES1), KS2 (for ES1+S2left arrow over right arrow ES1S2), and KS (for E+S1S2left arrow over right arrow ES1S2) Using published experimental data obtained on the Drosophila enzyme, we derive rough estimates for the intrinsic equilibrium constants KS1 ( approximately 2.5x10(6) M-1) and KS2 ( approximately 10(4) M-1) and for Ks. The huge value found for Ks, about 5x10(16) M-1, suggests that the ternary complex bears a close resemblance with a transition state complex, and is consistent with the notion of a capture of the crossover by a protein clamp. We give a theoretical description of analogues of tight DNA crossovers which consist of two DNA segments stabilized by a covalent crosslinking. Such analogues are predicted to bind the enzyme with a high affinity and should be useful tools for the study of the enzyme.

Symmetry and chirality in topoisomerase II-DNA crossover recognition.

Several experimental data support the notion that the recognition of DNA crossovers play an important role in the multiple functions of topoisomerase II. Here, a theoretical analysis of the possible modes of assembly of yeast topoisomerase II with right and left-handed tight DNA crossovers is performed, using the crystal coordinates of the docking partners. The DNA crossovers are assumed to be clamped into the central hole of the enzyme. Taking into account the rules for building symmetric ternary complexes and the structural constraints imposed by DNA-DNA and protein-DNA interactions, this analysis shows that two geometric solutions could exist, depending on the chirality of the DNA crossovers. In the first one, the two DNA segments are symmetrically recognized by the enzyme while each single double helix binds asymmetrically the protein dimer. In the second one, each double helix is symmetrically recognized by the protein around its dyad axis, while the two DNA segments have their own binding modes. The finding of potential DNA-binding domains which could interact with the crossovers provides structural supports for each model. The structural similarity of a loop containing a cluster of conserved basic residues pointing into the central hole of topoisomerase II and the second DNA-binding site of histone H5 which binds DNA crossover is of particular interest. Each solution, which is consistent with different sets of experimental data found in the literature, could either correspond to different functions of the enzyme or different steps of the reaction. This work provides structural insights for better understanding the role of chirality and symmetry in topoisomerase II-DNA crossover recognition, suggests testable experiments to further elucidate the structure of ternary complexes, and raises new questions about the relationships between the mechanism of strand-passage and strand-exchange catalyzed by the enzyme.

Mouse immunoglobulin A: nucleotide sequence of the structural gene for the alpha heavy chain derived from cloned cDNAs.

The cDNAs complementary to mouse immunoglobulin alpha heavy chain mRNAs have been cloned into the PstI site of the plasmid vector pBR322. Recombinant plasmids have been identified by hybrid-arrested translation and purification of alpha heavy chain mRNA on DNA-DBM filters. The nucleotide sequence of the inserts encodes the constant and 3' untranslated regions of the alpha heavy chain mRNA. The CH3 domains of human and mouse alpha chains are highly homologous, including a 36 amino acid fragment not reported in the protein sequence (Robinson and Appella, 1980). As in the case of the mu secreted heavy chain, the alpha heavy chain contains a carboxy terminal piece of 20 amino acids.

Isolation of a cDNA clone for a catalytic subunit of Torpedo marmorata acetylcholinesterase.

We have constructed a cDNA library from Torpedo marmorata electric organ poly(A+) RNA in the lambda phage expression vector lambda gt11. This library has been screened with polyclonal anti-acetylcholinesterase antibodies. One clone, lambda AChE1, produced a fusion protein which was recognized by the antibodies and which prevented the binding of native acetylcholinesterase in an enzymatic immune assay. These results indicate that lambda AChE1 contains a cDNA insert coding for a part of a catalytic subunit of Torpedo acetylcholinesterase. The 200-base-pair cDNA insert hybridized to three mRNAs (14.5, 10.5 and 5.5 kb) from Torpedo electric organs. These mRNAs were also detected in Torpedo electric lobes.

Kinetics of chromosome condensation in the presence of topoisomerases: a phantom chain model.

We discuss the requirement of type II DNA topoisomerase in the process of mitotic chromosome condensation. Using a known model describing the collapse of homopolymers, we propose that the compaction process necessitates a change in the topological state (i.e., a self-knotting) of the chromosomal chain. We argue that the enzymes are necessary to reach the compact metaphase state in a time interval that is much smaller than the time expected in the uncatalyzed process. The folding process is such that the potential entanglement points are localized at particular regions of the chromosome known as the scaffold-associated regions. The concentration of entanglements in the metaphase chromosome is related to the average size of the radial loops. A phantom chain model for the condensation process, in which each potential entanglement point is dealt with by a topoisomerase II molecule, is proposed.

DNA renaturation at the water-phenol interface.

We study the renaturation of complementary single-stranded DNAs in a water-phenol two-phase system, with or without shaking. In very dilute solutions, each single-stranded DNA is strongly adsorbed at the interface at high salt concentrations. The adsorption of the single-stranded DNA is specific to phenol and relies on stacking and hydrogen bonding. We establish the interfacial nature of DNA renaturation at high salt, either with vigorous shaking (in which case the reaction is known as the Phenol Emulsion Reassociation Technique or PERT) or without. In the absence of shaking, the renaturation involves a surface diffusion of the single-stranded DNA chains. A comparison of PERT with other known renaturation reactions shows that PERT is the most efficient one and reveals similarities between PERT and the renaturation performed by single-stranded nucleic acid binding proteins. The most efficient renaturation reactions (either with PERT or in the presence of condensing agents) occur in heterogeneous systems, in contrast with standard thermal renaturation, which takes place in the bulk of a homogeneous phase. This work highlights the importance of aromaticity in molecular biology. Our results lead to a better understanding of the partitioning of nucleic acids, and should help to design improved extraction procedures for damaged nucleic acids. We present arguments in favor of interfacial scenarios involving phenol in prebiotic chemistry.

Cyclization of globular DNA. Implications for DNA-DNA interactions in vivo.

The rate of cyclization of lambda DNA varies over more than 6 orders of magnitude, from 3.2 x 10(-7) s-1 to 2 s-1, in a Tris-EDTA buffer as a function of spermidine concentration. This variation is strictly correlated with the conformation of the chain. The highest rates are obtained when the chain is collapsed into a dense globular state. The effective concentration of the chain ends in the reaction is then 87 000-fold greater than in the random coil state. These results show that DNA globularity must be taken into account to understand biological processes involving intramolecular DNA-DNA interactions.

A liquid crystalline phase in spermidine-condensed DNA.

Over a large range of salt and spermidine concentrations, short DNA fragments precipitated by spermidine (a polyamine) sediment in a pellet from a dilute isotropic supernatant. We report here that the DNA-condensed phase consists of a cholesteric liquid crystal in equilibrium with a more concentrated phase. These results are discussed according to Flory's theory for the ordering of rigid polymers. The liquid crystal described here corresponds to an ordering in the presence of attractive interactions, in contrast with classical liquid crystalline DNA. Polyamines are often used in vitro to study the functional properties of DNA. We suggest that the existence of a liquid crystalline state in spermidine-condensed DNA is relevant to these studies.

Anaphase chromatid motion: involvement of type II DNA topoisomerases.

Sister chromatids are topologically intertwined at the onset of anaphase: their segregation during anaphase is known to require strand-passing activity by type II DNA topoisomerase. We propose that the removal of the intertwinings involves at the same time the traction of the mitotic spindle and the activity of topoisomerases. This implies that the velocity of the chromatids is compatible with the kinetic constraints imposed by the enzymatic reaction. We show that the greatest observed velocities (about 0.1 microns s-1) are close to the theoretical upper bound compatible with both the diffusion rate (calculated here within a probabilistic model) and the measured reaction rate of the enzyme.

DNA aggregation induced by polyamines and cobalthexamine.

We have studied the precipitation of short DNA molecules by the polycations spermidine, spermine, and cobalthexamine. The addition of these cations to a DNA solution leads first to the precipitation of the DNA; further addition resolubilizes the DNA pellet. The multivalent salt concentration required for resolubilization is essentially independent of the DNA concentration (between 1 microM/ml and 1 mg/ml) and of the monovalent cation concentration present in the DNA solution (up to 100 mM). The DNA aggregates are anisotropic; those obtained in the presence of the polyamines spermidine and spermine generally contain a cholesteric liquid crystalline phase that flows spontaneously. In contrast this phase is never seen in the presence of cobalthexamine. We propose that the ability of polyamines to condense DNA in fluid structures is an essential feature of their biological functions.

cDNA sequences of Torpedo marmorata acetylcholinesterase: primary structure of the precursor of a catalytic subunit; existence of multiple 5'-untranslated regions.

cDNA clones coding for a catalytic subunit of acetylcholinesterase were isolated from cDNA libraries constructed from Torpedo marmorata electric organ. The nucleotide sequence of the cloned cDNAs codes for a 599-amino acid precursor containing a 24-amino acid signal peptide. This primary structure has been compared with the sequences of Torpedo californica and Drosophila melanogasta acetylcholinesterases, and with that of human butyrylcholinesterase. Genomic blot experiments carried out with cDNA restriction fragments used as hybridization probes are in agreement with the existence of a single gene coding for the different catalytic subunits of Torpedo acetylcholinesterase. Unexpectedly, we observed multiple 5'-untranslated regions, which may contain several initiation codons.

Forces on chromosomal DNA during anaphase.

In the course of anaphase, the chromosomal DNA is submitted to the traction of the spindle. Several physical problems are associated with this action. In particular, the sister chromatids are generally topologically intertwined at the onset of anaphase, and the removal of the intertwinings results from a coupling between the enzymatic action of type II DNA topoisomerases and the force exerted by the spindle. We propose a physical analysis of some of these problems: 1) We compare the maximum force the spindle can produce with the force required to break a DNA molecule, and define the conditions compatible with biological safety during anaphase. 2) We show that the behavior of the sister chromatids in the absence of type II DNA topoisomerases can be described by two distinct models: a chain pullout model accounts for the experimental observations made in the budding yeast, and a model of the mechanical rupture of rubbers accounts for the nondisjunction in standard cases. 3) Using the fluctuation-dissipation theorem, we introduce an effective protein friction associated with the strand-passing activity of type II DNA topoisomerases. We show that this friction can be used to describe the situation in which one chromosome passes entirely through another one. Possible experiments that could test these theoretical analyses are discussed.

Structural relationships among mouse and human immunoglobulin VH genes in the subgroup III.

The mouse VHIII subgroup is composed of four families which share sequence homology. We isolated a VH germ-line genomic clone, which cross hybridizes with a cDNA probe from one of these families, derived from a myeloma secreting an antigalactan antibody. We report here the nucleotide sequence of the cross hybridizing gene and show that very likely it has an anti-sheep red blood cell specificity. Comparison of its nucleotide sequence with those of the three other VHIII families shows that these genes share segmental homologies of variable lengths. This suggests that interchanges of sequence blocks between VH genes could be an important evolutionary mechanism for diversifying the germ-line repertoire. The strong homology (82%) with human VHIII genes suggests that efficient antibody sequences are strongly conserved. This conservation of homology is particularly striking when compared to the more limited homology (63%) between mouse and human C kappa genes.

Synthesis in vitro of precursors of the catalytic subunits of acetylcholinesterase from Torpedo marmorata and Electrophorus electricus.

We translated poly(A-rich messenger RNA prepared from the electric organs of Electrophorus electricus and Torpedo marmorata in a reticulocyte lysate system. In the case of Electrophorus, which appears to contain only one type of acetylcholinesterase catalytic subunit, an anti-(Electrophorus acetylcholinesterase) antiserum precipitated a single 65-kDa polypeptide from the products translation obtained in vitro. In the case of Torpedo, where a number of distinct catalytic subunits corresponding to different fractions of the enzyme have been described, an anti-(Torpedo acetylcholinesterase) antiserum precipitated two main polypeptides, 61 kDa and 65 kDa, both of which could be displaced by unlabelled purified Torpedo acetylcholinesterase. Synthesis in vitro thus appears to produce a single type of precursor of the acetylcholinesterase catalytic subunit for Electrophorus, and at least two distinct precursors for Torpedo, suggesting that several mRNAs code for the catalytic subunits in the latter species.

Structure of the constant and 3' untranslated regions of the murine Balb/c gamma 2a heavy chain messenger RNA.

The complete sequence for the constant and 3' untranslated regions of a mouse gamma 2a immunoglobulin heavy chain mRNA is reported. The sequence is 1093 nucleotides long coding for the CH1 (amino-acids 118-214), the Hinge (215-230), the CH2 (231-340) and the CH3 (341-447). The 3' untranslated region is 103 nucleotides long preceding the poly(A). The nucleotide sequence predicts as in the case for gamma 1 and gamma 2b heavy chains an additional lysine residue before the termination codon. This sequence has been compared to the corresponding sequences of gamma 1 and gamma 2b heavy chain mRNAs. These sequences are respectively 75% and 84% homologous. The CH2 domains of gamma 2a and gamma 2b are 95% homologous at the nucleotide level. The cross-over point of a gamma 2a - gamma 2b heavy chain variant is located in a segment of 73 perfectly matching nucleotides. The 3' non coding regions of gamma 2a and gamma 2b are 89% homologous.

Mouse heavy chain variable regions: nucleotide sequence of a germ-line VH gene segment.

We have constructed a library of Balb/c mouse embryo DNA in the vector Charon 4A. The library was searched for sequences homologous to the VH region of a cloned cDNA of the UPC10 heavy chain mRNA. In this paper, we describe the structure and the partial nucleotide sequence of one of such clones (VH441). The nucleotide sequence of this germ-line gene indicates that it encodes amino-acids 1-98 of the X44 and J601 galactan-binding VH regions, but that it differs from the UPC10 VH segment by four single base changes. The VH gene appears to contain a 101 bases long intervening sequence within a precursor sequence identical to the precursor sequence of UPC10. The 3' non coding sequence of the V gene contains the two conserved sequences found in embryonic V DNA segments, CACAGTG and ACATGAACC, separated by 23 nucleotides and a sequence CACTGTG separated by 33 nucleotides from the first heptamer.

Correlation between D region structure and antigen-binding specificity: evidences from the comparison of closely related immunoglobulin VH sequences.

A VH region gene is generated from three gene segments, VH, D and JH, separated on the germline chromosome and rearranged during differentiation to generate an active VH gene. The sequence and length variations of the D region are an important source of the antibody diversification. To investigate their role in the antigen recognition, VH sequences of three myeloma proteins (ABE48, UPC10 and MOPC173) having different antigen specificities and whose VH segments were expected to be highly homologous have been determined: cDNA clones containing the structural gene sequences for the three proteins have been constructed. The nucleotide sequences of the three VH regions have been determined. The deduced amino-acid sequences are compared to those of four other myeloma proteins (J539, X44, X24 and T601). The seven proteins have highly homologous VH segments. The comparison points out the correlation existing between the D-region structure and the antigen-binding specificity.

Accelerated cyclization of lambda DNA.

In the presence of spermidine, the DNA molecule of the bacteriophage lambda undergoes a coil-globule transition. We report here that the cyclization of this molecule in its globular state is greatly accelerated (by more than 10(4)-fold) in comparison with the cyclization reaction taking place in the coil conformation.