Mechanism of transformation in Pichia methanolica yeast: transforming and nontransforming genes

Two types of genes were found in the study of transformation in yeast Pichia methanolica: transforming (Trg) and nontransforming (Ntg) genes. Transforming genes (P-ADE7,4 and S-LEU2), as linear DNA molecules, can transform competent cells with high efficiency inversely proportional to the molecule size. Nontransforming genes (P-ADE5 and H-LEU2) transform P. methanolica cells at an extremely low rate even when they are combined with transforming genes. The analysis showed that linear DNA molecules with Trg and Ntg can be either rearranged and integrated in random sites of the recipient genome or form circular plasmids, which are capable of autonomous replication irrespective of the presence of specific replicative elements.     

Excluded volume effect in unzipping DNA with a force

A double stranded DNA molecule when pulled with a force acting on one end of the molecule can become either partially or completely unzipped depending on the magnitude of the force F. For a random DNA sequence, the number M of unzipped base pairs goes as M approximately (F - Fc)(-2) and diverges at the critical force Fc with an exponent chi = 2. We find that when excluded volume effect is taken into account for the unzipped part of the DNA, the exponent chi = 2 is not changed but the critical force Fc is changed. The force versus temperature phase diagram depends on only two parameters in the model, the persistence length and the denaturation temperature. Furthermore a scaling form of the phase diagram can be found. This scaling form is parameter independent and depends only on the spatial dimension. It applies to all DNA molecules and should provide a useful framework for comparison with experiments.     

Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: polynucleotide binding and cooperativity

We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5′-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex.     

Conformational properties of the iso-1-cytochrome C denatured state: dependence on guanidine hydrochloride concentration

Production of seven single surface histidine variants of yeast iso-1-cytochrome c allowed measurement of the apparent pK(a), pK(a)(obs), for histidine-heme loop formation for loops of nine to 83 amino acid residues under varying denaturing conditions (2 M to 6 M guanidine hydrochloride, gdnHCl). A linear correlation between pK(a)(obs) and the log of the loop size is expected for a random coil, pK(a)(obs) proportional to k log(n), where k is a scaling factor and n is the number of monomers in the loop. For small loops of nine, 16, and 22 monomers, no dependence of pK(a)(obs) on loop size was observed at any denaturant concentration indicating effects from chain stiffness. For larger loops of 37, 56, 72, and 83 monomers, the dependence of pK(a)(obs) on log(n) was linear and the slope of that dependence decreased with increasing concentration of denaturant. The scaling factor obtained at 5 M and 6 M gdnHCl for the larger loop sizes was approximately -2.0, close to the value of -2.2 expected for a random coil with excluded volume. However, scaling factors obtained under less harsh denaturing conditions (2 M to 4.5 M gdnHCl) deviated strongly from that expected for a random coil, being in the range -3 to -4. The gdnHCl dependence of pK(a)(obs) at each loop size was also evaluated to obtain denaturant m-values. Short loops where chain stiffness dominates had similar m-values of approximately 0.25 kcal/mol M. For larger loops m-values decrease with increasing loop size indicating that less hydrophobic area is sequestered when larger loops form. It is known that the earliest events in protein folding involve the formation of simple loops. The data from these studies provide direct insight into the relative probability with which loops of different sizes will form, as well as the factors which affect loop formation.     

StpA protein from Escherichia coli condenses supercoiled DNA in preference to linear DNA and protects it from digestion by DNase I and EcoKI

The nucleoid-associated protein, StpA, of Escherichia coli binds non-specifically to double-stranded DNA (dsDNA) and apparently forms bridges between adjacent segments of the DNA. Such a coating of protein on the DNA would be expected to hinder the action of nucleases. We demonstrate that StpA binding hinders dsDNA cleavage by both the non-specific endonuclease, DNase I, and by the site-specific type I restriction endonuclease, EcoKI. It requires approximately one StpA molecule per 250–300 bp of supercoiled DNA and approximately one StpA molecule per 60–100 bp on linear DNA for strong inhibition of the nucleases. These results support the role of StpA as a nucleoid-structuring protein which binds DNA segments together. The inhibition of EcoKI, which cleaves DNA at a site remote from its initial target sequence after extensive DNA translocation driven by ATP hydrolysis, suggests that these enzymes would be unable to function on chromosomal DNA even during times of DNA damage when potentially lethal, unmodified target sites occur on the chromosome. This supports a role for nucleoid-associated proteins in restriction alleviation during times of cell stress.     

Translocation of Escherichia coli recA protein from a single-stranded tail to contiguous duplex DNA

Duplex DNA with a contiguous single-stranded tail was nearly as effective as single-stranded DNA in acting as a cofactor for the ATPase activity of recA protein at neutral pH and concentrations of MgCl2 that support homologous pairing. The ATP hydrolysis reached a steady state rate that was proportional to the length of the duplex DNA attached to a short 5' single-stranded tail after a lag. Separation of the single-stranded tail from most of the duplex portion of the molecule by restriction enzyme cleavage led to a gradual decline in ATP hydrolysis. Measurement of the rate of hydrolysis as a function of DNA concentration for both tailed duplex DNA and single-stranded DNA cofactors indicated that the binding site size of recA protein on a duplex DNA lattice, about 4 base pairs, is similar to that on a single-stranded DNA lattice, about four nucleotides. The length of the lag phase preceding steady state hydrolysis depended on the DNA concentration, length of the duplex region, and the polarity of the single-stranded tail, but was comparatively independent of tail length for tails over 70 nucleotides in length. The lag was 5-10 times longer for 3' than for 5' single-stranded tailed duplex DNA molecules, whereas the steady state rates of hydrolysis were lower. These observations show that, after nucleation of a recA protein complex on the single-stranded tail, the protein samples the entire duplex region via an interaction that is labile and not strongly polarized.     

Microinjected Tetrahymena rDNA ends are not recognized as telomeres in Xenopus eggs

Telomeres are essential structures that stabilize the ends of eukaryotic chromosomes and allow complete replication of linear DNA molecules. We examined the structure and replication of telomeres by observing the fate of the linear extrachromosomal rDNA of Tetrahymena after injection into unfertilized Xenopus eggs. The rDNA replicated efficiently as a linear extrachromosomal molecule, increasing in mass 30-50-fold by 15-20 h after injection. In addition, the molecules increased in length by addition of up to several kilobases of DNA to their termini. Sequence analysis demonstrated that the added DNA bore no resemblance to known telomeres. The junction between the rDNA and added DNA was apparently random, indicating that the addition reaction did not involve a site-specific recombination or integration event. Surprisingly, Southern blot analysis showed that the added DNA did not derive from Xenopus DNA, but rather from co-purifying and therefore co-injected Tetrahymena DNA. The nonspecific ligation of random DNA fragments to the rDNA termini suggests that microinjected Tetrahymena rDNA ends are not recognized as telomeres in Xenopus eggs.     

The Listeria monocytogenesiap gene as an indicator gene for the study of PrfA-dependent regulation

The Bacillus subtilis 168 RecR protein bound to duplex DNA in the presence of ATP and divalent cations (Mg²⁺ and Zn²⁺) was visualized by electron microscopy as a nearly spherical particle. A RecR homomultimer is frequently located at the intersection of two duplex DNA strands in an interwound DNA molecule, generating DNA loops of variable length. Two individual DNA molecules bound to the same protein are seen at a very low frequency, if at all. The association of RecR with the intersection of two duplex DNA strands is more often seen in supercoiled than with relaxed or linear DNA. The RecR protein displays a slight but significant preference for negatively supercoiled over linear DNA. The minimum substrate size for RecR protein is about 150 bp in length. A possible mechanism for RecR function in DNA repair is discussed.     

Limit theorems for the population size of a birth and death process allowing catastrophes

The linear birth and death process with catastrophes is formulated as a right continuous random walk on the non-negative integers which evolves in continuous time with an instantaneous jump rate proportional to the current value of the process. It is shown that distributions of the population size can be represented in terms of those of a certain Markov branching process. The ergodic theory of Markov branching process transition probabilities is then used to develop a fairly complete understanding of the behaviour of the population size of the birth-death-catastrophe process.Research done while on leave at Colorado State University from the University of Western Australia and partially supported by N.S.F. grant DMS-8501763     

Simulations of nucleation and early growth stages of protein crystals.

Analysis of known protein crystal structures reveals that interaction energies between monomer pairs alone are not sufficient to overcome entropy loss related to fixing monomers in the crystal lattice. Interactions with several neighbors in the crystal are required for stabilization of monomers in the lattice. A microscopic model of nucleation and early growth stages of protein crystals, based on the above observations, is presented. Anisotropy of protein molecules is taken into account by assigning free energies of association (proportional to the buried surface area) to individual monomer-monomer contacts in the lattice. Lattice simulations of the tetragonal lysozyme crystal based on the model correctly reproduce structural features of the movement of dislocation on the (110) crystal face. The dislocation shifts with the speed equal to the one determined experimentally if the geometric probability of correct orientation is set to 10(-5), in agreement with previously published estimates. At this value of orientational probability, the first nuclei, the critical size of which for lysozyme is four monomers, appear in 1 ml of supersaturated solution on a time scale of microseconds. Formation of the ordered phase proceeds through the growth of nuclei (rather then their association) and requires nucleations on the surface at certain stages.     

Site-specific recombination by the bacteriophage P1 lox-Cre system. Cre-mediated synapsis of two lox sites

The bacteriophage P1-encoded recombinase Cre forms a simple DNA-protein complex at the specific recognition site loxP. Furthermore, Cre is able to mediate a synaptic union of two loxP sites. When two loxP sites are on the same linear DNA molecule, Cre binds the two sites together to form a circular protein-DNA complex. These complexes can be resolved into a linear DNA molecule and a closed circular DNA molecule, the end products of site-specific recombination.     

Mechanisms of Transformation in Yeast Pichia methanolica: Transforming and Nontransforming Genes

Two types of genes were found in the study of transformation in yeastPichia methanolica: transforming (Trg) and nontransforming (Ntg) genes. Transforming genes (P-ADE7,4and S-LEU2), as linear DNA molecules, can transform competent cells with high efficiency inversely proportional to the molecule size. Nontransforming genes (P-ADE5 and H-LEU2) transform P. methanolica cells at an extremely low rate even when they are combined with transforming genes. The analysis showed that linear DNA molecules with Trg and Ntg can be either rearranged and integrated in random sites of the recipient genome or form circular plasmids, which are capable of autonomous replication irrespective of the presence of specific replicative elements.     

Microscopic model of protein crystal growth

A microscopic, reversible model to study protein crystal nucleation and growth is presented. The probability of monomer attachment to the growing crystal was assumed to be proportional to the protein volume fraction and the orientational factor representing the anisotropy of protein molecules. The rate of detachment depended on the free energy of association of the given monomer in the lattice, as calculated from the buried surface area. The proposed algorithm allowed the simulation of the process of crystal growth from free monomers to complexes having 10(5) molecules, i.e. microcrystals with already formed faces. These simulations correctly reproduced the crystal morphology of the chosen model system--the tetragonal lysozyme crystal. We predicted the critical size, after which the growth rate rapidly increased to approximately 50 protein monomers. The major factors determining the protein crystallisation kinetics were the geometry of the protein molecules and the resulting number of kinetics traps on the growth pathway.     

Statistical analyses support power law distributions found in neuronal avalanches

The size distribution of neuronal avalanches in cortical networks has been reported to follow a power law distribution with exponent close to -1.5, which is a reflection of long-range spatial correlations in spontaneous neuronal activity. However, identifying power law scaling in empirical data can be difficult and sometimes controversial. In the present study, we tested the power law hypothesis for neuronal avalanches by using more stringent statistical analyses. In particular, we performed the following steps: (i) analysis of finite-size scaling to identify scale-free dynamics in neuronal avalanches, (ii) model parameter estimation to determine the specific exponent of the power law, and (iii) comparison of the power law to alternative model distributions. Consistent with critical state dynamics, avalanche size distributions exhibited robust scaling behavior in which the maximum avalanche size was limited only by the spatial extent of sampling ("finite size" effect). This scale-free dynamics suggests the power law as a model for the distribution of avalanche sizes. Using both the Kolmogorov-Smirnov statistic and a maximum likelihood approach, we found the slope to be close to -1.5, which is in line with previous reports. Finally, the power law model for neuronal avalanches was compared to the exponential and to various heavy-tail distributions based on the Kolmogorov-Smirnov distance and by using a log-likelihood ratio test. Both the power law distribution without and with exponential cut-off provided significantly better fits to the cluster size distributions in neuronal avalanches than the exponential, the lognormal and the gamma distribution. In summary, our findings strongly support the power law scaling in neuronal avalanches, providing further evidence for critical state dynamics in superficial layers of cortex.     

Analysis of the gel electrophoresis of looped protein-DNA complexes by computer simulation

The theory of mass transport coupled to reversible interactions under chemical kinetic control forms the basis of a numerical model that has been applied to systems such as lac repressor-lac operator DNA, in which a protein binds in two different modes to linear DNA carrying two specific binding sites. Three complexes may be formed: (1) a linear 1:1 complex with one protein molecule bound to one site on the DNA molecule; (2) a 1:1 complex in which a single protein molecule is bound to both sites simultaneously, thereby inducing a large DNA loop; and (3) a 2:1 linear complex in which two protein molecules are bound in tandem, each occupying a single site. The computational model affords a quantitative numerical simulation of the observed gel electrophoretic patterns produced by titration of the DNA with protein and provides new insights into the shape and nature of the patterns. In particular, the patterns may represent unimodal or bimodal reaction zones. Nevertheless, analysis of the peaks in the patterns obtained at low DNA and high protein concentration provides essential information as to the stoichiometry of the complexes and satisfactory estimates of association constants. The theory thus provides the experimenter with guidelines for quantitative evaluation of the results of gel retardation assays of the particular system under investigation, once protein-induced DNA (or RNA) loops have been established by independent physical or chemical methods. It is suggested that these insights might also find application to systems involving the binding of two or three different proteins to DNA with loop formation.     

Probing protein-DNA interactions by unzipping a single DNA double helix

We present unzipping force analysis of protein association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding "event" revealed information about the spatial location and dynamic nature of the protein-DNA complex. The capacity of UFAPA to spatially locate protein-DNA interactions is demonstrated by noncatalytic restriction mapping on a 4-kb DNA with three restriction enzymes (BsoBI, XhoI, and EcoRI). A restriction map for a given restriction enzyme was generated with an accuracy of approximately 25 bp. UFAPA also allows direct determination of the site-specific equilibrium association constant (K(A)) for a DNA-binding protein. This capability is demonstrated by measuring the cation concentration dependence of K(A) for EcoRI binding. The measured values are in good agreement with previous measurements of K(A) over an intermediate range of cation concentration. These results demonstrate the potential utility of UFAPA for future studies of site-specific protein-DNA interactions.     

Studies of DNA dumbbells. II. Construction and characterization of DNA dumbbells with a 16 base-pair duplex stem and Tn end loops (n = 2, 3, 4, 6, 8, 10, 14).

The preparation and characterization of DNA dumbbells that contain the 16 base-pair duplex sequences 5'G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C3' (set 1) and 5'G-C-A-T-C-A-T-C-G-A-T-G-A-T-G-C3' (set 2) are reported. The dumbbells of set 1 have the duplex stem nucleated on both ends by Tn (n = 2, 3, 4, 6, 8, 10, and 14) loops. The dumbbells of set 2 have Tn (n = 2, 4, 8, 10) end loops. For the molecules of set 1, effects of end loop size on the electrophoretic mobility, CD and UV absorbance spectra, and cleavage by restriction enzymes, were investigated. Effects of loop size on the CD spectra and restriction enzyme cleavage of the molecules of set 2 were also examined. Optical melting curves of the molecules of set 1 were collected as a function of sodium ion concentration from 30 to 120 mM. These investigations revealed that as loop size decreases, the electrophoretic mobilities, rates of enzyme cleavage, and optical melting temperatures increase. For end loops with at least three T's the observed increases are inversely proportional to loop size. The behavior of the dumbbell with T2 end loops departs from this linear dependence and is anomalous in every experimental context. For molecules with end loops comprised of at least four T's CD spectra were virtually indistinguishable. However, these spectra differed considerably from the CD spectrum of the T2-looped molecule. The CD spectrum of the dumbbell with T3 end loops displayed features common to the dumbbells with larger loops and T2 end loops. Thermodynamic evidence that the terminal G.C base pairs (bps) nucleating the T2 end loops were intact was obtained from a comparison of the melting temperature of this molecule with that of a DNA dumbbell containing the 14 central bps of the set 1 duplex sequence linked instead by end loops comprised of the four base sequence, C-T-T-C. The tm of this latter molecule was determined to be 9 degrees C less than that of the former dumbbell assumed to contain a 16-bp stem and T2 end loops.(ABSTRACT TRUNCATED AT 400 WORDS)