Proteins searching for their target on DNA by one-dimensional diffusion: overcoming the “speed-stability” paradox

The sequence dependence of DNA-protein interactions that allows proteins to find the correct reaction site also slows down the 1D diffusion of the protein along the DNA molecule, leading to the so-called “speed-stability paradox,” wherein fast diffusion along the DNA molecule is seemingly incompatible with stable targeting of the reaction site. Here, we develop diffusion-reaction models that use discrete and continuous Gaussian random 1D diffusion landscapes with or without a high-energy cut-off, and two-state models with a transition to and from a “searching” mode in which the protein diffuses rapidly without recognizing the target. We show the conditions under which such considerations lead to a predicted speed-up of the targeting process, and under which the presence of a “searching” mode in a two-state model is nearly equivalent to the existence of a high-energy cut-off in a one-state model. We also determine the conditions under which the search is either diffusion-limited or reaction-limited, and develop quantitative expressions for the rate of successful targeting as a function of the site-specific reaction rate, the roughness of the DNA-protein interaction potential, and the presence of a “searching” mode. In general, we find that a rough landscape is compatible with a fast search if the highest energy barriers can be avoided by “hopping” or by the protein transitioning to a lower-energy “searching” mode. We validate these predictions with the results of Brownian dynamics, kinetic Metropolis, and kinetic Monte Carlo simulations of the diffusion and targeting process, and apply these concepts to the case of T7 RNA polymerase searching for its target site on T7 DNA.     

Chloroplast maintenance and partial differentiation in vitro

Tissue homogenates, etioplasts, and developing chloroplasts were prepared from cucumber (Cumucis sativus L.) cotyledons in tris-sucrose. They were incubated aerobically in the dark or in the light at pH 7.7 in the presence or absence of a cofactor mixture containing coenzyme A, glutathione, potassium phosphate, methyl alcohol, magnesium, nicotinamide adenine dinucleotide, and adenosine triphosphate. These cofactors were previously shown to be essential for protochlorophyll and chlorophyll biosynthesis. Ultrastructural changes were monitored by electron microscopy. The following observations were made. (a) Crude homogenates contained agents which degraded etioplasts and developing chloroplasts. (b) Added cofactors were essential for the maintenance of the membrane structure; they were also implicated in the transformation of the prolamellar body in the absence and presence of light. (c) Light pretreatment of the cotyledons improved the maintenance of the developing chloroplast membranes during subsequent in vitro incubation. (d) In the presence of the cofactors, grana formation appeared to take place in the absence of nuclear-cytoplasmic control.     

Molecular cloning of two linked loci that increase the transformability of transformation-deficient mutants of Haemophilus influenzae.

A plasmid containing a 13.3-kb insert (pER194) was isolated from an EcoRI genomic library of Haemophilus influenzae on the basis of its ability to increase the transformability of the transformation-deficient mutants Com-78 and Com-101. The plasmid failed to increase the transformability of the Rec-1 and Rec-2 mutants, indicating that the mutations producing the Com-78 and Com-101 phenotypes are distinct from those giving rise to the Rec-1 and Rec-2 phenotypes. The physical mapping of the cloned fragment on the H. influenzae chromosome was found to be consistent with the genetic mapping of the Com-101 trait. A 2.8-kb EcoRI-BglII subfragment, representing one end of the 13.3-kb clone, was found to increase the transformation frequency of the Com-78 and Com-101 mutants when supplied in trans, indicating that the subfragment carries one or more loci required for chromosomal transformation. The corresponding region of the Com-101 chromosome was determined by hybridization analysis to contain a 0.3-kb insertion, suggesting that the Com-101 strain may contain an insertion mutation at this locus. A 3.0-kb EcoRI-MluI subfragment, representing the other end of the 13.3-kb EcoRI fragment, was found to increase the transformation frequency of the Com-101 mutant but not of the Com-78 mutant, suggesting that the Com-101 phenotype results from a complex genotype involving mutations at two or more transformation-related loci. This conclusion is consistent with data indicating that the Com-101 trait can be genetically separated into at least two components.     

Correlation between the number of adult male Pectinophora gossypiella (Saund.) (Lep., Gelechidae) catches on pheromone traps and the rate of infestation in fruiting bodies of cotton plants by young larvae in three regions of central Greece

In order to evaluate the influence of the number of catches in pheromone-baited traps on the percentage of larval infestation, six delta traps equipped with sex attractant were placed in each of three regions in Central Greece (Farkadona, Farsala, Almyros) in cotton fields from 20 June until 30 September 1995. The collection of fruiting bodies took place weekly and the counting of adults in the traps was carried out each day. The population fluctuation in all three regions was similar with their peak during the first weeks of August. In Farkadona the infestation level was low (1% at the first sampling of August) with a maximum of 9% in the last sampling of September. In Farsala and Almyros, the infestation level was already high (10% and above) in early August. There was a significant positive linear correlation between the number of moth catches and the infestation percentage from first- and second-stage larvae on the first (R = 0694) and second (R = 0.7399) boll-feeding generations.     

ONTOGENETIC NICHE SHIFT IN THE BRACHIOPOD TEREBRATALIA TRANSVERSA: RELATIONSHIP BETWEEN THE LOSS OF ROTATION ABILITY AND ALLOMETRIC GROWTH

Abstract: Many articulated brachiopods experience marked life habit variations during ontogeny because they experience their fluid environment at successively higher Reynolds numbers, and they can change the configuration of their inhalant and exhalant flows as body size increases. We show that the extant brachiopod Terebratalia transversa undergoes a substantial ontogenetic change in reorientation governed by rotation around the pedicle. T. transversa′s reorientation angle (maximum ability to rotate on the pedicle) decreases during ontogeny, from 180 degrees in juveniles to 10–20 degrees in individuals exceeding 5 mm, to complete cessation of rotation in individuals larger than 10 mm. Rotation ability is substantially reduced after T. transversa achieves the adult lophophore configuration and preferred orientation with respect to ambient water currents at a length of 2.5–5 mm. We hypothesize that the rotation angle of T. transversa is determined mainly by the position of ventral and dorsal points of attachment of dorsal pedicle muscles relative to the pedicle. T. transversa shows a close correlation between the ontogenetic change in reorientation angle and ontogeny of morphological traits that are related to points of attachment of dorsal pedicle muscles, although other morphological features can also limit rotation in the adult stage. The major morphological change in cardinalia shape and the observed reduction of rotation affect individuals 2.5–10 mm in length. The position of ventral insertions of dorsal pedicle muscles remains constant, but contraction of dorsal pedicle muscles is functionally handicapped because dorsal insertions shift away from the valve midline, rise above the dorsal valve floor, and become limited by a wide cardinal process early in ontogeny (<5 mm). The rate of increase of cardinal process width and of distance between dorsal pedicle muscle scars substantially decreases in the subadult stage (5–10 mm), and most of the cardinalia shell traits grow nearly isometrically in the adult stage (>10 mm). T. transversa attains smaller shell length in crevices than on exposed substrates. The proportion of small‐sized individuals and population density is lower on exposed substrates than in crevices, indicating higher juvenile mortality on substrates prone to grazing and physical disturbance. The loss of reorientation ability can be a consequence of morphological changes that strengthen substrate attachment and maximize protection against biotic or physical disturbance (1) by minimizing torques around the pedicle axis and/or (2) by shifting energy investments into attachment strength at the expense of the cost involved in reorientation.     

Specific mode of interaction between components of model pulmonary surfactants using computer simulations

Atomistic molecular dynamics simulations and structural bioinformatics tools enable the identification of the exact mode of interaction between model pulmonary surfactant components. Two nanosecond long simulations of the N-terminal region of human surfactant protein-B (SP-B(1-25)) in dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) monolayers of different lipid surface densities reveal the preferential affinity of SP-B(1-25) for anionic phospholipids. In particular, arginine 12 and lysine 24 interact strongly and with high specificity with the phosphate group of the DPPG lipids, stabilizing the position, the orientation, and the secondary structure of the peptide in the monolayer. The peptide lies at an oblique angle to the interfacial plane, ranging between 47 degrees and 62 degrees, increasing with decreasing lipid surface density. In DPPC monolayers the interaction is largely determined by hydrophobic interactions. The non-specific nature of DPPC-SP-B(1-25) interactions allows for significant flexibility in the topology of the peptide in the lipid matrix. Bioinformatics tools are employed to generalize the simulation results to the sequences of SP-B(1-25) in other organisms. The importance of specific residues, and the role of the largely helical and amphiphilic nature of the peptide in the functionality of SP-B(1-25) are established. The synergy of classical mechanics tools with bioinformatics methods greatly enhances the molecular-level interpretation of pulmonary surfactant action and facilitates the development of design rules for synthetic surfactant analogues.     

Bacterial fingerprinting by flow cytometry: bacterial species discrimination.

BACKGROUND: A flow cytometric measurement (FCM) technique has been developed to size DNA fragments. Individual fragments of a restriction digest of genomic DNA, stained with an intercalating dye, are passed through an ultrasensitive cytometer. The measured fluorescence intensity from each fragment is proportional to the fragment length. METHODS: The isolation of bacterial genomic DNA and digestion by restriction enzymes were performed inside an agarose plug. Rare cutting enzymes were employed to produce a manageable number of DNA fragments. Electroelution was used to move the DNA fragments from the agarose plug into a solution containing polyamines to protect the DNA from shear-induced breakage. The DNA was stained with the bisintercalating dye thiazole orange homodimer and introduced into our ultrasensitive flow cytometer. A histogram of the fluorescence intensities (fingerprint) was constructed. RESULTS: Gram-positive Bacillus globigii and gram-negative bacteria Escherichia coli and Erwinia herbicola were distinguished by the fingerprint pattern of restriction fragments of their genomic DNA. DNA sizes determined by FCM are in good agreement with pulsed-field gel electrophoresis (PFGE) analysis. Flow cytometry requires only picogram quantities of purified DNA and takes less than 10 min for data collection and analysis. When the total sample preparation time is included, the analysis times for PFGE and FCM are similar ( approximately 3 days). CONCLUSIONS: FCM is an attractive technique for the identification of bacterial species. It is more sensitive and potentially much faster than PFGE.