The "megaprimer" method of site-directed mutagenesis

We describe a simple and efficient method of mutagenesis which we term the "megaprimer" method. The method utilizes three oligonucleotide primers to perform two rounds of polymerase chain reaction. In the method, the product of the first polymerase chain reaction is used as one of the polymerase chain reaction primers (a "megaprimer") for the second polymerase chain reaction. When a phage promoter and a translational initiation signal are attached to the appropriate oligonucleotide primer, the mutant protein can be generated without any in vivo manipulations. To illustrate the method, two mutations in the catalytic domain of the human factor IX gene have been generated. The substitution of megaprimers for oligonucleotide primers may have utility in other polymerase chain reaction-based methods.     

Anomalies in sedimentation. I. Stability theory of sedimenting entanglements in the "tight-bending" limit

A simple model is introduced to investigate the stability of a sedimenting entanglement. The sedimenting entanglement is represented by a sedimenting sieve. Solvent can pass through it, but single-chain molecules that flow into it become entangled and their flow decreases or, if permanent entanglements form, ceases entirely. With this model we are able to find the conditions under which the mass of a sedimenting entanglement remains constant, grows or decays to a stable value, grows beyond limit, or decays to the mass of a single chain. The theory is applied to the sedimentation of small concentrations of large chain molecules in solutions of small chain molecules in solutions of small chain molecules for the case in which the entanglements are long-lived. Equations are derived which, (1) give the stable entanglement mass as a function of rotor speed and concentration and, (2) for a given concentration predict the rotor speed at which the entanglement mass grows without limit. Numerical results for small concentrations of T2 DNA sedimenting in solutions of T7 DNA are presented.     

Mitochondrial uptake of bridged bis-methylpyridinium aldoximes and induction of the "petite" phenotype in yeast

The 3,3'-[omega,omega'-alkanediylbis(oxy)]bis[2- (hydroxyimino)methyl]-1-methylpyridinium derivatives bearing a linking chain of 4, 5 and 6 methylene groups are accumulated in mitochondria with increasing efficiency under the effect of the electrical potential. Accumulation does not take place with derivatives carrying a 2 and 3 methylene-long linking chain. The uptake process is saturable. The efficiency of the various derivatives to induce the "petite" phenotype in yeast reflects the uptake rate observed with purified mitochondria.     

The "sticky chain": a kinetic model for the deformation of biological macromolecules

The deformation behavior of certain biologic macromolecules is modeled by the "sticky chain," a freely jointed chain with weak bonds between subsequent joints. Straining the chain leads to thermally assisted breaking of the weak bonds, yielding a characteristic shape of the force-elongation curve, usually with a pronounced plateau, but sometimes displaying a pseudo-Hookean behavior over a wide range of deformations. The number of individual links is assumed to be large, so the stochastic time evolution of the individual events can be approximated by a differential equation. The cases of individual and collective bond breaking are treated and formulae given for various measurable quantities. A threshold strain rate is found, below which the deformation force no longer depends on the deformation velocity. The method is applied to experimental results for the deformation of single molecules like titin or DNA and the results agree with the parameters deduced from the same experiments by the original authors using Monte Carlo (MC) calculations. Despite its intrinsic continuous character, the model, therefore, is applicable even for the deformation of macromolecules with only a few discrete unfolding elements, yielding physical quantities from experimental results using simple formulae instead of a host of MC computations.     

On the controversy over "fast" and "slow" helix-coil transition rates in polypeptides

The controversy over “fast” and “slow” helix-coil transition rates in polypeptides is discussed. The “slow” results are derived from the assumption that multiple NMR spectra of α-CH and NH groups arise from chemical exchange. In this paper it is shown that such spectra may be obtained without invoking chemical exchange. The multiplicity arises from the difference in helicity of amino acid residues near the ends of the chain by comparison with amino acid residues nearer the middle, and from a polydispersity in molecular weight. As a consequence of this analysis, support is given to the “fast” transition rates.     

Mining the bovine genome with the "Bovine SNP Retriever"

Online resources for the bovine genome analysis are provided at the most important Web sites. Nonetheless, retrieval of single-nucleotide polymorphism (SNP)-related information is not always easy when searches must focus on complementary features. In this work, we present the Bovine SNP Retriever: a user-friendly tool for bovine SNP retrieval that also facilities the retrieval of SNP-related information within user-selected quantitative traits loci regions and reverse electronic polymerase chain reaction analysis on the bovine genome. The Bovine SNP Retriever is available at http://www.itb.cnr.it/ptp/bovine_snp_retriever/.     

New developments of the "lock-in" modified cycloSal-d4TMPs

New developments of the "lock-in" modified cycloSal-d4TMP are reported. Novel prodrugs with variations in the linker chain were introduced. The synthesis, hydrolysis properties in different media (PBS, CEM/0- and liver extracts) and the antiviral activities against HIV are shown.     

A "wringing" endorsement for myosin phosphorylation in the heart

On average, the human heart beats 100,000 times a day and it is in a person's best interest to have the heart move blood as efficiently as possible. For example, imagine a wet rag: squeezing the rag in your fist does not remove as much water as wringing the same rag between two hands. Thus, in hearts as in rags, torsional wringing, as opposed to squeezing, more thoroughly empties the heart of blood. Recent reports indicate that the activity and the distribution of myosin light chain kinase (MLCK) in the myocardium is key to this process. MLCK-dependent phosphorylation of the regulatory light chain (R-LC), a subunit of the myosin molecule, may lead to increased force and power of contraction. It is the asymmetric distribution of MLCK in the myocardium that allows for torsional wringing rather than squeezing. Specifically targeting MLCK expression in the heart might, in the future, lead to promising therapies that counteract cardiomyopathy.     

"Tissue" transglutaminase contributes to the formation of disulphide bridges in proteins of mitochondrial respiratory complexes

In this study we provide the first in vivo evidences showing that, under physiological conditions, "tissue" transglutaminase (TG2) might acts as a protein disulphide isomerase (PDI) and through this activity contributes to the correct assembly of the respiratory chain complexes. Mice lacking TG2 exhibit mitochondrial energy production impairment, evidenced by decreased ATP levels after physical challenge. This defect is phenotypically reflected in a dramatic decrease of motor behaviour of the animals. We propose that the molecular mechanism, underlying such a phenotype, resides in a defective disulphide bonds formation in ATP synthase (complex V), NADH-ubiquinone oxidoreductase (complex I), succinate-ubiquinone oxidoreductase (complex II) and cytochrome c oxidase (complex IV). In addition, TG2-PDI might control the respiratory chain by modulating the formation of the prohibitin complexes. These data elucidate a new pathway that directly links the TG2-PDI enzymatic activity with the regulation of mitochondrial respiratory chain function.     

Autocatalytic nature of "slow virus infections"

A concept that considers the causative nature of the so-called "slow virus infections", causing syndromes of spongiform encephalopathies in man and animals as a chain autocatalytic process is put forward. According to this concept, PrP(27-30) protein, isolated recently from the brains of scrapie-infected animals, is a C-terminal domain of the normal protein component of brain tissue which is a latent zimogen. Certain clinical and experimental data are discussed within the framework of this concept. Exogenous proteinases are presumed to be capable of triggering such a chain autocatalytic process in the brains of susceptible animals. Indeed, in one of our experiments, a subtoxic dose of pronase injected into mouse brain induced the development of a syndrome indistinguishable from spongiform encephalopathy in its clinical and pathomorphological manifestations. The probable role of neuron-specific proteins of intermediate filaments in such pathological processes is discussed. It seems possible that spongiform encephalopathies are particular cases of pathological processes that have catalytic nature. Presumably, the Alzheimer disease has such a catalytic causative nature.     

Application of "blue native" electrophoresis in the studies of mitochondrial respiratory chain complexes in physiology and pathology

"Blue Native" polyacrylamide gel electrophoresis (BN-PAGE), originally described by Sch?gger and von Jagow in 1991, is an elegant method to study protein complexes from mitochondrial membranes. BN-PAGE, commonly used in molecular biology to study composition of protein complexes and protein-protein interactions, enables separation of respiratory chain complexes keeping their properties and enzymatic activities unchanged. BN-PAGE, supplemented by other methods, e.g. in gel activity assay, SDS-PAGE (as a first or second dimension) can be successfully adapted for diagnosis of mitochondrial diseases connected with abnormalities of the respiratory chain. Therefore, to make a correct diagnosis of the deficiency of respiratory chain complexes, other methods, as histochemical colorimetric reactions allowing evaluation of the OXPHOS catalytic activity in individual cells and spectrophotometric technique should be used simultaneously with BN-PAGE.     

Far from equilibrium synthesis of "prebiotic" polymers

The existing experimental data on prebiotic nonenzymic biopolymer synthesis is used to construct a model which shows autocatalysis and highly nonlinear behavior. It is shown that far from thermodynamic equilibrium such systems may exhibit multiple steady states favoring the enhancement of the polymer population in the medium. The role of step by step chain propagation and template directed autocatalysis are studied and contrasted.     

"Oxygen regulation" of the respiratory chain composition in the yeast Debaryomyces hansenii under multiple stress

It was shown that two stress factors, hypoxia and hyperosmotic shock, if applied simultaneously to the yeast Debaryomyces hansenii, display an antagonistic mode of interaction, which results in an increased degree of halophily of this microorganism under microaerobic conditions. Studies of the effects of respiration inhibitors (sodium azide and salicyl hydroxamic acid, SHA) and of the pattern of changes in the composition of the respiratory chain of Debaryomyces hansenii under the stated stress conditions led to the suggestion of three (or four) chains of electron transfer functioning simultaneously in the cell: the classical respiratory chain involving cytochrome-c oxidase, an alternative respiratory chain involving a cyanide- and azide-resistant oxidase, and additional respiratory chains involving oxidases resistant to salt, azide and SHA. Thus, the antagonistic mode of interaction between hypoxia and hyperosmotic shock results from the redirection of the electron flow from the salt-susceptible respiratory systems to the salt-unsusceptible ones encoded by "the hypoxia genes" and activated (induced) under microaerobic conditions.     

An energetic evaluation of a "Smith" collagen microfibril model.

An energy minimized three-dimensional structure of a collagen microfibril template was constructed based on the five-stranded model of Smith (1968), using molecular modeling methods and Kollman force fields (Weiner and Kollman, 1981). For this model, individual molecules were constructed with three identical polypeptide chains ((Gly-Pro-Pro) _n , (Gly-Prop-Hyp) _n , or (Gly-Ala-Ala) _n , wheren=4, 12, and 16) coiled into a right-handed triple-helical structure. The axial distance between adjacent amino acid residues is about 0.29 nm per polypeptide chain, and the pitch of each chain is approximately 3.3 residues. The microfibril model consists of five parallel triple helices packed so that a left-handed superhelical twist exists. The structural characteristics of the computed microfibril are consistent with those obtained for collagen by X-ray diffraction and electron microscopy. The energy minimized Smith microfibril model for (Gly-Pro-Pro)₁₂ has an axial length of about 10.2 nm (for a 36 amino acid residue chain), which gives an estimated D-spacing (234 amino acids per chain) of approximately 66.2 nm. Studies of the microfibril models (Gly-Pro-Pro)₁₂, (Gly-Pro-Hyp)₁₂, and (Gly-Ala-Ala)₁₂ show that nonbonded van der Waals interactions are important for microfibril formation, while electrostatic interactions contribute to the stability of the microfibril structure and determine the specificity by which collagen molecules pack within the microfibril.     

A role for recombination in the production of "free-loader" lambda bacteriophage particles

Density-labeled crosses were performed with bacteriophage lambda under conditions which diminish DNA duplication. The production of viable phage containing fully conserved parental DNA was found to be dependent upon the action of the genetic recombination systems. The production of phage containing DNA with one newly synthesized chain was less dependent upon recombination. The production of phage with chromosomes both of whose chains were synthesized following infection show little, if any, dependence on recombination. One can speculate that some step in the maturation process of bacteriophage lambda is inseparable from the reduction of lambda DNA to the monomeric rods characteristic of lambda virions.     

Detection of the Cryptosporidium parvum "human" genotype in a dugong (Dugong dugon)

The Cryptosporidium "human" genotype was identified in a paraffin-embedded tissue section from a dugong (Dugong dugon) by 2 independent laboratories. DNA sequencing and polymerase chain reaction/restriction fragment length polymorphism analysis of the 18S ribosomal RNA gene and the acetyl CoA synthethase gene clearly identified the genotype as that of the Cryptosporidium variant that infects humans. This is the first report of the human Cryptosporidium genotype in a nonprimate host.     

"Portraying" of plant genomes using polymerase chain reaction amplification of ribosomal 5S genes.

Nontranscribed spacers of plant genes coding for ribosomal 5S RNA were amplified using the polymerase chain reaction. Primers were synthesized that were complementary to 3' (direct) and 5' (reverse) ends of the coding region and that are universal for higher plants. The patterns of polymerase chain reaction products are species and, sometimes, variety specific. The use of this approach for identification of barley 5S genes in chromosome-addition lines of wheat is discussed. This principle can be applied for the "portraying" of other tandem repetitive genes containing divergent regions.     

Prospects for realizing a structure of the spiral-turn-spiral type in a "zinc finger"

The realization of two models for Zn-finger structure--DNA-binding domain was estimated by taking into account the geometric and energetic requirements of polypeptide chain folding. The following structures were taken as models: antiparallel beta-structure and helix-turn-helix motif. The latter hasn't been considered earlier as the possible structure for Zn-finger. The preference was given to the above-mentioned models on the base of the available experimental data. It was shown that the helix-turn-helix motif for which the angle between axes of alpha-helices doesn't exceed 40 degrees cannot be excluded from the list of the assumed models for Zn-finger. There is a suggestion that the helix-turn-helix motif is likely to be characteristic for the majority of DNA-binding domains in proteins.     

A "helix-scissors" mechanism for the hinge-bending conformational change in phosphoglycerate kinase

X-ray studies of phosphoglycerate kinase (EC 2.7.2.3, PGK) have shown that the enzyme's single polypeptide chain is organized into two separate domains that correspond to the N- and C-terminal halves of the chain. Substrate binding studies and the incorporation of the complete amino acid sequence of horse-muscle PGK into its X-ray model suggest that the C-domain is an ADP/ATP binding unit and that the N-terminal domain contains the phosphoglycerate binding site and the active site located in a prominent cluster of positively charged residues. Because the distance between these two sites is 12-15 A, a hinge-bending of 10 degrees--20 degrees has been proposed to bring the two sites together for catalysis. Independent solution studies of yeast PGK have shown that the radius of gyration decreases significantly on the formation of the ternary complex. This change has been interpreted in terms of a 9 degrees--12 degrees rotation about a hinge in the interdomain region that brings the two domains together. We suggest here a structural basis for the proposed hinge-bending that involves the rotation of the two helices that form the domain interface about their contact normal carrying their respective domains with them.