Improving yeast two-hybrid screening systems.

Yeast two-hybrid (Y2H) screening methods are an effective means for the detection of protein-protein interactions. Optimisation and automation has increased the throughput of the method to an extent that allows the systematic mapping of protein-protein interactions on a proteome-wide scale. Since two-hybrid screens fail to detect a great number of interactions, parallel high-throughput approaches are needed for proteome-wide interaction screens. In this review, we discuss and compare different approaches for adaptation of Y2H screening to high-throughput, the limits of the method and possible alternative approaches to complement the mapping of organism-wide protein-protein interactions.     

The monoclonal antibody mAB 1A binds to the excitation--contraction coupling domain in the II-III loop of the skeletal muscle calcium channel alpha(1S) subunit

Interactions of the II-III loop of the voltage-gated Ca(2+) channel alpha(1S) subunit with the Ca(2+) release channel (RyR1) are essential for skeletal-type excitation-contraction (EC) coupling. Here, we characterized the binding site of the monoclonal alpha(1S) antibody mAB 1A and used it to probe the structure of the II-III loop in chimeras with different EC coupling properties. Phage-display epitope mapping of mAB 1A revealed a minimal consensus binding sequence X-P-X-X-D-X-P. Immunofluorescence labeling of (1S), alpha(1C), alpha(1D), and of II-III loop chimeras expressed in dysgenic myotubes established that mAB 1A reacted specifically with amino acids 737-744 in the II-III loop of alpha(1S), which is within the domain (D734-L764) critical for bidirectional coupling with RyR1. Comparing mAB 1A immunoreactivity with known structural and functional properties of II-III loop chimeras in which the non-conserved skeletal residues were systematically mutated to their cardiac counterparts indicated a correlation of mAB 1A immunoreactivity and skeletal-type EC coupling.     

Limitations of a two-step moment method for mapping linked multiple QTL

As an alternative to multiple-interval mapping a two-step moment method was recently proposed to map linked multiple quantitative trait loci (QTLs). The advantage of this moment method was supposed to be its simplicity and computational efficiency, especially in detecting closely linked QTLs within a marker bracket, but also in mapping QTLs in different marker intervals. Using simulations it is shown that the two-step moment method may give poor results compared with multiple-interval mapping, irrespective of whether the QTLs are in the same or in different marker intervals, especially if linked QTLs are in repulsion. The criteria of comparison are number of identified QTLs, likelihood ratio test statistics, means and empirical standard errors of the QTL position and QTL effects estimates, and the accuracy of the residual variance estimates. Further, the joint conditional probabilities of QTL genotypes for two putative QTLs within a marker interval were derived and compared with the unmodified approach ignoring the non-independence of the conditional probabilities.     

Efficient synthesis of ruthenium complexes of the type (R-bpy)2RuCl2 and [(R-bpy)2Ru(L-L)]Cl2 by microwave-activated reactions (R: H, Me, tert-But) (L-L: substituted bibenzimidazoles, bipyrimidine, and phenanthroline)

Complexes of the type (R-bpy)₂RuCl₂ (R: H, Me, tert-but) were synthesised by microwave-activated reactions of [Ru(cod)Cl₂]_n with substituted 2,2′-bipyridines in dimethylformamide as the solvent. The complexes were isolated in high yields and high purity from the reaction mixture. Microwave-assisted or thermal reaction of the (R-bpy)₂RuCl₂ solutions with substituted bibenzimidazoles, 1,10 phenanthroline or bipyrimidine in dmf/water mixtures resulted in the formation of mixed ligand complexes of the type [(R-bpy)₂Ru(L-L)]Cl₂. The complexes were characterised by NMR spectroscopy and MS. Furthermore, their photochemical and electrochemical properties were investigated and the solid state structure of (4-tert-butyl-bpy)₂RuCl₂ (3), [(4-tert-butyl-bpy)₂Ru(tetramethylbibenzimidazole)](PF₆)₂ (4), and [(4-tert-butyl-bpy)₂Ru(bipyrimidine] (PF₆)₂ (5) was determined by X-ray diffraction analysis of single crystals.     

Weak interionic interactions in 2-bromoimidazolium derivatives

The 2-bromoimidazolium bromide [ImBr]Br (5, Im=2,3-dihydro-1,3-diisopropyl-4,5-dimethylimidazol-2-ylidene) is prepared from Im (4) and bromine. From 4 and CBr₄, the adduct [ImBr]Br · CBr₄ (6) is obtained. 5 reacts with TeBr₄ to give the salt [ImBr][TeBr₅] (7). The crystal structures of 5-7 reveal the presence of weak interionic Br to Br interactions which are discussed on comparison with the structure of [ImI]I (8) and similar compounds.     

A Leishmania donovani gene that confers accelerated recovery from stationary phase growth arrest

We have isolated a gene, LdGF1, from the protozoan parasite Leishmania donovani. Overexpression of this gene confers a strong selective advantage in liquid culture after stationary phase growth arrest. We could show that recombinant L. donovani or Leishmania major, when overexpressing LdGF1, recover faster from a stationary phase growth arrest than control parasite strains. While no advantage of LdGF1 overexpression could be observed in log phase cultures or after a hydroxyurea-induced S-phase growth arrest, recovery from a cell cycle arrest due to serum deprivation was faster in LdGF1-overexpressing strains. This was found to be due to an accelerated release from a G₁ cell cycle arrest. By contrast, in a BALB/c mouse infection system, overexpression of LdGF1 in L. major resulted in reduced virulence. We conclude that increased levels of LdGF1 are beneficiary during recovery from G₁ cell cycle arrest, but pose a disadvantage inside a mammalian host. These results are discussed in the context of the observed loss of virulence during in vitro passage of Leishmania parasites.     

Tropomyosin exon 6b is troponin-specific and required for correct acto-myosin regulation

The specificity of tropomyosin (Tm) exon 6b for interaction with and functioning of troponin (Tn) has been studied using recombinant fibroblast Tm isoforms 5a and 5b. These isoforms differ internally by exons 6a/6b and possess non-muscle exons 1b/9d at the termini, hence they lack the primary TnT(1)-tropomyosin interaction, allowing study of exon 6 exchange in isolation from this. Using kinetic techniques to measure regulation of myosin S1 binding to actin and fluorescently labeled Tm to directly measure Tn binding, we show that binding of Tn to both isoforms is similar (0.1-0.5 microm) and both produce well regulated systems. Calcium has little effect on Tn binding to the actin.Tm complex and both exons produce a 3-fold reduction in the S1 binding rate to actin.Tm.Tn in its absence. This confirms previous results that show exon 6 has little influence on Tn affinity to actin.Tm or its ability to fully inhibit the acto-myosin interaction. Thin filaments reconstituted with Tn and Tm5a or skeletal Tm (containing exon 6b) show nearly identical calcium dependence of acto-myosin regulation. However, Tm5b produces a dramatic increase in calcium sensitivity, shifting the activation mid-point by almost an order of magnitude. This shows that exon 6 sequence and, hence, Tm structure in this region have a significant effect upon the calcium regulation of Tn. This finding supports evidence that familial hypertrophic cardiomyopathy mutations occurring adjacent to this region can effect calcium regulation.     

Disposition of calcium release units in agarose gel for an optimal propagation of Ca2+ signals

Clusters of calcium-loaded sarcoplasmic reticulum (SR) vesicles in agarose gel were previously shown to behave as an excitable medium that propagates calcium waves. In a 3D-hexagonal disposition, the distance between neighboring spheres (which may stand for SR vesicles) is constant and the relationship between distance and vesicular protein concentration is expected to be nonlinear. To obtain a distribution of SR vesicles at different protein concentrations as homogeneous as possible, liquid agarose gels were carefully stirred. Electron micrographs, however, did not confirm the expected relationship between inter-SR vesicle distance and vesicular protein concentration. Light micrographs, to the contrary, resulted in a protein concentration-dependent disposition of clusters of SR vesicles, which is described by a linear function. Stable calcium waves in agarose gel occurred at SR vesicle protein concentrations between 7 and 16 g/l. At lower protein concentrations, local calcium oscillations or abortive waves were observed. The velocities of calcium waves were optimum at approximately 12 g/l and amounted to nearly 60 microm/s. The corresponding distance of neighboring calcium release units was calculated to be approximately 4 microm. The results further show that calcium signaling in the described reaction-diffusion system is optimal in a relatively small range of diffusion lengths. A change by +/-2 microm resulted in a reduction of the propagation velocity by 40%. It would appear that 1), the distance between calcium release units (clusters of ryanodine receptors in cells) is a sensitive parameter concerning propagation of Ca2+ signals; and 2), a dysfunction of the reaction-diffusion system in living cells, however, might have a negative effect on the spreading of intracellular calcium signals, thus on the cell's function.     

Physical and functional interaction of the Werner syndrome protein with poly-ADP ribosyl transferase

Werner's syndrome is a rare disease of premature ageing. The WRN gene product defective in this disorder belongs to the RecQ helicase family and is thought to be involved in DNA metabolism. Another protein, which plays an important role in both DNA replication and repair, is the poly-ADP ribosyl transferase. Here we demonstrate an interaction of these two proteins resulting in ADP-ribosylation of the WRN protein. These results imply that WRN is involved in DNA replication and in DNA repair.