Rational design of an active avidin monomer

Homotetrameric chicken avidin that binds four molecules of biotin was converted to a monomeric form (monoavidin) by mutations of two interface residues: tryptophan 110 in the 1 --> 2 interface was mutated to lysine and asparagine 54 in the 1 --> 4 interface was converted to alanine. The affinity for biotin binding of the mutant decreased from K(d) approximately 10(-15) m of the wild-type tetramer to K(d) approximately 10(-7) m, which was studied by an optical biosensor IAsys and by a fluorescence spectroscopical method in solution. The binding was completely reversible. Conversion of the tetramer to a monomer results in increased sensitivity to proteinase K digestion. The antigenic properties of the mutated protein were changed, such that monoavidin was only partially recognized by a polyclonal antibody whereas two different monoclonal antibodies entirely failed to recognize the avidin monomer. This new monomeric avidin, which binds biotin reversibly, may be useful for applications both in vitro and in vivo. It may also shed light on the effect of intersubunit interactions on the binding of ligands.     

An unknown mechanism promotes somatic incompatibility in Ceratobasidium bicorne

Strains of Ceratobasidium bicorne (anamorph uninucleate Rhizoctonia), causing root dieback in nursery-grown conifer seedlings, were fruited in the laboratory and the pairing interactions among sibling, single-basidiospore progeny were investigated. No mating reactions were observed. Instead, a high frequency of somatic incompatibility was observed in progeny pairings, indicated by a killing reaction in hyphal anastomosis and by formation of a demarcation line. The F1 progeny also could be fruited, and the level of somatic incompatibility within the F2 progeny remained high, even if lower than in the F1 progeny. The interaction types in pairings within a family of progeny were similar in all respects to those between field isolates, indicating that the species is homothallic. The uninucleate condition of vegetative cells and the basidial characteristics would indicate homokaryotic fruiting, but the possibility of pseudohomothallism remains. We currently are not able to provide an explanation for the mechanism promoting somatic incompatibility in this species, but it seems likely that the classic heterogenic model of somatic incompatibility recognized in basidiomycetes is not applicable here. Alternative mechanisms are discussed.     

Localization of a gene for peripheral arterial occlusive disease to chromosome 1p31

Peripheral arterial occlusive disease (PAOD) results from atherosclerosis of large and medium peripheral arteries, as well as the aorta, and has many risk factors, including smoking, diabetes, hypertension, and hyperlipidemia. PAOD often coexists with coronary artery disease and cerebrovascular disease. Cross-matching a population-based list of Icelandic patients with PAOD who had undergone angiography and/or revascularization procedures with a genealogy database of the entire Icelandic nation defined 116 extended families containing 272 patients. A genomewide scan with microsatellite markers revealed significant linkage to chromosome 1p31 with an allele-sharing LOD score of 3.93 (P=1.04 x 10(-5)). We designate this locus as "PAOD1." Subtracting 35 patients with a history of stroke increased the LOD score to 4.93. This suggests that, although PAOD and other vascular diseases share risk factors, genetic factors specific to subtypes of vascular disease may exist.     

Cell lineage dynamics in stratified shoot apical meristems

We devise a stochastic and spatially explicit model for the dynamics of the initials cells in a stratified shoot apical meristem (SAM). The meristem is composed of three layers with seven initials per layer. We investigate the probability and number of divisions for a mutant lineage to either reach fixation or becoming purged through selection or drift. In contrast to previous studies our results show that the functional organization of the initials in stratified SAMs acts as an efficient purging mechanism particularly of deleterious mutations. All mutants are rapidly purged when deleterious. The probability of fixation for mutants with a higher fitness than the wild type increases linearly up to 70%. The median number of divisions to fixation of both genotypes is insensitive to the mutant's fitness. The median number of divisions to wild-type fixation is less than 100, with the upper quartile below 200. The largest number of divisions to wild-type fixation are in the order of 100 000 divisions. Our results indicate that the spatial organization of SAM enables the efficient purging of mutant lineages, particularly if they are deleterious. On the other hand, long-lived chimeric stages are common when mutant lineages succeed to overcome the initial numerical disadvantage.     

Degradation of humic acids by the litter-decomposing basidiomycete Collybia dryophila

The basidiomycete Collybia dryophila K209, which colonizes forest soil, was found to decompose a natural humic acid isolated from pine-forest litter (LHA) and a synthetic (14)C-labeled humic acid ((14)C-HA) prepared from [U-(14)C]catechol in liquid culture. Degradation resulted in the formation of polar, lower-molecular-mass fulvic acid (FA) and carbon dioxide. HA decomposition was considerably enhanced in the presence of Mn(2+) (200 microM), leading to 75% conversion of LHA and 50% mineralization of (14)C-HA (compared to 60% and 20%, respectively, in the absence of Mn(2+)). There was a strong indication that manganese peroxidase (MnP), the production of which was noticeably increased in Mn(2+)-supplemented cultures, was responsible for this effect. The enzyme was produced as a single protein with a pI of 4.7 and a molecular mass of 44 kDa. During solid-state cultivation, C. dryophila released substantial amounts of water-soluble FA (predominantly of 0.9 kDa molecular mass) from insoluble litter material. The results indicate that basidiomycetes such as C. dryophila which colonize forest litter and soil are involved in humus turnover by their recycling of high-molecular-mass humic substances. Extracellular MnP seems to be a key enzyme in the conversion process.     

Correlative motions and memory effects in molecular dynamics simulations of molecules: principal components and rescaled range analysis suggest that the motions of native BPTI are more correlated than those of its mutants

In this work MD simulations of the native bovine pancreatic trypsin inhibitor (BPTI) and 16 mutants were done in vacuum in order to study memory effects in the mutants using principal component analysis (PCA) and the rescaled range analysis (Hurst exponents). Both PCA and the rescaled range analysis support our previous proposition, based on PCA of lysozyme, that the motions of a native protein are more correlated than those of mutants. The methods are compared, the nature and applications of the rule and the role of the long-range correlations in MD time series (i.e. memory) are discussed in the context of collective motions.     

<Emphasis Type="Italic">In silico</Emphasis>discovery of gene-coding variants in murine quantitative trait loci using strain-specific genome sequence databases

Background  

The identification of genes underlying complex traits has been aided by quantitative trait locus (QTL) mapping approaches, which in turn have benefited from advances in mammalian genome research. Most recently, whole-genome draft sequences and assemblies have been generated for mouse strains that have been used for a large fraction of QTL mapping studies. Here we show how such strain-specific mouse genome sequence databases can be used as part of a high-throughput pipeline for the in silico discovery of gene-coding variations within murine QTLs. As a test of this approach we focused on two QTLs on mouse chromosomes 1 and 13 that are involved in physical dependence on alcohol.     

Altered affinity of CBF beta-SMMHC for Runx1 explains its role in leukemogenesis

Chromosomal translocations involving the human CBFB gene, which codes for the non-DNA binding subunit of CBF (CBF beta), are associated with a large percentage of human leukemias. The translocation inv(16) that disrupts the CBFB gene produces a chimeric protein composed of the heterodimerization domain of CBF beta fused to the C-terminal coiled-coil domain from smooth muscle myosin heavy chain (CBF beta-SMMHC). Isothermal titration calorimetry results show that this fusion protein binds the Runt domain from Runx1 (CBF alpha) with higher affinity than the native CBF beta protein. NMR studies identify interactions in the CBF beta portion of the molecule, as well as the SMMHC coiled-coil domain. This higher affinity provides an explanation for the dominant negative phenotype associated with a knock-in of the CBFB-MYH11 gene and also helps to provide a rationale for the leukemia-associated dysregulation of hematopoietic development that this protein causes.     

Characterization of three fragments that constitute the monomers of the human lysyl hydroxylase isoenzymes 1-3. The 30-kDa N-terminal fragment is not required for lysyl hydroxylase activity

Lysyl hydroxylase (LH) catalyzes the formation of hydroxylysine in collagens; three human isoenzymes have been cloned so far. We report here on the purification of all three recombinant isoenzymes to homogeneity from the medium of cultured insect cells, and we demonstrate that they are all homodimers. Limited proteolysis experiments identified two main protease-sensitive regions in the monomers of about 80-85 kDa, corresponding to three fragments A-C (from the N to C terminus), with molecular masses of about 30, 37, and 16 kDa, respectively. Fragment A was found to play no role in LH activity as a recombinant B-C polypeptide constituted a fully active hydroxylase with K(m) values for cosubstrates and the peptide substrate that were identical to those of the full-length enzyme. LH3, but not LH1 and LH2, has also been reported recently (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., and Myllyl?, R. (2000) J. Biol. Chem. 275, 36158-36163) to possess collagen glucosyltransferase activity. We confirm this highly surprising finding here and extend it by demonstrating that LH3 may also possess trace amounts of collagen galactosyltransferase activity. All the glucosyltransferase and galactosyltransferase activity of LH3 was found to reside in fragment A, which played no role in the hydroxylase activity of the polypeptide. This fragment is about 55% identical and 80% similar to the corresponding fragments of LH1 and LH2. However, the levels of the glycosyltransferase activities are so low that they may be of little biological significance. It is thus evident that human tissues must have additional glycosyltransferases that are responsible for most of the collagen glycosylation in vivo.