Variance Heterogeneity in Saccharomyces cerevisiae Expression Data: Trans-Regulation and Epistasis

Here, we describe the results from the first variance heterogeneity Genome Wide Association Study (VGWAS) on yeast expression data. Using this forward genetics approach, we show that the genetic regulation of gene-expression in the budding yeast, Saccharomyces cerevisiae, includes mechanisms that can lead to variance heterogeneity in the expression between genotypes. Additionally, we performed a mean effect association study (GWAS). Comparing the mean and variance heterogeneity analyses, we find that the mean expression level is under genetic regulation from a larger absolute number of loci but that a higher proportion of the variance controlling loci were trans-regulated. Both mean and variance regulating loci cluster in regulatory hotspots that affect a large number of phenotypes; a single variance-controlling locus, mapping close to DIA2, was found to be involved in more than 10% of the significant associations. It has been suggested in the literature that variance-heterogeneity between the genotypes might be due to genetic interactions. We therefore screened the multi-locus genotype-phenotype maps for several traits where multiple associations were found, for indications of epistasis. Several examples of two and three locus genetic interactions were found to involve variance-controlling loci, with reports from the literature corroborating the functional connections between the loci. By using a new analytical approach to re-analyze a powerful existing dataset, we are thus able to both provide novel insights to the genetic mechanisms involved in the regulation of gene-expression in budding yeast and experimentally validate epistasis as an important mechanism underlying genetic variance-heterogeneity between genotypes.     

Proteolytic Processing Causes Extensive Heterogeneity of Tissue Matrilin Forms

The matrilins are a family of multidomain extracellular matrix proteins with adapter functions. The oligomeric proteins have a bouquet-like structure and bind to a variety of different ligands whereby the avidity of their interactions is dependent on the number of subunits and domains present. Here we show the contribution of post-translational proteolytic processing to the heterogeneity of matrilins seen in tissue extracts and cell culture supernatants. A cleavage site after two glutamate residues in the hinge region close to the C-terminal coiled-coil oligomerization domain is conserved among the matrilins. Cleavage at this site yields molecules that lack almost complete subunits. The processing is least pronounced in matrilin-1 and particularly complex in matrilin-2, which contains additional cleavage sites. Replacement of the hinge region in matrilin-4 by the matrilin-1 hinge region had no marked effect on the processing. A detailed study revealed that matrilin-4 is processed already in the secretory pathway and that the activation of the responsible enzymes is dependent on proprotein convertase activity. Matrilin-3 and -4, but not matrilin-1 subunits present in matrilin-1/-3 hetero-oligomers, were identified as substrates for ADAMTS4 and ADAMTS5, whereas ADAMTS1 did not cleave any matrilin. A neo-epitope antibody raised against the N terminus of the C-terminal cleavage product of matrilin-4 detected processed matrilin-4 in cultures of primary chondrocytes as well as on cartilage sections showing that the conserved cleavage site is used in vivo.The matrilins form a four-member family of modular, multisubunit matrix proteins, which are expressed in cartilage and many other forms of extracellular matrix (for review, see Ref. 1). They participate in the formation of fibrillar or filamentous structures (2–7) and mediate interactions between collagen-containing fibrils (8, 9) and other matrix constituents like aggrecan (10), small leucine-rich proteoglycans (9), or COMP (11). Matrilins form homo- and hetero-oligomers by their C-terminal coiled-coil domain. In addition, the subunits contain epidermal growth factor-like and von Willebrand factor A (VWA)²-like domains, where the latter are presumably the major ligand binding domains (11). Mutations in matrilin-3 in humans cause different forms of chondrodysplasia (12–14) and are also linked to the development of hand osteoarthritis (15) and intervertebral disc degeneration (16).Proteolytic processing of extracellular matrix proteins plays both physiological and pathophysiological roles. Proteolysis is a major post-translational modification used to modify the function of proteins. Tissue homeostasis requires a well balanced synthesis and degradation of extracellular matrix proteins, specifically mediated by protease families like matrix metalloproteinases (17), ADAMs (18), or ADAMTSs (19). The development of degenerative diseases is often accompanied by an activation of such proteases. In addition, the cleavage sometimes releases protein fragments that have completely new functions (20, 21).Determination of which extracellular proteases cleave which substrates is crucial to understand the physiological function of both (22). Physiological cleavage has been described for most members of the matrilin family (4–6), but was not yet extensively studied. The adapter function of the matrilins may be modulated by physiological proteolysis that causes the loss of single subunits and thereby decreases the binding avidity (5). Interestingly, an earlier identified cleavage site in the hinge region of matrilin-4, N-terminal of the coiled-coil, is conserved throughout the matrilin family (5) and it was recently shown that matrilin-3 is cleaved by ADAMTS4 in vitro at this site (23). Here we studied matrilin processing in some detail and identified another member of the ADAMTS family, ADAMTS5, as being able to cleave matrilin-3 and -4. Such cleavage is likely to alter the cohesion of the extracellular matrix.     

Size and structure characterization of ethylhydroxyethyl cellulose by the combination of field-flow fractionation with other techniques. Investigation of ultralarge components

Ethylhydroxyethyl cellulose (EHEC) of three different viscosity classes (EHEC I, II, and III) was analyzed by programmed cross-flow asymmetrical flow field-flow fractionation coupled to multiangle light scattering and refractive index detectors to determine their size and molar mass distribution. Two size populations were detected in the two lower viscosity classes, EHEC I and II, one high molar mass and one ultrahigh molar mass (UHM). The two covered molar masses from 10(4) up to 10(9) g X mol(-1). The highest viscosity class EHEC III was less size-dispersed covering molar masses from 5 x 10(5) to 5 x 10(7) g.mol(-1). Filtering of the EHEC II solution removed small amounts of compact UHM material. Enzyme treatments were performed on EHEC II to further characterize it. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and anion ion-exchange chromatography coupled to pulsed amperometric detection showed that the UHM component contained EHEC.     

Heterogeneity of Collagen VI Microfibrils: STRUCTURAL ANALYSIS OF NON-COLLAGENOUS REGIONS*

Collagen VI, a collagen with uncharacteristically large N- and C-terminal non-collagenous regions, forms a distinct microfibrillar network in most connective tissues. It was long considered to consist of three genetically distinct α chains (α1, α2, and α3). Intracellularly, heterotrimeric molecules associate to form dimers and tetramers, which are then secreted and assembled to microfibrils. The identification of three novel long collagen VI α chains, α4, α5, and α6, led to the question if and how these may substitute for the long α3 chain in collagen VI assembly. Here, we studied structural features of the novel long chains and analyzed the assembly of these into tetramers and microfibrils. N- and C-terminal globular regions of collagen VI were recombinantly expressed and studied by small angle x-ray scattering (SAXS). Ab initio models of the N-terminal globular regions of the α4, α5, and α6 chains showed a C-shaped structure similar to that found for the α3 chain. Single particle EM nanostructure of the N-terminal globular region of the α4 chain confirmed the C-shaped structure revealed by SAXS. Immuno-EM of collagen VI extracted from tissue revealed that like the α3 chain the novel long chains assemble to homotetramers that are incorporated into mixed microfibrils. Moreover, SAXS models of the C-terminal globular regions of the α1, α2, α4, and α6 chains were generated. Interestingly, the α1, α2, and α4 C-terminal globular regions dimerize. These self-interactions may play a role in tetramer formation.     

Crystallinity and morphology in films of starch, amylose and amylopectin blends

Films of potato starch, amylose, and amylopectin and blends thereof were prepared by solution casting and examined using X-ray diffraction, light microscopy, transmission electron microscopy, and differential scanning calorimetry. Amylose films had a relative crystallinity of about 30% whereas amylopectin films were entirely amorphous. Blending of amylose and amylopectin resulted in films with a considerably higher degree of crystallinity than could be predicted. This is explained by cocrystallization between amylose and amylopectin and possibly by crystallization of amylopectin. The crystallized material gave rise to an endotherm detected with differential scanning calorimetry. The enthalpy and peak temperature of the transition also increased as the water content decreased. When the amylose proportion in the blends was low, separate phases of amylose and amylopectin were observed by light microscopy. At higher amylose proportions, however, the phase separation was apparently prevented by amylose gelation and the formation of a continuous amylose network. The amylose network in the films, observed with transmission electron microscopy, consisted of stiff strands and open pores and became less visible as the amylose proportion decreased. The water content of the films was dependent on the microstructure and the crystallinity.     

Catalytic convergence of manganese and iron lipoxygenases by replacement of a single amino Acid

Lipoxygenases (LOXs) contain a hydrophobic substrate channel with the conserved Gly/Ala determinant of regio- and stereospecificity and a conserved Leu residue near the catalytic non-heme iron. Our goal was to study the importance of this region (Gly(332), Leu(336), and Phe(337)) of a lipoxygenase with catalytic manganese (13R-MnLOX). Recombinant 13R-MnLOX oxidizes 18:2n-6 and 18:3n-3 to 13R-, 11(S or R)-, and 9S-hydroperoxy metabolites (～80-85, 15-20, and 2-3%, respectively) by suprafacial hydrogen abstraction and oxygenation. Replacement of Phe(337) with Ile changed the stereochemistry of the 13-hydroperoxy metabolites of 18:2n-6 and 18:3n-3 (from ～100% R to 69-74% S) with little effect on regiospecificity. The abstraction of the pro-S hydrogen of 18:2n-6 was retained, suggesting antarafacial hydrogen abstraction and oxygenation. Replacement of Leu(336) with smaller hydrophobic residues (Val, Ala, and Gly) shifted the oxygenation from C-13 toward C-9 with formation of 9S- and 9R-hydroperoxy metabolites of 18:2n-6 and 18:3n-3. Replacement of Gly(332) and Leu(336) with larger hydrophobic residues (G332A and L336F) selectively augmented dehydration of 13R-hydroperoxyoctadeca-9Z,11E,15Z-trienoic acid and increased the oxidation at C-13 of 18:1n-6. We conclude that hydrophobic replacements of Leu(336) can modify the hydroperoxide configurations at C-9 with little effect on the R configuration at C-13 of the 18:2n-6 and 18:3n-3 metabolites. Replacement of Phe(337) with Ile changed the stereospecific oxidation of 18:2n-6 and 18:3n-3 with formation of 13S-hydroperoxides by hydrogen abstraction and oxygenation in analogy with soybean LOX-1.     

Prime-boost vaccination with rBCG/rAd35 enhances CD8⁺ cytolytic T-cell responses in lesions from Mycobacterium tuberculosis-infected primates

To prevent the global spread of tuberculosis (TB) infection, a novel vaccine that triggers potent and long-lived immunity is urgently required. A plasmid-based vaccine has been developed to enhance activation of major histocompatibility complex (MHC) class I–restricted CD8+ cytolytic T cells using a recombinant Bacille Calmette-Guérin (rBCG) expressing a pore-forming toxin and the Mycobacterium tuberculosis (Mtb) antigens Ag85A, 85B and TB10.4 followed by a booster with a nonreplicating adenovirus 35 (rAd35) vaccine vector encoding the same Mtb antigens. Here, the capacity of the rBCG/rAd35 vaccine to induce protective and biologically relevant CD8⁺ T-cell responses in a nonhuman primate model of TB was investigated. After prime/boost immunizations and challenge with virulent Mtb in rhesus macaques, quantification of immune responses at the single-cell level in cryopreserved tissue specimen from infected organs was performed using in situ computerized image analysis as a technological platform. Significantly elevated levels of CD3⁺ and CD8⁺ T cells as well as cells expressing interleukin (IL)-7, perforin and granulysin were found in TB lung lesions and spleen from rBCG/rAd35-vaccinated animals compared with BCG/rAd35-vaccinated or unvaccinated animals. The local increase in CD8⁺ cytolytic T cells correlated with reduced expression of the Mtb antigen MPT64 and also with prolonged survival after the challenge. Our observations suggest that a protective immune response in rBCG/rAd35-vaccinated nonhuman primates was associated with enhanced MHC class I antigen presentation and activation of CD8+ effector T-cell responses at the local site of infection in Mtb-challenged animals.     

Metabolism and effect of prostaglandin H2 in adipose tissue.

Prostaglandin H2 (PGH2) inhibited noradrenaline induced cyclic AMP accumulation in isolated rat fat cells in a dose-dependent manner. IC50 was 10-25 ng/ml both in the absence and in the presence of theophylline. The degree of inhibition produced by PGH2 increased with time of incubation. A stable PGH2 analog did not inhibit cyclic AMP accumulation. PGH2 was rapidly converted by isolated fat cells to PGD2, PGE2 and PGF2alpha' but no formation of thromboxane B2 was found either in vitro or in vivo. PGE2 was a more potent inhibitor than PGH2 of noradrenaline induced cyclic AMP accumulation. PGD2 enhanced cyclic AMP accumulation in a limited concentration interval, while PGF2alpha was essentially uneffective. Our results suggest that PGH2 is an inhibitor of cyclic AMP formation in isolated rat fat cells only after conversion to PGE2. A physiological role for PGH2 as a modulator of lipolysis is considered unlikely.     

Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis

The intracellular bacterium Francisella tularensis is the causative agent of tularemia and poses a serious threat as an agent of bioterrorism. We have developed a highly effective molecular subtyping system from 25 variable-number tandem repeat (VNTR) loci. In our study, multiple-locus VNTR analysis (MLVA) was used to analyze genetic relationships and potential population structure within a global collection of 192 F. tularensis isolates, including representatives from each of the four subspecies. The VNTR loci displayed between 2 and 31 alleles with Nei's diversity values between 0.05 and 0.95. Neighbor-joining cluster analysis of VNTR data revealed 120 genotypes among the 192 F. tularensis isolates, including accurate subspecies identification. F. tularensis subsp. tularensis (type A) isolates showed great diversity at VNTR loci, while F. tularensis subsp. holarctica (type B) isolates showed much lower levels despite a much broader geographical prevalence. The resolution of two distinct clades within F. tularensis subsp. tularensis (designated A.I and A.II) revealed a previously unrecognized genetic division within this highly virulent subspecies. F. tularensis subsp. holarctica appears to have recently spread globally across continents from a single origin, while F. tularensis subsp. tularensis has a long and complex evolutionary history almost exclusively in North America. The sole non-North American type A isolates (Slovakian) were closely related to the SCHU S4 strain. Significant linkage disequilibrium was detected among VNTR loci of F. tularensis consistent with a clonal population structure. Overall, this work greatly augments the study of tularemia ecology and epidemiology, while providing a framework for future forensic analysis of F. tularensis isolates.     

ProbeMaker: an extensible framework for design of sets of oligonucleotide probes

Background  

Procedures for genetic analyses based on oligonucleotide probes are powerful tools that can allow highly parallel investigations of genetic material. Such procedures require the design of large sets of probes using application-specific design constraints.