The aim of this work is to find semi-rigid domains within large proteins as reference structures for fitting molecular dynamics trajectories. We propose an algorithm, multistage consensus clustering, MCC, based on minimum variation of distances between pairs of Cα-atoms as target function. The whole dataset (trajectory) is split into sub-segments. For a given sub-segment, spatial clustering is repeatedly started from different random seeds, and we adopt the specific spatial clustering with minimum target function: the process described so far is stage 1 of MCC. Then, in stage 2, the results of spatial clustering are consolidated, to arrive at domains stable over the whole dataset. We found that MCC is robust regarding the choice of parameters and yields relevant information on functional domains of the major histocompatibility complex (MHC) studied in this paper: the α-helices and β-floor of the protein (MHC) proved to be most flexible and did not contribute to clusters of significant size. Three alleles of the MHC, each in complex with ABCD3 peptide and LC13 T-cell receptor (TCR), yielded different patterns of motion. Those alleles causing immunological allo-reactions showed distinct correlations of motion between parts of the peptide, the binding cleft and the complementary determining regions (CDR)-loops of the TCR. Multistage consensus clustering reflected functional differences between MHC alleles and yields a methodological basis to increase sensitivity of functional analyses of bio-molecules. Due to the generality of approach, MCC is prone to lend itself as a potent tool also for the analysis of other kinds of big data.

A key role in cell-mediated immunity is dedicated to the major histocompatibility complex (MHC) molecules that bind peptides for presentation on the cell surface. Several in silico methods capable of predicting peptide binding to MHC class I have been developed. The accuracy of these methods depends on the data available characterizing the binding specificity of the MHC molecules. It has, moreover, been demonstrated that consensus methods defined as combinations of two or more different methods led to improved prediction accuracy. This plethora of methods makes it very difficult for the non-expert user to choose the most suitable method for predicting binding to a given MHC molecule. In this study, we have therefore made an in-depth analysis of combinations of three state-of-the-art MHC-peptide binding prediction methods (NetMHC, NetMHCpan and PickPocket). We demonstrate that a simple combination of NetMHC and NetMHCpan gives the highest performance when the allele in question is included in the training and is characterized by at least 50 data points with at least ten binders. Otherwise, NetMHCpan is the best predictor. When an allele has not been characterized, the performance depends on the distance to the training data. NetMHCpan has the highest performance when close neighbours are present in the training set, while the combination of NetMHCpan and PickPocket outperforms either of the two methods for alleles with more remote neighbours. The final method, NetMHCcons, is publicly available at www.cbs.dtu.dk/services/NetMHCcons , and allows the user in an automatic manner to obtain the most accurate predictions for any given MHC molecule.

House mice (Mus domesticus) communicate using scent-marks, and the chemical and microbial composition of these \'extended phenotypes\' are both influenced by genetics. This study examined how the genes of the major histocompatibility complex (MHC) and background genes influence the volatile compounds (analysed with Gas Chromatography Mass Spectrometry or GC/MS) and microbial communities (analysed using Denaturating Gradient Gel Electrophoresis or DGGE) in scent-marks produced by congenic strains of mice. The use of Consensus Principal Components Analysis is described and shows relationships between the two types of fingerprints (GC/MS and DGGE profiles). Classification methods including Support Vector Machines and Discriminant Partial Least Squares suggest that mice can be classified according to both background strain and MHC-haplotype. As expected, the differences among the mice were much greater between strains that vary at both MHC and background loci than the congenics, which differ only at the MHC. These results indicate that the volatiles in scent-marks provide information about genetic similarity of the mice, and support the idea that the production of these genetically determined volatiles is influenced by commensal microflora. This paper describes the application of consensus methods to relate two blocks of analytical data.

Mice expressing the H2b haplotype are resistant to infection with Theiler\'s murine encephalomyelitis virus (TMEV), which causes chronic demyelination in susceptible mice. The prominent cytolytic T-lymphocyte (CTL) response to the VP2 antigen encoded by TMEV led us to the identification of a class I-binding peptide derived from the VP2 antigen. Escherichia coli transformants overexpressing a series of 11 overlapping VP2 protein fragments were subjected to lysis and alkali digestion, and the resultant peptide pools were tested for their abilities to sensitize RMA-S targets for lysis by CTLs. The source of effector CD8+ T cells for the assays was either freshly harvested central nervous system-infiltrating lymphocytes (CNS-IL) or CNS-IL-derived VP2-specific CTL clones and lines. A 10-residue peptide at VP2 positions 121 to 130 (VP2(121-130)) (FHAGSLLVFM) was identified that sensitized targets for lysis and formed stable complexes with H2Db class I molecules. The VP2(121-130) peptide sensitized target cells for lysis by freshly harvested CNS-IL CTLs at femtomolar concentrations. Despite its relative high level of biological activity, the VP2(121-130) peptide is distinguished from other Db-binding peptides by its lack of an asparagine residue at position five, which had been previously proposed to be a requirement for Db-peptide complexing.

Interferons (IFNs) augment expression of major histocompatibility class I genes in many cells. To study the effect of IFNs on transcription of class I genes, we prepared and tested activity of chloramphenicol acetyltransferase (CAT) hybrid genes in which the cat gene is under the control of the 5\' flanking region of the murine H-2Ld gene. NIH 3T3 cells transiently transfected with a cat construct having the sequence from position -210 to -134 showed a four- to fivefold increase in CAT activity when treated with IFN-alpha/beta. This sequence contains the IFN consensus sequence (ICS) shared among IFN-inducible genes, as well as the class I regulatory element (CRE) that controls up and down regulation of class I gene expression. To determine the precise sequence requirement for the IFN action, the ICS and CRE were independently placed upstream of the class I or a heterologous simian virus 40 promoter, and CAT activity was tested. The ICS, but not the CRE, enhanced activity of both promoters by about twofold upon exposure to IFN-alpha/beta, although greater responses were noted when the ICS and CRE were combined. These results demonstrate that the ICS alone is capable of enhancing promoter activity in response to IFN-alpha/beta treatment and that the CRE exerts a synergistic effect. Further, we show that the ICS functions as an inducible enhancer since it acts regardless of its orientation and distance in the simian virus 40 promoter.

IFN-gamma induces the expression of the MHC class II-associated invariant chain (IN) protein in a variety of cells of nonlymphoid origin. Here we analyze the transcription from the murine invariant chain gene and delimit the cis-acting sequences which confer IFN-gamma responsiveness in human fibroblasts. The major start of transcription of the gene is located 29 bp 3\' of a TATA box and 85 bp 5\' of the single ATG codon which opens the reading frame. To identify the regulatory elements of the murine IN promoter which respond to IFN-gamma, the 5\' flanking region of the gene including its capsite and 85 bp of coding region have been cloned in front of the bacterial chloramphenicol acetyl transferase (CAT) gene. Examination of this construct and various 5\' and 3\' deletion mutants for IFN-gamma inducibility in transient transfection assays revealed that DNA sequences between -261 and -189 were essential and sufficient for the induction. Removal of sequences between -215 and -189 reduced inducibility of the IN-promoter and abolished the capacity of the element to transmit inducibility to a heterologous promoter. Single or multiple base changes in other parts of the element also abolished inducibility. Cotransfection of a 350 molar excess of the IFN-gamma response element with an inducible IN-CAT chimeric construct blocked inducibility, suggesting positive regulation. A protein binding to the central part of the IFN-gamma response element was detectable in gel retardation experiments; it was active only in extracts from IFN-gamma-treated cells.

Class II MHC proteins play central roles in controlling immune cell repertoire and responses. These roles depend on precise regulation of the level and cell-type specificity of class II gene expression. Instances of both coordinate and non-coordinate regulation of the multiple class II genes have been described. A 1.3 kb region of the class II MHC gene A alpha has previously been shown to activate transcription in a cell-type specific fashion that correlated with the expression of A alpha. The mouse A alpha gene differs from other class II MHC genes in that its conserved X region also contains the CRE/ATF DNA motif TGACGTCA. Substitution mutations were introduced into the 1.3 kb region such that the CRE/ATF (X2) motif was altered, but not the adjacent X1 or Y box motifs. Controls confirmed that these mutations eliminated the binding of nuclear proteins to the CRE/ATF motif and reduced transcriptional activity as much as mutation of the Y box. In addition, a new positive transcription element was identified far upstream from the conserved X-Y region, centered on position -970. The sequence of this region does not resemble previously described transcription elements or other MHC class II 5\' flanking sequences. The activity of this element was absolutely dependent on the presence of the X-Y region. These data are most consistent with a model in which functionally important sequences unique to a single class II MHC gene can be intimately interposed between conserved MHC transcription elements, and non-consensus elements upstream from the conserved region contribute to control of A alpha.