The HCP5 single-nucleotide polymorphism: a simple screening tool for prediction of hypersensitivity reaction to abacavir

The HLA-B 5701 allele is predictive of hypersensitivity reaction to abacavir, a response herein termed "ABC-HSR." This study of 1,103 individuals infected with human immunodeficiency virus assessed the usefulness of genotyping a HCP5 single-nucleotide polymorphism (SNP), rs2395029, in relation to ABC-HSR. In populations with European ancestry, rs2395029 is in linkage disequilibrium with HLA-B 5701. The HCP5 SNP was present in all 98 HLA-B 5701-positive individuals and was absent in 999 of 1005 HLA-B 5701-negative individuals. rs2395029 was overrepresented in 25 individuals with clinically likely ABC-HSR, compared with its frequency in 175 ABC-tolerant individuals (80% vs. 2%, respectively; P < .0001). Therefore, HCP5 genotyping could serve as a simple screening tool for ABC-HSR, particularly in settings where sequence-based HLA typing is not available.

A Simple Algorithm for Unique Representation of Chemical Structures - Cyclic/ Acyclic Functionalized Achiral Hydrocarbons

An algorithm, based on ‘vertex priority values’ has been proposed to uniquely sequence and represent connectivity matrix of chemical structures of cyclic/ acyclic functionalized achiral hydrocarbons and their derivatives. In this method ‘vertex priority values’ have been assigned in terms of atomic weights, subgraph lengths, loops, and heteroatom contents. Subsequently the terminal vertices have been considered upon completing the sequencing of the core vertices. This approach provides a multilayered connectivity graph, which can be put to use in comparing two or more structures or parts thereof for any given purpose. Furthermore the basic vertex connection tables generated here are useful in the computation of characteristic matrices/ topological indices, automorphism groups, and in storing, sorting and retrieving of chemical structures from databases.

Implicit task sequence learning

Implicit task sequence learning (TSL) can be considered as an extension of implicit sequence learning which is typically tested with the classical serial reaction time task (SRTT). By design, in the SRTT there is a correlation between the sequence of stimuli to which participants must attend and the sequence of motor movements/key presses with which participants must respond. The TSL paradigm allows to disentangle this correlation and to separately manipulate the presences/absence of a sequence of tasks, a sequence of responses, and even other streams of information such as stimulus locations or stimulus-response mappings. Here I review the state of TSL research which seems to point at the critical role of the presence of correlated streams of information in implicit sequence learning. On a more general level, I propose that beyond correlated streams of information, a simple statistical learning mechanism may also be involved in implicit sequence learning, and that the relative contribution of these two explanations differ according to task requirements. With this differentiation, conflicting results can be integrated into a coherent framework.

Origins and evolution of VNTR loci: The apolipoprotein B 3$\sp\prime$ VNTR

I studied the apolipoprotein (apo) B 3$\sp\prime$ variable number tandem repeat (VNTR) and did computer simulations of the stepwise mutation model to address four questions: (1) How did the apo B VNTR originate? (2) What is the mutational mechanism of repeat number change at the apo B VNTR? (3) To what extent are population and molecular level events responsible for the determination of the contemporary apo B allele frequency distribution? (4) Can VNTR allele frequency distributions be explained by a simple and conservative mutation-drift model? I used three general approaches to address these questions: (1) I characterized the apo B VNTR region in non-human primate species; (2) I constructed haplotypes of polymorphic markers flanking the apo B VNTR in a sample of individuals from Lorrain, France and studied the associations between the flanking-marker haplotypes and apo B VNTR size; (3) I did computer simulations of the one-step stepwise mutation model and compared the results to real data in terms of four allele frequency distribution characteristics.^ The results of this work have allowed me to conclude that the apo B VNTR originated after an initial duplication of a sequence which is still present as a single copy sequence in New World monkey species. I conclude that this locus did not originate by the transposition of an array of repeats from somewhere else in the genome. It is unlikely that recombination is the primary mutational mechanism. Furthermore, the clustered nature of these associations implicates a stepwise mutational mechanism. From the high frequencies of certain haplotype-allele size combinations, it is evident that population level events have also been important in the determination of the apo B VNTR allele frequency distribution. Results from computer simulations of the one-step stepwise mutation model have allowed me to conclude that bimodal and multimodal allele frequency distributions are not unexpected at loci evolving via stepwise mutation mechanisms. Short tandem repeat loci fit the stepwise mutation model best, followed by microsatellite loci. I therefore conclude that there are differences in the mutational mechanisms of VNTR loci as classed by repeat unit size. (Abstract shortened by UMI.) ^

Non B-DNA structures and genomic instability associated with myotonic dystrophy type 2 (CCTG).(CAGG) repeats

There are many diseases associated with the expansion of DNA repeats in humans. Myotonic dystrophy type 2 is one of such diseases, characterized by expansions of a (CCTG)•(CAGG) repeat tract in intron 1 of zinc finger protein 9 (ZNF9) in chromosome 3q21.3. The DM2 repeat tract contains a flanking region 5' to the tract that consists of a polymorphic repetitive sequence (TG)14-25(TCTG)4-11(CCTG) n. The (CCTG)•(CAGG) repeat is typically 11-26 repeats in persons without the disease, but can expand up to 11,000 repeats in affected individuals, which is the largest expansion seen in DNA repeat diseases to date. This DNA tract remains one of the least characterized disease-associated DNA repeats, and mechanisms causing the repeat expansion in humans have yet to be elucidated. Alternative, non B-DNA structures formed by the expanded repeats are typical in DNA repeat expansion diseases. These sequences may promote instability of the repeat tracts. I determined that slipped strand structure formation occurs for (CCTG)•(CAGG) repeats at a length of 42 or more. In addition, Z-DNA structure forms in the flanking human sequence adjacent to the (CCTG)•(CAGG) repeat tract. I have also performed genetic assays in E. coli cells and results indicate that the (CCTG)•(CAGG) repeats are more similar to the highly unstable (CTG)•(CAG) repeat tracts seen in Huntington's disease and myotonic dystrophy type 1, than to those of the more stable (ATTCT)•(AGAAT) repeat tracts of spinocerebellar ataxia type 10. This instability, however, is RecA-independent in the (CCTG)•(CAGG) and (ATTCT)•(AGAAT) repeats, whereas the instability is RecA-dependent in the (CTG)•(CAG) repeats. Structural studies of the (CCTG)•(CAGG) repeat tract and the flanking sequence, as well as genetic selection assays may reveal the mechanisms responsible for the repeat instability in E. coli, and this may lead to a better understanding of the mechanisms contributing to the human disease state. ^

Comportamiento a fatiga de uniones a solape simple con adhesivo epoxi de acero y acero prepintado

(SPA)Las propiedades mecánicas de las uniones adhesivas no solo dependen de la naturaleza y propiedades de los adhesivos; hay muchos otros parámetros que influyen directamente en el comportamiento de las uniones adhesivas. Algunos de los más significativos son: el acabado superficial de los materiales, área y espesor de la capa adhesiva, un diseño adecuado, secuencia de aplicación, propiedades químicas de la superficie y preparación de los sustratos antes de aplicar el adhesivo. La resistencia mecánica de las uniones adhesivas está íntimamente relacionada con el proceso de rotura, directamente dependiente del mecanismo cohesión-adhesión. El objeto de este trabajo es analizar la influencia de dos acabados superficiales diferentes, sobre el comportamiento a fatiga de uniones con adhesivo epoxi. Los resultados muestran que la compatibilidad química entre el adhesivo y la capa prepintada mejora la adhesión de las uniones y la resistencia mecánica frente a cargas estáticas y dinámicas. Se ha obtenido el límite de fatiga para las uniones de ambas superficies. (ENG)The mechanical properties of adhesive joints depend not only on the nature and properties of the adhesives; there are many parameters which influence the behaviour of the adhesive joints. Some of more significant parameters are: the surface conditions of materials, area and thickness of adhesive layer, compatibility of the design, applying sequence, chemical properties of surface and the surface treatments of substrates prior to be bonded. The mechanical resistance of adhesive joints is interrelated to the fracture process depended on cohesion-adhesion mechanism. The aim of this paper is analyse the influence of two different surfaces on the fatigue behaviour of epoxy adhesive joints. The results show that chemical compatibility of adhesive and paint improve adhesion of joints and the mechanical resistance against static and dynamic loads. The fatigue limited has been obtained for joints with both surfaces.

Computerized polymorphic marker identification: Experimental validation and a predicted human polymorphism catalog

A computational system for the prediction of polymorphic loci directly and efficiently from human genomic sequence was developed and verified. A suite of programs, collectively called pompous (polymorphic marker prediction of ubiquitous simple sequences) detects tandem repeats ranging from dinucleotides up to 250 mers, scores them according to predicted level of polymorphism, and designs appropriate flanking primers for PCR amplification. This approach was validated on an approximately 750-kilobase region of human chromosome 3p21.3, involved in lung and breast carcinoma homozygous deletions. Target DNA from 36 paired B lymphoblastoid and lung cancer lines was amplified and allelotyped for 33 loci predicted by pompous to be variable in repeat size. We found that among those 36 predominately Caucasian individuals 22 of the 33 (67%) predicted loci were polymorphic with an average heterozygosity of 0.42. Allele loss in this region was found in 27/36 (75%) of the tumor lines using these markers. pompous provides the genetic researcher with an additional tool for the rapid and efficient identification of polymorphic markers, and through a World Wide Web site, investigators can use pompous to identify polymorphic markers for their research. A catalog of 13,261 potential polymorphic markers and associated primer sets has been created from the analysis of 141,779,504 base pairs of human genomic sequence in GenBank. This data is available on our Web site (pompous.swmed.edu) and will be updated periodically as GenBank is expanded and algorithm accuracy is improved.

Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs

Unmethylated CpG dinucleotides in particular base contexts (CpG-S motifs) are relatively common in bacterial DNA but are rare in vertebrate DNA. B cells and monocytes have the ability to detect such CpG-S motifs that trigger innate immune defenses with production of Th1-like cytokines. Despite comparable levels of unmethylated CpG dinucleotides, DNA from serotype 12 adenovirus is immune-stimulatory, but serotype 2 is nonstimulatory and can even inhibit activation by bacterial DNA. In type 12 genomes, the distribution of CpG-flanking bases is similar to that predicted by chance. However, in type 2 adenoviral DNA the immune stimulatory CpG-S motifs are outnumbered by a 15- to 30-fold excess of CpG dinucleotides in clusters of direct repeats or with a C on the 5′ side or a G on the 3′ side. Synthetic oligodeoxynucleotides containing these putative neutralizing (CpG-N) motifs block immune activation by CpG-S motifs in vitro and in vivo. Eliminating 52 of the 134 CpG-N motifs present in a DNA vaccine markedly enhanced its Th1-like function in vivo, which was increased further by the addition of CpG-S motifs. Thus, depending on the CpG motif, prokaryotic DNA can be either immune-stimulatory or neutralizing. These results have important implications for understanding microbial pathogenesis and molecular evolution and for the clinical development of DNA vaccines and gene therapy vectors.

Folding protein models with a simple hydrophobic energy function: The fundamental importance of monomer inside/outside segregation

The present study explores a “hydrophobic” energy function for folding simulations of the protein lattice model. The contribution of each monomer to conformational energy is the product of its “hydrophobicity” and the number of contacts it makes, i.e., E(h⃗, c⃗) = −Σi=1N cihi = −(h⃗.c⃗) is the negative scalar product between two vectors in N-dimensional cartesian space: h⃗ = (h1, … , hN), which represents monomer hydrophobicities and is sequence-dependent; and c⃗ = (c1, … , cN), which represents the number of contacts made by each monomer and is conformation-dependent. A simple theoretical analysis shows that restrictions are imposed concomitantly on both sequences and native structures if the stability criterion for protein-like behavior is to be satisfied. Given a conformation with vector c⃗, the best sequence is a vector h⃗ on the direction upon which the projection of c⃗ − c̄⃗ is maximal, where c̄⃗ is the diagonal vector with components equal to c̄, the average number of contacts per monomer in the unfolded state. Best native conformations are suggested to be not maximally compact, as assumed in many studies, but the ones with largest variance of contacts among its monomers, i.e., with monomers tending to occupy completely buried or completely exposed positions. This inside/outside segregation is reflected on an apolar/polar distribution on the corresponding sequence. Monte Carlo simulations in two dimensions corroborate this general scheme. Sequences targeted to conformations with large contact variances folded cooperatively with thermodynamics of a two-state transition. Sequences targeted to maximally compact conformations, which have lower contact variance, were either found to have degenerate ground state or to fold with much lower cooperativity.

Sequence variation in the Fanconi anemia gene FAA

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive syndrome associated with chromosomal instability, hypersensitivity to DNA crosslinking agents, and predisposition to malignancy. The gene for FA complementation group A (FAA) recently has been cloned. The cDNA is predicted to encode a polypeptide of 1,455 amino acids, with no homologies to any known protein that might suggest a function for FAA. We have used single-strand conformational polymorphism analysis to screen genomic DNA from a panel of 97 racially and ethnically diverse FA patients from the International Fanconi Anemia Registry for mutations in the FAA gene. A total of 85 variant bands were detected. Forty-five of the variants are probably benign polymorphisms, of which nine are common and can be used for various applications, including mapping studies for other genes in this region of chromosome 16q. Amplification refractory mutation system assays were developed to simplify their detection. Forty variants are likely to be pathogenic mutations. Seventeen of these are microdeletions/microinsertions associated with short direct repeats or homonucleotide tracts, a type of mutation thought to be generated by a mechanism of slipped-strand mispairing during DNA replication. A screening of 350 FA probands from the International Fanconi Anemia Registry for two of these deletions (1115–1118del and 3788–3790del) revealed that they are carried on about 2% and 5% of the FA alleles, respectively. 3788–3790del appears in a variety of ethnic groups and is found on at least two different haplotypes. We suggest that FAA is hypermutable, and that slipped-strand mispairing, a mutational mechanism recognized as important for the generation of germ-line and somatic mutations in a variety of cancer-related genes, including p53, APC, RB1, WT1, and BRCA1, may be a major mechanism for FAA mutagenesis.

Chromosomal arm replacement generates a high level of intraspecific polymorphism in the terminal inverted repeats of the linear chromosomal DNA of Streptomyces ambofaciens

The chromosomal DNA of the bacteria Streptomyces ambofaciens DSM40697 is an 8-Mb linear molecule that ends in terminal inverted repeats (TIRs) of 210 kb. The sequences of the TIRs are highly variable between the different linear replicons of Streptomyces (plasmids or chromosomes). Two spontaneous mutant strains harboring TIRs of 480 and 850 kb were isolated. The TIR polymorphism seen is a result of the deletion of one chromosomal end and its replacement by 480 or 850 kb of sequence identical to the end of the undeleted chromosomal arm. Analysis of the wild-type sequences involved in these rearrangements revealed that a recombination event took place between the two copies of a duplicated DNA sequence. Each copy was mapped to one chromosomal arm, outside of the TIR, and encoded a putative alternative sigma factor. The two ORFs, designated hasR and hasL, were found to be 99% similar at the nucleotide level. The sequence of the chimeric regions generated by the recombination showed that the chromosomal structure of the mutant strains resulted from homologous recombination events between the two copies. We suggest that this mechanism of chromosomal arm replacement contributes to the rapid evolutionary diversification of the sequences of the TIR in Streptomyces.

Control of alternative pre-mRNA splicing by distributed pentameric repeats

Multiple copies of the hexamer TGCATG have been shown to regulate fibronectin pre-mRNA alternative splicing. GCATG repeats also are clustered near the regulated calcitonin-specific 3′ splice site in the rat calcitonin/CGRP gene. Specific mutagenesis of these repeats in calcitonin/CGRP pre-mRNA resulted in the loss of calcitonin-specific splicing, suggesting that the native repeats act to enhance alternative exon inclusion. Mutation of subsets of these elements implies that alternative splicing requires a minimum of two repeats, and that the combination of one intronic and one exonic repeat is necessary for optimal cell-specific splicing. However, multimerized intronic repeats inhibited calcitonin-specific splicing in both the wild-type context and in a transcript lacking endogenous repeats. These results suggest that both the number and distribution of repeats may be important features for the regulation of tissue-specific alternative splicing. Further, RNA containing a single repeat bound cell-specific protein complexes, but tissue-specific differences in protein binding were not detected by using multimerized repeats. Together, these data support a novel model for alternative splicing regulation that requires the cell-specific recognition of multiple, distributed sequence elements.

Peptides containing glutamine repeats as substrates for transglutaminase-catalyzed cross-linking: Relevance to diseases of the nervous system

Many proteins contain reiterated glutamine residues, but polyglutamine of excessive length may result in human disease by conferring new properties on the protein containing it. One established property of a glutamine residue, depending on the nature of the flanking residues, is its ability to act as an amine acceptor in a transglutaminase-catalyzed reaction and to make a glutamyl–lysine cross-link with a neighboring polypeptide. To learn whether glutamine repeats can act as amine acceptors, we have made peptides with variable lengths of polyglutamine flanked by the adjacent amino acid residues in the proteins associated with spinocerebellar ataxia type 1 (SCA1), Machado–Joseph disease (SCA3), or dentato-rubral pallido-luysian atrophy (DRPLA) or those residues adjacent to the preferred cross-linking site of involucrin, or solely by arginine residues. The polyglutamine was found to confer excellent substrate properties on any soluble peptide; under optimal conditions, virtually all the glutamine residues acted as amine acceptors in the reaction with glycine ethyl-ester, and lengthening the sequence of polyglutamine increased the reactivity of each glutamine residue. In the presence of transglutaminase, peptides containing polyglutamine formed insoluble aggregates with the proteins of brain extracts and these aggregates contained glutamyl–lysine cross-links. Repeated glutamine residues exposed on the surface of a neuronal protein should form cross-linked aggregates in the presence of any transglutaminase activated by the presence of Ca2+.

The N- and C-terminal regions of RBP-J interact with the ankyrin repeats of Notch1 RAMIC to activate transcription

The evolutionarily-conserved DNA-binding protein RBP-J directly interacts with the RAM domain and the ankyrin (ANK) repeats of the Notch intracellular region (RAMIC), and activates transcription of downstream target genes that regulate cell differentiation. In vitro binding assays demonstrate that the truncated N- and C-terminal regions of RBP-J bind to the ANK repeats but not to the RAM domain. Using an OT11 mouse cell line, in which the RBP-J locus is disrupted, we showed that RBP-J constructs mutated in the N- and C-terminal regions were defective in their transcriptional activation induced by either RAMIC or IC (the Notch intracellular region without the RAM domain) although they had normal levels of binding activity to DNA and the RAM domain. The studies using chimeric molecules between RBP-J and its homolog RBP-L showed that the N- and C-terminal regions of RBP-J conferred the IC- as well as RAMIC-induced transactivation potential on RBP-L, which binds to the same DNA sequence as RBP-J but fails to interact with RAMIC. Taken together, these results indicate that the interactions between the N- and C-terminal regions of RBP-J and the ANK repeats of RAMIC are important for transactivation of RBP-J by RAMIC.

BAliBASE (Benchmark Alignment dataBASE): enhancements for repeats, transmembrane sequences and circular permutations

BAliBASE is specifically designed to serve as an evaluation resource to address all the problems encountered when aligning complete sequences. The database contains high quality, manually constructed multiple sequence alignments together with detailed annotations. The alignments are all based on three-dimensional structural superpositions, with the exception of the transmembrane sequences. The first release provided sets of reference alignments dealing with the problems of high variability, unequal repartition and large N/C-terminal extensions and internal insertions. Here we describe version 2.0 of the database, which incorporates three new reference sets of alignments containing structural repeats, trans­membrane sequences and circular permutations to evaluate the accuracy of detection/prediction and alignment of these complex sequences. BAliBASE can be viewed at the web site http://www-igbmc.u-strasbg.fr/BioInfo/BAliBASE2/index.html or can be downloaded from ftp://ftp-igbmc.u-strasbg.fr/pub/BAliBASE2/.