A method for DNA sequencing by hybridization with oligonucleotide matrix.

A new technique of DNA sequencing by hybridization with oligonucleotide matrix (SHOM) which could also be applied for DNA mapping and fingerprinting, mutant diagnostics, etc., has been tested in model experiments. A dot matrix was prepared which contained 9 overlapping octanucleotides (8-mers) complementary to a common 17-mer. Each of the 8-mers was immobilized as individual dot in thin layer of polyacrylamide gel fixed on a glass plate. The matrix was hybridized with the 32P-labeled 17-mer and three other 17-mers differing from the first one by a single base change. The hybridization enabled us to distinguish perfect duplexes from those containing mismatches in 32 out of 35 cases. These results are discussed with respect to the applicability of the approach for sequencing. It was shown that hybridization of DNA with an immobilized 8-mer in the presence of a labeled 5-mer led to the formation of a stable duplex with the 5-mer only if the 5- and the 8-mers were in continuous stacking making a perfect nicked duplex 13 (5+8) base pairs long. These experiments and computer simulations suggest that continuous stacking hybridization may increase the efficiency of sequencing so that random or natural coding DNA fragments about 1000 bases long could be sequenced in more than 97% of cases. Miniaturized matrices or sequencing chips were designed, where oligonucleotides were immobilized within 100 x 100 micron dots disposed at 100 micron intervals. Hybridization of fluorescently labeled DNA fragments with microchips may simplify sequencing and ensure sensitivity of at least 10 attomoles per dot. The perspectives and limitations of SHOM are discussed.     

Characterization of a trp RNA-binding attenuation protein (TRAP) mutant with tryptophan independent RNA binding activity

TRAP (trp RNA-binding attenuation protein) is an 11 subunit RNA-binding protein that regulates expression of genes involved in tryptophan metabolism (trp) in Bacillus subtilis in response to changes in intracellular tryptophan concentration. When activated by binding up to 11 tryptophan residues, TRAP binds to the mRNAs of several trp genes and down-regulates their expression. Recently, a TRAP mutant was found that binds RNA in the absence of tryptophan. In this mutant protein, Thr30, which is part of the tryptophan-binding site, is replaced with Val (T30V). We have compared the RNA-binding properties of T30V and wild-type (WT) TRAP, as well as of a series of hetero-11-mers containing mixtures of WT and T30V TRAP subunits. The most significant difference between the interaction of T30V and WT TRAP with RNA is that the affinity of T30V TRAP is more dependent on ionic strength. Analysis of the hetero-11-mers allowed us to examine how subunits interact within an 11-mer with regard to binding to tryptophan or RNA. Our data suggest that individual subunits retain properties similar to those observed when they are in homo-11-mers and that individual G/UAG triplets within the RNA can bind to TRAP differently.     

Spectrum structures and biological functions of 8-mers in the human genome

The spectra of k-mer frequencies can reveal the structures and evolution of genome sequences. We confirmed that the trimodal spectrum of 8-mers in human genome sequences is distinguished only by CG2, CG1 and CG0 8-mer sets, containing 2,1 or 0 CpG, respectively. This phenomenon is called independent selection law. The three types of CG 8-mers were considered as different functional elements. We conjectured that (1) nucleosome binding motifs are mainly characterized by CG1 8-mers and (2) the core structural units of CpG island sequences are predominantly characterized by CG2 8-mers. To validate our conjectures, nucleosome occupied sequences and CGI sequences were extracted, then the sequence parameters were constructed through the information of the three CG 8-mer sets respectively. ROC analysis showed that CG1 8-mers are more preference in nucleosome occupied segments (AUC > 0.7) and CG2 8-mers are more preference in CGI sequences (AUC > 0.99). This validates our conjecture in principle.     

Enhanced Regulatory Sequence Prediction Using Gapped k-mer Features

Oligomers of length k, or k-mers, are convenient and widely used features for modeling the properties and functions of DNA and protein sequences. However, k-mers suffer from the inherent limitation that if the parameter k is increased to resolve longer features, the probability of observing any specific k-mer becomes very small, and k-mer counts approach a binary variable, with most k-mers absent and a few present once. Thus, any statistical learning approach using k-mers as features becomes susceptible to noisy training set k-mer frequencies once k becomes large. To address this problem, we introduce alternative feature sets using gapped k-mers, a new classifier, gkm-SVM, and a general method for robust estimation of k-mer frequencies. To make the method applicable to large-scale genome wide applications, we develop an efficient tree data structure for computing the kernel matrix. We show that compared to our original kmer-SVM and alternative approaches, our gkm-SVM predicts functional genomic regulatory elements and tissue specific enhancers with significantly improved accuracy, increasing the precision by up to a factor of two. We then show that gkm-SVM consistently outperforms kmer-SVM on human ENCODE ChIP-seq datasets, and further demonstrate the general utility of our method using a Naïve-Bayes classifier. Although developed for regulatory sequence analysis, these methods can be applied to any sequence classification problem.     

Predicting Peptide Structures in Native Proteins from Physical Simulations of Fragments

It has long been proposed that much of the information encoding how a protein folds is contained locally in the peptide chain. Here we present a large-scale simulation study designed to examine the extent to which conformations of peptide fragments in water predict native conformations in proteins. We perform replica exchange molecular dynamics (REMD) simulations of 872 8-mer, 12-mer, and 16-mer peptide fragments from 13 proteins using the AMBER 96 force field and the OBC implicit solvent model. To analyze the simulations, we compute various contact-based metrics, such as contact probability, and then apply Bayesian classifier methods to infer which metastable contacts are likely to be native vs. non-native. We find that a simple measure, the observed contact probability, is largely more predictive of a peptide''s native structure in the protein than combinations of metrics or multi-body components. Our best classification model is a logistic regression model that can achieve up to 63% correct classifications for 8-mers, 71% for 12-mers, and 76% for 16-mers. We validate these results on fragments of a protein outside our training set. We conclude that local structure provides information to solve some but not all of the conformational search problem. These results help improve our understanding of folding mechanisms, and have implications for improving physics-based conformational sampling and structure prediction using all-atom molecular simulations.     

Immunodominant epitopes in herpes simplex virus type 2 glycoprotein D are recognized by CD4 lymphocytes from both HSV-1 and HSV-2 seropositive subjects

In human recurrent cutaneous herpes simplex, there is a sequential infiltrate of CD4 and then CD8 lymphocytes into lesions. CD4 lymphocytes are the major producers of the key cytokine IFN-gamma in lesions. They recognize mainly structural proteins and especially glycoproteins D and B (gD and gB) when restimulated in vitro. Recent human vaccine trials using recombinant gD showed partial protection of HSV seronegative women against genital herpes disease and also, in placebo recipients, showed protection by prior HSV1 infection. In this study, we have defined immunodominant peptide epitopes recognized by 8 HSV1(+) and/or 16 HSV2(+) patients using (51)Cr-release cytotoxicity and IFN-gamma ELISPOT assays. Using a set of 39 overlapping 20-mer peptides, more than six immunodominant epitopes were defined in gD2 (two to six peptide epitopes were recognized for each subject). Further fine mapping of these responses for 4 of the 20-mers, using a panel of 9 internal 12-mers for each 20-mers, combined with MHC II typing and also direct in vitro binding assay of these peptides to individual DR molecules, showed more than one epitope per 20-mers and promiscuous binding of individual 20-mers and 12-mers to multiple DR types. All four 20-mer peptides were cross-recognized by both HSV1(+)/HSV2(-) and HSV1(-)/HSV2(+) subjects, but the sites of recognition differed within the 20-mers where their sequences were divergent. This work provides a basis for CD4 lymphocyte cross-recognition of gD2 and possibly cross-protection observed in previous clinical studies and in vaccine trials.     

Rapid synthesis of long-chain deoxyribooligonucleotides by the N-methylimidazolide phosphotriester method.

A modified phosphotriester method has been employed for the efficient chemical synthesis of long-chain deoxyribooligonucleotides. During the course of this work, a general and rapid procedure was developed for the preparation of 24-62-mers in solution. Preparative reversed phase column chromatography on silanized silica gel was used to purify triester intermediates starting from 10-mers. The rapid synthesis of 32-mer and 42-mer on glass and silica gel supports using suitably protected 2-8-mer blocks as coupling units has been also accomplished. In particular, a convenient procedure for the solid-phase synthesis of oligonucleotide blocks bearing 3'-terminal phosphodiester groups is described.     

Enhancer and promoter element interactions dictate cyclic adenosine monophosphate mediated and cell-specific expression of the glycoprotein hormone alpha-gene

The glycoprotein hormone alpha-gene is preferentially expressed in placental cell lines, but it is also expressed in several other cell lines indicating that the differential activity of the alpha-gene regulatory elements in various cell types is more quantitative than qualitative. The 5'-flanking region of the alpha-gene contains several distinct DNA regulatory sequences including an upstream regulatory element [(URE) -181 to -150 base pairs (bp)] that stimulates basal expression and an 18 bp twice-repeated cAMP-responsive element [(CRE) -146 to -111 bp]. We constructed an array of fusion genes containing the URE and/or the CRE linked to different truncated promoters [alpha-gene, somatostatin (SRIF), glucagon, Simian Virus 40]. These constructions were transiently expressed in placental, fibroblast, or islet cell lines to identify regulatory sequences involved in cell-specific expression as well as interactions between the URE, the CRE, and different promoter elements. The URE, CRE, and alpha-promoter elements contribute approximately 3-, 6-, and 5-fold, respectively, to preferential expression in JEG-3 cells. In JEG-3 cells, the URE is strictly dependent on the CRE for activity, but it functions in a promoter-independent manner. In contrast, the CRE is markedly promoter dependent. When linked to heterologous enhancers, the alpha-promoter is more active in JEG-3 cells than in other cell lines, thereby contributing substantially to preferential expression in placental cells. Although the CREs derived from the alpha and SRIF genes both activate expression of the alpha promoter, only the alpha CRE activates the SRIF promoter in JEG-3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of competence development in Haemophilus influenzae

Development of competence for DNA uptake by the bacterium Haemophilus influenzae is tightly regulated, and expression of the cell's complement of competence genes is absolutely dependent on the cAMP-CRP complex. A second regulator of competence may maximize competence under starvation conditions. Several investigators have recently identified a consensus sequence (competence regulatory element, CRE) in the promoter regions of some competence genes and have proposed that this may be a binding site for Sxy (TfoX), a putative positive regulator of competence. However, a scoring method that reliably ranks candidate binding sites according to affinity for the cognate binding protein predicts that the cAMP-CRP complex will bind CRE sequences with high affinity. Moreover, the predicted Sxy protein lacks recognizable DNA-binding motifs and has not been shown to bind DNA. No other consensus sequences (putative binding sites) were identified in the promoter regions of competence genes. These observations suggest that the proposed competence-specific regulatory elements are in fact CRP-binding sites, and highlight the central role of cAMP-an established bacterial mediator of the response to nutritional stress-in competence regulation. Minor sequence elements uniquely conserved in the set of CRE sequences are predicted to reduce CRP affinity, and a model is suggested in which a secondary regulator of competence genes may interact with CRP under certain conditions to stabilize the initiation complex.