Sequence Analysis

An Epstein-Barr virus deletion mutant associated with fatal http://bloodjournal.hematologylibrary.org/content/97/4/835.full.html Updated information and services can be found at: (372 articles) Plenary Papers (577 articles) Immunotherapy Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests

Abstract: This chapter investigates the issue of the role of the computer,in musical analysis. Starting with a survey of the main approaches in computer analysis, we focus on the particular problem of Jazz chord sequences harmonic analysis. We propose a theory of chord sequence analysis, based on an explicit conceptual hierarchy of analysis objects. We discuss the implementation,of the theory

We report the progression of splenic marginal zone lymphoma (SMZL) with circulating villous lymphocytes to Burkitt lymphoma with the presence of a t(8;14)(q24;q32) followed by a highly aggressive course. While the initial indolent lymphoma had an IgM λ immunophenotype the Burkitt lymphoma was IgM κ-positive. Immunoglobulin heavy chain gene (IGH) sequence analysis showed no identity between the two clones. We conclude that Burkitt lymphoma can occur in patients with SMZL, although not necessarily of identical clonal origin.

PH-20 is a glycoprotein located on the surface of the sperm plasma membrane and on the inner acrosomal membrane. The best understood function of sperm surface PH-20 is its hyaluronidase activity, which results in hydrolysis of the hyaluronic acid-rich cumulus matrix during sperm penetration of this extracellular oocyte investment. In this study, we investigated whether alterations in the secondary and tertiary structures of sperm surface PH-20 would affect its enzyme activity. Proteins were isolated from the sperm plasma membrane by treatment of living cells with phosphatidylinositol-specific phospholipase C (PI-PLC). PH-20 was purified from the PI-PLC released proteins by immunoaffinity chromatography. Two-dimensional electrophoresis of purified PH-20 revealed 6 isoforms with isoelectric points ranging from 5.1 to 6.0. Removal of the N-linked glycans from PH-20 with N-glycosidase F shifted the molecular weight from 64 kd to approximately 54 kd, its deduced molecular weight based on sequence analysis, suggesting that most if not all, of the potential N-glycosylation sites are linked to oligosaccharides. The lectins Con A and PSA recognized purified sperm surface PH-20 after Western blotting, suggesting that mannose is a major sugar within or at the terminal end of the linked glycan. The lectins UEA and LPA did not recognize PH-20 Western blot, suggesting that fucose and sialic acid are not terminal sugars of sperm surface PH-20. Deglycosylation of sperm surface PH-20 resulted in a complete loss of its hyaluronidase activity. The reduction of disulfide bonds with ␤-mercaptoethanol or dithiothreitol also resulted in loss of enzyme activity. We conclude that the hyaluronidase activity of sperm surface PH-20 is dependent on structural features established by sulfhydryl linkages, as well as glycosylation.

We determined the frequency distribution of Actinomyces spp. recovered in a routine clinical laboratory and investigated the clinical significance of accurate identification to the species level. We identified 92 clinical strains of Actinomyces, including 13 strains in the related Arcanobacterium-Actinobaculum taxon, by 16S rRNA gene sequence analysis and recorded their biotypes, sources, and disease associations. The clinical isolates clustered into

Two hemolysins, Sticholysin I (St I) and Sticholysin II (St II) were puri®ed from the sea anemone Stichodactyla helianthus combining gel ®ltration and ion exchange chromatography. The amino acid composition of both cytolysins was determined revealing a high proportion of glycine, lysine, tyrosine and non-polar amino acids (alanine, leucine and valine). Cysteine was not found in either polypeptide. Molecular masses of St I and St II were 19401 and 19290 Da, respectively. N-terminal sequence analysis of St I and St II showed a high homology between them suggesting they are isoforms of the same cytolysin. Compared with other sea anemone cytolysins, St I and St II contain a 22 amino acid insertion fragment also present in Eq T II/Tn C and probably in CaT I and Hm T and absent in C III, the major hemolysin previously reported in this anemone. 7

Troxacitabine (Troxatyl; BCH-4556; (-)-2'-deoxy-3'-oxacytidine), a deoxycytidine analogue with an unusual dioxolane structure and nonnatural L-configuration, has potent antitumor activity in animal models and is in clinical trials against human malignancies. The current work ...

Background: Molecular techniques are established as routine in virological laboratories and virus typing through (partial) sequence analysis is increasingly common. Quality assurance for the use of typing data requires harmonization of genotype nomenclature, and agreement on target genes, depending on the level of resolution required, and robustness of methods. Objective: To develop and validate web-based open-access typing-tools for enteroviruses and noroviruses. Study design: An automated web-based typing algorithm was developed, starting with BLAST analysis of the query sequence against a reference set of sequences from viruses in the family Picornaviridae or Caliciviridae. The second step is phylogenetic analysis of the query sequence and a sub-set of the reference sequences, to assign the enterovirus type or norovirus genotype and/or variant, with profile alignment, construction of phylogenetic trees and bootstrap validation. Typing is performed on VP1 sequences of Human enterovirus A to D, and ORF1 and ORF2 sequences of genogroup I and II noroviruses. For validation, we used the tools to automatically type sequences in the RIVM and CDC enterovirus databases and the FBVE norovirus database. Results: Using the typing-tools, 785(99%) of 795 Enterovirus VP1 sequences, and 8154(98.5%) of 8342 norovirus sequences were typed in accordance with previously used methods. Subtyping into variants was achieved for 4439(78.4%) of 5838 NoV GII.4 sequences. Discussion and conclusions: The online typing-tools reliably assign genotypes for enteroviruses and noroviruses. The use of phylogenetic methods makes these tools robust to ongoing evolution. This should facilitate standardized genotyping and nomenclature in clinical and public health laboratories, thus supporting inter-laboratory comparisons.

Telomerase is a ribonucleoprotein enzyme that can elongate telomeric DNA, which is thought to be required for the development of cellular immortality and oncogenesis in mammals. We examined telomerase activity in tissues and primary cultured lymphoid cells of adult penaeid shrimps. Using the telomeric repeat amplification protocol (TRAP), we studied the characteristics of a putative novel telomerase in Penaeus japonicus. This telomerase could be inactivated by heating or treatment with RNase A or proteinase K. At elongation, this telomerase required dATP, dGTP, and dTTP, but not dCTP, as substrates. Sequence analysis of the TRAP product revealed that this telomerase synthesized (TTAGG) n repeated sequences. The activity of this telomerase was decreased but still readily detectable in 100 ng of protein extract from lymphoid tissue. The telomerase activity was detected in all examined tissues including testis, ovary, lymphoid, heart, hepatopancreas, and muscle. The highest telomerase activity was in the extract of ovarian tissues. In primary cultured lymphoid cells, the telomerase activity was retained. Thus, primary cultured lymphoid cells of Penaeus japonicus possess one of the factors necessary for cell line establishment.

One of the central problems in modern biology is to identify the complete set of interactions among the proteins in a cell. The structural interaction of proteins and their domains in networks is one of the most basic molecular mechanisms for biological cells. Structural evidence indicates that, interacting pairs of close homologs usually interact in the same way. In this article, we make use of both homology and inter-domain linker region knowledge to predict interaction between protein pairs solely by amino acid sequence information. High quality core set of 150 yeast proteins obtained from the Database of Interacting Proteins (DIP) was considered to test the accuracy of the proposed method. The strongest prediction of the method reached over 70% accuracy. These results show great potential for the proposed method.

~ A novel approach to genetic sequence analysis is presented. This approach, based on compression of algorithms, has been launched simultaneously by Grumbach and Tahi, Milosavijevic and Rivals. To reduce the description of an object, a compression algorithm replaces some regularities in the description by special codes. Thus a compression algorithm can be applied to a sequence in order to study the presence of those regularities all over the sequence. This paper explains this ability, gives examples of compression algorithms already developed and mentions their applications. Finally, the theoretical foundations of the approach are presented in an overview of the algorithmic theory of information.

Multiple endocrine neoplasia type 1 (MEN 1) is characterised by the combination of tumours of the parathyroid, endocrine pancreas and anterior pituitary glands. In 1988 the MEN 1 gene was mapped to chromosome 11q13 and it was cloned in 1997. This gene contains 10 exons and extends across 9 Kb of genomic DNA; it encodes for a product of 610 amino acid named menin whose function is unknown. We have studied 10 unrelated MEN 1 kindreds by a complete sequencing analysis of the entire gene; mutations were identified in nine of them: five deletions, one insertion, two nonsense mutation and a complex alteration consisting of a deletion and an insertion that can be explained by a hairpin loop model. Two of the mutations have been previously described; the other seven were novel, and they were scattered throughout the coding sequence of the gene. As in previous series, no correlation was found between phenotype and genotype.

Bioremediation is a possible mechanism for clean-up of hydrocarbon-contaminated soils in the Antarctic. Microbes indigenous to the Antarctic are required that degrade the hydrocarbon contaminants found in the soil, and that are able to survive and maintain activity under in situ conditions. Alkane- degrading bacteria previously isolated from oil-contaminated soil from around Scott Base, Antarctica, grew on a number of n-alkanes from hexane (C6) through to eicosane (C20) and the branched alkane pristane. Mineralization of 14C-dodecane was demonstrated with four strains. Representative isolates were identified as Rhodococcus species using 16S rDNA sequence analysis. Rhodococcus spp. strains 5/14 and 7/1 grew at −2°C but numbers of viable cells declined when incubated at 37°C. Both strains appear to have the major cold-shock gene cspA. Partial nucleotide sequence analyses of the PCR-amplified cspA open reading frame from Rhodococcus spp. strains 5/14 and 7/1 were approximately 60% identical to cspA from Escherichia coli.

A search for noncanonical variants of the gypsy retrotransposon ( MDG4 ) in the genome of the Drosophila melanogaster strain G32 led to the cloning of four copies of the poorly studied 7411-bp gtwin element. Sequence analysis showed that gtwin belongs to a family of endogeneous retroviruses, which are widespread in the Drosophila genome and have recently been termed insect erantiviruses. The gtwin retrotransposon is evolutionarily closest to MDG4, as evident from a good alignment of their nucleotide sequences including ORF2 (the pol gene) and ORF3 (the env gene), as well as the amino acid sequences of their protein products. These regions showed more than 75% homology. The distribution of gtwin was studied in several strains of the genus Drosophila. While strain G32 contained more than 20 copies of the element, ten other D. melanogaster strains carried gtwin in two to six copies per genome. The gtwin element was not detected in D. hydei or D. virilis. Comparison of the cloned gtwin sequences with the gtwin sequence available from the D. melanogaster genome database showed that the two variants of the mobile element differ by the presence or absence of a stop codon in the central region of ORF3. Its absence from the gtwin copies cloned from the strain G32 may indicate an association between the functional state of ORF3 and amplification of the element.

Bacillomycin Lc, a newantifungal antibiotic of the iturin class, was isolated from a strain of Bacillus subtilis as a set of five congeners. The structure as determined by chemical and spectrometric analyses has been shown to differ from that of bacillomycin L by sequence changes from aspartate-1 to asparagine-1 and from glutamine-5 to glutamate-5. The five congeners differ from each other only in the structure of the aliphatic side chain of the constituent /?-amino acid. The hydrophobicity of the /?-amino acid affects the antifungal activity of the congener, as activity increased in the order of increased congener retention on a reversed-phase HPLCcolumn.

The promoter region of the murine branched-chain α-ketoacid dehydrogenase E2 subunit (dihydrolipoyl transacylase) gene was isolated and characterized. Sequence analysis of the promoter-regulatory region showed the presence of two inverted ‘CAAT box’ sequences, the most proximal being −42 to −48 bp upstream from the determined transcription initiation site, but no TATA-box sequences, similar to the human BCKAD E2 gene. The boundary of the minimum promoter sequence appeared to reside just inclusive of this first inverted CAAT sequence, but minigene transfer analysis demonstrated that the promoter proximal region between −65 and −140 bp is likely to be extremely important for controlling regulated changes in E2 RNA expression. The regulatory effect of this region may be modulated by a number of other upstream regions which were identified within the 7.0 kb sequence examined.

Transcription of the rat ␣ 1B adrenergic receptor (␣ 1B AR) gene is controlled by three promoters (P1, P2, and P3), which generate 2.3-, 2.7-, and 3.3-kb transcripts, respectively. The expression of the 2.3-kb mRNA species is tissue-specific. To explore the underlying mechanism, the P1 promoter was analyzed. DNase I footprinting of the P1 promoter yielded three protected regions: P1f1(؊49 to ؊62); P1f2 (؊73 to ؊90), and P1f3 (؊95 to ؊115). Sequence analysis of P1f3 revealed the presence of an SV40 core C enhancer-like element. In gel mobility shift assays, P1f3 was found to bind a sequence specific protein, which was competed away by a SV40 core C enhancer consensus oligonucleotide. Mutations of this enhancer-like core sequence within P1f3 significantly reduced specific protein binding to P1f3 and inhibited P1 promoter activity. The distribution of the protein which binds to P1f3 is restricted. These findings suggest that the P1 promoter is controlled by a cell-type-specific transcription factor, which may account for the tissuespecific expression of 2.3-kb rat ␣ 1B AR mRNA species.

Two adult sisters affected by cystic fibrosis were both shown to carry two different alterations within exon 11 of the CFTR gene, the nonsense mutation G542X and the missense mutation G551D. Both patients exhibit a relatively benign clinical course. In the described patients, G542X functions as a "mild" allele and is, in this respect, dominant to the "severe" G551D.

A nonmotile, nonspore-forming, Gram-negative, aerobic, small rod-shaped bacterium, isolated from soil, was identified as Chryseobacterium gleum on the basis of 16S rRNA gene sequence analysis. It was observed to grow luxuriously at pH 9 and tolerate highly alkaline environment up to pH 12. Orange red color was a peculiar character of these cells which on purification obtained 60-80 mg/l and found to be sphingosine type of sulfonolipid "sulfobacin A" on the basis of infrared, nuclear magnetic resonance, and mass spectral data. Inhibition of sulfobacin A synthesis by incorporation of L-cycloserine in culture growth medium suggested presence of serine palmitoyl transferase which is one of the important enzymes involved in its biosynthesis. Sulfobacin A from C. gleum LMG P-22264 exhibited cytotoxicity against four cell lines tested. Maximum activity against human mammary adenocarcinoma cells was indicative of its potential as an anticancer agent.

RNA folding is a kinetic process governed by the competition of a large number of structures stabilized by the transient formation of base pairs that may induce complex folding pathways and the formation of misfolded structures. Despite of its importance in modern biophysics, the current understanding of RNA folding kinetics is limited by the complex interplay between the weak base-pair interactions that stabilize the native structure and the disordering effect of thermal forces. The possibility of mechanically pulling individual molecules offers a new perspective to understand the folding of nucleic acids. Here we investigate the folding and misfolding mechanism in RNA secondary structures pulled by mechanical forces. We introduce a model based on the identification of the minimal set of structures that reproduce the patterns of force-extension curves obtained in single molecule experiments. The model requires only two fitting parameters: the attempt frequency at the level of individual base pairs and a parameter associated to a free energy correction that accounts for the configurational entropy of an exponentially large number of neglected secondary structures. We apply the model to interpret results recently obtained in pulling experiments in the three-helix junction S15 RNA molecule (RNAS15). We show that RNAS15 undergoes force-induced misfolding where force favors the formation of a stable non-native hairpin. The model reproduces the pattern of unfolding and refolding force-extension curves, the distribution of breakage forces and the misfolding probability obtained in the experiments.

摘要

本研究以生命歷程之研究取徑探討台灣民眾「轉至成人」(transition to adulthood)的歷程，嘗試回答以完成學業、開始工作、初次結婚以及初次生育所交織而成的轉至成人歷程呈現出哪些典型的類型、這些類型之轉至成人重要生命事件呈現出什麼樣的「持續期」(duration)、「順序」(order)與「時間間隔」(interval)、人口特徵如何影響轉至成人歷程類型，以及隨著時序展開，轉至成人歷程呈現出什麼樣的出生世代變遷。本研究以台灣社會變遷基本調查第三期第二次東亞比較調查與東亞社會階層與社會流動研究兩筆資料合併後進行分析，總分析樣本為7,248筆。
本研究使用能處理多重且動態生命事件的「多軌道序列分析」尋找轉至成人歷程類型，分別在1935-55、1956-67、1968-84出生世代找出4、3、4個轉至成人歷程類型，分析結果顯示各類型間的變異性隨著出生世代逐步增加，轉至成人重要生命事件的發生年齡也呈現出越來越晚的趨勢。
此外，研究也發現轉至成人歷程之持續期隨著出生世代逐漸延長，轉至成人生命事件的順序卻仍然穩固地呈現初次結婚、生育事件發生在完成學業與開始工作之後，而初次生育也持續地發生在初次結婚之後，順序的變動僅出現在完成學業與開始工作之間。時間間隔的分析則顯示轉至成人歷程延長最主要的因素來自於初次結婚事件的延後，而非初次生育，也就是在早期出生世代中結婚事件阻擋轉至成人歷程延長的效果隨著出生世代減弱了。
在人口特徵對於轉至成人歷程類型的影響效果探討上，研究發現性別和家庭社經地位對於轉至成人歷程類型的影響效果不但顯著，而且在不同出生世代都持續著。族群的影響效果則僅在最早出生世代較為明確，隨著出生世代越晚，族群間的轉至成人歷程差異逐漸縮小。


Abstract
This study explores the transition to adulthood in Taiwan adopting the life course approach. Using school completion, first full-time job, first marriage, and first birth to define transition to adulthood, we try to reveal the typology of transition to adulthood in Taiwan, the duration, order, and time interval of the life events of transition to adulthood, the influence of demographics on transition to adulthood, and the cohort change of transition to adulthood spanning from 1935-84. Combining data from two surveys, Second year, third round of Taiwan Social Change Survey and Social Stratification and Social Mobility in Three Countries, 7,248 life courses are analyzed.
Using Multichannel Sequence Analysis, which is capable of handling multiple dynamics life events, we find 4, 3, and 4 types of transition to adulthood in three cohort span, 1935-55, 1956-67, and 1968-84. Not only has the variability of these types increased overtime, life events of transition to adulthood occur later throughout the cohort span.
In addition, the study shows the duration of transition to adulthood extends over time, but the order of that remains mostly normative, with first marriage and first birth preceded by school completion and first full-time job and first birth preceded by first marriage. The order change only occurs between school completion and first full-time job. From the analysis of time interval of life events of transition to adulthood, the study discloses that the delay of first marriage, not first birth, is the main reason of the prolongation of transition to adulthood. This shows that first marriage’s effect on stopping transition to adulthood from extending in the early cohort has weakened in the later cohorts.
The effect of demographics on transition to adulthood varies. Sex and family socioeconomic status persist to have significant effect on transition to adulthood in different cohorts, while ethnic group has significant effect only in the early cohort, meaning the difference of transition to adulthood among ethnic groups has diminished over time.

The lipocalins are a family of extracellular proteins that bind and transport small hydrophobic molecules. They are found in eubacteria and a great variety of eukaryotic cells, in which they play diverse physiological roles. We report here the detection of two new eukaryotic lipocalins and a phylogenetic analysis of 113 lipocalin family members performed with maximum-likelihood and parsimony methods on their amino acid sequences. Lipocalins segregate into 13 monophyletic clades, some of which are grouped in well-supported superclades. An examination of the GϩC content of the bacterial lipocalin genes and the detection of four new conceptual lipocalins in other eubacterial species argue against a recent horizontal transfer as the origin of prokaryotic lipocalins. Therefore, we rooted our lipocalin tree using the clade containing the prokaryotic lipocalins. The topology of the rooted lipocalin tree is in general agreement with the currently accepted view of the organismal phylogeny of arthropods and chordates. The rooted tree allows us to assign polarity to character changes and suggests a plausible scenario for the evolution of important lipocalin properties. More recently evolved lipocalins tend to (1) show greater rates of amino acid substitutions, (2) have more flexible protein structures, (3) bind smaller hydrophobic ligands, and (4) increase the efficiency of their ligand-binding contacts. Finally, we found that the family of fatty-acid-binding proteins originated from the more derived lipocalins and therefore cannot be considered a sister group of the lipocalin family.

The unfolding of a protein by the application of an external force pulling two atoms of the protein can be detected by atomic force and optical tweezers technologies as have been broadly demonstrated in the past decade. Variation of the applied force results in a modulation of the free-energy barrier to unfolding and thus, the rate of the process, which is often assumed to have single exponential kinetics. It has been recently shown that it is experimentally feasible, through the use of force clamps, to estimate the distribution of unfolding times for a population of proteins initially in the native state. In this Letter we show how the analysis of such distributions under a range of forces can provide unique information about the underlying free-energy surface such as the height of the free-energy barrier, the preexponential factor and the force dependence of the unfolding kinetics without resorting to ad hoc kinetic models.

We reported previously that human geneMAGE-1 directs the expression of a tumor antigen recognized on a melanoma by autologous cytolytic T lymphocytes. Probing cosmid libraries with aMAGE-1 sequence, we identified 11 closely related genes. The analysis of hamster-human somatic cell hybrids indicated that the 12MAGE genes are located in the q terminal region of chromosome X. LikeMAGE-1, the 11 additionalMAGE genes have their entire coding sequence located in the last exon, which shows 64%-85% identity with that ofMAGE-1. The coding sequences of theMAGE genes predict the same main structural features for allMAGE proteins. In contrast, the promoters and first exons of the12 MAGE genes show considerable variability, suggesting that the existence of this gene family enables the same function to be expressed under different transcriptional controls. The expression of eachMAGE gene was evaluated by reverse transcription and polymerase chain reaction amplification. Six genes of theMAGE family includingMAGE-1 were found to be expressed at a high level in a number of tumors of various histological types. None was expressed in a large panel of healthy tissues, with the exception of testis and placenta.

Background: Sulphadoxine and pyrimethamine are anti-folate drugs that show synergistic anti-malarial effect. Point mutations in dihydrofolate reductase (dhfr) and dihydropteorate synthatase (dhps) cause anti-folate drug resistance phenotype in human malaria parasites. This study presents pattern of point mutations in dhfr/dhps genes among Indian sub-continent.

The cytolytic seed protein enterolobin from seeds ofEnterolobium contortisiliquumwas purified by using FPLC on a Mono Q column giving a single peak in capillary electrophoresis. The complete amino acid sequence of the plant cytolysin was determined by an automated method, yielding a molecular mass of 54,806 Da. Databank searches and sequence alignment demonstrated a high degree of sequence identity and

The cadherins comprise a family of single-pass transmembrane proteins critical for cell-cell adhesion in vertebrates and invertebrates. The recently determined structure of the whole ectodomain from C-cadherin suggests that the adhesion of cadherins presented by juxtaposed cells is mediated by a strand-swapped dimer in which core hydrophobic elements are exchanged between the partner molecules. Sequence analysis suggests that several cadherin subfamilies share this adhesive mechanism. Recent work has shed new light on the molecular basis of cadherin adhesion, although understanding the specificity of these interactions remains a major challenge.

The present study presents the complete genomic sequence of Nile tilapia (Oreochromis niloticus) transferrin and functional studies of transferrin in relation to saltwater resistance. The gene consists of a total of 17 exons separated by 16 introns. The complete coding domain sequence is 2085 bp and the gene spans more than 7 kb of genomic DNA. The gene was compared to other species using phylogenetic analyses and protein alignment.

The prothoracicotropic hormone (PTTH) of Drosophila melanogaster is a modulator of ecdysteroid (molting hormone) synthesis and was isolated and characterized from extracts of whole larvae (approximately 4 x 10(5) larvae). The purification protocol included delipidation, salt-extraction, heat treatment, conventional column chromatography, and HPLC, and yielded about 50 microg of pure hormone. Biological activity was followed using a ring gland in vitro assay in which ecdysteroidogenesis by control ring glands as measured by radioimmunoassay was compared with ring gland incubations containing active fractions. The molecular weight of the purified PTTH was 45 kDa and N-terminal amino acid sequence analysis indicated that those analyzed sequences displayed no significant homology with known peptides or peptide hormones, including PTTH from the silkmoth, Bombyx mori. Western blot analysis indicated that the native form of Drosophila PTTH was a single 66-kDa polypeptide with N-linked carbohydrate chains and intrachain disulfide bonds. The purified 45-kDa peptide is the deglycosylated form, a result of glycosidase activity present during preparation of the PTTH extract. The deglycosylated form shows heterogeneity, presumably as a result of varying degrees of deglycosylation at the N terminus.

A two-stage neural network has been used to predict protein secondary structure based on the position speci®c scoring matrices generated by PSI-BLAST. Despite the simplicity and convenience of the approach used, the results are found to be superior to those produced by other methods, including the popular PHD method according to our own benchmarking results and the results from the recent Critical Assessment of Techniques for Protein Structure Prediction experiment (CASP3), where the method was evaluated by stringent blind testing. Using a new testing set based on a set of 187 unique folds, and three-way cross-validation based on structural similarity criteria rather than sequence similarity criteria used previously (no similar folds were present in both the testing and training sets) the method presented here (PSIPRED) achieved an average Q 3 score of between 76.5 % to 78.3 % depending on the precise de®nition of observed secondary structure used, which is the highest published score for any method to date. Given the success of the method in CASP3, it is reasonable to be con®dent that the evaluation presented here gives a fair indication of the performance of the method in general.

GasCan is a specialized and unique database of gastric cancer protein encoding genes expressed in human and mouse. The features that make GasCan unique are availability of gene information, availability of primers for each gene, with their features and conditions given that are useful in PCR amplification, especially in cloning experiments and to make it more unique built in programmed sequence analysis facility is provided that analyze gene sequences in database itself, resulting sequence analysis information can be valuable for researchers in different experiments. Furthermore, DNA sequence analysis tool is provided that can be access freely. GasCan will expand in future to other species, genes and cover more useful information of other species. Flexible database design, expandability and easy access of information to all of the users are the main features of the database. The Database is publicly available at http://www.gastric-cancer.site40.net.

Filarial nematode parasites are a serious cause of morbidity in humans and animals. Identification of filarial infection using traditional morphologic criteria can be difficult and lead to misdiagnosis. We report on a poly- merase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based method to detect and differen- tiate a broad range of filarial species in a single PCR. The first internal transcribed

From 1,000 randomly selected colonies from cDNA libraries derived from murine spinal cord subtracted against white matter by means of suppression subtractive hybridization, 220 clones were identified as differentially expressed by dot blot analysis. Sequence analysis by the BLAST programming identified 140 unique genes. (1) The percentage of known sequences from myelin and other glial sources was reduced by approximately 75% over previous, similar subtractions employing visual cortex as the driver. (2) Differentially expressed genes tended to reflect existing expectations concerning structure and function of the spinal cord. (3) About 35% of all genes differentially expressed in the spinal cord in this study are also known to be differentially expressed for this structure as tabulated in the UniGene database. (4) About 33% of all genes differentially expressed in the present study are recorded as not present when measured in the spinal cord according to the UniGene database indicating that present techniques are not recording about a third of differentially expressed genes in this structure. (5) About 15% of all differentially expressed genes are for unknown, putative or hypothetical protein products. (6) About 4% of all differentially expressed genes are novel expressed sequence tags for the mouse. The current study demonstrates the importance of reducing the presence of glial associated sequences when comparing brain regions. It is concluded that the persistence of some myelin sequences in the spinal cord when white matter is used as the driver indicates that myelination is more active in this structure than for those areas represented by white matter and corpus callosum.

The utility of polymorphism analysis was determined for differentiation of the following subspecies of the Gram-positive plant pathogenic bacterium, Clavibacter michiganensis: C. m. subsp. michiganensis, C. m. subsp. sepedonicus, C. m. subsp. insidiosus C. m. subsp. nebraskensis, and C. m. subsp. tessellarius. Specific primers designed for amplification of the housekeeping genes recA, rpoB, and rpoD generated 827-, 1037-, and 862-bp DNA fragments, respectively. PCR products obtained from 40 C. michiganensis strains were analysed using RFLP with four restriction endonucleases, and those PCR products with specific RFLP patterns were sequenced. The genotypes discriminated after PCR–RFLP were specific for each subspecies and also allowed for differentiation of C. m. subsp. michiganensis strains. Sequence analysis of the recA, rpoB, and rpoD gene fragments also distinguished C. michiganensis subspecies and was useful for phylogenetic analysis of all subspecies. For rapid, inexpensive, and effective differentiation of the five subspecies in this research, we recommend the amplification of recA and/or rpoD gene fragments and digestion of the PCR products with the restriction endonuclease FnuDII.

Background: Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the aand b-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species. Methodology/Principal Findings: As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications.

The genes, cDNA alpES1 and alpES1, encoding Aspergillus clavatus ES1 alkaline protease were amplified from complementary DNA (cDNA) and genomic DNA, respectively, cloned in pCR ® II-TOPO plasmid and then sequenced. Sequence analysis of the cDNA alpES1 gene revealed an open reading frame (ORF) of 1212 bp encoding a pre-pro-protein of 403 amino acid residues consisting of a 21-aa signal peptide, a 100-aa pro-peptide and a 282-aa mature protein with a calculated molecular weight of 28.5 kDa. Compared to the cDNA alpES1 gene, the alpES1 gene contained three introns, which had 53, 57 and 54 bp, respectively. The cDNA alpES1 gene was then sub-cloned in pET-30b(+) and expressed in Escherichia coli BL21 (DE3). The purified recombinant protease had a molecular weight of about 32 kDa estimated by SDS-PAGE. Kinetic parameters, K m and k cat values of the recombinant AlpES1 for casein, were 0.23 mM and 12.38 min −1 , respectively. The catalytic efficiency (k cat /K m ) was 53.82 min −1 mM −1 .

Restriction fragment length polymorphism (RFLP) and sequence analyses of the PCR-amplified 16S-23S rDNA intergenic spacer (ITS) were used for differentiating Acidithiobacillus thiooxidans strains from other related acidithiobacilli, including A. ferrooxidans and A. caldus. RFLP fingerprints obtained with AluI, DdeI, HaeIII, HinfI and MspI enabled the differentiation of all Acidithiobacillus reference strains into species groups. The A. thiooxidans strains investigated (metal mine isolates) yielded identical RFLP patterns to the A. thiooxidans type strain (ATCC 19377(T)), except for strain DAMS, which had a distinct pattern for all enzymes tested. Fourteen A. ferrooxidans mine strains were assigned to 3 RFLP groups, the majority of which were grouped with A. ferrooxidans ATCC 23270(T). The spacer region of one representative strain from each of the RFLP groups obtained was subjected to sequence analysis, in addition to eleven additional A. thiooxidans strains isolated from sediment and water samples, and A. caldus DSM 8584(T). The tRNA(IIe) and tRNA(Ala) genes, present in all strains analyzed, showed high sequence similarity. Phylogenetic analysis of the ITS sequences differentiated all three Acidithiobacillus species. Inter- and infraspecific genetic variations detected were mainly due to the size and sequence polymorphism of the ITS3 region. Mantel tests showed no significant correlation between ITS sequence similarity and the geographical origin of strains. The results showed that the 16S-23S rDNA spacer region is a useful target for the development of molecular-based methods aimed at the detection, rapid differentiation and identification of acidithiobacilli.

The basic theory of Markov chains has been known to mathematicians and engineers for close to 80 years, but it is only in the past decade that it has been applied explicitly to problems in speech processing. One of the major reasons why speech models, based on Markov chains, have not been developed until recently was the lack of a method for optimizing the parameters of the Markov model to match observed signal patterns. Such a method was proposed in the late 1960's and was immediately applied to speech processing in several research institutions. Continued refinements in the theory and implementation of Markov modelling techniques have greatly enhanced the method, leading to a wide range of applications of these models. It is the purpose of this tutorial paper to give an introduction to the theory of Markov models, and to illustrate how they have been applied to problems in speech recognition.

The recently reported crystal structures of the membrane-embedded proton-dependent c-ring rotors of a cyanobacterial F 1 F o ATP synthase and a chloroplast F 1 F o ATP synthase have provided new insights into the mechanism of this essential enzyme. While the overall features of these crings are similar, a discrepancy in the structure and hydrogen-bonding interaction network of the H + sites suggests two distinct binding modes, potentially reflecting a mechanistic differentiation. Importantly, the conformation of the key glutamate side chain to which the proton binds is also altered. To investigate the nature of these differences, we use molecular dynamics simulations of both c-rings embedded in a phospholipid membrane. We observe that the structure of the c 15 ring from Spirulina platensis is unequivocally stable within the simulation time. By contrast, the proposed structure of the H + site in the chloroplast c 14 ring changes rapidly and consistently into that reported for the c 15 ring, indicating that the latter represents a common binding mode. To assess this hypothesis, we have remodeled the c 14 ring by molecular replacement using the published structure factors. The resulting structure provides clear evidence in support of a common binding site conformation and is also considerably improved statistically. These findings, taken together with a sequence analysis of csubunits in the ATP synthase family, indicate that the so-called proton-locked conformation observed in the c 15 ring may be a common characteristic not only of light-driven systems such as chloroplasts and cyanobacteria but also of a selection of other bacterial species.