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PubMed1995

Sequence analysis of the MAGE gene family encoding human tumor-rejection antigens.

The MAGE multigene family, which includes the MAGE-1 and -3 genes that encode tumor-rejection antigens on HLA-A1 recognized by cytotoxic T-lymphocytes (CTL), is preferentially expressed at the mRNA level on human malignant cells, but not on normal cells. However, little is known about the MAGE-4, -41 and -6 genes. In this study, we have amplified 1040 bp (MAGE-1), 1061 bp (MAGE-3 and -6) and 1064 bp (MAGE-4 and -41) cDNA fragments, including the entire coding sequences (927-951 bp), using the reverse transcription-polymerase chain reaction (RT-PCR) method followed by nucleotide (nt) sequencing. One member had greater than 80 or 66% homology with the other members at the nt or deduced amino acid (aa) levels, respectively. Higher homology was found between MAGE-3 and -6 (98% at the nt level) and also between MAGE-4 and -41 (98%). The results of this investigation demonstrated high homology, as well as the clear differences between the members of the MAGE family at the coding sequence level.

Amino Acid Sequence↗

PubMed2002

Crystal structure of recombinant human interleukin-22.

Interleukin-22 (IL-10-related T cell-derived inducible factor/IL-TIF/IL-22) is a novel cytokine belonging to the IL-10 family. Recombinant human IL-22 (hIL-22) was found to activate the signal transducers and activators of transcription factors 1 and 3 as well as acute phase reactants in several hepatoma cell lines, suggesting its involvement in the inflammatory response. The crystallographic structure of recombinant hIL-22 has been solved at 2.0 A resolution using the SIRAS method. Contrary to IL-10, the hIL-22 dimer does not present an interpenetration of the secondary-structure elements belonging to the two distinct polypeptide chains but results from interface interactions between monomers. Structural differences between these two cytokines, revealed by the crystallographic studies, clearly indicate that, while a homodimer of IL-10 is required for signaling, hIL-22 most probably interacts with its receptor as a monomer.

Amino Acid Sequence↗

PubMedDate not supplied

Cloning of the human homolog of mouse transmembrane tryptase.

BACKGROUND: Three functionally distinct tryptases have been identified in the mouse, one of which encodes an unusual protease that possesses a membrane-spanning domain located in its C terminus. METHODS AND RESULTS: Using the deduced nucleotide sequence of this mouse transmembrane tryptase (mTMT) gene in a polymerase chain reaction approach, cDNAs were isolated from a number of tissues which encode its human homolog. The amino acid sequences of hTMT and mTMT are 74% identical, and the human tryptase also has the novel membrane-spanning domain. CONCLUSION: The discovery that the human genome contains a large number of homologous, but distinct, tryptase genes suggests that the individual members of this family of proteases evolved to carry out discrete functions in mast cell-mediated allergic reactions.

Amino Acid Sequence↗

PubMed2004

C2C12 co-culture on a fibroblast substratum enables sustained survival of contractile, highly differentiated myotubes with peripheral nuclei and adult fast myosin expression.

We describe a simple culture method for obtaining highly differentiated clonal C2C12 myotubes using a feeder layer of confluent fibroblasts, and document the expression of contractile protein expression and aspects of myofibre morphology using this system. Traditional culture methods using collagen- or laminin-coated tissue-culture plastic typically results in a cyclic pattern of detachment and reformation of myotubes, rarely producing myotubes of a mature adult phenotype. C2C12 co-culture on a fibroblast substratum facilitates the sustained culture of contractile myotubes, resulting in a mature sarcomeric register with evidence for peripherally migrating nuclei. Immunoblot analysis demonstrates that desmin, tropomyosin, sarcomeric actin, alpha-actinin-2 and slow myosin are detected throughout myogenic differentiation, whereas adult fast myosin heavy chain isoforms, members of the dystrophin-associated complex, and alpha-actinin-3 are not expressed at significant levels until >6 days of differentiation, coincident with the onset of contractile activity. Electrical stimulation of mature myotubes reveals typical and reproducible calcium transients, demonstrating functional maturation with respect to calcium handling proteins. Immunocytochemical staining demonstrates a well-defined sarcomeric register throughout the majority of myotubes (70-80%) and a striated staining pattern is observed for desmin, indicating alignment of the intermediate filament network with the sarcomeric register. We report that culture volume affects the fusion index and rate of sarcomeric development in developing myotubes and propose that a fibroblast feeder layer provides an elastic substratum to support contractile activity and likely secretes growth factors and extracellular matrix proteins that assist myotube development.

Adult↗

PubMed1993

Rapid production of full-length, infectious geminivirus clones by abutting primer PCR (AbP-PCR).

The application of the polymerase chain reaction (PCR) method of DNA amplification for the isolation of full-length, infectious clones of geminiviruses is described. Non-overlapping, abutting 20-mer oligonucleotide primers were used to produce a linear product from the circular geminivirus genomic template. Clones of African cassava mosaic virus (ACMV) DNA A, obtained by this method, were infectious following mechanical inoculation (in the presence of ACMV DNA B) onto Nicotiana benthamiana. Normal ACMV symptoms resulted and typical geminate viral particles were detected by electron microscopy. The use of PCR for the detection and production of full-length, infectious geminivirus clones is discussed.

Amino Acid Sequence↗

PubMed2002

Microarray synthesis through multiple-use PCR primer design.

A substantial percentage of the expense in constructing full-genome spotted microarrays comes from the cost of synthesizing the PCR primers to amplify the desired DNA. We propose a computationally-based method to substantially reduce this cost. Historically, PCR primers are designed so that each primer occurs uniquely in the genome. This condition is unnecessarily strong for selective amplification, since only the primer pair associated with each amplification need be unique. We demonstrate that careful design in a genome-level amplification project permits us to save the cost of several thousand primers over conventional approaches.

Algorithms↗

PubMed2003

An ENSEMBLE machine learning approach for the prediction of all-alpha membrane proteins.

MOTIVATION: All-alpha membrane proteins constitute a functionally relevant subset of the whole proteome. Their content ranges from about 10 to 30% of the cell proteins, based on sequence comparison and specific predictive methods. Due to the paucity of membrane proteins solved with atomic resolution, the training/testing sets of predictive methods for protein topography and topology routinely include very few well-solved structures mixed with a hundred proteins known with low resolution. Moreover, available predictors fail in predicting recently crystallised membrane proteins (Chen et al., 2002). Presently the number of well-solved membrane proteins comprises some 59 chains of low sequence homology. It is therefore possible to train/test predictors only with the set of proteins known with atomic resolution and evaluate more thoroughly the performance of different methods. RESULTS: We implement a cascade-neural network (NN), two different hidden Markov models (HMM), and their ensemble (ENSEMBLE) as a new method. We train and test in cross validation the three methods and ENSEMBLE on the 59 well resolved membrane proteins. ENSEMBLE scores with a per-protein accuracy of 90% for topography and 71% for topology, outperforming the best single method of 7 and 5 percentage points, respectively. When tested on a low resolution set of 151 proteins, with no homology with the 59 proteins, the per-protein accuracy of ENSEMBLE is 76% for topography and 68% for topology. Our results also indicate that the performance of ENSEMBLE is higher than that of the best predictors presently available on the Web.

Algorithms↗

PubMed2001

Nucleotide sequence and polymerase chain reaction/restriction fragment length polymorphism analyses of Aleutian disease virus in ferrets in Japan.

Two ferrets with spontaneous Aleutian disease (AD) were found in Japan. The diagnosis was verified by polymerase chain reaction (PCR) amplification of part of the capsid gene specific to AD virus (ADV). The nucleotide sequences (365 bp in length) of the amplified fragments from the 2 ferrets differed by a single nucleotide, producing an amino acid alteration. Compared with other types of ADV, these isolates had 96% sequence similarity to a published ferret ADV (FADV) in contrast to <91% homology to various types of mink ADV (MADV). The phylogenetic tree of ADVs indicates that these 2 isolates and the published FADV belong to the same genetic group and definitely are divergent from MADVs. The predicted amino acid sequence of the hypervariable segment in the capsid gene was conserved among the 3 types of FADV. These results indicated that the 2 isolates found in Japan were new DNA types of FADV and could have been derived from FADV(s). A restriction fragment length polymorphism (RFLP) method to distinguish the ferret types of ADV from the mink types of ADV was developed on the basis of differences in their nucleotide sequences. Digestion of the PCR products with Afal or ScaI provided different cleavage patterns for FADV and MADV. This PCR/RFLP analysis of the ADV capsid gene will be a valuable asset for diagnosis of this virus infection in ferrets.

Aleutian Mink Disease↗

PubMed2005

Evaluation of methods for predicting the topology of beta-barrel outer membrane proteins and a consensus prediction method.

BACKGROUND: Prediction of the transmembrane strands and topology of beta-barrel outer membrane proteins is of interest in current bioinformatics research. Several methods have been applied so far for this task, utilizing different algorithmic techniques and a number of freely available predictors exist. The methods can be grossly divided to those based on Hidden Markov Models (HMMs), on Neural Networks (NNs) and on Support Vector Machines (SVMs). In this work, we compare the different available methods for topology prediction of beta-barrel outer membrane proteins. We evaluate their performance on a non-redundant dataset of 20 beta-barrel outer membrane proteins of gram-negative bacteria, with structures known at atomic resolution. Also, we describe, for the first time, an effective way to combine the individual predictors, at will, to a single consensus prediction method. RESULTS: We assess the statistical significance of the performance of each prediction scheme and conclude that Hidden Markov Model based methods, HMM-B2TMR, ProfTMB and PRED-TMBB, are currently the best predictors, according to either the per-residue accuracy, the segments overlap measure (SOV) or the total number of proteins with correctly predicted topologies in the test set. Furthermore, we show that the available predictors perform better when only transmembrane beta-barrel domains are used for prediction, rather than the precursor full-length sequences, even though the HMM-based predictors are not influenced significantly. The consensus prediction method performs significantly better than each individual available predictor, since it increases the accuracy up to 4% regarding SOV and up to 15% in correctly predicted topologies. CONCLUSIONS: The consensus prediction method described in this work, optimizes the predicted topology with a dynamic programming algorithm and is implemented in a web-based application freely available to non-commercial users at http://bioinformatics.biol.uoa.gr/ConBBPRED.

Algorithms↗

PubMed2002

Automated identification of single nucleotide polymorphisms from sequencing data.

Single nucleotide polymorphisms (SNPs) provide abundant information about genetic variation. Large scale discovery of high frequency SNPs is being undertaken using various methods. However, the publicly available SNP data are not always accurate, and therefore should be verified. If only a particular gene locus is concerned,locus-specific polymerase chain reaction amplification may be useful. Problem of this method is that the secondary peak has to be measured. We have analyzed trace data from conventional sequencing equipment and found an applicable rule to discern SNPs from noise. We have developed software that integrates this function to automatically identify SNPs. The software works accurately for high quality sequences and also can detect SNPs in low quality sequences. Further, it can determine allele frequency, display this information as a bar graph and assign corresponding nucleotide combinations. It is very useful for identifying de novo SNPs in a DNA fragment of interest.

Algorithms↗

PubMed1993

HLA-DQB1 and -DQA1 typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours.

In the present study PCR primers were designed for detecting all phenotypically expressed DQB1 and DQA1 allelic variability, 19 and 10 alleles, respectively, by PCR amplification with sequence-specific primers (PCR-SSP). For DQB1 typing, each sample was amplified by a first set of 14 PCR primer pairs, followed in some cases by two to six additional PCR reactions. The first 14 primer pairs allowed the identification/separation of all but a few of the recently described DQB1 alleles: DQB1*0504, DQB1*0605, DQB1*0606 and DQB1*0607 would not be identified; DQB1*0603 and DQB1*0608; and DQB1*0301 and DQB1*0304, respectively, would not be distinguished. Therefore an additional set of eight DQB1 primer pairs was used for a complete DQB1 typing, including all homozygous and heterozygous combinations. For DQA1 typing, 12 PCR reactions were performed per sample, 10 for detecting variability within the second exon and two for identifying first exon polymorphism. All homozygous and heterzoygous combinations of DQA1 alleles could be resolved by these primer pairs. In addition, four primer mixes were designed for determining codon 57 of the HLA-DQB1 gene. Thirty cell lines and 120 individuals were investigated by the DQB1 and DQA1 PCR-SSP technique, as well as with the HLA-DQ beta 57 primers. The concordance between PCR-SSP typing and assigning DQB1 and DQA1 alleles from TaqI DRB-DQA-DQB RFLP analysis was 100%. The reproducibility was 100% in 30 samples investigated on two separate occasions. Amplification patterns, investigated in 15 nuclear families, segregated according to dominant Mendelian inheritance. DQB1 and DQA1 PCR-SSP typing can be performed in 2 hours, including DNA extraction, PCR amplification and post-amplification processing. The method is technically simple and the typings are easy to interpret. The cost for typing one individual is low and is independent of the number of samples analyzed simultaneously, i.e. the technique is well-suited for routine clinical use.

Adult↗

PubMed2005

Secondary structure assignment that accurately reflects physical and evolutionary characteristics.

BACKGROUND: Secondary structure is used in hierarchical classification of protein structures, identification of protein features, such as helix caps and loops, for fold recognition, and as a precursor to ab initio structure prediction. There are several methods available for assigning secondary structure if the three-dimensional structure of the protein is known. Unfortunately they differ in their definitions, particularly in the exact positions of the termini. Additionally, most existing methods rely on hydrogen bonding, which means that important secondary structural classes, such as isolated beta-strands and poly-proline helices cannot be identified as they do not have characteristic hydrogen-bonding patterns. For this reason we have developed a more accurate method for assigning secondary structure based on main chain geometry, which also allows a more comprehensive assignment of secondary structure. RESULTS: We define secondary structure based on a number of geometric parameters. Helices are defined based on whether they fit inside an imaginary cylinder: residues must be within the correct radius of a central axis. Different types of helices (alpha, 3(10) or pi) are assigned on the basis of the angle between successive peptide bonds. beta-strands are assigned based on backbone dihedrals and with alternating peptide bonds. Thus hydrogen bonding is not required and beta-strands can be within a parallel sheet, antiparallel sheet, or can be isolated. Poly-proline helices are defined similarly, although with three-fold symmetry. CONCLUSION: We find that our method better assigns secondary structure than existing methods. Specifically, we find that comparing our methods with those of others, amino-acid trends at helix caps are stronger, secondary structural elements less likely to be concatenated together and secondary structure guided sequence alignment is improved. We conclude, therefore, that secondary structure assignments using our method better reflects physical and evolutionary characteristics of proteins. The program is available from http://www.bioinf.man.ac.uk/~lovell/segno.shtml.

Algorithms↗

PubMed1995

Sequence analysis of one major basic beta-crystallin (beta Bp) of amphibian lenses: evolutionary comparison and phylogenetic relatedness between beta- and gamma-crystallins.

beta Bp-Crystallin, a major basic beta-crystallin of vertebrate eye lens, is developmentally regulated during the process of amphibian metamorphosis. In order to facilitate the determination of the primary sequence of this ubiquitous crystallin present in all vertebrate species, cDNA mixture was synthesized from the poly(A)+ mRNA isolated from bullfrog eye lenses. A protocol of Rapid Amplification of cDNA Ends (RACE) was used to amplify cDNAs encoding beta Bp-crystallin by polymerase chain reaction (PCR). PCR-amplified product corresponding to beta Bp-crystallin was then ligated into pGEM-T vector and then transformed into E. coli strain JM109. One complete full-length reading frame of 615 base pairs, which covers a deduced protein sequence of 205 amino acids, including the universal initiating methionine, was revealed by automatic nucleotide sequencing with a fluorescence-based dideoxynucleotide chain-termination method. It shows 83, 74, 78 and 80 percent sequence similarity to the homologous beta 2 crystallins of chicken, rat, bovine, and human species, respectively, revealing the close structural relationship among beta Bp-crystallins even from remotely related species. In this study phylogenetic trees based on nucleotide and protein sequences of various beta- and gamma-crystallins from different vertebrate classes are constructed using a combination of distance matrix and approximate parsimony methods, which corroborate the previous supposition that beta- and gamma-crystallins form a superfamily with a common ancestry.

Amino Acid Sequence↗

PubMed2005

Expression studies on glucose-6-phosphate dehydrogenase in sea bream: effects of growth hormone, somatostatin, salinity and temperature.

In the present study, the gene encoding hepatic glucose-6-phosphate dehydrogenase (G6PDH) was cloned and characterized from silver sea bream (Sparus sarba). The deduced amino acid sequence from sea bream g6pdh shared 78-84% homology with deduced amino acid sequences from previously cloned teleost g6pdh genes. A reverse transcriptase polymerase chain reaction (RT-PCR) coupled with radioisotope hybridization method was used for assessment of g6pdh expression and it was found that administration of growth hormone to sea bream increased g6pdh transcript and G6PDH activity whereas injections of somatostatin decreased both of these parameters. It was also found that sea bream maintained at an isoosmotic salinity (12 ppt) and cold temperature (12 degrees C) displayed upregulated g6pdh expression and enhanced G6PDH activtity. The results from this study demonstrate that g6pdh expression can be mediated by both hormonal and environmental factors in teleosts.

Acclimatization↗

PubMed1998

Physical linkage of the B29/Ig-beta (CD79B) gene to the skeletal muscle, sodium-channel, and growth hormone genes in rat and human.

In the region between the polyadenylation site of the rat skeletal muscle (SkM) Na-channel gene and the 5' end of the growth hormone (GH) gene, a gene coding for B-cell-specific membrane protein B29/Ig-beta was found and noted to have the same orientation as the Na-channel and GH genes. Rat B29/Ig-beta gene was 3.1 kb in length with six exons and was separated by 3.3 and 9.3 kb from Na-channel and GH genes, respectively. Rat B29/Ig-beta protein comprised 228 amino acids, and its amino acid sequence was 85 and 69% identical with the mouse and human counterparts, respectively. With the long-area PCR method, genomic DNA connecting human SkM Na-channel (SCN4A) and B29/Ig-beta (CD79B) genes and CD79B and GH (GH1) genes was amplified, and the physical linkage of SCN4A/CD79B/ GH1 genes in the human genome was established. The human CD79B gene was separated by 6.3 and 10.5 kb from the SCN4A and GH1 genes, respectively.

Amino Acid Sequence↗

PubMed1995

Partial nucleotide sequencing of six subtype 2c hepatitis C viruses detected in Italy.

The great majority of 121 hepatitis C virus (HCV) isolates obtained from 117 Italian patients with community-acquired infection could readily be typed by genotype-specific PCR. Subtype 1b was dominant (74 isolates); subtypes 2b, 2a, and 1a followed, with 19, 14, and 8 isolates, respectively. The six isolates that remained untyped by this method were classified as subtype 2c on the basis of sequence analysis of PCR amplicors obtained from the core and NS5 genes. These findings indicate that HCV subtype 2c has a relatively high prevalence in Italy. Sequencing the core region from positions 160 to 259 is sufficient to distinguish subtype 2c from other known HCV genotypes.

Adolescent↗

PubMed2005

A method for the prediction of GPCRs coupling specificity to G-proteins using refined profile Hidden Markov Models.

BACKGROUND: G- Protein coupled receptors (GPCRs) comprise the largest group of eukaryotic cell surface receptors with great pharmacological interest. A broad range of native ligands interact and activate GPCRs, leading to signal transduction within cells. Most of these responses are mediated through the interaction of GPCRs with heterotrimeric GTP-binding proteins (G-proteins). Due to the information explosion in biological sequence databases, the development of software algorithms that could predict properties of GPCRs is important. Experimental data reported in the literature suggest that heterotrimeric G-proteins interact with parts of the activated receptor at the transmembrane helix-intracellular loop interface. Utilizing this information and membrane topology information, we have developed an intensive exploratory approach to generate a refined library of statistical models (Hidden Markov Models) that predict the coupling preference of GPCRs to heterotrimeric G-proteins. The method predicts the coupling preferences of GPCRs to Gs, Gi/o and Gq/11, but not G12/13 subfamilies. RESULTS: Using a dataset of 282 GPCR sequences of known coupling preference to G-proteins and adopting a five-fold cross-validation procedure, the method yielded an 89.7% correct classification rate. In a validation set comprised of all receptor sequences that are species homologues to GPCRs with known coupling preferences, excluding the sequences used to train the models, our method yields a correct classification rate of 91.0%. Furthermore, promiscuous coupling properties were correctly predicted for 6 of the 24 GPCRs that are known to interact with more than one subfamily of G-proteins. CONCLUSION: Our method demonstrates high correct classification rate. Unlike previously published methods performing the same task, it does not require any transmembrane topology prediction in a preceding step. A web-server for the prediction of GPCRs coupling specificity to G-proteins available for non-commercial users is located at http://bioinformatics.biol.uoa.gr/PRED-COUPLE.

Algorithms↗

PubMed2001

Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations.

Cassette replacement of acyltransferase (AT) domains in 6-deoxyerythronolide B synthase (DEBS) with heterologous AT domains with different substrate specificities usually yields the predicted polyketide analogues. As reported here, however, several AT replacements in module 4 of DEBS failed to produce detectable polyketide under standard conditions, suggesting that module 4 is sensitive to perturbation of the protein structure when the AT is replaced. Alignments between different modular polyketide synthase AT domains and the Escherichia coli fatty acid synthase transacylase crystal structure were used to select motifs within the AT domain of module 4 to re-engineer its substrate selectivity and minimize potential alterations to protein folding. Three distinct primary regions of AT4 believed to confer specificity for methylmalonyl-CoA were mutated into the sequence seen in malonyl-CoA-specific domains. Each individual mutation as well as the three in combination resulted in functional DEBSs that produced mixtures of the natural polyketide, 6-deoxyerythronolide B, and the desired novel analogue, 6-desmethyl-6-deoxyerythronolide B. Production of the latter compound indicates that the identified sequence motifs do contribute to AT specificity and that DEBS can process a polyketide chain incorporating a malonate unit at module 4. This is the first example in which the extender unit specificity of a PKS module has been altered by site-specific mutation and provides a useful alternate method for engineering AT specificity in the combinatorial biosynthesis of polyketides.

Acyltransferases↗