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Results for “alignment chaining method”

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PubMed1998

Rapid and efficient cloning of cDNAs encoding Krüppel-like zinc finger proteins by degenerate PCR.

To isolate cDNAs encoding Krüppel-like zinc finger proteins consisting of several hundred members, most of which are yet to be identified, from a limited number of available cells, we developed a rapid and efficient zinc finger gene cloning method based on reverse transcription-polymerase chain reaction (RT-PCR) using tagged, degenerate oligonucleotide primers corresponding to the conserved H/C link followed by the reverse blue selection to identify clones containing properly amplified fragments. More than 5x103 blue colonies were obtained from only 1ng of total RNA. Eighty-eight out of 89 clones, which were randomly picked up from blue colonies and sequenced, encoded 60 different zinc fingers with the expected structure, and among them, only four have been previously described. Furthermore, it was possible to rapidly select clones that were differentially expressed in a tissue and stimulation-specific manner by a differential screening of the zinc-finger cDNA library using probes consisting of distinct sets of the zinc-finger PCR products. These results indicate that our PCR-based method is quite efficient and suitable for analyzing not only zinc finger genes but also other large gene families, especially when the available cells are very limited.

Amino Acid Sequence↗

PubMed2001

A dependent-rates model and an MCMC-based methodology for the maximum-likelihood analysis of sequences with overlapping reading frames.

We present a model and methodology for the maximum-likelihood analysis of pairwise alignments of DNA sequences in which two genes are encoded in overlapping reading frames. In the model for the substitution process, the instantaneous rates of substitution are allowed to depend on the nucleotides occupying the sites in a neighborhood of the site subject to substitution at the instant of the substitution. By defining the neighborhood of a site to extend over all sites in the codons in both reading frames to which a site belongs, constraints imposed by the genetic code in both reading frames can be taken into account. Due to the dependency of the instantaneous rates of substitution on the states at neighboring sites, the transition probability between sequences does not factorize and therefore cannot be obtained directly. We present a Markov chain Monte Carlo procedure for obtaining the ratio of two transition probabilities between two sequences under the model considered, and we describe how maximum-likelihood parameter estimation and likelihood ratio tests can be performed using the procedure. We describe how the expected numbers of different types of substitutions in the shared history of two sequences can be calculated, and we use the described model and methodology in an analysis of a pairwise alignment of two hepatitis B sequences in which two genes are encoded in overlapping frames. Finally, we present an extended model, together with a simpler approximate estimation procedure, and use this to test the adequacy of the former model.

Base Sequence↗

PubMed2000

Classification of transmembrane protein families in the Caenorhabditis elegans genome and identification of human orthologs.

The complete genome sequence of the nematode Caenorhabditis elegans provides an excellent basis for studying the distribution and evolution of protein families in higher eukaryotes. Three fundamental questions are as follows: How many paralog clusters exist in one species, how many of these are shared with other species, and how many proteins can be assigned a functional counterpart in other species? We have addressed these questions in a detailed study of predicted membrane proteins in C. elegans and their mammalian homologs. All worm proteins predicted to contain at least two transmembrane segments were clustered on the basis of sequence similarity. This resulted in 189 groups with two or more sequences, containing, in total, 2647 worm proteins. Hidden Markov models (HMMs) were created for each family, and were used to retrieve mammalian homologs from the SWISSPROT, TREMBL, and VTS databases. About one-half of these clusters had mammalian homologs. Putative worm-mammalian orthologs were extracted by use of nine different phylogenetic methods and BLAST. Eight clusters initially thought to be worm-specific were assigned mammalian homologs after searching EST and genomic sequences. A compilation of 174 orthology assignments made with high confidence is presented.

Amino Acid Sequence↗

PubMed1999

Comparative analysis and serovar-specific identification of multiple-banded antigen genes of Ureaplasma urealyticum biovar 1.

Ureaplasma urealyticum is a causative agent of nongonococcal urethritis and is implicated in the pathogenesis of several other diseases. The species is divided into 14 serovars and two biovars, of which biovar 1 is most commonly isolated from clinical specimens. Reported associations between individual serovars and diseases have been difficult to confirm because of practical difficulties with serotyping. The multiple-banded antigen (MBA) is the predominant U. urealyticum antigen recognized during infections in humans and probably has a significant role in virulence. The 5' end of the MBA gene is relatively conserved but contains biovar, and possibly serovar, specificity. The 5' ends of the MBA genes of standard strains of U. urealyticum biovar 1, consisting of serovars 1, 3, 6, and 14, were amplified, cloned into pUC19, and sequenced to identify serovar-specific differences. The 5' end of the MBA gene sequence of serovar 3 was identical with the previously published sequence and differed by only three bases from that of serovar 14. Significant differences between the MBA gene sequences allowed biovar 1 to be divided into two subgroups, containing serovars 3/14 and serovars 1 and 6, respectively, using primers UMS-125-UMA269 and UMS-125-UMA269'. Serovars 1 and 6 were distinguished by restriction enzyme analysis of the amplicon and/or by PCR specific for serovar 6. These methods were used to identify and type U. urealyticum in 185 (46.3%) of 400 genital specimens from women. Biovar 1 was detected in 89.2% and biovar 2 in 18.3% of positive specimens. Of 165 specimens containing U. urealyticum biovar 1, 22.2% contained more than one serovar and 46.7, 46.1, and 25.5% contained serovars 1, 3/14, and 6, respectively. U. urealyticum was found in a significantly higher proportion of pregnant women than in sex workers and other women attending a sexually transmissible diseases clinic (P < 0.01). The methods described are relatively rapid, practicable, and specific for serotyping isolates and for direct detection and identification of individual serovars in clinical specimens containing U. urealyticum biovar 1.

Antigens, Bacterial↗

PubMed1999

GB virus C/hepatitis G virus groups and subgroups: classification by a restriction fragment length polymorphism method based on phylogenetic analysis of the 5' untranslated region.

A phylogenetic tree based on 150 5' untranslated region sequences deposited in GenBank database allowed segregation of the sequences into three major groups, including two subgroups, i.e., 1, 2a, 2b, and 3, supported by bootstrap analysis. Restriction site analysis of these sequences predicted that HinfI and either AatII or AciI could be used for genomic typing with 99.4% accuracy. cDNA sequencing and subsequent alignment of 21 Argentine GB virus C/hepatitis G virus strains confirmed restriction fragment length polymorphism patterns theoretically predicted. This method may be useful for a rapid screening of samples when either epidemiological or transmission studies of this agent are carried out.

5' Untranslated Regions↗

PubMed2006

Molecular diagnosis of avian nephritis: preliminary report.

Kidney samples from chickens diagnosed with acute nephritis and gout were subjected to histological and electron microscopic examination. The investigations revealed cytoplasmic inclusion bodies in the tubular epithelial cells containing round virions of about 30 nm in diameter. Since avian nephritis virus (ANV) is known as a potential causative agent of the so-called baby chick nephropathy, an RT-PCR assay was developed for the molecular detection of ANV-specific nucleic acid in the specimen. The specificity of the assay was confirmed by direct sequencing of the amplicon obtained in the reaction. The nucleotide sequence of the PCR product showed 92% identity with the reference ANV sequence deposited in the GenBank database. After having been validated on some other suspicious cases of avian nephritis, the PCR method described in this study can be a potential tool for routine diagnostic examination of samples submitted from cases of gout and nephropathy in chickens.

Acute Disease↗

PubMed1993

Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction.

A ribosomal DNA-polymerase chain reaction (PCR) method has been developed for species identification of individuals of the five most widespread members of the Anopheles gambiae complex, a group of morphologically indistinguishable sibling mosquito species that includes the major vectors of malaria in Africa. The method, which is based on species-specific nucleotide sequences in the ribosomal DNA intergenic spacers, may be used to identify both species and interspecies hybrids, regardless of life stage, using either extracted DNA or fragments of a specimen. Intact portions of a mosquito as small as an egg or the segment of one leg may be placed directly into the PCR mixture for amplification and analysis. The method uses a cocktail of five 20-base oligonucleotides to identify An. gambiae, An. arabiensis, An. quadriannnulatus, and either An. melas in western Africa or An. melas in eastern and southern Africa.

Animals↗

PubMed2003

A PCR method based on 16S rRNA sequence for simultaneous detection of the genus Listeria and the species Listeria monocytogenes in food products.

The genus Listeria comprises six closely related species, of which only Listeria monocytogenes is a human pathogen. The rapid and sensitive detection of L. monocytogenes is important in the food industry as well as in medical diagnosis. In this study, a PCR-based method for the rapid, specific, and sensitive detection of L. monocytogenes in food products was developed. The PCR is based on DNA sequences and primer pairs that are found within the 16S subunit of the rRNA gene and are specific to the Listeria genus and to L. monocytogenes within the Listeria genus. The primers for the Listeria genus and for L. monocytogenes were used in the same reaction mix for their simultaneous detection. In addition, a pair of bacterial primers universal to any bacterial DNA at the 16S subunit of the rRNA gene were developed as a positive control. For the detection of Listeria and L. monocytogenes in food products, the method includes selective enrichment for Listeria followed by DNA extraction and a specific PCR reaction. The method detects 1 to 5 CFU in a 25-g sample in < or = 24 h. It can be easily incorporated into the routine screening of diverse food products and readily adapted for clinical use.

Base Sequence↗

PubMed2003

Mapping of the Plasmodium falciparum multidrug resistance gene 5'-upstream region, and evidence of induction of transcript levels by antimalarial drugs in chloroquine sensitive parasites.

The Plasmodium falciparum multidrug resistance gene, pfmdr1, has been shown to be involved in the mediation of the parasite's response to various antimalarial drugs. Previous studies of pfmdr1 expression have shown that transcript levels are increased in drug-resistant isolates. However, a detailed examination of the transcriptional regulation of this gene has not been completed. The aim of this study was to map the 5' UTR of pfmdr1, and to examine the transcriptional profile of the gene in sensitive parasites treated with four different antimalarial drugs. RT-PCR and 5'-RACE mapping showed that the 5' UTR has a length of 1.94 kb. A putative promoter has been identified via transient transfection. Northern analysis revealed a 2.1- to 2.7-fold increase in pfmdr1 expression in 3D7 parasites treated with 50 nM chloroquine for 6 h, confirming results from Serial Analysis of Gene Expression. 3D7 parasites were subsequently treated with experimentally derived IC50 concentrations of mefloquine, quinine and pyrimethamine. pfmdr1 transcript levels specifically increased 2.5-fold at 6 h in mefloquine-treated parasites and threefold in parasites treated with quinine for 30 min. There was no evidence of transcript induction in pyrimethamine-treated parasites. This is the first evidence of induction of pfmdr1 expression in sensitive cells; and suggests a novel method of transcriptional control for this gene.

5' Untranslated Regions↗

PubMed1995

Automatic RNA secondary structure determination with stochastic context-free grammars.

We have developed a method for predicting the common secondary structure of large RNA multiple alignments using only the information in the alignment. It uses a series of progressively more sensitive searches of the data in an iterative manner to discover regions of base pairing; the first pass examines the entire multiple alignment. The searching uses two methods to find base pairings. Mutual information is used to measure covariation between pairs of columns in the multiple alignment and a minimum length encoding method is used to detect column pairs with high potential to base pair. Dynamic programming is used to recover the optimal tree made up of the best potential base pairs and to create a stochastic context-free grammar. The information in the tree guides the next iteration of searching. The method is similar to the traditional comparative sequence analysis technique. The method correctly identifies most of the common secondary structure in 16S and 23S rRNA.

Algorithms↗

PubMed2001

Diversity in the structural chemistry of (phosphine)gold(I) 1,3,4-thiadiazole-2,5-dithiolates (bismuthiolates I).

A series of dinuclear (phosphine)gold(I) complexes of the ambidentate 1,3,4-thiadiazoledithiolate ligand (SSS) were prepared in high yield from the corresponding (phosphine)gold(I) chlorides and K(2)(SSS) in methanol. While mononuclear components (R(3)P)AuCl with R(3) = Ph(3), Ph(2)Py, or Me(3) (1-3) gave open-chain complexes, the dinuclear components ClAu(Ph(2)P-E-PPh(2))AuCl with E = (CH(2))(6), (C(5)H(4))Fe(C(5)H(4)), or 1,4-CH(2)C(6)H(4)CH(2) afforded cyclic complexes (4-6). The products have been characterized by analytical and spectroscopic methods, and the crystal structures of 1-4 have been determined by single-crystal X-ray techniques. Crystals of 1 [(CH(2)Cl(2))(2)] and 2 (CH(2)Cl(2)) contain the molecules aggregated in strings with long and probably very weak intermolecular Au.S contacts. The P-Au-S groups are aligned parallel head-to-tail and shifted in opposite directions to reduce steric conflicts, thus ruling out aurophilic Au...Au bonding. By contrast, in crystals of 3 (CH(2)Cl(2)) with smaller tertiary phosphine ligands, the molecules are aggregated via short [3.0089(3) and 3.1048(5) A] and probably strong aurophilic bonding to give a two-dimensional network with tetranuclear units formed from four (Me(3)P)AuS moieties of four different molecules as the connecting elements. In these tetranuclear units [(Me(3)P)AuS-](4), the P-Au-S axes are rotated against each other ("crossed swords") by 108.5 degrees (P2-Au2...Au2'-P2') or 116.9 degrees (P2-Au2...Au1'-P1'), respectively, to minimize steric conflicts. There is also significant bending of the P-Au-S axes to bring the metal atoms closer together: P1-Au1-S1 = 171.88(8) degrees and P2-Au2-S2 = 165.52(8) degrees. In the crystals of the cyclic complex 4 which contain no solvent molecules, the molecular units are aggregated in strings with short closed-shell interactions between the gold atoms of neighboring molecules [3.1898(3) A]. Because of the metallocyclic structure, the shielding of the gold atoms is reduced to allow aurophilic bonding as the P-Au-S groups are rotated against each other (crossed) by a dihedral angle P-Au...Au-P of 74.6 degrees.

Journal Article↗

PubMed2006

Thickness dependence of microstructure in semiconducting films of an oligofluorene derivative.

The measurement and optimization of microstructure development in organic semiconductor films is valuable because microstructure in many cases critically impacts electronic performance. We demonstrate a general method to measure microstructure thickness dependence in thin films using surface-sensitive near edge X-ray absorbance fine structure (NEXAFS) spectroscopy. The method is applied to an oligofluorene derivative DDFTTF, which consists of a fluorene-bithiophene-fluorene core that is end-substituted with linear dodecyl groups. The substrate-relative orientations of the aromatic core and the aliphatic end chains are independently determined, and comparing these orientations to terrace heights from atomic force micrographs proves that the end chains are interdigitated or folded. By measuring microstructure development from 6 to 150 nm, we find that DDFTTF exhibits two different preferential microstructures: one with large terraces within which molecules exhibit a strongly vertical orientation, and one with much smaller domains within which molecules exhibit a mildly horizontal orientation. The relative distribution of these two preferential microstructures depends on the distance of the domains from the substrate and the substrate temperature during deposition. The utility of this method is tested using a lamination technique to measure the saturation hole mobility at the top and bottom interface of DDFTTF films. We find that local microstructures with greater pi orbital alignment in the source-drain plane correlate directly to better local saturation hole mobilities.

Crystallography, X-Ray↗

PubMed2006

Possible compartmentalization of hepatitis C viral replication in the genital tract of HIV-1-coinfected women.

BACKGROUND: We estimated the prevalence of hepatitis C virus (HCV) in cervical cytobrush samples from HCV/human immunodeficiency virus (HIV)-coinfected women and analyzed the HCV quasi species in both cytobrush and plasma samples. Possible compartmentalization of viral quasi species in the genital tract and plasma was evaluated by comparison of genetic heterogeneity and use of phylogenetic analysis. METHODS: Paired plasma and cytobrush samples were obtained from 85 HCV/HIV-coinfected women. The presence of HCV in cytobrush samples was evaluated by reverse-transcription polymerase chain reaction of the 5' untranslated region. Viral quasi species were analyzed by cloning and sequencing the highly variable region-1 in 8 patients. RESULTS: HCV was detected in 27% of cytobrush samples. The composition of viral quasi species was different in the 2 body compartments at both the nucleotide and amino acid level. In fact, the mean complexity was significantly lower in cytobrush samples, and a similar trend was observed for the other parameters of heterogeneity. Phylogenetic analysis and amino acid alignment identified several viral variants that were unique to each body compartment. CONCLUSIONS: Our data suggest that the genital and plasma quasi species represent distinct subpopulations, which possibly reflects compartmentalized viral replication. Alternatively, cell carriers harboring viral quasi species in the genital tract that are distinct from those in plasma could transfer the virus through the barrier separating the 2 body sites.

5' Untranslated Regions↗

PubMed2000

Length distribution of the peptidoglycan chains in the sacculus of Escherichia coli.

The stress-bearing fabric of bacteria is made of peptidoglycan. This crosslinked fabric is formed from disaccharide pentapeptide units that are transported through the cytoplasmic membrane and then polymerized in two directions: (i) to form oligoglycan chains; and (ii) to cross link these chains by tail-to-tail bonds from the muropeptides to the protruding peptides of other chains. The distribution of the glycan chain lengths is reminiscent of the "most probable distribution of polymer chemistry. Of course, the process is more complex than solely the random addition of units to growing chains. The complexity precludes mathematical analysis, but computer modeling of the Monte Carlo type is capable of including a range of possibilities. At each time point a specified number of disaccharides are singly added to the muramic acid residue ends of existing chains chosen at random. The transfer is in exchange for the cleavage of pyrophosphate bactoprenol that transported the disaccharide pentapeptide through the membrane. The progam then selects, again at random, which chain to cleave and between which two disaccharides of the chain the cleavage event is to occur. The cleavage generates an N -acetyl 1,6 anhydro-muramic acid end and a non-reducing N -acetyl glucosamine end. The simulation can be modified so that the program does not cleave off a disaccharide next to either end of the chain. Comparisons are shown with the experimental results of Obermann & H]oltje (1994. Microbiology140, 79-87.) They obtained their data by taking the results with normal growing cells and subtracting the similar data from minicells to estimate the chain length distribution in the cylinder part of the cell. In its most basic form the computer simulation has only one fitted parameter, K, which is the number of disaccharides added to the murein for every internal cleavage event. In this form the fitting to the experimental results is poor. One possible reason for this is that the tension on the chains, and therefore the probability of being cleaved by autolysins varies with orientation of the chain on the cylinder surface. It is well known that the tension in the cylindrical wall is twice as large in the circumferential direction as in the axial one, so one class would consist of those chains aligned longitudinally, subject to lower stress, and would have a higher energy of activation for autolysis than chains aligned circumferentially. A good fit is obtained on the assumption that there are only two classes of chains; one more likely to be cleaved than the other. The key point is that only two processes: adding of disaccharide pentapeptides at random to glycan chains and cleavage between the disaccharides at random, together with the assumption that the wall is less easily hydrolysed in the axial direction is sufficient to account for the experimental distribution.

Cell Wall Skeleton↗

PubMed1997

DISTREE: a tool for estimating genetic distances between aligned DNA sequences.

MOTIVATION: Substitution rates estimated from aligned DNA data can be used as genetic distances to investigate the phylogenetic relationship of those sequences. For this purpose, a Markov model of nucleotide substitution has to be assumed that describes this process most adequately. RESULTS: A program is presented that estimates substitution rates and their standard errors for a variety of Markov models. The model introduced by Hasegawa et al. (J. Mol. Evol., 22, 160-174, 1985) is the only one for which distances and standard deviations need to be calculated numerically, since analytical formulae cannot be derived. Each model is implemented in two different variants: (i) assuming rate homogeneity or (ii) starting from Gamma-distributed substitution rates across sequence sites. The estimation of heterogeneous substitution rates is based on a method suggested by Tamura and Nei (Mol. Biol. Evol., 10, 512-526, 1993). All required parameters are estimated from sequence data, hence the user is not asked to supply any additional input. One goal of the program is to support the user when choosing a particular model that describes most adequately the evolution of the given data set. For this purpose, a more detailed analysis of this model fit is provided. Phylogenetic trees reconstructed from the inferred distances using the neighbor-joining algorithm are also available.

Algorithms↗

PubMed2003

Ab initio modelling of the N-terminal domain of the secretin receptors.

G protein coupled receptors of the secretin family are activated by peptide hormones of about 30 residues in length. There is considerable sequence homology within both the hormone and receptor families. The receptors possess in addition to the integral membrane domain a characteristic extracellular domain of about 120 residues in length, having conserved cysteine residues, which are involved in disulphide bridge formation, and tryptophanes, which have been shown to be critical for hormone binding. This extracellular domain does not have detectable homology to any known protein fold. In order to be able to propose a structure for this domain we have used ab initio prediction methods combined with constraints based on experimental results for the disulphide connectivity. The results of computational tools for predicting secondary structure and accessibility, together with ligand binding and mutational data and other structural considerations were used in the ab initio protein folding programs DRAGON and GADGET and also the simpler program RAMBLE, which was able to explore different permutations of disulphide bond connectivity, tryptophan side chain orientation and chain topology. The methods generated a limited number of plausible models but no single unique solution was found under the constraints. One of these was refined into a full atomic model that contained a possible peptide binding site comprising the most conserved residues.

Amino Acid Sequence↗

PubMed2000

Molecular characterization of interstitial cells isolated from human heart valves.

BACKGROUND AND AIM OF THE STUDY: Myofibroblasts have been described as possessing certain characteristics of both fibroblasts and skeletal myocytes. These cells are of mesenchymal origin, were first described in wound healing, and have been found in many tissues. Myofibroblasts from other tissues have been shown to contract and to express sarcomeric (muscle) genes. In addition, these cells express certain regulatory (transcription factor) genes. The specific alignment of the cells may, at least in part, be governed by tissue polarity signals transmitted by members of the frizzled family of vertebrate tissue polarity genes. The aim of the present study was to characterize interstitial cells, with regard to the expression of myofibroblasts markers, isolated from the human heart valves. The expression of muscle structural, regulatory and tissue polarity genes has been undertaken with a view to understanding the development and contribution of interstitial cells to valve function and structure. METHODS: Interstitial cells were isolated and cultured from aortic, pulmonary, tricuspid and mitral valves of recipient hearts obtained during transplantation. Specific oligonucleotide primer pairs suitable for polymerase chain reaction (PCR) were designed for the genes of interest. Total RNA was extracted from the cultured cells and reverse transcriptase-PCR was used to determine gene expression. RESULTS: Cells from the four valve types were found to express various muscle structural genes. These include the thin filament sarcomeric genes for the cardiac isoforms of troponin T, I and C. Evidence was also found for expression of beta-myosin heavy chain (beta-MHC), alpha-MHC and cardiac myosin light chain 2 (MLC2) in these cells. The tissue polarity genes frizzled 2 (fz2) were expressed in all four valve types analyzed. CONCLUSION: Interstitial cells express a number of genes whose products may have functional significance for heart valves. These include members of the contractile apparatus such as MHC and troponins. The presence of members of the frizzled family, which specify the orientation of cell polarization, in these cells could indicate that interstitial cells are not randomly arranged in the valve tissue. Therefore, interstitial cells isolated from the human heart valves express a number of functionally important genes, suggesting a role in their specialized function.

Contractile Proteins↗

PubMed2003

Development of a PCR-based assay for rapid and reliable identification of pathogenic Fusaria.

Identification of Fusarium species has always been difficult due to confusing phenotypic classification systems. We have developed a fluorescent-based polymerase chain reaction assay that allows for rapid and reliable identification of five toxigenic and pathogenic Fusarium species. The species includes Fusarium avenaceum, F. culmorum, F. equiseti, F. oxysporum and F. sambucinum. The method is based on the PCR amplification of species-specific DNA fragments using fluorescent oligonucleotide primers, which were designed based on sequence divergence within the internal transcribed spacer region of nuclear ribosomal DNA. Besides providing an accurate, reliable, and quick diagnosis of these Fusaria, another advantage with this method is that it reduces the potential for exposure to carcinogenic chemicals as it substitutes the use of fluorescent dyes in place of ethidium bromide. Apart from its multidisciplinary importance and usefulness, it also obviates the need for gel electrophoresis.

Base Sequence↗