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Results for “third generation sequencing”

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At least 865 records · Page 48Linked to original sources
PubMed1994

Purification of E. coli-synthesized Pan proteins and development of a Pan-specific monoclonal antibody.

The helix-loop-helix (HLH) transcription factors, Pan-1 (E47) and Pan-2 (E12), are produced by the mechanism of alternative transcript splicing. Pan-1 and Pan-2 were expressed in Escherichia coli, and a purification scheme was developed. Purified Pan-2 was used to immunize Smith-Webster mice and a hybridoma was generated that produced a monoclonal antibody (Yae) that specifically recognized both native and denatured Pan-1 and Pan-2. Deletion mapping and sequence transfer studies have localized the determinant recognized by the Yae antibody to the region 195-208 of Pan-2. This region is conserved in Pan-1 and Pan-2. The Yae antibody recognized in vitro-synthesized ITF-1, a third E2A (Pan) gene product also produced by the mechanism of alternative RNA splicing, but did not recognize the related HLH proteins, ITF-2, REB alpha, or REB beta. By Western blot assay of pancreatic acinar cells, the Yae antibody detected a single protein species of 72 kD that comigrated with in vitro-synthesized Pan-1 and Pan-2.

Animals↗
PubMed2005

Familial hemiplegic migraine presenting as recurrent encephalopathy in a Native Indian family.

BACKGROUND: Familial hemiplegic migraine (FHM) is an autosomal dominant disorder, which can result from mutations in the CACNA1A (FHM1) and ATP1A2 (FHM2) genes. Typically, FHM presents with an aura of hemiplegia accompanied by a moderate-to-severe headache. FHM can be associated with other neurological findings including coma and seizures. METHODS: We describe the clinical and genetic features of a two-generation, seven-member Native Indian family with recurrent encephalopathy and FHM. RESULTS: Two of the three affected family members presented initially with encephalopathy, the third family member presented with classic episodes of migraine and hemiparesis. The CACNA1A gene locus was excluded in this family by haplotype analysis and no mutations were identified in the coding region of the ATP1A2 gene by direct sequencing. CONCLUSIONS: This emphasizes the genetic and clinical heterogeneity in familial hemiplagic migraine FHM and highlights the need to consider the diagnosis of FHM in cases of recurrent encephalopathy.

Adult↗
PubMed2005

[DHA-1 type cephalosporinase accompanied with SHV-12 prevailed in clinical isolates of Klebsiella pneumoniae].

OBJECTIVE: To study the prevalence, phenotype and genotype of the AmpC and ESBLs-producing clinical isolate of Klebsiella pneumoniae. METHODS: The clinical isolates of Klebsiella pneumoniae were examined by standard disk diffusion susceptibility tests, three-dimensional methods, isoelectric focusing (IEF) and microdilution methods. The conjugation experiment, multiplex PCR and DNA sequencing methods were used for further analysis. RESULTS: Four out of a total of 86 isolates tested were shown to be highly AmpC-producing by three-dimensional method. IEF showed that these strains produced a AmpC like beta-lactamase with a PI of 7.8, and DNA sequencing showed that the gene which expressed this AmpC like beta-lactamase was identical to DHA-1, a plasmid mediated cephalosporinase gene. These strains also produced an ESBL like beta-lactamase with a PI of 8.2 and the gene which expressed this beta-lactamase was identical to SHV-12. These strains were resistant not only to most of the third generation cephalosporins, but also to cefepime. However they were still susceptible to carbapenem. CONCLUSIONS: Highly AmpC-producing DHA-1 accompanied with SHV-12 in Klebsiella pneumoniae was reported here for the first time. They result in a significant rise in antibiotic resistance, which is regarded as a great challenge for clinical antibiotic therapy.

Bacterial Proteins↗
PubMed1990

The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes.

Human annexin V (PP4), a member of the family of calcium, membrane binding proteins, has been crystallized in the presence of calcium and analysed by crystallography by multiple isomorphic replacement at 3 A and preliminarily refined at 2.5 A resolution. The molecule has dimensions of 64 x 40 x 30 A3 and is folded into four domains of similar structure. Each domain consists of five alpha-helices wound into a right-handed superhelix yielding a globular structure of approximately 18 A diameter. The domains have hydrophobic cores whose amino acid sequences are conserved between the domains and within the annexin family of proteins. The four domains are folded into an almost planar array by tight (hydrophobic) pair-wise packing of domains II and III and I and IV to generate modules (II-III) and (I-IV), respectively. The assembly is symmetric with three parallel approximate diads relating II to III, I to IV and the module (II-III) to (I-IV), respectively. The latter diad marks a channel through the centre of the molecule coated with charged amino acid residues. The protein has structural features of channel forming membrane proteins and a polar surface characteristic of soluble proteins. It is a member of the third class of amphipathic proteins different from soluble and membrane proteins.

Amino Acid Sequence↗
PubMed2003

Crystal structure of Swine vesicular disease virus and implications for host adaptation.

Swine vesicular disease virus (SVDV) is an Enterovirus of the family Picornaviridae that causes symptoms indistinguishable from those of foot-and-mouth disease virus. Phylogenetic studies suggest that it is a recently evolved genetic sublineage of the important human pathogen coxsackievirus B5 (CBV5), and in agreement with this, it has been shown to utilize the coxsackie and adenovirus receptor (CAR) for cell entry. The 3.0-A crystal structure of strain UK/27/72 SVDV (highly virulent) reveals the expected similarity in core structure to those of other picornaviruses, showing most similarity to the closest available structure to CBV5, that of coxsackievirus B3 (CBV3). Features that help to cement together and rigidify the protein subunits are extended in this virus, perhaps explaining its extreme tolerance of environmental factors. Using the large number of capsid sequences available for both SVDV and CBV5, we have mapped the amino acid substitutions that may have occurred during the supposed adaptation of SVDV to a new host onto the structure of SVDV and a model of the SVDV/CAR complex generated by reference to the cryo-electron microscopy-visualized complex of CBV3 and CAR. The changes fall into three clusters as follows: one lines the fivefold pore, a second maps to the CAR-binding site and partially overlaps the site for decay accelerating factor (DAF) to bind to echovirus 7 (ECHO7), and the third lies close to the fivefold axis, where the low-density lipoprotein receptor binds to the minor group of rhinoviruses. Later changes in SVDV (post-1971) map to the first two clusters and may, by optimizing recognition of a pig CAR and/or DAF homologue, have improved the adaptation of the virus to pigs.

Adaptation, Physiological↗
PubMed1998

NaN, a novel voltage-gated Na channel, is expressed preferentially in peripheral sensory neurons and down-regulated after axotomy.

Although physiological and pharmacological evidence suggests the presence of multiple tetrodotoxin-resistant (TTX-R) Na channels in neurons of peripheral nervous system ganglia, only one, SNS/PN3, has been identified in these cells to date. We have identified and sequenced a novel Na channel alpha-subunit (NaN), predicted to be TTX-R and voltage-gated, that is expressed preferentially in sensory neurons within dorsal root ganglia (DRG) and trigeminal ganglia. The predicted amino acid sequence of NaN can be aligned with the predicted structure of known Na channel alpha-subunits; all relevant landmark sequences, including positively charged S4 and pore-lining SS1-SS2 segments, and the inactivation tripeptide IFM, are present at predicted positions. However, NaN exhibits only 42-53% similarity to other mammalian Na channels, including SNS/PN3, indicating that it is a novel channel, and suggesting that it may represent a third subfamily of Na channels. NaN transcript levels are reduced significantly 7 days post axotomy in DRG neurons, consistent with previous findings of a reduction in TTX-R Na currents. The preferential expression of NaN in DRG and trigeminal ganglia and the reduction of NaN mRNA levels in DRG after axonal injury suggest that NaN, together with SNS/PN3, may produce TTX-R currents in peripheral sensory neurons and may influence the generation of electrical activity in these cells.

Amino Acid Sequence↗
PubMed2005

"A posteriori" modification of carbosilane dendrimers and dendrons: their activation in core and branch positions.

The introduction of phenyl groups at different points on carbosilane dendrimers allows their acidolytic conversion to highly reactive triflato groups which in turn are readily substituted by anionic nucleophiles. Core phenylated first-fourth generation dendrimers were synthesized from tri(allyl)phenylsilane by an alternating sequence of hydrosilylation and allylation steps. Similarly, carbosilane dendrimers containing phenyl-Si groups at the branching points and in the periphery were prepared from tetraallylsilane which was hydrosilylated with PhHSiCl2. Reaction of the phenylated dendrimers with triflic acid in toluene cleanly gave the silyl triflate derivatives, provided that the correct stoichiometry of the reagents was used. In the presence of a large excess of triflic acid the SiMe3-end groups are slowly converted to SiMe2(OTf)-units. The proof of concept was provided by the fixation of a {Ph2PCH2} group using the lithiated diphenylphosphinomethanide Ph2PCH2Li, obtained by cleavage of Ph3SnCH2PPh2 with PhLi, as well as a lithiated ether-alcohol functionalized triphos derivative to the core of a third generation carbosilane dendrimer.

Journal Article↗
PubMed1997

Nucleotide compositional constraints on genomes generate alanine-, glycine-, and proline-rich structures in transcription factors.

Correlation between amino acid composition and nucleotide composition is examined. Class III POU transcription factors having higher third GC contents showed higher contents of alanine, glycine, and proline residues encoded by GC-rich nucleotides, and vice versa. This correlation was observed even among various types of transcription factors from vertebrates and invertebrates regardless of functional and structural constraints inherent to each protein. Furthermore, reptile class III POU sequences revealed no evolutionary directionality increasing the GC contents from cold- to warm-blooded vertebrates.

Alanine↗
PubMed1999

Cloning and analysis of unique human glutaminase isoforms generated by tissue-specific alternative splicing.

Three human glutaminase (hGA) isoforms were identified, two of which represent isoforms previously unidentified in any species. One isoform contains an open reading frame with high homology with the rat kidney-type glutaminase, suggesting that this isoform represents the human kidney-type glutaminase, hKGA. A second isoform, termed hGAC, contains an open reading frame that matches hKGA except for a unique COOH-terminal amino acid sequence. In addition, a third human glutaminase isoform was identified from a computer search and on further analysis was found to represent an additional unique isoform, hGAM. hKGA is expressed predominantly in brain and kidney but not in liver, hGAC is expressed principally in cardiac muscle and pancreas but not in liver or brain, and hGAM is expressed solely in cardiac and skeletal muscle. hGAC is the predominant isoform expressed by a human breast cancer cell line that exhibits a high rate of glutamine utilization and glutaminase activity. Genomic Southern analysis as well as isolation and analysis of five glutaminase genomic clones suggested that all three hGA isoforms originate from the same locus and therefore represent mRNA species that are produced by tissue-specific alternative splicing of a single pre-mRNA. Furthermore, an RT-PCR assay was developed that can be used to easily differentiate between hKGA and hGAC mRNA species.

Alternative Splicing↗
PubMed1995

The cytoplasmic domain of the Drosophila cell adhesion molecule neuroglian is not essential for its homophilic adhesive properties in S2 cells.

Drosophila neuroglian is a transmembrane glycoprotein that has strong structural and sequence homology to the vertebrate L1 gene family of cell adhesion molecules (Bieber, A.J., Snow, P.M., Hortsch, M., Patel, N.H., Jacobs, J.R., Traquina, Z.R., Schilling, J., and Goodman, C.S. (1989) Cell 59, 447-460. Two different neuroglian protein forms that are generated by a differential splicing process are expressed in a tissue-specific fashion by embryonic and larval cells (Hortsch, M., Bieber, A.J., Patel, N.H., and Goodman, C.S. (1990) Neuron 4, 697-709). The two neuroglial polypeptides differ only in their cytoplasmic domains. Both of these neuroglian species, when transfected into the expressed in Drosophila S2 cells, induce the calcium-independent, homophilic aggregation of transformed cells. A third artificial neuroglian protein form was constructed by substituting the neuroglian transmembrane segment and cytoplasmic domains with the glycosyl phosphatidylinositol attachment signal of the Drosophila fasciclin I protein. This cDNA construct generates a glycosyl phosphatidylinositol-anchored form of neuroglian, which retains the ability to induce homophilic cell aggregation when expressed in S2 cells, and was able to interact with both of the two naturally occurring neuroglian polypeptides. These results demonstrate that neuroglian mediates a calcium-independent, homophilic cell adhesion activity and that neither cytoplasmic neuroglian domains nor a direct interaction with cytoskeletal elements is essential for this property.

Amino Acid Sequence↗
PubMed1991

Distinct calcium channels are generated by alternative splicing and are differentially expressed in the mammalian CNS.

A number of pharmacologically and electrophysiologically distinct voltage-dependent Ca2+ channels have been identified in mammalian neurons. Two rat brain Ca2+ channel alpha 1 subunits (rbC-I and rbC-II) have been isolated by molecular cloning and shown to be highly related (95%) to the cardiac dihydropyridine-sensitive Ca2+ channel. The rbC-II protein is distinct from rbC-I in that it contains a 3 amino acid insert in the putative cytoplasmic loop between domains II and III and a 28 amino acid substitution corresponding to the third transmembrane segment (S3) of the fourth domain. We show that rbC-I and rbC-II transcripts are generated by alternative splicing and that they are differentially expressed in the rat CNS.

Amino Acid Sequence↗
PubMed2000

[The research development of single nucleotide polymorphism].

Single nucleotide polymorphism(SNP) is a novel set of polymorphism based on the single nucleotide variations at the genomic level, which distributes widely across the human genome and has high polymorphism information content (PIC). Now, tens of thousands SNPs have been discovered among the human genome and are introduced by many biological medicine networks. With the development of identification and analysis technique for SNP, especially the combination with DNA chips, they, as the third generation of genetic markers, are gradually fitable for fine-scale mapping of diseases, especially the complex diseases susceptibility genes and will eventually substitute for the most commonly used microsatellite markers presently.

Genetic Markers↗
PubMed1999

Alternative splicing regulates the production of ARD-1 endoribonuclease and NIPP-1, an inhibitor of protein phosphatase-1, as isoforms encoded by the same gene.

ARD-1 is an endoribonuclease identified initially as the product of a human cDNA that complements mutations in rne, a gene that encodes Escherichia coli ribonuclease E. NIPP-1 was identified in bovine nuclear extracts as an inhibitor of protein phosphatase-1. Earlier work has shown that the protein-coding sequence of ARD-1 is identical to the carboxy-terminal third of NIPP-1. However, whether ARD-1 is present in eukaryotes as a distinct entity has been unclear, as neither ARD-1-specific transcripts nor ARD-1 protein were detected in mammalian cells in earlier studies. Here we show that ARD-1 exists in human cells as a discrete protein, and that the ARD-1 and NIPP-1 peptides are isoforms encoded by a single gene and the same alternatively spliced precursor RNA. A retained intron containing multiple translation stop codons that are configured to terminate translation and initiate nonsense-mediated decay, limits the production of cellular ARD-1 protein. Our results establish the process by which functionally disparate ARD-1 and NIPP-1 peptides are generated from the protein-coding sequence of the same gene in human cells.

Alternative Splicing↗
PubMed2002

Improvement of distance-based phylogenetic methods by a local maximum likelihood approach using triplets.

We introduce a new approach to estimate the evolutionary distance between two sequences. This approach uses a tree with three leaves: two of them correspond to the studied sequences, whereas the third is chosen to handle long-distance estimation. The branch lengths of this tree are obtained by likelihood maximization and are then used to deduce the desired distance. This approach, called TripleML, improves the precision of evolutionary distance estimates, and thus the topological accuracy of distance-based methods. TripleML can be used with neighbor-joining-like (NJ-like) methods not only to compute the initial distance matrix but also to estimate new distances encountered during the agglomeration process. Computer simulations indicate that using TripleML significantly improves the topological accuracy of NJ, BioNJ, and Weighbor, while conserving a reasonable computation time. With randomly generated 24-taxon trees and realistic parameter values, combining NJ with TripleML reduces the number of wrongly inferred branches by about 11% (against 2.6% and 5.5% for BioNJ and Weighbor, respectively). Moreover, this combination requires only about 1.5 min to infer a phylogeny of 96 sequences composed of 1,200 nucleotides, as compared with 6.5 h for FastDNAml on the same machine (PC 466 MHz).

Computer Simulation↗
PubMed1979

Hepatitis B viral DNA molecules have cohesive ends.

Hepatitis B virus DNA made fully double stranded by a virion DNA polymerase reaction could be converted from circular to linear molecules by heating in 10 mM NaCl at 77 degrees C or in 100 mM NaCl at 90 degrees C for 15 min. Heat-generated linear hepatitis B virus DNA was reannealed to circular molecules by incubating in higher salt concentrations. The identity of the molecular forms was established by their electrophoretic mobility and appearance in electron micrographs. Recircularization was blocked by reacting linear molecules with nuclease S1 or avian myeloblastosis virus reverse transcriptase. These results suggest that the heated linear DNA had single-stranded ends with complementary nucleotide sequences. It also suggests that a discontinuity or nick is present in each strand of the circular DNA molecule after the single-stranded region is made double stranded by the virion DNA polymerase reaction. The difference in contour length by electron microscopy of circular and linear molecules spread under aqueous conditions suggested that the discontinuities in the two strands were about 270 base pairs apart. The amount of nucleotide incorporated into the ends of heat-generated linear hepatitis B virus DNA by reverse transcriptase suggested that the single-stranded ends were about 305 bases in length. This fully double-stranded linear DNA was cleaved with EcoRI or HpaI restriction endonuclease. The sum of the two fragments generated by each totaled 3,510 base pairs, 310 base pairs greater than the contour length of circular hepatitis B virus DNA which represents a third estimate of the distance between the discontinuities in the two DNA strands of circular DNA. Restriction endonuclease cleavage also indicated that the ends of heated linear DNA which correspond to the discontinuities in the two strands of the circular DNA are at unique sites in the DNA with respect to the restriction sites.

Base Sequence↗
PubMed1991

Mutations within the Ddc promoter alter its neuron-specific pattern of expression.

The Drosophila dopa decarboxylase gene (Ddc) is expressed in a reproducible set of approximately 150 neurons, and in a subset of the glia of the third instar larva's central nervous system (CNS). Expression in this pattern requires a cell type-specific neuronal enhancer/glial repressor region located 1000 bp from the transcriptional start site, and specific sequences within the promoter. We have used mutagenesis in vitro and P-element-mediated transformation to examine the role of the promoter, particularly its major CNS activator sequence (element I), in the generation of the wildtype expression pattern. Immunohistological analysis of these transgenic strains demonstrates that particular deletion mutations shift the site of transgene expression to a set of neurons which do not express Ddc at detectable levels in wild-type larvae. Transgene expression in these strains may be driven by a previously undetected activator sequence. Our data also suggest that glial expression may be driven by the same activator sequences that drive expression in the hypoderm.

Animals↗
PubMed2004

A state-space model of the hemodynamic approach: nonlinear filtering of BOLD signals.

In this paper, a new procedure is presented which allows the estimation of the states and parameters of the hemodynamic approach from blood oxygenation level dependent (BOLD) responses. The proposed method constitutes an alternative to the recently proposed Friston [Neuroimage 16 (2002) 513] method and has some advantages over it. The procedure is based on recent groundbreaking time series analysis techniques that have been, in this case, adopted to characterize hemodynamic responses in functional magnetic resonance imaging (fMRI). This work represents a fundamental improvement over existing approaches to system identification using nonlinear hemodynamic models and is important for three reasons. First, our model includes physiological noise. Previous models have been based upon ordinary differential equations that only allow for noise or error to enter at the level of observation. Secondly, by using the innovation method and the local linearization filter, not only the parameters, but also the underlying states of the system generating responses can be estimated. These states can include things like a flow-inducing signal triggered by neuronal activation, de-oxyhemoglobine, cerebral blood flow and volume. Finally, radial basis functions have been introduced as a parametric model to represent arbitrary temporal input sequences in the hemodynamic approach, which could be essential to understanding those brain areas indirectly related to the stimulus. Hence, thirdly, by inferring about the radial basis parameters, we are able to perform a blind deconvolution, which permits both the reconstruction of the dynamics of the most likely hemodynamic states and also, to implicitly reconstruct the underlying synaptic dynamics, induced experimentally, which caused these states variations. From this study, we conclude that in spite of the utility of the standard discrete convolution approach used in statistical parametric maps (SPM), nonlinear BOLD phenomena and unspecific input temporal sequences must be included in the fMRI analysis.

Adult↗
PubMed1999

Structure and diversification of the bovine immunoglobulin repertoire.

Our understanding of the basis to immunoglobulin formation in cattle has benefited substantially from the application of molecular biology over the past decade. It is now established that both the lambda light chain and heavy chain repertoires are founded upon the frequent expression of single gene families and subgroups of segments which are of conserved sequence. It is likely that a functional kappa locus exists in the bovine genome but this isotype comprises as few as 5% of bovine light chains. Similarly, alternative but non-expressed V(H) gene families are present posing intriguing but unresolved questions about the regulation of immunoglobulin synthesis. The heavy chain frequently bears a third complementarity-determining region which is atypically long but the processes which expand this region of the reading frame and its contribution to the interaction with antigen remain matters of speculation. Opportunities exist to map the major immunoglobulin loci and to define the membership and sequence diversity of the gene families which dominate each repertoire. However, it is already evident that cattle cannot generate significant diversity from rearrangement and junctional imprecision alone. Elucidation of the mechanism(s), dynamics and tissue distribution of immunoglobulin diversification in cattle, thus, remain key challenges in this branch of veterinary immunology.

Animals↗