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Results for “second generation sequencing”

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At least 253 records · Page 14Linked to original sources

PubMed2006

TspMI, a thermostable isoschizomer of XmaI (5'C/CCGGG3'): characterization and single molecule imaging with DNA.

TspMI, a thermostable isoschizomer of XmaI from a Thermus sp., has been characterized. The enzyme was purified to homogeneity using Cibacron-Blue 3GA agarose, Heparin agarose, SP sephadex C50, and Mono-Q fast protein liquid chromatography and was found to be a homodimer of 40 kDa. Restriction mapping and run-off sequencing of TspMI-cleaved DNA ends depicted that it cleaved at 5'C/CCGGG3' to generate a four-base, 5'-CCGG overhang. The enzyme was sensitive to methylation of second and third cytosines in its recognition sequence. TspMI worked optimally at 60 degrees C with 6 mM Mg(2+), no Na(+)/K(+), and showed no star activity in the presence of 25% glycerol. The enzyme could efficiently digest the DNA labeled with a higher concentration of YOYO-I (one dye molecule to one nucleotide), making it a useful candidate for real-time imaging experiments. Single molecule interaction between TspMI and lambda DNA was studied using total internal reflection fluorescence microscopy. The enzyme survived 30 polymerase chain reaction (PCR) cycles in the presence of 10% glycerol and 0.5 M trehalose without any activity loss and, hence, is suitable for incorporation in restriction-endonuclease-mediated selective-PCR for various applications.

Binding Sites↗

PubMed1993

Post-transfusional anti-HCV-negative non-A non-B hepatitis (II) serological and polymerase chain reaction analysis for hepatitis C and hepatitis B viruses.

Hepatitis C virus (HCV) is a major etilogical agent of post-transfusional and sporadic acute and chronic hepatitis in various geographical areas. However, anti-HCV seroconversion was uncommon in a recent study of patients with post-transfusional hepatitis in Paris, France (N. Asar et al., companion paper). The aim of the present study was to detect viral markers, in particular HCV RNA and hepatitis B virus (HBV) DNA, in these patients. A combination of second-generation assays for anti-HCV antibodies and the polymerase chain reaction were used to identify HCV RNA and HBV DNA sequences in serum samples collected before and after transfusion from patients who developed non-A, non-B hepatitis. Eighteen cases of acute, post-transfusional, non-A, non-B hepatitis were identified in the prospective clinical survey. Only three of these 18 subjects developed anti-HCV antibodies in second-generation tests. HCV RNA was identified in the serum of these three subjects but in none of the others. Two patients who were anti-HCV-negative had polymerase chain reaction evidence of HBV DNA. Known viral markers were not identified in 13 of the 18 patients with acute post-transfusional non-A, non-B hepatitis. These results raise the issue of HCV strains or 'non-A, non-B, non-C' viruses not identified by current HCV and HBV markers and implicated in post-transfusional hepatitis in France.

Base Sequence↗

PubMed2026

Scion-based drought stress memory affects potato response to water deficit.

A scion-based stress memory signal, which was derived from drought-primed potato plants, was transmitted to new potato plants generated through vegetative reproduction. This affected potato tuber yield. Drought is one of the most significant threats to agricultural productivity worldwide. The cultivated potato (Solanum tuberosum L.) is a crop species that is sensitive to drought stress. This study investigated the impact of scion-based drought stress memory on tuber yield, physiological parameters, gene expression, and DNA methylation in the vegetative progeny of grafted plants. The tuber progeny plants remembered the drought stress signal transmitted from the drought-primed scion. Significant changes were observed in the expression of genes, primarily those related to photosynthetic metabolic pathways, as well as those associated with chromatin remodeling, DNA repair, and the plant's response to abiotic stresses. The gene expression landscape corresponded with variability in chlorophyll fluorescence parameters. In the first and the second generation of vegetatively propagated plants, scion-based memory had a positive effect on tuber yield. This was achieved by buffering the decline in yield caused by drought, as compared to plants grown under control conditions. Whole-genome bisulfite sequencing analysis revealed no correlation between changes in DNA methylation and gene expression. Drought-induced alterations in DNA methylation were erased in the second progeny generation. We propose that there is a direct causal relationship between scion-based memory of drought stress and photosynthetic efficiency, as well as potato tuber productivity.

Solanum tuberosum↗

PubMed1992

Protein-DNA interactions at a yeast replication origin.

An understanding of the protein-DNA interactions in vivo at origins of DNA replication in eukaryotes is essential to delineate the mechanism of initiation of DNA synthesis and its control in the cell cycle. In the yeast Saccharomyces cerevisiae, a family of sequences known as autonomously replicating sequences (ARSs) function as origins of bidirectional DNA replication on plasmids and, in several instances, also in their normal chromosomal location. Here we use nucleotide resolution genomic footprinting to investigate the association of proteins with ARS1. Nuclease protection patterns indicate that at least two different cellular factors interact with functional elements in ARS1. The first seems to be ARS-binding factor 1. The second seems to be a novel protein that generates extensive protection over the essential ARS consensus sequence and phased DNaseI-sensitive sites across a functionally important flanking sequence. Hypersensitivity of this region to cleavage by copper phenanthroline indicates that it is under torsional strain, analogous to that produced at transcriptional start sites by assembly of an initiation complex. The protection in situ is similar to that generated by the origin recognition complex (ORC) protein.

Base Sequence↗

PubMed2004

A genetic polymorphism in the coding region of the gastric intrinsic factor gene (GIF) is associated with congenital intrinsic factor deficiency.

Congenital intrinsic factor (IF) deficiency is a disorder characterized by megaloblastic anemia due to the absence of gastric IF (GIF, GenBank NM_005142) and GIF antibodies, with probable autosomal recessive inheritance. Most of the reported patients are isolated cases without genetic studies of the parents or siblings. Complete exonic sequences were determined from the PCR products generated from genomic DNA of five affected individuals. All probands had the identical variant (g.68A>G) in the second position of the fifth codon in the coding sequence of the gene that introduces a restriction enzyme site for Msp I and predicts a change in the mature protein from glutamine(5) (CAG) to arginine(5) (CGG). Three subjects were homozygous for this base exchange and two subjects were heterozygous, one of which was apparently a compound heterozygote at positions 1 and 2 of the fifth codon ([g.67C>G] + [g.68A>G]). The other patient, heterozygous for position 2, had one heterozygous unaffected parent. Most parents were heterozygous for this base exchange, confirming the pattern of autosomal recessive inheritance for congenital IF deficiency. cDNA encoding GIF was mutated at base pair g.68 (A>G) and expressed in COS-7 cells. The apparent size, secretion rate, and sensitivity to pepsin hydrolysis of the expressed IF were similar to native IF. The allelic frequency of g.68A>G was 0.067 and 0.038 in two control populations. This sequence aberration is not the cause of the phenotype, but is associated with the genotype of congenital IF deficiency and could serve as a marker for inheritance of this disorder.

Adult↗

PubMed1994

Mitochondrial minicircle DNA supports plasmid replication and maintenance in nuclei of Trypanosoma brucei.

In a search for trypanosome DNA sequences that permit replication and stable maintenance of extrachromosomal elements, a 1-kilobase-pair (kbp) fragment from a mitochondrial kinetoplast DNA (kDNA) minicircle of Trypanosoma brucei was isolated and characterized. The plasmid pTbo-1, carrying the kDNA element, is maintained in T. brucei as a supercoiled concatemer containing approximately seven to nine pTbo-1 monomer units (5.6 kbp each) in a head-to-tail orientation. The concatemer is found in approximately one copy per cell when procyclic trypanosomes are cultured in the presence of 100 micrograms of hygromycin per ml; however, in the absence of continuous hygromycin selection, the plasmid is lost from the population with a t1/2 of approximately 8.7 days (17 cell generations). A second unrelated kDNA minicircle was also able to serve as an autonomously replicating sequence (ARS) element in T. brucei, suggesting that this is a general property of kDNA minicircles. Replication of mitochondrial DNA in the nucleus may be due to either a specific consensus sequence (such as in yeast ARS elements) or nonspecific sequence characteristics (such as the degree of A&T-richness or bent DNA).

Animals↗

PubMed1998

Engineering bidentate macromolecular inhibitors for trypsin and urokinase-type plasminogen activator.

Ecotin, a dimeric serine protease inhibitor from Escherichia coli, is a novel platform for inhibitor design. An approach using the three-dimensional structure of the ecotin-trypsin complex to guide combinatiorial design efforts was taken to create potent bidentate ecotin inhibitors for trypsin and human urokinase-type plasminogen activator (uPA). The ecotin surface loop that was redesigned is composed of residues 67 to 70 (60 s loop), and binds to the target protease at a region 25 A from the enzyme active site. Two ecotin phage display libraries were constructed to exploit the binding interactions at the 60 s loop. The ecotin 60X4 library, in which residues 67 to 70 of ecotin were randomized, was panned against rat and bovine trypsin in parallel for four rounds. Panning against bovine trypsin resulted in enrichment of ecotin phage but did not yield a consensus sequence. Panning against rat trypsin resulted in enrichment as well as the ecotin consensus sequence WGFP at positions 67 to 70. The variant ecotin encoded by this sequence inhibited rat trypsin at 80 pM, a 12-fold improvement over ecotin wild-type (WT). A second generation library, ecotin M84R+60X4 including an additional methionine to arginine substitution at position 84 in the primary binding site of ecotin, was generated for panning against uPA and rat trypsin. Panning against rat trypsin resulted in enrichment but no consensus sequence. Panning against uPA resulted in enrichment as well as the different ecotin consensus sequence WGYR at positions 67 to 70. Ecotin M84R+D70R bound to uPA at 50 pM, a 56,000-fold increase in binding compared to ecotin WT. Furthermore, ecotin M84R+D70R achieved a 13,680-fold preference of specificity towards uPA versus rat trypsin. The fact that the 60 s loop of ecotin plays different roles in binding to trypsin and uPA suggests this site can be used to introduce specificity and potency for other members of the serine proteases with a chymotrypsin fold.

Animals↗

PubMed2005

A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases.

Chemical genetic analysis of protein kinases involves engineering kinases to be uniquely sensitive to inhibitors and ATP analogs that are not recognized by wild-type kinases. Despite the successful application of this approach to over two dozen kinases, several kinases do not tolerate the necessary modification to the ATP binding pocket, as they lose catalytic activity or cellular function upon mutation of the 'gatekeeper' residue that governs inhibitor and nucleotide substrate specificity. Here we describe the identification of second-site suppressor mutations to rescue the activity of 'intolerant' kinases. A bacterial genetic selection for second-site suppressors using an aminoglycoside kinase APH(3')-IIIa revealed several suppressor hotspots in the kinase domain. Informed by results from this selection, we focused on the beta sheet in the N-terminal subdomain and generated a structure-based sequence alignment of protein kinases in this region. From this alignment, we identified second-site suppressors for several divergent kinases including Cdc5, MEKK1, GRK2 and Pto. The ability to identify second-site suppressors to rescue the activity of intolerant kinases should facilitate chemical genetic analysis of the majority of protein kinases in the genome.

Amino Acid Substitution↗

PubMed1997

Conserved mutations in human ferritin H pseudogenes: a second functional sequence or an evolutionary quirk?

This paper describes a search for a second functional human ferritin H gene in a collection of genomic clones. Nine new H-like sequences have been mapped to chromosomes 1p22-31, 1q32-42, 2q32-33, 3q21-23, 13q12, 14, 17p11-pter and X. These were examined for evidence of possible functionality by sequencing and by searching for possible introns. All except an uncharacterized sequence on chromosome 13 appear to be processed pseudogenes. However, nearly all share several conserved differences with the known functional sequence. These differences occur at regions of unusual structure. It is not known whether these sequences are derived from a second functional gene or from site-specific mutations in the generation of pseudogenes from the known functional gene. We also show that several hominoids contain H gene families with similar complexities to humans and that most of the human genes have counterparts in chimpanzees and gorillas.

Animals↗

PubMed1997

A method for genome comparisons and hybridization studies using known megabase-scale DNA sequences as a reference.

We present a method for genome comparisons and high-resolution hybridization analyses using megabase stretches of known DNA sequences as a reference. The method employs two-dimensional gel electrophoresis, separating genomic segments cut with different restriction endonucleases in the first and second dimensions, to generate filters suitable for image analysis and repeated nucleic acid hybridizations. The corresponding two-dimensional pattern is computed from the reference nucleotide sequence and matched to the observed pattern, thereby identifying each fragment on the filter; at the same time the technique uncovers discrepancies from the reference sequence. This permits genome comparisons as well as automated identification and quantification of hybridization patterns with various probes. The technique is illustrated by an analysis of Saccharomyces cerevisiae chromosome IX.

Chromosome Walking↗

PubMed1997

Splicing by overlap extension by PCR using asymmetric amplification: an improved technique for the generation of hybrid proteins of immunological interest.

Major histocompatibility complex (MHC) proteins play a central role in the immune recognition of antigen. The generation of hybrid MHC molecules has been of great value in elucidating the structure: function relationships of these key glycoproteins. In this report, the generation of cDNAs coding for seven such hybrid proteins is described. We have used the technique of splicing by overlap extension by the polymerase chain reaction (SOE by PCR) [Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. and Pease, L.R. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61-68] to generate intermediate products of each of the components of the hybrid, tipped with a small sequence of the other, and then mixed these products in a second-stage PCR to produce the final spliced product. Where we were unable to generate final product, we introduced an additional step of asymmetric PCR synthesis to generate an excess of those strands which would anneal in the final PCR and found this to be effective. We noted a significant but manageable mutation rate, possibly contributed to by the tendency of DNA polymerase to add additional non-templated nucleotides [Hu, G. (1993) DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3' end of a DNA fragment. DNA Cell Biol. 12, 763-770]. To avoid this, we modified our protocol to include a stage of blunting our intermediate products with T4 DNA polymerase prior to mixing them in the final PCR. We present this system as an effective mechanism to splice DNA.

Animals↗

PubMed1994

Selective cleavage of bcr-abl chimeric RNAs by a ribozyme targeted to non-contiguous sequences.

Conventionally designed ribozymes may be unable to cleave RNA at sites which are inaccessible due to secondary structure. In addition, it may also be difficult to specifically target a conventionally designed ribozyme to some chimeric RNA molecules. Novel approaches for ribozyme targeting were developed by using the L6 bcr-abl fusion RNA as a model. Using one approach, we successfully directed ribozyme nucleation to a site on the bcr-abl RNA that is distant from the GUA cleavage site. These ribozymes bound to the L6 substrate RNA via an anchor sequence that was complementary to bcr sequences. The anchor was necessary for efficient cleavage as the anchor minus ribozyme, a conventionally designed ribozyme, was inefficient at catalyzing cleavage at this same site. The effect of anchor sequences on catalytic rates was determined for two of these ribozymes. Ribozymes generated by a second approach were designed to cleave at a CUU site in proximity to the bcr-abl junction. Both approaches have led to the development of a series of ribozymes specific for both the L6 and K28 bcr-abl chimeric RNAs, but not normal abl or bcr RNAs. The specificity of the ribozyme correlated in part with the ability of the ribozyme to bind substrate as demonstrated by gel shift analyses. Secondary structure predictions for the RNA substrate support the experimental results and may prove useful as a theoretical basis for the design of ribozymes.

Base Sequence↗

PubMed2005

Manifestation of function-follow-form in cultured neuronal networks.

We expose hidden function-follow-form schemata in the recorded activity of cultured neuronal networks by comparing the activity with simulation results of a new modeling approach. Cultured networks grown from an arbitrary mixture of neuron and glia cells in the absence of external stimulations and chemical cues spontaneously form networks of different sizes (from 50 to several millions of neurons) that exhibit non-arbitrary complex spatio-temporal patterns of activity. The latter is marked by formation of a sequence of synchronized bursting events (SBEs)--short time windows (approximately 200 ms) of rapid neuron firing, separated by longer time intervals (seconds) of sporadic neuron firing. The new dynamical synapse and soma (DSS) model, used here, has been successful in generating sequences of SBEs with the same statistical scaling properties (over six time decades) as those of the small networks. Large networks generate statistically distinct sub-groups of SBEs, each with its own characteristic pattern of neuronal firing ('fingerprint'). This special function (activity) motif has been proposed to emanate from a structural (form) motif--self-organization of the large networks into a fabric of overlapping sub-networks of about 1 mm in size. Here we test this function-follow-form idea by investigating the influence of the connectivity architecture of a model network (form) on the structure of its spontaneous activity (function). We show that a repertoire of possible activity states similar to the observed ones can be generated by networks with proper underlying architecture. For example, networks composed of two overlapping sub-networks exhibit distinct types of SBEs, each with its own characteristic pattern of neuron activity that starts at a specific sub-network. We further show that it is possible to regulate the temporal appearance of the different sub-groups of SBEs by an additional non-synaptic current fed into the soma of the modeled neurons. The ability to regulate the relative temporal ordering of different SBEs might endow the networks with higher plasticity and complexity. These findings call for additional mechanisms yet to be discovered. Recent experimental observations indicate that glia cells coupled to neuronal soma might generate such non-synaptic regulating currents.

Action Potentials↗

PubMed1999

Molecular and Immunologic Characterization of Gynogenetic Channel Catfish (Ictalurus punctatus).

: Second-generation gynogenetic channel catfish were characterized by molecular and immunologic assays to determine if they were isogenic at major histocompatibility complex loci. Southern blot analyses, using channel catfish MHC class II B and class I A gene probes, revealed identical banding patterns among second-generation gynogenetic fish. In contrast, banding patterns from outbred fish differed not only from gynogenetic animals, but also among themselves. Nucleotide sequence analysis of the MHC class II beta(1) domain, which encompasses the peptide binding region, from four randomly selected gynogenetic fish showed a single DNA sequence. In contrast, analysis of the same region from three outbred fish showed sequences that differed not only among themselves, but also from those of gynogenetic animals. In cytotoxic assays, peripheral blood leukocytes from outbred fish lysed both gynogenetic and allogeneic targets, whereas those from gynogenetic fish lysed only allogeneic targets. Taken together, these results suggest that this group of second-generation gynogenetic channel catfish is isogenic at MHC loci and may provide an excellent system with which to study cell-mediated immunity in teleosts.

Journal Article↗

PubMed1993

Cloning and expression of alternative transcripts of a novel neuroendocrine-specific gene and identification of its 135-kDa translational product.

Monoclonal antibodies RNL-2 and RNL-3 were previously shown to react with four 35-45-kDa proteins, expressed only in small cell lung carcinoma NCI-H82 cells, but to stain a subset of neuroendocrine tissues and neoplasms (Broers, J. L. V., Mijnheere, E. P., Klein Rot, M., Schaart, G., Sijlmans, A., Boerman, O. C., and Ramaekers, F. C. S. (1991) Cancer 67, 619-633). We used RNL-2 and RNL-3 to isolate cDNA sequences that code for proteins containing the two corresponding epitopes and utilized such cDNAs to develop second generation antibodies. Using these antibodies, we identified a novel 135-kDa protein. The corresponding cDNAs were found to belong to a previously unknown gene with a neuroendocrine-specific expression pattern, tentatively designated NSP gene. NSP transcription appeared to result in mRNAs of 3.4 and 1.8 kilobases (kb). In the NCI-H82 cells only, an apparently aberrant transcript of 2.3 kb was found. cDNAs containing coding sequences of the 3.4-, 2.3-, and 1.8-kb transcripts were isolated, and nucleotide sequence analysis revealed extensive sequence overlap and open reading frames for proteins of 776, 356, and 208 amino acids, respectively. The three deduced proteins all have a common carboxyl-terminal part with two large hydrophobic regions. Transfection of the complete coding sequences of the 3.4-kb transcript resulted in the production of a protein with an apparent molecular mass of 135 kDa. This protein is predicted to be highly negatively charged (calculated pI of 4.35), to be rich in proline and serine, and to contain multiple potential phosphorylation sites.

Alternative Splicing↗

PubMed2001

Genetic organization of plasmid pXF51 from the plant pathogen Xylella fastidiosa.

The sequence of plasmid pXF51 from the plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, has been analyzed. This plasmid codes for 65 open reading frames (ORFs), organized into four main regions, containing genes related to replication, mobilization, and conjugative transfer. Twenty-five ORFs have no counterparts in the public sequence databases, and 7 are similar to conserved hypothetical proteins from other bacteria. A pXF51 incompatibility group has not been determined, as we could not find a typical replication origin. One cluster of conjugation-related genes (trb) seems to be incomplete in pXF51, and a copy of this sequence is found in the chromosome, suggesting it was generated by a duplication event. A second cluster (tra) contains all genes necessary for conjugation transfer to occur, showing a conserved organization with other conjugative plasmids. An identifiable origin of transfer similar to oriT from IncP plasmids is found adjacent to genes encoding two mobilization proteins. None of the ORFs with putative assigned function could be predicted as having a role in pathogenesis, except for a virulence-associated protein D homolog. These results indicate that even though pXF51 appears not to have a direct role in Xylella pathogenesis, it is a conjugative plasmid that could be important for lateral gene transfer in this bacterium. This property may be of great importance for future development of transformation techniques in X. fastidiosa.

Amino Acid Sequence↗

PubMed1992

Mutations recovered in the Chinese hamster aprt gene after exposure to carboplatin: a comparison with cisplatin.

Platinum-based compounds such as cisplatin and carboplatin are currently used for the treatment of a variety of solid tumors. Their primary mode of action involves the production of cross-links in DNA. These compounds induce mutations in bacterial as well as in mammalian cells. Previously we determined the sequence specificity and mutational outcome in the Chinese hamster aprt gene in mutants isolated after exposure to cisplatin. The second-generation platinum drug, carboplatin, is of clinical relevance because it displays different and less toxic effects. Here we report the mutagenic specificity of this related compound. Mutations recovered after exposure to carboplatin display the same preference for sequences that contain 5'-AGG-3', 5'-AGA-3' and 5'-GAG-3' as was found for cisplatin. We thus conclude that the mutagenic outcome of exposure to carboplatin and cisplatin respectively, is similar, if not identical.

Adenine Phosphoribosyltransferase↗

PubMed1996

Gene structure of a chlorophyll a/c-binding protein from a brown alga: presence of an intron and phylogenetic implications.

A Laminaria saccharina genomic library in the phage EMBL 4 was used to isolate and sequence a full-length gene encoding a fucoxanthin-chlorophyll a/c-binding protein. Contrary to diatom homologues, the coding sequence is interrupted by an intron of about 900 bp which is located in the middle of the transit peptide. The deduced amino acid sequence of the mature protein is very similar to those of related proteins from Macrocystis pyrifera (Laminariales) and, to a lesser extent, to those from diatoms and Chrysophyceae. Seven of the eight putative chlorophyll-binding amino acids determined in green plants are also present. Alignments of different sequences related to the light-harvesting proteins (LHC) demonstrate a structural similarity among the three transmembrane helices and suggest a unique ancestral helix preceded by two beta-turns. The beta-turns are conserved in front of the second helices of the chlorophyll a/c proteins more so than in chlorophyll a/b proteins. Phylogenetic trees generated from sequence data indicate that fucoxanthin-chlorophyll-binding proteins diverged prior to the separation of photosystem I and photosystem II LHC genes of green plants. Among the fucoxanthin-containing algae, LHC I or II families could not be distinguished at this time.

Amino Acid Sequence↗