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Results for “third generation sequencing”

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At least 163 records · Page 9Linked to original sources

PubMed1999

Identification of AUF-1 ligands reveals vast diversity of early response gene mRNAs.

Cell activation is associated with diverse and widespread changes in gene expression at both the transcriptional and post-transcriptional levels. AUF1 is a recently described cytoplasmic protein which likely participates in the post-transcriptional regulation (PTR) of AU-rich (ARE) mRNAs including those coding for cytokines and proto-oncogenes. Individual mRNAs subject to AUF1-mediated PTR can be predicted if AREs are present or the mRNA in question interacts in vitro or in vivo with AUF1. However, there are few, if any, general approaches for characterizing the overall repertoire of mRNAs subject to PTR by AUF1. In an effort to identify these mRNAs, we incubated total mRNA from mitogen-activated peripheral blood mono-nuclear cells (PBMCs) with AUF1 in vitro. AUF1-mRNA complexes were retarded on membranes, bound mRNAs eluted with high salt, and either used to generate a cDNA library or rebound to AUF1 a second or third time prior to elution and cDNA library construction. We have obtained partial nucleotide sequences from 130 clones which shows that the AUF1 selected libraries are rich in mRNAs containing 3' untranslated region AREs including a large number of early response gene cDNAs. As a test of the validity of this method, we also show that a randomly selected, novel mRNA contained in the library is stabilized upon cell activation.

Chromatography, Affinity↗

PubMed1994

Multiplex PCR assay specific for the multidrug-resistant strain W of Mycobacterium tuberculosis.

In 1991, a multidrug-resistant strain of Mycobacterium tuberculosis was isolated from eight people with tuberculosis at a state correctional facility in New York. This strain, which is designated strain W (IS6110 restriction fragment length polymorphism type 212072), was resistant to isoniazid, rifampin, ethambutol, streptomycin, kanamycin, ethionamide, and rifabutin. Since that outbreak, the W strain has been associated with outbreaks in five hospitals in the New York City area and is a continuing public health problem in the area. To be able to identify this strain rapidly, we developed a multiplex PCR assay which targets a direct repeat of IS6110 with a 556-bp intervening sequence (NTF-1). The amplification generates two amplicons from strain W, which indicate the presence and orientation of the NTF-1 sequence between the direct repeat of IS6110, and a third amplicon, which serves as an internal PCR control. The assay was evaluated with 193 isolates of M. tuberculosis, and all 48 strain W isolates among those 193 isolates were correctly identified.

Antitubercular Agents↗

PubMed1996

The t(5;17) variant of acute promyelocytic leukemia expresses a nucleophosmin-retinoic acid receptor fusion.

We have studied an acute promyelocytic leukemia (APL) patient with a variant t(5;17)(q32;q12). This translocation fuses the gene for the nucleolar phosphoprotein nucleophosmin (NPM) to the retinoic acid receptor alpha (RARA). Two alternatively spliced transcripts are expressed, which differ in 129 bases immediately upstream of the RARA sequence. The NPM sequences contained in the shorter NPM-RAR cDNA are identical to the NPM sequences contained in the NPM-ALK fusion gene expressed in t(2;5) lymphomas. The RARA sequences are the same as the RARA sequences found in the PML-RAR and PLZF-RAR fusion seen in t(15;17) and t(11;17) APL, respectively. Both NPM-RAR transcripts fuse NPM and RARA sequence in the same reading frame, to generate translation products of 57 kD and 62 kD. Both NPM-RAR proteins are expressed in the patient's leukemic cells, along with wild-type RARA derived from the uninvolved allele. In transcriptional assays using a retinoic acid response element reporter construct, both NPM-RAR fusion proteins act as retinoic acid-dependent transcriptional activators. This case defines a third class of APL rearrangements, all of which generate fusion proteins of RARA.

Animals↗

PubMed1993

A major difference between the divergence patterns within the lines-1 families in mice and voles.

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.

Animals↗

PubMed1993

Mechanisms of oncogenesis in patients with familial retinoblastoma.

In an analysis of mutations in the RB1 gene in three patients, selected at random, who had a positive family history of tumours, we identified mutations, in constitutional cells, involving exons 3, 13 and 17 of the RB1 gene. We used SSCP and PCR sequencing to screen affected individuals and other members of their families. In two cases the mutations were 2 bp and 1 bp deletions identified in exons 3 and 17 respectively. The third mutation was a 1 bp insertion in exon 13. All three mutations lead to the generation of downstream premature stop codons as a result of frameshift changes, although the mutation in exon 3 possibly affects the splicing mechanism. The sites within the RB1 gene where these mutations occur contain interspersed repetitive DNA sequences, direct and inverted repeat sequences and/or dyad symmetrical elements suggesting that these areas promote the appropriate local sequence environment for the generation of deletions and insertions in the RB1 gene.

Amino Acid Sequence↗

PubMed1992

Recognition of a tetranucleotide loop of signal recognition particle RNA by protein SRP19.

The interaction of protein SRP19 with the RNA component of human signal recognition particle (SRP) was studied by site-directed mutagenesis of the SRP RNA. The effects of nucleotide changes in the tetranucleotide loop (tetraloop) of helix 6 showed that SRP19 recognizes a tetraloop in a sequence-specific manner. Adenosine 149 at the third position of the tetraloop was essential for binding. In contrast, changes of the base at the second position had no effect. Mutations that disrupt or compensate individual SRP RNA helices were generated to investigate the importance of base pairing and to identify other binding sites. Considerable base pairing was essential in helix 6. Another SRP19-binding site was located in the distal part of helix 8. The primary sequences of the tetraloop-binding protein SR19 and of bacterial ribosomal protein S15 are shown to be similar.

Amino Acid Sequence↗

PubMed1991

Use of high coverage reference libraries of Drosophila melanogaster for relational data analysis. A step towards mapping and sequencing of the genome.

Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been generated. Also, a jumping library has been created by a new method that takes advantage of methylation differences between genomic DNA and vector. Thirdly, two cDNA libraries have been picked. All these libraries have been arrayed on high-density in situ filters, each containing 9216 clones. As a reference system, such filters are distributed and identified clones are provided. Single-copy probes have identified on average 1.4 cosmids per genome equivalent. Together with cytogenetically mapped yeast artificial chromosomes, the libraries are also being used for physically mapping the genome, mainly by oligonucleotide fingerprinting and pool hybridizations. cDNA clones are further examined by a partial sequencing analysis by oligomer hybridization.

Animals↗

PubMed1994

A molecular phylogeny of the gopher tortoises, with comments on familial relationships within the Testudinoidea.

Sequences from the mitochondrial cytochrome b gene were obtained to examine molecular phylogenetic relationships among the North American gopher tortoises. Data from 352 aligned positions generated a single most-parsimonious tree for each of three analytical approaches: (1) equal weighting, all substitutions; (2) equal weighting, third position changes limited to transversions; and (3) transversions weighted 10 times transitions. Identical topologies for the resulting trees depict the gopher tortoises as a monophyletic group comprising two well-defined clades. Observed sequence divergence (7.0%) between the agassizii (Gopherus agassizii; G. berlandieri) and the polyphemus (G. flavomarginatus; G. polyphemus) clades suggests an Early Miocene separation of these lineages. The cytochrome b phylogeny complements some previous systematic interpretations, including formal taxonomic recognition of the two distinct groups, but is at odds with the most recent morphological analysis. Additional sequence comparisons of selected testudinoid (batagurid, emydid, and testudinid) taxa yielded a phylogeny consistent with a morphologically based hypothesis demonstrating close phylogenetic affinities between the Testudinidae and the "Bataguridae."

Animals↗

PubMed1998

Hybrid membrane IgM with the transmembrane region of I-A alpha facilitates enhanced presentation of distinct epitopes to T cells.

The role of B cell Ag receptors (membrane Ig or mIg) in the efficient Ag presentation to T cells, including the requirement of mIgM-associated Ig alpha/Igbeta, remains unclear. We report here that mIgM, substituted with greater than two-thirds of the NH2-terminal A alpha transmembrane (TM) regions of the MHC class II molecule, are capable of mediating the efficient presentation of specific Ag to some (Group 1) but not all (Group 2) T cell hybridomas. In contrast, the generation of epitopes recognized by the Group 2 hybridomas can be mediated only by the wild-type mIgM. Tyrosine phosphorylation appears to be necessary for the enhanced Ag presentation to Group 2 hybridomas, while it does not for Group 1 hybridomas. In addition, differential sensitivity of Ag processing to leupeptin, different duration required for epitope generation/presentation, as well as the involvement of distinct epitopes for stimulation of these groups of T cell hybridomas were observed. These results suggest that transport of the mIgM/Ag complexes to an endocytic compartment(s) for generation of certain T cell epitopes may be mediated by the N-terminal TM sequence of mIgM, independent of Ig alpha/Igbeta association. This function can be replaced by two-thirds of the NH2-terminal TM region of A alpha chain of class II molecules.

Amino Acid Sequence↗

PubMed1997

Construction of a gene encoding the insect bactericidal protein attacin. Studies on its expression in Escherichia coli.

Attacin, a bactericidal small protein is produced by the giant silk moth Hyalophora cecropia. This paper deals with our efforts to clone the attacin cDNA in a bacterial vector to express it in Escherichia coli and produce the protein in sufficient amount, for further studies. We chose two inducible expression vector/bacterial cell systems: pPL-lambda/N99cI+ cells which is able to be induced by nalidixic acid, and pET3d/BL21(DE3) cells carrying a T7 RNA polymerase gene which is IPTG-inducible. After cloning in the pPL-lambda system and under no addition of the inducer, isolated transformants carried this plasmid with at least 2 concurrent deletions that drastically affected attacin expression, even though attacin gene seems to be intact as deduced by its PCR amplification. It was concluded that basal attacin expression occurred in this system and bacterial growth was limited. Plasmid deletions may have emerged by selection pressure as a way to avoid bactericidal expression and allow bacteria survival. The second cloning attempt was done in pET3d vector/BL21 cells, that should not express the cloned sequence (they lack T7 RNA polymerase gene). Transformed BL21 cells gave 3 recombinant plasmids, 2 of them presented a C deletion that generated an early stop signal in the attacin coding region. The third clone, pET-ATT18, carrying an intact gene, was transferred to BL21(DE3)-IPTG inducible cells in order to be expressed. Attacin was undetectable in stained gels or by Western blot analysis. However, expression was visualized in grown cells after 30 min of IPTG induction and 5 min of [35S]-methionine labeling, as a 22.5 kDa protein band by using gel electrophoresis and fluorography. This low level of expression drastically affected bacterial growth. Considering that attacin has no lytic activity, these results suggest that this molecule should block bacterial growth directly at the cytoplasm by an unknown mechanism, since no signal peptide coding sequence was incorporated in this gene construction, precluding periplasmic or external destination of this protein.

Anti-Bacterial Agents↗

PubMed1993

Cloning of the rat aortic smooth muscle Na+/Ca2+ exchanger and tissue-specific expression of isoforms.

The amino acid sequences of two isoforms of the rat aortic smooth muscle Na+/Ca2+ exchanger have been deduced by cloning and sequencing the cDNAs. These isoforms are identical in nucleotide sequence except that one has a 23-amino acid insertion at amino acid position 570. They are highly homologous to the canine cardiac exchanger except for the NH2-terminal portion and part of the large central hydrophilic domain (amino acid residues 570-631). They are 902 and 925 (with the insertion) amino acid long with calculated molecular masses of 100,676 and 103,200 (with the insertion), respectively, if the NH2-terminal 32-amino acid residues are eliminated as a cleaved signal sequence. Amplification of the variable region (amino acids 570-631) of the exchanger by means of the reverse transcriptase-polymerase chain reaction and DNA sequencing revealed that many isoforms of the exchanger are expressed in different rat tissues. The two clones isolated in this study are the predominant isoforms expressed in aorta, stomach, liver, and kidney. In cardiac and skeletal muscles, another isoform is dominant, which is equivalent to the canine cardiac exchanger. In brain, a third type is predominantly expressed. Alignment of the nucleotide sequences of these isoforms and Southern blot analysis of rat genomic DNA suggested that each isoform is generated through alternative splicing of the primary transcript.

Alternative Splicing↗

PubMedDate not supplied

The mutation causing the common apolipoprotein A-IV polymorphism is a glutamine to histidine substitution of amino acid 360.

Apolipoprotein (apo) A-IV is a protein involved in the metabolism of chylomicrons and high density lipoproteins. This protein displays genetic polymorphism due to two main codominant alleles, A-IV1 and A-IV2. We have identified the mutation that leads to this polymorphism. It is caused by a single-base substitution of guanine for thymine in the third base of codon 360. This substitution leads to a glutamine to histidine change. Direct sequencing of amplified DNA from eight subjects in a three-generation pedigree has demonstrated that the guanine to thymine substitution can explain the apo A-IV polymorphism. In 32 unrelated individuals, a correspondence between apo A-IV phenotype determined by isoelectric focusing and genotype determined with Fnu4HI digestion of amplified DNA could be demonstrated. The enzyme lecithin:cholesteryl acyltransferase (LCAT) is activated by apo A-IV. Under our in vitro conditions, the isoprotein apo A-IV 1-1 is a better LCAT activator than is the isoprotein apo A-IV 2-2. A knowledge of the molecular mechanism underlying the apo A-IV polymorphism will help to elucidate the mechanisms involved in LCAT activation.

Amino Acid Sequence↗

PubMed1997

Characterization and amino acid sequence of the OXY-2 group beta-lactamase of pI 5.7 isolated from aztreonam-resistant Klebsiella oxytoca strain HB60.

Klebsiella oxytoca strain HB60 is highly resistant to cefoperazone and aztreonam (MICs = 128 mg/L). It produces a chromosomally encoded beta-lactamase of pI 5.7 which was highly efficient against penicillins, first-generation cephalosporins and cefoperazone, a non-oxyimino third-generation cephalosporin. Aztreonam and oxyimino broad-spectrum cephalosporins were less good substrates. The beta-lactamase activity was susceptible to inhibition by clavulanic acid (IC50 = 1 microM). The enzyme purified to homogeneity had a specific activity towards benzylpenicillin of 3670 U/mg. The 263 amino acid residues of the protein were sequenced by Edman degradation of proteolytic peptides. The beta-lactamase was shown to belong to the OXY-2 group as it had only one amino acid substitution (Asn for Asp at ABL position 197) compared with the beta-lactamase (pI 5.2) from the aztreonam-susceptible K. oxytoca strain SL911 and two substitutions (Ala223 for Val and Asp255 for Asn) compared with the beta-lactamase (pI 6.4) from the aztreonam-resistant K. oxytoca strain D488. These three OXY-2-group enzymes behave in the same way towards beta-lactam antibiotics. The variability in the resistance of these K. oxytoca strains would thus seem to be due to variation in the level of production of the beta-lactamases rather than to structural alteration of the enzymes.

Amino Acid Sequence↗

PubMed1990

Detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin hypervariable region specific oligonucleotide probes.

To develop a sensitive and specific assay for minimal residual disease in acute lymphoblastic leukemia (ALL), we exploited the enormous diversity of genomic sequences created by immune receptor gene rearrangements. To isolate clone-specific sequences, we first synthesized oligonucleotides that match conserved variable (VH) and joining (JH) sequences flanking the third hypervariable region (HVR3) in the rearranged immunoglobulin heavy chain (IgH) locus. In polymerase chain reactions (PCR), these primers were then used to amplify the intervening HVR3 segments from leukemic DNA samples. Of 12 B-lineage ALLs studied, ten generated one or more fragments of the size expected for HVR3 gene segments. Thus, this single pair of amplimers was sufficient to isolate HVR3 sequences from a majority of acute lymphoblastic leukemias. To verify that the amplified fragments originated from HVR3 alleles and to assess their diversity, we sequenced 7 PCR products derived from 6 leukemias. In addition to elements of recognized D segments, each of the 7 fragments contained novel VH-D and D-JH junctional sequences, including N nucleotides, not known to be present in the germline. Each sequence was unique, and allele-specific oligonucleotide probes hybridized only to HVR3 segments from which the probes were derived. Therefore, as anticipated, these HVR3 segments appeared to possess the diversity required to serve as clonal markers for leukemic populations. To demonstrate that these amplified HVR3 alleles could serve as the basis for a sensitive and specific assay to detect rare leukemic cells, we analyzed in detail one pre-B leukemia that had rearranged 2 IgH alleles. The HVR3 sequences were shown to be linked to rearranged JH-containing restriction fragments in digests of genomic DNA, establishing their origin in the leukemic cells. We synthesized oligonucleotides corresponding to the unique junctional sequences in the HVR3 segments. Using these novel amplimers in an allele-specific amplification and hybridization procedure, we showed that this assay can detect 10 leukemic cells in a background of 10(6) normal blood mononuclear cells. In contrast, the leukemic HVR3 sequences were not detected in extracts of normal or unrelated remission leukemic leukocytes. We conclude that the assay for specific IgH HVR3 sequences is a realistic strategy for detection of minimal residual disease in B-lineage ALL.

Alleles↗

PubMed1990

Molecular structure of microtubule-associated protein 2b and 2c from rat brain.

Full length cDNA clones encoding microtubule-associated proteins (MAP) 2b and 2c from rat brain have been isolated and sequenced. The cDNA fragments spanning the coding regions for both MAP2b and MAP2c were assembled and expressed in Escherichia coli. The mobility of these bacterial expressed proteins in sodium dodecyl sulfate gels is identical to that of MAP2b and MAP2c from rat brain. The protein sequence of rat MAP2b has been compared to the full length sequence from mouse and the partial sequence from human high molecular weight MAP2. This comparison has revealed that MAP2b is composed of several highly conserved domains flanked by domains with extensive sequence divergence. Two of the conserved domains, found either at the NH2 or COOH terminus, overlap with the binding domain for the regulatory subunit of the cAMP-dependent protein kinase II and the microtubule-binding domain, respectively. A third homologous domain of unknown function lies in a central region of MAP2b. Secondary structure prediction suggests that the portion of MAP2b which extends from the microtubule surface is composed of an extensive number of alpha-helices separated by small turns which may account for the extended yet flexible structure of MAP2. Interestingly, the 4000-base pair deletion from the middle of MAP2b which generates MAP2c not only removes these helices, but also this third highly conserved MAP2b domain.

Amino Acid Sequence↗

PubMed1992

Differential splicing in the extracellular region of fibroblast growth factor receptor 1 generates receptor variants with different ligand-binding specificities.

We have cloned a genomic region of the murine fibroblast growth factor (FGF) receptor 1 (FGFR1) gene that includes three alternative exons for the third immunoglobulinlike domain in the extracellular region of the receptor. The mRNA of one of these splice variants encodes a secreted receptor that lacks transmembrane and cytoplasmic sequences as well as a portion of the third immunoglobulinlike domain. Highest levels of mRNA encoding this variant were found in brain, skeletal muscle, and skin. We expressed this form of FGFR1 in CHO cells and showed that the recombinant secreted protein binds acidic FGF. We also discovered a novel alternative exon in the third immunoglobulinlike domain that encodes part of a transmembrane FGFR1 mRNA. This exon is highly homologous to the corresponding region of the keratinocyte growth factor receptor. Transcripts including this exon were present at highest levels in the skin. We cloned an FGFR1 cDNA which includes this exon and expressed this receptor variant in L6 rat skeletal muscle myoblasts. The new receptor variant had a 50-fold-lower affinity for basic FGF than does the published FGFR1 variant, whereas both forms of receptor bound acidic FGF with high affinity. These results show that the third immunoglobulinlike domain plays an important role in determining the binding specificities for different FGFs. Our data provide the first evidence that differential splicing in the extracellular region of a receptor gene generates receptor variants with different ligand-binding specificities.

Amino Acid Sequence↗

PubMed1997

Free radical induced polymerization of synthetic peptides into polymeric immunogens.

Free radical induced polymerization of vinyl monomers such as the acryloyl peptides described here is a facile and rapid reaction used routinely, for example, in the polymerization of acrylamide and bisacrylamide for the assembly of polyacrylamide gels. The technology allows the incorporation of many of the same or different peptide determinants into a single polymer chain. In this study large polymers containing multiple copies of peptides representing T- and B-cell determinants of influenza haemagglutinin were constructed. The determinants retained antigenicity after the polymerization procedure and the polymers were highly immunogenic; the levels of antibody obtained after a single dose of polymeric immunogen were at least as great as those achieved only after repeated doses of the equivalent monomeric peptide. The technology has a wide range of potential applications, not the least significant of which is the construction of designer immunogens for third generation vaccine candidates.

Amino Acid Sequence↗

PubMed1991

P300 generation by novel somatosensory stimuli.

Event-related potentials (ERPs) to task-relevant target and task-irrelevant novel stimuli were recorded in a somatosensory discrimination task. Subjects pressed a button to mechanical taps of the fifth finger (targets, P = 0.12), randomly interposed in sequences of taps to the second finger (standards, P = 0.76). Two types of infrequent novel stimuli were delivered; one was a mechanical tap to the third or fourth finger (tactile novels, P = 0.06), another was an electric shock at the wrist (shock novels, P = 0.06). Correctly detected targets generated a parietal maximal P300 (P3b, latency 335 msec). Shock novels generated a central maximal P300 with a shorter peak latency (298 msec) than the P3b. Tactile novels generated a P300 with a scalp distribution comparable to the shock novels. Unlike the P3b, P300 amplitude to both the shock and tactile novel stimuli habituated by 20-30% across the first several stimulus presentations. These results indicate that, similar to the auditory and visual modality, task-irrelevant novel somatosensory stimuli generate a novelty P300 ERP. Differences in scalp distribution, latency and habituation characteristics suggest that the novelty P300 may have contributions from intracranial generators independent from target P300 sources.

Adolescent↗