SEARCH · PubMed Health

Results for “second generation sequencing”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 865 records · Page 48Linked to original sources

PubMed1997

Integration of 101 DNA markers across human Xp11 using a panel of somatic cell hybrids.

One hundred and one DNA markers previously assigned to the short arm of the human X chromosome were localized on a hybrid mapping panel consisting of ten radiation-reduced, and four classical somatic cell hybrids. Of the 101 DNA markers, 16 are genes, two are pseudogenes, 13 are expressed sequence tags, 32 are simple tandem repeats (STRs), four are restriction fragment length polymorphisms, one is a variable number of tandem repeats, and 33 are sequence tagged sites (STSs). Three of these markers, two STSs and one STR, were generated from the products of an inter-Alu PCR library of a radiation-reduced hybrid containing Xp11.4-->p11.22 as its only human DNA content. A second STR was isolated from a region-specific cosmid containing the gene ZNF21. The 101 DNA markers fell into 22 bins based on their retention on the hybrids of this panel, which, in combination with YAC contig data, could be further resolved into 24 bins. This hybrid map of Xp11 has an average resolution of approximately 0.8 Mb.

Animals↗

PubMed1991

Ethanol euthanasia and its effect on the binding of antibody generated against an immunogenic peptide construct.

Mice were immunised with an immunogenic peptide construct CKNNNSTNSGI coupled to diphtheria toxoid as a carrier. This peptide sequence contains the epitope STNS which is the target of inhibitory monoclonal antibodies directed against the second merozoite surface antigen of Plasmodium falciparum. Antisera raised against the peptide construct were taken using an injection of 70 per cent ethanol or sodium pentobarbitone as methods of euthanasia and these methods compared by determining their effects on the binding specificity of the antibody to the antigen using the immunological criteria of immunofluorescence, immunoblotting criteria of immunofluorescence, immunoblotting and ELISA assays. There was no significant decrease in antibody binding with either sodium pentobarbitone, or ethanol with a final concentration of less than 30 per cent in mouse antisera. Antisera with an added ethanol concentration of 40 to 60 per cent relaxed antibody conformation and this raises the possibility of using the differential effects of ethanol as a tool in mapping antigenic fine structure of a range of antibodies directed against defined epitopes. The cross-reactive response of non-specific antibodies in polyclonal antisera was lowered at the suggested dosage for ethanol euthanasia. Ethanol has immense potential as an alternative method of euthanasia when barbiturate drugs, such as sodium pentobarbitone, are unavailable in specific experimental protocols. This may especially aid research workers in developing countries involved in vaccine development, antibody production and subsequent serological analysis.

Amino Acid Sequence↗

PubMed1995

DNA sequence preferences at sites cleaved by human DNA topoisomerase II in response to novel quinolone derivatives.

We have examined the DNA cleavage site specificity of human type II DNA topoisomerase in the presence of each of five novel quinolone derivatives. Each quinolone derivative inhibited the human enzyme, inducing double-strand breaks with a four-base stagger. Break sites generated in response to each derivative had a predominance of C in the 3'-terminal position. Consensus sequences derived for cleavage sites induced by each derivative were strikingly similar, not only at the 3'-terminal position, but also at additional positions on either side of the broken phosphodiester bond. Analysis of these consensus sequences yielded information about possible interactions of specific substituents on the quinolone derivatives with DNA and/or topoisomerase. Comparison of the quinolone-based consensus sequences with those derived for cleavage sites generated by the human type II topoisomerase in the presence of either m-AMSA or VM-26, or in the absence of drug, provided compelling evidence that DNA cleavage sites include two domains: one which interacts with drug and a second, larger domain which interacts with topoisomerase.

Amsacrine↗

PubMedDate not supplied

DNA fingerprinting of Trypanosoma vivax isolates rapidly identifies intraspecific relationships.

Using the polymerase chain reaction and arbitrarily selected oligonucleotide primers of 10 or 11 bases, we have amplified DNA sequences from Trypanosoma vivax parasites isolated from South America and Africa. On the basis of polymorphisms in the DNA fingerprints generated by three of the primers, the parasites could be separated into two major groups, one comprising T. vivax isolates from Kenya and the second including all the other T. vivax parasites (from Colombia, The Gambia, Nigeria and Uganda). One of these three primers (ILo 525) also gave isolate-specific DNA fingerprints for the parasites tested, which will allow the use of this technique both in the species identification and discrimination of T. vivax parasites.

Animals↗

PubMed1995

A sequence property approach to searching protein databases.

Currently available sequence alignment programs are generally not capable of detecting functional and structural homologs in the twilight zone of sequence similarity, i.e. when the sequence identity falls below about 25%. Here we attempt to detect such weak similarities using an approach based on a notion of protein sequence similarity radically different from that used in sequential alignment. The approach defines protein sequence dissimilarity (or distance) as a weighted sum of differences of compositional properties such as singlet and doublet amino acid composition, molecular weight, isoelectric point (protein property search or PropSearch). With PropSearch, either single sequences can be used for a database query, or multiple sequences can be merged into an "average" sequence reflecting the average composition of a protein family. First, we show that members of structural protein families have a low mutual PropSearch distance when the weights are optimized to discriminate maximally between structural families. Second, we demonstrate the results of database searches using the PropSearch method. Such searches are very rapid when scanning a preprocessed database and do not require alignments. In cases in which conventional alignment tools fail to detect similarities, PropSearch can be used to generate hypotheses about possible structural or functional relationships between a new sequence and sequences in the database.

Algorithms↗

PubMed1986

Alternative RNA splicing generates transcripts encoding a thorax-specific isoform of Drosophila melanogaster myosin heavy chain.

Genomic and cDNA sequencing studies show that transcripts from the muscle myosin heavy-chain (MHC) gene of Drosophila melanogaster are alternatively spliced, producing RNAs that encode at least two MHC isoforms with different C termini. Transcripts encoding an MHC isoform with 27 unique C-terminal amino acids accumulate during both larval and adult muscle differentiation. Transcripts for the second isoform encode one unique C-terminal amino acid and accumulate almost exclusively in pupal and adult thoracic segments, the location of the indirect flight muscles. The 3' splice acceptor site preceding the thorax-specific exon is unusually purine rich and thus may serve as a thorax-specific splicing signal. We suggest that the alternative C termini of these two MHC isoforms control myofilament assembly and may play a role in generating the distinctive myofilament organizations of flight muscle and other muscle types.

Amino Acid Sequence↗

PubMed2004

The left end of IS2: a compromise between transpositional activity and an essential promoter function that regulates the transposition pathway.

Cut-and-paste (simple insertion) and replicative transposition pathways are the two classical paradigms by which transposable elements are mobilized. A novel variation of cut and paste, a two-step transposition cycle, has recently been proposed for insertion sequences of the IS3 family. In IS2 this variation involves the formation of a circular, putative transposition intermediate (the minicircle) in the first step. Two aspects of the minicircle may involve its proposed role in the second step (integration into the target). The first is the presence of a highly reactive junction formed by the two abutted ends of the element. The second is the assembly at the minicircle junction of a strong hybrid promoter which generates higher levels of transposase. In this report we show that IS2 possesses a highly reactive minicircle junction at which a strong promoter is assembled and that the promoter is needed for the efficient completion of the pathway. We show that the sequence diversions which characterize the imperfect inverted repeats or ends of this element have evolved specifically to permit the formation and optimal function of this promoter. While these sequence diversions eliminate catalytic activity of the left end (IRL) in the linear element, sufficient sequence information essential for catalysis is retained by the IRL in the context of the minicircle junction. These data confirm that the minicircle is an essential intermediate in the two-step transposition pathway of IS2.

Base Sequence↗

PubMed1992

Patterns of serological markers in transfusion-transmitted hepatitis C virus infection using second-generation HCV assays.

A semiautomated dot blot assay and cDNA polymerase chain reaction (PCR) were used to study longitudinal anti-hepatitis C virus (HCV) recognition patterns in relation to presence of HCV-RNA in transfusion recipients and their infectious donors. In 9 recipients, 4 different patterns of HCV infection were observed: (A) persistent HCV carriage accompanied by chronic hepatitis in 6, (B) acute resolved hepatitis, but persistent HCV replication in one, and (C) continuous HCV replication without hepatitis in one and (D) acute resolved hepatitis with clearance of infection in one. This last self-limited infection was characterized by the disappearance of HCV-RNA as well as anti-HCV reactivity. In contrast, antibody reactivity persisted in 7 of 8 patients with chronic HCV infection who could be followed until 1990. Seven of the 9 recipients developed antibodies to all recombinant peptides in dot blot assay; one became positive for anti-C33 and anti-core and one developed anti-core only. The sequence of appearance of antibodies differed among individual patients. In 7 patients with full anti-HCV recognition patterns, the sequence of events was (mean and limits in days after transfusion): onset of hepatitis at day 50 (22-74), seroconversion of anti-C33 at day 91 (59-129), anti-core at day 133 (54-203), and anti-C100 at day 143 (59-365). The incorporation of C33 and core proteins, in addition to C100, in the second generation anti-HCV ELISA enhanced the detection rate in the HCV-infected transfusion recipients from 7/9 (78%) to 9/9 (100%).(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers↗

PubMed1982

Diverse mechanisms in the generation of human beta-tubulin pseudogenes.

The sequence of two human beta-tubulin pseudogenes is described. One contains an intervening sequence but lacks sequences encoding the 55 N-terminal amino acids of the polypeptide chain. A second has no introns but has a polyadenylate signal and an oligoadenylate tract at its 3' end, and it is flanked by a short direct repeat. These sequences have arisen by different mechanisms, including one that probably involves reverse transcription of a processed messenger RNA and reintegration of the complementary DNA copy into the genome.

Base Sequence↗

PubMed1997

Analysis of immunoreceptor tyrosine-based activation motif (ITAM) binding to ZAP-70 by surface plasmon resonance.

The signaling function of the T cell antigen receptor (TCR) is mediated via CD3 polypeptides, the cytoplasmic sequences of which bear conserved immunoreceptor tyrosine-based activation motifs (ITAM). ITAM are defined by two YxxL/I sequences separated by a six-eight amino acid long spacer. Upon antigen recognition, ITAM become phosphorylated on both tyrosine residues, creating a high affinity binding site for the tandem SH2 domains found in the protein tyrosine kinase ZAP-70. Using surface plasmon resonance, we further dissected the sequences required for the binding of ZAP-70 to each TCR-associated ITAM. First, we generated protein tyrosine phosphatase-resistant ITAM peptide analogs, in which difluorophosphonomethyl phenylalanyl (F2p) replaced both phosphotyrosines, and showed that those protein tyrosine phosphatase-resistant analogs bind ZAP-70 with high affinity, establishing a rational strategy for the design of novel pharmacological tools capable of interfering with TCR signaling function. Second, we substituted the five amino acids separating the two YxxL/I sequences of the CD3 zeta 1 ITAM with a non-peptidic linker made up of gamma-amino butyric acid units and demonstrated that the length of this intervening sequence rather than its chemical composition is essential for high affinity binding of phosphorylated ITAM to the ZAP-70 SH2 domains.

Amino Acid Sequence↗

PubMed1992

Sequence evolution in mitochondrial ribosomal and ND-1 genes in lepidoptera: implications for phylogenetic analyses.

A 2,256-bp sequence of the mitochondrial genome of a lepidopteran (Spodoptera frugiperda) contains tRNAs for valine and leucine, the 16S rRNA, and three-quarters of the ND-1 presumptive protein-coding gene. A 64-bp stretch of unknown function was located between the rRNA and leucine tRNA. Sequence divergence in the 16S rRNA obtained from alignment with published insect sequences is consistent with phylogenetic hypotheses, in that Diptera and Lepidoptera are more closely related to each other (24% sequence divergence) than either is to Hymenoptera (31%). Within the ND-1 gene, sequences for four additional Lepidoptera were generated for a 314-bp region and contrasted with published sequences for the locust and Drosophila. Sequence divergence in this region was consistent with accepted phylogenetic relationships, but results of parsimony analyses were not. Cladograms consistently recovered accepted higher level relationships (monophyly of Lepidoptera), despite high homoplasy, but were unable to resolve superfamily and family relationships within Lepidoptera, regardless of the outgroup or character subset analyzed. Character analysis indicated that homoplasy was decreased at higher levels when first- and second-codon sites were used exclusively. At the lowest level (families), resolution was enhanced by inclusion of third-codon sites. Inability of molecular data to recover a well-established phylogeny may be rectified by additional characters or taxa, but it is clear that homoplasy is sufficiently high to caution against the acceptance of relationships generated with this molecular region that are not extremely robust.

Amino Acid Sequence↗

PubMed2002

Proteolytic processing of the hepatitis B virus e antigen precursor. Cleavage at two furin consensus sequences.

The Hepatitis B virus P22 protein is a nonstructural protein that is the precursor of the 17-kDa secreted e antigen (HBeAg). The mature HBeAg is obtained after the removal of the C-terminal region of P22, a process which involves a proprotein convertase. Our studies show first that the protease could cleave P22 at the C-terminal side of Arg(167) or Arg(154) and second, that the maturation process can be either done in one step or in two steps with the generation of a processing intermediate (P20). Our data also demonstrate that the removal of the P22 C terminus, which occurs mainly in the trans-Golgi network, can also be achieved after exocytosis. Keeping in mind this characteristic and the amino acid sequence of the cleavage sites, we concluded that furin is involved in the maturation of the HBeAg. In addition, we show that in our experimental system, the HBeAg is a 164-amino acid protein and not a 159-amino acid protein as previously reported.

Amino Acid Sequence↗

PubMed1999

Innate antibody catalysis.

Catalysis by antibodies is often assumed to require immunization with artificial haptens, which are proposed to stimulate adaptive immune processes and enable the development of catalytic sites with the ability to bind the transition state. Contrary to this assumption, we describe here a serine protease-like catalytic triad in an antibody light chain raised by immunization with vasoactive intestinal peptide (VIP), the structure and function of which is inherited via a germline V(L) gene. The serine protease mechanism was evident from loss of the catalytic activity by site directed mutagenesis at a framework region residue Asp1 (present study) and at two complementarity determining region residues Ser27a and His 93 (Gao, Q-S., Sun, M., Rees, A., Paul, S., 1995. Site-directed mutagenesis of proteolytic antibody light chain. J. Mol. Biol. 253, 658-664). All three catalytic residues (Ser27a, His93, Asp1) are also present in the germline counterpart of the mature V(L) gene, but the mature and germline sequences differ by four amino acids remote from the catalytic site. Reversion mutations were introduced at these amino acids in the mature light chain (His27 d:Asp, Thr28e:Ser, Ile34:Asn, Gln96:Trp; Kabat numbering, germline encoded residues shown second), generating the germline configuration of the protein. The germline light chain expressed peptidase activity, determined by assaying the cleavage of VIP and a synthetic protease substrate, Pro-Phe-Arg-Methylcoumarinamide. Differences between the kinetic constants for the mature and germline light chains were marginal. Diisopropylfluorophosphate, a serine protease inhibitor, blocked the peptidase activity of the germline light chain, suggesting the presence of the catalytic triad in a functional state. Like the mature light chain, the germline protein preferentially cleaves peptide bonds on the C-terminal side of basic residues. We conclude that the catalytic activity of certain antibodies is an innate function, originating over the course of phylogenetic evolution of the V(L) genes, as opposed to somatic processes.

Amino Acid Sequence↗

PubMed2003

Signal-anchor domains of proteins of the outer membrane of mitochondria: structural and functional characteristics.

We have studied the topogenesis of a class of mitochondrial outer membrane proteins that expose a hydrophilic domain to the cytosol and are anchored to the membrane by a single transmembrane domain in the N-terminal region. To determine the role of these latter sequences in the targeting and insertion of such proteins we took two approaches. First, a functional complementation assay was used to define the structural elements that together with the anchor domain make up the topogenic signal. Moderate hydrophobicity of the transmembrane domain was found to be the most important requirement. Variants with a scrambled sequence of the membrane-spanning segment were only partially functional suggesting that specificity in the amino acid sequence is also of considerable importance. A net positive charge at both flanking regions of the transmembrane domain contributes to the efficiency of targeting and membrane integration but is not an essential structural feature of this signal. Second, chimeras of Tom20, Tom70, and OM45 were generated that contained the cytosolic domain of Tom20 or Tom70 and the anchor domain of one of the other members of the class. These hybrid proteins were able to rescue the growth of cells lacking Tom20 or Tom70. Thus, anchor domains of outer membrane proteins are functionally interchangeable. They play only a minor role in the specific function of these proteins, but have a decisive role in topogenic signaling.

Amino Acid Sequence↗

PubMed2005

Depletion of CD25+ cells from human T-cell enriched fraction eliminates immunodominance during priming with dendritic cells genetically modified to express a secreted protein.

The ability of dendritic cells (DCs), genetically modified with one of two types of plasmid DNA vaccines to stimulate lymphocytes from normal human donors and to generate antigen-specific responses, is compared. The first type, also called "secreted" vaccine (sVac), encodes for the full length of the human prostate-specific antigen (PSA) with a signal peptide sequence so that the expressed product is glycosylated and directed to the secretory pathway. The second type, truncated vaccines (tVacs), encodes for either hPSA or human prostate acidic phosphatase (hPAP), both of which lack signal peptide sequences and are retained in the cytosol and degraded by the proteasomes following expression. Monocyte-derived dendritic cells are transiently transfected with either sVac or one of two tVacs. The DCs are then used to activate CD25+-depleted or nondepleted autologous lymphocytes in an in vitro model of DNA vaccination. Lymphocytes are boosted following priming with transfected DCs, peptide-pulsed DCs or monocytes. Their reactivity is tested against tumor cells or peptide-pulsed T2 target cells. Both tVacDCs and sVacDCs generate antigen-specific cytotoxic T-cell responses. The immune response is restricted towards one of the three antigen-derived epitopes when priming and boosting is performed with sVacDCs. In contrast, tVac-transfected DCs prime T cells towards all antigen-derived epitopes. Subsequent repeated boosting with transfected DCs, however, restricts the immune response to a single epitope due to immunodominance. While CD25+ cell depletion prior to priming with sVacDCs alleviates immunodominance, cotransfection of dendritic cells with GITR-L does so in some but not all cases.

Animals↗

PubMed1984

Multiple origins of transcription for the human placental lactogen genes.

Transcriptional activity of the three human placental lactogen (hPL) genes was compared in vitro and their relation to the hPL RNA species obtained from term placenta was analyzed. We used in vitro transcription system containing the HeLa cell crude extract as the source for RNA polymerase II and initiation factors. Gene fragments of identical length of 0.96 kilobase pair made from hPL1, hPL3, and hPL4 each contained the 5' flanking sequence of 497 base pairs, the first exon, the first intron, and a portion of the second exon. More than 90% transcripts of the hPL1 template were 470 nucleotides long, indicating that transcription was initiated at the proposed cap site. hPL3 and hPL4 genes generated heterogeneous RNA products of about 430, 470, 520, and 680 nucleotides suggesting that multiple start points were recognized for RNA synthesis in vitro. alpha-Amanitin sensitivity of transcription indicated that the DNA-dependent RNA synthesis was carried out by RNA polymerase II. These results show that hPL1, hPL3, and hPL4 genes have functional promoters and multiple initiation sites for transcription. Primer extension analysis of the 5' termini of hPL RNA isolated from term placenta shows that 82-83% of the transcripts are initiated at a region 29 base pairs downstream from a "TATA" sequence. This origin is observed in vitro for the transcript 470 nucleotides long. An additional upstream initiation region (-53) accounts for 8% of transcripts in term placenta and corresponds to the origin for the in vitro transcript of 520 nucleotides. At least three other sites 15, 23, and 39 base pairs downstream from the major cap site are functional in vivo. The initiation site at +40 is utilized preferentially for transcription from hPL3 and hPL4 genes in vitro. We have mapped the different transcription origins on hPL genes.

Base Sequence↗

PubMed1991

Localization of the calmodulin- and the actin-binding sites of caldesmon.

Expression of the C-terminal third of chicken gizzard caldesmon in Escherichia coli, using the Nagai vector (Nagai, K., and Thøgersen, H.V. (1987) Methods Enzmol. 153, 461-481), produces a cII-caldesmon fusion protein (27 kDa) with caldesmon sequence beginning at Lys579. Degradation during purification yields five peptides with molecular masses of 24, 22, 19 (two peptides), and 15 kDa. The 24-kDa peptide begins at Phe581; the 22-kDa peptide begins at Leu597, the two 19-kDa peptides begin at Phe581 and Val629, respectively; the 15-kDa peptide also begins at Val629. We estimate that the 15-kDa and one of the 19-kDa peptides end near Leu710. Site-directed mutagenesis was used to produce truncated peptides with known C termini; one peptide (17 kDa) terminates at Asn675. Digestion of the fragments with chymotrypsin generates a second 15-kDa fragment that begins at Ser666 (15K'). All of the peptides, with the exception of 15K', bind Ca(2+)-calmodulin-Sepharose and share a common 37-amino acid peptide between Val629 and Ser666, suggesting this contains the calmodulin binding site. Comparison with published sequences (Takagi, T., Yazawa, M., Ueno, T., Suzuki, S., and Yagi, K. (1989) J. Biochem. (Tokyo) 106, 778-783 and Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238) for other calmodulin-binding fragments further restricts the binding site to 7 residues, Trp-Glu-Lys-Gly-Asn-Val-Phe, between Trp659 and Ser666. All of the fragments, except the two 15-kDa peptides, co-sediment with F-actin, indicating that there are two segments in the C-terminal third of caldesmon that can interact with F-actin: one between Leu597 and Val629, the other between Arg711 and Pro756. Although separated in the primary sequence, these domains may interact with the calmodulin-binding region in the folded structure.

Actins↗

PubMed1994

Cloning of a cDNA encoding the dense granule protein GRA3 from Toxoplasma gondii.

GRA3 is a 30-kDa protein located inside the dense granules of Toxoplasma gondii. Following invasion and exocytosis of dense granules within the parasitophorous vacuole, GRA3 becomes associated with the parasitophorous vacuolar membrane (PVM) and extensions of the PVM which protrude into the cytoplasm. A partial cDNA encoding GRA3 was isolated from a Toxoplasma gondii expression library using polyclonal and monoclonal antibodies to the mature GRA3 protein of tachyzoites. Antibodies affinity purified using the cloned fusion protein reacted with a 30-kDa band on immunoblots and recognized dense granules, the PVM, and PVM extensions by immunofluorescence staining of infected cells. Northern blot analysis indicated the major transcript was of a slightly larger size, and the complete cDNA encoding GRA3 was subsequently obtained. Southern blot analysis suggests that GRA3 is present as a single copy. The cDNA encodes two methionines at the N-terminus followed by an open reading frame with a hydrophobic region of 22 amino acids flanked by charged residues consistent with a signal sequence. Four shorter hydrophobic regions occur but are insufficient to span the membrane. No significant homology was detected to other proteins, including other dense granule proteins. In vitro translation of RNA generated from the cDNA containing either one or two of the N-terminal methionines yielded peptides with apparent M(r) of 35,000 and 37,000 respectively. Translation of RNA from the cDNA containing only the second initiation site in the presence of dog pancreas microsomes resulted in reduction of 4 kDa, sufficient to account for removal of the putative signal sequence.

Amino Acid Sequence↗