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Results for “second generation sequencing”

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At least 667 records · Page 37Linked to original sources

PubMed2000

Members of the Arabidopsis HRT/RPP8 family of resistance genes confer resistance to both viral and oomycete pathogens.

Turnip crinkle virus (TCV) inoculation onto TCV-resistant Arabidopsis leads to a hypersensitive response (HR) controlled by the dominant gene HRT. HRT is a member of the class of resistance (R) genes that contain a leucine zipper, a nucleotide binding site, and leucine-rich repeats. The chromosomal position of HRT and its homology to resistance gene RPP8 and two RPP8 homologs indicate that unequal crossing over and gene conversion may have contributed to HRT evolution. RPP8 confers resistance to an oomycete pathogen, Peronospora parasitica. Despite very strong similarities within the HRT/RPP8 family, HRT and RPP8 are specific for the respective pathogens they detect. Hence, the HRT/RPP8 family provides molecular evidence that sequence changes between closely related members of multigene families can generate novel specificities for radically different pathogens. Transgenic plants expressing HRT developed an HR but generally remained susceptible to TCV because of a second gene, RRT, that regulates resistance to TCV. However, several transgenic plants that overexpressed HRT produced micro-HRs or no HR when inoculated with TCV and were resistant to infection. Expression of the TCV coat protein gene in seedlings containing HRT resulted in massive necrosis and death, indicating that the avirulence factor detected by the HRT-encoded protein is the TCV coat protein.

Amino Acid Sequence↗

PubMed1992

The human cord blood antibody repertoire. Frequent usage of the VH7 gene family.

We have previously demonstrated a consistent preferential usage of a small set of VH, DH and JH gene segments in second-trimester fetal liver. To examine the extent of heavy chain repertoire diversification in newborns, we generated an unrestricted cDNA library from cord blood mononuclear cells and sequenced the variable domains of randomly isolated Cmu+ transcripts. We found this set of transcripts to be enriched for JH4, 5 and 6, whereas previously reported fetal transcripts preferentially expressed JH3 and 4. The cord blood transcripts used a number of different DH gene segments, whereas fetal transcripts were enriched for DHQ52. Of the thirteen cord blood sequenced, three were members of the newly described VH7 family which to date has not been detected in fetal liver. Indeed, only one of the isolated VH gene segments had been previously observed in fetal transcripts. As a result of enhanced N-region addition and use of longer DH and JH gene segments, both the sequence diversity and range of potential antigen-binding structures of cord blood complementarity-determining region 3 domains was vastly expanded. Thus, the repertoire bias evident in fetal liver was no longer apparent in this more mature population of cord blood B cells.

Amino Acid Sequence↗

PubMed1997

Distribution and characterization of natriuretic peptide receptors in the gills of the spiny dogfish, Squalus acanthias.

The distribution and nature of natriuretic peptide receptors (NPR) in the gills of dogfish, Squalus acanthias, were examined by tissue section autoradiography, competition analysis, protein electrophoresis, guanylate cyclase (GC) assays, and molecular cloning. Specific NP binding occurred on the gill filaments, but not on the interbranchial septum or gill arch. The binding was densest on the efferent edge of the gills. Higher resolution light-microscopic examination of emulsion-coated sections showed that specific binding occurred mainly on the secondary lamellae and filament body and not on the arterial circulation. At least two types of NPR were revealed. One is linked to GC since NP binding stimulates the production of cGMP. The GC receptor may be similar to the NPR-B mammalian receptor since only pCNP stimulated cGMP production. The second receptor is not linked to GC and binds the specific ligand C-ANF [rat des(Gln18, Ser19, Gly20, Leu21, Gly22)]. The sequence of a cDNA generated using primers based on conserved regions of vertebrate NPR-C had considerable homology with mammalian and eel NPR-C and eel NPR-D. The presence of GC-linked NPR and NPR-C/ NPR-D suggests that the gills are an important target organ for NP action.

Amino Acid Sequence↗

PubMed1993

Identification of the fragment photoaffinity-labeled with azidodansyl-rhizoxin as Met-363-Lys-379 on beta-tubulin.

The rhizoxin (RZX)-binding site on porcine brain tubulin was investigated by photoaffinity labeling with the 5-azido-1-naphthalene sulfonyl (azidodansyl) derivative of RZX, nor-rhizoxin-22-al-5'-azidonaphthalene-1'-sulfonylhydrazo ne (azidodansylrhizoxin: Adan-RZX). Upon ultraviolet irradiation, Adan-RZX generates a highly reactive nitrene, which irreversibly binds to an amino acid residue(s) near the RZX-binding site. The label was found to be on beta-tubulin. Enzymatic digestion of the labeled tubulin generated only one major fluorescent peak on C18 reverse phase HPLC analysis. The labeled site(s) was mapped by using various combinations of highly specific peptidases in succession. That is, the labeled fragment generated by the first peptidase was purified by HPLC and exposed to a second peptidase; if the retention time in HPLC changed after the second digestion, the fragment generated in the first digestion must have contained the recognition site(s) of the second enzyme. From the results of these successive digestions and the known polypeptide sequences, we could identify the labeled fragment as Met-363-Lys-379 of beta-tubulin. This peptidase combination technique should be widely applicable.

Affinity Labels↗

PubMed1997

Is schizophrenia the price that Homo sapiens pays for language?

The dichotomy between schizophrenia and manic-depressive illness is, as E. Kraepelin suspected, flawed; no unequivocal separation can be achieved. There are no categories of psychosis, but only continua of variation. However, the definition of nuclear symptoms by K. Schneider reveals the fundamental characteristics of the core syndrome--it is independent of the environment and constant in incidence across populations that have been separated for thousands of years. The associated genetic variation must be as old as Homo sapiens and represent a component of diversity that crosses the population as a whole. The fecundity disadvantage that accompanies the syndrome requires a balance in a substantial and universal advantage; this advantage, it is proposed, is the speciation characteristic of language; language and psychosis have a common evolutionary origin. Language, it is suggested, originated in a critical change on the sex chromosomes (the 'speciation event'--the genetic change that defined the species) occurring in East Africa between 100 and 250 thousand years ago that allowed the two hemispheres to develop with a degree of independence. Language can be understood as bi-hemispheric with one component function--a linear output sequence--confined to the dominant hemisphere--and a second--parallel distributed sampling occurring mainly in the non-dominant hemisphere. This mechanism provides an account of the generativity of language. The significance of nuclear symptoms is that these reflect a breakdown of bi-hemispheric coordination of language, perhaps specifically of the process of 'indexicalisation' (the distinction between 'I' and 'you') of self- versus other-generated references. Nuclear symptoms can be described as 'language at the end of its tether'; the phenomena and population characteristics of the nuclear syndrome of schizophrenia thus yield clues to the origin of the species.

Biological Evolution↗

PubMed2004

Synthesis of cyclic peptidosulfonamides by ring-closing metathesis.

N-Protected beta-aminoethanesulfonyl chlorides (2a-e) were used in the preparation of sulfonamides 4, 8, 11a-c, and 15. Ring-closing metathesis of sulfonamides 4 and 8 did not lead to the expected nine-membered cyclic peptidosulfonamides. In contrast, the allylated peptidosulfonamides 11a-c and 15 turned out to be suitable precursor systems for ring-closing metathesis using second-generation Grubbs catalyst and nine-membered cyclic peptidosulfonamides were obtained in 47-60% yields. The possibility for incorporation of these cyclic peptidosulfonamides into a peptide sequence was illustrated by the incorporation of an amino acid on the "S"- or "N"-terminus leading to 16 and 18-20, respectively. A model of cyclic peptidosulfonamide 16 hints at an extended-like structure.

Journal Article↗

PubMed2006

Alternative splicing of the first intron of the steroid receptor RNA activator (SRA) participates in the generation of coding and noncoding RNA isoforms in breast cancer cell lines.

The Steroid Receptor RNA Activator 1 (SRA1) has originally been described as a noncoding RNA specifically activating steroid receptor transcriptional activity. We have, however, identified, in human breast tissue, exon- 1 extended SRA1 isoforms containing two initiating AUG codons and encoding a protein we called SRAP. We recently reported a decreased estrogen receptor activity in breast cancer cells overexpressing SRAP, suggesting antagonist roles played by SRA1 RNA and SRAP. SRA1 appears to be the first example of a molecule active both at the RNA and at the protein level. No data are currently available regarding the mechanisms possibly involved in the generation of coding and noncoding functional SRA1 RNAs. Using 5'-Rapid Amplification of cDNA Extremities (5'-RACE), we have herein identified several putative transcription initiation sites surrounding the second methionine codon and used to generate coding SRA1 transcripts. In the process, we also identified an alternatively spliced noncoding SRA1 transcript still containing an intron-1 sequence. Using targeted RT-PCR approaches, we confirmed the presence in breast cancer cell lines of SRA1 RNAs containing a full as well as a partial intron-1 sequence and established that the relative proportion of these RNAs varied within breast cancer cell lines. Using a "minigene" strategy, we also showed that artificial RNAs containing the SRA1 intron-1 sequence are alternatively spliced in breast cancer cell lines. Interestingly, the splicing pattern of the minigene products parallels the one of the endogenous SRA1 transcripts. Altogether, our data suggest that the primary genomic sequence in and around intron-1 is sufficient to lead to a differential splicing of this intron. We propose that alternative splicing of intron-1 is one mechanism used by breast cancer cells to regulate the balance between coding and functional noncoding SRA1 RNAs.

Alternative Splicing↗

PubMed1999

Functional features of the bovine enterovirus 5'-non-translated region.

The bovine enterovirus (BEV) serotypes exhibit unique features of the non-translated regions (NTRs) which separate them from the other enteroviruses. Their most remarkable property is an additional genome region of 110 nt located between the 5'-cloverleaf and the internal ribosome entry site (IRES). This genome region has the potential to form an additional cloverleaf structure (domain I*) separated from the 5'-cloverleaf (domain I) by a small stem-loop (domain I**). Other characteristics involve the putative IRES domains III and VI. In order to investigate the features of the 5'-NTR, several full-length coxsackievirus B3 (CVB3) cDNA plasmids with hybrid 5'-NTRs were engineered. After exchange of the CVB3 cloverleaf with the BEV1 genome region representing both cloverleafs, a viable virus chimera was generated. Deletion of domain I** within the exchanged region also yielded viable virus albeit with reduced growth capacity. Deletion of sequences encoding either the first or the second BEV cloverleaf resulted in non-infectious constructs. Hybrid plasmids with exchanges of the IRES-encoding sequence or the complete 5'-NTR were non-infectious. Transfection experiments with SP6 transcripts containing 5'-NTRs fused to the luciferase message indicated that IRES-driven translation is enhanced by the presence of the CVB3 cloverleaf and both BEV1 cloverleaf structures, respectively. Deletion of either the first or the second BEV cloverleaf domain reduced but did not abolish enhanced luciferase expression. These results suggest that the substitution of two putative BEV cloverleaf structures for the putative coxsackieviral cloverleaf yields viable virus, while BEV sequences encoding the IRES fail to functionally replace CVB3 IRES-encoding sequences.

5' Untranslated Regions↗

PubMed1999

Expression of the pseudorabies virus latency-associated transcript gene during productive infection of cultured cells.

Like other alphaherpesviruses, pseudorabies virus (PrV) exhibits restricted gene expression during latency. These latency-associated transcripts (LATs) are derived from the region located within 0.69 to 0.77 map units of the viral genome. However, the presence of such viral RNAs during a productive infection has not been described. Although several transcripts originating between 0.706 to 0.737 map units have been detected in PrV-infected cultured cells, their relationship to the LATs has not been examined. Therefore, to determine if any correlation exists between PrV LAT gene expression in the natural and laboratory systems, transcription from the LAT gene region during lytic infection of cultured neuronal and nonneuronal cells was evaluated. A Northern blot assay using single-stranded RNA probes complementary to the spliced in vivo 8. 4-kb largest latency transcript (LLT) detected 1.0-, 2.0-, and 8. 0-kb poly(A) RNAs in all PrV-infected cells lines. The 1.0- and 8. 0-kb transcripts partially overlapped the first and second exons of the LLT, respectively. In contrast, portions of both LLT exons comprised the 2.0-kb RNA sequence, which lacked the same intron as the LLT. Generation of this transcript began about 243 bp downstream of the LLT initiation site and terminated near the junction of BamHI fragments 8' and 8. Its synthesis was inhibited by cycloheximide but not by cytosine beta-D-arabinofuranoside, which suggests that the 2. 0-kb RNA is not an immediate-early gene product. Thus, although the PrV LAT gene is transcriptionally active during a productive infection of cultured cells, the resulting RNAs are distinctive from the LLT.

Animals↗

PubMed1991

In vivo sequence variation of the human immunodeficiency virus type 1 env gene: evidence for recombination among variants found in a single individual.

To assess in vivo sequence heterogeneity of the human immunodeficiency virus type 1 (HIV-1) env gene, we used the polymerase chain reaction to amplify proviral sequences present in peripheral blood mononuclear cell DNA of a patient with acquired immunodeficiency syndrome (AIDS). The amplified env gene fragment (575 bp) contains the first hypervariable region and part of the first conserved region. Eleven and twelve clones were sequenced, respectively, from specimens collected two months apart. Notable heterogeneity was observed among sequences recovered from both specimens. Also, the proviral population recovered from the first specimen varied significantly from that found in the second specimen. Both specimens contained forms with and without an 18 bp duplication. The presence or absence of this duplication, in addition to several point mutations, appear to define two molecular groups evolving in parallel within this patient. Several genotypes which had sequences characteristic of both groups occurred primarily in the second specimen; these can best be explained by multiple recombinational events between representatives of the two groups during reverse transcription. This study demonstrates that recombination may contribute significantly to the generation of diversity among HIV variants within a single individual.

Adult↗

PubMed1985

A novel strategy for constructing clustered point mutations.

We have modified the synthetic linker mutagenesis procedure (1,2) in order to facilitate both the construction and the analysis of deletions, insertions and clustered point mutations generated in DNA in vitro. The protocol as originally described by McKnight and Kingsbury (1) involved attaching a synthetic linker sequence to each 5' or 3' deletion endpoint in DNA. We have designed specific plasmid vectors that can be used to generate nested sets of deletion mutations in the DNA being analyzed. The utility of these vectors is that a linker sequence of choice can be inserted at the endpoint of a deletion in a single intramolecular ligation without the use of synthetic linker DNA. In a second modification of the original procedure, we have adopted a rapid method for sequencing supercoiled plasmid DNAs from 10 ml cultures by primer extension. The site-directed mutagenesis strategy outlined here is suited for studying regulatory regions of DNA, such as origins of DNA replication, transcriptional promoters, enhancer elements, and activator binding sites. We have used this rapid and efficient strategy to generate deletions, insertions, and clustered point mutations in the transcriptional control region of a gene encoding the major human ribosomal RNAs.

Base Sequence↗

PubMed1997

Enhanced immunogenicity of HIV-1 vaccine construct by modification of the native peptide sequence.

Viral proteins are not naturally selected for high affinity major histocompatibility complex (MHC) binding sequences; indeed, if there is any selection, it is likely to be negative in nature. Thus, one should be able to increase viral peptide binding to MHC in the rational design of synthetic peptide vaccines. The T1 helper peptide from the HIV-1 envelope protein was made more immunogenic for inducing T cell proliferation to the native sequence by replacing a residue that exerts an adverse influence on peptide binding to an MHC class II molecule. Mice immunized with vaccine constructs combining the more potent Th helper (Th) epitope with a cytotoxic T lymphocyte (CTL) determinant developed greatly enhanced CTL responses. Use of class II MHC-congenic mice confirmed that the enhancement of CTL response was due to class II-restricted help. Thus, enhanced T cell help is key for optimal induction of CTL, and, by modification of the native immunogen to increase binding to MHC, it is possible to develop second generation vaccine constructs that enhance both Th cell activation and CTL induction.

AIDS Vaccines↗

PubMed1999

PCR method for generating multiple mutations at adjacent sites.

Procedures to introduce point mutations, restriction sites and insert or delete DNA fragments are very important tools to study protein function. We describe here two-step PCR-based method for generating single or multiple mutations, insertions and deletions in a small region of the sequence. In the first step, a unique restriction site is introduced near the part of DNA sequence to be changed, without changing the amino acid sequence. For this step, one of the methods already described can be used. In the second step, mutations are introduced using mutagenic primers containing the unique restriction site from the first step at the 5' end, paired with a universal primer crossing another unique restriction site present originally in the sequence. The method is very simple, economic and rapid. In comparison with the traditional in vitro mutagenesis methods, one can generate large numbers of mutated plasmids in hours.

Gene Deletion↗

PubMed1996

Cystic fibrosis mutation detection by hybridization to light-generated DNA probe arrays.

We have combined photochemistry and photolithography with solid-phase DNA synthesis chemistry to form a new technology that makes high density oligonucleotide probe array synthesis possible. Hybridization to these two-dimensional arrays containing hundreds or thousands of oligonucleotide probes provides a powerful DNA sequence analysis tool. Two types of light-generated DNA probe arrays have been used to test for a variety of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. One array, made up of 428 probes, was designed to scan through the length of CFTR exon 11 and identify differences from the wild type reference sequence. The second type of array contained 1480 probes chosen to detect known deletions, insertions, or base substitution mutations. The validity of the probe arrays was established by hybridizing them with fluorescently labeled control oligonucleotide targets. Characterized mutant CFTR genomic DNA samples were then used to further test probe array hybridization specificity. Finally, ten unknown patient samples were genotyped using the CFTR probe array assay. The genotype assignments were identical to those obtained by PCR product restriction fragment analysis. Our results show that light-generated DNA probe arrays are highly effective in analyzing complex mutation and polymorphism patterns in a relatively large gene such as CFTR.

Base Sequence↗

PubMed1994

Sources of ambiguity in nucleic acid sequence alignment.

The discussion of molecular sequence alignment is becoming more prominent in studies of molecular systematics and evolution. As the basis for initial homology statements, alignment is crucial to comparative molecular biology. Although fundamental, alignment is not a process which necessarily yields objective, precise results. Ambiguities can appear in alignment due to a number of factors. Three such sources of ambiguity are discussed here. These are ambiguity in the establishment of alignment parameters, pair-wise order and individual "path" variation. The first arises from the necessary but empirically untestable assumptions of gap costs and other factors which are required to align sequences objectively. The second is due to the possible existence of non-unique solutions to the same alignment parameters in heuristic and exact solutions. The third is a result of multiple optimal paths within single alignments, potentially generating huge numbers of equally costly but unique alignments. Some of the problems with and several possible solutions to the difficult situation of non-unique alignments are discussed.

Algorithms↗

PubMed2004

Eimeria tenella sporozoites and merozoites differentially express glycosylphosphatidylinositol-anchored variant surface proteins.

Little is known about glycosylphosphatidylinositol (GPI)-linked surface proteins in the coccidian parasite Eimeria tenella. Examination of 28,550 EST sequences from the sporozoite and second merozoite developmental stages of the parasite led to the identification of 37 potential GPI-linked variant surface proteins, termed EtSAGs. Analysis of the complete nucleotide sequences of 23 EtSAG genes separated them into two multi-gene families. All the predicted EtSAG proteins (which vary in length from 228 to 271 residues) have an N-terminal hydrophobic signal peptide, a C-terminal hydrophobic GPI signal-anchor peptide and an extracellular domain organised around six cysteine residues, the positions of which are conserved within each family. Using specific antibodies against a small number of recombinant-expressed EtSAGs, the surface localisation and GPI-anchorage of members of both families was confirmed experimentally. Expression of EtSAGs is differentially regulated between the oocyst/sporozoite and second generation merozoite stages, with only one expressed specifically in the sporozoite, a small number expressed in both stages and the majority expressed specifically in the second generation merozoite. Preliminary data support a model in which multiple variant surface antigens are co-expressed on individual parasites, rather than a model of antigenic switching. The biological role(s) of EtSAGs and the effect(s) that expression of a complex repertoire of variant surface antigens by the second generation merozoite has on host adapted immunity are unknown.

Amino Acid Sequence↗

PubMed2006

Origin of the prevalent SFTPB indel g.1549C > GAA (121ins2) mutation causing surfactant protein B (SP-B) deficiency.

The SFTPB gene indel g.1549C > GAA (121ins2) accounts for about 2/3 of the mutant alleles underlying complete surfactant protein B deficiency. It is unclear, however, whether its prevalence is due to recurrent mutation or a founder effect. The underlying mutational mechanism was therefore sought through the analysis of local DNA sequence complexity. A relatively complex two-step process was proposed: the first step involving slipped mispairing mediated by a direct repeat and generating an AGAA micro-insertion, the second step involving hairpin loop resolution resulting in a CA micro-deletion. The possibility of a founder effect was then assessed by typing 8 intragenic SNPs in 17 independent 121ins2 chromosomes from 10 probands, with parental non-121ins2 chromosomes serving as controls. The 121ins2 chromosomes were assigned to three discrete haplotypes, whilst control chromosomes were distributed between 10 of the 11 observed parental haplotypes. The 121ins2 mutation was in strong and significant linkage disequilibrium (LD) with the tightly linked marker g.1580T/C (|D'| = 1; P approximately 0.024), although only moderate LD was found with the rest of the locus (|D'| approximately 0.54; P approximately 0.136). Data on haplotype structure and the locus LD pattern, obtained from 81 independent Western-European chromosomes, were consistent with the three mutation-bearing haplotypes having originated from a common ancestor by recombination. Interestingly, all families harboring the 121ins2 indel had ancestors from a region of Northwestern Europe populated by Frankish/Saxon migration. Taken together, these data are consistent with the view that an indel mutation occurred on a relatively common SFTPB haplotype and now accounts for the majority of (and possibly all) extant 121ins2 chromosomes.

Base Sequence↗

PubMed1987

Amino acids at the site of V kappa-J kappa recombination not encoded by germline sequences.

Murine V kappa-J kappa recombination is characterized by a maintenance of size at the site of recombination and the use of nucleic acids found only in germline sequences. This is in contrast to heavy chain VH-D-JH assembly where random nucleotides are added at the recombination sites to produce considerable size variation, even though the heptamer/nonomer recombination sequences are identical in both kappa and heavy chain genes. We have examined the origin of an unusual amino acid, Ile, found at the site of V kappa-J kappa recombination in antigalactan antibodies, by sequence analysis of the corresponding rearranged and germline genes. Results indicate that the Ile codon can be generated by use of a single nucleotide 3' of the V kappa segment in combination with the second and third nucleotides of the first codon of J kappa 5 or J kappa 4. However, several antigalactan antibodies express Ile in combination with J kappa 2. An Ile codon cannot be generated by recombination in any reading frame between germline V kappa and J kappa 2 segments. These results suggest that the origin of the Ile codon in lines using J kappa 2 may represent a novel even in murine light chain assembly, possibly similar to the de novo addition of nucleotides observed in heavy chain gene recombination.

Amino Acid Sequence↗