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Results for “third generation sequencing”

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At least 253 records · Page 14Linked to original sources

PubMed1997

Identification of two nucleotide-binding domains on the PB1 subunit of influenza virus RNA polymerase.

Influenza virus RNA polymerase is a multifunctional and multisubunit enzyme consisting of three viral P proteins, PB1, PB2, and PA. We have previously shown that radioactive 8-azido GTP (8-N3 GTP) was photo-crosslinked specifically to the PB1 subunit. Here we confirmed the specific crosslinking of PB1 using oxidized GTP and further identified the GTP analogue binding domains after proteolytic cleavage of the crosslinked PB1 with V8 protease. The cleavage pattern of PB1 was determined by analysis of the amino-terminal proximal sequence of fragments generated in the presence of increasing concentrations of V8 protease. The GTP-crosslinking was identified in three fragments: two adjacent fragments, P6 starting from residue 179 and Pllb starting from residue 298; and the third fragment, P11c, starting from residue 458. Thus, we propose that two GTP-binding sites exist in the PB1 subunit, i.e., the amino terminal-proximal site I located at the boundary between P6 and Pllb, and the carboxy terminal proximal site II on P11c fragment. The locations of GTP-binding sites I and II are close to those of sequence motif A and motif D, respectively, conserved among RNA-dependent RNA polymerases. Of the two fragments forming site I, the crosslinking of 8-N3 GTP is higher to P11b, while that of oxidized GTP is higher to P6, suggesting that the ribose and guanine moieties of GTP bound in this binding pocket face P6 and P11c, respectively. From the V8 concentration dependent change in proteolytic cleavage pattern, it is likely that the two GTP-binding sites on PB1 protein are located on structurally different domains. The existence of two GTP-binding sites is discussed in relation to the binding sites for substrates, primers, and products.

Amino Acid Sequence↗

PubMed1989

Functionally important regions of the factor IX gene have a low rate of polymorphism and a high rate of mutation in the dinucleotide CpG.

We have recently described genomic amplification with transcript sequencing (GAWTS), a three-step procedure that allows direct genomic sequencing. By GAWTS more than 100,000 bp of sequence have been generated from eight regions of the factor IX gene, which include the putative promoter region, the coding region, and the splice junctions. All eight regions were examined in 20 unrelated normal individuals of defined ethnicity and subsequently in 22 hemophiliacs in different families. The following three major conclusions emerge: (1) The rate of polymorphism in these eight regions of functional significance has been measured in an X-linked gene, and it is about one-third of the average rate observed for intronic and intergenic sequences on the X chromosome. The rate is low enough that the causative mutation should be the only sequence change seen in the overwhelming majority of hemophiliacs. (2) Transitions of CpG account for 31% (5/16) of the distinct mutations and for 38% (5/13) of the single-base changes. The rate of transitions at CpG is elevated by an estimated 77-fold, presumably owing to lack of repair of thymidine generated by the spontaneous deamination of 5-methylcytidine. (3) High-quality, reproducible sequence data can be obtained on a time scale that makes direct carrier testing and prenatal diagnosis feasible.

Amino Acid Sequence↗

PubMed1992

Novel Tay-Sachs disease mutations from China.

We describe three HEXA mutations associated with infantile Tay-Sachs disease (TSD) in three unrelated nonconsanguineous Chinese families. Novel mutations were found in two of these families. The third is a previously reported mutation (G-->A transition at nt 1444) (Nakano et al., 1988). Direct sequencing of PCR products identified a novel insertion of an A after nt 547 in family 1. This change generates an early termination codon 6 bp downstream from the insertion site. Allele-specific oligonucleotide hybridization confirmed homozygosity in the proband. Single strand conformational polymorphism analysis and direct sequencing of amplified exon 13 revealed a T-->C transition at nt 1453 with the corresponding amino acid substitution W485R in the second family. This mutation creates an Fnu4HI restriction site. The proband is homozygous for this allele. When the site-specific mutagenized alpha cDNA carrying the T-->C transition at nt 1453 was expressed in COS 1 cells hexosaminidase S activity was not detectable above background. A G-->A transition at nt 1444 (exon 13) corresponding to the E482K substitution was found in the third family. This mutation occurs at a CpG dinucleotide. It has been reported in an Italian TSD proband and causes defective intracellular transport of the alpha-subunit from the rough endoplasmic reticulum to the Golgi apparatus.

Animals↗

PubMed1999

Splicing of a retained intron within ROMK K+ channel RNA generates a novel set of isoforms in rat kidney.

The renal outer medulla K+ channel (ROMK) family of K+ channels may constitute a major pathway for K+ secretion in the distal nephron. To date, four main isoforms of this gene have been identified in the rat that differ only in their NH2-terminal amino acids and that share a common "core exon" that determines the remaining protein sequence. Using RT-PCR, we have identified a new set of ROMK isoforms in rat kidney that are generated by the deletion of a region within the ROMK core sequence that is identifiable as a typical mammalian intron. This splicing event was shown to be reproducible in vitro by detection of deleted ROMK mRNA in Madin-Darby canine kidney (MDCK) cells stably transfected with the gene for ROMK2. Translation of the deletion variant of ROMK2 was confirmed in vitro and visualized in MDCK cells following transient transfection with an enhanced green fluorescent protein tag. The deletion in this core region is predicted to generate hydrophilic proteins that are approximately one-third of the size of native ROMK and lack membrane-spanning domains.

Amino Acid Sequence↗

PubMed1997

Analysis of VH gene rearrangements from synovial B cells of patients with rheumatoid arthritis reveals infiltration of the synovial membrane by memory B cells.

The VH gene (Variable gene segments of the heavy chain locus) repertoire can be investigated by DNA analysis of rearranged immunoglobulin VH genes, which also allows for an indirect estimation of antibody selection by analysis of somatic mutations. Using a polymerase chain reaction (PCR) it is also possible to analyse these genes in small numbers of cells or even single cells. This approach was chosen to investigate germinal centre like lymphocyte follicles in the synovial membranes of two patients with rheumatoid arthritis (RA) in order to analyse the local humoral immune response in RA. Individual B-cell aggregates of synovial membrane of two patients with RA were isolated by micromanipulation from microscopic slides. VH-DH-JH (variable, diversity, and joining segments of the heavy chain locus) rearrangements in all possible VH-JH combinations were amplified from these B cell foci, cloned and subjected to sequence analysis. Sequence analysis revealed that most of the rearranged VH genes were somatically mutated with at least 1% (range 1.3-14.9%) somatic mutations and therefore were derived from antigen-selected memory B cells. Intraclonal diversity in one-third of the clones indicated the generation of memory B cells in the synovial membrane and characterized the synovial membrane as lymphatic tissue where secondary immune responses to an as yet unknown antigen take place.

Adult↗

PubMed1994

Ocular findings associated with a Cys39Arg mutation in the Norrie disease gene.

OBJECTIVE: To diagnose the carriers and noncarriers in a family affected with Norrie disease based on molecular analysis. DESIGN: Family members from three generations, including one affected patient, two obligate carriers, one carrier identified with linkage analysis, one noncarrier identified with linkage analysis, and one female family member with indeterminate carrier status, were examined clinically and electrophysiologically. Linkage analysis had previously failed to determine the carrier status of one female family member in the third generation. Blood samples were screened for mutations in the Norrie disease gene with single-strand conformation polymorphism analysis. The mutation was characterized by dideoxy-termination sequencing. RESULTS: Ophthalmoscopy and electroretinographic examination failed to detect the carrier state. The affected individuals and carriers in this family were found to have a transition from thymidine to cytosine in the first nucleotide of codon 39 of the Norrie disease gene, causing a cysteine-to-arginine mutation. Single-strand conformation polymorphism analysis identified a patient of indeterminate status (by linkage) to be a noncarrier of Norrie disease. CONCLUSION: Ophthalmoscopy and electroretinography could not identify carriers of this Norrie disease mutation. Single-strand conformation polymorphism analysis was more sensitive and specific than linkage analysis in identifying carriers in this family.

Adult↗

PubMed1980

Genetic transmission of retroviral genes and cellular oncogenes.

Several different families of retrovirus genome have been found to exist, each in multiple copies, in the cellular DNA of rodents and primates. There are at least four distinct families of genome in rodents: two type C families, the MTV family and another related to mouse type A particles. In primates there are also at least two families of endogenous type C virogenes and a third type D virogene family. Both in rodents and in primates, the virus-related sequences constitute almost 0.1% of the cellular genome. We have been able to generate transforming viruses, starting with endogenous mouse 'helper' type C viruses by passing them through chemically transformed mouse cells and selecting for variant viruses that have acquired the ability to induce normal cells to display anchorage-independent growth. These viruses produce both sarcomas and carcinomas in the animal; clones that produce only pulmonary carcinomas have also been selected. These presumably have arisen by recombination between the helper and 'transforming' sequences derived from the cells. Moloney sarcoma virus-transformed cells produce a new peptide, called sarcoma growth factor (SGF), that makes normal cells take on some of the properties of transformed cells. Studies with temperature-sensitive viral mutants show that the production of SGF is under the control of the transforming genes of the sarcoma virus.

Animals↗

PubMed1983

Sequences complementary to cellular deoxypolypyrimidines are localized in the 3' end of L-cell mRNA.

Many eukaryotic genomes have been shown to contain long pyrimidine tracts. In mouse L-cells, at least some of these pyrimidine tracts are transcribed and form a significant portion of the poly(A)+ RNA sequences. In this study the results from three different experimental methods indicate that the sequences complementary to polypyrimidines are localized to the 3' end of mRNA molecules. First, polypyrimidines reacted preferentially with the 3'-end fragments of mRNA generated by limited alkaline cleavage. Second, digestion of [3H]mRNA-polypyrimidine hybrids with RNase H released 3'-end fragments of mRNA which averaged only 250 nucleotides (NT) in length. Third, polypyrimidine tracts were isolated from cDNA which averaged only 200-250 NT extending from the 3' end of the corresponding mRNAs. These data suggest that the sequences transcribed from pyrimidine tracts are quite close to the 3' end of the cellular messages in which they occur, probably within the terminal untranslated region.

Animals↗

PubMed1992

The use of hybrid-selected template increases the specificity of the polymerase chain reaction.

An efficient method for generating full-length DNA sequences from apparently unsuccessful polymerase chain reactions (PCR) has been developed. In cases where nonspecific background interferes with detection of the PCR product, a second amplification is performed using a nested set of primers. The internal fragment of DNA amplified in this reaction is then blotted to a membrane and used to hybrid-select the desired DNA from the initial amplification. This DNA is eluted and used as the template for a third round of PCR. The re-use of the original primers from the initial reaction enables the final PCR to generate full-length DNA. This technique was used to clone a full-length gene segment 8 from a mutant influenza A/WSN/33 (H1N1) virus after initial PCR attempts had failed.

Base Sequence↗

PubMed2002

Synthesis of poly(alkyl aryl ether) dendrimers.

Poly(alkyl aryl ether) dendrimers of up to four generations composed of a phloroglucinol core, branching components, and pentamethylene spacers are synthesized by a divergent growth methodology. A repetitive synthetic sequence of phenolic O-alkylation and O-benzyl deprotection reactions are adopted for the synthesis of these dendrimers. The peripheries of the dendrimers contain 6, 12, 24, and 48 phenolic hydroxyl groups, either in the protected or unprotected form, for the first, second, third, and fourth generations, respectively. Because of the presence of hydrophilic exterior and relatively hydrophobic interior regions, alkaline aqueous solutions of these dendrimers are able to solubilize an otherwise insoluble pyrene molecule and these supramolecular complexes precipitate upon neutralization of the aqueous solutions.

Journal Article↗

PubMed1997

Multiple transcripts of the murine immunoglobulin epsilon membrane locus are generated by alternative splicing and differential usage of two polyadenylation sites.

The human C epsilon gene produces a number of alternatively spliced heavy chain transcripts of which some encode functional IgE isoforms. We now show that differentially processed epsilon mRNA variants also exist in the mouse and are generated by differential polyadenylation and alternative splicing of primary epsilon chain transcripts. The two poly(A) sites of the mouse membrane transcripts were identified in the present study by RACE-PCR analysis. The first poly(A) site is located 743 nt downstream from the beginning of the second membrane exon (M2) and contains the same non-consensus AGTAAA signal sequence as the single poly(A) site of the human membrane transcripts. The second poly(A) site is located almost 500nt further downstream and is characterized by an AAGAAA hexamer. This poly(A) site contains a (G+T) rich element downstream to the site of cleavage and polyadenylation and is preferentially utilized by the membrane epsilon transcripts. Additional diversity of epsilon transcripts is generated by alternative splicing between the last constant region exon (CH4) and the two membrane exons (M1 and M2). The alternatively spliced transcripts include two variants that skip the first membrane exon and encode epsilon heavy chains that lack the transmembrane domain. The third variant is generated by splicing to an internal site in M2 and codes for a membrane isoform that is 10 amino acids shorter in the cytoplasmic domain than the classical membrane IgE. Although little amino-acid sequence homology exists between the murine epsilon chain isoforms and their human counterparts, the pattern of splicing is rather conserved between the two species.

Alternative Splicing↗

PubMed1992

Cloning of an apparent splice variant of the rat N-methyl-D-aspartate receptor NMDAR1 with altered sensitivity to polyamines and activators of protein kinase C.

Molecular cloning identified complementary DNA species, from a rat ventral midbrain library, encoding apparent splice variants of the N-methyl-D-aspartate (NMDA) receptor NMDAR1 (which we now term NR1a). Sequencing revealed that one variant, NR1b, differs from NR1a by the presence of a 21-amino acid insert near the amino end of the N-terminal domain and by an alternate C-terminal domain in which the last 75 amino acids are replaced by an unrelated sequence of 22 amino acids. NR1b is virtually identical to NR1a in the remainder of the N- and C-terminal domains, at the 5' and 3' noncoding ends, and within the predicted transmembrane domains and extracellular and cytoplasmic loops. These findings suggest that the two forms of the receptor arise by differential splicing of a transcript from the same gene. Sequencing of other clones indicates the existence of a third variant, NR1c, identical to NR1b in its C terminus but lacking the N-terminal insert. NR1b RNA injected into Xenopus oocytes generated functional homomeric NMDA channels with electrophysiological properties distinct from those of NR1a homomeric channels. NR1b channels exhibited a lower apparent affinity for NMDA and for glutamate. NR1b channels exhibited a lower affinity for D-2-amino-5-phosphonovaleric acid and a higher affinity for Zn2+. The two receptor variants showed nearly identical affinities for glycine, Mg2+, and phencyclidine. Spermine potentiation of NMDA responses, prominent in oocytes injected with rat forebrain message, was also prominent for NR1a receptors, but was greatly reduced or absent for NR1b receptors. Treatment with the protein kinase C activator phorbol 12-myristate 13-acetate potentiated NMDA responses in NR1b-injected oocytes by about 20-fold; potentiation of NMDA responses in NR1a-injected oocytes was much less, about 4-fold. These findings support a role for alternate splicing in generating NMDA channels with different functional properties.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1994

The analytic third: working with intersubjective clinical facts.

In this paper, two clinical sequences are presented in an effort to describe the methods by which the analyst attempts to recognise, understand and verbally symbolize for himself and the analysand the specific nature of the moment-to-moment interplay of the analyst's subjective experience, the subjective experience of the analysand and the intersubjectively-generated experience of the analytic pair (the experience of the analytic third). The first clinical discussion describes how the intersubjective experience created by the analytic pair becomes accessible to the analyst in part through the analyst's experience of 'his own' reveries, forms of mental activity that often appear to be nothing more than narcissistic self-absorption, distractedness, compulsive rumination, daydreaming and the like. A second clinical account focuses on an instance in which the analyst's somatic delusion, in conjunction with the analysand's sensory experiences and body-related fantasies, served as a principal medium through which the analyst experienced and came to understand the meaning of the leading anxieties that were being (intersubjectively) generated.

Adult↗

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↗

PubMed1995

Identification of Zucchini yellow mosaic potyvirus by RT-PCR and analysis of sequence variability.

A reverse transcription-polymerase chain reaction (RT-PCR) method was used to identify Zucchini yellow mosaic virus (ZYMV) in leaves of infected cucurbits. Oligonucleotide primers which annealed to regions in the nuclear inclusion body (NIb) and the coat protein (CP) genes, generated a 300-bp product from ZYMV and also from the closely related watermelon mosaic virus type 2 (WMV-2). However, no product was obtained from papaya ringspot potyvirus which also infects cucurbits. ZYMV and WMV-2 were differentiated using a third primer which was complementary to a sequence in the 3'-untranslated region; a 1186-bp amplified product was obtained for ZYMV only. Nucleotide sequence analysis of the 300-bp fragments of Australian ZYMV and WMV-2 strains revealed 93.7-100% sequence identity between ZYMV strains. Multiple sequence alignments indicated that the nucleotide sequence which codes for the N-terminus of the CP was 74-100% identical for different isolates of ZYMV. The Australian isolate of WMV-2 was 43-46% identical to all isolates of ZYMV and was 84.6% identical to a Florida isolate of WMV-2.

Amino Acid Sequence↗

PubMed2003

Locating sequence on FPC maps and selecting a minimal tiling path.

This study discusses three software tools, the first two aid in integrating sequence with an FPC physical map and the third automatically selects a minimal tiling path given genomic draft sequence and BAC end sequences. The first tool, FSD (FPC Simulated Digest), takes a sequenced clone and adds it back to the map based on a fingerprint generated by an in silico digest of the clone. This allows verification of sequenced clone positions and the integration of sequenced clones that were not originally part of the FPC map. The second tool, BSS (Blast Some Sequence), takes a query sequence and positions it on the map based on sequence associated with the clones in the map. BSS has multiple uses as follows: (1) When the query is a file of marker sequences, they can be added as electronic markers. (2) When the query is draft sequence, the results of BSS can be used to close gaps in a sequenced clone or the physical map. (3) When the query is a sequenced clone and the target is BAC end sequences, one may select the next clone for sequencing using both sequence comparison results and map location. (4) When the query is whole-genome draft sequence and the target is BAC end sequences, the results can be used to select many clones for a minimal tiling path at once. The third tool, pickMTP, automates the majority of this last usage of BSS. Results are presented using the rice FPC map, BAC end sequences, and whole-genome shotgun from Syngenta.

Chromosomes, Artificial, Bacterial↗

PubMed1994

Genomic imprinting: lessons from mouse transgenes.

Genomic imprinting is a non-Mendelian form of inheritance that results in an expression difference between the two parental alleles of an autosomal locus. The study of mouse transgenes has provided us with descriptions of a variety of imprinting or parent-of-origin effects, thereby anticipating similar inheritance phenomena in non-transgenic mice. Many mouse transgenes exhibit parent-of-origin behavior only on mixed strain backgrounds, whereas others are imprinted on inbred strain backgrounds. In the former cases, the parent-of-origin effects are due to strain-specific modifiers of DNA methylation and expression. These are inherited in a parent-specific fashion and exert their effects after fertilization. In the latter cases, true germline transgene imprinting, the creation of an imprinted locus occurs in a series of sequential steps. First, there is an erasure of the imprint from the previous generation in both male and female fetal germ cells. Second, upon completion of gametogenesis, distinctive methylation patterns have been placed on the transgene sequences of the two mature gametes. Third, only one of these inherited patterns is maintained in the early, pre-implantation embryo. The pattern of the other parental allele is erased. Finally, the methylation pattern of the alleles evolve in the later stages of development, but nonetheless the methylation difference (imprint) of the locus persists. Transgene imprinting behaviors, either on mixed strain backgrounds and on inbred genetic backgrounds, have counterparts in endogenous genetic phenomena.

Alleles↗

PubMed2026

Genomic epidemiology of clinically critical antibiotic resistance in Salmonella enterica causing bloodstream infections across six Chinese provinces, 1994-2023.

Clinically critical antibiotic-resistant Salmonella enterica (S. enterica) causing bloodstream infections remains a public health challenge. Here, we aim to reveal the emergence and trends of clinically important antibiotic resistance in S. enterica causing bloodstream infections using 833 isolates from six Chinese provincial-level administrative areas during 1994-2023. We identified 48 serovars and 64 sequence types (STs). Overall, 8.52% of 833 isolates were resistant or had decreased susceptibility to ciprofloxacin, 4.32% and 6.84% reported resistance or decreased susceptibility to third- and fourth-generation cephalosporins (3GCs and 4GCs), 1.80% reported resistance to fosfomycin, and 2.16% reported resistance to azithromycin. Across these six regions, azithromycin and fosfomycin resistance is increasing, as is decreased susceptibility or resistance to ciprofloxacin, 3GCs, and 4GCs, especially among younger children and elderly people. Clinically prioritized antibiotic resistance also varies by region, serovar, and age group. S. Paratyphi A genotype 2.3.3 strains are mainly divided into 2 lineages distributed in Guangxi and Shanghai. Within the scope of this passive surveillance dataset, S. Typhi genotype 4.3.1.2.1 was identified as the earliest documented case among the collected isolates. Our retrospective and longitudinal genomic epidemiology study provides critical data for the formulation of treatment guidelines and policies for bloodstream infections and for the monitoring and control of antimicrobial resistance.

Humans↗