SEARCH · PubMed Health

Results for “Clone tracing”

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 19 recordsLinked to original sources

PubMed2025

SpaceBar enables clone tracing in spatial transcriptomic data.

We report a cellular barcoding strategy, SpaceBar, that enables simultaneous clone tracing and spatial transcriptomics profiling. Our approach uses a library of 96 synthetic barcode sequences that can be robustly detected by imaging based spatial transcriptomics (seqFISH), delivered such that each cell is labeled with a combination of barcodes. We used these barcodes to label melanoma cells in a tumor xenograft model and profiled both clone identity and spatial gene expression in situ. We developed a gene scoring metric that quantifies how strongly gene expression is driven by intrinsic cellular cues or extrinsic environmental signals. Our framework distinguishes between clonal dynamics and environmentally-driven transcriptional regulation in complex tissue contexts.

Journal Article↗

PubMed2003

Random priming PCR strategy to amplify and clone trace amounts of DNA.

Here we report a new methodology to study trace amounts of DNA of unknown sequence using a two-step PCR strategy to amplify and clone target DNA. The first PCR is carried out with a partial random primer comprised of a specific 21-nucleotide 5' sequence, a random heptamer, and a 3' TGGC clamp. The second PCR is carried out with a single 19-nucleotide primer that matches the specific 5' sequence of the partial random primer. Using human and Mycoplasma genitalium DNA as examples, we demonstrated the efficiency of this approach by effectively cloning target DNA fragments from 1 pg DNA sample. The cloning sensitivity could reach 100 fg target DNA templates. Compared to the strategy of first adding adapter sequences to facilitate the PCR amplification of unknown sequences, this approach has the advantage of allowing for the amplification of DNA samples in both natural and denatured forms, which provides greater flexibility in sample preparation. This is an efficient strategy to retrieve sequences from trace DNA samples from various sources.

Cloning, Molecular↗

PubMed1981

Cloning of alpha 2u globulin cDNA using a high efficiency technique for the cloning of trace messenger RNAs.

An extremely high-efficiency technique is described for cloning double-stranded (ds) cDNAs in Escherichia coli. The method, which uses two synthetic oligonucleotide linkers rather than one, results in approx. 200--500 recombinant clones per ng of ds cDNA. This technique was used to clone a cDNA comprising 95% of the full length of the mRNA of alpha 2u globulin, a male rat liver protein, which represents approx. 1% of hepatic messenger RNA. The cloned probe was applied to study the complex hormone controls of alpha 2u globulin mRNA in male and female rats.

Alpha-Globulins↗

PubMed2006

Pharmacologic characterization of the cloned human trace amine-associated receptor1 (TAAR1) and evidence for species differences with the rat TAAR1.

The hemagglutinin-tagged human trace amine-associated receptor1 (TAAR1) was stably coexpressed with rat Galpha(s) in the AV12-664 cell line, and receptor activation was measured as the stimulation of cAMP formation. After blockade of endogenously expressed alpha2- and beta-adrenoceptors with 2-[2-(2-methoxy-1,4-benzodioxanyl)]-imidazoline hydrochloride (2-methoxyidazoxan, RX821002) and alprenolol, respectively, the resulting pharmacology was consistent with that of a unique receptor subtype. beta-Phenylethylamine (beta-PEA), the putative endogenous ligand, gave an EC50 of 106 +/- 5 nM in the assay. For a series of beta-PEA analogs used to explore the pharmacophore, small substituents at ring positions 3 and/or 4 generally resulted in compounds having lower potency than beta-PEA, although several were as potent as beta-PEA. However, small substituents at ring position 2 resulted in a number of compounds having potencies as good as or better than beta-PEA. A number of nonselective antagonists known to share affinity for multiple monoaminergic receptors were evaluated for their ability to inhibit beta-PEA stimulation of the human TAAR1. None had an IC50 <10 microM. For comparison, the rat TAAR1 receptor was expressed in the AV12-664 cell line. A number of agonist compounds had significantly different relative potencies between the rat and human TAAR1, demonstrating a significant species difference between the rat and human TAAR1. The TAAR1 receptor exhibits a pharmacologic profile uniquely different from those of classic monoaminergic receptors, consistent with the structural information that places them in a distinct family of receptors. This unique pharmacologic profile suggests the potential for development of TAAR-selective agonists and antagonists to study their physiologic roles.

Animals↗

PubMed2025

Paired Single-Cell Transcriptome and DNA Barcode Detection in Zebrafish Using ScarTrace.

ScarTrace is a CRISPR/Cas9-based genetic lineage tracing method that allows for uniquely barcoding the DNA of single cells at a target GFP sequence during developing zebrafish embryos. Single cells from barcoded adult zebrafish can be isolated from various tissues (e.g., marrow, brain, eyes, fins), and their transcriptome and barcode sequences are captured by single-cell cDNA amplification and genomic DNA nested PCR, respectively. Computationally, cell type and barcode identification permit clone tracing and lineage tree reconstruction of tissues to unravel fate decisions during embryogenesis.

Animals↗

PubMed1997

A new dynamic tool to perform assembly of expressed sequence tags (ESTs).

MOTIVATION: Expressed Sequence Tags (ESTs) are short single-pass DNA sequences obtained from either ends of cDNA clones. To exploit these sequences efficiently, a dynamic Web-tool has been developed which uses these data to perform fast virtual cloning of cDNAs. RESULTS: Starting with a query sequence, the user is able to identify related ESTs and extend the sequence of interest step by step, possibly to a full-length transcript. Graphical views of the clustering are used to monitor the progress of a particular 'cloning' project. Potential open reading frames are detected by positional base preference, and hyperlinks to other Worldwide Web sites allows the user to retrieve information relevant to each EST in a cluster (e.g. sequence traces, clone size, plate position). Apart from cDNA cloning, this tool also provides a mechanism for collating gene families and polymorphism sites.

Algorithms↗

PubMed2002

Subtyping of Salmonella typhimurium by pulsed-field gel electrophoresis and comparisons with phage types and resistance types.

One-hundred and sixty-eight Salmonella enterica subsp. enterica serotype Typhimurium isolates have been analysed by phage typing, by pulsed-field gel electrophoresis and for their antimicrobial susceptibility. Those independent strains, isolated from food animal production including cattle, poultry and pig sectors have been collected by the French non human Salmonella network, during the first semester in 1999. Isolates encompassed 14 phage types. The majority of S. Typhimurium isolates was found to be definitive phage type DT104, representing 39% of all isolates. Other phage types were mainly DT8, PT U302, DT120, DT193 and DT135. Forty-six pulsotypes were obtained using Xbal restriction enzyme, and amongst them, ten were associated to the DT104 phage type. A major pulsotype (px1), was represented by 79% of DT104 isolates and was also found among DT120. Forty-eight percent of isolates showed a classic DT104 resistance profile to ampicillin, streptomycin, chloramphenicol, tetracycline, sulfonamides (ASCTSu). Among this resistance type, 84% were DT104 and 12% were DT120. Some pulsotypes were found associated to this resistant type. The pulsed field gel electrophoresis showed to be a useful typing method for discrimination of S. Typhimurium strains and for tracing clone through different sectors of origin in order to control their spread.

Bacterial Typing Techniques↗

PubMed2000

Membrane gamma-glutamyl transpeptidase activity of melanoma cells: effects on cellular H(2)O(2) production, cell surface protein thiol oxidation and NF-kappa B activation status.

The metabolism of glutathione by membrane-bound &ggr;-glutamyl transpeptidase (GGT) has been recently recognized as a basal source of hydrogen peroxide in the extracellular space. Significant levels of GGT activity are expressed by malignant tumours, and in melanoma cell lines they were found to correlate with the malignant behaviour. As hydrogen peroxide and other oxidants can affect signal transduction pathways at several levels, the present study was aimed to verify: (i) the occurrence of GGT-dependent production of hydrogen peroxide in melanoma cells; (ii) the effects of GGT-dependent prooxidant reactions on known redox-sensitive cellular targets, i.e. protein thiols, the nuclear transcription factor NF-kappa B and p53. Two melanoma Me665/2 cell clones, exhibiting traces of (clone 2/21) or high (clone 2/60) GGT activity, were studied. The occurrence of GGT-dependent production of hydrogen peroxide was apparent in 2/60 cells, in which it was accompanied by lower levels of cell surface protein thiols. In 2/60 cells, GGT expression was also associated with higher levels of NF-kappa B activation, as compared to GGT-poor 2/21 cell clone. Indeed, stimulation or inhibition of GGT activity in 2/60 cells resulted in progressive activation or inactivation of NF-kappa B, respectively. An analysis of the p53 gene product indicated lack of protein expression in 2/60 cells, whereas a mutant protein was highly expressed in 2/21 cells. Taken together, these results indicate that the expression of GGT activity can provide melanoma cells with an additional source of hydrogen peroxide, and that such prooxidant reactions are capable to modify protein thiols at the cell surface level. In addition, GGT expression results in an up-regulation of the transcription factor NF-kappa B, which could explain the higher metastatic behaviour reported for GGT-rich melanoma cells as compared to their GGT-poor counterparts.

Blotting, Western↗

PubMed1991

The origin of a morphologically unidentifiable human supernumerary minichromosome traced through sorting, molecular cloning, and in situ hybridisation.

A supernumerary minichromosome has been detected in a severely malformed patient. Attempts at identifying the marker by conventional approaches were unsuccessful. The physical isolation of the minichromosome by fluorescence activated sorting, molecular cloning of its DNA, and in situ hybridisation experiments performed with single copy DNA probes allowed us to show that it was derived from a rearrangement involving the centromere and the proximal region of the short arm of chromosome 9.

Abnormalities, Multiple↗

PubMed1989

Aspartate and asparagine tRNA genes in wheat mitochondrial DNA: a cautionary note on the isolation of tRNA genes from plants.

We have identified genes encoding a "native" tRNA(Asp) (trnD-GTC) and a "chloroplast-like" tRNA(Asn) (trnN-GTT) on opposite strands and 633 bp apart within a sequenced 1640 bp RsaI restriction fragment of wheat mtDNA. The trnD gene has been found previously at a different location in wheat mtDNA (P.B.M. Joyce et al. (1988) Piant Mol. Biol. 11, 833-843); the duplicate copies of this gene are identical within the coding and immediate flanking regions (9 bp downstream and at least 68 bp upstream), after which obvious sequence similarity abruptly disappears. The trnN gene is identical to its homolog in maize ctDNA; continuation of sequence similarity beyond the coding region suggests that this gene originated as promiscuous ctDNA that is now part of the wheat mitochondrial genome. In the course of this work, we have encountered some unexpected similarities between tRNA gene regions from wheat mitochondria and other sources. Detailed analysis of these similarities leads us to suggest that trnN genes reportedly from petunia nuclear DNA (N. Bawnik et al. (1983) Nucleic Acids Res. 11, 1117-1122) and lupine mtDNA (B. Karpińska and H. Augustyniak (1988) Nucleic Acids Res. 16, 6239) are, in fact, from petunia mtDNA and lupine ctDNA, respectively, whereas a putative wheat nuclear tRNA(Ser) (trnS-TGA) gene (Z. Szwekowska-Kulińska et al. (1989) Gene 77, 163-167) is actually from wheat mtDNA. In these instances, it seems probable that the DNA samples used for cloning contained trace amounts of DNA from another sub-cellular compartment, leading to the inadvertent selection of spurious clones.

Base Sequence↗

PubMed2001

Trace amines: identification of a family of mammalian G protein-coupled receptors.

Tyramine, beta-phenylethylamine, tryptamine, and octopamine are biogenic amines present in trace levels in mammalian nervous systems. Although some "trace amines" have clearly defined roles as neurotransmitters in invertebrates, the extent to which they function as true neurotransmitters in vertebrates has remained speculative. Using a degenerate PCR approach, we have identified 15 G protein-coupled receptors (GPCR) from human and rodent tissues. Together with the orphan receptor PNR, these receptors form a subfamily of rhodopsin GPCRs distinct from, but related to the classical biogenic amine receptors. We have demonstrated that two of these receptors bind and/or are activated by trace amines. The cloning of mammalian GPCRs for trace amines supports a role for trace amines as neurotransmitters in vertebrates. Three of the four human receptors from this family are present in the amygdala, possibly linking trace amine receptors to affective disorders. The identification of this family of receptors should rekindle the investigation of the roles of trace amines in mammalian nervous systems and may potentially lead to the development of novel therapeutics for a variety of indications.

Amino Acid Sequence↗

PubMed1998

Antigen direction of specific T-cell clones into gingival tissues.

This study was performed to investigate T-cell traffic to periodontal tissues during infection with a periodontal pathogen Actinobacillus actinomycetemcomitans (Aa). Rowett rat T-cell clones, A3 (CD4+ CD8-, alpha beta TCR+, NKRP-1-, specific to Aa) and G2 (CD4- CD8-, alpha beta TCR+, NKRP-1+, which reacts to Aa, Gram-negative and -positive bacteria), both expressed the same prominent adhesion molecules (LFA-1, VLA-4) to the same extent. Binding of both T-cell clones to rat endothelial cells in vitro was blocked by antibody to VLA-4. Rowett rats were infected with Aa and infused with Aa-stimulated, isogenic T-clone lymphocytes that had been labelled in vitro with 125IUdR. Radioactivity associated with recovery of clone A3, but not G2, was significantly elevated in the gingivae of infected rats, suggesting migration to infected animals' gingival tissues. Migration of radioactive Aa-specific A3 clone cells traced by autoradiography reached a maximum at 24 hr (1.2% of total lymphocytes as radiolabelled cells in infected gingiva versus 0.6% in noninfected), indicating an apparent antigen-directed retention in infected rats' gingival tissues. The G2 clone was not retained in the gingival tissues (0.20% of total lymphocytes as radiolabelled cells in infected gingiva versus 0.26% in non-infected). However, the possibility of A3 retention directed by inflammation or tissue-selective homing could not be excluded. In further experiments, other adoptively transferred T-clone lymphocytes [clones G23 (Th1) and F13 (Th2)] with specificity for the 29,000 MW outer membrane protein of Aa with the same prominent adhesion molecules could be recovered from rat gingivae previously challenged with this antigen. However, transferred T-clone lymphocytes [clone G26 (Th1)] with specificity for a different Aa antigen were not recovered. Therefore, the dynamics of cell entry into periodontal lesions vary for activated T lymphocytes with different antigenic specificities, indicating the significance of antigen in lymphocyte traffic to periodontal tissues.

Actinobacillus Infections↗

PubMed2002

A trace amine, tyramine, functions as a neuromodulator in Drosophila melanogaster.

The tyramine receptor (TyrR) is a G protein-coupled receptor for trace amines, cloned in Drosophila melanogaster, and claimed to be either an octopamine receptor or a tyramine receptor. We previously reported that in the larval neuromuscular junctions, the modulatory effect on the excitatory junction potentials of tyramine is distinctly different from that of octopamine. The effect of tyramine but not of octopamine was selectively abolished in the TyrR mutant hono, suggesting that this gene encodes a receptor for tyramine, and not for octopamine. We examined whether there was a gene-dosage effect of this tyramine modulation using combinations of hono, deficiency (Df) and wild-type alleles. The tyramine effect was observed in hono heterozygotes (+/hono), which showed intermediate levels of response, but was not seen in +/Df or hono/Df hemizygotes. While these further suggest that tyramine is the true ligand, it is possible that the gene-dosage effect is only evident above some threshold of gene expression levels. Immunohistochemical staining using an anti-tyramine antibody identified tyramine-containing neurons in the larval central nervous system, some of which were distinct from the octopamine-containing neurons. Taken together, these results strongly suggest that tyramine functions as a neuromodulator.

Animals↗

PubMed2000

Repertoire dynamics of autoreactive T cells in multiple sclerosis patients and healthy subjects: epitope spreading versus clonal persistence.

Autoantigen-specific T-lymphocytes are present in patients with autoimmune disease and in normal subjects. Little is currently known about the temporal variation (dynamics) of the immune repertoire of these autoreactive T cells. We analysed the long-term variation of the immune repertoire of T cells specific for myelin basic protein (MBP) in five untreated patients with multiple sclerosis and four normal control subjects over a mean observation period of 6 years. MBP-specific CD4(+) T-cell lines were selected with purified human MBP, and their epitope specificity was mapped with overlapping synthetic peptides. Three distinct patterns of repertoire development were observed. (i) Two patients and three control subjects maintained a broad epitope response with fluctuations over time. (ii) Two patients initially showed a focused response that broadened over the course of 6 years; this finding could be described as intramolecular epitope spreading. (iii) In one patient and one control subject, a strikingly focused response, which was directed to a cluster of nested epitopes in the MBP region 83-102, persisted over time. T-cell receptor Vbeta sequence analysis allowed us to trace individual clones of MBP-specific T cells for up to 7 years in the peripheral circulation in four of the five patients and three of the four controls, suggesting that the long-term persistence of MBP-specific T-cell clones is a common feature of the T-cell repertoire not unique to multiple sclerosis. The persisting MBP-specific T-cell clones were not detectable in the blood of one of the patients by complementarity-determining region (CDR)-3 spectratyping, indicating that their frequency does not exceed 1 in 5000 T cells. The temporal characteristics of the MBP-specific T-cell repertoire described here are relevant to therapeutic strategies targeting autoantigen-specific T cells in multiple sclerosis and other autoimmune diseases.

Adult↗

PubMed1999

Epidemiological typing of Streptococcus pneumoniae from various sources in Sweden and India using Box A PCR fingerprinting.

Epidemiological typing of Streptococcus pneumoniae is essential to determine strain relatedness and also to trace resistant clones. The novel Box A PCR assay was used for characterization of S. pneumoniae isolates from two Scandinavian countries and to compare those from India on the Asian continent. In addition, the assay was employed to determine the clonality of 25 pneumococcal strains from an outbreak in a day-care centre in Linköping, Sweden. All 25 showed a unique pattern with 100% homology for 24 of them, thereby establishing the clonal nature of the outbreak. The pneumococcal strains involved in the outbreak belonged to serotype 9 and were resistant to penicillin, with an MIC value of 2 mg/l. Thirty-eight genotypes were obtained when the Box A results were analysed by computer (Molecular Analyst Software with GelCompar). The discriminatory index of the method was D=0.98, which indicates excellent performance. No major segregation of strains from the different geographical locations was observed when a lower level of similarity was used for typing (80%, 13 types). However, at the level chosen for genotyping, 95% (38 genotypes) there was a clear geographical segregation. No correlation between genotype and serotype was seen as strains from a common place of origin were most often of different serotypes. Computer-assisted analysis of the results of Box A PCR typing facilitated the evaluation.

DNA Fingerprinting↗

PubMed2002

Polyclonal long-term repopulating stem cell clones in a primate model.

Hematopoietic bone marrow stem cells generate differentiated blood cells and, when transplanted, may contribute to other organs, such as the brain, heart, and liver. An understanding of in vivo clonal behavior of stem cells will have important implications for cellular and gene therapy. For the first time, we have directly demonstrated the derivation of circulating peripheral blood cells from individual stem cell clones. We analyzed the clonal composition of retrovirus-marked peripheral blood leukocyte populations in 2 different primate models by a novel direct genomic sequencing technique allowing the identification of vector insertion sites. More than 80 contributing long-term hematopoietic clones were identified in individual rhesus macaque peripheral blood transplant recipients and more than 25 different clones in a baboon marrow transplant recipient. Up to 5 insertion sequences from each animal were used to trace the long-term contribution of stem cell clones in these primate models. Continuous and mostly pluripotent contributions of peripheral blood leukocytes from each of the traced clones could be detected for the entire follow-up period of 23 to 33 months. Our study provides direct molecular evidence for a polyclonal, multilineage, and sustained contribution of individual stem cells to primate hematopoiesis.

Animals↗

PubMed2005

Growth and trace metal accumulation of two Salix clones on sediment-derived soils with increasing contamination levels.

The growth and metal uptake of two willow clones (Salix fragilis 'Belgisch Rood' and Salix viminalis 'Aage') was evaluated in a greenhouse pot experiment with six sediment-derived soils with increasing field Cd levels (0.9-41.4 mg kg-1). Metal concentrations of eight elements were measured in roots, stems and leaves and correlated to total and soil water metal concentrations. Dry weight root biomass, number of leaves and shoot length were measured to identify eventual negative responses of the trees. No growth inhibition was observed for both clones for any of the treatments (max. 41.4 mg kg-1 Cd, 1914 mg kg-1 Cr, 2422 mg kg-1 Zn, 655 mg kg-1 Pb), allowing their use for phytoextraction on a broad range of contaminated sediments. However, dry weight root biomass and total shoot length were significantly lower for S. viminalis compared to S. fragilis for all treatments. Willow foliar Cd concentrations were strongly correlated with soil and soil water Cd concentrations. Both clones exhibited high accumulation levels of Cd and Zn in aboveground plant parts, making them suitable subjects for phytoextraction research. Cu, Cr, Pb, Fe, Mn and Ni were found mainly in the roots. Bioconcentration factors of Cd and Zn in the leaves were highest for the treatments with the lowest soil Cd and Zn concentration.

Biodegradation, Environmental↗

PubMed1990

Comparative analytical studies on recombinant and native human somatotropin.

In polyacrylamide gel electrophoresis and isoelectric focusing, somatotropin produced by recombinant DNA technology is as heterogeneous as highly purified native pituitary somatotropin. This heterogeneity is not attributable to different degrees of deamination of a single molecular species. In addition to the main protein of 22 kDa, the cloned products contain traces of interchain disulphide dimers of somatotropin. The quantitative amino acid analyses of the three cloned somatotropins investigated are neither identical nor do they correspond to the analysis of the native pituitary hormone. Moreover, there are discrepancies between the amino acid compositions of the hormones studied and the generally recognized amino acid analysis for human somatotropin.

Amino Acids↗