Carbohydrate-encapsulated gold nanoparticles for rapid target-protein identification and binding-epitope mapping.
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Binding Sites↗
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The cholinergic innervation of the rat fascia dentata: identification of target structures on granule cells by combining choline acetyltransferase immunocytochemistry and Golgi impregnation.
A monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme, was used to study cholinergic synapses on identified (Golgi stained) granule cells in the rat fascia dentata. Choline acetyltransferase immunocytochemistry was applied to 40-microns Vibratome sections cut perpendicular to the longitudinal axis of the hippocampus. Light microscopy revealed fine varicose ChAT-immunoreactive axons in all layers of the fascia dentata, i.e., in the stratum moleculare, the stratum granulosum, and the subgranular polymorph zone. Most fibers were observed in the vicinity of granule cell bodies where they ran mainly parallel to the granular layer. Next, the immunostained Vibratome sections were sandwiched between small pieces of Parafilm and piled to form a block that was covered with agar and Golgi stained. After that, the sections were separated by cutting away the agar and removing the Parafilm. Sections containing well-impregnated granule cells were gold-toned (Fairén et al., '77), embedded in Araldite, and subjected to ultrathin sectioning for electron microscopy. A total of 14 gold-toned granule cells were examined in the electron microscope for synaptic contacts with cholinergic afferents. Choline acetyltransferase-immunoreactive axon terminals were observed that established symmetric synaptic contacts with the cell bodies and dendritic shafts of the gold-toned identified granule cells. Two types of contact were observed on spines arising from gold-toned granule cell dendrites. Immunoreactive terminals established asymmetric synaptic contacts with the head of small spines and symmetric contacts with the stalk of large, complex spines. The boutons forming asymmetric synaptic contacts with the cup-shaped spine head of the complex spines were not found to be immunoreactive. Our results demonstrate that cholinergic fibers to the rat fascia dentata establish characteristic types of synaptic contact with different postsynaptic elements of granule cells, suggesting a complex function of this afferent system.
Animals↗
Identification of target genes within an amplicon at 14q12-q13 in esophageal squamous cell carcinoma.
Comparative genomic hybridization studies have revealed frequent amplification of the 14q12-q13 region in esophageal squamous cell carcinoma (ESC) cell lines. To identify genes targeted for amplification, we first defined the minimal common region of amplification using fluorescence in situ hybridization in affected ESC cell lines. The amplicon covered about 6 Mb, between markers D14S1034 and L18528. Then we screened 32 ESC cell lines to discern amplifications and expression levels of 26 expressed sequence tags (ESTs) that had been localized to the amplified region. Five known genes (BAZ1A, SRP54, NFKBIA, MBIP, and HNF3A) and two uncharacterized ESTs (GenBank Accession numbers AA991861 and AA167732) within the amplicon showed amplification and consequent overexpression. Two of these transcripts were amplified in three of the primary ESCs we examined. Our findings suggest that these seven genes are candidate targets of the amplification mechanism and therefore may be associated, together or separately, with development and progression of ESC.
Blotting, Western↗
Sequence-specific DNA binding by p53: identification of target sites and lack of binding to p53 - MDM2 complexes.
An immune selection procedure was employed in order to isolate p53 binding sites from mouse genomic DNA. Two DNA clones capable of tight specific interaction with wild type p53 were subjected to further characterization. In both cases, the p53 binding regions displayed a high degree of sequence homology with the consensus binding site defined for human genomic DNA. One of the clones was found to be derived from the LTR of a retrovirus-like element (a member of the GLN family). The region encompassing the GLN LTR p53 binding site could confer p53 responsiveness upon a heterologous promoter. Furthermore, the expression of the endogenous, chromosomally integrated GLN elements was significantly induced upon activation of wild type p53 in cells harboring a temperature sensitive p53 mutant. Finally, it was demonstrated that p53 - MDM2 complexes fail to bind tightly to such a p53 binding site. This may contribute to the inhibition by MDM2 of p53-mediated transcriptional activation.
Animals↗
Identification of target genes regulated by PAX3 and PAX3-FKHR in embryogenesis and alveolar rhabdomyosarcoma.
PAX3 is a transcription factor important for neural, muscle, and facial development in vertebrates. To identify genes regulated by PAX3, we used a cyclic amplification and selection of targets (CASTing) strategy to isolate cis-regulatory elements bound by PAX3. CASTing libraries were constructed with mouse DNA fragments bound by mouse PAX3, and human genomic DNA fragments bound by human PAX3 and the fusion protein PAX3-FKHR. Approximately 1000 clones were sequenced from each of these three libraries. Numerous putative targets of PAX3 and PAX3-FKHR were identified and six genes, Itm2A, Fath, FLT1, TGFA, BVES, and EN2, were examined closely. The genomic DNA fragments near these genes contain PAX3 binding sites and confer PAX3-dependent regulation. The expression levels of these genes correlate with the PAX3 expression levels in mouse embryos or with PAX3-FKHR expression levels in rhabdomyosarcoma cell lines, and indicate they may be part of the PAX3 regulatory circuitry during embryogenesis and tumor formation.
Animals↗
Identification of target promoters for the Bacillus subtilis sigma X factor using a consensus-directed search.
The promoter selectivity of RNA polymerase (RNAP) can be altered by the association with alternative sigma subunits. Bacillus subtilis hosts a multitude of sigma factors, several of which coordinate the complex developmental program culminating in endospore formation. Genome sequencing has revealed an unanticipated seven new sigma factors of the highly divergent extracytoplasmic function (ECF) sub-family. Virtually nothing is known regarding either the promoter selectivity or the target genes for these newly identified sigma factors. We have used saturation mutagenesis to define a promoter consensus for recognition by one such ECF sigma factor, sigma X. The resulting consensus sequence was used to identify candidate sigma X target sites. Three newly identified sigma X-dependent promoters precede genes encoding regulatory proteins: an AbrB homolog (Abh), a putative response regulator aspartate phosphatase (RapD), and a regulator of autolysin expression (LytR). sigma X also contributes to the expression of CsbB, a putative membrane-bound glucosyl transferase that is partially controlled by the sigma B stress response sigma factor. Since LytR modulates the expression of the major autolytic amidase and CsbB may function in peptidoglycan synthesis or modification, we suggest that sigma X participates in the regulation of peptidoglycan synthesis and turnover.
Bacillus subtilis↗
Identification of target cells by immunohistochemical detection of covalently rearranged estradiol in rehydrated paraffin sections.
Estradiol is released from the binding niche of the receptor and covalently arrested in the molecular vicinity by the Mannich reaction during target fixation in acetic acid/formaldehyde. The exposed steroid is freely accessible for appropriate antibodies. It can be visualized in sections by the second antibody/enzyme technique in high resolution and without enhancements.
Animals↗
Identification of target mRNAs for the clock-controlled RNA-binding protein Chlamy 1 from Chlamydomonas reinhardtii.
An endogenous clock regulates the temporal expression of genes/mRNAs that are involved in the circadian output pathway. In the green alga Chlamydomonas reinhardtii, a clock-controlled RNA-binding protein (Chlamy 1) was identified recently, which represents an analog of the circadian trans-acting factor CCTR from the phylogenetically diverse alga Gonyaulax polyedra. In order to identify in C. reinhardtii target mRNAs that can be recognized by Chlamy 1, gel mobility-shift assays and UV-crosslinking experiments were carried out, and revealed that this protein interacts specifically with the 3' untranslated regions of several mRNAs and recognizes them all via a common cis-acting element, composed of at least seven UG repeats. By using competition assays, it was found that the affinity of Chlamy 1 is highest for mRNAs whose products are key components of nitrogen and CO2 metabolism. Since the activities of enzymes involved in nitrogen metabolism vary in a temporal pattern that is opposite in phase to that of Chlamy 1 binding activity, the protein may repress the translation of the cognate mRNAs.
3' Untranslated Regions↗
Yeast and drug discovery.
Advanced genetic techniques, along with the high degree of conservation of basic cellular processes, have made the yeast Saccharomyces cerevisiae a valuable system for identification of new drug targets, target-based and non-target-based drug screening, and detailed analysis of the cellular effects of drugs. Yeast also presents a convenient system for antifungal drug discovery because it is closely related to Candida albicans, a major human pathogen. Many yeast genes remain poorly characterized, and most of the sophisticated techniques in yeast have been in widespread use less than a decade - a period shorter than the typical cycle from target identification to marketing approval for a new drug. It is likely that most of the benefits of yeast in discovery and development of therapeutic compounds have yet to be realized.
Antifungal Agents↗
Identification of target cells for estrogen in the pars distalis of the armadillo by immunocytochemistry.
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Animals↗
Role of host antibody in the chemotherapeutic action of praziquantel against Schistosoma mansoni: identification of target antigens.
We have demonstrated previously in a mouse model that effective chemotherapy against Schistosoma mansoni with praziquantel (PZQ) is dependent upon an intact host antibody response. In the same study, it was found that worms recovered from PZQ-treated animals display surface-bound antibodies. In order to identify the target antigens of the antibodies involved in the synergy between PZQ and the immune response, monoclonal antibodies (mAbs) and polyclonal antisera recognizing different tegumental components were tested by indirect immunofluorescence (IF) assay for their ability to bind in vitro to the surface of 6-week-old schistosomes perfused from nude (athymic) mice 1 h after PZQ treatment. Nude mice were used as hosts because worms from these animals were found to lack bound anti-schistosome antibodies. Only 5 of the 21 antibodies tested reacted with drug-treated worms. This indicated that the damage caused by PZQ to the schistosome tegument is restricted to specific tegumental components. Of the positive reactions, one group of antibodies gave IF patterns different from, whereas the other group gave IF reactions similar to those seen with worms perfused from immunologically intact mice. Antibodies against a schistosome esterase and alkaline phosphatase produced reaction patterns in the former category. In contrast, two out of three monoclonal antibodies recognizing different epitopes on a 200-kDa glycoprotein abundant in worm tubercles gave IF patterns very similar to those observed on schistosomes from drug-treated, intact mice. The biological significance of these reactions was confirmed by demonstrating that transfer of one of the positive monoclonal antibodies to 6-week-infected, B cell-depleted (mu-suppressed) mice reconstitutes the efficacy of PZQ treatment to normal levels. The above results suggest that the antibodies involved in the mechanism of action of PZQ react with a limited set of antigens. Furthermore, they implicate the 200-kDa tubercle protein as a major target of this response in naturally infected hosts.
Animals↗
Identification of target genes of the transcription factor HNF1beta and HNF1alpha in a human embryonic kidney cell line.
Hepatocyte nuclear factor 1beta (HNF1beta, TCF2) is a tissue-specific transcription factor whose mutation in humans leads to renal cysts, genital malformations, pancreas atrophy and maturity onset diabetes of the young (MODY5). Furthermore, HNF1beta overexpression has been observed in clear cell cancer of the ovary. To identify potential HNF1beta target genes whose activity may be deregulated in human patients, we established a human embryonic kidney cell line (HEK293) expressing HNF1beta conditionally. Using Flp recombinase, we introduced wild type or mutated HNF1beta at a defined chromosomal position allowing a most reproducible induction of the HNF1beta derivatives upon tetracycline addition. By oligonucleotide microarrays we identified 25 HNF1beta-regulated genes. By an identical approach, we identified that the related transcription factor HNF1alpha (TCF1) affects only nine genes in HEK293 cells and thus is a less efficient factor in these kidney cells. The HNF1beta target genes dipeptidyl peptidase 4 (DPP4), angiotensin converting enzyme 2 (ACE2) and osteopontin (SPP1) are most likely direct target genes, as they contain functional HNF1 binding sites in their promoter region. Since nine of the potential HNF1beta target genes are deregulated in clear cell carcinoma of the ovary, we propose that HNF1beta overexpression in the ovarian cancer participates in the altered expression pattern.
Angiotensin-Converting Enzyme 2↗
Genotyping of enteroaggregative Escherichia coli and identification of target genes for the detection of both typical and atypical strains.
Enteroaggregative Escherichia coli (EAggEC) is an important cause of diarrhea worldwide, and there is a need for better detection methods in diagnostic laboratories. The aims of this study were i) to characterize strains of EAggEC by assigning each isolate a genotypic profile and (ii) to determine target genes for the detection of both typical and atypical EAggEC. The heterogeneity of the EAggEC group makes selection of a single target gene difficult. The plasmid-encoded genes, aat, aggR, and aap, are all appropriate targets for the detection of typical EAggEC. Of the chromosomally encoded genes, aaiA would be the most suitable target gene to identify typical and atypical EAggEC. The astA gene, encoding the enteroaggregative heat stable toxin, although not specific for EAggEC, may be used effectively in combination with other specific EAggEC genes. A polymerase chain reaction test based on the detection of characteristic EAggEC virulence genes, such as aat, astA, and aaiA, would improve EAggEC diagnosis.
Bacterial Typing Techniques↗
Identification of target genes for the Xenopus Hes-related protein XHR1, a prepattern factor specifying the midbrain-hindbrain boundary.
The midbrain-hindbrain boundary (MHB) acts as a local organizer in the development of the CNS in vertebrates. Previously, we identified an MHB-specific bHLH-WRPW transcriptional repressor gene, Xenopus Hes-related 1 (XHR1), which is initially expressed in the presumptive MHB (pre-MHB) region at the early gastrula stage. To better understand the gene cascades involved in MHB formation, we investigated the genes downstream from XHR1 by differential screening using a Xenopus cDNA macroarray and a dexamethasone (DEX)-inducible, dominant-negative transcriptional activator construct of XHR1 (XHR1-VP16-GR). Among the newly identified candidate target genes of XHR1 were Enhancer of split-related genes (ESR1, ESR3/7, and ESR9) and Xenopus laevis cleavage 2 (XLCL2). XHR1-VP16-GR induced the expression of the ESR genes and XLCL2 as well as Xdelta1, Xngnr1, and XHR1 itself in the presence of DEX even after pretreatment with the protein synthesis inhibitor, cycloheximide. This suggests that these genes are direct targets of XHR1. XHR1-knockdown experiments with antisense morpholino oligos and ectopic expression of wild-type XHR1 revealed that XHR1 is necessary and sufficient to repress ESR genes in the pre-MHB region. Misexpression of the ESR genes in the pre-MHB region repressed the MHB marker gene, Pax2, suggesting that the repression of the ESR genes by XHR1 is at least partly required for the early development of the pre-MHB. Our data also show that XHR1 is not activated by Notch signaling, differing from ESR genes. Taken together, we propose a model in which XHR1 defines the pre-MHB region as a prepattern gene by repressing those possible direct target genes.
Animals↗
Would Chlamydia trachomatis recombinant ribonucleic acid be a better target for identification?
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Chlamydia Infections↗
Identification of target genes of the lymphoid-specific transcription factor Oct2.
The Oct2 transcription factor is expressed predominantly in B lymphocytes and plays an essential role during the terminal phase of B cell differentiation. The regulatory regions of several genes specifically expressed in B cells contain functional binding sites for Oct2. Nevertheless, none of the genes originally thought to be regulated by Oct2 were affected in their expression in Oct2-deficient B cells. In an attempt to find such elusive Oct2 target genes and to understand the molecular function of Oct2 in B cell development, we isolated cDNAs for Oct2 target genes. So far, we have identified five potential targets for Oct2: the membrane glycoprotein CD36, the cysteine-rich secreted protein 3 (CRISP-3), a mouse homolog of the human monocyte/neutrophil elastase inhibitor (mEI) and two unknown cDNA sequences Nov1 and Nov2. These target genes show quite distinct expression patterns demonstrating that transcription factors in addition to Oct2 are involved in their regulation. Whereas CD36 and mEI were expressed in all hematopoetic cell lines containing Oct2,. CRISP-3 is pre-B cell-specific, Nov1 is plasma B cell-specific and Nov2 is B cell-specifically expressed.
Animals↗
Genomics-based identification of targets in pathogenic bacteria for potential therapeutic and diagnostic use.
The availability of numerous complete microbial genome sequences has profoundly altered our understanding of a number of fundamental biological processes. For example the enzymes involved in aminoacyl-tRNA (AA-tRNA) synthesis, the key process responsible for the accuracy of protein synthesis, have been found to be highly species-specific. In particular, a number of pathogens contain certain pathways of AA-tRNA synthesis that are unrelated to those found in their mammalian hosts. Since AA-tRNA synthesis is indispensable for cell viability, the discovery of pathogen-specific pathways and enzymes presents novel therapeutic and diagnostic targets. Here we will review recent advances in the elucidation of AA-tRNA synthesis pathways and discuss the possible pharmaceutical exploitation of these discoveries. In particular, the integration of genomic and biochemical approaches to identify novel targets for the treatment of Chlamydial infections and the diagnosis and treatment of Lyme disease will be presented.
Amino Acyl-tRNA Synthetases↗
Single gene target bacterial identification. groEL gene sequencing for discriminating clinical isolates of Burkholderia pseudomallei and Burkholderia thailandensis.
Proper identification of Burkholderia pseudomallei and Burkholderia thailandensis is crucial in guiding clinical management of patients with suspected melioidosis, as more than 99% of cases of melioidosis are caused by B. pseudomallei, whereas B. thailandensis is only responsible for causing less than 1% of the cases. However, the difference between the 16S ribosomal RNA gene sequences of B. pseudomallei and that of B. thailandensis is only 1%, and is therefore not discriminative enough for distinguishing the 2 species confidently. In this study, we amplified and sequenced the groEL genes of 7 strains of B. thailandensis and 6 strains of B. pseudomallei, and compared the sequences with 7 other groEL gene sequences of Burkholderia species. BLAST analysis revealed that the putative protein encoded by the groEL gene of B. thailandensis has 99.6%, 99.5%, 98.4%, 98.5%, and 96.5% amino acid identity with the groEL of B. pseudomallei, B. mallei, B. cepacia, B. vietnamiensis, and B. fungorum respectively. The amino acid sequences of GroEL of the strains of B. thailandensis and B. pseudomallei all showed >99.5% amino acid identity with each other. The nucleotide sequence of the groEL gene of any of the strains of B. thailandensis showed >99.8% nucleotide identity with that of any of the other strains of B. thailandensis, and the nucleotide sequence of the groEL gene of any of the strains of B. pseudomallei showed >99.5% nucleotide identity with that of any of the other strains of B. pseudomallei. However, the nucleotide sequence of any of the strains of B. thailandensis showed <97.6% nucleotide identity with any of the strains of B. pseudomallei. The amino acid sequences of GroEL of the 20 strains of Burkholderia species all showed >96% amino acid identity with each other. Furthermore, the nucleotide sequence of the groEL genes of the 2 strains of B. cepacia showed >99.5% nucleotide identity with each other, and the nucleotide sequence of the groEL gene of B. mallei showed >99.5% nucleotide identity with any of the strains of B. pseudomallei. The groEL gene sequence is therefore good for distinguishing between B. thailandensis and B. pseudomallei, and the GroEL amino acid and groEL nucleotide sequences of this single gene locus may potentially be useful for a 2-tier hierarchical identification of medically important Burkholderia at the genus and species levels respectively.
Base Sequence↗