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PubMed1997

ETS target genes: identification of egr1 as a target by RNA differential display and whole genome PCR techniques.

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing's sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.

3T3 Cells↗

PubMed1991

Effect of extraneous targets on identification of color-coded targets.

Subjects matched the color of a vertical target line, presented in 1 of 10 colors, to a set of 10 colors. The target line was presented either in isolation or together with 5 additional colored lines. Neither mean reaction time nor the number of matching errors increased when the extraneous lines were present, and the types of color confusions did not change systematically.

Aerospace Medicine↗

PubMed2000

Stochastic heuristic algorithms for target motif identification (extended abstract).

Target motifs are motifs that are "close" to one or more substrings in each sequence in one given set of sequences but are far from every substring in another given set of sequences. Target motifs have pharmaceutical applications; unfortunately, the problem of identifying target motifs is NP-hard and is thus unlikely to have efficient optimal solution algorithms. In this paper, we propose a set of simple modifications to the Gibbs Sampling heuristic for finding motifs which allows this heuristic to detect target motifs. We also present the results of several experiments relative to both simulated and real datasets which suggest that this modified heuristic is good at detecting target motifs under a variety of conditions.

Algorithms↗

PubMed1998

Vectors for transcription factor cloning and target site identification by means of genetic selection in yeast.

We describe the construction of a number of vectors that can be used in yeast genetic selection systems for cloning of transcription factors or other DNA-binding proteins and for identification of the target sites recognized by transcription factors. For transcription factor cloning we have designed an integration vector with two HIS3 reporter gene cassettes to stably integrate reporter gene constructs at the non-essential yeast PDC6 locus. This set of plasmids was tested in a one-hybrid assay with the rice transcription factor Oshox1, a member of the class of homeodomain leucine zipper proteins. A hybrid protein of Oshox1 and the Gal4 transcriptional activation domain was shown to specifically activate a reporter gene construct with upstream Oshox1 binding sites, which had been integrated at the PDC6 locus using the described vector system. Target site identification by genetic selection in yeast employs a transcriptional activator construct and a library of genomic or random DNA fragments upstream of a reporter gene. We have constructed two variants of a bacteriophage lambda vector which facilitates the construction of the required reporter gene library because of high cloning efficiency and easy conversion into a yeast/Escherichia coli shuttle vector library by Cre-loxP-mediated automatic subcloning. Tests with Oxhox1 as transcriptional activator demonstrated the usefulness of the deprived reporter gene vector.

Bacteriophage lambda↗

PubMed2005

Phenotype-first screening for the identification of novel drug targets.

Modern drug discovery is predominantly a target-driven process, where success is intricately linked to the selection of an appropriate molecular target. Ideally, there is conclusive functional evidence that a selected target is disease-relevant and, furthermore, suitable for drug development. Phenotype-first screening is a highly attractive approach for target identification because it offers the unique possibility to analyse entire genomes in an unbiased fashion for disease-related phenotypes. Various studies have demonstrated that phenotype-first screening can be successfully applied to the identification of drug targets, thus establishing this approach as a valuable tool for future target discovery efforts.

Animals↗

PubMed2005

Genome-wide identification of the regulatory targets of a transcription factor using biochemical characterization and computational genomic analysis.

BACKGROUND: A major challenge in computational genomics is the development of methodologies that allow accurate genome-wide prediction of the regulatory targets of a transcription factor. We present a method for target identification that combines experimental characterization of binding requirements with computational genomic analysis. RESULTS: Our method identified potential target genes of the transcription factor Ndt80, a key transcriptional regulator involved in yeast sporulation, using the combined information of binding affinity, positional distribution, and conservation of the binding sites across multiple species. We have also developed a mathematical approach to compute the false positive rate and the total number of targets in the genome based on the multiple selection criteria. CONCLUSION: We have shown that combining biochemical characterization and computational genomic analysis leads to accurate identification of the genome-wide targets of a transcription factor. The method can be extended to other transcription factors and can complement other genomic approaches to transcriptional regulation.

Amino Acid Motifs↗

PubMed1982

Identification of human CML target. HLA-B locus (B12) antigen variants defined by CTL generated between B locus-identical (B12) responder-stimulator pairs.

The genetics of human CML targets were studied by seven CTL generated in combinations iun which the responder/stimulator difference was limited to one (or two) HLA-A, -B, or -C antigens. Unstimulated peripheral blood lymphocytes were used as targets. CTL sensitized against antigens A11, Aw31, or B17 lysed all cells bearing the respective target from a large panel of cells from unrelated individuals. Hence, at least one CTL clone was directed against the HLA antigen molecule. However, all CTL also exerted cross-kill to cells not sharing the stimulating HLA antigen. For two CTL, the target of the cross-kill was not clarified. Five CTL were found where the cross-kill was directed against antigen HLA-B12 (Bw44 and Bw45). All these cTL were generated in R/S pairs identical for B locus antigens (Bw44/Bw35 heterozygotes). The individual CTL lysed different parts of the panel of B12-positive target cells. The interpretation is that these CTL detect subtypes of HLA antigens, but alternative possibilities are also considered. Four B12 subtypes are described, tentatively designated as B12-related CML targets. Identification of HLA-B-related CML targets represent CML "typing" of HLA-antigen differences that were not detected serologically. The subtypes can now be tested for their possible functional significance.

B-Lymphocytes↗

PubMed1999

Predictive smooth pursuit eye movements during identification of moving acuity targets.

Repetitive, brief target ramp movements every few seconds lead to anticipatory acceleration before each ramp onset and anticipatory deceleration before ramp offset. We assessed whether identifying novel changes in the pursuit target would alter this pattern of anticipatory pursuit. Without target identification (TI), anticipatory acceleration increased when intervals between ramps were regular, rather than random. It increased further when, between ramps, the target was invisible rather than stationary and visible. Anticipatory deceleration increased when the target was expected to stop rather than disappear at ramp offset. For TI trials, the pursuit target changed briefly into a Landolt C acuity target that had to be identified. Compared to no TI, anticipatory acceleration decreased when a stationary C always appeared just before ramp onset. It increased when a moving C appeared just after ramp onset, but only when the target was invisible between ramps. Anticipatory deceleration was reduced when a moving C appeared just before ramp offset, but did not increase when a stationary C appeared just after ramp offset. The changes were significant, but of small magnitude, suggesting that predictive pursuit, especially with a visible target between ramps, cannot be greatly influenced by attempts to selectively improve acuity at a particular phase of the stimulus.

Adult↗

PubMed2003

Identification of Drosophila MicroRNA targets.

MicroRNAs (miRNAs) are short RNA molecules that regulate gene expression by binding to target messenger RNAs and by controlling protein production or causing RNA cleavage. To date, functions have been assigned to only a few of the hundreds of identified miRNAs, in part because of the difficulty in identifying their targets. The short length of miRNAs and the fact that their complementarity to target sequences is imperfect mean that target identification in animal genomes is not possible by standard sequence comparison methods. Here we screen conserved 3' UTR sequences from the Drosophila melanogaster genome for potential miRNA targets. The screening procedure combines a sequence search with an evaluation of the predicted miRNA-target heteroduplex structures and energies. We show that this approach successfully identifies the five previously validated let-7, lin-4, and bantam targets from a large database and predict new targets for Drosophila miRNAs. Our target predictions reveal striking clusters of functionally related targets among the top predictions for specific miRNAs. These include Notch target genes for miR-7, proapoptotic genes for the miR-2 family, and enzymes from a metabolic pathway for miR-277. We experimentally verified three predicted targets each for miR-7 and the miR-2 family, doubling the number of validated targets for animal miRNAs. Statistical analysis indicates that the best single predicted target sites are at the border of significance; thus, target predictions should be considered as tentative until experimentally validated. We identify features shared by all validated targets that can be used to evaluate target predictions for animal miRNAs. Our initial evaluation and experimental validation of target predictions suggest functions for two miRNAs. For others, the screen suggests plausible functions, such as a role for miR-277 as a metabolic switch controlling amino acid catabolism. Cross-genome comparison proved essential, as it allows reduction of the sequence search space. Improvements in genome annotation and increased availability of cDNA sequences from other genomes will allow more sensitive screens. An increase in the number of confirmed targets is expected to reveal general structural features that can be used to improve their detection. While the screen is likely to miss some targets, our study shows that valid targets can be identified from sequence alone.

3' Untranslated Regions↗

PubMed2000

Protein expression strategies for identification of novel target proteins.

Identification of new target proteins is a novel paradigm in drug discovery. A major bottleneck of this strategy is the rapid and simultaneous expression of proteins from differential gene expression to identify eligible candidates. By searching for a generic system enabling high throughput expression analysis and purification of unknown cDNAs, we evaluated the YEpFLAG-1 yeast expression system. We have selected cDNAs encoding model proteins (eukaryotic initiation factor-5A [eIF-5A] and Homo sapiens differentiation-dependent protein-A4) and cDNA encoding an unknown protein (UP-1) for overexpression in Saccharomyces cerevisiae using fusions with a peptide that changes its conformation in the presence of Ca2+ ions, the FLAG tag (Eastman Kodak, Rochester, NY). The cDNAs encoding unknown proteins originating from a directionally cloned cDNA library were expressed in all three possible reading frames. The expressed proteins were detected by an antibody directed against the FLAG tag and/or by antibodies against the model proteins. The alpha-leader sequence, encoding a yeast mating pheromone, upstream of the gene fusion site facilitates secretion into the culture supernatant. EIF-5A could be highly overexpressed and was secreted into the culture supernatant. In contrast, the Homo sapiens differentiation-dependent protein-A4 as well as the protein UP-1, whose cDNA did not match to any known gene, could not be detected in the culture supernatant. The expression product of the correct frame remained in the cells, whereas the FLAG-tagged proteins secreted into the supernatant were short, out-of-frame products. The presence of transmembrane domains or patches of hydrophobic amino acids may preclude secretion of these proteins into the culture supernatant. Subsequently, isolation and purification of the various proteins was accomplished by affinity chromatography or affinity extraction using magnetizable beads coated with the anti-FLAG monoclonal antibody. The purity of isolated proteins was in the range of 90%. In the case of unknown cDNAs, the expression product with the highest molecular mass was assumed to represent the correct reading frame. In summary, we consider the YEpFLAG-1 system to be a very efficient tool to overexpress and isolate recombinant proteins in yeast. The expression system enables high throughput production and purification of proteins under physiological conditions, and allows miniaturization into microtiter formats.

Blotting, Western↗

PubMed2006

In silico identification of potential therapeutic targets in the human pathogen Helicobacter pylori.

Availability of genome sequences of pathogens has provided a tremendous amount of information that can be useful in drug target and vaccine target identification. One of the recently adopted strategies is based on a subtractive genomics approach, in which the subtraction dataset between the host and pathogen genome provides information for a set of genes that are likely to be essential to the pathogen but absent in the host. This approach has been used successfully in recent times to identify essential genes in Pseudomonas aeruginosa. We have used the same methodology to analyse the whole genome sequence of the human gastric pathogen Helicobacter pylori. Our analysis revealed that out of the 1590 coding sequences of the pathogen, 40 represent essential genes that have no human homolog. We have further analysed these 40 genes by the protein sequence databases to list some 10 genes whose products are possibly exposed on the pathogen surface. This preliminary work reported here identifies a small subset of the Helicobacter proteome that might be investigated further for identifying potential drug and vaccine targets in this pathogen.

Anti-Bacterial Agents↗

PubMed2001

Microarrays for studying the host transcriptional response to microbial infection and for the identification of host drug targets.

It is essential to understand the molecular basis of a host's response to microbial infection so that disease and tissue damage can be prevented. Modulation of host RNA expression is a critical set of molecular changes that occur upon infection. Global analysis of gene expression should provide an understanding of host RNA transcriptional changes that occur upon host-pathogen interaction. This series of articles focuses on the use of microarrays for analyzing the transcriptional response of a host to microbial infection and for drug target identification.

Animals↗

PubMed1991

Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence.

Point mutagenesis of the nuclear targeting sequence of nucleoplasmin has identified two interdependent basic domains. These are separated by 10 intervening "spacer" amino acids that tolerate point mutations and some insertions. Amino acids in both basic domains are required for nuclear targeting, and the transport defect of a mutation in one domain is amplified by a simultaneous mutation in the other. Therefore, these basic domains are interdependent. A strikingly similar motif of two clusters of basic residues is seen in the nuclear targeting sequence of Xenopus N1. It is also conserved in the related nucleolar protein NO38. Several other short sequences known to be necessary for nuclear targeting fall within a similar motif.

Amino Acid Sequence↗

PubMed1998

Drug target validation and identification of secondary drug target effects using DNA microarrays.

We describe here a method for drug target validation and identification of secondary drug target effects based on genome-wide gene expression patterns. The method is demonstrated by several experiments, including treatment of yeast mutant strains defective in calcineurin, immunophilins or other genes with the immunosuppressants cyclosporin A or FK506. Presence or absence of the characteristic drug 'signature' pattern of altered gene expression in drug-treated cells with a mutation in the gene encoding a putative target established whether that target was required to generate the drug signature. Drug dependent effects were seen in 'targetless' cells, showing that FK506 affects additional pathways independent of calcineurin and the immunophilins. The described method permits the direct confirmation of drug targets and recognition of drug-dependent changes in gene expression that are modulated through pathways distinct from the drug's intended target. Such a method may prove useful in improving the efficiency of drug development programs.

Calcineurin↗

PubMed2006

Direct and simultaneous identification of Mycobacterium tuberculosis complex (MTBC) and Mycobacterium tuberculosis (MTB) by rapid multiplex nested PCR-ICT assay.

The Mycobacterium tuberculosis (MTB) shows different virulence and host infection range from other members of the M. tuberculosis complex (MTBC). Differential identification of MTB from MTBC is thus important in certain occasions. The currently commercially available molecular assays which use either IS6110 or 16S rDNA fragment as identification targets are mainly designed for identifying MTBC but not for MTB. Comparative genomic DNA analysis has provided valuable information on regions of difference (RD) present in MTB but not in other members of the MTBC. RD9 region is further suggested to be a potential target for differential identification of MTB from MTBC. In this study, using IS6110 and Rv3618 (belong to RD9) as the specific identification targets for MTBC and MTB, respectively, we developed and tested a multiplex nested PCR-ICT (immuno-chromatography test) assay for simultaneously and directly detecting not only MTBC but also MTB from 1500 clinical sputum specimens. The results were compared with traditional culture and biochemical identification results together with patients' clinical assessments. This assay showed a 95.5% sensitivity, 97.9% specificity, 2.1% false positive rate and 4.5% false negative rate towards detection of MTBC, and a 93.0% sensitivity, 99.8% specificity, 0.2% false positive rate and 7.0% false negative rate for detection of MTB. This detection system shows great potential in clinical application.

Chromatography↗

PubMed2025

Accurate serotype identification of Streptococcus pneumoniae using nanopore Cas9-targeted serotype identification (nCATSerotyping).

Streptococcus pneumoniae (pneumococcus) is a leading cause of community-acquired pneumonia and invasive diseases, particularly among children and the elderly. The introduction of pneumococcal conjugate vaccines has significantly reduced invasive pneumococcal disease, but the prevalence of non-vaccine serotypes and newly emerging serotypes is increasing globally. Thus, accurate serotyping is essential for epidemiological surveillance and the development of next-generation multivalent pneumococcal vaccines. Conventional serotyping methods, including multiplex polymerase chain reaction (mPCR), monoclonal antibody (mAb) assays, and Quellung reaction using rabbit antisera, are limited by serotype coverage and cross-reactivity, making the detection of new or emerging serotypes challenging. In this study, we developed a nanopore Cas9-targeted serotyping (nCATSerotyping) platform, which employs Cas9-mediated enrichment of the capsular polysaccharide synthesis locus followed by Oxford Nanopore sequencing. Applying this method to 276 clinical pneumococcal isolates collected in South Korea (2018-2020), we achieved a serotyping success rate of 97.10% (268/276), significantly outperforming conventional methods such as mAb and mPCR, which identified only 76.45% (211/276) of isolates. Whole-genome sequencing of the remaining eight non-typeable isolates revealed them to be non-pneumococcal (oral streptococci), confirming 100% accuracy for S. pneumoniae serotyping. Importantly, our method identified emerging and underrepresented serotypes, including serotype 13 and null capsule clade strains. nCATSerotyping offers a rapid, accurate, and comprehensive solution for pneumococcal serotyping, with significant advantages in identifying novel and non-typeable strains. This scalable platform will be a valuable tool for global serotype surveillance and next-generation multivalent pneumococcal vaccine development.IMPORTANCEAccurate pneumococcal serotyping is critical for vaccine development and epidemiological surveillance, particularly as non-vaccine serotypes emerge following widespread pneumococcal conjugate vaccine implementation. Current serotyping methods face significant limitations in coverage and accuracy, identifying around 76% of pneumococcal isolates and failing to detect emerging serotypes like serotype 13 and null capsule clades. The nanopore Cas9-targeted serotyping platform addresses these critical gaps by achieving 100% serotyping accuracy for confirmed Streptococcus pneumoniae isolates while identifying previously undetectable strains that conventional methods missed. This comprehensive approach is essential for monitoring vaccine effectiveness, understanding serotype replacement patterns, and informing next-generation vaccine development strategies. Furthermore, the identification of misclassified oral streptococci highlights the diagnostic precision needed for accurate pneumococcal surveillance, ensuring that epidemiological data accurately reflect true pneumococcal disease burden and serotype distribution patterns.

Streptococcus pneumoniae↗

PubMed2004

Object substitution and its relation to other forms of visual masking.

Three experiments compared letter identification accuracy over a wide range of target-mask intervals and mask types, including metacontrast, random dot noise, four surrounding dots, digits and letters. These comparisons were motivated by object substitution theory which makes three general predictions about visual masking: (1) very different looking backward masks will be equivalent in their effects when spatial attention is distributed, such that target identification is delayed, (2) masks will differ most in their effects on target identification when they are temporally integrated with the target, and (3) backward masking will be minimized when attention can be pre-focused on the spatial location of the target and the mask does not interfere with target identification. Results strongly supported the predictions and pointed to a new understanding of masking based on the separate processes of object formation and object substitution.

Attention↗

PubMed2025

Identification of potential cell surface targets in patient-derived cultures toward photoimmunotherapy of high-grade serous ovarian cancer.

Tumor-targeted, activatable photoimmunotherapy (taPIT) has shown promise in preclinical models to selectively eliminate drug-resistant micrometastases that evade standard treatments. Moreover, taPIT has the potential to resensitize chemo-resistant tumor cells to chemotherapy, making it a complementary modality for treating recurrent high-grade serous ovarian cancer (HGSOC). However, the established implementation of taPIT relies on the overexpression of EGFR in tumor cells, which is not universally observed in HGSOCs. Motivated by the need to expand taPIT applications beyond EGFR, we conducted mRNA-sequencing and proteomics to identify alternative cell surface targets for taPIT in patient-derived HGSOC cell cultures with weak EGFR expression and lacking expression of other cell surface proteins commonly reported in the literature as overexpressed in ovarian cancers, such as FOLR1 and EpCAM. Our findings highlight TFRC and LRP1 as promising alternative targets. Notably, TFRC was overexpressed in 100% (N = 5) of the patient-derived HGSOC models tested, whereas only 60% of models had high EpCAM expression, suggesting that future larger cohort studies should include TFRC. While this study focuses on target identification, future work will expand the approaches developed here to larger HGSOC biopsy repositories and will also develop and evaluate antibody-photosensitizer conjugates targeting these proteins for taPIT applications.

Humans↗