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PubMed1996

Chemoreceptors expressed in taste, olfactory and male reproductive tissues.

We have identified three genes encoding previously uncharacterized chemoreceptors expressed in rat sensory and reproductive tissues using a reverse transcriptase polymerase chain reaction strategy. Degenerate oligonucleotides designed from conserved sequences in the rat olfactory receptor gene family were used to amplify candidate receptor gene products expressed in taste tissue. Sequence analysis of three distinct clonal isolates revealed that the gene products from taste bud were 30-75% identical to previously identified olfactory receptor genes. The genomic coding sequences predicted protein structures with seven membrane spanning regions that have strong conservation relative to other members of the G-protein-coupled olfactory receptor gene family. Transcripts for each of the three gene products were detected exclusively in taste, olfactory and male reproductive tissue. Sequence analysis of the polymerase chain reaction products confirmed that identical transcripts were expressed in all three tissues. These findings are the first demonstration that identical olfactory receptor-like gene are expressed in three distinct tissues.

Amino Acid Sequence↗
PubMed1995

Identification and tissue distribution of a novel variant of 11 beta-hydroxysteroid dehydrogenase 1 transcript.

A novel variant of 11 beta-hydroxysteroid dehydrogenase 1 (11 beta-HSD1) mRNA was identified from the ovine liver by reverse transcription-polymerase chain reaction (RT/PCR), and was named 11 beta-HSD1C mRNA. Sequence analysis of the RT-PCR product revealed that 11 beta-HSD1C mRNA was the product of an alternative exon-splicing within the 11 beta-HSD1 gene in which exon 5 was spliced out. Although it caused a deletion of 48 amino acids in the deduced 11 beta-HSD1 protein, this alternative splicing did not result in a shift within the predicted open reading frame of 11 beta-HSD1 cDNA. Thus, 11 beta-HSD1C mRNA was predicted to code for a protein of 244 amino acids. Using RT-PCR, we also examined the expression of 11 beta-HSD1C mRNA in ovine fetal organs and in maternal myometrium, endometrium, chorion, amnion and placenta. The 11 beta-HSD1C mRNA was expressed ubiquitously, similar to 11 beta-HSD1A mRNA, but at a lower abundance. Furthermore, since levels of 11 beta-HSD1C mRNA were directly related to those of 11 beta-HSD1A mRNA, there is no tissue-specificity for this shorter transcript and the only factor regulating its production appears to be 11 beta-HSD1A mRNA itself. To determine whether 11 beta-HSD1C mRNA encoded a functional enzyme, we inserted the cDNA into the expression vector pRc/CMV, and transfected the construct into Chinese hamster ovary cells. The transfected cells expressed a mRNA of expected size but contained no detectable 11 beta-HSD activity. When combined with cellular extracts of 11 beta-HSD1A cDNA transfected cells, they also did not alter either the dehydrogenase or reductase activity. The functional significance of the 11 beta-HSD1 transcript lacking exon 5 (11 beta-HSD1C mRNA) remains to be determined.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2003

Molecular cloning, functional expression, and gene silencing of two Drosophila receptors for the Drosophila neuropeptide pyrokinin-2.

The database of the Drosophila Genome Project contains the sequences of two genes, CG8784 and CG8795, predicted to code for two structurally related G protein-coupled receptors. We have cloned these genes and expressed their coding parts in Chinese hamster ovary cells. We found that both receptors can be activated by low concentrations of the Drosophila neuropeptide pyrokinin-2 (CG8784, EC(50) for pyrokinin-2, 1x10(-9)M; CG8795, EC(50) for pyrokinin-2, 5 x 10(-10)M). The precise role of Drosophila pyrokinin-2 (SVPFKPRLamide) in Drosophila is unknown, but in other insects, pyrokinins have diverse myotropic actions and are also initiating sex pheromone biosynthesis and embryonic diapause. Gene silencing, using the RNA-mediated interference technique, showed that CG8784 gene silencing caused lethality in embryos, whereas CG8795 gene silencing resulted in strongly reduced viability for both embryos and first instar larvae. In addition to the two Drosophila receptors, we also identified two probable pyrokinin receptors in the genomic database from the malaria mosquito Anopheles gambiae. The two Drosophila pyrokinin receptors are, to our knowledge, the first invertebrate pyrokinin receptors to be identified.

Amino Acid Sequence↗
PubMed2026

Internal Bayesian precision modulates the neural representation of social attention: Disentangling implicit and explicit components via model-informed multivariate EEG analysis.

Social attention integrates sensory cues with high-level cognitive expectations, yet the generative mechanisms through which implicit orienting and explicit belief-driven modulation interact remain poorly understood. This ambiguity complicates the distinction between specialized social modules and domain-general attentional processes. We combined a dynamic cueing task with hierarchical Bayesian modeling and model-informed multivariate EEG decoding to address this. Behavioral results revealed a computational double dissociation: symbolic arrow cues elicited heterogeneous strategies, whereas averted gaze recruited a consistent, surprise-driven computational phenotype. At the neural level, time-resolved decoding and temporal generalization revealed a critical representational shift starting approximately 400 ms post-cue. Initial activity related to physical cue features was rapidly replaced by stable neural templates of predicted spatial intent. Crucially, topographical activation patterns showed that this intentional template, characterized by a lateralized temporo-occipital distribution, emerged exclusively under high internal certainty. Furthermore, partial representational similarity analysis demonstrated that late-stage neural manifolds were overwhelmingly organized around integrated spatial goals rather than isolated sensory or motivational signals. These findings suggest that social attention is a specialized generative process, where internal certainty modulates the transformation of social perceptions into actionable top-down intentions.

Bayesian computational modeling↗
PubMed2003

Molecular identification of the first insect proctolin receptor.

The website of the Drosophila Genome Project (www.flybase.org) contains the sequence of an annotated gene CG6986, which is predicted to code for a G protein-coupled receptor. We cloned the cDNA of this gene and expressed it in Chinese hamster ovary cells. Screening of a neuropeptide library revealed that the expressed receptor was specific for the neuropeptide proctolin (EC(50), 6x10(-10)M). Proctolin (RYLPT) was the first invertebrate neuropeptide to be fully sequenced (already in 1975) and occurs with identical structure in both crustaceans and insects, where it has myo- and neurostimulatory actions. Northern blots showed that the Drosophila proctolin receptor was only weakly expressed in embryos, larvae, pupae, and in the thoraces and abdomina of adult flies, but strongly in the heads of adult animals. The Drosophila receptor reported here is the first invertebrate proctolin receptor to be identified.

Amino Acid Sequence↗
PubMed1988

cDNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis.

By the differential hybridization technique, we isolated a cDNA clone, MK1, whose RNA level increased in early stages of retinoic acid-induced differentiation of embryonal carcinoma cells. The amount of MK1 RNA progressively decreased in the later stages of the differentiation. In mouse embryos, MK1 RNA was abundant in mid-gestation stages (Day 8 to Day 11) and decreased thereafter. The corresponding RNA was 1.0 killobase in size. From the nucleotide sequence, MK1 gene was predicted to code a polypeptide of molecular weight 9,971, which was rich in basic amino acids.

Animals↗
PubMed2000

Genetic characterization of angiomatoid fibrous histiocytoma identifies fusion of the FUS and ATF-1 genes induced by a chromosomal translocation involving bands 12q13 and 16p11.

This case report documents the first karyotypic, fluorescence in situ hybridization, and genetic analysis of an angiomatoid fibrous histiocytoma that arose and recurred in the arm of a 5.5-year-old girl. Complex rearrangements between chromosomes 2, 12, 16, and 17 were noted, as well as deletion in the long arm of chromosome 11. Flow cytometry revealed a normal cell population. The t(12;16) site was further investigated using reverse transcriptase-polymerase chain reaction. We found that the FUS (also known as TLS) gene from 16p11 combined with the ATF-1 gene from 12q13 to generate a chimeric FUS/ATF-1. The FUS gene is rearranged in the t(12;16)(q13;p11) that characterizes myxoid liposarcoma and in acute myeloid leukemia with t(16;21)(p11;q22), while the ATF-1 gene is rearranged in the t(12;22)(q13;q12) found recurrently in clear cell sarcomas (malignant melanoma of soft parts). Thus, the FUS/ATF-1 gene in angiomatoid fibrous histiocytoma is predicted to code for a protein that is very similar to the chimeric EWS/ATF-1 found in clear cell sarcoma.

Activating Transcription Factor 1↗
PubMed1999

Alternative-splicing of serotonin receptor isoforms in the pharynx and muscle of the parasitic nematode, Ascaris suum.

Pharyngeal pumping is essential for nematode feeding and survival and is dramatically stimulated by serotonin (5-HT). In the present study, a cDNA pool was prepared from poly A + RNA isolated from pharynxes dissected from the parasitic nematode, Ascaris suum, and was used as a template for RT-PCR with degenerate primers designed from sequences conserved in 5-HT receptors from a variety of sources. A putative 5-HT receptor cDNA (AS1) was identified which exhibited most identity to the 5-HT2 family of receptors. AS1 was 1925 nucleotides, did not appear to be trans-spliced and contained a 3' untranslated region of 127 nucleotides with a polyadenylation signal (ATTAAA) and a short poly A+ tail. The coding region predicted a protein of 532 amino acids with a molecular weight of 60 176. When AS1 was transiently expressed in COS-7 cells, isolated membranes exhibited the high affinity, saturable binding of [125I]LSD. More importantly, [125I]LSD binding was inhibited by 5-HT, but not other biogenic amines, supporting the identification of AS1 as a 5-HT receptor. Additional cDNAs identical, in part, to AS1 were also identified. AS1deltaIV lacked a predicted 42 amino acids at the carboxy terminus of the third intracellular loop, while AS2 and AS3 contained different COOH-termini, regions implicated in G-protein coupling in other heptahelical receptors. A portion of the gene (5htn) encoding AS1 also was cloned and sequenced. This genomic fragment was about 10 kb, contained the entire AS1 open reading frame and included eight exons and seven introns. From this analysis, it appears that these different AS cDNAs were generated by alternative-splicing, AS1deltaIV from the deletion of exon IV, and AS2 and AS3 from the use of alternative sites within exon VII as 5' splice acceptor sites for exon VIII. Using RT-PCR and primers specific for each of the isoforms, AS1 -3 appeared to be expressed in pharynx, while only AS1 and AS2 were present in body wall muscle. More importantly, the deletion of exon IV appeared to be associated exclusively with AS1 in pharynx and AS2 in muscle.

Alternative Splicing↗
PubMed2001

Development and testing of radionuclide transport models for fractured rock: examples from the Nagra/JNC Radionuclide Migration Programme in the Grimsel Test Site, Switzerland.

The joint Swiss National Co-operative for the Disposal of Radioactive Waste (Nagra)/Japan Nuclear Cycle Development Institute (JNC) Radionuclide Migration Programme has now been on-going for over a decade in Nagra's Grimsel Test Site (GTS). The main aim of the programme has been the direct testing of radionuclide transport models in as realistic manner as possible. Although it will never be possible to fully test these models due to the large time and distance scales involved, tests of the model assumptions in scaled down but otherwise realistic conditions will contribute to developing confidence in the predictive power of the models. In this paper, the Nagra/JNC approach is highlighted with examples from a large programme of field, laboratory and natural analogue studies based around the GTS. The successes and failures are discussed as in the general approach to the thorough testing of predictive transport codes which will be used in repository performance assessment (PA). Some of the work is still on-going and this represents the first presentation of a unique set of results and conclusions.

Geological Phenomena↗
PubMed2001

Characterisation of a collagen gene subfamily from the potato cyst nematode Globodera pallida.

We have isolated two full-length genomic DNA sequences, which encode the cuticle collagen proteins GP-COL-1 and GP-COL-2, from the potato cyst nematode Globodera pallida. A third, partial collagen gene ORF termed gp-col-t(t=truncated) has also been isolated and appears to represent an unexpressed pseudogene. The gp-col-1 and gp-col-2 genes both contain three short (<97 bp) introns which disrupt coding regions predicted to specify proteins with molecular weights of 33 and 32.7 kDa respectively. All three sequences show high similarity to each other and to the previously isolated G. pallida cDNA clone gp-col-8. The conserved pattern of cysteine residues and non-(Gly-X-Y)(n) region sequence similarity observed in all four G. pallida genes suggests that these molecules form part of the same subfamily of collagens. Southern analysis indicates that this subfamily is likely to contain further members. The G. pallida collagen sequences show striking similarity to twelve genes from Caenorhabditis elegans which collectively represent the recently classified Group 1a collagen subfamily. No data exists on the function of this subfamily in C. elegans. gp-col-1 and gp-col-2 are developmentally regulated with transcripts of both genes detected in adult virgin and gravid females but not in pre-parasitic second stage juveniles. A similar expression pattern is observed for the Group 1a collagen lemmi 5 from Meloidogyne incognita perhaps indicating a generic link between subfamily and function during the various changes in cuticular structure which accompany nematode growth and reproduction. Immunochemical studies indicate that the GP-COL-1 protein is specifically located in the hypodermis of G. pallida adult females.

Amino Acid Sequence↗
PubMed2002

In vitro selection of high affinity HspR-binding sites within the genome of Helicobacter pylori.

The major chaperone genes of Helicobacter pylori are negatively regulated by HspR, a homologue of the repressor of the dnaK operon of Streptomyces coelicolor. Using an in vitro selection and amplification approach we identified two new chromosomal binding sites of the HspR protein. Both binding sites were characterized by footprinting analysis with purified HspR protein. Intriguingly, these HspR binding sites are located at the 3prime prime or minute ends of two genes coding for predicted proteins with functions unrelated to those of chaperones. This suggests that H. pylori HspR may regulate the expression of genes encoding proteins with diverse functions. Nucleotide sequence alignment of HspR-binding sites highlights conserved nucleotides extending outside the previously proposed consensus binding sequence with structural features predicting geometry of HspR binding as an oligomer.

Bacterial Proteins↗
PubMed2002

Cysteine and tyrosine-rich 1 (CYYR1), a novel unpredicted gene on human chromosome 21 (21q21.2), encodes a cysteine and tyrosine-rich protein and defines a new family of highly conserved vertebrate-specific genes.

A novel human gene has been identified by in-depth bioinformatics analysis of chromosome 21 segment 40/105 (21q21.1), with no coding region predicted in any previous analysis. Brain-derived DNA complementary to RNA (cDNA) sequencing predicts a 154-amino acid product with no similarity to any known protein. The gene has been named cysteine and tyrosine-rich protein 1 gene (symbol cysteine and tyrosine-rich 1, CYYR1). The CYYR1 messenger RNA was found by Northern blot analysis in a broad range of tissues (two transcripts of 3.4 and 2.2 kb). The gene consists of four exons and spans about 107 kb, including a very large intron of 85.8 kb. Analysis of expressed sequence tags shows high CYYR1 expression in cells belonging to the amine precursor uptake and decarboxylation system. We also cloned the cDNA of the murine ortholog Cyyr1, which was mapped by a radiation hybrid panel on chromosome 16 within the region corresponding to that containing the respective human homolog on chromosome 21. Sequence and phylogenetic analysis led to identification of several genes encoding CYYR1 homologous proteins. The most prominent feature identified in the protein family is a central, unique cysteine and tyrosine-rich domain, which is strongly conserved from lower vertebrates (fishes) to humans but is absent in bacteria and invertebrates.

Amino Acid Sequence↗
PubMed1997

Sequence analysis and expression of the polyhedrin gene of Choristoneura fumiferana cytoplasmic polyhedrosis virus (CfCPV).

The segmented double-stranded RNA genome of Choristoneura fumiferana cytoplasmic polyhedrosis virus (CfCPV) was extracted, polyadenylated, reverse-transcribed into cDNA and cloned. The cDNA clones that hybridized to the smallest genomic segment (segment 10) were identified, and its nucleotide sequence was determined. Genome segment 10 of CfCPV was found to be 1171 nucleotides in length with a single open reading frame in one strand capable of coding a predicted protein of 258 residues (Mr of 29,795), consistent with an apparent Mr of 30.5 kDa determined by SDS-PAGE of purified polyhedrin. Comparison of the nucleotide and amino acid sequences of the polyhedrin gene of CfCPV with those of other CPVs and with several nuclear polyhedrosis viruses revealed no particular homology. Analysis of the hydrophilic profiles and predicted secondary structures of Bombyx mori (BmCPV), Euxoa scandens (EsCPV) and CfCPV indicated the presence of seven similar regions located at the amino terminus of the polyhedrin polypeptide of the three viruses. The expression of the cloned CfCPV polyhedrin gene in Escherichia coli demonstrated that this polyhedrin has the property of self-assembly, since the production of crystal-like occlusion with a well-defined crystalline lattice structure was observed.

Animals↗
PubMed2000

Molecular cloning, characterization and expression of a novel retinal clusterin-like protein cDNA.

A novel gene expressed predominantly in retina, but detected at a conspicuously lower level in retina of canine progressive rod cone degeneration (prcd), has been identified by suppression subtractive hybridization and retinal cDNA library screening. The characterized region of cDNA of the novel gene includes 1017 nucleotides of coding sequence predicted to encode a protein of 338 amino acids (M(r) 39389), 791 nucleotides of 5'-untranslated region (UTR), and 300 nucleotides of 3'-UTR including the poly(A)(+) tail. Multiple transcripts were detected in retina by Northern blot analysis, and a lower level of expression was observed in brain and liver by RT-PCR. The transcript appears to be developmentally regulated with a burst in gene expression at a time period (34 postnatal days) that coincides with the photoreceptor differentiation phase of retinal development. The deduced amino acid sequence from the cDNA of the novel gene has 24% identity and 48% similarity with the multifunctional glycoprotein clusterin. Hence, the putative gene product from the novel transcript has been named clusterin-like protein 1 (CLUL1). The human homologue of CLUL1 cDNA has 84 and 70% identity at the level of nucleotides and amino acids, respectively, with the characterized canine cDNA. The presence of a stretch of 128 amino acids in the putative human CLUL1, not detected in canine CLUL1, suggests alternate splicing events. An STS database search revealed that the human homologue of CLUL1 maps to chromosome 18p, a location not yet reported to harbor an RP locus. Tissue-specific expression of CLUL1 in retina, and its lower abundance in different forms of PRA suggest that this novel gene may represent an as-yet unidentified locus for a retinal disorder.

Amino Acid Sequence↗
PubMed1997

UNIFOLIATA regulates leaf and flower morphogenesis in pea.

BACKGROUND: The vegetative phenotype of the pea mutant unifoliata (uni) is a simplification of the wild-type compound leaf to a single leaflet. Mutant uni plants are also self-sterile and the flowers resemble known floral meristem and organ identity mutants. In Antirrhinum and Arabidopsis, mutations in the floral meristem identity gene FLORICAULA/LEAFY (FLO/LFY) affect flower development alone, whereas the tobacco FLO/LFY homologue, NFL, is expressed in vegetative tissues, suggesting that NFL specifies determinacy in the progenitor cells for both flowers and leaves. In this paper, we characterised the pea homologue of FLO/LFY. RESULTS: The pea cDNA homologue of FLO/LFY, PEAFLO, mapped to the uni locus in recombinant-inbred mapping populations and markers based on PEAFLO cosegregated with uni in segregating sibling populations. The characterisation of two spontaneous uni mutant alleles, one containing a deletion and the other a point mutation in the PEAFLO coding sequences, predicted that PEAFLO corresponds to UNI and that the mutant vegetative phenotype was conferred by the defective PEAFLO gene. CONCLUSIONS: The uni mutant demonstrates that there are shared regulatory processes in the morphogenesis of leaves and flowers and that floral meristem identity genes have an extended role in plant development. Pleiotropic regulatory genes such as UNI support the hypothesis that leaves and flowers derive from a common ancestral sporophyll-like structure. The regulation of indeterminancy during leaf and flower morphogenesis by UNI may reflect a primitive function for the gene in the pre-angiosperm era.

Alleles↗
PubMed2001

A new hybrid algorithm for ECG signal compression based on the wavelet transformation of the linearly predicted error.

This paper describes a hybrid technique based on the combination of wavelet transform and linear prediction to achieve very effective electrocardiogram (ECG) data compression. First, the ECG signal is wavelet transformed using four different discrete wavelet transforms (Daubechies, Coiflet, Biorthogonal and Symmlet). All the wavelet transforms are based on dyadic scales and decompose the ECG signals into five detailed levels and one approximation. Then, the wavelet coefficients are linearly predicted, where the error corresponding to the difference between these coefficients and the predicted ones is minimized in order to get the best predictor. In particular, the residuals of the wavelet coefficients are uncorrelated and hence can be represented with fewer bits compared to the original signal. To further increase the compression rate, the residual sequence obtained after linear prediction is coded using a newly developed coding technique. As a result, a compression ratio (Cr) of 20 to 1 is achieved with percentage root-mean square difference (PRD) less than 4%. The algorithm is compared to an alternative compression algorithm based on the direct use of wavelet transforms. Experiments on selected records from the MIT-BIH arrhythmia database reveal that the proposed method is significantly more efficient in compression. The proposed compression scheme may find applications in digital Holter recording, in ECG signal archiving and in ECG data transmission through communication channels.

Algorithms↗
PubMed1999

Translational infidelity and human cancer: role of the PTI-1 oncogene.

Several components of the eukaryotic protein synthesis apparatus have been associated with oncogenic transformation of cells. Altered expression of translation elongation factor 1 alpha (EF-1 alpha), a core component of protein synthesis and closely related sequences have been linked with transformed phenotypes by several independent studies, in diverse systems. A dominant acting oncogene, prostate tumor inducing gene-1 (PTI-1) has provided further evidence for this link. PTI-1 appears to be a hybrid molecule with components derived from both prokaryotic and eukaryotic origins. The predicted protein coding moiety represents an EF-1 alpha molecule, truncated N-terminal to amino acid residue 68 and having six additional point mutations. This coding sequence is fused to a 5' untranslated region (UTR) showing strongest homology to ribosomal RNA derived from Mycoplasma hyopneumoniae. Expression studies using the cloned cDNA in nude mouse tumor formation assays have confirmed the oncogenic nature of the molecule. A broad spectrum of tumor derived cell lines, from varied tissue sources and blood samples from patients having confirmed prostate carcinoma, all scored positive for expression of PTI-1, while corresponding normal tissues or blood samples were negative. Based on its near identity to EF-1 alpha, it is proposed that PTI-1 represents a new class of oncogene whose transforming capacity probably arises through mechanisms including: (i) protein translational infidelity, resulting in the synthesis of mutant polypeptides due to loss of proofreading function during peptide chain elongation, (ii) by its association with and alteration of the cytoskeleton, (iii) by impinging on one particular or several different signal transduction pathways through its properties as a G-protein.

Amino Acid Sequence↗
PubMed1998

Molecular cloning of a novel heat induced/chilling tolerance related cDNA in tomato fruit by use of mRNA differential display.

Chilling injury was circumvented by heat-treating mature green tomatoes (Lycopersicon esculentum, cv. Mountain Springs) at 42 degrees C for two days prior to storing them at 2 degrees C for one or two weeks, whereas fruits stored at 2 degrees C without preheating developed typical chilling injury symptoms and failed to ripen at 20 degrees C. Using mRNA differential display and screening of the cDNA libraries, we have cloned from tomato fruit a full-length HCT1 cDNA (heat induced/chilling tolerance related). The protein ( 17.6 kDa) predicted from coding region of HCT1 cDNA has high identity with class II cytosolic small HSPs. The gene corresponding to HCT1 cDNA was termed as LeHSP 17.6. Southern-blot hybridization indicates that LeHSP 17.6 belongs to a two-member gene family. Northern blot analysis indicates the heat-induced transcript of the LeHSP 17.6 remains up-regulated during subsequent exposure of the fruit to chilling temperatures for at least one week and upon transfer to ripening temperatures for one day. Fruits which were only chilled show a low level of expression of the LeHSP 17.6 transcript. We hypothesize that LeHSP 17.6 may be involved in protecting the cell from metabolic dysfunctions leading to ripening failure caused by chilling injury. This is the first report of a class II cytosolic smHSPs encoding gene in tomato.

Amino Acid Sequence↗