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At least 883 records · Page 49Linked to original sources
PubMed2006

Genomic organization of the T-cell receptor gamma gene and PCR detection of its clonal rearrangement in canine T-cell lymphoma/leukemia.

Because the T-cell receptor gamma (TCRgamma) gene is rearranged at an early stage of T-cell development in both TCRalphabeta and TCRgammadelta lineages, it has been preferentially targeted to detect T-cell clonality in human lymphoma/leukemia. We isolated 22 independent cDNA clones encoding canine TCRgamma and the following analysis of nucleotide sequences using the dog genome database revealed that the canine TCRgamma locus contains at least four repertories of variable genes that can be organized into two distinct subgroups and six repertories of joining genes belonging to two distinct subgroups according to the nucleotide sequence similarity. The findings allowed us to design PCR primers that were directed to the conserved or specific nucleotide sequences for each subgroup of variable and joining genes. By using four different combinations of primers, a PCR-based analysis was performed on cell samples collected from T-cell lymphoma/leukemia and B-cell lymphoma cases and hyperplastic and normal lymph nodes. All cell samples from 11 T-cell malignancy cases exhibited clonal amplification by two out of four primer combinations. This finding was considered to be valuable in PCR-based analysis for detecting T-cell clonality in canine lymphoma/leukemia.

Amino Acid Sequence↗
PubMed2005

Expressed sequence tags from life cycle stages of Trichinella spiralis: application to biology and parasite control.

While the approach taken to date to study Trichinella spp., involves mainly characterization of individual genes of interest, we initiated a genomics approach as an antecedent to more complete genome sequencing. Our approach involves use of expressed sequence tags (ESTs) obtained from three life cycle stages of Trichinella spiralis; adult worms (AD), mature muscle larvae (ML) and immature L1 larvae (immL1, also known as newborn larvae) () to improve the technical capacity for research on Trichinella spp. and to generate information that will aid prospective development of relevant hypotheses. In this review, we will summarize findings of our EST analysis and discuss how they relate to topics mentioned above. The foundation laid by this data will also contribute toward development of a more substantial genomic database and technical capacity to dissect molecular interactions between vertebrate hosts and Trichinella spp.

Animals↗
PubMed2004

Computational discovery of DNA motifs associated with cell type-specific gene expression in Ciona.

Temporally and spatially co-expressed genes are expected to be regulated by common transcription factors and therefore to share cis-regulatory elements. In the ascidian Ciona intestinalis, the whole-genome sequences and genome-scale gene expression profiles allow the use of computational techniques to investigate cis-elements that control transcription. We collected 5' flanking sequences of 50 tissue-specific genes from genome databases of C. intestinalis and a closely related species Ciona savignyi. We searched for DNA motifs over-represented in upstream regions of a group of co-expressed genes. Several motifs were distributed predominantly in upstream regions of photoreceptor, pan-neuronal, or muscle-specific gene groups. One muscle-specific motif, M2, was distributed preferentially in regions from -200 to -100 bp relative to the translational start sites. Promoters of muscle-specific genes of C. intestinalis were isolated, connected with a green fluorescent protein gene (GFP), and introduced into C. intestinalis embryos. In muscle cells, these promoters specifically drove GFP expression, which mutations of the M2 sites greatly reduced. When M2 sites were located upstream of a basal promoter, the reporter GFP was specifically expressed in muscle cells. These results suggest the validity of our computational prediction of cis-regulatory elements. Thus, bioinformatics can help identify cis-regulatory elements involved in chordate development.

Animals↗
PubMed2005

Fog1 is required for cardiac looping in zebrafish.

To further our understanding of FOG gene function during cardiac development, we utilized zebrafish to examine FOG's role in the early steps of heart morphogenesis. We identified fragments of three fog genes in the zebrafish genomic database and isolated full-length coding sequences for each of these genes by using a combination of RT-PCR and 5'-RACE. One gene was similar to murine FOG-1 (fog1), while the remaining two were similar to murine FOG-2 (fog2a and fog2b). All Fog proteins were able to physically interact with GATA4 and function as transcriptional co-repressors. Whole-mount in situ hybridization revealed fog1 expression in the heart, the hematopoietic system, and the brain, while fog2a and fog2b expression was restricted to the brain. Injection of zebrafish embryos with a morpholino directed against fog1 resulted in embryos with a large pericardial effusion and an unlooped heart tube. This looping defect could be rescued by co-injection of mRNA encoding murine FOG-1, but not by mRNA encoding FOG-1 lacking the FOG repression motif. Taken together, these results demonstrate the importance of FOG proteins for zebrafish cardiac development and suggest a previously unappreciated role for FOG proteins in heart looping that is dependent on the FOG repression motif.

Animals↗
PubMed2005

The HIN-200 family: more than interferon-inducible genes?

The HIN-200 family was initially grouped together based on their hemopoietic expression, interferon-inducibility, nuclear localization, and characteristic 200 amino-acid domains. In this review, we performed a comprehensive search of genome databases and determined the location of previously characterized and predicted genes within the human, mouse, and rat HIN-200 loci. Several novel proteins were predicted in the mouse and rat. We also discuss recent advances in our understanding of this family of proteins and highlight the most important findings. In addition to a role in interferon biology, there is now good evidence supporting a role for these proteins as regulators of cell proliferation and differentiation. The activity of HIN-200 proteins is not restricted to the hemopoietic system as they are expressed and can function in a variety of other cells and tissues. The importance of HIN-200 proteins in disease now is beginning to be understood as they appear to be involved in autoimmunity and may act as tumor suppressor proteins.

Amino Acid Sequence↗
PubMed2006

Genes coding for intermediate filament proteins closely related to the hagfish "thread keratins (TK)" alpha and gamma also exist in lamprey, teleosts and amphibians.

The "thread keratins (TK)" alpha and gamma so far have been considered highly specialized intermediate filament (IF) proteins restricted to hagfish. From lamprey, we now have sequenced five novel IF proteins closely related to TKalpha and TKgamma, respectively. Moreover, we have detected corresponding sequences in EST and genomic databases of teleosts and amphibians. The structure of the TKalpha genes and the positions of their deduced amino acid sequences in a phylogenetic tree clearly support their classification as type II keratins. The genes encoding TKgamma show a structure typical for type III IF proteins, whereas their positions in phylogenetic trees favor a close relationship to the type I keratins. Considering that most keratin-like sequences detected in the lancelet also exhibit a gene structure typical for type III IF proteins, it seems likely that the keratin gene(s) originated from an ancient type III IF protein gene. According to EST analyses, the expression of the thread keratins in teleost fish and amphibians may be particularly restricted to larval stages, which, in conjunction with the observed absence of TKalpha and TKgamma genes in any of the available Amniota databases, indicates a thread keratin function closely related to larval development in an aquatic environment.

Amino Acid Sequence↗
PubMed2005

Duplication of growth hormone receptor (GHR) in fish genome: gene organization and transcriptional regulation of GHR type I and II in gilthead sea bream (Sparus aurata).

Nucleotide sequences encoding for functional growth hormone receptors (GHR) are now available in salmonids (coho and masu salmon) and other fish orders. Several authors have hypothesized a divergent evolution of salmonid GHRs, but the recent finding that a trout cDNA is related to non-salmonid (GHR type I) rather than to salmonid GHRs (GHR type II) points out a possible duplication of actively transcribed GHR genes. To address this issue, we search by RT-PCR for GHR type II in trout, gilthead sea bream, European sea bass, and turbot. Both in trout and gilthead sea bream, a cDNA sequence with all the characteristic features of masu and coho salmon GHRs (GHR type II) was found. Similarly, in European sea bass, a cDNA encoding for the intracellular domain of GHR type II was reported. No positive results were found in turbot, but searches in genome databases of fugu and zebrafish identified DNA sequences with a significant similarity to fish GHR type I and II, which are more related each other than to GHRs of tetrapods. Gene organization is, however, highly conserved through the evolution of vertebrates, and eight exons homologous to exons 2 and 4-10 of mammals were found in fish GHRs. Transcriptional regulation of GHR type I and II was also addressed by means of real-time PCR assays in gilthead sea bream. In liver and adipose tissue, GHR type I was the most abundant transcript, but the expression of GHR type I and II was practically equal in skeletal muscle. Both in liver and adipose tissue, the two GHRs were down-regulated by fasting. In skeletal muscle, the expression of GHR type I remained unaltered in fasted fish, whereas a 2- to 3-fold increase was found for GHR type II. All this provides suitable evidence for a tissue-specific regulation of fish GHRs, but further research is needed to determine whether these duplicated genes are evolved in a new or redundant fashion.

Amino Acid Sequence↗
PubMed2006

Molecular cloning of prepro-thyrotropin-releasing hormone cDNA from medaka (Oryzias latipes).

The cDNA encoding prepro-thyrotropin-releasing hormone (ppTRH) in a teleost, medaka (Oryzias latipes) was isolated and characterized. The medaka ppTRH cDNA codes for 270 amino acid residues including eight TRH progenitor sequences (-Lys/Arg-Arg-Gln-His-Pro-Gly-Lys/Arg-Arg-). In silico analyses of the medaka genome database predicted that the structure of the medaka ppTRH gene is similar to the ppTRH genes of the other vertebrate species studied to date; consisting of three exons and two introns. Identity of the medaka ppTRH with the other vertebrates is rather low except the sockeye salmon. A molecular phylogenic tree showed that the ppTRH sequences reflected the predicted pattern of species classification. RT-PCR analysis demonstrated ppTRH gene expression in the brain and retina. These results gave some insight into the molecular evolution of ppTRH and physiological functions of TRH in vertebrates.

Amino Acid Sequence↗
PubMed2005

Characterization of structure and expression of the Dzip1 gene in the rat and mouse.

A transcript encoding a rat homologue of DZIP1 (DAZ-interacting protein) was isolated from testis RNA. Like human DZIP1, it contains a C(2)H(2) zinc finger domain. A predicted mouse homologue of DZIP1 was found in the GenBank database. Genome analysis indicated that while DZIP1 and mouse Dzip1 contain 22 and 20 exons, respectively, the rat sequence was intronless, confirmed by PCR on genomic DNA. This rat Dzip1 sequence is homologous to mouse Dzip1 exons 1-6 and DZIP1 exons 5-9. As this rat sequence was shorter than DZIP1 it was designated rat Dzip1S. The rat genome also contained a further predicted homologue of DZIP1 displaying conserved linkage homology with mouse Dzip1 and DZIP1. This sequence, if expressed, is the true rat homologue of DZIP1, designated rat Dzip1. Rat Dzip1S mRNA was present in all tissues examined by qualitative RT-RCR, and in situ hybridization of rat testis confirmed that expression of rat Dzip1S mRNA was confined to the spermatogenic lineage, specifically premeiotic spermatogonia.

Adaptor Proteins, Signal Transducing↗
PubMed1998

A novel pathway for cytochromes c biogenesis in chloroplasts.

The cytochromes c are a useful model for the study of the pathways and mechanisms of assembly of the cofactor-containing components of energy transducing membranes. Genetic analyses have identified proteins that are required for the assembly of c-type cytochromes in mitochondria, bacteria and chloroplasts. The components of the pathway operating in fungal and animal mitochondria, i.e. the cytochrome (cyt) c and c1 heme lyases in the intermembrane space, were identified over a decade ago through the study of cytochrome deficiencies in Neurospora crassa and Saccharomyces cerevisiae. More recently, a large number of membrane or membrane-associated components were identified in various alpha- and gamma-proteobacteria as c-type cytochrome assembly factors; they comprise an assembly pathway that is evolutionarily and mechanistically distinct from that in fungal and animal mitochondria. The components function not only in the lyase reaction but also in the delivery and maintenance of the substrates in a state that is suitable for reaction in the bacterial periplasm. Yet a third pathway is required for cytochrome maturation in chloroplasts. Genetic analyses of Chlamydomonas reinhardtii ccs mutants, which are pleiotropically deficient in both the membrane-anchored cytochrome f and the soluble cytochrome c6, revealed a minimum of six loci, plastid ccsA and nuclear CCS1 through CCS5, that are required for the conversion of the chloroplast apocytochromes to their respective holo forms. Sequence analysis of the cloned ccsA and Ccs1 genes indicates that the predicted protein products are integral membrane proteins with homologues in cyanobacteria, some gram-positive bacteria (Bacillus subtilis, Mycobacterium spp.), beta-proteobacteria (Neisseria spp.) and an epsilon-proteobacterium (Helicobacter pylori). CcsA and Ccs1 require each other for accumulation in vivo and are therefore proposed to function in a complex, possibly with the products of some of the other CCS loci. A tryptophan-rich motif, which has been proposed to represent a heme binding site in bacterial cytochrome biogenesis proteins (CcmC and CcmF), is functionally important in plastid CcsA. As is the case for CcmC and CcmF, the tryptophan-rich sequence is predicted to occur in a loop on the p-side of the membrane, where the heme attachment reaction occurs. Conserved histidine residues in the CcsA and Ccs1 may serve as ligands to the heme iron. A multiple alignment of the tryptophan-rich regions of the CcsA-, CcmC- and CcmF-like sequences in the genome databases indicates that they represent three different families.

Amino Acid Sequence↗
PubMed2002

Genetic analysis of four novel peroxisome proliferator activated receptor-gamma splice variants in monkey macrophages.

Peroxisome proliferator activated receptor-gamma (PPAR-gamma) is abundantly expressed in atherosclerotic lesions and is implicated in atherogenesis. The existence of three splice variants, PPAR-gamma 1, PPAR-gamma 2, and PPAR-gamma 3 has been established. Using monocyte-derived macrophages from cynomolgus monkeys, we demonstrate here the identification of two new PPAR-gamma exons, exon C and exon D, which splice together with already established exons A1, A2, and B in the 5(') terminal region to generate four novel PPAR-gamma subtypes, PPAR-gamma 4, -gamma 5, -gamma 6, and -gamma 7. PPAR-gamma 4 and gamma 5 were detected only in macrophages whereas gamma 6 and gamma 7 were expressed both in macrophages and adipose tissues. None of these novel isoforms were detected in muscle, kidney, and spleen from monkeys. We found sequences identical to exons C and D in the human genome database. These and all PPAR-gamma exons known to date are encoded by a single gene, located from region 10498 K to 10384 K on human chromosome 3. We cloned and expressed PPAR-gamma 1, PPAR-gamma 4, and PPAR-gamma 5 proteins in yeast using the expression vector pPICZB. As expected, all recombinant proteins showed a molecular weight of approximately 50 kDa. We also investigated the effect of a high-fat diet on the level of macrophage PPAR-gamma expression in monkeys. RT-PCR showed a significant increase in total PPAR-gamma and ABCA1 mRNA levels in macrophages of fat-fed monkeys (n=7) compared to those maintained on a normal diet (n=2). However, none of the novel isoforms seemed to be induced by fat-feeding. We used tetracycline-responsive expression vectors to obtain moderate expression of PPAR-gamma 4 and -gamma 5 in CHO cells. In these cells, expression of PPAR-gamma 5 but not -gamma 4 repressed the expression of ABCA1. Neither isoform modulated the expression of lipoprotein lipase. Our results suggest that individual PPAR-gamma isoforms may be responsible for unique tissue-specific biological effects and that PPAR-gamma 4 and -gamma 5 may modulate macrophage function and atherogenesis.

Alternative Splicing↗
PubMed2002

SPAP2, an Ig family receptor containing both ITIMs and ITAMs.

This study reports cloning and characterization of SPAP2, a novel transmembrane protein. The extracellular portion of SPAP2 contains six immunoglobulin-like domains and its intracellular segment has two immunoreceptor tyrosine-based activation motifs (ITAMs) and two immunoreceptor tyrosine-based inhibition motifs (ITIMs). We also identified four alternatively spliced products. Sequence alignment with the genomic database revealed that the SPAP2 gene contains 16 exons and is localized at chromosome 1q21. PCR analyses demonstrated that SPAP2 mRNA is expressed in restricted human tissues including the kidney, salivary gland, adrenal gland, uterus, and bone marrow. Tyrosine-phosphorylated SPAP2 is specifically associated with SH2 domain-containing tyrosine kinases Syk and Zap70 and SH2 domain-containing tyrosine phosphatases SHP-1 and SHP-2. Site-specific mutagenesis studies revealed that tyrosyl residues 650 and 662 embedded in the ITIMs are responsible for the binding of Syk and Zap70 while tyrosyl residues 692 and 722 embedded in the ITIMs are involved in interactions with SHP-1 and SHP-2. Finally, recruitment of SHP-1 to the tyrosine-phosphorylated ITIMs led to a marked activation of the enzyme.

Alternative Splicing↗
PubMed2002

Human epidermis is a novel site of phospholipase B expression.

Phospholipase B (PLB) is an enzyme that displays both phospholipase A(2) and lysophospholipase activities. Analysis of human epidermis homogenates indicated the presence of a 97 kDa PLB protein, as well as a phospholipase A(2) activity, both being enriched in the soluble fraction. Immunolabelling and in situ hybridization experiments showed that this enzyme is expressed in the different layers of epidermis with an accumulation at the dermo-epidermis junction. RT-PCR data indicated that PLB is specifically expressed in natural and reconstructed epidermis. By 3'-RACE-PCR and screening of human genome databases, we obtained a 3600 bp cDNA coding for human PLB highly homologous to already described intestinal brush border PLBs. These data led us to conclude that the soluble PLB corresponds to a proteolytic cleavage of the membrane anchored protein. Altogether, our results provide the first characterization of human PLB which should play an important role in epidermal barrier function.

Amino Acid Sequence↗
PubMed2002

Molecular cloning and functional expression of a Drosophila corazonin receptor.

The insect adipokinetic hormones (AKHs) constitute a large family of neuropeptides that mobilize lipids and sugar from the insect fat body during energy-requiring activities such as flight. We have previously identified the first insect AKH receptors from the fruitfly Drosophila melanogaster and the silkworm Bombyx mori (Staubli et al., PNAS 2002, 99: 3446-3451). Here, we have cloned the cDNA of a Drosophila G protein-coupled receptor that was closely related to the first Drosophila AKH receptor both with respect to amino-acid sequence and gene structure. We have subsequently expressed this orphan receptor in Chinese hamster ovary cells and identified Drosophila corazonin as the endogenous ligand for the receptor. Corazonin increases heart beat in some insects, but its function in Drosophila is unknown. These results are intriguing, because not only are the Drosophila AKH and corazonin receptors structurally and evolutionarily related, but also are their preprohormones, which suggests a co-evolution of ligands and receptors. The Drosophila corazonin receptor is expressed in embryos, larvae, pupae, and adult flies. Furthermore, a receptor that is structurally very similar to the Drosophila corazonin receptor can be found in the genomic database from the malaria mosquito Anopheles gambiae.

Amino Acid Sequence↗
PubMed2003

Molecular identification of a Drosophila G protein-coupled receptor specific for crustacean cardioactive peptide.

The Drosophila Genome Project website (www.flybase.org) contains the sequence of an annotated gene (CG6111) expected to code for a G protein-coupled receptor. We have cloned this receptor and found that its gene was not correctly predicted, because an annotated neighbouring gene (CG14547) was also part of the receptor gene. DNA corresponding to the corrected gene CG6111 was expressed in Chinese hamster ovary cells, where it was found to code for a receptor that could be activated by low concentrations of crustacean cardioactive peptide, which is a neuropeptide also known to occur in Drosophila and other insects (EC(50), 5.4 x 10(-10)M). Other known Drosophila neuropeptides, such as adipokinetic hormone, did not activate the receptor. The receptor is expressed in all developmental stages from Drosophila, but only very weakly in larvae. In adult flies, the receptor is mainly expressed in the head. Furthermore, we identified a gene sequence in the genomic database from the malaria mosquito Anopheles gambiae that very likely codes for a crustacean cardioactive peptide receptor.

Amino Acid Sequence↗
PubMed2003

Novel human G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) are important mediators of signal transduction and targets for pharmacological therapeutics. Novel receptor-ligand systems have been discovered through the identification and analysis of orphan GPCRs (oGPCRs). Here we describe the discovery of seven novel human genes encoding oGPCRs. Each novel oGPCR gene was discovered using customized searches of the GenBank genomic databases with previously known GPCR-encoding sequences. The expressed genes can now be used in assays to determine endogenous and pharmacological ligands. GPR133, GPR134, GPR135, GPR136, and GPR137 share identities with a prostate-specific odorant-like GPCR-encoding gene (PSGR). GPR138 and GPR139 share identities with an odorant-like gene derived from human erythroid cells. Transcripts encoding GPR133, GPR134, GPR135, GPR136, and GPR137 were detected in various CNS tissues. The expression of odorant-like genes in non-olfactory tissues requires further clarification, which may be achieved through the search for endogenous cognate ligands for these and other oGPCRs.

Amino Acid Sequence↗
PubMed2003

Reconstitution of archaeal ribonuclease P from RNA and four protein components.

Ribonuclease P (RNase P) is an endonuclease responsible for generating the 5(') end of matured tRNA molecules. A homology search of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 genome database revealed that the four genes, PH1481, PH1601, PH1771, and PH1877, have a significant homology to those encoding RNase P protein subunits, hpop5, Rpp21, Rpp29, and Rpp30, of human, respectively. These genes were expressed in Escherichia coli cells, and the resulting proteins Ph1481p, Ph1601p, Ph1771p, and Ph1877p were purified to apparent homogeneity in a set of column chromatographies. The four proteins were characterized in terms of their capability to bind the cognate RNase P RNA from P. horikoshii. All four proteins exhibited the binding activity to the RNase P RNA. In vitro reconstitution of four putative RNase P proteins with the in vitro transcripted P. horikoshii RNase P RNA revealed that three proteins Ph1481p, Ph1601p, and Ph1771p, and RNase P RNA are minimal components for the RNase P activity. However, addition of the fourth protein Ph1877p strongly stimulated enzymatic activity, indicating that all four proteins and RNase P RNA are essential for optimal RNase P activity. The present data will pave the way for the elucidation of the reaction mechanism for archaeal as well as eukaryotic RNase P.

Animals↗
PubMed2003

High-throughput expression, purification, and characterization of recombinant Caenorhabditis elegans proteins.

Modern proteomics approaches include techniques to examine the expression, localization, modifications, and complex formation of proteins in cells. In order to address issues of protein function in vitro using classical biochemical and biophysical approaches, high-throughput methods of cloning the appropriate reading frames, and expressing and purifying proteins efficiently are an important goal of modern proteomics approaches. This process becomes more difficult as functional proteomics efforts focus on the proteins from higher organisms, since issues of correctly identifying intron-exon boundaries and efficiently expressing and solubilizing the (often) multi-domain proteins from higher eukaryotes are challenging. Recently, 12,000 open-reading-frame (ORF) sequences from Caenorhabditis elegans have become available for functional proteomics studies [Nat. Gen. 34 (2003) 35]. We have implemented a high-throughput screening procedure to express, purify, and analyze by mass spectrometry hexa-histidine-tagged C. elegans ORFs in Escherichia coli using metal affinity ZipTips. We find that over 65% of the expressed proteins are of the correct mass as analyzed by matrix-assisted laser desorption MS. Many of the remaining proteins indicated to be "incorrect" can be explained by high-throughput cloning or genome database annotation errors. This provides a general understanding of the expected error rates in such high-throughput cloning projects. The ZipTip purified proteins can be further analyzed under both native and denaturing conditions for functional proteomics efforts.

Animals↗