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Results for “Functional characterization”

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PubMed1999

Genetic redundancy and gene fusion in the genome of the Baker's yeast Saccharomyces cerevisiae: functional characterization of a three-member gene family involved in the thiamine biosynthetic pathway.

Redundancy is a salient feature of all living organisms' genome. The yeast genome contains a large number of gene families of previously uncharacterized functions that can be used to explore the functional significance of structural redundancy in a systematic manner. In this work, we describe results on a three-member gene family with moderately divergent sequences (YOL055c, YPL258c and YPR121w ). We demonstrate that two members are isofunctional and encode a hydroxymethylpyrimidine phosphate (HMP-P) kinase (EC 2.7.4.7), an activity required for the final steps of thiamine biosynthesis, whose genes were not previously known in yeast. In addition, we show that the three genes are each composed of two distinct domains, each corresponding to individual genes in prokaryotes, suggesting gene fusion during evolution. The function of the carboxy-terminal part of the proteins is not yet understood, but it is not required for HMP-P kinase activity. Expression of all three genes is regulated in the same way. Several other examples of gene fusions exist in the same biosynthetic pathway when eukaryotic genes are compared with prokaryotic ones.

Amino Acid Sequence↗

PubMed2000

Isolation and functional characterization of the chick M5 muscarinic acetylcholine receptor gene.

The chick is a widely used system for study of the actions of muscarinic acetylcholine receptors in the cardiovascular, visual, and nervous systems. We report the isolation and functional analysis of the gene encoding the chick M5 muscarinic receptor. RT-PCR analysis indicates that the M5 receptor is expressed at low levels in embryonic chick brain and heart. When expressed in stably transfected Chinese hamster ovary cells, the M5 receptor exhibits high-affinity binding to muscarinic antagonists and mediates robust activation of phospholipase C activity.

Amino Acid Sequence↗

PubMed2002

Identification, tissue expression, and functional characterization of Otx3, a novel member of the Otx family.

Transcription factors containing a homeodomain play an important role in the organogenesis of vertebrates. We have isolated a novel homeodomain transcription factor, Otx3, which is structurally and functionally related to Otx1 and Otx2, transcription factors that are critical in brain morphogenesis. Mouse Otx3 is a protein composed of 376 amino acids. Otx3 mRNA was expressed in mouse embryos from 10.5 to 13.5 days postcoitum (dpc) and in adult cerebellum as assessed by Northern blotting. Whole-mount in situ hybridization of mouse embryos from 9.5 to 11.5 dpc revealed strong expression of Otx3 mRNA in the diencephalon, mesencephalon, metencephalon, myelencephalon, and developing eye, indicating an expression pattern largely overlapping but distinct from those of Otx1 and Otx2. In addition, Otx3 was shown by electrophoretic mobility shift assay to bind to the TAATCC motif, the consensus binding sequence for Otx1, Otx2, and Crx. Results of a transcription reporter assay suggest that Otx3 functions as a transcription repressor by binding to this motif. These results suggest that Otx3 is a novel member of the Otx family and may be involved in the development of the central nervous system.

Amino Acid Sequence↗

PubMed2006

Functional characterization of AAA family FtsH protease of Mycobacterium tuberculosis.

FtsH is a membrane-bound ATP-dependent zinc-metalloprotease which proteolytically regulates the levels of specific membrane and cytoplasmic proteins that participate in diverse cellular functions, and which therefore might be of critical importance to a human pathogen such as Mycobacterium tuberculosis. As the substrates of MtFtsH in mycobacteria are not known, we examined whether recombinant MtFtsH could complement the lethality of a DeltaftsH3::kan mutation in Escherichia coli and elicit proteolytic activity against the known substrates of E. coli FtsH, namely heat shock transcription factor sigma(32) protein, protein translocation subunit SecY and bacteriophage lambdaCII repressor protein. The MtFtsH protein could not only efficiently complement lethality of DeltaftsH3::kan mutation in E. coli, but could also degrade all three heterologous substrates with specificity when expressed in ftsH-null cells of E. coli. These observations probably reveal the degree of conservation in the mechanisms of substrate recognition and cellular processes involving FtsH protease of M. tuberculosis and E. coli.

ATP-Dependent Proteases↗

PubMed2000

Cloning and functional characterization of the early-lymphocyte-specific Pb99 gene.

The Pb99 gene is specifically expressed in pre-B cells and thymocytes and not in mature B and T cells or nonlymphoid tissues, implying that it may function in early lymphoid development. We have previously described the cloning of an incomplete cDNA for Pb99. Here we report the isolation of full-length cDNAs and genomic clones for the murine Pb99 gene and the mapping of its location to mouse chromosome 8. Sequence analyses of different Pb99 cDNA clones suggest that there may be at least three forms of the Pb99 protein generated by differential processing of the Pb99 transcript. The cDNA with the longest open reading frame encodes a putative protein that has seven hydrophobic domains similar to those of seven membrane-spanning proteins, such as the classical G protein-coupled receptors. To directly address the role of the Pb99 protein in lymphoid development, Pb99-deficient mice were generated by gene targeting, and lymphocyte development in these mice was analyzed.

Amino Acid Sequence↗

PubMed2004

Functional characterization of a Trypanosoma brucei TATA-binding protein-related factor points to a universal regulator of transcription in trypanosomes.

Transcriptional mechanisms remain poorly understood in trypanosomatid protozoa. In particular, there is no knowledge about the function of basal transcription factors, and there is an apparent rarity of promoters for protein-coding genes transcribed by RNA polymerase (Pol) II. Here we describe a Trypanosoma brucei factor related to the TATA-binding protein (TBP). Although this TBP-related factor (TBP-related factor 4 [TRF4]) has about 31% identity to the TBP core domain, several key residues involved in TATA box binding are not conserved. Depletion of the T. brucei TRF4 (TbTRF4) by RNA interference revealed an essential role in RNA Pol I, II, and III transcription. Using chromatin immunoprecipitation, we further showed that TRF4 is recruited to the Pol I-transcribed procyclic acidic repetitive genes, Pol II-transcribed spliced leader RNA genes, and Pol III-transcribed U-snRNA and 7SL RNA genes, thus supporting a role for TbTRF4 in transcription performed by all three nuclear RNA polymerases. Finally, a search for TRF4 binding sites in the T. brucei genome led to the identification of such sites in the 3' portion of certain protein-coding genes, indicating a unique aspect of Pol II transcription in these organisms.

Amino Acid Sequence↗

PubMed1995

Localization and functional characterization of rat kidney-specific chloride channel, ClC-K1.

To investigate the physiological role of a kidney-specific chloride channel (ClC-K1), we sought to determine its exact localization by immunohistochemistry and its functional regulation using Xenopus oocyte expression system. The antiserum specifically recognized a 70-kD protein in SDS-PAGE of membrane protein from rat inner medulla and an in vitro translated ClC-K1 protein. Immunohistochemistry revealed that ClC-K1 was exclusively localized to the thin limb of Henle's loop in rat inner medulla. In comparison with the immunostaining with anti-aquaporin-CHIP antibody that only stains the descending thin limb of Henle's loop (tDL), ClC-K1 was found to be localized only in the ascending limb (tAL) which has the highest chloride permeability among nephron segments. Immunoelectron microscopy confirmed that the staining of ClC-K1 in tAL was observed in the region of both apical and basolateral plasma membranes. Expressed chloride current in Xenopus oocytes by ClC-K1 cRNA was regulated by extracellular pH and extracellular calcium. Furosemide inhibited the expressed current (Ki = 100 microM), whereas N-ethyl-maleimide stimulated the current. These functional characteristics were consistent with the in vitro perfusion studies of chloride transport in tAL. The localization and the functional characteristics described here indicate that ClC-K1 is responsible for the transepithelial chloride transport in tAL.

Animals↗

PubMed1989

Structural and functional characterization of the human immunodeficiency virus rev protein.

Expression of the human immunodeficiency virus (HIV) rev protein is required for expression of virus structural proteins. Site-directed mutagenesis was used to localize regions important for Rev function. We found that proteins with single amino acid substitution mutations concentrated within the amino termini and midportion of Rev were for the most part nonfunctional. Indirect immunofluorescence revealed that Rev was localized predominantly in the nucleolus. However, a deletion mutant that lacked the basic stretch of amino acids comprising residues Arg-Arg-Arg-Arg-Trp accumulated in the cytoplasm and was no longer functional. Consistent with this observation, a beta-galactosidase fusion protein containing this basic rich peptide at its amino termini was targeted to the nucleus. These observations indicate that the HIV rev protein has a distinct nuclear localization sequence and suggest that Rev-mediated regulation of gene expression involves nuclear events.

Amino Acid Sequence↗

PubMed1997

Functional characterization of mutant androgen receptors from androgen-independent prostate cancer.

Mutations in the androgen receptor (AR), that alter steroid hormone specificity have been identified in a series of androgen-independent prostate cancers. To address the functional properties of these mutant ARs that may have contributed to their selection in vivo, responses to a series of steroid hormones and antiandrogens were assessed. CV-1 cells were cotransfected with wild-type or mutant ARs and a luciferase reporter plasmid regulated by an androgen-responsive element. Dose-response curves were analyzed for 5alpha-dihydrotestosterone, the most active androgen in normal prostate, and androstenedione, a major androgen derived from the adrenals. Although the mutant ARs responded to both of these steroids, the responses were equivalent to or less than the wild-type AR. In contrast, responses to flutamide, a competitive antagonist of the wild-type AR, were markedly increased by three of the mutations. Similar responses were observed with a second antiandrogen, nilutamide. Bicalutamide, another antiandrogen related to flutamide, remained an antagonist for these mutant ARs. Finally, flutamide was observed to be a weak partial agonist of the wild-type AR in this system. These results indicate that flutamide used in conjunction with androgen ablation therapy for prostate cancer may select for tumor cells with flutamide-inducible ARs.

Amino Acid Substitution↗

PubMed1989

Lymph node macrophage heterogeneity: the phenotypic and functional characterization of two distinct populations of macrophages from rat lymph node.

The isolation and characterization of lymph node macrophages (M phi) has shown a hitherto unknown heterogeneity. Two types of M phi were distinguished by morphology, monoclonal antibody staining and functional assays. The type I M phi failed to express surface Ia even when activated, a characteristic which has only previously been reported for splenic marginal zone M phi; despite studies suggesting an antigen presentation role for the M phi, the failure to express surface Ia would seem to eliminate an interaction with T helper cells for the type I M phi in the lymph node. In contrast, the type I M phi, other characteristics of clustering with activated B cells in vitro, the colocalization of the type I M phi and activated B cells in situ, the specific uptake of thymus-independent type 2 antigens and the failure to undergo respiratory burst activity all suggest a M phi-B cell interaction, possibly of a trophic nature. The defective microbicidal activity of the type I M phi may have been compensated for by the type II M phi, which expresses both strong respiratory burst activity and surface Ia expression when freshly isolated. However, unlike the inflammatory M phi the activated phenotype of the type II M phi did not appear to be interferon-gamma dependent because type II M phi could also be isolated from nude rat lymph nodes.

Animals↗

PubMed1995

Immunological and functional characterization of Mycobacterium leprae protein antigens: an overview.

A major focus of leprosy research in the last 10 years has been the identification and characterization of antigens of Mycobacterium leprae that interact with antibodies and T cells of the host's immune response. Through the combined efforts of many different laboratories, a substantial number of protein antigens have been identified and characterized. In this MicroReview we present an updated list of M. leprae protein antigens, and, with emphasis on recent developments, summarize what is known regarding their functional and immunological features.

Antigens, Bacterial↗

PubMed1993

Human and murine dermis contain dendritic cells. Isolation by means of a novel method and phenotypical and functional characterization.

Dendritic cells (DC) comprise a system of cells in lymphoid and nonlymphoid organs that are specialized to present antigens and to initiate primary T cell responses. The Langerhans cell of the epidermis is used as a prototype for studies of DC in the skin. We have characterized a population of DC in human dermis, one of the first examples of these cells in nonlymphoid organs other than epidermis. To identify their distinct functions and phenotype, we relied upon the preparation of enriched populations that emigrate from organ explants of dermis. The dermal cells have the following key features of mature DC: (a) sheet-like processes, or veils, that are constantly moving; (b) very high levels of surface MHC products; (c) absence of markers for macrophages, lymphocytes, and endothelium; (d) substantial expression of adhesion/costimulatory molecules such as CD11/CD18, CD54 (ICAM-1), B7/BB1, CD40; and (e) powerful stimulatory function for resting T cells. Dermal DC are fully comparable to epidermis-derived DC, except for the lack of Birbeck granules, lower levels of CD1a, and higher levels of CD36. DC were also detected in explants of mouse dermis. We conclude that cutaneous DC include both epidermal and dermal components, and suggest that other human nonlymphoid tissues may also serve as sources of typical immunostimulatory DC.

Animals↗

PubMed2000

Molecular cloning and functional characterization of a new modulatory cyclic nucleotide-gated channel subunit from mouse retina.

Cyclic nucleotide-gated (CNG) channels play a key role in olfactory and visual transduction. Native CNG channels are heteromeric complexes consisting of the principal alpha subunits (CNG1-3), which can form functional channels by themselves, and the modulatory beta subunits (CNG4-5). The individual alpha and beta subunits that combine to form the CNG channels in rod photoreceptors (CNG1 + CNG4) and olfactory neurons (CNG2 + CNG4 + CNG5) have been characterized. In contrast, only an alpha subunit (CNG3) has been identified so far in cone photoreceptors. Here we report the molecular cloning of a new CNG channel subunit (CNG6) from mouse retina. The cDNA of CNG6 encodes a peptide of 694 amino acids with a predicted molecular weight of 80 kDa. Among the CNG channel subunits, CNG6 has the highest overall similarity to the CNG4 beta subunit (47% sequence identity). CNG6 transcripts are present in a small subset of retinal photoreceptor cells and also in testis. Heterologous expression of CNG6 in human embryonic kidney 293 cells did not lead to detectable currents. However, when coexpressed with the cone photoreceptor alpha subunit, CNG6 induced a flickering channel gating, weakened the outward rectification in the presence of extracellular Ca(2+), increased the sensitivity for L-cis diltiazem, and enhanced the cAMP efficacy of the channel. Taken together, the data indicate that CNG6 represents a new CNG channel beta subunit that may associate with the CNG3 alpha subunit to form the native cone channel.

Amino Acid Sequence↗

PubMed1989

Purification of chicken C3 and a structural and functional characterization.

A major plasma protein from chicken, analogous to mammalian complement component C3, was purified by the removal of plasminogen, precipitation with polyethyleneglycol, and ion-exchange chromatography. Purification was guided by a rabbit antiserum specific to chicken C3. The yield of native C3 was 27%, and purity and functional activity was assessed by SDS-PAGE, immunoprecipitation techniques, and the ability of the purified C3 to restore the haemolytic activity of C3-depleted chicken serum. Monoclonal antibodies were raised against purified chicken C3. These antibodies were characterized and used to prepare an immunosorbent column to deplete chicken plasma specifically of C3. Chicken C3 has a mol.wt of 185,000-195,000 and a two-chain structure with an alpha chain (118,000) and beta chain (68,000). Complement activation leads to changes in the electrophoretic mobility of chicken C3 and to a decrease in mol.wt to 144,000 corresponding to the release of a 15,000 C3a and a 34,000 C3d/C3dg fragment. Chicken C3 exists in multiple molecular forms with pI values of 6.4-6.6. A genetic polymorphism of chicken C3 based on electrophoretic mobility has not yet been detected after analysis of more than 500 individuals. The function of chicken C3 is dependent on a reactive thioester because treatment of purified chicken C3 with methylamine causes functional inactivation of C3.

Animals↗

PubMed2005

Identification and bioactivities of IFN-gamma in rainbow trout Oncorhynchus mykiss: the first Th1-type cytokine characterized functionally in fish.

IFN-gamma is one of the key cytokines in defining Th1 immune responses. In this study, an IFN-gamma homologue has been identified in rainbow trout Oncorhynchus mykiss, and its biological activities have been characterized. The trout IFN-gamma cDNA is 1034 bp in length and translates into a 180-aa protein. The first intron of the trout IFN-gamma gene contains highly polymorphic GACA minisatellites and 44-bp DNA repeats, giving rise to at least six alleles. IFN-gamma is structurally conserved among vertebrates, and a signature motif has been identified. A nuclear localization sequence known to be crucial for IFN-gamma biological activities is also present in the C-terminal region of the trout IFN-gamma. The IFN-gamma expression was induced in head kidney leukocytes by stimulation with PHA or poly(I:C) and in kidney and spleen of fish injected with poly(I:C). rIFN-gamma produced in Escherichia coli significantly stimulated gene expression of IFN-gamma-inducible protein 10 (gammaIP-10), MHC class II beta-chain, and STAT1, and enhanced respiratory burst activity in macrophages. Deletion of 29-aa residues from the C terminus containing the nuclear localization sequence motif resulted in loss of activity with respect to induction of gammaIP-10 in RTS-11 cells. Moreover, IFN-gamma-induced gammaIP-10 expression was completely abolished by the protein kinase C inhibitor staurosporine, and partially reduced by U0126, a specific inhibitor for ERKs. Taken together, the present study has demonstrated for the first time a functional IFN-gamma homologue in a fish species, strongly suggesting a conserved Th1 immune response is most likely present in lower vertebrates.

Amino Acid Sequence↗

PubMed1988

Structural and functional characterization of the short acidic transcriptional activation region of yeast GCN4 protein.

Derivatives of the yeast GCN4 transcription factor containing acidic regions of 35 to 40 amino acids fused directly to the DNA-binding domain are fully functional in vivo. High resolution deletion analysis and proteolytic mapping suggest that the activation region is a repeated structure composed of small units acting additively. Acidic character is a feature of the structural motif, possibly a dimer of alpha-helices from two GCN4 monomers, that may be important for interactions with the basic transcriptional machinery.

Amino Acid Sequence↗

PubMed2006

Structural and functional characterization of the conserved salt bridge in mammalian paneth cell alpha-defensins: solution structures of mouse CRYPTDIN-4 and (E15D)-CRYPTDIN-4.

alpha-Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell alpha-defensin cryptdin-4 (Crp4) and a mutant (E15D)-Crp4 peptide, in which a conserved Glu(15) residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar bactericidal activities and stability to proteolysis. These findings support the conclusion that the function of the conserved salt bridge in Crp4 is not linked to bactericidal activity or proteolytic stability of the mature peptide.

Amino Acid Sequence↗

PubMed2005

Molecular and functional characterization of an I(h)-channel from lobster olfactory receptor neurons.

We isolated a cDNA named PAIH encoding a member of the I(h)-channel family expressed in olfactory receptor neurons (ORNs) of the spiny lobster Panulirus argus. Functional expression of recombinant PAIH in HEK293 cells generated a slowly activating, noninactivating inward current under whole-cell voltage-clamp to hyperpolarizing voltage steps, the amplitude and activation rate of which increase with increasing hyperpolarization. The channel is weakly selective for K+. Intracellular cAMP or cGMP shifts activation of the current to less negative potentials in a concentration-dependent manner. Finally, the channel is blocked by the I(h)-channel blocker ZD7288. An I(h)-channel sharing the properties of the recombinant channel occurs in cultured lobster ORNs. PAIH immunoreactivity localizes the protein to the transduction compartment of the ORNs in situ, and selectively applying the blocker to the transduction compartment reduces spontaneous activity in the ORN. Collectively, these results implicate for the first time a functional role for an I(h)-channel in olfactory signal transduction.

8-Bromo Cyclic Adenosine Monophosphate↗