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Here, a complete set of recombinant fission yeast strains that coexpress each of the 57 human cytochrome P450 (CYP) enzymes together with their natural human electron transfer partner(s) was cloned. This strain collection was tested with two luminogenic probe substrates, and 31 human CYPs (including the orphan enzymes CYP2A7, CYP4A22 and CYP20A1) were found to metabolize at least one of these. Since other substrates are known for the remaining enzymes, all human CYPs are now shown to be active. Interestingly, CYP5A1 was found for the first time to work on a substrate other than prostaglandin H2 , and, moreover, to catalyze an aliphatic hydroxylation reaction that consumes molecular oxygen. Also, the ability of CYP11A1 to catalyze an aryl hydroxylation is another unexpected result.

Non-inactivating or slowly inactivating proton-gated cation channels are thought to play an important role in the perception of pain that accompanies tissue acidosis. We have identified a novel human proton-gated cation channel subunit that has biphasic desensitisation kinetics with both a rapidly inactivating Na+-selective and a sustained component. The protein shares 84% sequence identity with the proton-gated cation channel rASIC3 (rDRASIC) from rat sensory neurones. The biphasic desensitisation kinetics and the sequence homology suggest that this novel clone (hASIC3) is the human orthologue of rASIC3 (rDRASIC). While rASIC3 (rDRASIC) requires very acidic pH (pH < 4.5) for activation of the sustained current, the non-inactivating hASIC3 current starts to be activated when the pH decreases to below pH 6. hASIC3 is an acid sensor and might play an important role in the detection of lasting pH changes in human. We localised the hASIC3 gene to the human chromosome 7q35, 6.4 cRad telomeric from the microsatellite AFMA082XC9.

High-level overexpression of G protein-coupled receptors GPCRs in mammalian cells remains a difficult task inspite of newly developed virus based expression systems. Here, we show that the functional expression level of the recombinant bradykinin receptor (B2R) in mammalian cells can be increased up to sixfold just by the addition of dimethylsulphoxide in the culture medium. Total expression level, cellular localization and binding affinity of the recombinant receptor for its endogenous ligand remains unaltered. The strategy presented here, with recombinant B2R as a case example, is applicable to other GPCRs and provides a generic tool to improve the functional expression level of recombinant GPCRs in mammalian cells.

We previously reported that expression of myotonic dystrophy (DM1) expanded CUG repeats impedes NGF-induced differentiation in a PC12 clone (CTG90 cells). Here, we present evidence for changes in the fractional contribution of distinct voltage-gated Ca2+ channels, key elements in neurotrophin-promoted differentiation, to the total Ca2+ current in the CTG90 cells. Patch-clamp recordings showed that the relative proportion of pharmacologically isolated Ca2+ channel types differed between control and CTG90 cells. Particularly, the functional expression of N-type channels was significantly reduced. Though quantitative real-time RT-PCR revealed that transcripts for the pore-forming subunit encoding the N-type channels remained unchanged, the protein level analyzed by semi-quantitative Western blotting was down-regulated in the CTG90 cells. These data suggest modifications in the processing of N-type Ca2+ channels in PC12 cells expressing the DM1 mutation.

Sensory rhodopsin I (SRI) from Halobacterium salinarum was functionally expressed in Escherichia coli and subsequently purified to homogeneity using a C-terminal His-tag anchor. Yields of 3–4 mg SRI/l cell culture can be obtained. The absorption and photocycle properties of SRI were similar if not indistinguishable from those of the homologously expressed SRI. A global fit analysis of the photocycle data and the calculation of the spectra of states provided strong evidence for the existence of an N-like intermediate.

We demonstrate that, in rat pituitary, peptidylglycine α-amidating enzyme was encoded by at least 5 distinct mRNAs. Southern blot and ribonuclease protection analyses revealed that the mRNAs arose through alternative splicing. A variant lacking the transmembrane domain-coding sequence was a major mRNA species for the enzyme in the pituitary. When the cDNAs were expressed in COS-7 cells, the variant was the most efficient in producing a secretory form (37 kDA) of the enzyme.

The anticodon of the tRNALys gene (trnK) in the liverwort, Marchantia polymorpha, was anificially converted to an amber anticodon. This mutant tRNALys(CTA) gene carrying either the intron or the C27-C43 mismatch at the anticodon-stem is not functional in Escherichia coli, but without both of them, it does work as a tRNALys amber suppressor.

Taste-modifying proteins are a natural alternative to artificial sweeteners and flavor enhancers and have been used in some cultures for centuries. The taste-modifying protein, miraculin, has the unusual property of being able to modify a sour taste into a sweet taste. Here, we report the use of a plant expression system for the production of miraculin. A synthetic gene encoding miraculin was placed under the control of constitutive promoters and transferred to lettuce. Expression of this gene in transgenic lettuce resulted in the accumulation of significant amounts of miraculin protein in the leaves. The miraculin expressed in transgenic lettuce possessed sweetness-inducing activity. These results demonstrate that the production of miraculin in edible plants can be a good alternative strategy to enhance the availability of this protein.

Neuroblastoma×glioma NG108-15 hybrid cells have been examined for the expression of opioid receptor-like receptor (ORL 1 ). [ 3 H]Nociceptin/orphanin FQ (OFQ) bound to the cell membrane specifically ( K d =3.6±0.6 nM) and inhibited forskolin-stimulated cAMP accumulation (EC 50 =0.72±0.3 nM). The responsiveness of NG108-15 cells to nociceptin/OFQ was blocked by pertussis toxin but not by naltrindole. The inhibitory activity of nociceptin/OFQ was significantly reduced after a prechallenge with the same peptide but was not influenced by DPDPE pretreatment, indicating acute and homologous desensitization of ORL 1 receptors. Naltrindole caused the overshoot of cAMP in DPDPE-pretreated cells but not in nociceptin/OFQ-pretreated cells. The results indicate that ORL 1 is functionally expressed and does not cross-interact with specific ligands of the δ opioid receptor in NG108-15 cells. © 1997 Federation of European Biochemical Societies.

Thiocyanate hydrolase (SCNase) is a cobalt-containing enzyme with a post-translationally modified cysteine ligand, gammaCys131-SO(2)H. When the SCNase alpha, beta and gamma subunits were expressed in Escherichia coli, the subunits assembled to form a hetero-dodecamer, (alphabetagamma)(4), like native SCNase but exhibited no catalytic activity. Metal analysis indicated that SCNase was expressed as an apo-form irrespective of the presence of cobalt in the medium. On the contrary, SCNase co-expressed with P15K, encoded just downstream of SCNase genes, in cobalt-enriched medium under the optimized condition (SCNase((+P15K))) possessed 0.86 Co atom/alphabetagamma trimer and exhibited 78% of the activity of native SCNase. SCNase((+P15K)) showed a UV-Vis absorption peak characteristic of the SCNase cobalt center. About 70% of SCNase((+P15K)) had the gammaCys131-SO(2)H modification. These results indicate that SCNase((+P15K)) is the active holo-SCNase. P15K is likely to promote the functional expression of SCNase probably by assisting the incorporation of cobalt ion.

A tetracycline-dependent inducible system was used to achieve controlled expression of the glutamate transporter 1 (GLT-1) in C6 glioma cells. Non-induced cells show modest glutamate uptake and, in the presence of l-cystine, these cells tend to release substantial amounts of glutamate. Overnight exposure to doxycycline increased d-[3H]-aspartate uptake, reaching similar capacity as observed in cultured astrocytes. Efficient clearance of exogenously applied glutamate was evidenced in these cells, even in the presence of l-cystine. The addition of glutamate (100μM) to the medium of non-induced cells significantly increased their proliferation rate, an effect that was blocked when the expression of GLT-1 was induced. This suggests that impaired glutamate uptake capacity in glioma cells indirectly contributes to their proliferation.

The immunosuppressant cyclosporin A (CsA) reduced the functional expression of Kir2.1 potassium channels in Xenopus oocytes in a dose-dependent manner with an IC50 of 11 μM when the oocytes were incubated with CsA after RNA injection. FK506 was less effective than CsA; cyclosporin H, a non-immunosuppressive derivative of CsA, did not have a significant effect. CsA did not impair protein synthesis since other potassium channel types (Kir1.1, Kv1.1, Kv1.4) were much less sensitive to CsA. Our results suggest that the functional expression of Kir2.1 channels is facilitated by the peptidyl-prolyl isomerase cyclophilin. The observations illustrate a new role of CsA in regulation of membrane ion transport, and may provide an alternative explanation for CsA-induced side effects in clinical use.

Triacylglycerols are the most important storage lipids in most plants and animals. Acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1.20) catalyzes the final step of the pathway of triacylglycerol synthesis and is the only step which is unique to this process. Diacylglycerol acyltransferase is required for the synthesis of storage oil in a wide range of oil-bearing seeds and fruits and in floral structures such as petals, anthers and pollen. We describe the first cloning and functional expression of a cDNA encoding diacylglycerol acyltransferase from a plant. The cDNA, cloned from Arabidopsis thaliana, encodes a 520 amino acid protein with a predicted molecular mass of 59.0 kDa which shares 38% amino acid sequence identity with diacylglycerol acyltransferase from mouse. When expressed in insect cell cultures, the protein catalyzes the synthesis of [14C]triacylglycerol from [14C]diacylglycerol and acyl-CoA. Primer extension analysis revealed that the transcription begins 225 bases before the translation start site, yielding an unusually long 5′ untranslated region. The gene is expressed in a wide range of tissues but most strongly in developing embryos and petals of flowers.

The transfer of xylose from UDP-xylose to the core β-linked mannose of N-linked oligosaccharides by β1,2-xylosyltransferase (XylT) is a widespread feature of plant glycoproteins which renders them immunogenic and allergenic in man. Here, we report the isolation of the Arabidopsis thaliana XylT gene, which contains two introns and encodes a 60.2 kDa protein with a predicted type II transmembrane protein topology typical for Golgi glycosyltransferases. Upon expression of A. thaliana XylT cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited XylT activity in vitro. Furthermore, the recombinant enzyme displayed XylT activity in vivo in the insect cells, as judged by the acquired cross-reaction of cellular glycoproteins with antibodies against the β1,2-xylose epitope. The cloned XylT cDNA as well as the recombinant enzyme are essential tools to study the role of β1,2-xylose in the immunogenicity and allergenicity of plant glycoproteins at the molecular level.

Low voltage-activated T-type calcium channels are encoded by a family of at least three genes, with additional diversity created by alternative splicing. This study describes the cloning of the human brain α1G, which is a novel isoform, Cav3.1c. Comparison of this sequence to genomic sequences deposited in the GenBank allowed us to identify the intron/exon boundaries of the human CACNA1G gene. A full-length cDNA was constructed, then used to generate a stably-transfected mammalian cell line. The resulting currents were analyzed for their voltage- and time-dependent properties. These properties identify this gene as encoding a T-type Ca2+ channel.

The cDNA and gene encoding human chondroitin 6-sulfotransferase (C6ST) have been cloned. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active sulfotransferase, which used as acceptor substrates polymer chondroitin, various chondroitin sulfate isoforms and chondroitin sulfate tetrasaccharides. The identification of the reaction products demonstrated that the enzyme transferred sulfate to position 6 of GalNAc in the GlcAβ1-3GalNAc but not the IdoAα1-3GalNAc nor the GlcAβ1-3GalNAc(4-O-sulfate) sequences. The human C6ST gene spans more than 20 kb and consists of three exons. The protein-coding domain of the C6ST gene is divided into two discrete exons.

Neurotransmitter transport systems are major targets for therapeutic alterations in synaptic function. We have cloned and sequenced a cDNA encoding the human type 2 glycine transporter GlyT2 from human brain and spinal cord. An open reading frame of 2391 nucleotides encodes a 797 amino acid protein that transports glycine in a Na+/Cl−-dependent manner. When stably expressed in CHO cells, human GlyT2 displays a dose-dependent uptake of glycine with an apparent Km of 108 μM. This uptake is not affected by sarcosine at concentrations up to 1 mM. Radiation hybrid analysis mapped the GlyT2 gene to D11S1308 (LOD=8.988) on human chromosome 11p15.1–15.2.

A 13-kDa tobacco plasma membrane protein was isolated from two-dimensional electrophoresis gels. After microsequencing, RT-PCR techniques and cDNA library screening allowed for the cloning of two cDNAs. These cDNAs encoded for the subunit G of the vacuolar H+-ATPase, the first one identified in plants. Analysis of mRNA distribution showed a maximum level in the leaves and in the stem of the apical part of the tobacco plant. Heterologous functional complementation of the yeast mutant (deltavma10::URA3) was achieved with the two cDNAs. After fractionation of microsomal membranes on linear sucrose gradient, Western blots were performed using antibodies against recombinant protein and three peaks were identified: one which comigrated with the tonoplast marker and the others at slightly higher density corresponding to endoplasmic reticulum and to plasma membrane fractions.

Neuronal nicotinic acetylcholine receptors (nAChR) are made from different combinations of subunits encoded by a diverse family of genes. However, the recently cloned alpha 7 gene codes for subunits that can form homooligomeric nAChR complexes when expressed in Xenopus oocytes. Electrophysiological studies reveal that these alpha 7-nAChR function as alpha-bungarotoxin (Bgt)-sensitive, quickly activating/inactivating ion channels with a unique pharmacological profile and an unusually high permeability to calcium ions. Although similar observations have been made in studies of Bgt-sensitive, functional nAChR subtypes that are naturally expressed in neuronal cells, all attempts until now to reconstitute functional alpha 7-nAChR in cell lines have failed. Here we report the successful use of SH-SY5Y human neuroblastoma cells, which naturally express low levels of endogenous alpha 7 transcripts, to stably overexpress heterologous rat nAChR alpha 7 transgenes. These transgenes are expressed as the appropriately-sized alpha 7 messages and protein, and stably transfected SH-SY5Y cells have over 30-times higher levels of specific Bgt binding sites than do wild-type cells. Whole cell current recordings confirm that transfected cells express functional nAChR that are sensitive to blockade by Bgt and display the typical physiological and pharmacological profiles of alpha 7-nAChR. We conclude that stable, functional expression of alpha 7 transgenes in a mammalian cell line has been achieved for the first time.

The synthetic cRNA encoding for the major T lymphocyte K+ channel (Kv1.3) was injected into Xenopus fertilized eggs. Somites from embryos of stage 20–22 (about 40 h post-fertilization at 19°C) were dissociated and myotomal muscle cells were cultured in vitro for 2 days. The whole cell configuration of the tight seal patch-clamp technique was used to record K+ channel activity in cultured myocytes. These myocytes have two endogenous delayed-rectifiers (sustained and transient) and an inward-rectifier K+ currents, all of which are insensitive to the scorpion toxin charybdotoxin. Cultured myocytes dissociated from embryos injected with the Kv1.3 cRNA expressed the exogenous Kv1.3 channel. The Kv1.3 channel was identified by its physiological (a very low recovery from inactivation) and its pharmacological properties (a high sensitivity to charybdotoxin). This work demonstrates that Xenopus cultured myotomal muscle cells represent a very efficient and practical assay system for the functional expression of cloned ion channels.

The different murine D2-type dopamine receptors (D2L, D2S, D3L, D3S, and D4) were expressed in Xenopus laevis oocytes. The D2-type receptors were all similarly and efficiently expressed in Xenopus oocytes and were shown to bind the D2 antagonist [125I]sulpride. They were all shown to activate Cl− influx upon agonist stimulation. Using the diagnostic inhibitor bumetanide, we were able to separate the Na+/K+/2Cl− cotransporter component of the Cl− influx from the total unidirectional Cl− influx. The D3L subtype was found to operate exclusively through the bumetanide-insensitive Cl− influx whereas the other D2-type receptors acted on the Na+/K+/2Cl− cotransporter as well. The pertussis toxin sensitivity of the receptor-activated chloride influx via the Na+/K+/2Cl− cotransporter varied between the various D2-type receptors showing that they may couple to different G proteins, and activate different second messenger systems.

The sodium and chloride coupled γ-aminobutyric acid (GABA) transporter purified from rat brain, belongs to a superfamily of neurotransmitter transporters. They are involved in the termination of synaptic transmission and are predicted to have 12 membrane spanning α-helices with both amino- and carboxyl-termini oriented toward the cytoplasm. In order to define the domains not required for functional expression, we have constructed and expressed a series of deletion mutants in GAT-1, the cDNA clone encoding for the transporter. Transporters truncated at either end until just a few amino-acids distance from the beginning of helix 1 and the end of helix 12, retain their ability to catalyze sodium and chloride-dependent GABA transport.

We have cloned from human retina a cyclic nucleotide-gated (CNG) ion channel that is distinct from the one found in rod photoreceptors. This channel protein is highly homologous to the CNG channel that recently has been cloned from bovine testis and kidney and has been shown to be present i retinal cone photoreceptors. When expressed in human embryonic kidney cells, the protein forms functional ion channels with properties broadly similar to those described for the cloned bovine channel. The gene for this channel resides on chromosome 2.

We have cloned a partial rat kidney cDNA that encodes a 72.5 kDa N terminal fragment of a third isoform of the M110 subunit of phosphatase 1. This new isoform contains an insert in the 542-597 position not present in the M110 previously cloned (Chen et al. (1994) FEBS Lett. 356, 51-55) from the same species. The encoded cDNA was expressed as a soluble GST-fusion protein in E. coli, and its ability to interact with native PP-1C was measured both in vitro and in permeabilized smooth muscle. In vitro, the fusion protein was capable of selectively binding PP-1C and increasing the substrate specificity of the phosphatase towards myosin 13.2 +/- 3.5-fold (S.E. of the mean, n = 3). In permeabilized smooth muscle pretreated with microcystin, the recombinant protein alone (1.0 microM) did not cause relaxation, but did significantly enhance the ability of PP-1C (0.3 microM) to relax the muscle. These findings show that the N terminal domain of the M110 subunit is the primary site for both PP-1C and myosin binding, and thereby determines myosin specificity. The presence of isoformic variation within this sequence may permit organ/cell specific regulation of phosphorylation sites.

The Leiurus quinquestriatus hebraeus alpha anti-insect toxin (Lqh alpha IT) cDNA was engineered into the Autographa californica Nuclear Polyhedrosis Virus (AcNPV) genome. Insect cells infected with the recombinant virus secreted a functional Lqh alpha IT polypeptide. Spodoptera littoralis and Heliothis armigera larvae injected with recombinant budded virus, showed typical intoxication symptoms. This recombinant virus showed enhanced insecticidal potency against H. armigera larvae compared with wild type AcNPV. The present expression system will facilitate: (1) the future elucidation of structural elements involved in its prominent anti-insect toxicity; and (2) the future design of genetically modified alpha toxins with improved anti-insect selectivity.

Glucose-dependent insulinotropic polypeptide (GIP) plays an important role in the regulation of postprandial insulin secretion and proinsulin gene expression of pancreatic beta-cells. This study demonstrates the molecular cloning of a cDNA for the GIP-receptor from a human insulinoma lambda gt11 cDNA library. The cloned cDNA encoded a seven transmembrane domain protein of 466 amino acids which showed high homology (41%) to the human glucagon-like peptide 1 (GLP-1) receptor. Homology to the GIP receptor from rat or hamster was 79% and 81%, respectively. When transfected stably into fibroblast CHL-cells a high affinity receptor was expressed which coupled to the adenylate cyclase with normal basal cAMP and increasing intracellular cAMP levels under stimulation with human GIP-1-42 (EC50 = 1.29 x 10(-13) M). The receptor accepted only human GIP 1-42 (Kd = 1.93 +/- 0.2 x 10(-8) M) and porcine truncated GIP 1-30 (Kd = 1.13 +/- 0.1 x 10(-8) M) as high affinity ligands. At 1 microM, exendin-4 and (9-39)amide weakly reduced GIP-binding (25%) whereas secretin, glucagon, glucagon-like peptide-1, vasoactive intestinal polypeptide, peptide histidine-isoleucine, and pituitary adenylyl cyclase activating peptide were without effect. In transfected CHL cells, GIP-1-42 did not increase intracellular calcium. Northern analysis revealed one transcript of human GIP receptor mRNA with an apparent size of 5.5 kb. The exact understanding of GIP receptor regulation and signal transduction will aid in the understanding of the incretin hormone's failure to exert its biological action at the pancreatic B-cell in type II diabetes mellitus.

A cDNA encoding a receptor for the oxytocin-related peptide isotocin has been identified by screening a lambda gt11 library constructed from poly(A)+ RNA of the hypothalamic region of the teleost Catostomus commersoni. The probe used was obtained by PCR amplification of white sucker genomic DNA using degenerate primers based on conserved sequences in the mammalian receptor counterparts. The full-length cDNA specifies a polypeptide of 390 amino acid residues that displays the typical hydrophobicity profile of a seven transmembrane domain receptor and which exhibits greatest similarity to mammalian oxytocin receptors. Oocytes that express the cloned receptor respond to the application of isotocin by an induction of membrane chloride currents indicating that it is coupled to the inositol phosphate/calcium pathway. The isotocin receptor (ITR) can also be activated by vasotocin, mesotocin, oxytocin and Arg-vasopressin, although these have lower potencies than isotocin. ITR-encoding mRNA has been detected in brain, intestine, bladder, skeletal muscle, lateral line, gills and kidney indicating that this receptor may mediate a variety of physiological functions.

We report the cloning and characterization of two isoforms of human alpha 1c-adrenoceptor cDNA (alpha 1c-2, alpha 1c-3). These isoforms are generated by alternative splicing and differ from the clone we previously isolated (alpha 1c-1) in their length and sequences of the C-terminal domain. Tissue distribution of mRNAs showed that these variants co-express with alpha 1c-1 in the human heart, liver, cerebellum and cerebrum. Despite the structural differences, functional experiments in transfected CHO cells showed that the three isoforms have similar ligand binding properties, and all couple with phospholipase C/Ca2+ signaling pathway.

Recent evidence suggests that a number of non-phagocytic cell types may contain a superoxide generating NADPH oxidase. Studies to date on cultured human fibroblasts have primarily concerned the identification of cytochrome b 558 , whilst expression of other NADPH oxidase components have not been addressed. In this study we have investigated the expression of NADPH oxidase with particular reference to the cytosolic factors p47- phox and p67- phox . Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that human fibroblasts express mRNA for p47- phox , p67- phox and p22- phox . Expression of the gp91- phox transcript was not detected, indicating that human fibroblasts may possess an NADPH oxidase isoenzyme. Western blot analysis of human fibroblast cytosol, using an anti-p47- phox antibody (JW-1), identified a 47 kDa protein. Cell-free reconstitution assays showed that fibroblast cytosol could initiate superoxide generation when mixed with either human fibroblast membranes (0.16 nmol superoxide/min/μg membrane protein), or resting human neutrophil membranes (0.20 nmol superoxide/min/μg membrane protein). These data indicate that the expression of p47- phox and p67- phox by human fibroblasts may contribute to the cells' generation of superoxide.

We have cloned a novel inward rectifier potassium channel from a rat brain cDNA library and designated it RB-IRK2. The rat brain cDNA library was screened using a fragment of the mouse macrophage IRK1 cDNA as a probe. The amino acid sequence of RB-IRK2 shares 70%, 40% and 45% identity to mouse IRK1, rat ROMK1 and rat GIRK1, respectively. Xenopus oocytes injected with cRNA derived from RB-IRK2 expressed a potassium current which showed inward-rectifying channel characteristics similar to the IRK1 current, but distinct from the ROMK1 or the GIRK1 currents. However, the localization of RB-IRK2 mRNA in rat tissues, assessed by the Northern blot analysis, differed from that of mouse IRK1. These results indicate that the IRK family is composed of multiple genes, which express in different tissues and therefore may play heterogenous functional roles in various organs, including rat central nervous system.

Using oligonucleotide primers derived from the vesicular monoamine transporters sequences, a cDNA predicted to encode the bovine chromaffin granule amine transporter has been cloned (b-VMAT2). Surprisingly, its structure is more similar to the rat brain transporter (VMAT2), than to the rat adrenal counterpart (VMAT1). Unlike rat VMAT1, bovine VMAT2 appears to be expressed both in the adrenal medulla and the brain, as judged by Northern analysis. After modification/deletion of the seven amino acids at the N-terminus of the protein it was expressed in a functional form. The order of affinity of the bovine VMAT2 transporter to substrates is: serotonin>dopamine = norepinephrine>epinephrine. Also, the recombinant bovine adrenal transporter is highly sensitive to tetrabenazine, in sharp contrast to the rat adrenal transporter. The findings indicate, therefore, a clear species variation in which structure and function of the bovine adrenal transporter resemble the rat brain protein, while its tissue distribution is distinct from both types of rat proteins. In addition, the predicted protein sequence is identical to the experimentally determined N-terminus sequence of the purified vesicular amine transporter [Stern-Bach et al. (1992) Proc. Natl. Acad. Sci. USA 89, 9730-9733].

We have cloned an inward-rectifier potassium channel from a mouse brain cDNA library, studied its distribution in the brain by in situ hybridization and determined the chromosomal localization of the gene. A mouse brain cDNA library was screened using a fragment of the mouse macrophage IRK1 cDNA as a probe. Two duplicate clones of approximately 5.5 kb were obtained. Xenopus ococytes injected with cRNA derived from the clone expressed a potassium channel with inwardly rectifying channel characteristics. The amino acid sequence of the clone was identical to that of IRK1 recently cloned from a mouse macrophage cell line. In situ hybridization study showed the mouse brain IRK1 to be generally distributed throughout the brain, but in particular subsets of neurons at high levels. The gene was placed in the distal region of mouse chromosome 11, which contains several uncloned neurological mutations. These results provide the first demonstration of the cloning and distribution of an inward rectifier potassium channel from the nervous system.

Sequences specific for cyclic nucleotide-gated channels (CNG channels) have been amplified by PCR from cDNA of heart, aorta, sinoatrial node, cerebellum, C-cells and kidney. The complete amino acid sequence of a CNG channel from rabbit aorta has been deduced by cloning and sequence analysis of the cDNA. Synthetic RNA derived from this cDNA induces the formation of a functional CNG channel in Xenopus oocytes.

Carotenoids with cyclic end groups are essential components of the photosynthetic membrane in all known oxygenic photosynthetic organisms. These yellow pigments serve the vital role of protecting against potentially lethal photo-oxidative damage. Many of the enzymes and genes of the carotenoid biosynthetic pathway in cyanobacteria, algae and plants remain to be isolated or identified. We have cloned a cyanobacterial gene encoding lycopene cyclase, an enzyme that converts the acyclic carotenoid lycopene to the bicyclic molecule beta-carotene. The gene was identified through the use of an experimental herbicide, 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA), that prevents the cyclization of lycopene in plants and cyanobacteria. Chemically-induced mutants of the cyanobacterium Synechococcus sp. PCC7942 were selected for resistance to MPTA, and a mutation responsible for this resistance was mapped to a genomic DNA region of 200 bp by genetic complementation of the resistance in wild-type cells. A 1.5 kb genomic DNA fragment containing this MPTA-resistance mutation was expressed in a lycopene-accumulating strain of Escherichia coli. The conversion of lycopene to beta-carotene in these cells demonstrated that this fragment encodes the enzyme lycopene cyclase. The results indicate that a single gene product, designated lcy, catalyzes both of the cyclization reactions that are required to produce beta-carotene from lycopene, and prove that this enzyme is a target site of the herbicide MPTA. The cloned cyanobacterial lcy gene hybridized well with genomic DNA from eukaryotic algae, thus it will enable the identification and cloning of homologous genes for lycopene cyclase in algae and plants.

We recently cloned from a mouse 3T3 cell cDNA library a cDNA with sequence similarity to the p42mapk protein and other members of the MAP kinase family. To determine with certainty which member of the family this clone encodes, we have expressed the cDNA in COS cells and characterized the protein product. When the pSV2MAP plasmid carrying the full-length clone was transfected into COS cells, a protein of 42,000 Da was expressed. This 42 kDa protein displayed chromatographic properties indistinguishable from the endogenous p42mapk, and could be separated from the closely related pp44. In addition, upon serum stimulation, the 42 kDa protein became tyrosine-phosphorylated and enzymatically active towards the substrate myelin basic protein. We conclude that this clone codes for a functional p42mapk protein kinase.

N-terminal deletion mutants of Na,K-ATPase α 1 isoforms initiating translation at Met 34 (α 1 T 1 ) or at Met 43 (α 1 T 2 ) were expressed in X. laevis oocytes. Compared to β 3 cRNA injected controls, the co-expression of α 1 wt, α 1 T 1 , α 1 T 2 with β 3 subunits results in a 2- to 3-fold increase of ouabain binding sites, parallelled by a concomitant increase in Na,K-pump current. The apparent K 1 2 for potassium activation of the α 1 T 2 /β 3 Na,K-pumps is significantly higher than that of the α 1 wt/β 3 or α 1 T 1 /β 3 Na,K-pumps expressed at the cell surface. Total deletion of the lysine-rich N-terminal domain thus allows the expression of active Na,K-pump but with distinct cation transport properties.

Alternative oxidase (AOX) plays a pivotal role in cyanide-resistance respiration in the mitochondria of plants, fungi and some protists. Here we show that AOX from thermogenic skunk cabbage successfully conferred cyanide resistance to human cells. In galactose medium, HeLa cells with mitochondria-targeted AOX proteins were found to have significantly less reactive oxygen species production in response to antimycin-A exposure, a specific inhibitor of respiratory complex III. These results suggest that skunk cabbage AOX can be used to create an alternative respiration pathway, which might be important for therapy against various mitochondrial diseases.

Two alpha subunits of the GABAA receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the alpha 3 subunit previously cloned from bovine brain, while the other polypeptide is a yet known subunit, termed alpha 5. When coexpressed with the beta 1 subunit in Xenopus oocytes the receptors containing the alpha 5 subunit revealed a higher sensitivity to GABA than receptors expressed from alpha 1 + beta 1 subunits or alpha 3 + beta 1 subunits (Ka = 1 microM, 13 microM and 14 microM, respectively). The alpha 5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the alpha 5 subunit was colocalized with the alpha 1 and alpha 3 subunits only in cerebral cortex and in the hippocampal formation the alpha 5 subunit may be part of distinct GABAA receptors in neuronal populations within the olfactory bulb.

The glutamate analogue 2-amino-4-phosphonobutyrate (APB) is known to activate a subtype of metabotropic glutamate receptor in the central nervous system, including the retina. In the present study, APB receptors were studied using the Xenopus oocyte expression system. No endogenous APB sensitivity was detected in control oocytes. In contrast, microinjection of mRNA, extracted from quail brain, into Xenopus oocytes resulted in the functional expression of APB receptors after 3–5 days incubation. Application of 50 μM-1 mM APB to injected oocytes voltage clamped at a holding potential of −60 mV produced a sustained outward current which was associated with a significant decrease in membrane conductance; the reversal potential was around −11 mV. The response to APB was dose-dependent and non-desensitizing. This is the first demonstration of the expression of a conductance-decreasing receptor mechanism in Xenopus oocytes.

Subsequent to our earlier report on the first purification of antiquitin protein from seabream liver and demonstration of its enzymatic activity [FEBS Letters 516 (2002) 183–186], we report herein the cloning of its full-length cDNA sequence. The open reading frame encodes a protein of 511 amino acids. Results of RT-PCR indicate that antiquitin is highly expressed in both the seabream liver and kidney. Transfection studies in cultured eukaryotic cells provided further evidence that it is a cytosolic protein. Bacterial expression of the enzyme was also performed. The purified recombinant protein was demonstrated to exhibit similar kinetic properties as the native enzyme.

Kcv (K+ Chlorella virus) is a miniature virus-encoded K+ channel. Its predicted membrane–pore–membrane structure lacks a cytoplasmic C-terminus and it has a short 12 amino acid (aa) cytoplasmic N-terminus. Kcv forms a functional channel when expressed in human HEK 293 cells. Deletion of the 14 N-terminal aa results in no apparent differences in the subcellular location and expression level of the Kcv protein. However, the truncated protein does not induce a measurable current in transfected HEK 293 cells or Xenopus oocytes. We conclude that the N-terminus controls functional properties of the Kcv channel, but does not influence protein expression.

Anti-neutrophil cytoplasmic autoantibodies recognizing conformational epitopes (c-ANCA) of proteinase 3 (PR3) from azurophil granules are a diagnostic hallmark in Wegener's granulomatosis (WG). Because a functional PR3 homologue has not been identified in rodents, it is difficult to assess immunopathological responses in rats or mice immunized with patients' derived c-ANCA or human PR3. Here we report the full length cDNA cloning and functional expression of murine PR3 in HMC-1 cells. Recombinant murine PR3 shows highly similar substrate specificities towards synthetic peptides and is inhibited by human α1-proteinase inhibitor like human PR3. However, neither human c-ANCA, rabbit sera nor mouse monoclonal antibodies to human PR3 recognize the murine homologue. Consequently, it is unlikely that disease observed in mice after immunization with c-ANCA or human PR3 is caused by pathogenic antibodies directed against mouse PR3. Recombinant human-mouse chimaeric variants will be a valuable new tool to localize the disease-specific immunodominant epitopes in human PR3.

Protein D2 forms the water-filled pore across the outer membrane of Pseudomonas aeruginosa and allows the penetration of imipenem. We cloned the protein D2 gene by the antibody screening technique. When the imipenem-resistant mutant lacking protein D2 harbored the plasmid with the cloned D2 gene, the mutant overproduced protein D2 in the outer membrane. These transformants exhibited fully-restored imipenem susceptibility. The results prove unequivocally that protein D2 forms the imipenem-permeable pore in the P. aeruginosa outer membrane.

Oxidation of methionine residues in proteins to methionine sulfoxide can be reversed by the enzyme peptide methionine sulfoxide reductase (MsrA, EC 1.8.4.6). We cloned the gene encoding a human homologue (hMsrA) of the enzyme, which has an 88% amino acid sequence identity to the bovine version (bMsrA). With dot blot analyses based on RNA from human tissues, expression of hMsrA was found in all tissues tested, with highest mRNA levels in adult kidney and cerebellum, followed by liver, heart ventricles, bone marrow and hippocampus. In fetal tissue, expression was highest in the liver. No expression of hmsrA was detected in leukemia and lymphoma cell lines. To test if hMsrA is functional in cells, we assayed its effect on the inactivation time course of the A-type potassium channel ShC/B since this channel property strongly depends on the oxidative state of a methionine residue in the N-terminal part of the polypeptide. Co-expression of ShC/B and hMsrA in Xenopus oocytes significantly accelerated inactivation, showing that the cloned enzyme is functional in an in vivo assay system. Furthermore, the activity of a purified glutathione-S-transferase-hMsrA fusion protein was demonstrated in vitro by measuring the reduction of [3H]N-acetyl methionine sulfoxide.

The DNA sequence encoding a novel human thioredoxin reductase has been determined. The protein is predicted to have 524 amino acids including a conserved -Cys-Val-Asn-Val-Gly-Cys catalytic site and a selenocysteine containing C-terminal -Gly-Cys-SeCys-Gly. The predicted molecular mass is 56.5. The newly identified TR sequence exhibits 54% identity to a previously reported human thioredoxin reductase and 37% identity to human glutathione reductase. Transient transfection of human embryonal kidney cells results in a 5-fold increase in thioredoxin reductase activity but no increase in glutathione reductase activity.

We report the cloning and functional expression of a novel K+ channel beta-subunit from human atrium, hKv beta 3. hKv beta 3 is highly homologous to the two beta-subunits cloned from rat brain, Kv beta 1 and Kv beta 2, but has an essentially unique stretch of 79 N-terminal residues. Upon expression in Xenopus oocytes, hKv beta 3 accelerates the inactivation of co-injected hKv1.4 currents and induces fast inactivation of non-inactivating co-injected hKv1.5 currents. By contrast, hKv beta 3 had no effect on hKv1.1, hKv1.2, or hKv2.1 currents. Thus, hKv beta 3 represents a third type of K+ channel beta-subunit which modulates the kinetics of a unique subset of channels in the Kv1 subfamily.

Four cytochrome P450-dependent enzymes, among them dihydroxypterocarpan 6a-hydroxylase (D6aH), are specifically involved in the elicitor-inducible biosynthesis of glyceollins, the phytoalexins of soybean. Here we report that CYP93A1 cDNA, which we isolated previously from elicitor-induced soybean cells, codes for a protein with D6aH activity. Analysis of the catalytic properties of recombinant CYP93A1 expressed in yeast, its NADPH dependency, stereoselectivity and high substrate affinity confirmed that D6aH is the physiological function of CYP93A1. It thus represents the first isoflavonoid-specific CYP to be characterized at the molecular level. In elicitor-treated soybean cells producing phytoalexins, increases in D6aH activity were correlated with elevated transcript levels which indicates that expression of the enzyme is regulated at the level of transcription. Therefore, CYP93A1 cDNA can be used as a specific molecular marker for the inducible defense response against pathogen attack.

Conventional kinesins are molecular motors that move towards the plus end of microtubules. In animal species, they have been shown to be remarkably conserved in terms of both their primary sequence and several physiological properties, including their velocity of movement. Here we report the cloning of Synkin, a homologue of conventional kinesin from the zygomycete fungus Syncephalastrum racemosum [Steinberg, Eur. J. Cell Biol. 73 (1997) 124–131] that is 4–5 times faster than its animal counterparts. Expression in bacteria yields a fully functional motor that moves at the same speed as the native motor isolated from fungal hyphae and has similar hydrodynamic properties. Its sequence is most closely related to that of two other fungal kinesins from Neurospora and Ustilago, and shares several biochemical properties with the Neurospora motor. Fungal kinesins therefore seem to form a conserved subfamily of conventional kinesins distantly related to animal kinesins. They may help to identify sequence features important for determining motor velocity.

Following the biophysical analysis of plant K+ channels in their natural environment, three members from the green branch of the evolutionary tree of life KAT1, AKT1 and KST1 have recently been identified on the molecular level. Among them, we focused on the expression and characterization of the Arabidopsis thaliana K+ channel KAT1 in the insect cell line Sf9. The infection of Sf9 cells with KAT1-recombinant baculovirus resulted in functional expression of KAT1 channels, which was monitored by inward-rectifying, K+-selective (impermeable to Na+ and even NH4+) ionic conductance in whole-cell patch-clamp recordings. A voltage threshold as low as −60 to −80 mV for voltage activation compared to other plant inward rectifiers in vivo, and to in vitro expression of KAT1 in Xenopus oocytes or yeast, may be indicative for channel modulation by the expression system. A rise in cytoplasmic Ca2+ concentration (up to 1 mM), a regulator of the inward rectifier in Vicia faba guard cells, did not modify the voltage dependence of KAT1 in Sf9 cells. The access to channel function on one side and channel protein on the other make Sf9 cells a suitable heterologous system for studies on the biophysical properties, post-translational modification and assembly of a green inward rectifier.