Molecular cloning and sequence analysis of cDNAs encoding porcine kidney D-amino acid oxidase

Complementary DNAs encoding D-amino acid oxidase (EC 1.4.3.3, DAO), one of the principal and characteristic enzymes of the peroxisomes of porcine kidney, have been isolated from the porcine kidney cDNA library by hybridization with synthetic oligonucleotide probes corresponding to the partial amino acid sequences. Analysis of the nucleotide sequences of two clones revealed a complete 3211-nucleotide sequence with a 5'-terminal untranslated region of 198 nucleotides, 1041 nucleotides of an open reading frame that encoded 347 amino acids, and a 3'-terminal untranslated region of 1972 nucleotides. The deduced amino acid sequence was completely identical with the reported sequence of the mature enzyme [Ronchi, S., Minchiotti, L., Galliano, M., Curti, B., Swenson, R. P., Williams, C. H. J., & Massey, V. (1982) J. Biol. Chem. 257, 8824-8834]. These results indicate that the primary translation product does not contain a signal peptide at its amino-terminal region for its translocation into peroxisomes. RNA blot hybridization analysis suggests that porcine kidney D-amino acid oxidase is encoded by three mRNAs that differ in size: 3.3, 2.7, and 1.5 kilobases. Comparison of the sequences of the two cDNA clones revealed that multiple polyadenylation signal sequences (ATTAAA and AACAAA) are present in the 3'-untranslated region, making the different mRNA species. The efficiency of 3' processing of the RNA was quite different between the two signal sequences ATTAAA and AACAAA. Southern blot analysis showed the presence of a unique gene for D-amino acid oxidase in the porcine genome.     

A lipid transfer protein that transfers lipid

Very few lipid transfer proteins (LTPs) have been caught in the act of transferring lipids in vivo from a donor membrane to an acceptor membrane. Now, two studies (Halter, D., S. Neumann, S.M. van Dijk, J. Wolthoorn, A.M. de Maziere, O.V. Vieira, P. Mattjus, J. Klumperman, G. van Meer, and H. Sprong. 2007. J. Cell Biol. 179:101-115; D'Angelo, G., E. Polishchuk, G.D. Tullio, M. Santoro, A.D. Campli, A. Godi, G. West, J. Bielawski, C.C. Chuang, A.C. van der Spoel, et al. 2007. Nature. 449:62-67) agree that four-phosphate adaptor protein 2 (FAPP2) transfers glucosylceramide (GlcCer), a lipid that takes an unexpectedly circuitous route.     

Cloning and sequence analysis of porcine myoglobin cDNA

Porcine myoglobin cDNA clones have been isolated from a cDNA library prepared from enriched heart-myoglobin mRNA. Sequence analysis revealed 59 nucleotides (nt) in the 5'-untranslated, 462 nt in the amino acid (aa)-coding, and 590 nt in the 3'-untranslated regions. The myoglobin cDNA showed a high G + C content (60%). When the nt sequence of the porcine myoglobin cDNA is compared with those of seal and human myoglobin cDNAs deduced from the corresponding genomic myoglobin genes [Blanchetot et al., Nature 301 (1983) 732-734; Weller et al., EMBO J. 3 (1984) 439-446; Akaboshi, Gene 33 (1985) 241-249], a high degree of homology is observed in the 5'-untranslated region and in parts of the 3'-untranslated region, as well as in the coding region.     

Cloning, expression and sequence homologies of cDNA for human gamma enolase

The nucleotide sequence of the human gamma-enolase mRNA was determined from recombinant cDNA clones. The sequence spans 2273 bp and includes the complete coding region of 1299 bp, a 5'-noncoding region of 74 bp and a 897-bp-long 3'-noncoding region containing a variant polyadenylation signal (ATTAAA). The deduced amino acid (aa) sequence is 433 aa long and shows a 97% similarity with rat gamma-enolase. Both the 5'- and 3'-untranslated regions are similar (82% and 68%, respectively) to the analogous regions of the rat gamma-enolase gene, suggesting that a strong selective pressure operates on noncoding segments of gamma-enolase mRNAs. The size of the gamma-enolase mRNA expressed in human brain is 2.4 kb. A crosshybridizing 1.5-kb message is detected in human skeletal muscle which may be derived from the beta-enolase-coding gene.     

Identification of GB virus C variants by phylogenetic analysis of 5'-untranslated and coding region sequences.

Phylogenetic analysis of 44 GB virus C (GBV-C) 5'-untranslated region (5'-UTR) sequences from 37 individuals suggested the presence of GBV-C genotypes (A. S. Muerhoff, J. N. Simons, T. P. Leary, J. C. Erker, M. L. Chalmers, T. J. Pilot-Matias, G. J. Dawson, S. M. Desai, and I. K. Mushahwar, J. Hepatol. 25:379-384, 1996) that correlated with geographic origin: type 1, 2a and 2b, and 3 isolates are found predominantly in West Africa, the United States and Europe, and Japan, respectively. We have extended our analysis to include 5'-UTR sequences from 129 globally distributed GBV-C isolates and sequences from the second envelope protein (E2) gene and nonstructural (NS) regions 3 and 5b from a subset of these isolates. Bootstrap analysis of a 157-nucleotide segment of the 5'-UTR from 129 sequences provided weak support for the existence of the four major groups of GBV-C isolates previously described, although phylogenetic analysis of a 374-nucleotide segment of the 5'-UTR from 83 isolates provided stronger support. Thus, the groups of GBV-C variants previously identified upon analysis of the entire 5'-UTR can be distinguished by analysis of the shorter, 374-nucleotide region from the 5'-UTR. In contrast, independent analysis of the E2, NS3, or NS5b region sequences does not identify groups of GBV-C variants that correlate with geographic origin. However, bootstrap analysis of these coding sequences, when linked to form colinear sequences, demonstrates that longer coding regions can produce GBV-C groupings that are similar to that determined from 5'-UTR sequence analysis. The inability to distinguish between GBV-C variants by using small segments of coding sequence suggests that the GBV-C genome is constrained. As a result of these constraints, there is a high degree of nucleotide and amino acid sequence conservation between isolates from widely separated geographic areas. Hence, substitutions at many nucleotide positions are not tolerated, so that substitutions at the positions which can change are saturated, thereby obscuring the evolutionary relationships.     

Regulation by thyrotropin-releasing hormone (TRH) of TRH receptor mRNA degradation in rat pituitary GH3 cells.

In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) down-regulates TRH receptor (TRH-R) mRNA (Fujimoto, J., Straub, R.E., and Gershengorn, M.C. (1991) Mol. Endocrinol. 5, 1527-1532), at least in part, by stimulating its degradation (Fujimoto, J., Narayanan, C.S., Benjamin, J.E., Heinflink, M., and Gershengorn, M.C. (1992) Endocrinology 130, 1879-1884). Here we show that TRH regulates RNase activity in GH3 cells and that specific mRNA sequences are needed for in vivo regulation of TRH-R mRNA by TRH. TRH affected RNase activity in a biphasic manner with rapid stimulation (by 10 min) followed by a decrease to a rate slower than in control lysates within 6 h. This time course paralleled the effects of TRH on degradation of TRH-R mRNA in vivo. The regulated RNase activity was in a polysome-free fraction of the lysates and was not specific for TRH-R RNA. A truncated form of TRH-R RNA that was missing the entire 3'-untranslated region (TRHR-R5) was more stable than full-length TRH-R RNA (TRHR-WT). In contrast to TRHR-WT mRNA, TRHR-R5 mRNA and TRHR-D9 mRNA, which was missing the 143 nucleotides 5' of the poly(A) tail, were not down-regulated by TRH in stably transfected GH3 cells as their rates of degradation were not increased. These data show that TRH regulates RNase activity in GH3 cells, that the 3'-untranslated region bestows decreased stability on TRH-R mRNA and that the 3' end of the mRNA is necessary for regulation by TRH of TRH-R mRNA degradation. We present an hypothesis that explains specific regulation of TRH-R mRNA degradation by TRH in GH3 pituitary cells.     

Bovine dopamine beta-hydroxylase cDNA. Complete coding sequence and expression in mammalian cells with vaccinia virus vector.

We have isolated cDNA clones for bovine dopamine beta-hydroxylase from an adrenal medulla cDNA library and have determined the complete coding sequence. The largest cDNA clone isolated from the library is 2.4 kilobase pairs (kb) and contains an open reading frame of 1788 bases, coding for a protein of 597 amino acids and Mr = 66,803. The predicted amino acid sequence of the bovine cDNA contains 85% identity with human dopamine beta-hydroxylase (Lamouroux, A., Vingny, A., Faucon Biquet, N., Darmon, M. C., Franck, R., Henry, J.P., and Mallet, J. (1987) EMBO J. 6, 3931-3937; Kobayashi, K., Kurosawa, Y., Fujita, K., and Nagatsu, T. (1989) Nucleic Acids Res. 17, 1089-1102). Northern blot analysis reveals that the cDNA hybridizes to an mRNA of 2.4 kb present in bovine adrenal medulla, but not in kidney, heart, or liver. In addition, the cDNA hybridizes to a second RNA species of 5.5 kb, which is 4-fold less abundant than the 2.4-kb RNA. In vitro translation of a synthetic RNA transcribed from the 2.4-kb cDNA produces a 68-kDa protein, which is specifically immunoprecipitated by antiserum to bovine dopamine beta-hydroxylase. The 2.4-kb cDNA was cloned into a vaccinia virus vector, and the recombinant virus was used to infect the rat pheochromocytoma PC12 and monkey BSC-40 fibroblast cell lines. In both cell lines, infection with recombinant virus produces a protein of Mr = 75,000, which reacts with antiserum to bovine dopamine beta-hydroxylase. These results indicate that the 2.4-kb cDNA contains the genetic information necessary to code for the bovine dopamine beta-hydroxylase subunit.     

Glomerular basement membrane. Identification of a fourth chain, alpha 4, of type IV collagen

The noncollagenous domain hexamer of collagen IV from bovine glomerular basement membrane was further investigated to determine the types of collagen chain from which subunits M2*b and M3 are derived. M2*b was shown to be a shorter form, containing 9 fewer residues, of M2*a which was previously established as the noncollagenous domain of a third chain, alpha 3, of collagen IV (Saus, J., Wieslander, J., Langeveld, J.P.M., Quinones, S., and Hudson, B.G. (1988) J. Biol. Chem. 263, 13374-13380). M3 was identified as the noncollagenous domain of a fourth chain, alpha 4, of type IV collagen, on the basis of additional sequence data together with previous findings. A comparison of the collagenous-noncollagenous junction regions of alpha 3(IV) and alpha 4(IV) chains with those of classical alpha 1(IV) and alpha 2(IV) chains reveals structural information which provides a potential strategy for molecular cloning of these novel chains. The results further reveal the complexity of electrophoresis patterns of the hexamer and potential ambiguities in using one-dimensional patterns to determine whether molecular defects of collagen IV occur in pathological processes affecting basement membranes.     

Cloning and expression profile of chicken radixin

Radixin is a member of the ERM (ezrin/radixin/moesin) family of cytoskeletal linkers. We have cloned chicken radixin as a 4.3 kb cDNA, which encodes an 80 kDa protein that is more than 98% identical to radixin from evolutionarily diverse species. High sequence homology (70-80%) also extends into the 3'-untranslated region (UTR) of the radixin gene. The 3'-UTR of moesin, but not ezrin, was also conserved, suggesting an essential, and possibly specific, regulatory function. A distinct pattern of radixin expression is seen in chicken tissues, suggesting a cell-type-specific function.     

Determination of the three-dimensional solution structure of the histidine-containing phosphocarrier protein HPr from Escherichia coli using multidimensional NMR spectroscopy.

We recorded several types of heteronuclear three-dimensional (3D) NMR spectra on 15N-enriched and 13C/15N-enriched histidine-containing phosphocarrier protein, HPr, to extend the backbone assignments [van Nuland, N. A. J., van Dijk, A. A., Dijkstra, K., van Hoesel, F. H. J., Scheek, R. M. & Robillard, G. T. (1992) Eur. J. Biochem, 203, 483-491] to the side-chain 1H,15N and 13C resonances. From both 3D heteronuclear 1H-NOE 1H-13C and 1H-NOE 1H-15N multiple-quantum coherence (3D-NOESY-HMQC) and two-dimensional (2D) homonuclear NOE spectra, more than 1200 NOE were identified and used in a step-wise structure refinement process using distance geometry and restrained molecular dynamics involving a number of new features. A cluster of nine structures, each satisfying the set of NOE restraints, resulted from this procedure. The average root-mean-square positional difference for the C alpha atoms is less than 0.12 nm. The secondary structure topology of the molecule is that of an open-face beta sandwich formed by four antiparallel beta strands packed against three alpha helices, resembling the recently published structure of Bacillus subtilis HPr, determined by X-ray crystallography [Herzberg, O., Reddy, P., Sutrina, S., Saier, M. H., Reizer, J. & Kapafia, G. (1992) Proc. Natl, Acad. Sci. USA 89, 2499-2503).     

Nucleotide sequence and glucocorticoid regulation of the mRNAs for the isoenzymes of rat aspartate aminotransferase

Cytosolic and mitochondrial aspartate aminotransferase cDNAs were cloned from a lambda gt11 rat liver cDNA library. The complete coding sequence and the 3' non-coding sequence of the cytosolic isozyme mRNA were obtained from two overlapping cDNA clones. Partial sequences of the mitochondrial enzyme cDNAs were found to be identical to the recently published complete sequence (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). A single mRNA (2.4 kb (kilobase pair] hybridizing to the mitochondrial cDNA probe was detected by Northern blot analysis, whereas the cytosolic cDNA probe labeled one major (2.1 kb) and two minor (1.8 and 4 kb) mRNAs. The 1.8-kb and the 2.1-kb cytosolic aspartate aminotransferase mRNAs differ in their 3' ends and probably result from the use of either of the two polyadenylation signals present in the 3' noncoding region of the major cytosolic aspartate aminotransferase mRNA. Glucocorticoid hormones increased the activity of cytosolic but not mitochondrial aspartate aminotransferase in both liver and kidney. The increase in the enzyme activity was accompanied by an increase in the amount of the three corresponding mRNAs, while the mitochondrial enzyme mRNA was not significantly modified.     

Conserved and unique sequences in the 3'-untranslated region of rat smooth-muscle alpha-actin mRNA

We have isolated a cDNA clone corresponding to rat smooth-muscle alpha-actin mRNA [Hsu and Frankel, J. Biol. Chem. 262 (1987) 9594-9600]. We present here the sequence of the 3'-untranslated region (3'-UTR) of the cDNA. By comparison with the reported sequence of the chicken gene, this 3'-UTR region contains a conserved 36-bp sequence and a unique 48-bp G + C-rich sequence. An RNA probe containing only the 3'-UTR of the cDNA was synthesized and shown to be specific for smooth-muscle alpha-actin message.     

Forced subunit assembly in alpha1beta2gamma2 GABAA receptors. Insight into the absolute arrangement

The major isoform of the gamma-aminobutyric acid type A (GABA(A)) receptor is thought to be composed of 2alpha(1), 2beta(2), and 1gamma(2) subunit(s), which surround the ion pore. Definite evidence for the subunit arrangement is lacking. We show here that GABA(A) receptor subunits can be concatenated to a trimer that can be functionally expressed upon combination with a dimer. Many combinations did not result in the functional expression. In contrast, four different combinations of triple subunits with dual subunit constructs, all resulting in the identical pentameric receptor gamma(2)beta(2)alpha(1)beta(2)alpha(1), could be successfully expressed in Xenopus oocytes. We characterized the functional properties of these receptors in respect to agonist, competitive antagonist, and diazepam sensitivity. All properties were similar to those of wild type alpha(1)beta(2)gamma(2) GABA(A) receptors. Thus, together with information on the crystal structure of the homologous acetylcholine-binding protein (Brejc, K., van Dijk, W. J., Klaassen, R. V., Schuurmans, M., van Der Oost, J., Smit, A. B., and Sixma, T. K., (2001) Nature 411, 269-276, we provide evidence for an arrangement gamma(2)beta(2)alpha(1)beta(2)alpha(1), counterclockwise when viewed from the synaptic cleft. Forced subunit assembly will also allow receptors containing different subunit isoforms or mutant subunits to be expressed, each in a desired position. The methods established here should be applicable to the entire ion channel family comprising nicotinic acetylcholine, glycine, and 5HT(3) receptors.