Molecular cloning and expression of cDNAs encoding human alpha-mannosidase II and a previously unrecognized alpha-mannosidase IIx isozyme.

Golgi alpha-mannosidase II (alpha-MII) is an enzyme involved in the processing of N-linked glycans. Using a previously isolated murine cDNA clone as a probe, we have isolated cDNA clones encompassing the human alpha-MII cDNA open reading frame and initiated isolation of human genomic clones. During the isolation of genomic clones, genes related to that encoding alpha-MII were isolated. One such gene was found to encode an isozyme, designated alpha-MIIx. A 5-kb cDNA clone encoding alpha-MIIx was then isolated from a human melanoma cDNA library. However, comparison between alpha-MIIx and alpha-MII cDNAs suggested that the cloned cDNA encodes a truncated polypeptide with 796 amino acid residues, while alpha-MII consists of 1144 amino acid residues. To reevaluate the sequence of alpha-MIIx cDNA, polymerase chain reaction (PCR) was performed with lymphocyte mRNAs. Comparison of the sequence of PCR products with the alpha-MIIx genomic sequence revealed that alternative splicing of the alpha-MIIx transcript can result in an additional transcript encoding a 1139-amino acid polypeptide. Northern analysis showed transcription of alpha-MIIx in various tissues, suggesting that the alpha-MIIx gene is a housekeeping gene. COS cells transfected with alpha-MIIx cDNA containing the full-length open reading frame showed an increase of alpha-mannosidase activity. The alpha-MIIx gene was mapped to human chromosome 15q25, whereas the alpha-MII gene was mapped to 5q21-22.

Cloning and expression of the cDNA of chicken cation-independent mannose-6-phosphate receptor.

We cloned and sequenced the 8767-bp full-length cDNA for the chicken cation-independent mannose-6-phosphate receptor (CI-MPR), of interest because, unlike its mammalian homologs, it does not bind insulin-like growth factor II (IGF-II). The cDNA encodes a protein of 2470 aa that includes a putative signal sequence, an extracytoplasmic domain consisting of 15 homologous repeat sequences, a 23-residue transmembrane sequence, and a 161-residue cytoplasmic sequence. Overall, it shows 60% sequence identity with human and bovine CI-MPR homologs, and all but two of 122 cysteine residues are conserved. However, it shows much less homology in the N-terminal signal sequence, in repeat 11, which is proposed to contain the IGF-II-binding site in mammalian CI-MPR homologs, and in the 14-aa residue segment in the cytoplasmic sequence that has been proposed to mediate G-protein-coupled signal transduction in response to IGF-II binding by the human CI-MPR. Transient expression in COS-7 cells produced a functional CI-MPR which exhibited mannose-6-phosphate-inhibitable binding and mediated endocytosis of recombinant human beta-glucuronidase. Expression of the functional chicken CI-MPR in mice lacking the mammalian CI-MPR should clarify the controversy over the physiological role of the IGF-II-binding site in mammalian CI-MPR homologs.

Cloning, expression, and function of TFC5, the gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor TFIIIB.

TFC5, the unique and essential gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been cloned. It encodes a 594-amino acid protein (67,688 Da). Escherichia coli-produced B" has been used to reconstitute entirely recombinant TFIIIB that is fully functional for TFIIIC-directed, as well as TATA box-dependent, DNA binding and transcription. The DNase I footprints of entirely recombinant TFIIIB, composed of B", the 67-kDa Brf, and TATA box-binding protein, and TFIIIB reconstituted with natural B" are indistinguishable. A truncated form of B" lacking 39 N-terminal and 107 C-terminal amino acids is also functional for transcription.

Molecular cloning, characterization, and functional expression of rat oxidosqualene cyclase cDNA.

A cDNA encoding rat oxidosqualene lanosterol-cyclase [lanosterol synthase; (S)-2,3-epoxysqualene mutase (cyclizing, lanosterol-forming), EC 5.4.99.7] was cloned and sequenced by a combination of PCR amplification, using primers based on internal amino acid sequence of the purified enzyme, and cDNA library screening by oligonucleotide hybridization. An open reading frame of 2199 bp encodes a M(r) 83,321 protein with 733 amino acids. The deduced amino acid sequence of the rat enzyme showed significant homology to the known oxidosqualene cyclases (OSCs) from yeast and plant (39-44% identity) and still retained 17-26% identity to two bacterial squalene cyclases (EC 5.4.99.-). Like other cyclases, the rat enzyme is rich in aromatic amino acids and contains five so-called QW motifs, highly conserved regions with a repetitive beta-strand turn motif. The binding site sequence for the 29-methylidene-2,3-oxidosqualene (29-MOS), a mechanism-based irreversible inhibitor specific for the vertebrate cyclase, is well-conserved in all known OSCs. The hydropathy plot revealed a rather hydrophilic N-terminal region and the absence of a hydrophobic signal peptide. Unexpectedly, this microsomal membrane-associated enzyme showed no clearly delineated transmembrane domain. A full-length cDNA was constructed and subcloned into a pYEUra3 plasmid, selected in Escherichia coli cells, and used to transform the OSC-deficient uracil-auxotrophic SGL9 strain of Saccharomyces cerevisiae. The recombinant rat OSC expressed was efficiently labeled by the mechanism-based inhibitor [3H]29-MOS.

Cloning and expression of Stat5 and an additional homologue (Stat5b) involved in prolactin signal transduction in mouse mammary tissue.

Prolactin (PRL) induces transcriptional activation of milk protein genes, such as the whey acidic protein (WAP), beta-casein, and beta-lactoglobulin genes, through a signaling cascade encompassing the Janus kinase Jak2 and the mammary gland factor (MGF; also called Stat5), which belongs to the family of proteins of signal transducers and activators of transcription (STAT). We isolated and sequenced from mouse mammary tissue Stat5 mRNA and a previously unreported member, which we named Stat5b (Stat5 is renamed to Stat5a). On the protein level Stat5a and Stat5b show a 96% sequence similarity. The 5' and 3' untranslated regions of the two mRNAs are not conserved. Stat5a comprises 793 amino acids and is encoded by a mRNA of 4.2 kb. The Stat5b mRNA has a size of 5.6 kb and encodes a protein of 786 amino acids. Both Stat5a and Stat5b recognized the GAS site (gamma-interferon-activating sequence; TTCNNNGAA) in vitro and mediated PRL-induced transcription in COS cells transfected with a PRL receptor. Stat5b also induced basal transcription in the absence of PRL. Similar levels of Stat5a and Stat5b mRNAs were found in most tissues of virgin and lactating mice, but a differential accumulation of the Stat5 mRNAs was found in muscle and mammary tissue. The two RNAs are present in mammary tissue of immature virgin mice, and their levels increase up to day 16 of pregnancy, followed by a decline during lactation. The increase of Stat5 expression during pregnancy coincides with the activation of the WAP gene.

Cloning and expression of two brain-specific inwardly rectifying potassium channels.

We have cloned two inwardly rectifying K+ channels that occur selectively in neurons in the brain and are designated BIRK (brain inwardly rectifying K+) channels. BIRK1 mRNA is extremely abundant and is enriched in specific brainstem nuclei, BIRK1 displays a consensus phosphate-binding loop, and expression in Xenopus oocytes has shown that its conductance is inhibited by ATP and adenosine 5'-[gamma-thio]triphosphate. BIRK2 is far less abundant and is selectively localized in telencephalic neurons. BIRK2 has a consensus sequence for cAMP-dependent phosphorylation.

kappa-Opioid receptor in humans: cDNA and genomic cloning, chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system.

Using the mouse delta-opioid receptor cDNA as a probe, we have isolated genomic clones encoding the human mu- and kappa-opioid receptor genes. Their organization appears similar to that of the human delta receptor gene, with exon-intron boundaries located after putative transmembrane domains 1 and 4. The kappa gene was mapped at position q11-12 in human chromosome 8. A full-length cDNA encoding the human kappa-opioid receptor has been isolated. The cloned receptor expressed in COS cells presents a typical kappa 1 pharmacological profile and is negatively coupled to adenylate cyclase. The expression of kappa-opioid receptor mRNA in human brain, as estimated by reverse transcription-polymerase chain reaction, is consistent with the involvement of kappa-opioid receptors in pain perception, neuroendocrine physiology, affective behavior, and cognition. In situ hybridization studies performed on human fetal spinal cord demonstrate the presence of the transcript specifically in lamina II of the dorsal horn. Some divergences in structural, pharmacological, and anatomical properties are noted between the cloned human and rodent receptors.

The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.

The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.

Molecular cloning and expression of the 32-kDa subunit of human TFIID reveals interactions with VP16 and TFIIB that mediate transcriptional activation.

Transcription factor TFIID consists of TATA binding protein (TBP) and at least eight TBP-associated factors (TAFs). As TAFs are required for activated but not basal transcription, we have proposed that TAFs act as coactivators to mediate signals between activators and the basal transcription machinery. Here we report the cloning, expression, and biochemical characterization of the 32-kDa subunit of human (h) TFIID, termed hTAFII32. We find that hTAFII32 is the human homologue of Drosophila TAFII40. In vitro protein-protein interaction assays reveal that as observed with Drosophila TAFII40, hTAFII32 interacts with the C-terminal 39-amino acid activation domain of the acidic transactivator viral protein 16 (VP16) as well as with the general transcription factor TFIIB. Moreover, a partial recombinant TFIID complex containing hTAFII32 was capable of mediating in vitro transcriptional activation by the VP16 activation domain. These findings indicate that specific activator-coactivator interactions have been conserved between human and Drosophila and provide additional support for the function of these interactions in mediating transcriptional activation.

Cloning and expression of a second photoreceptor-specific membrane retina guanylyl cyclase (RetGC), RetGC-2.

One of the membrane guanylyl cyclases (GCs), RetGC, is expressed predominantly in photoreceptors. No extracellular ligand has been described for RetGC, but it is sensitive to activation by a soluble 24-kDa protein (p24) and is inhibited by Ca2+. This enzyme is, therefore, thought to play a role in resynthesizing cGMP for photoreceptor recovery or adaptation. By screening a human retinal cDNA library at low stringency with the cytoplasmic domains from four cyclases, we cloned cDNAs encoding a membrane CG that is most closely related to RetGC. We have named this GC RetGC-2, and now term the initially described RetGC RetGC-1. By in situ hybridization, mRNA encoding RetGC-2 is found only in the outer nuclear layer and inner segments of photoreceptor cells. By using synthetic peptide antiserum specific for each RetGC subtype, RetGC-2 can be distinguished from RetGC-1 as a slightly smaller protein in immunoblots of bovine rod outer segments. Membrane GC activity of recombinant RetGC-2 expressed in human embryonic kidney 293 cells is stimulated by the activator p24 and is inhibited by Ca2+ with an EC50 value of 50-100 nM. Our data reveal a previously unappreciated diversity of photoreceptor GCs.

Cloning, baculovirus expression, and characterization of a second mouse prolyl 4-hydroxylase alpha-subunit isoform: formation of an alpha 2 beta 2 tetramer with the protein disulfide-isomerase/beta subunit.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer, the beta subunit of which is a highly unusual multifunctional polypeptide, being identical to protein disulfide-isomerase (EC 5.3.4.1). We report here the cloning of a second mouse alpha subunit isoform, termed the alpha (II) subunit. This polypeptide consists of 518 aa and a signal peptide of 19 aa. The processed polypeptide is one residue longer than the mouse alpha (I) subunit (the previously known type), the cloning of which is also reported here. The overall amino acid sequence identity between the mouse alpha (II) and alpha (I) subunits is 63%. The mRNA for the alpha (II) subunit was found to be expressed in a variety of mouse tissues. When the alpha (II) subunit was expressed together with the human protein disulfide-isomerase/beta subunit in insect cells by baculovirus vectors, an active prolyl 4-hydroxylase was formed, and this protein appeared to be an alpha (II) 2 beta 2 tetramer. The activity of this enzyme was very similar to that of the human alpha (I) 2 beta 2 tetramer, and most of its catalytic properties were also highly similar, but it differed distinctly from the latter in that it was inhibited by poly(L-proline) only at very high concentrations. This property may explain why the type II enzyme was not recognized earlier, as an early step in the standard purification procedure for prolyl 4-hydroxylase is affinity chromatography on a poly(L-proline) column.

Cloning and expression of the large zebrafish protocadherin gene, Fat

The cadherin superfamily members play an important role in mediating cell-cell contact and adhesion (Takeichi, M., 1991. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251, 1451-1455). A distinct subfamily, neither belonging to the classical or protocadherins includes Fat, the largest member of the cadherin super-family. Fat was originally identified in Drosophila. Subsequently, orthologues of Fat have been described in man (Dunne, J., Hanby, A. M., Poulsom, R., Jones, T. A., Sheer, D., Chin, W. G., Da, S. M., Zhao, Q., Beverley, P. C., Owen, M. J., 1995. Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule. Genomics 30, 207-223), rat (Ponassi, M., Jacques, T. S., Ciani, L., ffrench, C. C., 1999. Expression of the rat homologue of the Drosophila fat tumour suppressor gene. Mech. Dev. 80, 207-212) and mouse (Cox, B., Hadjantonakis, A. K., Collins, J. E., Magee, A. I., 2000. Cloning and expression throughout mouse development of mfat 1, a homologue of the Drosophila tumour suppressor gene fat [In Process Citation]. Dev. Dyn. 217, 233-240). In Drosophila, Fat has been shown to play an important role in both planar cell polarity and cell boundary formation during development. In this study we describe the characterization of zebrafish Fat, the first non-mammalian, vertebrate Fat homologue to be identified. The Fat protein has 64% amino acid identity and 80% similarity to human FAT and an identical domain structure to other vertebrate Fat proteins. During embryogenesis fat mRNA is expressed in the developing brain, specialised epithelial surfaces the notochord, ears, eyes and digestive tract, a pattern similar but distinct to that found in mammals. (c) 2005 Elsevier B.V. All rights reserved.

Cloning and functional expression of venom prothrombin activator protease from Pseudonaja textilis with whole blood procoagulant activity

The snake venom group C prothrombin activators contain a number of components that enhance the rate of prothrombin activation. The cloning and expression of full-length cDNA for one of these components, an activated factor X (factor Xa)-like protease from Pseudonaja textilis as well as the generation of functional chimeric constructs with procoagulant activity were described. The complete cDNA codes for a propeptide, light chain, activation peptide (AP) and heavy chain related in sequence to mammalian factor X. Efficient expression of the protease was achieved with constructs where the AP was deleted and the cleavage sites between the heavy and light chains modified, or where the AP was replaced with a peptide involved in insulin receptor processing. In human kidney cells (H293F) transfected with these constructs, up to 80% of the pro-form was processed to heavy and light chains. Binding of the protease to barium citrate and use of specific antibodies demonstrated that gamma-carboxylation of glutamic acid residues had occurred on the light chain in both cases, as observed in human factor Xa and the native P. textilis protease. The recombinant protease caused efficient coagulation of whole citrated blood and citrated plasma that was enhanced by the presence of Ca2+. This study identified the complete cDNA sequence of a factor Xa-like protease from P. textilis and demonstrated for the first time the expression of a recombinant form of P. textilis protease capable of blood coagulation.

Molecular cloning and characterization of the mouse Na+ sulfate cotransporter gene (Slc13a4): structure and expression

Sulfate is an essential ion required for numerous functions in mammalian physiology. Due to its hydrophilic nature, cells require sulfate transporters on their plasma membranes to allow entry of sulfate into cells. In this study, we identified a new mouse Na+-sulfate cotransporter (mNaS2), characterized its tissue distribution and determined its cDNA and gene (Slc13a4) structures. mNaS2 mRNA was expressed in placenta, brain, lung, eye, heart, testis, thymus and liver. The mouse NaS2 cDNA spans 3384 nucleotides and its open frame encodes a protein of 624 amino acids. Slc13a4 maps to mouse chromosome 6131 and contains 16 exons, spanning over 40 kb in length. Its 5'-flanking region contains CART- and GC-box motifs and a number of putative transcription factor binding sites, including GATA-1, MTF-1, STAT6 and HNF4 consensus sequences. This is the first study to define the tissue distribution of mNaS2 and resolve its cDNA and gene structures, which will allow us to investigate mNaS2 gene expression in vivo and determine its role in mammalian physiology.