Cloning and sequencing an ovine interleukin-4-encoding cDNA

We have cloned a cDNA containing the complete coding sequence of ovine(ov) interleukin 4 (IL4) by the polymerase chain reaction using primers based on the 5' and 3' untranslated regions of the human IL4 gene. RNA was isolated from phorbol myristate acetate- and calcium ionphore A23187-stimulated mesenteric lymph node cells. The ovIL4 cDNA is 535 bp in length and contains an open reading frame of 408 nucleotides (nt) coding for a 15.1-kDa IL4 precursor of 135 amino acids (aa). Cleavage of the putative signal peptide of 22 aa yields the mature form of 13.2 kDa. Analysis of the mature aa sequence shows two potential N-linked glycosylation sites and six Cys residues. Ovine and bovine IL4 are shorter than human, mouse and rat IL4, because of a 51-nt deletion in the coding region. Comparison of the predicted aa sequence shows that ovIL4 shares 92, 57, 37 and 42% identity with the bovine, human, mouse and rat IL4s, respectively.     

Identification and characterisation of a human calmodulin-stimulated phosphodiesterase PDE1B1

A cDNA encoding a calmodulin-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE) was isolated from a human brain cDNA library. The cDNA, designated HSPDE1B1, encoded a protein of 536 amino acids that shared 96% sequence identity with the bovine "63 kDa" calmodulin-stimulated PDE. The recombinant protein had cyclic nucleotide phosphodiesterase activity that was stimulated approximately 2-fold by Ca2+/calmodulin and preferred cGMP as substrate. In addition, the enzymatic activity of HSPDE1B1 was inhibited by phosphodiesterase inhibitors with potencies similar to that displayed toward the bovine PDE1 enzymes: IBMX approximately equal to 8-methoxymethyl-IBMX > vinpocetine approximately equal to zaprinast > cilostamide > rolipram. HSPDE1B1 mRNA was found predominantly in the brain. Lower mRNA levels were found in heart and skeletal muscle. In situ hybridisation of brain revealed expression of HSPDE1B1 predominately in neuronal cells of the cerebellum, hippocampus and caudate. The HSPDE1B1 gene was mapped to human chromosome 12. A partial genomic sequence of HSPDE1B1 was isolated and shown to contain two splice junctions that are conserved in the rat PDE4 and the Drosophila dunce genes.     

A novel glutathione peroxidase in bovine eye. Sequence analysis, mRNA level, and translation

Bovine ciliary body contains a selenium-independent glutathione peroxidase (GPX) with a molecular mass of about 100 kDa that is composed of four identical subunits and exhibits no glutathione S-transferase activity. In this study, we isolated cDNA clones and determined the nucleotide sequence to deduce the primary structure of the enzyme. The cDNA contained 672 base pairs encoding a polypeptide with an estimated molecular mass of 25,064 Da. Translation of bovine ciliary mRNA produced a protein which was immunologically indistinguishable from GPX and showed high enzyme activity. The encoded amino acid sequence of the protein was 95% identical with that of a human keratinocyte gene product expressed in response to keratinocyte growth factor. It also showed sequence identity to bacterial alkyl hydroperoxide reductases and thiol specific antioxidant enzymes. GPX mRNA level was highest in the ciliary body, followed by the retina and iris. In various rat organs, the level of GPX mRNA was highest in the lung, followed by the muscle, liver, eye, heart, testis, thymus, kidney, and spleen. A very low level of mRNA was detected in the brain. Enzyme-linked immunosorbent assay with an antibody raised against the NH2-terminal sequence of GPX detected GPX protein in all rat tissues examined.     

Lack of correlation between apoptosis and DNA single-strand breaks in X-irradiated human peripheral blood mononuclear cells in the course of ageing

Inositol(1,3,4,5)tetrakisphosphate (InsP4) and phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) are two potential second messengers with a still largely unknown mode of action. We recently cloned the 42 kDa protein p42IP4 previously purified from pig cerebellum, which binds InsP4 (Kd approximately 2 nM) and PtdInsP3 with comparable affinities (Stricker et al., FEBS Lett. 405 (1997) 229). The protein p42IP4 (pig) is highly homologous to centaurin-alpha, a larger protein of 46 kDa, derived from a rat brain cDNA library clone (Hammonds-Odie et al., J. Biol. Chem. 271 (1996) 18859). Here we investigated whether also p42IP4 is expressed in rat brain and how it might be related to centaurin-alpha. When we carried out RT-PCR using mRNA from brain of rats of different ages we obtained several clones corresponding to p42IP4, but not to centaurin-alpha. The existence of p42IP4 in rat brain is supported by the following findings: (1) biochemical analysis of the purified rat brain protein shows inositol phosphate ligand affinities identical to those of the protein from other species; (2) Western blot analysis of rat brain membrane fractions using a peptide-specific antiserum revealed only the 42 kDa protein (p42IP4), but did not give evidence for the occurrence of a larger 46 kDa centaurin-alpha-like protein in rat brain; and (3) the amino acid sequences deduced from p42IP4 cDNA are highly homologous in several species and are confirmed by protein fragment microsequences. Thus, p42IP4 from rat brain which has two pleckstrin homology domains is a protein largely conserved between different species and most likely has an important function in inositol phosphate or inositol lipid signal transduction.     

Molecular characterization and localization of human metabotropic glutamate receptor type 4

Oligonucleotides of consensus sequences from rat metabotropic glutamate receptor (mGluR) genes were synthesized and used to amplify human DNA by the polymerase chain reaction (PCR). Five unique human sequences homologous to these rat receptor genes were isolated including mGluR4. A human cerebellum cDNA library was screened using this amplified mGluR4 sequence as a probe and yielded clones which between them contained the complete coding sequence for human mGluR4. The coding sequence is very similar to the equivalent rat gene (90% DNA sequence identity and 97% predicted protein sequence identity). The mGluR4 cDNA was transfected in Chinese hamster ovary (CHO) cells and stable clonal cell lines were isolated. Stimulation of the expressed receptor by L-2-amino-4-phosphonobutyrate (L-AP4), L-glutamate or (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) resulted in a reduction of forskolin-stimulated cyclic AMP (cAMP) with EC50 values of 0.2, 13 and 90 microM respectively. Quisqualate had little effect at concentrations up to 1 mM. In Northern blots mGluR4 mRNA appears to be brain-specific, and shows a distinct distribution (excluding the cerebellum), being expressed in the thalamus, hypothalamus and caudate nucleus. In situ hybridization studies on human brain sections confirmed this general pattern of distribution. The strongest mGluR4 mRNA signal was found in the cerebellar granule cells consistent with the reported distribution of mGluR4 in the rat brain. The major difference from the rat brain is the presence in the human brain of mGluR4 mRNA in the caudate nucleus and putamen.     

Physicians'' patterns of using a computerized collection of guidelines for primary care

Conserved amino acid motifs are found in numerous expressed genes. Proteins and peptides with functional relationships may be identified using probes designed to hybridize with these motifs. An oligonucleotide probe was prepared to match the sequence of the expected active region of a frog corticotropin-releasing factor-like peptide sauvagine and used to screen a sheep brain cDNA library. A novel 1331-bp cDNA encoding a putative 328-residue protein with a theoretical mass of 36 kDa was identified. The presence of a strong signal sequence indicates that it is a secreted protein. The amino- and carboxy-terminal regions are characterized by several potential phosphorylation sites and binding motifs, suggesting a role in intracellular signal transduction. Although the protein possesses a 7-residue sequence identical to that found in sauvagine, its overall primary structure most closely resembles those of the alpha-carbonic anhydrases (alpha-CAs). Moreover, the detection of the human and mouse orthologues in the EST databases, together with an evolutionary analysis, indicates that the protein represents a new member of the alpha-CA gene family, which we designate carbonic anhydrase-related protein XI (CA-RP XI), encoded by CA11 (human) and Car11 (mouse, rat). The human CA11 gene appears to be located between the secretor type alpha(1,2)-fucosyltransferase gene cluster (FUT1-FUT2-FUT2P) and the D-site binding protein gene (DBP) on chromosome 19q13.3. Despite potentially inactivating changes in the active-site residues, CA-RP XI is evolving very slowly in mammals, a property indicative of an important function, which has also been observed in the two other "acatalytic" CA isoforms, CA-RP VIII and CA-RP X, whose functions are unknown.     

A second form (beta isoform) of nucleoside diphosphate kinase from rat. Isolation and characterization of complementary and genomic DNA and expression

Complementary and genomic clones for a second form (the beta isoform) of rat nucleoside diphosphate kinase were isolated. Structural studies revealed that nucleotide and deduced amino acid sequences of the beta isoform were quite similar to those of the alpha isoform (identities were 82 and 89%, respectively), which were delineated in our previous study (Kimura, N., Shimada, N., Nomura, K., and Watanabe, K. (1990) J. Biol. Chem. 265, 15744-15749). The gene encoding the beta isoform, covering 10 kilobases and comprising five exons, was located in tandem in the immediate vicinity (at a 3-kilobase distance) of the 5' upstream of the alpha isoform gene which was recently reported from this laboratory (Ishikawa, N., Shimada, N., Munakata, Y., Watanabe, K., and Kimura, N. (1992) J. Biol. Chem. 267, 14366-14372), suggesting their generation by gene duplication. The exon-intron junctions were exactly conserved between the two genes. Southern blot analyses showed that unidentified fragments cross-reacted with the beta isoform cDNA probe besides those containing the genuine gene, and at least two of them were identified as possible processed pseudogenes. Northern and dot blot hybridization studies demonstrated that the alpha isoform was more expressed than the beta isoform in rat tissues examined except brain, from which the isoform designation was derived. These results suggest independent expression and specific roles of these isoforms in the cell. Comparative studies between rat and human isoforms indicate that the isoforms could have differentiated before the two species evolutionally diverged.     

Brain-specific receptor-type protein-tyrosine phosphatase RPTP beta is a chondroitin sulfate proteoglycan in vivo

By using polyclonal antiserum, which recognizes multiple proteoglycan core proteins, we isolated a cDNA species for an unknown chondroitin sulfate proteoglycan in bovine brain. Unexpectedly, DNA sequencing revealed that the cDNA encodes an open reading frame highly homologous to the human receptor-type protein-tyrosine phosphatase, RPTP beta. To prove that RPTP beta is a proteoglycan, we raised three polyclonal antibodies against extracellular and cytoplasmic domains of human RPTP beta. These antibodies have been shown to react with a smear band ranging from 350 to 500 kDa in human brain extracts. Digestion with chondroitinase ABC eliminated this smear and gave rise to a 310/300-kDa doublet band that was not detected without digestion, indicating that almost all of the RPTP beta molecules in the brain contain chondroitin sulfate chains. In the cerebellum, immunofluorescence staining of chondroitinase-treated sections revealed pericellular localization of RPTP beta in the external and internal granular layers. These data establish that RPTP beta is expressed constitutively as a chondroitin sulfate proteoglycan in the brain, and suggest that chondroitin sulfates may be an essential component for the physiological function of RPTP beta in vivo.     

A G-protein-coupled 130 kDa phospholipase C isozyme, PLC-beta 4, from the particulate fraction of bovine cerebellum

A 130 kDa PLC isozyme was purified from the particulate fraction of bovine cerebellum. This PLC was recognized by a polyclonal antiserum generated against the purified 97 kDa PLC-beta 4. Reconstitution of the purified 130 kDa PLC with the membranes of C6 Bu-1 cells in the presence of GTP gamma S or AlF4- resulted in PLC activation as well as the association of PLC with the membranes. Both the association and activation were revoked when the membrane was washed with 2 M KCl. The 97 kDa PLC-beta 4 did not associate with membranes. These data suggest that the 130 kDa PLC is the intact form of PLC-beta 4 the activity of which is likely to be regulated by a G-protein on the membrane.     

Characterization of a novel platelet-derived growth factor-associated protein

A novel mitogen-associated protein was isolated from a rat neural retina cell line. The protein copurified with platelet-derived growth factor (PDGF)-A and was therefore termed PDGF-associated protein (PAP). cDNAs corresponding to the protein were characterized from both rat and human cDNA libraries. PAP binds to PDGF with low affinity and enhances the mitogenic effect of PDGF-A but lowers the mitogenic activity of PDGF-B. PAP mRNA is abundant in the brain of newborn rats and is found in several other tissues.     

Molecular cloning of a full-length cDNA for human type 3 adenylyl cyclase and its expression in human islets

The GK (Goto-Kakizaki) rat is a lean model of type 2 diabetes in which the diabetic state was spontaneously induced. We recently demonstrated the presence in GK rats of two functional point mutations in the promoter region of the type 3 adenylyl cyclase (AC3) gene that resulted in overexpression of AC3 mRNA associated with increased cAMP generation. The AC3 gene promoter mutations are the first molecular changes to be described in any specific gene in the GK rat. Here we report cloning of a full-length cDNA encoding human AC3 from a human fetal brain cDNA library using a PCR-based screening method. This 4142-bp cDNA predicts an open reading frame encoding 1144 amino acids containing putative 12 transmembrane-spanning domains which are typically found in other mammalian AC isoforms. Comparison of the translated amino acid sequence of the AC3 gene between human and rat shows 95% homology. Using RT-PCR, clear AC3 expression was detected in isolated human islets as well as a cDNA panel containing templates from eight different tissues (brain, heart, kidney, liver, lung, pancreas, placenta, and skeletal muscle). This wide distribution of AC3 expression may involve a number of physiological and pathophysiological metabolic processes.     

Immunolocalization of phospholipase C isoforms in rat kidney

BACKGROUND: Phospholipase C (PLC) is an important factor in signal transduction because this enzyme is activated by several hormones and growth factors. Eight PLC isoforms have been described raising the possibility that different cells express a single isoform or activate specific isoforms in different cells. Therefore, the goal of this study was to determine which PLC isoforms are expressed in specific regions of rat kidney. METHODS: Western blot analysis was performed in microdissected nephron segments of rat kidney, while immunohistochemical analysis was performed on whole rat kidney slices using PLC isoform-specific antibodies. RESULTS: All three families of PLC isoforms (beta, gamma, and delta) were present throughout the cortical and medullary regions of the kidney. Only the PLC-beta1 isoform was observed in the brush border of the proximal tubule, but all isoforms were present in glomeruli and in the cytoplasm of tubular epithelial cells. In addition, only the PLC-gamma1 isoform was expressed in the internal elastic lamina of the renal artery, while vasa recta expressed PLC-beta1 most intensely. Medullary thick ascending limbs showed an intense level of expression of all three isoforms. CONCLUSION: Multiple PLC isoforms are present in glomeruli, renal tubules, and renal vasculature in vivo, but with some segment-specific differences. These findings suggest that the response of a specific cell is not determined by expression of only one PLC isoform, with the exception of the brush border of the proximal tubule and the renal arteries. Instead, the presence of multiple PLC isoforms in specific regions of the kidney suggests that hormonal regulation in vivo involves mechanisms beyond cell-specific isoforms of PLC.     

Coexistence of two beta subunit isoforms in the same gamma-aminobutyric acid type A receptor

Three novel subunit-specific antisera to the beta1, beta2, and beta3 subunits of rat gamma-aminobutyric acid type A (GABAA) receptors have been used to study the native receptor in the rat brain. Affinity-purified anti-beta1, anti-beta2, and anti-beta3 antibodies recognized in immunoblots protein bands of 57, 55, and 57 kDa, respectively. Quantitative immunoprecipitation of solubilized GABAA receptors from various rat brain regions showed that the beta2 subunit was the most abundant isoform in cerebellum (in 96% of the GABAA receptors) and cerebral cortex (64%) but that it was the least abundant isoform in hippocampus (44%). The beta3 subunit was found most abundant in hippocampus (64%) followed by cerebral cortex (48%) and cerebellum (33%). The beta1 subunit was present in a very small proportion of the cerebellar GABAA receptors (3%), but it was present in a high proportion of the GABAA receptors from the hippocampus (49%) and cerebral cortex (32%). Quantitative receptor immunoprecipitation or immunopurification followed by immunoblotting experiments have revealed the existence of colocalization of two different beta subunit isoforms in a significant proportion of the brain GABAA receptors. Thus, in the rat cerebral cortex 33% of the GABAA receptors have both beta2 and beta3 subunits, and 19% of the receptors have both beta1 and beta3 subunits. The extent of colocalization of beta subunit isoforms varied among brain regions, being highest in hippocampus and lowest in cerebellum. These and other results taken together suggest that the number of alpha, beta, and gamma subunits (stoichiometry) in the brain GABAA receptor pentamers might not be unique. It might vary depending on receptor type.     

The human glycine receptor beta subunit: primary structure, functional characterisation and chromosomal localisation of the human and murine genes

The inhibitory glycine receptor (GlyR) is a pentameric receptor comprised of alpha and beta subunits, of which the beta subunit has not been characterised in humans. A 2106 bp cDNA, isolated from a human hippocampal cDNA library, contained an open reading frame of 497 amino acids which encodes the beta subunit of the human GlyR. The mature human GlyR beta polypeptide displays 99% amino acid identity with the rat GlyR beta subunit and 48% identity with the human GlyR alpha 1 subunit. Neither [3H]strychnine binding nor glycine-gated currents were detected when the human GlyR beta subunit cDNA was expressed in the human embryonic kidney 293 cell line. However, co-expression of the beta subunit cDNA with the alpha 1 subunit cDNA resulted in expression of functional GlyRs which showed a 4-fold reduction in the EC50 values when compared to alpha 1 homomeric GlyRs. Glycine-gated currents of alpha 1/beta GlyRs were 17-fold less sensitive than homomeric alpha 1 GlyRs to the antagonists picrotoxin, picrotoxinin and picrotin, providing clear evidence that heteromeric alpha 1/beta GlyRs were expressed. The beta subunit appears to play a structural rather than ligand binding role in GlyR function. Fluorescence in situ hybridisation was used to localise the gene encoding the human GlyR beta subunit (GLRB) to chromosome 4q32, a position syntenic with mouse chromosome 3. In situ hybridisation using the human GlyR beta subunit cDNA showed that the murine GlyR beta subunit gene (Glrb) maps to the spastic (spa) locus on mouse chromosome 3 at bands E3-F1. This is consistent with the recent finding that a mutation in the murine GlyR beta subunit causes the spa phenotype. It also raises the possibility that mutations in the human beta subunit gene may cause inherited disorders of the startle response.     

Nucleoside diphosphate kinase from bovine retina: purification, subcellular localization, molecular cloning, and three-dimensional structure

The biochemical and structural properties of bovine retinal nucleoside diphosphate kinase were investigated. The enzyme showed two polypeptides of approximately 17.5 and 18.5 kDa on SDS-PAGE, while isoelectric focusing revealed seven to eight proteins with a pI range of 7.4-8.2. Sedimentation equilibrium yielded a molecular mass of 96 +/- 2 kDa for the enzyme. Carbohydrate analysis revealed that both polypeptides contained Gal, Man, GlcNAc, Fuc, and GalNac saccharides. Like other nucleoside diphosphate kinases, the retinal enzyme showed substantial differences in the Km values for various di- and triphosphate nucleotides. Immunogold labeling of bovine retina revealed that the enzyme is localized on both the membranes and in the cytoplasm. Screening of a retinal cDNA library yielded full-length clones encoding two distinct isoforms (NBR-A and NBR-B). Both isoforms were overexpressed in Escherichia coli and their biochemical properties compared with retinal NDP-kinase. The structures of NBR-A and NBR-B were determined by X-ray crystallography in the presence of guanine nucleotide(s). Both isoforms are hexameric, and the fold of the monomer is similar to other nucleoside diphosphate kinase structures. The NBR-A active site contained both a cGMP and a GDP molecule each bound at half occupancy while the NBR-B active site contained only cGMP.