The origin of 15R-prostaglandins in the Caribbean coral Plexaura homomalla: Molecular cloning and expression of a novel cyclooxygenase

The highest concentrations of prostaglandins in nature are found in the Caribbean gorgonian Plexaura homomalla. Depending on its geographical location, this coral contains prostaglandins with typical mammalian stereochemistry (15S-hydroxy) or the unusual 15R-prostaglandins. Their metabolic origin has remained the subject of mechanistic speculations for three decades. Here, we report the structure of a type of cyclooxygenase (COX) that catalyzes transformation of arachidonic acid into 15R-prostaglandins. Using a homology-based reverse transcriptase–PCR strategy, we cloned a cDNA corresponding to a COX protein from the R variety of P. homomalla. The deduced peptide sequence shows 80% identity with the 15S-specific coral COX from the Arctic soft coral Gersemia fruticosa and ≈50% identity to mammalian COX-1 and COX-2. The predicted tertiary structure shows high homology with mammalian COX isozymes having all of the characteristic structural units and the amino acid residues important in catalysis. Some structural differences are apparent around the peroxidase active site, in the membrane-binding domain, and in the pattern of glycosylation. When expressed in Sf9 cells, the P. homomalla enzyme forms a 15R-prostaglandin endoperoxide together with 11R-hydroxyeicosatetraenoic acid and 15R-hydroxyeicosatetraenoic acid as by-products. The endoperoxide gives rise to 15R-prostaglandins and 12R-hydroxyheptadecatrienoic acid, identified by comparison to authentic standards. Evaluation of the structural differences of this 15R-COX isozyme should provide new insights into the substrate binding and stereospecificity of the dioxygenation reaction of arachidonic acid in the cyclooxygenase active site.

Organization and expression of the double-stranded RNA genome of Helminthosporium victoriae 190S virus, a totivirus infecting a plant pathogenic filamentous fungus.

The complete nucleotide sequence, 5178 bp, of the totivirus Helminthosporium vicotoriae 190S virus (Hv190SV) double-stranded RNA, was determined. Computer-assisted sequence analysis revealed the presence of two large overlapping ORFs; the 5'-proximal large ORF (ORF1) codes for the coat protein (CP) with a predicted molecular mass of 81 kDa, and the 3'-proximal ORF (ORF2), which is in the -1 frame relative to ORF1, codes for an RNA-dependent RNA polymerase (RDRP). Unlike many other totiviruses, the overlap region between ORF1 and ORF2 lacks known structural information required for translational frameshifting. Using an antiserum to a C-terminal fragment of the RDRP, the product of ORF2 was identified as a minor virion-associated polypeptide of estimated molecular mass of 92 kDa. No CP-RDRP fusion protein with calculated molecular mass of 165 kDa was detected. The predicted start codon of the RDRP ORF (2605-AUG-2607) overlaps with the stop codon (2606-UGA-2608) of the CP ORF, suggesting RDRP is expressed by an internal initiation mechanism. Hv190SV is associated with a debilitating disease of its phytopathogenic fungal host. Knowledge of its genome organization and expression will be valuable for understanding its role in pathogenesis and for potential exploitation in the development of biocontrol measures.

Purification, molecular cloning, and expression of the mammalian sigma1-binding site.

Sigma-ligands comprise several chemically unrelated drugs such as haloperidol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum. These so-called sigma-receptors are believed to mediate various pharmacological effects of sigma-ligands by as yet unknown mechanisms. Based on their opposite enantioselectivity for benzomorphans and different molecular masses, two subtypes are differentiated. We purified the sigma1-binding site as a single 30-kDa protein from guinea pig liver employing the benzomorphan(+)[3H]pentazocine and the arylazide (-)[3H]azidopamil as specific probes. The purified (+)[3H]pentazocine-binding protein retained its high affinity for haloperidol, pentazocine, and ditolylguanidine. Partial amino acid sequence obtained after trypsinolysis revealed no homology to known proteins. Radiation inactivation of the pentazocine-labeled sigma1-binding site yielded a molecular mass of 24 +/- 2 kDa. The corresponding cDNA was cloned using degenerate oligonucleotides and cDNA library screening. Its open reading frame encoded a 25.3-kDa protein with at least one putative transmembrane segment. The protein expressed in yeast cells transformed with the cDNA showed the pharmacological characteristics of the brain and liver sigma1-binding site. The deduced amino acid sequence was structurally unrelated to known mammalian proteins but it shared homology with fungal proteins involved in sterol synthesis. Northern blots showed high densities of the sigma1-binding site mRNA in sterol-producing tissues. This is also in agreement with the known ability of sigma1-binding sites to interact with steroids, such as progesterone.

Cloning and expression of human deoxyguanosine kinase cDNA.

A human cDNA sequence homologous to human deoxycytidine kinase (dCK; EC 2.7.1.74) was identified in the GenBank sequence data base. The longest open reading frame encoded a protein that was 48% identical to dCK at the amino acid level. The cDNA was expressed in Escherichia coli and shown to encode a protein with the same substrate specificity as described for the mitochondrial deoxyguanosine kinase (dGK; EC 2.7.1.113). The N terminus of the deduced amino acid sequence had properties characteristic for a mitochondrial translocation signal, and cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein size of 28 kDa. Northern blot analysis determined the length of dGK mRNA to 1.3 kbp with no cross-hybridization to the 2.8-kbp dCK mRNA. dGK mRNA was detected in all tissues investigated with the highest expression levels in muscle, brain, liver, and lymphoid tissues. Alignment of the dGK and herpes simplex virus type 1 thymidine kinase amino acid sequences showed that five regions, including the substrate-binding pocket and the ATP-binding glycine loop, were also conserved in dGK. To our knowledge, this is the first report of a cloned mitochondrial nucleoside kinase and the first demonstration of a general sequence homology between two mammalian deoxyribonucleoside kinases. Our findings suggest that dCK and dGK are evolutionarily related, as well as related to the family of herpes virus thymidine kinases.

Detection of JC virus DNA sequence and expression of the viral oncoprotein, tumor antigen, in brain of immunocompetent patient with oligoastrocytoma.

We describe molecular and clinical findings in an immunocompetent patient with an oligoastrocytoma and the concomitant presence of the human papovavirus, JC virus (JCV), which is the etiologic agent of the subacute, debilitating demyelinating disease, progressive multifocal leukoencephalopathy. Histologic review revealed a glial neoplasm consisting primarily of a moderately cellular oligodendroglioma with distinct areas of a fibrillary astrocytoma. Immunohistochemical analysis revealed nuclear staining of tumor cells with antibodies against the viral oncoprotein [tumor antigen (T antigen)], the proliferation marker (Ki67), and the cellular proliferation regulator (p53). Using primers specific to the JCV control region, PCR yielded amplified DNA that was identical to the control region of the Mad-4 strain of the virus. PCR analysis demonstrated the presence of the genome for the viral oncoprotein, T antigen, and results from primer extension studies revealed synthesis of the viral early RNA for T antigen in the tumor tissues. The presence of viral T antigen in the tumor tissue was further demonstrated by immunoblot assay. To our knowledge, this is the first report of the presence of JCV DNA, RNA, and T antigen in tissue in which viral T antigen is localized to tumor cell nuclei and suggests the possible association of JCV with some glial neoplasms.

Molecular cloning of violaxanthin de-epoxidase from romaine lettuce and expression in Escherichia coli.

Plants need to avoid or dissipate excess light energy to protect photosystem II (PSII) from photoinhibitory damage. Higher plants have a conserved system that dissipates excess energy as heat in the light-harvesting complexes of PSII that depends on the transthylakoid delta pH and violaxanthin de-epoxidase (VDE) activity. To our knowledge, we report the first cloning of a cDNA encoding VDE and expression of functional enzyme in Escherichia coli. VDE is nuclear encoded and has a transit peptide with characteristic features of other lumen-localized proteins. The cDNA encodes a putative polypeptide of 473 aa with a calculated molecular mass of 54,447 Da. Cleavage of the transit peptide results in a mature putative polypeptide of 348 aa with a calculated molecular mass of 39,929 Da, close to the apparent mass of the purified enzyme (43 kDa). The protein has three interesting domains including (i) a cysteine-rich region, (ii) a lipocalin signature, and (iii) a highly charged region. The E. coli expressed enzyme de-epoxidizes violaxanthin sequentially to antheraxanthin and zeaxanthin, and is inhibited by dithiothreitol, similar to VDE purified from chloroplasts. This confirms that the cDNA encodes an authentic VDE of a higher plant and is unequivocal evidence that the same enzyme catalyzes the two-step mono de-epoxidation reaction. The cloning of VDE opens new opportunities for examining the function and evolution of the xanthophyll cycle, and possibly enhancing light-stress tolerance of plants.

Primary structure and expression of a pathogen-induced protease (PR-P69) in tomato plants: Similarity of functional domains to subtilisin-like endoproteases.

A 69-kDa proteinase (P69), a member of the pathogenesis-related proteins, is induced and accumulates in tomato (Lycopersicon esculentum) plants as a consequence of pathogen attack. We have used the polymerase chain reaction to identify and clone a cDNA from tomato plants that represent the pathogenesis-related P69 proteinase. The nucleotide sequence analysis revealed that P69 is synthesized in a preproenzyme form, a 745-amino acid polypeptide with a 22-amino acid signal peptide, a 92-amino acid propolypeptide, and a 631-amino acid mature polypeptide. Within the mature region the most salient feature was the presence of domains homologous to the subtilisin serine protease family. The amino acid sequences surrounding Asp-146, His-203, and Ser-532 of P69 are closely related to the catalytic sites (catalytic triad) of the subtilisin-like proteases. Northern blot analysis revealed that the 2.4-kb P69 mRNA accumulates abundantly in leaves and stem tissues from viroid-infected plants, whereas the mRNA levels in tissues from healthy plants were undetectable. Our results indicate that P69, a secreted calcium-activated endopeptidase, is a plant pathogenesis-related subtilisin-like proteinase that may collaborate with other defensive proteins in a general mechanism of active defense against attacking pathogens.

Molecular cloning and expression of a cyclic AMP-activated chloride conductance regulator: a novel ATP-binding cassette transporter.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-regulated, cAMP-activated chloride channel located in the apical membrane of many epithelial secretory cells. Here we report cloning of a cAMP-activated epithelial basolateral chloride conductance regulator (EBCR) that appears to be a basolateral CFTR counterpart. This novel chloride channel or regulator shows 49% identity with multidrug resistance-associated protein (MRP) and 29% identity with CFTR. On expression in Xenopus oocytes, EBCR confers a cAMP-activated chloride conductance that is inhibited by the chloride channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamine)benzoic acid, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Northern blot analysis reveals high expression in small intestine, kidney, and liver. In kidney, immunohistochemistry shows a conspicuous basolateral localization mainly in the thick ascending limb of Henle's loop, distal convoluted tubules and to a lesser extent connecting tubules. These data suggest that in the kidney EBCR is involved in hormone-regulated chloride reabsorption.

Cloning and expression of apoptosis inhibitory protein homologs that function to inhibit apoptosis and/or bind tumor necrosis factor receptor-associated factors.

Baculovirus inhibitors of apoptosis (IAPs) act in insect cells to prevent cell death. Here we describe three mammalian homologs of IAP, MIHA, MIHB, and MIHC, and a Drosophila IAP homolog, DIHA. Each protein bears three baculovirus IAP repeats and an N-terminal ring finger motif. Apoptosis mediated by interleukin 1beta converting enzyme (ICE), which can be inhibited by Orgyia pseudotsugata nuclear polyhedrosis virus IAP (OpIAP) and cowpox virus crmA, was also inhibited by MIHA and MIHB. As MIHB and MIHC were able to bind to the tumor necrosis factor receptor-associated factors TRAF1 and TRAF2 in yeast two-hybrid assays, these results suggest that IAP proteins that inhibit apoptosis may do so by regulating signals required for activation of ICE-like proteases.

A yeast artificial chromosome-based map of the region of chromosome 20 containing the diabetes-susceptibility gene, MODY1, and a myeloid leukemia related gene.

We have generated a physical map of human chromosome bands 20q11.2-20q13.1, a region containing a gene involved in the development of one form of early-onset, non-insulin-dependent diabetes mellitus, MODY1, as well as a putative myeloid tumor suppressor gene. The yeast artificial chromosome contig consists of 71 clones onto which 71 markers, including 20 genes, 5 expressed sequence tags, 32 simple tandem repeat DNA polymorphisms, and 14 sequence-tagged sites have been ordered. This region spans about 18 Mb, which represents about 40% of the physical length of 20q. Using this physical map, we have refined the location of MODY1 to a 13-centimorgan interval (approximately equal to 7 Mb) between D20S169 and D20S176. The myeloid tumor suppressor gene was localized to an 18-centimorgan interval (approximately equal to 13 Mb) between RPN2 and D20S17. This physical map will facilitate the isolation of MODY1 and the myeloid tumor suppressor gene.

The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

Spatial integration and cortical dynamics.

Cells in adult primary visual cortex are capable of integrating information over much larger portions of the visual field than was originally thought. Moreover, their receptive field properties can be altered by the context within which local features are presented and by changes in visual experience. The substrate for both spatial integration and cortical plasticity is likely to be found in a plexus of long-range horizontal connections, formed by cortical pyramidal cells, which link cells within each cortical area over distances of 6-8 mm. The relationship between horizontal connections and cortical functional architecture suggests a role in visual segmentation and spatial integration. The distribution of lateral interactions within striate cortex was visualized with optical recording, and their functional consequences were explored by using comparable stimuli in human psychophysical experiments and in recordings from alert monkeys. They may represent the substrate for perceptual phenomena such as illusory contours, surface fill-in, and contour saliency. The dynamic nature of receptive field properties and cortical architecture has been seen over time scales ranging from seconds to months. One can induce a remapping of the topography of visual cortex by making focal binocular retinal lesions. Shorter-term plasticity of cortical receptive fields was observed following brief periods of visual stimulation. The mechanisms involved entailed, for the short-term changes, altering the effectiveness of existing cortical connections, and for the long-term changes, sprouting of axon collaterals and synaptogenesis. The mutability of cortical function implies a continual process of calibration and normalization of the perception of visual attributes that is dependent on sensory experience throughout adulthood and might further represent the mechanism of perceptual learning.

Gene conversions and unequal crossovers between CYP21 (steroid 21-hydroxylase gene) and CYP21P involve different mechanisms.

Most cases of congenital adrenal hyperplasia, the inherited inability to synthesize cortisol, are caused by mutations in the steroid 21-hydroxylase gene (CYP21). Steroid 21-hydroxylase deficiency is unusual among genetic diseases in that approximately 95% of the mutant alleles have apparently been generated by recombination between a normally active gene (CYP21) and a linked pseudogene (CYP21P). Approximately 20% of mutant alleles carry DNA deletions of 30 kb that have presumably been generated by unequal meiotic crossing-over, whereas 75% carry one or more mutations in CYP21 that are normally found in the CYP21P pseudogene. These latter mutations are termed "gene conversions," although the mechanism by which they are generated is not well understood. To assess the frequency at which these different recombination events occur, we have used PCR to detect de novo deletions and gene conversions in matched sperm and peripheral blood leukocyte DNA samples from normal individuals. Deletions with breakpoints in a 100-bp region in intron 2 and exon 3 were detected in sperm DNA samples with frequencies of approximately 1 in 10(5)-10(6) genomes but were never detected in the matching leukocyte DNA. Gene conversions in the same region occur in approximately 1 in 10(3)-10(5) genomes in both sperm and leukocyte DNA. These data suggest that whereas deletions occur exclusively in meiosis, gene conversions occur during both meiosis and mitosis, or perhaps only during mitosis. Thus, gene conversions must occur by a mechanism distinct from unequal crossing-over.

Interleukin 1 beta suppresses transforming growth factor-induced inorganic pyrophosphate (PPi) production and expression of the PPi-generating enzyme PC-1 in human chondrocytes.

Articular cartilage chondrocytes have the unique ability to elaborate large amounts of extracellular pyrophosphate (PPi), and transforming growth factor beta (TGF beta) appears singular among cartilage regulatory factors in stimulating PPi production. TGF beta caused a time and dose-dependent increase in intracellular and extracellular PPi in human articular chondrocyte cultures. TGF beta and interleukin 1 beta (IL-1 beta) antagonistically regulate certain chondrocyte functions. IL-1 beta profoundly inhibited basal and TGF beta-induced PPi elaboration. To address mechanisms involved with the regulation of PPi synthesis by IL-1 beta and TGF beta, we analyzed the activity of the PPi-generating enzyme NTP pyrophosphohydrolase (NTPPPH) and the PPi-hydrolyzing enzyme alkaline phosphatase. Human chondrocyte NTPPPH activity was largely attributable to plasma cell membrane glycoprotein 1, PC-1. Furthermore, TGF beta induced comparable increases in the activity of extracellular PPi, intracellular PPi, and cellular NTPPPH and in the levels of PC-1 protein and mRNA in chondrocytes as well as a decrease in alkaline phosphatase. All of these TGF beta-induced responses were completely blocked by IL-1 beta. Thus, IL-1 beta may be an important regulator of mineralization in chondrocytes by inhibiting TGF beta-induced PPi production and PC-1 expression.

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.