Truncated RanGAP encoded by the Segregation Distorter locus of Drosophila.
Segregation Distorter (SD) in Drosophila melanogaster is a naturally occurring meiotic drive system in which the SD chromosome is transmitted from SD/SD+ males in vast excess over its homolog owing to the induced dysfunction of SD+-bearing spermatids. The Sd locus is the key distorting gene responsible for this phenotype. A genomic fragment from the Sd region conferred full distorting activity when introduced into the appropriate genetic background by germline transformation. The only functional product encoded by this fragment is a truncated version of the RanGAP nuclear transport protein. These results demonstrate that this mutant RanGAP is the functional Sd product. (+info)
Cloning and expression of a novel chicken sulfotransferase cDNA regulated by GH.
We have used mRNA differential display to compare gene expression in normal and GH receptor-deficient dwarf chickens, and report here the characterization of one differentially expressed gene, which shows significant sequence identity to the sulfotransferase gene family. Partial cDNA clones were isolated from a chicken liver cDNA library and an additional sequence was obtained using 5' rapid amplification of cDNA ends. A complete cDNA probe hybridizes to three transcripts (2.4, 2.0 and 1.45 kb) on Northern blots of chicken liver RNA, which differ in the length of the 3' untranslated region. All three transcripts are expressed at higher levels in normal vs dwarf chickens, as expected for a GH-regulated gene. The expression of this sulfotransferase mRNA was also detected in skeletal muscle, but not other tissues. The administration of GH to chickens increased the hepatic expression within 1 h, suggesting this sulfotransferase could be directly regulated by GH. Sulfotransferase activity, using estradiol or corticosterone as substrate, is detected in cells transfected with an expression vector containing the full-length cDNA. The sequence of this sulfotransferase does not show significant similarity with any subfamily of the sulfotransferases and its endogenous substrate is presently unknown. However, we speculate that GH activation of sulfotransferase activity could play a role in reducing concentrations of growth-antagonistic steroid hormones in GH target tissues. These results demonstrate the usefulness of differential display in this model system to identify genes that play a role in mediating GH action. (+info)
Induction of selected lipid metabolic enzymes and differentiation-linked structural proteins by air exposure in fetal rat skin explants.
The epidermal permeability barrier of premature infants matures rapidly following birth. Previous studies suggest that air exposure could contribute to this acceleration, because: (i) development of a structurally and functionally mature barrier accelerates when fetal rat skin explants are incubated at an air-medium interface, and (ii) occlusion with a water-impermeable membrane prevents this acceleration. To investigate further the effects of air exposure on epidermal barrier ontogenesis, we compared the activities of several key enzymes of lipid metabolism and gene expression of protein markers of epidermal differentiation in fetal rat skin explants grown immersed versus air exposed. The rate-limiting enzymes of cholesterol (HMG CoA reductase) and ceramide (serine palmitoyl transferase) synthesis were not affected. In contrast, the normal developmental increases in activities of glucosylceramide synthase and cholesterol sulfotransferase, responsible for the synthesis of glucosylceramides and cholesterol sulfate, respectively, were accelerated further by air exposure. Additionally, two enzymes required for the final stages of barrier maturation and essential for normal stratum corneum function, beta-glucocerebrosidase, which converts glucosylceramide to ceramide, and steroid sulfatase, which desulfates cholesterol sulfate, also increased with air exposure. Furthermore, filaggrin and loricrin mRNA levels, and filaggrin, loricrin, and involucrin protein levels all increased with air exposure. Finally, occlusion with a water-impermeable membrane prevented both the air-exposure-induced increase in lipid enzyme activity, and the expression of loricrin, filaggrin, and involucrin. Thus, air exposure stimulates selected lipid metabolic enzymes and the gene expression of key structural proteins in fetal epidermis, providing a biochemical basis for air-induced acceleration of permeability barrier maturation in premature infants. (+info)
Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1 entry.
Chemokine receptors and related seven-transmembrane-segment (7TMS) receptors serve as coreceptors for entry of human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV) into target cells. Each of these otherwise diverse coreceptors contains an N-terminal region that is acidic and tyrosine rich. Here, we show that the chemokine receptor CCR5, a principal HIV-1 coreceptor, is posttranslationally modified by O-linked glycosylation and by sulfation of its N-terminal tyrosines. Sulfated tyrosines contribute to the binding of CCR5 to MIP-1 alpha, MIP-1 beta, and HIV-1 gp120/CD4 complexes and to the ability of HIV-1 to enter cells expressing CCR5 and CD4. CXCR4, another important HIV-1 coreceptor, is also sulfated. Tyrosine sulfation may contribute to the natural function of many 7TMS receptors and may be a modification common to primate immunodeficiency virus coreceptors. (+info)
Molecular cloning and characterization of a human uronyl 2-sulfotransferase that sulfates iduronyl and glucuronyl residues in dermatan/chondroitin sulfate.
A partial-length human cDNA with a predicted amino acid sequence homologous to a previously described heparan sulfate iduronyl 2-sulfotransferase (Kobayashi, M., Habuchi, H., Yoneda, M., Habuchi, O., and Kimata, K. (1997) J. Biol. Chem. 272, 13980-13985) was obtained by searching the expressed sequence-tagged data bank. Northern blot analysis was performed using this homologous cDNA as a probe, which demonstrated ubiquitous expression of messages of 5.1 and 2.0 kilobases in a number of human tissues and in several human cancer cell lines. Since the human lymphoma Raji cell line had the highest level of expression, it was used to isolate a full-length cDNA clone. The full-length cDNA was found to contain an open reading frame that predicted a type II transmembrane protein composed of 406 amino acid residues. The cDNA in a baculovirus expression vector was expressed in Sf9 insect cells, and cell extracts were then incubated together with 3'-phosphoadenosine 5'-phospho[35S]sulfate and potential glycosaminoglycan acceptors. This demonstrated substantial sulfotransferase activity with dermatan sulfate, a small degree of activity with chondroitin sulfate, but no sulfotransferase activity with desulfated N-resulfated heparin. Analysis of [35S]sulfate-labeled disaccharide products of chondroitin ABC, chondroitin AC, and chondroitin B lyase treatment demonstrated that the enzyme only transferred sulfate to the 2-position of uronyl residues, which were preponderantly iduronyl residues in dermatan sulfate, but some lesser transfer to glucuronyl residues of chondroitin sulfate. (+info)
Crystal structure of the sulfotransferase domain of human heparan sulfate N-deacetylase/ N-sulfotransferase 1.
Heparan sulfate N-deacetylase/N-sulfotransferase (HSNST) catalyzes the first and obligatory step in the biosynthesis of heparan sulfates and heparin. The crystal structure of the sulfotransferase domain (NST1) of human HSNST-1 has been determined at 2.3-A resolution in a binary complex with 3'-phosphoadenosine 5'-phosphate (PAP). NST1 is approximately spherical with an open cleft, and consists of a single alpha/beta fold with a central five-stranded parallel beta-sheet and a three-stranded anti-parallel beta-sheet bearing an interstrand disulfide bond. The structural regions alpha1, alpha6, beta1, beta7, 5'-phosphosulfate binding loop (between beta1 and alpha1), and a random coil (between beta8 and alpha13) constitute the PAP binding site of NST1. The alpha6 and random coil (between beta2 and alpha2), which form an open cleft near the 5'-phosphate of the PAP molecule, may provide interactions for substrate binding. The conserved residue Lys-614 is in position to form a hydrogen bond with the bridge oxygen of the 5'-phosphate. (+info)
Reconstitution of functional L-selectin ligands on a cultured human endothelial cell line by cotransfection of alpha1-->3 fucosyltransferase VII and newly cloned GlcNAcbeta:6-sulfotransferase cDNA.
Recently, we proposed sialyl 6-sulfo Lewis X as a major carbohydrate-capping group of the L-selectin ligands on high endothelial venules in human lymph nodes. In this study we succeeded in reconstituting functional L-selectin ligands on a cultured human endothelial cell line, ECV304, by transfecting the alpha1-->3fucosyltranseferase VII (Fuc-T VII) and newly cloned GlcNAcbeta:6-sulfotransferase (6-Sul-T) cDNAs. The ECV304 cells transfected with Fuc-T VII cDNA expressed conventional sialyl Lewis X detected with specific antibodies including 2H5, whereas the cells transfected with 6-Sul-T cDNA expressed sialyl 6-sulfo lactosamine as well as MECA-79-defined carbohydrate determinants, but these singly transfected cells failed to express sialyl 6-sulfo Lewis X, as detected with the antisialyl 6-sulfo Lewis X mAb G152. Sialyl 6-sulfo Lewis X appeared only on the cells that were cotransfected with both 6-Sul-T and Fuc-T VII cDNAs. Significant adhesion of L-selectin-expressing cells was seen only to the doubly transfected ECV304 cells and was inhibited by G152. No adhesion was observed to the cells transfected either with 6-Sul-T or with Fuc-T VII cDNA alone. The mRNAs of the two enzymes were expressed or were inducible upon interleukin 1 stimulation in human endothelial cells. These results indicate that a set of carbohydrate determinants synthesized by the concerted action of the two enzymes, as typically represented by the sialyl 6-sulfo Lewis X-capping group, serves as an essential component of the ligand for L-selectin and that the reagents 2H5 and MECA-79, utilized in earlier studies to detect L-selectin ligand on high endothelial venules, recognize two different aspects of the same set of synthetic products. (+info)
Structure and function of a cysBJIH gene cluster in the purple sulphur bacterium Thiocapsa roseopersicina.
A gene cluster containing homologues of the genes cysB, cysJI and cysH was found in the genome of the sulphur-oxidizing purple bacterium Thiocapsa roseopersicina. The nucleotide sequence indicated four open reading frames encoding homologues of 3'-phosphoadenylylsulphate (PAPS) reductase (CysH), sulphite reductase flavoprotein (CysJ) and haem protein (CysI) subunits, and a transcriptional regulator (CysB). Genes cysJIH are separated by a short cis-active intergenic region from cysB which is transcribed divergently. cysB encodes a polypeptide of 35.9 kDa consisting of 323 amino acid residues with 40% identity to the CysB regulator from enterobacteria. cysH encodes a protein with 239 amino acid residues and a calculated mass of 27.7 kDa; cysJ encodes a protein with 522 amino acid residues and a mass of 57.8 kDa; and cysI encodes a protein with 559 amino acid residues and a mass of 62.3 kDa. The cysJIH gene products have been expressed and used for complementation of cys mutants from Escherichia coli Biochemical analysis. The gene product CysH is a thioredoxin-dependent PAPS reductase (EC 18.104.22.168). It was repressed under photoautotrophic growth using hydrogen sulphide as electron donor and derepressed under conditions of sulphate deficiency. Products of the cysJI genes were identified as the two subunits of NADPH-sulphite reductase (EC 22.214.171.124). cysJ encoded the flavoprotein, with > or = 39% identity to the protein from E. coli, and cysI encoded the haem protein, with > or = 53% identity. A cysI clone was used to complement the corresponding mutant from E. coli and to express enzymically active methylviologen-sulphite reductase. (+info)