Structure and evolution of a pair-rule interaction element: runt regulatory sequences in D. melanogaster and D. virilis.
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Pair-rule genes serve two important functions during Drosophila development: they first initiate periodic patterns, and subsequently interact with each other to refine these patterns to the precision required for definition of segmental compartments. Previously, we described a pair-rule input region of the runt gene. Here we further characterize this region through the use of reporter gene constructs and by comparison with corresponding sequences from Drosophila virilis. We find that many but not all regulatory properties of this '7-stripe region' are functionally conserved. Moreover, the similarity between these homologous sequences is surprisingly low. When compared to similar data for gap gene input element, our data suggest that pair-rule target sequences are less constrained during evolution, and that functional elements mediating pair-rule interactions can be dispersed over many kilobases. (+info)
A cluster of oppositely imprinted transcripts at the Gnas locus in the distal imprinting region of mouse chromosome 2.
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Imprinted genes tend to occur in clusters. We have identified a cluster in distal mouse chromosome (Chr) 2, known from early genetic studies to contain both maternally and paternally imprinted, but unspecified, genes. Subsequently, one was identified as Gnas, which encodes a G protein alpha subunit, and there is clinical and biochemical evidence that the human homologue GNAS1, mutated in patients with Albright hereditary osteodystrophy, is also imprinted. We have used representational difference analysis, based on parent-of-origin methylation differences, to isolate candidate imprinted genes in distal Chr 2 and found two oppositely imprinted genes, Gnasxl and Nesp. Gnasxl determines a variant G protein alpha subunit associated with the trans-Golgi network and Nesp encodes a secreted protein of neuroendocrine tissues. Gnasxl is maternally methylated in genomic DNA and encodes a paternal-specific transcript, whereas Nesp is paternally methylated with maternal-specific expression. Their reciprocal imprinting may offer insight into the distal Chr 2 imprinting phenotypes. Remarkably, Gnasxl, Nesp, and Gnas are all part of the same transcription unit; transcripts for Gnasxl and Nesp are alternatively spliced onto exon 2 of Gnas. This demonstrates an imprinting mechanism in which two oppositely imprinted genes share the same downstream exons. (+info)
Cloning of the mouse phospholipid hydroperoxide glutathione peroxidase gene.
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15-Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases (PH-GPx) are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes appears to be important for the cellular peroxide tone regulating the expression of redox sensitive genes. In contrast to lipoxygenases the molecular biology of PH-GPx is less well investigated. In this study we cloned the PH-GPx cDNA from a mouse fibroblast cDNA library and the PH-GPx gene from a mouse genomic library. The gene spans approximately 4 kb which includes 1 kb of 5'-flanking region and consists of seven exons and six introns. The immediate promoter region does not contain a TATA box but there are binding sites for several transcription factors which also occur in the porcine gene. Our investigations provide useful tools for future targeted gene disruption studies. (+info)
TM7XN1, a novel human EGF-TM7-like cDNA, detected with mRNA differential display using human melanoma cell lines with different metastatic potential.
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We have identified a novel 3845 bp cDNA differentially expressed in a human melanoma metastasis model. Northern blot analysis showed expression in the poorly and intermediately metastasizing cell lines and a marked downregulation in the highly metastatic cell lines. Using RT-PCR expression was also seen in several other tumor cell lines and normal cell types of human origin. cDNA sequence analysis revealed an ORF of 687 amino acids containing seven putative transmembrane domains C-terminally and a long N-terminus. The gene was mapped to 16q13. Highest homology was observed with members of the EGF-TM7 subfamily of the secretin/calcitonin receptor family. We propose the delineation of a subfamily of TM7 proteins, LN-TM7, containing seven transmembrane proteins with a long N-terminal extracellular part. (+info)
Cloning and functional characterization of a new multispecific organic anion transporter, OAT-K2, in rat kidney.
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We have isolated a cDNA coding a new organic anion transporter, OAT-K2, expressed specifically in rat kidney. The OAT-K2 cDNA had an open reading frame encoding a 498-amino acid protein (calculated molecular mass of 55 kDa) that shows 91% identity with the rat kidney-specific organic anion transporter, OAT-K1. Reverse transcription-coupled polymerase chain reaction analyses revealed that the OAT-K2 mRNA was expressed predominantly in the proximal convoluted tubules, proximal straight tubules, and cortical collecting ducts. When expressed in Xenopus oocytes, OAT-K2 stimulated the uptake of hydrophobic organic anions, such as taurocholate, methotrexate, folate, and prostaglandin E2, although its homolog OAT-K1 transported methotrexate and folate, but not taurocholate and prostaglandin E2. In MDCK cells stably transfected with the OAT-K1 and OAT-K2 cDNAs, each transporter was localized functionally to the apical membranes and showed transport activity similar to that in the oocyte. Moreover, the efflux of preloaded taurocholate was also enhanced across the apical membrane in OAT-K2 transfectant. The taurocholate transport by OAT-K2-expressing cells showed saturability (Km = 10.3 microM). Several organic anions, bile acids, cardiac glycosides, and steroids had potent inhibitory effects on the OAT-K2-mediated taurocholate transport in the transfectant. These findings suggest that the OAT-K2 participates in epithelial transport of hydrophobic anionic compounds in the kidney. (+info)
Halobacterial rhodopsins.
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Following the discovery of the bacteriorhodopsin proton pump in Halobacterium halobium (salinarum), not only the halorhodopsin halide pump and two photosensor rhodopsins (sensory rhodopsin and phoborhodopsin) in the same species, but also homologs of these four rhodopsins in strains of other genera of Halobacteriaceae have been reported. Twenty-eight full (and partial) sequences of the genomic DNA of these rhodopsins have been analyzed. The deduced amino acid sequences have led to new strategies and tactics for understanding bacterial rhodopsins on a comparative basis, as summarized briefly in this article. The data discussed include (i) alignment of the sequences to qualify/characterize the conserved residues; (ii) assignment of residues that cause differences in function(s)/properties; and (iii) phylogeny of the halobacterial rhodopsins to suggest their evolutionary paths. The four kinds of rhodopsin in each strain are assumed, on the basis of their genera-specific distributions, to have arisen by at least two gene-duplication processes during evolution prior to generic speciation. The first duplication of the rhodopsin ancestor gene yielded two genes, each of which was duplicated again to give four genes in the ancestor halobacterium. The bacterium carrying four rhodopsin genes, after accumulating mutations, became ready for generic speciation and the delivery of four rhodopsins to each species. The original rhodopsin ancestor is speculated to be closest to the proton pump (bacteriorhodopsin). (+info)
Characterization of two new channel protein genes in Arabidopsis.
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Aquaporins, small channel proteins, found in a variety of organisms are members of the major intrinsic protein (MIP) superfamily and have been shown to facilitate water transport when expressed in Xenopus oocytes. We isolated two Arabidopsis cDNAs, SIMIP and SITIP, that encode protein homologues of the MIP superfamily. SIMIP exhibits a high degree of sequence homology to PIP3 and MIP1, and thus may belong to the plasmamembrane intrinsic protein (PIP) subfamily, whereas salt-stress inducible tonoplast intrinsic protein (SITIP) is highly homologous to VM23 and gamma-TIP, and therefore may belong to the TIP subfamily. Expression studies revealed that the two genes showed a different expression pattern. The SIMIP gene was expressed in a tissue-specific manner, for example, its highest transcript level is found in flowers, relatively low levels in siliques, and very low level in leaves and roots. In contrast, SITIP was expressed in nearly equal amounts in all the tissues we examined. Also, the expression of SIMIP and SITIP showed a temporal regulation pattern. For example, the highest expression level was at 1 week after germination. In addition, the transcript levels of SIMIP and SMTIP were increased upon NaCl and ABA treatments. The biological function of the 2 genes were investigated using two NaCl stress-sensitive yeast mutant strains. The mutant yeast cells expressing these 2 genes were more resistant to high NaCl conditions. The results suggest that the proteins encoded by these genes may be involved in the osmoregulation in plants under high osmotic stress such as under a high salt condition. (+info)
Cloning, sequence analyses, expression, and distribution of ampC-ampR from Morganella morganii clinical isolates.
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Shotgun cloning experiments with restriction enzyme-digested genomic DNA from Morganella morganii 1, which expresses high levels of cephalosporinase, into the pBKCMV cloning vector gave a recombinant plasmid, pPON-1, which encoded four entire genes: ampC, ampR, an hybF family gene, and orf-1 of unknown function. The deduced AmpC beta-lactamase of pI 7.6 shared structural and functional homologies with AmpC from Citrobacter freundii, Escherichia coli, Yersinia enterocolitica, Enterobacter cloacae, and Serratia marcescens. The overlapping promoter organization of ampC and ampR, although much shorter in M. morganii than in the other enterobacterial species, suggested similar AmpR regulatory properties. The MICs of beta-lactams for E. coli MC4100 (ampC mutant) harboring recombinant plasmid pACYC184 containing either ampC and ampR (pAC-1) or ampC (pAC-2) and induction experiments showed that the ampC gene of M. morganii 1 was repressed in the presence of ampR and was activated when a beta-lactam inducer was added. Moreover, transformation of M. morganii 1 or of E. coli JRG582 (delta ampDE) harboring ampC and ampR with a recombinant plasmid containing ampD from E. cloacae resulted in a decrease in the beta-lactam MICs and an inducible phenotype for M. morganii 1, thus underlining the role of an AmpD-like protein in the regulation of the M. morganii cephalosporinase. Fifteen other M. morganii clinical isolates with phenotypes of either low-level inducible cephalosporinase expression or high-level constitutive cephalosporinase expression harbored the same ampC-ampR organization, with the hybF and orf-1 genes surrounding them; the organization of these genes thus differed from those of ampC-ampR genes in C. freundii and E. cloacae, which are located downstream from the fumarate operon. Finally, an identical AmpC beta-lactamase (DHA-1) was recently identified as being plasmid encoded in Salmonella enteritidis, and this is confirmatory evidence of a chromosomal origin of the plasmid-mediated cephalosporinases. (+info)