The mouse sulfate anion transporter gene Sat1 (Slc26a1): Cloning, tissue distribution, gene structure, functional characterization, and transcriptional regulation by thyroid hormone

Sulfate (SO42-) is required for bone/cartilage formation and cellular metabolism. sat-1 is a SO42- anion transporter expressed on basolateral membranes of renal proximal tubules, and is suggested to play an important role in maintaining SO42- homeostasis. As a first step towards studying its tissue-specific expression, hormonal regulation, and in preparation for the generation of knockout mice, we have cloned and characterized the mouse sat-1 cDNA (msat-1), gene (sat1; Slc26a1) and promoter region. msat-1 encodes a 704 amino acid protein (75.4 kDa) with 12 putative transmembrane domains that induce SO42- (also oxalate and chloride) transport in Xenopus oocytes. msat-1 mRNA was expressed in kidney, liver, cecum, calvaria, brain, heart, and skeletal muscle. Two distinct transcripts were expressed in kidney and liver due to alternative utilization of the first intron, corresponding to an internal portion of the 5'-untranslated region. The Sa1 gene (similar to6 kb) consists of 4 exons. Its promoter is similar to52% G+C rich and contains a number of well-characterized cis-acting elements, including sequences resembling hormone responsive elements T3REs and VDREs. We demonstrate that Sat1 promoter driven basal transcription in OK cells was stimulated by tri-iodothyronine. Site-directed mutagenesis identified an imperfect T3RE at -454-bp in the Sat1 promoter to be responsible for this activity. This study represents the first characterization of the structure and regulation of the Sat1 gene encoding a SO42-/chloride/oxalate anion transporter.

Molecular tools for studying puberty in the grey mullet, Mugil cephalus: cloning of cDNAs that regulate reproductive function

The cDNAs coding for the brain GnRHs (AY373449-51), pituitary GH, SL and PRL, and liver IGFs (AY427954-5) were isolated. Partial cDNA sequences of the brain (Cyp19b) and gonadal (Cyp19a) aromatases have also been obtained. These tools would be utilized to study the endocrine regulation of puberty in the grey mullet.

Identification and cloning of the gene encoding BmpC: an outer-membrane lipoprotein associated with Brachyspira pilosicoli membrane vesicles

The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the B. pilosicoli outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from B. pilosicoli strain 95-1000 cells by osmotic lysis in dH(2)O followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; p = 1.18 g CM-3) and a low-density fraction (LDMV; rho=1.12 g cm(-3)). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa B. pilosicoli OM protein) and was used as a source of antigens to produce mAbs. Five B. pilosicoli-specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the B. pilosicoli OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a B. pilosicoli 95-1000 genome library with the mAb and was expressed in Escherichia coli. Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of E. coli strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of B. pilosicoli isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within B. pilosicoli.

Optimal cloning for finite distributions of coherent states

We derive optimal cloning limits for finite Gaussian distributions of coherent states and describe techniques for achieving them. We discuss the relation of these limits to state estimation and the no-cloning limit in teleportation. A qualitatively different cloning limit is derived for a single-quadrature Gaussian quantum cloner.

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.

Pollination of Australian Macrozamia cycads (Zamiaceae): Effectiveness and behavior of specialist vectors in a dependent mutualism

Complementary field and laboratory tests confirmed and quantified the pollination abilities of Tranes sp. weevils and Cycadothrips chadwicki thrips, specialist insects of their respective cycad hosts, Macrozamia machinii and M. lucida. No agamospermous seeds were produced when both wind and insects were excluded from female cones; and the exclusion of wind-vectored pollen alone did not eliminate seed set, because insects were able to reach the cone. Based on enclosure pollination tests, each weevil pollinates an average 26.2 ovules per cone and each thrips 2.4 ovules per cone. These pollinators visited similar numbers of ovules per cone in fluorescent dye tests that traced insect movement through cones. Fluorescent dye granules deposited by Cycadothrips were concentrated around the micropyle of each visited ovule, the site of pollen droplet release, where pollen must be deposited to achieve pollination. In contrast, Tranes weevils left dye scattered on different areas of each visited ovule, indicating that chance plays a greater role in this system. Each weevil and 25 thrips delivered 6.2 and 5.2 pollen grains, respectively, on average, to each visited ovule per cone, based on examination of dissected pollen canals. In sum, the pollination potential of 25 Cycadothrips approximates that of one Tranes weevil.

Characterization of the highly efficient sucrose isomerase from Pantoea dispersa UQ68J and cloning of the sucrose isomerase gene

Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35 degrees C, and it produced a high ratio of isomaltulose to trehalulose (> 22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50 degrees C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35 degrees C and produced a lower ratio of isomaltulose to trehalulose (< 8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the K-m was 39.9 mM, the V-max was 638 U mg(-1), and the K-cat/K-m was 1.79 x 104 M-1 s(-1), compared to a K-m of 76.0 mM, a V-max. of 423 U mg(-1), and a K-cat/K-m of 0.62 x 104 M-1 s(-1) for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose.

Human SULT1A genes: Cloning and activity assays of the SULT1A promoters

The three human SULT1A sulfotransferase enzymes are closely related in amino acid sequence (>90%), yet differ in their substrate preference and tissue distribution. SULT1A1 has a broad tissue distribution and metabolizes a range of xenobiotics as well as endogenous substrates such as estrogens and iodothyronines. While the localization of SULT1A2 is poorly understood, it has been shown to metabolize a number of aromatic amines. SULT1A3 is the major catecholamine sulfonating form, which is consistent with it being expressed principally in the gastrointestinal tract. SULT1A proteins are encoded by three separate genes, located in close proximity to each other on chromosome 16. The presence of differential 5′-untranslated regions identified upon cloning of the SULT1A cDNAs suggested the utilization of differential transcriptional start sites and/or differential splicing. This chapter describes the methods utilized by our laboratory to clone and assay the activity of the promoters flanking these different untranslated regions found on SULT1A genes. These techniques will assist investigators in further elucidating the differential mechanisms that control regulation of the human SULT1A genes. They will also help reveal how different cellular environments and polymorphisms affect the activity of SULT1A gene promoters.

Cloning and sequence analysis of pituitary prolactin cDNA from the northern brown bandicoot (Isoodon macrourus)

The nuclectide sequence for pituitary prolactin cDNA from the marsupial bandicoot (Isoodon macrourus) was determined by reverse transcription-polymerase chain reaction and 5'/3' rapid amplification of cDNA ends. The deduced amino acid sequence showed high sequence identity with brushtail possum prolactin (95%) and all of the expected structural features of a quadruped prolactin. A prolactin gene tree was constructed and rates of evolution calculated for bandicoot, possum, opossum and several mammalian and non-mammalian prolactins. Bootstrap analysis provided strong support for marsupials as a sister group with eutherian mammals and weak support for opossum and bandicoot as an independent grouping from the brushtail possum. The rates of molecular evolution for marsupial prolactins were comparable to the slow rate seen in the majority of quadruped prolactins that have been sequenced. (c) 2005 Elsevier Inc. All rights reserved.

Mapping the I-3 gene for resistance to Fusarium wilt in tomato: application of an I-3 marker in tomato improvement and progress towards the cloning of I-3

Fusarium wilt of tomato, caused by the fungal pathogen, Fusarium oxysporum f. sp. lycopersici (Fol), is an economically damaging disease that results in huge losses in Australia and other countries worldwide. The I-3 gene, which confers resistance to Fol race 3, has been described in wild tomato, Lycopersicon pennellii, accessions LA716 and PI414773. We are pursuing the isolation of I-3 from LA716 by map-based cloning. We have constructed a high-resolution map of the I-3 region and have identified markers closely flanking I-3 as well as markers co-segregating with I-3. In addition, construction of a physical map based on these markers has been initiated. This review describes the context of our research and our progress towards isolating the I-3 gene. It also describes some important practical outcomes of our work, including the development and use of a PCR-based marker for marker-assisted selection for I-3, and the finding that the I-3 gene from LA716 is different to that from PI1414773, which we have now designated I-7. Tomato varieties combining I-3 and I-7 have been developed and are currently being introduced into commercial production to further safeguard tomato crops against Fusarium wilt.

Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins

Failure to express soluble proteins in bacteria is mainly attributed to the properties of the target protein itself, as well as the choice of the vector, the purification tag and the linker between the tag and protein, and codon usage. The expression of proteins with fusion tags to facilitate subsequent purification steps is a widely used procedure in the production of recombinant proteins. However, the additional residues can affect the properties of the protein; therefore, it is often desirable to remove the tag after purification. This is usually done by engineering a cleavage site between the tag and the encoded protein that is recognised by a site-specific protease, such as the one from tobacco etch virus (TEV). In this study, we investigated the effect of four different tags on the bacterial expression and solubility of nine mouse proteins. Two of the four engineered constructs contained hexahistidine tags with either a long or short linker. The other two constructs contained a TEV cleavage site engineered into the linker region. Our data show that inclusion of the TEV recognition site directly downstream of the recombination site of the Invitrogen Gateway vector resulted, in a loss of solubility of the nine mouse proteins. Our work suggests that one needs to be very careful when making modifications to expression vectors and combining different affinity and fusion tags and cleavage sites: (c) 2006 Elsevier Inc. All rights reserved.