Cloning and functional characterization of a second urea transporter from the kidney of the Atlantic stingray, Dasyatis sabina

2006 ◽  
Vol 291 (3) ◽  
pp. R844-R853 ◽  
Author(s):  
Michael G. Janech ◽  
Wayne R. Fitzgibbon ◽  
Mark W. Nowak ◽  
Donald H. Miller ◽  
Richard V. Paul ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Single Gene ◽  
Functional Characterization ◽  
Amino Acid Residues ◽  
Urea Transporter ◽  
Coding Region ◽  
Acid Protein ◽  
Atlantic Stingray ◽  
Rapid Amplification

The cloning of cDNAs encoding facilitated urea transporters (UTs) from the kidneys of the elasmobranchs indicates that in these fish renal urea reabsorption occurs, at least in part, by passive processes. The previously described elasmobranch urea transporter clones from shark (shUT) and stingray (strUT-1) differ from each other primarily because of the COOH-terminus of the predicted strUT-1 translation product being extended by 51-amino acid residues compared with shUT. Previously, we noted multiple UT transcripts were present in stingray kidney. We hypothesized that a COOH terminally abbreviated UT isoform, homologous to shUT, would also be present in stingray kidney. Therefore, we used 5′/3′ rapid amplification of cDNA ends to identify a 3′UTR-variant (strUT-1a) of the cDNA that encodes (strUT-1), as well as three, 3′UTR-variant cDNAs (strUT-2a,b,c) that encode a second phloretin-sensitive, urea transporter (strUT-2). The 5′UTR and the first 1,132 nucleotides of the predicted coding region of the strUT-2 cDNAs are identical to the strUT-1 cDNAs. The remainder of the coding region contains only five novel nucleotides. The strUT-2 cDNAs putatively encode a 379-amino acid protein, the first 377 amino acids identical to strUT-1 plus 2 additional amino acids. We conclude that 1) a second UT isoform is expressed in the Atlantic stingray and that this isoform is similar in size to the UT previously cloned from the kidney of the dogfish shark, and 2) at least five transcripts encoding the 2 stingray UTs are derived from a single gene product through alternative splicing and polyadenylation.

scholarly journals Functional domains of a negative regulatory protein, GAL80, of Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (7) ◽  
pp. 3009-3017 ◽  
Author(s):  
Y Nogi ◽  
T Fukasawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Regulatory Protein ◽  
Subcellular Fractionation ◽  
Functional Domains ◽  
Amino Acid Residues ◽  
Coding Region ◽  
Interaction Domain ◽  
Repression Domain

To study the functional domains of a transcriptional repressor encoded by the GAL80 gene of Saccharomyces cerevisiae, we constructed various deletion and insertion mutations in the GAL80 coding region and determined the ability of these mutations to repress synthesis of galactose-metabolizing enzymes as well as the capacity of the mutant proteins to respond to the inducer. Two regions, from amino acids 1 to 321 and from amino acids 341 to 423, in the total sequence of 435 amino acids were required for repression. The internal region from amino acids 321 to 340 played a role in the response to the inducer. The 12 amino acids at the carboxy terminus were dispensable for normal functioning of the GAL80 protein. Using indirect immunofluorescence and subcellular fractionation techniques, we also found that two distinct regions (amino acids 1 to 109 and 342 to 405) within the putative repression domain were capable of directing cytoplasmically synthesized Escherichia coli beta-galactosidase to the yeast nucleus. In addition, three gal80 mutations were mapped at amino acid residues 183, 298, and 310 in the domain required for repression. On the basis of these results, we suggest that the GAL80 protein consists of a repression domain located in two separate regions (amino acid residues 1 to 321 and 341 to 423) that are interrupted by an inducer interaction domain (residues 322 to 340) and two nuclear localization domains (1 to 109 and 342 to 405) that overlap the repression domains.

scholarly journals A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily

2013 ◽  
Vol 289 (3) ◽  
pp. 1377-1387 ◽  
Author(s):  
Jagdeep Kaur ◽  
Elena Olkhova ◽  
Viveka Nand Malviya ◽  
Ernst Grell ◽  
Hartmut Michel
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ligand Binding ◽  
Isothermal Titration Calorimetry ◽  
Functional Characterization ◽  
Homology Model ◽  
Docking Studies ◽  
Site Directed Mutagenesis ◽  
Titration Calorimetry ◽  
Amino Acid Residues

Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed.

scholarly journals Molecular Cloning and Bioinformatics Analysis of DQA Gene from Mink (Neovison vison)

2019 ◽  
Vol 20 (5) ◽  
pp. 1037
Author(s):  
Zhaobin Fan ◽  
Houfeng Zhang ◽  
Min Rong ◽  
Dongmei Meng ◽  
Zhenxing Yu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mechanisms ◽  
Signal Sequence ◽  
Amino Acid Sequences ◽  
Neovison Vison ◽  
Random Coil ◽  
Amino Acid Residues ◽  
Coding Region ◽  
Full Length Sequence

In the present study, we cloned, sequenced, and explored the structural and functional characteristics of the major histocompatibility complex (MHC)-DQA gene from mink (Neovison vison) for the first time. The full-length sequence of DQA gene was 1147-bp-long, contained a coding region of 768-bp, which was predicted to encoding 255 amino acid residues. The comparison between DQA from mink (Neovison vison) and other MHC-DQA molecules from different animal species showed that nucleotide and encoded amino acid sequences of the mink DQA gene exhibited high similarity with the ferret (Mustela pulourius furo). Phylogenetic analysis revealed that mink (Neovison vison) DQA is grouped with that of ferret (Mustela pulourius furo). The cloned sequence contained a 23-amino acid NH2-terminal signal sequence with the signal peptide cutting site located in amino acids 23–24, and had three Asn-Xaa-Ser/Thr sequons. Three cysteine residues were also identified (Cys-85, Cys-121, and Cys-138). The 218 to 240 amino acids were predicted to be the transmembrane domains. The prediction of the secondary structure revealed three α-helixes and fourteen β-sheets in Neovison vison DQA protein, while random coil was a major pattern. In this study, the whole CDS sequence of Neovison vison DQA gene was successfully cloned, which was valuable for exploring the function and antiviral molecular mechanisms underlying the molecule. The findings of the present study have laid the foundation for the disease resistance and breeding of mink.

Molecular and functional characterization of a urea transporter from the kidney of the Atlantic stingray

2003 ◽  
Vol 284 (5) ◽  
pp. F996-F1005 ◽  
Author(s):  
Michael G. Janech ◽  
Wayne R. Fitzgibbon ◽  
Ruihua Chen ◽  
Mark W. Nowak ◽  
Donald H. Miller ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Fold Increase ◽  
Urea Transporter ◽  
Acid Protein ◽  
Urea Uptake ◽  
Atlantic Stingray ◽  
Urea Reabsorption ◽  
Additional Sequence ◽  
Elasmobranch Fishes ◽  
High Degree

In general, marine elasmobranch fishes (sharks, skates, and rays) maintain body fluid osmolality above seawater, principally by retaining large amounts of urea. Maintenance of the high urea concentration is due in large part to efficient renal urea reabsorption. Regulation of renal urea reabsorption also appears to play a role in maintenance of fluid homeostasis of elasmobranchs that move between habitats of different salinities. We identified and cloned a novel 2.7-kb cDNA from the kidney of the euryhaline Atlantic stingray Dasyatis sabina (GenBank accession no. AF443781 ). This cDNA putatively encoded a 431-amino acid protein (strUT-1) that had a high degree of sequence identity (71%) to the shark kidney facilitated urea transporter (UT). However, the predicted COOH-terminal region of strUT-1 appears to contain an additional sequence that is unique among cloned renal UTs. Injection of strUT-1 cRNA into Xenopusoocytes induced a 33-fold increase in [14C]urea uptake that was inhibited by phloretin. Four mRNA bands were detected in kidney by Northern blot: a transcript at 2.8 kb corresponding to the expected size of strUT-1 mRNA and bands at 3.8, 4.5, and 5.5 kb. Identification of a facilitated UT in the kidney of the Atlantic stingray provides further support for the proposal that passive mechanisms contribute to urea reabsorption by elasmobranch kidney.

Functional domains of a negative regulatory protein, GAL80, of Saccharomyces cerevisiae

1989 ◽  
Vol 9 (7) ◽  
pp. 3009-3017
Author(s):  
Y Nogi ◽  
T Fukasawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Regulatory Protein ◽  
Subcellular Fractionation ◽  
Functional Domains ◽  
Amino Acid Residues ◽  
Coding Region ◽  
Interaction Domain ◽  
Repression Domain

To study the functional domains of a transcriptional repressor encoded by the GAL80 gene of Saccharomyces cerevisiae, we constructed various deletion and insertion mutations in the GAL80 coding region and determined the ability of these mutations to repress synthesis of galactose-metabolizing enzymes as well as the capacity of the mutant proteins to respond to the inducer. Two regions, from amino acids 1 to 321 and from amino acids 341 to 423, in the total sequence of 435 amino acids were required for repression. The internal region from amino acids 321 to 340 played a role in the response to the inducer. The 12 amino acids at the carboxy terminus were dispensable for normal functioning of the GAL80 protein. Using indirect immunofluorescence and subcellular fractionation techniques, we also found that two distinct regions (amino acids 1 to 109 and 342 to 405) within the putative repression domain were capable of directing cytoplasmically synthesized Escherichia coli beta-galactosidase to the yeast nucleus. In addition, three gal80 mutations were mapped at amino acid residues 183, 298, and 310 in the domain required for repression. On the basis of these results, we suggest that the GAL80 protein consists of a repression domain located in two separate regions (amino acid residues 1 to 321 and 341 to 423) that are interrupted by an inducer interaction domain (residues 322 to 340) and two nuclear localization domains (1 to 109 and 342 to 405) that overlap the repression domains.

Vector expression of adenovirus type 5 E1a proteins: evidence for E1a autoregulation

1985 ◽  
Vol 5 (10) ◽  
pp. 2684-2696
Author(s):  
D H Smith ◽  
D M Kegler ◽  
E B Ziff
Keyword(s):  
Amino Acid ◽  
Simian Virus 40 ◽  
Adenovirus Type ◽  
Simian Virus ◽  
Mutant Form ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Acid Protein ◽  
Dna Replication Origin ◽  
E1a Protein

We transiently expressed adenovirus type C E1a proteins in wild-type or mutant form from plasmid vectors which have different combinations of E1a and simian virus 40 enhancer elements and which contain the DNA replication origin of SV40 and can replicate in COS 7 cells. We measured the levels of E1a mRNA encoded by the vectors and the transition regulation properties of the protein products. Three vectors encoded equivalent levels of E1a mRNA in COS 7 cells: (i) a plasmid encoding the wt 289-amino acid E1a protein (this complemented the E1a deletion mutant dl312 for early region E2a expression under both replicative and nonreplicative conditions); (ii) a vector for the wt 243-amino acid E1a protein (this complemented dl312 weakly and only under conditions of high multiplicities of dl312); (iii) a mutant, pSVXL105, in which amino acid residues-38 through 44 of the 289-amino acid E1a protein (which includes two highly conserved residues) are replaced by 3 novel amino acids (this also complemented dl312 efficiently). A fourth vector, mutant pSVXL3 with which linker substitution shifts the reading frame to encode a truncated 70-amino acid fragment from the amino terminus of the 289-amino acid protein, was unable to complement dl312. Surprisingly, pSVXL3 overexpressed E1a mRNA approximately 30-fold in COS 7 cells in comparison with the other vectors. The pSVXL3 overexpression could be reversed by cotransfection with a wt E1a vector. We suggest that wt E1a proteins regulate the levels of their own mRNAs through the recently described transcription repression functions of the 289- and 243-amino acid E1a protein products and that pSVXL3 fails to autoregulate negatively.

Cloning and functional characterization of an uncoupling protein homolog in hummingbirds

2001 ◽  
Vol 5 (3) ◽  
pp. 137-145 ◽  
Author(s):  
CLAUDIA R. VIANNA ◽  
THILO HAGEN ◽  
CHEN-YU ZHANG ◽  
ERIC BACHMAN ◽  
OLIVIER BOSS ◽  
...  
Keyword(s):  
Uncoupling Protein ◽  
Expression System ◽  
Functional Characterization ◽  
Pectoral Muscle ◽  
Mitochondrial Fraction ◽  
Mrna Levels ◽  
Coding Region ◽  
Reading Frame ◽  
Rapid Amplification

The cDNA of an uncoupling protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird uncoupling protein (HmUCP) cDNA was amplified from pectoral muscle (flight muscle) using RT-PCR and primers for conserved domains of various known UCP homologs. The rapid amplification of cDNA ends (RACE) method was used to complete the cloning of the 5′ and 3′ ends of the open reading frame. The HmUCP coding region contains 915 nucleotides, and the deduced protein sequence consists of 304 amino acids, being ∼72, 70, and 55% identical to human UCP3, UCP2, and UCP1, respectively. The uncoupling activity of this novel protein was characterized in yeast. In this expression system, the 12CA5-tagged HmUCP fusion protein was detected by Western blot in the enriched mitochondrial fraction. Similarly to rat UCP1, HmUCP decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3′,3-dihexyloxacarbocyanine iodide. The HmUCP mRNA is primarily expressed in skeletal muscle, but high levels can also be detected in heart and liver, as assessed by Northern blot analysis. Lowering the room’s temperature to 12–14°C triggered the cycle torpor/rewarming, typical of hummingbirds. Both in the pectoral muscle and heart, HmUCP mRNA levels were 1.5- to 3.4-fold higher during torpor. In conclusion, this is the first report of an UCP homolog in birds. The data indicate that HmUCP has the potential to function as an UCP and could play a thermogenic role during rewarming.

The C-terminal sequence of amino acid residues of κ-casein isolated from buffalo's milk

1967 ◽  
Vol 34 (1) ◽  
pp. 85-88 ◽  
Author(s):  
M. H. Abd El-Salam ◽  
W. Manson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Peptide Chain ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Carboxypeptidase A ◽  
Phosphorous Content ◽  
Single Peptide

SummaryWhen κ-casein from buffalo's milk was treated with carboxypeptidase A (EC 3. 4. 2. 1),4 amino acids, valine, threonine, serine and alanine were released from the protein in a manner consistent with the view that they originate in the C-terminal sequence of a single peptide chain. The amounts produced suggest a minimum molecular weight for buffalo κ-casein of approximately 17000, in agreement with the value calculated from the phosphorous content on the basis of the presence of 2 phosphorus atoms/molecule. A comparison is made with the C-terminal sequence reported for bovine κ-casein.

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