NHE3 in an ancestral vertebrate: primary sequence, distribution, localization, and function in gills

2005 ◽  
Vol 289 (5) ◽  
pp. R1520-R1534 ◽  
Author(s):  
Keith P. Choe ◽  
Akira Kato ◽  
Shigehisa Hirose ◽  
Consuelo Plata ◽  
Aleksandra Sindić ◽  
...  
Keyword(s):  
Complete Sequence ◽  
Coding Region ◽  
Degenerate Primers ◽  
Acid Protein ◽  
Rapid Amplification ◽  
And Function ◽  
Apical Membranes ◽  
Carboxyl Tail ◽  
Ancestral Vertebrate ◽  
Amino Acid Protein

In mammals, the Na+/H+ exchanger 3 (NHE3) is expressed with Na+/K+-ATPase in renal proximal tubules, where it secretes H+ and absorbs Na+ to maintain blood pH and volume. In elasmobranchs (sharks, skates, and stingrays), the gills are the dominant site of pH and osmoregulation. This study was conducted to determine whether epithelial NHE homologs exist in elasmobranchs and, if so, to localize their expression in gills and determine whether their expression is altered by environmental salinity or hypercapnia. Degenerate primers and RT-PCR were used to deduce partial sequences of mammalian NHE2 and NHE3 homologs from the gills of the euryhaline Atlantic stingray ( Dasyatis sabina). Real-time PCR was then used to demonstrate that mRNA expression of the NHE3 homolog increased when stingrays were transferred to low salinities but not during hypercapnia. Expression of the NHE2 homolog did not change with either treatment. Rapid amplification of cDNA was then used to deduce the complete sequence of a putative NHE3. The 2,744-base pair cDNA includes a coding region for a 2,511-amino acid protein that is 70% identical to human NHE3 (SLC9A3). Antisera generated against the carboxyl tail of the putative stingray NHE3 labeled the apical membranes of Na+/K+-ATPase-rich epithelial cells, and acclimation to freshwater caused a redistribution of labeling in the gills. This study provides the first NHE3 cloned from an elasmobranch and is the first to demonstrate an increase in gill NHE3 expression during acclimation to low salinities, suggesting that NHE3 can absorb Na+ from ion-poor environments.

scholarly journals Frequent Occurrence of an Intron 4 Mutation in Multiple Endocrine Neoplasia Type 1

2002 ◽  
Vol 87 (6) ◽  
pp. 2688-2693 ◽  
Author(s):  
Jeremy J. O. Turner ◽  
Poloko D. Leotlela ◽  
Anna A. J. Pannett ◽  
Simon A. Forbes ◽  
J. H. Duncan Bassett ◽  
...  
Keyword(s):  
Splice Site ◽  
Splice Site Mutation ◽  
Pancreatic Tumors ◽  
Coding Region ◽  
Site Mutation ◽  
Autosomal Dominant Disorder ◽  
Acid Protein ◽  
Intron 4 ◽  
Amino Acid Protein

MEN1 is an autosomal dominant disorder characterized by parathyroid, pituitary, and pancreatic tumors. The MEN1 gene is located on chromosome 11q13 and encodes a 610-amino acid protein. MEN1 mutations are of diverse types and are scattered throughout the coding region, such that almost every MEN1 family will have its individual mutation. To further characterize such mutations we ascertained 34 unrelated MEN1 probands and undertook DNA sequence analysis. This identified 17 different mutations in 24 probands (2 nonsense, 2 missense, 2 in-frame deletions, 5 frameshift deletions, 1 frameshift deletional-insertion, 3 frameshift insertions, 1 donor splice site mutation, and a g→a transition that resulted in a novel acceptor splice site in intron 4). The intron 4 mutation was found in 7 unrelated families, and the tumors in these families varied considerably, indicating a lack of genotype-phenotype correlation. However, this intron 4 mutation is the most frequently occurring germline MEN1 mutation (∼10% of all mutations), and together with 5 others at codons 83–84, 118–119, 209–211, 418, and 516, accounts for 36.6% of all mutations, a finding that indicates an approach for identifying the widely diverse MEN1 mutations.

scholarly journals Molecular Cloning and Characterization of a cDNA Encoding Sedoheptulose-1,7-bisphosphatase from Mulberry (Morus alba var. multicaulis)

2008 ◽  
Vol 57 (1-6) ◽  
pp. 152-157 ◽  
Author(s):  
X. Ji ◽  
Y. Gai ◽  
J. Ma ◽  
C. Zheng ◽  
Z. Mu
Keyword(s):  
Morus Alba ◽  
Evolutionary Analysis ◽  
Comparison Analysis ◽  
Reading Frame ◽  
Acid Protein ◽  
Fructose 1,6 Bisphosphatase ◽  
Rapid Amplification ◽  
Molecular Evolutionary Analysis ◽  
Amino Acid Protein

Abstract A full-length cDNA encoding sedoheptulose-1,7-bisphosphatase (SBPase; EC 3.1.3.37) was cloned from mulberry (Morus alba var. multicaulis) by rapid amplification of cDNA ends (RACE). The cDNA consisted of 1,527 nucleotides with an open reading frame (ORF) of 1,179 nucleotides encoding a 393 amino acid protein of approximately 42.6 kDa. Sequence comparison analysis showed that mulberry SBPase (MSBPase) had high homology to other plant counterparts. Phylogenetic and molecular evolutionary analysis revealed that MSBPase fell into plant SBPase group. Moreover, SBPase and fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) shared 28-32% identical residues, suggesting that the two enzymes originated from the same evolution branch. Molecular modeling indicated that each subunit of MSBPase was composed of α-helices and β-sheets joined by turns and loops, and folded into a structure of hexahedron shape which was very similar to FBPase.

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.

A putative H+-K+-ATPase in the Atlantic stingray, Dasyatis sabina: primary sequence and expression in gills

2004 ◽  
Vol 287 (4) ◽  
pp. R981-R991 ◽  
Author(s):  
Keith P. Choe ◽  
Jill W. Verlander ◽  
Charles S. Wingo ◽  
David H. Evans
Keyword(s):  
Primary Sequence ◽  
Coding Region ◽  
Double Labeling ◽  
Protein Isolates ◽  
Tissue Sections ◽  
Dasyatis Sabina ◽  
Acid Protein ◽  
Atlantic Stingray ◽  
Full Length Sequence ◽  
Amino Acid Protein

In mammals, the gastric H+-K+-ATPase (HKα1) mediates acid secretion in the stomach and kidneys. Like mammals, elasmobranchs also secrete acid from their stomachs, but unlike mammals they primarily use their gills for systemic acid excretion instead of their kidneys. The purpose of this study was to determine if an HKα1 orthologue exists in an elasmobranch (Atlantic stingray, Dasyatis sabina), to determine if it is expressed in gills and, if so, to localize its expression and determine if its expression is regulated during hypercapnia or freshwater acclimation. A polyclonal antibody made against an HKα1 peptide detected HKα1 immunoreactivity in protein isolates and tissue sections of stingray stomachs and gills. Immunohistochemistry demonstrated that HKα1 immunoreactivity was present in a subpopulation of epithelial cells in both organs. Double-labeling experiments in the gills showed that HKα1 immunoreactivity occurred in Na+-K+-ATPase-rich cells and not in V-type H+-ATPase-rich cells. RT-PCRs were used to deduce the primary sequence of a putative H+-K+-ATPase from the stomach of Atlantic stingrays. The 3,421-base pair cDNA includes a coding region for a 1,025-amino acid protein that is over 80% identical to HKα1 of mammals. RT-PCRs were then used to demonstrate that this transcript is also expressed in the gills. To our knowledge, this is the first H+-K+-ATPase sequence reported for any elasmobranch and the first full-length sequence for any fish. We also provide the first evidence for its expression in the gills of any fish and demonstrate that its expression increased during freshwater acclimation but not exposure to hypercapnia.

MEI4, a meiosis-specific yeast gene required for chromosome synapsis

1992 ◽  
Vol 12 (3) ◽  
pp. 1340-1351
Author(s):  
T M Menees ◽  
P B Ross-MacDonald ◽  
G S Roeder
Keyword(s):  
Fusion Gene ◽  
Blot Hybridization ◽  
Coding Region ◽  
Yeast Saccharomyces Cerevisiae ◽  
Type Locus ◽  
Chromosome Synapsis ◽  
Acid Protein ◽  
Mutant Strains ◽  
Rad50 Gene ◽  
Amino Acid Protein

The MEI4 gene product is required for meiotic induction of recombination and viable spore production in the yeast Saccharomyces cerevisiae. DNA sequence analysis shows that the MEI4 gene encodes a 450-amino-acid protein bearing no homology to any previously identified protein. The MEI4 coding region is interrupted by a small intron located near the 5' end of the gene. Efficient splicing of the MEI4 transcript is not dependent on the MER1 protein, which is required for splicing the transcript of another meiotic gene, MER2. Expression of a mei4::lacZ fusion gene is meiosis-specific and depends on both heterozygosity at the mating-type locus and nutrient limitation. Northern (RNA) blot hybridization analysis suggests that MEI4 gene expression is regulated at the level of transcription. A functional MEI4 gene is not required for meiotic induction of transcription of the MER1, MER2, MEK1, RED1, SPO11, or RAD50 gene. Cytological analysis of mei4 mutant strains during meiotic prophase demonstrates that the chromosomes form long axial elements that fail to undergo synapsis. The meiosis II division is delayed in mei4 strains.

scholarly journals Assembly and Function of the RNA Editing Complex in Trypanosoma brucei Requires Band III Protein

2002 ◽  
Vol 22 (9) ◽  
pp. 3194-3203 ◽  
Author(s):  
Catherine E. Huang ◽  
Sean F. O'Hearn ◽  
Barbara Sollner-Webb
Keyword(s):  
Rna Editing ◽  
Guide Rna ◽  
Insertion And Deletion ◽  
Cell Extracts ◽  
Zinc Finger Motifs ◽  
Acid Protein ◽  
Mitochondrial Transcripts ◽  
Insertion Sites ◽  
And Function ◽  
Amino Acid Protein

ABSTRACT Trypanosome RNA editing, the posttranscriptional insertion and deletion of U residues in mitochondrial transcripts, is catalyzed by a protein complex containing seven distinct proteins. In this study, we cloned the gene for band III, a 555-amino-acid protein with two separate zinc finger motifs. We prepared antibodies that showed band III protein cofractionates with the previously characterized band IV protein throughout the purification of the editing complex and is not found free or in other protein associations; therefore, it is a true constituent of the editing complex. Double-stranded RNA interference efficiently depleted band III protein and demonstrated that band III expression is essential for growth of procyclic trypanosomes and for RNA editing. These depleted cell extracts were deficient specifically in guide RNA-directed endonuclease cleavage at both U deletion and U insertion sites and in the activity of the band IV ligase, but they retained the 3′-U-exonuclease and terminal-U-transferase activities as well as band V ligase of the editing complex. Loss of band III protein also resulted in almost complete loss of the band IV ligase protein and altered sedimentation of the band V ligase. These data indicate that band III is either the RNA editing endonuclease or a factor critical for cleavage activity in the editing complex. They also demonstrate that band III is required for proper assembly of the editing complex.

scholarly journals The structure of a highly conserved picocyanobacterial protein reveals a Tudor domain with a novel tRNA binding function

2019 ◽  
Author(s):  
Katherine M Bauer ◽  
Rose Dicovitsky ◽  
Maria Pellegrini ◽  
Olga Zhaxybayeva ◽  
Michael J Ragusa
Keyword(s):  
Amino Acid ◽  
Amino Acid Level ◽  
Computational Docking ◽  
Binding Function ◽  
Acid Protein ◽  
Tudor Domain ◽  
And Function ◽  
Positively Charged ◽  
Amino Acid Protein

ABSTRACTCyanobacteria of the Prochlorococcus and marine Synechococcus genera are the most abundant photosynthetic microbes in the ocean. Intriguingly, the genomes of these bacteria are very divergent even within each genus, both in gene content and at amino acid level of the encoded proteins. One striking exception to this is a 62 amino acid protein, termed Prochlorococcus/SynechococcusHyper Conserved Protein (PSHCP). PSHCP is not only found in all sequenced Prochlorococcus and marine Synechococcus genomes but it is also nearly 100% identical in its amino acid sequence across all sampled genomes. Such universal distribution and sequence conservation suggests an essential cellular role of the encoded protein in these bacteria. However, the function of PSHCP is unknown. We used Nuclear Magnetic Resonance (NMR) spectroscopy to determine its structure. We found that 52 of the 62 amino acids in PSHCP form a Tudor domain, while the remainder of the protein is disordered. NMR titration experiments revealed that PSHCP has only a weak affinity for DNA, but an 18.5 fold higher affinity for tRNA, hinting at an involvement of PSHCP in translation. Computational docking and mutagenesis studies identified a positively charged patch surrounding residue K30 that serves as the primary docking site for tRNA on PSHCP. These results provide the first insight into the structure and function of PSHCP and suggest a new function for Tudor domains in recognizing tRNA.

scholarly journals MEI4, a meiosis-specific yeast gene required for chromosome synapsis.

1992 ◽  
Vol 12 (3) ◽  
pp. 1340-1351 ◽  
Author(s):  
T M Menees ◽  
P B Ross-MacDonald ◽  
G S Roeder
Keyword(s):  
Fusion Gene ◽  
Blot Hybridization ◽  
Coding Region ◽  
Yeast Saccharomyces Cerevisiae ◽  
Type Locus ◽  
Chromosome Synapsis ◽  
Acid Protein ◽  
Mutant Strains ◽  
Rad50 Gene ◽  
Amino Acid Protein

The MEI4 gene product is required for meiotic induction of recombination and viable spore production in the yeast Saccharomyces cerevisiae. DNA sequence analysis shows that the MEI4 gene encodes a 450-amino-acid protein bearing no homology to any previously identified protein. The MEI4 coding region is interrupted by a small intron located near the 5' end of the gene. Efficient splicing of the MEI4 transcript is not dependent on the MER1 protein, which is required for splicing the transcript of another meiotic gene, MER2. Expression of a mei4::lacZ fusion gene is meiosis-specific and depends on both heterozygosity at the mating-type locus and nutrient limitation. Northern (RNA) blot hybridization analysis suggests that MEI4 gene expression is regulated at the level of transcription. A functional MEI4 gene is not required for meiotic induction of transcription of the MER1, MER2, MEK1, RED1, SPO11, or RAD50 gene. Cytological analysis of mei4 mutant strains during meiotic prophase demonstrates that the chromosomes form long axial elements that fail to undergo synapsis. The meiosis II division is delayed in mei4 strains.

Cloning and expression of apyrase gene from Ancylostoma caninum in Escherechia coli

2014 ◽  
Vol 60 (1) ◽  
Author(s):  
Ying Qiao ◽  
Theerakamol Pengsakul
Keyword(s):  
Recombinant Protein ◽  
Molecular Size ◽  
Cloning And Expression ◽  
Host Immune System ◽  
Ancylostoma Caninum ◽  
Acid Protein ◽  
Partial Cdna ◽  
And Function ◽  
Phosphate Groups ◽  
Amino Acid Protein

AbstractApyrase encoding metal-ions activated plasma membrane protease is present in animal and plant tissues. This enzyme can hydrolyze ADP and ATP pyrophosphate bond, resulting in AMP and free phosphate groups, and plays an important role for insects and parasites to evade host immune system. However localization and function of apyrase in the canine hookworm, Ancylostoma caninum, remains unknown. To analyze apyrase gene in A. caninum (a eukaryotic parasitic hookworm), a pair of primers was designed according to the previous EST data. The full-length cDNA of apyrase gene was amplified from A. caninum by RT-PCR. The partial cDNA of apyrase encodes 249 amino acid protein was expressed in Escherechia coli. The recombinant protein was induced to express under proper conditions and the molecular size was as expected. The recombinant protein was purified. The transcripts of apyrase in different stages of A. caninum were analyzed by the Real-time PCR assay, and Immuno-localization assays were used to research the protein expression in different stages of A. caninum

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