Cloning and characterization of an electrogenic Na/HCO3− cotransporter from the squid giant fiber lobe

2007 ◽  
Vol 292 (6) ◽  
pp. C2032-C2045 ◽  
Author(s):  
Peter M. Piermarini ◽  
Inyeong Choi ◽  
Walter F. Boron
Keyword(s):  
Giant Axon ◽  
Predicted Amino Acid Sequence ◽  
Giant Fiber ◽  
Reading Frame ◽  
Excitable Membranes ◽  
Current Voltage ◽  
Partial Cdna ◽  
Partial Cdna Clone ◽  
Current Voltage Relationship

The squid giant axon is a classic model system for understanding both excitable membranes and ion transport. To date, a Na+-driven Cl-HCO3− exchanger, sqNDCBE—related to the SLC4 superfamily and cloned from giant fiber lobe cDNA—is the only HCO3−-transporting protein cloned and characterized from a squid. The goal of our study was to clone and characterize another SLC4-like cDNA. We used degenerate PCR to obtain a partial cDNA clone (squid fiber clone 3, SF3), which we extended in both the 5′ and 3′ directions to obtain the full-length open-reading frame. The predicted amino-acid sequence of SF3 is similar to sqNDCBE, and a phylogenetic analysis of the membrane domains indicates that SF3 clusters with electroneutral Na+-coupled SLC4 transporters. However, when we measure pHi and membrane potential—or use two-electrode voltage clamping to measure currents—on Xenopus oocytes expressing SF3, the oocytes exhibit the characteristics of an electrogenic Na/HCO3− cotransporter, NBCe. That is, exposure to extracellular CO2/HCO3− not only causes a fall in pHi, followed by a robust recovery, but also causes a rapid hyperpolarization. The current-voltage relationship is also characteristic of an electrogenic NBC. The pHi recovery and current require HCO3− and Na+, and are blocked by DIDS. Furthermore, neither K+ nor Li+ can fully replace Na+ in supporting the pHi recovery. Extracellular Cl− is not necessary for the transporter to operate. Therefore, SF3 is an NBCe, representing the first NBCe characterized from an invertebrate.

Gene Cloning, Nucleotide Sequencing, and Purification and Characterization of the Low-Specificityl-Threonine Aldolase from Pseudomonas sp. Strain NCIMB 10558

1998 ◽  
Vol 64 (2) ◽  
pp. 549-554 ◽  
Author(s):  
Ji-Quan Liu ◽  
Saeko Ito ◽  
Tohru Dairi ◽  
Nobuya Itoh ◽  
Michihiko Kataoka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Significant Loss ◽  
Site Directed Mutagenesis ◽  
Nucleotide Sequencing ◽  
Predicted Amino Acid Sequence ◽  
Amino Acid Residues ◽  
Purification And Characterization ◽  
Reading Frame ◽  
Threonine Aldolase

ABSTRACT A low-specificity l-threonine aldolase (l-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia colicells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5′-phosphate as a coenzyme, is strictlyl specific at the α position, whereas it cannot distinguish between threo and erythro forms at the β position. In addition to threonine, the enzyme also acts on various other l-β-hydroxy-α-amino acids, includingl-β-3,4-dihydroxyphenylserine,l-β-3,4-methylenedioxyphenylserine, andl-β-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificityl-TA from Saccharomyces cerevisiae,l-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity l-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of thel-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5′-phosphate of the enzyme to catalyze the reversible aldol reaction.

Molecular cloning of the cDNA for ligatin

1989 ◽  
Vol 93 (2) ◽  
pp. 227-232
Author(s):  
E.R. Jakoi ◽  
A.L. Brown ◽  
Y.S. Ho ◽  
R. Snyderman
Keyword(s):  
Consensus Sequence ◽  
Blot Hybridization ◽  
Rabbit Antiserum ◽  
Acceptor Site ◽  
Protein Fusion ◽  
Reading Frame ◽  
Pair Sequence ◽  
Partial Cdna ◽  
Partial Cdna Clone ◽  
Trafficking Receptor

We describe the first isolation and sequence of a partial cDNA clone encoding ligatin, a trafficking receptor for phosphoglycoproteins. The clone was isolated from a human U937 promonocyte lambda gt11 cDNA library using rabbit antiserum to rat ileal ligatin. RNA blot hybridization revealed that the intact receptor transcript in human cells is 2.4 kilobases (kb). DNA sequencing together with expression of protein fusion products in Escherichia coli demonstrated that the cloned segment begins with a 1.2 kb open reading frame potentially encoding a 7.5 × 10(3) Mr section of the 10 × 10(3) Mr receptor followed by a 3′ tail of 948 bases. The 225 bases of coding sequence correspond to the carboxyl region of ligatin and contain a potential acceptor site for asparagine-linked glycosylation. Neither a poly(A) sequence nor polyadenylation signal was found at the 3′ end of the clone. In the 3′ untranslated region there is a paired consensus sequence (TGAGnnnTTTTTCA) that is analogous to a conserved 12 base-pair sequence present in clusters in several growth-controlled mRNAs, including those for c-fos and beta-actin. The identity of this clone as ligatin was confirmed immunologically using antisera to an encoded fusion protein and three independent regions of its derived sequence. In addition, one of these antibodies produced a punctate immunofluorescence pattern within the cytosol of U937 cells in a similar fashion to anti-ligatin serum.

scholarly journals Identification and Initial Characterization of the Murine Gammaherpesvirus 68 Gene M3, Encoding an Abundantly Secreted Protein

1999 ◽  
Vol 73 (5) ◽  
pp. 4524-4529 ◽  
Author(s):  
Victor van Berkel ◽  
Karen Preiter ◽  
Herbert W. Virgin ◽  
Samuel H. Speck
Keyword(s):  
Secreted Protein ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Murine Gammaherpesvirus ◽  
Amino Terminal ◽  
Partial Cdna ◽  
Rapid Amplification ◽  
Gammaherpesvirus 68 ◽  
Partial Cdna Clone ◽  
Murine Gammaherpesvirus 68

ABSTRACT Several viruses, including members of the gammaherpesvirus family, encode proteins that are secreted into the extracellular environment. We have identified an abundant 44-kDa secreted protein that is present in the supernatant of fibroblasts infected with murine gammaherpesvirus 68 (γHV68; also referred to as MHV-68) but not in that of uninfected fibroblasts. Sequence analysis of the amino terminus and of internal peptides revealed that this protein is encoded by the γHV68 M3 open reading frame (ORF). The amino-terminal sequence of the secreted protein starts at residue 25 of the M3 ORF, consistent with the first 24 residues functioning as a signal peptide. Northern blot analysis revealed a single abundant ∼1.4-kb early-late lytic transcript encoded by the M3 ORF. Analysis of a partial cDNA clone and subsequent analyses of products of rapid amplification of cDNA ends coupled with S1 nuclease protection assays demonstrate that the M3 protein is encoded by an unspliced, polyadenylated mRNA initiating at bp 7294 and terminating at bp 6007 of the γHV68 genome. The 3′ end of the M3 transcript maps 9 bp downstream of a consensus polyadenylation signal. Thus, the predicted M3 ORF is a functional gene that encodes an abundant secreted protein which is a candidate for interacting with host cellular receptors or cytokines.

scholarly journals Cloning and Characterization of a Cryptic Haloacid Dehalogenase from Burkholderia cepacia MBA4

1999 ◽  
Vol 181 (19) ◽  
pp. 6003-6009 ◽  
Author(s):  
Jimmy S. H. Tsang ◽  
Laiju Sam
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Burkholderia Cepacia ◽  
Amino Acid Sequences ◽  
Predicted Amino Acid Sequence ◽  
Haloacid Dehalogenase ◽  
Reading Frame ◽  
Relative Molecular Weight

ABSTRACT Burkholderia cepacia MBA4 has been shown to produce a single dehalogenase batch culture. Moreover, other cryptic dehalogenases were also detected when the cells were grown in continuous culture. In this paper, we report the cloning and characterization of one of the cryptic dehalogenases in MBA4. This cryptic haloacid dehalogenase, designated Chd1, was expressed constitutively in Escherichia coli. This recombinant Chd1 had a relative molecular weight of 58,000 and existed predominantly as a dimer. The subunits had a relative molecular weight of 27,000. Chd1 exhibited isomer specificity, being active towards thel-isomer of 2-monochloropropionic acid only. The structural gene, chd1, was isolated on a 1.7-kb PstI fragment. This fragment contains a functional promoter, because expression of chd1 in E. coli is orientation independent. The nucleotide sequence of this fragment was determined and characterized. An open reading frame of 840 bp encoding a putative peptide of 280 amino acids was identified. This corresponds closely with the size of the subunit. The nucleotide sequence of chd1 did not show any homology with those of other dehalogenase genes. Comparison of the predicted amino acid sequence, however, shows significant homology, ranging from 42 to 50%, with the amino acid sequences of many other dehalogenases. Chd1 is unusual in having a long leader sequence, a property of periplasmic enzymes.

Characterization of a partial cDNA clone detected by imidazoline receptor-selective antisera

1998 ◽  
Vol 72 (2-3) ◽  
pp. 98-110 ◽  
Author(s):  
Tina R. Ivanov ◽  
Julia Cay Jones ◽  
Monique Dontenwill ◽  
Pascal Bousquet ◽  
John E. Piletz
Keyword(s):  
Cdna Clone ◽  
Imidazoline Receptor ◽  
Partial Cdna ◽  
Partial Cdna Clone

scholarly journals Isolation of the Histone Lysine Methyltransferase Gene PhSDG8 and Characterization of Function in Shoot Branching

2020 ◽  
Vol 145 (5) ◽  
pp. 299-307
Author(s):  
Lili Dong ◽  
Tongrui Liu ◽  
Di Gao ◽  
Jing Li ◽  
Jie Qian
Keyword(s):  
Nicotiana Sylvestris ◽  
Predicted Amino Acid Sequence ◽  
Branch Number ◽  
Reading Frame ◽  
Histone Lysine Methyltransferase ◽  
Methyltransferase Gene ◽  
Localization Analysis ◽  
Lysine Methyltransferase ◽  
Branch Development

Petunia (Petunia ×hybrida) is an important ornamental plant, and its branch development has become a hot research topic. In this study, PhSDG8, an ortholog of SET domain group 8 (SDG8), was cloned from the petunia cultivar Mitchell Diploid. It had an open reading frame (ORF) of 5070 bp and encoded 1689 amino acids, with Suppressor variegation 3–9, Enhancer of zeste, Trithorax (SET), Zinc finger-cysteine and tryptophan conserved (Zf-CW), associated with SET (AWS) and Post SET domains. The predicted amino acid sequence of PhSDG8 was most closely related to Nicotiana sylvestris ASHH2 (NsASHH2). Expression analysis revealed that PhSDG8 expressed highest in the stems and lowest in the axil. Subcellular localization analysis showed that PhSDG8 was localized in the nucleus. Overexpression of PhSDG8 reduced the branch number of Arabidopsis thaliana sdg8-2. The silencing of PhSDG8 resulted in an increase in the number of branches of petunia and significant upregulation of PhUGT74E2. These results suggested that PhSDG8 may be a candidate gene for regulating the branching of petunia.

scholarly journals Identification of a Sterol Δ7 Reductase Gene Involved in Desmosterol Biosynthesis in Mortierella alpina 1S-4

2007 ◽  
Vol 73 (6) ◽  
pp. 1736-1741 ◽  
Author(s):  
Shuo Zhang ◽  
Eiji Sakuradani ◽  
Sakayu Shimizu
Keyword(s):  
Mortierella Alpina ◽  
Open Reading Frame ◽  
Predicted Amino Acid Sequence ◽  
Control Strain ◽  
Gene Encoding ◽  
Reading Frame ◽  
African Clawed Frog ◽  
Reductase Gene ◽  
Clawed Frog

ABSTRACT Molecular cloning of the gene encoding sterol Δ7 reductase from the filamentous fungus Mortierella alpina 1S-4, which accumulates cholesta-5,24-dienol (desmosterol) as the main sterol, revealed that the open reading frame of this gene, designated MoΔ7SR, consists of 1,404 bp and codes for 468 amino acids with a molecular weight of 53,965. The predicted amino acid sequence of MoΔ7SR showed highest homology of 51% with that of sterol Δ7 reductase (EC 1.3.1.21) from Xenopus laevis (African clawed frog). Heterologous expression of the MoΔ7SR gene in yeast Saccharomyces cerevisiae revealed that MoΔ7SR converts ergosta-5,7-dienol to ergosta-5-enol (campesterol) by the activity of Δ7 reductase. In addition, with gene silencing of MoΔ7SR gene by RNA interference, the transformant accumulated cholesta-5,7,24-trienol up to 10% of the total sterols with a decrease in desmosterol. Cholesta-5,7,24-trienol is not detected in the control strain. This indicates that MoΔ7SR is involved in desmosterol biosynthesis in M. alpina 1S-4. This study is the first report on characterization of sterol Δ7 reductase from a microorganism.

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