Abstract
The nucleotide sequence of the gltS gene coding for an Na+/glutamate symport carrier of Escherichia coli B has been determined, and the amino acid sequence of the carrier protein was deduced. The predicted glutamate carrier consists of 401 amino acids with a molecular weight of 42,455. A Shine-Dalgarno sequence and putative promoter sequences were found in the 5'-flanking region of the putative gltS gene. The predicted protein is very hydrophobic (73% nonpolar amino acids), and judging from its hydropathy profile, the protein is composed of 12 hydrophobic membrane-spanning segments with a mean length of 21.6 residues/segment. A typical rho-independent transcription termination signal was found downstream of the gltS gene. We found a conserved alignment of 5 amino acid residues (Gly42--Ala82-X-X-X-X-Leu87-X-X-X-Gly91-Arg92 ), which commonly exists in four Na+ symport carrier proteins, the glutamate carrier, and the proline carrier of E. coli, and the Na+/glucose co-transporters of rabbit and human intestines. We propose that this consensus sequence (or motif) may play an important role in cation recognition or binding in the Na+/solute symport reaction.
Highlights
Arg”), which commonly exists in four Na+ symport carrier proteins, the glutamate carrier, and the proline carier of E. coli, and the Na+/glucose co-transporters of rabbit and human intestines
We report the complete nucleotide sequence of the gltS gene, including the 5’- and 3’-flanking regions, the deduced amino acid sequence, and a putative structure of the Na+/glutamate symport carrier
Nucleotide Sequence of the gltS Gene-As described in the previous paper, the structural gene for the Na+/glutamate symport carrier, gltS, was cloned, and the gene was located on a 3.2-kilobase DNA fragment between the EcoRI site and the MluI site of pDES7004
Summary
DNA Manipulation-Preparation of plasmid DNA, digestion with restriction endonucleases, ligation with T4 DNA ligase, transformation with recombinant plasmids, and agarose gel electrophoresis were as described previously [21]. Preparation of singlestranded templates for DNA sequencing was carried out according to the standard method [20] using pUC118 and helper phage M13K07. The. EcoRI-digested pDES7004 and a EcoRI fragment from pH159 carrying the kanamycin-resistance gene were ligated with T4 DNA ligase, and the resultant plasmid was named pDES7005. SmaI, XbaI-digested pUC118 and EcoRI, SmaI-digested pUC118, respectively These two resultant plasmids were named pDES7006. The EcoRI-MluI fragment carrying the gltS gene was cloned into pUC118 in both directions. Chemicals-Restriction endonucleases, T4 DNA ligase, Klenow fragment, and a deletion kit for DNA sequence were obtained from.
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