The DNA sequence and derived amino-acid sequence of a 5618-base region in the 74-min area of the Escherichia coli chromosome has been determined in order to locate the structural gene, nirB, for the NADH-dependent nitrite reductase and a gene, cysG, required for the synthesis of the sirohaem prosthetic group. Three additional open reading frames, nirD, nirE and nirC, were found between nirB and cysG.Potential binding sites on the NirB protein for NADH and FAD, as well as conserved central core and interface domains, were deduced by comparing the derived amino-acid sequence with those of database proteins. A directly repeated sequence, which includes the motif -Cys-Xaa-Xaa-Cys-, is suggested as the binding site for either oneThe nirD gene potentially encodes a soluble, cytoplasmic protein of unknown function. No significant similarities were found between the derived amino-acid sequence of NirD and either NirB or any other protein in the database. If the nirE open reading frame is translated, it would encode a 33-amino-acid peptide of unknown function which includes 8 phenylalanyl residues.The product of the nirC gene is a highly hydrophobic protein with regions of amino-acid sequence similar to cytochrome oxidase polypeptide 1.Two genes essential for the major NADH-dependent nitrite reductase activity are located in the 74-min region of the Escherichiu coli K-12 chromosome [l]. They are nirB, the structural gene for the nitrite reductase apoprotein, and cysG, which is required for sirohaem synthesis. The prosthetic groups of nitrite reductase are FAD, an iron-sulphur cluster and sirohaem which is also found in the NADPH-dependent sulphite reductase. Mutants defective in the cysG gene are unable to grow without cysteine, or to reduce nitrite rapidly to ammonia [2,. The cysG product has recently been purified and characterized (C. Roessner, personal communication). It catalyses two methylation reactions in the conversion of uroporphyrinogen I11 into sirohaem.We have previously located the promoter, transcription and translation start points of the nirB gene and reported the DNA sequence and derived amino-acid sequence of the first 89 bases of nirB and its 5'-regulatory region [4]. We now report Correspondence to J. A. Cole,
The human brain voltage-gated Na+ channel type IIA alpha subunit was cloned and stably expressed in Chinese hamster ovary cells and its biophysical and pharmacological properties were studied using whole-cell voltage-clamp. Fast, transient inward currents of up to -8,000 pA were elicited by membrane depolarization of the recombinant cells. Channels activated at -50 mV and reached maximal activation at -10 mV to 0 mV. The reversal potential was 62 +/- 2 mV which is close to the Na+ equilibrium potential. The half-maximal activation and inactivation voltages were -24 +/- 2 mV and -63 +/- 1 mV, respectively. Currents were reversibly blocked by tetrodotoxin with a half-maximal inhibition of 13 nM. The effects of four commonly used anti-convulsant drugs were examined for the first time on the cloned human type IIA channel. Lamotrigine and phenytoin produced concentration- and voltage-dependent inhibition of the type IIA currents, whereas, sodium valproate and gabapentin (up to 1 mM) had no effect. These results indicate that recombinant human type IIA Na+ channels conduct tetrodotoxin-sensitive Na+ currents with similar properties to those observed in recombinant rat brain type IIA and native rat brain Na+ channels. This stable cell line should provide a useful tool for more detailed characterization of therapeutic modulators of human Na+ channels.
The anaerobically-regulated nirB promoter was used to express heterologous genes in Escherichia coli. Under anaerobic conditions the promoter was able to express tetanus toxin fragment C at approximately 20% total cell protein (tcp) and the Bordetella pertussis antigen pertactin at greater than 30% tcp. These levels are comparable to those obtained for the same products using the tac promoter. The nirB promoter is very well regulated, giving almost two orders of magnitude increase in fragment C on complete removal of oxygen.
The 74-min region of the Escherichiu coli chromosome includes five open reading frames of known sequence. The first and last of these genes, nirB and cysG, are transcribed in the same direction and both are essential for NADH-dependent nitrite reductase activity. The functions of the other genes, nirD, nirE and nirC, which are located between nirB and cysG, are unknown. The nirB gene is transcribed from a promoter which is anaerobically induced, expression being dependent on the transcription activator protein, Fnr. Here we show that the nirD, nirE, nirC and cysG genes are also expressed from the nirB promoter. After subcloning cysG, a second promoter was located less than 100 bases upstream of cysG. Two groups of transcription start points separated by 40 bases were detected in this region by S1 mapping. Rates of transcription from the isolated cysG promoter were the same during aerobic growth and anaerobic growth in the presence or absence of nitrite. However, when the nirB gene and its promoter were cloned back upstream from the cysG promoter, the rate of transcription was higher during anaerobic growth than during aerobic growth and was further induced by nitrite. These increases were totally dependent on a functiona1,fnr gene and were shown by S1 mapping experiments to be due to transcriptional readthrough from the Fnr-dependent nirB promoter.No promoter activity was associated with DNA fragments between the BumHI site located within the Nterminal coding region of the nirB gene and the cysG promoter located at the C-terminus of nirC.Sirohaem is one of the prosthetic groups of both the NADH-dependent nitrite reductase and the NADPH-dependent sulphite reductase of Escherichiu coli [I]. Sulphite reductase is an essential enzyme in the synthesis of cysteine from sulphate. Mutants defective in the cysG gene are unable to grow without cysteine either aerobically or anaerobically because the cysG product is essential to convert uroporphyrinogen 111 into sirohaem. As the NADH-dependent nitrite reductase is the most active enzyme for reducing nitrite to ammonia in E. coli, cysG mutants are also defective in nitrite reduction [2].The cysG gene is located extremely close to nirB, the structural gene for the nitrite reductase apoprotein, but is separated from it by three open reading frames which, in the preceding paper, we have designated nirD, nirE and nirC [3]. The third of these open reading frames, nirC, encodes a 28.5-kDa polypeptide with regions of amino acid sequence similar to cytochrome oxidase polypeptide 1 [3].The nirB gene is not expressed during aerobic growth. Transcription of nirB during anaerobic growth is dependent upon a functional Fnr protein and is further induced by nitrite (4, 51. As nirB and cysG are transcribed in the same direction (from nirB towards cysG), the aims of the experiments deCorrespondence to J. A. Cole,
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