The significance of split genes to developmental genetics.
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Aging↗
Biomedical subjects
A Danchin
Publications and source records attributed to A Danchin.
Membrane integration of carbohydrate transport in bacteria.
A system is described which permits bacteria to couple their intermediary metabolism to the available carbon supply. This system transports carbohydrates vectorially, with concomitant phosphorylation. A phosphorylation cascade is responsible for the coupling with metabolism through control of permeation of other carbon sources and synthesis of modulators such as cAMP.
Bacteria↗
Escherichia coli and Saccharomyces cerevisiae adenylate cyclases: a case of phylogenetic convergence?
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Adenylyl Cyclases↗
Yeast adenylate cyclase catalytic domain is carboxy terminal.
Subcloning of DNA fragments from the gene coding for yeast adenylate cyclase has permitted, after complementation studies in S. cerevisiae cdc35 mutants as well as E. coli cya mutants, to identify the sequence coding for the catalytic domain of the protein. No homology is found between the yeast cyclase catalytic domain and the homologous domain found in E. coli adenylate cyclase. Analysis by Northern blotting of yeast polyA mRNA has shown the existence of multiple transcriptional products of the gene. A putative origin of a major transcript (3.5 kb) would allow synthesis of a ca. 100,000 dalton protein exhibiting cyclase activity in its carboxy terminal domain, and having 7 repeats of 17 amino acids at its amino terminal end. Several note-worthy features, including the possibility of transcriptional control by the general control of amino acids biosynthesis, are present at this putative origin. Data are presented suggesting that a much longer gene product might also be synthesized from the CDC35 gene. Neither the gene organization nor the amino acid sequence of the protein does display any homology with the adenylate cyclase gene and protein of Escherichia coli. This suggests a case of evolutionary convergence for the synthesis of cAMP in prokaryotes and eukaryotes.
Adenylyl Cyclases↗
Protein export in prokaryotes and eukaryotes: indications of a difference in the mechanism of exportation.
Investigation of possible variations between prokaryotic and eukaryotic signal sequences of exported proteins has revealed unexpected differences. Apart from the known similarities (presence of a core hydrophobic sequence preceded by a positively charged amino terminus and followed by a flexible structure), we have found that the core is much more rigid in eukaryotic signals than in their prokaryotic counterparts, and that at both ends the constraints are much more stringent in bacteria than in human cells. The differences have been summarized as a set of 17 criteria describing noteworthy features discriminating between the two classes of signal peptides. The program we used permitted each class of sequences to be learned; Escherichia coli sequences were well learned (i.e., they could be recognized by the programs as having common features), whereas human sequences were found to exhibit a much wider variation. Thus it was possible to propose a consensus in the case of the bacterial peptides, but none (or a much looser one) in the case of the human sequences. Two sequences were exceptional among the E. coli signal peptides, those of lipoprotein and plasmid-borne beta-lactamase, suggesting that they have special origins or destinations. Finally, the differences found strongly suggest that the mode of secretion is rather different in the two types of organisms, in spite of the common features of the signal sequences.
Amino Acid Sequence↗
2-Ketoglutarate as a possible regulatory metabolite involved in cyclic AMP-dependent catabolite repression in Escherichia coli K12.
2-Ketoglutarate--unlike any other derivative of the citric acid cycle--was found to strongly repress catabolite-sensitive genes, such as the lactose operon (lac) or the tryptophanase gene (tna), when added to cells grown in glycerol. 2-ketoglutarate affects the expression of these genes by decreasing cyclic AMP synthesis. Such inhibition of cyclic AMP synthesis requires the presence of enzyme III, a component of the phosphoenol pyruvate:sugar phosphotransferase transport system (PTS). Thus, it is proposed that 2-ketoglutarate is one of the catabolite repressors postulated by Magasanik in 1961. In addition, by studying the effect of 2-ketoglutarate in various mutants, we show the existence of a cyclic AMP-independent catabolite repression mechanism whose mediator is synthesized from 2-ketoglutarate.
Cyclic AMP↗
Analysis of the ptsH-ptsI-crr region in Escherichia coli K-12: nucleotide sequence of the ptsH gene.
The nucleotide sequence of an Escherichia coli DNA segment containing the ptsH gene and the first 162 nucleotides of the ptsI gene encoding, respectively, Hpr and enzyme I of the phosphoenolpyruvate-dependent glycose phosphotransferase system (PTS), was determined. The ptsH promoter was localized using the S1 mapping technique. A nucleotide sequence very similar to the consensus binding site for cAMP receptor protein was found in the -35 region of the ptsH promoter. The ptsH gene is transcribed in the same direction as the ptsI gene and the crr gene (encoding enzyme IIIGlc of the PTS). Analysis of the nucleotide sequence substantiates the notion that the ptsH-ptsI-crr genes constitute a polycistronic operon.
Base Sequence↗
The complete nucleotide sequence of the adenylate cyclase gene of Escherichia coli.
The complete nucleotide sequence of the cya gene from E. coli was determined. The gene encodes a polypeptide consisting of 848 amino acid residues with a calculated molecular weight of 97,542. The deduced protein structure reveals that cyclase is comprised of two domains, an amino-terminal region exhibiting catalytic activity and a carboxy-terminal region possibly carrying regulatory function. The frequent appearance of rare codons in the beginning of the gene as well as the sequence duplication in the promoter-initiator region suggest possible regulation(s) at the translational level. An unknown gene (cyaX) which seems to code for a very hydrophobic protein was found following the cya gene. Sequence analysis suggests that the cyax is a part of the cya operon.
Adenylyl Cyclases↗
Identification of the Escherichia coli cya gene product as authentic adenylate cyclase.
The Escherichia coli cya gene has been fused in the same register with the lacZ gene. The corresponding hybrid cya-lacZ gene is expressed as a bifunctional protein that exhibits both adenylate cyclase and beta-galactosidase activities, thus proving that cya is the structural gene for adenylate cyclase. The hybrid protein was purified to homogeneity and has been used to raise antibodies that recognize wild-type adenylate cyclase. Finally, the protein has been submitted to amino acid sequence analysis. It has been found that the first ten amino acids fit the predicted sequence obtained from DNA sequence analysis, thus substantiating the prediction that the cya translation initiation codon is UUG .
Adenylyl Cyclases↗
Antibodies against a fused 'lacZ-yeast mitochondrial intron' gene product allow identification of the mRNA maturase encoded by the fourth intron of the yeast cob-box gene.
Several missense or nonsense mutations have been localized in the fourth intron open reading frame (ORF) of the yeast mitochondrial cytochrome b gene. These results and the phenotypes of mutants strongly suggested that a mRNA maturase, controlling the expression of both cytochrome b and cytochrome oxidase subunit I (COXI) genes, is encoded in this ORF. To investigate more directly the biosynthesis of mRNA maturase we raised antibodies against a part of the putative ORF translation product. For that purpose we inserted a fragment of the ORF sequence, in phase, into the C-terminal EcoRI site of lacZ gene. The hybrid gene was then expressed in Escherichia coli under the control of either the wild-type lac promoter or the thermoregulated lambda system PR/cI857. The hybrid protein was partially purified and antibodies were raised against it. These antibodies recognized a mitochondrially coded protein, p27, in intron mutants, whereas no such protein was detected in the wild-type cell. These results demonstrate that the p27 protein, previously shown to be associated with the mRNA maturase activity, is actually translated from the intron ORF. The autoregulated mRNA maturase synthesis model is discussed in relation to these results.
Base Sequence↗
Antibodies against synthetic oligopeptides allow identification of the mRNA-maturase encoded by the second intron of the yeast cob-box gene.
Genetic and biochemical evidence has strongly suggested that several introns located in yeast mitochondrial genes specifying apocytochrome b or cytochrome oxidase encode trans-acting proteins (termed mRNA-maturases) responsible for splicing the cognate intron and maturation of the mRNA. We have chemically synthesized three oligopeptides, predicted from the DNA sequence of the open reading frame (ORF) present in the second intron of the cob-box gene, and raised antibodies against them. These antibodies have allowed us to identify a protein of 42 kd as the product translated from the ORF of the wild-type intron. In two splicing-deficient mutants this protein is replaced by shorter polypeptides whose lengths and antigenic properties are in full agreement with the positions of TAA codons established by the DNA sequence of the intron's ORF.
Antibodies, Fungal↗
Transcriptional control of polarity in Escherichia coli by cAMP.
In Escherichia coli, 3'5'-adenosine cyclic monophosphate (cAMP) and its receptor protein (CAP) are known to be involved in the control of transcription initiation of catabolic operons. In previous papers we have shown that the cAMP-CAP complex is also involved as a modulator of polarity in polycistronic transcription units. Furthermore we showed that there exists a functional relationship between this complex and the transcription termination protein, Rho. In this work, we measured mRNA synthesis corresponding to the promoter proximal and distal parts of the lac and gal operons by DNA-RNA hybridization. We show that in these operons the main polarity effect is essentially transcriptional and the cAMP-CAP complex decreases polarity by interfering with premature transcription termination.
Base Sequence↗
Role of 2-ketobutyrate as an alarmone in E. coli K12: inhibition of adenylate cyclase activity mediated by the phosphoenolpyruvate: glycose phosphotransferase transport system.
2-ketobutyrate and its analogues were found to inhibit strongly and transiently the rate of beta-galactosidase synthesis in Escherichia coli K12. This effect was ascribed to a strong and transient inhibition of the adenylate cyclase activity. By using pts mutants, we showed, in agreement with our previous results (Daniel et al. 1983), that the likely target of 2-ketobutyrate and its analogues is the phosphoenolpyruvate: glycose phosphotransferase transport system (PTS). Furthermore, evidence for such a cascade effect caused by 2-ketobutyrate and its analogues allowed us to corroborate our previous proposal (Daniel et al. 1983) that 2-ketobutyrate, a precursor of isoleucine, acts as an E. coli alarmone monitoring the passage from anaerobic to aerobic growth conditions.
Adenylyl Cyclase Inhibitors↗
Metabolic alterations mediated by 2-ketobutyrate in Escherichia coli K12.
We have previously proposed that 2-ketobutyrate is an alarmone in Escherichia coli. Circumstantial evidence suggested that the target of 2-ketobutyrate was the phosphoenol pyruvate: glycose phosphotransferase system (PTS). We demonstrate here that the phosphorylated metabolites of the glycolytic pathway experience a dramatic downshift upon addition of 2-ketobutyrate (or its analogues). In particular, fructose-1,6-diphosphate, glucose-6-phosphate, fructose-6-phosphate and acetyl-CoA concentrations drop by a factor of 10, 3, 4, and 5 respectively. This result is consistent with (i) an inhibition of the PTS by 2-ketobutyrate, (ii) a control of metabolism by fructose-1,6-diphosphate. Since fructose-1,6-diphosphate is an activator of phosphoenol pyruvate carboxylase and of pyruvate kinase, the concentration of their common substrate, phosphoenol pyruvate, does not decrease in parallel.
Butyrates↗
Analysis of the ptsH-ptsI-crr region in Escherichia coli K-12: evidence for the existence of a single transcriptional unit.
A cosmid containing the ptsH, ptsI and crr genes of Escherichia coli coding for three components of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) has been isolated. The products of these genes were identified using the maxicell technique. The cloning of the PTS region as a whole allowed us to map and order ptsH, ptsI and crr genes in a 3.8-kb DNA fragment and determine that ptsI and crr are transcribed from a common promoter. They, therefore, constitute a single transcriptional unit which is likely to include also the ptsH gene. In addition, the crr gene may have a secondary promoter located inside ptsI.
Base Sequence↗
Two functional domains in adenylate cyclase of Escherichia coli.
To study the regulation of Escherichia coli adenylate cyclase, plasmids that carry part of the cya gene, as well as plasmids containing hybrid genes having part of cya fused to part of lacZ, were constructed. This allowed us to propose that the enzyme is made of at least two well-defined domains. The NH2 terminus carries the ATP leads to cAMP catalytic activity, whereas the COOH-terminal end corresponds to a regulatory polypeptide domain.
Adenylyl Cyclases↗
Regulation of adenylate cyclase synthesis in Escherichia coli: nucleotide sequence of the control region.
The regulatory region of the cya gene from Escherichia coli has been characterized by nucleotide sequence analysis and genetic approaches. Two promoters, P1 and P2, organized in that order with respect to the beginning of the cya open reading frame, were identified. Using cya-lac operon and protein fusions, it was possible to show that both promoters are active in vivo. P1 activity seemed sensitive to catabolite repression whereas activity of the stronger promoter, P2, did not respond to inhibition by glucose. No effect of cAMP or its receptor, catabolite activator protein (CAP), could be found although the DNA sequence reveals a consensus CAP site downstream of P2. The 548 nucleotides situated at the 3' end of the sequence carry an open reading frame which can tentatively be assigned to the beginning of adenylate cyclase. Among noteworthy features of the corresponding sequence are an UUG codon as the putative start site of cyclase, and a long hydrophobic stretch of amino acids resembling leader peptides in secreted or membrane proteins.
Adenylyl Cyclases↗