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PubMed1993

Nucleotide pool-sensitive selection of the transcriptional start site in vivo at the Salmonella typhimurium pyrC and pyrD promoters.

Expression of the Salmonella typhimurium pyrC and pyrD genes is regulated in response to fluctuations in the intracellular CTP/GTP pool ratio. The repressive mechanism involves the formation of a stable secondary structure (hairpin) at the 5' ends of the transcripts that precludes translational initiation by sequestering sequences required for ribosomal binding. The potential for hairpin formation is controlled through CTP/GTP-modulated selection of the transcriptional start site. Substitution of nucleotides in the region of transcriptional initiation has revealed that selection of the transcriptional start point in vivo depends on the nucleotide context within the initiation region and the nucleoside triphosphate pool ratios. For maximal control in response to CTP/GTP pool ratios, the wild-type CCGG start site motif appears to be optimal. Changing the -35 region in the pyrC promoter to the consensus sequence, or replacement of the pyrC promoter with the lac promoter from Escherichia coli, has served to illustrate that the ability of the RNA polymerase to select the initiation site in response to the intracellular nucleoside triphosphate pools is not promoter specific but is determined by the kinetic properties of the initiating RNA polymerase during the formation of the first phosphodiester bond of the transcript.

Base Sequence↗
PubMed1998

Glucose repression of maltase and methanol-oxidizing enzymes in the methylotrophic yeast Hansenula polymorpha: isolation and study of regulatory mutants.

Regulation of the synthesis of maltase and methanol-oxidizing enzymes by the carbon source has been analyzed in the methylotrophic yeast Hansenula polymorpha. Maltase was shown to be responsible for the growth of H. polymorpha not only on maltose, but also on sucrose. The affinity of maltase towards maltase substrates decreased in the order: 4-nitrophenyl glucoside (PNPG) < sucrose < maltose. Mutants with glucose repression-insensitive synthesis of alcohol oxidase and maltase were obtained from H. polymorpha by mutagenesis and subsequent selection on methanol medium in the presence of 2-deoxy-D-glucose. One of the isolated mutants, L63, was studied in more detail. Mutant L63 was recessive and monogenic and it was not deficient in hexokinase. Its analysis revealed that H. polymorpha most probably has a repressor protein that in the presence of glucose can down-regulate expression of both maltase and enzymes of methanol oxidation.

Alcohol Oxidoreductases↗
PubMed1984

Correlation between oxysterol binding to a cytosolic binding protein and potency in the repression of hydroxymethylglutaryl coenzyme A reductase.

Support for the role of a cytosolic oxysterol-binding protein in the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was obtained by correlating the relative binding affinities of a wide range of oxysterols to their potency in suppressing HMG-CoA reductase activity in mouse fibroblast cell cultures. Forty-seven oxysterols encompassing a 100-fold range of activity in both assays were tested and the two parameters were closely correlated for 35 of the sterols. Twelve sterols showed poor binding when compared to their ability to suppress HMG-CoA reductase activity in cell cultures. Among these were seven sterols with a ketone function at C-3. For this group, the discrepancy could be explained by their rapid conversion within cells to the 3 beta-hydroxy derivatives which have a much higher affinity for the binding protein. One sterol with 3-keto-4-ene grouping was not reduced to its 3 beta-hydroxy derivative in cells and thereby showed no discrepancy in the two assays. The remaining five sterols exhibiting discordant activities in the two tests contained 4,4-dimethyl moieties and were relatively weak suppressors of HMG-CoA reductase activity. Cellular metabolism of these sterols was not detected. Possible reasons for their apparent inactivity in the binding assay are discussed.

Animals↗
PubMed1998

A repressor function for telomerase activity in telomerase-negative immortal cells.

Human telomerase, a ribonucleoprotein that adds TTAGGG repeats onto telomeres and compensates for their shortening, is repressed in most normal human somatic cells. Human somatic cells are considered to have a limited proliferation capacity because of the telomere shortening. Although immortalization of somatic cells is often associated with telomerase reactivation, there are some immortal cells in which telomerase activity is undetectable. In these cells, telomeres may be maintained by an unknown mechanism other than telomerase reactivation. To examine the genetic regulation of telomerase activity, we constructed hybrids between immortal cells with (HepG2) and without (KMST6) telomerase activity. These two cell lines had relatively short and long telomeres, respectively. The hybrid cells continued to proliferate without detectable telomerase activity even after 100 population doublings. Telomerase-positive subpopulations occasionally appeared after serial passages. Southern blot analysis revealed that the hybrids had long terminal restriction fragments similar to that of KMST6, regardless of telomerase activity, and fluorescence in situ hybridization with a telomeric probe showed high-intensity hybridization signals on telomeres, indicating relatively long telomeric repeats. These results suggest that the telomerase-negative immortal cells contain a gene or genes functioning as a telomerase repressor and maintain telomere length by a dominant mechanism other than telomerase reactivation.

Cells, Cultured↗
PubMed1997

A multicomponent insulin response sequence mediates a strong repression of mouse glucose-6-phosphatase gene transcription by insulin.

Glucose-6-phosphatase (G6Pase) catalyzes the final step in the gluconeogenic and glycogenolytic pathways. The transcription of the gene encoding the catalytic subunit of G6Pase is stimulated by glucocorticoids, whereas insulin strongly inhibits both basal G6Pase gene transcription and the stimulatory effect of glucocorticoids. To identify the insulin response sequence (IRS) in the G6Pase promoter through which insulin mediates its action, we have analyzed the effect of insulin on the basal expression of mouse G6Pase-chloramphenicol acetyltransferase (CAT) fusion genes transiently expressed in hepatoma cells. Deletion of the G6Pase promoter sequence between -271 and -199 partially reduces the inhibitory effect of insulin, whereas deletion of additional sequence between -198 and -159 completely abolishes the insulin response. The presence of this multicomponent IRS may explain why insulin potently inhibits basal G6Pase-CAT expression. The G6Pase promoter region between -198 and -159 contains an IRS, since it can confer an inhibitory effect of insulin on the expression of a heterologous fusion gene. This region contains three copies of the T(G/A)TTTTG sequence, which is the core motif of the phosphoenolpyruvate carboxykinase (PEPCK) gene IRS. This suggests that a coordinate increase in both G6Pase and PEPCK gene transcription is likely to contribute to the increased hepatic glucose production characteristic of patients with non-insulin-dependent diabetes mellitus.

Animals↗
PubMed2001

Regulatory functions of serine-46-phosphorylated HPr in Lactococcus lactis.

In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of Lactococcus lactis participates not only in carbon catabolite repression of an operon encoding a beta-glucoside-specific EII and a 6-P-beta-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a ptsH1 mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with L. lactis vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as methyl beta-D-thiogalactoside (TMG) and 2-deoxy-D-glucose (2-DG). In vivo experiments with the ptsH1 mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and ptsH1 mutant strains, whereas TMG expulsion was slowed in the ptsH1 mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.

Bacterial Proteins↗
PubMed2000

Regulation of transcription of the mph(A) gene for macrolide 2'-phosphotransferase I in Escherichia coli: characterization of the regulatory gene mphR(A).

The synthesis of macrolide 2'-phosphotransferase I [Mph(A)], which inactivates erythromycin, is inducible by erythromycin. The expression of high-level resistance to erythromycin requires the mph(A) and mrx genes, which encode Mph(A) and an unidentified protein, respectively. We have studied the mphR(A) gene, which regulates the inducible expression of mph(A). An analysis of the synthesis of Mph(A) in minicells and results of a complementation test indicated that mphR(A) is located downstream from mrx and that its product, MphR(A), represses the production of Mph(A). DNA sequencing indicated that the mph(A), mrx, and mphR(A) genes exist as a cluster that begins with mph(A) and that the deduced amino acid sequence of MphR(A) can adopt an alpha-helix-turn-alpha-helix structure. To study the regulation of gene expression by MphR(A), we performed Northern blotting and primer extension. A transcript of 2. 9 kb that corresponded to the transcript of mph(A) through mphR(A) was detected, and its level was elevated upon exposure of cells to erythromycin. Gel mobility shift assays and DNase I footprinting indicated that MphR(A) binds specifically to the promoter region of mph(A), and the amount of DNA shifted as a results of the binding of MphR(A) decreased as the concentration of erythromycin was increased. These results indicate that transcription of the mph(A)-mrx-mphR(A) operon is negatively regulated by the binding of a repressor protein, MphR(A), to the promoter of the mph(A) gene and is activated upon inhibition of binding of MphR(A) to the promoter in the presence of erythromycin.

Amino Acid Sequence↗
PubMed1995

The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition.

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of beta-galactosidase with symmetric variants of alpha- and beta-centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the alpha-centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the alpha-centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. A180, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for alpha-centered trp operator variants with exchanges in positions 3, 4 and 5.

Amino Acid Sequence↗
PubMed1992

Positive regulation of the LPD1 gene of Saccharomyces cerevisiae by the HAP2/HAP3/HAP4 activation system.

The LPD1 gene of Saccharomyces cerevisiae, encoding lipoamide dehydrogenase (LPDH), is subject to catabolite repression. The promoter of this gene contains a number of motifs for DNA-binding transcriptional activators, including three which show strong sequence homology to the core HAP2/HAP3/HAP4 binding motif. Here we report that transcription of LPD1 requires HAP2, HAP3 and HAP4 for release from glucose repression. In the wild-type strain, specific activity of LPDH was increased 12-fold by growth on lactate, 10-fold on glycerol and four- to five-fold on galactose or raffinose, compared to growth on glucose. In hap2, hap3 and hap4 null mutants, the specific activities of LPDH in cultures grown on galactose and raffinose showed only slight induction above the basal level on glucose medium. Similar results were obtained upon assaying for beta-galactosidase production in wild-type, or hap2, hap3 or hap4 mutant strains carrying a single copy of the LPD1 promoter fused in frame to the lacZ gene of Escherichia coli and integrated at the URA3 locus. Transcript analysis in wild-type and hap2 mutants confirmed that the HAP2 protein regulates LPD1 expression at the level of transcription in the same way as it does for the CYC1 gene. Site-directed mutagenesis of the putative HAP2/HAP3/HAP4 binding site at -204 relative to the ATG start codon showed that this element was required for full derepression of the LPD1 gene on non-fermentable substrates.

Base Sequence↗
PubMed1968

Control of amino sugar metabolism in Escherichia coli and isolation of mutants unable to degrade amino sugars.

1. Growth of Escherichia coli on glucosamine results in an induction of glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] and a repression of glucosamine 6-phosphate synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16); glucose abolishes these control effects. 2. Growth of E. coli on N-acetylglucosamine results in an induction of N-acetylglucosamine 6-phosphate deacetylase and glucosamine 6-phosphate deaminase, and in a repression of glucosamine 6-phosphate synthetase; glucose diminishes these control effects. 3. The synthesis of amino sugar kinases (EC 2.7.1.8 and 2.7.1.9) is unaffected by growth on amino sugars. 4. Glucosamine 6-phosphate synthetase is inhibited by glucosamine 6-phosphate. 5. Mutants of E. coli that are unable to grow on N-acetylglucosamine have been isolated, and lack either N-acetylglucosamine 6-phosphate deacetylase (deacetylaseless) or glucosamine 6-phosphate deaminase (deaminaseless). Deacetylaseless mutants can grow on glucosamine but deaminaseless mutants cannot. 6. After growth on glucose, deacetylaseless mutants have a repressed glucosamine 6-phosphate synthetase and a super-induced glucosamine 6-phosphate deaminase; this may be related to an intracellular accumulation of acetylamino sugar that also occurs under these conditions. In one mutant the acetylamino sugar was shown to be partly as N-acetylglucosamine 6-phosphate. Deaminaseless mutants have no abnormal control effects after growth on glucose. 7. Addition of N-acetylglucosamine or glucosamine to cultures of a deaminaseless mutant caused inhibition of growth. Addition of N-acetylglucosamine to cultures of a deacetylaseless mutant caused lysis, and secondary mutants were isolated that did not lyse; most of these secondary mutants had lost glucosamine 6-phosphate deaminase and an uptake mechanism for N-acetylglucosamine. 8. Similar amounts of (14)C were incorporated from [1-(14)C]-glucosamine by cells of mutants and wild-type growing on broth. Cells of wild-type and a deaminaseless mutant incorporated (14)C from N-acetyl[1-(14)C]glucosamine more efficiently than from N[1-(14)C]-acetylglucosamine, incorporation from the latter being further decreased by acetate; cells of a deacetylaseless mutant showed a poor incorporation of both types of labelled N-acetylglucosamine.

Bacteriolysis↗
PubMed1999

The amino-terminal 100 residues of the nitrogen assimilation control protein (NAC) encode all known properties of NAC from Klebsiella aerogenes and Escherichia coli.

The nitrogen assimilation control protein (NAC) from Klebsiella aerogenes or Escherichia coli (NACK or NACE, respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (hut) operon and to repress transcription of the glutamate dehydrogenase (gdh) operon in K. aerogenes. Truncated NAC polypeptides, generated by the introduction of stop codons within the nac open reading frame, were tested for the ability to activate hut and repress gdh in vivo. Most of the NACK and NACE fragments with 100 or more amino acids (wild-type NACK and NACE both have 305 amino acids) were functional in activating hut and repressing gdh expression in vivo. Full-length NACK and NACE were isolated as chimeric proteins with the maltose-binding protein (MBP). NACK and NACE released from such chimeras were able to activate hut transcription in a purified system in vitro, as were NACK129 and NACE100 (a NACK fragment of 129 amino acids and a NACE fragment of 100 amino acids) released from comparable chimeras. A set of NACE and NACK fragments carrying nickel-binding histidine tags (his6) at their C termini were also generated. All such constructs derived from NACE were insoluble, as was NACE itself. Of the his6-tagged constructs derived from NACK, NACK100 was inactive, but NACK120 was active. Several NAC fragments were tested for dimerization. NACK120-his6 and NACK100-his6 were dimers in solution. MBP-NACK and MBP-NACK129 were monomers in solution but dimerized when the MBP was released by cleavage with factor Xa. MBP-NACE was readily cleaved by factor Xa, but the resulting NACE was also degraded by the protease. However, MBP-NACE-his6 was completely resistant to cleavage by factor Xa, suggesting an interaction between the C and N termini of this protein.

Bacterial Proteins↗
PubMed1977

Cro regulatory protein specified by bacteriophage lambda. Structure, DNA-binding, and repression of RNA synthesis.

The Cro protein specified by bacteriophage lambda is a repressor of the genes expressed early in phage development and is required for a normal late stage of lytic growth. We have purified Cro protein to virtual homogeneity and analyzed its structure and properties as a DNA-binding protein and repressor of RNA synthesis. To confirm that the protein is the product of the cro gene, we have also shown that a missense mutation in the cro gene leads to a product that is more temperature- and salt-sensitive in its DNA-binding property. As purified, Cro protein is a dimer of identical subunits of molecular weight 8600. The purified protein binds to lambda-DNA carrying the specific binding sites (operators oL and oR) with an estimated dissociation constant of 10(-10) M to 10(-11) M; there is also weaker binding to other sites on DNA, as found for other DNA-binding regulatory proteins. In a purified transcription system, the Cro protein is an effective and specific repressor of RNA synthesis from the N and cro genes; thus Cro is an autorepressor which regulates its own synthesis. A comparison of the properties of the two lambda repressor proteins, cI and Cro, indicates that cI is a "strong repressor" specialized for complete turnoff of lytic functions needed for the maintenance of lysogeny, whereas Cro is a "weak repressor" specialized for a gradual turnoff of early viral genes that potentiates the late stage of lytic development.

Amino Acids↗
PubMed1999

Rapid repression of maize invertases by low oxygen. Invertase/sucrose synthase balance, sugar signaling potential, and seedling survival.

We show here that invertase gene expression and the invertase-sucrose (Suc) synthase ratio decrease abruptly in response to low oxygen in maize root tips. In addition to aiding in the conservation of carbon and possibly ATP, this response has the potential to directly affect sugar signaling relative to carbon flux. Experiments were motivated by the potential for a reduced invertase/Suc synthase balance to alter the impact of respiratory and/or membrane carbon flux on sugar signaling. Maize (Zea mays L.) seedlings with 5-cm primary roots were exposed to anoxic (0% [v/v] O2), hypoxic (3% [v/v] O2), and aerobic conditions. Rapid repression of the Ivr1 and Ivr2 maize invertases by low oxygen was evident in root tips within 3 h at both the transcript and activity levels. The speed and extent of this response increased with the degree of oxygen deprivation and differed with genotypes. This decrease in expression also contrasted markedly to that of other genes for respiratory Suc metabolism, such as Suc synthases, which typically increased or remained constant. Although previous work showed that the contrasting effects of sugars on Suc synthase genes were reflected in their regulation by hypoxia and anoxia, the same was not observed for the differentially sugar-responsive invertases. Theoretically advantageous reductions in the invertase/Suc synthase balance thus resulted. However, where this response was extreme (an Oh43 inbred), total sucrolytic capacity dropped below an apparent minimum and root tip viability was reduced. Paradoxically, only Oh43 seedlings showed survival levels >80% (versus <50%) after extreme, long-term stress, suggesting a possible advantage for multiple means of reducing sink activity. Overall, our results demonstrate a rapid change in the regulation and balance of invertases and Suc synthases that could have an immediate impact on limiting the extent of Suc cleavage and reducing the extent of concomitant, hexose-based sugar signaling under low oxygen.

Enzyme Repression↗
PubMed1999

Tissue-specific regulation of the ecto-5'-nucleotidase promoter. Role of the camp response element site in mediating repression by the upstream regulatory region.

We have isolated the 5' region of the ecto-5'-nucleotidase (low K(m) 5'-NT) gene and established that a 969-base pair (bp) fragment confers cell-specific expression of a CAT reporter gene that correlates with the expression of endogenous ecto-5'-NT mRNA and enzymatic activity. A 768-bp upstream negative regulatory region has been identified that conferred lymphocyte-specific negative regulation in a heterologous system with a 244-bp deoxycytidine kinase core promoter. DNase I footprinting identified several protected areas including Sp1, Sp1/AP-2, and cAMP response element (CRE) binding sites within the 201-bp core promoter region and Sp1, NRE-2a, TCF-1/LEF-1, and Sp1/NF-AT binding sites in the upstream regulatory region. Whereas the CRE site was essential in mediating the negative activity of the upstream regulatory region in Jurkat but not in HeLa cells, mutation of the Sp1/AP-2 site decreased promoter activity in both cell lines. Electrophoretic mobility shift assay analysis of proteins binding to the CRE site identified both ATF-1 and ATF-2 in Jurkat cells. Finally, phorbol 12-myristate 13-acetate increased the activity of both the core and the 969-bp promoter fragments, and this increase was abrogated by mutations at the CRE site. In summary, we have identified a tissue-specific regulatory region 5' of the ecto-5'-NT core promoter that requires the presence of a functional CRE site within the basal promoter for its suppressive activity.

5'-Nucleotidase↗
PubMed1992

Analysis of glucose repression in Saccharomyces cerevisiae by pulsing glucose to a galactose-limited continuous culture.

In this study, glucose repression in Saccharomyces cerevisiae was analysed under defined physiological conditions, at both the molecular and physiological levels, by pulsing glucose to a galactose-limited continuous culture. During this pulse of glucose, the galactose feed was kept constant. Directly after the glucose pulse, carbon dioxide production increased while oxygen consumption remained constant, demonstrating that the surplus of glucose had been consumed by means of fermentation. The direct accumulation of galactose in the medium after the glucose pulse indicated that the consumption of galactose had been stopped instantaneously. Galactose uptake experiments revealed that the galactose transporter was still present but apparently was incapable of galactose uptake, which could be due to inhibition of the galactose transporter by glucose. The total concentration of cAMP increased from 5 nmol g-1 at t = 0 to 25 nmol g-1 at t = 1.5 min. After 2 min the concentration of cAMP gradually decreased again to the normal level. Within 2 min after the addition of glucose, the transcription of the GAL genes and SUC2 was inhibited. In addition, the transcription of the HXK1 gene, encoding hexokinase isoenzyme 1, was also inhibited, which demonstrates that the HXK1 gene is regulated at the transcriptional level comparable with invertase.

Adenosine Triphosphate↗
PubMed1998

Structure-based redesign of corepressor specificity of the Escherichia coli purine repressor by substitution of residue 190.

Guanine or hypoxanthine, physiological corepressors of the Escherichia coli purine repressor (PurR), promote formation of the ternary PurR-corepressor-operator DNA complex that functions to repress pur operon gene expression. Structure-based predictions on the importance of Arg190 in determining 6-oxopurine specificity and corepressor binding affinity were tested by mutagenesis, analysis of in vivo function, and in vitro corepressor binding measurements. Replacements of Arg190 with Ala or Gln resulted in functional repressors in which binding of guanine and hypoxanthine was retained but specificity was relaxed to permit binding of adenine. X-ray structures were determined for ternary complexes of mutant repressors with purines (adenine, guanine, hypoxanthine, and 6-methylpurine) and operator DNA. These structures indicate that R190A binds guanine, hypoxanthine, and adenine with nearly equal, albeit reduced, affinity in large part because of a newly made compensatory hydrogen bond between the rotated hydroxyl side chain of Ser124 and the exocyclic 6 positions of the purines. Through direct and water-mediated contacts, the R190Q protein binds adenine with a nearly 75-fold higher affinity than the wild type repressor while maintaining wild type affinity for guanine and hypoxanthine. The results establish at the atomic level the basis for the critical role of Arg190 in the recognition of the exocyclic 6 position of its purine corepressors and the successful redesign of corepressor specificity.

Adenine↗
PubMed1993

Negative regulation of sigma 54-dependent dctA expression by the transcriptional activator DctD.

In Rhizobium meliloti, the presence of the C4-dicarboxylate transport protein DctA is required for symbiotic N2 fixation in alfalfa root nodules. Expression of dctA is inducible and is mediated by a sensor and activator gene pair encoded by dctB and dctD. In the presence of C4-dicarboxylates, the DCTB sensor protein is believed to phosphorylate and activate DCTD, which in turn activates transcription at the sigma 54-dependent dctA promoter. Here, we present evidence that in addition to activating dctA transcription, DCTD can also repress expression of dctA. By employing an ntrC allele, ntrC283, whose product appears to activate dctA transcription independently of DCTD, we found that while ntrC283 leads to constitutive dctA expression in the absence of dctB and dctD, in a dctB+ dctD+ ntrC283 background high-level expression of dctA occurred in succinate but not in glucose-grown cells. This result suggested that in uninduced cells, inactive DCTD binds to the dctA promoter and prevents its activation by NTRC283. Consistent with the latter interpretation was the observation that overexpression of DCTD from a plasmid promoter prevents dctA expression and results in a Dct- phenotype. Moreover the Dct- phenotype resulting from the overexpression of dctD was dominant to ntrC283. Results from studies of the ability of ntrC283 to suppress the Dct- phenotype of dctB alleles, together with the finding that the Fix- phenotype of a particular dctB allele was dctD dependent, suggest that in particular dctB alleles, sufficient dctD transcription occurs such that the resulting inactive DCTD prevents activation of dctA transcription by NtrC283 or alternate symbiotic regulators. The latter suggestion is supported by the observation that in symbiosis, R. meliloti strains in which DCTD was overexpressed formed nodules which failed to fix nitrogen.

Alleles↗
PubMed2004

Repression of nodal expression by maternal B1-type SOXs regulates germ layer formation in Xenopus and zebrafish.

B1-type SOXs (SOXs 1, 2, and 3) are the most evolutionarily conserved subgroup of the SOX transcription factor family. To study their maternal functions, we used the affinity-purified antibody antiSOX3c, which inhibits the binding of Xenopus SOX3 to target DNA sequences [Development. 130(2003)5609]. The antibody also cross-reacts with zebrafish embryos. When injected into fertilized Xenopus or zebrafish eggs, antiSOX3c caused a profound gastrulation defect; this defect could be rescued by the injection of RNA encoding SOX3DeltaC-EnR, a SOX3-engrailed repression domain chimera. In antiSOX3c-injected Xenopus embryos, normal animal-vegetal patterning of mesodermal and endodermal markers was disrupted, expression domains were shifted toward the animal pole, and the levels of the endodermal markers SOX17 and endodermin increased. In Xenopus, SOX3 acts as a negative regulator of Xnr5, which encodes a nodal-related TGFbeta-family protein. Two nodal-related proteins are expressed in the early zebrafish embryo, squint and cyclops; antiSOX3c-injection leads to an increase in the level of cyclops expression. In both Xenopus and zebrafish, the antiSOX3c phenotype was rescued by the injection of RNA encoding the nodal inhibitor Cerberus-short (CerS). In Xenopus, antiSOX3c's effects on endodermin expression were suppressed by injection of RNA encoding a dominant negative version of Mixer or a morpholino against SOX17alpha2, both of which act downstream of nodal signaling in the endoderm specification pathway. Based on these data, it appears that maternal B1-type SOX functions together with the VegT/beta-catenin system to regulate nodal expression and to establish the normal pattern of germ layer formation in Xenopus. A mechanistically conserved system appears to act in a similar manner in the zebrafish.

Amino Acid Sequence↗