Negative Control Element Research Articles

Upregulation of human heme oxygenase gene expression by Ets-family proteins

Overexpression of human heme oxygenase-1 has been shown to have the potential to promote EC proliferation and angiogenesis. Since Ets-family proteins have been shown to play an important role in angiogenesis, we investigated the presence of ETS binding sites (EBS), GGAA/T, and ETS protein contributing to human HO-1 gene expression. Several chloramphenicol acetyltransferase constructs were examined in order to analyze the effect of ETS family proteins on the transduction of HO-1 in Xenopus oocytes and in microvessel endothelial cells. Heme oxygenase promoter activity was up-regulated by FLI-1ERGETS-1 protein(s). Chloramphenicol acetyltransferase (CAT) assays demonstrated that the promoter region (-1500 to +19) contains positive and negative control elements and that all three members of the ETS protein family were responsible for the up-regulation of HHO-1. Electrophoretic mobility shift assays (EMSA), performed with nuclear extracts from endothelial cells overexpressing HHO-1 gene, and specific HHO-1 oligonucleotides probes containing putative EBS resulted in a specific and marked bandshift. Synergistic binding was observed in EMSA between AP-1 on the one hand, FLI-1, ERG, and ETS-1 protein on the other. Moreover, 5'-deletion analysis demonstrated the existence of a negative control element of HHO-1 expression located between positions -1500 and -120 on the HHO-1 promoter. The presence of regulatory sequences for transcription factors such as ETS-1, FLI-1, or ERG, whose activity is associated with cell proliferation, endothelial cell differentiation, and matrix metalloproteinase transduction, may be an indication of the important role that HO-1 may play in coronary collateral circulation, tumor growth, angiogenesis, and hemoglobin-induced endothelial cell injuries.

Negative control elements of the cell cycle in human tumors

The retinoblastoma protein and p53 are both cell-cycle regulators and are, directly or indirectly, inactivated in the majority of human tumors. Recent studies have provided new mechanistic insights into how these proteins regulate cell growth in response to various intracellular and extracellular signals.

In vivo definition of the functional origin of leading strand replication on the lactococcal plasmid pFX2.

The lactococcal plasmid pFX2 belongs to a family of plasmids, whose prototype is the streptococcal plasmid pMV158, that replicates by the rolling circle mechanism. Determination of the nucleotide sequence of the repX gene of pFX2 allowed us to make some minor corrections in the published sequence, and to show that the repX gene is identical to the rep gene of plasmid pWV01. We have established pFX2 in Escherichia coli and in Streptococcus pneumoniae. In the latter host, we have defined in vivo the nick site introduced by the RepX protein. Plasmid pFX2 and the pMV158 derivative pLS1 exhibit a moderate degree of incompatibility in S. pneumoniae. Cloning of the double strand origin (dso) of pFX2 into a high-copy-number plasmid that is compatible with the pMV158 replicon led to an increase in incompatibility toward pLS1. Plasmids pFX2 and pLS1 exhibit homologies in their Rep proteins and in their dso sequences, but not in their negative control elements. Thus, the observed incompatibility indicates that cross-recognition of Rep proteins and dso takes place.

Overexpression Beadex mutations and loss-of-function heldup-a mutations in Drosophila affect the 3' regulatory and coding components, respectively, of the Dlmo gene.

LIM domains function as bridging modules between different members of multiprotein complexes. We report the cloning of a LIM-containing gene from Drosophila, termed Dlmo, which is highly homologous to the vertebrate LIM-only (LMO) genes. The 3' untranslated (UTR) of Dlmo contains multiple motifs implicated in negative post-transcriptional regulation, including AT-rich elements and Brd-like boxes. Dlmo resides in polytene band 17C1-2, where Beadex (Bx) and heldup-a (hdp-a) mutations map. We demonstrate that Bx mutations disrupt the 3'UTR of Dlmo, and thereby abrogate the putative negative control elements. This results in overexpression of Dlmo, which causes the wing scalloping that is typical of Bx mutants. We show that the erect wing phenotype of hdp-a results from disruption of the coding region of Dlmo. This provides molecular grounds for the suppression of the Bx phenotype by hdp-a mutations. Finally, we demonstrate phenotypic interaction between the LMO gene Dlmo, the LIM homeodomain gene apterous, and the Chip gene, which encodes a homolog of the vertebrate LIM-interacting protein NLI/Ldb1. We propose that in analogy to their vertebrate counterparts, these proteins form a DNA-binding complex that regulates wing development.

Ryanodine receptors regulate arterial diameter and wall [Ca2+] in cerebral arteries of rat via Ca2+-dependent K+ channels.

1. The effects of inhibitors of ryanodine-sensitive calcium release (RyR) channels in the sarcoplasmic reticulum (SR) and Ca2+-dependent potassium (KCa) channels on the membrane potential, intracellular [Ca2+], and diameters of small pressurized (60 mmHg) cerebral arteries (100-200 micron) were studied using digital fluorescence video imaging of arterial diameter and wall [Ca2+], combined with microelectrode measurements of arterial membrane potential. 2. Ryanodine (10 microM), an inhibitor of RyR channels, depolarized by 9 mV, increased intracellular [Ca2+] by 46 nM and constricted pressurized (to 60 mmHg) arteries with myogenic tone by 44 micron (approximately 22 %). Iberiotoxin (100 nM), a blocker of KCa channels, under the same conditions, depolarized the arteries by 10 mV, increased arterial wall calcium by 51 nM, and constricted by 37 micron (approximately 19 %). The effects of ryanodine and iberiotoxin were not additive and were blocked by inhibitors of voltage-dependent Ca2+ channels. 3. Caffeine (10 mM), an activator of RyR channels, transiently increased arterial wall [Ca2+] by 136 +/- 9 nM in control arteries and by 158 +/- 12 nM in the presence of iberiotoxin. Caffeine was relatively ineffective in the presence of ryanodine, increasing [calcium] by 18 +/- 5 nM. 4. In the presence of blockers of voltage-dependent Ca2+ channels (nimodipine, diltiazem), ryanodine and inhibitors of the SR calcium ATPase (thapsigargin, cyclopiazonic acid) were without effect on arterial wall [Ca2+] and diameter. 5. These results suggest that local Ca2+ release originating from RyR channels (Ca2+ sparks) in the SR of arterial smooth muscle regulates myogenic tone in cerebral arteries solely through activation of KCa channels, which regulate membrane potential through tonic hyperpolarization, thus limiting Ca2+ entry through L-type voltage-dependent Ca2+ channels. KCa channels therefore act as a negative feedback control element regulating arterial diameter through a reduction in global intracellular free [Ca2+].

Prohibitin, a putative negative control element present in Pneumocystis carinii.

Little is known about the molecules involved in the regulation of Pneumocystis carinii replication and development in vitro and in vivo. We describe in this report the identification of a P. carinii gene encoding the P. carinii prohibitin protein. In mammals, the prohibitin gene product has been shown to negatively regulate cell proliferation. A cDNA clone encoding the P. carinii prohibitin gene was isolated from a P. carinii cDNA library and identified on the basis of amino acid sequence homology with prohibitin from mammalian sources. Southern blot analysis confirmed that the prohibitin cDNA clone was of P. carinii origin. Western blot analysis of total P. carinii protein indicated that the prohibitin gene is transcribed and translated in vivo. The P. carinii prohibitin gene was expressed in vivo in human fibroblasts and shown to arrest the cell cycle in the G1 phase. The results obtained suggest a potential role for P. carinii prohibitin in the regulation of P. carinii proliferation and development.

The apolipoprotein A-I/C-III/A-IV gene cluster: ApoC-III and ApoA-IV expression is regulated by two common enhancers

Genetic, epidemiological and clinical evidence have clearly demonstrated the importance of the human apolipoprotein (apo) A-I/C-III/A-IV gene cluster in lipid metabolism and heart attack. The transcriptional regulation of these genes determines the level of the encoded proteins and therefore influences the concentration of triglycerides and cholesterol. Here, we analyze the existence of transcription control elements in the 6.6 kb apoC-III/A-IV intergenic region and their influence on the expression of both genes. Two main positive common control elements were found to modulate apoC-III and apoA-IV expression in HepG2 and in Caco-2 cells: the previously described apoC-III enhancer, located 0.8 kb upstream from the cap site of the gene, and a newly detected activating region located in the center of the intergenic sequence. The activity of both elements is highly increased by the hepatic and intestinal transcription factor HNF-4. Analysis of a 641 bp fragment containing the central element showed that it has the properties of a tissue-specific enhancer. Liver nuclear proteins interact with seven DNA binding sites present in this enhancer and HNF-4 specifically interacts with one of these sites. A third positive element, situated immediately upstream from the apoA-IV minimal promoter, is also activated by HNF-4; however, this element is not involved in apoC-III expression. In addition, two negative regions were identified, one located near the apoA-IV gene and the other one between the apoC-III enhancer and the newly identified central enhancer. In conclusion, negative and positive control elements are located in the apoC-III/A-IV intergenic region, including two enhancers important for the expression of the two genes. These results add new evidence that common regulatory elements for the expression of the apoA-I, apoC-III and apoA-IV genes are interspersed throughout the cluster.

Structural analysis of the proximal region of the microtubule-associated protein 1B promoter.

Microtubule-associated protein 1B (MAP1B) is a major cytoskeletal protein expressed early during development of the nervous system. Previous analysis of the MAP1B gene has identified two alternative promoters that can independently regulate neuron-specific expression of MAP1B. To further characterize the MAP1B promoters, we performed DNase I hypersensitivity assays in vivo over a range of 8.5 kb surrounding the transcription initiation sites. These studies identified a DNase I-hypersensitive site that was present in brain but not liver nuclei at the proximal region of the MAP1B promoter, located between the two transcription initiation sites. Fine mapping by S1 nuclease sensitivity localized two adjacent sites in the proximal promoter region that contained three symmetrical inverted repeats. Electrophoresis mobility shift assays showed that proteins present in nuclear extracts can bind two consensus regulatory elements present within the proximal promoter region, Sp1 and cyclic AMP response element. In addition, there was a specific nuclear protein binding activity with two common sequences, a "neuronal motif " and a TCC repeat motif. This binding activity was much more abundant in liver than in brain nuclear extracts, suggesting that it may represent a negative control element in the tissue-specific expression of the MAP1B gene.

The proximal promoter region of the gene encoding human 17beta-hydroxysteroid dehydrogenase type 1 contains GATA, AP-2, and Sp1 response elements: analysis of promoter function in choriocarcinoma cells.

The 5'-flanking region from -78 to +9 in the HSD17B1 gene serves as a promoter, and an HSD17B1 silencer element is located in position -113 to -78. In the present studies, we have characterized three regulatory elements in the proximal 5'-flanking regions of the gene, using electrophoretic mobility shift assays and reporter gene analysis. First, nuclear factors recognized by antibodies against Sp1 and Sp3 were found to bind the Sp1 motif in the region from -52 to -43. Mutation of the Sp1-binding site decreased the promoter activity to 30% in JEG-3 cells and to 60% in JAR cells, suggesting that binding to the Sp1 motif has a substantial role in the complete functioning of the HSD17B1 promoter. Second, the binding of AP-2 to its motif in the region from -62 to -53 led to reduced binding of Sp1 and Sp3, and furthermore, mutation of the AP-2 element increased promoter activity to 260% in JEG-3 cells. The data thus implied that AP-2 can repress the function of the HSD17B1 promoter by preventing binding to the Sp1 motif. Finally, GATA factors, GATA-3 in particular, were demonstrated to bind their cognate sequence in the HSD17B1 silencer region, and mutations introduced into the GATA-binding site increased transcriptional activity to the level seen in constructs not containing the silencer element. Thus, GATA-3 seems to prevent transcription in the constructs, and hence, the GATA motif also may operate as a negative control element for HSD17B1 transcription.

The Proximal Promoter Region of the Gene Encoding Human 17 -Hydroxysteroid Dehydrogenase Type 1 Contains GATA, AP-2, and Sp1 Response Elements: Analysis of Promoter Function in Choriocarcinoma Cells

The 5′-flanking region from −78 to +9 in the HSD17B1 gene serves as a promoter, and an HSD17B1 silencer element is located in position −113 to −78. In the present studies, we have characterized three regulatory elements in the proximal 5′-flanking regions of the gene, using electrophoretic mobility shift assays and reporter gene analysis. First, nuclear factors recognized by antibodies against Sp1 and Sp3 were found to bind the Sp1 motif in the region from −52 to −43. Mutation of the Sp1-binding site decreased the promoter activity to 30% in JEG-3 cells and to 60% in JAR cells, suggesting that binding to the Sp1 motif has a substantial role in the complete functioning of the HSD17B1 promoter. Second, the binding of AP-2 to its motif in the region from −62 to −53 led to reduced binding of Sp1 and Sp3, and furthermore, mutation of the AP-2 element increased promoter activity to 260% in JEG-3 cells. The data thus implied that AP-2 can repress the function of the HSD17B1 promoter by preventing binding to the Sp1 motif. Finally, GATA factors, GATA-3 in particular, were demonstrated to bind their cognate sequence in the HSD17B1 silencer region, and mutations introduced into the GATA-binding site increased transcriptional activity to the level seen in constructs not containing the silencer element. Thus, GATA-3 seems to prevent transcription in the constructs, and hence, the GATA motif also may operate as a negative control element for HSD17B1 transcription.

Identification of an intronic enhancer that nullifies upstream repression of SPARC gene expression.

The SPARC gene 5'flanking sequence has been shown to contain enhancer elements, but also negative control elements immediately upstream of the enhancer elements. Although these 5'enhancer elements are active in F9 and PYS-2 cells, their activities are nullified by the 5'repressor activity. In the present study we have identified within intron 1 between nucleotides (nt) +5000 and +5150 of the SPARC gene an enhancer element that bound to two transcription factors of 48 and 52 kDa and between nt +5000 and +5523 a DNase I hypersensitive site. Furthermore, a region containing the 3'intron 1 enhancer element, together with the 5'enhancer elements, neutralized the 5'repressor activity and stimulated efficient transcription. The resulting SPARC promoter activity is about equal in F9, differentiated F9 and PYS-2 cells. We consistently found that the rate of SPARC transcription is nearly the same in F9 and PYS-2 cells. Association of the 3'enhancer element in intron 1 with the DNase I hypersensitive site suggests that both play a role in regulating SPARC expression in vivo .

Regulation of the inducible acetamidase gene of Mycobacterium smegmatis.

The inducible acetamidase of Mycobacterium smegmatis NCTC 8159 is expressed at high levels in the presence of a suitable inducer, such as acetamide. The gene and 1.5 kb of upstream sequence had previously been sequenced. A further 1.4 kb of upstream sequence has now been determined, containing an additional ORF on the opposite strand to the acetamidase gene. This ORF has significant homologies to genes encoding regulatory proteins involved in amidase expression in other organisms. Restriction fragments from the 4 kb region were subcloned into a promoter-probe shuttle vector to locate the approximate region of the acetamidase promoter and investigate the mechanism of regulation. An inducible promoter was found to lie in the 1.4 kb region situated 1.5 kb upstream from the acetamidase coding region. Expression of the acetamidase was studied at the protein and mRNA levels. Using immunoblotting, induction of the enzyme was demonstrated in minimal medium containing succinate plus acetamide, but not in a richer medium (Lemco broth) plus acetamide, confirming that regulation of acetamidase expression is mediated by both positive and negative control elements. After induction by acetamide, an increase above basal level could be detected after 1 h for both protein levels (using ELISA) and mRNA levels (using Northern blot analysis), indicating that control of expression is at the mRNA level. The size of the mRNA transcript detected was approximately 1.2 kb, the size of the acetamidase coding region. Since no promoter was identified immediately upstream of the coding region, this raises the possibility that a larger, primary transcript (possibly polycistronic) is cleaved to produce a stable form encoding the acetamidase protein.

Endocytosis of the rat somatostatin receptors: subtype discrimination, ligand specificity, and delineation of carboxy-terminal positive and negative sequence motifs.

Endocytosis of the five rat somatostatin receptor subtypes (SSTR1-5) was investigated in transfected HEK cells by biochemical ligand binding assays and confocal microscopic analysis. Phenylarsine oxide-sensitive internalization of SSTR1-3 is dependent on SST-14 or SST-28, whereas only the octacosapeptide triggers this reaction with SSTR5. SSTR4 is not internalized with either SST. Internalized SSTR3 is cycled back to the plasma membrane while endocytosed rho-Ala1-SST-14 remains inside the cell. Delineation of sequence motifs responsible for internalization of SSTR3 revealed multiple serines and a threonine (Ser-341, Ser-346, Ser-351, and Thr-357) within the carboxy-terminal tail of which Ser-351 and Thr-357 were the most effective ones. Chimeras in which various segments of the carboxyl terminus of SSTR4 were replaced by the corresponding regions of SSTR3 were internalized as long as they contain the Ser/Thr motif. However, this internalization reaction was suppressed when the chimeras were extended by the carboxyl terminus of SSTR4 (residues 320-384), suggesting the presence of a negative control element in that region. Step-wise truncation of the carboxyl terminus of wild-type SSTR4 revealed a motif of three amino acid residues Glu-Thr-Thr (SSTR4-330-332) that is responsible for preventing internalization and may be important in regulating endocytosis of this receptor subtype.

Programmed cell death in bacteria: translational repression by mRNA end-pairing.

The hok/sok and pnd systems of plasmids R1 and R483 mediate plasmid maintenance by killing plasmid-free cells. Translation of the exceptionally stable hok and pnd mRNAs is repressed by unstable antisense RNAs. The different stabilities of the killer mRNAs and their cognate repressors explain the onset of translation in plasmid-free cells. The full-length hok and pnd mRNAs are inert with respect to translation and antisense RNA binding. We have previously shown that the mRNAs contain two negative translational control elements. Thus, the mRNAs contain upstream anti-Shine-Dalgarno elements that repress translation by shielding the Shine-Dalgarno elements. The mRNAs also contain fold-back-inhibition elements (fbi) at their 3' ends that are required to maintain the inert mRNA configuration. Using genetic complementation, we show that the 3' fbi elements pair with the very 5' ends of the mRNAs. This pairing sets the low rate of 3' exonucleolytical processing, which is required for the accumulation of an activatable pool of mRNA. Unexpectedly, the hok and pnd mRNAs were found to contain translational activators at their 5' ends (termed tac). Thus, the fbi elements inhibit translation of the full-length mRNAs by sequestration of the tac elements. The fbi elements are removed by 3' exonucleolytical processing. Mutational analyses indicate that the 3' processing triggers refolding of the mRNA 5' ends into translatable configurations in which the 5' tac elements base pair with the anti-Shine-Dalgarno sequences.

Molecules inhibiting neurite growth: a minireview.

Molecules and activities which repulse growing neurites or induce growth cone collapse and long-lasting growth inhibition have been defined over the last 10 years. Recently, specific guidance roles for developing axons and pathways could be associated with such repulsive effects. A high molecular weight membrane protein located in CNS myelin acts as potent neurite growth inhibitor and may play a role as a negative control element for sprouting, neurite growth and regeneration, and for the plasticity of the adult CNS. Interestingly, some guidance molecules can have positive, growth-promoting as well as negative, repulsive effects for specific types of neurons. These results underline the complex mechanisms involved in neurite guidance which depends on the interpretation of combinations of incoming signals by particular growth cones.

Characterization of the human XPA promoter

We cloned the human xeroderma pigmentosum group A gene ( XPA) and characterized the XPA promoter ( pXPA) by transient cat expression. The pXPA is extraordinarily weak in human fibroblasts (1% of RSV-LTR) and appears to function without any of the usual promoter elements. Regions containing positive and negative control elements were localized.

Promoter region of the mouse cyclin-dependent kinase 5-encoding gene

While cyclin-dependent kinases, such as CDC2 and CDK2, are key regulators of cell-cycle progression, cyclin-dependent kinase 5 (CDK5) is highly expressed in mature neurons with no evident cell-cycle regulation. The 5′-region of the mouse CDK5 gene was isolated and sequenced. The isolated clone included exons 1 through 7. The 5′-flanking region has a high G+C content. There is no TATA box around the transcriptional start points ( tsp), as determined by primer extension analysis. One CCAAT box, one AP-1-binding site, two AP-2-binding sites, and one cAMP-responsive element are located upstream from the tsp. Promoter/ cat fusion assays showed that the 5.8-kb fragment of this 5′-flanking sequence possessed the promoter activities expressing cat in rat PC12 pheochromocytoma cells. The effect of deletions of the promoter suggested the presence of two negative control elements located from −2.9 kb to −546 bp, and from −212 to −155 upstream from the 5′ end of the tsp. Two positive elements from bp −300 to −212 and from −155 to −41 were also detected. In the element from bp −300 to −212, there was a putative NF-IL6-binding sequence. Thus, the CDK5 promoter region contains multiple positive and negative cis-acting regulatory elements, an arrangement which suggests that the regulation of transcription of CDK5 is under complex control.

Gene Expression After Uninephrectomy in Rat: Simultaneous Expression of Positive and Negative Growth Control Elements

Purpose Compensatory renal growth after contralateral nephrectomy consists largely of hypertrophy of the renal cortex. The signals initiating compensatory renal growth are as yet unknown. Materials and Methods Using a technique of quantitative dot-blot hybridization, we examined the expression of a number of genes after contralateral nephrectomy in the rat in an effort to establish a characteristic “fingerprint” which might shed light on the mechanism of compensatory renal growth. Results Negative growth control elements, including transcriptional repressors (WT-1, junB, myn, HNF-1, p53, RB, Dr1, gas2, gadd153) were induced 1.7 to 4.7-fold within 1 hour after uninephrectomy, as were positive growth control elements (egr-1, c-jun, cyclin C, cyclin E; 1.9-to 4.2-fold induction). The steady state mRNA levels for heat shock genes hsp70, hsp86 and hsp90 beta, as well as extracellular matrix genes fibronectin and collagen I (alpha 1 chain) were also increased 1 hour after uninephrectomy. In addition, 2 Na sup + -specific exchangers (NHE-1 and Na/Ca) were induced within 1 hour after uninephrectomy. Conclusions These results suggest the coordinate expression of positive and negative growth control elements early in this physiologic model of organ growth.

The upstream region of the FOX3 gene encoding peroxisomal 3-oxoacyl-coenzyme A thiolase in Saccharomyces cerevisiae contains ABF1- and replication protein A-binding sites that participate in its regulation by glucose repression.

Expression of the FOX3 gene, which encodes yeast peroxisomal 3-oxoacyl-coenzyme A thiolase, can be induced by oleate and repressed by glucose. Previously, we have shown that induction was mediated by an oleate response element. Just upstream of this element a negatively acting control region that mediated glucose repression was found. In order to study this negative control region, we carried out DNA-binding assays and analyzed phenotypes of mutations in this region and in the trans-acting factor CAR80, which is identical to UME6. DNA-binding assays showed that two multifunctional yeast proteins, ABF1 and RP-A, interacted with the negative control element independently of the transcriptional activity of the FOX3 gene. ABF1 and RP-A, the latter being identical to BUF, were able to bind to DNA independently of one another but also simultaneously. The phenotypes of mutations in either DNA-binding sites of ABF1, RP-A, or both, which affected the DNA binding of these factors in vitro, indicated that these sites and the proteins that interact with them participate in glucose repression. The involvement of the RP-A site in glucose repression was further supported by our observation that the CAR80 gene product, which is required for repression mediated by the RP-A site, was essential for maintenance of glucose repression. In addition to the RP-A site in the FOX3 promoter, similar sequences were observed in other genes involved in peroxisomal function. RP-A proved to bind to all of these sequences, albeit with various affinities. From these results it is concluded that the ABF1 and RP-A sites are being required in concert to mediate glucose repression of the FOX3 gene. In addition, coordinated regulation of expression of genes involved in peroxisomal function in response to glucose is mediated by proteins associated with the RP-A site, probably RP-A and CAR80.

Mechanism of post-segregational killing by hok-homologue pnd of plasmid R483: Two translational control elements in the pnd mRNA

The pnd system of plasmid R483 mediates plasmid stabilization by killing of plasmid-free cells. The pnd mRNA is very stable and can be translated into PndA protein, a cell toxin which kills the cells from within by damaging the cell membrane. Translation of the pnd mRNA is inhibited by the PndB antisense, a small labile RNA of 63 nt. The rapid decay of the PndB antidote leads to onset of PndA synthesis in plasmid-free segregants or after addition of rifampicin. Surprisingly however, the full-length pnd mRNA was found to be translationally inactive whereas a 3'-end truncated version of it was found to be active. We have therefore suggested previously, that the 3'-end of the full-length pnd mRNA encodes a fold-back inhibitory sequence (fbi), which prevents its translation. Here we present an analysis of the metabolism of the pnd mRNAs. A mutational analysis shows that single point mutations in the fbi motif results in more rapid truncation. The fbi mutations could not be complemented by second-site mutations in either of the pndA or pndC Shine-Dalgarno (SD) elements. Surprisingly, mutations in the pndC SD element also lead to a more rapid truncation. The effect of these latter mutations was, however, complemented by mutations in a proposed anti-SD element upstream of the pndC SD. Mutations in the anti-SD element were lethal. These results show, that the pnd mRNA contains two negative control elements, one located in its very 3'-end (fbi), and one located just upstream of the pndC SD region (the anti-SD element). These observations add to the complexity of the induction scheme previously proposed to explain activation of pndA expression in plasmid-free cells: In addition to its negative effect of translation, the fbi structure also maintains a reduced processing rate in the 3'-end of the mRNA. This permits the accumulation of a reservoir of pnd mRNA, which can be activated by 3'-end processing in plasmid-free cells. The anti-SD may prevent translation of the pnd mRNA during transcription, thus preventing detrimental synthesis of toxin.