Cytological diagnosis of genital ureaplasma urealyticum and its importance in cervical inflammation.

OBJECTIVE: Cervical smear cytology, which is a gynecological cervical cancer screening test, can provide information about the presence of pathogenic microorganisms or the inflammation they cause. Among them, Ureaplasma urealyticum (Uu), which is a subspecies of Mycoplasma was held responsible for high-grade cervical intraepithelial lesions and malignancy due to long-lasting complicated vulvovaginitis clinic. We aimed at investigating the role of Uu in the inflammatory process of the cervix and to describe the cytological features that enable it to be recognized microscopically in cervical smear test. PATIENTS AND METHODS: Cervical smear and mycoplasma culture data of 123 women with complicated vulvovaginitis findings were evaluated. According to the Uu culture results, women were divided into two groups: the Uu-positive (n=59) and the Uu-negative group (n=64). The groups were compared in terms of cervical smear results, macroscopic view of the cervix, and secondary cytological evaluation results. RESULTS: The presence of inflammatory signs (83.1%) in the Uu-positive group was observed to be 83.1%, whereas 67.2% in the Uu-negative group, and the difference between the two groups was found to be significant (p=0.04). Besides, the difference in aggregated polymorphonuclear leukocytes (PMNL) between Uu-positive group (59.3%) and Uu-negative group (40.6%) was statistically significant (p=0.04). Similarly, nuclear atypia of epithelial cells in the Uu-positive group (33.9%) was observed to be higher than in the Uu-negative group (17.2%) (p=0.03). CONCLUSIONS: Uu causes inflammation of the cervix and cervical intraepithelial lesions. Aggregated PMNL observed in cervical smear cytology may be one of the findings that will give clues for Uu.

The effect of growth hormone addition protocols to poor ovarian responders in in vitro fertilization cycles.

OBJECTIVE: In vitro fertilization failure (IVF) is high in women with poor ovarian response or non-responder. For this reason, the addition of adjuvant treatments to IVF protocols has come to the fore. We assessed to investigate the effects of adjuvant GH therapy initiated in the mid-luteal phase on IVF success in poor ovarian response or non-responder women. PATIENTS AND METHODS: A retrospective study was performed in 93 poor ovarian response or non-responder women from a single center. GH treatment was added (GH-plus group) in the mid-luteal phase of the previous menstrual cycle to 47 of the women who underwent controlled ovarian stimulation with the flexible antagonist protocol. 46 women, as another group, were applied to a flexible antagonist-only protocol (GH-free group). The IVF outcome results were evaluated and compared within the groups. RESULTS: The number of retrieved oocytes was statistically significantly higher in the GH-plus group (2.28±1.975) than in the GH-free group (1.24±1.728) (p=0.01). Although statistically insignificant (p=0.55), the clinical pregnancy rate was higher in the GH-plus group [(8/47), 17%] than in the GH-free group [(5/46, 11%]. The cancellation rate was statistically significantly higher in the GH-free group (65.2%) than in the GH-plus group (44.7%) (p=0.04). No oocyte retrieved cycle rate was higher in the GH-free group (56%) than in the GH-plus group (25%) (p=0.002). CONCLUSIONS: Adjuvant GH therapy administration to IVF protocol in the mid-luteal phase gives poor ovarian response or non-responder women a chance to have a baby.

Hippocampal corticotropin releasing hormone: pre- and postsynaptic location and release by stress.

Neuropeptides modulate neuronal function in hippocampus, but the organization of hippocampal sites of peptide release and actions is not fully understood. The stress-associated neuropeptide corticotropin releasing hormone (CRH) is expressed in inhibitory interneurons of rodent hippocampus, yet physiological and pharmacological data indicate that it excites pyramidal cells. Here we aimed to delineate the structural elements underlying the actions of CRH, and determine whether stress influenced hippocampal principal cells also via actions of this endogenous peptide. In hippocampal pyramidal cell layers, CRH was located exclusively in a subset of GABAergic somata, axons and boutons, whereas the principal receptor mediating the peptide's actions, CRH receptor 1 (CRF1), resided mainly on dendritic spines of pyramidal cells. Acute 'psychological' stress led to activation of principal neurons that expressed CRH receptors, as measured by rapid phosphorylation of the transcription factor cyclic AMP responsive element binding protein. This neuronal activation was abolished by selectively blocking the CRF1 receptor, suggesting that stress-evoked endogenous CRH release was involved in the activation of hippocampal principal cells.

Roles of glutamate synthase, gltBD, and gltF in nitrogen metabolism of Escherichia coli and Klebsiella aerogenes.

Mutants of Escherichia coli and Klebsiella aerogenes that are deficient in glutamate synthase (glutamate-oxoglutarate amidotransferase [GOGAT]) activity have difficulty growing with nitrogen sources other than ammonia. Two models have been proposed to account for this inability to grow. One model postulated an imbalance between glutamine synthesis and glutamine degradation that led to a repression of the Ntr system and the subsequent failure to activate transcription of genes required for the use of alternative nitrogen sources. The other model postulated that mutations in gltB or gltD (which encode the subunits of GOGAT) were polar on a downstream gene, gltF, which is necessary for proper activation of gene expression by the Ntr system. The data reported here show that the gltF model is incorrect for three reasons: first, a nonpolar gltB and a polar gltD mutation of K. aerogenes both show the same phenotype; second, K. aerogenes and several other enteric bacteria lack a gene homologous to gltF; and third, mutants of E. coli whose gltF gene has been deleted show no defect in nitrogen metabolism. The argument that accumulated glutamine represses the Ntr system in gltB or gltD mutants is also incorrect, because these mutants can derepress the Ntr system normally so long as sufficient glutamate is supplied. Thus, we conclude that gltB or gltD mutants grow slowly on many poor nitrogen sources because they are starved for glutamate. Much of the glutamate formed by catabolism of alternative nitrogen sources is converted to glutamine, which cannot be efficiently converted to glutamate in the absence of GOGAT activity. Finally, GOGAT-deficient E. coli cells growing with glutamine as the sole nitrogen source increase their synthesis of the other glutamate-forming enzyme, glutamate dehydrogenase, severalfold, but this is still insufficient to allow rapid growth under these conditions.

Differential and age-dependent expression of hyperpolarization-activated, cyclic nucleotide-gated cation channel isoforms 1-4 suggests evolving roles in the developing rat hippocampus.

Hyperpolarization-activated cation currents (I(h)) are found in several brain regions including thalamus and hippocampus. Important functions of these currents in promoting synchronized network activity and in determining neuronal membrane properties have been progressively recognized, but the molecular underpinnings of these currents are only emerging. I(h) currents are generated by hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCNs). These channel proteins are encoded by at least four HCN genes, that govern the kinetic and functional properties of the resulting channels. Because of the potential impact of I(h)-mediated coordinated neuronal activity on the maturation of the functional hippocampal network, this study focused on determining the expression of the four members of the HCN gene family throughout postnatal hippocampal development at both the regional and single cell level.The results of these experiments demonstrated that HCNs 1, 2 and 4 are differentially expressed in interneuronal and principal cell populations of the rat hippocampal formation. Expression profiles of each HCN isoform evolve during postnatal development, and patterns observed during early postnatal ages differ significantly from those in mature hippocampus. The onset of HCN expression in interneurons of the hippocampus proper precedes that in the dentate gyrus, suggesting that HCN-mediated pacing activity may be generated in hippocampal interneurons prior to those in the hilus. Taken together, these findings indicate an age-dependent spatiotemporal evolution of specific HCN expression in distinct hippocampal cell populations, and suggest that these channels serve differing and evolving functions in the maturation of coordinated hippocampal activity.

Novel and transient populations of corticotropin-releasing hormone-expressing neurons in developing hippocampus suggest unique functional roles: a quantitative spatiotemporal analysis.

Robust physiological actions of the neuropeptide corticotropin-releasing hormone (CRH) on hippocampal pyramidal neurons have been demonstrated, which may contribute to synaptic efficacy and to learning and memory processes. These excitatory actions of the peptide, as well as the expression of the CRH receptor type that mediates them, are particularly prominent during early postnatal life, suggesting that endogenous CRH may contribute to processes involved in maturation of hippocampal circuitry. To further elucidate the function(s) of endogenous CRH in developing hippocampus, we used neurochemical and quantitative stereological methods to characterize in detail CRH-expressing neuronal populations during postnatal hippocampal differentiation. These experiments revealed progressively increasing numbers of CRH-expressing neurons in developing hippocampus that peaked on postnatal day 11-18 and then declined drastically to adult levels. These cells belonged to several discrete populations, distinguished by GAD67 mRNA expression, morphology, and distinct spatiotemporal distribution profiles. Importantly, a novel population of Cajal-Retzius-like CRH-expressing neurons was characterized that exists only transiently in early postnatal hippocampus and is positioned to contribute to the establishment of hippocampal connectivity. These findings suggest novel, age-specific roles for CRH in regulating early developmental events in the hippocampal formation.

Growth inhibition caused by overexpression of the structural gene for glutamate dehydrogenase (gdhA) from Klebsiella aerogenes.

Two linked mutations affecting glutamate dehydrogenase (GDH) formation (gdh-1 and rev-2) had been isolated at a locus near the trp cluster in Klebsiella aerogenes. The properties of these two mutations were consistent with those of a locus containing either a regulatory gene or a structural gene. The gdhA gene from K. aerogenes was cloned and sequenced, and an insertion mutation was generated and shown to be linked to trp. A region of gdhA from a strain bearing gdh-1 was sequenced and shown to have a single-base-pair change, confirming that the locus defined by gdh-1 is the structural gene for GDH. Mutants with the same phenotype as rev-2 were isolated, and their sequences showed that the mutations were located in the promoter region of the gdhA gene. The linkage of gdhA to trp in K. aerogenes was explained by postulating an inversion of the genetic map relative to other enteric bacteria. Strains that bore high-copy-number clones of gdhA displayed an auxotrophy that was interpreted as a limitation for alpha-ketoglutarate and consequently for succinyl-coenzyme A (CoA). Three lines of evidence supported this interpretation: high-copy-number clones of the enzymatically inactive gdhA1 allele showed no auxotrophy, repression of GDH expression by the nitrogen assimilation control protein (NAC) relieved the auxotrophy, and addition of compounds that could increase the alpha-ketoglutarate supply or reduce the succinyl-CoA requirement relieved the auxotrophy.

Enhanced CREB phosphorylation in immature dentate gyrus granule cells precedes neurotrophin expression and indicates a specific role of CREB in granule cell differentiation.

Differentiation and maturation of dentate gyrus granule cells requires coordinated interactions of numerous processes. These must be regulated by protein factors capable of integrating signals mediated through diverse signalling pathways. Such integrators of inter and intracellular physiological stimuli include the cAMP-response element binding protein (CREB), a leucine-zipper class transcription factor that is activated through phosphorylation. Neuronal activity and neurotrophic factors, known to be involved in granule cell differentiation, are major physiologic regulators of CREB function. To examine whether CREB may play a role in governing coordinated gene transcription during granule cell differentiation, we determined the spatial and temporal profiles of phosphorylated (activated) CREB throughout postnatal development in immature rat hippocampus. We demonstrate that CREB activation is confined to discrete, early stages of granule cell differentiation. In addition, CREB phosphorylation occurs prior to expression of the neurotrophins BDNF and NT-3. These data indicate that in a signal transduction cascade connecting CREB and neurotrophins in the process of granule cell maturation, CREB is located upstream of neurotrophins. Importantly, CREB may be a critical component of the machinery regulating the coordinated transcription of genes contributing to the differentiation of granule cells and their integration into the dentate gyrus network.

Nitrogen regulatory protein C-controlled genes of Escherichia coli: scavenging as a defense against nitrogen limitation.

Nitrogen regulatory protein C (NtrC) of enteric bacteria activates transcription of genes/operons whose products minimize the slowing of growth under nitrogen-limiting conditions. To reveal the NtrC regulon of Escherichia coli we compared mRNA levels in a mutant strain that overexpresses NtrC-activated genes [glnL(Up)] to those in a strain with an ntrC (glnG) null allele by using DNA microarrays. Both strains could be grown under conditions of nitrogen excess. Thus, we could avoid differences in gene expression caused by slow growth or nitrogen limitation per se. Rearranging the spot images from microarrays in genome order allowed us to detect all of the operons known to be under NtrC control and facilitated detection of a number of new ones. Many of these operons encode transport systems for nitrogen-containing compounds, including compounds recycled during cell-wall synthesis, and hence scavenging appears to be a primary response to nitrogen limitation. In all, approximately 2% of the E. coli genome appears to be under NtrC control, although transcription of some operons depends on the nitrogen assimilation control protein, which serves as an adapter between NtrC and final sigma(70)-dependent promoters.

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.

Two roles for the leucine-responsive regulatory protein in expression of the alanine catabolic operon (dadAB) in Klebsiella aerogenes.

The lrp gene, which codes for the leucine-responsive regulatory protein (Lrp), was cloned from Klebsiella aerogenes W70. The DNA sequence was determined, and the clone was used to create a disruption of the lrp gene. The lack of functional Lrp led to an increased expression of the alanine catabolic operon (dad) in the absence of the inducer L-alanine but also to a decreased expression of the operon in the presence of L-alanine. Thus, Lrp is both a repressor and activator of dad expression. Lrp is also necessary for glutamate synthase formation but not for the formation of two other enzymes controlled by the nitrogen regulatory (Ntr) system, glutamate dehydrogenase and histidase.

The nac (nitrogen assimilation control) gene from Escherichia coli.

The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonella typhimurium by Southern blotting, using a probe from the Klebsiella aerogenes nac (nacK) gene. The E. coli nac gene (nacE) was isolated from a cosmid clone by complementation of a nac mutation in K. aerogenes. nacE was fully functional in this complementation assay. DNA sequence analysis showed considerable divergence between nacE and nacK, with a predicted amino acid sequence identity of only 79% and most of the divergence in the C-terminal half of the protein sequence. The total predicted size of NAC(E) is 305 amino acids, the same as for NAC(K). A null mutation, nac-28, was generated by reverse genetics. Mutants bearing nac-28 have a variety of phenotypes related to nitrogen metabolism, including slower growth on cytosine, faster growth on arginine, and suppression of the failure of an Ntr-constitutive mutant to grow with serine as sole nitrogen source. In addition to a loss of nitrogen regulation of histidase formation, nac-28 mutants also showed a loss of a weak repression of glutamate dehydrogenase formation. This repression was unexpected because it is balanced by a NAC-independent activation of glutamate dehydrogenase formation during nitrogen-limited growth. Attempts to purify NAC(E) by using methods established for NAC(K) failed, and NAC(E) appears to be degraded with a half-life at 30 degrees C as short as 15 min during inhibition of protein synthesis.

Alanine catabolism in Klebsiella aerogenes: molecular characterization of the dadAB operon and its regulation by the nitrogen assimilation control protein.

Klebsiella aerogenes strains with reduced levels of D-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of nitrogen. The alanine catabolic operon of Klebsiella aerogenes, dadAB, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of dadA strains. The operon has a high similarity to the dadAB operon of Salmonella typhimurium and the dadAX operon of Escherichia coli, each of which codes for the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for E. coli and S. typhimurium, the dad operon of K. aerogenes is activated by the Ntr system, mediated in this case by the nitrogen assimilation control protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position -44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein.

Genetic analysis, using P22 challenge phage, of the nitrogen activator protein DNA-binding site in the Klebsiella aerogenes put operon.

The nac gene product is a LysR regulatory protein required for nitrogen regulation of several operons from Klebsiella aerogenes and Escherichia coli. We used P22 challenge phage carrying the put control region from K. aerogenes to identify the nucleotide residues important for nitrogen assimilation control protein (NAC) binding in vivo. Mutations in an asymmetric 30-bp region prevented DNA binding by NAC. Gel retardation experiments confirmed that NAC specifically binds to this sequence in vitro, but NAC does not bind to the corresponding region from the put operon of Salmonella typhimurium, which is not regulated by NAC.

Two roles for the DNA recognition site of the Klebsiella aerogenes nitrogen assimilation control protein.

The nitrogen assimilation control protein (NAC) binds to a site within the promoter region of the histidine utilization operon (hutUH) of Klebsiella aerogenes, and NAC bound at this site activates transcription of hutUH. This NAC-binding site was characterized by a combination of random and directed DNA mutagenesis. Mutations that abolished or diminished in vivo transcriptional activation by NAC were found to lie within a 15-bp region contained within the 26-bp region protected by NAC from DNase I digestion. This 15-bp core has the palindromic ends ATA and TAT, and it matches the consensus for LysR family transcriptional regulators. Protein-binding experiments showed that transcriptional activation in vivo decreased with decreasing binding in vitro. In contrast to the NAC-binding site from hutUH, the NAC-binding site from the gdhA promoter failed to activate transcription from a semisynthetic promoter, and this failure was not due to weak binding or greatly distorted protein-DNA structure. Mutations in the promoter-proximal half-site of the NAC-binding site from gdhA allowed this site to activate transcription. Similar studies using the NAC-binding site from hut showed that two mutations in the promoter proximal half-site increased binding but abolished transcriptional activation. Interestingly, for symmetric mutations in the promoter-distal half-site, loss of transcriptional activation was always correlated with a decrease in binding. We conclude from these observations that if the binding in vitro reflects the binding in vivo, then binding of NAC to DNA is not sufficient for transcriptional activation and that the NAC-binding site can be functionally divided in two half-sites, with related but different functions.

Activation of transcription initiation from the nac promoter of Klebsiella aerogenes.

The nac gene of Klebsiella aerogenes encodes a bifunctional transcription factor that activates or represses the expression of several operons under conditions of nitrogen limitation. In experiments with purified components, transcription from the nac promoter was initiated by sigma 54 RNA polymerase and was activated by the phosphorylated form of nitrogen regulator I (NRI) (NtrC). The activation of the nac promoter required a higher concentration of NRI approximately P than did the activation of the Escherichia coli glnAp2 promoter, and both the promoter and upstream enhancer element contributed to this difference. The nac promoter had a lower affinity for sigma 54 RNA polymerase than did glnAp2, and uninitiated competitor-resistant transcription complexes formed at the nac promoter decayed to competitor-sensitive complexes at a greater rate than did similar complexes formed at the glnAp2 promoter. The nac enhancer, consisting of a single high-affinity NRI-binding site and an adjacent site with low affinity for NRI, was less efficient in stimulating transcription than was the glnA enhancer, which consists of two adjacent high-affinity NRI-binding sites. When these binding sites were exchanged, transcription from the nac promoter was increased and transcription from the glnAp2 promoter was decreased at low concentrations of NRI approximately P. Another indication of the difference in the efficiency of these enhancers is that although activation of a nac promoter construct containing the glnA enhancer was relatively insensitive to subtle alterations in the position of these sites relative to the position of the promoter, activation of the natural nac promoter or a nac promoter construct containing only a single high-affinity NRI approximately P binding site was strongly affected by subtle alterations in the position of the NRI approximately P binding site(s), indicating a face-of-the-helix dependency for activation.

Repression of the Klebsiella aerogenes nac promoter.

Transcription of the nitrogen-regulated nac promoter of Klebsiella aerogenes requires sigma54 RNA polymerase, is activated by the phosphorylated form of the transcription factor nitrogen regulator I (NRI) (NtrC), and is repressed by the product of the nac gene, Nac. Nac protects a large portion of the nac control region, extending from positions -130 to -70, from digestion by DNase I. This site(s) lies immediately upstream from the site at which sigma 54 RNA polymerase binds, is downstream of a high-affinity binding site for the transcriptional activator NRI approximately P, and partially overlaps a low-affinity NRI approximately P-binding site. Binding of Nac to the DNA resulted in bending of the DNA but did not interfere with the binding of sigma 54 RNA polymerase to the promoter or with the binding of NRI approximately P to either the high-affinity site or low-affinity site. Furthermore, transcription assays with various wild-type and mutant templates suggested that Nac did not exclude NRI approximately P from either the low- or high-affinity sites, nor did Nac interfere with the ability of the polymerase to form the open complex when the binding sites for NRI approximately P were moved to different locations upstream from the promoter. Rather, Nac seemed to repress by an antiactivation mechanism in which the interaction of the NRI approximately P, bound at its normal sites, with sigma 54 RNA polymerase, bound to the promoter, was prevented.

Activation of the Escherichia coli lacZ promoter by the Klebsiella aerogenes nitrogen assimilation control protein (NAC), a LysR family transcription factor.

A chimeric promoter with the nitrogen assimilation control protein binding site from hutUp of Klebsiella aerogenes fused to the lacZ core promoter from Escherichia coli was built and cloned in a lacZ reporter plasmid. This construct showed a 14-fold increase of beta-galactosidase activity upon nitrogen limitation. Primer extension experiments showed that the nitrogen assimilation control protein activates lacZp1 in a position-dependent manner.

The nitrogen assimilation control protein, NAC, is a DNA binding transcription activator in Klebsiella aerogenes.

A 32-kDa polypeptide corresponding to NAC, the product of the Klebsiella aerogenes nac gene, was overexpressed from a plasmid carrying a tac'-'nac operon fusion and purified to near homogeneity by taking advantage of its unusual solubility properties. NAC was able to shift the electrophoretic migration of DNA fragments carrying the NAC-sensitive promoters hutUp, putPp1, and ureDp. The interaction between NAC and hutUp was localized to a 26-bp region centered approximately 64 bp upstream of the hutUp transcription initiation site. Moreover, NAC protected this region from DNase I digestion. Mobility shift and DNase I protection studies utilizing the putP and ureD promoter regions identified NAC-binding regions of sizes and locations similar to those found in hutUp. Comparison of the DNA sequences which were protected from DNase I digestion by NAC suggests a minimal NAC-binding consensus sequence: 5'-ATA-N9-TAT-3'. In vitro transcription assays demonstrated that NAC was capable of activating the transcription of hutUp by sigma 70-RNA polymerase holoenzyme when this promoter was presented as either a linear or supercoiled DNA molecule. Thus, NAC displays the in vitro DNA-binding and transcription activation properties which have been predicted for the product of the nac gene.

Roles of catabolite activator protein sites centered at -81.5 and -41.5 in the activation of the Klebsiella aerogenes histidine utilization operon hutUH.

The Klebsiella aerogenes hutUH operon is preceded by a promoter region, hut(P), that contains two divergent promoters (hutUp and Pc) which overlap and are alternately expressed. In the absence of the catabolite gene activator protein-cyclic AMP (CAP-cAMP) complex, Pc is predominantly expressed while hutUp is largely repressed. CAP-cAMP has the dual effect of repressing transcription from Pc while simultaneously activating transcription from hutUp. DNA deletion mutations in this region were used to identify DNA sequences required for transcription of these two promoters. We showed that inactivation of Pc by DNA deletion did not result in activation of hutUp in vitro or in vivo. In addition, Escherichia coli CAP mutants that are known to bind and bend DNA normally but are unable to activate various CAP-dependent promoters were also unable to activate hutUp in vivo. These results invalidate an indirect activation model by which CAP-mediated repression of Pc in itself would led to activation of hutUp. Gel retardation asays with various deletion mutations of hut(P) and DNase I protection analyses revealed a high-affinity CAP binding site (CAP site 1) centered at -81.5 relative to the hutUp start of transcription and a second low-affinity CAP site (CAP site 2) centered at about -41.5. CAP site 1 is essential for activation of hutUp. Although CAP site 2 by itself is unable to activate hutUp in vivo under catabolite-activating conditions, it appears to be required for maximal transcription from a site centered at -41.5, does not activate hutUp suggests that the role of CAP-cAMP at the weaker CAP site may be different from that of other promoters containing a similarly positioned site. We propose that CAP directly stimulates the activity of RNA polymerase at hutUp and that this reaction is completely dependent on a naturally occurring CAP site centered at -81.5 and also involves a second CAP site centered at about -41.5 for maximal activation.