Pa Promoter Research Articles

Identification of a xylose‐inducible promoter and its application for improving vitamin B12 production in Sinorhizobium meliloti

Sinorhizobium meliloti 320 is a vitamin B12 (VB12 ) high-producing strain that has been isolated and identified in our previous study. Because the regulatory toolbox for S.meliloti is limited, we searched for new genetic components and identified the two xylose-inducible promoters PA and PB based on a promoter-probe vector with a green fluorescent protein (GFP) as reporter. Compared with the ParaA promoter from S.meliloti, both promoters exhibited higher induced expression and lower basal expression. Subsequently, the influence of glucose or sucrose on the expression of GFP driven by these three promoters was assayed. Glucose repressed all three promoters, and the expression of ParaA was the lowest in the presence of glucose. Although sucrose repressed the expression of PA by 35% and improved the expression of ParaA by 16%, the expression level of PA was the highest and was 13% higher than that of ParaA . Lastly, we overexpressed the hemA gene in the C4 pathway using the PA promoter in S.meliloti 320, and the VB12 production of the engineered strain increased by 11%. The VB12 production was further increased by 11% by adding 0.1% sodium succinate to the culture medium.

STAT2-dependent induction of RNA adenosine deaminase ADAR1 by type I interferon differs between mouse and human cells in the requirement for STAT1

Expression of adenosine deaminase acting on RNA1 (ADAR1) is driven by alternative promoters. Promoter PA, activated by interferon (IFN), produces transcripts that encode the inducible p150 ADAR1 protein, whereas PB specifies the constitutively expressed p110 protein. We show using Stat1−/−, Stat2−/− and IRF9−/− MEFs that induction of ADAR1 p150 occurs by STAT2- and IRF9-dependent signaling that is enhanced by, but not obligatorily dependent upon, STAT1. Chromatin immunoprecipitation analysis demonstrated STAT2 at the PA promoter in IFN-treated Stat1−/− cells, whereas IFN-treated wild-type cells showed both STAT1 and STAT2 bound at PA. By contrast, with human 2fTGH cells and mutants U3A or U6A, ADAR1 induction by IFN was dependent upon both STAT1 and STAT2. These results suggest that transcriptional activation of Adar1 by IFN occurs in the absence of STAT1 by a non-canonical STAT2-dependent pathway in mouse but not human cells.

Insights on the regulation of the phenylacetate degradation pathway from Escherichia coli.

The paa genes for phenylacetic acid (PA) catabolism encode the best characterized aerobic hybrid route involved in the bacterial degradation of aromatic compounds. Here, we demonstrate that the divergent paaZ and paaA-K catabolic operons of Escherichia coli are regulated by two genes, paaXY, that form a distinct transcriptional unit driven by the Px promoter. In vivo and in vitro approaches using purified PaaX regulatory protein revealed that this regulator is able to bind and inhibit the activity of Px in a phenylacetyl-coenzyme A (PA-CoA) dependent manner. The autoregulation of paaXY is due to the competition between PaaX and RNA polymerase for binding to the regulatory Px promoter. Whereas a similar mechanism of repression mediated by PaaX was shown to occur at the catabolic Pz promoter; the catabolic Pa promoter is inhibited by PaaX by a mechanism that does not involves competition with RNA polymerase. We have shown for the first time that the paaY gene product is essential for an efficient growth in PA. Purified PaaY was shown to be a trimer in solution with a broad thioesterase activity stimulated by some metals. This thioesterase activity will allow the detoxification of some CoA-intermediates that block the aerobic catabolism of PA, as previously suggested, but also will avoid the accumulation of some CoA derivatives that could behave as antagonists of the inducer effect caused by PA-CoA on the PaaX repressor for an efficient expression of the paa genes. This regulatory function mediated by PaaY constitutes an additional regulatory checkpoint that makes the circuit that controls the transcription of the paa genes more complex than previously thought, and it could represent a general strategy present in most bacterial paa gene clusters that also harbour the paaY gene.

Mg2+‐modulated KMnO4 reactivity of thymines in the open transcription complex reflects variation in the negative electrostatic potential along the separated DNA strands

There is still a controversy over the mechanism of promoter DNA strand separation upon open transcription complex (RPo) formation by Escherichia coli RNA polymerase: is it a single or a stepwise process controlled by Mg2+ ions and temperature? To resolve this question, the kinetics of pseudo-first-order oxidation of thymine residues by KMnO4 in the -11 ... +2 DNA region of RPo at the lambdaP(R) promoter was examined under single-hit conditions as a function of temperature (13-37 degrees C) in the absence or presence of 10 mm MgCl2. The reaction was also studied with respect to thymidine and its nucleotides (TMP, TTP and TpT) as a function of temperature and [MgCl2]. The kinetic parameters, (ox)k and (ox)E(a), and Mg-induced enhancement of (ox)k proved to be of the same order of magnitude for RPo-lambdaP(R) and the nucleotides. Unlike the complex, (ox)E(a) for the nucleotides was found to be Mg-independent. The isothermal increase in (ox)k with increasing [Mg2+] was thus interpreted in terms of a simple model of screening of the negative charges on phosphate groups by Mg2+ ions, lowering the electrostatic barrier to the diffusion of MnO4- anions to the reactive double bond of thymine. Similar screening isotherms were determined for the oxidation of two groups of thymines in RPo at a consensus-like Pa promoter, differing in the magnitude of the Mg effect. Together, the findings show that: (a) the two DNA strands in the -11...+2 region of RPo-lambdaP(R) are completely separated over the whole range of temperatures investigated (13-37 degrees C) in the absence of Mg2+ (b) Mg2+ ions induce an increase in the rate of the oxidation reaction by screening negatively charged phosphate and carboxylate groups; and (c) the observed thymine reactivity and the magnitude of the Mg effect reflect variation in the strength of the electrostatic potential along the separated DNA strands, in agreement with the current structural model of RPo.

GvpE- and GvpD-mediated transcription regulation of the p-gvp genes encoding gas vesicles in Halobacterium salinarum.

The transcription of the 14 p-gvp genes involved in gas vesicle formation of Halobacterium salinarum PHH1 is driven by the four promoters pA, pD, pF and pO. The regulation of these promoters was investigated in Haloferax volcanii transformants with respect to the endogenous regulatory proteins GvpE and GvpD. Northern analyses demonstrated that the transcription derived from the pA and pD promoters was enhanced by GvpE, whereas the activities of the pF and pO promoters were not affected. Similar results were obtained using promoter fusions with the bgaH reporter gene encoding an enzyme with beta-galactosidase activity. The largest amount of specific beta-galactosidase activity was determined for pA-bgaH transformants, followed by pF-bgaH and pD-bgaH transformants. The presence of GvpE resulted in a severalfold induction of the pA and pD promoter, whereas the pF promoter was not affected. A lower GvpE-induced pA promoter activity was seen in the presence of GvpD in the pA-bgaH/DE(ex) transformants, suggesting a function of GvpD in repression. To determine the DNA sequences involved in the GvpE-mediated activation, a 50-nucleotide region of the pA promoter was investigated by 4-nucleotide scanning mutagenesis. Some of these mutations affected the basal transcription, especially mutations in the region of the TATA box and the putative BRE sequence element, and also around position -10. Mutant E, harbouring a sequence with greater identity to the consensus BRE element, showed a significantly enhanced basal promoter activity compared to wild-type. Mutations not affecting basal transcription, but yielding a reduced GvpE-mediated activation, were located immediately upstream of BRE. These results suggested that the transcription activation by GvpE is in close contact with the core transcription machinery.

Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

Regulation of the mhp Cluster Responsible for 3-(3-Hydroxyphenyl)propionic Acid Degradation in Escherichia coli

The mhp gene cluster from Escherichia coli constitutes a model system to study bacterial degradation of 3-(3-hydroxyphenyl)propionic acid (3HPP). In this work the regulation of the inducible mhp catabolic genes has been studied by genetic and biochemical approaches. The Pr and Pa promoters, which control the expression of the divergently transcribed mhpR regulatory gene and mhp catabolic genes, respectively, show a peculiar arrangement leading to transcripts that are complementary at their 5'-ends. By using Pr-lacZ and Pa-lacZ translational fusions and gel retardation assays, we have shown that the mhpR gene product behaves as a 3HPP-dependent activator of the Pa promoter, being the expression from Pr constitutive and MhpR-independent. DNase I footprinting experiments and mutational analysis mapped an MhpR-protected region, centered at position -58 with respect to the Pa transcription start site, which is indispensable for MhpR binding and in vivo activation of the Pa promoter. Superimposed in the specific MhpR-mediated regulation of the Pa promoter, we have observed a strict catabolite repression control carried out by the cAMP receptor protein (CRP) that allows expression of the mhp catabolic genes when the preferred carbon source (glucose) is not available and 3HPP is present in the medium. Gel retardation assays revealed that the specific activator, MhpR, is essential for the binding of the second activator, CRP, to the Pa promoter. Such peculiar synergistic transcription activation has not yet been observed in other aromatic catabolic pathways, and the MhpR activator becomes the first member of the IclR family of transcriptional regulators that is indispensable for recruiting CRP to the target promoter.

Transcriptional regulation of human Gal [formula omitted]1,3GalNAc/Gal [formula omitted]1, 4GlcNAc [formula omitted]2,3-sialyltransferase (hST3Gal IV) gene in testis and ovary cell lines

The mRNA expression of sialyltransferase genes is regulated in a cell-type-specific manner. The mRNAs of human Galβ1, 3GalNAc/Galβ1, 4 GlcNAc α2,3-sialyltransferase gene (hST3Gal IV) consist of six isoforms, type A1, A2, B1, B2, B3, and BX. These mRNAs are transcribed from different promoters, pA, pB1, pB2, pB3, and pBX, respectively. Type B mRNAs are expressed in several cells, whereas type A mRNAs are specifically expressed in testis, ovary, and placenta, suggesting that pA promoter activity is especially high in these tissues. We show herein germ-cell specific transcriptional regulation of the hST3Gal IV pA promoter. Using a luciferase assay, pA promoter activity is shown to be high in testis and ovary cell lines. We identified the enhancer region of the pA promoter, located at nt −520 to −420. These results suggest that this element plays a critical role in germ-cell specific regulation of the pA promoter. The results of site-directed mutagenesis suggest that AP2 and c-Ets sites in this region are involved in pA promoter activity, which in turn suggests that the hST3Gal IV gene is regulated in a tissue-restricted fashion at the level of transcription.

Imatinib (STI571) Provides Only Limited Selectivity for CML Cells and Treatment Might Be Complicated by Silent BCR-ABL Genes

Very promising results have been obtained in clinical trials on chronic-phase chronic myeloid leukemia (CP-CML) patients treated with imatinib mesylate (IM; Gleevec®, STI571), a BCR-ABL tyrosine kinase inhibitor. However, we found that IM caused considerable inhibition of normal hematopoietic progenitor cells upon treating control bone marrow (BM) cultures. In vitro IM treatment gave a decrease in the yield and size of colonies from BM of untreated CP-CML patients that was only two to three times that from the normal samples. Moreover, about 30% of myeloid progenitors (CFU-GM) from CML BM still formed colonies in the presence of IM, most of which had BCR-ABL RNA. About half of these treated colonies also displayed methylation of the internal ABL Pa promoter, a CML-specific epigenetic alteration, which was used in this study as a marker for BCR-ABL translocation-containing cells. However, ~5-8% of the treated or the untreated CML BM-derived colonies had no detectable BCR-ABL RNA by two or three rounds of RT-PCR despite being positive for the internal standard RNA and displaying hallmarks of CML, either t(9;22)(q34;q11) or ABL Pa methylation. Our results indicate that IM is only partially specific for CML progenitor cells compared to normal hematopoietic progenitor cells and suggest that some CML cells may have a silent BCR-ABL oncogene that could interfere with therapy.

Effect of A(n) tracts within the UP element proximal subsite of a model promoter on kinetics of open complex formation by Escherichia coli RNA polymerase.

In the open transcription complex (RPo), Escherichia coli RNA polymerase sigma(70) and alpha subunits are known to be in contact with each other and with the promoter region overlapping the -35 hexamer and the proximal part of the UP element. To probe the effect of A(n) DNA bending tracts in this region on initiation of transcription, kinetics of the formation of RPo by Escherichia coli RNA polymerase at two groups of synthetic consensus-like promoters bearing single DNA bending tracts (i). A(5 )within the proximal subsite region of the UP element (promoters Pk and Pl) and (ii). A(5)(Pg) or A(8)(Pm) in the region including the downstream end of the proximal UP subsite and the -35 consensus hexamer was studied in vitro using the fluorescence-detected abortive initiation assay. The kinetic data obtained demonstrate that the overall second-order rate constant k(a) of RPo formation is: (i.by almost one order of magnitude larger at Pk and Pl, relative to that at a control unbent promoter, and mainly due to a higher value of the equilibrium constant, K(1), of the initial closed complex; and (ii). several-fold smaller at Pg and Pm owing to a strongly decreased value of K(1). For Pm, the latter parameter was found to be dependent exponentially on four Mg(2+) ions, as compared with the seven ions remaining in equilibrium with the initial closed complex at the parent Pa promoter. This indicates that promoter region bearing a stiff A(8).T(8) fragment of B -DNA forms a smaller number of ionic contacts with the alpha subunit. These findings provide a new insight to and support the present model of interactions between RNA polymerase alpha and sigma(70) subunits with the proximal UP subsite and the -35 region of promoters.

Effect of A(n) tracts within the UP element proximal subsite of a model promoter on kinetics of open complex formation by Escherichia coli RNA polymerase.

In the open transcription complex (RPo), Escherichia coli RNA polymerase sigma(70) and alpha subunits are known to be in contact with each other and with the promoter region overlapping the -35 hexamer and the proximal part of the UP element. To probe the effect of A(n) DNA bending tracts in this region on initiation of transcription, kinetics of the formation of RPo by Escherichia coli RNA polymerase at two groups of synthetic consensus-like promoters bearing single DNA bending tracts (i). A(5 )within the proximal subsite region of the UP element (promoters Pk and Pl) and (ii). A(5)(Pg) or A(8)(Pm) in the region including the downstream end of the proximal UP subsite and the -35 consensus hexamer was studied in vitro using the fluorescence-detected abortive initiation assay. The kinetic data obtained demonstrate that the overall second-order rate constant k(a) of RPo formation is: (i.by almost one order of magnitude larger at Pk and Pl, relative to that at a control unbent promoter, and mainly due to a higher value of the equilibrium constant, K(1), of the initial closed complex; and (ii). several-fold smaller at Pg and Pm owing to a strongly decreased value of K(1). For Pm, the latter parameter was found to be dependent exponentially on four Mg(2+) ions, as compared with the seven ions remaining in equilibrium with the initial closed complex at the parent Pa promoter. This indicates that promoter region bearing a stiff A(8).T(8) fragment of B -DNA forms a smaller number of ionic contacts with the alpha subunit. These findings provide a new insight to and support the present model of interactions between RNA polymerase alpha and sigma(70) subunits with the proximal UP subsite and the -35 region of promoters.

Mg2+ does not induce isomerization of the open transcription complex of Escherichia coli RNA polymerase at the model Pa promoter bearing consensus -10 and -35 hexamers.

The kinetics and thermodynamics of the formation of the transcriptional open complex (RPo) by Escherichia coli RNA polymerase at the synthetic Pa promoter bearing consensus -10 and -35 recognition hexamers were studied in vitro. Previously, this promoter was used as a control one in studies on the effect of DNA bending by An x Tn sequences on transcription initiation and shown to be fully functional in E. coli (Loziński et al., 1991, Nucleic Acids Res. 19, 2947; Loziński & Wierzchowski, 1996, Acta Biochim. Polon. 43, 265). The data now obtained demonstrate that the mechanism of Pa-RPo formation and dissociation conforms to the three-step reaction model: bind-nucleate-melt, commonly accepted for natural promoters. Measurements of the dissociation rate constant of Pa-RPo as a function of MgCl2 concentration allowed us to determine the number of Mg2+ ions, nMg approximately/= 4, being bound to the RPo in the course of renaturation of the melted DNA region. This number was found constant in the temperature range of 25-37 degrees C, which indicates that under these conditions the complex remaines fully open. This observation, taken together with the recent evidence from KMnO4 footprinting studies that the length of the melted region in Pa-RPo at 37 degrees C is independent of the presence of Mg2+ ions (Lozinski & Wierzchowski, 2001, Acta Biochim. Polon. 48, 495), testifies that binding of Mg2+ to RPo does not induce its further isomerization, which has been postulated for the lambdaP(R)-RPo complex (Suh et al., 1992, Biochemistry 31, 7815; 1993, Science 259, 358).

Effect of Mg2+ on kinetics of oxidation of pyrimidines in duplex DNA by potassium permanganate.

Potassium permanganate oxidation of pyrimidine bases is often used to probe single-stranded regions in functional DNA-protein complexes. However, so far reactivity of these bases in double-stranded DNA has not been studied quantitatively. We have investigated the kinetics of oxidation of pyrimidines in supercoiled pDS3 plasmid dsDNA by quantitative KMnO4 footprinting, in connection with parallel studies on the effect of Mg2+ on kinetics of oxidation of individual thymines in the single-stranded region of the open transcription complex of Escherichia coli RNA polymerase at a cognate Pa promoter contained in this plasmid. Rate constants of oxidation for pyrimidines, kj, in selected regions of pDS3 DNA, including Pa promoter, were determined under single-hit reaction conditions in the absence and presence of 10 mM MgCl2. Their values appeared to be sequence-dependent and were: (i) the largest for Ts in 5'TA3' and 5'TC3' steps, while 2-4 times smaller for 5'-adjacent ones in TT(A,G,C) and TTT(A) runs, (ii) for Cs in 5'TC3' steps 2-4 fold smaller than for adjacent Ts, and (iii) in the presence of Mg2+ generally larger by a sequence-dependent factor: in 5'TC3' steps of about 2 and 4 for Ts and Cs, respectively, in 5'TA3' steps of TTA and TTTA sequences for 3'-terminal Ts of about 3, while for their 5'-neighbors o tinctly smaller value of about 2. Comparison of kj data for corresponding Ts located between +1 and -10 regions of Pa promoter in dsDNA and in ssDNA form in the open transcription complex, reported elsewhere, demonstrates that reactivity of pyrimidines in dsDNA is by 2-3 orders of magnitude smaller. The effect of Mg2+ in dsDNA is interpreted in terms of electrostatic barrier to diffusion of MnO4- on DNA surface, which is lowered by diffusive binding of these ions to backbone phosphates, involving also sequence-specific contacts with bases in the minor and major grooves of B-DNA.

Mg2+ ions do not induce expansion of the melted DNA region in the open complex formed by Escherichia coli RNA polymerase at a cognate synthetic Pa promoter. A quantitative KMnO4 footprinting study.

Footprinting studies of prokaryotic open transcription complexes (RPO), based on oxidation of pyrimidine residues by KMnO4 and/or OsO4 at a single oxidant dose, have suggested that the extent of DNA melting in the transcription bubble region increases in the presence of Mg . In this work, quantitative KMnO4 footprinting in function of the oxidant dose of RPO, using Escherichia coli RNA polymerase (E(sigma)70) at a fully functional synthetic promoter Pa having -35 and -10 consensus hexamers, has been used to determine individual rate constants of oxidation of T residues in this region at 37degrees C in the absence of Mg2+ and in the presence of 10 mM MgCl2, and to evaluate therefrom the effect of Mg2+ on the extent of DNA melting. Population distributions of end-labeled DNA fragments corresponding to oxidized Ts were quantified and analyzed according to the single-hit kinetic model. Pseudo-first order reactivity rate constants, ki, thus obtained demonstrated that Mg2+ ions bound to RPO merely enhanced the reactivity of all 11 oxidizable thymines between the +3 and -11 promoter sites by a position-dependent factor: 3-4 for those located close to the transcription start point +1 in either DNA strand, and about 1.6 for those located more distantly therefrom. On the basis of these observations, we conclude that Mg2+ ions bound to RPO at Pa do not influence the length of the melted DNA region and propose that the higher reactivity of thymines results mainly from lower local repulsive electrostatic barriers to MnO4 diffusion around carboxylate binding sites in the catalytic center of RPO and promoter DNA phosphates.

Muscle-regulated expression and determinants for neuromuscular junctional localization of the mouse RIalpha regulatory subunit of cAMP-dependent protein kinase.

In skeletal muscle, transcription of the gene encoding the mouse type Ialpha (RIalpha) subunit of the cAMP-dependent protein kinase is initiated from the alternative noncoding first exons 1a and 1b. Here, we report that activity of the promoter upstream of exon 1a (Pa) depends on two adjacent E boxes (E1 and E2) in NIH 3T3-transfected fibroblasts as well as in intact muscle. Both basal activity and MyoD transactivation of the Pa promoter require binding of the upstream stimulating factors (USF) to E1. E2 binds either an unknown protein in a USF/E1 complex-dependent manner or MyoD. Both E2-bound proteins seem to function as repressors, but with different strengths, of the USF transactivation potential. Previous work has shown localization of the RIalpha protein at the neuromuscular junction. Using DNA injection into muscle of plasmids encoding segments of RIalpha or RIIalpha fused to green fluorescent protein, we demonstrate that anchoring at the neuromuscular junction is specific to RIalpha subunits and requires the amino-terminal residues 1-81. Mutagenesis of Phe-54 to Ala in the full-length RIalpha-green fluorescent protein template abolishes localization, indicating that dimerization of RIalpha is essential for anchoring. Moreover, two other hydrophobic residues, Val-22 and Ile-27, are crucial for localization of RIalpha at the neuromuscular junction. These amino acids are involved in the interaction of the Caenorhabditis elegans type Ialpha homologue R(CE) with AKAP(CE) and for in vitro binding of RIalpha to dual A-kinase anchoring protein 1. We also show enrichment of dual A-kinase anchoring protein 1 at the neuromuscular junction, suggesting that it could be responsible for RIalpha tethering at this site.

Detailed Mapping of Methylcytosine Positions at the CpG Island Surrounding the Pa Promoter at the bcr-abl Locus in CML Patients and in Two Cell Lines, K562 and BV173

ABSTRACTChronic myelogenous leukemia (CML) is associated with a translocation of the protooncogene c-abl from chromosome 9 to chromosome 22, where it fuses to proximal exons of the bcr gene. The expression of the hybrid gene bcr-abl is regulated by the bcr promoter and results in a translation product with high tyrosine kinase activity. In most CML cases, one of two abl promoters (Pa) is nested within the bcr-abl transcription unit, but appears to be usually silent. Recently, de novo methylation of the Pa region and its correlation with disease progression were reported. As these previous studies were limited to the use of methylation-sensitive restriction endonucleases, our aim here was to obtain a complete map of methylcytosines and its variants in CML patients and in model cell lines. To achieve this, bisulfite conversion of cytosines (but not methylcytosines) to uracils in genomic DNA was employed. After modification, the region of interest was PCR-amplified and the products were cloned and sequenced. The results show methylation at a high level and in a homogenous pattern in the BV173 cell line, corresponding to the translocated abl alleles. Variant methylation observed in K562 cells correlates with multiple bcr-abl loci and an intact chromosome 9. Patients that were methylation-positive in restriction analysis showed sporadic and heterogenous occurrence of methylcytosines in bisulfite modification assays. Corresponding results were obtained using a quantitative Southern analysis of the extent of methylation. We conclude that restriction analysis combined with PCR is able to find rare cases of hypermethylation, e.g., for diagnostic purposes, but does not reflect the dominating level of methylation in Ph-positive cells.

Catabolism of Phenylacetic Acid in Escherichia coli: CHARACTERIZATION OF A NEW AEROBIC HYBRID PATHWAY

The paa cluster of Escherichia coli W involved in the aerobic catabolism of phenylacetic acid (PA) has been cloned and sequenced. It was shown to map at min 31.0 of the chromosome at the right end of the mao region responsible for the transformation of 2-phenylethylamine into PA. The 14 paa genes are organized in three transcription units: paaZ and paaABCDEFGHIJK, encoding catabolic genes; and paaXY, containing the paaX regulatory gene. The paaK gene codes for a phenylacetyl-CoA ligase that catalyzes the activation of PA to phenylacetyl-CoA (PA-CoA). The paaABCDE gene products, which may constitute a multicomponent oxygenase, are involved in PA-CoA hydroxylation. The PaaZ protein appears to catalyze the third enzymatic step, with the paaFGHIJ gene products, which show significant similarity to fatty acid beta-oxidation enzymes, likely involved in further mineralization to Krebs cycle intermediates. Three promoters, Pz, Pa, and Px, driven the expression of genes paaZ, paaABCDEFGHIJK, and paaX, respectively, have been identified. The Pa promoter is negatively controlled by the paaX gene product. As PA-CoA is the true inducer, PaaX becomes the first regulator of an aromatic catabolic pathway that responds to a CoA derivative. The aerobic catabolism of PA in E. coli represents a novel hybrid pathway that could be a widespread way of PA catabolism in bacteria.

Identification of the in vivo promoters of bacteriophages S13 and ϕX174 and measurement of their relative activities

Regions of bacteriophages ϕX174 and S13 that contain putative promoter sequences were amplified by the polymerase chain reaction (PCR) and cloned into the reporter vector pKO-1. Assays of galactokinase activity revealed in vivo promoter activity in those constructs containing the promoter sequences with transcription initiation (+1) sites at nucleotide positions 45, 982, 1823, and 5211. These were identical in location to sequences with in vitro promoter activity and to the three known promoters PA, PB, and PD. P5211 is the location of a new, fourth, promoter. A site with a +1 position at nucleotide 4876, previously shown to initiate RNA synthesis in an in vitro run-off transcription assay, had no in vivo promoter activity. To investigate whether flanking sequences had effects on promoter activity, restriction fragments of ϕX174 and S13 that encompass the in vivo promoters were cloned into the reporter vector pKO-1. The PA and P5211 promoter constructs showed dramatic effects with increases in activity of up to 7 times that shown with the PCR-generated promoter constructs. The ϕX174 PB promoter construct had a 50% decrease in activity compared with the PCR-generated PB clone. While the data showed that in most instances promoter activity is affected by the flanking sequences in which the promoter is embedded, no general pattern correlating flanking sequences and promoter activity could be discerned. Additional evidence that the promoter sequence regions were active in vivo promoters was obtained by S1 nuclease mapping experiments. Initiation of RNA synthesis was shown at positions 45, 982, and 5211.Key words: promoters, phage S13, phage phiX174, in vivo.

Identification of the in vivo promoters of bacteriophages S13 and phi X174 and measurement of their relative activities.

Regions of bacteriophages phi X174 and S13 that contain putative promoter sequences were amplified by the polymerase chain reaction (PCR) and cloned into the reporter vector pKO-1. Assays of galactokinase activity revealed in vivo promoter activity in those constructs containing the promoter sequences with transcription initiation (+1) sites at nucleotide positions 45, 982, 1823, and 5211. These were identical in location to sequences with in vitro promoter activity and to the three known promoters PA, PB, and PD. P5211 is the location of a new, fourth, promoter. A site with a +1 position at nucleotide 4876, previously shown to initiate RNA synthesis in an in vitro run-off transcription assay, had no in vivo promoter activity. To investigate whether flanking sequences had effects on promoter activity, restriction fragments of phi X174 and S13 that encompass the in vivo promoters were cloned into the reporter vector pKO-1. The PA and P5211 promoter constructs showed dramatic effects with increases in activity of up to 7 times that shown with the PCR-generated promoter constructs. The phi X174 PB promoter construct had a 50% decrease in activity compared with the PCR-generated PB clone. While the data showed that in most instances promoter activity is affected by the flanking sequences in which the promoter is embedded, no general pattern correlating flanking sequences and promoter activity could be discerned. Additional evidence that the promoter sequence regions were active in vivo promoters was obtained by S1 nuclease mapping experiments. Initiation of RNA synthesis was shown at positions 45, 982, and 5211.