A DNA expression array to detect toxic stress response in European flounder (Platichthys flesus).

As a first stage in developing a DNA array-based approach to investigating the effects of pollutants on an environmentally relevant European fish species, we have constructed a 160-gene custom microarray for European flounder. Degenerate primers were used to amplify 110 different fragments of stress-related and other genes from European flounder cDNA and genomic DNA. Additionally, 22 fragments were obtained by suppressive subtractive hybridisation (SSH). These fragments were cloned and sequenced, then, with additional control genes, used to create a cDNA microarray for flounder. After optimisation of the arraying process, hepatic mRNA was isolated from flounder caught in the polluted Tyne and relatively unpolluted Alde estuaries. Fluorescent cDNA probes were synthesised from the mRNA and used in dual-colour hybridisations to the microarray. A number of transcripts were differentially expressed between Tyne and Alde female flounder but these changes were not significant, due to high inter-individual variation. However, in comparisons between Tyne and Alde male flounder, 11 transcripts were found to significantly differ in expression (P<0.05). Seven transcripts were more highly expressed in the Tyne male fish (CYP1A, UDPGT, alpha-2HS-glycoprotein, dihydropyrimidine dehydrogenase, Cu/Zn SOD, aldehyde dehydrogenase and paraoxonase). Four transcripts (Elongation factor 1 (EF1), EF2, Int-6 and complement component C3) were found to be significantly less abundant in the Tyne male fish. Selected genes were assayed by real-time PCR, then normalised to alpha-tubulin. These assays confirmed the significance of the array results for CYP1A, UDPGT and EF1, but not for Cu/Zn SOD. This study provides a link between traditional single-gene biomarker studies and the emerging field of eco-toxicogenomics, demonstrating the utility of microarray studies on environmentally sampled, non-model organisms.

Characterisation of the connexin32 promoter and changes in response element complexes in rat liver and hepatocytes during culture associated with oxidative stress.

Hepatic gap junctional intercellular communication (GJIC), mediated principally by connexin 32, provides a mechanism for regulating multicellular activities between neighbouring cells. The control of Cx32 gene expression at the transcriptional level has been investigated in rat liver tissue and in primary rat hepatocytes during culture. Several response elements have been identified and characterised using the electrophoretic mobility shift assay. Nuclear protein extract prepared from rat primary hepatocytes cultured for 2 h gave a larger number of DNA-protein complexes than observed with extracts from liver in vivo, including complexes containing Sp1. In contrast, nuclear extracts prepared from primary rat hepatocytes cultured for 96 h, and subject to oxidative stress, gave altered DNA-protein complexes when compared to those from hepatocytes cultured for 2 h. These results indicate that culture conditions, known to cause a loss of connexin expression, can modulate the transcription of Cx32 in hepatocytes by affecting the regulatory trans/cis-interactions of redox-sensitive zinc finger proteins within the promoter.

Positioning of region 4 of the Escherichia coli RNA polymerase sigma(70) subunit by a transcription activator.

A DNA cleavage reagent, specifically tethered to residue 581 of the Escherichia coli RNA polymerase sigma(70) subunit, has been used to investigate the location of sigma(70) region 4 in different complexes at the galp(1) promoter and the effect of the cyclic AMP receptor protein. The positions of DNA cleavage by the reagent are not affected by the cyclic AMP receptor protein. We conclude that transcription activation at the galp(1) promoter by the cyclic AMP receptor protein does not involve major conformation changes in or repositioning of sigma(70) region 4.

Open complex formation during transcription initiation at the Escherichia coli galP1 promoter: the role of the RNA polymerase alpha subunit at promoters lacking an UP-element.

We have studied the role of the C-terminal domain of the alpha subunit (alphaCTD) of Escherichia coli RNA polymerase during transcription initiation at promoters lacking an UP-element. The temperature requirement for open complex formation was used as an indication of the kinetics of this process. We have previously shown that alphaCTD is required for transcription initiation at low temperature at the galP1 promoter, a promoter containing an UP-element. DNase I footprinting has been used to reveal the structure of open promoter complexes and the temperature requirement for open complex formation has been determined using potassium permanganate as a probe. In this work we show that, although alphaCTD is not absolutely required for transcription initiation at promoters lacking an UP-element, it does play a role during transcription initiation. This role is independent of the sequence of the promoter upstream from the -35 region and does not require stable alphaCTD-DNA interactions as determined by DNase I footprinting. The role of alphaCTD at promoters lacking an UP-element is discussed.

Organization of open complexes at Escherichia coli promoters. Location of promoter DNA sites close to region 2.5 of the sigma70 subunit of RNA polymerase.

A cysteine-tethered DNA cleavage agent has been used to locate the position of region 2.5 of sigma70 in transcriptionally competent complexes between Escherichia coli RNA polymerase and promoters. In this study we have engineered sigma70 to introduce a unique cysteine residue at a number of positions in region 2.5. Mutant proteins were purified, and in each case, the single cysteine residue used as the target for covalent coupling of the DNA cleavage agent p-bromoacetamidobenzyl-EDTA.Fe (FeBABE). RNA polymerase core reconstituted with tagged sigma derivatives was shown to be transcriptionally active. Hydroxyl radical-based DNA cleavage mediated by tethered FeBABE was observed for each derivative of RNA polymerase in the open complex. Our results show that region 2.5 is in close proximity to promoter DNA just upstream of the -10 hexamer. This positioning is independent of promoter sequence. A model for the interaction of this region of sigma with promoter DNA is discussed.

Region 2.5 of the Escherichia coli RNA polymerase sigma70 subunit is responsible for the recognition of the 'extended-10' motif at promoters.

At some bacterial promoters, a 5'-TG-3' sequence element, located one base upstream of the -10 hexamer element, provides an essential motif necessary for transcription initiation. We have identified a mutant of the Escherichia coli RNA polymerase sigma70 subunit that has an altered preference for base sequences in this 'extended -10' region. We show that this mutant sigma70 subunit substantially increases transcription from promoters bearing 5'-TC-3' or 5'-TT-3' instead of a 5'-TG-3' motif, located one base upstream of the -10 hexamer. The mutant results from a single base pair substitution in the rpoD gene that causes a Glu to Gly change at position 458 of sigma70. This substitution identifies a functional region in sigma70 that is immediately adjacent to the well-characterized region 2.4 (positions 434-453, previously shown to contact the -10 hexamer). From these results, we conclude that this region (which we name region 2.5) is involved in contacting the 5'-TG-3' motif found at some bacterial promoters: thus, extended -10 regions are recognized by an extended region 2 of the RNA polymerase sigma70 subunit.

Identification and characterization of inositol 1,4,5-trisphosphate receptors in rat testis.

PCR analysis and immunoblotting with isoform specific antibodies was used to identify the presence of type I, II and III inositol 1,4,5-trisphosphate receptors (InsP3Rs) in rat testis. PCR analysis also revealed that rat testis express both forms of the S1 splice variant (S1+ and S1-), but only the S2- from of the S2 splice variant of the type I InsP3 receptor. PCR analysis was also used to identify InsP3R isoform expression at a cellular level using myoid, Sertoli and germ cells derived from the testis of Wistar rats. The extent of [3H]-InsP3 binding was found to be 9 times lower for testicular microsomes than for cerebellar microsomes, with a Bmax of 1.4 pmoles/mg protein compared to 12.5 pmoles/mg protein for cerebellar microsomes. The Kd for InsP3 binding to its receptor in testicular microsomes was 60 +/- 10 nM which was similar to that found for cerebellar microsomes (80 +/- 20 nM). InsP3-induced Ca2+ release (IICR) in testicular microsomes was found to have an EC50 (concentration which causes a half-maximal response) of 0.5 +/- 0.03 microM, also similar to that seen for cerebellar microsomes (0.3 microM). Maximal IICR occurred at about 20 microM InsP3, with up to 4% of total intracellular Ca2+ stores being mobilized as compared to between 10-30% for cerebellar microsomes. Time resolved IICR using stopped-flow spectrofluorimetry, showed the kinetics of IICR for this testis preparation to be monophasic with a maximum rate constant of 0.15 s-1 at 30 microM InsP3. The rate constants are 7 times slower than values for cerebellar microsomes under similar conditions (approximately 1 s-1) and taken together with the binding data support the proposal that the receptor density/Ca2+ store is approximately 8 times lower than seen in cerebellar microsomal vesicles. The pharmacological properties as assessed using heparin and InsP3 analogues also confirmed similar behaviour for testicular InsP3Rs and cerebellar InsP3Rs.

Temperature-dependence of open-complex formation at two Escherichia coli promoters with extended -10 sequences.

We have studied the formation of open complexes between purified RNA polymerase from Escherichia coli and DNA fragments carrying the galP1 promoter, a promoter with an extended -10 region. Unusually, these complexes are formed readily at low temperatures. This low-temperature opening is unaffected by deletions of either upstream or downstream promoter sequences. We conclude that low-temperature open-complex formation is due to specific base sequences in and just upstream of the extended -10 region. In contrast, open complexes are not formed at low temperatures with DNA fragments carrying the E. coli cysG promoter, which also has an extended -10 region. This demonstrates that an extended -10 sequence alone is not sufficient for low-temperature opening. Additionally, we report the temperature dependence of a hybrid galP1-cysG promoter, the related galP2 and galP3 promoters and a derivative of galP1 with an improved -10 hexamer sequence.

Overexpression of human cardiac troponin-I and troponin-C in Escherichia coli and their purification and characterisation. Two point mutations allow high-level expression of troponin-I.

We have overexpressed human cardiac troponin-I in Escherichia coli. Initially, protein expression was not detected in the bacterial cell extracts. Systematic deletion of the N-terminal region of the protein generated a series of truncated mutants which were expressed at varying levels in the bacteria. This allowed us to narrow the problem down to the first five codons in the gene sequence. In order to achieve expression at high levels, two base changes were required, in the second and the fourth codons of the cDNA sequence. The codon changes, (Ala2) GCG-->GCC and (Gly4) GGG-->GGT, do not alter the coding potential of the DNA. We have also overexpressed the human cardiac isoform of troponin-C. Both proteins were purified using ion-exchange chromatography and have been proved to be biologically active. The recombinant troponin-I was able to bind to a troponin-C affinity column in the presence of 9 M urea in a calcium-dependent manner. The calcium-dependent troponin-I-troponin-C complex between both recombinant proteins was also demonstrated by alkaline-urea gel electrophoresis. In addition, troponin-I inhibited the acto-S1 Mg-ATPase activity; this inhibition was potentiated by the presence of tropomyosin and was reversed by the addition of troponin-C to the system. Biological activity was also demonstrated in vivo in that the recombinant proteins were able to restore the calcium-dependent force generation to calcium-insensitive skinned muscle fibres.

Cloning, sequencing and analysis of the structural gene and regulatory region of the Pseudomonas aeruginosa chromosomal ampC beta-lactamase.

The chromosomal gene from Pseudomonas aeruginosa encoding beta-lactamase has been cloned, and the sequence determined and compared with corresponding sequences of beta-lactamases from members of the enterobacteriaceae. Upstream of the beta-lactamase gene is an open reading frame which we postulate encodes a regulatory protein, AmpR. We identified a helix-turn-helix region in AmpR and a putative AmpR-binding site.

Transcriptional activation of the Klebsiella pneumoniae nifLA promoter by NTRC is face-of-the-helix dependent and the activator stabilizes the interaction of sigma 54-RNA polymerase with the promoter.

Activation of transcription at the Klebsiella pneumoniae nifLA promoter requires the phosphorylated form of the positive control protein NTRC, together with RNA polymerase modified by the alternative sigma factor sigma 54. Dimethylsulphate and potassium permanganate were used as probes to analyse the interaction of NTRC and sigma 54-RNA polymerase with supercoiled nifLA promoter DNA in vitro. In contrast to the glnAp2 promoter, sigma 54 holoenzyme did not protect guanine residues in the nifLA promoter from methylation in the absence of the activator. We propose that NTRC stabilizes the interaction of sigma 54-RNA polymerase with the -24, -12 region, in addition to its role in catalysing open complex formation. Phosphorylated NTRC binds to two sites located greater than 100 nucleotides upstream of the -24, -12 region; it also induces hyper-methylation of a G residue at -23. Enhanced methylation at -23 is not co-operative with the binding of activator to the upstream sites and may account for the ability of NTRC, when present at high concentration, to activate transcription in the absence of the upstream binding sites. The insertion of spacer mutations at -86 indicates that transcriptional activation of the nifLA promoter at low NTRC concentrations is face-of-the-helix dependent, both in vivo and in vitro. We propose that correct positioning of activator molecules at the upstream binding sites stabilizes the interaction of sigma 54-RNA polymerase with the downstream region via the formation of a DNA loop.

The role of activator binding sites in transcriptional control of the divergently transcribed nifF and nifLA promoters from Klebsiella pneumoniae.

The regulatory region spanning the divergently transcribed nifF and nifLA promoters contains a NIFA-specific upstream activator sequence (UAS) located around +59, and two NTRC binding sites centred at -142 and -163 with respect to the nifLA transcription start site. We have constructed mutations in each of these binding sites and examined their role in transcriptional activation of the divergently transcribed promoters. Analysis of a mutation at +60 confirms that the UAS is required for efficient NIFA-mediated activation of nifF transcription. This sequence is also required for maximal activation of the nifLA promoter. Mutations at -169 and -148, within the two NTRC binding sites, reduce activation of the nifLA promoter by NTRC in vivo and lower the affinity of the activator for these sites in vitro. Phosphorylation of NTRC by NTRB is required for efficient binding of NTRC to these sites.