Selection of scFv phages specific for chloramphenicol acetyl transferase (CAT), as alternatives for antibodies in CAT detection assays.

Reporter gene assays are commonly used in applied toxicology to measure the transcription of genes involved in toxic responses. In these reporter gene assays, transgenic cells are used, which contain a promoter-operator region of a gene of interest fused to a reporter gene. The transcription of the gene of interest can be measured by the detection of the reporter protein. Chloramphenicol acetyl transferase (CAT) is frequently used as a reporter protein in mammalian reporter gene assays. Although CAT can be measured by different detection systems, like enzymatic and immune assays, most of these tests are expensive, time-consuming and labor-intensive. The excellent characteristics of phages, like their high affinity and specificity, their fast, cheap and animal-friendly manufacturing process with low batch-to-batch variations and their stability, make them appropriate as alternatives for antibodies in detection assays. Therefore, in this study single-chain variable fragment (scFv) phages were selected with affinity for CAT. Several scFv phages were selected that showed affinity towards CAT in a screening ELISA. Surface plasmon resonance analyses showed that the tested scFv phages have an affinity for CAT with a dissociation constant (K(d)) around 1 µM. The selected scFv phages in this study could be used as capture elements in a highly sensitive sandwich ELISA to detect CAT concentration as low as 0.1 ng ml⁻¹ or 4 pM. This low detection limit demonstrates the potential of the scFv phages as an alternative for capturing antibodies in a highly sensitive detection test to measure CAT concentrations in reporter gene assays.

Interactions between two MerR regulators and three operator/promoter regions in the mercury resistance module of Bacillus megaterium.

The mercury resistance module of Bacillus transposon TnMERI1 is regulated by three operator/promoter regions (O/P merB3, O/P merR1, and O/P merR2) and two regulatory proteins (MerR1 and MerR2) encoded by the module itself. To clarify the roles of MerR1 and MerR2 in the regulatory mechanism, both proteins were overexpressed and purified. MerR1 bound the regulatory regions O/P merB3 and O/P merR1, with a preference for O/P merB3 as measured on in vitro gel shift assays. However, MerR2 bound O/P merR2, as revealed by gel shift and restriction endonuclease protection assays. The transcriptional start sites of O/P merB3 and O/P merR2 were determined by rapid amplification of 5'-cDNA ends (5'-RACE) in the TnMERI1 original host, Bacillus megaterium strain MB1. Real-time reverse transcription polymerase chain reaction (RT-PCR) assays showed that O/P merB3 and O/P merR1 were induced in the presence of Hg2+ but not O/P merR2. It was concluded that MerR1 regulates O/P merB3 and O/P merR1, while MerR2 regulates O/P merR2.

Identification and characterization of a DeoR-specific operator sequence essential for induction of dra-nupC-pdp operon expression in Bacillus subtilis.

The deoR gene located just upstream the dra-nupC-pdp operon of Bacillus subtilis encodes the DeoR repressor protein that negatively regulates the expression of the operon at the level of transcription. The control region upstream of the operon was mapped by the use of transcriptional lacZ fusions. It was shown that all of the cis-acting elements, which were necessary for full DeoR regulation of the operon, were included in a 141-bp sequence just upstream of dra. The increased copy number of this control region resulted in titration of the DeoR molecules of the cell. By using mutagenic PCR and site-directed mutagenesis techniques, a palindromic sequence located from position -60 to position -43 relative to the transcription start point was identified as a part of the operator site for the binding of DeoR. Furthermore, it was shown that a direct repeat of five nucleotides, which was identical to the 3' half of the palindrome and was located between the -10 and -35 regions of the dra promoter, might function as a half binding site involved in cooperative binding of DeoR to the regulatory region. Binding of DeoR protein to the operator DNA was confirmed by a gel electrophoresis mobility shift assay. Moreover, deoxyribose-5-phosphate was shown to be a likely candidate for the true inducer of the dra-nupC-pdp expression.

Construction and characterization of a stably transformed HeLa cell line in which the expression of bovine herpesvirus 1 ICP0 (BICP0) is induced by tetracycline.

To explore the effects BICP0 (a principal transactivator of BHV-1 gene expression) on viral promoter elements, we established a cell line in which the expression of BICP0 is regulated by tetracycline. A hybrid promoter containing reiterated copies of the tet-operator (tet-O) and a minimal herpesviral alpha gene transinducing factor (alpha TIF) responsive element (minimal human cytomegalovirus immediate early promoter) was fused to the BICP0 gene and used to transform a HeLa cell line which expressed a fusion protein consisting of the repressor of the tet-O and the transactivating domain of alpha TIF. Simultaneously, the hygromycin resistance gene was transfected to select cells in media containing either hygromycin alone or both hygromycin and tetracycline. Immunofluorescent assays indicated that BICP0 was synthesised in the transformed cell lines solely upon induction of the gene by tetracycline removal. Only cells which had been kept constantly in medium containing tetracycline were able to synthesise BICP0 upon induction. Induced cell lines transactivated the native BICP0 promoter as well as the herpes simplex virus thymidine kinase promoter and the long terminal repeat sequences of human immunodeficiency virus in a dose dependent manner. These cell lines may help to further explore the functions of BICP0 as well as to investigate the molecular basis of interactions between herpes- and retroviruses.

Specificity of action of a herpes virus VP16/tetracycline-dependent trans-activator in mammalian cell cultures.

In this work, we have studied the activity of a tetracycline modulatable trans-activator (tTA) generated by fusing the DNA binding domain of the tetracycline repressor to the trans-activation domain of the Herpes simplex virus protein 16 (HSV VP16) (plasmid pUHD15-1Neo). In the three different cell lines studied (HTC, rat hepatoma; T47D, human breast cancer; SK-N-BE, human neuroblastoma), the expression of the luciferase gene under the control of a tetracycline operator sequence (plasmid pUHC13-3) was used as a control of the incorporation and the functionality of the trans-activator. Clones selected from these cells responded in a time and dose-dependent manner to the withdrawal of tetracycline. In all these clones, the tTA trans-activator not only modulates the activity of the luciferase gene, but also modulates the activity of a number of endogenous proteins, including C/EBP beta, the glucocorticoid receptor (GR), and SP1. In the transfected cells, the level of these transcription factors was strongly inhibited in the presence of tetracycline and was highly increased after tetracycline removal. Electrophoresis mobility shift assay (EMSA) and footprint experiments proved that the induced proteins are perfectly efficient in binding the DNA. Their transcriptional activity was also determined. In HTC/A9 cells, the level of the chloramphenicol acetyltransferase (CAT) expression driven by the promoter of the alpha 1-glycoprotein (AGP) gene was strongly enhanced at 72-84 hr following removal of tetracycline from the growth media. The accumulation of the endogenous AGP mRNA also increased at 84 hr. In the T47D/TA11 and SK-N-BE/C2.6 cells, a general activation of protein synthesis was also evidenced.

A basic tail increases repression by dimeric lac repressor.

Tetrameric Lac repressor achieves cooperative repression by binding simultaneously to O1 and to one of the auxiliary operators O2 or O3, thereby forcing the intervening DNA into a loop. Dimeric Lac repressor is not able to form DNA loops and consequently shows no cooperative repression. We constructed a dimeric Lac repressor mutant which exhibits increased repression to the lac operon that does not depend on specific operator-repressor-operator loops. This Lac repressor carries a synthetic tail of basic residues attached to its C terminus. With this construct, we observe an increase of the in vivo repression upon addition of auxiliary lac operators to a chromosomal lac operon controlled by O1. This suggests that the basic tail enables dimeric Lac repressor to enhance its repression by additional non-specific DNA contacts.

Interaction of the Bacillus subtilis glnRA repressor with operator and promoter sequences in vivo.

In vivo dimethyl sulfate footprinting of the Bacillus subtilis glnRA regulatory region under repressing and derepressing conditions demonstrated that the GlnR protein, encoded by glnR, interacts with two sites situated within and adjacent to the glnRA promoter. One site, glnRAo1, between positions -40 and -60 relative to the start point of transcription, is a 21-bp symmetrical element that has been identified as essential for glnRA regulation (H. J. Schreier, C. A. Rostkowski, J. F. Nomellini, and K. D. Hirschi, J. Mol. Biol. 220:241-253, 1991). The second site, glnRAo2, is a quasisymmetrical element having partial homology to glnRAo1 and is located within the promoter between positions -17 and -37. The symmetry and extent of modifications observed for each site during repression and derepression indicated that GlnR interacts with the glnRA regulatory region by binding to both sites in approximately the same manner. Experiments using potassium permanganate to probe open complex formation by RNA polymerase demonstrated that transcriptional initiation is inhibited by GlnR. Furthermore, distortion of the DNA helix within glnRAo2 occurred upon GlnR binding. While glutamine synthetase, encoded by glnA, has been implicated in controlling glnRA expression, analyses with dimethyl sulfate and potassium permanganate ruled out a role for glutamine synthetase in directly influencing transcription by binding to operator and promoter regions. Our results suggested that inhibition of transcription from the glnRA promoter involves GlnR occupancy at both glnRAo1 and glnRAo2. In addition, modification of bases within the glnRAo2 operator indicated that control of glnRA expression under nitrogen-limiting (derepressing) conditions included the involvement of a factor(s) other than GlnR.

Stereochemical effects of L-tryptophan and its analogues on trp repressor's affinity for operator-DNA.

We have employed a filter binding assay to help study the mechanism by which bound L-tryptophan enables the Escherichia coli trp repressor to bind its operators. We have prepared variants of the trp repressor using structural analogues of the natural corepressor, L-tryptophan, and measured the affinity of these variants for a 20-base pair oligonucleotide duplex containing a symmetrical idealization of the trp operator from the E. coli trpEDCBA operon. By normalizing for each analogue's previously determined affinity for the trp aporepressor, we have estimated the extent to which each of the functional groups of bound L-tryptophan contributes to operator affinity. We discuss the likely role of these functional groups in the context of the crystal structures of the inactive, unliganded trp aporepressor, the liganded, active repressor, an inactive pseudorepressor (Pseudorepressors are formed by analogues of L-tryptophan that bind at the tryptophan-binding site but form near isomorphs of the repressor that have poor affinity for operator-DNA.) and the trp repressor/operator complex. We find that the alpha-amino group and an unsubstituted amino (-NH-) nitrogen of L-tryptophan's indole ring are essential for operator affinity. The former properly orients the corepressor and the latter interacts directly with the DNA. The alpha-carboxyl group, on the other hand, greatly enhances but is not essential for operator binding. The alpha-carboxylate's role, which is dependent on the corepressor's orientation in the binding pocket, is apparently to position the guanidinium group of Arg-84 for favorable contacts with the operator's sugar-phosphate backbone.

Interaction of the trp repressor from Escherichia coli with a constitutive trp operator.

The interaction of the trp repressor from Escherichia coli with a 20 bp fragment of DNA (CGTACTGATT.AATCAGTACG) corresponding to a mutant trp operator was studied by c.d. in the presence and absence of the co-repressor, L-tryptophan, and as a function of the concentration of K+ and Na+ ions. The affinity of the repressor for the mutant operator in the presence of tryptophan is about three orders of magnitude lower than the wild-type sequence. Binding in the absence of tryptophan is about 100-fold weaker than to the wild-type. The dependence of the dissociation constant on the concentration of K+ or Na+ is weak [d(log Ks)/d(log[M+]) = 2.5], and independent of the cation, indicating that electrostatic interactions are not as important for this repressor as for others.

Recombination by resolvase is inhibited by lac repressor simultaneously binding operators between res sites.

The Tn3 resolvase requires that the two recombination (res) sites be aligned as direct repeats on the same molecule for efficient recombination to occur. To test whether resolvase must contact the DNA between res sites as predicted by tracking models, we have determined the sensitivity of recombination to protein diffusion blockades. Recombination between two res sites is unaffected either by lac repressor or bacteriophage T7 RNA polymerase being bound between them. Yet recombination is inhibited by lac repressor if the res site is bounded by a lac operator on both sides. We demonstrate that lac repressor will bind to more than one DNA site under the conditions used to assay recombination. This result suggests that lac repressor can inhibit resolvase by forming a DNA loop that isolates a res site topologically. These results do not support a tracking model for resolvase but suggest that the structure and topology of the DNA substrate is important in the formation of a synapse between res sites.