Regulation of ferulic catabolic genes in Pseudomonas fluorescens BF13: involvement of a MarR family regulator.

In Pseudomonas fluorescens BF13, the cluster of genes essential for degradation of ferulic to vanillic acid (ech, vdh and fcs) is expressed in ferulic but not in succinic-grown cells. In the upstream region, we identified a gene, ferR, encoding a protein homologous to transcriptional regulators of the MarR family. A ferR knockout mutant (BF13-89) showed a 3.5-fold increase in expression of an ech-reporter gene fusion compared with the parent strain in succinic-grown cells, indicating that the ferR gene product negatively regulates expression of the ferulic catabolic operon in P. fluorescens BF13. Consistent with the increased expression of the catabolic genes in the ferR mutant, BF13-89 showed a shorter (relative to its FerR(+) parent) lag phase during carbon source shift from succinic to ferulic acid. However, expression of ech-lacZ fusion did not increase in BF13-89 grown in the presence of ferulic acid, indicating that FerR has a second function as transcriptional activator. Expression of ech-lacZ in a feruloyl-CoA synthetase-deficient strain revealed unambiguously that FerR-mediated activation of the ferulic catabolic operon is dependent on the thioester product of the feruloyl-CoA synthetase reaction.

A poplar plastocyanin mutant suitable for adsorption onto gold surface via disulfide bridge.

Aiming to achieve stable immobilization for a redox-active cupredoxin protein onto a gold substrate and its consequent molecular level monitoring by Scanning Tunnelling Microscopy (STM), we introduced a disulphide bridge within poplar plastocyanin, while avoiding the perturbation of its active site. We selected and modified residues Ile-21 to Cys and Glu-25 to Cys by structurally conservative mutagenesis. Optical absorption spectroscopy (UV-Vis), electron paramagnetic resonance (EPR), and resonance raman scattering (RRS) results indicate that the active site of the Ile21Cys, Glu25Cys plastocyanin (PCSS) to a large extent retains the spectroscopic properties of the wild-type protein. Furthermore, the redox midpoint potential of the couple CuII/CuI in PCSS, determined by cyclic voltammetry was found to be +348 mV close to the wild-type value. The STM images display self-assembled PCSS molecules immobilised onto gold substrate. Moreover, the full potentiostatic control of the electron transfer reaction during STM imaging, suggests that the adsorbed molecule maintains essentially its native redox properties.

Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.).

The monoclonal antibody DLT15, specific for thymocytes and peripheral T-cells of the teleost fish Dicentrarchus labrax (sea bass), was used to purify immunoreactive cells from blood and gut-associated lymphoid tissue. The purification was performed by immuno-magnetic sorting of leucocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by cytofluorimetric analysis. Following a single step, the percentage of DLT15-purified cells was 88 +/- 10% for gut-associated lymphoid tissue and 79 +/- 18% for blood leucocytes. DLT15-purified cells from gut-associated lymphoid tissue were employed for RNA extraction and cDNA synthesis. In RT-PCR experiments using as primers degenerate oligonucleotides corresponding to the peptide sequence MYWY and VYFCA of the trout TcR beta chain, a 203 bp product was amplified. When sequenced, the cDNA was found to show 60% nucleotide identity to the trout TcRV beta 3. By 3'-RACE the cDNA was elongated to obtain the TcR constant region, with high similarity to other fish TcR sequences. These results strongly suggest that cells recognised by DLT15 are putative T lymphocytes.

Nucleoli, rRNA genes and ITS region in Posidonia oceanica (L.) Delile.

The maximum number of nucleoli was counted in interphase nuclei of Posidonia oceanica, and a restriction pattern of nuclear rDNA was obtained after digestion with four restriction endonucleases and Southern hybridization. P. oceanica has only one type of ribosomal gene whose size was estimated to be 18.5 kbp long. The nucleotide sequence of the entire ITS region was also determined by direct sequencing of PCR amplified DNA fragments. The sequence of the ITS region was aligned with those of homologous regions of other monocots available in literature, and phylogenetic trees were obtained.

The human beta 2-adrenergic receptor expressed in Schizosaccharomyces pombe retains its pharmacological properties.

We have developed a rapid and efficient expression system to study the human beta 2 adrenergic receptor (hu beta 2AR) in the fission yeast Schizosaccharomyces pombe. This was achieved by cloning the hu beta 2AR gene, modified by replacement of the 5' untranslated and a small part of the N-terminal coding sequence (first 14 amino acids) with the corresponding region of the yeast Saccharomyces cerevisiae STE2 (alpha-factor receptor) gene. The gene was then placed under the control of a S. pombe constitutive promoter for alcohol dehydrogenase (adh). Hu beta 2AR expression was assessed by immunoblot analysis of the chimeric protein with an anti-STE2 serum raised against a dodecapeptide homologous to the N-terminal amino acids of STE2 and ligand binding was assayed using [125I]cyanopindolol. We demonstrate here that the chimeric receptor expressed in S. pombe exhibits the same characteristic ligand specificity and affinity as that of the authentic hu beta 2AR. This system constitutes a convenient alternative to existing methods for studying seven transmembrane domain receptors due to its simplicity and high reproducibility.

Functional relationship among TATA sequences, gene induction and transcription initiation in the beta-galactosidase, LAC4, gene from Kluyveromyces lactis.

In the 5' non-coding region of the beta-galactosidase, LAC4, gene of Kluyveromyces lactis, three TATA-like sequences are present at -230, -170 and -142 from the ATG translation start site. By means of deletion mutations in the TATA region, at least two of these TATA sequences, those at -230 and -142, were shown to be required for normal gene expression. Evidence is presented for a functional hierarchy and cooperation between these TATA sequences. The deletion or a change in the position of the TATA sequences affects both beta-galactosidase induction and the location of RNA initiation sites. The TATA sequence at -230 alone is sufficient for correct gene induction when it is moved to a position 41 bp from the major RNA initiation sites located around -110; the -142 TATA alone contributes only partly to gene induction. We suggest a functional distinction between these two related regulatory sequences. This functional distinction might be established by sequence differences and/or targets of unlike specific DNA binding protein(s). A conformational analysis of the LAC4 promoter showed that under torsional stress the functional elements UAS, TATA boxes RNA initiation sites and ATG can be detected as P1-sensitive sites. Possible functions of DNA structural alterations on gene expression are discussed.

Positive regulation of the beta-galactosidase gene from Kluyveromyces lactis is mediated by an upstream activation site that shows homology to the GAL upstream activation site of Saccharomyces cerevisiae.

In contrast to the Escherichia coli lac operon, the yeast beta-galactosidase gene is positively regulated. In the 5'-noncoding region of the Kluyveromyces lactis LAC4 gene, we mapped an upstream activation site (UAS) that is required for induction. This sequence, located between positions -435 and -326 from the start of translation, functions irrespective of its orientation and can confer lactose regulation to the heterologous CYC1 promoter. It is composed of at least two subsequences that must act in concert. One of these subsequences showed a strong homology to the UAS consensus sequence of the Saccharomyces cerevisiae GAL genes (E. Giniger, S. M. Varnum, and M. Ptashne, Cell 40:767-774, 1985). We propose that this region of homology located at about position -426 is a binding site for the product of the regulatory gene LAC9 which probably induces transcription of the LAC4 gene in a manner analogous to that of the GAL4 protein.

Topological properties of DNA domains and interaction with RNA polymerase II.

We have analyzed the relationship between the alterations of the DNA structure induced by topological constraint and the template properties of promoters in vitro. A cause-effect relationship has been defined in several instances. Experimental protocols have been developed for the study of the topological properties of RNA polymerase II promoters. The goal of these studies is the definition of the intrinsic structural informations of DNA.