Uncoupling of mitochondrial oxidative phosphorylation by thallium.

The mechanism of integration of λ$\text{bio}$ll, which is deleted of all the known λ recombination genes, was studied using $\text{bio}$ deleted hosts as recipients. The presence of $\text{rec}$BC DNase and $\text{exo}$I in the recipient cells affected the fate of λ$\text{bio}$ll DNA. In nine of ten $\text{imm}\text{λ}{}^{\mathrm{+}}$ transductants, insertion of the λ$\text{bio}$ll genome took place somewhere between J and N and the remaining one had abnormally permuted prophage λ. In this lysogen (#42), the sequence of prophage genes was similar to that of vegetative phage λ. The properties of lysogen #42 were compared with those of other lysogens.     

‘ATG vectors’ for regulated high-level expression of cloned genes in Escherichia coli

A plasmid cloning vector system has been constructed that allows for the production of large quantities of foreign proteins or fragments thereof, in an unfused state. These vectors provide strong regulated trp-lac fusion promoters and the lacZ ribosome-binding site (RBS) followed by an ATG translation initiation codon at an appropriate distance from the RBS. The ATG codon is located within a unique NcoI restriction site (CCATGG). Digestion with NcoI exposes the ATG for fusion. Gene fragments lacking a prokaryotic RBS and/or ATG start codons can be inserted in several ways. Expression experiments using a truncated cI gene of bacteriophage A or a large portion of the coding region of the Herpes simplex virus type l glycoprotein D gene have been performed. The results of these studies show that the vectors are useful for the high-level expression of prokaryotic and eukaryotic genes in Escherichia coli.     

Toxicity symptoms caused by high expression of Tet represser in tomato (Lycopersicon esculentum Mill. L.) are alleviated by tetracycline

As a first step towards transferring a tetracycline (Tc)-inducible gene expression system to tomato, we have transformed tomato plants with the Tn10-encoded tet repressor gene (tetR). Homozygous transformed plants with high expression of tetR mRNA show a deleterious phenotype, having reduced shoot dry weights and leaf chlorophyll content, an even more marked reduction in root dry weight and leaf size, and altered photosynthetic physiology. It appears that TetR protein exerts its toxicity only when expressed beyond a threshold level and by interacting with a process that is non-limiting under slow growth conditions. The deleterious phenotype was almost completely reversed by the application of 1 mg dm^?3 Tc to plants grown in sand. The possiblity is discussed that TetR causes these symptoms by binding to a specific DNA sequence functioning as a Tet operator. The effect of Tc on growth and physiology in wild-type plants grown in sand or rockwool is described. Tc at 0.1 mg cm^?3 had no effect. Tc at 1 mg dm^?3 caused a small reduction in root growth, while 5 and 20 mg dm^?3 Tc caused large reductions in growth and photosynthetic parameters.     

Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.     

Efficiency of the tetracycline-dependent gene expression system: complete suppression and efficient induction of the rolB phenotype in transgenic plants

We have investigated the use of the tetracycline-dependent gene expression system to regenerate and propagate tobacco plants transformed with a gene whose product — when highly expressed — interferes with regeneration and/or further reproduction. Plants transformed with the Agrobacterium rhizogenes rolB gene under the control of the tetracycline-dependent expression system were phenotypically indistinguishable from wild type owing to efficient repression of the promoter. Induction of the rolB gene with tetracycline led to high-level expression of the rolB mRNA, which resulted in extremely stunted plants with necrotic and wrinkled leaves that did not develop a floral meristem. Upon cessation of tetracycline treatment healthy shoots developed even from severely affected meristems. Data on the dose response of the rolB phenotype as a function of tetracycline concentration demonstrate that the tetracycline-dependent gene expression system can be used to modulate the manifestation of a particular phenotype.     

High pressure conditions stimulate expression of chloramphenicol acetyltransferase regulated by the lac promoter in Escherichia coli

Abstract Recombinant plasmids with the chloramphenicol acetyltransferase (CAT) structural gene behind several kinds of promoters were tested for expression in Escherichia coli during growth at atmospheric pressure (0.1 MPa) and at high pressure (30 MPa). Expression of the CAT gene from the lac promoter was remarkably activated (approx. 78-fold) by high pressure in the absence of the inducer isopropyl-β-d-thiogalactopyranoside (IPTG). The stimulation of the CAT activity by the lac promoter at high pressure did not simply result from an increased plasmid copy number, because the CAT activities from the other promoters and β-lactamase activities were unaffected at high pressure.     

Strong and regulated promoters in the cyanobacterium Anabaena PCC 7120

Abstract The strengths of several promoters were assessed in the cyanobacterium Anabaena PCC 7120 by fusing them to luxAB , encoding bacterial luciferase. Two promoters, P _tac and P _psbA , with sequences nearly identical to consensus Escherichia coli σ ⁷⁰ promoters, gave as high or higher expression than the strong Anabaena promoter, P _rbc . P _npt , the natural promoter driving expression of the kanamycin-resistance determinant from Tn5, was poorly expressed in Anabaena . The Lac repressor partially repressed expression from P _tac , permitting regulated expression in Anabaena after induction with isopropyl thiogalactoside to a level 4–5-fold higher than without inducer.