Properties of cloned promoters

Strong early bacteriophage T7 promoters A2 and A3 and also A2 and lac UV5 promoters with altered segments downstream the initiation of RNA start point were cloned using specially constructed plasmid vectors pBRS188 and PBRS240. The relative signal strengths of these promoters in vivo and in vitro were evaluated and the kinetic parameters of their interaction with RNA polymerase were determined. It has been shown that the nucleotide sequence of the transcribed region plays a significant role in specific promoter-RNA polymerase interaction and that the rate-limiting step of RNA synthesis initiation is different for various promoters.     

Deletion analysis of pollen-expressed promoters

Summary We have used microprojectile bombardment of tobacco pollen to study the DNA sequences involved in the expression of pollen-expressed genes. Promoter-reporter gene fusions constructed with the promoters of three different pollen-expressed genes from tomato (LAT52, LAT56, and LAT59) and either theβ-glucuronidase or luciferase reporter genes were assayed by bombarding hydrated tobacco pollen with the gene constructs precipitated onto tungsten microprojectiles. Reporter gene expression can be assayed within 30 min, with the maximal level of expression between 6 and 12 h after bombardment. By constructing and assaying promoter deletion derivatives, we have been able to delimit regions of the promoters that are necessary for high level expression in pollen. We also demonstrate that results with this transient expression system parallel the expression levels seen in pollen from stably transformed transgenic plants. The microprojectile bombardment assay can be used to rapidly test constructs for pollen expression beforeAgrobacterium-mediated plant transformation. Furthermore, it may be possible to adapt the microprojectile bombardment technique to achieve stable transformation of pollen. Presented in the Session-in-Depth Genetic Transformation and Genetic Analysis Using Microprojectile Bombardment at the 41st Annual Meeting of the Tissue Culture Association, Houston, Texas, June 10–13, 1990. This work was supported by the U.S. Department of Agriculture, Washington, DC, ARS CRIS 5335-22230-002-00D, and by the NSF Center for Plant Developmental Biology, UC-Berkeley, DIR-8719933.     

Electrostatic potentials of tumour promoters

Molecular-modelling studies and quantum-chemical calculations have been undertaken on a variety of structurally unrelated tumour promoters. The modelling studies reveal some broad areas of agreement in the associated electrostatic potentials for the molecules TPA, teleocidin, aplysiatoxin and ingenol. Azulene-type derivatives were used as models for inactive and active tumour promoters in the quantum chemical studies. The results obtained from these calculations of the electrostatic potentials show very little difference between active and inactive compounds, thus suggesting that other factors are of importance in these systems.     

Cloning of bacteriophage T5 promoters

Summary Bacteriophage T5 was subjected to combined hydrolysis with the restriction endonuclease PstI and HindIII and the resulting fragments were inserted into the plasmid pBR322. Selection of transformants for Ap^s-Tc^r-phenotype made it possible to screen the hybrid plasmids that contained promoter sequences in the cloned fragments.Two PstI/HindIII fragment, 720 bp (51% of the T5 DNA length) and 1,200 bp (70%) were cloned in this study. Tc^r levels for these plasmids were as high as 18 g/ml and 75 g/ml, respectively. The presence of Escherichia coli RNA polymerase binding sites on both fragments was shown using the nitrocellulose filter assay. These binding sites are situated between 35 bp and 95 bp from the HindIII cleavage site on the 1,200 bp fragment; and within 420 bp from the HindIII site on the 720 bp fragment.Abbreviations Ap ampicillin - Tc tetracycline - bp base pairs - NTPs nucleoside triphosphates - PBB polymerase binding buffer     

Thiol-sensitive promoters of Escherichia coli.

Mu dX(lac) insertion mutants of Escherichia coli CSH50 in which the expression of the lacZ gene was sensitive to the presence of exogenous 1-thioglycerol or dithiothreitol were isolated. Both stimulatory and inhibitory mutants were found. The existence of several thiol-sensitive promoters suggests that exogenous thiols may provoke global stress responses in E. coli.