This research has investigated the effect that altered supercoiling has on in vivo expression from bacterial promoters. In vivo expression was measured using the pKO system developed by McKenney et al. (1982). Supercoil levels were decreased through the use of the antibiotic nalidixic acid and were increased using a topoisomerase I deletion mutant. Galactokinase expression from a series of related hybrid promoters, the trp promoter flanked by oligonucleotide blocks, the trp promoter flanked by linker sections and the trp promoter flanked by a gyrase binding site was measured after incubation with nalidixic acid. Expression from the pBR322 tet promoter was inhibited by 50% under these conditions. Expression from the lacUV5, trp, and tettrp promoters was essentially unaffected. A 2-fold stimulation of expression was observed from the trplac and the trptet promoters. Expression from the trp promoter when flanked by upstream or downstream oligonucleotide blocks was similar to the values observed from control plasmids. The presence of a gyrase binding site (Kirkegaard and Wang, 1981) also exerted little influence on overall expression after a decrease in supercoiling.
To further study control of expression from the trp promoter, a series of plasmids was constructed which contained a synthetic lac operator at defined positions either upstream or downstream of the promoter. Downstream lac repressor binding diminished the levels of expression, while upstream binding had little effect on expression. Placement of the lac operator farther downstream decreased the level of repression observed. Plasmids were also constructed containing two lac operators, one upstream at -39 or -52 and an additional operator downstream. Galactokinase expression from these plasmids was decreased more than in those plasmids with only one lac operator.
In addition a system was developed that combined the M13 bacteriophage system of Messing (1981) and the pKO system of McKenney et al. (1982) in order to measure short term changes in transcription. Gene fragments from the (beta)-lactamase gene of pBR322 and the galactokinase gene of pKO-1 were cloned into M13mp8. Single strand DNA from these constructions will hybridize with mRNA transcribed from pKO derivatives, allowing measurement of transient changes in transcription.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/15905 |
| Date | January 1985 |
| Creators | HERRIN, GEORGE LEMON, JR. |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | application/pdf |
Page generated in 0.0118 seconds