Genetic associations of interleukin-2 levels in the chicken

http://www.worldcat.org/oclc/1599118

Identification and characterization of mycoplasma promoters

The development of a reporter gene system for mycoplasmas was sought to be able to examine gene regulation in mycoplasmas. Initial studies were designed to examine whether the Escherichia coli lacZ gene could be used as a reporter gene in mycoplasmas. The test system examined the ability of the Mycoplasma capricolum rrnA P2 or E. coli rrnB P1 promoters to initiate the transcription of trp[superscript]\u27-lacZYA in Acholeplasma ISM1499. The results showed that the M. capricolum, but not the E. coli promoter, could promote the expression of lacZ. Because the test system was based on a translational fusion that used the E. coli translational apparatus, the promoter probe vector pISM2050 was constructed to increase expression of lacZ in Acholeplasma. This vector was constructed so that randomly cloned mycoplasma DNA inserts must possess both the transcriptional and translational regions in order to drive lacZ expression. Only ten percent of the randomly cloned chromosomal DNA inserts from Acholeplasma ISM1499 that gave blue colony formation on 5-bromo-4-chloro-3-indolyl-[beta]-D-galactoside (X-gal) containing media in E. coli continued to demonstrate [beta]-galactosidase activity in Acholeplasma. This suggested that the A + T rich mycoplasmal sequences were giving pseudo promoter activity in E. coli. A Tn4001lac promoter probe vector was also developed. The insertion of lacZ into an IS256 arm of Tn4001, however, resulted in a ten-fold drop in transformation frequency as compared to the wild type. A derivative of Tn4001lac, Tn4001.2065, can be used to rescue adjacent chromosomal sequences that have demonstrated promoter activity. Thirteen Acholeplasma ISM1499 chromosomal DNA fragments containing promoter activity were identified and seven were mapped by primer extension. The sequence of the -10 region of the Acholeplasma promoter was similar to the consensus E. coli promoter, but the -35 region was more variable. The transcript levels of these lacZ fusion constructs in acholeplasma correlated with levels of [beta]-galactosidase activity. In conclusion, these studies show that the study of gene regulation can now be performed in mycoplasmas, and preliminary evidence suggests that the mycoplasma promoter is similar to the E. coli consensus promoter