Loretta Leive

Release of lipopolysaccharide by EDTA treatment of E., coli

Abstract The treatment of E. , coli with ethylenediaminetetraacetate which causes an increase in permeability also results in the rapid release of 35 to 50% of the cell wall lipopolysaccharide. Since the same conditions produce negligible loss of several other cell components, it is possible that the release of lipopolysaccharide is a direct result of EDTA action on the cell surface. This treatment may be a useful tool in the isolation of chemically pure lipopolysaccharide.

Actinomycin sensitivity in Escherichiacoli produced by EDTA

Abstract E. coli can be made sensitive to the action of actinomycin by brief treatment with EDTA. Cells thus treated are not osmotically fragile and are viable. It is anticipated that such preparations will be of value in studying RNA metabolism and related problems in E. coli

Synthesis, utilization and degradation of lactose operon mRNA in Escherichia coli

Abstract The action of inducer, and the kinetics of transcription, utilization, and degradation of lactose operon messenger RNA have been studied. Cells of Escherichia coli were made sensitive to actinomyein by brief treatment with EDTA, and then induced. The levels of β-galactosidase and thiogalactoside transacetylase were assayed at various times before and after the termination of induction by removal of inducer or addition of actinomyein. The results support the following conclusions. 1. (1) β-Galactosidase messenger RNA and transacetylase messenger RNA are degraded at approximately the same rate. If it is assumed that this operon produces a polycistronic messenger, then both ends are degraded at approximately the same rate. 2. (2) Inducer does not affect the rate of translation or the rate of degradation of β-galactosidase messenger RNA. 3. (3) After induction, 2.5 minutes (at 30 °C) are required for β-galactosidase induction to become insensitive to actinomyein. A total of four minutes is required for transacetylase induction to become insensitive to actinomyein. These results suggest that inducer action and the transcription of β-galactosidase take 2.5 minutes, while an additional 1.5 minutes is required to complete transcription of the entire operon. 4. (4) After the appropriate cistron has been transcribed, the remaining events culminating in appearance of either β-galactosidase or transacetylase take approximately 1.5 to 2 minutes. The results suggest that synthesis of β-galactosidase proceeds while transacetylase messenger RNA is still being formed. If this messenger RNA is polycistronic, it can be postulated that it is transcribed, translated and degraded simultaneously.

RNA degradation and the assembly of ribosomes in actinomycin-treated Escherichia coli

The fate of various fractions of RNA in Escherichia coli following inhibition of RNA synthesis by aetinomycin was studied. Cultures were made sensitive to the drug by brief treatment with EDTA. Following addition of aetinomycin, cells growing in minimal medium at 37°C lose 1·5 to 3% of their RNA with a half-life of approximately 1·5 minutes. This labile fraction appears to be messenger RNA because: (a) its loss is paralleled by loss of the in vivo capacity to make protein, and (b) sucrose gradients reveal that it sediments between 5 and 15 s, while during its disappearance other RNA, even if newly formed, is stable. The time course of incorporation of RNA into ribosomes was followed by adding aetinomycin to cells containing pulse-labeled RNA and determining the sedimentation properties in sucrose gradients of particles containing newly synthesized ribosomal RNA. RNA destined to appear in 50 s ribosomes first sediments at a much lower molecular weight, less than 30 s; then it forms an entity sedimenting at 40 to 45 s, and thereafter becomes part of a 50 s ribosome. This sequence provides evidence for a 40 to 45 s intermediate in the synthesis of 50 s ribosomes.

Some effects of inducer on synthesis and utilization of β-galactosidase messenger RNA in actinomycin-sensitive Escherichiacoli

Abstract The initial events in β-galactosidase induction were studied in Escherichia coli made sensitive to actinomycin by brief treatment with ethylenediaminetetraacetate. Experiments performed in the presence and absence of actinomycin or inducer lead to the following conclusions: (a) The synthesis of specific, non-DNA bound messenger RNA requires 2.5 minutes of the 4 minute interval between inducer addition and enzyme production. (b) The rate of destruction and the rate of utilization of this messenger RNA are independent of the presence or absence of inducer.