Originally, RNAse I contaminating most preparations of ribosomes caused much trouble in the study of ribosomes and their constituents. One might ask how the ribosomes could function in vivo without degradation if they are so quickly degraded in vitro. The answer is that RNAse I is located in the cell’s periplasmic space. There it does no harm to the ribosomes until the cells are lysed and it is released, whereupon it adventitiously binds tightly to ribosomes and degrades their RNA. The degradation is rapid at room temperature or above, but even occurs at temperatures near 0°.

Molecular biologists used two sensible approaches to solve the RNAse problem. The first was a classic case of applying genetics to solve a biochemical problem—isolate an RNAse I- mutant. This was not a trivial task because no genetic selection was apparent for permitting the growth of just RNAse I- mutants, nor was any physiological trait likely to reveal the desired mutants. The only known characteristic of the desired mutants would be their lack of RNAse I in the periplasmic space. The obvious solution to the problem of isolating the desired mutant under the circumstances, but apparently not one used before, was merely to use a brute-force approach and score several thousand candidate colonies from a heavily mutagenized culture for absence of the enzyme.

To minimize the work of scoring, the mutagen had to be highly effective. Fortunately, nitrosoguanidine can induce multiple mutations into each cell. As a result, any mutant lacking a nonessential gene activity can be found in a population of a few thousand candidates from nitrosoguanidine-mutagenized cultures. The work required to perform conventional RNAse I assays on several thousand different cultures is large. Therefore Gesteland devised two simple scoring methods. In one, the whole cells from individual colonies grown from a mutagenized culture were resuspended at 42° in buffer containing radioactive ribo somal RNA and EDTA. The high temperature and the EDTA released the RNAse from the cells without lysing them. Then, after an incubation in the presence of radioactive RNA, the undegraded RNA was precipitated by addition of acid, and its radioactivity was determined. Several hundred colonies per day could be assayed for the ability of their RNAse I to degrade the RNA. The second scoring method used a clever plate technique in which duplicate plates contained the colonies to be tested. One was overlaid with several milliliters of agar containing a high concentration of tRNA and EDTA and was incubated at 42°. During a few hours of incubation, those colonies containing RNAse I digested the tRNA to short oligonucleotides. The mutant colonies lacking RNAse I could not digest the tRNA in their immediate vicinity. Then the plate was flooded with concentrated HCl. The acid precipitated the tRNA, leaving the plate opaque except in the areas surrounding colonies containing RNAse I. The desired RNAse I- colonies lacked cleared halos and could easily be detected.

The second approach for elimination of RNAse I problems was biological. A number of different bacterial strains were examined for this enzyme. Strain MRE600 was found to lack the enzyme. Therefore this strain has been used as a source of ribosomes in some structural and assembly studies.

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مواضيع ذات صلة

Experiments with in vitro Ribosome Assembly

The Global Structure of Ribosomes

Assaying for Sequence Requirements of Gene Rearrangements

The D Regions in H Chains

The J Regions

Many Copies of V Genes and Only a Few C Genes

P Element Hopping by Chromosome Rescue

P Elements and Transformation

Retrotransposons in Higher Cells

Hopping by Tn10

Membrane Proteins

In vitro Transposition, Threading or Global Topology?