Related Experiment Videos
Isolation of ribosome particles from meningopneumonitis organisms
Abstract:
In ribonucleic acid (RNA) extracted by phenol and sodium dodecyl sulfate from purified reticulate bodies of meningopneumonitis (MP) organisms, 21S, 16S, and 4S RNA were found by sucrose density gradient sedimentation analysis. When purified reticulate bodies were homogenized by sonic treatment or by treatment with sodium deoxycholate and were fractionated by differential centrifugation, more than 50% of the RNA was recovered in the fraction which was sedimented by centrifugation at 105,000 x g for 2 hr, but not at 13,000 x g for 20 min. From homogenates prepared in this manner, 50S and 30S particles containing RNA were isolated by sucrose density gradient centrifugation. These 50S and 30S particles were also found in lysates of cytoplasmic fractions of infected cells which were labeled by (32)P during 17 to 17.5 hr or 15 to 18 hr after infection. The synthesis of 50S and 30S particles was not inhibited by actinomycin D. When infected cells were homogenized in the presence of 0.01 or 0.02 m MgCl(2), 70S particles were isolated instead of 50S and 30S particles. When dialyzed against low concentrations of MgCl(2), the 70S particles dissociated to 50S and 30S particles. The base ratio of the 70S particles is very similar to that of 16S plus 21S RNA. The characteristics of the 70S, 50S, and 30S particles suggest that these are ribosome particles, similar to bacterial ribosomes.
Insights
Researchers identified 50S and 30S ribonucleic acid (RNA) particles in meningopneumonitis organisms, suggesting they are ribosome components. These particles, similar to bacterial ribosomes, were found to be magnesium-dependent and resistant to actinomycin D.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Meningopneumonitis (MP) organisms are obligate intracellular bacteria.
- Understanding the molecular composition of MP organisms is crucial for comprehending their biology and pathogenesis.
Purpose of the Study:
- To characterize the ribonucleic acid (RNA) components and associated particles within purified reticulate bodies of MP organisms.
- To investigate the nature and assembly of these RNA-containing particles and their potential similarity to bacterial ribosomes.
Main Methods:
- Extraction and purification of RNA from MP organisms using phenol and sodium dodecyl sulfate.
- Sucrose density gradient centrifugation and differential centrifugation to isolate and analyze RNA and particulate fractions.
- Labeling of infected cells with (32)P to track RNA synthesis.
- Investigation of the effect of magnesium chloride (MgCl2) concentration on particle stability and dissociation.
Main Results:
- Identified 21S, 16S, and 4S RNA species in purified MP organisms.
- Isolated 50S and 30S RNA-containing particles from homogenized MP organisms.
- Demonstrated that these 50S and 30S particles are also present in infected host cell cytoplasm.
- Observed that 70S particles form in the presence of higher MgCl2 concentrations and dissociate into 50S and 30S particles upon dialysis against lower MgCl2 concentrations.
- Found that 50S and 30S particle synthesis is not inhibited by actinomycin D.
Conclusions:
- The isolated 50S and 30S particles, along with the 70S particles, exhibit characteristics consistent with ribosome subunits and intact ribosomes, respectively.
- These findings strongly suggest that MP organisms possess ribosome particles analogous to those found in bacteria.
- The magnesium-dependent nature of particle assembly and dissociation provides insights into ribosome biogenesis in these organisms.