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Cold-sensitive mutants of Drosophila melanogaster defective in ribosome assembly
Abstract:
Thirteen X-linked, cold-sensitive lethal, female-sterile mutants of Drosophila melanogaster located at eight separate loci were screened for their ability to assemble ribosomes at the restrictive temperature of 17 degrees. Females were labelled with 3H-uridine for either 2 or 20 hours at 17 degrees. A mitochondria-free extract was prepared and analyzed by means of sucrose gradient centrifugation. Four of the mutants, l(1)TW-2cs, l(1)HM16cs, l(1)HM23cs, and l(1)HM20cs, had a lower ratio of cpm in the 40S subunit to cpm in the 60S subunit (40S:60S ratio) than wild type with a 2-hour label. The same was true of a 20-hour label of l(1)TW-2cs, l(1)HM16cs, and l(1)HM23cs, which are allelic, resulted in a 40S:60S ratio higher than wild type. Four other cs mutants were found to have less drastic effects on ribosome assembly. The ribosomal subunits of mutants l(1)HM16cs and l(1)HM20cs sediment at the same rate as their wild-type counterparts. The same is true for the RNA in their ribosomal particles. Sucrose gradient analysis of ribosomes from cold-sensitive lethal, female-sterile mutants appears to be an effective method for finding mutants that affect ribosome assembly.
Insights
This study identifies four Drosophila melanogaster mutants that disrupt ribosome assembly. Analyzing ribosome subunit ratios reveals defects in cold-sensitive lethal mutants, aiding in the discovery of new genetic factors.
Area of Science:
- Developmental Biology
- Genetics
- Molecular Biology
Background:
- Ribosome biogenesis is crucial for protein synthesis and cellular function.
- X-linked, cold-sensitive lethal, and female-sterile mutations in Drosophila melanogaster can impact essential cellular processes like ribosome assembly.
Purpose of the Study:
- To investigate the ribosome assembly efficiency of thirteen X-linked, cold-sensitive lethal, female-sterile Drosophila mutants at a restrictive temperature.
- To identify specific mutants with defects in ribosome subunit production or maturation.
Main Methods:
- Drosophila melanogaster females were exposed to 3H-uridine labeling at 17 degrees Celsius.
- Mitochondria-free extracts were prepared and analyzed using sucrose gradient centrifugation to assess ribosome subunit ratios (40S:60S).
Main Results:
- Four mutants (l(1)TW-2cs, l(1)HM16cs, l(1)HM23cs, and l(1)HM20cs) exhibited altered 40S:60S ratios, indicating defects in ribosome assembly.
- Specific allelic mutants showed varying impacts on the 40S:60S ratio depending on labeling duration.
- The ribosomal subunits and RNA from mutants l(1)HM16cs and l(1)HM20cs were structurally similar to wild-type.
Conclusions:
- Sucrose gradient analysis of ribosome assembly is an effective method for identifying mutants affecting this process.
- Several cold-sensitive lethal, female-sterile mutants in Drosophila melanogaster possess defects in ribosome biogenesis.