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Purification and properties of biologically active rainbow trout testis protamine mRNA
Abstract:
At least two classes of protamine mRNA are present in both trout testis polysomal RNA and RNA from the postribosomal supernatant fraction of trout testis hormogenate both of which direct the synthesis of protamine in a Krebs II ascites S-30. One contains poly(A) tracts and the other is devoid of poly(A). Sucrose gradient analyses showed that the poly(A) containing protamine mRNA (poly(A) (+)) sedimented IN THE 6 S region with a shoulder in the 4 S region while the protamine mRNA devoid of poly(A) (poly(A) (-)) appeared to sediment at about 4 S and could not be resolved from tRNA. Analysis of the poly(A) (+) protamine mRNA by boundary sedimentation in an analytical ultracentrifuge showed a sedimentation coefficient of 5.7 S, a value which gives rise to an estimate of 165 to 170 nucleotides per molecule. The poly(A) (+) protamine mRNA migrated as a single species in formamide-containing polyacrylamide gels and its mobility in relation to markers of tRNA (4 S) and 5 S RNA was consistent with its sedimentation velocity of 6 S. The RNA present in the major band on an aqueous polyacrylamide gel was extracted and shown to code for protamine in a wheat germ cell-free system.
Insights
Two types of protamine messenger RNA (mRNA) were identified in trout testis, differing in their poly(A) tail presence. Both mRNA classes direct protamine synthesis, with poly(A)-containing mRNA being larger.
Area of Science:
- Molecular Biology
- RNA Biology
- Gene Expression
Background:
- Protamine synthesis is crucial for sperm development.
- Messenger RNA (mRNA) molecules carry genetic information for protein synthesis.
- The presence or absence of poly(A) tails on mRNA can affect its stability and translation.
Purpose of the Study:
- To investigate the characteristics of protamine mRNA in trout testis.
- To determine if different classes of protamine mRNA exist.
- To analyze the role of poly(A) tracts in protamine mRNA.
Main Methods:
- Isolation of polysomal RNA and postribosomal supernatant RNA from trout testis.
- In vitro protein synthesis using Krebs II ascites S-30 system.
- Sucrose gradient centrifugation and analytical ultracentrifugation.
- Polyacrylamide gel electrophoresis (PAGE).
Main Results:
- Two classes of protamine mRNA were found: one with poly(A) tracts (poly(A)(+)) and one without (poly(A)(-)).
- Poly(A)(+) protamine mRNA sedimented around 6 S, while poly(A)(-) mRNA sedimented around 4 S.
- Poly(A)(+) protamine mRNA was estimated to be 165-170 nucleotides long and migrated as a single species on gels.
- Extracted RNA from the major band coded for protamine in a wheat germ cell-free system.
Conclusions:
- Trout testis contains distinct populations of protamine mRNA based on poly(A) tail presence.
- These mRNA classes differ in size and sedimentation properties.
- Both classes are functional in directing protamine synthesis.