Related Experiment Videos
Translation of protamine mRNA in a rabbit reticulocyte cell-free system
Abstract:
Protamine mRNA isolated from the microsomal and postribosomal supernatant fractions of trout testis in poly A(+) (polyadenylated RNA)and poly A (-) (RNA devoid of poly A(+)) forms (GEDAMU, L. & Dixon, G.H. (1976) J. Biol. Chem. 251, 1446-1454 and 1455-1463) was translated in the heterologous rabbit reticulocyte cell-free system; the products were shown to be identical in mobility with authentic protamine by polyacrylamide and starch gel electrophoresis. Chromatography, on carboxymethyl cellulose (Whatman CM-52), of the labelled polypeptide products synthesized in this cell-free system in the presence of poly A (+) and poly A(-) mRNA fractions also showed that [14C]arginine was incorporated into all three protamine components resolved in this system, but there was an unequal and variable incorporation of label into the three components with different preparations of mRNA. These results were interpreted as showing that the population of subcomponents of the protamine mRNA coding for the three different protamine polypeptides varied in batches of trout testis at differing stages of development. In addition, the proportion of mRNA components varied between the poly A(+) and poly A(-) editions of the mRNA, and it appeared that the poly A(-) mRNA fraction might represent the product of deadenylation of an earlier population of poly A(+) mRNA.
Insights
Trout testis mRNA, both polyadenylated RNA and RNA devoid of poly A, was translated to produce protamine. Protamine mRNA subcomponent populations varied, suggesting developmental differences in trout testis.
Area of Science:
- Molecular Biology
- Biochemistry
- RNA Biology
Background:
- Protamine is a basic protein crucial for sperm DNA packaging.
- Trout testis protamine exists as multiple components.
- Messenger RNA (mRNA) populations can vary in composition.
Purpose of the Study:
- To investigate the translation products of trout testis protamine mRNA.
- To analyze the composition and variation of protamine mRNA subcomponents.
- To explore the role of polyadenylation in protamine mRNA.
Main Methods:
- Isolation of poly A(+) and poly A(-) protamine mRNA from trout testis.
- In vitro translation using a rabbit reticulocyte cell-free system.
- Analysis of translation products via gel electrophoresis and carboxymethyl cellulose chromatography.
Main Results:
- Cell-free translation yielded products identical to authentic protamine.
- [14C]arginine incorporation varied unequally among three protamine components.
- Protamine mRNA subcomponent populations differed between mRNA types and varied with testis development.
Conclusions:
- Protamine mRNA composition varies with trout testis developmental stage.
- Polyadenylated RNA (poly A(+)) and RNA devoid of poly A (poly A(-)) fractions contain different mRNA proportions.
- Poly A(-) mRNA may result from the deadenylation of poly A(+) mRNA.