Isolation and Cell-free Translation of Total Messenger RNA from Germinating Castor Bean Endosperm

Abstract

Polyadenylated RNA was isolated from the total RNA fraction extracted from the endosperm tissue of 3-day-old castor bean seedlings by affinity chromatography on oligo(dT)-cellulose. This polyadenylated RNA was efficiently translated into protein when added to a messenger RNA-dependent cell-free system derived from rabbit reticulocytes. Characterization of the translational products by electrophoresis followed by autoradiography established that numerous discrete polypeptides were formed with molecular weights ranging from 10,000 to over 100,000. Immunoprecipitation in the presence of antiserum raised in rabbits against the total glyoxysomal matrix proteins showed that these proteins accounted for 15 to 20% of the total translational products.Attempts to reconstitute rough endoplasmic reticulum by the addition of washed castor bean microsomal membranes to the translational system were unsuccessful, these membranes severely inhibiting protein synthesis. Canine pancreatic microsomes could be added to endosperm messenger RNA-dependent reticulocyte lysates at relatively high concentrations while still allowing significant protein synthesis.The uptake of water by dry seeds initiates a rapid transcription of RNA and ensuing protein synthesis which permits metabolic activity during germination (11,12). In the endosperm of germinating castor bean seedlings, this metabolic activity is predominantly associated with the conversion of stored triglycerides into carbohydrate (1). We have used this tissue to examine events surrounding the synthesis of gluconeogenic enzymes and the organelles (in particular, the glyoxysomes) which house them (5-7). This tissue offers a particularly attractive system for the study of organelle biogenesis, not only because of the rapid and massive formation of the organelles, but also because these syntheses occur in the absence of cell division or net protein accumulation.In order to reach a better understanding of these cellular processes, the isolation of mRNAs and their subsequent translation in a cell-free protein-synthesizing system are potentially important. The control of translation of specific mRNAs and mechanisms of post translational modification can be studied and the relevance of such events in terms of organelle biogenesis and protein segregation can be assessed.In Proteinase K was added (200 jig/ml final concentration) and the mixture was stirred for 10 min at room temperature. An equal volume of phenol-chloroform (1:1) was added and after a further 10-min stirring the phases were separated by centrifugation and the aqueous phase was removed. The organic phase was extracted with a half-volume of 20 mm Tris-HCl (pH 9.0), 2 mM EDTA, and the combined aqueous phases were reextracted twice with equal volumes of phenol-chloroform. The final aqueous phase was adjusted to 0.3 M NaCl and the nucleic acids were precipitated with 2.5 volumes of ethanol at -20 C overnight. The nucleic acid pellet, collected by centrifugation at 10,000g and 2 C for 10 min, was visibly contaminated with ...

Phytohormone control of translatable RNA populations in sexual organogenesis of the dioecious plant Mercurialis annua L. (2n = 16)

“…The total RNA preparation obtained was clear, rapidly dissolved and did not subsequently clog the affinity chromatography column. The absorption spectra and the yield (2 mg total RNA per g fresh weight, or 0.2%) were in agreement with published data (5). Total RNA was fractionated on a column of oligo-(dT)-cellulose, generally yielding 1% poly(A) RNA, regardless of the sex.…”

Cellular Distribution of Polysome Populations of Castor Bean Endosperm

“…Endogenous nucleases from numerous plant sources frequently have been reported to be a problem during polysome isolations. Recently, Lord and co-workers (4) have reported degradation problems when attempting to isolate intact polysomes from castor bean endosperm.…”

“…The same study (4) found that a substantial proportion of the poly(A)+ RNA was engaged with bound ribosomes. Additionally, poly(A)+ RNA increased in phase with cellular organelle content (4). The fact that all organelles proliferate rapidly and that the modes of segregation and synthesis of their various matrix enzymes may be analogous, strongly suggests that the polyribosome populations may be uniquely and discretely segregated on the ER.…”

“…Several studies indicate that glyoxysome accretion (1)(2)(3)9), membrane biosynthesis (13), and glyoxysomal enzyme synthesis (12) are rapidly taking place on the ER during the early stages of germination. A recent study (4) indicates that glyoxysomal protein synthesis accounts for about 15-20%o of the total protein synthesis. The same study (4) found that a substantial proportion of the poly(A)+ RNA was engaged with bound ribosomes.…”

“…A recent study (4) indicates that glyoxysomal protein synthesis accounts for about 15-20%o of the total protein synthesis. The same study (4) found that a substantial proportion of the poly(A)+ RNA was engaged with bound ribosomes. Additionally, poly(A)+ RNA increased in phase with cellular organelle content (4).…”