The fraction of poly(A)-containing RNA isolated from ripening bean (Phaseolus vulgaris) cotyledons that sedimented at 16 S in linear logarithmic sucrose gradients was at least as active a messenger as viral RNA when added to a cell-free protein-synthesizing system from wheat germ. The major products synthesized in vitro were polypeptides of about 47,000 and 43,000 daltons, corresponding to two of the three subunits of G1 protein, the most abundant bean seed storage protein. No trace of the largest (53,000 daltons) subunit was found among the polypeptides synthesized in vitro. Proof that the 47,000- and 43,000-dalton polypeptides coded by the 16S RNA were indeed subunits of G1 protein was obtained by immunoprecipitation with monovalent antibody to G1 protein and by electrophoretic mapping of peptides on acrylamide gels after digestion of mixtures of authentic protein and radioactive translation products with protease V8, chymotrypsin, and trypsin. The subunits synthesized in vitro were slightly smaller than the native subunits, probably because they lacked the sugar residues present on the holoprotein.

During cell division and during the induction of tubulin synthesis that accompanies flagellar regeneration in Chlamydomonas reinhardi, four tubulin mRNAs of discrete molecular sizes are produced. During induction two beta tubulin mRNAs (2.47 kb and 2.34 kb) and two alpha tubulin mRNAs (2.26 kb and 2.13 kb) are synthesized in high abundance and in a closely coordinated fashion. Combined data from restriction enzyme mapping (i.e., Southern analysis) of genomic DNA and of Charon 30 recombinant clones bearing inserts of Chlamydomonas tubulin genes provide direct evidence for four distinct tubulin genes in this organism. Dot-blot analysis of the level of hybridization of a 32p nick-translated beta tubulin cDNA to genomic DNA from gametic cells and to a clone containing the beta 1 tubulin gene indicate that each beta 1 tubulin gene is present in one copy per cell. Additional hybridization experiments employing fragments of cDNA clones which selectively anneal to either the 3' or 5' portions of the two alpha tubulin genes or to one or both of the two beta tubulin genes suggest that each tubulin gene is actively transcribed to give rise to one of the four tubulin mRNAs. These observations further suggest that at most four basic types of tubulin subunits are produced by Chlamydomonas and that the heterogeneity of tubulin subunits reported to exist in the flagellar axoneme must arise as a result of post-translational modification.

As seeds of the French bean (Phaseoluts tulgaris, L. cv.Tendergreen) mature, a single protein, GI globulin (analogous to legumin), represents the majority of protein synthesized. Washed polysomes extracted from developing cotyledons had little endogenous activity in amino acid in-corporation, but on addition of cell-free extracts from wheat germ, active incorporation was obtained, the level being similar to that with viral RNA as nmessenger. The M1g2+ optimum for protein synthesis in the presence of bean polysomes was 6 mM compared with 4 mM for synthesis of viral polypeptides in the wheat germ system. Using T-2 toxin as an inhibitor, it was shown that 29% of the incorporation depended on initiation events. Electrophoretic analysis of the total polypeptide products of cell-free synthesis gave a disperse profile. Centrifugation to remiiove polysome-bound peptides after 60 minutes incubation gave a supernatant having a product with the same electrophoretic mobility as GI globulin and containing 26%7G of the radioactivity present in the gel. Protein eluted from this peak was subjected to re-electrophoresis and shown to consist of the three polypeptide subunits characteristic of GI globulin.It has been appreciated for some years that the developing seed should be a useful experimental system for studies on protein synthesis (5, 6). The onset of rapid synthesis of protein at a well defined developmental stage has been documented for several legumes, including Vicia (5), Pisum (2), and Phlaseolus (11,12). An additional advantage to protein studies is the fact that during this growth phase relatively few molecular species are synthesized and accumulated in the maturing cotyledons of these plants. The Ltd., Elmhurst, Ill. 60126). One gram fresh weight of cotyledons was used per 3 ml of buffer. After four 20-sec bursts at full speed, the homogenate was filtered through four layers of acetate taffeta cloth. The filtrate was centrifuged (4 C) at 20,000 rpm for 40 min in a Spinco 60 Ti rotor. The supernatant was layered onto a 5-ml sucrose cushion (1.5 M sucrose, 20 mm KCl, 5 mm Mg acetate in 50 mm tris-acetate, pH 8.5) and centrifuged for 2 hr at 39,000 rpm. After two quick rinses with sterile distilled H20, the pellet (polysomes) was suspended gently in 10 mm tris-acetate, pH 8.5, containing 20 mm KCl, 1 mm Mg acetate, and 20' (v,'v)

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