--Julia Bailey-Serres and Michael Freeling

A heterologous in vitro translation system which uses maize polyribosomes and wheat germ translation factors has been made. The system faithfully elongates proteins from mRNAs which have already initiated translation on polysomes, but does not efficiently promote initiation of translation. Attempts to make a heterologous system capable of initiating translation have shown that wheat germ elongation factors, but not initiation factors, are compatible with maize ribosomes.

The system capable of in vitro, run-off translation of mRNAs present on polysomes was constructed as follows: polysomes were extracted from maize seedling roots in a buffer containing 400mM KOAc, 20mM HEPES (pH 7.6), 35mM MgOAc and 5mM BME; concentrated by centrifugation at 100 x g through a buffered 2M sucrose cushion (Lincoln et al., PNAS 84:2783-2792, 1986); and resuspended in translation buffer (100mM KOAc, 20mM HEPES (pH 7.6), 3.5mM MgOAc, 5mM BME). Wheat germ extract from Promega Biotec was made 130mM KOAc and centrifuged at 100xg to obtain the S-100 supernatant fraction. This fraction should contain factors sufficient for initiation and elongation of translation and lack wheat germ ribosomes. Run-off translation of polysomal RNA was carried out in 25ul reactions containing: 50-100ug polysomal protein in translation buffer, 2-4ul wheat germ S-100 fraction, 2ul 1mM amino acids minus methionine, 1.23ul [35S-methionine (1200 Ci/mmole) and sufficient KOAc to maintain a concentration of 100mM. The reaction was carried out at 25 C for 1hr and translation products were analyzed by SDS-PAGE.

To test for initiation of protein synthesis the reaction mix included poly A+ mRNA (1ug) encoding CAT (in vitro synthesized and analog-capped pSP6CAT-A+ per Callis et al., 1988), and the presence of CAT activity was tested. No initiation of translation was observed when polysomes extracted in 400mM KOAc, which should lack initiation factors, were combined with the 130mM KOAc treated wheat germ S-100 fraction, which should contain initiation factors. Also, no initiation of translation was observed when polysomes extracted in 100mM KOAc, which should maintain initiation factors, were combined with 100mM KOAc treated wheat germ S-100 fraction, which should contain elongation factors but not initiation factors (these elute at 120mM KOAc). Since the maize polysomes and wheat germ translation factors appeared to be incompatible for the initiation of translation, the effect of maize translation factors on the total wheat germ translation system was tested. A maize S-100 fraction was eluted from polysomes isolated in 20mM KOAc (at 20mM KOAc both initiation and elongation factors should remain associated with polysomes). Addition of this fraction to the total wheat germ extract dramatically inhibited the initiation of translation by the extract. In conclusion, certain factor(s) required for initiation of translation are unique to maize and others are unique to wheat germ. Nevertheless, run-off of maize polysomes by the wheat germ S-100 fraction is efficient and has proven useful.

Return to the MNL 63 On-Line Index
Return to the Maize Newsletter Index
Return to the MaizeGDB Homepage