Cloning of cDNA for pyruvate, Pi dikinase from maize leaves.

To obtain molecular probes for studies of gene regulation in photosynthetic tissues of maize, we have cloned DNA complementary to poly(A)+RNA extracted from green leaves by insertion into plasmid pBR322 and transformation of E. coli, strain RR1. Colonies were screened by sequential hybridization with 32P-labeled single stranded cDNA synthesized from pooled aliquots of poly(A)+RNA fractionated by sucrose density centrifugation. Among the clones bearing cDNA homologous to high molecular weight poly(A)+RNA, we identified one with an insert of 440 base pairs homologous to mRNA for pyruvate, Pi dikinase, a C-4 carbon cycle protein localized in mesophyll cells of the leaf. Our work indicates that the dikinase subunits are synthesized in the cytoplasm as precursors approximately 13,000 daltons larger than the mature peptide subunits. Leaves of seedlings illuminated during growth have higher levels of pyruvate, Pi dikinase mRNA than leaves of dark-grown plants.

Light-stimulated synthesis of NADP malic enzyme in leaves of maize.

Illumination of etiolated maize plants for 80 h brings about a 15-20-fold increase in activity of NADP malic enzyme (EC 1.1.1.40). Increases in NADP malic enzyme protein and in the level of translatable mRNA for this protein occur simultaneously with the activity increase. Radiolabeled amino acids are also incorporated into NADP malic enzyme during this time. These results are consistent with the conclusion that an increase in NADP malic enzyme activity during greening results from de novo synthesis of NADP malic enzyme protein. Polyadenylated RNA extracted from greening maize leaves directs the synthesis in vitro of a protein 12,000 daltons larger than NADP malic enzyme purified from corn leaves. This protein is a precursor of NADP malic enzyme because 1) both the precursor and mature NADP malic enzyme are immunoprecipitated by antibody made against NADP malic enzyme purified from corn leaves, 2) both NADP malic enzyme protein and the level of mRNA for the precursor increase during greening, and 3) peptide maps of the precursor and of mature NADP malic enzyme are very similar. Mature NADP malic enzyme and its precursor (synthesized in vitro) both migrate on sodium dodecyl sulfate-polyacrylamide gradient gels as doublet bands. Peptide analyses show all bands to be structurally related.

Light-stimulated increase of translatable mRNA for phosphoenolpyruvate carboxylase in leaves of maize.

Polyadenylated RNA has been isolated from maize leaves at various times during greening of etiolated seedlings and used to prime the wheat germ cell-free translation system. Levels of translatable messenger RNA for phosphoenolpyruvate carboxylase increase with the length of the illumination period. The pattern of the increase in translatable mRNA for the enzyme is similar to that of the increase in phosphoenolpyruvate carboxylase protein previously observed in intact tissues. Phosphoenolpyruvate carboxylase synthesized in vivo migrates as a doublet band on gradient sodium dodecyl sulfate-polyacrylamide gels. A similar doublet has been seen occasionally with the products of cell-free translation.

Evidence for Light-stimulated Synthesis of Phosphoenolpyruvate Carboxylase in Leaves of Maize.

Illumination (22,000 lumens per meter(2)) of etiolated maize plants for 80 hours brings about a 5-fold increase in phosphoenolpyruvate carboxylase activity per unit of protein. An increase in carboxylase protein and incorporation of [(35)S]methionine into the protein occurs simultaneously with the activity increase. In green plants, the level of phosphoenolpyruvate carboxylase protein and enzyme activity is dependent on the intensity of light during growth. These results are consistent with the conclusion that the activity increase results from light-stimulated de novo synthesis of phosphoenolypyruvate carboxylase protein.

Studies on termination of protein synthesis in wheat germ.

Oligophenylalanines are soluble in m-cresol, but oligophenylalanyl-tRNAs are not. This differential solubility can be used to assay oligophenylalanines released from tRNA during their synthesis by wheat germ extracts. When poly U is the message, virtually no free product appears. When poly A U (A < U) is used, a considerable amount of oligophenylalanines are released. The fraction of product released is approximately constant with time, implying that a steady-state is not achieved between initiation and release. The dependence of release on various reaction variables and the effects of several inhibitors on release indicate that the reaction is probably catalyzed by peptidyl transferase, in accord with the mechanisms described for mammals and prokaryotes.

Genetic analysis of alcohol dehydrogenase isozymes in pearl millet (Pennisetum typhoides).

Pearl millet (Pennisetum typhoides) produces three ADH isozymes, sets I, II, and III, with set III being expressed only in anaerobically treated seeds of seedlings. Variant strains have been identified which produce ADH isozymes with altered electrophoretic mobilities for sets I and II but not for set III activity. Based on genetic analysis of these variants and on dissociation-reassociation experiments, we propose that the three ADH isozymes are dimers of subunits coded by two structural genes, Adh1 and Adh2, with set I being a homodimer specified by Adh1, set III a homodimer specified by Adh2, and set II a heterodimer formed between the products of Adh1 and Adh2.

Studies of storage proteins of higher plants: I. Concanavalin a from three species of the genus canavalia.

Concanavalin A, the lectin of the Jack bean, Canavalia ensiformis, was extracted and compared with homologous proteins from Canavalia gladiata and Canavalia maritima. All proteins were bound to Sephadex G-100 and eluted from the gel with buffered glucose solution. Quantitative recoveries indicated that large quantities (23 to 28% of dry seed protein) of these lectins are synthesized by all three species. Antibody preparations made against C. ensiformis lectin failed to discriminate among the three proteins; the pattern of the precipitin bands indicated identical antigenic determinants in the Ouchterlony double-diffusion assay. Native and sodium dodecyl sulfate polyacryl-amide gel electrophoresis also failed to distinguish differences in the proteins. The storage protein active in carbohydrate binding is composed, in each case, of identical subunits. However, the amino acid composition of the subunit chains from the three sources is not identical. In particular, the lectins from C. ensiformis and C. gladiata contain two methionine residues per protein subunit, while only one methionine residue is found in the C. martima lectin. Cyanogen bromide cleavage of the purified subunit from C. maritima yieded two fragments with molecular weights estimated at 20,400 and 4,600, respectively. Amino acid analysis of the separated fragments indicated that the methionine residue at position 130 in C. ensiformis is absent in the lectin from C. maritima.

Multiple gene sites for 5S and 18 plus 28S RNA on chromosomes of Glyptotendipes barbipes (Staeger).

Ribosomal RNAs (28 + 18S and 5S) and 4S RNA extracted from the chironomid Glyptotendipes barbipes were iodinated in vitro with (125)I and hybridized to the salivary gland chromosomes of G. barbipes and Drosophila melanogaster. Iodinated 18 + 28 S RNA labeled three puffed sites with associated nucleoli on chromosomes IR, IIL, and IIIL of G. barbipes and the nucleolar organizer of Drosophila. Labeled 5S RNA hybridized to three sites on chromosome IIIR, two sites on chromosome IIR and one site in a Balbiani ring on chromosome IV of Glyptotendipes. Most of the label produced by this RNA was localized seven bands away from the centromere on the right arm of chromosome III, and we consider this to be the main site complementary to 5S RNA in the chironomid. This same RNA preparation specifically labeled the 56 EF region of chromosome IIR of Drosophila which has been shown previously to be the only site labeled when hybridized with homologous 5S RNA. Hybridization of G. barbipes chromosomes with iodinated 4S RNA produced no clearly localized labeled sites over the exposure periods studied.

The Coding Properties of Lysine-accepting Transfer Ribonucleic Acids from Black-eyed Peas.

Lysine-accepting transfer RNA from ungerminated and germinated embryo axes of black-eyed peas (Vigna sinensis L. Savi) was fractionated on benzoylated diethylaminoethyl cellulose and reverse phase Freon columns. Cochromatography indicated the presence of two similar lysyl transfer RNA fractions in each tissue. Ribosome binding studies revealed that the larger of the two fractions in each case is specific for the AAG codon, while the smaller one recognizes AAA and AAG. Possible implications of this difference in quantities of isoacceptors in translation of genetic information are discussed.