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.
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 svnthesized by all three species. Antibody preparations made against C. ensiformlis lectin failed to discriminate among the three proteins; the pattern of the precipitin bands indicated identical antigenic determinants in the Ouchterlony doublediffusion assay. Native and sodium dodecyl sulfate polyacrylamide 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. ensiforrnis 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 puri. fied subunit from C. maritimtia 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.During seed germination storage proteins are hydrolyzed, and the amino acids are transported into the growing seedling axis. However, certain storage proteins, called lectins and found largely in the cotyledons of legumes, have the capacity to bind specific carbohydrates. The functional significance of this property is unknown, but there have been several reports indicating that lectins have an affinity for the cell surface of species of Rhizobium and may, therefore, facilitate the entry of these symbiotic, nitrogen-fixing organisms into the root cortex (8,18).Relatively little is known about the mechanism of higher plant protein synthesis in comparison to that of microorganisms and animal tissues (38). A cell-free system from higher
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.
Ribosomal RNAs (28 + 18S and 5S) and 4S RNA extracted from the chironomid Glyptotendipes barbipes were iodinated in vitro with 125I and hybridized to the salivary gland chromosomes of G. barbipes and Drosophila melanogaster. Glyptotendipes. Most of the label produced by this RNA was localized seven bands away from the centromere on the right arm of chromosome llI, 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.
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