Gerard Francis Barry

Regulation of the Amount of Starch in Plant Tissues by ADP Glucose Pyrophosphorylase

Starch, a major storage metabolite in plants, positively affects the agricultural yield of a number of crops. Its biosynthetic reactions use adenosine diphosphate glucose (ADPGlc) as a substrate; ADPGlc pyrophosphorylase, the enzyme involved in ADPGlc formation, is regulated by allosteric effectors. Evidence that this plastidial enzyme catalyzes a rate-limiting reaction in starch biosynthesis was derived by expression in plants of a gene that encodes a regulatory variant of this enzyme. Allosteric regulation was demonstrated to be the major physiological mechanism that controls starch biosynthesis. Thus, plant and bacterial systems for starch and glycogen biosynthesis are similar and distinct from yeast and mammalian systems, wherein glycogen synthase has been demonstrated to be the rate-limiting regulatory step.

Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants.

Synthesis of the phytohormone ethylene is believed to be essential for many plant developmental processes. The control of ripening in climacteric fruits and vegetables is among the best characterized of these processes. One approach to reduce ethylene synthesis in plants is metabolism of its immediate precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). Soil bacteria containing an enzyme, ACC deaminase, were identified by their ability to grow on ACC as a sole nitrogen source. The gene encoding ACC deaminase was cloned and introduced into tomato plants. Reduction in ethylene synthesis in transgenic plants did not cause any apparent vegetative phenotypic abnormalities. However, fruits from these plants exhibited significant delays in ripening, and the mature fruits remained firm for at least 6 weeks longer than the nontransgenic control fruit. These results indicated that ACC deaminase is useful for examining the role of ethylene in many developmental and stress-related processes in plants as well as for extending the shelf life of fruits and vegetables whose ripening is mediated by ethylene.

Adenosine 5'-diphosphate-glucose pyrophosphorylase from potato tuber. Significance of the N terminus of the small subunit for catalytic properties and heat stability.

cDNAs encoding the large subunit and a possibly truncated small subunit of the potato tuber (Solanum tuberosum L.) adenosine 5[prime]-diphosphate-glucose pyrophosphorylase have been expressed in Escherichia coli (A.A. Iglesias, G.F. Barry, C. Meyer, L. Bloksberg, P.A. Nakata, T. Greene, M.J. Laughlin, T.W. Okita, G.M. Kishore, J. Preiss, J Biol Chem [1993] 268: 1081–1086). However, some properties of the transgenic enzyme were different from those reported for the enzyme from potato tuber. In this work, extension of the cDNA was performed to elongate the N terminus of the truncated small subunit by 10 amino acids. This extension is based on the almost complete conservation seen at the N-terminal sequence for the potato tuber and the spinach leaf small subunits. Expressing the extended cDNA in E. coli along with the large subunit cDNA yielded a transgenic heterotetrameric enzyme with similar properties to the purified potato tuber enzyme. It was also found that the extended small subunit expressed by itself exhibited high enzyme activity, with lower affinity for activator 3-phosphoglycerate and higher sensitivity toward inorganic phosphate inhibition. It is proposed that a major function of the large subunit of adenosine 5[prime]-diphosphate-glucose pyrophosphorylases from higher plants is to modulate the regulatory properties of the native heterotetrameric enzyme, and the small subunits major function is catalysis.

On the evolution of Tn21-like multiresistance transposons: Sequence analysis of the gene (aacC1) for gentamicin acetyltransferase-3-I(AAC(3)-I), another member of the Tn21-based expression cassette

SummaryThe aminoglycoside-3-O-acetyltransferase-I gene (aacC1) from R plasmids of two incompatibility groups (R1033 [Tn1696], and R135) was cloned and sequenced. In the case of R1033, it was shown that theaacC gene is coded by a precise insertion of 833 bp between theaadA promoter and its structural gene in a Tn21 related transposon (Tn1696). This insertion occurs at the same target sequence as that of the OXA-1 β-lactamase gene insertion in Tn2603. Upstream of theaacC gene, we found an open reading frame (ORF) which is probably implicated in the site-specific recombinational events involved in the evolution of this family of genetic elements. These results provide additional confirmation of the role of Tn21 elements as naturally occurring interspecific transposition and expression casssettes.

A broad-host-range shuttle system for gene insertion into the chromosomes of Gram-negative bacteria

A deletion derivative of transposon Tn7 containing the Escherichia coli lacZY genes as a selectable marker for insertion of foreign DNA into the chromosomes of soil bacteria was improved to facilitate the cloning of additional genes and their insertion by this element. This report describes a series of plasmid vectors that enable this cloning to be carried out in small, high-copy, narrow host-range plasmids. The final Tn element can then be easily moved (by transposition) without further use of restriction enzymes, to plasmids suitable for delivering it to the bacterial chromosome. The very high specificity for insertion of Tn7 into single locations in bacterial chromosomes has been exploited in the construction of a shuttle system for delivering these Tn7 elements.

AN ISOAMYLASE WITH NEUTRAL PH OPTIMUM FROM A FLAVOBACTERIUM SPECIES : CLONING, CHARACTERIZATION AND EXPRESSION OF THE IAM GENE

Abstract The gene encoding an isoamylase with neutral pH optimum (iam) from a Flavobacterium species was cloned using a PCR probe generated from highly conserved regions of amylolytic enzymes. Active isoamylase was expressed from a 4.9-kb Pst I fragment in Escherichia coli, and was detected in the extracellular medium by a plate assay. The iam nucleotide sequence has an open reading frame of 2334 nucleotides (778 amino acids) with a GC content of 69%. Sequence analysis suggests that transcriptional control of the Flavobacterium sp. iam gene is mediated through the product of a malT regulatory gene. The deduced amino acid sequence of iam contained an N-terminal signal peptide of 32 amino acids, and was 61% homologous with Pseudomonas amyloderamosa isoamylase. The mature enzyme, which was engineered for overexpression in E. coli and purified to homogeneity, has a relative molecular mass of 83 kDa, a pH optimum of 6–7, and a highest rate of hydrolysis for glycogen (but did not cleave pullulan). Polyclonal antiserum generated from purified donor isoamylase cross-reacted with crude and purified recombinant isoamylase from E. coli. This is the first report of the cloning, characterization, and sequence of an novel isoamylase that has a neutral pH optimum. A compari son of the sequence of Flavobacterium sp. iam with acidic isoamylase from Pseudomonas sp. identified putative residues which may be associated with the pH for optimal activity of isoamylases.

PROSPECTS FOR THE PRODUCTION OF CEREALS WITH IMPROVED STARCH PROPERTIES

The dominant pathway for the synthesis of starch involves three enzymes; ADPglucose pyrophosphorylase (ADPGlc PPase; EC 2.7.7. 27), which catalyzes the synthesis of ADPglucose; starch synthase (EC 2.4.1.21), which transfers the glucosyl portion of ADPglucose to a maltodextrin primer for synthesis and elongation of the α-1, 4 glucosyl chain and the branching enzyme (EC; 2.4.1.18) which transfers a portion of the elongated α-1, 4 glucosyl chain to form the α-1, 6 branch points present in amylopectin (and to small extent, in amylose). The physical properties of the starch in a plant are dependent on the properties and catalytic activities of the three enzymes mentioned above and alteration of the enzyme amounts and their properties will in turn, affect the properties of the starch synthesized. Recent results demonstrate that transformation of certain plants with a bacterial ADPGlc PPase gene can dramatically increase their starch content. It is quite possible that alteration of the proportion or amounts of branching enzyme and/or starch synthase would alter the structure and physical properties of the starch synthesized. To this end, the purified branching isoenzymes and soluble starch synthase isozymes are characterized to determine their specific roles in the synthesis of amylopectin. Preliminary experiments suggest that maize BE I isoenzyme is mainly involved in synthesis of the B chains of amylopectin while BE IIa and IIb are involved in the synthesis of the A chains.