ANALISIS DE RIESGOS Y PUNTOS CRÍTICOS DE CONTROL

The overall aim of the work described in this thesis was to determine whether the observed differences in allosteric properties of wheat leaf and wheat endosperm ADP-glucose pyrophosphorylase activities are the result of organ-specific gene expression. In the first part of this work, it was intended that the ADP-glucose pyrophosphorylase activity would be purified from developing wheat endosperm. It was envisaged that, in addition to providing Information on the subunit structure and N-terminal amino acid sequence of the endosperm enzyme, the purified protein would be injected into rabbits for the preparation of a specific immune serum. Western-blot analysis of wheat endosperm and wheat leaf soluble protein would follow, as a means of comparing the subunit structures of the wheat endosperm and wheat leaf ADP-glucose pyrophosphorylase enzymes.

A subsequent part of the study was the isolation and characterisation of cDNA clones encoding the wheat endosperm and wheat leaf ADP-glucose pyrophosphorylases. These cDNAs were to be

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used Initially In hybridisation analyses to compare the organisation of genes and sizes of mRNA transcripts encoding both ADP-glucose pyrophosphorylase Isoenzymes. Determination of the nucleotide sequences and derived amino acid sequences of wheat leaf and endosperm ADP-glucose pyrophosphorylases would follow the gene organisation studies. The identification of the primary translation products of wheat endosperm and wheat leaf ADP-glucose pyrophosphorylase mRNAs was considered to be important in this analysis; it was envisaged that a comparison of the sizes of nascent and mature polypeptides, coupled with nucleotide sequence analysis of full-length cDNA clones, would assist in determining whether different transit peptide sequences are involved in the transport of proteins to chloroplasts and amyloplasts. It was of particular importance to identify amino acid sequences in the wheat leaf and wheat endosperm ADP-glucose pyrophosphorylase isoenzymes which are related to the substrate, inhibitor and activator binding sites of other ADP-glucose pyrophosphorylase enzymes. A comparison of these sequences between wheat leaf and wheat endosperm Isoenzymes will be of considerable use in helping us to understand the different kinetics of these enzymes.

42 1. MATERIALS

A., Chemicals. btochemlcals. radiochemlcals and enzymes

All materials used were of the highest analytical grade available. The source of specific materials Is shown below under the supplier's name.

Aldrich Chemical Co Ltd, Gillingham, Dorset: Methyl sulfoxide, HPLC grade (DMSO)

Amersham International pic, Amersham, Buckinghamshire: Adenosine 5'- [7-32P]triphosphate (3000 Ci/mmol); amino acid mixture minus methionine (1/<M each amino acid); 2'- deoxyadenosine

5'-[a-3 5S ]thiotriphosphate (>1000 Ci/mmol) ; 2 ’-deoxyadenosine 5'-[a-3 2P]triphosphate (>400 Ci/mmol); 2'-deoxycytidlne 5 [a-32P]triphosphate (>400 Ci/mmol); cDNA synthesis system, a-D-[U-1 4C]glucose-1-phosphate (>150 mCi/mmol); Hybond-N nylon blotting membranes; L - [35S]methionine (>800 Ci/mmol); [14C] methylated protein molecular weight markers for gel

electrophoresis (10-50 fiCi/mg protein); T4 polynucleotide kinase; Protein A, 1 25I-labelled with Bolton and Hunter reagent, affinity purified (>30 mCi/ mg protein); Rabbit Ig, 1 2SI-labelled whole antibody (from donkey); Rabbit reticulocyte lysate, amino acid depleted; Restriction enzyme buffers (sterile 10X stocks).

Analytichem International, Harbor City, CA90710, U.S.A: Bond Elut SAX Extraction Cartridges.

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BDH Chemicals Ltd, Poole, Dorset:: Acetic acid; acetone; acrylamide, Grade 1 'Electran’; ammonia; ammonium acetate; boric acid; bromophenol blue, 'Electran'; chloroform; di-potasslum hydrogen orthophosphate; ethylenediamlne tetra-acetlc acid; Folin and Clocalteu's phenol reagent; D-glucose; N-glycylglycine; hydrochloric acid; hydrogen peroxide (100 volume); N-2- hydoxyethylplperazine-N'-2-ethanesulphonic acid; magnesium chloride hexahydrate; methanol; N,N'-methylenebisacrylamide.Grade 1 'Electran'; PAGE blue 83, 'Electran'; phenylmethane sulphonyl fluoride; polyethylene glycolG000 (PEGSOOO); potassium acetate; potassium chloride; potassium dlhydrogen orthophosphate; potassium tartrate; sodium acetate; sodium chloride, sodium dodecyl sulphate; sodium.hydroxide; sodium nitrite; N,N,N',N'-tetra methylethylenediamine, Grade 1 'Electran'; Tris

(hydroxymethyl)methylamine; urea.

Bethesda Research Laboratories (U.K.) Ltd, Cambridge, Cambridgeshire: 5-bromo-4-chloro-3-indoly1 -0-D-galactoside (X-gal); dithiothreitol; deoxyribonuclease (ribonuclease-free); formamide; glycerol; isopropylthio-0-galactoside (IPTG) ; Nick translation reagent kit; Nonidet P40; phenol, redistilled nucleic acid grade; proteinase K; ribonuclease (deoxyribonuclease - free); sucrose (nuclease-free).

Bio-Rad Laboratories Ltd, Watford, Hertfordshire: Bio-gel A150 (100-200 mesh); mixed bed resin AG501-X8 (20-50 mesh).

Bio-Yeda Ltd, Kiryat Welzmann, Rehovot, Israel: Horseradish peroxidase conjugated anti-rabbit IgG, from goat.

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Boehrlnger Corporation (London) Ltd, Lewes, Sussex: Adenosine- 5'-triphosphate; albumin, fraction V from bovine serum; alkaline phosphatase from calf intestine, molecular biology quality; di-ammonium 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS); glucose GOD-Perid assay kit; glucose-6-phosphate dehydrogenase; Klenow fragment of DNA polymerase; oxaloacetic acid; phosphoglycerate mutase; transfer ribonucleic acid (tRNA).

Calbiochem Biochemicals, San Diego, California, U.S.A: Lysozyme, egg white.

Collaborative Research Inc, Bedford, HA 01730, U.S.A: Oligo (dT)-cellulose Type 2.

Dlfco Laboratories Ltd, Detroit, Michigan, U.S.A: Bactoagar; bactotryptone; tryptlease; yeast extract.

Flsons Scientific Apparatus, Loughborough, Leicestershire: Caesium chloride.

Fluka AG, Buchs, Switzerland: Glyoxal.

New England Blolabs, Beverly, MA 01915-9990, U.S.A: EcoRI methylase; s-adenosylmethionine.

NEN Research Products, Stevenage, Hertfordshire: EN^HANCE autoradiography enhancer; GeneScreen Plus nylon blotting membrane.

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Oxold Ltd, Basingstoke, Hants: Phosphate-buffered saline (Dulbecco'A').

Pharmacia Fine Chemicals, Uppsala, Sweden: Agarose; dextran sulphate; £coRI linkers [5'-d(GGAATTCC)-3']; high molecular weight gel filtration calibration kit; lambda DNA-ffindlll digest, DNA size markers; low molecular weight gel electrophoresis calibration kit; methyl-7-guanosine cap analogue [m^G(5*)ppp(5')G]; mono Q HR 5/5 ; prepacked PD10 columns; protein A sepharose 6MB; restriction enzymes; sephadex G-50 fine; sepharose CL-4B; single-strand binding protein (Escherichia coll); superose HR 10/30; T4 DNA llgase, FPLC pure; T4 RNA ligase (RNase and DNase free, from T4- infected E.coll).

Schleicher and Schuell, Dassel, West Germany: BA 85/20 nitrocellulose filter discs.

Sigma Chemical Company Ltd, Poole, Dorset: Adenosine 5'-diphosphoglucose; adenosine 2' and 31 -monophosphoric acid; ampicillin; deoxycholic acid; ethidlum bromide; ficoll (molecular weight 400,000); a-D-glucose-1,6-diphosphate; glutathione; heparin (from Porcine sp. intestinal mucosa); hexadecyltrlmethylammonium bromide (CTAB); imidazole; 0-mercaptoethanol; a-nicotinamide adenine dinucleotide, reduced form; 0- nicotinamide adenine dinucleotide 3'-phosphate; octyl phenoxy polyethoxyethanol (Triton X-100); o-phenylenediamine; polyoxyethylene sorbltan monolaurate (Tween 20); polyvinyl pyrrolidone; sodium azide; sodium deoxycholate; trichloroacetic acid; urea hydrogen peroxide.

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Stratagene Cloning Systems,(U.K. supplier NBL Ltd, Cramllngton, Northumberland): DSK-[35S]-deoxynucleotlde S'-triphosphate dideoxy sequencing system; gigapack gold in vitro packaging kit; in vitro transcription kit.

B. Bacterial, plasmid and bacteriophage stocks

Bacteria, plasmid and bacteriophage stocks are shown in Table II.1 with the genotype, literature reference and supplier.

C.Plant material

Wheat (Triticum aestivum c.v. Hardier) seeds were obtained from the National Seed Development Organisation, Cambridge.