Summary
The genomic sequence of the potato gene for starch granule-bound starch synthase (GBSS; “waxy protein”) has been determined for the wild-type allele of a monoploid genotype from which an amylose-free (amf) mutant was derived, and for the mutant part of the amf allele. Comparison of the wild-type sequence with a cDNA sequence from the literature and a newly isolated cDNA revealed the presence of 13 introns, the first of which is located in the untranslated leader. The promoter contains a G-box-like sequence. The deduced amino acid sequence of the precursor of GBSS shows a high degree of identity with monocot waxy protein sequences in the region corresponding to the mature form of the enzyme. The transit peptide of 77 amino acids, required for routing of the precursor to the plastids, shows much less identity with the transit peptides of the other waxy preproteins, but resembles the hydropathic distributions of these peptides. Alignment of the amino acid sequences of the four mature starch synthases with the Escherichia coli glgA gene product revealed the presence of at least three conserved boxes; there is no homology with previously proposed starch binding domains of other enzymes involved in starch metabolism. We report the use of chimeric constructs with wild-type and amf sequences to localize, via complementation experiments, the region of the amf allele in which the mutation resides. Direct sequencing of polymerase chain reaction products confirmed that the amf mutation is a deletion of a single AT basepair in the region coding for the transit peptide. Premature termination of translation as a result of this frameshift mutation results in a small peptide. However, a protein reacting with anti-GBSS serum, slightly larger than the wild-type mature GBSS, can be detected in a membrane fraction from amylose-free tubers. A possible explanation for this phenomenon will be discussed.
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References
Bevan M (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12:8711–8721
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Brown JWS (1986) A catalogue of splice junction and putative branch point sequences from plant introns. Nucleic Acids Res 14:9549–9559
Buzby JS, Yamada T, Tobin EM (1990) A light-regulated DNAbinding activity interacts with a conserved region of a Lemna gibba rbcS promoter. Plant Cell 2:805–814
Camirand A, St-Pierre B, Marineau C, Brisson N (1990) Occurrence of a copia-like transposable element in one of the introns of the potato starch phosphorylase gene. Mol Gen Genet 224:33–39
Campbell WH, Gowri G (1990) Codon usage in higher plants, green algae, and cyanobacteria. Plant Physiol 92:1–11
Chambers SP, Prior SE, Bartsow DA, Minton NP (1988) The pMTL nic − cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68:139–149
Echt GS, Schwartz D (1981) Evidence for the inclusion of controlling elements within the structural gene at the waxy locus in maize. Genetics 99:275–284
Frischauf AM, Lehrach H, Poustka A, Murray N (1983) Lambda replacement vectors carrying polylinker sequences. J Mol Biol 170:827–842
Furukawa K, Tagaya M, Inoye M, Preiss J, Fukui T (1990) Identification of lysine 15 at the active site in Escherichia coli glycogen synthase. J Biol Chem 265:2086–2090
Gilmartin PM, Sarokin L, Memelink J, Chua N-H (1990) Molecular light switches for plant genes. Plant Cell 2:369–378
Grosovsky AJ, de Boer JG, de Jong PJ, Drobetsky EA, Glickman BW (1988) Base substitutions, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells. Proc Natl Acad Sci USA 85:185–188
Guiltinan MJ, Marcotte WR, Quatrano RS (1990) A plant leucine zipper protein that recognizes an abscisic acid response element. Science 250:267–271
Hanley BA, Schuler MA (1988) Plant intron sequences: evidence for distinct groups of introns. Nucleic Acids Res 16:7159–7176
Hergersberg M (1988) Molekulare Analyse des waxy Gens aus Solanum tuberosum und Expression von waxy antisense RNA in transgenen Kartoffeln. PhD Thesis, University of Cologne
Higgins DG, Sharp PM (1989) Multiple sequence alignment program. Comput Appl Biosci 5:151–153
Hovenkamp-Hermelink JHM, Jacobsen E, Ponstein AS, Visser RGF, Vos-Scheperkeuter GH, Bijmolt EW, Vries JN de, Witholt B, Feenstra WJ (1987) Isolation of an amylose-free starch mutant of the potato (Solanum tuberosum L.). Theor Appl Genet 75:217–221
Jacobsen E, Hovenkamp-Hermelink JHM, Krijgsheld HT, Nijdam H, Pijnacker LP, Witholt B, Feenstra WJ (1989) Phenotypic and genotypic characterization of an amylose-free starch mutant of the potato. Euphytica 44:43–48
Jefferson R, Goldsbrough A, Bevan M (1990) Transcriptional regulation of a patatin-1 gene in potato. Plant Mol Biol 14:995–1006
Keegstra K, Olsen LJ, Theg SM (1989) Chloroplastic precursors and their transport across the envelope membrane. Annu Rev Plant Physiol Plant Mol Biol 40:471–501
Klösgen RB, Gierl A, Schwarz-Sommer Z, Saedler H (1986) Molecular analysis of the waxy locus of Zea mays. Mol Gen Genet 203:237–244
Kumar A, Larsen CE, Preiss J (1986) Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli ADP-glucose: α-1,4-glucan,4-glucosyltransferase as deduced from the nucleotide sequence of the glgA gene. J Biol Chem 261:16256–16259
Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132
Lavintman N, Tandecarz J, Carceller M, Mendiara S, Cardini CE (1974) Role of uridine diphosphate glucose in the biosynthesis of starch. Mechanism of formation and enlargement of a glucoproteic acceptor. Eur J Biochem 50:145–155
van der Leij FR, Visser RGF, Oosterhaven K, van der Kop DAM, Jacobsen E, Feenstra WJ (1991) Complementation of the amylose-free starch mutant of potato (Solanum tuberosum L.) by the gene encoding granule-bound starch synthase. Theor Appl Genet, in press
Leloir LF, Rongine de Fekete MA, Cardini CE (1961) Starch and oligosaccharide synthesis from uridine diphosphate glucose. J Biol Chem 236:636–641
Lipman DJ, Pearson WR (1985) Rapid and sensitive protein similarity searches. Science 227:1435–1441
Liu XJ, Prat S, Willmitzer L, Frommer WB (1990) Cis regulatory elements directing tuber-specific and sucrose-inducible expression of a chimeric class I patatin promoter/GUS gene fusion. Mol Gen Genet 223:401–406
Maas C, Schaal S, Werr W (1990) A feedback control element near the transcription start site of the maize Shrunken gene determines promoter activity. EMBO J 9:3447–3452
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035–10038
Okagaki RJ, Wessler SR (1988) Comparison of non-mutant and mutant waxy genes in rice and maize. Genetics 120:1137–1143
Preiss J (1990) Biology and molecular biology of starch synthesis and its regulation. In: Miflin BJ (ed) Oxford Surveys of Plant Molecular and Cell Biology, vol 7. Oxford University Press, UK, in press
Putterill JJ, Gardner RC (1989) Initiation of translation of the β-glucuronidase reporter gene at internal AUG codons in plant cells. Plant Sci 62:199–205
Rohde W, Becker D, Salamini F (1988) Structural analysis of the waxy locus from Hordeum vulgare. Nucleic Acids Res 16:7185–7186
Rohde W, Becker D, Kull B, Salamini F (1990) Structural and functional analysis of two waxy gene promoters from potato. J Genet Breed 44:311–315
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schulze-Lefert P, Dangl JF, Becker-André M, Hahlbrock K, Schulz W (1989) Inducible in vivo DNA footprints define sequences necessary for UV light activation of the parsley chalcone synthase gene. EMBO J 8:651–656
Shannon JC, Garwood DL (1984) Genetics and physiology of starch development. In: Whistler RL, BeMiller JN, Paschall JF (eds) Starch: chemistry and technology, 2nd edn. Academic Press, Orlando, pp 25–86
Shure M, Wessler S, Fedoroff N (1983) Molecular identification and isolation of the Waxy locus in maize. Cell 35:225–233
Smith AM (1990) Evidence that the “waxy” protein of pea (Pisum sativum L.) is not the major starch-granule-bound starch synthase. Planta 182:599–604
Springer B, Bellmann R, Werr W (1990) Formation of composite nucleoprotein complexes near the transcription start of the Shrunken gene from maize. Mol Gen Genet 221:94–101
Svensson B, Jespersen H, Sierks MR, MacGregor EA (1989) Sequence homology between putative raw-starch binding domains from different starch-degrading enzymes. Biochem J 264:309–311
Tacke E, Prüfer D, Salamini F, Rohde W (1990) Characterization of a potato leafroll luteovirus subgenomic RNA: differential expression by internal translation initiation and UAG suppression. J Gen Virol 71:2265–2272
Takahashi Y, Niwa Y, Machida Y, Nagata T (1990) Location of the cis-acting auxin-responsive region in the promoter of the par gene from tobacco mesophyll protoplasts. Proc Natl Acad Sci USA 87:8013–8016
Vancanneyt G, Rosahl S, Willmitzer L (1990) Translatability of a plant-mRNA strongly influences its accumulation in transgenic plants. Nucleic Acids Res 18:2917–2921
Vasil V, Clancy M, Ferl RJ, Vasil IK, Hannah LC (1989) Increased gene expression by the first intron of maize Shrunken-1 locus in grass species. Plant Physiol 91:1575–1579
Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
Visser RGF, Hergersberg M, van der Leij FR, Jacobsen E, Witholt B, Feenstra WJ (1989) Molecular cloning and partial characterization of the gene for granule-bound starch synthase from a wildtype and an amylose-free potato (Solanum tuberosum L.). Plant Sci 64:185–192
Vos-Scheperkeuter GH, de Boer W, Visser RGF, Feenstra WJ, Witholt B (1986) Identification of granule-bound starch synthase in potato tubers. Plant Physiol 82:411–416
Wang Z, Wu Z, Xing Y, Zheng F, Guo X, Zang W, Hong M (1990) Nucleotide sequence of rice waxy gene. Nucleic Acids Res 18:5898
Wessler SR, Varagona MJ (1985) Molecular basis of mutations at the waxy locus of maize: Correlation with the fine structure genetic map. Proc Natl Acad Sci USA 82:4177–4181
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of M13mp18 and pUC19 vectors. Gene 33:103–119
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van der Leij, F.R., Visser, R.G.F., Ponstein, A.S. et al. Sequence of the structural gene for granule-bound starch synthase of potato (Solarium tuberosum L.) and evidence for a single point deletion in the amf allele. Molec. Gen. Genet. 228, 240–248 (1991). https://doi.org/10.1007/BF00282472
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DOI: https://doi.org/10.1007/BF00282472