Summary
The deposition of zein protein in maize endosperm is under the control of several regulatory loci. The isolation of DNA sequences corresponding to Opaque-2 (O2), one of such loci, is described in this paper. The mutable allele, o2-m5 was first induced moving the Ac transposable element present at the wx-m7 allele to the O2 locus. Genetic data suggest that a functional Ac element is responsible for the observed somatic mutability of o2-m5. The isolation of genomic clones containing flanking sequences corresponding to the O2 gene was possible by screening an o2-m5 genomic libary with a probe corresponding to internal Ac sequences usually absent in the defective element Ds. Out of 27 clones isolated with homology to the central part of Ac element, only clones 6IP and 21IP generated a 2.5 kb internal fragment size of an active Ac element when digested with PvuII restriction enzyme. A sequence representing a XhoI fragment of 0.9 kb lying, in the 6IP clone, adjacent to the Ac elements, was subcloned and utilized to prove that it corresponded to a part of the O2 gene. To obtain this information we made use of: (1) DNAs from several reversions originating from the unstable (o2mk-(r) allele, which, when digested with SstI, showed a correct 3.4 kb fragment typical of non-inserted alleles of the O2 locus; and (2) recessive alleles of the O2 locus which were devoid of a 2.0 kb mRNA, present on the contrary in the wild type and in other zein regulating mutants different from O2.
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References
Behrens U, Fedoroff N, Laird A, Müller-Neumann M, Starlinger P, Yoder J (1984) Cloning of Zea mays controlling element Ac from the wx-m7 allele. Mol Gen Genet 194:346–347
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Burr B, Burr FA (1976) Zein synthesis in maize endosperm by polyribosomes attached to protein bodies. Proc Natl Acad Sci USA 73:515–519
Clewell DB, Helinski DR (1969) Super-coiled circular DNA-protein complex in E. coli: purification and induced conversion to an open circular DNA form. Proc Natl Acad Sci USA 62:1159–1166
Come KC, Schmidt RJ, Burr B, Burr F (1987) Advantages and limitations of using Spm as a transposon tag. Proc. International Symp. on Plant Transposable Elements, August 22–26, 1987, Univ. of Wisconsin, Madison, WI
Dellaporta SL, Chomet PS (1985) The activation of maize controlling elements. In: Hohn B, Dennis ES (eds) Genetic flux in plants. Springer Verlag, Wien, pp 169–216
Fedoroff N, Wessler S, Shure M (1983) Isolation of the transposable maize controlling elements Ac and Ds. Cell 35:235–242
Fedoroff N, Furtek D, Nelson OE (1984) Cloning of the bronze locus in maize by a simple and generalizable procedure using the transposable controlling element Ac. Proc Natl Acad Sci USA 81:3825–3829
Frischauf AM, Lehrach H, Poutska P (1983) Lambda replacement vectors carrying polylinker sequences. J Mol Biol 170:827–842
Gianzza E, Righetti PG, Pioli F, Galante E, Soave C (1976) Size and charge heterogeneity of zein in normal and opaque-2 maize endosperms. Maydica 21:1–17
Heidecker G, Messing J (1986) Structural analysis of plant genes. Annu Rev Plant Physiol 37:439–466
Kloppstech K, Schweiger HG, (1976) In vitro translation of poly(A) RNA from Acetabularia. Cytobiogie 13:394–400
Larkins BA, Hurkman WI (1978) Synthesis and deposition of zein in protein bodies of maize endosperm. Plant Physiol 62:256–263
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Marotta R, Ponziani G, Motto M, Hartings H, Gierl A, Di Fonzo N, Soave C, Bianchi A, Salamini F (1986) Genetic instability of the shrunken and waxy loci in the o2-m(r)-Bg strain of maize. Maydica 31:131–151
McClintonck B (1951) Chromosome organization and genetic expression. Cold Spring Harbor Symp Quant Biol 16:13–47
Mertz ET, Bates LS, Nelson OE (1964) Mutant gene that changes protein composition and increase lysine content of maize endosperm. Science 145:279–280
Montanelli C, Di Fonzo N, Marotta R, Motto M, Soave C, Salamini F (1984) Occurrence and behaviour of the components o2-m(r)-Bg system of maize controlling elements. Mol Gen Genet 197:209–218
Motto M, Marotta R, Di Fonzo N, Soave C, Salamini F (1986) Ds-induced alleles at the opaque-2 locus of maize. Genetics 112:121–132
Müller-Neumann M, Yoder J, Starlinger P (1984) The sequence of the Ac element in Zea mays. Mol Gen Genet 198:19–24
Pedersen K, Bloom KS, Anderson JN, Glover UV, Larkins BA (1980) Analysis of the complexity and frequency of zein genes in the maize genome. Biochemistry 19:1644–1650
Peterson PA (1987) Mobile elements in plants. CRC Crit Rev Plant Sci 6:105–208
Pohlman RF, Fedoroff N, Messing J (1984) The nucleotide sequence of the maize controlling element Activator. Cell 37:635–643
Rigby PW, Dieckman M, Rhodes C, Berg P (1977) Labelling of deoxyribonucleic acid to high specific activity in vitro nick translation with DNA polymerase. J Mol Biol 113:237–251
Salamini F (1980) Genetic instability at the Opaque-2 locus of maize. Mol Gen Genet 179:497–507
Schmidt RJ, Burr FA, Burr B (1987) Transposon tagging and molecular analysis of the maize regulatory locus opaque-2. Science 238:960–963
Soave C, Salamini F (1984) Organization and regulation of zein genes in maize endosperm. Philos Trans R Soc Lond [Biol] 304:341–347
Soave C, Tardani L, Di Fonzo N, Salamini F (1981) Zein level in maize endosperm depends on a protein under control of the opaque-2 and opaque-6 loci. Cell 27: 403–410
Wienand U, Saedler H (1987) Plant transposable elements: unique structures for gene tagging and gene cloning: In: Hohn J, Schell J (eds) Plant gene research, vol IV, Plant DNA infectious agents. Springer, Wien, pp 205–227
Yamamoto KR, Alberts BM, Benzinger R, Lawhorne L, Treiber G (1970) Rapid bacteriophage sedimentation in the presence of polythylene glycol and its application to large-scale virus purification. Virology 40:734–740
Yanish-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119
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Communicated by H. Saedler
This paper is dedicated to the memory of R. Marotta, who actively participated in the realization of this work
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Motto, M., Maddaloni, M., Ponziani, G. et al. Molecular cloning of the o2-m5 allele of Zea mays using transposon marking. Mol Gen Genet 212, 488–494 (1988). https://doi.org/10.1007/BF00330854
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DOI: https://doi.org/10.1007/BF00330854