Abstract
Five cDNA clones (ADR6, ADR11-1, ADR11-2, ADR12-1 and ADR12-2), representing three families of auxin down-regulated (ADR) genes were isolated and characterized. These were isolated by screening a λZap cDNA library with the partial cDNA clones p6, p11 and p12, isolated earlier (Baulcombe and Key, J Biol Chem 255: 8907–8913, 1980). Hybrid-select translation of ADR6, ADR11-2 and ADR12-2 clones produced polypeptides of 33 kDa 22.5 kDa and a 6 and 7 kDa respectively, when analyzed by SDS-PAGE. ADR6 and ADR12-2 gave one and two spots, respectively, on an IEF-SDS 2D gel. ADR11-2 probably encodes a basic protein as it was only resolved on non-equilibrium pH gradient gel electrophoresis (NEPHGE). Genomic Southern blot analysis of ADR6, ADR11 and ADR12 suggests that each represents a small multigene family. The RNA levels corresponding to ADR6, ADR11 and ADR12 decrease in response to applied auxin by 100-, 15- and 10-fold, respectively (Baulcombe and Key, 1980). Runoff transcription, done in the presence and absence of auxin, showed that the rate of transcription of the genes corresponding to ADR6, ADR11-2 and ADR12-2 was reduced in the presence of auxin, but the decrease was small relative to the decrease in the cytoplasmic levels of these mRNAs, in response to auxin. A comparative analysis of the influence of auxin on in vitro transcription and steady state RNA levels corresponding to these ADR cDNAs suggests that the decrease in rate of transcription due to auxin is not enough to account for the auxin-induced decrease in the steady state levels. Northern analysis showed developmental and organ/tissue-specific response of these ADR genes. Furthermore, the expression of the genes corresponding to ADR6 and ADR12-1 appears to be upregulated by light, whereas the gene corresponding to ADR11 appears to be down-regulated by light.
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References
Baulcombe DC, Key JL: Polyadenylated RNA sequences which are reduced in concentration following auxin treatment of soybean hypocotyls. J Biol Chem 255: 8907–8913 (1980).
Key JL: Modulation of gene expression by auxin. Bioessays 11: 52–68 (1989).
Reddy ASN, Poovaiah BW: Molecular cloning and sequencing of a cDNA for an auxin-repressed mRNA: correlation between fruit growth and repression of the auxin-regulated gene. Plant Mol Biol 14: 127–136 (1990).
Reddy ASN, Costa M, An G, Poovaiah BW: Fruit specific expression of a reporter gene that is fused to 5′ region of an auxin-repressed gene. Third International Congress of Plant Molecular Biology, Abstract 850 (1991).
Chengappa S, Reddy ASN, Croissant-Sych Y, Patil S, Poovaiah BW: Isolation of an auxin-repressed cDNA using antibodies produced against a 52 kD protein from strawberry fruit. Third International Congress of Plant Mol Biol, Abstract 851 (1991).
Hong JC, Nagao RT, Key JL: Characterization and sequence analysis of a developmentally regulated putative cell wall protein gene isolated from soybean. J Biol Chem 262: 8367–8376 (1987).
Feinberg A, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13 (1983).
Frischamp AM, Garoff H, Lehrach H: A subcloning strategy for DNA sequence analysis. Nucl Acid Res 8: 5541 (1980).
Gantt JS, Key JL: Coordinate expression of ribosomal protein mRNA following auxin treatment of soybean hypocotyls. J Biol Chem 260: 6175–6181 (1985).
Schagger H, vonJagow G: Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166: 368–379 (1987).
O'Farrell PH: High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250: 4007–4021 (1975).
O'Farrell P, Goodman MM, O'Farrell PH: High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12: 1133–1142 (1977).
Kimpel JA, Nagao RT, Goekjian V, Key JL: Regulation of the heat shock response in soybean seedlings. Plant Physiol 94: 988–995 (1990).
Halward TM, Stalker HT, LaRue EA, Kochert G: Genetic variation detectable with molecular markers among unadapted germ-plasm resources of cultivated peanut and related wild species. Genome 34: 1013–1020 (1991).
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
Conner TW, Goekjian VH, LaFayette PR, Key JL: Structure and expression of two auxin-inducible genes from Arabidopsis. Plant Mol Biol 15: 623–632 (1990).
Lutcke HA, Chow KC, Mickel FS, Moss KA, Kern HF, Scheele GA: Selection of AUG initiation codons differ in plants and animals. EMBO J 6: 43–48 (1987).
Kozak M: The scanning model for translation: An update. J Cell Biol 108: 229–241 (1989).
Bassuner R, Baumlein H, Huth A, Jung R, Wobus U, Rapoport TA, Saalbach G, Muntz K: Abundant embryonic mRNA in field bean (Vicia faba L.) codes for a new class of seed proteins: cDNA cloning and characterization of the primary translation product. Plant Mol Biol 11: 321–334 (1988).
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Datta, N., LaFayette, P.R., Kroner, P.A. et al. Isolation and characterization of three families of auxin down-regulated cDNA clones. Plant Mol Biol 21, 859–869 (1993). https://doi.org/10.1007/BF00027117
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DOI: https://doi.org/10.1007/BF00027117