Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein.
Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, we started by isolating poly(A)(+) RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor because: (i) it is rich in proline and poor in leucine, and (ii) the message appears to be more abundant when carrot tissue is wounded. From a cDNA library constructed with poly(A)(+) RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A)(+) RNA encoding this 33-kDa peptide. We isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pDC5 as a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clone pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization and DNA sequence analysis indicate that pDC5 is a hybrid clone corresponding to two RNA species. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding.
Department of Biology, Washington University, St. Louis, MO 63130.
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Chen
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Varner