MOLECULAR CHARACTERIZATION OF THE PATATIN GENE FAMILY OF POTATO (EXPRESSION, STRUCTURE)
Abstract
The major proteins in potato tubers are a family of 40Kd glyco-proteins of unknown function named patatin. Poly(A)('+) mRNA from membrane-bound polyribosomes of developing tubers was used to construct a cDNA library from which two classes of patatin clones were isolated. Sequence analysis of pGM01 and pGM203, full-length cDNA clones of each class, revealed the clones to be 98% homologous, corresponding to the two major species of patatin identified by polypeptide sequence analysis. Both clones encode proteins of 363 amino acids with an additional 23 amino acid hydrophobic signal sequence at the amino terminus. Northern blot analysis of tuber RNA using cDNA probes showed patatin mRNA to be approximately 1550 nucleotides in length. The mRNA was not detectable in polyribosomal or total RNA of stems or leaves. A genomic library was constructed with DNA from the cultivar "Superior" and five different patatin genomic clones were isolated. Two clones, SB6B and SA10C, appeared to represent intact genes present in the tetraploid genome at approximately twenty copies each. Sequence analysis revealed SB6B and SA10C to be 5.4Kb and 4.8Kb in length, respectively. The introns vary greatly in length and have evolved primarily by insertion/deletion events. Neither gene encodes the cDNAs we described and both have frame-shift mutations in central exons. However, the genes have normal consensus sequences for promoter elements, polyadenylation signals, and intron splice sites and appear transcriptionally competent. If active, they would encode truncated patatin proteins approximately one-half their normal size. Two dimensional western blots reveal no such low molecular weight proteins, but resolve as many as eighteen patatin isoforms in tubers. S1 nuclease analysis demonstrates the apparent absence of transcripts from either SB6B or SA10C and shows the patatin mRNAs detectable in roots to be a subset of the patatin mRNAs in tubers. Though apparently minimally mutated, it seems SB6B and SA10C have been silenced during their recent evolution, possibly due to problems of mRNA stability.
Degree
Ph.D.
Subject Area
Molecular biology|Botany
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