Abstract
Partial amino acid sequences of a 49 kDa apyrase (ATP diphosphohydrolase, EC 3.6.1.5) from the cytoskeletal fraction of etiolated pea stems were used to derive oligonucleotide DNA primers to generate a cDNA fragment of pea apyrase mRNA by RT-PCR and these primers were used to screen a pea stem cDNA library. Two almost identical cDNAs differing in just 6 nucleotides within the coding regions were found, and these cDNA sequences were used to clone genomic fragments by PCR. Two nearly identical gene fragments containing 8 exons and 7 introns were obtained. One of them (H-type) encoded the mRNA sequence described by Hsieh et al. (1996) (DDBJ/EMBL/GenBank Z32743), while the other (S-type) differed by the same 6 nucleotides as the mRNAs, suggesting that these genes may be alleles. The six nucleotide differences between these two alleles were found solely in the first exon, and these mutation sites had two types of consensus sequences. These mRNAs were found with varying lengths of 3′ untranslated regions (3′-UTR). There are some similarities between the 3′-UTR of these mRNAs and those of actin and actin binding proteins in plants. The putative roles of the 3′-UTR and alternative polyadenylation sites are discussed in relation to their possible role in targeting the mRNAs to different subcellular compartments.
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Abbreviations
- cDNA:
-
complementary DNA
- CSB:
-
cytoskeleton-stabilizing buffer
- DTT:
-
dithiothreitol
- EDTA:
-
ethylenediaminetetraacetic acid
- HEPES:
-
N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
- PMSF:
-
phenylmethylsulfonyl fluoride
- RT-PCR:
-
reverse transcription — polymerase chain reaction
- SDS:
-
sodium dodecyl sulfate
- Tris:
-
tris(hydroxymethyl)aminomethane
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Sequence data from this article were deposited with the DDBJ/EMBL/GenBank Data Libraries under Accession Nos. Genomic sequences of pea apyrase: AB023621, AB030444, AB030445, AB038554, AB038555. cDNA sequences of pea apyrase: AB022319, AB027614, AB038668, AB038669.
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Shibata, K., Abe, S. & Davies, E. Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum). Acta Physiol Plant 23, 3–13 (2001). https://doi.org/10.1007/s11738-001-0016-y
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DOI: https://doi.org/10.1007/s11738-001-0016-y