Sequence variability in avocado sunblotch viroid (ASBV)
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Nucleic Acids Research
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Volume 16 Number 20 1988
Sequence variability in avocado sunblotch viroid (ASBV) Vicente Pallas, Isabel Garcfa-Luque, Esteban Domingo1 and Ricardo Flores Instituto de Agroquimica y Tecnologia de Alimentos (CSIC), Jaime Roig, 11, 46010 Valencia and 'Centro de Biologfa Molecular (CSIC-UAM), Facultad de Ciencias, Universidad Aut6noma, Cantoblanco, Madrid Accession no. X13000 Submitted September 1, 1988
Avocado sunblotch viroid (ASBV) holds a unique position among viroids. From a structural point of view it has a limited sequence homology with other members of this group (1), and functionally, ASBV is the only viroid for which it has been demostrated that RNA transcripts generated in vitro from cDNA clones are able to be processed at specific sites in the isence of enzymes (2). Although sequence heterogeneity among different isolates of ASBV has been suggested (3,4), only the sequence of an Australian isolate of this viroid has been published so far (1). Using two different experimental approaches, we have determined the complete nucleotide sequence of a Spanish isolate of ASBV obtained from an avocado tree cv. 'Fuerte' displaying the symtoms of the disease. With base-specific RNases we determined all the sequence except residues no. 1 to 8, following the numbering proposed previously (1), without finding any difference between both isolates. By cloning of cDNA fragments (synthesized by means of an oligonucleotide primer complementary to positions no. 42 to 57) inserted into the Sma I site of bacteriophage M13mpl8 and M13mpl9 DNA, and sequencing by the Tffeoxynucleotide chain termination method, the following differences were observed: the U at position no. 3 was substituted by an A in two clones, and in one of them the U no. 5 was replaced by an A; in another clone the A no. 7 was absent, and finally in another one the stretch of oligo A (positions no. 122-128) had nine insted of seven residues. Therefore, all the sequence heterogeneity was observed in the two end loops of the molecule (Fig. 1), but not in its central part which appears to be involved in the self-processing of ASBV plus and minus RNA species. The high sequence homology (p98%) of these two isolates of so distant geographic localizations, suggests a comnion origin for boths of them. W.A)
(U.U U AI I u
A uAuU GAG A !U GAA AAGA.AGAGAGGAUA GA AAAACAUAGU GAC
GG UU UU UUUCU G G A AC/A G I6 UU UAC A LA/AU GAG CGA GGGAGAGA GGAG CA GGAGAACGAUUU CAG UCA AUCCG CAAACC A
A CUC GCU UA UUCUCU CUUA GC-UCACUUGA MA GUC AGA-AGGGU GAAAGG U UAA UUU-UUUU GUUACUUCUAU CU AUUUGGAACG CUG G UU U A AA 4A U C AA U U U C AA C CU A G c CA aU A u U AC I
Fig. 1. Secondary structure model of ASBV as proposed in (1). Residues different from this sequence are indicated by arrowheads ( *).
Acknowledgements We thank Dr. C. Escarmis and S. de la Luna their helpful advice, M. Davila and M. Climent their assistance, and Dr. J.M. Farre for providing the avocado tissue. V.P. was the recipient of a fellowship of the Generalitat Valenciana. This work was partially supported by a grant of the Comisi6n Asesora de Investigaci6n Cientifica y Tecnica of Spain. References 1. Symons, R.H. (1981). Nucleic Acids Res., 9, 6527-6537. 2. Hutchins, C.J., Rathjen, P.D., Foster, A.C. and Symons, R.H. (1986). Nucleic Acids Res., 14, 3627-3640. 3. Barker, .MT., TEnnes, J.L., Murphy, P.J. and Symons, R.H. (1985). J. Virol. Meth., 10, 87-98. 4. Palukailtis P.7Rakowski, A.G., Alexander, D.McE. and Symons, R.H. (1981). Ann. appl. Biol., 98, 439-449. 94 IRL Press Limited, Oxford, England.