Nudeotkle Sequence of Cucumber Pale Fruit Virotd: Homology to Hop Stunt Viroid

Double stranded cDNA of cucumber pale fruit viroid (CPFV) has been cloned by the method of Okayama and Berg (Mol.Cell.Biol.2,161-170 (1982)) and the complete nucleotide sequence was established. The covalently closed circular molecules of single-stranded CPFV RNA consists of 303 nucleotides. The nucleotide sequence of CPFV was compared with the previously established sequence of hop stunt viroid (HSV), which consists of 297 nucleotides (Ohno ejt a_l. Nucleic Acid Res.11,6185-6197 (1983)). CPFV differs from HSV in the nucleotide sequence at 16 positions which include 8 exchanges, 7 insertions and 1 deletion. Both viroids share about 95% sequence homology. Considering the pathogenic properties of both viroids together, it is concluded that CPFV is a cucumber isolate of HSV. INTRODUCTION Viroid are infectious low molecular weight RNA species causing serious disease on higher plants (1). Eight viroids have been sequenced so far and shown to form highly base-paired unique rod-like secondary structures. Comparative sequence analysis of these viroids shows that potato spindle tuber viroid (PSTV) (2), citrus exocortis viroid (CEV) (3), chrysanthemum stunt viroid (CSV) (3,4), tomato planta macho viroid (TPMV) (5), and tomato apical stunt viroid (TASV) (5), belonging to "PSTV group", share 60-80% sequence homology (6). On the other hand, coconut cadang-cadang viroid (CCCV) (7), avocado sunblotch viroid (ASBV) (8) and hop stunt viroid (HSV) (9) are distantly related to "PSTV group". Cucumber pale fruit viroid (CPFV) and HSV were found to be the causal agents of cucumber pale fruit disease (10) and hop stunt disease (11-13), respectively. They were found independently in different host plants, cucumber (Cucumis © IRLPrsn Limited, Oxford, England. 3427 Nucleic Acids Research sativus L.) and hop (Humulus lupulus L.), in the separated countries. Recently, we have reported that although both viroids were not distinguishable by their pathogenic properties (14), they could separate each other by polyacrylamide gel electrophoresis and CPFV was larger than HSV by only several nucleotides (15). In this paper, we describe molecular cloning of cDNA of CPFV and the complete nucleotide sequence. HSV and CPFV share very high sequence homology. MATERIALS AND METHODS Enzymes. PolyA polymerase was prepared from E.coli B/r (16). AMV reverse transcriptase was purchaced from Seikagaku Kogyo Co., E.coli DNA polymerase I, E.coli RNase H and terminaldeoxynucleotidyl transferase were purchased from BRL. E.coli DNA ligase was obtained from New England Biolabs. Restriction enzymes, T4 polynucleotide kinase and E.coli alkaline phosphatase were from Takara Shuzo Co., Kyoto. Purification of CPFV. CPFV, kindly provided by Dr.H.L.Sanger (Max-Planck Inst.), was maintained and propagated in cucubmber plants (C.sativus cv. Suyo). Cucumber pale fruit viroid RNA was purified by the method described by Uyeda et̂ aj^. (17). For the final steps of the purification, CPFV RNA was separated from cellular RNAs by 15% polyacrylamide gel electrophoresis under non-denaturing condition. CPFV RNA which contained both circular and linear molecules, was eluted from the gel, absorbed to and eluted from DEAE-cellulose (Whatman DE-32) to remove gel contaminants and used for dot blot hybridization analysis and molecular cloning. Dot blot hybridization. Sequence homology between HSV and CPFV was tested preliminary by dot blot hybridization analysis (18). 3plabelled pHSV-A60 (9), with a specific activity of 10 cpm/pg DNA, was prepared by nick-translation (19) and used as prove for hybridization. Molecular cloning of CPFV. The purified CPFV RNA was treated with E. coli alkaline