Plant Molecular Biology 17: 175-177, 1991. © 1991 Kluwer Academic Publishers. Printed in Belgium. Update section Sequence Nucleotide sequence analysis of a protein gene of peanut stunt virus cDNA clone encoding the coat 175 R. A. Naldu 1, G. B. Collins 2 and S. A. Ghabrial 1. Departments of Plant Pathology1 and Agronomy 2, University of Kentucky, Lexington, K Y 40546-0091, USA (*author for correspondence) Received 15March 1991; accepted 29March 1991 Peanut stunt virus (PSV, a cucumovirus) is an important pathogen of many major crops world- wide [6]. PSV is a single-stranded, positive-sense RNA plant virus with a tripartite genome consist- ing of RNA 1, 2 and 3. RNA 4, which is also encapsidated, is a subgenomic RNA that contains the coat protein gene [6]. As a part of a project aimed at producing coat protein-mediated trans- genic resistance to PSV in plants, we have cloned PSV RNA 4. Methods for isolation of viral RNA from isolate PSV-V and for purification of RNA 4 from polyacrylamide gels were as described by Naidu et al. [2]. Purified RNA 4 was first poly- adenylated [5] and then cDNA copies of poly- adenylated RNA 4 were synthesized by the method of Gubler and Hoffman [ 1] using oligo- (dT)12_18 primer. The ds-cDNA was made blunt- ended with T4 DNA polymerase and ligated into the phagemid vector pUCll9 at the Sma I site. Clones containing RNA 4 sequences were identi- fied by Southern hybridization using 32p-labelled cDNA to PSV-RNA 4 as a probe. Recombinant cDNA clones with inserts of about 1 kb in length (estimated full length of RNA 4) were selected. The insert in one such clone, designated pPSV4-43, was subcloned into the transcription vector pBluescript II KS + (Stratagene, La Jolla, CA). The resultant plasmid, referred to as pBPSV4-43, was generated by digesting pPSV4-43 with Bam HI/Eco RI and ligating into the pBluescript vector linearized with the same enzymes. The plasmid pBPSV4-43 contained the PSV cDNA sequence downstream of the bac- teriophage T3 promoter. Cell-free transcription using T3 polymerase and translation of the tran- scripts in a rabbit reticulocyte lysate system revealed that the principal translation product co- migrated with authentic PSV capsid protein, as determined by SDS-PAGE. For DNA sequence analysis, single-stranded DNA templates representing both strands of PSV cDNA were produced as follows. Plasmid pPSV4-43 was digested with Bam HI/Eco RI and ligated into the phagemid vector pUCll8 line- arized with the same enzymes. The resulting plas- mid, pPSV4-34 contained the same PSV cDNA insert as pPSV4-43, but in the reverse orientation. Single-stranded DNA, generated from pPSV4-43 and pPSV4-34 with the aid of the helper phage M 13K07, were sequenced using the dideoxy chain termination method [4]. A directed sequencing protocol using progressive oligonucleotide prim- ers was followed [3]. For each DNA strand, universal primer and three synthetic oligonucle- otide primers were used for sequencing. The three oligonucleotide primers used for sequencing the plus strand (generated from pPSV4-43) were complementary to nucleotides 225 -242, 485-501, and 754-770. The three oligonucle- otides primers used in sequencing the strand with The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X56544.