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Title:Characterization and nucleotide sequence of apple mosaic virus coat protein gene
Author(s):Alrefai, Rudaina Hussein
Doctoral Committee Chair(s):Korban, Schuyler S.
Department / Program:Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Agriculture, Agronomy
Biology, Molecular
Agriculture, Plant Pathology
Abstract:Apple mosaic virus (ApMV) (Idaho strain) was purified from infected cucumber cotyledons. Virions 3 and 4 (top components in sucrose gradient) were isolated and RNA was extracted. A double stranded copy DNA (cDNA) was synthesized from in vitro polyadenylated RNA3 and cloned into a pBluescript T-vector. The inserts of four clones from the 3$\sp\prime$-end of RNA3 were sequenced and the 3$\sp\prime$-primer was designed to synthesize cDNA of RNA3. The DNA was cloned into the plasmid vector pUC118. A set of overlapping cDNA clones was used to determine the nucleotide sequence of RNA4. The 5$\sp\prime$-end of RNA4 was mapped using the primer extension technique. ApMV RNA4 was found to be 983 nucleotides in length containing open reading frame (ORF) of a 662 nucleotides, which was flanked by 5$\sp\prime$ and 3$\sp\prime$-nontranslated sequence of 55 and 265 nucleotides, respectively. The ORF encodes for a coat protein of a 25,085 Mr. The sequence contains several inverted repeats that could form hair-pin structures. In addition, two palindromic sequence were found in the ORF with a third palindrome in the 3$\sp\prime$-nontranslated region. These structures may be involved in the recognition of RNA by viral replicase or in genome activation by the coat protein. The amino-terminus of ApMV coat protein is basic and rich in Cys, whereas the carboxy-terminus has an even distribution of basic and acidic amino acids, similar to other ilarviruses.
To transform plants with ApMV coat protein gene an Agrobacterium-mediated transformation system was developed for apple "Royal Gala" plants. Kanamycin completely inhibited regeneration of untransformed leaf sections on a 1/2 Murashige and Skoog (MS) regeneration medium. Apple leaf sections were transformed by Agrobacterium strains C58Z707:pZA-7 and EHA101:pZA-7 and calli were produced on a 1/2MS selection medium.
Issue Date:1993
Rights Information:Copyright 1993 Alrefai, Rudaina Hussein
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9314835
OCLC Identifier:(UMI)AAI9314835

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