Identification and elimination of an aberrant splice product from cDNAs encoding the human adenovirus type 5 E4orf6 protein.
Growing awareness of the central role of the E4orf6 protein in regulating the infectious cycle of human adenoviruses has led to greatly intensified efforts to define its functions and mechanisms of action. Many workers employ cDNAs to generate plasmid or viral vectors to express E4orf6 in the absence of other viral products. In addition to the normal 34-kDa product, such vectors consistently produce a polypeptide of about 8 kDa. In the present report we show that this protein is produced by an aberrant mRNA utilizing the 5' splice donor site used normally by the virus to produce the E4orf6/7 product, which shares 58 residues with E4orf6. This amino terminal coding sequence is linked to a 3' sequence via a novel splice acceptor site in an alternative reading frame of the E4orf6 cDNA. The 5' donor site was altered by PCR-directed mutagenesis to yield a construct that produces high levels of E4orf6 in the absence of the 8-kDa polypeptide. This construct should eliminate some of the problems encountered previously using the wild-type E4orf6 coding sequence.
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Department of Biochemistry, McGill University, Montreal, Quebec, H3G 1Y6, Canada.
Chu-Pham-Dang
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Branton
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D017504:Adenovirus E4 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000260:Adenoviruses, Human / Q000235:genetics / Q000378:metabolism*; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion
D017504:Adenovirus E4 Proteins; D018076:DNA, Complementary; D011994:Recombinant Proteins