Recombinant baculoviruses as vectors for identifying proteins encoded by intron-containing members of complex multigene families.
Using a transfer vector derived from Bombyx mori nuclear polyhedrosis virus (BmNPV), we have constructed recombinant baculoviruses that contain complete silk moth chorion chromosomal genes encoding high-cysteine proteins under the control of the polyhedrin promoter. Silk moth tissue culture cells infected with these recombinant viruses were found to contain abundant RNA sequences of sizes similar to those of the authentic chorion mRNAs. Chorion transcripts present in infected cells were initiated almost exclusively at the cap site of the polyhedrin start site. Primer extension and RNase protection experiments revealed that a considerable proportion of the resultant transcripts were spliced at the same sites as those utilized in follicular cells for the production of functional chorion mRNA. Electrophoretic analysis and immunoprecipitation of the proteins of host cells infected with the recombinant viruses revealed the presence of the corresponding chorion proteins. We conclude that baculovirus vectors can be used for expressing efficiently not only cDNAs or simple genes devoid of intervening sequences but also intron-containing chromosomal genes. Thus, recombinant baculoviruses offer a powerful alternative to hybrid-selected translation, particularly when the identification of proteins encoded by members of complex multigene families is required.
Iatrou
K
K
Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, AB, Canada.
Meidinger
R G
RG
Goldsmith
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D012831:Bombyx; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004527:Egg Proteins / Q000235:genetics*; D005796:Genes; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics*; D007438:Introns; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D011401:Promoter Regions, Genetic; D012326:RNA Splicing; D013539:Simian virus 40 / Q000235:genetics; D014158:Transcription, Genetic
D000952:Antigens, Polyomavirus Transforming; D004527:Egg Proteins; D008565:Membrane Proteins; D015345:Oligonucleotide Probes; C015753:chorion proteins