Identification of a very early promoter of insect Hz-1 virus using a novel dual-expression shuttle vector.
Very early promoters of viruses control the proper cascade expression of viral genes and are essential for completion of virus life cycles. These promoters are usually rare and weak and do not encode structural proteins. As a result, they are difficult to identify. In order to identify and clone the very early promoters of a large eukaryotic DNA virus, the Hz-1 virus, a novel cloning strategy was applied. This strategy is based on a dual-expression shuttle vector containing a promoter-less lacZ gene. Insertion of eukaryotic promoters upstream permits the efficient expression of LacZ in bacteria cells. The function of the putative promoters was then confirmed by their proper expression in insect cells. The first two productive infection-specific promoters of Hz-1 virus, contained within the shuttle vectors pTSV-2-129 and pTSV-2-49, were cloned from the HindIII-K and HindIII-A fragments of the Hz-1 viral genome, respectively. By primer extension analysis, an immediate and constitutive expression of the promoter in clone pTSV-2-129 was detected after viral infection. Identification of the productive infection-specific promoters has laid down important groundwork for future studies on the molecular mechanism of the transcriptional switch between productive and persistent infections of Hz-1 virus.
Lee
S T
ST
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China.
Yu
S M
SM
Hsu
E L
EL
Chao
Y C
YC
D000426:Alcohol Dehydrogenase / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004330:Drosophila / Q000235:genetics; D004926:Escherichia coli; D005260:Female; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D010053:Ovary; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D018411:Spodoptera; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis
D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase; D001616:beta-Galactosidase