Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacterial luciferase, derived from a fusion of the luxA and luxB genes of Vibrio harveyi, has been expressed at very high levels in caterpillars and insect cells. The coding sequence for luciferase was inserted into vectors developed in our laboratory which were designed to expedite screening of recombinant virus. These vectors contained the beta-galactosidase indicator gene under control of immediate early (IE1), early (ETL), or very late (P10) promoters and a cloning site for inserting the fused luciferase gene next to the polyhedrin promoter. Recombinant baculoviruses containing the luciferase gene as well as the beta-galactosidase gene could be easily selected when Bluo-gal (beta-galactosidase indicator) was included in the plaque assays. Using cells derived from the fall armyworm (Spodoptera frugiperda), luciferase was strongly expressed very late in infection (48-72 h). The bacterial luciferase assay was sufficiently sensitive that light production could be detected from an extract of a single cell. In addition, live insects, including the cabbage looper (Trichoplusia ni) and saltmarsh caterpillar (Estigmene acrea) were infected by mixing recombinant baculovirus into their diet. Cabbage loopers (with an average wet weight of 223 mg) produced at least 195 micrograms of active luciferase and levels of synthesis peaked between 96-120 h. The results indicate that bacterial luciferase may be used as a reporter of gene expression in insects.
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PMID:Bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae. 130 4

The region of the Autographa californica nuclear polyhedrosis virus (AcMNPV) encompassing the EcoRI T fragment (29.0 to 30.1 map units) was characterized by DNA sequencing, transcriptional mapping, and site-directed mutagenesis. The largest transcript from this region, an early 1.7-kilobase (kb) poly(A)+ RNA, encompassed three tandem, nonoverlapping open reading frames (ORFs). The largest of these ORFs, ETL, was proximal to the 5' end of the transcript and had the capacity to encode a 28-kilodalton (kDa) polypeptide. A recombinant virus, vETL beta gal, containing the Escherichia coli beta-galactosidase (beta gal) gene fused to the N-terminal two-thirds of the ETL ORF, produced blue plaques in the presence of a chromogenic indicator of beta gal and wild-type levels of polyhedra in cell culture. This recombinant was also infectious in insect larvae by oral administration of occluded virus. Comparison of vETL beta gal and wild-type viral proteins pulse-labeled at various times postinfection (p.i.) revealed (i) absence of a virus-induced 28-kDa polypeptide, (ii) early expression of a large (approximately 130-kDa) polypeptide which may be the ETL-beta gal fusion protein, (iii) a delay in expression of early 35 and 40-kDa polypeptides, and (iv) a 4- to 6-h delay in the expression of late proteins in vETL beta gal-infected cells. Cycloheximide did not inhibit synthesis of the 1.7-kb RNA but did inhibit its shutoff, which occurs at 12 h p.i. in the absence of inhibitors. Thus, the ETL gene product is apparently an early 28-kDa protein which is necessary, directly or indirectly, for timely expression of many other AcMNPV genes. The promoter-leader regions of the 1.7-kDa transcript showed significant sequence similarities to the leader of the AcMNPV IE-1 gene. The middle ORF within the 1.7-kb transcript, ETM, would encode a hydrophobic polypeptide of 113 amino acid residues. ETS, a small ORF within and proximal to the 3' end of the 1.7-kb transcript, was also transcribed as a set of smaller (approximately 0.5-kb) RNAs initiated heterogeneously in the region between ETL and ETS and persisting throughout infection.
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PMID:Characterization of an early gene accelerating expression of late genes of the baculovirus Autographa californica nuclear polyhedrosis virus. 329 91