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)

We have developed a genetic screen for temperature-sensitive mutations in the very late transcription apparatus of the Autographa californica nuclear polyhedrosis virus. This method starts with the BacPAKS virus, which has the Escherichia coli lacZ gene under the control of the very late polyhedrin promoter. The desired mutants are temperature-sensitive for beta-galactosidase production and can be complemented by wild-type virus, which lacks the lacZ gene. Two mutants created by nitrosoguanidine mutagenesis and identified by this screen, and one mutant identified by another screen, have been mapped by marker rescue to the viral protein kinase 1 gene (pk-1). The protein kinase genes of these three mutant viruses have been sequenced, revealing the same point mutation in two of them and a different point mutation in the other. In each case, a single amino acid is changed: In two mutants, XF4 and XF5, Asp 92 is changed to Asn; in the other mutant, KT800, Thr 204 is changed to lle. Northern blotting of RNA made in cells infected by these three mutant viruses has shown that the accumulation of very late transcripts (lacZ and p10) is temperature-sensitive, but that accumulation of at least one late transcript (vp39) is not temperature-sensitive. Nuclear run-on transcription assays with two of the mutants indicate that very late transcription is somewhat temperature-sensitive, although this defect is not as pronounced as the temperature-sensitivity detected by Northern blotting. Transcription of at least one late gene (vp39) is not temperature-sensitive in cells infected by these two mutants. Thus, it appears that the viral protein kinase-1 is involved in very late gene expression. Some of this effect is at the transcription level, but some may also be exerted at the posttranscription level.
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PMID:Temperature-sensitive mutations in the protein kinase-1 (pk-1) gene of the Autographa californica nuclear polyhedrosis virus that block very late gene expression. 886 93

A new, versatile baculovirus vector was developed for the generation of recombinants in the yeast Saccharomyces cerevisiae and for the expression of foreign proteins in both insect larvae and in insect cells. This vector is based on Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) and exploits the 10-kDa protein promoter (p10) for the expression of the foreign gene. The p10 locus was used for the insertion of a yeast-selectable marker system (ARS-URA-URA3) and of a gene for screening and titration of recombinants in insect cells (beta-galactosidase). The polyhedron-positive phenotype of this vector is maintained allowing its use in insect larvae, by feeding polyhedra, and in insect cells, by infecting with budded virus. The generation of this baculovirus vector requires a single recombination step in yeast prior to infection of insect cells, but has the advantage over the vector designed previously (Patel et al., A new method for the isolation of recombinant baculovirus, Nucleic Acids Research 20 (1992) 97-104) that these vectors can also be used in insects.
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PMID:Generation of a p10-based baculovirus expression vector in yeast with infectivity for insect larvae and insect cells. 939 40

A special recombinant of Autographa californica multicapsid nuclear polyhedrosis virus (AcNPV) was designed to study the early histopathological events of baculovirus infection in Spodoptera exigua larvae. This recombinant contained a Drosophila melanogaster heat shock 70 promoter driving an Escherichia coli beta-galactosidase (Lac-Z) reporter gene to monitor the presence of early viral gene expression and a second reporter gene, the E. coli beta-glucuronidase (GUS) gene, under control of the very late AcNPV p10 promoter to monitor viral replication. In S. exigua larvae, permissive Spodoptera spp. cultured cells, and nonpermissive D. melanogaster cultured cells early viral gene expression was indicated by the appearance of Lac-Z as early as 3 hr p.i. Late viral gene expression was indicated by the appearance of GUS and occurred only in the permissive cultured cells and larvae. Early and late viral gene expression could be detected simultaneously using differential enzyme histochemistry. Analysis of infected S. exigua larvae revealed that midgut columnar cells and, at a low frequency, midgut regenerative cells were the primary sites of infection. Parental nucleocapsids were apparently transported through columnar cells to underlaying regenerative cells before virus replication and progeny production. Infection of tissues beside the midgut epithelium was not detected prior to viral replication within the midgut, suggesting that infection of the midgut is an important prelude to systemic infection.
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PMID:Passage of Autographa californica nuclear polyhedrosis virus through the midgut epithelium of Spodoptera exigua larvae. 1183 15

Macroautophagic activity is most directly and precisely measured by a cargo sequestration assay. Long-lived, cytosolic proteins that are degraded exclusively by the autophagic-lysosomal pathway, such as lactate dehydrogenase (LDH) are suitable as endogenous sequestration probes. Autophagic sequestration is measured as transfer of the protein from the soluble (cytosolic) to the sedimentable (organelle-containing) cell fraction, using leupeptin or other proteinase inhibitors to block inactivation and degradation of the protein inside autophagic vacuoles. A convenient separation method is electrodisruption of the cells, followed by sedimentation of the organelle fraction through a Nycodenz density cushion. A promising variant of the cargo assay is to use a protein probe that is processed by the autophagic-lysosomal pathway so as to generate an intravacuolar fragment. Because there is no cytosolic background, subcellular fractionation is unnecessary, allowing the use of the autophagic fragment assay to measure autophagic activity in whole cells. In hepatocytes, a small fragment, p10(BHMT), made by autophagic processing of the enzyme betaine:homocysteine methyltransferase, thus accumulates in an autophagy-dependent manner in the presence of leupeptin. Autophagic sequestration can also be measured by using exogenous cargo probes, such as radiolabeled di- and trisaccharides, which can be loaded into the cytosol of hepatocytes by reversible electrodisruption or mechanical stress. Raffinose is the preferable probe for measurement of autophagic activity, whereas sucrose (which can be hydrolyzed in amphisomes and lysosomes by added endocytosed invertase) and lactose (which is hydrolyzed in lysosomes by the endogenous beta-galactosidase) are useful for dissection of the various steps in the autophagic-lysosomal pathway and for studying autophagic-endocytic interactions. Furthermore, the intralysosomal hydrolysis of autophagocytosed lactose can be measured in whole cells (as formation of the hydrolysis product, galactose), thus providing a background-free assay (autophagic lactolysis) of the overall autophagic-lysosomal pathway.
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PMID:Sequestration assays for mammalian autophagy. 1920 Aug 76


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