title: A novel fluorescent transcriptional reporter for cell-based microarray assays.
abstract: Cell-based microarrays have been used for a wide variety of assays including gain-of-function, loss-of-function and compound screening. Many of these assays have employed fluorescent proteins as reporters. These fluorescent reporter proteins can be monitored in living cells but have low sensitivity of detection compared to enzymatic reporters. Here we have described a novel transcriptional reporter assay using the alkaline phosphatase reporter enzyme and a fluorescent substrate (ELF-97) to screen for gain-of-function mutations in the type-I cGMP-dependent protein kinase (PRKG1). We have identified a constitutively active mutant of this enzyme in which a conserved Glu at position 81 was mutated to Lys.
authors.affiliation: Molecular, Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI, USA. tanyamm@umich.edu
authors.forename: Tanya M
authors.initials: TM
authors.lastname: Redmond
authors.forename: Michael D
authors.initials: MD
authors.lastname: Uhler
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004396:Coloring Agents; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D005240:Feasibility Studies; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D007202:Indicators and Reagents; D008239:Lysine / Q000378:metabolism; D009943:Organophosphorus Compounds; D052999:Quinazolinones; D013379:Substrate Specificity; D046888:Tissue Array Analysis / Q000379:methods*
chemical_list: D004396:Coloring Agents; D007202:Indicators and Reagents; D009943:Organophosphorus Compounds; D052999:Quinazolinones; C079177:2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone; D017869:Cyclic GMP-Dependent Protein Kinases; D000469:Alkaline Phosphatase; D008239:Lysine