ABSTRACT
The Aspergillus alc regulon encodes a transcription factor, ALCR, which regulates transcription from cognate promoters such as alcA(p). In the presence of suitable chemical inducers, ALCR activates gene expression from alcA(p). The alc regulon can be transferred to other species and can be used to control the expression of reporter, metabolic and developmental genes in response to low-level ethanol exposure. In this paper, we describe a versatile system for targeting the alc regulon to specific cell types in Arabidopsis by driving ALCR expression from the GAL4 upstream activator sequence (UAS). Large numbers of Arabidopsis lines are available in which GAL4 is expressed in a variety of spatial patterns and, in turn, drives the expression of any gene cloned downstream of the UAS. We have used a previously characterized line that directs gene expression to the endosperm to demonstrate spatially restricted ethanol-inducible gene expression. We also show that the domain of inducible gene expression can easily be altered by crossing the UAS::ALCR cassette into different driver lines. We conclude that this gene switch can be used to drive gene expression in a highly responsive, but spatially restricted, manner.
Subject(s)
Arabidopsis/genetics , DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Gene Expression Regulation, Plant/drug effects , Transcription Factors/metabolism , Genes, Plant , RegulonABSTRACT
How cells achieve their final sizes is a pervasive biological question. One strategy to increase cell size is for the cell to amplify its chromosomal DNA content through endoreduplication cycles. Although endoreduplication is widespread in eukaryotes, we know very little about its molecular mechanisms. Successful progression of the endoreduplication cycle in Arabidopsis requires a plant homologue of archaeal DNA topoisomerase (topo) VI. To further understand how DNA is endoreduplicated and how this process is regulated, we isolated a dwarf Arabidopsis mutant, hyp7 (hypocotyl 7), in which various large cell types that in the wild type normally endoreduplicate multiple times complete only the first two rounds of endoreduplication and stall at 8C. HYP7 encodes the RHL1 (ROOT HAIRLESS 1) protein, and sequence analysis reveals that RHL1 has similarity to the C-terminal domain of mammalian DNA topo IIalpha, another type II topo that shares little sequence homology with topo VI. RHL1 shows DNA binding activity in vitro, and we present both genetic and in vivo evidence that RHL1 forms a multiprotein complex with plant topo VI. We propose that RHL1 plays an essential role in the topo VI complex to modulate its function and that the two distantly related topos, topo II and topo VI, have evolved a common domain that extends their function. Our data suggest that plant topo II and topo VI play distinct but overlapping roles during the mitotic cell cycle and endoreduplication cycle.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Arabidopsis/metabolism , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/metabolism , Nuclear Proteins/metabolism , Ploidies , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Archaeal Proteins , Base Sequence , Cell Cycle , Cell Size , Cloning, Molecular , DNA/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Phenotype , Protein Binding , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The ALCR/alcA (alc) two-component, ethanol-inducible gene expression system provides stringent control of transgene expression in genetically modified plants. ALCR is an ethanol-activated transcription factor that can drive expression from the ALCR-responsive promoter (alcA). However, the alc system has been shown to have constitutive expression when used in plant callus or cell suspension cultures, possibly resulting from endogenous inducer produced in response to lowered oxygen availability. To widen the use of the alc system in plant cell culture conditions, the receptor domain of the rat glucocorticoid receptor (GR) was translationally fused to the C terminus of ALCR to produce ALCR-GR, which forms the basis of a glucocorticoid-inducible system (alc-GR). The alc-GR switch system was tested in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells using a constitutively expressed ALCR-GR with four alternative alcA promoter-driven reporter genes: beta-glucuronidase, endoplasmic reticulum-targeted green fluorescent protein, haemagglutinin, and green fluorescent protein-tagged Arabidopsis (Arabidopsis thaliana) Arath;CDKA;1 cyclin-dependent kinase. Gene expression was shown to be stringently dependent on the synthetic glucocorticoid dexamethasone and, in cell suspensions, no longer required ethanol for induction. Thus, the alc-GR system allows tight control of alcA-driven genes in cell culture and complements the conventional ethanol switch used in whole plants.
Subject(s)
Arabidopsis Proteins/genetics , Gene Expression Regulation, Plant , Genes, Switch , Nicotiana/genetics , Plant Proteins/genetics , Animals , Arabidopsis/genetics , Base Sequence , DNA Primers , Ethanol/pharmacology , Green Fluorescent Proteins/genetics , Solanum lycopersicum/genetics , Plants, Genetically Modified , RNA, Plant/genetics , RNA, Plant/isolation & purification , Rats , Reverse Transcriptase Polymerase Chain Reaction , Transcription FactorsABSTRACT
In a proteomics-based screen for proteins interacting with cyclin-dependent protein kinase (CDK), we have identified a novel CDK complex containing the eukaryotic translation initiation factor, eIF4A. Reciprocal immunoprecipitations using antibodies against eIF4A indicate that the interaction is specific. The CDKA-eIF4A complex is abundant in actively proliferating and growing cells but is absent from cells that have ceased dividing. The CDKA-eIF4A complex contains kinase activity that is sensitive to the CDK-specific inhibitor roscovitine. This interaction points to a possible molecular mechanism linking cell proliferation with translational control.
Subject(s)
Cell Division/physiology , Cyclin-Dependent Kinases/metabolism , Eukaryotic Initiation Factor-4A/metabolism , Protein Biosynthesis/physiology , Arabidopsis/cytology , Arabidopsis/enzymology , Blotting, Western , Cyclin-Dependent Kinases/chemistry , Enzyme Inhibitors/pharmacology , Eukaryotic Initiation Factor-4A/chemistry , Precipitin Tests/methods , Protein Kinase Inhibitors , Protein Kinases/metabolism , Purines/pharmacology , Roscovitine , Nicotiana/cytology , Nicotiana/enzymologyABSTRACT
Controlled gene expression in time and space is a powerful tool for the analysis of gene function during plant development. Here, we report ethanol inducible gene expression in defined sub-domains of the shoot apical and floral meristems. For this, expression of an ethanol-regulated transcription factor, ALCR, is restricted to precise domains using specific promoters. Gene expression activation is followed using reporters under the control of the alcA promoter, which responds to ALCR only in the presence of the ethanol. We demonstrate that precise control of spatially limited gene expression can be achieved. The kinetics of reporter gene activation and inactivation following a pulse of ethanol induction shows that the system is dynamic and suitable for precise temporal control of expression. The system is both flexible and robust, permitting simultaneous expression of two genes in a given domain or, conversely, the expression of a gene in two separate domains. We also show that this strategy can be applied to mis-express genes with developmental roles, by manipulating expression of the SHOOT MERISTEMLESS (STM) and CYCLIN D3;1 (CYCD3;1) genes during plant development.
