Control of gene expression in eukaryotes

Details Control of gene expression in eukaryotes Control of gene expression in eukaryotes

1999-12-22

2003-12-09

McKelvey, Terry (Department: 1636)

Chemistry: molecular biology and microbiology

Plant cell or cell line, per se ; composition thereof;...

Plant cell or cell line, per se, contains exogenous or...

C435S320100

Reexamination Certificate

active

06660524

ABSTRACT:

1. INTRODUCTION
This invention relates to an inducible gene expression system, particularly but not exclusively eukaryotes, such as plants, for example.
2. BACKGROUND TO THE INVENTION
Manipulation of plants to improve certain characteristics requires the control of expression of foreign or endogenous genes in plant tissues. Such manipulation relies on the availability of mechanisms to control gene expression as required. It is therefore advantageous to have the choice of a variety of different promoters so that the most suitable promoter may be used. A range of promoters is known to be operative within plants.
Within the promoter there are several defined domains which are necessary for the function of the promoter. The first of these domains is located immediately upstream of the structural gene and forms the core promoter region, about 70 base pairs immediately upstream of the genes. This region contains the CAAT and TATA boxes and represents a transcription initiation sequence which defines the transcription start site for the gene. A series of regulatory sequences upstream of the core promoter sequence constitute the remainder of the promoter. The regulatory sequences determine the expression levels, the spatial and temporal pattern of expression and possible expression under inductive conditions.
The control of expression of heterologous genes in plant cells is important for the successful genetic manipulation of plants to alter and/or improve phenotypic characteristics. Promoters and/or regulatory sequences from bacteria, viruses, fungi and plants have been used to control gene expression in plants. In some cases it will be desirable to control the time and/or extent of the expression of introduced genetic material in plants, plant cells or tissue. The ability to regulate the expression of transgenes provides several important advantages: (1) regulation of expression of gene(s) that might interfere with the transformation and regeneration process (Roeder et al., 1994, McKenzie et al., 1998), (2) reversible control of gene expression at a specific time (e.g. manipulation of carbon metabolism by Caddick et al., 1998 and secondary product formation by Sommer et al., 1998), (3) control of growth and development (e.g. flowering, plant fertility, cell wall formation), (4) control of genes that respond to environmental signals (e.g. attack by pathogens, such as, for example, nematodes, arachnids or aphids), (5) expression of selectable marker genes and (6) expression of recombinase proteins at specific time points. Each of these applications can use the inducible gene expression system and novel sequences of the present invention.
2.1 Known Regulatable Gene Expression Systems in Plants
A few plant genes are known to be induced by a variety of internal and external factors including plant hormones, heat/cold shock, chemicals, pathogens, lack of oxygen and light. Few of these systems have been described in detail.
Ideally a chemically inducible activating promoter in a 5′ regulatory region should have low background activity in the absence of an inducer and demonstrate high expression in the presence of an inducer. A chemically inducible repressing promoter in a 5′ regulatory region should have low background activity in the presence of an inducer and demonstrate high expression in the absence of an inducer. The activator/repressor should also only allow control of the transgene. This renders the use of most endogenous promoters unsuitable and favors the use of those better characterized regulatory elements of model organisms distant in evolution, such as yeast,
E. coli
, Drosophila or mammalian cells, that respond to signals that are usually not encountered in higher plants. These characteristic regulatory elements are, however, less advantageous in their operation than the system proposed in the present invention.
On this basis, two different concepts of gene control can be realized, namely promoter-repressing systems and promoter-activating systems.
2.2 Promoter-repressing Systems
The repression principle is based on the sterical interference of a protein with the proteins important for transcription. It is a common mechanism in bacteria, for example LexA, Lac and Tet, but occurs much less frequently in higher eukaryotes. Two bacterial repressor/operator systems (Lac and Tet) have been used to control the activity of promoters transcribed by RNA polymerase II. Gatz and Quail (1988) taught the use of the Tn10-encoded Tet repressor/operator with a cauliflower mosaic virus 35S promoter in a transient plant expression system. Frohberg et al., (1991) and Gatz et al., (1991, 1992) characterised the effect of placing Tet operator sequences at different positions in a CaMV 35S promoter. U.S. Pat. No. 5,723,765 and International Patent Application, Publication No. WO 96/04393 disclosed use of the Tet repressor system for the inducible expression of the Cre recombinase in transgenic plants. Wilde et al., (1992) used the Lac repressor/operator system for the inducible expression from a chlorophyll a/b binding protein promoter in protoplasts of stably transformed plants.
2.3 Promoter-activating Systems
A second approach for the construction of a chemically inducible system is to use transcriptional activators from higher eukaryotes. The mammalian glucocorticoid receptor (GR), which activates eukaryotic expression only in the presence of steroids has been used by Picard et al., (1988) in
Schizosaccharomyces pombe
. Schena et al., (1991) have shown that transcription from a target promoter containing GR-binding sites was strictly dependent on the addition of steroids in transiently transformed tobacco cells. Lloyd et al., (1994) have used a fusion of the steroid receptor protein with the maize transcription factor R to complement an Arabidopsis mutant in a steroid inducible fashion. Aoyama and Chua (1997) disclosed use of a chimeric transcription factor consisting of the DNA-binding domain of the yeast transcription factor Ga14, the transactivating domain of the herpes viral protein Vp16 and the receptor domain of the rat glucocorticoid receptor to induce the expression of a reporter gene in transgenic plants through the application of steroids.
International Patent Application, Publication No. WO 96/27673 describes the use of a steroid receptor system in transgenic plants using chimeric GR receptors with Vp16 and C1 transcriptional activation domains and Ga14 DNA binding domains.
Another eukaryotic ligand-dependent activator is Ace1, a copper-dependent transcriptional activator from yeast. Mett et al., (1993) have shown that Ace1 regulates the expression of a suitable target promoter (CaMV 35S-90 bp promoter containing the Ace1 binding site) in transgenic plants. McKenzie et al., (1998) used a similar system (Ace1 binding sites with a CaMV 35S-40 bp promoter) to investigate copper-inducible activation of the ipt gene in transgenic tobacco.
AlcR is the specific activator of the
Aspergillus nidulans
ethanol-utilisation pathway, mediating the induction of its own transcription and that of the structural genes alcA and aldA. AlcR is a DNA binding protein that recognises specific binding sites in structural gene promoters (Kulmburg et al., 1992, Fillinger & Felenbok 1996). Felenbok (1991) used the AlcA-AlcR system for the expression of recombinant proteins in Aspergilli. The ethanol inducible gene switch was used by Caddick et al., (1998) to manipulate carbon metabolism in transgenic plants and also by Salter et al (1998) to examine the induction of a chloramphenicol acetyltransferase (CAT) reporter construct by ethanol. This system has also been used in International Patent Application, Publication No. WO 93/21334 for the inducible activation of a chimeric alcA/CaMV 35S promoter in transgenic plants.
2.4 Fusion Proteins
A third strategy is based on the construction of fusion proteins between transcriptional transactivation domains and bacterial repressor proteins such as the Lac and the Tet repressor. Weinmann et al (1994) used a tetracycline controlled transactivator (the vir

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