DNA constructs and plants incorporating them

Details DNA constructs and plants incorporating them DNA constructs and plants incorporating them

1998-03-17

2003-08-12

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C800S288000, C800S298000, C800S317000, C800S317300, C800S320000, C800S320100, C435S412000, C435S414000, C435S419000, C435S430000, C435S468000

Reexamination Certificate

active

06605754

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to DNA constructs and plants incorporating them. In particular, it relates to expression cassettes and promoter sequences for the expression of genes in plants.
2. Description of Related Art
Gene expression is controlled by regions upstream (5′) of the protein encoding region, commonly referred to as the “promoter”. A promoter may be constitutive, tissue-specific, developmentally-programmed or inducible.
Manipulation of crop plants to improve characteristics (such as productivity or quality) requires the expression of foreign or endogenous genes in plant tissues. Such genetic manipulation therefore relies on the availability of means to control gene expression as required; for example, on the availability and use of suitable promoters which are effective in plants. It is advantageous to have the choice of a variety of different promoters so that the most suitable promoter may be selected for a particular gene, construct, cell, tissue, plant or environment. A range of promoters are known to be operative in plants.
Within the promoter region there are several domains which are necessary for full function of the promoter. The first of these domains lies immediately upstream of the structural gene and forms the “core promoter region” containing consensus sequences, normally 70 base pairs immediately upstream of the gene. The core promoter region contains the characteristic CAAT and TATA boxes plus surrounding sequences, and represents a transcription initiation sequence which defines the transcription start point for the structural gene. The precise length of the core promoter region is indefinite but it is usually well-recognisable. Such a region is normally present, with some variation, in all promoters. The base sequences lying between the various well-characterised “boxes” appear not to be of great importance.
The presence of the core promoter region defines a sequence as being a promoter: if the region is absent, the promoter is non-functional. Furthermore, the core promoter region is insufficient to provide full promoter activity. A series of regulatory sequences upstream of the core constitute the remainder of the promoter. The regulatory sequences determine expression level, the spatial and temporal pattern of expression and, for an important subset of promoters, expression under inductive conditions.
SUMMARY OF THE INVENTION
Several naturally-occurring promoters and associated gene expression systems are known. The best characterised regulatory systems are those of bacteria in which the specific interactions between DNA-binding proteins (repressors) and the target DNA sequences (operators) results in the negative repression of gene activity.
The alcA/alcR gene activation system from the fungus
Aspergillus nidulans
is also well characterised. The ethanol utilization pathway in
A nidulans
is responsible for the degradation of alcohols and aldehydes. Three genes have been shown to be involved in the ethanol utilization pathway. Genes alcA and alcR have been shown to lie close together on linkage group VII and aldA maps to linkage group VIII (Pateman J H et al, 1984, Proc. Soc. Lond, B217:243-264; Sealy-Lewis H M and Lockington R A, 1984, Curr. Genet, 8:253-259). Gene alcA encodes ADHI in
A nidulans
and aldA encodes AldDH, the second enzyme responsible for ethanol utilization. The expression of both alcA and aldA are induced by ethanol and a number of other inducers (Creaser E H et al, 1984, Biochemical J, 255:449-454) via the transcription activator alcR. The alcR gene and a co-inducer are responsible for the expression of alcA and aldA since a number of mutations and deletions in alcR result in the pleiotropic loss of ADHI and aldDH (Felenbok B et al, 1988, Gene, 73:385-396; Pateman et al, 1984; Sealy-Lewis & Lockington, 1984). The ALCR protein activates expression from alcA by binding to three specific sites in the alcA promoter (Kulmberg P et al, 1992, J. Biol. Chem, 267:21146-21153).
The alcR gene was cloned (Lockington R A et al, 1985, Gene, 33:137-149) and sequenced (Felenbok et al, 1988). The expression of the alcR gene is inducible, autoregulated and subject to glucose repression mediated by the CREA repressor (Bailey C and Arst H N, 1975, Eur. J. Biochem, 51:573-577; Lockington R A et al, 1987, Mol. Microbiology, 1:275-281; Dowzer C E A and Kelly J M, 1989, Curr. Genet, 15:457-459; Dowzer C E A and Kelly J M, 1991, Mol. Cell. Biol, 11:5701-5709). The ALCR regulatory protein contains 6 cysteines near its N terminus coordinated in a zinc binuclear cluster (Kulmberg P et al, 1991, FEBS Letts, 280:11-16). This cluster is related to highly conserved DNA binding domains found in transcription factors of other ascomycetes. Transcription factors GAL4 and LAC9 have been shown to have binuclear complexes which have a cloverleaf type structure containing two Zn(II) atoms (Pan T and Coleman J E, 1990, Biochemistry, 29:3023-3029; Halvorsen Y D C et al, 1990, J. Biol. Chem, 265:13283-13289). The structure of ALCR is similar to this type except for the presence of an asymmetrical loop of 16 residues between Cys-3 and Cys-4. ALCR positively activates expression of itself by binding to two specific sites in its promoter region (Kulmberg P et al, 1992, Molec. Cell. Biol, 12:1932-1939).
The regulation of the three genes, alcR, alcA and aldA, involved in the ethanol utilization pathway is at the level of transcription (Lockington et al, 1987; Gwynne D et al, 1987, Gene, 51:205-216; Pickett et al, 1987, Gene, 51:217-226).
There are two other alcohol dehydrogenases present in
A nidulans
. ADHII is present in mycelia grown in non-induced media and is repressible by the presence of ethanol. ADHII is encoded by alcB and is also under the control of alcR (Sealy-Lewis & Lockington, 1984). A third alcohol dehydrogenase has also been cloned by complementation with a adh-strain of
S cerevisiae
. This gene alcC, maps to linkage group VII but is unlinked to alcA and alcR. The gene, alcC, encodes ADHIII and utilizes ethanol extremely weakly (McKnight G L et al, 1985, EMBO J, 4:2094-2099). ADHIII has been shown to be involved in the survival of
A nidulans
during periods of anaerobic stress. The expression of alcC is not repressed by the presence of glucose, suggesting that it may not be under the control of alcR (Roland L J and Stromer J N, 1986, Mol. Cell. Biol, 6:3368-3372).
In summary,
A nidulans
expresses the enzyme alcohol dehydrogenase I (ADH1) encoded by the gene alcA only when it is grown in the presence of various alcohols and ketones. The induction is relayed through a regulator protein encoded by the alcR gene and constitutively expressed. In the presence of inducer (alcohol or ketone), the regulator protein activates the expression of the alcA gene. The regulator protein also stimulates expression of itself in the presence of inducer. This means that high levels of the ADH1enzyme are produced under inducing conditions (ie when alcohol or ketone are present). Conversely, the alcA gene and its product, ADH1, are not expressed in the absence of inducer. Expression of alcA and production of the enzyme is also repressed in the presence of glucose.
Thus the alcA gene promoter is an inducible promoter, activated by the alcR regulator protein in the presence of inducer (ie by the protein/alcohol or protein/ketone combination). The alcR and alcA genes (including the respective promoters) have been cloned and sequenced (Lockington R A et al, 1985, Gene, 33:137-149; Felenbok B et al, 1988, Gene, 73:385-396; Gwynne et al, 1987, Gene, 51:205-216).
Alcohol dehydrogenase (adh) genes have been investigated in certain plant species. In maize and other cereals they are switched on by anaerobic conditions. The promoter region of adh genes from maize contains a 300 bp regulatory element necessary for expression under anaerobic conditions. However, no equivalent to the alcR regulator protein has been found in any plant. Hence the alcR/alcA type of gene regulator system is not known in plants. Constitutive e

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