Gene expression

Details Gene expression Gene expression

1998-08-13

2001-07-03

Benzion, Gary (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C435S069100, C435S320100, C435S419000, C435S468000, C536S024100, C800S288000, C800S294000, C800S298000

Reexamination Certificate

active

06255558

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a nucleic acid sequence optimised for expression in a plant cell, and to vectors and plant cells comprising the nucleic acid sequence.
BACKGROUND OF THE INVENTION
In yeast there is a gene called GAL4, the product of which acts as a transcriptional activator (Johnston 1987 Microbiol. Rev. 51, 458-476). The GAL4 gene is quite large (3 Kb) and the encoded polypeptide comprises an N-terminal DNA-binding domain, a C terminal domain having a transcriptional activator function, and an intervening glucose-responsive element “GRE” (GAL4 being repressed in the presence of glucose).
The GAL4 protein has been quite well characterised. Amino acid residues 1-147 of the protein bind to DNA in a sequence-specific manner (Keegan et al 1986 Science 231, 699-704). The transcriptional activation function is associated with two short portions of the C terminal domain (Ma & Ptashne 1987 Cell 48, 847-853). The DNA sequence to which GAL4 binds has been identified as a 17 mer, which must be present as a repeat for optimal GAL4 binding (Giniger et al, 1985 Cell 40, 767-774), and may be referred to as a GAL4-responsive “upstream activation sequence” (UAS): GAL4 binds to the UAS 5′ of a gene by means of the DNA-binding domain, and the C-terminal domain causes up-regulation of transcription of the gene.
Recently, a two-element system has been developed for directing gene expression in
Drosophila melanogaster
. Brand and Perrimon (1993 Development 118: 401-415,) randomly inserted the gene encoding the GAL4 into the Drosophila genome using a P-element based vector.
A large number of stable Drosophila lines were generated which each express GAL4 in a particular pattern, dependent on adjacent genomic DNA sequences. A chosen target gene could then be cloned under the control of GAL4 upstream activation sequences (UAS); separately transformed, and maintained silently in the absence of GAL4. Genetic crossing of this single line with any of the library of GAL4-containing lines allowed activation of the target gene in many different tissue and cell types, and the phenotypic consequences of mis-expression, including those lethal to the organism, could be conveniently studied. In addition, the library of GAL4-containing flies has become an increasingly characterised and shared resource, and which provides a common entry point for various “reverse” genetic techniques.
A similar system operable in plants would be highly desirable. However, expression of heterologous eukaryotic genes in plants has proved highly problematical in the past (see, for example, Green Fluorescent Protein) and efficient expression of GAL4 appears equally difficult, and it has been suggested that this is due to inefficient translation of GAL4 mRNA in plants (Reichel et al, 1995 Plant Cell Reports 14, 773-776). Ma et al, (1988 Nature 334, 631-633) were able to obtain transient expression of modified, functional GAL4 derivatives in tobacco-leaf protoplasts but could not demonstrate the presence of functional, full-length wild-type GAL4. Similarly, transient expression of GAL4 derivatives has been demonstrated in maize protoplasts (McCarty et al, 1991 Cell 66, 895-905) and when introduced by biolistic methods, in maize aleurone tissues or embryogenic calli (Goff et al, 1991 Genes & Dev. 5, 298-309; Goff et al, 1992 Genes & Dev. 6, 864-875). Hitherto however, there have been no reports of stable, efficient expression of functional GAL4 or derivaties thereof in a plant cell.
It is an aim of the present invention to provide a novel expression system operable in plants, utilising a modified portion of the GAL4 gene.
SUMMARY OF THE INVENTION
In a first aspect the invention provides a nucleic acid sequence, expressible in a plant cell, encoding at least an effective portion of a GAL4 DNA-binding domain, the sequence having an A/T base content substantially reduced relative to the wild-type yeast sequence.
The A/T content of the wild-type yeast sequence encoding the DNA-binding domain of GAL4 is about 59%. The % A/T base content of the sequence of the invention encoding the effective portion of the GAL4 DNA-binding domain will be understood to be substantially reduced when it is less than 50%. Preferably the A/T content is less than 45%, and more preferably less than 40%. The sequence of the invention may be made, for example, by site-directed mutagenesis, or be made de novo by chemical synthesis.
An “effective portion” of the DNA-binding domain is a portion sufficient to retain most (i.e. over 50%) of the DNA-binding activity of the full length DNA-binding domain. Typically the “effective portion” will comprise at least two thirds of the full length sequence of the DNA-binding domain. Conveniently the nucleic acid sequence will encode substantially all of the GAL4 DNA-binding domain (i.e. amino acid residues 1-147 of the yeast polypeptide), although a substantially smaller portion (about 75 amino acid residue) is quite adequate to retain most of the DNA-binding activity (Kraulis et al, 1992 Nature 356, 448-450; and Marmorstein et al, 1992 Nature 356, 408-414). In one particular embodiment, the sequence will comprise the nucleotide sequence shown in the 5′ portion (nucleotides 1 to about 460) of
FIG. 1
, which sequence has a substantially reduced A/T content (38%), relative to the wild-type yeast sequence, yet encodes an identical amino acid sequence.
The GAL4 DNA-binding domain has no transcriptional activation function in its own right. Thus, in preferred embodiments, the sequence encoding the effective portion of the GAL4 DNA-binding domain will be operably linked to one or more other nucleic acid sequences, which sequences may be structural (i.e. encode functional polypeptides) and/or regulatory. Typically the sequence encoding the DNA-binding domain will be operably linked (e.g. fused in-frame) to a sequence encoding a peptide or polypeptide with a regulatory function, preferably a transcriptional activator. The transcriptional activator may be the activation domain of GAL4 protein, the sequence encoding which should preferably be optimised for expression in plants (e.g. by reducing the A/T content thereof). Alternatively, the transcriptional activator could be any one of a number of such proteins known to be active in plants, which will be well-known to those skilled in the art, such that the sequence of the invention encodes a chimeric polypeptide, comprising at least an effective portion of the GAL4 DNA-binding domain and a transcriptional activation domain. A particularly suitable transcriptional activator domain is that obtainable from herpes simplex virus (HSV) VP-16, (see Greaves and O'Hare 1989 J. Virol 63, 1641-1650; VP, being also known as VMW65). Other transcriptional activation domains include certain peptides encoded by
E. coli
genomic DNA fragments (Ma & Ptashne 1987 Cell 51, 113-119) or synthetic peptides designed to form amphiphilic &agr;-helix (Giniger & Ptashne 1987 Nature 330, 670-672). A common feature appears to be the requirement for excess charge, either positive (Gill & Ptashne 1987 Cell 51, 121-126) or, more especially for plant activation domains, excess negative charge (Estruch et al, 1994 Nucl. Acids Res. 22, 3983-3989). With this in mind, the person skilled in the art could readily synthesise, or find naturally-occurring sequences, which encode peptides or polypeptides with transcriptional activation activity in plants. Such activator sequences may, in any event, be modified for optimal activity in a plant cell.
In a further aspect the invention provides a nucleic acid construct, comprising the nucleic acid sequence defined above. In one particular embodiment, the nucleic acid construct could be used as an “enhancer-trap” to “fish” for plant enhancer sequences (Cf. Sundaresan et al, 1995 Genes & Dev. 9, 1797-1810). In such an embodiment, the construct will preferably include right and left Ti-DNA, to allow for random, stable insertion into the genome of a plant cell host. The construct will preferably comprise a naive plant promoter sequence, by which

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