Sequences from auxin-induced gene products targeting fusion...

Details Sequences from auxin-induced gene products targeting fusion... Sequences from auxin-induced gene products targeting fusion...

1998-04-22

2001-04-24

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C536S024100, C536S023600, C536S023720, C536S023400, C800S290000, C800S295000, C800S298000, C800S288000, C435S069100, C435S468000, C435S410000, C435S419000, C435S069700, C435S069800

Reexamination Certificate

active

06222095

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to plant genetic engineering. In particular, it relates to new methods for targeting desired recombinantly expressed polypeptides for degradation in vivo.
BACKGROUND OF THE INVENTION
The cultivation of plants has been a major focus of human activity for millennia. The development of techniques for introducing new genes into plants has now added a new dimension to this activity. Genetic engineering permits the introduction into plants of a desirable traits, such as pest or disease resistance, improved taste, improved storage characteristics, or improved ripening characteristics. Efforts are even underway to engineer plants which can act as factories to produce bulk chemicals, human therapeutics, and other commercially valuable products.
While techniques to introduce heterologous genes into plants and to obtain their expression are well known, they do not as yet permit as much control over expression as might be desired. Typically, to ensure the introduced gene is expressed, it is put under the control of a strong promoter. This may be acceptable or even desirable if the goal is merely the accumulation of high levels of the gene product. In some cases, however, the gene product may be toxic to the plant at high levels. In other instances, it may be desirable that the gene only be expressed at certain times during the plant's development, and not linger. It would therefore be desirable to have additional ways to modulate levels of gene product expression in plants.
A number of peptide regions of some short-lived proteins of organisms other than plants have been shown to operate as transferable, dominant degradation signals. Mitotic cycling contain a conserved nine amino acid sequence called the “destruction box” which is necessary for their cell cycle specific degradation (Glazer et al., 1991). A 54 amino acid peptide from sea urchin cyclic B containing the destruction box is sufficient for targeting a reporter protein for cell cycle-specific proteolysis (Glotzer et al., 1991). Two different peptide regions which were sufficient for targeting &bgr;-galactosidase for rapid degradation were identified in the yeast transcriptional repressor, MAT&agr;2 (Hochstrasser and Varshavsky, 1990). One consists of the N-terminal 67 amino acid residues and targets proteins containing it for degradation via the ubiquitin pathway (Chen et al., 1993). The N-terminal transmembrane domain of mammalian 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-R) and of one of the yeast homologs (HMG-R-2) is necessary for its feedback regulated degradation and sufficient for targeting a reporter protein for feedback regulated degradation (Chun et al., 1990; Hampton and Rine, 1994). In response to serum deprivation, the motif KFERQ (SEQ ID NO:6) and similar peptides operate as degradation signals by targeting proteins for import into mammalian lysosomes (Dice, 1990). However, in the case of the yeast transcription factor GCN4, no small region was found to be sufficient for targeting a fusion protein for degradation suggesting that there may be a tertiary structural component to the degradation signal(s) contained within GCN4 (Kornitzer et al., 1994).
In plants a number of gene families referred here to as primary auxin response genes are known to be expressed early in plant tissues in response to the plant hormone auxin. The primary response genes are activated without a requirement for de novo protein synthesis and can be divided into 5 families based on amino acid identity and auxin induction kinetics: Aux/IAA, SAR—small auxin-unregulated, GH3-like, amino cyclopropane-1-carboxylic acid synthase (ACS), and glutathione-S-transferase (GH2/4-like) (see, Abel et al., Bases 18:647-654 (1996) and Abel et al.
Plant Physiol.
111:9-17 (1996) for reviews of these plant gene families).
None of these protein sequences is known to work with respect to sequences expressed in plants. Thus, the identification of transferable peptide signals that specifically target proteins in plant cells for degradation would be desirable. The present invention provides these and other advantages.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules comprising a polynucleotide sequence encoding a primary auxin response protein degradation signal linked to a heterologous eukaryotic polynucleotide sequence encoding a target polypeptide. Typically, the primary auxin response protein is an Aux/IAA protein, for example from
Pisum sativum.
A preferred Aux/IAA protein degradation signal is from PSIAA6 (SEQ ID NO:5) or PSIAA4. Fragments of the full length polypeptides can also be used.
In some embodiments, the Aux/IAA protein degradation signal includes a heterologous nuclear localization signal, for example from squash leaf curl virus BR1 protein. The polynucleotide encoding the Aux/IAA protein degradation signal can be positioned either 3′ or 5′ to the polynucleotide encoding the target polypeptide. In many embodiments, it is positioned 5′.
The invention also provides transgenic plants comprising an expression cassette comprising a polynucleotides of the invention. The particular plant used is not critical to the invention.
The invention further provides methods of targeting a recombinantly expressed target polypeptide in a plant for degradation. The method involve introducing into the plant a heterologous expression cassette comprising a polynucleotide sequence encoding an IAA protein degradation signal linked to a heterologous polynucleotide sequence encoding the target polypeptide, whereby a fusion protein comprising the IAA protein degradation signal linked to the target polypeptide is expressed in the plant. The recombinant expression cassette can be introduced into the plant using genetic engineering techniques or by traditional breeding methods.


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