Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters fat – fatty oil – ester-type wax – or...
Patent
1995-07-11
1999-03-09
McElwain, Elizabeth F.
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters fat, fatty oil, ester-type wax, or...
800250, 536 236, 435 691, 4351721, 4351723, 435410, 435418, 435419, A01H 500, C12N 1582, C12N 500
Patent
active
058803297
ABSTRACT:
Disclosed are PAP mutants having reduced phytotoxicity compared to wild-type PAP, and which retain wild-type PAP biological activity in plants. One group of PAP mutants is characterized by at least one amino acid substitution in the N-terminus of mature PAP, such as the Glycine 75 residue or the Glutamic acid 97 residue. Another group of preferred PAP mutants is characterized by mutations such as truncations in the C-terminal region of mature PAP. PAP mutants having from at least about 26 to about 76 mature PAP amino acids (not counting the 29-amino acid C-terminal extension of wild-type PAP) exhibit reduced phytotoxicity and retain PAP biological activity in plants. The disclosed PAP mutants may include the 22-amino acid N-terminal signal sequence and/or the C-terminal extension of wild-type PAP.
Also disclosed are DNA molecules encoding the PAP mutants. The DNAs can be operably linked to a promoter functional in given host cells such as plants, and stably transformed into a vector functional in said cells. Procaryotic or eucaryotic hosts, e.g., yeast or plants, stably transformed with a mutant PAP-encoding DNA are further disclosed, as well as protoplasts stably transformed with the DNAs. Transgenic plants and seed containing the DNAs are also provided. The transgenic plants exhibit broad spectrum virus resistance. They include monocots, such as cereal crops, and dicot plants.
Further disclosed is a method for identifying a PAP mutant having reduced phytotoxicity and which retains PAP biological activity. The method involves the steps of providing a eucaryotic cell stably transformed with a mutagenized PAP-encoding DNA molecule, wherein the DNA molecule is operably linked to an inducible promoter functional in eucaryotic cells. The thus-transformed cell is cultured in a suitable medium, and after a predetermined time, an inducer is added to the medium to cause expression of the DNA molecule. A determination is then made as to whether any cultured cells survive the induction of expression of the DNA molecule. The presence of which indicates the presence of a PAP mutant having reduced phytotoxicity so that the biological activity of the PAP mutant encoded by the mutagenized DNA can then be determined. Any PAP mutants which also exhibit broad spectrum virus resistance in an in vivo or in vitro assay would be considered as PAP mutants which retain PAP biological activity in plants. Isolated and purified PAP mutants identified by the aforesaid process are also provided.
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McElwain Elizabeth F.
Rutgers The State University
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