Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-05-01
2001-01-23
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300, C536S024310, C536S024320
Reexamination Certificate
active
06177245
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to the manipulation of the enzymatic activity responsible for the conversion of protoporphyrinogen IX to protoporphyrin IX in a biosynthetic pathway common to all eukaryotic organisms. In one aspect, the invention is applied to the development of herbicide resistance in plants, plant tissues and seeds. In another aspect, the invention is applied to the development of diagnostics and treatments for deficiencies in this enzymatic activity in animals, particularly humans.
BACKGROUND OF THE INVENTION
I. The Protox Enzyme and its Involvement in the Chlorophyll/Heme Biosynthetic Pathway
The biosynthetic pathways which leads to the production of chlorophyll and heme share a number of common steps. Chlorophyll is a light harvesting pigment present in all green photosynthetic organisms. Heme is a cofactor of hemoglobin, cytochromes, P450 mixed-function oxygenases, peroxidases, and catalases (see, e.g. Lehninger,
Biochemistry
. Worth Publishers, New York (1975)), and is therefore a necessary component for all aerobic organisms.
The last common step in chlorophyll and heme biosynthesis is the oxidation of protoporphyrinogen IX to protoporphyrin IX. Protoporphyrinogen oxidase (referred to herein as “protox”) is the enzyme which catalyzes this last oxidation step (Matringe et al.,
Biochem. J
. 260: 231 (1989)).
The protox enzyme has been purified either partially or completely from a number of organisms including the yeast
Saccharomyces cerevisiae
(Labbe-Bois and Labbe, In
Biosynthesis of Heme and Chlorophyll
, E. H. Dailey, ed. McGraw Hill: New York, pp. 235-285 (1990)), barley etioplasts (Jacobs and Jacobs,
Biochem. J
. 244: 219 (1987)), and mouse liver (Dailey and Karr,
Biochem
. 26: 2697 (1987)). Genes encoding protox have been isolated from two prokaryotic organisms,
Escherichia coli
(Sasarrnan et al.,
Can. J. Microbiol
. 39: 1155 (1993)) and
Bacillus subtilis
(Dailey et aL,
J. Biol. Chem
. 269: 813 (1994)). These genes share no sequence similarity; neither do their predicted protein products share any amino acid sequence identity. The
E. coli
protein is approximately 21 kDa, and associates with the cell membrane. The
B. subtilis
protein is 51 kDa, and is a soluble, cytoplasmic activity.
Presently, too little is known about the protox enzyme to allow isolation of protox encoding genes from higher eukaryotic organisms (i.e. animals, plants and all other multicellular nucleate organisms other than lower eukaryotic microorganisms such as yeast, unicellular algae, protozoans, etc.) using known approaches.
In particular, many of the standard techniques for isolation of new proteins and genes are based upon the assumption that they will be significantly similar in primary structure (i.e. amino acid and DNA sequence) to known proteins and genes that have the same function. Such standard techniques include nucleic acid hybridization and amplification by polymerase chain reaction using oligonucleotide primers corresponding to conserved amino acid sequence motifs. These techniques would not be expected to be useful for isolation of eukaryotic protox genes using present structural information which is limited to prokaryotic protox genes since there is no significant structural similarity even among the known prokaryotic protox genes and proteins.
Another approach that has been used to isolate biosynthetic genes in other metabolic pathways from higher eukaryotes is the complementation of microbial mutants deficient in the activity of interest. For this approach, a library of cDNAs from the higher eukaryote is cloned in a vector that can direct expression of the cDNA in the microbial host. The vector is then transformed or otherwise introduced into the mutant microbe, and colonies are selected that are phenotypically no longer mutant.
This strategy has worked for isolating genes from higher eukaryotes that are involved in several metabolic pathways, including histidine biosynthesis (e.g. U.S. patent application Ser. No. 08/061,644 to Ward et al., incorporated by reference herein in its entirety), lysine biosynthesis (e.g. Frisch et al.,
Mol. Gen. Genet
. 228: 287 (1991)), purine biosynthesis (e.g. Aimi et al.,
J. Biol. Chem
. 265: 9011 (1990)), and tryptophan biosynthesis (e.g. Niyogi et al.,
Plant Cell
5: 1011 (1993)). However, despite the availability of microbial mutants thought to be defective in protox activity (e.g.
E. coli
(Sasannan et al.,
J. Gen. Microbiol
. 113: 297 (1979)),
Salmonella typhimurium
(Xu et al.,
J. Bacteriol
. 174: 3953 (1992)), and
Saccharomyces cerevisiae
(Camadro et al,.
Biochem. Biophys. Res. Comm
. 106: 724 (1982)), application of this technique to isolate cDNAs encoding eukaryotic protox enzymatic activity is at best unpredictable based on the available information.
There are several reasons for this. First, the eukaryotic protox cDNA sequence may not be expressed at adequate levels in the mutant microbe, for instance because of codon usage inconsistent with the usage preferences of the microbial host. Second, the primary translation product from the cloned eukaryotic coding sequence may not produce a functional polypeptide, for instance if activity requires a post-translational modification, such as glycosylation, that is not carried out by the microbe. Third, the eukaryotic protein may fail to assume its active conformation in the microbial host, for instance if the protein is normally targeted to a specific organellar membrane system that the microbial host specifically lacks. This last possibility is especially likely for the plant protox enzyme, which is associated in the plant cell with organelles not present in microbial hosts used in the complementation assay. In particular, the plant protox enzyme is associated with both the chloroplast envelope and thylakoid membranes (Matringe et al.,
J. Biol. Chem
. 267:4646 (1992)), and presumably reaches those membrane systems as a result of a post-translational targeting mechanism involving both an N-terminal transit sequence, and intrinsic properties of the mature polypeptide (see, e.g. Kohorn and Tobin,
Plant Cell
1: 159 (1989); Li et al.,
Plant Cell
3: 709 (1991); Li et al.,
J. Biol. Chem
. 267: 18999 (1992)).
II. Involvement of the Protox Gene in Animal/Human Disease Conditions
The protox enzyme is known to play a role in certain human disease conditions. Patients suffering from variegate porphyria, an autosomal dominant disorder characterized by both neuropsychiatric symptoms and skin lesions, have decreased levels of protox activity (Brenner and Bloomer,
New Engl. J. Med
. 302: 765 (1980)). Due to the lack of knowledge regarding the human protox enzyme and its corresponding gene, options for diagnosing and treating this disorder are presently very limited.
III. The Protox Gene as a Herbicide Target
The use of herbicides to control undesirable vegetation such as weeds or plants in crops has become almost a universal practice. The relevant market exceeds a billion dollars annually. Despite this extensive use, weed control remains a significant and costly problem for farmers.
Effective use of herbicides requires sound management. For instance, time and method of application and stage of weed plant development are critical to getting good weed control with herbicides. Since various weed species are resistant to herbicides, the production of effective herbicides becomes increasingly important.
Unfortunately, herbicides that exhibit greater potency, broader weed spectrum and more rapid degradation in soil can also have greater crop phytotoxicity. One solution applied to this problem has been to develop crops which are resistant or tolerant to herbicides. Crop hybrids or varieties resistant to the herbicides allow for the use of the herbicides without attendant risk of damage to the crop. Development of resistance can allow application of a herbicide to a crop where its use was previously precluded or limited (e.g. to pre-emergence use) due to sensitivity of the crop to the herbicide. For example, U.S. Pat. No. 4,761,373 to Ander
Volrath Sandra
Ward Eric R.
Einsmanh Juliet C.
Fredman Jeffrey
Lebel Edouard G.
Meigs J. Timothy
Novartis Finance Corporation
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