Patent Grant
7666642
Nematodes (derived from the Greek word for thread) are active, flexible, elongate, organisms that live on moist surfaces or in liquid environments, including films of water within soil and moist tissues within other organisms. While only 20,000 species of nematode have been identified, it is estimated that 40,000 to 10 million actually exist. Some species of nematodes have evolved as very successful parasites of both plants and animals and are responsible for significant economic losses in agriculture and livestock and for morbidity and mortality in humans (Whitehead (1998) Plant Nematode Control. CAB International, New York).
Nematode parasites of plants can inhabit all parts of plants, including the roots, developing flower buds, leaves, and stems. Plant parasites are classified on the basis of their feeding habits into the broad categories: migratory ectoparasites, migratory endoparasites, and sedentary endoparasites. Sedentary endoparasites, which include the root knot nematodes (Meloidogyne) and cyst nematodes (Globodera and Heterodera) induce feeding sites and establish long-term infections within roots that are often very damaging to crops (Whitehead, supra). It is estimated that parasitic nematodes cost the horticulture and agriculture industries in excess of $78 billion worldwide a year, based on an estimated average 12% annual loss spread across all major crops. For example, it is estimated that nematodes cause soybean losses of approximately $3.2 billion annually worldwide (Barker et al. (1994) Plant and Soil Nematodes: Societal Impact and Focus for the Future. The Committee on National Needs and Priorities in Nematology. Cooperative State Research Service, US Department of Agriculture and Society of Nematologists). Several factors make the need for safe and effective nematode controls urgent. Continuing population growth, famines, and environmental degradation have heightened concern for the sustainability of agriculture, and new government regulations may prevent or severely restrict the use of many available agricultural anthelmintic agents.
The situation is particularly dire for high value crops such as strawberries and tomatoes where chemicals have been used extensively to control soil pests. The soil fumigant methyl bromide has been used effectively to reduce nematode infestations in a variety of these specialty crops. It is however regulated under the U.N. Montreal Protocol as an ozone-depleting substance and is scheduled for elimination in 2005 in the United States (Carter (2001) California Agriculture, 55(3):2). It is expected that strawberry and other commodity crop industries will be significantly impacted if a suitable replacement for methyl bromide is not found. Presently there are a very small array of chemicals available to control nematodes and they are frequently inadequate, unsuitable, or too costly for some crops or soils (Becker (1999) Agricultural Research Magazine 47(3):22-24; U.S. Pat. Nos. 6,048,714). The few available broad-spectrum nematicides such as Telone (a mixture of 1,3-dichloropropene and chloropicrin) have significant restrictions on their use because of toxicological concerns (Carter (2001) California Agriculture, Vol. 55(3):12-18).
Fatty acids are a class of natural compounds that have been investigated as alternatives to the toxic, non-specific organophosphate, carbamate and fumigant pesticides (Stadler et al. (1994) Planta Medica 60(2):128-132; U.S. Pat. Nos. 5,192,546; 5,346,698; 5,674,897; 5,698,592; 6,124,359). It has been suggested that fatty acids derive their pesticidal effects by adversely interfering with the nematode cuticle or hypodermis via a detergent (solubilization) effect, or through direct interaction of the fatty acids and the lipophilic regions of target plasma membranes (Davis et al. (1997) Journal of Nematology 29(4S):677-684). In view of this general mode of action it is not surprising that fatty acids are used in a variety of pesticidal applications including as herbicides (e.g., SCYTHE by Dow Agrosciences is the C9 saturated fatty acid pelargonic acid), as bactericides and fungicides (U.S. Pat. Nos. 4,771,571; 5,246,716) and as insecticides (e.g., SAFER INSECTICIDAL SOAP by Safer, Inc.).
The phytotoxicity of fatty acids has been a major constraint on their general use in agricultural applications (U.S. Pat. No. 5,093,124) and the mitigation of these undesirable effects while preserving pesticidal activity is a major area of research. The esterilication of fatty acids can significantly decrease their phytotoxicity (U.S. Pat. Nos. 5,674,897; 5,698,592; 6,124,359). Such modifications can however lead to dramatic loss of nematicidal activity as is seen for linoleic, linolenic and oleic acid (Stadler et al. (1994) Planta Medica 60(2): 128-132) and it may be impossible to completely decouple the phytotoxicity and nematicidal activity of pesticidal fatty acids because of their non-specific mode of action. Perhaps not surprisingly, the nematicidal fatty acid pelargonic acid methyl ester (U.S. Pat. Nos. 5,674,897; 5,698,592; 6,124,359) shows a relatively small “therapeutic window” between the onset of pesticidal activity and the observation of significant phytotoxicity (Davis et al. (1997) J Nematol 29(4S):677-684). This is the expected result if both the phytotoxicity and the nematicidial activity derive from the non-specific disruption of plasma membrane integrity. Similarly the rapid onset of pesticidal activity seen with many nematicidal fatty acids at therapeutic concentrations (U.S. Pat. Nos. 5,674,897; 5,698,592; 6,124,359) suggests a non-specific mechanism of action, possibly related to the disruption of membranes, action potentials and neuronal activity.
Ricinoleic acid, the major component of castor oil, provides another example of the unexpected effects esterification can have on fatty acid activity. Ricinoleic acid has been shown to have an inhibitory effect on water and electrolyte absorption using everted hamster jejunal and ileal segments (Gaginella et al. (1975) J Pharmacol Exp Ther 195(2):355-61) and to be cytotoxic to isolated intestinal epithelial cells (Gaginella et al. (1977) J Pharmacol Exp Ther 201(1):259-66). These features are likely the source of the laxative properties of castor oil, which is given as a purgative in humans and livestock, e.g., as a deworming agent. In contrast, the methyl ester of ricinoleic acid is ineffective at suppressing water absorption in the hamster model (Gaginella et al. (1975) J Pharmacol Exp Ther 195(2):355-61).
The macrocyclic lactones (e.g., avermectins and milbemycins) and delta-toxins from Bacillus thuringiensis (Bt) are chemicals that in principle provide excellent specificity and efficacy and should allow environmentally safe control of plant parasitic nematodes. Unfortunately, in practice, these two approaches have proven less effective for agricultural applications against root pathogens. Although certain avermectins show exquisite activity against plant parasitic nematodes these chemicals are hampered by poor bioavailability due to their light sensitivity, degradation by soil microorganisms and tight binding to soil particles (Lasota & Dybas (1990) Acta Leiden 59(1-2):217-225; Wright & Perry (1998) Musculature and Neurobiology. In: The Physiology and Biochemistry of Free-Living and Plant-parasitic Nematodes (eds R. N. Perry & D. J. Wright), CAB International 1998). Consequently, despite years of research and extensive use against animal parasitic nematodes, mites and insects (plant and animal applications), macrocyclic lactones (e.g., avermectins and milbemycins) have never been commercially developed to control plant parasitic nematodes in the soil.
Bt delta-toxins must be ingested to affect their target organ, the brush border of midgut epithelial cells (Marroquin et al. (2000) Genetics. 155(4): 1693-1699). Consequently they are not anticipated to be effective against the dispersal, non-feeding, juvenile stages of plant parasitic nematodes in the field. These juvenile stages only commence feeding when a susceptible host has been infected. Thus, Bt delta-toxins nematicides may need to penetrate the cuticle in order to be effective. In addition, soil mobility of a relatively large 65-130 kDa protein—the size of typical Bt delta-toxins—is expected to be poor and delivery in planta is likely to be constrained by the exclusion of large particles by the feeding tube of certain plant parasitic nematodes such as Heterodera (Atkinson et al. (1998) Engineering resistance to plant-parasitic nematodes. In: The Physiology and Biochemistry of Free-Living and Plant-parasitic Nematodes (eds R. N. Perry & D. J. Wright), CAB International 1998).
Many plant species are known to be highly resistant to nematodes. The best documented of these include marigolds (Tageles spp.), rattlebox (Crotalaria spectabilis), chrysanthemums (Chrysanthemum spp.), castor bean (Ricinus communis), margosa (Azardiracta indica), and many members of the family Asteraceae (family Compositae) (Hackney & Dickerson. (1975) J Nematol 7(1):84-90). The active principle(s) for this nematicidal activity has not been discovered in all of these examples and no plant-derived products are sold commercially for control of nematodes. In the case of the Asteraceae, the photodynamic compound alpha-terthienyl has been shown to account for the strong nematicidal activity of the roots. Castor beans are plowed under as a green manure before a seed crop is set. However, a significant drawback of the castor plant is that the seed contains toxic compounds (such as ricin) that can kill humans, pets, and livestock and is also highly allergenic.
There remains an urgent need to develop environmentally safe, target-specific ways of controlling plant parasitic nematodes. In the specialty crop markets, economic hardship resulting from nematode infestation is highest in strawberries, bananas, and other high value vegetables and fruits. In the high-acreage crop markets, nematode damage is greatest in soybeans and cotton. There are however, dozens of additional crops that suffer from nematode infestation including potato, pepper, onion, citrus, coffee, sugarcane, greenhouse ornamentals and golf course turf grasses.
Nematode parasites of vertebrates (e.g., humans, livestock and companion animals) include gut roundworms, hookworms, pinworms, whipworms, and filarial worms. They can be transmitted in a variety of ways, including by water contamination, skin penetration, biting insects, or by ingestion of contaminated food.
In domesticated animals, nematode control or “de-worming” is essential to the economic viability of livestock producers and is a necessary part of veterinary care of companion animals. Parasitic nematodes cause mortality in animals (e.g., heartworm in dogs and cats) and morbidity as a result of the parasites' inhibiting the ability of the infected animal to absorb nutrients. Parasite-induced nutrient deficiency results in diseased livestock and companion animals (i.e., pets), as well as in stunted growth. For instance, in cattle and dairy herds, a single untreated infection with the brown stomach worm can permanently stunt an animal's ability to effectively convert feed into muscle mass or milk.
Two factors contribute to the need for novel anthelmintics and vaccines for control of parasitic nematodes of animals. First, some of the more prevalent species of parasitic nematodes of livestock are building resistance to the anthelmintic drugs available currently, meaning that these products will eventually lose their efficacy. These developments are not surprising because few effective anthelmintic drugs are available and most have been used continuously. Presently a number of parasitic species has developed resistance to most of the anthelmintics (Geents et al. (1997) Parasitology Today 13:149-151; Prichard (1994) Veterinary Parasitology 54:259-268). The fact that many of the anthelmintic drugs have similar modes of action complicates matters, as the loss of sensitivity of the parasite to one drug is often accompanied by side resistance—that is, resistance to other drugs in the same class (Sangster & Gill (1999) Parasitology Today Vol. 15(4): 141-146). Secondly, there are some issues with toxicity for the major compounds currently available.
Human infections by nematodes result in significant mortality and morbidity, especially in tropical regions of Africa, Asia, and the Americas. The World Health Organization estimates 2.9 billion people are infected with parasitic nematodes. While mortality is rare in proportion to total infections (180,000 deaths annually), morbidity is tremendous and rivals tuberculosis and malaria in disability adjusted life year measurements. Examples of human parasitic nematodes include hookworm, filarial worms, and pinworms. Hookworm is the major cause of anemia in millions of children, resulting in growth retardation and impaired cognitive development. Filarial worm species invade the lymphatics, resulting in permanently swollen and deformed limbs (elephantiasis) and invade the eyes causing African Riverblindness. Ascaris lumbricoides, the large gut roundworm infects more than one billion people worldwide and causes malnutrition and obstructive bowl disease. In developed countries, pinworms are common and often transmitted through children in daycare.
Even in asymptomatic parasitic infections, nematodes can still deprive the host of valuable nutrients and increase the ability of other organisms to establish secondary infections. In some cases, infections can cause debilitating illnesses and can result in anemia, diarrhea, dehydration, loss of appetite, or death.
While public health measures have nearly eliminated one tropical nematode (the water-borne Guinea worm), cases of other worm infections have actually increased in recent decades. In these cases, drug intervention provided through foreign donations or purchased by those who can afford it remains the major means of control. Because of the high rates of reinfection after drug therapy, vaccines remain the best hope for worm control in humans. There are currently no vaccines available.
Until safe and effective vaccines are discovered to prevent parasitic nematode infections, anthelmintic drugs will continue to be used to control and treat nematode parasitic infections in both humans and domestic animals. Finding effective compounds against parasitic nematodes has been complicated by the fact that the parasites have not been amenable to culturing in the laboratory. Parasitic nematodes are often obligate parasites (i.e., they can only survive in their respective hosts, such as in plants, animals, and/or humans) with slow generation times. Thus, they are difficult to grow under artificial conditions, making genetic and molecular experimentation difficult or impossible. To circumvent these limitations, scientists have used Caenorhabidits elegans as a model system for parasitic nematode discovery efforts.
C. elegans is a small free-living bacteriovorous nematode that for many years has served as an important model system for multicellular animals (Burglin (1998) Int. J. Parasitol., 28(3): 395-411). The genome of C. elegans has been completely sequenced and the nematode shares many general developmental and basic cellular processes with vertebrates (Ruvkin et al. (1998) Science 282: 2033-41). This, together with its short generation time and ease of culturing, has made it a model system of choice for higher eukaryotes (Aboobaker et al. (2000) Ann. Med. 32: 23-30).
Although C. elegans serves as a good model system for vertebrates, it is an even better model for study of parasitic nematodes, as C. elegans and other nematodes share unique biological processes not found in vertebrates. For example, unlike vertebrates, nematodes produce and use chitin, have gap junctions comprised of innexin rather than connexin and contain glutamate-gated chloride channels rather than glycine-gated chloride channels (Bargmann (1998) Science 282: 2028-33). The latter property is of particular relevance given that the avermectin class of drugs is thought to act at glutamate-gated chloride receptors and is highly selective for invertebrates (Martin (1997) Vet. J. 154:11-34).
A subset of the genes involved in nematode specific processes will be conserved in nematodes and absent or significantly diverged from homologues in other phyla. In other words, it is expected that at least some of the genes associated with functions unique to nematodes will have restricted phylogenetic distributions. The completion of the C. elegans genome project and the growing database of expressed sequence tags (ESTs) from numerous nematodes facilitate identification of these “nematode specific” genes. In addition, conserved genes involved in nematode-specific processes are expected to retain the same or very similar functions in different nematodes. This functional equivalence has been demonstrated in some cases by transforming C. elegans with homologous genes from other nematodes (Kwa et al. (1995) J. Mol. Biol. 246:500-10; Redmond et al. (2001) Mol. Biochem. Parasitol. 112:125-131). This sort of data transfer has been shown in cross phyla comparisons for conserved genes and is expected to be more robust among species within a phylum. Consequently, C. elegans and other free-living nematode species are likely excellent surrogates for parasitic nematodes with respect to conserved nematode processes.
Many expressed genes in C. elegans and certain genes in other free-living nematodes can be “knocked out” genetically by a process referred to as RNA interference (RNAi), a technique that provides a powerful experimental tool for the study of gene function in nematodes (Fire et al. (1998) Nature 391(6669):806-811; Montgomery et al. (1998) Proc. Natl. Acad Sci USA 95(26):15502-15507). Treatment of a nematode with double-stranded RNA of a selected gene can destroy expressed sequences corresponding to the selected gene thus reducing expression of the corresponding protein. By preventing the translation of specific proteins, their functional significance and essentiality to the nematode can be assessed. Determination of essential genes and their corresponding proteins using C. elegans as a model system will assist in the rational design of anti-parasitic nematode control products.
The invention features nucleic acid molecules encoding Meloidogyne incognita, Heterodera glycines, Diroilaria immitis, Strongyloides stercoralis and Rhabditella axei fatty acid desaturases and other nematode fatty acid desaturase-like proteins. M. incognita is a Root Knot Nematode that causes substantial damage to several crops, including cotton, tobacco, pepper, and tomato. H. glycines, referred to as Soybean Cyst Nematode, is a major pest of soybean. D. immitis (dog heartworm) and S. stercoralis (human threadworm) are mammalian parasites. R. axei is a free-living nematode that serves as a model for parasitic nematodes. In part, the fatty acid desaturase-like nucleic acids and polypeptides of the invention allow for the identification of a nematode species, and for the identification of compounds that bind to or alter the activity of fatty acid desaturase-like polypeptides. Such compounds may provide a means for combating diseases and infestations caused by nematodes, particularly those caused by M. incognita (e.g., in tobacco, cotton, pepper, or tomato plants), H. glycines (e.g., in soybeans), D. immitis (e.g., in dogs) and S. stercoralis (e.g., in humans).
The invention is based, in part, on the identification of cDNAs encoding M. incognita fatty acid desaturases (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3). These 1194, 1242 and 1260 nucleotide cDNAs have 1191, 1239 and 1161 nucleotide open reading frames encoding 397, 413 and 387 amino acid polypeptides (SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10) respectively.
The invention is also based, in part, on the identification of a cDNA encoding H. glycines fatty acid desaturase (SEQ ID NO: 4). This 1488 nucleotide cDNA has a 1167 nucleotide open reading frame encoding a 389 amino acid polypeptide (SEQ ID NO: 11).
The invention is also based, in part, on the identification of a partial cDNA fragment encoding D. immitis fatty acid desaturase (SEQ ID NO: 5). This 1068 nucleotide cDNA has a 867 nucleotide open reading frame encoding a 289 amino acid polypeptide (SEQ ID NO: 12).
The invention is also based, in part, on the identification of a cDNA encoding S. stercoralis fatty acid desaturase (SEQ ID NO: 6). This 1221 nucleotide cDNA has a 1104 nucleotide open reading frame encoding a 368 amino acid polypeptide (SEQ ID NO: 13).
The invention is also based, in part, on the identification of a cDNA encoding R. axei fatty acid desaturase (SEQ ID NO: 7). This 1233 nucleotide cDNA has a 1122 nucleotide open reading frame encoding a 374 amino acid polypeptide (SEQ ID NO: 14).
In one aspect, the invention features novel nematode fatty acid desaturase-like polypeptides. Such polypeptides include purified polypeptides having the amino acid sequences set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 and 14. Also included are polypeptides having an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14. The purified polypeptides can be encoded by a nematode gene, e.g., a nematode gene other than a C. elegans gene. For example, the purified polypeptide has a sequence other than SEQ ID NO: 32 (C. elegans fatty acid desaturase). The purified polypeptides can further include a heterologous amino acid sequence, e.g., an amino-terminal or carboxy-terminal sequence.
Also featured are purified polypeptide fragments of the aforementioned fatty acid desaturase-like polypeptides, e.g., a fragment of at least about 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 325, 350, 375, 395, 400 amino acids. Non-limiting examples of such fragments include: fragments from about amino acid 1 to 50, 1 to 75, 1 to 92, 1 to 96, 1 to 100, 1 to 125, 1 to 399, 51 to 100, 92 to 150, 96 to 150, 92 to 400, 200 to 300, and 1 to 380 of SEQ ID NO: 8, 9, 10, 11, 12, 13 and 14. The polypeptide or fragment thereof can be modified, e.g., processed, truncated, modified (e.g. by glycosylation, phosphorylation, acetylation, myristylation, prenylation, palmitoylation, amidation, addition of glycerophosphatidyl inositol), or any combination of the above. These various polypeptide fragments can be used for a variety of purposes, including for the eliciting of antibodies directed against a fatty acid desaturase-like polypeptide.
In another aspect, the invention features novel isolated nucleic acid molecules encoding nematode fatty acid desaturase-like polypeptides. Such isolated nucleic acid molecules include nucleic acids comprising, consisting of or consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7. Also included are isolated nucleic acid molecules having the same sequence as or encoding the same polypeptide as a nematode fatty acid desaturase-like gene (other than C. elegans fatty acid desaturase-like genes).
Also featured are: 1) isolated nucleic acid molecules having a strand that hybridizes under low stringency conditions to a single stranded probe of the sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7 or their complements and, optionally, encodes polypeptides of between 375 and 425 amino acids and preferably have Δ12 fatty acid desaturase activity; 2) isolated nucleic acid molecules having a strand that hybridizes under high stringency conditions to a single stranded probe of the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7 or their complements and, optionally, encodes polypeptides of between 375 and 425 amino acids and preferably have Δ12 fatty acid desaturase activity; 3) isolated nucleic acid fragments of a fatty acid desaturase-like nucleic acid molecule, e.g., a fragment of SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7 that is about 230, 435, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1450 or more nucleotides in length or ranges between such lengths; and 4) oligonucleotides that are complementary to a fatty acid desaturase-like nucleic acid molecule or a fatty acid desaturase-like nucleic acid complement, e.g., an oligonucleotide of about 10, 15, 18, 20, 22, 24, 28, 30, 35, 40, 50, 60, 70, 80, or more nucleotides in length. Exemplary oligonucleotides are oligonucleotides which anneal to a site located between nucleotides about 1 to 24, 1 to 48, 1 to 60, 1 to 120, 24 to 48, 24 to 60, 49 to 60, 61 to 180, 145 to 165, 165 to 185, 1260 to 1280, 1281 to 1300, 1301 to 1320, 1321 to 1340, 1341 to 1360, 1361 to 1380, 1381 to 1400, 1401 to 1420, 1421 to 1456 of SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7. Such nucleic acid fragments are useful for detecting the presence of fatty acid desaturase-like mRNA. Nucleic acid fragments include the following non-limiting examples: nucleotides about 1 to 200, 100 to 300, 200 to 400, 300 to 500, 400 to 600, 500 to 700, 600 to 800, 700 to 900, 800 to 1000, 900 to 1100, 1000 to 1200, 1100 to 1300, 1200 to 1400, 1300 to 1456 of SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7. Also within the invention are nucleic acid molecules that hybridize under stringent conditions to nucleic acid molecules comprising SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7 and comprise 3,000, 2,000, 1,000 or fewer nucleotides and preferably encode a polypeptide having Δ12 fatty acid desaturase activity and/or have a sequence corresponding to that of a naturally occurring nematode. The isolated nucleic acid can further include a heterologous promoter operably linked to the fatty acid desaturase-like nucleic acid molecule.
A molecule featured herein can be from a nematode of the class Araeolaimida, Ascaridida, Chromadorida, Desmodorida, Diplogasterida, Monhysterida, Mononchida, Oxyurida, Rhigonematida, Spirurida, Enoplia, Desmoscolecidae, Rhabditida, or Tylenchida. Alternatively, the molecule can be from a species of the class Rhabditida, particularly a species other than C. elegans.
In another aspect, the invention features a vector, e.g., a vector containing an aforementioned nucleic acid. The vector can further include one or more regulatory elements, e.g., a heterologous promoter. The regulatory elements can be operably linked to the fatty acid desaturase-like nucleic acid molecules in order to express a fatty acid desaturase-like nucleic acid molecule. In yet another aspect, the invention features a transgenic cell or transgenic organism having in its genome a transgene containing an aforementioned fatty acid desaturase-like nucleic acid molecule and a heterologous nucleic acid, e.g., a heterologous promoter.
In still another aspect, the invention features an antibody, e.g., an antibody, antibody fragment, or derivative thereof that binds specifically to an aforementioned polypeptide. Such antibodies can be polyclonal or monoclonal antibodies. The antibodies can be modified, e.g., humanized, rearranged as a single-chain, or CDR-grafted. The antibodies may be directed against a fragment, a peptide, or a discontinuous epitope from a fatty acid desaturase-like polypeptide.
In another aspect, the invention features a method of screening for a compound that binds to a nematode fatty acid desaturase-like polypeptide, e.g., an aforementioned polypeptide. The method includes providing the nematode polypeptide; contacting a test compound to the polypeptide; and detecting binding of the test compound to the nematode polypeptide. In one embodiment, the method further includes contacting the test compound to a mammalian or plant fatty acid desaturase-like polypeptide; and detecting binding of the test compound to the mammalian or plant fatty acid desaturase-like polypeptide. A test compound that binds the nematode fatty acid desaturase-like polypeptide with at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold affinity greater relative to its affinity for the mammalian (e.g., a human) or plant fatty acid desaturase-like polypeptide can be identified.
Alternatively, the compounds can bind to the mammalian or plant fatty acid desaturase with affinity similar to that of the nematode fatty acid desaturase, but not be significantly detrimental to a plant and/or animal.
The invention also features methods for identifying compounds that alter the activity of a nematode fatty acid desaturase-like polypeptide. The method includes contacting the test compound to the nematode fatty acid desaturase-like polypeptide and detecting a fatty acid desaturase-like activity. A decrease in the level of fatty acid desaturase-like activity of the polypeptide relative to the level of fatty acid desaturase-like activity of the polypeptide in the absence of the test compound is an indication that the test compound is an inhibitor of the fatty acid desaturase-like activity. In still another embodiment, the method further includes contacting a nematode fatty acid desaturase polypeptide with a test compound such as an allosteric inhibitor, a substrate analog, other analogs, or other types of inhibitors that prevent binding of the fatty acid desaturase-like polypeptide to other molecules or proteins (“fatty acid desaturase-like polypeptide binding partners”). A change in activity or fatty acid desaturase-like polypeptide binding of proteins normally bound by the fatty acid desaturase is an indication that the test compound is an inhibitor of the fatty acid desaturase-like activity or is an inhibitor of the interaction of the fatty acid desaturase-like polypeptide with one of its binding partners. Such inhibitory compounds are potentially selective agents for reducing the viability, growth, development or reproduction of a nematode expressing a fatty acid desaturase-like polypeptide, e.g., M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei. These methods can also include contacting the test compound with a plant or mammalian (e.g., as human) fatty acid desaturase-like polypeptide; and detecting a fatty acid desaturase-like activity of the plant or mammalian fatty acid desaturase-like polypeptide. A compound that decreases nematode fatty acid desaturase activity to a greater extent than it decreases a plant or mammalian fatty acid desaturase-like polypeptide activity is a candidate selective inhibitor of nematode viability, growth or reproduction. A desirable compound can exhibit 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or greater selective activity against the nematode polypeptide. Any suitable assay can be used to measure fatty acid desaturase activity, including that of Banas et al. (Physiology, Biochemistry and Molecular Biology of Plant Lipids, Williams et al., eds., Kluwer Academic, Dordrecht, Netherlands, 1996, pages 57-59).
Another featured method is a method of screening for a compound that alters an activity of a fatty acid desaturase-like polypeptide or alters binding or regulation of other polypeptides by fatty acid desaturase. Thus, the activity of the fatty acid desaturase can be measured directly by monitoring the substrate or product of the fatty acid desaturase or indirectly by measuring the activity (e.g., monitoring the substrate or product) of an enzyme that acts downstream of fatty acid desaturase. Thus, the methods of the invention include providing a fatty acid desaturase polypeptide; contacting a test compound to the polypeptide; and detecting a fatty acid desaturase-like activity of the polypeptide or the activity of polypeptides bound or regulated by the fatty acid desaturase, wherein a change in activity of the fatty acid desaturase-like polypeptide or other downstream polypeptides relative to the fatty acid desaturase-like activity of the polypeptide or downstream polypeptides in the absence of the test compound is an indication that the test compound alters the activity of the polypeptide(s). Similarly, the method includes providing the polypeptide; contacting a test compound to the polypeptide; and detecting a fatty acid desaturase-like activity or the resulting unsaturated fatty acid products, wherein a change in activity of fatty acid desaturase-like polypeptides or the resulting unsaturated fatty acid products relative to the fatty acid desaturase-like activity of the polypeptide or the level of fatty acid products in the absence of the test compound is an indication that the test compound alters the activity of the polypeptide(s).
The methods of the invention can include contacting the test compound to a plant or mammalian (e.g., a human) fatty acid desaturase-like polypeptide and measuring the fatty acid desaturase-like activity of the plant or mammalian fatty acid desaturase-like polypeptide or other polypeptides affected or regulated by the fatty acid desaturase or the resulting unsaturated fatty acid products. A test compound that alters the activity of the nematode fatty acid desaturase-like polypeptide (or the level of fatty acid products) at a given concentration and that does not substantially alter the activity of the plant or mammalian fatty acid desaturase-like polypeptide, downstream polypeptides, or level of fatty acid products at the same given concentration can be identified. Thus, the methods of the invention can be used to identify candidate compounds that are relatively selective for one or more nematode fatty acid desaturase-like polypeptides relative to one or more mammalian and or plant fatty acid desaturase-like polypeptides. An additional method includes screening for both binding to a fatty acid desaturase-like polypeptide and for an alteration in the activity of a fatty acid desaturase-like polypeptide.
The methods of the invention include the identification of compounds that inhibit both nematode and plant fatty acid desaturase-like polypeptides. Such compounds can be useful for treatment of prevention of nematode infection of plants because plants are often not significantly impaired by inhibition of the activity of a fatty acid desaturase-like polypeptide. Moreover, such inhibitors can be administered to a mammal for treatment or prevention of infection by a nematode.
Yet another featured method is a method of screening for a compound that alters the viability or fitness of a transgenic cell or organism (e.g., a nematode). The transgenic cell or organism has a transgene that expresses a fatty acid desaturase-like polypeptide. The method includes contacting a test compound to the transgenic cell or organism and detecting changes in the viability or fitness of the transgenic cell or organism. This alteration in viability or fitness can be measured relative to an otherwise identical cell or organism that does not harbor the transgene.
The invention also features compounds that are relatively selective inhibitors of one or more nematode fatty acid desaturase-like polypeptides relative to one or more plant or animal fatty acid desaturase-like polypeptides. The compounds can have a Ki for a nematode fatty acid desaturase that is 10-fold, 100-fold, 1,000-fold or more lower than for a plant or animal fatty acid desaturase-like polypeptides, e.g., a host plant or host animal of the nematode. The invention further features relatively non-selective inhibitors as well as completely non-selective inhibitors.
Also featured is a method of screening for a compound that alters the expression of a nematode nucleic acid encoding a fatty acid desaturase-like polypeptide or nucleic acid encoding a nematode fatty acid desaturase-like polypeptide, e.g., a nucleic acid encoding a M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase-like polypeptide. The method includes contacting a cell, e.g., a nematode cell, with a test compound and detecting expression of a nematode nucleic acid encoding a fatty acid desaturase-like polypeptide, e.g., by hybridization to a probe complementary to the nematode mRNA encoding an fatty acid desaturase-like polypeptide or by contacting polypeptides isolated from the cell with a compound, e.g., antibody that binds a fatty acid desaturase-like polypeptide.
In yet another aspect, the invention features a method of treating a disorder (e.g., an infection) caused by a nematode, e.g., M. incognita, H. glycines, D. immitis or S. stercoralis, in a subject, e.g., a host plant or host animal. The method includes administering to the subject an effective amount of an inhibitor of a fatty acid desaturase-like polypeptide activity or an inhibitor of expression of a fatty acid desaturase-like polypeptide. Non-limiting examples of such inhibitors include: an antisense nucleic acid (or PNA) to a fatty acid desaturase-like nucleic acid, an antibody to a fatty acid desaturase-like polypeptide, an analog of a natural substrate of a fatty acid desaturase, a fatty acid, or a small molecule identified as a fatty acid desaturase-like polypeptide inhibitor, e.g., an inhibitor identified by a method described herein.
In another aspect, methods for desaturating fatty acids to Δ12 fatty acids are provided. Such methods can include the steps of: (a) providing a cell that harbors a fatty acid desaturase polypeptide; and (b) growing the cell under conditions in which the fatty acid desaturase polypeptide desaturates a fatty acid to produce a corresponding Δ12 unsaturated fatty acid.
In still another aspect, methods of inhibiting nematode (e.g., M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei) Δ12 fatty acid desaturase(s) are provided. Such methods can include the steps of: (a) providing a nematode that expresses a Δ12 fatty acid desaturase-like enzyme; (b) contacting the nematode with fatty acid analogs or other compounds that inhibit the enzyme. Also provided are methods of rescuing the effect of the inhibitor. Such methods comprise the steps of: (a) inhibiting the enzyme and (b) providing Δ12 unsaturated fatty acids exogenously.
In another aspect, methods of reducing the viability or fecundity or slowing the growth or development or inhibiting the infectivity of a nematode using a fatty acid analog or inhibitor of a fatty acid desaturase are provided. Such methods comprise the steps of (a) providing a nematode that expresses a Δ12 fatty acid desaturase; (b) contacting the nematode with specific inhibitory fatty acid analogs or inhibitors of a Δ12 fatty acid desaturase; (c) reducing the viability or fecundity of the nematode. Also provided are methods of rescuing the effect of the fatty acid desaturase inhibitors or other inhibitors. Such methods can involve contacting the nematode with Δ12 unsaturated fatty acids exogenously.
In another aspect, methods of inhibiting a Δ12 fatty acid desaturase using RNA interference methods are provided. Such methods comprise the steps of (a) providing a nematode that contains a Δ12 fatty acid desaturase like gene; (b) contacting the nematode with double stranded RNA (dsRNA). Such methods can be used to reduce viability or fecundity, to slow growth or development, or to inhibit infectivity. In another aspect, methods of rescuing the effect of RNA interference by supplying specific Δ12 unsaturated fatty acids are provided.
The methods of the invention include a method for identifying an inhibitor of a fatty acid desaturase-like polypeptide, the method comprising: (a) providing a cell expressing a fatty acid desaturase-like polypeptide; (b) contacting the cell with a test compound; (c) measuring the fatty acid desaturase-like polypeptide activity of the cell, wherein a change in fatty acid desaturase-like polypeptide activity of the cell relative to the fatty acid desaturase-like polypeptide activity of the cell in the absence of the test compound is an indication that the test compound alters the activity of the fatty acid desaturase-like polypeptide.
The methods of the invention further include a method for identifying an inhibitor of a fatty acid desaturase-like polypeptide, the method comprising: (a) providing a nematode expressing a fatty acid desaturase-like polypeptide; (b) contacting the nematode with a test compound; (c) measuring the fatty acid desaturase-like polypeptide activity of the nematode, wherein a change in fatty acid desaturase-like polypeptide activity of the nematode relative to the fatty acid desaturase-like polypeptide activity of the nematode in the absence of the test compound is an indication that the test compound alters the activity of the fatty acid desaturase-like polypeptide.
Another method for identifying an inhibitor of a fatty acid desaturase-like polypeptide comprises: (a) providing a cell expressing a fatty acid desaturase-like polypeptide; (b) contacting the cell with a test compound; (c) measuring the viability of the cell in the presence of the test compound; and (d) comparing the viability of the cell in the presence of the test compound to the viability of the cell in the presence of the test compound and a product of the fatty acid desaturase-like polypeptide, wherein greater viability in the presence of the test compound and the product compared to viability in the presence of the test compound is an indication that the test compound alters the activity of the fatty acid desaturase-like polypeptide. The invention features a method for identifying an inhibitor of a fatty acid desaturase-like polypeptide, the method comprising: (a) providing a nematode expressing a fatty acid desaturase-like polypeptide; (b) contacting the nematode with a test compound; (c) measuring the viability or the fecundity of the nematode in the presence of the test compound; and (d) comparing the viability or fecundity of the nematode in the presence of the test compound to the viability or fecundity of the nematode in the presence of the test compound and a product of the fatty acid desaturase-like polypeptide, wherein greater viability or fecundity in the presence of the test compound and the product compared to viability or fecundity in the presence of the test compound is an indication that the test compound alters the activity of the fatty acid desaturase-like polypeptide. In preferred embodiments the product is linoleic acid.
The invention also features inhibitors identified by the screening methods disclosed herein.
The invention features a method for reducing the viability, growth, or fecundity of a nematode, the method comprising exposing the nematode to an agent that inhibits the activity of a fatty acid desaturase-like polypeptide (e.g., a Δ12 fatty acid desaturase) and a method for protecting a plant from a nematode infection, the method comprising applying to the plant or to seeds of the plant an inhibitor of a nematode fatty acid desaturase-like polypeptide. The invention also features a method for protecting a mammal from a nematode infection, the method comprising administering to the mammal an inhibitor of a nematode fatty acid desaturase-like polypeptide (e.g., a Δ12 fatty acid desaturase). In preferred embodiments the inhibitor does not significantly inhibit the activity of a fatty acid desaturase-like polypeptide expressed by the plant or at least does not do so to the extent that the growth of the plant is impaired.
A “purified polypeptide”, as used herein, refers to a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide can constitute at least 10, 20, 50 70, 80 or 95% by dry weight of the purified preparation.
An “isolated nucleic acid” is a nucleic acid, the structure of which is not identical to that of any naturally occurring nucleic acid, or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule but is not flanked by both of the nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones in a DNA library such as a cDNA or genomic DNA library. Isolated nucleic acid molecules according to the present invention further include molecules produced synthetically, as well as any nucleic acids that have been altered chemically and/or that have modified backbones.
Although the phrase “nucleic acid molecule” primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” refers to the sequence of the nucleotides in the nucleic acid molecule, the two phrases can be used interchangeably.
The term “substantially pure” as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules. The substantially pure polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
The “percent identity” of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLASTN and BLASTX programs (version 2.0 and 2.1) of Altschul et al. (1990). J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs for the determination of percent identity of amino acid sequences or nucleotide sequences, the default parameters of the respective programs can be used. The programs are available on the Internet at: www.ncbi.nlm.nih.gov.
As used herein, the term “transgene” means a nucleic acid sequence (encoding, e.g., one or more subject polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic plant, animal, or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic plant, animal, or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the plant's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and other nucleic acid sequences, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
As used herein, the term “transgenic cell” refers to a cell containing a transgene.
As used herein, a “transgenic plant” is any plant in which one or more, or all, of the cells of the plant includes a transgene. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by T-DNA mediated transfer, electroporation, or protoplast transformation. The transgene may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
As used herein, the term “tissue-specific promoter” means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as a leaf, root, seed, or stem.
As used herein, the terms “hybridizes under stringent conditions” and “hybridizes under high stringency conditions” refer to conditions for hybridization in 6× sodium chloride/sodium citrate (SSC) buffer at about 45° C., followed by two washes in 0.2×SSC buffer, 0.1% SDS at 60° C. or 65° C. As used herein, the term “hybridizes under low stringency conditions” refers to conditions for hybridization in 6×SSC buffer at about 45° C., followed by two washes in 6×SSC buffer, 0.1% (w/v) SDS at 50° C.
A “heterologous promoter”, when operably linked to a nucleic acid sequence, refers to a promoter which is not naturally associated with the nucleic acid sequence.
As used herein, an agent with “anthelmintic activity” is an agent, which when tested, has measurable nematode-killing activity or results in reduced fertility or sterility in the nematodes such that fewer viable or no offspring result, or compromises the ability of the nematode to infect or reproduce in its host, or interferes with the growth or development of a nematode. In the assay, the agent is combined with nematodes, e.g., in a well of microtiter dish having agar media or in the soil containing the agent. Staged adult nematodes are placed on the media. The time of survival, viability of offspring, and/or the movement of the nematodes are measured. An agent with “anthelminthic activity” can, for example, reduce the survival time of adult nematodes relative to unexposed similarly staged adults, e.g., by about 20%, 40%, 60%, 80%, or more. In the alternative, an agent with “anthelminthic activity” may also cause the nematodes to cease replicating, regenerating, and/or producing viable progeny, e.g., by about 20%, 40%, 60%, 80%, or more.
As used herein, the term “binding” refers to the ability of a first compound and a second compound that are not covalently linked to physically interact. The apparent dissociation constant for a binding event can be 1 mM or less, for example, 10 nM, 1 nM, 0.1 nM or less.
As used herein, the term “binds specifically” refers to the ability of an antibody to discriminate between a target ligand and a non-target ligand such that the antibody binds to the target ligand and not to the non-target ligand when simultaneously exposed to both the given ligand and non-target ligand, and when the target ligand and the non-target ligand are both present in molar excess over the antibody.
As used herein, the term “altering an activity” refers to a change in level, either an increase or a decrease in the activity, (e.g., an increase or decrease in the ability of the polypeptide to bind or regulate other polypeptides or molecules) particularly a fatty acid desaturase-like or fatty acid desaturase activity (e.g., the ability to introduce a double bond at the Δ12 position of a fatty acid). The change can be detected in a qualitative or quantitative observation. If a quantitative observation is made, and if a comprehensive analysis is performed over a plurality of observations, one skilled in the art can apply routine statistical analysis to identify modulations where a level is changed and where the statistical parameter, the p value, is, for example, less than 0.05.
In part, the nematode fatty acid desaturase proteins and nucleic acids described herein are novel targets for anti-nematode vaccines, pesticides, and drugs. Inhibition of these molecules can provide means of inhibiting nematode metabolism, growth, viability, fecundity, development, infectivity and/or the nematode life-cycle.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
Described below is the identification of M. incognita, H. glycines, D. immitis, S. stercoralis and R. axei fatty acid desaturase cDNAs (SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7) and the polypeptides they encode (SEQ ID NO: 8, 9, 10, 11, 12, 13 and 14). The fatty acid desaturases are Δ12 fatty acid desaturases. Also described below are experiments demonstrating that the fatty acid desaturase is essential for nematode viability. Also described below are inhibitors of the fatty acid desaturase. Certain sequence information for the fatty acid desaturase genes described herein is summarized in Table 1, below.
M. incognita
M. incognita
M. incognita
H. glycines
D. immitis
S. stercoralis
R. axei
Unsaturated fatty acids are essential to the proper functioning of biological membranes. At physiological temperatures, polar glycerolipids that contain only saturated fatty acids cannot form the liquid-crystalline bilayer that is the fundamental structure of biological membranes. The introduction of an appropriate number of double bonds (a process referred to as desaturation) into the fatty acids of membrane glycerolipids decreases the temperature of the transition from the gel to the liquid-crystalline phase and provides membranes with necessary fluidity. Fluidity of the membrane is important for maintaining the barrier properties of the lipid bilayer and for the activation and function of certain membrane bound enzymes. There is also evidence that unsaturation confers some protection to ethanol and oxidative stress, suggesting that the degree of unsaturation of membrane fatty acids has importance beyond temperature adaptation. Unsaturated fatty acids are also precursors of polyunsaturated acids (PUFAs) arachidonic and eicosapentaenoic acids in animals, which are important sources of prostaglandins. These molecules are local hormones that alter the activities of the cells in which they are synthesized and in adjoining cells, mediating processes in reproduction, immunity, neurophysiology, thermobiology, and ion and fluid transport.
The ability of cells to modulate the degree of unsaturation in their membranes is primarily determined by the action of fatty acid desaturases. Desaturase enzymes introduce unsaturated bonds at specific positions in their fatty acyl chain substrates, using molecular oxygen and reducing equivalents from NADH (or NADPH) to catalyze the insertion of double bonds. In many systems, the reaction uses a short electron transport chain consisting of NAD(P)H, cytochrome b5 reductase, and cytochrome b5, to shuttle electrons from NAD(P)H and the carbon-carbon single bond to oxygen, forming water and a double bond (C═C). Many cukaryotic dcsaturases are endoplasmic reticulum (ER) bound non-heme diiron-oxo proteins which contain three conserved histidine-rich motifs and two long stretches of hydrophobic residues. These hydrophobic alpha helical domains are thought to position the protein with its bulk exposed to the cytosolic face of the ER and to organize the active site histidines to appropriately coordinate the active diiron-oxo moiety.
While most eukaryotic organisms, including mammals, can introduce a double bond into an 18-carbon fatty acid at the Δ9 position, mammals are incapable of inserting double bonds at the Δ12 or Δ15 positions. For this reason, linoleate (18:2 Δ9,12) and linolenate (18:3 Δ9,12,15) must be obtained from the diet and; thus, are termed essential fatty acids. These dietary fatty acids come predominately from plant sources, since flowering plants readily desaturate the Δ12 and the Δ15 positions. Certain animals, including some insects and nematodes, can synthesize de novo all their component fatty acids including linolcate and linolenate (Watts and Browse (2002) Proc Natl Acad Sci USA, 99(9):5854-9; Borgeson et al. (1990) Biochim Biophys Acta. 1047(2):135-40; Cripps et al. (1990) Arch Biochem Biophys. 278(1):46-51). The nematode C. elegans, for example, can synthesize de novo a broad range of polyunsaturated fatty acids including arachidonic acid and eicosapentacnoic acids, a feature not shared by either mammals or flowering plants (Tanaka et al. (1999) Eur J. Biochem. 263(1):189-95).
The C. elegans desaturase gene fat-2 has been expressed in S. cerevisiae and shown to be a Δ12 fatty acid desaturase. This enzyme introduces a double bond between the 12th and the 13th carbons (from the carboxylate end) and can convert the mono-unsaturated oleate (18:1 Δ9) and palmitoleate (16:1 Δ9) to the di-unsaturated linoleate (18:2 Δ9,12) and 16:2 Δ9,12 fatty acids, respectively.
The nematode Δ12 enzymes are potentially good targets for anti-nematode compounds for several reasons. Firstly, the enzymes appear to be phylogenetically diverged from their homologs in plants, having less than 40% pairwise sequence identity at the amino acid level and phylogenetic analyses demonstrate clustering of nematode Δ12 and ω-3 desaturases away from homologs in plants. Experiments with both transgenic Arabidopsis and soybeans reveal that plants can tolerate significant reductions in Δ12 fatty acid desaturase activity, suggesting that inhibitors of desaturases would likely not be toxic to plants (Singh et al. (2000) Biochem. Society Trans. 28: 940-942; Lee et al. (1998) Science 280:915-918). In addition, as mentioned above, mammals are thought not to have A 12 fatty acid desaturases. Thus, inhibitors of the enzyme are likely to be non-toxic to mammals. Importantly, as detailed herein, a fatty acid desaturase of nematodes has been shown to be essential to their viability, both through inhibitor and RNA-mediated interference studies. Thus, Δ12 fatty acid desaturases could serve as ideal targets for anti-nematode control, as inhibitors of the enzyme could specifically target nematodes while leaving their animal and plant hosts unharmed.
Numerous analogs of fatty acids exist and some may act as specific inhibitors of enzymes such as desaturases that act on fatty acids, a fact that could be exploited for development of anti-nematode compounds. Sterculic acid, a cyclopropenoid fatty acid analog of oleic acid, is a potent inhibitor of Δ9 fatty acid desaturases (Schmid & Patterson (1998) Lipids 23(3):248-52; Waltermann & Steinbuchel (2000) FEMS Microbiol Lett. 190(1):45-50). It has also been speculated that cyclopropenoid analogs of linoleic acid may similarly inhibit Δ12 fatty acid desaturases (Dulayymi et al. (1997) Tetrahedron 53(3): 1099-1110). It is worth noting however that malvalate, a Δ8 cyclopropene fatty acid, seems to be equally inhibitory to 69 desaturases in some systems, (Schmid & Patterson (1998) Lipids 23(3):248-52), demonstrating how difficult it is to predict inhibitory profiles for some fatty acid analogs. Thia fatty acid analogs (i.e., sulfur containing fatty acids) are also potential inhibitors of fatty acid desaturases (Skrede et al. (1997) Biochim Biophys Acta 1344(2): 115-131; Hovik et al. (1997) Biochim Biophys Acta 1349(3):251-256) as are trans fatty acids (Choi et al. (2001) Biochem Biophys Res Commun 284(3):689-93). However, the specificity and pesticidal activity of these analogs is again difficult to predict (Beach et al. (1989) Mol Biochem Parasitol 35(1):57-66).
Certain fatty acids are also specific receptor antagonists (Yagaloff (1995) Prostaglandins Leukot Essent Fatty Acids 52(5):293-7).
Other analogs of linoleic acid that may also be specific Δ12 inhibitors include the epoxy fatty acid (vernolic acid), the acetylenic fatty acid (crepenynic acid), 12-oxo-9(Z)-octadecenoic acid methyl ester or the hydroxy fatty acids (ricinoleic and ricinelaidic acid). Inhibitors that interfere with Δ12 fatty acid desaturase activity are expected to be toxic to nematodes. Importantly, fatty acid analogs such as ricinoleic, ricinelaidic, vernolic and crepenynic acid methyl esters do not appear to be toxic (or are very much less toxic) to at least some plants and are predicted not to be toxic (or are very much less toxic) to at least some animals, including mammals. Such fatty acid analogs could potentially be used in the development of nematode control agents.
Although previously expressed in plants, fatty acid analogs such as crepcnynate, ricinoleate and vernolate acids were not thought to be specific inhibitors of the endogenous plant Δ12 desaturase (Broun & Somerville (1997) Plant. Physiol. 113:933-942; Singh et al. (2000) Biochem. Society Trans. 28(6): 940-942). Changes in the ratio of oleate to linoleate in plants expressing the genes for these analogs was instead attributed to a negative interaction between the enzymes involved (Singh et al. (2001) Planta 212: 872-879). Addition of ricinoleate exogenously to Nervospora crassa results in a significant decrease in oleate (C18:1) and an increase in linolenate (C18:3) again providing no indication that compounds like ricinoleate were in fact specific Δ12 desaturase inhibitors (Goodrich-Tanrikulu et al. (1996) Appl Microbiol Biotechnol. 46(4):382-7).
We made the surprising discovery that methyl esters of certain fatty acid analogs (e.g., ricinoleate, vernolate) are nematicidal and have activity consistent with that of specific inhibitors of nematode Δ12 desaturases. The fatty acid methyl esters show significantly enhanced activity over other eighteen carbon fatty acid esters such as oleate, elaidate and linolcate. In contrast to short chain seemingly non-specific pesticidal fatty acid esters such as laurate and pelargonate, the fatty acid analogs that are predicted Δ12 desaturase inhibitors show dramatically reduced phytoxicity and can therefore be used effectively while minimizing undesirable damage to non-target organisms.
Fatty acid-based analogs or other types of inhibitors may be supplied to plants exogenously, through sprays for example. It is also possible to provide inhibitors through a host organism or an organism on which the nematode feeds. For example, a host cell that does not naturally produce an inhibitor of the M. incognita, H. glycines, D. immitis and S. stercoralis fatty acid desaturase-like polypeptides can be transformed with enzymes capable of making inhibitory analogs and provided with appropriate precursor chemicals exogenously. Alternatively, the active inhibitors and precursors can be made endogenously by the expression of the appropriate enzymes. In addition, yeast or other organisms can be modified to produce inhibitors. Nematodes that feed on such organisms would then be exposed to the inhibitors.
In one embodiment, transgenic cells and/or organisms could be generated that produce enzymes active on fatty acids (e.g., desaturating, hydroxylating, conjugating, and/or epoxygenating enzymes). Such enzymes may be expressed, for example, in plants, vertebrates, and/or nematodes. These enzymes may produce fatty acids, analogs, or other inhibitors that can then act as specific inhibitors for other enzymes such as a fatty acid desaturase (e.g., a Δ12 epoxygenase from Crepis palaestina produces vernolic acid, a Δ12 desaturase inhibitor, in transgenic Arabidopsis) (Singh et. al. (2000) Biochem. Society Trans. 28:940-942; Lee et al. (1998) Science 280:915-918).
More generally, a recombinant expression vector capable of expressing an enzyme active on fatty acids could be transformed into a host cell of an organism that is parasitized by a parasitic nematode, (M. incognita, H. glycines. D. immitis or S. stercoralis, for example). Fatty acid analogs that act as inhibitors of (M. incognita, H. glycines, D. immitis or S. stercoralis Δ12 fatty acid desaturases, for example, can then be produced in the host cell or organism. In this manner, Δ12 fatty acid desaturases from feeding parasitic nematodes (e.g., M. incognita, H. glycines, D. immitis or S. stercoralis) could be rendered inactive by the fatty acid analog.
In another embodiment, a recombinant expression vector harboring a Δ12 fatty acid desaturase-like polypeptide from, for example, M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei, can be used to produce a recombinant fatty acid desaturase polypeptide that is functional in a cell, plant or animal, and that can desaturate fatty acids that are normally produced by the cell, plant or animal or that are provided exogenously to the cell, plant or animal to the corresponding Δ12 fatty acid. In this way, a cell, plant or animal can be produced that has a higher proportion of Δ12 unsaturated fatty acids than an otherwise similar cell, plant, or animal lacking the recombinant fatty acid desaturase polypeptide. In this way, a cell, plant or animal that has increased resistance to a Δ12 fatty acid desaturase inhibitor can be produced.
A recombinant Δ12 fatty acid desaturase, e.g., a M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase, may also be useful for producing lipids having a higher proportion of Δ12 unsaturated fatty acids, whether by means of recombinant expression in a cell or in an industrial process using purified nematode Δ12 fatty acid desaturase polypeptide. Such lipids are useful as food oils, as nutritional supplements, and as chemical feedstocks, for example.
The following examples are therefore to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All of the publications cited herein are hereby incorporated by reference in their entirety.
TBLASTN searches of a database of nematode EST sequences (McCarter et al. (1999) Washington University Nematode EST Project) with C. elegans Δ12 fatty acid desaturase (FAT-2) gene sequence (GenBank® Accession No: AAF63745: ID No: 7546993) identified two EST's (AW783527 and AW871151) that are predicted to encode at least a portion of a Δ12 Fatty Acid Desaturase-like enzymes (Δ12 FAT) in one nematode species, M. incognita.
Full Length Δ12 Fatty Acid Desaturase-like cDNA Sequences
The plasmid clone corresponding to the M. incognita EST sequence AW783527 was obtained from the Genome Sequencing Center (St. Louis, Mo.). This plasmid clone was designated Div249. The cDNA insert in the plasmid was sequenced in its entirety. Unless otherwise indicated, all nucleotide sequences determined herein were sequenced with an automated DNA sequencer (such as model 373 from Applied Biosystems, Inc.) using processes well known to those skilled in the art. Primers used for sequencing are listed in Table 1 (see below). Partial sequence data for the M. incognita Δ12 FAT was obtained from Div249, including nucleotide sequence for codons 90-397 and additional 3′ untranslated sequence. The clone lacked the first 89 codons of the M. incognita Δ12 FAT, as well as the 5′ untranslated region.
The following methods were used to obtain the full-length nematode Δ12 FAT gene and to determine its complete sequence. First, RNA was obtained from plant parasitic nematodes, which are maintained on greenhouse pot cultures depending on nematode preference. Root Knot Nematodes (Meloidogyne sp) were propagated on Rutgers tomato (Burpee). Total RNA was isolated using the TRIZOL reagent (Gibco BRL). Briefly, 2 ml of packed worms were combined with 8 ml TRIZOL reagent and solubilized by vortexing. Following 5 minutes of incubation at room temperature, the samples are divided into smaller volumes and spun at 14,000×g for 10 minutes at 4° C. to remove insoluble material. The liquid phase was extracted with 200 μl of chloroform, and the upper aqueous phase was removed to a fresh tube. The RNA was precipitated by the addition of 500 μl of isopropanol and centrifuged to pellet. The aqueous phase was carefully removed, and the pellet was washed in 75% ethanol and spun to re-collect the RNA pellet. The supernatant was carefully removed, and the pellet was air dried for 10 minutes. The RNA pellet was resuspended in 50 μl of DEPC-H20 and analyzed by spectrophotometry at 260 and 280 nm to determine yield and purity. Yields could be 1-4 mg of total RNA from 2 ml of packed worms.
To obtain the missing 5′ sequence of the M. incognita Δ12 FAT gene, the 5′ RACE technique was applied, and SL1 PCR was performed using first strand cDNA from M. incognita as a template. Briefly, SL1 PCR utilizes the observation, that unlike most eukaryotic mRNAs, many nematode mRNA molecules contain a common leader sequence (5′ ggg ttt aat tac cca agt ttg a 3′; SEQ ID NO: 17) transpliced to their 5′ ends. If this sequence is present on the 5′ end of a cDNA, that cDNA can be amplified using PCR with a primer that binds to the SL1 transpliced leader and a gene-specific primer near the 3′ end of the cDNA.
Briefly, following the instructions provided by Life Technologies cDNA synthesis kit, first strand cDNA synthesis was performed on total nematode RNA using SuperScript™ II Reverse Transcriptase and an oligo-dT primer (which anneals to the natural poly A tail found on the 3′ end of all eukaryotic mRNA). RNase H was then used to degrade the original mRNA template. Following degradation of the original mRNA template, the first strand cDNA was directly PCR amplified without further purification using Taq DNA polymerase, a gene specific primer (FAT10, SEQ ID NO: 19) designed from known sequence that anneals to a site located within the first strand cDNA molecule, and the SL1 primer, which is homologous the 5′ end of the cDNA of interest. Amplified PCR products were then cloned into a suitable vector for DNA sequence analysis. This procedure was performed to obtain clone Div864. This clone contained codons 1-120 in addition to 5′ untranslated sequences. Taken together, clones Div249 and Div864 contain sequences comprising the complete open reading frame of the Δ12 FAT gene from M. incognita.
To obtain the complete Δ12 fatty acid desaturase gene from M. incognita on one clone, primers FAT30 and FAT31 were designed to amplify the complete open reading frame. Following PCR amplification, several independent clones were obtained. DNA sequence analysis revealed that two very similar but distinct fatty acid desaturase genes had been identified. Along with the gene sequence reported for SEQ ID NO: 1 (1191 nucleotide ORF, 397 amino acid polypeptide,
In order to obtain the H. glycines Δ12 fatty acid desaturase gene, the 5′ RACE technique was applied, and SL1 PCR was performed using first strand cDNA from H. glycines as a template (cDNA synthesis explained above). The first strand cDNA was directly PCR amplified using the SL1 primer and a gene specific degenerate primer (FAT33, SEQ ID NO: 24) designed to anneal to region of strong homology shared across many nematode desaturase genes. Amplified PCR products were then cloned into a suitable vector for DNA sequence analysis. This procedure was performed to obtain clone Div1870. This clone contained codons 1-193 in addition to 5′ untranslated sequences. To obtain the 3′ sequence of the gene, the 3′ RACE technique was applied . The first strand cDNA was directly PCR amplified using a gene specific primer (FAT44, SEQ ID NO: 25) designed from known sequence that anneals within the first strand cDNA molecule of interest, and an oligo dT primer, which is homologous to the 3′ end of the cDNA of interest. This procedure was performed to generate clone Div2724, which contains codons 144-389 in addition to 3′ untranslated sequences. Taken together, clones Div1870 and Div2724 contain sequences comprising the complete open reading frame of the Δ12 fatty acid desaturase gene of H. glycines. The predicted Δ12 fatty acid desaturase polypeptide reported for SEQ ID NO: 11, (1167 nucleotide ORF, 389 amino acid polypeptide,
In an attempt to obtain the D. immitis Δ12 fatty acid desaturase gene, first strand cDNA, using D. immitis cDNA as a template (cDNA synthesis described above), was directly PCR amplified, using a gene-specific degenerate primer (FAT35, SEQ ID NO: 26) designed to anneal to a region of strong homology shared across many nematode desaturase genes, and another gene-specific degenerate primer (FT05, SEQ ID NO: 27), which was predicted to be homologous to a region near the 5′ end of the cDNA of interest. Amplified PCR products were then cloned into a suitable vector for DNA sequence analysis. This procedure was performed to obtain clone Div3228, which contains codons 1-224, which correspond to codons 78-301 of the C. elegans fatty acid desaturase (SEQ ID NO: 32). To acquire the missing 3′ sequence of D. immitis Δ12 fatty acid desaturase, the 3′ RACE technique was applied using a gene-specific primer (FAT 12, SEQ ID NO: 28) designed to a known sequence that anneals to a site located within the first strand cDNA molecule, and an oligo dT primer, which is homologous to the 3′ end of the cDNA of interest. Amplified PCR products were then cloned into a suitable DNA vector for sequence analysis. This procedure was performed to obtain the clone Div3230, which contained the codons 196-289, which correspond to codons 273-376 of the C. elegans fatty acid desaturase (SEQ ID NO: 32), in addition to 3′ untranslated sequences. Taken together, clones Div3228 and Div3230 comprise approximately 80% of the complete D. immitis Δ12 FAT open reading frame. The 5′ end sequence of this gene has yet to be completed. The partial Δ12 fatty acid desaturase polypeptide sequence reported for SEQ ID NO: 12 (867 nucleotide ORF, 289 amino acid polypeptide,
Plasmid clone, Div3013, corresponding to the S. stercoralis EST sequence (GenBank® Identification No: 9830288) was obtained from the Genome Sequencing Center (St. Louis, Mo.). The cDNA insert in the plasmid was sequenced in its entirety. Full sequence data for the S. stercoralis Δ12 fatty acid desaturase was obtained from Div3013, including nucleotide sequence for codons 1-368, (the full open reading frame) and additional 5′ and 3′ untranslated sequences. The predicted gene sequence reported for SEQ ID NO: 6 (1104 nucleotide ORF, 368 amino acid polypeptide,
In order to obtain the sequence of the R. axei Δ12 fatty acid desaturase gene, first strand cDNA from R. axei was directly PCR amplified, using a gene-specific degenerate primer (FAT35, SEQ ID NO: 26) designed to anneal to region of strong homology shared across many nematode desaturase genes, and another gene-specific degenerate primer (FAT34, SEQ ID NO: 29) designed to anneal to region of strong homology shared across many nematode desaturase genes near the 3′ end of the cDNA of interest. Amplified PCR products were then cloned into a suitable DNA vector for sequence analysis. This procedure was performed to obtain the clone Div1843, which contained the codons 185-301 (an internal fragment missing both the 5′ and 3′ ends of the gene). In order to obtain the missing 5′ sequence of the R. axei Δ12 fatty acid desaturase gene, the 5′ RACE technique was applied and SL1 PCR was performed using first strand cDNA from R. axei as a template (cDNA synthesis described above). The first strand cDNA was directly PCR amplified using a gene specific primer (FAT40, SEQ. ID. NO. 30) designed from the known sequence of clone Div 1843, that anneals to a site located within the cDNA of interest, and the SL primer, which is homologous to the 5′ end of many nematode cDNAs. Amplified PCR products were then cloned into a suitable vector for DNA sequence analysis. This procedure was performed to obtain the clone Div2026, which contained the codons 1-199, in addition to 5′ untranslated sequences. To obtain the missing 3′ sequence of the gene, the 3′ RACE technique was applied using a gene specific primer (FAT42, SEQ ID NO: 31) designed from the known sequence of clone Div1843, that anneals to a site located within the cDNA of interest, and an oligo dT primer, which is homologous to the 3′ end of the cDNA of interest. Amplified PCR products were then cloned into a suitable vector for DNA sequence analysis. This procedure was performed to obtain clone Div2149. This clone contained codons 246-374 in addition to 3′ untranslated sequences. Taken together, clones Div1843, Div2149, and Div2026 contain sequences comprising the complete open reading frame of the Δ12 FAT gene from R. axei. The predicted Δ12 fatty acid desaturase polypeptide gene sequence reported for SEQ ID NO: 7, (1122 nucleotide ORF, 374 amino acid polypeptide,
#codon numbering is based on SEQ ID NO:32
Characterization of M. incognita, H. glycines, D. immitis. S. stercoralis and R. axei Δ12 Fatty Acid Desaturase Genes.
The similarity between the Δ12 fatty acid desaturase proteins from M. incognita, H. glycines, D. immitis, S. stercoralis and R. axei from C. elegans is presented as a multiple alignment generated by the ClustalX multiple alignment program as described below (
The similarity between M. incognita, H. glycines, D. immitis, S. stercoralis and R. axei Δ12 fatty acid desaturase-like sequences and other sequences was also investigated by comparison to sequence databases using BLASTP analysis against nr (a non-redundant protein sequence database available on the Internet at www.ncbi.nlm.nih.gov) and TBLASTN analysis against dbest (an EST sequence database available on the Internet at www.ncbi.nlm.nih.gov; top 500 hits; E=1e-4). The “Expect (E) value” is the number of sequences that are predicted to align by chance to the query sequence with a score S or greater given the size of the database queried. This analysis was used to determine the potential number of plant and vertebrate homologs for each of the nematode fatty acid desaturase-like polypeptides described above. While M. incognita (SEQ ID NO: 1, 2 and SEQ ID NO:3), H. glycines (SEQ ID NO:4), D. immitis (SEQ ID NO:5), S. stercoralis (SEQ ID NO:6) and R. axei (SEQ ID NO:7) fatty acid desaturase-like cDNA sequences had numerous plant hits, they had no vertebrate hits in nr or dbest having sufficient sequence similarity to meet the threshold E value of 1e-4 (this E value approximately corresponds to a threshold for removing sequences having a sequence identity of less than about 25% over approximately 100 amino acids). Accordingly, the M. incognita, H. glycines, D. immitis, S. stercoralis and R. axei fatty acid desaturase-like enzymes of this invention do not appear to share significant sequence similarity with the more common vertebrate fatty acid desaturase enzymes such as the Δ6 fatty acid desaturase of Homo sapiens (a member of the FAD family of desaturases, GenBank Accession No. NP—068373), the Δ5 fatty acid desaturase of Homo sapiens (also a member of the FAD family, GenBank Accession No. NP—037534) or other mammalian fatty acid dcsaturases.
On the basis of the lack of similarity to vertebrates, the D. immitis and S. stercoralis fatty acid desaturase-like enzymes (e.g., Δ12 fatty acid desaturase) are useful targets of inhibitory compounds selective for some nematodes over their hosts (e.g., humans, animals). While at least some plants have fatty acid desaturases that are somewhat homologous to those in parasitic nematodes, including M. incognita and H. glycines, other criteria make them promising targets for the control of plant parasitic nematodes, including the fact that at least in some cases, plants can tolerate significant reductions in fatty acid desaturase activity while, as demonstrated below, nematodes can not.
Functional predictions were made using four iterations of PSI-BLAST with the default parameters on the nr database. PSI-BLAST searches and multiple alignment construction with CLUSTALX demonstrated that the C. elegans gene (GenBank® Accession No: AAF63745) was a member of the fatty acid desaturase family. Reciprocal blast searches and phylogenetic trees confirm that the nucleotide sequences in M. incognita, H. glycines, D. immitis, S. stercoralis and R. axei do encode orthologs of the C. elegans gene and therefore are also likely fatty acid desaturase proteins with the same activity. Protein localizations were predicted using the TargetP server available on the Internet at: www.cbs.dtu.dk/services/TargetP. The M. incognita fatty acid desaturase (SEQ ID NO: 8, 9 and SEQ ID NO:10), H. glycines (SEQ ID NO:11), D. immitis (SEQ ID NO:12), S. stercoralis (SEQ ID NO:13) and R. axei (SEQ ID NO: 14) polypeptides are likely to be cytoplasmic. In addition, they are predicted to have four transmembrane regions, consistent with the model that they are membrane bound.
RNA Mediated Interference (RNAi)
A double stranded RNA (dsRNA) molecule can be used to inactivate a Δ12 fatty acid desaturase (Δ12 FAT) gene in a cell by a process known as RNA mediated-interference (Fire ct al. (1998) Nature 391:806-811, and Gönezy et al. (2000) Nature 408:331-336). The dsRNA molecule can have the nucleotide sequence of a Δ12 FAT nucleic acid described herein or a fragment thereof. The dsRNA molecule can be delivered to nematodes via direct injection, or by soaking nematodes in aqueous solution containing concentrated dsRNA, or by raising bacteriovorous nematodes on E. coli genetically engineered to produce the dsRNA molecule.
RNAi by injection: To examine the effect of inhibiting Δ12 FAT activity, a Δ12 dsRNA was injected into the nematode, basically as described in Mello et al. (1991) EMBO J. 10:3959-3970. Briefly, a plasmid was constructed that contains a portion of the C. elegans Δ12 FAT gene sequence, specifically a fragment 651 nucleotides long, containing the entire first exon and terminating just before the conserved intron splice junction between the first exon and first intron. This construct encodes approximately the first 217 amino acids of the C. elegans Δ12 FAT gene. Primers were used to specifically amplify this sequence as a linear dsDNA. Single-stranded RNAs were transcribed from these fragments using T7 RNA polymerase and SP6 RNA polymerase (the RNAs correspond to the sense and antisense RNA strands). RNA was precipitated and resuspended in RNAse-free water. For annealing of ssRNAs to form dsRNAs, ssRNAs were combined, heated to 95° for two minutes then allowed to cool from 700 to room temperature over 1.5-2.5 hours.
DsRNA was injected into the body cavity of 15-20 young adult C. elegans hermaphrodites. Worms were typically immobilized on an agarose pad and injected with 2-5 μl of dsRNA at a concentration of 1 mg/ml. Injections were performed with visual observation using a Zeiss Axiovert compound microscope equipped with 10× and 40×DIC objectives, for example. Needles for microinjection were prepared using a Narishige needle puller, stage micromanipulator (Leitz) and a N2-powered injector (Narishige) set at 10-20 p.s.i. After injection, 200 μl of recovery buffer (0.1% salmon sperm DNA, 4% glucose, 2.4 mM KCl, 66 mM NaCl, 3 mM CaCl2, 3 mM HEPES, pH 7.2) were added to the agarose pad and the worms were allowed to recover on the agarose pad for 0.5-4 hours. After recovery, the worms were transferred to NGM agar plates seeded with a lawn of E. coli strain OP50 as a food source. The following day and for 3 successive days thereafter, 7 individual healthy injected worms were transferred to new NGM plates seeded with OP50. The number of eggs laid per worm per day and the number of those eggs that hatch and reach fertile adulthood were determined. As a control, Green Fluorescent Protein (GFP) dsRNA was produced and injected using similar methods. GFP is a commonly used reporter gene originally isolated from jellyfish and is widely used in both prokaryotic and eukaryotic systems. The GFP gene is not present in the wild-type C. elegans genome and, therefore, GFP dsRNA does not trigger an RNAi phenotype in wild-type C. elegans. The C. clegans Δ12 FAT RNAi injection phenotype presented as a strongly reduced F1 hatch-rate, with the few surviving individuals arrested in an early larval stage.
RNAi by feeding: C. elegans can be grown on lawns of E. coli genetically engineered to produce double stranded RNA (dsRNA) designed to inhibit Δ12 FAT expression. Briefly, E. coli were transformed with a genomic fragment of a portion of the C. elegans Δ12 FAT gene sequence, specifically a fragment 651 nucleotides long, containing the entire first exon and terminating just before the conserved intron splice junction between the first exon and first intron. This construct encodes approximately the first 217 amino acids of the C. clegans Δ12 FAT gene. The 651 nucleotide genomic fragment was cloned into an E. coli expression vector between opposing T7 polymerase promoters. The clone was then transformed into a strain of E. coli that carries an IPTG-inducible T7 polymerase. As a control, E. coli was transformed with a gene encoding the Green Fluorescent Protein (GFP).
Feeding RNAi was initiated from C. elegans eggs or from C. elegans L4s. When feeding RNAi was started from C. elegans eggs at 23° C. on NGM plates containing IPTG and E. coli expressing the C. elegans Δ12 FAT or GFP dsRNA, the C. elegans Δ12 FAT RNAi feeding phenotype presented as partially sterile F1 individuals and dead F2 embryos. When feeding RNAi was started from C. elegans L4 larvae at 23° C. on NGM plates containing IPTG and E. coli expressing the C. elegans Δ12 FAT or GFP dsRNA, the C. elegans RNAi feeding phenotype presented as partially sterile P0 individuals with developmentally arrested, sterile F1 nematodes.
C. elegans cultures grown in the presence of E. coli expressing dsRNA and those injected with dsRNA from the Δ12 FAT gene were strongly impaired indicating that the fatty acid desaturase-like gene provides an essential function in nematodes and that dsRNA from the fatty acid desaturase-like gene is lethal when ingested by or injected into C. elegans.
Rescue of C. elegans Δ12 FAT RNAi Feeding Phenotype by Linoleic Acid Methyl Ester
The C. elegans Δ12 fatty acid desaturase (Fat2 protein) converts the mono-unsaturated oleic acid to the di-unsaturated fatty acid linoleic acid. The 612 FAT RNAi prevents expression of the Δ12 fatty acid desaturase, which is predicted to cause a decrease in levels of linoleic acid in the nematode, leading to arrested development and death. Addition of 3 mM linoleic acid methyl ester to the NGM media used for the RNAi experiment brings about a partial rescue of the Δ12 FAT RNAi feeding phenotype. Addition of 3 mM oleic acid methyl ester does not rescue the Δ12 FAT RNAi feeding phenotype (see Table 3 below).
C. elegans Δ12 FAT RNAi feeding phenotypes (starting
Inhibitor Studies
Vernolic acid and ricinoleic acid are naturally occurring plant-produced fatty acid homologs that we predict to be specific inhibitors of Δ12 FAT enzymes. The addition of these compounds to living cultures of C. elegans is expected to mimic the effects of the Δ12 FAT RNAi experiments since, in each case, the phenotype observed should derive from the inhibition of the nematode Δ12 FAT. To explore this possibility, C. elegans cultures were started from eggs on NGM plates containing their E. coli food source and one of either 3 mM ricinolcic acid methyl ester or 3 mM vernolic acid methyl ester. Total eggs layed and hatch-rates of F1 and F2 individuals were followed and compared to nematode cultures grown in the presence of control fatty acids oleic acid methyl ester and linoleic acid methyl ester. C. elegans L4 larvae were added to NGM plates containing OP50 E. coli and one of the following methyl esters: oleic acid, linolcic acid, ricinoleic acid, vernolic acid or none. C. elegans L4 larvae growing on plates containing ricinoleic acid (
Identification of Additional Fatty Acid Desaturase-Like Sequences
A skilled artisan can utilize the methods provided in the example above to identify additional nematode fatty acid desaturase-like sequences, e.g., fatty acid desaturase-like sequences (including Δ12 fatty acid desaturase sequences) from nematodes other than M. incognita, H. glycines, D. immitis, S. stercoralis, R. axei and/or C. elegans. In addition, nematode fatty acid desaturase-like sequences can be identified by a variety of methods including computer-based database searches, hybridization-based methods, and functional complementation.
Database Identification A nematode fatty acid desaturase-like sequence can be identified from a sequence database, e.g., a protein or nucleic acid database using a sequence disclosed herein as a query. Sequence comparison programs can be used to compare and analyze the nucleotide or amino acid sequences. One such software package is the BLAST suite of programs from the National Center for Biotechnology Institute (NCBI; Altschul et al. (1997) Nuc. Acids Research 25:3389-3402). A fatty acid desaturase-like sequence of the invention can be used to query a sequence database, such as nr, dbest (expressed sequence tag (EST) sequences), and htgs (high-throughput genome sequences), using a computer-based search, e.g., FASTA, BLAST, or PSI-BLAST search. Homologous sequences in other species (e.g., humans and animals) can be detected in a PSI-BLAST search of a database such as nr (E value=10, H value=1e-2, using, for example, four iterations; http://www.ncbi.nlm.nih.gov/). Sequences so obtained can be used to construct a multiple alignment, e.g., a ClustalX alignment, and/or to build a phylogenetic tree, e.g., in ClustalX using the Neighbor-Joining method (Saitou et al. (1987) Mol. Biol. Evol. 4:406-425) and bootstrapping (1000 replicates; Felsenstein (1985) Evolution 39:783-791). Distances may be corrected for the occurrence of multiple substitutions [Dcorr=−ln(1-D-D2/5) where D is the fraction of amino acid differences between two sequences](Kimura (1983) The Neutral Theory of Molecular Evolution, Cambridge University Press).
The aforementioned search strategy can be used to identify fatty acid desaturase-like sequences in nematodes of the fobllowing non-limiting, exemplary genera:
Plant Parasitic Nematode Genera Include:
Afrina, Anguina, Aphelenchoides, Belonolaimus, Bursaph elenchus, Cacopaurus, Cactodera, Criconema, Criconemoides, Cryphodera, Ditylenchus, Dolichodorus, Dorylaimus, Globodera, Helicotylenchus, Hemicriconemoides, Hemicycliophora, Heerodera, Hirschmanniella, Hoplolaimus, Hypsoperine, Longidorus, Meloidogvne, Mesoanguina, Nacobbus, Nacobbodera, Panagrellus, Paratrichodorus, Paratylenchus, rarylenchus, Pterorylenchus, Punctodera, Radopholus, Rhadinaphelenchus, Rotylecnchulus, Rotylenchus, Scutellonemna, Subanguina, Thecavermiculatus, Trichodorus, Turbatrix, Tylenchorhynchus, Tylenchulus, Xiphinema.
Animal and Human Parasitic Nematode Genera Include:
Acanthocheilonema, Aelurostrongylus, Ancylostoma, Angiostrongylus, Anisakis, Ascaris, Ascarops, Bunostomunm, Brugia, Capillaria, Chabertia, Cooperia, Crenosoma, Cyalthostonme species (Small Strongyles), Dictyocaulus, Dioctophyma, Dipetalonema, Dirofiliaria, Dracunculus, Draschia, Elaneophora, Enterobius, Filaroides, Gnathostoma, Gonylonema, Habronema, Haemonchus, Hyostrongylus, Lagochilascaris, Litomosoides, Loa, Mammomonogamus, Mansonella, Muellerius, Metastrongylid, Necator, Nemiatodirus, Nippostrongylus, Oesophagostomum, Ollulanus, Onchocerca, Ostertagia, Oxyspirura, Oxyuris, Parafilaria, Parascaris, Parastrongyloides, Parelaphostrongylus, Physaloptera, Physocephalus, Protostrongyhlus, Pseudoterranova, Setaria, Spirocerca, Stephanurus, Stephanofilaria, Strongyloides, Strongylus, Spirocerca, Syngamus, Teladorsagia, Thelazia, Toxascaris, Toxocara, Trichinella, Trichostrongylus, Trichuris, Uncinaria, and Wuchereria.
Particularly Preferred Nematode Genera Include:
Plant parasitic: Anguina, Aphelenchoides, Belonolaimus, Bursaphelenchus. Ditylenchus, Dolichodorus, Globodera, Heterodera, Hoplolaimus, Longidorus, Meloidogyne, Nacobbus, Pratylenchus, Radopholus, Rotylenchus, Tylenchulus, Xiphinema.
Animal & Human parasitic: Ancylostoma, Ascaris, Brugia, Capillaria, Cooperia, Cyathostome species, Diclyocaulus, Dirofiliaria, Dracunculus, Enterobius, Haenonchus, Necator, Nematodirus, Oesophagostomumn, Onchocerca, Ostertagia, Oxyspirura, Oxyuris, Parascaris, Strongyloides, Strongylus, Syngamus, Teladorsagia, Thelazia, Toxocara, Trichinella, Trichostrongylus, Trickhuris, and Wuchereria.
Particularly Preferred Nematode Species include:
Plant parasitic: Anguina tritici, Aphelenchoides fragariae, Belonolaimus longicaudattus, Bursaphelenchus xylophilus, Ditylenchus destructor, Ditylenchus dipsaci, Dolichodorus heterocephalous, Globodera pallida, Globodera rostochiensis, Globodera tabacum, Heterodera avenae, Heterodera cardiolata, Heterodera carotae, Heterodera cruciferae, Heterodera glycines, Heterodera major, Heterodera schachtii, Heterodera zeae, Hoplolaimus tylenchiformis, Longidorus sylphus, Meloidogyne acronea, Meloidogyne arenaria, Meloidogyne chitwoodi, Meloidogyne exigua, Meloidogyne graminicola, Meloidogyne hapla, Meloidogyne incognita, Meloidogyne javanica, Meloidogyne nassi, Nacobbus batatiformis, Pratylenchus brachyurus, Pratylenchus coffeae, Pratylenchus penetrans, Pratylenchus scribneri, Pratylenchus zeae, Radopholus similis, Rotylenchus reniformis, Tvlenchulus semipenetrans, Xiphinema americanum.
Animal & Human parasitic: Ancylostoma braziliense, Ancylostoma caninutm, Ancylostoma cevlanicunm, Ancylostoma duodenale, Ancylostomna tubaefbrme, Ascaris suum, Ascaris lumbrichoides, Brugia malayi, Capillaria bovis, Capillaria plica, Capillaria feliscati, Cooperia oncophora, Cooperia punctata, Cyathostome species, Dictyocaulus filaria, Dictyocaulus viviparus, Dictyocaulus arnfieldi, Dirofiliaria immitis, Dracunculus insignis, Enterobius vermicularis, Halemonchus contortus, Haemonchus placei, Necator amnericanus, Nematodirus helvetianus, Oesophagostomum radiatum, Onchocerca volvulus, Onchocerca cervicalis, Ostertagia ostertagi, Ostertagia circurmcincta, Oxvuris equi, Parascaris equortum, Strongyloides stercoralis, Strongylus vulgaris, Strongylus edentatlus, Syngamus trachea, Teladorsagia circumcincta, Toxocara cati, Trichinella spiralis, Trichostrongylus axei, Trichostrongylus colubriformis, Trichuris vulpis, Trichuris suis, Trichurs trichiura, and Wuchereria bancrofti.
Further, a fatty acid desaturase-like sequence can be used to identify additional fatty acid desaturase-like sequence homologs within a genome. Multiple homologous copies of a fatty acid desaturase-like sequence can be present. For example, a nematode fatty acid desaturase-like sequence can be used as a seed sequence in an iterative PSI-BLAST search (default parameters, substitution matrix=Blosum62, gap open=11, gap extend=1) of a nonredundant database such as wormpep (E value=1e-2, H value=1e-4, using, for example 4 iterations) to determine the number of homologs in a database, e.g., in a database containing the complete genome of an organism. A nematode fatty acid desaturase-like sequence can be present in a genome along with 1, 2, 3, 4, 5, 6, 8, 10, or more homologs.
Hybridization Methods A nematode fatty acid desaturase-like sequence can be identified by a hybridization-based method using a sequence provided herein as a probe. For example, a library of nematode genomic or cDNA clones can be hybridized under low stringency conditions with the probe nucleic acid. Stringency conditions can be modulated to reduce background signal and increase signal from potential positives. Clones so identified can be sequenced to verify that they encode fatty acid desaturase-like sequences.
Another hybridization-based method utilizes an amplification reaction (e.g., the polymerase chain reaction (PCR)). Oligonucleotides, e.g., degenerate oligonucleotides, are designed to hybridize to a conserved region of a fatty acid desaturase-like sequence. The oligonucleotides are used as primers to amplify a fatty acid desaturase-like sequence from template nucleic acid from a nematode, e.g., a nematode other than M. incognita, and/or C. elegans. The amplified fragment can be cloned and/or sequenced.
Complementation Methods A nematode fatty acid desaturase-like sequence can be identified from a complementation screen for a nucleic acid molecule that restores fatty acid desaturase-like activity to a cell lacking a fatty acid desaturase-like activity. Routine methods can be used to construct strains (i.e., nematode, yeast, bacterial strains) that lack fatty acid desaturase activity. For example, a nematode strain mutated at the fatty acid desaturase gene locus can be grown (i.e., rescued) on supplements such as Δ12 unsaturated fatty acids. Such a strain can be transformed with nematode cDNAs predicted to encode fatty acid desaturases. Strains can be identified in which fatty acid desaturase activity is restored by selecting for those transgenic lines that exhibit growth in the absensce of supplemental Δ12 unsaturated fatty acids. The plasmid harbored by the rescued strain can be recovered to identify and/or characterize the inserted nematode cDNA that provides fatty acid desaturase-like activity when expressed. Similarly, a bacterial and/or yeast strain can be used as a selection system, whereby the Δ12 fatty acid desaturase gene(s) can be mutated using, for example, phage transduction (Clark et al. (1983) Biochem. 22:5897-5902; Simon et al. (1980) J. Bacteriology 142:621-632). The mutant cell line can be sustained on exongenous unsaturated fatty acids. A strain lacking Δ12 fatty acid desaturase gene(s) can be transformed with a plasmid library expressing nematode cDNAs. Strains can be identified in which fatty acid desaturase activity is restored, i.e., that can grow in the absence of exogenous fatty acids. In still another embodiment, a microorganism (i.e., a yeast strain) that naturally does not contain a Δ12 fatty acid desaturase can be transformed with plasmids expressing nematode genes. Transformed strains that exhibit Δ12 fatty acid desaturase activity can be identified using, GC analysis to measure fatty acid composition. Those clones that convert oleic acid to linoleic acid can be selected, for example (Sakurdani et al. (1999) Eur. J. Biochem. 261:812-820.
Full-length cDNA and Sequencing Methods The following methods can be used, e.g., alone or in combination with another method described herein, to obtain full-length nematode fatty acid desaturase-like genes and determine their sequences.
Plant parasitic nematodes are maintained on greenhouse pot cultures depending on nematode preference. Root Knot Nematodes (Meloidogyne sp) are propagated on Rutgers tomato (Burpee), while Soybean Cyst Nematodes (Heterodera sp) are propagated on soybean. Total nematode RNA is isolated using the TRIZOL reagent (Gibco BRL). Briefly, 2 ml of packed worms are combined with 8 ml TRIZOL reagent and solubilized by vortexing. Following 5 minutes of incubation at room temperature, the samples are divided into smaller volumes and spun at 14,000×g for 10 minutes at 4° C. to remove insoluble material. The liquid phase is extracted with 200 μl of chloroform, and the upper aqueous phase is removed to a fresh tube. The RNA is precipitated by the addition of 500 μl of isopropanol and centrifuged to pellet. The aqueous phase is carefully removed, and the pellet is washed in 75% ethanol and spun to re-collect the RNA pellet. The supernatant is carefully removed, and the pellet is air dried for 10 minutes. The RNA pellet is resuspended in 50 μl of DEPC-H20 and analyzed by spectrophotometry at λ260 and 280 nm to determine yield and purity. Yields can be 1-4 mg of total RNA from 2 ml of packed worms.
Full-length cDNAs can be generated using 5′ and 3′ RACE techniques in combination with EST sequence information. The molecular technique 5′ RACE (Life Technologies, Inc., Rockville, Md.) can be employed to obtain complete or near-complete 5′ ends of cDNA sequences for nematode fatty acid desaturase-like cDNA sequences. Briefly, following the instructions provided by Life Technologies, first strand cDNA is synthesized from total nematode RNA using Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) and a gene specific “antisensc” primer, e.g., designed from available EST sequence. RNase H is used to degrade the original mRNA template. The first strand cDNA is separated from unincorporated dNTPs, primers, and proteins using a GlassMAX Spin Cartridge. Terminal deoxynucleotidyl transferase (TdT) is used to generate a homopolymeric dC tailed extension by the sequential addition of dCTP nucleotides to the 3′ end of the first strand cDNA. Following addition of the dC homopolymeric extension, the first strand cDNA is directly amplified without further purification using Taq DNA polymerase, a gene specific “antisense” primer designed from available EST sequences to anneal to a site located within the first strand cDNA molecule, and a deoxyinosine-containing primer that anneals to the homopolymeric dC tailed region of the cDNA in a polymerase chain reaction (PCR). 5′ RACE PCR amplification products are cloned into a suitable vector for further analysis and sequencing.
The molecular technique, 3′ RACE (Life Technologies, Inc., Rockville, Md.), can be employed to obtain complete or near-complete 3′ ends of cDNA sequences for nematode fatty acid desaturase-like cDNA sequences. Briefly, following the instructions provided by Life Technologies (Rockville, Md.), first strand cDNA synthesis is performed on total nematode RNA using SuperScript™ Reverse Transcriptase and an oligo-dT primer that anneals to the polyA tail. Following degradation of the original mRNA template with RNase H, the first strand cDNA is directly PCR amplified without further purification using Taq DNA polymerase, a gene specific primer designed from available EST sequences to anneal to a site located within the first strand cDNA molecule, and a “universal” primer which contains sequence identity to 5′ end of the oligo-dT primer. 3′ RACE PCR amplification products are cloned into a suitable vector for further analysis and sequencing.
Nucleic Acid Variants
Isolated nucleic acid molecules of the present invention include nucleic acid molecules that have an open reading frame encoding a fatty acid desaturase-like polypeptide. Such nucleic acid molecules include molecules having: the sequences recited in SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7; and sequences coding for the fatty acid desaturase-like proteins recited in SEQ ID NO: 8, 9, 10, 11, 12, 13 and/or 14. These nucleic acid molecules can be used, for example, in a hybridization assay to detect the presence of a M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei nucleic acid in a sample.
The present invention includes nucleic acid molecules such as those shown in SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7 that may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions, or insertions. Nucleotide insertional derivatives of the nematode gene of the present invention include 5′ and 3′ terminal fusions as well as intra-sequence insertions of single or multiple nucleotides. Insertional nucleotide sequence variants are those in which one or more nucleotides are introduced into a predetermined site in the nucleotide sequence, although random insertion is also possible with suitable screening of the resulting product. Deletion variants are characterized by the removal of one or more nucleotides from the sequence. Nucleotide substitution variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide inserted in its place. Such a substitution may be silent (e.g., synonymous), meaning that the substitution does not alter the amino acid defined by the codon. Alternatively, substitutions are designed to alter one amino acid for another amino acid (e.g., non-synonymous). A non-synonymous substitution can be conservative or non-conservative. A substitution can be such that activity, e.g., fatty acid desaturase-like activity, is not impaired. A conservative amino acid substitution results in the alteration of an amino acid for a similar acting amino acid, or amino acid of like charge, polarity, or hydrophobicity, e.g., an amino acid substitution listed in Table 3 below. At some positions, even conservative amino acid substitutions can disrupt the activity of the polypeptide.
The current invention also embodies splice variants of nematode fatty acid desaturase-like sequences.
Another aspect of the present invention embodies a polypeptide-encoding nucleic acid molecule that is capable of hybridizing under conditions of low stringency (or high stringency) to the nucleic acid molecule put forth in SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7 or their complements.
The nucleic acid molecules that encode for fatty acid desaturase-like polypeptides may correspond to the naturally occurring nucleic acid molecules or may differ by one or more nucleotide substitutions, deletions, insertions, and/or additions. Thus, the present invention extends to genes and any functional mutants, derivatives, parts, fragments, naturally occurring polymorphisms, homologs or analogs thereof or non-functional molecules. Such nucleic acid molecules can be used to detect polymorphisms of fatty acid desaturase genes or fatty acid desaturase-like genes, e.g., in other nematodes. As mentioned below, such molecules are useful as genetic probes; primer sequences in the enzymatic or chemical synthesis of the gene; or in the generation of immunologically interactive recombinant molecules. Using the information provided herein, such as the nucleotide sequence SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7, a nucleic acid molecule encoding a fatty acid desaturase-like molecule may be obtained using standard cloning and a screening techniques, such as a method described herein.
Nucleic acid molecules of the present invention can be in the form of RNA, such as mRNA, or in the form of DNA, including, for example, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. The nucleic acids may be in the form of RNA/DNA hybrids. Single-stranded DNA or RNA can be the coding strand, also referred to as the sense strand, or the non-coding strand, also known as the anti-sense strand.
Expression of Fatty Acid Desaturase-Like Polypeptides
One embodiment of the present invention includes a recombinant nucleic acid molecule, which includes at least one isolated nucleic acid molecule depicted in SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7, inserted in a vector capable of delivering and maintaining the nucleic acid molecule into a cell. The DNA molecule may be inserted into an autonomously replicating factor (suitable vectors include, for example, pGEM3Z and pcDNA3, and derivatives thereof). The vector nucleic acid may be a bacteriophage DNA such as bacteriophage lambda or M13 and derivatives thereof. The vector may be either RNA or DNA, single- or double-stranded, prokaryotic, eukaryotic, or viral. Vectors can include transposons, viral vectors, episomes, (e.g., plasmids), chromosomes inserts, and artificial chromosomes (e.g. BACs or YACs). Construction of a vector containing a nucleic acid described herein can be followed by transformation of a host cell such as a bacterium. Suitable bacterial hosts include, but are not limited to, F. coli. Suitable eukaryotic hosts include yeast such as S. cerevisiae, other fungi, vertebrate cells, invertebrate cells (e.g., insect cells), plant cells, human cells, human tissue cells, and whole eukaryotic organisms. (e.g., a transgenic plant or a transgenic animal). Further, the vector nucleic acid can be used to generate a virus such as vaccinia or baculovirus.
The present invention also extends to genetic constructs designed for polypeptide expression. Generally, the genetic construct also includes, in addition to the encoding nucleic acid molecule, elements that allow expression, such as a promoter and regulatory sequences. The expression vectors may contain transcriptional control sequences that control transcriptional initiation, such as promoter, enhancer, operator, and repressor sequences. A variety of transcriptional control sequences are well known to those in the art and may be functional in, but are not limited to, a bacterium, yeast, plant, or animal cell. The expression vector can also include a translation regulatory sequence (e.g., an untranslated 5′ sequence, an untranslated 3′ sequence, a poly A addition site, or an internal ribosome entry site), a splicing sequence or splicing regulatory sequence, and a transcription termination sequence. The vector can be capable of autonomous replication or it can integrate into host DNA.
In an alternative embodiment, the DNA molecule is fused to a reporter gene such as β-glucuronidase gene, β-galactosidase (lacZ), chloramphenicol-acetyltransferase gene, a gene encoding green fluorescent protein (and variants thereof), or red fluorescent protein firefly lucifcrase gene, among others. The DNA molecule can also be fused to a nucleic acid encoding a polypeptide affinity tag, e.g. glutathione S-transferase (GST), maltose E binding protein, protein A, FLAG tag, hexa-histidine, or the influenza HA tag. The affinity tag or reporter fusion joins the reading frames of SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7 to the reading frame of the reporter gene encoding the affinity tag such that a translational fusion is generated. Expression of the fusion gene results in translation of a single polypeptide that includes both a nematode fatty acid desaturase-like region and a reporter protein or affinity tag. The fusion can also join a fragment of the reading frame of SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7. The fragment can encode a functional region of the fatty acid desaturase-like polypeptides, a structurally intact domain, or an epitope (e.g., a peptide of about 8, 10, 20, or or more amino acids). A nematode fatty acid desaturase-like nucleic acid that includes at least one of a regulatory region (e.g., a 5′ regulatory region, a promoter, an enhancer, a 5′ untranslated region, a translational start site, a 3′ untranslated region, a polyadenylation site, or a 3′ regulatory region) can also be fused to a heterologous nucleic acid. For example, the promoter of a fatty acid desaturase-like nucleic acid can be fused to a heterologous nucleic acid, e.g., a nucleic acid encoding a reporter protein.
Suitable cells to transform include any cell that can be transformed with a nucleic acid molecule of the present invention. A transformed cell of the present invention is also herein referred to as a recombinant or transgenic cell. Suitable cells can either be untransformed cells or cells that have already been transformed with at least one nucleic acid molecule. Suitable cells for transformation according to the present invention can either be: (i) endogenously capable of expressing the fatty acid desaturase-like protein or; (ii) capable of producing such protein after transformation with at least one nucleic acid molecule of the present invention.
In an exemplary embodiment, a nucleic acid of the invention is used to generate a transgenic nematode strain, e.g., a transgenic C. elegans strain. To generate such a strain, nucleic acid linked to a C. elegans promoter is injected into the gonad of a nematode, thus generating a heritable extrachromosomal array containing the nucleic acid (see, e.g., Mello et al. (1991) EMBO J. 10:3959-3970). The transgenic nematode can be propagated to generate a strain harboring the transgene. To identify specific inhibitors of the M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei A12 FAT2, the C. elegans Δ12 FAT gene can be “knocked out” by continuous growth on E. coli engineered to produce dsRNA homologous to the C. elegans Δ12 FAT gene (described earlier). Nematodes of the strain can be used in screens to identify inhibitors specific for a M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase-like gene.
In another embodiment, a nucleic acid of the invention can be cloned behind a yeast-specific transcription promoter can be used to generate a transgenic yeast strain, such as Saccharomyces cerevisiae. The S. cerevisiae strain can be transformed using the lithium acetate procedure (Ito et al. (1983) J. Bacteriology 153:163-168; Sakuradani (1999) Eur J. Biochem. 261:812-820). Such a strain can be used to identify inhibitors specific for a M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase-like polypeptide.
Production of Fatty Acid Desaturase-Like Polypeptide Substrates and Inhibitors
In still another embodiment, a nucleic acid of the invention can be used to generate a transgenic plant such as Arrabidopis thaliana, a model legume Medicago truncatula, or any plant of interest, e.g., a nematode host. For example, the fatty acid desaturase like-gene can be cloned into a vector under the control of the cauliflower mosaic virus (CaMV) 35S promoter/nopaline synthase terminator cassette (Baulcombe et al. (1986) Nature 321:446-449) which can then be introduced into an Agrobacterium strain by the freeze thaw method. Agrobacterium-mediated transformation can be accomplished by the planta-vacuum-infiltration method (Bouchez et al. (1993) C. R. Acad. Sci. Paris 316:1188-1193) and transformed transgeneic plant lines can be selected (Spychalla et al. (1997) Proc. Natl. Acad. Sci. 94:1142-1147). Such a plant line can be used to identify inhibitors specific for M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase-like polypeptides or other plant fatty acid desaturase-like polypeptides. Fatty acid desaturase can be expressed in soybean and/or soybean somatic embryos using, for example, particle bombardment method of transformation (Finer et al. (1991) In Vitro Cell. Dev. Biol. 27:175-182; Cahoon et al. (1999) Proc. Natl. Acad. Sci. USA 96: 12935-12940). It is also desirable to generate plants with increased resistance to inhibitors of fatty acid desaturase-like polypeptides by providing the plant with a transgene expressing a fatty acid desaturase. A transformed cell that harbors a M. incognita fatty acid desaturase polypeptide may naturally produce products of Δ12 fatty acid desaturases (e.g. linoleic acid). In this circumstance, the cell may produce a higher proportion of Δ12-desaturated fatty acids than an otherwise similar cell lacking the M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase polypeptide.
If the host cell does not naturally produce a substrate for fatty acid desaturase, one or more substrates can be provided exogenously to cells transformed with an expressible fatty acid desaturase polynucleotide (e.g., by topical application (Spychalla et al. (1997) Proc. Natl. Acad. Sci. USA 94:1142-1147)), or fatty acid desaturase can be co-expressed in cells together with one or more cloned genes that encode polypeptides that can produce substrate compounds from precursor compounds in such cells.
A transformed cell may also be engineered that harbors a polypeptide that produces inhibitors of the Δ12 fatty acid desaturase-like gene of nematodes. For example, a cell may be engineered to produce a Δ12 hydroxylase, a Δ12 acetylenase, and/or a Δ12 epoxygenase.
Such polypeptides may produce fatty acid analogs that inhibit the nematode Δ12 fatty acid desaturase-like polypeptide (i.e., ricinolcic acid, crepenynic acid, and vernolic acid). Such genes may be linked to a root-specific promoter and transformed into a plant, for example. Examples of suitable genes include: Crepsis palacstina Δ12 fatty acid epoxygenase (GenBank® Accession No. CAA76156; produces vernolic acid); Crepis alpina Δ12 fatty acid acetylenase (GenBank® Accession No. CAA76158; produces crepenynic acid); Ricinus Communis oleate 12-hydroxylase (GenBank® Accession No. AAC49010; produces ricinoleic acid); Momordica charantia Δ12 oleic desaturase-like protein (GenBank® Accession No. AAF05916; produces alpha-eleostearic acid); and Impatiens balsamina Δ12 oleic acid desaturase-like protein (GenBank® Accession No. AAF05915; produces alpha-eleostearic acid).
Oligonucleotides
Also provided are oligonucleotides that can form stable hybrids with a nucleic acid molecule of the present invention. The oligonucleotides can be about 10 to 200 nucleotides, about 15 to 120 nucleotides, or about 17 to 80 nucleotides in length, e.g., about 10, 20, 30, 40, 50, 60, 80, 100, 120 nucleotides in length. The oligonucleotides can be used as probes to identify nucleic acid molecules, primers to produce nucleic acid molecules, or therapeutic reagents to inhibit nematode fatty acid desaturase-like protein activity or production (e.g., antisense, triplex formation, ribozyme, and/or RNA drug-based reagents). The present invention includes oligonucleotides of RNA (ssRNA and dsRNA), DNA, or derivatives of either. The invention extends to the use of such oligonucleotides to protect non-nematode organisms (for example e.g., plants and animals) from disease by reducing the viability of infecting namatodes, e.g., using a technology described herein. Appropriate oligonucleotide-containing therapeutic compositions can be administered to a non-nematode organism using techniques known to those skilled in the art, including, but not limited to, transgenic expression in plants or animals.
Primer sequences can be used to amplify a fatty acid desaturase-like nucleic acid or fragment thereof. For example, at least 10 cycles of PCR amplification can be used to obtain such an amplified nucleic acid. Primers can be at least about 8-40, 10-30 or 14-25 nucleotides in length, and can anneal to a nucleic acid “template molecule”, e.g., a template molecule encoding a fatty acid desaturase-like genetic sequence, or a functional part thereof, or its complementary sequence. The nucleic acid primer molecule can be any nucleotide sequence of at least 10 nucleotides in length derived from, or contained within sequences depicted in SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7 and their complements. The nucleic acid template molecule may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, plant cell, fungal cell, or bacterial cell. A primer can be chemically synthesized by routine methods.
This invention embodies any fatty acid desaturase-like sequences that are used to identify and isolate similar genes from other organisms, including nematodes, prokaryotic organisms, and other eukaryotic organisms, such as other animals and/or plants.
In another embodiment, the invention provides oligonucleotides that are specific for a M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase-like nucleic acid molecule. Such oligonucleotides can be used in a PCR test to determine if a M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei nucleic acid is present in a sample, e.g., to monitor a disease caused M. incognita, H. glycines, D. immitis or S. stercoralis.
Protein Production
Isolated fatty acid desaturase-like proteins from nematodes can be produced in a number of ways, including production and recovery of the recombinant proteins and/or chemical synthesis of the protein. In one embodiment, an isolated nematode fatty acid desaturase-like protein is produced by culturing a cell, e.g., a bacterial, fungal, plant, or animal cell, capable of expressing the protein, under conditions for effective production and recovery of the protein. The nucleic acid can be operably linked to a heterologous promoter, e.g., an inducible promoter or a constitutive promoter. Effective growth conditions are typically, but not necessarily, in liquid media comprising salts, water, carbon, nitrogen, phosphate sources, minerals, and other nutrients, but may be any solution in which fatty acid desaturase-like proteins may be produced.
In one embodiment, recovery of the protein may refer to collecting the growth solution and need not involve additional steps of purification. Proteins of the present invention, however, can be purified using standard purification techniques, such as, but not limited to, affinity chromatography, thermaprecipitation, immunoaffinity chromatography, ammonium sulfate precipitation, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, and others.
The fatty acid desaturase-like polypeptide can be fused to an affinity tag, e.g., a purification handle (e.g., glutathione-S-reductase, hexa-histidine, maltose binding protein, dihydrofolate reductases, or chitin binding protein) or an epitope tag (e.g., c-myc epitope tag, FLAG™ tag, or influenza HA tag). Affinity tagged and epitope tagged proteins can be purified using routine art-known methods.
Antibodies Against Fatty Acid Desaturase-like Polypeptides
Recombinant fatty acid desaturase-like gene products or derivatives thereof can be used to produce immunologically interactive molecules, such as antibodies, or functional derivatives thereof. Useful antibodies include those that bind to a polypeptide that has substantially the same sequence as the amino acid sequences recited in SEQ ID NO: 8, 9, 10, 11, 12, 13 and/or 14, or that has at least 80% similarity over 50 or more amino acids to these sequences. In a preferred embodiment, the antibody specifically binds to a polypeptide having the amino acid sequence recited in SEQ ID NO: 8, 9, 10, 11, 12, 13 and/or 14. The antibodies can be antibody fragments and genetically engineered antibodies, including single chain antibodies or chimeric antibodies that can bind to more than one epitope. Such antibodies may be polyclonal or monoclonal and may be selected from naturally occurring antibodies or may be specifically raised to a recombinant fatty acid desaturase-like protein.
Antibodies can be derived by immunization with a recombinant or purified fatty acid desaturase-like gene or gene product. As used herein, the term “antibody” refers to an immunoglobulin, or fragment thereof. Examples of antibody fragments include F(ab) and F(ab′)2 fragments, particularly functional ones able to bind epitopes. Such fragments can be generated by proteolytic cleavage, e.g., with pepsin, or by genetic engineering. Antibodies can be polyclonal, monoclonal, or recombinant. In addition, antibodies can be modified to be chimeric, or humanized. Further, an antibody can be coupled to a label or a toxin.
Antibodies can be generated against a full-length fatty acid desaturase-like protein, or a fragment thereof, e.g., an antigenic peptide. Such polypeptides can be coupled to an adjuvant to improve immunogenicity. Polyclonal serum is produced by injection of the antigen into a laboratory animal such as a rabbit and subsequent collection of sera. Alternatively, the antigen is used to immunize mice. Lymphocytic cells are obtained from the mice and fused with myelomas to form hybridomas producing antibodies.
Peptides for generating fatty acid desaturase-like antibodies can be about 8, 10, 15, 20, 30 or more amino acid residues in length, e.g., a peptide of such length obtained from SEQ ID NO: 8, 9, 10, 11, 12, 13 and/or 14. Peptides or epitopes can also be selected from regions exposed on the surface of the protein, e.g., hydrophilic or amphipathic regions. An epitope in the vicinity of the active or binding site can be selected such that an antibody binding such an epitope would block access to the active site or prevent binding. Antibodies reactive with, or specific for, any of these regions, or other regions or domains described herein are provided. An antibody to a fatty acid desaturase-like protein can modulate a fatty acid desaturase-like activity.
Monoclonal antibodies, which can be produced by routine methods, are obtained in abundance and in homogenous form from hybridomas formed from the fusion of immortal cell lines (e.g., myclomas) with lymphocytes immunized with fatty acid desaturase-like polypeptides such as those set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13 and/or 14.
In addition, antibodies can be engineered, e.g., to produce a single chain antibody (see, for example, Colcher et al. (1999) Ann N Y Acad Sci 880: 263-80; and Reiter (1996) Clin Cancer Res 2: 245-52). In still another implementation, antibodies are selected or modified based on screening procedures, e.g., by screening antibodies or fragments thereof from a phage display library.
Antibodies of the present invention have a variety of important uses within the scope of this invention. For example, such antibodies can be used: (i) as therapeutic compounds to passively immunize an animal in order to protect the animal from nematodes susceptible to antibody treatment; (ii) as reagents in experimental assays to detect presence of nematodes; (iii) as tools to screen for expression of the gene product in nematodes, animals, fungi, bacteria, and plants; and/or (iv) as a purification tool of fatty acid desaturase-like protein; (v) as fatty acid desaturase inhibitors/activators that can be expressed or introduced into plants or animals for therapeutic purposes.
An antibody against a fatty acid desaturase-like protein can be produced in a plant cell, e.g., in a transgenic plant or in culture (see, e.g., U.S. Pat. No. 6,080,560).
Antibodies that specifically recognize a M. incognita. H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase-like proteins can be used to identify a M. incognita. H. glycines, D. immitis, S. stercoralis or R. axei nematodes, and, thus, can be used to monitor a disease caused by M. incognita, H. glycines, D. immitis or S. stercoralis.
Nucleic Acids Agents
Also featured are isolated nucleic acids that are antisense to nucleic acids encoding nematode fatty acid desaturase-like proteins. An “antisense” nucleic acid includes a sequence that is complementary to the coding strand of a nucleic acid encoding a fatty acid desaturase-like protein. The complementarity can be in a coding region of the coding strand or in a noncoding region, e.g., a 5′ or 3′ untranslated region, e.g., the translation start site. The antisense nucleic acid can be produced from a cellular promoter (e.g., a RNA polymerase II or III promoter), or can be introduced into a cell, e.g., using a liposome. For example, the antisense nucleic acid can be a synthetic oligonucleotide having a length of about 10, 15, 20, 30, 40, 50, 75, 90, 120 or more nucleotides in length.
An antisense nucleic acid can be synthesized chemically or produced using enzymatic reagents, e.g., a ligase. An antisense nucleic acid can also incorporate modified nucleotides, and artificial backbone structures, e.g., phosphorothioate derivative, and acridine substituted nucleotides.
Ribozymes: The antisense nucleic acid can be a ribozyme. The ribozyme can be designed to specifically cleave RNA, e.g., a fatty acid desaturase-like mRNA. Methods for designing such ribozymes are described in U.S. Pat. No. 5,093,246 or Haselhoffand Gerlach (1988) Nature 334:585-591. For example, the ribozyme can be a derivative of Tetrahymena L-19 IVS RNA in which the nucleotide sequence of the active site is modified to be complementary to a fatty acid desaturase-like nucleic acid (see, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).
Peptide Nucleic acid (PNA): An antisense agent directed against a fatty acid desaturase-like nucleic acid can be a peptide nucleic acid (PNA). See Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23) for methods and a description of the replacement of the deoxyribose phosphate backbone for a pseudopeptide backbone. A PNA can specifically hybridize to DNA and RNA under conditions of low ionic strength as a result of its electrostatic properties. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.
RNA Mediated Interference (RNAi): A double stranded RNA (dsRNA) molecule can be used to inactivate expression of a fatty acid desaturase-like gene in a cell by a process known as RNA mediated-interference (RNAi; e.g., Fire et al. (1998) Nature 391:806-811, and Gönezy et al. (2000) Nature 408:331-336). The dsRNA molecule can have the nucleotide sequence of a fatty acid desaturase-like nucleic acid described herein or a fragment thereof. The molecule can be injected into a cell, or a syncitia, e.g., a nematode gonad as described in Fire et al., supra.
In one embodiment, dsRNA can be introduced by soaking of nematodes. Double-stranded RNA may be introduced to nematodes by directly soaking individual worms in a solution of dsRNA. Soaking of C. elegans in a solution of dsRNA can be accomplished essentially as described in Tabar et al. ((1998) Science 282:430-1)). Briefly, Hermaphrodite L4-stage C. elegans are washed twice in siliconized tubes with approximately 1 ml M9 buffer (5 g/L NaCl, 11.32 g/L Na2 HPO4.7H2O, 3 g/L KH2 PO4, 1 mM MgSO4). In each wash, the worms are allowed to settle for 10 minutes and most of the supernatant removed. Between five and twenty worms in minimal volume (5-10 ul) are transferred to a fresh siliconized tube and an equal volume of specific dsRNA (resuspended in sterile, RNase-free water) is added. The final concentration of dsRNA is generally between 0.1 and 3.0 mg/ml. Up to 10% (v/v) lipofectin (Gibco-BRL) may be added to the mix. The mixture is incubated for 10 to 30 hours at a constant temperature between 15° and 23° C. The worms are then transferred individually to NGM-agar plates containing a lawn of E. coli (such as strain OP50), incubated at constant temperature between 15° and 23° C. and scored daily for phenotypes of the worms and their progeny for at least four consecutive days.
Screening Assays
Another embodiment of the present invention is a method of identifying a compound capable of altering (e.g., inhibiting or enhancing) the activity of fatty acid desaturase-like molecules. This method, also referred to as a “screening assay,” herein, includes, but is not limited to, the following procedure: (i) contacting an isolated fatty acid desaturase-like protein with a test inhibitory compound under conditions in which, in the absence of the test compound, the protein has fatty acid desaturase-like activity; and (ii) determining if the test compound alters the fatty acid desaturase-like activity or alters the ability of the fatty acid desaturase to regulate other polypeptides or molecules e.g., the ability of the enzyme to desaturate fatty acids. Suitable inhibitors or activators that alter a nematode fatty acid desaturase-like activity include compounds that interact directly with a nematode fatty acid desaturase-like protein, perhaps but not necessarily, in the active or binding site. They can also interact with other regions of the nematode fatty acid desaturase protein by binding to regions outside of the active site or site responsible for regulation, for example, by allosteric interaction.
In one embodiment, an M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase-like polypeptide is expressed in a yeast cell, for example in S. cerevisiae, as has been described for a C. elegans FAT-2-like polypeptide (Peyo-Ndi (2000) Archives of Biochemistry and Biophysics 376:399-408). Overall fatty acid composition from wild-type and fatty acid desaturase-harboring yeast can than be assessed using, for example, gas-chromotography-mass spectrometry techniques (GC-MS). Optimally, an increase in Δ12 unsaturated fatty acids would be concomitant with introduction of an M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase-like polypeptide into the yeast strain. Test compounds can then be added to the yeast strain and fatty acid composition can be measured. A test compound that alters fatty acid composition, particularly decreases Δ12 unsaturated fatty acids in the yeast strain harboring the M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase-like polypeptides would be considered candidate compounds.
In one embodiment, an M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase-like polypeptide is expressed in a eukaryotic or plant cell, for example in Chinese hamster ovary cells or rabbit skin cells. Overall fatty acid composition from wild-type and fatty acid desaturase-harboring eukaryotic cells can than be assessed using, for example, gas chromatography-mass spectrometry techniques (GC-MS). Optimally, an increase in Δ12 unsaturated fatty acids would be concomitant with introduction of an M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase-like polypeptide into the cells. Test compounds can then be added to the eukaryotic cells and fatty acid composition can be measured. A test compound that alters fatty acid composition, particularly decreases Δ12 unsaturated fatty acids in the cells harboring the M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase-like polypeptides would be considered candidate compounds.
Similarly, a M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase-like polypeptides can be expressed in plants, for example in soybean, cotton, tobacco, potato, M. truncatula, and/or Arabidopsis. Plants used for transformation may have a functional Δ12 fatty acid desaturase gene or may be deficient in Δ12 fatty acid desaturase activity. Relative amounts of fatty acids can be measured and compared. Compounds that alter fatty acid compositions, particularly those that decrease Δ12 unsaturated fatty acids, would be candidate compounds.
Compounds: A test compound can be a large or small molecule, for example, an organic compound with a molecular weight of about 100 to 10,000; 200 to 5,000; 200 to 2000; or 200 to 1,000 daltons. A test compound can be any chemical compound, for example, a small organic molecule, a carbohydrate, a lipid, an amino acid, a polypeptide, a nucleoside, a nucleic acid, or a peptide nucleic acid. Small molecules include, but are not limited to, metabolites, metabolic analogues, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds). Compounds and components for synthesis of compounds can be obtained from a commercial chemical supplier, e.g., Sigma-Aldrich Corp. (St. Louis, Mo.). The test compound or compounds can be naturally occurring, synthetic, or both. A test compound can be the only substance assayed by the method described herein. Alternatively, a collection of test compounds can be assayed either consecutively or concurrently by the methods described herein. A compound may be an analog. Specifically, inhibitors may be analogs of fatty acids, for example, cyclypropenoid analogs of linoleic acid. Epoxy fatty acids (vernolic acid), acetylenic fatty acids (crepenynic acid) and hydroxy fatty acids (ricinoleic acid) may be used as inhibitors. Analogs such as α-elostearic acid with conjugated double bonds may be acceptable inhibitors. These may be expressed in plants (e.g. a Δ12 epoxygenase from C. palaestina produces vernolic acid in transgenic Arabidopsis (Singh et. al., supra).
Compounds can also act by allosteric inhibition or by preventing the fatty acid desaturase from binding to, and thus, acting on its target, i.e., a fatty acid.
A high-throughput method can be used to screen large libraries of chemicals. Such libraries of candidate compounds can be generated or purchased, e.g., from Chembridge Corp. (San Diego, Calif.). Libraries can be designed to cover a diverse range of compounds. For example, a library can include 10,000, 50,000, or 100,000 or more unique compounds. Merely by way of illustration, a library can be constructed from heterocycles including pyridines, indoles, quinolines, furans, pyrimidines, triazines, pyrroles, imidazoles, naphthalenes, benzimidazoles, piperidines, pyrazoles, benzoxazoles, pyrrolidines, thiphenes, thiazoles, benzothiazoles, and morpholines. It may be cyclypropenoid analogs of linoleic acid, epoxy fatty acids (vernolic acid), acetylenic fatty acids (crepenynic acid), and/or hydroxy fatty acids (ricinoleic acid). Analogs such as α-elostcaric acid with conjugated double bonds may be acceptable inhibitors. A library can be designed and synthesized to cover such classes of chemicals, e.g., as described in DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA. 91:11422; Zuckennann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Eng. 33:2059; Carell et al. (1994) Angew Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.
Organism-based Assays: Organisms can be grown in microtiter plates, e.g., 6-well, 32-well, 64-well, 96-well, 384-well plates. In one embodiment, the organism is a nematode. The nematodes can be genetically modified. Non-limiting examples of such modified nematodes include: 1) nematodes or nematode cells (M. incognita, H. glycines, D. immitis, S. stercoralis, R. axei, and/or C. elegans) having one or more fatty acid desaturase-like genes inactivated (e.g., using RNA mediated interference); 2) nematodes or nematode cells expressing a heterologous fatty acid desaturase-like gene, e.g., a fatty acid desaturase-like gene from another species; and 3) nematodes or nematode cells having one or more endogenous fatty acid desaturase-like genes inactivated and expressing a heterologous fatty acid desaturase-like gene, e.g., a M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase-like gene as described herein.
A plurality of candidate compounds, e.g., a combinatorial library, can be screened. The library can be provided in a format that is amenable for robotic manipulation, e.g., in microtitre plates. Compounds can be added to the wells of the microtiter plates. Following compound addition and incubation, viability and/or reproductive properties of the nematodes or nematode cells are monitored.
The compounds can also be pooled, and the pools tested. Positive pools are split for subsequent analysis. Regardless of the method, compounds that decrease the viability or reproductive ability of nematodes, nematode cells, or progeny of the nematodes are considered lead compounds.
In another embodiment, the compounds can be tested on a microorganism, (e.g., a yeast and/or bacterium). For example, a M. incognita, H. glycines, D. innmmitis, S. stercoralis or R. axei fatty acid desaturase gene can be expressed in S. cerevisiae, as has been described for a C. elegans FAT-2 like polypeptide (Peyo-Ndi (2000) Archives of Biochem and Biophysics 376: 399-408). The generation of such strains is routine in the art. As described above for nematodes and nematode cells, the cell lines can be grown in microtitre plates, each well having a different candidate compound or pool of candidate compounds. Growth is monitored during or after the assay to determine if the compound or pool of compounds is a modulator of a nematode fatty acid desaturase-like polypeptide.
In another embodiment fatty acid composition of the organisms can be determined, using, for example, GC-MS. Fatty acid methyl esters can be formed by a transesterification reaction and extracted. GC-MS can be performed using a Hewlet Packard 6890 gas chromatograph interfaced with a Hewlet Packard 5973 mass selective detector, for example. Retention times of resulting methyl esters can be compared with those of known samples (Cahoon, supra). In another embodiment, ethyl esters can be extracted with hexane and analyzed by GLC through a 50 m by 0.32 mm CP-Wax58-CB fused silica column (Chrompack) (Singh, supra; Lee, supra). In another embodiment, if a M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase gene is expressed in a microorganism (i.e., yeast), fatty acid methyl esters can be prepared using methanolic HCl as described (Reed et al. (2000) Plant Physiol. 122:715-720; Meesapyodsuk, (2000) Biochemistry 39:11948-11954).
In Vitro Activity Assays: The screening assay can be an in vitro activity assay. For example, a nematode fatty acid desaturase-like polypeptide can be purified as described above. The polypeptide can be disposed in an assay container, e.g., a well of a microtitre plate along with an appropriate substrate. A candidate compound can be added to the assay container, and the fatty acid desaturase-like activity is measured. Optionally, the activity is compared to the activity measured in a control container in which no candidate compound is disposed or in which an inert or non-functional compound is disposed. Fatty acid composition can be determined, using, for example, GC-MS. Fatty acid methyl esters can be formed by a transesterification reaction and extracted. GC-MS can be performed using a Hewlet Packard 6890 gas chromatograph interfaced with a Hewlet Packard 5973 mass selective detector, for example. Retention times of resulting methyl esters can be compared with those of known samples (Cahoon, supra). In another embodiment, ethyl esters can be extracted with hexane and analyzed by GLC through a 50 m by 0.32 mm CP-Wax58-CB fused silica column (Chrompack) (Singh, supra; Lee, supra).
In Vitro Binding Assays: The screening assay can also be a cell-free binding assay, e.g., an assay to identify compounds that bind a nematode fatty acid desaturase-like polypeptide. For example, a nematode fatty acid desaturase-like polypeptide can be purified and labeled. The labeled polypeptide is contacted to beads; each bead has a tag detectable by mass spectroscopy, and test compound, e.g., a compound synthesized by combinatorial chemical methods. Beads to which the labeled polypeptide is bound are identified and analyzed by mass spectroscopy. The beads can be generated using “split-and-pool” synthesis. The method can further include a second assay to determine if the compound alters the activity of the fatty acid desaturase-like polypeptide.
Optimization of a Compound: Once a lead compound has been identified, standard principles of medicinal chemistry can be used to produce derivatives of the compound. Derivatives can be screened for improved pharmacological properties, for example, efficacy, pharmacokinetics, stability, solubility, and clearance. The moieties responsible for a compound's activity in the above-described assays can be delineated by examination of structure-activity relationships (SAR) as is commonly practiced in the art. One can modify moieties on a lead compound and measure the effects of the modification on the efficacy of the compound to thereby produce derivatives with increased potency. For an example, see Nagarajan et al. (1988) J. Antibiot. 41:1430-8. A modification can include N-acylation, amination, amidation, oxidation, reduction, alkylation. esterification, and hydroxylation. Furthermore, if the biochemical target of the lead compound is known or determined, the structure of the target and the lead compound can inform the design and optimization of derivatives. Molecular modeling software is commercially available (e.g., Molecular Simulations, Inc.). “SAR by NMR,” as described in Shuker et al. (1996) Science 274:1531-4, can be used to design ligands with increased affinity, by joining lower-affinity ligands.
A preferred compound is one that interferes with the function of a fatty acid desaturase-like polypeptide and that is not substantially toxic to plants, animals, or humans. By “not substantially toxic” it is meant that the compound does not substantially affect the respective animal, or human fatty acid desaturase proteins or fatty acid desaturase activity. Thus, particularly desirable inhibitors of M. incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase do not substantially inhibit fatty acid desaturase-like polypeptides or fatty acid desaturase activity of vertebrates, e.g., humans for example. Other desirable compounds do not substantially inhibit to fatty acid desaturase activity of plants.
Standard pharmaceutical procedures can be used to assess the toxicity and therapeutic efficacy of a modulator of a fatty acid desaturase-like activity. The LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population can be measured in cell cultures, experimental plants (e.g., in laboratory or field studies), or experimental animals. Optionally, a therapeutic index can be determined which is expressed as the ratio: LD50/ED50. High therapeutic indices are indicative of a compound being an effective fatty acid desaturase-like inhibitor, while not causing undue toxicity or side-effects to a subject (e.g., a host plant or host animal).
Alternatively, the ability of a candidate compound to modulate a non-nematode fatty acid desaturase-like polypeptide is assayed, e.g., by a method described herein. For example, the inhibition constant of a candidate compound for a mammalian fatty acid desaturase-like polypeptide can be measured and compared to the inhibition constant for a nematode fatty acid desaturase-like polypeptide.
The aforementioned analyses can be used to identify and/or design a modulator with specificity for nematode fatty acid desaturase-like polypeptide over vertebrate or other animal (e.g., mammalian) fatty acid desaturase-like polypeptides. Suitable nematodes to target are any nematodes with the fatty acid desaturase-like proteins or proteins that can be targeted by a compound that otherwise inhibits, reduces, activates, or generally affects the activity of nematode fatty acid desaturase proteins.
Inhibitors of nematode fatty acid desaturase-like proteins can also be used to identify fatty acid desaturase-like proteins in the nematode or other organisms using procedures known in the art, such as affinity chromatography. For example, a specific antibody may be linked to a resin and a nematode extract passed over the resin, allowing any fatty acid desaturase-like proteins that bind the antibody to bind the resin. Subsequent biochemical techniques familiar to those skilled in the art can be performed to purify and identify bound fatty acid desaturase-like proteins.
Agricultural Compositions
A compound that is identified as a fatty acid desaturase-like polypeptide inhibitor can be formulated as a composition that is applied to plants, soil, or seeds in order to confer nematode resistance. The composition can be prepared in a solution, e.g., an aqueous solution, at a concentration from about 0.005% to 10%, or about 0.01% to 1%, or about 0.1% to 0.5% by weight. The solution can include an organic solvent, e.g., glycerol or ethanol. The composition can be formulated with one or more agriculturally acceptable carriers. Agricultural carriers can include: clay, talc, bentonite, diatomaceous earth, kaolin, silica, benzene, xylene, toluene, kerosene, N-methylpyrrolidone, alcohols (methanol, ethanol, isopropanol, n-butanol, ethylene glycol, propylene glycol, and the like), and ketones (acetone, methylethyl ketone, cyclohexanone, and the like). The formulation can optionally further include stabilizers, spreading agents, wetting extenders, dispersing agents, sticking agents, disintegrators, and other additives, and can be prepared as a liquid, a water-soluble solid (e.g., tablet, powder or granule), or a paste.
Prior to application, the solution can be combined with another desired composition such as another anthelmintic agent, germicide, fertilizer, plant growth regulator and the like. The solution may be applied to the plant tissue, for example, by spraying, e.g., with an atomizer, by drenching, by pasting, or by manual application, e.g., with a sponge. The solution can also be distributed from an airborne source, e.g., an aircraft or other aerial object, e.g., a fixture mounted with an apparatus for spraying the solution, the fixture being of sufficient height to distribute the solution to the desired plant tissues. Alternatively, the composition can be applied to plant tissue from a volatile or airborne source. The source is placed in the vicinity of the plant tissue and the composition is dispersed by diffusion through the atmosphere. The source and the plant tissue to be contacted can be enclosed in an incubator, growth chamber, or greenhouse, or can be in sufficient proximity that they can be outdoors.
If the composition is distributed systemically thorough the plant, the composition can be applied to tissues other than the leaves, e.g., to the stems or roots. Thus, the composition can be distributed by irrigation. The composition can also be injected directly into roots or stems.
A skilled artisan would be able to determine an appropriate dosage for formulation of the active ingredient of the composition. For example, the ED50 can be determined as described above from experimental data. The data can be obtained by experimentally varying the dose of the active ingredient to identify a dosage effective for killing a nematode, while not causing toxicity in the host plant or host animal (i.e. non-nematode animal).
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
This application is a divisional of U.S. application Ser. No. 12/061,072, filed Apr. 2, 2008, which is a divisional of U.S. application Ser. No. 11/060,008, filed Feb. 17, 2005, now abandoned which is a divisional of U.S. application Ser. No. 10/243,468, filed Sep. 13, 2002, now U.S. Pat. No. 6,875,595 which claims priority to U.S. provisional application Ser. No. 60/322,003, filed Sep. 13, 2001, all of which are herein incorporated by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| 6194167 | Browse et al. | Feb 2001 | B1 |
| Number | Date | Country | |
|---|---|---|---|
| 20090203127 A1 | Aug 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60322003 | Sep 2001 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12061072 | Apr 2008 | US |
| Child | 12354282 | US | |
| Parent | 11060008 | Feb 2005 | US |
| Child | 12061072 | US | |
| Parent | 10243468 | Sep 2002 | US |
| Child | 11060008 | US |
