Genes encoding desaturases and their applying

FIELD: genetic engineering, biotechnology, biochemistry, agriculture, food industry, medicine.

SUBSTANCE: invention relates to the transformation of plant with nucleic acid encoding enzyme Δ6-desaturase in C. elegans that results to preparing a plant with enhanced content of gamma-linolenic acid and resistance to cold. Desaturase extracted from the plant can be used for preparing a drug used for treatment of disorder in body associated with deficiency of gamma-linolenic acid in it.

EFFECT: valuable biological properties of genes and desaturases.

36 cl, 9 dwg, 2 ex

 

The present invention relates, inter alia, to new desaturases and their use.

Over the last few years of higher plants, most significantly from Arabidopsis thaliana was identified a number of microsomal and soluble fatty acid desaturases. This was the result of the combined genetic and biochemical approach to obtaining and complementaly mutant lines of Arabidopsis defective in desaturation or elongation of fatty acids (Somerville S., Browse J. (1996) Trends Cell. Biol., 6, 148-1153). The importance of this approach is confirmed by the isolation and characterization of genes encoding microsomal desaturase, such as desaturase Δ12(Okuley J., et al., (1994), Plant Cell 6, 147-158) and Δ15(Arondel, V., et al., (1992), Scin 258, 1353-1355) (encoded by the genes FAD2 and FAD3, respectively), enzymes that have previously been intractable using classical methods of cleaning due to their hydrophobicity. The allocation of these and related enzymes, such as Δ12the hydroxylase from Ricinus communis (van de Loo F N et al., (1995) Proc. Natl. Acad. Sci. USA 92, 6743-6747), allowed the identification of a number of conservative motifs in plant microsomal desaturases, most notably, the so-called “his-tag” boxes (Shanklin, J et al., (1997) Proc. Natl. Acad. Sci. USA 92, 6743-6747). Proteins containing these motifs can be classified as enzymes containing di-iron center (Shanklin, J. et a., (1997) Proc. Natl. Acad. Sci. USA 94, 2981-2986).

In WO 93/11245 (Du Pont) describes the various nucleic acid fragments encoding desaturase, in particular Δ12and Δ15desaturase that have been isolated from various plants. Recently from a plant of the borage (Borago officinalis), which accumulates γ-linolenovuyu acid (GLA), has identified cDNA clone using vysokopatogennogo PCR against the his-tag motives. In the US 5614393 (Rhone-Poulenc Agrochimie) is described and claimed nucleotide sequence Δ6desaturase borage. Although in the description of the invention assumes that a nucleic acid encoding Δ6desaturase, can be selected by the expert from animal cells easily, suitable animal cells is not offered (as opposed to fungal and bacterial cells), and there is no description of allocation of such nucleic acids from animal cells. Using heterologous expression in transgenic tobacco has been shown that the isolated cDNA clone encodes microsomal Δ6desaturase (O. Sayanova et al., (1997) Proc. Natl. Acad. Sci. USA 94, 4211-4216). Desaturation in Δ6the position is an unusual modification in higher plants, found only in a small number of species, such as borage, evening primrose (Oenothera spp.) and red currant (Ribes spp.), that accumulate Δ6-n is saturated fatty acids GLA and octadecatetraenoic acid (OTK:18: 4Δ6,9,12,15also known as stereonova acid) in the seeds and/or leaves.

GLA is a vegetable fatty acid of great importance and widely used in the treatment of several medical conditions, including eczema and mastalgia. Argue that the use of GLA restores lost Δ6unsaturated fatty acids or meet increased demand (Horrobin, D.F. (1990) Rev. Contemp. Pharmakother. 1: 1-45).

In order to link features figure 5 to show in simplified form the path of metabolism, which, as it occurs in some organisms (including humans), and which involved Δ6desaturase. You can see that GLA may be synthesized in vivo from linoleic acid under the action Δ6desaturase, and that GLA can be used for the synthesis of di-Homo-GLA, which can be transformed into arachidonic acid under the influence of Δ5desaturase. Arachidonic acid is a precursor of various important eicosanoids including prostaglandins and leukotrienes). Δ6desaturase also turns α-linoleic acid in Rev. Thus, it is clear that Δ6desaturase is the first compulsory stage in the biosynthesis pathway of these biologically active molecules (see figure 5).

The sequence previously isolated microsomal Δ6desaturase Boer is cnica differs from previously characterized plant microsomal desaturases/hydroxylase fact, it contains an N-terminal extension, which shows homology with cytochrome b5as well as the fact that the third (closest to the C-end) his-tag Boxing differs from the consensus (Shanklin J et al, (1997) Proc. Natl. Acad. Sci. USA 94, 2981-2986) H-X-H-H-H-glutamine, replacing the first histidine. This was also observed in the case of Δ6desaturase cyanobacterium Synechocystis (GenBank ID; L11421). In WO 93/06712 (Rhone Poulenc Agrochimie) describes the selected nucleic acid encoding a Δ6desaturase isolated from Synechocystis, and claimed bacterial Δ6desaturase and their application.

Although Δ6desaturation of fatty acids is an unusual modification in higher plants, consider that in animals it is normal. Essential fatty acid linoleic acid (18:2Δ9,12) desaturases to GLA with Δ6-desaturase that is the first stage in the path of the biosynthesis of eicosanoids (which include the prostaglandins and leukotrienes). This leads to rapid metabolism of GLA (up to di-Homo-GLA and arachidonic acid; 20:3 Δ8,11,14and 20:4 Δ5,8,11,14, respectively). Therefore, accumulation of GLA is usually not observed.

The worm nematode Caenorhabitis elegans is extremely useful due to the fact that his genetics are well studied, and it has many similarities with the higher animals, as, for example, with people who mi, and, therefore, is extremely useful in the development of desaturases for use in such animals.

According to the present invention features a polypeptide having desaturase activity, which contains the amino acid sequence shown in figure 1.

Amino acid sequence shown in figure 1, is a sequence of Δ6desaturase available worm nematode Caenorhabitis elegans. This is important because prior to the present invention is not reported successful sequencing or purification of animal Δ6desaturase. As C.elegans does not maintain the GLA, the allocation of a Δ6desaturase was unexpected in order to highlight gene desaturases.

Desaturase according to the invention differs significantly from the known desaturases. Homology between Δ6desaturases according to the invention and microsomal Δ12and Δ15the desaturases from Arabidopsis, is described in WO 93/11245 is 24% and 16%, respectively, as determined using the BESTFIT program. Gene Δ6desaturase of the present invention shows 21%identity with desaturase FAT-1 C.elegans described in article Spychalla, J.P. et al. Proc. Natl. Acad. Sci. 94, 1142-1147. The sequence homology between Δ6desaturases of the present invention and Δ6Synechochocystis, describe the Noi in WO 93/06712, is only 23%.

According to another aspect of the invention, it is thus proposed that a dedicated animal Δ6desaturase.

Amino acid sequence shown in figure 1, is significant due to the fact that it has a very low level of sequence identity with Δ6Desaturate borage (the only other eukaryotic Δ6Desaturate sequenced to the present invention). Indeed, this level of sequence identity below 32%. At such a low level of identity can be expected that these two polypeptide will have completely different functions. Suddenly that they both have activity D6desaturase.

The present invention, however, is not limited to Δ6desaturase having the sequence shown in figure 1. It also includes other desaturase with at least 32%sequence identity with this sequence. Preferred polypeptides of the present invention have at least 40%or, more preferably, at least 50%amino acid sequence identity with this sequence. More preferably the degree of sequence identity is at least 75%. The identity of posledovatelno and at least 90%, at least 95% or at least 99% are most preferred.

For the purposes of the present invention, the sequence identity (either amino acid or nucleotide) can be determined by using the program “BESTFIT” Wisconsin Sequence Analysis Package GCG 8.0.

Where there is a high degree of sequence identity can be relatively small differences in amino acid sequence. So, for example, may be less than 20, less than 10 or even less than 5 differences.

Fragments of the polypeptides described above are also within the scope of the present invention provided that they have desaturase activity, i.e. they have the ability to introduce a double bond in the substrate at a certain position, as determined by GC-MS (gas chromatography - mass spectrometry). What is the lowest limit activity? These fragments preferably have a length of at least 100 amino acids. More preferably, these fragments have a length of at least 150 amino acids.

In summary, the polypeptide of the present invention has desaturase activity:

a) contains the amino acid sequence shown in figure 1;

b) has one or more than one amino acid deletions, insertions, or replacements relative to the polypeptide as defined, is Elena in (a) above, but along with that has at least 32%amino acid sequence identity; or

C) is a fragment of a polypeptide as defined in (a) or (b) above, which has a length of at least 100 amino acids.

The term “polypeptide” is used here in a General sense to indicate that a particular molecule contains many amino acids bonded together by peptide bonds. Therefore, the scope of this term includes substances that are in the literature sometimes referred to as peptides, polypeptides or proteins.

Preferably the polypeptide of the present invention must have a domain of cytochrome. Domain cytochrome can be defined as transferring electrons domain, which contains a prosthetic heme group. Preferably there is a domain of cytochrome b. More preferable is the domain of cytochrome b5(preferably it includes the motif N-R-G-G-X15-R-X3-6-N, where X represents any amino acid). The domain of cytochrome b5present in the amino acid sequence as Δ6desaturase borage and Δ6desaturase C.elegans shown in figb. The domain of cytochrome b5preferred is N-terminal domain - that is, it is closer to the N-terminal region of desaturase than to the C-terminal region. This status is made by contrast with other desaturases. For example, yeast Δ9Desaturate with the C-terminal domain of cytochrome b5and with vegetable Δ12and Δ15the desaturases, which have no domain of cytochrome b5.

The polypeptide of the present invention preferably has one or more than one his-tag box (most preferably three). One of them might have a replacement H→Q. (She gives variant his-tag box, which is conserved in several species of animals/plants).

The polypeptides of the present invention can have any spatial specificity, including CIS/TRANS activity, although it is preferable that they represented desaturase front end, introducing a double bond between C3 and C7 positions, measured from COOH (Δ end) of this group. The specialist is able to easily distinguish different desaturase by defining different positions of double bonds introduced by these desaturases. This can be done using known analytical techniques, such as using gas chromatography and mass spectrometry.

Especially preferred desaturases according to the invention are Δ6desaturase.

Preferably, these desaturase found in nature by one or more than one organism, which does not maintain the GLA (the EU is ü, where GLA can be produced, but is not generally found in connection with the fact that she very quickly undergoes metabolism). Such desaturase can occur in nature by one or more than one animal. These desaturase found in nature by one or more than one nematodes, such as C.elegans.

To better understand the scope of the present invention, now more details will be discussed polypeptides in the volume of each of the above (a), (b) and (C).

Polypeptides in volume (a)

The polypeptide in (a) may consist of the amino acid sequence shown in figure 1, or can have additional N-terminal and/or an additional C-terminal amino acid sequence.

Additional N-terminal or C-terminal sequence can be enabled for different reasons, and methods to incorporate such additional sequences are well known in the art. Such techniques include using techniques of gene cloning, in accordance with which the molecules of nucleic acids are ligated together, and then used for expression of the polypeptide in a suitable host.

Additional sequences can be enabled in order to change the characteristics of a specific polypeptide. This can be useful for improving the expression of the if expression regulation in separate systems of expression. For example, the additional sequence may provide some protection from proteolytic cleavage.

Additional sequences may also be useful for changing the properties of the polypeptide to facilitate its identification or purification. For example, it may be a signal sequence to direct the transport of the polypeptide to a specific location within the cell or export the polypeptide from the cell. For a variety of expression systems can use different signal sequences.

In another example, the inclusion of additional polypeptide sequence sew with a group that can be allocated by using affinity chromatography. This grouping can be an epitope, affinity column can contain immobilized immobilized antibodies or antibody fragments that bind to a specified epitope (preferably with a high degree of specificity). The resulting protein can usually be eluted from the column by addition of a suitable buffer.

Additional N-terminal or C-terminal sequence may, however, be present simply as a result of the specific technique used to obtain the polypeptide of the present invention, and should not provide any competitive advantage is to maintain primary characteristic.

Polypeptides in the amount of (b)

Now, turning to the polypeptide defined in (b) above, it should be understood that they are variants of the polypeptides of the data in (a) above.

In the amino acid sequence of the polypeptide that has a desired property, it is often possible to make various changes to get variants which do have this property. Such variants of the polypeptides described in (a) above, are within the scope of the present invention and are discussed in more detail in sections (i)to(iii) below. They include allelic and nehalennia options.

(i) Replacement

A variant example of the present invention is a polypeptide as defined in (a) above, characterized by replacement of one or more than one amino acid of one or more than one other amino acid.

The expert knows that different amino acids have similar characteristics. One or more than one amino acid of the polypeptide is often possible to replace one or more than one other amino acid without eliminating the desired property of the polypeptide (for example, desaturase activity).

For example, the amino acids glycine, alanine, valine, leucine and isoleucine can often replace each other (amino acids having aliphatic side chains). Of these possible substitutions preferably, glycine and alanine usage is followed to replace each other (because they have relatively short side chains), as valine, leucine and isoleucine was used to replace each other (because they have larger aliphatic side chains that are hydrophobic). Other amino acids, which often can be replaced each other, include phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulfur-containing side chains).

On replacement of this nature are often referred to as “conservative” or “semi-conservative” amino acid substitutions.

(ii) Deletions

Deletions of amino acids may be preferred because it is possible to reduce the total length and the molecular weight of the polypeptide while retaining the desired activity. This can give the opportunity to reduce the amount of polypeptide required for a particular purpose.

(iii) Insertions

You can also obtain insertions of amino acids in relation to the polypeptide as defined in (a) above. This can be done to change the nature of the polypeptide (for example, to facilitate the identification, purification or expression).

Polypeptides comprising the amino acid changes (C the exchange, deletions or insertions relative to the sequence of a polypeptide as defined in a) above can be obtained using any suitable technique. For example, a nucleic acid sequence comprising a desired change in the sequence can be obtained using site-directed mutagenesis. Then this sequence can be used to allow expression of the polypeptide, with a corresponding change in its amino acid sequence.

Polypeptides in volume (in)

As discussed above, is often the preferred reduction in length of the polypeptide. Therefore, the characteristic (C) of the present invention encompasses fragments of the polypeptides (a) or (b) above, which have a length of at least 100 amino acids, but which may not be as long as a full-sized polypeptide shown in figure 1. Preferably, these fragments have a length of at least 200, at least 300 or at least 400 amino acids.

Now the different uses of the polypeptides of the present invention will be described only with the help of an example.

The polypeptides of the present invention can be used, inter alia, upon receipt of useful molecules. For example, Δ6desaturase can be used to obtain γ-linolenic acid (GLA), or upon receipt of IU is aboliton, in respect of which the GLA is a precursor. For example, octadecatetraenoic acid (TCI; 18:4 Δ6,9,12,15), a member of the n-3 (or ω-3) fatty acids, can be produced by Δ6-desaturation α-linolenic acid.

GLA, QCD and their metabolites are useful in medicine. They can be used in the manufacture of drugs for the treatment of disorders that involve the deficiency of GLA or metabolite formed from GLA in vivo (for example, eicosanoid). Disorders that can be treated include eczema, mastalgia, hypercholesterolemia, atherosclerosis, coronary heart disease, diabetic neuropathy, viral infections, acne, hypertension, cirrhosis and cancer.

These metabolites can be produced in vivo in a suitable host or in vitro.

When the metabolite to produce in vitro, desaturase of the present invention and its substrate are usually obtained separately, and then combine when you want to produce this metabolite. Thus, the scope of the present invention is included a method of obtaining GLA or QCD, where Δ6desaturase of the present invention is used for the conversion of the substrate linoleic acid or substrate α-linolenic acid GLA or QCD, respectively.

When the metabolite to produce in vivo in an organism such as a plant or animal, the substrate is La desaturase of the present invention will typically be implemented by corresponding non-human organism. Therefore, the production of the metabolite in vivo can be accomplished by embedding the gene encoding desaturase of the present invention, in the body and provide opportunities for expression of desaturase this organism. This desaturase then can act on its substrate. Thus, it should be understood that the polypeptides of the present invention can be used to provide activity desaturase in organisms that normally would not have such activity, or to enhance desaturase activity in organisms, already possessing some desaturase activity. If you want a useful metabolite can be cleaned from such a body. Alternative this organism can be used directly as a source metabolite. Specific methods of cloning, which can be used to obtain transgenic organisms with desaturase activity, are discussed next.

The polypeptides of the present invention can also be used as indicators of the transformation of the body. For example, if the body is intended for transformation, does not have a defined desaturase, and nucleic acid intended for use in transformation encodes desaturase, after trial transformation can conduct analysis to determine p is outstay whether this desaturase or not. So, if Δ6desaturase you can analyze for the presence of GLA and GLA can serve as a simple marker for the presence of functional transgenic cassette containing a sequence encoding a Δ6desaturase.

Following the application of the present invention is to obtain antibodies. In the scope of the present invention includes antibodies that bind to the polypeptides of the present invention.

Preferred antibodies specifically bind to the polypeptides of the present invention and, therefore, they can be used for purification of such polypeptides. (For example, they can be mobilitat and use to bind to the polypeptides of the present invention. These polypeptides can then be eluted by washing with a suitable eluent under appropriate conditions).

The antibody or its derivative in the scope of the present invention can be used in diagnosis. For example, analyses of the binding of such antibodies or derivative, can be applied to determine whether certain desaturase or not. This is useful in the diagnosis of disorders that arise due to the absence of functional desaturase.

Antibodies in the scope of the present invention may be monoclonal or polyclonal.

Monoclonal antibodies can be obtained from a hybrid. You can create them using the merge myeloma cells and spleen cells, which produce the desired antibody, to create an immortal cell line. So, you can use the well-known method of Kohler &Milstein (Nature 256, 52-55 (1975)) or variations of this method.

Methods of obtaining monoclonal and polyclonal antibodies that bind to a particular polypeptide, are well developed in the art. They are discussed in standard textbooks on immunology, for example in Roitt et al., Immunology second edition (1989), Churchill Livingstone, London.

In addition to the complete antibodies, the present invention includes derivatives thereof, which are capable of contacting the polypeptides of the present invention. Thus, the present invention includes antibody fragments and synthetic design. Examples of fragments of antibodies and synthetic constructions are given Dougall et al., in Tibtech 12 372-379 (September, 1994).

Antibody fragments include, in the example, Fab, F(ab’)2and Fv fragments. (They are discussed, for example, in Roitt et al. (see above)). Fv fragments can be modified with the formation of the synthetic constructs, known as single-chain Fv (scFv) molecule. It includes a peptide linker covalently connecting Vhand Vlareas that contribute to the stability of the molecule. Other synthetic structures that can be used include CDR peptides. They are synthetic peptides containing the antigen-binding determinants. You can also use peptide mimetics. These molecules are typically organic ring limited conformations that resemble the structure of the CDR loops, and which include interacting with antigen side chains.

Synthetic constructs include chimeric molecules. So, for example, humanized (or primaryservername) antibodies or their derivatives are within the scope of the present invention. Example gumanitarnogo antibody is an antibody having a frame areas of a person, but hypervariable regions of rodents.

Synthetic constructs also include molecules containing additional grouping, which provides the molecule with some desired property in addition to binding to the antigen. For example, this is ruppirovka can be a label (e.g. a fluorescent or radioactive label). Alternative it can be a pharmaceutically active agent.

In the scope of the present invention also includes nucleic acid molecules.

Such molecules are nucleic acids:

a) encode the polypeptide of the present invention; or

b) complementary molecules as defined in (a) above; or

C) hybridize to molecules as defined in (a) or (b) above.

These nucleic acid molecules and their use are discussed in more detail below.

The polypeptides of the present invention can be encoded by a great variety of molecules, nucleic acids, whereas the well-known degeneracy of the genetic code. All these molecules encoding nucleic acids are included in the scope of the present invention. Preferred encoding nucleic acid molecule encoding the polypeptide shown in figure 1. They include molecules of nucleic acids containing the coding sequence shown in figure 1, and degenerate variants.

Molecules of nucleic acids can be used directly. Alternative you can build them into vectors.

Nucleic acids or vectors containing them can be used when cloning. You can enter them in inhuman masters, to ensure the expression of the polypeptides of the present invention, the COI is lsua techniques well-known specialists in this field of technology. Alternative you can use the cell-free expression systems.

Methods cloning, expression and purification of polypeptides are well known to the specialist. Various such methods are described in standard textbooks such as Sambrook et al. (Molecular Cloning 2ndEdition, Cold Spring Harbor Laboratory Press (1989)); Old & Primrose (Principles of Gene Manipulation, 5thEdition, Blackwell Scientific Publications (1994)); and Stryer (Biochemistry, 4thEdition, W H Freeman and Company (1995)).

Using a suitable expression system, it is possible to produce the polypeptide in the desired form. For example, the polypeptides can be produced by microorganisms, such as bacteria or yeast, cultured insect cells (which can infect a baculovirus or mammalian cells (such as cells of the Chinese hamster ovary (Cho)).

However, the preferred hosts are plants or material reproduction of plants, such as sunflower, canola, sunflower, cereals, including corn, tobacco, beans, including peanuts and soy, safflower, oil palm, coconut palm and other palms, cotton, sesame, mustard, flax, castor bean, borage and evening primrose, or the material of reproduction.

Technology for the production of plants or material reproduction of plants is now well developed. She briefly reviewed the tsya, for example, in WO 96/21022. Desaturase isolated from animals successfully expressed in plants. For example, Spychalla, J.P. et al. (see above) describes the expression of desaturase C.elegans in transgenic Arabidopsis. In addition, in EP 0550162 (Pioneer Hi-Bred International, Inc.) describes chimeric gene construct encoding a Δ9desaturase isolated from rats, and plants transformed with this construct to obtain fatty acids. Desaturase described in this publication has only 22%identity with Δ6desaturases of the present invention.

Specific techniques that can be used are discussed below. Of course, it should be understood that such techniques are not limiting.

(i) a Vector system based on Agrobacterium tumefaciens

These vector systems include systems based on Ti, such as pGV3850, in which T-DNA is neutralized. Preferably there is a selective marker (such as a token, which provides resistance to antibiotics).

You can also use an intermediate vector (PV). They tend to be small in size and, therefore, they are usually easier to manipulate than with large vectors based on Ti. PV as a whole represent vectors derived from T-DNA, cloned in plasmid vectors, isolated from E. coli such as pBR322. PV is often defectio conjugation, and so to give mobility PV can be used capable of conjugation plasmid (such as pRK2013) so that PV can be transformed into the recipient Agrobacterium. Then in Agrobacterium may occur homologous recombination in vivo to give the ability to integrate PV in the resident been disarmed Ti-plasmid with the aim of obtaining cointegrate, which is able to autonomously replicate in Agrobacterium.

Another alternative is the use of binary Ti vectors. In this case, the modified area of the T-DNA carrying the foreign DNA can be done with a small plasmid that replicates in E. coli (for example pRK252). Then, this plasmid (sometimes called mini-Ti or micro-Ti) can conjugative move through technovillage pairing in A.tumefaciens, which contains a compatible vir gene (provided the TRANS-function vir).

Binary vectors can be used even without Ti sequences. In this case, in order to facilitate the transfer of plasmids, it is possible to use bacterial functions mob and oriT. In addition, it might provide TRANS-function vir.

Vector system, discussed above, can be used for gene transfer into plants using a Protocol (Horsch et al. (Science 227, 1229-31 (1985)) or its variants. In this case, from the leaves of dicotyledonous plants the composter you can cut a small CI is CI, to sterilize the surface, and then place them in the environment, having in its composition A.tumefaciens, which contains the recombinant T-DNA, in which the foreign gene that is designed to move, accompanied by a selective marker (e.g. peo genome). Then these discs can be cultivated for 2 days, and then transferred to the medium for selection of the selective marker (For selective marker peo this can be done using cultivation, using a medium containing kanamycin). A.tumefaciens can be killed by using a medium containing carbenicillin. Shoots will develop from callus normal within 2-4 weeks. They can then be cut off and transplanted into the environment, inducing the roots, and when they become large enough, transplant them into the soil.

(ii) a Vector system based on Agrobacterium rhizogenes

These systems include Ri derived plasmids. Ri T-DNA, as a rule, considered to be harmless, and therefore, these plasmids can be considered equivalent neutralized Ti plasmids. Developed cointegration PV system, based on the Ri plasmid.

(iii) System transformation based on protoplasts of plants

Suitable methods are described in “Plant Gene Transfer and Expression Protocols, ed. H.Jones, Human Press Methods in Molecular biology, 49, 1995.

Transformation of plants can be facilitated by removing vegetative cell article the NOC with the formation of protoplasts. These cell wall can be removed by any suitable means, including mechanical destruction or processing cellulolyticus and pectinolytic enzymes. Then the protoplasts can be separated from other components by centrifugation, and then to transform protoplasts of heterologous DNA can be used methods such as electroporation. In appropriate culture conditions, these transformed protoplasts will increase new cell wall, and share. You can then induce shoots and roots, and can be formed shoots.

(iv) Transfection using biolistics

For delivery of DNA or RNA in plant cells can be used high-speed microarray carrying these DNA or RNA. It is possible to obtain a wide variety of transgenic plants and is suitable for both monocotyledonous and dicotyledonous plants. For example, you can use gold or tungsten particles coated with DNA or RNA. Suitable apparatus for throwing these micronarrative include devices based powder, the apparatus on the basis of the electric discharge and pneumatic devices.

(v) System based viruses

Vectors of plant DNA viruses include viruses cauliflower mosaic (which infect a number of dicotyledonous) and geminivirus (which infec who demonstrate a wide range of dicotyledonous and monocotyledonous). Plant RNA viruses are the majority and include mosaic virus of fire (which infects a number of Graminae, including barley) and tobacco mosaic virus (which infects tobacco plants).

From the preceding description it should be understood that the molecules of nucleic acids encoding polypeptides of the present invention, can be cloned and expressed in a wide variety of organisms.

In addition to nucleic acid molecules encoding the polypeptides of the present invention (which is here referred to as “encoding” of a molecule of nucleic acid), the present invention also includes nucleic acid molecule, they are complementary. For example, both strands of double-molecule nucleic acid included in the scope of the present invention (they are related to each other or not). Also included in the mRNA molecule and the complementary DNA (such as cDNA molecules).

Molecules of nucleic acids, which can gibridizatsiya with one or more than one nucleic acid molecule of the issues discussed above, are also covered by the present invention. Such nucleic acid molecules here referred to as “hybridization” of a molecule of nucleic acid.

Hybridomas the nucleic acid molecule of the present invention can have a high degree of identity is the beginning of the sequence the entire length of the molecule with the nucleic acid in the amount of (a) or (b), above (for example, at least 50%, at least 75% or at least 90%sequence identity).

As should be clear to experts in the field of technology, the higher the degree of sequence identity, which has given the single-stranded nucleic acid molecule with another single-stranded molecule of nucleic acid, the higher the probability that in suitable conditions, it will gibridizatsiya with a single-stranded nucleic acid molecule that is complementary to another of the single-stranded nucleic acid molecule.

Preferably, hybridization of the molecule according to the present invention had a length of at least 10 nucleotides, and preferably has the length of at least 25, at least 50, at least 100 or at least 200 nucleotides.

Preferred hybridization molecules hybridize under strict conditions of hybridization. One example of stringent hybridization conditions is when the attempt hybridization is carried out at a temperature from about 35°up to about 65°using saline solution, which is about 0.9-molar. However, the specialist should be able to vary these parameters accordingly to take into account variables such as probe length, structure foundations, type presets the relevant ions, etc.

Most preferably, hybridization of the nucleic acid molecule of the present invention hybridize to a DNA molecule that has the coding sequence shown in figure 1, with an equivalent RNA, or a sequence complementary to any of the above molecules.

Hybridization of the nucleic acid molecule can be useful, for example, as probes or primers.

The probes can be used to clean and/or identify nucleic acid. For example, they can be used to identify the presence of all gene desaturase or its area, and, therefore, they are useful in the diagnosis.

The primers are useful for amplifying the nucleic acids or their sections, for example, by using PCR methods.

In addition to use as probes or primers, hybridization of the nucleic acid molecule of the present invention can be used as antisense molecules to change the expression of the polypeptides of the present invention by binding to complementary nucleic acid molecules. (Typically, this can be achieved by providing nucleic acid molecules that bind to RNA molecules that normally must be transmitted, thereby preventing the broadcast of the accounts for the formation of duplexes).

Hybridization of nucleic acid molecules can also be obtained in the form of ribozymes. Ribozymes can also be used to regulate expression by binding them with molecules of RNA and cleavage of these molecules, which consist of specific target sequence recognized by these ribozymes.

From the preceding discussion it should be understood that a large number of nucleic acids are included in the scope of the present invention. Unless the context indicates otherwise, the nucleic acid molecule of the present invention can, thus, possess one or more than one of the following characteristics:

1. They can be a DNA or RNA (including variants of naturally occurring DNA or RNA structures, which are not found in nature Foundation and/or are not found in nature skeletons).

2. They can be single-stranded or Dantewada.

3. They can be provided in recombinant form, that is covalently cross-linked to a heterologous 5’ and/or 3’ flanking sequence with the formation of chimeric molecules (e.g. vector), which does not occur in nature.

4. They can be presented without the 5’ and/or 3’ flanking sequences, which are commonly found in nature.

5. They can be represented in essentially pure form, nab the emer using probes for isolation of cloned molecules, with the desired target sequence, or by using methods of chemical synthesis. Thus, they can be presented in a form that is essentially free of impurities proteins and/or other nucleic acids.

6. They can be equipped with introns (for example in full-length gene) or do not contain introns (such as cDNA).

Now the present invention will be described only with the help of example with reference to the accompanying graphical materials figure 1-6.

Figure 1 shows the DNA sequence and set it amino acid sequence of the full-size cDNA pCeD6.1 C.elegans. Position the N-terminal domain of cytochrome b5and the variant of the third his-tag box are underlined. Deduced amino acid sequence of this cDNA is identical to the sequence predicted for residues 1-38 and 68-473 W08D2.4.

On figa shows the comparison of the set of amino acid sequences of cDNA CeD6.1 C.elegans and predicted protein W08D2.4 C.elegans (MywormD6=CeD6.1; cew08d2=OPC (open reading frame) W08D2.4).

On FIGU shows a comparison of the established amino acid sequence Δ6desaturase borage (Sayanova et al. (1997) Proc. Natl. Acad. Sci. USA 94, 4211-4216) and cDNA CeD6.1 C.elegans (Boofd6=Δ6desaturase Borago officinatis; ceeld6=CeD6.1).

On IgA and 3C shows the methyl esters of total lipids S.cerevisiae, grown in inducing conditions (linoleic and galactose).

Figure 4 shows the GC-MS analysis of the peak identified in yeast carrying pYCeD6.1.

Figure 5 shows a simplified version of the metabolism of n-6 essential fatty acids in mammals.

On figa and 6B shows the esters of fatty acids and methyl leaves material, both from the control and transgenic plants of Arabidopsis (A)and transgenic Arabidopsis plants expressing Δ6desaturase C.elegans (B).

Example 1 - the Selection gene Δ6desaturase and expression in yeast

In the database sequences NCBI EST conducted the search of amino acid sequences, using the known Δ6desaturase fatty acids borage (Sayanova et al. (1997), see above) and limiting the search to sequences containing variant his-tag box Q-X-X-H-H.

Were identified EST C.elegans. They were further characterized using the database search plans EST C.elegans (laboratory of Prof. Ikuhara (Y.Kohara) (National Institute of Genetics, Mishima, Japan); the DNA Database of Japan)to identify the appropriate cosignee clones.

Plan EST C.elegans partial cDNA clone of 448 BP, designated as yk436b12, was identified through these searches, and this clone was used for screening cDNA libraries C.elegans (mixed stage; also supplied Ave is F. Of there). It showed that the clone yk436b12 was homologous to a portion of a gene that is present on cosmides W08D2 (Z70271 catalog number Genbank), which forms a part of chromosome IV. Using forecasting proteins Genefinder (Wilson R et al. (1994) Nature 368, 32-38) it was predicted that the grounds 21-2957 of Comedy W08D2 encode ORFS of 473 residue which is interrupted by 5 introns. Wilson R et al. describe the sequence of chromosome III C.elegans. Was identified and further purified a number of positive clones, and sequencing confirmed that the full-size clones encode a transcript, probably transcribed from a gene, designated W08D2.4, cosmides W08D2, as identified through database searches of genes sequenced using the plan of the C.elegans genome.

The study predicted polypeptide (designated W08D2.4 in Sanger Centre Nematode Sequencing Project, Hinxton, UK) revealed that he had several characteristics reminiscent of the microsomal desaturase fatty acids, including three his-tag box. However, this predicted protein sequence indicated the presence of N-terminal domain, similar to cytochrome b5containing the diagnostic motif H-P-G-G found in the proteins of the cytochrome b5(Lederer F. (1994) Biochimie 76, 674-692). Because Δ6desaturase allocated by the inventors of borage, also contained N domain b 5it was indicated that W08D2.4 can encode Δ6desaturase.

A more thorough study of this sequence revealed the presence of a third variant his-tag box with the replacement of H→Q (again, as was observed in Δ6desaturase borage). The degree of similarity between W08D2.4 and Δ6Desaturate borage is less than 52% and, therefore, is low. The figure is less than 31%obtained for identity, is also low.

Because W08D2.4 is encoded by a gene that contains a lot of (6) introns, it was necessary to allocate the full-size cDNA to verify the sequence predicted by Genefinder program, as well as to enable the expression of ORS to determine the encoded function.

Screening of the cDNA library was carried out by EST insert yk436b12 (courtesy of Professor I. of There) and identified a number of positive plaques. Then, they were purified to homogeneity, cut out and sequenced the largest inserts (~1450 BP) obtained from saved fahmid. Thus confirmed that the cDNA identified by the authors of the invention, homologous really W08D2.4, and 5’ and 3’ ends of cDNA equivalent to the bases 9 and 3079 sequence of Comedy W08D2.

Because the initiating code ATG, which, as predicted by Genefinder program, is the start of the gene product W082.4, indeed represents the first methionine in the cDNA clone, the inventors have ascertained that they have identified is really a full-sized cDNA. The DNA sequence and set it amino acid sequence of one representative cDNA clone (named pCeD6.1; length 1463 BP) is shown in figure 1, this set of amino acid sequence identical predicted for W08D2.4 in most parts of the protein. Position the N-terminal domain of cytochrome b5and the variant of the third his-tag box are underlined. Installed amino acid sequence of this cDNA is identical to the sequence predicted for residues 1-38 and 68-473 W08D2.4.

However, the DNA sequence encoding residues 38-67 (Y-S-I......L-Y-F), predicted for W08D2.4, this cDNA clone is not present. This means that the installed amino acid sequence CeD6.1 actually has a length of 443 amino acids in contrast to the sequence predicted for W08D2.4, which has a length of 473 residue. The only other difference between these two amino acid sequences is to replace M→V residue 401, which is the result of replacing the Foundation of A→G (base 1211). These two sequences are compared on Figo, as well as compares the installed Amin is acid sequence Δ 6desaturase borage and installed amino acid sequence CeD6.1 (figb). It is likely that additional sequence predicted for W08D2.4 is the consequence of an incorrect forecast internetzone boundaries.

Note the presence of the motif H-P-G-G cytochrome b5on the N-end (encoded by bases 96-108) and replace N→Q in the third his-tag box (encoded by bases 1157-1172).

The coding sequence W08D2.4 been built into the yeast expression vector pYES2 by PCR. For injection sites nl and Sacl on the 5’ and 3’ ends respectively, used nucleotides with protruding 5’ ends. The correctness of the constructs was checked by transcription and translation in vitro using the TnT system (Promega).

Specifically, then, as a matrix for PCR amplification of the full predicted coding sequence (the length of 443 amino acid residue) used the clone pCeD6.1 and cloned into the yeast expression vector pYES2 (Invitrogen) to obtain pYCeD6. The correctness of this PCR-generated sequences were checked by transcription/translation of this plasmid in vitro, using the promoter of the T7 RNA polymerase is present in pYES2.

Using a combined system of transcription/translation Promega TnT, generated products broadcast and analyzed them using electrophoresis, itag (polyacrylamide gel) with LTOs (sodium dodecyl sulphate) and autoradiography according to the manufacturer's instructions. Thus revealed (data not shown), which plasmid pYCeD6 generates the product of ~55 kDa, whereas in the control (pYES2) failed to get any protein products, which indicates that the design was correct.

The resulting plasmids were introduced into yeast (S.cerevisiae) using the lithium acetate method (Guthrie C., Fink, G.R. (1991) Meths Enz. 194) and induced transgene expression by the addition of galactose. The yeast was added 0.2 M linoleate (sodium salt) in the presence of 1% tergitol NP-40.

Transformation and selection of yeast able to grow on oracularities environment, revealed yeast colonies carrying the recombinant plasmid pYCeD6 through a selective marker URA3, which carries pYES2. Expression pYCeD6 was achieved through the induction of the GAL promoter that is present in pYES2. This was carried out after the cells were grown overnight with raffinose as a carbon source, and the medium was supplemented with the addition of linoleate (18:2) in the presence of low levels of detergent. This latest addition was required because the normal substrate for Δ6desaturation are fatty acids 18:2 that are not commonly found in S.cerevisiae.

Total fatty acids of yeast analyzed by GC of the methyl esters. Confirmation of the presence of GLA was performed using GC-MS (Sayanova et al. (1997), see above).

In greater detail, is to ultural then allowed to continue to grow after induction, however, taking aliquots for analysis by GC. When methyl esters of total fatty acids was isolated from yeast carrying plasmid pYCeD6 and grown in the presence of galactose, and analyzed linoleate by GC, an additional peak was observed (figure 3). Figure 3 panel a shows the yeast transformed with control (empty) vector pYES2, panel B presents the yeast transformed pYCeD6.1. Total methyl esters of fatty acids are identified as 16:0 (peak 1), 16:1 (peak 2), 18:0 (peak 3), 18:1 (peak 4), 18:2 (peak 5; added exogenous). An additional peak (6) panel B corresponds to 18:3 GLA arrow. He had the same retention time as the standard for true GLA, which indicates that techenie yeast capable of Δ6-desaturation of linoleic acid. In none of the control samples (transformation pYES2) such peaks were not observed. The identity of this additional peak was confirmed by GC-MS, which positively identified this compound as GLA (figure 4). In experiment 4, the sample was analyzed using mass spectra, as previously (O. Sayanova et al. (1997) Proc. Natl. Acad. Sci. USA 94, 4211-4216), and these data were used to search in the library of profiles. This sample was identified as GLA. Shows the comparison of the mass spectra of the new peak (a) and genuine GLA (B); visual and computer studies is revealed, that they are identical. This confirms that CeD6.1 encodes Δ6desaturase C.elegans, and that this cDNA should probably transcribed from a gene, which, as predicted, encodes ORS W08D2.4, although installed amino acid sequence CeD6.1 30 residues less than the sequence W08D2.4.

Example 2 - Expression Δ6desaturase C.elegans in plants

The coding sequence Δ6the desaturases C.elegans was subcloned into the plant expression vector pJD330, which contains the viral 35S promoter and Nos terminator. Then the cassette, or the promoter/coding sequence/the terminator was subcloned into the plant binary vector transformation pBin19, and the resulting plasmid was introduced into Agrobacterium tumefaciens. Then the Agrobacterium strain used for transformation of Arabidopsis by using vacuum infiltration of inflorescences. Seeds were collected and placed on plates with selective medium containing kanamycin. As pBin19 confers resistance to the antibiotic, will grow only transformed plant material. Resistant lines were identified and subjected to samoobsledovaniu to obtain homozygous material. The material leaves were analyzed at the profiles of fatty acids, using the same method as the one that was used for the expression of desaturase nematodes in d is Oziah. Methyl esters of fatty acids were separated by GC, and new peaks, shown in Fig.6, identified by comparison with known standards and GC-MS. In (B) shows two new peaks, which were identified as γ-linolenic acid (peak 1) and octadecatetraenoic acid (peak 2). They represent the products Δ6-desaturation of fatty acids precursors linoleic acid and α-linolenic acid, respectively.

The inventors have shown that C.elegans cDNA (CeD6.1) encodes Δ6desaturase, and that this sequence is identical with the predicted OPC W08D2.4 except insert 30 residues present in the N-terminal region of the latter protein. It is unclear whether a set of amino acid sequence predicted for CeD6.1, variant splicing W08D2.4, or it is the result of an erroneous prediction of intron/exon connections program Genefinder. However, it is clear that CeD6.1 encodes Δ6desaturase.

It seems that the OPC encoded by this sequence C.elegans, related Δ6desaturase fatty acids in higher plants, previously identified by the authors of the invention (Sayanova et al. (1997), see above), in that they both contain N-terminal domains show homology with cytochrome b5. Demonstrated that Mick is Somalia desaturase fatty acids using free microsomal cytochrome b 5as its electron donor (Smith, M. A. et al. (1990) Biochem. J. 272, 23-29, M.A. Smith et al. (1992) Biochem. J. 287, 141-144), and it seems that the vast majority of the identified sequences for these enzymes do not contain this additional domain of cytochrome b5(Okuley J. et al. (1994) Plant Cell. 6, 147-158, Aronel V. et al. (1992) Science 258, 1353-1355 and Napier, J.A. et. al. (1997) Biochemical J., 328: 717-8).

Until the present invention was aware of only two examples of desaturases, containing the domain of cytochrome b5and one is a Δ6desaturase borage, and the other is a yeast microsomal Δ9(OLE1) desaturase (Napier, J.A. et al. (1997) Biochemical J., see above, and Mitchell AG, Martin CE (1995) J. Biol. Chem. 270, 29766-29772). OLE1, however, contains a C-terminal domain of cytochrome b5(Napier J.A. et al, (1997) Biochemical J, in press, and Mitchell AG, Martin CE (1995) J. Biol. Chem. 270, 29766-29772). The reason cytochrome b5perhaps that Δ6desaturase is desaturase “front end”. (Desaturation “front end” can be defined as the final reaction desaturation and chain fatty acids, usually to introduce a double bond between the previously existing connection and Δ-carboxy end groups (Mitchell AG, Martin CE (1995) J. Biol. Chem. 270, 29766-29772 and Aitzetmuller K., Tseegsuren, N (1994) J. Plant Physiol. 143, 538-543).

In any case, now is the fact that as variant his-tag box, and the N-terminal house the cytochrome b 5are conservative both in plants and in animals, as evidenced by their presence in both Δ6the desaturases, borage and nematodes.

Thus, the invention can enable the identification of other Δ6the desaturases and other desaturases “front end”that need to be identied by the presence of these motifs.

1. The selected polypeptide having desaturase activity that:

a) has the amino acid sequence shown in figure 1;

b) has one or more than one amino acid deletions, insertion or substitution relative to the polypeptide as defined in (a) above, but has with him at least 95%amino acid sequence identity; or

C) is a fragment of a polypeptide as defined in (a) or (b) above, which has a length of at least 100 amino acids.

2. The polypeptide according to claim 1, characterized in that it has a domain of cytochrome.

3. The polypeptide according to claim 2, characterized in that it has a domain of cytochrome b5.

4. The polypeptide according to any one of claims 1 to 3, characterized in that it has at least one his-tag box.

5. The polypeptide according to any one of claims 1 to 4, characterized in that it has three his-tag box.

6. The polypeptide according to any one of claims 1 to 5, characterized in that it is the way the th desaturase, that introduces a double bond between an existing double bond and carboxypropyl fatty acids.

7. The polypeptide according to any one of claims 1 to 6, characterized in that it is a Δ6-desaturase.

8. The polypeptide according to any one of claims 1 to 7, characterized in that it occurs naturally in the body, which does not accumulate γ-linolenic acid (GLA).

9. The polypeptide according to any one of claims 1 to 8, characterized in that it occurs in nature in eukaryotes.

10. The polypeptide according to any one of claims 1 to 9, characterized in that it occurs in nature in the animal.

11. The polypeptide according to any one of claims 1 to 10, characterized in that it occurs in nature in the nematode.

12. The polypeptide according to any one of claims 1 to 11, characterized in that it occurs in nature Caenorhabitis elegans.

13. The polypeptide according to claim 1, characterized in that it comprises the amino acid sequence shown in figure 1, or of its plot.

14. The polypeptide according to any one of claims 1 to 13 for receiving or selecting antibodies.

15. The polypeptide according to any one of claims 1 to 13 for use in medicine.

16. The polypeptide according to any one of claims 1 to 13 for the preparation of drugs for the treatment of disorders that involve the deficit γ-linolenic acid or metabolite formed in vivo from γ-linolenic acid (GLA).

17. The polypeptide according to item 16, wherein the metabolite is eicosanoid.

p> 18. The polypeptide according to any one of p-17, for the treatment of disorders that represent eczema, mastalgia, hypercholesterolemia, atherosclerosis, coronary heart disease, diabetic neuropathy, viral infections, acne, liver cirrhosis, hypertension and cancer.

19. Token transformation of the body containing the polypeptide according to any one of claims 1 to 13.

20. Marker according to claim 19, wherein the organism is a plant.

21. The antibody or its derivative which specifically binds to a polypeptide according to any one of claims 1 to 13 and obtained with the use of this polypeptide.

22. The antibody or its derivative according to item 21 for the diagnosis of disorders that arise due to the absence of functional desaturase.

23. The way to assess whether the organism polypeptide according to any one of claims 1 to 13 or not, which determine whether this organism polypeptide that binds to an antibody or derivative according to item 21 or not.

24. The method according to item 23, wherein the organism is a human.

25. The method according to item 23 or 24, characterized in that it is carried out preferably in vitro.

26. The method of obtaining the GLA, in which for the conversion of linoleic acid into GLA use of the polypeptide according to any one of claims 1 to 13.

27. The method of obtaining octadecatetraenoic acid (OTC), where to turn α-linolenic acid in QCD ISOE is isout polypeptide according to any one of claims 1 to 13.

28. The selected nucleic acid molecule that:

(a) encodes a polypeptide according to any one of claims 1 to 13, or

b) is the complement of the nucleic acid molecule as defined in (a) above.

29. The nucleic acid molecule according p for use as a probe or as a primer.

30. The nucleic acid molecule according p to obtain the body that accumulates GLA or metabolite formed from GLA in the body.

31. The nucleic acid molecule according p to obtain the body that is cold.

32. A vector containing the nucleic acid molecule according p.

33. Vector on p to obtain the body that accumulates GLA or metabolite formed from GLA in the body.

34. Vector on p to obtain the body that is cold.

35. The method of producing the polypeptide according to any one of claims 1 to 13, wherein the host containing a nucleic acid molecule according p or vector for p, and the host is preferably a plant such as oilseed rape culture, sunflower, cereals, including corn, tobacco, beans, including peanuts and soy, safflower, oil palm, coconut palm and other palms, cotton, sesame, mustard, hemp, castor-oil plant, borage and evening primrose, or the material for the propagation of plants incubated under conditions that calls the non expression of the specified polypeptide, and then clear the specified polypeptide.

36. The way to create a host containing a nucleic acid molecule according p or vector for p, and the host is preferably a plant such as oilseed rape culture, sunflower, cereals, including corn, tobacco, beans, including peanuts and soy, safflower, oil palm, coconut palm and other palms, cotton, sesame, mustard, hemp, castor-oil plant, borage and evening primrose, or material for propagation, in which the nucleic acid according p or vector for p injected into the body to ensure its transformation.



 

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