A method for the identification or selection of eukaryotic cells

 

(57) Abstract:

Introduction to the population of eukaryotic cells a nucleotide sequence that encodes a protein such as an enzyme mannose-6-phosphate-isomerase, when the nutrient medium contains a compound toxic to cells or inhibiting their growth, such as mannose, adding reagent that reduces the toxicity or inhibitory effect, identifies and discriminates transformed cells without danger of destruction of the untransformed cells in the population and without joint introduction of genes for resistance to antibiotics and herbicides (positive selection). Positive selection also simplifies used vector design, because the same gene can be used as both a reporter and selective gene. 8 C. p. F.-ly, 4 Il., 9 table.

The object of the present invention is a method for the selection of genetically transformed cells that have the desired nucleotide sequence, by providing the transformed cells a selective advantage. The selective advantage possessed by the transformed cells, may be due to their enhanced compared sesta, growth factor or energy source.

It is known that in the case when the genetic material must be introduced into a population of cells by transformation, successfully transformed only a certain number of cells. Identification and separation of transformed cells traditionally produced with the use of "negative selection", in which the transformed cells are able to survive and grow, while untransformed cells are Rostova inhibition or possibly even die from substance to which the transformed cells are resistant due to their transformation.

For example, in the case of transformation of a population of plant cells selection of transformed cells is usually based on the presence of transformed cells "selective gene, which imparts resistance to the antibiotic or herbicide. Selective gene, which in itself may not perform any useful function in the transformed plant (and may even be undesirable for plants), pair or introduced together with the desired gene, which must be entered in the plant, so that both genes are introduced into a population of cells or, more correctly, set the cells are cultivated in the environment, containing an antibiotic or herbicide to which the genetically transformed cells are resistant due to the presence in them of selective gene that allows the identification of transformed cells, because normal cells that do not contain the resistance gene to an antibiotic or herbicide, are Rostova inhibition or perish.

Such methods negative selection have a number of disadvantages. For example, normal cells may die due to the presence of antibiotics or herbicides in the growth environment. As a result, in the case when the population of cells represents an organized tissue, there is a danger that can kill not only the untransformed cells, but also transformed cells due to the fact that the death of the untransformed cells may interrupt the supply of nutrients to the transformed cells, or due to the fact that the oppressed or the perishing of the untransformed cells can release toxic compounds.

Another disadvantage of negative selection is that the presence of nonessential gene, e.g. the gene encoding resistance to the antibiotic, is, how safe is it to introduce antibiotic resistance genes in plants and microorganisms. This concern is especially significant in relation to food plants and microrganisms, which are not intended for use in closed systems (e.g. microrganism intended for use in agriculture), as well as in respect of mikroorganizmov, which are designed for use in closed systems, but may accidentally penetrate beyond them.

Another disadvantage of negative selection is that the plant tissue or cells treated with toxic substances, become more susceptible to bacterial infection. This poses a problem when used as a vector for Agrobacterium transformation, as the treated tissue or cells sometimes overgrown with bacteria, even when the use of antibiotics to prevent bacterial growth.

In addition, the selection of cells or tissues with the use of negative selection requires strict selection time of expression of the introduced genes in relation to the selection process. If transgenic cells will handle toxic compound before will happen the expression of detoxification or pens, how transgenic and necroshine cells will be killed. If the selection is performed too late, the selection of transgenic cells or tissues may prevent, for example, the formation of shoots or callus from untransformed cells or tissues, which will create a barrier to the penetration of the compounds used for selection of transformed cells.

The above drawbacks are eliminated at least to a significant extent by the method according to the present invention (referred to as "positive selection" or a combined positive/negative selection"), which makes possible the identification and isolation of genetically transformed cells without danger of destruction of the untransformed cells in the population and without the necessity of joint introduction of genes for resistance to antibiotics or herbicides. In addition to the fact that eliminate the need for the genes of antibiotic - or herbicidetolerant, the way of positive selection according to the present invention is often much more effective than the traditional negative selection and combination of positive and negative selection provides the frequency of selection of transgenic shoots the same, if the applicatio positive selection gives the advantage a single gene can be used as a reporter gene and selective gene, which leads to simplification of vector designs, greater stability of these structures and a 100% correlation between the expression of reporter and selective genes.

Positive selection also can eliminate the foregoing problems in the timing, as selective compounds can be produced due to the action of gene products formed as a result of expression of the introduced gene on specific substrates. Therefore, the selective connection can accumulate as a consequence of the selective expression of the gene as a selective action you receive when producing a sufficient number of selective compounds.

According to the present invention offers a method for the identification or selection from a population of eukaryotic cells, cultured in a medium containing at least one connection of cells with metabolic advantage resulting from their transformation, in which:

(a) cells transform nucleotide sequence or added together nucleotide sequence, one of which is stimulates the activity of a gene, encoding this protein a;

b) the compound is mannose or xylose or a derivative or a precursor or substrate for the protein, or it can be metabolized by the transformed cells in this substrate, with the specified connection is not mannose in the case when the protein is mannose-6-phosphate-isomerase.

The invention also includes a method for the identification or selection from a population of eukaryotic cells, cultured in a medium containing at least one compound of the cells, which have a metabolic advantage resulting from their transformation, in which:

(a) cells transform nucleotide sequence or co-introduced nucleotide sequence, one of which contains an area that: (a) encodes a protein involved in the metabolism of the compounds, and/or (b) regulates the activity of the gene encoding this protein a;

b) in medium was added reagent that reduces the toxicity of the compounds to the cells.

Preferably, in the case where the reagent, reducing toxicity, added to the culture medium, the join would be mannosamine-6-phosphate-isomerase.

Cells that have "metabolic advantage", among other things capable of faster growth compared with cells that do not have this advantage, and/or capable to utilize substrates (such as predecessors of nutrients and other), which are cells that do not possess this advantage, unable to dispose of, and/or is able to detoxify substances that are toxic or any abscopal otherwise the growth of cells that do not possess the specified advantage.

A protein that participates in the metabolism of compounds" is usually, but not exclusively, the enzyme that may be responsible, directly or indirectly, for the production or utilization of the compounds or its derivatives or precursors. Protein may also participate in the metabolism of compounds, if he connects with it, migrates from one site to another within cells or tissue or organism or otherwise, isolates it, thereby changing its local availability.

Region nucleotide sequence, "which regulates the activity of the gene encoding the protein may alter the level of expression of the endogenous gene by virtue of the fact that she allrazolam plot length up to 10000 etc. O. on the other hand, indirect regulation may be the result of changes in the binding of RNA polymerase with the promoter and a structural gene encoding a protein, or complementary binding of a nucleotide sequence with at least part of a structural gene, thereby lowering the amount of protein in the cell.

Under "derivative" of mannose or xylose see any connection, is able to be disposed of, to communicate, to be a substrate or product of any protein that is involved directly or indirectly in the metabolism of mannose or xylose. In the case of mannose to the number of such derivatives include carbohydrates such as glucose or galactose, which may have been exposed epimers with the formation of mannose or its derivatives or precursors. The term "derivative" also includes the remains of mannose or xylose, having one or more hydroxyl groups with which residues are linked by covalent or ionic bond. Among such related balances include ester, ether group, amino group, aminogroup, phosphate group, sulfate group, carboxyl group, carboxialkilnuyu groups and their combinations. Derivatives of mannose or xylose can also include predestine xylose.

The term "cell" in the context of the invention includes the protoplasts, the term "population" includes tissue, organ or part of the population of individual cells in/on the substrate or the whole organism, e.g. a plant.

The present invention also includes transformed cells, which selectrow with the use of the method according to the invention, and such transformed cells, which are plant cells, and plants, the seed or seeds obtained from such cells. Among the plants that can be ofselection according to the invention include: fruits, including tomatoes, mangoes, peaches, apples, pears, strawberries, bananas and melon; field crops such as canola, sunflower, tobacco, sugar beet, grain, fine grain, such as wheat, barley and rice, corn and cotton, vegetables such as potatoes, carrots, lettuce, cabbage and onions.

The most preferred plants are sugar beets, and corn.

The application of this method of positive selection in vitro is most appropriate, for example, applied to the transformation carried out on whole plants or parts of plants in which the plants or parts thereof have been transformed, t the th damage to adjacent normal cells. Therefore, the transformed cells have a selective advantage compared with nontransgenic cells (for example, the ability to form shoots), but untransformed cells not inflicted any damage in the sense that they can be damaged or killed, as in the case of negative selection with antibiotics or herbicides.

"The selective advantage possessed by the transformed cells can usually be the difference or advantage, allowing to identify transformed cells simple visual way, i.e. without the use of special analysis to determine the presence of the marker gene.

The population of cells can be grown in a medium containing at least one compound, which may be inactive and which is directly or indirectly activated in transformed cells, this compound is inactive in normal cells or less active in normal cells compared to transformed cells so that the transformed cells have a selective advantage, allowing for the selection of the cell pop enevitsa available for transformed cells due to the expression or transcription of the nucleotide sequence, while such a connection is not available for the untransformed cells or is less accessible to the untransformed cells, whereby transformed cells acquire a selective advantage.

In the case where the polypeptide encoded by the nucleotide sequence, directly activates the inactive connection in transformed cells, normal cells may contain endogenous or to produce a certain amount of a specified polypeptide, which often can be an enzyme. In such cases there is no need to "inactive connection" was completely inactive in normal cells, as may be sufficient to allow connection or nutrient was only significantly less active in normal cells compared to transformed cells. In other words, the qualitative difference between transformed and untransformed cells against activation initially inactive connection may be a sufficient criterion for breeding. In such cases, cells can be added inhibitors or substrates that cancerpreventing to zamecatljivo the production of this active inhibitor in the amount when the specified native enzyme is essentially completely inhibited.

Cells can also be transformed simultaneously introduced nucleotide sequence that can encode permease or other transport factor, which allows the connection to pass through the cell membrane and act in transformed cells or penetrate through the other (organello) membrane, so that the activation of inactive compounds include selective absorption of compounds transformed cells, and absorption of the untransformed cells are impossible or occurs to a lesser extent. Instead, in order to facilitate the penetration of compounds into the cell, simultaneously introduced nucleotide sequence may instead send your product in an area in which localized inactive connection, such as the plasma membrane or in the vacuole or the endoplasmic reticulum.

In the case when two nucleotide sequences are jointly introduced into cells, they can be paired with each other or can be entered together in such a way that the presence of a single nucleotide sequence in the cell indicates prisutstvovalo are thus, a typical, although not a required part of the same genetic structure and can be entered using the same vector.

Since it is necessary to introduce the nucleotide sequence expressibility in transformed cells, genetic structure, containing two nucleotide sequences, usually has a regulatory sequence that promotes expression of the nucleotide sequences, for example, known promoters and transcription terminators. Thus, jointly introduced nucleotide sequence is usually associated with a promoter, which can be constitional or regulatory.

The described methods can also be applied when two nucleotide sequences is administered independently. This can be accomplished, for example, by using the same bacteria for the introduction of both genes and the introduction of a relatively large number of copies of the desired nucleotide sequence into a cell, whereby a relatively high probability that the cells, which, as shown, Express simultaneously introduced nucleotide sequence will also be sudnow, causing joint gene expression in the same cell, has, as you might expect, low probability, and improved frequency selection taking place during implementation of the method of positive selection, as should therefore be expected, are of particular advantage in such systems.

The connection used for the purposes of breeding, can also have both positive and negative effects. For example, mannose in sufficiently high concentrations is toxic to most plants, but in cells containing enzymes metabolizing mannose, its negative impact limitowana, and cells also get the advantage in the ability to utilize mannose as a source of carbohydrates. In this case one connection and one gene together to provide a combined system of positive and negative selection, although such a system can be created with the use of two or more genes, which together are responsible for the inhibition of the negative impacts of the connection and for the manifestation of the positive effect of the compound in transformed cells.

In a further implementation of the method, the expression and transcription of the nucleotide pacientes connection in transformed cells, whereby transformed cells can be identified or ofselection from untransformed cells.

In a further implementation of the method of transformed cells can be ofselection by applying a combination of positive selection and negative selection, the nucleotide sequence in transformed cells is usually administered together with an additional nucleotide sequence that encodes resistance to at least one component selected from the group consisting of toxins, antibiotics and herbicides, and the environment in which cells were cultured contains at least one component selected from the group consisting of toxins, antibiotics and herbicides, to which the transformed cells are resistant. Preferably, the nucleotide sequence was administered together with at least two different selective genes.

Preferably, the connection was mannose or xylose. As was shown above, the connection, however, may be a derivative of mannose, for example, mannose-6-phosphate, or a derivative of xylose, such as xilosoft or predecessor mannose or the military to introduce them. Such a nucleotide sequence may encode genes, giving viral, fungal, bacterial resistance or resistance to nematodes.

Cells can be transformed by bacteria such as bacteria of the genus Agrobacterium, sensitive to the specified connection, so that the selection of transformed cells using the specified connection acquires the advantage that reduces the danger politinformation infection of transformed cells by the bacterium. It should be noted that cells can be transformed by any acceptable known methods, including electroporation, microinjection, application microsurgey gun and the transformation of Riand Ti-plasmids. Transformed cells in the respective cases can be regenerated into whole plants, in which recombinant DNA is stably integrated into the genome.

Preferably, the protein was an enzyme involved in the metabolism of mannose or xylose. Among such enzymes include xylose isomerase, phosphomonoesterase, such as mannose-6-phosphate-isomerase; mannose-1-phosphate-isomerase; postmenopause, annotationes that prevrashatesa and mannose-1-phosphatase; and permease, which are involved in the transport of mannose, or a derivative or precursor, in a cage.

Reagent in order to decrease the toxicity of compounds to cells, is usually a derivative of glucose, such as methyl-3-O-glucose or phlorizin.

The present invention will be hereinafter disclosed, based on consideration of the following text together with the accompanying illustrations, in which:

In Fig. 1 presents a scheme of obtaining BcII/HindIII restriction fragment containing the coding region phosphomonoesterase E. coli;

in Fig. 2 shows the structure of plasmid EPL [1];

in Fig. 3 presents the structure of the binary plasmid pBKL4 [2];

in Fig. 4 shows the structure of plasmid pBKL4 containing man A gene that is built between the GUS gene and NPTII gene.

Construction of binary plasmids p(BKL4-mannose) containing coding sequences phosphomonoesterase E. coli

Gene phosphomonoesterase E. coli (EC 5.3.2.8) comes from the plasmid pGS63 [3] (Fig. 1), designs, derived from pBR322, in which the region between the unique > PST and HindIII sites was substituted by section E. coli chromosome carrying the structural gene (man A) phosphomonoesterase and a fragment of a neighboring gene fumarase (fum (A). So nogo clone pUC18 was Legerova > PST /BamHI fragment (2466, etc., n), containing full > PST /HindIII chromosomal fragment and the site of pBR322 length 357, etc. ad that led to the formation of pDO18 (see Fig. 1).

pDO18 cleaved by restriction enzyme HindIII, and the resulting unavailable 3' ends finished using polymerase maple. Linearized DO18 plasmid filled with Hindlll site (HindIII*) (see Fig. 1) were cleaved by restriction enzyme BcII and received BcII-HindIII*fragment length 1218, etc., ad, containing the coding region phosphomonoesterase, clone in plasmid pEnhanced-Peter-Linker (reinforced linker Peter) (pEPL), which was first subjected to restriction by SmaI, and then by BamHI. The resulting plasmid is called p(EPL-mannose).

pEPL design of pCaMVCN [4, 5], in which the CAT gene was deleted by restriction on > PST. A small linker (linker: > PST -BamHI-BaII- > PST) built-in > PST the site of this plasmid, resulting in a gain plasmid, referred to as pLise (pL). pL subjected to restriction by HincII and BgIII, and the resulting fragment containing the 35S promoter and NOS terminator, clone to another pL plasmid subjected to restriction by EcoRV and BgIII. As EcoRV and HincII sites are with blunt ends. The resulting structure is called pEnhanced-Lise (pEL). pEL is significantly different from CaMVCN fact that it contains a variant of the 35S promoter and tandem duplication 250 base pairs is Yu approximately ten times higher, than natural 35S promoter [6] . pEL is subjected to restrictions in > PST and BgIII, thereby removing NOS terminator, and instead embed CaMV terminator (DW2t). Finally, > PST site located between the power 3e5S promoter and CaMV terminator, embed the linker (>PST -BamHI-SmaI-SacI-SaII-Sphi). This plasmid is called pEPL (see Fig. 2).

p(EPL-mannose), subject to the restriction on Hindlll to highlight fragment containing the full enhanced 35S promoter, the coding region phosphomonoesterase E. coli and CaMV terminator. The selected clone fragment in the HindIII site of the binary vector pBKL4 (Fig. 4). The resulting plasmid is called p(BKL-mannose). Hindlll site in pBKL4 is located between the gene of resistance to kanamycin and genome-glucuronidase (GUS) (see Fig.3). Chimeric minasny gene, the gene for resistance to kanamycin (NPTII) and the GUS gene have each its own promoter and terminator. In Fig. 4 shows p(BKL-mannose) design containing the chimeric gene phosphomonoesterase built between GUS and NPTII genes plasmids pBLK4.

Design p(BKL-mannose) was isolated from E. coli and transformed it Agrobacterium tumefaciens strain LBA4404, which contains a disarmed helper plasmid pAL4404 [7,8], the method of freezing and thawing [9].

The sequence of the structural gene (man A), Cory shoots of Solanum tuberosum 'Saturna', 'Bintje' or 'Dianella' support, as described [10], LS the substrate (see below) with the addition of 2 uM of silver thiosulfate at a temperature of 25oC and cyclic shift light: 16 h light/8 h darkness. The original culture was perseval through 20-40 days. The leaves were separated from stems and cut them into nodal segments (approx. 0.8 cm), containing one node.

Inoculation of tissue potatoes

Cups for cocultivation contain LS substrate (sucrose 30 g/l), agar (8 g/l), 2,4-dichlorophenoxyacetic acid (2.0 mg/l) and TRANS-zeatin (0.5 mg/l).

Shoots of about 40-day-old cultures of shoots (height approx. 5-6 cm) cut into internodal segments (approx. 0.8 cm). The segments are placed into liquid LS-substrate (LS-medium) containing Agrobacterium tumefaciens transformed so that it contains the binary vector carrying the genes, which are intended for introduction into the cells of the potato. Among such genes include, for example, genes encoding glucuronidase (GUS), the NPTII gene, encoding resistance to the antibiotic kanamycin and/or genes encoding proteins involved in the metabolism of mannose, for example, mannose-6-phosphate-isomerase, manasaputras, postmenopause and so on (see below).

Agrobacterium is grown during the course the Oia (0.10 g/l); mannitol (10.0 g/l) and yeast extract (0.40 g/l) containing appropriate antibiotics (corresponding resistance gene of Agrobacterium strain), up until the optical density at 660 nm (OD-660) reaches a value of approximately 0.8. Then the suspension is centrifuged, and the cells resuspended in LS-environment so that the OD value of 0.5.

The above internodal segments then incubated in suspension resuspending Agrobacterium for 30 minutes and then the excess bacteria is removed from the segments by blotting them with sterile filter paper.

Co-cultivation of segments of shoots and Agrobacterium

The segments of the shoots will cocultivated the bacterium within 72 hours on filter paper on LS-substrate (as described above) in Petri dishes, covered with white tissue paper. This substrate will be referred to herein as "substrate for securityservice". The substrate and the segments are covered with sterile filter paper and Petri dishes incubated at 25oC and cyclic shift light: 16 h light/8 h darkness.

The washing procedure

After 48 hours of cocultivation segments shoots transferred to LS-medium containing 800 mg/l of carbenicillin. These transplanted leccia transformed tissue

Washed thus the segments are then transferred to LS-substrate (as before) with the difference that the concentration of TRANS-zeatin was 1 mg/l, and substrate type gibberelline acid (0.1 mg/l) and carbenicillin (800 mg/l), valid also add kanamycin sulfate (50 mg/l) and/or mannose (0-20 g/l) and/or sucrose (0-20 g/l). This substrate is hereinafter referred to as "substrate for selection/regeneration". Segments inoculant on fresh substrate at intervals of two weeks or so, as described below. After 2-4 weeks of segments develop shoots, with the formation of new shoots lasts about 3-4 months.

Rooting regeneriruyuschim shoots

Regeneriratia shoots transferred to rooting medium, representing LS-substrate with the addition of carbenicillin (500 mg/l).

Transfer regeneriruyuschim shoots in the soil

Re-rooted regeneriratia sprouts (plants) (height approximately 2-3 cm) are transplanted from rooting in the soil and placed in castillou the camera at the 21oC with a cyclic light: 16 hours light/8 hours dark and 200-400 uE/m2/s. After the plants are fairly well accustomed, their transfer to the greenhouse, where they grow on the tee transformants

Transgenic genotypes regenerierung escape control:

(a) by conducting NPTII assays as described [II];

(b) by conducting GUS analysis on the enzyme expressed by the conjunction of the input genome-glucuronidase according to [12];

(C) by analyzing the mRNA expression introduced gene encoding the enzyme, for example, phosphomonoesterase involved in mannose metabolism, or by measuring the activity of the enzyme.

Example 1

Regeneriratia plants receive, as described above, with the difference that the segments of the shoots will not cocultured with bacteria, and subsequent washing procedure is not carried out. The number regeneriruyuschim shoots determine on the 40th day from the beginning of the experiment. Table 1 presents the results of inhibition of mannose regeneration of shoots from stem segments potatoes, which were not transformed Agrobacterium. From table 1 it is evident that mannose inhibits regeneration of such shoots, and sucrose stimulates such regeneration. Generally speaking, mannose cannot be used as a source of carbohydrates for most plant species. Adding to the plants mannose she is metabolized and accumulates mannose-6 is the quantity of the transformed substance depends on the activity of the isomerase. This fructose-6-phosphate can be utilized by plants, but in General, high levels of mannose (regardless of whether there is or there is no other source of carbohydrates) is toxic to plants. Thus, as can be seen from table 1, the formation of shoots completely inhibited when the concentration of mannose is 5-10 g/l, regardless of the availability of sucrose, even if it is present in high concentrations.

Example 2

Regenerated plants receive, as described above. Agrobacterium, which will coincourt segments of shoots was transformed by the construction of p(BKL-mannose), obtained as described above, so that the bacterium is vector containing genes encoding GUS and mannose-6-phosphate-isomerase.

Transgenic shoots (GUS+) selectrow on the basis of their ability to metabolize mannose in the presence of the reagent (methyl-3-O-glucose), which reduces the toxicity of mannose for shoots. Shoots, which GUS+, selectyou on the basis of their ability to grow in the presence of mannose in a concentration of about 5 g/L.

Also carried out control experiments in which Agrobacterium used for transformation of segments shoots, carrying the vector, similar to p(BKL-mannose) with the difference that the data shoots grow in the presence of 5 g/l mannose and 20 g/l sucrose GUS+ transformants were not obtained.

Example 3

Conduct the following experiment, in which the Agrobacterium used for transformation of segments shoots, vector is similar to p(BKL-mannose) with the difference that it does not have the gene encoding mannose-6-phosphate-isomerase. This vector contains the gene encoding NPTII, which is able to give the transformed cells resistant to kanamycin. Accordingly GUS+ transformants selectrow on the basis of their resistance to kanamycin, was present at a concentration of 50 mg/L. In this last breeding GUS+ genotype had lower compared to example 2, the part of the selected cells.

Example 4

Repeat example 3 with the difference that Agrobacterium transformed with the plasmid p(BKL-mannose), a GUS+ transformants are selected on the basis of their ability to grow on kanamycin (50 mg/ml). In this case smaller than in example 3, the part of the selected shoots is GUS+.

Example 5

Perform a standard technique for tobacco leaf disk, as described in example 13 [13] , with the difference that eliminate the Agrobacterium inoculation and stage cocultivation. As cytokinins applied benzyladenine (1 mg/l), and the carbohydrate content is represented below. The number regenerierbare is no inhibits the regeneration of shoots in the presence of sucrose and in addition, D-xylose is not utilized as a source of carbohydrates. D-xylulose is a good source of carbohydrates during regeneration of shoots.

Xylose can be converted into xylulose using xylose isomerase. In accordance with this functional xylose isomerase gene, as well as structural gene under the control of suitable promoters and terminators, introducing into plants or their parts, or their cells, and transgenic plants (or parts of them, or their cells) selectrow on their ability to use xylose as a source of carbohydrate.

Example 6

The explants receive and process as described above in paragraph "Selection of transformed tissue", with the difference that in the substrate for selection/regeneration was not added kanamycin or carbenicillin, and plant tissue is transformed. Thus, the only stage subculturing is the transfer of explants with substrate for cocultivation on the substrate for selection/regeneration, enriched with xylose concentrations listed below. The number regeneriruyuschim shoots register after 12 weeks.

From the results shown in table 3, it is seen, Thu and, in addition, D-xylose is not a source of carbohydrates in plants that are necroshine the enzyme or protein metabolizing xylose. Moreover, D-xylose (5 g/l), added to the substrates in the absence of sucrose, called regeneration 2.2 shoots per Explant after 9 weeks.

Example 7

Repeat example 5, with the difference that the substrate for selection/regeneration enriched methyl-3-O-glucose (ISH) in the concentrations shown in table 4. The percentage of surviving explants were recorded after 8 weeks.

From the results shown in table 4, it is seen that the joint processing COULD inhibit the toxic effect of mannose on sensitive plant tissue. Because mannose toxic in concentrations that are optimal for connections that are sources of carbohydrates, the addition COULD create the opportunity for enrichment of the substrate carbohydrates in the form of mannose in optimal concentrations. This makes it possible to use mannose as an agent of positive selection in the absence of other sources of carbohydrates.

Example 8

Repeat example 7, with the difference that the substrate for the regeneration/selection contains mannose (15 g/l), methyl-3-O-glucosylated transgenic gene mannose-6-phosphate-isomerase. After 21 days otsilindrovannye shoots are harvested. All the collected shoots analyze on the subject of expression jointly introduced gene-glucuronidase, and then calculate the total number (two charges) transgenic shoots expressing glucuronidase (GUS+), explants, as a share of shoots, expressing glucuronidase (GUS+) of the total number atzelektronik shoots (table 5).

From the results shown in table 5, it is seen that when the joint addition of mannose and methyl-3-O-glucose selection of transgenic shoots are possible even at high concentrations of mannose and the absence of other sources of carbohydrates.

Example 9

Repeat example 8, with the difference that COULD replace floridino. From table 6 it is seen that when the joint addition of mannose and phloridzin possible selection of 100% of transgenic shoots at high concentrations of mannose and the absence of other sources of carbohydrates. This is an example of how symbiotic growth on minimal medium and the production of exudates can be minimized by adding an inhibitor of glucose transport.

Example 10

From table 7 it is seen that connection, non-mannose, can be approx mannose-6-phosphate isomerase from E. coli.

Example 11

Sugar beet transform the so-called cot-pet (SEM-ger) [14] , in which as explants use cotyledons, including the petioles. The cuttings are separated from the seeds, germinated and vynashivalsya for 4-7 weeks during the 12oC in a light regime of 16 h day/8 h night. The cotyledons were cut into 2-3 mm below the node, separated and cultured either in the dark or exposed to light in the presence or absence of xylose. From table 8 it is seen that the xylose utilized sugar beet as a source of carbohydrates in the presence of light but not in darkness, suggesting that based on the xylose-positive breeding of sugar beet, transgenic, among other things, gene xylose-isomerase, should be carried out in the dark.

Example 12

Get the explants, transgenic, among other things, gene mannose-6-phosphate isomery, and the substrate for selection/regeneration type mannose and sucrose, as shown in table 9. The number regeneriruyuschim shoots register after 11 weeks. Table 9 presents the number regeneriruyuschim shoots on substrates containing methyl-3-O-glucose as a percentage of shoots, regeneriruyuschim on substrates without mannose and methyl-3-O-glucose.
Transgenic fabric: - 1,4 shoots/Explant

Fabric wild type: - 1.7 shoot/Explant

It should be noted that the present invention is not limited to the above examples. For example, tissue-specific expression of the gene encoding the enzyme involved in the metabolism of mannose or xylose, or a metabolic derivative of mannose or xylose or predecessor mannose or xylose, can be applied to control the growth regulation of these tissues during their cultivation on the substrate, which is a modulator of the specified enzyme. Moreover, mannose or xylose (including their derivatives or precursors) can be used as a selective herbicide for crops with a selective advantage that have been transformed through the introduction of genes encoding proteins involved in the metabolism of xylose or mannose or their precursors or derivatives thereof.

It should also be borne in mind that the application of the method according to the invention may include:

a) eukaryotic organisms as a whole or in certain types of tissues/cells reduced levels of fructose-6-phosphate or its derivatives, with the introduction of the gene encoding, for example, the th/cells elevated levels mannose-6-phosphate or its derivatives, with the introduction of the gene encoding, for example, phosphomonoesterase;

C) eukaryotic organisms that possess all or some types of cells increased phosphomonoesterase activity due to the introduction of the gene encoding phosphomonoesterase in these cells.

Literature

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2. Nielsen, K. K. et al., Mol. Plant Microbe Interact. 6, pp. 495-506 (1993).

3. Miles, H. et al., Gene 32, pp. 41-48 (1984).

4. Fromm et al., Proc. Natl. Acad. Sci. USA 82, p. 5824 (1985).

5. Fromm et al., Nature 319, p. 791 (1986).

6. Kay et al., cience 236, pp. 1299-1302 (1987).

7. Hoekema et al., Nature 303, pp. 179-180 (1983).

8. Ooms et al., Plasmid 7, pp. 15-29 (1982).

9. Holsters division et al., Mol. Gen. Genet. 163, pp. 181-187 (1978).

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13. PCT/92/00252.

14. Application PCT/DK 92/00108.

1. A method for the identification or selection of eukaryotic cells with metabolities advantage resulting from their transformation, from a population of cells cultured in/on a medium containing at least one toxic for cells or inhibitory otherwise the cell growth connection, otlichayushiesya sequence, containing region, which encodes a protein involved in the metabolism of the compounds, and/or regulates the activity of a gene encoding this protein, on Wednesday, add a reagent that reduces the toxicity of the compounds to the cells or the effect of inhibiting the growth of cells of the connection.

2. The method according to p. 1, characterized in that the said compound is mannose, and nucleotide or co-introduced nucleotide sequence encodes mannose-6-phosphate-isomerase.

3. The method according to p. 2, characterized in that at least one of these nucleotide sequences contains DNA, which is modified so that the codons preferred for the body, which enter the above sequence, the expression of the modified thus DNA in the specified organism leads to the formation of a protein essentially similar to the protein produced by expression of the unmodified DNA in the body, for which the components of the sequence encoding the protein, are endogenous.

4. The method according to any of paragraphs.1-3, characterized in that the specified connection is inactive in normal cells or less irovannyh cells, than for the untransformed cells, and the expression or transcription of the specified nucleotide sequence increases the availability of the compounds to cells or increases the activity of the enzyme endogenously present in the cell, so that the activity of the specified enzyme in transformed cells more activity in untransformed cells, or the expression or transcription of the specified nucleotide sequence results in the blocking of metabolism specified connection or block in the synthesis of the compounds in transformed cells, whereby transformed cells can be separated from untransformed cells.

5. The method according to any of paragraphs.1-4, characterized in that the cells transformed nucleotide sequence that encodes an exogenous promoter capable of homologous recombination with a DNA sequence in/near a structural gene, which is able to be controlled by the specified promoter.

6. The method according to any of paragraphs.1-5, characterized in that the said compound is mannose.

7. The method according to any of paragraphs.1-6, characterized in that the specified soedineniya fact, that this protein is an enzyme.

9. The method according to p. 8, characterized in that said enzyme is selected from the group consisting of photochromes, focusareas, phosphatases, SharePort, zacharopoulos and photochromes.

 

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FIELD: biotechnology.

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36 cl, 4 tbl, 13 ex

FIELD: chemistry.

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FIELD: chemistry.

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24 cl, 2 dwg, 2 tbl, 3 ex

FIELD: chemistry.

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