The nucleic acid sequence encoding a protein having a delta-5,7 sterol, delta-7 reductase activity (options), protein from arabidopsis thaliana with a specified activity (options), the expression vector, a method of cloning a nucleic acid, a diagnostic probe, the method of obtaining protein, methods of recovery of sterol (options), the method of producing pregnenolone (options), the transformed yeast strain and method of detecting failure of the delta-5,7 sterol, delta-7 reductase

 

(57) Abstract:

The invention relates to genetic engineering. Cloning of the nucleotide sequence in yeast allows to obtain a protein having a Delta-5,7 Sterol, Delta-7 reductase activity. Recovery of Sterol in position C-7 can be used to obtain pregnenolone and hydrocortisone, and the use of appropriate hybridization probe for detecting failure in humans Delta-5,7 Sterol, Delta-7 reductase, such as congenital insufficiency, and in patients suffering from various diseases, for example with the clinical manifestation of the syndrome RSH/SLO. 21 N. and 10 C.p. f-crystals, 30 tab., table 2.

The present invention relates to DNA sequences encoding protein A. tagliani, having a Delta-5,7 Sterol, Delta-7 reductase activity, protein Delta-7 Ed to the way they are received, to a transformed yeast strains and their application.

Delta-5,7 Sterol, Delta-7 reductase (E. C. 1.3.1.21) is a microsomal enzyme whose presence was detected through its action in the homogenates of rat liver (M. E. Dempsey with co-workers. Methods Enzymol., 15, 501-514, 1969),Visim from NADPH and provides in vitro recovery of 7-dehydrocholesterol in cholesterol.

Sterols are essential components of membranes in eukaryotes, however, depending on the type, they have different structure. In eukaryotic cells, such as yeast, the main Sterol is ergosterol, which contains unsaturated double bond at position C-5 and C-7, the branched side chain at position C-24 unsaturated bond at position C-22, while in mammals, cholesterol differs unsaturated bond in position C-5 and a saturated side chain. Sitosterol, stigmasterol and campesterol, which represent the most common plant sterols, have branched out, but saturated side chain, and so, like sterols vertebrates, have no bonds in position C-7. The enzyme that causes this difference in the structure of the Sterol nucleus, is a Delta-5,7 Sterol, Delta-7 reductase.

Delta-5,7 Sterol, Delta-7 reductase has never been purified to homogeneity, and the literature found only a partial description of treatment (above M. E. Dempsey with co-authors, M. Taton with co-authors). When this protein was not considered from the standpoint of its physical and chemical properties. No information regarding protein sequence grip 7-dehydrocholesterol in humans has been described in connection with the syndrome RSH/Smith-Lemley-Opitz (SLO) (J. M. Opitz coauthors. Am. J. Med. Genet., 50, 326-338, 1994).

Thus, the cloning of a cDNA that encodes a Delta-5,7 Sterol, Delta-7 reductase, which may allow to identify the appropriate sequence of the protein, as well as to characterize a human gene or to identify their innate failure, cannot be produced by conventional methods using, for example, methods of hybridization and/or immunological detection. Therefore, it is particularly important to search for new methods of screening, allowing cloning, in particular, in the absence of information about the protein.

Ergosterol, the main Sterol of fungal membranes, includes a pair of conjugated double bonds in position to 5.7, allowing connection of the family of polyene, such as nystatin, have antimicotics action (R. Bittman with co-workers, J. Biol. Chem., 260, 2884-2889, 1985). The strong dependence of the action of nystatin on the membrane concentration of unsaturated sterols in position With-5,7 allowed to take in S. cerevisiae mutant strains relative to the accumulated sterols (S. W. Molzahn with co-workers, J. Gen. Environ., 72, 339-348, 1972). Thus, mutants egg and egg accumulate sterols, not having conjugated double bonds in position With-5,7 due nedostatocno is s (Century Arthinton with co-workers. Gene, 102, 39, 1991). Such mutants are viable, as ergosterol, which is the major natural yeast Sterol, can under certain conditions be replaced by a different sterols-substitutes, including cholesterol.

The advantage of increasing the resistance to nystatin yeast strains enriched in sterols that have no double bonds in position to 5.7, was used by the inventors for cloning cDNA that encodes a heterologous protein having a Delta-5,7 Sterol, Delta-7 reductase activity, a screening method that uses metabolic interference in S. Cerevisiae. However, the success of this approach, the advantage of which is its independence from the knowledge of the DNA sequences or protein, and detecting, based solely on the enzymatic activity, it is impossible to count because of the numerous difficulties of a technical nature, with which it is associated.

The first limitation is due to the fact that the action of nystatin is not fully understood (L. W. Parks with co-authors, CRC Critical Reviews in Microbiology, 301-304, 1978). For example, you can anticipate a weak specificity nistatina selection due to its indirect nature, which results in domnich mutations leading to sustainability, not dependent metabolic pathways of sterols. So cells that have been transformed by the Bank, can Express heterologous genes providing resistance to nystatin, not dependent metabolic pathways of sterols.

Another example of the expected limitations refers to the fact that the heterologous protein may have weak activity in the cell, leading to the absence or weakening of resistance to nystatin, for example, one of the following reasons: 1) weak expression of the gene encoding Delta-5,7 Sterol, Delta-7 reductase; 2) low activity or no activity of the protein due to poor collapsed or because of incorrect subcellular addressing; 3) protein plants does not recognize the yeast sterols as substrates or 4) sterols that may be substrates, present in esterified form or accumulate in vesicles and are not in contact with the enzyme. Thus, it is possible to foresee that the accumulated thus sterols are not exposed to Delta-5,7 Sterol, Delta-7 reductase, which, in the case of eukaryotes, is localized in the microsomes.

The present invention relates to the cloning of a cDNA A. hoist the Red, according to the method of cloning, performed in yeast due to metabolic interference-based resistance to nystatin. Protein Delta-7 Ed ensures the recovery of sterols containing unsaturated bond at position C-7, by means of biological recovery, which is also used as a good solution to the problem of selective reduction in position C-7 of sterols or steroids possessing an unsaturated double bond in position to 5.7, which is impossible when using methods of chemical recovery.

The present invention also relates to transformed the yeast cells expressing the Delta-7 Ed, which surprisingly accumulate products, saturated in position C-7, and possibly saturated in position C-22, which, in contrast to ergosterol, are substrates of the enzyme cleavage of the side chain of cholesterol, i.e. the cytochrome P450The SCC.

These unexpected properties allow the use of transformed yeast, which is the subject of the present invention, in the method of obtaining sterols or steroids, representing industrial and/or pharmacological interest, in particular to obtain pregnenolone by the UB>450SCC (P450SCC) in the presence of adenotonsillectomy (ADR) and adrenodoxin (ADX). Transformed yeast, which is the subject of the present invention can also be used in the method of obtaining the intermediate steroids path metabolize cholesterol hydrocortisone in mammals and other vertebrates. Such use has the advantage that it allows to get hydrocortisone or its intermediate products by means of biological oxidation, which does not require the use as the source substrate exogenous Sterol, such as cholesterol that gets around the problem of penetration of Sterol in yeast, proofed for exogenous sterols in terms of aerobiosis has already been described (R. T. Lorentz with co-workers, J. Bacteriology, 981-985, 1986).

The present invention relates also to the use of nucleotide sequences obtained using the method of cloning, which is the subject of the present invention. The sequence of the RNA or DNA encoding a Delta-5,7 Sterol, Delta-7 reductase, can be used in the diagnosis or treatment of diseases associated with the gene product of the Delta-5,7 Sterol, Delta-7 reductase. For example, it is believed that the syndrome RSH/SLO. Thus, the sequence of human DNA can be used as a probe for diagnosing the failure of the Delta-5,7 Sterol, Delta-7 reductase, as well as in gene therapy to correct the specified deficiencies.

Thus, the object of the present invention is a nucleic acid sequence comprising a sequence encoding a protein having a Delta-5,7 Sterol, Delta-7 reductase activity and specified nucleic acid may be DNA or RNA and first cDNA.

Delta-5,7 Sterol, Delta-7 reductase activity can be detected, for example, using an enzymatic test in vitro (in vitro), described below in the experimental part.

The object of the present invention in the first place is the cDNA sequence, in which the coding sequence encodes a protein A. tagliani, having a Delta-5,7 Sterol, Delta-5,7 reductase activity and having a sequence of nucleotides of the sequence SEQ ID No. 1 (see the end of the text), as well as allelic variants of this sequence.

The above sequence of cDNA encoding a protein, listaselect cDNA, which may be obtained, for example, by cloning in yeast, based on the Bank of the expression of A. thaliana using the screening method, based on the emergence of resistance of yeast to nystatin, subject to the conditions detailed below.

The knowledge of the above nucleotide sequences SEQ ID No. 1 allows to reproduce the present invention by conventional methods, well known in the art, for example, by performing a chemical synthesis or screening genomic Bank or Bank cDNA using synthesized oligonucleotide probes using the methods of hybridization or amplification by PCR (polymerase chain reaction).

The object of the present invention is a DNA sequence encoding a protein having a Delta-5,7 Sterol, Delta-7 reductase activity and hybridizers with the nucleotide sequence SEQ ID No. 1 under conditions of medium or high temperature hybridization, or possessing with the sequence identity of 60% or greater.

Sequence hybridizers detectable in the sequence SEQ ID No. 1, hybridizing under normal conditions with the X6, with the subsequent washing, or less high temperature hybridization, for example, at a temperature of +42°C for 24 h in 20% solution of formamide, SSCX6 with subsequent washing in the well-known normal conditions (T. Maniatis coauthors, Molecular cloning. Cold Spring Harbor Laboratory Press, 1969).

The percentage identity of a nucleotide sequence may be determined using, for example, the BLAST program (basic local alignment search tool) (S. F. Altschul with co-workers, J. Mol. Biol., 215, 403-410, 1990) on the NCBI server.

The present invention relates to a DNA sequence that encodes a protein having a Delta-5,7 Sterol, Delta-7 reductase activity, amplified by PCR, using as a nucleating oligonucleotides encoding the consensus sequence having the amino acid sequence SEQ ID No. 3:

in which XAA in position 7 represents the amino acid TRP, or Tyr and XAA in position 12 is a GIS or Liz.

The above sequence of SEQ ID No. 3 corresponds to a new consensus sequence, which was determined to be the result of the comparison of amino acid sequences between the new sequence of SEQ ID who educts, with a specific action in a position other than the position C-7, or receptors for lamina, as described in detail below in the experimental part. On the basis of information derived from the amino acid sequence of SEQ ID no 3, may be identified and synthesized the seed, consisting of not more than 45 nucleotides, which in combination with the second priming oligo-dT (17 nucleotides) as the opposite sequence will allow, through the use of a set of PCR (for example, set Stratagene), to amplify DNA encoding the protein having a Delta-5,7 Sterol, Delta-7 reductase activity.

The present invention relates also to a protein of A. thaliana, having a Delta-5,7 Sterol, Delta-7 reductase activity and amino acid sequence of SEQ ID No. 2 (see the end of the text), as well as allelic variants and analogues of the sequence.

Under the alleles and analogues understand the sequence modified by substitution, deletion or addition of one or several amino acids, provided that these products will retain the same function. Modified sequences can, for example, be obtained by the method of directed Mut is tsya protein A. thaliana, having a Delta-5,7 Sterol, Delta-7 reductase activity, and the above-mentioned amino acid sequence SEQ ID No. 2, referred to as the Delta-7 Ed.

The present invention relates also to a protein having a Delta-5,7 Sterol, Delta-7 reductase activity and amino acid sequence identical to the sequence SEQ ID No. 2 approximately 60 percent or more.

The percent identity can be determined using, for example, the above program BLAST.

The present invention relates also to a protein having a Delta-5,7 Sterol, Delta-7 reductase activity and cross-immunoreactivity with protein A. thaliana above-mentioned protein Delta-7 Ed.

Protein can be detected, for example, by immunoassay using antisera directed against the protein Delta-7 Ed received well-known methods.

One aspect of the present invention relates to a protein having a Delta-5,7 Sterol, Delta-7 reductase activity, such as obtained by expression in a cell host containing a certain above the DNA sequence and, first of all, the protein of A. thaliana, such as obtained by expression in a cell is Q ID No. 2.

When the protein, which is the subject of the present invention, is produced by expression in a cell host, this is done using methods of genetic engineering and cell culture, well known to experts.

The expression can be carried out in prokaryotic cells-hosts, such as E. coli, or in eukaryotic cells-owners, for example, in the cell of a mammal, an insect cell or a yeast, comprising a sequence encoding a protein Delta-7 Ed, which is the subject of the present invention, which is preceded by an appropriate promoter.

The obtained recombinant protein can be glycolytically and neglikolizirovanny.

The present invention relates, in particular, to the protein of the subject invention, such as obtained by expression in yeast.

The present invention also relates to an antibody directed against a protein having above a certain Delta-5,7 Sterol, Delta-7 reductase activity. The specified antibody may be a polyclonal antibody or a monoclonal antibody obtained by methods well known to specialists.

The present invention relates tangermuende the specified vector.

The expression vectors are well-known vectors providing for the expression of the protein under the control of an appropriate promoter. In the case of prokaryotic cells, the promoter can be, for example, the lac promoter, the trp promoter, the tac promoter; the promoter-lactamase or the promoter PL. In mammalian cells the promoter may be the SV40 promoter or the promoters of the adenovirus. Baculovirus vectors can also be used for expression in insect cells. In the case of yeast cells the promoter can be, for example, the PGK promoter, the ADH promoter, the promoter SUS or the promoter GAL10/CYC1.

Cells-owners can be prokaryotic cells or eukaryotic cells. Prokaryotic cells are, for example, E. coll. Bacillus or Streptomyces. Eukaryotic cell hosts include yeast and filament fungi, and cells of higher organisms, such as mammalian cells or insect cells. Mammalian cells can be cells Cho hamster or Cos cells monkeys. The insect cells are, for example, SF9 cells. The yeast cells can be, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe or Kluyveromyces lactis.

The object of the present invention is also a method of cloning a nucleic kikosi method of screening, selected among:

- resistance of a microorganism to nystatin or similar connection, the toxicity of which depends on the presence of sterols containing unsaturated bond at position C-7

hybridization of nucleic acid with the nucleotide sequence of the above sequence SEQ ID No. 1,

- identification of nucleic acid information by additional DNA sequences isolated arbitrarily,

- a direct expression of the protein and subsequent immunological detection using antibodies directed against a protein having the above amino acid sequence SEQ ID No. 2.

Under microorganism understand yeast, such as S. cerevisiae, S. pombe, or K. lactis.

Compounds similar to nystatin contain, for example, amphotericin b or filipin.

Under the hybridization understand hybridization at medium or high temperature hybridization in normal conditions, well known to experts (above T. Maniatis with co-authors).

The identification information can be carried out by, for example, using the above program BLAST.

Description example casanuova below, in the experimental part.

The object of the present invention is also a sequence of nucleic acid, DNA or RNA, such as obtained above method of cloning. The nucleic acid sequence may be a prokaryotic or eukaryotic origin, depending on the material, on the basis of which was carried out cloning, for example, of human origin.

The object of the present invention is a host cell transformed by a vector containing the DNA sequence obtained using the above-described method of cloning. The host-cell can be a prokaryotic cell or eukaryotic cell. Examples of host cells and vectors cited above.

The object of the present invention is primarily a host cell selected among yeast or filament of a fungus transformed by a vector containing a DNA sequence which is the subject of the present invention as defined above, or a DNA sequence, such as obtained by the method described above cloning.

One of the objects of the present invention relates also to method the formed cage-master, which is the subject of the present invention, and allocate the expressed protein and, primarily, to the way in which the host-cell are transformed yeast in which the coding sequence of the DNA is under the control of the promoter of the yeast.

One of the objects of the present invention relates also to a method of recovery in vitro Sterol, unsaturated in position C-7, which is incubated for Sterol that is to be restored, with the protein obtained by the method described above, after which the recovered Sterol can be selected.

One of the objects of the present invention relates also to a method of recovery in vitro exogenous Sterol, unsaturated in position C-7, which is incubated for Sterol transformed with the host-cell, which is the subject of the present invention, with the possibility of allocating the received recovered Sterol. The host-cell can be a prokaryotic cell or eukaryotic cell, in particular a yeast or filament fungus.

One of the objects of the present invention also relates to a method for recovery of in vivo endogenous unsaturated in position C-7 of Sterol, which cultivar or filament of a fungus, with the possibility of subsequent allocation of accumulated restored Sterol.

The present invention relates, primarily, to a certain above recovery method in vitro or in vivo, which received the restored Sterol is a substrate of the enzyme cleavage of the side chain of cholesterol (P450SCC), and, first of all, relates to a method for recovery in vivo, in which the endogenous Sterol, irreparably damaged, is ergosta 5,7 diene 3-ol, ergosta 5,7,24(28) triene 3-ol or ergosta 5,7,22 triene 3-ol or a mixture.

The object of the present invention is also a method of obtaining pregnenolone in which cultured transformed cell hosts, which are the subject of the present invention, selected among yeast or filament of a fungus, with the possibility of subsequent allocation of accumulated restored endogenous Sterol (accumulated restored endogenous sterols) in position C-7, and then incubated restored sterols in the presence of P450SCC and possibly in the presence of adenotonsillectomy (ADR) and adrenodoxin (ADX), with the possibility of allocating the received pregnenolone.

The subject of this izaberete Vlada yeast.

The present invention relates also to method of obtaining pregnenolone, which is cultivated yeast, transformed with one or more vectors, providing coexpression protein having a Delta-5,7 Sterol, Delta-7 reductase activity, and P450SCC, and possibly ADR and ADX, with the possibility of allocation of free or esterified of pregnenolone.

The present invention relates, in particular, the above-described method, in which the cultured transformed yeast, coexpression protein having a Delta-5,7 Sterol, Delta-7 reductase activity, P450SCC, ADR and ADX, and, mainly, to the above method, in which a protein having a Delta-5,7 Sterol, Delta-7 reductase activity is protein A. tagliani, Delta-7 Ed, and, primarily, to the above method, in which the yeast strain is a strain EC01/pCD63.

Examples of obtaining pregnenolone in accordance with the present invention are provided below in the experimental part.

Transformed yeast used for implementing the above method of producing pregnenolone may be, for example, yeast, cotransformation very protein, having a Delta-5,7 Sterol, Delta-7 reductase activity and expression vector of cytochrome P450SCC and possibly ADX and ADR. A description of known vectors in the expression of cytochrome P450S and/or ADX is, for example, in patent application EEC EP 0340878, and also below in the experimental part.

Used transformed yeast can also be yeast, in which the DNA sequence encoding a protein having a Delta-5,7 Sterol, Delta-7 reductase activity, integrated into the private gene locus, for example in the ADE2 locus, and in which ergosterol is the main Sterol in terms of the expression of the Delta-7 reductase. The resulting “integrated” yeast can then be transformed using the integrating expression cassettes or expression vectors containing the DNA sequence encoding the cytochrome P450SCC and possibly ADX and ADR.

An example of the construction of yeast strains producing in vivo pregnenolone or complex exploitedby ether are provided below in the experimental part.

Thus, the object of the present invention is also strain transformed yeast coexpression protein having a Delta-5,7 of STERI the new, in particular the above yeast strain, in which a protein having a Delta-5,7 Sterol, Delta-7 reductase activity is a protein of A. thaliana, Delta-7 Ed and, first of all, yeast strain, denoted as EC01/D63, the detailed construction of which are provided below in the experimental part.

The present invention also applies to the sequence of human DNA, such as obtained by the above method clone used as a probe for the diagnosis of congenital lack of Delta-5,7 Sterol, Delta-7 reductase. The lack of Delta-5,7 Sterol, Delta-7 reductase inhibitor includes, for example, the lack of 7-dehydrocholesterol, causing abnormally low levels of cholesterol in the plasma.

The present invention relates also to a method for identifying shortage Delta-5,7 Sterol, Delta-7 reductase, comprising incubation of a sample containing human genomic DNA using the probe indicated above, in standard hybridization and detection of fixing or not fixing the probe to genomic DNA, no fixing or restoring it, pointing to a congenital lack of the Delta-5,7 Sterol, Delta-7 reductase.

The method according izobretali the newborn, as well as in patients suffering from various diseases, particularly in patients with clinical manifestation of the syndrome RSH/SLO.

The accompanying drawings illustrate certain aspects of the present invention:

In Fig. 1 presents the General profile of sterols extracted from clone F22, resistant to nystatin, obtained by the screening of the Bank of A. thaliana in yeast FY 1679. The analysis was carried out by RP-HPLC at 205 nm or 285 nm (Fig. 1A) or by the method of CPG by comparison with the untransformed yeast FY1679 (Fig 1B).

In Fig. 2 shows the restriction map of the fragment is Not the first plasmid pF22 and strategy to determine the sequence of bases in DNA.

In Fig. 3 presents the nucleotide sequence of cDNA Delta-7 Ed (SEQ ID No. 1) and corresponding amino acid sequence (SEQ ID No. 2).

Fig. 4 illustrates the measurement of in vitro activity of Delta-5,7 Sterol, Delta-7 reductase microsome fraction FY1679 transformed inducible expression vector Delta-7/V8”. The analysis is performed by CPG and shows the transformation of the substrate 7-dehydrocholesterol (Tg=16,528) cholesterol (TR=15,887) in the presence of endogenous sterols of ergosta 5,22 Dien 3-ol (TR=16,682); ergosterol (TR=17,249); Argostoli (Fig. 5A); ergosta 5,24 (28) Dien 3-ol (5B) or ergosta 5,22 Dien 3-ol (5C), purified on the basis of the transformed yeast expressing Delta-7 Ed, followed by incubation in the presence of P450SCC, ADX and ADR. The analysis is performed by means of CPG comparison with a control cleavage of cholesterol (solid line).

In Fig. 6 shows the construction of an integrative plasmid D7 that integrate cDNA encoding a Delta-7 Ed (Sterol C reductase), in the ADE2 locus.

Fig. 7 represents the analysis of CPG total sterols extracted by saponification with alkali from strain FY1679 and integrated strain ELR01, cultured in the presence of galactose.

In Fig. 8 schematically shows the construction of a Shuttle vector E. coli-S. cerevisiae V13 containing the specific site of Sal I (Sa) mnogocentrovom sector clone.

In Fig. 9 presents the stages of building integrative plasmids pCD62-1 and pCD62-2 to enable integrating the expression cassette of ADR in the intergenic area leu2 and sl1.

SCM1 and SCM2: multiple cloning sites.

In Fig. 10 shows the strategy of building strain CDR01.

In Fig. 11 shows the steps of constructing a plasmid expression RSO containing both kayh of cells (cell lysate) or culture medium (environment), isolated from strain EC01/D63 after induction by galactose for 9 h (Fig. 12A) or 24 h (Fig. 12B).

In Fig. 13 shows the structure of plasmid pTG 7457.

In Fig. 14 shows the structure of plasmid pTG 7453.

In Fig. 15 shows the structure of plasmid pTG 10014.

In Fig. 16 shows the structure of plasmid pTG 10004.

In Fig. 17 shows the structure of plasmid pTG 10031.

In Fig. 18 shows the structure of plasmid pTG 10033.

The following examples illustrate the present invention, however, limiting it.

EXAMPLE 1: Cloning of a cDNA that encodes a Delta-5,7 Sterol, Delta-7 reductio (Delta-7 Ed) A. tagliani

A - Bank Screening expression A. tagliani in yeast

The original Bank of cDNA expression represents the Bank described M. Minet with co (Plant J., 2, 417-422, 1992), which was prepared on the basis of A. thaliana mRNA during germination stage two leaves, the cDNA of which is limited by the Not I site were included in the Bst section XI cassette expression Shuttle vector for E. coli/S. cerevisiae pFL61. This cassette contains sequences of the promoter and terminator of the gene phosphoglycerate (PGA). The replication origin, etc is the sector in E. coli is based on the plasmid pUC19.

Yeast strain FY 1679 (Mat), which is an isogenic strain strain S288C and described A. Thierry with co-authors (Yeast, 6, 521-534, 1990), was transformed with the cDNA Bank using the method based on the use of Liliaceae described D. Gietz with co-authors (Nucleic Acids Res., 20, 1425, 1992).

Cells were deposited on the surface of the synthetic environment SGI containing 7 g/l yeast nitrogen base” (Difco), 1 g/l of Baccaratcasinoonline (Difco), 20 g/l glucose, 20 mg/l tryptophan and lacking uracil. Received 105prototrophic transformed to uracil, which were grouped and re-deposited on the surface of the same synthetic medium without uracil and containing 2 or 5 μg/ml of nystatin; 5×104cells on the Cup. Thus were enumerated 106cells for each concentration of nystatin. After 3 days incubation at a temperature of +28°With the received approximately 100 clones were grown at a concentration of nystatin in 2 µg/ml and reached a group of 5 clones, Sterol composition of which was subjected to analysis of high-performance method obraniakowi liquid chromatography (denoted further RP-HPLC),SS="ptx2">B - Analysis of sterols accumulated in the clone F22

Total sterols yeast is obtained using the method of saponification with alkali, described L. Parks with co-authors (Methods in Enzymol, 111, 333-346, 1985), then it is analyzed using RP-HPLC and/or gas chromatography (denoted CPG).

Received Sterol residue dissolved in a mixture of ethanol - tetrahydrofuran - water (65:10:25 by volume), and then analyzed by the method of column chromatography RP-HPLC on silica With 18 Applied Biosystems (100×2.1 mm) with a flow rate of 1 ml/min and at a temperature of +55°C with a linear gradient of methanol in water (from 50% to 100% over 18 min) and photometric analysis at 205 nm and 285 nm in comparison with standard values for ergosterol, campestrini and cholesterol.

Sterol composition also analyze the method CPG on capillary column SE-30 Alltech (30 m × 0.32 mm) using helium as carrier gas at a temperature of +280°C and +310°, respectively, for injector and detector, with the initial temperature rise to +110°C to +245°C at a speed of 45°C/min, and then 3°C/min to achieve temperature +280°C.

The analysis method RP-HPLC (Fig. 1A) and the analysis of CPG (Fig. 1B) show the profile of sterols accumulated is shaped strain FY 1679 and its replacement by two major sterols in the same number, which do not absorb at 285 nm and, therefore, no longer have the dual conjugate unsaturated bond according to the analysis method RP-HPLC.

In Fig. 1A campestrin (Sigma) (24-To-ergosta 5-ene 3-ol) contains approximately 35% dehydroreticuline (24-3-ergosta 5-ene 3-ol).

In the cDNA Cloning of the Delta-7 Ed

Plasmids derived from clone F22, were amplified in E. coli in accordance with the method described by J. N. Strathern with co-authors (Methods in Enzymol., 194, 319-329, 1991), and split Not I. Were obtained fragment of about 600 base pairs (p. O.) and a fragment of 1.6 thousand p. O. Strain FY1679 was transformed, respectively, each of the above fragments. Sterol composition of each clone of the transformed yeast was subjected to analysis as described above, and allowed to identify plasmids containing the gene responsible for changing the Sterol profile. Identified in this way plasmid was named pF22.

Mr. sequencing cDNA Delta-7 Ed

The insert cDNA pF22 was subcloned in the website Not I vector pUC9N derived from pUC9 (Pharmacia), in which the plot Eco RI multiple cloning site is replaced by a box plot of the Not I restriction while maintaining the phase considered is the current plot Not I and internal EcoRI sites, PvuII or Hindi II, respectively, were subjected to subclavian in the plasmid pBLuescript (Stratagene). The nucleotide sequence was determined by the method of Sanger DNA polymerase, Sequenase (set Stratagene) on both strands, using direct and inverse seed pUC9, T3 and T7 pBLuescript or specific nucleating inferred from nucleotide sequences of cDNA.

Preparation of aggregate obtained sequences gives the nucleotide sequence of cDNA Delta-7 Ed A. thaliana (SEQ ID No. 1) shown in Fig. 3. It includes 1496 nucleotides, ending with a polyadenylation sequence. It is an open reading frame beginning with the initial methionine from nucleotide 76 and ending with the termination codon with a nucleotide 1366. As a result, there is an open phase read from 1290 nucleotides encoding a protein of 430 amino acids. The coding region cDNA Delta-7 Ed encodes a protein Delta-7 Ed deduced amino acid sequence of which (SEQ ID No. 2) is shown in Fig. 3. The protein sequence contains 430 amino acids with a calculated molecular mass of 49,286 kDa.

A sample of E. coli strain DH5-1 containing cDNA Delta-7 Ed in the vector pUC9N (designation: cDNA del the Astaire) number 1-1535.

D - Determination of the consensus sequence SEQ ID no 3

By information retrieval sequences in the databases (Genbank and EMBL) revealed that the protein Delta-7 Ed A. thaliana has some sequence similarity with other Sterol-inhibitors, in particular with the Sterol C-14 reductase and Sterol C-24 (28) reductase in S. cerevisiae, which are described respectively in R. T. Lorentz co-authors (DNA Cell Biol., 11, 685-692, 1992) and W. Chen and co-authors (Yeast, 7, 305-308, 1991), as well as with the product of the gene sts 1+S. pombe, the description of which give M. Shimanuki with co (Mol. Biol. Cell, 3, 263-273, 1992) and Sterol C-14 reductase in Neurospora crassa (No. H in the EMBL database). In addition, the protein Delta-7 Ed has similarities to 400 amino acids C-terminal part of the receptor lamina In chicken and with an end section of the corresponding receptor of a person described in N. J.Worman with co-authors (J. Cell Biol., 1ll, 1535-1542, 1990) and in E. Schuler with co-authors (J. Biol. Chem., 269, 11312-11317, 1994).

Were placed a series of sequences, proving the identity between the amino acid sequence SEQ ID No. 2, derived from cDNA obtained above Delta-7 Ed, a sequence of three yeast steinrucken and two receptors lamina In, then was ODA CLASS="ptx2">

where XAA at position 1 represents the amino acid TRP or Tyr and XAA in position 12 is a GIS or Liz to obtain oligonucleotides used as seed for amplification by PCR of new sequences in the genomic DNA or cDNA encoding the protein having a Delta-5,7 Sterol, Delta-7 reductase activity.

E - the expression of the protein Delta-7 Ed in yeast

a) construction of the inducible expression vector Delta-7/u in yeast

The deletion of the non-coding areas cDNA P22 was carried out by the method of PCR amplification using the following specific oligonucleotides:

5' TGCGGAGTCC TGGGCGGAAC THTATS 3' (SEQ ID No. 4) and

5' TIGHTTTT CAAAAAT CHHATH 3' (SEQ ID NO. 5),

who were determined to enter the site enzyme BamH I directly after the initial codon and site Kpn I directly below the stop codon.

cDNA was amplified using 1 ng plasmid “cDNA Delta red/UC9N” in the presence of 2 units of Pfu DNA polymerase and 0.2 μm each of the above nucleating with the following amplification conditions: +94°C, 10 C, +52°C, 50 C, +74°, 90; 33 cycle using niiccee of Wamn I and I and the PKK switching in the areas of Wamn I and the PKK I Shuttle vector E. Coli/S. cerevisiae pYeDP 1/8-2 (designation: V8), described in C. Cullin with co-authors (Gene, 65, 203-217, 1988). V8 includes marker selection and contains the URA3 cassette expression in yeast containing the promoter GAL10/CYC1 and termination sequence of the PGK gene. The resulting vector, denoted by Delta-7/V8, allows inducible expression of the galactose protein Delta-7 Ed.

b) obtaining the protein Delta-7 Ed

Yeast strain FY 1679 (Mata) was transformed with the above plasmids Delta-7/V8 method using litigated described in D. Gietz with co-authors (cited above).

Transformed yeast were cultivated at a temperature of +27°C in the above-described selective medium SGI, but in which the glucose concentration is 5 g/l, to obtain the saturation cell density (D600Nm=12). Then the culture was diluted by adding volume full environment YP (10 g/l yeast extract (Difco) and 10 g/l of bactopeptone (Difco)), followed by the addition of ethanol (1/5%) as carbon source. When growth is allowed to reach a cell concentration of at least 7×107cells/ml, the expression of the Delta-7 Ed was raised Downey environment, SLI, appropriate environment SGI, in which glucose is replaced by galactose (20 g/l) up to obtain a cell concentration of 2×107cells/ml

C) Enzymatic test In vitro Delta-5,7 Sterol, Delta-7 reductase activity

Expression of the protein Delta-7 Ed was detected using an enzymatic test described M. Taton with co-authors (Biochem. Bioph. Res. Commun., 181, 465-473, 1991), however, without a system of regeneration of NADPH, microsome or cytosolic cell preparations of the above-induced yeast.

Cell fractions were obtained by mechanical crushing of induced cells and isolation of fractions by the method of ultracentrifugation, as described by R. Urban co-authors (Eur. J. Biochem., 222, 843-850, 1994). Cells are harvested and then washed twice using buffer. THOSE KCl (Tris-Hcl 50 mm, pH 7.4; 1 mm EDTA, KCl 0.1 M) and suspended in lysis buffer TE-sorbitol 0,6 M Then added glass beads with a diameter of 0.45-0.5 mm (Braun) until the surface of the cell suspension, followed by stirring for 5 minutes at a temperature of +4°C. the Cell lysate on the surface of the collect, and glass beads washed three times using lisanova buffer. The lysate and wash are combined and centrifuge holds the exploration fluid is centrifuged for 1 h at 100000 g at a temperature of +4°C. The precipitate containing the microsome fraction and the supernatant, which represents the cytosolic fraction is collected separately.

Received microsome fraction or cytosolic fraction, respectively, incubated for 90 min at a temperature of +37°C in buffer Tris/Hcl, 100 mm at pH of 7.3, containing as substrate 7-dehydrocholesterol 150 μm, emulsified with tween 80 (1.5 g/l) and in the presence of 2 mm NADPH. Sterols extracted by adding 3 volumes of a mixture of methanol and dichloromethane (50:50 by volume), and then perform the analysis of CPG, comparing with standard products.

The formation of cholesterol (TR=15,887 min) on the basis of 7-dehydrocholesterol (TR=16,528 min) is shown in Fig. 4 microsome fraction (3.5 mg/ml protein), obtained as described above, on the basis of yeast FY 1679, the transformed vector Delta-7/VS, induced for 3 h, and endogenous sterols which have a longer time of trapping (TR=16,682 min for ergosta 5-22 Dien 3-ol; TR=17,249 min for ergosterol and TR=17,664 min for ergosta 5-ene 3-ol).

The results indicate that the protein Delta-7 Ed on the one hand, is expressed in the transformed yeast and vo endogenous sterols yeast unsaturated in position C-7.

Yeast strains, the main sterols which have a double bond at position C-5,7, were transformed by the vector Delta-7/V8, obtained in Example 1 and then were cultured and induced as described in Example 1. Endogenous sterols, which profile is analyzed by the method CPG, were extracted and purified by the method RP-HPLC as described in Example 1, using a preparative C18 column (100×4.6 mm), and then identified by IR-, UV-, CM -, and NMR methods. Accordingly, we used the following three strains:

A strain FY1679 described in Example 1;

- Mutant strain EGD denoted PLC 1051, characterized by lack of Sterol C-22 desaturase, which was obtained by crossing strain FY1679 and the original strain ro described in S. W. Morzahn with co-authors (J. Gen. Environ., 72, 339-348, 1972) and which accumulates ergosta 5,7-Dien 3-ol;

- Double mutant strain EGD,5, denoted by the PLC 1451, characterized by lack of Sterol C-22 desaturase (EGD) and Sterol C-24 (28) reductase (EGD), was obtained by crossing strain FY1679 and strain ro described in S. W. Morzahn with co-authors (listed above) who received spontaneous resistance to nystatin in the result is 4 (28) triene 3-ol. The resulting haploid strain containing the double mutation egg, egg, grows in the presence of galactose and incapable of fermenting substrate, and auxotrophic for uracil, tryptophan and histidine. Strains PLC 1051 and PLC 1451 were filed in NCCM February 10, 1995 under the numbers 1-1536 and 1-1537 respectively.

Basic reduced sterols, respectively, identified on the basis of the three above-mentioned transformed strains listed in the table below:

EXAMPLE 3: Getting pregnenolone in vitro by cleavage of endogenous sterols yeast, restored in position C-7

Pregnenolone get through enzymatic test cleavage of the side chain of cholesterol in vivo, described in Wada and co-authors (Arch. Biochem. Biophys., 290, 376-380, 1991), in which 260 μm Sterol, restored in position C-7 obtained in Example 2 is subjected to incubation in 150 μl of phosphate buffer 10 mm, pH 7.4, 100 mm Nad, tween 20 and 0.3% in the presence of 140 nm adenotonsillectomy, of 1.16 μm adrenodoxin and 0.68 μm cytochrome P450SCC of animal origin, derived from the adrenal glands of a bull, as described, for example, D. W. Seybert with co-authors (J. Biol. Chem., 254, 12088-12098, 1979).

The reaction zaposlednie one volume mixture of methanol and dichloromethane (50:50 by volume). Sterols are extracted by the method of CPG as described in Example 1.

In Fig. 5 shows respectively that ergosta 5-ene 3-ol (Fig. 5A), ergosta 5,24(28) Dien 3-ol (Fig. 5B) or ergosta 5,22 Dien 3-ol (Fig. 5C) is a substrate of cytochrome P450SCC and leads to a product having the time of trapping (TR), identical to TR pregnenolone obtained in the same conditions by cleavage of cholesterol.

The results obtained indicate that the transformed yeast expressing Delta-7 Ed, accumulate sterols used directly to obtain pregnenolone by biological oxidation in vitro.

EXAMPLE 4: Construction of yeast strains producing in vivo pregnenolone or complex exploitedby ether

And Construction of strain ELR01 containing a cassette for the expression of the Delta-7 Ed A. thaliana, integrated into the ADE2 locus haploid strain FY1679 mating type a(FY1679 Mat)

a) Constructing an integrative plasmids Raphaelite-7 (D7)

Construction of plasmids pAD7 was conducted, as shown in Fig. 6. The BglII fragment (2244 p. O.), containing the ADE2 gene of S. cerevisiae was isolated from the plasmid pASZ 11 (A. Stotz with co-workers. Gene, 95, 91, 1990) and included in the vector pBluescriptII KS+ (Stratagene) is m linearized at the unique and dephosphorylating the website Stu I.

A fragment of approximately 2,44 thousand base pairs, containing the promoter GAL10/CYCl encoding phase Delta-7 Ed (Sterol C reductase) and the PGK terminator (tPGK), was obtained from the plasmid Delta-7/V8, obtained in Example 1E by PCR, using as seed the following oligonucleotides:

5' GATTACCA ATTCHA AAC 3' (SEQ ID No. 6).

5' AGARTTHA AGATCH SIZEAAA 3' (SEQ ID NO. 7)

identified for conjugation, respectively, with the end of the 3' gene terminator tPGK and the end of the URA3 gene. Were used for plasmid Delta-7/VS as a matrix (80 ng), the above oligonucleotides (0.5 μm each), native Pfu DNA polymerase (1 unit in the buffer recommended by the company Stratagene) with the following amplification conditions: 35 cycles; 1 min at a temperature of +95°C; 5 s at +95°C; 30 s at +56°C; 4 min 30 s at +70°C). Then the obtained fragment amplification was cloned with a clear limitation in the above linearized plasmid pBS-ADE2 to obtain plasmids D7 in which the NotI fragment- > PST from approximately 4720 p. O. contains a gene ADE2, not interrupted by the expression cassette of the Delta-7 Ed.

b) Chromosomal integration into the yeast strain FY1679 Mat

The NotI fragment- > PST (4720 p. O.) is informatsii method, using litigated described in D. Gietz with co-authors (cited above).

Transformants which integrated the specified fragment by homologous recombination were selected by their resistance to nystatin, and this phenomenon is caused by the expression of the Delta-7 Ed by transformation of the yeast Delta-5,7 Sterol Delta-5 sterols.

Transformed cells were subjected to incubation for 4 h at a temperature of +28°C in complete medium YP described in Example 1E, containing glucose (20 g/l) and an additional adenine (20 mg/l). They were then concentrated and applied on a minimum SLI-agar medium (1 g/l of Baccaratcasinoonline; 7 g/l yeast nitrogen base”; 20 g/l galactose; 20 mg/l adenine; 20 g/l agar, and then incubated for one night at a temperature of +28°C for the induction of gene expression of the Delta-7 Ed. No Aracinovo supplements allows you to limit the growth of cells. Clones were collected, grouped and plotted on a complete environment YP containing galactose (20 g/l) and an additional adenine (20 mg/l), and in the presence of increasing concentrations of nystatin (0 μg/ml, 1 μg/ml, 2 μg/ml, 5 μg/ml, 20 μg/ml, respectively). On the fourth day it was received irenesany to capacity with minimal WO-medium (7 g/l yeast nitrogen base without amino acids; 20 g/l glucose), enriched with uracil, leucine, tryptophan, histidine and adenine (20 mg/l each).

Auxotroph to adenine, caused by violation of the ADE2 gene was subsequently confirmed by the absence of growth above the minimum WO-environment enriched with uracil, leucine, tryptophan, histidine, but lacking adenine.

The presence of the expression cassette in the genome of the yeast was verified by amplification by PCR-based genomic DNA of the obtained clones using the seed, including the abovementioned sequences SEQ ID No. 6 and SEQ ID No. 7.

The functionality of the integrated gene Delta-7 Ed was confirmed by the analysis method CPG content of sterols accumulated in yeast and extracted by saponification with alkali in accordance with the method described in Example 1B, using 5-meter capillary column SE-30 (Alltech).

The analysis revealed a modified profile containing saturated sterols in position C-7 in those cases where the clones were cultured in the presence of galactose. The resulting strain, designated ELR01, accumulate ergosta 5 Yong-3-ol and ergosta 5,22 Dien 3-ol instead of ergosta 5, 7, 22 triene 3-ol (ergosterol), the basic degree which induces gene Delta-7 Ed in cases when it is cultivated in the presence of galactose, due to trascriptional control promoter GAL10/CYC1. Although the unit of expression of the Delta-7 Ed has the same transcriptional direction as the ADE2 gene, no expression of the Delta-7 Ed not detected in the analysis of the content of sterols method CPG, when the cultivation is carried out in the presence of glucose due to repression of the promoter GAL10/ CYC1.

B - Construction strain CDR01 containing the expression cassette Mature forms bullish adenotonsillectomy (ADRm), integrated between LEU2 loci and SPL1 haploid strain FY1679 mating type alpha (Mat alpha)

a) Construction of Shuttle vector E. coli - S. cerevisiae V13

Vector V13 corresponds to the vector V8 (C. Cullin al. cited previously), including the marker URA3 selection and cassette expression in yeast containing the promoter GAL10/CYC1 (pG/C), in which an additional plot Sal I (Sa) was introduced into the multiple cloning site, in accordance with the circuit construction shown in Fig. 8.

Vector V8 was cleaved with restriction enzymes Hind III and BamH I, and the resulting fragment BamH I - Hind III (1722 p. O.) containing the URA3 gene and the promoter GAL10/CYC1 (also called “ur-Gal”), was subcloned between segment “ur-Gal was then subjected to amplification by PCR on the basis of 30 ng of the obtained plasmid pUC18/“ur Gal”, denatured for 30 s at a temperature of +95°C under the following conditions: 30 cycles; 5 temperature (+86°C; 10 s at +95°C; 40 s at 38°C; 5 s at +55°C and 2 min at +74°C, 2 units Taq DNA polymerase (Boehrunger) in the buffer of the manufacturer and 1 μm of each of the blades comprising the following nucleotide sequences:

5' GGATCC of GTCHAT ATTACHT THATTTT TGCG 3' (SEQ ID 8) and

5' TEAACHER GZCAT 3' (SEQ ID NO. 9)

The seed SEQ ID No. 8 includes one plot of Wamn I GGATCC identical to the plot fragment “ur Gal”, 3 consecutive nucleotide at the site of UMN I, not hybridizers matrix, one plot Sal I GTCHAT and one sequence homologous to the sequence of the promoter GAL10/CYC1. The seed SEQ ID No. 9 conjugated with the previous sequence plot Hind III multiple cloning site of the vector pUC18. Fragment Hind III - BamH I from 1722 p. O. obtained after amplification, was split Xho I m Barn HI, releasing a fragment of 254 p. O., containing the promoter GAL10/CyC1 (pG/C), which was then subcloned in the vector V8, split by enzymes BamH I and Xho I.

The resulting vector V13 contains the restriction sites that allows you to easily subclinical K0The SCC.

On the basis of the vector V13 were respectively obtained as described below, the vector V13-ADR, vector V13-ADX and vector V13-SCC10:

) Construct a vector V13-ADR

One piece of Sal I - I of the PKK 1478 p. O. containing cDNA encoding ADRm, was isolated from the plasmid pGBADR-2 described in Example 25 of the patent application EEC EP 340878, and subcloned into the corresponding sites of vector V13 to obtain vector V13-ADR.

) Construct a vector V13-ADX

Fragment Sal BamH I from 390 p. O. containing cDNA encoding ADXm, was isolated from the plasmid pGBADX-1 described in Example 23 the patent application EEC EP 340878, and subcloned into the corresponding sites of vector VI3 to obtain vector V13-ADX.

) Construct a vector V13-SCC10

Fragment Sal I - EcoR I from 1554 p. O. containing cDNA encoding P450S, was isolated from the plasmid pGBSCC-10, described in Example 6 of the patent application EEC EP 340878, and subcloned into the corresponding sites of the vector VI 3 to obtain the vector V13-SCC10.

C) Designing integrative plasmids pCD62-1 and pCD62-2

Construction of plasmids pCD62-1 and pCD62-2 was carried out, as shown in Fig. 9.

) Construction of plasmids pFL26CD

the General leu2 gene from the end of the 5' gene spl1 (denoted s11) (C. Kolman with co-workers, J. Bacteriol., 175, 1433, 1993), by the following method.

Two fragments of DNA from 704 p. O. and 347 p. O., containing, respectively, the end of the 5' gene ieu2 and the end of the 3' gene sl1, were synthesized by PCR using a seed having the following sequences nucleotides:

5' THOUGHTS ACUTRACK GATT 3' (SEQ ID No. 10) and

5' GTGGTGGTGG CCCCCCCCCC TRATATA THTAI 3' (SEQ ID NO. 11)

for amplification of a fragment from 704 p. O. and nucleotide sequences

5' CAAGCAGCAG of GZZZZZ AAAAAAH GTGT 3' (SEQ ID No. 12) and

5' TZTZTZ TAGATZ TATG 3' (SEQ ID NO. 13)

for amplification of a fragment from 347 p. O.

Priming SEQ ID No. 11 and SEQ ID No. 12 include a sequence GGGGG corresponding to the Not I site and 3 reasons not konjugierte with the matrix. The seed SEQ ID No. 10 conjugated with a sequence located at 536 p. O. above the stop-codon leu2, and the seed SEQ ID No. 13 includes a sequence located at 194 p. O. above the stop-codon sl1.

To start the fragments of 704 p. O. and 347 p. O. amplified by PCR using as template a plasmid pFL26 and as enzyme - Pfu DNA-p is x fragment konjugierte 20 p. O. at the level of ends generated by priming SEQ ID No. 11 (fragment from 704 p. O.) and priming SEQ ID No. 12 (a fragment of 347 p. O.) on the basis of the end of the 5'; these 20 p. O. correspond to 20 to the first nucleotide of each of the blades, respectively.

The product resulting from the conjugation of DNA fragments from 704 p. O. (1 ng) and 347 p. O. (2 ng) was amplified using nucleating SEQ ID No. 10 and SEQ ID No. 13 in compliance with the following conditions: 30 cycles; 10 s at a temperature of +95°C, 5 s at +60°C, 1 min at +45°C, 5 s at +65°C and 2 min at +72°C, followed by one cycle of 7 min at a temperature of +72°C; 50 pmol of each seed and 1 unit of Pfu DNA polymerase in 50 ál reaction buffer (Stratagene). The result was obtained amplificatory a fragment of 1031 p. O., including the site of rupture Not I. Then the specified fragment was cleaved by enzymes Bst XI and Nsi I and the resulting fragment of 920 p. O., containing a Not I site, was included in place of the original fragment Bst XI - Nsi I plasmids pFL26 to obtain plasmids pFL26CD, the map of which is shown in Fig. 9a.

) Construction of plasmids D60

Obtaining plasmids DP10036

Fragment Sal I - BamH I from 390 p. O. containing cDNA encoding a Mature bull adrenodoxin (ADXm), was isolated from the plasmid is vtoroy are inducible promoter GAL10/CYC1 and terminator terPGK. Plasmid pTG10033, the map of which is shown in Fig. 18 and which corresponds to the expression vector pTG10031 (Fig. 17) containing the CYC1 promoter and terPGK, in which CYC1 promoter was replaced with the promoter GAL10/CYC1, was obtained in accordance with the methodology described below.

Thus obtained plasmid, denoted pDP10034 contains an expression cassette ADX, i.e. the gene encoding ADXm, under the transcriptional control of the GAL10/CYC1 and terPGK. Further, the term “expression cassette” is used for all genes in a transcription based on GAL10/CYC1 and terPGK.

Fragment Hind III 3593 p. O. containing the marker URA3 selection and the expression cassette ADR, was isolated from the plasmid V13-ADR cleaved by the restriction enzyme Hind III, and then entered into the corresponding site of plasmid pDP10034 cleaved by the restriction enzyme Hind III. The resulting plasmid, denoted pDP10036 contains expression cassettes ADX and ADR, separated from one another marker URA3 (Fig. 9b).

Obtaining plasmids D6

Fragment Afl III - ACC I from 2770 p. O., containing the cartridge ADR, was isolated by partial cleavage of plasmid pDP10036 the enzymes Afl III and ACC I, with a clear limitation due to processing fragment Klena (Stratagene). In the resulting plasmid, denoted pCD60, the expression cassette ADR is limited on two sides by areas Not I, one of which is located at 209 p. O. above the site of ligation Afl III/Sma I and comes from a subcloned fragment, and the other comes from the multiple cloning site (SCM1) plasmid pBlue-Script KS+ (Fig. 9b).

) Construction of plasmids pCD62-1 and pCD62-2

The Not I fragment from 2558 p. O. isolated from plasmid pCD60 cleaved by the restriction enzyme Not I, was then subcloned into the unique Not I site of plasmid pFL26CD. Depending on the direction of embedding fragments were obtained two plasmids, denoted pCD62-1 and pCD62-2 (Fig. 9b).

Plasmid pCD62-l expression cassette ADR oriented in transcriptional direction leu2 gene, while the specified orientation in the case of plasmids pCD62-2 has the opposite direction.

For further designing was selected plasmid pCD62-1.

b) Chromosomal integration into the yeast strain FY1679 (Matalpha)

Plasmid pCD62-1 includes areas homologous chromosomal locus of strain FY1679. These zones correspond to fragments Qg1 II - l I from 1060 p. O. (A), EcoR I - Not I from 707 p. O. () and Not I - Qg1 II of 602 p. O. (C), respectively, as shown in Fig. 10.-) (R. S. Sikorski and co-authors. Genetics, 122, 19, 1989).

Plasmid pCD62-l was linearized by cleavage with restriction enzyme b I, the area of the gap which is located outside of the homologous areas, after which it was introduced by transformation into strain FY1679 (Matalpha) using a method that uses Liliaceae (D. Gietz al. cited above).

The ability to repairbot cellular DNA with yeast gap repair”) and selection of recombinants with phenotype LEU2+allowed to take at the first stage, two types of recombinants: the first type is obtained as a result of homologous recombinations at the level of fragments a and b, and the second type was obtained as a result of homologous recombinations at the level of fragments a and C. Only the last type of recombination ensure integration of the expression cassette ADR in addition to the restoration of the phenotype LEU2+.

To select the specified second type of clone was performed screening by PCR using the above seed SEQ ID No. 10 and the seed that contains the following nucleotide sequence:

5' TAZETTA CCTTTGTGGT 3' (SEQ ID NO. 14)

with those who Amma FY1679 (Matalpha).

In the specified screening of the seed with SEQ ID No. 14 conjugated exclusively with a sequence that encodes a ADRm and the seed SEQ ID No. 10 conjugated with chromosomal sequence (Fig. 10).

Reaction amplification was performed using as template genomic DNA (20 ng) isolated from strain FY1679 (Matalpha) (C. Hoffman with co-authors, Gene, 57, 267, 1987), one unit of Taq DNA polymerase (Boehrunger), 50 pmol of each of the blades and 30 cycles of the PCR (10 s at a temperature of +95°C, 1 min at +55°C, 3 min at +72°C), under normal conditions, recommended by the provider.

Amplification resulted in the selection of 2.9 thousand p. O., in the case of integration of the expression cassette. Otherwise, there was no indication of product amplification.

Selected strain FY1679 (Matalpha) containing an integrated expression cassette ADR were identified CDR01.

Expression of ADR specified strain was identified on the basis of cytosolic cellular fractions obtained in accordance with the Protocol described in Example 1E, by immunoabsorbent protein recognized by antibodies anti-ADR.

Functionality ADR expressed strain CDR01, was confirmed in struggles in Example 3, in which purified ADR (0.28 pmol) was replaced by cytosolic cell fraction strain CDR01 content 100 µg total protein. It was observed bioconversion of cholesterol in pregnenolone about 25%, which is comparable with the level of conversion obtained with purified ADR.

In Designing a diploid strain IS, coexpressing Delta-7 Ed A. tagliani and ADRm

Diploid strain AS was obtained by crossing haploid strains CDR01 and ELR01 obtained as described above, in accordance with the Protocol described in G. Sprague and co-authors (Methods in Enzymology, 194, 77, 1991).

The first selection in the above-described minimal selective medium enriched with uracil, tryptophan and histidine (20 mg/l each), but lacking leucine, allowed us to isolate the diploid clones LEU2+(prototrophic a sign that indicates the presence of the expression cassette ADR). Then these clones were tested for resistance to nystatin at 5 µg/ml (a sign of resilience, indicates the presence of the cassette in the expression of Delta-7 Ed on a rigid synthetic SLI-agar medium, the description of which is cited above.

Thus, among the clones that are resistant to nystatin, was proizvod and pregnenolone 3-acetate

a) Constructing a plasmid expression pCD63

Construction of plasmids pCD63 was conducted, as shown in Fig. 11.

The Not I fragment from 4961 p. O., containing the expression cassette ADX, marker URA3 selection and the expression cassette ADR, was isolated from the plasmid pDP10036 obtained as described above, and split the restriction enzyme Not I, and then cloned at the Not I site in the multiple cloning site of the plasmid pFL45L (N. Bonneaud with co-workers. Yeast, 7, 609, 1991). The resulting vector, denoted pDP10037 shown in Fig. 11.

On the one hand, plasmid pDP10037 was linearized by cleavage enzyme TthlllI, the cleavage site which is located in a gene, encoding a ADRm.

On the other hand, a fragment of Pvu II - EcoRV from 3476 p. O., containing the expression cassette P450SCC and the end of the 5' marker URA3, was cleared on the basis of plasmids V13-SCC10 obtained previously and split by enzymes Pvu II and EcoRV.

Both linear, respectively obtained DNA have homologous regions, which correspond, on the one hand, the end of the 5' URA3 gene and the promoter GAL10/CYC1 and, on the other hand, the terminator terPGK, as shown in Fig. 11a.

These two fragments were then introduced into yeast strain FY1679 (MA Is S="ptx2">The following selection prototrophic recombinants for uracil and tryptophan (URA3+TRP1+) allowed us to identify clones in which double-strand break generated by the absorption of the restriction enzyme Tth111I, was reparieren as a result of integration of the expression cassette P450SCC by homologous recombination.

Selection of recombinants (U3+, TRP1+) was carried out on minimal selective medium WO enriched with leucine, histidine and adenine (20 mg/l each), but lacking uracil and tryptophan. On the basis of the 50 United clones total DNA was extracted by the method described in C. Hoffman and co-authors (Gene, 57, 267, 1987), and then introduced by electroporation into the strain E. coli XL1-Blue (Stratagene). Clones transformed by the plasmid generated by “the gift of repair, were selected on rich medium LB (tripton 1% extracted from yeast to 0.5%, NaCl 1%) containing 50 mg/l ampicillin. Based on one of the selected clones by the method described in J. Sambrook and co-authors (Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989) was extracted plasmid, denoted pCD63. The obtained plasmid D63 contains expression cassettes ADX and P450SCC separated from one another marketed pCD63 was introduced into the above strain IS by transformation method, using Liliaceae (D. Gietz coauthors, cited previously). Then the transformed yeast were cultivated on minimal medium WO, described above, and lacking uracil, tryptophan, adenine and leucine, but with the addition of histidine (20 mg/l).

Thus was selected strain, denoted EC01/pCD63. The sample strain designation EC01/pCD63 was filed February 10, 1995 at the CNCM under number 1-1538.

C) Obtaining in vivo pregnenolone and pregnenolone 3-acetate

Strain EC01/pCD63 was cultivated at a temperature of +28°With aerobiosis (130 rpm) in a 3-liter Erlenmeyer flask before reaching the stationary phase of growth (D0600 = 12 to 13) in the selective medium SGI described in Example 1A, and in which the glucose concentration is 5 g/L. Then the culture was diluted by addition of one volume of the above-described full environment YP and the subsequent addition of ethanol (0.5% by volume) as the carbon source. Upon reaching a new stationary phase of growth (D0600 = 12 to 13) in culture add galactose (20 g/l) in the form of a concentrated solution (500 g/l), in order to simultaneously induce the expression of genes encoding ADXm, ADRm, P450SCC and Delta-7 Ed, which are respectively under pusica respectively in the cells and in the supernatant of the culture, in accordance with the following method.

After 9 h and 24 h induction samples were taken of the cultures of 50 ml To separate cells from culture media each sample was subjected to centrifugation (4000 g, 10 min, +4°C).

On the one hand, the cells are lysed by mechanical grinding in the presence of glass beads according to the method indicated in Example 1E. On the basis of the thus obtained cell lysate sterols are then extracted by adding one volume of hexane.

On the other hand, the sterols present in the culture medium, extracted directly by adding one volume of hexane.

Content extracted sterols analyze method CPG as described in Example 1, in comparison with standard products.

The results obtained after 9 hours or after 24 hours of induction, are presented respectively in Fig. 12A and 12B.

Fig. 12A with the results obtained after 9 hours of induction shows:

- the presence in the cell lysate of the basic compounds with the same time retaining that standard pregnenolone (TR=11,8 min), while in time of retention pregnenolone n is endogenous sterols in position C-7 (ergosta 5-ene 3-ol and ergosta 5,22 Dien 3-ol), identified respectively with TR=18 min and TR=17 min Saponification of cell lysate before analysis leads to the presence of a basic compound, camiglibose with pregnenolone. This gives you the opportunity to confirm that the accumulated connection, TR is equal to 11.8 min, corresponds to pregnenolone;

- significant absence in the culture medium of pregnenolone or its acetate.

Fig. 12B with the results obtained after 24 hours of induction, shows:

- the presence in the cell lysate negligible amounts of pregnenolone (TR=10,2 min) and pregnenolone (TR=12 min), and reduced yeast endogenous sterols (TR=17 min and TR=18 min). Cholesterol (TR=16,2 min) is the internal standard added before extraction;

- the presence in the culture medium mainly pregnenolone and small amounts of pregnenolone.

The experiments conducted in parallel with the strain IS transformed with control plasmid, such as indicated previously pFL45L, did not show any peak corresponding to pregnenolone, free or in the form of acetate.

Designed identification of sterols shows that yeast sohanny sterols, after induction in the presence of galactose with significant production after 9 hours of induction.

These results show, on the one hand, the effective mobilization of reduced endogenous sterols in regulation s-1 strain IS and, on the other hand, the efficiency of binding reactions section of the side chain of endogenous sterols.

Preparation Example 4: Construction of plasmids pTG10033

1. A derivative of pUC19 with a new multi-site alonsomania:

Vector cloning MMR (C. Yanish-Peron with co-authors, Gene, 33, 103, 1985) was subjected to mutagenesis using the following oligonucleotide:

5' GCGCCCCGCC GTCGTT AGTRG 3' SEQ ID NO 15

for the introduction of the Not I site in the sequence of a truncated gene lac I and to obtain plasmids M13TG724.

Then in section EcoR I plasmids M13TG724 was introduced polylinker containing sites EcoR I, SnaB I and Not I, using the following oligonucleotides:

5' ATTGGGCC GTGTGT 3' SEQ ID no 16

5' ALCATECH TCGCGCCTCT 3' SEQ ID NO 17

to obtain plasmids M13TG7244, which was observed modification of the inserts during a stage of amplification. Insert plasmids M13TG7244 has SL is, and the plot lacZ plasmid pUC19 in italics.

After cleavage of plasmid M13TG7244 the enzymes EcoR I and Sst I was introduced polylinker containing areas Mlu I and Avr II, using the following oligonucleotides:

5' CAACCCGACC TAGG 3' SEQ ID no 18

5' ATTRSTART OF ACHCHHI CT 3' SEQ ID NO 19

After cleavage by the enzyme Pvu II received fragment Pvu II was subcloned in pUC19 (C. Yanish-Perron al. cited above) to obtain plasmids pTG7457 (Fig. 13).

2. Subclavian PGK terminator

pUC19 was split by enzymes of Wamn I and EcoR I and introduces a new polylinker of UMN I, Sst I, using the following oligonucleotides:

5' GITSCHLAG RATCATCHER TCCTGGGGCC 3' SEQ ID NO. 20,

5' AGARTTHA ATCCTCTCCG GUTTERING ATTATCH 3' SEQ ID NO 21

5' TTTGGTCTAT ATGCAGTG of TCHARACTER 3' SEQ ID no 22

5' OUTTTTTTT GTGTGT AAGCTGCGGC 3' SEQ ID NO 23

Thus was obtained a plasmid pTG7453 (Fig. 14), which was then split by enzymes of Wamn I and Sst I. the Areas of polylinker between AMN I and Sst I were put into derivative obtained above plasmids pTG7457 and cleaved by enzymes restrictiv I, Not I, SnaB I, Pvu I.

Specified new plasmid was cleaved with restriction enzymes Bgl II and Hind III, and the fragment Bgl II - Hind III, containing the PGK promoter (R. A. Hitzeman with co-workers. Nucleic Acids Res., 10, 7791, 1982; G. Loison with co-workers. Yeast, 5, 497, 1989) was introduced into it to obtain plasmids PTG10014 (Fig. 15).

3. Subclavian promoters

a) the Promoter CYC1

Lots of polylinker between BamH I and Sst I plasmids pTG7453 were introduced in the derived plasmids pTG745, as described above. New resulting plasmid was cleaved with restriction enzyme SnaB I, and then to obtain the plasmid pTG7503 was introduced fragment Rsa I-Dra I from 456 p. O. plasmid pEMBL8 (L. Dente with co-workers. Nucleic Acid Res., 11, 1645, 1983) containing the replication origin of phage fl.

Fragment BamH I Hind III from 0,78 thousand base pairs in the plasmid pGBSCC-9 obtained in Example 6 of the patent application EEC EP 0340378 containing the CYC1 promoter S. cerevisiae, polylinker and lactase terminator K. lactis, was subcloned in the plasmid pTG7503 cleaved by the restriction enzymes Hind III and BamH I to obtain plasmids pTG10004 (Fig. 16).

Then the sites Xho I and Mlu I CYC1 promoter were removed by the method of site-directed mutagenesis of double-stranded DNA plasmids pTG10004 using the following oligonucleotides:

5' Gzhel cleaved with restriction enzymes Sal I and Xho I and then was introduced plot Mlu I, using the following oligonucleotides:

5' TCHOCHKES ZHTHG 3' SEQ ID No. 25 and

5' TCPACCESS GTCC 3' SEQ ID NO 26

to obtain plasmids pTG10006.

b) the Promoter GAL10/CYC1

Plasmid pYeDPl/8-2 (C. Cullin with co-workers. Gene, 203, 1988) was revealed using the restriction enzyme Xho I. Created connecting the ends were folded using a fragment maple DNA polymerase, and then the plasmid was re-Legerova. Thus obtained plasmid pTG10010, in which the promoter GAL10/CYC1 did not contain a section Xho I, is used as template for amplification by PCR.

4. Construction of expression vector G10031

The remainder of the coding sequence of lacZ was removed in plasmid pTG7503 using directed mutagenesis using the following oligonucleotides:

5' THHGTTG TTTTTTTT TGCCTGGGGC 3' SEQ ID NO 27

to obtain plasmids G7549.

The lacZ promoter present in the plasmid pTG7549 was then subjected to deletions using the following oligonucleotides:

5' GGCCCCAACA CCAAA 3' SEQ ID No. 28 and

5'ITTTTTT TTGC 3' SEQ ID NO 29

which are then entered into a plasmid, p is smidi pTG7553.

One piece of UMN I, Mlu I, containing the CYC1 promoter, was obtained on the basis of plasmids pTG10006 cleaved by enzymes of Wamn I and Mlu I, and one piece of Mlu I, Hind III, containing the PGK promoter was isolated from plasmid pTG10015 cleaved by restriction enzymes Mlu I and Hind III. On these two fragments was performed ligation, and the resulting ligation product was introduced into a plasmid pTG7553, pre-split by enzymes Mlu I and Hind III.

The following oligonucleotide:

5' HATZLACHA CGCGGCGCGG 3' SEQ ID NO. 30,

hybridized with the following oligonucleotide:

5' CGCGGCGCGG CHATCHAT 3' SEQ ID NO. 31,

forming linker BamH I, Mlu I, containing lots Cla I and Not I, was added and subjected to legirovanie to obtain expression vector pTG10031 (Fig. 17).

Fragment amplificatory by PCR, obtained above from the plasmid pYeDP1/8-2, was cleaved with restriction enzymes Cla I or Sal I, and then was introduced into a plasmid pTG10031, previously cleaved with the same enzymes to obtain the plasmid G10033 (Fig. 18).

1. The nucleic acid sequence encoding a protein having a Delta-5,7 Sterol, Delta-7 reductase activity and having the nucleotide sequence SEQ ID No. 1 or a nucleotide sequence, which is an allelic variant of the sequence SEQ ID No. 1 and having the specified activity.

2. The DNA sequence encoding a protein having a Delta-5,7-Sterol, Delta-7 reductase activity, which hybridized sequence defined in paragraph 1, under conditions of average temperature of hybridization corresponding to hybridization at 42°C for 12 h in 50% solution of formamide, SSC X6, followed by rinsing, or who possesses with the sequence identity of 60% or more.

3. The DNA sequence encoding a protein having a Delta-5,7-Sterol, Delta-7 reductase activity, amplificatory by PCR, using as a nucleating oligonucleotides encoding the consensus sequence having the amino acid sequence of SEQ ID No. 3:

in which XAA in position 7 is the amino acid is TRP or Tyr and XAA in position 12 is composed with the amino acid sequence of SEQ ID No. 2 or a sequence which its option and having the specified activity.

5. Protein of A. thaliana, having a Delta-5,7-Sterol, Delta-7 reductase activity and having the amino acid sequence of SEQ ID No. 2, referred to as the Delta-7 Ed.

6. The protein having a Delta-5,7-Sterol, Delta-7 reductase activity and having the amino acid sequence that has identity with the sequence SEQ ID No. 2 under item 5 to 60% and more.

7. The protein having a Delta-5,7-Sterol, Delta-7 reductase activity and cross-immunoreactivity with protein A. thaliana under item 5, encoded by the DNA sequence under item 2.

8. The protein having a Delta-5,7-Sterol, Delta-7 reductase activity, obtained by expression in a yeast cell containing the DNA sequence according to any one of paragraphs.1-3.

9. Protein of A. thaliana, having a Delta-5,7-Sterol, Delta-7 reductase activity, obtained by expression in a yeast cell containing the DNA sequence encoding amino acid sequence SEQ ID No. 2.

10. The protein expression vector containing the DNA sequence according to any one of paragraphs.1-3.

11. The method of cloning a nucleic acid that encodes a protein having deltaic acids, obtained on the basis of the Bank of protein expression with the specified activity from A. thaliana by their hybridization with the nucleotide sequence SEQ ID No. 1.

12. The DNA sequence encoding a protein having a Delta-5,7-Sterol, Delta-7 reductase activity, cloned by the method according to p. 11.

13. The DNA sequence under item 12, characterized in that it is human DNA.

14. Probe for the diagnosis of congenital deficiency of the Delta-5,7-Sterol, Delta-7-reductase, which is a sequence of DNA of the person under item 13.

15. A method of obtaining a protein having a Delta-5,7 Sterol, Delta-7 reductase activity, wherein the cultured cell host transformed by the vector according to p. 10 or vector containing a DNA sequence according to p. 12, and produce downregulation of protein.

16. The method according to p. 15, wherein the host-cell are transformed yeast in which the coding sequence of the DNA is under the control of the promoter of the yeast.

17. The recovery method in vitro Sterol, unsaturated in position C-7, characterized in that incubated Sterol that is to be restored, with the protein obtained is placed in vitro by p. 17, characterized in that the restored Sterol is a substrate of the enzyme cleavage of the side chain of cholesterol (P450SCC).

19. The method of recovery in vivo exogenous Sterol, unsaturated in position C-7, characterized in that incubated Sterol with the host-cell transformed by the vector according to p. 10 or vector containing a DNA sequence according to p. 12, with the possibility of allocating the received recovered Sterol.

20. The method of recovery in vivo under item 19, characterized in that the restored Sterol is a substrate of the enzyme cleavage of the side chain of cholesterol (P450SCC).

21. The method of recovery in vivo endogenous Sterol, unsaturated in position C-7, characterized in that the cultivated strain-host transformed by the vector according to p. 10 or vector containing a DNA sequence according to p. 12, with the possibility of subsequent allocation of accumulated restored Sterol.

22. The recovery method in vivo in p. 21, characterized in that the restored Sterol is a substrate of the enzyme cleavage of the side chain of cholesterol (P450SCC).

23. The recovery method in vivo in p. 22, characterized in, cohost 5,7,22 triene 3-ol, or their mixture.

24. The method of producing pregnenolone, wherein the cultured cell host transformed by the vector according to p. 10 or vector containing a DNA sequence according to p. 12, with the possibility of subsequent allocation of accumulated endogenous Sterol (accumulated endogenous sterols), restored in position C-7, incubated restored sterols in the presence of P450SCC and possibly in the presence of adenotonsillectomy (ADR) and adrenodoxin (ADX) with the possibility of allocating the received pregnenolone.

25. The method according to p. 24, wherein the host-cell is yeast.

26. The method of producing pregnenolone, characterized in that the cultivated yeast, transformed with one or more vectors, providing coexpression protein having a Delta-5,7-Sterol, Delta-7 reductase activity, P450SCC and possibly ADR and ADX, with the possibility of allocation of free or esterified pregnenolone.

27. The method of producing pregnenolone under item 26, characterized in that the culturing the transformed yeast coexpression protein having a Delta-5,7 Sterol, Delta-7 reductase activity, P450S the heat activity is protein A. thaliana Delta-7 Ed.

29. The method according to p. 28, wherein the yeast strain is a strain EC01/pCD63.

30. Transformed yeast strain EC01/pCD63 Saccharomyces cerevisiae, coexpression protein A. thaliana Delta-7 Ed on p. 5, P450SCC, ADR and ADX and nakaplivali free or esterified pregnenolone.

31. Method of detecting failure of the Delta-5,7-Sterol, Delta-7-reductase, characterized in that carry out the incubation of a sample containing human genomic DNA with a probe under item 14 in standard hybridization and detection of fixing or not fixing the probe to genomic DNA, with no fixation or poor fixation point to an innate lack of Delta-5,7-Sterol, Delta-7-reductase.



 

Same patents:

The invention relates to genetic engineering and can be used for therapeutic purposes, in particular in the treatment of neoplastic processes

The invention relates to biotechnology, in particular biotechnology crops

The invention relates to the field of biochemistry and can be used when conducting any tests that require allocation of nucleic acids from complex samples, in particular in medical diagnostics, forensic examination and the examination of food products

The invention relates to medicine and can be used in molecular diagnostics

The invention relates to the field of medicine and relates to a method of diagnosing individual sensitivity of the brain to ischemia
The invention relates to biotechnology, in particular the production of steroids

The invention relates to biotechnology, namely microbiological obtaining1,2-corticosteroids
The invention relates to a method for isolation and purification of budesonide is a highly effective hormonal bronchodilator

The invention relates to genetic engineering and can be used in plant breeding
Up!