Biologically active animal fatty acid Δ5-desaturase, dna sequence encoding the same, cloning end expressing vectors containing said sequence, method for production of polyunsaturated fatty acids, method for converting of dihomo-γ-linolenic acid to arachidonic acid, probe (variants), and method for detection of Δ5-desaturase using the same

FIELD: biotechnology, in particular gene engineering, pharmaceutical and food processing industry.

SUBSTANCE: DNA sequence (1341 n.p.) encoding fatty acid -desaturase (447 amino acid residue, 57 kD) of nematode Caenorhabditis elegants is isolated and characterized. Obtained DNA-sequence is expressed in bacterium and yeast cells to produce Biologically active enzyme recombinant form. Said recombinant form is capable to catalyze conversion of dihomo-γ-linolenic acid to arachidonic acid and eicosatetraenoate to eicosapentaenoate.

EFFECT: method for large-scale production of polyunsaturated fatty acid.

15 cl, 4 dwg, 1 ex

 

This invention relates to DNA sequences coding for Δ5-desaturase fatty acids encoded Δ5-fatty acid desaturases and use Δ5-fatty acid desaturases.

Polyunsaturated fatty acids are important physiologically because of their specific health-promoting activities and biomedicinsk in respect of their potential pharmaceutical use in the treatment of certain disease conditions.

Polyunsaturated fatty acids are precursors of two large classes of metabolites: prostanoids (which include prostaglandin and thromboxanes and leukotrienes. Δ5-Desaturase fatty acids catalyzes the conversion of di-Homo - γ-linolenic acid (DPC) in arachidonic acid (AA) and eicosatetraenoate (THESE) in eicosapentaenoate (EPE), by introducing a double bond in position Δ5-carbon of the respective substrates, and exists in the form associated with the membrane of the endoplasmic reticulum protein in its native state.

Arachidonic acid is a 20-membered carbon chain with 4 double bonds, and plays an important role in human metabolism, as it is a precursor for the synthesis of prostaglandins are fatty acids with 20-membered carbon chain which contain 5-membered carbon to which ICO. Prostaglandins are modulators of hormone action, and the potential effects of prostaglandins include stimulation of inflammation, regulation of blood flow to individual organs, control of ion transport through some membrane and modulation of synaptic transmission. Prostaglandins are also potentially applicable as a contraceptive, because of their ability to suppress the secretion of progesterone. Therefore, the ability to modulate prostaglandin synthesis by controlled levels of expression of the synthesis of the precursor polyunsaturated fatty acids is very important both medical and industrial.

The increasing importance of polyunsaturated fatty acids in the food and pharmaceutical industries has led to increased demand, which exceeded the levels of modern production and additional sources of high quality cheap polyunsaturated fatty acids.

Modern industrial sources of polyunsaturated fatty acids include selected seed plants, marine fish and selected mammals, and traditional processing methods for the extraction of polyunsaturated fatty acids from these sources include solvent extraction, demanganization, the formation of adducts of urea and distillation. However, presents the sources and EUT disadvantages, related to seasonal and climatic differences in the levels of production, as well as the lack of vegetable and fish sources and high prices of refining low-grade oils. High prices associated with insufficient levels of production, slowed the development of suitable industrial applications of polyunsaturated fatty acids.

Large efforts have been spent on the development of alternative sources of polyunsaturated fatty acids, and studies have been conducted to characterize the compound of genes and encoded proteins of their biosynthesis. Engineering the biosynthesis of polyunsaturated fatty acids, such as oil crops, has many advantages for the production of large quantities, for example, γ-linolenate (CH), di-Homo - γ-linolenate (DGP), arachidonic acid (AA), eicosapentaenoate (EPE) and docosahexaenoate (DGE). Practical implementation of this was illustrated by gene expression Δ6-desaturase borage in tobacco, which was the product of CH and octadecatetraenoic acid 18:4 (Soyanova et.al. (1997), PNAS 94, 9411-9414), Since most of the biosynthetic genes for the synthesis of polyunsaturated fatty acids become available, this provides an opportunity for producing at least GL, AK, EPE, DGE in oilseed crops, as well as monitoring type sobrang the lipid. The benefits that would be derived from such crops include cheap and supported reserve required in large scale polyunsaturated fatty acids, given the profile of polyunsaturated fatty acids to meet the specific nutritional needs, and in the fine chemical industry getting unusual fatty acids with the desired degree and to the provisions of unsaturation.

An additional approach to obtain polyunsaturated fatty acids is the utilization of biosynthetic capacity of the lower organisms, such as algae, bacteria, fungi (including phykomycets), which can synthesize a whole range of polyunsaturated fatty acids and can be grown on an industrial scale. Genetic transformation of these organisms will facilitate the excretion overproducing strains and manipulation of polyunsaturated profile by engineering.

Were cloned fungal Δ5and Δ6-desaturase fatty acids and their sequences discussed in WO 98/46763, WO 98/46764, WO 9846765.

The metabolism of polyunsaturated fatty acids is of the highest importance in human metabolism. These acids through eicosanoids, are fundamental for the proper maintenance of homeostasis and coupled with serious physiological and pathophysiological syndromes.

Unexpected is Annaud writers of the invention was isolated from soil dwelling filamentous fungus of the class zygomycetes, Mortierella alpina, and characterized the DNA sequence encoding a functionally active Δ5-desaturase fatty acids.

However, the inventors have unexpectedly isolated from the nematode, Caenorhabditis elegans, and characterized the DNA sequence encoding a functionally active Δ5-desaturase fatty acids. This DNA sequence encoding a functionally active Δ5-desaturase fatty acids, considered to be probably more closely related to Δ5-Desaturate fatty acids person than any of the so far selected sequences of the gene Δ5-desaturase fatty acids.

As well as the potential beneficial effects to humans from the polypeptide encoded by the DNA sequences according to the invention, the DNA sequence according to the invention can facilitate the cloning of the equivalent gene in humans and, thus, to contribute to overproduction sequence of human DNA and make it possible biomedical use in the treatment of some human diseases.

Vegetable and mushroom desaturase are mainly integral membrane polypeptides, which complicates their treatment and subsequently the characteristics of traditional methods. Therefore, for a better understanding of lipid metabolism selected molecular the s methods includes the use of mutants and transgenic plants.

According to the first aspect of the invention proposed a dedicated animal Δ5-desaturase fatty acids and its functionally active part.

According to the second aspect of the invention proposed dedicated Δ5-desaturase fatty acids .elegans.

According to a third aspect of the invention proposed a DNA sequence according to the first or second aspect of the invention, which includes at least part of the sequence represented in SEQ.2, and equivalents of this sequence or parts of this sequence that encode functionally active Δ5-desaturase fatty acid due to the degeneracy of the genetic code. Preferably, the DNA sequence derived from the DNA sequence of the nematode Caenorhabditis elegans.

Preferably, the gene encoding Δ5-desaturase fatty acids encoded by the cloned gene has a length of 1341 BP (base pairs). Protein with a calculated molecular mass of 57 kDa has a length of 447 amino acids.

Alternatively, the DNA sequence encodes a functionally active Δ5-desaturase fatty acids and comprises at least part of the sequence represented in SEQ.1, and equivalents of this sequence or parts, e is th sequence, which encode functionally active Δ5-desaturase fatty acid due to the degeneracy of the genetic code. Preferably, the DNA sequence derived from the DNA sequence Mortierelfa alpina.

Preferably, the gene encoding Δ5-desaturase fatty acids encoded by the cloned gene has a length of 1338 BP Protein with a calculated molecular mass of 57 kDa has a length of 447 amino acids.

Preferably, the DNA sequence according to the third aspect of the invention is functionally active in a mammal.

Preferably, the DNA sequence is expressed in the mammal.

Preferably, the DNA sequence is expressed in humans.

Preferably, the DNA sequence obtained by modification of functionally active natural gene encoding a Δ5-desaturase fatty acids.

Preferably, the modification includes modification of chemical, physical or biological methods without loss of catalytic activity of the enzyme it encodes.

Preferably, the modification improves the catalytic activity of the enzyme it encodes.

Preferably, the biological modification includes methods of recombinant DNA and methods of forced evolution.

Preferably, metadrenaline evolution is a DNA shuffling.

According to a fourth aspect of the invention proposed a polypeptide encoded by the DNA sequence according to the third aspect of the invention.

Preferably, at least part of the polypeptide has the sequence presented in SEQ.3, or functionally active equivalent of this sequence or parts of this sequence. Alternatively, at least part of the polypeptide has the sequence presented in SEQ.4, or functionally active equivalent of this sequence or parts of this sequence.

Preferably, the polypeptide catalyzes the conversion of di-Homo - γ-linolenic acid to arachidonic acid.

Preferably, the polypeptide is modified without loss of catalytic activity of the encoded polypeptide.

Preferably, the polypeptide is modified in such a way as to enter a specific degree of saturation of the substrate at a specific position in the molecular structure of the substrate.

According to the fifth aspect of the invention proposed a vector containing a DNA sequence of any part of a DNA sequence according to the third aspect of the invention.

According to the sixth aspect of the invention, a method for obtaining a polyunsaturated fatty acid, in which the substrate is brought into contact with Δ5-desaturase fat sour is on the first or second aspect of the invention or a polypeptide according to the fourth aspect of the invention.

According to the seventh aspect of the invention, a method for conversion of di-Homo - γ-linolenic acid arachidonic acid, which catalyze the conversion Δ5-Desaturate fatty acids on the first or second aspect of the invention or the polypeptide according to the fourth aspect of the invention.

According to the eighth aspect of the invention proposed a body designed for producing high levels of the polypeptide according to the fourth aspect of the invention.

According to the ninth aspect of the invention proposed a body designed for producing high levels of the product of the reaction catalyzed Δ5-Desaturate fatty acids on the first or second aspect of the invention or the polypeptide according to the fourth aspect of the invention.

Preferably, the body is designed to implement the method according to the sixth or seventh aspect of the invention.

Preferably, the organism is a microorganism.

Preferably, the microorganism is selected from algae, bacteria and fungi.

Preferably, mushrooms include phykomycets. Alternatively, the microorganism is a yeast.

Alternatively, the organism is a plant. Preferably, the plant is selected from oil-bearing plants.

Preferably, oilseed plant which I selected from oilseed rape, sunflower, cereals, including maize, tobacco, legumes, including peanuts and soy, safflower, oil palm, coconut and other palm, cotton, sesame, mustard, flax, castor, borage and evening primrose.

According to the tenth aspect of the invention proposed seed or other reproductive material, originating from the organism to the ninth aspect of the invention.

Preferably, the organism is a mammal.

According to the eleventh aspect of the invention proposed multienzyme path, where the path includes Δ5-desaturase fatty acids on the first or second aspect of the invention.

According to the twelfth aspect of the invention the proposed connection is obtained by transformation of the substrate, where the specified transformation catalyze Δ5-Desaturate fatty acids on the first or second aspect of the invention.

According to the thirteenth aspect of the invention proposed an intermediate compound obtained by the reaction catalyzed Δ5-Desaturate fatty acids on the first or second aspect of the invention.

According to the fourteenth aspect of the invention proposed a food product or dietary Supplement containing polyunsaturated fatty acid obtained by the method according to the sixth aspect of the invention.

According to the fifteenth aspect is subramania offered a pharmaceutical drug, containing polyunsaturated fatty acid obtained by the method according to the sixth aspect of the invention.

According to the sixteenth aspect of the invention proposed prostaglandins synthesized by biosynthetic comprising catalytic activity Δ5-desaturase fatty acids on the first or second aspect of the invention.

According to the seventeenth aspect of the invention, a method of modulation of prostaglandin synthesis by controlling the levels of expression of the DNA sequence according to the third aspect of the invention.

According to the eighteenth aspect of the invention proposed probe, which includes the entire DNA sequence according to the third aspect of the invention or part thereof or equivalent RNA sequence.

According to the nineteenth aspect of the invention proposed a probe that includes the entire polypeptide-Δ5-desaturase fatty acids according to the fourth aspect of the invention or part thereof.

According to the twentieth aspect of the invention, a method for allocating Δ5-desaturases using the probe according to the nineteenth aspect of the invention.

It is possible that the gene according to the invention can be transformed into human cells and used in methods of gene therapy at a suitable level in vivo to provide the patient a continual supply of fatty acids to build emich enzymes in polyunsaturated fatty acids. This could be an effective preventive treatment, for example, for patients suffering from high cholesterol levels or other medical conditions, where the introduction of polyunsaturated fatty acids may have beneficial effects for preventing disease.

However, either the entire DNA sequence of the invention or part thereof or the entire polypeptide sequence of the invention or part thereof could be used as probes to search for research or diagnostic purposes.

Hereinafter the invention will be described by a single example with reference to the accompanying graphics, SEQ.1-4 and 1-4, in which:

SEQ.1 represents the cDNA sequence encoding Δ5-desaturase fatty acids from Mortierella alpina;

SEQ.2 is a cDNA sequence encoding a Δ5-desaturase fatty acids from .elegans;

SEQ.3 represents a peptide sequence obtained by translation of the gene sequence SEQ.1;

SEQ.4 represents a peptide sequence obtained by translation of the gene sequence SEQ.2;

Figure 1 is an alignment of the gene encoding Δ5-desaturase fatty acids Mortierella alpina with different *6-desaturases and Δ5-desaturase;

<> Figure 2 is an alignment of the gene encoding Δ5-desaturase fatty acids Δ6-Desaturate .elegans and mushroom Δ5-Desaturate of .alpina;

Figure 3 is a obtained by gas chromatography, the chromatogram of the methyl esters of fatty acids from the induced yeast cells transformed transformed genome Δ5-desaturase fatty acids Mortierella alpina, and reinducing yeast cells transformed;

Figure 4 is a obtained by gas chromatography, the chromatogram of the methyl esters of fatty acids from the induced yeast cells transformed transformed genome Δ5-desaturase fatty acids C.elegans, and reinducing yeast cells transformed.

Cloning and sequencing of the gene Δ5-desaturase fatty acids from Mortierella alpina

The DNA sequence of the invention encoding Δ5-desaturase fatty acids, was cloned using PCR in combination with matrices from a library of cDNA and primers designed in a specific way. The function of DNA sequences, namely the conversion of di-Homo - γ-linolenic acid (DGPC) in arachidonic acid (AA) and eicosatetraenoate (THESE) in eicosapentaenoate (EPE), was examined by expression of the corresponding DVS is in yeast.

Gene Δ5-desaturase fatty acids from Mortierella alpina cloned methods polymerase chain reaction (PCR)using cDNA from Mortierella alpina as template and degenerate oligonucleotide primers (EAP)designed a specific way, as shown below, based on the first and third his-tag boxes vegetable Δ12and Δ15-desaturases, previously identified by Senkinom (Shanklin, J, Whittle E J & Fox, BG Biochemistry. 33, 12787-12794 (1994)).

Degenerate oligonucleotide primers (EAP),

5'-GCGAATTA(A/T)TIGGICA(T/C)GA(T/C)TG(T/C)GICA-3'

5'-GCGAATTCATIT(G/T)IGG(A/G)AAIA(G/A)(A/G)TG(A/G)TG-3'

where I represents inosine and Eco RI sites are underlined.

PCR amplification was performed completely traditionally fuser for reactions programmed to execute the program: 2 min at 94°C, then 45 seconds at 94°C, 1 minute at 55°and 1 minute at 72°C for 32 cycles, followed by elongation at 72°C for an additional 10 minutes. The products of PCR amplification were separated in a 1-percentage agarose gels.

A number of PCR products, amplified in cDNA matrix from Mortierella alpina included product length 660 BP, which was purified from the gel, cloned in the vector pGEM-T (PromegaRTM) and transformed into the expression host Escherichia coli DH 5α.

To sequence product length 660 BP designed the primers (P) and performed the amplification of fragments by PCR using the cloned fragment length 660 gel as a matrix and sequence-specific primers (P), based on sequence product length 660 BP

Delta direct

5'-GATGCGTCTCACTTTTCA-3'

Delta In reverse

5'-GTGGTGCACAGCCTGGTAGTT-3'

The products of this PCR amplification was purified from the gel and used as probes for screening cDNA library Mortierella alpina. Probe to find words were hybridisable with 25 of the 3.5×105proven phage clones and one clone, as was shown by restriction analysis, had the expected size is 1.5 so P.N. (thousands of base pairs). This clone, designated L11, were selected for further analysis. Sequence analysis of the L11 revealed an open reading frame length 1338 BP encoding a polypeptide of 446 amino acids. When he continued to analyze protein and genomic databases using the program GCG8 (Devereux J. et al. Nucleic Acids. Res. 12, 387-395 (1984)), L11 showed a low degree of identity (20%) gene Δ6-desaturase of species Synechocystis PCC6803 (Figure 1).

Figure 1 is aligned sequences have the following registration numbers:

S54259 Δ12-desaturase Spirulina registration number H

S54809 Δ6-desaturase Spirulina registration number H

S68358 estimated desaturase of sphingolipids registration number H

S35157 Δ6-desaturase Synechocysti registration number L 11421

PBOR6 Δ6-desaturase borage registration number U79010

FU2 Δ5-desaturase registration number AF054824

In addition, although all three his-tag Boxing, characteristic desaturase enzymes present in the transmitted sequence, the third his-tag Boxing, localized in the sequence in position 1159 BP, contains option QXXHH. The translated sequence also contains N-the end of the cytochrome b5-like heme-binding domain, which includes EHHPGG-fragment, although this feature was previously found only on the other end of fungal desaturases.

Southern blotting genomic DNA

Sequence-specific primers designed to sequence L11 between the his-tag boxes 1 and 3 sequences L11, used in PCR reactions to amplify a segment with a length of 660 BP sequence L11. The PCR product length of 660 BP was purified from the gel and did blot-hybridization on Southern restricion genomic DNA from Mortierella alpina and Mucor circinelloides, using as a probe a fragment length of 660 BP Results indicate that the gene encoding Δ5-desaturase fatty acids according to the invention, presented in Mortierella alpina unique copy and apparently absent in Mucor circinelloides. In addition, Mucor circinelloides does not detect the I Δ 5-desaturase activity.

Expression of the cloned gene Mortierella alpina, encoding Δ5-desaturase fatty acids

In order to confirm that the sequence L11 encodes the enzyme - Δ5-desaturase fatty acids, cDNA was subcloned into the yeast expressing the vector pYES2 provided by Invitrogen™under control of the GAL4 promoter-polymerase to yield the plasmids pYES2/L11. The expression of L11 was checked by transcription-translation pYES2/L11 in vitro using a coupled system of transcription and translation Promega™. Received products broadcast labelled35S-methionine, which were separated by electrophoresis in SDS page (polyacrylamide gel) in the presence of SDS (sodium dodecyl sulfate) and visualized by exposure to autoradiographic film. The calculated molecular mass of the product was 55-60 kDa, and the control plasmid without insert, pYES2, unable to give any labeled product broadcast.

The construct pYES2/L11 transformed in the yeast, Saccharomyces cerevisiae, and were grown on medium YCA depleted uracil. The transformants were selected because of the presence of breeding marker URA3, carrying pYES2/L11, and the expression of L11 was induced by adding galactose to a final concentration of 1% mm. The cultures were grown overnight in the presence of 0.5 mm di-Homo - γ-linolenate,detergent (1%tergitol, NP-40) and 2%raffinose. Aliquot samples were taken at time t=0, t=4 hours and t=16 hours. All fatty acids yeast analyzed by GC (gas chromatography) methyl esters. Lipids from induced and reinducing control samples were transmaterial 1M HCl in methanol at 80°C for 1 hour. Methyl esters of fatty acids (MASK) were extracted in hexane. Analysis by GC MASK was performed using a gas chromatograph Hewlett Packard series 58804, equipped with an attached capillary column 25 M × 0.32 mm RSL-VR and a flame ionization detector.

When the methyl esters of the fatty acids isolated from yeast carrying the plasmids pYes2/L11 and grown in the presence of galactose and di-Homo - γ-linolenic acid, was analyzed by GC, found additional peak (see figure 3). This additional peak had the same retention time as the standard authentic arachidonic acid (Sigma), showing that transgenic yeast capable of decaturian di-Homo - γ-linolenic acid in Δ5-position. Such peaks were not detected in the control samples (transformation pYes2), Figure 3. Identity additional peak was confirmed by GC-MS (gas chromatography-mass spectrometry) (Kratos MC80RFA operating at voltage ionization 70 eV with a scan range of 500-40 far is he), which accurately identified this compound as arachidonic acid.

This demonstrates that the DNA sequence of the Mortierella alpina encodes a functionally active polypeptide involved in the synthesis of arachidonic acid in the presence of galactose and di-Homo - γ-linolenic acid.

Cloning and sequencing of the gene Δ5-desaturase fatty acids C.elegans

Previously, the inventors have identified mushroom Δ5and Δ6-desaturase fatty acids, which differed from the previously identified microsomal desaturases from both vegetable and animal species. This difference was caused by the presence of the N-terminal extension segment, which showed homology with electron-donor protein cytochrome b5.

While the characteristics of mushroom Δ5-desaturase fatty acids Mortierella alpina and Δ6-desaturase fatty acids C. afegans (located in cosmides W08D2 (registration number Z70271)), the inventors identified a related sequence in cosmides T13F2.1 (registration number Z81122), also containing DNA .elegans, which probably encodes desaturase fatty acids.

Analysis of the sequences using the program Genefinder (Wilson, R. et al (1994) Nature, 368, 32-38)) showed that Comedy W08D2 and T13F2 contain overlapping part is key. However, it was found that cosmid T13F2, contains an open-reading frames (ORFS), designated T13F2.1, which contains the N-terminal domain of cytochrome b5(specific diagnostic fragment His-Pro-Gly-Gly), and three his-tag Boxing is characteristic of all microsomal desaturases. Moreover, this alleged desaturase contained a third variant his-tag box with H→Q replacement instead of the first histidine in the fragment of His-X-X-His-His. This glutamate substitution is present both in plants and in animals Δ6-fatty acid desaturases, and mushroom Δ5desaturase fatty acids from .alpina.

The overlap between kominami T13F2 and W08D2 made possible the establishment of spatial proximity LFS proposed desaturase T13F2.1 with Δ6-Desaturate fatty acids, indicating that the two sequences are tandemly on chromosome IV, separated 990 bases from the predicted stop codon T13F2.1 before initiating methioninamide triplet Δ6-desaturase fatty acids.

Because sequence analysis of the predicted ORFS T13F2.1 separated adjacent introns, heterologous functional expression of the genomic DNA is impossible. Therefore, polymerase chain reaction was used to amplify a clone containing a partial cDNA corresponding to large before is shown the exon at the 5'-end OCR T13F2.1 using the following primers: CE direct and BEHOLD reverse

CE direct

5' - ATGGTATTACGAGAGCAAGA-3'

CE reverse

5' -TCTGGGATCTCTGGTTCTTG-3'

After initial denaturation at 94°C for 2 minutes, amplification was performed in 32 cycles of: 45 seconds at 94°C, 1 minute at 55°and 1 minute at 72°s, followed by final elongation at 72°C for an additional 10 minutes.

The DNA fragment of the correct predicted size (which is visualized in 1%agarose gel) amplified, band on the gel was cut out, and DNA was purified and ligated directly from pGEM-T (Promega), and the resulting plasmid was transformed into cells of E. coli DH 5α. Plasmid DNA was purified for sequencing using the set of Qiagen QIAprep miniprep, and the nucleotide sequence of the insert was determined by automated sequencing using a DNA sequencing machine (ABI-377.

In order to allocate the full coding site corresponding OPC T13F2.1, this dedicated PCR amplificatory fragment length 233 BP used for screening the cDNA library of the mixed phase .elegans, which was constructed in λiZapll Prof. Yuji Kohara-Mishima, Japan. Screening was performed using standard methods (Sambrook et al (1989) Molecular Cloning. A Laboratory Manual), using as a probe the cloned PCR product. DNA fragments were labeled with radioactive isotope α[32R] dCTP (deoxycytidylate), using ready-made re Klenow mixture for labeling DNA (Pharmacia). Of the 1.4×105plaque-forming units tested for hybridization with a fragment length of 233 BP 5 plaques gave positive answers and were cut from cups with agar and suirvey in SM buffer. The resulting phage suspension was checked for the presence of T13F2.1 by PCR amplification using CE direct and BEHOLD reverse. One clone, designated L4, cleaned 2 additional cycles of cultivation and screening by hybridization at 65°using fragment length 233 P.N., highlighted by PCR. Plasmid L4 was isolated from λ-clone L4 by excision and sequenced both strands of cDNA inserts using DNA-sequencing machine (ABI-377 Perkin-Elmer.

The resulting DNA sequence is presented in SEQ.2, and the predicted amino acid sequence presented in SEQ.4.

Functional analysis of L4 in yeast

Full coding section L4 (encoding 447 amino acids) amplified by PCR using the primers shown below YCED direct and YCED reverse, which also included flanking HindIII and BamHI sites of restriction:

YCED direct

5'-GCGAAGCTTAAAATGGTATTACGAGAGCAAGAGC-3'

(annealing with the initiating methionine are shown in bold, the restriction site HindIII underlined)

YCED reverse

5'-GCGGGATCCAATCTAGGCAATCTTTTTAGTCAA-3'

(annealing to the complement of the stop codon is shown in bold, the restriction site BamHI underlined).

Amplificatory the PCR product containing the full coding section L4, ligated with yeast expressing vector pYES2 (Invitrogen), down from the GAL1 promoter, using the restriction sites HindIII and BamHI (enzymes provided by Boehringer Mannheim). The resulting construct, designated pYES2/L4, transformed into cells of E. coli and the accuracy obtained using PCR inserts in the plasmids pYES2/L4 confirmed in vitro by coupled transcription-translation using the TNT system (Promega). The resulting products broadcast marked35S-methionine, were separated by electrophoresis in SDS page in the presence of SDS and visualized by autoradiography.

Product broadcast received with pYES2/L4, had a molecular weight of approximately Mr=57 000, whereas the control vector, pYES2 without insert, did not give the product broadcast.

For functional analysis of the coding section L4 recombinant plasmid was transformed into S.cerevisiae cells DBY746 method of treatment with lithium acetate (Elble R. (1992) Bio Techniques 13 18-20). Cells were cultured overnight in a medium containing raffinose as a source of carbon enriched by the addition of either linoleic acid (18:2Δ9,12), or di-Homo - γ-linolenic acid (C20:33Δ8,11,14in the presence of 1%tergitol (as described Napier et al (1998) Biochem. J. 330 611-614). These fatty acids do not have S.cerewsiae, but serve as specific substrates for either Δ6-or Δ5-desaturase, respectively. The expression of the coding section L4 from the GAL1 promoter vector was induced by adding galactose to 1%. The cultivation was continued for 16 hours prior to sampling aliquot samples for analysis of fatty acids by GC. All fatty acids extracted from yeast cultures were analyzed by gas chromatography (GC) methyl esters. Lipids were transmaterial 1M HCl in methanol at 80°C for 1 h, then methyl esters of fatty acids (MASK) were extracted in hexane. Analysis by GC MASK was performed using a gas chromatograph Hewlett Packard series A equipped with attached capillary column 25 M × 0.32 mm RSL-500 BP and a flame ionization detector. Fatty acids were identified by comparison with retention time of standards MARK (Sigma). The relative percentage of fatty acids was calculated from the areas under the peaks. Arachidonic acid was identified by GC-MS using Krats MC80RFA operating at voltage ionization 70 eV with a scan range of 500-40 daltons. Figure 4 shows the result of analysis by GC of the methyl esters of fatty acids of the transformed yeast strains. An additional peak is detected on the scan obtained with induced pYES2/L4, grown in the presence of di-Homo - γ-linolenic acid, compared with the control containing an empty vector. This peak was absent in reinducing cultures grown on medium with di-Homo - γ-linolenic acid and, in addition, it is important to note that pYES2/L4 grown in the presence of linoleic acid, is not able to accumulate any new peaks, indicating that this fatty acid is not a substrate for the enzyme encoded by cDNA .elegans. The retention time of an additional peak is identical to the retention time of an authentic standard methyl-arachidonic acid. Fatty acid derived from di-Homo - γ-linolenic acid, optionally characterized by GC-MS (gas chromatography-mass spectrometry and identified as arachidonic acid. Therefore, the results show that transformed with plasmid pYES2/L4 yeast cells acquired functional Δ5-desaturase activity and were able to synthesize arachidonic acid from di-Homo - γ-linolenic acid serving as substrate. Δ5-Desaturase in transformed yeast, apparently, was an effective catalyst.

This demonstrates that the DNA sequence of .elegans encodes a functionally active polypeptide involved in the synthesis of arachidonic acid in p is outstay galactose and di-Homo - γ -linolenate.

1. Biologically active animal Δ5-desaturase fatty acids, characterized by the amino acid sequence shown in SEQ ID NO:4, or a portion exhibiting catalytic activity inherent in the full-form of the enzyme.

2. Biologically active animal Δ5-desaturase fatty acids according to claim 1, originating from Caenorhabditis elegans.

3. The DNA sequence encoding a biologically active Δ5-desaturase fatty acids and characterized by nucleotide sequence that essentially corresponds to either SEQ ID NO:2, or part thereof, or the equivalent of this sequence or parts determined by the degeneracy of the genetic code.

4. The DNA sequence according to claim 3, originating from the DNA sequence of the nematode Caenorhabditis elegans.

5. The DNA sequence according to claim 3 or 4, where the DNA sequence is functionally active in a cell of a mammal.

6. The DNA sequence according to claim 5, where the DNA sequence is expressed in the cell of a mammal.

7. The DNA sequence according to claim 6, where the DNA sequence is expressed in the human cell.

8. Biologically active #x00394; 5-desaturase characterized by the amino acid sequence encoded by the DNA according to claim 3.

9. The cloning vector containing the DNA sequence according to claim 3 or any part thereof.

10. Expressing the vector containing the DNA sequence according to claim 3.

11. A method of obtaining a polyunsaturated fatty acid, in which the substrate representing di-Homo - γ-linolenic acid or eicosatetraenoic lead in contact with Δ5-Desaturate fatty acids according to any one of claims 1, 2 or 8.

12. The way of transformation of di-Homo - γ-linolenic acid to arachidonic acid in which the specified transformation catalyze Δ5-Desaturate fatty acids according to any one of claims 1, 2 or 8.

13. The probe nucleotide sequence that corresponds to the DNA sequence according to claim 3, equivalent RNA sequence or a part of one of these sequences.

14. Probe, the nucleotide sequence is determined from the amino acid sequence of Δ5-desaturase according to any one of claims 1, 2 or 8.

15. How to identify Δ5-desaturase using the probe indicated in paragraph 13 or 14.



 

Same patents:

FIELD: biotechnology, genetic engineering.

SUBSTANCE: inventions represent sets of primers, pairs of primers and individual primers used in detection and/or identification of transgene DNA sequences in vegetable material and products comprising thereof, methods for detection and/or identification with their using and a device for realization of one of methods. Primers are complementary to 5 typical marker genes and regulatory DNA sequences - gus gene from microorganism E. coli, nptII gene from transposon Tn5, 35S-promoter site in cauliflower mosaic virus, terminator sites in nos gene and ocs gene from A. tumefaciens that comprises most often in genetic constructions of the most abundant in the world market of transgene plant strains and have nucleotide sequences given in SEQ ID Nos. 1-10. Methods involve DNA extraction from vegetable material and products comprising thereof, carrying out asymmetric or symmetric polymerase chain reaction (PCR), among them the multiplex reaction, with taking part of extracted DNA and set of primers, separation of amplified reaction products and their following detection and/or identification by their hybridization with oligonucleotides immobilized on biological microchip or by gel-electrophoresis method, or by using amplifying agent in real time regimen. Original selection of primers and addition of correcting nucleotide exchanges in their sequences provide similarity in their thermodynamic parameters and complete compatibility in sets that provides their effective working in the similar reaction conditions and enhance specificity, sensitivity and reliability of methods for detection and/or identification of transgene DNA sequences with their sharing significantly. Using in method in hybridization analysis of PCR results with specially developed biological microchips BM "Tressigen"-aminated glasses with oligonucleotides immobilized directly on their surface with nucleotide sequences given in SEQ ID Nos. 11-15, and apparatus-program complex "Degmigen" enhances specificity, economy and technological effectiveness of the process significantly. Inventions can be used in monitoring biological safety of nutrient, fodder foodstuffs and other goods of mass consumption.

EFFECT: improved method for analysis, valuable properties of primers.

54 cl, 5 dwg, 1 tbl, 5 ex

FIELD: biotechnology, genetic engineering, virology.

SUBSTANCE: invention proposes a method for isolating virus IRT KRS in polymerase chain reaction. Method involves carrying out polymerase chain reaction with primers B1 and B2 followed by differentiation of detected DNA in case of positive result in PCR reaction. Result is considered to be positive if PCR product corresponds to size of fragment consisting of 464 pair bases. For differentiation PCR product is treated with (restriction) endonuclease Sac II. As result, the presence of one fragment with length 464 pair bases corresponds to the vaccine strain TK-A and two fragments of size 343 and 121 pair bases correspond to epizootic strains and isolates.

EFFECT: improved detecting method and analysis.

1 dwg, 3 tbl, 6 ex

FIELD: genetic engineering, medicinal genetics.

SUBSTANCE: invention relates to the constructed plasmid DNA alpha-R-12 based on vector pBR325 and fragment of alphoidal DNA of human lymphocytes prepared from Eco RI restricts of the total fraction of human alphoidal DNA in hybridization on chromosomes in situ under special conditions of enhanced rigidity. Using the invention allows carrying out the reliable identification of human 6-th chromosome in norm and in pathology.

EFFECT: improved method for chromosome analysis.

4 ex

FIELD: veterinary science, virology.

SUBSTANCE: method for evaluation of semen from bull-sires for contamination with cattle infectious rhinotracheitis virus involves taking semen samples, combining semen samples obtained from bull-sire for one month and their analysis as a single sample. The analysis is carried out by method of molecular hybridization. For evaluation of semen from rejected bull-sires the semen samples are taken obtained for all period of their exploitation. Method provides reducing labor intensity and time for analysis and enhanced sensitivity of analysis in assay of semen contamination with indicated virus.

EFFECT: improved method for evaluation of semen.

2 cl, 1 tbl, 4 ex

FIELD: biochemistry, molecular biology.

SUBSTANCE: invention proposes variants of a method for detecting the sequence in DNA- or RNA-"target" that is based on carrying out the primer splicing reaction under conditions providing the definite limit of the product size (isometric primer splicing). Indicated conditions involve using at stage for primer splicing hybridized preliminary with the corresponding sequence site - a "target", the reaction mixture, wherein at least one of types of dNTP (deoxynucleoside triphosphate) is absent or replaced with the corresponding terminating substrate. The usage of proposed method provides significant enhancing sensitivity of analysis and to reducing time for its carrying out. Invention can be used in diagnostic tests and analytical systems for any designations suggesting detection of specific nucleic acid sequence in the sample.

EFFECT: improved detecting method.

44 cl, 2 dwg, 2 tbl, 1 ex

FIELD: molecular biology, medicine.

SUBSTANCE: invention relates to methods (variants) for identification of P[8] genotype rotavirus subtypes. Claimed method includes rotavirus RNA isolation followed by reverse transcription thereof. Further single-round polymerase chain reaction is carried out followed by analysis of obtained fragments in agarose gel. For identification of P[8]-1 subtype 5'-cca ttt att tga atc gtt a-3' primer is used, and for identification of P[8]-1 subtype 5'-cca ttt atc tga atc att a-3' primer is used. Presence of desired subtype is detected on the base of specific fragment with size of 410 n.p. Method of present invention is useful in detection of P[8] genotype rotavirus of various subtypes for monitoring of rotavirus strain circulation and investigation of their variability, abundance, etc.

EFFECT: simplified and accelerated method for strain differentiation.

2 cl, 2 ex

FIELD: molecular biology, biochemistry, diagnostic systems useful in detection of certain genes as well as known or unknown gene mutation in biological samples.

SUBSTANCE: invention relates to method for characterization of nucleic acid molecule sequence by detection of variation presence (or absence) in this sequence and characterization of detected variations. Claimed method includes enzymatic copying of sequence being interested in presence of at least one kind of dNTF with modified base which represents DNA-glycosilase substrate; treatment of obtained copy with DNA-glycosilase and further with agent cleaving the sequence to form fragments having OH-group on 3'-end4 incubation of said fragments in presence of matrix nucleic acid and enzyme being capable to provide tailing thereof; and extension product analysis.

EFFECT: testing and diagnosis methods with improved effectiveness.

20 cl, 13 dwg, 4 ex

FIELD: biotechnology, biochemistry.

SUBSTANCE: method for preparing DNA preparation involves separation of components from preliminary disrupted bacterial cells by electrophoresis in U-shaped tube and selection of DNA-containing fraction on interface of buffer and sample. Applying the invention allows simplifying the process for preparing the preparation of bacterial DNA for carrying out polymerase chain reaction. Invention can be used in laboratory and scientific practice for preparing the preparation of bacterial DNA.

EFFECT: improved preparing method.

4 ex

FIELD: biotechnology.

SUBSTANCE: the present innovation deals with analyzing a sample of porcine genetic material for the presence of quantitative tags locus (QTL) localized in chromosome 2 Sus scrofa, in position 2p1.7 as it was established during mapping. One should select swine with desired genotypic properties.

EFFECT: higher efficiency of selection.

21 cl, 10 dwg, 4 ex

FIELD: biochemistry, molecular biology, in particular analytical systems based on DNA PCR-amplification.

SUBSTANCE: invention relates to composition for amplification reaction, comprising two heat-resistant enzymes, wherein one (major) component represents DNA polymerase, which is usually applied in PCR method, and the second (minor) component represents 3'-exonuclease from Archeoglobus fulgidus (Afu III exonuclease).

EFFECT: method for PCR-amplification with improved accuracy and increased validity of obtained test-system data.

10 cl, 13 dwg, 2 tbl, 13 ex

FIELD: biotechnology, medicine, food industry.

SUBSTANCE: method for preparing conjugated linoleic acid involves creature and incubation of reaction mixture comprising microorganisms able to transform linoleic acid to conjugated linoleic acid and micelles formed by a surface-active substance and linoleic acid. Product prepared by this method comprises at least 75% of cis-9, trans-11-isomer of conjugated linoleic acid. Applying the invention provides preparing the enhanced yield of conjugated linoleic acid.

EFFECT: improved preparing method.

17 cl, 8 tbl, 1 dwg, 8 ex

FIELD: organic chemistry, chemical technology, pharmacy.

SUBSTANCE: invention relates to the improved method for isolating and purifying pravastatin or its pharmacologically acceptable salt from impurities of pravastatin analogues. Method involves extraction of pravastatin or its pharmacologically acceptable salt comprising impurities of pravastatin analogues with organic solvent of the formula: CH3CO2R wherein R represents alkyl comprising 3 or 4 carbon atoms. Also, invention relates to the improved method for isolating or purifying pravastatin or its pharmacologically acceptable salt that involves decomposition of impurities by using inorganic acid or that involves removing compound of the formula (I): by using inorganic solvent. Also, invention relates to the composition comprising pravastatin sodium salt and compound of the formula (I) taken in the amount 0.1 wt.-% or less with respect to the amount of pravastatin sodium salt. Method provides preparing the end product of the high purity degree.

EFFECT: improved purifying method.

18 cl, 1 tbl, 8 ex

FIELD: food-processing industry.

SUBSTANCE: method involves mixing dried fish farce, cabbage, tomato, flour of bean, onion, carrot, sorghum groats, ground pike's pyloric appendage, edible salt, CO2-extracts of dill and celery, rape oil and preparation of Mortierella strangulata micromycet biomass produced by predetermined process; packing mixture.

EFFECT: increased content of poly-unsaturated fatty acids and vitamins and nutritive substances in readily accessible form.

FIELD: food-processing industry.

SUBSTANCE: method involves mixing dried fish farce, cabbage, tomato, flour of bean, onion, carrot, sorghum groats, ground pike's pyloric appendage, edible salt, CO2-extracts of dill and celery, rape oil and preparation produced from Mortierella pusilla micromycet biomass obtained by predetermined process; packing mixture.

EFFECT: provision for obtaining of product with increased content of poly-unsaturated fatty acids, vitamins and nutritive substances in readily accessible form.

FIELD: food-processing industry.

SUBSTANCE: method involves mixing dried fish farce, cabbage, tomato, flour of bean, onion, carrot, sorghum groats, ground pike's pyloric appendage, edible salt, CO2-extracts of dill and celery, rape oil and preparation produced from Mortierella sclerotiella micromycet biomass obtained by predetermined process; packing mixture.

EFFECT: provision for obtaining of product with increased content of poly-unsaturated fatty acids, vitamins and nutritive substances in readily accessible form.

FIELD: food processing industry, in particular gerontological diet products.

SUBSTANCE: invention relates to producing of gerontological diet products. Claimed product is prepared by mixing of dried fish forcemeat, cabbage, tomato, kidney flour, onion, carrot, sorghum grain, grinded pike pyloric appendage, table salt, CO2-extracts from dill and celery, rape oil and preparation obtained from biomass of Mortierella gracilis micromycete according to claimed technology, followed by mixture packaging.

EFFECT: gerontological diet product with increased content of multiunsaturated fat acids and vitamins, as well as nutrients in more digestible form.

FIELD: canned food industry, in particular, production of gerodietary canned food.

SUBSTANCE: method involves preparing fish farce; mixing fish farce with cabbage, tomato, bean, onion, carrot, groats of sorghum, edible salt, CO2-extracts of dill, celery and Mortierella nantahalensis micromycet biomass, and rape-seed oil; cutting receipt mixture; packing; rolling and sterilizing.

EFFECT: harmonic organoleptical properties, increased digestion and wider range of gerodietary foods.

The invention relates to the production technology gerodietetic products
The invention relates to the production technology gerodietetic products
The invention relates to the production technology gerodietetic products

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: method involves selection of signal sequence suitable for the effective expression of Leu-hirudine in E. coli cells by the polymerase chain reaction-screening method. Method involves construction of a protein as a precursor of hirudine based on the selected signal sequence of surface membrane protein from Serratia marcescens, oprF protein from Pseudomonas fluorescens or fumarate reductase from Shewanella putrifaciens by joining the Leu-hirudine amino acid sequence with C-end of selected signal sequence. Prepared precursor of Leu-hirudine is used in a method for preparing Leu-hirudine. Invention provides preparing Leu-hirudine by the direct secretion in E. coli cells with the high yield. Invention can be used in preparing the hirudine precursor.

EFFECT: improved preparing method.

4 cl, 1 dwg, 2 tbl, 12 ex

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