Pcupl-aβ-sup35mc hybrid gene for analysis of factors regulating production, aggregation and disaggregation of human amyloid β (aβ) peptide in yeast system

FIELD: biotechnology, medicine.

SUBSTANCE: Pcup1-Aβ-SUS35MC hybrid gene is produced on the base of CUP1 promoter, sequence encoding of full-length human amyloid beta (Aβ) peptide, sequence encoding of M and C domains of Sup35 protein of Saccharomyces cerevisiae, and translation terminating sequence.

EFFECT: method for scale screening of Aβ-peptide aggregation regulating agents.

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The invention relates to genetic engineering and biomedical industry and represents the nucleotide sequence of the DNA encoding the hybrid gene is Aβ-SUP35MC.

The invention is of great practical, medical and moral aspect, as regards, in particular, not only the problem of increasing the average duration of human life, but also his mental health. With increasing life expectancy in developed countries naturally increases the number of people suffering from neurodegenerative diseases. In this regard, the study of the mechanisms of neurodegenerative diseases and the search for ways of their treatment are one of the priorities of modern Biomedicine. Greatest practical interest in the study of Alzheimer's disease (one of the most common forms of senile marasmus)that is forming in the tissues of the Central nervous system of the so-called amyloid plaques, the main component of which is a peptide Andβ. Andβthe peptide is a product of one of the two ways of proteolysis of the protein RDA (Amiloid Precusor Protein) (1) - Wisniewski T., Ghiso j, Frangione Century Bilology of Aβamyloid in Alzheimer's desease. // Neurobiology of desease. 1997. 4: 313-328. There is reason to believe that the formation of amyloid plaques is a result of the violation is of rocessing, that can lead to increased production Andβ-peptide (2) - Citron M., Westaway D., Xia, W., Carlson, G. et al. Mutant presenilins of Alzheimer's disease increase production of 42-residue amyloid beta-protein in both transfected cells and transgenic mice. // Nat Med. 199 V3. Page 67-72; (3) - Suzuki n, Cheung T.T., X.D. Cai, A. Odaka et al. An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants. // Science. 1994. 264. P.1336-1340; (4) - Tamaoka A., A. Odaka, Y. Ishibashi, M. Usami et al. APP717 missense mutation affects the ratio of amyloid beta protein species (A beta 1-42/43 and a beta 1-40) in familial Alzheimer's disease brain. J Biol Chem. 1994. 269(52). P. 32721-32724. In the oligomeric isoforms Andβthe peptide represented as β-layers and causes neurotoxic effect (5) - Price D.L., Sisodia S.S. Mutant genes in familial Alzheimer's disease and transgenic models. Annu Rev Neurosci. 1998. V21. P.479-505. People of middle age, this disease is quite rare, however, after 85 years of Alzheimer's suffers every third. According to forecasts of the Nobel laureate Stanley of Prusiner by 2025 year the number of Americans suffering from the disease, will be 10 million, and 2050 50 million (6) - Prusiner S.B. Shattuck lecture - neurodegenerative deseases and prions. // N Eng J Med. 2001. V.344. No.20. P.1516-1526. Alzheimer's disease inevitably leads to mental and physical activity and, ultimately, has a fatal outcome. To date, there are no ways of dealing with this incurable disease. In this regard, the study of biological aspects of this disease and finding medicinalfunds against Alzheimer's extremely relevant.

Characterization of the hybrid gene.

Hybrid gene Pcupl-Aβ-SUP35MC for analysis of factors governing the production, aggregation and disaggregatio peptide amyloid β (Aβ) man in the yeast system, having a size 2493 base pairs, consists of the following elements:

Xhol fragment-BamHl size 477 BP, representing the sequence, inducible CUPl-promoter for the expression of sequences in a yeast cell. The level of expression of sequences under the control of the CUPl-promoter depends on the concentration of copper ions in the medium. With increasing concentration of copper ions increases the level of expression of sequences that are controlled CUPl-promoter;

BamHI-BamHl fragment size 135 base pairs, representing the sequence, encoding amyloid beta peptide (Aβ-peptide) of a person, size 40 amino acids, flanked TATA box and the start-codon (ATO) with the 5'-end;

BamHl-Sacl fragment size 1879 base pairs, representing the sequence, encoding M and C domains of protein Sup35 yeast Saccharomyces cereviceae, flanked by a BamHl restriction site to the 5'and sequences, transcription termination and translation, and a Sad site to the 3'end. The functions of the M domain is unknown. Since the domain is an auxiliary factor in the termination of translets and eRF3 (7) - Zhouravleva, G., Frolova L., Le Goff x, Le Guellec, R., hige-Vechtomov, S., Kisselev L., Philippe M. Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRFl and eRF3. // EMBO J. 1995. 14:4065-4072. Inactivation With domain leads to a decrease in the accuracy of the translation termination and induction of nonsense-suppression.

The mutual position of elements of the hybrid gene PCUPl-Aβ-SUP35MC presented in figure 1.

Figure 1 lists the restriction enzymes cut sites (Xhol, BamHl, BamHl and Sad), flanking elements of the hybrid gene (CUPl-promoter, a sequence encoding Aβthe peptide of human rights and the fragment encoding M and C domains of the yeast SUP35 gene). Figures represent the number of nucleotides coding circuit, followed by the corresponding restriction enzyme hydrolyzes the sequence.

Elements (sequence, encoding Aβ-peptide and Sup35MC) are under the control of CUPl-promoter, flanked by restriction sites and represent a single open reading frame.

In a specific example of a gene integrated in the centromeric plasmid [PCUPl-Aβ-SUP35MC(UBA3)], which contains bacterial and yeast origin replication, resistance gene ampicillin - AmpR gene and the URE3.

The nucleotide sequence of the hybrid gene Pcupl-Aβ-SUP35MC:

Getting a hybrid gene Pcupl-Aβ-SUP35MC and an example implementation.

Placentas is required, coding Andβthe peptide was obtained using the standard method of RT-PCR from total RNA of brain abortus (Protocol below). The primers contain regulatory sequences (TATA-box and the ATG codon that initiates transcription), the sites of enzyme BamHl and Sad and sequences complementary 5' and 3' ends Andβcoding 40 amino acids.

The nucleotide sequence of primers:

F-primer: 5' GGGTCCAC GGATCC TAT ATG TCT GATGCAGAATTCCGACAT 3'

R-primer: 5' GTTATAAA GGATCC GACAACACCGCCCACCAT 3'

To obtain the single-stranded Copiii cDNA was carried out reverse transcription reaction:

Total RNA from brain abortus - 1 μl (1 μg/μl)

Primer F - 1 μl (100 ng)

Add:

5 × buffer - 4 μl

dNTP - 3 μl (2mM)

Polymerase chain reaction:

cDNA - 2 μl

10 × buffer - 5

dNTP - 2 μl (2mM)

primer F -1 μl (100 ng)

primer R - 1 μl (100 ng)

H2O - 38 μl

polymerase "Vent" - 1 μl

Polymerase chain reaction:

Phase 1 94° 1 min

Phase 2 94° 1 min

41° 1 min 1 cycle

72° 1 min

Phase 3 94° 1 min

63° 1 min 40 cycles

72° 1 min

Plasmid [Psup35-SUP35MC(URA3)] (Werzyn et al., 2001) was hydrolysed using restricted Xhol and BamHl that made it possible to replace the SUP35 promoter to the coding sequence of the CUP1 promoter (nucleotide sequence of CUPl promoter presents). The ligation reaction was carried out according to the standard Protocol of the firm from Promega. Thus was constructed plasmid [PCUPlSUP35MC(URA3)].

Amplificatory sequence encoding Aβthe peptide was verified by sequencing (firm helix) and, then, using a ligating built into plasmid [Pcupl-SUP35MC(URA3)] BamHl sites and Sad, located behind CUPl-promoter. We received centromeric plasmid [Pcupl-Aβ-SUP35MC(URA3)] contains the sequence of the hybrid gene Aβ-SUP35MC under control of the inducible CUPl-promoter, bacterial and yeast origin replication, resistance gene ampicillin - AmpR gene and the URE3.

An example of using a hybrid gene Rcupl-Aβ-SUP35MC

Search agents that block the aggregation Andβ-peptide, it is planned to use a yeast test systems, which will allow large-scale screening of proteins, peptides and chemical agents that block and (or) prevent the aggregation of Aβ-peptide. A key element of such a test system is claimed hybrid gene Rcupl-Aβ-SUP35MC. This hybrid gene is expressed in a yeast strain and encodes a hybrid protein Aβ-Sup35MC. The sequence corresponding to the M and C domains of the yeast protein Sup35, performs the function of the actor translation termination. Amino acid sequence of Aβ-peptide person is able to aggregate with each other, which leads to aggregation and partial inactivation of the hybrid protein Ap-Sup35MC. Used yeast strain has a deletion of the chromosomal copy of the gene SUP35 and marked nonsense-mutation adel-14. Aggregation and inactivation of Aβ-Sup35MCp reduce the accuracy of the translation termination, which leads to suppression of nonsense mutations adel-14 and detected by the criterion of "growth - no growth on selective medium without adenine". Thus, the use of hybrid gene Rcupl-Aβ-SUP35MC allows to detect aggregation Andβ-peptide person on the phenotypic level. This allows you to quickly and cheaply conduct primary large-scale search for proteins, peptides and chemical compounds that block and prevent pathogenic aggregation Andβ-peptide. In particular, it is planned to conduct a full screening of cDNA libraries from human brain and to identify proteins that control the oligomerization Andβ-peptide. Such proteins can be considered as potential therapeutics against Alzheimer's disease.

Other test systems

The applicant did not know of the patent and scientific literature, a similar test system enabling the above large-scale screening of agents is s, blocking aggregation Andβ-peptide. Search the factors controlling aggregation Andβ,conducted experiments on mammals, cell culture, or in the in vitro system.

In particular, transgenic line of mice that carry the gene ARR human and prone to the development of neurodegenerative diseases result from the removal of Aβ-peptide (8) - Sturchler-Pierrat Ch., Abramowski D., Duke, M., K. Wiederhold et al. Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology. // Proceeding of National Academy of Sciences of USA. 1997. Vol.94, P.13287-13292). Testing each potential drug requires experiment with a whole group of animals and lasts at least one year.

Experiments with cell cultures and in the in vitro system require the use of biochemical methods (9) - Pike C.J., Walencewicz AJ, C.G. Glabe, Cotman C.W. In vitro aging of beta-amyloid protein causes peptide aggregation and neurotoxicity. Brain Research. 1991 Nov 1; 563(1-2), P.311-314); (10) - Lorenzo, A., B.A. Yankner Beta-amyloid neurotoxicity requires fibril formation and is inhibited by congo red. Proc Nati Acad Sci USA. 1994 Dec 6; 91(25), P.12243-12247). It is obvious that these approaches do not allow large-scale screening of agents that block the formation of amyloid.

The analysis was carried out using the standard method of differential centrifugation followed by Western-blot hybridization (MA and Lindquist, 1999, Nature Cell Biol, 1(6): 358-361).

Total protein was isolated from strain accharomyces cerevisiae, containing the hybrid gene Pcupl-Aβ-SUP35MC, and divided using ultracentrifugation in sucrose gradient in the different fractions. Top (lightest) fraction contains protein in a Monomeric isoforms. In all other fractions (sedimentary) protein can be detected only if it forms in the cells of high molecular weight aggregates due to aggregation. After that the protein obtained from all of the fractions denaturiruet to the Monomeric state and were applied to polyacrylamide gel for conducting protein electrophoresis. Proteins transferred from gel to nitrocellulose membrane and carried out Western blot hybridization with monoclonal antibodies (4F Sigma) to aβthe peptide. In all analyzed fractions revealed specific hybridization signal in the region of 80-90 kDa, which corresponds to the size of the protein Andβ-Sup35MC. On the second track specific signal corresponds to Monomeric protein fractions Aβ-Sup35MC. The presence of a specific signal on the 2nd, 3rd, 4th and 5th tracks indicates the presence in yeast cells aggregates Aβ-Sup35MC of different sizes (figure 2).

Since a large fraction of the protein is detected in sediment fractions, it can be argued that protein Aβ-Sup35MC aggregates in the yeast cell. Because of the shortened protein Sup35MC not capable of aggregation (Ter-Avanesyan et al., 199, Genetics, 137:671-676), it can be argued that the aggregation due to the presence in the composition of the hybrid protein sequence Aβ-peptide.

The list of references

1. Wisniewski T., Ghiso j, Frangione Century Bilology of Ar amyloid in Alzheimer's desease. // Neurobiology of desease. 1997. 4: 313-328.

2. Citron M., Westaway D., Xia, W., Carlson, G. et al. Mutant presenilins of Alzheimer's disease increase production of 42-residue amyloid beta-protein in both transfected cells and transgenic mice. // Nat Med. 199 V3. Page 67-72.

3. Suzuki n, Cheung T.T., X.D. Cai, A. Odaka et al. An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants. // Science. 1994. 264. P.1336-1340.

4. Tamaoka A., A. Odaka, Y. Ishibashi, M. Usami et al. APP717 missense mutation affects the ratio of amyloid beta protein species (A beta 1-42/43 and a beta 1-40) in familial Alzheimer's disease brain. J Biol Chem. 1994. 269(52). P.32721-32724.

5. Price D.L., Sisodia S.S. Mutant genes in familial Alzheimer's disease and transgenic models. Annu Rev Neurosci. 1998. V21. P.479-505.

6. Prusiner S.B. Shattuck lecture - neurodegenerative deseases and prions. // N Eng J Med. 2001. V.344. No.20. P.1516-1526].

7. Zhouravleva, G., Frolova L., Le Goff x, Le Guellec, R., Inge-Vechtomov, S., Kisselev L., Philippe M. Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3. // EMBO J. 1995. 14:4065-4072.

8. Sturchler-Pierrat Ch., Abramowski D., Duke, M., K. Wiederhold et al. Iwo amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology. // Proceeding of National Academy of Sciences of USA. 1997. Vol.94, P.13287-13292.

9. Pike C.J., Walencewicz AJ, C.G. Glabe, Cotman CW. In vitro aging of beta-amyloid protein causes peptide aggregation and neurotoxicity. Brain Research. 1991 Nov 1; 563(1-2), P.311-314.

10. Lorenzo A., B.A. Yankner Beta-amyloid neurotoxicity requires fibril formation and is inhibited by congo red. Proc Nati Acad Sci USA. 1994 Dec 6; 91(25), P.12243-12247.

Hybrid is Yong P cupl-Aβ-SUP35MC for analysis of factors governing the production, aggregation and disaggregatio peptide amyloid β (Aβ) man in the yeast system, consisting of Xhol fragment-BamHl size 477 base pairs, representing a sequence of CUPl-promoter, BamHl-BamHl fragment size 135 base pairs, representing a sequence encoding a full-sized amyloid beta peptide (Aβ-peptide) of a person, size 40 amino acids (see nucleotide sequence presented in the sequence listing under the number "2"), flanked TATA box and the start-codon (ATG) with 5'-end, BamHl-Sacl fragment size 1879 base pairs, representing the sequence, encoding M and C domains of protein Sup35 yeast Saccharomyces cerevisiae, flanked by a BamHl restriction site to the 5'and the sequence termination broadcast and website Sad with the 3'-end.



 

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