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WO1996012008A1 - Synthese de genes resistant a la methylase - Google Patents

Synthese de genes resistant a la methylase Download PDF

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Publication number
WO1996012008A1
WO1996012008A1 PCT/US1995/013536 US9513536W WO9612008A1 WO 1996012008 A1 WO1996012008 A1 WO 1996012008A1 US 9513536 W US9513536 W US 9513536W WO 9612008 A1 WO9612008 A1 WO 9612008A1
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WO
WIPO (PCT)
Prior art keywords
gene
methylase
resistant
lad
mrkl
Prior art date
Application number
PCT/US1995/013536
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English (en)
Inventor
Thomas R. Skopek
Original Assignee
Merck & Co., Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Publication of WO1996012008A1 publication Critical patent/WO1996012008A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • the present invention relates to a general approach for the synthesis of genes resistant to mammalian CpG methylase enzyme.
  • Methylation of DN A in mammalian cells is a natural process involved in the control of gene expression.
  • Methyl groups are added to cytosine residues in CpG sequences by the enzyme CpG methylase. This enzyme converts the cytosine residues in CpG sequences to 5-methylcytosine.
  • Virtually all methylation in mammalian DNA is in the form of 5-methylcytosine in CpG sequences. The presence of a high level of methylation in a gene results in suppression of the gene's expression.
  • 5-Methylcytosine has been shown to be highly promutagenic since it can spontaneously deaminate to form thymine in the DNA. Upon replication of the DNA, the newly formed thymine will base pair with adenosine, resulting in a GC ⁇ AT transition mutation. Foreign genes often become highly methylated when introduced into mammalian cells, resulting in inactivation and enhanced rates of mutation. Therefore, the presence of 5 -methyl cytosine is undesirable in many recombinant DNA experiments which involve the introduction of foreign genes into whole animals or into cells in culture media. Prior attempts to addess this problem have included using
  • 5-azacytidine to replace cytidine in the DNA.
  • 5AzC inactivates the CpG methylase enzyme when it attempts to react with 5AzC in the DNA, thus indirectly protecting other cytosine residues in the DNA from methylation by the methylase enzyme.
  • 5AzC is extremely toxic, most likely due to its nonspecific nature. It is expected that the addition of 5AzC leads to the demethylation of all sequences, including those sequences which must remain methylated to maintain the viability of the cell or organism. Thus, this approach does not provide a reliable, long-term solution to the problem.
  • methylase-resistant genes can be synthesized by removal of the highly methylated CpG sequences. This is accomplished by the introduction of specific alterations in the coding sequence of a given gene which destroys the 5'-CpG-3' methylase recognition sequences but which retain the original coding information of the gene.
  • the present invention contemplates a method for the synthesis of methylase-resistant genes by removal of CpG sequences through the introduction of specific alterations in the coding sequence of a given gene which destroys methylase recognition without altering the original coding information of the gene. By substituting for the CpG sequences, the problem with high background mutation rates due to deamination of 5-methylcytidine residues would be lessened.
  • transgenic animals such as mice carrying bacterial reporter genes for mutagenesis such as in the Big Blue® mouse system from Stratagene and the MutaMouseTM system from Hazelton Washington.
  • Transgenic animal models are also being developed for the study of disease etiology and the use of these methylase-resistant genes could increase the possibility of success by circumventing the problem of methylation of inserted gene sequences.
  • this invention may be useful relative to DNA vaccines.
  • the efficacy of DNA vaccines is dependent on the continued expression of the desired protein product.
  • the use of methylase-resistant genes may prevent gene inactivation caused by methylation. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention contemplates a method for the synthesis of methylase-resistant genes by removal of CpG sequences through the introduction of specific alterations in the coding sequence of a given gene which destroys methylase recognition without altering the original coding information of the gene.
  • the CpG sequence When the CpG sequence is in the first and second position of the codon, it codes for the amino acid arginine (CGX). Arginine is also coded by the codons AGG and AGA and these codons can replace the unwanted CGX codons.
  • the invention has been demonstrated through the use of new genes, designated the mrkl-mrklX genes, which code for the protein product of the Escherichia coli lad gene.
  • the protein product forms the basis of a mutational selection scheme which is important in the mutation research field.
  • Strain Y1088 [el4"(mcrA), ⁇ (lac)U169, supE, supF, hsdR, metB, trpR, tonA21, proC::Tn5(KanO [pMC9Ampr, Tet r ]] and strain AG1 [recAl , endAl , gyrA96, thi-1 , hsdR 17 (rfc- ⁇ ik-i-), supE44, relAl ] were obtained from Stratagene (La Jolla, CA).
  • Strain ⁇ 5 [ara, thi, strA, val r , galE, ⁇ prolac, ⁇ 80dlac ⁇ (tonB-lacI) was the generous gift of Dr. Roel Schaaper (National Institute of Environmental Health Studies, Research Triangle Park, NC). Strain ⁇ 5 possesses an intact lacZ gene, but the lad gene has been completely removed by deletion .
  • Plasmid pBR322 was obtained from GIBCO/BRL (Bethesda, MD). Plasmid pMC9 is a pBR322-derived vector containing the lad ⁇ gene. pMC9 was isolated from strain Y1088.
  • DNA was synthesized using standard ⁇ -cyanoethyl phosphoramidite chemistry and assembled by Midland Certified Reagent Co. (Midland TX). Mrkl was synthesized in sections as a series of four double-stranded cassettes (A-D).
  • Cassette A contained the EcoRl -Sphl region
  • B contained the Sphl-Kpnl region
  • C contained the Kpnl-Pstl region
  • D contained the Pstl-Sall region .
  • Each cassette was assembled by ligating overlapping, complimentary oligonucleotides which ranged inlength from 50-150 nucleotides.
  • the flanking regions of each cassette contained added restriction enzyme sites to facilitate cloning of the cassette and assembling the final gene construct. The cassettes were then assembled by cutting with the appropriate flanking restriction enzyme and systematically attaching one cassette to another with ligase.
  • the coding region of the wild-type E. coli lad gene contains 95 occurrences of the CpG dinucleotide in its nontranscribed strand. Of these 95 CpG sequences, 37 are associated with XCG codons (where X is any base), 40 are associated with contiguous XXC_GXX codons, and 18 are associated with arginine codons, CGX.
  • the first two types of CG sequence were removed by altering the wobble (third) base of the CG -containing codon(s).
  • the 18 CG-containing arginine codons (CGX) could have been removed by substitution of AGA or AGG arginine codons; however, these were left in the prototype gene since AGA and AGG codon usage in E. coli is rare.
  • the mrkll sequence is identical to the mrkl sequence with the exception that six CGT arginine codons (bases 64-66, 103-105, 151- 153, 256-258, 301-303, and 352-354) were replaced with AGG arginine codons.
  • the first three arginine codons were located in the EcoR 1 -Sphl region (cassette A), and the remaining three were located in the Sphl- Kpnl region (cassette B)(see mrkl synthesis above).
  • the sequences in cassettes A and B were individually modified using PCR amplification with mismatched primers.
  • both sense and antisense primers containing the desired sequence alteration were synthesized; these covered and extended approximately 20 nucleotides beyond the region spanned by the three arginine codons in the cassette.
  • Each "mutagenic" primer was paired with a short downstream primer flanking the vector/insert junction for PCR amplification.
  • the two PCR reactions for each cassette were run for several cycles and then combined.
  • the pooled sample was then heated and allowed to anneal. This generated long, overlapping duplexes containing recessed 3' ends; these were extended in a subsequent polymerase step.
  • This product was then diluted into another PCR reaction and amplified with the two short flanking primers to produce the full length, modified casette sequence.
  • the full length sequence was cloned into pUC18 and sequenced to verify its structure.
  • the modified casette A and B were then used with casette C and D to assemble the complete mrkll gene.
  • the mrkl and mrkll constructs contain a promoter with consensus -35 and -10 regions (separated by 17 bases), a translation enhancer from gene 10 of bacteriophage T7 and a consensus Shine- Dalgamo sequence.
  • Gene 10 of bacteriophage T7 is disclosed in Olins, P.O. and Rangwala, S.H. (1989) J. Biol. Chem. 264, 16973-16976.
  • the mrklll sequence was generated by replacing the promoter region of mrkll with the lad ⁇ promoter.
  • the lad ⁇ promoter sequence was generated by PCR amplification of plasmid pMC9 using two PCR primers homologous to this region.
  • the upstream primer (5'- CCGG AATTCG ACCATCG AATGGTG-3 ' contained an EcoRl site at its 5' end; the 3' end was homologous to lad bases -87 to -74 (see Figure 1 ).
  • the downstream primer (5 '-CCCAT_ATGCACCCTGAA-3') contained a Ndel site at its 5' end; the 3' end was homologous to bases -27 to -4.
  • the PCR product was cut with EcoRl and Ndel and used to replace the EcoRl -Ndel promoter-containing fragment of mrkll.
  • MrklV-MrklX were produced from mrkll by PCR amplification with a primer containing one or two mismatches in the -35 region of the promoter and a primer homologous to the end of the coding region. Mismatched primers used were:
  • the downstream primer was 5'-TCGGTCGACTATTACTGGCC-3'.
  • the mrkl -IX gene products were digested with EcoRl and Sail and cloned into pBR322 by replacing the EcoR l -Sail fragment of the tetracycline resistance gene.
  • the sequence of all final mrk constructs was verified with an Applied Biosystems automated DNA sequencer using the fluorescent dideoxy sequencing method.
  • mrkl-mrklX gene construct in plasmid pBR322 were introduced into E. coli strain ⁇ 5 [ara, thi, strA, val r , galE, ⁇ prolac, ⁇ 80dlac ⁇ (tonB-/ ⁇ c/)], obtained from Roel Schaaper (NIEHS, Research Triangle Park, NC).
  • ⁇ 5 has the lad gene deleted from its chromosome but has an intact and functional lacZ gene.
  • the lacZ gene encodes the enzyme ⁇ -galactosidase which cleaves the chromogenic-substrate X-gal (source) to produce a blue color.
  • Active lad gene product represses the expression of the lacZ gene (in the absence of inducer of the lactose operon). If repressed by lad gene product, the lacZ gene is not expressed and the cells do not produce ⁇ - galactosidase and white colonies are seen in the presence of X-gal. Since ⁇ 5 has the lad gene deleted, the lacZ gene is expressed and it forms blue colonies in the presence of X-gal. As a further test, the experiment was also performed in the presence of a specific inducer of the lacZ gene, LPTG. IPTG binds tightly and specifically to the lad protein; when bount to IPTF, the lad protein can no longer repress the lacZ gene and ⁇ -galactosidase is produced.
  • LPTG a specific inducer of the lacZ gene
  • WESTERN BLOTS ⁇ 5 cells carrying unmodified pBR322, pBR322 containing lacfl, and pBR322 containing mrkl - IX were analyzed by Western blot (1 ) using polyclonal antiserum to lad repressor protein.
  • Bacteria were grown in NZY broth with ampicillin and diluted to an O.D.600 of 0.5. Aliquots of bacterial suspension (0 - 2.5ml) were boiling for 10 min. in sample buffer containing 1 % SDS and 350 mM 2-mercaptoethanol. Solubilized proteins were separated by polyacrylamide gel electrophoresis (PAGE) using pre-cast 10-20% polyacrylamide gels (ISS, Natick, MA).
  • the proteins were transferred to nitrocellulose using an ISS semi-dry electroblotter at 1 milliamp./cm ⁇ for 1.5 hr.
  • the nitrocellulose was air dried for 5 minutes and incubated 1 hr at room temperature in tris buffered saline (TBS) containing 5% non-fat dry milk to block non-specific protein binding sites on the nitrocellulose.
  • TBS tris buffered saline
  • a 1 :2000 dilution of rabbit polyclonal antiserum to lad protein (Stratagene, La Jolla, CA) was added to the blots in TBS containing 1 % nonfat dry milk and incubated overnight at 4°C. Blots were washed 3 times in TBS with 0.1% tween for 10 minutes each.
  • Detection of lad antibody was done as described in ECL detection kit (Amersham Life Science, Arlington Heights, IL.). Horseradish peroxidase linked anti-rabbit Ig was diluted 1 :2000 in TBS + 1% non-fat dry milk and added to the blot for 1 hr at room temperature. Following the incubation, the blots were washed in TBS with 0.1 % tween. The blots were visualized on Reflection film (NEN) following the addition of ECL detection reagent. Film exposure was generally less than one minute.
  • Plasmids pBR322, pMC9 (pBR322 carrying the lad q gene), and pBR322 carrying mrk I through mrk IX were transformed into E. coli strain ⁇ 5 (deleted for lad). Cells carrying unmodified pBR322 or mrk III developed blue colonies on plates containing ampicillin and X-gal while strains carrying the lad ⁇ gene or other mrk derivatives produced white colonies. These results suggested that cells carrying the lad ⁇ gene or mrk derivatives, except mrkll I, were expressing lad protein at a level that was capable of repressing the lacZ gene.
  • ATGTTTCTGC CAAAACCCGT GAAAAAGTGG AAGCAGCCAT GGCAGAACTG AATTACATCC 240
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • ATGTTTCTGC CAAAACCAGG GAAAAAGTGG AAGCAGCCAT GGCAGAACTG AATTACATCC 240

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Abstract

L'invention se rapporte à un procédé de synthèse de gènes résistant à la méthylase. Le procédé consiste à effectuer des modifications spécifiques dans la séquence de codage d'un gène donné qui détruit les séquences de reconnaissance de la méthylase sans modifier les informations de codage d'origine du gène. L'invention se rapporte également à de nouveaux gènes, appelés mrkI-mrkIX, qui ont été synthétisés pour illustrer le procédé.
PCT/US1995/013536 1994-10-13 1995-10-10 Synthese de genes resistant a la methylase WO1996012008A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US32223994A 1994-10-13 1994-10-13
US322,239 1994-10-13

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WO1996012008A1 true WO1996012008A1 (fr) 1996-04-25

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1419257A1 (fr) * 2001-07-27 2004-05-19 Geneinvent Bbl AB Vecteurs resistants a la methylation
WO2007128685A1 (fr) * 2006-05-02 2007-11-15 Chromagenics B.V. Sélection de cellules hôtes exprimant une protéine à de hauts niveaux
US7723500B2 (en) 1994-07-15 2010-05-25 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20110052666A1 (en) * 2009-09-03 2011-03-03 Medtronic, Inc. Compositions, Methods, and Systems for SIRNA Delivery
US8039230B2 (en) 2004-11-08 2011-10-18 Chromagenics B.V. Selection of host cells expressing protein at high levels
US8263393B2 (en) 2002-12-20 2012-09-11 Chromagenics B.V. Means and methods for producing a protein through chromatin openers that are capable of rendering chromatin more accessible to transcription factors
US8771984B2 (en) 2004-11-08 2014-07-08 Chromagenics B.V. Selection of host cells expressing protein at high levels
US8999667B2 (en) 2004-11-08 2015-04-07 Chromagenics B.V. Selection of host cells expressing protein at high levels
US9228004B2 (en) 2004-11-08 2016-01-05 Chromagenics B.V. Selection of host cells expressing protein at high levels

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993017123A1 (fr) * 1992-02-27 1993-09-02 Ohio University Test de la mutagenicite a l'aide de genes rapporteurs avec frequences de methylation modifiees

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993017123A1 (fr) * 1992-02-27 1993-09-02 Ohio University Test de la mutagenicite a l'aide de genes rapporteurs avec frequences de methylation modifiees

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7723500B2 (en) 1994-07-15 2010-05-25 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
EP1419257A1 (fr) * 2001-07-27 2004-05-19 Geneinvent Bbl AB Vecteurs resistants a la methylation
US8263393B2 (en) 2002-12-20 2012-09-11 Chromagenics B.V. Means and methods for producing a protein through chromatin openers that are capable of rendering chromatin more accessible to transcription factors
US8039230B2 (en) 2004-11-08 2011-10-18 Chromagenics B.V. Selection of host cells expressing protein at high levels
US8771984B2 (en) 2004-11-08 2014-07-08 Chromagenics B.V. Selection of host cells expressing protein at high levels
US8999667B2 (en) 2004-11-08 2015-04-07 Chromagenics B.V. Selection of host cells expressing protein at high levels
US9228004B2 (en) 2004-11-08 2016-01-05 Chromagenics B.V. Selection of host cells expressing protein at high levels
WO2007128685A1 (fr) * 2006-05-02 2007-11-15 Chromagenics B.V. Sélection de cellules hôtes exprimant une protéine à de hauts niveaux
CN101437946B (zh) * 2006-05-02 2012-07-04 科罗迈吉尼科斯公司 选择高水平表达蛋白质的宿主细胞
US20110052666A1 (en) * 2009-09-03 2011-03-03 Medtronic, Inc. Compositions, Methods, and Systems for SIRNA Delivery

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