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WO1992001811A1 - Analyse des mutations de ctp-synthetase donnant lieu a une resistance a la polychimiotherapie - Google Patents

Analyse des mutations de ctp-synthetase donnant lieu a une resistance a la polychimiotherapie Download PDF

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WO1992001811A1
WO1992001811A1 PCT/GB1991/001243 GB9101243W WO9201811A1 WO 1992001811 A1 WO1992001811 A1 WO 1992001811A1 GB 9101243 W GB9101243 W GB 9101243W WO 9201811 A1 WO9201811 A1 WO 9201811A1
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mutation
ctp
dna
assay method
assay
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Mark Meuth
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Cancer Research Horizons Ltd
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Imperial Cancer Research Technology Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • CTP synthetase is also a key regulatory point as its activity is subject to activation by GTP and inhibition by CTP, and mutant cell strains altered in this regulation (ie insensitive to inhibition by CTP) display a complex phenotype: i) increased intracellular pools of CTP and dCTP, ii) multidrug resistance, and iii) an increased rate of spontaneous mutation.
  • MDR multidrug resistance phenotype
  • AML acute myelogenous leukemia
  • ALL acute ly phocytic leukemia
  • non-Hodgkin's lymphomas 5-fluorouracil (5-FU)
  • DNA alkylating agents such as cyclophosphamide (used against high grade lymphoma, ALL), melphalan (used against myeloma) and chlorambucil (used against low grade lymphoma, CLL) .
  • One aspect of the invention provides a genetic assay method comprising the steps of (1) obtaining a polynucleotide having the same nucleotide sequence as the CTP synthetase gene, or a relevant part thereof, of the tumor cell of an organism and (2) assaying that polynucleotide for a mutation in exons 4, 5 or 7 or for a mutation in an intron which controls the splicing of any of the said exons, the mutation being indicative of the CTP synthetase encoded by the said gene having deficient regulation by CTP.
  • the "relevant part" of the CTP synthetase gene is any part which contains the region(s) in which the mutation is being sought.
  • the assay is capable of detecting mutations in the polynucleotide which give rise to amino acid changes in the region 110-236, especially in the sub- regions 110-120, 148-166 and 230-236 and particularly at amino acid positions 114, 152, 154, 157, 161, 164 or 235.
  • the polynucleotide may be genomic DNA, cDNA or mRNA or it may have been cloned or amplified from any of these. (In the case of RNA, of course, the sequence will be the same as the CTP gene but with uracil bases in place of thymidine bases.) The only important criterion is that the polynucleotide being assayed should represent the region of the CTP synthetase gene in which the said mutation is being sought. DESCRIPTION OF PREFERRED EMBODIMENTS
  • the assay technique may be chosen from those which are available and convenient in the art at the time. Currently, these include sequencing the nucleotide sequence by techniques such as the Sanger or Maxam/Gilbert methods; differential hybridization of an oligonucleotide probe designed to hybridize at the relevant positions of either the wild-type or mutant sequence; denaturing gel electrophoresis following digestion with an appropriate restriction " enzyme, preferably following amplification of the relevant DNA regions; SI nuclease sequence analysis; non-denaturing gel electrophoresis, preferably following amplification of the relevant DNA regions; conventional RFLP (restriction fragment length polymorphism) assays; or DNA amplification using oligomicleotides which are matched for the wild-type region and unmatched for the mutant region or vice versa.
  • sequencing the nucleotide sequence by techniques such as the Sanger or Maxam/Gilbert methods; differential hybridization of an oligonucleotide probe designed to hybridize at the relevant positions of either the wild-
  • a non-denaturing gel is used to detect differing lengths of fragments resulting from digestion with an appropriate restriction enzyme.
  • the DNA is usually amplified before digestion, for example using the polymerase chain reaction (PCR) method disclosed in Saiki et al and modifications thereof. Otherwise 10-100 times more DNA would need to be obtained in the first place, and even then the assay would work only if the restriction enzyme cuts the nucleic acid infrequently.
  • PCR polymerase chain reaction
  • an "appropriate restriction enzyme” is one which will recognise and cut the wild-type sequence and not the mutated sequence or vice versa.
  • Various enzymes are disclosed below as specific examples, but any enzyme which cuts at the same place (an “isoschizomer") or which recognises the same sequence and cuts the DNA at a point within or adjacent the sequence will be suitable; more are being discovered all the time. It is convenient if the enzyme cuts nucleotides only infrequently, in other words if it recognises a sequence which occurs only rarely.
  • Restriction enzymes useful in connection with the mutations described above include, for example, Sphl (GCATGC) and StuI (AGGCCT) .
  • GCATGC Sphl
  • AGGCCT StuI
  • One of the specific mutations found removes the Sp ⁇ l site.
  • Msp ⁇ and TaqI sites may be created in mutations.
  • the amplification or not of the DNA is used as the basis of the assay, rather than just as a means of generating enough DNA for the assay to be performed. If the primer matches the wild-type sequence, then wild-type DNA will be amplified and mutant DNA will not. If the primer matches a mutant sequence (it is convenient for the PCR primer to have the mutation as its 3'-most nucleotide), then matching mutant DNA will be amplified whereas wild-type and non-matching mutant DNA will not. The former approach is more indicative of the presence of a mutation at the relevant site but the latter approach is more informative regarding the nature of the mutation and is more sensitive. A positive reaction can be confirmed by using a primer for amplification of the opposite strand.
  • the nucleic acids, usually DNA rather than RNA, which are assayed may be obtained from any appropriate tumor cell of the body, for example a biopsy sample from a solid tumor or a white blood cell obtained from blood samples taken from patients at presentation or during or after chemotherapy.
  • the mutations appear to be tumor-specific and to arise during tumor development. They may be enriched during a first round of drug therapy.
  • the DNA is extracted by known techniques and a specific region of the CTP synthetase sequence is amplified using the PCR. In an RFLP-based assay, it is then digested with the restriction enzyme and subjected to PAGE (polyacry1amide gel electrophoresis).
  • the gel is stained and photographed to reveal a pattern of fragments indicative of whether the patient is MDR.
  • the stain will usually comprise a labelled probe which will hybridise to one or more of the nucleotide fragments in question, the label being any agent which can be visualized, for example a radioactive atom.
  • the whole procedure, using current technology, takes about 5 hours.
  • the phenotype may develop as a result of spontaneous mutation in a target cell or as a result of the mutagenic action of chemotherapeutic agents.
  • Cells with the mutation have a growth advantage in the normal cellular milieu or during chemotherapy and are therefore selected for.
  • a kit may be provided, according to another aspect of the invention, to perform the assay.
  • a kit in which the assay is based on selective amplification by PCR will typically contain the primer(s) needed for the PCR amplification and also control nucleic acid for both MDR and non-MDR phenotypes, so that the results of the assay can be analyzed more readily.
  • An RFLP kit will typically contain restriction enzyme(s) and probe(s) and, optionally, PCR primers for general (non-selective) amplification of the DNA prior to RFLP analysis.
  • the assays of the invention will be valuable in relation to human medicine and may be used prior to treatment with a drug which is not metabolised correctly in the MDR phenotype.
  • the drug may be any of those mentioned above, especially araC.
  • a different drug the metabolism of which is unaffected or affected desirably by the MDR phenotype, may be administered.
  • the mutations may be important in tumor progression and metastasis and detecting them may therefore be useful for monitoring tumor progression.
  • successive assays may be performed on the same tumor to monitor its progress, the treatment being adapted appropriately.
  • the proportion of cells having the MDR phenotype may indicate the development of the tumor and its metastatic potential, hence indicating the need for an antimetastasis treatment.
  • the assay will reveal whether the mutation is one which affects splicing of the said exon or exons.
  • mutations of interest are those which do not reduce the CTP synthetase activity to a fatally low level but which reduce the inhibition by CTP.
  • mutant CTP synthetases themselves (rather than mutations in the gene) by sequencing the said regions of the protein.
  • this is not nearly as feasible as the assays described above based on nucleotide sequences.
  • monoclonal antibodies specific for the mutant or for the wild-type enzyme.
  • Such monoclonal antibodies can be prepared by known techniques, for example those disclosed in H. Zola “Monoclonal Antibodies” (CRC Press 1988) or J.G.R Hurrell: “Monoclonal Hybridoma Antibodies” (CRC Press 1982), which are incorporated herein by reference.
  • a further aspect of the invention provides a method of treating a patient with a drug, resistance to which is affected by the presence or absence of the MDR phenotype, the method comprising (1) determining whether a tumor cell of the patient has a significant mutation in exons 4, 5 or 7 encoding amino acid residues 110 to 236 of CTP synthetase or in an intron which controls splicing of any of the said exons and (2) devising a dosage regimen of the said drug for the patient according to whether there is such a mutation.
  • the said dosage regimen may be zero; an alternative drug or radiotherapy may be used instead.
  • Figure 1 shows the wild- type human CTP synthetase cDNA sequence
  • Figure 2 shows Chinese Hamster Ovary CTP synthetase cDNA
  • Figure 3 shows a first peptide sequence from Chinese Hamster Ovary CTP synthetase
  • Figure 4 shows a second peptide from Chinese Hamster Ovary CTP synthetase
  • Figure 5 shows a third peptide sequence from Chinese Hamster Ovary CTP synthetase.
  • the cytidine auxotrophic strain CR"2 originally isolated from the Toronto Pro" CHO cell line (Kelsall and Meuth, 1988), was grown in ⁇ -minimal essential medium ( -MEM, GIBCO) supplemented with 7.5% fetal calf serum, 10 ⁇ M thymidine and 20 ⁇ m cytidine. When CTP synthetase proficient transfectants were selected, 5% dialysed fetal calf serum was used for the cytidine deficient medium. Chromosome and DNA mediated gene transfer
  • Metaphase chromosomes were prepared essentially as described by Lewis et al , (1980). Colcemid was added to a 11 exponentially growing culture of HeLa cells to a final concentration of 50 ng/ml. After 18 h cells were harvested and resuspended in 100 ml of ice cold 75 mM KC1 for 30 min. After recentrifugation, the cells were then resuspended in 2 ml ice cold 15 mM HEPES(Na) pH 7.0, 3mM CaCl 2 , and 1% NP-40 for disruption by a Dounce homogenizer (10 strokes, on ice).
  • the homogenate was centrifuged at 200 g to remove debris and the resulting supernatant was then centrifuged at 1500 g for 20 min at 4°C. The pellet obtained was washed once with ice cold homogenization buffer without detergent. This chromosome preparation was then resuspended in 10 ml of gene transfer buffer (25 mM HEPES(Na) pH 7.1, 140 mM NaCl, and 0.75 mM Na 2 HP0 4 ) and 0.5 ml of 2.5 M CaCl 2 was added.
  • gene transfer buffer 25 mM HEPES(Na) pH 7.1, 140 mM NaCl, and 0.75 mM Na 2 HP0 4
  • the resultant co-precipitate was immediately added to recipient (CR"2) cells (2-3 x 10 6 /10 cm dish) in growth medium supplemented with antibiotics (0.05 mg/ml gentamycin and 1 ⁇ g/ml amphotericin B, Sigma), 15 mM HEPES(Na) pH 7.1, and 0.1% polyethylene glycol (MW1500).
  • antibiotics 0.05 mg/ml gentamycin and 1 ⁇ g/ml amphotericin B, Sigma
  • 15 mM HEPES(Na) pH 7.1 15 mM HEPES(Na) pH 7.1
  • polyethylene glycol MW1500
  • Recipient cells were exposed to chromosome or DNA-calcium phosphate co-precipitate for 8 - 16 h, treated with 10% DMSO in the last 30 min, and then allowed to recover in nonselective growth medium for 24 h. Treated cells were trypsinized, washed in PBS twice and plated at a density of 2 - 5 x 10 5 /10 cm dish in the absence of cytidine. Colonies appearing after 2 - 4 weeks were picked for further analysis.
  • a human lambda gtll cDNA library prepared with RNA isolated from human testis (Clontech), was screened with the conserved 3.3 kb unique human DNA fragment common to all our transfectants. Positives obtained were then used for DNA sequence analysis.
  • RNA prepared from HeLa cells for RACE PCR Rapid amplification of cDNA end polymerase chain reaction: Frohman et al . , 1988
  • RACE PCR rapid amplification of cDNA end polymerase chain reaction: Frohman et al . , 1988
  • oligonucleotide primer nucleotides 784—> 765 or 5'- CAACATGGCAGAACATTGAT-3' , SEQ14
  • super RT Anglian Biotec Ltd. UK
  • the product was A tailed by nucleotidyl transferase (BRL) and the opposite DNA strand was synthesized using a (dT)17 adaptor and the Tag polymerase (Perkin Elmer Cetus).
  • the resultant double-stranded DNA was then amplified using the adaptor and a nested primer (nucleotides 758 --> 739 or 5'-TCCTTCACTGATGTGTCAAG-3' , SEQl5) by PCR.
  • the product of this reaction was digested with Sail, and Bglll and cloned into the M13 vectors mpl8 and 19. The clone with the longest insert (700bp) was sequenced.
  • Sequencing was performed by the dideoxy chain termination method using [ - 35 S]dATP. Polymerization reactions using Sequenase (USB) were primed by either the 17mer universal primer or appropriate internal primers. Sequencing reactions were electrophores . ed on 5% polyacrylamide denaturing gels.
  • the Chinese hamster (CHO) CTP synthetase deficient cytidine auxotroph CR" 2 was treated with DNA or mitotic chromosomes co-precipitated with calcium phosphate. After recovery and expression, the cells were plated in selective medium (lacking cytidine) to allow formation of colonies acquiring the human CTP synthetase gene. The frequency of such colonies in the first round of transfer was ⁇ 10 6 . When purified DNA was used instead of isolated chromosomes, the frequency of colony formation was about 10" 7 . In control cultures (no added DNA or chromosomes) no colonies were observed (frequency ⁇ 10 ⁇ 7 ).
  • DNAs purified from several of the isolates were digested with the restriction endonuclease Ec ⁇ Rl. Digests were fractionated by electrophoresis on agarose gels and transferred to nitrocellulose filters. These filters were then hybridized with labelled human Alu repeat BLUR 8. DNA purified from the colonies recovered after CMGT contained a smear of fragments by hybridizing with the BLUR 8 probe. Colonies derived from DNA transfers, on the other hand, showed no human Alu containing DNA (data not shown) .
  • chromosomes isolated from one of the transfectants were used to transfect CR ⁇ 2 for a second round of transfection and chromosomes purified from a secondary transfectant (PC91C) were subsequently used in a third round.
  • the frequencies of colonies growing in the absence of cytidine in the latter two rounds were somewhat lower than the first (1 - 2 x 10" 7 ), but DNA purified from these survivors clearly contained human Alu sequences. It also appeared that the colonies obtained from these further rounds .of CMGT had progressively fewer human Alu containing fragments.
  • DNA obtained from one of the isolates (91C12) was digested with EcoRI , size fractionated and ligated to .EcoRI cleaved DNA from the lambda vector NM1149.
  • the recombinant phage libraries derived were screened for human ⁇ iu-bearing fragments: 120 independent isolates were eventually picked and cloned inserts from these recombinants were used to screen a panel of primary, secondary and tertiary transfectants. Presumably any fragments retained by all the strains would be closely linked with the CTP synthetase structural gene. Of the 120 fragments screened in this manner only two were common to the entire panel.
  • RNA prepared from human cells was fractionated and probed on Northern blots with the labelled 3.3 kb fragment. A clear band of about 3 kb hybridized with this probe. In contrast RNA prepared from the CTP synthetase deficient hamster strain showed no band.
  • RACE polymerase chain reaction RACE PCR
  • An oligonucleotide complementary to the sequence just downstream of the Bglll site of our cDNA was used as primer for reverse transcription of the CTP synthetase message in RNA prepared from HeLa cells. Transcripts were then A tailed and a second (nested) oligonucleotide complementary to the cDNA together with a tail-specific oligonucleotide (also bearing a Sail site for subsequent cloning) were used to amplify the products generated from reverse transcription.
  • RACE PCR RACE polymerase chain reaction
  • DNA or RNA is purified by standard techniques from white blood cells of patients with acute leukemia or from solid tumor cells. DNA can be used directly in polymerase chain reaction techniques to detect the mutant gene but RNA is first reverse transcribed using a suitable oligonucleotide primer (for example TCAGCCATCTCTTTCCATTT, SEQ16) 3' of the critical region and reverse transcriptase (super RT, Yalen Biotec Ltd., UK) as recommended by the manufacturer. The region of interest is then specifically amplified using a suitable 5' oligo- nucleotide (for example CACAGATGCAATCCAGGAGT, SEQ17) in combination with nested 3' oligos, for example as follows:
  • a suitable oligo- nucleotide for example CACAGATGCAATCCAGGAGT, SEQ17
  • exon 4 5' primer: GTAACTATGAGCGGTTCCTT (SEQl8) 3' primer: AGTTCTCTCTTTTGACCTTG (SEQl9)
  • exon 5 5' primer: CACAGATGCAATCCAGGAGT (SEQ20) 3' primer: GGAACTAGACTGACGTGGAT (SEQ21)
  • exon 7 5' primer: CAAGGTCAAAAGAGAACT (SEQ22) 3' primer: CTCTAACAACAAGGGGACTC (SEQ23)
  • exon 4 5' primer: GCAACAACTTAGACATCTGC (SEQ24) 3' primer: ACTCCTGGATTGCATCTGTG (SEQ25)
  • exon 7 5' primer: GTTCAACAGGGGAACAGAAG (SEQ26) 3' primer: ATTTACTCACTTGTTCAGGC (SEQ27)
  • thermostable DNA polymerase Perkin Elmer Cetus or one of the new, more accurate thermostable polymerases (Vent TM DNA polymerase, New England Biolabs) is used to amplify the DNA through 30 cycles of denaturation (95°, 15 sec), reannealing (52°, 15 sec), and elongation (72°, 1 min.).
  • the critical mutation can then be detected in the resulting product (200-300 bp in the reactions templated by the cDNA) by any of the following techniques: 1)
  • the double-stranded product is first digested with one or more suitable restriction endonucleases (e.g. Sph I and Stu I) having sites within this region of the wild- tyP* 3 gene. Mutations falling in the recognition sequences will eliminate the sites and the reactions will retain a full-length fragment after fractionation on agarose gels.
  • suitable restriction endonucleases e.g. Sph I and Stu I
  • the double-stranded product may be bound to nitrocellulose in dot or slot blots and mutation-specific oligonucleotides (see below) end-labelled with 32 P or fluorescently-labelled nucleotides may be hybridized with the bound DNA followed by a stringent washing protocol (twice for 10 min in 2 x SSC/0.2% SDS followed by 30-60 min in 3M tetramethyl-ammonium chloride, 50 mM Tris HC1 (pH 7.5), 2 mM EDTA, 0.3% SDS at 61°C) . DNA samples having the mutation will be labelled while those lacking the mutations will be negative.
  • the wild-type oligonucleotide may be hybridized to a similar filter as a positive control. This approach is preferred as, at least in our hands, it is quick and sensitive.
  • the double-stranded PCR product may be produced using end-labelled primers.
  • a formamide-dye mixture is then added to an aliquot of the product before being fractionated on a non-denaturing polyacrylamide gel in tris-borate buffer containing 10% glycerol as described by Orita et al (1989). Under such conditions mutant molecules run aberrantly and are easily detectable when compared to the wild-type control.
  • Alternative systems of electrophoresis have been described (e.g. Kogan &
  • the denatured-reannealed product may also be subjected to chemical treatment that results in cleavage at mismatches (Montandon et al (1989). In the latter case the reaction products are then fractionated by standard polyacrylamide gels and any digested products are apparent as smaller fragments.
  • DNA can also be used as a starting material in which case the regions amplified from the above priming oligonucleotides are larger as they include introns.
  • the PCR reaction is identical except that the elongation time will be extended to 2 min.
  • the mutation in exon 4 is such that intron sequences must be used as 5' primer.
  • the products of the reaction are analysed for the critical mutations as described above except in the case of the non-denaturing polyacrylamide gel.
  • oligonucleotides amplifying only the exon bearing the mutations eg AGCTTGGTGGAACCGTG (SEQ36) and CTGGGGAACTAGACTGAC (SEQ37) producing a fragment of 119 bp for exon 5, for example
  • the critical region may be sequenced (as described previously, Phear et al 1989) using an 32 P end-labelled primer to detect and identify the mutations.
  • An alternative approach is to use mutant specific oligonucleotides, which bear the mutant specific nucleotide at the very 3' end of the primer, in the polymerase chain reaction together with an upstream or downstream oligonucleotide.
  • mutant oligonucleotides from the opposite strand may be used together with a downstream primer yielding 202 to 228 bp fragments in the case of RNA.
  • DNA-based confirmation assays can be devised using intron sequences.
  • the oligonucleotides to be used are: Reaction 1 (mutant nucleotide in bold and underlined)
  • GTAACTATGAGCGGTTCCTT (SEQ38) mutant GCATCTGTCATATGAGGGAA (SEQ39)
  • CAAGGTCAAAAGAGAACT SEQ46

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Abstract

On analyse les mutations de cellules cancéreuses dans les exons (4, 5 et 7) du gène de CTP-synthétase ou dans un intron régissant son épissage, au moyen de procédés se basant sur RFLP ou PCR, etc., pour révéler la présence du phénotype de résistance à la polychimiothérapie (MDR).
PCT/GB1991/001243 1990-07-25 1991-07-25 Analyse des mutations de ctp-synthetase donnant lieu a une resistance a la polychimiotherapie Ceased WO1992001811A1 (fr)

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GB9016287.6 1990-07-25
GB909016287A GB9016287D0 (en) 1990-07-25 1990-07-25 Assay for genetic abnormality
US68297591A 1991-04-11 1991-04-11
US682,975 1991-04-11

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0486621A4 (en) * 1989-08-09 1993-03-31 The Children's Medical Center Corporation Nmda oxidizing agents for protecting neurons from injury
CN103540570A (zh) * 2013-10-30 2014-01-29 南京工业大学 一种定点突变改造的三磷酸胞苷合成酶

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EP0333465A2 (fr) * 1988-03-18 1989-09-20 Baylor College Of Medicine Détection de mutations utilisant des oligonucléotides d'amorce compétitives
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WO1987005943A1 (fr) * 1986-03-28 1987-10-08 Board Of Trustees Of University Of Illinois Compositions et procedes permettant d'obtenir des clones contenant des sequences d'adn associees a une resistance des cellules humaines a une multiplicite de medicaments
EP0333465A2 (fr) * 1988-03-18 1989-09-20 Baylor College Of Medicine Détection de mutations utilisant des oligonucléotides d'amorce compétitives
WO1990002203A1 (fr) * 1988-08-19 1990-03-08 Scanlon Kevin J Detection de la progression et de la resistance aux medicaments de tumeurs chez l'homme

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Title
Biological Abstracts, volume 79, 1985, (Phil. PA, US) J.D. McLaren et al.: "The kinetics and feedback inhibition of CTP synthetase in wild-type and mutant Chinese hamster cells", see abstract 30797 *
Chemical Abstracts, volume 100, no. 15, 9 April 1984, (Columbus, Ohio, US) M. Trudel et al.: "Biochemical characterization of the hamster thymutator gene and its revertants", see page 135, abstract 115791j, & J. Biol. Chem. 1984, 259(4), 2355-9 *
The EMBO Journal, volume 9, no. 7, July 1990, Oxford University Press, M. Yamauchi et al.: "Molecular cloning of the human CTP synthetase gene by functional complementation with purified human metaphase chromosomes", pages 2095-2099, see page 2098, final paragraph - page 2099 (cited in the application) *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0486621A4 (en) * 1989-08-09 1993-03-31 The Children's Medical Center Corporation Nmda oxidizing agents for protecting neurons from injury
CN103540570A (zh) * 2013-10-30 2014-01-29 南京工业大学 一种定点突变改造的三磷酸胞苷合成酶
CN103540570B (zh) * 2013-10-30 2015-09-23 南京工业大学 一种定点突变改造的三磷酸胞苷合成酶

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