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US20040248126A1 - Method of detecting toxic substance - Google Patents

Method of detecting toxic substance Download PDF

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Publication number
US20040248126A1
US20040248126A1 US10/487,439 US48743904A US2004248126A1 US 20040248126 A1 US20040248126 A1 US 20040248126A1 US 48743904 A US48743904 A US 48743904A US 2004248126 A1 US2004248126 A1 US 2004248126A1
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US
United States
Prior art keywords
process according
mrna
genes
gene
yeast
Prior art date
Legal status (The legal status 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 status listed.)
Abandoned
Application number
US10/487,439
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English (en)
Inventor
Hitoshi Iwahashi
Yuko Momose
Emiko Kitagawa
Junko Takahashi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Daikin Industries Ltd
Original Assignee
Individual
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 Individual filed Critical Individual
Assigned to NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY, DAIKIN INDUSTRIES, LTD. reassignment NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: IWAHASHI, HITOSHI, KITAGAWA, EMIKO, MOMOSE, YUKO, TAKAHASHI, JUNKO
Publication of US20040248126A1 publication Critical patent/US20040248126A1/en
Assigned to DAIKIN INDUSTRIES, LTD. reassignment DAIKIN INDUSTRIES, LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY
Priority to US11/772,057 priority Critical patent/US20070287155A1/en
Priority to US12/554,386 priority patent/US8192962B2/en
Abandoned legal-status Critical Current

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Classifications

    • 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
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates to biology-based processes for detecting toxic substances, and specifically to processes for detecting toxic substances which comprises detecting mRNA that is transcribed in the presence of test materials by a particular gene from yeast.
  • Japan are marketed in Japan.
  • the devices for evaluation based on emission intensity of luminous bacteria are commercially available (MICROTOX, azur, Co., USA; LUMIS, drlange, Co. Germany).
  • MICROTOX azur, Co., USA
  • LUMIS drlange, Co. Germany
  • the invention of the present application relates a process for detecting a toxic substance, which comprises detecting mRNA that is expressed in the presence of a test material by a cell comprising a yeast gene that is selected from a group consisting of the following, or a gene that is homologous to the yeast genes and is derived from other species, wherein the mRNA corresponds to said yeast gene or said homologous gene thereof:
  • yeast genes and the amino acid sequences of the corresponding protein are disclosed in public databases such as MIPS in Germany: Kunststoff Information Center for Protein Sequence, and SGD in USA: Saccharomyces Genome Database, and are known via the internet.
  • a gene(s) that is (are) homologous to the yeast genes means genes that comprise a base sequence having a homology of 50% or more, preferably 80% or more to the base sequences of yeast genes, and that encode a protein having the same functions as the proteins encoded by the yeast genes.
  • Cells as used herein may be either eukaryotic or prokaryotic cells as long as they comprise the genes as described above.
  • Preferred cells are yeast cells.
  • mRNA is detected through reverse transcription-PCR.
  • Reverse transcription-PCR is well known in the art. See, for example, NAKABEPPU Yusaku, et al.: Cell Technology, suppl. Tips series, Modified PCR Tips, Shujunsha Co.Ltd., 1999, pp25-43.
  • the invention comprises:
  • Test materials comprising a chemical substance to be tested are added to the medium, and the cells are incubated further for several hours. Concentrations of the test materials are selected to not lead to cell death. Test materials may comprise one or more chemical substances.
  • a polynucleotide having a poly T structure which is immobilized on the surface of magnetic beads or latex beads is used to trap the mRNA, and then the mRNA is washed and eluted with spine column in view of the fact that a poly A chain is attached to the 3′ terminus of mRNA, which is readily conducted using a commercially available kit such as Oligotex-dT30 ⁇ Super>mRNA Purification Kit, Takara.
  • the mRNA is reverse-transcribed with a reverse transcriptase (Super Script II Reverse Transcriptase; catalogue No. 18064-014, GibcoBRL) using fluorescence-labeled nucleotides.
  • labeled cDNAs that are introduced with fluorescence-labeled nucleotides during the reverse transcription are obtained.
  • Reverse transcriptases and nucleotides that are labeled with Cy3-dUTP or Cy5-dUTP are also commercially available (for example, Super Script II Reverse Transcriptase; catalogue No. 18064-014, GibcoBRL). Fluorescence-labeled cDNAs are detected with a fluorophotometer.
  • RT-PCR is a procedure for detection and quantitative determination of an intended RNA in a form of the amplified cDNA, which comprises reverse-transcribing mRNA into cDNA with a reverse transcriptase, and conducting PCR using the cDNA as a starting material as well as specific primer sets and a thermostable DNA polymerase.
  • a microarray containing the gene as described above may be prepared to hybridize them with the cDNAs, thereby facilitating the efficient detections, which is a preferred embodiment of the present invention.
  • the mRNA is detected by northern blotting.
  • Northern blotting is well-known in the art (OGATA Nobukuni, NOJIMA Hiroshi: Genetic Engineering Keywords Book, revised 2nd ed., Yodosha, cojp, 2000, pp299-301).
  • the process comprises
  • Procedures of northern blotting comprises electrophoresing the RNA, transferring the pattern to a filter, and hybridizing it with a specific probe labeled with an isotope, thereby analyzing the presences and the amount of the mRNA in a sample as well as the length of the same.
  • a microarray containing the gene as described above may be prepared to hybridize them with the cDNAs, thereby facilitating the efficient detections.
  • the detection of mRNA is conducted by detecting the production of a polypeptide encoded by the yeast gene as described above.
  • the production of a polypeptide may be detected for example using an antibody directed to the polypeptide.
  • Toxic substances to be detected according to the present invention include arsenic, cadmium, mercury, 4-nitroquinolin-N-oxide, 2,4,5-trichlorophenol, y-hexachlorocyclohexane, manganese ethylenebis(dithiocarbamate), 2,4,5,6-tetrachloro-1,3-benzenedicarbonitrile, tetramethylthiuram disulfide, Zinc N,N′-ethylenebis(dithiocarbamate), gingerol, acrolein, and dimethylsulfoxide, all of which are mutagenic.
  • Yeast Saccharomyces cereuisiae S288C ( ⁇ SUC2mal mel gap2 CUP1)
  • YPD medium yeast extract 1%, polypepton 2%, glucose 2%
  • One of toxic chemical substances was added to the cells at logarithmic growth phase, and the cells were further incubated for two hours.
  • Cells were cultured without any chemical substance in the same condition, and were used as control. Concentrations of the chemical substances were defined to inhibit the growth of the yeast but not lead to death.
  • the culture was centrifuged to collect the cells.
  • sodium acetate buffer 50 mM sodium acetate, 10 mM EDTA, 1% SDS
  • the mixture was shaken at 65° C. for five minutes, followed by returning to room temperature, and obtaining the supernatant, of which the procedures were repeated two times.
  • 1 ⁇ 2 amount of a solution of phenol/chloroform was added, and the mixture was centrifuged to give a supernatant, which was added with an equal amount of chloroform, and the mixture was centrifuged to give a supernatant.
  • a polynucleotide having a poly T structure which was immobilized on the surface of latex beads was used to trap the mRNA, and then the mRNA was washed and eluted with spine column (Oligotex-dT30 ⁇ Super>mRNA Purification Kit, Takara).
  • Reverse transcription of the mRNA was conducted with a reverse transcriptase (Super Script II Reverse Transcriptase; catalogue No. 18064-014, GibcoBRL) using fluorescence-labeled nucleotides to give cDNAs that were introduced with Cy3-dUTP or Cy5-dUTP during the reverse transcription.
  • the labeled cDNAs were dissolved in TE buffer (10 mM Tris-HCl/1 mM EDTA, pH8.0), and the solution was dropped on the DNA chip containing the whole genes of yeast (DNA Chip Research Inc. Japan) so that the cDNAs were hybridized on the DNA chip at 65° C. for over 12 hours.
  • the fluoresces intensity of the DNA chip was read with a scanner, and the ratio relative to the fluorescence intensity resulting from the absence of chemical substance was estimated as the following, which is shown in Tables 1 to 9:
  • the ⁇ ⁇ level ⁇ ⁇ of ⁇ ⁇ expressed ⁇ ⁇ m ⁇ ⁇ RNA in ⁇ ⁇ the ⁇ ⁇ presence ⁇ ⁇ of ⁇ ⁇ chemical ⁇ ⁇ substance The ⁇ ⁇ level ⁇ ⁇ of ⁇ ⁇ expressed ⁇ ⁇ m ⁇ RNA in ⁇ ⁇ the ⁇ ⁇ absence ⁇ ⁇ of ⁇ ⁇ chemical ⁇ ⁇ substance TABLE 1 Unknown protein yeast genes yeast The level of expressed mRNA in the presence of chemical substance/ genes (1) (2) (3) (4) (5) (7) (8) (9) (10) YCR102C 3.4 3.2 2.4 1.1 0.7 0.4 1.1 4.9 9.8 61.5 YDL218W 3.5 1.2 2.6 1.4 0.7 0.9 1.1 1.5 72.5 12.6 YDR533C
  • the tables show that the expressed mRNA of about 700 of 2400 unknown yeast genes was induced by any one of toxic chemical substances such as heavy metals, agricultural chemicals, surfactants (Table 1), as well as the expressed mRNA of 167 mitochondria-located genes (Table 2), 52 DNA repair genes (Table 3), 161 energy genes (Table 4), 142 transport facilitation genes (Table 5), 90 stress protein genes (Table 6), 142 metabolism genes (Table 7), 60 detoxification genes (Table 8), and 507 genes belonging to other category (Table 9).
  • toxic chemical substances such as heavy metals, agricultural chemicals, surfactants (Table 1)
  • Table 2 the expressed mRNA of 167 mitochondria-located genes
  • Table 3 52 DNA repair genes
  • 161 energy genes Table 4
  • 142 transport facilitation genes Table 5
  • 90 stress protein genes Table 6
  • 142 metabolism genes Table 7
  • 60 detoxification genes Table 8
  • 507 genes belonging to other category Table 9
  • the ⁇ ⁇ level ⁇ ⁇ of ⁇ ⁇ expressed ⁇ ⁇ m ⁇ ⁇ RNA in ⁇ ⁇ the ⁇ ⁇ presence ⁇ ⁇ of ⁇ ⁇ chemical ⁇ ⁇ substance The ⁇ ⁇ level ⁇ ⁇ of ⁇ ⁇ expressed ⁇ ⁇ m ⁇ ⁇ RNA in ⁇ ⁇ the ⁇ ⁇ absence ⁇ ⁇ of ⁇ ⁇ chemical ⁇ ⁇ substance .
  • “Intensity” indicated in the rightmost column of Tables 1 to 9 is a value of the level of expressed mRNA of each gene in control cells as divided by the average level of the expressions of the whole genes. When the intensity is low, the error of measurement may be possibly large. The detection is considered more precise when expression magnification (expressed mRNA in the presence of chemical substance/expressed mRNA in the absence of chemical substance) is larger.
  • Yeast gene as used in the processes according to the invention is selected, of which the intensity is preferably 0.3 or more, more preferably 0.5 or more, and of which the expression magnification is preferably 3 or more, more preferably 5 or more.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
US10/487,439 2001-08-24 2002-08-23 Method of detecting toxic substance Abandoned US20040248126A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US11/772,057 US20070287155A1 (en) 2001-08-24 2007-06-29 Processes for detecting toxic substances
US12/554,386 US8192962B2 (en) 2001-08-24 2009-09-04 Processes for detecting toxic substances

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2001-255380 2001-08-24
JP2001255380A JP4475373B2 (ja) 2001-08-24 2001-08-24 毒性物質の検出方法
PCT/JP2002/008494 WO2003018791A1 (en) 2001-08-24 2002-08-23 Method of detecting toxic substance

Related Child Applications (1)

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US11/772,057 Abandoned US20070287155A1 (en) 2001-08-24 2007-06-29 Processes for detecting toxic substances
US12/554,386 Expired - Fee Related US8192962B2 (en) 2001-08-24 2009-09-04 Processes for detecting toxic substances

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Country Status (4)

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US (3) US20040248126A1 (ja)
EP (2) EP1426438A4 (ja)
JP (1) JP4475373B2 (ja)
WO (1) WO2003018791A1 (ja)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090246755A1 (en) * 2003-12-02 2009-10-01 Daikin Industries, Ltd. Method of examining chemical using gene-disrupted strain

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004357575A (ja) * 2003-06-04 2004-12-24 Daikin Ind Ltd 微生物の循環式培養方法
CA2739022A1 (en) * 2008-10-13 2010-04-22 Wabash National, L.P. Foldable mobile storage container

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU3931300A (en) * 1999-03-31 2000-10-16 Rosetta Inpharmatics, Inc. Methods for identifying pathway-specific reporters and target genes, and uses thereof
JP3446042B2 (ja) 2000-02-28 2003-09-16 独立行政法人産業技術総合研究所 化学物質の微生物学的同定方法
JP4022610B2 (ja) * 2000-04-07 2007-12-19 独立行政法人産業技術総合研究所 化学物質の毒性評価方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090246755A1 (en) * 2003-12-02 2009-10-01 Daikin Industries, Ltd. Method of examining chemical using gene-disrupted strain
US8173387B2 (en) 2003-12-02 2012-05-08 Daikin Industires, Ltd. Method of examining chemical using gene-disrupted strain

Also Published As

Publication number Publication date
EP1426438A1 (en) 2004-06-09
US8192962B2 (en) 2012-06-05
WO2003018791A1 (en) 2003-03-06
EP1847601A3 (en) 2008-01-16
JP2003061673A (ja) 2003-03-04
EP1847601A2 (en) 2007-10-24
EP1426438A4 (en) 2005-01-26
US20070287155A1 (en) 2007-12-13
US20100075324A1 (en) 2010-03-25
JP4475373B2 (ja) 2010-06-09

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