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Patents owned by Toyota Central R&D Labs. Inc.

Technology overview

Toyota Central R&D Labs. Inc. is a laboratory owned by companies in the Toyota Motors Group. Main R&D activities are centered towards motor vehicle development, and is divided into five main sectors; energy and environment, safety and human engineering, mechanical engineering, system engineering and electronics, and materials.  The related patents described in this section discloses a recombinant gene comprising an SOS gene and a luciferase gene, and a method to detect mutagenic substances using microorganisms transformed with the recombinant gene.  Examples in the specification of this patent family explain production of a umuC-luc gene fusion using plasmid pSK1002 (containing umuD,C-lacZ; Shinagawa et al., Gene 23:167-74 (1983) and the luc gene of the firefly Photinus pyralis (on Pica Gene™ Cassette vector; Toyo Ink MFG. Co. Ltd, Japan), which was introduced into S. typhimuriumTA1535 and E. coli CH26.

Details of patent documents

Patent or Publication no. Title, Independent Claims and Summary Assignee and licensing information
EP 649905

  • Earliest priority – 22 Oct 1993
  • Filed – 21 Oct 1994
  • Granted – 23 Sept 1998
  • Expected expiry – 21 Oct 2014
 Title – Methods of detection of mutagens using luminescence gene

Claim 1
A recombinant gene comprising

  • an SOS gene expressed when a DNA is damaged and a gene expressing luciferase activity positioned downstream of the SOS gene.
Claim 4
A host microorganism transformed with a recombinant gene comprising

  • an SOS gene expressed when a DNA is damaged and a gene expressing luciferase activity positioned downstream of the SOS gene.
Claim 10
A method for detecting or quantitating a mutagenic substance in a sample, comprising the steps of:

  • culturing a host microorganism transformed with a recombinant gene comprising an SOS gene expressed when a DNA is damaged and a gene expressing luciferase activity positioned downstream of the SOS gene, in a medium to which the sample is added; and
  • measuring a luminescence generated by expression of the gene expressing luciferase activity.
Claim 16
A recombinant gene comprising

  • an SOS gene expressed when a DNA is damaged and genes expressing luciferase activity and an enzyme which catalyzes the production of a substrate for the luciferase, positioned downstream of the SOS gene.
Claim 21
A host microorganism transformed with a recombinant gene comprising

  • an SOS gene expressed when a DNA is damaged and genes expressing luciferase activity and an enzyme which catalyzes the production of a substrate for the luciferase positioned downstream of the SOS gene.
Claim 26
A method for detecting or quantitating a mutagenic substance in a sample, comprising the steps of:

  • culturing a host microorganism transformed with a recombinant gene comprising an SOS gene expressed when a DNA is damaged and genes expressing luciferase activity and an enzyme which catalyzes the production of a substrate for luciferase, positioned downstream of the SOS gene, in a medium to which the sample is added; and
  • measuring a luminescence generated by expression of the genes expressing luciferase activity and an enzyme which catalyzes the production of a substrate for luciferase.

The claims are generally drawn towards:

  • a recombinant gene comprising an SOS gene and a gene expressing luciferase (claim 1)
  • a host microorganism transformed with a recombinant gene comprising an SOS gene and a gene expressing luciferase (claim 4)
  • a method for detecting or quantitating a mutagenic substance in a sample (claim 10, 26)
  • a recombinant gene comprising an SOS gene, a gene expressing luciferase and an enzyme which catalyzes the production of a substrate for the luciferase (claim 16)
  • a host microorganism transformed with a recombinant gene comprising an SOS gene, a gene expressing luciferase and an enzyme which catalyzes the production of a substrate for the luciferase (claim 21)

Definitions extracted from the specification are:

  • SOS gene – may be any SOS gene which is expressed when a DNA is damaged and which contains so called SOS box
  • Gene encoding luciferase – there is no definition for this term. The description states that ‘various genes can be used’
  • Luciferase substrate – a long chain aldehyde or the like
  • Host microorganism – any microorganism which allows SOS gene being expressed when a DNA is damaged by a genotoxic substance such as a mutagenetic substance, in other word, host microorganism may be those having the components of SOS response
  • Enzyme catalyzing the production of a substrate for the luciferase – AND(P)H:FMN reductase, fatty acid reductases, or the like
  • Transformed – conventional procedures used for transformation of microorganisms such as bacteria may be used
  • Measuring (in claim 10) – cultured microbial cells are collected, and the cell wall is disrupted to release an expression product
  • Measuring (in claim 26) – the cultured medium per se generates the luminescence if the sample contained a genotoxic substance such as a mutagenic substance. Therefore, immediately after the culturing the luminescence can be measured

Comments:

Independent claim 1 of EP 649905 is relatively broad in two aspects:

  1. It is a ‘product claim’, therefore there is no limit on utility.
  2. There is no restriction on the type of SOS gene nor the gene expressing luciferase activity (in contrast to claim 1 of US 5702883, see below).

The technology was enabled by transforming E. coli (strain CSH26) and S. typhimurium (strain TA1535) wtih a umuC-luc fusion protein-producing luminescence vector, which  was subsequently tested for luminescence against 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), 4-nitroquinoline 4-oxide (4NQO), 1-nitropyrene (1-NP), 2-aminoanthracene (2-AA) and benzo[a]pyrene (Bap). Construction of a vector containing both lux and gene coding for the enzyme that catalyzes the substrate for luciferase was built from pUCD620 (Kado et al., Plant Molecular Biology Reporter, 1987, 5, 225), the SOS gene umuD,C added, and introduced into S. typhimurium TA1535.

Designated contracting States at the time of grant are: Switzerland, Germany, France, United Kingdom, Liechtenstein

 Toyota Central R&D Labs Inc.

41-1, AZA YOKOMICHI, OAZA NAGAKUTE NAGAKUTE-CHO, AICHI-GUN, AICHI, JAPAN

Note: all enquiries are through a standard mailing form provided on their website:

http://www.tytlabs.co.jp/eindex.html
US 5702883

  • Earliest priority – 22 Oct 1993
  • Filed – 21 Oct 1994
  • Granted – 30 Dec 1997
  • Patent expired – 1 Feb 2006
 Title – Methods of detection of mutagens using luminescence gene

Claim 1
A method for identifying a mutagenic substance, comprising the steps of:

  • culturing Escherichia or Salmonella transformed with recombinant DNA comprising

(i) an Escherichia or Salmonella SOS gene and
(ii) a gene encoding luciferase isolated from Photobacterium phosphoreum, Photobacterium leiognethi, Vibrio splendidus, Vibrio cholerae, Photinus pyralis, Vibrio harveyi or Vibrio fischeria; wherein
said gene encoding luciferase is positioned downstream of said SOS gene such that when said SOS gene is expressed, then said gene encoding luciferase is also expressed;

  • contacting said culture with a substance to be tested wherein said Escherichia or Salmonella in said culture is provided with a luciferase substrate; and
  • determining whether said substance is mutagenic by measuring the amount of luminescence in said culture.

The claims are generally drawn towards:

  • a method for identifying a mutagenic substance, comprising culturing Escherichia or Salmonella transformed with recombinant DNA comprising an SOS gene and a gene encoding luciferase (claim 1)

Definitions extracted from the specification are:

  • SOS gene – may be any SOS gene which is expressed when a DNA is damaged and which contains so called SOS box
  • Gene encoding luciferase – there is no definition for this term. The description states that ‘various genes can be used’
  • luciferase substrate – a long chain aldehyde or the like

Comments:

US 5702883 expired due to non-payment of maintenance fees.
Remarks Two related patents were granted in Japan:

  1. JP 3277426 – Transgenic Salmonella containing a SOS gene, gene expressing luciferase activity and luciferase substrate gene (expected expiry 15 Feb 2014);
  2. JP 3277436 – Transgenic Salmonella containing a SOS gene and gene expressing luciferase activity (expected expiry 26 Sept 2014).

Search strategy

This patent was identified as reference to WO 1997/41251 titled ‘Recombinant nucleic acid sequences and methods for determining both genotoxicity and mutagenicity of a sample and the kinetics of the genetoxicity’ filed by VITO.