Method for Verifying and/or Identifying Hormonally Effective Substances

• US 20110256579A1
• Filed: 12/22/2009
• Published: 10/20/2011
• Est. Priority Date: 12/22/2008
• Status: Abandoned Application

• Current Assignee: Technische Universitat Munchen
• Original Assignee: Technische Universitat Munchen
• Inventors: Zierau, Oliver, Lehmann, Sylvi, Diel, Patrick, Vollmer, Günter, Rataj, Felicitas, Ostermann, Kai, Gross, Annett, Rodel, Gerhard
• Application Number:
  US13/141,342
• Publication Number:
  US 20110256579A1
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: C12Q 1/025 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics antimicrobial activity C12Q1/18
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

The invention relates to a method for verifying and/or identifying hormonally effective substances using the pheromone system of yeast, to yeast cells for carrying out the method, and to the use thereof. The method according to the invention comprises the following steps: a) adding yeast culture medium or buffer to a sample which presumably contains a hormonally effective substance, b) thereafter contacting the sample with haploid yeast cells that possess features 1 through 3: 1. the DNA sequence coded for a heterologous hormone receptor is placed under the control of a constitutive promoter, 2. the DNA sequence coded for a yeast pheromone is placed under the control of a promoter that can be regulated by the hormone receptor, 3. a gene that is coded for a receptor for the yeast pheromone is constitutively expressed, wherein the activation of the hormone receptor by the hormonally effective substance leads to the expression and secretion of the yeast pheromone, and c) the physiological and/or morphological changes of haploid yeast cells are detected.

Claims

1-26. -26. (canceled)

27. A method for verifying and/or identifying hormonally effective substances, comprising the steps of:

a) to a sample that comprises presumably a hormonally active substance a yeast culturing medium or buffer is added,

b) the sample to which the yeast culturing medium or buffer has been added is contacted with haploid yeast cells that comprise the following features 1 to 3, wherein either all features 1 to 3 are realized by a single type of haploid yeast cells or the features 1 and 2 are realized by a first type of haploid yeast cells and the feature 3 by a second type of haploid yeast cells;

1. the DNA sequence that codes for a heterologous hormone receptor is subjected to the control of a constitutive promoter and is functionally expressed;

2. the DNA sequence coding for a yeast pheromone is subjected to the control of a promoter that can be regulated by the heterologous hormone receptor;

3. a gene that codes for a receptor for the yeast pheromone is constitutively and functionally expressed,

 

wherein the activation of the heterologous hormone receptor by the hormonally active substance causes the expression of the DNA sequence coding for the yeast pheromone and the secretion of the pheromone from the haploid yeast cell; and

c) a morphological change and/or a physiological change of the haploid yeast cells are detected.

28. The method according to claim 27, wherein the morphological change of the haploid yeast cells is a directed cell wall growth caused by the action of the yeast pheromone.

29. The method according to claim 27, wherein the morphological change of the haploid yeast cells is detected by a light microscope.

30. The method according to claim 27, wherein the morphological change of the haploid yeast cells is detected by a physical measurement of turbidity.

31. The method according to claim 27, wherein the morphological change of the haploid yeast cells is a cell cycle arrest caused by the action of the yeast pheromone.

32. The method according to claim 27, wherein the physiological change of the haploid yeast cells comprises the expression of a specific gene.

33. The method according to claim 32, wherein the specific gene codes for a marker protein.

34. The method according to claim 32, wherein the specific gene codes for a fluorescent protein.

35. The method according to claim 32, wherein the specific gene codes for a protein that fluoresces green (GFP), yellow (YFP), blue (BFP), cyan (CFP), or red (dsRed).

36. The method according to claim 27, wherein the hormone receptor is an intracellular hormone receptor.

37. The method according to claim 27, wherein the hormone receptor is a nuclear hormone receptor.

38. The method according to claim 27, wherein the hormone receptor is a steroid receptor.

39. The method according to claim 27, wherein the hormone receptor is selected from androgen receptors and estrogen receptors.

40. The method according to claim 27, wherein the constitutive promoter is selected from the group consisting of ADH1, GPD, TEF2, CYC, PGK1, PGK1, SPT15, PMA1, SUF14, GAPFL, TPI1 and TDH3 promoters.

41. The method according to claim 27 for detecting hormonally active substances.

42. The method according to claim 41 for detecting hormonally active substances in urine samples, blood samples, and serum samples.

44. The method according to claim 27 for evaluating loading of environmental samples with hormonally active substances.

45. The method according to claim 44 wherein the environmental samples are water samples or tissue samples.

46. The method according to claim 27 for evaluating hormonal activity of synthetic or natural substances.

47. The method according to claim 27 for screening substance libraries or complex substance mixtures.

48. The method according to claim 27 for identifying potentially hormonally active substances for pharmaceutical application.

49. The method according to claim 27 for toxicological screening of waste water, drinking water, packaging materials, food stuffs or food supplements with regard to undesirable hormonal ingredients.

50. The method according to claim 27 for screening with respect to use of hormones in fattening of animals.

43. The method according to 42 for detecting doping agents in urine samples.

51. Haploid yeast cells with the following features:

a DNA sequence that codes for a heterologous hormone receptor is subjected to the control of a constitutive promoter and is functionally expressed;

a DNA sequence that codes for a yeast pheromone is subjected to the control of a promoter that can be regulated by the heterologous hormone receptor;

wherein the activation of the heterologous hormone receptor by the hormonally active substance causes the expression of the DNA sequence coding for the yeast pheromone and the secretion of the yeast pheromone from the haploid yeast cell.

52. The haploid yeast cells according to claim 51, constitutively and functionally expressing a gene that codes for a receptor of the yeast pheromone.

53. The haploid yeast cells according to claim 51 for detecting hormonally active substances.

54. The haploid yeast cells according to claim 53 for detecting hormonally active substances in urine samples, blood samples, and serum samples.

56. The haploid yeast cells according to claim 51 for evaluating loading of environmental samples with hormonally active substances.

57. The haploid yeast cells according to claim 56 wherein the environmental samples are water samples or tissue samples.

58. The haploid yeast cells according to claim 51 for evaluating hormonal activity of synthetic or natural substances.

59. The haploid yeast cells according to claim 51 for screening substance libraries or complex substance mixtures.

60. The haploid yeast cells according to claim 51 for identifying potentially hormonally active substances for pharmaceutical application.

61. The haploid yeast cells according to claim 51 for toxicological screening of waste water, drinking water, packaging materials, food stuffs or food supplements with regard to undesirable hormonal ingredients.

62. The haploid yeast cells according to claim 51 for screening with respect to use of hormones in fattening of animals.

55. The haploid yeast cells according to 53 for detecting doping agents in urine samples.