Patent Application: US-66836500-A

Abstract:
test agents are screened for their effects on mitochondria using a bioassay . mutant cells with mutations in their mitochondria are readily detectable . agents which induce such mutations are thereby identified as potential disease causing agents .

Description:
it is a discovery of the present invention that changes in the mitochondrial genome can be readily detected using a simple set of screening steps . thus agents , whether chemical or physical , can be readily tested for their effects on mitochondrial genomes . as validation of the principle of the invention , the chemotherapeutic agent adriamycin has been tested in the assay system and has been found to induce mutations in the mitochondrial genome . the bioassay of the invention can be used in a forward or a backward direction , i . e ., to either find agents which induce mutations in mitochondrial genomes or which suppress the phenotype of such mutations . any eukaryotic cells can be used in the bioassay system , although yeast cells are preferred . adenine auxotrophs are used because they accumulate a red pigment which can be detected visually . alternatively an optical sensing machine can be used to distinguish between red and white colonies . the red pigment accumulation typically occurs under culture conditions which employ glucose ( dextrose ). when such adenine auxotrophs are grown on glucose most cells appear red , but rare cells will be observed that appear white . contacting the cells with a test agent prior to the culturing on glucose may increase the ratio of the population of cells which appear white . an agent which increases the ratio is a candidate mitochondrial dna damaging agent . to confirm that the agent is damaging the mitochondrial dna , the detected white cells can be tested for the ability to grow on a non - fermentable carbon source , such as glycerol , succinate , acetate , pyruvate , fumarate , or lactate . cells with damaged mitochondrial dna will not grow on such carbon sources . in one embodiment of the invention the cells used are yeast cells . the yeast cells can be of any genus and species , but saccharomyces cerevisiae are preferred due to the extensive genetic knowledge accumulated in the art about this species . the adenine mutation can be in any gene of the adenine pathway which causes the cell to accumulate red pigment . suitable genes include ade1 and ade2 . when performing the bioassay in the reverse direction to identify agents which enhance mitochondrial function , one begins with cells which are both adenine auxotrophic and mitochondrial function deficient . cells are then assayed to detect the ratio of cells which appear red and which can grow on a non - fermentable carbon source . suppressor mutations may be induced by test agents either on the mitochondrial genome or on the nuclear genome . alternatively , epigenetic mechanisms may be responsible for phenotypic reversion . under some circumstances it may be desirable to confirm that the mutation is on the mitochondrial genome . one method for so doing is to mate or fuse the putative mitochondrial dysfunctioning cell with a cell which has no mitochondrial genome . failure to change the mitochondrial phenotype upon mating or fusing suggests that the agent induced a mitochondrial mutation . the mitochondrial phenotype can be tested on either a non - fermentable medium or on a glucose medium and scored for failure to grow or red pigment accumulation , as discussed above . kits may be formulated for practicing the present invention . the kits may be for practicing the forward or backward direction of the bioassay . the kits will contain in one or more associated containers a sample of cells which are auxotrophic for adenine , medium containing a fermentable and medium containing a non - fermentable carbon source . instructions for carrying out the invention may be contained as a package insert , or as a computer readable medium , or as a reference to a published paper or website . preferably all components of the kit will be in a single container . depending on whether the kit is for the forward or backward bioassay , the cells will contain functional or non - functional mitochondria . the above disclosure generally describes the present invention . a more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only , and are not intended to limit the scope of the invention . this example demonstrates the effect of adriamycin on the generation of cells defective in mitochondrial function . the yeast s . cerevisiae strain yph499 ( a ura3 - 52 , lys2 - 801 , ade2 - 101 , trpl - 63 , his3 - 200 , leul - 1 ) was grown in ypd to log phase . cell were then harvested and suspended in water containing various concentration of adriamycin for one hour . in order to test whether white colonies were defective in mitochondrial function , we transferred the colonies to an agar media containing glycerol ( nonfermentable carbon source ). utilization of glycerol by an eukaryotic cell requires intact mitochondrial function ( 2 ). as shown in fig1 b , when randomly chosen white colonies were patched on glycerol plates , none were able to grow indicating a defect in mitochondrial function . as shown in fig2 exposure of yeast cells resulted in increase number of white colonies with increasing dose of adriamycin . the number of surviving colonies that were white reached about 30 % at 150 μglml concentration . these results indicate that mitochondrial dysfunction can be measured by red to white color change and that adriamycin induced mitochondrial dysfunction . this example analyzes the nature of mitdna in white cells after adriamycin treatment . dna was stained with a fluorescent dye , 4 , 6 - diamidino - 2 - phenylindole ( dapi )( 2 ). fig3 a demonstrates that both the nuclear and mitdna were stained in wild type yeast cells . however , when several white colonies obtained after treatment with adriamycin were stained , it produced two types of dna staining 1 ) colonies whose cells lost their mitdna ( fig3 c , only nuclear dna is stained ) and 2 ) colonies whose cells contained the mitdna ( fig3 d , both nuclear and mitdna are stained ). in this study , a known yeast strain lacking mitdna ( rho 0 ) served as a negative control ( fig3 b , only nuclear dna is stained ). the genetic approach employed involved mating haploid white colonies generated after adriamycin treatment ( shown in fig2 “ a ” mating type ) with a haploid rho 0 strain ( of opposite mating type “ α ” yph 500 ( ura3 - 52 , lys2 - 801 , ade2 - 101 , trpl - 63 , his3 - 200 , leu2 - 1 ) lacking mitdna ( 3 ). the diploid cells ( a total of 143 ) were tested for growth on glycerol medium . if the white cells contained a mutation in a nuclear gene affecting the mitochondrial function , it can be expected that diploid cells will grow on glycerol because a nuclear defect can be complemented with rho 0 ( because it contains intact nuclear dna but lacks mitdna ). when diploid cells were tested in this manner , none grew on glycerol medium ( fig4 ). together , these studies indicate that adriamycin preferentially damages mitdna . this preferential damage by adriamycin can result either in complete loss of mitdna ( due to severe damage ) and / or mutations in mitdna ( due to minor damage fig3 ). this example demonstrates that the colorimetric method can be used to measure mitochondrial dysfunction induced by physical environmental agents like ultra violet light ( uv ), and chemical agents like hydrogen peroxide ( h 2 o 2 ) and methyl methane sulphonate ( mms ). fig5 indicates that while h 2 o 2 did not result in increased white colonies , uv and mms did . these white colonies when tested do not grow on glycerol indicating a defect in mitochondrial function . the microscopic and genetic tests ( as described above ) revealed that white colonies either contained mutant mitdna or lacked mitdna at all ( fig6 ). 1 . singh k k ( 1998 ) mitochondrial dna mutations in aging , disease and cancer . springer , n . y . pp1 - 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