WO1990009452A1

WO1990009452A1 - Yeast system for detection of aneuploidy and identification of targets

Info

Publication number: WO1990009452A1
Application number: PCT/US1990/000616
Authority: WO
Inventors: Michael A. Resnick; Seymour Fogel; Friedrich Zimmermann
Original assignee: The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce
Priority date: 1989-02-15
Filing date: 1990-02-08
Publication date: 1990-08-23
Also published as: IL93386A0; AU5181890A

Abstract

A sensitive system based on yeast for detection of rare aneuploidy events and identification of targets is described. Methods for detecting chemically induced chromosomal changes are also described.

Description

"Yeast system for detection of Aneuploidy and Identification of Targets"

The present invention provides a unitary genetic system for assessing chemically induced aneuploidy and chromosome loss utilizing the yeast Saccharomyces cerevisiae. The system of the present invention allows the detection of rare aneuploidy events due to chromosome gain, loss or missegregration and the determination of whether the aneuploidy or chromosome loss is due to chemical action on DNA or nonDNA targets in actively growing cells.

A review of various aneuploidy detection systems can be found in the book entitled "Aneuploidy Etiology and Mechanisms", edited by Dellarco et al, 1985, Plenum Press, New York. However, it should be noted that the systems heretofore designed to examine chemically induced aneuploidy in mammalian cells or other cell systems have several disadvantages some of which are noted below:

a. Lack of sensitivity

b. Not possible to identify targets for

aneuploidy induction

c. Not possible to identify events at the

genetic level

d. Not possible to readily distinguish

chromosome gain vs chromosome loss

e. Lack of ability to analyze meiotic

aneuploidy

f. Difficulty of genetic manipulation and

lengthy time for experiments.

Summary of the Invention

It is, furthermore, an object of the present invention to provide a single unified yeast system which enables sensitive detection of genetic events such as chromosome mutation, recombination and the like.

It is a further object of the present invention to provide a single yeast strain in which chromosome gain or loss can be detected in mitotic cells and chromosome gain can be detected in meiotic cells and in which permeability to a variety of chemicals is made possible by permeability mutations.

It is another object of the present invention to provide a yeast system in which the effects of chemical agents on chromosome stability and distribution can be reliably determined in mitotically growing as well as meiotically developing cells.

It is still a further object of the present invention to provide a yeast strain which is modifiable to allow inclusion of mammalian tubulin genes or segments thereof as well as other components of the segregation apparatus and the determination of the effect of chemical agents on the relationship between tubulin function and chromosomal changes.

It is yet another object of the present invention to provide means for determining whether the target for chemically induced changes in chromosomal stability and distribution is a DNA or a non-DNA entity, such as tubulin.

Other objects and advantages will become evident from the following detailed description of the invention.

Detailed Description of the Invention

The above and various other objects and advantages of the present invention are achieved by a genetically modified strain of Saccharomyces cerevisiae which allows sensitive detection of aneuploidy and chromosomal loss.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed herein are standard methodologies well known to one of ordinary skill in the art.

1. General design of the chromosome loss/gain/permeable strain referred to as LGP;

The yeast strain employed is a diploid; however, if meiotic aneuploidy is to be assessed, this strain has the capability for undergoing high levels or meiosis.

Chromosome α contains a "SELECTABLE MUTATION" that results in resistance to some agent such as a protein synthesis inhibitor. Chromosome α also has a centromere linked recessive mutation "a" and another identifiable recessive mutation (b) on the opposite chromosomal arm. There is a homologous chromosome α that has wild type genes for these three markers. Loss of the homologous chromosome or missegregation results in the expression of the recessive resistance gene and confirmation is provided by the expression of the other recessive mutations. If recessive mutation is very near the centromere there may be no need for recessive mutation "b". Also contained on chromosome α and the homologous chromosome are two GENE DOSAGE genes. At least one of these GENE DOSAGE genes, gene C, provides the opportunity to select cells with increased gene dosage. The presence of an additional chromosome α is signalled by the greater number of the selectable GENE DOSAGE C genes. The additional chromosome is confirmed by an associated increase in the number of copies of the second gene dosage gene D. It is, therefore possible to detect chromosome loss, missegregation or chromosome gain events within the same chromosome. The GENE DOSAGE genes could be on another chromosome B in which case chromosome gain would be measured for chromosome B and chromosome loss or missegregation would be measured for chromosomeα,

In order to monitor DNA perturbations other genetic systems are included. These include a system for detecting intragenic recombination, particularly gene conversion, that invloves mutations "e" and "f" in a common gene such that recombination will result in a wild type form of the GENE CONVERSION GENE system; the gene conversion events can be selected. Also included is a MUTATION GENE SYSTEM that allows for the detection and selection of mutational events by reversion of mutation "g".

In order to allow a variety of chemicals to be examined, the strain includes a PERMEABILITY GENE MUTATION that is homozygous if recessive (or a single copy of a dominant mutation); this allows for the uptake of chemicals not normally taken up by cells, such as large molecules.

If human or animal tubulin gene, or parts thereof, are incorporated in yeast, the strsin may contain a complete or partial TUBULIN REPLACEMENT GENE. This is achieved by replacing one or more of the yeast tubulin genes by a corresponding human or animal tubulin gene such that some or all of the required tubulin of the cell coded by a NATURAL TUBULIN GENE is replaced by whole or partial (as a chimeric protein) human or animal cell tubulin. Partial replacement means a chimeric tubulin formed between the natural yeast tubulin and the human or animal cell tubulin; this results in a functional fusion of two tubulin genes of different origin. The corresponding natural yeast tubulin gene is rendered inactive by mutation "i". Similar methodologies may be applied for other components of the chromosome segregational apparatus including centromere proteins, centriole proteins and proteins associated with microtubules.

Chromosomes α and α

SELECTABLE RECESSIVE CENTRORECESSIVE GENE GENE

RECESSIVE MUTATION MERE MUTATION DOSAGE DOSAGE α

MUTATION A B GENE GENE α

C D

+ + + GENE GENE

DOSAGE DOSAGE α GENE GENE C D

(NOTE: the gene dosage genes could be on another

CHROMOSOME beta)

OTHER GENES: They need not be on the same chromosome

GENE MUTATION GENE CONVERSION mutation g

GENE

mutation e

GENE MUTATION GENE CONVERSION mutation g

GENE

mutation f

PERMEABILITY NATURAL TUBULIN

GENE GENE

mutation h mutation i

PERMEABILITY NATURAL TUBULIN

GENE GENE

mutation h mutation i

TUBULIN REPLACEMENT GENE

2. Synthesis of a New Component.

Combining a chromosome loss and gain systems into one LGP strain.

Systems based in yeast for detecting aneuploidy either due to chromosome gain (BR1669) or due to chromosome loss (D61.M) have been separately described (Zimmermann et al, 1985, Mut. Res. 149, 339; and Whittaker et al, 1986, Yeast 2:421. Standard genetic techniques are used to combine the genetic features of the D61.M and the BR1669 systems into one unified LGP system. It is preferred that the system be sensitive to copper in order to confirm chromosome gain with the BR1669 system.

Placing gene dosage genes on chromosome PC and OC so that chromosome loss and gain can be measured for the same chromosome.

A region of chromosome VIII (in BR1669) of yeast contains the arg4-8 mutation and a single copy of the cupl gene. These genes have been used to monitor aneuploidy due to chromosome gain and the methods are briefly described in Whittaker, et al, 1986, (Yeast, 2:421). A plasmid "cassette" containing the chromosome gain detecting genes, arg4-8. and cupl of BR1669, is then made. The arg4-8 and cupl regions are placed on integrating plasmid such that standard yeast molecular biology methods involving "targetting" can be used to integrate it into the desired place in the genome. Since this cassette is designed to allow targeting to any chromosome using standard yeast molecular biology techniques, it has now become possible to detect aneuploidy for any desired chromosome. It is necessary, however, to remove the corresponding resident ARG4 and cupl gene sequences. This system can also be included in artificial chromosomes that have been introduced into yeast where the artificial chromosomes have been derived from other non-yeast elements (such as human chromosomal DNA or other DNA) Other genetic systems can be developed with similar features. Aneuploidy induction can occur by effects of chemicals on DNA, tubulin or other targets in the LGP strain. Examples of such chemicals are propionitrile, benomyl and some neurotoxins, such as 2,5 hexanedione.

The genetic markers used to signal aneuploidy and methods of treatment have been described for the D61.M and the BR1669 strain systems. These are summarized in Zimmermann et al supra for D61.M strain and in Whittaker et al, Molecular General Genetics, for the BR1669 strain. (Also see "Detection of the indicators of chromosome gain..." and " ... of chromosome loss ..." described below. The combined fusion system of the present invention includes these features. Chemical treatment conditions have been defined as have the methods for confirmation of aneuploidy. Chemicals that induce aneuploidy can be separated into those that act on DNA or another target. Propoinitrile and beromyl are specific to non-DNA targets. Fumaric acid dinitrile appears to act on both. DNA acting chemicals are expected to cause, in addition to aneuploidy, recombination and/or mutation. Chemicals suspected of acting on tubulin and other parts of the segregational apparatus, can be investigated further by the system of the present invention to ascertain the site of action. Cells can be treated with the agent and disruption of tubulin can be determined using antibody to the tubulin. Standard antibody staining procedures can be used to determine loss or change in tubulin resulting from chemical effects (Kilmartin et al, 1982, J. Cell Biol. 93: 576-582). Because the level of sensitivity for detecting aneuploidy with yeast is so high (10^-5) and cells are permeable or can be made permeable by mutation (Sugino et al, 1981) to a broad range of chemicals, it is possible to identify many chemicals that induce aneuploidy by acting on targets other than DNA particularly tubulin (including human or human/yeast chimeric tubulin). .Levels of aneuploidy induction are expected to reflect levels of interaction with the tubulin. Also the chemicals can be designed to maximize a desired response (including genetic response) with the tubulin. The induction of aneuploidy via tubulin or microtubule alterations requires that cells are in a growth phase. Since it is desired that chemotherapeutic agents act preferentially on growing cells, the combined system provides a good model for developing such agents. It also provides a means for determining the aneuploidogenic effects of many chemicals that may be hazardous to human health.

The system of the present invention also allows the determination of the potential of chemicals to act as neurotoxins. Several solvents that cause peripheral neuropathies have recently been shown to cause aneuploidy in yeast, either as single chemicals or as combinations of chemicals. These include methyl ethyl ketone and 2,5 hexanedione. These chemicals, which may affect tubulin aggregation in vitro, have often been shown to affect axonal transport. The system of the present invention enables the design of safer chemicals or combinations. Detection of the indicators or chromosome gain. Arg+ and copper resistance, with the LGP fusion strain.

Aneuploidy due to chromosome gain can be readily detected among the meiotic products and mitotically growing cells of strain BR1669. These features are also incorporated into the LGP strain in accordance with the present invention. Ordinary euploid cells carrying the temperature sensitive arg4-8 allele (12 cM from CEN VIII), as well as the linked cupI^s gene are auxotrophic for arginine and inhibited in their growth by 0.01 mM CuSO₄. Genetically comparable hyperploid cells, i.e. either disomic haploids or trisomic diploids, carrying these marker loci in each genome, exhibit prototrophy with respect to the arginine requirement (at 30º, but not at 36º) and enhanced copper resistance (Whittaker et al, supra). By using appropriate defined media, euploid cells and aneuploids can be readily distinguished according to their differential growth responses. It is noted here that the ratio of arg4-8 alleles/haploid genome is two in disomes generated by the meiotic system and is 1.5 in trisomes arising in the mitotic system

Preparation of Cultures.

Approximately 200 cells, derived from each of ten independent colonies of BR1669 grown on YEPAD are suspended in 20 ml or YPA and incubated on a reciprocal shaker at 30ºC. After approximately 45 hr, the titer is 4-8 × 10⁷ cells/ml. A 2 ml sample is taken, washed once with 10 ml water, diluted and plated on synthetic complete and selective (SCarg- + 0.1 ug/ml arg) media (see below). The remaining 18 ml cultures are stored at 4ºC until the arginine "prototrophic" papillae are counted 7 days later. A culture exhibiting the median arginine "prototrophy" frequency is used as the starting material in subsequent chemical trials and is stored for up to 4 weeks.

Phenotypic Class Arg- Arg- Arg- Cup (0.01mM)

24º 30º 36º 30º

Hyperploids.

(i.e. trisomes + + - + and disomes)

Site revertants

Some 2nd site + + + - revertants

2nd site

revertants. + + - -

Euploids

2º losses of + - - - chr. VIII.

Petites

Phenotypic classes observed upon characterization of papillae arising on SCarg-+0.1 ug/ml arg at 30ºC,

Mitotic drug treatment protocol.

1. Dilute the cold-stored starter culture (<4 weeks old) 1:10 into prewarmed YPA in a 125 ml erlenmeyer. Measure the culture's Klett reading (i.e. absorbance). Incubate, shaking (300 rpm) at 30ºC.

2. When the Klett reading has doubled (+ 5 units) perform the following:

i) dilute a 100 ul portion of the culture

1:10⁴ & 1:10⁵ and plate to two SC plates. ii) using a Finpipette, dispense 2 ml alliquots into Sarsdedt tubes (#60.540). Add appropriate volumes of drug to the 2 ml cultures using a positive displacement pipette (check calibration by weighing 10 × 100 ul alliquots of water before use) .

Include an untreated control and solvent control (if necessary).

3. The drug-treated cultures are then incubated according to one of the following protocols:

i) Continuous 16 hr Regimen. Following drug addition, incubate the Sarsdedt tubes at a 45º angle at 30ºC, shaking at 300 rpm for 16 hours,

ii) Cold Shock. After adding drug, incubate at 30ºC for 4 hrs as described above_* Then, remove the cultures from the 30º incubator and incubate at 0ºC for 16 hrs as follows: Place plastic beakers in an ice bucket and fill with ice - in, underneath and around the beakers. Place the Sarsdedt tubes in the ice-containing beakers and add water until the tubes begin to float. Add more ice until the beakers are full, securely close the ice-bucket's lid and place in the 4ºC room for 16 hours. After cold shock, vortex the Sarsdedt tubes briefly and place in a 30ºC water bath for 2 mins. Then, place the tubes in the shaking incubator at 30º for 4 hours.

4. After 4 hours, remove from the incubator and add 8 ml of sterile H₂O. Vortex mix, spin, discard the supernatant and resuspend the pellet in 10 ml H₂O. Vortex mix, spin, discard the supernatant and resuspend the pellet in 2 ml H₂O.

5. The undiluted (10º) cell suspension is plated on SCarg- + 0.1 ug/ml arg (0.15 ml/plate; 3 replicates). In addition, 5 × 10^-1 (0.5 ml of "10º" + 0.5 ml H₂O) 10^-1 (0.5 ml "10º" + 4.5 ml H₂O) and 5 × 10 ^-2(0.5 ml "10^-1" + 0.5ml HO)₂ dilutions are plated on this selective medium. 10-4 & 10-5 dilutions are plated on SC (i.e. non-selective medium) for viable titer determinations.

6. The SCarg- + 0.1 ug/ml arg plates and SC plates are incubated at 30ºC. The selective plates are sealed in plastic sacks and counted after 7 days incubation. The SC plates are not sealed and counted after 4-5 days. Meiotic System.

To assess both mitotic and meiotic aneuploidy in the very same diploid culture, a portion of the untreated diploid culture described above is incubated with shaking to late log phase (i.e. approximately 4 × 10⁷ cells/ml). The culture is centrifuged and the pellet suspended in KAc sporulation medium to a final density of approximately 2 x 10⁷ cells/ml. Aliquots (2 ml) of the cell suspension are distributed into 50 ml Corning centrifuge tubes (#25335). Drug additions are made at this point. Solvent and untreated (i.e. zero addition) controls are also included. Optimal sporulation results when this suspension is vigorously aerated by shaking 400 rpm) at 30ºC for 3 days. Typically, between 80-90% of untreated cells form asci.

Following exposure to the KAc sporulation medium for 3 days, the sporulation efficiency (i.e. the no. diploids: no. asci) is determined in each sample under Phase Contrast optics.

The cultures are harvested and washed twice in 35 ml water. The pellets are suspended in 2 ml water and 0.5 ml of Zymolase 100-T solution is added to digest ascal walls. After incubation for 16 hr at 30ºC shaking at 200 rpm, the digested cultures are sonicated using a Bronwill SoosoniklV sonicator (equipped with a microprobe) for two 30 second periods at 60% power. This treatment disrupts remaining diploid cells and dispenses the spores. The sonicated preparations are then washed twice with 35 ml 1.5% (w/v) Tergitol NP-40 to remove cellular debris. Finally, the pellets are suspended in 4 ml of Tergitol and sonicated as above just prior to plating. A non-ionic detergent is used since aqueous spore suspensions tend to aggregate due to the hydrophobic nature of their walls. The resulting spore suspension contains less than 2% of spores in clumps and diploid cells are not observed.

This 5 × 10^-1 dilution is plated on Scarg-+ 0.1 ug/ml arg (0.15 ml/plate; 3 replicates). In addition, 10^-1 (0.5 ml of "5×l0^-1" + 2.0 ml Tergitol) and 5×10^-2 (0.5 ml of "5×10^-1" + 4.5 ml Tergitol) dilutions are plated on selective medium. 10 and 10^-5 dilutions are plated (in triplicate; 0.15ml/plate) on SC. Note that the diluent used in meiotic platings is Tergitol.

The Scarg- + 0.1 ug/ml plates and Sc plates are incubated at 30ºC. The selective plates are sealed in plastic sacks and counted after 10 days incubation. The

SC plates are not sealed and are counted after 4-5 days.

Plating Technique

Inoculation of the SCarg- + 0.1 ug/ml arg plates with an even, well-spread inoculum is essential for the quantification of arginine "prototrophic" colonies. The sample to be plated is drawn into a 0.5 ml serological pipette (Corning #7077-5X). Samples (0.15 ml) are run out onto the center of the Petri dish. Using a Petri dish turntable (Fisher #08-758), the inoculum is spread across the plate using the pipette tip.

Aneuploid Selection.

After 7 days (mitotic system) or 10 days (meiotic system) incubation at 30ºC, arginine "prototrophs" are counted, collected and tested for chromosome VIII hyperploidy (see below). The colonies arising on SCarg- + 0.1 ug/ml arg medium display a distinct morphology that is generally distinguishable from that of euploid colonies arising on synthetic complete medium.

The number of viable plating units is determined by the dilutions plated on SC.

Phenotypic Characterization of Presumptive Chromosome Gain Aneuploids.

Colonies are picked from the selective medium and patched onto the marked positions of synthetic complete plates, carrying impressions previously made by the 32 prongs of a multiple inoculation device. Both BR1669 (negative control) and BR1669-9.2 (trisomic positive control) are included (or haploid derivatives thereof). This master plate is incubated at 30ºC for 2- 3 days, when material from it is transferred to the "pronger" by dipping the shafts into the synthetic complete grown patch and then into a flat bottom half area microtiter dish (Costar #3696), whole wells contain

50 ul of sterile water. The cells are suspended by repeated agitation of the device within the wells. The multiple inoculation device is then removed from the microtiter dish, and the inoculum droplets on the end of the prongs are transferred to the diagnostic medium. The device is then re-immersed in the microtiter dish, removed and another plate inoculated. This operation is repeated until each diagnostic plate (see below) is inoculated. Plates are incubated at 30º (unless otherwise specified) and scored after 1-3 days.

In this fashion, both the arginine requirement

(on SCarg- at 24º, 30º and 36ºC) and the copper resistance phenotypes (on SCcup - all levels) are scored with optimal reliability. SC, YEPAD, glycerol and KAc are also included, mating type is determined in haploids.

The various phenotypic classes observed upon characterization of the papillae arising on SCarg- + 0.1 ug/ml arg at 30ºC are summarized in Table ii. Both arg4-8 revertants and chromosome VIII hyperloids (i.e. trisomes and disomes) are arginine independent on pronging to SCarg- at 30ºC. However, only the bόna fide hyperloids are phenotypically more copper resistant, due to the concomitant increase in gene dosage of the copper resistance gene, cupl. also located on chromosome VIII. Hence, inoculation of synthetic complete medium supplemented with 0.01 mM CuSo₄ (SCcup) as described above provides confirmation of chromosome VIII hyperploidy. By enumerating the copper-sensitive arginine prototrophs, the mutagenic effects of test chemicals are determined.

In summary, both the mitotic and meiotic systems are competent to detect chromosome VIII hyperploidy in addition to site and second site reversion at arg4-8. Genetic Analysis of Aneuploids.

Confirmation of chromosome VIII hyperploidy is provided by genetic analysis. Tetrad analysis of trisomic diploids (2n+1) arising in a mitotic trial should reveal a 2⁺:2 cosegregation for arginine prototrophy at 30ºC and enhanced copper resistance.

Disomic haploids (n+1) arising meiotically can be mated to a related copper-sensitive haploid bearing the arg4-9 mutation. Tetrads are dissected from each hybrid and the segregation for copper resistance: copper sensitivity noted. arg4-8 and arg4-9 display allelic complemenatation, and each allele individually and each allele pair generate a distinct phenotype as outlined below. A trisomic segregation pattern for arginine phenotype (at 24º, 30º, and 36ºC) confirms that the arg4-8 parent was a bona fide disome (i.e. arg4-8/arg4-8/arg4-9..

Media and Conditions

Allele/Allele pair Arg24º Arg30º Arg36º arg4-8 + - - arg4-8/arg4-8 + + - arg4-9 - - - arg4-8/arg4-9 + + +

Preparation of Ball Ground Media. The ball-ground medium provided contains the ingredients listed in Table III. Note that this is SCarg- i.e. it contains no L-arginine HCl.

i) Synthetic Complete (SC). Combine 27.7 -g medium and 15.4 g Phytager in an Erlenmeyer. Add 1.0 ml of a 30 mg/ml L-arginine HCl solution. Bring up to 1 liter with deionized, glass distilled water. Mix throughly to dissolve the constituents. Autoclave. Dispense 20-30 ml medium per plate.

ii) Scarg- + 0.1 ug/ml arg. Combine 27.7 g medium and 15.4 g Phytager in an Erlenmeyer. Bring up to 1 liter with deionized, glass distilled water. Mix the ingredients to dissolve and then autoclave. After autoclaving add 0.33 ml of 0.3 mg/ml L-arginine HCl solution. Dispense

precisely 20 ml of medium per plate.

iii) SCcup. Combine 27.7 g medium and 15.4 g

Phytager. Add 1.0 ml of a 30 mg/ml L-arginine HCl solution. Bring up to 1 liter with deionized, glass distilled water, mix and autoclave. When the medium has cooled to approximately 60º - 70ºC add the following volumes of 5 mM CuSo₄ 5H₂O:

To prepare Add

0.0050 mM SCcup 1. 0 ml 5mM CuSo₄ per liter 0.0075 " " 1. 5 ml " " "" " 0.0100 " " 2.0 ml " " "

0.0125 " " 2 .5 ml " " " " 0.0150 " " 3 .0 ml " " " " 0.0175 " " 3 .5 ml " " " " 0.0200 " " 4 .0 ml " " " "

Dispense precisely 20 ml of medium per plate. The L-arginine HCl solution is prepared as follows:

Prepare a stock solution of 3.0 mg/ml L-arginine

(HCl in deionized, glass distilled water. Filter sterilize. Add 10 ml of this 3.0 mg/ml solution to 90 ml deionized, glass distilled water. Filter sterilize this

0.3 mg/ml working solution and store at 4ºC.

The CuSo₄5H₂O solution is prepared as follows:

Prepare a stock solution of 50 mM CuSo₄ 5H₂O in deionized, glass distilled water (1.25 g/100 ml). Filter sterilize. Add 10 ml of this stock to 90 ml deionized, glass distilled water. Filter sterilize this 5 mM working solution and store at room temperature (22º - 25ºC).

As soon as the plates have cooled, they are inverted and left overnight at room temperature. Excess water is then shaken from the lids and the plates are stored "lid-down", sealed in plastic sacks.

The day before plating, the plates are removed from the sacks and allowed to dry "lids-down" overnight at room temperature.

Final Conc. of AA.

Regular Additions per liter media × 50 ×100

Adenine HCl 38 mg 0.22 mM 1.9 g 3.8 g

L-Arginine HCl⁺ 30 mg 0.14 mM 1.5 g 3.0 L-Histidine

(monohydrochloride) 20 mg 0.10 mM 1.0 g 2.0

L-Leucine 40 mg 0.30 mM 2.0 g 4.0

L-Lysine

(monohydrochloride) 40 mg 0.20 mM 2.0 g 4.0 L-Methionine 20 mg 0.13 mM 1.0 g 2.0

L-Phenylalanine 50 mg 0.30 mM 2.5 g 5.0 D,L-Threonine 300 mg 2.50 mM 15.0 g 30.0 L-Tryptophan 30 mg 0.15 mM 1.5 g 3.0 L-Tyrosine 20 mg 0.10 mM 1.0 g 2.0 Uracil 20 mg 0.18 mM 1.0 g 2.0 L-Valine 50 mg 0.43 mM 2.5 g 5.0 L-Isoleucine 20 mg 0.15 mM 1.0 g 2.0

Glucose 20 g 1000 g 2000 g

YNB w/o A.A. or Ammonium Sulfate 85 g 170 g Ammonium Sulfate 250 g 500 g Note: This medium contains no L-arginine HCL.

Other Media and Solutions Required.

I) YEPAD (solid)

2% (w/y) glucose

2% (w/y) Difco Bacto Peptone

1% (w/y) Difco Bacto yeast extract

1.5% (w/y) Difco Bacto agar

160 mg/l L-Adenine HCl.

II) YPA (liquid)

2% (w/y) glucose

2% (w/y) Difco Bacto Paptone

1% (w/y) Difco Bacto yeast extract

L-adenine HCl 30 mg/1

L-arginine HCl 30 mg/1

D, L-threonine 300 mg/1

Uracil 20 mg/1 iii) Zymolyase 100-T (Miles Laboratories).

Prepare a 0.4 mg/ml solution in sterile water. Use immediately — do not store,

iv) 1.5% (v/v) Tergitol NP40 (sigma T7631).

Prepare a 10% (v/v) stock solution by heating the undiluted Tergitol (i.e by microwave) and diluting 1:10 with water. Take 60 ml of this 10% (v/v) stock and add to 340 ml sterile water to give a 1.5% working solution.

Strain Maintenance.

A sample of the fusion strain (2n) and a (2n+l) strain BR1669-9.2 should be stored at -70ºC. For routine use, these strains are spread for single colonies on YEPAD, incubated at 30ºC for 4-5 days and stored, sealed at 40º. Every week, a colony is picked from this cold- stored master plate and spread on a fresh YEPAD plate for incubation at 30ºC. The cold-stored master should not be kept for more than 1 month. At this time, fresh material from the -70ºC freezer should be used to establish a new master plate. Testing the selective (SCarg- + 0.1 ug/ml arg) Medium

The selective properties of the SCarg- + 0.1 ug/ml arg medium may be readily tested as follows:

i) Take a colony of the fusion strain and BR1669- 9.2 (trisome) growing on YEPAD and suspend separately in 1 ml of sterile water.

ii) Perform a hemocytometer count on the cell suspensions.

iii) Dilute the suspensions such that approximately 200 cells will be plated onto growth media, iv) Plate on SC and SCarg- + p.l ug/ml arg.

v) Incubate at 30 ºC for 7 days.

The BR1669-9.2 culture gives rise to colonies at 100% efficiency on SCarg- + 0.1 ug/ml arg relative to Sc. Brl669, however, fails to give rise to macrocolonies on the selective medium.

This procedure should be performed whenever a new batch of selective medium is prepared.

Detection of the indicators of chromosome loss, cycloheximide resistance, with the LGP fusion strain.

Chromosome loss can be readily detected, among mitotically growing cells of D61.M. These features are incorporated into the LGP strain. The genotype of the strain is as shown below.

Strain D61.M forms red colonies on media with reduced amounts of adenine; in the synthetic media the supplementation is 4 mg/1 adenine (free base). In the complex YEPD media adenine is limiting if Difco yeast extract is being used. Other products have more adenine and pigmentation of colonies is weak. This strain is sensitive to 1.6 ppm cycloheximide, it requires adenine and isoleucine for growth. All the other markers are recessive and not expressed: hisX, leu1, trp5 cause requirements for histidine, leucine and tryptophan. Markers MAL2 and SUCX allow strain D61.M to ferment maltose and sucrose respectively. Markers malO and sucO are inactive "alleles" which cannot support the utilization of maltose and sucrose respectively, they are recessive whereas MAL2 and SUCX are dominant. The homozygous condition of ilv1-92/ilv1-92 causes the requirement for isoleucine, and this can be used as a reverse mutation test with a certain degree of mutagen specificity. Expression of the two mating type alleles and the other recessive markers can be used to investigate the correlation between the unmasking of the linked recessive alleles cyh2 - leu1 and ade6 with the unmasking of recessive markers on different chromosomes. Detection of mitotic chromosome loss:

Recessive cyh2 is the selective marker. Its expression can be stored on a medium with 1.6 ppm cycloheximide in a YEPD medium. The color marker on the other side of the centromere is ade6. Its expression changes the usually red pigmentation of colonies to an unpigmented white. Expression of these two markers alone is not sufficient evidence for chromosome loss. It is critically important to test for a simultaneous expression of centromere marker leu1. This is done by simply picking white colonies from the cycloheximide containing selective medium onto a medium without leucine. It is assumed that the appearance of white resistant colonies expression leu1 is indicative for the loss of the chromosome carrying the respective dominant alleles: CYH2, LEU1, and ADE6. This is a correct assumption if certain criteria are met: 1. More than 30% of white resistant colonies should express leu1. 2. Expression of leu1 among red cycloheximide resistant colonies should not exceed more than 10%.

Factors interfering with the system:

Certain respiratory deficient mutants in strain D61.M turn white on the selective YEPD cycloheximide medium. They are characterized by a slightly glossy appearance, larger then average colony size and they can be sectored red and white. They turn red on a synthetic medium. Strong induction of mitotic recombination and point mutation by agents like EMS (ethyl methane sulfonate) will generate numerous white resistant colonies which are genuinely white because of the expression of ade6, but they very rarely are luelr. They are due to double events of point mutation, mitotic gene conversion or mitotic crossing-over. Induction of these types of genetic effects is easily detected by an increase in the frequencies of red resistant colonies.

Preparation of Cultures:

The frequencies of red resistant colonies vary between 15-30 × 10^-5 colony forming units. This relatively high frequency contrasts with the much less frequent incidence of white resistant colonies requiring leucine which varies between 0.20 - 2.00 × 10^-6 per colony forming units. Therefore, it is critically important to select cultures for a normal and low spontaneous background of resistant cells. Ten parallel cultures are started by inoculating about 200 cells per 5 ml portion of liquid YEPD in metal capped test tubes. These are placed on a reciprocal shaker and incubated at 28ºC for about 36 - 40 hr. At this time, titers have reached about 3 × 10⁷ cells/ml. Samples of 0.1 ml are then plated onto one plate each of a YEPD medium containing 1.6 ppm cycloheximide. The parallel cultures are then stored in a refrigerator until the plates can be read and the cultures used. Cultures up to an age of two weeks are typically used. The test plates can be read after about 5 - 6 days, and the cultures giving unusually high colony counts are discarded. In practice, cultures which give more than 600 colonies per plate are rejected.

Preparing Cultures for Treatment:

It is expected that aneuploidy can only be induced when growing cells are exposed to mutagens. Tested cultures are diluted 1 : 10 into fresh YEPD medium and shaken for 3 -4 hr when they will have reached a titer of between 1 -2 × 10⁷ cells/ml as determined by a hemocytometer count where all cells and buds of a size larger than 1/3 of a normal cell are counted as an individual cell even if they are found in clusters of up to four cells. Chemicals are then added to culture volumes of uniformly 2 ml. Incubation is then carried out on a shaker at 28ºC overnight (about 16 hrs). Titers are then determined by hemocytometer counts and cultures- if necessary-adjusted down to about 2 × 10⁷ cells/ml in order to avoid over-crowding of plates with resistant colonies. In the case of agents inducing mitotic recombination, 2 × 10⁷ cells/ml can be much too high and additional dilutions before plating on the selective cycloheximide medium may be indicated. Additional dilutions are required for determining viable titers on the basis on colony forming units. Routine 1/1000, 1/10,000 dilution for plating on the synthetic complete medium is employed. Washing of cells is not necessary with most of the volatile chemicals. However, for routine work, cultures have to be washed before plating. This can be done by adding an isotonic saline to the 2 ml treatment culture. Make use of 10 ml centrifuge tubes and add 8 ml of this solution, spin in a table top centrifuge, discard the supernatant, add another 10 ml of the saline, spin again and finally resuspend the cells either in the original volume of 2 ml or even less in order to concentrate the cell suspension in the case of strong growth inhibition.

Much stronger effects in the induction of mitotic chromosome loss are often obtained when the treatment is interrupted by an intermittent incubation of the cultures containing the agent to be tested in ice - usually overnight - before the incubation at 28ºC is continued for another period of 4 hr. The standard protocol is:

4 hr at 28 ºC - overnight (12-16 hours) in an ice bath - 4 hr at 28 ºC - washing - plating. Alternatively, continuous incubation at reduced growth temperature (about 16C-20C) can be used. Evaluation of experiments.

The following data have to be recorded:

On the non-selective synthetic medium:

Count all the colonies - record white colonies separately.

On the selective cycloheximide medium:

Count separately the red colonies, test 50 from the control for expression of leu1. Count separately the white colonies.

Further tests with the white resistant colonies: Decide whether all the white colonies can be tested for the expression of leu1. If there are too may white colonies on the cycloheximide medium, a representative sample has to be tested - either all white colonies from one plate, or all white colonies from a given sector of a plate.

Testing the white resistant colonies:

Master plates can be made on a cycloheximide medium and replica plated 24 hr later onto a synthetic medium lacking leucine and a YEP medium with glycerol (3%) instead of glucose to determine respiratory deficient mutant, additional replica plates could be synthetic minus tryptophan, synthetic minus histidine, YEP maltose medium with 2 ppm Antimycin A where colonies expressing ma10 cannot grow, lawn plates on YEPD with a MATa and another with MAT tester. These plates are replica plated after another 24 hr onto a synthetic minimal medium in order to detect isolates expressing one of the two MAT alleles.

The minimum additional test is for the expression leu1.

This can also be achieved by directly picking white resistant colonies onto a synthetic medium without leucine.

The category of white resistant colonies is then broken up in four categories:

1. Total count of white resistant colonies 2. Number of leucine requiring white colonies

3. Number of white colonies not requiring

leucine

4. Number of colonies turning red on the

synthetic medium most of which do not require leucine. The respiratory deficient nature of such colonies has to be determined if this category becomes numerous. The full testing protocol with a master plate and replica plating on a YEP glycerol medium is then indicated.

To be computed:

1. Viable titer as plating units per ml. This value is arrived at my multiplying the number of colonies on the non-selective medium with the dilution factor aώ an additional factor to arrive at a total plated volume of 1.00 ml.

The calculation in the case of five plates used would be: Number of colonies per five plates × 2 (to arrive at a plating volume of 1.00) × dilution factor.

2. Resistant red colonies per 10⁵ plating units. This will indicate how may cell in the population express only cyh2. If their incidences increase definitely above the control level, indication mitotic recombination and/or point mutation is indicated.

3. Resistant white colonies expressing the leucine requirement per 10⁵ plating units. They are considered to represent cases of mitotic chromosome loss if their relative frequencies among the white resistant colonies exceed 30%. Additional calculation may be indicated especially if there is evidence for the induction of mitotic recombination/point mutation.

1. Compute the relative frequencies of white colonies or colonies with white sectors on the non-selective medium in order to quantitate events leading to the expression of ade6.

2. Determine the frequencies of red resistant colonies expressing leu1. This allows to quantitate the genetic events occurring in the centromere - leu1 interval.

3. Computer the compounded frequencies of white non-selected × frequencies of leucine-requiring red resistant colonies. The actual frequencies of white resistant colonies expressing leu1 should be 20 times higher than the compounded frequencies.

Interpretation of Data:

A precaution required with the test design is that chromosome loss and multiple events of mitotic recombination or point mutation and even chromosomal segment losses generate the same types expressing ade6, leu1 and cyh2. However, the decision is simple and straight forward if there is no indication for the induction of mitotic recombination and/or point mutation. This is the case when the relative frequencies of the red resistant colonies remain at control level.

Induction of mitotic recombination and/or point mutation requires careful interpretation. There is a distinct possibility that mitotic recombinational events could be clustered around the centromere. Therefore, a safety margin has been taken into the interpretation. i.e. Mitotic chromosome loss is indicated if the actually observed frequencies of white and leucine-requiring resistant colonies are 20 times higher than the compounded frequencies of white non-selective colonies times the leucine-requiring red resistant colonies.

Special Precautions:

Diploid stains tend to sporulate in exhausted media, in plain buffers and even in distilled water.

Therefore, incubation periods in plain buffers or water should be restricted to periods of not more than four hours.

Aneuploidy can be induced in a variety of organisms by cold shock alone. Yeast cells are no exception to this even though the frequencies are so low that the effects of cold shock-induced aneuploidy are not readily detected. Frequent shifting of cultures between the refrigerator and room temperature should be avoided.

Having described the various methodologies, the advantages and application of the invention are now listed:

1. The invention provides a unified system for selectively detecting chromosomal gain, loss and aneuploidy in mitotically growing or meiotically developing cells using genetic methods.

2. The system enables the detection of chemicals that induce changes in chromosomal number as well as chromosomal loss. Included among the agents are neurotoxins (such as methyl ethyl ketone, 2,5 hexanedione etc.,), tumor or antitumor agents (such as nocadozole, propinoitrile, carbendizim and the like). 3. The system allows the detection of chemically induced changes by a broad range of chemical agents due to the presence of a permeability mutation. Methodologies for obtaining permeability mutations are well described in the art and should be known to one of ordinary skill in the art.

4. The system allows the detection of chemically induced chromosomal changes for any chromosome and for artificial chromosomes. The methods for identifying changes for any chromosome comprises the development of a "signalling cassette" that includes gene dosage markers. The transference of the cassette of genetic information to any chromosome is accomplished by standard molecular biology techniques well known in the art.

5. The system enables the discrimination between DNA and non-DNA targets for chemically induced chromosomal changes. This is accomplished by determining whether chemicals also induce recombination and/or mutation as indicators of DNA damage.

6. The system enables the assessment of tubulins other than yeast tubulin as targets for chemically induced chromosomal changes. This is accomplished by replacing relevant tubulin genes. Similarly, segregational components other than tubulins can be examined.

7. The system enables the screening of agents that may act as neurotoxins. Examples include methyl ethyl ketone and 2,5 hexanedione.

8. The system enables the screening of agents that may affect productive meiosis thereby affecting human reproduction. This is due to commonality of meisis in yeast and human meiocytes. Examples include carbendizim and nocadozole.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

For example a modification of the fusion strain described herein includes the chromosome gain features including the arg4-8 and cup1 alleles, but instead of detecting chromosome loss using genetic markers on chromosome VII, the chromosome loss is detected as loss of chromosome V. The selection marker is canR which provides resistance to canavanine and the associated mutant genetic markers for chromosome loss detection are yra3 and trp2 or his1.

Claims

WHAT IS CLAIMED IS:

1. A diploid S. cerevisiae fusion stain with genetic markers having the following properties:

(a) enabling the genetic detection of chromosomal gain and loss in mitotic cells and chromosomal gain meiotic cells arising naturally or as a result of exposure to chemical or physical agent;

(b) enabling differentiation between DNA versus non-DNA targets of chemically induced chromosomal changes; or (c) enabling detection of induced chromosomal changes when a component in said fusion strain is derived from another species, said component including synaptonemal complex, centromere, kinetochore, microtubular protein or tubulin.

2. A method for detecting chemically induced chromosomal changes, comprising the steps of:

(a) exposing the fusion strain of claim 1 to a chemical whose effect on the chromosome changes is to be determined, during mitotic growth or meiotic development; (b) then placing the fusion strain from step (a) to a suitable selective medium;

(c) thereafter examining plated colonies of step (b) for putative changes in chromosome number by conventional methodologies.

3. A method for distinguishing DNA vs non-DNA targets of chemically induced chromosomal changes, comprising performing the steps of claim 2 and then determining genetic changes indicative of mutation and recombination using suitable genetic markers.

4. A method for detecting changes in an artificial chromosome, comprising the steps of claim 3 and finding signalling mutations or genes in another chromosome due to transference of genetic signalling cassette.

5. A method for detecting chromosomal changes resulting after manipulation of a cellular component, comprising the steps of claim 3 and determining normal growth and viability of the fusion strain indicating no effect of cell component manipulation, or only after determining that cell component manipulation had little effect on growth and viability, finding changes in the chromosome number indicative of effects resulting from cell component manipulation.

6. A method for detecting chromosomal changes resulting from incorporation of a cellular component from another species, comprising the steps of:

(a) identifying the incorporation of said cellular component by conventional genetic or molecular techniques, and then determining chromosomal changes by the method of claim 5.

7. The fusion strain of claim 1 further comprising signalling mutations or genes transferred from genetic signalling cassettes identified in another homologous, heterologous or artificial chromosome.

8. The fusion strain of claim 1 being homozygous for arg4-8 and cup1 and heterozygous for cyh2 leu1 ade6 and exhibiting greater than 50% meiosis in a suitable growth medium.

9. The fusion strain of claim 1 being homozygous for arg4-8 and cupl and heterozygous for canR, ura3 and trp2 or his1.