Detection of Heritable Mutations as Quantitative Changes in Protein ...

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Mar 3, 1987 - nitrosourea. Proteins from the livers of approximately. 800 mice were separated by two-dimensional electro- phoresis, and abundances were ...
THEJOURNAL OF BIOLOGICAL

CHEMISTRY

Vol. 262, No. 26, Issue of September 15, pp. 12764-12767,1987 Printed in U.S.A.

Detection of Heritable Mutations as Quantitative Changes in Protein Expression* (Received for publication, March 3, 1987)

Carol S. GiomettiS, M. Anne Gemmell, Sharron L. Nance, Sandra L. Tollaksen, and John Taylor From the Division of Biological and Medical Research, Argonne National Laboratory, Argonne,Illinois 60439-4833

A computerized search for the ,appearance of herit- chemicals (1,2) or ionizing radiation (3). Detection of quanablemutations (as indicated bychangesinprotein titative protein changes that reflect either point mutationsor expression) wasconducted onthree setsof mice, whose gene deletions, however, has been hampered by the inability sires hadbeen either untreated,exposed to 3 gray units to obtain quantitative measurements from the large number of gamma radiation, or treated with 150 mg/kg ethyl- of two-dimensional electrophoresis patternsrequired for munitrosourea. Proteins from the livers of approximately tation screening(4). Anderson et al. (5) have shown that two800 mice were separatedby two-dimensional electro- dimensional electrophoresis, coupled with computerized data phoresis, and abundances were measured by using im- analysis,candetect a 50% reduction in proteinamount, age analysis techniques. Heritable mutations were de- provided thatthebackgroundquantitativevariationsare tected by the appearance of new proteins or by the small. However, the contribution of individual sample variaof normally occur- bility, both experimental and biological, to the overall quanquantitative decrease in abundance ring proteins. Measurements of the variability of the titative datadispersion is currently unknown. The magnitude protein abundance indicate that at least 48 proteins of such variability may well determine the feasibility of ultiare consistentenoughtobeusedinsearcheswhen mately using two-dimensional electrophoresis protein sepamutations are expected to result ina 50% reduction in rations to screen human samples for the occurrence of induced thenormalamount of protein. New proteinswere found in four offspring from ethylnitrosourea-treated mutations following exposure to known or suspected mutamice, and in each case a nearby spot was found to be gens. We report here the results of a mutagenesis study inwhich significantly diminished. These mutations were confirmed in subsequent generations.A computer-assisted heritable mutations, represented as altered protein expressearch detected three of these mutations on the basis sion, were detected by computer-assisted screening of twoof the abundance decrease alone. Theseresults indicate dimensional electrophoresis protein patterns. This studywas of two-dimenthat two-dimensional electrophoresis can be used to designed to assess the quantitative capabilities the application detect mutations reflected as quantitative changes in sional electrophoresis and to evaluate possible protein expression, provided that the proteins to be of this technique to mutation studies in humans. To minimize monitored are quantitatively stable when samples from quantitative variabilitydue to genetic heterogeneity and thus different individuals are compared. concentrate on quantitative variability introduced by samplingandnongenetic biological factors (e.g. age, diet), we chose to use inbred strainsof mice for our initial study. Thus, the results presentedhere represent the simplest casefor the Exposure toa mutagen can cause both point mutations and application of two-dimensional electrophoresis to screening small chromosomal deletions. The combination of a gamete for mutations that cause quantitative protein changes and carrying a point mutation with a gamete carrying the unal- serve as a foundation for humanstudies in which genetic tered gene could resultinan offspring that expresses an heterogeneity will contribute additional quantitative variabilaltered protein togetherwith the normal proteina t 50% of its ity (5-7). normal abundance. A gamete in which a structural gene has This studyincluded 797 offspring fromuntreated male mice been deleted could, if combined with a gamete carrying the or male mice treated with either ethylnitrosourea or gamma normal gene, result in an offspring that expresses the corre- radiation. Ethylnitrosourea-induced mutations, previously sponding gene product at 50% of itsnormalabundance. shown to cause the appearance of new protein spots in twoTherefore,detection of quantitativealterations in protein dimensional electrophoresis patterns of mouse liver proteins expressioncould, theoretically, be used to measure genetic with a corresponding decrease in the intensityof an adjacent changes that canbe tested for heritability and to provide data spot ( 2 ) , allowed the detection of rare quantitative protein for estimation of mutation rates. alterations in two-dimensional electrophoresis patterns tobe Two-dimensionalelectrophoresishas beenusedsuccessvalidated. The ability to detect radiation-induced mutations fully to detect qualitative protein changes indicative of point could then be realistically assessed. mutations (1, 2) and gene deletions (3) induced by toxic EXPERIMENTALPROCEDURES



A N D RESULTS’

* This work was supported by the United States Department of Energy, Office of Health and Environmental Research, under Contract W-31-109-ENG-38. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. 2 T o whom reprint requests should be addressed: Division of Biological and Medical Research, Argonne National Laboratory, 9700 S. Cass Ave., Argonne, IL 60439-4833.

DISCUSSION

The results of this study demonstrate that quantitative two-dimensional electrophoresis can be used to detect muta-



Portions of thispaper(including“ExperimentalProcedures,” “Results,” Figs. 1-5, Table I, and additional references) are presented in miniprint at the end of this paper. The abbreviations used are: CV,

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tions that cause an altered gene resulting in the expression of gels with subsequent comparison of average spot volumes. significantly a variant protein together witha 50% reduction in the abun- Biological factors, on the other hand, contribute variability seen in theliver proteins dance of the normal protein. The detection of such mutations to the normal quantitative mice, as quantitative changes in protein expression is, however, of inbred mice. A subpopulation of liver proteins in male limited by thebackgroundquantitativevariationineach for example, has been found to fluctuate in abundance as a protein monitored. The detection of three out of four ethyl- function of sexual maturity (data not shown). Some of the nitrosourea-induced mutations based on quantitative changesobserved quantitative variability may be a function of the in normal liver proteins demonstrates this limitation and setsliver itself,beinga tissue inwhich protein metabolism is the present detection thresholdof the two-dimensiona~elec- responsive t o hormonalcontrols,diet,andlorcircadian trophoresis system. rhythm. Another tissue orcell type may, therefore, be better T h e use of two-dimensional electrophoresis to detect mu- suited to two-dimensional electrophoresis mutagenesis studtations that cause the total loss of one genecopy must still be ies. Careful evaluation andmodification of sampling protocol validated. For this experiment, in order to simplify the mu- should, however, produce an increase in the number of protation searchprotocol, the assumptionwas made that thetoss teins that can be monitored for quantitative protein changes of one gene copy would result ina 50% reduction in the in animal studies. synthesis of the amount of the corresponding protein. The The applicability of the two-dimensional electrophoresis possibility exists, however, that intracellularregulatory mech- approach to mutation detection in humans remains t o be anisms may cause compensatory synthesis of proteins in order determined. One consideration is that, unlike themouse systo maintain normal concentrations. Given the constraint of tem, nongenetic, or biological variables are not easily conquantitative reproducibility defined by our present data, such trolled among humansubjects. Normal genetic differencesare compensatory mechanisms must be investigated, since quanalso expected to introduce additional background quantitative titative changes of less than 50% that could be significant indicators of mutation might otherwise beignored. The anal- variation since, when different mouse strains are compared, ysis of protein expression in tissues from heterozygous carriers more genetically regulated quantitative than qualitative protein differences are found (6, 7). Although estimates of the of known gene inversion or deletion mutations (available as occurrence of qualitative genetic variants l i e . protein polymouse stocks) or in cultured cell lineswith inducedgene beenmade (9-11), no deletions should demonstrate whether or not such mutations morphisms) in human samples have similar studies have been done to evaluategenetically influare detectableby two-dimensional electrophoresis. The absence of significant changes in liver protein expres- enced quantitative protein variability. Finally, human samsion among 369 offspringfrom irradiated males may be a ples for genetic studies are limited t o those tissues or body by relatively noninvasive methods, reflection of (a)the influenceof cellular c o m p e n s a t o ~mech- fluids that can be obtained anisms that maskedgene deletions or ( b ) the limited number Le. serum, urine, peripheralblood cells, or skin fibroblasts. Of of proteins that had the quantitative stability required for these, only the blood cells or fibroblasts produce two-dimendetection of a 50% decrease in protein abundance.Assuming sional electrophoresis patterns comparablein simplicity and that monitoring the48 protein spots with coefficient of vari- resolution to those of the mouse liver pattern (12-16). The ation values of no more than 15% would have p e ~ i t t e d applicabilit~of two-dimensional electrophoresis to mutation detection of a 50% reduction in expression and that eachof studies utilizing human material should, therefore, be based the 48 protein spots represents an independent gene locus, on the resultsof studies that measure the quantitative variaand given a specific locus mutation rate of 2.7 X per gray bility of proteins expressed in human cells (e.g. platelets or unit perlocus as representativeof the response to single doses skinfibroblasts)as a function of both nongenetic (intraof gamma radiation (8), the expected mutation yield in this individual variability) andgenetic (interindividua~ variability) study would have been about one event in the 369 gametes factors. Such studieswould define a subpopulation of proteins screened following exposure to3 gray units. Thus,zero events that have the quantitative stabilityrequired for the detection is well within the limits of expectation for the number of of heritable mutations and allow a more realistic assessment individuals screened. If more protein spots with low levels of of the feasibility of using two-dimensional electrophoresis for normalvariability could be monitored, t,he probability of human mutation studies. detecting a quantitative protein variant in a sample of 400 Two-dimensional electrophoresis, together with computerindividuals would obviously become more feasible. assisted data analysis, can be used t o detect mutations as In the mouse model system, optimization of the number of quantitative alterations in protein expression. The use of twoprotein spots ina two-dimensional electrophoresis pattern dimensional electrophoresis for detecting mutations has an that can be monitored for quantitative changes may only importantadvantage over other technologies suchasthe require stricter control of quantitative variability introduced emerging DNA methods. By analyzing proteins expressed in by both technical inconsistencies and nongenetic biological the offspring of exposed individuals, survivablemutations are factors. Anderson et al. ( 5 ) have demonstrated that. minor being monitored. In animal models, the impact of such mudifferences in sample loading, electrophoresis, staining and tations on the well-being of carriers inseveralsucceeding destaining,andcomputer imaging actuallyintroduce very little quantitative variation into the two-dimensional electro-generations can be assessed, including the consequences of phoresis patternsof mouse liver proteins. Such variation couldcarrying the mutation as a homozygous trait. In addition, having no correlabe limited further by the analysis of each sample on multiple rather than identifying DNA damage and tion between thedamageand metabolic functions in the ~ . ~ _ _ _ ~ coefficient of variation; ENU, ethylnitrosourea; ZDE, two-dimen- organism, the identificationof alterations in protein expressional electrophoresis. Miniprintis easily read with the aid of a sion allows the identificationof the specific lesion, via amino standard magnifying glass. Full size photocopies are available from acid sequencing back to the DNA level. Thus, two-dimenthe Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, sional electrophoresis measures damage to functional DNA MD 20814. Request Document No. 87 "657, cite the authors, and include a check or money order for $4.40 per set of photocopies. Full rather than totalDNA. Methods arenow being developed for size photocopies are also included in the microfilm edition of the the identification of the altered peptide in the proteins discussed in this paper (17, 18)with a view toward amino acid Journal that is available from Waverly Press. "

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sequencing and characterization of the mutation at the gene level. Acknowledgments-We thank D. Grahn for his invaluable guidance in the areas of mouse genetics and radiation biology throughout the course of this experiment and for his assistance with the statistical analysis of the data. We also acknowledge N.Anderson, L. Anderson, and F. Giere for their assistance with the experimental design and treatment of the mice, and G. Spicer for his assistance scanningtwodimensional electrophoresis gels. REFERENCES 1. Klose, J. (1975) Humungenetik 26,231-243 j,A. S., Grant, F. J. & Heddle, J. A. (1983) 2. Marshall, R. R., h

Can. J. Genet. Cytol. 25,457-466 3. Baier, L. J., Hanash, S. M. & Erickson, R. P. (1984) Proc. Natl. Acud. Sci. U. S. A . 81,2132-2136 4. Neel, J. V., Rosenblum, B. B., Sing, C.F., Skolnick, M.M., Hanash, S. M. & Steinberg, S. (1984) in Two-dimensional Gel Electrophoresis of Proteins (Celis, J. E. & Bravo, R., eds) pp. 259-306, Academic Press, Inc., New York 5. Anderson, N. L.,Nance, S. L., Tollaksen, S. L., Giere, F.A. &

S u p p l e m e n t a r y I(.rerlal t o : DETECTION OF HERITABLE MUTATIONS AS QUANTITATIVE CHANCES I N PROTEIN EXPRESSION

Anderson, N. G. (1985) Electrophoresis 6,592-599 6. Klose, J. & Feller, M. (1981) Biochern. Genet. 19,859-870 7. Jungblut, P. & Klose, J. (1985) Biochem. Genet. 23, 227-245 8. Searle, A. G. (1974) Adu. Radiut. Biol. 4, 131-207 9. McConkev. E. H.. Tavlor. B. J. & Phan,. D. (1979) . . Proc. Nutl. Acad. Sii. U. S. A. 76,6500-6504 10. Walton, K. E., Styer, D. & Gruenstein, E. I. (1979) J. Biol. Chern. 254,7951-7960 11. Hanash. S. M.. Baier. L. J.. Welch. D.. Kuick. R. & Galteau. M. ' (1986j Am. J. Hum: Genet. 39, 317-328 12. Anderson, N. L. & Anderson, N. G. (1977) Proc. Nutl. A d . Sci. U. S. A . 74,5421-5425 13. Anderson, N. G., Anderson, N. L. & Tollaksen, S. L. (1979) Clin. Chem. 25,1199-1210 14. Edwards, J. J., Anderson, N. G., Nance, S. L. & Anderson, N. L. (1979) Blood 53, 1121-1132 15. Willard-Gallo, K. E. (1984) Ann. N. Y. Acud. Sci. 428,201-222 16. Gemmell, M. A. & Anderson, N. L.(1982) Clin. Chem. 28,10621066 17. Zhang, J.-S., Giometti, C. S. & Tollaksen, S. L.(1986) in Electrophoresis '86 (Dunn, M., ed) pp. 621-625, VCH Verlagsgesellschaft, Weinheim 18. Giometti, C. S. & Zhang, J.-S. (1986) in Electrophoresis '86 (Dunn, M., ed) pp. 670-673, VCH Verlagsgesellschaft, Weinheim

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