Abstract
Several bioanalytical techniques including electrofocusing, two-dimensional technique comprising electrofocusing and electrophoresis, and chromatography have been evaluated and improved to provide a method for detecting gene mutations in mice.
The use of 1 mm gels for both electrofocusing and electrophoresis in the two-dimensional technique resulted in better resolution, shorter separation time, and reduced costs. An electrophoretic apparatus for the second direction electrophoresis has been designed which eliminated cutting of a flat gel into strips prior to electrophoresis. These changes reduced the total time for a two-dimensional separation to 4 h.
The scope and application of isoelectric focusing in detecting gene mutations in mice have been improved by the agar contact replica method. This method allows the simultaneous analysis of several genetic loci besides facilitating automation of evaluation, documentation, and storage of zymograms.
Preliminary studies on the detection of heritable metabolic disorders of amino acid metabolism in mice show a potential for such studies in estimating mutation rates in mice. Some possible techniques for such studies and their implications are discussed.
Zusammenfassung
Mehrere bioanalytische Techniken (Elektrofokussierung, eine die Elektrofokussierung und Elektrophorese kombinierende zweidimensionale Technik, Chromatographie) wurden miteinander verglichen und verbessert, um eine neue Methode zum Nachweis von Genmutationen bei Mäusen zu entwickeln.
Die Verwendung 1 mm dicker Gele sowohl zur Elektrofokussierung als auch zur Elektrophorese ergeben höhere Auflösung, kürzere Trennzeit und verminderte Kosten. Ein elektrophoretischer Apparat für die Elektrophorese in der zweiten Richtung wurde konstrukturell so verändert, daß das Schneiden des flachen Gels in Streifen vor der Elektrophorese überflüssig wird. Diese Veränderungen vermindern die Gesamtzeit einer zweidimensionalen Trennung auf 4h.
Die Anwendungsmöglichkeit der isoelektrischen Fokussierung zum Nachweis von Genmutationen bei Mäusen wurde wesentlich durch die „agar contact replica method“ vergrößert. Diese Modifikation gestattet die gleichzeitige Untersuchung verschiedener genetischer Loci und erleichtert die Automatisierung der Auswertung, Dokumentation und Aufbewahrung der Zymogramme.
Vorversuche zum Auffinden erblicher Störungen des Proteinstoffwechsels bei Mäusen zeigen ein großes Potential für derartige Untersuchungen zur Bestimmung der Mutationsrate bei Mäusen. Einige mögliche Techniken für diese Untersuchungen und ihre Bedeutung werden diskutiert.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Arlinger, L.: Analytical isotachophoresis — principle of separation and detection. Protides of Biological Fluids 22, 661–668 (1974)
Arvidson, S., Wadström, T.: Detection of proteolytic activity after isoelectric focusing in polyacrylamide gel. Biochim. biophys. Acta (Amst.) 310, 418–420 (1973)
Berry, H. K.: Screening for genetic disorders. Fed. Proc. 34, 2134–2139 (1975)
Bhakdi, S., Knüffermann, H., Hoelzl-Walloch, D. F.: Separation of EDTA-extractable erythrocyte membrane proteins by isoelectric focusing linked to electrophoresis in sodium dodecyl sulfate. Biochim. biophys. Acta (Amst.) 345, 448–457 (1974)
Catsimpoolas, N.: Isoelectric focusing and isotachophoresis of proteins. Separation Sci. 8, 71–121 (1973)
Doane, W. W.: Quantitation of amylase in Drosophila separated by acrylamide gel electrophoresis. J. exp. Zool. 164, 363–378 (1967)
Everaerts, F. M.: Isotachophoresis. J. Chromatogr. 65, 3–12 (1972)
Free, A., Free, H.: Urinalysis in clinical laboratory practice. Cleveland: CRC Press 1975
Harris, H.: The Principle of human biochemical genetics, 2nd Ed., pp. 310–313. Amsterdam: North-Holland 1975
Ivarie, R. D., O'Farrell, P. H.: A steroid response analyzed by two-dimensional electrophoresis. Paper presented at the International Symposium on Electrofocusing and Isotachophoresis at Hamburg, August 2–4, 1976
Klose, J.: Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Hum. Genetik 26, 231–243 (1975)
Klose, J.: The protein-mapping-method employed to test for chemically induced point mutations in mice. Arch. Toxicol. 38, 53–60 (1977)
Kopwillem, A., Lundin, H.: Analytical isotachophoresis. X. Amino acid analysis. LKB Application Note 183 (1974)
Latner, A. L.: Isoelectric focusing in liquid and gels as applied to clinical chemistry. Adv. clin. Chem. 17, 193–250 (1975)
Malling, H. V., Valcovic, L. R.: Electrophoretic mobility assay in the mouse. Arch. Toxicol. (in press) (1977)
Mohrenweiser, H. W., Burkhart, J. G., Feuers, R. J., Kane, A. C., May, J. B., McAninch, J. E.: Development of an in vivo mammalian microlesion detection system. Paper presented at 7th Annual Meeting of American Environmental Mutagen Society at Atlanta on March 12–15, 1976
Narayanan, K. R.: Detection of biochemical variants in mice. GSF-Bericht M 199, 27 (1974)
Narayanan, K. R.: Fingerprinting as a tool for biochemical characterization of different strains of mice. GSF-Bericht B 626, 44 (1975)
Narayanan, K. R., Raj, A. S.: A method for simultaneous analysis of several genetic loci in mice. In: Electrofocusing and isotachophoresis (B. J. Radola, D. Graesslin, eds.), pp. 221–231. Berlin: de Gruyter 1977
Nyhan, W. L.: Patterns of clinical expression and genetic variation. In: Heritable disorders of amino acid metabolism. Patterns of clinical expression and genetic variation (W. L. Nyhan, Ed.), pp. 3–14. New York: Wiley 1974
O'Farrell, P. H.: High resolution two-dimensional electrophoresis of proteins. J. biol. Chem. 250, 4007–4021 (1975)
Pretsch, W., Narayanan, K. R.: Untersuchung erblicher Stoffwechselstörungen bei der Maus. GSF-Bericht PG 22, 39–40 (1976)
Raj, A. S.: Isozyme patterns of esterases and malate dehydrogenases in different strains of mice. GSF-Bericht B 626, 50–52 (1975)
Russell, L. B., Russell, W. L., Popp, R. A., Vaughan, C., Jacobson, K. B.: Radiation-induced mutations at mouse hemoglobin loci. Proc. nat. Acad. Sci. (Wash.) 73, 2843–2846 (1976)
Schmidt, L.: The biochemical detection of metabolic diseases: Screening tests and a systematic approach to screening. In: Heritable disorders of amino acid metabolism. Patterns of clinical expression and genetic variation (W. L. Nyhan, Ed.), pp. 675–697. New York: Wiley 1974
Scriver, C. R.: Inborn errors of amino-acid metabolism. Brit. med. Bull. 25, 35–41 (1969)
Shih, V. E.: Laboratory techniques for the detection of hereditary metabolic disorders, pp. 11–76. Cleveland: CRC Press 1973
Siciliano, M. J., Humphrey, R. M.: Electrophoretic approach to mutagenesis in mammalian cell lines. Mutation Res. 31, 323 (1975)
Valcovic, L. R., Malling, H. V.: An approach to measuring germinal mutations in the mouse. Environ. Hlth Perspect. 6, 201–205 (1973)
Valcovic, L. R., Malling, H. V.: Mutation detection in the biochemical specific locus system. Mutation Res. 31, 338 (1975)
Vesterberg, O.: Isoelectric focusing of proteins in polyacrylamide gels. Biochim. biophys. Acta (Amst.) 257, 11–19 (1972)
Vesterberg, O., Eriksson, R.: Detection of fibrinolytic activity by a zymogram technique after isoelectric focusing. Biochim. biophys. Acta (Amst.) 285, 393–397 (1972)
Wadström, T., Smyth, C. J.: Some application of a zymogram method in combination with isoelectric focusing in polyacrylamide gels. In: Isoelectric focusing (J. P. Arbuthnott, J. A. Beeley, Ed.), pp. 152–177. London: Butterworths 1975
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Narayanan, K.R. Detection of biochemical mutants in mice. Arch. Toxicol. 38, 61–73 (1977). https://doi.org/10.1007/BF00293664
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00293664