Abstract
Four electrophoretic and/or enzyme-activity variants of murine LDH-A subunit (Ldh1 a-m1Neu, Ldh1 a-m5Neu, Ldh1 a-m6Neu, Ldh1 a-m9Neu), induced by procarbazine hydrochloride or ethylnitrosourea (ENU), were analyzed at the DNA level. The exons of the Ldh1 gene from homozygous mutants were amplified by PCR and sequenced. Three mutations resulted from nucleotide substitutions in exon 5: the transitions A→G at codons 216 (Ldh1 a-m5Neu) and 225 (Ldh1 a-m6Neu), and the transversion G→C (Ldh1 a-m1Neu) at codon 222. The mutations resulted in the replacements of Glu by Gly (Ldh1 a-m5Neu), Gln by Arg (Ldh1 a-m6Neu) and Asp by His (Ldh1 a-m1Neu). The fourth base substitution, the transition T→C (Ldh1 a-m9Neu), has been found at the GT donor splice site following the first exon; this mutation affected the efficiency of transcription. All ENU-induced mutations were A/T→G/C transitions. The mutation events could be correlated with the biochemical and physiological alterations observed in affected mice.
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Aebi, M., Hornig, H., Padgett, R.A., Reiser, J., Weissmann, C. (1986). Sequence requirements for splicing of higher eukaryotic nuclear pre-mRNA. Cell 47, 555–565.
Charles, D.J., Pretsch, W. (1981). A mutation affecting the lactate dehydrogenase locus Ldh-1 in the mouse—I. Genetical and electrophoretical characterization. Biochem. Genet. 19, 301–309.
Charles, D.J., Pretsch, W. (1987). Linear dose-response relationship of erythrocyte enzyme-activity mutations in offspring of ethylnitrosourea-treated mice. Mutat. Res. 176, 81–91.
Chatterjee, B., Gimbel, W., Görblich, T., Werner, T. (1993). HPLC-purification of PCR-products for direct sequencing. Trends Genet. 9, 406.
Chung, F.-Z., Tsujibo, H., Bhattacharyya, U., Sharief, F.S., Li, S.S.-L. (1985). Genomic organization of human lactate dehydrogenase-A gene. Biochem. J. 231, 537–541.
Davis, L.G., Dibner, M.D., Battey, J.F. (1986). Basic Methods in Molecular Biology, (New York: Elsevier Science Publishing Co., Inc.).
Eventoff, W., Rossmann, M.G., Taylor, S.S., Torff, H.-J., Meyer, H., Keil, W., Kiltz, H.-H. (1977). Structural adaptations of lactate dehydrogenase isozymes. Proc. Natl. Acad. Sci. USA 74, 2677–2681.
Fukasawa, K.M., Li, S.S.-L. (1987). Complete nucleotide sequence of the mouse lactate dehydrogenase-A functional gene: comparison of the exon-intron organization of dehydrogenase genes. Genetics 116, 99–105.
Hiraoka, B.Y., Sharief, F.S., Yang, Y.-W., Li, W-H., Li, S.S.-L. (1990). The cDNA and protein sequences of mouse lactate dehydrogenase B. Molecular evolution of vertebrate LDH genes A (muscle), B (heart) and C (testis). Eur. J. Biochem. 189, 215–220.
Hull, J., Shackleton, S., Harris, A. (1993). Abnormal mRNA splicing resulting from three different mutations in the CFTR gene. Hum. Mol. Genet. 2, 689–692.
Li, S.S.-L. (1989). Lactate dehydrogenase isoenzymes A (muscle), B (heart) and C (testis) of mammals and the genes coding for these enzymes. Biochem. Soc. Transact. 17, 304–307.
Li, S.S.-L., Tiano, H.F., Fukasawa, K.M., Yagi, K., Shimizu, M., Sharief, F.S., Nakashima, Y., Pan, Y.-C.E. (1985). Protein structure and gene organization of mouse lactate dehydrogenase-A isozyme. Eur. J. Biochem. 149, 215–225.
Markert, C.L., Shakelee, J.B., Whitt, G.S. (1975). Evolution of a gene: Multiple genes for LDH isozymes provide a model of the evolution of gene structure, function and regulation. Science 189, 102–114.
Merkle, S., Pretsch, W. (1992). Characterization of two electrophoretic lactate dehydrogenase-A mutants in Mus musculus. Biochem. Genet. 30, 49–59.
Musick, W.D.L., Rossmann, M.G. (1979). The structure of mouse testicular lactate dehydrogenase isoenzyme C4 at 2.9 Å resolution. J. Biol. Chem. 254, 7611–7620.
Pan, Y.-C.E., Sharief, F.S., Okabe, M., Huang, S., Li, S.S.-L. (1983). Amino acid sequence studies on lactate dehydrogenase C4 isozymes from mouse and rat testes. J. Biol. Chem. 258, 7005–7016.
Pastink, A., Vreeken, C., Nivard, M.J.M., Searles, L.L., Vogel, E.W. (1989). Sequence analysis of N-ethyl-N-nitrosourea-induced vermilion mutations in Drosophila melanogaster. Genetics 123, 123–129.
Pretsch, W. (1989). Eight independent Ldh-1 mutations of the mouse recovered in mutagenicity experiments: Biochemical characteristics and chromosomal localization. Genet. Res., 53, 101–104.
Pretsch, W., Charles, D.J. (1984). Detection of dominant enzyme mutants in mice: Model studies for mutations in man. In Monitoring Human Exposure to Carcinogenic and Mutagenic Agents, IARC Scientific Publications No. 59, A. Berlin, M. Draper, K. Hemminki, H. Vainio, eds. (Lyon: International Agency for Research on Cancer), pp. 361–369.
Pretsch, W., Merkle, S., Favor, J., Werner, T. (1993). A mutation affecting the lactate dehydrogenase locus Ldh-1 in the mouse. II. Mechanism of the LDH-A deficiency associated with hemolytic anemia. Genetics 135, 161–170.
Richardson, K.K., Richardson, F.C., Crosby, R.M., Swenberg, J.A., Skopek, T.R. (1987). DNA base changes and alkylation following in vivo exposure of Escherichia coli to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea. Proc. Natl. Acad. Sci. USA 84, 344–348.
Sakai, I., Sharief, F.S., Li, S.S.-L. (1987a). Molecular cloning and nucleotide sequence of the cDNA for sperm-specific lactate dehydrogenase-C from mouse. Biochem. J. 242, 619–622.
Sakai, I., Sharief, F.S., Pan, Y.-C.E., Li, S.S.-L. (1987b). The cDNA and protein sequences of human lactate dehydrogenase B. Biochem. J. 248, 933–936.
Skopek, T.R., Walker, V.E., Cochrane, J.E., Craft, T.R., Cariello, N.F. (1992). Mutational spectrum at the Hprt locus in splenic T cells of B6C3F1 mice exposed to N-ethyl-N-nitrosourea. Proc. Natl. Acad. Sci. USA 89, 7866–7870.
Takeno, T., Li, S.S.-L. (1989). Human testicular lactate dehydrogenase-C gene is interrupted by six introns at positions homologous to those of LDH-A (muscle) and LDH-B (heart) genes. Biochem. Biophys. Res. Commun. 159, 579–583.
Zdarsky, E., Favor, J., Jackson, I.J. (1990). The molecular basis of brown, an old mouse mutation, and of an induced revertant to wild type. Genetics 126, 443–449.
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Sandulache, R., Pretsch, W., Chatterjee, B. et al. Molecular analysis of four lactate dehydrogenase-A mutants in the mouse. Mammalian Genome 5, 777–780 (1994). https://doi.org/10.1007/BF00292012
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DOI: https://doi.org/10.1007/BF00292012