The Use of Mutator for Gene-Tagging: Cross-Referencing between Transposable Element Systems
A Mutator system, resulting in an approximately 30-fold increase in spontaneous mutation rate in maize, was first described by Donald S. Robertson in 1978 (15). This phenomenon was associated with the movement of a transposon, when a Mutator-derived mutant allele of the Adh1 locus was shown to contain a DNA insertion (24). The transposon, called Mu1, was found to be 1,367 bp in length, to have nearly perfect terminal inverted repeats, and to denerate a 9-bp direct repeat of the Adh1 target site upon insertion (2). Using this initial correlation between a mutant phenotype arising from a Mutator line and the presence of a particular DNA sequence (Mu1) within the mutated gene, it seemed reasonable to assume that other Mutator-induced mutations might also be due to the same DNA sequence. If true, the Mu1 sequence might be used as a hybridization probe to molecularly identify and clone the mutated gene by a process termed gene-tagging. Since the Mu1 sequence is present in multiple copies in the genome of Mutator-de rived plants (2,3), the problem would really be one of identifying which Mu1-hybridizing clone also contained DNA of the mutated gene of interest.
Such a gene-tagging approach was used successfully to clone the Mutator-induced a-Mum2 allele of the maize A1 gene (13). Approximately 35 different Mu1-hybridizing recombinant phage clones prepared from plant material homozygous for the recessive a-Mum2 mutation were screened. One clone was identified as containing at least a portion of the sought gene, for this DNA fragment contained an En-transposon insertion when plant material containing an En-induced mutable A1 allele was cloned. Such cross-referencing or identification of the sought gene by using more than one transposon-induced mutant allele is common in gene-tagging experiments (see Ref. 23 for review).
The Mu1-hybridizing element within the a-Mum2 allele was found to be of similar size and structure as Mu1 (13). Further characterization of the A1 gene transcription unit (22) places the Mu1-like element in opposite orientation with respect to the direction of transcription as compared to the original Mu1 element in the Adh1 gene. But, like that element, it generates a 9-bp duplication of target site sequence (Fig. 1).
Therefore the Mutator system seemed to have several characteristics which would lend themselves to a more extensive gene-tagging program: mutants are generated at an exceptionally high rate, with approximately 35% of the new mutants exhibiting a mutable phenotype indicative of a transposon insertion (15); there was a correlation (albeit limited) between a Mutator-derived mutant and the presence of the Mu1 DNA sequence within the mutated gene (13,24); and, finally, that sequence was small in size and of a relatively well-defined structure, thus facilitating cloning.
The following is a preliminary report of a gene-tagging experiment begun in the summer of 1985 using the Mutator system. The basic premise of the experiment is quite simple and a nontargeted approach to gene-tagging (23). It relies upon the assumption that many of the “new” mutants derived from a Mutator line are just as interesting as those already named and mapped to chromosome position (perhaps even allelic), and that, just as in the case of the Adh1 and A1 mutants described above, insertion of a Mu1-like transposon into a gene may be the cause of the mutant phenotype. This latter postulate may not be true for every mutant; however, a Mu1-like element has recently been found in the bz1-mu1 allele of the maize Bz gene (25), two other independent mutations of the Adh1 gene (19), a Mutator-induced mutant of the Sh1 gene (20), and a bz2-mu1 allele of the Bz2 gene (Walbot et al., this Volume).
The problem then becomes one of identifying mutant phenotypes of interest and quickly screening those mutants for the presence of Mu1-hybridizing fragments closely linked with the mutant phenotype. As in any gene-tagging experiment, the final identification of the mutant gene fragment is not an easy task and may rely upon further studies of the mutant plant phenotype, other mutant alleles (cross-referencing), and perhaps complementation of the mutation through transformation.
KeywordsTransposable Element Mutant Phenotype Mutator System Adh1 Gene Zebra Stripe
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- 3.Bennetzen, J.L. (1984) Transposable element Mu1 is found in multiple copies only in Robertson’s Mutator maize lines. J. Molec. Appl. Genet. 2:519–524.Google Scholar
- 4.Bennetzen, J.L. (1987) Covalent DNA modification and the regulation of Mutator element transposition in maize. Molec. Gen. Genet. (in press).Google Scholar
- 6.Coe, Jr., E.H., D.A. Hoisington, and M.G. Neuffer (1987) Linkage map of corn (maize). In Maize Genetics Cooperative Newsletter, E. Coe, ed. Dept. of Agronomy and U.S. Department of Agriculture, University of Missouri, Columbia, Missouri, 61:116-147.Google Scholar
- 10.Neuffer, M.G., L. Jones, and M.S. Zuber (1968) The Mutants of Maize. Crop Sci. Soc. Amer., Madison, Wisconsin, 74 pp.Google Scholar
- 12.Neuffer, M.G., D.A. Hoisington, and V. Walbot (1985) The lesion mutants of maize. In Plant Genetics, M.F. Freeling, ed. Alan R. Liss, Inc., New York, pp. 830–833.Google Scholar
- 14.Redei, G.P. (1974) Economy in mutation experiments. Z. Pflanzenzuechtg. 73:87–96.Google Scholar
- 18.Robertson, D.S. (1986) New Information on the timing of Mu’s mutator activity. In Maize Genet. Coop. Newsletter 60:12–14.Google Scholar
- 20.Rowland, J., and J. Strommer (1986) Insertion of Robertson’s Mutator in an exon affects transcript stability. Maize Genet. Coop. Newsletter 60:17.Google Scholar
- 23.Shepherd, N.S. (1988) Transposable elements and gene-tagging. In Plant Molecular Biology: A Practical Approach, C.H. Shaw, ed. IRL Press, London (in press).Google Scholar
- 25.Taylor, L.P., V.L. Chandler, and V. Walbot (1986) Insertion of 1.4 kb and 1.7 kb Muelements into the Bronze-1 gene of Zea mays L. Maydica 31:31–45.Google Scholar