Isolation and molecular analysis of Mu-induced bz1 and sh1 mutants

--Kristine Hardeman, Susan Belcher and Vicki Chandler

To establish what types of transposable elements were contributing to the mutation rate in our Mutator stocks, experiments were undertaken to trap elements capable of transposing into genes for which molecular probes were available. Three different Mutator stocks were used in this experiment. One, which was the original Mutator stock used to generate B-Peru mutants in this lab, had a typically high number (30-50) of Mu1-hybridizing sequences. To increase the probability of isolating non-Mu1 elements, two other stocks were used that contained b-Peru mutable alleles and had been screened for low (1-4) Mu1 copy number (Chandler and Turks, MNL 62:58-59).

These three Bz1 Sh1 Mutator stocks were crossed with bz1 sh1 tester stocks and the progeny were scored for bz1 and sh1 mutants (Table 1). Only the progeny from the B-Peru Mutator source were fully scored for sh1 mutants because the other stocks contained dent, which made scoring for sh1 mutants difficult. However, a small number of ears from the b-Peru mutable Mutator sources were sufficiently plump to score for sh1 mutants and 1 mutant was recovered. All putative bz1 and sh1 mutants obtained were again crossed to the bz1 sh1 tester, and only those that transmitted the mutant phenotype were scored as mutants.

Table 1. Crosses to generate bz1 and sh1 mutants.
 
  female parent   male parent
B-Peru Mu: Mu, B-P, Bz1 Sh1, r-g X b, bz1 sh1, R-g
  purple, plump   bronze, shrunken
b-Perumu5: b, bz1 sh1, R-g X b-Perumu5/b, Bz1 Sh1, r-g
  bronze, shrunken    colorless with purple sectors, plump
b-Perumu218: b, bz1 sh1, R-g X b-Perumu218/b, Bz1 Sh1, r-g
  bronze, shrunken   colorless with purple sectors, plump

The F1 progeny from each cross should be purple, plump unless a mutation event occurred: stable bz1 mutations would be bronze and plump, unstable bz1 mutations would be bronze with purple sectors and plump, and sh1 mutations would be purple and shrunken.

The b-Perumu5 and b-Perumu218 stocks had to be used as male onto bz1 sh1 ears because of the R alleles they contained. The bz1 sh1 tester stocks contained the R-g allele and the b-Perumu5 and b-Perumu218 stocks contained the colorless r-g allele. If R-g pollen is crossed onto r-g ears, the resulting kernels would be mottled due to incomplete expression of R-g, which would have obscured the mutant phenotype we were searching for. However, if r-g pollen was crossed onto R-g ears, solid purple kernels would be obtained which could be scored for exceptional bronze kernels representing insertions into the bz1 gene. Self contaminants were recognized as bronze, shrunken kernels and discarded.

Table 2 lists the total number of kernels scored, the number of mutants isolated, and the frequency at which the mutants were recovered. All of the mutants were due to independent events. Despite the large difference in the Mu1 copy number in the three Mutator stocks used, the frequency at which mutations were recovered at a single gene (bz1) was not significantly different in the three stocks.

Table 2. Number of mutants isolated.
 
   
Total Scored
Mutants
 
Mu Source Mu1 copy # #ears #kernels bz1 sh1 Mutation Frequency
B-Peru Mu 30-50 587 76,313 5 8 Bz1: 6.6 x 10-5
            Sh1: 1.0 x 10-4
b-Perumu5 1-4 535 137,044 8 - Bz1: 5.8 x 10-5
b-Perumu218 1-4 646 215,355 7 1 Bz1: 3.3 x 10-5

Several of the Mu-induced bz1 and sh1 mutants recovered have been molecularly analyzed. Table 3 lists the Mutator source and apparent sizes of the inserts found in the Bz1 and Sh1 genes as deduced from Southern blots. The results show that, although similar Mutator stocks were used to generate the bz1 and sh1 mutants, very different types of elements, based on size, inserted into the two genes.

Table 3:. Molecular results of insertion sizes of mutant.
 
Mutator Source Size of insert in Bz1 Size of Insert in Sh1
B-Peru 2 mutants: 1.2-1.4kb 4 mutants: ~1.4kb 
    ~4.0kb
    ~5.0kb
    undetermined
b-Perumu5 5 mutants: 800bp none
b-Perumu218 1 mutant: 800bp 1 mutant: ~3.0kb

One of the bz1 mutants derived from the b-Perumu5 Mutator source has been cloned. The insert is ~800bp and hybridizes to both Mu1 terminal and Mu1 internal probes. The Mu1 hybridization, the insert size, and restriction pattern suggest that the element is a deletion derivative of Mu1.

Numerous insertions into bz1 have been isolated from Mutator stocks and molecularly characterized in other laboratories. Interestingly, almost all of the inserts have been Mu1, or the Mu1-related element Mu1.7 (W.E. Brown, D.S. Robertson and J.L. Bennetzen, personal communication; L.P. Taylor, V.L.Chandler, V. Walbot, Maydica 31, 1986). The only exception we are aware of is the bz1-rcy allele which contains a Mu related element referred to as r-cy:Mu7 that only shares the 220bp Mu termini with Mu1 (P. Schnable, personal communication). The insertion in the bz1 mutant that we have cloned appears to be a deletion derivative of Mu1. The remaining insertions in the bz1 mutants that we have molecularly analyzed also appear to be either Mu1 or the Mu1-deletion described above. The Mutator stocks which were used to generate these mutants resulted in sh1 mutants with insertions that, based on size, hybridization, and restriction mapping, appear to be different from Mu1. This finding, as well as the result from other labs of almost every Mu-induced bz1 mutant being due to the insertion of a Mu1 or Mu1.7 element, suggests that the bz1 gene may contain a hotspot for Mu1 insertion.

Further molecular studies of the Mu-induced bz1 and sh1 mutants are in progress. We are particularly interested in determining if the elements inserted into these alleles contain Mu termini, and if so, if they represent previously uncharacterized Mu elements. Alternatively, the insertions may represent other families of transposable elements.


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