40. Screening of Mutator-related sequence in rice

Ryuji ISHIKAWA^1,2, Damon LISCH² and Michael FREELING²

1) Faculty of Agriculture, Hirosaki University, Hirosaki, 036 Japan

2) Dept. of Plant Biology, University of California, Berkeley, CA 94720, USA

Mutator is one of transposable elements in maize. Standard Mutator strains of maize having 100 copies of this element show a mutation rate 30 to 50 times higher than that of the background. The Mutator family carries unique characteristics, i.e., terminal inverted sequences are quite long consisting of about 220 bp and conserved, but the internal regions are different between families with some exceptions. Recently, a regulatory element for the system, called MuDR, was cloned (Chomet et al. 1991). Blot hybridization using an internal fragment of MuDR as a probe revealed that sequence homology exists between several monocot plants including rice. Based on this observation, we attempted to clone Mutator-related sequences from the rice genome.

Three different probes given in Table 1 were used to screen rice genomic libraries derived from IR36. Of them, probe EndMu includes the entire part of one terminal inverted element. Probe Mu* or Rice* (homologous each other, explained below) has cartain internal sequences commonly found among most monocot plants (Lisch unpubl.data). In the case of maize Mutator, two major species of transcripts have been identified, a large transcript of 2.8 kb and a small one of 0.9 kb. Probe H-H corresponds to the large transcript. All of them hybridized to homologous sequences in the rice genome under a low stringency condition. In addition, other kinds of probes were prepared by PCR amplification to screen Mu-related sequences from rice. Primers used for PCR were designed so as to include the 3' terminal sequences of each two transcripts in MuDR. The primer pair enabled to amplify the internal region in MuDR, named Mu* (Chomet et al. 1991). Using DNA from two rice cultivars, Taichung 65 and IR36, as templates, a DNA fragment of about 440 bp was amplified, which was named Rice*. Although the size of Rice* fragment was only about half of that (Mu*) amplified from maize, the Rice* probe hybridized to a part of MuDR. The size difference is not unexpected, since the Mu* region is highly mutable in maize.

From the rice genomic library, 4 clones were selected based on their hybridization survey to EndMu probe, among which E5, H1 and *5 clones hybridized to Mu*, but H-H probe hybridized only H1 clone (Table 1). The most promising of these, the H1 clone, also hybridized to the Rice* probe. Taken together, we tentatively conclude that the H1 clone contains the Mutator homologue sequence of rice. We are now in the process of sub-cloning and sequencing this clone.

Table 1.  Characterization of clones
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                                                   Probe
Clone      Size (kb)          _________________________________________________
                                   EndMu           Mu*            H-H
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E4            13                     +              -              -
E5            14                     +             ++              -
H1            14                     +              +              ++
*5            18                     +             ++              -
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+ and ++ show positive signals for respective probes.

References

Chomet, P., D. Lisch, K. J. Hardeman, V. L. Chandler and M. Freeling, 1991. Identification of a regulatory transposon that controls the Mutator transposable element system in maize. Genetics 129: 261-270.