We have analyzed the structure of the shrunken (Sh) locus, encoding endosperm sucrose synthase, in a strain containing an unstable recessive mutation (sh-m5933) caused by Ds. In addition, we have analyzed the structure of the Sh locus in nine spontaneous Sh revertants of sh-m5933.
The sh-m5933 allele contains a ca. 30 Kb insertion in the Sh locus, of which more than 15 kb are present in our genomic clone. That part of the 30 kb inserted DNA not contained in our clone was analyzed by restriction mapping using the blotting technique and probes derived from the 5'-end of the sucrose synthase gene. The restriction pattern of the first 7 kb of uncloned insertion DNA is similar to that of the cloned part of the insertion. The patterns can be explained by the following: (a) the 30 kb insertion terminates in long inverted repeats and (b) the first 1 kb segment adjacent to the 3'-end of the sucrose synthase gene is missing from the same position of the DNA adjacent to the 5'-end of the sucrose synthase gene. It will be shown below (see next report) that most of the DNA inserted into the sucrose synthase gene is not the element Ds.
In addition to the long insertion in the sucrose synthase gene, another aberration is present in sh-m5933. Part of the 30 kb insert DNA, including the uncloned junction with the 5'-end of the sucrose synthase gene and a large segment of DNA adjacent to it, are duplicated in sh-m5933. This duplication must be located on the same chromosome, as it does not segregate from the mutation upon crossing.
In nine spontaneous sh revertants, the 30 kb insertion within the sucrose synthase gene is excised and the structure of the wild type sucrose synthase gene is restored. The large duplicated segment that includes part of the insertion and part of the sucrose synthase gene is, however, retained. In one of the nine revertants, an alteration in the duplication has occurred. The data show that a 2 kb segment of insertion DNA near the junction with the sucrose synthase gene has been deleted.
The retention of the long duplication in the revertants explains the observation that all of the revertants show phenotypes associated with the presence of Ds in the vicinity of the sucrose synthase gene. Only in one of the revertants is a different pattern obtained. This is the same revertant that has suffered a deletion within the duplicated DNA segment at the junction of the 5'-part of the sucrose synthase gene and part of the insertion. The correlation between an altered pattern of chromosome breakage and the loss of DNA adjacent to the junction is an indication that this DNA is a part of Ds responsible for the chromosome breakage pattern. This work is detailed in Courage-Tebbe et al., Cell 34:383, 1983.
U. Courage-Tebbe, H. P. Doring, N. Fedoroff,* and P. Starlinger
*Carnegie Institution of Washington, Baltimore, MD
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