We recently succeeded in cloning the viviparous-1 locus by transposable element tagging using the Robertson's Mutator-induced vp-mum1 allele isolated by Philip Stinard and Donald Robertson at Iowa State. In the course of confirming our clone, pVPM1B (Figure 1), we have done some preliminary molecular analysis of several vp mutant alleles. Figure 2 shows a Southern blot of total DNA restriction digests from normal and mutant plants of each stock probed with sequences flanking the Mu insertion in pVPM1B (see Fig. 1). In every case we detect differences between the mutant and wildtype alleles. In all cases, the wildtype alleles segregating in these stocks resemble those found in W22. At least two of these mutants, vp-w1 and vp-w2, arose spontaneously in W22 inbred stocks. In addition, further restriction analysis (not shown) indicated the W22 allele as the probable progenitor of vp-muml and vp-w3. Therefore, in these cases direct comparisons can be made. The rearrangements we see in these stocks, particularly the deletion in vp-w2, are convincing evidence that we have cloned vp. We can summarize the data as follows:
vp-mum2: This mutable allele should contain a Mutator insertion at vp. The normal plant in this case is a heterozygote. The apparent incomplete digestion of the mutant DNA by BamHI is possibly due to DNA methylation associated with Mu element inactivation. (In other mum2 plants we see complete digestion.) Note that we see different size shifts with EcoRI and BamHI. Based on these and other polymorphisms detected by the righthand half of our clone we conclude that the wildtype (W22-like) allele segregating in this stock is not the progenitor of vp-mum2. However, using these polymorphisms we have identified the probable progenitor allele in the parental Mutator stock (obtained from Donald Robertson). Relative to that allele we see an approximate 1.4kbp BamHI size polymorphism in vp-mum2 consistent with a Mu1-size insertion.
vp-R: This is the standard mutant allele described by Robertson (Genetics 40:745). Although the progenitor is unknown, this mutant is clearly different from the wildtype W22 allele.
vp-w3: This apparently stable mutant arose in one of O. Nelson's Ac Ds stocks. In digests with at least 4 enzymes including the 2 shown we see a consistent 2kbp size shift in the mutant restriction fragment. Analysis of the parental stock (a gift of O. Nelson) revealed only the W22 wildtype allele. We conclude that this allele contains a 2kbp insertion, possibly a Ds element. We have confirmed that this stock contains active Ac by crossing it to a sh2-m (no Ac) stock. However, we see no evidence of vp mutability
vp-w1: This mutant arose spontaneously in a homogeneous W22 background. One parent carried R-st, thus it may have been induced by the I-R transposon of R-st J.L. Kermicle, personal communication). Interestingly, the rearrangement in this allele is detected in digests with EcoRI and SstI (not shown), which cut to the right of the Mu insertion in pVPM1B (Fig. 1), but not with BamHI (The normal plant is heterozygous). Therefore, the lesion must occur beyond the left border of pVPM1B, and the vp gene must extend beyond our clone. The data are consistent with either a large insertion containing several of the relevant restriction sites or a large deletion (>10kbp).
vp-w2: This allele also arose spontaneously in an R-st W22 stock. In this mutant the entire probe sequence has been deleted (at least 1kbp). We have confirmed this in digests with 4 different enzymes. However, sequences to the right of the 0.5kpb PstI fragment in pVPM1B are detected in vp-w2 DNA (not shown), therefore one breakpoint of the deletion occurs within the cloned region, roughly 2kbp from the site of the Mu1 insertion in vp-mum1.
Figure 3 summarizes our conclusions from the Southern data. Curiously, all of the mutants for which we have firm data map toward the left end of pVPM1B. Nonetheless, probes from the right end hybridize to the largest vp transcript.
We are indebted to Hugo Dooner for sharing his extensive collection of up mutants and to Stinard and Robertson for their vp-mum1 and vp-mum2 stocks.
Figure 1. Restriction map of pVPM1B. Bs - BstEII; Hf = HinfI; P = PstI; S = SstI; Bg = BglII; R = EcoRI.
Figure 2. Southern blot analysis of vp mutant stocks.
Figure 3. A physical map of vp. This schematic shows the approximate positions of 5 vp mutations relative to our clone. The drawing corresponds to the wildtype allele found in W22, which is apparently the progenitor of all the mutants shown with the exception of vp-mum2. B=BamHI; Bg - BglII; R - EcoRI.
Donald R. McCarty, Christian B. Carson and S. Caprice Simonds
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