We have been using the transposable element Robertson's Mutator as an insertional mutagen and "transposon tag" to clone the B locus. The B-Peru allele was used as the target for our Mutator experiments since it causes pigment expression in the kernel. Four mutations that have a variegated phenotype have been isolated from Mutator stocks and their phenotypes and segregation properties are described in a separate report (V. Chandler, S. Belcher and D. Turks, this issue).
Our cloning efforts were initially focused on the b-Perumu5 allele because it had a reversion pattern more characteristic of Mu1-induced mutations: small somatic revertant sectors and infrequent germline revertants. Our strategy has been to identify the particular Mu element linked to b-Perumu5 using Southern blot analysis of DNA from plants segregating the mutant phenotype. We first used an internal Mu1 probe that will recognize both Mu1 and Mu1.7 elements, because insertions of these elements are responsible for most of the molecularly characterized Mutator-induced mutations. There are only 2 restriction fragments homologous to this internal Mu1 probe in the non-Mutator b tester stock. We determined that there were 10 to 20 Mu1-like elements and 10 to 20 Mu1.7-like elements in our b-Perumu5 plants. We screened for the plants containing the lowest number of Mu elements and outcrossed these to a non-Mutator b r-g tester (W23/K55). After several generations of outcrosses, the total number of Mu1 and Mu1.7 elements had been diluted to 4 to 10 copies. In these lineages, it was possible to unambiguously identify the only Mu1-homologous restriction fragment that segregated with the b-Perumu5 allele.
Using standard protocols, the Mu1-homologous restriction fragment that segregated with the b-Perumu5 phenotype was isolated from a total genomic library. An 850bp Hinfl restriction fragment adjacent to the Mu1 element was subcloned and used as a hybridization probe to examine the progenitor B-Peru stock and other b-Perumu5 plants. This sequence recognized a single 5.4kbp BglII fragment in the progenitor B-Peru stock and a 6.8kbp BglII fragment in the B-Perumu5 stock, suggesting that a 1.4kbp Mu1 element had inserted into this region of the genome.
A combined molecular and genetic analysis was then done to determine if the sequences adjacent to the cloned Mu1 element represented the B-Peru gene. RFLP mapping placed this sequence to within 4 map units of the B locus on the short arm of chromosome 2 (T. Helentjaris, personal communication). This confirmed our previous segregation analysis that suggested the cloned Mu1 element was tightly linked to the B locus. If the Mu1 element that inserted into this chromosomal region is responsible for the b-Perumu5 phenotype, there should be a 100% correlation between the presence of the 6.8kbp BglII fragment (Mu1 element insertion) and the spotted phenotype. We used Southern blots to examine DNA from 38 individuals that derived from the original b-Perumu5/b kernel. Thirty-five individuals fit the above criteria, but 3 exceptions that transmitted the spotted phenotype and no longer contained the Mu1 element at this site were identified. Other RFLP markers were then used to demonstrate that these exceptional individuals had undergone recombination events separating the Mu1 element insertion from the B-Peru locus. This demonstrated that the Mu1 element we cloned was not responsible for the b-Perumu5 phenotype.
These data suggested that we had cloned a sequence that maps close to B, but is not part of the B locus. Our data further demonstrated that neither a Mu1.7 nor a Mu1 element was responsible for the b-Perumu5 phenotype, as we had cloned and characterized the only restriction fragment homologous to Mu1 that mapped to chromosome 2S. This was initially surprising as most of the molecularly characterized Mutator-induced mutations have been caused by the insertion of a Mu1 element. However, during the past two years, several exceptions have been reported in addition to our b-Perumu5 allele.
Using probes specific for all characterized Mu elements we have investigated whether any of these elements were inserted into the b locus, generating the b-Perumu5 allele. Karen Oishi and Mike Freeling provided a probe specific for Mu3, the element that inserted into Adh1, and Sue Wessler provided a probe specific for the Mu element that inserted into the wx-mum5 mutation isolated by Don Robertson. We have also used probes specific for 4 other Mu elements, Mu4, Mu5, Mu6, and Mu7, that were cloned from maize stocks by cross-hybridization with Mu terminal probes (Talbert, Kelly, and Chandler, this issue). All 6 of these elements are homologous to Mu1 termini, but share no internal homology with each other or with Mu1 and Mu1.7. Each probe recognized 2 to 8 unique restriction fragments, but none of these fragments segregated with the b-Perumu5 allele.
A similar analysis has been performed with plants segregating the b-Perumu216 and b-Perumu218 alleles. We have not identified any restriction fragments homologous to any cloned Mu element cosegregating with these two mutant alleles. After 4 successive outcrosses, the b-Perumu220 allele still contains numerous Mu1 and Mu1.7 elements (40-80), so it has not been possible to determine if a Mu1 -homologous fragment is cosegregating with this mutant allele. However, none of the restriction fragments homologous to the other 6 elements segregate with the b-Perumu220 allele.
Our results demonstrate that 3 unstable mutations isolated from Mutator stocks are not caused by previously characterized Mu elements. All maize lines contain additional copies of Mu termini that are not part of characterized Mu elements, so it is likely that additional Mu elements remain to be discovered. One or more of these may be responsible for the B-Peru alleles we have isolated. Alternatively, another type of element unrelated to Mu elements by sequence similarity may be responsible for these mutations. Genetic tests and Southern blots suggest an Ac element is not responsible for the B-Peru alleles. Experiments are in progress to test for Spm activity.
We are trying to "trap" the element(s) transposing in these stocks by screening for mutations in genes for which we have probes available. We have crossed b-Perumu5 and b-Perumu218 plants (that contain only a few Mu1 and Mu1.7 elements) to testers for bz1, sh1, and a1. Multiple, independent insertions into each gene have been isolated at frequencies ranging from 5 X 10-3 to 10-5. This winter the newly isolated mutations will be characterized to determine what kind of insertion element they contain. If new elements are found they will be cloned, and we will determine whether any of these elements are responsible for the B-Peru mutations. If so, we will use these elements as probes to clone the B-Peru gene.
Vicki L. Chandler and Devon Turks
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