Cloning of the Bz2 locus of Zea mays using the transposable element Ds as a gene tag

Summary The Bz2 locus of Zea mays has been cloned, utilizing the presence of the transposable element Dissociation (Ds) at the locus as a gene tag. The Ds element inserted in the bz2-m allele was identified among many members of the Ac/Ds family in a Southern blot analysis of a population segregating for bz2-m and Bz2. After cloning a DNA fragment from the bz2-m allele, sequences flanking the Ds insertion were shown to be Bz2-specific and were used to isolate a homologous fragment from a wild-type Bz2 line. The Ds insertion in the bz2-m allele was found to be a Ds2 element identical to the Ds insertion in adh1-2F11.     

Genetic Fine Structure of the BRONZE Locus in Maize

The bronze (bz) locus in maize, located in the short arm of chromosome 9 (9S), is the structural gene for the anthocyanin biosynthetic enzyme UFGT. The gene has been cloned and its physical map has been oriented relative to the centromere of 9S. We report here the genetic fine structure mapping of several biochemically characterized EMS-induced bz-E mutations, derived from the Bz-W22 isoallele, and Ds insertion bz-m mutations, derived from the Bz-McC isoallele. Two UFGT(-), CRM(+ ) mutants (bz-E2 and bz-E5), which genetically identify coding sequences in the gene, and three UFGT(-), CRM(- )bz-E mutants were mapped against the Ds insertion mutants bz-m1 and bz-m2(DI) by selecting Bz intragenic recombinants from heterozygotes of the type bz-E/bz-m . The exclusive occurrence of one recombinant outside marker class allowed the unambiguous placement of the mutants in a genetic fine structure map. Peculiarly, the two CRM(+)bz-E mutants lie upstream of the three CRM(-)bz-E mutants and at a considerable genetic distance. The UFGT allozymes encoded by the progenitor alleles Bz-W22 and Bz-McC differ in two properties, thermal stability and activity. The sites responsible for these properties were mapped as unselected markers among the Bz intragenic recombinants. The thermal stability site, which also identifies a coding region of the gene, mapped very close to the CRM(+)bz-E mutant sites. The site responsible for variation in activity, which probably identifies a region involved in regulation of expression of the bz locus, mapped at the 5' or proximal end of the locus. It was found to be inseparable from the Ds insertion in bz-m1 that lies very close to the 5' end of the transcribed region.-Evidence was obtained that the insertion of Ds within the bz gene has a suppressing effect on intragenic recombination. Additional data are also presented supporting our observation that Ds affects the pattern of intragenic recombination at bz.-Based on the total genetic length of the bz gene and on the physical size of the transcribed region, we estimate that one unit of recombination at bronze corresponds to 14 kb of DNA. This estimate is more than 100 times smaller than the average value for the whole genome and implies that there may be regions, such as bronze, that serve as hotspots for recombination.     

Molecular analysis of the loss of somatic instability in the bz2::mu1 allele of maize

Summary Multiple genetic and epigenetic changes were detected within one plant generation at the bz2:: mu1 mutable allele in a population of 118 plants. Loss of somatic instability in bz2::mu1 was usually correlated with methylation of the Mu1 transposable element; in 6 plants, somatic instability was lost as a result of mutations in bz2::mu1. This is a surprisingly high frequency of mutation per allele (2.5%) for the Mutator family, for which germinal revertants occur at a frequency of about 10^–4 per gamete. One germinal excision event was found that contained an 8 by deletion, frameshift mutation in Bronze-2. The three other mutants described occurred as a result of abortive transposition, in which 75–77 by deletions were generated at the junction between Bronze-2 and Mu1. We discuss the possible mechanisms, and the role of host factors in abortive transposition in maize.     

Developmental and genetic aspects of Mutator excision in maize

The regulation of excision of Mu elements of the Mutator transposable element family of maize is not well understood. We have used somatic instability of Mu receptor elements from the Bronze 1 and Bronze 2 loci to monitor the frequency and the timing of excision of Mu elements in several tissues. We show that spot size in the aleurone of a bz2::mu1 stock varies between one to approximately 256 cells. This indicates that excision events begin eight divisions prior to full aleurone differentiation and end after the last division of the aleurone. We show that excision is equally biased for late events in all other tissues studied. A locus on chromosome 5 has been identified that affects spot size, possibly by altering the timing of Mu excision. Using somatic excision as an assay of Mutator activity, we found that activity can change in small sectors of the tassel; however, there are no overall activity changes in the tassel during the period of pollen shedding. We also report the recovery of germinal revertants for the bz1::mu1 and bz2::mu1 alleles. One of these revertant alleles was characterized by Southern blot analysis and found to be similar to the progenitor of the mutable allele.     

Recombination occurs uniformly within the bronze gene, a meiotic recombination hotspot in the maize genome.

The bronze (bz) gene is a recombinational hotspot in the maize genome: its level of meiotic recombination per unit of physical length is > 100-fold higher than the genome's average and is the highest of any plant gene analyzed to date. Here, we examine whether recombination is also unevenly distributed within the bz gene. In yeast genes, recombination (conversion) is polarized, being higher at the end of the gene where recombination is presumably initiated. We have analyzed products of meiotic recombination between heteroallelic pairs of bz mutations in both the presence and absence of heterologies and have sequenced the recombination junction in 130 such Bz intragenic recombinants. We have found that in the absence of heterologies, recombination is proportional to physical distance across the bz gene. The simplest interpretation for this lack of polarity is that recombination is initiated randomly within the gene. Insertion mutations affect the frequency and distribution of intragenic recombination events at bz, creating hotspots and coldspots. Single base pair heterologies also affect recombination, with fewer recombination events than expected by chance occurring in regions of the bz gene with a high density of heterologies. We also provide evidence that meiotic recombination in maize is conservative, that is, it does not introduce changes, and that meiotic conversion tracts are continuous and similar in size to those in yeast.     

The frost gene of Neurospora crassa is a homolog of yeast cdc1 and affects hyphal branching via manganese homeostasis

The Neurospora crassa mutant frost has a hyperbranching phenotype that can be corrected by adding Ca(2+), suggesting that characterization of this gene might clarify the mechanism of Ca(2+)-dependent tip growth. The wild-type allele was cloned by sib selection using protoplasts from arthroconidia. RFLP analysis revealed that the cloned DNA fragment mapped to the fr locus. The nucleotide sequence of genomic and cDNA was determined. The deduced amino acid sequence showed homology to the Saccharomyces cerevisiae CDC1 protein, implicated in manganese homeostasis. The fr mutant was sensitive to Mn(2+), and a revertant allele whose product differs by one amino acid was tolerant to Mn(2+). Mn(2+) depletion induced the wild-type strain to hyperbranch, resulting in a morphology similar to that of fr. The fr mutant was also sensitive to calcineurin inhibitors. These results suggest that fr is involved in Mn(2+) homeostasis and point to a role for Mn(2+) in Neurospora branching.     

Reactivation of the Mutator transposable element system following gamma irradiation of seed

Summary The bz2-mu1 allele contains a 1.4 kb Mu element insertion in the open reading frame of the bronze-2 locus. This insertion suppresses gene activity. In an active Mutator line, however, the bz2-mu1 allele shows high somatic instability resulting in numerous purple spots of full gene activity against a beige background in the aleurone tissue of the kernel; restoration of gene activity results from excision of the Mu element. In contrast, in plants with an inactive Mutator system, uniformly bronze kernels are found, and the Mu element at bz2-mu1 is stabilized. Accompanying a loss of somatic instability, this Mu element, as well as the Mu elements elsewhere in the genome, have an increased level of DNA modification. Spontaneous reactivation of somatic instability in inactive Mutator lines rarely occurs; however, reactivation can be induced with gamma irradiation. Reactivated plants regain both the spotted kernel phenotype indicative of element excision from the bz2-mu1 reporter allele and diagnostic restriction sites within the Mu elements indicative of a hypomethylated state. The reactivated plants transmit these characters to their progeny. These data support the hypothesis that genomic shock can elicit cryptic transposable element activities in maize. Possible mechanisms for inactivation and reactivation of the Mutator transposable element system are also discussed.