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.     

Is the Suppressor-Mutator Element Controlled by a Basic Developmental Regulatory Mechanism?

We report the results of genetic studies on derivatives of two different alleles of the maize a locus with an insertion of the Suppressor-mutator (Spm) transposable element in which the element is inactive, but can be reactivated readily. We present evidence that the mechanism that determines whether the element is in an active or inactive phase has two genetically distinguishable components. One determines whether or not the element is genetically active (the phase setting) and the other determines the stability of the setting in development, its heritability, and its phase in the next generation (the phase program). We show that the element's phase can be reset in a reproducible pattern during plant development. We also show that the Spm element can be reprogrammed to undergo a subsequent phase change without a concomitant phase change. The capacity to reset and reprogram the Spm element is differentially expressed within the plant in a pattern that is correlated with the developmental fate of apical and lateral meristems, suggesting the involvement of a basic developmental determination mechanism.     

Genetic and molecular analysis of the Spm-dependent a-m2 alleles of the maize a locus

The Suppressor-mutator (Spm) transposable element family of maize consists of the fully functional standard Spm (Spm-s) and many mutant elements. Insertion of an Spm element in or near a gene can markedly alter its expression, in some cases bringing the gene under the control of the mechanisms that regulate expression of the element. To gain insight into such mechanisms, as well as to enlarge our understanding of the Spm element's genetic organization, we have analyzed derivatives of a unique Spm insertion at the maize a locus in which the gene is co-expressed and co-regulated with the element. We describe the genetic properties and the structure of the a locus and Spm element in 9 strains (collectively designated the a-m2 alleles) selected by McClintock from the original a-m2 allele for heritable changes affecting either the Spm element or expression of the a gene. Most of the mutations are intra-element deletions within the 8.3-kb Spm element; many alter both Spm function and expression of the gene. Spm controls a gene expression in alleles with internally deleted, transposition-defective Spm elements and element ends contain the target sequences that mediate Spm's ability to activate expression of the gene. We argue that the properties of the a-m2 alleles reflect the operation of an element-encoded positive regulatory mechanism, as well as a negative regulatory mechanism that affects expression of the element, but appears not to be mediated by an element-encoded gene product.     

Reactivation of a silenced minimal Mutator transposable element system following low-energy nitrogen ion implantation in maize

In maize, Mutator transposable elements are either active or silenced within the genome. In response to environmental stress, silenced Mutator elements could be reactivated, leading to changes in genome structure and gene function. However, there is no direct experimental evidence linking environmental stress and Mutator transposon reactivation. Using a maize line that contains a single inactive MuDR and a lone nonautonomous Mutator element, a Mu1 insertion in the recessive reporter allele a1-mum2 in an inactive Mutator background, we directly assessed Mutator reactivation following low-energy nitrogen ion implantation. We observed that N⁺ implantation decreased cytosine methylation in MuDR terminal inverted repeats and increased expression of mudrA and mudrB. Both changes were associated with increased transpositional activity of MuDR through reactivation of the inactive minimal Mutator transposable element system. This study provides direct evidence linking environmental stress agents and Mutator transposon mobilization in maize. In addition, the observed changes to DNA methylation suggest a new mechanism for mutations by low-energy ion implantation.     

The mop1 (mediator of paramutation1) mutant progressively reactivates one of the two genes encoded by the MuDR transposon in maize

Transposons make up a sizable portion of most genomes, and most organisms have evolved mechanisms to silence them. In maize, silencing of the Mutator family of transposons is associated with methylation of the terminal inverted repeats (TIRs) surrounding the autonomous element and loss of mudrA expression (the transposase) as well as mudrB (a gene involved in insertional activity). We have previously reported that a mutation that suppresses paramutation in maize, mop1, also hypomethylates Mu1 elements and restores somatic activity to silenced MuDR elements. Here, we describe the progressive reactivation of silenced mudrA after several generations in a mop1 background. In mop1 mutants, the TIRA becomes hypomethylated immediately, but mudrA expression and significant somatic reactivation is not observed until silenced MuDR has been exposed to mop1 for several generations. In subsequent generations, individuals that are heterozygous or wild type for the Mop1 allele continue to exhibit hypomethylation at Mu1 and mudrA TIRs as well as somatic activity and high levels of mudrA expression. Thus, mudrA silencing can be progressively and heritably reversed. Conversely, mudrB expression is never restored, its TIR remains methylated, and new insertions of Mu elements are not observed. These data suggest that mudrA and mudrB silencing may be maintained via distinct mechanisms.     

Spotting factor (Spf) from the Spotted-dilute system in maize is a member of the En/Spm controlling element family

The Spotted-dilute controlling element system in maize involves an autonomous Spotting factor (Spf), and a receptor at the r1 locus haplotype R1-r(spotted dilute2). Its relationship with other maize transposable element systems is poorly characterized. Through development of a genetic tester that carries receptors for both the Spotted-dilute and the En/Spm controlling element systems, we determined that both receptors respond equally to Spf and En/Spm and that Spf is therefore a member of the En/Spm family of controlling elements.     

Inheritance of Mutator Activity in ZEA MAYS as Assayed by Somatic Instability of the bz2-mu1 Allele

Mutator lines of maize were originally defined by their high forward mutation rate, now known to be caused by the transposition of numerous Mu elements. A high frequency of somatic instability, seen as a fine purple spotting pattern on the aleurone tissue, is characteristic of Mu-induced mutable alleles of genes of the anthocyanin pathway. Loss of such somatic instability has been correlated with the de novo, specific modification of Mu element DNA. In this report the presence or loss of somatic instability at the bz2-mu1 allele has been monitored to investigate the inheritance of the Mutator phenomenon. The active state is labile and may become weakly active (low fraction of spotted kernel progeny) or totally inactive (no spotted kernel progeny) during either outcrossing to non-Mutator lines or on self-pollination. In contrast, the inactive state is relatively permanent with rare reactivation in subsequent crosses to non-Mutator lines. Cryptic bz2-mu1 alleles in weakly active lines can be efficiently reactivated to somatic instability when crossed with an active line. However, in reciprocal crosses of active and totally inactive individuals, strong maternal effects were observed on the inactivation of a somatically unstable bz2-mu1 allele and on the reactivation of cryptic bz2-mu1 alleles. In general, the activity state of the female parent determines the mutability of the progeny.     

A maize cryptic Ac-homologous sequence derived from an Activator transposable element does not transpose.

Summary Sequences sharing homology to the transposable element Activator (Ac) are prevalent in the maize genome. A cryptic Ac-like DNA, cAc-11, was isolated from the maize inbred line 4Co63 and sequenced. Cryptic Ac-11 has over 90% homology to known Ac sequences and contains an 11 by inverted terminal repeat flanked by an 8 by target site duplication, which are characteristics of Ac and Dissociation (Ds) transposable elements. Unlike the active Ac element, which encodes a transposase, the corresponding sequence in cAc-11 has no significant open reading frame. A 44 by tandem repeat was found at one end of cAc-11, which might be a result of aberrant transposition. The sequence data suggest that cAc-11 may represent a remnant of an Ac or a Ds element. Sequences homologous to cAc-11 can be detected in many maize inbred lines. In contrast to canonical Ac elements, cAc-11 DNA in the maize genome is hypermethylated and does not transpose even in the presence of an active Ac element.     

The wp mutation of Glycine max carries a gene-fragment-rich transposon of the CACTA superfamily

We used soybean (Glycine max) cDNA microarrays to identify candidate genes for a stable mutation at the Wp locus in soybean, which changed a purple-flowered phenotype to pink, and found that flavanone 3-hydroxylase cDNAs were overexpressed in purple flower buds relative to the pink. Restriction fragment length polymorphism analysis and RNA gel blots of purple and pink flower isolines, as well as the presence of a 5.7-kb transposon insertion in the wp mutant allele, have unequivocally shown that flavanone 3-hydroxylase gene 1 is the Wp locus. Moreover, the 5.7-kb insertion in wp represents a novel transposable element (termed Tgm-Express1) with inverted repeats closely related to those of other Tgms (transposable-like elements, G. max) but distinct in several characteristics, including the lack of subterminal inverted repeats. More significantly, Tgm-Express1 contains four truncated cellular genes from the soybean genome, resembling the Pack-MULEs (Mutator-like transposable elements) found in maize (Zea mays), rice (Oryza sativa), and Arabidopsis thaliana and the Helitrons of maize. The presence of the Tgm-Express1 element causing the wp mutation, as well as a second Tgm-Express2 element elsewhere in the soybean genome, extends the ability to acquire and transport host DNA segments to the CACTA family of elements, which includes both Tgm and the prototypical maize Spm/En.     

Molecular and Genetic Characterization of Mu Transposable Elements in Zea Mays: Behavior in Callus Culture and Regenerated Plants

Active Mutator lines of maize (Zea mays L.) have a high mutation rate and contain multiple hypomethylated 1.4-kb and 1.7-kb Mu transposable elements. Correlated with the inactivation of the Mutator system, these Mu elements cease to transpose and become more methylated. To determine whether the shock of tissue culture can affect Mutator activities, F1 progenies of outcrosses between active or inactive Mutator stocks and inbred line A188 were used to initiate embryogenic callus cultures. HinfI restriction digestion of genomic DNA isolated from 3-5-month-old cultures demonstrated that there is a very good correlation between the modification state of Mu elements in the cultures and the Mutator parent. Despite the dedifferentiation and rapid proliferation characteristic of tissue culture, the Mutator activity state is relatively stable during an extended tissue culture period. Cultures established from inactive Mutator lines were not reactivated; cultures established from active lines maintained a high Mu copy number, and most Mu elements remained unmodified. In contrast, weakly active Mutator parents gave rise to cultures in which Mu element modification could switch between low and high methylation during the culture period. Evidence for transposition was investigated with EcoRI digestion of genomic DNA isolated at different times during culture. The appearance of novel Mu-hybridizing fragments and a strong background hybridization are interpreted as evidence that transposition events occur during culture. Plants regenerated from such active cultures transmitted Mutator activity to their progeny.     

Amplification of genomic sequences flanking transposable elements in host and heterologous plants: a tool for transposon tagging and genome characterization.

The isolation of sequences flanking integrated transposable elements is an important step in gene tagging strategies. We have demonstrated that sequences flanking transposons integrated into complex genomes can be simply and rapidly obtained using the polymerase chain reaction. Amplification of such sequences was established in a model system, a transgenic tobacco plant carrying a single Ac element, and successfully applied to the cloning of a specific Spm element from a maize line carrying multiple Spm hybridizing sequences. The described utilization of methylation sensitive restriction enzymes (including those with degenerate recognition sequences) in the generation of templates for amplification will simplify the cloning and mapping of genomic sequences adjacent to transposable elements.