Sequence, Genomic Distribution and DNA Modification of a Mu1 Element from Non-Mutator Maize Stocks

The increased mutation rate of Mutator stocks of maize has been shown to be the result of transposition of Mu elements. One element, Mu1, is present in 10-60 copies in Mutator stocks and approximately 0-3 copies in non-Mutator stocks. The sequence, structure and genomic distribution of an intact Mu1 element cloned from the non-Mutator inbred line B37 has been determined. The sequence of this element, termed Mu1.4-B37, is identical to Mu1 and it is flanked by 9-bp direct repeats indicative of a target site duplication. Mu1.4-B37 is not in the same genomic location in all stocks, which further suggests that it transposed into its genomic location in B37. We previously reported that in genomic DNA this element is modified such that certain methylation-sensitive restriction enzymes will not cut sites within the element. This is similar to that observed for Mu elements in Mutator stocks that have lost activity. We report herein that the Mu1.4-B37 element loses its modification and becomes accessible to digestion when placed in an active Mutator stock by genetic crosses. This suggests that factors conditioning unmodified elements are dominant in the initial cross between Mutator and non-Mutator stocks. In F2 individuals that have subsequently lost Mutator activity the Mu1.4-B37 element again becomes modified as do most of the Mu elements in the stock. Thus, the modification state of the Mu1.4-B37 element and the other Mu1-like elements correlates with Mutator activity. We hypothesize that factor(s) within an active Mutator stock may inhibit the modification of Mu elements, and that this activity is missing in non-Mutator stocks and may become limiting in certain Mutator stocks resulting in DNA modification.     

Mu transposable elements are structurally diverse and distributed throughout the genusZea

Summary The Robertson's Mutator stock of maize exhibits a high mutation rate due to the transposition of theMu family of transposable elements. All characterizedMu elements contain similar 200-bp terminal inverted repeats, yet the internal sequences of the elements may be completely unrelated. Non-Mutator stocks of maize have a 20–100-fold lower mutation rate relative to Mutator stocks, yet they contain multiple sequences that hybridize to theMu terminal inverted repeats. Most of these sequences do not cohybridize to internal regions of previously clonedMu elements. We have cloned two such sequences from the maize line B37, a non-Mutator inbred line. These sequences, termedMu4 andMu5, have an organization characteristic of transposable elements and possess 200-bpMu terminal inverted repeats that flank internal DNA, which is unrelated to other clonedMu elements.Mu4 andMu5 are both flanked by 9-bp direct repeats as has been observed for otherMu elements. However, we have no direct evidence that they have recently transposed because they have not been found in known genes. Although the internal regions ofMu4 andMu5 are not related by sequence similarity, both elements share an unusual structural feature: the terminal inverted repeats extend more than 100 bp internally fromMu-similar termini. The distribution of these elements in maize lines and related species suggests thatMu elements are an ancient component of the maize genome. Moreover, the structure of theMu termini and the fact thatMu termini are found flanking different internal sequences leads us to speculate thatMu termini once may have been capable of transposing as independent entities.     

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.     

The Mu Transposable Elements of Maize: Evidence for Transposition and Copy Number Regulation during Development

The Mu transposon of maize exists in a highly mutagenic strain called Robertson's Mutator. Plants of this strain contain 10-50 copies of the Mu element, whereas most maize strains and other plants have none. When Mutator plants are crossed to plants of the inbred line 1S2P, which does not have copies of Mu, the progeny plants have approximately the same number of Mu sequences as did their Mutator parent. Approximately one-half of these copies have segregated from their parent and one-half have arisen by transposition and are integrated into new positions in the genome. This maintenance of copy number can be accounted for by an extremely high rate of transposition of the Mu elements (10-15 transpositions per gamete per generation). When Mutator plants are self-pollinated, the progeny double their Mu copy number in the first generation, but maintain a constant number of Mu sequences with subsequent self-pollinations. Transposition of Mu and the events that lead to copy number maintenance occur very late in the development of the germ cells but before fertilization. A larger version of the Mu element transposes but is not necessary for transposition of the Mu sequences. The progeny of crosses with a Mutator plant occasionally lack Mutator activity; these strains retain copies of the Mu element, but these elements no longer transpose.     

Characterization of bz1 mutants isolated from mutator stocks with high and low numbers of Mu1 elements

The high frequency of mutations in Mutator stocks of maize is the result of transposition of Mu elements. Nine different Mu elements that share the 220 bp Mu terminal inverted repeats have been described. Mu1 elements have been found inserted into most of the molecularly characterized mutant alleles isolated from Mutator stocks, and most Mutator stocks contain a high number of Mu1 elements (10-60). However, it is clear that additional Mu elements, which share the Mu1 termini but have unrelated internal sequences, can also transpose in Mutator stocks. We were interested in comparing the mutation frequency and type of elements that inserted into a particular locus when Mutator stocks with differing numbers of Mu1 elements were utilized. Furthermore, previous studies with Mu-induced mutations have demonstrated that the element that inserted most frequently was Mu1. Therefore, to try to obtain Mu elements different from Mu1 we utilized a stock that had a low number (3-6) of Mu1 elements as well as a Mutator stock with a more typical number of Mu1 elements (20-60). Utilizing both stocks, we isolated numerous mutants at one gene, Bronze 1 (Bz1), and compared the type of elements inserted. In this paper we report that both the high and low Mu1 stocks produced bz1 mutants at frequencies characteristic of Mutator stocks, 6.6 and 4.3 x 10(-5), respectively. We describe the isolation of 20 bz1 mutations, and the initial molecular characterization of eight unstable mutations: two from the high Mu1 stock and six from the low Mu1 stock. The six alleles isolated from the low Mu1 stock appear to contain deleted Mu1 elements, and the two alleles isolated from the high Mu1 stock contain elements very similar to Mu1. When the mutants from the low Mu1 stocks were examined, it was found that the Mu1-related elements increased from 3-6 copies to 9-20 copies in one generation. The high number of Mu1-related elements was maintained in subsequent outcrosses. This spontaneous activation and amplification of Mu1-related elements occurred in at least 1% of the low Mu1 plants.     

Two Maize Genes Are Each Targeted Predominantly by Distinct Classes of Mu Elements

The Mutator transposable element system of maize has been used to isolate mutations at many different genes. Six different classes of Mu transposable elements have been identified. An important question is whether particular classes of Mu elements insert into different genes at equivalent frequencies. To begin to address this question, we used a small number of closely related Mutator plants to generate multiple independent mutations at two different genes. The overall mutation frequency was similar for the two genes. We then determined what types of Mu elements inserted into the genes. We found that each of the genes was preferentially targeted by a different class of Mu element, even when the two genes were mutated in the same plant. Possible explanations for these findings are discussed. These results have important implications for cloning Mu-tagged genes as other genes may also be resistant or susceptible to the insertion of particular classes of Mu elements.     

High-throughput linkage analysis of Mutator insertion sites in maize

Insertional mutagenesis is a cornerstone of functional genomics. High-copy transposable element systems such as Mutator ( Mu ) in maize ( Zea mays ) afford the advantage of high forward mutation rates but pose a challenge for identifying the particular element responsible for a given mutation. Several large mutant collections have been generated in Mu -active genetic stocks, but current methods limit the ability to rapidly identify the causal Mu insertions. Here we present a method to rapidly assay Mu insertions that are genetically linked to a mutation of interest. The method combines elements of MuTAIL (thermal asymmetrically interlaced) and amplification of insertion mutagenized sites (AIMS) protocols and is applicable to the analysis of single mutants or to high-throughput analyses of mutant collections. Briefly, genomic DNA is digested with a restriction enzyme and adapters are ligated. Polymerase chain reaction is performed with TAIL cycling parameters, using a fluorescently labeled Mu primer, which results in the preferential amplification and labeling of Mu -containing genomic fragments. Products from a segregating line are analyzed on a capillary sequencer. To recover a fragment of interest, PCR products are cloned and sequenced. Sequences with lengths matching the size of a band that co-segregates with the mutant phenotype represent candidate linked insertion sites, which are then confirmed by PCR. We demonstrate the utility of the method by identifying Mu insertion sites linked to seed-lethal mutations with a preliminary success rate of nearly 50%.     

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.     

Sequence similarity of putative transposases links the maize Mutator autonomous element and a group of bacterial insertion sequences.

The Mutator transposable element system of maize is the most active transposable element system characterized in higher plants. While Mutator has been used to generate and tag thousands of new maize mutants, the mechanism and regulation of its transposition are poorly understood. The Mutator autonomous element, MuDR, encodes two proteins: MURA and MURB. We have detected an amino acid sequence motif shared by MURA and the putative transposases of a group of bacterial insertion sequences. Based on this similarity we believe that MURA is the transposase of the Mutator system. In addition we have detected two rice cDNAs in genbank with extensive similarity to MURA. This sequence similarity suggests that a Mutator-like element is present in rice. We believe that Mutator, a group of bacterial insertion sequences, and an uncharacterized rice transposon represent members of a family of transposable elements.     

Somatic excision of the Mu1 transposable element of maize.

The Mu transposons of the Robertsons's Mutator transposable element system in maize are unusual in many respects, when compared to the other known plant transposon systems. The excision of these elements occurs late in somatic tissues and very rarely in the germ line. Unlike the other plant transposons, there is no experimental evidence directly linking Mu element excision and integration. We have analyzed the excision products generated by a Mu1 transposon inserted into the bronze 1 locus of maize. We find that the excision products or 'footprints' left by the Mu1 element resemble those of the other plant transposable elements, rather than those of the animal transposable element systems. We also find some novel types of footprints resembling recombinational events. We suggest that the Mu1 element can promote intrachromosomal crossovers and conversions near its site of insertion, and that this may be another mechanism by which transposons can accelerate the evolution of genomes.     

Mu1-Related Transposable Elements of Maize Preferentially Insert into Low Copy Number DNA

The Mutator transposable element system of maize was originally identified through its induction of mutations at an exceptionally high frequency and at a wide variety of loci. The Mu1 subfamily of transposable elements within this system are responsible for the majority of Mutator-induced mutations. Mu 1-related elements were isolated from active Mutator plants and their flanking DNA was characterized. Sequence analyses revealed perfect nine base target duplications directly flanking the insert for 13 of the 14 elements studied. Hybridizational studies indicated that Mu1-like elements insert primarily into regions of the maize genome that are of low copy number. This preferential selection of low copy number DNA as targets for Mu element insertion was not directed by any specific secondary structure(s) that could be detected in this study, but the 9-bp target duplications exhibited a discernibly higher than random match with the consensus sequence 5'-G-T-T-G-G/C-A-G-G/A-G-3'.