Molecular characterization of Mutator systems in maize embryogenic callus cultures indicates Mu element activity in vitro

Summary Active Mutator lines of maize (Zea mays L.) are characterized by their ability to generate new mutants at a high rate and by somatic instability at Mutator-induced mutant alleles. Mutagenically active lines with fewer than ten Mu elements per diploid genome have not been observed. Alteration of Mutator activity has been shown to correlate with the state of modification of Hinfl restiction sites that lie within inverted terminal repeats of Mu elements. To determine whether active Mutator systems can be established and maintained in culture, copy number and modification state of Mu elements were investigated in embryogenic callus lines derived from F₁S of crosses of active Mutator stock with the inbred lines A188 and H99. All callus lines studied maintain high Mu-element copy numbers, and more than half show a continued lack of modification at the Mu element Hinfl sites; thus, parameters associated with mutagenic activity in planta are present in some, but not all, callus lines. Mutator activity was then tested directly by restriction fragment analysis of subclonal populations from A188/Mu ² and H99/Mu ² embryonic cultures. Novel Mu-homologous restriction fragments occurred in 38% of the subpopulations which contained unmodified Mu elements, but not in control cultures containing modified, genetically inactive Mu elements. We conclude that Mu elements from active Mutator parents can remain transpositionally active in embryogenic cell culture. Active Mutator cell lines may be useful for the production of mutations in vitro.     

Concomitant regulation of Mu1 transposition and Mutator activity in maize

Summary The mutagenic activity of the maize transposable element system Mutator can be lost by outcrossing to standard, non-Mutator lines or by repetitive intercrossing of genetically diverse Mutator lines. Lines losing Mutator mutagenic activity in either manner retain high copy numbers (10–15 per diploid genome) of the Mutator-associated Mu transposable elements. Frequent transposition of Mu1-related elements is observed only in active Mutator lines, however. The loss of Mutator activity on intercrossing is correlated with an increase in the copy number of Mu1-like elements to 40–50 per diploid genome, implying a self-encoded or self-activated negative regulator of Mu1 transposition. The outcross loss of Mutator activity is only weakly correlated with a low Mu element copy number and may be due to the loss of a positive regulatory factor encoded by a subset of Mu1-like elements. Transposition of Mu elements in active Mutator lines generates multiple new genomic positions for about half the elements each plant generation. The appearance of Mu1-like elements in these new positions is not accompanied by equally high germinal reversion frequencies, suggesting that Mu1 may commonly transpose via a DNA replicative process.     

Somatic Mutator activity expression is dependent on the strength of Cy trans-active signals in maize

Summary Two receptor element alleles (vp-rcy and bz-rcy) that respond to the trans-active element (Cy) controlling Mutator activity were used to analyze the strength of trans-active signals from Cy elements derived from a Mutator active line. Evidence is presented that the Mutator population tested consists mainly of a class of weak Cy elements designated as Cy:Mu. When Cy:Mu element are present in only a few copies, the strength of the combined transposition signal is weak. It is only when these active elements have a high copy number that the overall transposition signal is sufficiently strong enough to elicit a high frequency of transposition events. This study seeks to investigate the nature of the trans-active signal from Cy:Mu elements. The implication of these results for molecular studies is discussed.Journal Paper No. J-13083 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project No. 2381     

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.     

Regulation of Mu element copy number in maize lines with an active or inactive Mutator transposable element system

Summary In the progeny of an active Mutator plant, the number of Mu elements increases on self-pollination and maintains the average parental Mu content on outcrossing to a non-Mutator line; both patterns of transmission require an increase in the absolute number of Mu elements from one generation to the next. The same average copy number of Mu elements is transmitted through the male and female, but there is wide variation in the absolute copy number among the progeny. In inactive Mutator plants —defined both by the loss of somatic instability at a reporter gene (bronze2-mu1) and by modification of the HinfI sites in the terminal inverted repeat sequences of Mu elements —the absolute copy number of Mu elements is fixed in the parent. Thus, in outcrosses Mu element number is halved, and on self-pollination Mu copy number is constant. Reactivation of somatic mutability at cryptic bz2-mu1 alleles in inactive individuals by crossing to an active line seems not to involve an increase in Mu element copy number transmitted by the inactive individual. These and other results suggest that increases in Mu copy number occur late in plant development or in the gametophyte rather than after fertilization.     

Outcross-dependent transpositions of copia-like mobile genetic elements in chromosomes of an inbred Drosophila melanogaster stock

Summary Transpositions of copia-like mobile genetic elements (MDG1, MDG3 and copia) were studied in crosses of the inbred maladaptive LA line with other laboratory lines made in order to replace specific chromosome pairs in the LA line. Individuals with various hybrid genotypes displayed changed chromosomal patterns of mobile elements compared with the parent LA chromosomes. Variability of the chromosomal molecular structure in hybrids was observed when crossing over was suppressed in the process of hybrid genome constructions. Multiple transposition events were detected in hybrid genomes carrying the second chromosomal pair of the LA line, but not if it was replaced by the second chromosome of the Swedish-b stock. No transpositions were detected in control crosses that did not involve the LA line. Outcross-dependent MDG1 transposition hot spots in the LA second chromosome were found to coincide with previously established hot spots for spontaneous transpositions in the LA line coupled with a fitness increase. The data obtained demonstrate that crosses involving inversions suppressing crossing over cannot guarantee that the chromosomal molecular content will remain the same: it can change as a result of mobile element trans-positions.     

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.     

Genetic studies on the loss of mu mutator activity in maize

Mutator activity of the Mu mutator system of maize can be lost by either outcrossing or inbreeding Mu stocks. The nature of these two kinds of Mu-loss phenomena was analyzed by testing the results of crossing Mu-loss stocks by active Mu lines. Outcross- Mu-loss stocks are capable of supporting Mu activity if crossed by an active mutator line. Inbred-Mu-loss stocks, however, inactivate the active Mu system contributed by a Mu line. Also, inbred- Mu-loss lines do not regain Mu activity after at least three generations of outcrossing to non-Mu stocks. These results suggest that, once the Mu system is inactivated by inbreeding, it remains inactivated for at least three generations of outcrossing. Further, once the system responsible for inactivation is established, it will, in turn, inactivate an active Mu system contributed by crossing with Mu plants. The outcross-Mu-loss does not seem to involve such an inactivation system. These results are interpreted in the light of recent evidence that Mu inactivation results from the modification of Mu 1 transposable elements involved in the Mu phenotype.     

Binding sites for maize nuclear proteins in the terminal inverted repeats of the Mul transposable element

Summary Nuclear protein extracts from Mu-active, Mu-inactive and non-Mutator lines of maize were used to identify the binding sites for maize nuclear proteins in the terminal inverted repeats (TIR) of the Mul transposable element. We found binding activities of nuclear proteins that specifically interact with both TIRs of the Mu1 element. DNase I footprinting was performed to localize the binding sites. We found that the nuclear proteins from Mu-active lines and non-Mu lines bound to the Mu1 TIR at two different sites, i.e. a 13 by sequence (CGGGAACGGTAAA, designated as site I) and another 8 by sequence (CGGCGTCT, designated as site II). However, the nuclear proteins from Mu-inactive lines bound only one of these sites, i.e. site I. Mobility shift assays with synthetic oligonucleotides containing site I and 11 respectively confirmed the specificities of these binding activities. Site I was shown to be an imperfect direct repeat of a hexamer binding site (CGGGAA CGGTAA). Oligonucleotides containing either of the hexamers showed specific binding activity to nuclear protein from both Mu-active and Mu-inactive lines. The possible role of these proteins in Mu transposition is discussed.     

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 use of transgenic plants to understand transposition mechanisms and to develop transposon tagging strategies

This review compares the activity of the plant transposable elements Ac, Tam3, En/Spm and Mu in heterologous plant species and in their original host. Mutational analysis of the autonomous transposable elements and two-element systems have supplied data that revealed some fundamental properties of the transposition mechanism. Functional parts of Ac and En/Spm were detected by in vitro binding studies of purified transposase protein and have been tested for their importance in the function of these transposable elements in heterologous plant species. Experiments that have been carried out to regulate the activity of the Ac transposable element are in progress and preliminary results have been compiled. Perspectives for manipulated transposable elements in transposon tagging strategies within heterologous plant species are discussed.     

Differential activity of the maize mutator Mu at different loci and in different cell lineages

Summary Mutator activity of the maize mutator (Mu) system varies for different loci. Mutation frequencies as high as 7.54x10^–5 and as low as 4.0x10^–6 are observed for 5 loci (i.e., y ₁,yg2, bz1, sh2, and wx). For the waxy locus, a higher mutation frequency is observed in Mu plants crossed as males than when Mu plants function as females. The frequency of unselected mutations also is found to be higher in Mu plants crossed as males than in the first-generation Mu plants crossed as females. The mutation frequency of the y1 locus, however, does not differ in the male or female crosses. Mu-induced mutation frequencies vary with respect to loci and, for some loci, may depend on other factors such as the sex of the Mu parent or the previous crossing history of the Mu parent. More limited data have been obtained for 4 additional loci(su1, c1, c2 and o2).Journal Paper No. J-11487 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2623     

Covalent DNA modification and the regulation of Mutator element transposition in maize

Summary Analyses of the multiple genomic Mu transposable elements in active Mutator lines with several C-methylation sensitive restriction enzymes indicate that Mu elements are undermodified compared with total maize nuclear DNA. Intercrossing of diverse Mutator lines leads to a discrete hypermodification of the Mu elements in a particular plant concurrent with a loss of mutagenic and transpositional potential. The modification events observed appear to be methylation of cytosine at the 5 position in the sequences 5-CG-3 and 5-CNG-3. Some potential C-methylation sites in Mu elements show a higher degree of methylation than others. Once established, the modified Mu state, like the loss of Mutator activity, is stable on outcrossing. Crosses between active Mutator lines with unmodified Mu elements and Mutator-loss lines with modified Mu elements show partial maternal dominance for the modification event. Mutator activity may also be lost thorugh outcrossing in a mechanism not associated with any detected modification events.     

Survey of Mariner-Like Elements in the Housefly, Musca Domestica

The presence of mariner-like elements in four strains of the housefly, Musca domestica, was surveyed by PCR. Using the inverted terminal repeat (ITR) sequences of the Mos 1element as primers, DNAs were successfully amplified from all strains of the housefly. Southern blot analysis indicated that these amplified DNAs were repetitive sequences in the genome of M. domestica. Sequence analyses of cloned PCR products showed that they were 45% identical to the Mos 1element. These fragments appeared to be nonfunctional, because they contained no intact open reading frame (ORF) capable of encoding transposase. We conclude that these DNAs are degraded mariner-like elements (MLEs) in M. domestica. Because these endogenous MLEs in M. domesticado not encode any functional proteins, they probably would not affect the behavior of mariner-based vectors if such were introduced into this species as transformation vectors.     

Dedifferentiation-mediated changes in transposition behavior make the Activator transposon an ideal tool for functional genomics in rice

There is an inverse relationship between the level of cytosine methylation in genomic DNA and the activity of plant transposable elements. Increased transpositional activity is seen during early plant development when genomic methylation patterns are first erased and then reset. Prolonging the period of hypomethylation might therefore result in an increased transposition frequency, which would be useful for rapid genome saturation in transposon-tagged plant lines. We tested this hypothesis using transgenic rice plants containing Activator (Ac) from maize. R₁ seeds from an Ac-tagged transgenic rice line were either directly germinated and grown to maturity, or induced to dedifferentiate in vitro, resulting in cell lines that were subsequently regenerated into multiple mature plants. Both populations were then analyzed for the presence, active reinsertion and amplification of Ac. Plants from each population showed excision-reinsertion events to both linked and unlinked sites. However, the frequency of transposition in plants regenerated from cell lines was more than nine-fold greater than that observed in plants germinated directly from seeds. Other aspects of transposon behavior were also markedly affected. For example, we observed a significantly larger proportion of transposition events to unlinked sites in cell line-derived plants. The tendency for Ac to insert into transcribed DNA was not affected by dedifferentiation. The differences in Ac activity coincided with a pronounced reduction in the level of genomic cytosine methylation in dedifferentiated cell cultures. We used the differential transposon behavior induced by dedifferentiation in the cell-line derived population for direct applications in functional genomics and validated the approach by recovering Ac insertions in a number of genes. Our results demonstrate that obtaining multiple Ac insertions is useful for functional annotation of the rice genome.These authors contributed equally to the work     

Recent amplification of miniature inverted-repeat transposable elements in the vector mosquito Culex pipiens: characterization of the Mimo family

We describe a new family of repetitive elements, named Mimo, from the mosquito Culex pipiens. Structural characteristics of these elements fit well with those of miniature inverted-repeat transposable elements (MITEs), which are ubiquitous and highly abundant in plant genomes. The occurrence of Mimo in C. pipiens provides new evidence that MITEs are not restricted to plant genomes, but may be widespread in arthropods as well. The copy number of Mimo elements in C. pipiens (1000 copies in a 540 Mb genome) supports the hypothesis that there is a positive correlation between genome size and the magnitude of MITE proliferation. In contrast to most MITE families described so far, members of the Mimo family share a high sequence conservation, which may reflect a recent amplification history in this species. In addition, we found that Mimo elements are a frequent nest for other MITE-like elements, suggesting that multiple and successive MITE transposition events have occurred very recently in the C. pipiens genome. Despite evidence for recent mobility of these MITEs, no element has been found to encode a protein; therefore, we do not know how they have transposed and have spread in the genome. However, some sequence similarities in terminal inverted-repeats suggest a possible filiation of some of these mosquito MITEs with pogo-like DNA transposons.     

Evidence for De Novo rearrangements of Drosophila transposable elements induced by the passage to the cell culture

The genomic distribution and the number of elements of eleven transposon families have been compared by the Southern technique between permanent cultured cells, larval salivary glands and the brains and whole flies of an inbred Drosophila line (inb-c) from which the cells were established. In cultured cells, changes in restriction patterns consistent with various types of rearrangements such as amplification, transposition and excision of the elements of copia, 1731, 412, 297 and mdg-4 transposon families are detected whereas B 104, G and blood elements appear stable. In previous reports these rearrangements were not detected among individuals of the inb-c line or among samples of somatic tissues, or in samples spanning years of maintenance of cultured cells. Hence, we believe that they have been induced de novo during the passage to the cell culture.     

<Emphasis Type="Italic">Anaconda</Emphasis>, a new class of transposon belonging to the <Emphasis Type="Italic">Mu</Emphasis> superfamily,has diversified by acquiring host genes during rice evolution

A new type of transposon, named Anaconda (Anac) has been found in rice (Oryza sativa). In this paper, we demonstrate that Anaconda elements have diversified by acquisition of host cellular genes, amplification of the elements, and substitution and deletion of short segments. We identified four Anaconda elements in studies of rice alternative oxidase (AOX) genes, and subsequently isolated an additional 23 elements based on the identity of their terminal inverted repeats (TIRs). The Anaconda elements have long TIRs (114–458 bp). They also have direct repeats of 9 or 10 bp in their flanking regions that are thought to have been generated upon transposition. These structural features reveal that the Anaconda elements belong to the Mu superfamily. The most prominent feature of the Anaconda elements is the high frequency with which they have acquired host cellular genes. Of the 27 elements found here, 19 appear to have sequences presumably derived from rice genes, for example, the genes for AOX1c (four elements), cytochrome P450 (five elements), l-asparaginase (five elements), and PCF8 (two elements). Four elements, AnacA1–A4, have both the AOX1c and P450 genes. One element, AnacB14, involves a gene similar to mudrA of maize MuDR. Database analyses revealed that the loci of 26 of the 27 Anaconda elements in the subspecies japonica are the same as those in the subspecies indica. This suggests that these elements were incorporated before the divergence of these two subspecies.