Mutator transposase is widespread in the grasses

Although the Mutator (Mu) system is well characterized in maize (Zea mays), very little is known about this highly mutagenic system of transposons in other grasses. Mutator is regulated by the MuDR class of elements, which encodes two genes, one of which, mudrA, has similarity to a number of bacterial transposases. Experiments in our laboratory, as well as database searches, demonstrate that mudrA sequences are ubiquitous and diverse in the grasses. In several species it is clear that multiple paralogous elements can be present in a single genome. In some species such as wheat (Triticum aestivum) and rice (Oryza sativa), mudrA-similar sequences are represented in cDNA databases, suggesting the presence of active Mu transposon systems in these species. Further, in rice and in sorghum, mudrA-like genes are flanked by long terminal inverted repeats, as well as the short host sequence direct repeats diagnostic of insertion. Thus, there is ample evidence that systems related to Mu in maize are at least potentially active in a wide variety of grasses. However, the mudrB gene, though important for Mu activity in maize, is not necessarily a component of Mu elements in other grasses.     

Mutator转座子及MULE在植物基因与基因组进化中的作用

Mutator（Mu）转座子是植物中已发现的转座最活跃的转座子,其高的转座频率及趋向于单拷贝功能基因转座的特性,使该转座子成为玉米功能基因克隆的主要方法.Mu转座子的同源类似因子广泛存在于被子植物基因组中,而且同一基因组中往往具有多种变异类型.它不仅具有其他DNA转座子在基因和基因组进化中的普遍作用,而且具有能够承载基因组内功能基因和基因片段的载体功能,这种载体Mu转座子（Pack-MuLEs）能够在基因组内移动众多的基因片段,从而对基因和基因组进化产生作用.Mu转座子的同源序列发生在水稻与狗尾草之间的水平转移提供了高等植物核基因水平转移的首个例证.对Mu转座子的了解促进了我们对动态基因组概念的认识.文章对Mutator转座子的发现、转座特征、基因标签应用等的研究进展进行了综述,对Mu转座子家族的同源序列进行了分类,讨论了该转座子在基因组进化中的作用,分析了应加强研究的问题.     

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.     

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.     

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'.     

Maize Mu transposons are targeted to the 5' untranslated region of the gl8 gene and sequences flanking Mu target-site duplications exhibit nonrandom nucleotide composition throughout the genome

The widespread use of the maize Mutator (Mu) system to generate mutants exploits the preference of Mu transposons to insert into genic regions. However, little is known about the specificity of Mu insertions within genes. Analysis of 79 independently isolated Mu-induced alleles at the gl8 locus established that at least 75 contain Mu insertions. Analysis of the terminal inverted repeats (TIRs) of the inserted transposons defined three new Mu transposons: Mu10, Mu 11, and Mu12. A large percentage (>80%) of the insertions are located in the 5' untranslated region (UTR) of the gl8 gene. Ten positions within the 5' UTR experienced multiple independent Mu insertions. Analyses of the nucleotide composition of the 9-bp TSD and the sequences directly flanking the TSD reveals that the nucleotide composition of Mu insertion sites differs dramatically from that of random DNA. In particular, the frequencies at which C's and G's are observed at positions -2 and +2 (relative to the TSD) are substantially higher than expected. Insertion sites of 315 RescueMu insertions displayed the same nonrandom nucleotide composition observed for the gl8-Mu alleles. Hence, this study provides strong evidence for the involvement of sequences flanking the TSD in Mu insertion-site selection.     

Burkholderia cenocepacia phage BcepMu and a family of Mu-like phages encoding potential pathogenesis factors

We have isolated BcepMu, a Mu-like bacteriophage whose host range includes human pathogenic Burkholderia cenocepacia (formally B. cepacia genomovar III) isolates, and determined its complete 36748 bp genomic sequence. Like enteric bacteriophage Mu, the BcepMu genomic DNA is flanked by variable host sequences, a result of transposon-mediated replication. The BcepMu genome encodes 53 proteins, including capsid assembly components related to those of Mu, and tail sheath and tube proteins related to those of bacteriophage P2. Seventeen of the BcepMu genes were demonstrated to encode homotypic interacting domains by using a cI fusion system. Most BcepMu genes have close homologs to prophage elements present in the two published Salmonella typhi genomes, and in the database sequences of Photorhabdus luminescens, and Chromobacterium violaceum. These prophage elements, designated SalMu, PhotoMu and ChromoMu, respectively, are collinear with BcepMu through nearly their entire lengths and show only limited mosaicism, despite the divergent characters of their hosts. The BcepMu family of Mu-like phages has a number of notable differences from Mu. Most significantly, the critical left end region of BcepMu is inverted with respect to Mu, and the BcepMu family of transposases is clearly of a distinct lineage with different molecular requirements at the transposon ends. Interestingly, a survey of 33 B.cepacia complex strains indicated that the BcepMu prophage is widespread in human pathogenic B.cenocepacia ET12 lineage isolates, but not in isolates from the PHDC or Midwest lineages. Identified members of the BcepMu family all contain a gene possibly involved in bacterial pathogenicity, a homolog of the type-two-secretion component exeA, but only BcepMu also carries a lipopolysaccharide modification acyltransferase which may also contribute a pathogenicity factor.     

Characterization of the maize Mutator transposable element MURA transposase as a DNA-binding protein.

The autonomous MuDR element of the Mutator (Mu) transposable element family of maize encodes at least two proteins, MURA and MURB. Based on amino acid sequence similarity, previous studies have reported that MURA is likely to be a transposase. The functional characterization of MURA has been hindered by the instability of its cDNA, mudrA, in Escherichia coli. In this study, we report the first successful stabilization and expression of MURA in Saccharomyces cerevisiae. Gel mobility shift assays demonstrate that MURA is a DNA-binding protein that specifically binds to sequences within the highly conserved Mu element terminal inverted repeats (TIRs). DNase I and 1,10-phenanthroline-copper footprinting of MURA-Mu1 TIR complexes indicate that MURA binds to a conserved approximately 32-bp region in the TIR of Mu1. In addition, MURA can bind to the same region in the TIRs of all tested actively transposing Mu elements but binds poorly to the diverged Mu TIRs of inactive elements. Previous studies have reported a correlation between Mu transposon inactivation and methylation of the Mu element TIRs. Gel mobility shift assays demonstrate that MURA can interact differentially with unmethylated, hemimethylated, and homomethylated TIR substrates. The significance of MURA's interaction with the TIRs of Mu elements is discussed in the context of what is known about the regulation and mechanisms of Mutator activities in maize.     

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.     

Horizontal transfer of a plant transposon

The majority of well-documented cases of horizontal transfer between higher eukaryotes involve the movement of transposable elements between animals. Surprisingly, although plant genomes often contain vast numbers of these mobile genetic elements, no evidence of horizontal transfer of a nuclear-encoded transposon between plant species has been detected to date. The most mutagenic known plant transposable element system is the Mutator system in maize. Mu-like elements (MULEs) are widespread among plants, and previous analysis has suggested that the distribution of various subgroups of MULEs is patchy, consistent with horizontal transfer. We have sequenced portions of MULE transposons from a number of species of the genus Setaria and compared them to each other and to publicly available databases. A subset of these elements is remarkably similar to a small family of MULEs in rice. A comparison of noncoding and synonymous sequences revealed that the observed similarity is not due to selection at the amino acid level. Given the amount of time separating Setaria and rice, the degree of similarity between these elements excludes the possibility of simple vertical transmission of this class of MULEs. This is the first well-documented example of horizontal transfer of any nuclear-encoded genes between higher plants.     

A highly conserved, small LTR retrotransposon that preferentially targets genes in grass genomes

LTR retrotransposons are often the most abundant components of plant genomes and can impact gene and genome evolution. Most reported LTR retrotransposons are large elements (>4 kb) and are most often found in heterochromatic (gene poor) regions. We report the smallest LTR retrotransposon found to date, only 292 bp. The element is found in rice, maize, sorghum and other grass genomes, which indicates that it was present in the ancestor of grass species, at least 50-80 MYA. Estimated insertion times, comparisons between sequenced rice lines, and mRNA data indicate that this element may still be active in some genomes. Unlike other LTR retrotransposons, the small LTR retrotransposons (SMARTs) are distributed throughout the genomes and are often located within or near genes with insertion patterns similar to MITEs (miniature inverted repeat transposable elements). Our data suggests that insertions of SMARTs into or near genes can, in a few instances, alter both gene structures and gene expression. Further evidence for a role in regulating gene expression, SMART-specific small RNAs (sRNAs) were identified that may be involved in gene regulation. Thus, SMARTs may have played an important role in genome evolution and genic innovation and may provide a valuable tool for gene tagging systems in grass.     

An efficient and accurate integration of mini-Mu transposons in vitro: a general methodology for functional genetic analysis and molecular biology applications.

Transposons are mobile genetic elements and have been utilized as essential tools in genetics over the years. Though highly useful, many of the current transposon-based applications suffer from various limitations, the most notable of which are: (i) transposition is performed in vivo, typically species specifically, and as a multistep process; (ii) accuracy and/or efficiency of the in vivo or in vitro transposition reaction is not optimal; (iii) a limited set of target sites is used. We describe here a genetic analysis methodology that is based on bacteriophage Mu DNA transposition and circumvents such limitations. The Mu transposon tool is composed of only a few components and utilizes a highly efficient and accurate in vitro DNA transposition reaction with a low stringency of target preference. The utility of the Mu system in functional genetic analysis is demonstrated using restriction analysis and genetic footprinting strategies. The Mu methodology is readily applicable in a variety of current and emerging transposon-based techniques and is expected to generate novel approaches to functional analysis of genes, genomes and proteins.     

Nested Insertions and Accumulation of Indels Are Negatively Correlated with Abundance of Mutator-Like Transposable Elements in Maize and Rice

Mutator-like transposable elements (MULEs) are widespread in plants and were first discovered in maize where there are a total of 12,900 MULEs. In comparison, rice, with a much smaller genome, harbors over 30,000 MULEs. Since maize and rice are close relatives, the differential amplification of MULEs raised an inquiry into the underlying mechanism. We hypothesize this is partly attributed to the differential copy number of autonomous MULEs with the potential to generate the transposase that is required for transposition. To this end, we mined the two genomes and detected 530 and 476 MULEs containing transposase sequences (candidate coding-MULEs) in maize and rice, respectively. Over 1/3 of the candidate coding-MULEs harbor nested insertions and the ratios are similar in the two genomes. Among the maize elements with nested insertions, 24% have insertions in coding regions and over half of them harbor two or more insertions. In contrast, only 12% of the rice elements have insertions in coding regions and 19% have multiple insertions, suggesting that nested insertions in maize are more disruptive. This is because most nested insertions in maize are from LTR retrotransposons, which are large in size and are prevalent in the maize genome. Our results suggest that the amplification of retrotransposons may limit the amplification of DNA transposons but not vice versa. In addition, more indels are detected among maize elements than rice elements whereas defects caused by point mutations are comparable between the two species. Taken together, more disruptive nested insertions combined with higher frequency of indels resulted in few (6%) coding-MULEs that may encode functional transposases in maize. In contrast, 35% of the coding-MULEs in rice retain putative intact transposase. This is in addition to the higher expression frequency of rice coding-MULEs, which may explain the higher occurrence of MULEs in rice than that in maize.