Mutator Transposons

Plant & Microbial Biology, Univ. of California, Berkeley

In recent years, geneticists are using extensively mutator elements, abbreviated to Mu, to clone, sequence and mutate most maize genes. This has been made possible following the isolation and cloning of the regulatory element, MuDR, constituting the two essential genes, MuDRA and MuDRB in the Mu element and the bioengineering of RescueMu. The latter is being extensively used to disrupt the genes of interest in maize and determine their function.

In the November 2002 issue of Trends in Plant Science (vol. 7: 498-504), Damon Lisch at the Dept of Plant and Microbial Biology, UCB, reviews the current status of mutator transposons and their potential use for unraveling gene functions in other plant groups, besides using the technique more intensively in maize genetics.

Factors that impeded the progress of research in mutator transposons: In the past there was no substantial progress in understanding the nature of the mutagenic plant transposons and their mode of function. Lack of progress was due to (a) difficulty to isolate and clone MuDR, the regulatory element of the system (b) complexity to engineer an Mu element and (c) non-availability of low copy number mutator lines. Success in resolving some of these difficulties has shed considerable light into the working mechanism of these autonomous bodies i.e., how they are integrated and excised from the genome, how they are applied to clone, sequence and mutate most maize genes.

Recent Information about Mu transposons: Recent studies have shown that (a) all maize Mu elements are flanked on both sides by ~220 bp long almost identical (97%) inverted repeats designated (terminal inverted repeats = TIRs), and (b) different classes of Mu elements differ from one another in their internal sequences, lying between the two TIRs and (c) their activities are coordinated by MuDR regulating genes, resident on an Mu element. MuDR elements are self-replicating i.e., autonomous and they participate for transposition of non-autonomous classes of Mu elements. The two genes that constitute MuDR elements are: mudrA and mudrB. Transcription of these two genes occurs from two ends proceeding inwards after being initiated from promoters located within the terminal inverted repeats. Because of this complexity, several proteins are encoded from each reading frame. Interestingly, the best-characterized mudrA transcript, encoded by a 120 kDa transposase (MURA), share a domain similarity with several bacterial transposases. On the other hand, there is no similarity between the major transcript of mudrB, encoding the 23 kDa protein MURB and any known sequences outside maize, making it difficult to determine its precise function.

Homologous MuDR sequences in maize line: Recent studies have also revealed that all maize lines carry homologous MuDR sequences (hMuDRs), sharing 80–99% identity to those of MuDR. The function of these elements have not been characterized although it is known hMuDRs are not associated with Mutator.

Functions of mudrA mudrB: Mutator activity is dependent upon the presence of mudrA gene. Mutators showing deletion of the full length of mudrA gene, undergo methyla- tion at their TIR ends and as a result all new excisions and insertions are stopped. On the other hand, the presence of only mudrA gene or 35S-promoter-driven mudrAcDNA in Mutators leads to hypomethylation of Mu1 elements, accompanied by late somatic excisions.

The functions of mudrB gene are not known. However, without mudrA, the Mutator can neither undergo hypomethylation nor can it cause excision or insertion.

Role of non-autonomous non-transposon host sequences: Mu elements capture non-autonomous non-transposon host sequences. The latter cause new mutations in Mutator lines. Arabidopsis and rice show Mu-like elements abbreviated to MULEs. They also behave in the same fashion i.e., causing mutation by gene capture.

The author describes a recently-constructed new class of Mu elements called RescueMu. Researchers at Stanford University first generated this kind of artificial transposon. It differs from its counterpart in that it replicates itself, instead of jumping from place to place in the chromosome as the plant grows. After replication, it passes on its copy to the egg and pollen. After insertion it does not move. The engineered RescueMu contained pBluescript (a commonly used plasmid vector), an antibiotic (ampicillin) resistant marker, ori (origin of replication) from Escherichia coli and a unique marker sequence from Rhizobium meliloti. The author is of the opinion that the new class of the synthetic Mu element will pave the way of quicker analysis of gene functions in maize as this technique does not involve sequencing of the part of the maize genome comprising retrotransposons. In addition, new DNA insertions may be excised, circularized, transformed into E. coli and sequenced. The last-mentioned feature helps researchers find the transposon into a new location along with the flanking sequence of the gene of interest.

The working mechanism of Mu-elements:Small revertant sectors appear as a result of excision of Mu elements from visible reporter genes positioned in the somatic tissue. Mu elements are excised from a chromosome site comprising a gene resulting in the gain of a trait which may be referred to as a revertant sector. Interestingly, excisions occur mostly in somatic and rarely in reproductive tissues, indicating that some unknown mechanism operates protecting the latter from Mu-induced mutations. However, when new mu-induced mutant genes are transmitted, the donor element is not lost. Furthermore, as the plant grows, duplications of elements present in small numbers such as MuDR may occur, as was demonstrated from the analysis of progeny for reciprocal crosses. In one direction, duplications were observed, while it was absent in the progeny of the reciprocal cross. The author points out that in a typical Mutator line only 20% of all new mutations occur before the commencement of meiosis. Of 80% of the mutations that occur in a single seed, 25% are postmeiotic. Recent findings about different classes of Mu elements reveal that their preferences for genes vary, although they mostly target the 5′ region of the gene. In other words, such preferences may be generic. For instance, frequent targets of Mu insertions is gl8, showing a strong preference for the 5′ region of the gene.

How is Mu-element inactivation process detected? Mu-element is inactivated when methylation of cytosines occurs at the 5′ prime end within TIRs lacking transposase. The process of methylation depends upon whether or not MuDR gene is present in the system. When the latter is lost as a result of segregation, methylation occurs. On the other hand, loss of methylation restores MuDR activity, suggesting that the absence of the transposase results in TIR methyla- tion. In some instances, transcriptional silencing may occur in the presence of potentially active MuDR elements by its methylation in more typical Mutator lines. This inactivity is more or less permanent unless reactivated through exposure to UV radiation or other potent sources. How is the transposition of the Mu element regulated? The author presents models explaining how the transposition of Mu-elements is regulated. One of the mechanism is the gap-repair model. The mechanism which operates in the reproductive tissue is different than that occurring in the somatic tissue. In reproductive tissues, the duplicated segment, derived from the sister chromatid, is used to repair gaps efficiently. At the late stage of the somatic tissue development, repair mechanism operates inefficiently, involving either ineffective template-mediate repair or just simple ligation, following the last S phase. The author further points out that since the gap is confined to a single strand, this process does not give rise to reversions in the tissue in question.

Evolution: While describing the course of evolution, the author points out that Mu elements are far more wide spread in plants than it was originally thought. Besides maize and Arabidopsis, these elements were discovered in rice, barley, sorghum, lotus, and practically in all major subfamilies of grasses. murdA-homologous sequences is a common feature in the above taxa. In fact, the author points out that a close look at EST databases will reveal that mudrA homologs occur in a large number of plant species. As mentioned earlier, these elements called MuLEs (mu-like-elements In order occur in a wide variety of taxa. The major difference between Mu’s and MuLEs is that the latter lack MuDRB gene. Interestingly, some active MuLE elements such as jittery in maize show more similar base sequences with Arabidopsis and rice than between different MuDR lines in maize.

In his concluding remarks, the author brings into focus the necessity to unraveling the mechanisms that control MuDR activity, particularly in relation to post-transcrip- tional modification that MuDR proteins undergo at various developmental stages. The author suggests that future research should be directed to gain insight into the detailed biochemical and molecular analysis of phenomena that classical genetic approaches have posed. Some other questions to be addressed to are: histone deacetyla- tion or DNase-I digestion and their association with gene silencing. The valuable information obtained from the isolation of genes effecting silencing inArabidopsis, Caenorhabditis elegans and Chlamydomonas reinhardtii might be helpful to understand the mechanism of silencing in maize.

 

Leave a Reply

Your email address will not be published. Required fields are marked *

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>