Molecular analysis of the Ubiquitous (Uq) transposable element system of Zea mays.

Summary The Uq transposable element of maize is the most widely dispersed among different maize populations and genetic testerstrains. Despite intensive genetic characterization, little is known about its molecular structure. In order to obtain information relevant to this topic, we have cloned and sequenced three ruq receptors. Surprisingly, they are all Ds1-like receptor types of the Ac-Ds transposon family. Based on our molecular data, we present a model to explain the functional differences associated with the differential expression of the Uq and Ac transposon systems.     

Tissue-specific accumulation of MURB, a protein encoded by MuDR, the autonomous regulator of the Mutator transposable element family.

The Mutator (Mu) system of transposable elements is highly mutagenic and can maintain high levels of activity through multiple generations due to frequent transpositions of both its autonomous and nonautonomous components. This family also shows pronounced developmental regulation. Most notable is the very low frequency of germinal reversions, despite the high levels of somatic transpositions and excisions, and the high frequency of germinally transmitted duplication events. Here, we report the production of antibodies raised against MURB, one of two proteins encoded by MuDR, the autonomous regulator of the Mu family. Immunolocalizations performed using anti-MURB antibodies reveal that this protein is present in specific tissues during male inflorescence development. Throughout much of development, MURB is detected at the highest levels in cell lineages that may find themselves in the germ line, but no MURB is detected in microspore mother cells. These cells are the direct precursors to pollen. Based on these observations as well as previous data, we discuss the relationship between the expression of MURB and developmental regulation of Mu activity.     

Excision of the En/Spm transposable element of Zea mays requires two element-encoded proteins.

An excision assay system for En/Spm was developed in transgenic tobacco. The characteristics of excision and integration are similar to the natural system of Zea mays. In this transgenic model system two En/Spm encoded trans-acting functions, TNPA and TNPD, are required for excision. A biochemical model for transposition is proposed that might also be applicable to other transposable elements.     

The role of subterminal sites of transposable element Ds of Zea mays in excision

Transposition depends on DNA sequences located at or near the termini of the transposon. In the maize transposable element Ds, these sequences were studied by site-directed mutagenesis followed by a transient excision assay in Petunia protoplasts. The transposase-binding AAACGG motifs found in large numbers in the element are important, but none of them is in itself indispensable, for excision. However, mutation of an isolated motif at the 3′ end considerably reduced excisability. The inverted termini were confirmed to be indispensable. Point mutations in regions outside the inverted termini of Ds and not located in the transposase-binding motifs had, in some cases, a pronounced effect on excision frequency. The implications of these findings are discussed.     

The bz-rcy allele of the Cy transposable element system of Zea mays contains a Mu-like element insertion

Summary The receptive component of theCy transposable element system (rcy: Mu7) at theBz locus ofZea mays L. is 2.2 kb and has long terminal inverted repeats. The insertion is flanked by a 9 bp duplication. In the presence of an autonomousCy element in the genome,rcy: Mu7 is excised frombz-rcy in a manner consistent with a model suggested previously. The termini ofrcy: Mu7 have 85% sequence similarity with theMu1 element ofZ. mays. This is consistent with the observation thatMu1 can behave genetically like a receptive component of theCy system.     

Genetic and molecular analysis of the Enhancer (En) transposable element system of Zea mays

A newly isolated, unstable mutation wx-844::En-1 of Zea mays was proven to be caused by the insertion of the autonomous transposable element En into the Waxy (Wx) gene. Molecular analysis revealed that En-1 is 8.4 kb long, has a 13-bp long perfect inverted repeat at its termini and generates a 3-bp target site duplication. En-1 is integrated into an intron located approximately in the middle of the transcribed region of the Wx gene. Structural evidence is presented indicating that a receptor component (Inhibitor) can arise by internal deletion of an autonomous En element.     

The putative transposase of transposable element Ac from Zea mays L. interacts with subterminal sequences of Ac.

The Ac-specific ORFa protein, overexpressed in a baculovirus system, specifically binds to several subterminal fragments of Ac. The 11 bp long inverted repeats of the transposable element are not bound by the ORFa protein. Major ORFa protein-binding sites were delineated on 60 and 70 bp long sequence segments that lie 100 bp inside of the 5' Ac terminus and 40 bp inside of the 3' terminus respectively. Within all strongly bound fragments, and particularly in these 60 or 70 bp long segments, the hexamer motif AAACGG is repeated several times in direct or inverted orientation. The ORFa protein binds to synthetic concatemers of this motif, whereas the mutant motif AAAGGG is not complexed. Methylation of the cytosine residues in the AAACGG motif and/or its complementary strand has pronounced effects: whereas one of the two hemimethylated sequences has a higher affinity to the ORFa protein than both unmethylated and holomethylated DNAs, the other hemimethylated DNA is virtually not complexed at all. The native ORFa protein binding sites are more complex than the AAACGG sequence: certain Ac and Ds1 fragments devoid of AAACGG motifs (but containing several similar sequences) are weakly bound by the ORFa protein.     

Analysis of sh-m6233, a mutation induced by the transposable element Ds in the sucrose synthase gene of Zea mays

The unstable allele sh-m6233 caused by insertion of the transposable element Ds into the sucrose synthase gene of maize, was cloned. The mutation is caused by the insertion of an ˜4 kb DNA segment, consisting of two identical Ds elements of ˜2000 bp length, of which one is inserted into the center of the other in inverted orientation. This structure is, at the level of restriction mapping and partial DNA sequencing, identical to the double Ds element found in a larger insert in the mutant allele sh-m5933. 8 bp of host DNA are duplicated upon insertion. In a revertant, a 6-bp duplication is retained.     

The En/Spm transposable element of Zea mays contains splice sites at the termini generating a novel intron from a dSpm element in the A2 gene

The A2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable elements rcy and dSpm as gene tags. The A2 gene encodes a putative protein of 395 amino acids and is devoid of introns. Two a2-m1 alleles, containing dSpm insertions of different sizes, were characterized. The dSpm element from the original state allele has perfect termini and undergoes frequent transposition. The element from the class II state allele is no longer competent to transpose. It has retained the 13 bp terminal inverted repeat but has lost all subterminal sites at the 5' end, which are recognized by tnpA protein, the most abundant product of the En/Spm transposable element system. The relatively high A2 gene expression of one a2-m1 allele is due to removal of almost all dSpm sequences by splicing. The slightly altered A2 enzyme is still functional as shown by complementation of an a2 mutant with the corresponding cDNA. The 5' and 3' splice sites are constituted by the termini of the dSpm element; it therefore represents a novel intron of the A2 gene.     

FB-NOF is a non-autonomous transposable element,expressed in Drosophila melanogaster and present only in the melanogaster group

Most foldback elements are defective due to the lack of coding sequences but some are associated with coding sequences and may represent the entire element. This is the case of the NOF sequences found in the FB of Drosophila melanogaster, formerly considered as an autonomous TE and currently proposed as part of the so-called FB-NOF element, the transposon that would be complete and fully functional. NOF is always associated with FB and never seen apart from the FB inverted repeats (IR). This is the reason why the FB-NOF composite element can be considered the complete element. At least one of its ORFs encodes a protein that has always been considered its transposase, but no detailed studies have been carried out to verify this.     

Imprinting of R-r, paramutation of B-I and Pl, and epigenetic silencing of MuDR/Mu transposons in Zea mays L. are coordinately affected by inbred background

The extent of imprinting at R-r, frequency of paramutation at B-Intense and Pl, and epigenetic silencing of Mu transposons were evaluated in the W23 and A188 inbred lines of maize. All types of epigenetic phenomena affecting these loci of the anthocyanin pathway occurred more frequently in the W23 inbred line. Absence of down-regulation was dominant in F1 hybrid progeny. Identical alleles programme lower anthocyanin accumulation in A188 than in W23, and A188 plants develop more rapidly than W23. The possibilities that specific genetic factors, intrinsic gene expression levels and/or the rapidity of the life cycle modulate epigenetic gene controls are discussed.     

Distribution of sequences related to the Bg transposable element of maize in Zea and related genera

Thirty-four accessions from Zea and 10 accessions from related genera were assayed for the presence of Bg, a transposable element originally found in maize (Zea mays ssp. mays). Bg-like sequences, identified as hybridizing bands on Southern blots, were visualized in all Zea accessions and were present in approximately equal numbers in teosinte and maize. With the exception of Tripsacum dactyloides, all accessions from related genera failed to hybridize with the Bg probes, even at reduced stringency. A comparison of the restriction patterns of related inbred lines revealed numerous common hybridizing fragments. An index of molecular similarity (MS) was used to determine the degree of similarity between pairs of inbred lines. Computed MS values endorse an inbred relationship and are in good agreement with published results of cluster analysis on these inbred lines.