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.     

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.     

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.     

Analysis of the zinc finger domain of TnpA, a DNA targeting protein encoded by mobilizable transposon Tn4555

The mobilizable transposon Tn4555, found in Bacteroides spp., is an important antibiotic resistance element encoding a broad spectrum beta-lactamase. Tn4555 is mobilized by conjugative transposons such as CTn341 which can transfer the transposon to a wide range of bacterial species where it integrates into preferred sites on the host chromosome. Selection of the preferred target sites is mediated by a DNA-binding protein TnpA which has a prominent zinc finger motif at the N-terminus of the protein. In this report the zinc finger motif was disrupted by site directed mutagenesis in which two cysteine residues were changed to serine residues. Elemental analysis indicated that the wild-type protein but not the mutated protein was able to coordinate zinc at a molar ration of 1/1. DNA binding electrophoretic mobility shift assays showed that the ability to bind the target site DNA was not significantly affected by the mutation but there was about a 50% decrease in the ability to bind single stranded DNA. Consistent with these results, electrophoretic mobility shift assays incorporating zinc chelators did not have a significant on affect the binding of DNA target. In vivo, the zinc finger mutation completely prevented transposition/integration as measured in a conjugation assay. This was in contrast to results in which a TnpA knockout was still able to insert into host genomes but there was no preferred target site selection. The phenotype of the zinc finger mutation was not effectively rescued by providing wild-type TnpA in trans. Taken together these results indicated that the zinc finger is not required for DNA binding activity of TnpA but that it does have an important role in transposition and it may mediate protein/protein interactions with integrase or other Tn4555 proteins to facilitate insertion into the preferred sites.     

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.     

Cloning of the Bz2 locus of Zea mays using the transposable element Ds as a gene tag

Summary The Bz2 locus of Zea mays has been cloned, utilizing the presence of the transposable element Dissociation (Ds) at the locus as a gene tag. The Ds element inserted in the bz2-m allele was identified among many members of the Ac/Ds family in a Southern blot analysis of a population segregating for bz2-m and Bz2. After cloning a DNA fragment from the bz2-m allele, sequences flanking the Ds insertion were shown to be Bz2-specific and were used to isolate a homologous fragment from a wild-type Bz2 line. The Ds insertion in the bz2-m allele was found to be a Ds2 element identical to the Ds insertion in adh1-2F11.     

The Spm (En) transposable element controls the excision of a 2-kb DNA insert at the wx allele of Zea mays

The waxy (Wx) locus of Zea mays was cloned from strains carrying the wild-type and wx^m-8 mutant alleles. The receptor component of the Suppressor-Mutator (Spm) controlling element system in the wx^m-8 allele was shown to be a 2 kb long insertion within the transcribed region of the Wx gene. The insertion, termed Spm-I8, is excised during somatic reversion events induced by the autonomous controlling element Enhancer (En), which is an equivalent to Spm. Integration of Spm-I8 into the Wx gene generates a 3-bp target site duplication. Spm-I8 has a 13 bp long inverted repeat at its termini. The ends of the element can be further folded to build a large double-stranded structure consisting of five perfectly matching double-stranded regions of 9–13 bp in length, interrupted by single-stranded loops. A comparison of the wild-type and wx^m-8 alleles revealed two additional insertions 6 (insert-1) and 0.25 (insert-2) kb in length. No En-induced excision of insert-1 and insert-2 could be detected so far. There is remarkable structure and sequence homology between Spm-I8 and the transposable elements Tam1 and Tam2 of Antirrhinum majus at their termini, reflecting a possible evolutionary and/or functional relationship between transposons in different plant species.     

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.     

Overproduction of the protein encoded by the maize transposable element Ac in insect cells by a baculovirus vector

Summary The polypeptide encoded in the Activator (Ac) element of Zea mays L. has been expressed in Spodoptera frugiperda insect cells using plasmids which carry the strong polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPVs with the Ac-cDNA integrated and under the control of the viral polyhedrin promoter have been isolated and their genomes have been partly characterized as to the location of the foreign DNA insert. Upon infection of S. frugiperda cells with the recombinant AcNPV, maize Ac element specific messenger RNAs, as well as a newly synthesized polypeptide with an apparent molecular weight of about 116 kDa, have been detected in extracts of recombinant infected cells. This polypeptide is absent from extracts of wild-type infected cells expressing the polyhedrin polypeptide which can be recognized by the presence of nuclear inclusion bodies. Recombinant infected cells lack this protein. The Ac specific polypeptide is detected by antisera, which have been raised against fusion proteins containing Ac sequences synthesized in Escherichia coli, both in immunoprecipitation and in Western blotting experiments. The Ac specific protein is a nuclear phosphoprotein and represents about 1%–2% of the newly synthesized protein.     

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.     

Molecular cloning of the a1 locus of Zea mays using the transposable elements En and Mu1

The a1 locus of Zea mays has been cloned using transposable elements as gene tags. The strategy was to make genomic libraries from maize stocks with a1 mutations induced either by En(Spm) or by Robertson's Mutator-system. These libraries were then screened with either Spm-I8 and En1, for the En-containing mutant, or with Mu1 for the Mu-induced mutation. There are many En and Mu1 hybridizing sequences present in the maize genome, however, by a process of cross-screening of the positives from the two libraries and by molecular analysis of the En-positive clones it was possible to identify clones in both libraries carrying all or part of the a1 gene.