Transposon Tagging Experiments Research Articles

The Maize High-Lysine Mutantopaque7Is Defective in an Acyl-CoA Synthetase-Like Protein

Maize (Zea mays) has a large class of seed mutants with opaque or nonvitreous endosperms that could improve the nutritional quality of our food supply. The phenotype of some of them appears to be linked to the improper formation of protein bodies (PBs) where zein storage proteins are deposited. Although a number of genes affecting endosperm vitreousness have been isolated, it has been difficult to clone opaque7 (o7), mainly because of its low penetrance in many genetic backgrounds. The o7-reference (o7-ref) mutant arose spontaneously in a W22 inbred, but is poorly expressed in other lines. We report here the isolation of o7 with a combination of map-based cloning and transposon tagging. We first identified an o7 candidate gene by map-based cloning. The putative o7-ref allele has a 12-bp in-frame deletion of codons 350-353 in a 528-codon-long acyl-CoA synthetase-like gene (ACS). We then confirmed this candidate gene by generating another mutant allele from a transposon-tagging experiment using the Activator/Dissociation (Ac/Ds) system in a W22 background. The second allele, isolated from ∼1 million gametes, presented a 2-kb Ds insertion that resembles the single Ds component of double-Ds, McClintock's original Dissociation element, at codon 496 of the ACS gene. PBs exhibited striking membrane invaginations in the o7-ref allele and a severe number reduction in the Ds-insertion mutant, respectively. We propose a model in which the ACS enzyme plays a key role in membrane biogenesis, by taking part in protein acylation, and that altered PBs render the seed nonvitreous.

Characterisation of Aft1 a Fot1/Pogo type transposon of Aspergillus fumigatus

In recent years the filamentous fungus Aspergillus fumigatus has become a significant cause of infection in man and as such has become the focus of much study. It is thought to be the leading mould pathogen in leukaemia and transplant patients and is responsible for mortality in a large number of individuals with immunological disorders. In an attempt to develop molecular mutagenesis tools for assessment of this organism, the genome of A. fumigatus was analysed to identify possible functional transposable elements. An apparently intact Fot1/Pogo type transposon with 65% identity to the active Tan1 element of Aspergillus niger was identified and designated Aft1. Aft1 is a 1.9 kb element present in multiple (>20) highly conserved copies. It encodes a 332 amino acid transposase which contains all the functional motifs required for transposition. In addition, the transposase was expressed in cultures grown at 37 °C in all three strains assessed and excision analysis suggests Aft1 may be active and of use in transposon tagging experiments. Southern hybridisation patterns indicate that Aft1 is widely distributed amongst clinical isolates of A. fumigatus with considerable variation in genomic localisation. A comprehensive analysis of the genomic localisation of Aft1 in the sequenced strain AF293 show that one insertion is 30 bases upstream of a predicted gene encoding a G-protein coupled receptor. Expression analysis indicates that this gene has been inactivated by the insertion.

The Ac/Ds Transposon System from Maize as a Tool for Generating Mutant Phenotypes in Tomato (Lycopersicon Esculentum)

ABSTRACTA non-targeted transposon tagging experiment was performed in tomato (Lycopersicon esculentum). The Ac/Ds transposon system from maize was used. To generate a transposon tagging population, tomato genotypes ET 570 and SLJ10512 were used. The ET 570 genotype contains a single homozygous T-DNA with Ds at the end of the long arm of chromosome 3. The SLJ10512 genotype carries a stabilized Ac that can activate Ds in trans. This tagging population was tested at the seedling level (Van der Biezen et al., 1996) and two phenotypic mutants were found that co-segregated with a Ds insertion: feebly (fb) and yellow jim (yj). In this work, performed under field conditions, all stages of individual plant development were screened for mutant phenotypes. Out of 269 families, with in total approximately 400 unique transposed Ds elements, 5 different phenotypes were observed. One of the phenotypes, fruit distortion, segregated in a Mendelian fashion. Plants with the fruit distortion phenotype did not produce seeds. In the case of the other 4 phenotypes, green spots, dark green leaves, dwarf and partially hairless plant only one phenotypic mutant plant per family was obtained and, thus, could not be classified. Phenotypic mutants, obtained from this transposon tagging experiment, that show linkage to the Ds insertion, might be used to isolate the genes responsible for the mutant phenotypes.

Ds elements on all five Arabidopsis chromosomes and assessment of their utility for transposon tagging.

The maize transposons Activator (Ac) and Dissociation (Ds) tend to transpose to sites close to their original position and can be efficiently used to transposon-tag genetically linked genes. To facilitate this approach, we describe the locations of seven T-DNAs carrying Ds elements, including at least one on each of the five chromosomes. For five of these T-DNAs, we have confirmed that the Ds element transposes preferentially to genetically linked sites. A large-scale transposon-tagging experiment was performed by activating Ds from eight chromosomal locations that included at least one on each of the five chromosomes. These experiments produced a total of 1132 F3 families that were predicted to carry around 870 independent Ds insertions. In these populations, 33 independently isolated mutants that were visibly different from wild-type were identified. Twenty-nine of these mutants were studied genetically, and 14 were not tagged with Ds because the element could be separated from the mutation by recombination. The remaining 15 mutations were possibly tagged because the transposon and the mutation were not separated by recombination. These experiments provide tools for transposon-tagging on each chromosome, and indicate that approximately 50% of identified mutations are likely to be tagged, thereby enabling cloning of the affected genes.

Distribution of unlinked transpositions of a Ds element from a T-DNA locus on tomato chromosome 4.

In maize, receptor sites for unlinked transpositions of Activator (Ac) elements are not distributed randomly. To test whether the same is true in tomato, the receptor sites for a Dissociation (Ds) element derived from Ac, were mapped for 26 transpositions unlinked to a donor T-DNA locus on chromosome 4. Four independent transposed Dss mapped to sites on chromosome 4 genetically unlinked to the donor T-DNA, consistent with a preference for transposition to unlinked sites on the same chromosome as opposed to sites on other chromosomes. There was little preference among the nondonor chromosomes, except perhaps for chromosome 2, which carried seven transposed Dss, but these could not be proven to be independent. However, these data, when combined with those from other studies in tomato examining the distribution of transposed Acs or Dss among nondonor chromosomes, suggest there may be absolute preferences for transposition irrespective of the chromosomal location of the donor site. If true, transposition to nondonor chromosomes in tomato would differ from that in maize, where the preference seems to be determined by the spatial arrangement of chromosomes in the interphase nucleus. The tomato lines carrying Ds elements at known locations are available for targeted transposon tagging experiments.

Suppression of recombination in wide hybrids of Petunia hybrida as revealed by genetic mapping of marker transgenes

In the course of a heterologous transposon tagging experiment in Petunia hybrida (n=7), 135 independent T-DNA loci were tested for linkage to the target genes Hf1 and Fl, which are located on the two largest chromosomes. Approximately one-third (47) of these T-DNA loci were linked to one of these two markers. Of these 47 linkedloci, 19 mapped within 1 cM of its marker, indicating a highly non-random genetic distribution of introduced loci. However, rather than non-random integration within both of the marked chromosomes, this probably reflects a suppression of recombination around these marker loci in the particular wide hybrids used for mapping. This hypothesis was tested by measuring recombination between linked T-DNAs in an inbred background. Inbred recombination levels were found to be at least 3-fold higher around the Hf1 locus and 12-fold higher around Fl compared to the wide hybrids. These findings may reflect the origin of P. hybrida by hybridization of wild species, and while relevant to genetic mapping in petunia in particular they may also have more general significance for any mapping strategies involving the use of wide hybrids in other species.

The homeotic Macho mutant of Antirrhinum majus reverts to wild-type or mutates to the homeotic plena phenotype.

Plants of Antirrhinum majus carrying the semidominant Macho alleles of the plena gene display carpelloid sepals and staminoid petals, but the two inner flower whorls of stamens and carpels are normal and produce fertile gametes. In the recessive plena mutant, in contrast, the two outer whorls are normal whereas the stamens are largely or entirely petaloid and the carpels sepaloid, thus producing weakly male-fertile or fully sterile lines. Two new plena and two new Macho alleles have been induced in transposon tagging experiments. Genetic and molecular analysis revealed that the two contrasting mutant phenotypes are caused by mutations in one and the same gene: Several wild-type plants appeared among 27,000 F1 plants of a cross between Macho female plants and wild-type males bearing the active transposons Tam1 and Tam3. One of these plants segregated plena mutants, three showed reversions to wild-type and another two segregated Macho plants, possibly representing somatic reversions. Additional evidence was provided by an allelism test of Macho x plena. Molecular analysis has independently corroborated the genetical results. Moreover, the double mutant Macho/deficiens shows only carpels and plena/deficiens only sepals, which is in accord with combinatorial models for homeotic flower formation presented recently.

Linked and unlinked transposition of a genetically marked Dissociation element in transgenic tomato.

We have introduced a genetically marked Dissociation transposable element (Dsneo) into tomato. In the presence of Ac transposase, Dsneo excised from an integrated T-DNA and reinserted at numerous new sites in the tomato genome. The marker genes of Dsneo (NPTII) and the T-DNA (HPT) facilitated identification of plants bearing transposon excisions and insertions. To explore the feasibility of gene tagging strategies in tomato using Dsneo, we examined the genomic distribution of Dsneo receptor sites, relative to the location of the donor T-DNA locus. Restriction fragment length polymorphism mapping of transposed Dsneo elements was conducted in two tomato families, derived from independent primary transformants each bearing Dsneo within a T-DNA at a unique position in the genome. Transposition of Dsneo generated clusters of insertions that were positioned on several different tomato chromosomes. Dsneo insertions were often located on the same chromosome as the T-DNA donor site. However, no insertion showed tight linkage to the T-DNA. We consider the frequency and distance of Dsneo transposition observed in tomato to be well suited for transposon mutagenesis. Our study made use of a novel, stable allele of Ac (Ac3) that we discovered in transgenic tomato. We determined that the Ac3 element bears a deletion of the outermost 5 base pairs of the 5'-terminal inverted repeat. Though incapable of transposition itself, Ac3 retained the ability to mobilize Dsneo. We conclude that a dual element system, composed of the stable Ac3 trans-activator in combination with Dsneo, is an effective tool for transposon tagging experiments in tomato.

Molecular cloning and characterization of iojap (ij), a pattern striping gene of maize.

Iojap (ij) is a recessive striped mutant of maize affecting the development of plastids in a local and position-dependent manner on the leaves. The ij-affected plastids are transmitted to some of the progeny even when the function of the nuclear gene is restored. Developmental defects during embryogenesis and leaf proliferation are other phenotypic characteristics of ij. The extent of striping and the degree of developmental arrest in ij depend upon genetic background. To understand the diverse and unique phenotypic expression of ij, a transposon tagging experiment has been conducted using Robertson's Mutator (Mu). A new ij mutant was obtained from crosses of the reference allele of (ij-ref) to Mu lines. Subsequent genetic and molecular studies showed that the mutant carried a new ij allele (ij-mum1) from the Mu lines and contained a Mu1 element that cosegregated with the iojap phenotype. A 6.0 kb EcoRI genomic DNA fragment containing the Mu1 element was cloned. ij-ref is unstable, and revertants (Ij-Rev) have been obtained. Using the flanking DNA from the genomic clone as a probe, DNA polymorphisms were detected between ij-ref and these revertants. Further, transcripts were restored to the normal level in Ij-Rev seedlings. Comparison of genomic DNA clones from ij-ref, ij-mum1 and Ij indicated that the ij-ref allele contained 1.5 kb of additional DNA related to a transposable element, Ds. Germinal and somatic revertant alleles were derived by excision of this 1.5 kb element from ij-ref. The structure of the Ij gene and the DNA sequence of its transcribed region were determined. The Ij gene encodes a 24.8 kDa protein that showed no significant sequence similarity with proteins listed in databases.

Transactivation of Ds by Ac-transposase gene fusions in tobacco.

To study regulation of the (Ds) transposition process in heterologous plant species, the transposase gene of Ac was fused to several promoters that are active late during plant development. These promoters are the flower-specific chalcone synthase A promoter (CHS A), the anther-specific chalcone isomerase B promoter CHI B and the pollen-specific chalcone isomerase A2 promoter CHI A2. The modified transposase genes were introduced into a tobacco tester plant. This plant contains Ds stably inserted within the leader sequence of the hygromycin resistance (HPT II) gene. As confirmed with positive control elements, excision of Ds leads to the restoration of a functional HPT II gene and to a hygromycin resistant phenotype. No hygromycin resistance was observed in negative control experiments with Ac derivatives lacking 5' regulatory sequences. Although transactivation of Ds was observed after the introduction of transposase gene fusions in calli, excision in regenerated plants was observed only for the CHS A- or CHI B-transposase gene fusions. With these modified transposase genes, somatic excision frequencies were increased (68%) and decreased (22%), respectively, compared to the situation with the Ac element itself (38%). The shifts in transactivation frequencies were not associated with significant differences in the frequencies of germinally transmitted excision events (approximately 5%). The relative somatic stability of Ds insertions bearing the CHI B-transposase gene fusion suggests the usefulness of this activator element for transposon tagging experiments.

Variable Patterns of Transposition of the Maize Element Activator in Tobacco.

The strategy to be followed in a transposon tagging experiment will be determined largely by the transposition pattern of the transposon in question. With a view to utilizing the maize element Activator (Ac) as a transposon tag in heterologous systems, we investigated the pattern of Ac transposition from six different loci in transgenic tobacco. We isolated germinal revertants from plants carrying mutable alleles of the antibiotic-resistant gene streptomycin phosphotransferase (SPT) and mapped the location of the transposed Ac (trAc) elements relative to the donor SPT gene. A comparison of the distributions of trAcs among the six loci revealed that, although the receptor sites for trAcs tend to be linked to the donor locus, the pattern of Ac transposition in tobacco displays surprising locus-to-locus variation. Some trAc distributions showed the same tight clustering around the donor locus previously seen in maize, whereas others were more dispersed. The possible meaning of these findings and their implication for transposon tagging in heterologous systems are discussed.

A transposon tagging strategy with Ac on plant cell level in heterologous plant species

The maize transposable element Ac can have a ‘late’ excision time during leaf development in certain transgenic tobacco plants. This was visualized with an assay based on Ac-excision restoring GUS-expression. Leaves of the described plants contain over 10 3 small blue spots, each of these spots representing an independent excision event. Leaves showing this ‘late’ excision phenomenon may be used for transposon tagging experiments at plant cell level. Plants which display ‘late’ Ac-excision do not detectably express GUS during the preceding callus phase, thus allowing transformants to be preselected for a ‘late’ timing of excision. To examine the applicability of this phenomenon a phenotypic selection assay for excision of Ac was used. Transformed calli containing Ac within the hygromycin resistance gene were regenerated and protoplasts isolated from leaves of regenerated plants were selected on hygromycin. Up to 0.8% of these protoplasts displayed hygromycin resistance. The hygromycin resistant derivatives analyzed were shown to represent independent transposition events. Ac-insertions which can be generated in this way may be used for transposon tagging experiments at cell level.