Spotting factor (Spf) from the Spotted-dilute system in maize is a member of the En/Spm controlling element family

The Spotted-dilute controlling element system in maize involves an autonomous Spotting factor (Spf), and a receptor at the r1 locus haplotype R1-r(spotted dilute2). Its relationship with other maize transposable element systems is poorly characterized. Through development of a genetic tester that carries receptors for both the Spotted-dilute and the En/Spm controlling element systems, we determined that both receptors respond equally to Spf and En/Spm and that Spf is therefore a member of the En/Spm family of controlling elements.     

Insertions of a Novel Class of Transposable Elements with a Strong Target Site Preference at the R Locus of Maize

The r locus of maize regulates anthocyanin synthesis in various tissues of maize through the production of helix-loop-helix DNA binding proteins capable of inducing expression of structural genes in the anthocyanin biosynthetic pathway. The complex r variant, R-r:standard (R-r), undergoes frequent mutation through a variety of mechanisms including displaced synapsis and crossing over, and intrachromosomal recombination. Here we report a new mechanism for mutation at the R-r complex: insertion of a novel family of transposable elements. Because the elements were first identified in the R-p gene of the R-r complex, they have been named P Instability Factor (PIF). Two different PIF elements were cloned and found to have identical sequences at their termini but divergent internal sequences. In addition, the PIF elements showed a marked specificity of insertion sites. Six out of seven PIF-containing derivatives examined had an element inserted at an identical location. Two different members of the PIF element family were identified at this position. The seventh PIF-containing derivative examined had the element inserted at a distinct position within r. Even at this location, however, the element inserted into a conserved target sequence. The timing of PIF excision is unusual. Germinal excision rates can range up to several percent of progeny. Yet somatic sectors are rare, even in lines exhibiting high germinal reversion rates.     

Gene Marker Loss Induced by the Transposable Element, En, in Maize

The En/Spm transposable element system in maize includes the functional element, En/Spm and the receptor element I/dSpm. An En receptor has been found that shows En-induced breakage. This En-responsive receptor (designated 1836518) is located on the short arm of chromosome 9, proximal to Wx. In the presence of En, markers distal to the receptor show a loss of gene expression. Kernels heterozygous for aleurone and endosperm marker genes have a variegated appearance. The hypothesis is advanced that this variegation represents a physical loss of the chromosome segments carrying the genes distal to the receptor position. It is the first case of an En-controlled breakage event.     

The structure and paramutagenicity of the R-marbled haplotype of Zea mays.

Paramutation is the meiotically heritable silencing of a gene that can occur in particular heterozygous combinations. The R-marbled (R-mb) haplotype is paramutagenic: it causes paramutable r1 haplotypes like R-r to become heritably silenced. R-mb was found to comprise three distinct r1 genes arranged as direct repeats. The most distal gene of R-mb, Scm, contains a novel transposable element, Shooter (Sho). Excision of the Sho element early in aleurone development results in the characteristic "marbled" aleurone pigmentation pattern conferred by R-mb. The effect of gene copy number on the paramutagenic strength of R-mb was tested. Paramutagenic strength of R-mb is directly correlated with r1 gene copy number. Paramutagenic strength of R-mb is directly correlated with r1 gene copy number. Paramutagenic strength of R-mb was not affected by removal, through crossing over, of the Sho transposon. Finally, R-mb does not appear to contain the transposable element, Doppia, which is associated with paramutability of R-r, and has been suggested to play a role in paramutagenicity of another paramutagenic haplotype, R-stippled.     

Diversity of LTR-retrotransposons and <Emphasis Type="Italic">Enhancer/Suppressor</Emphasis><Emphasis Type="Italic">Mutator</Emphasis>-like transposons in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Cassava (Manihot esculenta Crantz), though a major world crop with enormous potential, is very under studied. Little is known about its genome structure and organisation. Transposable elements have a key role in the evolution of genome structure, and can be used as important tools in applied genetics. This paper sets out to survey the diversity of members of three major classes of transposable element within the cassava genome and in relation to similar elements in other plants. Members of two classes of LTR-retrotransposons, Ty1/copia-like and Ty3/gypsy-like, and of Enhancer/Suppressor Mutator (En/Spm)-like transposons were isolated and characterised. Analyses revealed 59 families of Ty1/copia, 26 families of Ty3/gypsy retrotransposons, and 40 families of En/Spm in the cassava genome. In the comparative analyses, the predicted amino acid sequences for these transposon classes were compared with those of related elements from other plant species. These revealed that there were multiple lineages of Ty1/copia-like retrotransposons in the genome of cassava and suggested that vertical and horizontal transmission as the source of cassava Mecops may not be mutually exclusive. For the Ty3/gypsy elements network, two groups of cassava Megyps were evident including the Arabidopsis Athila lineage. However, cassava En/Spm-like elements (Meens) constituted a single group within a network of plant En/Spm-like elements. Hybridisation analysis supported the presence of transposons in the genome of cassava in medium (Ty3/gypsy and En/Spm) to high (Ty1/copia) copy numbers. Thus the cassava genome was shown to contain diverse members of three major classes of transposable element; however, the different classes exhibited contrasting evolutionary histories.     

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.     

The wp mutation of Glycine max carries a gene-fragment-rich transposon of the CACTA superfamily

We used soybean (Glycine max) cDNA microarrays to identify candidate genes for a stable mutation at the Wp locus in soybean, which changed a purple-flowered phenotype to pink, and found that flavanone 3-hydroxylase cDNAs were overexpressed in purple flower buds relative to the pink. Restriction fragment length polymorphism analysis and RNA gel blots of purple and pink flower isolines, as well as the presence of a 5.7-kb transposon insertion in the wp mutant allele, have unequivocally shown that flavanone 3-hydroxylase gene 1 is the Wp locus. Moreover, the 5.7-kb insertion in wp represents a novel transposable element (termed Tgm-Express1) with inverted repeats closely related to those of other Tgms (transposable-like elements, G. max) but distinct in several characteristics, including the lack of subterminal inverted repeats. More significantly, Tgm-Express1 contains four truncated cellular genes from the soybean genome, resembling the Pack-MULEs (Mutator-like transposable elements) found in maize (Zea mays), rice (Oryza sativa), and Arabidopsis thaliana and the Helitrons of maize. The presence of the Tgm-Express1 element causing the wp mutation, as well as a second Tgm-Express2 element elsewhere in the soybean genome, extends the ability to acquire and transport host DNA segments to the CACTA family of elements, which includes both Tgm and the prototypical maize Spm/En.     

The behaviour of the autonomous maize transposable element En/Spm in Arabidopsis thaliana allows efficient mutagenesis

The behavior of the autonomous maize transposable element En/Spm of maize was studied in Arabidopsis. Transgenic Arabidopsis plants carrying En-1 elements were propagated for 12 generations using a single seed descent procedure. The distribution and activity of the En-1 element was monitored using Southern DNA hybridisations in generations 1, 6 and 12. In the first generation the highest number of En-1 insertions per line was 7, which increased to 20 in generation 12. The average number of En-1 insertions increased only slightly in the population, due to a gradual accumulation of segregants that lost the transposable element. During the development of the En-1 mutagenised population the element remained active even in the high-copy lines. In situ hybridisation demonstrated that multiple En-1 insertions were distributed over all Arabidopsis chromosomes. From the initial En-1 mutagenised populations many unstable gene mutations were recovered, indicating that En-1 can be used as a efficient tool for gene tagging in Arabidopsis.     

Amplification of genomic sequences flanking transposable elements in host and heterologous plants: a tool for transposon tagging and genome characterization.

The isolation of sequences flanking integrated transposable elements is an important step in gene tagging strategies. We have demonstrated that sequences flanking transposons integrated into complex genomes can be simply and rapidly obtained using the polymerase chain reaction. Amplification of such sequences was established in a model system, a transgenic tobacco plant carrying a single Ac element, and successfully applied to the cloning of a specific Spm element from a maize line carrying multiple Spm hybridizing sequences. The described utilization of methylation sensitive restriction enzymes (including those with degenerate recognition sequences) in the generation of templates for amplification will simplify the cloning and mapping of genomic sequences adjacent to transposable elements.