Recombination between Components of a Mutable Gene System in Maize

An unstable component (I-R) of the R-stippled allele interacts with a linked modifier which enhances stippled's expression ( M-st) to delete the intervening segment. The precision of deletion formation suggests a recombinational basis, specifically unequal crossing over between I-R and M-st. Four deletions were selected as losses of R function from plants homozygous for R-st and M-st. Also lost are stippled's near-colorless seed phenotype, its paramutagenicity and a closely linked gene, Inhibitor of striate. The deletion chromosomes, missing a 6-cM segment, are transmitted normally by ovules but in reduced frequency by pollen. Homozygous and heteroallelic combinations of the deletions confer defective seed lethality. The four did not differ detectably in transmissibility or breakpoint termini. The recurrence of deletions that have the same termini is explained by recombination between I-R and M-st. The homology between M-st and I-R, and their presence in the same chromosome arm, favors the view that M-st originated by I-R transposition.     

Genetic characterization of a mutable allele,R-marbled (R-mb), in maize (Zea mays L.)

TheR-marbled (R-mb) allele in maize confers a distinct pattern of anthocyanin pigmentation in the aleurone. We investigated the genetic mechanism involved in the formation and variability of this pattern. Wide variation was observed in the extent of anthocyanin pigmentation in marbled kernels. Progeny testing of different expressions resulted in distinct segregation profiles, indicating that the somatic variegation has a genetic basis. Drastic reduction in penetrance and expressivity was noticed whenR-mb was transmitted in a single dose through the pollen parent. Analysis of the colored kernels fromR-mb, including discordant endosperm-embryo phenotypes, showed that only germinal reversions fromR-mb toR-sc (self-colored) were transmissible. Unlike other pattern alleles at theR locus, viz.R-stippled (R-st) andR-Navajo (R-nj),R-mb reverts toR-sc at a higher frequency. No dominance-recessiveness relation was found among the three pattern alleles. Reciprocal-cross differences occurred whenR-mb was crossed withR-nj orR-st, but the interaction ofR-mb withR-st was not entirely similar to that withR-nj. The characteristic variegation pattern due toR-mb is attributed to the action of a transposable genetic element on the basis of somatic and germinal instability, occurrence of discordant endosperm-embryo phenotypes, and genetic analysis ofR-mb/R-st andR-mb/R-nj heterozygotes. The distinct genetic behaviour ofR-mb, in comparison withR-st andR-nj, indicates specificity of the controlling element operating at this allele.     

Use of Ac as an insertional mutagen in Arabidopsis

A pilot-scale transposon mutagenesis experiment using a modified autonomous Activator (Ac) element, AcΔNael, was carried out in Arabidopsis thaliana. Four different transformants carrying Ac elements in different and defined genomic locations were used to generate 1000 plants carrying approximately 500 independent germinal transposition events. These plants were then selfed and the 1000 families screened in tissue culture and soil for phenotypic mutants. Fifty different families segregated mutations in their progeny. Preliminary Southern blot analysis of 29 families which segregated mutant progeny, showed that 28 had a transposed Ac. Six of the families were further tested for linkage between the transposed Ac and the mutant phenotype, and instability of the putatively tagged locus. Two of the mutants were shown to be tagged as they were tightly linked to a transposed Ac, and somatic and germinal reversion was associated with loss of Ac. One other mutant locus was shown to be closely linked to a transposed Ac, and therefore was likely to be tagged. The remaining three mutations were not tagged as they were not linked to a transposed Ac. In two of the tagged mutants Ac had transposed to closely linked sites, while in a third mutant the co-segregating Ac had transposed to a site which was not tightly linked to the donor T-DNA. Multiple insertions into the DIF1 locus were found, due to the preferential transposition of Ac to a linked site.     

Transposition of Ac from the P Locus of Maize into Unreplicated Chromosomal Sites

We have analyzed donor and target sites of the mobile element Activator (Ac) that are altered as a result of somatic transposition from the P locus in maize. Previous genetic analysis has indicated that the two mitotic daughter lineages which result from Ac transposition from P differ in their Ac constitution at the P locus. Both lineages, however, usually contain transposed Ac elements which map to the same genetic position. Using methylation-sensitive restriction enzymes and genomic blot analysis, we identified Ac elements at both the donor P locus and Ac target sites and used this assay to clone the P locus and to identify transposed Ac elements. Daughter lineages were shown to be mitotic descendants from a single transposition event. When both lineages contained Ac genetic activity, they both contained a transposed Ac element on identical genomic fragments independent of the genetic position of the target site. This indicates that in the majority of cases, Ac transposition takes place after replication of the donor locus but before completion of replication at the target site.     

A chromosome replication pattern deduced from pericarp phenotypes resulting from movements of the transposable element, modulator, in maize

Modulator (Mp) was mapped after it transposed from the P locus on chromosome 1 by studying 105 light variegated/red twin sectors on medium variegated pericarp ears. Sixty-one percent of the receptor sites were detectably linked to P, and these showed an asymmetry of distribution adjacent to P. No transpositions were mapped in the 4 map units proximal to P, whereas 23 cases mapped to the same length distal to P. The remaining transpositions of Mp on chromosome 1, both proximal and distal to P, were equally scattered. It has previously been shown that when Modulator transposes it replicates at the P locus and a second time at the receptor site. The pattern of transposition adjacent to P is consistent with a hypothesis that a replicon initiation site is situated proximal to P; that Modulator transposes at the time of replication; that it is not able to transpose into a replicated region but only into a replicating one. No difference in distribution of receptor sites was found when the Modulator was detected vs. not detected in the red co-twins by testing with a Dissociation element.     

The I--R system of hybrid dysgenesis in Drosophila melanogaster: are I factor insertions responsible for the mutator effect of the I--R interaction?

Summary When Drosophila melanogaster males coming from a class of strains known as inducer are crossed with females from the complementary class (reactive), a quite specific kind of sterile female (SF) is obtained that exhibits other dysgenic traits such as non-disjunctions and non-randomly distributed mutations. This syndrome is caused by the interaction of the I factor linked to inducer chromosomes with the maternally inherited reactive state R. This I-R interaction is also responsible for chromosomal contamination that is likely to result from very frequent I factor insertions into reactive chromosomes. Such insertions might be responsible for the I-R induced mutations and some data concerning this hypothesis are reported here.Out of nine I-R-generated mutants one, the white ^IR1 (w ^IR1) allele, is closely linked to an I factor, which maps either at the site of the mutation or within less than 0.02 map units. In addition, w ^IR1 is somewhat unstable when transmitted through SF females.In contrast, the typical I factor does not seem to be associated with any of the eight other mutants as judged by their inability to induce the female sterility characteristic of the I-R syndrome. The possibility is discussed that most of I-R-induced mutations are nevertheless caused by insertions of either undetectable I factors or other transposable elements, not related to I, whose transposition is dependent on the I-R interaction.     

Successive transposition explosions in Drosophila melanogaster and reverse transpositions of mobile dispersed genetic elements

Transposition outbursts occur in the destabilized Drosophila melanogaster strain ct^MR2 carrying a mutation in the locus cut induced by an insertion of mdg4. While the distribution of mobile genetic elements remained unchanged in the great majority of germ cells, in a few cells numerous transpositions had occurred involving mdg (copia-like), fold-back and P-elements. We used in situ hybridization to analyze the distribution of five families of mdg elements in the X-chromosome during several consequent mutational changes in D. melanogaster. Each of them was accompanied by many changes in mdg localization, all of which occurred in one and the same cell. Thus, we could observe the series consisting of up to five successive transposition explosions leading to an almost complete change in the distribution of the mdg elements tested. We also found that in the course of successive transposition explosions, mdg elements often inserted into those sub-sections of the X-chromosome where they had previously been located. This phenomenon, designated as reverse directed transposition, was studied in more detail on insertion into the locus yellow. The rate of reverse transposition of the same mdg element to the corresponding locus was 10–100 times as high as that of primary insertion. In some cases, `the transposon shuttle' into and out of the locus was observed. The existence of `transposition memory' partially explains the specificity of mdg localization in closely related strains as well as the co-ordinated behaviour of different mdg elements in independent transposition explosions. The evolutionary significance of transposition explosions and directed reverse transposition (transposon shuttle) is discussed.     

Distribution of Unlinked Receptor Sites for Transposed Ac Elements from the Bz-M2(ac) Allele in Maize

We have shown before that the Ac element from the maize bz-m2(Ac) allele, located in the short arm of chromosome 9 (9S), transposes preferentially to sites that are linked to the bz donor locus. Yet, about half of the Ac transpositions recovered from bz-m2(Ac) are in receptor sites not linked to the donor locus. In this study, we have analyzed the distribution of those unlinked receptor sites. Thirty-seven transposed Ac (trAc) elements that recombined independently of the bz locus were mapped using a set of wx reciprocal translocations. We found that the distribution of unlinked receptor sites for trAs was not random. Ten trAcs mapped to 9L, i.e., Ac had transposed to sites physically, if not genetically, linked to the donor site. Among chromosomes other than 9, the Ac element of bz-m2(Ac) appeared to have transposed preferentially to certain chromosomes, such as 5 and 7, but infrequently to others, such as 1, the longest chromosome in the maize genome. The seven trAc elements in chromosome 5 were mapped relative to markers in 5S and 5L and localized to both arms of 5. We also investigated the transposition of Ac to the homolog of the donor chromosome. We found that Ac rarely transposes from bz-m2(Ac) to the homologous chromosome 9. The clustering of Ac receptor sites around the donor locus has been taken to mean that a physical association between the donor site and nearby receptor sites occurs during transposition. The preferential occurrence of 9L among chromosomes harboring unlinked receptor sites would be expected according to this model, since sites in 9L would tend to be physically closer to 9S than sites in other chromosomes. The nonrandom pattern seen among the remaining chromosomes could reflect an underlying nuclear architecture, i.e., an ordering of the chromosomes in the interphase nucleus, as suggested from previous cytological observations.     

Developmental profile of P element transposition inDrosophila somatic cells

Transposition of the P element duringDrosophila ontogenesis was monitored. A modified P element was transposed by the PΔ2-3 transposase source. P elements inserted into the genome were cloned by the plasmid rescue at various developmental stages of the G1 hybrid to trace events in somatic cells. The transposed elements were directly counted by analyzing RFLP of genomic DNA fragments flanking the P elements. Transposition began from the late embryonic stage, but occurred rarely. Frequent transposition was observed from the late third instar to early pupal stage. From these results, transposition of the P element would appear to be affected by the developmental state of somatic host cells.     

TED,an Autonomous and Rare Maize Transposon of the Mutator Superfamily with a High Gametophytic Excision Frequency

Mutator (Mu) elements, one of the most diverse superfamilies of DNA transposons, are found in all eukaryotic kingdoms, but are particularly numerous in plants. Most of the present knowledge on the transposition behavior of this superfamily comes from studies of the maize (Zea mays) Mu elements, whose transposition is mediated by the autonomous Mutator-Don Robertson (MuDR) element. Here, we describe the maize element TED (for Transposon Ellen Dempsey), an autonomous cousin that differs significantly from MuDR. Element excision and reinsertion appear to require both proteins encoded by MuDR, but only the single protein encoded by TED. Germinal excisions, rare with MuDR, are common with TED, but arise in one of the mitotic divisions of the gametophyte, rather than at meiosis. Instead, transposition-deficient elements arise at meiosis, suggesting that the double-strand breaks produced by element excision are repaired differently in mitosis and meiosis. Unlike MuDR, TED is a very low-copy transposon whose number and activity do not undergo dramatic changes upon inbreeding or outcrossing. Like MuDR, TED transposes mostly to unlinked sites and can form circular transposition products. Sequences closer to TED than to MuDR were detected only in the grasses, suggesting a rather recent evolutionary split from a common ancestor.     

ISLpl1 Is a Functional IS30-Related Insertion Element in Lactobacillus plantarum That Is Also Found in Other Lactic Acid Bacteria

We describe the first functional insertion sequence (IS) element in Lactobacillus plantarum. ISLpl1, an IS30-related element, was found on the pLp3 plasmid in strain FB335. By selection of spontaneous mutants able to grow in the presence of uracil, it was demonstrated that the IS had transposed into the uracil phosphoribosyltransferase-encoding gene upp on the FB335 chromosome. The plasmid-carried IS element was also sequenced, and a second potential IS element was found: ISLpl2, an IS150-related element adjacent to ISLpl1. When Southern hybridization was used, the copy number and genome (plasmid versus chromosome) distribution data revealed different numbers and patterns of ISLpl1-related sequences in different L. plantarum strains as well as in Pediococcus strains. The ISLpl1 pattern changed over many generations of the strain L. plantarum NCIMB 1406. This finding strongly supports our hypothesis that ISLpl1 is a mobile element in L. plantarum. Database analysis revealed five quasi-identical ISLpl1 elements in Lactobacillus, Pediococcus, and Oenococcus strains. Three of these elements may be cryptic IS, since point mutations or 1-nucleotide deletions were found in their transposase-encoding genes. In some cases, ISLpl1 was linked to genes involved in cold shock adaptation, bacteriocin production, sugar utilization, or antibiotic resistance. ISLpl1 is transferred among lactic acid bacteria (LAB) and may play a role in LAB genome plasticity and adaptation to their environment.     

Mobile elements and transposition events in the cut locus of Drosophila melanogaster

We have cloned from the Oregon R strain of Drosophila melanogaster a 240 kb segment of DNA that contains the cut (ct) locus, and characterized the region for the presence of repetitive elements. Within this region at least five copies of the suffix element were detected, as well as several putatively novel mobile elements. A number of mutations obtained from the unstable ct ^MR2 strain and its derivatives were mapped within the cut locus. Comparison between parental and daughter strains indicates that frequently two or more independent transposition events involving the cut locus occur simultaneously within a single germ cell, thus providing a molecular basis for the transposition explosion phenomenon.     

Plant color effects of certain anomalous forms of theRrallele in maize

Summary The genetically changes forms ofR ^r arising regularly in maize plants heterozygous for the stippled, light stippled, and marbled alleles, and previously shown to influence aleurone color differentially, were found to affect coleoptile and seedling leaf sheath color also. Failure to demonstrate corresponding differential effects on root pigmentation possibly was due to inadequacy of the testing procedure. Two of the modified forms ofR ^r were observed to reduce glume and anther color also. The experimental results imply that theR locus as a region is involved in the phenomenon.Paper No. 677 from the Department of Genetics, College of Agriculture, University of Wisconsin.     

Characterization of the new insertion sequence IS91 from an alpha-hemolysin plasmid of Escherichia coli

Summary IS91 is a 1.85 kb insertion sequence originally resident in the -hemolytic plasmid pSU233. The element was transposed sequentially from this plasmid to pA-CYC184, to R388, and to pBR322. Both cointegrates and simple insertions of the element were obtained. A detailed restriction enzyme map of the element is presented. This does not bear any relationship to the maps of previously described insertion sequences. Furthermore, hybridization between these sequences and IS91 could not be demonstrated.Deletion derivatives of IS91 were constructed which are unable to transpose. However, their transposition can be complemented in Trans by wild-type elements. One of these deletion derivatives has been genetically labeled with a kanamycin resistance marker from Tn5. When this new element was complemented for transposition, only about 2% of the transposition products were cointegrates. Thus, the behavior of IS91 is better explained by transposition models that allow direct transposition.Part of this work was carried out by E. Diaz-Aroca as a requirement for her degree in Sciences. The work is published (Esmeralda Diaz-Aroca, Tesina de Licenciatura, Universidad Autónoma de Madrid, 1983) and it contains the complete details of procedures and results of the cloning experiments and the restriction maps of the plasmids shown in this work. It is available from the authors upon request     

Frequency and distance of transposition of a modifiedDissociation element in transgenic tobacco

Effective transposon tagging with theAc/Ds system in heterologous plant species relies on the accomplishment of a potentially high transposon-induced mutation frequency. The primary parameters that determine the mutation frequency include the transposition frequency and the transposition distance. In addition, the development of a generally applicable transposon tagging strategy requires predictable transposition behaviour. We systematically analysedDs transposition frequencies andDs transposition distances in tobacco. An artificialDs element was engineered with reporter genes that allowed transposon excision and integration to be monitored visually. To analyse the variability ofDs transposition between different tobacco lines, eight single copy T-DNA transformants were selected. Fortrans-activation of theDs elements, differentAc lines were used carrying an unmodifiedAc ⁺ element, an immobilizedsAc element and a stableAc element under the control of a heterologous chalcone synthas (chsA) promoter. With allAc elements, eachDs line showed characteristic and heritable variegation patterns at the seedling level. SimilarDs line-specificity was observed for the frequency by whichDs transpositions were germinally transmitted, as well as for the distances of theDs transpositions. ThesAc element induced transposition ofDs late in plant development, resulting in low germinal transposition frequencies (0.37%) and high incidences of independent transposition (83%). The majority of theseDs elements (58%) transposed to genetically closed linked sites (10 cM).     

Transposition of the maize autonomous element Activator in transgenic Nicotiana plumbaginifolia plants

The maize autonomous transposable element Ac was introduced into haploid Nicotiana plumbaginifolia via Agrobacterium tumefaciens transformation of leaf disks. All the regenerated transformants (R0) were diploid and either homozygous or heterozygous for the hygromycin resistance gene used to select primary transformants. The Ac excision frequency was determined using the phenotypic assay of restoration of neomycin phosphotransferase activity and expression of kanamycin resistance among progeny seedlings. Some of the R0 plants segregated kanamycin-resistant seedlings in selfed progeny at a high frequency (34 to 100%) and contained one or more transposed Ac elements. In the primary transformants Ac transposition probably occurred during plant regeneration or early development. Other R0 transformants segregated kanamycin-resistant plants at a low frequency (≤ 4%). Two transformants of this latter class, containing a unique unexcised Ac element, were chosen for further study in the expectation that their kanamycin resistant progeny would result from independent germinal transposition events. Southern blot analysis of 32 kanamycin-resistant plants (R1 or R2), selected after respectively one or two selfings of these primary transformants, showed that 27 had a transposed Ac at a new location and 5 did not have any Ac element. Transposed Ac copy number varied from one to six and almost all transposition events were independent. Southern analysis of the R2 and R3 progeny of these kanamycin-resistant plants showed that Ac continued to transpose during four generations, and its activity increased with its copy number. The frequency of Ac transposition, from different loci, remained low (≤ 7%) from R0 to R3 generations when only one Ac copy was present. The strategy of choosing R0 plants that undergo a low frequency of germinal excision will provide a means to avoid screening non-independent transpositions and increase the efficiency of transposon tagging.     

EVOLUTIONARY STEPS AND TRANSPOSABLE ELEMENTS IN DROSOPHILA MELANOGASTER: THE MISSING RP TYPE OBTAINED BY GENETIC TRANSFORMATION

The I-R and P-M hybrid dysgenesis systems in Drosophila melanogaster have been interpreted as due to recent invasions of the genome by the I and P mobile genetic elements. Temporal and geographical surveys have never shown individuals harboring P sequences but devoid of active I elements. We describe here the successful genetic transformation by autonomous P elements of embryos initially devoid of active I elements and any P sequences. The results demonstrate that P elements may invade the genome of Drosophila melanogaster in the absence of active I elements. Using gel blotting, in situ hybridization techniques, and genetic experiments, we have monitored the behavior of newly introduced P elements in several transformed lines over 30 generations. The switch of cytotype from M to P occurred very slowly and the number of P copies simultaneously increased to about 25. These RP lines possess the properties required to induce P-M hybrid dysgenesis but totally retain the R cellular state. Consequently, this new mobile element combination presents a strong reciprocal post-zygotic isolation with IM strains due to both P-M and I-R hybrid dysgenesis systems. This genomic incompatibility could be considered as a first step toward speciation in Drosophila populations.