Genetic analysis of instability in Petunia hybrida

Summary In crossing experiments with Petunia hybrida, new mutations, some unstable, have been found in descendants of plants having an unstable allele of the anthocyanin gene An1. One of the unstable mutations affecting the new anthocyanin gene An11 was genetically analyzed, and it was subsequently established in which step of anthocyanin synthesis that An11 is involved. The discovery of new, unstable mutations at other loci indicates that in Petunia also a relation exists between unstable mutations and the presence of transposable elements in the genome. It was demonstrated that reverted alleles (an1 ^+/+) originating from unstable An1 alleles are less stable than the original wild-type allele An1, and that reversions do not increase the chances of occurrence of new, stable or unstable mutations at other loci. These results provide additional arguments in favour of the hypothesis posed in an earlier paper that reversions of unstable An1 alleles are not the result of excision of the inserted transposable element, but are due to the repair of secondary mutations induced by the insert in the regulatory region of the locus. Consequently, a reverted allele still contains the inserted element that may again induce mutations leading to inactivation of An1.     

Genomic regulation of transposable elements in Drosophila

Transposable elements are a major source of genetic change, including the creation of novel genes, the alteration of gene expression in development, and the genesis of major genomic rearrangements. They are ubiquitous among contemporary organisms and probably as old as life itself. The long coexistence of transposable elements in the genome would be expected to be accompanied by host-element coevolution. Indeed, the important role of host factors in the regulation of transposable elements has been illuminated by recent studies of several systems in Drosophila. These include host factors that regulate the P element, a host mutation that renders the genome permissive for gypsy mobilization and infection, and newly induced mutations that affect the expression of transposon insertion mutations. The finding of a type of hybrid dysgenesis in D. virilis, in which multiple unrelated transposable elements are mobilized simultaneously, may also be relevant to host-factor regulation of transposition.     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

Identification of putative nonautonomous transposable elements associated with several transposon families inCaenorhabditis elegans

Putative nonautonomous transposable elements related to the autonomous transposons Tc1, Tc2, Tc5, andmariner were identified in theC. elegans database by computational analysis. These elements are found throughout theC. elegans genome and are defined by terminal inverted repeats with regions of sequence similarity, or identity, to the autonomous transposons. Similarity between loci containing related nonautonomous elements ends at, or near, the boundaries of the terminal inverted repeats. In most cases the terminal inverted repeats of the putative nonautonomous transposable elements are flanked by potential target-site duplications consistent with the associated autonomous elements. The nonautonomous elements identified vary considerably in size (from 100 by to 1.5 kb in length) and copy number in the available database and are localized to introns and flanking regions of a wide variety ofC. elegans genes. Correspondence to: W. Belknap     

Mu1-Related Transposable Elements of Maize Preferentially Insert into Low Copy Number DNA

The Mutator transposable element system of maize was originally identified through its induction of mutations at an exceptionally high frequency and at a wide variety of loci. The Mu1 subfamily of transposable elements within this system are responsible for the majority of Mutator-induced mutations. Mu 1-related elements were isolated from active Mutator plants and their flanking DNA was characterized. Sequence analyses revealed perfect nine base target duplications directly flanking the insert for 13 of the 14 elements studied. Hybridizational studies indicated that Mu1-like elements insert primarily into regions of the maize genome that are of low copy number. This preferential selection of low copy number DNA as targets for Mu element insertion was not directed by any specific secondary structure(s) that could be detected in this study, but the 9-bp target duplications exhibited a discernibly higher than random match with the consensus sequence 5'-G-T-T-G-G/C-A-G-G/A-G-3'.     

The use of transgenic plants to understand transposition mechanisms and to develop transposon tagging strategies

This review compares the activity of the plant transposable elements Ac, Tam3, En/Spm and Mu in heterologous plant species and in their original host. Mutational analysis of the autonomous transposable elements and two-element systems have supplied data that revealed some fundamental properties of the transposition mechanism. Functional parts of Ac and En/Spm were detected by in vitro binding studies of purified transposase protein and have been tested for their importance in the function of these transposable elements in heterologous plant species. Experiments that have been carried out to regulate the activity of the Ac transposable element are in progress and preliminary results have been compiled. Perspectives for manipulated transposable elements in transposon tagging strategies within heterologous plant species are discussed.     

Direct determination of the influence of extreme temperature on transposition and structural mutation rates of Drosophila melanogaster mobile elements

Two sets of mutation accumulation lines, one reared at 28°C and the other at 24°C, were compared for their transposition and rearrangement rates of eleven transposable element families. The changes affecting mobile elements were analysed by the Southern technique and in situ hybridization. No differences were found between treated and control lines. The role of the host genotype in transposition control and the significance of structural mutations in transposable element dynamics are discussed.     

Gene tagging in Petunia hybrida using homologous and heterologous transposable elements

In this paper we describe the current state of knowledge of transposable element systems in Petunia hybrida. The main features of the unstable An1 alleles are discussed. The data on derivative (un)stable alleles at different loci are summarized. A simple strategy is outlined for random versus directed gene tagging using endogenous and heterologous elements. The progress in the cloning of endogenous elements is presented.     

Independent transposition of multiple Ac elements in the same transgenic tomato cell

To effectively use transposable elements for the genetic manipulation of plant species lacking well characterized endogenous elements, it is important to evaluate the behavior of known transposable elements following their introduction into heterologous host species. One critical parameter concerns the timing of transposition in relation to the development of the transgenic host since this will affect the frequency with which transposition events are captured in the gametes. In order to examine whether different elements in the same cell are differentially active during development, we used Southern hybridizations to assess the activity of Activator (Ac) elements in progeny plants derived from a tomato transformant carrying five Ac'x at two loci. All of the elements at one locus transposed in the primary transformant at a developmental stage resulting in the transmission of newly transposed elements to the next generation. In contrast, one or more of the Ac's at the second locus were not active at this stage and were transmitted to the next generation at the original donor T-DNA insertion site. These elements were, however, transpositionally active in somatic tissue. These results demonstrated that individual transposable elements in the same transformed cell can be differentially activated during development.     

DNA transposon-based gene vehicles - scenes from an evolutionary drive

DNA transposons are primitive genetic elements which have colonized living organisms from plants to bacteria and mammals. Through evolution such parasitic elements have shaped their host genomes by replicating and relocating between chromosomal loci in processes catalyzed by the transposase proteins encoded by the elements themselves. DNA transposable elements are constantly adapting to life in the genome, and self-suppressive regulation as well as defensive host mechanisms may assist in buffering ‘cut-and-paste’ DNA mobilization until accumulating mutations will eventually restrict events of transposition. With the reconstructed Sleeping Beauty DNA transposon as a powerful engine, a growing list of transposable elements with activity in human cells have moved into biomedical experimentation and preclinical therapy as versatile vehicles for delivery and genomic insertion of transgenes. In this review, we aim to link the mechanisms that drive transposon evolution with the realities and potential challenges we are facing when adapting DNA transposons for gene transfer. We argue that DNA transposon-derived vectors may carry inherent, and potentially limiting, traits of their mother elements. By understanding in detail the evolutionary journey of transposons, from host colonization to element multiplication and inactivation, we may better exploit the potential of distinct transposable elements. Hence, parallel efforts to investigate and develop distinct, but potent, transposon-based vector systems will benefit the broad applications of gene transfer. Insight and clever optimization have shaped new DNA transposon vectors, which recently debuted in the first DNA transposon-based clinical trial. Learning from an evolutionary drive may help us create gene vehicles that are safer, more efficient, and less prone for suppression and inactivation.     

Super-unstable mutations associated with P-M hybrid dysgenesis in Drosophila melanogaster

Super-unstable mutations occasionally appear either in natural populations of Drosophila melanogaster or in P-M hybrid dysgenesis. We found that they may be reproducibly obtained with a high frequency from crosses between males from the % MathType!MTEF!2!1!+-% feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaWaaubiaeqale% qabaWaaubiaeqameqaleaacaGGQaaaoeaacaWGHbGaam4raaaaa0qa% aiaadEhaaaaaaa!3A01!\[\mathop w\nolimits^{\mathop {aG}\nolimits^* } \] strain and females from the w ^aG* strain or its derivatives. Super-unstable mutations in the ocelliless, singed, white, yellow and other loci have been obtained. Each super-unstable mutation gives rise to a large family of new super-unstable mutations with a wide range of phenotypic expression. Mutations with the same phenotype often differ in the specificity of their potential for further mutation. As a rule, a super-unstable mutation is associated with a specific reversible mutation and paired alleles are formed in this way. Other mutations are usually irreversible, but new mutations of these may also form paired alleles. Active transposase encoded by transposable P elements is necessary to maintain super-instability. Finally, some preliminary molecular data are discussed which suggest that this type of super-instability is a result of interaction between P elements and a novel mobile element, designated as X.     

Transposable elements and fitness in Drosophila melanogaster

Transposable elements constitute a significant fraction of the Drosophila melanogaster genome. The five families of moderately repeated transposable elements identified to date occupy dispersed and variable genomic locations, but have relatively constant copy numbers per individual. What effect to these elements have on the fitness of the individuals harboring them? Experimental evidence relating to this question is reviewed. The relevant data fall into two broad categories. The first involves the determination of the distribution of transposable elements in natural populations, by restriction mapping or in situ hybridization, and the comparison of the observed distribution with different theoretical expectations. The second approach is to study directly the effects of new transposable element-induced mutations on fitness. The P family of transposable elements is a particularly efficient mutagen, and the results of experiments in which initially P-free chromosomes are contaminated with P elements are discussed with regard to P-induced fitness mutations.     

Identification and characterization of the duplicate rice sucrose synthase genes <Emphasis Type="Italic">OsSUS5</Emphasis> and <Emphasis Type="Italic">OsSUS7</Emphasis> which are associated with the plasma membrane

Systematic searches using the complete genome sequence of rice (Oryza sativa) identified OsSUS7, a new member of the rice sucrose synthase (OsSUS) gene family, which shows only nine single nucleotide substitutions in the OsSUS5 coding sequence. Comparative genomic analysis revealed that the synteny between OsSUS5 and OsSUS7 is conserved, and that significant numbers of transposable elements are scattered at both loci. In particular, a 17.6-kb genomic region containing transposable elements was identified in the 5′ upstream sequence of the OsSUS7 gene. GFP fusion experiments indicated that OsSUS5 and OsSUS7 are largely associated with the plasma membrane and partly with the cytosol in maize mesophyll protoplasts. RT-PCR analysis and transient expression assays revealed that OsSUS5 and OsSUS7 exhibit similar expression patterns in rice tissues, with the highest expression evident in roots. These results suggest that two redundant genes, OsSUS5 and OsSUS7, evolved via duplication of a chromosome region and through the transposition of transposable elements.     

Transposable elements in individual genotypes of Drosophila simulans

Transposable elements are abundant, dynamic components of the genome that affect organismal phenotypes and fitness. In Drosophila melanogaster, they have increased in abundance as the species spread out of Africa, and different populations differ in their transposable element content. However, very little is currently known about how transposable elements differ between individual genotypes, and how that relates to the population dynamics of transposable elements overall. The sister species of D. melanogaster, D. simulans, has also recently become cosmopolitan, and panels of inbred genotypes exist from cosmopolitan and African flies. Therefore, we can determine whether the differences in colonizing populations are repeated in D. simulans, what the dynamics of transposable elements are in individual genotypes, and how that compares to wild flies. After estimating copy number in cosmopolitan and African D. simulans, I find that transposable element load is higher in flies from cosmopolitan populations. In addition, transposable element load varies considerably between populations, between genotypes, but not overall between wild and inbred lines. Certain genotypes either contain active transposable elements or are more permissive of transposition and accumulate copies of particular transposable elements. Overall, it is important to quantify genotype‐specific transposable element dynamics as well as population averages to understand the dynamics of transposable element accumulation over time.     

A selection cartridge for rapid detection and analysis of spontaneous mutations including insertions of transposable elements in Enterobacteriaceae

Summary We present a method that allows positive selection and rapid analysis of mutations in Enterobacteriaceae. Mutations are detected in a 2630 bp selection cartridge inserted in two different bacterial mutlicopy plasmid vectors. Spontaneous mutations in Escherichia coli, Enterobacter cloacae and Citrobacter freundii include insertions, deletions and point mutations. The small size of the target sequence facilitates rapid analysis of DNA rearrangements by cleavage with restriction enzymes and of any type of mutation by DNA sequence analysis. While in E. coli insertions of the mobile elements IS1, IS2 and IS5 were readily found, insertions of putative new transposable elements were detected in Enterobacter cloacae. The selection cartridge can thus serve as a tool for studying the spectrum of insertion mutations in Enterobacteriaceae and probably other Gramnegative bacteria, and the dependency of this spectrum on physiological and environmental factors and the host's genetic background can be investigated.     

Horizontal transfer of P elements and other short inverted repeat transposons

Evidence for horizontal transfer of the P family of transposable elements in the genus Drosophila is reviewed and evaluated, along with observations consistent with the recent invasion of Drosophila melanogaster by these elements. Some other examples of horizontal transfer involving other groups of transposable elements having short inverted terminal repeats are also briefly described. The sequential mechanistic steps likely to be involved in a horizontal transfer event are explored, including the requirement for suitable interspecific vectors or carriers. Finally, the frequency and significance of horizontal transfer of transposable elements are briefly discussed within an evolutionary framework.     

Transposable elements controlling genetic instabilities in mammals

It is proposed that the instabilities in gene action of some alleles at certain loci in the mouse (e.g., a, c, H-2 Mi, p, pe, T, Va, W), which do not seem to conform to traditional hypotheses of gene action, are better interpretable in the light of modern studies of transposable DNA elements (insertion sequences and transposons of prokaryotic organisms; controlling elements of maize; transposable controlling elements of Drosophila). Some phenotypic evidence in the mouse in support of this hypothesis is presented for the a, Mi, p, and W loci, which affect pigmentation.     

A genetic analysis of mutations recovered from tomato following Agrobacterium-mediated transformation with the maize transposahle elements Activator and Dissociation

Summary The maize autonomous transposable element, Activator (Ac), and the nonautonomous element Dissociation (Ds), were introduced into the tomato cultivars VF36 and VFNT Cherry by Agrobacterium-mediated transformation. Progeny families from 145 primary transformants were scored at the seedling stage for phenotypically variant individuals. When VF36 was transformed, 20% of families had progeny with aberrant phenotypes. The mutation frequency in VFNT Cherry transformants was lower; in this cultivar 7% of the transformants had progeny segregating for seedling mutations. The majority of the mutations showed monogenic inheritance in the R₁ population, suggesting that the mutations occurred early in the transformation/regeneration process. One mutation, however, was recovered at low frequency in the R₁, suggesting a late mutagenesis event. When tomato was transformed with either the Ac or Ds elements, no differences in mutation frequencies were observed. Since Ac is transpositionally active in tomato transformants while Ds is not, these numbers indicate that the mutation frequency inherent to the transformation process is higher than the mutational activity of Ac. These results demonstrate that efficient gene tagging using heterologous transposable elements will require screening for transposon-induced mutations in later generations.     

The splicing of transposable elements and its role in intron evolution

Recent studies have demonstrated that transposable elements in maize and Drosophila are spliced from pre-mRNA. These transposable element introns represent the first examples of recent addition of introns into nuclear genes. The eight reported examples of transposable element splicing include members of the maize Ac/Ds and Spm/dSpm and the Drosophila P and 412 element families. The details of the splicing of these transposable elements and their relevance to models of intron origin are discussed.     

Paramutation in maize

Paramutation is a heritable change in gene expression induced by allele interactions. This review summarizes key experiments on three maize loci, which undergo paramutation. Similarities and differences between the phenomenology at the three loci are described. In spite of many differences with respect to the stability of the reduced expression states at each locus or whether paramutation correlates with DNA methylation and repeated sequences within the loci, recent experiments are consistent with a common mechanism underlying paramutation at all three loci. Most strikingly, trans-acting mutants have been isolated that prevent paramutation at all three loci and lead to the activation of silenced Mutator transposable elements. Models consistent with the hypothesis that paramutation involves heritable changes in chromatin structure are presented. Several potential roles for paramutation are discussed. These include localizing recombination to low-copy sequences within the genome, establishing and maintaining chromatin domain boundaries, and providing a mechanism for plants to transmit an environmentally influenced expression state to progeny.