Evolution of Ac and Dsl elements in select grasses (Poaceae)

We present data on evolution of the Ac/Ds family of transposable elements in select grasses (Poaceae). An Ac-like element was cloned from a DNA library of the grass Pennisetum glaucum (pearl millet) and 2387 bp of it have been sequenced. When the pearl millet Ac-like sequence is aligned with the corresponding region of the maize Ac sequence, it is found that all sequences corresponding to intron II in maize Ac are absent in pearl millet Ac. Kimura's evolutionary distance between maize and pearl millet Ac sequences is estimated to be 0.429±0.020 nucleotide substitutions per site. This value is not significantly different from the average number of synonymous substitutions for coding regions of the Adh1 gene between maize and pearl millet, which is 0.395±0.051 nucleotide substitutions per site. If we can assume Ac and Adh1 divergence times are equivalent between maize and pearl millet, then the above calculations suggest Ac-like sequences have probably not been strongly constrained by natural selection. The level of DNA sequence divergence between maize and pearl millet Ac sequences, the estimated date when maize and pearl millet diverged (25–40 million years ago), coupled with their reproductive isolation/lack of current genetic exchange, all support the theory that Ac-like sequences have not been recently introduced into pearl millet from maize. Instead, Ac-like sequences were probably present in the progenitor of maize and pearl millet, and have thus existed in the grasses for at least 25 million years. Ac-like sequences may be widely distributed among the grasses. We also present the first 2 Dsl controlling element sequences from teosinte species: Zea luxurians and Zea perennis. A total of 10 Dsl elements had previously been sequenced from maize and a distant maize relative, Tripsacum. When a maximum likelihood network of genetic relationships is constructed for all 12 sequenced Dsl elements, the 2 teosinte Dsl elements are as distant from most maize Dsl elements and from each other, as the maize Dsl elements are from one another. Our new teosinte sequence data support the previous conclusion that Dsl elements have been accumulating mutations independently since maize and Tripsacum diverged. We present a scenario for the origin of Dsl elements.     

Molecular evolutionary characterization of an Activator (Ac)-like transposable element sequence from pearl millet (Pennisetum glaucum) (Poaceae)

We present data on the evolution of the Ac/Ds family of transposable elements in select grasses (Poaceae). A defective Ac-like element was cloned from a DNA library of the grass Pennisetum glaucum (pearl millet) and its entire 4531 bp sequence has been determined. When the pearl millet Ac-like sequence is aligned with the maize Ac sequence, it is found that there is approximately 70% DNA similarity in the central region spanning most of maize Ac exon II and all of exon III. In addition, there are two smaller regions of similarity at the Ac terminii. Besides these three major structural similarities, Pennisetum Ac has two large regions, one 5 and one 3, that show little similarity to Zea Ac. Furthermore, most of the sequences corresponding to intron II in maize Ac are absent in pearl millet Ac. Kimura's evolutionary distance between the central region of maize and pearl millet Ac sequences is estimated to be 0.429±0.020 nucleotide substitutions per site. This value is not significantly different from the average number of synonymous substitutions for coding regions of the Adh1 gene between maize and pearl millet, which is 0.395±0.051 nucleotide substitutions per site. If we assume Ac and Adh1 divergence times are equivalent between maize and pearl millet, then the above calculations suggest Ac-like sequences have probably not been strongly constrained by natural selection. Conserved DNA and amino acid sequence motifs are also examined. The level of DNA sequence divergence between maize and pearl millet Ac sequences, the estimated date when maize and pearl millet diverged (25–40 million years ago), coupled with their reproductive isolation/lack of current genetic exchange, all support the theory that Ac-like sequences have not been recently introduced into pearl millet from maize. Instead, Ac-like sequences were probably present in the progenitor of maize and pearl millet and have thus existed in the grasses for at least 25 million years.     

Distribution of sequences related to the Bg transposable element of maize in Zea and related genera

Thirty-four accessions from Zea and 10 accessions from related genera were assayed for the presence of Bg, a transposable element originally found in maize (Zea mays ssp. mays). Bg-like sequences, identified as hybridizing bands on Southern blots, were visualized in all Zea accessions and were present in approximately equal numbers in teosinte and maize. With the exception of Tripsacum dactyloides, all accessions from related genera failed to hybridize with the Bg probes, even at reduced stringency. A comparison of the restriction patterns of related inbred lines revealed numerous common hybridizing fragments. An index of molecular similarity (MS) was used to determine the degree of similarity between pairs of inbred lines. Computed MS values endorse an inbred relationship and are in good agreement with published results of cluster analysis on these inbred lines.     

The binding motifs for Ac transposase are absolutely required for excision of Ds1 in maize

A reverse genetic system for studying excision of the transposable elementDs1 in maize plants has been established previously. In this system, theDs1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of theAc element, excision ofDs1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences requiredin cis for excision ofDs1. TheDs1 element contains theAc transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5′ subterminal region). We showed that mutation of these motifs abolished completely the excision capacity ofDs1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3′) subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.     

Implications for the cis-requirements for Ds transposition based on the sequence of the wxB4 Ds element

Summary The nucleotide sequence of the 1494 by wxB4 Ds element is presented. A comparison with previously characterized Ds elements reveals several novel features. This element has less Ac terminal sequence than other Ac-like Ds elements. The left terminus contains 398 by of Ac sequence interrupted by a transposon-like DNA insertion, leaving only 317 by of contiguous Ac sequence. The right terminus has 259 by of Ac terminal sequence. The interior of the element contains sequences not found in other cloned members of the Ac/Ds family. We suggest that the role of this non-Ac DNA is to separate the Ac termini by a minimum distance and may be a cis requirement for Ds transposition in maize.Abbreviations Ac activator - Adh1 alcohol dehydrogenase 1 - Ds dissociation - RFLP restriction fragment length polymorphism - Spm suppressor mutator - Wx waxy     

A maize cryptic Ac-homologous sequence derived from an Activator transposable element does not transpose.

Summary Sequences sharing homology to the transposable element Activator (Ac) are prevalent in the maize genome. A cryptic Ac-like DNA, cAc-11, was isolated from the maize inbred line 4Co63 and sequenced. Cryptic Ac-11 has over 90% homology to known Ac sequences and contains an 11 by inverted terminal repeat flanked by an 8 by target site duplication, which are characteristics of Ac and Dissociation (Ds) transposable elements. Unlike the active Ac element, which encodes a transposase, the corresponding sequence in cAc-11 has no significant open reading frame. A 44 by tandem repeat was found at one end of cAc-11, which might be a result of aberrant transposition. The sequence data suggest that cAc-11 may represent a remnant of an Ac or a Ds element. Sequences homologous to cAc-11 can be detected in many maize inbred lines. In contrast to canonical Ac elements, cAc-11 DNA in the maize genome is hypermethylated and does not transpose even in the presence of an active Ac element.     

Binding ofNicotiana nuclear proteins to the subterminal regions of theAc transposable element

Specific binding ofNicotiana nuclear protein(s) to subterminal regions of theAc transposable element was detected using gel mobility shift assays. A sequence motif (GGTAAA) repeated in both terminal regions ofAc, was identified as the protein binding site. Mutation of two nucleotides in this motif was sufficient to abolish binding. Based on a series of competition assays, it is deduced that there is cooperative binding between two repeats, each similar to the GGTAAA motif. The binding protein is probably similar to a previously characterized maize protein which binds to a GGTAAA-containing motif located in the ends ofMutator. Moreover, we show that DNA fromDs1 competes for protein binding toAc termini, and we show, by sequence analysis, that GGTAAA binding sites are present in the terminal region ofTgm1, Tpn1, En/Spm, Tam3 andDs1-like elements. This suggests that the binding protein(s) might be involved in the transposition process.     

Characterization of theAc/Ds behaviour in transgenic tomato plants using plasmid rescue

We describe the use of plasmid rescue to facilitate studies on the behaviour ofDs andAc elements in transgenic tomato plants. The rescue ofDs elements relies on the presence of a plasmid origin of replication and a marker gene selective inEscherichia coli within the element. The position within the genome of modifiedDs elements, rescued both before and after transposition, is assigned to the RFLP map of tomato. Alternatively to the rescue ofDs elements equipped with plasmid sequences,Ac elements are rescued by virtue of plasmid sequences flanking the element. In this way, the consequences of the presence of an (active)Ac element on the DNA structure at the original site can be studied in detail. Analysis of a library ofAc elements, rescued from the genome of a primary transformant, shows thatAc elements are, infrequently, involved in the formation of deletions. In one case the deletion refers to a 174 bp genomic DNA sequence immediately flankingAc. In another case, a 1878 bp internalAc sequence is deleted.     

Somaclonal variation in a maize inbred line is not associated with changes in the number or location of Ac-homologous sequences

Summary Somaclonal variation (tissue culture-induced mutations) may result, in some instances, from the activation of transposable elements. This study was conducted to determine whether somaclonal variants in the Zea maize L. inbred line FR27rhm were associated with movement of the transposable element Activator (Ac). Ten variants, seven of which from genetic analyses fit a single recessive gene model and three which did not due to a low number of mutant plants, were selected for analysis. Total DNA from these and from uncultured FR27rhm seedlings were examined by Southern blot analysis using the internal 1.6-kb Hind III fragment derived from the cloned Ac7 element as a probe. By using a restriction endonuclease which does not cut within the element, the number and distribution of the copies of Acrelated sequences in the FR27rhm genome could be determined. From the number of bands seen in the blots, we conclude that the FR27rhm inbred contains large numbers of Ac -related sequences. However, the pattern of bands seen in the ten variants and in the uncultured seedlings were identical, indicating that there had been no movement of any of the Acrelated sequences to cause the tissue culture-induced mutations.Contribution from Department of Agronomy, University of Illinois, Urbana, IL 61801, supported by funds from the Illinois Agricultural Experiment Station and Illinois Foundation Seeds     

Analysis of a case of somatic instability in a strain of maize from India

A case of somatic instability affecting aleurone colour in a strain of maize from India with flint background was analysed. The somatic instability is localized to theC ¹ (Inhibitor) allele ofC locus on the short arm of chromosome 9. Molecular tests indicated thatAc is not present in the Indian stock and the evidence is consistent with the involvement of theEn (Spm) transposable element in the instability. The presence of theEn (Spm)-like element in the stock would suggest that these elements have been present in the maize genome for a long time. A new allele ofshrunken (sh1) gene with a somewhat unorthodox breeding behaviour is also described.     

Genetic and molecular analysis of tissue-culture-derived Ac elements

Summary Our previous experiments on maize (Zea mays L.) plants regenerated from tissue culture revealed genetic activity characteristic of the transposable element Activator (Ac) in the progeny of 2–3% of the plants tested, despite the lack of Ac activity in the progenitor plants. The objective of the present study was to determine whether the presence of Ac activity in tissue-culture-derived plants was associated with changes in the number or structure of Ac-homologous DNA sequences. Families segregating for Ac activity were obtained by crossing plants heterozygous for Ac activity onto Ac-responsive tester plants. A DNA probe derived from a previously isolated Ac sequence was used to examine the Ac-homologous sequences within individual progeny seedlings of segregating families and noncultured control materials. All plants tested had six or more Ac-homologous DNA sequences, regardless of whether Ac activity was present. In the segregating progeny of one tissue-culturederived plant, a 30-kb Ac-homologous SstI restriction fragment and a 10-kb Ac-homologous BglII restriction fragment were found to cosegregate with Ac activity. We propose that these fragments contained a previously silent Ac sequence that had been activated during tissue culture. Although one or more Ac sequences were often hypomethylated at internal PvuII and HpaII sites in plants with Ac activity, hypomethylation was not a prerequisite for activity. Reduced methylation at these sites may have been a result rather than a cause of Ac activity.     

Molecular and functional characterization of Slide, anAc-like autonomous transposable element from tobacco

A new transposable element of tobacco, Slide, was isolated from thetl mutant line, which shows somatic instability, after its transposition into a locus encoding nitrate reductase (NR). The Slide-124 element is 3733 bp long and its coding sequences show similarities with conserved domains of the transposases ofAc, Tam3 andhobo. Excision from the NR locus is detectable in somatic leaf tissues and Slide mobility is triggered by in vitro tissue culture. Slide excision events create footprints similar to those left byAc and Tam3. Tobacco lines derived from thetl mutant line seem characterized by unmethylated copies of a few members of the highly repetitive Slide family. Slide mobility was monitored in transient expression assays. In wild-type tobacco protoplasts, the complete Slide element, as well as a defective copy, is able to excise. The complete Slide element, but not the defective version, is able to excise in protoplasts of the heterologous species lettuce (Lactuca sativa). These results show that Slide carries the functions required for its own mobility, and represents the first autonomousAc-like element characterized inSolanaceae species.     

Binding sites for maize nuclear proteins in the subterminal regions of the transposable elementActivator

Genetic data suggest that transposition of the maize elementActivator (Ac) is modulated by host factors. Using gel retardation and DNase I protection assays we identified maize proteins which bind to seven subterminal sites in both ends ofAc. Four DNase I-protected sites contain a GGTAAA sequence, the other three include either GATAAA or GTTAAA. The specificity of the maize protein binding toAc was verified by using a synthetic fragment containing four GGTAAA motifs as probe and competitor in gel retardation assays. All seven binding sites are located within regions requiredin cis for transposition. A maize protein binding site with the same sequence has previously been identified in the terminal inverted repeats of the maizeMutator element. Thus, the protein, that recognizes this sequence is a good candidate for a regulatory host factor forAc transposition.     

Distribution of Bs1 retrotransposons in Zea and related genera

Summary Thirty-eight accessions from Zea and 20 accessions from related genera were probed for the presence of Bs1, a retrotransposon originally found in maize. All maize and teosinte plants tested show the presence of Bs1 in one to five densely hybridizing bands. The mean copy numbers of Bs1 elements among the maize and teosinte accessions were similar: 2.92 and 3.25, respectively, with no large differences between any subgroups. Most exotic maize samples exhibited two common bands of 7.8 kb and 4.7 kb. Section Zea teosintes (but not teosintes of section Luxuriantes) also show the presence of a common band of the same size as the smaller common band in maize. At reduced stringency, Tripsacum dactyloides exhibited a single hybridizing band at 6.9 kb. Results argue for the evolution of maize from a mexicana or parviglumis teosinte, and the evolution of the Bs1 element within the tribe Andropogoneae. Additionally, recombinant inbred lines were probed for the presence of Bs1, in order to map the chromosomal locations of Bs1 elements in four different maize lines. Two of the recombinant inbred parental lines had an element (Bs1-F) on chromosome 5, while the other two lines had an element (Bs1-S) on chromosome 8. Restriction site polymorphisms have apparently arisen in the vicinity of Bs1-S since its insertion. Segregation analysis of other lines was also performed; the data indicate that Bs1 has the distribution expected of a transposable element, different locations in different lines, and not that of a fixed gene locus. However, the common bands in the Zea mays lines and the recombinant inbred data imply that Bs1 is not highly mobile.     

Spontaneous chromosome inversions of guatemalan teosintes (Zea mexicana)

Two paracentric inversions,In3 andIn9, were found in the F1 hybrids of maize and Florida teosinte and these inversions were contributed by the teosinte parent. The length ofIn3 was equivalent to about 35 percent of the length of the long arm of chromosome 3, while that ofIn9, about 60 percent of the length of the short arm of chromosome 9.There were also two paracentric inversions,In1 andIn9, in the F1 hybrids of maize and Jutiapa teosinte and these inversions were inherited from the teosinte parent. The length of theIn1 occupied 22 percent of the total length of the long arm of chromosome 1, while that ofIn9, 60 percent of the total length of the short arm of chromosome 9.Only one paracentric inversion,In9, was identified in the F1 hybrids of maize and Lake Retana teosinte, and this inversion was also from the teosinte parent. As length and location are considered, thisIn9 is the same as theIn9's of Florida teosinte and Jutiapa teosinte.At anaphases I and II of the microsporocyte divisions of the F1 hybrids, evidences of crossovers within the inverted segments, such as bridges and fragments, were obtained for all of these inversions. The interchromosome effect ofIn3 of Florida teosinte, and that ofIn1 of Jutiapa teosinte on the frequency of crossovers within the inverted segment ofIn9's are discussed.Chromosome inversions have probably accompanied the divergence of geographical races of teosinte. This might also be true for the race diversities of maize. The absence ofIn9 in certain teosinte races of southern Mexico and northern Guatemala is accounted for by the substitution of maize chromosome for this inversion.     

Molecular cloning of the o2-m5 allele of Zea mays using transposon marking

Summary The deposition of zein protein in maize endosperm is under the control of several regulatory loci. The isolation of DNA sequences corresponding to Opaque-2 (O2), one of such loci, is described in this paper. The mutable allele, o2-m5 was first induced moving the Ac transposable element present at the wx-m7 allele to the O2 locus. Genetic data suggest that a functional Ac element is responsible for the observed somatic mutability of o2-m5. The isolation of genomic clones containing flanking sequences corresponding to the O2 gene was possible by screening an o2-m5 genomic libary with a probe corresponding to internal Ac sequences usually absent in the defective element Ds. Out of 27 clones isolated with homology to the central part of Ac element, only clones 6IP and 21IP generated a 2.5 kb internal fragment size of an active Ac element when digested with PvuII restriction enzyme. A sequence representing a XhoI fragment of 0.9 kb lying, in the 6IP clone, adjacent to the Ac elements, was subcloned and utilized to prove that it corresponded to a part of the O2 gene. To obtain this information we made use of: (1) DNAs from several reversions originating from the unstable (o2mk-(r) allele, which, when digested with SstI, showed a correct 3.4 kb fragment typical of non-inserted alleles of the O2 locus; and (2) recessive alleles of the O2 locus which were devoid of a 2.0 kb mRNA, present on the contrary in the wild type and in other zein regulating mutants different from O2.This paper is dedicated to the memory of R. Marotta, who actively participated in the realization of this work     

The binding motifs forAc transposase are absolutely required for excision ofDs1 in maize

A reverse genetic system for studying excision of the transposable elementDs1 in maize plants has been established previously. In this system, theDs1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of theAc element, excision ofDs1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences requiredin cis for excision ofDs1. TheDs1 element contains theAc transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5′ subterminal region). We showed that mutation of these motifs abolished completely the excision capacity ofDs1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3′) subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.     

Preferential transposition ofAc to linked sites in Arabidopsis

We have investigated the pattern of transposition of an intact, 4.6-kbAc element inArabidopsis thaliana. Because the trans-acting transposition function (transposase) ofAc is not fully penetrant in Arabidopsis, it is not possible to use it as a diagnostic feature to scoreAc genetically, as has been done in maize and tobacco. Instead, the presence or absence of a transposedAc (trAc) was monitored by Southern blots. Germinal transpositions from the marker SPT::Ac were selected using a streptomycin germination assay and scored for the presence of atrAc. Segregation of thetrAc element and the SPT donor locus was scored in the F₂ progeny of the germinal revertants, and the recombination fraction between thetrAc element and SPT was estimated by the method of maximum likelihood. We have found that, as in maize and tobacco, receptor sites fortrAcs in Arabidopsis tend to be linked to theAc donor locus.     

Transposition of the maize transposable element Ac in Solanum tuberosum

Summary The maize transposable element Ac has been introduced into potato via the T-DNA (transferred DNA) of Agrobacterium tumefaciens. Ac was inserted within the untranslated leader region of a neomycin phosphotransferase II (NPT-II) gene such that excision restored NPT-II activity. Two approaches to monitor Ac excision were used. (i) Using an Agrobacterium strain harbouring plasmid pGV3850::pKU3, leaf discs were selected on kanamycin (Km) after exposure to Agrobacterium. (ii) Using a strain containing plasmid pGV3850HPT::pKU3, the leaf discs were selected on hygromycin (Hm) and the resulting shoots were checked for NPT-II expression. Thirteen kanamycin resistant shoots transformed with pGV3850::pKU3 were isolated, suggesting that Ac had excised from the NPT-II gene. Out of 43 hygromycin resistant shoots transformed with pGV3850HPT::pKU3, 22 expressed the NPT-II gene, indicating that Ac had undergone excision in approximately 50% of the hygromycin resistant shoots. Southern analysis revealed that all kanamycin resistant plants contained the DNA restriction fragments expected when Ac excises from the NPT-II gene. The presence of Ac at new locations within the genomic DNA of several transformants was also detected.