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.     

Molecular evolution of human T-cell leukemia virus

Summary In light of previous data, which suggested thatAc-like sequences might have undergone a significant radiation in the recent past, I examined the copy number ofAc-like sequences in representatives of all theZea taxa, both maize and teosinte. The maize and teosinte samples contained approximately equal numbers ofAc-like sequences. FewAc-like sequences were in unmethylated regions of DNA. Unmethylated elements were distributed randomly among both maize and teosinte lines. The appearance in a line of a discrete band resulting from digestion with one methylation-sensitive restriction enzyme was correlated with the appearance of discrete bands with other methylation-sensitive bands. This suggests that individualAc-like elements are occasionally demethylated in many sites. No unmethylated element having restriction fragments of the lengths predicted from the publishedAc sequence was seen in the approximately 326 elements examined.     

Tuareg, a novel miniature-inverted repeat family of pearl millet (Pennisetum glaucum) related to the PIF superfamily of maize

Miniature-inverted repeat transposable elements (MITEs) are abundantly repeated in plant genomes and are especially found in genic regions where they could contribute regulatory elements for gene expression. We describe with molecular and cytological tools the first MITE family reported in pearl millet: Tuareg. It was initially detected in the pearl millet ortholog of Teosinte-branched1, an important developmental gene involved in the domestication of maize. The Tuareg family was amplified recently in the pearl millet genome and elements were found more abundant in wild than in domesticated plants. We found that they shared similarity in their terminal repeats with the previously described mPIF MITEs and that they are also present in other Pennisetum species, in maize and more distantly related grasses. The Tuareg family may be part of MITEs activated by PIF-like transposases and it could have been mobile since pearl millet domestication. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. O. Robin contributed the FISH and fiber-FISH hybridizations.     

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.     

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     

Feature article

Domesticated maize emerged from human selection, exploitation, and cultivation of natural recombinants between two wild grasses that had novel characteristics desired by humans for food. Crossing experiments reconstructing prototypes of ancient archaeological specimens demonstrate how the simple flowering spike of the wild relatives of maize was transformed into the prolific grain-bearing ear within a few generations of intergenomic recombination between teosinte andTripsacum. The high degree of morphological similarities of segregating intercross progeny to archaeological specimens from Tehuacán, Oaxaca, and Tamaulipas provides strong support for this evolutionary scenario. Comparative genomic analysis of maize, teosinte, andTripsacum confirms that maize has inherited unique polymorphisms from aTripsacum ancestor and other unique polymorphisms from a teosinte progenitor. This supports the hypothesis thatTripsacum introgression provided the mutagenic action for the transformation of the teosinte spike into the maize ear. This model for the origin of maize explains its sudden appearance, rapid evolutionary trajectory, and genesis of its spectacular biodiversity.     

Cereal prolamin evolution and homology revealed by sequence analysis

Prolamin mixtures were isolated from oats, rice, normal and high-lysine sorghum, two varieties of pearl millet, two strains of teosinte, and gamma grass and subjected to NH₂-terminal amino acid sequence determinations. In each case (except for rice, whose prolamins apparently have blocked or unavailable NH₂-terminal residues), primarily a single sequence was observed despite significant heterogeneity, suggesting that prolamin homology in each cereal arose through duplication and mutation of a single ancestral gene. Comparisons were then made to prolamin sequences previously determined for wheat, corn, barley, and rye. Within genera, different varieties or subspecies exhibited few differences, but more distantly related genera, subtribes, and tribes showed increasingly large differences. Within the subfamily Festucoideae, no homology was apparent between prolamins of oats and those of the subtribe Triticinae (including wheat, rye, and barley, for which prolamin homology was previously demonstrated). Within the subfamily Panicoideae, corn was shown to be closely related to teosinte but more distantly to Tripsacum. Sorghum was shown to have diverged less from corn than had millet. These comparisons demonstrate that prolamin sequence analyses can successfully predict and clarify evolutionary relationships of cereals.     

Knob heterochromatin homology in maize and its relatives

Summary We have characterised the major DNA sequence component of knob heterochromatin in maize, teosinte andTripsacum. Sequence analysis of this DNA gives strong support to the proposal that maize originated by selection of variants in teosinte. In situ hybridization has confirmed that this repeating DNA sequence, which is the major component of maize knob heterochromatin, is also the major component of knobs in teosinte,Zea diploperennis andTripsacum. In Southern blot hybridizations the repeat has a similar basic organization in all taxa;Tripsacum, however, is differentiated from maize and teosinte by a number of sequence features. Maize and teosinte knob heterochromatin are indistinguishable with regard to the distribution of mutations in the 180-bp repeat and the presence and organization of a 202-bp variant sequence. The knob DNA sequence was not detectable in three species ofCoix, an Old World genus of the Maydeae.Within the repeat unit is a 27-bp region that shows no sequence changes in maize, teosinte orTripsacum. The remainder of the repeat unit has randomly distributed nucleotide changes. The presence of the conserved sequence region suggests that knob DNA may have a functional role in the nucleus.     

Different mechanisms generating sequence variability are revealed in distinct regions of the hydroxyproline-rich glycoprotein gene from maize and related specie*

Summary The sequences of the genes coding for a hydroxyproline-rich glycoprotein from two varieties of maize (Zea mays, Ac1503 and W22), a teosinte (Zea diploperennis) and sorghum (Sorghum vulgare) have been obtained and compared. Distinct patterns of variability have been observed along their sequences. The 500 by region immediately upstream of the TATA box is highly conserved in theZea species and contains stretches of sequences also found in the sorghum gene. Further upstream, significant rearrangements are observed, even between the two maize varieties. These observations allow definition of a 5 region, which is common to the four genes and is probably essential for their expression. The 3 end shows variability, mostly due to small duplications and single nucleotide substitutions. There is an intron present in this region showing a high degree of sequence conservation among the four genes analyzed. The coding region is the most divergent, but variability arises from duplications of fragments coding for similar protein blocks and from single nucleotide substitutions. These results indicate that a number of distinct mechanisms (probably point mutation, transposon insertion and excision, homologous recombination and unequal crossing-over) are active in the production of sequence variability in maize and related species. They are revealed in different parts of the gene, probably as the result of the different types of functional constraints acting on them, and of the specific nature of the sequence in each region.The sequences reported in this paper have been deposited in the EMBL/GenBank Database (Bolt, Beranek, and Newman Laboratories, Cambridge, Mass., and EMBL, Heidelberg), accession nos. M36635 (maize Ac1503), X63134 (maize W22), X64173 (teosinte) and X56010 (sorghum)     

Nucleotide Polymorphism in the Adh1 Locus of Pearl Millet (Pennisetum Glaucum) (Poaceae)

We investigated nucleotide polymorphism in the Adh1 locus of pearl millet (Pennisetum glaucum) (Poaceae) by determining the DNA sequence of 20 alleles from 10 individuals. The individuals were sampled from throughout pearl millet's indigenous range and represent both wild and cultivated accessions. Our results indicated that there is little nucleotide polymorphism in the Adh1 locus. Estimates of per site nucleotide polymorphism did not differ significantly between cultivated and wild millet accessions. We compared nucleotide polymorphism in pearl millet Adh1 with nucleotide polymorphism in maize (Zea mays) Adh1 and conclude that the maize Adh1 sample is more polymorphic. Increased polymorphism in maize Adh1 may be attributable, in part, to faster substitution rates in the maize lineage. Analysis suggests that substitution rates in the maize Adh1 lineage are ~1.7 times faster than substitution rates in the millet Adh1 lineage.     

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.     

Linkage of the alcohol dehydrogenase structural genes in pearl millet (Pennisetum typhoides)

Pearl millet produces three ADH isozymes, Sets I, II, and III. Naturally occurring ADH electrophoretic variants affecting Sets I and II isozymes but not III have been previously described. Analysis of such variants led to the identification of the Adh1 structural gene. The existence of a second Adh structural gene was inferred from dissociation-reassociation studies of Set II. In the present report, a naturally occurring variant affecting the electrophoretic mobility of Sets III and II but not Set I is described. Analysis of this variant confirms the existence of a second structural gene, Adh2. Crosses utilizing this Adh2 marker reveal a dissimilarity with maize and other plants such as sunflower and narrow-leafed lupins. Adh1 and Adh2 of pearl millet do not segregate independently; indeed, no recombinants have been observed. This is the first major difference encountered in an otherwise remarkably similar genetic and environmental control of the ADH isozymes in maize and millet. The organization of the Adh genes of pearl millet may reflect a more primitive arrangement than that of maize.This work was supported by a PHS National Research Service Award Training Grant in Genetics to the Biology Department of the University of Oregon.     

Crossing maize with sorghum,Tripsacum and millet: the products and their level of development following pollination

Summary Maize was crossed with sorghum, Tripsacum and millet with the aim of introgressing desirable alien characteristics into maize. The products of crosses were analyzed as to their level of differentiation following pollination; their further development on artificial culture medium was compared. In spite of a stimulation rate close to 5%, no evidence of hybridization between maize and sorghum or millet could be obtained. The plants recovered proved to be of maternal origin. However, with an appreciable frequency, stimulation leading to hypertrophic growth of nucellar tissue was observed. This phenomenon is bound to pollination, never occurring in non-pollinated ears. In crosses involving Tripsacum, more than 140 true hybrids were isolated. The influence of the genotypes used as well as factors such as climatic conditions or in vitro techniques are discussed. Except for one haploid maize plant, all the plants recovered proved to be classical hybrids, most of them showing the expected complement of chromosomes from each parent (10 + 36 chromosomes), a few others being slightly hyperploid (2n = 47 to 50 chromosomes). No non-classical hybrids constituted by a nonreduced female gamete and a reduced male gamete were obtained.     

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.     

Molecular analysis of the Ubiquitous (Uq) transposable element system of Zea mays.

Summary The Uq transposable element of maize is the most widely dispersed among different maize populations and genetic testerstrains. Despite intensive genetic characterization, little is known about its molecular structure. In order to obtain information relevant to this topic, we have cloned and sequenced three ruq receptors. Surprisingly, they are all Ds1-like receptor types of the Ac-Ds transposon family. Based on our molecular data, we present a model to explain the functional differences associated with the differential expression of the Uq and Ac transposon systems.     

Molecular Evolution of the Ac/Ds Transposable-Element Family in Pearl Millet and Other Grasses

We report an Ac-like sequence from pearl millet (Pennisetum glaucum) and deletion derivative Ac-like sequences from pearl millet and another grass species, Bambusa multiplex. Sequence relationships between the pearl millet and maize Ac elements suggest the Ac/Ds transposable-element family is ancient. Further, the sequence identity between the Bambusa Ac-like sequence and maize Ac implies that the Ac/Ds transposable-element family has been in the grass family since its inception. The Ac-like sequences reported from pearl millet and maize Ac are statistically heterogeneous in pair-wise distance comparisons to each other. Yet, we are unable to discriminate between differential selection or ectopic exchange (recombination and conversion) between nonidentical transposable element homologues, as the cause of the heterogeneity. However, the more extreme heterogeneity exhibited between the previously described pearl millet element and maize Ac seems likely to derive from ectopic exchange between elements with different levels of divergence.     

Natural variants of pearl millet (Pennisetum typhoides) with altered levels of set II alcohol dehydrogenase activity

Two linked genes, Adh1 and Adh2, specify three sets of ADH isozymes in pearl millet. Set I is a homodimer specified by Adh1, Set III is a homodimer specified by Adh2, and Set II is a heterodimer consisting of one ADH1 subunit and one ADH2 subunit. Dry seeds exhibit only Sets I and II. Anaerobic treatment of seeds greatly increases the activity of Sets I and II and causes the Set III isozymes to be expressed. In the investigation reported here, the ADH zymogram phenotypes of 112 inbred pearl millet lines were analyzed. Two kinds of naturally occurring ADH variant strains were observed: in the low-activity variant, Set II activity is low in the dry seed, and no Set III activity is present upon anaerobic treatment. In the high-activity variant, Set II activity is high and Set III isozymes are expressed in the dry seed. The mutation in the high-activity strain appears to affect the product of Adh2 and not the product of Adh1. Dominance tests show that the mutations in both types of variant strains act in cis. These observations and linkage tests indicate that the mutations are closely linked to or at the Adh2 locus.This work was supported by a PHS National Research Service Award Training Grant in Genetics to the Biology Department of the University of Oregon.     

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.     

<Emphasis Type="Italic">Mutator</Emphasis>-like elements identified in melon,Arabidopsis and rice contain ULP1 protease domains

The transposon Mutator was first identified in maize, and is one of the most active mobile elements in plants. The Arabidopsis thaliana genome contains at least 200 Mutator-like elements (MULEs), which contain the Mutator-like transposase gene, and often additional genes. We have detected a novel type of MULEs in melon (CUMULE), which, besides the transposase, contains two ubiquitin-like specific protease-like sequences (ULP1). This element is not present in the observed location in some melon cultivars. Multiple copies of this element exist in the Cucumis melo genome, and it has been detected in other Cucurbitaceae species. Analysis of the A. thaliana genome revealed more than 90 CUMULE-like elements, containing one or two Ulp1-like sequences, although no evidence of mobility exists for these elements. We detected various putative transposable elements containing ULP1-like sequences in rice. The discovery of these MULEs in melon and Arabidopsis, and the existence of similar elements in rice and maize, suggest that a proteolytic function may be important for this subset of the MULE transposable elements. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Nucleotide sequence data reported are available in the GenBank database under the accession number AY524004.