Gene structure dynamics and divergence of the polygalacturonase gene family of plants and fungus.

Whole copies of the polygalacturonase (PG) genes from rice (Oryza sativa subsp. japonica) and a filamentous fungus (Aspergillus oryzae) were isolated. The orthologs of the rice PGs were also retrieved from other plant species. The 106 plant PGs analyzed were divided into 5 clades, A, B, C, D, and E. The fungus PGs were classified into 3 clades, of which one formed a loose cluster with clade E of the plant PGs. Four domain motifs (I, II, III, IV) were identified in all PGs. Motifs II and III were split by introns such as G/DDC and CGPGHGIS/IGSLG, respectively. In plant PGs there were 446 introns in total and 3.98 introns per gene. Intron phase distribution was 65.5% for phase 0, 19.7% for phase 1, and 14.8% for phase 2 in plant PGs. In the PGs of A. oryzae there were 37 introns of phase 0 (59.5%), phase 1 (24.3%), and phase 2 (16.2%), with 2.47 introns per gene. The 5 clades of plant PGs were divided into 3 basic gene structure lineages. Intron positions and phases were conserved among the PGs in the first 2 lineages. The third lineage consisted of PGs of clade E, which also carried highly conserved introns at different positions from other PGs. Intron positions were not as highly conserved in fungus PGs as in plant PGs. The introns in the current PGs have been present since before the divergence of monocots from dicots. The results obtained show that differential losses of introns created gene diversity, which was followed by segmental and tandem duplication in plant PGs.

Terminal-repeat retrotransposons in miniature (TRIM) are involved in restructuring plant genomes.

A new group of long terminal repeats (LTR) retrotransposons, termed terminal-repeat retrotransposons in miniature (TRIM), are described that are present in both monocotyledonous and dicotyledonous plant. TRIM elements have terminal direct repeat sequences between approximately 100 and 250 bp in length that encompass an internal domain of approximately 100-300 bp. The internal domain contains primer binding site and polypurine tract motifs but lacks the coding domains required for mobility. Thus TRIM elements are not capable of autonomous transposition and probably require the help of mobility-related proteins encoded by other retrotransposons. The structural organization of TRIM elements suggests an evolutionary relationship to either LTR retrotransposons or retroviruses. The past mobility of TRIM elements is indicated by the presence of flanking 5-bp direct repeats found typically at LTR retrotransposon insertion sites, the high degree of sequence conservation between elements from different genomic locations, and the identification of related to empty sites (RESites). TRIM elements seem to be involved actively in the restructuring of plant genomes, affecting the promoter, coding region and intron-exon structure of genes. In solanaceous species and maize, TRIM elements provided target sites for further retrotransposon insertions. In Arabidopsis, evidence is provided that the TRIM element also can be involved in the transduction of host genes.

A novel hybrid open reading frame formed by multiple cellular gene transductions by a plant long terminal repeat retroelement.

The discovery that vertebrate retroviruses could transduce cellular sequences was central to cancer etiology and research. Although not well documented, transduction of cellular sequences by retroelements has been suggested to modify cellular functions. The maize Bs1 transposon was the first non-vertebrate retroelement reported to have transduced a portion of a cellular gene (c-pma). We show that Bs1 has, in addition, transduced portions of at least two more maize cellular genes, namely for 1,3-beta-glucanase (c-bg) and 1,4-beta-xylan endohydrolase (c-xe). We also show that Bs1 has maintained a truncated gag domain with similarity to the magellan gypsy-like long terminal repeat retrotransposon and a region that may correspond to an env-like domain. Our findings suggest that, like oncogenic retroviruses, the three transduced gene fragments and the Bs1 gag domain encode a fusion protein that has the potential to be expressed. We suggest that transduction by retroelements may facilitate the formation of novel hybrid genes in plants.

Tc8, a Tourist-like transposon in Caenorhabditis elegans.

Members of the Tourist family of miniature inverted-repeat transposable elements (MITEs) are very abundant among a wide variety of plants, are frequently found associated with normal plant genes, and thus are thought to be important players in the organization and evolution of plant genomes. In Arabidopsis, the recent discovery of a Tourist member harboring a putative transposase has shed new light on the mobility and evolution of MITEs. Here, we analyze a family of Tourist transposons endogenous to the genome of the nematode Caenorhabditis elegans (Bristol N2). One member of this large family is 7568 bp in length, harbors an ORF similar to the putative Tourist transposase from Arabidopsis, and is related to the IS5 family of bacterial insertion sequences (IS). Using database searches, we found expressed sequence tags (ESTs) similar to the putative Tourist transposases in plants, insects, and vertebrates. Taken together, our data suggest that Tourist-like and IS5-like transposons form a superfamily of potentially active elements ubiquitous to prokaryotic and eukaryotic genomes.

Population dynamics of an Ac-like transposable element in self- and cross-pollinating arabidopsis.

Theoretical models predict that the mating system should be an important factor driving the dynamics of transposable elements in natural populations due to differences in selective pressure on both element and host. We used a PCR-based approach to examine the abundance and levels of insertion polymorphism of Ac-III, a recently identified Ac-like transposon family, in natural populations of the selfing plant Arabidopsis thaliana and its close outcrossing relative, Arabidopsis lyrata. Although several insertions appeared to be ancient and shared between species, there is strong evidence for recent activity of this element family in both species. Sequences of the regions flanking insertions indicate that all Ac-III transposons segregating in natural populations are in noncoding regions and provide no evidence for local transposition events. Transposon display analysis suggests the presence of slightly higher numbers of insertion sites per individual but fewer total polymorphic insertions in the self-pollinating A. thaliana than A. lyrata. Element insertions appear to be segregating at significantly lower frequencies in A. lyrata than A. thaliana, which is consistent with a reduction in transposition rate, reduction in effective population size, or reduced efficacy of natural selection against element insertions in selfing populations.

Sequence and analysis of the Arabidopsis genome.

The comprehensive analysis of the genome sequence of the plant Arabidopsis thaliana has been completed recently. The genome sequence and associated analyses provide the foundations for rapid progress in many fields of plant research, such as the exploitation of genetic variation in Arabidopsis ecotypes, the assessment of the transcriptome and proteome, and the association of genome changes at the sequence level with evolutionary processes. Nevertheless, genome sequencing and analysis are only the first steps towards a new plant biology. Much remains to be done to refine the analysis of encoded genes, to define the functions of encoded proteins systematically, and to establish new generations of databases to capture and relate diverse data sets generated in widely distributed laboratories.

Survey of transposable elements from rice genomic sequences.

Oryza sativa L. (domesticated rice) is a monocotyledonous plant, and its 430 Mb genome has been targeted for complete sequencing. We performed a high-resolution computer-based survey for transposable elements on 910 Kb of rice genomic DNA sequences. Both class I and II transposable elements were present, contributing 19.9% of the sequences surveyed. Class II elements greatly outnumbered class I elements (166 versus 22), although class I elements made up a greater percentage (12.2% versus 6.6%) of nucleotides surveyed. Several Mutator-like elements (MULEs) were identified, including rice elements that harbor truncated host cellular genes. MITEs (miniature inverted-repeat transposable elements) account for 71.6% of the mined transposable elements and are clearly the predominant type of transposable element in the sequences examined. Moreover, a putative Stowaway transposase has been identified based on shared sequence similarity with the mined MITEs and previously identified plant mariner-like elements (MLEs). Members of a group of novel rice elements resembling the structurally unusual members of the Basho family in Arabidopsis suggest a wide distribution of these transposons among plants. Our survey provides a preview of transposable element diversity and abundance in rice, and allows for comparison with genomes of other plant species.

Mutator-like elements in Arabidopsis thaliana. Structure, diversity and evolution.

While genome-wide surveys of abundance and diversity of mobile elements have been conducted for some class I transposable element families, little is known about the nature of class II transposable elements on this scale. In this report, we present the results from analysis of the sequence and structural diversity of Mutator-like elements (MULEs) in the genome of Arabidopsis thaliana (Columbia). Sequence similarity searches and subsequent characterization suggest that MULEs exhibit extreme structure, sequence, and size heterogeneity. Multiple alignments at the nucleotide and amino acid levels reveal conserved, potentially transposition-related sequence motifs. While many MULEs share common structural features to Mu elements in maize, some groups lack characteristic long terminal inverted repeats. High sequence similarity and phylogenetic analyses based on nucleotide sequence alignments indicate that many of these elements with diverse structural features may remain transpositionally competent and that multiple MULE lineages may have been evolving independently over long time scales. Finally, there is evidence that MULEs are capable of the acquisition of host DNA segments, which may have implications for adaptive evolution, both at the element and host levels.

Molecular characterization of the Abp1 5'-flanking region in maize and the teosintes.

Auxin-binding protein 1 subsp. mays (ABP1) has been suggested as a receptor mediating auxin-induced cell expansion and differentiation. In maize (Zea mays), ABP1 is encoded by a single gene, Abp1. The TATA and CAAT promoter elements as well as the transcriptional start site were previously identified and all were found to be located within a transposable element (TE), Tourist-Zm11. In this study we report the cloning and characterization of the Abp1 5'-flanking region in maize and its wild relatives, the teosintes. We provide evidence for insertion polymorphism corresponding to Tourist-Zm11 and two novel TEs, Batuta and Pilgrim. Despite this polymorphic structure, the Abp1 core promoter in maize and the teosintes is conserved, is located downstream of the TE insertions in the 5'-flanking region, and is TATA-less. We discuss the potential evolutionary impact of these TEs on the regulation of Abp1 gene expression.

Transposon diversity in Arabidopsis thaliana.

Recent availability of extensive genome sequence information offers new opportunities to analyze genome organization, including transposon diversity and accumulation, at a level of resolution that was previously unattainable. In this report, we used sequence similarity search and analysis protocols to perform a fine-scale analysis of a large sample ( approximately 17.2 Mb) of the Arabidopsis thaliana (Columbia) genome for transposons. Consistent with previous studies, we report that the A. thaliana genome harbors diverse representatives of most known superfamilies of transposons. However, our survey reveals a higher density of transposons of which over one-fourth could be classified into a single novel transposon family designated as Basho, which appears unrelated to any previously known superfamily. We have also identified putative transposase-coding ORFs for miniature inverted-repeat transposable elements (MITEs), providing clues into the mechanism of mobility and origins of the most abundant transposons associated with plant genes. In addition, we provide evidence that most mined transposons have a clear distribution preference for A + T-rich sequences and show that structural variation for many mined transposons is partly due to interelement recombination. Taken together, these findings further underscore the complexity of transposons within the compact genome of A. thaliana.

Identification and characterization of 14 transposon-like elements in the noncoding regions of members of the Xa21 family of disease resistance genes in rice.

The rice disease resistance gene Xa21, which encodes a receptor-like kinase, is a member of a multigene family. Based on comparisons of genomic sequences of seven family members, seventeen transposon-like elements were identified in the 5' and 3' flanking regions and introns of these genes. Sequence characterization revealed that these elements are diverse, showing similarity to maize Ds, CACTA and miniature inverted repeat-like elements, as well as novel elements. Only two elements were located in presumed coding regions, indicating that integration of transposable elements at the Xa21 disease resistance locus occurred preferentially in noncoding regions.

A computer-based systematic survey reveals the predominance of small inverted-repeat elements in wild-type rice genes.

Several recent reports indicate that mobile elements are frequently found in and flanking many wild-type plant genes. To determine the extent of this association, we performed computer-based systematic searches to identify mobile elements in the genes of two "model" plants, Oryza sativa (domesticated rice) and Arabidopsis thaliana. Whereas 32 common sequences belonging to nine putative mobile element families were found in the noncoding regions of rice genes, none were found in Arabidopsis genes. Five of the nine families (Gaijin, Castaway, Ditto, Wanderer, and Explorer) are first described in this report, while the other four were described previously (Tourist, Stowaway, p-SINE1, and Amy/LTP). Sequence similarity, structural similarity, and documentation of past mobility strongly suggests that many of the rice common sequences are bona fide mobile elements. Members of four of the new rice mobile element families are similar in some respects to members of the previously identified inverted-repeat element families, Tourist and Stowaway. Together these elements are the most prevalent type of transposons found in the rice genes surveyed and form a unique collection of inverted-repeat transposons we refer to as miniature inverted-repeat transposable elements or MITEs. The sequence and structure of MITEs are clearly distinct from short or long interspersed nuclear elements (SINEs or LINEs), the most common transposable elements associated with mammalian nuclear genes. Mobile elements, therefore, are associated with both animal and plant genes, but the identity of these elements is strikingly different.

LTR-retrotransposons and MITEs: important players in the evolution of plant genomes.

Retrotransposons are an abundant and ancient component of plant genomes, yet recent evidence indicates that element activity in many modern plants is restricted to times of stress. Stress activation of plant retrotransposons may be a significant factor in somaclonal variation, in addition to providing an important means to isolate new active elements. Long terminal repeat retrotransposons and a second class of elements we have called miniature inverted-repeat transposable elements (MITEs) have recently been found to be associated with the genes of diverse plants where some contribute regulatory sequences. Because of their sequence diversity and small size, MITEs may be a valuable evolutionary tool for altering patterns of gene expression.

Transposon-mediated chromosomal rearrangements and gene duplications in the formation of the maize R-r complex.

R-r controls the production of anthocyanin pigment in plant parts and the aleurone layer of seeds through the production of a family of related transcriptional activating proteins of the helix-loop-helix type. The R-r complex comprises a series of repeated, homologous components arranged in both direct and inverted orientations. These include the P component, a simple R gene that confers pigmentation of plant parts, and the S subcomplex that consists of a truncated inactive R gene called q, and two functional R genes, S1 and S2, that pigment the aleurone. The S genes are arranged in an unusual inverted head-to-head orientation. The identity of each functional component was confirmed by microprojectile bombardment of intact maize tissues with cloned genomic DNA and by analysis of in vivo mRNA populations. Sequence analysis suggests that the S subcomplex was derived through the rearrangement of a simple P-like progenitor element. At the rearrangement breakpoints, features typical of the CACTA family of transposable elements were found. The location and arrangement of these CACTA element sequences implies that this element may have mediated the chromosomal rearrangements that led to the formation of the R-r complex. The unusual structure of R-r explains much of the meiotic instability of the complex.

Stowaway: a new family of inverted repeat elements associated with the genes of both monocotyledonous and dicotyledonous plants.

Members of a new inverted repeat element family, named Stowaway, have been found in close association with more than 40 monocotyledonous and dicotyledonous plant genes listed in the GenBank and EMBL nucleic acid data bases. Stowaway elements are characterized by a conserved terminal inverted repeat, small size, target site specificity (TA), and potential form stable DNA secondary structures. Some elements are located at the extreme 3' ends of sequenced cDNAs and supply polyadenylation signals to their host genes. Other elements are in the 5' upstream regions of several genes and appear to contain previously identified cis-acting regulatory domains. Although the Stowaway elements share many structural features with the recently discovered Tourist elements, the two families share no significant sequence similarity. Together, the Stowaway and Tourist families serve to define an important new class of short inverted repeat elements found in possibly all flowering plant genomes.

Mobile inverted-repeat elements of the Tourist family are associated with the genes of many cereal grasses.

Tourist was originally described as a 128-bp insertion mutation in the maize wx-B2 allele. Subsequent analysis revealed that Tourist elements are in the introns or flanking sequences of 11 maize genes and a single barley gene. In this study we report that Tourist elements are frequently associated with the wild-type genes of two other grasses, rice and sorghum. Six of 35 rice and 5 of 8 sorghum complete gene sequences reported to date contain Tourist elements. Furthermore, 11 additional maize genes have been found to contain Tourist elements, bringing the current total of elements associated with maize genes to 23. Sequence comparison of Tourist elements has led to the identification of four subfamilies, designated A-D. Evidence is presented for the recent mobility of elements in three of these subfamilies and in three of the four grass species. These data suggest that Tourist elements are highly repetitive in the genomes of some and perhaps all members of the grasses.

Tourist: a large family of small inverted repeat elements frequently associated with maize genes.

The wx-B2 mutation results from a 128-bp transposable element-like insertion in exon 11 of the maize Waxy gene. Surprisingly, 11 maize genes and one barley gene in the GenBank and EMBL data bases were found to contain similar elements in flanking or intron sequences. Members of this previously undescribed family of elements, designated Tourist, are short (133 bp on average), have conserved terminal inverted repeats, are flanked by a 3-bp direct repeat, and display target site specificity. Based on estimates of repetitiveness of three Tourist elements in maize genomic DNA, the copy number of the Tourist element family may exceed that of all previously reported eukaryotic inverted repeat elements. Taken together, our data suggest that Tourist may be the maize equivalent of the human Alu family of elements with respect to copy number, genomic dispersion, and the high frequency of association with genes.

Molecular analysis of the maize wx-B3 allele indicates that precise excision of the transposable Ac element is rare.

The somatic and germinal behavior of the maize wx-B3 mutation indicates that this Ac allele rarely reverts. Endosperms containing wx-B3 display tiny and infrequent Wx revertant sectors while no significant reversion is detected when wx-B3 pollen is stained with I/KI. Previous studies of other transposable element alleles that revert infrequently have implicated low levels of element excision. Unlike these other alleles, the wx-B3 Ac element is indistinguishable from fully active Ac elements with respect to its structure, and its ability to transpose from the Wx gene or to trans-activate a Ds element. Characterization of somatic and germinal excision events lead us to conclude that excision of the wx-B3 Ac element almost always produces null alleles. Furthermore, the excellent correlation between the position of the wx-B3 mutation on the physical and genetic maps indicates that the Ac insertion is the only lesion of wx-B3. As a result, precise excision of this Ac should restore Wx function. The fact that revertant sectors and pollen grains are rare indicates that precise excision of Ac is also rare. The finding that the wx-B3 reversion frequency is comparable whether wx-B3 is hemizygous or over a wx allele with a wild-type insertion site illustrates a fundamental difference between the excision mechanisms of Ac and Drosophila P elements.