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.

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.

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.

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.

In vitro synthesis of cellulose II from a cytoplasmic membrane fraction of Acetobacter xylinum.

The cytoplasmic and outer membranes of Acetobacter xylinum (ATCC 53582) were isolated by discontinuous sucrose density ultracentrifugation. Both lysozyme (EC 3.2.1.17) and trypsin (EC 3.4.21.4) were required for efficient crude membrane separation. Primary dehydrogenases and NADH oxidase were used as cytoplasmic membrane markers, and 2-keto-3-deoxyoctulosonic acid was used to identify the outer membranes. Cellulose synthetase (UDP-glucose:1,4-beta-D-glucan 4-beta-D-glucosyltransferase; EC 2.4.1.12) activity was assayed as the conversion of radioactivity from UDP-[(14)C]glucose into an alkali-insoluble beta-1,4-D-[(14)C]glucan. This activity was predominantly found in the cytoplasmic membrane. The cellulose nature of the product was demonstrated by (i) enzymatic hydrolysis followed by TLC, (ii) methylation analysis followed by TLC, and (iii) GC/MS. Further, the weight-average and number-average degree of polymerization of the in vitro product, determined by high-performance gel permeation chromatography, were 4820 and 5270, respectively. In addition, x-ray diffraction analysis indicated that the in vitro product is cellulose II, which is in contrast to the in vivo product-namely, cellulose I.

Oryzalin, a dinitroaniline herbicide, binds to plant tubulin and inhibits microtubule polymerization in vitro.

The effects of oryzalin, a dinitroaniline herbicide, on chromosome behavior and on cellular microtubules (MTs) were examined by light microscopy and immunogold staining, respectively, in endosperm cells from Haemanthus katherinae Bak. Brief treatments with 1.0·10(-8) M oryzalin reduced markedly the migration rate of anaphase chromosomes and 1.0·10(-7) M oryzalin stopped migration abruptly. Oryzalin (1.0·10(-7) M) depolymerized MTs and prevented the polymerization of new MTs at all stages of the mitotic cycle. The chromosome condensation cycle was unaffected by oryzalin. Endothelial cells from the heart of Xenopus leavis showed no chromosomal or microtubular rearrangements after oryzalin treatment. The inhibition by oryzalin of the polymerization of tubulin isolated from cultured cells of Rosa sp. cv. Paul's scarlet was examined in vitro by turbidimetry, electron microscopy and polymer sedimentation analysis. Oryzalin inhibited the rapid phase of taxol-induced polymerization of rose MTs at 24°C with an apparent inhibition constant (K i ) of 2.59·10(6) M. Shorter and fewer MTs were formed with increasing oryzalin concentrations, and maximum inhibition of taxol-induced polymerization occurred at approx. 1:1 molar ratios of oryzalin and tubulin. Oryzalin partially depolymerized taxol-stabilized rose MTs. Ligand-binding experiments with [(14)C]oryzalin demonstrated the formation of a tubulin-oryzalin complex that was time- and pH-dependent. The tubulin-oryzalin interaction (24°C, pH 7.1) had an apparent affinity constant (K app) of 1.19·10(5) M(-1). Oryzalin did not inhibit taxol-induced polymerization of bovinebrain MTs and no appreciable binding of oryzalin to brain tubulin or other proteins was detected. The results demonstrate pharmacological differences between plant and animal tubulins and indicate that the most sensitive mode of action of the dinitroaniline herbicides is the direct poisoning of MT dynamics in cells of higher plants.

Resistance of Rosa microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin.

The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Paul's scarlet) by colchicine and the binding of colchicine to tubulin were examined in vitro and compared with data obtained in parallel experiments with bovine brain tubulin. Turbidimetric measurements of taxol-induced polymerization of rose microtubules were found to be sensitive and semiquantitative at low tubulin concentrations, and to conform to some of the characteristics of a nucleation and condensation-polymerization mechanism for assembly of filamentous helical polymers. Colchicine inhibited the rapid phase of polymerization at 24°C with an apparent inhibition constant (K i) of 1.4·10(-4) M for rose tubulin and an apparent K i=8.8·10(-7) M for brain tubulin. The binding of [(3)H]colchicine to rose tubulin to form tubulin-colchicine complex was mildly temperature-dependent and slow, taking 2-3 h to reach equilibrium at 24°C, and was not affected by vinblastine sulfate. The binding of [(3)H]colchicine to rose tubulin was saturable and Scatchard analysis indicated a single class of low-affinity binding sites having an apparent affinity constant (K) of 9.7·10(2) M(-1) and an estimated molar binding stoichiometry (r) of 0.47 at 24°C. The values for brain tubulin were K=2.46·10(6) M(-1) and r=0.45 at 37°C. The binding of [(3)H]colchicine to rose tubulin was inhibited by excess unlabeled colchicine, but not by podophyllotoxin or tropolone. The data demonstrate divergence of the colchicine-binding sites on plant and animal tubulins and indicate that the relative resistance of plant microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin.

Inhibition of plant cell proteolytic activities that degrade tubulin.

The requirement for proteinase inhibitors during the chromatographic isolation of tubulin from cultured cells of rose (Rosa sp. cv. Paul's scarlet) was examined by NadodecylSO4-polyacrylamide gel electrophoresis, electron microscopy and immunoblotting. Tubulin fractions isolated in the absence of proteinase inhibitors showed substoichiometric ratios of alpha-subunit to beta-subunit, and low molecular weight polypeptides, one (approximately 32 Kd) of which coassembled with polymers. Electron microscopy revealed polymorphic structures, including C- and S-shaped ribbons and free protofilaments. Immunoblotting experiments with IgGs to the individual alpha- and beta-subunits showed that some of the low molecular weight polypeptides were fragments of proteolytically degraded subunits. The use of low micromolar concentrations of the synthetic proteinase inhibitors leupeptin hemisulfate and pepstatin A protected tubulin from endogenous proteolytic activities during the isolation procedure and resulted in increased tubulin purity.

Tubulins from different higher plant species are immunologically nonidentical and bind colchicine differentially.

We have initiated immunological and drug-binding studies on the tubulins from different higher plant species. Antibodies were raised against electrophoretically separated rose (Rosa sp.) tubulin alpha- and beta-subunits and characterized by immunoblot autoradiographic assays. Each IgG preparation bound to its antigen and cross-reacted differentially with the respective tubulin subunits from an alga, sea urchin, rabbit, and cow. Antigenic determinants were shared more among the beta-subunits than among the alpha-subunits from these organisms. Tubulins were isolated from cultured cells of carrot (Daucus carota) and hibiscus (Hibiscus rosa-senensis). Immunoautoradiography and quantitation of cross-reactivity on blots showed nonidentity among homologous subunits from rose, carrot, hibiscus, and alga tubulins, with more antigenic differences among alpha-subunits than among beta-subunits. Comparative colchicine-binding assays showed that rose and hibiscus tubulins bound 33% and 65%, respectively, of the colchicine bound by carrot tubulin and that higher plant tubulins bound much less colchicine than bovine brain tubulin under identical conditions.