Susan R. Wessler

The Waxy (Wx) locus in maize determines the amylose content of pollen and endosperm tissue. There are several mutant alleles of the locus caused by insertion of transposable controlling elements. In the present study, we have used the properties of controlling element alleles to identify the Wx locus and its gene product, with the subsequent objective of isolating the elements causing the mutations. We present evidence that the Wx locus encodes a starch granule-bound 58 kd polypeptide that is synthesized in vitro as a 65 kd precursor. We describe the isolation of recombinant plasmids containing cDNA inserts homologous to Wx mRNA and a recombinant lambda phage containing a genomic Eco RI fragment encompassing most or all of the Wx transcription unit. We show that a mutation caused by the controlling element Dissociation (Ds) is attributable to an insertion of approximately 2.4 kb at the Wx locus.

Shaping Plant Evolution Amborella trichopoda is understood to be the most basal extant flowering plant and its genome is anticipated to provide insights into the evolution of plant life on Earth (see the Perspective by Adams ). To validate and assemble the sequence, Chamala et al. (p. 1516 ) combined fluorescent in situ hybridization (FISH), genomic mapping, and next-generation sequencing. The Amborella Genome Project (p. 10.1126/science.1241089 ) was able to infer that a whole-genome duplication event preceded the evolution of this ancestral angiosperm, and Rice et al. (p. 1468 ) found that numerous genes in the mitochondrion were acquired by horizontal gene transfer from other plants, including almost four entire mitochondrial genomes from mosses and algae.

Previous studies have suggested that the R locus of maize is responsible for determining the temporal and spatial pattern of anthocyanin pigmentation in the plant. In this report we demonstrate that three members of the R gene family, P, S, and Lc, encode homologous transcripts 2.5 kilobases in length. The structure of one R gene, Lc, was determined by sequencing cDNA and genomic clones. The putative Lc protein, deduced from the cDNA sequence, is composed of 610 amino acids and has homology to the helix-loop-helix DNA-binding/dimerization motif found in the L-myc gene product and other regulatory proteins. It also contains a large acidic domain that may be involved in transcriptional activation. Consistent with its proposed role as a transcriptional activator is our finding that a functional R gene is required for the accumulation of transcripts of at least two genes in the anthocyanin biosynthetic pathway. We discuss the possibility that the diverse patterns of anthocyanin pigmentation conditioned by different R genes reflect differences in the R gene promoters rather than their gene products.

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.

Restriction endonuclease fragments containing part of the Waxy (Wx) locus have been cloned from strains with insertion mutations at the locus caused by the controlling elements Activator (Ac) and Dissociation (Ds). Evidence is presented that the genetically defined Ac element corresponds to a 4.3 kb insertion, while the two Ds elements correspond to 4.1 kb and 2.0 kb insertions, all near the 3' end of the Wx transcription unit. The 4.1 kb Ds is almost completely homologous to the Ac element, differing by a central deletion of less than 0.2 kb. The 2.0 kb Ds element is homologous to the ends of the Ac element. Sequences homologous to the ends of the Ac element are present in many copies in the genomes examined, while there are ten or fewer copies of a sequence with homology to the center of the cloned Ac element. The Ac element at the Wx locus can be distinguished structurally from the other Ac-like sequences in the genome.

All known active plant retrotransposons are largely quiescent during development but activated by stresses, including wounding, pathogen attack and cell culture. This may reflect a survival strategy based on plant biology, or retrotransposons could be the stress-induced generators of genomic diversity proposed by McClintock.

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.

Cut-and-paste DNA transposable elements are major components of eukaryotic genomes and are grouped into superfamilies (e.g., hAT, P) based on sequence similarity of the element-encoded transposase. The transposases from several superfamilies possess a protein domain containing an acidic amino acid triad (DDE or DDD) that catalyzes the "cut and paste" transposition reaction. However, it was unclear whether this domain was shared by the transposases from all superfamilies. Through multiple-alignment of transposase sequences from a diverse collection of previously identified and recently annotated elements from a wide range of organisms, we identified the putative DDE/D triad for all superfamilies. Furthermore, we identified additional highly conserved amino acid residues or motifs within the DDE/D domain that together form a "signature string" that is specific to each superfamily. These conserved residues or motifs were exploited as phylogenetic characters to infer evolutionary relationships among all superfamilies. The phylogenetic analysis revealed three major groups that were not previously discerned and led us to revise the classification of several currently recognized superfamilies. Taking the data together, this study suggests that all eukaryotic cut-and-paste transposable element superfamilies have a common evolutionary origin and establishes a phylogenetic framework for all future cut-and-paste transposase comparisons.

Meiotic recombination rates can vary widely across genomes, with hotspots of intense activity interspersed among cold regions. In yeast, hotspots tend to occur in promoter regions of genes, whereas in humans and mice, hotspots are largely defined by binding sites of the positive-regulatory domain zinc finger protein 9. To investigate the detailed recombination pattern in a flowering plant, we use shotgun resequencing of a wild population of the monkeyflower Mimulus guttatus to precisely locate over 400,000 boundaries of historic crossovers or gene conversion tracts. Their distribution defines some 13,000 hotspots of varying strengths, interspersed with cold regions of undetectably low recombination. Average recombination rates peak near starts of genes and fall off sharply, exhibiting polarity. Within genes, recombination tracts are more likely to terminate in exons than in introns. The general pattern is similar to that observed in yeast, as well as in positive-regulatory domain zinc finger protein 9-knockout mice, suggesting that recombination initiation described here in Mimulus may reflect ancient and conserved eukaryotic mechanisms.

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.

In cultivated rice two wild-type alleles, Wxa and Wxb, predominate at the waxy locus, which encodes granule-bound starch synthase. The activity of Wxa is 10-fold higher than that of Wxb at the level of both protein and mRNA. Wxb has a +1G to T mutation at the 5' splice site of the first intron. Sequence analysis of Wxb transcripts revealed that splicing occurs at the mutant AG/UU site and at two cryptic sites: the first is A/GUU, one base upstream of the original site and the second is AG/GU found approximately 100 bases upstream of the mutant splice site. We introduced single base mutations to the 5' splice sites of both Wxa and Wxb, fused with the gus reporter gene and introduced them into rice protoplasts. Analysis of GUS activities and transcripts indicated that a G to T mutation in Wxa reduced GUS activity and the level of spliced RNA. Conversely, a T to G mutation of Wxb restored GUS activity and the level of spliced RNA to that of wild-type Wxa. These results demonstrated that the low level expression of Wxb results from a single base mutation at the 5' splice site of the first intron. It is of interest that the Wxb allele of rice carrying the G to T mutation of intron 1 has been conserved in the history of rice cultivation because there is a low amylose content of the seed caused by this mutation.

The wx-K mutation results from the insertion of a copia-like retrotransposon into exon 12 of the maize waxy gene. This retrotransposon, named Hopscotch, has one long open reading frame encoding all of the domains required for transposition. Computer-assisted database searches using Hopscotch and other plant copia-like retroelements as query sequences have revealed that ancient, degenerate retrotransposon insertions are found in close proximity to 21 previously sequenced plant genes. The data suggest that these elements may be involved in gene duplication and the regulation of gene expression. Similar searches using the Drosophila retrotransposon copia did not reveal any retrotransposon-like sequences in the flanking regions of animal genes. These results, together with the recent finding that reverse-transcriptase sequences characteristic of copia-like elements are ubiquitous and diverse in plants, suggest that copia-like retrotransposons are an ancient component of plant genomes.

Despite the prevalence of transposable elements in the genomes of higher eukaryotes, what is virtually unknown is how they amplify to very high copy numbers without killing their host. Here, we report the discovery of rice strains where a miniature inverted-repeat transposable element ( mPing ) has amplified from ≈50 to ≈1,000 copies in four rice strains. We characterized 280 of the insertions and found that 70% were within 5 kb of coding regions but that insertions into exons and introns were significantly underrepresented. Further analyses of gene expression and transposable-element activity demonstrate that the ability of mPing to attain high copy numbers is because of three factors: ( i ) the rapid selection against detrimental insertions, ( ii ) the neutral or minimal effect of the remaining insertions on gene transcription, and ( iii ) the continued mobility of mPing elements in strains that already have >1,000 copies. The rapid increase in mPing copy number documented in this study represents a potentially valuable source of population diversity in self-fertilizing plants like rice.

Transposable elements are ubiquitous in plant genomes, where they frequently comprise the majority of genomic DNA. The maize genome, which is believed to be structurally representative of large plant genomes, contains single genes or small gene islands interspersed with much longer blocks of retrotransposons. Given this organization, it would be desirable to identify molecular markers preferentially located in genic regions. In this report, the features of a newly described family of miniature inverted repeat transposable elements (MITEs) (called Heartbreaker ), including high copy number and polymorphism, stability, and preference for genic regions, have been exploited in the development of a class of molecular markers for maize. To this end, a modification of the AFLP procedure called transposon display was used to generate and display hundreds of genomic fragments anchored in Hbr elements. An average of 52 markers were amplified for each primer combination tested. In all, 213 polymorphic fragments were reliably scored and mapped in 100 recombinant inbred lines derived from a cross between the maize inbreds B73 × Mo17. In this mapping population, Hbr markers are distributed evenly across the 10 maize chromosomes. This procedure should be of general use in the development of markers for other MITE families in maize and in other plant and animal species where MITEs have been identified.

The Activator (Ac) element at the waxy locus (wx-m7 allele) has the ability to undergo changes in its genetic activity and cycles between an active and inactive phase. Comparison of active Ac elements at several loci and the inactive Ac at wx-m7 by Southern blot analysis revealed that the inactive Ac sequence was not susceptible to digestion by the methylation sensitive enzyme PvuII while active elements were susceptible to PvuII digestion. Restriction digest comparisons between the clones of the active and inactive Ac elements were indistinguishable. Further analyses with the enzymes SstII and the methylation sensitive and insensitive isoschizomers EcoRII and BstNI showed the inactive Ac sequence was methylated at these sites, whereas the active Ac was hypomethylated. Although the active Ac at the wx-m7 allele in different genetic backgrounds showed differences in the Ac DNA modification pattern, at least a fraction of genomic DNA contained Ac sequences that were unmethylated at all of the internal sites we assayed. These data may suggest a role for DNA modification in the ability of Ac to transpose from the waxy locus and to destabilize unlinked Ds elements.

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.

The temporal and spatial patterns of anthocyanin pigmentation in the maize plant are determined by the presence or absence of the R protein product, a presumed transcriptional activator. At least 50 unique patterns of pigmentation, conditioned by members of the R gene family, have been described. In this study, microprojectiles were used to introduce into maize cells a vector containing the transcription unit from one of these genes (Lc) fused to a constitutive promoter. This chimeric gene induces cell autonomous pigmentation in tissues that are not normally pigmented by the Lc gene. As a reporter for gene expression studies in maize, R is unique because it can be quantified in living tissue simply by counting the number of pigmented cells following bombardment. R may also be useful as a visible marker for selecting stably transformed cell lineages that can give rise to transgenic plants.

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.

Cot-based sequence discovery represents a powerful means by which both low-copy and repetitive sequences can be selectively and efficiently fractionated, cloned, and characterized. Based upon the results of a Cot analysis, hydroxyapatite chromatography was used to fractionate sorghum ( Sorghum bicolor ) genomic DNA into highly repetitive (HR), moderately repetitive (MR), and single/low-copy (SL) sequence components that were consequently cloned to produce HRCot, MRCot, and SLCot genomic libraries. Filter hybridization (blotting) and sequence analysis both show that the HRCot library is enriched in sequences traditionally found in high-copy number (e.g., retroelements, rDNA, centromeric repeats), the SLCot library is enriched in low-copy sequences (e.g., genes and “nonrepetitive ESTs”), and the MRCot library contains sequences of moderate redundancy. The Cot analysis suggests that the sorghum genome is approximately 700 Mb (in agreement with previous estimates) and that HR, MR, and SL components comprise 15%, 41%, and 24% of sorghum DNA, respectively. Unlike previously described techniques to sequence the low-copy components of genomes, sequencing of Cot components is independent of expression and methylation patterns that vary widely among DNA elements, developmental stages, and taxa. High-throughput sequencing of Cot clones may be a means of “capturing” the sequence complexity of eukaryotic genomes at unprecedented efficiency. [Online supplementary material is available at www.genome.org . The sequence data described in this paper have been submitted to the GenBank under accession nos. AZ921847 - AZ923007 . Reagents, samples, and unpublished information freely provided by H. Ma and J. Messing.]

A 314-bp DNA element called Heartbreaker-hm1 ( Hbr-hm1 ) was previously identified in the 3′ untranslated region of a mutant allele of the maize disease resistance gene HM1 . This element has structural features of miniature inverted-repeat transposable elements (MITEs) and is a member of a large family of approximately 4,000 copies in the maize genome. Unlike previously described MITEs, most members of the Hbr family display over 90% sequence identity. This, coupled with the insertion of an Hbr element into an allele of the HM1 gene, suggested that this family might have spread recently throughout the genome. Consistent with this view is the finding that Hbr insertion sites are remarkably polymorphic. Ten of ten loci containing Hbr elements were found to be polymorphic for the presence or absence of Hbr among a collection of maize inbred lines and teosinte strains. Despite the fact that over 80% of the maize genome contain moderate to highly repetitive DNA, we find that randomly chosen Hbr elements are predominantly in single or low copy regions. Furthermore, when used to query both the public and private databases of plant genes, over 50% of the sequences flanking these Hbr elements resulted in significant “hits.” Taken together, these data indicate that the presence or absence of Hbr elements is a significant contributory factor to the high level of polymorphism associated with maize genic regions.

Abstract Stowaway is a superfamily of miniature inverted repeat transposable elements (MITEs) that is widespread and abundant in plant genomes. Like other MITEs, however, its origin and mode of amplification are poorly understood. Several lines of evidence point to plant mariner-like elements (MLEs) as the autonomous partners of the nonautonomous Stowaway MITEs. To better understand this relationship, we have taken advantage of the nearly complete genome sequences of two rice subspecies to generate the first inventory of virtually all MLEs and Stowaway families coexisting in a single plant species. Thirty-four different MLEs were found to group into three major clades and 25 families. More than 22,000 Stowaway MITEs were identified and classified into 36 families. On the basis of detailed sequence comparisons, MLEs were confirmed to be the best candidate autonomous elements for Stowaway MITEs. Surprisingly, however, sequence similarity between MLE and Stowaway families was restricted to the terminal inverted repeats (TIRs) and, in a few cases, to adjacent subterminal sequences. These data suggest a model whereby most of the Stowaway MITEs in rice were cross-mobilized by MLE transposases encoded by distantly related elements.

Recent studies of rice miniature inverted repeat transposable elements (MITEs), largely fueled by the availability of genomic sequence, have provided answers to many of the outstanding questions regarding the existence of active MITEs, their source of transposases (TPases) and their chromosomal distribution. Although many questions remain about MITE origins and mode of amplification, data accumulated over the past two years have led to the formulation of testable models.

The molecular basis for the low level expression of three alleles of the maize waxy (Wx) gene has been described. Each allele contains a retrotransposon in intron sequences. These insertions represent previously undescribed elements, and their association with three wx alleles indicates that retrotransposon elements are important agents of spontaneous mutation in maize. For each allele, element sequences are spliced from pre-mRNA with the surrounding intron even though the insertions increase intron length by approximately 40- to 60-fold. In addition, despite differences in element sequences, insertion sites, and relative orientations, each element disrupts long-range splice site recognition leading to novel Wx transcripts where exons both upstream and downstream of the insertion site are skipped. The expression of wx alleles with large insertions in introns provides support for studies that indicate that the primary cis requirement for maize introns is the splice donor and acceptor sites.

In addition to discovering TEs, McClintock also uncovered disparate ways that TEs can alter genetic information. At one end of the spectrum she found that TEs could restructure genomes through element-mediated chromosomal rearrangements. At the other end she and others found they could generate new alleles by inserting into and around genes and altering their expression. Thus, the presence and extraordinary abundance of TEs in eukaryotic genomes promote a myriad of genome-altering events.

Miniature inverted repeat transposable elements (MITEs) are widespread in eukaryotic genomes, where they can attain high copy numbers despite a lack of coding capacity. However, little is known about how they originate and amplify. We performed a genome-wide screen of functional interactions between Stowaway MITEs and potential transposases in the rice genome and identified a transpositionally active MITE that possesses key properties that enhance transposition. Although not directly related to its autonomous element, the MITE has less affinity for the transposase than does the autonomous element but lacks a motif repressing transposition in the autonomous element. The MITE contains internal sequences that enhance transposition. These findings suggest that MITEs achieve high transposition activity by scavenging transposases encoded by distantly related and self-restrained autonomous elements.

With the advent of genome sequencing projects, vast amounts of DNA sequence, from a wide variety of plant and animal species, have become available for analysis. Miniature inverted-repeat transposable elements (MITEs), with their high copy number, distinct structural features (target site duplications [TSDs] and terminal inverted repeats [TIRs]), and compact stature, are relatively easy to mine from DNA sequence databases. Comparison of the transposase-encoding Tc elements and the numerous MITE families suggested possible scenarios for the origin of MITEs in Caenorhabditis elegans . Several groups independently discovered an abundant family of short (80-bp) palindromic elements, called Mrs or Made1 . The TIRs are 80 to 100% similar to those of mariner -like elements (MLEs) that are dispersed in the genome. These were the first MLEs identified in mammals and were subsequently grouped into the Hsmar1 family. Plant MITEs related to pogo -like transposons have only been identified in Arabidopsis . Phylogenetic analyses of over 100 plant MLE transposase sequences revealed the existence of multiple and divergent lineages of MLE transposases. Together these results provide an explanation for the proliferation, diversity, and success of Stowaway MITEs in plant genomes. P instability factor (PIF) is an active DNA element family first discovered as multiple mutagenic insertions into the maize R gene. Several MITE families described from plants, animals, and fungi do not share any structural or sequence features with known DNA transposon families. The proliferation of nonautonomous elements has been hypothesized to lead to the extinction of the cognate autonomous element through titration of active transposase.

Miniature inverted-repeat transposable elements (MITEs) are widespread and abundant in both plant and animal genomes. Despite the discovery and characterization of many MITE families, their origin and transposition mechanism are still poorly understood, largely because MITEs are nonautonomous elements with no coding capacity. The starting point for this study was P instability factor (PIF), an active DNA transposable element family from maize that was first identified following multiple mutagenic insertions into exactly the same site in intron 2 of the maize anthocyanin regulatory gene R. In this study we report the isolation of a maize Tourist-like MITE family called miniature PIF (mPIF) that shares several features with PIF elements, including identical terminal inverted repeats, similar subterminal sequences, and an unusual but striking preference for an extended 9-bp target site. These shared features indicate that mPIF and PIF elements were amplified by the same or a closely related transposase. This transposase was identified through the isolation of several PIF elements and the identification of one element (called PIFa) that cosegregated with PIF activity. PIFa encodes a putative protein with homologs in Arabidopsis, rice, sorghum, nematodes, and a fungus. Our data suggest that PIFa and these PIF-like elements belong to a new eukaryotic DNA transposon superfamily that is distantly related to the bacterial IS5 group and are responsible for the origin and spread of Tourist-like MITEs.

Transposable elements (TEs) are the major component of plant genomes where they contribute significantly to the >1,000-fold genome size variation. To understand the dynamics of TE-mediated genome expansion, we have undertaken a comparative analysis of the TEs in two related organisms: the weed Arabidopsis thaliana (125 megabases) and Brassica oleracea ( approximately 600 megabases), a species with many crop plants. Comparison of the whole genome sequence of A. thaliana with a partial draft of B. oleracea has permitted an estimation of the patterns of TE amplification, diversification, and loss that has occurred in related species since their divergence from a common ancestor. Although we find that nearly all TE lineages are shared, the number of elements in each lineage is almost always greater in B. oleracea. Class 1 (retro) elements are the most abundant TE class in both species with LTR and non-LTR elements comprising the largest fraction of each genome. However, several families of class 2 (DNA) elements have amplified to very high copy number in B. oleracea where they have contributed significantly to genome expansion. Taken together, the results of this analysis indicate that amplification of both class 1 and class 2 TEs is responsible, in part, for B. oleracea genome expansion since divergence from a common ancestor with A. thaliana. In addition, the observation that B. oleracea and A. thaliana share virtually all TE lineages makes it unlikely that wholesale removal of TEs is responsible for the compact genome of A. thaliana.

The AP2 DNA binding domain was thought to be plant specific because of its presence in plant, but not animal, transcriptional regulators, particularly members of the AP2/ERF family. Two recent studies have identified the AP2 domain in bacteria, bacteriophage and a ciliate as part of proteins that also encode site-specific endonucleases. The association of AP2 with an enzyme known to catalyze its own movement within populations and between species explains the unusual distribution of AP2 and, as such, adds to a growing list of phenomena where mobile DNA has promoted evolutionary novelty.

More than 40 mutant alleles of the waxy (Wx) locus of maize are available for molecular analysis. Previous studies have examined the nature of phenotypically unstable Wx mutant alleles caused by insertion of the maize transposable activator (Ac) and dissociation (Ds) elements. In this study we have used Southern blot analysis to characterize the locus in 22 strains harboring wx alleles with stable mutant phenotypes. Of these mutations, 17 are of spontaneous origin, 4 were induced by gamma rays, and 1 was induced by ethyl methanesulfonate. Of these 22 alleles, we find that 13 have either insertions or deletions within the Wx transcription unit. The insertions range in size from 150 base pairs to 6.1 kilobases. For 4 of the 6 deletions identified, the two breakpoints are within the Wx gene. For 9 other alleles we can detect no obvious lesions within or around the transcription unit. Evidence is presented that the insertions and deletions result in the mutant phenotype and are not polymorphisms. This conclusion is based on two findings: (i) a survey of inbred lines revealed only a single instance of polymorphism within the transcription unit, whereas all of the lesions described alter the transcription unit; and (ii) there is an excellent correlation between the position of these lesions on the physical map and their relative position on a fine structure genetic map of the locus.

Miniature inverted-repeat transposable elements (MITEs) are short, nonautonomous DNA elements that are widespread and abundant in plant genomes.Most of the hundreds of thousands of MITEs identified to date have been divided into two major groups on the basis of shared structural and sequence characteristics: Tourist-like and Stowaway-like.Since MITEs have no coding capacity, they must rely on transposases encoded by other elements.Two active transposons, the maize P Instability Factor (PIF ) and the rice Pong element, have recently been implicated as sources of transposase for Tourist-like MITEs.Here we report that PIF-and Pong-like elements are widespread, diverse, and abundant in eukaryotes with hundreds of element-associated transposases found in a variety of plant, animal, and fungal genomes.The availability of virtually the entire rice genome sequence facilitated the identification of all the PIF/ Pong-like elements in this organism and permitted a comprehensive analysis of their relationship with Tourist-like MITEs.Taken together, our results indicate that PIF and Pong are founding members of a large eukaryotic transposon superfamily and that members of this superfamily are responsible for the origin and amplification of Tourist-like MITEs.Sequence data from this article have been deposited with the conclusion that a related element in the rice genome, EMBL/GenBank Data Libraries under accession nos.AY362792called Pong, was the most likely source of TPase mobiliz-AY362819.

Mutations caused by the insertion of members of the Ac or Spm family of transposable elements result in a great diversity of phenotypes. With the cloning of the mutant genes and the characterization of their products, the mechanisms underlying phenotypic diversity are being deciphered. These mechanisms include (i) imprecise excision of transposable elements, which can result in the addition of amino acids to proteins; (ii) DNA methylation, which has been correlated with the activity of the element; (iii) transposase-mediated deletions within elements, which can inactivate an element or lead to a new unstable phenotype; and (iv) removal of transcribed elements from RNA, which can facilitate gene expression despite the insertion of elements into exons. An understanding of the behavior of the maize elements has provided clues to the function of cryptic elements in all maize genomes.

The precursor of the ribulose-1,5-bisphosphate carboxylase small subunit and other proteins from Chlamydomonas reinhardtii are efficiently transported into chloroplasts isolated from spinach and pea. Thus, similar determinants specify precursor-chloroplast interactions in the alga and vascular plants. Removal of all or part of its transit sequence was found to block import of the algal small subunit into isolated chloroplasts. Comparison of available sequences revealed a nine amino acid segment conserved in the transit sequences of all small subunit precursors. A protease in the vascular plant chloroplasts recognized this region in the Chlamydomonas precursor and produced an intermediate form of the small subunit. We propose that processing of the small subunit precursor involves at least two proteolytic events; only one of these has been evolutionarily conserved.

The waxy (wx) locus of maize encodes an enzyme responsible for the synthesis of amylose in endosperm tissue. The phenotype of the Dissociation (Ds) insertion mutant wx-m1 is characterized by endosperm sectors that contain different levels of amylose. We have cloned the Wx gene from this allele and from two germinal derivatives, S5 and S9, that produce intermediate levels of amylose. The Ds insertion in wx-m1 is in exon sequences, is 409 bp in length and represents an example of a class of Ds elements that are not deletion derivatives of the Activator (Ac) controlling element. The two germinal derivatives, S5 and S9, lack the Ds element but contain an additional 9 and 6 bp, respectively, at the site of Ds insertion. The level of Wx mRNA and Wx protein in S5 and S9 is essentially the same as in normal endosperm tissue but Wx enzymatic activity is reduced. Thus, the lesions in S5 and S9 lead to the addition of amino acids in the Wx protein, resulting in Wx enzymes with altered specific activities. This work supports the notion that the maize transposable elements may serve a function in natural populations to generate genetic diversity, in this case, proteins with new enzymatic properties.

In some instances, insertion of maize transposable elements into exons does not result in the total loss of enzymatic activity. In other instances, messenger RNAs of wild-type size are encoded by genes known to contain the maize transposable element Dissociation (Ds) in exons. To understand how Ds is processed from RNA, a study was made of transcripts encoded by two alleles of the maize waxy (wx) gene containing Ds insertions in exon sequences. The analysis was carried out in strains where the Ds element could not excise from the wx gene. Despite insertions of 4.3- and 1.5- Ds elements, the predominant transcripts encoded by these two genes were wild type in size. For both alleles, DNA sequencing of complementary DNAs revealed that the Ds elements had been spliced in a similar manner. Splicing was accomplished by the utilization of multiple 5′ donor splice sites in the Ds termini and a 3′ acceptor site within the wx gene adjacent to the Ds element. The net effect in both cases was the removal of most of the Ds element from the messenger RNA.

An active miniature inverted repeat transposable element (MITE), mPing, was discovered by computer-assisted analysis of rice genome sequence. The mPing element is mobile in rice cell culture and in a few rice strains where it has been amplified to >1,000 copies during recent domestication. However, determination of the transposase source and characterization of the mechanism of transposition have been hampered by the high copy number of mPing and the presence of several candidate autonomous elements in the rice genome. Here, we report that mPing is active in Arabidopsis thaliana, where its transposition is catalyzed by three sources of transposase from rice: the autonomous Ping and Pong elements and by a cDNA derived from a Ping transcript. In addition to transposase, the product of a second element-encoded ORF of unknown function is also required for mPing transposition. Excision of mPing in A. thaliana is usually precise, and transposed copies usually insert into unlinked sites in the genome that are preferentially in or near genes. As such, this will be a valuable assay system for the dissection of MITE transposition and a potentially powerful tagging system for gene discovery in eukaryotes.

The nucleotide sequence of the control region of the leu operon of Salmonella typhimurium was determined. A prominent feature of this region is a signal for termination of transcription. In vitro, transcription does terminate at this site, yielding a leader RNA of about 160 nucleotides as a major product. This leader RNA is potentially translatable into a peptide containing 28 amino acids, 4 of which are adjacent leucine residues. Several regions of base complementarity exist within the leader, positioned such that pairing of one region precludes pairing of another. The position of the four leucine codons relative to two regions of base complementarity suggest a model for the regulation of the leu operon similar to that proposed by Yanofsky and coworkers for the trp operon. In addition, a third region of base complementarity was identified which, when incorporated into the model, explains why premature termination is the usual outcome when transcription is initiated in vitro by purified RNA polymerase.

Abstract Background Cis-regulatory sequences control gene expression through the coordinated action of transcription factors and their associated partners. Both genetic and epigenetic perturbation of cis-regulatory sequences can lead to novel patterns of gene expression. Phased genome assemblies now enable the local dissection of linkages between cis-regulatory sequences, including their epigenetic state, and gene expression to further characterize gene regulation in heterozygous genomes. Results We assembled a locally phased genome for a mandarin hybrid named ‘Fairchild’ to explore the molecular signatures of allele-specific gene expression. With genome phasing, genes with allele-specific expression were paired with haplotype-specific chromatin states, including levels of chromatin accessibility, histone modifications, and DNA methylation. We found that 30% of variation in allele-specific expression could be attributed to haplotype associated factors, with allelic levels of chromatin accessibility and three histone modifications in gene bodies having the most influence. Structural variants in promoter regions were also associated with allele-specific expression, including specific enrichments of hAT and MULE-MuDR DNA transposon sequences. Mining of cis-regulatory sequences underlying regions with allelic variation in chromatin accessibility revealed a paternally-associated sequence motif bound by ERF48, a target of the Polycomb repressive complex 2 (PRC2), and sequence similarity of this motif corresponded to local levels of H3K27me3, a signature of PRC2 activity. Conclusions Using a locally phased assembly of a heterozygous citrus cultivar, we dissected the interplay between genetic variants and molecular phenotypes with the goal of revealing functional cis-regulatory sequences and exploring the evolution of gene regulation.

Complete and partial sequences of mariner-like elements (MLEs) have been reported for hundreds of species of animals, but only two have been identified in plants. On the basis of these two plant MLEs and several related sequences identified by database searches, plant-specific degenerate primers were derived and used to amplify a conserved region of MLE transposase genes from a variety of plant genomes. Positive products were obtained for 6 dicots and 31 monocots of 54 plant species tested. Phylogenetic analysis of 68 distinct MLE transposase sequences from 25 grass species is consistent with vertical transmission and rapid diversification of multiple lineages of transposases. Surprisingly, the evolution of MLEs in grasses was accompanied by repeated and independent acquisition of introns in a localized region of the transposase gene.

The largest component of plant and animal genomes characterized to date is transposable elements (TEs). The availability of a significant amount of Lotus japonicus genome sequence has permitted for the first time a comprehensive study of the TE landscape in a legume species. Here we report the results of a combined computer-assisted and experimental analysis of the TEs in the 32.4 Mb of finished TAC clones. While computer-assisted analysis facilitated a determination of TE abundance and diversity, the availability of complete TAC sequences permitted identification of full-length TEs, which facilitated the design of tools for genomewide experimental analysis. In addition to containing all TE types found in previously characterized plant genomes, the TE component of L. japonicus contained several surprises. First, it is the second species (after Oryza sativa) found to be rich in Pack-MULEs, with >1000 elements that have captured and amplified gene fragments. In addition, we have identified what appears to be a legume-specific MULE family that was previously identified only in fungal species. Finally, the L. japonicus genome contains many hundreds, perhaps thousands of Sireviruses: Ty1/copia-like elements with an extra ORF. Significantly, several of the L. japonicus Sireviruses have recently amplified and may still be actively transposing.

The transposable Dissociation (Ds) element of maize was first discovered as a site of high-frequency chromosome breakage. Because both Ds-mediated breakage and transposition require the presence of the Activator (Ac) element, it has been suggested that chromosome breakage may be the outcome of an aberrant transposition event. This idea is consistent with the finding that only complex structures containing multiple Ds or Ac and Ds elements have been correlated with chromosome breakage. In this report, we describe two chromosome-breaking maize alleles that contain pairs of closely linked but separate Ds elements inserted at the Waxy locus. A polymerase chain reaction assay was utilized to isolate intermediates in the breakage process. The DNA sequence of these intermediates reveals deletions and base pair changes consistent with transposon footprints that may represent the junctions between fused sister chromatids. These results provide direct molecular evidence that chromosome breakage is the result of aberrant transposition events.

Abstract R and B genes and their homologues encode basic helix-loop-helix (bHLH) transcriptional activators that regulate the anthocyanin biosynthetic pathway in flowering plants. In maize, R/B genes comprise a very small gene family whose organization reflects the unique evolutionary history and genome architecture of maize. To know whether the organization of the R gene family could provide information about the origins of the distantly related grass rice, we characterized members of the R gene family from rice Oryza sativa. Despite being a true diploid, O. sativa has at least two R genes. An active homologue (Ra) with extensive homology with other R genes is located at a position on chromosome 4 previously shown to be in synteny with regions of maize chromosomes 2 and 10 that contain the B and R loci, respectively. A second rice R gene (Rb) of undetermined function was identified on chromosome 1 and found to be present only in rice species with AA genomes. All non-AA species have but one R gene that is Ra-like. These data suggest that the common ancestor shared by maize and rice had a single R gene and that the small R gene families of grasses have arisen recently and independently.

Previous genetic and structural evidence indicates that the maize R gene encodes a nuclear transcriptional activating factor. In-frame carboxyl- and amino-terminal fusions of the R gene to the reporter gene encoding β-glucuronidase (GUS) were sufficient to direct GUS to the nucleus of the transiently transformed onion (Allium cepa) epidermal cells. Further analysis of chimeric constructs containing regions of the R gene fused to the GUS cDNA revealed three specific nuclear localization sequences (NLSs) that were capable of redirecting the GUS protein to the nucleus. Amino-terminal NLS-A (amino acids 100–)109, GDRRAAPARP) contained several arginine residues; a similar localization signal is found in only a few viral proteins. The medial NLS-M (amino acids 419–)428, MSERKRREKL) is a simian virus 40 large T antigen-type NLS, and the carboxyl-terminal NLS-C (amino acids 598–)610, MISESLRK-AIGKR) is a mating type α-2 type. NLSs M and C are independentlysufficient to direct the GUS protein to the nucleus when it is fused at the amino terminus of GUS, whereas NLS-A fused to GUS partitioned between the nucleus and cytoplasm. Similar partitioning was observed when localization signals NLS-A and NLS-C were independently fused to the carboxy-terminal portion of GUS. A sequential deletion of the localization signals indicated that the amino-terminal and carboxyl-terminal fusions of R and GUS were redirected to the nucleus only when both NLS-A and -M, or NLS-C and -M, were present. These results indicate that multiple localization signals are necessary for nuclear targeting of this protein. The conservation of the localization signals within the alleles of R and similar proteins from other organisms is also discussed.

Abstract The waxy gene, which is responsible for the synthesis of amylose in endosperm and pollen, is genetically well characterized in many grasses including maize and rice. Homology between the previously cloned maize waxy gene and the rice gene has facilitated our cloning of a 15-kb HindIII fragment that contains the entire rice gene. A comparison of the restriction maps of the maize and rice genes indicates that many restriction sites within translated exons are conserved. In addition, the rice gene encodes a 2.4-kb transcript that programs the in vitro synthesis of a 64-kD pre-protein which is efficiently precipitated with maize waxy antisera. We demonstrate that these gene products are altered in rice strains containing mutant waxy genes. Southern blot analysis of 16 rice strains, ten containing waxy mutations, reveals that the waxy gene and flanking restriction fragments are virtually identical. These results contrast dramatically with the high level of insertions and deletions associated with restriction fragment length polymorphism and spontaneous mutations among the waxy alleles of maize.

We have determined the structure of five spontaneous deletions within the maize waxy (Wx) gene. Of these, four were found in spontaneous wx mutants (wx-B, wx-B1, wx-B6, wx-C4) and include exon sequences; the fifth is restricted to an intron and represents a restriction fragment length polymorphism of a nonmutant allele (Wx-W23). The deletions, which range in size from 60 to 980 base pairs (bp), cluster in a G+C-rich region of approximately 1000 bp that is capable of forming stable secondary structures. Most striking is our finding that all of the alleles have DNA insertions (filler DNA) of 1-131 bp between the deletion endpoints. For three of the five deletions, the filler DNA and sequences at the deletion termini appear to be derived from sequences near one deletion endpoint. A previously reported spontaneous deletion of the maize bronze gene (bz-R) also contains filler DNA. The association of filler DNA with maize deletion endpoints contrasts dramatically with the rarity of similar events in animal germ-line and bacterial mutations.

The DNA sequence of 306 base-pairs preceding and within the first structural gene of the leu operon of Escherichia coli, leuA, was determined. A prominent feature of this sequence is a signal for transcription termination. In vitro, transcription does terminate at this site, yielding a leader RNA of about 165 nucleotides. This leader RNA is potentially translatable into a peptide containing 28 amino acids, four of which are adjacent Leu residues. Several regions of base complementarity exist within the leader RNA. When the nucleotide sequences of the leu leaders of E. coli and Salmonella typhimurium are compared these segments are conserved while other segments vary appreciably. The highly conserved regions are those thought to play a functional role in attentuation control of leu operon expression.

Abstract Insertional mutagenesis of legume genomes such as soybean (Glycine max) should aid in identifying genes responsible for key traits such as nitrogen fixation and seed quality. The relatively low throughput of soybean transformation necessitates the use of a transposon-tagging strategy where a single transformation event will produce many mutations over a number of generations. However, existing transposon-tagging tools being used in legumes are of limited utility because of restricted transposition (Ac/Ds: soybean) or the requirement for tissue culture activation (Tnt1: Medicago truncatula). A recently discovered transposable element from rice (Oryza sativa), mPing, and the genes required for its mobilization, were transferred to soybean to determine if it will be an improvement over the other available transposon-tagging tools. Stable transformation events in soybean were tested for mPing transposition. Analysis of mPing excision at early and late embryo developmental stages revealed increased excision during late development in most transgenic lines, suggesting that transposition is developmentally regulated. Transgenic lines that produced heritable mPing insertions were identified, with the plants from the highest activity line producing at least one new insertion per generation. Analysis of the mPing insertion sites in the soybean genome revealed that features displayed in rice were retained including transposition to unlinked sites and a preference for insertion within 2.5 kb of a gene. Taken together these findings indicate that mPing has the characteristics necessary for an effective transposon-tagging resource.

Class 2 transposable elements (TEs) are the predominant elements in and around plant genes where they generate significant allelic diversity. Using the complete sequences of four grasses, we have performed a novel comparative analysis of class 2 TEs. To ensure consistent comparative analyses, we re-annotated class 2 TEs in Brachypodium distachyon, Oryza sativa (rice), Sorghum bicolor and Zea mays and assigned them to one of the five cut-and-paste superfamilies found in plant genomes (Tc1/mariner, PIF/Harbinger, hAT, Mutator, CACTA). We have focused on noncoding elements because of their abundance, and compared superfamily copy number, size and genomic distribution as well as correlation with the level of nearby gene expression.Our comparison revealed both unique and conserved features. First, the average length or size distribution of elements in each superfamily is largely conserved, with the shortest always being Tc1/mariner elements, followed by PIF/Harbinger, hAT, Mutator and CACTA. This order also holds for the ratio of the copy numbers of noncoding to coding elements. Second, with the exception of CACTAs, noncoding TEs are enriched within and flanking genes, where they display conserved distribution patterns, having the highest peak in the promoter region. Finally, our analysis of microarray data revealed that genes associated with Tc1/mariner and PIF/Harbinger noncoding elements have significantly higher expression levels than genes without class 2 TEs. In contrast, genes with CACTA elements have significantly lower expression than genes without class 2 TEs.We have achieved the most comprehensive annotation of class 2 TEs to date in these four grass genomes. Comparative analysis of this robust dataset led to the identification of several previously unknown features of each superfamily related to copy number, element size, genomic distribution and correlation with the expression levels of nearby genes. These results highlight the importance of distinguishing TE superfamilies when assessing their impact on gene and genome evolution.

Transposable elements (TEs) are dynamic components of genomes that often vary in copy number among members of the same species. With the advent of next-generation sequencing TE insertion-site polymorphism can be examined at an unprecedented level of detail when combined with easy-to-use bioinformatics software. Here we report a new tool, RelocaTE, that rapidly identifies specific TE insertions that are either polymorphic or shared between a reference and unassembled next-generation sequencing reads. Furthermore, a novel companion tool, CharacTErizer, exploits the depth of coverage to classify genotypes of nonreference insertions as homozygous, heterozygous or, when analyzing an active TE family, as rare somatic insertion or excision events. It does this by comparing the numbers of RelocaTE aligned reads to reads that map to the same genomic position without the TE. Although RelocaTE and CharacTErizer can be used for any TE, they were developed to analyze the very active mPing element which is undergoing massive amplification in specific strains of Oryza sativa (rice). Three individuals of one of these strains, A123, were resequenced and analyzed for mPing insertion site polymorphisms. The majority of mPing insertions found (~97%) are not present in the reference, and two siblings from a self-crossed of this strain were found to share only ~90% of their insertions. Private insertions are primarily heterozygous but include both homozygous and predicted somatic insertions. The reliability of the predicted genotypes was validated by polymerase chain reaction.

To understand the success strategies of transposable elements (TEs) that attain high copy numbers, we analyzed two pairs of rice (Oryza sativa) strains, EG4/HEG4 and A119/A123, undergoing decades of rapid amplification (bursts) of the class 2 autonomous Ping element and the nonautonomous miniature inverted repeat transposable element (MITE) mPing Comparative analyses of whole-genome sequences of the two strain pairs validated that each pair has been maintained for decades as inbreds since divergence from their respective last common ancestor. Strains EG4 and HEG4 differ by fewer than 160 SNPs and a total of 264 new mPing insertions. Similarly, strains A119 and A123 exhibited about half as many SNPs (277) as new mPing insertions (518). Examination of all other potentially active TEs in these genomes revealed only a single new insertion out of ∼40,000 loci surveyed. The virtual absence of any new TE insertions in these strains outside the mPing bursts demonstrates that the Ping/mPing family gradually attains high copy numbers by maintaining activity and evading host detection for dozens of generations. Evasion is possible because host recognition of mPing sequences appears to have no impact on initiation or maintenance of the burst. Ping is actively transcribed, and both Ping and mPing can transpose despite methylation of terminal sequences. This finding suggests that an important feature of MITE success is that host recognition does not lead to the silencing of the source of transposase.

Transposable elements (TEs) shape genome evolution through periodic bursts of amplification. In this study prior knowledge of the mPing/Ping/Pong TE family is exploited to track their copy numbers and distribution in genome sequences from 3,000 accessions of domesticated Oryza sativa (rice) and the wild progenitor Oryza rufipogon. We find that mPing bursts are restricted to recent domestication and is likely due to the accumulation of two TE components, Ping16A and Ping16A_Stow, that appear to be critical for mPing hyperactivity. Ping16A is a variant of the autonomous element with reduced activity as shown in a yeast transposition assay. Transposition of Ping16A into a Stowaway element generated Ping16A_Stow, the only Ping locus shared by all bursting accessions, and shown here to correlate with high mPing copies. Finally, we show that sustained activity of the mPing/Ping family in domesticated rice produced the components necessary for mPing bursts, not the loss of epigenetic regulation.

Since the advent of methodologies to analyze the content of whole genomes (e.g., renaturation kinetics and Cot analysis), it has been known that a large fraction of eukaryotic genomes is highly repetitive (1, 2). Recent computer-assisted analysis of several sequenced eukaryotic genomes, including Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and humans, has demonstrated that most repetitive DNA is composed of or derived from transposable elements (TEs). In the human genome, for example, TEs are the single most abundant component, accounting for over 40% of the total DNA (3). Although this amount of TEs is viewed as a hindrance to those engaged in the determination and assembly of DNA sequence, the availability of both complete and partial eukaryotic genome sequences is providing TE biologists with a bonanza of raw material that is being used to understand how genomes evolve.

The R/B genes of maize encode a family of basic helix-loop-helix proteins that determine where and when the anthocyanin-pigment pathway will be expressed in the plant. Previous studies showed that allelic diversity among family members reflects differences in gene expression, specifically in transcription initiation. We present evidence that the R gene Lc is under translational control. We demonstrate that the 235-nt transcript leader of Lc represses expression 25- to 30-fold in an in vivo assay. Repression is mediated by the presence in cis of a 38-codon upstream open reading frame. Furthermore, the coding capacity of the upstream open reading frame influences the magnitude of repression. It is proposed that translational control does not contribute to tissue specificity but prevents overexpression of the Lc protein. The diversity of promoter and 5' untranslated leader sequences among the R/B genes provides an opportunity to study the coevolution of transcriptional and translational mechanisms of gene regulation.

We previously reported that three alleles of the maize waxy (wx) gene were alternatively spliced as a result of the insertion of retrotransposons into intronic sequences. In addition, inefficient splicing of element sequences with the surrounding intron produced wild-type transcripts that presumably were responsible for the observed residual gene expression in the endosperm. In this study, we report that one of these alleles, wxG, has a tissue-specific phenotype with 30-fold more WX enzymatic activity in pollen than in the endosperm. Quantification of wxG-encoded transcripts in pollen and the endosperm demonstrates that this difference can be accounted for by tissue-specific differences in RNA processing. Specifically, there is approximately 30-fold more correctly spliced RNA in pollen than in the endosperm. Based on an analogy to similar examples of tissue-specific alternative splicing in animal systems, we hypothesize that the tissue-specific phenotype of the wxG allele may reflect differences in the concentration of splicing factors in these tissues.

Abstract A new and unusual family of LTR elements, Dasheng, has been discovered in the genome of Oryza sativa following database searches of ~100 Mb of rice genomic sequence and 78 Mb of BAC-end sequence information. With all of the cis-elements but none of the coding domains normally associated with retrotransposons (e.g., gag, pol), Dasheng is a novel nonautonomous LTR element with high copy number. Over half of the ~1000 Dasheng elements in the rice genome are full length (5.6–8.6 kb), and 60% are estimated to have amplified in the past 500,000 years. Using a modified AFLP technique called transposon display, 215 elements were mapped to all 12 rice chromosomes. Interestingly, more than half of the mapped elements are clustered in the heterochromatic regions around centromeres. The distribution pattern was further confirmed by FISH analysis. Despite clustering in heterochromatin, Dasheng elements are not nested, suggesting their potential value as molecular markers for these marker-poor regions. Taken together, Dasheng is one of the highest-copy-number LTR elements and one of the most recent elements to amplify in the rice genome.

Mariner-like elements (MLEs) are DNA transposons found throughout the plant and animal kingdoms.A previous computational survey of the rice (Oryza sativa) genome sequence revealed 34 full length MLEs (Osmars) belonging to 25 distinct families.This survey, which also identified sequence similarities between the Osmar elements and the Stowaway superfamily of MITEs, led to the formulation of a hypothesis whereby Stowaways are mobilized by OSMAR transposases.Here we investigate the DNAbinding activities and specificities of two OSMAR transposases, OSMAR5 and OSMAR10.Like other mariner-like transposases, the OSMARs bind specifically to the terminal inverted repeat (TIR) sequences of their encoding transposons.OSMAR5 binds DNA through a bipartite N-terminal domain containing two functionally separable helix-turn-helix motifs, resembling the paired domain of Tc1-like transposases and PAX transcription factors in metazoans.Furthermore, binding of the OSMARs is not limited to their own TIRs; OSMAR5 transposase can also interact in vitro with TIRs from closely related Osmar elements and with consensus TIRs of several Stowaway families mined from the rice genome sequence.These results provide the first biochemical evidence for a functional relationship between Osmar elements and Stowaway MITEs and lead us to suggest that there is extensive cross-talk among related but distinct transposon families co-existing in a single eukaryote genome.

Cot-based cloning and sequencing (CBCS), a synthesis of Cot analysis, DNA cloning and high-throughput sequencing, promises to accelerate the study of eukaryotic genomes. In particular, CBCS will (1) permit efficient gene discovery in species with substantial quantities of repetitive DNA, (2) allow the sequence complexity (i.e. all the unique sequence information) of large genomes to be elucidated at a fraction of the cost of shotgun sequencing, and (3) enhance genome sequencing efforts by facilitating capture of low-copy sequences not secured by EST sequencing. CBCS should accelerate comparative genomics research, especially in large genomes such as those of many crops.

The R locus of maize is one of several genes that condition the red and purple anthocyanin pigments throughout the body of the plant and seed. Many alleles of the R locus have been described, each determining a different pattern of pigment distribution in the plant. Genetic studies have lead to the proposal that R is a complex locus containing several similar but functionally distinct modules that regulate anthocyanin expression in specific organs or tissues. We report on the isolation of a segment of DNA from the R locus using the controlling element Activator (Ac)as a transposon tag. Molecular evidence is presented confirming the complex organization of this regulatory locus. The identification of cross-hybridizing restriction fragments corresponding to the tissue-specific R components suggests that these elements, while functionally divergent in tissue-specificity, have retained significant DNA homology and may have evolved from a common ancesteral component at R.

Abstract Anthocyanin pigmentation patterns in different plant species are controlled in part by members of the myc-like R regulatory gene family. We have examined the molecular evolution of this gene family in seven plant species. Three regions of the R protein show sequence conservation between monocot and dicot R genes. These regions encode the basic helix-loop-helix domain, as well as conserved N-terminal and C-terminal domains; mean replacement rates for these conserved regions are 1.02 x 10(-9) nonsynonymous nucleotide substitutions per site per year. More than one-half of the protein, however, is diverging rapidly, with nonsynonymous substitution rates of 4.08 x 10(-9) substitutions per site per year. Detailed analysis of R homologs within the grasses (Poaceae) confirm that these variable regions are indeed evolving faster than the flanking conserved domains. Both nucleotide substitutions and small insertion/deletions contribute to the diversification of the variable regions within these regulatory genes. These results demonstrate that large tracts of sequence in these regulatory loci are evolving at a fairly rapid rate.

The magellan transposable element is responsible for a spontaneous 5.7-kb insertion in the maize wx-M allele. This element has the sequence and structural characteristics of a Ty3/gypsy-like retrotransposon. The magellan element is present in all Zea species and Tripsacum andersonii; it is absent, however, in the genomes of all other Tripsacum species analyzed. The genetic distances between magellan elements suggest that this retrotransposon is evolving faster than other Zea nuclear loci. The phylogeny of magellan within Zea and T. andersonii also reveals a pattern of interspecies transfers, resulting in the movement of magellan subfamilies between different species genomes. Interspecific hybridization may be a major mechanism by which this retrotransposon invades and establishes itself in new taxa.

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Transposable element (TE) polymorphisms are important components of population genetic variation. The functional impacts of TEs in gene regulation and generating genetic diversity have been observed in multiple species, but the frequency and magnitude of TE variation is under appreciated. Inexpensive and deep sequencing technology has made it affordable to apply population genetic methods to whole genomes with methods that identify single nucleotide and insertion/deletion polymorphisms. However, identifying TE polymorphisms, particularly transposition events or non-reference insertion sites can be challenging due to the repetitive nature of these sequences, which hamper both the sensitivity and specificity of analysis tools.We have developed the tool RelocaTE2 for identification of TE insertion sites at high sensitivity and specificity. RelocaTE2 searches for known TE sequences in whole genome sequencing reads from second generation sequencing platforms such as Illumina. These sequence reads are used as seeds to pinpoint chromosome locations where TEs have transposed. RelocaTE2 detects target site duplication (TSD) of TE insertions allowing it to report TE polymorphism loci with single base pair precision.The performance of RelocaTE2 is evaluated using both simulated and real sequence data. RelocaTE2 demonstrate high level of sensitivity and specificity, particularly when the sequence coverage is not shallow. In comparison to other tools tested, RelocaTE2 achieves the best balance between sensitivity and specificity. In particular, RelocaTE2 performs best in prediction of TSDs for TE insertions. Even in highly repetitive regions, such as those tested on rice chromosome 4, RelocaTE2 is able to report up to 95% of simulated TE insertions with less than 0.1% false positive rate using 10-fold genome coverage resequencing data. RelocaTE2 provides a robust solution to identify TE insertion sites and can be incorporated into analysis workflows in support of describing the complete genotype from light coverage genome sequencing.

Significance Transposable elements (TEs) represent the largest component of the genomes of higher eukaryotes. Among this component are some TEs that have attained very high copy numbers, with hundreds, even thousands, of elements. By documenting the spread of mPing elements throughout the genomes of a rice population, we demonstrate that such bursts of amplification generate functionally relevant genomic variations upon which selection can act. Specifically, continued mPing amplification increases the number of tightly linked elements that, in turn, increases the frequency of structural variations that appear to be derived from aberrant transposition events. The significance of this finding is that it provides a TE-mediated mechanism that may generate much of the structural variation represented by pan-genomes in plants and other organisms.

A 128-bp insertion into the maize waxy-B2 allele led to the discovery of Tourist, a family of miniature inverted repeat transposable elements (MITEs). As a special category of nonautonomous elements, MITEs are distinguished by their high copy number, small size, and close association with plant genes. In maize, some Tourist elements (named Tourist-Zm) are present as adjacent or nested insertions. To determine whether the formation of multimers is a common feature of MITEs, we performed a more thorough survey, including an estimation of the proportion of multimers, with 30.2 Mb of publicly available rice genome sequence. Among the 6600 MITEs identified, >10% were present as multimers. The proportion of multimers differs for different MITE families. For some MITE families, a high frequency of self-insertions was found. The fact that all 340 multimers are unique indicates that the multimers are not capable of further amplification.

Abstract Lc, a member of the maize (Zea mays)R/B gene family, encodes a basic helix-loop-helix transcriptional activator of the anthocyanin biosynthetic pathway. It was previously shown that translation of the Lc mRNA is repressed by a 38-codon upstream open reading frame (uORF) in the 5′ leader. In this study, we report that a potential hairpin structure near the 5′end of the Lc mRNA also represses downstream translation in the rabbit reticulocyte in vitro translation system and in transient transformation assays. Base pairing of the hairpin is important for repression because its destabilization increases translation of the uORF and the downstream ORF. However, translation of the uORF is not required for the hairpin-mediated repression. Instead, the uORF and the 5′-proximal hairpin mediate two independent levels of repression. Although the uORF represses downstream translation due to inefficient reinitiation of ribosomes that translate uORF, the hairpin inhibits ribosome loading at the 5′ end of the mRNA.

To regulate Waxy (Wx) gene expression by introducing antisense genes, we connected the 2.3 kb Wx cDNA having 450 bp of the Wx first intron in reverse orientation to rice Wx and maize alcohol dehydrogenase1 (Adh1) promoters and used these constructs to transform rice plants. Of 10 independent transgenic lines analysed, four lines showed various degrees of reduction in amylose and WAXY (WX) protein levels in the endosperm. In two transgenic lines, complete absence of amylose was observed which made the seeds opaque white like glutinous rice (amylose-deficient waxy (wx) mutant). In one of the transgenic lines, A1 line, the presence of the antisense Wx gene cosegregated with reduction of amylose content in the endosperm. In the same line, a reduction in the level of endogenous Wx mRNA was observed in immature endosperm. Interestingly, this reduction was observed only with mature spliced transcripts but not with unspliced transcripts. Reduced amylose synthesis was also observed in pollen grains of four transgenic lines. These results suggest that integrated antisense Wx gene caused a reduction in amylose synthesis in endosperms and pollen grains of transgenic rice carrying the antisense Wx cDNA. These results indicate that manipulation of starch and other carbohydrates in rice grain is possible using antisense genes.

We report the first comprehensive analysis of transposable element content in the compact genomes (approximately 20 Mb) of four species of Entamoeba unicellular protozoans for which draft sequences are now available. Entamoeba histolytica and Entamoeba dispar, two human parasites, have many retrotransposons, but few DNA transposons. In contrast, the reptile parasite Entamoeba invadens and the free-living Entamoeba moshkovskii contain few long interspersed elements but harbor diverse and recently amplified populations of DNA transposons. Representatives of three DNA transposase superfamilies (hobo/Activator/Tam3, Mutator, and piggyBac) were identified for the first time in a protozoan species in addition to a variety of members of a fourth superfamily (Tc1/mariner), previously reported only from ciliates and Trichomonas vaginalis among protozoans. The diversity of DNA transposons and their differential amplification among closely related species with similar compact genomes are discussed in the context of the biology of Entamoeba protozoans.

Maize R genes encode a small family of transcriptional activators of several structural genes in the anthocyanin biosynthetic pathway. The 5′ leader region of most R genes contains a 38-codon upstream open reading frame (uORF) that previously was shown to be responsible for the repression of downstream gene expression in a transient transformation assay. In this study, we report that the 5′ leader also can repress translation of the downstream luciferase gene both in the rabbit reticulocyte translation system and in transgenic rice plants. The ability to visualize the uORF peptide after in vitro translation permits quantification of both products of dicistronic mRNAs. Similarly, the construction of transgenic rice plants expressing wild-type and mutant constructs permits the quantification and correlation of steady state mRNA levels and reporter gene activities. Using these assays, we demonstrate directly that translation of the uORF is required for repression, that increasing translation of the uORF peptide decreases downstream gene expression, and that repression is unaffected by either subtle or gross changes in the uORF peptide. Rather, we find that ribosomes that translate the uORF reinitiate inefficiently and that the intercistronic sequence downstream of the uORF mediates this effect.

Dasheng is one of the highest copy number long terminal repeat elements and one of the most recent elements to amplify in the rice (Oryza sativa) genome. However, the absence of any significant coding capacity for retroviral proteins, including gag and pol, suggests that Dasheng is a nonautonomous element. Here, we have exploited the availability of 360 Mb of rice genomic sequence to identify a candidate autonomous element. RIRE2 is a previously described gypsy-like long terminal repeat retrotransposon with significant sequence similarity to Dasheng in the regions where putative cis factors for retrotransposition are thought to be located. Dasheng and RIRE2 elements have similar chromosomal distribution patterns and similar target site sequences, suggesting that they use the same transposition machinery. In addition, the presence of several RIRE2-Dasheng element chimeras in the genome is consistent with the copackaging of element mRNAs in the same virus-like particle. Finally, both families have recently amplified members, suggesting that they could have been co-expressed, a necessary prerequisite for RIRE2 to serve as the source of transposition machinery for Dasheng. Consistent with this hypothesis, transcripts from both elements were found in the same expressed sequence tag library.

Abstract The Louisiana iris species Iris brevicaulis and I. fulva are morphologically and karyotypically distinct yet frequently hybridize in nature. A group of high-copy-number TY3/gypsy-like retrotransposons was characterized from these species and used to develop molecular markers that take advantage of the abundance and distribution of these elements in the large iris genome. The copy number of these IRRE elements (for iris retroelement), is ∼1 × 105, accounting for ∼6–10% of the ∼10,000-Mb haploid Louisiana iris genome. IRRE elements are transcriptionally active in I. brevicaulis and I. fulva and their F1 and backcross hybrids. The LTRs of the elements are more variable than the coding domains and can be used to define several distinct IRRE subfamilies. Transposon display or S-SAP markers specific to two of these subfamilies have been developed and are highly polymorphic among wild-collected individuals of each species. As IRRE elements are present in each of 11 iris species tested, the marker system has the potential to provide valuable comparative data on the dynamics of retrotransposition in large plant genomes.

Crop loss due to soil salinization is an increasing threat to agriculture worldwide. This review provides an overview of cellular and physiological mechanisms in plant responses to salt. We place cellular responses in a time- and tissue-dependent context ...Read More

The formation of hybrid zones between nascent species is a widespread phenomenon. The evolutionary consequences of hybridization are influenced by numerous factors, including the action of natural selection on quantitative trait variation. Here we examine how the genetic basis of floral traits of two species of Louisiana Irises affects the extent of quantitative trait variation in their hybrids. Quantitative trait locus (QTL) mapping was used to assess the size (magnitude) of phenotypic effects of individual QTL, the degree to which QTL for different floral traits are colocalized, and the occurrence of mixed QTL effects. These aspects of quantitative genetic variation would be expected to influence (1) the number of genetic steps (in terms of QTL substitutions) separating the parental species phenotypes; (2) trait correlations; and (3) the potential for transgressive segregation in hybrid populations. Results indicate that some Louisiana Iris floral trait QTL have large effects and QTL for different traits tend to colocalize. Transgressive variation was observed for six of nine traits, despite the fact that mixed QTL effects influence few traits. Overall, our QTL results imply that the genetic basis of floral morphology and color traits might facilitate the maintenance of phenotypic divergence between Iris fulva and Iris brevicaulis, although a great deal of phenotypic variation was observed among hybrids.

Although quantitative traits loci (QTL) analysis has been widely performed to isolate agronomically important genes, it has been difficult to obtain molecular markers between individuals with similar phenotypes (assortative mating). Recently, the miniature inverted-repeat transposable element mPing was shown to be active in the japonica strain Gimbozu EG4 where it had accumulated more than 1000 copies. In contrast, most other japonicas, including Nipponbare, have 50 or fewer mPing insertions in their genome. In this study we have exploited the polymorphism of mPing insertion sites to generate 150 PCR markers in a cross between the closely related japonicas, Nipponbare × Gimbozu (EG4). These new markers were distributed in genic regions of the whole genome and showed significantly higher polymorphism (150 of 183) than all other molecular markers tested including short sequence repeat markers (46 of 661). In addition, we performed QTL analysis with these markers using recombinant inbred lines derived from Nipponbare × Gimbozu EG4, and successfully mapped a locus involved in heading date on the short arm of chromosome 6. Moreover, we could easily map two novel loci involved in the culm length on the short arms of chromosomes 3 and 10.

Most of our understanding of plant genome structure and evolution has come from the careful annotation of small (e.g., 100 kb) sequenced genomic regions or from automated annotation of complete genome sequences. Here, we sequenced and carefully annotated a contiguous 22 Mb region of maize chromosome 4 using an improved pseudomolecule for annotation. The sequence segment was comprehensively ordered, oriented, and confirmed using the maize optical map. Nearly 84% of the sequence is composed of transposable elements (TEs) that are mostly nested within each other, of which most families are low-copy. We identified 544 gene models using multiple levels of evidence, as well as five miRNA genes. Gene fragments, many captured by TEs, are prevalent within this region. Elimination of gene redundancy from a tetraploid maize ancestor that originated a few million years ago is responsible in this region for most disruptions of synteny with sorghum and rice. Consistent with other sub-genomic analyses in maize, small RNA mapping showed that many small RNAs match TEs and that most TEs match small RNAs. These results, performed on ∼1% of the maize genome, demonstrate the feasibility of refining the B73 RefGen_v1 genome assembly by incorporating optical map, high-resolution genetic map, and comparative genomic data sets. Such improvements, along with those of gene and repeat annotation, will serve to promote future functional genomic and phylogenomic research in maize and other grasses.

Abstract Background PIF/Harbinger is the most recently discovered DNA transposon superfamily and is now known to populate genomes from fungi to plants to animals. Mobilization of superfamily members requires two separate element-encoded proteins (ORF1 and TPase). Members of this superfamily also mobilize Tourist -like miniature inverted repeat transposable elements (MITEs), which are the most abundant transposable elements associated with the genes of plants, especially the cereal grasses. The phylogenetic analysis of many plant genomes indicates that MITEs can amplify rapidly from one or a few elements to hundreds or thousands. The most active DNA transposon identified to date in plants or animals is mPing , a rice Tourist -like MITE that is a deletion derivative of the autonomous Ping element. Ping and the closely related Pong are the only known naturally active PIF/Harbinger elements. Some rice strains accumulate ~40 new mPing insertions per plant per generation. In this study we report the development of a yeast transposition assay as a first step in deciphering the mechanism underlying the amplification of Tourist -MITEs. Results The ORF1 and TPase proteins encoded by Ping and Pong have been shown to mobilize mPing in rice and in transgenic Arabidopsis . Initial tests of the native proteins in a yeast assay resulted in very low transposition. Significantly higher activities were obtained by mutation of a putative nuclear export signal (NES) in the TPase that increased the amount of TPase in the nucleus. When introduced into Arabidopsis , the NES mutant protein also catalyzed higher frequencies of mPing excision from the gfp reporter gene. Our yeast assay retains key features of excision and insertion of mPing including precise excision, extended insertion sequence preference, and a requirement for two proteins that can come from either Ping or Pong or both elements. Conclusions The yeast transposition assay provides a robust platform for analysis of the mechanism underlying transposition catalyzed by the two proteins of PIF/Harbinger elements. It recapitulates all of the features of excision and reinsertion of mPing as seen in plant systems. Furthermore, a mutation of a putative NES in the TPase increased transposition both in yeast and plants.

The dynamic genome is a concept associated with the discovery of transposable elements by Barbara McClintock. Her Nobel lecture concluded with a challenge to biologists considering this issue (1). She wrote,

Abstract The molecular basis for the unusual phenotype conditioned by the waxy(Wx)-m5 Ds allele has been elucidated. Unlike most Ds alleles, Wx-m5 is phenotypically wild-type in the absence of Ac. We find that the Wx-m5 gene contains a 2-kb Ds element at -470 relative to the start of Wx transcription, representing the most 5' insertion of any transposable element allele characterized to date in plants. Despite its wild type phenotype, Wx-m5 has reduced levels of Wx enzymatic activity indicating that Ds insertion influences Wx gene expression. In the presence of Ac, Wx-m5 kernels have sectors of null expression on a wild-type background and give rise to stable wx and unstable wx-m germinal derivatives. Seventeen of 20 derivatives examined are wx-m alleles and at least 15 of these appear to result from intragenic transposition of Ds from -470 to new sites within the Wx gene. Three wx-m alleles contain two Ds elements, one at -470 and a second in Wx coding sequences. Surprisingly, only 3 out of 20 derivatives are stable wx mutants and these have sustained gross rearrangements of Wx and flanking sequences. For most other maize transposable element alleles somatic sectors and germinal derivatives usually arise following element excision or deletions of element sequences. In contrast, element insertion following intragenic transposition is apparently responsible for most of the somatic sectors and germinal derivatives of Wx-m5.

There are six examples of maize transposable elements that are spliced from pre-mRNA. These represent the first introns that have been added to nuclear genes in recent years. All six are members of the Ac/Ds or Spm/dSpm family of elements and all have been inserted into exons of active genes within the last twenty-five years. The structure of these element-introns and the sequences involved m their splicing are presented. These examples illustrate how active transposable elements can also function as introns and how they may evolve into stable introns.

The Tc1/mariner transposable element superfamily is widely distributed in animal and plant genomes. However, no active plant element has been previously identified. Nearly identical copies of a rice (Oryza sativa) Tc1/mariner element called Osmar5 in the genome suggested potential activity. Previous studies revealed that Osmar5 encoded a protein that bound specifically to its own ends. In this report, we show that Osmar5 is an active transposable element by demonstrating that expression of its coding sequence in yeast promotes the excision of a nonautonomous Osmar5 element located in a reporter construct. Element excision produces transposon footprints, whereas element reinsertion occurs at TA dinucleotides that were either tightly linked or unlinked to the excision site. Several site-directed mutations in the transposase abolished activity, whereas mutations in the transposase binding site prevented transposition of the nonautonomous element from the reporter construct. This report of an active plant Tc1/mariner in yeast will provide a foundation for future comparative analyses of animal and plant elements in addition to making a new wide host range transposable element available for plant gene tagging.

Gene families compose a large proportion of eukaryotic genomes. The rapidly expanding genomic sequence database provides a good opportunity to study gene family evolution and function. However, most gene family identification programs are restricted to searching protein databases where data are often lagging behind the genomic sequence data. Here, we report a user-friendly web-based pipeline, named TARGeT (Tree Analysis of Related Genes and Transposons), which uses either a DNA or amino acid 'seed' query to: (i) automatically identify and retrieve gene family homologs from a genomic database, (ii) characterize gene structure and (iii) perform phylogenetic analysis. Due to its high speed, TARGeT is also able to characterize very large gene families, including transposable elements (TEs). We evaluated TARGeT using well-annotated datasets, including the ascorbate peroxidase gene family of rice, maize and sorghum and several TE families in rice. In all cases, TARGeT rapidly recapitulated the known homologs and predicted new ones. We also demonstrated that TARGeT outperforms similar pipelines and has functionality that is not offered elsewhere.

Large lecture classes and standardized laboratory exercises are characteristic of introductory biology courses. Previous research has found that these courses do not adequately convey the process of scientific research and the excitement of discovery. Here we propose a model that provides beginning biology students with an inquiry-based, active learning laboratory experience. The Dynamic Genome course replicates a modern research laboratory focused on eukaryotic transposable elements where beginning undergraduates learn key genetics concepts, experimental design, and molecular biological skills. Here we report on two key features of the course, a didactic module and the capstone original research project. The module is a modified version of a published experiment where students experience how virtual transposable elements from rice (Oryza sativa) are assayed for function in transgenic Arabidopsis thaliana. As part of the module, students analyze the phenotypes and genotypes of transgenic plants to determine the requirements for transposition. After mastering the skills and concepts, students participate in an authentic research project where they use computational analysis and PCR to detect transposable element insertion site polymorphism in a panel of diverse maize strains. As a consequence of their engagement in this course, students report large gains in their ability to understand the nature of research and demonstrate that they can apply that knowledge to independent research projects.

Abstract A few families of retrotransposons characterized by the presence of long terminal repeats (LTRs) have amplified relatively recently in maize and account for >50% of the genome. Surprisingly, none of these elements have been shown to cause a single mutation. In contrast, most of the retrotransposon-induced mutations isolated in maize are caused by the insertion of elements that are present in the genome at 2-50 copies. To begin to understand what limits the amplification of this mutagenic class of LTR-retrotransposons, we are focusing on five elements previously identified among 17 mutations of the maize waxy gene. One of these elements, Stonor, has sustained a deletion of the entire gag region and part of the protease domain. Missing sequences were recovered from larger members of the Stonor family and indicate that the deletion probably occurred during retrotransposition. These large elements have an exceptionally long leader of 2 kb that includes a highly variable region of ∼1 kb that has not been seen in previously characterized retrotransposons. This region serves to distinguish each member of the Stonor family and indicates that no single element has yet evolved that can attain the very high copy numbers characteristic of other element families in maize.

The origin of maize has been a topic of interest to both biologists and archaeologists. During the twentieth century, the view point that maize is a domesticated form of teosinte received convincing support from biological data and is now broadly accepted among biologists familiar with the issues and data. There is no support of any kind for an alternative view that maize is a hybrid of the grasses Zea diploperennis and Tripsacum .

Abstract Genetic mapping studies provide insight into the pattern and extent of genetic incompatibilities affecting hybridization between closely related species. Genetic maps of two species of Louisiana Irises, Iris fulva and I. brevicaulis, were constructed from transposon-based molecular markers segregating in reciprocal backcross (BC1) interspecific hybrids and used to investigate genomic patterns of species barriers inhibiting introgression. Linkage mapping analyses indicated very little genetic incompatibility between I. fulva and I. brevicaulis in the form of map regions exhibiting transmission ratio distortion, and this was confirmed using a Bayesian multipoint mapping analysis. These results demonstrate the utility of transposon-based marker systems for genetic mapping studies of wild plant species and indicate that the genomes of I. fulva and I. brevicaulis are highly permeable to gene flow and introgression from one another via backcrossing.

Opportunities for large numbers of undergraduates to engage in authentic research experiences are limited in many large public institutions. These large public institutions serve the vast majority of students who are historically underrepresented in STEM fields, such as first-generation, low-income students of color. Although a course-based undergraduate research experience (CURE) is one scalable approach to providing such opportunities, there is limited evidence about the impact of participation, particularly for students historically underrepresented in science. This study provides evidence of the influence of student participation in a CURE on undergraduate science course grades using an experimental design and multiple years of data from students at a Hispanic-serving institution. Course grades were compared for five different science courses across five cohorts of students participating in a CURE ( n = 935) and a similar group of students who did not participate in the CURE ( n = 1,144). CURE students had significantly higher overall grades in a lecture course directly related to the CURE even after statistically adjusting for demographic and academic characteristics. Implications for CUREs as a model for improving science knowledge and achievement for students typically underrepresented in STEM fields are discussed.

Transposable elements are mobile sequences found in nuclear genomes and can potentially serve as molecular markers in various phylogenetic and population genetic investigations. A PCR-based method that utilizes restriction site variation of element copies within a genome is developed. These patterns of site variation, referred to as transposon signatures, are useful in differentiating between closely related groups. Signature data using the magellan retrotransposon, for example, is useful in examining relationships within the genus Zea and Tripsacum. This method allows transposable elements, or even other multiple-copy nuclear DNA sequences, to be generally utilized as molecular markers in discriminating between other closely related species and subspecies.

Three mutations, each causing constitutive expression of the Salmonella typhimurium leu operon, were cloned into phage vector lambda gt4 on EcoRI DNA fragments carrying all of that operon except for part of the promoter-distal last gene. Sequence analysis of DNA from these phage demonstrated that each contains a single base change in the leu attenuator. Transcription of mutant DNA in vitro resulted in transcription beyond the usual site of termination. The level of beta-IPM dehydrogenase, the leuB enzyme, was elevated 40-fold in a strain carrying one of these mutations, and starvation of this strain for leucine had little effect on the amount of activity expressed. Using a strain with a wild-type promoter-leader region of the leu operon, the rates of synthesis and degradation of leu leader RNA and readthrough RNA (leu mRNA) were measured by DNA-RNA hybridizations with specific DNA probes. The rate of synthesis of the leu leader was about the same in cells grown with excess or with limiting leucine. On the other hand, the rate of synthesis of leu mRNA was 12-fold higher for cells grown in limiting leucine as opposed to excess leucine. The rate of degradation of these RNA species was the same under both conditions of growth. Thus, the variation in expression of the leu operon observed for cells grown in minimal medium is, for the most part, not caused by control over the frequency of initiation or by the differential stability of these RNA species. Rather, the variation is a direct result of the frequency of transcription termination at an attenuator site. These results taken together suggest that transcription attenuation is the major mechanism by which leucine regulates expression of the leu operon of S. typhimurium for cells growing in a minimal medium.

Abstract Cis -regulatory elements (CREs) are critical in regulating gene expression, and yet our understanding of CRE evolution remains a challenge. Here, we constructed a comprehensive single-cell atlas of chromatin accessibility in Oryza sativa , integrating data from 104,029 nuclei representing 128 discrete cell states across nine distinct organs. We used comparative genomics to compare cell-type resolved chromatin accessibility between O. sativa and 57,552 nuclei from four additional grass species ( Zea mays, Sorghum bicolor, Panicum miliaceum , and Urochloa fusca ). Accessible chromatin regions (ACRs) had different levels of conservation depending on the degree of cell-type specificity. We found a complex relationship between ACRs with conserved noncoding sequences, cell-type specificity, conservation, and tissue-specific switching. Additionally, we found that epidermal ACRs were less conserved compared to other cell types, potentially indicating that more rapid regulatory evolution has occurred in the L1 epidermal layer of these species. Finally, we identified and characterized a conserved subset of ACRs that overlapped the repressive histone modification H3K27me3, implicating them as potentially critical silencer CREs maintained by evolution. Collectively, this comparative genomics approach highlights the dynamics of cell-type-specific CRE evolution in plants.

Physiologia PlantarumVolume 103, Issue 4 p. 581-586 Transposable elements associated with normal plant genes Susan R. Wessler, Susan R. Wessler S. R. Wessler (e-mail sue@dogwood.botany.uga.edu), Depts of Botany and Genetics, Life Sciences Building, The Univ. of Georgia, Athens, GA 30602, USA.Search for more papers by this author Susan R. Wessler, Susan R. Wessler S. R. Wessler (e-mail sue@dogwood.botany.uga.edu), Depts of Botany and Genetics, Life Sciences Building, The Univ. of Georgia, Athens, GA 30602, USA.Search for more papers by this author First published: 17 January 2002 https://doi.org/10.1034/j.1399-3054.1998.1030418.xCitations: 27Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume103, Issue4August 1998Pages 581-586 RelatedInformation

Transposable element (TE) display was shown to be a highly specific and reproducible method of detecting the insertion sites of TEs in individuals of the African malaria mosquito, Anopheles gambiae, and its sibling species, A. arabiensis. Relatively high levels of insertion polymorphism were observed during the TE display of several families of miniature inverted-repeat TEs (MITEs) that have variable copy numbers. The genomic locations of selected insertion sites were identified by matching the sequences of their corresponding bands in a TE display gel to specific regions of the draft A. gambiae genome assembly. We discuss different scenarios in which TE display will provide powerful dominant and co-dominant genetic markers to study the behaviour of TEs in A. gambiae populations and to illustrate the complex population genetics of this intriguing disease vector. We suggest that TE display can also provide tools for a phylogenetic analysis of the A. gambiae complex.