Structural analysis of Tpn1, a transposable element isolated from Japanese morning glory bearing variegated flowers

The 6.4 kb transposable element Tpn1 belonging to the En/Spm family was found within one of the DFR (dihydroflavonol-4-reductase) genes for anthocyanin biosynthesis in a line of Japanese morning glory (Pharbitis nil) bearing variegated flowers. Sequencing of the Tpn1 element revealed that it is 6412 by long and carries 28-bp perfect terminal inverted repeats. Its subterminal repetitive regions, believed to be the cis-acting sequences for transposition, show striking structural features. Twenty-two copies of the 10-bp sequence motif GACAACGGTT can be found as direct or inverted repeats within 650 by of the 5′ end of the element, and 33 copies of the sequence motif lie within 800 by of the 3′ terminus. All these 22 copies of the sequence motif near the 5′ terminus and 30 copies in the 3′ terminal region are arranged as inverted repeats and 3–8 by AT-rich sequences are detected between these inverted repeats. In addition, four copies of 122-bp tandem repeats and six copies of 104-bp tandem repeats are present in the 5′ and 3′ subterminal repetitive regions, respectively. No large open reading frame characteristic of autonomous elements of the En/Spm family can be detected within the element. The results are discussed with respect to heritable changes in flower variegation in this line of Japanese morning glory.     

Isolation of a Suppressor-mutator/Enhancer-like transposable element, Tpn1, from Japanese morning glory bearing variegated flowers.

The Japanese morning glory has an extensive history of genetic studies. Many mutants in the colors and shapes of its flowers and leaves have been isolated since the 17th century, and more than 200 genetic loci have been localized for the 10 linkage groups. They include over 20 mutable loci, several with variegated flower phenotypes. In a line of Japanese morning glory bearing variegated flowers called flecked, a transposable element of 6.4 kb, termed Tpn1, was found within one of the anthocyanin biosynthesis genes encoding dihydroflavonol-4-reductase (DFR). The 6.4-kb element carries 28-bp perfect terminal inverted repeats, the outer 13 bp being identical to those of the maize transposable element Suppressor-mutator/Enhancer. It is flanked by 3-bp direct repeats within the second intron of the DFR gene, 9 bp upstream of the third exon. When somatic and germinal excision occurs, it produces excision sequences characteristic of plant transposable elements. Cosegregation data of the variegated flower phenotype and the DFR gene carrying Tpn1 indicated that the mutable phenotype is due to excision of Tpn1 from the DFR gene. Sequences homologous to Tpn1 are present in multiple copies in the genome of Japanese morning glory.     

Structural analysis of Tpn1, a transposable element isolated from Japanese morning glory bearing variegated flowers

The 6.4 kb transposable element Tpn1 belonging to the En/Spm family was found within one of the DFR (dihydroflavonol-4-reductase) genes for anthocyanin biosynthesis in a line of Japanese morning glory (Pharbitis nil) bearing variegated flowers. Sequencing of the Tpn1 element revealed that it is 6412 by long and carries 28-bp perfect terminal inverted repeats. Its subterminal repetitive regions, believed to be the cis-acting sequences for transposition, show striking structural features. Twenty-two copies of the 10-bp sequence motif GACAACGGTT can be found as direct or inverted repeats within 650 by of the 5 end of the element, and 33 copies of the sequence motif lie within 800 by of the 3 terminus. All these 22 copies of the sequence motif near the 5 terminus and 30 copies in the 3 terminal region are arranged as inverted repeats and 3–8 by AT-rich sequences are detected between these inverted repeats. In addition, four copies of 122-bp tandem repeats and six copies of 104-bp tandem repeats are present in the 5 and 3 subterminal repetitive regions, respectively. No large open reading frame characteristic of autonomous elements of the En/Spm family can be detected within the element. The results are discussed with respect to heritable changes in flower variegation in this line of Japanese morning glory.     

Insertion of a novel transposable element in the tyrosinase gene is responsible for an albino mutation in the medaka fish, Oryzias latipes

In the medaka fish (Oryzias latipes) many mutants for body color have been isolated. A typical example is the recessive oculocutaneous albino mutant i, which has amelanotic skin and red-colored eyes with no tyrosinase activity. To cast light on the molecular basis of the albino mechanism, we performed Southern blot analysis of genomic DNA from the mutant with an authentic tyrosinase gene probe; the results demonstrate that an extra 1.9 kb fragment is present inside the first exon. The insertion is responsible for the oculocutaneous albinism. About 80 copies of this fragment are present in the genomes of albino-i and wild-type fish; these repeated sequences are here designated Tol1 elements and the particular element found in the tyrosinase gene of albino-i is denoted Tol1-tyr. The nucleotide sequence of Tol1-tyr shows that the fragment (i) carries terminal inverted repeats of 14 bp, and (ii) is flanked by duplicated 8 by segments of the host chromosome. These are properties of DNA-mediated transposable elements. Comparison of the nucleotide sequence of Tol1-tyr with other sequences in DNA databases, with special attention to sequences of transposable elements known to date, did not reveal any similarity. Thus, Tol1 constitutes a hitherto unknown family of DNA transposable elements.     

Insertion of a novel transposable element in the tyrosinase gene is responsible for an albino mutation in the medaka fish,Oryzias latipes

In the medaka fish (Oryzias latipes) many mutants for body color have been isolated. A typical example is the recessive oculocutaneous albino mutant i, which has amelanotic skin and red-colored eyes with no tyrosinase activity. To cast light on the molecular basis of the albino mechanism, we performed Southern blot analysis of genomic DNA from the mutant with an authentic tyrosinase gene probe; the results demonstrate that an extra 1.9 kb fragment is present inside the first exon. The insertion is responsible for the oculocutaneous albinism. About 80 copies of this fragment are present in the genomes of albino-i and wild-type fish; these repeated sequences are here designated Tol1 elements and the particular element found in the tyrosinase gene of albino-i is denoted Tol1-tyr. The nucleotide sequence of Tol1-tyr shows that the fragment (i) carries terminal inverted repeats of 14 bp, and (ii) is flanked by duplicated 8 by segments of the host chromosome. These are properties of DNA-mediated transposable elements. Comparison of the nucleotide sequence of Tol1-tyr with other sequences in DNA databases, with special attention to sequences of transposable elements known to date, did not reveal any similarity. Thus, Tol1 constitutes a hitherto unknown family of DNA transposable elements.     

The effect of E. coli host strain on the consensus sequence of regions of the human L1 transposon.

We have used highly methylation tolerant host strains to clone hyper- and hypo-methylated genomic elements from different regions of the same family of long interspersed repetitive elements from human DNA, specifically the 1.8 kilobase (kb) and 1.2kb KpnI fragments from members of the L1 family of transposable elements in which respectively some 18% and 2.7% of cytosines are methylated in vivo in human spleen DNA. The consensus of the DNA sequences of the ends of 13 clones from the hypomethylated region of human L1 agreed exactly with the consensus derived previously from clones made using conventional host strains. However the sequences of 18 of our clones from the 5' end of the hypermethylated region differed significantly from the sequences of clones made using conventional hosts (P less than 0.0001). The 5' region of the 1.8kb L1 region is a CpG island which, in human somatic tissue, appears to be maintained in a highly methylated state, including methylation at sites other than CpG dinucleotides. The consensus sequence of this region also has features suggestive of a previously unrecognized open reading frame.     

Characterization of a pollen-specific cDNA from sunflower encoding a zinc finger protein.

The maize transposable element Activator (Ac) carries subterminal CpG-rich sequences which are essential for the transposition of the element. It has previously been shown that the methylation of certain sequences contained in this region can alter their ability to interact with the Ac-encoded protein. The novel hypothesis that the methylation of subterminal Ac sequences is required for transposition was tested. Approximately 150 bp of the 5' subterminal region of the Ac element was examined for the presence of 5-methylcytosines by the ligation-mediated polymerase chain reaction (LMPCR)-aided genomic sequencing method. The methylation status of 22 and 39 cytosines on either strand of the DNA were analysed in each of five different transgenic tobacco cultures carrying transposable Ac sequences. Ten micrograms of tobacco DNA were used for each base-specific cleavage reaction before amplification by LMPCR. All but one of the cytosines were unmethylated. Only a minor fraction of the Ac molecules was methylated at one cytosine residue. It is concluded that DNA methylation at the tested Ac sequences is not required for the transposability of Ac or Ds elements in tobacco cells.     

Characterization of transposable elements in the genome of rice (Oryza sativa L.) using Representational Difference Analysis (RDA)

Representational Difference Analysis was applied to characterize genomic differentiations between rice ( Oryza sativa) and foxtail millet ( Setaria italica) and subsequently to identify rice transposable elements. Rice was used as the tester and millet as the driver. A total of eleven, non-redundant, positive clones were isolated from the library. Their analysis revealed that they all represent dispersed repetitive DNA sequences. In addition, homology searches using the BLAST procedure showed that they correspond to seven distinct rice transposable elements. Three had been previously identified as gypsy-like retroelements ( Retrosat1, RIRE3 and RIRE8). The remaining four are novel: we named them hipa (a CACTA-like transposon), houba (a copia-like retroelement), hopi and dagul (two gypsy-like retroelements). The RDA clones were used as probes in Southern hybridization experiments with genomic DNAs of several species from the family Poaceae. The results suggest that the genomic differentiations associated with the activity of these transposable elements are of relatively recent origin. In addition, comparison of the hybridization patterns obtained for several Oryza species suggests that several independent amplifications of these transposable elements might have occurred within the genus.     

New transposable elements identified as insertions in rice transposon Tnr1

Tnr1 (235 bp long) is a transposable element in rice. Polymerase chain reactions (PCRs) done with a primer(s) that hybridizes to terminal inverted repeat sequences (TIRs) of Tnr1 detected new Tnr1 members with one or two insertions in rice genomes. Six identified insertion sequences (Tnr4, Tnr5, Tnr11, Tnr12, Tnr13 and RIRE9) did not have extensive homology to known transposable elements, rather they had structural features characteristic of transposable elements. Tnr4 (1767 bp long) had imperfect 64-bp TIRs and appeared to generate duplication of a 9-bp sequence at the target site. However, the TIR sequences were not homologous to those of known transposable elements, indicative that Tnr4 is a new transposable element. Tnr5 (209 bp long) had imperfect 46-bp TIRs and appeared to generate duplication of sequence TTA like that of some elements of the Tourist family. Tnr11 (811 bp long) had 73-bp TIRs with significant homology to those of Tnr1 and Stowaway and appeared to generate duplication of sequence TA, indicative that Tnr11 is a transposable element of the Tnr1/Stowaway family. Tnr12 (2426 bp long) carried perfect 9-bp TIRs, which began with 5'-CACTA- -3' from both ends and appeared to generate duplication of a 3-bp target sequence, indicative that Tnr12 is a transposable element of the En/Spm family. Tnr13 (347 bp long) had 31-bp TIRs and appeared to generate duplication of an 8-bp target sequence. Two sequences, one the transposon-like element Crackle, had partial homology in the Tnr13 ends. All five insertions appear to be defective elements derived from autonomous ones encoding the transposase gene. All had characteristic tandem repeat sequences which may be recognized by transposase. The sixth insertion sequence, named RIRE9 (3852 bp long), which begins with 5'-TG- -3' and ends with 5'- -CA-3', appeared to generate duplication of a 5-bp target sequence. These and other structural features indicate that this insertion is a solo LTR (long terminal repeat) of a retrotransposon. The transposable elements described above could be identified as insertions into Tnr1, which do not deleteriously affect the growth of rice cells.     

Cloning and characterization of variable-sized gypsy mobile elements in Drosophila melanogaster

A cosmid genomic library from a known gypsy-induced forked mutation, f1, was screened by 32P-labeled gypsy transposable element. Of more than 250 positive clones we randomly selected 21 for in situ hybridization to wild-type polytene chromosomes. Two clones hybridized to region 15F on the X-chromosome, the cytological position of forked. A third clone hybridized to at least 17 sites on the chromosomes indicating the presence of repetitive sequences in the gypsy flanking DNA. All clones labeled the centromeric regions heavily. Ten clones, including the two hybridizing at 15F, were chosen for further analysis, and restriction mapping allowed us to place them into three groups: (1) full-length, (2) slightly diverging, and (3) highly diverging gypsy elements. Group (2) is missing the XbaI site in both their long terminal repeats (LTRs) as well as the middle HindIII site; four of these gypsy elements also have a approximately 100-bp deletion at the 5' LTR. The group (3) gypsy transposons are missing one LTR and also have highly diverging DNA sequences. The restriction analyses further imply that most of these different gypsy elements are present in more than one copy in the genome of the f1 stock used in this study. The results raise intriguing questions regarding the significance of transposable elements in evolution and biological functions.     

ISWpi1 from Wolbachia pipientis defines a novel group of insertion sequences within the IS5 family

Insertion sequences are transposable elements that can represent substantial proportions of prokaryotic genomes and play a substantial role in shaping host genome evolution. As such, evaluating and understanding insertion sequence diversity is an important task to fulfill, because it is expected to yield new insight into the evolution of bacterial transposable elements and contribute to improve genome annotations. Here, I characterized an insertion sequence, termed ISWpi1, for which the taxonomic distribution appears to be restricted to the obligate intracellular alpha-Proteobacterium Wolbachia pipientis. ISWpi1 exhibits approximately 46% identity at the amino acid level with members of the IS1031 group of insertion sequences from the IS5 family. However, the IS1031 group is characterized by a transposase gene encoded by a single open reading frame, whereas the ISWpi1 transposase gene consists of two overlapping open reading frames presumably translated as a single protein via programmed translational frameshifting. Such structure suggests that ISWpi1 may instead be related to the IS427 group of insertion sequences from the IS5 family. Altogether, these data indicate that ISWpi1 extends the known spectrum of diversity of the IS5 family, and I propose to define a novel group of insertion sequences within the IS5 family typified by ISWpi1. Probable transpositional activity, relevant insertion site preferences and taxonomic specificity make ISWpi1 a promising tool for experimentally manipulating W. pipientis bacteria, especially in light of the increasing interest in developing these bacteria as tools for controlling insect disease vectors and agricultural pests.     

Structure and unusual characteristics of a new family of transposable elements in the sea urchin Strongylocentrotus purpuratus.

The transposable element family TU of the sea urchin Strongylocentrotus purpuratus, a higher eucaryote, has recently been described (D. Liebermann, B. Hoffman-Liebermann, J. Weinthal, G. Childs, R. Maxson, A. Mauron, S.N. Cohen, and L. Kedes, Nature [London] 306:342-347, 1983). A member of this family, TU4, has an insertion, called ISTU4, of non-TU DNA. ISTU4 is a member of a family of repetitive sequences, which are present in some 1,000 copies per haploid S. purpuratus genome (B. Hoffman-Liebermann, D. Liebermann, L.H. Kedes, and S.N. Cohen, Mol. Cell. Biol. 5:991-1001, 1985). We analyzed this insertion to determine whether it is itself a transposable element. The nucleotide sequence of ISTU4 was determined and showed an unusual structure. There are four, approximately 150 nucleotides long, imperfect direct repeats followed by a single truncated version of these repeats. This region is bounded at either side by approximately 100-nucleotide-long sequences that are not related to each other or to the repeats. Nucleotide sequences at the boundaries of ISTU4-homologous and flanking regions in five genomic clones show that ISTU4 represents a family of sequences with discrete ends, which we call Tsp elements. We showed that the genomic locus that carries a Tsp element in one individual was empty in other individuals and conclude that Tsp elements are a new and different type of transposable element. Tsp elements lack two features common to most other transposable elements: Tsp integration does not result in the duplication of host DNA, and there are no inverted repeats at their termini, although short inverted repeats are present at a distance from the termini.     

Homology between a region of the human retinoblastoma gene and L1 family repetitive sequences

Five clones that hybridized weakly with the human retinoblastoma (Rb) gene were obtained by screening a human genomic library in a non-stringent condition. The DNAs of two of these clones were partially sequenced and found to contain a region with considerable homology to part of the Rb gene. These two clones were found to contain L1 family repeating sequences. This finding is discussed in relation with possible functions of the L1 family. As the L1 family is transposable, the Rb gene may be inactivated by recombination at this homologous region. Another possibility related with the DNA binding properties of Rb and L1 family proteins is also discussed.     

Fungal transposable elements and genome evolution

The transposable elements (TEs) identified in fungal genomes reflect the whole spectrum of eukaryotic transposable elements. Most of our knowledge comes from species representing different ecological situations: plant pathogens, industrial, and field strains, most of them lacking the sexual stage. A number of changes in gene structure and function has been shown to be TE-mediated: inactivation of gene expression upon insertion within or adjacent to a gene, DNA sequence variation through excision and probably extensive chromosomal rearrangements due to recombination between members of a particular family. Moreover, TEs may have other roles in evolution related to their ability to be horizontally transferred and to capture and transpose chromosomal host sequences, thus providing a mechanism for dispersing sequences to new sites. However, the activity of transposable elements and consequently their proliferation within a host genome can be affected, in some fungal species which undergo meiosis, by silencing processes. Our understanding of the biological effects of TEs on the fungal genome has increased dramatically in the past few years but elucidation of the extent to which transposons contribute to genetic variation in nature, providing the flexibility for populations to adapt successfully to environmental changes is an important area for future research. This revised version was published online in August 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">Pearl</Emphasis>, a Novel Family of Putative Transposable Elements in Bivalve Mollusks

Abstract While genome sequencing projects have discovered numerous types of transposable elements in diverse eukaryotes, there are many taxa of ecological and evolutionary significance that have received little attention, such as the molluscan class Bivalvia. Examination of a 0.7-MB genomic sequence database from the cupped oyster Crassostrea virginica revealed the presence of a common interspersed element, CvA. CvA possesses subterminal inverted repeats, a tandemly repeated core element, a tetranucleotide microsatellite region, and the ability to form stable secondary structures. Three other less abundant repetitive elements with a similar structure but little sequence similarity were also found in C. virginica. Ana-1, a repetitive element with similar features, was discovered in the blood ark Anadara trapezia by probing a genomic library with a dimeric repeat element contained in intron 2 of a minor globin gene in that species. All of these elements are flanked by the dinucleotide AA, a putative target-site duplication. They exhibit structural similarity to the sea urchin Tsp family and Drosophila SGM insertion sequences; in addition, they possess regions of sequence similarity to satellite DNA from several bivalve species. We suggest that the Crassostrea repetitive elements and Ana-1 are members of a new MITE-like family of nonautonomous transposable elements, named pearl. Pearl is the first putative nonautonomous DNA transposon to be identified in the phylum Mollusca.