Characterization of Tpn1 family in the Japanese morning glory: En/Spm-related transposable elements capturing host genes

Some mutant phenotypes are known to be unstable somatically and germinally due to the insertion of transposable elements in the Japanese morning glory (Ipomoea nil). Several transposable elements that cause mutable phenotypes have recently been isolated. All of these elements show characteristic features of the En/Spm (Enhancer/Suppressor-mutator) or CACTA family. They carry common 28 bp terminal inverted repeats and subterminal repetitive regions and are known as the Tpn1 family. All of these elements are thought to be non-autonomous and mobilized by unidentified autonomous element(s). Using a probe corresponding to the subterminal region, we isolated many genomic Tpn clones, 120 of which were classified into 28 types based on their restriction maps. The copy number of the Tpn1 family was estimated to be between 500 and 1,000 copies per haploid genome. We then determined the complete sequences of 28 representative clones from each Tpn type. Most Tpn elements showed a high degree of similarity to plant genes in their internal sequences, suggesting that the Tpn1 family captured host gene sequences during the process of evolution. Detailed analyses of Tpn104 in comparison with an orthologous host gene InAP2B confirmed this assumption.     

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.     

Somatic mutations caused by excision of the transposable element, Tpn1, from the DFR gene for pigmentation in sub-epidermal layer of periclinally chimeric flowers of Japanese morning glory and their germinal transmission to their progeny

Pigmentation in flowers of Japanese morning glory is intense in the epidermal layer, lighter in the subepidermis, and much lighter in the internal tissues; by contrast coloration in stems occurs only in the sub-epidermal layer. The a-3 ^f mutant of Japanese morning glory bears white flowers with normal-colored flecks and sectors, and its variegation also occurs in leaves and stems. The mutable line can produce chimeric flowers pigmented uniformly in the sub-epidermal tissue and variegated in the epidermal layer, and stems of these flowers are also pigmented. Since they give selfed progeny that segregate to give a ratio of three germinal revertants bearing fully colored flowers to one flecked mutant, it has been [OR Imai (1934) has] postulated that somatic mutations in the sub-epidermal layer can be transmitted to the next generation and that the germ cells in the reproductive organs must form from the cells of the sub-epidermal layer. Recently, we found that the 6.4-kb En/Spm-related transposable element, Tpn1, resides within the DFR-B gene for anthocyanin biosynthesis in the mutable a-3 ^f line. To test whether somatic mutations caused by Tpn1 excision from the DFR-B gene in the subepidermis of periclinally chimeric flowers are transmissible to their progeny, we have examined the structure of the DFR-B region in the germinal revertants derived from the chimeric flowers and compared the sequences generated by the somatic excision of Tpn1 in periclinally chimeric flowers with those in their germinal revertants. Our results confirm that somatic mutations caused by Tpn1 excision from the DFR-B gene in the sub-epidermal tissue of chimeric flowers can be transmitted to their progeny, which results in the generation of germinal revertants.     

The transposable element En/Spm-encoded TNPA protein contains a DNA binding and a dimerization domain

The En/Spm-encoded TNPA protein binds to 12-bp DNA sequence motifs that are present in the sub-termini of the transposable element. DNA binding of TNPA to monomeric and dimeric forms of the binding motif was analyzed by gel retardation and cross-linking studies. A DNA binding domain at the N-terminal and a dimerization domain at the C-terminal portion of TNPA were localized using deletion derivatives of TNPA. These domains are novel since no apparent homology has been found in the data bases. The stoichiometry of the TNPA-DNA complexes was analyzed. A special complex is formed with a tail-to-tail dimeric DNA binding motif, most probably involving two DNA-bound TNPA molecules that interact via their dimerization domains. In redox reactions the requirement for one or two disulfide bonds for DNA binding of TNPA was shown. The implications of these findings for the excision mechanism of En/Spm are discussed.     

Floral-specific polypeptides of the Japanese morning glory

Polypeptides from stems, leaves, sepals, corollas, stamens and pistils of the Japanese morning glory (Ipomoea nil Roth (Pharbitis nil Chois.)) were separated by one- and two-dimensional gel electrophoresis and visualized by silver staining. The majority of polypeptides were expressed in two or more organs, while those specific to only one organ were comparatively rate. Among the polypeptides of the former class were two which appeared to be floral-specific. A 46-kDa (kilodalton) polypeptide was expressed in corollas, stamens and pistils, whereas a 32-kDa polypeptide was observed only in extracts prepared from reproductive organs. Polypeptide spots from the various organs were compared with those from leaves, and it was found that sepals and stems shared 40–50% of their polypeptides with leaves, whereas corollas, stamens and pistils shared 20% or less. The latter organs shared 120 polypeptides or roughly 15% of those identified in the floral extracts. Floralorgan-specific polypeptides comprised nearly 10% of the total floral polypeptides identified.Abbreviations kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

DNA sequence of the transposable element IS1

Summary The nucleotide sequence of an IS1 element recently transposed into the lacI gene is reported. This sequence is nearly identical to one previously reported for another IS1 element (Ohtsubo and Ohtsubo, 1978). The implications of this similarity are discussed. The sizes of potential polypeptides encoded in the IS1 DNA have been determined and possible roles for these peptides in the illegitimate recombination events mediated by the element are considered.     

Terminal repeats of the Drosophila transposable element copia: nucleotide sequence and genomic organization

We have determined the nucleotide sequence of the terminal regions of two members of the copia sequence family of D. melanogaster. The first 276 bp at one end of a copia element are repeated in direct orientation at its other end. The direct repeats on a single copia element are identical to each other, but they differ by two nucleotide substitutions between the two elements which were examined; this suggests that during transposition only one direct repeat of the parent element is used as a template for both direct repeats of the transposed element. Each direct repeat itself contain a 17 bp imperfectly matched inveted terminal repetition. The ends of copia show significant sequence homology both to the yeast Ty1 element and to the integrated provirus of avian spleen necrosis virus, two other eucaryotic elements known to insert at many different chromosomal locations. Analysis of the genomic organization of the direct repeat sequence demonstrates that it seldom, if ever, occurs unlinked to an entire copia element.     

A transcriptional terminator sequence in the prokaryotic transposable element IS1

The prokaryotic transposable element IS1 is known to exert a strong polar effect upon integration into an operon. To elucidate this polar effect, we constructed a plasmid which has an IS1 integrated between the 5' half of the tet gene for tetracycline resistance and the cat structural gene for chloramphenicol resistance. The cat gene is expressed by the tet promoter and the presence of IS1 in orientation I, in which the IS1 transposase genes insA and insB are in the same orientation as the cat gene, reduced the cat expression. By introducing deletions or insertions within the IS1 sequence, we were able to map a rho-dependent terminator TIS1A between the insA and insB genes. Translational interruption between these ins genes is important for TIS1A to be an active terminator.