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 characterization of the dihydroflavonol 4-reductase B (DFR-B) gene in the sweet potato.

A genomic fragment containing the dihydroflavonol 4-reductase B (DFR-B) gene was cloned from the sweet potato (Ipomoea batatas) and its nucleotide sequence was analyzed. The exons and flanking regions were highly homologous to those of previously reported DFR-B genes of the Japanese morning glory, whereas the introns and the intergenic region were less conserved. In addition to the sequences of three miniature inverted-repeat transposable elements (MITEs) and one direct repeat previously reported in the DFR-B gene of Japanese morning glory, two mobile element-like sequences were newly identified in the sweet potato DFR-B gene. At least four allelic sequences were found to exist by amplification of the DFR-B gene from various sweet potato cultivars. One of these allelic sequences had a 2-kb deletion in the intergenic region and was observed in the cultivars with high anthocyanin content in their storage roots.     

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.     

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.     

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.     

Binding ofNicotiana nuclear proteins to the subterminal regions of theAc transposable element

Specific binding ofNicotiana nuclear protein(s) to subterminal regions of theAc transposable element was detected using gel mobility shift assays. A sequence motif (GGTAAA) repeated in both terminal regions ofAc, was identified as the protein binding site. Mutation of two nucleotides in this motif was sufficient to abolish binding. Based on a series of competition assays, it is deduced that there is cooperative binding between two repeats, each similar to the GGTAAA motif. The binding protein is probably similar to a previously characterized maize protein which binds to a GGTAAA-containing motif located in the ends ofMutator. Moreover, we show that DNA fromDs1 competes for protein binding toAc termini, and we show, by sequence analysis, that GGTAAA binding sites are present in the terminal region ofTgm1, Tpn1, En/Spm, Tam3 andDs1-like elements. This suggests that the binding protein(s) might be involved in the transposition process.     

猪α-1,3-半乳糖转移酶基因打靶载体的构建

目的:构建猪α-1,3-半乳糖转移酶(GGTA1)基因的正负筛选打靶载体。方法:以原代猪胚胎成纤维细胞基因组DNA为模板,采用长程PCR方法扩增出GGTA1基因的2条片段;以长约2kb包含部分第9外显子的片段为同源短臂,在XbaⅠ和ClaⅠ位点插入pLoxP质粒正筛选标记neo基因的3'端;以长约5.4kb包括部分第8外显子、全部第8内含子及部分第9外显子的片段为同源长臂,于NotⅠ位点插入该质粒中neo基因的5'端;2.7kb的负筛选标记tk基因位于载体中同源短臂的3'端外侧。结果与结论:酶切、PCR及测序结果表明,同源臂被正确连接至质粒pLoxP,成功构建了猪GGTA1基因正负筛选打靶载体pSL/GT。     

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.     

Gene duplication and mobile genetic elements in the morning glories

We review gene duplication and subsequent structural and functional divergence in the anthocyanin biosynthesis genes in the Japanese and common morning glories and discuss their evolutionary implications. These plants appear to contain at least six copies of the CHS gene and three tandem copies of the DFR gene. Of these, the CHS-D and DFR-B genes are mainly responsible for flower pigmentation and mutations in these genes confer white flowers. We compared the genomic sequences of these duplicated genes between the two morning glories and found small mobile element-like sequences (MELSs) and direct repeats (DRs) in introns and intergenic regions. The results indicate that the MELS elements and DRs play significant roles in divergence after gene duplication. We also discuss DNA rearrangements occurring before and after speciation of these morning glories. DNA transposable elements belonging to the Ac/Ds or En/Spm families have acted as major spontaneous mutagens in these morning glories. We also describe the structural features of the first Mu-related element found in the morning glories and polymorphisms found in the same species.     

Structure and sequence analysis of the human activin beta A subunit gene.

The cloned genomic DNA containing the human activin beta A subunit gene were analyzed by restriction endonuclease mapping, Southern blotting and DNA sequencing. The activin gene is composed of two exons interrupted by the 9-kb intron. The TATA, CCAAT and CT-stretch sequences were found in the 5'-flanking region of the gene. An intronic sequence contained SV40 enhancer core element in the vicinity of the exon 1. In the 3'-flanking region, we identified eight consensus polyadenylation sequences, five ATTTA motifs, CA element consisting of (CA)14, AP-1 binding site and two SV40 enhancer core elements. A dot matrix analysis revealed the high degree of conservation between the human and rat sequences within the 3'-flanking region, suggesting a possible functional significance.     

Identification of <Emphasis Type="Italic">r</Emphasis> mutations conferring white flowers in the Japanese morning glory (<Emphasis Type="Italic">Ipomoea nil</Emphasis>)

The wild-type Japanese morning glory [Ipomoea nil (L.) Roth.] exhibits blue flowers with red stems, and spontaneous r mutants display white flowers with green stems. We have identified two r mutations, r1-1 and r1-2, that are caused by insertions of Tpn1-related DNA transposable elements, Tpn3 (5.6 kb) and Tpn6 (4.7 kb), respectively, into a unique intron of the CHS-D gene, which is responsible for flower and stem pigmentation. Both Tpn3 and Tpn6, which belong to the En/Spm or CACTA superfamily, are nonautonomous elements lacking transposase genes but containing unrelated cellular DNA segments including exons and introns. Interestingly, r1-2 contains an additional 4-bp insertion at the Tpn3 integration site in r1-1, presumably a footprint caused by the excision of Tpn3. The results strengthen the previous notion that Tpn1 and its relatives are major spontaneous mutagens for generating various floriculturally important traits in I. nil. Since I. nil has an extensive history of genetic studies, molecular identification of classical spontaneous mutations would also facilitate reinterpretation of the abundant classical genetic data available. An erratum to this article can be found at     

Cloning and molecular analysis of the Arabidopsis gene Terminal Flower 1

The Arabidopsis gene Terminal Flower 1 (TFL1) controls inflorescence meristem identity. A terminal flower (tfl1) mutant, which develops a terminal flower at the apex of the inflorescence, was induced by transformation with T-DNA. Using a plant DNA fragment flanking the integrated T-DNA as a probe, a clone was selected from a wild-type genomic library. Comparative sequence analysis of this clone with an EST clone (129D7T7) suggested the existence of a gene encoding a protein similar to that encoded by the cen gene which controls inflorescence meristem identity in Antirrhinum. Nucleotide sequences of the region homologous to this putative TFL1 gene were compared between five chemically induced tfl1 mutants and their parental wild-type ecotypes. Every mutant was found to have a nucleotide substitution which could be responsible for the tfl1 phenotype. This result confirmed that the cloned gene is TFL1 itself. In our tfl1 mutant, no nucleotide substitution was found in the transcribed region of the gene, and the T-DNA-insertion site was located at 458 bp downstream of the putative polyadenylation signal, suggesting that an element important for expression of the TFL1 gene exists in this area. Received: 14 November 1996 / Accepted: 29 November 1996