Screening of Transgenic Plants by Amplification of Unknown Genomic DNA Flanking T-DNA

Spertini, D.; Béliveau, Catherine; Bellemare, Guy

doi:10.2144/99272st01

Cited by 55 publications

(44 citation statements)

References 5 publications

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“…The junction fragments containing the Tol2 ends and the genomic DNA were cloned either by inverse PCR (Kawakami 2004;Kawakami et al 2004b) or by adaptor ligation PCR (Kotani et al 2006). The adaptor ligation PCR method described previously (Siebert et al 1995;Spertini et al 1999) was applied to our system as follows. The genomic DNA from transgenic fish is digested with MboI, BglII, BamHI, SpeI, XbaI, or NheI.…”

Section: Methodsmentioning

confidence: 99%

Functional Dissection of the Tol2 Transposable Element Identified the Minimal cis-Sequence and a Highly Repetitive Sequence in the Subterminal Region Essential for Transposition

2006

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The Tol2 element is a naturally occurring active transposable element found in vertebrate genomes. The Tol2 transposon system has been shown to be active from fish to mammals and considered to be a useful gene transfer vector in vertebrates. However, cis-sequences essential for transposition have not been characterized. Here we report the characterization of the minimal cis-sequence of the Tol2 element. We constructed Tol2 vectors containing various lengths of DNA from both the left (59) and the right (39) ends and tested their transpositional activities both by the transient excision assay using zebrafish embryos and by analyzing chromosomal transposition in the zebrafish germ lineage. We demonstrated that Tol2 vectors with 200 bp from the left end and 150 bp from the right end were capable of transposition without reducing the transpositional efficiency and found that these sequences, including the terminal inverted repeats (TIRs) and the subterminal regions, are sufficient and required for transposition. The left and right ends were not interchangeable. The Tol2 vector carrying an insert of .11 kb could transpose, but a certain length of spacer, ,276 but .18 bp, between the left and right ends was necessary for excision. Furthermore, we found that a 5-bp sequence, 59-(A/G)AGTA-39, is repeated 33 times in the essential subterminal region. Mutations in the repeat sequence at 13 different sites in the subterminal region, as well as mutations in TIRs, severely reduced the excision activity, indicating that they play important roles in transposition. The identification of the minimal cis-sequence of the Tol2 element and the construction of mini-Tol2 vectors will facilitate development of useful transposon tools in vertebrates. Also, our study established a basis for further biochemical and molecular biological studies for understanding roles of the repetitive sequence in the subterminal region in transposition.

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Section: Methodsmentioning

confidence: 99%

Functional Dissection of the Tol2 Transposable Element Identified the Minimal cis-Sequence and a Highly Repetitive Sequence in the Subterminal Region Essential for Transposition

2006

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“…This yielded a product encoding a partial open reading frame (ORF) with extensive sequence similarity to F3959H. We used primers based on this new pea sequence together with primers based on the Medicago sequence for adaptorligation PCR (Spertini et al, 1999), which enabled us to isolate genomic DNA sequences and a larger cDNA product including a TAG stop codon. Amplification and sequencing of a single PCR product, using primers at the 59 and 39 ends of the surmised contig, confirmed that a 1,548-bp cDNA encoded a cytochrome P450 monooxygenase 515 amino acids long.…”

Section: The B Mutant Lacks Delphinidin and Petunidinmentioning

confidence: 99%

“…Products were separated by electrophoresis on a 1% agarose gel in 13 Tris-borate/EDTA buffer. A 794-bp sequence obtained from this fragment was used to design additional primers for the amplification of 3,231-bp genomic DNA using successive rounds of adaptor ligation PCR (Spertini et al, 1999). The genomic DNA sequence was used to design primers pinkmtF1 and 39extR for the amplification of a 1,595-bp cDNA clone, minus the ATG start codon and extending 50 bp beyond the TAG stop codon.…”

Section: Lc-msmentioning

confidence: 99%

The b Gene of Pea Encodes a Defective Flavonoid 3′,5′-Hydroxylase, and Confers Pink Flower Color

et al. 2012

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The inheritance of flower color in pea (Pisum sativum) has been studied for more than a century, but many of the genes corresponding to these classical loci remain unidentified. Anthocyanins are the main flower pigments in pea. These are generated via the flavonoid biosynthetic pathway, which has been studied in detail and is well conserved among higher plants. A previous proposal that the Clariroseus (B) gene of pea controls hydroxylation at the 59 position of the B ring of flavonoid precursors of the anthocyanins suggested to us that the gene encoding flavonoid 39,59-hydroxylase (F3959H), the enzyme that hydroxylates the 59 position of the B ring, was a good candidate for B. In order to test this hypothesis, we examined mutants generated by fast neutron bombardment. We found allelic pink-flowered b mutant lines that carried a variety of lesions in an F3959H gene, including complete gene deletions. The b mutants lacked glycosylated delphinidin and petunidin, the major pigments present in the progenitor purple-flowered wild-type pea. These results, combined with the finding that the F3959H gene cosegregates with b in a genetic mapping population, strongly support our hypothesis that the B gene of pea corresponds to a F3959H gene. The molecular characterization of genes involved in pigmentation in pea provides valuable anchor markers for comparative legume genomics and will help to identify differences in anthocyanin biosynthesis that lead to variation in pigmentation among legume species.

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“…We used the iPCR method (Hui et al, 1998;Spertini et al, 1999) to isolate the flanking sequences of T-DNA, One microgram of genomic DNA was digested with 10 units of PstI or ClaI in 50 L for 10 h. After the enzymes were heat inactivated, the samples were ethanol precipitated and dissolved in 50 L of ligation buffer, then ligated at 8°C for 12 h, using 1 unit of T4 DNA ligase (Boehringer Mannheim/Roche, Basel). Nested PCR was performed to amplify the flanking sequence.…”

Section: Ipcrmentioning

confidence: 99%

Generation and Analysis of End Sequence Database for T-DNA Tagging Lines in Rice

et al. 2003

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We analyzed 6,749 lines tagged by the gene trap vector pGA2707. This resulted in the isolation of 3,793 genomic sequences flanking the T-DNA. Among the insertions, 1,846 T-DNAs were integrated into genic regions, and 1,864 were located in intergenic regions. Frequencies were also higher at the beginning and end of the coding regions and upstream near the ATG start codon. The overall GC content at the insertion sites was close to that measured from the entire rice (Oryza sativa) genome. Functional classification of these 1,846 tagged genes showed a distribution similar to that observed for all the genes in the rice chromosomes. This indicates that T-DNA insertion is not biased toward a particular class of genes. There were 764, 327, and 346 T-DNA insertions in chromosomes 1, 4 and 10, respectively. Insertions were not evenly distributed; frequencies were higher at the ends of the chromosomes and lower near the centromere. At certain sites, the frequency was higher than in the surrounding regions. This sequence database will be valuable in identifying knockout mutants for elucidating gene function in rice. This resource is available to the scientific community at http://www.postech.ac.kr/life/pfg/risd.Insertional mutagenesis is one of the most useful methods for analyzing gene function. When foreign DNA is inserted into a gene, it not only creates a mutation but also tags the affected gene, facilitating its isolation and characterization (Azpiroz-Leehan and Feldmann, 1997). Transposons and T-DNA have been used most widely as an insertional mutagen (Mathur et al., 1998;Wisman et al., 1998; Krysan et al., 1999;Parinov et al., 1999;Speulman et al., 1999;Tissier et al., 1999). It is believed that T-DNA insertion is a random event and that the inserted sequences are stable through multiple generations (Azpiroz- Leehan and Feldmann, 1997;Parinov and Sundaresan, 2000). Insertional mutant pools have been constructed in Arabidopsis and used for functional analysis of a number of genes (Feldmann, 1991; Koncz et al., 1992; Azpiroz-Leehan and Feldmann, 1997; Bechtold and Pelletier, 1998; Krysan et al., 1999; Galbiati et al., 2000;Parinov and Sundaresan, 2000; Bouché and Bouchez, 2001;Sessions et al., 2002;Szabados et al., 2002). The procedure for T-DNA insertional mutagenesis has also been applied to rice (Oryza sativa) using the Agrobacterium tumefaciensmediated transformation method (Hiei et al., 1994). Jeon et al. (2000) have reported the construction of over 20,000 T-DNA-tagged rice lines. A T-DNA insertional mutagen can be modified to trap a gene by inserting a reporter gene, such as gus (␤-glucuronidase), next to the T-DNA border (Sundaresan et al., 1995; Jeon et al., 2000;Springer, 2000). Approximately 5% to 10% of the mutagenized lines are GUS positive, demonstrating the efficiency of this gene-trapping system (Chin et al., 1999; Jeon et al., 2000).Completion of the genome sequencing for both Arabidopsis and rice has provided new reverse genetic means for assigning biological functions to sequenced genes (Kumar...

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Screening of Transgenic Plants by Amplification of Unknown Genomic DNA Flanking T-DNA

Cited by 55 publications

References 5 publications

Functional Dissection of the Tol2 Transposable Element Identified the Minimal cis-Sequence and a Highly Repetitive Sequence in the Subterminal Region Essential for Transposition

Functional Dissection of the Tol2 Transposable Element Identified the Minimal cis-Sequence and a Highly Repetitive Sequence in the Subterminal Region Essential for Transposition

The b Gene of Pea Encodes a Defective Flavonoid 3′,5′-Hydroxylase, and Confers Pink Flower Color

Generation and Analysis of End Sequence Database for T-DNA Tagging Lines in Rice

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