A general method to isolate genes tagged by a high copy number transposable element

Souer, Erik; Quattrocchio, Francesca; Vetten, Nick de; Mol, J.A.; Koes, Ronald

doi:10.1046/j.1365-313x.1995.7040677.x

Cited by 45 publications

(37 citation statements)

References 26 publications

(33 reference statements)

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“…DNA extraction, DNA gel blot analysis, polymerase chain reaction (PCR) amplification, and sequence analyses were performed as previously described (Souer et al, 1995;de Vetten et al, 1997). The flanking sequence of the dTph1 element in an2-W82 (jaf41) was identified by established procedures (Souer et al, 1995;de Vetten et al, 1997) and used to screen a petal cDNA library and a genomic library of P. hybrida V26.…”

Section: Nucleic Acid Analysismentioning

confidence: 99%

“…The flanking sequence of the dTph1 element in an2-W82 (jaf41) was identified by established procedures (Souer et al, 1995;de Vetten et al, 1997) and used to screen a petal cDNA library and a genomic library of P. hybrida V26. an2 cDNAs from other lines were isolated by reverse transcription (RT)-PCR, and crucial regions were sequenced in at least two independently amplified products and in many cases by sequencing the corresponding region in PCR products amplified from genomic DNA.…”

Section: Nucleic Acid Analysismentioning

confidence: 99%

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Molecular Analysis of the anthocyanin2 Gene of Petunia and Its Role in the Evolution of Flower Color

et al. 1999

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The shape and color of flowers are important for plant reproduction because they attract pollinators such as insects and birds. Therefore, it is thought that alterations in these traits may result in the attraction of different pollinators, genetic isolation, and ultimately, (sympatric) speciation. Petunia integrifolia and P. axillaris bear flowers with different shapes and colors that appear to be visited by different insects. The anthocyanin2 ( an2 ) locus, a regulator of the anthocyanin biosynthetic pathway, is the main determinant of color differences. Here, we report an analysis of molecular events at the an2 locus that occur during Petunia spp evolution. We isolated an2 by transposon tagging and found that it encodes a MYB domain protein, indicating that it is a transcription factor. Analysis of P. axillaris subspecies with white flowers showed that they contain an2 Ϫ alleles with two alternative frameshifts at one site, apparently caused by the insertion and subsequent excision of a transposon. A third an2 ؊ allele has a nonsense mutation elsewhere, indicating that it arose independently. The distribution of polymorphisms in an2 ؊ alleles suggests that the loss of an2 function and the consequent changes in floral color were not the primary cause for genetic separation of P. integrifolia and P. axillaris. Rather, they were events that occurred late in the speciation process, possibly to reinforce genetic isolation and complete speciation. INTRODUCTIONFlowers are the structures containing the male and female sex organs of angiosperms. Flowers of diverse species display a wide range of different morphologies and pollination strategies. For instance, flowers of wind-pollinated species usually possess small and inconspicuous petals or no petals at all, whereas flowers of insect-pollinated plants usually possess large, brightly colored, and patterned petals that serve as visual signals and a landing site for visiting insects.Recent experiments suggest that the wide variety of plant and flower morphologies may have depended on the evolution of a relatively small number of genes. First, mutations at single loci, usually isolated by breeders or researchers, are sufficient to cause fundamental alterations in inflorescence architecture (Doebley et al., 1997;Souer et al., 1998). Similarly, the different shapes, colors, and color patterns of naturally occurring Mimulus (monkeyflower) spp are due to alterations at only a few (major) loci (Bradshaw et al., 1995).Second, even very different inflorescence and flower architectures appear to be determined by genes that often encode conserved proteins but that differ in their expression patterns (reviewed in Doebley and Lukens, 1998).The isolation of key regulatory loci and analysis of the molecular alterations that have taken place in them will provide new insights into the evolution and diversification of flower morphology. The biosynthesis of anthocyanin flower pigments is particularly suited for such studies, because it is a well-defined biochemical pathway that is being...

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Section: Nucleic Acid Analysismentioning

confidence: 99%

Section: Nucleic Acid Analysismentioning

confidence: 99%

Molecular Analysis of the anthocyanin2 Gene of Petunia and Its Role in the Evolution of Flower Color

et al. 1999

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“…Application of similar methods may enable identification of function of the petunia dif genes. The development of a straightforward method for cloning genes tagged by the multicopy Tphl transposon (Souer et al, 1995) should facilitate the cloning of other petunia genes involved in anthocyanin synthesis.…”

Section: Isolation Of Flavonoid Modification Genesmentioning

confidence: 99%

Genetics and Biochemistry of Anthocyanin Biosynthesis

Holton¹,

Cornish²

1995

The Plant Cell

705

448

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“…To isolate a fragment of the an1 locus, we cloned dTph1 flanking sequences unique for an1 -W138 plants by a combination of inverse polymerase chain reaction (PCR) and differential screening of amplification products (Souer et al, 1995). This yielded four genomic fragments that contained a dTph1 insertion in six different an1 -W138 plants but not in plants homozygous for two independent An1 ϩ reversion al- leles.…”

Section: Isolation Of the An1 Genementioning

confidence: 99%

“…dTph1 flanking sequences were amplified by inverse polymerase chain reaction (PCR), using dTph1 specific primers and cloned into M13 phage to obtain a library of dTph1 flanking sequences (Souer et al, 1995). Duplicate plaque lifts, taken on Hybond-N membranes (Amersham), were hybridized to 32 P-labeled dTph1 flanking sequences generated by inverse PCR amplification of circularized RsaI-digested DNA from a second an1-W138 homozygote (ϩ probe) or a plant homozygous for an An1 ϩ revertant allele (Ϫ probe).…”

Section: Molecular Analysis Of the An1 Locusmentioning

confidence: 99%

anthocyanin1 of Petunia Encodes a Basic Helix-Loop-Helix Protein That Directly Activates Transcription of Structural Anthocyanin Genes

Spelt¹,

Quattrocchio²,

Mol³

et al. 2000

The Plant Cell

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162

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The petunia loci anthocyanin1 ( an1 ), an2 , an4 , and an11 are required for the transcription of anthocyanin biosynthetic genes in floral organs. The an2 and an11 loci were recently cloned and shown to encode a MYB-domain transcriptional activator and a cytosolic WD40 protein, respectively. Here, we report the isolation of an1 by transposon tagging. an1 encodes a new member of the basic helix-loop-helix family of transcription factors that is functionally and evolutionarily distinct from JAF13, the apparent petunia ortholog of maize RED1 and snapdragon DELILA. We provide genetic evidence that the transcription factors encoded by an1 , an2 , and an4 operate in an unexpectedly complex regulatory hierarchy. In leaves, ectopic expression of AN2 induces an1 expression, whereas in anthers, an1 expression depends on an4 , encoding (or controlling) a MYB protein that is paralogous to AN2. Experiments with transgenic plants expressing a post-translationally controlled AN1-GLUCOCORTICOID RECEPTOR fusion protein indicated that independent of protein synthesis, AN1 directly activates the expression of the dfrA gene encoding the enzyme dihydroflavonol 4-reductase and of Pmyb27 encoding a MYB-domain protein of unknown function.

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A general method to isolate genes tagged by a high copy number transposable element

Cited by 45 publications

References 26 publications

Molecular Analysis of the anthocyanin2 Gene of Petunia and Its Role in the Evolution of Flower Color

Molecular Analysis of the anthocyanin2 Gene of Petunia and Its Role in the Evolution of Flower Color

Genetics and Biochemistry of Anthocyanin Biosynthesis

anthocyanin1 of Petunia Encodes a Basic Helix-Loop-Helix Protein That Directly Activates Transcription of Structural Anthocyanin Genes

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