The methylation patterns of chromosomal integration regions influence gene activity of transferred DNA in Petunia hybrida

Pröls, Felicitas; Meyer, Peter

doi:10.1111/j.1365-313x.1992.00465.x

Cited by 23 publications

(26 citation statements)

References 38 publications

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“…Previous studies have shown that certain regions exist in the plant genome where integration of transgenes does not result in expression of the ®nal protein product. Such regions include the heterochromatic areas located near the centromere of the chromosomes (ProÈ ls and Meyer 1992;Ye and Signer 1996). Simon and Knowles (1993) reported that integration of transgenes in telomeric regions, which are transcriptionally active areas in the plant genome, can also lead to gene silencing.…”

Section: Discussionmentioning

confidence: 98%

Transgene expression in rice engineered through particle bombardment: molecular factors controlling stable expression and transgene silencing

Kohli

Gahakwa

Vain

et al. 1999

Planta

131

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Transgenic rice (Oryza sativa L.) lines were generated through particle-bombardment-mediated transformation. Hygromycin phosphotransferase (hpt) was used as a selectable marker gene on co-integrate plasmids containing either one or two unselected genes, the Bialaphos-resistance gene (bar) coding for phosphinothricin acetyltransferase and the b-glucuronidase gene (gusA), respectively. Transformants were analyzed to determine possible correlation between expression, integrated transgene copy number and/or complexity of integration patterns. We observed that an increase in transgene copy number did not always lead to a concomitant decrease in expression levels or to silencing through co-suppression. Transgenic lines with four to ®ve copies of integrated transgenes expressed the protein product of both unselected genes stably and at levels comparable to transformants with one or two copies. In the majority of lines we analyzed, expression patterns of the two unselected genes were similar. In lines where transgene silencing was observed, this was independent of position eects. In speci®c cases, silenced transgenes could be reactivated by treatment with 5-azacytidine, suggesting methylation of cytosine residues. We report that methylation of cytosines may not spread to adjacent regions, hence other transgenes in the vicinity of the silenced transgene remain active. By comparing the structure of transgenic loci with expression patterns of introduced genes, we concluded that the integrity of integrated transgenes was a major factor in the onset of silencing. We observed that the presence of truncated sequences of transgenes capable of generating incomplete transcripts, resulting in aberrant RNA species, may be responsible for silencing.

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Section: Discussionmentioning

confidence: 98%

Transgene expression in rice engineered through particle bombardment: molecular factors controlling stable expression and transgene silencing

Kohli

Gahakwa

Vain

et al. 1999

Planta

131

View full text Add to dashboard Cite

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“…The literature shows that, even in single-copy T-DNA plants, many factors are responsible for the induction of low transgene expression. Beside the T-DNA integration position itself, linkage to repetitive sequences [44], the presence of neighboring highly methylated sequences [45], and the absence of MAR sequences near the integration site [26] are implicated as causative factors for the induction of low transgene expression. Additionally, an abrupt change in AT content [19] and the presence of vector backbone sequences [46] may reduce transgene expression.…”

Section: Discussionmentioning

confidence: 99%

Single-copy T-DNAs integrated at different positions in the Arabidopsis genome display uniform and comparable β-glucuronidase accumulation levels

Buck

Windels²,

Loose³

et al. 2004

CMLS, Cell. Mol. Life Sci.

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This study aimed at determining whether transgene expression variability is observed in single-copy T-DNA plants and whether it can be correlated with the T-DNA integration position. Among a population of 135 Arabidopsis thaliana transformants, selected on the basis of antibiotic resistance marker expression, 21 single-copy T-DNA transformants were identified and characterized. In 19 of these 21 lines, 35S-beta-glucuronidase transgene expression, measured in two subsequent generations, was similar. This observation means that the intra-transformant variability was as high as the inter-transformant variability. Integration into an intergenic or genic region, into an exon or intron, in sense or antisense orientation, did not result in differential transgene expression. Remarkably, single-copy transformants were not always the highest expressers, implying that low transgene expression is not always induced by multicopy transformants. In only 2 of the 21 single-copy plants was the transgene expression more than 20-fold lower. However, characteristics of the insertion position in one of these lines did not differ significantly when compared to high-expressing lines. In the remaining line, methylation of the transgene was clearly demonstrated. In conclusion, screening for single-copy T-DNA transformants greatly enriches for stable and high transgene expression, because the integration position is not a major determinant of transgene expression variability in Arabidopsis.

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“…Even in the true transformants a differential GUS expression was observed, as a strong expression was seen in some seedlings while a weak expression was observed in others. This could be due to different copy numbers of the transgene in the individual plants (Hobbs et al 1993;Tjokrokusumo et al 2000) or to methylation in the chromosomal integration regions (Prols and Meyer 1992).…”

Section: Selection Of Transformed Plants Through Gus Gene Expressionmentioning

confidence: 99%

Improvement of cotton fiber quality by transforming the acsA and acsB genes into Gossypium hirsutum L. by means of vacuum infiltration

Wang

Zhao

et al. 2004

Plant Cell Reports

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A novel method for the genetic transformation of cotton pollen by means of vacuum infiltration and Agrobacterium-mediated transformation is reported. The acsA and acsB genes, which are involved in cellulose synthesis in Acetobacter xylinum, were transferred into pollen grains of brown cotton with the aim of improving its fiber quality by incorporating useful prokaryotic features into the colored cotton plants. Transformation was carried out in cotton pollen-germinating medium, and transformation was mediated by vector pCAMBIA1301, which contains a reporter gene beta-glucuronidase (GUS), a selectable marker gene, hpt, for hygromycin resistance and the genes of interest, acsA and acsB. The integration and expression of acsA, acsB and GUS in the genome of transgenic plants were analyzed with Southern blot hybridization, PCR, histochemical GUS assay and Northern blot hybridization. We found that following pollination on the cotton stigma transformed pollen retained its capability of double-fertilization and that normal cotton seeds were produced in the cotton ovary. Of 1,039 seeds from 312 bolls pollinated with transformed pollen grains, 17 were able to germinate and grow into seedlings for more than 3 weeks in a nutrient medium containing 50 mg/l hygromycin; eight of these were transgenic plants integrated with acsA and acsB, yielding a 0.77% transformation rate. Fiber strength and length from the most positive transformants was 15% greater than those of the control (non-transformed), a significant difference, as was cellulose content between the transformed and control plants. Our study suggests that transformation through vacuum infiltration and Agrobacterium mediated transformation can be an efficient way to introduce foreign genes into the cotton pollen grain and that cotton fiber quality can be improved with the incorporation of the prokaryotic genes acsA and acsB.

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The methylation patterns of chromosomal integration regions influence gene activity of transferred DNA in Petunia hybrida

Cited by 23 publications

References 38 publications

Transgene expression in rice engineered through particle bombardment: molecular factors controlling stable expression and transgene silencing

Transgene expression in rice engineered through particle bombardment: molecular factors controlling stable expression and transgene silencing

Single-copy T-DNAs integrated at different positions in the Arabidopsis genome display uniform and comparable β-glucuronidase accumulation levels

Improvement of cotton fiber quality by transforming the acsA and acsB genes into Gossypium hirsutum L. by means of vacuum infiltration

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