Premature Polyadenylation at Multiple Sites within a<i>Bacillus thuringiensis</i> Toxin Gene-Coding Region1

Diehn, Scott H.; Chiu, Wan-Ling; Rocher, E. Jay De; Green, Pamela J.

doi:10.1104/pp.117.4.1433

Cited by 58 publications

(45 citation statements)

References 44 publications

(64 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…PCR products for each segment were cloned and then assembled by standard cloning methods to generate the complete synthetic B.t.-toxin-coding region. in BMS cells, the cryIA(c)-coding region and rbcS-E9 3Ј UTR contained in a fragment excised from p1335 with BglII and ClaI were inserted into p1138 (Diehn et al, 1998) containing the 2X35S and ADH1 intron cut with BamHI and ClaI to yield p1345 (Fig. 2).…”

Section: Gene Constructionsmentioning

confidence: 99%

“…Expression of the internal reference ␤-globin gene in BY-2 cells was from p1185 (Fig. 2) (Diehn et al, 1998).…”

Section: Gene Constructionsmentioning

confidence: 99%

“…2). The wild-type cryIA(c)-coding region and rbcS-E9 3Ј UTR were excised from p995 (Diehn et al, 1998) with BbuI and BamHI and substituted for the synthetic cryIA(c)-coding region in p1345 cut with BbuI and BamHI to yield p1346 (Fig. 2).…”

Section: Gene Constructionsmentioning

confidence: 99%

“…The plasmid p1204 (Diehn et al, 1998) shown in Figure 2 containing the truncated cryIA(c)-coding region flanked by 2X35S (Diehn et al, 1998) and the 3Ј UTR from the pea Rubisco small subunit rbcS-E9 gene was used for transient expression of B.t.-toxin genes in BY-2 cells. The synthetic cryIA(c)-coding region was excised from p1334 (see previous section) with BbuI and BamHI and substituted for the wild-type cryIA(c)-coding region in p1204, which was also cut with BbuI and BamHI to generate p1335 (Fig.…”

Section: Gene Constructionsmentioning

confidence: 99%

“…PCR products of the correct size were cut out of low-melting-point agarose gels and cloned after removal of terminal adenosines into the EcoRV site of a Bluescript II SK(Ϫ) vector with a modified multiple cloning site (p948) (Diehn et al, 1998). Clones containing inserts were screened by dideoxy sequencing (Sanger et al, 1977) for PCR products with no or only one to two sequence errors.…”

Section: Synthetic Bacillus Thuringiensis (Bt)-toxin Gene Constructionmentioning

confidence: 99%

See 4 more Smart Citations

Direct Evidence for Rapid Degradation ofBacillus thuringiensis Toxin mRNA as a Cause of Poor Expression in Plants1

Rocher

Vargo-Gogola²,

Diehn

et al. 1998

Plant Physiology

Self Cite

View full text Add to dashboard Cite

It is well established that the expression of Bacillus thuringiensis (B.t.) toxin genes in higher plants is severely limited at the mRNA level, but the cause remains controversial. Elucidating whether mRNA accumulation is limited transcriptionally or posttranscriptionally could contribute to effective gene design as well as provide insights about endogenous plant gene-expression mechanisms. To resolve this controversy, we compared the expression of an A/Urich wild-type cryIA(c) gene and a G/C-rich synthetic cryIA(c) B.t.-toxin gene under the control of identical 5 and 3 flanking sequences. Transcriptional activities of the genes were equal as determined by nuclear run-on transcription assays. In contrast, mRNA half-life measurements demonstrated directly that the wildtype transcript was markedly less stable than that encoded by the synthetic gene. Sequences that limit mRNA accumulation were located at more than one site within the coding region, and some appeared to be recognized in Arabidopsis but not in tobacco (Nicotiana tabacum). These results support previous observations that some A/U-rich sequences can contribute to mRNA instability in plants. Our studies further indicate that some of these sequences may be differentially recognized in tobacco cells and Arabidopsis.Gene transfer into plants has become a relatively simple and routine process in many species. However, successful integration of a transgene into the plant genome does not automatically result in expression. For example, introduction of an additional copy of an endogenous gene can trigger cosuppression mechanisms, resulting in silencing of both the introduced and the endogenous genes (for review, see Meyer and Saedler, 1996). In the case of foreign genes transferred into plants, it is not uncommon to find that the introduced gene is incompatible with plant geneexpression mechanisms. Bacillus thuringiensis (B.t.)-toxin genes are perhaps the most widely known example of foreign genes for which it has been difficult to obtain useful levels of expression in transgenic plants (Diehn et al., 1996). Although a variety of mechanisms controlling gene expression have been blamed for poor B.t.-toxin expression, the exact cause remains controversial because of the limited amount of data gathered to address the problem. This is in part due to the inherent difficulties associated with studying a gene expressed at very low levels. It is also due to the fact that, for practical applications in agriculture, this problem can be circumvented by the brute-force approach of constructing highly modified synthetic versions of B.t.-toxin genes that yield high levels of expression. Nevertheless, elucidating the mechanisms that limit B.t.-toxin expression could make the design of new genes more efficient and provide insight about steps in gene expression that are relevant to endogenous plant genes.A widely observed characteristic of B.t.-toxin gene expression in plants has been that little or no mRNA accumulates, even when transcription is under the control of strong pr...

show abstract

Section: Gene Constructionsmentioning

confidence: 99%

“…Expression of the internal reference ␤-globin gene in BY-2 cells was from p1185 (Fig. 2) (Diehn et al, 1998).…”

Section: Gene Constructionsmentioning

confidence: 99%

Section: Gene Constructionsmentioning

confidence: 99%

Section: Gene Constructionsmentioning

confidence: 99%

Section: Synthetic Bacillus Thuringiensis (Bt)-toxin Gene Constructionmentioning

confidence: 99%

See 3 more Smart Citations

Direct Evidence for Rapid Degradation ofBacillus thuringiensis Toxin mRNA as a Cause of Poor Expression in Plants1

Rocher

Vargo-Gogola²,

Diehn

et al. 1998

Plant Physiology

Self Cite

View full text Add to dashboard Cite

show abstract

The Story of Bollgard® Cotton

Purcell

Oppenhuizen

Wofford

et al. 2004

Handbook of Plant Biotechnology

View full text Add to dashboard Cite

Bollgard® cotton is one of the great success stories for agricultural biotechnology. It broke new ground as a commercially successful transgenic plant product that could protect itself from damage from insect pests. This provided farmers with a dramatic new technology that offered significant benefits over the chemical insecticides that were being used. Delivering this product took many years of discovery and development and required significant scientific advances. Since its introduction in 1996, the use of Bollgard® cotton has expanded rapidly. It is now a valuable tool for agriculture with commercial growings in nine countries on five continents. Bollgard® cotton provides economic, environmental and social benefits for large and smallholder farmers around the globe.

show abstract

BtCrops: A Novel Insect Control Tool

Rie¹

2002

Applications and Systematics of Bacillus and Relatives

View full text Add to dashboard Cite

Premature Polyadenylation at Multiple Sites within aBacillus thuringiensis Toxin Gene-Coding Region1

Cited by 58 publications

References 44 publications

Direct Evidence for Rapid Degradation ofBacillus thuringiensis Toxin mRNA as a Cause of Poor Expression in Plants1

Direct Evidence for Rapid Degradation ofBacillus thuringiensis Toxin mRNA as a Cause of Poor Expression in Plants1

The Story of Bollgard® Cotton

BtCrops: A Novel Insect Control Tool

Contact Info

Product

Resources

About