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Identification of a plastid intercistronic expression element (IEE) facilitating the expression of stable translatable monocistronic mRNAs from operons.

Zhou F, Karcher D, Bock R - Plant J. (2007)

Bottom Line: At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur.As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs.We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Molekulare Pflanzenphysiologie (MPI-MP), Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany.

ABSTRACT
Most plastid genes are part of operons and expressed as polycistronic mRNAs. Many primary polycistronic transcripts undergo post-transcriptional processing in monocistronic or oligocistronic units. At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur. As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs. We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts. Separation of foreign genes by this element facilitates transgene stacking in operons, and thus will help to expand the range of applications of transplastomic technology.

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Related in: MedlinePlus

Seed assays to confirm homoplasmy of Nt-pZF transplastomic plants and test for antibiotic resistanceSeeds from the wild-type, a selfed transplastomic line and reciprocal crosses between the transplastomic line and the wild-type were germinated on antibiotic-free medium, medium with spectinomycin (Spec; 500 mg l–1), medium with kanamycin (Kan; 400 mg l–1) and medium with both drugs (Spec + Kan). Maternal inheritance of both antibiotic resistances confirms transgene localization in the plastid genome; lack of segregation in the T1 generation confirms homoplasmy of the transplastomic lines.
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fig03: Seed assays to confirm homoplasmy of Nt-pZF transplastomic plants and test for antibiotic resistanceSeeds from the wild-type, a selfed transplastomic line and reciprocal crosses between the transplastomic line and the wild-type were germinated on antibiotic-free medium, medium with spectinomycin (Spec; 500 mg l–1), medium with kanamycin (Kan; 400 mg l–1) and medium with both drugs (Spec + Kan). Maternal inheritance of both antibiotic resistances confirms transgene localization in the plastid genome; lack of segregation in the T1 generation confirms homoplasmy of the transplastomic lines.

Mentions: To ultimately confirm homoplasmy of the transplastomic lines, seeds were obtained from transplastomic plants that had either been selfed or reciprocally crossed to the wild-type. With these seeds, inheritance assays were performed, which represent the most sensitive available test to assess homoplasmy (Bock, 2001; Maliga, 2004). As expected, lack of segregation of spectinomycin resistance in the T1 generation demonstrated homoplasmy (Figure 3, and data not shown) and confirmed uniparentally maternal transgene inheritance, as is typical of a plastid-encoded trait.


Identification of a plastid intercistronic expression element (IEE) facilitating the expression of stable translatable monocistronic mRNAs from operons.

Zhou F, Karcher D, Bock R - Plant J. (2007)

Seed assays to confirm homoplasmy of Nt-pZF transplastomic plants and test for antibiotic resistanceSeeds from the wild-type, a selfed transplastomic line and reciprocal crosses between the transplastomic line and the wild-type were germinated on antibiotic-free medium, medium with spectinomycin (Spec; 500 mg l–1), medium with kanamycin (Kan; 400 mg l–1) and medium with both drugs (Spec + Kan). Maternal inheritance of both antibiotic resistances confirms transgene localization in the plastid genome; lack of segregation in the T1 generation confirms homoplasmy of the transplastomic lines.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230500&req=5

fig03: Seed assays to confirm homoplasmy of Nt-pZF transplastomic plants and test for antibiotic resistanceSeeds from the wild-type, a selfed transplastomic line and reciprocal crosses between the transplastomic line and the wild-type were germinated on antibiotic-free medium, medium with spectinomycin (Spec; 500 mg l–1), medium with kanamycin (Kan; 400 mg l–1) and medium with both drugs (Spec + Kan). Maternal inheritance of both antibiotic resistances confirms transgene localization in the plastid genome; lack of segregation in the T1 generation confirms homoplasmy of the transplastomic lines.
Mentions: To ultimately confirm homoplasmy of the transplastomic lines, seeds were obtained from transplastomic plants that had either been selfed or reciprocally crossed to the wild-type. With these seeds, inheritance assays were performed, which represent the most sensitive available test to assess homoplasmy (Bock, 2001; Maliga, 2004). As expected, lack of segregation of spectinomycin resistance in the T1 generation demonstrated homoplasmy (Figure 3, and data not shown) and confirmed uniparentally maternal transgene inheritance, as is typical of a plastid-encoded trait.

Bottom Line: At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur.As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs.We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Molekulare Pflanzenphysiologie (MPI-MP), Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany.

ABSTRACT
Most plastid genes are part of operons and expressed as polycistronic mRNAs. Many primary polycistronic transcripts undergo post-transcriptional processing in monocistronic or oligocistronic units. At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur. As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs. We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts. Separation of foreign genes by this element facilitates transgene stacking in operons, and thus will help to expand the range of applications of transplastomic technology.

Show MeSH
Related in: MedlinePlus