Limits...
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.

Show MeSH

Related in: MedlinePlus

Generation of plastid-transformed plants to test putative intercistronic processing elements in vivo(a) Map of the targeting region in the tobacco plastid genome (ptDNA). Genes above the lines are transcribed from left to right; genes below the line are transcribed in the opposite direction. The transgenes are targeted to the intergenic spacer between the trnfM and trnG genes.(b) Construction of plastid transformation vectors (pZF series) integrating an operon of two transgenes (nptII and yfp) into the tobacco plastid genome, along with the selectable marker gene for chloroplast transformation, aadA (Svab and Maliga, 1993). Relevant restriction sites used for cloning and RFLP analysis are indicated, sites lost by ligation of heterologous ends are shown in parentheses. Prrn, rRNA operon promoter; TpsbA, 3′ UTR of the psbA gene; Prrn-T7g10, rRNA operon promoter fused to the leader sequence of bacteriophage T7 gene 10; TrbcL, 3′ UTR of the rbcL gene; IEE (putative processing element); SD, Shine–Dalgarno sequence; Trps16, 3′ UTR of the rps16 gene.(c) Southern blot confirming plastid transformation and assessing homoplasmy of transplastomic lines. Digestion with BamHI produces fragments of approximately 4.5 kb in the wild-type and approximately 8 kb in all transplastomic lines. This size difference corresponds exactly to the combined size of the three integrated transgenes. Note that the probe (a radiolabeled ycf9 fragment) detects a faint wild-type-like band in all transplastomic lines that has been shown previously to come from promiscuous chloroplast DNA in the tobacco nuclear (or mitochondrial) genome (Ruf et al., 2000).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230500&req=5

fig02: Generation of plastid-transformed plants to test putative intercistronic processing elements in vivo(a) Map of the targeting region in the tobacco plastid genome (ptDNA). Genes above the lines are transcribed from left to right; genes below the line are transcribed in the opposite direction. The transgenes are targeted to the intergenic spacer between the trnfM and trnG genes.(b) Construction of plastid transformation vectors (pZF series) integrating an operon of two transgenes (nptII and yfp) into the tobacco plastid genome, along with the selectable marker gene for chloroplast transformation, aadA (Svab and Maliga, 1993). Relevant restriction sites used for cloning and RFLP analysis are indicated, sites lost by ligation of heterologous ends are shown in parentheses. Prrn, rRNA operon promoter; TpsbA, 3′ UTR of the psbA gene; Prrn-T7g10, rRNA operon promoter fused to the leader sequence of bacteriophage T7 gene 10; TrbcL, 3′ UTR of the rbcL gene; IEE (putative processing element); SD, Shine–Dalgarno sequence; Trps16, 3′ UTR of the rps16 gene.(c) Southern blot confirming plastid transformation and assessing homoplasmy of transplastomic lines. Digestion with BamHI produces fragments of approximately 4.5 kb in the wild-type and approximately 8 kb in all transplastomic lines. This size difference corresponds exactly to the combined size of the three integrated transgenes. Note that the probe (a radiolabeled ycf9 fragment) detects a faint wild-type-like band in all transplastomic lines that has been shown previously to come from promiscuous chloroplast DNA in the tobacco nuclear (or mitochondrial) genome (Ruf et al., 2000).

Mentions: To identify a minimum sequence element sufficient for triggering processing of polycistronic transcripts into stable and translatable monocistronic mRNAs, we decided to test sequences derived from the two major processing sites mapped upstream and downstream of psbH in vivo by chloroplast transformation. To this end, we constructed a plastid transformation vector with two transgenes linked together in an operon: the kanamycin resistance gene nptII and the gene for the yellow fluorescent protein, yfp (Figure 2a,b). The two coding regions are separated by a sequence encoding a stem–loop structure (TrbcL) (Figure 2b) to ensure transcript stability of the mRNA from the first cistron after processing, two restriction sites suitable for integrating potential intercistronic processing elements, and a Shine–Dalgarno sequence to mediate translation initiation at the second cistron. For both major processing sites (Figure 1b–d), we constructed two chloroplast transformation vectors (Table 1). Vectors pZF75 and pZF77 contain the complete secondary structures in which the cleavage sites are embedded. This corresponds to sequence elements from −25 to +25 with respect to the psbT–psbH processing site (Figure 1b,d; vector pZF75) and −14 to +14 with respect to the psbH–petB processing site (Figure 1c,d; vector pZF77). In addition, we used two shorter sequences that included only the stem–loop up to the first bulge in the stem structure (Figure 1d). These sequence elements correspond to nucleotide positions −15 to +15 with respect to the psbT–psbH processing site (Figure 1b,d; vector pZF74) and −8 to +8 with respect to the psbH–petB processing site (Figure 1c,d; vector pZF76). Finally, a fifth construct containing no putative processing element between the nptII and yfp cassettes was transformed as a control (pZF73; Table 1).


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)

Generation of plastid-transformed plants to test putative intercistronic processing elements in vivo(a) Map of the targeting region in the tobacco plastid genome (ptDNA). Genes above the lines are transcribed from left to right; genes below the line are transcribed in the opposite direction. The transgenes are targeted to the intergenic spacer between the trnfM and trnG genes.(b) Construction of plastid transformation vectors (pZF series) integrating an operon of two transgenes (nptII and yfp) into the tobacco plastid genome, along with the selectable marker gene for chloroplast transformation, aadA (Svab and Maliga, 1993). Relevant restriction sites used for cloning and RFLP analysis are indicated, sites lost by ligation of heterologous ends are shown in parentheses. Prrn, rRNA operon promoter; TpsbA, 3′ UTR of the psbA gene; Prrn-T7g10, rRNA operon promoter fused to the leader sequence of bacteriophage T7 gene 10; TrbcL, 3′ UTR of the rbcL gene; IEE (putative processing element); SD, Shine–Dalgarno sequence; Trps16, 3′ UTR of the rps16 gene.(c) Southern blot confirming plastid transformation and assessing homoplasmy of transplastomic lines. Digestion with BamHI produces fragments of approximately 4.5 kb in the wild-type and approximately 8 kb in all transplastomic lines. This size difference corresponds exactly to the combined size of the three integrated transgenes. Note that the probe (a radiolabeled ycf9 fragment) detects a faint wild-type-like band in all transplastomic lines that has been shown previously to come from promiscuous chloroplast DNA in the tobacco nuclear (or mitochondrial) genome (Ruf et al., 2000).
© Copyright Policy
Related In: Results  -  Collection

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

fig02: Generation of plastid-transformed plants to test putative intercistronic processing elements in vivo(a) Map of the targeting region in the tobacco plastid genome (ptDNA). Genes above the lines are transcribed from left to right; genes below the line are transcribed in the opposite direction. The transgenes are targeted to the intergenic spacer between the trnfM and trnG genes.(b) Construction of plastid transformation vectors (pZF series) integrating an operon of two transgenes (nptII and yfp) into the tobacco plastid genome, along with the selectable marker gene for chloroplast transformation, aadA (Svab and Maliga, 1993). Relevant restriction sites used for cloning and RFLP analysis are indicated, sites lost by ligation of heterologous ends are shown in parentheses. Prrn, rRNA operon promoter; TpsbA, 3′ UTR of the psbA gene; Prrn-T7g10, rRNA operon promoter fused to the leader sequence of bacteriophage T7 gene 10; TrbcL, 3′ UTR of the rbcL gene; IEE (putative processing element); SD, Shine–Dalgarno sequence; Trps16, 3′ UTR of the rps16 gene.(c) Southern blot confirming plastid transformation and assessing homoplasmy of transplastomic lines. Digestion with BamHI produces fragments of approximately 4.5 kb in the wild-type and approximately 8 kb in all transplastomic lines. This size difference corresponds exactly to the combined size of the three integrated transgenes. Note that the probe (a radiolabeled ycf9 fragment) detects a faint wild-type-like band in all transplastomic lines that has been shown previously to come from promiscuous chloroplast DNA in the tobacco nuclear (or mitochondrial) genome (Ruf et al., 2000).
Mentions: To identify a minimum sequence element sufficient for triggering processing of polycistronic transcripts into stable and translatable monocistronic mRNAs, we decided to test sequences derived from the two major processing sites mapped upstream and downstream of psbH in vivo by chloroplast transformation. To this end, we constructed a plastid transformation vector with two transgenes linked together in an operon: the kanamycin resistance gene nptII and the gene for the yellow fluorescent protein, yfp (Figure 2a,b). The two coding regions are separated by a sequence encoding a stem–loop structure (TrbcL) (Figure 2b) to ensure transcript stability of the mRNA from the first cistron after processing, two restriction sites suitable for integrating potential intercistronic processing elements, and a Shine–Dalgarno sequence to mediate translation initiation at the second cistron. For both major processing sites (Figure 1b–d), we constructed two chloroplast transformation vectors (Table 1). Vectors pZF75 and pZF77 contain the complete secondary structures in which the cleavage sites are embedded. This corresponds to sequence elements from −25 to +25 with respect to the psbT–psbH processing site (Figure 1b,d; vector pZF75) and −14 to +14 with respect to the psbH–petB processing site (Figure 1c,d; vector pZF77). In addition, we used two shorter sequences that included only the stem–loop up to the first bulge in the stem structure (Figure 1d). These sequence elements correspond to nucleotide positions −15 to +15 with respect to the psbT–psbH processing site (Figure 1b,d; vector pZF74) and −8 to +8 with respect to the psbH–petB processing site (Figure 1c,d; vector pZF76). Finally, a fifth construct containing no putative processing element between the nptII and yfp cassettes was transformed as a control (pZF73; Table 1).

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