Plant Molecular Biology 11:589-600 (1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands
589
Transcription of ten ribosomal protein genes from tobacco chloroplasts: a compilation of ribosomal protein genes found in the tobacco chloroplast genome Chikara Ohto, Keita Torazawa, Minoru Tanaka, Kazuo Shinozaki and Masahiro Sugiura*
Center for Gene Research, Nagoya University, Chikusa, Nagoya 464-01, Japan (*author for correspondence) Received 7 June 1988; accepted in revised form 10 August 1988
Key words: chloroplast, codon usage, intron, ribosomal protein gene, transcription, tobacco Abstract
Transcription of rps2, rps4, rps7, rpsll, rps14, rpsl5, rpsl8, rpl20, rp133 and rpi36 from the tobacco chloroplast genome has been studied. Northern blot analysis has revealed that all these genes are transcribed in the chloroplast. Multiple transcripts were detected for all the genes and amounts of the transcripts were quite different among the ten genes. These ten ribosomal protein genes together with the ten other ribosomal protein genes published previously were compiled and compared. Four out of the twenty genes contain introns, possible secondary structures of which are presented.
Introduction
Tobacco chloroplast ribosomes are 70S in size and contain 23S, 16S, 5S and 4.5S rRNAs [30] and 5 8 - 6 2 ribosomal proteins [6]. All the rRNAs are known to be encoded in the chloroplast genome [30]. Analyses o f the synthesis of ribosomal proteins in isolated chloroplasts have shown that a chloroplast genome encodes about one-third of the ribosomal proteins [9, 10, 21]. The electrophoretic patterns and molecular weight frequency distributions of the proteins o f tobacco chloroplast ribosomes are quite similar to those of the Escherichia coli ribosomes [6]. Determination of the primary structure o f ribosomal protein L12 from spinach chloroplasts [3] and immunological studies [2, 8] showed also the similarity of chloroplast and E. coli ribosomal proteins. These led us to search for chloroplast ribosomal protein genes through their homology with E. coli counterparts. The tobacco rpsl9 was the first potential gene for a ribosomal protein to be
identified in a chloroplast genome [29], and subsequently additional putative chloroplast genes for ribosomal proteins have been reported in a variety of plants (e.g. [19, 20, 28]). Some of them contain introns (e.g. [401). Twenty different open reading frames (ORFs) potentially coding for proteins homologous to E. coli ribosomal proteins have been found in the tobacco chloroplast genome [27, 31], and ten of the ORFs have been shown to be transcribed in tobacco chloroplasts [26, 35, 39]. Here we present transcription patterns of the other ten ORFs. We also present a compilation of the potential genes for ribosomal proteins from tobacco chloroplast DNA and proposed secondary structures of their introns.
Materials and methods
Recombinant plasmids pTBa2, pTBa4, pTB30, pTBa3, pTS9, pTS8, pTP9, and pTB12, which contain
590
rpsl5, rps2, rpsl4, rps4, rp133 + rpsl8, rpl20, rp136 + rpsll, and rps7 of Nicotiana tabacum cv. Bright Yellow 4 chloroplasts, respectively, have been described [32]. [ot32p]-labeled single-stranded D N A probes were synthesized on recombinant M 1 3 m p l 0 / l l and M13mp18/19 DNAs derived from the above plasmids using [ct3zp]dCTP and Klenow fragment. The gene-specific single-stranded D N A probes were separated by polyacrylamide gel electrophoresis in 8 M urea after digestion with appropriate restriction enzymes. Total tobacco chloroplast R N A was prepared as described [34]. R N A (1 #g/lane) was fractionated by 1.1% agarose gel containing 6% formaldehyde [17] and transferred to nylon membrane sheets (Nitran NY13) by capillary attraction with 20× SSC. The sheets were then dried for 1 h at room temperature and for 2 h at 80 °C in vacuo. Prehybridization and hybridization were performed according to the instruction manual for GeneScreen (DuPont). D N A sequence data were analyzed with an N E C PC98XA computer using the G E N E T Y X program and a microVAXII computer using the U W G C G and I D E A S programs.
Results and discussion D N A sequencing o f the entire tobacco chloroplast genome revealed the presence of 20 different ORFs potentially coding for polypeptides homologous to E. coli ribosomal proteins [27, 31]. rp136 is a new gene found through homology with E. coli rpmJ [38]. Figure 1 shows a m a p of these putative genes (rpl for 50S subunit proteins and rps for 30S subunit proteins). Transcription of 10 out of the 20 ORFs has been analyzed previously, and all ten were found to be expressed in tobacco chloroplasts [26, 35, 39]. Northern blot hybridization was performed to detect transcripts from the remaining ten putative genes for ribosomal proteins: rps2, rps4, rps7, rpsll, rpsl4, rpsl5, rpsl8, rpl20, rp133, and rp136. Total tobacco chloroplast R N A was electrophoresed in 1.1070 denaturing agarose gels and transferred to nylon membrane sheets. The R N A blot was hybridized with each of the gene-specific single-stranded D N A probes (Fig. 2). All the D N A probes hybridized to multiple R N A bands, indicating that the ten ORFs
lr~ ; ~
Jl~rpI2*
~.55 55
Fig. 1. Location of ribosomal genes on the tobacco chloroplast genome. Bold lines show the inverted repeat. Asterisks indicate split genes. Genes shown inside the circle are transcribed clockwise and genes shown outside the circle are transcribed counterclockwise.
are transcribed in tobacco chloroplasts. The rps2 probe hybridized to at least six R N A bands ranging from 4.8-1.5 kb. The presence of long transcripts suggests cotranscription of rps2 with the downstream atp genes (atpI, atpH, atpF and atpA) as reported for the spinach and pea rps2 and atp genes [13]. rpsl4 is transcribed as a major 5.2 kb R N A in which the upstream psaA and psaB sequences are contained [18]. The rps4 probe hybridized to several R N A bands of 4 . 6 - 0.7 kb, suggesting that rps4 is cotranscribed with its adjacent genes. The rp133 and rpsl8 probes hybridized to two comm o n R N A bands of 1.7 kb and 1.3 kb, indicating that these two genes are cotranscribed. The 0.7 kb R N A detected by the rpsl 8 probe may be a processed and monocistronic m R N A for CS18. At least five R N A bands of 3 . 8 - 0 . 7 kb were detected with the rpl20 probe, rpl20 is likely to be cotranscribed with the upstream 5'-rpsl2 gene [31, 39]. The rp136 and rpsl 1 probes hybridized to at least five c o m m o n faint R N A bands of 2.5-1.0 kb. These are likely to be parts of the rp123 gene cluster and cotranscribed with the upstream eight ribosomal protein genes and the downstream rpoA gene. The rps7 probe hybridized to 2.0, 1.3 and 0.9 kb R N A bands and rps7 is
591
Fig. 2. Northern blot analysis of transcripts from rps2, rpsl4, rps4, rp133, rpsl8, rpl20, rp136, rpsll, rps7 and rpsl5. Total chloroplast RNA (1 #g/lane) was fractionated in formaldehyde-agarose gels. The probes used are indicated as pentagons below the gene maps: rps2, 532 bases(b), Nar I-Aha II fragment; rpsl4, 288 b, Eco RI-Bst NI fragment; rps4, 148 b, Eco T14I fragment; rp133, 268 b, Dra I-Bal I fragment; rpslS, 265 b, Nco I-Sal I fragment; rpl20, 367 b, Nsp V-CIa 1 fragment; rp136, 126 b, Alu I-Rsa I fragment; rpsll, 299 b, Acc II-Acc I fragment; rps7, 384 b, Bgl II-Bam HI fragment; rpslS, 288 b, Taq I fragment. These probes had similar specific activities. Exposure period was 2 days (using an intensifying screen, IS) for rps2, 5 h for rpsl4, 7 days (IS) for rps4, 1 day for rp133 and rpslS, 7 days (IS) for rpl20, 17 days (IS) for rp136, rpsll and rpsl5, and 1 day for rps7. RNA sizes (kb) were estimated by reference to positions of the RNA size marker (BRL).
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589
Transcription of ten ribosomal protein genes from tobacco chloroplasts: a compilation of ribosomal protein genes found in the tobacco chloroplast genome Chikara Ohto, Keita Torazawa, Minoru Tanaka, Kazuo Shinozaki and Masahiro Sugiura*
Center for Gene Research, Nagoya University, Chikusa, Nagoya 464-01, Japan (*author for correspondence) Received 7 June 1988; accepted in revised form 10 August 1988
Key words: chloroplast, codon usage, intron, ribosomal protein gene, transcription, tobacco Abstract
Transcription of rps2, rps4, rps7, rpsll, rps14, rpsl5, rpsl8, rpl20, rp133 and rpi36 from the tobacco chloroplast genome has been studied. Northern blot analysis has revealed that all these genes are transcribed in the chloroplast. Multiple transcripts were detected for all the genes and amounts of the transcripts were quite different among the ten genes. These ten ribosomal protein genes together with the ten other ribosomal protein genes published previously were compiled and compared. Four out of the twenty genes contain introns, possible secondary structures of which are presented.
Introduction
Tobacco chloroplast ribosomes are 70S in size and contain 23S, 16S, 5S and 4.5S rRNAs [30] and 5 8 - 6 2 ribosomal proteins [6]. All the rRNAs are known to be encoded in the chloroplast genome [30]. Analyses o f the synthesis of ribosomal proteins in isolated chloroplasts have shown that a chloroplast genome encodes about one-third of the ribosomal proteins [9, 10, 21]. The electrophoretic patterns and molecular weight frequency distributions of the proteins o f tobacco chloroplast ribosomes are quite similar to those of the Escherichia coli ribosomes [6]. Determination of the primary structure o f ribosomal protein L12 from spinach chloroplasts [3] and immunological studies [2, 8] showed also the similarity of chloroplast and E. coli ribosomal proteins. These led us to search for chloroplast ribosomal protein genes through their homology with E. coli counterparts. The tobacco rpsl9 was the first potential gene for a ribosomal protein to be
identified in a chloroplast genome [29], and subsequently additional putative chloroplast genes for ribosomal proteins have been reported in a variety of plants (e.g. [19, 20, 28]). Some of them contain introns (e.g. [401). Twenty different open reading frames (ORFs) potentially coding for proteins homologous to E. coli ribosomal proteins have been found in the tobacco chloroplast genome [27, 31], and ten of the ORFs have been shown to be transcribed in tobacco chloroplasts [26, 35, 39]. Here we present transcription patterns of the other ten ORFs. We also present a compilation of the potential genes for ribosomal proteins from tobacco chloroplast DNA and proposed secondary structures of their introns.
Materials and methods
Recombinant plasmids pTBa2, pTBa4, pTB30, pTBa3, pTS9, pTS8, pTP9, and pTB12, which contain
590
rpsl5, rps2, rpsl4, rps4, rp133 + rpsl8, rpl20, rp136 + rpsll, and rps7 of Nicotiana tabacum cv. Bright Yellow 4 chloroplasts, respectively, have been described [32]. [ot32p]-labeled single-stranded D N A probes were synthesized on recombinant M 1 3 m p l 0 / l l and M13mp18/19 DNAs derived from the above plasmids using [ct3zp]dCTP and Klenow fragment. The gene-specific single-stranded D N A probes were separated by polyacrylamide gel electrophoresis in 8 M urea after digestion with appropriate restriction enzymes. Total tobacco chloroplast R N A was prepared as described [34]. R N A (1 #g/lane) was fractionated by 1.1% agarose gel containing 6% formaldehyde [17] and transferred to nylon membrane sheets (Nitran NY13) by capillary attraction with 20× SSC. The sheets were then dried for 1 h at room temperature and for 2 h at 80 °C in vacuo. Prehybridization and hybridization were performed according to the instruction manual for GeneScreen (DuPont). D N A sequence data were analyzed with an N E C PC98XA computer using the G E N E T Y X program and a microVAXII computer using the U W G C G and I D E A S programs.
Results and discussion D N A sequencing o f the entire tobacco chloroplast genome revealed the presence of 20 different ORFs potentially coding for polypeptides homologous to E. coli ribosomal proteins [27, 31]. rp136 is a new gene found through homology with E. coli rpmJ [38]. Figure 1 shows a m a p of these putative genes (rpl for 50S subunit proteins and rps for 30S subunit proteins). Transcription of 10 out of the 20 ORFs has been analyzed previously, and all ten were found to be expressed in tobacco chloroplasts [26, 35, 39]. Northern blot hybridization was performed to detect transcripts from the remaining ten putative genes for ribosomal proteins: rps2, rps4, rps7, rpsll, rpsl4, rpsl5, rpsl8, rpl20, rp133, and rp136. Total tobacco chloroplast R N A was electrophoresed in 1.1070 denaturing agarose gels and transferred to nylon membrane sheets. The R N A blot was hybridized with each of the gene-specific single-stranded D N A probes (Fig. 2). All the D N A probes hybridized to multiple R N A bands, indicating that the ten ORFs
lr~ ; ~
Jl~rpI2*
~.55 55
Fig. 1. Location of ribosomal genes on the tobacco chloroplast genome. Bold lines show the inverted repeat. Asterisks indicate split genes. Genes shown inside the circle are transcribed clockwise and genes shown outside the circle are transcribed counterclockwise.
are transcribed in tobacco chloroplasts. The rps2 probe hybridized to at least six R N A bands ranging from 4.8-1.5 kb. The presence of long transcripts suggests cotranscription of rps2 with the downstream atp genes (atpI, atpH, atpF and atpA) as reported for the spinach and pea rps2 and atp genes [13]. rpsl4 is transcribed as a major 5.2 kb R N A in which the upstream psaA and psaB sequences are contained [18]. The rps4 probe hybridized to several R N A bands of 4 . 6 - 0.7 kb, suggesting that rps4 is cotranscribed with its adjacent genes. The rp133 and rpsl8 probes hybridized to two comm o n R N A bands of 1.7 kb and 1.3 kb, indicating that these two genes are cotranscribed. The 0.7 kb R N A detected by the rpsl 8 probe may be a processed and monocistronic m R N A for CS18. At least five R N A bands of 3 . 8 - 0 . 7 kb were detected with the rpl20 probe, rpl20 is likely to be cotranscribed with the upstream 5'-rpsl2 gene [31, 39]. The rp136 and rpsl 1 probes hybridized to at least five c o m m o n faint R N A bands of 2.5-1.0 kb. These are likely to be parts of the rp123 gene cluster and cotranscribed with the upstream eight ribosomal protein genes and the downstream rpoA gene. The rps7 probe hybridized to 2.0, 1.3 and 0.9 kb R N A bands and rps7 is
591
Fig. 2. Northern blot analysis of transcripts from rps2, rpsl4, rps4, rp133, rpsl8, rpl20, rp136, rpsll, rps7 and rpsl5. Total chloroplast RNA (1 #g/lane) was fractionated in formaldehyde-agarose gels. The probes used are indicated as pentagons below the gene maps: rps2, 532 bases(b), Nar I-Aha II fragment; rpsl4, 288 b, Eco RI-Bst NI fragment; rps4, 148 b, Eco T14I fragment; rp133, 268 b, Dra I-Bal I fragment; rpslS, 265 b, Nco I-Sal I fragment; rpl20, 367 b, Nsp V-CIa 1 fragment; rp136, 126 b, Alu I-Rsa I fragment; rpsll, 299 b, Acc II-Acc I fragment; rps7, 384 b, Bgl II-Bam HI fragment; rpslS, 288 b, Taq I fragment. These probes had similar specific activities. Exposure period was 2 days (using an intensifying screen, IS) for rps2, 5 h for rpsl4, 7 days (IS) for rps4, 1 day for rp133 and rpslS, 7 days (IS) for rpl20, 17 days (IS) for rp136, rpsll and rpsl5, and 1 day for rps7. RNA sizes (kb) were estimated by reference to positions of the RNA size marker (BRL).
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(241 a.a.)
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