The Plant Journal 52 Transcription of plastid genes …sugita-g/pub/KabeyaTPJ(2007).pdfJournal...

Transcription of plastid genes is modulated by twonuclear-encoded a subunits of plastid RNA polymerasein the moss Physcomitrella patens

Yukihiro Kabeya†, Yuki Kobayashi‡, Hiromichi Suzuki, Jun Itoh and Mamoru Sugita*

Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan

Received 15 June 2007; revised 18 July 2007; accepted 23 July 2007.

*For correspondence (fax +81 52 789 3080; e-mail [email protected]).†Present address: Miyagishima Initiative Research Unit, Frontier Research System, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.‡Present address: Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-0032, Tokyo, Japan.

The nucleotide sequences reported in this paper have been submitted to the DDBJ/EMBL/GenBank database under accession numbers AB110071 (PpRpoA2 gene)

and AB110072 (PpRpoA2 cDNA).

Summary

In general, in higher plants, the core subunits of a bacterial-type plastid-encoded RNA polymerase (PEP) are

encoded by the plastid rpoA, rpoB, rpoC1 and rpoC2 genes. However, an rpoA gene is absent from the moss

Physcomitrella patens plastid genome, although the PpRpoA gene (renamed PpRpoA1) nuclear counterpart is

present in the nuclear genome. In this study, we identified and characterized a second gene encoding the

plastid-targeting a subunit (PpRpoA2). PpRpoA2 comprised 525 amino acids and showed 59% amino acid

identity with PpRpoA1. Two PpRpoA proteins were present in the PEP active fractions separated from the

moss chloroplast lysate, confirming that both proteins are a subunits of PEP. Northern blot analysis showed

that PpRpoA2 was highly expressed in the light, but not in the dark, whereas PpRpoA1 was constitutively

expressed. Disruption of the PpRpoA1 gene resulted in an increase in the PpRpoA2 transcript level, but most

plastid gene transcript levels were not significantly altered. This indicates that transcription of most plastid

genes depends on PpRpoA2-PEP rather than on PpRpoA1-PEP. In contrast, the transcript levels of petN, psbZ

and ycf3 were altered in the PpRpoA1 gene disruptant, suggesting that these are PpRpoA1-PEP-dependent

genes. These observations suggest that plastid genes are differentially transcribed by distinct PEP enzymes

with either PpRpoA1 or PpRpoA2.

Keywords: a subunit, chloroplast, plastid-encoded RNA polymerase, Physcomitrella, transcription.

Introduction

Plastids are semi-autonomous organelles that possess their

own genetic information. The components of photosynthe-

sis complexes and the translational and transcriptional

apparatus are encoded separately by plastid and nuclear

genomes. The nuclear-encoded components synthesized in

the cytoplasm are imported post-translationally into the

plastids and assembled with the plastid-encoded compo-

nents (Martin and Herrmann, 1998).

Plastids of higher (seed) plants contain two distinct DNA-

dependent RNA polymerases: the plastid-encoded plastid

RNA polymerase (PEP), and the nuclear-encoded plastid

RNA polymerase (NEP) (Maliga, 1998; Hess and Borner,

1999). The PEP enzyme comprises a core complex aabb¢b¢¢

encoded as plastid genes rpoA, rpoB, rpoC1 and rpoC2.

Transcription initiation by PEP is required for multiple

nuclear-encoded r factors, which recognize the bacterial-

type promoter sequence of the photosynthesis genes while

containing canonical –10 and –35 elements (Shiina et al.,

2005). PEP activity is severely inhibited by tagetitoxin, an

inhibitor of prokaryote RNA polymerase (Mathews and

Durbin, 1990). In contrast, NEP preferentially transcribes

housekeeping genes such as rpoB, rpl23 and clpP. The NEP

promoter resembles the plant mitochondrial promoter

sequence and differs completely from the PEP promoter

(Kapoor and Sugiura, 1999; Liere and Maliga, 1999). In

general, genes for non-photosynthetic components are

730 ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd

The Plant Journal (2007) 52, 730–741 doi: 10.1111/j.1365-313X.2007.03270.x

transcribed by NEP during the early stage of plastid differ-

entiation and development. Subsequent transcription of the

genes for photosynthesis-related components is directed by

PEP (Hajdukiewicz et al., 1997). Several lines of evidence

indicated that a plastid-localized bacteriophage-type RNA

polymerase (RpoT) is an NEP (Lerbs-Mache, 1993; Hedtke

et al., 1997; Liere et al., 2004). NEP activity is not affected by

tagetitoxin.

We have reported previously that the rpoA gene is absent

from the plastid genome of the moss Physcomitrella patens,

and we have identified a nuclear counterpart, PpRpoA

(Sugiura et al., 2003). The loss of rpoA from the plastid

genome is a general occurrence in the arthrodontous

mosses, suggesting that the rpoA gene was lost from the

plastid genome and transferred to the nucleus during the

evolutionary history of the mosses (Sugita et al., 2004;

Goffinet et al., 2005). However, it is unclear whether the

nuclear PpRpoA gene really encodes the a subunit of PEP

and is required for the function of PEP. The RNA polymerase

core enzyme of Escherichia coli is assembled in the

sequence: a fi aa fi aab fi aabb¢, indicating that the a sub-

unit plays a key role in the assembly of the core enzyme

(Kimura and Ishihama, 1995).

In this study, we identified a second PpRpoA gene

(PpRpoA2), and showed that both PpRpoA and PpRpoA2

proteins were fractionated with PEP enzyme activity. The

two PpRpoA genes were differently expressed under differ-

ent light and dark conditions. We disrupted the PpRpoA1

gene by homologous recombination and characterized the

disruptant with respect to plastid gene expression. Disrup-

tion of the PpRpoA1 gene resulted in the alteration of several

chloroplast genes at the transcript level. We discuss this

transcription modulation, which appears to be mediated by

the two a subunits of PEP in Physcomitrella.

Results

Identification and characterization of the PpRpoA2 gene

To search for PpRpoA paralog(s) we performed a tBLAST

search using the query as the amino acid sequence of the

PpRpoA against the expressed sequence tag (EST) database

at PHYSCObase (http://moss.nibb.ac.jp) (Nishiyama et al.,

2003). End sequences of an EST clone pphf35o11 (DDBJ/

EMBL/GenBank accession nos BJ947461 and BJ958286)

were found to encode a partial coding sequence homolo-

gous to PpRpoA. We then isolated and sequenced the

cognate cDNA. The encoded protein comprised 525 amino

acid residues, which showed 59.1% identity with PpRpoA.

Therefore, the newly identified sequence was designated

as PpRpoA2 and the previously identified PpRpoA as

PpRpoA1. No other homologous sequences were found

in this analysis. Comparison of the PpRpoA2 cDNA and

the corresponding genomic sequences in PHYSCObase

(gnl|ti|870055012, gnl|ti|870076393, gnl|ti|86233862,

gnl|ti|713796403, gnl|ti|692455034 and gnl|ti|846052148)

revealed that the PpRpoA2 gene comprises seven exons

and six introns (Figure 1a). The first and sixth introns are

located in the 5¢- and 3¢-untranslated regions, respectively.

The intron insertion positions are not conserved between

PpRpoA1 and PpRpoA2 genes (Figure 1c). As shown in

Figure 1b, PpRpoA2 protein has a sequence that is homo-

logous to a part of HSP70 at the N-terminal region. PpRpoA2

is highly homologous to the sequences of known plastid-

encoded RpoAs and a cyanobacterium RpoA. Functional

amino acid residues were determined in the a subunit of

E. coli (Kimura and Ishihama, 1995). These are involved in

the dimerization of a subunits (45Arg at residue 45), in the

assembly of aab (48Leu) and in the core complex formation

of aabb¢ (86Lys and 173 Val). The two PpRpoA proteins also

have these conserved amino acid residues (Figure 1c), and

therefore are predicted be able to assemble the core PEP

enzyme.

PpRpoA2 has the N-terminal extension sequence, which is

predicted to specify plastid targeting (a score of 0.863) by the

TARGETP program for protein sorting (Emanuelsson et al.,

2000). To examine the cellular localization of PpRpoA2, we

constructed the plasmid PpRpoA2-gfp encoding a chimeric

protein of the N-terminal 125 amino acid residues fused to

sGFP and introduced it to the moss protonemal protoplasts.

Green fluorescence of PpRpoA2-GFP was localized in the

chloroplasts (Figure 1d, panel a) as was that of PpRpoA1-

GFP (Figure 1d, panel c). This clearly indicates that PpRpoA2

is a plastid-localized protein. This strongly suggests that the

two PpRpoA proteins function as an a subunit of the PEP

enzyme in the Physcomitrella plastids.

PpRpoA1 and PpRpoA2 proteins are components

of PEP enzyme

Amino acid sequence identity and the domain structures of

PpRpoA proteins strongly suggested that PpRpoA1 and

PpRpoA2 are the a subunit of PEP. To confirm this, we

investigated whether PpRpoA1 and PpRpoA2 proteins are

contained in PEP active fractions separated by anion

exchange column chromatography. As shown in Figure 2a

and Figure S1, transcriptional active fractions 21–26 were

eluted with about 0.4 M KCl, and these transcriptional

activities were severely inhibited by the addition of tageti-

toxin, an inhibitor of PEP transcription activity. Western blot

analysis showed that PpRpoA1 and PpRpoA2 are detected as

40-kDa and 50-kDa bands, respectively, in the PEP active

fraction 25, but not in inactive fractions 11 and 31

(Figure 2b). The cross-reaction of the antibodies against the

respective PpRpoA proteins with the recombinant proteins

was estimated to be less than 5% (Figure 2c). This result

indicates that both PpRpoA1 and PpRpoA2 proteins are

bona fide components of the functional PEP enzyme.

Nuclear-encoded a subunits of PEP in moss 731

ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 730–741

(a) (b)

(c)

(d)

732 Yukihiro Kabeya et al.


PpRpoA1 and PpRpoA2 genes are differentially

expressed in the protonemata

To investigate the transcript levels of PpRpoA1 and

PpRpoA2 in the protonemata, we performed Northern blot

analysis. The transcript levels of PpRpoA1 accumulated at

substantial levels and showed low-amplitude fluctuation for

all RNA samples (Figure 3), as reported previously (Ichikawa

et al., 2004). The transcript level of PpRpoA2 decreased

significantly after 24 h in the dark, and its transcript declined

to 20% of its level in constant light (Figure 3, 4 L versus 3LD).

Upon transfer of the moss protonemata back into light, these

transcripts accumulated until they were restored to the

control level (Figure 3, 3LDL). This profile was similar to that

of Lhcb2 (encoding light-harvesting chlorophyll a/b binding

protein), a known light-responsive gene. This result indi-

cates that expression of the PpRpoA2 gene is differentially

regulated in a light-dependent manner.

Targeted disruption of the PpRpoA1 gene

To investigate further the precise role of PpRpoA1 and

PpRpoA2 gene products, we attempted to generate either

(a)

(b)

(c)

Figure 2. Preparation of plastid-encoded RNA polymerase active fractions

from the isolated chloroplasts and immunoblot detection of PpRpoA1 and

PpRpoA2.

(a) Fractionation of transcription activity by anion exchange chromatography.

Fractions were eluted with a linear KCl gradient. Transcription activities of

each fraction were measured as incorporation of [a-32P]UTP into the

synthesized RNA. The broken line indicates the concentration of KCl. Squares

indicate transcription activity without tagetitoxin and closed circles indicate

transcription activity with tagetitoxin.

(b) Immunoblot detection of PpRpoA1 and PpRpoA2 in the eluted fractions.

Aliquots (10 ll) of fractions 11 (lanes 1 and 4), 25 (lanes 2 and 5), and 35 (lanes

3 and 6), were separated using 10% PAGE, and then subjected to

immunodetection using anti-PpRpoA1 or anti-PpRpoA2 antisera.

(c) Evaluation of the cross-reaction of PpRpoA1 and PpRpoA2 with the

respective anti-PpRpoA antisera. A series of diluted (100, 25 or 5 ng) Thio-

PpRpoA1 or His-PpRpoA2 was loaded in 10% PAGE and then subjected to

immunodetection.

Figure 3. Transcript levels of the PpRpoA1 and PpRpoA2 genes in the moss

protonemata. Total RNA (15 lg) was separated on a 1.2% formaldehyde-

containing agarose gel and subjected to northern blot analysis. RNA was

extracted from 4-day-old protonemata grown under continuous illumination

(4 L), from 4-day-old protonemata treated for 1 day in darkness before

harvesting (3LD) and 3LD protonemata treated for a further 1 day under light

conditions (3LDL). Ethidium bromide-staining gel was shown as a loading

control (rRNA). Sizes of detected transcripts are indicated on the right of the

panels.

Figure 1. (a) Diagrams of the PpRpoA1 and PpRpoA2 genes. Boxes indicate exons.

(b) Diagrams of the PpRpoA1 and PpRpoA2 proteins. The black boxes indicate the C-terminal domain of the a subunit (aCTD), the gray boxes indicate the N-terminal

domain of the a subunit (aNTD) and the white box indicates a part of the HSP70 protein.

(c) Alignment of a subunit homologs of PpRpoA2 (AB293564) with PpRpoA1 (AB110072), Glycine max RpoA (Gm_rpoA; DQ317523), Nicotiana sylvestris (Ns_rpoA;

NC007500), Oryza sativa (Os_rpoA; X15901), fern Adiantum capillus-veneris (Ac_rpoA; NC004766), liverwort Marchantia polymorpha (Mp_rpoA; X04465), red alga

Cyanidiaschyzon merolae (Cm_rpoA; NC004799) and cyanobacterium Synechococcus elongatus PCC 6301(sy_rpoA; AP008231). The black and white arrowheads

indicate the positions of introns in PpRpoA1 and PpRpoA2, respectively. The asterisks indicate the functional amino acid residues, which were identified in the E. coli

a subunit (Kimura and Ishihama, 1995).

(d) Localization of PpRpoA-GFP fusion proteins. Physcomitrella protoplasts were transformed with PpRpoA2-GFP fusion plasmids PpRpoA2-gfp (a and b) and

PpRpoA1-gfp (c and d). a and c, fluorescence of GFP (green) using the cube U-MNIBA (Olympus, http://www.olympus-global.com); b and c, fluorescence of

chlorophyll (red) using the cube U-MWIG.



PpRpoA1 or PpRpoA2 knock-out mosses. Targeted disrup-

tion of the PpRpoA1 gene was achieved by inserting an nptII

cassette into the HindIII site within exon 2 of the plasmid

(Figure 4a). As shown in Figure 4b, we isolated eight G418-

resistant mosses and performed Southern blot analysis to

verify the targeted disruption. Probing with PpRpoA1 cDNA

detected the predicted 6.1-kb EcoRV signal in the wild-type

moss. In contrast, an 8.1-kb signal appeared in the G418-

resistant moss lines (#14, #22, #23 and #24), corresponding

to the 2.0-kb nptII cassette integrated into the PpRpoA1

locus. A uniform population of the transformed moss gen-

ome in the transgenic moss was verified further by PCR

analysis (Figure 4b). In contrast, all transformants for con-

struction of the PpRpoA2 disruptant possessed both the

wild-type and the nptII cassette-inserted PpRpoA2 gene,

representing heteroplasmic moss lines (Figure S2). Among

(a)

(b)

(e)

(c)

(d)

Figure 4. Generation of the PpRpoA1 gene disruptant and phenotype of the disruptant.

(a) The genomic structure of the wild-type and the PpRpoA1 disruptant. The expected fragment sizes after EcoRV digestion of genomic DNA for the DNA-blot

analyzed are shown. Primers (P-F, forward and P-R, reverse) and the expected fragment sizes for PCR analysis are also shown.

(b) DNA-blot analysis of G418-resistant mosses. Total DNA from wild-type (WT) and eight independent G418-resistant mosses (#11 to #24) were digested with EcoRV

and hybridized with the PpRpoA1 cDNA or ntpII probes. PCR analysis showed that 1.9-kb and 3.9-kb fragments were derived from the wild-type and G418-resistant

mosses, respectively.

(c) Total RNA (15 lg) from 4-day-old protonemata of the wild type or transgenic line #22 was subjected to Northern blot analysis with the PpRpoA1 cDNA probe.

(d) Total cellular protein (50 lg) from wild type and line #22 was subjected to Western blot analysis. To control for loading, antiserum detected against tobacco

chloroplast RNA-binding protein cp28 was used. The PpRpoA1 protein (40 kDa) was detected in WT but not in line #22.

(e) Morphology of the wild type and the PpRpoA1 disruptant protonemal colonies under continuous light conditions. Scale bars = 1 cm (protonema colony) and

1 mm (leafy shoot). Colonies were grown for 18 days and average diameters and SD of 10 colonies are plotted. The length of the leafy shoots from the wild type and

disruptant #22 was also measured and the SD are shown.



the PpRpoA1-disruptant mosses, the #22 transgenic moss

was selected as the representative PpRpoA1 disruptant and

was characterized further.

Both the transcript and the gene product of PpRpoA1 were

not detected by RNA-blot and immunoblot analyses in the

#22 transgenic moss (Figures 4c,d). This result clearly indi-

cates that PpRpoA1 protein is absent from the PpRpoA1

disruptant. The PpRpoA1 disruptant displayed the green

phenotype like the wild-type mosses, but showed slightly

retarded growth (Figures 4d,e). In the continuous light

condition, the colony size was smaller in the PpRpoA1

disruptant than in the wild type. The mean length of the leafy

shoot of the PpRpoA1 disruptant was the same as that of

wild type until the 34-day-old adult gametophore stage.

Thereafter, the disruptant leafy shoots grew slowly and were

somewhat smaller than those of the wild-type moss. Thus,

the phenotypic characters did not differ significantly

between the disruptant and wild-type mosses.

Effect of PpRpoA1 disruption on the plastid gene expression

To examine the effect of PpRpoA1 disruption on the

expression of PpRpoA2, the transcript level of PpRpoA2 was

measured by Northern blot analysis (Figure 5a). The

PpRpoA2 transcript level in the PpRpoA1 disruptant was

twice that in the wild type. To further examine the steady-

state transcript levels of the plastid genes in the PpRpoA1

disruptant, we performed plastid DNA microarray analysis.

In the 4-day-old protonemata grown under constant light

conditions, most plastid genes including psaA, psbA, psbD

and rrn16 were expressed at similar levels in the wild type

and in the PpRpoA1 disruptant, but some tRNA levels

increased in the disruptant (Table 1).

To confirm the microarray analysis results, we performed

RNA blot hybridization (Figure 5). The transcripts of psaA,

psbA, psbD, chlN, atpF, ycf4, trnL-UAG, trnfM-CAU and

rrn16 accumulated at similar levels in the wild type and in

the PpRpoA1 disruptant under both light and dark condi-

tions (Figure 5b). This result was consistent with that of the

array analysis. In addition, the transcripts of six genes (atpB,

ycf2, matK, rpoC1, chlB and psaM) accumulated greatly and

at similar levels in the wild type and in the disruptant grown

under constant light, whereas their transcripts declined to

faint levels in dark conditions (Figure 5c). In contrast, petN

and ycf3 transcript levels decreased to 40% and 20% of the

wild-type level, respectively, under constant light conditions

(Figure 5d, 4L lanes). In addition, psbZ transcript level in the

disruptant decreased to 25% of wild-type level under dark

conditions (Figure 5d, 3LD lanes), although it was

unchanged under constant light conditions (4 L lanes). The

six tRNAs accumulated at 2–10-fold higher levels in the

disruptant than in the wild-type protonemata grown under

(a)

(d)

(e)

(b) (c)

Figure 5. Transcript levels of the PpRpoA2 gene and plastid genes in the wild type and the PpRpoA1 disruptant. Total RNA was extracted from the wild type (WT) or

the PpRpoA1 disruptant (DA1) of 4-day-old protonemata grown under continuous illumination (4 L) and 4-day-old protonemata treated for 1 day in darkness before

harvesting (3LD).

(a) Transcripts of three nuclear genes, PpRpoA1, PpRpoA2 and Lhcb2 were detected by Northern blot analysis.

(b)–(e) Transcripts of plastid genes were detected using plastid gene-specific probes. Positions of RNA markers were indicated on the right of panels.



Table 1 Genes whose expression was affected by the PpRpoA1 disruptant

4L 3LD 4L 3LD

Gene Name log2(DA1/WT)* log2(DA1/WT)* Gene Name log2(DA1/WT)* log2(DA1/WT)*

rps18/psaJ 0.08 � 0.04 )0.21 + 0.29 rpoB-a )0.11 + 0.08 0.76 + 1.613’-rps12 0.16 + 0.04 )0.10 + 0.23 rpoB-b )0.14 + 0.07 )0.27 + 0.645’-rps12 0.00 + 0.04 )0.03 + 0.31 rpoC1-e2 )0.08 + 0.09 1.11 + 0.59aadA 0.62 + 1.05 ND rpoC2-a )0.06 + 0.10 0.56 + 0.59accD )0.10 + 0.08 )0.46 + 0.33 rpoC2-b )0.34 + 0.08 NDActin )0.96 + 0.18 ND rps11/rps8 )0.57 + 0.05 0.91 + 0.43atpA 0.02 + 0.08 )0.55 + 0.23 rps14 )0.57 + 0.09 )0.21 + 0.55atpB )0.08 + 0.02 1.52 + 0.20 rps15 )0.79 + 0.07 )0.35 + 1.22atpE 0.08 + 0.05 1.42 + 0.39 rps2 )0.61 + 0.03 1.46 + 0.55atpF 0.01 + 0.01 )0.93 + 0.30 rps3/rps19 )0.51 + 0.02 0.39 + 0.13atpH 0.26 + 0.06 )0.55 + 0.32 rps4 )0.93 + 0.11 0.67 + 0.19atpI )0.84 + 0.03 )0.24 + 0.42 rps7 0.07 + 0.10 )0.34 + 0.19cemA (ycf10) )0.86 + 0.03 0.54 + 0.32 rrn )2.26 + 0.07 )0.89 + 0.32chlB 0.07 + 0.09 1.02 + 0.29 rrn16 1.24 + 0.04 0.00 + 0.57chlL )0.30 + 0.09 )0.16 + 0.78 rrn23 )0.25 + 0.07 0.28 + 0.70chlN )0.05 + 0.05 1.03 + 0.38 rrn4.5/rrn5 0.59 + 0.11 )0.25 + 0.49clpP )0.06 + 0.06 )0.56 + 0.23 spacer+ORF19 0.28 + 0.07 )0.36 + 0.78matK 0.25 + 0.08 1.49 + 0.23 trnA-UGC )0.20 + 0.02 0.98 + 0.17ndhA )0.71 + 0.06 )0.51 + 0.29 trnC-GCA 0.17 + 0.12 NDndhB 0.01 + 0.06 0.23 + 0.22 trnE-Y-D ND NDndhC )0.02 + 0.13 0.26 + 0.55 trnF-GAA 1.33 + 0.49 )1.72 + 0.67ndhD )0.53 + 0.10 )0.94 + 0.31 trnfM-CAU )0.91 + 0.40 NDndhE/ndhI )0.36 + 0.07 )0.39 + 0.13 trnG-UCC 1.71 + 0.09 NDndhF )0.45 + 0.04 )0.67 + 0.50 trnG-GCC 0.55 + 0.05 NDndhH )0.83 + 0.08 )0.55 + 0.16 trnH-GUG 0.35 + 0.82 NDndhJ )0.02 + 0.10 0.30 + 0.60 trnI-GAU 0.18 + 0.07 0.66 + 0.32ndhK 0.02 + 0.04 0.46 + 0.35 trnI-CAU ND NDnptII 0.37 + 0.60 4.33 + 0.19 trnK )0.52 + 0.14 )0.02 + 0.37ORF40/ORF197 0.79 + 0.04 ND trnL-UAA 0.25 + 0.11 NDpBS 0.82 + 0.82 ND trnL-CAA ND )0.14 + 0.74petA )0.40 + 0.05 0.59 + 0.32 trnL-UAG )0.74 + 1.24 NDpetB )0.15 + 0.06 )0.68 + 0.16 trnM-CAU 0.29 + 0.06 0.03 + 0.59petD 0.08 + 0.09 )0.60 + 0.13 trnN-GUU )1.50 + 5.00 NDpetG/petL 0.37 + 0.08 )1.16 + 0.27 trnP-UGG/W-CCA 2.36 + 0.02 0.24 + 0.08psaA )0.70 + 0.07 0.12 + 0.56 trnQ-UUG 2.66 + 0.04 )0.74 + 0.40psaB )0.25 + 0.05 )0.18 + 0.14 trnR-ACG 0.16 + 0.29 )2.50 + 1.78psaC 0.50 + 0.11 )0.28 + 1.11 trnR-CCG ND )0.74 + 0.21psaI 0.00 + 0.11 )0.52 + 0.17 trnS-GCU 0.65 + 0.03 )0.40 + 0.50psaM 1.41 + 0.22 )0.70 + 0.31 trnS-GUA 0.57 + 0.05 )0.62 + 0.93psbA 0.11 + 0.08 )0.56 + 0.21 trnS-UGA 2.64 + 0.10 )0.79 + 0.20psbB )0.05 + 0.09 )0.61 + 0.26 trnT-GGU ND )1.89 + 1.42psbC )0.22 + 0.09 )0.38 + 0.12 trnT-UGU )0.80 + 0.62 NDpsbD )0.16 + 0.08 )0.56 + 0.16 trnV-UAC )0.18 + 0.05 )0.02 + 0.40psbE/F/L/J 0.13 + 0.02 )0.28 + 0.24 trnV-GAC )2.11 + 1.15 NDpsbH/T )0.02 + 0.04 )0.49 + 0.20 ycf12 1.07 + 0.11 NDpsbK/psbI 0.60 + 0.04 )0.27 + 0.26 ycf1-a )0.88 + 0.03 0.63 + 2.02psbM 0.76 + 0.11 )0.70 + 0.31 ycf1-b )0.79 + 0.05 )0.12 + 1.37pseudotrnV ND )1.79 + 1.20 ycf2-a 1.01 + 0.06 1.40 + 0.87rbcL 0.08 + 0.04 )0.50 + 0.46 ycf2-b 1.31 + 0.06 )0.10 + 0.69rpl14/rps16 )0.61 + 0.03 0.38 + 1.20 ycf3 )0.74 + 0.07 0.34 + 0.19rpl2/rpl23 )0.87 + 0.08 0.57 + 0.46 ycf4 )0.86 + 0.04 1.09 + 0.36rpl20 0.77 + 0.05 0.37 + 0.84 ycf6 (petN) 0.57 + 0.16 )1.88 + 0.12rpl21/rpl32 )0.57 + 0.19 0.44 + 1.45 ycf66 )0.06 + 0.07 )0.46 + 0.40PpRpoA1 )0.87 + 0.24 ND ycf9 (psbZ) 0.17 + 0.07 )1.44 + 0.66

*Values shown are means � SD of data from six spots.Hybridization signals for the wild type (WT) and the PpRpoA1 disruptant (DA1) were measured to estimate the signal intensity (log10 [D1*WT]) andratio of disruptant to wild type (log2 [D1/WT]). The hybridization was repeated twice with Cy3 or Cy5 dye swapping. The reliability of thehybridization signals was evaluated by the intensity of the signal (log10 [D1*WT] >4), and the dispersion among six spots on two slide glasses (SDof log2 [D1/WT] should be <0.5). Genes whose expression was judged to be altered are shaded in gray. 4L, four-day-old protonemata grown underconstant light condition. 3LD, four-day-old protonemata treated for one day in darkness before harvesting. ND, Not detected.



constant light conditions (Figure 5e). In dark-treated proto-

nemata, however, their transcript levels were the same in the

disruptant and in the wild type (Figure 5e).

Disruption of PpRpoA1 does not affect the

expression of plastid r factor genes

The effect of PpRpoA1 gene disruption on the plastid gene

expression may be caused by the modulation of the

expression of plastid r factors. To investigate this possibil-

ity, the transcript levels of three genes, PpSig1, PpSig2 and

PpSig5, encoding the plastid r factor were measured by

reverse transcriptase-polymerase chain reaction (RT-PCR) in

the wild type and the PpRpoA1 disruptant (Figure 6). The

three r factor genes were highly expressed in the light (4 L

and 3LDL) and were low in the dark (3LD). This expression

profile was similar to those of the PpRpoA1 and PpRpoA2

genes. Although there could be other differences at the

protein level, at least the transcript levels of the three PpSig

genes were not significantly different between the wild type

and the PpRpoA1 disruptant.

Discussion

In this study, we identified the second nuclear gene

PpRpoA2 encoding the PEP a subunit. Both PpRpoA2 and

PpRpoA1 proteins were detected immunologically in the

tagetitoxin-sensitive PEP active fractions. This biochemical

property confirms that the nuclear-encoded RpoA consti-

tutes the core PEP enzyme.

Northern blot analysis (Figure 3) indicated that expression

of the PpRpoA2 gene is regulated tightly in a light-depen-

dent manner, as is the light-responsive gene Lhcb2, whereas

PpRpoA1 is expressed constitutively in both light and dark

conditions. This suggests that the two PpRpoA proteins play

different roles in the transcription of plastid genes. Although

disruption of the PpRpoA1 gene resulted in the slightly

retarded growth of protonemal colonies, expression of most

plastid genes, including psbA, rrn16 or psaA, was not

affected by disruption of PpRpoA1. This implies that

PpRpoA1 is dispensable to plastid function and that

PpRpoA2 plays a central role in plastid transcription. An

alternative possibility is that PpRpoA2 merely complements

the loss of PpRpoA1 function. We prefer the first suggestion

because PpRpoA1 disruptants were obtained easily, but

PpRpoA2 was not disrupted.

The most interesting finding is that the expression of

petN, psbZ, ycf3 and several tRNA genes was altered in the

PpRpoA1 disruptant (Figure 5). Of these genes, three (petN,

psbZ and ycf3) can be categorized as PpRpoA1-PEP-depen-

dent genes. Thus the modulation of transcription may be

mediated by two a subunits of PEP in the moss Physcomit-

rella. In the wild-type moss chloroplasts the PEP enzyme

comprises PpRpoA1 (a1 subunit) or PpRpoA2 (a2 subunit), or

both, together with bb¢b¢¢, and may exist as three isoforms

(a1a1bb¢b¢¢, a1a2bb¢b¢¢ and a2a2bb¢b¢¢). In contrast, in the

PpRpoA1 disruptant, PEP exists presumably as a uniform

complex of a2a2bb¢b¢¢. The lower petN, psbZ and ycf3

transcript levels in the PpRpoA1 disruptant indicates that

the three genes are transcribed predominantly by PpRpoA1-

PEP (a1a1bb¢b¢¢). Interestingly, transcript levels of some tRNA

genes were very low in the light and high in the dark

(Figure 5e). The transcript levels of those tRNA genes were

enhanced significantly by PpRpoA1 disruption even under

light conditions (Figure 5e). This might be caused by over-

expression of PpRpoA2. Perhaps their transcript levels may

be modulated by different stability under diurnal day and

night control in the disruptant.

In E. coli the a subunit is required for transcription

activation by protein factors, and for interaction with DNA-

activation elements (Kimura and Ishihama, 1995). Sigma

factors are the most important determinants for the selec-

tion and initiation of transcription of plastid genes (Shiina

et al., 2005), and recognize the promoter consisting of –10

and –35 elements. PpRpoA proteins must interact with some

r factor bound to the promoter. To compare the promoter

sequences of the PpRpoA1-dependent or -independent

genes, we used a primer extension experiment to identify

putative transcription initiation sites of the moss plastid

genes (Figure S3). As shown in Figure 7, PpRpoA1-indepen-

dent genes have canonical –35 and –10-like elements, and

PpRpoA1-dependent genes (petN and ycf3) also have a

canonical –35 element and an extended –10 sequence,

GAT(G/A)TATATA(T/A)AT. The other putative PpRpoA1-

dependent gene, psbZ, has a sequence TCGGCCA that is

also found in the upstream region of the ycf3 ) 253. Among

the three Physcomitrella plastid r factor genes, the PpSig1

Figure 6. Semi-quantitative reverse transcriptase-polymerase chain reaction

analysis of PpRpoA1, PpRpoA2 and plastid r factor transcript levels in the wild

type and the PpRpoA1 disruptant. Total RNA was extracted from the wild type

or the PpRpoA1 disruptant of 4-day-old protonemata grown under continuous

illumination (4 L), 4-day-old protonemata treated for 1 day in darkness before

harvesting (3LD) and 3LD protonemata treated for a further 1 day under light

conditions (3LDL), reverse-transcribed, and amplified by PCR using a primer

set specific to each PpRpoA, PpSig or PpActin3 gene.



and PpSig2 genes are expressed throughout the day, and

their fluctuations suggest very low amplitude diurnal

rhythms of mRNA levels. In contrast, PpSig5 mRNA showed

a very high amplitude diurnal rhythm with peaks observed in

the light phases (Ichikawa et al., 2004). Although similar

results were also observed in this study (Figure 6), these

plastid r factors are unlikely to be responsible for the

alteration of plastid gene expression in the disruptant.

However, we cannot exclude the possibility that alternation

of protein levels or phosphorylation states of PpSigs influ-

ences the steady-state transcript levels of plastid genes in

the disruptant. Therefore, we hypothesize that different

combinations of the two a subunits with a certain r factor

(rather than PpSig1, PpSig2 and PpSig5), or that some

transcription factors interact with an upstream element or

promoter of each plastid gene to modulate the strength of

transcription activity. PpRpoA2 possesses a portion of DnaK/

HSP70, which may provide an additive function to PpRpoA2

as an a subunit. We speculate that PpRpoA2 protein is able

to facilitate or interfere with the r or other transcription

factors.

Previous studies of tobacco plants have demonstrated

that photosynthesis genes are transcribed by PEP, that some

plastid genes such as atpB and rrn16 are transcribed by both

PEP and NEP, and that most housekeeping genes are

transcribed by NEP (Hajdukiewicz et al., 1997). A relative

increase in NEP activity was observed in PEP-deficient

tobacco (Krause et al., 2000; Legen et al., 2002). We did not

construct PEP-deficient mosses in this study, and therefore

we cannot conclude whether NEP exists in the P. patens

chloroplasts. Double knock-out mutants of PpRpoA1 and

PpRpoA2 are needed to address this question. Alternatively,

disruption of plastid genes rpoB, rpoC1 or rpoC2 may also

help address this issue. We attempted to construct plastid

rpo gene knock-out mosses, but we obtained only hetero-

transplastomic lines, which possessed both the wild-type

plastomes and the rpo gene-disrupted plastome (unpub-

lished data). In the moss P. patens, the haploid gametophyte

dominates the life cycle, represented by the filamentous

protonema (juvenile gametophytes) and the leafy moss

plant (adult gametophyte). Plastid ontogeny in mosses

differs distinctly from that in vascular plants (Reski, 1998).

This may be the reason why this moss developed such a

unique system for plastid transcription that is unlike higher

plants.

Experimental procedures

Plant material

Physcomitrella patens (Hedew.) Bruch & Schimp subsp. patensTan was grown at 25�C under continuous illumination at30 lmol m)2 sec)1 on the minimal medium (BCD medium) sup-plemented with 0.5% glucose, 1 mM CaCl2 and 5 mM diammonium(+)-tartrate agar plate as described previously (Sugiura and Sugita,2004).

Isolation and sequence analysis of cDNA

cDNA encoding PpRpoA2 was prepared using primers cA2.F(5¢-TCTCTCCTGCAGGCCTCTTCACCTCTAC-3¢) and cA2.R(5¢-GCCTGTCAGGCTCCATCTCTAAGTGGTTTC-3¢) designed fromsequences of an EST clone (pphf35o11). Sequencing was per-formed with an ABI PRISM 3100 sequencer and the DYEnamicET Terminator Cycle Sequencing Kit (GE Healthcare, http://www.gehealthcare.com) using appropriate sequencing primers.Alignments of amino acid sequences were constructed by theCLUSTALX program, version 1.81 (Thompson et al., 1994).

Figure 7. Alignment of nucleotide sequences of

the region upstream of the 5¢-ends of PpRpoA1-

independent and -dependent gene transcripts.

The underlines indicate the position correspond-

ing to the 5¢-end of the major transcript. The

arrowheads indicate the initiation nucleotide of

mature tRNAs. The r70-type promoter in Escher-

ichia coli comprising TTGACA (–35 element) and

TATAAT (–10 element) are indicated by the gray

box. The conserved element specific to the

PpRpoA1-dependent promoter is boxed.



Construction of the PpRpoA2-GFP fusion gene

and moss transformation

A DNA fragment encoding the N-terminal 125 amino acid residuesof PpRpoA2 was amplified from the cDNA as above with primersA2GFP.F (5¢-CCCGTCGACCACCATGGCAACTGTCATGGGCGC-3¢)and A2GFP.R (5¢-ACGTGTCGACGGCCTTTTCTGCAGCTTCTGTAA-3¢). The PCR product was digested with SalI and inserted into theSalI-cleaved CaMV35S-sGFP(S65T)-nos3¢ (Chiu et al., 1996) tocreate the PpRpoA2-gfp construct. The reporter construct wasintroduced into the protoplasts prepared from the 3-day-old proto-nemata. As a positive control, the PpRpoA-gfp construct was alsoused for transformation (Sugiura et al., 2003).

Chloroplast isolation and anion

exchange chromatography

Intact moss chloroplasts were isolated from 4-day-old protonemalcells as described previously (Kabeya and Sato, 2005). To preparethe chloroplast lysate, intact chloroplasts were resuspended inbuffer 1 (10 mM Tris–HCl, pH 8.0, 1 mM EDTA, 5 mM DTT, 1 M KCl)and incubated for 15 min on ice. An equal volume of buffer 2 (50 mM

Tris–HCl, pH 8.0, 10 mM MgCl2, 2 mM DTT, 20% sucrose, 50% glyc-erol) was added to the lysed plastids, and (NH4)2SO4 was added tothe plastid lysates to a final concentration of 10%. The mixture wasstirred for 30 min on ice and then centrifuged at 50 000 g for 1 h.(NH4)2SO4 was added to the supernatant to a final concentration of60%. The pellet was resuspended in buffer 3 (20 mM HEPES, pH 8.0,100 mM KCl, 12.5 mM MgCl2, 2 mM EDTA) and dialyzed with 10volumes of buffer 3 containing protease inhibitor cocktail (Roche,http://www.roche.com) for 16 h. The supernatant was loaded onto aMini-Q FPLC column (GE Healthcare). The column was washed withbuffer 3, eluted with a 30-ml liner gradient of 0.1–0.4 M KCl, and thento 1.0 M KCl, and fractions (1 ml) were collected.

Measurement of transcription activity

Transcription activity was measured as incorporation of [a-32P]UTP.The reactions were carried out in a total volume of 60 ll containingtranscription buffer (60 mM Tris–HCl, pH 8.0, 10 mM MgCl2), 80 URNase inhibitor (Takara, http://www.takara-bio.com), 2% glycerol,2 mM DTT, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 5 lM [a-32P]UTP(at a specific radioactivity 0.16 GBq lmol)1), 6 lg denatured calfthymus DNA as a template and 10 ll dialyzed supernatants of eachfraction, as described above. The reaction mixtures were incubatedin the presence or absence of tagetitoxin (10 lM) for 30 min at 25�Cand then 10-ll aliquots were spotted onto DEAE paper (DE-81;Whatman, http://www.whatman.com). After successive washingwith 5% Na2PO4, water and ethanol, the radioactivity was deter-mined by scintillation counting.

Preparation of recombinant PpRpoA proteins

and Western blot analysis

A 1068-bp DNA fragment encoding the amino acid residues 90–450of PpRpoA1 were amplified from the PpRpoA1 cDNA (Sugiura et al.,2003) using specific primers rA1.F (5¢-GACGTACTAGCTT-GGACAAAAGCT-3¢) and rA1.R (5¢-ATTGCATAATGGATTGTTCT-CAG-3¢). The PCR product was inserted into the expression vectorpBAD/Thio-TOPO (Invitrogen, http://www.invitrogen.com) and theresultant plasmid pBAD-A1 was obtained. A 1146-bp DNA fragment

encoding the amino acid residues 143–525 of PpRpoA2 was ampli-fied from the PpRpoA2 cDNA (this study) using specific primersrA2.F (5¢-AGTAGATCTACTACCACTGCGGACGGACCCATG-3¢) andrA2.R (5¢-AGTGTCGACCTACTACGTCCTGCAGTGACTTTGCAG-3¢).The PCR product was digested with SalI and BglII, and inserted intoBamHI and SalI sites of the expression vector pQE-30 (Qiagen, http://www.qiagen.com) and the resultant plasmid pQE-A2 was gener-ated. pBAD-A1 and pQE-A2 was transformed into E. coli LMG194and M15/pRep4 cells, respectively. The overexpression and purifi-cation of thioredoxin-tagged PpRpoA1 protein (Thio-PpRpoA1) orHis-tagged PpRpoA2 protein (His-PpRpoA2) with Ni-NTA agarosewas carried out according to the manufacturer’s instructions. Puri-fied recombinant proteins were used to immunize rabbits. Poly-clonal antisera were obtained and used in the immunoblot analysis.For immunoblot analysis, sodium dodecylsulphate–polyacryl-amide-gel electrophoresis and blotting were carried out using a 10%polyacrylamide gel as described previously (Kabeya et al., 2002).Anti-tobacco chloroplast RNA-binding protein cp28 antibody wasused as the control (Nakamura et al., 1999).

Isolation and gel-blot analysis of DNA and RNA

Total DNA and RNA were isolated from the protonemata asdescribed previously (Hattori et al., 2007). RNA was extracted fromthe protonemata grown under different light and dark conditions, asindicated. For Southern blot analysis, DNA was digested withrestriction enzymes, separated on 1% agaraose gel, and blottedonto a Hybond N+ membrane (GE Healthcare). The membrane washybridized for 15 h at 65�C with a [32P]-labeled DNA probe andwashed at 65�C. For RNA gel-blot analysis, total RNA (15 lg) wassubjected to electrophoresis in 1.2% formaldehyde-containingagarose gel, and transferred to Hybond N+ membranes. Hybridiza-tion and detection were carried out as described using digoxigenin-labeled DNA probes (Kabeya et al., 2002). As plastid gene-specificDNA probes, DNA fragments spotted on the moss plastid DNAmicroarray (Nakamura et al., 2005) were used. A PpRpoA1 cDNAprobe (a NotI-digested 1414-bp 5¢ RACE-cDNA clone; Sugiura et al.,2003) and an nptII gene cassette probe (a HindIII-digested fragmentfrom pMBL6, a gift from Dr Jesse Machuka of the PhyscomitrellaEST Programme) were used. A PpRpoA2 probe was amplified withthe primers rA2.F and rA2.R, and a Lhcb2 probe was prepared usingPCR with primers Lhcb2.F (5¢-TAACGGTGAGTTCGCTGGTGAC-3¢)and Lhcb2.R (5¢-GTTCATGTCAATAGTCTAGTTC-3¢).

Construction for PpRpoA gene disruption

and moss transformation

A 2511-bp region containing the PpRpoA1 gene was amplifiedfrom P. patens genomic DNA with A1_KO.F (5¢-GTTAACAAAA-CATACAATGTAAAG-3¢) and A1_KO.R (5¢-AATGCGGTGG-TAAACTGGTCTCTG-3¢), and cloned into the pGEM-T Easy(Promega, http://www.promega.com) to generate pYK-DA1. Thechimeric nptII gene cassette from pMBL6, which consists of thecauliflower mosaic virus 35S promoter, the nptII coding region andthe 35S terminator, was excised as a 1961-bp HindIII fragment. ThenptII cassette was inserted into the HindIII site in the exon 2 of thePpRpoA1 gene in pYK-DA1, either with the same (pYK-DA1-1) oropposite (pYK-DA1-2) direction of transcription as PpRpoA1. Forconstruction of the PpRpoA2 disruptant, a 4440-bp DNA fragment(AB293563) was amplified with A2_KO.F (5¢-ATGCGGCCGCTTGT-AGATGATAATACCTCAATTCCGA-3¢) and A2_KO.R (5¢-ATGCGGCC-GCGCAAATAGTAAGACGTCCAGTAAGA-3¢). The PCR fragment



was digested with NotI and cloned into the NotI site of pBlueScriptIISK+ to generate pYK-DA2. The nptII cassette was inserted into theNcoI sites in the exon 2 to exon 4 of the PpRpoA2 gene in pYK-DA2.

Transformation was performed essentially according to theprocedure of Nishiyama et al. (2000). NotI-BstXI-digested pYKD-1or NotI-digested pYKD-2 (30 lg) was incubated at 45�C for 5 minwith protonemal protoplasts in polyethleneglycol (PEG) 6000. PEG-treated protoplasts were incubated at 25�C in the dark, and then inprotoplast regeneration medium for 3 days under continuous light.Regenerated protoplasts were transferred to BCDATG mediumcontaining 50 lg ml)1 geneticin (G418) to select transformants. Forverification of PpRpoA1 disruptants, PCR was performed using P-F(5¢-GTGAGAGGATTGAGACTGGTG-3¢) and P-R (5¢-TAGCCATAGA-TCAATAAAACAACC-3¢). For verification of PpRpoA2 disruption,PCR was performed using A2C.F (5¢-TAAGAGGAATTCGACTGTAG-TTGCG-3¢) and A2C.R (5¢-CGTTTGTGTGATCAATCATCCACG-3¢).

Microarray analysis

Plastid DNA microarray analysis was performed as described pre-viously (Nakamura et al., 2005). Fluorescence cDNA probes weregenerated by direct incorporation of Cy3- or Cy5-dUTP (GE Health-care) during reverse transcription. Briefly, 10 lg of plastid RNA fromwild-type and PpRpoA1 disruptant mosses was annealed with amixture of 216 plastid gene primers (1 pmol each) at 70�C for 5 min.Reverse transcription, purification of cDNA probes, hybridizationand washing were performed as described by Nakamura et al.(2005). Fluorescent images were visualized and analyzed withGENEPIX 4000 and accompanying software (Axon Instruments,http://www.axon.com).

Semi-quantitative RT-PCR

DNA-free total cellular RNA (5 lg) was reverse-transcribed usingSuperScript II (Invitrogen) with oligo(dT)15 primer, and first-strandcDNA was synthesized as previously described (Aoki et al., 2004).PCR was performed using the first-strand cDNA and appropriateprimer set as follows: A1-RT.F (5¢-TGGTCTCTGCTATAGAAGGT-TCGAATTCTA-3¢) and A1-RT.R (5¢-CTCATCAGAGTCACTGCGGAT-CACAAGCTC-3¢) for PpRpoA1, A2-RT.F (5¢-TGAAGGACAGTCAA-TCCGTACTGAGGCTTA-3¢) and A2-RT.R (5¢-AATGGAGATACGGC-AAATCGAGCGTAATGC-3¢) for PpRpoA2. The primers for PCRanalysis of PpSig (Ichikawa et al., 2004) were as follows:5¢-AAATCCGGCAGTCCGTCTGCTCGT-3¢ and 5¢-ACTGATGCTCTCT-AGTGACA-3¢ for PpSig1, 5¢-GTTGAATTGGATACAGAGGCT-3¢ and5¢-GCTCCTGAACCAGCATTCGCTTTG-3¢ for PpSig2, 5¢-CAAGTGGC-TGAGGATCAGCAAGT-3¢ and 5¢-TTGGCGCGTTGGATATTCACTCT-3¢ for PpSig5. As a control, an actin3 gene sequence was amplifiedusing primers (5¢-CGGAGAGGAAGTACAGTGTGTGGA-3¢ and5¢-ACCAGCCGTTAGAATTGAGCCCAG-3¢, Aoki et al., 2004). A cycleof PCR consisted of 30-s denaturation at 94�C, 30-s annealing at 55�Cand 40-s extension at 72�C. The optimal cycle number of PCR was30 cycles for PpRpoA1 and PpRpoA2, 32 for PpSig1, 35 for PpSig2and PpSig5, and 26 for PpActin3. PCR products were subjected to2% agarose gel electrophoresis.

Acknowledgements

We thank Dr Jesse Machuka for pMBL6 as part of the PhyscomitrellaEST Programme at the University of Leeds and Washington Uni-versity (St Louis, MO, USA). We also thank Dr Shin-ya Miyagishimafor kindly supporting this work, and Dr Takahiro Nakamura and

Dr Setsuyuki Aoki for valuable discussions. This work wassupported by a Grant-in-aid from the Ministry of Agriculture,Forestry and Fisheries (Bio-Design Project), and by a Research Grantfrom the DAIKO FOUNDATION (Nagoya). YK was a recipient ofa Japan Society for Promotion of Science (JSPS) Post-doctoralFellowship.

Supplementary Material

The following supplementary material is available for this articleonline:Figure S1. Chromatography of the chloroplast lysate on MiniQcolumn.Figure S2. Generation of the PpRpoA2 gene disruptant.Figure S3. Mapping of the 5¢-ends of plastid gene transcripts.This material is available as part of the online article from http://www.blackwell-synergy.com.

Supplementary Reference

Sambrook, J., Fritsch, E.F., and Manniatis, T. (1989) MolecularCloning: A Laboratory Mannual, 2nd ed. NY: Cold Spring HarborLaboratory Press.

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