August 1992
Volume 308. number 3, 258-260 FEBS 11439 O 17,92 Federation of Earol.w.an Uioehemieal Societies 0014:~793/92/$~.00
Tobacco chloroplast ribosomes contain a homologue of E. coli ribosomal protein L28 Fumiaki Yokoi and Masahiro Sugiura Center for Gone l~exeareh. Nagoxa Univer#lO', Nagoya 464-01. $ttpa~# Received 12 May 1992; revi~d version received 7 July 1992 The ~nes for ribosomal proteins I ~ and L33 constitute an opcron (rpm/~63 in ~'. chit. but in plant ~loroplasts L33 is en¢od~ by tl'm chloroplast DNA and L28 s~ms to be e~oded by the nuclear Ilenom¢, A 15 kDa protein was i~iated from the ~0 S subunit of ~bae.¢o chloroplast ribo~rrmc and its N.t©mfinal amino acid sequence was determined, A eDNA for this protein was cloned and analyzed, The eDNA enood¢~ a 151 amino add protein consistinil of a predicted transit.pcptide of 74 amino acids and a mature protein of 77 amino acids, Tlu~ mature protein is homolog,ou~ to E. cull L28, hen~ we named it chloroplast I..2tt (CL28). This is the first report on the preu:n~ ofnn B, ¢oli L.2Z.like protein in another ori~nism. Chloroplast Ribosomal protein: CI.28; Tobacco
I. I N T R O D U C T I O N Chloroplast ribosomes are 70 S in size similar to those of E. coli and contain 3-5 different rgNAs and about 60 ribosomal protein species (reviewed in [1,2]), Determination of the entire chloroplast DNA sequen~ and analysis of chloroplast ribosomal proteins revealed the pre~ence of 21 chloroplast loc". for r:bosomal proteins in tobacco [3-5], including rp/33 encoding the ribosomal protein L33 (CL33), This gone has been 1o :alized in the large single copy region of tobacco chloroplast DNA. based on its homology to E. coli L33 [3]. However no O R F corresponding to £. coli ribosomal protein L28 has been found in tobacco chloroplast D N A , although in E, coli the genes encoding L28 and L33 o~ur together in a single operon O'pmBG) [6]. Since E. coil mutants missing L28 are viable [.7], it is possible that chloroplast ribosomes lack an E. coil L28-type protein. The primary structure of L28 has only been reported in E. coil [8]. To ch~k whether the gone encoding chloroplast L28, if there is one, is encoded by the nuclear genome, we have attempted to isolate the corresponding chloroplast ribosomal protein from tobacco leaves and to clone its eDNA. Sequence analysis revealed in fact the presence of an E, coli L28-type protein in tobacco chloroplast ribosomes, suggesting that L28 has a function in ribosomes.
Correspondence address: M, Sugiara, Center for Oene R~earch, Na. goya University, Chikusa, Nagoya, 464.01, Japan, Fax: (81) (52) 782 3849,
258
2. MATERIAI.S A N D METHODS 2.1. [xalalimt of rilmsm.,I prateitt CL28 Chloroplast ribosomal subunits *.,.'ere prepared from mature to. bacco ieav~ (Hlrolia.tt ralmr~m!vat'. Bright Yellow 4) by a l~,~viourJy reported method [9]. Ribosomal proteins were fractionated by reverse. pha.~ chromatography (Pharnmei.'t FPLC PRO.RPC HRS/10) fol. iowr.d by SDS.polyacrylamidc l~l el¢ctrophore~h; (details will I~: pub. lishcd elsewhere). Alter being ¢l¢¢troblotted on a polpAnylidene di. fluoride membrane, tl'm proteins ~:ra stain©d with Coomaraie R3S0 stain (Pharmacia). A 15 kDa protein band was cut out and its N. terminal amino acid sequeru:¢was deterrninexi with a gas.phat¢ pro. rein ~¢quen~r (Applied IliosyRemz 490A.120A).
t OL
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GGAAtqAAGTC ~AAC A GGGCAA.ACAA 0G ' ~ 6 " ~ C J ~ ' r e A KA ¢¢A ¢ A~ e ACA A A AAA~rTG¢ K K It 11 R & K Y S II It X H K T K K I. Q
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31], AAT'TT~TA A A¢¢ T'r¢'.,AATA¢AA G.41:~ATT'TaG ~ ( ~ . * C A ~ ¢ AA~'.C~rTA'~9~'TGA AA¢ F V rl I. Q Y R I ~ ~ g A G K It Y Y It t,
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~CACT~AT'~TT~ATGCC~CAAAAA~AC~AC~AA~A~ ]; ]i~CA~'~G~r~TT~eA~
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1.41 tSi
Fig, 1, Nucleotid¢ and deri~l amino acid sequences of a eDNA encoding tobacco chloroplast ribosomal protein CL28. The triangle indicates the proem.sing site. The asterisk rcprc~:nts the stop eodon, The conrer,'ed sequen¢.¢ flanking the start ¢odon in dlcot plant,~ is uad:rlined,
Publiahed by Elset,/¢r$ci¢nc¢ Pul)llshersB, i/,
Volume 308, nurn~r 3
FEBS LETTERS l
iO
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Ausutt 1992
40
60
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¢LIB
RRICFicTGIf0KINRAI~IwgHgNW'rlU~LGiCVN IAWI¢~;WWlCAGKg~/I~LRLS?g/LIKTI gKNOLDAVASICAGZD~|KI¢ It
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Fil, 2, Comparison of the predicted amino acid ~.qucnc¢ of tobacco chloroplast ribosomal protein CI.2~ (CL28) with that of E, coal ribolmrnal protein L28 (EL28), Asteri~a indicate identical a~ino acidl. The N.tcrmina! amino acid u~;iuenra of the tTB protein ~ ~own above C!.,28.
2,2, Cteni~&and ~¢qu¢;leh~ of the eDNA encodin~ CL2¢ A H, ta&tt'um leaf eDNA library wai ¢otlttt'ucled by Dr, R,F,
Whittier usin8 at eDNA synth~is kit in ,,!,8t10 (Amertham), An olillon;tfl¢otide probe ($'.TT(TC)TTICCIGT(AG)AAIGGI(GC)(AT)IATIC-Y) was syntht~i~..d usin8 an Appli~ Bios~tems 318A synthe.
sizcr basedon the amino acid sequence (KIXPFTGKK). Screcniol of the libra~ was carried out accordinl to the instruction manual from Amcnharn (eDNA synthesis kit ABtI0)and [10], TI~ hybridization tempemtt~'~ was 41PC, Recombinant ADNA ~ pmiatrcd by a minilynte method [l 1], The inert was d i ~ t e d with E¢oRi and subeloncd
3
E.coli
O
Q,-3 O 3:
0
-3 0
40
80
Residue Number Fitl, 3, Hydropathy plots of tobacco CL2B and E, coli L28, Hydropathy was ~lculatcd with an 1 I.point window according to Kyte and Doolittle [t6].
259
Volume 308. numl~r 3
FEBS LETTERS
into the Bluescript plasmid. SK+ (Stratallen¢). Th¢ nucleotid¢ r~qucn¢¢ was d¢temlined by the did¢oxy ¢hain termination method [12] using dca~a.lcqucnu~ sequencing kits (United Slat~ Biochemical).
3, RESULTS A N D DISCUSSION
3,1, Isolation of ribosomal proteh~ CL,28 Reverse-phase chromatography of 50 S ribosomal proteins from tobacco leaves produced at least 12 sharp peaks (details will be published elsewhere), The fourth peak was resolved t'urther into 4 bands by SDS-PAGE. A 15 kDa protein (band 17B) ca the membrane was subjected to protein microsequencing. Twenty-one amino acids of the N.terminus were analyzed and 19 of these residues were clearly identified (Fig. 2), 3.2. Sequ¢,ce of'the CL2t] eDNA A eDNA clone was isolated using an oligonuclcotide. which was synthesized based on the N-terminal amino acid sequence of the 15 kDa protein. This e D N A was sequenced and contains an insert of 671 bp with a reading frame of 151 amino acid residues (Fig. l), This reading frame contains three possible initiation codons (residues l. 4 and 15). The first A T G codon occurs in the sequence A A A A A T G G C , which matches the consensus sequence flanking functional start ¢odons in dicot plants [ 13]. This suggests that the first A T G codon is the functional initiation codon. The 21 amino acids in the 15 kDa protein which were determined by amino acid sequencing match the predicted sequence of residues 75-95. Thus, the mature protein is 77 amino acids long. The preceding peptide is probably 74 amino acids in length, of which 38 are hydrophobic (51%) and 9 ar¢ basic (12%). This motif is similar to the amino acid composition of other transit pcptidcs [14], suggesting that the pre~ding sequen~ is a transit peptid=. The size of the predicted mature protein is the same as that of E. coli L28 (77 amino acids). It is a basic protein (12 lysinc residues out of 77) and has a calculated molecular weight of 8924. Discrepancies between observed and calculated molecular weights are often found in ribosomal proteins (e.g. [4]), Th= mature protein has 36% amino acid identity to E, cots L28 (Fig. 2) and their hydropathy profiles arc similar (Fig. 3). Therefore, we conclude that the chloroplast protein is a homologue of E.coli L28 and designate it CL28. The function of L28 is not clear because mutants which lack L28 are viable in E. co[i [7]. The primary
260
August 1992
structure of L28 has only been reported in E. coil [8]. The tobacco CL28 sequence is the second report of'this protein. Rat and yeast l~Ss have no homology with E, carl L28. Based on the conserved structure between E, coil I28 and tobacco CL28. L28 is thought to have a function as yet unclear ir~ ribosomes. In E, coil the L28 gen¢ (rpmB) is cotranscril:~d with the L33 gent (rpmG) [6]. in land plants the CL33 sene is located in the chicroplast genome ill while the CL28 gene is in the nucleus, This suggests a chloroplast to nucleus gene relocation during evolution (e,8, [l 5D, ..lrk,~,'lcdj~¢,w,ls: We thank Dis. M. Sugita. T. Waka~.~gi -~ndY. Li I'or discussions and u~ful SUSllmtions. Ms. C. Sugi|a for techni~l
assistance :rod Dr, R.F. Whilti¢~ for the tobacco leaf eDNA library construction. We al~ express our fltank,: to Dr, R. l.[¢rrmafm for critical rcadin~ of 1h¢ m,,nuscrip|,
REFERENCES [ll Susiurn, M. (19119)Annu, Rev. Cell i~;,a~.S. $1-70. [2l M.ch¢. R, (1990) Plant $ci. 72, 1-12. [3] Shinozaki, R., Chine. M,. Tanaka, M,, Wakasugi. T,. H.yashida, N,. Mutsubayashi, T,, Zaita, N,, Cltunwongse, J.. Obokata, J,, Shinoxaki. K.Y.. Ohio, C,. Toruz,'twa, K., Mcns, O,Y.. Sugita, M., Deno, H.. Kamotmshira, T,. Yamada. g,, Kusuda, L, Takaiwa, F., Kate, A,. Tohdoh, N,. Shimuda. H, and SUlliura. M. 0986) EMBO J S, 2043-2049. [4] Yokel, F.. VaHilcvu, A.. Hayashida, N,. Torazawa, K,, Wakasugi. T. and SuBiuPa. M. (1990) FEBS Left. 2"/6. I~K-g0, iS} Sugiura. M.. Tora=wa. K. and Wak-SulBi, T., in: Translational Apparatus of Phn|osynth¢fi¢ Organcll=s (R. Mach¢, E, Stutz and A.R. Subramanian. Eds,), Springer, Berlin, 1991. pp, $9-69. [6] Late,J,S., An, G,, Frie,',en, J,D, and l~no, K, (19~1)Mot, Gen,
G=net. I~, 21~-223. [7] Dobbs. E.R. ¢1979)J. l.]actcriol. 140,7341-737, [tq Wittmann.Liehold, B, and Marzinzig, E, 09?7) FEllS l.¢tt, xl, 214-217. [9] Cap¢l, M.S. and Bourque. D.P,(1982).I, Uiol. Chore,257,"/746'/?$3. [10] Wallace. R.B. nnd Miyada. C.G. 098-/) MethodsEnzymol. 152. 432-442. [I I] Manfioletti, G, and Schneider,C. (1988)Nucleic Acids Rcs. 16. 21173-211~1, [12] Sangar. F.. Nicklcn. S. and Coulson, A.R. (19-/7) Prec. Natl. Acad. Sci, USA 74. 54~3-5467, [13] Cavener. D.R.. :rod Ray. S.C. (Ig91) Nucleic Acids Rcs. 19.318.%
3192, [14] Hcijn=, G,. Hirai, T,, Kl~sgen, R,13. Stcppuhn..I,, Bruce. B., Keclpltra. K. ,rod Hcrrm:,nn. R. (1991) Plant Mol. Biol. Rcp, 9, 104-126, [15] Smookcr,P.M,, Sdu;nidt,J, and Subramani:m,A,E. U991) Bio. ¢himi¢ 73. 845-851. [161 Kyt¢. J. and Doolittle. R.F. (1982)J. Mol. Biol. 157. 10S-132.
Volume 308. number 3, 258-260 FEBS 11439 O 17,92 Federation of Earol.w.an Uioehemieal Societies 0014:~793/92/$~.00
Tobacco chloroplast ribosomes contain a homologue of E. coli ribosomal protein L28 Fumiaki Yokoi and Masahiro Sugiura Center for Gone l~exeareh. Nagoxa Univer#lO', Nagoya 464-01. $ttpa~# Received 12 May 1992; revi~d version received 7 July 1992 The ~nes for ribosomal proteins I ~ and L33 constitute an opcron (rpm/~63 in ~'. chit. but in plant ~loroplasts L33 is en¢od~ by tl'm chloroplast DNA and L28 s~ms to be e~oded by the nuclear Ilenom¢, A 15 kDa protein was i~iated from the ~0 S subunit of ~bae.¢o chloroplast ribo~rrmc and its N.t©mfinal amino acid sequence was determined, A eDNA for this protein was cloned and analyzed, The eDNA enood¢~ a 151 amino add protein consistinil of a predicted transit.pcptide of 74 amino acids and a mature protein of 77 amino acids, Tlu~ mature protein is homolog,ou~ to E. cull L28, hen~ we named it chloroplast I..2tt (CL28). This is the first report on the preu:n~ ofnn B, ¢oli L.2Z.like protein in another ori~nism. Chloroplast Ribosomal protein: CI.28; Tobacco
I. I N T R O D U C T I O N Chloroplast ribosomes are 70 S in size similar to those of E. coli and contain 3-5 different rgNAs and about 60 ribosomal protein species (reviewed in [1,2]), Determination of the entire chloroplast DNA sequen~ and analysis of chloroplast ribosomal proteins revealed the pre~ence of 21 chloroplast loc". for r:bosomal proteins in tobacco [3-5], including rp/33 encoding the ribosomal protein L33 (CL33), This gone has been 1o :alized in the large single copy region of tobacco chloroplast DNA. based on its homology to E. coli L33 [3]. However no O R F corresponding to £. coli ribosomal protein L28 has been found in tobacco chloroplast D N A , although in E, coli the genes encoding L28 and L33 o~ur together in a single operon O'pmBG) [6]. Since E. coil mutants missing L28 are viable [.7], it is possible that chloroplast ribosomes lack an E. coil L28-type protein. The primary structure of L28 has only been reported in E. coil [8]. To ch~k whether the gone encoding chloroplast L28, if there is one, is encoded by the nuclear genome, we have attempted to isolate the corresponding chloroplast ribosomal protein from tobacco leaves and to clone its eDNA. Sequence analysis revealed in fact the presence of an E, coli L28-type protein in tobacco chloroplast ribosomes, suggesting that L28 has a function in ribosomes.
Correspondence address: M, Sugiara, Center for Oene R~earch, Na. goya University, Chikusa, Nagoya, 464.01, Japan, Fax: (81) (52) 782 3849,
258
2. MATERIAI.S A N D METHODS 2.1. [xalalimt of rilmsm.,I prateitt CL28 Chloroplast ribosomal subunits *.,.'ere prepared from mature to. bacco ieav~ (Hlrolia.tt ralmr~m!vat'. Bright Yellow 4) by a l~,~viourJy reported method [9]. Ribosomal proteins were fractionated by reverse. pha.~ chromatography (Pharnmei.'t FPLC PRO.RPC HRS/10) fol. iowr.d by SDS.polyacrylamidc l~l el¢ctrophore~h; (details will I~: pub. lishcd elsewhere). Alter being ¢l¢¢troblotted on a polpAnylidene di. fluoride membrane, tl'm proteins ~:ra stain©d with Coomaraie R3S0 stain (Pharmacia). A 15 kDa protein band was cut out and its N. terminal amino acid sequeru:¢was deterrninexi with a gas.phat¢ pro. rein ~¢quen~r (Applied IliosyRemz 490A.120A).
t OL
¢4 A.4~4~ ¢ ~ GAT'rTf */'(k4AC CA,~4 A ~,Q A ' t ~
A A~
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CA A A A A~ H A
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CAACAATGGTGGCAGGT&TA^GCCTC&G^C;GACCAGT'qA"~AGT~CAG~C&~ " T H V A G I | L it G P V H S • tl R T F S
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41
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cT'r CCCA ATT~=.AG7GGTC'r ~U4r,~AT'r TC6.A~.'~ C¢¢ h t ~ i # e4TATC'/'TCATCAQCTCCAC S Q ~ II G 6 K | S • T It F | | S S A I'
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31], AAT'TT~TA A A¢¢ T'r¢'.,AATA¢AA G.41:~ATT'TaG ~ ( ~ . * C A ~ ¢ AA~'.C~rTA'~9~'TGA AA¢ F V rl I. Q Y R I ~ ~ g A G K It Y Y It t,
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lilt
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1.41 tSi
Fig, 1, Nucleotid¢ and deri~l amino acid sequences of a eDNA encoding tobacco chloroplast ribosomal protein CL28. The triangle indicates the proem.sing site. The asterisk rcprc~:nts the stop eodon, The conrer,'ed sequen¢.¢ flanking the start ¢odon in dlcot plant,~ is uad:rlined,
Publiahed by Elset,/¢r$ci¢nc¢ Pul)llshersB, i/,
Volume 308, nurn~r 3
FEBS LETTERS l
iO
~,o
$O
Ausutt 1992
40
60
(IO
'tO
I?B
ZR~XPlMrOI~SNRANKV|HSH
gLiB
$ IWP,..QVTOKIqlPVTO~NfiIflA LNA?I~I~P,~LI~NLI'II1"1R7t~/18 B g,Jf W I,RVflAgOl'lRVI DIt~ i DTVLAgLBAliOglg't
¢LIB
RRICFicTGIf0KINRAI~IwgHgNW'rlU~LGiCVN IAWI¢~;WWlCAGKg~/I~LRLS?g/LIKTI gKNOLDAVASICAGZD~|KI¢ It
it
IIII
•
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Illl
Illl
4
1
Ill
lit"
It
Ill
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I
It
II
I
II
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Fil, 2, Comparison of the predicted amino acid ~.qucnc¢ of tobacco chloroplast ribosomal protein CI.2~ (CL28) with that of E, coal ribolmrnal protein L28 (EL28), Asteri~a indicate identical a~ino acidl. The N.tcrmina! amino acid u~;iuenra of the tTB protein ~ ~own above C!.,28.
2,2, Cteni~&and ~¢qu¢;leh~ of the eDNA encodin~ CL2¢ A H, ta&tt'um leaf eDNA library wai ¢otlttt'ucled by Dr, R,F,
Whittier usin8 at eDNA synth~is kit in ,,!,8t10 (Amertham), An olillon;tfl¢otide probe ($'.TT(TC)TTICCIGT(AG)AAIGGI(GC)(AT)IATIC-Y) was syntht~i~..d usin8 an Appli~ Bios~tems 318A synthe.
sizcr basedon the amino acid sequence (KIXPFTGKK). Screcniol of the libra~ was carried out accordinl to the instruction manual from Amcnharn (eDNA synthesis kit ABtI0)and [10], TI~ hybridization tempemtt~'~ was 41PC, Recombinant ADNA ~ pmiatrcd by a minilynte method [l 1], The inert was d i ~ t e d with E¢oRi and subeloncd
3
E.coli
O
Q,-3 O 3:
0
-3 0
40
80
Residue Number Fitl, 3, Hydropathy plots of tobacco CL2B and E, coli L28, Hydropathy was ~lculatcd with an 1 I.point window according to Kyte and Doolittle [t6].
259
Volume 308. numl~r 3
FEBS LETTERS
into the Bluescript plasmid. SK+ (Stratallen¢). Th¢ nucleotid¢ r~qucn¢¢ was d¢temlined by the did¢oxy ¢hain termination method [12] using dca~a.lcqucnu~ sequencing kits (United Slat~ Biochemical).
3, RESULTS A N D DISCUSSION
3,1, Isolation of ribosomal proteh~ CL,28 Reverse-phase chromatography of 50 S ribosomal proteins from tobacco leaves produced at least 12 sharp peaks (details will be published elsewhere), The fourth peak was resolved t'urther into 4 bands by SDS-PAGE. A 15 kDa protein (band 17B) ca the membrane was subjected to protein microsequencing. Twenty-one amino acids of the N.terminus were analyzed and 19 of these residues were clearly identified (Fig. 2), 3.2. Sequ¢,ce of'the CL2t] eDNA A eDNA clone was isolated using an oligonuclcotide. which was synthesized based on the N-terminal amino acid sequence of the 15 kDa protein. This e D N A was sequenced and contains an insert of 671 bp with a reading frame of 151 amino acid residues (Fig. l), This reading frame contains three possible initiation codons (residues l. 4 and 15). The first A T G codon occurs in the sequence A A A A A T G G C , which matches the consensus sequence flanking functional start ¢odons in dicot plants [ 13]. This suggests that the first A T G codon is the functional initiation codon. The 21 amino acids in the 15 kDa protein which were determined by amino acid sequencing match the predicted sequence of residues 75-95. Thus, the mature protein is 77 amino acids long. The preceding peptide is probably 74 amino acids in length, of which 38 are hydrophobic (51%) and 9 ar¢ basic (12%). This motif is similar to the amino acid composition of other transit pcptidcs [14], suggesting that the pre~ding sequen~ is a transit peptid=. The size of the predicted mature protein is the same as that of E. coli L28 (77 amino acids). It is a basic protein (12 lysinc residues out of 77) and has a calculated molecular weight of 8924. Discrepancies between observed and calculated molecular weights are often found in ribosomal proteins (e.g. [4]), Th= mature protein has 36% amino acid identity to E, cots L28 (Fig. 2) and their hydropathy profiles arc similar (Fig. 3). Therefore, we conclude that the chloroplast protein is a homologue of E.coli L28 and designate it CL28. The function of L28 is not clear because mutants which lack L28 are viable in E. co[i [7]. The primary
260
August 1992
structure of L28 has only been reported in E. coil [8]. The tobacco CL28 sequence is the second report of'this protein. Rat and yeast l~Ss have no homology with E, carl L28. Based on the conserved structure between E, coil I28 and tobacco CL28. L28 is thought to have a function as yet unclear ir~ ribosomes. In E, coil the L28 gen¢ (rpmB) is cotranscril:~d with the L33 gent (rpmG) [6]. in land plants the CL33 sene is located in the chicroplast genome ill while the CL28 gene is in the nucleus, This suggests a chloroplast to nucleus gene relocation during evolution (e,8, [l 5D, ..lrk,~,'lcdj~¢,w,ls: We thank Dis. M. Sugita. T. Waka~.~gi -~ndY. Li I'or discussions and u~ful SUSllmtions. Ms. C. Sugi|a for techni~l
assistance :rod Dr, R.F. Whilti¢~ for the tobacco leaf eDNA library construction. We al~ express our fltank,: to Dr, R. l.[¢rrmafm for critical rcadin~ of 1h¢ m,,nuscrip|,
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