TitleNUCLEOTIDE SEQUENCE ANALYSIS OFCHLOROPLAST DNA FROM A LIVERWORT,MARCHANTIA POLYMORPHA L.( Dissertation_全文 )

Author(s) Fukuzawa, Hideya

Citation Kyoto University (京都大学)

Issue Date 1986-11-25

URL http://dx.doi.org/10.14989/doctor.k3630

Right

Type Thesis or Dissertation

Textversion author

Kyoto University

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INTRODUCTION

CHAPTER 1

CONTENTS

,Molecular cloning of promoters functional in Escherichia coli from chloroplast DNA 7

CHAPTER II Structure and gene organization of the chloroplast genome -~ 19

11-1 Transfer RNA genes

11-2 Genes for photosynthetic polypeptides

II-3 Genes for ribosomal proteins and cf.. subunit of RNA polymerase (rpoA)

37

--- 44

55

11-4 Putative gene ndh3 and unidentified open reading frames --- 61

CHAPTER III Split gene for chloroplast ribosomal protein S12 63

REFERENCES 73

SUMMARY 86

LIST OF PUBLICATIONS 88

ACKNOVILEDGMENT 90

I

DNA

1. coli

IPTG

IR

LSC

.t:1. polvmorpha

00

ONPG

.. ORF

RNA

Rubisco

SO sos sse Tris

bp

kb

.. kd mRNA

rRNA

tRNA

atp

infA

ndh

pet

psa

psb

rbcL

rpl

rpo

rps

rrn

trn

ABBREVIATIONS

deoxyribonucleic acid

Escherichia coli

isopropyl-p-q-thiogalactopyranoside

inverted repeat

large single copy

Marchantia polymorpha L .

optical density

o-nitrophenyl-~D-galactopyranoside

open reading frame

ribonucleic acid

ribulose-l,5-bisphosphate carboxylase/oxygenase

Shine-Oalgarno

sodium dodecyl sulfate

small single copy

tris(hydroxymethyl)aminomethane

base pairs

kilobase pairs

kilodaltons

messenger RNA

ribosomal RNA

transfer RNA·

Gene symbols

genes for subunits of H+-ATP synthase

gene for initiation factor 1

ORFs homologous to human mitochondrial NAOH

dehydrogenase

genes for photoelectron transfer polypeptides

genes for photosystem I chlorophyll ~ apoproteins

genes for photosystem II chlorophyll ~ apoproteins

gene for the large subunit of Rubisco

genes for 50S subunit of ribosomal proteins

genes for subunits of RNA polymerase

genes for 70S subunit of ribosomal proteins

genes for ribosomal RNAs

genes for transfer RNAs

n

INTRODUCTION

The chloroplast is the organelle of plants and green algae that has the

photosynthetic apparatus and its own house keeping machinery. Chloroplasts and

other plastids contain their own autonomously replicating DNA genome that is

composed by double stranded covalently closed circular DNA. In 1960s, DNA molecules

were visualized in chloroplasts by electronmicroscopic analysis (Ris and Plaut 1962,

Sager and Ishida 1963). In 1976~ maize chloroplast DNA was analyzed by rest~iction

enzymes and physically mapped to be a circle of about 140,000 nucleotide pairs

(Bedbrook et~. 1976). This included two large inverted repeat regions (IRA and

IRS) of 22 kb coding for a set of ribosomal RNA genes. These IR regions were

separated by large and small single copy regions (lSC and SSC regions). Chloroplast

DNA of most plants and green algae has chromosomes of this general structure except

for a few legumes (Pisum sativum and Vicia faba) and Euglena gracilis. Pea

chloroplast DNA has no large repeat regions and Euglena gracilis chloroplast DNA has

tandemly repeated regions coding for ribosomal RNA operons. Among chloroplast DNAs

of the inverted repeat type from green plants, one of the smallest size is that of

Marchantia polymorpha (121 kb) with 10 kb IR sequences (Ohyama et~. 1983), whereas

that of Chlamydomonas reinhardii is the largest (195 kb) with 21 kb IR sequences

(Rochaix and Malnoe 1978).

The majority of proteins present in chloroplasts are encoded by nuclear DNA, but

the rest are encoded by chloroplast DNA and synthesized by the chloroplast

transcription-translation machinery. The nucleotide sequences of many chloroplast

genes from various plant species have been determined-(Crouse et~. 1985). The

first chloroplast gene sequence to be determined for a chloroplast protein was that

for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL)

(McIntosh et~. 1980). Since that time, a sizable number of plastid genes for

proteins have been sequenced as summarized in Table 1. However, the complete

sequence has not been determined as yet for any species of plants.

-:-1-

Gene

rbcL

psaA

psaB

psbA

I N

I

psbB psbC

pshO

psbE

psbF

psbG

Table 1. Genes for proteins coded by chloroplast genome.

Protein product

Ribulose-l.5-bisphosphate carboxylase/oxygenase

PS 1. P700 apaprotein

PS I. P70D apaprotein

PS II. ·"32 kd" protein

PS II. P6BO "51 kd" protein PS II. "44 kd" protein

PS I I. "02 ·po 1ypept ide"

PS II. cytochrome b-559

PS II. cytochrome b-559 (URF39)

PS II. G-protei n

Plant source

Maize Spinach Tobacco Anabaena Chlamydomonas reinhardii Synechcoccus Anacycti~ nidulans Rhodospiri1lum rubum Barley Euglena gracilis

·l1aize Spinach r~aize Spinach Nicotian" debneyi Spinach Am"ranthus hybridus Soybean Anabaena Chlamydomonas reinhardii Pea Solanum nigrum Euglena gracilis Nicatiana tabacum Mustard ---Spinach Spinach Spinach Chlamydomonas reinhardii Pea Spinach Spinach Spinach Oenothera hookeri Nicotiana tabacum Wheat ---Spinach Oenothera hooker; Nicotiana tabacum Wheat ---Maize

Reference

McIntosh ei.al. 19BO Zurawski et aT. 1981 Si nozak i &Sugi ura 1982 Curtis & Haselkorn 1983 Oron et al. 1983 ReicheTt~ Delaney 1983 Shinozaki et a1. 1983 Nargang et-al~ 1984 Zurawsk fet 81.1984 Gingrich &HaT1ick 1985 Fisch et al. 1985 Kirsch-et!il. 1986 Fisch e~a~ 1985 Kirsch-et!i1. 1986 Zurawsk~e~al. 1982 Zurawski et aT. 1982 Hirshberg~ McIntosh1983 Spielmann & Stutz 1983 Curtis & Haselkorn 1984 Erickson et al. 1984 Oishi et aT.-- 1984 GoloubTnoff et al. 1984 Keller & Stutz -- 1984 Sugita & sugiura 1984 Link & Langridge 1984 I~orri s & Herrmann 1984 Alt et al. 1984 Holschu~et al. 1984 Rochaix e~a~ 1984 Rasmussen-e~al. 1984 Alt et al.- -- 1984 HolsCiiu~et a1. 1984 Herrmann et aT. 1984 Carrillo et aT. 1986 Carrillo et aT. 1985 Hird et a~ -- 1986 Herrmannet a1. 1984 Carrillo et aT. 1986 Carrillo et aT. 1986 Hird et a~ -- 1986 Steinmetzet .B. 1986

w

Table 1 (continued). ------ ----------Gene

petA

petB petD

atpA atpB

atpE

atpF

atpH .

atpI

rp12

rpl16 rps4 rps7 rpsll rps12 rps14

rps16 rps19

infA tufA rpoA rpoB

Protein product

Cytochrome f preprotein

Cytochrome b6 Cytochrome b6/f complex, subunit 4

H+-ATP synthase, alpha subunit ~-ATP synthase, behta subunit

~-ATP synthase, epsilon subunit

~-ATP synthase, subunit

~-ATP synthase, subunit III

H+-ATP synthase, subunit IV

50S ribosomal protein 2

50S ribosomal prot~in 16 30S ri bos omal protei n 4 30S ribosomal protein 7 30S ribosomal protein 11 30S ribosomal protein 12 305 ribosomal protein 14

305 ribosomal protein 16 30S ribosomal protein 19

-initiation factor 1 Elongation factor Tu

Plant source

Pea Spinach wheat Oenothern hookeri Spinach Pea Spinach Tobacco 11aize Spinach Tobacco Barley Maize Spinach Tobacco Barley Wheat TODacco Wheat Spinach Tobacco Pea

Nicotiana debneYi Spi nach . Spirodela o1igorhiza Maize Eug 1 ena oraci 1 i s· Spinach Euglena gracilis Marcnantia polyrnorpha Spinach Tobacco Tobacco Nic'otiana debneYi Spinach Spinach Euglena gracilis Spinach Tobacco

Reference ------------_._----

Hi lley et a l. Alt et af.­Willey et ~. Tyagi & Herrmann Heinemeyer et al. Phillips & Gray Heinemeyer et al. Deno et al.- -Krebberset a1. Zurawski et af. Shinozaki~ Sugiura Zurawski & Clegg Krebbers et a l. Zurawski et aT. Shinozakietal. Zurawski&Clegg Bi rd et Ii l. Shinozakiet al. Howe et a l~ c­Alt- eta-';­Deneeta1. CozenS-at ~.

. Zurawsk iet a l. Zurawski et af. Pes no et aT.­Subramanian et al. Montandon & Stutz l1ul1er et al. Montandon.~Stutz Umesono et al. Kirsch eta"f:'" Shi n'ozaki et a l. Sugita & Sugiura Zuraws ki et a 1. Zurawski et aT. l-1uller eta1:-'" Montandon ~Stutz Muller lit al. Ohm~ et al~

1984 1984 1984 1986 1984 1984 1984 1983 1982 1982 1983 1984 1982 1982 1983 1984 1985 1986 1982 1983 1984 1986

1984 1984 1986 1983 1984 1986 1984 1984 1986 1986 1983 1984 1984 1986 1983 1986 1986

RNA polymerase 0/ subunit RNA polymerase f3 subunit -----_. ._--'-_. ----------

The chloroplast DNAs from common bean, soybean, the fern Dsmunda and the algae

Chlamydomonas reinhardii have been shown to exist as a 50:50 mixture of two

genetically identical but physically distinct molecules called isomer differing only

for in the relative orientation of their single copy regions (Palmer 1983). In the

case of ~. polymorpha, isomers have been identified by fragmentation using PstI, and

southern hybridization analysis of the chloroplast DNA with cloned DNA fragments as

shown in Fig. 1. 32P-Labeled DNA fragments involved in either single copy region

(probe 1) or inverted repeat (probe 2) hybridized to the chloroplast DNA segments

generated by BamHI or PstI restriction enzyme (Fig. 2). Probe 1 containing a DNA

segment of an inverted ,repeat hybridizea with the four largest fragments (Pl, Pl',

P2'and P2') produced by PstI. On the other hand, probe 2 containing a DNA segment

of a single copy region only hybridized with two fragments (Pl and Pl'). So in the

~. polymorpha chloroplasts two types of DNA molecules exist in an equal amount. It

is interesting to know whether the events on DNA replication have any relationsh,ip

to the formation of isomers. But the mechanisms of flip-flop recombination have not

been clarified yet.

The chloroplast biogenesis depends on the coordinative gene expression of both

the chloroplast genome and the nuclear genome. But the interaction between

chloroplast and nuclear genetiC systems has not been clarified. In this study, the

author has intended to reveal the mechanisms of gene expressio~ of the chloroplast

genome on th~ basis of the molecular biological methods as follows.

Chapter I; Chloroplast transcriptional promoters were cloned and characterized

using the I. coli gene fUSion method. Chapter II; The primary structure of the ~.

polymorpha chloroplast DNA was determined and genes for transfer RNAs,

photosynthetic polypeptides, ribosomal proteins, and 0( subunit of RNA polymerase

were identified. Chapter III; An unique system for gene expression that may be

called trans-splicing was found in the biogenesis of ribosomal protein 512.

-4-

Bam HI Pst I 1 2 1 2

91 -1 I "I PI~I B2- Pl r ~t:.-

94-

1 - Pl-II-I • • - -

• •

Figure 1. Southern hybridization of the probes containing DNA segments of an

inverted repeat region and a single copy region. Chloroplast DNA fragments

digested by BamHI and PstI restriction enzymes were electrophoresed in 0.6%

agarose gels (each left lane), transferred to nylon membrane filters and

hybridized with 32P-labeled probes . Filters were hybridized with the probe

containing the Bg16 segment of a single-copy region (black box in the right

figure and Lane 1) and probe 2 containing the Bg21 fragment of an inverted

repeat (Lane 2) . Autoradiograms are shown right side of each electrophoretic

pattern .

- 5-

Isom~rs i nchloroplastDNA from ~. polymorRha

Figure 2. Isomers and their restriction maps of the chloroplast DNA. kinds of molectlles (form I and II) of the chlot'1oplast DNA are shown.

Two

IR and L5 indiCate inverted-I"epeat-sequences containing ribosomal RNA genes, the gene

for the large subunit Of rlbulose"':-l,~5"'bisphosphate carboxylase/oJ:(ygenase.

respectively.

CHAPTER I Molecular cloning of promoters functional in Escherichia coli

from chloroplast DNA

Chloroplasts have their own transcriptional and translational systems including

unique transfer RNAs, ribosomal RNAs, RNA polymerase and ribosomal proteins

different from those of the cytoplasm. Determination of the locations of

transcriptional and translational regulatory regions on the chloroplast genome would

lead to a basic understanding of the mechanisms of gene expression in chloroplasts.

It has been suggested that mechanisms of gene expression in chloroplasts are similar

to those in I. coli (Kidd and Bogorad 1979. \~hitfeld and Bottomley 1983. Gruissem et

~. 1983, Gruissem and Zurawski 1985). If this is the case, I. coli plasmid vectors

can be used to clone chloroplast DNA fragments containing transcriptional start

signals which can function in ~. coli. Casadaban et ~. constructed a plasmid in

which (3-galactosidase activity was expressed by insertion of exogenous DNA fragments

having promoter sequences, ribosomal binding sites. and translation start codons in

the right frame, and showed that the strength of promoters and ribosome binding

activity can be assayed by measuring the enzyme activity (Casadaban et~. 1980).

In this chapter, the author shows an evidence that some of the chloroplast

promoters can be expressed in 1. coli by using gene fusion techniques with the

~-galactosidase gene. The nucleotide sequence of the DNA fragment with the highest

enzyme activity in ~. coli was determined and the location of the promoter was

mapped on the chloroplast genome. To find out whether the chloroplast DNA fragment

in fact initiates transcription in 1. coli as well as in chloroplasts, Sl nuclease

mapping experiments were done. The mechanisms of gene expression in chloroplasts·

are discussed.

MATERIALS AND METHODS

DNA isolation. restriction endonuclease digestion. and hybridization

Chloroplast DNA was isolated from liverwort, ~ .. polymorphasuspension culture by,

the method describes previously (Ohyama, et~. 1982) with some-modifications.

-7-

Chloroplasts were isalatei fromcu·ltured cells byus'ing blender containing sea sand,

(20-25 mesh). Restriction digestion, hybridization and plasmid DNA isolation were

carried out by the procedure d'escribed by Maniatis et ~.(1982).

Cloning of chloroplast DNA fragments

Chloroplast DNA was cTeaved by EcoRI. Sau3A, BglII, Sma I and BamHI restriction

endonuc"1eases~'Restri cted ch 1 orop 1 ast DNA fragments were 1 i gated into

dephosphorylated EcoRI, ·BamHI and SmaI sites of plasmid pMC1403 (Casadaban et ~.

1980). The ligated DNA was used to transform competent cells of ~. coli strain

MC1061 (~D139, .d{ar-a, leti)7697, AlacX74, illU-, £@}K-, hsr-, hsm-, strA)

(Casadaban and cohen 1980). Transformed cells were selected on lactose MacConkey

agar plates containing 100 }Jg/mg of ampicillin. Red colonies using lactose (lac+)

and resistant· to ampicillin were picked up and cultured in L-broth in the presence

of ampicillin.

f3-Ga 1 actosidaseassay

~. co"; cells harboring recombinant plasmids were grown to 2-5 x 108 cells/ml

and ~-galactosidase activities were assayed as described by Platt et ~.(1972).

DNA sequenci rig . , '

The nucleotide sequence was determined by'the dideoxychain termination method

(Sanger 'et aT. 1977) using M13 phages mp10 and mpl1 (Messing 19B3). For size

markers in 51·nuclease ~appi~g, a HincIl-EcoRI fragment (see Fig. 3) was labeled'

with [o32p]-ATP (3000 Ci/mmol, Amersham) at the 5' terminus by T4 polynucleotide

kinase after alkali phosphatase treatment, and sequenced by the method of Maxam and

Gi 1 bert (1977).

RNA isolation and Slnutlease mapping

Chloroplast RNA' Was extracted from ·chloroplast peliets by phenol chloroform

- -S·,

extraction described by Yamano et ~.(1984). ~. coli total RNA was extracted by the

SDS-hot phenol method and S1 mapping was performed by the procedure described by

Aiba et .5U..(1981) using 5' end labeled EcoRI-Sau3A fragment (105- 6 cpm/)Jg) and 100

}lg of RNA.

RESULTS

Cloning and characterization of chloroplast DNA fragments having promoter

function

Eleven recombinants were selected On lactose MacConkey agar plates containing

ampicillin as red colonies showing fi-galactosidase production in ~. coli. The

(3-ga lactosidase assays of recombi nants' showed a' variety of 1 evels of the act; vities

summarized in Table 1. The recombinants can be gr6uped in three tategories by

comparing with IPTG induced (3-galactosidase activity in ~. coli wild strain W3110:

" a group with a slightly higher level of the enzyme activity such as recombinants

harboring plasmids pMP901-903; 2, a group with a rather low level of the activity

such as recombinants carrying plasmids pMP921, pMP954-956; and 3. a group with a

very low level of the activlty such as recombinants carrying plasmids pMP910-912.

pMP953.

Restriction analysis of recombinant plasmids (pMP901-903) with high levels

(3-galactosidase activity showed that they had the hme 5.1 kb DNA .fragment, which

corresponded to Ec8 fragment (see Fig. 3). To confirm the inserted ,DNA fragment in

the plasmid pMC1403 was generated from.11. polymorphachloroplast DNA and to find its

site on the chloroplast physical maps," plasmid 'pMP903 was lab~led with [d.32p]-dCTP . '. ':.

using the nick translation reaction and hybridized with chloroplast DNA fragments on

nitrocellulose filter (Fig. 1). 32P-LabeledpMP903'hybfidized io th~ c~loroplast

DNA fragments Ba2. Ba4, Ba6 and Ball (Fig. lB). The 1.1 kb BamHI' fragment derived

from the 5.1 kb EcoRI fragment with an additional 10 bp of pMC1403 was recloned into

pMC1403 and the recombinant plasmid was named pMP904 (Fig. 2). Southern

hybridization with chloroplast DNA fragments showed that the inserted DNA fragment

-9'--:"

Table 1. ~~alactosidase activity and copy numbers in clones containing

recombinant plasmids.

{J-Ga Illel osidasc Relative 1-1051 slrains Plasmids aClivity umounts of

(units)" plasmidsh

W3110 (- IPTG) <II W3110 (+ IPTG) 344±28 MCI061 <5 MCI061 pMCI403 <6 1.0

MCI061 pMP901 361 ±20 O.9±O.1 pMP902 328±27 0.9±O.1 pMP903 360±23 O.9±O.1 pMP904< 408±39 I.D± D.I pMP905" 365±20 O.9±0.1 pMP906< 308± 33 1.0±O.1

MCI06J pMP954d 200± 14 1.0±O.1 pMP955d 267± II D.9±O.1 pMP956J 233±24 O.8±O.2 pMP921 253±2D 1.1 ±O.I

MClO61 pMP953'· 49± 7 D.7±D.2 pMP9JD 17± 3 D.9±D.1 pMP911 73± 8 0.9±D.I pMP912 56± II 1.0±O.1

a) Numbers are averages of three experiments and expressed as units per mg of

total protein (Plattet ~. 1972). b) Plasmid copy numbers were measured by the method of Projan et~. (1983)

and expressed as the number relative to pMC1403 in £. coli strain MC1061 as 1. O.

c) Derived from pMP903 (see Figure 2) d) Containing rbcL gene. e) Containing (3 subunit gene of H+-ATP synthase.

-10-

(1.1 kb) of pMP904 hybridized to Ec4 (located on the other IR region), Ec8, BaG and

Ball (see Fig. le). These results and no cleavage ~ite of EcoRl on the Ball

fragment indicate that the cloned DNA fragment originates from the Ba6 fragment and

is located on the large single copy region close to the inverted· repeat (IR) (Fig.

3). As the clone harboring plasmid pMP904 still expressed the high level of the

enzyme activity, the internal HincII-EcoRI fragment was further subcloned into

pMC1403 (Fig.2). The recombinant plasmid, named pMP905, kept its high level of

~-galactosidase production. However, the recombinant plasmid pMP906. which was

constructed by deletion of HincII-AluI fragment (position at 3 to 348) from pMP905

(see Fig. 4), gave slightly less activity than plasmids pMP904 and pMP905 (Table 1).

Nucleotide sequence of a promoter and its downstream region in chloroplast DNA

As the HincII-EcoRI chloroplast DNA fragment inserted' pMP905 appeared to have a

transcriptional and translational start signals, the nucleotide sequence between two

HincI! sites was determined by the strategy' shown in Fig. 3. The nucleotide

sequence of the 1192 bp chloroplast DNA fragment and junction region between the

chloroplast DNA fragment and the lac'Z gene of pMP905 is shown in Fig. 4A and 4B,

respectively.

51 nuclease mapping

The position in the sequence corresporiding to the 51 end of the mRNA was

determined by an Sl nuclease mapping procedure. The 533 bp HincII-EcoRI fragment

(Position at 4 to 536 in Fig. 4A) was labeled at the 51 ends with [~32PJ-ATP and

digested by Sau3A restriction enzyme, generating two fragments, 66 nucleotides long

and 467 nucleotide long. The 32P-labeled 467 bp fragment was hybridized with either

~. £2li RNA or chloroplast RNA at 370C~ Hybridized DNA-RNA molecules were digested

by Sl nuclease and the length of the Sl nuclease~resistant DNA fragment was measured

by electrophoresis with a Maxam-Gilbert sequence ladder generated from the 467. bp

fragment as shown in Fig. 5. The 51 end of the mRNA from both~. coli and

-11-

A B C 1 234 5 1 234 5 1 234 5

3 .-89 3 -B9 3 4 "89 4 Ec4- , -Bg4 _Bg6 -B97 Ba6_

Ec EcB- _

Boll- -- Ball-

-Bg21

Figure 1. Southern hybridization of chloroplast DNA fragments with plasmids

pMP903 (8) and pMP904 (C). Panel A shows agarose gel electrophoresis stained

by ethidium bromide. Lanes 1, 2, 3, 4 and 5 correspond to the electrophoretic

patterns of digests by EcoRI, EcoRI/BamHI, BamHI, BamHI/BglII and BglII

restriction enzymes, respectively.

pMP903

0 s B

1~ Bam HI ! ~

-Z B d igestion

~ y

E H

pMP904 "Os s HnclI, B~l ur

y 'z B digest ion

~tion topMC1403

pMP905 ( Sma!. BamHls ites)

~s 'z B

Figure 2. Construction of plasmids pMP9Q4 and pMP905 from pMP903 containing

promoter regions. Heavy lines indicate chloroplast DNA segments. The

structural gene of ~-ga1actosidase is shown as IZ.

-12-

Ball

Bat.

;/~\ /" "?l\ BaG'

MpCt -DNA , , , '\,

'\ , ,

165

BamHI Ba4 EcoRI Ec8 Hincll

5.1 kb

, , , '\

'\, '\, , ,

'\

Ha He

SOD 1000 --------~p- •

------~.- -.~-----------• •

Figure 3. Location of the DNA fragments having promoter function on the physical map of chloroplast DNA. and the 'sequencing' strategy. IR indicates

inverted repeat region c6ntainihg ribosomai'RNA operon~'including 16S and 23S

rRNA genes. LS and (3 indicate the genes for the large subunit of ribulose-

1, 5-bi sphosphate carbo~yl ase!oxygena'se and for the (3 stibuni~of H+.:...ATP synthase. respectiv~ly. A, E,'Ha, H~ and S iridicate cleavage sites of th~

restriction enzymes AluI, EcoRI, HaeUI, HincII and Sau3A, r~spectively.

Bottom arrows indicate sequencing strategies using the M13 phagesmplO and mpll.

-13-

A IU.nI:II s.uJi\ ,. . -" ~ . -'" . ," " '., .. . . ..

.S." CTT(;"C'C"I'C"I'GMA'%'CiTC'TCATCAT.MMTTCTCTTCT-MA'TAAATACTTAT'%'TTT'lTt'Cc:t'AATC'rAc(:'\'rCATAMnc~TTCAATTTrT'I'tTATCCA.uCMlr:"t'C1'~ 1lC . . '. .. .------, (------. . .. ;.------ ---::-' (----------.

c:rACACC'M';'ACTTCA,"""atT"'1'''fA~cri-rrrrMa..n~T'''GAcAATTTttrAeiT'''~A:.~~::~;AiTrn~~ATi\C;:~~~T~

AluE . . . . .. . '. . . . ,. . .... CJ.t:T>T.TTCA.M.TAC ........ O.CATMAt:TCJ.CAGM~MAG.~~~:~~t:T .... =AI~TCJ.CJ.CAC.TMGt:TCACCt:TMCC

. . .'. /. -3: . . '~T~. . . ~TrI'T1'A'fC"M"~T1\CATAT ... GA~i\AAAG.A..o\MA1"AT'f'G~AA~AMTM.MTCTl'ATAM~ ... 'tCCM.TTM'TAT'CT~r.ArrAA.TAAA~ •• ..:, t ... ~. .--____ ,_ (--____ ... .__ _ ____ ., (--

tMA xlaICAu,)- £.coRI Et::c-Rl

AA:TAT"T~C1r.·TCCATINC1'I;;,:",~AAAGo.tt~A"t"T'CA,.AATTGcr;gMTij!AcACCi'iC#~CA'l·cCllITriA.v.A~-rd"MnCAAG-rMT .... TA'rCT"l"ci ____ MM. . "" ;. & - If - r. I( K P T H H .,.. R 1 tI R f" rf '$ C V H If" to 'J: -,: L 'N S S .rf ISS

(ORr~~11) . . ,D 1'TAT ..... TATWTATAAmi-rrATAT...-rnrni.io.CAM~~~ ... TACAC"TC"i6AA~rnATAMTT~T"GM.i.T ... G~TI.'fO.AA.nCMTT'T~M~ATAA L T I H [ I FYI" L r H Jt, V. K S S'c I 0 c· t. 1: 'X, ,. t 'N 'I R H .R Y' 0 I-' c r 'I{ !:; II! t .-.. • ___ -.:.--. .. ____ '1 ____ •. - +~M_) t ___ •· ... ___ 1- c_

A'I"'i'T~'l"CM-io/..CT"fA.AGiACCCACT"cTrAc:Ac.v.AA.i.ACAATTCcttrATTAto~1:'C.AC1'AT"'GTTHGAWc..r.ATAmi:rrc.'M'rM.u.CACA.Vr.TAAA.w.,..TCGAT --~.pt ""0 V I:: \' P V·L 't' .e X T· 1 R···t.·. to t K ·N· _0 Y S l' -0 V 'PI I D B If: JC 1"·0 l K It V'1 IDRFfiD2~' . " .. .. '

TCi\A~M'l'C'n'.v..,.Gt'1'ATAJ\~AMtAc7cA~T;'OG1''''CO"rucAGGJ.TJ.T'''~AiA.\AC'CiMTC.\riAn.AAAn E-' r r H V ~ Y ~ S ~ .~ ~. II K L P ~ ~ It K It leT r ~. G Y T, V'~ .Y·K R H 11K 1.

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GOA.TC""rocTrAni:rAmCA1TA~"TAAAT~T'M'T""tt,i.,CGTTTT~CA1"ACCTATi .. u-f"'TATCC'ca.tA-CGTTT~T"'T'CC"'GcnAT~CGCCJ.CGCA.CCCCTM·C: "ODO Q S G 't SIP L I" S " 1(.', ....... . 'IC~'O]'~' .. H i\" I R 1 'i 'R' It. Y T peT R n

, HinctI ~ .~. ~ ;,; ~ ~ . . . '.' .,.

CCAI'CtGTAC:CTA.u.Tn'C""TOA.AATAGT"rMA~GCCACAMA.oU.M1'"TAACATA'tMTAMClA"I"";T1'AMAMCcrccAMCAACAGAGGAATCATMCMG1"CMC :3' R S V P ~ f D·C [ V It C ~l·P.~ ~ K L .T· y. N 'K H ! ~ K' C R N' H R ·C [ '1 T S' Q

SO-UkD teaRt Sad B4mHI

MTAT~TCc:A.TCGc:TC.v.TCGnAAAGc.ACea...CTCA;~~G~f"""·TTC""·-:-C:-:=r:-1 :::1:"' .-,"'.cC--C-O--TC-::C--r.I1'T--"!"· .---__ -_-'-::,.-.-:.-:}--; ------. . H Ai!:,., L It. R' P. T H' N' \II R. I .P G· D p V V L

(CU'01'" ." ........ - -.. --'.. -

•••

Figure 4. Nucleotide sequence of a DNA fragment having promoter function

derived from liverWort chloroplast DNA. Nucleotide sequence of the 1192 bp

HincIIfpagme~t (Fig. 3) is shown in (A). Possible stem, ~nd loop 'structure~

are ,sho~n ,by broken. lines with. arrows. Bold vert.icalilrro\,/s indicate the

start sites of ~RNA. trans~riPtipn i~~. coli Cis well as in Ghloroplasts. The

reg; OI1~ of 1~'-35", ,"-10", Shine-Da 1 gCirn9 sequence ( SP) ,and genes for

tRNAIle(CAU)' and,p?"g~l,a~tQsiclase~ are boxed~ Deduted a~;rlO acid sequences of

ORF601. 602. ,and 6Q~ are ~hown undTr,th~. nlicleo~id7 sequencesl:>Y!iingle letter

symbols. Doub.lE1" under.lines indicate termination codons. N-Terminal region of

thefllsed'protein. between' ORF60; ~nd ~-ga'actO!)idasecoded by pMP903-'-.906 i~ shown in (B).

C G 3 ' 5'

+ +

j j 1'\ 123G C TAG 1

-G C T

~1 A G G T A T A

\ l T T A A

A L/

B 5 ' 3 '

Figure 5. S1 nuclease mapping by ~. coli RNA (A) and chloroplast RNA (8) of the

promoter region. The Sl nuclease protected DNA fragments (lanes 1, 2 and 3) were

electrophoresed in parallel with the Maxam-Gilbert sequence ladders. Lane 1 (A),

and lanes 1, 2 and 3 (8) correspond to the concentration of Sl nucleases, 5000, 50,

500 and 5000 units per reaction, respectively. Sl mapping in lane 1 (8) should read

to be A, because of the smiling pattern of the gel electrophoresis. An arrow

indicates the direction of the transcription.

chloroplast was thus mapped on the sequence to be 45-46 nucleotides upstream from

the ATG translational start codon of ORF601 (Fig. 4).

DISCUSSION

From~. polymorpha chloroplast DNA, DNA fragments were cloned functional in I. coli transcription and translation system. Several chloroplast genes have been

cloned into I. coli plasmids. With the rbcL gene, its expression was observed in an

in Yi!rQ coupled transcriptional translational system derived from E. coli. Gatenby

-15-

et al. reported that rbcL genes from maize and wheat chloroplasts were expressed in

I. coli (1981). Kong et al. reported the cloning of promoter-containing restriction

fragments from Nicotiana chloroplast DNA and location of the fragments on the

chloroplast genome (1984).

In this study 11 recombinants were obtained which were selected on lactose

MacConkey plates as red colonies. These clones, howev~r,varied in their enzyme

activity (Table 1). As plasmid copy numbers were not much different in each clone,

those variation may reflect the efficiency of the transcriptional start signals and

ribosome binding activities in the chloroplasts. For ihstance, a recombinant

carrying plasmid pMP954-956 (containing the rbcL promoter) had a rather high level

of the enzyme activity as expected. A recombinant harboring plasmid pMP953

(containing the promoter region of the ~subunit gene of H+-ATP synthase) showed

quite a low level of the activity. These results coincide with the fact that the

mRNA ~ynthesis of the (3 subunit gene is considerably lower than that of rbcL gene

(Shinozaki et~. 1983). Therefore, the efficiency of I. coli transcriptional

system may reflect that of the transcription in chloroplasts.

The plasmid pMP905, carrying a promoter region of an unidentified open reading

frame named ORF601, gave the highest level of enzyme activity in £. coli. Analysis

of the nucleotide sequence of the promoter and its downstream region revealed that a

translational initiation codon (ATG) of ORF601 was found 38 bp upstream from the

EcoRI site and its open reading frame was fused to the lac'Z gene in the right frame

(see Fig. 4B). And 12 bp upstream from ATG codon, a sequence (TAAaaAG) partially

complementary to the 3' end of £. coli 16S rRNA (Shine-Oalgarno (SO) sequence)

(Shine and Oalgarno 1974) was found. There was a typical sequence for the

transcriptional promoter signal (TATAAT), called the Pribnow-box(Pribn"ow 1975) or

"-10" region at 53 bp upstream from the ATG codon. There was an unique sequence

(aTTGAat) at 82 bp upstream; called the "-35" region which is thought to be a RNA

polymerase recognition site ih I. coli (Takanami et ~. 1976). In addition, three

possible stem-loop structures can be formed between the "-35" region and SO-like

-16""-

sequence as indicated by underlining with arrows in Fig. 4A. But at the position

493-566 a tRNA gene, whose anticodon was CAU, was identified by forming typical

secondary structure as shown in Fig. 6. This tRNA gene has 94.6% homology (79/84)

with spinach chloroplast isoleucine tRNA gene (Francis and Oudock 1982) located in

IR regions. So this tRNA gene was confirmed to code isoleucine tRNA inM.

polymorpha chloroplasts. This highly active promoter may be for tRNAI1 e(C*AU) and

for the downstream ORFs in the LSC region. Three open reading frames were

identified downstream from the promoter region, and designated ORF601, ORF602 and

ORF603. Their possible gene products were estimated to be 73, 91 and >52 amino acid

residues, respectively. A typical SO sequence (AGGAG) was found seventeen bp

upstream from the ATG codon of ORF602, and a stem structure can be formed between

tOhe SO sequence and ATG codon as reported in tb"e Chlamydomonas rbcL gene (Oron et

~. 1983). This stem structure may have a role to let the SO sequence close to the

ATG codon. ORF602 and ORF603 were identified to be putative genes for proteins

corresponding to I. coli ribosomal protein L23 and L2, respectively. Detail data

are presented in chapter II.

Nucleotide sequence analysis revealed that the organization of this chloroplast

promoter was similar to that of I. coli promoters. Furthermore, these results of 51

mapping using in vivo transcripts from both I. coli and chloroplasts showed that the

transcription starts at almost the same position downstream from the promoter

region. So the gene fusion method described °here could be a powerful technique to

clone and characterize the promoters on the chloroplast genome.

-17-:-

A (3') g A

·(5') G-C C-G A-U U-A. C-G C-G A-U U

U UGUCC U A UA G I I I I I A

G AGUCG ACAGG U C G II leu

AAAGC U UU A . U

C CG A C-G A C-G A-U A-U

C A U A .C A U

Figure 6. Secondary structure of !!. polymorphachloroplasttRNAlle(C*AU)

.deduced from the DNA sequenc~. The nucleotides GGA .at the 3' terminus are not

encoded by the chloroplast genome. C* is putative hypermodified base.

~18-

Chapter II Structure and gene organization of the chloroplast genome

To understand the genetic system in the chloroplast, the nucleotide sequence of

the liverwort, ~. polymorpha chloroplast DNA was determined. The~. polymorpha

chloroplast DNA has been physically mapped previously (Ohyama et ~. 1983). The

gene for the large subunit of ribulose-1.5-bisphosphate carboxylase/oxygenase has

been mapped on the chloroplast genome by heterologous .hybridization with tobacco

rbcL gene. The genes for ribosomal RNAs: 235, 165, 5S and 4.5S have been also

localized in the inverted repeat regions (Ohyama et al. 1983,Yamano et~. 1984,

Yamano et~. 1985). The overall gene organization deduced from the complete

nucleotide sequence is described by Ohyama et~. (1986). In this study, properties

~nd characterization of genes on the LSC region (from psbG to 16S .rRNA gene: 30,600

bp) deduced from the nucleotide sequence are presented and discussed. The region

analyzed in this study is shown as a black box with physical ~aps in Fig. 1. In the

region described here, putative genes for seven tRNAs, ten photosynthetic

polypeptides, thirteen ribosomal proteins and 0( subunit of RNA polymerase were

identified. In addition, an open reading frame (ORF) was found to show significant

amino acid sequence homology to a subunit of NADH dehydrogenase.in human

mitochondria.

MATERIALS AND METHODS

Chloroplast DNA was isolated from cell suspension culture of ~. polymorpha as

described previously (Ohyama et !l. 1982). Chloroplast DNA was cloned into £. coli

plasmid vectors: pBR322, pKC7, pUC13 •. pUC18 and pUC19. Recombinant plasm1ds used

for the nucleotide sequencing were summarized in Table 1. The. locations of

chloroplast DNA fragments used in sequence determination are shown in Fig. 2. Each

plasmid was sonicated (Deininger 1983) by TOMY handy sonicator and randomly cloned

into SmaI or HincII sites of phage mp18 and mpl9 (Perron et!l. 1981). Recombinant

phages containing chloroplast DNA fragments were screened by dot-hybridization with

the chloroplast DNA (Hu and Messing 1982). Obtained shotgun libraries were used for

-19-

.... ··.'.:11

",

_,I.

Nip Ct-DNA

", ",

'F;gu~el~' RestriC:ti6n m~p of ttieM~polymo~pha chloroplast DNA. Narh)w lines

: . with a rrovtheads ~ns idethe.Ci rcLi lar map i ndi c.atei nverted' rElpeat regjons.. LS

'. illdicp.tes the. site ·pt the gene for th.e l.arge sU,bunitof ribulose-l)5-

. bispho~phat~ c~rboxYlas~/oxyge~~se (rbc'L). Th~ sequenced region in thi~ study

is shown as a black box:

:': "

. ('

.. ' ~

-.. r .' r, ~. " " , , ;

-20-

·f

• 'l>OC 1

L,IVERWOR,"

CHLOROPl!AST DNA

LSC

Figure 2. Gene organization of the chloroplast genome from a liverwort. M. ~rpha and sequenced restriction fragments. Thick lines indicate the

inverted repeats (IRA and IRS). sse and Lse indicate the small single-copy region and large single-copy region. respectively. Genes shown outside the '

map are transcribed anticlockwise~ and tnose inside are· transcribed clockwise.

The tRNA genes are i dent,ifi ed by tlie qne-~ etter am~ no aci d code wHh thei r

anticodons g1ven dn parentheses. Asterisks indicate genes having introns in

their sequences (Ohyama et EJ. 1986).. Restriction fragments used in sequence determination are shown outside the genetic map.

-21-

Table 1. Recombinantplasmids used for the nucleotide sequence determination.

'-'---Pl~sm;d-Fra;;;nt;)(k~)----~-P~;~tiorib)---Vec;~;---cl~~i~;-";ite"-------~~~-"";''':'''~~--~-'. -----------:--~-..:..-~..:..----. -----.--.,...----:-----~:.....-----

pMP593 B 10 g (3.8) 51611 - 55380 pKC7 BglII pMP228 B5 (10.3) 47155 - 57496 pBR322 BamHI pMP452 Bg8 (6.2) 55380 ,... 61569 pKC7 Bg1 II pMP708 P8. (5.3) 57bB2·~ .. 62.368 . pUC18 Pst! pMP7T3 ~g13 (2.9) 61569 ~64474 pUC18 BamHI pMP727 P6 (7.0) .'62368 - 69315 pBR322 PstI pMP310 8g5 .. (7.5) 65513 -73011 pKC7 BglII pMP71 0 Pl0 (3.7)' 69804 - 73521 pUC18 PstI,' pMP376 8g3 (11.4) 73011 .,... 84425 pKC7 BglII pMP?06 86 (5.8) 76397 -' 82182 pBR322 BamHI

~- ...... -------..;.---, . -~----....;.....;--.-:.:----, ...... --=-----:'-- ....... ---------~------------.~'":"":"':"".--;;...----a) corre~pond to chloroplast DNA fragments generated by restriction enzymes;

Bg1 II (Bg), BamHI (B)andPstI (P). , b) counted from the:first nucleotide of LSCregion next to IRA'

DNA sequence determination by the chain termination method (Sanger et~. 1977)

using universal primers M3 and M4 (Takara shuzo Ltd.) and buffer gradient gels

(Biggin et al. 1983). The DNA sequence data ,read from 'autoradiogral1)s were handled ~- ,

by personal computer PC-980l usirig the software DNASIS (HITACHI SK Ltd.).ORFs

deduced from the nucleotide sequences were searched for amino acid sequence

homologies with. protein data base (NBRF release 6.0) using the search algorithm

des.crl bed by Wil bur and l ipman (1983). Previ ous ly pub 1 i shed amino acid sequ~nces of

polypeptides in other p1ant species were also used to homology search.

RESULTS

Gen~otgani:zat;on in ':tile 1SC re!j;on (psbtr16SrRNA gerie)

Thenucleotide,seq~~nce .(30~600 bp) from the B9111 site (position 5161l) to

BamHI site. (82188) covering the junction (JLBJ between LSCand IRB reg.ions was

determined~ 'A' computer searcn of the DNA sequence led to the identification'cif ORFs

that"b~gin with the tr~nslati~ni~iti~tion codon AUG and: end at an'either

termiriationcodon(UAA, UAG,andUGA). Thegene.organizatiori deduced from the

nucleotide sequence are shown schematically in Fig. 3. The L!?C region sequenced in

this 'stu-dy was divided into seven blocks (Fig. 3 A-G) depending on the directions of

thetrans'ahonal:ori~ntation (showri by horizontal arrows in Fig. 3). The

=22-

III a. iii

I·

...J U -e

a .0 o 0: i

'" ~ '" 0

D I +·---------=E~--~~~

., ;;; '" ..,

~ u.

'" o. ·0·

• B

III ~ .. C.

F

a. __ _ ··-""" .. ···~ .. ····-··JLB

G

< iii ~-

c !; ~I.L UJ !;; Il. u..:.o .D Ii..

.0:: a: rn.~ c: . 0'0 c. c. 0 11._ ..

G

Figure 3. Detail gene organization of the region sequenced in this study.

The coding regions of genes are indicated .bybold lines. Introns (interVening

sequences) are shown as hatched box. Genes shown on lines a.re transcribed to the right side, and those under lines are transcribed to the left sid~~ The

sequence files are indicated by arrows with the names of sequence fHes. J'LB

indicates the junction site between LSC r~g;ona!1d IRB region.

Each. line indicates 10 kb long.

--,-23-

Table 2. list of identified genes and open reading frames. and their loci on the chloroplast genome.

Gene From To Length Amino acid M.W. Comments (bp) residue

(A5. atpB .:.. 55846 54368 1479 492 53179.3 88.4% (Spi) 62.8% (Eco) atpE - 54362 53955 409 135 15054.3 63.0% (Spi) 22.2% (Eco) trnM(CAU) + 53801 53874 74 94.6% (Tab)

*trnV(UAC) - 53652 53051 602 91.14 (Tob) (Exon 1) 53652 53616 37 (intron) 53615 53086 530 (Exon 2) 53085 53051 35

ndh3 - 52877 52515 363 120 14188.7 30.8% (mit) psbG -52524 . 51793 732 243 27609.6 62.1% (Mz) ORF169 - 51742 51233 510 169 20084.8

( B) 1428 rbcl +56355 57782 475 52790.0 90.5% (Spi)

trnR(CCG) + 57877 57950 74 ORF315 + 58065 59015 951 316 35826.3 ORF36b + 59193 59303 111 36 4017 .8 ORF184 + 59525 60079 555 184 21533.1 ORF434 . + 60151 61455 1305 434 51866.2 petA + 61641 62503 963 320 33482.6 78.8% (Spi)

(C) ORF40 - 62916 52794 123 40 4101.8 12.6% (Cya) ORF38 - 63152 63036 117 38 4479.1 psbF - 63293 63174 120 39 4468.3 89.14 (Spi) psbE - 63554 63303 252 83 9493.5 89.2% (Spi) ORF42a - 63584 63556 129 42 ' 5101.9

(D) ORF31 + 64152 64247 96 31 3465.4 ORF37 +'64370 64483 114 37 4075.9 trnW(CCA) - 54626 64553 74 93.4% (Spi) trnP(UGG) - 54788 64715 74 93.4% (Sp1) ORF42b + 55027 65155 129 42 4746.5 rp133 + 65273 6547D 198 65 7782.1 36.9% (Eco) rps18 + 65498 65725 228 75 8879.5 34.7% (Eco)

(E) rplZO - 66157 65807 351 116 12773.0 45.6% (Eco)

*rps12A - 67057 1 7 123 13797.0 70.27. (Eco) 91.9% (Tob) (Exon 1) 67057 66944 111 38 trans-split (intron ?) 66943 7 7, - (Exon 2,3 coded oppasit strand)

'*ORF203 - 68640 67130 1511 203 22685.0 (Exlin1) 68640 68570 71 - (87.07. homologous to spinach ~-gene) (intron) 68569 68052 518 (Exon 2) 68051 67760 292 (intl"on) 67759 67379 381 (Exon3)

(F) ·67378 67130 249

psbB + 69026 70552 1527 508 ' 56191. 5 BB.2% (Spi) ORF35 + 70669 70776 108 35 3958.8 ORF27 + 70B12 70955 84 27 326B.0 ORF74 + 71092 71316 225 74 792B.3

*pctB + 71424 72566 1143 215 24306.5 85.07. (Spi) (Exon 1) 71424 71429 6 (intron) 71430 71924 495 (Exon 2) 71925 12566 642

*petD + 72715 73690 976 160 17413.5 95.6% (Spi) (Exon 1) 72715 72722 8 (intron) 72723 73215 493 (Exon 2) 73216 73690 475

-~-----~---------------- ----------------~~- .........

-24~

Table 2 (continued).

Gene From To Length Amino ecid M.W. Comments (bp) residue

(G) rpoA - 74824 73802 1023 340 39240.2 25.61 (Eco), 54.11 (Spf) rpsll - 75249 74857 393 130 14172.5 51.5% (Eco), 72.3% (Spi) secX - 75413 75300 114 37 4521.5 62.2% (Eco), 86.5:! (Spi) infA - 75686 75450 237 78 8978.4 56.4% (Eco), 60.3;:: (Spi) rps8 - 76171 75773 399 132 14921.4 45.5% (Eco) I:"p114 - 76621 76253 369 122 13496.6 58.2% (Eco)

*rp116 - 77685 76719 967 143 16149.8 53.8% (Eco), 72.0% (Spir) (Exon ]) 77685 77677 9 (intron) 77676 77142 535 (Exon 2) 77141 76719 423

rps3 - 78396 77743 654 217 25055.0 40.6% (Eco) rp122 - 78B04 78445 360 119 13580.8 37.8% (Eco) rps19 - 79100 78822 279 92 10553.3 63.0% (Eco), 83.77. (Spi)

*rp12 - 80514 79137 i378 277 31162.9 48.4% (Eco). 58.57. (Spi) (Exon 1) 80514 80118 397 (intron) 80117 79574 544 (£xon 2) 79573 79137 437

rp123 - 80B25 B0550 276 91 10768.5 29.7% (Eco) trnI(C*AU) - 81057 80984 74 93.27. (Spi) C*; modified base trnV(GAC) + 81814 81885 72 95.8% (Spi) in IR region

Amino acid sequence homologies are ~alcu'ated as -(identical residue number)

(reSidue number of liverwort product)

Homo logy pe'rcentages wi th gene products of Spi nach (Sp1), I . .£Ql! (Eco). Tobacco (Tob). human mitochondria (mit), Mai~e (Mz). Cyanella (Cya), and Spirodela oligorhiza (Spir) . are shown jn comments.

-25-

A TcmMCAcCAGcmGMTcCMCACCTGcmAGTCTcCGmGTGGTGACATMGTCCCTCCCTAcAMTcTAATMTAnmGcMcGATGAGGTCTGCTCGATMw.TTTTT 56291 OKYGAKFGYGAKTETQPSH GGAGG <rt>c:L

+- <---- --~

TrCTAATTnTCmTGTATAAJ..w.TTTTTrMGTAAAMmTTTCcMTAATAAAACi.TTATTATTGTATATTGrrrTrrATATGTMTGCMCCTAGCTATTGTArTAnAAATAA 56171 +--- __ <--- - - - ---+ <TAACAT <lACGn

AmTAnArirrrrrirArTGATACACArTGACmAAcMTTmCAGTATATAGAnTAAATATATAi"ATATATATATATATATATATATATGAGTATrATATATCTATATCTATAT 56051 +-- -- <-- - --

ATATATATATAcATATATAToomATATATTTTCTCATrAAmAGTATCAAAAmMTCTATCTM;TTMcncMAAATAnMi:AAw.GmAAATATATArTrmCTMG 55931 '--...,----~ TTGTtA> mAAT>

iHAGmmTrATTAnAATrGAmArrTGATACACMTATTTTTTTTATTATAATTlCATTATTAAcTMCTTmATmAW .. W.CAMnnrTAGCTTTTaaMTGTClACA 5S!l11 .~~ H K T PI F L A F G H 5 T

i:TTGTTGCTMAAATATAGGMGTATTACTWaTTATTGGTCCCGTATTAGATGTTGCCTrrrCTCCAGGGAAAATGCcTAATAmATMCTCTTTMTrGTTAAAGATCAAAATTCA 55691 l V A K H I G 5 I· T' Q Y I G P V L 0 V A F 5 P G K H P ~ I Y II S L 1 V K 0 Q ~ 5

GC TGGTGMGAM TT M TGrT ACTTGTGAAGTTCMCM nGTT AGGA!\A T MCAMGTMGAGCTGm;CTATGAGIGCGACAGA looM TGA TGcGGGin ATGAMGrT A TTGA TACT 55571 AGE E 1 N V T C E V Q Q L L G II II K V R A V A H 5 A TOG H H R G H K V lOT

GGTGCTCCnTMCAGTTCcTGTAGGAGAAGCGACTCTTCGACGAATTTTTMTGTTTTAGGAGAACCTGTAGATMCTTAl;GACCTGTAGMGnACTACAACAmCcTATTCATAGA 55451 GAPlTVPVGEATLGRIFHVLGEPVOPlLGl'VEVTTTFPIHR

GCTGCTCCTGCTTTTACTcMTTAGATACcAMTTATCTATrmGAAAcAGGMTTAAAGTAGTAGATCTnrAGCTCCTrATCGTCGTGGAGGMAAATrGGATTArrTGGAGGAGCT 55331 II A P AFT Q LOT K LSI F E T G 1 K V VOL LAP Y R R G G K 1 G L FG G A

GGTGTAGGAMMCAGnCnATTATGGAATrMTTMTMCATCTTGAMaCACATGGAGGTGmCAGTAmGGAGGAaTAGGG~CTCGTaMaGAMTGATCmACATG 5521 I G V G K T V L I H ELI PI ~ ILK A /I G G V 5 V F GG V G E R T REG " 0 L Y H

GAMTGAMcMTCTAMGTMTAMTGMCAAAA.TAmCAGAATCMMcTTGCTTTM;mATGGTcMATGAATaMCCACCAGGCGCTCGTATGAGAGTTGGGTTMCAGCmA 55091 EHKES~V1IIEQIl15ESKVALVYGQIIIIEPPGARHRVGLTAL

ACTATGGCTcAGTAmTCGTGATGTTMTAAACAAGATGTACTTTTATTrATTGATAATATTmCGTTTrGTTCAAGcAcGTTCAGAAcmCTCCTTTATTAGGTAcMTGCCGTCT 54971 Til A E Y FRO V II K Q 0 V L L FlO /I I F R F V QA G S E V SAL l G R II P S

GCAGTAGGATATCMCCMCTTTMGTACAiiAMTGGGAACmACP.AGAMGAATMCrTCTACAMAGMcciATCMrTACTTCTATTCAAaCTGmATGTACCTGCGGATGAmA 54851 AVGYQPTLSTEHGTLQERITSTKEGSITSIQAVYVPAOOl

ACAGATCCGGcrcCTCCMCMCTTTTGCTCAmAGATGCCACTACTGTATTATCTAGAGGmAGCAGCrAAAGGAArTrATCCTGCTGTAGATCcrrTAGATTCMCTrCTACMTG 54731 TOP A PAT T F A II lOA T T V L 5 R G L A A K G I Y P A V 0 P LOS T 5 T H

TrACMCCTTGGATTGTAGGTGAAGMCATTATGAAACTGCGCMGGAGTMMCAGACmACMCGATACAMGMTTACMGATATTATTGCTAITCnGGmAGATGMTTATCT S4Gl I LQPI/IVGEEHYETAQGVKQTLQRYKElQOIIAILGLOELS

iiAAGAAGA ICG m AACTGTAGCMGAGCACGCAMA T AGi.MGA TTm;' TCACMCCTTrCTTTCT AGCAGAAGnm ACAGCTTCGCCAGGAAAA T A TCT MGTCTY AGAGAAACT S4 4 91 EEDRLTVARARKIERFLSQPFFVAEVFTGSPGKYVSlRET

ATAAAAGGAmCAMTGATTCmCGGGAGAATTAGATAGCCTTCCTcMCMGCArrrTAmAGTAGMMTATAGATGAAGCTACTGCAMA.GCAGCTACmAcMcTGGAGAGT 54371 I I( GF Q HI L S GEL OS l P E Q A f Y L V G" 1 0 EAT A K A AT L Q V E S

AtpE. GGAG

TMAAATTATGCTAAATCTTCGTATCATGGCTCCTMTCcMTTGmGoMTTCGGAl'I\TrCMGAAArTATTTTATcMCGAATAGTGGaCAMTTGoMTACTACCTAACCATGCTT 54251 II l II l R I II A P II R 1 V 1/ II SOl Q Ell L 5 T II 5 G Q I GIL P II HAS

CAGTmMcTGCmAGATATAGGAATTGTCAAAATACGCCTTMTGATCMTGGTCTACTATGGCATTMTGGGTGGrTrrGCTATGATrGACAATMTMmAACTATTTTAGTTA 54131 VLTALDIGIVKIRLIIDQIISTHAlIIGGfAHIOII,,"LTILVIl

ATGATGCTGAMMGCTAGTGAMTAGATTATCMGAAGcACMGAAACrmCAMMGCrAAAAcAMmAGMGAAGCAGAAGGTMCAAMAAMi.GMATCGAAGCTCTATTAG S4011 OAEKASEIOYQEAQETfQKAKTIIlE[AEGII~KK[IEALLV

TTmAAMGAoCTMAGCAi.cATTAGAAGCMTCMTATGaCATCAMGTrATAAATTAMTAATTMrTMTAATTAAAAAmATArTAGATGCCACTrmCTGGcATCTAATATA 53891 f K R A K A R LEA r II HAS K L -- +--> <-~ 1-1 - __ ~ __

MTT~cnACcTACTATTGGATrTGAACCAATGACTCTCGci:GTATGAMGCGATACTCTAAACCACTGAGTrMGTAGGTATTTTATTTcAMmATTATMcTATATA 53771 --+ J '-AUGGAUGAUAACCUAAACUUGGUUACUGAGAGCGGCADACUUUCGCUAUGAGAUUUliCUGACUCAAUUCAUCCA-5 ' <Mel-atl < 1AATAT

ATMCTTTTMTGCAAGATMcTAAAAAAMTTGAATCTAAACCTTGAcMTAAATAATMAAAAaTATATATATATATTGTATAGAAMTMTACTMTATMCTCGTcAGTATTMAG 53651 <TACCTT TTGTAT> TATAAC> s'-AG

+-><---l- V.HJAC,

GGCTATAGCTCAGCGGTA(lPi;CGCCTCGrrTACACGTGCGCcMTGCTTATCAMMmrmTCGArrTGTCGATTCACMcTMMGTmCTMnTrrGTGAMTAGAAAATGTC 53531 GGWADAGCUCAGCGGUAGAGCGCCUCGWUACACgugyg •••••••••••••••• (1 n tron) ••••••••••••••••••••••••••••••••••••••••••••••••••••••••

TTACTCmci.rTTAATCArTGAGAAAAATAGCCTGACAcMATMmCTATTAmArTrGAMTTAcAnmAGTTGATATGGTTMmmCTmAATGTTArTACATGATGA 53411 •••••••••••••••••••••••••••••••••••••••••••••••••••••••• (lntron) ••••••••••••••••••••••••••••••••••••••••••••••••••••••••

GAA TT ACGGGGAACTCAAGA T A TTCTTTTTTrGCm ATaAAATTTT MGG TG TAT AAAA mCA TA TT A TrTT AGCAAci.GAMCTCTTT A TTGAGTMA TCCA TGT MAAAACAMCC 53291 ........................................................ (lnlron) ...................................................... ..

TMGTCM TAmGATMnTrrGAAAAAcTnGGGATTGTATTAAMrrTmAGAArrTrMGCAMCGcAACCATCTlATIATTMci.MAAAAAGci.TGGAAAATMCTAAATTM 53171 ........................................................ (lntron) ....................................................... ..

Figure 4A.

-26-

CACTTAGTTMTAMCGAGCCW1CCA1AAAMCATGCATGITCCGTrCITMAGCAGTTCITMITTMAAcMCTGrTrrACCGAGMTGTCTACGGTrCAAATCCGTATAGCCCTA 53051 ................ rog ccg-•• g •• -g ••• ~uuC.u9 • ...,9~ uuy •••••••••••••••••••••••••• cU'YYrayCGAGAAUGUCUACGGUUCAMtJCCGUAUAGCCCI)J\-J'

AATfTCTfTrTTTTTATAAMTGMMMGGTATACITCATCATAMAGATTAGTTMTMAAmMCTAAAMATCTMAGITCMATAAcTACAATTATMTMTMITGACCMTT 52931 <~---t +---- --- <----

TAMCITrrTTmATAMCnGAMTTATATATATTTmAGCAGGrnTrMTGfTTTTAcrrCAAA.t..i.TA1GATTArrrrITCaTArTTfTATIMTAATTAGrrrTTTCTCMTAC 52611 _________ --, ndh3. AGGAGG H f L L Q KYO Y r f V f L L I I S f f S I L

TAATTrrTTCITTGTCAMATGGATAGCACCTATMATMAGGACCTGAMAAmACAMTTATGAATCAGGTATAGAACCGATGGGAcAAcCrTGTArTCMmCAMTTCGATATT 52691 I F S L 5 K \I I A P I f1 K G P E K f T S YES G I E P H G E A C I Q f Q I R Y Y

ATATGmGCTTTAGfTTTTGTMTTmcATGTAGAAAcAGTTmCmATCCITGGI;CTATGAGTrrTrATMTTTTGGTATATCATCTTfTATIcAAGCTfTMTTrHATTTTM 52571 H f A L V f V I f 0 VET V FLY PI/A M 5 F Y f1 F GIS S F J E A l I F J L I

rTTTMTTATTGGTTTACTATATGCATGGci.M.w;GAGcACTAGMTGGTCTIAMmCMATTfTTTAcTIGTGAAAi.TAGTTTAGAGGATMCTCTACMCTATGCITMAAATTC 52451 LIIGLVYAIIRKGALEIIS~

p.bG, /\IlGAG H V L II F X F F 1 C· l' 11 S LED " S T T H l X II S

TATAGMTCTTCTTTTATTMCAMACTCrTACAAATICMTTATTTTAACMCmTAATGATTfTTCTAATTGGGCTMlACTTTCTAGTCTATGGCCACTCCIDATGGTACMGTTG 52331 I E.5 S· Fill K T L T 11 S [ J L T T F fl 0 F S II II A R l 5 S L 1/ P l L Y G T S C

TfGmfATTiw.TTTGCATCATTMTTGGTTCA[GATT[GATmGATC~TfATGGmAGTACCTAGATCCAGCCCTMlACMGCAGATrrGATMTMCAGCTGGTACrGTMCTAT 52211 Cf J E FA S L I G S R F 0 fOR Y a L v P R SSP R Q A 0 l 1 1 fAG TV T H

GMAATGGCTCCTTCmAGTTAGATTGTATGAACAMTGCCTGAACCAMATATGTAArTGCAATGGGTGCGTGTACTATrACTGGAGGCAfGTTfAGT.;'CAGATTCATATACTACAGT 52091 KHAPSLVRLYEQMPEPKYVIAMGACTITGGHfSTOSYTTV

TCGTGGGGTAGATAAATTAATrCCTGTGGATATTTATTTAi:CTGCATGTCCGCCTAMCcAGAAGCMTTATAGATGCTATMTAAMCrTCGTAAAMAATAGCTCMcAMTAfATGA 51911 RGVOKLIPVOIYlPGCPPxPEAIIOAIIKLRKxlAQEIYI:

~ TCC TT AAAAMGGAACCAcA TTfTTT ACTIT AM TCA TCM TTCAA TTrrrmCAM Ti:! AGACAA TCCAMACT AAcTTccTCAAACCAA mrrccM TeT AAAAA 51651 E K K ILK K GT R F f T L 1/ 11 Q f 1/ f f $ 11 LOll P K L T $ 5 II Q f F Q S K K

MCTTCTAMGTmATTAGAAACATCmMCAmAAAGMAAGGAAM mATAAATATMccTmAcrmGACTAAMAAAMTAAM.w.GTAAAAATAATATimAAACAIT 51731 T 5 K V L LET S L T F K EKE f1 L ~ +-- -> <- --> ORf169. H L 11' J

nmMATAACAATMI"AA;..TACAAGG.Ai:Gm"'TCTj\mG(mMnAAGCMAATTTAAMC"'CA~CCTH~TmIil\TmWGGAAT ... Cii-.AAcm ... CA~TTkATCT 51611 l K fl fl /j 1/ K J Q G R LSI 1/ L I K " fl L K " R P L G F 0 Y Q G [ E T L Q IRS

. 8g1l1

B TGTAGACArTCCAAAAGCTAAAMATHGITncATMAATAAAAAaTTAGTTAATMTGAAATTATMTMAMMATATTGTGTATcAAATAMTCMTTMTAATMMAAAACTAC 55930

T S H G F A. L F H T K H •• lpe

crrAG.W.Ai..ATATATArrTAAACTTTTTCTTAATAHTnGAAGTTAACTAGATAGArTAMrmGATACTAMTTAATGACAMATATATAMmGATATATATcrATATATATAT 56050 <TAATTT <ACTGTT 1-- -----

ATATAGATATAGATATATAATACTCATATATATATATATATATATATATATATATAmAMTCTATATACTGAAAAATTGTTMGGTcMTCiTGTATcAATMAAMAAATAATAMAT 56170 -- - ~--. -----+

TT A m M T M TACM TAGCTAGGTTGCA IT ACA TAT AJ.MAACM TATACAA T M TM TGrm A iTA nGGAAAAM Trnr ACTT AAAMA TTmTATACAAMGi.AMA TT AeM 56290 TrGCAT. TACMT. +- - - - -----, ...,.-- ---. ~

AAAAATTnTATCGAGCAcACCTCATCCTTGCAAGAATATrATTAGAmGTAGGGAGGGACTTATGTcACCACAAACGGAGACTAAAGCAGGTGnccATTCAAAGcTGcTGITAMcA 56410 roc!> GGAGG H S P Q T E T K A G V G f K A G V K D

t-- ---.

TTATCGATTMCITATTAcACTCCCGATIATGAGACCMGGATACCGATATTTTAGCAGCAmAGAATGACTCCTCAGCCrGGAGITCCAGCGGAAcAAGCAGGCMCGCAGITGCTGC 56530 Y R L T Y Y T P 0 YET K 0 T 0 I L A A F R H T P Q P G V P A E E A G fl A V A A

TGAATCTTCMCTGGTACATGGACTACAGTnGGACTGATGGTCTTACTMCCTTGATCimATAAAGGTCGATGCfATGATATTGACCCTGTTCCTGcAGAAGAAAATCAATATAITGC 56650 E SST G T Ii T T V Ii TOG l T II LOR Y K G RC Y 0 [ 0 P V P GEE Il Q Y J A

TTATGTAGCTTATCCm...GAmATTTcAAGAAGGGTCTGITACAAATATGmACTTWnGTAGGTAATGTAmGGGmAMGCmAAGAGcGTTACGTCITGAAGATTT.uG 56770 YVAYPlOLfEEGSVTIIHFTSIVGIIYFGFKALRALRLEOLR

MTTCCTCcAGCnACACAAAAACTfTCcAAcGTCCTCcTCATGGTAITCAAonGAGAGAGATAAATIWCAAATATGGTCGTCCT1TAnAGGATGTACTATTAAACCAMATTAGG 56890 I P PAY T K T f Q G P P II G I Q V E R 0 K L II K Y G R P L L G C T I K P K l G

mATCTGCTAAAAATTATGGTCGAGCTGTATATGAATGTCTTCCTGGTGGACTTGArrTIACTAAAGATGATGAAAAcGTAAAnCTcAACCAmATGCGnGGAGAGATCCITTCrT 57010 L S A K " Y G R A V Y EeL R G G l 0 F TKO 0 E N. V H S Q P F H R W R 0 R F L

A TTTal ACcAGAAGCTA TTTAT AM TCTcAAGCAGAAACTGGAGAAATci..AAcGACA IT Am AM TGeT ACTGCAGGr Au. TGTGAAcAM TGCT AAMAGAGCAGCA TGTGCTAGAcA 57130 FVAEAIYK5QAETGEIKGIIYLHATAGTCEEHLKRAACARE

GITAGGTGTACCMTIGHATGCACGATTACTTAACTGGtGGmCACTGCAAATACTAi;rCTGGCTrrTrATTGCCGTGACMTOOmACITCTTCATATTCACCGTGCMTGCATGC 57250 l G V P I V H If 0 .y l T G G F T A II T S l A· F Y C R 0 " G L L L HIll R A H " A

AGnATTGATAGACAAAAAMTCATGGTATACATTTCCGTGTATTAGCAi.AAGCmACGTATGTCTGGTGGAGATCATATTCACGCTcGTACTGTTGTAccTAAACT1iiMcGAGACcG 57370 V lOR Q K 1/ H G J I! f R V L A K A L R H S G G 0 II I HAG T V v' G K L E GO R

TCAAGT MCTTT AGGmcGTAGA m ACITCGTGA TGACTATA TTGAAi.AAGA TAGAAGTCGTGGTA mAmCAcAi:AAGA nGGGTrrCmACcTGGTGfTTTCCCTGT ACCA TC 51490 Q V T L G f Y 0 L L ROD Y J E K 0 R S R G [ Y f' TQ 0 1/ V S LPG V f P V A .5

Figure 4A. and. 48. I _ ':. ~: :

-27-

TGGTGGGATCCATCmGGCArATGCCTGCITTAACTuW,:mTTGGAGATCACTCTGTmAr.Ai.nl:CCTGGTccMemAGGTcATCe'rTGGGGTMCGCACCTGGTGCACITGC 57610 G G I' 11 V 1/ H H j> A' L TEl F G· DDS V l Q F G GaT l a H P II Gil A P ·G· A V A

TAACCCAGTTTCGlTAGAAGCITCeaTAcAAGCAcGTMTGAAGGTCGTi:ATCITGCTCGTCAAGCAAATGAAATTATTCGCCAAGCTTGTAAGTGCAGTCCTGAGTTATCTGCTGCITG 57730 IIRVSlEACVQARJ/EGRDLAREGIIE r 1 REACKWSPELSAAC

TGAAAmG~TTMAmGMrTrGATAiTArTGATACmGTAAMTMAGi-AGATAmrATCTTMAAAmrGTAATTTrCTTTTmTATCTCAGArTrCAGATAAAA 57B50 E . 1 11 K ElK F E F 0 1 lOT L .~- +--~. <---+ +-- > <---

AAAAGAAAATTACAAAATTnAAAATGGGitTGTAGCTcicTGGATTAGAGCTCATGGTTCCIlAATCATGAAGTCMGcGTTCGAATCCCTTCTAACCCTnnCTTArTrlGAATATrT 57970 ___ ~_~'_~>5'_GeGUUUGUAGCUCACUGGAUUAGAGCUCAUGGUUCCGAAUCAUGAAGUCAAGGGUUCCAAUCCCUUCUMCCCU-3'

At<J'-CCG>

TTGTATMioAA.wo.AATTGTTcTATrATTMATTACITAAATTMAATTi-mCATATATATTTTTTTT~GCATTrfTTTATGTcmAATGAArTccmGAAGA 58090 DRF316. GAGe H S t Ii II 1/ FED

TAAACGAAcATTTGGTGGAitAATTCGCGCrmATTc.Ww.cCTACTmCGATATATrmAGTGAAAGACAAAAAGATCGATATAitMAATTGACACTACTAAcGCATTATGCAC 56210 K R R f G,G L 1 G A fiE KAT KG Y I F S ERE K 0 R Y r KID T T X G L 1/ T

TAGATGTGAi:MnaCGAAMTATGTTATATGITACATTiTTGAGAwAATAAACGMmGTGAAGMTGTGGATATCAmACAAATGAGTAGTAcAGAAAGAATTGMcrmMT 5BJJO R C D 'I CEil H l Y V R f L R Q 'I K RIC E E C G Y H L Q H SST E R 1 ELL I

TGATCGTGGTACTTGGTATCCAATGGATGMGATATGAcTGCTCCAGATGTTCTTAMrTnCTGATGAAGATTCTTATMAAATCGAAitGCTTTTTATCAMMCcAACTGGTTTAAC 58450 D R G T .11 Y P H <0 E 0 H TAR D V L K f S 0 E D S Y K /I RIA F Y Q r. R T G L T

iGATGCMTTCMACACGcATAGGTCAArTAMCGGTArTCCTAfrGCATrGGMGTTATGMTTTTcMTTTATGGGCGcJAGTATGGMTCTGTAGTAGGTGAAAAAATTACTCGTCT 5B570 DAIQTGIGQllIGIPIALGVIIDfQFHGGSHGSVVGEKITRL

TATTGAATATGCTACTAGAGCArCAATGCCATTMTTATAGTATGTTCrTCTGGTGCAGCACGCATGcMcMGGAACATrAAGCTTAATGCAMTGGCTMAATTTCrTCGGTTTTGcA 58590 lEY A T R A. S H p. l 1 1 V C SSG GAR II Q E G T l S l H Q H A K ISS V L Q

MTTCATcMGCCCAAAMAMTTACTTTATATAGCTATTCTTACCTATCCTACAACAGGAGGAGTTACiGCAAGmTGcTATGTTAGGGGATAfTArTATTGCTGAGCCAAAAGCTTA 58810 I II Q A Q K R L l Y 1 AIL T Y P T T G G V T A. S F G H L G 0 I I I A £ P KAY

TATTGCATTTGCAGCAAAA.i.GAGTTATTaMCMACmACMCAAAAAATACCAGATc;GTTTTCAAGrTGCAGMTCATTAmGATC~TGGTTTACTTCAmAAfTGTTCCAAGAAA 5Jl93D I A FAG. K R V I.E Q T l R Q Kip 0 G F Q v A E S l FOil G L L 0 l 1 V P R II

TCTrTTAAAAcGTGTTrrAAGTGAMrnl:TGAATTATATAACGCTGCTCCTTGTAAAAMmcAAMitccTTTTTTMATMTTTTGTTA(lACmTAGTAmTAGTAGmTTTT 59050 L L K G V L S ElF ELY /I A A P C K K F Q NSF F K -- +-

TTAATTCAAAnmAATMAATATTATATTTTATTATTATTATTTATTAAAATAAGATATAATTTTTAmAGTTTAGTmAMTAAMTATAGTATATATTATATTTATTTTmn 59110 -----,.> <------~- ~-~ --->

ATTGTATTTTMGGTAnrTnATGACAGCnCITAmACCTTCTATTTTTGTTCCrrTAGTTGGATTAATTTTTCCTGCTATTACTATGGCTTCATTATTTATAfATAITGAACMcA 59290 ORf36b> AGG < H . T A S Y L PSI F V P l V G L I F PAl T HAS L FlY 1 E Q 0

TGAAATTTTATAMTAAATTGGAGACTAAMATITnnl:TACTTITTATMAATATATATATGITATATATCAATTnl:nGTGATATATAITCTATMTACmGTc.i..aMGTMAAA 59410 ElL --- +--~-- <--- ~ - ------

AMTTITTAATTTAITGTTATTAITATAATATTGATTATTTTTATATTcAA,w.TMTCMTAmmTrATCMCArTATTATTCAACTAGTTTAGcAGACATTCrrTTGTTATGAAT 59530 ---r t----> ORFl84. AGCAG fI II

TTACAAGTGCACCATATTAMOTAGATfTTATAATAGGATCTCGAAGAATMGTMTTTTrGTTGGGCrmATTCmTAmGGTGcATTAGGTmTTTmGTTGCATTTTCTAGT 59650 l Q v 0 II 1 R V 0 F I I G S R R r S II F C \I A F 1 L L F GAL G F F F.V G F S S

TAffiGCAAAAAGATTTMnCCTTHTTATcAGeTGAACAAATTnATTTATCCCAcAAGGAAITGTAATGTGTTmATGGTATTGcAGGrITATTTATTAGTTTTTATTTATGGTGC 59770 YlQKOLIPfLSAEQILflPQGIVI'ICfYGIAGLflSfflllC

ACTATTTGrTGGAATGTCcGTAGTCCCTATMTAAAmGATAAACAAAMGGAATATmCTATTITTCGTTGGGGArTrCCTGGAAAMATCGTCGTATTITTATTcMTTmAAn .59890 TIC \I Ii V G S G Y /I K F '0 K Q x G 1 F S 1 F R II G F P G X /I R R r F 1 Q F L I

AAAGATATTCAATCAATACGAATCGAAGTTCMGAAGGTitTTTATCTCGTCGCGTTCrTTATATMAAATAAAAGGTcMCCAGATATACCTTTAAGTAGAATTGAAGAATATTTTAcA 60010 ~ a ! Q SIR tI ( V q '£ G f L 5 R R V l r I K I K G Q P ~ I P L S R ! E £ f f r TT AAGAGAAA TGGMGA r AAAGCTGCTGAGTT AGCTCGTTTTTT AAAAGmCTA TTcAAGGTA m AAACTTmA TT ACGTCTTTTTi-r AT AAM TAT MMA TATGCTGmTTTT A 60130 LREMEDKAAElARfLKVSIEGI- _. <-->

GCAAA TT AT AM TAGTrnl: ATGMGAA6M TTTT AGTT i. TTGGCGAA TnnCA TCAcA TTTTrCCTCTrCCA TA TTGitem AGAAA.o.AGCA TAT WGCCAGT ,w,CGTATAc.w. 60250 Dlll'434. H K K N F S Y W R I F Ii II I f ALP res l E K ArK ASK R I Q K

AAATAAAGWGAITATTTTTTGTATMAAATA1ACTTTTTTCATCAAAicGTTCTTGGCACTCTATTcitrrrTATATAGATACAGAATTAAATMTTCTGTTITTAAMTATAmcA 60370 1 KKDYfLYKlI1 LfSSKRSIIQSllFYI DTEllIlISVFKJ YLS

GTCTTHAcAATATAAATTAAGmGTGGitAATTCAGcTTTTTCTAATTrTTTCTrTAmTT~TTCAAA.i.TTTCATTTAATTCTACCAMTATTAAT~ 60490 l t E Y K l 5 L I.' l 1 Q L f l I f 5 L f f K K U S K f {J L I L PilI II £ KKK K

AGAGAAAMTAAACAc.w.ATTAt;CTTGcATTAGAGCTACTCTAAATGAmAGNlAGrTGCAGACGTTACTAmATTnCTTcmTTTATcmAcATAi.MM~TAArT 60510 R K 1 /I R K L A \I 1 RAT l /I D L E 5 II R R Y Y l F S 5 f L S L 0 K K E XII II f

TTTCTTTTTTACAMTGAAAAGTTCTACATrGACAGCTATAGCTTATcMTCTATAGGTCTTGTACCACGITCTATMcACGAACTTmCMGAmAAAGCACAGTTAACAAATCMT 50730 5 F l Q ,II I( 5 S R ·L T A I AYE S I G L V P RS I T RTf SR f /( A E l T II Q S

CAAGTTCGcTrGTA TT A.v.Ac.i.A m AGGrr AGCAAAA TATCMGCGTTGcCncrr'r Ai:AGTATA TTcGCTGm A ninr A TTCCrTTAGGAGmciTnTmrTCAAAM TaCT . 50B 50 S S l V L KEf R l A K Y Q A LAS L Q Y I G C 'L F F I P L G V S f F f Q K C F

Figure 4B (continued).

TTTT /lGAGCCCTGGII TTcAAM TTGGTGGM TA m AreM TCTCAAAITn-rrrGIICTICA me' "GoA AGAA .. AGcTrr MAAAMCTTCMGAAA nGAAGAAl:TImTCCTT Ai; 60970 L E P II I Q II \I \I fl' [ Y Q S Q [ F l T 5 r (f E E K A L K K L Q E I ['E L FII L 0

ATMAGlAA;GACAlllnu.TCAAAc.MJ..i.TAtMTTGcAAGAmGACTMAGAMTTCACtMCAAAW!CGAIITTi.GTTCAAATTrATAATAA1~TAGTATTAAAATTGrmAt 6\OSO K V H T Y 5 S II" K I Q L Q D L T K E I II Q Q T [ E L V Q [ Y II " D S, [ K [ V L "

ATTTGCTAACTGIITCTCAmGGmATTACTTTAAGTTGTTTATTTAmn,GCAAAAGAACGTCTTGITATTTTAMnCTTGGGCTCMGMTTGrTrTArAGCTTAAGCGIITACGA 61210 L LTD L I II FIT l S C l F 1 L G K E R L V I l" S \I A Q ELF.Y S L 5,0 T H

TGAAAGCTTTTTTTIITTcrrHATTAACTGArrTATGTATTGGATTTCA1TCW;TCATGGTTGGGAMTIGTAATAAGC1CTTGTTTMw-CAnnw.rmmCATAATAAACATG 6;"G K A r F 1 L L L T 0 Lei G F Ii S P II G II E [ V ISS C L E H F G f V " II K II V

TAATTTCGTGTTTTGTTTeMCllmCCAGTAATTTTAGACACAGTCTrTAMTAmGATTTTTeGTCATTTAAATCGTATATCGCCrTCcATTGTAGw.crTATCATACTATGAATG 61450 I S C F V S T F P V 1 LOT v F K Y L I F RilL II R 1 S P 5, I V A T Y H T H II E

AATAAAAMTrCMAArrTIAGGTCTTTnTGTTTACATfMATAAAAITATTTCATATI!",mATATIMMGTAGAAMTTTTTCmATTTATTt.TrATTGTTAiU-TAA1GGCt.G {,\510 .i..~)o (-. +---~:> ,,---t- +-> <-.

ATCTTGTAAMTTGIIGTAGmAAACAATAAAACTATTCTrAAAAATTATTTGAAATAMTAATCTAACTATGCAAAAcAGAAACTTTMTAACTTGIIrTATCAAATGGGCCATTCGArT' 6\690 +----> <--4 +--> <----> H Q II R II F II II L 1 I K \I A I R l

p.tA>

AATTTCCATAATGIITTATTATAAAIACAATATTTTCGTcATCTATTTCAGAAGCCTTTCCTAmATGcACAACAAGGrTATGIIAAATCCACGIIGMGcTACTGGIICGTATTGTATGTGC 61810 IS, J K I 1 I Il T I F W 5 SIS E A F P 'I Y A Q Q G Y' E " PRE A T G R 1 V C A

TAATTGTCAmAGCT~CCGGTTGATAnGIIAGnCCCCAATCTi;TTTTACCAMCACAG1GrriGAGGCAGHGTCAAAATTCCTTATGIITATGCAAATAAAACMGTACTTGC 61930 II C Il L A K K P VOl E V P Q S V L P 1/ T V F E A V V KIP Y 0 H Q I l Q v l A

T AA TGGT ~GTTCTh AM TGTTGGAGCAGTTCT TA mTACCAGAAGGTTncAA TT ACCTCcTrCTGII TCGIIA TTCCTCCTGAAA TGAAAGAi.AAAA TTGST AA TCTTTnrT 61050 II G K K G 5 L Il V G A V l I L PEG F E lAP S 0 RIP P E H K E K I G II l F f

TCAACCCTATAGTAATGAT~TATmAGTAAIAGGTCCAGTTCCAGGA,W.M.UATAGTGAAATGGTTTTTi:CAATTCTCTCTCCAGIITCcAGClAClAAcAMGAAGcru. 62110 Q P Y S fl 0 K K /I I LV) C P v P G K K Y S E M V F P I l S POP A T Il K E A 11

TTTrrrAAMTATCCAATTTATGrTGGTGGTAATAGACGGAGAGGACAGAmATCCTGATGGMGTAMAGTAATAATACAGTTTATAATGCTTCAArTACAGGllAAAGTAAGTAAAAT 62290 F L K Y P J Y V G G II R G R G Q 1 y, P 0 G 5 K S 1/ II T V Y " A SIT G K V S K J

TTTTCGTAMct..w.GGGTGGGTATGAAATAACAATTGlliGATATTTC~TGGTCATMAGTTGTTGATATTTClGCTGCAGGIICCAGMCTTATTArTrCAGMGGTGAGCTTGTGIIA 62410 F R K E KG G Y E J TID 0 ISO G Il K V V D ) S A A G P El J J S E GEL V K

AGIAGIITcAACCTTTAACTAATAATcCAAi.TGTAGGTGCGmGGTCMGG1GIITGCTGi.AGTAGTACTTCAAGATCCATrACGlATTcAAGGTCTTTTATTATTTTTTGGATCACHAT 62530 v D Q P l 'T I' II PI/V G G F G Q GOA E V V L Q 0 P l R 1 Q C l L L F F G S V I

TTTAIlCACAAAIATTTTTAi;HCTTAAAMGAAACAATriGAAAAAGTACAATTAGCAGAGATGAATTmAAm~TAGTAAATrAAGcrAATi.TTMTACTAmAATAAAM 62650 L A Q J F L V L KKK Q F E K V Q L A EM " F - +'---- -> <----~ +

AAIGGAAAAATGGllACATTCATTTTGGTTAATGTTCCArTTTtCCATTTGTTi1AAAAAMATATATAAACTTTTTCccAGmAAATAAACAGTAAAGIITrCMTAnTITAT/lTTCAM' 62770 ----------~--,

AAAATTATATrrTTTGTTTTGTATTATAAAat.TGATCCTAA1CCAGllATATGMCCAlAAAAAAAGIITACCTACTAAACCGIITCACAAGGATACCAGClACAGTACCCATrMCCACAM 6Z890' <--~ _. L SSG L G S Y S G Y F F [ G V l G I V l I G A V T GIL ~ l

<OIlf~O

c .. . . .. .. .. .. .. .. .. .. .. AClAACGCT AA lCTT AAAGCACCGA TT AAI>MAAGAAAA T MCl AA TT A TTGT AAGCAT MAMA TeCT AA T MCMT MAAAAATT AAG TATAAA TACCAAA HA H AT AT AAAA TTTT 64091 V l A L T lAG I l F L f Y S J [. l L H AGGA <ORF31 '

---> .. .. .. .. .. .. .. .. . .. .. .. CAAATAAAAATGGTTTITTTATATATAAAGIITATATATATATAATmmTATTMTGllAAATTATACTTAATATAATATTAATATATATTATTATATATATATTATTTTGllCAAAAAC 63971

---- -- - - __ <TAlMT <AClCH +- - --- --->

TATTTAAAATAGATTGAAAAi.AA.w.AAeTTAAATAMTTAAGTAATATAAAAAATAAnTATTll1TJli-AGTGTAGIITAATTATTTCGGGIITAGIICcAATCTGTAAATATTACATATT 63851 +-- -> <-----+ +-- -> <--

.. .. .. ... .. .. .. .. .. .. .. .. GTGCAmGMCTTCGTTMmATTmATATAmATATATATAATTGTAATACATCTAmCATATAATTATTAATTAAATTMTAAmATTGTTTTAACTTATTTMmTTTTG 6)731 + TTGJ\AC, TATATT> > <---~

AA TTT ATCTTGCTGCGT AAAi.AGAACA TT AGCTATACT MGTT AGTATGCTrCAAAAA TACCTTTGGTA T AAAAACAAcAACCT AACAGGGm AAMGTAA TTTTCAGGAAGTTTTT AA 63611 1lIIf42a> Ht Q K Y L 1/ Y K " PI II LTG F K S " F E £ V F " ,

TCCTCTTATTrrTCGGATGllmCCCCCTTGmAAAAAAi.ATATAlGGAGClAACATGTCTGGllAATACGcGAGAcCCTCCTTTTCCloATATAATTACCAGTATTAGATATTGGCTM 63491' P L 1 F G K IS P L f K K II 11/ S -H S G H T G E R P fAD fiT SIR Y \I V J

p.bE> GGAG

fCCA T AGCA TCACTATACCTTCTTT A m A nGCAGGTTGGTT A mGTcAcCACAGGGrTCCmATGil TGTGTTCGGAi.GTCCTCGTCCAAATGAA TA TrrCACAGi.AAACCGACAAG 63371 IISITIPSlflAGlIlFVSTGLAynVfGSPRPIIEYFIEHRQE

AAGTACCACT AA 1 AACTGGCCcTTTTAATTCCTTAGAACAAATTGII lGAAmAtMAI; TCCTTTT AGGllGcCATTAA TcACTATACATAGAACTTATCcAATTlTTACcGTAAGil TGeT 6)251 V P t [ T G R F "5 l E Q IDE F T K S F - H TID R T Y P 1FT. V RilL

, p<bF. AGGAGe

TAGCCGfTCACGGIITTAGCTGTACCTACAGfTTTCTTTTTAcGTGCAATATCAGCAATGcAGTTTATTcAA.a.c...TMmTAAAAAAAArTrAGAACTATGACACAACCAAAICCAAACA ,53131 A V II G' L A V P T V F F L G A [ 5 A H Q F I Q R - O!!f38>H T Q P " P " K

Figure 48 and 4C.

-29-

AACAAAGTGTAG,.v.TTAMTi;GTACTAGCCrCTATTGGOOTTrATTAi:TMTATTTimcTrGCTGnTrATrrrCTMITAmmTMTTAMAArrITTTTTAAATMTTCTTTAC 63011 OSVEl~RTSlYWGlllIFVlAVLFSHYFFH-

+- ~-> (~-~t

i:nATcT~ci-mrTCATTATTTGTA.mGmATGiTriGAGTTATMCrAmMTAA.v.TTTIGAA.i.AGGAGTA.MnCMTGGCcAATACTACCGGAAGGGTTCC 62891 f-'.--> (_-+ ORF40> AGGAG II A 11 T T G R V P

TnGTGGCTMTCGGTACTGTACCTGGTATCCTTGTIlATcGcTTTAilTAGGTATCTTmTTATGGTTCATATTCTGGArTAGGATCATCmATMTAcMAACA.llAAAATArMTTTT 62171 l II L 1 G T V A Gil V 1 G l V G I, F f Y G S Y 5 G l G 5 S l -.. +--> (-

rTTGMT AT MAM TA rrGAi. TCm ACTGm A TTT MACTCCGAAAAAGm ATATA rinnnr AACAAA TGGAAAM TGGMCA IT MCCAAAA TGMTGrrcCA nTTTCCA TT' 62651 -to +--> <--+ +-:- ~-> <-- --t I (

rrmATTAAAIAGTATTMTATTAGmMmACTArrimAAATTMAMTTCATCTCTGCTMTTGTACTmTcAA...TTGmCnTTTMGMCTMAAATAmGTGCrMA 62531 + (_ ---+ • ...., f H II E A l Q V KEF Q KKK l V L r 1 Q A l

<petA

D rrAAAAACrTCCTCMAA rTACrrrTMACcCTaTTAGGrrGTTGTTTTTATACCAAAGGTA TmTWGCA TACT MClT AGTATAGCr AA rem ri-m ACGCAGi:AAGA TAM IT 6373()

II F VEE f II S K F G T L 11 " II K Y 1/ l Y K Q l II <1JRHZ.

CAA.MA.MrfA.MTMGTTMAACMTMATTATTMmAATTAATAAnATATGAAATAIlATGTATTACMTTATATATATAAATATATAAAAATAAATTAACGAAGfTCAAATGCAC 63850 _> <--+ <TTATAT (CAAGTT +

AATATGTAATATTTACAGATrGGTCTATCi:CIlAAATAArTATCTACACTAMMMAATAAATTArrrrTTATATTACrTAATTTAmMGT II fill jill i CMTcTATTTIMATA 63970 <-- ~.. t----""- -) <- ~-..:..-....t- +~

GTTTTTGTcMAATMTA!ATATATMTMTATATATTMTATTATATTAAGTATAArrTrCATTMTIlAMAMATTATATATATATATCTTTATATATAAMMACcATrrrrATTTG 64090 TTGTCA, TMTAT> +----- - - -- ----

<-- --- -+

AAMrTTTATATMTMmGGTATTTAfAcTTMTTrrnTATTGTTATTAGGAmrTIATGCTTAcAATMTTAGTTATTTTCTTTTmMTCGGTaCmMCAnAGCGTTAGT 64210 <-- -- t AGIlA II L Til 5 Y F L r L I GAL T L A L V

ORF3!>

TTTATTTAriGGGITAAATMAATACMCTrAmAAAAAATMmAAAAAAGGITAmMAmCAnGTATTTCTCw.CTTTTrTrGAGAITCATAGTAAACTACMTACTMCT 64330 L fiG L H ~ I Q L I ~ +---,' <--,--+ +--, <-+ +-- --

MATTAGTTArrATTrCAGTrMT~TGGnGMGi:nrGTTGTCTGGMnGni-rAGGCTTMnCCTATMCmACnGGAnAmGTAACTGCGTATCTC 64450 -> <-- --+ ORF37> H V E A L ,L S G I V l G LIP I T L L G L F V T A Y L

CAATATCGACGTGGTIlATcAAITAGATCrTIMTTGAAMGTCMmTTGTmTAAGTCCTCCCmATAGGIlAGGITmAmTAnMAAAMAATTCACGCTCTGTAGGAmG 64570 Q, Y R R G 0 Q L 0 L - +- ----, <-- ~~ 3'-GUGCIlAGACAUCCUMAC

AACCTACIlACATTAGGTTrTGGAGACCTACGTTCTACCaAACTGMCTAA.v.GCGcnATrACTTATTAATTAGTATTMTATGMTTATATTTATAmATATACATAMTATATATAT 64690 UUGGAUGCUGUAAUCCAAAACCUCUGGAUGCAAGAUGGCUUGACUUIlAUUUUCGCG-5,' +--- - --- ---> <---

<T.-p--(CA

ATMATATATMTAccnAilMGGTAGGcATIlACAGllArTCIlAACCTGCGACATTTTGTACCCAAAAe.o.McGCGCTACCw.CTGCGcTACATCCCTAMcmTTTTATCTATCTGTA 64610 3' -AUCCCUACUGUCCUMGCUUGGACGCUGUAAAACAUGGGUUUUGUUUGCGCIlAUGGUUUIlACGCGAUGUAGGGA-5' <Prn-UGG < T

--- ---- - ---+

TTGTACITmrrrrrrTTeTTTGCCTAcnATTTTACnACTATATATATATATrITnrrCTTilAAAAGATAAAAAGMMAATATATTMAMATrrAmMAACMAAAAnTn 64930 MCAT <ACGIlAT _, <-' --> T

<----TGHTATTMTCTAGTTMCATMTTATGTGTAGTATATACTATATATMTATATATAAAMTGCMATGTTATAAAAAA.v....GGAGTMmMAATGCMGATGTMAAACATATCrT 65050 TGm, CATMT> 1 >( ORF~Zb>' AGIlAG M Q 0 V K T Y L

TCTACTGCACCTGTTrr AGCr ACA ITGTGGTTTGGGnrTI AGCTGGGrrGTT M TTGAA... TT M TCGITrmrCCAGA TeeTTT AGITCTTCCA TTT TIn MCA mAAAGT MAiA 65170 STAPYlATLWfGfLAGLLIEIHRFFfOALVlPfF-

AATGTCAAAGTMATIlACTTrrcATATTAAGTGTTMTTAArnATTAmTMTMTATIrrmMTAsGTGGTATAMCCTMAAmCMATAGAATTATGGCTAA.v.GTAAAIlAT 65290 ----+' .,,133, H A K 5 K 0

~------- -> <--~

ATAAGAGTci.CMTTMITrAllAATGTArTMTTGTGcr~TIlAT~GG{lTAmCTAGATATACrACCCAAAAo\MTCGTCGAA...TACACCMTrCIlATTccAA 65410 I R Y T 1 H l E C I II C A Q II 0 E K R K K G ( 5 R Y T T Q K 11 R R II T P 1 R L E -. IT AAMAAA r TTrG TrGTTA TTGT M T MAw ACTA TTi:ACAAAGMA T MAAAM T AAAAA m MAGcm AT MAA TTT AGTT A IGMCMA TCT iIAAAGA rcrrcTCGTAGGCGT 65530 L K K FCC Y C II KilT .I H K ElK K - .".18> II H K S K ,R S S R R R

ATGCCACCcA TT AGA TCAGGAIlAAA T AA riGA IT AT AAAM TAl AAGTTj ACTTCGTCM man AilTGAGCMGGAA.i.M TA ITA TeT AIlACGGA TaM T AIlA TTGACTTCAAAGcM 55650 II P P IRS G r 1 J D Y K /I I S l l R R F V S £ Q G K I L 5 R R Il II R L T S K Q

CAACGTTTATrMCTATAGCMTTAAACaAcCTCGTGrrTIAGcmGTTACCTTmTAMTMCIlAAAATTAATTTATCATTAmAnAATATACAGTTTTTTTAriAAACCTCCCC '65770 Q R l L T I A I K R A R V L ALL P F l H HEn' ._ _

GGMmAITTTmMTT~TCCGIlAGAGGmnnrAnc-rGTMTMTAnmAATrATTGTcGA.i.MAcwAmATcTAATATAGCTATTTcAaCTAGAAnTrrCTAmM, 55890 -, <' 1 ~ E Til II K liT 5 f C f K 0 l I A I Q A l ( K R H L

<",'20

Figure 4C and 4D.

-30'-

E ACCTGCCCAACCAGAAACTAMGCAGTATGCATTAMTcMCAGCGATTAAGCGACCTGGATCAmMcACAACTGTATGAACACGATACCAAGGTAMCCCATAAAAATACCccrnC 69011

G II \I G S V L A T /I H l /I V A [ L R GPO 'fI L V v T H V R Y \I P L 'C H <p;bB GGA

TCAAAGAGAA n AGACGCTA TGT AACTTTTTTGCA m AAM mATT AA IT AM TAGTTWCCCTmITACTCA TCCWAGGCAAcA T MCMMCT M TAT A mm ACCAA TA 6B891 <TMm <TGAGTA +--. <--+

+-. <--+

MCGTMGCACAAACACmACCATTTCTATCmAGGATMCAATGGAGAGATTGGTCCCATITmArTnACTTCAAnmAmATrCTATCTAGACACTAGACAMTAMTAM 66711 +- ~~-.' <----.. I -> (- ----

+--

iAAAAmn~TAmATMMnGTATTCTAmTATAGi.AAAACTATAi:ATMTMAT~TMATAGMmCAmi-rACGTnTTi:T!',m.TAGMGAcTAmfGmGTGGM 66651 TTGTAT> TATMT. GGA

~TGCCTATTGGTGTTCCGAAi.CTTCCTTrTCGTCTCCCAGGAGAAGAAGATGCTGHTGGi.TTGACGTATMTGCGCCTTATTCAATAnTrAGTTATATGcMAGAATC 68531 ORfZ03> H PIG V P K V P F R LPG E E 0 II V II [ 0 V y' gu9Y9 .............. (tntron) ••••••••••••

CGTCAITmGCAGACrAMCTCTTTrTTATrCACTTAMmCAAAMTATATCAAATTrTTAMCCGTGAATTTATATIAMAAAATTCATTATMAATrCTATGGTTMTTMAATA 6641 I ........................................................ (lntron) ........................................................ .

AATAMGTATTMAACTTCrITCAATTCrrTCATAATMcTMATMACMTAmMAATrTTATMArTCAAAMTTAITTCmATATGfACAAATAATACTCAGAGMAmnrA 6B291 ........................................................ (lntron) ....................................................... .

iGAAGTAGAACATAAACCfMCGATTTnTATTCMAACTATmATAMTMGAAATATTTATTGTTTMGAAAMAATATATATATcAATAAATAAAMATMTGTTcAATTAGCAM 68111 ........................................................ (lntron) ....................................................... ..

GTAGCAMATIGMCTACAATrfCTAAAAAiw.GCTMTTTTTACAATAACTTMGCTGTATGCCCTTAMAAGTGCTTOTACACTTTTATAAGAMAAAATMTMAArTATCTTAATC 68051 ............................ 0-.0- •• I .. I ................ 0- ...... ragce-9-bug~.)-----c;JaOla-UUC8.U91J-c99wuy ••••••••• ~ ~ •• o. ~ .......... CU{Jyy-y-ay

AATCGACTTTATCGTGAMcATTACTmrITAGCCCAACAAGTAGATGACGAAATAGCAMTCMCTTAnGGTATTATGATGTACCTTAATGGAGAAcATGAAAGTAMGATATGTAC 61931 II R l Y R E R L l FLO Q Q V ODE 1 A IIQ L [ G I H H Y L II G E U E S K 0 H Y

TrATATATWTTCTCCTGGTGGTGCfGTTITAGCTGGMTnCTGTnAfGATGCGATCWmGTTGTACCTGATGTICATACAATTTGTATGGGATIAGCTGCTTcAATGGGCTCT 61811 L Y IllS P G G A V lAG I S V Y D A H Q F V V P 0 V tl TIC H G L A ASH G S

nTATTTTMCAGGAGGAGAMTTACTAAACGTATAGCACTACefCACGCTrfCTGCCAATGAfHTTTTATGTCTGCACWAMAGGTAAAAATMCATCACATATATATTTTTATAC 67691 f J l T G· G E [ T K RIA L P tj A 9u9Y9 ................... (lot"'n) ................................... ..

AAMAT~TATAMTTArAnri:rmAAGTTTATTCTAGCGITATAAACTMTAATTAAAMTMAnmMTMTTMAi..McTrTGcMTTGCTCMTIMTATTTTC 67511 ................. ; ...................................... (lnlron) ....................................................... .

ITTAmAGcAATAGAGCTATCATAATAAAAMAGATACrTnMATTATATMTf.mATMATTTTTAMTATAAAAMAMTATATATATATATAArTAAAATAGAGCrCTAfGCAA 67451 ........................................................ (1ntron) ........................................... r.gcc9~.u9 •• -. . .. .. . .. . . .. .. .. .. CTMMATGCATGTACACTTCGmCAmATTTTTTTAATAAAAAAAATMAAAATTGMTAmATTMTAGGGTTATGATTCATCAACCTGCTAGTTCnATTATGATGGACAAGCT 67331 '"') ••• -uuC.u9U-t:99uuY ............................. , ........... cu.n-y-.yR ~ H 1 \I Q ~ ASS Y YOu \) A

GcAGAATGTATTATCGMGci.GAAGAAGnTrGAAACTTCGTGATTGTATTAeTMAGTTTATGTACAAAilMCTGGTAMCCTTTATGGGTMTTTCTGAAGATATGGAAAGAGATCrT 61211 GEe I H E A' E E V L K L ROC I T K y. Y Y Q R T G K P l W V I SED HER 0 V

mATGTCAGCAAAAGAAG~CTTTATGGTATTGTAcACTTAGTTGCTATAGAAAAcAATTCTACTAnAAAMTTAGITmMAcAAAMAAmTArnGTTATGCTTAGGm 61091 F H SA K E A K l Y C I V 0 t V A I Ell 11 S T 1 K II ~ +__---. ATCCAMCTMAAAATTTTCCATATAAGTTACAATcecTAi:TATTCMcAATTAATTMMATAAAAGAcAACCCATccMMTAGAAcMAATCACCAGCCCTTAMccATGC"TCM 66971 <-- ---~ rpsl2A> H P T I Q.Q L [ R II K R Q PIE 11 R T K SPA L K C C P Q

CCTAGAGGAGTATCTACTAGA.GTGTATGTGCGACTTGTTTAAATCAMAACGTTMMAmAAAGATCAMATTGCATAAMATTTTTTTATTTTMTAACCTMAGATATAGTATCTA 66B51 R R G V C T R V Y qu9l'11 ............... (Intran 7) +--. <--+ +-.-. TTGTTGmAGATACAATTTATAGmCCrTrGGTGCAMTCCAATCATCnAAGmAGGATAGMMCCAmCTCAAAGGGTAGCGACrGATTCTcMTCCCTTAAGCGAGMAm 66731

<--> +--- -- -- -- - ->

TATTMAAAATTTTTGCATMTATAATArTACHATATMCCGTMMACGAAACTGMCGGTCAGCTATICAGCGMCCnCAAATMcATGCCGTTAAnMTAAAAAAAACAAmT 66Gll -->

TGAMCTmi:irAGTGmCATTMTAAAAMAGCTTCAAATCAGAMTIATACAMTMCTGATATTATCMTATATAITATATATTACAAGCTTCceTATATAGAMCGACCTATTC 66491 --> +--.<-->

+---- <-- --- ----+

GCAM TOC TmGGT A TTTTITTTTT ATAci. T AAGAAAACGAAGAAA mm AT AGCAA rCT MGMAA TAAAA T AAAACmTrT A TT AT AAAAA TTGT AGA f!ATAGr AAGCAMCT 66251 ~-> <--> I- --> <--->

GCAATMAAAAATAmATTGMAATCGATGTTTTGATATAAAAAMTAci.CACACACAAAnmGAATAATTMMCcAGTATATACAGCAATGACTAGAGTTAMCCTGGTTATGTA 66131 +--> <--+ rplll). H T R V K R G Y V

GCACGMMCGcCGTMAAATATTCTTACGCnACATCTcGAmCMGcAACTCATTCcMACTTTTTAGAACTGCTAATCAACAAGGAATGAGAGCArTAGCATCATcTCATCGCGAT 66011 ARK R !I K II I L T L- T S 0 F Q G T II sx L F R r A tl Q Q G H R A LAS S H R. 0

AGAGGTAAAC~TCTTAGACGriTATGGATTAC:TCGAGTTMTGCAGCCGcMGAGATAATGGAAmCCTATAATMATTMTTGAATAmATATMAMAAMATrCTT 65891 R C. K R K Rill R R l ~ I T R V II A A A ROIl CIS Y '11 K LIE Y l Y KKK I L

TrMATAGAA.W.TTCTAGCTCA.AATAGCTATATTAGATAMTTTTGrni:rCGACAATAATTAAAMTAnATTAC~TAAMMAACCTCrCeGGAi-MTTMMAAATMATTce 65771 l II R K I l A Q [ A I l 0 K f C F S T I I K II I I T E -.

Figure 4E.

-31-

G.GGGAGGmMT~CTGrATAnAAT..w.TAA1GATAAATTMITnCGTTAnj-AAAAAAGGTMcAAAGCTAAAACACGAGcTCGmMTTGCTATAGTTMTAAACGTTG 65651 -.---t . _tIE'NIlLFPLLALVRARKIAITLLRQ

<'Ps1B

F . . . . .. . . . . . . . GATTCTTTCCATATAACTAAAATATTGAATAAGGCGCATTATACGTCMTCCAAACAGCATCTTCTTCTCCTGGGAGACGAAAAGGAACTTTCGGAACACCAATAGGCATTTrrTTTTTC 68650· ••••••••••• (1nlron) ••••• : ......... 9Y9u9 Y V 0 I II V A DEE G P L R F P V K P V G I P H <ORFZ03

TTCCACAAACAAAATACTCfrCTATMTAAAAAAACGTAMMTGMATTCTATTTATTTATTTATTATATATAGTTTTTCTATMMIAGAATACAATmATAAATATAAAAATTrrr 66770 AGG ., ',TAATAT . <TATGTT

+- --- ~- -~- --- ---> nTATrrATTTGTCTAGTGTCTAGATAaMTAMTAAAMTTGAAGTMMTAAAAAATilGGACCAATCrCTCCATTGrTATCCTAAAcATAGMATGCTAAACTGmGTGCTTACGrT 68890 __ • __ _)I . (-- '. . +----> ,-~- ---r ---fo.

TATTGGTAAMAATATATTAuTmcnArGTTGccmGGGATGAGTAAAAMGGGTTG.AACTAmMTTMTMArTnMATGcMMMcTTAcATAGCGTCTMTTmmGA 69010 +---> ,-. ATGAGT> TTTAAT.

+--> '-·-r GAAAClGGGT A nTITATGcGmACCTTGGTATCGTGTrU. TACAGTTG rGn AM TGA TCCAGGTCGCTTAA TCGCTGTrCA m AA TGCA TAC recTTT AGTTTCTGGTTGGGCAGGT 69130

AGG p$bBl- H G L P II r R Y liT V V L 110 P GR L I A V H L H II TAL V S G WAG

TCTATGGCrTrATATGAATIAGCTGnTITGATCCTTCTGATCCAGTTCTrGATCCMTinGGAGACAAGGCATGmGTTATACCmTATGACTCGTTrAGGAATMCGAAATCCTGG 69250 S II A L .y E LAY FOP S D P V LOP H U R Q G II F V I P F H T R L G 1 T K S \I

GGGGGTTGIlAGTATTACAcGAGAAACTGTTACTAACGCAGGTATCTGGAGTTATGMGcAuTAGCTGCAGTACATATTGITTTATCAGGATTACTTTrrrTGGCAGCTAmGGCATTGG 69370 G G II SIT G ( T V T II A G I W S Y E G V A A V fl I V L S G L L F L A A 1 \I H \I

GTGTATTGGGAmAGAAcTGTITCGTGATGAAcGTACAilGTAAGccncmAGAmAccTAAAATrTnGGMTTcATTTGTTTCTTTCTGMGTAcmcnTIGcnTIGGAGcA 69490 V Y \I 0 L ELF ROE, R T G K P S l D L P KIF G I tI L ,F L S G V L C f A F G A

TTTCATGTMCTGGmATTrGGTCCTGcMTATGGArrTCTGATCCTTATGGATTAACAGGAAAAGTACAACCTGTAGCrCCTGCTTGGGGTGCTGAAGGTTTTGATCCrrTTGTACCT 69610 FHVTGLFGPGIIIISDPYGLTGKYOPVAPAIIGAEGFOPFYP

GGAGGAATTGCTTCTCATcATATTGCTGcAl;GTATTTTAGGMTATTAGCTGGmGmCATCTTAGTGTTCGTCCTCCTCAAAGATTATATAAAGGATrACGTATGGGAAATGTTcM 69730 G G I A Sil 11 1 A A GIL GIL A G L Fil L S V R P P Q R L Y K G L R H G II Y E

ACAGTTTTATCCAGTAGTATrGCAGCTGTrTTTTTlGCTGCTTTTGTTGTrCCGGGAAcTATGTGGTACGGTTCTGCAGCAACTCCAArTGMTTAmGGTCCTACTCGTTACCMTGG 69853 TV L S S,S 1 A A V F F A A F V V A G T H \I Y GSA AT PIE L f G P TRY Q II

.. .. .. • • .. • • .. r • •

GATCAAGGATTTTTTCAGCMGAAATAGATCGAAGAATTCGCTCTAGTMAGCAGAAAAmMGTTTATCAGAAGCTTCCTCTAAAATTCCTGAAAMTTAGCTTlTTATGATTATATT 69970 o Q G F F 0 Q E lOR R IRS S K A E II L S L S ( A II SKI P E K L A F Y 0 Y I

.. .. . .. .. .. .. .. .. .. .. .. GGTAATAATCCTGCTMAGGAGGATTATTTAGAGCTGGAGCGATGGATAATGGAGATGGTATAGCAGTTGGTTGGTTAGGCCATGCAGTTTTTAAAGATAMGMGGAAATGAGCTTTTC 70090 GIl H P A K G G L F RAG A HOI/ G 0 G,I A Y G \I L GilA Y F K D KEG II ELF

.. .. . . .. .. .. .. .. .. .. .. GTTCGTCGTATGCCTACTTTTTTTGAMCTTTTCCAGTTGTmGGTAGATGAACMGGMTTGTTAGAGCTGATGTTCCAmAGAAGAGCAGAATCGAAGTATAGTGTTGAACMGTA 70210 VRRMPTffETFPVVLVDEQGIYRAOVPFRRAESKYSYEQV

GGTGTAACTGTTGAATTTTATcGTGGTaMCTTGATCCGGTTAGTTTTAGTGACCCTGCMCIIGTCAMAAATATGCTAGACGCGCTcAATTAGGTGAAAmTTGAATTTGATCGTGcr 70330 GYTVEFYGGELOGVSFSOPATVKKYARRAQLGEIFEFORA

ACTTTAAAA TCGGATGGTGTTTTTCGMGTAGTCCAAGAOOTTGGmACnTICCTCATGCTACATTTGCTCTTCrrfrTTTCTTTCCTCIITAmGG!:ATGGTGCTAGMCATTGTTT 70450 T L K 5 0 G V FRS 5 P R. G II F T F GilA T F ALL F F F G II I W H GAR T L f

AGAGATGTTTTTGCAGGMITGATCCTGATnAGATGCTWGTGGAAmGGAGCGmCAGAAATTAGGAGATCCMCAACMAAAGACAAGTAATATMMTATATTTTATATCm 70570 R 0 V FAG I 0 PO LOA Q V E F G A F Q K L GOP T T K R Q V 1 ~

+-- -- - ----

TAAATAMTAAMTTTTTAGTACAGnTImw.cTAAAMTATTATTTMTTAGTACGAMGTTATcTGCAMTTAmAccTMTATACAMTATATGGMGCIITTAGmATACAT 70590 ---- -----,; <--- ._- - - - - ~ ORF35> H E A L ·V Y T F

nTIGTTGGTAGGTAcmAcGAATCATTrmTTGCTATrmmWGAACCACCTMAGTACCAAGTAMGGAAim..o.TMMCGTTMTATTcAATTAGTAArTrMTAnAM 70Bl0 L L V C T L G 1 [ F F A 1 F F REP P K V P S K G K, K ~ t----- -> ,---~

TTACTAATTTTGGACTAAlGACTTTTTAGTffAAAAAGT!:ATTAGTCcMMTTAGTCrTCATGTTCTTUMTGGATcTCTMGTTCAITAGAAGGTTGTCCAMTGCGGTATAMGAG 70930 --------->, I H F F K WIS· K FIR R l S K C G I K S -----r ORFZ7.

CATMCCAGTMAGCTTATMGTAAACAAGATATGAAGATAGCCACMAAGTTGCAGnTCCATTGTTAOOTAGTTCCAMATAATGGTATATTmMTGTATAnTITrAATATMTA 71050 ITS KAY Ii: .... ~~ t(-. -t .+--:-- <_~

GTACAAAAAimAATMATCTCMCTAATCTGATMGrrTTATGGCTAcACAMTMTTGATGACACTCCrAAAACMAAGGMAMAAAGTGGTATAGGTGATATATTAAAACCATTM 71170 0I!f74. HAT Q 1 I DOT P K T K G K K S GIG D I L. K P L II

A1TCAGAGTATGGAAAAGTGGCTCCTGGTTGGGGAACrACrccrcnATGGGTATTATGi.TGGCTCnrTrGCAGTTT ITrTAGTTGTTATTTTAGMCrTTATMTTCCTCTGmTGT 71293 S E Y .G K V A P G II G T T, P L, H C 1 H HAL F A V FLY V I L E L T II S S Y L L

TAf;ATGGAGTTTCIIGtrAGnGGTAATAMTMAAATT~TGMTTGciGCTTjnTIAGCAGCAATTCATTrffirAAnTIAGGTAGmMTTGTGTMTTATTAAAnc~ 71410 o G V S V SW -- +----,--. ---. > (-- I +-

Figure 4E and 4F.

-32-

AGGATTTnWTArGGGTiiTGCGTCTTGTGTAMTAMTCTATATTTATAAiACAAAATAACTTGTTACrGATATATTAAATATT~TTrniTnGTTAMTGTTTACAAAT 71530 AGGA p<>tB> H G 9"9Y9 ••••••••••••••••••••••••••••••• (lntron) ••••••••••••••••••••••••••••••• ~ ••••••••••••••••••••••••• -)0<---+

TTGTTAGCAmAMCCAcA.w.AATGAAAA.w:TTAMCTATGATTAATrTTTATAMmATTAGTTATACTTCGTTATCAATATAAi.AAAATTMrTATATGCATTMTCAAATGTA 71650 ....................................................... ( Intron) •••••••••••••••••••••••••••••••••••• , •••••••••••••••••••••

TGAAAATGTnATAAAATATAAAAAATGATMAAAAAGATrTTCACTCAnCTATCmrTTTfAGTCATCGGAGTTTMTAAAAATCTACCCTTTMTACTAATTATTAAGATTTAMC 71770 ........................................................ (Intron) ........................................ ; •••••••••••••••••

AAGAAAA T AMAAAAAA T AMAAGA TTCCTCAAAAAAAAACA TATATAT AAACTTGAGA T AAAAACAAAM TAT AM rrTrrncm MGCTCT MeAn AT AM TM TCA TTT ACCCT 71890 ... .o .............. of' I .............................................................. (intron). 0- ..................................... ... bgcclJ-au911i:1-----g~aoCll--uucIl1l9u--cg9u

TTTTCGACGGCGAACTrrAilTAAccTATCTCAATAAAGTAiACGATTGGmGAAGAGcGTCTTGAGArTCAAGCGATTGCAGATGATATAACMGTAAATATGTTCCTCCACATGTTM 72010 "y ••.••••••.•••••••••••• C".yy-y-.y~ V Y 0 \I fEE R LEI Q A I A 0 0 ITS K Y V P P II V II

TATrTTTTATTGTTTAGGAGGrATTACrrTMmGmTrrAGTTCAAGTAGCTACTcGCTTTeCTATGACTTTnArTATCGTCCTACrGTMCTcMCCITTTTCATCTCTTCAArA 72130 I f Y C l G G I T LTC f L V Q V A T G f A II T FY Y R pry TEA F S S V Q Y

CATTATGACTGAAGTAMrTrrGGATGGcTTATTCGCTCAGTTCATCGcTGGTCAGCAAGTATGATGGrTTTMTGATcATTTTACATAmTTCGTGrTrATCTMCAGGAccTTTTAA 72250 1 H T E V II F G 1/ L 1 R S V II R \I S ASH II V L II II I L II 1 f R V Y LTG C f K

AAAACCTCcGGM TT MCrTGGGTT ACTGGTGTT A TTTT AGCAGTTTTAACTGTATcrrirCGTGTT AcAccTTA TTCm ACCTTeccA TCAM TTCGn A nGGGcAilrr AAAA TTGT 72310 K P R £ L T 1/ V T G V 1 .L A V LTV S F G V T G Y S L P 1/ 0 Q I G Y 1/ A V K 1 Y

MCTGGrG r ACCAGMGcM TTCCAA T M TrGGA TCTCcTn AGTTGAGTr A TTACGccilAAcTGT MGTGTTGGTCAA TCGACA TT MCrCGA TTTT AT AGTTTACA TACTTTTGTATT 72490 T G V PEA I PI 1 G S P L VEL L R G S V S V G QS T L T R f Y S L" T F V L

GccrCTTTTAACTGCAATATrrATGTTMTGCACTTTTTMTGATTCGTMACAAGGTAmCAGGTCcGTTATAMTTACGTAMTTTATTACAAAATAAAMAGTTTAAATACTMrT 72610 P L L T A I f H L H II F L H 1 R K Q GIS G P L - t-->

+---> <---. TCA meCA i A TTTT ATGcA TTTTTTTTTTCTA mGAAi.cTTcTTTTT AGAGAAA TGeT AAAAAAAmTTTTTMT AGATATTTTATi.AAccAAAAT,w,n ATGGGi.GTGTGTGACi: 7Z 730

<_ +-> <----;- AGGA petiJ> H G V 9"9YG •••

TTAmAATAATMTTTGAGTTATAeAGAMTTATTTAATATCTGTTAcATAAAATTTAATMGATTAT[iTATTTTTATCCCAATTArrTrrnrAGTAAAAACTTGGGTrATMTGrrT 72850 ••••••••••••••••••••••••••••••••••••••••••••••••••••••• (lntron) ......................................................... .

TTCATTCT AAM rrrrrni-cTA TGA TCA TrrrrGAA T AilTAAAGACTTCGTT AM TeeM TAM TT 1\ rTrCGAA TA rrTCAAAA TilT AT AAGAAAGA TAGTATTAAAAA TACA TTCA IT 72970 ••••••••••••••••••••••••••••••••••••••••••••••••••••••• (lntran) •••••••••••••••••••••••••••• : ••••••••••••••••• , ••••••••••

TCTGTTGTGi I iii 1111 i ; TTACTATCGGccTAAAAAAAAGATCTMTiw..w..w.AACAAAATTArTAATAAGTTmmTTTTATAAAAAAAATAAAGACAATTCMMMATcA ·73090 ••••••••••••••••••••••••••••••••••••••••••• ; ••••••••••• (lnlron) •••••••••••••••••••••••• , ••••••••••••••••••••• , ••••••••••

AAAATTAMCrrGMTTATGAACATAAAGTrrnTTTGATrMAAAArrTCATTAATGrTGGACCCGGATGATATTAAAnATCATGTCCGATTCmGGGGQGACrrrTrrMTCTAC!: 73Z10 ••••• ' ........ I •••• ~ .......... I ................. I .. (intron) ......... 0 ....... r-.l!i9CC9 ... 4ugal!l..........gai~ul-uuclugu-cgguuy ••••• I .......... ~ •• I •• CU.lI.Y)'

TTMTMCAi.AAAAACCTcATTTAAGTGATCCTATATTACGAGCTAMrTAGCAAAAGGTATGGGACATAATTATTATOOTGAGeCTGCTrCCCCAMCGATCTTTrATATATTmCc.i. 73330 -1-'1 T K K POL SOP I L R A K l II K G H G II II Y Y G EPA \I P II D L t Y I F P

GTAGTTATrTTACGTACTATrGeCTGTACTGTTccm...cCTGTTTTAGMCmCAATGATTGGTGAACCTGCAAATCCTTTTGCAAeTCCTTTAGAAATTTTACCAGAATGGTArTrT 73450 VVILGTIACTVGLAVLEPSIIIGEPAHPFATPLEILPEI/YF

mCCAGTTmCAMTACTrCGTACGGTAcCTMTAMCnTrAGGTCTACTTTTMTGGCTGCTGTACCTGCAGGArTATTMCAGrTCCTTTTTTAGAMATGTTAATAAATTTCAG 73570 fPYfQILRTVPIIKLLGVLLMAAVPAGLLTVPfLEHVNKFQ

MTceTTTTimcGTcCAGTAGCTACTAcAcTIITTmAATAGGTACTGTCGTAGCTcrTrGGTTIIGGAATTGGAeCTGCrrrAccTArTGATAAATCmGACTTTAGGTTTGTTTTAA 73690 liP f R R P V A T T V f L Ie T V V A L \I L GIG A ALP lOX S L T L G L F ~

+-

MTATATArTTTTTTTATAAAACTAGAAATMGGmGAMTTTTTTACTMAAAGTAAi.AAAmc.W.CCTTAmCTAGTTTTATAAAACGTTTTTCAATCCAATTACCTMAGATA 73810 ->(- II - l Y

<rpoA

G Buill • . . l. . . . . . . • . GTCCGCCACTGGAAACACCACTAGGATCCTTCCCGTIICGACTTGCATGTGITAAGCATGCCGCCAGCGTTCATCCTGAGCCAGGATCAMCTCTCCATCAGATTCATMTTATATTAm 82091 CACCCGGUGACCUU\JGlIGGUGAUCCUAGGMGGGCAUGCUGMCGUAtACAAUUCGUACGGCGGUCGCAAGUAGGACUCGGUCCUAGUlJUGAGACGuACUCU-S' <165 riUIA

Tr ACrr AT AGCTTCCTTTTTC6TAAACAAAGCAGA mcAA... TCGTCrrcCA TCCCAAGAGA TAGA T AACTr .nom A rTrrrCATTtACrrCA TATTAilcnGMGCTCA TiTeT AGi . 81971 . <TCA

ATACCCATACCrACCCTATTATGTCAATCCCACAAGCCTCTn-CMTMcMGMAACMCAAATCAAAATGCTTTAACTATTTTTAGGGATAATtAGGrTCGMCTGATGAcTTCCACC 81851 TAT <ACAGTT 3' -AUCCCUAtlUAG1JCCAAGCUUGACUACUGMGGUGG

ACGTCAAGGTGATACTCTACCGCTGAGTTAjAICCCTATTCr~rTAAAATCTGAi:TTCTAAAAAATATATTArrTATAGATMTATATTTTTTCTATTTTCATT 81731 UGCAGUUCCACUAUGAGAUGGCGACUCAAUAlIAGCGA-5' <V41-<W: <TTATAT <fM

CAAATTmTAMcTrAMTCTCAAAMTTTAAGAAACTTATGCAACCArTMTTATTTcATATMTTATATTACtACcrTACCAATCCAAAw..wJ.TACTATTTAGTAmMTATA 61611 GTT +--> <_ <TMTAT <TAGGTT _, <---+ <-

Figure 4F and 46.

-33-

CrrnGTrTAi:~GACAAAAi.AA TAT AAAriAAAM m AT A TTTT ATM r A.w.w.A TT AG TTGAcciciGAM TG1CTCA TGA T MM TTGTCTTCT AM TAM TACTTA TTmm 81491 ~_> (~ +---:),(--' I-

+-> <-+ +--> <--+

eel All TeTAcGA TCA ~ AMTIOOTTCAA iTmtn ATGCAAACAGrrTiil AAAMAt.GeTACACtn AAtnCAAJ>AAaTT ATATACGTTITTleCrTrm MCA TI AAAA T AGAC 81371 +----> <~ . .....---:-> <-'-, ..... --><-- ~I-

AAnTrTTAcATAmGmTATACCTATAITmAGrrrTnATACGTAGGTAMTAMAA,w.CACTATATTCMMATAGAAAAAAMGACATAAAcTCACAGMCcAAAGCAAAM 81251 j--- ~-- ----> <- -' - - -- --'---+ .....

TmrAAAAMTMAMAnCCACTAAAATi;rATMTCAcACAGATAAGcTCACGCTMci:CGTCAMnITrATGTTAAAMAATACATATA~GAAMATMTTGATTC 61131 <-'-' -I- - - +---> <-I- '

lR <- J LB -. LSC

AAm~TAAMTGnATMACTATMTCAT'cCMITMTATCTATTATIMTAMTMTATA1MAAAGCATCCATGGCTGMTGGTTAMGCACCCMCTCATMTTGGCGMT 61011 HTMG> TATMT> nD-CAU. 5'-GCAUCCAUGGCUGMUGGUUAMGCACCCMCUCAUMUUGGCGMU

TCACAGGTTCAATTeCTGnGGATGCAmAMMMTTcMTTCMGTAATATATCnCTnATATATAAATATMnTTnATATACTTmMCAMGTMAMGTTCTATACAGTG 60891 UCACAGGUUCAAUUCCUGUUGGAUGCA-3" . _. <-----+

TCTAAMMAITTATAMn;\TAGAMTAcATATCAAATTGMmMGG.i.GAMTTATAAAmATGMTCMGTTMGTACCCAGTACITACAGAAA.D.AACMTTCGTi'TATTAGAM 80771 . AGCAG rp123> Ii I) Q V K Y ? V L T E K T I R L l E J:

AMATCAGTATAGmT(J,\TGTCMTATTuATTCAMTAIW.CACAMTMAAMATGGAITGMCmTCmMTGTTAMGTTATMGTGTMATAGTCATCGTCTTCCMMAAM 80551 II Q Y S f 0 V' /I I 0'5 II K T ,Q I K K U I ELF F )) V K V I S V fI S II R L P KKK

~TAcGTACGAcAAcAGGATATACTGTTCGrrATMACGMTcATTATMMriGCMTCTGGTrArri:;ATTi:CATTAnCTi:AMTMATMAAMATTnATTACaTm 60531 K K I G T T TG' ,Y T V R Y K R M I I K L Q S G Y 5 I P L f 511 K .~

ACA TAccrAT AA TT ATATCGCCA TAcimTATATCGAGCTTATACGCcAGGCACGCGT AACCGATCTGTACCT AMmcA TGAM T AGIT AM roTCAGCCACAAAAAAAA TT MCA T A 60411 rp12> H A J R L Y RAY T P G T R I) R S V P K FOE I V K C Q P Q K K L 1 Y

TMTAMc.\TATTAAAAMGGTCGAMCAACAGAGGMTcATMCMGTcAACACCCAGcAGGTGGACAcAMAGACmATCGMAAATAGATTTTCMCGAMTAAAAMTATATMC 60Z91 II K 1I I K'K G' R " " R '0 I ITS Q II R G G G II K R L Y R' KID F Q R I) K Y. Y 1 T

.. .. .. .. .. .. .. .. .. .. .. .. TGGGAAAATTAAAACTATAGAGTATGACCCAMTCGTAIITACATATATTTGTCTAAITAATTATGMGATGGTGAMMCCATATATTTTATATCCACGTGGCATfAMTTAGATGACAC 80)7) G ~ I K T I. E Y O' P II R " T Y I C' L I II YEO G E K R Y . I L Y P R G' IX L DDT

MTTAmCTAGTGAAGAAGCACCTATmMTTGGAMTACeCTAeCrrTGAGTGCGGmGMTTATATAmACGTcGTCGGMATAACCGACTMcAMTMACTTATAMTCTAT 80051 I ISS E E It P [ l I G H T l P L Tg"9Y\h .............. (lntron) ..................................... :.

CACTM TCCAP.GMA TTGGAAAGACCTT MAAcGAMCTMMAGGAJoJo.M TAGGCMGTGAAAAAGGTTm M TAT AT AAM TMAAAAACTTCAAl\!iATA TTA TM Tis. TGGAMA TT 79931 ••••••••••••••••••••••• ~ .................... ,: ••••••••• (Intron) ••••••••••••••••••••••••••••••••••.••••••••••••••••• : ••••

TmAMGCATTAMGTMTATATMMTAGGAMCMTTTTATTCAMcAMTTTATAATMTMAMGTrACmTAITATGATTTGTAGGTCAMGACAMm~ 19B11 •••••••••••••• : .......................................... (Introo) ......................................................... .

GAMcTMTTATGCTTCCTAAGTTATATMAATAmMAGCGTAMTAAATAAAGTCATccmCTGATGCTAAAiGAATATCATAAGCCAGATGATGcAMAMcCMGGACGTAMA 79691 ..................... ,., ........................... · ...... (Intron) .......... , .............................................. .

AACCAAGGACGGTAAAAMCTAMTTTmAAMCGTCTAGAAAAcCTGTA TGCITGAM.W.GCITGTACAGmGGGA.i.GAGAITTTMTATMMM mAAMTCTAtnCMCCA 79511 ..... 111" I ..... I ............... 0- ................. 1 rI:l9Ctg-i::.,Jga~-ga.l!I.a--:--uuc:a.u9U---c99uuy • .............. 11 ................ 0.0 ............... CUOYYY-l!!lY II

ATATGCCA TTAGGTACTGCTA TTCACMTAITGAM T McACCTGGMMGGTGGACAA ITAGT MilAGcAGCCGGAACTGT AGCMMA ITA TTGCAMi.GAAGGACAGTr AGrr ACAe H P L G T AI fl HIE I T P G ~ G G Q L V R A A G T V A K I [ ~ KEG Q L V T L

TACGCTTACCTrCAGGAGMATrAGATTAATCTCTCJ!.4.AAATGmAGcMCMTAGGAci.AATTCGMATGTTGAlGTAMTMlTTMGMTAGGTAAAGCAGGGTCAAAACCTTGGT R L P S· G [ I R l J 5 Q K C L A T I G Q I G II V 0 V II )1 l RIG X A G S K Rill

TAGGTAMCcACCAMAGTTAGAGGAGT AGITATGMTCcTATAGATCACCCTCACGGTGGTGGAGAAGGTAGAGCACCcATTGGTAGAAA.w.ACCATTAACTCCTTGcGGTCATCCiG GKRPKVRGVVHIlPIOIIPlIGGGEGRAP[GRKKPLTPIIGIIPA

CAcTTGGMAi.AGMGTAGAAMMTMTAAATATAGCGATACrCTTATTCTTCGTCGTCGTAAAMTAGCTMGCTAAAAMAGAMGAAAMTAMGGATAGTTGGcMTGACACCTT L G K l! S R K I) II Y. Y SOT l r l R R R Y. " 5 ... rpsl9> AGGA H T R S

i:MTAAAAAAAGGTCCrmGTAGCTGATcAmATT~TAGAAAATCTTMCITMMAAAGMw.MAMTMTMTMCATGaTCTCGAGci.TCTACAATTGTACCTACAA I K K G P F V A 0 II L L K K I E II l )1 L K K E K K I I I T II S R A S T ,1 V P T H

TGATTGGTCATACMTAGCTGTTCATMTGGACMGMCATTTACCMrrTATATMCAGATCGTATGGITGGTCACAMTrAGGAGAAITCGCTCCTACTCGAACTTTTCGAGGACACG IGHTIAVllllnQElJlPIYlTORHVGIlKLGEFAPTRTfRGIiA

CAAA.AAATGATMAAAATCCCGTCGTTMITAGGAGArMITnrMTGci.AAcTAATAcTTCTMlAAAAMATCCGTGCrGTTGCTAAACATATACAT;'~GTCTCCAcATAMGTACG K )/ 0 K K S R R -- AGGAG rp122> H Q T· 'II T SilK K I R A V A K II I II H S P H K V R

MGAGT AGTT AGTCAAA TTCGTCGTCG7TCIT ATGAACMGCACTTATGA r A IT AGAGITT ATGCCGTATCGAGCA TGcM TeCAA fA miCAA TTACTrTCA TCTGCAGCTGCAM TCe R V V S Q r R G RS Y E Q A L Ii I l E r Ii PY R A ell P I L Q L L S 5 A A A II A

TMTCATMITTTGGATTMGTAAAACAMCTTAmATAAGTGAMTTcMGTAMTAAAGGMcnnTnAMAGAmCMCCMcAGCTCAAGGACGTGGCTATCCTATACACM II II II f G L S K T II L F I 5 E J Q V " K G T F F K R F Q P R A Q G R GYP I H K

ACCTACTTGTCATATMCrArrGTACTGAATATTmCCTAAATAAAAMAATTGAAAMi.mGTTMTATMTlT~TATATATATGGGA~TMACCCACTTGG P T CHI T I V L II I l P K """ +---><- __ rps3, H G Q K I II P l G

mT AGACTTGGT A T MCAcAAM TCACCGCrCA TA TTGGTnGCAAACAAMM TA TTci' AMGTTTTTGMGAAGA TMAAMATACGTGACTGTA TTGMTT ATATGTACAAAMCA r R L G IrQ 1/ H R S Y \I F " I) K K Y S K V FEE D K K I ROC I ELY v Q Y. II

Figure 4G (continued).

-34-

79451

79331

)921J

79091

76971

16851

78731

78611

78491

18371

78251

TATAAAAAATTCTTCMATTATeGAeGAArTeCTCGTGTTGMATTAAM!w..w.CAGATnMTTcMilTTGMATATATACAGGATTTCCTGCmATrAGTAGMAGCCGAGGTCIl 18131 I K H S 5 " ~ G G, I A R V E I ~ R K T D l I Q v E I , T C F P ~ l L V E S R G Q

AGGAA TTGMCM IT MAA rTMA TeT ACAAM Til TA TTA TCTTCAGMcA 'r AGMGACTCCGM TGACTTT M TCGMA TTGCCMACCCr IICGGAGAACCMMII ncTTGCMAMA 78011 G I E Q L K LII Y Q 'Il I l S, 5 E 0 R R L R II T LIE I. A K P Y G E P K I' L II K K

AATTGCmAAMTTAGAMGTAGGGTTGCrmAGIICcMCAATGMAAAAGCCATTcMTTAGCAMAi.MGGAMTATAMAGGMTTMMTACMATAGCAGGTAGACTTMTGG 7)891 I A L K L E S R V A F R R T H K K A J E L A K K G /I I K G I K J Q I II G R L II G

AcCTGAAATTGCTCGTGTTcAATGGGCACcAGMGGTAGAGTTCCmACMACMTMGAGCACGMTTAATTATTGCTATTACGCAGCTCAMCMTTfACGGAGTArrllGGMTCAA 77771, A E JAR V E \I ARE G R V P L Q T I R II R I II Y C Y Y II A Q T I Y G V LG 1 K

AGmGGATATTTCMGATci.AGAATMTTAmnrrcMTCMATCIICTTTMTTATr.AAmMCATMAMMMATrGCrATGCTTAGTGTGTGACrCGmAmCAMATGTT 77651 V W I F Q 0 E E ~ rp115> H LS 9ugY9 •••••• (lntron) .......

t---~- <- ------+

ACTTAAAAAAi:AMATTGMACTCTAGmATACTAGMMTMTTTAToArmATATTAGMMTArN...r.ACACrTTci:~CMATTrrcTTGTGAAGCG"""""AMCTMTC 77531' •••••••••••••••••••••••••••••••••••••••••••••.••••••••• (I.teon) ...................... .' ................................. .

CATMMATTGTAGGGmTTGTTATAGTATrMAACGcMMAMTAAGAGCTTTATTTTMTAAMACTMGAMATTAAMilGMMi.MAAGcmATTATAGAMi.MAAcCIM 77411 ....................................................... (IAtron) ••••••••••••••••• , ...................................... .

ACAMATGTATMMTCATAAAMCGMGcAATCTATAMTMTAAMACTrrmGTATfTTTAmATCAGATAGGATGGCGAAAAAAACCMAMTAAATTTGMATAACTTAMAT 71291 ....................................................... (Intron) ........................................................ .

i.cMAmMAmATTACAAATMMAATTATTMAIiAGTAMTATTCGCCCGTGGArrTrTTTATTTTATATAMTTTATTCATGAccAGCCGGATcMTCAMATTTCATGTCCGGT 77111 ........................................................ 0- ............................... (1nLron) ......... 0- ................... • rllgC:~9-4~9"4--g44a-lJu'c4U9U-C~H)U

TnGMGTAGCGATCAMTCGACTATMCCCTMMGMcAAMmCGTMACMCATTGTGGAMmAMAGGMTATCTACTCGAGGTMTGTTATATGTTTTGGcMAmcCGC 71051 "y ................. cuoyy-y-.yP K R T K F R K Q II C GilL K GIS T R G /I V I C F G r. F P L

TTcAAGCACTCGAGCCCTCTTGGATMCArCrCGACAMTAGMGCAGGTCGCAGAGCTATMCTCGCTACCCTCGTCGAGGTGGTAMrTATGGATTCGTATAmCCTGATMACCM 76931 QALEPSWITSRQIEAGRRAITRYARRGGKtWIRIFPDKPJ

TrACTATTCcACCTGCAGMACACGMTGGGATCGGGTMAGGATCTCCAGMTATTGGGTAGCTGTAGrTMACCTGG...AAMTACmATGAMTTAGTGGCGTATCTGMAATATTG 16811 T I R P A ErR H G 5 G K G 5 P f Y W V A V V K P G K I LYE I S G V SEll I A

CrAGAGCTGCGATGAMATTGCAGCATATAAMTGCCGATACGTACTCMTrrATTACMCATCTAGmAMTMMAACMGAMTATAilMMMTTACTMTTAGITMTTATATA 16691 R A A H K I A A Y K H P I R T Q f J T T S S l U K K Q E I -- +--><---+

AATTTTAMTATTMAATTGGCCCTCCCTAATCCATCCATTrrAGGGGGGGGATT~TGATTCAACcTCAMCTTArTrMATGTTGi:AGATMTAGTGGAGCTCGA 76511 t-~> <-~t l----> <----t rp1H> H I Q P Q T Y L /I V A 0' 11 S GAR .. • .. .. .. • .. • .. 0- .. •

AMCTMTGTGCATTCGAGTTATAGGMCGAGTMTCGAMATATGCAMTATTGGTGATATTATTATTGCTGTTGTTMAGMGCAGTGCCAMTATGcCTATTMMAATCCGAAATT 76451 K l II C I R V I G T S Il R K Y A II I G 0 II! A V V K E A V P 1I HPJ K r. S E I

GTMliAGCTGTMTTGTACGTACGTGTMAiiMTTTAMCGMATMTGGATcCATMTN...r.AmGAToATMTGCAGcAuTTGTTATTMTCMGAAGGMATCCAMAGGMCTCGA 16331 V R A V I V R T C KEf K R II /I G S I I K F 0 0 II A A V V I II Q E G II P K G T R

GTTTTlGGTCCAATTGCTAoAGMTTMGAGMTCTMrrTrACTMAATAGmCGTTAGCTCCAGMGTmATMATAMATAmAhnrATMATAMTMAAGACTTATMM 76211 V F G P I ARE L RES !l F T KJV S L A PE V L - +-----> <-------+ +--

TAmATTTTATATTTTTCAATTMmMGGAGTAmATGGGGMTGATACAATTGCGMTATGATMCCTCMTAAGAMTGCAMrTrAGGGAMATMMACAGrTCMGTACeT 760~1 -> <----t rp,8> AGIiAG H G 1/ 0 T I A II' HIT SIR II A /I L G K I K T V Q V P

GCTACTMTATMCTAGAMTATTGCMMi.TTcmnCMGAAGGnrTATAGATMCTrrATTGATAATAMCAMATACTAMGATATTTTMmTAMTCTAA.IIATATcAAGGG 75971 A T I( I T R 1/ J A K 1 l' F Q E G F J 0 /I F J 0 U K Q II TKO I L I L U t K" Y Q G

~TCTTATATAACAAcmAi.GACGMTTAGTMACCAGG~TTMGMTATATTCTMTcATMAGMArTCCMAAGrrTrAGGTGGMTGGGMTTGTMrremec 15851 KKKKSYI TTtRR I SKPGLR IYSIIIIKEJ PKVlGGHGI V IlS

ACGTCTCGAGGMTTATGAcAGATCGAGMGCTCGACMAAAMilATTGGGGGCGMCrTTrATGTTATGTATGGTAAmmATMAAMTmA~TAGTTACTTACTATC 15731 TS R G I H TOR EAR Q K K J G GEL L C Y V W -

-+----~<-- ..

GTTTTTATTAATGTTGGnTATTMMAGcAGATTCTTCITTTMTGGAGAMCMMAITMTTGATATGGMGGTGTTGTTATAGAATCAcnCCTMTGCMCAmCGAGTTTATT 75611 AGGAG InfA> H E K Q K L 1 () H E G V V I E S L PI/A T f R V Y l

TAGATMTGGATGTATAGTATrMCACATATATCAGGMAAATCCGACGAAATTATATTCGMTATTACCCGGAGATAGAGTAMAGTccAATTMGTCCTrATGAmAACTAMGGTC 15491 o II G C I V L Till 5 G K I R R II Y 1 R I LPG, 0 R V K VEt Spy 0 l T K C R

GTATMCTTATAGACTTCGTGCAMATCTTCAMTMTTAAAAMTTAMilMAAAMMTrAGAGATTAAATAmATcAAMTCCGCGCTTCTGrrcci.MAAmCTGMAATTCTC 75371 I T Y R L R A K S S II II - GAG .cd> II K I R A S Y R K J CEil C R

GATTMTTCGACGCCGAAGACGMTTATGGTAGTTTGTTCTMTCCAMAc...CMACAMGACMGGTT~GrirAMTMMACACATATMAATATATACATATAGTAMT 75251 l I R R R R R I H V V C S 1/ P K 11 K Q R Q G ~ - rpo,l1>

TATGCCAAMTCTGTAAAMAMTTMfTTACGTMAGcMMCGTAGCITACCTAMccAGTTATTCATATTCMGCCAGCTTTMTMTACMTTGTAACTGTTACAGATATTAGAGG 75131 H P K S V K K I II L R K G K R R L P KG V I II 1 Q A S'F II I(T I V T V TO 1 R G

GCMGTCCmCATGGTcTTCrGCTGGTGCTrGCGGAmAMGGTACMAMAAAGTACCCCAmGCcGCTCMACCGCrGCAGAMATGCTATTCGGATATTMTTGATCMGGTAT ,75011 QVVSWSSAGACGFKGTKKSTPFA"QTA"EIIAJRllIDQGH

iiAMc.w;CGGMcTTATGATrAGTGGTCcAGGACCAGGcAGAGATACGGc...TTACGAGcMTTCGTCccAGTGGTATMTACTTAGmrGTACGTGACGTMCTCCCATGCCTCATM 7~691 K Q A E V H J S G P G P G ROT A L R A, I R R S G I I l S f V R D V T P HP H II

Figure 4G (continued).

-35-

TGGATGTAGAi:CACCTAllAAAAAGACGTGTATMATAAAAAAAACTAm~TATTAATArGAnCMGATGAMTAAAAGmCTAcTCMAi:Am.CAGTGGAAGTGTATT 14111 G C R P P R K R R V ~ . ~ H I Q 0 ElK V S T Q r. L Q II. K C 1

GAATCTAAAATAGAAAGTAMcGTCnCrrrATAGTCGTTTCGCTAmcACCmTAGAAAAGGTCMGCCAATACAGnGGMTAGCTATGCGTAGAGCGTTACTTAATGMATTGAA 74651 E 5 K 1 E S K R L L Y 5 R f A I S p f R K G Q A II T V G 1 A H R R ALL II £1 E

GGAGCTICTAnACATACGCTMMTAAA.MMt;TAAMcATGAATATTeMCAATMTAGcmACMGAATCTATTCATGATATATTAATTMmAAMGAMTTGrTnAAMAGT 74531 GAS! T VA K 1 I: K V K II E Y S T I I G L Q E 51 II D III II L K E I V L I: S

GAATCrnTcMCCTCMMAGI;ATATATTiCAGm·TAGGACCTMAMAATAACTGCTCAAGATATTAAAGGGCCTTcTTGTATTAAcATTATGATAATAGCCCAATATATAGCAACT 14411 £ 5 FE P Q. X A Y I 5 V l G P X K I r A Q D J K G P SCI K 1 H I 1 A Q Y I II T

TTAAACAAAcATATmATTAcMATTGAATrMATATTci.MAAcATCGTGGATATCGTATTGAAMCTTACAMAATATCAAGAAGGnrAmcCAGTGGATCCTGTnrfATGCCA 74291 L II K 0 ! L LEI E l II I f K 0 R G V R I E II L Q K Y Q E G L f P V 0 A V F H P

ATACGAAATGOOTTATAGTGTTCATTCrrTTGMAGTuAllAMMAArTAMGAMTACrrTTTCTTuAMTCTGCACTCATGGMGITTGACrCCAAAAGMGCTCrTTATGMGCT 74171 1 !I II A II Y S V· II S F ESE K K IKE I L F LEI ~ TOG S L T P K E A LYE A

TCTCGMAmAATTGATTTATTTATTCCTTrMTTMTTCAGAAAAMAAllAMMAATTnGGMTAGAAAAAACAAATGAATCAMTATGTCTTATTTTCCnTTcMTCTGTATCA 74051 5 R /I LID ~ F I P l 1 /I S E K K E K II F G I E K TilE S Ii H S Y F P F 0 S V S

CTGGATATTci.A.AAMTGACWAGATGtTGCTTTlAAACATATATTTArTGATCAACTAGAATTACCTGCCAGAGCATATMTTGTCTTAAAMAGTAMTGTGCATAcAATAGCAGAT 73931 L 0 I E KilT K 0 V A F K II I FlO Q L E L PAR A Y II elK K V II V II T I A D

TT A TT ACACTATAGTGAAGA TCA m M n MAA n MAM rrnGGAAAAMA TCAGT AGAACAAGnrTGGAAGCA TT AMAAAACG rTrnCAA TCeM TT ACCT AAAAA T AMAA T 13811 l LilY S E 0 0 L II: ! K N F G K K S V E Q V LEA L K K R F S I Q L P K fl r. II

lATCTTTAGGTAAHGGAlTGAAAMCGTTTrATMAACTAGMATMGGnTGMATTTTTTACTTTTTAcTAAAAMrTTCAMCCTTATTTCTAGTTTTATAAMAAMTATATATT 73691 Y L -

Figure 4G (continued).

Figure 4. Nucleotide sequence of the chloroplast DNA. The nucleotide

position numbers are counted from the 5 1-terminal nucleotide next to the

inverted repeat IRA' Dots are put on every ten nucleotide. Amino acid sequences deduced from the nucleotide sequences are shown under the DNA

sequence by one letter symols. Stop codons are shown by double underlines.

Putative stem-loop structures are shown by broken lines with arrow heads.

Predicted promoter sequences and Shine-Dalgarno sequences are shown under the

DNA sequence. Transfer RNA sequences are shown under the DNA sequence.

Introns are shown by dots under the DNA sequence with 51 and 3 1 terminal

consensus sequences (gagyg and ragccg.augaa •• gaaa •• uucaugu.c99uUYi r represents· a or 9. and y re~resents c or u).

-36-

nucleotide sequences of each block are shown in Fig. 4 A-G. The amino acid

sequences of ORFs and the nucleotide sequences of transfer RNAs deduced from the DNA

sequence are shown below the nucleotide sequences. Introns in ORFs are predicted in

one tRNA gene (valine~UAC) and six protein coding sequences for petB, petD, rp12,

rpl16. and rps12 genes and ORF203 by the presence of the 5' consensus sequence

(GUGYGj Y represents C or T). and 3' consensus secondary structures with the common

sequences (RAGCCG.AUGAA •• GAAA •• UUCAUGU.CGGUUYj R represents A or G) characteristic

to group II introns found in fungal mitochondrial genes and Euglena gracilis

chloroplast genes (Michel and Dujon 1983. Keller and Michel 1985). Identified genes

and open reading frames (ORFs), and their loci on the chloroplast genome are

summarized in Table 2. Genes are categorized into three groups: 11-1 transfer RNA

genes; 11-2 genes for photosynthetic polypeptides: 11-3 genes for ribosomal proteins

and subunits of RNA polymerase. In the section 11-4, unidentified open reading

frames are discussed. Detail characterization of these genes are described

following sections.

11-1 Transfer RNA·genes

As previously mentioned. chloroplasts contain genes for their own rRNAs. They

probably also contain genes for all of their tRNAs. They show high homology with

the corresponding genes of I. coli. Genes for numerous tRNAs have been sequenced

and mapped on chloroplast chromosomes (Crouse et ~. 1985).

RESULTS

From the DNA sequence, tRNA genes were predicted as regions that have higher GC

content than spacer regions between ORFs as shown in Fig. 5. Seven tRNA genes were

located by searching for the T-jbloop consensus sequence (GTTCRA) and identified by

constructing the clover-leaf structures as shown in Fig. 6.

-37-

lW ',' ". '. , . atpB . ' .. §i- -- -; '. . - ~ @'-....~~~ ... ,-_f ..... ~ ./, ... ...., "\. "e" ,.,.,.,' • -r' '.-- .'---e=Y.~ ,.r-.,

D 1 III :;ro zoo .:00 H\J &\J ";OJ ;;m :'\:IJ itl.'\J 1'100 1m.)

'!;~~"--'~~. 1~ li~ i~oo It.OO l€W liOO u:ru lK\) a'CO 2100 :?:a'J 2m) 2~OO

'~~~~~~ _.::.'0 "~~ ·.m = -,"00 _.m.(UJ ,I(,J !~(\' "'('0 3.:00 ;~oo !~ro

ORF184 ---

Figure 5. GC content and corresponding g~nes. GC content was plotted by

calculation in average 30 nucleotides. The coding sequences are shown by bold

lines with names of genes.

-38-

Figure 6. Secondary structures of tRNAs deduced from the DNA sequences.

The 3'-terminal eGA nucleotides are not coded by the chloroplast geno~e. The insertion site of an intron in the tRNAValCUAC) is shown by an arrow head.'

~39-

Valine and isoleucine tRNA genes (tnnV-GAC and trnI-e*AU)

A gene for tRNAVal(GAC) was found at position 81814 to 81885. The 5' terminus

of 16S ribosomal RNA gene was mapped at the position 82109 by comparing with maize

and tobacco chloroplast 16S rRNA genes (Schwarz and Kossel 1980, Tohdoh and Sugiura

1982). It was shown that the primary transcript of the 16S rRNA does not include

tRNAVal(GAC) (Strittmatter et ~. 1985). In the liverwort putative promoter

sequences were indiVidually located upstream from 16S rRNA gene and valine tRNA

gene. A tRNA gene (81057-80984) is located on the opposite DNA strand 756 bp apart

from the 5' terminal end of the trnV-GAC gene. The unmodified anticodon is CAU

complementary to the methionine codon AUG. The nucleotide sequence shows 43.2% and

54.1% homologies with the spinach initiator methionine tRNA(CAU) (Calagan et ~.

1980) and elongator methionine tRNA(CAU) (Pirtle et~. 1982), respectively, but

exhibits 93.2% homology. with the spinach chloroplast isoleucine tRNA(C*AU). In

addition, the tRNA gene in liverwort has extra mismatching within the anticodon stem

as seen in the case of spinach isoleucine tRNA(C*AU) (Kashdan MA et~. 1982 and

Francis et~. 1982). Therefore, coding sequence for this tRNA can be tRNA gene

(trnI-C*AU) highly modified in the first nucleotide of the anticodon.

Arginine tRNA gene (trnR-CCG)

A tRNA gene (57877-57950) was found 94 bp downstream from the termination codon

of the rbcL gene. The tRNA has the anticodon of eCG that can recognize CGG arginine

codon. A pair of mismatching nucleotides (U-U) was found in the amino acyl stem

(see Fig. 6B). The liverwort trnR-ACG gene in the IR region also have two

mismatching nucleotides (U-U and U-U) in its amino acyl stem (Kohch; et~. 1986).

Tryptophan and proline tRNA genes (trnW-CCA and trnP-UGG)

Coding sequences for two tRNAs were found at pOSitions 64626-64553 and 64788-

64715 near the psbE gene in Fig. 40. Their secondary structures showed the

anticodon triplets, eCA and UGG, pairing with codons for tryptophan UGG and proline

-40-

CCA, respeC£ively(Fig. 6C and 60). Therefore these tRNAs were identified to be

tryptophan tRNA and proline tRNA. The tRNATrp(CCA) and tRNAPrO(UGG) show 93.4% and

93.4% sequence homologies with spinach chloroplast tRNATrp(CCA) and tRNAPro(UGG)­

(Canaday et ~.1981 and Francis et~. 1982), respectively. The genes for these

tRNAs were d~sidnated trnW-CCA and trnP-UGG. Their coding sequ~nces were separated

by 88 bp spacer region. A significant promoter sequence for these genes ;s present

20.bp upstream from th~ 5t end of the proline- tRNA gene, but not in tbe spacer region

between two tRNA genes. Two tRNA genes must be co-transcribed in a primary

transcript and processed into mature tRNA molecules, although 42 bp stem structure

in the spacer region can be formed as described in tRNAArg(eCG).

Elongator methionine and valine tRNA genes (trnM-CAU and trnV-UAC)

In the 8g1Il fragment 8g10, 83 bp apart from atpE coding region, a tRNA.gene

(53801-53874) was found on the opposite strand. The anticodon of the tRNA was AUG.

The tRNA showed sequence homology with spinach elongatormethionine tRNA (94.8%),

and with initiator methionine tRNA (46.8%). Therefore this tRNA gene was confirmed

to be elongator methionine tRNA gene. A tRNA gene (53652-53051), whose anticodon

was UAe was found 148 bp apart from trriM-CAU. This putative valine tRNA gene was

split by 530 bp intron. at the junction between anticodon stem and loop as shown in

Fig. 6F.

DISCUSSION

Thirty two species of tRNA genes have been identified and mapped on the

liverwort chloroplast genome (Ohyama et ll. 1986). Those identified tRNAs are

listed in the codon table as shown in Table 3. The tRNAs encoded by chloroplast

genome are sufficient to read all codons taking into account an exterided wobbling

and modification in the anticodons. No tRNA gene would encode a 3'-terminal eeA

nucleotides. Five species of tRNA genes (trnI-GAU, trnV-UAC, trnA-UGC, trnK-UUU and

trnG-UCC) have been found to be split by group II introns. A tRNA gene (trnL-UAA)

-41-

Table 3. Codon table and unmodified anticodons of tRNAs coded by

Marchantia polymorpha chloroplast genome.

codon anticodon codon anticodon codon anticodon codon anticodon

UUU} U~~ VAU} UGU} Phe Tyr Cys

UUC GM UCC ' GGA UAC GUA UGC GCA

.UM* Ser

UUA} .UCA UGA UM Ter UGA .Ter

. Leu UUG eM UCG UAG Ter UGG Trp CCA

am} em} CAU} eGUj ACG His

CUC CCC (GGG) CAC GUG CGC Leu Pro Arg

CUA UAG CCA UGG CM}

UUG CGA Gin

CUG CCG CAG CGG CCG

AOUl Am} MUl CGU}

GAO* Asn Ser

AVC ·ile ACC GGU Me GUU AGe GCU Thr

UUU* AVA CAV ACA UGU AM }LYS

AG.l\} ueu ( Met CAU Arg

AUG fMet' ACG MG AGG CAU

GOO} GC"} GAV} GGUl . Asp

GUe . GAe Gec. GAC GUC GGC Gee Val

UAC* Ala

UGC* Gly

UCC* GVA . GCA GAA}

vve GGA Glu

GUG GCG GAG GGG

AUG codon is an initiation codon. Termination codons (UAA, UAG and UGA)

are indicated by Ter. Asterisks indicate presence of introns in the

~oding sequences •.

-42-

is interrupted by group I intron. Five species of tRN~ genes (trnV-GAG, trnI-GAU,

trnA-UGC, trnR-AGG, trnN-GUU) have been identified in each inver,ted repeat reg.ion

(see Fig. 2).

Three kinds of arginine tRNA genes have been identified on the chloroplast

genome. Dup 1 i cated genes, trnR-AGGs are 1 oca 11 zed in IR regi ons (Kohchi et a 1.

1986) and 'a trnR-:UGU gene was mapped near the 3' side of the atpA gene coding for

o(subunit ~f H+-ATP synthase (Umesono et £1.. 1986, see Fig. 2). In liverwort

ch19roplast, genome, however, the trnArg(GGG) gene, which has not been found in the

.chloroplasts. of any species of .plants,., was identified near the 3' end of the rbcL

gene. Arginine codons in the codon table are separated into two boxes including AGR

and GGNcodons (see Table 3). Godons, AGA and AGG, can be read by tRNAArg(UCU)

using ~/u wobbling of the third letter. The GGU and GGG codons .can also be

recognized by tRNAArg(AGG) and tRNAArg(GGG), respectively. However, GGG and GGA·

codons could not be read by tRNAArg(AGG) and tRNAAr9(GGG) without modification of

the first letter of ACG and GGG anticodons. In I. coli there are also three species,

of arginine tRNAs having anticodons ACG, GCG and UGU. Therefore these results

indicate that the mechanisms of the codon-anticodon recognition in chloroplasts is

similar to those in I. coli. In addition, unpaired nucleotides ;n amino acyl stem

were also reported in chloroplast tRNAAr9(ACG) molecules of liverwort (Kohchi et £1.

1986), Euglena gracil is (Ha 11 i ck et £1. 1984)', Spi rode 1 a 01 i gorhi za (Keus et £1..

1984) Pelargonium zonale (Hellmund et £1.. 1984), and tobacco (Kato et £1. 1985).

These inc;:omplete structures of amino acyl stems may alter the codon-anticodon

recognition. A promoter sequence is not detected in the 94 bp spacer region between

rbcL andtrnR coding regions. :Instead, there are two stem structures consist of 8

and 3,2 bp ,long (dG= -44.9 kcal) that may function as intracistronic termination or

RNA processing signals (Fig. 4B).If it is so, the trnR .gene may be co-transcribed

with rbcl gene and processed into mature tRNA.

-43-

II-2 Genes for photosYnthetiC polypeptides

The most important l"ole'of chloroplasts is photosynthesis. L;verwo'rt

chloroplast genome ;s smaller (121 kb) than those in other higher plants {Palmer .§1

!!.l. 1985}. Nevertheless it is interesting to know whether the liverwort chloroplast , ,

genome has necessary sets of genes for photosynthetic polypeptides; Events in

photosynthesis take place on the thylakoid' membrane as well as in stroma of the

chloroplasti. The thylakoid membrane contains re~ction centers (called photosystems

I ~nd II). cytochrome b6/f complex and coup~ing factors. A cytochrome b6/f complex

in chloroplasts operates in an electron transfer chain of a plastoquinol­

plastocyanin OXidoreductase between photosystem II and I. The complex has four

major polypeptides. Three of them. cytochrome f, cytochrome b6 and 17 kd subunit 4.

are synthesized in the chloroplasts, whereas the Rieske FeS protein is synthesized

in the cytoplasms (Hauska 1985). The genes for cytochrome b6 and the 17 kd subunit

4 are separated by 1 kbon the chloroplast genome and are transcribed as a common

precursor mRNA (Alt et El. 1983). The gene for cytochrome f is located distantly

from others and translated into a preprotein larger th~n mature cytochrome f.

suggesting that processing occurs during insertion from the ribosomes in thestronia '

into thethylakoid membrane CAlt et El. 1983. 14hilley et El. 1984. Alt and Herrmann

1984 ).

RESULTS

Genes for photosynthetic polypeptides were identified by compartng amino acid

sequences of the ORFs with those of photosynthetic proteins reported previously iri

other species of pl~nts. The amino acid sequence alignments of the identified

photosynthetic proteins are shown in Fig. 7 A"'-K. On the LSC region'sequElnced in

this' study, there are ten genes for photosynthetic proteins; the large subunit of

r1bulose-l.57 bisphosphate carboxylase/oxygenase (rbcL), 51 kd-P~680 chlorophyll ~

apoprotein (psbB). cytochrome b-559 polypeptides (psbE and psbF). cytochrome f

preprotein (petA). cytochrome b6 (petB). cytochrome b6/f complex subunit 4 (petD)

-44-

CA) rbel

L 1y~ntort Splnoch Tob.a-c:co &1 •• A. n1du~.!Ins

(8) !,sh8

LhcN'Drt

Splnoch

(el p:b£

lIvoj;'(l,t Splnoch lClb~-C'co OonoUlOr.

(0) p.bF

t hen-ort 51' lnoch Tob.acco OJ1nother.

(E) p.bG

MSPQT ETKAGVGFKAGI'I:OYRL TYVTP OYi:T~DTO I LMFRKTPOPGVPAEU,GllAV MESSTGnmvWrOGllllLORYKGRCYO I DPVPGWIQY I AY ::::::::: 5: E::::::: :r-:::,:: E::: l::::::: ::VS,,:: ::P::: :A:::::::::::::::::::,,:::::::: :II:E: :A:::::: :C: :::::::: :5:::,::: ,E:~:::::: E:Q:::::::::: 'V,: ,,,: ,P:: ::An:::::::,,:::::::: :s:: ,::: ::,R:ER:V::KO::::: :::::::: :5:,::::::: :1;:::::: E,:::: i:,::: ::V:: ,l: ::P: ,::A:::::: :A::,:::,:::: ,5::::: '" :H,E: :::OPD:: :C, :-:X:QSA:-: y,:::,:: k,:::,::, TP:" ,l:,:: :1'5::,::: ,D:,:Ao I:::::,::::::: ::L: :D.'I:,,: :K: :I/,E: :():: ::5: 1':1'

VAVPLOLfEEGSVTlIIIFT5iVGlIVFGFKAlRALRlEOLnipPAI'TKTroGPPIIGIQVERDKLijKYGRPLlGCTI~PKlGi.sAKIIYGRAV;'ECU!GGlDFj ~;;;i};;;;;;;;;;;;;;;;;;;;;;;:;;;;;;;;; ;;~; ;~;;;;;;;;;;;;;;;;; ~;;; ;;;;;;;;;;;;;;; ;;;; ;;;;;;;; ;;;; ;;; : : : : :: : : , : : : : : : : : : : : :: : : :: : : : : : : :: : , : , : :: : , : 5: : :: : , : R:II:: , : :: : ,II:: , : : : :: : , :: : , , : : ::: , :: : C:: : : :: : : :: : I:::::::::::,:: Il:::::::::::: I :S::::: I:F:V: lV::::::::::,:: ::l,:::: ,:11:::::::,::::::: ::.::::::::::::::

. KOOEIIVlISQP FIIRWRORFLfVAEA I YKSQA[TGE I KGIIVLIIAl AGT CEEMl~RMCAREi.GVP I Villl[)YL TGGfT AlITSLAfYCRDIIGLLLH! IIRA!IIIAV ::.,:,:::::;:,:::,:: :e:: :L, :A::::::::,:::::,,::: :U:M::: Vf:':'::':::,,:::::I:::, T:511::::::,:::::::::::

~~:; ~;; ~ ~;: ~ ~:; ~~ ~~:~~ ~ ~~~ :~~:;~ ~;;;;;;;;;;;;; ;~;; l;~;~~; ;Q;;;;; ;:~;;;;;:;;;; ;~;S~; ;;;;;;;;; ~;;;;;;; ::,:: I: ::::Q:,:::::: ::0:,11: :::::::::::: ::V: :P:::: :11: ::EF:K: ::H:: I:: :F::A:::: :: T:: 1:1/, :: ::V:::::::::::

I DRQKIIHG I irrRVLAKAlRilSGGDH I ~AG TVVGnEGDRQVTLGFVDLLiWOY! E~DR5RG I YFlQOW'ISLPGVF PVASGG I HVWIlIIPAi. TE I fGDOSVl. ::::,:: :Ii:::::::::: l:::::: :5::::,::: ,[:01:::::::,::: :T:::::,::::: :S,,:T: ::l:::,:::,,:::::,,::,:::,,:

;;;; ~;; ;~; ;~;;;;;; ;;;; ;;~; ;~;;;;;;;; ;~~~:;;;;;;;;;; ;~~;?~;;; ;;;; ;;; ;;;~; ;;i;;;;;;;;;;;;;;;;; ;~;~;;;; :: ::R::::::::: ::C: :l::::: LIS: II:::::: ,KAS:::,: ::1i,E:lI::A::,: : V;:, " ::A:Ii::: L:: :.:::::,:::::, V:,,:::,::

QFGGGT lGIIPWGllAPGA V NmVSLEACVOAAl1 EGRDlAAi:CIIE II REACKwSPELSMCi: I W~£ j ~f Eri)/ J on E::,,::::::::::::::::: A,,:::,:::::::::::, :T:::: :T::,:: :A:::: V:,:::, ::PAII,: V ::::::::::::,:::,:::: :A:::" K,:,::::, :0:::::::::':::::: A::: :V:: ::V: II:MV:V:DK E::::,::::::, :fI::A::: :A,::::,:::::,,::: :VQ-: ,KII,::, :A: ,A::,::: :D::: O:KAII:: I E:::::::::::, :L: :1::: :A::::,::::::.:: :Y:: :GO:l:: :G::::, ,A: :lPl::::::: : £TIl: ~:

475 ~75 90.S~ 477 90.91 47.4 87. n 472 80.6t

IIGLPWYRVllivvWOPCRLi AVIIUlHTAL V 5G1/AcSIW. YELAVFDPSD?VLDPHlI!1QGHFY I PFHTRLG I TKSllG~1I5 irCHVIIIAGi II$YEG"iMvH

~ ~;.w~~~il~~~~ilo~~~~ ~ S~ill ~I~ ~~ ~~~~~~~ ~ ~~~ ~~o~ ~~~~~~~~~~L; ~}HTRL~ i il,Sii~~1 i ~~Gi 1 ~OP s i WSYEGVAGAI'I

IVlSOLLfLMIWllW'IYWDLELfROERTGKPSLOLPKIFGlllLFlSGVLCFAfGAFlIVTi;LFGPGIWISDPYGLTG~VQPVA!'AWGAEOFOPFV~GGIA5

HI! I flAG I LG i LAGlFIIL5 VRPPQRL Y~Glfu..GIIV HVlSSS I M vrr MF VVAGl!1lIYGSfIA TP I EUGPTRVQWOQGFrQQE 1 ORR I RSSKAEIILSLS E

;Iil i :J.GTLb \ U.W~L~~ks~Q~L ~~~L~:II ~TVL~S~ I!J.~ ~~ !J.~~~i~~~ r ~p I WGPiR~Q'~bQGY }&l~ I Y~RVSAGLAi:ilQsd AIISK I P EKlAfYDY 1 GIIII?AKGGLFRAGA VO~GOG I AVGWlGilA VFKDXEGII ELfVRIIli? TFFETF PVVL VOEQG I VRIIOVPFRRAE SKYSV EQVGVTVE

~~skIH~U.~~bYiGili,pIlGGUiJ.GSIIoiIGbGi~~~l~iIP'}R~~~GRt~~~R~p~~m~p~~~Iix;~i~iJ.~~P~RiJ.ES~Y~~iQ~G~iVE fYGGElOOV5fSOPArVKKrARIW)LG£I;ErDRArlKsix;vfRSSPRG~nFGllArfJ\i.lFffGII11IHGAijTlFROVfJiGIDPOlDAQVETGAFQnGD

FYGGElHE~h~v~~~X~~1R~Qml}h!JJ.Eg~~fg~~RGwn~!;:~5r~W~f~~I:d~5~TlfkDvr~IDPDlDVQv[r~n~I~D PTTKRQVI 508 :::: :: PTTAAQGV .s08 88.2%

I(SGIlTGERPf NIl ! T5 J Roo.! lIS I T I PSlr J AGlILH5TGLA Y~VFGSPRP/jEYrrEIIRQEVPL I TGRfllSlfQl Of rTKSF :: ,5:·,: ,5::::::::::::,:::::,::::::::::,::::::::::::::::: ::S: :GI:::::: :0:: ::l: ::SR:: :: :5::: :s:::::::::::::::::::::::::::::::::::::::::::::::: :5: :GI:::::: :DP:: :l: ::SII:: :: :5: :C: $:::::::::::::::::::::::::::::::::::::::::::::: :::5::GI:::::: :D::::l: ::SR::

HT! DRTYPI Fr~RWLA VlIGLAVPTV Ff LaA 1 SAHQFHiR :::::::::::::::: I" ::.i::: s: ::S::::::::G : ;;:::;;::;;:::: I:::::::;;::: S;;::::::G : : : :::::: :: ::: ::: : ::: :::: 5:,.: S:: ::" ::G

39 ·39 ·89.·77 39 92.3% 39 9Z.J%

83 63 69. ,X 83 88.07 83 88.07

llvc ..... ort IIVL11FXFfTCEIISlEDIIsrnlKJISl ESSFIII~TLTIISI i l TttuofsiniARLSSLWPli. YGT5CCFlEFASL IGSRFOFORYGl VPR5SPRQMlL I ITA ••• :: h =:: : := :: ::: ::::::n:::::::-;n::::::::n:::n:::a:::::::::;::::; ::

Ko I,. HVl TEYSEKKKKEGKOS I n 1 II-Sll £FP llOQTSSII$VI STTPIiDlSIIW$RlSSlWP II YGTSCCf I m.st I GSRFOFORYGLVPRSSPRQAOlI t fA

GTVTIIKMPSl VRLY[WE:PKYV I AHGACr I TGG/(FSTUSYTTVRGVOKL J PVOI YLPGCPPK PUll I itA II KtR~K r AQ(! Y(EK~ J i.KKGTRfHlU

. . . . ~QfllF F511lDNPKL T -SSllQF FQSXKTSXVl1ETSlJ FXEXElIL 243

TRHKLYVRRSTHTG~YEQELlYQ;'PS~LOISSETmsbvsSYKLVH 248 62. IX

Figure 7 (continued).

-45--.

L1'veriiort Sp~h'.e~"" ' , Pca .

. :Oenotherll.'

, MQI/RilftliIL i I ~WA I RU 5 iM III ilT i niss I SEAFP i-r AQQGrfliPREA tGii IVCAIICHLAK!:BVD I EVPQSVLPIITV~EAVV!: I PYDflQ1 KQvt:IIl/GK ::TI: TfSW:KtQIT; s: ,: SL: LVI: T~i::AII;Y::f:::::::,:; ,i:: i i:: i::: ::11:::::: i: :JI: ': :ll:::: :':":R':::: :L:::::::: ::T:,:AFSWiK: EIT: s: :VLUI:YI.: TRAPiill:Yi :F::: :'::::: i i.: iii:" i:: i: i :lli:::::: :":Ai: ill:::::: :"R::, :': :V::: :':::: " ",HK:TfSW:~iElTiS: :LSLM:Yl:TRT::ill: Y::f::: i:::: :":::::::: i:::: :11:;:: i::: :A::: 0::: i:: iR::: :R:V:::::::: :E: ::TfSWVi:EO~IT:S: :VS:I1:YV: TRT:::II:Y::F:::::"::: ii::::: i i:;: :;:5:::::: :~i :,,:: :D:::: ::LRi:~,::::l::,:':~::::

. KGSLIIVG~vLlLPEGFELAPSDi!IPPEllKEKIi;lIlFFQPYSllllmILViGPVPGKr.YSEllVFPILSPri~AtliKEA!lFlKyprivGGIIRi>RGQIYPDGSK :;G:::::::::::::::: :P:: :5:: i:: :M:': :S::S:RPH:Q:::.:::::: :Q::: ,'IT:: ::Ai: ii :r.:DV:::: ,::: ii:::::::::: i,i:.

~::A:: :::: i::::::::: iPII:LS:Q1:::: :::S::S:RpT::::::, :::::::::: IT::::::::: iKRDVY:':: ::Li:::: ::,:::::::::

~;g~~[~~;; ~,;;;; ~;; ~ ~~~; ;~;; ~;;~; ;;~*; ;~~~~~~~;~ ~; ~ ~ ~ ;;;~;;; ~ l~1; ~;11 ~;; ;K;~~; iii; ii; iii; ;;; ~; ;;;;;; ~ SlIIiTVYl/ASi TGKVSKI FR~t:KGGYE I Tl 00 I SOOIlKWO I SA;!GPEl i i SEGELVKVoQPl Tl/iIPI/VilGFGqGOAEVVl.QDPLR j QG Ll.LffGSV I LAO

. ·.1 iI:/itm~1r1~ml~ 111 ~11!11~1~1'11m:~ 1 !~il1! m~ l! 11f~ ~ll i ~11111111 ~ ~ 1! 1111~ ll! i!!~! i 11~lmlllll IFl~LKKK.QFmQLAEIlNi= : :: :: :: ::::: ::: s: ::: ;?;; ;;;;;;;; ;;;~;;~,; ,v::':, i:,',,'::::: ,Y:'::":

320 , 320 78.8% 320 75.92 3.18 . 78;.1% 320. 79.1%

(G) petB"

L. "lven.ort, MGKVVDWFEERLEIQAIAODITSKVVPPHVl/lfYCLGGlitr.CFLVQVATGFMTFYYRPTVTEAFSSVQrIHTEVIIFGWlIRSVHRWSASfUiVU-L'IILH

, ~IGSK/IVSRFRR~RMI ~~~~~~~~:,t~,I~~~m:~[~~g&o~i}~~~~t~~Rmio~~AgQ~i~~~~~I}&~~m&iIR~~~;~~L~iUI Spin.ch

(Ii) 'petD

l1verwort Spfn.cli Po.

(I) alpS

Liverwort Spinach Tob.cco l1.i,e E. coif

I fRVVl TGGFKKPREL TlIVTGVI LAVL TVSrGVTGYSlPilOQ I GYHAVK i VTGVPEAIP i IGSPl VELLRGSVSVGQSTi. TRFYS LllTFVLPLL TA I FHi.

vF~t~1. T~~f~~~REt~tT~t\lLG~1. TASfG~T~YS!'P~~ ~ G~H~ v~ It~GVPO~1 "VI 6SPt. tEL LRGS.\SVaQS TI. TRFmiM~I.~W ~ v}~~. 215

: ~: -: : :-::: : : : : : :- .. M1IFLMlRKQGISGPL, 211 .86.0%.

. , ~.

'1IGmKPOLSDP1 LRAKLAKGMGilNYVGEPAWPIIDLL Y I FPVVlLGT I ACr.VGLAVLEPSM I GEPAI;PFATPLE I LPEWYf ~PVf91 LRTVPIIKLLGVLl. ::::::" :1/' :V:,::: ,,::::::::::::::::::::::::::::: :11: :,::::::::::::: D:::,::,,:::::::::::::::::::::::::

: : : : : -:J:: V: : : :: :.::: : : :: : :;, : : ::: : ::: : :.:: : n: : : : II: :: : : :: : : :: : : ;: 0.: :: : :: : : :: , : " :,:: : ,; : : :: : :: : , :::

I-IMVPAGLtTv~FLElmIKfQIIPFRRPVAnVFLlGtvvAi.WLGIGMLPIDr.sLTLGLF "S::::: ,;" ,::,:,,: ,::: ::::::::: :V,:::::::,: ::T;::::::::,::' :VS~::: :,::::::::: :":::::::::::::: :"::::::: :,: '':,:: ::J::: E::::: ,,: '"

160 139 (160) 95.6t 139 93.1%

H~'rIlFLAfGHSTL VAKIl I GS] TQV I GPVLilV AFSPGKHPN I YIISLI VKoQIISAG'EE II/VTCEVQQLLmlilKYR~ Vi\fISATOGI1BRGMKV i DTGAP L TVPV HRIIIPTTSOP:V, ::EX: :L:R,A: I:::: :11:: ,p,::::::: :A::: :GROT: :QPM:::::::::::: ;R::,:,::: :,': LT::: E::,::::: S:::

'I<RIIIPTTSGS:V:: :EK:,:P,RVV, I::::::::: r::,:,:,: :A:V:QGRO:V:QP:: :A::: :~::::: :R::: I:::; :E: LT::: E::::::: IS::: I<RTIIPTTSRP: I:: lEE: SV, R:D: I::::: : IT: P: ::L:Y: ::A:::: SRDT:OKQ:::: :::::::::: R:::: ::::: E: L:::: E:::: :T:: S:::

MAT: K:V,:: :A: Y:: E: PQDAV,RV:OA: E:OIIG: ER--LVL::: :Q: :GGI::TI: :GSS: :LR:':LO:K: LEII: IE:::

,(;EATlGRIFi,vLGEPVDl/LGPVEy.mFPiHRMPAfTQl.OTKLSIFETGIKYVOLLAPVRRGGKIGLFGGAGVGKTVLiMELIIIIIILXilliGGYSVFGGV

.~ ~~~; ~~; ~ t;;~; ;;?~ ;~~~; ;~~ i ~:~;; ~;~;;~~ ~,::~~\~; ;r;;;I; ;~;;;;;; ;,;; ;;;;;; ;;;;; ;.; ;;.;;;;; ;~;;;; ~,;;;;~;; iG::: :.:::::::: :.1::::: :OTSA:::,: :5:: :':IE,:::.::::::::: :.:::;::::::: :i·::,:.::,::: i,: :'",: :::A::::: n: ,: t: :K:::: ::M::::::: :MK:E1GEEERIIAl'::,: : SYEE:SIISQELL: ::::, I: :MC:FAK:, £V:~,::: i: :~::: : WI: ::: R: :AIE:$:Y:: ,Ai:

GERTREGIWLYMEMKESKVillEQlllSESKVALVYGQMIIEPPGARHRVGlTALTMAEYFRilVfIKQOVLLFiol/lfRFVQAGSEVSALLGriilP.SAVGYQPTl. ::::: ::::,:,:: i: :G:::::: :Ai::::::::::::::::::::::::::,:::::::: E::,,:,::::::,:,:,;:,:::,::, :~::::: i: i:,

:: ~:~ ~ ~ ~~}:~~ :~m:: b~~~~; ;: I::: ::: ~:: :m: ~l: :~::: :i~~ l mm::,::~: ::~~~}~: :~:::!: ::::::::::: ::: STEMGTLQEiUTSTKEGSITsIQAVYVPAilDLTDPJlPATTfAillOATTV(SRGLMKGIYPAVOPLOSrSTMlQPWIVGEEHVETAQGVKQTLQRYXELQ ::: ::s::::,::::::,::: :~::::,::::::::::::::::::::::::::::::::::::":::,:::, :.:R:::,:::, I: ,R: :E::,::: ::: ,:: ::S:::':::::::.::::',:,::::::,,:::::::::::::::::::::::::::,:::::::: i:::::: :R:::::::::: :R:::::::::::: ::: ::5:::::::: :K::::::::::,:::::::::::::::::: f:::::::: :5,,:::::::::::::::: :R:: :11:::::: :R,: E::::: ,::: ~E: ::V, i:::::: :T, U::Y::,:::::,::: :S::: ii::::: :V:: ::QI:Sl: ::~::::: :,::' :RQ:D: lV::Q:: :0:: R::QSI:: ::Q::K

OIIAI LGLOELSEEORL TV ARARK I ERFLSQPFFVAEVFTGSPGKYVSLRETl KGFQfll LSGELOSLPEQAFYL VGII IOEATAKM TLQVES ,::::::::::::::;::::::::::::::::::;::::::::::, :G;A::~:R:: :t:::: i: :::':,::::;::::,,:::: :MIILEH: ,KlKK

i::: :::~::::::: m~:!~ ~ ~ :~::: i ~ i ~i! ~.i 1 ~.iii i:i i :;~'~~i ;~{ ~ k~i~l: i~:~:~'i : : : ~,~:ii1 ~~ : mi~~t~~: :~t~~

Figure 1 (continued).

~92 498 BB.4l 498 87.4~ 498 a6.2~ 460 62.B~

(J) .tpE

L 1 ,.",or ~, ,HUILRI MPNR] VWIISD I Qf: II LSTI15GQi G I LPIIHASVLTALD I G I V~ i RL-lIoQHsmLHGGF AMI OWWL TJ L VlliiAEtASEIDYQEAQETFQKAK ,Spl"o'ck' 'IHdiCVLTi" is:,: ,EVI::::::,: i ,', "V: f::, PTA,: V:,:, lR'" :-,::: L, L, ,', ,'::: R:G:': Er:','::;,:: :RG,D:::P::':"Q:.LEEE, 'Tob.ceo Hh, :SVLT::::: :0: EVE, :V:: ,,',::::::::: ,PIA: :V,:: :LR:::-::, :L::: :,:::: :R:G: :H:V:::::: :G:O: :P::: :Q:LEL:E Mol z. MK::: YVlT: k:: r;OCEVK:,,:: ::,:: :': :V::!:: PIli: :V,M:PLR::: L:::: L,AV:W5: P,R: V:: Ell:, G,:,: LGiO:: PE" ,QALEhE 'E. '0';11 'HAMJYII: DVV5~EQQljfSGLVE,K:,QVTGSE' Elf, y,G: :Pb, :IKP:MIR,VKQIIGllEEfIY:S: :'ILEVQfG: ViJ;ADl: IRG(JOL: EAR:lIEAKR,:E ,-

.. ;. . T/ILEEAEGIIKKKE IEALLVFKRhKARLEAItlflASKL 135 A:: RK:: :KRQ: -:: :!l:ALR:: RT,V"SiTl:5 13~ 63.0.

·fI, VKK::: RRQ:~:: :fl:ALR:: RT,V:::: PI, 133 63,'7%' A"SK: "T: ~t-V"K:ALR"RT: l"V:WIPPSII 137 51.9Z EHISSSH: DVOY~AQ:SAEL:A: I:Q: RLSS ,133 22.2~,

(1:) ndh3

ORF 120 MFLLQKYOYFfVfLL II SfFS I L I FSLSKW lAP I flKGPEKFTS YESG I EPMGEACi QFQ iRYYl1FALVFV I FOVETV fl.:'" VPWAMSFYilFG I S SF I E~~ flu. mit. URfJ Hlf ~ All i.Hi flTLLAll.LHI I TF~LP9L~GYM~~S~P~~C~FD~~SP ~RVP hI1KH.LVA I TFLlfDLE.1 AlI.LPLpiliiLQTT~L-P LI\~SSL . .

IfIUlIIGLVY!\f/R-KGALEfiS ,lZ0

L~il i~~SlAYE~'LQkG-[O~TE 114 30.8X

Figurf! 7., Amino acidsequenc;e ali9I:lments of photosynthetic polypeptides.

T-he ami no acid sequences are s,hown by or:re letter codes. Identi cal ami noC!-cid

residues areshCiwn by colons, 'and deleted residues are shown by dashes. The

am;'nQ ad; d residue n'umbers and sequencehcimol og; e's with liverwort gene

pr:qducts ate: indicated at,theend of; sequences "

-47-

and ~ and € subunits of H+-ATP synthase (atpB and atpE). The amino acid sequences

of the photosynthetic proteins in liverwort chloroplasts exhibit high homologies

(78.8%-95.6%) to those of spinach chloroplasts except for atpE (63.0% homologous to

spinach atpE) and psbG (62.1% to maize).

Gene for the large subunit of ribulose-l.5-bisphosphate carboxylase/oxygenase' (rhcL)

The rbcL gene was previous'ly mapped in the Bam5 fragment by heterologous

hybridization with the tobacco chloroplast rbcL gene (Ohyama et £1. 1983). An

ORF475 (56355-57782) shows high amino acid sequence homology with rbcL of spinach

(90.5%) (Zurawski et~. 1981), tobacco (90.5%) (Shinozaki and Sugiura 1982), maize

(87.6%) ·(McInt.osch et £1. 1980) and &. nidulance (80.6%) (Shinozaki and Sugiura

1983) (Fig. 7A). The rbcL gene was localized 508 bp apart on the opposite strand to

the atpB gene coding for (3 subunit of H+-ATP synthase. In the spacer region between

atpB andrbcL genes, typical stem-loop structures can be formed including 45 bp AT­

rich stem structure (dG= -44.9 kcal).

Genes for photosystem II P-680 chlorophyll.~ apoprotein (psbB). and for cytochrome

b-559 polypeptides (psbE and psbF)

The amino acid sequence of an ORF508 (69026-70552) shows 88.2% homology to that

of the spinach 51 kd photosystem II P-680 chlorophyll ~ apoprotein (Morris and

Herrmann 1984). The amino acid sequence alignment with that of spinach is shown in

Fig. 7B.

ORF83 (63554-63303) and ORF39 (63293~53174) are located close together (10 bp

apart). The ORf83 shows 89.2% amino acid sequence homology to that of the spinach

apocytochrome b-559 polypeptide, which is psbE gene product (Herrmann et £1. 1984).

The ORF39 shows 89.7% amino acid sequence homology to the b-559 URF39 gene product

of spinach (Herrmann et~. 1984). The psbE and psbF genes are proceeded by the

typical Shine-Dalgarno-like sequences (GGAGG and AGGAGG, respectively). The psbF

gene terminates by a TAG stop codon that overlap to two nucleotides of the SO

-48-

sequence for psbE. Their sequence' alignments are shown'. in Fig. 7C and 7D .•

Gene 'for photo system II G-protein (psbG)

Recently Steinmetz et ~.ic:lentified a new protein (248 amino-ac;id residues

long) associated with the photosystem II· complex and analyzed.the fine structure of

its gene, psbG, on the maize .chloroplast genome (.1986). The 11. polymorpha'

counterpart of psbG gene (52524-51793; encoding 243 amino acids, 27.6 kd) was

identified and the predicted amino acid sequences were compared as shown in Fig. 7E.

Unlike other photosystem II polypeptides,. the psbGproteins are significantly

diverse iri N~ and C-terminal portions; in average they are only 62.1% .homologous.

whereas the central portions (maize, amino acid residue number.36-182; 11. polymorpha

37-183) are 91.9% identical. In 11. 'polyrnorpha, .the psbG gene overlaps with the last

seven nucleotides of the preceding ndh3.gene •.

Gene for cytochrome f preprotein (petA)

An ORF320 (61641-62603) shows high degree of amino acid sequence homologies.to

those of the cytochrome f preprotein'of spinach (78.8%) (Alt et ll. 1984), pea

(75.9%) (Willey et~. 1984), Oenothera hooker; (78.1%) (Tyagi et~. 1986), and

wheat (79.1%) (I~illeyet·~. 1984). The ORF320 can be liverwort cytochrome f

preprotein gene' (petA) whose molecular weight ;s.33.4 kd. The amino acid sequence

alignments compared with those of other plant species are shown in Fig. 7F. The N­

terminal 35 amino acid sequence of liverwort cytochrome f·polypeptides.sho ..... s

relatively lower homology (40.0%) to that of spinach •. On'the other. hand, the

remaining sequence of 285 residues gives . high homology. (83. 5% with spinach

cytochrome f mature protein). However,the hydrophobic·characteristics of. the N­

terminal region are conserved in petA gene products indicating that N-terminal -35

amino acid residues may be functional as a signal peptide. The molecular ~eight of

liverwort mature cytochrome f polypeptide is 31.3 kd.

-49-

Genes for cytochrome b6 (petB) and for cytochrome b6/f complex subunit 4 (petO)

ORF162 (72078-72566) can be postulated by using initiation codon AUG (72078-

72080). The C-terminal amino acid sequence was found to be homologous to that of

the spinach cytochrome b6 polypeptide (petS) (Heinemeyer et EJ.. 1984). However the

N-tefminal portion including initiation codon is quite different from that of

spinach petS gene. Instead, a consensus sequence for the 3' end of the intron

(RAGCCG.AUGAA •• GAAA •• UUCAUGU.CGGUUY) was found in the beginning (71859-71924) of the

ORF162. A consensus sequence specific to the 5' end of the intron (GUGYG) was also

located further upstream of the ORF162. This indicates that the liverwort petS gene

(71424-72566) is interrupted by 495 bp intron (71430-71924) and codes a polypeptide

of 215· amino acid residues (Fig. 4F and 7G).

The amino acid sequence of ORF139 (73271-73690) shows 95.6% and 93~1%

homologies to cytochrome b6/f complex subunit 4 of spinach (Heinemeyer etEJ.. 1984)

and pea (Phillips et EJ.. 1984), respectively (Fig. 7H). However, the consensus

sequences for an intron (493 bp: 72723-73215) are also present in petO gene as in

the petS gene. The putative secondary structures of intronsin petS and petD genes

are shown in Fig. 8. The initiation codon af the petD gene can be extended to the

upstream methionine codon (72715) as shown in Fig. 4F. The molecular weight of the

liverwort petO gene product can be estimated to be 17.4 kd(160 amino acid residues)

considering presence of an intron in the coding sequence. On the other hand, intron

specific sequences can also be identified on th~ ONA sequences of the spinach and

pea genes, although they do not describe the presence of introns in the coding

sequences for petO gene. If this is true, the amino acid sequences of the 5'

extended region of the petO genes were highly conserved in the three species of

plants (95.6% homologous to spinach petO protein, see Fig. 4F). A sequence motif

(AGGA) for the ribosome binding siles are present 9 bp upstream from initiation

codons of each petO and petS gene. '

-50-

6 C G U G

5'--CAAGGAUUUUUGAAUAUGGGU~ AG6A MetGly

petB>

G U G U G

5'--AAAGGAAAAUGGAUUAUGGGAGU~ AGGA MetGlyVa

petD>

A A li U A G A U U U ,A U U A U A U A A 6 C U '6 U A A G C­! ~ .. ~ . : : :: A U U U G 6 C A U U U U U C

~ ~ ~ G G ~ ~ A ~ ~ ~ U

'~ ~ ~ ~ ~ ~ ~ A ~ ~ i U A A A 'A -U

_~AAAGUAUAC6AUUGG--3' -LysVa nyrA~pTrp

A A U A U U A : U

U U : A G A : U U G : C U U : A 6 GAG C eGG A : U • • •• ••• G

- U C U U A,G C C' U U U '6 6 G G 6 G A C U U U ... : : • : : U U U C cue U A AU U A A A U

..,. AACAAAAAAACCT -3 I lThrLysLysPhc

Figure 8. Secondary structures of introns in petB and petD precursor mRHA molecules. The Shihe-Dalgarno sequences and amino acid se!luences' are shown

under the messenger. RNA molecules. Underlined nucleotide A is putative branch

point in the lariat form. The junction sites -between exons and introns are

indicated by arrow heads.

-51'-

Genes for ~and S subunits of ~-ATP synthase (atpB and atpE)

A gene cluster,atpB (55846-54368) - atpE (54362-53955), was localized 508 bp

, apart from rbcl in head to head on the opposite DNA strand. The nucleotide-binding

subunit (3 encoded for byatpB gene contains 492 amino acids (Higgins et!!l. 1985).

The~. polymorpha atpB amino acid sequence showed 88.4% homology to spinach atp8

gene product (Zurawski et!!l. 1982), 86.2% to maize (Krebbers et i!l. 1982), 62.8% to

~. coli (Saraste et!!l. 1981). In contrast. 11. polymorpha e subunit (135 amino acid

residues), product of atpE, is less homologous to that of spinach (63.0%, Zurawski

et!!l. 1982), maize (51. 9%, Krebbers et!!l. 1982) and ~. col i (22.2%. Sarasteeti\..

1981). Although the Shine-Dalgarno sequence of atpE gene was overlapped with the

atpB coding region in spinach and maize, the coding sequences of these two genes of

11. polymorpha were not overlapped as shown in Fig. 9.

DISCUSSION

The comparison of amino acid sequences of photosynthetic polypeptides between

liverwort and other higher plant chloroplasts reveals the highly conserved amino

acid sequences (78.8-95.6% with spinach photosynthetic polypeptides) during the

events of chloroplast evolution. In contrast, it is interesting to note less amino

acid sequence conservation in transcriptional and translational polypeptides

including d. subunit of RNA polymerase (54%), ribosomal proteins (46-68%) and

initiation factor 1 (65%). These results suggest that chloroplast ribosomal

proteins and RNA polymerases may have changed faster than photosynthetic

polypeptides,ir ,the event of evolution.

Previous studies showed that several plants have two rbcl mRNA transcripts:

(Erion ;985). It has been shown that the smaller mRNA transcripts are produced by

processing of the primary rbcl transcripts (Mullet et!!l. 1985). Although the

spacer region between rbcl and atpB coding regions show less sequence homology with

those of other plant species, several regions are conserved in land plant

-52-

J", ''';.: :·,i '.:,\ :i-,

" ; 1 ~ .J l 10. < •• , '. -.,

MAAGCcMiv."IIC'l"l"TAA~GCCTTMTCi;G"GGG1'GIIT;'1'GGCMTGllrnACCACcri;GIICG!CGTci.GCGCIIc;'/lGchIlCA.v.TGTIc-rer ' KIIKKL ••• GGJlGG HIIMTYlIl,OVVSAEOQI-IFS

(atpnp . (atpE»

GCT ACTGCJuUJ\GCJ\GCT Ai:Tn IICMGTGc"GIIGTT AIIA.V.'l'1'IITGCTi .. \II TCT1'CGT~ TCATGGCTCcr All TCGMm'l'T'TCGM'rTcc II T II K II II T L (I V E S }! L " L 11 I Ii " P II R I V '" ,~ 5' ("tpal> - GGAG (;;tpur;-- --- ---

'l h-

Figure 9. Comparison of nucleotide sequence of spacer region between atpB a~d atpE genes. The i denti ca 1 amirlO aci d res; dues between 11- po lyinorpha 'atpBE and ,n

~pin~ch atpBE proteins are undeilined. Shine-Dalgarno sequences are shown

u~der the ,nucleotide sequen~es by bold letters.

·J5 .11) S/) rmw; .... W!C~ .. AATAATGT1T ..... ·~Al11nATCGAGCAGACCT .. ·lTGTI,G"..GACGGACTJ ATG ---- - --

-65.,-

TOEl.i.,cc:o

.3.\ ·lD r ,RIM. 5/)

C.(;lTGC·· .. W~C!ATAATGAIG!1T· ••••• Ml100T'':rAG:.CCI •.• TrGIA,GGGAGGGATI i AIG

-18 •• - --111s.- -368'- 1:,.

·J5 ·10 i-7 "RNA Sll G(;lIGC •••• aiACAAiI.AToATGTTl· .... ·AAI iT ,IGICC,\"T~GAC:I .. • T1GI~GGGAGt",,(;ACTi ~IG . = - =

·31 ·10 r.·R1I~ r_R.~~ S!l GS; IGC· .··lAlACAATI.A1GATGT11 ...... AAlioc~ATCr,!~"G'CCc .. ·llGTAGGG~~L.(;ACn ~lG = = - =

--23B'p- -J68P-

Figure 10. Comparison of the leader sequences of rbcl gene. The promoter

sequences referred to II-lO" and "-35", and Shine-Dalgarno sequences are double

underlined. The conserved sequences in the 51 leader regions of plant

chloroplast rbcL gene ~re indicated by thick underlines.

-53-

11 ;

chloroplasts. The promoter sequences for rbcL and atpB referred to 11_1011 and "-35"

regions, and the Shine-Oalgarno sequence (GGAGG) for rbcL were conserved. In

addition, a sequence motif (TCGAG) was found 38-45 bp apart from the SD sequence of

rbcL by comparing the DNA sequences as shown in Fig. 10. Interestingly,' one of the

51 terminal sites of processed rbcL mRNAs are located 8-12 bp upstream from this

conserved sequence. motif in maize and spinach. The distance from the rb~L promoter

to SO sequence in liverwort rbcL is shorter than those in other plant chloroplast

rbcL, but the distances between promoter regions and this newly identified sequence

motifs ~~re not conserved in land plants. This sequence motif may have some roles

on the t,:,ans1~tio,nal regulation of rbcL gene expression.

In the'l iverwort cytochrome f preprotein (petA), the N-termina 1 regi on .(1:-~5

ami no aci d res idue) shows rather low homology (40%) wi th correspondi ng'reg;:oris of

those from pea and spinach chloroplasts. The N-terminal 35 amino acid residues of

the pea and spinach cytochrome f proteins have been shown to be removed as a signal

peptide for localization into the thylakoid membrane (Willey et ~. 1983).

The liverwort chloroplast petB and petD genes were found to 'be interrupted by

introns classified as group II intron in fungal mitochondria (Keller and Michel

1985). Introns were not reported in the spinach chloroplast petB and petD genes and

it has been suggested that the psbB, petB and petD genes are co-transcribed in a

primary transcript following intercistronic splicing (Westhoff 1985). A similar

gene organization as in the liverwort genome can be interpreted in the spinach

nucleotide sequence presented by Heinemeyer et~. (1984) by application of intron

consensus sequences (Keller and Michel 1985, Ohyama et~. 1986). The molecular

weight calculated from the deduced amino acid sequences of the pe~D (17.~kd) agrees

reaio~~bli with that (lj.5 kd)' of the spinach subunit 4.polypeptide determined; by

50S-polyacrylamide gel electrophoresis (Alt et~. 1983). Findings of introris'in

liverwort petS and petD genes and absence of promoter sequences and termination

signals in the spacer regions between psbS, petB and petD genes suggest that these

three genes are transcribed in a 7.3 kb common precur.sor mRNA. Further study to

-54-"-

identify the splicing junction between their exons ;'s in progress.

11-3 Genes for ribosomal proteins and ~ subunit of RNA polymerase (rpoA)

Gene expression in the chloroplasts shows many similarities with those in

prokaryot i c ce 11 s such as 1. coli. Ri bosoma 1 RNAs (rRNA), trans fer RNAs (tRNA) and

ribosomal proteins involving the transcription and translation in chloroplasts have

prokaryotic features (Whitfeld and Bottomley 1983, Kozak 1983). Chloroplast

ribosomes playa fundamental role not only in chloroplast biogenesis but also in

photosynthesis, since several photosynthetic proteins are made on chloroplast

ribosomes (Ellis 1981). Chloroplast ribosomes share many structural and functional

similarities in common with prokaryotic ribosomes including subunit sedimentation

coefficients, mode of action, size and sequences of rRNAs (Bartsch et~. 1982, .

Shmidt et~. 1985, Bartsch 1985). The similarities in the translational systems

are so extensive that initi~tion and elongation factors. even ribosomal subunits of

f. coli and chloroplast can be interchanged (Graves and Spremulli 1983, Schmidt et

!l . .1983). Some of the cDNA coding for the chloroplast ribosomal protein have been

obtained from pea (Gantt and Key 1986).

The genes for chloroplast ribosomal proteins; S4 (Subramanian at~. 1983), S7

(Montandon and Stutz 1984), S11 (Muller et EJ.. 1986), S12 (Montandon and Stutz

1984), S16 (Shinozaki et EJ.. 1986), S19 (Sugita and Sugiura 1983),L2 (Zurawski et

~. 1984), S14 (Umesono et~. 1984, Kirsch at~. 1986), and L16 (Posnoet~.

1986) have been identified by amino acid ~equence homology with the corresponding I. coli ribosoma) proteins. One ·third of chloroplast ribosomal proteins are, thought to

be encoded by chloroplast genome (Filho at~. 1981) but some of the re~ts are shown

to be encoded by nuclear genome as a precursor polypeptides (Schm~dt et ll. 1985).

It is not so far: clarified how many genes for ribosomal proteins are encoded by

chloroplast genome.

Two different types of RNA polymerase activities appear to be present in

chloroplasts (Greenberget~ •. 1984). One;s associated with a transcriptionally

-55-

active chromosome and is preferentially active in rRNA synthesis (Br.iat and Mache

1980). The other ;s readily extracted in soluble form active in tRNA and mRNA

transcription '(Gruissem et ~.-1983). The site' of ,synthesis of these RNA

polymerases have not proved to be either in cytoplasms or in chloroplasts (Lerbs rt

~. 1985, Muller et~. 1986). ' The solub1e RNA polymerase from maize (Kidd and

Bogorad, 1979) and pea (Tewari and Goel 1983) has been shown to have subunit,

structure.

RESULTS

ThirteenORFs shows significant am;no-a~id sequence homologies with respective

~. coli ribosomal proteins (l2, U4, U6, L20, l22. L23, L33. $3, 58, 511,512, 518,

and 519). The amino acid sequence alignments withccirresponding ~. coli ribosomal

proteins are shown in Fig. 11 A-M. Percentages of the amino acid sequence homology

of liverwort ORFs to~. coli ribosomal protein genes range from 25.3% (L23) to 70.2%

(512).

A large cluster of ribosomal protein genes was found on the LSG region next to

the Junction site (JLB) (Fig. 3G and 4G). The organization of the gene cluster was

(trnI)-L23-35bp-L2-36bp~S19-17bp-L22-48bp-S3-57bp~L16-97bp~L14-81bp~S8-86bp-(infA)-

36bp-(secX)-50bp-si1-32bp-(rpoA). The putative infA gene product exhibits 56.4% and

60.3% amino acid sequence homologies to the~. coli initiation factor 1 (Pon et .5!l.

1979) and the spinach infA product (Muller et.5!l. 1986). respectively (Fig. 11-0).

The secX is the putativegene'for unknown polypeptide expressed ;n ~. coli (Gerett;

et.5!l. 1983). The amino acid sequence shows 62.2% homology to the~. coli secX

protein and also shows 86.5% homology to the spinach open reading frame

corre spond i og to the 1; verwort secX prote i n eMu 11 er et El. 1986) (Fig. 11 P) • The

rpoA is the putative gene for~ subunit of RNA polymerase whose amino acid sequence

shows homologies to those of I. coli (25.6%, Meek et~. 1984) and spinach (54.1%.

Muller et ll. 1985) (Fig. 11N). Spacer regions between their genes in the cluster

appears to be. less- than 97 bplong. No'- significant prokaryotic promoter .. sequences

-56-

(A) rp 12 (LZ)

U"CNort Spinach hf.Q]l

l-lAl~L YRAYTPGTRIIRSVPKFDE I VKCQPQKKL TYtIKHIKKGRmlRGI rTSQHRGGGHKRL YRKI DFQRilKKYITGKI KTI EYDPIlRIITYICLlI/YEOGE : ::11: :KiS:SS:: :GA:QVKSIIPRtItlLlSGQRRCG---:':: :A:::: :AR:;:::::::::::: :R:: E:D:V:: :V:::'::: :'::Il:: :,::Il:G:::' :: VVKCKPTS:: R: HVVKVVIIP: LH:GK: FAP: LEKUSKSG':::: II: R:: TR: I::: : : Q": : I V: : K:: : DG: PAVVERL:: : :: :S"II:A: VL:K: "

KRYIL VPRGi KLODT IISSEEAP I L1G1ITLPL TrlHPLGTi\llllH EI TPGKGGQLVRMGTVAK IIAKEGQlVTLRLPSGE 1 RLlSQKCU.T IGQI GIIVOV :::: :11:: :AJlG::: V:Gi: V: :KH: :A::: :0::::::::::::: L: R::: :1\::: :A::: L::: ::KSA::K::: ::V:::: KII: S: :V:,:V:: :G: R::: : A: K: l:AG: O:Q: GVO:A: KP: ::: :MR: I: V: STY: :V: 11K:: :::: : A: S::: YVO: V:RD:AY::::: R:: :M: KVEAD: R:: L:EV: :AEII

III1LR I GKAGSKRWLGKRPKVRGVVMI1P I Oi'PIIGGGEGRAPI GRKKPLTPWGIIPAlGKRSRKIlIlKYSOTL i LRRRKtlS :QK: L: R: : :::: :::: : V::: ::::: V::: T: VVK: GPOL'-: E: A:O:L: V1UILEEEVEKGlIIIVI lLLFVOVVI1RKEIIEIlRISFFVFT MLRVL::: :M:: R: V: : T:: :iA::: V::: G: :11: :: II-F: -: If: V: ::,: VOlK: :KT: S:-: -T: KF:V::: SK

277 287 ,58;5%, 272 4S.4%

(B) rp114 (Ll4)

L f"CNort HIQPQiYLIiVAmlSGARKlHCIRVI GTSIIRKY/\/lIGDII iAVYKEAVPIIMPI KKSEIVRAV IVRTCKEFKRlItIG511 KFDDIlAAVV IIIOEG-IIPKGTRVF : :: :: :::::::::: .: :-: ! : ! : :! : : = : .. ...... .. .. .. ~ ........ .. ............... ..

hf.Q]l HI QEQTHl/IVAOllSGARRVHCIKVLGGSHRRYAGVGO II KIT I KEAI PRGKVKr.GOVLKAVVVRTKKGVRRPDGSViRFDG;lACVLL~W1SEQP IGTill F · .

GPIARELRESUFTK1VSLAPEVl 122 ............ .. .. ...... . GPVTRELRSEKFHKI1SlAPEVL 121 58.2%

(C) rp116 (Ll6)

l!veNort Splrodcl~

.L.~

MLSP KRTKfRKQHCG ilL KG i STRGllV 1 C fGK FPLQAL E P SW 1 TSRQ I EAG R RA 1 TRY ARRGGKlW [R I FPOKP I T I R PAET RMGSG KGSPEYl-IV'AVVKPG ::::: ::R::: ::R:RJ-I: :K:Y:: :H:S::RYA:::V: :A:: :A::: :::::: :S:::::::: l:V:L: ::::V:L:::::::::: :::::: ::5'::::: : :Q:::::: ::M: K: RIlR: LAQGTO:S-: :S:G:K:VGRGRL:A:::::::: :H: :AVK:O:: I:: :V::::::: EK: LAV:: :K:: :IIV::::: llQ::

.. . .. .. K I L Y£ISGVSnnARMNKIMYKHPIRTQF JTTSSLtlKKQEI R::: : : G: ::: TV: : T:: lL:: 5:::::: ::: lEE :V:: :MO: :P: EL:: E: F:L: :A: L:: K:T:V: KTVM

143 135 72.0% 136 53.8%

(D) rp 120 (LZO)

L t VCNort flTRYKRGYVARKR'RKIi I L TL TSGFQGTfISi:LFRTAIIQQGMRALASSIlRDRGKRKRIILRRLWITRVIlAAAi!DNG 1 SYHKLi EYL YKKK I LltlRK I LAQ (Ai .. ..... ........ .. ...... , .......................... .. .. ..... ........ ...... ...... .. .... .... .. ... .. ..... ...... .... .... . ...... .

.L. f.Q]l HARV):RGV J ARARIIKK I LKQAKGrYGARSRYYRVAFQAV I KAGQYAYRDRRQRKRQFRQLIIIAR IIIAAARQlIG 1 SYSKF IIIGLKKASVE I ORK (LADI AV !

lOKFCFSTlI Kill ITE 116

FOKVAfTALVEKAKAALA liS 46.6%

(E) rp 122 (LZ2)

t fVCNort HQTI4TSHKKi RAV~~~ I ~M:""~PH~~~R~VSQ~ ~~RSYE~~~H~ ~EFMPYR~CNP I LQL~S~~;I~II~~F~LSKTtI;F i SE ~ Q~IIK~TFF~~FQ:~~ .L. ~ MET IAKHRHARSSAQKVRLVAOLlRGKKVSQALDIl iYTIIKKMVLVKKVLESAI AIIAEHIIOGAD I DOLKViKI F.VOEGP5MKRIMPRAKG

· . RGYPHIKPTCH ITlVLHI LPK .......... .. .. .. ... .. RAORIlKRTSHTTV\,VSDR

119

110 37;8%

(F) rp 123 (LZ3) I- • .. .. .. .. .. .. ..

l t veNort MIIQVKYPVLTEKT J RLLEK-IIQYSFOVNI OSflKTOI KKWI ELFFIIVKV I SVIISHRLPKKKKK IGTTTGYTVRYKRMI IKLQSGYS I PLFSflK 91 . :.. .... : ': : ,:': .......... ......: : .. - ... .

.L. ~ HI REERLLKVLRAPIIVSEKASTAHEKSIH I VLKVAKOATKAE J~MVOKLFEVEVEVVIITLVVKGKVKRHGQR1GRRSO-KKA YVTLKEGONLOFVGGAEK 100 25.3%

(GJ rp133 (L33) .. .. .. .. .. ..

LlvcNort MAKSKO I RVTI IILECI NCAQIIOEKRKKG I SRYTTQKHRRIITP I RLELKKFCCYWKHTI HKEJ KK 65 ...... '" ......... . .. .... . ............. . MAK-GJREKIKL----VSSAGTGIlFVTTTKHKRTKPEKlELKKFOpVVRQHVYIKEAKIK 54 36.9%

. ~ . . (II) rps3 (S3)

.. .. I- I- I ~ .. .. .. ,; .. ..

L I VCNort HGQKWPLGFRlG I TQIIHRSYIIFAIIKKY5KVFEE-DKKJ ROC I-EL YVQKH I Ktl5SIIYGG IARVE I KRKiDLlQVEI YTGFPAlLVESRGQG I EQLKLII .... ... ... . .......... . ....... .... .... .... . HGQKVHPtlG I RLG I VKPWIISTWFAtlTKEFADIILOSDFKVRQYL TKELAKASVS-,--- RI VI ERPAKSI RVTI HTARPG 1 V J GKKG-EOVEKL-

.. .. .. .. .. .. '.- -. . .. VQII I LSSEDRRLRMTLI E I AKPYGEPKI LAKKI ALKLESRVAFRRTM~KAI ELAKKGN I KG I KI 01 AGRLNGAE IARVEIIAREGRVPLQTl RAR( IIyen,

=-.. .. ... :::: ::: :::: ...... ::': ::::::: ::- ::::::: : n : : RKVVAOIAGVPAQI NIAEVRKPELOAKLVAOSI TSQlERRVHFRRAHKRAVQIlr.HR lGAKGIKVEVSGRLGGAEIARTEIIYREGRVPLIlTLRADIOYNTS

· . MQTIYGVLGIKVlIl FQDEE ............... .. .. ... . ...... .

217 , . '} :. ~ I', ,~

EAHTTYGVIGVKVWIFKGEI LGGMMVEQPEKPMOPKK(jQRKGRK 233 40.6%

Figure 11 (continued).

-57-

(1) rpsB (sa)

L t verwort I'.GllOT IIII/Hi TS I RNIINLGK IKTVQVPIITNITRfll AKllFQEGF lOlln iJIIKQrmo r l i l/ILKY -QGKKXKSY r TTLRRI SKPGLR I YSIIIJKE I PKVlG

h £.'2.ll HSHQOP I ~O~L ~R I ~;IGQMIIKIIIIVmpSSK LXVIII ~IIV~KE~GF I EOFKVEG-o~kpE~E ~ Tl.~~FQ~~'\VVts I QRV---SRPG~R [~KRKD~L~~~I-IA . GMGIV/LSTSRGlMTDREARQKKIGGELLCYI'II 132 .... !::: :::: -:::. ::: ::: GlGIIIVVSTSKGVHTDRIIIIRQIIGLGGE IICYVII 130 45.5%

(J) rpsll (511)

liverwort Spin.eh I:.~

HPXSVKKI NLRKGKRRLPKGV I HIQASFllilTl ViVIDI RG~VVSWSSIIG~CGFKGTKKSTPF IIIIQTMEHIII R iLl DQGMKQAEVHI SG? HAXP1PXIGSRRNGR:SS::SIIRKI::::: :V:::::::::::: :V:; R:: ::11:; :T:: :R::: RG:::::::: :G::: :TVVE:: :QR:::: :K::

:A:APJRARK:VR:QVS-D: :11: :11::::::::: I:: RQ:III1LG:AT: :GS:: R: SR:::::::: V;:: RCIIDAVKEY: I :IIL::: VX:: .. .. I ..

GPGRDTALRAlRRSG1ILSFVROVTPMPIINGCRPPRKRRV SL: : : II::::: ::::: L: :::: II:::: :: : : : ::: X:: : : :: ::ESTI:: L1IM: FRlTHlT:::: I:::::: ::X::::

130 138 72.3% 129 51.5%

(1::) rpsll (Sll)

Ltve ..... ort Tob.eeo L. gr~ci lis h coli

.. .. .. . .. . . . . .. MPTlQQL I RflKRQP I EflRTKSPAlXGCPQRRGVCTRVVnTPKKPIISIILRK I ARVRl TSGFE I TAY I PG I GHNLQEIISVVL VRGGRVKDlPGVRYli II RG : :: : K::::: T:: : : R: V::: : : : R: : : : : :: T:::: : : I: ::: : : : : :: : V:: :::: : : : : : : : : : : : : : : : : : : : : : :: : :::::: : : : : :.: : : : V: : :: :LEH:T:SP:KK:XRK:::::::::: :K:AI :11::::::::::::::: :YT::: :$: :L:V::::::::::::::;::: I:::::::::: ;K: :V::: : II: VII: : V: KP :ARKVIIKSNV: :: EA,: : K: : ::: :::: :: : : ::: ;: ::: ve: : : : : II: : : V: S:: G: E: :::: :: : : I: I: : : : : : : :: ::: :: TV: :

· . TLOAVGVKDRQQGRSKYGVKKSK : ::::: :: :: : :: : : :::::: P: C:: : AS::~: KUII: : :::;:: P: PK II:: CS:::: :K:II:::::: :RP:A

lZ3 123 91.9% 125 73.6% 124 70.2%

(L) rpsl8 (SIB) .. • • • .. .. 0-

l iVCl"ort HHKSI(RSSRRRMPp IRSGEI IOYKIIISLLRRFVSEQGK I LSRRMIIRLTSXQQRLL T1AIXRARVLALLPFUlNEII 75 .. .. .. .. .......... " .. .

h £.'2.ll MARYFRRRKFCRfTIIQGVQEiOYKOiATLXflYITESGKiVPSRITGTRIIKyQRQLIIR;'ii:RARYLSLLPYTDRHQ 75 34. n (H) rps19 (519)

Llvervort Splnllch Tob~ceo

hE!!.!.!. (N) rpolI

L jv"rvort Spinoch h~

CO) in(1I

Liv"rvort SpiMcn hill!.

(P) seeX

Llvcntart Spinllch h£2!!

.. . .. . . .. . .. .. MTRS I KKGPFVIIDHLlKK I EIIUJLKKEKK III TlISRAST! VP-TMI GHT IIIVHNGQEHLP I Y ITORHVGHKLGEFIIPTRTFRGHIIKIIDKKSRR :: ::l: :11::: :11:: :R:: :/(: :/(:A:: E: :V:::::::: 1:-:::::::: I:: :R::::::::::::::::::::: :W::::::: :11:::: ::: :l::II::: :11::::: :ox: :T:A::E::V:::::::: I :-:::::::: I: ::K:::::::: :5::::::::::: :lIl::: :::S:flR::: :P:: L::::: IOL:::: :V:KAVESGD: :PlR::: :-::: F:DR::: t:::::: :RO:V:VFV:: E:::::::::::: ::Y::: :A: K:AKKK

92 9l 83.7% 92 BO. 4~ 92 63.0%

• • • .. ... .. • • 'I- •

M lQOEI KVSTQTLQWKC I ESKI ESKRLLYSRFAI SPFRKGQAIITVGIAMRRIILLNEI EGASliY AKIKKV.KHEYSTI IGLQESI IIDIllllLKEI VlKSES : VREK: R: ::::::: :: V:: RTO:: C: II: G:: Il:: lH: : : : 0: I: : : : : : : :: G::: : TC:: R:: SE: I P:Q: : : : l: I: : : V: E: : H: : : ::: : R: Nl

HQGSV: EFL:PRlVO: :QVS5THAKVTlE:LER:FGH: L:II: L:: I:: SSl1P: CAV: EVE:OG: L::::: KE:V: :D:LE:: l:: :GLAVRVQG

FEPQKAYlSVLGPKKITlIQOll<GPSClKIil1lAQYIATlilKDI LLEI ELill EKD-RGYRI ENLQKYQ£GLFPVDAVfMP I RIIIINYSVHSFESEKKI KE i YGTCE: S: C: R: : RGV:::: : Il: PYVE: VKIIT:H: : S:IEP: D: C:G:Ql: RN~:: : II: KAPIINF: D: S:: I:: t: : :V: : V: II: 1: :YGIIGNEKQ:: KDEVILTLNKS: IGPV:: II: : TIIDGDVE: VKP-: IIVICHLT: WAS: SHR: XVQRG:: :VPASTRIHS: EOERPIGRLLVO-: C:: -~V:RIAYllV: A

LFlEI WTOGSt TPKEAL YEASRIIL 10LFIPLIIISEKKEKilFGI EKHlESiIMSYfPFQSV5LDI EKMTKOVIIFKH I FIOQl.ELPARAYNCLKKVIIVHT lAO ::: : : :: H: : :: ::: :::: : ::::::: L:: FLIIA: EIIV-: LEOIIQIIKV: LPL-: T: H!lRLAE: R: 11K: KI: L: F: : : : : : : : : P: I: : : : : : S: I: : LL: ARV:OR: : LOKlVI :-M:ltIG-T: :-PEEA: RRAATlLIIEQL: AFVOLRDVRQ: EV-KE:: PEf: Pil-LRPV: 0:: : TV: SA: :: :AEAI: Y: G:

LLHYSEODL i K I KflFGKKSVEQVLEALKKRFS IQLPKNKim : :NN:QE::: :H:II: RIEK: K: IFGT:E: H: V: D:-:::R :VQRT:VE: l: TP:L:!: :LTEIXOV:AS:GlSLGMRLE: WPPIISlflOE

340 335 '54.1% 329 25.6%

MEKQKl r OMEGVV I ESLPNA TFRVYLOIIGC I VL TH 1 SGKi RRIIY IRIlP(iDRVKVELSPYDL TXGR ITYRlRIIKSSIIII :KE: :W:-II:: LIT:::: :GH:W:R:: :£DPI:GYV:: R:: :5S:::::::::: I:V: R: :S:R::: I::: :11: D::O :A: EOII: EMO: T: l: T::: TM::: E: E: :IIV: Til::: :: !-I: K: :::: :T: :/(: T: ::T::: :5::: :VF: S:

· . . MKlRASVRKICEIICRLIRRRRRIHVVCSN-PKHKQRQG ::::::: :P: ::K:: ::: ::G:: I: I:: :-:::::::: ::V::::K:L:R::KIVK:OGV:R: I: :AE::::::::

37 37 86.5% 38 62.2:t

Figure 11 (continued).

-58-

78 77 60.3:;: 72 56.41:

(Q)Ollfl84

Llva .. ort ORF184 . HllLQVDH I RVDFJ IGSRRI StlFeWAF I LLFGALGFfFVGFSSYLQKDLI PFLSAEQI L,jPQGIVHCFViaAGLFlSFVL

Euglena ORF149 11NLRDltIIIHTLSKtlEtHkIlKQr.QItILPKIL~QEIKElmKI i r.WfY-lI1VM~l~G I~~L 1~~1 ~~~I Glm~; Y~LD~SEr I ~F~Q!;h~c~~~TC~1 L~~I/IQ WCTlCWIlVGSGYHKFDKQKGI F51 FRWGFPGKIIRRI F IQFllKDIQSI RMEVQEGfLSRRVL VI Ki KGQPDIPLSR (EEYFTLREMEOKAAELARFLKVSI EG I ,184

.... ••• .. .. .. ......... 0-.. .... .. .. .. ..... '. .... . .. J SI ILNGVGEGYtlEFtIKEl/ILMTI YRKGKQGKt/SDltl,1 TY5LKD I VKE

(RJ 0llF203 . . Liverwort ORF203 (Exon I) HPIGVPKVPFRLPGEEDAVWI DV'-( intran)

::::::-:-:!:: ;::::: : :: SplMch X-gene (E~an 1) MPIGVPKVPFRSPGEEOASWVDV-( intran) 87.DZ

(5) ORF40 .. • • 0-

MIIllTTGRYPlWl IGTVAGI l YIGlVGIFFYGSYSGLGSSL ................ •••••• 0-0- ...... . : : : : : : :: ~:: = : : : HAIHGGR I PLlILVATVAGLAAIGVLGI fFYGGYSGLGSSI

40

4072.6%

149 ·38.3<:

Figure 11. Amino acid sequence alignments of ribosomal related proteins and subunit. of RNA polymerase. The amino ,acid sequences are .shown by one letter

codes. Identical amino acid residues are shpwn by colons, and deleted residues are shown by dashes. The amino acid residue numbers' and sequence'

homologies with liverwort gene products are indicated at the end of sequences.

-59-

were. observed between them. Another ribosomal protein gene cluster is localized

between' trnP-UGG and psbB as the order of trnp-'124bp-L33-27bp-S18-81 bp-L20~786bp-, .

S12( exonl),-72bp-ORF203-385bp-psbB. ' The rps12 g~ne (Exon 1) is .loca 1 ized 72 bp

downstream from TAG stop codon of ORF203. The rest of the exons are located far

apart on the different DNA strand indicating trans-splicing mechanism for the gene

expression (Fukuzawa et~. 1986). Detail discussion on the trans-split gene rps12

are ;n Chapter III. The coding sequences for ribosomal protein L2 and L16 are

interrupted by 545 bp and 534 bp group II introns, respectively (Fig. 4F).

DISCUSSION,

In the liverwo~t chloroplast genome, nucleotide seqtience rev~aled a large

cluster of genes coding for ribosomal and related proteins (trnI-L23~L2-S19-L22-S3-

L16~L14~S~-infA-secX-S11-rpoA) on the LSC region near the J LS• Upstream from the

gene for L23 ~ibosomal protein, there is an isoleucine tRNA(C*AU) gene whose

promoter highly functioned in 1. coli as well as in chloroplasts (Fukuzawa et ~.

1985). The length of the gene cluster from the 51 end of isoleucine tRNA gene to 31

end of rpoA gene is approximately 7.3 kb. No promoter sequence can not be found in

the short (less than 97 bp) spacers between ribosomal protein genes indicating that

the gene cluster may be transcribed into a single precursor RNA from the trnI to

rpoA genes. Furthermore. this cluster has similar order to the clusters reported in

the 1. coli ribosomal protein operons such as the S10 operon (Zurawski and Zurawski

1985 ). ~ operon (Cerretti et ~. 1983), and alpha operon (Bedwell et~. 1985)

(S10-L3-L4-L23-L2-S19-L22-S3-L16-L29-S17. L14-L24-LS-S14-S8-L6-L1B-SS-L30-secY-secX,

and S13-S11-S4-rpoA-L17, respectively). Two additional clusters of ribosomal

protein genes are seen in the orders of S12{Exon1)-L20 and S18-L33 between trnP-UGG

and psbB genes. The genes for S12 (from the 39th amino acid residue to C-terminal

end) and S7 ribosomal protein also have been clustered as seen in l. coli str-operon

(S12-S7-EFG-EFTu ' Post gl~. 1978, Post and Nomura 1980). In contrast, the

chloroplast rps2, rps4. rps14, rps15 and rp121 genes are scattered throughout the

-60-

liverwort chloroplast genome.

The rp12 genes of spinach and Nicotiana debneyi has been· located in the inverted

repeat regions •. In H. debneyi, but not in spinach, rp12 is interrupted by a 666 bp

intran (Zurawski et~. 1984)., However. liverwort rp12 gene is located on the LSC

region just outside of the IRB region and has 544 bp intron. Introns are also

present in the rpl16 gene of Spiradela oligorhiza (interrupted by 1411 bp intron)

(Posno et al. 1986). The first exonof genes for L16 ribosomal protein encodes for

the first three amino acid residues (Met-Leu-Ser) of N7terminal. It is interesting

that the products of ribosomal protein genes encoded in the chloroplast genome are

important components in the initial stage of ribosome and rRNA assembly (Dorne et

~. 1984).

A putative gene for ~ subunit of RNA polymerase is located at the 31 end of the

gene cluster for ribosomal proteins. Genes for ~ and ~' subunits of RNA polymerase

(rpoB, rpoCl and rpoC2) are located as a single operon at 50 kb apart from rpoA gene

. (Umesono et~. 1986). An ORF homologous to the i. coli sigma subunit of RNA,

polymerase is not found on the liverwort chloroplast genome.

II~ The putative gene ndh3 and unidentified open reading frames

, It is reported that the t1,. polymorpha chloroplast genome contained a set of

homologues of mammalian mitochondrial "URF" genes (Ohyama et~. 1986). In this

region, a gene named ndh3 corresponding to human mitochondrial URF3 was identified

by amino acid sequence comparison (Fig. 7K). The mitochondrial URF3 gene code for

the- component .of respi'ratory-chain NADH dehydrogenase complex (Chomyn et~. 1985).

An ORF120· (52877-52515) is located at the upstream region of psbG with an overlap of

seve~ nucleotides~ This ORF dose not contain an intron and the product (120 amino

acid residues, 14.2 kd) is similar in size to a human mitochondrial URF3 protein

(114 amino acid residues, Anderson et ~. 1981) sharing 30.8% homology. Actually

proteins of chloroplast ndh genes are not yet identified, but a 3' half of ndh3 gene

would be also conserved in maize chloroplast genome; the published sequence of maize

-61'-

psbG gene contains its 5' flanking region of 158 nucleotides (Steinmetz et ~.

1986). If twoG residues (1ocated at -68 and.-96 in their numbering) are deleted,

the region from -157 to +7 ·will encode a polypeptide similar to the last 54 amino

acids of the M. polymorpha ndh3 product (85.2% identical) as well as ~noverlapPing

to the downstream psbG gene. There has been no previous "report of the presence of

ndh ge~es in chloropla~t·genome. However, an NADH-plastoquinone-(PQ) oXidoredtictase

activity has been detected in the chloroplasts of Chlamydomonas reinhardii (Bennoun

1982), thus it is possible that this ORF encodes one of subunits of the NADH-PQ

oxidoreductase.

Fifteen significant ORFs, which do not show any homologies with previously

reported genes, were located on the sequenced region in this study (see Table 2).

Amino Acid sequences of two unidentified open reading frames in liverwort

chloroplast genome show significant homologies with those of unidentified frames

reported in other kinds of chloroplast genome. An ORF184 (59525-60079) shows 38.3%

local homology to the ORF149 located at the next to the gene for elongation factor

Tu of Euglena chloroplasts (Fig. 11Q). The Euglena ORF149 does not terminate of its

stop codon but follow intron sequence (Montandon and Stutz 1983). It is interesting

to compare the C-terminal region of liverwort ORF184 (position 128 to 184 amino acid

residue) with the corresponding Euglena ORF in the Exon 4 described by them. The

first exon (68640-68570) of the ORF203 shows 20 out of 23 amino acid identity with

an reading frame in spinach X-gene on the opposite strand of psbB gene as shown in

Fig. llR. It is reasonable to beHeve that the open reading frames conserved in two

kinds of chloroplasts would code .polypeptide having an unknown function. In

addition. an ORF40 (62916-62794) showed 72.6% homology with cyanella Cyanophora

paradoxa ORF40, which ;s not proved to have any function (Fig. 11S, Bryant personal

communication).

-62-'-

CHAPTER III The split gene for chloroplast ribosomal protein S12

Introns (intervening sequences) in a chloroplast RNA gene have been reported;

the 235 rRNA gene of Chlamydomonas reinhardii (Rochaix and Malone 1978, Rochaix et

~. 1985); the tRNA genes. trnI-GAU and trnA-UGC, in the 165-235 rDNA spacer region

of lea mays (Koch et~. 1981) and Nicotiana tabacum (Takaiwa and Sugiura 1982); as

well as the chloroplast tRNA genes trnL-UAA (Steinmetz et~. 1983a, Bonnard et ~.

1984), trnK-UUU (5ugita at ~. 1985), trnG-UCC (Deno et~. 1984a, Quigley and Weil

1985) and trnV-UAC (Deno et~. 1982. Krebbers et~. 1984. Zurawski and Clegg

1984). Introns within chloroplast protein genes also have been reported in several

genes of Euglena gracilis; for the large subunit of ribulose-1,5-bisphosphate

carboxylase/oxygenase (rbcL) (Koller et~. 1984), the elongation factor Tu (tufA)

(Montandon and Stutz 1983), and the 32-kd protefn (psbA) (Karabin et~. 1984.

Keller and Michel 1985). The gene for the 32-kd-protein of £. reinhardii also has

introns (Erickson et~. 1984) as does the- gene for the H+-ATP synthase subunit I

(atpF) of wheat (Bird et~. 1985). Zurawski et~. (1984) reported that the

chloroplast ribosomal protein l2 (rp12) in Nicotiana debneyi has a single intran.

Several genes in the chloroplast DNA from the liverwort. M. polymorpha- are shown to

have introns in their coding sequences (see Fig. 2 in. chapter II).

Hallick et~. (1985) and Fromm et~. (1986) reported that the reading frame of

the ribosomal protein 512 in li. tabacum is interrupted by two introns, but they

described only the second one. During nucleotide sequencings of the chloroplast DNA

from the liverwort, M .. polymorpha, however, the first exon with the 51 intron

boundary sequence was found on the opposite strand of the chloroplast DNA. In this

chapter, the complex structure of the putative gene for chloroplast ribosomal

protein 512 from the liverwort. M. polymorpha is presented. which has threeexons

split into different DNA strands. The mechanisms of the expression of this

unusually organized gene will be discussed.

-63-

Iff, /JlJlymorplla Ct - DNA t pSDB '" I rNll"

I"NZO

Figure 1. Locations of coding regions for chloroplast ribosomal protein S12

on physical maps of chloroplast DNA from a liverwort. M. polymorpha. Exon 1

(rps l2A) wa·siocated on the 8g 1 II fragment (8g5) i and the di recti on of its

transcription was clockwise indicated by an atrow. BY cohtrast, exons 2 and 3

(rps12B and C) ·were found. on the BamHI fragment (Ball), and their

transcription being in the opposite di:ection from that of exon 1 (counter

clockWise). The abbreviations rps12. rps7 ahd ~p120 are for genes of

riboso~~l prbtei~s S12~ S7 and L20. The site of the gerie for the l~rge

subunit of ribulose-l,S-bisphosphate carboxylase/oxygenase (rbcL) is shown.

IRA and IRg indicate a set of inverted repeats, and SSC and LSC indicate the

small single copy and large single copy regions. respectively.

-64-

MATERIALS AND METHODS

Chloroplast DNA fragments. the BamHI (Ball) and BglII (Bg5) fragments. were

cloned into the respective plasmids, pBR322 and pKC7, as described by Maniatis et

!l. (1982). A physical map of the chloroplast DNA for BamHI fragments has been

described previously (Ohyama et~. 1983, Umesono et~. 1984). The location of the

6g5 fragment on that map was determined by restriction analysis and Southern

hybridization (Fig, 1). Methods for the sequence determination are described in

chapter II.

RESULTS AND DISCUSSION

At first a coding region for ribosomal protein 512 on. the .BamHI fragment (Ball)

was identified using Southern hybridization with·an g. gracilis probe (provided by

Drs. Montandon and Stutz). The Ball fragment· was mapped at the junction (J lA)

between the inverted repeat (IRA) and the large single copy (lSC) region (Fig. 1).

DNA sequence analysis of the Ball fragment revealed that the coding sequence for the

ribosomal protein S12 wa~ found, however, the N-terminal 38 amino acids of the

protein was missing in this coding regi9n.

By amino acid homology search between g. coli 512 (Post et~. 1978) protein and

open reading frames deduced from the nucleotide sequence data files. the missing N­

terminal 38 amino-acid sequence was foun~ on the BglII fragment ·(~g5) appro~imately

60 kb away on the opposite DNA strand (Fig. 1). ~omplete nucleotide sequences of

the coding regions for ribosomal protein 512; rps12A -and rps12B-C, including the

fl an king regi ons, are -shown in F; g. 2. Exons 1: and 2 were fo 11 owed by a consensus

sequence (GTGCG) of the 51 boundary regions found in fungal mitochondrial gr:oup II

introns and g. gracilis introns (Michel ·and Dujon 1983). \ In addition,· much conserved

sequences, RAGCCG.AUGAA •• GAAA •• UUCAUGU.CGGUUY, were found in'the 1ntrons 75

nucleotides upstream from exon 2 and 61 nucleotides upstream from exon 3. This

consensus sequence has been present in all the introns found so far in chloroplast

genes of ~. gracilis (Keller and Michel 1985). The secondary structures of introns

-65-

A ORF~03 (Exon3)

mATGTCAGcAAMGMGCAAMCTTTATGGTAtTGTAGAmAGTTCCTATAGMMCAATTCTACTATTMAAATTi.crITTTAAACMAMMTTITATTTCTTAicGTTACGnr 120 F ~ SilK E A K L Y G ! V D L v· A I t N N S T I K 11 +------>(- ---+

rps12A .(t.onl) .

ATCCMACTMMMTmGCATATMGrTA TGCCTACTATTCMCAATTMTTACAMTMMCACMCCCATCci.MATMMcAAMTCACCAGCCCTTAMC(;ATGCCCTCM 240 ? TIL I R UK R PIE U R T K SPA L K G CPO

CGTMACGAGTATCTACTAGAGTGTA TGCGACTTCTTrMATCAJ.MilccTTMAAATTTMACATcAAAATTCCATMMATTTTTTTATTTTMTAACGTAMGATATAGTAICTA 360 R R- G V C T R V Y U~Y9 (S' 'nt.-on) , f-' (----T _,

TTGTTGTTTAcATAtAATTTATAGTTTCCTTTGGTGCAMTCCMTCATCTTMCmAGcATAGAAAACCATTTCTcr.AAGGGTAGCGACTGATTCTcAATCCCTTMGCCAGMATrT qeo (--I-

TATTMAAAATTTTTCCATAATATMTArTACTTATATMCCGTMMACGAMCTGMCGGTCAGCTATrCACCGMCCTTCAAATMCATCCCGTTMTTMTMAMMACMTTrT 600 +->

TGAMcrniTTTAGTGTrTCATTMTMAAMAGCTTcAMTCAGAMTrATA(;AMTAACTGATATTATCMTATATATTATATATTACMGCTTCGGTATATACMAGGACCTATTC 720 +-----:.- <-----t

. Gr:.GMGGAGAMCTATAci.M~GGAi.TGCATMTTmCTTAGTTAMGGTCCTATcci:TTMTT~CTMGMGGTATCATACCTAMMMTTATTATTMGcAAGCTiATI,GTA 840 +--> <--I-

GCMATCCmTGGTArnTrTTTTTATACAIMGMMCGMGAMTrTTTTATAGcMTCTMGAAAATMMTMAACTTTTTTArTAlAAMATTGIAGATTATAGIMGCMACT 960 +- --> (---+

GCMTMMAMIATTTATTGMMICGATGTTTTGATATM.MAMTACACACACAcr.AATTTTTGMTMTTMMCGAGTATATAcAGCMTGACTAGAGTTMACGTGGTTATGTA lOBO +-> (_. -~ rp120> H T R V K R G Y V

GCACGMMCGGCGTAMMTATTCTTACGCTTACATCTGGATTTCMGGMCTCATTCGMACTTTTThGMCTGCTMTCMCMGcM.TGAGAGCATTAGCATCATCTCATCGCGAT 1200 ARK R R· KilT L T l T S G F Q G T II 5 K L F R T A II Q Q G ~ R A LAS 5 )I R D

AGACGTMACcMMAGAAATCTTAGACGTrrATGGATTACTCGAGTTMTGCAGCCGcM.GAGATMTGGMmCCTATMTMATTMTTGMTArTTATAT~TTCTT 1320 RGKRKRIILRRLWITRVIIAAARDIIGISYIIKLltYLYKKKIL

TTMATAGllAAMrrcTAGCTCAMTAGCTATATTAGATAMmTGTrTTTCGACMTMTTAMMTATTATTACAcAATAMMMACCTtTCCGG~TAATTAMMMTAMncc 1440 L II R K I L A Q 1 A I L 0 K fer S Til K II I ! T E --. _. __

GGGGAGGTTiMTAMAAAACTGTATATTAATAMTMTGATMATTMTmCGTTArTTAMAMGGTMCMAGCTMAACACGAGCTCGTTTMnGCTATACTTAATMACGTTG 1560 - ---IIEIIIIlFl'LLALVRARKIAITLLRQ

<rp.ll1

Figure 2. Complete nucleotide sequences near exon 1 (A). and exons 2 and 3

(B) of the rps12 gene. The exons of rps12 gene are boxed, and the consensus

sequences of their 5' intron boundary regions (gugyg, y represents C or T

residues), as well as tho'se of the near 3' intron boundary regions (ragccg­

augaa-gaaa--uucaugu-cgguuy. and cuayy(-)y-ay, r represents A or G residues)

are shown under the nucleo£ide sequences. J LA stands for a junctibn of ~n

inverted repeat (IRA) and a large single copy region (LSC). Amino acids are

expr~ssed as one letter symbols under the nucleotide sequences. Possible stem

structure for the transcription termination are shown by bold arrows.

-66:-

B Inyorl~d Rep •• t -_ Lorgo Singlo Copy

J£,A

TCAMTHTiATGTTMMW.TACATATi.GMGMMAMGMAAATAATTGmGAAiTTllGAMTAAMTGTTATAMCTA~~TCA1rTGMCGACMGCCGTATGMATGAAAA TTAAGA,. TATAAT> -----l"'o!I.9ccog-augbb-g1l4'O-

ep.IZB (EXOH 2) (3' loteM)

TATCAAGTA(GGTTTTGTAMGTGACAATTrAGGTMCTiAITTGTCAACTTTTC CTACAACAcCAAivv.AACCMACTCTGCCTTACGMAAATAGClW.cTTAGACTMCClCTG -uut.U?U-C99uuY---------------cu.yy-y-. 'T T P K K P N 5 A L R X 1 h R V R L T S G

GATTTGAMTTACTGCATATATTCCAGGTATTGGCCATAAITTGCAAG ..... CATTCAGTTGnTTGGTMGr.GGAGGAAGGGTCAMGATTTACCTGGTCTMGATATU.TATTATTAGAG F E I T A YIP GIG II H l Q [ II 5 V V L VR G G R V K 0 L ~ G V R Y Il I I ~ G

GMtACTGGATGCTGTAGcAGTAAMGATCGTCAACAAGGGCGTTCT iGCGTTGTATATTATMTCTAHMMTGTATCATTTTAGATACCTMrri"TTGCTGATAATATGTMAA o A V G V K 0 R G R S ugyg-(Inlrcn) ----------------

MTAGCfMCCAGTGATTAAMTTTACArTTTMAACGci..w.A.MGCAGcCfATATGTArATMMTMMTAMATATTATCTATArTATATACTATACAATATCT"cGGTTTTATTT ~------------(I"tron)

ATAGTTAAAATAMAATTTMcTTTTcccTTAcnmMTTCAMATMMMAAnrTACTTHTTAGAACAAGTTMMTAAATAGCAA.w.TAAAMt..ATHATTTTTATACMTA -------------------(Inlron)

TTTTTATAMTAMCCTMGGAHTTTTATTTMCGATTATAhMTACMGATTTCCAATAGTAMACACTGGAAACCcATACTCMTTAAAAGTGAGTWCATCMTAAMTTMAcA -----( Inleoo)

rps12C (UOII 3) .. .. .. .. • • .. .. 4 • • •

120

240

350

460

600

720

B40

ATGTMMAGCCGTATICGTTGMAATAGGATGTACGGTTTGGIIGGGAGATMMAMTCCACCCTAC TATGGAGTMMMfHCAAN AMmMMTMCTCTTMATMMAA 950 ---ragc-cg-ZlIugo!4-gl!Ula.-UUC5ugu-cgguuy cuayyy-a Y G V :K K S ~

ATTAACTTTMTIAmArTATTATGTCACGTMAAGTAnGCAGAAAAACAAGTTGcMMCCTGATCCAATATATCGGMTCGATTAGTTMTATCrTAGTTMTCGTATTTTAMM 1080 ~a. II 5 R K S I A E K Q ~ A K POP I Y R I/R LVI/ II LVI/ R ILK II

ATG~TCATTAGCrTATCGGATTi:mATAAAGi:TATGMAMTATAAMCM.MAAr:AJi.AAAAJw.TcCATTAmGTATTACGTCMGCAGTTCGMMGTMCTCClMCG 1200 G K K S LAY R I l Y K A 1\ 'K 1/ I K Q K T K K 1/ P L r V L R Q A Y R K V T PI/V

• .. • • 0- • .. • • • • ..

TCACAGTCAMGCMGACCCATCGATGGATCCACTTATCAAGTTCCACTAGAMTTAMTCTACACAAGGAMGGCATTAGCCATTCGTTGGCTATTAGGAGCCTCACGGAAACGCTCAG 1320 lYKARRIDGSTYQVPlEIKSTQGKALAJRlIllGASRKRSG

.. .. .. .. .. .. .. .. . .. .. . GTCMMTATGGCTTTTAMCTTAGTTATGMTTMTTGACGCAGCCAGAGATAATGGAATTGCTATICGTMAAAAGMGAAACICATMAATGGCAGMGCTMTAGAGCTTTTGCTC 1440

Q II 1\ ArK L S Y ELIDA II R 0 1/ G I A IRK K E E T H K H A E A 1/ R A f A H

AmlCGTTAAATMAAACGTATAMTTArAMAAAAu.ATTTTTATTGrATIGAAATATGCTITMTAmmATTAnACAMTATnCAATACMTMAAATTCrTTTAGTTTTTT 1560 r R .... __ __ _ .. ---------

Figure 2 (continued).

-67-:-:

U G

A A A A G A

C V A G A U

"")~ " A A A

A A U G G C AAGCCGUAU A

~~U~G~~~ GA

u G U U G G U A A A G U G A C A A" U A U G

C G U G

: = : ; ~ :: :. -: t: ~. A U U U U CAe U G U U A U A" C A U V C C -

5'-" --ACUAGAGUGUAU,," ThrArgValTyr

."..., ACUACMCACCA--3' TyrThrThrPro

" £Xon 1 " Exon 2

A A U A A A U

U A A A U A U A " A G A A

A : U A C C : G C : G U:A V:A AA C:G A:U G A A:U C:G U A U:AAA:U U V A AGe G A C G

"CA G A V G Ace AAGCCGUAU A

/. ~ ; ~ ~ ~""~ D t u ~ ~ b l ~ G U U ACCUUU G

G AU " ~ ~GGGAGAUA\

C~~~~~~" ~CCCACCU\/ G ""C-C ."..., MUAUGGAGUA-3' G ysTyrGlyVal U G E.cn 3

5' -CMGGGCGUUCUA~ "GlnGlyArgScrL

Ellon 2

Figure 3. Possibly secondary structures of introns in rps12 gene. Intron­

exon boundaries are shown by arrow heads. Two distinct RNA "molecules

containing exonl and exon2 are placed close together according to group II

introns specific stem-loop structure. Amino acid sequence are shown under the

RNA sequences by three letter.codes.

-68~

A ORF203 rps12A rp120

psbB

1" i • • .c 1 ..

8gl11 (8g 5 ) B9111

B JLA rps 12B-C rps7

168 trnV . ! . .. f • ( I~

1 kb -BamHI (Ba11 ) BamHI

Figure 4. Gene organizations near the rps12 gene in exon 1 (A) and exons2

and 3 (B). Arrows indicate the direction of transcription. The abbreviations

rps12, rps7 and rp120 are the same as in Fig. 1. The symbol, psbB, represents

the gene for the ,P680 chlorophyll ~ apoprotein in photosystem rI. JLA stands

for a junction of an inverted repeat (IRA) and a large single copy region

(LSC) •

-69-

E'. 9n.a ci.Li.-I>

('I, po.lYllloilpha

E; coli.

E.. 91laei..li.-I>

('I. polY11lon.pha

E. coli.

'10 '20 30 40 50 60 + + + ... + +

MPTLEHLTRSPRKKIKRKTKSPALKGCPQKRAICMRVYTTTPKKPNSALRKVTRVRLSSG *** * * * * *********** * * *~*********** **** ** MPTIQQLIRNKRQPIENRTKSPALKGCPQRRGVCTRVY~TTPKKPNSALRKIARVRLTSG

* * ** * * *** *** ********************* ***** * MATVNQLVRKPRARKVAKSNVPALEACPQKRGVCTRVYT~PKKPNSALRKVCRVRLTNG

70 80 90 100 110 120 + + + + + +

LEVTAYIPGIGHNLQEHSVVLIRGGRVKDLPGVKYHVIRGCLDAASVKNRKNARSKYGVKKPKPK * ****************** *********** ** *** *** ** * ******** * --­FEITAYIPGIGHNLQEHSVVLVRGGRVKDLPGVRYHIIRGTLDAVGVKDRQQGRSKYGVKKSK

** * ** * ********* * ************** ** ** ***** * ******* * FEVTSYIGGEGHNLQEHSVILIRGGRVKDLPGVRYHYVRGALDCSGVKDRKQARSKYGVKRPKA

Figure 5. Amino acid sequences for the ribosomal protein S12 from M. polymorpha compared with· those from~. coli and~. gracilis. Vertical arrow

heads indicate sites of splicing junctions in the ribosomal protein S12 from

M. polymorpha. Asterisks denote amino acids that are identical between the

two proteins. Amino acids are expressed as one letter symbols.

-70-

between exons are shown in Fi.g. 3 •. Splice Junctions are indicated by arroW.heads

deduced from·the conserved sequences and secondary structures. The putative

branching nucleotide (adenine) is shown by underline in Fig. 3.

The gene organizations deduced from DNA sequences nea~ coding regions for

ribosomal protein S12 are shown in Fig. 4. ORFs corresponding to the ribosomal

proteins 57 and L20 were identified by their amino acid sequence homologies, 42.6%

and 46.6%, for the respective ~. coli ribosomal proteins (Post and Nomura 1980,

Hittmann and Seib 1979). In case of the ribosomal protein 514 of ]1. po1ymorpha

amino acid homology was 45% to that of ~. coli (Umesono et~. 1984). By contrast,

the amino acid sequence of chloroplast ribosomal protein 512 from]1. po1ymorpha

showed markedly higher homologies, 73.6% to that of ~. gracilis (Montandon and 5tutz

1984) and 70.2% to that of ~. coli (Post et ~. ·1978) (Fig. 5). The amino acid

sequence of the ribosomal protein 512 from]1. polymorpha near the splice junctions

(arrow heads in Fig~ 5) showed ~n even higher homology to sequences from~. gracilis

and ~. coli. both of which have no intron. This highly conserved amino acid.

sequence suggests that the chloroplast ribosomal protein 512 may have an .essential

role in the ribosomal function during protein synthesis in chloroplasts and that

rps12 gene may have a regulating function in the coordinative biogenesis of

chloroplast ribosome.

The ORF203, which was interrupted by two introns (518 bp and 380 bp), was

detected further upstream from exon 1 of rps12 gene (Fig. 3A). An reading frame

from the ATG codon (67372) to the TAG stop codon (67130) in the third exon of ORF203

was previously called ORF80 because of assuming no intron. Although the exon-i~tron

boundary sequences of ORF203 are a little diverged from the consensus sequences,

introns are correctly excised from the precursor messenger RNA including rps12A ~nd

rp120 coding sequences (Kohchi and Umes.ono unpublished dat~).. In a.ddition. first

exon of ORF203 (68640-68570) shows 20 out of 23 amino acid residues identity (87.0%)

with an reading frame in spinach X-gene next to psbB gene, which is shown to be

transcribed in vivo.

-71-:--

Exon 1 of ribosomal protein' 512 with the 5' intron boundary sequence .was

followed by a coding sequence of ribosomal protein L20(Fig. 2A) 786 bpapart.

Following the coding re~ion' of ribo~omal protein 512 in exon 3 isa ribosomal

protein S7 coding region (Fig. 2B). Close linkage of ribosomal protein S7 and S12

genes also exists in I. gracilis' (Montandon and Stutz 1983) and I. coli str-operon

(Post et ~. 1978, Post and Nomura 1980).

Transcription fo~ e~oris 2 and 3t as well as for the ribosomalprotein·S7 gene,

is initiated by a tyj:dcal'prokaryotic promoter sequence (...:35 and -10 regions) found

upstream (Fig. 28). S1 mappings showed that this promoter was highly active in

chloroplasts as well as in' I. coli (Fukuzawa et~. 1985). Northern hybridizations

also' showed the active transcrlption for exon 1. If the split gene described here

provides active mRNA that ca~ld be translated ihto mature S12 protein, there must be

a rejoining of exons at the RNA or DNA level. The DNA r~arrangement of these coding

regions is not observed in chloroplast genome by restriction analysis. Results of

Sl mappings and Northern hybridizations suggest that transcription units for exons 2

and 3 are independent of the unit for exon 1. Therefore, active mRNA for ribosomal

protein 512 was thought to be formed post-transcriptionally by a mechanism such as

that of trans-sp 1; cing descri bed by 501 nick (1985) and Konarsk et al. (1985). An

further investigation of the transcription and splicing mechanisms for the split

gene rps12 would clarify this complex gene organization and expression ·in the

chloro·plast.

After the publication 6f these results, the nucleotide sequences of the tobacco

ch 1 orop 1 ast ri bosoma'l protei n S12 was reported to be trans-sp 1 it as in the case of

liverwort (Torazawa et ll. 1986). This suggest that trans-split: rps12 ·gene on the

chloroplast genome may be a general feature 'in plant chloroplasts and has some

regulatory;function in chloroplast biogenesis •.

-72-

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~85':""

SU~Y

CHAPTER I Molecular cloning of promoters functional in Escherichia coli

from chloroplast DNA

DNA fragments cloned from chloroplast DNA of a liverwort, Marchantia polymorpha,

functional in £. coli as transcriptional promoters using gene fusion to the £. coli.

lac'Z gene. A recombinant plasmid gave as high a level of~-galactosidase activity

as when it was induced by IPTG in £. coli wild type strain W3110. The inserted

chloroplast DNA fragment was sequenced and mapped at the terminus of the inverted

repeat region upstream from the 165 ribosomal RNA gene. The direction of the

transcription from this promoter was opposite from that of 165 ribosomal RNA gene.

This highly active promoter was for trnI-C*AU gene and clustered genes for ribosomal

proteins. 51 nuclease mappings using both chloroplast and £. coli RNAs showed that

the transcription starts at almost the same position downstream from the consensus

Pribnow-box-like region. This clone also had a higher activity of j1-galactosidase

in £. coli than those containing promoters of rbcL and the P subunit gene of H+-ATP

synthase. Two clusters of genes for ribosomal proteins were identified downstream

from this highly active promoters.

CHAPTER II Structure and gene organization of the chloroplast genome

. The nucleotide sequence of the large single copy region (psbG-16S rRNA gene;

30,600 bp) of the chloroplast DNA from a liverwort, ~. polymorpha was determined.

This region encodes genes for seven tRNAsi tRNAVal(GAC), tRNAIle(C*AU),

tRNAArg(CCG), tRNAPro(UGG), tRNATrp(CCA), tRNAMet(CAU). tRNAVal(UAC), ten

photosynthetic polypeptides; the large subunit of ribulose-l.5~bisphosphate

carboxylase/oxygenase (rbcL), 51 kd photosystem II chlorophyll ~ apoprotein (psbB).

apocytochrome b-559 polypeptides (psbE and psbF), cytochrome f preprotein (petA),

cytochrome b6 polypeptide (petB) and cytochrome b6/f complex subunit 4 polypeptide

(petD), f and E subunits of H+-ATP synthase (atpB and atpE). photosystem II G­

protein (psbG). and ribosomal proteins (L2, Ll4, L16, L20, L22. L23. L33, 53, 58,

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511, 512, 518, 518 and S19), initiation factor 1 (infA) and ~ subunit of RNA

polymerase (rpoA). Interestingly, functionally related genes are clustered as

follow: (1) A ribosomal protein gene cluster involving transcriptional and

translational machinery, trnI~L23-L2-S19-L22-S3-L15-L14-S8~infA-secX-S11~rpoA, was

found at the terminus of the large single copy region next to the inverted repeat

region (IRB). (2) A cluster of photosynthetic genes, psbB-ORF35-0RF27-0RF74-petB­

petD is located next to the ribosomal protein gene cluster. (3) A cluster incl~ding

photosynthetic genes rbcL-trnR-ORF316-0RF36b-ORF184-0RF434-petA;. was also found in

large single copy region. Introns (intervening sequences) were found in coding

sequences for ribosomal protein genes (rp12, rpl16 and rps12), tRNAVa1 (UAC) gene and

photosynthetic genes (petB and petD). Interestingly, an open reading frame was

found to show significant amino acid sequence homology to ·a subunit of NADH

dehydrogenase in human mitochondria.

CHAPTER III Split gen~ for chloroplast ribosomal protein S12

A coding sequence corresponding to the ~. coli ribosomal protein 512 gene

(rps12) was f~und to be split into three exons. Strikingly, the first exon with the

5' intron boundary sequence was located on the opposite strand of the chloroplast

DNA (121 kb, Circular molecule) approximately 50 kb away from the rest of the exons.

The amino acid sequence deduced from the DNA sequence was highly homologous to the

sequences of the 512 ribosomal protein of ~. coli (70.2%), and Euglena gracilis

chloroplasts (73.6%). As the DNA rearrangement of these coding regions is not

observed, the active messenger RNA for ribosomal 'protein S12 is thought to be formed

post-transcriptionally such as that of trans-splicing. This may be the first

identification of an example for in vivo trans-splicing.

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LIST OF PUBLICATIONS

(a) Ohyama K, Wetter LR, Yamano Y, Fukuzawa H, Komano T (1982) "A simple met.hod for isola:tion of chloroplast DNA from Marchantia

polymorpha L. cell suspension culture."

Agric Biol Chem 46:237-242

(b) Ohyama K, Yamano Y, ~u~uzawa H. Komano T. Yamagishi H. Fujimoto S. Sugiura M

,(1983 ) "Physi<;:al mappings of chloroplast DNA from liverwort Marchantia po 1 ymorpha L. ce 11 suspens i on cultures. 11

Mol Gen Genet 189:i-9

(c) Tanaka A. Yamano Y. Fukuzawa H. Ohyama K. Komano T (1984) "in vitro DNA synthesis by chloroplasts isolated from Marchantia polymorpha L. cell suspension cultures. It

Agric Biol Chern 48:1239-1244

Cd) Yamano Y. Kohchi T, Fukuzawa H. Ohyama K. Komano T (1985)

ItNuc1eotide sequence of chloroplast 4:5S ribosomal RNA from a leafy liverwort. Jungermannia subulata, and a thalloid liverwort, Marchantia polymorpha,"

FEBS Letter 185:203-207

(e) Fukuzawa H. Uchida Y. Yamano Y, Ohyama K, Komano T (1985)

IIMole'cular cloning of promoters functional in Escherichia coli from

chloroplast DNA of a liverwort Marchantia polymorpha. 1I

Agric. Biol. Chern. (1985) 49j2725-2731

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(f) Fukuzawa H, Kohchi T, Sano T, Shirai H. Umesono K, Ohyama K, Ozeki H. Komano T

(1986) "Structure and organization of Marchantia po1ymorphach1oroplast genome

(III) LSC region (rbcL"-J LB ) having gene clusters of ribosomal proteins

and photosynthetic polypeptides.",

in preparation.

(g) Fukuzawa H, Kohchi T, Shirai H, Ohyama K; Umesono K, Inoktichi H, Ozeki H (1986)

"Coding sequences for chloroplast ribosomal protein S12 from the

liverwort. Marchantia polymorpha, are separated far apart on the different DNA strands."

FEBS Letter (1986) 198:11-15

(h) Ohyama K, Fukuzawa H, Kohchi T, Shirai H, Sa no T,Sane S, Umesono K; Shiki S,

Takeuchi M, Chang Z, Aota Si Inokuchi H~ Oz~ki H (1986)

"Chloroplast gene organization deduced from complete sequence of

liverwort Marchantia polymorpha chloroplast DNA."

Nature 322:572-574

Chapter I is described in reference (e).

Chapter II is described in reference (f).

Chapter III is described in reference ~g).

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ACKNOWLEDGMENT

The author wishes to express his grateful acknowledgment to Professor Tohru

Komano, Faculty of Agriculture, Kyoto University, for his continuous encouragement

throughout this work, and for critical reading of this manuscript.

The author would like to express his great gratitude to Dr. Kanji Ohyama,

Laboratory of Plant Molecular Biology, Research Center for Cell and Tissue Culture,

Faculty of Agriculture, Kyoto University, for allowing to use his facilities and his

kind guidance throughout this study.

The author thanks to Professor Haruo Ozeki, Faculty of Science, Kyoto

University, and also to Drs. Kazuhiko Umesono, Yoshiaki Yamano, Yuko Uchida, for

their helpful suggestion and valuable discussion.

The author thanks to Dr. Toshimichi Ikemura, National Institute of Genetics and

Dr. Minoru Kanehisa, Chemical research institute, Kyoto University, for their kind

support to computer analysis using data base.

The author is indebted to Mr. Takayuki Kohchi. Mr. Tohru Sano. Mr. Hiromasa

Shirai. Mr. Shigehiro Asano, Mr. Yoshinori Yamashita and all the members of the

laboratory of Plant Molecular Biology, Research Center for Cell and Tissue Culture,

and the laboratory of Biochemistry, Department of Agricultural Chemistry, Faculty of

Agriculture, Kyoto University, for their kind assistances in this study.

9f-rcPr Hideya Fukuzawa

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