Chloroplast Molecular Genetics of <i>Chlamydomonas</i>: a Tool for Studying the Function, Synthesis and Assembly of the Photosynthetic Apparatus

Proceedings Chapter

Reference

Chloroplast Molecular Genetics of Chlamydomonas : a Tool for Studying the Function, Synthesis and Assembly of the Photosynthetic

Apparatus

ROCHAIX, Jean-David, et al .

Abstract

The green unicellular alga Chlamydomonas reinhardtii offers interesting possibilities for studying the function, synthesis and assembly of the photosynthetic apparatus. Chloroplast and nuclear genes coding for subunits of photosystem II, a major multimolecular membrane-associated photosynthetic complex, have been isolated and characterized.

Examination of these genes and of their expression in wild-type cells and in chloroplast and nuclear photosystem II mutants has revealed a complex regulatory circuitry between chloroplast and nucleocytoplasmic compartments. One of the photosystem II subunits which is involved in herbicide binding has been found to have distinct single amino acid substitutions in several herbicide resistant mutants. Some of these mutations do not appreciably affect photosynthetic yield and may therefore be of agronomic interest.

ROCHAIX, Jean-David, et al . Chloroplast Molecular Genetics of Chlamydomonas : a Tool for Studying the Function, Synthesis and Assembly of the Photosynthetic Apparatus. In: Alačević, Marija & Hranueli, Daslav. Genetics of Industrial Microorganisms: proceedings of the Fifth International Symposium on the Genetics of Industrial Microorganisms . Zagreb : PLIVA, 1987. p. 285-293

Available at:

http://archive-ouverte.unige.ch/unige:150719

Disclaimer: layout of this document may differ from the published version.

1 / 1

1

- --- -- ---

#

;1

.:~ ~

~ -

~\ .,,

<~ : _:... ...

r,

-.,,,.

~,.!

·!I ·i ,~I

~-.I

Fifth lntematronal Symposium on the Genetics of Industrial Microorganlsms&s~

M. AJet evic .• D. Hranueli. Z. Toman (eds.)

Chloroplast Molecular Genetics of Ch/amydomonas: a Tool for Studying the Function, Synthesis and Assembly of the

Photosynthetic Apparatus

J.D. Rochaix,

s.

Mayfield, J. Erickson, P. Malnoe and M. Kuchka

Departments of Molecular and Plant Biology, University of Geneva, Geneva, Switzerland

SUMMARY

The green unicellular alga Chlamydomonas .re inhardtii offers interesting possibilities for studying the f unction , synthesis and assembly of the photosynthetic apparatus.

Chloroplast and nuclear genes coding for ^subunit~ of photo- system II, a major multimolecular membrane-associated photo- synthetic complex, have been isolated and characterized.

Examination of these genes and of their expression in wild-type cells and in chloroplast and nuclear photosystem II mutants has revealed a complex regulatory circuitry between chloroplast and nucleocytoplasmic compartments. One of the photosystem II subunits which is involved in

herbicide binding has been found to have distinct single amino acid substitutions in several herbicide resistant mutants. Some of these mutations do not appreciably affect photosynthetic yield and may therefore be of agronomic interest.

INTRODUCTION

The biosynthesis of the photosynthetic apparatus in higher plants and algae is achieved through the cooperation of two distinct genetic systems located in the chloroplast and nucleocytoplasmic compartments. Most photosynthetic complexes consist of subunits some of which are encoded by the chloroplast genome and translated on chloroplast

ribosomes while others are encoded by the nuclear genome, translated on cytoplasmic ribosornes and imported into the chloroplast where they assemble with their chloroplast partner polypeptides into functional complexes. Our work in recent years has been aimed at understanding the molecular basis of the cooperative interplay between these two

cellular compartments (cf. fig. 3).

A brief de~cription of photo'synthesis may be helpful for understanding the following (cf. fig. 1). Light energy is captured and converted into chemical energy by photo- system II and photosystem I which act in series and which are connected by an electron transport chain. Photosystem II (PSII) generates a strong oxidant capable of splitting water into molecular oxygen, protons and electrons.

Electrons are then channelled along the electron transport chain and they ultimately reduce NADP. Electron transport is

285

:·~ ·

~~

-.,, ::Q . .. ,.--

-~

--:.~-~-·-'""

,,

~~. ·i

t

'·

..

,

j

• 4

-- . - - --- ^{- ---- --}

286

CHLOROPLAST MOLECULAR GENETICS OF CHLAMYDOMONAS coupled with an influx of protons into the thylakoid

vesicles thereby creating a proton gradi.ent which is used to produce ATP. Both NADP and ATP are utilized to drive the reactions of the Calvin cycle whose net result is the fixation of

co

2 and the production of carbohydrates.

It is essential to choose a system which allows for a combined genetic and biochemical-molecular approach. The green unicellular alga Chlamydomonas r e i n hardtii a ppears to be a powerful model sys tem in this r e spec t. An important

featu~e is that photosynthetic function is dispensable in this organism provided a reduced carbon source such as acetate is added to the growth medium. It is therefore possible to isolate acetate requiring mutants that are unable to grow with CO as the unique carbon source. Indeed numerous nuclear and

c~loroplast

mutants of this sort have been isolated which are affected either in the photo- synthetic apparatus or in the chloroplast protein synthesizing system.

As higher plants, C. reinhardtii contains three genetic systems located in the nuc leocytoplasm, chloroplast and mitochondria whose major features are summarized in Table I.

Although the complexity of the chloroplast DNA is rather modest, 190 kb, this DNA represents 14% of the cellular DNA mass which implies that it exists in 80 copies per cell. It is easy to distinguish between nuclear, chloroplast and mitochondrial mutations based on their distinctive

segregation pattern during meiosis. While nuclear mutations are inherited according to Mendelian rules, chloroplast

mutati~ns are preferentially inherited uniparentally from the mt parent (1). Interestingly, mitochondrial genes appear to be inherited uniparent,ally from the mt- parent

( 2) • ·--

Light +--Strama.---...

NAOP·- 1 NA~PH

. I lro2l+2H•

+---- - - - -- Chloroplast membranu - - -- ---it

Fig . 1 . Primary r eaction s o f photosynthesis . Chl,

chlorophyll antenna ; PSI! , PSI , pho tosystems I I , I; ETC, electron t r ansport c hain . C02 fixation ( ¹⁾ and ATP s ynthe s i s

( 2 ) are catalyzed by ribulose b isphosphate carboxylase- o xygenase and ATP synthase , respectively.

·~ ....

~-: ..,

..

( ^{. .. l}

~ ... · ':"!

h ,~

~ ~

' ~

^J

~ ~

_J

~

~~~~~~~~~~~~~~~~~~~~

....

___

^...

^--·

^·-.

^{--- - -- ---- --}

.MYDOMONAS d to

- a

~ to Ln

foed :i.ve

:iet le I.

!:"

. It DNA

ions '.)m t

he sis t

J

i _J J

•

ROCHAIX et al.

Table 1. Genetic systems of Chlamy domonas reinhardtii

DNA Complexity (kb) %mass Copy Inheritance number

nuclear 7-9xl0 4 (99.7%) 85 1 Mendelian chloroplast 190 (0.3%) 14 50-80 uni parental

maternal mitochondrial 16 (0.02%) 1 50-80 uni parental

*

paternal

*

This has only been demonstrated in crosses between C. reinhardtii and~- smithii (2).

Here we review our recent work on PSII in which we have taken advantage of several chloroplast and nuclear mutants which specifically affect PSII function and assembly.

Photosystem II ^consist~ of the core complex embedded in the thylakoid membrane, the oxygen evolving complex and the associated light harvesting system (fig. 2). The exact polypeptide composition of PSII is still unknown. At least 11 distinct polypeptides could be recognized in the PSII core complex of C. r einhardti i (3). The core also contains the reaction center chlorophyll P680, several core antenna chlorophylls, pheophytin, iron, cytochrome b559, two

acceptor plasto-quinones QA and QB and a primary electron donor Z (Fig.2). The major core proteins (47K, 43K, Dl, D2, .____

cyt b 559) are all encoded by the chloroplast genome and their genes ^(psbB~ psbC, psbA, psbD, psbE respectively) have been sequenced in several systems (4,5). The oxygeh evolving system of PSII includes 3 extrinsic proteins of 33, 24 and 18 kd. They are located on the lumen side of the thylakoids and are all encoded by nuclear genes and synthesized as larger precursors in the cytoplasm (6,7).

RESULTS AND DISCUSSION -

We have started our studies on PSII with the two core proteins Dl and D2 and their genes. Increased interest in these two proteins has arisen recently because of the similarity between the reaction centers of PSII and of purple photosyn t hetic bact eria (8 , 9) . In both cases a

pheophytin acts as inter mediate electron accept or and a very similar electron .accepting quinone iron c omplex exists . A structural bomology between Dl , D2 and the L , M subunit s of the bacterial reaction center has been noted (10 ) . Michel an~ coworkers have recently determined by X-ray

Crlstallographic analysis the molecu l ar structure at 3A r cs'_'l~t tion of the reaction center o f Rhodopseudomonas

v1r1d1s (8) . Based on the structur~l a nd functiona l homo l ogy

287

- r ":""' t

~. *'"

,_

-f"'- -~

~ .,

• '!" ·~- .:~ :·~

~

.

··.

1

1'

--~"?;.

-

^{. ··-- ---}

^{- -}

^----- ·---···- - - - - . ·--··---- --- - -

288

CHLOROPLAST MOLECULAR GENETICS OF CHLAMYDOMONAS

oxygen evolution , ~

, ,

H2') ~ Z-+ P680-+Pheophytin -+ QA-+Oe

Fig. 2. Photosystem II. LHCP, Light harvesting chlorophyll protein complex. QA Q , primary and secondary electron acceptors. Z primary

e~ectron

donor. See text for further explanations.

between the two bacterial and PSII subunits, it has been proposed that D2 a nd Dl a re the apoproteins of the stabl e primary a nd secondary electron acceptors of PSII a nd that they ^c~n be folded with 5 t ransmembrane domains to f o rm ^{a - -~}

core with c hlorophyll , pheophytin a nd quinone which is v ery similar t o the bacterial reaction c enter (8 ). This model does not however agree completely with protease digestion data on thylakoid membranes (11). ·

The genes of Dl and 02, psbA and psbD, respectively, have been localized on the chloroplast genome of ·

f·

reinhardtii (cf. fig. 3) and their nucleotide sequences.

have been determined (12, 13). Since the psbA gene is . located in the inverted repeat of the chloroplast genome, it is present in two copies. •

It is well established that Dl is the herbicide binding protein (14). Analysis of psbA from mutants with different levels of resistance and cross-resistance to the herbicides atrazine, diuron and bromacil (15) has allowed us to

identify four residues on the 352 amino acid Dl polypeptide which are changed in these mutants (val 219 ~ ser: phe 255•

tyr, ser 264+ala, leu ^27S~phe (16, 17, J. Erickson, M.

Rahire and J.D. Rochaix, unpublished results). These results have added new insights i n t o the struc ture-function

relationship o f t he Dl p ro t e in. They r evea l that mutations affecting four different amino acid re sidues of the Dl protein are cor re lated with different type s and levels of herbicide resi s tance. Intere stingly the mutations affecting V219, F255 and L275 do not · alter electron transport and

! '

'

J

f ;

~ l

i

j

·l

I

j

_~ '

i

-1 !

1 ₁

i

^~

_~

1

1

-~ l

AMYDOMONAS

hyll er

n le at a very on 1

y, ces e, it ndi ren"t:

cide s tide 5- s ults :::>ns '.)f

ting

I

~ l

~

ROCHAIX et al.

289

hence photosynthetic yield, (15) while the mutation

affecting S264 does (16). Thus , the first three changes may be of use in genetic engineering of herbicide resistance.

In collaboration with P. Bennoun's group i n Paris we have examined several uniparental mutants blocked in PSI!

functio n. Among 78 chloroplast PSI! mutants examined , 68 were unable 3

50

synthesize Dl 4 (as measured ^by pulse labelling cells with s - sulf ate or C -acetate) (18). Analysis of the chloroplast genome of 22 o f these mutants revea l ed that they all have deleted both copies of psbA . Although these mutants are able t o synthesize and t o integrat e the other PSII

polypeptides i n the thylakoid membranes , they are unable t o assemble a stable f unctional PSII c omplex (18). Among the few o ther PSI! mutants that do have an intact psbA gene, one is unable to produce 02. We have found recently that in this mutant a 46 bp stretch of the 0 2 gene , psbD, has been

duplicated . Of special interest is the fact that this mutant is also unable to synthesize Dl, suggesting that D2 may be involved in the control of translation of Dl (13).

By probing a cDNA library from

c.

reinhardtii in the expression vector Agtll with antibodies (obtained from N. H.

Chua) against the three nuclear encoded polypeptides from the o xygen e vo l ving c omple x , c DNA clones correspond ing to · these three polypeptides were isolated. The genes of the 33

(OEEl) and 24kd (OEE2) proteins have been sequenced and s hown to code for p r oteins of 290 and 245 amino acids,

~e spectively (20 , unpublished result s ) . Comparison of the

?rotein sequence s derived from the DNA sequences with the

amino terminal ends of the proteins has revea led that the ! two transit peptides of the two proteins comprise 52 and 57 ^{I .~}

amino acids, respectively.

l

An important advantage of C. reinhardtii is that it

t

allows one to easily isolate chloroplast and nuclear mutants

! •:

₁

a ffected in PSII function based on their altered -- i ',, fluorescence properties (19). We have examined in some · detail several chloroplast and nuclear PSII mutants by

dete rmining the steady state levels of mRNA and p4oteins of most of the PSI! components. In some cases the rates of

prote~g synthesis ^wef~ established by pulse labelling cells with S-sulfate or c-acetate. The picture which emerges from these studies (13, 18, 20, unpublished results) can be summarized as follows.

Chloroplast mutations which prevent the synthesi s of e i the r of the PSII core c omponents (43K , 0 1 , D2) lead to t he destabilization and degradation of the core c omp lex

prote ins. Interestingly the PSI! c o re proteins whose genes are no t aff ect ed are still synthe~ized at wild-type levels and are integrated into the thylakoid ·membra ne ,but they do not accumulate stably (18 ). It t herefore appears that the

sto ~chiometric accumulatio n of the PSII core polypeptide s is achieved at the post- translatio nal level. An exception i s the mutant whic h has a lesion i n its p s bD gene . Here both

~he 02 an~ Dl polypeptides are no l o nger s ynthesized (13) . 1he proteins from the oxygen e vo l ving c omplex acc umulate i n the se mutants to variable amounts ranging betwee n 10 and 100 % o f wild-type levels . It is no t yet known whether these

·--.., ·, .... '<(f"

: ..

~ ^~

~ _{... ~ .. ~ "\}

...

^~

,.·.;:;;:

-&-'-

.,...,.;~ '

:;:i:#-'1:'""° ..

_{... ~ 7'J·}

·~··

"

•.

"

• • · ~ ";;":.-f.,

290

CHLOROPLAST

psbB

CHLOROPLAST MOLECULAR GENETICS OF CHLAMYDOMONAS

Cll _Q 0 Cll >

c Cll

NUCLEO-CVTOPLASM

NUCLEUS

R1 R2

~---

Fig . 3. Synthesis and a ssembl y o f PS II . e t DNA, c hlorop l a st DNA with the genes o f t he core PSII polype ptides 47K (Psb B), Dl (psbA) D2 (psbDl a nd 43K (ps bC ) i n t he thy l a ko id membrane

(darkened ). The nuclear genes of the proteins from t he o xygen evolving ^~omplex OEEl (psb I) , OEE2 (ps b I I) and OEE3

(psbIII ) are indicated . The s e proteins a r e syn thesi zed a s precursors on cytoplasmic ribosomes. Rl, R2 and R3 refer to nuclear genes whose products regulate the translation of chloroplast proteins. T, translation on chloroplast (left) and cytoplasmic ribosomes (right) •

proteins are still bound to the thylakoid membrane or whether they are free in the thylakoid lumen. In contrast, mutations in the nuclear gene of the OEE2 protein of the oxygen evolving complex which abolish the expression of this gene, do not affect the synthesis and accumulation of the other polypeptides of the oxygen evolving complex and of the PSII core polypeptides (20). Mutations affecting the OEEl protein gene have a more complex phenotype. While the other polypeptides of the oxygen evolving complex are present in normal amounts, the accumulation of the core polypeptides is reduced to about 25% of wild-type suggesting that the OEEl protein plays some role in stabilizing the PSI! complex.

l

1

4

j

~::.-~

~- 1

1

... ^--~

1

~~~~~~~~~~~- -

---

.AMYDOMONAS i.SM

]- --

! 0

_I

I

,

~ E3

I

_'

plast PsbB), mbrane

OEE3 er to as of eft)

ast, .f this he

of the the EEl other

t in

des is OEEl x.

i

i j l

ROCHAIX et al.

291

Nuclear mutations of considerable interest are those which prevent specifically the synthesis

of

defined

chloroplast encoded PSII core polypeptides. Mutants of this type include F34 which is unable to produce the 43 kd

polypeptide (21) and UVl-8 and XRl-1 which do not synthesize the Dl and D2 proteins. The phenotype of these mutants has been shown to be due to single mutations (21, M. Kuchka, unpublished results). Since the steady state levels of the mRNAs of the missing polypeptides in these mutants are as in wild-type, the mutations either interfere with the

translation of these proteins or render them highly

unstable. The genes affected in these mutants appear to play

~ regulatory role in PSII synthesis. The results of this PSII analysis are summarized in fig. 3 where several regulatory steps are indicated. From this preliminary analysis it appears that the nucleus exerts a major control on the synthesis of the chloroplast encoded PSII

polypeptides principally at the translational and/or post-translational level.

PROSPECTS

The photosynthetic apparatus of C. reinhardtii

resembles closely that of higher piants. It is especially noteworthy that the nucleotide sequences of the PSII core polypeptide genes have been highly conserved. It appears therefore legitimate to extrapolate the r.esults obtained with herbicide-resistant mutants from C. reinhardtii to plants. Because it has now been possible to merge the biophysical, biochemical, molecular and genetic approaches in this alga, it is an excel lent model system for studying the complex regulatory circuitry between chloroplast and nucleocytoplasmic compartments which is involved in the synthesis of PSII.

A transformation system has been established for

£·

reinhardtii that allows one to introduce foreign DNA ...

stably into the cells and autonomously replicating plasmids have been constructed (22, 23, 24). It is hoped that

improvements in the transformation efficiency will soon allow us to use this system for isolating regulatory genes of the sort described above by complementing_ the appropriate mutations with wild type DNA libraries.

ACKNOWLEDGEMENTS

We thank

o.

Jenni for drawings and photography. This work was supported by Swiss National Fund grant 3.587.084.

REFERENCES

l. Gillham, N.W. (1978) Organelle Heredity, Raven Press, New York.

2. Boynton, J.E., Palmer, J.D., Harris, E.H. and Gillham, N.W. (1984). Inheritance and molecular divergence of chloroplast and mitochondrial genomes in crosses between the interfertile algae species. Chlamydomonas smithii and~· reinhardtii. Genetics 107, No. 3, Sl3.

I

t

~

~

~ ;

i

^I

l.! _r1

~ · I

'" l

i; ^l

. -T-,,

:<

~

·.· .. ^{; ~}

-' ~

"

'·

.

292

CHLOROPLA.ST MOLECULA.R GENETICS OF CHLAMYDOMONAS 3. Delepelaire, P. (1984) Partial characterization of the

biosynthesis and integration of the photosystem II reaction centers in the thylakoid membrane of Chlamydomonas -reinhardtii . EMBO J.

lr

701-706.

4.

5.

6.

7.

8.

Whitfeld , P . R. a nd Bot t omley, W. (1983) Organization and structure of chloroplast genes. Ann. Rev. Plant • Physiol. 34, 279-310.

Rochaix, J:O. (1986) Molecular Genetics of chloroplasts and mitochondria in the unicellular green alga

Chlamydomonas. FEMS Microbiology Reviews. In press.

Murata, N. and Miyao, M. (1985) Extrinsic membrane protein in the photosynthetic oxygen-evolving complex.

Trends Biochem. Sci. 10, 122-124.

Westhoff, P., Jansson,-C., Klein-Hitpab, L., Berzborn, R., Larsson, C., Bartlett, S.G. (1985) Intracellular coding sites of polypeptides associated with photo- synthetic oxygen evolution of photosystem II. Plant Molec. Biol. 4, 137-146.

Deisenhofer, J~, Epp,

o.,

Miki, K., Huber, R. and Michel, H. (1985) X-ray structure analysis at 3A resolution of a membrane protein complex: folding of the protein subunits in the photosynthetic

reaction center from Rhodopseudomonas viridis . Nature 318, 618-624.

9. Trebst, A. (1986) The topology of the plastoquinone and herbicide binding peptides of photosystem II in the thylakoid membrane. Z. Naturforsch. 412, 240-245.

10. Hearst, J.E. and Sauer, K. ( 19 8 4) Protein sequenc·e homologies between portions of the L and M subunits of reaction centers of Rhodopseudomonas capsulata and the QB protein of c hloroplast thylakoid membranes:

A proposea relation to quinone b inding sites . Z. Naturforsch. 39c, 421-424.

11. Marder, J.B., Goloubinoff, P. and Edelman, M. (1984) Molecular architecture of the rapidly metabolized 32kDa protein of PSI!: Indication for COOH-terminal processing of a chloroplast menlbrane polypeptide.

J. Biol. Chem. 259, 3900-3908.

12. Erickson, J.M., Rahire, M. and Rochaix, J.D. (1984) EMBO J. 2753-2762. Chlamydomonas reinhardtii gene for the 32000 mol. wt. protein of phptosystem II contains four large introns and is located entirely within the chloroplast inverted repeat. EMBO J. 3,

2753-2762. -

13. Erickson, J.M., Rahire, M., Malnoe, P., Girard- Bascou, J., Pierre, Y., Bennoun, P. and Rochaix, J.D. (1986) Lack of the D2 protein in a

Chlamvdomonas reinhardtii psbD mutant affects photo- s ystem II stability and Dl expression. EMBO J. 5,

1745-1754. -

14. Pfister, K., Steinback, K.E., Gardner, G., Arntzen, C.J. (1981) Photoaffinity labeling of an herbicide receptor protein in chloroplast membranes. Proc.

Natl. Acad. Sci. USA. 78, 981-985.

'

t ^..

_~

- .

^I ~ I

-~

~

·i

1

!

....

,1YDOMONAS 1e

>ts

~x . i,

.

·e

•.nd

.s:

. - -

----·-

^·--

--- ^- .

I

~

'

I

' ' f:

'·

ROCHAIX et al.

15. Galloway, R. and Mets ,

L:"

(1984) Atraz ine, bromacil and diuron res i stance in Chlamydomonas : A single non- mendelian genetic locus controls the structure of the thylakoid binding site. Plant Physiol. 74, 469-474.

16. Erickson, J . M., Rahire, M. , Bennoun , P . ,l5elepelaire, P. , Diner, B. and Rochaix , J . D. (1984) Herbicide resistance in Chlamydomonas reinhardtii re sults from a mutation in the chloroplast gene for the 32k dalton protein in photosystem II. Proc . Natl. Acad. Sci. USA 81 , 3617-3621.

17. Erick son, J . M., Rahire, M., Rochaix, J.D. and Mets, L.

(1985) Herbicide Resistance and Cross-Resistance : Changes at Three Distinct Sites in the Herbicide- Binding Protein. Science 228, 204-207.

18. Bennoun, P., Spiere r -Her z , ~, Erickson, J., Girard- Bascou, J., Pierre, Y., Delosme, M. and Rochaix, J.D.

(1986) Characterization of photosystem II mutants of Chlamydomonas reinhardtii lacking the psbA gene.

19.

20.

21.

22 . 23.

Plant . Mclee . Biol. 6, 15 1-160.

Bennoun, P. and Delepelaire, P. (1982) Isolation of photosynthesis mutants in Chlamydomonas . In Methods in chloropl ast molecular biology . Edelman et al.

eds . pp. 25-38. Elsevier Biomedi cal Press .

Mayfield, S.P., Rahire, M. , Frank, G., Zuber, H. and Rochaix, J . D. (1986). Expression of the nuclear OEE2 gene is required for high levels of photosynt hetic oxygen evolution in Chlamydomonas reinhardtii.

Submitted.

Chua, N. H. and Bennoun, P. (1975) Thylakoid membrane polypeptides of Ch.lamydornonas reinhardtii : Wild-type and mutant strain deficient in photosystem II reacti0n center . Proc . Natl. Acad . Sci. USA 72 , 2175- 217 9.

Rochaix, J . D. and van Dillewijn, J . 11982) Transforma- tion of the green unicellula r alga Chlamydomonas reinhardtii with yeast DNA . Nature 296 , 70-72.

Rochaix , J . D., Rahire, M. and van Dillewijn , J . (1984 ) Construction and Characterization of

Autonomousl y Replicating Plasmids in the Green' Uni- cellular Alga Chlamydornonas reinhardt ii. Cell 36,

925-931 . .

24. Hasnain, S . E., Manavathu , E. K. and Leung, W.L . (1985) DNA mediated Transfor mation of Chlamydomonas

reinhardtii cells. Use of Arninoglycos i de 3 ' -phospho- transfera s e as a selectable marker. Mo).. Cell . Biol.

~' 364 7- 365 0.

293