Chlamydomonas
631
gene, transcription of the chimeric gene
was strongly increased after cells had been
deflagellated, although the level of mRNA
from the chimeric genes was only 5 to
10% of that from the endogenous
β
2
tubu-
lin gene.
3.2
Chloroplast Transformation
Chloroplast transformation of
C.
rein-
hardtii
can be achieved when cells are
bombarded with DNA-coated tungsten
particles from a particle gun. Chloroplast
mutants carrying a defective photosyn-
thetic gene are usually transformed with
theco
r
respond
ingw
i
ld
-
typegene
,wh
ich
integrates into the chloroplast genome
by homologous recombination. Alterna-
tively, nonphotosynthetic markers such
as ribosomal RNA genes with mutations,
conferring resistance to streptomycin and
spectinomycin, can be used for selec-
tion. Resistance to these antibiotics can
also be obtained with the bacterial
aadA
(aminoglycoside adenyl transferase) gene
fused to a chloroplast promoter and 5
0
leader region. These tools have opened
the door for genetic engineering of the
chloroplast genome. It is now possible to
perform chloroplast gene disruptions and
site-directed mutagenesis, and to insert
and express foreign genes (e.g. chimeric
GUS constructs) at speciFc sites in the
chloroplast genome.
4
Chlamydomonas as a Model System
4.1
Function and Assembly of the
Photosynthetic Apparatus
The primary reactions of photosynthe-
sis occur at the thylakoid membranes,
in which light energy is collected and
converted into chemical energy through
charge separations across the membrane
and a series of complex oxidoreduction re-
actions. Ultimately, the process leads to the
formation of an electrochemical gradient
across this membrane and the production
of ATP and NADPH, both of which are
required to drive the Calvin cycle, which
results in CO
2
Fxation and the synthe-
sis of carbohydrates. ±our multimolecular
complexes are involved in these primary
reactions: photosystem II and photosys-
tem I and their associated light-harvesting
systems; the cytochrome
b
6
f
complex; and
ATP synthetase (±ig. 5). Table 2 lists some
of the major subunits of photosystems II
and I and their genes.
As in higher plants, the biosynthesis
of the photosynthetic apparatus of
C. rein-
hardtii
occurs through the concerted action
of two genetic systems located in the nu-
cleus and chloroplast respectively. Several
subunits of the photosynthetic complexes
are encoded by the chloroplast genome and
translated on 70S chloroplast ribosomes.
The remaining subunits are encoded by
nuclear genes and translated on cytosolic
80S ribosomes as precursors with a tran-
sit peptide at the N-terminal end, which
targets the protein to the chloroplast com-
partment. Upon import of the protein
into the chloroplast, the transit peptide
is cleaved by a stromal peptidase. Proteins
targeted to the thylakoid lumen have a
bipartite transit sequence: the N-terminal
part acts as a chloroplast transit sequence
and is cleaved in the stroma; the C-terminal
part always contains a hydrophobic region
that is required for translocation across
the thylakoid membrane, and this region
is cleaved by a second protease, which is
tightly associated with the thylakoids. In
the Fnal steps, chloroplast- and nuclear-
encoded subunits are assembled either in
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