634
Chlamydomonas
the terminal electron acceptors of pho-
tosystem I. One possibility is that the
clearing system, which recognizes mis-
folded protein complexes and degrades
them subsequently, works more efFciently
in
C. reinhardtii
than in cyanobacteria.
Site-directed mutagenesis of chloroplast
genes encoding subunits of photosystem
II, photosystem I, and the cytochrome
b
6
f
complex through chloroplast transforma-
tion has been performed extensively and
has provided new and important insights
into the structure–function relationship of
these complexes.
While chloroplast transformation occurs
in all cases examined through homolo-
gous recombination and allows one to
perform gene disruptions or site-directed
mutagenesis in the chloroplast genome,
nuclear transformation appears to lead, in
most cases, to random insertions within
the nuclear genome. Attempts to dis-
rupt nuclear genes through homologous
recombination have not yet succeeded.
However, it has been possible to use
transforming DNA as a random mutagen
and, thus, to inactivate speciFc nuclear
genes by using suitable screens. This was
Frst achieved with the nuclear
atp
Cgene
encoding the
CF1
γ
subunit of ATP syn-
thetase. Wild-type cells were transformed
with a nonfunctional homologue of the
atp
C gene in the presence of carrier DNA.
After enrichment for photosynthetic mu-
tants and an immunoscreen, a mutant was
isolated that lacked the
γ
-subunit because
its
atp
C gene had been rearranged. Sim-
ilarly, cells from the
arg
7mu
t
an
tw
e
r
e
cotransformed with the argininosuccinate
lyase gene and the
psa
± gene that had been
mutated by three small internal deletions.
Transformants were selected for arginine
prototrophy and screened for fluorescence
patterns typical of photosystem I mutants,
and by DNA analysis. One transformant
that was obtained lacked
Psa
±andhadsuf-
fered a large rearrangement at the
Psa
±
locus. It appears, therefore, that transfor-
mation is a valuable tool for disrupting
speciFc nuclear genes of
C. reinhardtii
,
provided suitable screens for the mutant
phenotype are available.
4.1.2
Nuclear Mutations Affecting
Posttranscriptional Steps in the Expression
of Chloroplast Genes
Posttranscriptional steps are important in
the control of chloroplast gene expres-
sion.
C. reinhardtii
offers unique advan-
tages for genetic analysis of posttranscrip-
tional events. Several nuclear mutations
destabilize speciFcally deFned chloroplast
mRNAs. Mutations of this type have
been reported for
psbB
,
psbD
,
atpA
,
atpB
,
and
rbcL
mRNA. In these mutants, all
other chloroplast transcripts accumulate
normally. It has been possible to iden-
tify the target region for
psbD
and
psbB
RNA degradation in nuclear mutants that
do not accumulate these mRNAs. ±or
this purpose, either the 5
0
or 3
0
un-
translated regions of these mRNAs were
fused to the reporter
aadA
, which confers
spectinomycin resistance. These chimeric
genes were introduced into the chloro-
p
l
a
s
tg
en
om
eo
faw
i
l
d
-
t
yp
es
t
r
a
inan
d
the transformants of mating-type
+
were
crossed to either of the original mutants of
mating-type
. Because of the uniparental
inheritance of the chloroplast genome, all
the offspring from these crosses inherit
the chloroplast chimeric gene, whereas the
nuclear mutations segregate 2 : 2. If the 5
0
untranslated region of
psbD
or
psbB
is suf-
Fcient to confer instability to the chimeric
RNA, there will be cosegregation between
spectinomycin sensitivity and photosyn-
thetic deFciency. This cosegregation has
indeed been shown to occur among the
progeny, and the loss of chimeric RNA in
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