630
Chlamydomonas
Selection is performed on medium lack-
ing arginine or ammonium, respectively,
by transformation with the corresponding
wild-type genes. It is also possible to trans-
form wild-type strains using markers that
confer resistance to several drugs such
as emetine, phleomycin, paromomycin,
and spectinomycin. The latter three are
of bacterial origin and need to be driven
by Chlamydomonas promoters for expres-
sion in the algal cells. Although several
foreign drug-resistant markers have been
used
successfully
in
Chlamydomonas
cells, efFcient expression of foreign genes
remains a difFcult task. It is also possi-
ble to introduce additional genes into the
nuclear genome by cotransformation. In
most cases, transformation occurs through
nonhomologous recombination, as the
transforming DNA appears to integrate at
random sites of the nuclear genome. This
property has been used successfully for
tagging genes. In this approach, new mu-
tations are induced through the integration
of the transforming DNA into nuclear
genes. The bacterial vector sequences in
the transforming DNA can then be used
as a probe for isolating the mutated
gene. Two mobile elements of
C. rein-
hardtii
– Gulliver, which resembles clas-
sical transposable elements, and
TOC1
,
which is related to retrotransposons – have
been characterized and can also be used
for nuclear gene tagging. The high nuclear
transformation yield has made it possi-
ble to perform gene-rescue experiments
by complementing nuclear mutations with
genomic cosmid libraries of
C. reinhardtii
.
While nuclear transformation is efFcient
when cloned
C. reinhardtii
nuclear genes
are used as the transforming DNA, it has
been difFcult to express foreign genes ef-
Fciently in this organism even when they
are fused to
C. reinhardtii
promoters and
3
0
untranslated regions of abundant mR-
NAs. It is not yet clear whether this is
due to the biased codon usage of nuclear
genes of
C. reinhardtii
, to methylation of
the foreign DNA sequences, or to other
factors. Codon bias is clearly important as
shown by the successful expression of G±P
(green fluorescent protein) when its gene
is reconstructed with the
C. reinhardtii
codon usage. Moreover, it is possible to
express chimeric genes in the nuclear
compartment, provided all gene sequences
originate from
C. reinhardtii
.T
r
an
s
c
r
ip
-
tional fusions between the
cabII-1
and the
nit-1
genes, coding for a light-harvesting
chlorophyll a/b binding protein and the
nitrate reductase respectively have been
introduced into the nuclear genome and
expressed. These chimeric constructs con-
sist of the
cabII-1
promoter region fused
at its transcriptional initiation site to the
nit-1
gene
.Inw
i
ld
-
typece
l
l
s
,exp
re
s
s
ion
of the
cabII
gene is strongly increased
in the light, whereas the
nit-1
gene is re-
pressed in the presence of ammonium and
is expressed in media containing nitrate.
Expression of the
nit-1
gene requires a
positive-acting regulator encoded by the
nit-2
gene. In the transformants, expres-
sion of the chimeric
cabII-1/nit-1
gene was
also stimulated by light. However, the
nit-1
mRNA accumulated to a much lower level
than the
cabII-1
mRNA. Expression could
be slightly stimulated by adding further up-
stream sequences of the
cabII-1
promoter
to the chimeric construct. Expression of
the chimeric gene occurred in the presence
of ammonium and no longer depended on
the
Nit-2
product. This is a useful prop-
erty since many mutations in
C. reinhardtii
have been isolated in a
nit-1 nit-2
genetic
background. Similar chimeric genes have
been produced with the tubulin
β
2
pro-
moter and the gene encoding periplasmic
arylsulfatase. As observed for the tubulin
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