638
Chlamydomonas
nonselective growth conditions. No homo-
plasmic suppressor segregants could be
obtained even after repeated cloning or
crosses under phototrophic growth condi-
tions. In contrast, photosynthetic-deFcient
segregants quickly became homoplasmic
under heterotrophic growth conditions.
The molecular basis of the heteroplasmic-
ity has been determined for a suppressor
of an amber (UAG) mutation. In this
suppressor, the original mutation is still
present in all chloroplast DNA copies.
However, 70% of the copies of tRNA
trp
have the tryptophan CCA anticodon
changed to the amber-speciFc CUA. Under
phototrophic growth conditions, therefore,
stable heteroplasmicity can arise as a bal-
anced polymorphism of suppressor and
wild-type alleles of a tRNA gene within
the chloroplast genome. In this case,
the suppressor allele restores ribulose
1,5-bisphosphate carboxylase, whereas the
wild-type allele is required for normal pro-
tein synthesis. When photosynthesis is not
required, the suppressor tRNA allele is lost
because of random segregation, giving rise
to homoplasmic photosynthetic-deFcient
segregants.
As mentioned earlier, it is possible to
disrupt chloroplast genes encoding com-
ponents of the photosynthetic apparatus
using transformation and the
aadA
expres-
sion cassette that confers spectinomycin
resistance in the chloroplast. The transfor-
mants that are obtained usually become
homoplasmic after a few cloning steps.
However, disruptions of chloroplast genes
that are essential under all growth con-
ditions never give rise to homoplasmic
transformants. In this case, stable hetero-
plasmicity is maintained as long as specti-
nomycin is present in the growth medium.
Stable heteroplasmicity can, therefore, be
used for identifying chloroplast genes en-
coding essential functions.
4.2
Function and Assembly of the Flagellar
Apparatus
Chlamydomonas reinhardtii
possesses two
flagella, located at the anterior end of the
cell, that are assembled on basal bodies.
During cell division, basal bodies migrate
to the interior of the cell and function
as centrioles by organizing the spindle
apparatus. The flagellar system of
Chlamy-
domonas
has proven to be particularly well
suited for studying microtubule assem-
bly and function, and motility. This is
because flagellar biosynthesis can be read-
ily synchronized, and numerous mutants
affected in the function and assembly of
the flagellar apparatus have been isolated.
These mutants can be separated into two
major classes: those with abnormal or no
motility, usually called
paralyzed mutants
(with the acronym pf), and those defective
in flagellar assembly (fla).
Extensive ultrastructural and biochemi-
cal studies have revealed that the flagellae
consistofasetofnineouterdoublets,each
consisting of two microtubules A and B,
and a central pair of microtubules (±ig. 8).
Outer and inner arms arise from A micro-
tubules of the outer doublets. The outer
arms, which comprise dyneins and large
multisubunit ATPases, extend toward the
B tubules, where they act to generate inter-
doublet sliding. Outer-arm dyneins consist
of
α
-,
β
-, and
γ
-heavy chains and of inter-
mediate and light chains. Partial cDNA
sequences of the
γ
-heavy chain and of an
unidentiFed heavy chain of
C. reinhardtii
have revealed that these proteins have at
least two conserved domains correspond-
ing to the ATP hydrolytic site and to a
region related to the microtubule binding
domain of the kinesin superfamily. A mu-
tant lacking outer arms could be rescued
by transformation with a genomic clone
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