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Instability of a yellow mutation in Chlamydomonas reinhardtii is not due to TOC1 elements
DAY, Anil, ROCHAIX, Jean-David
Abstract
yc4 is an unstable yellow-in-the-dark mutation found in the unicellular green alga Chlamydomonas reinhardtii. yc4 reverts to the wild-type green-in-the-dark phenotype at the frequency of 1x10⁻⁴ revertants per cell division. We have tested whether mutation and reversion of yc4 is due to the insertion and excision of the C. rein-hardtii transposon TOC1.
Southern blots showed differences in the distribution of TOC1 elements when the original mutant yc4 strain, which contained over 30 TOC1 elements, was compared with green-in-the-dark-revertants. However, analysis of the yc4 mutation in a different genetic background containing half the number of TOC1 elements showed that reversion of yc4 is not associated with loss of TOC1 elements.
DAY, Anil, ROCHAIX, Jean-David. Instability of a yellow mutation in Chlamydomonas
reinhardtii is not due to TOC1 elements. Current Genetics , 1990, vol. 18, no. 2, p. 171-174
DOI : 10.1007/BF00312607
Available at:
http://archive-ouverte.unige.ch/unige:143388
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Instability of a yellow mutation in Chlamydomonas reinhardtii
is not due to TOC1 elements
A. Day* and J.-D. Rochaix
Departments of Molecular and Plant Biology, Sciences II, University of Geneva, 30, Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland Received May 1, 1990
Summary. yc4 is an unstable yellow-in-the-dark mutation found in the unicellular green alga Chlamydomonas rein- hardtii, yc4 reverts to the wild-type green-in-the-dark phenotype at a frequency of 1 x 10 -4 revertants per cell division. We have tested whether mutation and reversion ofyc4 is due to the insertion and excision of the C. rein- hardtii transposon TOC1. Southern blots showed differ- ences in the distribution of TOC 1 elements when the orig- inal mutant yc4 strain, which contained over 30 TOCI elements, was compared with green-in-the-dark-rever- tants. However, analysis of the yc4 mutation in a differ- ent genetic background containing half the number of TOCI elements showed that reversion ofyc4 is not asso- ciated with loss of TOC1 elements.
Key words: Chlamydomonas reinhardtii- Yellow muta- tion - Transposon
Introduction
In micro-organisms, genetic instabilities which result in loss or gain of a pigment have a striking consequence:
single colonies are sectored into triangles of a different colour. Instability of a trait can arise as a consequence of a number of processes including: a high overall mutation rate (in Miller 1972); localized hot spots that are due to frameshift mutations (DeBoer and Ripley 1984), single- base substitutions (Cambareri et al. 1989; DeBoer and Ripley 1984), gene conversion (Klein and Petes 1981), tandem duplications (Erickson et al. 1986), excision of transposable elements (Martin et al. 1985) and genetic switches (Zieg et al. 1977); and finally loss of plasmid DNA (Hieter et al. 1985). In C. reinhardtii, a total of eight genetic loci that confer a yellow phenotype in the dark
* Present address: Genetics Laboratory, Biochemistry Department South Parks Road, Oxford OX13QU, UK
Offprint requests to: A. Day at his present address
have been identified (Harris 1984). Amongst these differ- ent yellow-in-the-dark mutations, genetic instability is a feature commonly associated with the yl locus (Sager 1955; Ford and Wang 1982). Some yl alleles have been reported to interconvert with their wild-type alleles at frequencies as high as 1 x 10 -3 (Ford and Wang 1982).
Figure I A shows single colonies of an unstable yellow mutant (yc4) grown in the dark; single colonies contain sectors of mutant yellow cells (inability to accumulate chlorophyll) which are separated by revertant green sec- tors; the yellow colour is due to carotenoids. A number of models have been proposed to explain the instability of yellow mutations (Ford and Wang 1982); these include excision of a transposable element from a green-in-the- dark locus. We have previously shown that reversion of the FUD44 mutation is due to incomplete excision of a transposable element, called TOC1, from an intron in the gene encoding the oxygen evolving enhancer 1 protein (Day et al. 1988). Based on this example, we decided to test whether the yc4 mutation is due to insertion of a TOC1 element and if reversion ofyc4 is due to excision of this TOC1 element. The model is shown in Fig. 1 B.
Since excision of TOC1 leaves behind a solo long termi- nal repeat (LTR), the TOCI insertion must be assumed to lie in a non-translated region of the green-in-the-dark gene. Excision could be due to a recombination event between the 217 bp repeat of TOC1 or due to a gene conversion event with a solo LTR located elsewhere in the genome (Fig. 1 C); both processes are implicated in the reversion of FUD44 although gene conversion is a much rarer event (1/39 versus 38/39, cases, see Day et al.
1988). The approximately ten-fold difference in reversion frequency between FUD44 (around 1 x 10 -5) and yc4 (around 1 x 10 -4) does not rule out this model since TOCI excision rates at different locations cannot be as- sumed to be equal.
Materials and methods
Media, strains and crosses. Chlamydomonas cells were grown in an acetate medium (Gorman and Levine 1965). cc735 was obtained
172
C
}~ EXCISION ~
B y G
~ ~ |
217 X I I217 123
237
217
237
~ ' I A ~ * ~
Fig. 1. A A single colony of yc4 containing a green-in-the-dark revertant sector. B A model for the instability of the yc4 locus. In this model, the yellow-in-the-dark phenotype (y) is proposed to be due to insertion of a 5.7 kbp TOCI element (Day et al. 1988) into the ye4 locus (indicated by a box). Excision of TOC1 allows re-expression of the yc4 locus producing a green-in-the-dark phenotype (G). C TOC1 excision is incomplete and leaves behind a solo LTR. The results of a previous study (Day et al.
1988) suggest that the major TOC1 excision pathway is due to a cross over event between the 217 bp repeats of TOC1 (shown on the left).
Gene conversion occurs at a lower frequency but is also implicated in TOC1 excision. The results of a gene conversion event between a deleted solo LTR containing an adenosine base in place of bases 122-508 and a complete TOCI element is shown on the right
from Dr. E. H. Harris at the Chlamydomonas culture collection, Duke University, Durham, NC. cc1952 (Gross et al. 1988) was from Dr. P.A. Lefebvre (University of Minnesota). 137c is our standard wild-type strain that remains green-in-the-dark, yc4 was isolated as an unstable yellow-in-the-dark mutation by P. Bennoun (Paris).
Genetic crosses were performed as described in Levine and Ebersold (1960). The segregation of the yc4 mutation was 2:2 in 12 complete tetrad products of a yc4 (rot +) x 137c (rot-) cross, yc4 was crossed to a stable yellow-in-the-dark mutant which was the subject of a previous study (Malnoe et al. 1988). The two yellow mutations do not appear to be linked because a total of 13 green and 11 yellow progeny were recovered amongst the products of nine incomplete tetrads.
Isolation of revertants and estimation of the reversion frequency of the ye4 mutation. Green revertants of ye4 were isolated by plating sam- ples from cultures grown in continous light or darkness on a solid medium and then allowing the plates to grow in the dark. Complete- ly green revertant colonies were obtained at similar frequencies from the light grown and dark grown cultures. Two methods were used to obtain an approximate estimate of reversion frequency. In the t~rst method 15,000 colonies were plated in the dark and inspect- ed after a period of growth. Four colonies were i/2 sectors of green and yellow. This means that, on dividing, four out of 15,000 freshly plated cells gave rise to green revertant cells. This gives a frequency of 3 x 10 -4 revertants per cell division. In the second method, 24 yellow colonies of around 1 mm or less in width were resuspend- ed in 200 gl of TAP medium and left overnight before plating a 1/10 of this volume in order to estimate total cell numbers and the number of revertants. Eight cultures which gave similar numbers of yellow colonies, and contained at least one green colony, were used in the analysis. The rate of reversion was calculated to be approxi- mately I x 10 .4 revertants per cell division from the equation:
r = aNjn(NtCa) (Luria and Delbruck 1943) where r = average num- ber of revertant colonies produced per culture (37.5), Nt=average number of colonies produced per culture (48,000), C = number of cultures (8), a=revertants per cell division/In 2.
DNA manipulations. Methods for DNA extraction, restriction en- zyme digestion, DNA electrophoresis, DNA blotting (Southern
1979 a) and DNA hybridization and sizing (Southern 1979 b) were as previously described (Day et al. 1988). Filters were washed in 0.1 x SSC, 0.1% SDS at 60~ (1 x SSCis0.15M NaC1,0.015M trisodium citrate, pH 7.5). A 4.1 kbp HindIII + PstI internal DNA fragment of TOCI (pTOCI.IR1) purified away from plasmid se- quences on agarose gels was used as a hybridization probe. Frag- ments were labelled by nick translation (Maniatis et al. 1982) or the random primer method of Feinberg and Vogelstein (1983).
Autoradiography andfluorography. Hybridization patterns were vi- sualized by a mixture of autoradiography and fiuorography achieved by exposing films to blots in cassettes containing intensify- ing screens (Ilford, fast tungstate) at -70~
Results and discussion
Figure 2 A shows a Southern blot bearing HindIII + SalI double digests of D N A from strain yc4 and four indepen- dent green revertants probed with a large internal TOC1 probe. Apart from dark multicopy bands each b a n d rep- resents one T O C I element. All four revertants (Fig. 2A, lanes 2 - 5 ) lack the 15 and 6.8 kbp bands that are found in the yc4 parent strain (Fig. 2A, lane 1). The absence of two, rather than one, T O C I b a n d in all four revertants was suprising since the model in Fig. 1 B predicts that only one specific b a n d should be lost: the b a n d contain- ing the TOC1 element that interrupts the yc4 locus. Pre- sumably, one or both of the TOC1 excision events that result in the loss of the 15 and 6.8 kbp bands is uncon- nected with reversion ofye4. The loss of two bands in the yc4 revertants (Fig. 2 A, lanes 2 - 5 ) is associated with a gain of one or two new TOC1 bands. The sizes of these new bands are indicated to the right of Fig. 2 A. Although this correlation between loss of TOC1 bands and gain of new TOC1 bands is suggestive of excision being related to
Fig. 2. Southern blot analysis of the distribution of the TOCI elements in
yc4
yellow-in-the-dark strains and green-in-the-dark revertants. The positions of the 21, 9.4, 6.6 and 5.0 kbp marker DNA fragments are shown to the left of each panel. In Figs. 2A and B band differences between lanes arearrowed.
A Hybridization pattern of TOC1 elements in
SalI+ HindIII
digests of DNA from the originalyc4
strain and revertants yc4-Rl to R4.B Hybridization pattern of TOC1 elements in
BamHI + HinarlII
digests of DNA from a green-in-the-dark strain and a mutant yellow-in-the-dark derivative. Thegreen-in-the-dark strain is a green product of a
yc4
x cc1952 cross, c Hybridization pattern of TOC1 elements inBamHI +HindIII
digests of DNA from four yellow-in-the-dark progeny of a yc4 x cc1952 cross (yc4-10, 11, 17, 19) and revertants (ye4-10R, IIR, 17R, 19R)integration, the relationship may be indirect and reflect a more general control on TOCI copy number; a correla- tion is also found between numbers of TOC1 bands lost in FUD44 and new bands gained in FUD44 revertants (Day et al. 1988).
In an effort to make the relationship betwen TOC1 excision and reversion of
yc4
clearer, the originalyc4
strain was crossed to a strain bearing two TOCl-related elements (cc1952). D N A from progeny arising from this cross were examined on Southern blots. A total of two complete tetrads (each containing two green and two yel- low products) and nine yellow progeny were studied.The yellow products of the
yc4
x cc1952 cross were unstable and gave rise to green sectors like the parentalyc4
strain. The TOC1 hybridization pattern in digests(BamHI + HindIII)
of D N A from four yellow progeny (Fig. 2 C, lanes 1-4) was compared with digests of D N A from green revertant derivatives (Fig. 2C, lanes 5-8).The results clearly show that the yellow to green transi- tion is not associated with the loss of TOC1 elements in any of the four derivatives of the
yc4
x cc1952 cross (Fig. 2 C). We conclude that the model shown in Fig. 1 B is incorrect and that excision of TOC1 is not the basis for the instability of theyc4
mutation. We have carried out a similar analysis on strain cc735 which contains an un- stable yl mutation (data not shown). All the 14BamHI
TOC1 bands in cc735 remain in two independent green revertants. This strengthens our conclusion that excision of a TOC1 element is not responsible for the instability of yellow mutations.Figure 2A shows that two bands present in the mu- tant
yc4
are absent in four revertants and this result needs explanation. Although a number of scenarios can be en- visaged where excision of particular TOC1 elements is in some way linked to reversion of the yellow-in-the-dark mutation, we prefer a simpler explanation. We believe that theyc4
strain gained the two TOC1 bands after thetime at which the revertants were isolated but before the time of D N A extraction.
On mitotic growth, one of the green products of the
yc4
x cc1952 cross gave rise to a yellow colony. D N A from the green parent colony is compared with D N A from its spontaneous mutant derivative in the Southern blot of Fig. 2 B. The green to yellow transition is associat- ed with loss of a 12.4 kbp band and gain of a 10.8 kbp TOC1 band. This result indicates that the presence of the unstableyc4
mutation is not required for TOC1 move- ment and is suggestive of a general background instabili- ty of TOC1 elements in the strains studied.Acknowledgements.
We thank E Ebener for art work and photogra- phy and Drs. P. Bennoun, E. H. Harris and P. A. Lefebvre for sending us C. reinhardtii strains. This work was supported by grant 3 328 086 from the Swiss National Foundation to J.-D.R.References
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