Article
Reference
Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library
PURTON, Saul, ROCHAIX, Jean-David
Abstract
We report the rescue of an arginine-requiring mutant (arg7-8) of Chlamydomonas reinhardtii by complementation using total DNA from a genomic cosmid library. Using the glass-bead transformation method of Kindle [8] four putative transformants able to grow in the absence of exogenous arginine were obtained from 3×109 treated cells. Southern blot analysis reveals that at least three of the clones have acquired an additional copy of the gene (ARG7) encoding argininosuccinate lyase (ASL). The arginine-independent phenotype is stable in the absence of selective pressure and high levels of ASL activity are detected in all four clones.
We conclude that these represent true transformants and that any stable nuclear mutant of Chlamydomonas could be rescued using this approach.
PURTON, Saul, ROCHAIX, Jean-David. Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library. Plant Molecular Biology , 1994, vol. 24, no. 3, p.
533-537
DOI : 10.1007/BF00024121
Available at:
http://archive-ouverte.unige.ch/unige:135743
Disclaimer: layout of this document may differ from the published version.
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Short communication
Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library
Saul Purton 1, >N and Jean-David Rochaix
Departments of Molecular Biology and Plant Biology, University of Geneva, 30 Quai-Ernest Ansermet, 1211-Geneva 4, Switzerland; 1Current address: Department of Biology, University College London, Gower Street, London WCIE 6BT, UK (*author for correspondence)
Received 26 August 1993; accepted in revised form 12 November 1993
Key words: arg7 mutant, Chlamydomonas, complementation, cosmid library
Abstract
We report the rescue of an arginine-requiring mutant (arg7-8) of Chlamydomonas reinhardtii by comple- mentation using total DNA from a genomic cosmid library. Using the glass-bead transformation method of Kindle [8] four putative transformants able to grow in the absence of exogenous arginine were ob- tained from 3 x 109 treated cells. Southern blot analysis reveals that at least three of the clones have acquired an additional copy of the gene (ARGT) encoding argininosuccinate lyase (ASL). The arginine- independent phenotype is stable in the absence of selective pressure and high levels of ASL activity are detected in all four clones. We conclude that these represent true transformants and that any stable nuclear mutant of Chlamydomonas could be rescued using this approach.
The green unicellular biflagellate alga Chlamy- domonas reinhardtii is an excellent model system for the study of such cellular processes as pho- tosynthesis, motility and metabolism [6]. Genetic studies have proved particularly fruitful since C. reinhardtii has a small, haploid nuclear genome and undergoes a simple sexual cycle. Conse- quently, a large repertoire of mutants exist and are maintained in laboratory collections around the world. Such mutants include those defective in the biogenesis of the photosynthetic, respiratory and flagellar apparatus, in cell cycle control, in light perception and phototaxis, and in various biosynthetic pathways [6]. The advent of a nuclear transformation system for C. reinhardtii
[3, 9] now makes possible the characterization of
these mutants at the molecular level. In the past few years the identity of a number of genetic loci has been confirmed by complementation of the mutant with a cloned gene [e.g. 3, 9, 13, 15].
Moreover, novel mutants have been generated by exploiting the random integration of transforming DNA into the C. reinhardtii nuclear genome, oc- casionally disrupting a functionally important gene. Such 'tagged' genes can then be isolated and characterized [ 16].
In the case of those mutants for which the iden- tity of the affected gene product is not apparent, and for which recreating the mutant phenotype using a tagging approach would be a lengthy pro- cedure, cloning by complementation is a highly desirable capability. In addition, such a proce-
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dure would allow the cloning of suppressor mu- tations, which could be isolated in no other way.
Given the small size of the C. reinhardtii nuclear genome (ca. 100 Mb [6]) and the high frequency of transformation obtained using the glass bead procedure of Kindle [8], complementation of a stable, single locus mutation using a genomic li- brary is, apriori, quite feasible. To date, the char- acterization of genomic clones for 21 nuclear genes of C. reinhardtii has been described. All contain at least one intron and many contain mul- tiple introns. Consequently, any strategy for iso- lating C. reinhardtii genes by complementation must take into account the likelihood of large genes spanning many kilobases. We have there- fore constructed a cosmid library of the C. rein- hardtii genome in order to maximize the chances of cloning intact genes suitable for complemen- tation. The large size ofcosmid clones (ca. 50 kb) is not a barrier to efficient transformation since both lambda and cosmid clones harbouring the ARG7 marker [3] give transformation rates of approximately one transformant per 2 x 105 arg7 recipient cells (unpublished data). As a first stage towards devising a routine procedure for cloning by complementation, we describe the rescue of an arg7 mutant to prototrophic growth using total DNA from the cosmid library.
The library was constructed in the 6.8 kb low- copy-number cosmid vector pPR691 [7] using high-molecular-weight (ca. 200 kb) total genomic DNA isolated from C. reinhardtii strain cwl5 (mt-) according to the method of Weeks et al.
[18]. The DNA was partially digested with the enzyme Mbo I and size-fractionated as described by DiLella and Woo [4]. Cloning into the Barn HI site of the double cos site vector was as described by Bates [ 1] and packaging in vitro in Gigapack II Gold packaging extract was according to the manufacturer's protocol (Stratagene, Jolly, CA).
The total number of independent clones in the library was determined as 2.7 x 105 by plating on the recA deletion strain DK1 [12]. The insert size of twelve randomly picked clones was deter- mined as 37-42 kb (data not shown). Cosmid DNA was prepared from the unamplified library by growing 1 x 105 clones in DK1 and purifying
the DNA on a caesium chloride gradient [14].
This number of clones represents ca. 40 genome equivalents and has a greater than 99.8~o prob- ability of representing a complete genomic library [19].
We routinely achieve 200-300 arg + colonies per transformation (i.e. treating 3 x 107 cells with 2 #g of DNA) when using a cosmid clone har- bouring theARG7 gene to rescue the mutant arg7- 8cwd (unpublished data). Based on this value and an average insert size in the library of 40 kb, the formula derived by Clarke and Carbon [2] pre- dicts that 30 such transformations using the cosmid library DNA are required to have a 95 ~o probability of complementing a mutation. We therefore conducted 50 glass bead transforma- tions [8] of the mutant arg7-Scwd using 2 #g of supercoiled DNA per transformation. As a result, we obtained four putative transformant colonies capable of arginine-independent growth. Clones of all four were obtained by streaking to single colonies on medium lacking arginine and total DNA was prepared [ 18]. Southern blot analysis of the four clones was carried out using probes specific for ARG7 (a 168 bp Sau 3A-Sac I frag- ment from exon 8 of the gene [database accession number X16619]) and for the cosmid vector.
Figure 1 shows the result of the Southern analysis. When Stu I-digested DNA from the four putative transformants (named T6, T14, T30 and T40) and from the untransformed strain arg7- 8cwd are probed with DNA specific for ARG7, the resident 15 kb Stu I fragment harbouring the gene (Fig. 2) is seen in all cases. A second frag- ment is also seen in the case ofT6, T14 and T30.
The size of this fragment is different in each trans- formant demonstrating that they are independent clones. We propose that T6, T14 and T30 repre- sent true transformants resulting from the inte- gration into the nuclear genome of cosmid clones harbouring the wild-type ARG7 gene. The ob- served difference in size of the Stu I fragments carrying the endogenous and introduced ARG7 genes suggest that the transforming DNA lacked the left-hand Stu I site shown in Fig. 2, or that integration into the genome was the result of a recombination event downstream of this site.
Fig. 1. Southern blot analysis of genomic DNA from arg7- 8cwd and the four arg + clones. The DNA was digested with Stu I and probed with a DNA fragment [14] from the ARG7 gene (left panel) or with the entire vector DNA (right panel).
Note: the increased intensity of the introduced ARG7 DNA relative to the endogenous fragment is an artefact of blotting and is not seen in repeated experiments.
Southern blot analysis of Sal I-digested genomic DNA from the clones reveals only a single 8.1 kb band in each case, demonstrating that the intro- duced ARG7 gene carrying the right-hand Stu I site (Fig. 2) is intact in the transformants (data not shown). Several studies have shown that transforming DNA integrates into the C. rein- hardtii nuclear genome almost exclusively via il- legitimate (non-homologous)recombination [3, 9, 15]. However, we have been unable to test whether or not the introduced DNA in the trans- formants is linked to the arg7 locus. In spite of
probe
_ , , , _
ARG7
2 kb Fig. 2. Physical map of the ARG7 locus showing the positron of the ARG7 gene (hatched box) and the location of restric- tion sites. The position of the probe used in the Southern blot analysis is indicated by an arrow.
several attempts, we have failed to back-cross any of the transformants with arg7-8.
The right-hand panel of Fig. 1 shows a re-probing of the Southern blot with radiolabelled pPR691 DNA. No hybridization of the probe with arg7-8cwdDNA is seen, but several pPR691- related Stu I fragments are detected in the ge- nomes of T6, T14 and T40. The presence of cosmid vector DNA in T40 would suggest that this clone is also a true transformant since the probability of arg7-8 reversion and transforma- tion by a random cosmid clone is extremely low.
Spontaneous reversion in the studies of arg7-8 was not detected in the studies Konvalinkova et al. [ 11 ] and we have never observed reversion as a consequence of the vortexing procedure used in the transformation method (unpublished data).
It is more probable that T40 is the result of complementation by a cosmid clone that retains the whole of the 15 kb Stu I fragment after inte- gration. Consequently, a Stu I digest fails to sep- arate the endogenous and introduced DNA in the Southern analysis. Since the pPR69 1 vector lacks any Stu I sites, the presence of more than one hybridizing band in each of the T6, T14 and T40 digests indicates that these transformants have arisen via one or more of the following events:
(1) integration as a result of recombination be- tween the vector sequence and nuclear DNA;
(2) duplication of vector DNA in the transfor- mant genome; (3) co-transformation with cosmid clones unrelated to ARG7. Further analysis is required to distinguish between these events. The complete absence of vector DNA in T30 indi- cates that integration of the cosmid DNA is the result of two recombination events, one on either side of the cloned ARG7 gene.
We have determined the enzyme activity of argininosuccinate lyase for the mutant and the four clones using the Sakaguchi assay as de- scribed by Farrell and Overton [5]. The results of two independent measurements are shown in Table 1. The clones display very high levels of ASL activity: from 115~o to 342~/o of wild-type levels. Since each transformant contains only one copy of the wild-type ARG7 gene, we can only speculate that such elevated levels of enzyme ac-
536
tivity result from 'positional effects' whereby cis- acting elements at the site of integration of the introduced gene influence its expression. Alterna- tively, trans-complementation at the protein level between the wild-type ASL subunit and the en- dogenous pool of subunits encoded by the arg7-8 gene may contribute to the increased enzyme ac- tivity.
Tablel. Specific activity of ASL in the transformants. Values for two independent experiments are expressed as a percent- age of the wild-type ASL specific activity (0.28/1tool arginine per mg total protein per hour).
Wild-type 100 % 100 %
arg7-8 0 0
T6 348 342
T14 117 115
T30 209 175
T40 142 150
We have demonstrated that complementation of nuclear mutants of C. reinhardtii using a ge- nomic library is feasible. Using the vector sequence in the transformant as a 'handle' on the complementing DNA, probes specific for this DNA could be generated using techniques such as plasmid rescue [16] or inverse PCR [17].
These probes would then be used to isolate the complementing clones from the cosmid library.
One complication to this strategy is the presence of additional vector DNA in the transformants as a result of co-transformation with unrelated cosmid clones. An alternative approach is the use of ordered libraries in which individual cosmid clones are pooled in ordered arrays representing nested subsets of the library. Such a strategy would allow the identification of complementing clones after several rounds of transformation using these subsets. Perhaps the most desirable strategy would be the use of a library built in a cosmid vector carrying a C. reinhardtii origin of DNA replication. Cosmid DNA replicating au- tonomously in C. reinhardtii could then be res- cued back to E. coli by packaging in vitro. Such a technique has been used successfully in the res-
cue of cosmids from transfected mammalian cells [e.g. 10]. These different strategies for cloning Chlamydomonas genes by complementation are under development in several laboratories and will no doubt result in major advances in our understanding of the many and varied mutant phenotypes observed in this useful alga.
Acknowledgements
We thank Dr N. Gumpel for critical reading of the manuscript, and Dr Reeves (University of Sidney, Australia) for the gift of the vector pPR691. This work was supported by grant 31.26345.89 from the Swiss National Foundation and by a Long-term EMBO Fellowship to S.P.
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