Proc. Natl. Acad. Sci. USA Vol. 89, pp. 91%-9200, October 1992 Genetics
Efficient mass transformation of Tetrahymena thermophila by electroporation of conjugants (ciliate/ribosomal protein L29 gene/rRNA gene/cydobeinmide/paromomycin)
JACEK GAERTIG* AND MARTIN A. GOROVSKY Department of Biology, University of Rochester, Rochester, NY 14627
Communicated by Joseph G. Gall, June 2, 1992 (received for review April 22, 1992)
advantage of injected rDNA (31). There are two kinds of rDNA vectors. "Somatic" vectors, based on the mature macronuclear rDNA, either recombine with the endogenous, linear rDNA molecules (28) or are maintained as circular plasmids (32, 33) when injected into macronuclei of vegetative cells. Processing vectors are plasmids containing the micronuclear rDNA. After microinjection into a developing macronucleus, the rDNA is excised from the processing vector and amplified (29). The total number of transformed cells that can be obtained by microinjection is low because it is time consuming and technically demanding. Electroporation of somatic cells (34, 35) gave transformation efficiencies that were so low that most investigators continue to use microinjection. The low efficiency of electrotransformation of somatic cells could be due to the high copy number of endogenous drug-sensitive rDNA molecules in the somatic macronucleus. Conjugating cells would seem to offer some advantages for electrotransformation. Young developing macronuclei (anlagen) contain only a few copies of endogenous genes, including the rRNA genes (36, 37). Thus, an exogenous sequence introduced at this stage might more easily reach a selectable level if replicated during endoreplication of the macronuclear genome and amplification of the rDNA. We describe a technique for electrotransformation of conjugating Tetrahymena that easily generates large numbers of transformants and fully substitutes for microinjection. This method should aid in the development of improved vectors, enabling cloning of genes by complementation in Tetrahymena, where a number of fundamental processes lacking in yeast (e.g., ciliogenesis, electrophysiological coordination of ciliary motility, genomic rearrangement, gene amplification, histone H1 function) are known to occur.
ABSTRACT Conjugating cells of the ciliate Tetrahymena thermophila were electroporated in the presence of plasmid DNA containing a paromomycin-resistant ribosomal RNA gene (rDNA). Cells were selected with paromomycin following 12-24 hr of growth on nonselective medium. Resistant cells appeared after 2-3 days. Processing vectors containing the micronuclear rDNA and somatic vectors containing the macronuclear gene transformed the cells, with the former yielding frequencies up to 900 transformants per ,ug of plasmid DNA. A ribosomal protein gene (pL29) conferring cycloheximide resistance also transformed conjugating cells. The transformation efficiency of the puasid containing only the rpL29 gene was increased by insertion of an rDNA replication origi and by cotransformation and preselection with an rDNA vector. These results indicate that electroporation can be used for the production of large numbers of transformed Tetrahymena.
Tetrahymena thermophila is a popular model for (largely separate) molecular and genetic studies, which have provided insights into self-splicing RNA (1), telomeres (2, 3), the genetic code (4), DNA sequence reorganization (5), and histone function and evolution (6). Genetic approaches (7) have been used to investigate fundamental cellular processes including endocytosis (8), exocytosis (9, 10), cell shape and size (11-13), pattern formation (14-16), cell cycle and cytokinesis (17), temperature-regulated expression of surface antigens (18, 19), and cilia biogenesis (20, 21). However, a high-frequency DNA-mediated transformation method is not available for Tetrahymena. Thus, cloning of genes by complementation of mutant phenotypes, a powerful approach in the characterization of genes in yeast (22), has not been possible. Current methods for DNA-mediated transformation in Tetrahymena utilize the rRNA gene (rDNA), which has a unique organization (see refs. 23 and 24). To understand that organization it is necessary to understand the nuclear dimorphism of ciliated protozoa. Ciliates have two distinct nuclei: the germ-line micronucleus and the somatic macronucleus. New macro- and micronuclei are formed during conjugation from the diploid micronucleus. Macronuclear development involves fragmentation, rearrangement, partial elimination, and endoreplication of the zygotic genome (5, 25, 26). In vegetative cells, most, if not all, ofthe transcriptional activity takes place in the macronucleus. During macronuclear formation, the single-copy micronuclear rDNA is amplified into 10,000 extrachromosomal, palindromic dimers (23). Tetrahymena can be transformed by microinjection of mutated extrachromosomal rDNA (27) or of plasmids containing a mutated rRNA gene (28, 29). Selection of transformants relies on a dominant paromomycin-resistance mutation in the 17S rRNA sequence (30) and on a replication
MATERIALS AND METHODS Strains, Cell Growth, and Conjugation. Strains CU427, ChxA/ChxA [cycloheximide-sensitive (cys), VI], CU428, Mpr/Mpr [6-methylpurine-sensitive (6-mps), VII], and B208611, ChxA+/ChxA+; Mpr+/Mpr+ (cys, 6-mps, II) were kindly provided by P. J. Bruns (Cornell University). All three strains are Pmr+IPmr+ [paromomycin-sensitive (pms)] and contain B-type rDNA. Cells were grown to late logarithmic phase in SPP medium (38) containing 1% peptone plus penicillin G (100 units/ml), streptomycin (100 jg/ml), and amphotericin B (0.025 Ag/ml) (GIBCO) at 300C with agitation. To induce conjugation (39) two strains of different mating types were washed with 10 mM Tris HCl (pH 7.5) and
starved for about 24 hr at 300C. Cells were mixed at a concentration of 3 x 105 cells per ml in 10 mM Tris-HCI (pH Abbreviations: rpL29, ribosomal protein L29 gene; pms, paromomycin-sensitive; pmr, paromomycin-resistant; cys, cycloheximidesensitive; cyr, cycloheximide-resistant. *On leave from the Institute of Zoology, Warsaw University, Warsaw, Poland.
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
9196
Genetics:
Gaertig and Gorovsky
Proc. Natl. Acad. Sci. USA 89 (1992)
7.5) to give a fluid depth of about 1 cm and incubated at 30TC without agitation. Electroporation. The following conditions were used unless changes are described. Ten to 12 hr after mixing, conjugants were pelleted (1100 X g for 2 min) and suspended in an equal volume of 10 mM Hepes (pH 7.5). Cells were washed and resuspended in Hepes buffer at 1.5 x 107 per ml. Electroporation was achieved with a capacitor-discharge-based Cell Porator (GIBCO/BRL) with 0.4 cm between electrodes. The resistance switch was set at the "High ohm" position, connecting a 10 £l resistor in parallel with the electroporation chamber. The chambers were lined with Hepes buffer just before use to fill the dead space between the electrodes and the chamber walls. Cell suspension (250 Al) and plasmid DNA (10-40 ,ug in 2-20 A.l of water) were mixed and immediately transferred to the electroporation chamber. A pulse was delivered at 350-400 V (field strength, 875-1000 V/cm) with a 330-p.F capacitor. Electroporations were at room temperature, without carrier DNA. Cells were suspended in S ml of culture medium 10-60 sec after electroporation and diluted 1:40, and 25 ILI was distributed into each well ofa 96-well microtiter plate and 175 Al ofculture medium was added per well. Ten-fold higher cell concentrations were plated for cotransformation experiments. After 12-24 hr of growth at 30TC, paromomycin (Humatin, 500.ug/ml in culture medium; Parke-Davis) was added to give 100 pug/ml. After 3-4 days, the wells without growing cells were counted and the number of transformants was calculated by using the Poisson distribution (39). Usually one or two plates were analyzed, corresponding to 1.2-2.4% of the total electroporated sample. Plasmid DNA. pDSH8 is a pUC19 derivative containing the micronuclear rDNA gene plus flanking sequences (40, 41). The rDNA of pDSH8 encodes a mutation in the rRNA that confers resistance to paromomycin (pmr) (30) and originates from a C3 strain and has a replication advantage over the host strain B type (31). Clone Tt947-L29A (40) is a pDSH8 derivative containing a version ofthe Tetrahymena ribosomal protein L29 (rpL29) gene conferring resistance to cycloheximide (cyr) (Fig. 1). prD4-1 (refs. 32 and 33; Fig. 1) contains a C3, pmr, 5' half ofthe macronuclear rRNA gene and a 3' half originating from a B strain, plus a duplication of a 1.9-kb fragment of the 5' nontranscribed spacer (32) to which the macronuclear rDNA replication origin has been mapped (42). TtL29A3 is a pUC19 derivative containing the rpL29 gene. CV21 and CV22 were constructed by insertion of a 1.9-kb 3
Pmr
pD5H8
U
-
--_
origin-containing BamHI fragment of prD4-1 into the BamHI site of TtL29A3 in either orientation (Fig. 1). Large quantities of plasmid DNA were isolated and precipitated as described (43,44). DNA was aliquoted and stored at -200C. Isolation of Total Tetrahymena DNA and Southern Blotting. Tetrahymena DNA was isolated (33) and pelletable carbohydrates were removed by centrifugation at 35,000 x g for 1 hr. Restriction enzyme digestions were according to the suppliers' instructions. Southern blots (45) were prepared on nylon membranes (Magnagraph; Micron Separations, Westboro, MA) and probes were labeled with [a-32P]dATP (46).
RESULTS Electroporation Induces Transformation of Tetrahymena Mediated by an rDNA Processing Vector. When a processing vector bearing the pmr micronuclear rDNA is injected into developing macronuclei, the rDNA is correctly processed and amplified, leading to the replacement of endogenous, pms rDNA (29). To determine whether such a vector could transform conjugants by electroporation, cells (CU428 and CU427) were mated for 11-12 hr and electroporated in the presence of 10-12 pug of pDSH8. Ten to 20% of the cells survived and completed conjugation. Electroporated cells were grown for 24 hr and then selected in medium with paromomycin at 100 pug/ml. Resistant cells appeared after 2-3 days. In independent experiments, 50-300 putative transformants were obtained per 1Lg of plasmid DNA (500-3600 total transformants per single experiment). All 142 independent pmr lines tested for resistance to higher concentrations of drug grew at 1000 gg/ml. All resistant lines tested displayed the processed pD5H8 restriction pattern; little or no endogenous B-type host DNA was detected (Fig. 2). This result is consistent with the transformation to pmr mediated by the pDSH8 plasmid. Optimization of Electrotransformation. Electroporation conditions. The highest transformation frequencies were obtained by using 875-1000 V/cm with the 330-tkF capacitor. No transformants were detected after electroporation using 875 V/cm and capacitors
Efficient mass transformation of Tetrahymena thermophila by electroporation of conjugants (ciliate/ribosomal protein L29 gene/rRNA gene/cydobeinmide/paromomycin)
JACEK GAERTIG* AND MARTIN A. GOROVSKY Department of Biology, University of Rochester, Rochester, NY 14627
Communicated by Joseph G. Gall, June 2, 1992 (received for review April 22, 1992)
advantage of injected rDNA (31). There are two kinds of rDNA vectors. "Somatic" vectors, based on the mature macronuclear rDNA, either recombine with the endogenous, linear rDNA molecules (28) or are maintained as circular plasmids (32, 33) when injected into macronuclei of vegetative cells. Processing vectors are plasmids containing the micronuclear rDNA. After microinjection into a developing macronucleus, the rDNA is excised from the processing vector and amplified (29). The total number of transformed cells that can be obtained by microinjection is low because it is time consuming and technically demanding. Electroporation of somatic cells (34, 35) gave transformation efficiencies that were so low that most investigators continue to use microinjection. The low efficiency of electrotransformation of somatic cells could be due to the high copy number of endogenous drug-sensitive rDNA molecules in the somatic macronucleus. Conjugating cells would seem to offer some advantages for electrotransformation. Young developing macronuclei (anlagen) contain only a few copies of endogenous genes, including the rRNA genes (36, 37). Thus, an exogenous sequence introduced at this stage might more easily reach a selectable level if replicated during endoreplication of the macronuclear genome and amplification of the rDNA. We describe a technique for electrotransformation of conjugating Tetrahymena that easily generates large numbers of transformants and fully substitutes for microinjection. This method should aid in the development of improved vectors, enabling cloning of genes by complementation in Tetrahymena, where a number of fundamental processes lacking in yeast (e.g., ciliogenesis, electrophysiological coordination of ciliary motility, genomic rearrangement, gene amplification, histone H1 function) are known to occur.
ABSTRACT Conjugating cells of the ciliate Tetrahymena thermophila were electroporated in the presence of plasmid DNA containing a paromomycin-resistant ribosomal RNA gene (rDNA). Cells were selected with paromomycin following 12-24 hr of growth on nonselective medium. Resistant cells appeared after 2-3 days. Processing vectors containing the micronuclear rDNA and somatic vectors containing the macronuclear gene transformed the cells, with the former yielding frequencies up to 900 transformants per ,ug of plasmid DNA. A ribosomal protein gene (pL29) conferring cycloheximide resistance also transformed conjugating cells. The transformation efficiency of the puasid containing only the rpL29 gene was increased by insertion of an rDNA replication origi and by cotransformation and preselection with an rDNA vector. These results indicate that electroporation can be used for the production of large numbers of transformed Tetrahymena.
Tetrahymena thermophila is a popular model for (largely separate) molecular and genetic studies, which have provided insights into self-splicing RNA (1), telomeres (2, 3), the genetic code (4), DNA sequence reorganization (5), and histone function and evolution (6). Genetic approaches (7) have been used to investigate fundamental cellular processes including endocytosis (8), exocytosis (9, 10), cell shape and size (11-13), pattern formation (14-16), cell cycle and cytokinesis (17), temperature-regulated expression of surface antigens (18, 19), and cilia biogenesis (20, 21). However, a high-frequency DNA-mediated transformation method is not available for Tetrahymena. Thus, cloning of genes by complementation of mutant phenotypes, a powerful approach in the characterization of genes in yeast (22), has not been possible. Current methods for DNA-mediated transformation in Tetrahymena utilize the rRNA gene (rDNA), which has a unique organization (see refs. 23 and 24). To understand that organization it is necessary to understand the nuclear dimorphism of ciliated protozoa. Ciliates have two distinct nuclei: the germ-line micronucleus and the somatic macronucleus. New macro- and micronuclei are formed during conjugation from the diploid micronucleus. Macronuclear development involves fragmentation, rearrangement, partial elimination, and endoreplication of the zygotic genome (5, 25, 26). In vegetative cells, most, if not all, ofthe transcriptional activity takes place in the macronucleus. During macronuclear formation, the single-copy micronuclear rDNA is amplified into 10,000 extrachromosomal, palindromic dimers (23). Tetrahymena can be transformed by microinjection of mutated extrachromosomal rDNA (27) or of plasmids containing a mutated rRNA gene (28, 29). Selection of transformants relies on a dominant paromomycin-resistance mutation in the 17S rRNA sequence (30) and on a replication
MATERIALS AND METHODS Strains, Cell Growth, and Conjugation. Strains CU427, ChxA/ChxA [cycloheximide-sensitive (cys), VI], CU428, Mpr/Mpr [6-methylpurine-sensitive (6-mps), VII], and B208611, ChxA+/ChxA+; Mpr+/Mpr+ (cys, 6-mps, II) were kindly provided by P. J. Bruns (Cornell University). All three strains are Pmr+IPmr+ [paromomycin-sensitive (pms)] and contain B-type rDNA. Cells were grown to late logarithmic phase in SPP medium (38) containing 1% peptone plus penicillin G (100 units/ml), streptomycin (100 jg/ml), and amphotericin B (0.025 Ag/ml) (GIBCO) at 300C with agitation. To induce conjugation (39) two strains of different mating types were washed with 10 mM Tris HCl (pH 7.5) and
starved for about 24 hr at 300C. Cells were mixed at a concentration of 3 x 105 cells per ml in 10 mM Tris-HCI (pH Abbreviations: rpL29, ribosomal protein L29 gene; pms, paromomycin-sensitive; pmr, paromomycin-resistant; cys, cycloheximidesensitive; cyr, cycloheximide-resistant. *On leave from the Institute of Zoology, Warsaw University, Warsaw, Poland.
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
9196
Genetics:
Gaertig and Gorovsky
Proc. Natl. Acad. Sci. USA 89 (1992)
7.5) to give a fluid depth of about 1 cm and incubated at 30TC without agitation. Electroporation. The following conditions were used unless changes are described. Ten to 12 hr after mixing, conjugants were pelleted (1100 X g for 2 min) and suspended in an equal volume of 10 mM Hepes (pH 7.5). Cells were washed and resuspended in Hepes buffer at 1.5 x 107 per ml. Electroporation was achieved with a capacitor-discharge-based Cell Porator (GIBCO/BRL) with 0.4 cm between electrodes. The resistance switch was set at the "High ohm" position, connecting a 10 £l resistor in parallel with the electroporation chamber. The chambers were lined with Hepes buffer just before use to fill the dead space between the electrodes and the chamber walls. Cell suspension (250 Al) and plasmid DNA (10-40 ,ug in 2-20 A.l of water) were mixed and immediately transferred to the electroporation chamber. A pulse was delivered at 350-400 V (field strength, 875-1000 V/cm) with a 330-p.F capacitor. Electroporations were at room temperature, without carrier DNA. Cells were suspended in S ml of culture medium 10-60 sec after electroporation and diluted 1:40, and 25 ILI was distributed into each well ofa 96-well microtiter plate and 175 Al ofculture medium was added per well. Ten-fold higher cell concentrations were plated for cotransformation experiments. After 12-24 hr of growth at 30TC, paromomycin (Humatin, 500.ug/ml in culture medium; Parke-Davis) was added to give 100 pug/ml. After 3-4 days, the wells without growing cells were counted and the number of transformants was calculated by using the Poisson distribution (39). Usually one or two plates were analyzed, corresponding to 1.2-2.4% of the total electroporated sample. Plasmid DNA. pDSH8 is a pUC19 derivative containing the micronuclear rDNA gene plus flanking sequences (40, 41). The rDNA of pDSH8 encodes a mutation in the rRNA that confers resistance to paromomycin (pmr) (30) and originates from a C3 strain and has a replication advantage over the host strain B type (31). Clone Tt947-L29A (40) is a pDSH8 derivative containing a version ofthe Tetrahymena ribosomal protein L29 (rpL29) gene conferring resistance to cycloheximide (cyr) (Fig. 1). prD4-1 (refs. 32 and 33; Fig. 1) contains a C3, pmr, 5' half ofthe macronuclear rRNA gene and a 3' half originating from a B strain, plus a duplication of a 1.9-kb fragment of the 5' nontranscribed spacer (32) to which the macronuclear rDNA replication origin has been mapped (42). TtL29A3 is a pUC19 derivative containing the rpL29 gene. CV21 and CV22 were constructed by insertion of a 1.9-kb 3
Pmr
pD5H8
U
-
--_
origin-containing BamHI fragment of prD4-1 into the BamHI site of TtL29A3 in either orientation (Fig. 1). Large quantities of plasmid DNA were isolated and precipitated as described (43,44). DNA was aliquoted and stored at -200C. Isolation of Total Tetrahymena DNA and Southern Blotting. Tetrahymena DNA was isolated (33) and pelletable carbohydrates were removed by centrifugation at 35,000 x g for 1 hr. Restriction enzyme digestions were according to the suppliers' instructions. Southern blots (45) were prepared on nylon membranes (Magnagraph; Micron Separations, Westboro, MA) and probes were labeled with [a-32P]dATP (46).
RESULTS Electroporation Induces Transformation of Tetrahymena Mediated by an rDNA Processing Vector. When a processing vector bearing the pmr micronuclear rDNA is injected into developing macronuclei, the rDNA is correctly processed and amplified, leading to the replacement of endogenous, pms rDNA (29). To determine whether such a vector could transform conjugants by electroporation, cells (CU428 and CU427) were mated for 11-12 hr and electroporated in the presence of 10-12 pug of pDSH8. Ten to 20% of the cells survived and completed conjugation. Electroporated cells were grown for 24 hr and then selected in medium with paromomycin at 100 pug/ml. Resistant cells appeared after 2-3 days. In independent experiments, 50-300 putative transformants were obtained per 1Lg of plasmid DNA (500-3600 total transformants per single experiment). All 142 independent pmr lines tested for resistance to higher concentrations of drug grew at 1000 gg/ml. All resistant lines tested displayed the processed pD5H8 restriction pattern; little or no endogenous B-type host DNA was detected (Fig. 2). This result is consistent with the transformation to pmr mediated by the pDSH8 plasmid. Optimization of Electrotransformation. Electroporation conditions. The highest transformation frequencies were obtained by using 875-1000 V/cm with the 330-tkF capacitor. No transformants were detected after electroporation using 875 V/cm and capacitors
