Non-Mendelian Mutation in Paramecium tetraurelia

The Paramecium tetraurelia mutant called d48 has a complete copy of the A surface protein gene in its micronuclei, but lacks the A gene in the macronucleus. Previous experiments have shown that microinjection of a plasmid containing the entire A gene or a large portion of the gene into the macronucleus of d48 rescued the cell line after formation of a new macronucleus (autogamy). Here we show that several different regions of the A gene can rescue d48, but 100% of the activity cannot be localized to a single, defined region. Inversion of a sequence contained within an A gene plasmid had no measurable effect on rescue efficiency and co-injection of two different plasmids results in enhancement of rescue activity despite the noncontiguous form f the DNA sequences. Both these results suggest that no specific product (RNA or protein) with defined end points is made from the rescuing fragment. A unique restriction site was created in the A gene and used to demonstrate that the injected DNA does not serve as a direct template for the swthesis of the new macronuclear DNA. Models to explain the action of the injected DNA are discussed. T HE free-living, single-celled eucaryote Paramecium has two types of nuclei: a highly polyploid macronucleus, which is transcriptionally active and therefore determines the phenotype of the cell, and two micronuclei, which are transcriptionally silent but contain the germline DNA. During sexual reproduction, either autogamy (self-fertilization) or conjugation (mating), the old macronucleus is destroyed and a new one is created from a copy of the micronuclear genome. It is during this process of macronuclear development that extensive cleavage, rearrangement, telomere addition and amplification of the macronuclear DNA occurs, resulting in two functionally distinct types of nuclei which differ in their DNA content (reviewed in BLACKBURN and KARRER 1986, YAO 1989). Differences between micronuclear and macronuclear DNA have been characterized in many ciliates, but little is understood about the molecules that interact with DNA to control these differences. An unusual Paramecium mutant, called d48, has been a useful model to investigate at least one component important in the formation of the Paramecium macronuclear genome. Originally generated by X-ray mutagenesis of Paramecium tetraurelia stock 51, the d48 mutant contains a normal copy of the A surface protein gene in its micronucleus, but the gene is either deleted or is present in extremely low copy numbers in its macronucleus so that the mutant is unable to express the A surface protein (EPSTEIN and FORNEY 1984, RUDMAN et al. 1991). The A gene is normally located 8-26 kilobases from the macronuclear telomere and in d48 a new macronuclear telomere is created at the 5' end of the gene (FORNEY and BLACKBURN 1988). Genetics 136: 1319-1324 (April, 1994) Genetic experiments have shown that differences between the cytoplasm of d48 and wild-type cells affect the processing of the A gene during macronuclear development. Unlike Mendelian mutations which segregate 1:l in the F2 generation, a cross between wild-type stock 51 (A') and d48 (A-) cells produces all A+ cells from wild-type cytoplasm and all Acells from d48 cytoplasm (EPSTEIN and FORNEY 1984; RUDMAN et al. 1991). The transfer of macronucleoplasm from a wild-type cell into the d48 mutant demonstrated that the old macronucleus is the source of the cytoplasmic determinant ( ~ ~ A R U M O T O 1986). Micronuclear transfer experiments have shown that the micronucleus of the d48 cell is completely normal. Transfer of a d48 micronucleus into an amicronucleate wild type cell results in a wild type cell line after autogamy, and the reciprocal transfer of a wild type micronucleus into an amicronucleate d48 cell has no effect on the d48 mutant phenotype after autogamy (KOBAYASHI and KOIZUMI 1990). Together these experiments indicate that cytoplasmic factors produced from the wild type macronucleus during sexual reproduction are necessary for proper incorporation of the A gene into the next macronucleus. Recent studies have shown that microinjection of a plasmid containing the entire A gene or even a portion of the gene into the d48 macronucleus results in permanent rescue of the mutation after the formation of a new macronucleus (YOU et al . 1991; JESSOP-MURRAY et al. 1991). To elucidate the molecular mechanism responsible for the d48 phenomenon, we have constructed several plasmids containing different portions of the A gene. These plasmids were microinjected into the d48 1320 Y. You, J. Scott and J. Forney macronucleus and their ability to rescue d48 after the formation of a new macronucleus was measured. MATERIALS AND METHODS Cell ines and culture: P. tetraurelia, stock 51 (ATCC 30303), and strain d48 of P. tetraurelia (derived from stock 51) were cultured in 0.25% wheat grass medium (Pines International, Lawrence, Kansas) buffered with 0.45 g of Na,HPO, per liter. In some cases, the medium was augmented with 5 mg of stigmasterol (Sigma) per liter. The sterilized medium was inoculated 24 to 48 hr before use with a nonpathogenic strain of Klebsiella pneumoniae (ATCC 27889). Induction of A surface protein expression, assessment of autogamy, harvesting of cells, and detection of the A surface protein by anti-sera testing have been described by SONNEBORN (1950). Cloned DNA: The nucleotide sequence positions shown in this paper begin numbering with the translation start of the A gene as +1 (GenBank accession number M65163). Plasmid pSAlOSX was previously constructed as described in You et al. (1991). It contains the entire A gene coding region as well as 1.5 kb of 5' upstream and 0.4 kb of 3' flanking DNA. pSA-K is a derivative of pSAlOXS and was constructed by sitedirected mutagenesis to create a KpnI site at position 884 without any amino acid changes. An inversion within the coding region (pSA-invl) was created by digesting pSA-K with BglII (2971) and BclI (4554), then religating the mixture of fragments and transforming into Escherichia coli host JM101. Clones containing the inverted region were identified by restriction enzyme digestion. To delete the BclI (4554EcoRI (7026) fragment, pSA-K was digested with BclI then EcoRI. The larger fragment was purified from an agarose gel by Elu-Quick and ligated with pUCl19. Two sitedirected mutations (T4251C and T4254C) were introduced into pSAlOXS to construct a plasmid with a unique SstII site (pSA-SstII) without changing the amino acid sequence of the A protein. pSA8.8R which was previously described (FORNEY et al. 1983) was digested with BgAI and ligated into pUCl19 to create two subclones pSA4.0-5' (-1034-2971) and pSA4.0-3' (2971-7026). pSB11.6 is a plasmid that contains the entire B gene coding region as well as 5' and 3' flanking sequences inserted into pUC119 (SCOTT et al. 1993). pSA2.1H (3092-5277) was previously described (You et al. 1991) Microinjection: Microinjection of supercoiled plasmid DNA into the d48 macronucleus was performed as previously described by GODISKA et al . (1987). Briefly, DNA was dissolved at a concentration of 1-2 mg/ml in either TE buffer (10 mM Tris-HC1,l mM EDTA) or microinjection buffer (1 14 mM KC1, 20 mM NaC1, 3 mM NaH,P04, pH 7.4). Approximately 3-6 pl of the DNA solution were injected into the d48 macronucleus using a glass microneedle 1-2-pm diameter at the tip. After microinjection, individual cells were transferred to fresh medium. Screening pre-autogamy injected cell for transformation: Cell lines were screened to identify transformants using a dot blot procedure obtained from Indiana University OESSOPMURRAY et al. 1991). Generally, 15-18 fissions after microinjection, about 2500 cells (approximately 400 ng of DNA) were denatured with 0.1 volume of 3 M NaOH at 60" for 30 min, neutralized with 7.5 M NH,OAc and transferred onto Nytran (Schleicher and Schuell Inc., Keene, New Hampshire). The filter was fixed at 80" for 30 min, then hybridized with the appropriate nick translated probe. Preparation of paramecium DNA: Approximately 100 ml cultures of paramecium (150,000 cells) were centrifuged and then resuspended in 0.4 ml of their own culture fluid and quickly added to 0.8 ml of lysing solution (1 % sodium dodecyl sulfate (SDS), 0.05 M disodium EDTA, 100 mM Tris-HC1, pH 9.5) at 65" for 10 min. The DNA was prepared from lysates by two rounds of phenolchloroform extraction and ethanol precipitation. The final DNA pellet was resuspended in 20 pl of TE. Southern blots, nick translations and hybridizations: Southern transfers onto Nytran or nitrocellulose filters were performed according to SAMBROOK et al. (1989). Before hybridization, filters were prewashed with 10 X Denhardt solution, 0.1% SDS, 0.2 M phosphate buffer and 5 X SET (1 X SET = 0.15 M NaC1,30 mM Tris, 2 mM EDTA) at 65" for 1 hr. The filters were then incubated with hybridization solution (1 X Denhardt's solution, 20 mM phosphate buffer, 5 X SET, 0.25% SDS) at 65" for 0.5 hr before adding the labeled probe. After 15-20 hr, filters were washed three times for 30 min each in 0.2 X SET, 0.1% SDS, 0.1% sodium pyrophosphate and 25 mM phosphate buffer at 65" for the first two washes and 70" for the last. The filters were exposed to Kodak X-Omat AR film at -70" with Cronex Lightning-Plus intensifier screens. Scoring for rescue of d48: In this report transformed cells will refer to cells that stably maintain the injected DNA through vegetative divisions, and rescued cells will refer to lines that have lost the injected DNA but express the A surface protein after formation of a new macronucleus (autogamy). Two methods were used to score cell lines after autogamy. In the daily isolation line method, the injected cell was placed in a depression well and each day a single cell from each depression slide was transferred to a new depression containing 0.8 ml of fresh culture fluid. Autogamy was assessed by staining the starved cell lines with acetocarmine. After autogamy, cell lines were placed at 34" to induce A expression. Lines able to express A after this treatment were considered to have been rescued. To correlate our results with KIM et al. (1994) we used another method in which the injected cell was placed in a depression slide, then after three days all the cells were moved to a tube. The tube was alternately fed and starved over a period of 4-6 weeks to induce autogamy. Cells were placed at 34" to induce A expression and scored with anti-A serum.