Copyright 0 1996 by the Genetics Society of America Mapping Three Classical Isozyme Loci in Tetrahymena: Meiotic Linkage of EstA to the ChxA Linkage Group Sally Lyman Allen,* Dawn Zeilingert and Eduardo Oriast *Department of Biology, University of Michigan, Ann Arbor, Mirhigan 48109-1048 and ?Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, California 9?106 Manuscript received July 1, 1996 Accepted for publication September 5, 1996 ABSTRACT We demonstrate a reliable method for mappingconventional loci and obtainingmeiotic linkage data for theciliated protozoan Tetrahymena thermophila.By coupling nullisomic deletion mapping with meiotic linkage mapping, loci known to be located on a particular chromosome or chromosome arm can be tested for recombination. This approach has been used to map three isozyme loci, EstA (Esterase A), EstB (Esterase B), and AcpA (Acid Phosphatase A), with respect to the ChxA locus (cycloheximide resistance) and 11 RAPDs (randomly amplified polymorphic DNAs). To assign isozyme loci to chromo- somes, clones of inbred strains C3 or C2 were crossed to inbred strain B nullisomics. EstA, EstB and AcpA were mapped to chromosomes IR, ?L and X,respectively. To test EstA and AcpA for linkage to known RAPD loci on theirrespective chromosomes, a panel of Round I1 (genomic exclusion) segregants from a B/C3 heterozygote was used. Using the "MAKER program, EstA was assigned to the ChxA linkage group on chromosome IR,and a detailed map was constructed that includes 10 RAF'Ds. AcpA (on X),while unlinked to all the RAPDs assigned to chromosome ? by nullisomic mapping, does show linkage to a RAF'D not yet assignable to chromosomes by nullisomic mapping. SOZYMES were among the first loci investigated ge- meiosis, with respect to ploidy or chromosomal mor- I netically in Tetrahymenathermophila (ALLEN 1960, phology (McCoy 1977). McCoy (1977) also detected 1961, 1965, 1968; ALLEN et al. 1963a,b; BORDENet ul. inbred strain differences with regard to a possible link- 1973). Seven loci were identified, each with two alleles: age of EstA and ChxA (cycloheximide resistance). He Esterase A (EstA),Esterase B (EstB), Acid Phosphatase observed linkage when inbred strains B and C2 were A (AcpA),Tyrosine Aminotransferase A ( TutA), NADP- crossed but not with crosses of inbred strains B and D. Isocitrate dehydrogenase A (IdhA),NADP-Malate dehy- In crosses involving the 40 loci known then, a total drogenase (MdhA), and Tetrazolium Oxidase A ( TzoA). ofsix potential linkage groups was found (DOERDER Genetically determined variation was observed among 1973; McCoy 1977). At least three of these linkage the inbred strains (Table 1) . groups have not stood the test of time: when mapping There arefive pairs of chromosomes in the micronu- with nullisomic strains, those supposedly linked mark- cleus of T. thermophila. With at least 40 loci identified ers mappedto different chromosomes (BRUNSand CAS by the early 1970's, linkage between some of these loci SIDY-HANLEY1993). might have been expected. No obvious meiotic linkages The nullisomic strains are a uniqueset of strains that were observed among six of the seven isozyme loci nor have proved very useful in genetic studies. They are with any of the other 30-35 loci, except for one case: characterized by having micronuclei (germ-line nuclei) EstA and mat (mating type locus) (ALLEN 1964; ALLEN that have lost both copies of one or more chromosomes and GIBSON1973; DOERDER1973). However, even this or chromosome arms (BRUNSet al. 1982, 1983). They linkage was disputed (MCCOY1977) since crosses involv- survive vegetativelybecause they are heterokaryons hav- ing different combinations of inbred strains did not ing normal somatic macronuclei. The macronucleus is behave uniformly with regard to recombination fre- the only nucleus active in gene expression and vegeta- quency between mat and EstA. It was suggested that tive reproduction, and therefore a population of cells crosses between certain inbred strains showing linkage can be propagated indefinitely while missing one or involved structural heterozygosity while crosses ofother more chromosomes in theirmicronuclei. During sexual strains not showing linkage for thesame markers lacked reproduction the micronuclei of conjugating cells un- this structural heterozygosity. However, no gross chro- dergo meiosis. When a nullisomic strain is crossed to a mosomal rearrangementscould be detectedduring normal strain, progeny with monosomic micronuclei are produced. Using this type of cross and a battery of nullisomic strains missing different chromosomes, Curresponding author Sally Lyman Allen, Department of Biology, University of Michigan, Ann Arbor, MI 48109-1048. genes can be assigned to their respective chromosomal E-mail: [email protected] location. To date over 100 different loci, >50 cloned Genetics 144: 1489-1496 (December, 1996) 1490 S. L. Allen, D. Zeilinger and E. Orias TABLE 1 Allelic constitution of inbred strains of T. thennophila Isozyme locus Inbred strain EstA EstB AcpA TatA 7ioAIdhA MdhA A B B A s s S F B B B B S S S F C3 C B A S S s F C2 C C B F F F F D B B A s S s S EstA, Esterase A, EstB, Esterase B; AcpA, Acid Phosphatase A TutA, Tyrosine Aminotransferase A IdhA, NADP-Isocitrate Dehydrogenase A; MdhA, NADP-Malate Dehydrogenase A TzoA, Tetrazolium Oxidase A. Alleles A, B and C refer to theparticular inbred strain in which the allele was first observed. Alleles Fand S refer to the relative migration after electrophoresis: S, slow; F, fast. sequences and 47 RAPDs (randomly amplified polymor- chromosome(s), is used to indicate the micronuclear compc- phic DNAs) have beenmapped to specific chromo- sition of each nullisomic line. Recently thawed stocks were used in all crosses. Clone C2-2671(C) was a gift from Dr. somes or chromosome arms using this technique THOMASNERAD at the American Type Culture Collection (BRUNSand CASSIDY-HANLEY 1993). (ATCC) and has the ATCC number 30389. The other clones Combining nullisomic deletion mappingwith recom- listed in Table 2 have been kept frozen in the ORIA~lab (as bination data allowed thedetermination of meiotic described by FLACKS1979) since shortly after their isolation linkage between the mat locus and the ribosomal RNA and initial characterization in the BRUNS'lab. Culture media: PP210 (nutritionallyrich, proteose-pep- gene (BLEYMANet al. 1992). Nullisomic mapping re- tone-based growth medium), 2% BP (bacterizedproteose vealed that both mat and the ribosomal RNA gene are peptone used for sequentially growing and starving cells) and located on the left arm of chromosome 2. Appropriate Dryl's buffered salts solution (used for starving cells) have crosses revealed meiotic linkage. The distance between previously been described (Omand BAUM1984). Toselect the two loci is estimated to be 34.3 cM (LYNCHet al. for progeny, cycloheximide (cycl) (Sigma) was added to PP210 medium at a final concentration of 15 pg/ml. 1995). Genetic procedures: Routine procedures for the mainte- This approach was used here to map three of the nance of strains and growth of cells have been previously isozyme loci, EstA,EstB and AcpA. They were first as- described (Owand BRUNS1975; ORLASand HAMII.TON signed to a particular chromosome, or chromosome 1979). For long-term maintenance, stocks obtained in this arm, by deletion mapping using crosses to 10 different work were frozen in liquid nitrogen byJuDrTH DODGEORUS. Unless otherwise specified all the work was done at 30". nullisomic strains. Linkage to known RAPD loci was Crosses: Cultures were sequentially grown, starved, mixed, then tested by appropriate crosses and, when found, refed, and cycloheximide-selected in Petri dishes (Oms and maps were constructed. EstA was assigned to the ChxA BRUNS1975). The progeny were then grown 5-6 days in linkage group previously located to 1R and built by Erlenmeyer flasks (100 ml in 250-ml flasks) before extracts mapping RAPD loci (BRICKNERet al. 1996).Here, a were prepared forisozyme phenotyping. Twoday-old cultures were used for preparing DNA when required for PCR and linkage group is defined as a group of loci such that RAPD testing. every member shows statistically significant linkage to Isozymephenotyping: Whole-cell extracts were made by at least one other member of the group. As more loci concentrating the cells by centrifugation and rinsing them are mapped, the ChxA linkage group should become with Dryl's salts solution. The cells were then frozen-thawed coextensive with the entire chromosome 1. No linkage six times in a dry-ice ethanol bath and stored at -20". Gelswere madein 12% hydrolyzed potatostarch was found between AcpA, located to chromosome X, (STARCHART,Smithville, TX: lots W560-2 and W562-2). The and six knownRAPD loci on chromosome 3, but it does starch buffer was 30 mM boric acid-Tris, pH 7.7 for the ester- show linkage to a RAPD not yet assigned to chromo- ases and pH 7.15 for the acid phosphatases. We had to lower somes by nullisomic mapping. EstB, known from this the pH to 7.15 to reveal the B allele of AcpA, which is very work to be located on chromosome X, has yet to be sensitive to pH compared to the A (C3) allele. The end-tray buffer was 0.3 M boric acid-Tris, pH 7.34 for both theesterases tested for linkage to these RAPD loci because inbred and acid phosphatases. Except for the pH of the buffers, strains B and C3 are not polymorphic for E&. details of the technique used for starch-gel electrophoresis followed published procedures (ALLEN 1960, 1964; ALI.EN et MATERIALSAND METHODS al. 1963a,b). Gels were run for 4 3/4 hours at 245V for the esterases and 225V for the acid phosphatases. Strains The isozyme genotypes for each of theinbred The gels were then sliced into pieces and marked; the strains of T. thennophila are listed in Table 1. pieces were placed in a tray (for the esterases) or test tubes The clones used (Table 2) were derived from various inbred (for theacid phosphatases), and anenzyme-specific substrate strains of T.