Copyright 0 1994 by the Genetics Society of America

A Temperature-Sensitive Mutation of the Temperature-Regulated SerH3 i-Antigen Gene of Tetrahymena thermophila:

Implications for Regulation of Mutual Exclusion

Gregory L. Lacrosse and F. Paul Doerder

Department of Biology, Cleveland State University, Cleveland, Ohio 44115 Manuscript received April 22, 1994

Accepted for publication June 6, 1994

ABSTRACT The Sergenes of Tetrahymena thermophila specify alternative forms of a major cell surface glycoprotein,

the immobilization or i-antigen (i-ag). Regulation of i-ag expression assures that at least one i-ag gene is expressed at all times. To learn more about the regulatory system and the possible role of i-ag itself, we studied SerH3-ts1, a temperature-sensitive allele of the temperature-regulated SerH3 gene normally ex- pressed from 20-36". In homozygotes grown at the nonpermissive temperature (>32"), H3 is not present on the cell surface, but the gene continues to be transcribed until its 36" cutoff. H3 formed at the permissive temperature is stable at nonpermissive temperatures, indicating that SerH3-tsl is temperature- sensitive for synthesis rather than function. At nonpermissive temperatures, the S i-ag is expressed in place of H3. This result suggests that normal H protein may play a role in regulating S expression. SerH3-tsl was isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . Sequencing of SerH3-tsl revealed a single A + G transition at nucleotide 473, resulting in the substitution of glycine for aspartate. The affected residue is conserved in the internal repeats comprising the H protein, and the charge difference correlates with changes in electrophoretic mobility of the H3 protein.

T EMPERATURE-sensitive mutations long have been useful in dissecting genetic phenomena. Here we

describe a temperature sensitive mutation of a tempera- ture regulated gene, the SerH3 surface protein gene of Tetrahymena thermophila. SerH alleles code for an im- munodominant surface protein called the immobiliza- tion antigen (i-ag) and are expressed between 20" and 36". SerH is one of a series of isoloci specifjmg alterna- tive forms of i-ag. Others include SerL expressed below 20°, SerT expressed above 36", SerS expressed in me- dium containing 200 mM NaCl, and S e d expressed in medium containing anti-H antibody. The Sergenes have been of experimental interest because of their "mutually exclusive" expression. As in Paramecium where similar i-ag genes are found, these genes constitute a reserve of genetic information from which only one, or sometimes a few, i-ag genes are selected for expression, primarily by environmental conditions. While the regulatory mecha- nism is not yet understood, it appears to be multilevel, involving transcription initiation (GILLEY et al. 1990), mRNA stability (LOVE et al. 1988), and, possibly, i-ag itself (CAPDEVILLE 1979; SMITH and DOERDER 1992b).

Recent attention has focused on the SerH3 gene, particularly its 36" cutoff. At this temperature SerH3 is regulated primarily by mRNA stability (LOVE et al. 1988). SerH3 is one of four ( SerHl through SerH4) al- lelic variants specifjmg antigenically distinct GPI-linked surface proteins (KO and THOMPSON 1992; RON et al. 1992) with M, of 44,000 (H2) to 52,000 (H3) (DOERDER and BERKOWITZ 1986). The SerH3 sequence (TONDRAVI

Genetics 138 297-301 (October, 1994)

et al. 1990) reveals a protein consisting of three nearly identical cysteine delineated periods. Such periods, con- sisting of -85 amino acids and 8 cysteines, also have been found in i-ags of Paramecium primaurelia (PRAT 1990) and P. tetraurelia (NIELSEN et al. 1991). Even though they appear to be a structurally conserved fea- ture, the periods diverge in amino acid sequence more than the amino and carboxyl termini of the protein (PRAT 1990; NIELSEN et al. 1991). Comparison of recently sequenced SerHl u. DM and F. P. DOERDER, unpub lished results) with SerH3 shows a similar situation. The cysteine periods therefore appear to be responsible for differences in antigenicity (PRAT 1990).

To explore further the temperature-sensitive nature of SerH, we characterized a mutation temperature sen- sitive for SerH3. We wanted to know which, if any, i-ag is expressed in the absence of H under nonpermissive conditions. If, as suspected, H protein and/or H mRNA has a regulatory function, this function might be iden- tified by a nonfunctional H protein. Also, since N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is the mutagen of choice in T . thermophila, we wanted to learn about the molecular nature of MNNGinduced lesions in this organism.

MATERIALS AND METHODS

Stock cell lines and culture conditions: Wild-type strains A ( S e r H l ) , B (SerH3) and B3 (SerH4) were originally o b tained from DAVID L. NANNEY. The RseD mutant was isolated by DOERDER (1986). CU399, a functional heterokaryon sensi- tive to cycloheximide (macronucleus) but homozygous for

298 G. L. Lacrosse and F. P. Doerder

cycloheximide resistance (micronucleus) was obtained from PETER J. BRUNS. A*III and B*VII are genomic exclusion strains (ALLEN et al. 1967; WEINDRUCH and DOERDER 1975; PITTS and DOERDER 1988), which yield 1:l genotypic ratios when crossed to heterozygotes. Cells were cultured in 10 X 150-mm test tubes containing 8 ml of PPY medium (1 % proteose peptone (Difco), 0.15% yeast extract (U.S. Biochemical Corp.), and 0.01 mM FeC1,). The RseD mutant, used as an Spositive control, was cultured in PPYS, which is PPY medium contain- ing 0.2 M NaCl. All stock cell lines were maintained at room temperature (22-24').

Isolation of SerH3-tsl mutation: Log phase cells of strain B were exposed to 10 pg/ml N-methyl-M-nitro-N-nitroso- guanidine for 3 hr, washed into BP (a 190 dilution of PPY inoculated with Klebsiella pneumoniae) for recovery of one or two fissions, and crossed with CU399 in order to bring micro- nuclear genes into expression in the macronucleus. After 10-15 fissions in the presence of cycloheximide to select for true conjugants, cultures were screened for temperature- sensitive mutations in a two-step process that takes advantage of macronuclear assortment (DOERDER 1986). First, anti-H3 was added to tube cultures (80 cultures in 16 X 100-mm tubes, -3-4 X IO5 cells) grown at 34" and resistant (nonimmobi- lized) cells swimming at the top were removed to fresh tubes. Second, the next day, the cultures were divided into two, one at 28" and one at 34", and the next day, these duplicate cultures were again serotyped with anti-H3. Putative temperature- sensitive cells were cloned from cultures that upon retest were immobilized at 28" but not at 34". Heterozygous isolate B2685-10 was inbred to homozygosity (B2779 and B2780) by genomic exclusion.

Temperature cutoff and temperature shift: To determine the upper limit of H3 expression in the mutant, stock cultures kept at room temperature (about 24") were used to inoculate fresh PPY tubes which were cultured overnight at temperatures of 31" through 34" in 0.5" intervals. Incubator temperatures were stabilized for at least 24 hr before cultures were placed in the incubator. The cultures were serotyped with H3 anti- serum the next day. For temperature shift experiments, cells were grown in PPY for 24 hr and washed into Tris, pH 7.4, buffer at the time of temperature shift (WILLIAMS et al. 1985). Every half hour following the shift, 750 pl of cells were removed and tested for the presence of H3 and/or S i-ag by immunofluorescence.

Immobilization and immunofluorescent assays: Immobili- zation and immunofluorescence assays were performed as pre- viously described (DOERDER 1981) with antisera listed in Table 1 of SMITH et al. (1992). All antisera were prepared in rabbits against purified i-ag and are specific for their homologous pro- teins. Immobilization reactions were considered positive if cells were slowed or immobilized compared to controls. Im- munofluorescence assays were considered positive if cells were uniformly red.

Reverse transcription polymerase chain reaction (RT-PCR): Reverse transcription was performed using Moloney murine leukemia virus reverse transcriptase according to manufactur- er's directions (Life Technologies, Inc.). The RNA template (purified according to SAMBROOK et al. 1989) was DNased with RQ1 DNase (Promega) (SAMBROOK et al. 1989). PCR reactions on cDNA templates were carried out in a Perkin-Elmer Cetus thermal cycler in 25yl volumes by adding all the reagents ex- cept the Tuq polymerase to 0.5-ml PCR tubes. The tubes were then heated to SO", after which the Taq DNA polymerase was added. The initial cycle consisted of denaturation at 94" for 5 min, annealing at 54" for 2 min and extension at 72" for 2 min. This was followed by 29 cycles at 94" for 1 min, 58" for 1 min and 72" for 1.5 min. Each PCR reaction contained the follow-

ing: 2 pl of 1.25 mM dNTPs, 2.5 pl of 25 p~ MgCl,, 2.5 pl of 1OX Taq buffer, 0.2 pl of Tuq DNA polymerase, 8.3 pl dH,O, 5 pl DNA or cDNA (about 1 pg) , and 2.5 pl of each (25 p ~ ) primer. The two primers were H3AT, 5"GTAAAACAA- AACTATAATAATTTGJ', a sequence beginning at the 3' nucleotide of the AUG start codon of SerH? and dTRI, 5 ' - C G C G A A T T C C T T T I T T l " T T T I - I T T - 3 ' , a se- quence complementary to poly(A).

Northern blot: Northern blots were prepared as previously described (HALLBERG et al. 1984; KRAUS et al. 1987) and were probed with pC6295, a subclone of pC6 specific for SerH? (KILE et al. 1988).

Cloning the SerH3-tsl gene: The SerH3- t s l macronuclear gene was cloned from both B2779 and B2780 by sequentially isolating the 9.4kb EcoRI and 2.2-kb HindIII fragments con- taining the SerH3 gene, as based on the restriction map of TONDRAW (1990). HindIII 2.2-kb fragments were ligated into pGEM-7Zf + plasmid vector (Promega), and recombinant plasmids were screened for the SerH3-tsl gene with SerH? cDNA clone pC6. Three positive clones were isolated. Each had the same restriction pattern as SerH?, and each served as a template for PCR amplification of appropriately sized DNA with SerW3-specific primers. Both strands were sequencedwith a Sequenase kit (U.S. Biochemical Corp.) using universal primers and primers internal to SerH?.

Immunodiffusion: Cell extracts for immunodiffusion were prepared as described by SMITH and DOERDER (1992a). Immu- noelectrophoresis tablets (Bio-Rad) (0.08 M Tris-HC1, pH '7.2, 0.15 M NaCl, 0.01 M sodium azide in dH,O) were used to pre- pare a 1% agarose gel poured on a 10 cm X 10-cm glass plate. A total of 6 p1 of undiluted anti-H3 (D91 as listed in Table 1 Of SMITH et al. (1992) and 6 pl of cell extract were loaded into wells. The gel was placed in a moist chamber for 24 hr in order for precipitin bands to develop.

Western blot: Purified native cell surface antigen (DOERDER and BEUOWITZ 1986) was subjected to polyacrylamide gel elec- trophoresis, 5% gel (0.09 M Tris, 0.08 M boric acid, pH 8.8). Western blots were prepared as described by SMITH et al. (1992).

RESULTS

Mendelian inheritance of SerH3-tsl: F, progeny of crosses between homozygous temperature sensitive strains (B2779 and B2780) and wild-type strains B (SerH3) and B3 (SerH4) were phenotypically normal at 34", indicating that the mutation is recessive. F, X F, crosses yielded a total of 43H:12ts F, progeny, an ac- ceptable 3:l ratio ( P > 0.5) (Table l ) , and genomic ex- clusion crosses (in which heterozygotes segregate 1:l genotypic/phenotypic ratios) segregated 23H: 16ts prog- eny, an acceptable 1:1 ratio ( P > 0.2). Segregation from SetH3 and SerH4 indicated that the mutation is at %H, hence the designation SerH3-tsl. Homozygous SerH3-tsl cells breed true in subsequent crosses (n = 95).

Upper limit of the permissive temperature range of SerH3-tsl expression: Nine mutant clones were grown overnight at various temperatures in 0.5" intervals. Cells grown >32" were not immobilized, whereas the same lines grown <31.5" were immobilized with anti-H3. As- says of cells grown between 31.6" and 31.9" at 0.1" in- tervals yielded mixed results. Because all clones were

TS SerH3 Mutation 299

TABLE 1

Mendelian inheritance of SerHStsl

Phenolyes ohsenfed at 34" Cross

(;cnot\prs crossctl 11 0 . H.7 1s H4

F, (cxpcctetl JH: I IS ratio) . S ~ r l ~ 3 / S ~ r H 3 - t . ~ 1 X SrrH3/SrrHjr- t t l 2764 23 7 SrrH4/SrrH3-l.rI X SrrH4/SerH3- / . sI 2790 5 'Lo

(;enomic exclusion (rxpected 1 H: 1 ts ratio) .Sr1-If3/.SrrH3-t.s I X I3*VII 2779 2 3 S~rl -I3 /SrrHjr- / rI X '\*I11 2780 10 6 SrrH3/ .SerH3- /sI X A*III 278 1 6 2 S r r H 4 / S e r H 3 - t s l X A * I I I 2 i X 3 5 )

positive at 531.5" and negative at ?32", the upper tem- perature limit of H3 expression in SerH3-tsl cells there- fore is taken as 32".

The i-ag expressed at the nonpermissive temperature: Nine homozygous mutant lines cultured at 34" were se- rotyped in log phase using antisera to all the known surface i-ag. Only a n t i s immobilized the cells. Control wild-type SerH3 cells were not immobilized. SerH3-tsI mutants therefore express S in place of H3 at nonper- missive temperatures. At 38" (T expression) S was present on both mutant and wild-type cells. This result is consistent with those of SMITH and DOERDER (1992a), who found that at 38" in strain B cells, S is coexpressed with T in all phases of the growth cycle.

Both wild-type and mutant cells grown to stationary phase (about 72 hr) at 28" were positive with antis. This result confirms those Of SMITH and DOERDER (19924 who found that there is variable coexpression of H and S in strain B cells in stationary phase. The coexpression of H and S i-ag in wild-type cells, and the expression of S in place of H3 at nonpermissive temperatures in mutant cells, suggests that H i-ag affects S expression.

SerH3-tsl is temperaturesensitive for synthesis: The effect of temperature on mutant H3 i-ag was assayed in u i m ~ by immunofluorescence and in vitro by immuno- diffusion. In the in uiuo assay, mutant B2780 and wild- type cells in PPY were shifted from 28" to 34" in starva- tion medium (Tris-HC1, pH 7.4, buffer) and assayed at intervals by immunofluorescence. The strains were in- distinguishable, with H3 being present on the surface for >3 hr after temperature shift. Assays with an t i s showed that S appeared on the surface of mutant cells, but not wild type, within 2 hr and therefore was co- present with HS. These results show that antigen re- placement is not quantitatively reciprocal, and, more importantly, that H3 formed at 28" is not antigenically altered upon temperature upshift.

Temperature sensitivity for synthesis was confirmed by immunodiffusion (Figure 1). For wild-type strain B, H3 protein was present at 28" and 34", but not at 38", which is consistent with the Northern analyses (below) aswell as previous experiments showing that the H3 pro- tein disappears after shifting the temperature to >36"

R

B-28 2780-28 2780-28 2780-34 2780-34 2780-38 2780-38

seD-NaCI B-28

B-28 O

B-28 O

B-28O B-34' B-34' B-38 O

B-38 O

RseD-N B-28'

laCl

FIGURE 1 .-Immunodiffusion analysis of HS expression in strain B (SerH3) and B2780 ( S ~ r H j r - l s I ) at permissive and nonpermissive temperatures. Cell extracts (see MA'rERIAIS ASD METHODS) were added to outer wells, and anti-H?i against pu- rified H3 was added to the inner wells. RseD expresses only S i-ag when cultured in 0.2 M NaCI. The gel shown in this figure was developed at 28". Identical results were observed on a du- plicate gel developed at 34".

(WILLIAMS et al. 1985). With B2780 mutant, H3 protein was present at 28" but not at 34" or higher. Significantly, identical results were obtained with the same immuno- diffusion experiment performed at 34". These results show that preformed H3 i-ag does not significantly change its antigenic conformation at high tempera- tures. Only newly formed H3 appears to be affected by temperatures above 32".

Level of defect in SerH3-tsl: To determine if the temperature sensitive phenotype of SerH3-tsl is caused by changes in the level of mRNA, a Northern blot, shown in Figure 2, was prepared using total RNA from both strain B and strain B2780, grown at temperatures of 28- 40". For strain B, SerH3 message was present up to 36". This corresponds well with published experiments show- ing the half-life of H3 mRNA to be ? 1 hr at temperatures below 36", and 5 3 min at temperatures above 36" (LOVE et nl. 1988). For mutant B2780, H3 transcript was de- tectable up to 36", well past the 32" cut off for protein. Upon prolonged exposure, a weak signal was seen with RNA from cells grown at 38".

These results were confirmed by RT-PCR. Full length H3 cDNA was obtained from wild-type (strain B) RNA at 28" and 34" but not at 38" (as expected). Full length H3

300 G. L. Lacrosse and F. P. Doerder

1 2 3 4 5 6 7 8 9 1 0

FIGURE 2.-Northern blot analysis H3 mRNA (arrowhead) in wild-type strain B (SerH3) and mutant strain B2780 (SerH3- t s l ) at permissive and nonpermissive temperatures. Equal amounts of total RNA (8 pg determined spectrophotometri- cally) was applied to each lane. Lanes 1-5, wild type; lanes 6-10, mutant. Lanes 1 and 6,28"; lanes 2 and 7,34"; lanes 3 and 8, 36"; lanes 4 and 9, 38"; lanes 5 and 10, 40".

cDNA was obtained from mutant RNA (B2780 and B2779) at 28" and 34", and, in reduced amounts, at 38" (gel not shown). These results suggest that SerH3 tran- script in mutant cells may be more stable than in wild type, a possibility which has not been further pursued.

Molecular lesion in SerH3-tsl: To identify the mo- lecular basis of the mutation responsible for the tem- perature sensitive phenotype, the SerH3-tsl gene from both €32780 and B2779 was cloned and sequenced. When the sequence was compared to the published SrrH3 sequence (TONDRAVI et al. 1990), a single A -+ G transition (Figure 3) was found at nucleotide position 473 in the coding region. Since no other change in the transcript region was found, it is reasonable to conclude that this single mutation is responsible for the loss of H3 protein under nonpermissive conditions.

The A + G transition results in a missense aspartate + glycine substitution. This amino acid change would make mutant H3 less negative than the wild type. In order to test this prediction, a Western blot of purified native wild-type and mutant H3 was prepared. Figure 4 shows that the predicted mobility difference was indeed observed.

DISCUSSION

SerH3-tsI is a temperature-sensitive allele of SerH3, itself a temperature-regulated gene coding for a major surface protein. S~rH3- tsI is temperature-sensitive for synthesis of the H3 i-ag at >32". The mutational lesion is an A- G transition resulting in an aspartate -+ glycine substitution. In the absence of H3 at nonpermissive tem- peratures, S is found on the surface.

The expression of S i-ag in place of H3 at nonper- missive temperatures is relevant to the mutually exclu- sive expression of ciliate i-ags. All cells, including mu- tants, appear never to be without an i-ag (SMITH and DOERDER 1992b), suggesting that the regulatory system is sensitive to the presence of i-ag and switches to an alternate when the i-ag is no longer synthesized. An ex-

2780 B G A T C G A T C

FICCRE 3.-Region o f scquencing gcl showing the A + G transition (arrowhead, nucleotide 473) in SerH3-tsI ofmutant B2780. Identical results were observed with B2779.

+ ts + ts

FIGURE 4.M'estern blot showing charge difference be- tween wild-type (+) and mutant HJ (ts) protein. Purified pro- tein was separated on a native gel, blotted on to nitrocellulose and probed with anti-H3 (see MATTERIAIS ..\sD METI-IODS). Blot shows duplicate lanes loaded with different amounts of sample.

haustive study of i-ag expression in wild-type and mutant cells indicates certain regulatory links (SMITH and DOERDER 1992b). One such link is between H and S. These i-ags are coexpressed in wild-type cells at station- ary phase, and all mutants defective in SerH expression express S togetherwith at least one other i-ag (SMITH and DOERDER 1992b). Significantly, as shown in this study, SerH3-tsl homozygotes express only S at the nonper- missive temperature. That an i-ag is expressed at all is significant because it is strong evidence that functional H protein plays a regulatory role by (somehow) inhib- iting S expression. Regulatory roles for i-ag protein have been postulated before (CAPDEYII.I.E 1979; SMITH and DOERDER 1992b), and further experiments are required to determine the precise regulatory role.

It is not clear how the aspartate + glycine missense mutation at residue 158 results in the temperature sen- sitivity of H3 protein. Temperature shift experiments showed that the H3 protein formed at permissive tem- peratures is antigenically unaltered at 34", whereas cells grown continuously at 34" have no detectable H3 protein. The mutational effect thus appears to be for synthesis rather than for function. As mentioned in the Introduction, H i-ags have three periods each with "85

TS SerH3 Mutation 301

amino acids and 8 cysteines. The high number of cys- teines suggests numerous disulfide linkages. Residue 158 lies within the first of these periods, and an aspartate is found at the same position in the other two periods. Perhaps at nonpermissive temperatures glycine inhibits the formation of disulfide linkages, resulting in the rapid destruction of the protein.

The mutagen MNNG is the only mutagen that yields significant numbers of germinal lethals in T. ther- mophila ( O w and BRUNS 1976). SerH3-tsl is the first MNNGinduced mutation to be sequenced. Studies in other systems have shown that the primary mutagenic effect of MNNG is a G -+ A transition (96% of cases) with lesser amounts of A 4 G transitions, A + T and G + C transversions, deletions and frameshift mutations (LUCCHESI et al. 1986; BURNS et al. 1987; GORDON et al. 1988,1990). There is also a preference for guanines that are flanked by a 5' guanine or adenine (BURNS et al. 1987; GORDON et al. 1988; SOCKETT et al. 1991). There are 26 such sites in the SerH3gene. The A+ G transition in SerH3-tsl is a rare type of MNNG mutation occurring when the methyl group of 04-methylthymine is not removed, thereby allowing it to mispair with guanine (KOHALMI and KUNZ 1988). The occurrence of this mu- tation, if indeed due to MNNG, may indicate the lack of appropriate transferase activity in T. thermophila. How- ever, sequence analysis of two MNNG induced muta- tions in the SerHl gene (SerHl-1 and SerHl-2) shows that both are the expected G + A transitions at GG hotspots (JOE D m , personal communication). It will be interesting to determine the mutational lesions of other MNNGinduced mutations in T. thermophila.

The authors thank JOE DEAK for critical review of the results. This work was supported by a Graduate College Student Research Award, Cleveland State University.

LITERATURE CITED

ALLEN, S. L., S. K FILE and S. L. KOCH, 1967 Genomic exclusion in Tetrahymena. Genetics 5 5 823-837.

BURNS, P. A., A. J. E. GORDON and B. W. GLICKMAN, 1987 Influence of neighboring base sequence on N-methyl-N'-nitro-N-nitro-

J. Mol. Biol. 194: 385-390. soguanidine mutagenesis in the lacl gene of Escherichia coli.

CAPDEVILLE, Y., 1979 Regulation of surface antigen expression in

J. Cell. Physiol. 99: 383-394. Paramecium primaurelia. 11. Role of the surface antigen itself.

DOERDER, F. P., 1981 Differential expression of immobilization an- tigen genes in Tetrahymena thermophila. 11. Reciprocal and non- reciprocal transfer of i-antigen during conjugation and expres- sion of i-antigen genes during macronuclear development. Cell Diff. 10: 299-307.

DOERDER, F. P., 1986 Dominant mutations regulating i-antigen ex- pression in Tetrahymena thermophila. J. Hered. 77: 202-204.

DOERDER, F. P., and M. S. BERKOWITZ, 1986 Purification and partial characterization of the H immobilization antigens of Tetrahy- mena thermophila. J. Protozool. 33: 204-208.

GILLEY, D., B. M. RUDMAN, J. R. PREER, JR. and B. POLISKY, 1990 Mul- tilevel regulation of surface antigen gene expression in Parame- cium tetraurelia. Mol. Cell. Biol. 10: 1538-1544.

GORDON, A. J. E., P. A. BURNS and B. W. GLICKMAN, 1988 N-Methyl- K-nitro-N-nitrosoguanidine induced mutation in a RecAstrain of Escherichia coli. Mutat. Res. 201: 219-228.

GORDON, A. J. E., P. A. BURNS and B. W. GUCKMAN, 1990 N-Methyl- K-nitro-N-nitrosoguanidine induced DNA sequence alteration: non-random components in alkylation mutagenesis. Mutat. Res. 233: 95-103.

HALLBERG, R. L., K W. KRAUS and R. C. FINDLY, 1984 Starved Tetra- hymena thermophila cells that are unable to mount an effective heat shock response selectively degrade their rRNA. Mol. Cell. Biol. 4 2170-2179.

&LE, J. P., H. D. LOVE, JR., C. A. HUBACH and G. A. BANNON, 1988 Re- producible and variable rearrangements of a Tetrahymena ther- mophila surface protein gene. Mol. Cell. Biol. 8: 5043-5046.

KO, Y.-G., and G. A. J. THOMPSON, 1992 Immobilization antigens from Tetrahymena thermophila are glycosyl-phosphatidylinositol- linked proteins. J. Protozool. 39: 719-723.

KOHALMI, S. E., and B. A. KUNZ, 1988 Role of neighboring bases and assessment of strand specificity in ethylmethanesulphonate and N-methyl-N-nitro-N-nitrosoguanidine mutagenesis in the SUP40 gene of Saccharomyces cerevisiae. J. Mol. Biol. 204: 561-568.

KRAUS, K. W., P. J. GOOD and R. L. HALLBERG, 1987 A heat shock- induced, polymerase 111-transcribed RNA selectively associates with polysomal ribosomes in Tetrahymena thermophila. Proc. Natl. Acad. Sci. USA &i: 383-387.

LOVE, H. D. J., A. ALLEN-NASH, Q. A. ZHAO and G. A. BANNON, 1988 mRNA stability plays a major role in regulating the temperature-specific expression of a Tetrahymena thermophila surface protein. Mol. Cell. Biol. 8: 427-432.

LUCCHESI, P., M. WWAY and M. G. MARINUS, 1986 Analysis of forward mutations induced by N-methyl-N'-nitro-N-nitro- soguanidine in the bacteriophage P22 mnt repressor gene. J. Bacteriol. 166: 34-37.

NIELSEN, E., Y. YOU and J. FORNEY, 1991 Cysteine residue periodicity is a conserved structural feature of variable surface proteins from Paramecium-tetraurelia. J. Mol. Biol. 222 835-841.

O m , E., and P. J. BRUNS, 1976 Induction and isolation of mutants in Tetrahymena. Methods Cell Biol. 12: 247-284.

PITTS, R. A,, and F. P. DOERDER, 1988 Genomic exclusion and other micronuclear anomalies are common in genetically defective clones of Tetrahymena thermophila. Genetics 120: 135-143.

PRAT, A., 1990 Conserved sequences flank variable tandem repeats in two alleles of the G surface protein of Paramecium primaurelia. J. Mol. Biol. 211: 521-535.

RON, A,, N. E. WILLIAMS and F. P. DOERDER, 1992 The immobiliza- tion antigens of Tetrahymena thermophila are glycoproteins. J. Protozool. 39: 508-510.

SAMBROOK, J., E. F. FRITSCH and T. MANIATIS, 1989 Molecular Cloning: A Laboratory Manual, Ed. 2. Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y.

SMITH, D. L., and F. P. DOERDER, 1992a Exceptions to mutual exclu- sion among cell surface i-antigens of Tetrahymena thermophila during salt stress and stationary phase. J. Protozool. 39: 628-635.

SMITH, D. L., and F. P. DOERDER, 1992b Multiple effects of mutation on expression of alternative cell surface protein genes in Tetra- hymena thermophila. Genetics 130: 97-104.

SMITH, D. L., M. S. BERKOWITZ, D. POTOCZAK, M. KRAusE, C. RAAB et al., 1992 Characterization of the T, L, I, S, M and P cell surface (immobilization) antigens of Tetrahymena thermophila: molecu- lar weights, molecular isoforms, and crossreactivity of antisera. J. Protozool. 39: 420-428.

S O C K E ~ , H., S. ROMAC and F. HUTCHINSON, 1991 Mutagenic specificity of N-methyl-N'-nitro-N-nitrosoguanidine in the gpt gene on a chromosome of Chinese hamster ovary cells and of Escherichia coli cells. Mol. Gen. Genet. 227: 252-259.

TONDRAVI, M. M., R. L. WILLIS, H. D. LOVE, JR. and G. A. BANNON, 1990 Molecular characterization of SerH3, a Tetrahymena ther-

Mol. Cell. Biol. 10: 6091-6096. mophila gene encoding a temperature-regulated surface antigen.

WEINDRUCH, R. H., and F. P. DOERDER, 1975 Agedependent micro-

Ageing Dev. 4 263-279. nuclear deterioration in Tetrahymenapyriformis, syngen 1. Mech.

WILLIAMS, N. E., F. P. DOERDER and A. RON, 1985 Expression of a cell surface immobilization antigen during serotype transformation in Tetrahymena thermophila. Mol. Cell. Biol. 5 1925-1932.

Communicating editor: S. L. ALLEN