Vol. 10, No. 4
MOLECULAR AND CELLULAR BIOLOGY, Apr. 1990, p. 1538-1544 0270-7306/90/041538-07$02.00/0 Copyright © 1990, American Society for Microbiology
Multilevel Regulation of Surface Antigen Gene Expression in Paramecium tetraurelia DAVID GILLEY, BERTINA M. RUDMAN, JOHN R. PREER, JR., AND BARRY POLISKY* Programii in Molecular, Cellutlar, and Dev'elopmental Biology, Department of Biology, Indiana University, Bloomington, Indiana 47405 Received 29 August 1989/Accepted 10 January 1990
A family of genes is responsible for production of surface antigenic components of Paramecium tetraurelia. These surface proteins are expressed in a mutually exclusive manner. Individuals rarely display more than one type. However, changes in environmental conditions can cause different surface proteins which replace preexisting types to be expressed. We investigated the nature of regulation of the genes for the A, C, and H surface antigens of P. tetraurelia. A system for in vitro run-on transcription was developed from crude Paramecium extracts and used in this analysis. The genes for surface antigens A and H were controlled at the level of transcription. However, the gene for surface antigen C demonstrated both transcriptional and posttranscriptional control, depending on the serotype being expressed. When animals expressed serotype A, the gene for surface antigen C was not transcribed. However, when animals expressed serotype H, the gene for surface antigen C was actively transcribed and stable surface antigen C mRNA was present in the cells, although surface antigen C was not detectable by serotype testing or by a salt-alcohol extraction method. The kinetics of transformation from serotype H to serotype C were determined by using the in vitro transcription system and monitoring steady-state RNA levels. During the transition, serotype A transcription was detected in run-on transcription experiments, although this RNA did not accumulate. The results indicate that serotype expression is controlled at several levels and that not all serotype genes are controlled in the same manner.
Surface antigen genes in Parcameciumn tetrauirelia constitute a family of distinct, unlinked loci which display mutually exclusive expression (3, 5, 7; for reviews, see references 11 and 15). In a particular individual, only a single type of surface antigen is present on the surface at any time. The surface antigens, also referred to as immobilization antigens, are large glycoproteins, from 250 to 300 kilodaltons, that make up about 50% of the protein in cilia or 3.5% of the total protein in P. tetrarairelia. Certain environmental conditions, such as the temperature and/or the composition of the culture medium, favor stable expression of a particular surface antigen. Animals that express a specific serotype are capable of switching or transforming to another serotype within 2 to 3 fissions after an environmental change. In P. tetraurelia stock 51, 11 distinct antigenic types have been found and designated serotypes A, B, C, D, E, G, H, I, J, N, and Q. By using their respective mRNAs to probe genomic libraries, several surface antigen genes have been isolated (4, 6). The mechanism of controlled expression of the surface antigen genes is not known, but it does not appear to function by chromosomal rearrangements (4), as in other systems (2, 10, 17). Changes at the DNA level have not been detected upon serotype switching, although very large or very small changes have not been ruled out. The surface antigen genes are amplified from one copy per haploid micronucleus to about 2,000 copies per macronucleus (13). The large copy number, along with the speed with which 100% of a large population can be switched to another serotype, indicates a highly regulated control mechanism. The copy number of the surface antigen genes in the macronucleus is not influenced by the expression of other serotypes. Therefore, the expression of the serotype genes is not controlled by changes in gene dosage (4). *
To investigate the regulation of the genes for surface antigens A, C, and H in P. tetrauirelia, an in vitro transcription system was developed so that nuclear run-on experiments could be performed. Two of the genes studied, those for surface antigens A and H, clearly showed transcriptional regulation, although the gene for C displayed either transcriptional or posttranscriptional control, depending on the serotype expressed. Therefore, the comprehensive control mechanism of surface antigen gene expression cannot be explained by a simple set of repressor-operator interactions acting at the transcriptional level. MATERIALS AND METHODS Paramecium. P. tetraiurelia stock 51 was the strain used in all experiments. Cultures were grown in Y medium, an infusion of 0.15% Cerophyl (Cerophyl Co., Kansas City, Mo.)-10 mg of yeast extract per ml-45 mg of Na,HPO4 per ml-0.1 mg of stigmasterol per ml. Y medium was inoculated with Klebsiella pneuinoniae (14) 24 h before use. Serotypes. Serotypes A, C, and H were maintained at 27, 19, and 13°C, respectively. Serotypes were identified as described by Sonneborn (14) with sera from the Sonneborn collection at Indiana University. In vitro nuclear run-on transcription. The following protocol was based on that of Love et al. (8). Packed cells (0.1 ml or 106 animals) were gently mixed with 0.9 ml of extraction buffer {0.1 M sucrose, 0.1 M KCI, 2.5 mM MgCl,, 2.5 mM EGTA [ethylene glycol-bis(3-aminoethyl ether)-N,N,N',N'tetraacetic acid], 10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] [pH 6.5], 1% Triton X-100} and placed on ice for 5 min. The cells were then washed twice with centrifugation for 2 min at 2,000 x g with extraction buffer lacking Triton X-100. Cytoskeletal frameworks were suspended to 1 ml in transcription buffer I (0.05 M Tris hydrochloride, 0.05 M KCI, 5 mM MgCl2, 1 mM spermidine, 1 mM spermine, 1 mM CaCl, 2 mM dithiothre-
Corresponding author. 1538
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itol, 0.1 M sucrose, 25% glycerol [pH 8.1]) and then used directly for in vitro transcription or frozen on liquid N, and stored at -70°C for later use. For in vitro transcription reactions, 50 [L1 of cytoskeletal frameworks was added to 25 ,ul of transcription buffer 11 (0.1 M Tris hydrochloride, 0.1 M KCl, 0.01 M MgCl2, 2 mM spermidine, 2 mM spermine, 4 mM putrescine, 6 mM dithiothreitol, 2 mM CaCl2, 1.2 mM aurintricarboxylic acid [pH 8.1])-2.5 ,u1 each of 20 mM ATP, CTP, and GTP-40 U of RNasin-125 ,uCi of [t_-32P]UTP and incubated at 25°C for 40 min. This mixture was extracted with phenol and then chloroform-isoamyl alcohol (24:1, vol/vol). Unincorporated counts were removed by passage over a G50 spin column. Hybridization conditions. Slot and Southern blots were hybridized at 42°C in 5x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-50 mM NaH2PO4 (pH 7.4)-50% formamide-0.5% sodium dodecyl sulfate-3 x Denhardt solution (9)-10% dextran sulfate (average molecular weight, 500,000)-denatured salmon sperm DNA at 200 ,ug/ml. Filters were washed twice with 2 x SSC-0. 1% sodium dodecyl sulfate at room temperature for 10 min and then washed twice with 0.1 x SSC-0. 1x sodium dodecyl sulfate at 68°C for 20 min. RNA isolation. RNA was prepared by lysing P. tetraurelia cells in guanidine hydrochloride and purifying it as previously described (12). Northern (RNA) blots. Total RNA was electrophoresed through 1% agarose-formaldehyde gels and transferred to Nytran nylon membranes (Schleicher & Schuell, Inc., Keene, N.H.) as previously described (9), except that RNA was UV cross-linked as recommended by the manufacturer. Protein analysis. Crude protein extracts were prepared by salt-alcohol extraction and electrophoresed as previously described (12). RESULTS Maps of the genes for surface antigens A, C, and H. Three genes for surface antigens A, C, and H in P. tetraiurelia stock 51 have been characterized previously at the molecular level (4, 6). Restriction maps of the macronuclear versions of these genes are shown in Fig. 1, along with the specifically identified subclones that were used in this study. In vitro nuclear run-on experiments. To determine the level of regulation of the three surface antigen genes, nuclear run-on experiments were performed. Cytoskeletal frameworks were made as described in Materials and Methods from populations of animals expressing each serotype. These cytoskeletal frameworks consist of detergent-treated cells with permeabilized cell membranes containing intact macronuclei. Microscopic inspection of the frameworks showed that most of the cytoplasmic contents had been removed. The frameworks were capable of incorporation of labeled ribonucleoside triphosphates into acid-insoluble material. The frameworks were used to elongate nascent RNA chains in vitro in the presence of [a-32P]UTP as described in Materials and Methods. Certain features of the in vitro system were investigated. Trichloroacetic acid-precipitable counts incorporated into the in vitro transcription reaction were sensitive to RNase A treatment (data not shown). The Paramecium RNA polymerase activity responsible for expression of the serotype genes within cytoskeleton frameworks (as shown below) was completely inhibited by addition of 10 ,ug of oa-amanitin per ml. This concentration of a-amanitin had no inhibitory effect on RNA polymerase I transcription of rRNA in this system (data not shown). We
A gere
A
S
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S
H
pSAi.4H
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C gene H
H
C pSC1 35H
H genie
H
a,Xi R1
,
Al pSHSR
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RI khb
FIG. 1. Partial restriction maps of the genes for surface antigens A, C, and H and the subclones used. The thick lines and arrows indicate the transcribed region of the gene for each surface antigen. The subclone(s) used is indicated below each map. All subclones were cloned into pUC8 (Bethesda Research Laboratories, Inc., Gaithersburg, Md.). Rl, EcoRI; H, Hindlll; X, Xhol; S, Sall. kb, Kilobase.
conclude that the RNA polymerase is a type II enzyme typically involved in transcription of eucaryotic genes. We used the in vitro-synthesized labeled RNA as a probe for hybridization to various cloned DNAs immobilized on nitrocellulose (Fig. 2). When this slot blot was probed with labeled RNA made by cytoskeletal frameworks from animals expressing serotype A (Fig. 2, slot blot 1), only the subclones specific for the gene for surface antigen A and the ot-tubulin clone hybridized to in vitro-synthesized RNA. Note that the single-stranded DNA making up the antisense strand of the gene for A (Al anti-) hybridized to the labeled RNA, but the single-stranded DNA making up the sense strand of the gene for A (Al sense) did not hybridize. Therefore, animals expressing serotype A do not transcribe the genes for the other two serotypes, C and H. Also, the cytoskeletal framework transcription system displays proper strand specificity, at least in the region of the gene for surface antigen A probed. The hybridized molecules were stripped from the slot blot and reprobed with labeled RNA synthesized in vitro by cytoskeletal frameworks from animals expressing serotype C (Fig. 2, slot blot 2). Only the subclones specific for the gene for surface antigen C and the gene for a-tubulin showed hybridization. Therefore, animals expressing the gene for serotype C do not transcribe the gene for serotype A or H. When the blot was reprobed with labeled RNA synthesized in vitro by cytoskeletal frameworks from animals expressing serotype H (Fig. 2, slot blot 3), the subclones specific for the genes for surface antigen C and a-tubulin hybridized. These results indicate that when animals express serotype H, the gene for H is transcribed as expected, but in addition, the gene for surface antigen C is rapidly transcribed. Surprisingly, the rate of transcription of the gene for C in H-expressing cells (Fig. 2, slot blot 3) normalized to a-tubulin was estimated to be 1.2 times the rate of transcription of the gene for C in C-expressing cells (Fig. 2, slot blot 2). The H-expressing cells used in this experiment were essentially in the stationary growth phase, dividing once every 14 days, while the C-expressing cells divided once per 2 days. Consequently, we examined the relative transcription rates of serotype genes in cells dividing every 48 h.
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MOL. CELL. BIOI.
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1
2
3
A
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Al
Al sense
0
Al anti-
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-
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4
n ..
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_
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FIG. 2. DNA slot blot analysis. In vitro-synthesized. labeled RNA from animals expressing serotypes A. C. and H was used to probe various cloned DNAs immobilized on nitrocellulose. Slot blots: 1, probed with labeled RNA from animals expressing serotype A; 2, probed with labeled RNA from animals expressing serotype C: 3, probed with labeled RNA from animals expressing serotype H. Slots: Al, pSA2SH DNA, a subclone from the 5' end of the gene for A; Al sense, pSA2SHs DNA, sense strand of pSA2SH; Al anti-. pSA2SHa DNA. antisense strand of pSA2SH: A2. pSA1.4H DNA. an internal subclone of the gene for A: C. pSC1.35H DNA. an internal subclone of the gene for C: H. pSH5R DNA, an internal subclone of the gene for H; X, lambda DNA; pT7/T3-18 DNA. a pUC plasmid derivative; ox-tubulin, pT2c DNA, a subclone containing all of the coding region of the gene for P. tetratieliai s-tubulin except about 100 nucleotides at the 3' end. Each slot contained 500 ng of DNA. The DNA contained in the subclones is described in Fig. 1.
Under these conditions, the rate of transcription of the gene for C in H-expressing cells was 43% of its rate in Cexpressing cells (see Fig. 5). These and other results are summarized in Table 1. The results indicate that a serotype gene that is not present on the surface can be highly transcribed and that its rate of transcription varies with the cell growth rate. To characterize the in vitro transcription system further, we determined whether transcription of the gene for surface antigen A in vitro was confined to the coding regions of the gene. Plasmid pSA14SB, containing the entire gene for A (Fig. 3C), was cut with HinidIII and Es oRI. The DNA fragments were resolved on a 0.1% agarose gel and transferred to nitrocellulose. The blot was then probed with labeled RNA synthesized in vitro from animals expressing TABLE 1. Run-on transcription DNA on
In vitro-labeled run-on RNA from serotype":
nitrocellulose (serotype)
pSAl.4H (A) pSC1.35H (C) pSH5R (H)
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aL-t
*__
-8.3
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FIG. 6. Northern analysis of various mRNAs present during the switch from serotype H to serotype C. Total RNA was isolated at eight times during the switch, resolved on a 1.0% agarose-formaldehyde gel, and transferred to nylon. The nylon membrane was then probed with various labeled. gene-specific probes. Blots: 1. probed with pSC1.35H; 2. probed with pSH5R; 3. probed with pT2c (gene for OL-tubulin). kb, Kilobases.
scribed during the first several hours after the switch. Transcription of the gene for A gradually decreased, but the gene continued to be transcribed at a low rate. The relative rates of transcription of the surface antigen genes following the shift are shown in Fig. 7A. A Northern blot was prepared for the serotype H to serotype C switch to determine the rates at which various mRNA species accumulated (Fig. 6 and 7B). At each of the eight time points, total RNA was isolated and then resolved on a 1.0% denaturing formaldehyde-agarose gel, transferred to a nylon membrane, and hybridized with the indicated probes. A cloned (x-tubulin DNA probe was used to normalize the RNA amounts loaded on the gel. When the blot was hybridized with a probe specific for the gene for surface antigen C (pSC1.35H), there was a 4- to 5-h lag in the increase of the steady-state C message from time zero and then a very rapid increase of the C message to a final steady-state level. When the Northern blot was probed with a probe specific for the gene for surface antigen H (pSH5R). the H mRNA level decreased exponentially after the shift and was undetectable 18 h after the switch. The RNA blot was also hybridized with a probe specific for the gene for surface antigen A (pSA1.4H). No stable A message was detected at any time, either before or after the shift (data not shown). Surface antigen analysis. Since serotype H animals contain surface antigen C mRNA, an effort was made to determine whether they contain C protein that is unable to participate in the surface protein immobilization reaction. Surface antigens were isolated by a salt-alcohol extraction method from each population and resolved on a sodium dodecyl sulfate4.5% polyacrylamide gel (Fig. 8). Paranme(ium surface antigens are large (250 to 300 kilodaltons) and abundant polypeptides and are easily resolved on such gels. For each of the three serotypes studied, there was no evidence of another surface antigen present except the homologous type. No surface antigen C protein was detectable when animals were serotype H, despite the high levels of surface antigen C mRNA found under these conditions. These results do not rule out the presence of C protein that cannot be extracted by salt-alcohol.
0
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: 0
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40
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time after shift (hrs) FIG. 7. Relative rate of transcription (A) and relative level of RNA (B) compared with those of the homologous type for the switch from serotype H (U) to serotype C (O) (see the text for a description of the experiment). A. Serotype A. The data were obtained by scanning slot (A) or Southern (B) blots and normalizing their densities to that of ox-tubulin. A value of 1 represents the rate of transcription or the level of RNA in the pure serotype.
DISCUSSION In this study, we investigated the regulation of the genes for surface antigens A, C. and H in P. tetiraielia. Expression of surface antigens in P. tetri-aielia is coordinately controlled and mutually exclusive (11). Expression of the genes for surface antigens A and H is controlled at the transcriptional level. The gene for surface antigen A was not
L
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4
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C
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m_
a
FIG. 8. Surface antigen proteins present in various serotypes. Salt-alcohol extracts of cells were electrophoresed in a sodium dodecyl sulfate-4.5% polyacrylamide gel and stained with Coomassie brilliant blue. kd. Kilodaltons.
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transcribed in nuclear run-on experiments when animals expressed serotypes C and H, and the gene for surface antigen H was not transcribed when animals expressed serotypes A and C. Further support for a transcriptional control mechanism for the gene for H was demonstrated when animals were switched from serotype H to serotype C (Fig. 5 and 6). After the animals were switched, the level of H mRNA dropped exponentially (Fig. 7B). This rate of decrease suggests that preexisting H mRNA is eliminated by cell division after the shift rather than by alterations in the rate of its degradation. The gene for surface antigen C demonstrated both transcriptional and posttranscriptional control. These modes of regulation were dependent on the serotype expressed. When animals expressed serotype A, expression of the gene for surface antigen C was controlled at the level of transcription, as determined by nuclear run-on experiments. When animals expressed serotype H, the gene for surface antigen C was actively transcribed and stable C mRNA was present, but no surface antigen C was detected. When animals expressed serotype H, the transcription rate of the gene for surface antigen C was dependent on the growth rate. At slow growth rates, the rate of transcription of the gene for C was approximately the same as when animals expressed serotype C (Table 1). At faster growth rates, stable C mRNA was still present but its rate of transcription dropped to about one-third of the rate observed in cells expressing serotype C. These results indicate transcriptional regulation of serotype C. However, the existence of large amounts of C mRNA in H-expressing cells suggests that posttranscriptional control is the predominate form of
regulation. A posttranslational mode of negative regulation of surface antigen C cannot be ruled out by our experiments. C mRNA could be translated and C protein could be quickly degraded. Alternatively, C protein might be segregated to a cell compartment other than the surface and then transported to the surface when serotype C expression conditions occur. We used a salt-alcohol extraction procedure to extract surface antigens. This procedure may not detect surface proteins in other cell compartments. Attempts to run total cell protein on one-dimensional sodium dodecyl sulfate-polyacrylamide gels did not reveal C protein, but these gels did not provide much resolution for the wide spectrum of proteins present in P. tetrair-elia. Further experiments are required to investigate these possibilities. It should be noted that early investigators concluded that P. tetralurelia was capable of producing immobilization antigens that never reach the cell surface (for a discussion of such "secondary antigens,"' see refer-
11). During the switch from serotype H to serotype C, we observed that the gene for surface antigen A was transiently transcribed at early time points but mRNA for the gene for A did not accumulate. Multiple surface antigens might be deregulated in a similar manner after a switch until one
ence
serotype dominates. Alterations in surface antigens have been described in other protozoa. Trypanosomes evade host immune defenses by altering their surface proteins, which are known as variant surface glycoproteins. Although the nature of regulation of antigenic switching in trypanosomes is unknown, certain features appear likely to be different from serotype switching in P. teteiaiuelia. Antigenic switching in trypanosomes can be regulated by DNA rearrangements, in which genes that encode variant surface glycoproteins may be transcriptionally activated following transposition to specific
1543
telomere-proximal "expression sites" (16). Switching is believed to be independent of external stimuli. Variant surface glycoprotein mRNAs are generated by tirans-splicing of transcripts produced by an oQ-amanitin-resistant RNA polymerase. In P. tetraiurelia, the surface antigen mRNAs are not generated by trans-splicing and we have shown that the RNA polymerase involved in their production is sensitive to a-amanitin in vitro. A more closely related system is the surface antigen system of Tetrahvmena thermophila. At least five distinct serotypes exist which are expressed in a mutually exclusive manner under different environmental conditions (1). One serotype, known as H3, is produced between 20 and 35°C but not at higher temperatures. Love et al. (8) have shown that the transcription rate for this gene does not change substantially following a temperature shift but the stability of the mRNA is greatly reduced following the shift. Thus, in T. thermophila and P. tetraiirelia, surface antigen expression can be regulated at posttranscriptional levels, although the features of the regulation may be different. ACKNOWLEDGMENTS We thank Louise B. Preer and Tim Fitzwater for assistance and helpful advice. D.G. is a predoctoral trainee supported by a Public Health Service Training grant in Molecular Biology from the National Institutes of Health. This work was supported by Public Health Service grant GM 31745 from the National Institutes of Health. LITERATURE CITED 1. Bannon, G. A., R. Perkins-Dameron, and A. Allen-Nash. 1986. Structure and expression of two temperature-specific surface proteins in the ciliated protozoan Tetrahvimena thermwopliil/a. Mol. Cell. Biol. 6:3240-3245. 2. Borst, P. 1986. Discontinuous transcription and antigenic variation in trypanosomes. Annu. Rev. Biochem. 55:701-732. 3. Epstein, L. M., and J. D. Forney. 1984. Mendelian and nonMendelian mutations affecting surface antigen expression in Pair-anecii,n tetraiurelia. Mol. Cell. Biol. 4:1583-1590. 4. Forney, J. D., L. M. Epstein, L. B. Preer, B. M. Rudman, D. J. Widmayer, W. H. Klein, and J. R. Preer, Jr. 1983. Structure and expression of genes for surface proteins in Paramecium. Mol. Cell. Biol. 3:466-474. 5. Gilley, D., J. R. Preer, Jr., K. J. Aufderheide, and B. Polisky. 1988. Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei. Mol. Cell. Biol. 8:4765-4772. 6. Godiska, R. 1987. Structure and sequence of the surface protein gene of Paraimeciu,m and comparison with related genes. Mol. Gen. Genet. 208:529-536. 7. Godiska, R., K. J. Aufderheide, D. Gilley, P. Hendrie, T. Fitzwater, L. B. Preer, B. Polisky, and J. R. Preer, Jr. 1987. Transformation of Paramecium by microinjection of a cloned serotype gene. Proc. NatI. Acad. Sci. USA 84:7590-7594. 8. Love, H. D., Jr., A. Allen-Nash, Q. Zhao, and G. A. Bannon. 1988. mRNA stability plays a major role in regulating the temperature-specific expression of a Tetrah.vmnena tlhermoplhila surface protein. Mol. Cell. Biol. 8:427-432. 9. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor. N.Y. 10. Meyer, T. F., N. Malawer, and M. So. 1982. Pilus expression in Neisseria gonorrholie(ae involves chromosomal rearrangements. Cell 30:45-52. 11. Preer, J. R., Jr. 1986. Surface antigens in Paa-mneiueuimi. p. 301-339. In J. G. Gall (ed.). Molecular biology of the ciliated protozoa. Academic Press. Inc.. New York. 12. Preer, J. R., Jr., L. B. Preer, and B. M. Rudman. 1981. mRNAs for the immobilization antigens of Paramecium. Proc. Natl. Acad. Sci. USA 78:6776-6778.
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13. Soldo, A. T., and G. A. Godoy. 1972. The kinetic complexity of Paramecium macronuclear deoxyribonucleic acid. J. Protozool. 19:673-678. 14. Sonneborn, T. M. 1950. Methods in the general biology and genetics of P. aurelia. J. Exp. Zool. 113:87-143. 15. Sonneborn, T. M. 1975. Paramecium aurelia. p. 469-594. In R.
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King (ed.), Handbook of genetics, vol. 2. Plenum Publishing Corp., New York. 16. Van der Ploeg, L. H. T. 1987. Control of variant surface antigen switching in trypanosomes. Cell 51:159-161. 17. Zieg, J., M. Hilmen, and M. Simon. 1978. Regulation of gene expression by site-specific inversion. Cell 15:237-244.
MOLECULAR AND CELLULAR BIOLOGY, Apr. 1990, p. 1538-1544 0270-7306/90/041538-07$02.00/0 Copyright © 1990, American Society for Microbiology
Multilevel Regulation of Surface Antigen Gene Expression in Paramecium tetraurelia DAVID GILLEY, BERTINA M. RUDMAN, JOHN R. PREER, JR., AND BARRY POLISKY* Programii in Molecular, Cellutlar, and Dev'elopmental Biology, Department of Biology, Indiana University, Bloomington, Indiana 47405 Received 29 August 1989/Accepted 10 January 1990
A family of genes is responsible for production of surface antigenic components of Paramecium tetraurelia. These surface proteins are expressed in a mutually exclusive manner. Individuals rarely display more than one type. However, changes in environmental conditions can cause different surface proteins which replace preexisting types to be expressed. We investigated the nature of regulation of the genes for the A, C, and H surface antigens of P. tetraurelia. A system for in vitro run-on transcription was developed from crude Paramecium extracts and used in this analysis. The genes for surface antigens A and H were controlled at the level of transcription. However, the gene for surface antigen C demonstrated both transcriptional and posttranscriptional control, depending on the serotype being expressed. When animals expressed serotype A, the gene for surface antigen C was not transcribed. However, when animals expressed serotype H, the gene for surface antigen C was actively transcribed and stable surface antigen C mRNA was present in the cells, although surface antigen C was not detectable by serotype testing or by a salt-alcohol extraction method. The kinetics of transformation from serotype H to serotype C were determined by using the in vitro transcription system and monitoring steady-state RNA levels. During the transition, serotype A transcription was detected in run-on transcription experiments, although this RNA did not accumulate. The results indicate that serotype expression is controlled at several levels and that not all serotype genes are controlled in the same manner.
Surface antigen genes in Parcameciumn tetrauirelia constitute a family of distinct, unlinked loci which display mutually exclusive expression (3, 5, 7; for reviews, see references 11 and 15). In a particular individual, only a single type of surface antigen is present on the surface at any time. The surface antigens, also referred to as immobilization antigens, are large glycoproteins, from 250 to 300 kilodaltons, that make up about 50% of the protein in cilia or 3.5% of the total protein in P. tetrarairelia. Certain environmental conditions, such as the temperature and/or the composition of the culture medium, favor stable expression of a particular surface antigen. Animals that express a specific serotype are capable of switching or transforming to another serotype within 2 to 3 fissions after an environmental change. In P. tetraurelia stock 51, 11 distinct antigenic types have been found and designated serotypes A, B, C, D, E, G, H, I, J, N, and Q. By using their respective mRNAs to probe genomic libraries, several surface antigen genes have been isolated (4, 6). The mechanism of controlled expression of the surface antigen genes is not known, but it does not appear to function by chromosomal rearrangements (4), as in other systems (2, 10, 17). Changes at the DNA level have not been detected upon serotype switching, although very large or very small changes have not been ruled out. The surface antigen genes are amplified from one copy per haploid micronucleus to about 2,000 copies per macronucleus (13). The large copy number, along with the speed with which 100% of a large population can be switched to another serotype, indicates a highly regulated control mechanism. The copy number of the surface antigen genes in the macronucleus is not influenced by the expression of other serotypes. Therefore, the expression of the serotype genes is not controlled by changes in gene dosage (4). *
To investigate the regulation of the genes for surface antigens A, C, and H in P. tetrauirelia, an in vitro transcription system was developed so that nuclear run-on experiments could be performed. Two of the genes studied, those for surface antigens A and H, clearly showed transcriptional regulation, although the gene for C displayed either transcriptional or posttranscriptional control, depending on the serotype expressed. Therefore, the comprehensive control mechanism of surface antigen gene expression cannot be explained by a simple set of repressor-operator interactions acting at the transcriptional level. MATERIALS AND METHODS Paramecium. P. tetraiurelia stock 51 was the strain used in all experiments. Cultures were grown in Y medium, an infusion of 0.15% Cerophyl (Cerophyl Co., Kansas City, Mo.)-10 mg of yeast extract per ml-45 mg of Na,HPO4 per ml-0.1 mg of stigmasterol per ml. Y medium was inoculated with Klebsiella pneuinoniae (14) 24 h before use. Serotypes. Serotypes A, C, and H were maintained at 27, 19, and 13°C, respectively. Serotypes were identified as described by Sonneborn (14) with sera from the Sonneborn collection at Indiana University. In vitro nuclear run-on transcription. The following protocol was based on that of Love et al. (8). Packed cells (0.1 ml or 106 animals) were gently mixed with 0.9 ml of extraction buffer {0.1 M sucrose, 0.1 M KCI, 2.5 mM MgCl,, 2.5 mM EGTA [ethylene glycol-bis(3-aminoethyl ether)-N,N,N',N'tetraacetic acid], 10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] [pH 6.5], 1% Triton X-100} and placed on ice for 5 min. The cells were then washed twice with centrifugation for 2 min at 2,000 x g with extraction buffer lacking Triton X-100. Cytoskeletal frameworks were suspended to 1 ml in transcription buffer I (0.05 M Tris hydrochloride, 0.05 M KCI, 5 mM MgCl2, 1 mM spermidine, 1 mM spermine, 1 mM CaCl, 2 mM dithiothre-
Corresponding author. 1538
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itol, 0.1 M sucrose, 25% glycerol [pH 8.1]) and then used directly for in vitro transcription or frozen on liquid N, and stored at -70°C for later use. For in vitro transcription reactions, 50 [L1 of cytoskeletal frameworks was added to 25 ,ul of transcription buffer 11 (0.1 M Tris hydrochloride, 0.1 M KCl, 0.01 M MgCl2, 2 mM spermidine, 2 mM spermine, 4 mM putrescine, 6 mM dithiothreitol, 2 mM CaCl2, 1.2 mM aurintricarboxylic acid [pH 8.1])-2.5 ,u1 each of 20 mM ATP, CTP, and GTP-40 U of RNasin-125 ,uCi of [t_-32P]UTP and incubated at 25°C for 40 min. This mixture was extracted with phenol and then chloroform-isoamyl alcohol (24:1, vol/vol). Unincorporated counts were removed by passage over a G50 spin column. Hybridization conditions. Slot and Southern blots were hybridized at 42°C in 5x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-50 mM NaH2PO4 (pH 7.4)-50% formamide-0.5% sodium dodecyl sulfate-3 x Denhardt solution (9)-10% dextran sulfate (average molecular weight, 500,000)-denatured salmon sperm DNA at 200 ,ug/ml. Filters were washed twice with 2 x SSC-0. 1% sodium dodecyl sulfate at room temperature for 10 min and then washed twice with 0.1 x SSC-0. 1x sodium dodecyl sulfate at 68°C for 20 min. RNA isolation. RNA was prepared by lysing P. tetraurelia cells in guanidine hydrochloride and purifying it as previously described (12). Northern (RNA) blots. Total RNA was electrophoresed through 1% agarose-formaldehyde gels and transferred to Nytran nylon membranes (Schleicher & Schuell, Inc., Keene, N.H.) as previously described (9), except that RNA was UV cross-linked as recommended by the manufacturer. Protein analysis. Crude protein extracts were prepared by salt-alcohol extraction and electrophoresed as previously described (12). RESULTS Maps of the genes for surface antigens A, C, and H. Three genes for surface antigens A, C, and H in P. tetraiurelia stock 51 have been characterized previously at the molecular level (4, 6). Restriction maps of the macronuclear versions of these genes are shown in Fig. 1, along with the specifically identified subclones that were used in this study. In vitro nuclear run-on experiments. To determine the level of regulation of the three surface antigen genes, nuclear run-on experiments were performed. Cytoskeletal frameworks were made as described in Materials and Methods from populations of animals expressing each serotype. These cytoskeletal frameworks consist of detergent-treated cells with permeabilized cell membranes containing intact macronuclei. Microscopic inspection of the frameworks showed that most of the cytoplasmic contents had been removed. The frameworks were capable of incorporation of labeled ribonucleoside triphosphates into acid-insoluble material. The frameworks were used to elongate nascent RNA chains in vitro in the presence of [a-32P]UTP as described in Materials and Methods. Certain features of the in vitro system were investigated. Trichloroacetic acid-precipitable counts incorporated into the in vitro transcription reaction were sensitive to RNase A treatment (data not shown). The Paramecium RNA polymerase activity responsible for expression of the serotype genes within cytoskeleton frameworks (as shown below) was completely inhibited by addition of 10 ,ug of oa-amanitin per ml. This concentration of a-amanitin had no inhibitory effect on RNA polymerase I transcription of rRNA in this system (data not shown). We
A gere
A
S
H
S
H
pSAi.4H
H H
H H
H
H
H
H
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H
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O
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X
H
PSA2SH
C gene H
H
C pSC1 35H
H genie
H
a,Xi R1
,
Al pSHSR
,i
RI khb
FIG. 1. Partial restriction maps of the genes for surface antigens A, C, and H and the subclones used. The thick lines and arrows indicate the transcribed region of the gene for each surface antigen. The subclone(s) used is indicated below each map. All subclones were cloned into pUC8 (Bethesda Research Laboratories, Inc., Gaithersburg, Md.). Rl, EcoRI; H, Hindlll; X, Xhol; S, Sall. kb, Kilobase.
conclude that the RNA polymerase is a type II enzyme typically involved in transcription of eucaryotic genes. We used the in vitro-synthesized labeled RNA as a probe for hybridization to various cloned DNAs immobilized on nitrocellulose (Fig. 2). When this slot blot was probed with labeled RNA made by cytoskeletal frameworks from animals expressing serotype A (Fig. 2, slot blot 1), only the subclones specific for the gene for surface antigen A and the ot-tubulin clone hybridized to in vitro-synthesized RNA. Note that the single-stranded DNA making up the antisense strand of the gene for A (Al anti-) hybridized to the labeled RNA, but the single-stranded DNA making up the sense strand of the gene for A (Al sense) did not hybridize. Therefore, animals expressing serotype A do not transcribe the genes for the other two serotypes, C and H. Also, the cytoskeletal framework transcription system displays proper strand specificity, at least in the region of the gene for surface antigen A probed. The hybridized molecules were stripped from the slot blot and reprobed with labeled RNA synthesized in vitro by cytoskeletal frameworks from animals expressing serotype C (Fig. 2, slot blot 2). Only the subclones specific for the gene for surface antigen C and the gene for a-tubulin showed hybridization. Therefore, animals expressing the gene for serotype C do not transcribe the gene for serotype A or H. When the blot was reprobed with labeled RNA synthesized in vitro by cytoskeletal frameworks from animals expressing serotype H (Fig. 2, slot blot 3), the subclones specific for the genes for surface antigen C and a-tubulin hybridized. These results indicate that when animals express serotype H, the gene for H is transcribed as expected, but in addition, the gene for surface antigen C is rapidly transcribed. Surprisingly, the rate of transcription of the gene for C in H-expressing cells (Fig. 2, slot blot 3) normalized to a-tubulin was estimated to be 1.2 times the rate of transcription of the gene for C in C-expressing cells (Fig. 2, slot blot 2). The H-expressing cells used in this experiment were essentially in the stationary growth phase, dividing once every 14 days, while the C-expressing cells divided once per 2 days. Consequently, we examined the relative transcription rates of serotype genes in cells dividing every 48 h.
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1
2
3
A
C
H
Al
Al sense
0
Al anti-
A2
-
C
P.
4
n ..
a
_
pT7Ir3-18
ac-tubufn
-
FIG. 2. DNA slot blot analysis. In vitro-synthesized. labeled RNA from animals expressing serotypes A. C. and H was used to probe various cloned DNAs immobilized on nitrocellulose. Slot blots: 1, probed with labeled RNA from animals expressing serotype A; 2, probed with labeled RNA from animals expressing serotype C: 3, probed with labeled RNA from animals expressing serotype H. Slots: Al, pSA2SH DNA, a subclone from the 5' end of the gene for A; Al sense, pSA2SHs DNA, sense strand of pSA2SH; Al anti-. pSA2SHa DNA. antisense strand of pSA2SH: A2. pSA1.4H DNA. an internal subclone of the gene for A: C. pSC1.35H DNA. an internal subclone of the gene for C: H. pSH5R DNA, an internal subclone of the gene for H; X, lambda DNA; pT7/T3-18 DNA. a pUC plasmid derivative; ox-tubulin, pT2c DNA, a subclone containing all of the coding region of the gene for P. tetratieliai s-tubulin except about 100 nucleotides at the 3' end. Each slot contained 500 ng of DNA. The DNA contained in the subclones is described in Fig. 1.
Under these conditions, the rate of transcription of the gene for C in H-expressing cells was 43% of its rate in Cexpressing cells (see Fig. 5). These and other results are summarized in Table 1. The results indicate that a serotype gene that is not present on the surface can be highly transcribed and that its rate of transcription varies with the cell growth rate. To characterize the in vitro transcription system further, we determined whether transcription of the gene for surface antigen A in vitro was confined to the coding regions of the gene. Plasmid pSA14SB, containing the entire gene for A (Fig. 3C), was cut with HinidIII and Es oRI. The DNA fragments were resolved on a 0.1% agarose gel and transferred to nitrocellulose. The blot was then probed with labeled RNA synthesized in vitro from animals expressing TABLE 1. Run-on transcription DNA on
In vitro-labeled run-on RNA from serotype":
nitrocellulose (serotype)
pSAl.4H (A) pSC1.35H (C) pSH5R (H)
A
C
H
1
1.0 x 10-2 1
C
2
H
3
aL-t
*__
-8.3
L51*
- 2.0
B
FIG. 6. Northern analysis of various mRNAs present during the switch from serotype H to serotype C. Total RNA was isolated at eight times during the switch, resolved on a 1.0% agarose-formaldehyde gel, and transferred to nylon. The nylon membrane was then probed with various labeled. gene-specific probes. Blots: 1. probed with pSC1.35H; 2. probed with pSH5R; 3. probed with pT2c (gene for OL-tubulin). kb, Kilobases.
scribed during the first several hours after the switch. Transcription of the gene for A gradually decreased, but the gene continued to be transcribed at a low rate. The relative rates of transcription of the surface antigen genes following the shift are shown in Fig. 7A. A Northern blot was prepared for the serotype H to serotype C switch to determine the rates at which various mRNA species accumulated (Fig. 6 and 7B). At each of the eight time points, total RNA was isolated and then resolved on a 1.0% denaturing formaldehyde-agarose gel, transferred to a nylon membrane, and hybridized with the indicated probes. A cloned (x-tubulin DNA probe was used to normalize the RNA amounts loaded on the gel. When the blot was hybridized with a probe specific for the gene for surface antigen C (pSC1.35H), there was a 4- to 5-h lag in the increase of the steady-state C message from time zero and then a very rapid increase of the C message to a final steady-state level. When the Northern blot was probed with a probe specific for the gene for surface antigen H (pSH5R). the H mRNA level decreased exponentially after the shift and was undetectable 18 h after the switch. The RNA blot was also hybridized with a probe specific for the gene for surface antigen A (pSA1.4H). No stable A message was detected at any time, either before or after the shift (data not shown). Surface antigen analysis. Since serotype H animals contain surface antigen C mRNA, an effort was made to determine whether they contain C protein that is unable to participate in the surface protein immobilization reaction. Surface antigens were isolated by a salt-alcohol extraction method from each population and resolved on a sodium dodecyl sulfate4.5% polyacrylamide gel (Fig. 8). Paranme(ium surface antigens are large (250 to 300 kilodaltons) and abundant polypeptides and are easily resolved on such gels. For each of the three serotypes studied, there was no evidence of another surface antigen present except the homologous type. No surface antigen C protein was detectable when animals were serotype H, despite the high levels of surface antigen C mRNA found under these conditions. These results do not rule out the presence of C protein that cannot be extracted by salt-alcohol.
0
0
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: 0
20
0
40
60
80
100
120
time after shift (hrs) FIG. 7. Relative rate of transcription (A) and relative level of RNA (B) compared with those of the homologous type for the switch from serotype H (U) to serotype C (O) (see the text for a description of the experiment). A. Serotype A. The data were obtained by scanning slot (A) or Southern (B) blots and normalizing their densities to that of ox-tubulin. A value of 1 represents the rate of transcription or the level of RNA in the pure serotype.
DISCUSSION In this study, we investigated the regulation of the genes for surface antigens A, C. and H in P. tetiraielia. Expression of surface antigens in P. tetri-aielia is coordinately controlled and mutually exclusive (11). Expression of the genes for surface antigens A and H is controlled at the transcriptional level. The gene for surface antigen A was not
L
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1
2
3
4
A
C
H
C
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a
FIG. 8. Surface antigen proteins present in various serotypes. Salt-alcohol extracts of cells were electrophoresed in a sodium dodecyl sulfate-4.5% polyacrylamide gel and stained with Coomassie brilliant blue. kd. Kilodaltons.
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transcribed in nuclear run-on experiments when animals expressed serotypes C and H, and the gene for surface antigen H was not transcribed when animals expressed serotypes A and C. Further support for a transcriptional control mechanism for the gene for H was demonstrated when animals were switched from serotype H to serotype C (Fig. 5 and 6). After the animals were switched, the level of H mRNA dropped exponentially (Fig. 7B). This rate of decrease suggests that preexisting H mRNA is eliminated by cell division after the shift rather than by alterations in the rate of its degradation. The gene for surface antigen C demonstrated both transcriptional and posttranscriptional control. These modes of regulation were dependent on the serotype expressed. When animals expressed serotype A, expression of the gene for surface antigen C was controlled at the level of transcription, as determined by nuclear run-on experiments. When animals expressed serotype H, the gene for surface antigen C was actively transcribed and stable C mRNA was present, but no surface antigen C was detected. When animals expressed serotype H, the transcription rate of the gene for surface antigen C was dependent on the growth rate. At slow growth rates, the rate of transcription of the gene for C was approximately the same as when animals expressed serotype C (Table 1). At faster growth rates, stable C mRNA was still present but its rate of transcription dropped to about one-third of the rate observed in cells expressing serotype C. These results indicate transcriptional regulation of serotype C. However, the existence of large amounts of C mRNA in H-expressing cells suggests that posttranscriptional control is the predominate form of
regulation. A posttranslational mode of negative regulation of surface antigen C cannot be ruled out by our experiments. C mRNA could be translated and C protein could be quickly degraded. Alternatively, C protein might be segregated to a cell compartment other than the surface and then transported to the surface when serotype C expression conditions occur. We used a salt-alcohol extraction procedure to extract surface antigens. This procedure may not detect surface proteins in other cell compartments. Attempts to run total cell protein on one-dimensional sodium dodecyl sulfate-polyacrylamide gels did not reveal C protein, but these gels did not provide much resolution for the wide spectrum of proteins present in P. tetrair-elia. Further experiments are required to investigate these possibilities. It should be noted that early investigators concluded that P. tetralurelia was capable of producing immobilization antigens that never reach the cell surface (for a discussion of such "secondary antigens,"' see refer-
11). During the switch from serotype H to serotype C, we observed that the gene for surface antigen A was transiently transcribed at early time points but mRNA for the gene for A did not accumulate. Multiple surface antigens might be deregulated in a similar manner after a switch until one
ence
serotype dominates. Alterations in surface antigens have been described in other protozoa. Trypanosomes evade host immune defenses by altering their surface proteins, which are known as variant surface glycoproteins. Although the nature of regulation of antigenic switching in trypanosomes is unknown, certain features appear likely to be different from serotype switching in P. teteiaiuelia. Antigenic switching in trypanosomes can be regulated by DNA rearrangements, in which genes that encode variant surface glycoproteins may be transcriptionally activated following transposition to specific
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telomere-proximal "expression sites" (16). Switching is believed to be independent of external stimuli. Variant surface glycoprotein mRNAs are generated by tirans-splicing of transcripts produced by an oQ-amanitin-resistant RNA polymerase. In P. tetraiurelia, the surface antigen mRNAs are not generated by trans-splicing and we have shown that the RNA polymerase involved in their production is sensitive to a-amanitin in vitro. A more closely related system is the surface antigen system of Tetrahvmena thermophila. At least five distinct serotypes exist which are expressed in a mutually exclusive manner under different environmental conditions (1). One serotype, known as H3, is produced between 20 and 35°C but not at higher temperatures. Love et al. (8) have shown that the transcription rate for this gene does not change substantially following a temperature shift but the stability of the mRNA is greatly reduced following the shift. Thus, in T. thermophila and P. tetraiirelia, surface antigen expression can be regulated at posttranscriptional levels, although the features of the regulation may be different. ACKNOWLEDGMENTS We thank Louise B. Preer and Tim Fitzwater for assistance and helpful advice. D.G. is a predoctoral trainee supported by a Public Health Service Training grant in Molecular Biology from the National Institutes of Health. This work was supported by Public Health Service grant GM 31745 from the National Institutes of Health. LITERATURE CITED 1. Bannon, G. A., R. Perkins-Dameron, and A. Allen-Nash. 1986. Structure and expression of two temperature-specific surface proteins in the ciliated protozoan Tetrahvimena thermwopliil/a. Mol. Cell. Biol. 6:3240-3245. 2. Borst, P. 1986. Discontinuous transcription and antigenic variation in trypanosomes. Annu. Rev. Biochem. 55:701-732. 3. Epstein, L. M., and J. D. Forney. 1984. Mendelian and nonMendelian mutations affecting surface antigen expression in Pair-anecii,n tetraiurelia. Mol. Cell. Biol. 4:1583-1590. 4. Forney, J. D., L. M. Epstein, L. B. Preer, B. M. Rudman, D. J. Widmayer, W. H. Klein, and J. R. Preer, Jr. 1983. Structure and expression of genes for surface proteins in Paramecium. Mol. Cell. Biol. 3:466-474. 5. Gilley, D., J. R. Preer, Jr., K. J. Aufderheide, and B. Polisky. 1988. Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei. Mol. Cell. Biol. 8:4765-4772. 6. Godiska, R. 1987. Structure and sequence of the surface protein gene of Paraimeciu,m and comparison with related genes. Mol. Gen. Genet. 208:529-536. 7. Godiska, R., K. J. Aufderheide, D. Gilley, P. Hendrie, T. Fitzwater, L. B. Preer, B. Polisky, and J. R. Preer, Jr. 1987. Transformation of Paramecium by microinjection of a cloned serotype gene. Proc. NatI. Acad. Sci. USA 84:7590-7594. 8. Love, H. D., Jr., A. Allen-Nash, Q. Zhao, and G. A. Bannon. 1988. mRNA stability plays a major role in regulating the temperature-specific expression of a Tetrah.vmnena tlhermoplhila surface protein. Mol. Cell. Biol. 8:427-432. 9. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor. N.Y. 10. Meyer, T. F., N. Malawer, and M. So. 1982. Pilus expression in Neisseria gonorrholie(ae involves chromosomal rearrangements. Cell 30:45-52. 11. Preer, J. R., Jr. 1986. Surface antigens in Paa-mneiueuimi. p. 301-339. In J. G. Gall (ed.). Molecular biology of the ciliated protozoa. Academic Press. Inc.. New York. 12. Preer, J. R., Jr., L. B. Preer, and B. M. Rudman. 1981. mRNAs for the immobilization antigens of Paramecium. Proc. Natl. Acad. Sci. USA 78:6776-6778.
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13. Soldo, A. T., and G. A. Godoy. 1972. The kinetic complexity of Paramecium macronuclear deoxyribonucleic acid. J. Protozool. 19:673-678. 14. Sonneborn, T. M. 1950. Methods in the general biology and genetics of P. aurelia. J. Exp. Zool. 113:87-143. 15. Sonneborn, T. M. 1975. Paramecium aurelia. p. 469-594. In R.
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King (ed.), Handbook of genetics, vol. 2. Plenum Publishing Corp., New York. 16. Van der Ploeg, L. H. T. 1987. Control of variant surface antigen switching in trypanosomes. Cell 51:159-161. 17. Zieg, J., M. Hilmen, and M. Simon. 1978. Regulation of gene expression by site-specific inversion. Cell 15:237-244.
