NONBEHAVIORAL SELECTION FOR PAWNS, MUTANTS O F PARAMECIUM AURELZA WITH DECREASED EXCITABILITY STANLEY J. SCHEIN Department of Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461 Manuscript received March 1, 1976 Revised copy received June 10, 1976 ABSTRACT
The reversal response in Paramecium aurelia is mediated by calcium which carries the inward current during excitation. Electrophysiological studies indicate that strontium and barium can also carry the inward current. Exposure to high concentrations of barium rapidly paralyzes and later kills wild-type paramecia. Following mutagenesis with nitrosoguanidine, seven mutants which continued to swim in the ‘high-barium’ solution were selected. All of the mutants show decreased reversal behavior, with phenotypes ranging from extremely non-reversing (‘extreme’ pawns) to nearly wild-type reversal behavior (‘partial‘ pawns). The mutations fall into three complementation groups, identical to the pwA, pwB, and pwC genes of KUNCet al. (1975). All of the p w A and pwB mutants withstand longer exposure to barium, the pwB mutants surviving longer than the p w A mutants. Among mutants of each gene, survival is correlated with loss of reversal behavior. Double mutants (A-B, A-C, B-C), identified in the exautogamous progeny of crosses between ‘partial’ mutants, exhibited a more extreme non-reversing phenotype than either of their singlemutant (‘partial’ pawn) parents.---Inability to reverse could be expected from an alteration in the calcium-activated reversal mechanism or in excitation. A normal calcium-activated structure was demonstrated in all pawns by chlorpromazine treatment. In a separate report (SCHEIN,BENNETTand KATZ 1976) the results of electrophysiological investigations directly demonstrate decreased excitability in all of the mutants, a decrease due to an altered calcium activation. The studies of the genetics, the survival in barium and the electrophysiology of the pawns demonstrate that the p w A and pwB genes have different effects on calcium activation.
T H E use of genetics to dissect a complex physiological process is very much in the tradition of modem genetics. I n the very least, the number of genes directly affecting the process may be counted and a clearer idea of its true complexity obtained. The mutants may direct further physiological dissection, and at best, may even assist in molecular characterization. Paramecium aurelia is well suited for the application of genetic techniques to the study of electrical excitability, a phenomenon characterized by HODGKIN and HUXLEY (1952) in terms of voltage-dependent ion conductances. P. aurelia has a convenient genetic system for the generation and analysis of mutants (SONNEBORN 1975a), and it is large enough for microelectrode insertion and electrophysiological characteriGenetics 84: 453-468 November, 1976
454
S. J. SCHEIN
zation (NAITOHand ECKERT 1968a). Also, it may be grown in pure culture in large quantities. I n an attempt to apply genetic techniques to the study of excitability, KUNG (19714 selected mutants of Paramecium aurelia, mutants deficient in their LC reversal response”. Previously, NAITOH( 1969), using glycerol-extracted paramecia and later, NAITOHand KANEKO(1972), using Triton X-100, had established that ciliary reversal was a response to increased calcium concentration. NAITOHand ECKERT(1968a, 1968b) had suggested that the rapid depolarization during excitation was due to a voltage-dependent increase in the membrane’s conductance to calcium. Evidence has since been presented (MACHEMER and ECKERT 1973) to show that the calcium which enters (through the calcium conductance) during excitation is also responsible for ciliary reversal. KUNG(1971b) selected mutants with defective-reversal response by picking individuals that could still swim up (paramecia are negatively geotactic) when exposed to an ionic environment which elicits frequent reversals and makes directed upward swimming impossible in the wild type. Mutants which could not reverse, which he called pawns, had indeed become inexcitable (KUNGand ECKERT 1972). In the present experiments mutants with altered excitability have been selected on the basis of the known electrophysiological properties of the calcium conductance. I n the biological preparations in which calcium is found to carry the inward ionic current during excitation, barium and strontium are also capable of entering (presumably through the same channel) and may even do so with greater ease than calcium. This result has been found in many electrophysiological preparations including Paramecium caudatum (NAITOH and ECKERT 1968b). Barium might be expected to have toxic properties and, as described in RESULTS, appropriate solutions can paralyze wild-type paramecia and later kill them. (Note, however, that the mechanism of neither the paralyzing nor the toxic effects of barium is known.) Mutants resistant to these effects of barium have been isolated. All mutants isolated have behavioral defects; however, in addition to mutants which have no reversal behavior remaining (“extreme” pawns), phenotypes intermediate between extreme pawn and wild-type (“partial” pawns) have been found. The mutations have been characterized genetically and shown to fall into three complementation groups, identical to pwA, pwB and pwC of KUNGet al. (1975). Also, pwB mutants show longer survival in barium than pwA mutants, which in turn, survive longer than the wild type. An effect of the mutations on the calcium-activated reversal structure has been eliminated by the ability of all the mutants to perform reversed swimming upon exposure to detergent concentrations of chlorpromazine. Finally, a direct electrophysiological measurement of excitability, the subject of a separate report (SCHEIN,BENNETT and KATZ1976), shows decreased excitability due to decreased inward calcium current (calcium activation) in all of the mutants. The studies of the genetics, the survival in barium and the electrophysiology of the pawns allow speculation which assigns to the pwA gene a role in “gating” and to the pwB gene a role affecting either the pore or the total number of channels.
PAWN MUTANTS O F PARAMECIUM
455
MATERIALS A N D METHODS
Cell strains. All strains were derived from a culture of wild type, syngen 4 (now called 1975)), stock 51s (non-kappa bearing), from C. KUNG. The P . tetraaurelicz (SONNENBORN PO (resistance to polyenes, in particular, amphotericin B) marker will be described elsewhere. Three pawns, p w A { 9 4 ) , pwB(95), and pwC(l31) were provided by KUNGfor the purpose of determining whether our mutations were in the same genes. Growth of cells. Growth medium was prepared as follows: Cerophyl (rye) grass powder (Ceropbyl Labxatories, Inc., Kansas City, MO. 64112) (5 gm/liter), NaH,PO, (5 mM), Na,HPO, (5 mM) and 15 liters of deionized water were autoclaved for 90 minutes. The mixture was allowed to settle for several days and then passed through cheesecloth and, by suction filtration, through Whatman #54 filter paper. The liquid was autoclaved again (60 minutes) and stored at 4”.From several hours to a day before inoculation with paramecia, sterile medium was ‘bacterized with Enterobacter aerogenes. This medium supports growth of P . aurelia to a density of between 5,000 and 10,00O/ml. All strains are kept in 2 ml tube cultures at room temperature and transferred once each week. Further details on the care and feeding of paramecia can be (1947, 1950, 1957, 1970), BEALE(1953) and HANSON (1974). found in SONNEBORN Mating. Mating was accomplished using the methods described in the above SONNEBORN references. In addition, the presumed ‘FI’ cultures were generally characterized phenotypically in order to (a) learn about complementation and the dominant or recessive nature of mutations, or ‘b) io dcmonstrate the hybrid character of the ex-conjugant progeny. The latter was necessary because not all visually observed matings result in exchange of genetic material. Following starvation and autogamy, sometimes a sample (0.1 ml) from the starved culture was placed into freshly bacterized 2 ml tubes. Usually, individual cells were cloned into either 2 ml tube cultures or the wells of a 96-well plate (Falcon Microtest I1 Plates, #3040, and Lids, #3041), depending on the requirements of subsequent characterizations. When the F2 had cleared the medium of bacteria, they were characterized phenotypically in order to learn whether complementing mutations were linked, or to demonstrate the expected close linkage between two mutations which did not complement. Autogamy- was usually assured by growing the F1 through at least 25 divisions prior to starvation; autogamy was ascertained by observing the expression of recessive markers in the F2. The marker mutations:
PO,
sp, bd
All three marker mutations are recessive. Amphotericin B resistance ( P O for polyene resistance). To 0.5 ml of cells in stationary phase in medium cleared of bacteria are added 50 pl of amphotericin B (250 pg/ml) . Amphotericin B is obtained as a liquid, Fungizone (Grand Island Biological Company, Long Island, New York), in which deoxycholate is present to solubilize the drug. Wild-type cells are all dead by ten minutes; cells in cultures that are marked with the amphotericin B resistance ( P O ) mutation continue to swim for more than 24 hours, although ten minutes is the usual checkpoint. Spinner ( s p ) . A mutant that swims forward normally but spins in place during ‘reversal’ (instead of swimming backward) was isolated in the first mutagenesis (described below), labeled spinner (sp) and used as a marker. The reversal response of the sp mutant is identical in frequency and duration to the wild type; excitability as measured electrophysiologically is also identical (SCHEIN,unpublished results). The presence of the sp trait may be determined only if cells can be made to exhibit reversal behavior. Non-pawns and partial pawns may be placed in Solution-S (see below-Reversal Behavior) and vigorous reversed swimming or spinning i n place can be observed. Extreme pawns must be treated with chlorpromazine (see below) to induce reversal behavior and permit s p characierization. Spinner has also been reported by KUNGet al. 1975). Body deformity ( b d ) . The pwA(94) and pwB(95) mutants obtained from KUNGwere marked with the body deformity mutation. In a culture of bd cells, many sickle-shaped cells are found; other less simply described morphological abnormalities are also seen. Mutagenesis. Four mutageneses with a one-hour treatment of 75, 50, 75 and 100 Fg/ml nitrosoguanidine (NTG) wei‘e done and the mutants which resulted labeled as the 100, 200, 300, and
456
S. J. SCHEIN
400 series. Mutagenesis followed the method reported by KUNG (1971a). Ex-autogamous death ranged from 30-60%. Reversal Behavior. Cells were tested within 24 hours after the medium had been cleared. Cells were transformed in a micropipette from the cleared growth medium to the test solutions (S and P-see below). Their reversal response was observed under a stereomicroscope. Under these conditions the cells receive both a chemical stimulus (NAITOH1968) and a short mechanical stimulus. Dryl’s solution (DRYL1959) (1 mM NaH,PO,, 1 mM Na,HPO,, 2 m M Na-citrate, 1.5 m M CaC1,) induces frequent reversals in the wild type. Test solution-S (for Stimulating), Dryl’s solution with the addition of BaC1, to 2 mM, is even more effective in causing reversal behavior. Ionic test: Barium paralysis. Within 15 seconds of transfer to Test solution-P (for Paralyzing: 1 m M Na, HPO,, 1 mM NaH,PO,, 2 m M Na-citrate, 0.1 mM CaCl,, 10 m M BaCl,, 16 m M NaCl), wild-type cells are paralyzed. Barium killing. These experiments used two protocols. The first used 15 ml of cells in growth medium (whose pH was adjusted to 6.8 with HC1) and then addition of BaC1, to 7.5 mM. The number of motile cells in samples was counted at each time-point. A motile cell is defined in these experiments as a cell which shows any motion. When ‘motile’ cells were cloned into medium, normal healthy cultures resulted from 83% of the clones; cloning non-motile cells into fresh medium did not revive any of them. Assay of the number of motile cells is therefore approximately equivalent to assay for survival. The second protocol involved a 4-hour adaptation of cells in 15 ml “.33-Dryl” (1 m M Na,HPO,, Im M NaH,PO,, 2 mM Na-citrate, .33 m M CaC1,) followed by addition of BaCl to 1 mM. At each time-point, a sample of each tube was removed and the number of motile paramecia counted. Chlorpromazine reuersal. 50 pl of a stock chlorpromazine (Thorazine) solution (4 mM) was added to .45 ml of a solution containing 1 m M NaH,PO,, 1 mM Na,HPO,, 2 m M Na-citrate, 0.1 mM or 1 mM CaCl,. Several paramecia were then transferred from growth medium t o the 400 pM chlorpromazine solution, and their swimming behavior was observed. RESULTS
The initial selection for cells which could not reverse swimming direction used chemotactic (20 mM NaC1,0.3 mM CaCl,, 1 m M Tris-HC1, p H 7.2) interference with galvanotaxis (paramecia swim to the anode (JAHN 1961) ) , a modification of KUNG’Schemotactic interference with geotaxis (KUNG1971a). One pawn mutant was isolated from the initial selection and labeled 100.
Selection of mutants by barium paralysis Further study revealed that, whereas wild-type cells are reproducibly paralyzed within 15 seconds following transfer to Solution-P, the swimming of the 100-pawn was unaffected. Cells from the second mutagenesis were therefore selected o n the basis of the resistance to the paralyzing effect of barium. Cells were concentrated in growth medium to 2 X 104/ml, 1 ml of 100 m M BaC1, was added to 9 ml of cells, the mixture vortexed for five seconds, spread in a trough, and any cells still swimming between 30 and 120 seconds later were picked out and cloned. Seven mutants, 202, 214, 314, 320, 325, 414, and 419 were isolated. After correction for ex-autogamous death etc., it is estimated that the seven new mutants came from 4 x105 exautogamous lines. 325 was slow growing and could not be mated, so it was discarded. 202 was lost at a later date in the study.
45 7
PAWN MUTANTS O F PARAMECIUM
Reversal behavior Table 1, part A describes the reversal behavior of strains upon exposure to Dryl's solution and Test Solution-S, a modification of Dryl's solution (BaCL is added to 2 mM) to make it more stimulating. The mutants are grouped according to gene, data for which classification will be presented below. The phenotypes of the pwA and pwB mutants span a wide range of behavior, from reversal behavior almost indistinguishable from wild type (pwB(314)) to completely absent reversal behavior. The pwC(380) mutant is not distinguished from the wild type in reversal behavior. Response to the paralyzing effect of barium Table 1, part A also describes the response of cells to Test Solution-P, a solution high in barium (10 mM) and low in calcium (0.1 mM) , a solution which paralyzed wild-type cells within from ten to fifteen seconds. In this case, the pwC(320) mutant is readily distinguished from wild type, as it continues swimming, albeit slowly, for over ten minutes. The extremely defective nature of the reversal response in pwB(100) is demonstrated by its complete lack of response; TABLE 1 Characterization of mutants ~
~
~
~
~
~~
~
~~~~~~
~~
~
~
Response t o test solutions
Strain
-___-
Pawn Phenotype
Dry1
A. wild type
wild type
>
RR 2' ( 1/sec 1
IR-45"; RRR 2' (4/sec)
NR
pwB(100) pwB(314)
extreme partial
NR RR-90"
pwA(Z14)
"extreme"
NR
pwA(414) pwA(419) pwC(320)
extreme extreme partial
NR NR RR
extreme extreme extreme extreme extreme
NR NR NR NR NR
B pwA(214)-pwB(314) pwA(414)-pwB(314) pwA(94)-p~B(314) p~A(214)-p~C(320) pwB(314)-p~C(320)
____-
So1n:S
> 2'
Explanation of symbols: RR-t: Repeating reversals for or greater than time t IR-t: Initial reversal, lasting time t RRR-t: Rapidly repeating reversals for or greater than time t P: Paralysis NR: No reversal or no response (S)WS: (Slow) wide spiral forward swimming
>
IR-45"; RRR 2' Hesitate; NR NR NR IR-45"; RRR 2'
>
>
NR NR NR NR NR
Soh.-P
IR-10";
P NR IR-6";
sws > IO' IR-5";
ws > IO' Hesitate-2" Hesitate-2" IR-2"; sws IO'
>
NR NR NR
ws-I
NR
Pi
202-sp 202-sp 214 214-sp 100-sp 100-po 320-sp
P’
214 119-po 414-s~ pwA(94)-bd 314.~0 pwB(Y5)-bd pwC(131)
extreme extreme extreme extreme partial extreme partials
Pawn phenotype
-
-
rp
-
60/0 75/0 69/05
48/0 54/0 72/0
53/0
pw/not pw
37/32
33/27
26/27 20/28 31/23 37/35
sp/not s p
PO
27/33 not done
24/24
po/not
34/41
35/37
bd/not bd
1 pw/not pw phenotype was characterized using S o h - P . Table 1 describes the response of the various pawns; 314 and 320 are partial pawns; the rest are extreme pawns. sp/not s p phenotype was characterized using So1n.S for the wild-type and partial pawns, chlorpromazine for extreme pawns. 3 po/not po phenotype was characterized as described in METHODS. 4 bd (body deformity) is characterized simply by looking at the cells. 5 Like 320, at room temperature, the “temperature-sensitive” pwC(131) is not paralyzed in So1n.-P.
pwC
pwB
pwA
Crosses between mutants of the same gene
TABLE 2A
E
$
4
?
320-s~
PWB X PWC 314-pO wild-type
wild-type wild-type wild-type
wild-type wild-type wild-type wild-type wild-type wild-type -
-
-
-
-
-
-
PO
F11,2,3,4
-
-
sp
-
-
-
bd
Crosses between mutants in different genes
Pawn phenotype
~~~~
24/89 44./1610 48/1 311 37/1412 36/21Q
46/ 14 36/12 46/11 63/220 27/gU
pw/not pw
F9,1.2,3,4
notdone 36/24 3 1 /30 not done 18/39
35/25 18/30 27/30 19/167 22/13
sp/not sp
21/36
17/18
27/33 27/21 23/34
po/not p a
26/25
16/16
bd/not bd
6 7
It was possible to phenotypically classify the 69 pawns as 26 214’s, 23 314’s, and 20 double-mutants (214-314). Only the 314 and non-pawn F2’s were classified with respect to the spinner trait. Of the 27 pawns, 9 were believed to be double-mutants (314-414). 3 Several likely double-mutants were chosen for genetic characterization. 1 0 No attempt was made to systematically sort 214-320 from 214. 11 It was difficult to identify which of the extreme pawns (32 extreme, 16 partial) were double-mutants; four were chosen for genetic characterization; three of the four were double-mutants. 12 14 of the 37 pawns were designated double-mutants (pwA(94)-320).
320-SP 320 320-SP
1 00-sp
P?.
O W A X P W C214 414-sp pwA(94)-bd
Cross
100-sp 100-sp 314 314-p0 S I 4-sp
P1
D W AX ~ w B214-SP 414-sp 419-po 214-sp 414-sp pwA(94)-bd
~~~~~~~~~~~~~~~~~
TABLE 2B
z
2 z
D
E
$
>
cd
r
0
2
Z
2
z
’ 2
460
S. J. SCHEIN
indications of slight residual responsiveness are also demonstrated in several of the mutants which were unresponsive in Dryl’s solution and Test Solution-S (pwA(414) for example). Rapid phenotypic separation of pawns from wild type is made possible by the tests described in Table 1. I n some cases, different pawn strains may be distinguished from each other. The ability to detect residual reversal behavior in most of the pawns allowed the identification in many cases of double mutants with even less reversal behavior than either of their complementing parents (Table 1,part B) . Complementation and linkage analysis The pawn and marker mutations are due to single gene, recessive mutations, (SCHEIN1976a). In all cases the F1 heterozygotes are phenotypically wild type, and the F2 segregation pattern shows no linkage between the markers and the pawn mutations. Table 2 lists crosses between (usually marked) pawns. Part 2a lists crosses between mutants of the same gene. Noncomplementation is shown between the 202, 214,414,419, and pwA(94) mutants, between the 100, 314 and pwB(95) mutants, and between the 320 and pwC(131) mutants. Part 2b lists crosses between mutants in different genes. Complementation and nonlinkage is shown between p w A and pwB mutants, between p w A and pwC mutants, and between pwB and pwC mutants. In some of the crosses involving complementing mutations the double-mutant F2 progeny, which should have been 25% of the total, could be identified by their greater behavioral defect than either of the single mutated parents. Table 1, part B describes the behavior of double mutants. Identification of the double mutants was not difficult in crosses where both parents had significant reversal behavior, examples being pwA(214) X pwB(314) and pwB(314) X pwC(320). Double mutants involving the “extreme” p w A (414) allele, such as pwA(414)pwC(320), were difficult to identify, but phenotypic characterization was still helpful. Table 3 lists the crosses which confirmed the double-mutant genotype of pwA(214)-pwB(314), pwB(314)-pwC(320), pwA(94)-pwB(314) and identified as well as confirmed the double-mutant genotype of pwA(414)-pwB(314) and pwA(414)-pwC(320). In all cases the F2 progeny were followed, giving the expected independent segregation of the pawn genes and the markers. The relative resistance to killing by barium
A semi-logarithmic plot of survival in barium (7.5 mM in growth medium) for each mutant strain is shown in Figure 1, parts a through g. All of the lines are plotted together in Figure l h and demonstrate several points. Firstly, the killing curve for the pwC(320) mutant, which cannot be distinguished from the wild type by observation of reversal behavior, is also indistinguishable from the wild type.
2
1
extreme partial extreme partial extreme partial extreme partial partial partial
Pawn phenotype
-
-
-
-
-
-
-
-
-
-
-
-
-
bd
-
PO
-
-
-
sp
44/0 24/0 75/0 not done2 51/0 52/0
87/0
63/0
75/0 78/0
pw/not p w
21/23 10/14 32/43 not done2 27/24 28/24
35/40 not done 33/30
s p / n o t sp
F21
not done 31/21
12/12
po/not p o
46/41
33/45
bd/not bd
Methods for Phenotypic characterization were identical to Table 2. Because the F1 differs phenotypically from both parents, proof of the proposed genotype did not require characterization of the F2.
p w A ( 2 1 4 ) - p w B ( 3 1 4 ) - ~pwA(419) ~ pwB(95)-bd pwA(94)-pwB(314) pwA(214)-sp pwB(95)-bd pwA(414) - p ~ B ( 3 1 4-SP ) pw.4 (214) p~B(100)-~0 pwA(414)-pwC(320) p~A(214)-~p pwC(320) -sp p ~ B ( 3 1 4 ) - p w C ( 3 2 0 ) - ~pWB(1OO)-Sp 0 pwC(320)-sp
Proposed genotype X Tester strain
F1'
Test crosses to prove double mutant genotypes
TABLE 3
z
H
8
5
v > 5 >
8
v)
3
>
z
2
B
A
I
\ m
\.
L 1
-
.
H
t
2
3 HUURS
9
5
FIGURE 1.-Each of figures l a through I g shows the results of three separate experiments. To cells in 15 m l of growth medium (pH adjusted to 6.8 with HCI) was added BaCl, to 7.5 mM. The number of motile cells/ml (ordinate) were counted a t each time p i n t (abscissa), The lines were computed to give a least-squares fit, and are all plotted together in Figure Ih. Motile cells are equivalent to living cells, tiommotile cells to dead cells.
PAWN’ MUTANTS O F PARAMECIUM
463
Secondly, p w A mutants demonstrate greater resistance to barium than the wild type, whose numbers are reduced 10-fold in 75 minutes. The corresponding h e for the extreme p w A mutants (414 and 419) is 150 minutes. Also, the p w A ( 2 1 4 ) mutant, which is not so extreme behaviorally and which has about 5% of the wild-type active calcium conductance (SCHEIN,BENNETT and KATZ1976), has a killing curve intermediate between the extreme pwA’s and the wild type. Thirdly, the extreme pwB(100) mutant shows the greatest resistance of all, four hours being required to get a 10% survival. The surprise of the study was the killing curve of the pwB(314), which displays considerable reversal behavior and has 25% of the wild-type active calcium conductance; its survival is like the extreme p w A mutants. T o confirm the high resistance of pwB(314) to barium, the study was repeated with a solution of defined composition (modified Dryl’s salt solution) brought to 1 mM BaC1,. The results of two experiments with p w A ( 2 1 4 ) , pwB(314) along with one experiment with the wild type, plotted in Figure 2a through 2c and summarized in Figure 2d, are entirely consistent with the conclusion from the first set of experiments: a pwB mutant is more resistant to the killing effect of barium than a p w A mutant with a similar or even greater behavioral defect.
FIGURE 2.-Figure 2a shows the result of one experiment; Figures 2b and 2c show the results of two separate experiments. To cells adapted in 1 mM Na,HPO,, ImM NaH,PO,, 2 mM Na-citrate, 0.33 CaC1, for 4 hour%BaCl, was added to ImM. The number of motile cells (ordinate) were counted at each time point (abscissa). The lines were computed to give a leastsquares fit, and all are plotted together in Figure 2d.
464
S. J. SCHEIN
Nondefective calcium-activated reversing structure I n the course of screening drugs for the generation of drug resistance marker mutations, the following peculiar response of the wild type to 400 pM chlorpromazine was observed. After an initial reversal, the normal response to transfer from growth medium to the salt-solution, the individual swims forward for about ten seconds, slows down to a stop, then swims backward for about three seconds before slowing down, settling to the floor of the chamber, and dying. With the omission of the initial reversal, the extreme pawn mutants respond in an identical fashion. Reversal behavior has been demonstrated in all of the pawns, showing that the calcium-sensitive structure responsible for reversal of ciliary beat is not affected by the mutation. The chlorpromazine test is rapid and was used routinely to identify the presence or absence of the spinner in extreme pawns. With chlorpromazine treatment, pawns without the spinner trait swim backward, pawns with the spinner trait spin in place. All of the sp identifications on extreme mutants in Tables 2 and 3 were done using chlorpromazine. Many of the strains so classified have since been tested genetically and no errors in sp assignment have been discovered. DISCUSSION
Isolation of pawn mutants by resistance to barium solutions KUNGand his associates have isolated over fifty unconditional pawn mutants by behavioral selection: chemotactic inhibition of geotaxis (KUNG1971a; CHANG et al. 1974). All mutations affected two unlinked genes, p w A and pwB. I n addition, of the five temperature-sensitive pawns which have been isolated (CHANG and KUNG1973), four were pwA mutants. The fifth demonstrated the existence of a third unlinked pwC gene. The mutants have been studied behaviorally (KUNG1971b; CHANGet al. 1974) and electrophysiologically (KUNGand ECKERT 1972; SATOWand KUNG1974). Study of pwB(d4-95) shows nearly 1972). Finally, a normal calcomplete loss of excitability (KUNGand ECKERT cium-sensitive reversal mechanism has been demonstrated in the mutants by reversed swimming after Triton X-100 treatment (KUNGand NAITOH1973). The first mutant (and the most extreme pawn phenotype) in the present study was isolated using chemotactic inhibition of galvanotaxis, a modification of KUNG’Sbehavioral selection. One property of this mutant-resistance to barium -suggested that an “ionic selection”, based on the ion-specific properties of the calcium channel, might focus specifically on mutations in excitability involving alterations in the calcium channel. Therefore the second set of (seven) mutants was isolated using a selection based on resistance to paralysis by barium. The behavioral analysis of the mutants showed that a significant fraction of the pawns are “partial” pawns. The genetic analysis showed that mutations of the same three genes as KUNG’S pwA, pwB and pwC were isolated. The selection yielded the second partial mutant of the pwB gene ( 3 1 4 ) discovered to date, and the only pwC mutant (320) selected on the basis of its properties at room temperature. The usefulness of partial mutants will be discussed below.
PAWN MUTANTS O F PARAMECIUM
465
Induction of reversal behauior b y chlorpromazine Chlorpromazine induces several seconds of reversed swimming before cells die. This result is similar to the induction of reversal following Triton X-100 treatment (NAITOHand KANEKO 1972). The mechanism of action of both chemicals is probably based on detergency. Ions, calcium in particular, are thus allowed to bypass the normal membrane permeability barriers and enter the cell, causing ciliary reversal. Evidence for chlorpromazine’s ability to increase membrane permeability has been found in another ciliate, Tetrahymena pyriformis (NATHAN and FRIEDMAN 1972). Chlorpromazine induction of reversal has been used in the present report to demonstrate a normal calcium-sensitive reversal mechanism in extreme pawns. It has also been used extensively in genetic characterization, to determine whether the extreme pawns also have spirmer trait. A phenotypic difference between pwA and pwB The survival of p w A and pwB mutants in barium solutions was prolonged over that of the wild type and, as might have been expected, among mutants of each gene, the greater the reduction in reversal behavior, the longer the survival. However, the resistance of pwB mutants appeared greater than that of p w A mutants. These killing curves are the first evidence that it is possible to phenotypically distinguish p w A from pwB mutants: For a given degree of loss of reversal response, pwB mutants are much more resistant to the toxic effects of barium than pwA mutants. In addition, barium killing represents a new ‘‘ionic selection” method which should specifically enrich the yield of pwB’s and, in particular, of the elusive partial pwB’s. The selection would also be useful for isolating conditional lethals. T h e ualue of partial pawns Partial pawns have already proven their value in several ways. Electrophysiological measurements directly confirmed the decreased active calcium conductance in the mutants. The partial pawns were useful in that a simple correlation between degree of loss of reversal behavior and the decrease in active calcium current was possible, as was observed (SCHEINet al. 1976). Further electrophysiological characterization of the alteration in calcium activation is possible only if some calcium activation is still present, as with partial pawns. In crosses between complementing pawns it was easy to distinguish the doublymutated F2 progeny from their singly-mutated parental-type and wild-type sibs. Proof of the double-mutant genotype of those so chosen is given in Table 3. An electrophysiollogical study of the double mutants, included in SCHEINet al. (1976) indicates that the defect in excitability is also greater in double mutants then in their parents. Voltage clamping will be required before further characterization of the interaction between the pawn genes is possible. The frequency of isolation of partial pawns is surprising, many partial pwA’s having been found, only two partial pwB’s. I n CHANGet al. (1974), of 22 pwA mutants, 5 were leaky 1, 13 were leaky 2, (pwA(214) would fit here), 2 were
466
S. J. SCHEIN
leaky 3 (pwA(202) would have fit here) and 2 were leaky 4 (equivalent to m y term “partial pawn”). None of the 14 pwB’s were at all leaky. Of the five temperature-sensitive pawns isolated (CHANGand KUNG1973) four were pwA, one was pwC, and pwB was conspicuous by its absence. The curious distribution of partial mutants is consistent with the model described below.
A model SCHEIN,BENNETT and KATZ(1976) found a second method for distinguishing pwB from pwA mutants: I n addition to the effect of the pawn mutations on calcium activation, a second electrical property of the membrane of P . aurelia, anomalous rectification, was reduced in pwB mutants. The reduction was also proportional to the reduction in active calcium conductance and the degree of behavioral deficit. This observation suggested to us that the pwB gene product is the “pore”, while the pwA gene product performs or affects the depolarizationsensitive function of the channel (the “gate”). While the model explains the electrophysiological data to date and is admittedly speculative, it provides a tentative explanation for the intriguing disparity between the frequencies of isolation of partial mutants in the pwA and pwB genes. Mutations in the “gate” molecule would result in alterations of voltage sensitivity or time course of the conductance change, alterations which would span a wide range of severity. One of perhaps several gating charges could be modified, or the fraction of the transmembrane potential which is traversed when the gating charges move could be decreased. Both sorts of mutations would change voltage sensitivity to varying degrees. The stiffness of the gate “hinge” might be increased; the speed of motion of the gating charges might be decreased. Both alterations would slow down the time course of the Conductance change. Indeed, many partial o r “leaky” mutants have been isolated in the putative “gate” gene, pwA. However, the normal ‘‘pore’’ exhibits ion selectivity, admitting the divalent ions Ca++, Sr++ and Ba++ but excluding Mg++, Mn++, monovalent cations and all anions. Investigations into the ion-selective properties of the sodium channel and the channel-blocking agents like tetrodotoxin have placed severe restraints on the size of the pore ( 3 x 5A) (HILLE1971). Likewise, the selectivity of the calcium channel presumably requires a structure of carefully tailored dimensions. Even small modifications of the molecule, changing the pore dimensions by perhaps a single Angstrolm, would be enough to completely close the pore. Thus, extreme mutants would be the rule, partial mutants the exception, as is the case in the putative ‘Lpore”gene, pwB. The differential killing by barium is consistent with the model. The calcium channels of a mutant with altered gates (pwA) would still be expected to open, though less frequently, and admit some barium. Regardless of the state of the gate (open or closed), the calcium channels of a mutant with a closed pore (pwB) would never admit barium. Finally, the process of autogamy has allowed measurement of the gradual loss of excitability which occurs when heterozygous (wild type/pawn) paramecia
PAWN MUTANTS O F PARAMECIUM
46 7
with wild-type excitability are “suddenly” changed to homozygous pawns with normal channels remaining in the membrane. These measurements have shown that the lifetime of the molecular target of the pwA and pwB genes is identical and remarkably long, between 5.5 and 8 days (SCHEIN1976b). These results are entirely consistent with the notion that the pwA and pwB genes affect a common structure, the calcium channel. The model may also be tested, since it predicts that voltage sensitivity and time course has been altered in pwA mutants, maximum active calcium conductance and perhaps ion selectivity in the pwB mutants. Electrophysiological studies utilizing voltage-clamp methods should allow measurement of these parameters and comparison with the wild type. I am especially grateful to DR. MEL COHNof the Salk Institute and DR. J. T. AUGUSTat the Albert Einstein College of Medicine for their support and encouragement in the early phases of DAVIDand this work. I am also deeply appreciative of the kindness shown me by DRS.CHARLES M. V. L. BENNETT,who have given generously of their time and advice. S. SCHEINwas supported by grants from the National Institutes of Health, GM11301 and 5T5GM1674. LITERATURE CITED
BEALE,G. H., 1954 The Genetics of Paramecium aurelia. Cambridge University Press, London. L. J. ROBLES,S. S. LUI and C. KUNG,1974 An extensive beCHANG,S.-Y., J. v . 4 ~HOUTEN, havioural and genetic analysis of the pawn mutants in Paramecium aurelia. Genet. Res. Camb. 23: 165-173. CHANG,S.-Y. and C. KUNG, 1973 Genetic analysis of heat-sensitive pawn mutants of Paramecium aurelia. Genetics 75: 49-59.
DRYI,,S., 1959 Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autogamy and conjugation. J. Protozool. 6:196. HANSON, E. D., 1974 Methods in the cellular and molecular biology of paramecium. pp. 319363. In: Methods in Cell Physiology, Vol. 8. Edited by D. M. PRESCOTT. Academic Press, New York.
HILLE, B.,1971 The permeability of the sodium channel to organic cations in myeoinated nerve. J. Gen. Physiol. 58: 599-619. HODGKIN, A. L. and A. P.HUXLEY, 1952 -4 quantitative description of membrane current and its application to conduction and excitation i n nerve. J. Physiol. 117: 500-544. JAHN,T. L., 1961 The mechanism of ciliary movement. I. Ciliary reversal and activation by electric curfent; the Ludloff phenomenon in terms of core and volume conductors. J. Protozool. 8: 369-380. KUNG,C., 1971a Genic mutants with altered system of excitation in Paramecium aurelia. 11. Mutagenesis, screening and genetic analysis of the mutants. Genetics 49: 29-45. -, 1971b Genic mutants with altered system of excitation i n Paramecium aurelia. I. Phenotypes of behavioral mutants. Z. vergl. Physiol. 71: 142-164, KUNG,C. and R. ECKERT,1972 Genetic modification of electric properties in an excitable membrane. Proc. Nat. Acad. Sci. U.S.A. 69:93-97. KUNG, C. and Y. NAITOH,1973 Calcium-induced ciliary reversal in the extracted models of “Pawn”, a behavioral mutant of paramecium. Science 179: 195-196. KUNG,C., S.-Y. CHANG,Y. SATOW, J. VAN HOUTEN and H. HANSMA, 1975 Genetic dissection of behavior in Paramecium. Science 188: 898-901..
468
S . J. SCHEIN
MACHEMER, H. and ECKERT,1973 Electrophysiological control of reversed ciliary beating in Paramecium. J. Gen. Physiol. 61: 572-587. NAITOH,Y., 1969 Control of the orientation of cilia by ATP, calcium and zinc in glycerolextracted Paramecium aurelh. J. Gen. Physiol. 53 :517-529. NAITOH,Y. and R. ECKERT,1968a Electrical properties of Paramecium caudatum: modification by bound and free cations. Z. vergl. Physiol. 61 : 497-452. -, 19681, Electrical properties of Paramecium caudatum: all-or none electrogenesis. Z. vergl. Physiol. 61 : 453-472. NAITOH,Y. and H. KANEKO,1972 ATP-reactivated Triton-extracted models of Paramecium: modification of ciliary movement by calcium ions. Science 176: 523-524.
H. A. and W. FRIEDMAN, 1962 Chlorpromazine affects permeability of resting cells of NATHAN, Tetrahymena pyriformis. Science 135: 793-794. SATOW, Y. and C. KUNG,1974 Genetic dissection of active electrogenesis in Paramecium aurelia. Nature 247: 69-71. SCHEIN,S. J., 1976a Electrical excitability in Paramecium aurelia. Ph.D. Thesis, Albert Einstein 1976b Calcium channel stability measured by gradual loss of College of Medicine. -, excitability in pawn mutants of Paramecium aurelia. J. Exp. Biol. (In press). SCHEIN,S. J., M. V. L. BENNETTand G. KATZ,1976 Altered calcium conductance in pawns, behavioral mutants oE Paramecium aurelia. J. Exp. Biol. (In press).
T. M., 1947 Recent advances in the genetics of Paramecium and Euplotes. Adv. SONNEBORN, Genet. 1: 263-358. -, 1950 Methods in the general biology and genetics of Para1957 Breeding systems, reproductive mecium aurelia. J. Exp. Zool. 113: 87-148. --, methods, and species problems i n Protozoa. pp. 155-324. In: The Species Problem. Edited by E. MAYR.AAAS, Washington, D.C. --, 1970 Methods i n Paramecium Research. pp. 241-339. In: Methods in Cell Physiology, Vol. 4. Edited by D. M. PRESCOTT. Academic Press, New York. --, 1975a Paramecium aurelia, pp. 469-594. In: Handbook of Genetics, Vol. 2. Edited by R. KING. Plenum Press, New York. -, 197513 The Paramecium aurelia complex of Fourteen Sibling Species. Trans. Amer. Micros. SOC.94: 155-178. Corresponding editor: S. ALLEN
The reversal response in Paramecium aurelia is mediated by calcium which carries the inward current during excitation. Electrophysiological studies indicate that strontium and barium can also carry the inward current. Exposure to high concentrations of barium rapidly paralyzes and later kills wild-type paramecia. Following mutagenesis with nitrosoguanidine, seven mutants which continued to swim in the ‘high-barium’ solution were selected. All of the mutants show decreased reversal behavior, with phenotypes ranging from extremely non-reversing (‘extreme’ pawns) to nearly wild-type reversal behavior (‘partial‘ pawns). The mutations fall into three complementation groups, identical to the pwA, pwB, and pwC genes of KUNCet al. (1975). All of the p w A and pwB mutants withstand longer exposure to barium, the pwB mutants surviving longer than the p w A mutants. Among mutants of each gene, survival is correlated with loss of reversal behavior. Double mutants (A-B, A-C, B-C), identified in the exautogamous progeny of crosses between ‘partial’ mutants, exhibited a more extreme non-reversing phenotype than either of their singlemutant (‘partial’ pawn) parents.---Inability to reverse could be expected from an alteration in the calcium-activated reversal mechanism or in excitation. A normal calcium-activated structure was demonstrated in all pawns by chlorpromazine treatment. In a separate report (SCHEIN,BENNETTand KATZ 1976) the results of electrophysiological investigations directly demonstrate decreased excitability in all of the mutants, a decrease due to an altered calcium activation. The studies of the genetics, the survival in barium and the electrophysiology of the pawns demonstrate that the p w A and pwB genes have different effects on calcium activation.
T H E use of genetics to dissect a complex physiological process is very much in the tradition of modem genetics. I n the very least, the number of genes directly affecting the process may be counted and a clearer idea of its true complexity obtained. The mutants may direct further physiological dissection, and at best, may even assist in molecular characterization. Paramecium aurelia is well suited for the application of genetic techniques to the study of electrical excitability, a phenomenon characterized by HODGKIN and HUXLEY (1952) in terms of voltage-dependent ion conductances. P. aurelia has a convenient genetic system for the generation and analysis of mutants (SONNEBORN 1975a), and it is large enough for microelectrode insertion and electrophysiological characteriGenetics 84: 453-468 November, 1976
454
S. J. SCHEIN
zation (NAITOHand ECKERT 1968a). Also, it may be grown in pure culture in large quantities. I n an attempt to apply genetic techniques to the study of excitability, KUNG (19714 selected mutants of Paramecium aurelia, mutants deficient in their LC reversal response”. Previously, NAITOH( 1969), using glycerol-extracted paramecia and later, NAITOHand KANEKO(1972), using Triton X-100, had established that ciliary reversal was a response to increased calcium concentration. NAITOHand ECKERT(1968a, 1968b) had suggested that the rapid depolarization during excitation was due to a voltage-dependent increase in the membrane’s conductance to calcium. Evidence has since been presented (MACHEMER and ECKERT 1973) to show that the calcium which enters (through the calcium conductance) during excitation is also responsible for ciliary reversal. KUNG(1971b) selected mutants with defective-reversal response by picking individuals that could still swim up (paramecia are negatively geotactic) when exposed to an ionic environment which elicits frequent reversals and makes directed upward swimming impossible in the wild type. Mutants which could not reverse, which he called pawns, had indeed become inexcitable (KUNGand ECKERT 1972). In the present experiments mutants with altered excitability have been selected on the basis of the known electrophysiological properties of the calcium conductance. I n the biological preparations in which calcium is found to carry the inward ionic current during excitation, barium and strontium are also capable of entering (presumably through the same channel) and may even do so with greater ease than calcium. This result has been found in many electrophysiological preparations including Paramecium caudatum (NAITOH and ECKERT 1968b). Barium might be expected to have toxic properties and, as described in RESULTS, appropriate solutions can paralyze wild-type paramecia and later kill them. (Note, however, that the mechanism of neither the paralyzing nor the toxic effects of barium is known.) Mutants resistant to these effects of barium have been isolated. All mutants isolated have behavioral defects; however, in addition to mutants which have no reversal behavior remaining (“extreme” pawns), phenotypes intermediate between extreme pawn and wild-type (“partial” pawns) have been found. The mutations have been characterized genetically and shown to fall into three complementation groups, identical to pwA, pwB and pwC of KUNGet al. (1975). Also, pwB mutants show longer survival in barium than pwA mutants, which in turn, survive longer than the wild type. An effect of the mutations on the calcium-activated reversal structure has been eliminated by the ability of all the mutants to perform reversed swimming upon exposure to detergent concentrations of chlorpromazine. Finally, a direct electrophysiological measurement of excitability, the subject of a separate report (SCHEIN,BENNETT and KATZ1976), shows decreased excitability due to decreased inward calcium current (calcium activation) in all of the mutants. The studies of the genetics, the survival in barium and the electrophysiology of the pawns allow speculation which assigns to the pwA gene a role in “gating” and to the pwB gene a role affecting either the pore or the total number of channels.
PAWN MUTANTS O F PARAMECIUM
455
MATERIALS A N D METHODS
Cell strains. All strains were derived from a culture of wild type, syngen 4 (now called 1975)), stock 51s (non-kappa bearing), from C. KUNG. The P . tetraaurelicz (SONNENBORN PO (resistance to polyenes, in particular, amphotericin B) marker will be described elsewhere. Three pawns, p w A { 9 4 ) , pwB(95), and pwC(l31) were provided by KUNGfor the purpose of determining whether our mutations were in the same genes. Growth of cells. Growth medium was prepared as follows: Cerophyl (rye) grass powder (Ceropbyl Labxatories, Inc., Kansas City, MO. 64112) (5 gm/liter), NaH,PO, (5 mM), Na,HPO, (5 mM) and 15 liters of deionized water were autoclaved for 90 minutes. The mixture was allowed to settle for several days and then passed through cheesecloth and, by suction filtration, through Whatman #54 filter paper. The liquid was autoclaved again (60 minutes) and stored at 4”.From several hours to a day before inoculation with paramecia, sterile medium was ‘bacterized with Enterobacter aerogenes. This medium supports growth of P . aurelia to a density of between 5,000 and 10,00O/ml. All strains are kept in 2 ml tube cultures at room temperature and transferred once each week. Further details on the care and feeding of paramecia can be (1947, 1950, 1957, 1970), BEALE(1953) and HANSON (1974). found in SONNEBORN Mating. Mating was accomplished using the methods described in the above SONNEBORN references. In addition, the presumed ‘FI’ cultures were generally characterized phenotypically in order to (a) learn about complementation and the dominant or recessive nature of mutations, or ‘b) io dcmonstrate the hybrid character of the ex-conjugant progeny. The latter was necessary because not all visually observed matings result in exchange of genetic material. Following starvation and autogamy, sometimes a sample (0.1 ml) from the starved culture was placed into freshly bacterized 2 ml tubes. Usually, individual cells were cloned into either 2 ml tube cultures or the wells of a 96-well plate (Falcon Microtest I1 Plates, #3040, and Lids, #3041), depending on the requirements of subsequent characterizations. When the F2 had cleared the medium of bacteria, they were characterized phenotypically in order to learn whether complementing mutations were linked, or to demonstrate the expected close linkage between two mutations which did not complement. Autogamy- was usually assured by growing the F1 through at least 25 divisions prior to starvation; autogamy was ascertained by observing the expression of recessive markers in the F2. The marker mutations:
PO,
sp, bd
All three marker mutations are recessive. Amphotericin B resistance ( P O for polyene resistance). To 0.5 ml of cells in stationary phase in medium cleared of bacteria are added 50 pl of amphotericin B (250 pg/ml) . Amphotericin B is obtained as a liquid, Fungizone (Grand Island Biological Company, Long Island, New York), in which deoxycholate is present to solubilize the drug. Wild-type cells are all dead by ten minutes; cells in cultures that are marked with the amphotericin B resistance ( P O ) mutation continue to swim for more than 24 hours, although ten minutes is the usual checkpoint. Spinner ( s p ) . A mutant that swims forward normally but spins in place during ‘reversal’ (instead of swimming backward) was isolated in the first mutagenesis (described below), labeled spinner (sp) and used as a marker. The reversal response of the sp mutant is identical in frequency and duration to the wild type; excitability as measured electrophysiologically is also identical (SCHEIN,unpublished results). The presence of the sp trait may be determined only if cells can be made to exhibit reversal behavior. Non-pawns and partial pawns may be placed in Solution-S (see below-Reversal Behavior) and vigorous reversed swimming or spinning i n place can be observed. Extreme pawns must be treated with chlorpromazine (see below) to induce reversal behavior and permit s p characierization. Spinner has also been reported by KUNGet al. 1975). Body deformity ( b d ) . The pwA(94) and pwB(95) mutants obtained from KUNGwere marked with the body deformity mutation. In a culture of bd cells, many sickle-shaped cells are found; other less simply described morphological abnormalities are also seen. Mutagenesis. Four mutageneses with a one-hour treatment of 75, 50, 75 and 100 Fg/ml nitrosoguanidine (NTG) wei‘e done and the mutants which resulted labeled as the 100, 200, 300, and
456
S. J. SCHEIN
400 series. Mutagenesis followed the method reported by KUNG (1971a). Ex-autogamous death ranged from 30-60%. Reversal Behavior. Cells were tested within 24 hours after the medium had been cleared. Cells were transformed in a micropipette from the cleared growth medium to the test solutions (S and P-see below). Their reversal response was observed under a stereomicroscope. Under these conditions the cells receive both a chemical stimulus (NAITOH1968) and a short mechanical stimulus. Dryl’s solution (DRYL1959) (1 mM NaH,PO,, 1 mM Na,HPO,, 2 m M Na-citrate, 1.5 m M CaC1,) induces frequent reversals in the wild type. Test solution-S (for Stimulating), Dryl’s solution with the addition of BaC1, to 2 mM, is even more effective in causing reversal behavior. Ionic test: Barium paralysis. Within 15 seconds of transfer to Test solution-P (for Paralyzing: 1 m M Na, HPO,, 1 mM NaH,PO,, 2 m M Na-citrate, 0.1 mM CaCl,, 10 m M BaCl,, 16 m M NaCl), wild-type cells are paralyzed. Barium killing. These experiments used two protocols. The first used 15 ml of cells in growth medium (whose pH was adjusted to 6.8 with HC1) and then addition of BaC1, to 7.5 mM. The number of motile cells in samples was counted at each time-point. A motile cell is defined in these experiments as a cell which shows any motion. When ‘motile’ cells were cloned into medium, normal healthy cultures resulted from 83% of the clones; cloning non-motile cells into fresh medium did not revive any of them. Assay of the number of motile cells is therefore approximately equivalent to assay for survival. The second protocol involved a 4-hour adaptation of cells in 15 ml “.33-Dryl” (1 m M Na,HPO,, Im M NaH,PO,, 2 mM Na-citrate, .33 m M CaC1,) followed by addition of BaCl to 1 mM. At each time-point, a sample of each tube was removed and the number of motile paramecia counted. Chlorpromazine reuersal. 50 pl of a stock chlorpromazine (Thorazine) solution (4 mM) was added to .45 ml of a solution containing 1 m M NaH,PO,, 1 mM Na,HPO,, 2 m M Na-citrate, 0.1 mM or 1 mM CaCl,. Several paramecia were then transferred from growth medium t o the 400 pM chlorpromazine solution, and their swimming behavior was observed. RESULTS
The initial selection for cells which could not reverse swimming direction used chemotactic (20 mM NaC1,0.3 mM CaCl,, 1 m M Tris-HC1, p H 7.2) interference with galvanotaxis (paramecia swim to the anode (JAHN 1961) ) , a modification of KUNG’Schemotactic interference with geotaxis (KUNG1971a). One pawn mutant was isolated from the initial selection and labeled 100.
Selection of mutants by barium paralysis Further study revealed that, whereas wild-type cells are reproducibly paralyzed within 15 seconds following transfer to Solution-P, the swimming of the 100-pawn was unaffected. Cells from the second mutagenesis were therefore selected o n the basis of the resistance to the paralyzing effect of barium. Cells were concentrated in growth medium to 2 X 104/ml, 1 ml of 100 m M BaC1, was added to 9 ml of cells, the mixture vortexed for five seconds, spread in a trough, and any cells still swimming between 30 and 120 seconds later were picked out and cloned. Seven mutants, 202, 214, 314, 320, 325, 414, and 419 were isolated. After correction for ex-autogamous death etc., it is estimated that the seven new mutants came from 4 x105 exautogamous lines. 325 was slow growing and could not be mated, so it was discarded. 202 was lost at a later date in the study.
45 7
PAWN MUTANTS O F PARAMECIUM
Reversal behavior Table 1, part A describes the reversal behavior of strains upon exposure to Dryl's solution and Test Solution-S, a modification of Dryl's solution (BaCL is added to 2 mM) to make it more stimulating. The mutants are grouped according to gene, data for which classification will be presented below. The phenotypes of the pwA and pwB mutants span a wide range of behavior, from reversal behavior almost indistinguishable from wild type (pwB(314)) to completely absent reversal behavior. The pwC(380) mutant is not distinguished from the wild type in reversal behavior. Response to the paralyzing effect of barium Table 1, part A also describes the response of cells to Test Solution-P, a solution high in barium (10 mM) and low in calcium (0.1 mM) , a solution which paralyzed wild-type cells within from ten to fifteen seconds. In this case, the pwC(320) mutant is readily distinguished from wild type, as it continues swimming, albeit slowly, for over ten minutes. The extremely defective nature of the reversal response in pwB(100) is demonstrated by its complete lack of response; TABLE 1 Characterization of mutants ~
~
~
~
~
~~
~
~~~~~~
~~
~
~
Response t o test solutions
Strain
-___-
Pawn Phenotype
Dry1
A. wild type
wild type
>
RR 2' ( 1/sec 1
IR-45"; RRR 2' (4/sec)
NR
pwB(100) pwB(314)
extreme partial
NR RR-90"
pwA(Z14)
"extreme"
NR
pwA(414) pwA(419) pwC(320)
extreme extreme partial
NR NR RR
extreme extreme extreme extreme extreme
NR NR NR NR NR
B pwA(214)-pwB(314) pwA(414)-pwB(314) pwA(94)-p~B(314) p~A(214)-p~C(320) pwB(314)-p~C(320)
____-
So1n:S
> 2'
Explanation of symbols: RR-t: Repeating reversals for or greater than time t IR-t: Initial reversal, lasting time t RRR-t: Rapidly repeating reversals for or greater than time t P: Paralysis NR: No reversal or no response (S)WS: (Slow) wide spiral forward swimming
>
IR-45"; RRR 2' Hesitate; NR NR NR IR-45"; RRR 2'
>
>
NR NR NR NR NR
Soh.-P
IR-10";
P NR IR-6";
sws > IO' IR-5";
ws > IO' Hesitate-2" Hesitate-2" IR-2"; sws IO'
>
NR NR NR
ws-I
NR
Pi
202-sp 202-sp 214 214-sp 100-sp 100-po 320-sp
P’
214 119-po 414-s~ pwA(94)-bd 314.~0 pwB(Y5)-bd pwC(131)
extreme extreme extreme extreme partial extreme partials
Pawn phenotype
-
-
rp
-
60/0 75/0 69/05
48/0 54/0 72/0
53/0
pw/not pw
37/32
33/27
26/27 20/28 31/23 37/35
sp/not s p
PO
27/33 not done
24/24
po/not
34/41
35/37
bd/not bd
1 pw/not pw phenotype was characterized using S o h - P . Table 1 describes the response of the various pawns; 314 and 320 are partial pawns; the rest are extreme pawns. sp/not s p phenotype was characterized using So1n.S for the wild-type and partial pawns, chlorpromazine for extreme pawns. 3 po/not po phenotype was characterized as described in METHODS. 4 bd (body deformity) is characterized simply by looking at the cells. 5 Like 320, at room temperature, the “temperature-sensitive” pwC(131) is not paralyzed in So1n.-P.
pwC
pwB
pwA
Crosses between mutants of the same gene
TABLE 2A
E
$
4
?
320-s~
PWB X PWC 314-pO wild-type
wild-type wild-type wild-type
wild-type wild-type wild-type wild-type wild-type wild-type -
-
-
-
-
-
-
PO
F11,2,3,4
-
-
sp
-
-
-
bd
Crosses between mutants in different genes
Pawn phenotype
~~~~
24/89 44./1610 48/1 311 37/1412 36/21Q
46/ 14 36/12 46/11 63/220 27/gU
pw/not pw
F9,1.2,3,4
notdone 36/24 3 1 /30 not done 18/39
35/25 18/30 27/30 19/167 22/13
sp/not sp
21/36
17/18
27/33 27/21 23/34
po/not p a
26/25
16/16
bd/not bd
6 7
It was possible to phenotypically classify the 69 pawns as 26 214’s, 23 314’s, and 20 double-mutants (214-314). Only the 314 and non-pawn F2’s were classified with respect to the spinner trait. Of the 27 pawns, 9 were believed to be double-mutants (314-414). 3 Several likely double-mutants were chosen for genetic characterization. 1 0 No attempt was made to systematically sort 214-320 from 214. 11 It was difficult to identify which of the extreme pawns (32 extreme, 16 partial) were double-mutants; four were chosen for genetic characterization; three of the four were double-mutants. 12 14 of the 37 pawns were designated double-mutants (pwA(94)-320).
320-SP 320 320-SP
1 00-sp
P?.
O W A X P W C214 414-sp pwA(94)-bd
Cross
100-sp 100-sp 314 314-p0 S I 4-sp
P1
D W AX ~ w B214-SP 414-sp 419-po 214-sp 414-sp pwA(94)-bd
~~~~~~~~~~~~~~~~~
TABLE 2B
z
2 z
D
E
$
>
cd
r
0
2
Z
2
z
’ 2
460
S. J. SCHEIN
indications of slight residual responsiveness are also demonstrated in several of the mutants which were unresponsive in Dryl’s solution and Test Solution-S (pwA(414) for example). Rapid phenotypic separation of pawns from wild type is made possible by the tests described in Table 1. I n some cases, different pawn strains may be distinguished from each other. The ability to detect residual reversal behavior in most of the pawns allowed the identification in many cases of double mutants with even less reversal behavior than either of their complementing parents (Table 1,part B) . Complementation and linkage analysis The pawn and marker mutations are due to single gene, recessive mutations, (SCHEIN1976a). In all cases the F1 heterozygotes are phenotypically wild type, and the F2 segregation pattern shows no linkage between the markers and the pawn mutations. Table 2 lists crosses between (usually marked) pawns. Part 2a lists crosses between mutants of the same gene. Noncomplementation is shown between the 202, 214,414,419, and pwA(94) mutants, between the 100, 314 and pwB(95) mutants, and between the 320 and pwC(131) mutants. Part 2b lists crosses between mutants in different genes. Complementation and nonlinkage is shown between p w A and pwB mutants, between p w A and pwC mutants, and between pwB and pwC mutants. In some of the crosses involving complementing mutations the double-mutant F2 progeny, which should have been 25% of the total, could be identified by their greater behavioral defect than either of the single mutated parents. Table 1, part B describes the behavior of double mutants. Identification of the double mutants was not difficult in crosses where both parents had significant reversal behavior, examples being pwA(214) X pwB(314) and pwB(314) X pwC(320). Double mutants involving the “extreme” p w A (414) allele, such as pwA(414)pwC(320), were difficult to identify, but phenotypic characterization was still helpful. Table 3 lists the crosses which confirmed the double-mutant genotype of pwA(214)-pwB(314), pwB(314)-pwC(320), pwA(94)-pwB(314) and identified as well as confirmed the double-mutant genotype of pwA(414)-pwB(314) and pwA(414)-pwC(320). In all cases the F2 progeny were followed, giving the expected independent segregation of the pawn genes and the markers. The relative resistance to killing by barium
A semi-logarithmic plot of survival in barium (7.5 mM in growth medium) for each mutant strain is shown in Figure 1, parts a through g. All of the lines are plotted together in Figure l h and demonstrate several points. Firstly, the killing curve for the pwC(320) mutant, which cannot be distinguished from the wild type by observation of reversal behavior, is also indistinguishable from the wild type.
2
1
extreme partial extreme partial extreme partial extreme partial partial partial
Pawn phenotype
-
-
-
-
-
-
-
-
-
-
-
-
-
bd
-
PO
-
-
-
sp
44/0 24/0 75/0 not done2 51/0 52/0
87/0
63/0
75/0 78/0
pw/not p w
21/23 10/14 32/43 not done2 27/24 28/24
35/40 not done 33/30
s p / n o t sp
F21
not done 31/21
12/12
po/not p o
46/41
33/45
bd/not bd
Methods for Phenotypic characterization were identical to Table 2. Because the F1 differs phenotypically from both parents, proof of the proposed genotype did not require characterization of the F2.
p w A ( 2 1 4 ) - p w B ( 3 1 4 ) - ~pwA(419) ~ pwB(95)-bd pwA(94)-pwB(314) pwA(214)-sp pwB(95)-bd pwA(414) - p ~ B ( 3 1 4-SP ) pw.4 (214) p~B(100)-~0 pwA(414)-pwC(320) p~A(214)-~p pwC(320) -sp p ~ B ( 3 1 4 ) - p w C ( 3 2 0 ) - ~pWB(1OO)-Sp 0 pwC(320)-sp
Proposed genotype X Tester strain
F1'
Test crosses to prove double mutant genotypes
TABLE 3
z
H
8
5
v > 5 >
8
v)
3
>
z
2
B
A
I
\ m
\.
L 1
-
.
H
t
2
3 HUURS
9
5
FIGURE 1.-Each of figures l a through I g shows the results of three separate experiments. To cells in 15 m l of growth medium (pH adjusted to 6.8 with HCI) was added BaCl, to 7.5 mM. The number of motile cells/ml (ordinate) were counted a t each time p i n t (abscissa), The lines were computed to give a least-squares fit, and are all plotted together in Figure Ih. Motile cells are equivalent to living cells, tiommotile cells to dead cells.
PAWN’ MUTANTS O F PARAMECIUM
463
Secondly, p w A mutants demonstrate greater resistance to barium than the wild type, whose numbers are reduced 10-fold in 75 minutes. The corresponding h e for the extreme p w A mutants (414 and 419) is 150 minutes. Also, the p w A ( 2 1 4 ) mutant, which is not so extreme behaviorally and which has about 5% of the wild-type active calcium conductance (SCHEIN,BENNETT and KATZ1976), has a killing curve intermediate between the extreme pwA’s and the wild type. Thirdly, the extreme pwB(100) mutant shows the greatest resistance of all, four hours being required to get a 10% survival. The surprise of the study was the killing curve of the pwB(314), which displays considerable reversal behavior and has 25% of the wild-type active calcium conductance; its survival is like the extreme p w A mutants. T o confirm the high resistance of pwB(314) to barium, the study was repeated with a solution of defined composition (modified Dryl’s salt solution) brought to 1 mM BaC1,. The results of two experiments with p w A ( 2 1 4 ) , pwB(314) along with one experiment with the wild type, plotted in Figure 2a through 2c and summarized in Figure 2d, are entirely consistent with the conclusion from the first set of experiments: a pwB mutant is more resistant to the killing effect of barium than a p w A mutant with a similar or even greater behavioral defect.
FIGURE 2.-Figure 2a shows the result of one experiment; Figures 2b and 2c show the results of two separate experiments. To cells adapted in 1 mM Na,HPO,, ImM NaH,PO,, 2 mM Na-citrate, 0.33 CaC1, for 4 hour%BaCl, was added to ImM. The number of motile cells (ordinate) were counted at each time point (abscissa). The lines were computed to give a leastsquares fit, and all are plotted together in Figure 2d.
464
S. J. SCHEIN
Nondefective calcium-activated reversing structure I n the course of screening drugs for the generation of drug resistance marker mutations, the following peculiar response of the wild type to 400 pM chlorpromazine was observed. After an initial reversal, the normal response to transfer from growth medium to the salt-solution, the individual swims forward for about ten seconds, slows down to a stop, then swims backward for about three seconds before slowing down, settling to the floor of the chamber, and dying. With the omission of the initial reversal, the extreme pawn mutants respond in an identical fashion. Reversal behavior has been demonstrated in all of the pawns, showing that the calcium-sensitive structure responsible for reversal of ciliary beat is not affected by the mutation. The chlorpromazine test is rapid and was used routinely to identify the presence or absence of the spinner in extreme pawns. With chlorpromazine treatment, pawns without the spinner trait swim backward, pawns with the spinner trait spin in place. All of the sp identifications on extreme mutants in Tables 2 and 3 were done using chlorpromazine. Many of the strains so classified have since been tested genetically and no errors in sp assignment have been discovered. DISCUSSION
Isolation of pawn mutants by resistance to barium solutions KUNGand his associates have isolated over fifty unconditional pawn mutants by behavioral selection: chemotactic inhibition of geotaxis (KUNG1971a; CHANG et al. 1974). All mutations affected two unlinked genes, p w A and pwB. I n addition, of the five temperature-sensitive pawns which have been isolated (CHANG and KUNG1973), four were pwA mutants. The fifth demonstrated the existence of a third unlinked pwC gene. The mutants have been studied behaviorally (KUNG1971b; CHANGet al. 1974) and electrophysiologically (KUNGand ECKERT 1972; SATOWand KUNG1974). Study of pwB(d4-95) shows nearly 1972). Finally, a normal calcomplete loss of excitability (KUNGand ECKERT cium-sensitive reversal mechanism has been demonstrated in the mutants by reversed swimming after Triton X-100 treatment (KUNGand NAITOH1973). The first mutant (and the most extreme pawn phenotype) in the present study was isolated using chemotactic inhibition of galvanotaxis, a modification of KUNG’Sbehavioral selection. One property of this mutant-resistance to barium -suggested that an “ionic selection”, based on the ion-specific properties of the calcium channel, might focus specifically on mutations in excitability involving alterations in the calcium channel. Therefore the second set of (seven) mutants was isolated using a selection based on resistance to paralysis by barium. The behavioral analysis of the mutants showed that a significant fraction of the pawns are “partial” pawns. The genetic analysis showed that mutations of the same three genes as KUNG’S pwA, pwB and pwC were isolated. The selection yielded the second partial mutant of the pwB gene ( 3 1 4 ) discovered to date, and the only pwC mutant (320) selected on the basis of its properties at room temperature. The usefulness of partial mutants will be discussed below.
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465
Induction of reversal behauior b y chlorpromazine Chlorpromazine induces several seconds of reversed swimming before cells die. This result is similar to the induction of reversal following Triton X-100 treatment (NAITOHand KANEKO 1972). The mechanism of action of both chemicals is probably based on detergency. Ions, calcium in particular, are thus allowed to bypass the normal membrane permeability barriers and enter the cell, causing ciliary reversal. Evidence for chlorpromazine’s ability to increase membrane permeability has been found in another ciliate, Tetrahymena pyriformis (NATHAN and FRIEDMAN 1972). Chlorpromazine induction of reversal has been used in the present report to demonstrate a normal calcium-sensitive reversal mechanism in extreme pawns. It has also been used extensively in genetic characterization, to determine whether the extreme pawns also have spirmer trait. A phenotypic difference between pwA and pwB The survival of p w A and pwB mutants in barium solutions was prolonged over that of the wild type and, as might have been expected, among mutants of each gene, the greater the reduction in reversal behavior, the longer the survival. However, the resistance of pwB mutants appeared greater than that of p w A mutants. These killing curves are the first evidence that it is possible to phenotypically distinguish p w A from pwB mutants: For a given degree of loss of reversal response, pwB mutants are much more resistant to the toxic effects of barium than pwA mutants. In addition, barium killing represents a new ‘‘ionic selection” method which should specifically enrich the yield of pwB’s and, in particular, of the elusive partial pwB’s. The selection would also be useful for isolating conditional lethals. T h e ualue of partial pawns Partial pawns have already proven their value in several ways. Electrophysiological measurements directly confirmed the decreased active calcium conductance in the mutants. The partial pawns were useful in that a simple correlation between degree of loss of reversal behavior and the decrease in active calcium current was possible, as was observed (SCHEINet al. 1976). Further electrophysiological characterization of the alteration in calcium activation is possible only if some calcium activation is still present, as with partial pawns. In crosses between complementing pawns it was easy to distinguish the doublymutated F2 progeny from their singly-mutated parental-type and wild-type sibs. Proof of the double-mutant genotype of those so chosen is given in Table 3. An electrophysiollogical study of the double mutants, included in SCHEINet al. (1976) indicates that the defect in excitability is also greater in double mutants then in their parents. Voltage clamping will be required before further characterization of the interaction between the pawn genes is possible. The frequency of isolation of partial pawns is surprising, many partial pwA’s having been found, only two partial pwB’s. I n CHANGet al. (1974), of 22 pwA mutants, 5 were leaky 1, 13 were leaky 2, (pwA(214) would fit here), 2 were
466
S. J. SCHEIN
leaky 3 (pwA(202) would have fit here) and 2 were leaky 4 (equivalent to m y term “partial pawn”). None of the 14 pwB’s were at all leaky. Of the five temperature-sensitive pawns isolated (CHANGand KUNG1973) four were pwA, one was pwC, and pwB was conspicuous by its absence. The curious distribution of partial mutants is consistent with the model described below.
A model SCHEIN,BENNETT and KATZ(1976) found a second method for distinguishing pwB from pwA mutants: I n addition to the effect of the pawn mutations on calcium activation, a second electrical property of the membrane of P . aurelia, anomalous rectification, was reduced in pwB mutants. The reduction was also proportional to the reduction in active calcium conductance and the degree of behavioral deficit. This observation suggested to us that the pwB gene product is the “pore”, while the pwA gene product performs or affects the depolarizationsensitive function of the channel (the “gate”). While the model explains the electrophysiological data to date and is admittedly speculative, it provides a tentative explanation for the intriguing disparity between the frequencies of isolation of partial mutants in the pwA and pwB genes. Mutations in the “gate” molecule would result in alterations of voltage sensitivity or time course of the conductance change, alterations which would span a wide range of severity. One of perhaps several gating charges could be modified, or the fraction of the transmembrane potential which is traversed when the gating charges move could be decreased. Both sorts of mutations would change voltage sensitivity to varying degrees. The stiffness of the gate “hinge” might be increased; the speed of motion of the gating charges might be decreased. Both alterations would slow down the time course of the Conductance change. Indeed, many partial o r “leaky” mutants have been isolated in the putative “gate” gene, pwA. However, the normal ‘‘pore’’ exhibits ion selectivity, admitting the divalent ions Ca++, Sr++ and Ba++ but excluding Mg++, Mn++, monovalent cations and all anions. Investigations into the ion-selective properties of the sodium channel and the channel-blocking agents like tetrodotoxin have placed severe restraints on the size of the pore ( 3 x 5A) (HILLE1971). Likewise, the selectivity of the calcium channel presumably requires a structure of carefully tailored dimensions. Even small modifications of the molecule, changing the pore dimensions by perhaps a single Angstrolm, would be enough to completely close the pore. Thus, extreme mutants would be the rule, partial mutants the exception, as is the case in the putative ‘Lpore”gene, pwB. The differential killing by barium is consistent with the model. The calcium channels of a mutant with altered gates (pwA) would still be expected to open, though less frequently, and admit some barium. Regardless of the state of the gate (open or closed), the calcium channels of a mutant with a closed pore (pwB) would never admit barium. Finally, the process of autogamy has allowed measurement of the gradual loss of excitability which occurs when heterozygous (wild type/pawn) paramecia
PAWN MUTANTS O F PARAMECIUM
46 7
with wild-type excitability are “suddenly” changed to homozygous pawns with normal channels remaining in the membrane. These measurements have shown that the lifetime of the molecular target of the pwA and pwB genes is identical and remarkably long, between 5.5 and 8 days (SCHEIN1976b). These results are entirely consistent with the notion that the pwA and pwB genes affect a common structure, the calcium channel. The model may also be tested, since it predicts that voltage sensitivity and time course has been altered in pwA mutants, maximum active calcium conductance and perhaps ion selectivity in the pwB mutants. Electrophysiological studies utilizing voltage-clamp methods should allow measurement of these parameters and comparison with the wild type. I am especially grateful to DR. MEL COHNof the Salk Institute and DR. J. T. AUGUSTat the Albert Einstein College of Medicine for their support and encouragement in the early phases of DAVIDand this work. I am also deeply appreciative of the kindness shown me by DRS.CHARLES M. V. L. BENNETT,who have given generously of their time and advice. S. SCHEINwas supported by grants from the National Institutes of Health, GM11301 and 5T5GM1674. LITERATURE CITED
BEALE,G. H., 1954 The Genetics of Paramecium aurelia. Cambridge University Press, London. L. J. ROBLES,S. S. LUI and C. KUNG,1974 An extensive beCHANG,S.-Y., J. v . 4 ~HOUTEN, havioural and genetic analysis of the pawn mutants in Paramecium aurelia. Genet. Res. Camb. 23: 165-173. CHANG,S.-Y. and C. KUNG, 1973 Genetic analysis of heat-sensitive pawn mutants of Paramecium aurelia. Genetics 75: 49-59.
DRYI,,S., 1959 Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autogamy and conjugation. J. Protozool. 6:196. HANSON, E. D., 1974 Methods in the cellular and molecular biology of paramecium. pp. 319363. In: Methods in Cell Physiology, Vol. 8. Edited by D. M. PRESCOTT. Academic Press, New York.
HILLE, B.,1971 The permeability of the sodium channel to organic cations in myeoinated nerve. J. Gen. Physiol. 58: 599-619. HODGKIN, A. L. and A. P.HUXLEY, 1952 -4 quantitative description of membrane current and its application to conduction and excitation i n nerve. J. Physiol. 117: 500-544. JAHN,T. L., 1961 The mechanism of ciliary movement. I. Ciliary reversal and activation by electric curfent; the Ludloff phenomenon in terms of core and volume conductors. J. Protozool. 8: 369-380. KUNG,C., 1971a Genic mutants with altered system of excitation in Paramecium aurelia. 11. Mutagenesis, screening and genetic analysis of the mutants. Genetics 49: 29-45. -, 1971b Genic mutants with altered system of excitation i n Paramecium aurelia. I. Phenotypes of behavioral mutants. Z. vergl. Physiol. 71: 142-164, KUNG,C. and R. ECKERT,1972 Genetic modification of electric properties in an excitable membrane. Proc. Nat. Acad. Sci. U.S.A. 69:93-97. KUNG, C. and Y. NAITOH,1973 Calcium-induced ciliary reversal in the extracted models of “Pawn”, a behavioral mutant of paramecium. Science 179: 195-196. KUNG,C., S.-Y. CHANG,Y. SATOW, J. VAN HOUTEN and H. HANSMA, 1975 Genetic dissection of behavior in Paramecium. Science 188: 898-901..
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MACHEMER, H. and ECKERT,1973 Electrophysiological control of reversed ciliary beating in Paramecium. J. Gen. Physiol. 61: 572-587. NAITOH,Y., 1969 Control of the orientation of cilia by ATP, calcium and zinc in glycerolextracted Paramecium aurelh. J. Gen. Physiol. 53 :517-529. NAITOH,Y. and R. ECKERT,1968a Electrical properties of Paramecium caudatum: modification by bound and free cations. Z. vergl. Physiol. 61 : 497-452. -, 19681, Electrical properties of Paramecium caudatum: all-or none electrogenesis. Z. vergl. Physiol. 61 : 453-472. NAITOH,Y. and H. KANEKO,1972 ATP-reactivated Triton-extracted models of Paramecium: modification of ciliary movement by calcium ions. Science 176: 523-524.
H. A. and W. FRIEDMAN, 1962 Chlorpromazine affects permeability of resting cells of NATHAN, Tetrahymena pyriformis. Science 135: 793-794. SATOW, Y. and C. KUNG,1974 Genetic dissection of active electrogenesis in Paramecium aurelia. Nature 247: 69-71. SCHEIN,S. J., 1976a Electrical excitability in Paramecium aurelia. Ph.D. Thesis, Albert Einstein 1976b Calcium channel stability measured by gradual loss of College of Medicine. -, excitability in pawn mutants of Paramecium aurelia. J. Exp. Biol. (In press). SCHEIN,S. J., M. V. L. BENNETTand G. KATZ,1976 Altered calcium conductance in pawns, behavioral mutants oE Paramecium aurelia. J. Exp. Biol. (In press).
T. M., 1947 Recent advances in the genetics of Paramecium and Euplotes. Adv. SONNEBORN, Genet. 1: 263-358. -, 1950 Methods in the general biology and genetics of Para1957 Breeding systems, reproductive mecium aurelia. J. Exp. Zool. 113: 87-148. --, methods, and species problems i n Protozoa. pp. 155-324. In: The Species Problem. Edited by E. MAYR.AAAS, Washington, D.C. --, 1970 Methods i n Paramecium Research. pp. 241-339. In: Methods in Cell Physiology, Vol. 4. Edited by D. M. PRESCOTT. Academic Press, New York. --, 1975a Paramecium aurelia, pp. 469-594. In: Handbook of Genetics, Vol. 2. Edited by R. KING. Plenum Press, New York. -, 197513 The Paramecium aurelia complex of Fourteen Sibling Species. Trans. Amer. Micros. SOC.94: 155-178. Corresponding editor: S. ALLEN
