Inter- and intrasyngenic variation of Paramecium hemoglobin—I. Paramecium aurelia complex

Comp. Biochem. Physiol. Vol. 75B, No. 3, pp. 415-420, 1983 Printed in Great Britain.

0305-0491/8353.00+ .00 © 1983 Pergamon Press Ltd

INTER- AND INTRASYNGENIC VARIATION OF PARAMECIUM HEMOGLOBIN--I. PARAMECIUM A URELIA COMPLEX ITARU USUK1 and TOSH1AKI IRIE* Department of Biology, College of General Education, Niigata University, Niigata 950-21, Japan (Received 4 November 1982) A b s t r a c t - - l . Hemoglobins (Hbs) from Paramecium aurelia complex showed a considerable degree of interand intrasyngenic variations by polyacrylamide gel electrophoresis (PAGE). 2. All of the Hbs from various stocks of different syngens were resolved into definite 12 variants, which gave expression to a difference in the mobility by PAGE. 3. An argument that the multiplicity of Paramecium Hb may be an artifact caused by endogenous proteases, was not supported by the facts obtained on a crude Hb sample prepared under the presence of antipain, leupeptin, pepstatin and phosphoramidon in sut~cient concentrations. 4. The Hb variants can be subdivided into two groups by their molecular weights, taking either value of 11,000 and 13,000.

INTRODUCTION

MATERIALS AND METHODS

The presence of a H b in Paramecium was reported by Sato and Tamiya (1937) and Keilin and Ryley (1953). Partial purification and characterization of the H b was made by Smith et al. (1962), who had found that the respiratory pigment is a monomeric protein with an extremely high oxygen affinity, as like as mammalian myoglobins. Recently it was reported that the Hb in P. tetraurelia is resolvable into distinct five variants, by the methods of P A G E and isoelectric focusing (Davis and Steers, 1976; Steers and Davis, 1979). Moreover, the chromatgraphically purified native oxy-Hb was reported to differ between two c o m m o n Paramecium species, P. primaurelia and P. caudatum : the former generated three heterogeneous components, while the later bore only one homogeneous component (Irie and Usuki, 1980). P. aurelia is a species complex which is subdivided into a number of varieties called syngens, and 14 syngens have been described (Sonneborn, 1975). On these ciliates some investigators have pointed out that certain mitochondrial and cytoplasmic enzymes give an expression to electrophoretic variants and most syngens differ in types of isozyme distribution ( T a i l 1968, 1970, 1978; Allen and Gibson, 1971; Allen and Golembiewski, 1972; Allen et al., 1973). This may suggest a possibility that P. aurelia complex generates a difference in Hb types among syngens. We thus planned to study the Hbs from various stocks of different syngens of P. aurelia complex, together with stocks of other Paramecium species. In this paper the results obtained on 14 stocks in eight syngens of P. aurelia are dealt with, as the first step in a comparative investigation of Paramecium Hb.

Animals and culture The ciliates used were P. primaurelia (syngen l, stocks 16 and 513), P. biaurelia (syngen 2, stock 7), P. triaurelia (syngen 3, stocks 37 and 152), P. tetraurelia (syngen 4, stocks 32, 51, 127 and 239), P. pentaurelia (syngen 5, stock 87), P. sexaurelia (syngen 6, stock 159), P. septaurelia (syngen 7, stocks 38 and 227) and P. octaurelia (syngen 8, stock 299). The culture medium was a diluted solution of fresh lettuce juice buffered with 0.1% Na2HPO4.12 H:O and supplemented with a mixture of 25 mg/1 of azolectin (Daigo Nutrit. Chem. Indust., Osaka), 25 mg/l of soybean lecithin (Wako Pure Chem. Indust., Osaka) and 0.5 mg/l of stigmasterol (Merck). The medium was inoculated with Enterobacter aerogens (IAM 1183, supplied from Institute of Applied Microbiology, University of Tokyo) for 15-20 hr before use. Each of the ciliates was grown for 4-5 days at 27°C.

*Present address: Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan. 415

Preparation of hemoglobin sample (a) Ordinary method. After Paramecia in each of the stocks were grown in about 250 I. of the media, they were harvested, then the packed cells obtained were homogenized using a Teflon-glass homogenizer. The homogenate supernatant was fractionated between 40 and 70% in saturation of ammonium sulfate, and the salt held in the precipitate was eliminated by passing through a Sephadex G-25 column (2.6 x 75cm) equilibrated with 10mM Tris-HCl buffer (pH 7.5) as described in a previous paper (Irie and Usuki 1980). In this study the resulting protein solution was lyophilized by use of a freeze-drying apparatus, in order to avoid any loss of Hbs in the solution. The lyophilizate obtained was dispersed into a small amount of deionized water and the insoluble substances which arose during lyophilization were centrifuged off at 27,000 g for 20 min. The red colored protein solution thus obtained was chromatographed on a column (2.6 × 95 cm) of Sephadex G-75 equilibrated with 10mM Tris-HCl buffer (pH 7.5). After the Hb fractions eluted were concentrated by a manner of freeze-drying, the pigment solution was re-chromatographed on the same column with the same buffer. The eluted Hb was concentrated again and stored in a deep freezer at -90°C until use.

416

ITARU USUK1 and TOSHIAKI IRII~

(b) Simplified method. To prepare a Hb sample under the presence of inhibitor for endogenous proteases, a red supernatant called the pressed juice, which was obtained by crushing packed Paramecia under high centrifugal force, was adopted. The pressed juice was prepared according to the method described by Koizumi and Kobayashi (1976) with a slight modification. That is, by centrifugation at 130g for a few min, Paramecia in 5 101. of the culture were concentrated, and the cells were suspended and washed repeatedly with the Dryl's saline solution (Dryl, 1959). Then, the cells were packed by centrifugation at 290g for a few rain and excess water on the cells was removed as far as possible. After the packed cells were maintained in an ice cold temperature, 50rag of Sephadex G-25 fine powder (Pharmacia Fine Chem.) and 100 id of inhibitor which was wanted to examine were added. Then the contents in a centrifugal tube (10 × 95 ram) were covered with a silicon oil (KF 50-100, sp. gr. 0.98-1.00, Shin-Etsu Kagaku, Japan). After the tubes were balanced with addition or subtraction of the silicon oil, centrifugation was carried out at 32,000 g for 60 rain at 0~ 2'C. The pressed juice was yielded about 0.5 ml/ml of the packed cells. The juice was collected and stored in liquid nitrogen or in a deep freezer at - 9 0 C . Protease inhibitors used were leupeptin, antipain, phosphoramidon (purchased from Peptide Instit., Protein Res. Found., Osaka) and pepstatin (the product of Sigma Chem. Co. ).

Eleelrophoresis and molecular weighl estimation Electrophoresis was carried out using 15'I,, polyacrylamide slab gel (150 × 170× 2mm) or disc gel (5 × 140ram) in a discontinuous buffer system (Davis, 1964). The Hb samples which were subjected to electrophoresis were converted previously to the cyanomet-form by addition of an equal volume of Tris H3PO 4 buffer (pH 6.9) containing 20 mM K3Fe(CN)6 and 40 mM KCN. Electric current applied was 75 mA for a slab gel and 2 mA per tube for disc gels, during about 2 hr at 17C. Densitometric analysis was performed with a Shimazu dualwavelength chromatoscanner CS-910. Molecular weight was estimated using 10 and 1501, polyacrylamide gel containing 0.1'~i~ sodium dodecyl sulfate (SDS), with or without 8 M urea, according to the procedures of Weber and Osborn (1969) and Swank and Munkres (1971). Samples of the individual Hb were obtained by cutting from the gels previously electrophoresed and they were treated with 200-400 p l of 0.01 M phosphate buffer (pH 7.2) containing 1!',o SDS and 5'~, mercaptoethanol or 0.01 M Tris H3PO 4 buffer (pH 6.8) containing I'% SDS, 8 M urea and 5",, mercaptoethanol at 40 C over a night. Occasionally, mercaptoethanol in the solution was replaced with 1°;, dithiothreitol. Of the sample, 5-10/11 was applied together with an aliquot of bromophenol blue. A constant current of 180 mA was applied for 8 hr at 26,27 C. As standard proteins for tool. wt, albumin (egg white, 45,000), pepsin (hog stomach mucosa, 34,700), trypsinogen (bovine pancreas, 24,000), fl-lactoglobulin (bovine milk, 18,400), cytochrome c (horse heart, type Ill, 12,400) and 7-chymotrypsin (bovine pancreas, type l l, B-chain 13,000, C-chain 11,000) purchased from Sigma Chem. Co. and myoglobin (sperm whale, 17,200) of the product of Miles Co. were used. RESULTS

Eleetrophoretic pattern Hbs from various stocks of different syngens were purified by twicefold gel filtration on Sephadex G-75. By this procedure H b was collected in a steep single peak of fractions which showed similar spectral properties to those of m a m m a l i a n Hbs and myoglobins, as reported in a previous p a p e r (Irie a n d Usuki, 1980).

Syngen 1

2

4

5

6

7

8

I

Hbl

Hb2 Hb3 Hb4

~

Hb5 Hb B

Hbz Hbe

3

I

I

i

I

I

I

~ d

~

I

I I

~

Hb9

Hblo Hb n H b ~2

I I

~ ~

16

I

~j~

7

152

51

87

159

38

299

Stock

Fig. 1. Hb profiles representing an intersyngenic variation among stocks of syngens 1 8. Electrophoresis was carried out using 15"ii polyacrylamide slab gel. The gel was unstained.

As s h o w n in Fig. 1, however, each of the Hbs isolated is resolved into several reddish b a n d s by P A G E , indicating a p o l y m o r p h i c form of H b in P. aurelia complex. In a d d i t i o n to this, some of the H b variants resolved a p p e a r in c o m m o n to m a n y syngens and others occur only in some syngens. While some variants are enriched in a syngen, in other syngens they are p o o r or completely absent. Thus, P. aurelia complex reveals a noticeable degree of intersyngenic variation of H b by P A G E . As s h o w n in Fig. 2, the electrophoretically resolvable H b c o m p o n e n t s from various stocks are in 12 definite variants according to their relative mobilities a n d designated from Hb~ to Hbt2 , in order of the migration from the slowest rate to the fastest rate. U n d e r standardization, the variants Hb~, Hb4 and Hbs are characteristic of syngens 3, 4 and 1, respectively. O n the other h a n d , most of the variants are joined with m a n y different syngens. F o r example, Hb~o occurs in c o m m o n to 7 syngens out of 8, with an exception of syngen 7 in which this variant is not detected. H b ~ is also distributed widely, in high c o n c e n t r a t i o n s for syngens 1, 3, 5 a n d 8 a n d in an intermediate or low c o n c e n t r a t i o n s for syngens 2, 4, 6 a n d 7. In Fig. 3 H b profiles from different stocks within a syngen are compared. There is a variation between stocks belonging to each of syngens 1,3, 4 and 7. For instance, stock 16 o f syngen 1 generates distinct three H b bands, while stock 513 o f the same syngen reveals only two a n d in the stock the b a n d c o r r e s p o n d i n g to H b 3 is indiscernible. Equally, stock 152 of syngen 3 bears six bands, but stock 37 exhibits no more than three, due to the indistinctness of Hb> Hb~ and Hbt2. As s h o w n in Fig. 4, the H b profiles are analyzed densitometrically. The results o b t a i n e d indicate that the intrasyngenic variation m a y be a t t r i b u t a b l e to a

417

Paramecium hemoglobin--I

®

Syngen

-(9

Electrophoresis

1

3

4

7

Hbl Hb~

J

1-16 k

Hbz Hb,i Hbn

II

Hb6 Hb? Hbe Hblo Hb ii 2-7 Hb12

16513

37.152

32.51.127.239 38.227

3-152

Stock

L.,,,

.J

Fig. 3. Hb profiles representing an intrasyngenic variation among different stocks belonging to each of syngens l, 3, 4 and 7. Electrophoresis was as in Fig. 1.

ItJ !

4-32

_)1 7-38

2 34 56 789 10 11 12 Hemoglobin

variant

Fig. 2. Densitometric Hb profiles representing definite 12 variants distinguished by the mobility by PAGE. Electrophoresis was carried out using 15~ polyacrylamide slab gel. Densitometry was performed with 420 nm in the sample beam and 600 nm in the reference beam, using an electrophosed unstained gel.

deviation of relative concentrations in each of the variants provided to the given syngen. In the case of syngen 4, for example, each of four stocks examined contains Hb3, Hb4, Hbs, Hb 7, Hbl0 and H b , in a variety of concentrations, although the amount of Hb~0 is predominant in common to all of the stocks. In Table 1, distribution and relative concentrations of Hb variants in stocks of syngens 1-8 are summerized. Effects o f protease inhibitor The multiplicity of Paramecium Hb may be a phenomenon which is ascribed to actions of endogenous proteases, since these enzymes can break down primary Hb molecule into some fragments. This possibility was tested by use of a crude Hb sample called the pressed juice, in the presence of powerful protease inhibitors, such as antipain, leu-

peptin, pepstatin and phosphoramidon in a sufficient concentration. In Fig. 5 some of the results obtained are shown. At first, the pressed juice, when it was prepared with the absence of any inhibitor, generated essentially similar Hb profile to that of the sample purified from the same stock of Paramecium. Secondly, treatment by inhibitors resulted in no change of the variants in a given stock. Among the inhibitors examined, pepstatin brought about a faint profile of Hb as a whole, probably due to an acidic conditions employed. Even in this case, the resulting profile remained similar in every respects to others. These facts support the view that the multiplicity of Hb in P. aurelia complex is of a primary nature, and is not artifact. Molecular weight Figure 6 shows the S D S - P A G E pattern of the 12 variants isolated from various stocks of different syngens. All of the variants examined are arranged on two lines in a slab gel. That is, six variants including Hb4, Hb 6, Hbs, Hbl0, Hbll and Hb]2 moved with a faster rate than other six, consisting of Hbl, Hb2, Hb3, Hbf, Hb7 and Hb9, regardless of stocks and syngens whose the variants have supplied. As shown in Fig. 7, the variants with faster migration correspond to the mol.wt of approx 11,000 and another group with slower migration is 13,000. DISCUSSION

It has already been reported that the sibling species of P. aurelia are provided with a multiple form of Hb, such as five variants in P. tetraurelia (stock 51, syngen 4) (Davies and Steers, 1976; Steers and Davis, 1979) and three variants composing of two majors and one minor in P. primaurelia (stock 16, syngen l) (Irie and Usuki, 1980). However, it is difficult to know whether

ITARU USUK] a n d TOSHIAKI IRIE

418

(~)

Electrophoresis~

(+)

!

Syngen

1

i i

Stock 16

i

:

I ~L

!

513

!!

'

Syngen

4

Stock 32 Syngen 3

~

152

j

'

JL_. i

Syngen

-

127

7 38

L

227

,

l lh J i l l

PIll

,

239

J , j ' 1 2 34 56 7 89 10 11 12

I

2 34 56 789 10 11 12 Hemoglobin

variant

Fig. 4. D e n s i t o m e t r i c H b profiles representing an i n t r a s y n g e n i c v a r i a t i o n a m o n g different stocks belonging to each o f syngens 1, 3, 4 and 7. E l e c t r o p h o r e s i s a n d d e n s i t o m e t r y were carried out as in Fig. 2. * In syngen 4 was identified to be a c y t o c h r o m e c by the spectral properties.

T a b l e 1. D i s t r i b u t i o n , relative c o n c e n t r a t i o n a n d mol. wt o f the H b v a r i a n t s detected from different stocks o f syngens 1 8

No. of Syngen

Stock

examples

1

2

3

16 513

3 5

----

1 1

3-5 1 2

2

7

4

18-24

13-17

3

37 152

5 7

4~5 5 8

1

1 2 3-7

7 6 7 7

--

4

32 51 127 239

5

87

4

--

6

159

3

--

7

38 227

3 2

8

299

1

Mol. wt

5 13 .--

9 13

4-5 14-22 8 12 22 34

Hb variant 6 7

4

5

0 1

1 2 0-1

0-1 I-2

1 2

t6,24

• 12-15 2-5

5-7

4~6 7-11 3-7 5-11

--

8

9

10

11

12

52 56 45 54

0 1

2-3 2-7

27--3i 37-42

5 1~4

2634

1

0 I

68

I 2

......

1 2 7 8 I-3 10--t5

3 6 22 24 0 I 0- 1

1~4

5 4 60 16, 19 34--38 24-27 42 50 29-32 36,43 6 8

--

30---32 41.-46

6 8

1 3

0 1 0 1

1 I

0-1 4-5

3

--

14-21

I-5

--

7--9

× l0 4

1.3

1.3

1.3

1.1

1.3

6~62 30

['18-2l] I 19-20J /

1.3

87 90

8- 12

~

9-11 31

9 10 13-14

36,41

31~34

0 1

I.I

I.I

I.I

/

0 1 I.I

I.I

1

32 37 45-53 24-33 25-30

1.3

E l e c t r o p h o r e s i s a n d d e n s i t o m e t r y were carried out as in Fig. 2. Hb~ and Hb~ in syngen 7 were undistinguishable.

Paramecium hemoglobin--I Hbl Hb2 Hb3 Hbs Hbs Hbr Hb9

Hbto Hblt Hbl~

a

c

d 2-7

e

f

a

b

c

d

e

f

3-152

Fig. 5. Hb profiles from a crude sample which was prepared by the pressed juice method under the presence or absence of protease inhibitors. The examples were concerned to stock 7 of syngen 2 and stock 152 of syngen 3. The pressed juice was prepared under the presence of (a) antipain (2 x 10-4 M in final concentration, pH 7.0), (b) leupeptin (2x 10-4M, pH 7.0), (c) phosphoramidon (2 × l0 -4 M, pH 7.0) and (d) pepstatin (10 -4 M, pH 2.8), and (e) without any inhibitor (pH 7.0). (f) was a purified sample prepared by the ordinary method, pHs used were regulated by phosphate buffer (pH 7.0) or biphthalateacetate buffer (pH 2.8), 0.02M in final concentration. Electrophoresis was carried out using 15~o polyacrylamide disc gels. The gels were stained by the benzidine reaction.

all of the variants reported are different or not, since mutual relationship between the variants from different species of Paramecium remains obscure. In this study the Hbs of P. aurelia complex were classified into 12 variants according to mobility by PAGE. The variants reported on P. tetraurelia (Davis and Steers, 1976; Steers and Davis, 1979) correspond to Hb3, Hbs, Hb7, Hbt0 and HbH, and that those of P. primaurelia (Irie and Usuki, 1980) fit to Hb8 and Hb]~ for the majors and Hb12 for the minor, although another variant Hb3 has been lost during the purification procedures employed. Hence, we can now describe that both of the syngens contain two common variants Hb3 and HbH and most of other variants detected differ each other. An inter- and intrasyngenic variation of certain mitochondrial and cytoplasmic enzymes in P. aurelia complex have been researched by some investigators (Tait, 1970, 1978; Allen et al., 1973). They have shown that, some of the isozyrne patterns are identical between different syngens, and that the frequency of intrasyngenic variation of the isozyme distribution is rare in most syngens (Tait, 1969, 1970; Allen and Gibons, 1971; Allen and Golembiewski, 1972; Allen et al., 1973). On the other hand, the Hbs in P. aurelia complex show a noticeable variation among stocks as well as syngens. Our examination leads to an interpretation that the intersyngenic variation of Hb is due to a difference in the combination of the variants distributed to the given syngens. Some authors think that a polymorphism of Paramecium Hb may be attributable to artifacts, derived from partial hydrolysis of Hb molecules in healthy

419

cells by an activation of endogenous proteases, This possibility, however, was not supported by the fact that all of the variants resolved from a purified preparation are recognized from a crude sample, which is prepared by omission of procedures for the purification of Hbs and under the influence of a serine-thiol protease inhibitors (antipain and leupeptin), metalloprotease inhibitor (phosphoramidon) and carboxyl protease inhibitor (pepstatin), respectively. This study makes clear that the electrophoretically resolvable Hb variants can be subdivided into two groups by their tool. wt, showing the value of approx 11,000 for six variants and 13,000 for another six variants on SDS-PAGE. Irie and Usuki (1980) have reported that the two major Hbs of P. primaurelia have the tool. wt of approx 11,000. This agrees well with the results in this study, since the two variants in the ciliate are identified to be Hbs and Hbll in the group with the mol. wt of 11,000. However, the variants in P. tetraurelia have been dealt with the value of 15,000 + 500, on the bases of S D S - P A G E

2

3

4

5

6

7

Hemoglobin

8

9

10 11 12

variant

Oa

Pp

Tg

Mb

Cc

Hb:~

Hb3

Hblo

Hbll

Fig. 6. SDS-PAGE pattern of the Hb variants representing two discordant migration rates (above) and a com-

parison of the mobility to authentic proteins with known mol. wt (below). Before the SDS treatments, Hb~-Hb5 and Hb~0-Hb~2 were extracted from the corresponding bands after cutting from previously electrophoresed 15% polyacrylamide disc gels, with Hb6-Hb9 were extracted from 10% disc gels. Then, the variants were run on 15% polyacrylamide slab gel, without urea. The gels were stained with 0.25~o Coomassie Brilliant Blue. Oa, ovoalbumin; Pp, pepsin; Tg, trypsinogen; Mb, myoglobin; Cc, cytochrome c.

ITARU USUKI and TOSHIAKI IRH!

420

4

A

1

"~,_0 3

2

-,-~2

3

B

4 5

5

"6

7

1 I

0

I

.2

I

I

.4

I

(

.6

I

.8

Mobility Fig. 7. Molecular weight estimation of the Hb variants in dependence of SDS-PAGE. The mobility of the variants was estimated using (A) 15'?,~igel and (B) 10°,ogel containing 8 M urea, after staining with 0.25~;; Coomassie Brilliant Blue• Arrow, HbL,_.~,~:~j~; Arrowhead, Hb4.6.sjO.llandl2. (1) albumin; (2) pepsin: (3) trypsinogen; (4)/t-lactoglobulin, (5) myoglobin; (6) cytochrome c; (7) B-chain of :~-chymotrypsin: (8) C-chain of :~-chymotrypsin.

and sedimentation equilibrium analysis (Davis and Steers, 1976; Steers and Davis, 1979). This value is apparently larger than those described in this paper. Namely, three variants corresponding to Hb3, Hb5 and H b 7 a r e in the members having a mol. wt of 13,000. The remaining two are Hb]0 and Hb] L, which belong to another group with the value of 11,000. Acknowledgements--We are grateful to Dr S. Koizumi and members of his laboratory, Miyagi College of Education, Sendal, for kind supply of the stocks of the sibling species of Paramecium aurelia and valuable advice for the preparation of the pressed juice of Paramecia cells.

REFERENCES

Allen S. L. and Gibson I. (1971) Intersyngenic variations in the esterases of axenic stocks of Paramecium aurelia. Biochem. Genet. 5, 161-181. Allen S. L. and Golembiewski P. A. (1972) Inheritance of esterases A and B in syngen 2 of Paramecium aurelia. Genetics 71, 469 475.

Allen S. L.. Farrow S. W. and Golcmbiewski P. A. (1973) Esterase variations between the 14 syngens of Paramecium aurclia under axenic growth. Genclic.~ 73, 561 573. Davis B. (1964) Disc elcctrophoresis I]. Method and application to human serum proteins..4nn. N. Y. Acad. 5"H. 121, 404 427. Davis R. H. Jr and Steers E. Jr (1976) Myoglobm from tile ciliate protozoan Paramecium aurelia. ('omp. Biochem. Physiol. 54B, 141 143. Dryl S. (1959) Antigenic transformation in Paramecium aurelia alter homologous antiserum lreatment during autogamy and conjugation. J. Proto=ool. 6(suppl.t. 25. lrie T. and Usuki 1. (I980) Disparity of native ox 5hemoglobin components isolated t?om Paramecium caudatum and Paramecium primaurelia. ('omp. Biochem. Physiol. 67B, 549- 554. Keilin S. and Ryley J. F. 11953) Haemoglobin m prolozoa. Nature, Lond. 172, 451. Koizumi S. and Kobayashi S. ([976) A sludy on the matekiller toxin by microinjection in Paramecium. Genet. Res. 27, 179 185. Sato T. and Tamiya H. (1937) Uber die Atmungsfarbstoffe yon Paramecium. Cvtologia (Fujii Jub. Vol.) 1133-1137. Smith M. tt.. George P. and Preer J. R. Jr (1962) Preliminary observations on isolated Paramecium hemoglobin. Arehs. Biochem. Biophys. 99, 313 318. Sonneborn T. M. (1975) The Paramecium aurelia complex of lburteen sibling species. Trans. Am. microsc. Soe. 94, 155 178. Steers E. Jr and Davis R. tt. Jr (1979) Purification and characterization of Ihc myoglobins of Paramecium telraurelia. Comp. Bioehem. Physiol. 62B, 393-402. Swank R. T. and Munkres K. D. (1971) Molecular weight amdysis of oligopeptidcs by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. 4na/w. Biochem. 39, 462 477. Tail A. (I 968) Genetic control of/~'-hydroxybutyrate deh~drogenasc in Paramecium aurclia. Nature, l,ond. 219, 941. Tail A. (1969) Syngen differences in electrophoretic mobility of cerlain enzymes ill Paramecium aurelia. J. Promzool. 16(suppl.) 28. Tait A. (197(t) Enzyme wmation between syngens in Paramecium aurelia. Biochem. Genet. 4, 461 470. Tail A. (I 978) Species identilicalion in prolozoa: Glucosephosphate isomerase variation in the Paramecium aure/ia group. Biochem. Genet. 16, 945 955. Weber K. and Osborn M. (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406M-412.