Inter- and intrasyngenic variation of paramecium hemoglobin—II. Paramecium caudatum, paramecium jenningsi and Paramecium multimicronucleatum

Comp. Biochem. Physiol. Vol. 75B, No. 3, pp. 421424, 1983 Printed in Great Britain.

0305-0491/8353.00+ .00 © 1983PergamonPress Ltd

INTER- A N D INTRASYNGENIC VARIATION OF P A R A M E C I U M HEMOGLOBIN--II. P A R A M E C I U M CA UDA TUM, P A R A M E C I U M J E N N I N G S I A N D PARAMECIUM MULTIMICRONUCLEA TUM ITARU USUKI and TOSH1AKIIRIE* Department of Biology, College of General Education, Niigata University, Niigata 950-21, Japan (Received 4 November 1982) Abstract-- 1. Paramecium caudatum contains a multiple form of hemoglobin (Hb), which is composed of high concentration of a major variant and only a small amount of several minor variants. 2. The electrophoretic mobility and molecular weight of the major variant in P. caudatum are identical to a well-characterized variant, Hb~0, in P. aurelia complex. 3. P. caudatum shows no intersyngenic variation of the Hb between syngen 1 and syngen 3. lntrasyngenic variation is not significant, although some deviations in the variant concentrations among different stocks are recognized. 4. P. jenningsi generates similar Hbs to those of P. caudatum. 5. The Hb in P. multimicronucleatum is characterized with a major variant corresponding to Hbll and several minor variants corresponding to Hb2 and Hb12 in P. aurelia complex.

INTRODUCTION

MATERIALS AND METHODS

In the preceding paper we reported that P. aurelia complex generates multiple Hbs, which are resolvable into 12 variants on the basis of the mobility by polyacrylamide gel electrophoresis (PAGE), and that its Hb profile exhibits considerable inter- and intrasyngenic variations, independent of a difference in the combination of variants distributed to syngens or a deviation in the relative concentration of individual variants among stocks within a syngen (Usuki and Irie, 1983). On the other hand, Irie and Usuki (1980) have found that P. caudatum (two stocks with the complementary mating types in syngen 3) reveals one homogeneous Hb. Steers et al. (1981) have also isolated a single form of Hb from P. caudatum (a clone in syngen 1). This information suggests an important difference of the Hbs between P. caudatum and P. aurelia complex. Molecular weight of P. caudatum Hb has been reported to be 13,500 in syngen 1 (Steers et al., 1981) and approx 11,000 in syngen 3 (Irie and Usuki, 1980). However, such discrepancy of the value does not form a solid foundation that Hb species varies between both of the syngens in P. caudatum. In this paper we describe the results of comparative research on Hbs from various stocks of different syngens in P. caudatum, together with some findings on the Hbs from P. jenningsi and P. multimicronucleatum, with an aim to increase our knowledge on Paramecium Hb.

The ciliates examined were four stocks of syngen 1 and eight stocks of syngen 3 in Paramecium caudatum, two stocks and their caryonide of syngen 2 in Paramecium multimicronucleatum, and one stock of syngen I in Paramecium jenningsL These are listed in Table 1. All of the methods used were the same as those described in the preceding paper (Usuki and Irie, 1983).

*Present address: Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan. 421

RESULTS Ten stocks of P. caudatum (4 in syngen 1 and 6 in syngen 3) were surveyed for the Hb profiles by PAGE. As shown in Fig, 1, all of the stocks examined generate a single definite Hb band with an identical mobility, irrespective of syngen difference in this species. Exactly similar Hb band was also obtained from two other stocks, Yg 2 known as a trichocyst non-discharge mutant (Takei and Hiwatashi, 1981) and CNR known as a behavioral mutant derived from ciliary non-reversal (Takahashi and Naitoh, 1978). However, it was found, by a densitometric analysis or the application of benzidin reaction, that the Hb profiles from all stocks of P. caudatum show several bands, comprising one major and two or three minors. Looking at the electrophoretic mobility, as seen in Fig. 2, the major Hb variant in P. caudatum corresponds to a well-characterized variant, Hb~0, in P. aurelia complex as reported in the previous paper (Usuki and Irie, 1983). While the minor variants in the former fit to Hb3, Hb 6 and HbH in the latter. As summarized in Table 2, relative concentration of the major variant (in correspondence to Hb~0) reaches more than 94%, whereas each of other variants remains only a few per cent in P. caudatum. There is

422

ITARU USUKI a n d TOSHIAK] IR]E

Table 1. Stocks used for the investigation Species

Syngcn

P. caud.

1

P. multi

2

P. jen.

1

Mating Type

Stock

Sa 9 YnK I Sa 6 YnK 2

1 I 1I 11

Nn21 1 GT 601 Sh 23 Ym la c103 Yd 1

V V V VI VI V, VI

Yg 2 CNR

V, VI V, VI

53 B TH 103

III IV

Geographic origin

Sendai, Japan Kofu. Japan Sendal Japan Kofu. Japan Nagoya, Japan Tsukuba. Japan Sendai. Japan Sakata, Japan Burgsteinfurt, Germany a progeny of Yt 1 (from Yamagata) × d m-t 3 (from Kyoto) a trichocyst non-discharge mutant* a cilia non-reversal mutant+ USA USA South India:]:

*Takei and Hiwatashi (1981). tTakahashi and Naitoh (1978). ++Diller and Earl (1958) and Sonneborn (1958).

no significant variation between both of the syngens examined, although some deviation in relative concentration of the individual variants is recognized among different stocks examined. As seen in Fig. 2 and Table 2, the Hb profile of P. jenningsi is a close resemblance to that of P. caudatum. Thus, the major variant in this ciliate corresponds to Hb Wof P. aurelia complex and its concentration is by far the highest, with an association of trace amounts of other variants, which correspond to H b 3, H b 6 a n d Hb~, as in P. caudatum.

aurelia complex. This variant was nearly 90°,o in relative concentration. The remaining other variants in P. multimicronucleatum fitted closely with Hb2, Hb3 and Hb~2, which differed obviously from those of P. caudatum. Among the minors, the variant which corresponds to Hb2 is most enriched, although the relative concentration is no more than approx 7%. In Fig. 3 the SDS-PAGE patterns of the major variants isolated from some stocks of P. caudatum, P. jenningsi and P. muhimicronucleatum are shown. The variants from different sources are arranged in a

P. multimicronucleatum showed several definite Hb bands by PAGE. The greatest amount shows faster migration than those of P. caudatum and P. ienningsi, and it corresponds to Hb~ in the variants of P. Hb~ Hb 5 Hb7

Hb,o

Hbl, Hb12

I

O

~

m

0

0

Q

~

~

0

O1

~ ,

+

co

.~

co

(D

~

0")

--<

< Z

0 --I

cn ~

0

CO <

~

--< 3

0 0

~" <

03 <

~ < +

<

ol

---t con

co

I

2UI~

<

<

<

<'S

<

_<

_ <-

+ --

-..l

-l-

I C

Ii d

oo

GO

M

I I

Syngen

1

Syngen

3

Fig. I. Hb profiles from four stocks of syngen 1 and six stocks of syngen 3 in P. caudatum, representing single definite bands with an identical mobility in various stocks of different syngens. Electrophoresis was carried out using a 15% polyacrylamide slab gel. The gel was unstained.

Fig. 2. Hb profiles from P. caudatum (C), P./enningsi (J) and P. multimicronucleatum (M), representing similarities and differences in the mobility of the variants on a 15'lj polyacrylamide slab gel. For a comparison, the Hb from stock 51 of P. tetraurelia (T) was run on the same gel. The gel was unstained.

423

Paramecium hemoglobin--II

Table 2. Relative concentration of Hb variants distributed to some stocks of P. caudatum, P. jenningsi and P. multimicronucleatum Variant

P. caudatum Yd 1 Yg 2

Sa 9

Hb 2 Hb 3 Hb 6 Hbl0 Hb u Hbl2

.

. 1

97 2 .

. . 1-2 2 trace 1 96-97 94 2 3 . .

P. jen.

CNR

53 B

. 1 1 94 4-5 .

trace trace 99 1

7 2 1 88 2

P. multi. TH 103 53 B × TH 103

6 2 --90 2

5 3 1 -88-89 2-3

Each of the variants was identified on the basis of mobility by PAGE, in the standard of those in P. aurelia complex as reported in the previous paper (Usuki and Irie, 1983). Variant concentrations were determined by a densitometric analysis, with 420 nm in the sample beam and 600 nm in the reference beam, using an electrophoresed unstained 15% polyacrylamide slab gel.

line, which coincides well with the mobility of the P. aurelia Hbs with a mol. wt of approx 11,000, as reported in the previous paper (Usuki and Irie, 1983).

DISCUSSION

It has been reported that P. caudatum contains a single form of Hb, in both of syngen 1 (Steers et al., 1981) and syngen 3 (Irie and Usuki, 1980). However, this study makes clear that the P. caudatum Hb is composed of several variants, which comprise a major variant at a high concentration and minor variants at a low concentration. This indicates that the Hb in P. caudatum is principally polymorphic, as

Oa

Mb Hb3

Cc

Hblo

I~

I'~" i

-,

-

O

~

--

I

+

o

C

J

M

T

Fig. 3. SDS-PAGE patterns of the major variants isolated from P. caudatum (C), P. jenningsi (J) and P. multimicronucleatum (M). Before the SDS-treatment, the sample Hbs were extracted from the corresponding bands after cutting from previously electrophoresed 15~0 polyacrylamide disc gels. For a comparison, a mixture of Hb 3 (with a mol. wt of 13,000) and Hb~0(with a mol. wt of 11,000) isolated from stock 51 of P. tetraurelia (T) was applied. The electrophoresis was performed using a 15~opolyacrylamide slab gel containing 0.1% SDS. The gel was stained with 0.25~o Coomassie Brilliant Blue. Oa, ovalbumin; Tg, tripsinogen from bovine pancreas; Mb, sperm whale myoglobin; Cc, cytochrome c from horse heart.

are the Hbs in P. aurelia complex reported in the preceding paper (Usuki and Irie, 1983). We inquired into the mutual relationship between the variants from different Paramecium species, on the basis of the mobility by P A G E and SDS-PAGE. This proved that the major variant in P. caudatum corresponds closely to Hb~0 in P. aurelia complex and the minors in the former fit to Hb3, Hb 6 and Hbu in the latter, regardless of the syngen-difference in this ciliate. Relative concentration of the individual variants resolved show some variations among the stocks examined, although the deviation observed is too small to evaluate a significant intrasyngenic variation of Hb. We have previously reported that P. aurelia complex exhibits considerable inter- and intrasyngenic variations of the Hbs (Usuki and Irie, 1983). In a connection with this, inter- and intrasyngenic variations of various enzymes are known in P. aurelia complex (Allen and Gibson, 1971; Allen et al., 1973; Tait, 1970, 1978) and in Tetrahymena pyriformis complex (Allen and Weremiuk, 1971; Borden et al., 1973). On the other hand, P. caudatum has been reported to have no inter- and intrasyngenic variation of lactate dehydrogenase, with an exception of one case (Agatsuma & Tsukii, 1980). This may offer an analogy with the Hb of P. caudatum. Steers et al. (1981) have described that the P. caudatum Hb has a mol. wt of 13,500, smaller than the value of 15,000 + 500 for the multiple Hbs of P. tetraurelia (Davis and Steers, 1976; Steers and Davis, 1979). Irie and Usuki (1980) have reported the value of approx 11,000 for the H b from P. caudatum and P. primaurelia. In this study it was demonstrated that the S D S - P A G E pattern has identical mobility of the major variants isolated from P. caudatum, P. jenningsi and P. multimicronucleatum, and that the mobility coincides well with those of the P. aurelia Hbs with a mol. wt of 11,000 (Usuki and Irie, 1983). P. jenningsi has been reported to hold a close connection with P. aurelia on morphological grounds (Diller and Earl, 1958; Sonneborn, 1958). It is an interesting fact that, however, this species generates an identical Hb to that of P. caudatum. That is, the H b from P. jenningsi is practically impossible to distinguish from the P. caudatum Hb by P A G E and S D S - P A G E , although we have found that isoelectric focusing brings about a slight difference between the major variants from both of the Paramecium species (Usuki and Irie, unpublished).

424

ITARU USUK[ and TOSHIAKI ]R1E

P. multimicronucleaturn reveals a multiplicity of H b by P A G E . The Hbs resolved are c o m p o s e d of a m a j o r variant c o r r e s p o n d i n g to H b ~ in P. aurelia complex, in association with certain m i n o r s c o r r e s p o n d i n g to H b > H b 3 and Hb~2. Such variant c o m p o s i t i o n in this species distinctly differs from those of P. caudatum, P. jenningsi and of m a n y syngens of P. aurelia complex as reported in the previous p a p e r (Usuki and Die, 1983). In addition to this, the m a j o r variant in P. multimicronucleatum is by far the p r e d o m i n a n t in relative c o n c e n t r a t i o n , reaching to nearly 907,i, a n d the H b shows no significant intrasyngenic variation. These facts may indicate t h a t P. multimicronucleatum H b is nearer to P. caudatum H b t h a n P. aurelia Hbs, a l t h o u g h further investigation on m a n y stocks and syngens is desired. A&nowledgements--We wish to thank Dr K. Hiwatashi, Biological Institute, Tohoku University, Sendai, and Dr K. Mikami, Miyagi College of Education, Sendai, for the kind supply of the stocks of P. caudatum and P. jenningsi and their valuable advice for the culture. We are also indebted to Dr Y. Takagi, Department of Biology, Nara Women's University, Nara, for the supply of the seed animals of P. multimicronueleatum. REFERENCES

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Allen S. L., Farrow S. W. and Golembiewski P. A. (1973) Esterase variations between the 14 syngens of Paramecium aurelia under axenic growth. Genetics' 73, 561 573. Borden D., Miller E. T.. Nanney D. k. and Whitt (5. S. (1973) The inheritance of enzyme variants for tyrosinc aminotransferase, NADP-dependent malate dehydrogenase, and telrazolium oxidase in Tetrahvmena pyri[ormis, syngen I. Gem,ties 74, 595 6(/3. Davis R. H. Jr and Steers E. Jr (1976) Myoglobin from the ciliate protozoan Paramecium aurelia. Comp. Biochcm. Physiol. 54B, 141 143. Diller W. F. and Earl P. R. (1958) Parameeium/ennings'i, n. sp. J. Protozool. 5, 155 158. lrie T. and Usuki I. (1980) Disparity of native oxyhemoglobin components isolated from Paramecium caudatum and Paramecium primaurelia. ('omp. Biochem. Physiol. 67B, 549--554. Sonneborn T. M. (1958) Classification of syngens of the Paramecium aurelia- multimieronueh,atum complex. J. Protozool. 5(suppl.), 17 18. Steers E. Jr and Davis R. H. Jr (1979) Purilication and characterization o[" the myoglobins of Paramecium tetraurelia. Comp. Bioehem. Physiol. 62B, 393 402. Steers E. Jr, Barnett A. and Lee C. E. (1981) Isolation and characterization of the hemoglobin from Paramecium eaudatum. Comp. Bioehem. Physiol. 70B, 185-191. Tait A. (1978) Species identification in protozoa: Glucosephosphate isomerase variation in the Paramecium aurelia group. Bioehem. Genel. 16, 945-955. Takahashi M. and Naitoh Y. (1978) Behavioral mutants of Parameeium eaudatum with defective membrane electrogenesis. Nature, Lond. 271, 656-659. Takei K. and Hiwatashi K. (1981) Trichocyst non-discharge mutants in Paramecium eaudatum and their genetic analysis (in Japanese). Jap. J. Proto=ool. 14, 44-45. Usuki 1. and Irie T. (1983) Inter- and intrasyngenic variation of Paramecium hemoglobin I. Parumecium aurelia complex. ('omp. Biochem. Physiol. 75B, 415-420.