9
Ann. Microbiol. (Inst. Pasteur) 1984, 135 A, 73-82 COMPARISON OF THE AMINO ACID COMPOSITIONS AND ANTIGENIC PROPERTIES OF SPIRALINS PURIFIED FROM THE PLASMA MEMBRANES OF DIFFERENT SPIROPLASMAS by H. Wr6blewski, D. Robic, D. Thomas and A. Blanchard Groupe de Recherches en Biologie cellulaire, Universitd de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex (France) SUMMARY. Spiralins were purified by agarose-suspension electrophoresis after extraction with detergents from the membranes of the following spiroplasmas: Spiroplasma cirri C189, S. cirri Maroc (R8A2), S. cirri Scaph and the honey-bee spiroplasma B88. The four proteins (molecular mass~26,000 daltons, as determined by sodium dode- eyl sulphate-pore gradient electrophoresis) showed very similar amino acid compo- sitions characterized by the absence of methionine and tryptophan and a high polarity index (>49%). When compared with the amino acid composition of S. citri membrane, the four spiralins had little or no histidine, a low content of glycine, leucine, tyrosine, phenylalanine and arginine, and a high content of threo- nine, alanine and valine. Comparison of the amino acid compositions according to the criteria described by Cornish-Bowden (Anal. Biochem., 1980, 105, 233-238) strongly suggests that all four spiralins are related. A crossed immunoelectropho- retical comparison, however, shows that though the three proteins purified from S. cirri strains (serogroup I-l) are antigenically similar, they do not seem to share common epitopes with spiralin from the honey-bee spiroplasma B88 (serogroup I-2). KEY-WORDS: Spiroplasma cirri, Spiralin, Amino acid; Antigenic properties, Polarity index, Similarity. INTRODUCTION. Spiralin is the most abundant protein in the membrane of Spiroplasma cirri C189, where it contributes up to 22% of the total protein fraction [16]. This amphi- philic molecule is capable of torming homooligomers in situ and has an apparent molecular mass of 26 kdaltons [13, 14, 16]. It is also the main antigen of the S. cirri membrane [11]. The function of this protein remains unknown. We have, however proposed a model suggesting a role in the determination of the cell shape of S. eitri Manuserit re~u le 8 aofit 1983. Correspondence should be sent to H. Wr6blewski.

Comparison of the amino acid compositions and antigenic properties of spiralins purified from the plasma membranes of different spiroplasmas

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Page 1: Comparison of the amino acid compositions and antigenic properties of spiralins purified from the plasma membranes of different spiroplasmas

Ann. Microbiol. (Inst. Pasteur) 1984, 135 A, 73-82

COMPARISON OF THE AMINO ACID COMPOSITIONS

AND ANTIGENIC P R O P E R T I E S

OF SPIRALINS P U R I F I E D

FROM THE PLASMA MEMBRANES

OF D I F F E R E N T SPIROPLASMAS

by H. Wr6blewski, D. Robic, D. Thomas and A. Blanchard

Groupe de Recherches en Biologie cellulaire, Universitd de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex (France)

SUMMARY.

Spiralins were purified by agarose-suspension electrophoresis after extraction with detergents from the membranes of the following spiroplasmas: Spiroplasma cirri C189, S. cirri Maroc (R8A2), S. cirri Scaph and the honey-bee spiroplasma B88. The four proteins (molecular mass~26,000 daltons, as determined by sodium dode- eyl sulphate-pore gradient electrophoresis) showed very similar amino acid compo- sitions characterized by the absence of methionine and tryptophan and a high polarity index (>49%). When compared with the amino acid composition of S. citri membrane, the four spiralins had little or no histidine, a low content of glycine, leucine, tyrosine, phenylalanine and arginine, and a high content of threo- nine, alanine and valine. Comparison of the amino acid compositions according to the criteria described by Cornish-Bowden (Anal. Biochem., 1980, 105, 233-238) strongly suggests that all four spiralins are related. A crossed immunoelectropho- retical comparison, however, shows that though the three proteins purified from S. cirri strains (serogroup I-l) are antigenically similar, they do not seem to share common epitopes with spiralin from the honey-bee spiroplasma B88 (serogroup I-2).

KEY-WORDS: Spiroplasma cirri, Spiralin, Amino acid; Antigenic properties, Polarity index, Similarity.

INTRODUCTION.

Spiralin is the most abundant protein in the membrane of Spiroplasma cirri C189, where it contributes up to 22% of the total protein fraction [16]. This amphi- philic molecule is capable of torming homooligomers in situ and has an apparent molecular mass of 26 kdaltons [13, 14, 16]. It is also the main antigen of the S. cirri membrane [11]. The function of this protein remains unknown. We have, however proposed a model suggesting a role in the determination of the cell shape of S. eitri

Manuserit re~u le 8 aofit 1983.

Correspondence should be sent to H. Wr6blewski.

Page 2: Comparison of the amino acid compositions and antigenic properties of spiralins purified from the plasma membranes of different spiroplasmas

74 H. W R O B L E W S K I AND COLL.

in conjunction with extrinsic proteins [12]. Some data suggest tha t these proteins may be the fibrils recently observed in several spiroplasmas [8, 9].

The goal of the present investigation was to purify spiralins from different spiroplasmas and to compare them with the protein first isolated from S. cirri C189, at both the chemical and antigenic levels.

~]ATERIALS AND METHODS.

Purification o] spiralins. The spiroplasmas were grown as previously described [16], except tha t the

concentration of foal serum in the culture medium was reduced to 5% (vol/vol). The cells were harvested by centrifugation (15,000 g, 15 min, 4 ~ C), dispersed into 50 mM Tris-hydrochloride buffer (pH 7.5) and disrupted by sonication (20 kcycles, 2 • 1 min, 0 ~ C). The membranes were pelleted by eentrifugation (40,000 g, 1 h, 4 ~ C), extensively washed with 50 mM Tris-hydrochloride buffer (pH 8.0) and depleted of the bulk of extrinsic proteins [14]. Spiralin was purified from these membranes by agarose-suspension eleetrophoresis in the presence of DOC, after extraction with detergents under non-denaturing conditions [16].

Sodium dodecgl sulphate pore-gradient eleclrophoresis (SDS-PGE). Linear gradients with polyacrylamide concentrations of 6-30% were used to

analyse samples by SDS-PGE. Bis-acrylamide concentration was 3.5% of total monomer concentration. Electrophoresis was carried out for 3,000 V/h in 40 mM Tris-20 mM acetate buffer (pH 7.4) containing 2 mM EDTA and 0.1% SDS.

Deoxgcholate-crossed immunoelcctrophoresis (DOC-CIE). Detergent-CIE [1, 5] was performed in 1-mm thick agarose gels cast on 7 • 7 cm

hydrophilic plastic sheets (Gel Bond film from Bio Bad). The agarose concentration was 1% (w/v) in veronal buffer pH 8.6 (I =0.03). To prevent spiralin precipitation, 13 mM D0C was incorporated into the gel and the eatholyte of the first directional electrophoresis [15]. Other experimental details were as described [6, 15]. Anti- membrane and anti-spiralin antibodies were elicited in rabbits by subcutaneous inoculation (twice a month for three months) of an emulsion made with a suspension of membranes (2 mg/ml) or a solution of pure spiralin (10 ~g/ml) and incomplete Freund's adjuvant, respectively.

Amino acid analysis. Amino acid analysis of the purified spiralins was performed as described

earlier [161 .

Test o/composition data. Similarity of amino acid compositions was estimated by the use of the index

of Marchalonis and Weltman [7] as described by Cornish-Bowden [4]. When compar- ing the compositions of two proteins A and B, the Marchalonis and Weltman index is:

SAQ=10 4 E (Xi~--Xi~) 2

DOC-GIE = crossed immunoelectrophoresis in the presence of sodium deoxy- cholate.

SDS-PGE = pore-gradient electrophoresis in the presence of sodium do-deeyl sulphate.

Spiralin B88 = spiralin f rom the honey-bee spiroplasma B88.

Spiralin C189 = spiralin f rom S. cirri C189. Spiralin 1R8A2 = spiralin f rom S. citri R8A2. Spiralin Scaph = spiralin f rom S. citri Scaph.

Page 3: Comparison of the amino acid compositions and antigenic properties of spiralins purified from the plasma membranes of different spiroplasmas

A M I N O A C I D C O M P O S I T I O N O F F O U R S P I R A L I N S 75

where 100 XiA and 100 XiB are t h e mole pe r cen t f r ac t ions of t he i th t y p e of amino acid in p ro te in A and p ro te in B, respec t ive ly . Therefore , t he smal le r the SAQ va lue , t h e h igher the s im i l a r i t y be tween the two pro te ins .

RESULTS.

The h o m o g e u e i t y of t he spi ra l ins pur i f ied f rom the m e m b r a n e s of S. cirri R8A2, S. cirri Scaph and of t he sp i rop la sma B88 was assessed b y S D S - P G E and b y DOC- CIE. Only one b a n d was de tec ted in S D S - P G E for each p ro t e in p r e p a r a t i o n , even wi th a high load of sample p rov ing the absence of m i n o r c o n t a m i n a n t s (fig. 1). E a c h spi ra l in had an a p p a r e n t molecu la r mass of a b o u t 26 kda l tons , i den t i ca l to t he figure ear l ier ob t a ined for sp i ra l in C189 [16]. Only one i m m u n o p r e c i p i t a t e was e v i d e n t in D O C - C I E when sp i ra l ins were run in a gel con ta in ing an t ibod ie s aga ins t t h e m e m b r a n e f rom which the p ro te in was i so la ted . F igu re 2 i l lus t ra tes t he resul t s o b t a i n e d wi th sp i ra l in f rom sp i rop lasma B88.

w

FIG. 1. - - SDS-PGE o[ spiroplasma membrane proleins and of purified spiralins. (1) 10 ~g of spiralin B88; (2)60 ~g of unfractionated membrane protein from the honey-bee

spiroplasma B88; (3) marker proteins: lactate dehydrogenase (36,000 daltons), catalase (60,000 daltons), serum albumin (67,000 daltons) and thyroglobufin (330,000 daltons); (4) mar- ker proteins: ~4actalbumin (14,400 daltons), trypsin inhibitor (20,100 daltons), carbonic anhydrase (30,000 daltons), serum albumin (67,000 daltons) and phosphorylase b (94,000 dal- tons); (5) 25 ~g of extrinsic proteins from spiroplasma B88 membrane; (6) 35 ~g of integral proteins from the spiroplasma B88 membrane; (7) 15 ~g of spiralin ]q8A2; (8) 15 ~zg of spiralin Scaph. All proteins were solubilized with 0.1 M SDS in the presence of 0.2 M 2-mercaptoethanol. Anode at bottom. Proteins were stained with Serva Blue lq. F ~ fibril protein.

Page 4: Comparison of the amino acid compositions and antigenic properties of spiralins purified from the plasma membranes of different spiroplasmas

76 H. W R O B L E W S K I AND COLL.

TABLE I. - - The amino acid composi t ion of the (( S. citri ~) C189 membrane and of four spiralins purified from different spiroplasmas.

S. citri C189

Amino membrane acid (tool %)

Spiralin (mol %)

C189 (*) I /8A2 Scaph B88

Asx 11.53 13.53 13.62 13.67 Thr 6.94 9.41 9.64 9.37 Ser 5.08 4.58 6.68 4.61 Glx 10.23 10.44 10.02 10.20 Pro 3.62 4.21 3.50 4.24 Gly 7.24 5.07 5.00 5.07 Ala 8.32 12.50 12.84 13.30 1/2 Cys 0.67 0.64 0.55 0.58 Yal 7.76 11.91 11.81 11.98 Met 1.65 0 0 0 Ile 8.10 6.86 6.35 6.39 L eu 7.43 4.92 4.79 4.88 Tyr 3.04 2.26 2.45 2.46 Phe 3.48 1.55 1.83 1.87 His 1.20 0.11 0 0 Lys 10.11 11.50 10.84 11.15 Arg 2.84 0.25 0 0.25 Trp 0.74 0 0 0 P I 47.93 49.82 50.80 49.25

11.37 10.85

5.47 9.42 3.10 5.86

13.99 0.00

11.30 0 5.32 4.40 2.18 2.36 0.60

13.12 0.65 0

51.48

(*) Data f rom Wroblewski et al. [16]. PI: polari ty index (tool % of polar amino acids: Asx, Thr, Ser, Glx, His, Lys, and Arg) [3].

The amino acid compositions of the unfractionated membrane of S. cih'i C189 and of the purified spiralins are shown in table I. The compositions of the four spiralins were similar and quite different from that of the membrane. The spiralins were characterized by the absence of methionine and tryptophan. The amount of arginine and histidine was negligible. Compared to the unfractionated membrane, the four spiralins had a low content of glycine, leucine, tyrosine and phenylalanine. Conversely, they had a high content of threonine, alanine and valine. It should also be noted tha t the spiralins had a polarity index > 4 9 % slightly higher than the membrane (~_48%).

To measure the similitaries between amino acid compositions, the Marchalonis and Weltman index [7] was calculated for each pair of proteins. The figures which were obtained are shown in table II. SAQ values ranged from 1.2 to 6.2 when spiralins isolated from the three S. cirri strains were compared. Somewhat higher values were obtained when spiralin B88 was compared to the spiralins from S. cirri strains (18.1 ~<S. AQ ~<19.8).

Fla. 2. - - DOC-CIE o[ spiralin and of unfractionated membrane of the honeg-bee spiroplasma B88.

Samples: 25 ~g of membrane protein (A) and 5 lxg of pure spiralin (B) solubilized wi th 0.2 M DOC in the presence of 0.2 M 2-mercaptoethanol . Size of the gel: 85 x 100 X 1 mm. Concentrat ion of ant i -spiroplasma B88 membrane serum: 5 ~ l . em -2. Anode: r ight (first direction) and top (second direction). Immunopree ip i ta tes were stained wi th Serva Blue t/.

Arrow points out of the spiralin immunoprecipi ta te .

Page 5: Comparison of the amino acid compositions and antigenic properties of spiralins purified from the plasma membranes of different spiroplasmas

ii!i}i!iiii!iiii !ii!!iii!ii!!~!

iiii' !iiiiiiiiiiii~iiii i}iiiil}iii!}ili} i!iiii}}i3ii!!i]!!!

~iiiii~iiii!ili!i!ili

ii{!ii~i!ii}lii!iill

i!iiiiiiiii!!iiii!ii~

!iiiiii~iiii!!iiii!

A

B

FI~. 2

Page 6: Comparison of the amino acid compositions and antigenic properties of spiralins purified from the plasma membranes of different spiroplasmas

A M I N O A C I D C O M P O S I T I O N O F F O U R S P I R A L I N S 79

TABLE II . - - SAQ va lues ob ta ined by c o m p a r i n g the a m i n o acid compos i t i ons

of f o u r sp i ra l ins f r o m di f ferent or igins .

B88 0 C189 19.8 0 R8A2 18.7 6.2 0 Scaph 18.1 1.2 5.8 0

B88 C189 R8A2 Scaph

A n t i g e n i c r e l a t i o n s h i p s were i n v e s t i g a t e d b y r u n n i n g t h e sp i ra l ins b y pa i r s in t a n d e m C I E in aga rose gels c o n t a i n i n g a n t i - s p i r a l i n C189 or a n t i - s p i r a l i n B 8 8 a n t i - bodies . F i g u r e 3 i l l u s t r a t e s an e x a m p l e of such a c o m p a r i s o n . T h e re su l t s a r e s u m m a - r i zed in t a b l e I I I . C ros s - r eac t ions ( p r e s e n t l y , r e a c t i o n s of c o m p l e t e fu s ion of i m m u n o p r e c i p i t a t e s ) were e v i d e n t b e t w e e n t h e sp i r a l in s i so l a t ed f r o m t h e S . c irr i s t ra ins , b u t n o t b e t w e e n t he se p r o t e i n s a n d sp i r a l in B88.

Fro. 3. - - Tandem crossed immunoelectrophoresis of two purified spiralins.

Samples: 3 ~g of spiralin C189 (left) and 4 ~g of spiralin R8A2 (right). Concentration of anti- spiralin C189 serum: 5 Ezl. cm -2. See legend to figure 2 for other experimental details. Observe reaction of complete fusion between immunoprecipitates. O = antigen wells (origins of antigen migration in the first directional electrophoresis).

TABLE I I I . - - A n t i g e n i c c ross - reac t iv i ty between spiralins as evidenced by D00-CIE.

B88 + C189 -- + R8A2 -- + + Scaph -- + +

B88 C189 R8A2 Scaph

+ indicates fusion of immunoprecipitates and therefore antigenic cross-reactivity.

-- indicates absence of cross-reactivity. NB. - - Positive reactions were always reactions of complete fusion.

Page 7: Comparison of the amino acid compositions and antigenic properties of spiralins purified from the plasma membranes of different spiroplasmas

80 H. W R O B L E W S K I AND COLL.

DISCUSSION.

Spiralins were purified from the membrane of three different strains of S. cilri and from the honey-bee spiroplasm B88. The homogeneity of the proteins was demonstrated by SDS-PGE and by DOC-CIE. The unique properties of their amino acid composition, and in particular, the absence of both methionine and t ryptophan, are further evidence of their high degree of purity.

Assuming a molecular weight of 26 kdaltons, each spiralin contains about 250 amino acid residues per tool. The SAQ values (1.2 to 19.8) are well under the critical value of 33.6 defined by the (( strong test )) of Cornish-Bowden [4] for proteins of this size. This indicates a strong similarity between the four spiralins. Interest- ingly, significantly smaller values (1.2~<S. AQ ~<6.2) were obtained when comparing S. cilri spiralins than when comparing them with spiralin B88 (18.1 ~S.AQ~19.8) .

The chemical similarities were in agreement with results of the antigenic comparisons, since cross-reactivity was observed between S. cilri spiralins, but not between them and spiralin B88. Reactions of complete fusion between immuno- precipitates of spiralins C189, R8A2 and Scaph suggest tha t these proteins share the same epitopes, which supports the classification of the corresponding strains in the species S. cilri (serogroup I-l) [2, 10]. The evidence of chemical similarity, but the apparent absence of shared epitopes between spiralin B88 and S. citri spiralins, is in agreement with the classification of the honey-bee spiroplasma B88 m the same serogroup as S. cirri strains but in a different subgroup (I-2) ]2].

This investigation suggests tha t anti-spiralin antibodies may prove to be very valuable tools not only in exploring the topofunctional properties of spiralin [12], but also for taxonomic purposes. This has already been anticipated in a serological comparison of spiroplasmas based upon the use of anti-membrane and anti- spiralin antibodies in growth and metabolism linhibition tests and in deformation tests [11]. Anti-spiralin antibodies should also be useful in the isolation of mem- brane surface mutants (spiroplasmas with modified spiralin ecto-epitopes) or for the inhibition of fast growing spiroplasmas in order to select slowly growing ones from, for example, plant or animal extracts.

RESUMs

COMPARAISON DE LA COMPOSITION EN AMINOACIDES ET DES PROPRIETIES ANTIGt~NIQUES DE SPIRALINES

PURIFIEES A PARTIR DE DIFFERENTS SPIROPLASMES

Quatre spiralines ont dt~ purifides par ~lectrophor~se sur suspension d'agarose apr~s extraction au moyen de ddtergents h part ir de la membrane des spiroplasmes suivants : Spiroplasma cilri C189, S. cilri Maroe (R8A2), S. cirri Scaph et le spiro- plasme B88 de l'abeille. Ces protSines poss~dent une m~me masse moldeulaire apparente (_~26 000 daltons) estimde par ~lectrophor~se sur gradient de pores en pr~senee de dod~eyl sulfate de sodium. Leurs compositions en acides aminSs sont tr~s voisines et caract~risdes par l'absence de m~thionine et de t ryptophane et par un indite de polarit5 dlevd (>49%) . Par rapport h la fraction protdique mem- branaire totale de S. cilri C189, les quatre spiralines purifi~es eontiennent peu ou pas d'histidine, peu de glyeine, de leueine, de tyrosine, de phdnylalanine et d'arginine ; en revanche, elles contiennent beaueoup de thr~onine, d'alanine et de valine. La eomparaison des compositions en aminoaeides selon les erit~res ddcrits par Cornish- Bowden (Anal. Biochem., 1980, 105, 233-238) sugg~re fortement que les quatre spiralines sont apparentdes. Une analyse immuno~leetrophordtique montre que si les trois protfiines purifi~es /~ part ir de souehes de S. cilri (sfirogroupe I-l) sont

Page 8: Comparison of the amino acid compositions and antigenic properties of spiralins purified from the plasma membranes of different spiroplasmas

AMINO ACID COMPOSITION OF FOUR SPIRALINS 81

antigdniquement similaires, elles ne semblent pas contenir d'dpitopes identiques h ceux de la spiraline du spiroplasme B88 de l'abeille (sdrogroupe I-2).

MOTS-CLI~S : Spiroplasma citri, Spiraline, Acide amind ; Propridtds antigdniques, Indice de polarit6 ; Similarit6.

ACKNOWLEDGEMENTS

This work was supported by the Centre National de la Recherche Scientifique (LA 256, Contrat CNRS-Universit6 and ATP (( Microbiologie )), Contrat N ~ 29.82.25).

Thanks are due to A. M. Touzalin in for skillful technical assistance and to D. Webb for critical reading of the manuscript.

REFERENCES

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[2] Bov]~, J. M., SAILLARD, C., JUNCA, P., DE GORcE-DUMAS, J. R., RICARD, B., NHAMI, A., WHITCOMB, R. F. , WILLIAMSON, D. & TULLY, J. G., Guanine- plus-cytosine content, hybrida tion percentages, and EcoRI restriction enzyme profiles of spiroplasmal DNA. Rev. in/ecl. Dis., 1982, 4, S129-S136.

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[5] JOHANSSON, K.-E. & HJEnT~N, S., Localisation of the Tween 20-soluble mem- brane proteins of Acboleplasma laidlawii by crossed immunoelectropho- resis. J. tool. Biol., 1974, 86, 341-348.

[6] JOHANSSON, K.-E. & WROBLEWSKI, H., Characterization of membrane proteins by crossed immunoelectrophoresis, in (( Methods in myeoplasmology )) (J. G. Tully & S. Razin), vol. I, (p. 257-267). Academic Press, London, New York, 1983.

[7] MARCHALONIS, J. J. & WELTMAN, J. K., Relatedness among proteins: a new method of estimation and its application to immunoglobulins. Comp. Biochem. Physiol., 1971, 38 B, 609-625.

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[10] WHITCOMB, R. F., TULLY, J. G., CLARK, T. B., WILLIAMSON, D. L. & Bov~, J. M., Revised serological classification of spiroplasmas, new provi- sional groups, and recommendations for serotyping of isolates. Curr. Microbiol., 1982, 7, 291-296.

[11] WHITCOMB, R. F., TULLY, J. G. & WROBLEWSKI, ]~t., Spiralin: maj or membrane protein specific to subgroup I-1 spiroplasmas. Curt. Microbiol., 1983, 8, 7-12.

[12] WR()BLEWSKI, H., Spiralin: its topomolecular anatomy and its possible func- tion in the Spiroplasma cirri cell membrane. Zbl. Backt., I. Abt. Orig., 1978, 241, 179-180.

[13] WROBLEWSKI, H., Amphiphilic nature of spiralin, the major protein of the Spiroplasma cilri cell membrane. J. Bact., 1979, 140, 738-741.

[14] WR6BLEWSKI, H., Electrophoretic analysis of the arrangement of spiralin and of other major proteins in isolated Spiroplasma cirri cell membranes. J. Bacl., 1981, 145, 61-67.

Ann. MicrobloL (Inst. Pasteur), 135.4, n ~ 1, 1984. 6

Page 9: Comparison of the amino acid compositions and antigenic properties of spiralins purified from the plasma membranes of different spiroplasmas

82 H. WROBLEWSKI AND COLL.

[15] WR6BLEWSKI, H., oloHANSSON, K.-E. & ]~URLOT, R., Crossed immuno- electrophoresis of membrane proteins from Acholeplasma laidlawii and Spiroplasrna cilri. Int. J. sgst. Bact., 1977, 27, 97-103.

[16] WR~)BLEWSKI, H., JOHANSSON, K.-E. & HJERT~N, S., Purification and characterization of spiralin, the main protein of the Spiroplasma cirri membrane. Biochim. biophgs. Acta (Amst.), 1977, 465, 275-289.