Histones from the Fruit Fly Ceratitis capitata : Isolation and Characterization

Eur. J. Biochem. 48, 53-61 (1974)

Histones from the Fruit Fly Ceratitis capitata Isolation and Characterization

Luis FRANCO, Francisco MONTERO, Jacinto M. NAVLET, Julian PERERA, and M. Carmen ROJO Departamento de Bioquimica, Facultad de Ciencias, Universidad Complutense, Madrid

(Received April 30, 1974)

1. Histones from the fruit fly Cerutitis cupitatu have been obtained and characterized by amino acid analysis and polyacrylamide gel electrophoresis. It has been found that the five major histone groups are present in this insect.

2. The fractionation of histones has been achieved by using acid or organic selective extractions. Cerutitis cupituta F1 exhibited a reduced electrophoretic mobility when compared with the mammalian corresponding histone. On the contrary, fly F2A1 mobility was similar to that of mammalian homologous histone. Some differences were found, however, in amino acid composition. It has been found that cysteine does not occur in fly F2A1, but this amino acid seemed to be present in the F3 histone, which aggregated under oxidizing conditions.

3. A strongly basic protein was isolated together with histones when pharate adult chromatin was used as starting material. Amino acid analysis of this protein revealed the presence of hydroxyproline (19 %), as well as high amounts of threonine (30 x) and arginine (23 %). It was found that this specific protein was not a nuclear component and it seemed to be located at the pharate adult envelope; the name of purarium-specific protein is proposed for this protein. Its strongly basic nature is judged to be the cause by which it became attached to chromatin during the isolation procedure.

Histones have been isolated from a wide variety of eukaryotic cells, from unicellular organisms to mammalian tissues. Certain invertebrate species have also been examined for their histone content, but present knowledge about the histone complement of lower animals is still far from complete. Research on the histone complement of insects seems to be of some interest, because of their situation in the context of biological evolution and, on the other hand, due to the well-defined developmental changes taking place during metamorphosis and differentiation of these organisms.

Lindh and Brantmark [l] first studied the basic nuclear proteins from the blowfly Culliphoru erythro- cephulu, but the identification of such proteins as histones was not easy because of the complex electrophoretic patterns obtained. More interpretable patterns were obtained by Pallotta and Berlowitz [2], working with both crickets (Achetu domesticu) and mealy bugs (Plunococcus citvi) and by Harris and Forrest [ 3 ] with the milkweed bug Oncopeltus dusciutus.

Dick and Johns [4] used a high-salt dissociation method to isolate chromatin basic proteins from

Drosophila melunoguster adults. Cohen and Gotchel [ 5 ] were able to obtain histones from polytene nuclei of D. melunoguster and they found a close similarity between these histones and those isolated from non- polytene nuclei. These authors also extracted F1, F2A, F2B and F3 fractions from Drosophilu nuclei by the method 1 of Johns [6]. More recently, Oliver et ul. have used a method based on the differential solubilization of histone perchlorates for fractionating Drosophilu histones [7]. The extent of cross-contamination between different fractions is less than 2 % [8], thus making possible a more accurate comparison of Drosophilu and higher animal histones.

The five major histone fractions found in mammalian tissues are also present in D. melunogastev chromatin, although some dissimilarities were found between fly and calf thymus histones in both electrophoretic behaviour and amino acid composition. The most important difference was found in the very lysine-rich histone; Drosophilu F1 exhibits a relatively low lysine content, as well as a low alanine content. This protein also moves with an electrophoretic mobility lower than that of the comparable thymus histone. Cohen

Eur. J. Biochem. 48 (1974)

54 Histones from the Fruit Fly C. capitata

and Gotchel interpreted this difference in terms of a 10 % increase in molecular weight [5], but Oliver and Chalkley [8] argued that mobility reduction is a consequence of both a 5% increase in molecular weight and a decreased charge density (23.6% of basic residues instead of 28.6 % in calf thymus FI).

Although present-day knowledge of insect (special- ly of Diptera) histones shows a close similarity to mammalian histones, comparisons between histone patterns from different developmental stages have not been widely studied. Recently, Oliver and Chalkley [9] have isolated histones from larval and adult purified chromatin. Their studies were conducted with two species of Drosophila: D. melanoguster and D. virilis. They found only minor differences in the quantitative analysis of histones : the relative amount of each fraction seemed to be species-specific and stage-specific to a very small extent. The more distinc- tive feature of larval histones was the presence of a specific basic protein moving somewhat faster than F1 in 15% polyacrylamide gels. In view of their findings, however, Oliver and Chalkley argued against this larval-specific protein being a histone [9]. The most convincing argument was that it seemed to be dissociated from chromatin at low salt concentra- tions (0.15 M NaCI).

The present report describes the isolation of histones from another Diptera species, Ceratitis capi- tutu. The biochemistry of development has been extensively studied in this species [lo-151 and its life cycle is now well established [16]. These facts have been the reasons for making C. capitata our material of choice. Studies now described have been mainly conducted at the pharate adult stage, although larval and adult animals have been used in some instances.

MATERIALS AND METHODS

Culture of Insects

Ceratitis capitata was grown as described else- where [17]. Insects were collected at the larval, pharate adult or adult stages and washed free of culture medium.

Isolation of Chromatin

Insects were homogenized in 10 vol. (w/v) of 0.075 M NaC1, 0.024 M EDTA solution at pH 8.0 (medium A) as described by Johns and Dick [4]. The homogenate was filtered through cheese cloth and centrifuged at 1500 x g for 15 min in the GSA angle head of a RC-2B Sorvall centrifuge. Pellets

were washed three times with medium A, four times with 0.05 M Tris-HC1 buffer, pH 8.0 and, finally, twice with 0.14 M NaCl solution. The final sediment, further referred to as crude chromatin, was immediately purified as described below, or extracted in order to obtain crude total histones or histone fractions. In some experiments medium B (0.075 M NaCI, 0.024 M EDTA, pH 6.5) or medium C (0.14 M NaC1, 0.01 M trisodium citrate [4]) was used instead of medium A.

Purification of Crude Chromatin

Crude chromatin was suspended in 0.05 M Tris- HC1 buffer pH 8.0 to a concentration of about 0.5 mg DNA per ml and the suspension was then sheared in a close-fitting teflon-glass Potter-Elvehjem homo- genizer. 10 ml of the sheared suspension were layered on the top of a discontinuous sucrose gradient made up in 50-ml centrifuge tubes with 15 ml of 2.0 M sucrose and 15 ml of 1.5 M sucrose, both in 0.05 M Tris buffer. The tubes were spun in the Sorvall HB-4 swinging bucket rotor for 3 h at 27000 x gav. Pellets were washed three times with 0.14 M NaCl solution by suspending and centrifuging at 12 000 x g,, for 15 min. The final pelleted material (partially purified chromatin) was gently suspended in Tris buffer.

Isolation of Total Histones

Acid Extraction. Acid extraction of histones either from crude or from partially purified chromatin was carried out with 0.25 N HC1 according to Johns [18]. Histones were precipitated from the clear supernatant with 8 vol. acetone, washed three times with acetone and dried under vacuum.

Salt Dissociation of Chromatin. Histones were prepared by the method of Dick and Johns [4], but a final concentration of 2 M NaCl was used by mixing equal volumes of chromatin suspension and 4.0M NaC1.

Fractionation Procedures

F1 histone was obtained by extraction of crude chromatin with 5 % perchloric acid according to Johns and Butler [19].

Extraction with 0.25 N HC1, 80% ethanol was performed by mixing an amount of crude chromatin equivalent to 30 mg DNA with 100 ml of the extraction medium. The following steps were carried out according to Johns [6]. F2B histone was obtained from the ethanol - HC1-insoluble material following method 2 of Johns [6].


L. Pranco, F. Montero, J. M. Navlet, J. Perera, and M. C. Rojo 55

The F2A histone fractions were obtained by treat- Table 1. Amino-acid composition of Ceratitis cauitata and ing the crude chromatin with 75 % ethanol, 10 ./, (w/v) guanidinium chloride by suspending an amount of chromatin equivalent to 20mg DNA in 100ml of medium. Subsequent steps were done according to Johns [20]. In order to obtain the F2A1 histone, the pooled guanidinium chloride extracts were mixed, under continuous stirring, with 1 vol. acetone, and the precipitate, mainly F2A2 histone, was spun down. Crude F2A1 was then precipitated by adding 1.5 vol. acetone to the clear supernatant, and was washed three times with acetone and dried under vacuum. The F2A1 histone was purified by a method based on that of Hoare [21]. Crude F2A1(500 mg) was dissolved in 30 ml of 0.01 N HC1, to which 1 ml of concentrated HCl was added. The solution was mixed, under continuous stirring, with 85 ml of cold acetone. The mixture was allowed to stand overnight at -20 "C and the precipitate which developed was spun down and washed twice with a mixture containing acetone, 0.01 N HC1 and concentrated HCI in the same pro- portions as that from which the protein was precipitated. This purification procedure was repeated once more, and the final precipitate (pure F2A1) was washed twice with acetone and dried under vacuum.

Oxidation of Histones

Oxidation of cysteine-containing histones was carried out by shaking a solution of arginine-rich histones in 6 M guanidinium chloride, 0.3 M Tris buffer, pH 8.3, according to Marzluff et al. [22].

Analytical Methods

Electrophoretic analyses were performed in 20 "/, polyacrylamide gels according to Johns [18]. For comparative purposes, double-gel electrophoresis was run as described by Johns and Forrester [23].

Proteins were hydrolyzed in vacuum-sealed Pyrex tubes with 6.0 N distilled HCl for 18 h at 105 "C, unless otherwise stated. Amino acid analysis was carried out in an Unichrom (Beckman) amino acid analyzer by the single-column procedure of Deveny [24]. Hydroxyproline-containing samples were run as described by Spackman [25].

RESULTS

Table 3 gives the amino acid composition of the whole acid-soluble proteins from crude and partially purified chromatin from C. capitata pharate adults.

Drosophila melanogaster iota1 histones A = acid-soluble protein from C. capitata crude chromatin. B = acid-soluble protein from C. capitatu partially purified chromatin. C = histones from D. melanogaster purified chromatin [XI. D = acid-soluble protein from salt-dissociated crude chromatin from C. capitata pharate adults. E = whole histone from salt-dissociated D. melanogaster chromatin [4]. F = as D, except that histones were precipitated with trichloroacetic acid (final concentration 18 %) instead of acetone

Amino acid Amount in :

A B C D E F

mo1/100 mol

Aspartic acid 11.0 10.1 8.2 8.2 8.8 8.9 Threonine 4.4 4.8 6.0 10.9 5.4 5.8 Serine 3.8 3.7 6.4 5.6 7.0 8.1 Glutamic

acid 17.5 16.9 11.1 9.0 12.4 10.1 Proline 4.2 3.5 4.6 5.5 4.7 5.8 GI ycine 8.1 6.7 8.5 7.6 8.5 7.2 Alanine 9.0 9.7 12.0 11.3 11.7 11.6 Half-

cystine trace trace 0.0 0.0 0.0 trace Valine 6.9 6.2 5.5 6.3 5.2 4.3 Methionine 1.2 0.7 0.4 trace 0.2 0.6 Isoleucine 5.7 4.4 4.3 3.0 3.3 3.5 Leucine 6.0 5.8 1.4 3.0 7.7 5.5 Tyrosine 1.4 0.9 1.9 0.3 1.2 1.7 Phenyl-

alanine 2.7 2.4 2.2 0.9 2.0 1.2 Lysine 9.3 14.1 12.4 16.0 16.2 17.3 Histidine 1.7 1.9 1.8 1.0 1.8 2.0 Arginine 7.1 8.2 7.3 11.2 5.0 6.4

Basiciacidic 0.6 0.9 1.1 1.6 1.1 1.3 LysiArg 1.3 1.7 1.7 1.4 2.8 2.7

Purification resulted in an increase in the basic/acidic amino acid ratio, as well as in the lysine/arginine ratio, but after purifying acidic amino acid values are still high. This fact, as well as the electrophoretic pattern of acid-soluble proteins from purified chromatin (Fig. 1 B), may be indications of the presence of non- histone contaminant proteins. Indeed, slow-moving proteins hardly can be regarded as histones and it can be concluded that gradient centrifugation of sheared chromatin is not a very efficient method to purify insect chromatin. When the method of Dick and Johns [4] was used, a cleaner electrophoretic pattern was obtained (Fig. 1 C). Double-gel electrophoresis against calf thymus whole histone was also carried out and the results are shown in Fig. 1 A. The electrophoretic pattern exhibited by the acid extracts shows the presence of seven major fractions which have been numbered as shown in Fig. 1. Fraction 1


56

F1- F2B-F3-

F2A2- F2A1-

Histones from the Fruit Fly C. capitatu

A B C Fig. 1. Electrophoresis of histones and acid-soluble proteins of Ceratitis capitata. (A) Double-gel electrophoresis of calf thymus (left) and C. capitata (right) whole histones. Fly histones were obtained by a modification of the method of Dick and Johns [4]. (B) Acid-soluble proteins from C. capitata

partially purified chromatin. (C) Acid-soluble proteins from salt-dissociated crude chromatin from C. capituta. The major histone bands have been numbered according to their mobility. Migration is from top (anode) to bottom

mobility corresponds to that of calf thymus F2A1. This fraction is hardly distinguishable, if at all, in preparations from salt-dissociated chromatin. This may be explained in terms of the restricted dissociation [26] and/or aggregation 1271 of F2A1 histone in high ionic strength media. The above interpretation is, of course, based only on the electrophoretic identity of fraction 1 and F2A1. Further evidences of this assumption will be established later on in this paper. Only traces of fraction 4 are present in histones obtained through salt-dissociation of chromatin, and fractions 3 , 5 and 6 account for most of the protein obtained. Finally, the slow-moving bands, as well as the high-molecular-weight excluded protein which remained at the gel surface could not be detected in histone preparations obtained by the method of Dick and Johns [4], even when gels were overloaded.

The most striking feature of histone preparations obtained from dissociated chromatin is its anomalous amino acid composition. The threonine content is abnormally high, and the analysis for arginine also gives too high values if the low extractability of arginine-rich histones is considered. Examination of Table 1, which also gives the composition of Droso- phila histones, is conclusive. A discussion of these abnormalities will be given later.

The above results did not depend on the method used for preparation of crude chromatin, since the use of either medium B or medium C (see Materials and Methods) led to similar results.

Very lysine-rich histones were extracted from chromatin with 5 % perchloric acid according to Johns and Butler [19]. When trichloroacetic acid (final concentration 18%) was added to the clear perchloric acid solution, a precipitate was formed. Electrophoresis of the precipitated material (Fig. 2A, D) showed the correspondence to fractions 6 and 7, and its composition closely resembles to that of F1 histones (Table 2). Whether both fractions 6 and 7, or only the major one, i.e. fraction 6, have to be taken as very lysine-rich histones cannot be decided at the present time, as attempts to purify fractions 6 and 7 have so far been unsuccessful. When chromatin from adult insects was used as a source for very lysine-rich histone preparations, similar results in both electrophoretic behaviour and amino acid composition were found (Fig. 2C and Table 2).

Unexpected results were, however, exhibited by pharate adult preparations at this stage. After remov- ing very lysine-rich histones from the perchlorate extract, considerable amounts of protein still remained in 18 % trichloroacetic acid solution. This protein

Eur. J . Biochem. 48 (1974)

L. Franco, F. Montero, J. M. Navlet, J. Perera, and M. C. Rojo 57

7-

6- 5-

3- 2-

A 8 c D

Fig. 2. Electrophoresis oj histones and proteins of C . capitata. (A) Double-gel electrophoresis of whole histone (left) and 5 % perchloric-acid-soluble, 18 % trichloroacetic-acid-insoluble protein from C. capitata pharate adult chromatin. The pattern shows the correspondence between this protein and fraction 6. (B) Double-gel electrophoresis of total histones (left) and 18 % trichloroacetic-acid-soluble, 30 % trichloroacetic-acid-insoluble protein from C. capitata pharate adult chromatin. (C) 5 % perchloric-acid-soluble, 18 trichloroacetic-acid-insoluble protein from C. capitata adult chromatin. (D) As (C), but from pharate adult chromatin

Table 2. Amino-acid cornposition of Ceratitis capitata very lysine-rich histones

Amino acid Amount in :

pharate adults adults

mo1/100 mol

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phen ylalanine Lysine Histidine Arginine

5.2 7.3

11.2 6.5 5.0 7.6

15.4 trace

5.7 0.5 2.3 3.6 1.3 0.8

23.0 2.3 2.1

5.5 7.1

10.6 5.5 7.6 7.5

16.6 0.0 6.0 0.0 3.0 4.0 1 .5 1 .o

22.1 0.5 1.5

Basic/acidic 2.3 Lys,'Arg 10.9

2.2 14.7

could only be precipitated by raising the trichloroacetic acid concentration to 30 %. No trace of fractions 6 and 7 was detected in the 18 % acid-soluble protein, which was clearly identified as fraction 5 (Fig. 2B). The amino acid analysis of this fraction (Table 3 ) showed its very unusual composition. First of all, the very high content of threonine and arginine should be noted; they roughly account for the 50% of the amino acid residues. The presence of hydroxyproline is also unforeseen, as this amino acid has mainly been found previously in merely structural proteins, such as collagen. In view of its composition, it is most unlikely that fraction 5 is a histone, in spite of its strongly basic nature. The presence of hydroxyproline, as mentioned, would suggest a structural role for this protein. This suggestion was reinforced by the fact that fraction 5 was not present at all in adult insect chromatin, and it could be detected in larval histone preparations only as a minor component. Thus, a plausible hypothesis might be the identification of fraction 5 with some component of the external envelopes of pharate adults. To prove this assumption, puparia remaining after adult emergence were collected, rinsed with distilled water and homogenized in medium C (see above). The insoluble material was exhaustively washed with 0.14 M NaCl and extracted with 5 % perchloric acid. This extract was clarified by filtration through a no. 4 sintered glass funnel and trichloroacetic acid to a final concentration of 30 % was added. The precipitate was washed, dried and analyzed as described above; its amino acid composition and electrophoretic mobility were identical to those of fraction 5 . It can be concluded from this experiment that fraction 5 is not a nuclear component, and it may represent some kind of structural protein located at any of the puparium envelopes.

The question arose as to how this puparium protein became attached to chromatin. Taking into account its extremely basic nature, as well as the extended conformation which its composition would suggest, the idea that fraction 5 could be removed in some way from the puparium and bound to chromatin either during homogeneization or in any subsequent step, was a very attractive one. In order to make this point clear, rat liver chromatin was prepared [27] and mixed with an aqueous solution of fraction 5 (0.14 mg fraction 5 per mg of chromatin DNA). Immediately after mixing rat chromatin solution lost its gel appearance and it could be spun down at low centrifugal fields. No protein remained in the supernatant, and extraction of the sediment with 5 % perchloric acid yielded a mixture of rat liver F1 and fraction 5 (Fig. 3).

Thus, fraction 5 can be considered as a contaminant of pharate adult whole histone preparations and it



Table 3. Amino-acid analyses of pharate adult ,fraction 5 (puparium-specific protein) after varying times of hydrolysis

Amino acid Amount after hydrolysis for:

18 h 24 h 36 h 48 h

Hydroxy- proline

Aspartic acid Threonine Serine Glutamic

acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Unknown” Tyrosine Phenylalanine Lysine Histidine Arginine

mo1/100 mol

17.0 1.8

28.9 2.7

1.7 3.8 2.3 2.9 0.0 2.7 0.0 0.0 0.0 9.8 0.0 0.0 2.5

trace 23.7

19.0 1.8

30.6 2.3

1.5 3.9 2.2 2.5 0.0 2.6 0.0 0.0 0.0 9.0 0.0 0.0 1.4

trace 23.1

19.8 2.0

30.2 2.3

1.7 3.9 2.4 2.5 0.0 2.5 0.0 0.0 0.0 8.4 0.0 0.0 1.5

trace 22.7

19.0 1.8

30.4 2.2

1.7 4.1 2.4 2.6 0.0 2.8 0.0 0.0 0.0 5.2 0.0 0.0 1.7

trace 26.0

a The amount of the unknown, ninhydrin-positive sub- stance, eluting just after leucine has been calculated by using an average factor.

accounts for the high threonine and arginine content found in histones obtained from salt-dissociated chromatin. On the other hand, the findings described above also provided a method for avoiding that contamination. For this purpose, the procedure of Dick and Johns [4] was followed, but trichloroacetic acid (final concentration 18 %) instead of acetone was used for the late precipitation of histones. Material obtained in this way did not possess fraction 5 , as revealed by electrophoretic analysis, and it exhibited an amino acid composition in good agreement with the one reported by Dick and Johns [4] for whole histone from D. rnelunoguster adults (Table 1).

Extraction of chromatin with ethanol- HCl as described under Materials and Methods, yielded fractions 1, 2 and 4, as well as some non-histone slow-moving proteins (Fig. 4B). When the ethanol - guanidinium chloride extraction was used, only fractions 1 and 2 were obtained, as Fig. 4C shows. These results gave a first indication about the nature of arginine-rich histones, provided that F3 is the only arginine-rich histone which cannot be extracted from chromatin with ethanol - guanidinium chloride [20]. The above results may be taken as a preliminary

5-

Rat liver’ F1 histone

-5

A 8 Fig. 3. Polyacryhmide-gel electrophoresis patterns showing the interuction between C . capitata fraction 5 (puparium- specijic protein) and chromatin. (A) 5 perchloric-acid- soluble, 30 trichloroacetic acid-insoluble proteins obtained from a preparation of rat liver chromatin to which a solution of fraction 5 was added prior to perchloric acid extraction. The figure shows the presence of fraction 5, indicating that it was previously attached to rat liver chromatin. (B) Fraction 5 from C. capitata pharate adults

evidence of fraction 4 being an arginine-rich histone similar to F3. In order to test this assumption, oxidation experiments were conducted as described above, using the ethanol - HC1-soluble histones (a preparation similar to that of Fig. 4B) as starting material. After completion of the reaction, the electrophoresis of oxidized histones showed the almost complete absence of band 4, while the intensity of the slow- moving band marked by an arrow in Fig. 4B, pro- portionally increased. It can therefore be concluded that fraction 4 corresponds to F3 histone, the slow- moving band being an oxidation product, due to the formation of intermolecular disulphide bridges.

Ethanol - guanidinium chloride extracts were used for F2A1 preparation. The histone fraction obtained according to the procedure described under Materials and Methods was identified as fraction 1 (Fig. 4D). Taking into account its electrophoretic mobility, similar to that of calf thymus F2A1 (see Fig. lA), the amino acid composition (Table 4) and the peculiar- ities of the isolation procedure, fraction 1 is judged to be the F2A1 histone.

The residual chromatin after extraction with ethanol-HC1 was used for the isolation of moderately

Eur. J . Biochem. 48 (1974)


7-

6- 5-

4- 3-

2- 1-

A B C D Fig. 4. Polyucrjlamide-gel electrophoresis patterns qf arginine- rich histones f i o m C . capitata. (A) Total histones from phardte adult chromatin. (B) Ethanol - HC1-soluble histones. The band marked by an arrow corresponds to the oxidation product of fraction 4 (F3). (C) Ethanol- guanidinium- chloride-soluble histones. (D) F2A1 histone. See the text for further details

Table 4. Amino-acid composition of fractions I (F2AI) and 3 (FZB) from C. capitata pharate adults

Amino acid Amount in:

fraction 1 fraction 3

mol/100 mol

Aspartic acid 7.7 7.9 Threonine 5.8 6.0 Serine 2.8 7.0 Glutamic acid 8.8 11.5 Proline 3.0 5.1 Glycine 12.4 8.5 Alanine 8.6 8.5 Half-cystine 0.0 0.7 Valine 7.2 6.0 Methionine 1.1 0.7 Isoleucine 5.5 4.0 Leucine 9.4 6.0 Tyrosine 3.6 2.8 Phenylalanine 2.8 2.3

Histidine 3.9 3.8 Lysine 8.6 11.1

Arginine 10.8 8.1

Basic/acidic 1.3 1.2 Lys/Arg 0.8 1.4

1- 6- 5-

Fig. 5. Double-gel electrophoresis of’ C. capitata total histones (left) and a crude preparation of n7oderately lysine-rich histone (right)

lysine-rich histone. By applying the method 2 of Johns, first described for calf thymus F2B isolation [6], a preparation was obtained in which the major component was identified as fraction 3 (Fig. 5). It is obvious from this figure that further purification procedures ought to be applied in order to isolate fraction 3 in a pure form. However, these data, as well as the amino acid composition of crude fraction 3 (Table 4) provide a criterion sufficient to identify the fly fraction 3 as a moderately lysine-rich histone.

DISCUSSION

The foregoing results corroborate the presence of the five major groups of histones in Diptera, in close agreement with the reports of other workers [4,5,7,8]. The identification of histone fractions has been made possible by applying methods similar to those used by Oliver and Chalkley [8] : electrophoretic mobility, solubility in 5 perchloric acid, ethanol - HCI and ethanol - guanidinium chloride. The composition of the isolated fractions has been used to identify each fraction. Their behaviour in high-ionic-strength sol- vents and the results obtained in the oxidation experiments have also been taken as additional evidence.

Fractions 1, 2 and 4 can be regarded as arginine- rich histones. Fraction 1 corresponds to F2A1 in its



solubility in either ethanol - HC1 or ethanol - guanidinium chloride, as well as in its amino acid analysis. It also has the same electrophoretic mobility as calf thymus F2A1. The conservation of F2A1 mobility among a wide diversity of organisms is now a well documented fact [29]. It has been sometimes taken as resulting from a structural conservation during biological evolution, and the very close relationships between the amino acid compositions and sequences of pea and calf thymus histone [30] support this idea. However, it has recently been found that yeast F2A1 composition differs from that of calf thymus homologous histone, in spite of their identical mobility [31]. Therefore, it is difficult to draw conclusions about the fly F2A1 structure based solely on electrophoretic mobility similarities. The Ceratitis capitata F2A1 is more acidic than the mammalian homologous histone and it exhibits other differences as well. The major divergences from calf thymus F2A1 are found in the contents of glycine and leucine. It should be noted that differences in these amino acids have also been found between yeast and calf thymus F2A1 [31]. These facts are of interest because of the current idea about the evolutionary stability of F2A1 histone

On the other hand, it should be noted that evidence for cysteine occurrence in C. capitata F2A1 has not been found by amino acid analysis or during the oxidation experiments. This lack of cysteine may be of some significance, because sea urchin F2A1 does possess cysteine and, therefore, this histone aggregates under oxidizing conditions [32]. Although the precise role of echynoderma F2A1 oxidation is not yet understood, research on the cysteine occurrence in F2A1 from lower animals may be of interest in the study of the evolutionary history of the F2A1 histone.

Fractions 2 and 4 correspond to mammalian histone fractions F2A2 and F3 respectively. C. cupi- tata F3 mobility is slightly smaller than that of calf thymus F3. These results disagree with those of Oliver and Chalkley [8], although these authors used a different electrophoretic procedure.

Both solubility in 5% perchloric acid and amino acid analysis led to the identification of fraction 6 as similar to mammalian F1. At the present time it is not possible to decide whether fraction 7 is a histone; if it were, it would represent a second class of lysine- rich histone. Reduced mobility seems to be a feature of Diptera F I , and present results are in agreement with those of Cohen and Gotchel [5] and of Oliver and Chalkley [8].

Fractionation methods also suggest that C. capi- tutu fraction 3 corresponds to mammalian F2B. Its composition, even from a crude preparation, is very similar to that of Drosophila homologous histone [8],

~ 9 1 .

although it has a reduced alanine content. However, a mobility shift towards the anode was observed in Ceratitis F2B when compared with the corresponding Drosophila fraction [5,8]. Comparisons, of course, cannot be firmly established due to the differences in electrophoretic methods.

Fraction 5 exhibits characteristic features which are entirely its own. As pointed out before, hydroxyproline has been found in some structural extended proteins but, as far as we know, it has never been encountered together with such a high content of both threonine and arginine. Moderately high amounts of threonine are present in some structural proteins or peptides from yeast cell wall [33] and arginine usually account for more than 60% of amino acid residues in protamines [34] ; however, the simultaneous presence of these amino acids at very high levels is (an unusual feature. Due to its location at the pharate adult envelope, we propose for fraction 5 the de- nomination of puparium-specific protein. The unknown peak obtained in amino acid analysis eluted with the same retention time as glucosamine and its amount clearly diminished as the hydrolysis time became longer (Table 3). Most of the structural cell wall proteins are carbohydrate-linked and thus, the ninhydrin-positive, unknown product of hydrolysis might correspond to the junction point between the polypeptide and carbohydrate chains.

The fact that chromatin can bind puparium-specific protein might provide a tool for studying chromatin structure and interactions, although more research on this subject would be desirable. Nevertheless, this property does not fully explain the presence of puparium-specific protein in histone preparations. Actually, the electrophoretic band for this protein is weaker when histones are extracted from chromatin with dilute acids (Fig. lB), and accounts for more than 30 % of the material obtained after salt dissociation of chromatin. Thus, an alternative hypothesis may be that puparium-specific protein is mainly removed from the puparium in the course of salt dissociation, their presence in final histone preparations arising as a consequence of cross-contamination between chromatin and puparium debris. This contamination may be explained in terms of the extremely basic nature of puparium-specific protein and may also account for the difficulties found in purifying pharate adult chromatin.

This work has been supported in part by a grant from the Znstituto de Estudios Nucleares, Spain. One of us (M.C.R.) is grateful to the Funducibn Juan March (Madrid) for a fellowship. We are also very indebted to Prof. A. M. Municio for his helpful advice and encouragement and to Dr E. W. Johns for his critical reading of the manuscript.



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Histones from the Fruit Fly Ceratitis capitata : Isolation and Characterization

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Transcript of Histones from the Fruit Fly Ceratitis capitata : Isolation and Characterization