Em. J. Biochem. 45,83--89 (1974)

Histones from Baker’s Yeast Isolation and Fractionation

Luis FRANCO, Ernest W. JOHNS, and Jacinto M. NAVLET

Departamento de Bioquimica, Facultad de Ciencias, Universidad Complutense, Madrid, and Chester Beatty Research Institute, London

(Received December 20, 1973)

1. Baker’s yeast histones have been isolated either by acid extraction of purified chromatin or by salt-dissociation of crude chromatin. These two methods gave similar results as judged by the amino-acid composition and electrophoretic behaviour of histones.

2 . Chemical fractionation methods have been used to isolate both, moderately lysine-rich and arginine-rich histones. Neither F1 nor F3 histones could be detected in yeast chromatin throughout these fractionation procedures. Results obtained in experiments of interactions between yeast whole histones ans calf thymus DNA also confirm the absence of a very lysine-rich histone in yeast.

3. The fractionation of arginine-rich histones was achieved by selective precipitation with acetone. F2AI and F2A2 histones were obtained, purified and characterised by electrophoresis and aniino acid analyses. The similarities between yeast and other organisms histone fractions are discussed.

Histones have been found in all multicellular or unicellular eukaryotic organisms examined to date. Their function, however, has been a rather contro- versial subject, as they seem to play a structural role as well as controlling transcription. Indeed, both of these roles may be closely correlated, the inhibi- tion of RNA synthesis being a consequence of the histone-induced superhelical structure of DNA [I].

Results obtained during the last decade indicate the conservative nature of histones. Although some cells or tissues possess unique histones (avian ery- throcytes [ 2 ] and trout testis [3]), the five major histone fractions are present in all higher organisms. Moreover, their properties are very similar. By study- ing the electrophoretic behaviour [4], as well as other physical [B] and chemical [6] properties of vertebrate histones, Chalkley and his colleagues were able to assess that arginine-rich histones are extremely conservative. This fact has been establish- ed, at least for the F2A1 histone, by comparing the primary structures of this histone isolated from both calf thymus and pea seedlings [7]. However, very lysine-rich histones seem to be more species- specific and some differences in electrophoretic mobility as well as in their microheterogeneity have been found in several organisms [4,8].

Histones also occur in unicellular eukaryotes, such as protozoa [9], green algae [lo] and yeasts [ I l l .

It is therefore of interest to carry out studies on unicellular organisms in order to elucidate the evolu- tionary history of histones. Also, such studies may result in a better knowledge of the role of histones in controlling gene expression and differentiation.

Tetrahymena histones have been reasonably well characterised by the work of Iwai and his colleagues [12, 131. They succeeded in fractionating Tetra- hymena whole histones by chromatography on carboxymethyl-cellulose and were able to prove that a second very lysine-rich histone occurs in addition to that found in higher organisms. On the other hand, a clear correlation was found between the F2 and F3 fractions from both Tetrahymena and calf thymus. This correlation is by no means an identity, because some differences were noted when comparisons between both types of histones were made. These differences were regarded as a conse- quence of both conservation and variation of histones during biological evolution [13].

Yeast histones have also been studied, although complete fractionation and characterization has not yet been achieved. Tonino and Rozijn first reported the occurrence of histones in yeast nuclei [l l , 141 and described a partial chromatographic fractionation procedure. They explained their results in terms of the absence of a very lysine-rich histone, but the chromatographic procedure did not alIow for a more

Eur. J. Biochem. 45 (1974)

84 Baker’s Yeast Histones

thorough characterization. Recently, Wintersberger et al. [15] have isolated histones from nuclei of expo- nentially growing Xaccharornyces cerevisiae cells. These histones resolved into three major bands when analyzed by polyacrylamide gel electrophoresis.

Chemical fractionation methods, that can be applied for bulk isolation of histone fractions, have not yet been used with chromatin from unicellular organisms. This paper deals with the application of these methods to the fractionation of baker’s yeast histones.

MATERIALS AND METHODS Isolation of Whole Histones

Acid Extraction. Acid extraction of purified chro- matin was carried out according to Tonino and Rozijn [14]. Histones were precipitated from the clarified 0.25 N HC1 solution by adding 8 volumes of acetone, washed three times with acetone and dried under vacuum.

Salt Dissociation of Chromatin. Crude chromatin, prepared as described by Tonino and Rozijn [14], was dissociated with aqueous 20°/, NaCl a t pH 7.0 for 2 h. Histones were isolated from the NaCl solu- tion by the procedure of Dick and Johns [16].

Fractionation of Yeast Histones Argininc-rich histones were extracted from purifi-

ed chromatin by a modification of the Method 2 of Johns [17]. Chromatin (20mg DNA) was washed twice with goo/, ethanol and thoroughly mixed with 10 ml ethanol-1.25 N HC1 (4: 1, v/v). Extraction was carried out by stirring the mixture for 18 h in the cold. The sediment was again extracted twice with 5 ml of the ethanol-HC1 reagent for 4 h each, and the extracts were combined and filtered through a no. 4 sintered glass funnel. Whole arginine-rich histones wcre then precipitated by adding 4 volumes of acetone.

I n order to further fractionate arginine-rich histones, the clear ethanol-HC1 extract was mixed, with continuous stirring, with 1.5 volumes of cold acetone. F2A2 histone which precipitated was spun down and washed once with a mixture of ethanol- 1.25 N HCl(4 : 1, v/v) to which 1.5 volumes of acetone had been added, then twice with acetone and dried under vacuum. F2A1 was recovered from the super- natant by adding 1.0 volume of acetone; it was washed three times with acetone and dried under vacuum. No evidence for F3 occurrence in yeast could be obtained in the course of this extraction, as will be discussed in more detail under Results.

Purification of F2A1 histone was achieved by adding 1.0 volume of acetone to a 0.6-mg/ml solution of crude F2A1 preparation in 0.1 N H,SO,. Pure F2A1 was precipitated in this way and was washed and dried as described above.

Lysine-rich histones were obtained from the ethanol-HC1 extraction sediment. In ordcr to remove all traces of arginine-rich histones, this sediment was further washed with the ethanol-HCl reagent until the absorbance a t 280 nm of thc supernatant dropped to zero. The sediment was then stirred for 3 h with 0.25 N HC1 (3.0 ml per mg DNA) and the extraction then repeated once with half this volume. After clarification, lysine-rich histones were precipitated from the pooled extracts with 8 volumes of acetone and were washed and dried as usual.

Ethanol - guanidinium chloride treatment of chromatin [ 181 resulted in an incomplete extraction of arginine-rich histones. Perchloric acid extraction of chromatin was carried out according to Johns and Butler [19].

Interactions between D N A and Histones Interactions between histones and DNA were

studied by the precipitation technique of Johns and Forrester [20]. Calf thymus DNA was used t,hrough- out these experiments. Calf thymus F2Al and F2A2 histones were obtained by the Method2 of Johns [171.

Analytical Methods Polyacrylamide gel electrophoresis was carried

out in 20°/, gels according to Johns [21]. Double-gel electrophoresis were run as described by Johns and Forrester [22]. Amino acid analyses were carried out, after acid hydrolysis of proteins, either by the method of Spackman et al. [23] using a Technicon Autoanalyser apparatus or by the single-column procedure of D6vAny [24] in an Unichrom (Beckman) amino acid analyser. No corrections for hydrolytic losses were made.

RESULTS

Fig. 1 A shows the electrophoretic pattern of yeast whole histones obtained by extraction of purifi- ed chromatin with 0.25 N HC1. The three major bands will be referred to as fractions a, b and c in order of increasing mobilities. Fraction c migrates with the same mobility as calf thymus F2Al histone, as shown by the double-gel electrophoresis (Fig. 1 B). The ot8her yeast histone fractions have no comparable mobilities with those of calf thymus. The electro- phoretic patterns also reveal some degree of hetero- geneity in the yeast histone fractions.

The electrophoretic comparison of yeast histones, obtained by the two alternative procedures outlined under Materials and Methods, is shown in Fig.2. The removal of fraction c from chromatin seems to be incomplete when histones are obtained by the salt-dissociation procedure. This low solubilization of fraction c in high-ionic-strength media, as well

Eur. J. Bioehem. 45 (1974)

L. Franco, E. W. Johns, and J. M. Navlet

Table 1. Amino-acid analyses of yeast whole histones

85

Amino acid Content in whole histone from

purified salt-dissociated chromatin chromatin

mo1/100 mol

Aspartic acid 6.8 7.6 T hreonine 6.3 6.3 Serine 7.8 7.9 Glutamic acid 9.6 10.0 Proline 3.6 4.1 Glycine 7.7 6.6 Alanine 11.5 11.5

Valine 5.5 5.3 Methionine trace 0.4 Isoleucine 5.7 5.6 Leucine 8.5 8.1 Tyrosine 2.0 2.9 Phenylalanine 2.4 2.1 Lysine 12.0 12.2 Histidine 1.9 2.0

8.9 7.3 Fig. 1. (A) Polyacrylamide-gel electrophoresis patterns of yeast whole histones. The sample (40 pg protein) was developed in

gel electrophoresis [22] of calf thymus (left) and yeast (right) L ~ ~ J A ~ ~ 1.3 1.6 whole histones. Migration is from top (anode) to bottom

Half-cystine 0.0 0.2

20°/, polyacrylamide gels according to Johns [21]. (B) Double- BjAa 1.4 1.2

a B/A stands for the basic over acidic amino acid ratio.

contrary, favoured under salt-dissociation condi- tions, which also make the heterogeneity of fractions a and b more evident (Fig.2).

Amino acid analyses reveal only minor differences between both samples of total yeast histones (Table i), and the results are consistent with those of Tonino and Rozijn [14]. The ratio lysine/arginine is enhanced when the salt-dissociation method is used. This can be explained as a consequence of the incomplete removal of arginine-rich histones, which is in accord- ance with the weaker intensity of band c (Fig.2).

Fig. 2. Double-gel electrophoresis of yeast whole histones obtnin- ed by the two methods described in the text. Left: 0.25 N HCI extraction from purified chromatin. Right: 0.25 N HCI soluble proteins from salt-dissociated crude chromatin. See Fig. 1 legend for electrophoresis conditions

as the identity of electrophoretic mobilities between calf t'hymus F2A1 and yeast fraction c may be taken as preliminary evidence of the similarity of both fractions. Extraction of yeast fraction a is, on the

Fractionation of Yeast Histones No protein was obtained when purified chromatin

was submitted to 5 O / / , perchloric acid extraction. It is known that this treatment results in the solubiliza- tion of the FI histone [I91 and, therefore, these results are in good agreement with those of Tonino and Rozijn, who first reported that yeast lacks a very lysine-rich histone. I n order to check the possib- ility that a very lysine-rich histone could have been removcd from chromatin during the 0.14M NaCl washing of yeast chromatin, the saline washings were examined for their histone content, Only traces of fraction a were found and F1-like proteins could not be detected.

Extraction of chromatin with ethanol-HC1, if carried out as described above, entirely removed fraction c, whereas no trace of fraction a could be detected in the ethanol-HC1 extracts, even when

Eur. J . Biochem. 45 (1974)

86 Baker’s Yeast Histones

Fig. 3. Polyacrylamide gel electrophoresis patterns of yeast whole histone and histone fractions. (A) Yeast total histone, 40 pg. (B) Residual histones after extraction of purified chromatin with ethanol-1.25N HC1 (4:1, v/v) [17], as described in the text; 20 pg protein was applied to the gel. (C) Ethanol-HC1 soluble histones from purified chromatin. The gel was overloaded to show the absence of fraction a. Electrophoreses were conducted as described in Pig. 1 A

electrophoretic gels were overloaded (Fig. 3). Frac- tion b was, however, prescnt in both the ethanol- HCl extract and residue. When extractions were performed with ethanol-HC1 volumes less than that described total separation of fractions a and c was not achieved, and cross-contamination took place. Fraction b, on the contrary, was never fully resolved, i.e., it is always partitioned between the ethanol-HC1 solution and the residue. For convenience, thc ethanol-HC1 insoluble part of fraction b will be fur- ther referred to as fraction bl , while the soluble material will be referred to as fraction b2. The above data can be taken as evidence of the arginine-rich nature of histones b2 and c.

The separation of both arginine-rich fractions (b2 and c) was achieved by using the selective pre- cipitation method as described above. Results are shown in Fig.4 and Table 2 gives the amino acid composition of fractions b2 and c. Compositions of some other arginine-rich histones from calf thymus and Tetrahymena pyriformis are also included for comparison. Both amino acid analysis and electro- phoretic mobility lead to the identification of yeast fraction c as F2AI. The behaviour of fraction c in the salt-dissociation experiments described above is also similar to calf thymus F2A1. The conclusion that fraction b2 is similar t o F2A2 can also be drawn, although the identification is from the similarities in amino acid composition rather than from electro- phoretic mobility.

As stat,ed above, no evidence for the occurrence of F3 in yeast was obtained throughout these fractio-

Fig.4. Polyncrylamide-gel electrophoresis patterns of yeast arginine-rich fractions. (A) Ethanol-HCI soluble histones (20 pg protein). (B) Second step of acetone precipitation of ethanol-HC1 soluble histones. The gel was charged with 15 pg protein which was identified as P2A1 histone (see the text for further details). (C) First acetone precipitation of ethanol-HC1 soluble histones. The protein applied to the gel (20 pg) wap, identified as F2A2 histone. (D) Double-gel electrophoresis of ethanol-HC1 soluble histones (left) and yeast tjotal histones (right)

nation procedures. No precipitate was formed during dialysis of the ethanol-HC1 extracts against ethanol. However, when very concentrated extracts were used, some precipitation took place. The protein so obtained moved in polyacrylamide gels to a position identical to that of yeast F2A2 (band b2). Moreover, precipitated material did not contain cysteine, and the ratio lysinelarginine was 1.2. This data cannot exclude the possibility that this protein is an F2A2 subfraction, different from the whole F2A2 obtained by the procedure outlined above, but i t strongly argues against its being an F3 fraction similar to that described for mammalian tissues.

On the other hand, fraction a can be identified as a moderately lysine-rich histone, because i t is not soluble in ethanol-HC1. Fractions a and b l together analyse as moderately lysine-rich histones (Table 3). The closest resemblances between calf thymus histones F2B and fractions a plus b l from yeast are in the content of basic amino acids and in the product glycine x arginine, which has previously been used as an identifying feature of histone frac- tions [25]. Attempts to further fractionate the group of yeast fractions a and b l have been unsuccessful.

Interactions between Histones and DNA

Fig. 5 A shows the precipitation curves of calf thymus DNA with calf thymus and yeast whole histones. When experiments were carried out with

Eur. J. Biochem. 45 (1974)

L. Franco, E. W. Johns, and J. M. Navlet 87

Table 2. Amino-acid composition of arginine-rich histones from yeast and other organisms

Amino acid Yeast fraction Tetruhymenn Calf thymus Yeast fraction Tetrahymena Calf thymus b2 fr. 1141 (13) FZ42 C fr. IV-C3 (13) F2A1

Aspartic acid Threonine Serine Qlutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine

4.5 4.4 4.8

10.2 4.6 7.4

12.8 t r 4.7

t r 4.9

11.1 2.2 2.9

12.0 1.6

11.7

8.0 5.6 7.0 8.0 4.5 9.5

11.8 0.0 5.5 1.3 5.2 9.8 2.4 3.1

10.3 2.1 6.3

6.6 3.9 3.4 9.8 4.1

10.8 12.9 0.0 6.3 0.0 3.9

12.4 2.2 0.9

10.2 3.1 9.4

7.5 5.1 5.6

11.0 2.6 9.8 7.2 0.0 6.3 0.6 6.1

10.4 2.2 2.5

10.2 1.7

11.2

6.3 6.2 6.4 7.0 2.1

10.1 10.1 0.0 7.4 1.1 4.7 6.6 2.7 3.3

12.0 2.3

12.1

4.9 6.9 2.0 5.9 1.0

16.7 6.9 0.0 8.8 1.0 5.9 7.8 3.9 2.0

10.8a 2.0

13.7

B/Ab 1.72 1.17 1.38 1.25 1.99 2.45 LYS/A% 1.03 1.63 1.08 0.91 0.99 0.79

a This figure includes the s-N-methyllysine content. b Ratio of basic to acidic amino acids.

Table 3. Amino-acid conaposition of yeast and call-thymus moderatelv lvsine-rich histones

Amino acid Yeast fractions Calf t,hymus a + b l F 2 3

Aspartic acid Threonine Serine Glutaniic acid Proline Glycine Alanine Valine Half-cystine Methionine lsoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine _ _ _ _ - ~ B/Aa

Qlv x Ara Lys/Arg

mol/lOO mol

9.0 6.0 7.5

10.4 3.8 6.9 8.3 4.9 0.7 0.7 5.2 6.4 3.1 2.9

15.8 2.7 5.8

4.8 6.4

11.2 8.0 4.8 5.6

10.4 7.2 0.0 1.6 4.8 4.8 4.0 1.6

16.0 2.4 6.4

1.3 1.9 2.7 2.5

40.0 36.0

100

90

80

70

60

50

40 30

~~ ~

a Ratio of basic to acidic aniino acids.

HistonelDNA ra t io

yeast histones, complete removal of DNA from solu- tion was only accomplished a t histonelDNA ratios higher than 1.3, whereas total precipitation of DNA with calf thymus histones was achieved a t a ratio

Fig.5. Precipitation curves of calf thymus DNA with whole histone ( A ) and F2A1 (B) . The amount of precipitated DNA was determined by the method of Johns and Forrester [20]. (0-0) Thymus histones; (o---a) yeast histones

Eur. J. Biochem. 45 (1974)

88 Baker’s Yeast Histones

of 1.1, according to the previously described values [26]. Yeast F2A1 (Fig.5B) and F2A2 (not shown in Fig. 5 ) histones behaved as thymus homologous histones did.

DISCUSSION

The foregoing experiments clearly confirm the absence of a very lysine-rich histone in baker’s yeast chromatin, previously reported by Tonino and Rozijn [11,14]. The behaviour of yeast whole histone in not precipitating DNA as efficiently as calf thymus whole histone also agrees with the absence of F1 -like histones in yeast chromatin, since F I is the most efficient histone fraction in this respect 1201.

The above results also pose a new question con- cerning the conservation of histone fractions in the course of biological evolution. F3 histone has been regarded as a very conservative one [4], although some evolutionary changes have been detected in it [6]. I ts absence in detectable amounts in yeast chromatin, however, might be an indication of its late origin in the course of evolution.

The yeast fraction b2 corresponds to mammalian F2A2. I ts composition is very close to that of the calf thymus fraction, although the yeast histone has a higher glycine content. The current view that F2A2 is essential in order to maintain the structure of DNA in chromatin [27] is, thus, reinforced.

Fraction c corresponds to F2Al. The amino acid composition of the yeast F2A1 histone, however, does not correspond precisely to that of the higher organisms homologous fraction. The main differences are seen in the acidic amino acids, glycine and leucine. It must be noted, however, that Tetra- hymena F2A1 histone also has a low glycine and a high acidic amino acid content [13] ; (see also Table 2 ) . In examining the data given in Table2, it can be seen that the more differentiated or evolved the organism, the higher the glycine and the lower the acidic amino acid content. Thus, F2A1 histone is no so highly conserved as previously thought. The knowl- edge of the primary structure of the F2A1 histone from unicellular organisms will help to establish the evolutionary history of histones. The amino acid sequence of yeast F2A1 is now under study, and preliminary data (Franco, L., Municio, A. M., Navlet, J. M., and Perera, J., unpublished) show some interesting differences from the calf thymus F2A1 histone. Basic residues are, for instance, clustered a t the C-terminal region rather than in the N-terminal one.

While the yeast fractions a and b l together may be considered as moderately lysine-rich histones, the characterization of the individual proteins is not so definite. The behaviour of fraction a in selective fractionation procedures indicates its F2B-like

nature, but the identification of fraction b l as a moderately lysine-rich histone is only tentative, and the possibility remains of its being a somewhat different histone. Very recently, Hsiang and Cole [28] have reported the presence of two different histones, having ratios of lysine/arginine of 2.3 and 1.2 in Neurospora crassa chromatin. These Neuro- spora histones have a high alanine content (12 -130/,) and their molecular weights are quite different (14000 for fraction C3 and 8000 for C4). Although no determination of molecular weights of yeast histones have been made, the relative similarity between a and b l electrophoretic mobilities may be indicative of only minor differences in their molecular weights. Nevertheless, selective separation of. both these yeast histones seems to be neccessary in order to confirm their properties and speculate on their bio- logical role.

Most of this work was carried out during the stay of one of us (L. I?.) a t the Chester Beatty Research Institute. We wish to thank to the Pundacidn Juan Narch (Madrid) whose financial support made possible this stay. We are also very indebted to Mrs D. Purkiss for amino acid analyses and to Prof. A. M. Municio for helpful discussions and en- couragement.

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L. Franco and J. M. Navlet, Departamento de Bioquimica, Facultad de Cienciaa, Universidad Complutense, Madrid-3, Spain

E. W. Johns, Chester Beatty Research Institute, Institute of Cancer Research, Royal Hospital, Fulham Road, London, Great Britain SW3 6BJ

Eur. J. Biochem. 45 (1974)