- Email: [email protected]
H4 histone in the macronucleus of Blepharisma japonicum (Protozoa, Ciliophora, Heterotrichida)
FEMS Microbiology Letters 149 (1997) 93^98
H4 histone in the macronucleus of Blepharisma japonicum (Protozoa, Ciliophora, Heterotrichida) Mariangela Salvini a *, Elisabetta Bini b , Annalisa Santucci c , Renata Batistoni ;
a b
d
Scuola Normale Superiore di Pisa, Pisa, Italy
é di Pisa, Pisa, Italy Dipartimento di Scienze dell'Ambiente e del Territorio, Universita
c d
é di Siena, Siena, Italy Dipartimento di Biologia Molecolare, Universita é di Pisa, Pisa, Italy Dipartimento di Fisiologia e Biochimica, Universita
Received 26 November 1996; revised 4 February 1997; accepted 4 February 1997
Abstract
Two clones, obtained by polymerase chain reaction from macronuclear DNA of the unicellular ciliated protist Blepharisma , were isolated and sequenced. They correspond to fragments of two different putative H4 histone genes. The existence of multiple H4 histone genes was also suggested by Southern blot hybridisation experiments employing one of the obtained clones as a probe. Two B. japonicum H4 protein fragments, which were directly sequenced, show differences in the amino acid sequences too. The comparison of the obtained B. japonicum H4 partial amino acid sequences with each other, and with H4 from other ciliates and from representative microbial and multicellular organisms, highlights the larger histone heterogeneity of lower eukaryotes compared to that observed in higher organisms. japonicum
Keywords :
Unicellular eukaryote; Heterotrich ciliate;
Blepharisma japonicum
1. Introduction
H4 is considered the most highly conserved com* Corresponding author. Present address: Dipartimento di Scienze dell'Ambiente e del Territorio, Universitaé di Pisa, Via Volta 4, 56100 Pisa, Italy. Tel.: +39 (50) 500840; fax: +39 (586) 881170; e-mail: [email protected] A, alanine; R, arginine; N, asparagine; D, aspartic acid; Q, glutamine; E, glutamic acid; G, glycine ; H, histidine; I, isoleucine; L, leucine; K, lysine; M, methionine; P, phenylalanine; T, threonine; Y, tyrosine; V, valine; oligo, oligodeoxyribonucleotide; PAGE, polyacrylamide gel electrophoresis ; AU, acetic acid-urea ; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/ 0.015 M Na3 citrate, pH 7.6 ; Taq polymerase, Thermus aquaticus polymerase; SSU-rRNA, small subunit ribosomal rRNA; LSU-rRNA, large subunit ribosomal rRNA Abbreviations :
; Macronucleus ; H4 histone
ponent of nucleosomal histones in the eukaryotic chromatin. Recently, H4 has been extensively investigated in the yeast Saccharomyces cerevisiae, because of its implication in an epigenetic mechanism of gene expression control [1]. The unicellular ciliated protists represent an interesting alternative genome model for investigating characteristics of H4. The evolutionary line of Ciliophora has produced a rich assortment of genetically distant species, which share an intriguing peculiarity: nuclear dualism. Two morphologically and functionally di¡erent nuclei, the macronucleus and the micronucleus, are present in the same cell. Only the micronucleus participates in sexual reproduction, ensuring the transmission of genetic information. Conversely,
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during the vegetative phase of the ciliate biological cycle, only the macronuclear DNA is actively transcribed, whereas the micronuclear DNA is silent (partially or totally ^ still open to discussion). The two nuclei show di¡erent histone components. Since the macronucleus derives from a micronucleus at the end of each sexual stage, the mechanisms which set up and preserve di¡erentiated histone complements must be ¢nely regulated ([2,3] and references therein). Data on H4 histone have been obtained only for a limited number of ciliate species: Tetrahymena ([3] and references therein), Oxytricha nova [4], Stylonychia lemnae [5], and Euplotes crassus [4,6]. No information is available, however, from heterotrich representatives that, on the basis of congruent phylogenies based on either SSU-rRNA or LSU-rRNA, are associated with karyorelictids in a clade that emerges as a sister group to all other ciliates [7]. In this paper we analyse the macronuclear H4 in Blepharisma japonicum, a heterotrich ciliate which is scarcely known from a molecular point of view [8,9]. The B. japonicum clone employed in this work has a steady mating type, without the tendency to change reversibly as is usually shown for this ciliate [10]. 2. Materials and methods
2.1. Histone extraction, amino acid composition and sequence analysis
The procedure for histone extraction and puri¢cation from B. japonicum (clone A5/3, albino, steady mating type I) isolated macronuclei was described in Salvini et al. [8]. H4 histone was obtained by preparative AU-PAGE electrophoresis. Determination of amino acid composition was carried out according to the `Dabs-Amino acid kit' (Beckman). T and H concentrations were gathered by extrapolating, at time 0, the values relative to three di¡erent hydrolysis times. Amino acid fragments of B. japonicum and E. crassus H4 were directly sequenced with Edman automated digestion by a protein microsequencer, model 470A, on line with a PTH amino acid analyser, model 120A and a control/data module 900A (Applied Biosystem).s
2.2. Analysis of PCR products
DNA was extracted from macronuclei isolated as above, with Iso Quick Kit (MicroProbe Corporation). In order to amplify H4 histone sequences from B. japonicum, two degenerate primers were selected sharing a similar annealing temperature of about 54³C. Primer A (5P-AATCTAGAAA(AG)GG(AT)CTGGG(ACT)AA-3P) was deduced by comparing the H4 amino acid fragments of B. japonicum with the corresponding regions of other ciliates (Fig. 1). The reverse primer C (5P-AGTCTAGA(AGCT)CCTTGTCT(CT)TT(GC)A-3P) was similarly chosen on the basis of the multiple sequence alignment in Fig. 1. A XbaI restriction site was added for cloning strategy in both of the oligos. PCR was performed with PCR Core Kit (Boehringer Mannheim), using a Perkin Elmer Termocycler (GenAmp 2400), in 100 Wl reactions, containing 2.5 U Taq polymerase, 10 ng macronuclear DNA, and 0.5 mM primer concentration. Ampli¢cation cycles were as follows: 1 cycle: 94³C, 4 min/37³C, 30 s/72³C, 30 s; 6 cycles: 94³C, 30 s/37³C, 30 s/72³C, 30 s; 30 cycles: 94³C, 30 s/54³C, 30 s/72³C, 30 s; ¢nal elongation: 74³C, 5 min. PCR products were analysed in 1% agarose gel, and the band corresponding to the expected size was excised from the gel and the DNA recovered using QIAquick Gel Extraction Kit (Promega). The eluted fragments, after XbaI digestion, were cloned in pGEM7Zf(+) and the clones were selected by Southern blot hybridisations with oligo C (5PAAGCCCGCTATCAGAAGA(TC)T(AG)GC-3P) (Fig. 1), used as a probe after labelling by DIG Oligonucleotide 3Q-End Labeling Kit (Boehringer Mannheim). Hybridisations were carried out at 38³C and washes were performed according to Boehringer Mannheim protocols. Both orientations of the two cloned DNA inserts were sequenced using the T7 polymerase kit (Pharmacia). 2.3. Restriction enzyme analysis of the H4 macronuclear DNA
Macronuclear DNA was completely digested with a number of restriction enzymes, electrophoresed in 2% agarose gel, and transferred onto Hybond-N ¢lters (Amersham) [11]. BjMH4-1 DNA, DIG-labelled with `DIG DNA Labelling Kit' (Boehringer Mann-
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M. Salvini et al. / FEMS Microbiology Letters 149 (1997) 93^98
heim), was used as a probe in the hybridisations, performed according to Boehringer Mannheim protocols. Washes were carried out in 1USSC plus 0.1% SDS, at 60³C. 3. Results and discussion
The amino acid composition of the puri¢ed H4 has been determined as a starting point of this study (Table 1). The H4 amino acid molar ratios in B. japonicum appear quite similar to the amino acid composition of H4 in T. thermophila. However, some di¡erences occur for speci¢c amino acids, according to the heterogeneity already observed for unicellular eukaryote histones on the basis of electrophoretic data [8]. We performed a direct sequencing of a part of the amino-terminal region of B. japonicum H4. Interestingly, this analysis revealed the presence of two H4 variants ^ hereafter referred to as H4(1) and H4(2) ^ which di¡er for the insertion of the S21 residue. Two variants, which di¡er from each other only for the
6
Fig. 1. Multiple alignment of amino acid H4 sequences from ciliates. For comparison, H4 sequences of some representative microbial and multicellular organisms are shown. The alignment was performed with Clustal W [15] and was manually improved by adding B. japonicum and E. crassus sequences. The ¢rst M residue is reported to indicate the complete amino acid sequences deduced from nucleotide sequences. The accession numbers of the sequences are indicated in brackets. indicates identical amino acids, - indicates gaps used to obtain maximum alignment; * indicates missing data. The sequences used to design the oligos are indicated with capital letters and boxed. H4(1): B. japonicum H4 protein fragment (P80737). H4(2): B. japonicum H4 protein fragment (P80738). BjMH4-1: amino acid sequence deduced from clone BjMH4-1 (X97995). BjMH4-2: amino acid sequence deduced from clone BjMH4-2 corresponding to a part of a B. japonicum H4 (X97996). Ec: E. crassus : Amino-terminal fragment: determined as described in Section 2 (P80739); carboxylic end fragment [6]. Tt: T. thermophila (X00417). On: O. nova (M24411). Sl: S. lemnae (X16018). Sc: S. cerevisiae (X00724); An: Aspergillus nidulans (X55549). Nc: Neurospora crassa (X01611). Pp: Physarum polycephalum (X00449). Vc: Volvox carteri (X06963). Ce: Caenorhabditis elegans (X15634). Dm: Drosophila melanogaster (X14215). Xl: Xenopus laevis (X00224). Hs: Homo sapiens (X60482). Ps: Pisum sativum (U10042). A comprehensive H4 sequence compilation can be found at website http://www.nc.nml.nih.gov/Baxevani/HISTONES/H4.html c
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96
for a H4 histone role, given that single amino acid substitutions
S. cerevisiae
in
the
H4
corresponding
region
of
nullify the H4 function in this organism
[1]. On the basis of the multiple amino acid H4 sequence alignment, we devised a PCR-based strategy to select H4 macronuclear DNA fragments in
B.
japonicum.
An ampli¢cation product, about 260 bp
in
was
length,
cloned.
Two
recombinant
clones,
BjMH4-1 and BjMH4-2, were selected by Southern blot hybridisation with oligo B, corresponding to the 35^42 amino acid region of
B. japonicum H4
(cf. Fig.
1). The two clones are 267 bp long and they di¡er from each other in 22 positions. Because the major-
B. japonicum macronuclear DNA with DIG-labelled BjMH4-1. HindIII (lane 1), BglII (lane 2), EcoRI (lane 3), AluI (lane 4), MboI (lane 5), DpnI (lane 6) and XbaI (lane 7). Molecular mass markers in kb are Fig. 2. Southern blot hybridisation of digested
shown on the right of the ¢gure.
ity of nucleotide replacements fall in the third position of the codons, the cloned genes encode two putative H4 fragments that di¡er from each other in only three amino acids (positions 29, 68, 83, Fig. 1). Two of the replacements are not conservative : a basic amino acid (K83) is replaced by an acid amino acid (E), and an apolar residue (I29) by a neutral
inversion of the R19 and K20 residues, have been reported in
T. pyriformis.
Since these variants are
absent in the closely related species
T. thermophila,
polar one (N). The amino acid deduced BjMH4-1 sequence perfectly matches the directly sequenced peptide fragment, lacking S21 (Fig. 1).
the two H4 histones that have been retained appear
The peculiar ciliate bias to use only AGA among
to have no distinct functions [3]. The sequenced frag-
six arginine codons has been con¢rmed in
ments share conserved parts with other unicellular
cum
B. japoniB.
H4, where AGA encodes all 14 R residues.
eukaryote H4 proteins (Fig. 1). In particular four lysine residues, essential for the functioning of the H4 A domain in the yeast
S. cerevisiae
[1], are highly
conserved. These residues ¢ll the positions 6, 9, 13, 17 in
B. japonicum
and the last one was identi¢ed as
Table 1 Comparison
a residue which can be acetylated in this organism. It is interesting to note that this residue belongs to a
of
T. thermophila
amino
acid
molar
ratios
of
B. japonicum
T. thermophilaa
S
2.5
pentapeptide region (GAKRH), named histone H4
T
7.6
signature, which has been conserved throughout evo-
R
14
12.7
lution [12]. This region appears to be only di¡erent
G
6.8
16
13.7
5.8
A
6.7
8.8
P
1.5
0.9
V
11.3
8.8
M
0
1.9
iant of this sequence has been found. On the whole,
I
3.6
4.9
the distribution of the amino acid di¡erences ob-
L
8
4.9
F
1.4
2.9
K
10.3
10.7
H
1.8
1.9
Y
3.7
3.9
in
B. japonicum,
due to the insertion of a K residue
(GAKKRH ; Fig. 1), and therefore in this respect
japonicum
served in
B.
represents the ¢rst organism where a var-
B. japonicum
is not random. Variations in
the number and in the composition of amino acids are grouped at the NH2 end. Moreover, a cluster of six residues is changed in the R22^N27 segment of
japonicum.
B.
This region, which also appears variable
in the other ciliates examined, might be important
N+D
5.8
5.8
E+
4.8
4.8
a
Deduced from Bannon et al. [14].
FEMSLE 7489 1-5-97
B. japonicum
H4
an
M. Salvini et al. / FEMS Microbiology Letters 149 (1997) 93^98
uses the universal genetic code, like higher eukaryotes, but unlike other ciliates, in which stop signal codons encode some amino acids, such as Q or C [7]. Indeed the eukaryotic terminal triplet UAA, representing one of the Q codons in other ciliates, is used as a stop signal in the B. japonicum K tubulin gene [9]. In agreement with this interpretation is the observation that deviant glutamine codons are not found in the sequenced parts of B. japonicum H4, where CAA encodes Q30. Moreover B. japonicum mRNA is correctly translated in an eukaryotic cellfree system for the in vitro translation, and this suggests that no deviation from a standard translation system occurs [13]. Even if an early origin of the deviant code followed by reversion in the Blepharisma and Euplotes lineages has been proposed [2], the apparently non-deviated code of Blepharisma could correspond to a pleisomorphic character trait. It is possible that ciliate ancestors used a code similar to the universal genetic code and that deviations have occurred independently several times within the phylum [7]. The amino acid sequences deduced from the two clones (BjMH4-1 and BjMH4-2) were compared with the directly sequenced H4(1) and H4(2) and with H4 sequences of some representative microbial and multicellular organisms. The BjMH4-1 and BjMH4-2 deduced amino acid sequences (89 aa) con¢rm the peculiar GAKKRH `H4 signature' motif, and a non-random distribution of amino acid di¡erences is observed at the NH2 end, and in the R22^ N27 and T48^L87 regions (Fig. 1). BjMH4-1 and BjMH4-2 amino acid sequences differ from the other ciliate H4 by 33.9% and 37.3%, respectively. These high percentage values re£ect the great di¡erences among ciliate H4 sequences and underline what appears to be a characteristic of microbial lineages. The divergent H4 proteins could be a result of the enormous genetic diversity within unicellular eukaryote groups, and/or the presence of reduced constraints in the chromatin organisation. Indeed a similar divergence in sequence is not found when multicellular eukaryote H4 proteins are compared with each other, even when representatives of very distant taxa are considered (cf. Fig. 1). The macronuclear B. japonicum DNA, digested with restriction enzymes that are not present in the two cloned sequences, was analysed by Southern blot japonicum
97
hybridisation, using the BjMH4-1 insert as a probe, in order to analyse H4 histone gene organisation (Fig. 2). Multiple hybridisation bands are observed with HindIII, EcoRI, AluI, BglII and XbaI. This result suggests that more than one H4 gene could be present in the B. japonicum macronuclear DNA, according to the di¡erent proteins identi¢ed. The presence of multiple copies of H4 genes could represent a typical feature of the ciliate macronucleus. In fact two H4 genes have been reported in S. lemnae [5] and Tetrahymena [2,14] and four genes are present in O. nova [4]. A single H4 gene has been found only in E. crassus, although the same authors do not exclude the presence of a second gene [4]. The multiple hybridisation bands observed in B. japonicum di¡er in intensity, probably due to a di¡erential H4 ampli¢cation in the polygenomic macronuclear DNA. In fact it is unknown if, during macronuclear development, all the DNA molecules are equally involved in the ampli¢cation process, or if a control mechanism exists that discriminates between genes. Such a mechanism is probably present in ciliates, as the extensive ampli¢cation of the macronuclear extra-chromosomal rDNA suggests ([2] and references therein). MboI and DpnI B. japonicum restricted DNAs reveal the presence of a single band, 1 kb and 4 kb in length, respectively (Fig. 2, lanes 5 and 6). These results suggest that a little cluster of non-tandemly repeated H4 genes could be placed on the same DNA molecule in the macronucleus. A detailed analysis of the H4 DNA region will reveal the overall number of H4 genes in B. japonicum and their arrangement. Moreover, a functional study could clarify whether the transcription control mechanism involving H4 in yeast represents a general process at least in lower eukaryotes that share some peculiarities, e.g. unicellular condition. In this context, understanding how fundamental a process is depends upon its utilisation in a variety of systems. Acknowledgments
We are grateful to Dr. M. Peccatori for amino acid composition measurements, and to Prof. F. Dini for his critical reading of the manuscript. This work was supported by MURST grants.
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98
References
ponicum
[1] Megee, P.C., Morgan, B.A. and Smith, M.M. (1995) Histone H4 and the maintenance of genome integrity. Genes Dev. 9, 1716^1727. [2] Prescott, D.M. (1994) The DNA of ciliated protozoa. Microbiol. Rev. 58, 233^267. [3] Gorowsky, M. (1986) Ciliate chromatin and histones. In : The Molecular Biology of Ciliate Protozoa (Gall, J., Ed.), pp. 227^ 262. Academic Press, New York. [4] Harper, D.S. and Jahn, C.L. (1989) Actin, tubulin and H4 histone genes in three species of hypotrichous ciliated protozoa. Gene 75, 93^107. [5] Wefes, I. and Lipps, H.J. (1990) The two macronuclear histone H4 genes of the hypotrichous ciliate
compared to that of
Tetrahymena pyriformis.
FEMS
Microbiol. Lett. 111, 301^308.
Stylonychia lemnae.
DNA Seq. 1, 25^32. [6] Harper, D.S. and Jahn, C.L. (1989) Di¡erential use of termination codons in ciliated protozoa. Proc. Natl. Acad. Sci. USA 86, 3252^3256. [7] Tourancheau, A.B., Tsao, N., Klobutcher, L.A., Pearlman, R.E. and Adoutte, A. (1995) Genetic code deviations in the ciliates : evidence for multiple and independent events. EMBO J. 14, 3262^3267. [8] Salvini, M., Bini, E., Pellegrini, S., Nobili, R., Piras, L. and Giorgi, F. (1993) Macronuclear chromatin of
[9] Liang, A. and Heckman, K. (1993)
Blepharisma
uses UAA as
a terminal codon. Naturwissenschaften 80, 225^226. [10] Miyake, A. and Harumoto, T. (1990) Asymmetrical cell division in
Blepharisma japonicum :
di¡erence between daughter
cells in mating-type expression. Exp. Cell Res. 190, 65^68. [11] Southern, E. (1975) Detection of speci¢c sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503^517. [12] PROSITE Entry PS00047. [13] Salvini, M., Santangelo, G. and Nobili, R. (1983) Preliminary results on RNA synthesis during cell union activation in
japonicum
B.
Suzuki (Protozoa Ciliata). Monitore Zool. Ital.
(N.S.) 17, 165^172. [14] Bannon, G.A., Bowen, J.K., Yao, M.C. and Gorowsky, M. (1984)
Tetrahymena
H4 genes : structure, evolution and organ-
isation in macro and micronuclei. Nucleic Acids Res. 12, 1961^1975. [15] Thompson,
J.D.,
Higgins,
D.G.
and
Gibson,
T.J.
(1994)
CLUSTAL W : improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionsspeci¢c gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673^4680.
Blepharisma ja-
FEMSLE 7489 1-5-97
H4 histone in the macronucleus of Blepharisma japonicum (Protozoa, Ciliophora, Heterotrichida) Mariangela Salvini a *, Elisabetta Bini b , Annalisa Santucci c , Renata Batistoni ;
a b
d
Scuola Normale Superiore di Pisa, Pisa, Italy
é di Pisa, Pisa, Italy Dipartimento di Scienze dell'Ambiente e del Territorio, Universita
c d
é di Siena, Siena, Italy Dipartimento di Biologia Molecolare, Universita é di Pisa, Pisa, Italy Dipartimento di Fisiologia e Biochimica, Universita
Received 26 November 1996; revised 4 February 1997; accepted 4 February 1997
Abstract
Two clones, obtained by polymerase chain reaction from macronuclear DNA of the unicellular ciliated protist Blepharisma , were isolated and sequenced. They correspond to fragments of two different putative H4 histone genes. The existence of multiple H4 histone genes was also suggested by Southern blot hybridisation experiments employing one of the obtained clones as a probe. Two B. japonicum H4 protein fragments, which were directly sequenced, show differences in the amino acid sequences too. The comparison of the obtained B. japonicum H4 partial amino acid sequences with each other, and with H4 from other ciliates and from representative microbial and multicellular organisms, highlights the larger histone heterogeneity of lower eukaryotes compared to that observed in higher organisms. japonicum
Keywords :
Unicellular eukaryote; Heterotrich ciliate;
Blepharisma japonicum
1. Introduction
H4 is considered the most highly conserved com* Corresponding author. Present address: Dipartimento di Scienze dell'Ambiente e del Territorio, Universitaé di Pisa, Via Volta 4, 56100 Pisa, Italy. Tel.: +39 (50) 500840; fax: +39 (586) 881170; e-mail: [email protected] A, alanine; R, arginine; N, asparagine; D, aspartic acid; Q, glutamine; E, glutamic acid; G, glycine ; H, histidine; I, isoleucine; L, leucine; K, lysine; M, methionine; P, phenylalanine; T, threonine; Y, tyrosine; V, valine; oligo, oligodeoxyribonucleotide; PAGE, polyacrylamide gel electrophoresis ; AU, acetic acid-urea ; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/ 0.015 M Na3 citrate, pH 7.6 ; Taq polymerase, Thermus aquaticus polymerase; SSU-rRNA, small subunit ribosomal rRNA; LSU-rRNA, large subunit ribosomal rRNA Abbreviations :
; Macronucleus ; H4 histone
ponent of nucleosomal histones in the eukaryotic chromatin. Recently, H4 has been extensively investigated in the yeast Saccharomyces cerevisiae, because of its implication in an epigenetic mechanism of gene expression control [1]. The unicellular ciliated protists represent an interesting alternative genome model for investigating characteristics of H4. The evolutionary line of Ciliophora has produced a rich assortment of genetically distant species, which share an intriguing peculiarity: nuclear dualism. Two morphologically and functionally di¡erent nuclei, the macronucleus and the micronucleus, are present in the same cell. Only the micronucleus participates in sexual reproduction, ensuring the transmission of genetic information. Conversely,
0378-1097 / 97 / $17.00 ß 1997 Published by Elsevier Science B.V. All rights reserved PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 0 6 1 - X
FEMSLE 7489 1-5-97
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M. Salvini et al. / FEMS Microbiology Letters 149 (1997) 93^98
during the vegetative phase of the ciliate biological cycle, only the macronuclear DNA is actively transcribed, whereas the micronuclear DNA is silent (partially or totally ^ still open to discussion). The two nuclei show di¡erent histone components. Since the macronucleus derives from a micronucleus at the end of each sexual stage, the mechanisms which set up and preserve di¡erentiated histone complements must be ¢nely regulated ([2,3] and references therein). Data on H4 histone have been obtained only for a limited number of ciliate species: Tetrahymena ([3] and references therein), Oxytricha nova [4], Stylonychia lemnae [5], and Euplotes crassus [4,6]. No information is available, however, from heterotrich representatives that, on the basis of congruent phylogenies based on either SSU-rRNA or LSU-rRNA, are associated with karyorelictids in a clade that emerges as a sister group to all other ciliates [7]. In this paper we analyse the macronuclear H4 in Blepharisma japonicum, a heterotrich ciliate which is scarcely known from a molecular point of view [8,9]. The B. japonicum clone employed in this work has a steady mating type, without the tendency to change reversibly as is usually shown for this ciliate [10]. 2. Materials and methods
2.1. Histone extraction, amino acid composition and sequence analysis
The procedure for histone extraction and puri¢cation from B. japonicum (clone A5/3, albino, steady mating type I) isolated macronuclei was described in Salvini et al. [8]. H4 histone was obtained by preparative AU-PAGE electrophoresis. Determination of amino acid composition was carried out according to the `Dabs-Amino acid kit' (Beckman). T and H concentrations were gathered by extrapolating, at time 0, the values relative to three di¡erent hydrolysis times. Amino acid fragments of B. japonicum and E. crassus H4 were directly sequenced with Edman automated digestion by a protein microsequencer, model 470A, on line with a PTH amino acid analyser, model 120A and a control/data module 900A (Applied Biosystem).s
2.2. Analysis of PCR products
DNA was extracted from macronuclei isolated as above, with Iso Quick Kit (MicroProbe Corporation). In order to amplify H4 histone sequences from B. japonicum, two degenerate primers were selected sharing a similar annealing temperature of about 54³C. Primer A (5P-AATCTAGAAA(AG)GG(AT)CTGGG(ACT)AA-3P) was deduced by comparing the H4 amino acid fragments of B. japonicum with the corresponding regions of other ciliates (Fig. 1). The reverse primer C (5P-AGTCTAGA(AGCT)CCTTGTCT(CT)TT(GC)A-3P) was similarly chosen on the basis of the multiple sequence alignment in Fig. 1. A XbaI restriction site was added for cloning strategy in both of the oligos. PCR was performed with PCR Core Kit (Boehringer Mannheim), using a Perkin Elmer Termocycler (GenAmp 2400), in 100 Wl reactions, containing 2.5 U Taq polymerase, 10 ng macronuclear DNA, and 0.5 mM primer concentration. Ampli¢cation cycles were as follows: 1 cycle: 94³C, 4 min/37³C, 30 s/72³C, 30 s; 6 cycles: 94³C, 30 s/37³C, 30 s/72³C, 30 s; 30 cycles: 94³C, 30 s/54³C, 30 s/72³C, 30 s; ¢nal elongation: 74³C, 5 min. PCR products were analysed in 1% agarose gel, and the band corresponding to the expected size was excised from the gel and the DNA recovered using QIAquick Gel Extraction Kit (Promega). The eluted fragments, after XbaI digestion, were cloned in pGEM7Zf(+) and the clones were selected by Southern blot hybridisations with oligo C (5PAAGCCCGCTATCAGAAGA(TC)T(AG)GC-3P) (Fig. 1), used as a probe after labelling by DIG Oligonucleotide 3Q-End Labeling Kit (Boehringer Mannheim). Hybridisations were carried out at 38³C and washes were performed according to Boehringer Mannheim protocols. Both orientations of the two cloned DNA inserts were sequenced using the T7 polymerase kit (Pharmacia). 2.3. Restriction enzyme analysis of the H4 macronuclear DNA
Macronuclear DNA was completely digested with a number of restriction enzymes, electrophoresed in 2% agarose gel, and transferred onto Hybond-N ¢lters (Amersham) [11]. BjMH4-1 DNA, DIG-labelled with `DIG DNA Labelling Kit' (Boehringer Mann-
FEMSLE 7489 1-5-97
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M. Salvini et al. / FEMS Microbiology Letters 149 (1997) 93^98
heim), was used as a probe in the hybridisations, performed according to Boehringer Mannheim protocols. Washes were carried out in 1USSC plus 0.1% SDS, at 60³C. 3. Results and discussion
The amino acid composition of the puri¢ed H4 has been determined as a starting point of this study (Table 1). The H4 amino acid molar ratios in B. japonicum appear quite similar to the amino acid composition of H4 in T. thermophila. However, some di¡erences occur for speci¢c amino acids, according to the heterogeneity already observed for unicellular eukaryote histones on the basis of electrophoretic data [8]. We performed a direct sequencing of a part of the amino-terminal region of B. japonicum H4. Interestingly, this analysis revealed the presence of two H4 variants ^ hereafter referred to as H4(1) and H4(2) ^ which di¡er for the insertion of the S21 residue. Two variants, which di¡er from each other only for the
6
Fig. 1. Multiple alignment of amino acid H4 sequences from ciliates. For comparison, H4 sequences of some representative microbial and multicellular organisms are shown. The alignment was performed with Clustal W [15] and was manually improved by adding B. japonicum and E. crassus sequences. The ¢rst M residue is reported to indicate the complete amino acid sequences deduced from nucleotide sequences. The accession numbers of the sequences are indicated in brackets. indicates identical amino acids, - indicates gaps used to obtain maximum alignment; * indicates missing data. The sequences used to design the oligos are indicated with capital letters and boxed. H4(1): B. japonicum H4 protein fragment (P80737). H4(2): B. japonicum H4 protein fragment (P80738). BjMH4-1: amino acid sequence deduced from clone BjMH4-1 (X97995). BjMH4-2: amino acid sequence deduced from clone BjMH4-2 corresponding to a part of a B. japonicum H4 (X97996). Ec: E. crassus : Amino-terminal fragment: determined as described in Section 2 (P80739); carboxylic end fragment [6]. Tt: T. thermophila (X00417). On: O. nova (M24411). Sl: S. lemnae (X16018). Sc: S. cerevisiae (X00724); An: Aspergillus nidulans (X55549). Nc: Neurospora crassa (X01611). Pp: Physarum polycephalum (X00449). Vc: Volvox carteri (X06963). Ce: Caenorhabditis elegans (X15634). Dm: Drosophila melanogaster (X14215). Xl: Xenopus laevis (X00224). Hs: Homo sapiens (X60482). Ps: Pisum sativum (U10042). A comprehensive H4 sequence compilation can be found at website http://www.nc.nml.nih.gov/Baxevani/HISTONES/H4.html c
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96
for a H4 histone role, given that single amino acid substitutions
S. cerevisiae
in
the
H4
corresponding
region
of
nullify the H4 function in this organism
[1]. On the basis of the multiple amino acid H4 sequence alignment, we devised a PCR-based strategy to select H4 macronuclear DNA fragments in
B.
japonicum.
An ampli¢cation product, about 260 bp
in
was
length,
cloned.
Two
recombinant
clones,
BjMH4-1 and BjMH4-2, were selected by Southern blot hybridisation with oligo B, corresponding to the 35^42 amino acid region of
B. japonicum H4
(cf. Fig.
1). The two clones are 267 bp long and they di¡er from each other in 22 positions. Because the major-
B. japonicum macronuclear DNA with DIG-labelled BjMH4-1. HindIII (lane 1), BglII (lane 2), EcoRI (lane 3), AluI (lane 4), MboI (lane 5), DpnI (lane 6) and XbaI (lane 7). Molecular mass markers in kb are Fig. 2. Southern blot hybridisation of digested
shown on the right of the ¢gure.
ity of nucleotide replacements fall in the third position of the codons, the cloned genes encode two putative H4 fragments that di¡er from each other in only three amino acids (positions 29, 68, 83, Fig. 1). Two of the replacements are not conservative : a basic amino acid (K83) is replaced by an acid amino acid (E), and an apolar residue (I29) by a neutral
inversion of the R19 and K20 residues, have been reported in
T. pyriformis.
Since these variants are
absent in the closely related species
T. thermophila,
polar one (N). The amino acid deduced BjMH4-1 sequence perfectly matches the directly sequenced peptide fragment, lacking S21 (Fig. 1).
the two H4 histones that have been retained appear
The peculiar ciliate bias to use only AGA among
to have no distinct functions [3]. The sequenced frag-
six arginine codons has been con¢rmed in
ments share conserved parts with other unicellular
cum
B. japoniB.
H4, where AGA encodes all 14 R residues.
eukaryote H4 proteins (Fig. 1). In particular four lysine residues, essential for the functioning of the H4 A domain in the yeast
S. cerevisiae
[1], are highly
conserved. These residues ¢ll the positions 6, 9, 13, 17 in
B. japonicum
and the last one was identi¢ed as
Table 1 Comparison
a residue which can be acetylated in this organism. It is interesting to note that this residue belongs to a
of
T. thermophila
amino
acid
molar
ratios
of
B. japonicum
T. thermophilaa
S
2.5
pentapeptide region (GAKRH), named histone H4
T
7.6
signature, which has been conserved throughout evo-
R
14
12.7
lution [12]. This region appears to be only di¡erent
G
6.8
16
13.7
5.8
A
6.7
8.8
P
1.5
0.9
V
11.3
8.8
M
0
1.9
iant of this sequence has been found. On the whole,
I
3.6
4.9
the distribution of the amino acid di¡erences ob-
L
8
4.9
F
1.4
2.9
K
10.3
10.7
H
1.8
1.9
Y
3.7
3.9
in
B. japonicum,
due to the insertion of a K residue
(GAKKRH ; Fig. 1), and therefore in this respect
japonicum
served in
B.
represents the ¢rst organism where a var-
B. japonicum
is not random. Variations in
the number and in the composition of amino acids are grouped at the NH2 end. Moreover, a cluster of six residues is changed in the R22^N27 segment of
japonicum.
B.
This region, which also appears variable
in the other ciliates examined, might be important
N+D
5.8
5.8
E+
4.8
4.8
a
Deduced from Bannon et al. [14].
FEMSLE 7489 1-5-97
B. japonicum
H4
an
M. Salvini et al. / FEMS Microbiology Letters 149 (1997) 93^98
uses the universal genetic code, like higher eukaryotes, but unlike other ciliates, in which stop signal codons encode some amino acids, such as Q or C [7]. Indeed the eukaryotic terminal triplet UAA, representing one of the Q codons in other ciliates, is used as a stop signal in the B. japonicum K tubulin gene [9]. In agreement with this interpretation is the observation that deviant glutamine codons are not found in the sequenced parts of B. japonicum H4, where CAA encodes Q30. Moreover B. japonicum mRNA is correctly translated in an eukaryotic cellfree system for the in vitro translation, and this suggests that no deviation from a standard translation system occurs [13]. Even if an early origin of the deviant code followed by reversion in the Blepharisma and Euplotes lineages has been proposed [2], the apparently non-deviated code of Blepharisma could correspond to a pleisomorphic character trait. It is possible that ciliate ancestors used a code similar to the universal genetic code and that deviations have occurred independently several times within the phylum [7]. The amino acid sequences deduced from the two clones (BjMH4-1 and BjMH4-2) were compared with the directly sequenced H4(1) and H4(2) and with H4 sequences of some representative microbial and multicellular organisms. The BjMH4-1 and BjMH4-2 deduced amino acid sequences (89 aa) con¢rm the peculiar GAKKRH `H4 signature' motif, and a non-random distribution of amino acid di¡erences is observed at the NH2 end, and in the R22^ N27 and T48^L87 regions (Fig. 1). BjMH4-1 and BjMH4-2 amino acid sequences differ from the other ciliate H4 by 33.9% and 37.3%, respectively. These high percentage values re£ect the great di¡erences among ciliate H4 sequences and underline what appears to be a characteristic of microbial lineages. The divergent H4 proteins could be a result of the enormous genetic diversity within unicellular eukaryote groups, and/or the presence of reduced constraints in the chromatin organisation. Indeed a similar divergence in sequence is not found when multicellular eukaryote H4 proteins are compared with each other, even when representatives of very distant taxa are considered (cf. Fig. 1). The macronuclear B. japonicum DNA, digested with restriction enzymes that are not present in the two cloned sequences, was analysed by Southern blot japonicum
97
hybridisation, using the BjMH4-1 insert as a probe, in order to analyse H4 histone gene organisation (Fig. 2). Multiple hybridisation bands are observed with HindIII, EcoRI, AluI, BglII and XbaI. This result suggests that more than one H4 gene could be present in the B. japonicum macronuclear DNA, according to the di¡erent proteins identi¢ed. The presence of multiple copies of H4 genes could represent a typical feature of the ciliate macronucleus. In fact two H4 genes have been reported in S. lemnae [5] and Tetrahymena [2,14] and four genes are present in O. nova [4]. A single H4 gene has been found only in E. crassus, although the same authors do not exclude the presence of a second gene [4]. The multiple hybridisation bands observed in B. japonicum di¡er in intensity, probably due to a di¡erential H4 ampli¢cation in the polygenomic macronuclear DNA. In fact it is unknown if, during macronuclear development, all the DNA molecules are equally involved in the ampli¢cation process, or if a control mechanism exists that discriminates between genes. Such a mechanism is probably present in ciliates, as the extensive ampli¢cation of the macronuclear extra-chromosomal rDNA suggests ([2] and references therein). MboI and DpnI B. japonicum restricted DNAs reveal the presence of a single band, 1 kb and 4 kb in length, respectively (Fig. 2, lanes 5 and 6). These results suggest that a little cluster of non-tandemly repeated H4 genes could be placed on the same DNA molecule in the macronucleus. A detailed analysis of the H4 DNA region will reveal the overall number of H4 genes in B. japonicum and their arrangement. Moreover, a functional study could clarify whether the transcription control mechanism involving H4 in yeast represents a general process at least in lower eukaryotes that share some peculiarities, e.g. unicellular condition. In this context, understanding how fundamental a process is depends upon its utilisation in a variety of systems. Acknowledgments
We are grateful to Dr. M. Peccatori for amino acid composition measurements, and to Prof. F. Dini for his critical reading of the manuscript. This work was supported by MURST grants.
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98
References
ponicum
[1] Megee, P.C., Morgan, B.A. and Smith, M.M. (1995) Histone H4 and the maintenance of genome integrity. Genes Dev. 9, 1716^1727. [2] Prescott, D.M. (1994) The DNA of ciliated protozoa. Microbiol. Rev. 58, 233^267. [3] Gorowsky, M. (1986) Ciliate chromatin and histones. In : The Molecular Biology of Ciliate Protozoa (Gall, J., Ed.), pp. 227^ 262. Academic Press, New York. [4] Harper, D.S. and Jahn, C.L. (1989) Actin, tubulin and H4 histone genes in three species of hypotrichous ciliated protozoa. Gene 75, 93^107. [5] Wefes, I. and Lipps, H.J. (1990) The two macronuclear histone H4 genes of the hypotrichous ciliate
compared to that of
Tetrahymena pyriformis.
FEMS
Microbiol. Lett. 111, 301^308.
Stylonychia lemnae.
DNA Seq. 1, 25^32. [6] Harper, D.S. and Jahn, C.L. (1989) Di¡erential use of termination codons in ciliated protozoa. Proc. Natl. Acad. Sci. USA 86, 3252^3256. [7] Tourancheau, A.B., Tsao, N., Klobutcher, L.A., Pearlman, R.E. and Adoutte, A. (1995) Genetic code deviations in the ciliates : evidence for multiple and independent events. EMBO J. 14, 3262^3267. [8] Salvini, M., Bini, E., Pellegrini, S., Nobili, R., Piras, L. and Giorgi, F. (1993) Macronuclear chromatin of
[9] Liang, A. and Heckman, K. (1993)
Blepharisma
uses UAA as
a terminal codon. Naturwissenschaften 80, 225^226. [10] Miyake, A. and Harumoto, T. (1990) Asymmetrical cell division in
Blepharisma japonicum :
di¡erence between daughter
cells in mating-type expression. Exp. Cell Res. 190, 65^68. [11] Southern, E. (1975) Detection of speci¢c sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503^517. [12] PROSITE Entry PS00047. [13] Salvini, M., Santangelo, G. and Nobili, R. (1983) Preliminary results on RNA synthesis during cell union activation in
japonicum
B.
Suzuki (Protozoa Ciliata). Monitore Zool. Ital.
(N.S.) 17, 165^172. [14] Bannon, G.A., Bowen, J.K., Yao, M.C. and Gorowsky, M. (1984)
Tetrahymena
H4 genes : structure, evolution and organ-
isation in macro and micronuclei. Nucleic Acids Res. 12, 1961^1975. [15] Thompson,
J.D.,
Higgins,
D.G.
and
Gibson,
T.J.
(1994)
CLUSTAL W : improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionsspeci¢c gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673^4680.
Blepharisma ja-
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