- Email: [email protected]
Several highly divergent histone H3 genes are present in the hypotrichous ciliate Stylonychia lemnae
FEMS Microbiology Letters 175 (1999) 45^50
Several highly divergent histone H3 genes are present in the hypotrichous ciliate Stylonychia lemnae Detlef Bernhard * Universitaët Leipzig, Institut fuër Zoologie, Spezielle Zoologie, TalstraMe 33, 04103 Leipzig, Germany Received 11 February 1999; received in revised form 30 March 1999; accepted 2 April 1999
Abstract In the protozoan Stylonychia lemnae 10 different histone H3 genes were discovered by polymerase chain reaction (PCR) amplification and sequence analysis. One of them is interrupted by a short intron sequence. These genes code for nine divergent histone H3 proteins. The genetic distances between some of these variants are very high. Most of the substitutions, as well as insertions/deletions, were found in the amino-terminal region. One variant shows an extremely elongated and altered Nterminus, which did not allow an unambiguous alignment with other histone H3 variants in this region. Hybridization experiments using the different H3 genes as probes indicate that even more histone H3 variants must exist in this species. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Histone H3; Ciliate ; Stylonychia lemnae
1. Introduction The ciliated protozoa are characterized by their nuclear dualism. The micronuclei function as germ line nuclei, and are mostly transcriptionally inactive. They divide mitotically during the vegetative growth of the cell and undergo meiosis before the conjugation of two mating cells, where haploid micronuclei are exchanged. The macronuclei are transcriptionally active and divide `amitotically' (for review see [1]). They originate from the zygotic micronucleus after conjugation in a complicated event which di¡ers among the ciliate groups. The most extreme pro-
* Tel.: +49 (341) 9736732; Fax: +49 (341) 9736749; E-mail: [email protected]
cesses of DNA rearrangements occur in hypotrichous ciliates. After mitosis of the synkaryon, one of the daughter nuclei di¡erentiates into the macronuclear anlagen. The chromosomes in the anlagen are ¢rst polytenized by multiple rounds of DNA replication, followed by a process of fragmentation and elimination of all nonmacronuclear-destined sequences. Telomeric repeats are then added to the remaining macronuclear-destined DNA molecules and in the ¢nal step, these short, linear gene-sized pieces are again ampli¢ed by further rounds of DNA replication. Compared to other eukaryotes, ciliates have highly variant histones H4 [2] and in the hypotrichous ciliate Euplotes crassus an unusual histone H3 was found [3]. Hybridization experiments to DNA of another hypotrichous ciliate, Stylonychia lemnae (for-
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 7 4 - 3
FEMSLE 8769 19-5-99
46
D. Bernhard / FEMS Microbiology Letters 175 (1999) 45^50
merly S. mytilus SyngenI), suggested the presence of six macronuclear H3 genes [4]. However, none of these genes was sequenced. In this study, the histone H3 genes from S. lemnae are further characterized by PCR ampli¢cation, cloning and sequencing of internal H3 fragments and one complete gene-sized piece containing an extremely divergent H3 variant.
2. Materials and methods 2.1. Ciliate species investigated and isolation of DNA Cultivation of S. lemnae and isolation of whole genomic DNA was done as described elsewhere [4]. 2.2. Ampli¢cation, cloning, and sequencing of histone H3 genes PCR primers for the ampli¢cation of histone H3 genes were based on published sequences of the ciliate Tetrahymena thermophila and di¡erent eukaryotic species. Both primers H3F (5P-GGCTAGAACTAAATAAACTGCTAGAAA-3P) and H3R (5PCTTCTGGCGAGTTGCATATCCT-3P) lie near the ends of the gene spanning 112 of the 136 amino acids compared to T. thermophila. PCR ampli¢cation was carried out in a ¢nal volume of 50 Wl containing: 50^200 ng of whole DNA; 0.1 WM of each primer; 10 mM Tris pH 8.3; 1.5 mM MgCl2 ; 50 mM KCl; 200 WM dNTPs; and 1 U Taq polymerase. After heating to 95³C for 5 min, 35 cycles were carried out: 95³C, 40 s; 42³C, 1 min; and 72³C, 1 min. Histone H3 gene probes were prepared by the same procedure, using 1^5 ng of plasmid DNA containing the cloned H3 genes as a template, 130 WM dTTP and 70 WM DIG-11-dUTP label. Ampli¢ed DNA was blunt-ended, phosphorylated and cloned in pUC18 SmaI/BAP using the SureClone ligation Kit (Pharmacia). Twenty di¡erent clones containing recombinant plasmid DNA were isolated. The complete sequence for both strands of each clone was determined using the Thermo Sequenase £uorescent labeled primer cycle sequencing kit, with 7-deazadGTP (Amersham) on an automated LI-COR DNA sequencer. The complete gene-sized piece of 1 kb containing an unusual histone H3 variant was isolated by cut-
ting out this macronuclear fraction from an 1% agarose gel. The eluted macronuclear DNA fraction served as template in two PCR reactions: primer H3R was combined with a telomere primer (5PAGATCTGCGGCCGC4 A4 C4 A4 CC-3P) and the telomere primer was combined with another forward primer H3F10 (5P-CAATGAGAACTAAGAGTGCAGC-3P). Ampli¢cation consisted of 40 cycles: 95³C, 1 min; 52³C, 1 min; 72³C, 1 min. The PCR products were cloned and sequenced as described above. 2.3. Southern hybridizations The DNA of S. lemnae (each sample 8 Wg) was separated on a 1% agarose gel and transferred to a nylon membrane by capillary transfer [5]. Histone H3-containing gene-sized pieces were detected using digoxigenin-labeled gene probes. Hybridization and detection was performed with the DIG Nucleic Acid Detection Kit (Boehringer Mannheim), following the Boehringer manual. The temperature of hybridization was 65³C, stringent washing was carried out in 0.1USSC, 0.1% SDS at 65³C.
3. Results and discussion 3.1. Sequence analysis of the isolated histone H3 clones For cloning of the histone H3 genes of S. lemnae two internal H3 primers were constructed and used for a PCR reaction with total DNA as template. The PCR product was cloned and 20 clones were randomly picked and sequenced. Nine di¡erent nucleotide sequences were found, coding for eight di¡erent histone H3 proteins (Fig. 1). The sequence of clone 5 contains a short intron of 68 bp between positions 50 (serine) and 51 (threonine). At the 5P- and 3P-splicing junctions of the intron the classical eukaryotic consensus sequences were found. A high degree of variation can be seen between the di¡erent histone H3 sequences. Some nucleotide sequences are very similar, e.g. clones 7 and 9 di¡er in only one nucleotide, resulting in the same amino acid sequence (STYLE7). Both sequences are also very similar to clones 2 and 3. Other sequences are highly
FEMSLE 8769 19-5-99
D. Bernhard / FEMS Microbiology Letters 175 (1999) 45^50
47
Fig. 1. Alignment of all deduced histone H3 amino acid sequences of S. lemnae (STYLE, EMBL database accession numbers Y16620, Y16628^Y16635) together with the E. crassus and T. thermophila histone H3 sequences. The K-helical regions are shaded. EUCR (v) is the`vegetative' and EUCR (p) the `polytene' histone H3 of E. crassus, TET31 is the major and TET33 the minor histone H3 variant of T. thermophila. Dots indicate the same amino acid as in the reference sequence, dashes indicate deletion/insertion.
divergent, showing variations in length from 112 to 114 amino acids and many substitutions (Fig. 1). Between two such protein sequences up to 23 positions can di¡er. However, as described for the histones H4 in ciliates [2], there are few changes in basic amino acid composition, and where they do occur they are mostly replaced by other basic amino acids. Among the H3 variants substitutions not only occur in the loop between helices 2 and 3, but also in the Khelix 1 and more frequently in the long helix 3 (Fig.
1). However, like the histones H4 of ciliates [2], most di¡erences are found in the amino-terminal region. Structural studies of nucleosomes have shown that the C-terminal domains of the four core histones are involved in both the histone-histone and the histoneDNA interactions [6,7]. The N-terminal domains of the histones protrude on the outside of the nucleosomes, where they play an important role in the activation or repression of transcription, DNA replication, nuclear division, and the maintenance of
FEMSLE 8769 19-5-99
48
D. Bernhard / FEMS Microbiology Letters 175 (1999) 45^50
Fig. 2. Hybridization with the di¡erent histone H3 probes to macronuclear DNA of S. lemnae. Lane numbers refer to clone numbers, e.g. 3 = clone 3. M: V-Marker EcoRI/HindIII digested.
genome integrity [8^10]. The great variability in the amino-terminal domains of these variants suggests that they may play di¡erent roles in the cell cycle of S. lemnae and might be expressed during di¡erent stages, as seen for the `vegetative' and `polytene' H3 variants in E. crassus [3]. 3.2. Macronuclear organization of the histone H3 genes Hybridization experiments were carried out in order to assign the di¡erent histone H3 genes to macronuclear gene-sized pieces. The cloned histone H3 sequences were labeled and hybridized individually to Stylonychia DNA. These probes hybridize strongly to macronuclear DNA of about 1.21 kb, 1.59 kb, 2.3 kb, 2.55 kb, and 3.94 kb (Fig. 2), as previously described [4]. The band at 1.59 kb reported by these authors appears to be somewhat smaller (about 1.55 kb) in this study. This signal always appears and is the only one when clones 2, 3, 7 and 9 are used as probes. Clone 6 gives an additional weaker signal at the position of about 2.3 kb. The nucleotide sequences of clones 2, 7, and 9 are nearly identical, di¡ering in only one or two nucleotides from each other. Gene-sized pieces of identical length with small variations in their nucleotide sequences were also detected in other ciliate genes
[11,12]. However, larger di¡erences exist between the sequences of clones 2, 7, and 9 and those of clones 3 and 6 (up to 12%). Thus, the prominent signal at about 1.55 kb might comprise gene-sized pieces of nearly identical length derived from di¡erent micronuclear precursors. Similarly, the probes of clones 1 and 5 reveal their strongest signals at 2.3 kb (Fig. 2). These sequences show a high degree of divergence, also indicating the presence of two di¡erent gene-sized pieces of nearly identical length. Additional hybridization bands are characteristic for single probes: a band at about 4 kb was only detected with clone 4. Also, the signals at about 1.2 and 2.55 are only found using clone 8 as a probe. Presumably, these are also two variants with similar sequences or they are products of alternative processing as found in other di¡erent-sized macronuclear molecules in hypotrichs [13,14]. Decreasing hybridization temperatures (50^55³C) yields further weak bands (not shown), including a signal at about 1 kb, which was also found previously [4]. Thus, additional H3 variants with clearly deviating nucleotide sequences are to be expected. 3.3. Isolation of the histone H3 gene on the 1-kb gene-sized piece In order to determine the sequence of the histone
FEMSLE 8769 19-5-99
D. Bernhard / FEMS Microbiology Letters 175 (1999) 45^50
H3 gene located on the gene-sized piece of 1 kb this class of macronuclear gene-sized pieces was used in PCR reactions with one of the internal primers and a telomere speci¢c primer. A PCR product was only obtained using the telomere primer with the primer H3R. Therefore, based on the sequence of this fragment a new primer H3F10 was constructed to amplify the end and the 3P £anking region of the gene together with the telomere primer. The sequence of this gene-sized piece revealed a histone H3-like protein. The N-terminal region is highly altered compared to other histones H3, making an alignment in this region nearly impossible (STYLE10 in Fig. 1). There are 42 additional amino acids, many substitutions and starting at position 14 a repeated motif of the amino acids lysine and glycine (KGKGKGKGKGK) is found. Despite many substitutions in the C-terminus an alignment in this region is unambiguous. The 5P-noncoding region spans 150 bp without the telomere sequences. A putative CCAAT box (3107) and two putative TATA boxes (319, 346) were found at a reasonable distance to the translation start. 3.4. Comparison with histone H3 variants from other ciliates The H3 genes show again the high genetic diversity within the ciliates also seen for rRNA (reviewed in [15]) and histone H4 genes [2]. Many di¡erences in the H3 amino acid sequences exist, not only compared to the oligohymenophorean T. thermophila, but also between the more closely related hypotrichous species E. crassus and S. lemnae. However, more surprising are the numerous histone H3 variants in this species. In the oligohymenophorean T. thermophila three H3 genes were found [16], two of which encode an identical, replicationdependent (major) H3 protein. The third gene encodes a replication-independent variant, which is expressed in nuclei of non-growing cells [17]. This quantitatively minor H3 variant has 16 substitutions compared to the major H3. None of the H3 variants of S. lemnae shows a higher sequence similarity with either the major or with the minor variants of Tetrahymena. In the hypotrichous ciliate E. crassus only two
49
di¡erent histones H3 were detected [3]. Transcripts of one gene accumulate only during the meiotic and early polytene stages of the sexual phase of the life cycle. The transcripts of the other gene were detected during vegetative growth and the ¢nal replication phase of the sexual phase. Thus, in contrast to the `vegetative' histone H3, the `polytene' histone H3 may play a fundamental role in the development of the macronucleus [3]. The `polytene' variant contains two insertions in the amino-terminal domain and also some substitutions. Only the histone-like protein `STYLE10' also has insertions in the amino-terminal region. However, there are no sequence similarities with the `polytene' variant of E. crassus. At present, why there are so many variants in S. lemnae remains an open question since the genome organization as well as the processes of DNA rearrangement during development of the new macronuclei are very similar in hypotrichs (reviewed in [1]). The great variability in the amino-terminal domain of all these variants indicates that they may also play di¡erent roles in the cell cycle of S. lemnae and might be expressed during di¡erent stages, as seen for the H3 variants in E. crassus [3]. Presumably, some variants may be speci¢c to the transcriptionally silent micronucleus because indications for a micronucleus-speci¢c histone H3 in S. lemnae were given [18]. These questions should be addressed by studying the expression of these genes. An analysis of the transcripts could also give clues as to why S. lemnae has so many variants compared to all other eukaryotes studied so far.
Acknowledgments I want to thank Martin Schlegel and Stan Theophilou for their critical reading of the manuscript.
References [1] Prescott, D.M. (1994) The DNA of ciliated protozoa. Microbiol. Rev. 58, 233^267. [2] Bernhard, D. and Schlegel, M. (1998) Evolution of histone H4 and H3 genes in di¡erent ciliate lineages. J. Mol. Evol. 46, 344^354. [3] Jahn, C., Ling, Z., Tebeau, C.M. and Klobutcher, L.A. (1997) An unusual histone H3 speci¢c for early macronuclear devel-
FEMSLE 8769 19-5-99
50
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
D. Bernhard / FEMS Microbiology Letters 175 (1999) 45^50 opment in Euplotes crassus. Proc. Natl. Acad. Sci. USA 94, 1332^1337. Elsevier, S.M., Lipps, H.J. and Steinbruëck, G. (1978) Histone genes in macronuclear DNA of the ciliate Stylonychia mytilus. Chromosoma 69, 291^306. Southern, E.M. (1975) Detection of speci¢c sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503^517. Arents, G., Burlingame, R.W., Wang, B.-C., Love, W.E. and Moudrianakis, E.N. (1991) The nucleosomal core histone ocî resolution: A tripartite protein assembly and a tamer at 3.1 A left-handed superhelix. Proc. Natl. Acad. Sci. USA 88, 10148^ 10152. Arents, G. and Moudrianakis, E.N. (1993) Topography of the histone octamer surface: Repeating structural motifs utilized in the docking of nucleosomal DNA. Proc. Natl. Acad. Sci. USA 90, 10489^10493. Hecht, A., Laroche, T., Strahl-Bolsinger, S., Gasser, S.M. and Grunstein, M. (1995) Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins : A molecular model for the formation of heterochromatin in yeast. Cell 80, 583^592. Megee, P.C., Morgan, B.A. and Smith, M.M. (1995) Histone H4 and the maintenance of genome integrity. Genes Dev. 9, 1716^1727. Santisteban, M.S., Arents, G., Moudrianakis, E.N. and Smith, M.M. (1997) Histone octamer function in vivo: mutations in the dimer-tetramer interfaces disrupt both gene activation and repression. EMBO J. 16, 2493^2506. Hicke, B.J., Celander, D.W., MacDonald, G.H., Price, C.M.
[12]
[13]
[14]
[15] [16]
[17]
[18]
and Cech, T.R. (1990) Two versions of the gene encoding the 41-kDa subunit of the telomere binding protein of Oxytricha nova. Proc. Natl. Acad. Sci. USA 87, 1481^1485. Gray, J.T., Celander, D.W., Price, C.M. and Cech, T.R. (1991) Cloning and expression of genes for the Oxytricha telomere-binding protein: speci¢c subunit interactions in the telomeric complex. Cell 67, 807^814. Herrick, G., Hunter, D., Williams, K. and Kotter, K. (1987) Alternative processing during development of a macronuclear chromosome family in Oxytricha fallax. Genes Dev. 1, 1047^ 1058. Klobutcher, L.A., Hu¡, M.E. and Gonye, G.E. (1988) Alternative use of chromosome fragmentation sites in the ciliated protozoan Oxytricha nova. Nucleic Acids Res. 16, 251^264. Schlegel, M. (1994) Molecular phylogeny of eukaryotes. TREE 9, 330^335. Thatcher, T.H., MacGa¡ey, J., Bowen, J., Horowitz, S., Shapiro, D.L. and Gorovsky, M.A. (1994) Independent evolutionary origin of histone H3.3-like variants of animals and Tetrahymena. Nucleic Acids Res. 22, 180^186. Bannon, G.A., Calzone, F.J., Bowen, J.K., Allis, C.D. and Gorovsky, M.A. (1983) Multiple, independently regulated, polyadenylated messages for histone H3 and H4 in Tetrahymena. Nucleic Acids Res. 11, 3903^3917. Schlegel, M., Muller, S., Ruder, F. and Buësen, W. (1990) Transcriptionally inactive micronuclei, macronuclear anlagen and transcriptionally active macronuclei di¡er in histone composition in the hypotrichous ciliate Stylonychia lemnae. Chromosoma 99, 401^406.
FEMSLE 8769 19-5-99
Several highly divergent histone H3 genes are present in the hypotrichous ciliate Stylonychia lemnae Detlef Bernhard * Universitaët Leipzig, Institut fuër Zoologie, Spezielle Zoologie, TalstraMe 33, 04103 Leipzig, Germany Received 11 February 1999; received in revised form 30 March 1999; accepted 2 April 1999
Abstract In the protozoan Stylonychia lemnae 10 different histone H3 genes were discovered by polymerase chain reaction (PCR) amplification and sequence analysis. One of them is interrupted by a short intron sequence. These genes code for nine divergent histone H3 proteins. The genetic distances between some of these variants are very high. Most of the substitutions, as well as insertions/deletions, were found in the amino-terminal region. One variant shows an extremely elongated and altered Nterminus, which did not allow an unambiguous alignment with other histone H3 variants in this region. Hybridization experiments using the different H3 genes as probes indicate that even more histone H3 variants must exist in this species. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Histone H3; Ciliate ; Stylonychia lemnae
1. Introduction The ciliated protozoa are characterized by their nuclear dualism. The micronuclei function as germ line nuclei, and are mostly transcriptionally inactive. They divide mitotically during the vegetative growth of the cell and undergo meiosis before the conjugation of two mating cells, where haploid micronuclei are exchanged. The macronuclei are transcriptionally active and divide `amitotically' (for review see [1]). They originate from the zygotic micronucleus after conjugation in a complicated event which di¡ers among the ciliate groups. The most extreme pro-
* Tel.: +49 (341) 9736732; Fax: +49 (341) 9736749; E-mail: [email protected]
cesses of DNA rearrangements occur in hypotrichous ciliates. After mitosis of the synkaryon, one of the daughter nuclei di¡erentiates into the macronuclear anlagen. The chromosomes in the anlagen are ¢rst polytenized by multiple rounds of DNA replication, followed by a process of fragmentation and elimination of all nonmacronuclear-destined sequences. Telomeric repeats are then added to the remaining macronuclear-destined DNA molecules and in the ¢nal step, these short, linear gene-sized pieces are again ampli¢ed by further rounds of DNA replication. Compared to other eukaryotes, ciliates have highly variant histones H4 [2] and in the hypotrichous ciliate Euplotes crassus an unusual histone H3 was found [3]. Hybridization experiments to DNA of another hypotrichous ciliate, Stylonychia lemnae (for-
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 7 4 - 3
FEMSLE 8769 19-5-99
46
D. Bernhard / FEMS Microbiology Letters 175 (1999) 45^50
merly S. mytilus SyngenI), suggested the presence of six macronuclear H3 genes [4]. However, none of these genes was sequenced. In this study, the histone H3 genes from S. lemnae are further characterized by PCR ampli¢cation, cloning and sequencing of internal H3 fragments and one complete gene-sized piece containing an extremely divergent H3 variant.
2. Materials and methods 2.1. Ciliate species investigated and isolation of DNA Cultivation of S. lemnae and isolation of whole genomic DNA was done as described elsewhere [4]. 2.2. Ampli¢cation, cloning, and sequencing of histone H3 genes PCR primers for the ampli¢cation of histone H3 genes were based on published sequences of the ciliate Tetrahymena thermophila and di¡erent eukaryotic species. Both primers H3F (5P-GGCTAGAACTAAATAAACTGCTAGAAA-3P) and H3R (5PCTTCTGGCGAGTTGCATATCCT-3P) lie near the ends of the gene spanning 112 of the 136 amino acids compared to T. thermophila. PCR ampli¢cation was carried out in a ¢nal volume of 50 Wl containing: 50^200 ng of whole DNA; 0.1 WM of each primer; 10 mM Tris pH 8.3; 1.5 mM MgCl2 ; 50 mM KCl; 200 WM dNTPs; and 1 U Taq polymerase. After heating to 95³C for 5 min, 35 cycles were carried out: 95³C, 40 s; 42³C, 1 min; and 72³C, 1 min. Histone H3 gene probes were prepared by the same procedure, using 1^5 ng of plasmid DNA containing the cloned H3 genes as a template, 130 WM dTTP and 70 WM DIG-11-dUTP label. Ampli¢ed DNA was blunt-ended, phosphorylated and cloned in pUC18 SmaI/BAP using the SureClone ligation Kit (Pharmacia). Twenty di¡erent clones containing recombinant plasmid DNA were isolated. The complete sequence for both strands of each clone was determined using the Thermo Sequenase £uorescent labeled primer cycle sequencing kit, with 7-deazadGTP (Amersham) on an automated LI-COR DNA sequencer. The complete gene-sized piece of 1 kb containing an unusual histone H3 variant was isolated by cut-
ting out this macronuclear fraction from an 1% agarose gel. The eluted macronuclear DNA fraction served as template in two PCR reactions: primer H3R was combined with a telomere primer (5PAGATCTGCGGCCGC4 A4 C4 A4 CC-3P) and the telomere primer was combined with another forward primer H3F10 (5P-CAATGAGAACTAAGAGTGCAGC-3P). Ampli¢cation consisted of 40 cycles: 95³C, 1 min; 52³C, 1 min; 72³C, 1 min. The PCR products were cloned and sequenced as described above. 2.3. Southern hybridizations The DNA of S. lemnae (each sample 8 Wg) was separated on a 1% agarose gel and transferred to a nylon membrane by capillary transfer [5]. Histone H3-containing gene-sized pieces were detected using digoxigenin-labeled gene probes. Hybridization and detection was performed with the DIG Nucleic Acid Detection Kit (Boehringer Mannheim), following the Boehringer manual. The temperature of hybridization was 65³C, stringent washing was carried out in 0.1USSC, 0.1% SDS at 65³C.
3. Results and discussion 3.1. Sequence analysis of the isolated histone H3 clones For cloning of the histone H3 genes of S. lemnae two internal H3 primers were constructed and used for a PCR reaction with total DNA as template. The PCR product was cloned and 20 clones were randomly picked and sequenced. Nine di¡erent nucleotide sequences were found, coding for eight di¡erent histone H3 proteins (Fig. 1). The sequence of clone 5 contains a short intron of 68 bp between positions 50 (serine) and 51 (threonine). At the 5P- and 3P-splicing junctions of the intron the classical eukaryotic consensus sequences were found. A high degree of variation can be seen between the di¡erent histone H3 sequences. Some nucleotide sequences are very similar, e.g. clones 7 and 9 di¡er in only one nucleotide, resulting in the same amino acid sequence (STYLE7). Both sequences are also very similar to clones 2 and 3. Other sequences are highly
FEMSLE 8769 19-5-99
D. Bernhard / FEMS Microbiology Letters 175 (1999) 45^50
47
Fig. 1. Alignment of all deduced histone H3 amino acid sequences of S. lemnae (STYLE, EMBL database accession numbers Y16620, Y16628^Y16635) together with the E. crassus and T. thermophila histone H3 sequences. The K-helical regions are shaded. EUCR (v) is the`vegetative' and EUCR (p) the `polytene' histone H3 of E. crassus, TET31 is the major and TET33 the minor histone H3 variant of T. thermophila. Dots indicate the same amino acid as in the reference sequence, dashes indicate deletion/insertion.
divergent, showing variations in length from 112 to 114 amino acids and many substitutions (Fig. 1). Between two such protein sequences up to 23 positions can di¡er. However, as described for the histones H4 in ciliates [2], there are few changes in basic amino acid composition, and where they do occur they are mostly replaced by other basic amino acids. Among the H3 variants substitutions not only occur in the loop between helices 2 and 3, but also in the Khelix 1 and more frequently in the long helix 3 (Fig.
1). However, like the histones H4 of ciliates [2], most di¡erences are found in the amino-terminal region. Structural studies of nucleosomes have shown that the C-terminal domains of the four core histones are involved in both the histone-histone and the histoneDNA interactions [6,7]. The N-terminal domains of the histones protrude on the outside of the nucleosomes, where they play an important role in the activation or repression of transcription, DNA replication, nuclear division, and the maintenance of
FEMSLE 8769 19-5-99
48
D. Bernhard / FEMS Microbiology Letters 175 (1999) 45^50
Fig. 2. Hybridization with the di¡erent histone H3 probes to macronuclear DNA of S. lemnae. Lane numbers refer to clone numbers, e.g. 3 = clone 3. M: V-Marker EcoRI/HindIII digested.
genome integrity [8^10]. The great variability in the amino-terminal domains of these variants suggests that they may play di¡erent roles in the cell cycle of S. lemnae and might be expressed during di¡erent stages, as seen for the `vegetative' and `polytene' H3 variants in E. crassus [3]. 3.2. Macronuclear organization of the histone H3 genes Hybridization experiments were carried out in order to assign the di¡erent histone H3 genes to macronuclear gene-sized pieces. The cloned histone H3 sequences were labeled and hybridized individually to Stylonychia DNA. These probes hybridize strongly to macronuclear DNA of about 1.21 kb, 1.59 kb, 2.3 kb, 2.55 kb, and 3.94 kb (Fig. 2), as previously described [4]. The band at 1.59 kb reported by these authors appears to be somewhat smaller (about 1.55 kb) in this study. This signal always appears and is the only one when clones 2, 3, 7 and 9 are used as probes. Clone 6 gives an additional weaker signal at the position of about 2.3 kb. The nucleotide sequences of clones 2, 7, and 9 are nearly identical, di¡ering in only one or two nucleotides from each other. Gene-sized pieces of identical length with small variations in their nucleotide sequences were also detected in other ciliate genes
[11,12]. However, larger di¡erences exist between the sequences of clones 2, 7, and 9 and those of clones 3 and 6 (up to 12%). Thus, the prominent signal at about 1.55 kb might comprise gene-sized pieces of nearly identical length derived from di¡erent micronuclear precursors. Similarly, the probes of clones 1 and 5 reveal their strongest signals at 2.3 kb (Fig. 2). These sequences show a high degree of divergence, also indicating the presence of two di¡erent gene-sized pieces of nearly identical length. Additional hybridization bands are characteristic for single probes: a band at about 4 kb was only detected with clone 4. Also, the signals at about 1.2 and 2.55 are only found using clone 8 as a probe. Presumably, these are also two variants with similar sequences or they are products of alternative processing as found in other di¡erent-sized macronuclear molecules in hypotrichs [13,14]. Decreasing hybridization temperatures (50^55³C) yields further weak bands (not shown), including a signal at about 1 kb, which was also found previously [4]. Thus, additional H3 variants with clearly deviating nucleotide sequences are to be expected. 3.3. Isolation of the histone H3 gene on the 1-kb gene-sized piece In order to determine the sequence of the histone
FEMSLE 8769 19-5-99
D. Bernhard / FEMS Microbiology Letters 175 (1999) 45^50
H3 gene located on the gene-sized piece of 1 kb this class of macronuclear gene-sized pieces was used in PCR reactions with one of the internal primers and a telomere speci¢c primer. A PCR product was only obtained using the telomere primer with the primer H3R. Therefore, based on the sequence of this fragment a new primer H3F10 was constructed to amplify the end and the 3P £anking region of the gene together with the telomere primer. The sequence of this gene-sized piece revealed a histone H3-like protein. The N-terminal region is highly altered compared to other histones H3, making an alignment in this region nearly impossible (STYLE10 in Fig. 1). There are 42 additional amino acids, many substitutions and starting at position 14 a repeated motif of the amino acids lysine and glycine (KGKGKGKGKGK) is found. Despite many substitutions in the C-terminus an alignment in this region is unambiguous. The 5P-noncoding region spans 150 bp without the telomere sequences. A putative CCAAT box (3107) and two putative TATA boxes (319, 346) were found at a reasonable distance to the translation start. 3.4. Comparison with histone H3 variants from other ciliates The H3 genes show again the high genetic diversity within the ciliates also seen for rRNA (reviewed in [15]) and histone H4 genes [2]. Many di¡erences in the H3 amino acid sequences exist, not only compared to the oligohymenophorean T. thermophila, but also between the more closely related hypotrichous species E. crassus and S. lemnae. However, more surprising are the numerous histone H3 variants in this species. In the oligohymenophorean T. thermophila three H3 genes were found [16], two of which encode an identical, replicationdependent (major) H3 protein. The third gene encodes a replication-independent variant, which is expressed in nuclei of non-growing cells [17]. This quantitatively minor H3 variant has 16 substitutions compared to the major H3. None of the H3 variants of S. lemnae shows a higher sequence similarity with either the major or with the minor variants of Tetrahymena. In the hypotrichous ciliate E. crassus only two
49
di¡erent histones H3 were detected [3]. Transcripts of one gene accumulate only during the meiotic and early polytene stages of the sexual phase of the life cycle. The transcripts of the other gene were detected during vegetative growth and the ¢nal replication phase of the sexual phase. Thus, in contrast to the `vegetative' histone H3, the `polytene' histone H3 may play a fundamental role in the development of the macronucleus [3]. The `polytene' variant contains two insertions in the amino-terminal domain and also some substitutions. Only the histone-like protein `STYLE10' also has insertions in the amino-terminal region. However, there are no sequence similarities with the `polytene' variant of E. crassus. At present, why there are so many variants in S. lemnae remains an open question since the genome organization as well as the processes of DNA rearrangement during development of the new macronuclei are very similar in hypotrichs (reviewed in [1]). The great variability in the amino-terminal domain of all these variants indicates that they may also play di¡erent roles in the cell cycle of S. lemnae and might be expressed during di¡erent stages, as seen for the H3 variants in E. crassus [3]. Presumably, some variants may be speci¢c to the transcriptionally silent micronucleus because indications for a micronucleus-speci¢c histone H3 in S. lemnae were given [18]. These questions should be addressed by studying the expression of these genes. An analysis of the transcripts could also give clues as to why S. lemnae has so many variants compared to all other eukaryotes studied so far.
Acknowledgments I want to thank Martin Schlegel and Stan Theophilou for their critical reading of the manuscript.
References [1] Prescott, D.M. (1994) The DNA of ciliated protozoa. Microbiol. Rev. 58, 233^267. [2] Bernhard, D. and Schlegel, M. (1998) Evolution of histone H4 and H3 genes in di¡erent ciliate lineages. J. Mol. Evol. 46, 344^354. [3] Jahn, C., Ling, Z., Tebeau, C.M. and Klobutcher, L.A. (1997) An unusual histone H3 speci¢c for early macronuclear devel-
FEMSLE 8769 19-5-99
50
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
D. Bernhard / FEMS Microbiology Letters 175 (1999) 45^50 opment in Euplotes crassus. Proc. Natl. Acad. Sci. USA 94, 1332^1337. Elsevier, S.M., Lipps, H.J. and Steinbruëck, G. (1978) Histone genes in macronuclear DNA of the ciliate Stylonychia mytilus. Chromosoma 69, 291^306. Southern, E.M. (1975) Detection of speci¢c sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503^517. Arents, G., Burlingame, R.W., Wang, B.-C., Love, W.E. and Moudrianakis, E.N. (1991) The nucleosomal core histone ocî resolution: A tripartite protein assembly and a tamer at 3.1 A left-handed superhelix. Proc. Natl. Acad. Sci. USA 88, 10148^ 10152. Arents, G. and Moudrianakis, E.N. (1993) Topography of the histone octamer surface: Repeating structural motifs utilized in the docking of nucleosomal DNA. Proc. Natl. Acad. Sci. USA 90, 10489^10493. Hecht, A., Laroche, T., Strahl-Bolsinger, S., Gasser, S.M. and Grunstein, M. (1995) Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins : A molecular model for the formation of heterochromatin in yeast. Cell 80, 583^592. Megee, P.C., Morgan, B.A. and Smith, M.M. (1995) Histone H4 and the maintenance of genome integrity. Genes Dev. 9, 1716^1727. Santisteban, M.S., Arents, G., Moudrianakis, E.N. and Smith, M.M. (1997) Histone octamer function in vivo: mutations in the dimer-tetramer interfaces disrupt both gene activation and repression. EMBO J. 16, 2493^2506. Hicke, B.J., Celander, D.W., MacDonald, G.H., Price, C.M.
[12]
[13]
[14]
[15] [16]
[17]
[18]
and Cech, T.R. (1990) Two versions of the gene encoding the 41-kDa subunit of the telomere binding protein of Oxytricha nova. Proc. Natl. Acad. Sci. USA 87, 1481^1485. Gray, J.T., Celander, D.W., Price, C.M. and Cech, T.R. (1991) Cloning and expression of genes for the Oxytricha telomere-binding protein: speci¢c subunit interactions in the telomeric complex. Cell 67, 807^814. Herrick, G., Hunter, D., Williams, K. and Kotter, K. (1987) Alternative processing during development of a macronuclear chromosome family in Oxytricha fallax. Genes Dev. 1, 1047^ 1058. Klobutcher, L.A., Hu¡, M.E. and Gonye, G.E. (1988) Alternative use of chromosome fragmentation sites in the ciliated protozoan Oxytricha nova. Nucleic Acids Res. 16, 251^264. Schlegel, M. (1994) Molecular phylogeny of eukaryotes. TREE 9, 330^335. Thatcher, T.H., MacGa¡ey, J., Bowen, J., Horowitz, S., Shapiro, D.L. and Gorovsky, M.A. (1994) Independent evolutionary origin of histone H3.3-like variants of animals and Tetrahymena. Nucleic Acids Res. 22, 180^186. Bannon, G.A., Calzone, F.J., Bowen, J.K., Allis, C.D. and Gorovsky, M.A. (1983) Multiple, independently regulated, polyadenylated messages for histone H3 and H4 in Tetrahymena. Nucleic Acids Res. 11, 3903^3917. Schlegel, M., Muller, S., Ruder, F. and Buësen, W. (1990) Transcriptionally inactive micronuclei, macronuclear anlagen and transcriptionally active macronuclei di¡er in histone composition in the hypotrichous ciliate Stylonychia lemnae. Chromosoma 99, 401^406.
FEMSLE 8769 19-5-99