Macronucleus Structure and Macronucleus Development in Hypotrichous Ciliates

Inl. J. De,. BioI. 40: 141-147 (1996) 141

Macronucleus structure and macronucleus development in hypotrichous ciliates

HANS J. LIPPS" and CLAUDINEEDER2 '/nstitut fur Zelfbiologie, Universitat WittenlHerdecke, Witten, and 2Max Delbruck Zentrum fUr Molekulare Medizin, Berlin, Germany

ABSTRACT In the course of macronuclear development of the hypotrichous ciliates all genetic information not required for normal growth of the cell is removed from the new macronucleus. This differentiation process involves DNA-splicing, excision of transposons, DNA-fragmentation, selective gene amplification and telomere addition. Since many of the processes observed during macronuclear development, such as DNA-transposition, DNA-rearrangement or selective DNA- amplification, may occur in differentiating cells of higher organisms, this biological system provides an unusual opportunity to study the ways in which DNA-sequences can be manipulated in a differentiating cell.

KEY WORDS: DNA-jJroressing, D~\:4-{mml}ositioll, DXA-Jragmentation, {p!mneres, minirhm!!losomes

Introduction ing of molecular mechanisms involved in the differentiation of a new macronucleus. The unicellular ciliated protozoa are among the most complex eucaryotic cells and have been studied for many years as mod- Macronuclear structure in hypotrichous ciliates el systems of cellular differentiation processes (for review see Grell, 1973; Jean. 1986; Gall, 1986). The DNA content and the kinetic complexity of macronuclei One characteristic of the ciliate cell is the occurrence of two and micronuclei from two hypotrichous ciliates are shown in morphological and functional different nuclei in one cell, Table 1. While micronuclear DNA reassociates as multiple DNA macronuclei and micronuclei. While macronuclei represent the components indicating the presence of repeated and non repeat- vegetative nucleus, making all the RNA required for normal ed sequences, the reassociation kinetics of macronuclear DNA growth of the cell, the micronuclei represent the generative follows a simple second order reaction kinetic. No repetitive DNA nuclei of the cell. After sexual reproduction (conjugation) a new could be found in the macronucleus and the kinetic complexity of macronucleus is formed by a micronuclear derivative while the its DNA is only about 10-14 times the complexity 01 E. coli DNA old macronucleus is resorbed. A schematic diagram of (for review see Kraut eta/., 1986; Prescott, 1994). These results macronuclear development in hypotrichous ciliates is shown in suggest that only part of the micronuclear DNA sequences are Figure 1. It includes an initial DNA synthesis phase leading to present in the macronuclear genome and that most of the chro- the formation of polytene chromosomes; these are subsequent- mosomal DNA, that is not essential for the vegetative life cycle ly degraded and up to over 90 % of their DNA is eliminated. A of the cells, is specifically eliminated in the course of macronu- second DNA synthesis phase then leads to the vegetative clear development. macronucleus (for review see Kraut et al.) 1986; Klobutcher and As early as 1971 Prescott and his coworkers reported that the Prescott, 1986; SteinbrOck, 1990; Prescott. 1994). This devel- DNA of macronuclei from hypotrichous ciliates is of low molecu- opmental process suggests that macronuclear development in lar weight when investigated by sedimentation analysis and elec- hypotrichous ciliates is accompanied by dramatic DNA reorga- tron microscopy. These results were later confirmed by agarose nization processes and recent work has demonstrated that this gel electrophoresis (Fig. 2) and probing the DNA with specific developmental process provides the opportunity to study basic gene probes (reviewed in: Klobutcher and Prescott. 1986). The biological phenomena such as specific DNA fragmentation, size of macronuclear DNA-molecules varies between about 0.3 DNA-elimination, DNA-transposition, DNA-splicing and de novo and 20 kb with an average size of 1-3 kb. Some fragments are addition of telomeric sequences (for review see Klobutcher and Jahn. 1991; Prescott. 1994). In this paper we will give a short Ablm'l'iatiml-l lISI'd in 111/5/mjln: IES, illTtTlla] eliminated ~equ{,I1("{,~; THE, overview of the molecular organization of the hypotrichous cili- telomere-bearing eJemeut; TEC, tran~po~otl Eup!otes uaSSlLS; Tee, te]otll- ate nuclei and review some recent progress in our understand- t:n: con mining clcmcnt.

"'Address for reprints: Institut fur Zellbiologie, Universitat Witten/Herdecke, Stockumer Str. 10, 0-58448 Witten, Germany. FAX: 2302.669220.

0214-62R2/96/S03.01J r.JI:BC Pr~" Prim.JinSp;!in 142 H.I Lipps ([lid C. Eder

0 so 100 amplified macronuclear DNA-sequence has been shown to pro- mote plasmid amplification in mouse cells and may represent, in hours vivo, a strong origin of replication (Wegner el a/., 1989). Detailed analysis of the subtelomeric regions from Styfonychia revealed a 0 0 highly ordered and conserved sequence organization and it has . been speculated that some of these subtelomeric sequences e may be involved in the DNA-fragmentation process during macronuclear differentiation (Maercker and Lipps, 1993). A ~Dolytenisation DNA degradation DNA amplifIcation schematic diagram of one macronuclear DNA-molecule from m Slylonychia is shown in Figure 3. ~m E 0 The DNA of the ciliate nuclei is associated with typical eucary- 0 ~m U otic histones. However, only small quantities of histone H 1 are 0 ~0 E ~c detected in macronuclear chromatin (for review see Kraut et al., ~0 0 ~ro L L ~u 1986; Gadilla el al., 1986). There is one report descnbing the ~2 m c u ro 0 m L ~~0 E absence of histone H3 in the micronucleus and the presence of a L C ~0 Uro 2: u D. t high molecular weight protein X in Slylonychia lemnae. In the C ~;;; ro course of macronuclear development this protein X is replaced by m ~0 "'"~Z > <:> "E "- a conventional histone H3 (Schlegel el al., 1990). Limited digestion of macronuclear and micronuclear chromatin with micrococ- Fig. 1, Schematic diagram of macronuclear development in hypotri- cal nuclease revealed a nucleosome repeat of 220 bp in the chous ciliates (modified after Ammermann et ai" 1974; Kraut et aI., macronuclei of Slylonychia and Oxylricha and a repeat length of 1976). 200 bp in the micronuclei of Slylonychia and 180 bp in the micronuclei of Oxytricha (for review see: Kraut et al., 1986). seen as prominent bands, indicating that certain DNA molecules Proteins specifically binding to telomeric DNA and to the 3' single- occur in higher copy numbers than others. In fact, it was shown stranded extension of telomeres have been identified in the for defined sequences, that the copy number of macronuclear macronucleus of several hypotrichous ciliates. A defined DNA- DNA-molecules varies between about 103 and 106 per macronu- protein complex in this region is formed with nucleosomes cleus, being always constant for each sequence (see e.g. phased inwards from this complex (Lipps el al., 1982; Gottschling Helftenbein 1985, Gonzelmann and Helftenbein, 1987; Baird and and Gech, 1984; Gottschling and Zakian, 1986; Steinhilber and Klobutcher, 1991). The banding pattern of macronuclear DNA Lipps, 1986; Price and Gech, 1987, 1989; Raghuraman and seems to be characteristic for different species and has been Gech, 1989; Pnce, 1990; Gray el al., 1991; Fang el al., 1993). used as a systematic criterion to identify hypotrichous ciliates Lysis of macronuclei under low ionic strength releases short (for review see Ammermann, 1990). chromatin fibers and the length distribution of these fibers corre- Taking all these results together it can be concluded that each sponds well to the size distribution of macronuclear DNA-mole- macronucleus contains between 10,000 to 20,000 different cules (Lipps el al., 1978; Butler el al., 1984; Gadilla et a/., 1986). DNA-molecules, each present in copy numbers between sever- Thus, under these conditions it would be possible to isolate and al hundred up to 106 per nucleus, i.e. one macronucleus con- characterize specific macronuclear "minichromosomes" in their tains several millions individual short DNA-molecules. chromatin configuration. However, when macronuclei are gently Sequence analysis of many macronuclear DNA-molecules lysed on a deionized water hypophase only long continuous 30 (forreview see Prescott, 1994) revealed that they always contain nm chromatin fibers are observed (Meyer and Lipps, 1981). one open reading frame, 3' and 5' non coding regions of various Possibly, telomere-protein-interactions may lead to a higher length and telomeric repeats consisting of blocs of 5'-C4A4 and a order chromatin organization in the macronucleus of hypotric- 3' protruding G4T4 tail which can adopt a special DNA-structure hous ciliates (Lipps el al., 1982). Antibodies against Z-DNA both in vilro and in vivo (e.g. Lipps, 1980; Oka el al., 1980; strongly react with the macronucleus but not with the micronu- Klobutcher el al.. 1981; Kaine and Spear, 1982; Lipps el al., cleus of Sly/onychia. When macronuclear DNA is nicked by gen- 1982; Pluta el al., 1982; Helftenbein, 1985; Gonzelmann and tle DNase I treatment no more binding of the anti-Z-DNA-anti- Helftenbein, 1987; Harper and Jahn, 1989; Sundquist and Klug, body is observed, suggesting that macronuclear DNA occurs in 1989; Williams el al., 1989; Wefes and Lipps, 1990; Fang and Gech, 1991; Kang el al., 1992). Introns are only very rarely found TABLE 1 in macronuclear genes (Brunen-Nieweler, 1991; Fang and Cech, 1991; Gray el a/., 1991) and, similar to other ciliated protozoan, DNA CONTENT AND DNA COMPLEXITY OF MACRONUCLEAR an unusual genetic code is found in many of these genes AND MICRONUCLEAR DNA OF TWO HYPOTRICHOUS CILIATES (Helftenbein, 1985; Horowitz and Gorovsky, 1985; Preer el al., DNA content (pg) Kinetic complexity 1985; Meyer et al., 1992). In the non-coding regions of macronuclear "gene-sized" DNA-molecules sequences which possibly Macro- Micro- Macro- Micro- can be related to gene expression and replication are found nucleus nucleus nucleus nucleus (Helftenbein el al., 1989). However, so far no replication origin Sty/onychia lemnae 788 13 3.1x101O 1.45x1012 has actually been mapped in macronuclear DNA-molecules. Oxytricha sp. 1t6 t .32 3.6x1010 1.5x1012 One sequence derived from one subtelomeric region of an over- MacrOlluc!ellr diflerentillfio1/ 143 a supercoiled configuration in vivo (Lipps at a/., 1982). The exact kb in vivo organization of DNA molecules in the macronucleus still needs to be examined.

DNA-sequence organization in the micronucleus 20-

Micronuclear DNA is contained in many, usually over hundred, small chromosomes and its DNA has chromosomal size. ~~ The main results coming from the molecular analysis of the 5,3 ___ ------.. micronuclear genome are: macronuclear precursor genes occur 5.0- in clusters separated from each other by long non-coding 4.2- regions, macronuclear precursor genes contain internal 3.6- sequences, (internal eliminated sequences, IES) not found in the macronucleus, they are not associated with telomeric sequences and a large percentage of the micronuclear genome consists of 2.0 ______transposon-like elements. 1.9~~_ Several highly repetitious sequence families have been char- 1.7- acterized in the micronucleus of different hypotrichous ciliates. 1.3 ------Fig. 2. Electrophoretic For example, a 160 bp tandemly arranged sequence family is pattern of macronucJear part of the highly repetitious sequences in Sly/onychia pusfu/afa 0.9- DNA of Sty/onychia lem- (Dawson el ai, 1983), repeats of about the same size have been ~- 0.8-- nae separated on detected in Oxy/richa fallax(Dawson elal., 1984). Large, over 11 agarose gels. (A) kb, DNA fragments belonging to a repetitive sequence family of Molecular weight stan- the chromosomal DNA of Oxytricha were characterized (Boswell dard (B) Macronuclear ef a/., 1983). DNA IC) Micronuclear Part of the micronuclear repetitious DNA-sequences are fam- A B c DNA. ilies of elements resembling transposons. Different classes of transposon-Jike elements have been described in the micronu- The evolution and possible biological function of all these trans- clei of various hypotrichous ciliates (for review see Klobutcher poson-like elements is not clear; however, at least part of the and Jahn, 1991). A schematic drawing showing the different TCE is transcribed at a specific stage during macronuclear classes is shown in Figure 4. In Eupfotas crassus two related development (Stoll el a/., 1993). families of transposon-like elements, TEC1 and TEC2 (Fig. 4 a) MacronucJear precursor sequences occur in the micronuclear were characterized in the micronuclear genome. About 30.000 genome in clusters of two to several genes. The individual clus- copies of these elements are found in the micronucleus and are ters are separated by long (over 10 kb) non-coding sequences. absent in the macronucleus. They have a size of about 5.3 kb Within a cluster, short spacer regions (0-600 bp) are tound with 700-725 bp terminal inverted repeats and a 5' TA 3' target between the different macronuclear destined sequences site duplication. Very often, these sequences interrupt macronu- (Boswell el a/., 1983; Klobutcher el a/., 1986; Jahn ef a/., clear precursor sequences (Jahn el al., 1988a,b, 1989; Baird ef 1988a,b; Jahn, 1991; Eder el a/., 1993). An interesting sequence a/., 1989; Krikau and Jahn, 1991; Tausta e/ al., 1991). Oxyfricha organization was described in a spacer region in Styfonychia tal/ax contains about 1.000 copies of the transposon-like ele- lemnae; since only few sequence data are available no general ment TBE-1 (telomere bearing element) in its micronuclear rules about spacer sequence organization can be made at this genome (Fig. 4b). It has an approximate size of 4 kb with 77 bp point (Eder el a/., 1993). In no case analyzed are telomeric terminal inverted repeats surrounded by a 3 bp direct repeat, repeats associated with macronuclear precursor sequences, so which appears to be the equivalent of a target site duplication. that these sequences must be added to macronuclear Telomeric C4A4-repeats are found at the ends of the inverted sequences in the course of macronuclear development. Very repeats (Herrick el a/., 1985; Hunter el a/., 1989). Both elements, often, macronuclear destined sequences are interrupted by short TEC and TBE, appear to encode transposase proteins (Doak ef non-coding sequences (internal eliminated sequences, IES). a/., 1993). Another telomere containing element (TCE) was Usually the different segments that comprise the macronuclear described in the micronucleus and the polytene chromosomes of DNA-molecules are arranged in a linear order (Klobutcher at al., Sly/onychia lemnae (Fig. 4c). About 1.500 copies of this element 1986; Jahn ef a/., 1988a,b; Jahn 1991; Bierbaum el a/., 1991; are found in the micronucleus. It has a size of about 7 kb and is Eder at al., 1993). However, in the case of the actin gene and the flanked by 2 kb direct repeats. Immediately adjacent to one of genes coding for telomere-binding proteins, the DNA-segments these repeats, an 18mer of the C4A4telomeric repeat sequence that will form the macronuclear DNA-molecules are scrambled in is localized. In addition, sequences homologous to a 2.6 kb the micronuclear genome implying an extensive reorganization macronuclear DNA-molecule are found in this element. This process during macronuclear development in order to create sequence family is highly conserved showing almost 90 % functional genes (Greslin et al., Mitcham et al., 1992; Prescott; homology to each other (Stoll el a/., 1993). It seems very likely 1992,1994). that many of the other repetitive sequence families in hypotric- The internal eliminated sequences (IES) interrupting hous ciliates will be shown to have transposon-like character. macronucJear precursor sequences are small (10-550 bp) --

144 H.i. Lipps and C. Eder sequences and composed of unique DNA. On average one IES A 5.3 kb is found per 1 kb of macronuclear precursor sequence. The gen- 4kb eral structure of a typicallES is shown in Figure 4 d. Almost all B of the IES are bordered by 2-7 bp direct repeats and very often imperfect inverfed repeats are found near the ends (Klobutcher 6.8 kb at a/., 1984; Herrick at a/., 1987; Ribas-Aparicio at a/., 1987; c Jahn at a/., 1988a,b; Baird at al., 1989; Hunter at al., 1989; 0. \-0.5 kl Bierbaum at a/., 1991; Eder et al., 1993). This sequence organi- D [E]] zation led to the suggestion that IES may be degenerate ver. sians of transposable elements (for review see: Klobutcher and Ddlrectrepeat Jahn 1991). Macronuclear differentiation I telomerlcsequence

While the morphological events occurring during macronu- &'SSSI homology to macronuclear DNA clear development are well described (for review see: Ammermann at al., 1974; Kraut at al., 1986), the molecular Inl,lertedrepeat processes (summarized in Fig. 5) are still poorly understood. ~ Macronuclear differentiation starts with the mitotic division of Fig. 4 Schematic diagrams of transposon-like elements and inter- the syncaryon. The factors determining which of the daughter nal eliminated sequences hypotrichous ciliates. (A) TEC, (8) TBE, (C) nuclei will develop into the new macronucleus are unknown in TCE, (D) IES (modified after Klobutcher and Jahn. 1991; Stoll et al., hypotrichous ciliates. The first DNA reduction event can occur 1993) as early as the first DNA synthesis phase (Fig. 1). In Sty/onychia /emnae only about 30% of the chromosomes enter are interconnected by a 10 nm chromatin axis. Later, these 30 the polytenization stage; the rest become pycnotic and eventu- nm chromatin loops are released from the axis in the form of ally disintegrate (Ammermann et al., 1974; Meyer and Lipps, chromatin rings and eventually become degraded while the 10 1981). The number of polytene chromosomes, having a 100- nm chromatin axis remains intact (Meyer and Lipps, 1980; 200 polyteny, has been estimated to be about 50-100 and the 1981). This morphological observation possibly can be related total number of bands in the polytene chromosomes is around to the en masse excision of transposon-like elements and 10.000-15.000 (Ammermann at al., 1974). In Oxytricha all chro- internal eliminated sequences in the course of polytene chromosomes become polytenized, the macronuclear anlage con- mosome formation. In Euplotes crassus the elements TEC1 tains about 120 polytene chromosomes with a total number of and TEC2 are eliminated from the genome during a discrete bands of about 10.000 (Spear and Lauth, 1976). So far, no time period and their excision precedes the removal of IES. comparison of sequence copy numbers has been made Both elements show similar excision mechanisms: They between the micronucleus and the polytene chromosomes of appear as free circles following excision. The junction of these the macronuclear anlagen. However, injection of macronuclear free circular forms consists of both copies of the target site precursor sequences into the macronuclear anlage suggest that direct repeats with a few, normally 10, additional nucleotides selective amplification of macronuclear destined sequences between them. This implies that excision involves staggered occurs during the first DNA-synthesis phase (Wen et al., 1995). cuts in the chromosome that are subsequently filled in and Sequences involved in the control of copy number have not yet therefore the two excision products share some of the same been described. nucleotides (Baird et al., 1989; Jahn at al., 1989; Tausta and In spread electron microscopical preparations of the devel- Klobutcher, 1989, 1990; Krikau and Jahn, 1991; Tausta et al., oping macronucleus it became apparent, that during formation 1991; Klobutcher and Jahn, 1991; Jaraczewski and Jahn, of the polytene chromosomes a dramatic reorganization of 1993; Klobutcher at al., 1993). The TBE-1 elements are also chromatin occurs. An increasing percentage of 30 nm chro- excised as circles but the junction contains only one direct matin fibers becomes organized into loop-like structures which repeat and no flanking DNA (Hunter et al., 1989; Williams et al., 1993). The degradation of the polytene chromosomes becomes evi- dent in electron microscopic thin sections by the appearance of membranous material surrounding the bands of the chromosomes. In later stages, bands are completely enclosed in vesi- cles (for review see Klobutcher and Prescott, 1986; Kraut et al., inl,lertedrepeat ~openreadlngrrame 1986). It is assumed that in this stage fragmentation of the ~ genome, degradation of DNA and eventually telomere addition occurs. In contrast to the holotrichous ciliate Tetrahymena very palindromiCsequences .. telomere 10 few data about DNA fragmentation and telomere addition exist in hypotrichous ciliates (for review see: Klobutcher and Jahn, Fig. 3. Schematic diagram of a gene-sized macronuclear DNA-mol- 1991). In Oxytricha multiple fragmentation can be used, ieading ecule from Sty/onychia /emnae (modified after Maercker and Lipps, 1993) to different size-versions of macronuclear DNA-molecules MaCl'Ollllclear differelltiatioll 145

IES Acknowledgments oc This work was supported by the Deutsche Forschungsgemeinschaft. spacer transposon

References ",'" ""'" i AMMERMANN. D-/{1990}. The contribution of hypotrichous ciliates to our under. standing of mQlecular biology and evolution of ciliates. Zool. Sci. 7: 13-22. fragmentation excision AMMERMANN, 0.," STEINBRUCK, G., V. BERGER, L. and HENNIG, W. (1974). The development of the macronucleus in the ciliated protozoan Sty/onychia mytilus. Chromosoma 4: 401-419. se1ect1ve 2mpllfication reorderlngorexons BAIRD, S,E. and KLOBUTCHER, L.A. (1989), Characterization of chromosome fragmentation in two protozoans and identification of a candidate fragmentation sequence in Euplotes crassus. Genes Dev. 3: 585-597. '"'''','''''''''''''''"" ""r BAIRD, S.E. and KLOBUTCHER, L.A. (1991). Differential DNA amplification and ...I' "'''''''''',','''''''''"'''''''''',''''''''' "''''''''''''','','''' copy number control in the hypotrichous ciliate Eupfotes crassus. J. Protozoal. 38: 136-140.

BAIRD, S.E" FINO, G.M., TAUSTA, S.L. and KLOBUTCHER, L.A. (1989). Micronuclear genome organization in Euplotes crassus. A transposon-like element is removed during macronuclear development. Mol. Cell. Bio/. 9: 3793-3807. BIERBAUM, P, DONHOFF, 1. and KLEiN, A. (1991). Macronuclear and micronu- \/telomere addition clear configurations of a gene encoding the protein synthesis elongation factor ET1-(.( in Stylonychia lemnae, Mol. Microbiol. 5: 1567-1575. Fig. 5 Molecular events occurring during macronuclear differentia- BOSWELL, R.E.. JAHN, C.L., GRESLlN, A.F. and PRESCOTT, D.M. (1983), tion in hypotrichous ciliates. Organization of gene and non-gene sequences in the micronuclear DNA of Oxytricha nova. Nucleic Acids Res. 11: 3201-3217. (Herrick et al., 1987; Baird and Klobutcher, 1989). In contrast, BUTLER, AP., LAUGHLIN, TJ., CADILLA, C.L, HENTY, J.M. and OLlNS, D.E. (1984), Physical structure of the gene-sized chromatin from the protozoan Eup/otes crassus uses only unique fragmentation sites. In this Oxytricha. Nucleic Acids Res. 12: 3201-3217. species a conserved sequence was found in the subtelomeric BRUNEN-NIEWELER, C., SCHMIDT, H.J, and HECKMANN, K. (1991). Two regions of macronuclear DNA-molecules which may be a candi- introns in the pheromone 3-encoding gene of Euplotes octocarinatus. Gene date for a cis-acting sequence directing the fragmentation 109: 233-237. process (Baird and Klobutcher, 1989). A systematic analysis of CADILLA, C.L., HARP, J., FLANAGAN, J.M., OLlNS, A.L and OLlNS, D.E. (1986). the subtelomeric regions of Sty/onychia macronuclear DNA-mol- Preparation and characterization of soluble macronuclear chromatin from the hypotrich Eupfotes eurystomus. Nuc/eic Acids Res. 14: 823-841 . ecules revealed a highly ordered and common sequence orga- CONZELMANN, K.K. and HELFTENBEIN, E. (1987). 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-- 146 H.J. Lipps and C. Eder

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