European Journal of PROTISTOLOGY · (Ciliophora, Frontoniidae): Its Systematic Implications 361 Use...

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European Journal of Formerly PROTISTOLOGICA PROTISTOLOGY Editorial Board: Κ. HAUSMANN (Managing Editor) Institut für Zoologie Freie Universität Berlin Königin-Luise-Straße 1-3 1000 Berlin 33 (West) B. S. C. LEADBEATER Department of Plant Biology University of Birmingham P.O. Box 363 Birmingham Β 15 2 TT (Great Britain) D. H. LYNN Department of Zoology University of Guelph Guelph, Ontario N 1 G 2 W 1 (Canada) D. J. PATTERSON (Review Editor) Department of Zoology University of Bristol Bristol BS8 1UG (Great Britain) Editorial Assistant: L. PLATT, Berlin (West) Board of Reviewers: A. ADOUTTE/Orsay (France) CH. F. BARDELE/Tübingen (West Germany) J. BOUIX/Montpellier (France) G. BRUGEROLLE/Clermont-Ferrand (France) E. U. CANNING/Ascot (Great Britain) G. CLEFFMANN/Gießen (West Germany) I. DESPORTES/Paris (France) J. F. DUBREMETZ/Villeneuve d'Ascq (France) J. FEBVRE/Villefranche (France) T. FENCHEL/Helsing0r (Denmark) B. J. FINLAY/Windermere (Great Britain) W. FOISSNER/Salzburg (Austria) J. GENERMONT/Paris (France) J. GRAIN/Clermont-Ferrand (France) A. GRfBECKI/Warszawa (Poland) M. HAUSER/Bochum (West Germany) I. B. HEATH/Downsview (Canada) K. HECKMANN/Münster (West Germany) K. W. JEON/Knoxville (USA) M. JERKA-DZIADOSZ/Warszawa (Poland) R. LARSSON/Lund (Sweden) P. DE PUYTORAC Groupe de Zoologie et Protistologie Universite de Clermont-Ferrand II Boite Postale 45 63170 Aubiere (France) Y. SHIGENAKA Faculty of Integrated Arts and Sciences Hiroshima University Higashisenda-machi, Naka-ku Hiroshima 730 (Japan) J. J. LEE/New York (USA) J. LOM/Ceske Budejovice (CSFR) P. A. LUPORINI/Camerino (Italy) H. MACHEMER/Bochum (West Germany) H. MEHLHORN/Bochum (West Germany) J. P. MIGNOT/Clermont-Ferrand (France) 0. MOESTRUP/Copenhagen (Denmark) M. MULISCH/Köln (West Germany) J. NILSSON/Copenhagen (Denmark) R. NOBILI/Pisa (Italy) R. K. PECK/Geneve (Switzerland) I. RAIKOV/Leningrad (USSR) W. REISSER/Göttingen (West Germany) P. SATIR/Bronx (USA) E. SCHNEPF/Heidelberg (West Germany) J. SCHREVEL/Paris (France) M. SLEIGH/Southampton (Great Britain) T. SUZAKI/Hiroshima (Japan) K. VICKERMAN/Glasgow (Great Britain) N. WILBERT/Bonn (West Germany) Volume 25 · 1989/90 Gustav FischerVerlag Stuttgart- New York

Transcript of European Journal of PROTISTOLOGY · (Ciliophora, Frontoniidae): Its Systematic Implications 361 Use...

Page 1: European Journal of PROTISTOLOGY · (Ciliophora, Frontoniidae): Its Systematic Implications 361 Use of Nile Red as a Rapid Measure of Lipid Content in Ciliates 369 Ρ salter iomonas

European Journal of Formerly PROTISTOLOGICA

P R O T I S T O L O G Y Editorial Board:

Κ. H A U S M A N N (Managing Editor) Institut für Zoologie Freie Universität Berlin Königin-Luise-Straße 1-3 1000 Berlin 33 (West)

B. S. C. LEADBEATER Department of Plant Biology University of Birmingham P.O. Box 363 Birmingham Β 15 2 TT (Great Britain)

D. H . L Y N N Department of Zoology University of Guelph Guelph, Ontario N 1 G 2 W 1 (Canada)

D. J. PATTERSON (Review Editor) Department of Zoology University of Bristol Bristol BS8 1UG (Great Britain)

Editorial Assistant: L. PLATT, Berlin (West)

Board of Reviewers:

A. ADOUTTE/Orsay (France) CH. F. BARDELE/Tübingen (West Germany) J. BOUIX/Montpellier (France) G. BRUGEROLLE/Clermont-Ferrand (France) E. U. CANNING/Ascot (Great Britain) G. CLEFFMANN/Gießen (West Germany) I . DESPORTES/Paris (France) J. F. DUBREMETZ/Villeneuve d'Ascq (France) J. FEBVRE/Villefranche (France) T. FENCHEL/Helsing0r (Denmark) B. J. FINLAY/Windermere (Great Britain) W. FOISSNER/Salzburg (Austria) J. GENERMONT/Paris (France) J. GRAIN/Clermont-Ferrand (France) A. GRfBECKI/Warszawa (Poland) M . HAUSER/Bochum (West Germany) I . B. HEATH/Downsview (Canada) K. HECKMANN/Münster (West Germany) K. W. JEON/Knoxville (USA) M . JERKA-DZIADOSZ/Warszawa (Poland) R. LARSSON/Lund (Sweden)

P. DE PUYTORAC Groupe de Zoologie et Protistologie Universite de Clermont-Ferrand I I Boite Postale 45 63170 Aubiere (France)

Y. SHIGENAKA Faculty of Integrated Arts and Sciences Hiroshima University Higashisenda-machi, Naka-ku Hiroshima 730 (Japan)

J. J. LEE/New York (USA) J. LOM/Ceske Budejovice (CSFR) P. A. LUPORINI/Camerino (Italy) H . MACHEMER/Bochum (West Germany) H . MEHLHORN/Bochum (West Germany) J. P. MIGNOT/Clermont-Ferrand (France) 0 . MOESTRUP/Copenhagen (Denmark) M . MULISCH/Köln (West Germany) J. NILSSON/Copenhagen (Denmark) R. NOBILI/Pisa (Italy) R. K. PECK/Geneve (Switzerland) I . RAIKOV/Leningrad (USSR) W. REISSER/Göttingen (West Germany) P. SATIR/Bronx (USA) E. SCHNEPF/Heidelberg (West Germany) J. SCHREVEL/Paris (France) M . SLEIGH/Southampton (Great Britain) T. SUZAKI/Hiroshima (Japan) K. VICKERMAN/Glasgow (Great Britain) N . WILBERT/Bonn (West Germany)

Volume 25 · 1989/90

Gustav FischerVerlag • Stuttgart- New York

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25/1 C o n t e n t s

RAUSMANN, Κ. and PATTERSON, D . J.

REVIEW NlLSSON, J. R.

ARTICLES SCHMAHL, G., MEHLHORN, Η. and TARASCHEWSKI, H .

LARSSON, J. I . R.

MACHEMER-RÖHNISCH, S. and MACHEMER, H .

RÖTTGER, R . , SCHMALJOHANN, R . and ZACHARIAS, Η.

AMMERMANN, D . , SCHLEGEL, Μ . and HELLMER, K . - H .

SODEIK, B. , BRIX, K. and STOCKEM, W .

BOOK REVIEWS

1 Editorial

2 Tetrahymena in Cy to toxicology: W i t h Special Reference to Effects of Heavy Metals and Selected Drugs

2 6 Treatment of Fish-Parasites 7. Effects of Sym. Triazinone (Toltrazuril) on Developmental Stages of Myxobolus sp. Bütschli, 1 8 8 2 (Myxosporea, Myxozoa): A Light and Electron Microscopic Study

3 3 On the Cytology and Life Cycle of Cougourdella polycentropi Weiser, 1 9 6 5 , wi th Comments on the Taxonomy of the Genus Cougourdella Hesse, 1 9 3 5 (Microspora, Cougourdellidae)

4 5 A Ca Paradox: Electric and Behavioural Responses of Paramecium Following Changes in External Ion Concentration

6 0 Endoreplication of Zygotic Nuclei in the Larger Foraminifer Heterostegina depressa (Nummulitidae)

6 7 North American and Eurasian Strains of Stylonychia lemnae (Ciliophora, Hypotrichida) Have a High Genetic Identity, but Differ in the Nuclear Apparatus and in their Mating Behavior

7 5 Sequestration of Microinjected Molecular Probes from the Cyto­plasm of Amoeba proteus

8 5 BIBEL, D . J.: Milestones in Immunology (GÖTZ, P.)

8 5 BROCK, T. D . : Robert Koch. A Life in Medicine and Bacteriol­ogy DUBOS, R.: Pasteur and Modern Sciences (ZÖLFFEL, Μ.)

8 5 FOISSNER, W. and FOISSNER, I . : Catalogus Faunae Austriae. Teil Ic: Stamm: Ciliophora (CORLISS, J. O.)

8 6 JONES, R and ILMAVIRTA, V . : Flagellates in Freshwater Ecosystems (FlNLAY, B. J.)

8 6 SALFELDER, K . - H . wi th SAUERTEIG, Ε . , N O V O A MONTERO, D . , DE LISCANO, T. R. and CARRERO GUERRA, J . E.: Protozoan Infections in Man (PIEKARSKI, G.)

8 7 SLEIGH, M . A. (ed.): Microbes in the Sea (PATTERSON, D . J.)

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A R T I C L E S

ENTZEROTH, R. and CHOBOTAR, B .

W I E M A N N , M . and GÖRTZ, H . - D .

OGDEN, C . G . and MEISTERFELD, R.

L O M , J., DYKOVÄ, L , KÖRTING, W . and KLINGER, H .

DOUGHTY, M . J .

HAUSER, M . , LINDENBLATT, J . and HÜLSMANN, Ν .

HELFTENBEIN, E. , CONZELMANN, Κ . K . , BECKER, K . F . and FRITZENSCHAF, Η .

BENITO, J . and FERNANDEZ, I .

LARSSON, J . I . R.

ISHIDA, M . , SHIGENAKA, Y. and TANEDA, K.

B O O K R E V I E W S

A N N O U N C E M E N T S

8 9 A Freeze-Fracture Study of the Host Cell-Parasite Interface during and after Invasion of Cultured Cells by Cystozoites of Sarcocystis muris

1 0 0 Release of the Endonucleobiotic Bacterium Holospora elegans from its Host Cell Paramecium caudatum

1 0 9 The Taxonomy and Systematics of Some Species of Cucurbitella, Difflugia and Netzelia (Protozoa: Rhizopoda); w i t h an Evaluation of Diagnostic Characters

1 2 9 Heterosporis schuberti n.sp., a New Microsporidian Parasite of Aquarium Fish

1 3 6 Differential Effects of Quin-2 and Quin 2 - A M on Ciliary Re­sponses of Paramecium caudatum at Moderate Calcium Levels

1 4 5 The Cytoskeleton of Reticulomyxa filosa Reticulopodia Contains Glu-Tubulin as a M a i n Component

1 5 8 Regulatory Structures of Gene Expression, DNA-Replication and DNA-Rearrangement in Macronuclear Genes of Stylonycbia lemnae, a Hypotrichous Ciliate

1 6 8 The Ultrastructural Identity of Protoentospora ptychoderae Sun, 1 9 1 0 (Apicomplexa, Coccidia), a Parasite of the Body Cavity of the Enteropneust Glossobalanus minutus (Kowalewsky, 1 8 6 6 ) : Growing Trophozoites

1 7 2 Light and Electron Microscope Studies on Jirovecia involuta sp. nov. (Microspora, Bacillidiidae), a New Microsporidian Parasite of Oligochaetes in Sweden

1 8 2 Studies on the Mechanism of Cell Elongation in Blepharisma japonicum I . A Physiological Mechanism H o w Light Stimulation Evokes Cell Elongation

1 8 7 DUBEY, J . P. and ΒΕΑΤΠΕ, C. P.: Toxoplasmosis of Animals and M a n (ENTZEROTH, R.)

1 8 7 PERSSON, G. and JANSSON, M . (eds.): Phosphorus in Freshwater Ecosystems (HILTON, J.)

1 8 7 SLEIGH, Μ . Α . : Protozoa and Other Protists (PATTERSON, D . J .)

1 8 8 BELL, G.: Sex and Death in Protozoa (HECKMANN, Κ.)

1 8 8 HIGGINS, R. P. and THIEL, Η. (eds.): Introduction to the Study of Meiofauna (PATTERSON, D. J.)

1 8 9 TAZAWA, M : Cell Dynamics, Supplementum 1 and 2 , Protoplasma (STOCKEM, W . )

1 9 0

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25/3 A R T I C L E S

BRUGEROLLE, G. and PATTERSON, D . J. 191

LUKES, J. and VÄVRA, J. 200

KERYER, G . and ADOUTTE, Α . , N G , S. F., 209 C O H E N , J., GARREAU DE LOUBRESSE, N . , ROSSIGNOL, M . , STELLY, N . and BEISSON, J.

RÖTTGER, R . , KRÜGER, R . and 226 DE RIJK, S.

HELLUNG-LARSEN, P., LEICK, V. , 229 TOMMERUP, N . and KRONBORG, D . SCHNEPF, E., WINTER, S., STORCK, I . 234 and QUADER, Η .

L O M , J. and DYKOVA, L 243

RADZIKOWSKI, S. and STEINBRÜCK, G.

M Ö H R , Μ . , RUTHMANN, Α . , EICHENLAUB-RITTER, U . , K Ü H N , S. and TRAUB, P.

KAMRA, K . and SAPRA, G. R .

O B I T U A R I E S

B O O K R E V I E W S

249

255

264

279

284

285

A N N O U N C E M E N T S

286

286

286

287

288

A Cytological Study of Aulacomonas submarina Skuja 1939, a Heterotrophic Flagellate w i t h a Novel Ultrastructural Identity Life Cycle of Amblyospora weisen n.sp.: (Microsporidia) in Aedes cantans (Diptera, Culicidae) Purification of the Surface Membrane-Cytoskeleton Complex (Cortex) of Paramecium and Identification of Several of its Protein Constituents

Trimorphism in Foraminifera (Protozoa)-Verification of an O l d Hypothesis Chemotaxis in Tetrahymena

A Complementary Experimental Study of Cell Division in the Dinoflagellate Prorocentrum micans Hrabyeia xerkophora n . gen. n . sp., a New Microsporidian w i t h Tailed Spores from the Oligochaete Nais christinae Kasparzak, 1973 Location of r D N A in the Heteromeric Macronucleus of Chilodo-nella steint Evidence for Intermediate-like Filaments in a Heterotrichous Ciliate

Partial Retention of Parental Ciliature During Morphogenesis of the Ciliate Coniculostomum monilata (Dragesco &c Njine, 1971) Njine, 1978 (Oxytrichidae, Hypotrichida) In Memoriam: Jean Cachon (1922-1989) (FEBVRE, J., FEBVRE-CHEVALIER, C. and GREUET, C.) In Memoriam: Wulf-Ulrich Berthold (1938-1989) (AMMERMANN, D.)

BOFFEY, S. A. and LLOYD, D. (eds.): Division and Segregation of Organelles GOEBEL, E. (ed.): Intracellular Bacteria MANTELL, S. H . , CAPMAN, G. P. and STREET, P. F. S. (eds.): The Chondriome Chloroplast and Mitochondrial Genomes SCANNERINI, S., SMITH, D. , BONFANTE-FASOLO, P. and G I A N I N A Z Z I -PEARSON, V. (eds.): Cell to Cell Signals in Plant, Animal and Microbial Symbiosis WICKNER, R. B., HINNEBUSCH, Α., LAMBOWITZ, A. M . , GUNSALUS, I . C. and HOLLANDER, A. (eds.): Extrachromosomal Elements in Lower Eukaryotes (PATTERSON, D. J.) BUETOW, D . E.: The Biology of Euglena. Volume I V : Subcellar Biochemistry and Molecular Biology (PATTERSON, D . J.) HALLEGRAEFF, G. M : Plankton: A Microscopic W o r l d (PATTERSON, D . J.)

LARSEN, J. and MOESTRUP, 0 . : Guide t i l Toksike og Potentielt Toksike Marine Alger; Guide to Toxic and Potentially Toxic Marine Algae (PATTERSON, D . J.) RONDANELLI, E. G. (ed.): Infectious Diseases Color Atlas Mono­graphs. 1 . Amphizoic Amoebae: Human Pathology (HÜLSMANN, Ν.)

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25/4 R E V I E W

HAACKE-BELL, Β . , HOHENBERGER-BREGGER, R . and PLATTNER, H .

A R T I C L E S

MILDER, R . , PLESSMANN CAMARGO, E . and FREYMULLER, E .

M I Y A K E , Α . , HARUMOTO, T . , SALVI, B . and RTVOLA, V .

TRIEMER, R . E . and O T T , D . W .

LARSSON, J . I . R .

Y A N O , J . and SUHAMA, M .

BENITO, J . and FERNANDEZ, I .

W I C K H A M , S. A . and L Y N N , D . H .

SERRANO, S., GUINEA, Α . , SOLA, A . and G I L , R .

COLE, Τ . Α . , FOK, Α . K . , U E N O , M . S and A L L E N , R . D .

BROERS, C . Α . M . , STUMM, C . K . , VOGELS, G . D . and BRUGEROLLE, G .

GROLI£RE, C . - A . , C H A K L I , R . , SPARAGANO, O . et PEPIN, D .

O B I T U A R Y

B O O K R E V I E W S

A C K N O W L E D G E M E N T S

A U T H O R I N D E X

S U B J E C T I N D E X

289 Trichocysts of Paramecium: Secretory Organelles in Search of their Function

306 Intracytoplasmic Flagellum in Trypanosomatids

310 Defensive Function of Pigment Granules in Blespharisma japoni-cum

316 Ultrastructure of Diplonema ambulator Larsen & Patterson (Euglenozoa) and its Relationship to Isonema

321 On the Cytology of Jirovecia caudata (Leger and Hesse, 1916) (Microspora, Bacillidiidae)

331 Nuclear Behavior in Triple-Conjugant Fusion Complexes of the Ciliate Stylonychia pustulata: Inhibition of Meiosis and Retention of the Macronucleus

338 Ultrastructural Observations on Merogony of Protoentospora ptychoderae Sun, 1910 (Apicomplexa, Coccidia) from the Entero-pneust Glossobalanus minutus (Kowalewsky, 1866)

345 Relations between Growth Rate, Cell Size, and D N A Content in Colpodean Ciliates (Ciliophora: Colpodea)

353 Morphology and Morphogenesis of Disematostoma colpidioides (Ciliophora, Frontoniidae): Its Systematic Implications

361 Use of Nile Red as a Rapid Measure of Lipid Content in Ciliates

369 Ρ salter iomonas lanterna gen. nov., sp. nov., a Free-living Amoe-boflagellate Isolated from Freshwater Anaerobic Sediments

381 Application de la Colonisation d'un Substrat Artificiel par les Cilies ä l'fitude de la Qualite des Eaux d'une Riviere

391 In Memoriam: Raymond Hovasse (1895-1989) ( D E PUYTORAC, P.)

396 AESCHT, E. and FOISSNER, W . : Catalogus Faunae Austriae. Teil la : Stamm: Rhizopoda (OGDEN, C. G.)

396 HEMLEBEN, C H . , SPINDLER, M . and ANDERSON, O. R.: Modern Planktonic Foraminifera (HOTTINGER, L.)

396 HUGHES, Μ. Ν and POOLE, R. K.: Metals and Microorganisms (NlLSSON, J . R.)

397 PIROZYNSKY, K. A. and HAWKSWORTH, D . L. (eds.): Coevolution of Fungi wi th Plants and Animals (HEATH, M . C.)

397 SANDGREN, C. J . (ed.): Growth and Reproductive Strategies of Freshwater Phytoplankton (HEANEY, S. I.)

398 SCHWEMMLER, W . : Symbiogenesis. A. Macromechanism of Evolu­tion (PATTERSON, D . J.)

399

400

401

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Europ.J.Protistol. 25, 158-167 (1989) October 27, 1989

European Journal of

P R O T I S T O L O G Y

Regulatory Structures of Gene Expression, DNA-Replication and DNA-Rearrangement in Macronuclear Genes of Stylonychia lemnae, a Hypotrichous Ciliate

Elke Helftenbein, Karl Klaus Conzelmann, Karl Friedrich Becker and Hariolf Fritzenschaf Institut für Biologie III, Abt. Zellbiologie, Universität Tübingen, Tübingen, FRG

SUMMARY

Seven transcriptionally active macronuclear D N A molecules of Stylonychia lemnae have been analyzed for potential regulatory sequences of gene expression, D N A replication and D N A rearrangement. Transcription initiation is mediated by a canonical T A T A - b o x or by T A T A -box-like sequences, and transcription efficiency may be regulated by gene-specific downstream promoter elements (DPE). Putative signals for R N A 3'-formation are represented by noncon-served palindromes located upstream of the mature R N A 3'-termini. Sequences indicating polyadenylation of the m R N A molecules are not detectable. A single palindrome of A- and T-residues is present within the 80 5'-terminal bases of each macronuclear D N A molecule and very likely functions as the replication origin. The 7 D N A molecules consist of several nonconserved inverted and direct repeats (IR, D R ) suggested to play a role in D N A rearrangement during macronuclear development.

Introduction

Recently several hundred eukaryotic genes transcribed by Pol I I have been cloned, sequenced and analyzed for consensus sequences involved in regulation of gene expres­sion [4, 5, 7, 29, 39]. Two types of nucleotide sequences involved in regulation of transcription initiation have been identified: promoters and enhancers [31]. Promoters are necessary for accurate and efficient initiation of transcrip­tion and are located within the 100 bp immediately upstream of the transcription start sites. A typical promot­er consists of several sequence elements. One of these represents the well-conserved TATA-box flanked by GC-stretches that ensures the accurate initiation. It is centred about 30 bases upstream of the transcription start site. The elements located further upstream, designated upstream promoter elements (UPEs), increase the rate of initiation [33]. UPEs differ gene- and tissue-specifically and act regardless of their orientation with respect to the TATA-box. Their regulating function is mediated by binding of

0932-4739/89/0025-0158$3.50/0

nuclear proteins, referred to as transcription factors. Enhancers, the second type of sequence elements regulat­ing gene expression, increase the rate of transcription of promoters and act in an orientation independent manner at great distances upstream and downstream of the transcription unit [46]. They also contain discrete D N A sequence elements specifically interacting with proteins. Transcription is typically initiated between 20 and 80 nucleotides upstream of the coding region preferentially at the motif CA (cap-site) followed by pyrimidines [7].

Formation of precise RNA 3'-termini generally occurs by two different mechanisms signalled by distinct D N A sequences. Accurate termination of transcription is one of these processes and seems to be signalled by T-stretches or AT-rich stem loops located downstream of the transcrip­tion termination site [14]. The second mechanism, referred to as RNA 3'-processing, includes 3'-cleavage of pre-mRNA molecules mainly followed by polyadenylation of the resulting mRNA molecules. These processes are pref­erentially signalled by a cluster of sequences beginning

© 1989 by Gustav Fischer Verlag, Stuttgart

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Regulatory Structures in Stylonychia lemnae · 159

with the highly conserved polyadenylation signal A A T A A A , located 10 to 20 bases upstream of the cleavage site CA (functioning as the polyadenylation site) and ending with G/T-clusters within 30 nucleotides down­stream of the cleavage site [5, 6,14, 27, 43]. In contrast to this set of sequences a different, well conserved sequence combination of a palindrome located immediately upstream of the cleavage site and of a downstream positioned nonanucleotide signals RNA 3'-cleavage with­out polyadenylation in the histone genes of higher eukar-yotes [45]. The same mechanism is also found in plastids of higher plants and is regulated by nonconserved inverted sequences located immediately upstream of the mature RNA 3'-termini [49].

The nucleotides preceding the coding regions [CCAC-CATG] seem to be involved in the initiation of translation invertebrates [28,29]. The A-residue atpos.-3 is strongly conserved in all organisms including plants and lower eukaryotes, whereas the Cs may be replaced by As, as it is found in Drosophila and yeast [9, 17].

A n interesting question is, whether these general eukar-yotic signals of gene expression are also existent and functioning in ciliates, a group of lower unicellular eukaryotes, showing very special features of genome organisation. Ciliates contain two morphologically and functionally different nuclei: the generative micronuclei playing a major role in sexual reproduction (conjugation) and showing no or little transcriptional activity in the vegetative cell cycle, and the somatic macronuclei provid­ing all RNA for vegetative cell growth [2, 3]. Macronuclei develop from micronuclei by complex species specific processes, referred to as macronuclear development. In the hypotrichous ciliate Stylonychia lemnae this macronuclear development includes chromosome polytenization, de­gradation of the giant chromosomes, selective DNA elimination, D N A rearrangement and DNA amplification [41]. The resulting transcriptionally active macronuclear genome is organized in about 15 000 different DNA molecules of 0.4 to 20 kbp in length, of which each is present in a distinct copy number of a few to more than 100 000 [3, 10, 18, 19, 30, 48]. Each DNA molecule is thought to represent a transcription and replication unit and consists of a single intronless coding region flanked by short (74 bp to 1000 bp) AT-rich (75%-80%) noncoding sequences terminating in telomeric inverted repeats of C4A4. The coding regions show, like those of some other ciliates, an altered genetic code using the "universal" stop codons TAA and TAG to specify glutamine [8,10,18,19, 22, 25, 36, 40].

We examined the noncoding sequences of 7 expressed macronuclear genes from Stylonychia lemnae for potential regulatory elements of gene expression, DNA replication and D N A rearrangement. Four of the investigated Stylo­nychia genes represent the two a- and the two ß-tubulin genes, each of them present in a high ( a r and βι 150 000) and in a low copy number (a 2- and ß 2 30 000) [10,18,19]. With respect to the biological function of the a- and ß-tubulins the coordinated expression of their genes is expected.

The other 3 cloned macronuclear DNA molecules represent transcriptionally active genes in all tested Stylo­

nychia strains, but the functions of their encoded proteins are unknown. The genes p4A3 and pob were chosen with respect to their special features in single Stylonychia strains. Parts of the coding region of p4A3 are repeated in several differently sized macronuclear DNA molecules of strain DO (Fritzenschaf and Helftenbein, in prep.) and the DNA molecule pob becomes overamplified during vegeta­tive growth in strain SP (Wegner et al., in prep.). The gene pmal was investigated as a representative of an extremely short macronuclear DNA molecule (unpublished). A com­parison of the detected presumptive regulatory sequences in these 7 macronuclear genes of Stylonychia and in the previously reported genes from other ciliates is dis­cussed.

Material and Methods

Cell cultivation and isolation of macronuclear DNA and RNA Cells of S. lemnae strain D O (collected in North Germany; origin of the cloned α 2 - , ß r , ß2-tubulin genes), strain SP (South Germany; origin of the α 1-tubulin gene, pob and p4A3) and 6-Schi (South Germany; origin of p m a l ) were cultivated in Pringsheim solution as described by Ammermann [1] and D N A was purified from isolated macronuclei [2] as previously de­scribed [48]. Total R N A was isolated by the procedure of Glisin [12] and contaminating D N A was eliminated by DNAse I digestion with 3000 units/ml at 3 7 ° C for 40 min.

Cloning and screening of macronuclear DNA molecules Macronuclear D N A was size-fractionated by preparative aga­

rose gel electrophoresis and cloned by G-C-tailing (a 1-tubulin gene) [18] or after Bal 31 digestion of the protruding 3'-ends as previously described [10]. Screening of recombinant plasmids was done by colony hybridization [16] with radioactive labelled c D N A probes of the a- and ß-tubulin genes [10, 18, 19] and of enriched radioactive labelled macronuclear D N A molecules con­taining the pob and p 4 A 3 genes.

DNA sequencing and Sj-mapping D N A sequencing was performed by the chemical method of

Maxam and Gilbert [32] or by the enzymatic procedure of Sanger et al. [44]. Sj-mapping experiments were carried out as described by Helftenbein [19].

Denaturing agarose gel electrophoresis and hybridization reactions For denaturing gels R N A and D N A was glyoxylated [34] and

gel electrophoresis and hybridization was done as previously described [10].

Results

Transcription initiation sites of the a- and ̂ -tubulin genes

Identification of the transcription start sites located about 40 bases upstream of the coding regions of the α 2-, ß r and ß2-tubulin genes (Fig. 1) have recently been published [10, 19]. The transcription initiation site of the

Page 8: European Journal of PROTISTOLOGY · (Ciliophora, Frontoniidae): Its Systematic Implications 361 Use of Nile Red as a Rapid Measure of Lipid Content in Ciliates 369 Ρ salter iomonas

Ρ * A 3

pob

C C C C A A A Ä C C C C A A A A C C C C

C C C C A A A A C C C C

C C C C A A A A C C C C A A A A C C C C A A A A C C C C

C C C C A A A Ä C C C C A A A A C C C C

A A A A C C C C

- A G A A C A G T G G G A T T C G G A G G G A A A T C A G T T A T A A T A A T A G T T C A A A C G A J T A A A T T A T A A A " Ä } T T T G A A T T A G T T A

A T A G C A

• A T G f T G A C T C A G | A T T G T A A A A T A T T T C A A G T A A A T A A G T A A . T A T A T A T C T A

G A A G G C T A A G A T A T A T A T T T A A A T A T A

C C C C A A A Ä C C C C A A A A C C C C ζ ^ C C T G T C G G A T T T C C T A G G G A T T T T G A T T A A G A A T T T A A A T T T A A A T T A A T A T G A A T T T A A T T G A A T T T A A C T G T C A G C T T

C C C C A A A A C C C C A A A A C C C C A A A A C C C C A A A A C C C C A A A A C C C C A A A A C C C C

O N Ο

m

X

Ts 7t η ο

β*

| p ° b

Jpmal

A G A T T C C C C T A T T C A A A A A T T A A G A G G G T C C T T C A T A T C C A T T C A A A T A T A T C T T A A A T A C T T A A A C T T A A C T A G T A A T T A T A A T T A T T T C A T T T T T T C A A A C A A A A T C A

T T G C C C } T A T A A A T | A G C G A A A T A A A G T G G G A A A T T A T A A C T T T A T T T T A T A T T A A

] C T A C T A A A T A G G T T T T A A C T C A A A T A A A A T C A A G G A C A A C J T A A A T T T T A A A T T J C A G A G C C T G A A T A T A A G G A T A C C A A T T

]

A A C A A A A T C A

T T A A G A A G T A T T A A T T A T T C T T A A T T T T C A

A C T C T T C A T C

A A T C T T C A T C

A A G T G A T C A G T A A A G T A A A A T T C A A T C C T G T C C A T C C G T T T G A A T T T A A T T G T J T A T A A A T l A T p C G C C C T C T C T A A C T A A T

P * A 3 ; J A G A T A T A T A A A T C A A G A A T A T C A T C A A T A A T A T T T T A A T T T A G G A T A T A T T A A C T T C T T C A G C C C | r A T A A ATJAAGCCAAT

T C T T A T T A A A T A T G T T G G T T A T A A A C T C A A A A A A C T T C T T T G A G T C T G G T G T G A A T C A G [ r A T A T A T T T

- 1 0 0

G G C T T G T T G f T T A T T A A A A A T T J G T T T T G A A T T G A A A T A G T T G T

- 9 0 - 8 0 - 7 0 - 6 0 - 5 0

A T A T A A T T C A A A T A A T T T T C

T A A C T T A A T T C A A T T A A T A A

G G T T A T T T T A A T A A T T T A T A

C A T T G T T A A A T T T T T A T A T T

A A A A C A A A A C

T C T A A A A A C A

A A T A T A A T T A

A A A T A A T T A G

2 0

A A A A C A A A C C

T C A A G T C A C A

T A T C A T A A A A

T A A C T A A T A T

G T T A A T A A A G

- 1 0

A T G

A T G

A T G

A T G

A T G

A T G

A T G

pob ^ A T A C C A T G T A T C C T T A G G A G T G G G A T T C A G G A G C T G A A A A A A T T T A A T A A A A T T T G A A T T A A A T C C A G C C A A A A T T T A A A T T T T A T G A A T T A A A T T G C C T A G G A A A A T T T T T A T C C A C A G G G C C

- 3 7 0 - 3 4 0

A A T T C G G T C C A A A T C C C G T T G G A C C A C C G G A A T T G G A T G G G T A T G C G C G C A C G C C C G T C A A T A T C G A T A C C G T A G A T T C C G A A T C T G G G C T C A G A C C C T A A C C A C C A G C A A T G G T G C A A A A G T T G A A A A C ^

- 2 7 0 - 2 6 0 - 2 5 0 - 2 4 0 " 2 3 0 - 2 2 0 - 2 1 0 - 3 3 0 - 2 8 0

Fig. 1. Nucleotide sequence of the 5'-noncoding regions. All sequences are aligned to their translation start sites ( A T G ) . Gs and Cs are marked by dots, TATA-boxes [31] and TATA-box- l ike sequences are boxed. Boxes with double lines show eukaryotic recognition sites for transcription factors [33]. Identical sequences are overlined and homologous sequences, representing presumptive gene-specific D P E s , are underlined by ^mm : transcription start sites; <·.·-·,--.ι: presumptive start sites of transcription; —><—: palindromes representing the replication origins; < > : sequence shown in the lower pare of the figure.

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^ 1 5 α Be 5

—I Η Η -FV SBgl Ε ß g l

Ö l

Sac W - >ac Bglll H i n d

f

Β 5 -

DNA fragment coding sequence protected fragment

α ι 2 9 0 bp 68 b 169 b

m RNA

- 1 0 1 b sets

f t * *

3 '

3 6 7 bp 81 b 1 0 3 b + 22 b

Fig 2 Si-mapping of the a r t u b u l i n gene. The reactions were carried out as described in Materials and Methods. (A) Restriction map of the α,-tubuhn gene and D N A fragments of the cloned molecule used in the Srprotection experiments. Open bars represent the coding region; thin lines indicate noncoding sequences and filled boxes represent the terminal repeats. Sa: S a d ; K : K p n ; Be: Bell ; S: Sali ; P: PstI; Pv: Pvu; Bgl: Bgll; E : Eco R I : Bg: Bglll. (B) summarized data of the S r protection experiments, b: bases; bp: base pairs. (C) Autoradiographs of the 5 ' and 3' protected D N A fragments separated in denaturing Polyacrylamid gels, which are calibrated by using radioactive products of a sequencing reaction as molecular size markers [19].

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162 · Ε. Helftenbein, Κ. Κ. Conzelmann, Κ. F. Becker and Η. Fritzenschaf

αι-tubulin gene was determined by Si-mapping experi­ments as shown in Fig. 2. The single protected DNA fragment of 169 bases demonstrates that transcription is precisely initiated, like in the other tubulin genes, but in a larger distance from the coding region at pos. - 101 (Fig. 1). Thus the length of the four mRNA leaders are in the range of those known from other eukaryotes [7]. However, the first two transcribed nucleotides (cap site) of all tubulin genes are not conserved (ο^: TT; αχ: ΤΑ; βι: CC; ß 2 : AT) and neither of them represents the eukaryotic cap site motif CA [7].

Common potential promoters of the 7 macronuclear genes

In the (X2- and ß2-tubulin genes and in the gene p4A3 a canonical TATA-box flanked by short GC-rich stretches is present 60 to 70 bases upstream of the coding regions (Fig. 1). The αϊ- and ßj-tubulin genes and the genes pob and pmal coding for still unidentified proteins contain in corresponding distances short AT-stretches similar to a TATA-box (TATA-box-like sequences), which are also flanked by GC-rich regions (Fig. 1). These two types of TATA-boxes are located in a classical distance of 25 to 30 bases upstream of the transcription start sites of the 4 tubulin genes. The GC-rich flanks represent regions with the highest GC-contents of the entire 5'-noncoding regions from all 7 genes, as shown for the tubulin genes in Fig. 3.

n t - 2 0 0 - 1 5 0 - 1 0 0 - 5 0 ATG Fig. 3. Base composition of the 5'-noncoding regions in the tubulin genes. The relative GC-contents are plotted at each base position and the base compositions were determined every base in a 5 bp range, using Pustell Sequence Analysis Programs, Interna­tional Biotechnologies, Inc. Bars represent TATA-boxes in the ctr and ß 2-tubulin genes and T A T A - b o x like sequences in the a\- and β ι-tubulin genes; arrows indicate the transcription start sites; nt: nucleotides.

Upstream of the two types of TATA-boxes neither the sequences of the UPEs found in higher eukaryotes nor conserved sequences functioning as potential ciliate spe­cific UPEs could be detected. One exception is given by the ß2-tubulin gene, which shows the recognition site of the Spl transcription factor in an unusually large distance from the TATA-box at pos. - 169 (Fig. 1).

Almost identical T- and Α-stretches interrupted by single C-residues are present in the transcribed regions of the tubulin genes, located very close to the translational start sites (12, 12, 2, 11 bp upstream of ATG in a 1 ? ct2, βι, ß2, respectively) (Fig. 1). In corresponding regions of the other 3 macronuclear genes similar but not identical sequences of more alternating As and Ts without single Cs are present. Neither these sequences nor those of the tubulin genes do resemble known eukaryotic signals involved in transcription or translation processes.

Putative regulatory sequences for initiation of translation

The pentanucleotide upstream of the ATG in the α-tubulin genes resembles the CCACC (ATG) of verte­brates [28, 29], whereas a transition to the pentanucleo-tides of Drosophila and yeast, prefering As [9, 17], is visible within the ß-tubulin genes. Pentanucleotides con­sisting almost completely of As are found in the other 3 macronuclear genes. The assumed indispensable A-residue in pos. - 3 is present in all genes except for the gene pob, where it is replaced by a T-residue.

Potential signals for the formation of mRNA 3'-termini

The recently reported transcribed parts of the 3'-noncoding regions from the 012-, ß r and ß2-tubulin genes [10, 19] are shown in Fig. 4. The identification procedure of the a]-tubulin mRNA trailer by Sj-mapping experiments is explained in Fig. 2, showing a single precisely defined 3'-terminus located 25 bases down­stream of the coding region (pos. TGA + 25 in Fig. 4). Such single well defined RNA 3'-termini were also found in the ß-tubulin genes ( ß i : T G A + 73; ß 2 : T G A + 57). However, two RNA 3'-termini located at slightly sepa­rated positions TGA + 48 and TGA + 44 were found in the (^-tubulin gene. Thus the tubulin gene trailers are unusually short in comparison to those of other euka­ryotes, which show lengths of several hundred to more than 1000 nucleotides [5].

The last two transcribed bases of all the tubulin genes are given by the dinucleotides TA or CA representing the eukaryotic polyadenylation site. However, the polyadeny­lation signal A ATA A A generally centred 15 bases upstream of the polyadenylation site is not present in either of the transcribed regions of the tubulin genes. Surprisingly such a sequence was detected in the c^-tubulin gene in the nontranscribed region starting at pos. TGA + 68 and in that of the ß rtubulin gene at pos. TGA + 175, +271 and + 282. Shortened versions of the eukaryotic GT-clusters normally located 15 bases downstream of the RNA 3' cleavage site are also present in the tubulin genes but in a

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J pob :J GEB

T G A

T G A

T G A

T G A

T G A

T G A

T G A

C C A T A C A T T C

G C A T A C A T A A

G A C A T T A G C T

T C T G C T T G A T

A G T A A G C A A C

I G G C C G A T T G A

G C A C C A C C A C

• 1 0

G C C C A C A C A G G C C T A A A C C A

G C C C T A A A A ' C C T A A C I A T A

G A A C A G C A C A C A G C A A C A A C

T T T T T A C A A A A T A A A G I T A

T T A T C T C T C A G G A A T G T A A A

C C A T A G G T C A A T T C C A A C T A

A C A C A G C T C T T A A G G T T C C A

4 2 0 + 3 0

A A C T A G A A C C

G G A G G C A C C G

A A A C C A T A T G

A A C G C T A A G T

A A A C C A T A A A

C T T T A T A C A A

T A A A A C T A C A

T A A C T T A T T A

C A T A T T C C A G

A A A T C T A T G T

A T T A T A T C T A

C A A T A T A A T A

T A T A T A T C C A

A T T A A C C T G C

A T T T A G T A T T

ACAAGCCTAT TTAAAAGCCT ATTAGAATAG

CAGTACCTGT TGCTTATCAT TTTATCTCCT

AACTAATAAT gTTTTAAATT CATGTATAGG

AAATGTTATT TGTCTTTTAA_ TTTCGACAAA

ΑAGGAGOAAG CCTACCTTGA GACAATGCAA

ATTAATAATC GATTCTGAAT CCT

• 1 2 0 + 1 3 0 + 1 4 0

1ST | P * * 3 : |

1 ^ 1

A A T T G T C T T T T A A T A T T T T T T G A G T C C A T G A T A T T T T T T T C C T C T G A A A T C G G A T T T C A T G G A T T T T C T C A A T T C C C C A T T A T T T A A T C G A T T C T A T C T C A A A —

T C T A T A C T T A T T C C Ä T T A T C C T T A T A T A A T C T A T A A C C C C A T A A G T T T T A G A A C A A C A T T T T C C C C T T T T T T T C A A A T A T A T T T C C T G G G T G C C A A C A T T T T C G T G G A T T C T T A A T T C A A A T C T T T

C A G A T A T A A A G A A T T G A A A A T G G C A G G A A A G A T C J A A T A A A J C T A G T T T A A A G C A A G A G A C T T T G C T T T C A C Τ A A A G A T A T T G T G A T A T G T G T C G A C A T T A T C A A G A A G A A T A T C C A A T A A C T T T A A T A A G A | A A T A A A | A A T C

A T C T G C G T A T A A G T T T C T A T C T C T C C A T C A A T T A T A T C C T A A T T C A G C C T A T A T T T C T A T A T C T T A G C A A G T T C T C A T C G A T A T A T A G T A T T T T A A T T C O C T T T C A C A C T Τ Τ Α Τ Τ Α Α Α Α Τ C A T G A T T T T C A T C T T T T A T C

A A A A T C T A T G G A A C C A A G G T C C A A G A A C C C A A G G T T G A A C A A G C T G A T C C A T T A G C T G A T G C T G A G A G A G A A T T G A A G C A A G C T G C T T A T C T T C A A G C T C T T T A A A A C A T C C A C G G A C A T T A C A A T G T C T A A T C T C C A A A

[P4A3J [ Pob

p - 1 :

AJfcATAAAJAAT TGATAAATAT AGATTTATTT AGAAAATTTA GATGAAAAAT CTAATTCATT

G G G G T T T T G G G G

G G G G T T T T G G G G

G G G G T T T T G G G G

G G G G T T T T G G G G

C G T T C A G A A A G G T A A C C C A C T T T C T G A T T T A G A G A G A G A A A T G A A A C A A G C A A C T T A C C T C G G A G G T A T ( j T A G A A C A T C T A C G G T C A T T A C G C C A A T Q T C A A O C T C O T C A ^ T C T A A A C C A A A G C C C A G C A ζ ) G G G G T T T T G G G G

G G G G T T T T G G G G

G G G G T T T T G G G G

Fig. 4. Nucleotide sequences of the 3'-noncoding regions. Al l sequences are aligned to their coding regions ( T G A ) ; sequences identical to the eukaryotic polyadenylation signal [5] are boxed and sequences similar to the GT-clusters present in the sequence set mediating R N A 3 '-processing in eukaryotes [5] are marked by " w . 4 M B : represent identified R N A 3'-termini and < S I 3 shows putative R N A 3'-termini; the suggested gene-specific signal T A A A C is underlined. The R N A 3 '-terminal inverted repeats are indicated by arrows. < > : additional nucleotide sequence, which is not shown and which does not contain potential regulatory signals.

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164 · Ε. Helftenbein, Κ. Κ. Conzelmann, Κ. F. Becker and Η. Fritzenschaf

larger distance of about 60 bases around pos. TGA + 120 (Fig. 4).

The mRNA 3 '-termini of the 4 tubulin genes are located within an identical sequence TAAAC and each of the 4 mRNA trailers contains a palindrome of a nonconserved sequence very close to its 3'-end (Fig. 4). Such a combina­tion of the sequence TAAAC and an immediately upstream located nonconserved palindrome is also present in the gene p4A3 between pos. T G A + 330 and TGA + 400 (Fig. 4), where the mRNA 3' terminus has been localized by preliminary experiments (data not shown). The other two cloned genes, pob and p m a l , do not contain the motif TAAAC but also show nonconserved palindromic sequences in their 3'-noncoding regions. The positions of the palindromes (pob: TGA + 6 to T G A + 22; p m a l : TGA + 69 to TGA + 115) (Fig. 4) are in good agreement with the mRNA lengths identified in Northern blots and preliminary S ι-mapping experiments (data not shown).

Potential hairpin structures or larger T-stretches down­stream of the RNA 3'-end could not be detected in any of the tubulin genes.

Inverted and direct repeats within the 7 macronuclear genes

Each of the 7 cloned genes contains an inverted repeat consisting only of A- and T-residues within the first 80 nucleotides of their 5'-noncoding regions (Fig. 1). Such potential hairpin structures are unique in each DNA molecule, except for the gene pob, which contains two additional inverted repeats of A- and T-residues in the 5'-noncoding region, beginning at pos. - 383 and - 169 (Fig. 1). These AT-structures show high homology to autonomously replicating sequences identified in mouse rDNA (50).

Short inverted repeats (IR) of 5 to 12 bases are localized at both ends of each DNA molecule and with different frequency within each molecule (Fig. 5). The lengths of the

100bp

4

q > ^

^ . i

+~± i

4P ^

*1 CC 2

P i

P 2

p4A3 pob pmal

Fig. 5. Schematic representation of inverted (IR) and direct repeats (DR) within the 7 macronuclear D N A molecules. Open bars represent coding regions and thin lines show noncoding sequences. IRs are shown by: and D R s by: D . Sequences of IRs or D R s are not identical and each comprises 5 to 12 nucleotides. represents sequences, which are not flanked by IRs or DRs .

4 H=

L

nucleotide stretches inside each IR varies extremely from about 20 bases in the 3 '-terminal IR of gene p4 A3 to about 580 bases in the 5'-terminal IR of the same gene. Adjacent IRs are located side by side or separated by 1 to 3 bases, or by direct repeats (DR) (Fig. 5). These short IRs and DRs might play a role in DNA rearrangement during macro­nuclear development, since such short nonconserved sequences are found to surround the several short parts of the macronuclear DNA molecules within the highmolecu-lar weight DNA of the micronucleus (Helftenbein and Richter, in prep).

Discussion

The detailed sequence analyses of the noncoding regions of 7 macronuclear genes from Stylonychia identified common features, some of which with similarities to known eukaryotic regulatory signals for gene expression, DNA replication and DNA rearrangement. Out of the conserved sequence set of typical eukaryotic promoters consisting of UPEs and a TATA-box flanked by GC-rich stretches [31] only the TATA-box element is present in 3 of the 7 genes. The other 4 genes contain at corresponding positions TATA-box-like sequences with GC-flanks on either side. Thus these sequences may also signal, like TATA-boxes in higher eukaryotes, accurate initiation of transcription in Stylonychia as found in the a r and β ι-tubulin genes (Fig. 1). The lacking of eukaryotic UPEs as well as the missing of conserved Stylonychia specific sequences suggest that the efficiency of transcription initiation may be regulated in Stylonychia differently compared with other eukaryotes.

Three different mechanisms of gene specific regulation of the transcription initiation in Stylonychia are conceiv­able. The first one might be represented by the sequence of the TATA-box itself, leading to a high transcription initiation rate of genes containing a canonical TATA-box, whereas the genes with only a TATA-box-like sequence may be transcribed with a lower efficiency. Such a mechanism could be realized in the a- and ß-tubulin genes, of which the highly amplified otp and β ι-tubulin genes (150000 copies) contain only TATA-box-like sequences and the less abundant a 2- and ß2-tubulin genes show canonical TATA-box sequences. Quantitative measure­ments of the transcription products of the 4 tubulin genes support this hypothesis (unpublished). A second regula­tion mechanism may be enabled by the conserved A- and T-rich sequences found immediately upstream of the coding regions within the mRNA leaders (Fig. 1). These sequences, which contain identical nucleotide stretches in the tubulin genes and slightly different nucleotides in the other 3 genes, could function as downstream promoter elements (DPEs) comparable to the UPEs found in higher eukaryotes [31]. Thus the assumed regulation mechanism should be mediated by the binding of these sequences to specific transcription factors. The third regulation mechanism of the transcription initiation efficiency is represented by the specific amplification of the genes during the macronuclear development occuring in Stylo-

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Regulatory Structures in Stylonychia lemnae · 165

nychia [10, 18, 19, 36, 48] and presumably in all hypotrichous ciliates.

The general eukaryotic sequence sets regulating tran­scription termination or RNA 3'-processing followed by polyadenylation are almost completely absent in the macronuclear genes of Stylonychia. We therefore assume that the formation of the distinct single RNA 3' termini of the tubulin genes is mediated by other motifs than the known ones. The detected 3'-terminal nonconserved pal­indromic sequences and the downstream adjacent con­served pentanucleotide TAAAC of the mRNA molecules of the tubulin and the p4A3 genes seem to represent such Stylonychia specific signals (Fig. 4). The existence of only palindromes upstream of the roughly determined RNA 3 '-ends of the genes pob and pma 1 led us to assume that in Stylonychia a potential hairpin structure of a noncon­served sequence in combination with the dinucleotides CA or TA located at the RNA 3 '-termini is generally sufficient for RNA 3'-formation. Similar signals were found for RNA 3 '-processing without polyadenylation in plastids of higher plants [49] and in histone genes of higher eukar­yotes [45]. Thus the investigated RNA molecules of Stylonychia may also lack polyadenylated regions.

However, the motif TAAAC present only in some genes (tubulin and p4A3) could represent an additional sequence necessary for accurate RNA 3'-processing as found in the tubulin genes, or it might act as a signal for a Stylonychia specific RNA 3' modification. Whether this assumed modification is generally different from the eukaryotic polyadenylation process remains to be investi­gated. Evidences for such a hypothesis are the poor enrich­ment of tubulin mRNA molecules by oligo dT-cellulose chromatography of total RNA (unpublished) and the identification of unusually short A stretches, which are only attached to about 0.2% of all RNA molecules (unpublished).

The existence of the species specific poorly conserved pentanucleotides preceding the coding regions [28, 29] in the 7 investigated genes, suggests that translation initiation is similarly regulated in Stylonychia and in higher eukar­yotes.

The discussed results lead to the conclusion that the 7 investigated macronuclear genes might have contained earlier in ciliate evolution the general eukaryotic promoter sequences, of which they maintained only the TÄTA-box or a TATA-box-like sequence inevitable for accurate transcription initiation. However, all eukaryotic upstream promoter elements acting as modulators of the transcrip­tion initiation efficiency were lost. The efficiency might instead be regulated by ciliate specific mechanisms, namely by the specific amplification of genes during macronuclear development and/or by gene specific DPEs, which might be created as a result of the very short noncoding regions (e.g., a 2 : 74 bp) (Fig. 1) of the macronuclear DNA molecules. From the sequence set acting in eukaryotic RNA 3'-formation only a few remnants could be detected in the 7 macronuclear genes. Such sequences are the polyadeny­lation site-motif CA and TA, conserved at all 7 mRNA 3'-termini, the polyadenylation signal AATAAA, present only in the α 2- and ß-tubulin genes but at strange distances

from the mRNA 3'-termini, and the shortened GT-clusters found only in some genes but also at unusual positions (Fig. 4). Whether CA/ΤΑ and AATAAA represent indeed significant signals within the very AT-rich (75%-80%) noncoding regions of the macronuclear DNA molecules is questionable. Thus it could not be decided, whether the ciliates possessed in the past the eukaryotic signals for RNA 3'-processing and polyadenylation, or whether the ancestors of ciliates had already evolved the assumed present mechanism of RNA 3'-formation without poly­adenylation like that in the eukaryotic histone genes [45] or plastids of plants [49].

These general assumptions of gene expression regula­tion in Stylonychia are supported by sequence analysis of other recently investigated ciliate genes. Although tran­scription is initiated in the actin gene of the hypotrichous ciliate Oxytricha nova divergently from Stylonychia at 3 distinct sites, two of the transcribed RNA molecules show leaders comparable in length to those found in Stylonychia and the gene contains also a TATA-box-like sequence with GC-rich flanks at a corresponding distance from the coding region [15]. In all of the other so far reported genes of Oxytricha without identified transcription start sites, such a TATA-box-like sequence arrangement is present between 70 and 90 nucleotides upstream of the coding regions [20, 24, 26]. Even the published sequences for histone and actin genes of Tetrahymena, a holotrichous ciliate, do share these characteristics [11, 21, 23, 37], whereas in the surface antigen genes of Paramecium, another holotrichous ciliate, the accurate initiation of transcription at a single site seems to be signalled differ­ently [13, 35]. In accordance with the 7 investigated genes of Stylonychia none of these other ciliate genes contains the eukaryotic sequence set involved in RNA 3'-formation followed by polyadenylation. Although the RNA trailers of the Tetrahymena histone genes are approximately 4 times longer than the trailers of genes from Stylonychia and Paramecium, in all of these genes only one single RNA 3'-terminus could be detected, preceded also by a short nonconserved IR, except for the histone Η41 gene of Tetrahymena [23]. However, the motif TAAAC found at the RNA 3'-termini in some genes of Stylonychia is not present in either of the few other investigated ciliate mRNA molecules. Thus TAAAC seems to be a potential gene- or species-specific signal.

The single AT-rich IR detected within the last 80 5'-terminal bases of each macronuclear DNA molecule represents very likely the origin of D N A replication, which is expected from electron microscopical investigations close to one end of each DNA molecule. This assumption was confirmed by testing the 3 reiterated potential hairpin structures of the gene pob (Fig. 1) for autonomous repli­cation in a heterologous mouse L-cell system (Helftenbein et al., submitted). These experiments demonstrate that each of the 3 palindromes initiates DNA replication and indicate that the exceptional reiteration of such structures in the gene pob mediates the observed overamplification of this macronuclear gene during vegetative cell-growth (Helftenbein et al., submitted). In all reported macronu­clear DNA molecules of the closely related ciliates Stylo-

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166 · Ε. Helftenbein, Κ. Κ. Conzelmann, Κ. F. Becker and Η. Fritzenschaf

nychia pustulata [38] and Oxytricha fallax and O. nova [15, 20, 24, 26] such a unique AT-rich IR is detected at corresponding positions in the 5'- or 3'-noncoding regions, presumably also functioning as the replication origin.

The short nonconserved IR sequences localized at the 5 '-and 3'-termini of each investigated macronuclear D N A molecule from Stylonychia, as well as the adjacent irregu­larly distributed ones (Fig. 5) may represent signals com­parable to the IRs found at the termini of transposable elements in higher eukaryotes [47]. This assumption is strongly supported by the finding of such structures in micronuclear DNA, where they mark the macronuclear D N A sequences (Helftenbein and Richter, in prep.). This result and the identified short macronuclear stretches within the micronuclear DNA are in strong contrast to the results reported for Oxytricha, showing macronuclear sequences of larger extents in the micronuclear genome. Instead of terminal IRs of macronuclear D N A stretches, short DRs have been found at one end of the maintained macronuclear sequence and at one end of the eliminated internal sequence in Oxytricha [26,42]. Thus in Oxytricha no signals of DNA rearrangement were detectable in macronuclear DNA molecules.

Acknowledgements

We would like to thank Prof. W. Seyffert for his generous help with computer analyses and Prof. D . Ammermann and Dr. Hel­mut Schmidt for their critical comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft.

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Key words: Hypotrichous ciliates - Macronuclear genes - Gene expression - DNA-replication -DNA-rearrangement

Elke Helftenbein, Institut für medizinische Virologie und Infektionsepidemiologie, Hölderlinplatz 10, D-7000 Stuttgart 1, F R G