The genes encoding pheromones 1 and 2 of Euplotes octocarinatus

Europ.]. Protisto!' 32, Supp!. I: 158-164 (1996) October 31, 1996

European Journal of

PROTISTOLOGY

The Genes Encoding Pheromones 1 and 2 of Euplotes octocarinatus Wolfgang Teckentrup, Jost Puppe, J. Christoph Weiligmann, Helmut J. Schmidt, and Klaus Heckmann Institut fOr Allgemeine Zoologie und Genetik, Universitiit MOnster, MOnster/Westf., Germany

SUMMARY The genes encoding pheromones 1 and 2 of Euplotes octocarinatus are located on 1761 bp and 1763 bp long macronuclear DNA molecules. Both molecules as well as the cDNAs corresponding to the genes located on these molecules were cloned and sequenced. The genes have the same general structure. In both cases, the sequences of the secreted pheromones are preceded by leader sequences which contain typical ER-signal sequences. Each of the two genes contains three introns, a long one interrupting the leader sequence and two short ones located in the sequence coding for the secreted pheromone. Both genes contain three in-frame TGA stop-codons, presumably encoding cysteines for these pheromones. Very similar or identical non-coding sequences suggest regulatory functions, and putative regulatory consensus sequences are present in these regions. The data of pheromones 1 and 2, deduced from their genes, complete previous studies on pheromones 3 and 4 of E. octocarinatus, and allow, by comparison, several general conclusions about the entire pheromone system of this species. Most importantly, all four pheromone genes have a highly similar gene structure with in-frame TGA codons, astonishingly similar or identical non-coding regions (including introns) but rather poor similarities between the secreted pheromone proteins.

Introduction Cells of the ciliated protozoon Euplotes octocarinatus communicate by water soluble signal substances before they enter conjugation [8, 9]. These pheromones or gamones have been isolated and were identified as polypeptides [26, 27]. They are secreted by mature cells and induce competent cells of other mating types to prepare for conjugation [13]. Induced cells change their surface properties [20], unite pairwise, undergo meiosis, and then cross-fertilize each other [14]. Afterwards the coconjugants separate and the exconjugants reorganize and give rise to new lines of asexually reproducing cells that may conjugate again once they have become sexually mature and happen to meet cells of complementary mating types. In contrast to E. raikovi and other marine Euplotes species [16] there are good reasons to assume that E. 0932-4739-96-0032-0158$3 .50-0

octocarinatus has a closed mating type system [7]. Until now only ten different mating types have been identified. They are determined by four codominant alleles (mr', mt-, mt ', rnr"], six mating types by the heterozygous combinations of these alleles, and four mating types by their homozygous combinations. Each of these four alleles governs the production of another pheromone or gamone (G 1, G 2 , G 3 and G 4 ). That the cells respond only to pheromones not synthesized by themselves is rationalized by assuming that mating competence is specified by pheromone-specific receptors and that cells express receptors only for those pheromones they do not synthesize themselves [9]. Pheromone 3 was the first E. octocarinatus pheromone which could be studied molecularly in depth, with very important unexpected results. First, the universal stop codon TGA was found three times in-frame in the pheromone 3 encoding sequence, and was shown © 1996 by Gustav Fischer Verlag

Pheromone Genes of E. octocarinatus . 159

to be translated as cysteine [17]. Second, comparisons of cDNA sequences with macronuclear genomic sequences resulted in the discovery of introns. They belong to the class of pre-mRNA introns and constitute the first example of introns in any Euplotes species and the first case of multiple introns in hypotrichous ciliates [3]. Subsequently, a study of the pheromone 4 gene of E. octocarinatus was added, including a report of its cDNA as well as its macronuclear genomic sequence [18]. The study reported here completes this investigation by including pheromones 1 and 2, again both with their respective cDNA and macronuclear genomic data, thus allowing a detailed comparison of the entir e pheromone system of E. octo carinatus. The most remarkable result from this comparison is the finding of a highly similar gene structure with very similar introns and non -coding regions, but rather poor similarities in the regions encoding for the secreted pheromone proteins themselves. Material and Methods Cells and culture conditions Strains 69(1)-VII (mr'mt '), 69(2) -VIII (mr-rnr') and 5(20)IV (mt-mr') were used in this study. They are descendants of the two interbreeding stocks 3-1 (rnt-rnt-] and l l -Il (mt -rnr'] of E. octocarinatus Carter, 1972, that had been used previously to study the mating type system and the mating type determination in th is species. For origin of the stocks, the mating type system of E. octocarinatus and the induction of mat ing by pheromones, see Heckmann and Kuhlmann [9]. The strains were grown at room temperature with the photosynthetic flagellate Chlorogonium elongatum as a food source. They were grown either in a diluted soil medium in Boveri dishes [9] or in CME [13] in Fernbach culture flasks or in 15 liter glass flasks. Chlorogonium was cultivated in 5 liter con ical flasks at 20 °C with aeration in an 12 hours light/ 12 hours dark cycle in SMC [5].

Chemicals Restr iction and DNA-modifiying enzymes were from Boehringer Mannheim and Gibco BRL. Taq DNA polymerase was obtained from Gibco BRL, Sequenase from USB, radiochemicals were from Amersham. The phagemid vector pT7T3 19U was from Pharmacia, the EcoRllinkers were obtained from New England Biolabs.

Preparation of DNA and southern blotting Preparation of genomic DNA of E. octocarinatus was carried out as prev iously described [18]. Tota l DNA was fractionated on 1 % agarose gels, blotted on Biodyne A nylon membranes (Pall, Frankfurt, Germany) and hybridized with radioactively labeled pheromone 3 cDNA according to established procedures [24].

Isolation of RNA Total RNA was isolated from 1-4 x 10 7 cells by the method of Pulissant and Houbedine [22]. Poly(A)+ RNA was prepared by affinity chromatography on oligo(dT)-cellulose (Gibco BRL) as described by Aviv and Leder [1], with the

exception that MOPS was used as buffer instead of Tris. Quality and quantity were determined spectrophotometrically by measuring absorption at 260 and 280 nm [24].

cDNA synthesis The pheromone 1 cDNA library was constructed in the vector }.gt10 by the method of Gubler and Hoffmann [6]. The cDNA was treated with Sl nuclease and ligated with EcoRI linkers prior to its insertion into the EcoRI site of the vector. The pheromone 1 gene was identified by plaque hybridization using a radioactively labeled part of the pheromone 3 cDNA and was isolated by standard techniques [24]. Pheromone 2 cDNA was made by reverse transcriptase PCR (RT-PCR). Two micrograms of poly(A) +-RNA of the E. octo carinatus strain 5(20)-IV were reverse transcribed using the oligonucleotide 5'-CCAAGCTTGGATCCGAATTCTTTTTTTTTTI I I I I I 1-3' as a primer with 200 U of M -MLV reverse transcriptase (Gibco BRL) as suggested by the manufacturer. The reverse transcription mix was cleared of residual primers and nucleotides by passing it over a Centr icon L 100 unit (Amicon) according to the manufacturer's instruct ions. A retentate of about 50,u1 was collected and the pheromone 2 cDNA was amplified by PCR using the oligonucleotide 5 '-CCAAGCTTGGATCCGAATTC-3 ' and the gene-specific prime r 5 '-CGGAATTCATGAAAGCCATTTTCAT-3'.

Construction of genomic libraries Genomic libraries were constructed from DNA of the E. octocarinatus strains 69(1)-VII and 69(2) -VIII in the phage Agt 10. Therefore the corresponding DNAs were separated on 2 % low-melting-point agarose gels. DNA molecules in the 1.6-2.0 kb size range were eluted from the gels using Qiaex (Qiagen) . The gene-sized molecules carrying the pheromone genes were suspected to be in this size range on the basis of prev ious southern blot hybridizations with pheromone 3 cDNA as a probe. The libraries were constructed and screened as previously described [18], again using labeled pheromone 3 cDNA as a probe.

Polymerase chain reaction The PCR was carried out in a total volume of 100 ,ul containing Tris in a final concentration of 10 mM (pH 8.3), 50 mM KCI, 200,uM of each dNTP and 2.5 U of Taq DNA polymerase. 10,u1 of first-strand cDNA were used with 2.5 mM MgCh and each primer in a final concentration of 0.25,uM for pheromone 2 cDNA amplification. Amplification of genomic DNA was performed using 50 ng DNA, 1.0 mM MgCI 2, 0.25,uM of the oligonucleotide 5'GCGAATTCCCCAAAACCCCAAAA-3', which is homologous to E. octocarinatus telomeres in its last 16 nucleotides, and 0.5,uM of the respective gene-specific primer. The sequences of the gene-specific primers 5 ' -GGATCCTACTTGTCTGTCTGAAG-3 ' and 5'-GCGAATTCATGAAAGCCATTTTCA-3 ' correspond to the 3 '- and 5'-end of the previously determined pheromone 1 cDNA. Amplification was achieved in a Perkin Elmer Cetus Thermal Cycler as prev iously described in the case of cDNA amplification [18]. For ampl ification of genomic DNA the reaction mixes were denatured at 94 °C for 4 min, followed by 30 cycles consisting of denaturation for 1 min at 94 "C, annealing for 1 min at 55 °C and 2 min of polymerase exten sion at 72 "C, Cycling was followed by a final extension step

160 . W. Teckentrup,

J. Puppe, J. c. Weiligmann,

H .]. Schmidt, and K. Heckmann

of 5 min. PCR products were electrophoresed on 1 % agarose gels and isolated using Qiaex (Qiagen).

A Met Lye Ala lIe Phe lIe lIe Leu Ala rle lieu Met Yal Thr GIn

Subcloning and sequencing

Ala

fhA

Lys

Met

Thr Set

INS

val Aso

Thr INS

Leu GIn Ser GIn

lIe GIn Ser tya Phe Glp Set Lye Asp GIn Leu Ala Ser

DNA fragments, obtained after digestion from the purified ), phage, were subcloned into the EcoRI site of the phagemid vector pT7T3 19U [24]. The PCR products were digested with restriction enzymes corresponding to the restriction sites incorporated with the primer before cloning in the vector pT7T3 19U. Double-stranded DNA sequencing was performed by the dideoxy chain termination method using Sequenase.

Thr

Phe

15 30 45

Glo Thr Ser Ser Lys Leu Lys Gly Glu Cys Asp Thr lle lle Pro

60

As p Phe Thr Gly Cys Aso Ala Aso Asp Asp Cys Pro Leu Ser Phe

75

Thr eya Ser Ala Thr Gly Aso Asp Lys Glu Leu eya Asp Ala Ile

90

Gly Glo Aso Val Va l Asp Met lle Phe Ala His Trp Ser Thr Cys

10 5

Trp Aso Thr Pro Leu Aso Cya Glu Ser Phe Ala Tyr Glo Thr Tyr 120 Ala lle Tyr Aso Ala Pro Glu Leu Cys Gly Cys Asp His Va l Asp

1 35

Gl u Glu Thr Trp Leu Glu lle Leu Asp Ser Va l Cys Pro Asp Lle 150 Asp

151

B

Results Met Lys Ala lIe Phe lIe lIe Leu Ala lIe Leu Met Ya l Thr GI n

In order to study the pheromone 1 and 2 genes of E. octocarinatus we constructed genomic libraries from DNA of the homozygous strains 69(1)-VII and 69(2)-VIII. The preparations of genomic DNA were known to contain mostly macronuclear DNA, but also, to a lesser degree, micronuclear DNA, mitochondrial DNA and DNA from symbiotic bacteria. The isolated DNAs ranged from 0.5 to larger than 23 kb when separated on native agarose gels. To determine the respective sizes of the macronuclear chromosomes carrying the corresponding pheromone genes, isolated DNAs were separated on 1 % agarose gels, blotted onto nylon membranes and hybridized with radioactively labeled pheromone 3 eDNA. In both cases hybri dization signals were obtained at approximately 1.7 kb. To obtain the pheromone 1 encoding gene, a library was constructed from total cell DNA, while the library for the isolation of the pheromone 2 gene was constructed from size fractionated DNA . For the partial library, genomic DNA of the strain 69(2)-VIII was separated by agarose gel electrophoresis and the DNA in the size range of 1.6-2.0 kb was eluted before cloning into the phage vector Agt 10. In both libraries 400000 pfu were screened with the labeled pheromone 3 eDNA, but only one hybridizing clone was found in the partial library. In the other library no positively reacting plaque could be detected. The positive clone was further purified by rescreening, analyzed by restrict ion endonuclease analysis, subcloned and sequenced . Sequence comparison clearly showed that its 1763 bp insert is the macronucleus chromosome carrying the pheromone 2 gene (EMBL accession no. X86554) . In order to identify the corre sponding transcript, we amplified the pheromone 2 eDNA by reverse transcriptase PCR (RT-PCD) from poly(A)+-RNA of moderately starved cells of the strain 5(20)-IV. Subcloning and sequencing of the PCR product revealed an open reading frame of 462 bp. The open reading frame contains three inframe TGA codons and codes for a 153-aa protein (Fig. 1a). Sequence comparison showed that the coding region of the pheromone 2 macronuclear chromosome is interrupted by three introns.

Ala Phe Lys Met

Thr

Ser Lys val Aso

Thr

15

We Leu GIn Ser GIn

30

lIe GIn Ser tya Phe GIn Set Lye Asp Lye Leu Ala Ser Thr Phe

45

Tbr Ser Ser GIn Leu Lys Asp Ser Cys Leu Aso Asp Pro Gl u

60

Glo Arg Phe Tyr Ile Thr Gly Cys Ser Aso Aso Pro Va l Cys Gly

75

Asp Ala Phe Asp eya Ser Ala Thr Gly Asp Asp Glu Glu Lys eya

90

GIn

As p Ala Va l Gly His Aso Va l Ile Asp Leu Phe Tyr Tyr Phe Trp 105 Gly Thr Cys Va l Aso Asp Tyr Ala Ser Cys Ile Met Phe Ala Ala

12 0

Th r Thr Tyr Aso Met Tyr Aso Gly Pro Glu Aso Cys Gly eya Thr

135

'lYr Va l Asp Tyr Glu Asp Trp Leu Asp Tyr Phe Asp Cy s Pro Ser 150 Phe Ser Gly

1 53

Fig. 1. Amino acid sequences of pheromones 1 and 2 of E. octocarinatus . A. Pheromone 1. B. Pheromone 2. The sequences are deduced from cloned cDNAs. The leader sequences are underlined. TGA encoded cysteines are in boldface .

We do not know why all attempts to isolate a pheromone 1 clone from a genomic library failed, but instead we obtained information about the pheromone 1 gene by screening a eDNA library of the strain 69(1)-VII with a pheromone 3 eDNA probe. We isolated a clone of about 600 bp that has a 456-bp open reading frame again with three in-frame TGA codons. This corresponds to a protein of 151 aa (Fig. Ib). To find out whether the coding sequence of the pheromone 1 gene is interrupted by introns as well and to get information about the non-coding sequences of this gene we amplified the pheromone 1 macronucleus chromosome with the help of peR technology. For this we used gene-specific primers that were synthesized on the basis of the determined eDNA sequence in combi nation with primers that are homologous to the telomere sequences (C4A 4 repeats) terminating all macronuclear DNA molecules (Fig. 2). The genespecific primers were used in excess in order to favour specific over nonspecific amplification that takes place by amplification of macronuclear molecules from telomere to telomere. Fig. 3 demonstrates that nonspecific amplification still occurred, but in lower quantities. Putative specific PCR products of 1.3 and 1.6 kb were cloned and sequenced. In each case three independently obtained PCR products were analyzed in order to recognize possible artefacts due to missing proofreading activity of Taq DNA polymerase. The complete

Pheromone Genes of E. octocarinatus . 161

evaluation of these data showed that the macronuclear chromosome carrying the pheromone 1 gene (EMBL accession no. X84894), having 1761 bp (including telomeres), is very similar to its pheromone 2 counterpart. Sequence comparison allowed the identification of three introns that show an unusually high degree of sequence homology with the introns of the pheromone 2 gene as well as with the genes of pheromones 3 and 4. Also noticeable are the complete identity of the 162 bp long 5'-non-coding regions of all four known macronuclear pheromone encoding genes and the high degree of homology in their 3'-non-coding regions. Table 1 summarizes sequence comparison data of all pheromone genes of E. octo carinatus. Table 2 shows the length of the various coding and non-coding parts of these genes. Discussion The present study is mainly a characterization of the pheromone 1 and 2 encoding genes of E. octocarinatus, but together with previously published data on pheromones 3 and 4 it fills the gaps to understand the general picture for a probably complete mating type system. All available information supports the hypothesis that E. octocarinatus has a closed multipolar mating type system with only four different pheromones [17]. All of them could be comparatively analyzed with the data on the pheromone 1 and 2 genes now at hand. Previously all four pheromones had been isolated and characterized as polypeptides [26, 27] and their N-terminal sequences were determined by Edman degradation. By comparison with the respective DNA sequences we should therefore determine precisely the parts corresponding to the secreted pheromones and those belonging to leaders. The homologies between the mature pheromones are astonishingly low. They are 38-50 % between phe romones 1-3 and onl y 18-21 % when these are compared with pheromone 4. This is in contrast to some very striking similarities in the structure of the pheromone genes (Fig. 2, Table 2). Most obviously, all of them have the same basic

T

structure with coding regions always subdivided into 4 exons and 3 introns, and are flanked by 5'- and 3 '-noncoding regions with typical C 4A4 telomeric repeats. All four genes are also located on macronuclear chromosomes of very similar length, namely about 1.7 kb . Furthermore, the corresponding proteins are similar in size, ranging from 85 amino acids for pheromone 4 to 101 amino acids for pheromone 2. The 5'-noncoding sequences in all four pheromone genes are completely conserved. Significant similarities exist for the 3 '-non-coding regions as well, but to a much lesser extent. Putative chromosome fragmentation sites with TTGAA and TTCAA consensus sequences [2] are present in all pheromone genes in their typical distance of 17 bp from the telomeres. Other consensus sequences are less obvious. Nevertheless, pheromones 1 and 2 show a sequence 5'-AATAAC-3' in their 3'-non coding region. A similar sequence is present in pheromone 3, but only pheromone 4 shows the sequence 5'-AATAAA-3' consistent with typical eukaryotic polyadenylation signals. A search for eukaryotic promoter elements yielded TATA boxes and TATA-like putative elements as well as inverted repeats capable of forming a stem-loop structure and a putative CAAT box. However, Helftenbein et al. [11] found for tubulin genes of Stylonychia lemnae that none of several similar putative promoter elements could be significantly ad dressed as a true promotor. The pheromones are synthesized as prepro-pheromones. The prosequence is preceded by a typical ERsignal sequence of 16 amino acids. It is identical for all four pheromones. Compared one by one, pheromones 1 and 2 have very similar pro-sequences and those of pheromones 2 and 3 are even identical. The respe ctive pheromone 4 sequence is identical only for the 23 N -terminal amino acids and diverges completel y afterwards. It is 10 amino acids shorter, too. Interestingly the leader seqences of all four pheromones are terminated by the amino acid lysine which probably is involved in the proteolytic processing of the pro-pheromone. An analysis of codon usages surprisingly revealed 1 to 3 in-frame TGA codons for all pheromone genes.

I 1

I 2

I 3

1.3 kb 1.6 kb

Fig. 2. Map of a macronuclear chromosome encoding a pheromone gene. The black boxes refer to the telomeres (T). The other boxed areas refer to the coding sequence of a pheromone. The open boxes stand for the prepro-sequence ; the stippled boxes stand for the sequence of the secreted pheromone. The connecting lines indicate 5'- and 3'-non-coding areas and the introns (I). The bars below the map indicate the 5 '-PCR-product (1.3 kb) and the 3'-PCR product (1.6 kb).

162 . W. Teckentrup,

J.

Puppe,]. C. Weiligmann, H. J. Schmidt, and K. Heckmann

1 2

3

bp

8-

218

177 -

1230000-

Fig. 3. Pheromone specific PCR products from genomic DNA. Lane 1: Molecular weight markers. Lane 2: 3'-fragment of the pheromone 1 gene (1.6 kb) and several unspecific products. Lane 3: 5 '-fragment of the pheromone 1 gene (1.3 kb) and unspecific products .

Such TGAs were previously shown to encode cysteines in pheromone 3 [17], and we assume the same role for the TGA codons of the other pheromone genes. There are three TGAs each in the pheromone 1,2 and 3 genes and one TGA in the pheromone 4 gene, at respective positions within the gene. All genes terminate with TAA which is apparently the only stop codon used by Euplates [21]. Heterologous in vitro translation studies with E. acto carinatus mRNA resulted in abundant long products [25]. It is therefore unlikely that TGA codons or any other stop codons are frequently used with coding functions in the Euplotes genome. Nevertheless, TGA codons are not unique for pheromone genes, and were also discovered for some other Euplates genes [12, 15]. All pheromones are rich in cysteines, and when compared, all cysteines are found at corresponding positions of the molecules. Pheromones 2 and 3 even agree with respect to their TGA encoded cysteine positions, whereas pheromone 1 has one change in this regard. Macronuclear genes of ciliates are usually intronfree, in particular for hypotrichous ciliates. However, the pheromone genes of E. octocarinatus contain three introns each, always two short ones and one long one. The long introns have sizes of 513-772 bp and are the longest introns of hypotrichous ciliates described so far. They all obey the GT/AG rule deduced for splice junctions of nuclear pre-mRNA introns of higher eukaryotes. The small introns have low G + C values, the large introns are similar to exons in this respect. There is, however, no evidence that any gene product is encoded by the large introns. Unlike introns from mammals typical polypyrimidine stretches and T runs [4] cannot be detected for these pheromone introns. On the other hand, putative branch point sequences are detectable. Although we cannot explain this, it is nevertheless worth pointing out that, in comparison, intron sequences are much more similar than exon sequences for these four genes (Tab. 1). Introns are usually less well preserved than exons, since intron mutations do not change any gene products. Conserved introns, as found here, could indicate certain regulatory functions associated with them, but there is no experimental evidence yet.

Table 1. Comparison of homologous regions of the four macronuclear chromosomes of Euplotes octocarinatus that carry the four pheromone encoding genes. Homologous parts of the chromosomes

5'-non-coding region 3'-non-coding region Sequence encoding the leader Sequence encoding the mature protein Intron 1 Intron 2 Intron 3

Degree of homology

GtlG 2

G]/G 3

GiG 3

G/G 4

GiG 4

GiG 4

100% 94% 99% 64% 95% 83% 84%

100% 77% 99% 51 % 94% 84% 87%

100% 80% 100% 64% 96% 95% 94%

100% 59% 59% 38% 76% 58% 51 %

100% 61 % 60% 36% 71% 50% 50%

100% 40% 60% 42% 67% 51 % 45%

Pheromone Genes of E. octocarinatus . 163 Table 2. Numbers of nucleotides of coding and non -coding regions of the four macronuclear chromosomes of Euplotes octocarinatus that carry four pheromone encoding genes. Macronuclear chromosomes 5 ' -non-coding * prepro-sequence mature pheromone Exon 1 Exon 2 Exon 3 Exon 4 Intron 1 Intron 2 Intron 3 3 ' -non-coding *

Gj

G2

G3

G4

162 bp 156bp 300bp 66bp 253 bp 48 bp 89 bp 514bp 65bp 72bp 495bp

162 bp 156bp 306bp 66bp 262bp 48bp 86bp 515 bp 63 bp 72bp 489bp

162bp 156bp 300bp 66bp 279bp 48bp 83 bp 511 bp 63 bp 72bp 482bp

162 bp 126bp 258bp 66bp 184 bp 42bp 92bp 772bp 64bp 146 bp 151 bp

-' Including 28 bp telomere sequences

Mating type pheromones and their genes have been studied for the distantly related marine ciliate Euplotes raikovi, too [23]. Interestingly, none of the more unusual features described above for E. octocarinatus is shared by E. raikovi. The sequences coding for the prepro proteins are intron-free, and do not contain any TGA-encoded cysteines [19]. This is an ideal example to show that phylogenetic distances in ciliates can be so great that even genes which should be homologous cannot be recognized as such within the same genus . Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (SFB 310, Teilprojekt Cl) and the Fonds der Chemischen Industrie.

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23

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24 25

26 27

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Key words: Amino acid sequences of pheromones 1 and 2 - Multiple introns - UGA codon translation - Leader peptide Klaus Heckmann, Institut fur Allg. Zoologie und Genetik, SchloBplatz 5, D-48149 Munster, Germany