Transcription Rates and Transcript Stabilities of Macronuclear Genes in Vegetative Euplotes crassus Cells

J. Euk. MicrobioL. 42(6), 1995, pp. 691-696 0 1995 by the Society of Protozoologists

Transcription Rates and Transcript Stabilities of Macronuclear Genes in Vegetative Euplotes crassus Cells

ANDREAS BRANDT and ALBRECHT IUEIN' Molecular Genetics, Department of Biology, Philipps- University, 0-35032 Marburg, Germany

ABSTRACT. In hypotrichous ciliates such as Euplotes crassus genes in the transcriptionally active macronucleus are present on individual minichromosomes which occur in gene-specific copy numbers. This different degree of gene amplification can be understood as a means to preset the expression potential of the respective genetic information. In addition, the actual steady state transcript amounts are governed by the transcription rates and transcript stabilities. To establish the relative effects of these three parameters the copy numbers of genes transcribed by the three different polymerases were determined. The transcript levels of growing or starving vegetative cells were then determined, and nuclear run-on assays were performed to determine the transcription rates of the genes in the different nutritional states. A weak correlation between the gene copy numbers and transcription rates was found. The transcripts of genes synthesized by RNA polymerase I1 exhibited different stabilities upon starvation of the cells, compared to the supposedly stable ribosomal 5s and 26s RNA. Refeeding of the cells after starvation also resulted in a differential response with respect to the accumulation of the transcripts of different genes transcribed by RNA polymerase 11, which can be interpreted in the context of the gene functions.

Supplementary key words. Ciliates, gene amplification, hypotnchous ciliates, minichromosomes.

ILIATED protozoa are characterized by nuclear dimor- C phism [29]. They contain generative, transcriptionally mostly inactive micronuclei (321 and vegetative, transcriptionally active macronuclei [ 171. These two different types of nuclei are derivatives of a diploid synkaryon which results from a fusion of haploid nuclei generated in the course of a sexual process called conjugation [30]. During conjugation two cells of com- patible mating types exchange these haploid meiosis products, which then fuse to yield the precursor of both the new micro- and macronuclei. In the macronuclear precursor, the Anlage, extensive processing of the chromosomes takes place resulting in the formation of gene-sized minichromosomes, which are differentially amplified and bounded by telomeres [31]. In Eu- plotes crassus the average copy number of the different macronuclear chromosomes is in the order of lo3 [2]. However, the range of amplification of the individual genes in hypotrichous ciliates is lo2 to lo5 [2, 3, 5 , 9, 21, 22, 341.

Gene amplification is known from other eukaryotic organisms also. In Drosophila the chorion genes [38], which encode a mass product needed in egg development, are amplified, corresponding to the need to increase the gene dosage in order to achieve high amounts of gene products. Therefore, it has been tempting to interpret the different gene amplification in the ciliate macronuclei as a major primary determinant of different gene expression levels [24]. In contrast, in normal diploid eukaryotic cells the primary expression control is on the transcriptional level, notably at the initiation step. Posttranscriptional control of gene product levels includes the differential RNA decay [6, 11, 371. While RNA polymerase I and I11 transcripts are generally stable, mRNA can have vastly different half lives which are also dependent on the physiological state of the cell.

Since it could not be expected that transcriptional control and differential RNA stabilities are of no importance in ciliates, we were interested to find out, which relative roles gene dosage, transcription rates and transcript stabilities would have in de- termining steady state transcript levels in cells of the hypotrichous ciliate E. crassus under different physiological conditions. We have therefore determined the copy numbers of macronuclear genes transcribed by the three different RNA polymerases. Subsequently, we have measured the steady state levels and, as far as possible, the transcription rates of these genes after feeding or starvation. We have indeed found a weak correlation between the copy numbers and the transcription rates. The transcripts differed with respect to their stabilities, which were assayed by quantitative Northern hybridization. Upon refeeding the pre-

I To whom correspondence should be addressed.

viously decaying mRNA reaccumulated with different kinetics, which we will discuss with respect to their functions. The transcription rates dropped for all polymerases during starvation which probably reflects the energy limitation under this condition.

MATERIALS AND METHODS Euplotes crassus and algal strains. E. crassus strains Livl and

Por3 [39] as well as the feeding organism Dunaliella tertiolecta were gifts from Dr. P. Luporini. The E. crassus cells used were exconjugants ofthe two strains which exhibited the same mating types as the parental strains.

Gene probes. The following gene probes were generously given to us by the mentioned colleagues. The histone H4 800 bp coding sequence (C. L. Jahn, pers. commun.) and the &tubulin gene [20], both cloned in pBluescript (Stratagene, Hei- delberg, Germany), were gifts of Dr. C. L. Jahn. The gene encoding the telomere binding protein [40] was given to us by Dr. C. Price. It was cloned into pT7/T3 a-19 (Life Technologies, Eggenstein, Germany). The 5s rRNA gene from Euplotes eu- rystomus [34], obtained from Dr. D. E. Olins was also cloned in the same vector plasmid. An evolutionarily highly conserved 2888 bp region (nucleotides 5372-8260 of the total chromosmal sequence) of the 26s rRNA gene from Tetrahymena termophila [14], cloned in pACYCl84 [35] was a gift of Dr. M.-C. Yao. The EF- la I and TBP encoding genes were isolated in our lab- oratory and cloned in pT7/T3 a- 19.

Culturing of Euplotes crassus. The culturing procedures followed those described earlier [36] with the following modifi- cations. The cells were grown in 30 x 20 x 5 cm polystyrene dishes at 22" C using the following medium: 500 mM NaC1,40 mM MgSO,, 20 mM KCl, 20 mM CaSO,, 1.2 pM NH,NO,, 4.6 nM FeCl, and 0.3 nM MnCl,. They were fed with Dunaliella tertiolecta grown under illumination in 10 1-bottles with aera- tion in the culturing medium with the addition of 10% sea water (Meeresbiologische Station Helgoland, Germany), 0.0 1% KNO, and 0.001% KH,PO,. Starvation was started by filtration of the cell suspension through 30 pm and 10 pm nylon gauze and resuspension of the collected cells in medium without algae. Refeeding was achieved by collecting the cells and resuspension in a culture of algal cells.

RNA preparation. RNA was prepared from E. crassus cells using a modification of the guanidinium isothiocyanate extraction method described before [8]. lo6 cells were lysed in 1 ml RNA Clean solution (AGS, Heidelberg, Germany). After the addition of 10% chloroform and 0.4% isoamyl alcohol the phases were separated by centrifugation. The RNA was precipitated

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692 J. EUK. MICROBIOL., VOL. 42, NO. 6. NOVEMBER-DECEMBER 1995

with isopropanol, washed with ethanol, and dissolved in H20/ 0.1% diethyl pyrocarbonate (H,O/DEPC). In order to resolve secondary structures the RNA was repeatedly heated to 65" C with vigorous mixing and cooled on ice. The solution was then centrifuged for 10 min at 15,000 g and the supernatant was stored at -80" C.

Northern hybridization. The RNA was separated on agarose gels containing 0.4% formamide [ 11. The gel was then transferred twice into H,O/DEPC. Downward capillary transfers [7] were performed onto Hybond N nylon membranes (Amersham, Braunschweig, Germany). The transferred RNA was fixed to the membrane by UV-irradiation. Prehybridization was done in 25 mM K-phosphate, pH 7.4, 5 x SSC (SSC = 0.15 M NaCI, 0.0 15 M Na-citrate), 5-fold concentrated Denhardt's solution and 50% formamide at 42" C for 4 h. Hybridization against 32P- labelled [ 15, 161 appropriate cloned restriction fragments from the respective genes was performed over night under the same conditions. The filters were washed twice with 2x SSC, 0.1% SDS at 42" C and twice with 0.1 x SSC, 0.1% SDS at 60" C.

Isolation of E. crussus DNA and DNA/DNA-hybridization. E. crassus was starved for 3 days and concentrated by filtration. After spinning the cells down they were resuspended in 10 vol- umes of 250 mM NaC1, 100 mM EDTA, 10 mM Tris/HCl pH 7.5, 0.5% SDS and lysed by incubation at 65" C for 15 min. The lysate was treated with 10 pg/ml RNAse for 30 rnin at 37" C followed by digestion with 200 pg/ml proteinase K at 50" C for 16 h. After phenol extraction, dialysis against TE (1 0 mM Tris/HCl pH 8, 1 mM EDTA) and ethanol precipitation the DNA was dissolved in TE. Southern hybridization was performed as described after downward capillary transfer of electrophoretically separated total DNA onto Hybond N nylon membranes. Cloned genes or fragments thereof were used as 32P-labelled probes. Labelling of the probes was done after their excision from the cloning vectors.

Isolation of macronuclei. All following steps were performed at 4" C. The cells were collected on 10 pm gauze, washed off in a small volume of medium and centrifuged for 5 rnin at 350 g. They were washed in 137 mM NaCl, 6.5 mM Na2HP0,, 2.7 mM KC1, 1.5 mM KH2P04, 5 mM MgCl,. After centrifugation as above they were resuspended in buffer A (1 00 mM Hepes pH 7.6, 10 mM KCl, 1.5 mM MgC12, 10% glycerol) for 15 rnin and spun down at 200 g for 5 min. The cells were transferred to a mortar and ground up in liquid nitrogen. The resulting powder was resuspended in TMG (10 mM Tris/HCl pH 7.5, 1 mM MgCl,, 10% glycerol) and centrifuged onto a layer of 40% sucrose in buffer A for 5 min at 600 g. The pelleted nuclei were resuspended in TMG containing 50 pdml PMSF, 50 pdml Leupeptin, 15 pdml Pepstatin, and 80 units RNAse Block (Stra- tagene, Heidelberg, Germany). They were stored in liquid nitrogen.

Nuclear run-on experiments. The previously described method [26] was modified as follows: The macronuclei were thawed on ice and spun down at 4" C for 5 rnin at 350 g. The nuclei obtained from 2-3 x lo6 cells were then resuspended in 50 p1 50 mM Tris/HCl pH 8, 50 mM (NH4),S04, 5 mM MgCI,, 1 mM CaCI,, 1 mM spermidine, 1 mM spermine, 100 mM sucrose, 25% glycerol. 50 p1 of nuclei were mixed with 25 pl 100 mM Tris/HCl pH 8, 100 mM (NH,),SO,, 10 mM MgC12, 2.5 mM CaCl,, 2.5 mM spermidine, 2.5 mM spermine, 1 mM MnCl,, 2.5 p1 each of 10 mM ATP, GTP and CTP, 120 U RNAse Block, 2 pl 1.25 M creatine phosphate, 1 unit creatine kinase (Boehringer, Mannheim, Germany), and I00 MCi a[32P]UTP (800 Ci/mmol, 40 mCi/ml). They were then incubated for 30 rnin at 28" C. This time is sufficient to complete the run-on transcription. The obtained labelled RNA was pu-

rified as described above. It was dissolved in H,O/DEPC and treated at 75" C for 3 rnin to remove secondary structure immediately before using it in filter hybridization against cloned gene probes. The DNA was dissolved in 10 x SSC and fixed to Hybond N nylon membranes with the help of a Slot Blot apparatus (Minifold 11, Schleicher and Schuell, Dassel, Germany). The DNA was fixed by UV-cross-linking.

Calculation of the expected ratios of signal strengths in nuclear run-on experiments for different gene lengths. It is first assumed that genes are transcribed at equal rates. The transcription corn- plexes would then be evenly spaced. We also assume that to a first approximation the incorporation of precursors into RNA in vitro reflects elongation ofalready initiated RNA chains, since reinitiation is a rare event under the chosen conditions. Under these premises the lengths of the genes determine the amount of label which a polymerase can incorporate in the following way: Let n be the minimum spacing of two transcribing complexes and m the relative gene length, i.e. the gene length divided by the minimal spacing, then

n x m [ l + (m - 1)/2] would be the summed total length of the transcribed RNA of a given gene reflected by the run-on incorporation of labelled precursors. This expression, which is the sum of an arithmetical series is derived assuming that the gene is loaded with evenly spaced polymerases at minimum spacing which would be ca- pable of finishing transcription without reinitiation. When m reaches high values, i.e. when genes are long, the ratio of the corresponding signal strengths for different genes approaches the ratio of the squares of the gene lengths.

RESULTS Copy numbers of macronuclear genes transcribed by the three

different RNA polymerases. In order to determine the copy numbers of different genes in the macronucleus of E. crassus which are transcribed by the three different RNA polymerases, quantitative filter hybridization experiments were performed. Figure 1 shows the results. The copy numbers of the ribosomal 5s and 26s rRNA genes were of the same order. It is apparent that there are two macronuclear chromosomes carrying 5s rRNA genes of equal copy number in the E. crassus macronucleus. The investigated genes transcribed by RNA polymerase I1 ranged from 5,000 (telomere binding protein and TATA binding protein) to 84,000 (P-tubulin) with histone H4 (1 2,000) and protein synthesis elongation factor EF-la I (22,000) in between. A copy number of 20,000 for the H4 gene has previously reported [23]. The deviation from the value arising from the present study could be the use of different strains. We have not aimed at finding genes with lower copy numbers in this study, because measurement of the transcript levels or even transcription rates would most likely have been very difficult. However, macronuclear genes do exist with very much lower copy numbers (< 500 per macronucleus) in E. crassus (e.g. the genes encoding the two largest subunits of RNA polymerase 11, J. Bender, unpubl. data).

Transcription rates of different macronuclear genes. The rate of transcription of a gene is determined by the frequency of transcription initiation and the polymerization rate of the RNA polymerase. Since the latter is a constant, a direct comparison of the total transcription rates of different genes transcribed by the same polymerase is a measure of the relative initiation fre- quencies. In addition, differential amplification proportionally increases the number of initiation sites, i.e. the number of promoters. The measurements of the total transcription rates for different genes can be done in nuclear run-on experiments. Nu-

BRANDT & KLEIN-TRANSCRIPTION OF MACRONUCLEAR GENES IN E. CRASSUS 693

Fig. 1. Copy number determination of macronuclear genes by quantitative Southern hybridization. Radioactive gene-specific probes were hybridized to known amounts of cloned gene sequences (left) given in amol ( l O - I * mol) or total cellular DNA (right). The vector plasmids containing the cloned sequences were linearized prior to electrophoresis. Their lengths were: EF-la I, 1.6 kb; 26s rRNA, 2.9 kb; 5s rRNA, 930 bp; TBP, 1.5 kb; TP, 1.6 kb; P-tubulin, 1.5 kb; histone H4,800 bp. The copy numbers were determined by comparison of the signal strengths on the basis of the published DNA amount per macronucleus in E. crassus [25] . Note the double band hybridizingwith the 5s rRNA probe, which indicates that there are two different 5s genes present in the macronucleus. The given average were obtained by comparison of at least two pairs of signal obtained with the cloned genes and the cellular DNA on the same film. This procedure corrects for occasional uneven DNA transfer during the blotting process. The figure is composed of autoradiographs of individual experiments for the different genes.

clei isolated from cells are incubated with labelled R N A precursors in vitro and the synthesized products a t e hybridized to excess filter-bound D N A of cloned genes. Transcription reinitiation occurs rarely in vitro [28]. Only with 5s rRNA genes has it been found that one polymerase can reinitiate, yielding up to three transcripts per gene within 40 min under optimal conditions [4 11. Therefore, the amount of product in most cases directly reflects the number of active polymerase molecules transcribing the genes a t the time of isolation of the nuclei, unless,

Fig. 2. Determination of transcription rates in nuclear run-on experiments. The reaction products of run-on experiments with nuclei isolated at the indicated time points during starvation or refeeding were hybridized to filter-bound excess cloned DNA (1 pmol) of the indicated genes. Binding of the DNA was done using a slot blot procedure. The different cloning vectors were also bound to the filters as controls. The top two rows show autoradiographs of the same 26s rRNA specific hybridizations after different exposure times (one and 10 days, respectively).

as seen in one case, regulated pausing interferes with the inter- pretation of the results [13]. Since at equal initiation rates an equal number of polymerase molecules per D N A length will be present on different genes, the amount of gene specific transcript formed in vitro is also dependent on the gene size. The first and last lanes o f Fig. 2 qualitatively shows the results of run-on experiments with nuclei obtained from cells which were fed with algae. The signals can be quantitatively evaluated by scanning the autoradiograms. For the comparison of the relative transcription rates of different genes their copy numbers and sizes have to be taken into accounts (compare Methods). Such results of four independent experiments are shown in Tab. 1. It can be seen that the 26s and 5s rRNA genes are transcribed a t equal rates. Compared to these genes, synthesized by R N A polymerase I and 111, respectively, the three genes transcribed by R N A polymerase I1 are transcribed less efficiently, indicating weaker promoters. The transcription rates of E F - l a I and P-tubulin are roughly equal but much lower than that of the gene encoding the TATA box binding protein TBP. Signals were not obtained

Table 1. Relative transcription rates of macronuclear genes as determined by nuclear run-on experiments.

Ratio expected Average Observed on the basis of

equal ratesb Compared gene pair ratio range"

26s RNA/5S RNA 252 83-490 270 P-Tub./SS RNA 3.5 0.5-7.1 700 8-Tub.lEF- 1 I 2.6 1.3-5.0 4.6 TBP/&Tubulin 7.6 4.7-16 0.06

a The results are based on four independent experiments, The expected ratios were calculated according to the formula given

in the Methods, taking into account the gene lengths and the copy numbers of the genes. It is assumed that on the average one polymerase molecule can transcribe a 120 bp 5 s rRNA gene. This length is also taken as the minimum spacing of transcribing polymerases. The gene lengths are 5S, 120 bp; 26S, 3.9 kb; EF-la I, 1.3 kb; TBP, 1.4 kb; P-tubulin, 1.5 kb. The copy numbers of the genes can be taken from Fig. 1.

694 J. EUK. MICROBIOL., VOL. 42, NO. 6, NOVEMBER-DECEMBER 1995

Fig. 3. Northern hybridization analysis of total RNA isolated from E. crussus cells at different time points of starvation and refeeding. Equal amounts of RNA were electrophoretically separated on denaturing agarose gels. Excess gene-specific 32P-labeled DNA probes were hybridized to the filter bound RNA. Note that the signal strengths can only be compared within experiments using the same probe due to the different specific activities of the probes.

for the two other tested genes with copy numbers below 15,000 per nucleus.

Influence of the nutritional state of the cells on transcription rates. Figure 2 shows that the nutritional state of the cells has an influence on the transcription rates of genes transcribed by any of the three polymerases. Starvation rapidly resulted in reduced transcription. However, transcription of all three polymerases did continue at low rates even after an 18 h starvation period. Refeeding resulted in a rapid recovery of the transcription rates seen in unstarved cells.

Transcript stabilities. The available steady state transcript amounts in the cell are determined by both the synthesis and decay rates. To assess the influence of RNA decay we performed Northern hybridization experiments. RNA was prepared from cells which were fed or starved for the same time periods as the ones used for the nuclear preparations for the run-on experiments just described. Labeled probes were then hybridized to the filter bound RNA. Figure 3 shows the results. Since equal

amounts of total RNA was loaded in the different lanes and the stable ribosomal RNA account for more than 90% of the RNA approximately even signals are seen for the 5s and 26s rRNA. The same was true for the @-tubdin and the TBP mRNA, which indicates its high stability after starvation. In contrast, the translation elongation factor EF- 1 a I mRNA decreased rapidly upon starvation and reaccumulated fast after refeeding. A third pattern was obtained with the two mRNA encoding structural proteins associated with DNA, the telomere binding protein (TP) and histone H4. Here, starvation led to a constant transcript level for several hours of starvation with subsequent decay. Remarkably, reappearance of elevated mRNA levels was delayed compared to the EF- l a I transcript.

DISCUSSION The aim of the present investigation was the analysis of the

possible contributions of gene dosage, transcription rates and transcript stabilities to the overall regulation of the steady state RNA concentrations in Euplotes crassus vegetative cells under varying physiological conditions.

Transcription rates in growing cells. Our results show that the total transcription rates of the two rRNA genes transcribed by RNA polymerases I or I11 are similar. This had to be expected because of their function as equimolar constituents of the ribosomes. The transcription rates of the genes transcribed by RNA polymerase I1 vary. The rates are generally lower than those of the two ribosomal RNA genes. With the exception of the TBP encoding gene discussed below, run-on transcription could only be detected for high copy genes, namely those encoding EF-la I (22,000 copies per macronucleus) and @-tubulin (84,000), whereas the transcription of the histone H4 gene (1 2,000) and the gene encoding the telomere binding protein (5,000) was not detectable. The transcription rate of the @-tubulin gene was higher than that of the gene encoding EF-la I, as had been expected on the basis of their different gene dosage under the condition of equal promoter strengths. Therefore, these results are in line with the assumption that the gene dosage does serve to preset the overall level of transcription. However, the observed high transcription rate of the gene encoding the TATA-binding transcription factor TBP is a notable exception. With a 17-fold lower copy number compared to the P-tubulin gene its transcription rate is still about 8-fold higher, which indicates a 160-fold higher transcription initiation frequency, when the different gene lengths are taken into account (Table 1). This in turn means that the promoter is much more efficient. Since the non-translated region of the TBP encoding gene is very short (63 bp between the telomere and the ATG codon) it is easy to look for known promoter elements. In fact a consensus TATAA-sequence is found directly next to the telomere (M. Gmpfer, unpubl. result). It has been noted before [19, 241 (J. Bender, pers. commun.) that transcription start sites are fre- quently found approximately 30 bp downstream of the telomere on E. crassus and E. octocarinatus macronuclear chromosomes. This has resulted in the suggestion that the telomere binding protein might play a role in the stabilization of a transcription initiation complex, especially since within the very short un- translated regions additional transcription factors could hardly have binding sites, for spatial reasons. So far, to our knowledge, the TBP gene is the only E. crassus gene which has a TATA- box immediately next to the telomere. If the telomere binding protein were to interact with TBP the adjacent position of the TATAA-sequence might result in a particularly efficient promoter.

Transcription rates are dependent on the physiological state of the cell. Upon starvation of the ciliate cells the transcription

BRANDT & KLEIN-TRANSCRIPTION OF MACRONUCLEAR GENES IN E. CRASSUS 695

rates of all three R N A polymerases decreased steadily. Refeed- ing of the cells resulted in a rather rapid increase. The easiest explanation for these observations is that the reversible energy limitation influences the formation or stability of the transcription complexes.

Different patterns of transcript stabilities. Under these conditions the differential transcript stabilities result in the regulation of differential steady state concentrations of the transcripts. T h e stable rRNAs served as internal standards. The P-tubulin and T B P transcripts were found t o have comparable high stabilities. While nothing is known about the mechanism of stabilization or destabilization of the TBP m R N A in other eukaryotes, it can be rationalized that it could belong to a class of transcripts the destabilization of which depends on its translation like P-tubulin mRNA. In other eukaryotic systems this message has been shown to decay under the influence of mo- nomeric tubulin only when it i s translated [ 181. Under conditions of energy limitation translation of the m R N A would be turned off. As a result the decay would be prevented. The apparently constant level in growing and starved cells would result from a much higher turnover o f the transcript in growing cells a t the observed high synthesis rates.

E F - l a 1 resembles household genes of higher eukaryotes i n that its transcript is destroyed upon starvation which results i n the cessation of protein synthesis. In these cases translation of the messenger protects it from nuclease attack [4, 10, 271. Our studies on the decay rates of the transcripts of the TP and H4 genes agree with those of a previous investigation focussing on changing transcript concentrations after conjugation [ 331 in which R N A concentrations in vegetative cells were measured 6 and 18 h after starvation prior to matings. In contrast to our results a decay of the P-tubulin transcript was observed in that study. We have no explanation for the discrepancy, even though we have used different strains.

The third class of stability behavior is seen in the case of the histone H 4 and telomere binding protein transcripts. These transcripts are stable for an extended period of starvation before they eventually decay. Notably, their reaccumulation which reflects their resynthesis, is delayed as compared to that of the E F - l a I transcript. This suggests a cell cycle regulation of their synthesis and/or stabilities. Both genes encode DNA-binding structural proteins which are massively required only during the S-phase. Due to the asynchrony of the growing cells a t the t ime of food withdrawal it can be assumed that only part of the population has passed the S-phase of the on-going cell cycle. This is certainly different after 18 h of starvation. The low level of transcription seen during starvation and in the first 2 hours after refeeding might indicate a cell cycle-independent histone replacement synthesis which has previously been described in Tetrahymena [42]. It is not surprising-but has not been seen before- that the transcript of the telomere binding protein follows the same stability pattern, given its similar function with respect of structuring the chomosomal DNA. The finding that histones can also interact with transcription factors [ 121 would add to the functional similarity between the two proteins, if the telomere binding protein is actually involved in transcription initiation, which, however, remains to be shown.

ACKNOWLEDGMENTS We thank P. Luporini for the gift of strains and C. L. Jahn,

D. E. Olins, C. Price and M.-C. Yao for supplying gene probes. We are grateful to H. Bestgen for photographic work and V. Florian for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft.

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Received 4-14-95, 6-21-95; accepted 6-23-95

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Preliminary Characterisation of Chlorarachniophyte Mitochondria1 DNA PAUL GILSON,’ ROSS WAUER and GEOFF McFADDEN

*Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, 3052, VIC, Australia

ABSTRACT. The division Chlorarachniophyta comprises amoeboflagellate protists with complex chloroplasts derived from the endosymbiosis of a eukaryotic alga. Analysis of chlorarachniophyte chromosomal DNAs by pulsed-field gel electrophoresis revealed an apparently linear 36-kb chromosome that could not be ascribed to either the host or endosymbiont nuclei. A single eubacterial-like small subunit ribosomal RNA gene is encoded on this chromosome and phylogenetic analyses places this gene within a clade of mitochondrial genes from other eukaryotes. High resolution in situ hybridization demonstrates that transcripts of the small subunit ribosomal RNA gene encoded by the 36-kb chromosome are exclusively located in the mitochondria. The 36-kb chromosome thus likely represents a linear mitochondria1 genome. Small amounts of an apparently dimeric (72 kb) form are also detectable in pulsed- field gel electrophoresis.

Supplementary key words. Chlorarachnion, endosymbiosis, in situ hybridization, linear mitochondrial DNA, rRNA.

HLORARACHNIOPHYTES are a group of unicellular C amoeboflagellate algae with unusual chloroplasts bounded by four membranes [15, 171. These so-called chloroplasts are actually drastically reduced eukaryotic endosymbionts, complete with a vestigial nucleus sandwiched between the second and third chloroplast membranes [22]. The endosymbiont has apparently lost many of the subcellular components (cytoskel- eton, Golgi apparatus, cell wall and mitochondria) typical of eukaryotic cells. The vestigial nucleus, known as the nucleo- morph, contains three small chromosomes which encode components for ribosomes located in the periplastidal space (a rem- nant of the endosymbiont’s cytoplasm situated between the second and third membranes surrounding the chloroplast) [22].

I To whom correspondence should be addressed.

Chromosomes belonging to the host nucleus and nucleo- morph have previously been identified on the basis of their ribosomal RNA genes and telomeric elements [9, 221. The nu- cleomorph contains three small chromosomes (95 kb, 140 kb, 145 kb) and the nucleus contains at least 16 chromosomes rang- ing in size from 325 kb to 2000 kb. Two chromosomes of sizes 36 kb and 72 kb do not hybridize to either nuclear or nucleo- morph telomeric probes suggesting they are not of eukaryotic origin and probably originate from either the chloroplast of the endosymbiont or mitochondrion of the host cell [9]. Sequence analyses and in situ hybridization data demonstrating a mitochondrial origin for these chromosomes are presented in this paper.

MATERIALS AND METHODS Organisms. Chlorarachniophyte strain CCMP 62 1 was ob-

tained from the Culture Collection of Marine Phytoplankton,

Documents

Transcription Rates and Transcript Stabilities of Macronuclear Genes in Vegetative Euplotes crassus Cells