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Volume 270, Number 13, Issue of March 31, 1995 pp. 7601-7608
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcription in Vitro of Tetrahymena Class II and Class III Genes (*)

(Received for publication, January 4, 1995)

Leif K. Larsen ,  Karsten Kristiansen (§)

From the Department of Molecular Biology, University of Odense, Campusvej 55, DK-5230 Odense M, Denmark

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A method for preparation of transcriptionally active nuclear extracts from the ciliated protozoan Tetrahymena thermophila is described. Cells were lysed in the presence of gum arabic, and nuclei were further purified in the presence of Ficoll 400. Highly concentrated nuclear extracts were prepared by ultracentrifugation of nuclei in a buffer containing potassium glutamate and spermidine. These extracts supported accurate transcription initiation of T. thermophila class II and III genes. Using the histone H3-II gene as a template, we demonstrated that physiologically induced changes in transcriptional activity in vivo were reflected in the transcriptional activity of the nuclear extract in vitro. By electrophoretic mobility shift assays, five conserved sequence elements in the upstream region of the histone H3-II gene were shown specifically to bind proteins in extracts from exponentially growing as well as from starved cells, and by UV cross-linking we further characterized the specific binding of two proteins to an oligonucleotide containing a conserved CCAAT box motif. Transcription competition experiments showed that addition of this oligonucleotide decreased transcription significantly. Competition with oligonucleotides corresponding to the two proximal conserved sequence elements almost completely abolished transcription of the H3-II gene suggesting that binding of transacting factors to these elements is crucial for initiation of transcription.

INTRODUCTION

In higher eukaryotes mRNA levels are controlled by a complex interplay of transcriptional and post-transcriptional mechanisms. In lower eukaryotes examples of post-transcriptional regulation of mRNA abundance have been described (Warner et al., 1993), but generally, transcriptional control of mRNA levels appears to prevail. The ciliated protozoans represent one of the earliest divergent branches of the eukaryotic lineage (Sogin et al., 1986) and have as model organisms been of considerable importance for studies of fundamental molecular mechanisms such as RNA self-splicing (Zaug et al., 1986) and telomere formation (Greider and Blackburn, 1987). A considerable number of protein-encoding genes from ciliates have been cloned and characterized, but no functional analyses of promoter regions have been reported. To date, analyses of promoter structures in Tetrahymena have been limited to mapping of DNase hypersensitive regions in the promoters of the L1 and S25 ribosomal protein genes (Nørgaard et al., 1992) and in the promoter of the histone H4-I gene (Pederson et al., 1986). Apart from these studies, identification of putative promoter elements in ciliates has been restricted to computer-assisted searches for sequence elements exhibiting similarity to known eukaryotic promoter elements (Brunk and Sadler, 1990).

Recently, transformation of Tetrahymena thermophila with protein-encoding genes was reported (Yao and Yao, 1991; Kahn et al., 1993; Gaertig et al., 1994), but transformation was in all cases accomplished by homologous recombination making this approach unsuitable as a general procedure for functional analyses of promoters in ciliates. An alternative approach would be to develop an in vitro transcription system specific for ciliate genes. Systems capable of accurate in vitro transcription are well established for higher eukaryotes (Manley et al., 1980; Dignam et al., 1983; Parker and Topol, 1984), but it has consistently been found difficult to achieve in vitro transcription of class II genes in extracts from lower eukaryotes (Lue and Kornberg, 1987).

Tetrahymena extracts capable of accurate transcription of rDNA by polymerase I have been described (Sutiphong et al., 1984; Matsuura et al., 1986), but transcription in vitro of ciliate class II genes has not been reported. In this report we describe the preparation of a nuclear extract from T. thermophila that supports accurate transcriptional initiation of exogenously added ciliate class II and class III genes. Extracts prepared from exponentially growing and starved cells, respectively, differed in their ability to transcribe the histone H3-II gene. By competition with double-stranded oligonucleotides, we demonstrated that four conserved sequence elements in the intergenic region between the divergently transcribed histone H3-II and H4-II genes are involved in the regulation of transcription of the histone H3-II gene. Interestingly, electrophoretic mobility shift assays revealed that these oligonucleotides bound different complements of proteins in extracts prepared from exponentially growing and starved cells, respectively.

MATERIALS AND METHODS

Plasmids

A partial genomic library was generated by ligating size fractionated EcoRI/HindIII fragments of T. thermophila macronuclear DNA into the selection vector pUN121 (Nilsson et al., 1983). A clone containing a 3.6-kilobase EcoRI/HindIII fragment harboring the entire H4-II histone gene (HHF2 according to the nomenclature of Thatcher et al.(1994)) and the 5`-end of the H3-II histone gene (HHT2) down to position +237 relative to the A in the ATG start codon was isolated by screening with a cDNA specific for histone H4-II. The 3.6-kilobase fragment was cloned into the EcoRI and HindIII sites of pBluescript KS+ (Stratagene). This plasmid was named pG78 RH3.6 (^1)(Fig. 1). A 449-bp (^2)EcoRI/BglII fragment isolated from pH3-II RH3.6 covering position -212 to position +237 in the H3-II gene was cloned into the BamHI and EcoRI sites of pBluescript KS+. This plasmid was named pH3-II RB449 (Fig. 1). The 351-bp KpnI/BglII fragment from pH3-II RH3.6 covering position -212 to position +139 in the H3-II gene was cloned into the KpnI and BamHI sites of pBluescript KS+. This plasmid was named pH3-II KB351 (Fig. 1).

Figure 1: The histone H3-II containing plasmids used in this study. Transcribed regions are shown in black. Polylinker regions are shaded. Closed arrows indicate the T3 or T7 RNA polymerase initiation sites in the pBluescript vectors. Open arrows indicate the H3-II and H4-II transcription initiation sites.


p5S rDNA SS256 contains a SalI fragment of a 5 S rRNA gene (subcloned from pBS-A4 obtained from R. Hallberg, Department of Biology, Syracuse University) with 145 bp of upstream sequences and the entire transcribed region except for the last 4 bp in the 3`-end of the gene. This fragment was cloned into the SalI site of pBluescript KS+.

Oligonucleotides

The following oligonucleotides were obtained from Otto Dahl, Chemical Laboratory II, University of Copenhagen: oligonucleotide 1A, 5`-TTGTTGTCGATAAAGAATT-3`; oligonucleotide 1B, 5`-TTTTTAATAAAATTCTTTATCG-3`; oligonucleotide 2A, 5`-CAGGATTATGCCAAACATTT-3`; oligonucleotide 2B, 5`-AGATCATTTGAAATGTTTGGC-3`; oligonucleotide 3A, 5`-AAATCATCCAATCAAAATTGTTC-3`; oligonucleotide 3B, 5`-GAGAAGATATGAACAATTTTG-3`; oligonucleotide 4A, 5`-GGATAAAATCTCAAAAATCTGAT-3`; oligonucleotide 4B, 5`-GAAAGGAAATCAGATTTTTGAGAT-3`; oligonucleotide 5A, 5`TTATTTAATTATCCAAATT-3`; oligonucleotide 5B, 5`-CTCCGAGCAATTTGGATAA-3`.

The oligonucleotides were annealed pairwise by mixing 50 µl each of oligo(A) and (B) (100 pmol/µl) with 6.8 µl of 1 M Tris-HCl, pH 7.5, and 1 µl of 100 mM EDTA. This mixture was boiled for 3 min and then cooled slowly to the annealing temperature (23 °C for oligonucleotides 1 and 2, 20 °C for oligonucleotides 3 and 5 and 33 °C for oligonucleotide 4). After annealing for 1 h, 1.7 µl of 1 M MgCl(2), 1.7 µl of 100 mM DTE, 17 µl of 1 mg/ml bovine serum albumin, 42.5 µl of dATP, dGTP, dCTP, and dTTP (2 mM each), and 16 units of T7 DNA polymerase were added. The fill-in reaction was allowed to proceed for 30 min at the annealing temperature. Five µl of 0.5 M EDTA and 16 µl of 3 M sodium acetate were added, and the reaction mixture was extracted with phenol/chloroform. For preparation of labeled oligonucleotides, 4 pmol were annealed in a volume of 10 µl and filled-in in the presence of [alpha-P]dCTP to a specific activity of 3000 Ci/mmol. The double-stranded oligonucleotides were precipitated by addition of 3 volumes of 99% ethanol and were subjected to electrophoresis in a native 12% polyacrylamide gel. Following electrophoresis the part of the gel containing the double-stranded oligonucleotide was detected by UV shadowing or autoradiography and isolated. The oligonucleotides were eluted by the addition of 200 µl of elution buffer (0.5% SDS, 250 mM sodium acetate, pH 8.0, 1 mM EDTA) followed by gentle shaking overnight at 37 °C. The supernatant was isolated, and 200 µl of elution buffer was added to the gel pieces followed by an additional incubation for 2 h. The supernatants were combined and extracted with phenol/chloroform. The oligonucleotides were precipitated with 3 volumes of ethanol, washed twice with 75% ethanol, dried, and dissolved in a solution of 25 mM sodium acetate, pH 8.0, 1 mM EDTA. The concentrations of the double-stranded oligonucleotides were determined by UV spectroscopy.

Nuclear Extract

T. thermophila, strain SB715 (Turkewitz et al., 1991) was grown in 0.75% proteose peptone, 0.75% yeast extract, 10 mM MgSO(4), 0.05 mM CaCl(2), 0.5 mM KH(2)PO(4), 0.1 mM ferri citrate for at least five generations to a density of approximately 4 times 10^5 cells/ml. The growth of the culture was monitored by counting the cells in a Coulter counter. Two liters of culture were mixed with crushed ice, and the cells were harvested by centrifugation in a Beckman JA10 rotor at 2,500 rpm for 3 min at 2 °C followed by washing in 800 ml of nuclei buffer (0.1 M sucrose, 10 mM Tris-HCl, pH 7.5, 3 mM CaCl(2), 1 mM MgCl(2), 10 mM NaCl, 0.1 mM EDTA, 1 mM DTE) and recentrifugation. The collected cells were resuspended in 60 ml of nuclei buffer with 4% gum arabic and inhibitors (0.01 TIU/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml antipain, 1 µg/ml pepstatin, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM beta-glycerophosphate) and lysed in a Potter-Elverhjem homogenizer with a tight-fitting Teflon pestle (Braun-Melsungen) at 1,500 rpm. The lysate was centrifuged at 3,500 rpm for 5 min at 2 °C in a Beckman JS13.1 swinging bucket rotor, and the supernatant was aspirated. The pelleted nuclei were resuspended in 60 ml of nuclei buffer with 4% gum arabic and inhibitors and recentrifuged as above. The nuclei were resuspended in 60 ml of nuclei buffer with 18% Ficoll 400 and inhibitors and centrifuged at 8,000 rpm for 10 min at 2 °C in a JS13.1 rotor. The supernatant was aspirated and the pellet recentrifuged for a few seconds. The last traces of supernatant were removed, and the nuclei were resuspended in 0.5 ml of extraction buffer (25 mM HEPES, pH 7.9, 1.8 M potassium glutamate, 4.5 mM spermidine, 3.3 mM magnesium acetate, 0.3 mM EDTA, 7 mM EGTA) per g of nuclei. The viscous suspension was transferred to 0.5-ml Eppendorf tubes, and extraction was allowed to proceed for 30 min before the tubes were floated on water and centrifuged at 25,000 rpm for 1 h at 2 °C in a Beckman SW28 rotor (100,000 times g). The supernatant was mixed with 0.3 volumes of 87% glycerol and frozen in aliquots in liquid nitrogen.

Determination of Protein Concentration

Protein concentrations were determined by UV spectroscopy as described by Warburg and Christian(1942).

Isolation of Total RNA

Total RNA was isolated from exponentially growing cells as described by Chomczynski and Sacchi (1987).

In Vitro Transcription using Nuclear Extract

For a standard transcription reaction, the following was mixed at room temperature in a total volume of 20 µl: 3.8 µl of transcription buffer (50 mM HEPES, pH 7.9, 7.5 mM spermidine, 16 mM magnesium acetate, 0.5 mM EDTA), 3 µl of 10 mM DTE, 3 µl of nucleotide mix (5 mM each of ATP, GTP, CTP, and UTP), 25 units of RNasin (Promega), 0.5 µg of template DNA. Transcription was initiated by addition of 5 µl of nuclear extract, allowed to proceed at 25 °C for 30 min, and then stopped by digestion with 20 units of DNase I (RNase-free, Boehringer) for 5 min. Proteins were digested by the addition of 100 µl of stop buffer (0.5% N-lauryl-sarcosine, 50 µg/ml proteinase K, 10 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10 mM EDTA, 0.1 mg/ml wheat germ tRNA) followed by incubation at 37 °C for 1 h. The synthesized RNA was precipitated by the addition of 170 µl of GdSCN precipitation mixture (2 volumes of isopropyl alcohol added to a solution of 6 M guanidine thiocyanate and 10 mM sodium citrate, pH 7, in water), washed twice with 75% ethanol, and dried gently in a Speed-vac concentrator.

Preparation of Antisense RNA by in Vitro Transcription

Linearized plasmids were transcribed with T3 or T7 RNA polymerase as described by the manufacturer (Promega). The transcripts were labeled with [alpha-P]UTP (specific activity 130 Ci/mmol). Following transcription the template (0.2-0.6 µg) was digested with 10 units of DNase I (Boehringer Mannheim, RNase-free) at 37 °C for 30 min, and 50 µl of stop buffer were added. After digestion at 37 °C for 30 min, the antisense transcripts were purified, either by phenol/chloroform extraction followed by ethanol precipitation or by precipitation using the GdSCN precipitation mixture. The transcripts were washed twice with 75% ethanol, dried gently, and finally dissolved to a specific activity of 10^5 counts/min/µl in water.

RNase Protection

An improved RNase protection procedure (^3)was followed. Briefly, total RNA or in vitro synthesized RNA was dissolved in 3 µl of antisense transcript solution and 24 µl of deionized formamide, and then 3 µl of hybridization buffer (400 mM PIPES, pH 6.4, 10 mM EDTA, 4 M NaCl) were added. The RNA was denatured by heating to 85 °C for 10 min and hybridized overnight at 37 °C. Three-hundred µl of RNase digestion mixture (300 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 700 units/ml RNase T1, 40 µg/ml RNase A) were added, and digestion was allowed to proceed for 1 h at 15 °C. The digestion was stopped by the addition of 20 µl of 10% N-lauryl-sarcosine, 10 µl of 10 mg/ml proteinase K, and 5 µg of tRNA. The mixture was incubated for 30 min at 37 °C, and the RNA was precipitated by the addition of 500 µl of GdSCN precipitation mixture, washed once with 75% ethanol, dried, and redissolved in 4 µl of formamide-dye mix (98% deionized formamide, 10 mM EDTA, pH 8.0, 0.05% bromphenol blue, 0.05% xylene cyanol). Two µl were loaded per lane on a sequencing gel.

Electrophoretic Mobility Shift Assay

The following was mixed at room temperature in a total volume of 6 µl: 1.5 µl of transcription buffer, 1 µl of 10 mM DTE, 2 µg of poly(dI-dC) (Boehringer Mannheim) and competitor as stated in the figure legends. Prebinding (Demczuk et al., 1991) was initiated by the addition of 2 µl of nuclear extract diluted to a concentration of 2.5 µg/µl (in a mixture of 963 µl of extraction buffer, 3 µl of 0.5 M DTE, 2034 µl of water, 900 µl of 87% glycerol). After 5-10 min of prebinding, the binding reaction was initiated by addition of 2 µl (10 fmol) of labeled double-stranded oligonucleotide. After 20 min of binding at room temperature, the reaction was loaded on a 5% polyacrylamide (acrylamide/bisacrylamide 38:2, w/w) gel containing 50 mM Tris, 380 mM glycine, 2 mM EDTA, pH 8.5. The gel had been pre-electrophoresed for 30 min at 13 V/cm using the same buffer as running buffer. Electrophoresis was continued at the same voltage until the bromphenol blue in a marker lane had migrated through two-thirds of the gel.

UV Cross-linking

Binding reactions were mixed as above in a total volume of 25 µl, incubated for 20 min, and then subjected to irradiation at 254 nm from a Camag lamp held at a distance of 5 cm for 15 min. Twenty-five µl of SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2.5% SDS, 10% glycerol, 5% beta-mercaptoethanol, 0.002% bromphenol blue) were added, the samples were boiled for 3 min and immediately loaded on a 10% polyacrylamide SDS gel. (Laemmli, 1970) with prestained markers (Pharmacia).

RESULTS

Preparation of Nuclear Extract

The standard procedure for preparation of nuclear extracts for in vitro transcription involves lysis of cells in hypotonic buffers followed by isolation of nuclei and extraction of nuclear proteins with 0.42 M potassium chloride (Dignam et al., 1983). A major problem in this approach is leakage of nuclear proteins during lysis of cells and during purification of nuclei (Shapiro et al., 1988; Lue and Kornberg, 1987). To minimize this problem, disruption of cells in an isotonic buffer and inclusion of polymers like gum arabic or Ficoll in the nuclei buffers have been employed (Lue and Kornberg, 1987). However, attempts to prepare active extracts from T. thermophila following these lines were unsuccessful. The resulting extracts did not support in vitro transcription of cloned T. thermophila genes, and furthermore, had very low protein concentrations caused in part by substantial losses of nuclear proteins during dialysis. Recently, Kamakaka et al.(1991) described the preparation of a soluble nuclear fraction with high protein concentration capable of efficient RNA polymerase II-dependent transcription. In this procedure nuclei are extracted with potassium glutamate and subjected to high speed centrifugation. Potassium glutamate, even in concentrations as high as 400 mM, does not inhibit transcription in vitro (Verdier et al., 1990; Kamakaka et al., 1991), and thus, the dialysis step necessary in the procedure of Dignam et al. (1983) can be omitted.

By using this procedure and by adding Ficoll during the preparation of nuclei, we were able to obtain nuclear extracts containing 20-80 mg of protein/ml. As described below, these extracts supported in vitro transcription of cloned genes from T. thermophila.

In Vitro Transcription of the Gene Encoding Histone H3-II

The divergently transcribed H3-II and H4-II genes are well suited for in vitro transcription experiments because the intergenic region can be expected to possess the elements necessary for initiation of transcription. Initial experiments employing the H3-II gene as a template showed that inclusion of promoter sequences up to position -213 (relative to the A in the ATG start codon, cf.Fig. 6) was sufficient for an efficient in vitro transcription as exemplified by the in vitro transcription of pH3-II RB449 shown in Fig. 2. Transcription with 5 µl of extract prepared from exponentially growing cells resulted in distinct bands (Fig. 2, lane 1) and was sensitive to alpha-amanitin at a concentration of 2 µg/ml (Fig. 2, lane 2), indicating that transcription was dependent on RNA polymerase II (Eichler and Corr, 1989). No transcription was observed when pBluescript was used as a template (Fig. 2, lane 3). Extracts prepared from cells starved for 24 h in 10 mM Tris-HCl, pH 7.5, were inactive (Fig. 2, lane 5). A 1:1 mixture of extracts from starved and exponentially growing cells exhibited a decrease in transcriptional activity that was roughly proportional to the reduction in amount of nuclear extract from the exponentially growing cells (Fig. 2, lane 6) indicating that the nuclear extract prepared from starved cells did not contain inhibitors that per se affected transcription in vitro. Under our standard conditions for in vitro transcription approximately 0.001 transcripts were generated per template.

Figure 6: The intergenic region of the divergently transcribed H3-II-H4-II histone genes. Thirty sequences in the EMBL data base were aligned using the PILEUP program of the GCG package (Deveraux et al., 1984). The consensus was displayed with the program PRETTY counting 20 conserved nucleotides as a consensus. The sequence of the intergenic region of T. thermophila is shown with the nucleotides identical to the consensus shown in uppercase. The ATG translation start codons are underlined. The oligonucleotides used for electrophoretic mobility shift assays and competition experiments are shaded, and the sites of transcription initiation are shown with open arrows. The sequence is numbered counting the A in the ATG start codon of the H3-II gene as +1.


Figure 2: In vitro transcription of the histone H3-II gene. The transcription products were detected with T3 RNA polymerase generated antisense transcripts of XbaI linearized pH3-II RB449. Lane 1, 0.5 µg pH3-II RB449 transcribed with 5 µl (165 µg) of nuclear extract; lane 2, as lane 1, but with 2 µg/ml alpha-amanitin; lane 3, 0.5 µg of pBluescript KS+ transcribed with 5 µl of nuclear extract; lane 4, as lane 1 but stopped immediately after the addition of nuclear extract; lane 5, 0.5 µg of pH3-II RB449 transcribed with 5 µl of nuclear extract from starved cells; lane 6, 0.5 µg of pH3-II RB449 transcribed with 2.5 µl of nuclear extract from starved cells and 2.5 µl of nuclear extract from exponentially growing cells; lane t, RNase protection with 40 µg of wheat germ tRNA; lane T, RNase protection with 40 µg of T. thermophila total RNA. Open and closed arrows indicate in vitro and in vivo transcribed RNA, respectively.


To demonstrate that the nuclear extract supported accurate transcription initiation, transcripts from a number of different templates were analyzed (Fig. 3). Analysis of total RNA isolated from exponentially growing T. thermophila showed that transcription of the H3-II gene was initiated at two sites mapping to position -60 (major transcription start point) and -46 (minor transcription start point) relative to the A in the translation start codon ATG (Fig. 3, lane T). The in vitro transcription with pH3-II RH3.6 and pH3-II RB449 as templates revealed that the same transcription start points were used in vitro (Fig. 3, lanes 1 and 2). Transcripts generated from pH3-II KB351 contain 14 nucleotides of the polylinker region in common with the labeled antisense RNA. Accordingly, transcription of pH3-II KB351 gave rise to protected products that were extended by 14 nucleotides (Fig. 3, lane 3). Addition of alpha-amanitin to 2 µg/ml abolished transcription (Fig. 3, lane 4). Thus, the in vitro transcription system displayed the expected alpha-amanitin sensitivity and utilized transcription start sites identical to those used in vivo. However, it is noteworthy that transcripts initiated at position -60 dominated in vivo, whereas transcripts initiated at position -46 were most abundant in vitro.

Figure 3: In vitro transcription of the histone H3-II gene. The transcription products were detected with T3 RNA polymerase generated antisense transcripts of XbaI linearized pH3-II KB351. Templates: lane 1, 0.5 µg of pH3-II RB449; lane 2, 0.5 µg of pH3-II RH3.6; lane 3, 0.5 µg of pH3-II KB351; lane 4, 0.5 µg of pH3-II KB351 + 2 µg/ml alpha-amanitin; lane 5, 0.5 µg of pH3-II KB351 linearized with XbaI; lane t, RNase protection with 40 µg of wheat germ tRNA; lane T, RNase protection with 40 µg of T. thermophila total RNA. Open and hatched arrows indicate in vitro transcribed RNA with no protected polylinker and in vitro transcribed RNA with a protected polylinker, respectively. Closed arrows indicate in vivo transcribed RNA.


We examined the influence of DNA topology by comparing in vitro transcription of a supercoiled template with a linearized template (Fig. 3, lanes 3 and 5). In this particular experiment, the linearized template was more efficiently transcribed than the supercoiled one, but other experiments showed no difference between supercoiled and linearized templates.

Optimization of in Vitro Transcription

Preliminary experiments had shown that the concentrations of Mg and spermidine had profound effect on the in vitro transcription activity. Hence, the effects of varying the concentration of Mg and spermidine were analyzed (Fig. 4). The maximal rate of transcription was obtained at 0.25 mM Mg and 1.5 mM spermidine. However, the highest proportion of accurately initiated transcripts was obtained at 2.5 mM Mg and 1.5-20 mM spermidine. Consequently, the combination 2.5 mM Mg and 1.5 mM spermidine was selected as a standard for in vitro transcription reactions. It should be noted that the results in Fig. 2and Fig. 3were obtained by using these concentrations of Mg and spermidine.

Figure 4: The effect of magnesium acetate and spermidine on in vitro transcription. Each in vitro transcription reaction contained 0.5 µg of pH3-II RB449 as template, and the transcription products were detected using T3 RNA polymerase generated antisense transcripts of XbaI linearized pH3-II RB449. To the reactions marked 0 mM MgAc, EDTA was added to a concentration of 10 mM to chelate Mg present in the extract. (For these reactions, magnesium acetate was added to a total concentration of 16 mM prior to the addition of DNase.) The lanes containing reactions with 0, 0.25 and 1.0 mM magnesium acetate were exposed for 14 h without an intensifying screen; the lanes containing reactions with 2.5 and 10 mM magnesium acetate were exposed for 50 h with an intensifying screen. Sper. spermidine.


In Vitro Transcription of Other Genes

The performance of the in vitro transcription system was investigated using other templates. Accurately initiated transcripts were found with the histone H4-I gene and the actin gene, but not with ribosomal protein genes (results not shown). The RNA polymerase III activity was analyzed using a 5 S rRNA gene as template. RNase protection of 5 S rRNA present in the extract (Fig. 5) or in total RNA (not shown) gave rise to multiple bands. These multiple bands can be explained by a combination of incomplete trimming by RNase A and by sequence heterogeneity in the 5 S rRNA genes of T. thermophila (Leuhrsen et al., 1980; Kumazaki et al., 1982). Thus, based on the known specificity of RNase A and T1, incomplete trimming of the sequence 5`-AAAAU-3` in the 3`-end of the labeled antisense transcript complementary to the 5`-ATTTT-3` sequence immediately upstream from the transcription start point will result in a ``protected'' antisense RNA that is five nucleotides longer than the full-length protected probe. Furthermore, an A/G heterogeneity in position 4 relative to the 5`-end of the 5 S rRNA, and a C/T heterogeneity in position 114 can be anticipated to give rise to protected fragments that deviate three nucleotides and one nucleotide, respectively, from a full-length protected probe. A C/T heterogeneity in position 2 cannot be detected with RNase A or T1. In vitro transcription of the cloned 5 S rRNA gene followed by RNase protection (Fig. 5, lane 1) gave rise to two products with a size difference of five nucleotides. The 206 nucleotides product marked by the upper asterisk in Fig. 5corresponds in size to that expected for a correctly initiated transcript, whereas the 211 nucleotide species in analogy with the results obtained by analysis of endogenous 5 S rRNA in all probability arose by incomplete trimming of the 3`-end of the antisense probe. Thus, the in vitro transcription system appears to support accurate initiation of transcription of the 5 S rRNA gene.

Figure 5: In vitro transcription of a 5 S rRNA gene. The transcription products were detected with T7 RNA polymerase-generated antisense transcripts of XhoI linearized p5SrRNA SS256. Lane 1, transcription of 0.5 µg of p5SrRNA SS256 (supercoiled); lane 2, control reaction with 0.5 µg of pBluescript KS+. Open and closed arrows indicate in vitro transcribed and endogenous RNA, respectively. Asterisks indicate the positions of correctly trimmed antisense probes that were complementary to the protecting endogenous 5 S rRNA and in vitro transcribed 5 S rRNA, respectively.


Identification of Regulatory Cis-acting Elements

The intergenic regions of the divergently transcribed H3-II and H4-II genes from 29 Tetrahymena species and Glaucoma chattoni have been cloned and aligned (Brunk and Sadler, 1990). In addition, we have performed a thorough analysis of the intergenic region using the Pileup program of the GCG package (Deveraux et al., 1984) and the Signal Scan program (Prestridge, 1991). These analyses revealed the presence of several conserved sequence elements. As conservation may indicate functional importance, the conserved elements were further characterized using the Signal Scan program. Brunk and Sadler noted that the sequences of two conserved regions, positions -160 to -149 and -192 to -203, conformed to the consensus for a CCAAT box. In addition, our analyses revealed that three additional conserved regions exhibit similarity with known cis-acting consensus sequences, a H4TF-1 consensus sequence GATTTC (Dailey et al., 1986) in position -176 to -171 which with one mismatch is repeated in an inverted orientation in position -178 to -183, a HiNF-A (van Wijnen et al., 1987) consensus sequence AGAAATG (one mismatch) in position -108 to -113, and an octamer binding sequence ATTTGCAT (one mismatch) (Sive and Roeder, 1986; Barberis et al., 1987; Staudt et al., 1988) in position -221 to -229.

To analyze the functional importance of the conserved elements, double-stranded oligonucleotides (Fig. 6) corresponding to these regions were prepared. Oligonucleotide 1 encompasses the transcription initiation site, oligonucleotide 2 the HiNF-A homology, oligonucleotide 3 one of the CCAAT boxes, oligonucleotide 4 the H4TF-1 consensus, and oligonucleotide 5 the octamer consensus site.

Electrophoretic mobility shift assays (Staudt et al., 1986) showed that all oligonucleotides bound protein(s) in the extracts prepared from exponentially growing and starved cells, respectively, and furthermore, reciprocal competition experiments demonstrated that the binding was specific for each oligonucleotide (Fig. 7). An unrelated oligonucleotide did not bind any protein in the nuclear extract (results not shown). The complexes formed with nuclear extracts from exponentially growing cells were clearly distinct from those formed with nuclear extracts prepared from starved cells (Fig. 7, and results not shown).

Figure 7: Electrophoretic mobility shift assay of oligonucleotides 1-5 with nuclear extract prepared from exponentially growing cells (A) or from cells starved for 24 h (B). Each individual oligonucleotide was subjected to competition with oligonucleotide 1-5 as indicated. Nuclear extract was preincubated with or without 5 nmol of competitor followed by the addition of 10 fmol of labeled oligonucleotide. Free probe and complexes were separated on native gels as described under ``Materials and Methods.'' C and O, indicate competitor and labeled oligonucleotide, respectively.


UV Cross-linking

Oligonucleotide 3 contains a canonical CCAAT motif. This motif was originally recognized as an important promoter element in several vertebrate genes and binds to a large family of transacting factors (Santoro et al., 1988). Interestingly, members of this family are structurally and functionally related to the yeast Hap2/Hap3 transcriptional regulators (Chodosh et al., 1988). From a phylogenetic point of view, it was therefore of interest to characterize in more detail the proteins which bind to the CCAAT containing oligonucleotide 3. Fig. 8shows that two proteins in the nuclear extract from exponentially growing cells could be specifically cross-linked to oligonucleotide 3. Competition with the non-related oligonucleotide 1 did not prevent cross-linking, whereas competition with unlabeled oligonucleotide 3 completely suppressed cross-linking to the labeled oligonucleotide 3 (lanes 2 and 3). No labeled species could be observed when the UV cross-linked reaction mixture was treated with proteinase K prior to electrophoresis (lane 4). The labeled species migrated as proteins of molecular weight 34,000 and 42,000. The migration of DNA-protein cross-linked molecules in SDS gels closely follows the log molecular weight migration relationship of pure proteins. Assuming that only one strand of the double-stranded oligonucleotide became covalently attached to each of the binding proteins, the masses of the proteins binding to oligonucleotide 3 can be estimated to be 23.5 and 31.5 kDa, respectively.

Figure 8: UV cross-linking of oligonucleotide 3 to proteins in nuclear extract prepared from exponentially growing cells. Lane 1, standard cross-linking reaction; lane 2, cross-linking in the presence of a 500-fold molar excess of oligonucleotide 1; lane 3, cross-linking in the presence of a 500-fold molar excess of oligonucleotide 3; lane 4, cross-linking followed by treatment with proteinase K; lane 5, cross-linking reaction with no nuclear extract. The migration of prestained markers is indicated to the right.


Transcription Competition

To investigate the functionality of the conserved sequence elements, transcription competition experiments were performed (Vaccaro et al., 1990; Hai et al., 1988; Verdier et al., 1990). Fig. 9shows how transcription of the H3-II gene was affected by competition with a 100-fold molar excess of each of the double-stranded oligonucleotides 1-5. Addition of oligonucleotide 1 or 2 resulted in a marked reduction in transcriptional activity, while addition of oligonucleotide 3 or 4 resulted in a minor reduction. Addition of oligonucleotide 5 only reduced transcription marginally. Thus, interference with binding of transacting factors to the two proximal conserved sequence elements resulted in a dramatic decrease in transcriptional activity suggesting that these regions are crucial at least for basal promoter activity. Of interest, oligonucleotide 1 encompasses an AT-rich region similar to those previously suggested to be part of a transcription initiation motif in Tetrahymena (Rosendahl et al., 1991; Hansen et al., 1991).

Figure 9: Oligonucleotide mediated competition of in vitro transcription. Transcription of 0.5 µg of pG78 RH3.6 in the presence of 12 pmol (100-fold molar excess) of oligonucleotide 1-5 as indicated. The transcription products were detected with T3 RNA polymerase generated antisense transcripts of XbaI linearized pH3-II RB449. Lane 1, in vitro transcription in the absence of competitor; lanes 2-6, in vitro transcription in the presence of a 100-fold molar excess of the double-stranded oligonucleotides 1-5 as indicated.


DISCUSSION

In this report we describe the preparation of a nuclear extract from the ciliated protozoan T. thermophila that supports accurate transcription initiation in vitro by RNA polymerase II and III. Previous attempts to achieve in vitro transcription of ciliate class II and III genes have been unsuccessful. The reasons for these failures are probably related to the preparation of the nuclear extract itself as well as to problems concerning the choice of suitable templates for in vitro transcription. Since no functional analyses of ciliate promoters have been performed so far, rational delimitation of a functional promoter has in most cases been impossible. Therefore, we decided to use clones of the histone H3-II gene as model templates for the development of a Tetrahymena based in vitro transcription system. In ciliates the genes encoding histones H3-II and H4-II are clustered and oriented in a head to head fashion with an intergenic region of approximately 345 bp (Brunk and Sadler, 1990). No introns are present in the two genes, and consequently, the intergenic region can be expected to contain the sequences necessary for at least basal transcription. Furthermore, the organization of the two histone genes has been shown to be conserved in a large number of ciliates making it possible to identify conserved sequence elements of possible regulatory importance (Brunk and Sadler, 1990; this report). For preparation of active nuclear extracts, minimization of nuclear leakage during isolation of nuclei was clearly of importance. Several ways of preparing nuclei were investigated, and the most successful combination was found to be disruption of the cells in a Potter-Elverhjem homogenizer in the presence of 4% gum arabic (Gorovsky, 1975), followed by a final purification of nuclei in a buffer containing 18% Ficoll 400 (Lue and Kornberg, 1987). Cell lysis in buffers containing Nonidet P-40 resulted in inactive extracts with very low concentrations of protein. Furthermore, the use of a potassium glutamate and spermidine containing buffer and centrifugation for the preparation of a nuclear extract with high concentration of protein according to the procedure of Kamakaka et al.(1991) were instrumental in obtaining active extracts. Finally, a number of observations has suggested that chloride ions inhibit the transcriptional activity of nuclear extracts (Lue and Kornberg, 1987; Shapiro et al., 1988; Verdier et al., 1990). Consequently, we substituted acetate for chloride, although we did not perform a systematic comparison of the performance of extracts prepared with chloride or acetate.

The transcriptional efficiency of the nuclear extract from T. thermophila was lower than those reported for metazoanderived systems (Shapiro et al., 1988; Kamakaka et al., 1991), but comparable to those reported for extracts prepared from yeast (Lue et al., 1989; Verdier et al., 1990) and Neurospora (Tyler and Giles, 1985). This relatively low transcriptional activity may be characteristic for extracts from lower eukaryotes, but the promoter organization and transcription start site patterns of the genes used as templates for in vitro transcriptions may well add to the low transcriptional efficiency. Thus, the genes from lower eukaryotes which have been used as templates for in vitro transcription initiate transcription from multiple start sites in vivo (Lue and Kornberg, 1987; Lue et al., 1989; Verdier et al., 1990; Tyler and Giles, 1985), and it is a general observation that such genes even in the more efficient extracts from mammalian cells normally are poorly transcribed in vitro (Farnham and Schimke, 1986; Osborne et al., 1987; Kageyama et al., 1988). Most if not all ciliate class II genes also seem to initiate transcription in vivo from multiple transcription start sites (Nielsen et al., 1986; Rosendahl et al., 1991; Hansen et al., 1991). In higher eukaryotes a substantial number of genes that initiate transcription from multiple start sites encode housekeeping proteins (Dynan, 1986). To this class of genes belongs the ribosomal protein genes which have been notoriously difficult to transcribe in vitro (Zahradka and Sells, 1988; Yoganathan et al., 1992; Chung and Perry; 1991). Like other genes encoding housekeeping proteins, these genes lack canonical TATA boxes in the promoter region. Interestingly, mutating an AT-rich region in the promoter of the S16 ribosomal protein gene to a canonical TATA box increased in vitro transcription dramatically (Chung and Perry, 1991). It is also noteworthy that in vitro transcription of other TATA-less mammalian genes from which transcription in vivo is initiated at multiple start sites failed to reflect the in vivo utilization of the individual start sites. Thus, minor in vivo start sites became the most prominent start site in vitro, whereas transcription from major in vivo transcription start sites became barely detectable in vitro (Farnham and Schimke, 1986; Osborne et al., 1987; Kageyama et al., 1988). The same phenomenon was observed by in vitro transcription of the T. thermophila histone H3-II gene, where the most abundant transcript was initiated at position -46, and the less abundant transcript at position -60, whereas the reverse was observed in vivo. Similarly, preferential utilization in vitro of minor in vivo transcription start sites was also observed with yeast extracts (Lue and Kornberg, 1987) and Neurospora extracts (Tyler and Giles, 1985). The reason for this difference in transcription start site utilization remains to be established, but it is obviously not a peculiarity of the Tetrahymena system.

The physiological variation in the rate of transcription of the histone H3-II gene according to the nutritional status of the cells (Bannon et al., 1983) is clearly reflected in the transcriptional activity of extracts prepared from exponentially growing and starved cells, respectively. Thus, extracts from starved cells did not support detectable transcription of the H3-II gene. The results obtained by mixing extract prepared from exponentially growing and starved cells suggested that the extract from starved cells did not contain grossly inhibitory substances. Rather, the lack of detectable transcriptional activity could be attributed to a lack of positively acting factors.

A computer-assisted comparison of the intergenic region between the H3-II and the H4-II genes in 30 different ciliates identified five conserved sequence elements. By electrophoretic mobility shift assays, oligonucleotides corresponding to each of these elements were found specifically to bind proteins in extracts from exponentially growing as well as starved cells. Interestingly, each oligonucleotide bound a different complement of proteins in extracts from exponentially growing cells and starved cells. Oligonucleotide 3 harbors a canonical CCAAT motif. By UV cross-linking, we showed that two proteins in the extract from exponentially growing cells bound to this oligonucleotide. The molecular masses of these proteins were estimated to 23.5 and 31.5 kDa, respectively. In mammals as well as in yeast, CCAAT motifs appear to be recognized by a large family of transacting factors that bind in the form of heterodimers to the target sequences. This pattern appears to be followed in T. thermophila, and furthermore, the molecular masses of the two binding proteins are comparable with those of the Hap2 and Hap3 proteins in yeast for which predicted molecular masses of 16 and 30 kDa, respectively, have been reported (Chodosh et al., 1988). The functional importance of the conserved sequence elements was assessed in transcription competition experiments. These experiments revealed that oligonucleotides 1 and 2, in particular, exerted a profound effect on transcriptional activity of the histone H3-II gene leading to an almost complete abrogation of H3-II transcription implying that factors binding to these two proximal elements are critical for transcription. Oligonucleotides 3 and 4 also significantly decreased transcription, whereas oligonucleotide 5 only affected transcription marginally. Thus, the CCAAT box-binding proteins in T. thermophila appear to be bona fide transacting factors, although less decisive than the factors binding to the two proximal elements.

The performance of the T. thermophila transcription system was investigated using other class II and class III T. thermophila genes as templates. Accurate initiation of transcription was achieved with the histone H4-I gene and the actin gene. However, in analogy with results obtained with other in vitro transcription systems, we were unable to detect specific initiation of transcription from either of four ribosomal protein genes. Using a 5 S rRNA gene as template, we demonstrated that the nuclear extract also supported accurate and efficient transcription of a class III gene. Of interest, the 5 S rRNA gene utilized in this study did not contain a canonical TATA box in the proximal upstream region. In N. crassa (Tyler, 1987), Drosophila (Sharp and Garcia, 1988), and Bombyx mori (Morton and Sprague, 1984), the presence of a TATA box approximately 25 base pairs upstream from the transcription initiation site was shown to be essential for efficient and accurate initiation of 5 S rRNA transcription in vitro. Interestingly, another 5 S rRNA gene cloned from T. thermophila (Pederson et al., 1984) possesses a canonical TATA box at position -30 to -26. Thus, it is possible that a TATA box may be dispensable for 5 S rRNA transcription in the T. thermophila system as it is in Xenopus derived systems, where no conserved sequences upstream from the transcription start site are necessary for accurate initiation (Sakunjo et al., 1980; Morton and Sprague, 1984). Although no pseudogenes have been identified in T. thermophila to date, formally it can not be excluded that the 5 S rRNA gene used in this study is a pseudogene which, in analogy with a human pseudogene lacking upstream promoter elements (Nielsen et al., 1993), can be transcribed in vitro albeit with a low efficiency.

FOOTNOTES

*
This work was supported by grants from the Danish Natural Science Research Council and the Protein Engineering Research Centre under the Danish Biotechnology Research and Development Programme. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: +45-66-15-86-00 (Ext. 2408); Fax: +45-65-93-17-70; kak{at}dou.dk.

(^1)
R. Hummel, unpublished results.

(^2)
The abbreviations used are: bp, base pair(s); DTE, dithioerythritol; rpm, revolutions/min; PIPES, 1,4-piperazinediethanesulfonic acid.

(^3)
N. E. Petersen, L. K. Larsen, H. Nissen, L. G. Jensen, M. Horder, N. Gregersen, and K. Kristiansen, manuscript submitted for publication.

ACKNOWLEDGEMENTS

We thank R. Hallberg for the plasmid pBS-A4.

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