Molecular and Cellular Biochemistry 76:123-131 (1987) © Martinus Nijhoff Publishers, Boston - Printed in the Netherlands

Original Article

Immunological relationships between Artemia RNA polymerases and between RNA polymerases II from different eukaryotic organisms

123

Victor Diaz, Miguel Quintanilla, Jesfis Cruces, Jaime Renart and Jesfis Sebastifin Instituto de Investigaciones Biornddicas del C.S.LC. and Departamento de Bioquimica de la Facultad de Medicina de la U.A.M., 28029-Madrid, Spain

Received 17 December 1986; accepted 26 February 1987

Key words: RNA polymerases, brine shrimp, antibodies, immunoblotting

Summary

Rabbit antibodies against Artemia RNA polymerase II have been raised and utilized to study the immunolog- ical relationships between the subunits from RNA polymerases I, II and III from this organism and RNA polymerase II from other eukaryotes. We describe here for the first time the subunit structure of Artemia RNA polymerases I and III. These enzymes have 9 and 13 subunitsrespectively. The anti-RNA polymerase II antibodies recognize two subunits of 19.4 and 18 kDa common to the three enzymes, and another subunit of 25.6 kDa common to RNA polymerases II and III. The antibodies against Artemia RNA polymerase II also react with the subunits of high molecular weight and with subunits of around 25 and 33 kDa of RNA polymerase II from other eukaryotes (Drosophila melanogaster, Chironomus thummi, triticum (wheat) and Rattus (rat)). This interspecies relatedness is a common feature of eukaryotic RNA polymerases.

Abbreviations: RNAp - RNA polymerase, DPT - diazophenylthioether, SDS - sodium dodecylsulfate.

Introduction

Eukaryotic RNAp are complex multisubunit en- zymes with specific transcriptional functions [1- 3]. Each of the three classes recognizes different types of promoter regions within the genome and requires different types of transcriptional factors involved in the specificity of gene expression [4]. In spite of this specificity, all RNAp must carry out a series of common tasks, including DNA and nucleoside triphosphate recognition, catalysis of the phosphodiester bond and RNA chain elonga- tion. The study of the relation between structure and function of RNAp subunits has been under-

taken by two main approaches: 1) the determina- tion of subunits immunologically related between different RNAp that could account for important conserved functions, and 2) the molecular cloning of the genes coding for each subunit.

Using immunological methods, cross-reacting subunits have been found between different RNAp from a given organism and between the same RNAp from different organisms (see for instance, ref. [5-7]), indicating the existence of highly con- served subunits, which are supossed to accomplish similar functions.

The genes for the large subunit of RNAp II from Drosophila melanogaster, yeast, mouse and hu-

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mans [8-12], as well as for some small subunits of yeast RNAp [13], have been isolated. These studies further support the idea that some subunits are highly conserved, since extensive homology has been found between yeast and D. melanogaster large subunits of RNAp II and E. coli RNA poly- merase /3' subunit [14, 15]. Moreover, the D. melanogaster gene is able to complement c~- amanintin sensitivity in mammalian cells [16].

Extensive characterization of RNAp from the crustacean Arternia has been carried out in our laboratory in the last years [17-19]. Different regu- lation of RNAp I, II and III expression during de- velopment of Artemia, makes it a suitable system to study many aspects of the regulation of RNAp gene expression as well as RNAp activity. These studies would require the isolation of the genes for the different subunits of RNAp.

In this paper we describe the purification and subunit structure of RNAp I and III from Artemia. In addition, we have obtained polyclonal antibod- ies againts RNAp II and have used them to define immunologically related subunits between the three RNAp from Artemia as well as between RNAp II from Artemia, D. melanogaster, Chironomus thummi, wheat and rat. The availability of these antibodies is an important step towards the isola- tion of RNAp II genes.

Experimental procedures

Organism and growth conditions

Artemia cysts were obtained from San Francisco Bay Brand Inc. Treatment of cysts and growth con- ditions of embryos and larvae were as described elsewhere [18].

RNAp assay

The RNAp assay was as reported by Osuna and Se- basti~in [18]. RNAp I and III were assayed using na- tive calf thymus DNA and RNAp II using heat- denatured DNA. One unit is defined as the amount of enzyme that catalyzes the incorporation of 1

nmol of UMP into acid-precipitable material in 10 min under the assay conditions.

Solubilization and separation of Artemia RNAp I and III

Newly hatched nauplii (45 g wet weight for RNAp I and 80 g for RNAp III) were homogenized with 3 vol of buffer B (50 mM Tris-C1, pH 7.5, 5 mM 2-mercaptoethanol, 0.2 mM EDTA, 20% glycerol, 0.1 mg/ml Soybean trypsin inhibitor). The homogenate was centrifuged at 30000xg for 20 rain. The pellet, containing the nuclear fraction, was solubilized by resuspension and homogeniza- tion in buffer B plus 0.35 M (NH4)2SO4. This homogenate was centrifuged at 105000×g for 2 hr, being the supernatant the soluble extract. The soluble extract was adjusted to 75 mM (NH4)2SO 4 by the addition of buffer B and incubated at 4 °C for 15 min, to precipitate DNA. It was then cen- trifuged at 30000 x g for 15 min, and the superna- tant passed through a DEAE-Sephadex column (130 ml) equilibrated in the same buffer, After washing, the column was eluted with a linear gra- dient (500 ml) from 0.075 to 0.6 M (NH4)2SO 4 in buffer B. The first peak, eluting at 0.15 M (NH4)2SO 4 and resistant to ~-amanitin at 1 t~g/ml [see ref. 18], contained RNAp I. The second peak, eluting at 0.24 M (NH4)2SO 4 and fully sensitive to o~-amanitin, was RNAp II. The third peak, also resistant to o~-amanitin and eluting at 0.3 M (NH4)2SO4, included RNAp III.

Purification of RNAp I

Second DEAE-Sephadex chromatography. Pooled RNAp I activity was diluted with 1 vol of buffer B and applied to a second DEAE-Sephadex column (40 ml) in the same conditions as described for the first DEAE-Sephadex chromatography, except that a 120 ml gradient was used. RNAp I activity eluted at 0.15 M (NH4)2SO 4.

Phosphocellulose chromatography. The pooled activity of the previous step was diluted with 1 vol of buffer B and applied to a phosphocellulose

column (25 ml) equilibrated in 75 mM (NH4)2SO 4 in buffer B. After washing, a gradient (90 ml) was applied between 0.075 M to 0.6 M (NH4)2SO 4 in buffer B. RNAp I activity eluted at 0.28 M

(NH4)2SO4. DNA-cellulose chromatography. The pooled ac-

tivity from the previous step was diluted with buff- er B to a final (NH4)2SO4 concentration of 50 mM, and applied to a DNA-cellulose column (5 ml) equilibrated with 50 mM (NH4)2SO 4 in buffer B with 8% instead of 20% glycerol. The column was washed with the above buffer and then eluted with 10 ml of 0.5 ml (NH4)2SO 4 in buffer B containing 8% glycerol.

Glycerol gradient centrifugation. The activity of the DNA-cellulose step (aprox. in 3 ml) was applied to the top of a glycerol gradient (8-25%) contain- ing 50 mM Tris-C1, pH 7.5, 5 mM 2-mercapto- ethanol, 0.2 mM EDTA, 0.5 M (NH4)2SO 4. The gra- dients were run in a SW27 rotor at 26000 rpm for 45 hr at 4°C.

Purification of RNAp III

Second DEAE-cellulose chromatography. The pooled RNAp III activity was diluted with 1 vol of buffer B and applied to a second DEAE-Se- phadex column (40 ml) equilibrated with 0.15 M (NH4)2SO 4 in buffer B. After washing, a gradient (160 ml) from 0.15 M to 0.6 M (NH4)2SO 4 in buff- er B was applied. RNAp III activity eluted at 0.3 M

(NH4)2SO 4. CM-Sephadex chromatography. The pooled ac-

tivity from the second DEAE-Sephadex chro- matography was diluted with 5 vol of buffer B and applied to a CM-Sephadex column (40 ml) equili- brated with 50 mM (NH4)2SO 4. After washing, a gradient (160 ml) from 0.05 M to 0.6 M (NH4)2SO 4 in buffer B was applied. RNAp III activity eluted at 0.15 M (NH4)2SO 4.

Phosphocellulose chromatography. The pooled activity from the previous step was diluted with 0.5 vol of buffer B and applied to a phosphocellulose column (30 ml) equilibrated with 0.1 M (NH4)2SO 4 in buffer B. After washing, a gradient (120 ml) from 0.1 M to 0.6 M (NH4)2SO 4 in buffer B was

125

applied. RNAp III activity eluted at 0.25 M

(NH4)2SO4. DNA-cellulose chromatography and glycerol gra-

dient centrifugation were carried out as described for RNAp I.

Purification of RNAp H

RNAp II was prepared from Drosophila melanogaster and Chironomus thummi according to Kramer and Bautz [20], from wheat germ (Triti- cum) by the method of Hodo and Blatti [21] and from rat liver (Rattus) as described by Weaver et al. [22].

Preparation of anti-Artemia RNAp H antisera

Antibodies against RNAp II were raised in white rabbits (New Zealand) by intradermal injection of 200 ~g of protein (purified from Artemia larvae as described by Cruces et al. [19]), in complete Freund's adjuvant, at several sites in the back and intramuscularly in the posterior legs, 3 times at 1-week intervals. Rabbits were boosted 5 and 7 weeks later in the same way with 300-400 ~g of protein in incomplete Freund's adjuvant. Rabbits were bled weekly after the last immunization.

When needed, the IgG fraction was purified by affinity chromatography on protein A-Sepharose [231.

Other methods

Gel electrophoresis was carried out according to Laemmli [24], except for electrotransfer, for which the Neville system was used [25]. DPT-paper was prepared according to Seed [26]. Electrotransfer of proteins to DPT-paper and incubation with antisera was performed according to Reiser and Wardaie [27]. Spot-immunodetection of RNAp was as described by Huet et al. [5]. Protein was deter- mined by a modification of the method of Brad- ford [28].

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Results

Purification of RNA polymerases I and III

Table 1 shows the summary of the purification of RNAp I and III. The yield for RNAp I was 20% while RNAp III was purified with 7% yield. The actual extent of purification could not be calculat- ed since it was impossible to discriminate between RNAp I and III in the soluble extracts. Artemia RNAp III is only 25% inhibited by c~-amanitin at 500/zg/ml, and at this concentration of the toxin, RNAp I is inhibited 10% (18). Purified RNAp III was also insensitive to high a-amanitin concentra- tions, a property in common with RNAp III from other arthropods like D. melanogaster [29] and Bombix mori [30]. The specific activity for RNAp III after the glycerol gradient step could not be cal- culated, due to the extremely low protein concentra-

Table L Purification of RNA polymerases I and III from Arte-

mia larvae.

Fraction Protein Total Specific Yield (mg) activity activity (%)

(units) (u/mg)

A. RNA polymerase I. 1 st DEAE-Sephadex 33 110 3.3 100 2 nd DEAE-Sephadex 15 93.5 6.2 85 Phosphocellulose 4.6 77.2 17 70 DNA-cellulose 0.8 23.6 30 21.5 Glycerol gradient 0.21 21.4 102 19.5

B. RNA polymerase III. 1 st DEAE-Sephadex 16.5 23 1,4 100 2 nd DEAE-Sephadex 3.5 22.5 6.4 97.5 CM-Sephadex 0.44 15.7 35.2 68 Phosphocellulose 0.28 10.7 38,2 46.3 DNA-cellulose 0.12 4.1 34,2 17.8 Glycerol gradient N.D. a 1.6 N.D. 7

a N.D.: not determined (see text)

Fig. 1. Subunit composition of Artemia RNAp I and III. Aliquots of glycerol gradient fractions from RNAp I and III purifications were precipitated with 5O7o triehloroacetic acid, washed with ethanol:ether (1:1) and resuspended in Laemmli sample buffer [24]. Samples were electrophoresed on a 8-27% polyacrylamide gradient gel. Lanes 1-3, RNAp I (enzymatic activity was present in lanes 1-2). Lanes 4 -6 , RNAp IH (enzymatic activity was present in lane 5). Molecular weight markers (in kDa) are shown in the middle of the figure; arrows show the size of the RNAp subunits (in kDa).

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tion in the purified enzyme preparation. Although neither RNAp was homogeneous, it

was possible to define their subunit structure, based on the comigration of RNAp activity and the different polypeptides in glycerol gradients (Fig. 1). Table 2 shows the calculated molecular weights of

each subunit. RNAp I contained 9 subunits while RNAp III had 13. The three RNAp have two subunits of the same electrophoretic mobility (19.4 and 18.5 kDa); RNAp I and III have a 41 kDa subunit, whereas RNAp II and III have subunits of 25.6 and 14.7 kDa.

Fig. 2. Reactivity of anti-Artemia RNAp II antibodies with Artemia RNAp. A: Immunodo t assay. 1 /zg of each RNAp was spotted

on nitrocellulose membranes and treated as described by Huet et al. [5], using immune and control IgGs, as stated. B: Inhibition of the activity. Aliquots of the three RNAp (0.018 units) were incubated with increasing amounts of IgG at 30 °C for 20 min in the buffer used for the assay. The reaction was started by the addition of a cocktail containing nucleoside tr iphosphates and DNA. Assay was as described in ref. 18.

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Antibodies against Artemia RNAp H

The specificity of the antibodies against RNAp II was checked in two ways. First, using the im- munodot technique (5); as shown in Fig. 2A, the

ant i -RNAp II IgG gives positive signal with the

three RNAp, whereas control serum does not. Sec- ond, we studied the effect of preincubation with

antibodies on the activity of the enzymes. Fig. 2B shows that RNAp II is strongly inhibited, whereas

RNAp I and I I I are inhibited to a much lesser ex- tent, much more IgG being needed to give this inhi- bition. As expected, control IgG did not inhibit the

activity of any RNAp. These experiments demonstrate that the antibod-

ies obtained can be a useful tool to study RNAp,

as they are specific for RNAp II but they also recognize the other two RNAp.

Recognition of Artemia RNAp subunits by the anti-RNAp H antibodies

Fig. 3 shows an immunoblott ing analysis of RNAp II. All except the smallest subunit (11.5 kDa) react-

ed with the anti serum. I f the same experiment is carried out with RNAp

I and I I I (Fig. 4), the antibodies recognize a small set of polypeptides: the common subunits of 19.4 and 18.5 kDa of the three RNAp, suggesting that they are the same polypeptide in the three enzymes. The same is true for subunit of 25.6 kDa, common to RNAp II and III. By contrast, the subunit of 14.7 kDa of RNAp III did not react, although the

antibodies recognize a subunit of the same elec-

trophoretic mobility in RNAp II.

Fig. 3. Specifity of anti-RNAp II antisera. Larval RNAp II (10 tzg) from the glycerol gradient step (see ref. 19) was electrophoresed using the Neville system in a 8-27% polyacrylamide gradient gel, and immunodetected as described in Materials and Methods. Inset: immunodetection of the two high molecular weight subunits of RNAp II. Purified enzyme (0.05 t~g) was electrophoresed as above in a 6-10% polyacrylamide gel.

129

Fig. 4. Specificity of anti-RNAp II antibodies towards the iso- lated subunit of Artemia RNAp. Fifteen/xg of each RNAp were electrophoresed in a 15°70 polyacrylamide gel, using the Neville system. Transfer to DPT-paper and immunodetection were as described in Methods. A, RNAp I; B, RNAp II; C, RNAp III. Numbers refer to the size of the cross-reacting subunits (in kDa).

Immunodetection of RNAp H from other eukaryotes

Ingles [31] demonstrated for the first time that anti- bodies against RNAp react with the same enzymes from other species. These results have been con- firmed for other systems in several laboratories

[1, 31.

Table2. Molecular weight of the subunits of Artemia RNA

polymerases

I II III

205

175 (172) 145 140

41 35

27.5 32.5

170

126 60 51 49 41

25.6 25.6 22

19.4 19.4 19.4 18.5 18.5 18.5

16.8

14.7 14.7 14

13.5

12.7

11.5

15

12.9

12.5 11.8

Numbers represent size in kDa of the corresponding subunits. RNAp II data are taken from ref. 19.

We have checked the reactivity of the antibodies against Artemia RNAp II towards other RNAp II. The results are shown in Fig. 5. These antibodies react with RNAp II from wheat germ, D. melanogaster, C. thummi, rat liver, and also with D. discoideum [32]. In all these systems, the high molecular weight subunits always react. In the small molecular weight subunit range, the antibod- ies cross react with different polypeptides, accord- ing to the system, although some generalizations can be drawn: a polypeptide of around 25 kDa reacts in all the systems studied so far, whereas a polypeptide of around 33 kDa reacts in all except wheat germ and D. discoideum [32].

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Fig. 5. Immunodetection of different RNAp II with anti Artemia RNAp II antibodies. Fifteen t~g of partially purified RNAp II from wheat germ (A), D. melanogaster (B), rat liver (C) and C tummi (D), were treated essentially as described for Fig. 4. Each polymerase was electrophoresed in a different gel along with Artemia RNAp II as control. Numbers refer to the size of the cross-reacting subunits (in kDa).

Discussion

We have partially purified RNAp I and III from Artemia . Difficulties in the total purification arises

from the presence of yolk granules in the nuclear

fraction of A r t e m i a extracts. These granules are

composed mainly of lipovitellin. In the naupliar

stage, lipovitellin is degraded giving rise to several

smaller polypeptides [33]. Nevertheless, we have as- signed the subunit structure of the enzymes based on the distribution of the different polypeptides in the glycerol gradient fractions relative to the RNAp activity (Fig. 1). A r t e m i a RNAp I have subunits with similar molecular weights to those from other systems [1, 3]. A r t e m i a RNAp III has a 60 kDa subunit which seems to be common to arthropods" in D. melanogas ter this subunit is of 62 kDa [20]. In

other systems there is a subunit in the 7 0 - 9 0 kDA range [3]. The two bands seen in Fig. 1 in the 70 kDa

region are present all over the gradient and are likely to be an artifact due to sulfhydryl reagents [34]. A

doublet of 49-51 kDa can also be seen in Fig. 1. A

subunit with variable molecular weight between 50

and 58 kDa has also been found in other systems [1]. The availability of polyclonal antibodies against

RNAp II has allowed us to detect cross-reacting subunits between A r t e m i a RNAp. The two detected subunits of 19.4 and 18.5 kDa, common to the three enzymes, present relative mobilities similar to those described in other systems. The same is true for the 25.6 kDa subunit common to RNAp II and III.

Purified RNAp II from four different species cross-reacted with ant i -Ar temia RNAp II antibod-

ies. A polypeptide of molecular weight around 25 kDa could be detected in all RNAp II tested. At least one of the two high molecular weight subunits from each RNAp II, also reacts with the antibod- ies. Although the 15% polyacrylamide gel used do not permit to distinguish which one, we are tempt- ed to assign this reactivity to the largest one, which has been shown to be the target for a-amanitin [35, 36], and presents sequence homology between different species [14, 15].

A further step in the study of the structural rela- tionships between subunits from the different RNAp in Artemia, will require the molecular clon- ing of their genes. In this sense, the antibodies we describe in this paper seems to be a good tool for the isolation of these genes either by screening of expression genotheques or polysome immuno- selection techniques.

Acknowledgements

We thank Leandro Sastre for helpfull discussions and Elvira Domlnguez for technical help. This in- vestigation was supported by grants from Comisi6n Asesora para la Investigaci6n Cientifica y T6cnica. M.Q. was a postodoctoral fellow of the Consejo Su- perior de Investigaciones Cientlficas, and V.D. a predoctoral fellow funded by the Fondo de Inves- tigaciones Sanitarias de la Seguridad Social.

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Address for offprints: Jaime Renart, lnstituto de Investiga- ciones Biom6dicas del C.S.I.C., Facultad de Medicina de la U.A.M., Arzobispo Morcillo 4, 28029-Madrid, Spain