SV40 DNA injected into <u>Xenopus</u> oocyte nuclei is transcribed by RNA polymerase B

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SV40 DNA injected into Xenopus oocyte nuclei is transcribed by RNA polymerase B

RUNGGER, Duri, HUBER, Jean-Pierre, TURLER, Hans

Abstract

SV40 DNA I, injected into Xenopus oocyte nuclei is transcribed. The SV40-specific RNA molecules migrate on sucrose gradients as do viral RNAs formed in infected green monkey cells but a variable proportion of RNA sequences complementary to SV40 DNA is also found in the light region of the gradients. All SV40-specific RNA species seem to be synthesized by RNA polymerase B as their synthesis is completely sensitive to low concentrations (0.1 μg/ml) of α-amanitin. Concomittantly, the formation of SV40-specific proteins (tumor antigens) is inhibited by injecting α-amanitin together with the SV40 DNA.

RUNGGER, Duri, HUBER, Jean-Pierre, TURLER, Hans. SV40 DNA injected into Xenopus oocyte nuclei is transcribed by RNA polymerase B. Cell Biology International Reports , 1979, vol. 3, no. 2, p. 183-188

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, Cell Biology International Reports, Vol. 3, No. 2, 1979

SV40 DNA INJECTED INTO XENOPUS OOCYTE NUCLEI IS TRANSCRIBED BY RNA POLYMERASE B

Duri Rungger*, Jean-Pierre Huber* and Hans Ti.irler+

*

Biologie animale, Universite de Geneve, 154, route de Malagnou CH-1224 Chene-Bougeries, Switzerland

183

+ Biologie moleculaire, Universite de Geneve, 30, quai E. Ansermet CH-1211 Geneve, Switzerland

ABSTRACT. SV40 DNA I, injected into Xenopus oocyte nuclei is trans- cribed. The SV40~specific RNA molecules migrate on sucrose gradients as do viral RNAs formed in infected green monkey cells but a vari-·

able proportion of RNA sequences complementary to SV40 DNA is also found in the light region of the gradients. All SV40-specific RNA species seem to be synthesized by RNA polymerase B as their synthe- sis is completely sensitive to low concentrations ( 0.1 µg/ml ) of a-amanitin. Concomittantly, the formation of SV40-specific proteins

(tumor antigens) is inhibited by injecting a-amanitin together with the SV40 DNA.

INTRODUCTION. Various DNA templates have been shown to be efficient- ly transcribed in Xenopus oocytes (Brown and Gurdon, 1977; Mertz and Gurdon, 1977; Kressmann et al., 1977, 1978). After injection of SS or tRNA genes, a predominant amount of the RNA synthesized on these templates was of correct size but some larger and smaller RNAs were also formed (Brown and Gurdon, 1977; Kressmann et al., 1977, 1978).

Structural genes, normally transcribed by RNA polymerase B, were transcribed faithfully enough to allow successive processing, inclu- ding splicing, and translation into normally sized polypetides such as Drosophila histones and SV40-specif ic proteins (DeRobertis and Mertz, 1977; Rungger and Ti.irler, 1978). The RNA synthesized on the injected SV40 DNA did show a clear band corresponding to SV40 19S mRNA but substantial amounts of smaller and larger RNA species were found hybridizing to SV40 DNA (Laskey et al., 1977). Similar studies on transcription of sea urchin histone genes have shown that, al- though some histone mRNA-like molecules appeared, the bulk of the newly synthesized RNA was distributed along the whole length of a gel (Kressmann et al., 1977).

0309-1651 /79/020183-06/$02 .00/0 ©1979 Academic Press Inc. (London) Ltd.

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No published data are available as yet on the type of RNA polyme- rase involved in transcription of injected templates. The results of the present study show that both, correctly sized and small RNA spe- cies, transcribe d in the oocytes from injected SV40 DNA, are synthe- sized by RNA polymerase B.

EXPERIMENTAL PROCEDURES. The injection technique includes centrifu- gation of the oocytes to facilitate nuclear injec t ion (Kressmann et al., 1977) and has been described in det ail el s ewhere (Rungger and Turler, 1978).

The SV40 DNA I was purified from SV40 i nfected g r een monkey CV.l cells (Flow Laboratories, Irvine, Scotland) according to Germond et al. (1974) and resuspended in injection buffer at a concentration of 0.5 µg/µl. Immediately before injection, 50 µCi of vacuum- dried a32P GTP were dissolved in 5 µl of this DNA solution. In the experimental series using a -amanitin, 15 µl of this mixture were divided into 5µ1 aliquots either used directly for injection, or transferred to tubes containing 2.5 ng, 25 ng respectively of vacuum-dried a-amani tin

(Boehringer, Ingelheirn, BRD). Injection of 20 nl thus resulted in identical final concentrations of 10 ng of DNA and 0.2 µCi a32P-GTP per oocyte nucleus, where as the concentration of a-amaniLin was ei- ther 0.1 µg/ml of nuclear volume (assumed to be 100 nl) or 0.1 µg/ml of total cell volume (1 µl).

After incubation for 18 hours, the oocytes were selected, washed in distilled water and homogenized in O.lM sodium acetate buffer pH5 containing 0.5 % sodium lauryl sulphate (SDS) and 4 µg/ml of polyvi- nyl sulphate. The RNA was purified by several extractions with cold phenol and finally chloroform (Rungger and Crippa, 1977). The alco- hol- precipitated and resuspended RNA was then fractionated on 15% - 30% sucrose gradients (16 hours, 24.000v~ SW4lrotor). The gradients were collected, aliquots of each fraction were TCA precipitated and counted, and the rest of each fraction was precipitated in alcohol and resuspended in 2xSSC for hybridization.

Boiled SV40 DNA was loaded onto nitrocellulose filters and the charged filter surface was mechanically cut into 3.5 mm squares, each loaded with 1 µg of DNA. The filters were used for hybridiza- tion (2xSSC, 650C, 18 hours) in microcap tubes containing 100µ1 of RNA solution. The RNAase-treated and washed filters were counted in a liquid scintillation counter.

For the analysis of SV40-specific proteins formed, subsets of the oocytes used to monitor RNA synthesis, were incubated for another 24 hours (18 - 42 hours after injection) in 300 µCi/ml of 35S methioni- ne. Processing by homogenization, immunoprecipitation with antibo- dies directed against SV40 tumor antigens (Ahmad-Zadeh et al., 1976), isolation of the immune-complexes on protein A sepharose and their analysis on SOS gels were done as described in detail previously

(Rungger and Turler, 1978).

•

Cell Biology International Reports, Vol. 3, No. 2, 1979 185 RESULTS AND DISCUSSION. The results presented in Fig. l show that oocytes injected with SV40 DNA synthesize RNA sequences complemen- tary to the injected template, whereas RNA isolated from oocytes, in- jected with GTP only, does not hybridize to SV40 DNA above the back- ground levels. The amount of SV40-specific RNA relative to total RNA synthesis was 0.9%. In the experiment shown in Fig.2 it was 5.4%.

This apparent variability is probably due to variation in the pre- dominant rRNA synthesis in the different batches of oocytes.

In both experimental series, the RNA synthesized on the injected SV40 DNA includes molecules of different sizes. As many a s 48% (Fig.

la) or 65 % (Fig. 2a) of the RNA molecules complementary to SV40 DNA sediment between 14S and 36S with distinct peaks at about 16S, 19S and 26S. These values correspond to the RNA species formed during early and late SV40 tra nscription in infected cells (for review s ee

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Fig.l. Size distribution of SV40-specific RNA sequences s ynthesized in 63 oocytes injected with SV40 DNA (~), a nd control RNA isolated from 54 oocytes injected with GTP (]?_). The position of SS, 18S and 28S RNA is indicated in~· Symbols: e-e total radioactivity incorpo- rated into RNA (scale on left ordinate) !--! RNAase resi s tant counts hybridized to SV40 DNA (scale on right ordinate) or ~-- ~ to blanc filters. The blancs are not shown in .e_ where they completely overlap with the SV40-filters.

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Fig.2. Inhibition of the synthesis of SV40- specific RNA sequences(~)

by a - amanitin (.e_, £._), in batches of 16 oocytes injected with either SV40 DNA (~), with SV40 DNA and 0. 1 µg of a-amanitin per ml of nucle- ar volume (£), or with SV40 DNA and 0.1 µg of a-amanitin per ml of total cell volume (£._).The symbols are explained in the legend to Fig.l. All blanc filters yavt less than Bcpm and are not shown.

~: autoradiograph of a gel loaded with aliquots of the immunopreci- pitable proteins from subsets (l3 each) of the oocyte-batches analy- zed for RNA ( in~, .e_, and £._).Slot 1: normally sized SV40 tumor antigens

(t,T) synthesized in the absence of a-amanitin (for RNA patterncf.~)

Slots 2 and 3: inhibition of SV40-specific proteins by low concentre:r tions of a-amanitin (cf. RNA pattern in b and c)

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Cell Biology International Reports, Vol. 3, No. 2, 1979

₁₈₇ Acheson, 1976). The 26S RNA is thought to correspond to a complete transcript of the SV40 genome. The remaining 35%, 52% respectively of the SV40-specific RNA sequences formed in the oocytesareshorter

(4S - lOS) than any known SV40 RNA in vivo.

The translational system of the oocyte seems to select for RNA molecules of correct size as the virus-specific proteins synthesized are all of normal size. There are no partial polypeptides detectable

(Fig 2d, slot 1). In this study we only monitored the formation of tumor antigens, but we have shown previously that, under the same conditions, all SV40-specific proteins are made (Rungger and Tiirler, 1978) .

The addition of a-amanitin to a final concentration of O.lµg/ml completely blocks the synthesis of all SV40-specific RNAs, including the small fragments (Fig. 2b and c). Concomittantly, the synthesis of both tumor antigens is suppressed (Fig. 2d, slots 2 and 3). Even though the predominant RNA polymerase C of the oocyte is able to use native DNA templates in vitro (Long et al., 1976), it seems not to be active on the injected SV40 DNA. Our results show that, in the oocyte, SV40 DNA is selectively read by RNA polymerase B as this is known at least for late transcription in vivo.

However, the data presented in this study do not proof yet that initiation and termination of transcription are specific on injected structural genes. Electron microscopic observations on spread trans- criptional complexes of injected Xenopus and Dytiscus rDNA (Trende- lenburg and Gurdon, 1978; Trendelenburg et al., 1978)have shown normal matrix units, indicating correct initiation, to occur on a few rDNA circles. On the other hand, short and irregularly arranged lateral fibrils and giant transcriptional complexes without appa- rent polarity were also observed. Injected sea urchin histone genes are selectively transcribed by RNA polymerase B but there is little specificity in either size or strand selectivity (Probst, Kressmann and Birnstiel, personal communication).

Different interpretations of the present results must thus be considered. The normally sized RNA species might represent the pro- duct of precise transcription, eventually followed by cleavage and splicing. It is however possible as well that they are formed by cleavage of aspecifically transcribed larger molecules, which seem to occur also in vivo (see Acheson, 1976). It is not clear either, whether the small RNA sequences observed are breakdown products of originally correctly sized molecules, or whether they represent short random transcripts. Further experiments are presently carried out to test whether initiation of transcription is specific enough in the oocyte system to allow a rough localization of initiation si- tes by injecting different restriction fragments of a given gene and by measuring their template activity.

ACKNOWLEDGEMENTS. We thank Consuelo Salomon for excellent technical assistance and O. Jenny for the graphs. This work was supported by Swiss National Foundation grants 3.300.0.78 and 3.128.0.77.

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2, 1979 Acheson,N.H. (1976).Transcription during productive infection with

polyoma virus and simian virus 40. Cell 8,1-12.

Ahmad-Zadeh,C.,Allet,B.,Greenblatt,J. and Weil,R. (1976).Two forms of simian-virus-40-specific T-antigen in abortive and lytic infection.

Proceedings of the National Academy of Sciences U. S .A. 73, 1097-1101.

Brown,D.D. and Gurdon,J.B. (1977).High-fidelity transcription of 5S DNAinjected into Xenopus oocytes. Proceedings of the National Academy of Sciences U.S.A.74,2064-2068.

DeRobertis,E.M. and Mertz,J.E. (1977).Coupled transcriptionand trans- lation of DNA injected into Xenopus oocytes. Cell 12,175-182.

Germond,J. E., Vogt, V. M. and Hirt, B. (1974). Characterization of the sin- gle-strand-specific nuclease S1activityon double-stranded super- coiled polyoma DNA. European Journal of Biochemistry 43, 591- 600.

Kressman,A.,Clarkson,S.G.,Pirotta,V. and Birnstiel,M.L. (1978) .Trans- cription of cloned tRNA gene fragments and subfragments injected into the oocyte nucleus of Xenopus laevis. Proceedings of the National Academy of Sciences U.S.A.75 , 1176-1180.

Kressmann,A.,Clarkson,S.G.,Telford,J.L. and Birnstiel,M.L. (1977).

Transcription of Xenopus tDNAmeth and sea urchin histone DNA in- jected into the Xenopus oocyte nucleus. Cold Spring Harbour Sym- posia on quantitative Biology 42,1077-1082.

Laskey,R.A. ,Honda,B.M. ,Mills,A.D. ,Morris,N.R. ,Wyllie,A.H. ,Mertz,J.E., DeRobertis,E.M. and Gurdon,J.B. (1977).Chromatin assembly and transcription in eggs and oocytes of Xenopus laevis. Cold Spring 1

Harbour Symposia on quantitative Biology 42,171-178.

Long,E. ,Dina, D. and Crippa,M. (1976) .DNA-dependent RNApolymerase C from Xenopus laevis ovaries. European Journal of Biochemistry 66, 269- 275.

Mertz,J.E.and Gurdon,J.B. (1977) .Purified DNAsare transcribed after injection into Xenopus oocytes. Proceedings of the National Aca- demy of . Sciences U.S.A.75,1176-1180.

Rungger, D. and Crippa , M. (1977). In vivo transcription of the spacer se- quences of rDNA in Xenopus. Experimental Cell Research 107, 227-237.

Rungger,D. and Tiirler,H. (1978) .DNAs of simian virus 40 and polyoma direct the synthesis of viral tumor antigens and capsid proteins in Xenopus oocytes. Proceedings of the National Academy of Scien- ces U.S.A.75, in press.

Trendel enburg,M.F. and Gurdon,J.B. (1978).Transcription of cloned Xe- nopus ribosomal genes visualised after injection into oocyte nu- clei. Nature 276,292-294.

Trendelenburg,M.F. ,Zentgraf,H. ,Franke,W.W. and Gurdon,J.B. (1978) Transcriptional patterns of amplified Dytiscus genes coding for ribosomal RNA after injection into Xenopus oocyte nuclei. Procee- dings of the National Academy of Sciences U.S.A.75,3791-3795.

Received: 19th December 1978 Accerted: 2nd January 1979

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