Physical state, expression and regulation of two glucocorticoid-controlled genes on bovine papilloma virus vectors

J. Mol. Biol. (1986) 187, 557-568

Physical State, Expression and Regulation of Two Glucocorticoid-controlled Genes on Bovine Papilloma Virus Vectors Patrick Matthiast, Uta Boeger, Ulrich Danesch Giinther Schiitz and Hans-Ulrich Bernard Institute of Cell and Tumor Biology German Cancer Research Center Im Neuenheimer Feld 280, D-6900 Heidelberg, (Received 7 August

1985, and in revised form

F.R.G.

15 October 1985)

We have studied the extrachromosomal maintenance and the transcription regulation of two glucocorticoid-inducible genes on bovine papilloma virus (BPV) vectors in cl27 mouse fibroblasts. These genetic elements were the rat tryptophan oxygenase (TOase) gene promoter, which is active in viva only in hepatocytes, and the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR). From both genes, fusions of the 5’-flanking region of the transcription unit to the bacterial gene for chloramphenicol acetyltransferase (CATase) were constructed. These fusion genes were inserted either into pCGBPV9, a BPV a vector containing “stabilizing” vector encoding G418 resistance, into pBPV-BVl, segments of the human /I-globin gene, or into a BPV construct, whose bacterial plasmid sequences could be removed before transfection. Five constructs of the two latter groups, selectable in cl27 cells only as foci, were normally maintained in the extrachromosomal state. In contrast, three out of five constructs based on pCGBPV9 and selectable for resistance against G418 were maintained in a high molecular weight form, most probably of intrachromosomal concatemeric nature, while the remaining two G418-resistant construct,s appeared alternatively in this or the extrachromosomal monomeric form. In contrast to its absence of expression in fibroblasts in vivo, the TOase gene element present on BPV vectors was found to be active in fibroblasts in these transfection experiments. As judged by CATase activities and for TOase also by mapping of the transcription start sites, transcription of both genes was under hormonal regulation. All BPV vectors proved to be useful tools in the study of these regulated genes, and in only one out of ten constructs was regulation atypical, possibly due to effects from flanking vector sequences.

1. Introduction

et al., 1975; Danesch et al., 1983). Run-on experiments performed with isolated liver nuclei demonstrated that the hormone acts by increasing the transcriptional rate of the TO gene (Danesch et al., 1983). Analysis of the chromatin structure around the 5’ end of the TO gene has revealed a number of tissue-specific DNase I-hypersensitive sites present only in hepatocytes but independent of the hormone response (Becker et al., 1984). These properties make the TO gene an interesting model in which to study tissue specificity and hormone dependence of gene expression. Experiments are in progress in our laboratory towards a molecular understanding of the mechanisms underlying this regulation, Since the TO gene consists of 12 exons and its t,otal size exceeds 20 kb, we have constructed a fusion gene between 5’-flanking regions of TO and the bacterial

Expression of the gene encoding the liver-specific enzyme tryptophan oxygenase is confined in a tissue-specific manner to hepatocytes (Feigelson & Killewich, 1979). Dexamethasone administration to adrenalectomized rats leads to a tenfold increase in enzyme activity, reflecting a corresponding increase in the steady-state level of TOase$ mRNA (Schiitz t Present address: Institut fur Molekularbiologie II, der Universitiit Zurich, Honggerberg, CH-8093 Zurich, Switzerland. $ Abbreviations used: TOase, tryptophan oxygenase; TO, gene for TOase; kb, lo3 bases; bp, base-pair(s); CATase, chloramphenieol a.cetyltransferase; CAT, gene for CATase; RI’V, bovine papilloma virus; MMTWLTR, mouse mammary tumor virus long terminal repeat. 0022-2836/86/040557-12 $03.00/O

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8 1986 Academic Press Inc. (London) Ltd.

558

P. Matthias

gene coding for the enzyme chloramphenicol acetyltransferase (Gorman et al., 1982). These fusion genes and its deletion derivatives are being tested in stable and transient transfection assays in cell lines. In this paper we describe experiments examining the behavior of such a fusion gene in an extrachromosomal vector. This particular approach was taken to create a controlled genetic environment for these genes following their stable introduction into eukaryotic cells, and to establish these genes in minichromosomes for the study of associated proteins and chromatin structure. To propagate the TO-CAT gene in an extrachromosomal form we used vectors derived from the bovine papilloma virus genome. BPV is one of few genetic elements that have the potential to maintain inserted genes in an extrachromosomal state in some rodent fibroblasts (for a review, see IXMaio, 1985). We have described the construction and behavior of pCGBPV9, a generally selectable vector derived from BPV by the insertion of the gene conferring resistance against G418 in t.ransfected cells (Matthias et al., 1983). While the presence of the G418-resistance gene and additional cosmid sequences still allowed BPV to replicate extrachromosomally in mouse cl27 cells, this was not found to be the case in several other cell lines tested, e.g. hepatoma cells (our unpublished results). Hence, this study is restricted to an analysis of TO promoter function in fibroblasts only. For comparative purposes, we included in our study a different hormonally regulated element, namely the long terminal repeat of the mouse mammary tumor virus. In studies from other groups it has been shown, that fusion of the MMTV-LTR to various marker genes confers hormonal regulation upon them after transfection into rodent fibroblast, indicating the presence of a glucocorticoid responsive element (Lee et al., 1981; Huang et aE., 1981; Hynes et al., 1981; Buet’ti & Diggelmann, 1981; Chandler et al., 1983). Hormonal regulation of the MMTV element has also been observed when it was present on a BPV vector (Ostrowski et al., 1983). We report that the TO-CAT fusion gene and a similar construct containing part of the MMTVLTR can be maintained on BPV in an extrachromosomal form. We also show that some of these constructs may integrate into the host cell genome as tandemly repeated elements. Which of these alternative patterns is observed does not seem to depend on the nature of the cloned segments alone but can also be affected by poorly understood factors such as the relative orientation of vector and insert (Matthias et al., 1983). Expression studies with these vectors show that the TO promoter is transcribed in all types of fibroblast in contrast to the situation in vivo, where expression is cell type-specific. A reduced range of hormonal regulation is observed, which does not seem to be influenced by flanking sequences of the vector. In contrast., while many MMTV constructs show a typical range of regulation, one shows a

et al.

modification of its expression, flanking elements. In most

possibly due to BPV cases the hormonal

inducibility does not seem to be influenced by the two alternative physical states of the BPV vectors.

2. Materials and Methods (a) Reagents Restriction endonucleases, bacteriophage T4 DNA ligase and T4 DNA polymerase were from BRL, Kornberg DNA polymerase I (Escher&&a co&) and calf intestine phosphatase (CIP) from Boehringer-Mannheim. radio-chemicals from Amersham, G418 (Geneticin) from Gibco. (b) Plasmid constructions All in vitro constructions were done according to standard procedures (Maniatis et al., 1982) with E. coli DHI (Hanahan, 1983) as the bacterial recipient. The general construction strategy was to fuse 2 glucocorticoid regulatable elements, namely the 5’-flanking sequences/ promoter region of the rat tryptophan oxygenase gene. or part of the MMTV-LTR to the bacterial CAT gene. This was done so as to make the CAT translation initiation codon the first AUG downstream from the in z&o transcription start of the TO or MMTV-LTR promoter. These 2 fusion genes, called TO-CAT and MMTV-CAT. were inserted into 3 BPV vectors, namely pCGBPV9 (Zinn et al., 1983) and (Matthias et al., 1983), pBPV-BVl ~230.8 (Sarver et al., 1982). The construction of the TO-CAT fusion gene will be published in detail (U. Danesch, unpublished results), but can be summarized as follows: a 1.9 kb BarnHI-HgaI fragment containing TO 5’-flanking sequences and extending into the gene up to position +50 was made blunt-ended with T4 polymerase at the HgaI site (position +59) and joined to synthetic XhoI linkers. The plasmid pAlO-CAT2 (Gorman et al., 1982). which contains the CAT gene was opened at its single Hind111 site, made blunt-ended with T4 polymerase and subsequently ligated to XhoI linkers. The TO fragment was then inserted into this modified pAlO-CAT2 between the newly created XhoI site and the BgZI site. Insertion of a synthetic BarnHI linker into the single Sal1 site of this plasmid permitted the reclamation of the TO-CAT fusion gene as a 3.7 kb BamHI fragment devoid of plasmid sequences. The MMTV-LTR was obtained as a 760 bp BamHIXhoI fragment, which was derived through several subcloning steps from the original HaeIII fragment encompassing bases -631 to + 125 around the 5’ start (R. Miksicek, personal comsite of transcription munication) of an endogenous unintegrated GR mouse mammary tumor virus (Buetti & Diggelmann, 1981). This segment was similarly fused to the XhoI-BamHI fragment containing the CAT cassette allowing the isolation of a 2.6 kb BamHI MMTVCAT fusion gene. The 3.7 kb BarnHI TO-CAT fragment and the 2.6 kb RamHI MMTV-CBT fragment were introduced in the following BPV vectors. pCGBPV9 is a prokaryotic-eukaryotic shuttle vector consisting of a segment of ColEl, a phage 1 cos site, and the complete BPV genome linearized at its Hind111 site. Its neomycin-resistance gene is expressed by a combined prokaryotic and eukaryotic promoter, to make it selectable in E. co& (kanamycin-plates) or mammalian cells (G418 medium). It was found previously that this vector normally replicates as an extrachromosomal and

Glucocorticoid-controlled HmdlIl

559

Genes

XhoI

pCGBPV-MMTV-CAT-A

(b)

BPV-TO-CAT1 p230,8-TO-CAT1

i’dI

Figure 1. Structure of vectors used in this study. (a) The central circle represents the vector pCGBPV9 (11.6 kb), into (2.6 kb) fusion genes were inserted. The “2” at the end whose 2 BumHI sites the TO-CAT (3.7 kb) or the MMTV-CAT of the plasmid designates the orientation of the insert, namely CAT and BPV transcription units pointing in opposite directions. (b) The central circle represents the vector pBPV-BVl (11.2 kb), orientation of the inserts designated as above. (c) p2308TOCAT (14.4 kb) is a construct, whose bacterial plasmid sequences can be removed before transfection by BumHI cleavage and recircularization with T4 DNA ligase, to yield the eukaryotic vector BPV-TOCAT1 (11.2 kb). Constructions are described in detail in Materials and Methods. Open bar, bacterial plasmid DNA; filled bar, BPV DNA; stippled bar, human /?-globin DNA; hatched bar, CAT coding sequences; TO and CAT segments are designated by letters: Arrows indicate the orientation of the various transcription units unrearranged monomer in G41 S-selected mouse c 127 fibroblasts, while a construct differing only in the orientation of the BPV part relative to the cosmid showed physical behavior tentatively identified as intrachromosomal tandem repeats (Matthias et al., 1983). pCGBPV9 has 2 BarnHI sites, one in the BPV genome, the other in the plasmid part. Either of these sites was removed individually by gap-filling with T4 polymerase, and the TO-CAT or MMTV-CAT fusion genes were introduced into the single remaining BumHI site of either of the 2 resulting vectors. Constructs containing the fusion gene within the plasmid part are indicated by the letter “A”, those with the fusion gene in BPV by “B”. The suffix “1” designates unidirectionality of the BPV and CAT transcription units: the index “2” denotes that the transcription units are in opposite orientations (Fig. 1(a)).

As a second recipient vector we chose pBPV-BVl (Zinn et at., 1983). This plasmid contains the 69q/, fragment encompassing all early genes of BPV necessary for episomal maintenance and for the transformed phenotype of recipient cells (Nakabayashi et al., 1983), a segment of pBR322 and 2.7 kb of the human p-globin gene. The latter element has been observed to stimulate high-efficiency transformation and episomal maintenance of inserted genes (DiMaio et al., 1982). The TO-CAT or the MMTV-CAT fusion genes were inserted in both orientations into the single BamHI site of this vector, within the /?-globin sequences to produce pBPV-BVlL TO-CAT-Al and 2 or pBPV-BVl-MMTVCAT-1 and 2 (Fig. l(b)). A third construct was based on the strategy of having a vector whose bacterial plastnid sequences could be removed before transfection. ~230.8 contains the loon&

560

P. Matthias

BPV genome as a BamHI fragment cloned into pML2d (Lusky & Botchan, 1981), but having 1 BumHI site replaced by a Sal1 linker (Sarver et al., 1982; P. Howley, personal communication). Into this Sal1 site TO--CAT was cloned as a 3.7 kb Sal1 fragment (reclaimed from plasmid pUD-TO-CAT24) containing a BumHI site close to the Sal1 fragment downstream from CAT (Matthias, 1985). Through BumHI cleavage, followed variably by in vitro ligation before infection, the vector BPV-TO-CAT1 devoid of bacterial plasmid sequences was obtained (Fig. l(c)). (c) Cell

et al.

1983). a 650nucleotide RNA probe complementary to the TO-CAT hybrid mRNA was synthesized from a plasmid containing the 650 bp EcoRI-PwuII fragment, of TO-CAT inserted into the SP62PL vector under reaction conditions described by Zinn et al. (1983) and Melton et al. (1984). A sample (5 fmol) of this probe (spec. act. 8.5 x lo7 cts/min per pmol) was hybridized to 20 +ag of total cellular RNA from transformants and subsequently digested with RNase A and T, under the conditions described by Zinn et al. (1983). The RNase-resistant products were displayed on sequencing gels.

culture

Mouse cl27 fibroblasts (Lowy et al., 1978) were grown and transfected according to published methods (Wigler et al., 1977; Matthias et al., 1983). (d) DNA isolation and Southern blot analysis Procedures for DNA isolation and Southern blot analysis followed protocols described by Maniatis et al. (1982) and Matthias et aZ. (1983). (e) Hormon.aZ induction, Fetal calf serum was stripped of endogenous steroid hormones by stirring it for 30 min in the presence of 50 mg activated charcoal/ml (Norit A; Serva). The charcoal was then pelleted by centrifugation and the serum was filtered. For hormonal induction experiments cells were in Dulbecco’s modified Eagle’s medium replated supplemented with stripped serum and after 1 day dexamethasone (stock solution 10e3 M in 100% EtOH) was added to a final concentration of 10d6 M. After incubation for a further 4 to 24 h, the cells were processed for CATase assay or RNA analysis (f) CATase activity determination The CATase assay was performed exactly as described by Gorman et al. (1982) with known amounts of proteins. Protein concentrations were determined using either the method of Lowry et al. (1951) or the method of Bradford (1976). Quantification of CATase activity was done by cutting out the radioactive spots from the thin-layer chromatography plate and counting them in a liquid scintillation counter (Beckman Instruments). (g) RNA isolation, S, nuclease and DNase “Lpping Total cellular RNA was extracted by the LiCl/urea procedure (Auffray & Rougeon, 1980) with minor modifications. Contaminating DNA was removed with 0.5 unit of RNase-free DNase I (prepared by treatment with macaloid: Picard & Schaffner. 1983), in 100 ~1 of a solution containing 20 mM-HEPES (pH 7.4), 5 mMMgCl,, 1 mM-CaCl, and 1 miw-MnCl, at 37°C 30 min. To stop the digestion, the RNA was extracted with phenol/ chloroform (1 : 1, v/v) once and precipitated by the addition of 0.1 vol. 8 M-LiCl and 2.5 vol. ethanol (100%). The RNA analysis by S, nuclease mapping with 5’-endlabeled probes (Weaver & Weissmann, 1979) was done exactly as previously described (Matthias et uZ., 1982) except that the hybridization temperature was 45°C in case of a double-stranded probe or 30°C in case of a single-stranded probe. For RNA analysis by RNase protection (Zinn et al.,

3. Results (a) Physical state of BP V vectors containing TO-CAT or MMT V-CAT fusion genes in mouse cl27 Jibroblasts

The section on plasmid constructions (section (b), Materials and Methods) describes our strategies for inserting TO-CAT and MMTV-CAT fusion genes in various ways into BPV-derived vectors. This multitude of constructs was chosen in order to understand, whether flanking sequences (e.g. the BPV enhancers) of the vector modify the expression characteristics of inserted genes, and to maximize the chance that at least some of the plasmids would show the physical behavior of extrachromosomal replication in cl27 cells. Table 1 gives a complete list of the six vectors containing TO-CAT and the four vectors with MMTV-CAT. Since in this study MMTV-CAT was included primarily as a control, only a few of the constructs with TO-CAT were duplicated with this insert. transformants derived from Mouse cl27 pCGBPV9 and its derivatives were selected for resistance to G418, while transformants containing all other vectors were selected for format.ion of foci. Tn most experiments, these conditions yielded between 100 and 1000 clones upon application of 1 ,ug DNA to lo6 cells. From all experiments a minimum of six transfectants was picked, grown to 10’ cells and analyzed for the physical state of the vectors in Southern blot experiments without and, in some cases, with restriction enzyme digestion. All vectors derived from pCGBPV9 that contain TO-CAT or MMTV-CAT have sizes of approximately

15 kb.

The

supercoiled

form

of

these

plasmids

runs, on 0.6% (w/v) agarose gels, well ahead of the linear chromosomal DNA, which has an average size of more than 50 kb in our standard total

cellular

DNA

preparations.

This can be shown

by controls that contain vector DNA isolated from E. co& mixed

with

high

molecular

weight

DNA

preparations (Fig. 2). Although the parental vector pCGBPV was almost exclusively maintained extrachromosomally in G418-resistant colonies, only a few of the recombinants

containing

this type of behavior.

In two out of 13 clones

containing

pCGBPV-MMTV-CAT-Al

of 12 clones containing the

vector

DNA

chromosomally, ticularly

in

inserts

retained

and nine out

pCGBPV-MMTV-CAT-A2

was found t,o replicate extrawhile in all other clones, parall clones containing the various

Glucocorticoid-controlled

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Genes

Table 1 Physical

state of BP V vectors in cl27 Jibroblasts Number of transfections done

Plasmid pCGl3PV9 pCGBPV-TO-CAT-A2 pCGBPV-TO-CAT-H pCGBPV-TO-CAT-B2 pBPV-BVl -TO-CAT-A 1 pBPV I-BV 1-TO-CAT-AZ BPV~TOKATl pCGBPV-MMTV-(IAT-Al pCGBPV-MMTV-C!AT-A2 pBPV-BVl-MMTV-CAT-Al pBPV-BVl-MMTV-CAT-AZ

Number of clones analyzed 52 6

10 6 6 6 14 13 12 6 2

Clones with extrachromosomal supercoiled 13PV monomers 46 0 0 0 6 6

10 2 9 5 2

Clones with hugh molecular weight BPV DNA 6 6

10 6 0 0 4

11 3

1 0

Clones selected for G418’ or focus formation were picked, grown to 10’ cells and total cellular DNA was isolated; 2 to 15 ng of DNA were run in 0.6% agarose gels with plasmid DNA of bacterial origin as size-marker for uncleaved supercoiled DNA. The DNA was blotted to nitrocellulose filters and hybridized against 32P-labeled pCGBPV9. The decision as to whether BPV homologous sequences were considered extrachromosomal monomeric or intrachromosomal tandem repeats was made according to the criteria outlined in the legends to Figs 2, 3 and 5.

pCGBPV-TO-CAT constructs, the vector was found associated with the high molecular weight cellular DNA (Table 1 and Fig. 2). The absence of extrachromosomal monomeric molecules carrying TO-CAT was quite unexpected, as the parent& vector is normally maintained in G41Sresistant cl27 colonies in this form. Formally

this observation could reflect any of a variety of different physical states of the vector DNA: extrachromosomal DNA of concatemeric or structure, intrachromosomally concatenated inserted DNA present either in dispersed sites or in one or a few sites in a concatemeric form. To distinguish between these possibilities we analyzed

Figure 2. Southern blot analysis of total cellular DNA from G418’-selected colonies containing pCGBPV---TO-CAT (a) Undigested DNA; (b) XhoI (a single-cutting enzyme) cleaved DNA; (c) BamHI (double-cutting enzyme) cleaved DNA: 15pg of undigested DNA were run on a 0.7% agarose gel or 2Oc(g of digested DNA on a 1.27; gel. The nitrocellulose filter containing the transferred DNA was hybridized against 32P-labeled pCGBPV-TO-CAT-B1 DNA. The clone series 90, 92 and 94 contain pCGBPV-TO-CAT-A2, B2 and Bl, respectively. The control lanes contain copy number equivalents of the plasmids of bacterial origin (1.6 pg plasmid/l pg chromosome1 DNA = 1 copy/diploid nucleus). In (a), the position of the visible high molecular weight DNA (HMW) is indicated, which showed essentially an vectors.

identical

position

in comparison

with

the autoradiographic

separately with Hue11 and EcoRI and then mixed). 19

distribution.

M in (c) is size marker

(pHSG336

digested

562

P. Matthias et al.

HMW

Figure 3. Southern blot analysis of total cellular DNA from G418’-selected colonies containing pCGBPVMMTV-CAT vectors. 627 series: pCGBPFMMTV-CATA2; 628 series: pCGBPV-MMTV-CAT-AI; the 3 lanes on the right contain 50 and 10 copy number equivalents/cell, with plasmid DNA of bacterial origin; and chromosomal D?r’A of cl27 containing BPV (ID13); 2 pg of undigested DNA was run on a 0.7% agarose gel. The nitrocellulose filter containing the transferred DKA was hybridized against 32P-labeled pCGBPV9.

the same DNAs after digestion with single or double-cutting enzymes. If the transfected DNA was present exclusively as extrachromosomal concatenates or concatemers, restriction digestion with a single or double-cutting restriction enzyme should yield only one or two DNA fragments, respectively. If, on the other hand, more fragments are generated than expected, the material is likely t’o be intrachromosomal. It can also be extrachromosomal, but must then comprise a variety of rearranged or recombined species. Figure 2(b) and (c) presents the results of a Southern blot analysis done with total cellular DNAs that’ have been digested either with XhoI (an enzyme cleaving once in the vector to give a 15 kb fragment) or with BamHI (an enzyme cleaving the vector into an 11.5 and a 3.7 kb fragment). Both digestions produce the expected fragment(s), though their relative intensities indicate that they are not necessarily present in equimolar amounts (e.g. 90.2, 94.4). In addition, a variety of other bands of various sizes and intensities are observed that differ between individual clones. Some of these bands have a single-copy equivalent intensity and could represent junction fragments between integrated copies of the pCGBPV-TO-CAT plasmids and the mouse cellular DNA. The observed pattern is consistent with one or several integration events, with several copies of the plasmid arranged in a tandem array at each int.egration site. By comparing the intensit.ies of the

expected fragments with those of the reconstructions the copy number can be estimated to be approximately 50 copies per cell (interestingly, a similar copy number is observed with BPV vectors replicating as extrachromosomal monomers). A somewhat different type of behavior is found for pCGBPV-MMTV-CAT-Al and -A2. In 11 out of 25 clones all or at least a fract,ion of the vector sequences behaved like the expected monomeric supercoil (Fig. 3). Obviously, the suitability of pCGBPV9 as an extrachromosomal vector can depend on the nature of the inserted genetic e1ement.s. It is unclear which part of the 1.9 kb segment stemming from the TO gene modifies BPV replication so as to disrupt extrachromosomal monomeric maintenance. In order to learn whether the mere presence of the TO-CAT segment would lead to similar effects in different BPV vectors, we tested constructs not containing bacterial plasmid sequences or containing additional sequences from the human /?-globin gene. The first type of construct was obtained by BamHI digestion of the plasmid p230+TO-CAT and subsequent isolation of the relevant fragment containing the BPV genome linked to the TO-CAT fusion gene (Fig. 1(c)). This DNA was then used for transfection, either as such or after recircularization with DNA ligase. Figure 4(a) shows the analysis of DNA of foci obtained with linear transfecting DNA derived from p230+TO-CAT, while Figure 4(b) shows the analysis of DNA of six foci generated by recircularized DNA. All DNA preparations from Figure 4(b) show a predominance of small ext’rachromosomal elements with five out of six having the migration behavior expected for an 11.7 kb monomeric

circle.

In contrast,

in Figure

4(a) DNAs

from only four out of seven foci show any extrachromosomal material. Only focus 227-l contains a single extrachromosomal species of the expected size, while focus 225.2 contains an equimolar amount of this and a second smaller extrachromosomal species. The other clones display an even more complex pattern of ext,rachromosomal and high molecular weight elements. Three of the DNAs, shown in Figure 4(b), that contain supercoiled species migrating at roughly 11.7 kb were chosen for restriction analysis. EcoRI digestion of intact BPV-TO-CAT1 DNA should yield four fragments with sizes of 7.0, 3.7, 0.74 and 0.2 kb. The three largest of these DNA fragments are visible in Figure 4(c), while the 200 bp fragment is missing because it ran out of the gel. We can conclude from these data that circularization of the DNA before transfection resulted in a large proportion

of foci establishing

the expected

extra-

chromosomal element, while this event is much less frequent when linear DNA is used. Figure 5 shows the analysis of total cellular DNA from 12 foci obtained with pBPV-BVl-TO-C!iZTAl and -2 that contain the stabilizing segment, of the globin gene (Zinn et aZ., 1983). The DNAs of ail 12 foci contain an extrachromosomal species

Glucocorticoid-controlled

(a) 225 ‘l----l 12345

563

Genes

Cc)

(b) 227

289

289 I

I 12

I

2

3

4

5

6

I

I 123

sm

“;”

-9.5 -6.6

t

”

-0.56

Figure 4. Southern blot analysis of DNA from focus-selected clones containing BPV-TO-CAT1 DNA. (a) Undigested Hirt supernatant DNA of 6 clones obtained after transfection with the linear BPV-TO-CAT1 BamHI fragment. An amount of DPU’Acorresponding to lo6 cells was loaded on each slot of an 0.6% agarose gel. (b) Undigested total cellular DNA (12 pg/slot) of 6 clones obtained after transfection with the in vitro ligated BPV-TO-CAT1 BamHI fragment preparation. The arrow indicates position of an 11.6 kb supercoiled circle. (c) EcoRI restriction analysis of the DNA of 3 foci shown in (b) in undigested form, showing the appropriate restriction pattern for BPV-TO-CAT1 (see the text). The rightmost lane indicates the position of size markers (sm; HindIII-digested phage 1 DNA). comigrating with the bacterial plasmid DNA. The molecules of the extrachromosomal structure present in these foci was analyzed further by digesting the DNAs with either BamHI or Hind111 and all six expected bands of appropriate size were detected. Similar data were obtained with the pBPV-BVl constructs containing MMTV-CAT (data not shown). (b) CATase expression under the control of the TO and MT V promoters Our expression studies were focused on three questions. We first asked whether the TO promoter, which is normally hepatocyte-specific, would be recognized in fibroblasts in transfection experiments; secondly, we asked whether this element would be appropriately regulated by glucocorticoids in comparison to the corresponding MMTV constructs, whose regulation was anticipated from published work (for a review, see Ringold, 1983). Finally, it was necessary to show whether CATase activities and their changes were reflecting the use of the same transcription start sites as are used in viva.

Several clones with each construct were grown up and extracts were prepared. Equivalent amounts of protein from each extract were used to determine CATase activities under conditions that were in the linear range of the reaction. Representative examples of CATase expression in individual clones are shown in Table 2. In all cases clones were only chosen if their DNA structure had been previously analyzed. Most clones containing the TO-CAT fusion gene showed significant amounts of CATase activity, which indicates that the TO promoter functions after transfection into cl27 fibroblasts. In spite of the encountered variability between independent clones, no systematic difference possibly stemming from the structure of the transfected vector can be read from these data. In order to determine whether the TO-CAT and the MMTV-CAT fusion genes were hormonally regulated in individual clones, cells were replated in medium containing stripped serum and after one day were induced with dexamethasone. Control plates were kept for the same period of time without added hormone. At the end of the induction period the cells were lysed and extracts

564

P. Matthias

IO

281

280

COpleS

I

I

2

3

4

5

6

I

2

:

3

‘.

4

6

5

,/’ ‘

Figure 5. Southern blot analysis of undigested total cellular DEA from foci obtained by transfection with pBPV-BVl-TO-CAT-AI and A2. A 12 pg sample of tot,al cellular DNA was run on a @79/, gel and probed with 32P-labeIed after transfer to nitrocellulose pCGBPV9.

The control lanes contain 200 pg and 20 pg of

pBPV-BVl-TO-CAT-AI. 1 copy of BPV per diploid

which correspond nucleus.

to

10 and

were assayed for CATase activity. The results are presented in Table 2, which also shows induction factors calculated from the ratio of induced to uninduced values. The induction is up to 2.0-fold for the TO promoter and up to 20-fold for the MMTV promoter. For pCGBPV-MMTV-CAT vectors a dependence on orientation of the hormonal stimulation was (see Table 2). The construct with observed orientation 1 gave, similar to both pBPV-BVlMMTV-CAT vectors, an approximate lo- to 20-fold induction. In contrast to this, orientation 2 was less inducible, most probably due to an increase in the basal level of expression. (c) Transcription of the TO-CAT gene initiates positions used in the hepatocyte in vivo

at

We next wanted to see whether the CAT expression observed in G418’ colonies was reflecting a correct usage of the TO promoter (i.e. start sites at positions + 1 and - 180, as used in viva) (Schmid et al., 1982), or whether it was due to aberrant transcription initiation. This was particularly

et al. important as BPV can produce significant amounts of read-through transcription (DiMaio, 1985). We therefore analyzed several RNA preparations from transformants containing the BPV-TO-CAT vector intra- (Fig. 6(a)) or extrachromosomally (Fig. 6(b)) by S, nuclease and RNase mapping (Weaver & Weissmann, 1979; Zinn et al., 1983), using a 5’-endlabeled DNA probe. As a probe, a 350-nucleotide single-stranded XhoI-Hind111 fragment 5’ asymmetrically labeled at the XhoI site was prepared. If correctly initiated TO-CAT mRNA is hybridized with this probe, a fragment of 64 nucleotides in length is expected to be protected from S, nuclease digestion. Figure 6(a) shows the result of such an experiment performed with several clones containing intrachromosomal tandem repeat copies of pCGBPVS-TO-CAT plasmids. The correct start site (es) at position + 1 is used in all clones presented while the correct upper start, site at position - 180 is not used. Instead, an “incorrect upper start” (ius) mapping approximately 20 bp upstream from the former is found with variable intensities in most clones (Fig. 6(a)). This band might reflect an S1 nuclease artefact, as it maps precisely to an A +T-rich region 200 bp upstream from the cap site (Schmid et aE., 1982). Hormonal induction is demonstrated by the fact that the RNA from cells induced with hormone (+ in Fig. 6(a)) protects more probe t’han RNA from control cells (- in Fig. 6(a)). Figure 6(b) shows analysis of RNA from foci containing BPV-TO-CAT1 extrachromosomally done by the RNase-protection procedure developed by Zinn et al. (1983) and Melt,on et al. (1984). In this method, an antisense RNA probe is prepared using the SP6 transcription system, which is then hybridized with the RNA sample to be test’ed. The RNA-RNA hybrids are subsequently digested with a combination of RNase A and RNase T,, and the resistant products are displayed on a sequencing gel. Such an analysis has been done with RNAs from several foci and the results of the analysis of RNA from two foci are presented in Figure 6(b). If the TO-CAT mRNA is correctly initiated at position + 1 a band of 213 nucleotides is expected to be protected. As seen in Figure 6(b), the RNA preparations

analyzed

protect

this band primarily.

Minor amounts of large products are also found. which correspond to either the correct or the site. The hormonal incorrect start upper inducibility was measured by excising the radioact.ive bands from the gel and counting them in a liquid scintillation counter. When glucocorticoids were present for four hours, the RNA from the TO-CAT fusion gene is fivefold induced and, after 24 hours, about 2.5-fold. As a positive control for

these induction experiments. a pool of G418’ Ltkcells stably transfected with the TO-CAT fusion gene was analyzed and found to show 3-3-fold induction. In such pools the TO-CAT fusion gene typically shows threeto sixfold induction by dexamethasone, as was measured by CATase assay or RNA analysis (U. Danesch, unpublished results).

Glucocorticoid-controlled

565

Genes

Table 2 Basal and hormonal@ induced expression of the CAT fusion genes in cl27 foci or G418’ colonies containing BP V-TO-CAT or BP V-MMT V-CAT DNA

Transferted

DNA

p(:GI~PV~TO-CAT-AZ pCGBPV-TO-CAT~BP I’(:(:UPV~TO~(:AT-BI pBPV~BGI-TO-CAT-AI III~P~~U\‘I~TO-(‘AT~A~ BPV~TO&(‘ATl pN:UP\‘-MMTV-CAT-Al pU:UP\‘-

MMTV-(:AT-A2

pUP4&BVlLMMT\‘YAT~Al PUP\‘-- I3V I pAMMT\--(!AT-A2

Clone no.

Protein (P&T)

90.2 90.6 92.2 92.5 94.2 94.4 280.1? 2t30.2t 2a1.1t 281.2? 225.2 227.1 628.8 628-Y 627.2 627.9 4.5 4.6 5.1 5-2

400 50 400 400 400 400

100 100 10 10 10 10 100 100 100 100

Reaction time (min)

CATase activity (40 conversion) + Dex

120 60 120 120 120 120

8.0 11.5 8.2 42.2 20.2 62.1

13.8 17.5 16.2 67.5 36.6 77.1

1.i 1.5 2.0 1% 1.8 1.2

60 60 30 30 30 30 60 60 60 60

0.8 2.8 5. 1 24 59.0 18.8 2.0 1.6 4.6 5.5

1.6 4.0 94.0 48.8 63.3 49.5 17.7 21.8 94.8 954

2.0 1.4 18.4 20.1 I.1

The Table gives 2 typical examples for each construct out of larger series tested. t This assay was not evaluated quantitatively, but inspection of the autoradiograph induction of CATase activity.

This type of analysis has also been extended to additional foci, including those derived from transfection with pBPV-BV-TO-CAT constructs. In all cases the correct start site was used and RKA of the appropriate size was det’ected.

4. Discussion (a) Experimental

Fold induction

- Dex

2.6

8.8 13.x 20.7 17.3 clearly showed

other cells, particularly rat hepatoma cells (our unpublished results). Owing to these limitations. our study was confined to cl27 cells. To compare our findings with a gene that has been found to be expressed in a regulated manner in fibroblasts we performed parallel experiments with an MMTVCAT fusion gene on BPV.

strategy

This paper represents part of a broad study done by our group towards the understanding of the regulatory cascade that governs the expression of t’he tryptophan oxygenase gene. This gene is transcribed in the rat only in hepatocytes beginning at’ day 10 after birth, and efficient transcription is also under the control of glucocorticoid hormones. To approach empirically an understanding of this regulatory cascade, we asked whether this gene can be st’udied in transfection experiments in fibroblasts and whether 1.9 kb of 5’-flanking sequence present in a fusion gene is sufficient for its regulation. Functional analysis of such a fusion gene would be greatly facilitated in a controlled genetic environment. Since integration into the chromosome is random. we tried to achieve this goal by insertion of the TO-CAT fusion gene into BPV-derived vectors with a potential for extrachromosomal replication. These vectors show extrachromosomal behavior only in cl27 and 3T3 fibroblasts and integrate as single copies into the chromosomal DNA of many

(b) Physical maintenancr In 90% of all G418’ cl27 clones containing pCGBPV9 this vector replicated as an extrachromosomal monomer (Table 1). We hoped that pCGBPV9 would show the same pr0pert.y aft.er insertion of exogenous transcription units. Unexpectedly, t’his was not consistently the case. All constructs containing the TO-CAT fusion gene inserted in either of two different sites gave D?u‘A species in transfectants corn&rating with high molecular weight, DNA, In contrast, very similar constructs in which 760 bp of MMTV replaced 1.9 kb of the TO sequence had a propensity to stay extrachromosomal, but with frequencies depending on the orientation of the insert. The h,vpothesis that TO-CAT could constitut’e an element interfering with extrachromosomal maintenance per se proved to be wrong, since in vitro religated BPV-TO-(:AT vectors or TO-CAT inserts in pHPV-HVI replicated as extrachromosomal monomers. The alternative to ext,rachromosomal monomeric replication was a cosegregation of BP\‘-homologous

P. Mat&as

et al.

414

212

(b)

Figure 6. Analysis of transcription

start sites of the TO-CAT fusion gene from (a) intra- or (b) extrachromosomal copies of BPV-TO-CAT vectors. (a) S, n&ease mapping analysis of RNAs from G418’ colonies. For every reaction 30 pg of total cellular RNA were hybridized with 30 fmol of the single-stranded HindIII-XhoI probe, end-labeled at the XhoI site (spec. act. 2.4 x lo6 cts/min per pmol, 5’ end). Above the slots the number of the clone is indicated, as in the text or in the Tables. +, Indicates RNA extracted from cells that had been induced for 24 h with 10m6 Mdexamethasone; -, indicates RNA extracts from uninduced cells; cs, correct start site at position + 1; ius. incorrect upper start site at position -200. Probe, indicates a lane where the probe alone has been loaded on the gel. The left and rightmost lanes of the gel contain end-labeled DNA fragments as size markers (phage fd replicative form DNA), cleaved with HinfI. (b) RNase mapping analysis of RNA from cl27 foci. The RNA of several foci was analyzed by RNase protection. For every reaction 20 /Lg of total cellular RNA were hybridized with 5 fmol of probe, except for lanes P and 225.2 (right of the Figure) where only 15 pg or 85 pg of RNA were used, respectively. The fragment corresponding to correctly initiated TO-CAT-RNA has a predicted size of 213 nucleotides, while RNA initiating at the correct upper start site would protect a fragment of 393 nucleotides. The numbers above the Figure designate the corresponding focus or pool (P). -, Indicates that the RNA came from uninduced cells, while + 24 and +4 indicate that the RNA originated from cells induced with lo6 M-dexamethasone for 24 or 4 h, respectively. The Probe lanes show 2 dilutions of the RNA probe used (775 nucleotides long, prepared from template pSPTO-Eco/Pvu). M lanes contain phage fd replicative form DNA, cleaned with Hid.

Glucocorticoid-controlled material with high molecular weight DNA. Such a signal could in principle stem from four different forms, namely extrachromosomal physical concatenated DNA, intracontamemeric or chromosomally integrated copies in dispersed sites, or intrachromosomally integrated tandem head-totail repeats. We believe that we normally observe the latter situation. In contrast to some other BPV vector systems (Karin et al., 1983), extensive shearing of the cellular DNA never released low molecular weight DNA species, as would have been expected from concatenated DNA (data not shown). Intrachromosomally dispersed sequences never produced full-length molecules on restriction digestion, but rather numerous novel bands, a prediction we could not. verify. BarnHI or XhoI restriction fragment analysis led us to conclude that we are dealing with concatemeric molecules. The observation of few extra bands of approximately one copy per genome equivalent suggests one or few insertion sites of these intrachromosomal concatemers. Similar observations have also been made by other groups using various BPV hybrid et al., molecules (Hsiung et al., 1984; Sambrook 1985: DiMaio et al., 1985). but have occasionally been interpreted in a different way (Meneguzzi et al.. 1984). The choice between the alternative modes of physical location of BPV vectors seems to be made neither by the nature of the vector nor by the inserted DNA alone, but is instead determined by poorly understood properties of the whole construct. These properties seem to determine a propensity towards either of the alternatives, since individual clones obtained during the same transfection experiment ma,y still contain the vector in either physical form.

(c) Expression of CAT from the TO and MMT 17 promoters We have shown that for transcription of the TOCAT fusion gene from intra- or extrachromosomal BPV vectors the same 5’ start site is used in cl27 cells as for transcription of the TO gene in hepatocytes. This proves that these fibroblasts contain t’he protein factors needed for correct initiat’ion of this gene and suggests that epigenetic factors are required to maintain the unexpressed state of the TO gene in fibroblasts in uivo. Our data suggest that BPV vector systems are suitable tools for the study of hormonally regulated genes. but several limitations and partially understood observations deserve t’o be discussed. All clones containing t’he same or comparable BPVTO-CAT or BPV-MMTV-CAT constructs show comparable levels of CATase activity at the uninduced or induced level, normally varying by less than one order of size from clone to clone. In this context, we do not believe that this is a significant’ difference. since repeated growth and extraction of the same clone gave similar variat,ions. Since most extrachromosomal

Genes

567

monomeric vectors show a comparable range of expression and regulation, we believe that effects from flanking vector sequences that may modify the expression of inserted genes are negligible and justify the assumption that the homogeneous physical environment on an extrachromosomal vector may help to establish a homogeneous chromatin environment around an inserted gene. However, clones containing pCGBPV-MMTVCAT-Al show an increased basal expression in comparison with pCGBPV-MMTV-CAT-AS. Since we have not mapped the CAT transcripts in these clones, we do not know whether they reflect RNAs starting at the correct site in the MMTV promoter. The BPV enhancer, active only in BPV-infected cells, has recently been located between the Hind111 and HpaI sites of BPV (P. Howley, personal communication). This leaves open the possibility that in these clones the basal level of MMTV transcription is under the influence of this BPV element. It has already been shown that intact MMTV DNA and fusion genes between MMTV-LTR and suitable test genes retain hormonal inducibility after transfection into eukaryotic cells (Hynes et al.. 1981; Buetti & Diggelmann, 1981; Lee et al., 1981; Huang et aZ., 1981). Our constructs with MMTVCAT confirmed data previously reported, which indicate that an MMTV-ras fusion gene present on BPV was maintained extrachromosomally in cl27 foci and that its transcription was under hormonal control (Ostrowski et al., 1983). In contrast to the strong glucocorticoid regulation of MMTV, a weaker but reproducible hormonal inducibility could be demonstrated for TO by either CATase assay or RNA mapping. Even though in some experiments a fivefold increase in TO-CAT-specific RNA upon hormone addition was found, the hormonal inducibility obtained in most cases was only up to 2.0.fold for CATase assay or RNA mapping. Tn rat hepatocytes, transcription of the TO gene is induced approximately tenfold by the addition of glucocorticoid hormones (Danesch et al., 1983)) while in stably transformed Ltkcells TO-CAT mRNA accumulates only three- to sixfold after glucocorticoid induction. Two glucocorticoid response elements have been identified in the 1900 bp fragment of the TO $-flanking segment contained in the fusion gene (V. Danesch, unpublished results). It’ is conceivable that regulation by glucocorticoid hormones on BPV vectors leads to a lower overall inducibility since the genes from all copies might contribute to the basal expression but only some might contribute to the induced level. Such a buffering effect may be compensated for in the MMTV-CAT fusion gene through the very high inducibility of this promoter (Chandler et al., 1983), whereas little or no induction is observed with genes regulated over a smaller range, such as metallothionin or somatotrophin (Pavlakis & Hamer, 1983; Karin et al., 1983; Kushner et al., 1982). A high

P. Matthias

568

inducibility has also been observed for the interferon gene in BPV (Zinn et al., 1983; Goudburn et al., 1985). Our cell lines containing TO-CAT and MMTVCAT fusion genes extrachromosomally maintained and regulated may now permit an analysis of their experiments structure. Preliminary chromatin suggest the presence in the extrachromosomally maintained TO-CAT fusion gene of the same DNase I-hypersensitive sites that are found in this gene in hepatocytes (Becker et al., 1984; U. Boeger, unpublished results). These experiments suggest that the DNA sequence per se is sufficient to induce a particular chromatin structure to give rise to DNase I-hypersensitive site, as has already been shown for the simian virus 40 enhancer (Fromm & Berg, 1983; Jongstra et al., 1984) and for MMTVLTR (Zaret & Yamamoto, 1984). Analysis of these extrachromosomal molecules will also be attempted with the genomic sequencing technique (Church & Gilbert, 1984) to examine protein-DNA interactions in the TO and MMTV promoter regions. The presence of approximately 50 copies per cell of the extrachromosomal fusion genes should greatly facilitate this type of analysis. We thank R. Miksicek for providing the MMTV-LTR subclone, R. Renkawitz and R. Miksicek for stimulating discussions, M. Cole and U. Joa for excellent typing of the manuscript. This work was supported by a grant (Schu 51/4-2) from the Deutsche Forschungsgemeinschaft to G.S.

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by P. Chambon