THE JOURNAL OF BlOLOGlCAL CHEMISTRY Vol. 257, No. 12, Issue of June 25, pp. 7254-7261. 1982 Prrnted in U.S.A.

Transcription of the Chicken a2 (Type I) Collagen Gene by Homologous Cell-free Extracts*

(Received for publication, February 16, 1982)

Glenn T. MerlinoS, Jaya Sivaswami Tyagi, Benoit de Crombrugghe, and Ira Pastan From the Laboratory of Molecular Biology, Division of Cancer Biology and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205

We have used two methods to detect specific tran- scription of the chicken a2 (type I) collagen gene in cell- free extracts derived from Rous sarcoma virus-trans- formed chicken embryo fibroblasts. The first method is a modification of the S1 nuclease mapping procedure which utilizes a DNA probe labeled with 32P at the 5’ end of the HindIII linker originally used to clone the collagen promoter region into pBR322. The probe dis- tinguishes newly made, specific RNA from endogenous RNA and nonspecific transcripts. Using this procedure we have found that chicken whole cell extracts support accurate initiation of transcription of the chicken a2 (type I) collagen DNA template. Addition of either cre- atine phosphate, GTP, or UTP to concentrations of approximately 3 to 5 m~ was found to stimulate RNA polymerase II transcription by 5- to 10-fold. The second method employs an avian myeloblastosis virus reverse transcriptase-catalyzed primer extension procedure, rendered in vitro-specific by use of a pBR322 fragment as primer. These two techniques should be useful for analyzing specific transcription in other types of cell- free extracts.

The collagens are a family of proteins found in most animal tissues, where they play various important structural roles as intracellular matrix constituents. Type I collagen, which con- sists of one a2 and two a1 polypeptide chains, is a prevalent protein in bone, tendon, and skin (1) and is enriched in cultured chicken embryo fibroblast cells. The gene coding for a2 (type I) collagen is 38 kilobases long. The coding informa- tion in this gene has been found to be subdivided into greater than 50 exons (2,3). The transcription start site at the extreme 5’ end of the a2 collagen gene has been located (2), and subcloned into the plasmid pBR322 (4) . Residing upstream of the start site (+I) is a TATA box (-33), a CAT box (-a), and sequences having the potential to form hairpin structures which may be important in regulating the transcription of the collagen gene.

Transcriptional control represents a critical element in the regulation of gene expression. A thorough understanding of the nature of this control must ultimately involve reproduc- tion of in vivo transcription events in reconstituted cell-free systems. Recently, significant progress has been made toward developing such in vitro systems using extracts derived from KB or HeLa cell lines (5, 6). With the exception of certain viral promoters, most class I1 eukaryotic genes have been

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisenent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. + Supported by a postdoctoral fellowship from the Arthritis Foun- dation.

studied in these transcription systems using a heterologous combination of polymerases, factors, and DNA templates ( 7 4 , thereby limiting the authenticity of transcriptional con- trol events. More recently, homologous cell-free systems have been described for the insects Drosophila and Bombyx (10, 11).

We have previously reported that the chick a2 (I) collagen gene is accurately and efficiently transcribed in HeLa cell extracts (4). We wanted to examine transcription of the chick a2 (I) collagen gene in extracts derived from both normal and Rous sarcoma virus-transformed CEF’ because transforma- tion by this virus results in a striking reduction in the synthesis of both the a2 (I) collagen protein and the RNA encoding (u2 (I) collagen (12-20). The latter suggests that RSV transfor- mation mediates the expression of the a2 (I) collagen gene in CEF by a transcriptional control mechanism.

As a fiist step in these studies we decided to use RSV- transformed CEF to prepare cell-free extracts because large amounts of these cells were readily available and because others have had success using extracts derived from trans- formed cells types (5, 6). Here we describe an homologous chicken system which uses extracts derived from RSV-trans- formed CEF in concert with the chick a2 (I) collagen pro- moter. Specific transcription can only be detected by using in vitro-specific S1 nuclease mapping and avian myeloblastosis virus reverse transcriptase-catalyzed primer extension. Using these procedures to examine RSV-transformed CEF extracts, we have found that chick RNA polymerase I1 is capable of accurately initiating transcription of the a2 (I) collagen gene in vitro.

EXPERIMENTAL PROCEDURES

Cells and Viruses-CEF were prepared and propagated in GM medium at 39 “C as previously described by Vogt (21). CEF were transformed with the Schmidt Ruppin-D strain of Rous sarcoma virus as described elsewhere (15). S3 HeLa cells, obtained from Meloy Laboratories, were cultured in 5% horse serum and harvested at a cell density of approximately 4.5 X 10’ celldml.

Preparation of Whole Cell Extracts-HeLa cell extracts to be used for in uitro transcription were prepared by a modification of the procedure of Manley et al. (6) , as detailed elsewhere (4). CEF and RSV-transformed CEF extracts were made in the same manner, except that 0.5-1.0% of aprotinin (Sigma) was included in the (NH&SO, pellet-resuspension buffer and in the final dialysis buffer (20 m~ 4-(2-hydroxyethyl)-1-piperazineethanesdfonic acid (pH 7.9), 100 m~ KCI, 12.5 mM MgC12, 0.1 mM EDTA, 2 mM dithiothreitol, 17% (v/v) of glycerol). The final protein concentration of RSV-trans- formed CEF extracts was between 25 and 35 mg/ml, lower than that of most HeLa cell extracts which were usually greater than 40 mg/ ml. Protein concentrations were determined using a y-globulin stan- dard in a Bio-Rad assay system (22).

In Vitro Transcription Reactions-RNA was synthesized in vitro

’ The abbreviations used are: CEF, chicken embryo fibroblast; RSV, Rous sarcoma virus.

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as previously described (4, 23), except that no radioactive precursor was included. Typically, reaction mixtures contained 13 r n ~ 4-(2- hydroxyethy1)-1-piperazineethanesulfonic acid (pH 7.9), 8 mM MgCL 65 m M KCl, 0.07 mM EDTA, 1.3 m~ dithiothreitol, 11% glycerol, 500 p~ ATP, CTP, GTP, and UTP, 50 pg/ml of template DNA, and 16.5- pl extract in a 25 pl reaction volume. Unless otherwise indicated, reaction mixtures were incubated for 30 min at 30 "C. Creatine phosphate, creatine phosphokinase (Sigma), a-amanitin, and actino- mycin D were utilized as described in the figure legends. Some reactions were run without added DNA template; however, template DNA was added after the reaction was stopped to maintain standard hybridization conditions.

Reactions were terminated and RNA prepared for S1 nuclease mapping or primer extension using conditions described by Weil et al. (5), except that reaction stop buffer contained 0.25 mg/ml of tRNA, and RNAs were ethanol-precipitated twice prior to hybridi- zation. To test RNA polymerase activity, UTP assays were performed using calf thymus DNA as described by Weil and Blatti (24).

Isolation and Labeling of DNA-The plasmid pCa2PRO-3, con- taining the chick a2 collagen gene promoter, was isolated and pre- pared as template for in vitro transcription as described elsewhere (4). All restriction endonuclease digestions were performed as sug- gested by the manufacturer (New England Biolabs). DNA fragments were electrophoretically fractionated and isolated using either low melting agarose gels (Marine Colloids) or electrophoretic elution. Isolated fragments were alkaline phosphatase-treated and T4 poly- nucleotide kinase-labeled as previously described (2). The "P-labeled DNA was digested with the appropriate restriction endonuclease, and the resulting fragments were separated on 7 M urea-polyacrylamide gels. For the 158- and 132-base pair fragments, a 7% polyacrylamide gel was used; while for the adenovirus-specific 452-base pair fragment, a 4% gel was utilized. The desired '*P-labeled DNA fragment was recovered from the acrylamide gel using the elution method of Maxam and Gilbert (25). After two ethanol precipitations, the DNA fragment was dissolved in water.

DNA sequence determination was according to the procedure of Maxam and Gilbert (26). The plasmid pSmaF, containing the ade-

a b c d e f g

0

c

FIG. 1. Autoradiogram of runoff RNA synthesized using the pCa2PRO-3 X Bam HI collagen DNA template. Labeled RNA was synthesized and isolated as described elsewhere (4). The RNA was denatured in 80% formamide and fractionated on a 7 M urea-4% polyacrylamide gel. a , 32P-labeled OX X Hae I11 markers; 6, synthesis of RNA transcripts using RSV-transformed CEF extracts; c, same as 6 + 1 g / m l of a-amanitin; d, same as b + 200 pg/ml of a-amanitin; e, same as b + 50 pg/ml of actinomycin D; f, synthesis of RNA transcripts using HeLa cell extracts; g, same as f + 1 pg/ml of a-amanitin. Arrows indicate the expected location of the correctly initiated 460-base RNA runoff transcript.

novirus 2 major late promoter, was a gift of P. A. Weil (University of Iowa).

SI Nuclease Mapping-SI nuclease mapping was performed by Weaver and Weissman's (27) modification of the procedure of Berk and Sharp (28). Unlabeled RNA, isolated from 25-p1 in vitro tran- scription reactions, was hybridized with between 0.01 and 0.03 pg of a DNA fragment (either pCa2PRO-3-derived Ava 11-Hind111 or pSmaF-derived Xho I-HindIII) which had been T, polynucleotide kinase-labeled at i ts 5' end (specific activity about 5 X lo6 cpm/pg of DNA), in 10 pl of 80% deionized formamide, 0.4 M NaCl, 0.04 M 1,4- piperazinediethanesulfonic acid (pH 6.4). 1 m~ EDTA at 38 "C. After 3 h, the hybridization mixture was diluted 10-fold into ice-cold S1 nuclease digestion buffer containing lo00 units/ml of BRL S1 nu- clease (2). After a 30-min incubation (40 "C), the reaction mixture was made 0.2 mg/ml with yeast tRNA, phenol-extracted, chloroform- extracted, and precipitated with ethanol. The nucleic acid pellet was suspended in 80% deionized formamide, 1 r n ~ EDTA, 10 m~ NaOH, bromphenol blue, xylene cyanol, heated to 90 "C for 5 min, quick- chilled, and subjected to 7 M urea-polyacrylamide gel electrophoresis (25). Radioactive bands were visualized by standard autoradiographic techniques.

Primer Extension-Primer extension was performed using a mod- ification of procedures described elsewhere (2.29). Unlabeled nucleic acids were isolated from 25-1.11 in vitro transcription reactions as described above, digested with iodoacetate-treated deoxyribonuclease I according to Lee and Roeder (30), and the resulting RNA was hybridized with 5 X lo4 cmp of a pBR322-specific 132-base Rsa I, HindIII DNA fragment, labeled at its Rsa I 5' end using T, polynu- cleotide kinase as described above. After 3 h at 38 "C, the nucleic acids were ethanol-precipitated and dissolved in 40 p1 of 50 mM Tris- HCl (pH 8.3), 50 r n ~ KCl, 6 mM MgC12, 10 m~ dithiothreitol, 20 pg/ ml of actinomycin D, and 1 m~ each of the four deoxyribonucleoside triphosphates. The primer was extended using approximately 1.5 units/pl of avian myeloblastosis virus reverse transcriptase (gift from J. W. Beard) a t 41 "C for 60 min. The nucleic acids were then denatured, phenolized, ethanol-precipitated, and electrophoresed on a 7 M urea-5.5% polyacrylamide gel as described elsewhere (2). Radio- active DNA fragments representing in vitro transcripts were visual- ized by standard autoradiographic techniques.

RESULTS

In Vitro-specific SI Nuclease Mapping-We had previ- ously demonstrated (4) that the promoter-containing 5' end of the chick a2 (type I) collagen gene functions accurately and efficiently as DNA template in a HeLa whole cell in vitro transcription system, prepared essentially as described by Manley et al. (6). Our next goal was to develop an homologous chicken cell in vitro transcription system to be used with the collagen gene, containing chick-specific RNA polymerase and regulatory factors. During preliminary experiments we at- tempted to analyze cell-free RNA from chicken extracts on denaturing polyacrylamide gels by "runoff" transcription techniques that were successful with HeLa or KB extracts (5, 6) using an enzymatically truncated DNA template. However, only a smear of 32P-labeled RNA products was observed (Fig. 1, lane 6) . The majority of this activity was resistant to both low and high ( 1 and 200 pg/ml) levels of a-amanitin, suggesting that it was the result of transcription by RNA polymerase I (Fig. 1, lanes c and d). This was in marked contrast to HeLa cell extracts, which exhibited little background activity (Fig. 1, lanes f and g) .

To detect specific RNA polymerase 11-catalyzed transcrip- tion within the chicken extracts, we decided to employ the more sensitive S1 nuclease mapping procedure (28). For cleaner, clearer results, it is desirable to use conditions where the DNA probe is labeled at one end, and then hybridized to cold RNA (27). The drawback of this approach is that the DNA probe cannot distinguish between the newly made in vitro-synthesized RNA transcripts and endogenous in vivo RNA. To circumvent this problem, we have modified the S1 nuclease mapping procedure by utilizing a DNA probe which is end-labeled at the HindIII linker originally used to clone

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the a2 collagen promoter DNA into pBR322. This procedure is schematized in Fig. 2. The collagen promoter-containing plasmid pCa2PRO-3, linearized by Bum HI digestion, was used as DNA template. The in vitro made RNA contained collagen-specific sequences, HindIII linker sequences, and pBR322 sequences. Endogenous RNA present in the extract, of course, contained no linker or pBR322 sequences. The DNA probe used for hybridization was a 158-base Ava II- HindIII fragment, 5”labeled at the HindIII site. By hybrid- izing to this site, only in vitro synthesized RNA was capable of protecting the radioactive DNA probe from S1 nuclease degradation. In vivo collagen RNA was therefore not visual- ized by this procedure.

Extracts were made from RSV-transformed CEF using a modification of the procedure of Manley et al. (6) (see “Experimental Procedures”). The extracts were used with Bum HI-cleaved pCa2PRO-3 DNA template to synthesize a2 collagen-specific RNA. This RNA was hybridized to the Ava 11-Hind111 probe described above, treated with S1 nuclease, and subjected to denaturing 7% polyacrylamide gel electro- phoresis. Fig. 3c shows that a single stranded DNA fragment of approximately 116 bases was protected from S1 nuclease digestion. This DNA fragment co-migrated with the fragment generated using the same procedure but with HeLa cell ex- tracts (Fig. 3b). Although the intensity of the bands shown for HeLa and RSV-transformed CEF are comparable, the activity of the HeLa extracts varies from preparation to preparation, and Fig. 3b represents a HeLa extract of relatively low activity. The S1 nuclease mapping procedure showed that protection of the 116-base DNA fragment by RNA from the RSV-trans- formed CEF extract was both DNA-dependent (Fig. 3g) and actinomycin D sensitive (Fig. 3f).

Accurate Collagen Gene Transcription Initiation in

I 1 16 n Fragment

Protected

1 Nothong Vtslble

Protected

FIG. 2. Schematic representation of modified, in uitmspe- cific S1 nuclease mapping. Plasmid pCa2PRO-3 DNA (thick dark lines), containing the a2 collagen gene promoter subcloned into pBR322 (thick dark wavy lines) via HindIII-specific linkers (cross- hatched lines), was used as template in an in vitro transcription reaction (a). The reaction was stopped, and total extract RNA (thin lines), consisting of newly synthesized cell-free RNA (A) and endog- enous extract RNA ( B ) , was isolated and hybridized (6 ) to the 158 base Aua 11-Hind111 DNA probe 5”32P-end-labeled (*) at the HindIII linker. Upon subsequent S1 nuclease digestion (c) , endogenous extract RNA did not protect the HindIII end-labeled linker (B) . However, cell-free RNA hybridized perfectly to, and protected the 5”labeled end. Properly initiated cell-free RNA therefore protected a 116-base 32P-end-labeled (*) DNA fragment which was visualized by denaturing polyacrylamide gel electrophoresis and subsequent autoradiography (d), while endogenous RNA protected no labeled DNA. The large white arrow represents the direction of transcription. + I indicates the start of transcription.

a b c d e f g h 194- , ”.

1- 118- - __t

72- II)

FIG. 3. S1 nuclease mapping of cell-free RNA transcripts synthesized from the a2 collagen gene template (pCa2PRO-3) by RSV-transformed CEF extracts. Cell-free RNA was hybridized at 38 “C for 3 h to the 5’-end-labeled 158-base collagen-specific DNA probe, and the resulting hybrids digested with S I nuclease at IO00 units/ml for 30 min (40 “C). The SI-resistant DNA was electropho- resed on a 7 M urea-7% polyacrylamide gel and visualized by standard autoradiographic techniques. a, 32P-labeled DX X Hue 111 markers; b, DNA protected from SI nuclease digestion by RNA from HeLa cell extracts using pCa2PRO-3 X Barn HI as template; c, DNA protected from S1 by RNA from chicken cell extracts using pCa2PRO-3 X Barn HI as template; d, same as c + 1 pg/d of a-amanitin; e, same as c + 200 pg/ml of a-amanitin; f, same as c + 50 pg/d of actinomycin D, g, same as c except no DNA template was added to the reaction; h, 158- base hybridization probe alone. Arrows indicate the location expected for the 116-base-protected DNA fragment.

Chicken Extracts-This analysis of DNA fragments allowed only an approximation of the in vitro transcription start site. To precisely locate this site, S1 nuclease-protected DNA fragments were electrophoresed on a denaturing 10% poly- acrylamide gel adjacent to a Maxam and Gilbert (26) sequence ladder of the 158-base mapping probe. Fig. 4 proves that the same two DNA fragments, within one nucleotide of each other, were protected by RNA from both HeLa (Fig. 4a) and RSV-transformed CEF extracts (Fig. 4 4 . When the 1.0-1.5 nucleotide adjustment was made to correct the difference in migration between the products of S1 nuclease digestion and Maxam and Gilbert chemical cleavage (31), transcription by extracts from both species was found to initiate at T and A in the coding sequence T T T A G C. This corroborated results obtained earlier using HeLa extracts, in which it was demon- strated by identification of the first and second bases associ- ated with the RNA cap structure, that the start site was at A in the above sequence (4) . This was the same transcription start site found in vivo (2).

To determine if RNA polymerase I1 was responsible for the observed transcription of the a2 collagen gene by RSV-trans- formed CEF extracts, low levels of a-amanitin were utilized. Unexpectedly, 1 p g / d of a-amanitin failed to completely inhibit the synthesis of collagen RNA, as evidenced by the protected 116-base DNA fragment shown in Fig. 3d. Further- more, high levels of a-amanitin (200 pg/ml), known to inhibit RNA polymerase I11 as well as I1 (32), also failed to eliminate the remaining transcription (Fig. 3e). The amount of resistant transcription, varying from preparation to preparation, was approximately 33% of the total 116-base specific transcription

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activity during a 30-min incubation period. There are two possible explanations for the presence of a-amanitin-resistant transcription products. One is that a-amanitin-resistant RNA polymerase I actually initiates in vitro at the same site on the

a b

=*

z c z

r r T A T A

c d

' GAG - G AG m

Gc - TC

AG - T AC

A GC '2t

AG

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8

FIG. 4. Determination of the in ui tm transcription start site of the chicken a2 collagen gene using RSV-transformed CEF extracts. SI-resistant DNA fragments were prepared as in Fig. 2, and electrophoresed on a 7 M urea-10% polyacrylamide gel. The 158- base 5'-end-labeled DNA probe was sequenced according to Maxam and Gilbert (26), and electrophoresed alongside the S1-protected DNA fragments in the same gel. a, DNA protected from S1 by RNA from HeLa cell extracts using pCa2PRO-3 X Barn HI as template; 6, Maxam and Gilbert reaction specific for G + A c, Maxam and Gilbert reaction specific for G; d, DNA protected from S1 by RNA from chicken cell extracts using pCa2PRO-3 X Barn HI as template. Arrow indicates the location expected for the 116 base-protected DNA fragment. + I marks the in uiuo start of a2 collagen gene transcription. Lane u is underexposed relative to lune d to aid in identifying the initiating nucleotide.

H3 1

250 n Fragment FIG. 5. Schematic representation of modified, in vitro-spe-

cific avian myeloblastosis virus reverse transcriptase-cata- lyzed primer extension. Plasmid pCa2PRO-3 (thick dark line), containing the a2 (I) collagen gene promoter subcloned into pBR322 (wauy lines) via Hind111 linkers (location marked by H3), was used as template in an in vitro transcription reaction (a). The reaction was stopped, and total extract RNA (thin lines) was isolated and hybrid- ized ( 6 ) to the 132-base Rsa I-Hind111 pBR322-specific DNA primer (thick wavy line) 5'-32P-end-labeled (*) at the Rsu I restriction site. Only cell-free RNA was capable of hybridizing to this pBR322-derived DNA fragment. The DNA primer was then extended by avian mye- loblastosis virus reverse transcriptase (c) to the 5' end of the RNA (hatched line). The labeled, extended DNA fragment was electropho- resed on a denaturing polyacrylamide gel and visualized by autora- diography (d). The large white arrow represents the direction of transcription. +1 indicates the start of transcription.

collagen DNA template as RNA polymerase 11. A second possibility is that the a-amanitin-resistant band shown in Fig. 3d is formed when the hybrid molecules consisting of the 158- base end-labeled probe and the longer end-to-end RNA po- lymerase I transcripts are digested preferentially by Sl nu- clease at the start site region, which is AT-rich in the chick a2 (I) collagen gene (2). Such an artifact in S1 nuclease mapping has been described by Hansen et al. (33). T o distin- guish between these two possibilities, we examined in vitro transcription using a modification of the primer extension procedure described by Wickens et al. (29).

I n Vitro-specific Primer Extension-Fig. 5 illustrates the strategy used for in vitro-specific primer extension. A 132- base pair pBR322-specific DNA primer labeled at the 5' end was hybridized to RNA synthesized in chicken extracts off the a2 (I) collagen gene template Bam HI-cleaved pCa2PRO-3. Because the primer was pBR322 DNA it could not hybridize to endogenous collagen RNA, but only to RNAs made in vitro off the pBR322-containing pCa2PRO-3 plasmid template. Avian myeloblastosis virus reverse transcriptase was used to extend the DNA primer to the end of the cell-free RNA, and the resulting DNAs were electrophoresed on a urea-5.5% polyacrylamide gel.

Fig. 6 shows that both HeLa and chicken extracts accurately initiated the synthesis of collagen RNA (lanes a and c). However, accurate transcription is completely inhibited by ru-amanitin in both extracts (Fig. 6, lanes 6 and e), suggesting that only RNA polymerase I1 is capable of initiating transcrip- tion of the a2 (I) collagen gene in vitro. It is likely, therefore, that the RNA polymerase I-specific band visible in Fig. 3 (lanes d and e) results from an S1 nuclease artifact.

To determine if specific factors were necessary for the accurate transcription displayed in Fig. 6 (lanes c and d), the naked a2 collagen gene template pCa2PRO-3 was transcribed

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Homologous Chicken in Vitro Transcription System

a b c d e f g h

I

FIG. 6. Primer extension using cell-free RNA transcripts synthesized off the a2 collagen gene template (pCa2PRO-3). Cell-free RNA, purified free of contaminating DNA by DNase I digestion, was hybridized at 38 "C for 3 h to the 5' end-labeled 132- base pBR322-specific DNA primer. The primer was extended by 1.5 units/pl of avian myeloblastosis virus reverse transcriptase for 60 min (41 "C). The resulting DNA fragments were electrophoresed on a 7 M urea-5.5% polyacrylamide gel and visualized by autoradiography. a, DNA extended by reverse transcription of RNA derived from HeLa cell extracts using pCa2PRO-3 X B u m HI as template; b, same as a + 200 pg/ml of a-amanitin; c, DNA extended by reverse transcription of RNA derived from RSV-transformed CEF cell extracts using pCa2PRO-3 X B u m HI as template + 5 m~ creatine phosphate; d, same as c but without creatine phosphate; e, same as d + 200 pg/ml of a-amanitin; f, m e as d + 50 pg/ml of actinomycin D; g, DNA extended by reverse transcription of RNA synthesized off the pCa2PRO-3 X B a m HI template by partially purified calf thymus RNA polymerase 11; h, overexposure of lane e. Markers a t left are in base pairs. Arrow indicates the expected location of the 250-base extended DNA fragment.

using partially puritied calf thymus RNA polymerase IL2 Fig. 6g shows that initiation of transcription occurred at a large number of sites, but not significantly from the correct in uiuo/ in vitro a2 (I) collagen gene transcription start site. This result indicates that chicken factors (or HeLa factors) are necessary for accurate a2 collagen gene transcription.

Optimization of the Chicken Cell-free System-Because creatine phosphate plus creatine phosphokinase addition was found to increase the efficiency of transcription in HeLa whole cell extracts (X), we determined what effect creatine phos- phate plus creatine phosphokinase would have on RNA tran- scription in RSV-transformed CEF extracts. The effect of creatine phosphate plus creatine phosphokinase on in vitro collagen gene transcription can best be seen in a 90-min time course. Fig. 7A shows that without creatine phosphate plus creatine phosphokinase addition most transcription of the a2 (I) collagen gene by RNA polymerase I1 occurred during the first 20 min; however, when creatine phosphate plus creatine phosphokinase was added, synthesis of collagen RNA tran- scripts by RNA polymerase I1 was dramatically enhanced. Moreover, active RNA polymerase I1 transcription continued throughout the entire 90 min. Further studies showed that creatine phosphate alone would stimulate RNA polymerase I1 activity 5 to 10 times, in agreement with the results of Handa et al. (34). A comparison of a2 collagen gene transcrip- tion with and without the addition of creatine phosphate alone was made using SI nuclease mapping and primer exten- sion. The results are autoradiographically displayed in Fig. 7B

J. S. Tyagi, G. T. Merlino, B. de Crombrugghe, and I. Pastan, manuscript in preparation.

(lanes a and b) and Fig. 6 (lanes d and c), respectively. Relatively high concentrations of creatine phosphate (2-10 mM) were necessary to stimulate transcription (Fig. 8). In contrast, addition of comparable concentrations of creatine or phosphoenolpyruvate had no effect on RNA polymerase I1 transcription (data not shown).

To determine if the chicken extracts would support the accurate transcription of other eukaryotic genes, and to see if that transcription would be enhanced by addition of creatine phosphate, the adenovirus 2 major late gene was examined by S1 nuclease mapping. The plasmid pSmaF, containing the promoter of the adenovirus 2 major late gene, was used as

A

2 70

TIME ( M i n )

B COLLAGEN ADENOVIRUS

a b c d e f

"

FIG. 7. Effect of creatine phosphate on transcription in RSV- transformed CEF extracts. Protected DNA fragments were gen- erated by SI nuclease mapping as described in Fig. 2. A, time course of in vitro transcription of the a2 collagen gene template in the presence (A) or absence (0) of 10 m~ creatine phosphate and 50 pg/ ml of creatine phosphokinase. Autoradiographic bands representing the 116-base, 32P-labeled SI nuclease-protected DNA fragments were used to quantitate the accumulation of accurately initiated collagen- specific RNA transcripts. The bands were scanned by a microdensi- tometer, and the areas under the resulting peaks were determined. These areas are expressed in the above ordinate as arbitrary tran- scription units. In this experiment background activity (a-amanitin- resistant transcription) was subtracted from all data points. B, auto- radiogram showing in vitro SI nuclease mapping of the a2 collagen gene (a-c) and the adenovirus 2 major late gene (d-f). S1 nuclease mapping of transcription of the adenovirus 2 gene (in plasmid pSmaF) was performed the same as for collagen, except that an adenovirus 2- specific 452-base 5' end-labeled probe was used for hybridization, and a 6% polyacrylamide gel was used for electrophoresis. a, DNA pro- tected from SI nuclease by RNA from chicken extracts using pCa2PRO-3 x B u m HI as template; 6, same as a + 5 mM creatine phosphate; c, same as a + 1 pg/ml of a-amanitin; d, DNA protected from SI by RNA from chicken extracts using pSmaF X Sma I as template; e, same as d + creatine phosphate/creatine phosphokinase; f, same as d + 1 pg/ml of a-amanitin. Arrows indicate the location expected for the collagen-specific 116-base-protected DNA fragment (to left) or the adenovirus-specific 197-base-protected fragment (to right).

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70r

CONCENTRATION ImMI

FIG. 8. Effect of various concentrations of creatine phos- phate, GTP, or UTP on a2 collagen gene transcription by RSV- transformed CEF extracts. The accumulation of collagen RNA transcripts was quantitated using S1 nuclease mapping as described in Fig. 6A, and this accumulation was expressed as arbitrary transcrip- tion units (ordinate). Transcription reaction times were 60 min for creatine phosphate (CP) (O), 30 min for GTP (M), and 30 min for UTP (A).

a b c d

c.

FIG. 9. Primer extension using cell-free RNA transcripts synthesized off the pCa2PRO-3 a2 collagen gene template. Extended DNA fragments were prepared and electrophoresed as described in Fig. 5. a, DNA extended by reverse transcription of RNA derived from RSV-transformed CEF cell extracts using pCa2PRO-3 X Bum HI as template; b, same as u + 3 m~ GTP and 3 mM UTP; c, DNA extended by reverse transcription of RNA derived from normal CEF cell extracts using pCa2PRO-3 X Barn HI as template + 5 m~ creatine phosphate; d, DNA extended by reverse transcription of RNA derived from Chinese hamster ovary cell extracts using pCa2PRO-3 X Barn HI as template + 5 m~ creatine phosphate. The Chinese hamster ovary extract was stored for greater than 1 year at -70 "C prior to usage here. Arrows indicate the expected location of the 250-base-extended DNA fragment.

template in chicken cell-free extracts. The transcription prod- ucts were isolated and hybridized to a denawred 452-base Xho I-Hind111 DNA fragment (map units 15.7 to 17.0) (35), "2P-labeled at the 5' end of the Hind111 restriction site. Be- cause the transcription start site was known (5,35), a 197-base single-stranded DNA fragment was expected to be protected from S1 nuclease by accurately initiated RNA transcripts. Fig.

7B, lane d, reveals that accurately initiated RNA transcripts were generated by the chicken extracts upon addition of the adenovirus 2 major late gene template. A comparison of lanes d and e reveals that creatine phosphate addition results in a marked increase in the intensity of the RNA polymerase II- specific band relative to the background bands, until it rep- resents the prominent transcription product. Accurate ade- novirus 2 major late gene in vitro transcription was completely sensitive to a-amanitin (Fig. 7, lane f ) . We conclude that as expected, the effect of creatine phosphate on chicken RNA polymerase I1 activity is of a more general nature.

To help optimize the chicken in vitro transcription system, increasing concentrations of the ribonucleoside triphosphates were added to the extracts and the effect on specific transcrip- tion assayed by S1 nuclease mapping as described above. Surprisingly, high concentrations (2-5 mM) of GTP and UTP were found to stimulate RNA polymerase 11-catalyzed colla- gen gene transcription by a factor of about 5 to 10 (Fig. 8). The effective concentration of these nucleotides is on the same order as that found for creatine phosphate (Fig. 8). Higher concentrations of GTP and UTP either did not stim- ulate, or were actually inhibitory. Comparable levels of ATP and CTP did not significantly enhance RNA polymerase I1 transcription (data not shown).

To prove that GTP and UTP stimulated accurate collagen gene transcription and not just end-to-end polymerization through the transcription start site, RNA generated by cell- free extracts containing Barn HI-cleaved pCa2PRO-3 in the presence or absence of 3 m~ each GTP plus UTP was hybrid- ized to the 132-base pBR322 primer, which was then extended by avian myeloblastosis virus reverse transcriptase. Fig. 9 (lanes a and 6 ) clearly demonstrates that the presence of GTP plus UTP stimulates accurate in vitro transcription. Microdensitometric scanning of this autoradiogram showed that the increase was approximately 6-fold.

DISCUSSION

We here describe an homologous chicken transcription sys- tem in which RNA synthesis is assayed either using a modi- fication of the S1 nuclease mapping procedure (28) or by primer extension. When runoff transcription techniques were intially utilized to analyze transcription of the a2 (I) collagen gene by chicken cell-free extracts only a smear of "P-labeled RNA was seen (Fig. 1). In contrast to HeLa cell extracts which are characterized by very low background activity, the major- ity of the transcription in the chicken extracts was the result of RNA polymerase I (Fig. 1). The RNA polymerase I behav- ior was inexplicable because transcription activity on crude calf thymus template DNA, as determined by UTP incorpo- ration assays, revealed that chick-specific extracts contained only 20% more RNA polymerase I activity than their HeLa counterpart. Furthermore, the smear of transcription activity was observed whether the collagen promoter in plasmid pCa2PRO-3 or the RSV promoter in plasmid pSRI (23) was utilized.

These results suggested that RNA polymerase I was initi- ating and terminating at many different sites on the DNA template. RNA polymerase I was removed from the chicken extracts using DEAE-cellulose and phosphocellulose ion ex- change chromatography in an attempt to visualize less active RNA polymerase I1 transcripts. However, still no specific runoff transcription products synthesized by the deficient extract were detected. Because removal of any part of the whole cell extract could result in the loss of important regu- latory factors, we decided to develop an assay which could detect specific transcripts made from unfractionated chicken extracts.

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7260 Homologous Chicken in Vitro Transcription System

The modified S1 nuclease mapping procedure appeared to be a good choice for several reasons. 1) A small 158-base DNA hybridization probe could be utilized which allowed for tran- scriptional evaluation in a narrow window, eliminating most of the random background activity associated with the 4.7- kilobase pair DNA template. 2) By using a 5’ end-labeled DNA probe (27), a mandatory termination point was estab- lished. Any RNA transcript terminating prior to the 5’ labeled end of the probe would not protect that label from S1 nuclease digestion. 3) The collagen gene transcription start site was located in the middle of the end-labeled probe, so that properly initiated RNA would protect a precisely sized 116- base DNA fragment, smaller than the 158-base probe. The probe which often self anneals rendering it resistant to S1 nuclease migrated as a 158-base fragment and therefore did not interfere with the analysis of transcription. 4) Finally, to distinguish between the newly made cell-free RNA transcripts and endogenous extract RNA, the 158-base DNA probe was end-labeled at the Hind111 linker originally used to subclone the a2 collagen promoter DNA into pBR322. The results, displayed in Fig. 3, reveal that the vast majority of the background activity was eliminated, allowing accurate analy- sis of in vitro transcription. Similarly, in vitro-specific primer extension was used to analyze transcription in the chicken homologous system. This method also has the advantages described for the S1 nuclease mapping procedure but is not subject to artifacts in AT-rich regions (see “Results”). When a pBR322 end-labeled DNA fragment was used as primer for avian myeloblastosis virus reverse transcriptase, specific cell- free transcripts could easily be detected (Fig. 6).

Because we have been successful in analyzing transcription in chicken extracts, we believe that modified S1 nuclease mapping and primer extension will be of value in analyzing other in vitro transcription systems. To test the applicability of these techniques, we have examined transcription of the chicken a2 (I) collagen gene in an extract derived from Chinese hamster ovary cells. Cell-free RNA from Chinese hamster ovary extracts was analyzed by avian myeloblastosis virus reverse transcriptase-catalyzed primer extension as described above. Fig. 9d reveals that these extracts are capable of accurately transcribing the chicken collagen gene. Analysis of these extracts by runoff transcription, however, revealed only a smear of activity similar to that observed for both normal CEF and RSV-transformed CEF extracts.

It is worth mentioning that these methods are extremely sensitive, and one must be wary of artifacts when investigating extracts whose transcriptional activity is relatively weak. This is evidenced by the formation of RNA polymerase I-specific bands of the correct size during S1 nuclease mapping of RNA synthesized by chicken cell-free extracts (Fig. 3, lanes d and

While optimizing this system, we found that relatively high concentrations of GTP and UTP were capable of stimulating in uitro collagen gene transcription. Because the molar con- centrations of GTP and UTP were much higher (2-5 a) than those expected to be sufficient for RNA polymerization, it is conceivable that these nucleotides were acting in vitro at some other level. Phosphorylation is one obvious candidate for a mechanism by which nucleotides can affect macromolec- ular functions. There is considerable support for the notion that a phosphorylation-dephosphorylation mechanism plays a role in regulating RNA polymerase activity both in vivo and in vitro (for review, see Ref. 36). However, the reason for the observed stimulation remains to be elucidated.

Although in uitro-specific S1 nuclease mapping and primer extension allowed detailed analysis of transcription by RSV- transformed CEF extracts, we have not yet been able to detect

e) .

accurate transcriptional initiation of the a2 (I) collagen gene in extracts derived from normal CEF. When cell-free RNA synthesized by normal CEF extracts was analyzed by primer extension as described above, no 250-base extended DNA fragment was formed, indicating that accurate transcription was not occurring (Fig. 9c). However, the CEF extracts were not deficient in RNA polymerase 11, as determined by UTP assays. There must, therefore, be additional reasons for the failure of normal CEF to exhibit accurate in vitro transcrip- tion. Groudine and Weintraub (37) have found that RSV transformation of CEF results in RNA accumulation from about lo00 new transcription units. It is therefore possible that relative to RSV-transformed CEF, normal CEF keep much tighter control over in vivo RNA polymerase II-cata- lyzed transcription; furthermore, this gross difference in gene regulation may be mirrored in vitro. We have identified an inhibitor of in vitro RNA polymerase I1 activity present in chicken embryos and in CEF.’ We are presently examining the relationship between this inhibitor and gene regulation in chicken cells.

Acknowledgments-We wish to thank P. A. Weil for the plasmid pSmaF, B. Lovelace and A. Harris for culturing the chicken cells, R. Coggin for typing the manuscript, and R. Steinberg for taking the photographs. We are grateful to I. Dawid, G. Khoury, C. McKeon, and H. Ohkubo for useful discussions concerning this project.

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G T Merlino, J S Tyagi, B de Crombrugghe and I Pastancell-free extracts.

Transcription of the chicken alpha 2 (Type I) collagen gene by homologous

1982, 257:7254-7261.J. Biol. Chem. 

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