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The Major Late Promoter of Adenovirus 2 (In Vitro Mutagenesis/RNA Polymerase Wi/RNA Cap Site/Eukaryotic Promoter) SHIU-LOK Hu* and JAMES L

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The Major Late Promoter of Adenovirus 2 (In Vitro Mutagenesis/RNA Polymerase Wi/RNA Cap Site/Eukaryotic Promoter) SHIU-LOK Hu* and JAMES L Proc. Nati Acad. Sci. USA Vol. 78, No. 2, pp. 820-824, February 1981 Biochemistry DNA sequence required for initiation of transcription in vitro from the major late promoter of adenovirus 2 (in vitro mutagenesis/RNA polymerase Wi/RNA cap site/eukaryotic promoter) SHIU-LOK Hu* AND JAMES L. MANLEYtt *Cold Sp ng Harbor Laboratory, Cold Spring Harbor, New York 11724; and tDepartment of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Communicated by A. D. Hershey, October24, 1980 ABSTRACT We have identified a region of the viral genome MATERIALS AND METHODS required for the initiation of transcription in vitro from the major late promoter of adenovirus 2. A fragment of the adenovirus ge- nome containing the cap site of the major late transcripts was in- Enzymes and Reagents. Phage T4 DNA ligase and restriction serted into plasmid pBR322 and cloned. Deletions were then gen- endonucleases Alu I and Ava I were purchased from New En- erated in vitro in and around the T-A-T-A-A-A-A sequence located gland BioLabs. Other restriction enzymes and nuclease BAL 25-31 nucleotides (positions -25 to -31) upstream from the cap 31 were obtained from Bethesda Research Laboratories (Rock- site. DNAs with these deletions were tested for their ability to ini- ville, MD). T4 polynucleotide kinase was obtained from P-L tiate transcription in vitro by the method ofManley et al [Manley, J. L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L. (1980) Proc. Biochemicals and calfintestine alkaline phosphatase from Boeh- NatL Acad. Sci USA 77, 3855-3859]. Whereas removal of se- ringer Mannheim. Synthetic EcoRI and HindIII linkers were quences upstream from position -47 or downstream from position obtained from Collaborative Research (Waltham, MA). ['y- -12 did not abolish transcription, deletions extending into, or be- 32P]ATP (>2000 Ci/mmol; 1 Ci = 3.7 x 10"° becquerels) was yond, the T-A-T-A-A-A-A sequence reduced transcription to less purchased from Amersham and [a-32P]UTP from ICN. All en- than 1/10th. Removal of the normal cap site slightly reduced, but zymatic reactions, unless otherwise stated, took place under did not abolish, transcription. These results indicate that the re- gion of the genome upstream of the cap site, with boundaries conditions specified by the manufacturer. within 15-17 nucleotides to either side of the T-A-T-A-A-A-A se- Construction of Late Promoter Clones. Ad2 DNA was iso- quence, is required for the initiation oftranscription in vitro from lated from purified virions grown in HeLa cells (11). One mi- the major late promoter ofadenovirus 2. crogram ofAd2 DNA was digested to completion with HindIII and the fragments produced were ligated (15'C, 16 hr) to 1 ,ug Control oftranscription constitutes an important step in the reg- of HindIII-cut DNA of plasmid pBR322. Transformation of ulation ofgene expression. Studies ofprokaryotic systems indi- Escherichia coli X1776 was performed according to unpublished cate that the initiation ofmRNA synthesis requires interactions procedures of D. Hanahan. Ampicillin-resistant colonies were between RNA polymerase and a specific region of DNA up- picked and screened for their plasmid inserts (12). Clones ofall stream from the gene to be transcribed, called the promoter (1). internal HindIII fragments of Ad2 DNA were obtained. The Similar attempts to identify promoters for eukaryotic genes HindIll C clone (pAd6) contains adenovirus sequences from have been handicapped by the lack ofsuitable genetic and bio- coordinate 8 to 17. The cap site of the major late transcripts is chemical systems. Recently, however, developments in recom- located 193 nucleotides from the HindIII site at 17. This clone binant DNA technology and the advent ofin vitro transcription was used as one ofthe late promoter clones for the construction systems offer an approach to the study ofthe molecular mecha- of deletion mutants. Another late promoter clone, called p44, nisms oftranscriptional controls in eukaryotic systems (2-5). was constructed by ligating the 430-base-pair (bp) Alu I frag- The major late transcription unit ofadenovirus type 2 (Ad2) is ment of pAd6 to the large fragment of EcoRI-HindflI-cut one of the best characterized in eukaryotes. During the late pBR322 DNA by using EcoRI and HindIII linkers (see Fig. 1). stage ofAd2 infection, the major viral transcript is initiated at a This clone contains adenovirus sequence complementary to the site around coordinate 16.5 and elongated toward the right end first 33 nucleotides of the major late transcript and about 400 of the genome (6, 7). This primary transcript is believed to be bp upstream ofthe cap site. The structures ofthese clones were processed into several families ofcytoplasmic messenger RNAs determined by restriction endonuclease analysis and, in the case that all have the same capped 5' end as the primary transcript of p44, by DNA sequencing. (7, 8). Twenty-five to 31 nucleotides upstream from the RNA cap Construction of Deletion Mutant Clones. Both pAd6 and site, there is a stretch of A+T-rich sequence (T-A-T-A-A-A-A) p04 were used as starting materials for the construction ofdele- that appears to be conserved in a number of eukaryotic genes tions upstream from the cap site. Three micrograms ofpAd6 or transcribed by RNA polymerase II (9). The similarity of this p04 DNA was linearized with Xho I and then digested with A+T-rich sequence and the "Pribnow box" ofprokaryotic pro- nuclease BAL 31 (0.5 unit in a 50-,ul reaction mixture) at 30°C moters has been noted (10). We report here experiments de- for 3-5 min. Nuclease reactions were stopped by dilution with signed to determine the role of this A+T-rich sequence in the 10 mM Tris HCl, pH 7.9/1 mM EDTA followed by extractions initiation oftranscription in vitro from the major late promoter with phenol and chloroform and precipitation with ethanol. The ofAd2. resulting fragments were ligated to 20-fold molar excess of The publication costs ofthis article were defrayed in part by page charge Abbreviations: Ad2, adenovirus type 2; bp, base pair(s); kb, kilobase(s). payment. This article must therefore be hereby marked "advertise- t Present address: Department of Biology, Columbia University, New nmnt" in accordance with 18 U. S. C. §1734 solely to indicate this fact. York, NY 10027. 820 Downloaded by guest on September 30, 2021 Biochemistry: Hu and Manley Proc. NatL Acad. Sci. USA 78 (1981) 821 FIG. 1. Cloning of the Ad2 major late promoter and the construction of deletion clones. The left side of the fig- ure illustrates the cloningoftheregion Xho I of Ad2 genome containing the major ; ' s late promoter. Structure 1 represents Ad: NrlA _j 4 #r _VVP~W 1 the parental clone, pAd6, used to con- Hind Xho I Hind. struct upstream deletion clones. Struc- -1 EcoRI HindIII ture 2 represents p44, an Alu I sub- Bal31 clone of Ad6, used to construct both Xho I, upstream and downstream deletion i clones. The scheme to pBR322 I f, - ~ .WA used construct -TT* ITYYv +M. .,_f --isM EcoRI HindIII Alu I Alu I HindIII EcoRI HindIII + EcoRI H upstream deletion clones from pAd6 is + + V linkers HindIII illustrated on the right side of the fig- EcoRI linkers HindlIII linkers ure. See text for details. Heavy solid lines represent adenovirus genome. XhoI I Wavy lines represent se- At EcoRl HindIII 1EcoRI Hind][IIHOPI pBR322 VEco quences. Restriction endonuclease sites 2 AepI /V ZZZv-*ZZ ZZ ZZ2> _ are indicated by upward or downward EcoRA HindIII arrows. The relevant positions of HindIII Hindr cleavage sites on Ad2 DNA are '_ A iLigate,' ,,' indicated in % Ad2 map units. The po- sition of the cap site and the direction of major late transcripts are indicated EcoRI HindIII by open arrows. polynucleotide kinase-treated EcoRI linkers and then redigest- templates and analyzed on a 1.4% agarose gel (Fig. 2, lanes ed with EcoRI. The DNA fragments were separated from un- 3-7). Transcripts of lengths 4.36, 3.7, 1.43, 0.65, and 0.37 kil- ligated linkers by passage through a Sepharose 4B column obases (kb) were observed for p44 DNA cut by EcoRI, Pst I, equilibrated with 0.15 M NaCl/10 mM Tris-HCl, pH 7.9/1 mM Ava I, HincHI, and BamHI enzymes, respectively. These are EDTA. Excluded fractions from the column were pooled and the sizes expected for transcripts that initiated at the Ad2 cap the DNA was precipitated by ethanol. This DNA was religated site, continued into pBR322 sequences, and terminated at the and used for the transformation of E. coli. Ampicillin-resistant end ofthe DNA fragment. These results indicate that transcrip- clones were selected and the clones with plasmids of appro- tion initiates accurately from the Ad2 major late promoter lo- priate sizes were identified (12). The extent of deletion was estimated by the size of fragments produced by Hae III diges- pBal E p356 tion and resolved by electrophoresis in 7 M urea/8% acrylamide 1 2 3 4 D 6 7 gels. Exact endpoints of the deletions were located by the DNA sequence determination method of Maxam and Gilbert (13). The procedure is summarized in Fig. 1. Plasmid p44 was used 4.36- as the starting material for the construction of deletions down- -3.61 stream from the cap site. The experimental design was analo- gous to that for the construction of 5' deletions, except that 1.75-u HindIII was used for the initial linearization of p44 and that, 1.43- _ after a brief digestion (0.5-1.5 min) with nuclease BAL 31, HindIII linkers were used to recircularize the plasmid.
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