A Specific DNA Sequence Controls Termination of Transcription in

MOLECULAR AND CELLULAR BIOLOGY, Apr. 1986, p. 1032-1043 0270-7306/86/041032-12$02.00/0 Copyright © 1986, American Society for Microbiology

Vol. 6, No. 4

A Specific DNA Sequence Controls Termination of Transcription in the Gastrin Gene KENZO SATO,' RYOJI ITO,' KWANG-HEE BAEK,1 AND KAN AGARWALl 2* Departments of Chemistry2 and Biochemistry and Molecular Biology,' The University of Chicago, Chicago, Illinois 60637 Received 16 October 1985/Accepted 24 December 1985

We located and characterized a downstream transcriptional regulatory element in the human gastrin gene by transferring the gastrin gene 3' fragment, from which the polyadenylation signal sequence was deleted, into the shuttle vector pSCAT10 at a site located immediately downstream from thI chloramphenicol acetyltransferase (CAT) gene and upstream from the simian virus 40 polyadenylation region. Study of CAT RNA derived from the hybrid plasmids, indicated regulation of transcription on the gasirim gene fragment. Analysis of deletion mutants generated from the 5' region of the fragment by CAT assay and by Si nuclease mapping of mRNAs indicated the possible involvement of an oligothymidylate-rich sequence in transcription regulation. Mapping of gastrin gene RNA 3' ends to the 5' side proximal to the oligothymidylate-rich sequence clearly demonstrated that this sequence is a transcriptional terminator element. This unique sequence, interspersed with one or two adenines, which also functions in an orientation-dependent manner, is located 192 nucleotides downstream from the gastrin gene polyadenylation site, and serves as a transcriptional termination signal.

Formation of proper 3' ends of transcripts are important regulatory events in eucaryotic and procaryotic gene expression. In Escherichia coli, termination of transcription by RNA polymerase generally occurs at specific sites on the DNA template and is modulated by the transcriptional termination factor rho, in vivo (44). In contrast, the termination sites for the majority of eucaryotic RNA polymerase II transcription units have not yet been defined (5). It is known, however, that RNA polymerase II transcripts undergo processing at their 3' ends by two different processing mechanisms. One of these mechanisms occurs during processing of histone nascent transcripts, which generally contain an inverted repeat sequence of about 30 to 40 bases downstream from the protein termination codon (4, 38). The mature histone mRNAs thus carry a unique stem-loop structure on their 3' ends. (12) The other mechanism has been observed in processing of transcripts coding for proteins other than histones. These transcripts possess a polyadenylated [poly(A)'] 3' terminus approximately 20 nucleotides downstream from an invariant hexanucleotide sequence, AAUAAA (14, 39). Since processing of both types of transcripts requires the presence of approximately 100 nucleotides past the processing site (31, 40), transcription must terminate near or past these required sequences. Indeed, from the study of transcriptional termination of the ,-globin (major) gene (8, 10, 22, 41), the ovalbumin gene (25), and the a-amylase Amy2a gene (18), it is implied that termination occurs within the region between 700 and 1,500 nucleotides, at 900 nucleotides, and at 2.5 kilobases (kb) and 4 kb, respectively, downstream from the polyadenylic acid [poly(A)] addition site. These studies, however, did not reveal specific or favored sites for transcription termination. The biological significance of transcription terminating far past the poly(A) processing site is unknown. There has been little evidence that a defined sequence of DNA causes transcription to terminate at a specific site. However, transcription termination in yeast appears to be linked to *

polyadenylation of mRNA. For some yeast genes, when the terminal mRNA sequence is CAATGCTITG, the alternative motif 'I'ITT'ATA has been proposed as a yeast terminator (20, 21). To understand the mechanism(s) by which DNA sequences regulate structure and expressioh of transiatable genes, we began studying the role of the 3' region'of the gastrin gene In transcriptional regulation in vivo. In this paper, we describe results of our studies which show that 3' regulation of transcription of the chloramphenicol, acetyltransferase (CAT) gene may be regulated by a 3' flanking region of the gastrin gene, when the latter is placed downstream from the CAT gene translation termination codon. The putative transcriptional 3' regulator elenient, as identified by analysis of deletion mutants and Si mapping of the in vivo transcripts, is composed of a unique stretch of oligothymidylates. The direct involvement of this sequence in transcriptional termination is based on the evidence that gastrin gene RNA 3' ends map to the 5' proximal region of this sequence.

MATERIALS AND METHODS Cells, plasmids, enzytnes and reagents. CV-1 cells were a gift of K. Subramanian and were grown in Dulbecco modified Eagle medium supplemented with glutamine (2 mM), penicillin (1,000 U/ml), streptomycin (100 ,tg/ml), and 10% fetal bovine serum. Plasmid pSV2CAT was a gift from G. Jay. Plasmid pMC1429 was a gift from M. Casadaban. All other plasmids used in these studies were constructed and isolated with E. coli LE392. T4 DNA ligase and DNA polymerase (Klenow fragment) were purchased from Boehringer Mannheim. Restriction enzymes were purchased from New England Biolaboratories or Boehringer Mannheim and were used as directed by the supplier. ExollI and Si nucleases were purchased from Bethesda Research Laboratories. CAT and o-nitrophenyl-pD-galactopyranoside were purchased from Sigma Chemical Co. All other chemicals were of the highest commercial grade available.

Corresponding author. 1032

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Transfection procedure. To introduce vector DNA into cells, the calcium phosphate precipitation procedure described by Gorman et al. (16) and modified by Luthman and Magnusson (27) was used. The DNA for transfection (total, 10 ,ug) was precipitated by the procedure described previously and combined with the medium containing 400 ,uM chloroquine; the total mixture was then added directly to the cells. After a 4-h incubation at 37°C in a CO2 incubator, the cells were washed four times with serum-free medium and then incubated at 37°C in 10% fetal bovine serum-containing medium until they were harvested. CAT and P-galactosidase assay. The cells were harvested at 48 h after transfection and assayed for CAT and galactosidase activities. The cell extract was prepared in 50 ,ul of 250 mM Tris hydrochloride (pH 7.8)-i mM phenylmethylsulfonyl fluoride-0.5% Nonidet P-40 by vortexing over a period of 20 min at 4°C. The cell extract was centrifuged for 10 min at 4°C, and the supernatant was used in the enzyme assays. For the CAT assay, 5 pAl of cell extract, 5 ,ul of 10 mM acetyl coenzyme A, 0.2 p.l of 100 mM chloramphenicol, and 40 p1 of 250 mM Tris hydrochloride (pH 7.8) were incubated at 37°C for 1 h. The reaction mixture was extracted with 400 p.l of ethylacetate, and the organic layer was evaporated. Unreacted chloramphenicol was separated from the mono- and diacetylated forms by the silica gel high pressure-liquid chromatography procedure described by Hartzell et al. (19), using chloroform-methanol (95:5). The peak for acetylated chloramphenicol was integrated by an automatic integrator. Values of percent acetylation within the linear range are indicated. Each transfection experiment was carried out in triplicate, and the values of CAT activities which deviated within 5% were considered valid and are reported here. For the p-galactosidase assay, the remaining 20 p.l of cell extract supernatant was added to 175 p.l of 10 mM KCI-1 mM MgSOt-100 mM sodium phosphate-50 mM 2-mercaptoethanol; the pH was adjusted to 7.5. This solution was preincubated at 37°C for 5 min and then 66 p.l of onitrophenyl-,3-D-galactopyranoside (4 g/liter in 100 mM sodium phosphate [pH 7.5]) was added, and the incubation was continued at 37°C for an additional 1 h. The reaction was terminated by addition of 86 p1l of 1 M Na2CO3, and absorbance at 420 nm was measured. Cloning of DNA fragments into M13 phage vectors. All of the 3' gastrin gene restriction fragments, varying in length from 100 to 1,000 nucleotides, were subcloned into mp8 and mp9, mplO and mpll, or mpl8 and mpl9, depending on the restriction site availability in the different M13 phage vectors. These M13 phage recombinants served as the insert source for deletion mutagenesis, and for subcloning into expression plasmids. Construction of deletion mutants. The deletion mutants involving the hexanucleotide AATAAA and surrounding regions were generated by linearization by Bcl of the replicative form (RF) DNA mpllO19, which contains the HindIII(2)-HindIII(3) fragment of the 3' region of the gastrin gene, followed by successive treatment with ExoIll and Si nucleases under controlled conditions of incubation temperature and time (17). The DNA ends were made blunt by treatment with Klenow fragment and circularized by T4 DNA ligase. Transfection of JM101 yielded phage, some of which were randomly selected for DNA isolation (32). The precise position of each deletion was determined by nucleotide sequence analysis. The deletion mutants involving the 3' regulatory region were generated by linearization of clone mp9703 (containing

Polll TERMINATION IN THE GASTRIN GENE

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the 4vaII-BanI fragment) by EcoRI, followed by digestion with slow-acting BAL 31 endonuclease for different times. The approximate extent of deletion, at a given time of incubation, was determined by electrophoresis of HindlIl digests on 5% polyacrylamide gels. The reaction mixture, which contained clones with approximately 50 to 150 nucleotides removed, was worked up by following a procedure described previously (26). The inserts of deletion mutants were excised by digestion with HindIII and separated and isolated by agarose gel electrophoresis and electroelution. The DNA ends were made blunt by treatment with Klenow fragment and ligated into SmaI-digested, calf intestinal alkaline phosphatase-treated mpl9 RF DNA. The resulting plaques from transfections of JM101 were randomly selected, and their DNAs were isolated. Nucleotide sequence analysis of phage DNAs established the precise positions of the deletions. Construction of expression plasmids. Two different types of pSCAT plasmids were constructed by deletion of the simian virus 40 (SV40) t-splice region from the pSV2CAT plasmid. Both pSCAT plasmids contained a polylinker region immediately downstream from the CAT gene. One of the constructs, pSCAT10, contained the SV40 polyadenylation region, while in the other, pSCAT6, this region was absent. The construction of all the plasmids described in this paper followed standard procedures of restriction digestion, DNA modification, ligation, and analysis of subclones (28). All of the subclones were characterized by detailed restriction endonuclease mapping, and in some cases, by njicleotide sequence analysis. The plasmid DNAs frohn thoroughly characterized clones were purified by equilibrium density centrifugation in CsCl before their use in DNA transfection assays.

Construction of plasmids pSCAT6 and pSCAT10 followed the strategy outlined in the legend to Fig. 1. The pSV2CAT plasmid was doubly digested with Hindlll and BamHI, and the resulting two fragments were isolated by agarose gel electrophoresis and electroelution. The snmall fragment that contained the CAT gene was further digested with MboI and isolated. This CAT gene fragment was ligated to the large Hindlll-BamHI fragment (see Fig. 1). The resulting plasmid, pSCAT6, was digested with BamHI, made blunt-ended with Klenow fragment, and ligated to the blunt-ended polylinker. Two types of plasmids were identified by restriction endonuclease mapping. They differed from each other in their polylinker orientation with respect to the CAT gene. For construction of pSCAT10 plasmids, the pSV2CAT plasmid was doubly digested with HpaI and HindlIl, and the large fragment containing the ampicillin gene was isolated. This fragment, the blunt-ended polylinker fragment, and the CAT gene fragment, isolated from pSCAT6 by following the steps indicated in the legend to Fig. 1, were ligated together. Extensive screening by restriction endonuclease mapping resulted in the identification of two plasmids, pSCAT10 pll and pSCAT10 p12, consistent with the structures shown in

Fig. 1.

Plasmids pSG24 and pSG12 were constructed by using the following strategy. The pSCAT10 plasmid was dQubly digested with HindIII and SmaI, and the large fragment lacking the CAT gene was isolated from the genomic DNA clone by agarose gel electrophoresis and electroelution. For construction of the pSG24 plasmid, the gastrin gene fragment, StuI-BanI was isolated, and the ends were made blunt and ligated to the blunt-ended large fragment. Extensive restriction endonuclease mapping resulted in the identification of the pSG24 plasmid. Similarly, for the construction of

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MOL. CELL. BIOL.

SATO ET AL.

the pSG12 plasmid, the gastrin gene fragment StuI-BanI was first inserted into M13 mpl9 DNA at the SmaI site and was then doubly digested with BclI and TthlllI. The resulting large fragment was isolated, made blunt-ended, and circularized by ligation. The mpl9 RF DNA was doubly digested with EcoRI and SaI, made blunt-ended and ligated to the large fragment isolated from plasmid pSCAT10 as described above. The resulting plasmid pSG12 was characterized by extensive restriction endonuclease mapping. RNA analysis. Total RNA was extracted from cells 24 h after transfection by the guanidine isothiocyanate procedure described by Chirgwin et al. (6). The DNA and proteins were removed from the RNA by centrifugation through 5.7 M CsCl (15, 37). Poly(A)-enriched RNA was isolated by chromatography on oligodeoxythymidylate cellulose (2). Both CAT poly(A)-enriched RNA and gastrin total RNAs were analyzed by S1 nuclease mapping (3). Three labeled probes were used for S1 analysis. Probe 1 corresponded to the SV40 poly(A) region, HpaI-BamHI fragment; probe 2 spanned the gastrin gene AvaII-BanI fragment, and probe 3 represented the gastrin gene fragment ApaI-HindIII(2). Single-stranded, labeled probes were prepared by using the primer extension-restriction enzyme digestion method (24). Briefly, 2 ,ug of recombinant M13 phage DNA, sequencing primer (0.5 pmol, 15-mer), dCTP, dTTP, dGTP (each 20 ,uM), and (a-32P)dATP (15 pmol, 3,000 Ci/mmol) were treated in 12 ,ul of sequencing buffer containing 1 U of Klenow fragment, at 23°C for 40 min before being chased by 0.5 mM dATP for 2 min. The labeled DNA was digested by the appropriate restriction endonuclease, and the strands were denatured and separated by electrophoresis on a 5% polyacrylamide gel as described by- Maxam and Gilbert (30). Hybridization of an appropriate amount of RNA and probe for S1 analysis was carried out in 50%o formamide at 50°C for 12 h. Each sample was then treated with 100 U of S1 in a total volume of 100 ,Jl. The S1 nuclease samples were analyzed by electrophoresis on 6% acrylamide-8 M urea gels. RESULTS

Experimental design. To identify the DNA region which regulates transcription of the gastrin gene downstream from the polyadenylation site, we have designed a strategy which allows the study of the regulation of transcription in vivo. This strategy involves placement of gastrin gene 3' fragment(s) in the pSCAT10 plasmid, between the CAT gene and the SV40 polyadenylation region. If the inserted DNA fragment, which lacks the gastrin polyadenylation region (11), contains a 3' transcriptional regulatory region, transfection of the hybrid plasmids into CV-1 cells should result in unstable, non-polyadenylated [poly(A)-] transcripts. On the other hand, if the fragment lacks a 3' transcriptional regulatory region, then relatively stable poly(A)+ transcripts should result. The poly(A)t transcripts can then be identified by a positive CAT assay, whereas poly(A)+ transcripts can be assumed from the absence of the CAT gene product. The assay for CAT enzyme activity in the transfected cells is rapid, simple, and highly sensitive and thus offers an attractive system for initial screening of the 3' transcriptional regulatory element. The problem of varying levels of CAT activity from experiment to experiment can be controlled by including a shuttle vector, pMC1924, which contains an easily selectable E. coli ,-galactosidase gene, in cotransfection of pSCAT10 and pSCAT6 plasmid derivatives. The ratio of the levels of CAT and ,-Gal activities from the same cell

extract supernatant should be similar from one transfection to another and thus should generate confidence in the levels of CAT activity observed. Once the presence of a 3' transcriptional regulatory region is indicated on a fragment, its location can be determined to within 100 to 200 base pairs by employing shorter DNA restriction fragments in these screening experiments. Stepwise deletion mutagenesis of the fragment should reveal the DNA sequence that may be involved in 3' transcription regulation. Si nuclease mapping of the 3' region of the CAT transcript should reveal the positional relationship of the regulatory sequence and the 3' end of the nascent transcript. In the event that CAT RNAs [poly(A)+ and poly(A)-] cannot be satisfactorily mapped due to their extreme lability, the CAT gene can be replaced by the gastrin gene lacking a polyadenylation signal in the plasmid constructs. Unlike CAT RNA, the gastrin RNA derived from these plasmids should exhibit greater stability in CV-1 cells, allowing mapping of their 3' ends. If gastrin

RNA 3' ends map to the 5' proximal site of the regulatory sequence and transcripts corresponding to the downstream region from the regulatory sequence (resulting from a processing event) are not found, then the results of these studies would strongly suggest, although do not prove, that the regulatory sequence is an RNA polymerase II transcriptional terminator. Construction of CAT gene expression plasmids lacking the SV40 t-splice region. It is possible that newly placed DNA fragments, downstream from the SV40 t-splice and upstream from the SV40 polyadenylation region, may cause a different and unexpected mode of transcript processing which may complicate the study of transcriptional termination. We chose therefore to construct a pSV2CAT plasmid derivative that lacked the entire SV40 t-splice region. The deletion of the SV40 t-splice region would simplify these studies and not affect the efficiency of CAT gene expression. The plasmids were constructed by employing the scheme illustrated in Fig. 1. Since pSCAT plasmids may have general use in studying 3' end processing of transcripts, a polylinker region was inserted immediately next to the 3' end of the CAT gene. The presence of the polylinker greatly simplifies the insertion and characterization of a DNA fragment in this region. As shown in Fig. 1, two sets of pSCAT plasmids were constructed; one set, pSCAT6 pll and p12, contained polylinkers in both orientations, and the other set, pSCAT10 pll and p12, contained the SV40 polyadenylation region in addition to the polylinker in both orientations. A brief description of the constructions of these plasmids was given above. The four plasmids were thoroughly characterized by constructing detailed restriction endonuclease maps. Isolation and characterization of deletion mutations surrounding the gastrin AATAAA sequence. The unique Bcll site, located two nucleotides upstream from the hexanucleotide sequence (Fig. 2), was employed in the generation of deletions on both sides of this restriction site (46). The BclI-digested M13 mpllO19 clone was subjected to successive treatments with ExoIllI and S1 nucleases under reaction conditions which removed only a few nucleotides. Several deletion mutants were identified by nucleotide sequence analysis. The nucleotide sequence of one such mutant is given in Fig. 2, which shows deletion of the AATAA in addition to 15 upstream nucleotides. This deletion mutant was chosen for further study. For construction of pSCAT10 and pSCAT6, plasmids containing gastrin gene 3' fragments (including and excluding the polyadenylation signal sequence), M13 RF DNAs containing these segments were cleaved at the appropriate restriction sites of the polylinker

Polll TERMINATION IN THE GASTRIN GENE

VOL. 6, 1986

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FIG. 1. Construction scheme for plasmids pSCAT10 (pll and p12) and pSCAT6 (p1l and p12). For deletion of the SV40 t-splice region and the SV40 polyadenylation region, pSV2CAT plasmid was doubly digested with HindIII and BamHI. The resulting large (3.4-kb) and small (1.6-kb) fragments were isolated. The CAT gene fragment, generated by further digestion of the small fragment with MboI, was isolated by agarose gel electrophoresis. Ligation of the CAT gene and large fragment resulted in the pSCAT6 plasmid. To insert a polylinker, the pSCAT6 plasmid was digested with BamHI, and the ends were made blunt with Klenow fragment and then ligated with blunt-ended polylinker containing the restriction sites as indicated. This construction generates two plasmids, pSCAT6 pll, containing the polylinker in one orientation and pSCAT6 p12 with an inverse orientation with respect to the CAT genes as indicated. For the construction of plasmid pSCAT10, which lacks only the SV40 t-splice region, plasmid pSV2CAT was doubly digested with HindIII and HpaI, and the resulting large fragnent was isolated. The CAT gene fragment was generated by linearization of pSCAT6 with BamHI, followed by repair of ends with Klenow fragment, further cleavage by HindIII, and then isolation of the CAT gene fragment. Ligation of the large ampicillin-gene-containing fragment, the CAT gene fragment, and the blunt-ended polylinker generated two sets of plasmids, pSCAT10 pll and pSCAT10 p12 as indicated.

regions. These fragments were purified and then ligated to the appropriately linearized plasmids pSCAT10 and pSCAT6. Expression analysis of pSCAT plasmids containing gastrin gene 3' fragments. To determine whether the levels of CAT activity were dependent on the presence of a polyadenylation signal (11), a comparison of the levels of CAT activity generated by the plasmids pSCAT6, pSCAT10, and pSV2CAT was made. As a control for transfection efficiencies and cell extraction procedures in most of the experiments described in this paper, an internal reference plasmid (pMC1924) containing the E. coli P-galactosidase gene (36) was included in each transfection. The same extract used to assay the CAT activity was assayed for P-galactosidase activity. The transfection efficiencies were very close to equal among all the samples, differing by no more than a factor of two (Fig. 3). Thus, the low CAT activity (less than 2%) from plasmid pSCAT6 and high CAT activity (greater than 90%) from plasmid pSCAT10, taking pSV2CAT activity as 100%, showed that the CAT activity generated by these plasmids is absolutely dependent on the presence of a polyadenylation signal. This is an important observation since our strategy is based on the dependence of

CAT activity on the presence of a polyadenylation signal region. Having established the suitability of the pSCAT10 and pSCAT6 plasmids for the study of 3' transcriptional regulatory regions, we inserted gastrin gene 3' fragments into the polylinker sites of these plasmids. The resulting hybrid plasmids were thoroughly characterized by detailed restriction endonuclease mapping and the levels of CAT activity directed by each of the plasmids were measured. Plasmids pSCAT10-103 and pSCAT6-23, as expected, generated similar levels of CAT activity, because in each case the 3'-end processing of transcripts is under the control of the gastrin polyadenylation sequence (Fig. 3). When the gastrin polyadenylation signal sequence was deleted from fragment I, the resulting plasmids, pSCAT10-111 and pSCAT6-33, generated less than 10o CAT activity (Fig. 3, table). These results were highly significant, considering that plasmid pSCAT10-111 contained an SV40 polyadenylation region next to the 3' end of gastrin gene fragment I, which lacks a polyadenylation signal region. A high level of CAT activity would be expected from this plasmid if the entire gastrin gene fragment and the SV40 polyadenylation region were transcribed. Since a very low level of CAT activity was

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AGCCCTGTC CCCTGAAAAA CTGATCAAAA ATAAA CTAGT TTCCAGTGGA TCAATGGACT GTGTC Bcl I TthI11I FIG. 2. Restriction endonuclease map of the human gastrin gene, an extended map of the HindIII(2) to EcoRI region, and the polyadenylation sequence region. The top line shows the restriction endonuclease map of the 7.8-kb DNA that contains the gastrin gene. The thick black lines identify exons; introns are represented by the thin line between the exons. The middle region of the figure shows an expanded restriction endonuclease map of the HindIII(2) to EcoRJ region. The restriction sites used in subcloning and the position of the polyadenylation signal sequence are indicated. The bottom portion of the figure shows the sequence surrounding the polyadenylation region. The open bar under the sequence shows the nucleotide regions that were deleted (for details see the text).

generated from plasmid pSCAT10-111, a 3' transcriptional regulatory region must be present on gastrin gene fragment I. Furthermore, the loss in ability to express the CAT gene product appears to be due to the presence of a 3' regulatory element on the gastrin gene fragment I, rather than to a variation in transfection efficiency. To determine whether additional 3' transcriptional regulatory regions are present on the other downstream region fragments, plasmids containing gastrin gene fragments II and III (Fig. 3) were constructed. The 82% CAT activity from plasmid pSCAT10135, which contains gastrin gene fragment II (see Fig. 3), is similar to the 88% activity from plasmid pSCAT10-103, which indicates the absence of a 3' transcriptional regulatory region on fragment II. Similar results were obtained when the plasmid containing fragment III was examined. The absence of a 3' transcriptional regulatory region on fragments II and III, which span a 1.4-kilobase-pair region, indicates that the low CAT activity generated by plasmid pSCAT10-111 is not artificial. To precisely locate the 3' transcriptional regulatory region, we constructed several pSCAT plasmids, containing DNA fragments, covering the entire region of gastrin gene fragment I. The low level (7%) of CAT activity generated by plasmid pSCAT10-176, which contains the AvaII-BanI fragment, indicates the presence of a transcriptional regulatory element on fragment V. When the AvaII-BanI fragment was inserted into pSCAT10 and pSCAT6, plasmids in reverse orientation with respect to the CAT gene, the resulting plasmids pSCAT10-175 and pSCAT6-161 showed 90% and less than 1% levels of CAT activity, respectively. These results further support the contention that fragment V contains a 3' transcriptional regulatory element which functions in an orientation-dependent manner. To further focus the location of the 3' transcriptional regulatory region on frag-

ment V, we constructed plasmids pSCAT10-275 containing the AvaII-BstN1 region and pSCAT10-289 containing the BstNl-BanI region. Low levels (7.2%) of CAT activity from pSCAT10-275, and high levels (78%) from pSCAT10-289 locate the 3' transcriptional regulatory region on fragment VI and not fragment VII. Therefore, a 3' transcriptional regulatory element is located on the AvaII-BstNl fragment. Identification of the gastrin gene 3' transcriptional regulatory sequence. Having experimentally demonstrated that 3' transcriptional regulation occurs on gastrin gene fragment I, we determined the entire nucleotide sequence of fragment I by the dideoxy sequencing method (42). Each strand of the fragment was sequenced twice. The sequence of the 606nucleotide-long fragment I is shown in Fig. 4. Examination of the sequence between the Avall and BstNl sites immediately reveals a stretch of oligothymidylates, occasionally interrupted by one or two adenylates. This stretch of 32 nucleotides (Fig. 4, boxed), which contains 27 T's and 5 A's, is unique and may be involved in 3' transcriptional regulation. A similar but shorter stretch of 15 nucleotides, composed of 13 T's, 1 G, and 1 A was found on the BanIHindIII(3) fragment. Like the sequence of 32 nucleotides, this sequence may also represent a 3' transcriptional regulatory element. Since both of the oligothymidylate-rich regions are similar in sequence composition, we chose to study further the 32-nucleotide region. To show involvement of the 32-nucleotide region of the AvaII-BstN1 fragment in 3' transcriptional regulation, we generated several deletion mutations spanning the entire oligothymidylate-rich region. For the construction of deletion mutants, the AvaII-BanI fragment was subcloned into M13 mp9, and the resulting cloned DNA was linearized with EcoRI (the polylinker EcoRI site is proximal to the Avall site in this clone), followed by a BAL 31 digestion under

VOL. 6, 1986

Polll TERMINATION IN THE GASTRIN GENE Hindlll

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FIG. 3. The restriction endonuclease map of the human gastrin gene 3' region, depiction of restriction fragments, and a table of CAT and 3-Gal activities of the plasmids. The top portion of the figure shows the restriction map of the gastrin gene 3' region. The open box shows the 3' exon and the position of the polyadenylation signal. The restriction sites used in the generation of fragments are indicated. The middle portion of the figure shows the restriction fragments and the corresponding regions. The lower part of the figure shows the CAT and ,8-Gal activities. One unit of CAT activity is defined as 1 nmol of chloramphenicol acetylated per min at 37°C. One unit of P-Gal activity is defined as 1 nmol of o-nitrophenyl released per min at 37°C. By taking plasmid pSCAT10-generated CAT activity as 100%o, CAT activity generated by other plasmids is given in percent.

controlled reaction conditions (45). The BAL 31 digested DNA was then digested with HindIII, followed by treatment of the ends to make them blunt, and finally the fragments were separated by agarose gel electrophoresis. A broad range of fragment lengths was eluted from the gel, ligated to M13 mpl9, and cleaved with SmaI. This strategy resulted in numerous clones which were initially screened by restriction digestion for the size of the deletion. A total of 16 mutant RF DNAs, containing deletions ranging from 50 to 150 base pairs (bp), were therefore selected for further characterization by nucleotide sequence analysis. Of the 16, 5 represented deletions covering the entire oligothymidylate-rich region. The deleted regions are shown in Fig. 5. For the construction of expression plasmids containing deletion mutants, the DNA inserts from these recombinant phages were

removed and inserted into the polylinker region of the pSCAT10 and pSCAT6 plasmids. Both pSCAT10 and pSCAT6 plasmids containing deletion mutant regions were examined for levels of CAT activity. Only values of CAT activity from the pSCAT10 plasmid derivatives are given in Fig. 5, because all pSCAT 6 plasmid derivatives, as expected, generated less than 2% CAT activity. The deletion of the region upstream from the oligothymidylate-rich region (nucleotides + 106 to + 177, plasmid pSCAT10-178) resulted in CAT activity levels of less than 10%, which indicates that the region upstream from the oligothymidylate-rich sequence is not involved in the 3' transcriptional regulation (Fig. 5). However, deletion of nucleotides from the beginning of the 5' end of the oligothymidylate region resulted in increasing levels of CAT

MOL. CELL. BIOL.

SATO ET AL.

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+500

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HindlIl

(3)

FIG. 4. Nucleotide sequence of the 3' region of the gastrin gene HindIII(2)-HindIII(3) fragment. The nucleotide numbers are indicated above the antisense strand, beginning with the poly(A) addition site. The nucleotides downstream from the poly(A) addition site are indicated by positive numbers. Major restriction sites that were used in the construction of expression vectors are underlined. The poly(A) signal sequence is shown by the dashed line. The 3' transcriptional regulatory sequence is enclosed in the box.

activity. For example, deletion of 5 and 16 nucleotides (plasmids pSCAT10-196 and pSCAT10-207) yielded 16 and 23% CAT activity, respectively. When 31 of the 32 nucleotides of the oligothymidylate-rich sequence were deleted (plasmid pSCAT10-222), nearly 80% CAT activity was generated. Further deletion of the entire oligothymidylate-rich sequence, and the first 13 nucleotides downstream from this sequence (plasmid pSCAT10-235), restores all of the CAT activity (greater than 95%). The CAT activities resulting from these deletions show that the oligothymidylate-rich

+150

+130

J6%~

-1+150

- --

VAOTC Avall +250

GTCT

sequence blocks continuation of transcription past this region. However, when the oligothymidylate region is removed, transcription proceeds on through the downstream region. Although deletion of the purine-rich sequence of 8 to 12 nucleotides proximal to the 3' end of the oligothymidylate-rich sequence increases levels of CAT activity (77 to 96%), the role of this sequence in 3' transcriptional regulation cannot be ascertained by these studies. Characterization of CAT gene transcripts. To determine whether polyadenylation of CAT transcripts was regulated

Is

a:: kGAGTAACC I I

:

I

I I II I

: rl=Tmcc!

As

Ni Plai

r GAGA

+350

---

IWAACCTC16cci

I IIA IT

X AI:

MATFACASOM Ba I

ldC AT at1v1t' MSi yact1vly

relait/16

j6 __ ce____l__ (I) uc1tS/1un

pSCAT1O-176 pSCAnO-178 pSCAnO-196 pSCAT1O-207

0.06

6.6

cell

0.46

0.07

9.3

0.60

0.12

16.0

0.46

0.17

23.3

0.52

pSCATO-222

0.60 0.74

77.7 95.8

0.60

SCT1O-235 pSCAnO

0.78

100.0

0.51

0.55

FIG. 5. The nucleotide sequence of the AvaII-BanI fragment, position of the deleted nucleotides, and a table of CAT and P-Gal activities of the plasmids. The 3' transcriptional regulatory sequence is shown in a box. The arrows pointing downwards indicate the position of the deletion, and the horizontal arrows indicate the beginning (arrow pointing to the right) and the end (arrow pointing to the left) of the deleted sequence. The lower portion of the figure shows a table of CAT and ,-Gal activities of the plasmids. The plasmids listed in the table are given in the order of deletion, beginning with the first arrow to the left of the sequence. Definitions of the CAT and n-Gal enzyme units are found in the legend to Fig. 3.

Polll TERMINATION IN THE GASTRIN GENE

VOL. 6, 1986

Bam HI

Hpa I Hind X(2)

SHind3M(3)1

Ban I

3E ~~~~~Ava