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Intervening Sequences in an Archaea DNA Polymerase Gene (Intron/Self-Splicing/Protein Splicing) FRANCINE B

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Intervening Sequences in an Archaea DNA Polymerase Gene (Intron/Self-Splicing/Protein Splicing) FRANCINE B Proc. Natl. Acad. Sci. USA Vol. 89, pp. 5577-5581, June 1992 Biochemistry Intervening sequences in an Archaea DNA polymerase gene (intron/self-splicing/protein splicing) FRANCINE B. PERLER*t, DONALD G. COMB*, WILLIAM E. JACK*, LAURIE S. MORAN*, BOQIN QIANG*t, REBECCA B. KUCERA*, JACK BENNER*, BARTON E. SLATKO*, DONALD 0. NWANKWO*, S. KAY HEMPSTEAD*, CLOTILDE K. S. CARLOW*, AND HOLGER JANNASCH§ *New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915; and §Woods Hole Oceanographic Institute, Woods Hole, MA 02543 Communicated by Charles C. Richardson, March 18, 1992 (receivedfor review January 29, 1992) ABSTRACT The DNA polymerase gene from the Archaea indicated expression constructs. Samples were analyzed by Thermococcus litoralis has been cloned and expressed in Esch- SDS/PAGE in 4-20Wo gels (ISS, Daiichi, Tokyo, Japan) with erichia coil. It is split by two intervening sequences (IVSs) that prestained markers (BRL), transferred to nitrocellulose (9), form one continuous open reading frame with the three poly- probed with anti-Vent DNA polymerase sera (10), and de- merase exons. To our knowledge, neither IVS is similar to tected using alkaline phosphatase-linked anti-mouse second- previously described introns. However, the deduced amino ary antibody as described by the manufacturer (Promega). acid sequences of both IVSs are similar to open reading frames Polymerase Assay. Polymerase activity was measured as present in mobile group I introns. The second IVS (IVS2) cpm incorporated into acid-insoluble DNA. Lysates were encodes an endonuclease, I-TH I, that cleaves at the exon prepared by resuspending Escherichia coli cell pellets in 20 2-exon 3junction after IVS2 has been deleted. IVS2 self-splices mM Tris-HCl, pH 8/50 mM NaCI/0.1% Triton X-100/bovine in E. cofi to yield active polymerase, but processing is abolished serum albumin (0.2 mg/ml), heating to 80TC for 20 min, and if the IVS2 reading frame is disrupted. Silent changes in the pelleting cell debris for 10 min at 12,000 x g at room DNA sequence at the exon 2-1VS2 junction that maintain the temperature. Assays were incubated at 720C in 10 mM original protein sequence do not inhibit splicing. These data KCI/10 mM (NH4)2SO4/20 mM Tris, pH 8.8/2 mM MgSO4/ suggest that protein rather than mRNA splicing may be 0.1% Triton X-100/[32P]dCTP (10-20 x 106 cpm/ml)/all four responsible for production of the mature polymerase. dNTPs (each at 33 uM)/activated DNA (0.2 mg/ml) (26). Library Construction and Screening. T. litoralis DNA par- In 1985, a species of extreme thermophile was isolated from tially digested with EcoRI was cloned into Agtll and AZapII a submarine thermal vent near Naples, Italy (1). This orga- (Stratagene) and libraries were screened with anti-Vent DNA nism, Thermococcus litoralis, can be cultured at up to 980C polymerase sera (9, 10). BamHI-digested T. litoralis DNA and contains a heat-stable DNA polymerase that we call Vent was cloned into ADash (Stratagene) and screened by hybrid- DNA polymerase (New England Biolabs). This paper de- ization to the 1.3-kilobase (kb) EcoRI insert from AV10-49 scribes the cloning, sequencing, and expression of the Vent (ref. 9). DNA polymerase gene¶ and the finding of two intervening DNA Sequencing. Both strands of the polymerase coding sequences (IVSs) that make up 55% of the polymerase gene, region (Fig. 1) were sequenced from two independent one of which, IVS2, encodes the I-Tli I (I, intron) endonu- sources: the three EcoRI fragments and subclones of the clease. 12-kb BamHI fragment from AV56-9 (11). To our knowledge, this is the first report of introns in Construction of a Recombinant Expressing Vent DNA Poly- protein coding genes of Archaea or eubacteria, although merase. Vent DNA polymerase was expressed in E. coli by introns have been found in protein coding genes of eubacte- The riophage (2). Previously described Archaea or eubacterial using the T7 expression system of Studier et al. (8). gene introns are mainly pre-tRNA or self-splicing introns in stable lacking IVS1 was created in a multistep ligation involving RNAs (2-5). sequences from (i) an Nde I site created (12) at the initiation Introns often contain open reading frames (ORFs) that are codon [base pair (bp) 2911 to the Pvu I site (bp 1720), (ii) a in-frame with either the 5' or 3' exon, but not with both exons. bridging synthetic double-stranded oligonucleotide with Pvu An intron in the Saccharomyces cerevisiae TFP1 gene forms I and Bsu36I termini (bp 1721-1772 joined to bp 3387-3412) a single ORF with the surrounding exons; the authors pro- (5 '-CGAAAAGAAAATGCTCGATTATAGGCAAAG- posed (6, 7) that this intron is spliced at the protein, not the GGCTATTAAATTGCTAGCAAACAGCTATTACGGC- mRNA, level. In the present study, we describe two introns TATATGGGGTACCC-3' and 3'-TAGCTTTTCTTTTAC- that form a single ORF with the surrounding exons. Further- GAGCTAATATCCGTTTCCCGATAATTTAAC- more, we present evidence indicating that the Vent DNA GATCGTTTGTCGATAATGCCGATATACCCCATGG- polymerase IVSs are removed either by protein splicing or by GATT-5'), and (iii) a Bsu36I (bp 3412) to BamHI (bp 5832) RNA splicing that requires I-Tli I as a maturase. fragment, where numbers in parentheses indicate coordi- nates. These gene segments were inserted into Nde I/BamHI-cut pAII17, a pETlic (8) derivative that had been MATERIALS AND METHODS modified to reduce basal expression from the T7 RNA Western Blots. Anti-Vent DNA polymerase sera was raised polymerase promoter by the upstream addition offour copies by immunizing mice with purified native T. litoralis DNA polymerase (New England Biolabs). Western blot analysis Abbreviations: ORF, open reading frame; IVS, intervening se- employed protein samples from isopropyl ,B-D-thiogalacto- quence; Pola, polymerase a. side-induced cultures of BL21(DE3)plysS (8) containing the tTo whom reprint requests should be addressed. tPresent address: Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, 5 Dong Dan San Tiao, Beijing, The publication costs of this article were defrayed in part by page charge China. payment. This article must therefore be hereby marked "advertisement" IThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M74198). 5577 Downloaded by guest on September 30, 2021 5578 Biochemistry: Perler et al. Proc. Natl. Acad. Sci. USA 89 (1992) FpPITEE R AUG H. ND A.V6-49 k-- FR AUG C RQ P N!"' .A V5 6-9 I I IVS12. WVSt- .:. IN IVS2. -- lk=::I>E.o||;o\-...1 AUG C R P FH r N ND pV1i6O- I V&s1. i & FIG. 1. Maps of Vent DNA polymerase AUGC C Rl P B N - clones. The genomic polymerase gene con- + + pNEB6B7 -_IVS2-_ tains two IVSs (open boxes) and three exons * (hatched boxes); dashes represent deleted se- AUG C R1 P quences. Only part of AV56-9 (12 kb) is pre- pAKK4 - sented. The polymerase start codon (AUG, bp .+ 291) and the termination codon (TAG, bp Properties of mutant clones: 5397) are indicated. IVS2 encodes I-Tli I en- donuclease. Production in E. coli of active AUG C R P N., Al TAPAA Vent DNA polymerase (Pol) or I-Tli I is indi- .Mi cated (ND, not determined). R, EcoRI; C, Cla pAKCi ..--- I; P, Pvu I; B, Bsu36I; N, Nde I; Bm, BamHI. AUG C R f) N CAJ Mutations are as follows (see Tables 1 and 2): N+AT/TAA, AT insertion at the Nde I site + + pAKG1 -'I_VS2_2 resulting in a termination codon (TAA); AUG C H I-' .212 N N+CAT, CAT insertion at the Nde I site; E2/12, location ofexon 2-IVS2junction silent + + pAKO *1-- t- IVS2 mutations. pV160-11 and pAKC1 fail to splice +_S_ .. At_ ... A. | +~~~~~~~~~~~~~~~~ in E. coli, whereas pNEB687, pAKG1, and KB 0 the pAKQ series do splice. of the rrnb transcription terminator from pRS415 (13). The pNEB687 digested with Cla I and Nsi I. The exon 2-IVS2 resulting construct was named pNEB687 (Fig. 1). junction was formed by direct ligation of the PCR product Deletion ofIVS2. The polymerase chain reaction (PCR) was blunt ends. The resulting constructs, the pAKQ series, were used to generate two fragments with termini near the planned sequenced (11) to verify alterations (Table 2). deletion. The PCR mixture contained Vent DNA polymerase buffer (New England Biolabs)/bovine serum albumin (0.1 mg/ml)/all four dNTPs (each at 0.2 mM)/pNEB687 (0.9 RESULTS Ag/ml)/primers (0.5 ug/ml)/Vent DNA polymerase (10 Cloning Vent DNA Polymerase. Vent DNA polymerase is units/ml). Amplification was carried out for 15 cycles of94TC extremely thermostable. It was purified by conventional for 0.5 minm 50TC for 0.5 min, and 72TC for 2 min by using a chromatography to yield a final preparation containing four Perkin-Elmer/Cetus thermal cycler. Both PCR fragments proteins detectable by Coomassie Blue staining. We identi- incorporated silent changes at bp 3518 (G -) A) and 3521 (T fied the 93-kDa (+5%) species as the polymerase by assaying -- A) to create a Sca I site thatjoined the fragments. The first proteins renatured after elution from a SDS/PAGE gel (ref. fragment extended from the EcoRI site (bp 1269) to the newly 15; H. M. Kong, R.B.K., and W.E.J., unpublished data). created Sca I site (bp 3518). The second fragment included The eluted polymerase also possesses a 3' -- 5' exonuclease the Sca I site to bp 3533, then skipped to bp 4704, and activity that enhances the fidelity of polymerization (16). continued to the HindIII site at bp 5058.
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