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Open Reading Frame Cloning: Identification, Cloning, and Expression of Open Reading Frame DNA (Lacz Gene Fusion/Frameshift Mutant/Hybrid Protein) MARK R

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Open Reading Frame Cloning: Identification, Cloning, and Expression of Open Reading Frame DNA (Lacz Gene Fusion/Frameshift Mutant/Hybrid Protein) MARK R Proc. NatL Acad. Sci. USA Vol. 79, pp,. 6598-6602, November 1982 Genetics Open reading frame cloning: Identification, cloning, and expression of open reading frame DNA (lacZ gene fusion/frameshift mutant/hybrid protein) MARK R. GRAY*, HILDUR V. COLOT*, LEONARD GUARENTEt, AND MICHAEL ROSBASH* *Department of Biology, Brandeis University, Waltham, Massachusetts 02254; and tDepartment of Biology, Massachusetts Institute ofTechnology, Cambridge, Massachusetts 02139 Communicated by Norman Davidson, July 14, 1982 ABSTRACT A plasmid was constructed that facilitates the galactosidase can carry a wide range ofprotein sequences at its cloning and expression ofopen reading frame DNA. A DNA frag- amino terminus and still retain substantial biological activity ment containing a bacterial promoter and the amino terminus of (2, 3). We have taken advantage ofthis property to create a lacZ the cl gene of bacteriophage A was fused to an amino-terminally vector for the cloning of open reading frames. We have intro- deleted version of the lacZ gene. An appropriate cloning site was duced a frameshift mutation by inserting a DNA linker at the inserted between these two fragments such that a frameshift mu- junction of a cdlacZ fused gene. As expected, this frameshift tation was introduced upstream of the lacZ-encoding DNA. This results in a relatively low level oflacZ activity. The DNA linker cloning vehicle produces a relatively low level of fi-galactosidase encodes a cloning site. Consequently, the vector is able to activity when introduced into Escherichia cola The insertion of screen for segments of open reading frame DNA, since a con- foreign DNA at the cloning site can reverse the frameshift mu- tation and generate plasmids that produce a relatively high level tinuous open reading frame sequence is a necessary condition of (3-galactosidase activity. A large fraction ofthese plasmids pro- forhigh-level expression ofthe lacZ sequence downstream from duce a fusion protein that has a portion of the A cI protein at the the insert. A successful construction results in the synthesis of amino terminus, the foreign protein segment in the middle, and a lacZ fusion polypeptide that includes the protein segment the IacZ polypeptide at the carboxyl terminus. The production of coded forby the inserted open reading frame. The vector is able a high level offi-galactosidase and a large fusion polypeptide guar- to identify, clone, and express open reading frame DNA in one antees the cloning ofa DNA fragment with at least one open read- step. ing frame that traverses the entirety ofthe fragment. Hence, the method can identify, clone, and express (as part ofa larger fusion METHODS AND MATERIALS polypeptide) open reading frame DNA from among a large col- lection of DNA fragments. Materials. Restriction enzymes, T4 DNA ligase, and BAL- 31 nuclease were purchased from New England BioLabs and The use of recombinant DNA technology has increased our Bethesda Research Laboratories. Calf intestine phosphatase understanding ofthe organization and expression ofeukaryotic was purchased from Boehringer-Mannheim; 5-bromo-4-chlo- DNA. While many goals can be achieved with existing tech- ro-3-indolyl f3-D-galactoside (XGal), o-nitrophenyl f3-D-galac- nology, certain features ofeukaryotic gene organization are still topyranoside (ONPG), and isopropyl f3-D-thiogalactoside attainable only with difficulty. In particular, the identification were purchased from Sigma. XGal plates and MacConkey agar of protein-coding segments within a large block of eukaryotic plates (Difco) were prepared as described by Miller (4). Sma DNA is quite challenging. One should be able to take advantage I/BamHI adapter was purchased from New England Biolabs. ofproperties that are unique or highly enriched in coding DNA. Antibodies. Rabbit anti-p-galactosidase was a gift of Beth Perhaps the most ubiquitous and specific feature ofcoding DNA Rasmussen, rabbit anti-cd was a gift ofJeffrey Roberts, and the frames. at the "WI-labeled IgG fraction of a goat anti-rabbit immunoglobulin is the presence ofopen reading Indeed, analysis Fc serum was a gift of Susan Lowey and Joan Press. DNA sequence level has proved invaluable in identifying genes Bacterial Strains. The lac deletion strain LG90 (F- Alac pro or gene segments. Although there are many examples ofgenes XIII) was used exclusively (2). in which relatively small exons are interrupted by relatively Plasmid DNA Preparation. Plasmid DNA was prepared by large and numerous introns, this characteristic appears to be standard CsCI/ethidium bromide centrifugation methods (5) or relatively infrequent in lower eukaryotes. Also, higher eukary- from minilysates prepared by using the alkaline lysis procedure otic genes have been analyzed that contain open reading frames (6). of substantial length. Plasmid Constructions. Plasmid DNA was digested with In this report, we describe a method for cloning pieces of commercial restriction enzymes as recommended by the sup- DNA based on this property. We have exploited the elegant pliers. Where indicated, restricted DNA was treated with suf- work of Beckwith and co-workers (1), who have demonstrated ficient phosphatase [10 mM Tris-HCl (pH 8.0), 30 min at 370C] the power and utility of gene and operon fusions to the Esch- to reduce self-ligation of a comparable amount of vector DNA erichia coli 3-galactosidase gene (the lacZ gene). Many ofthese by a factor of at least 1/100. DNA was sonicated in 5 ml of TE studies rely on the fact that the enzyme, ,3-galactosidase (f3-D- buffer (10 mM Tris-HCl, pH 8.3/1 mM EDTA)/0.2 M NaCl galactoside galactohydrolase, EC 3.2.1.23) is often biologically with six 30-sec bursts at maximum power, concentrated on a active when it carries other protein segments at its amino ter- 0.2-ml column of DEAE-cellulose, eluted with TE buffer/I M minus. Indeed, it appears that in yeast, as well as in E. coli, /3- NaCl, and precipitated with ethanol. DNA was resuspended in TE buffer, digested lightly with BAL-31 nuclease (sufficient to The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: XGal, 5-bromo-4-chloro-3-indolyl &D-galactoside; bp, ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. base pair(s). 6598 Downloaded by guest on October 2, 2021 Genetics: Gray et aL Proc. NatL Acad. Sci. USA 79 (1982) 6599 remove approximately 25 nucleotides from each end ofa similar quantity of control restriction enzyme-digested DNA frag- A ments) at 30'C, and fractionated by size on 10% acrylamide/ Tris borate/EDTA (TBE) gels alongwith appropriate molecular weight markers (7). Ligations. Cohesive-end ligations were carried out as follows: 100 ng of plasmid vector and various amounts of eukaryotic DNA were ligated for 3 hr at 220C in a final volume of 100 ILI with 0.1 (Bethesda Research Laboratories) unit ofT4 DNA li- gase. Blunt-end ligations were typically carried out as follows: 500 ng ofplasmid vector and various amounts ofpotential insert Hind III DNA were ligated overnight at 160C in a final volume of 10 1ul with 1 unit ofT4 DNA ligase. XcI leu pMR1 AAacCAAGOTGGArCC3GGAGCWTGQCTG (in frayfe) Transformation. Bacterial transformation into LG90was car- lacZ ried out by standard procedures (8), with the following modi- HindrE (Bam) Iac I fications. All ligated DNA samples were phenol extracted and XcI Ieu pMR2 ethanol precipitated prior to transformation, which caused a AAGICCAIAG rTMCTLG (out of frame) reproducible 5- to 10-fold increase in transformation efficiency. I- - -_ lacZ- Transformed cells (1 ml) were spread on the appropriate indi- Hind I lacI cator plates containing ampicillin at 25 ,g/ml. Colonies were scored for phenotype on MacConkey agar plates after 24 hr at B 370C. If the plates were crowded, the colony phenotype was scored at 48 hr. Hybrid Protein Analysis. Three-milliliter samples of cells (out of frame) from a saturated culture (grown in L broth containing ampicillin lacZ- at 50 pug/ml) were centrifuiged, the pellet was suspended in 150 A1 of 1.2-fold concentrated sample buffer (9) and heated at 1000C for 3 min, and the viscosity was reduced by repeated passages through a 22-gauge syringe needle. Between 4 and 8 ,ul ofthe protein samples were electrophoresed in 7.5% acryl- amide gels (9). Protein blotting was carried out according to published procedures (10). f-Galactosidase Assays. These were carried out according to Miller (4) except that .cells were grown in L broth. RESULTS XcI Hindu Barm Bam ' leu Sma lad Ourinitial hypothesis was that aframeshift mutation placed near Sma lac I the amino terminus of an open reading frame sequence that contains the lacZ sequence at its carboxyl terminus would pro- FIG. 1. Construction of vectors. (A) Plasmids pKB252 (11), pLG200, duce a plasmid that would be phenotypically lacZ- when trans- and pLG400 (12) were manipulated by standard procedures to produce formed into E. coli. Furthermore, the insertion offoreign DNA the appropriate fragments for ligation. Briefly, pKB252 was digested at or near the frameshift could correct the frameshift mutation with Hae m/Hindm and the largest fragment was purified. pLG200 (orpLG400) was digested with Sma I/Hindu andthe largestfragment and generate a lacZ+ insert DNA-containing plasmid. The two was purified. These two fragments were ligated together and ampi- constructions shown in Fig. 1A were designed to test this hy- cillin-resistant colonies were selected. The restriction sites of the re- pothesis. A segment of the ci gene from bacteriophage A ter- sultant plasmids were verified. pMR1 was derived by using pLG400 minating at a HindIII site at codon 157 was fused to a lacIZ frag- and pMR2 was derived by using pLG200. Both plasmids, when trans- ment. The cl gene fragment also contains two lacpromoters that formed into LG90, form blue colonies on XGal plates.
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