EXPRESSION OF ENZYMATICALLY ACTIVE REVERSE TRANSCRIPTASE

Abstract: A plasmid which, when introduced into a suitable host cell and grown under appropriate conditions, effects expression of a gene on the plasmid and production of a polypeptide having reverse transcriptase activity. The plasmid is a double-stranded DNA molecule which includes in a 5' to 3' order the following: a DNA sequence which includes an inducible promoter; a DNA sequence which includes an ATG initiation codon; the central portion of the Moloney murine leukemia virus (MuLV) pol gene, said central portion including a DNA sequence which encodes the polypeptide having reverse transcriptase activity; a DNA sequence which contains a gene associated with a selectable or identifiable phenotypic trait which is manifested when the vector is present in the host cell; and DNA sequence which contains an origin of replication from a bacterial plasmid capable of autonomous replication in the host cell. The invention also concerns a method for recovering purified enzymatically-active polypeptide having reverse transcriptase activity, the polypeptide being encoded by the plasmid pB6 B15.23, from a suitable host cell e.g. E. coli HB101 producing the polypeptide. Finally, the invention concerns use of the polypeptide to prepare complementary DNA (cDNA).

Description: EXPRESSION OF ENZYMATICALLY ACTIVE REVERSE TRANSCRIPTASE Background Of The Invention This invention was made with government support under Grant Number CA 30488 from the National Cancer Insti¬ tute of the United States Department of Health and Human Services. The U.S. government has certain rights in this invention. Throughout this application various publications are referenced by number within parentheses. Full cita¬ tions for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entire¬ ties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. In the early stages of the retroviral life cycle/ viral RNA is copied to form a double-stranded DNA, which is integrated into host DNA to generate the provirus (for review, 1) . The synthesis of the proviral DNA is cata¬ lyzed by the enzyme reverse transcriptase, which may efficiently utilize either RNA or DNA templates for DNA synthesis by the elongation of a primer bearing a paired 3' hydroxyl terminus. Inherent in the same protein is a second activity, RNAse H, which degrades RNA present as a duplex RNA:DNA hybrid. The viral pol gene encodes many enzymatic activities which partici¬ pate in various steps of the life cycle. The pol gene product is initially expressed as a polyprotein Pr200 gag ~P° 1 (2, 3), containing sequences encoded by the aaa gene fused to sequences encoded by the pfii. gene; proteolytic processing is required to remove the gag sequences and to excise the mature products from the pol sequences. In the murine retroviruses, such as Moloney urine leukemia virus (MuLV) , the pal sequences are processed to three nonoverlapping products: a small protein en¬ coded by the 5* end of the gene, probably the protease needed for g_-_3. and £_2_L processing; reverse transcrip¬ tase, the largest protein from the middle portion of the precursor; and a protein at the 3' end, apparently involved in integration of the provirus. The viral MuLV reverse transcriptase ' has been purified (4,5) and shown to be a monomer (4,5) of molecular weight between 70,000 to 80,000 daltons. The purified protein was shown to have a nuclease activity (RNAse H) which degraded RNA contained in an RNA-DNA hybrid (4,5). Reverse transcriptase is widely used as a means of producing complementary DNA (cDNA) copies of messenger RNA (mRNA) molecules. These cDNAs may be inserted into expression vectors which are used to transform cells so that the resulting cells produce a desired polypeptide encoded by the original mRNA. The only disclosure in the art concerning production of a polypeptide having reverse transcriptase activity by bacteria transformed with genetically engineered vec¬ tors involves the shotgun cloning into Escherichia coli of total genomic DNA isolated from the cells of warm¬ blooded vertebrate animals, e.g. fowl liver cells, [Japanese patent publication no. 56087600] . Reverse transcriptase produced by and isolated from, virions is commercially available. However, it is quite expensive due to the low abundance of the pol gene product in the virions. To overcome this problem, the present invention uses a modified region of the MuLV pol gene which is inserted into a plasmid, its transcription being controlled by an inducible promoter. The modifications to the in¬ serted gene fragment result in the production of a polypeptide with reverse transcriptase activity. Additionally, the present invention describes a method of isolating the non-naturally occurring polypeptide whereby a novel combination of column chromatography techniques is employed, including phosphocellulose and polyribocytidylie acid-agarose chromatography. Summary Of The Invention The invention concerns a double-stranded DNA plasmid which, when expressed in a suitable host cell, produces a polypeptide having reverse transcriptase activity, the plasmid comprising in 5' to 3' order: a DNA sequence which includes an inducible promoter; a DNA sequence which includes an ATG initiation codon; the central portion of the Moloney murine leukemia virus (MuLV) ' ol gene, said central portion including a DNA sequence which encodes the polypeptide having re¬ verse transcriptase activity; a DNA sequence which contains a gene associated with a selectable or identifiable phenotypic trait which is manifested when the vector is present in the host cell; and a DNA sequence which contains an origin of replication from a bacterial plasmid capable of autonomous replica¬ tion in the host cell. The plasmid of this invention may be introduced into a suitable host cell where the gene may be expressed under suitable conditions. In a presently preferred embodiment, the plasmid is pB6B15.23 and the host cell is an Escherichia coli HB101 cell (deposited together under ATCC No. 39939) . Suitable inducible promoters are ones which are induced when the host cell is grown in a medium deficient in one or more amino acids. One such inducible promoter is the Trp operon of £. coli. The invention also concerns a method for recovering the polypeptide having reverse transcriptase activity in purified form after it is produced in the suitable host cell. The method comprises disrupting the host cells, recovering soluble material, then recovering from the soluble material the polypeptide in purified form, e.g. by chromatography on a series of chromatographic columns. 10 Host cells containing the plasmid of this invention have been used to produce a polypeptide having reverse transcriptase activity and characterized by being en¬ coded by the plasmid pB6B15.23. _ _ This invention also concerns uses of the novel ' polypep¬ tide having reverse transcriptase activity. One such use comprises contacting of an RNA molecule with the polypeptide under suitable reverse transcribing condi¬ tions so as to produce a DNA molecule which is complex 2Q entary to the RNA molecule. 25 30 35 Brief Description of the Drawings Fig. 1. Construction of trpE-pol gene fusions. The central portion of the pol gene was excised from a cloned copy of the M-MuLV genome by cleavage with Sac I plus Hind III and inserted into the polylinker sequence of the expression vector pATHl. The resulting plasmid, pSHl, expressed a 124,000 dalton fusion protein and substantial levels of active reverse transcriptase. In a second step, the bulk of the trpE sequences and varying amounts of the a gene were deleted, and bac¬ terial clones were screened for increased levels of reverse transcriptase activity. The highest-level producer, pSHNBδ, encoded an 89,000-dalton protein. ' The DNA sequence in the region of the deletion, along with the predicted amino acid sequence of the encoded protein, is indicated. Fig. 2. Construction of plasmid pB6B15.23 expressing stable reverse transcriptase The complete genome of M-MuLV as a linear provirus is shown at the top. Long Terminal Repeats (LTRs) and the regions encoding the gag, pol . and env precursors are indicated in boxes. Plasmid pSHNBδ (6) contained the region of the pol gene from Sac I to Hind III (from 2.95 to 5.4 on the map (7)) inserted 3' of the DNA encoding the N-terminus of the £ιp£ gene. The posi¬ tions of the origin of replication (ori) and the gene conferring ampicillin resistance (amp) are indicated. pSHNBδ was linearized with Hind III, digested with Bal 31 nuclease, and ligated with T4 DNA ligase; the DNA products were used to transform HB101, and the result- ing colonies were screened for reverse transcriptase as described in Experimental Procedures. The characteris¬ tics of plasmid pB6B15.23 are summarized at the bottom. The numbers in parentheses refer to the maps of M-MuLV and pBR322 as described by Sutcliffe (7 and 8, respec¬ tively) . The resulting gene fusion consists of an open reading frame encoding 728 amino acids. The first 18 amino acids at the N-terminus are encoded by the trpE gene, followed by 7 amino acids which are .encoded by the pol gene but are not part of the M-MuLV reverse transcriptase (S. Oroszlan, personal communication) . The subsequent 694 amino acids are encoded by the pol gene, and the terminal 9 amino acids by pBR322. The sequence of the 3* terminus of the gene was determined by the method of Maxam and Gilbert (9) after 5' end labeling with polynucleotide kinase at the Bgl II site. The DNA sequence and the deduced amino acid sequence are indicated at the bottom. Fig. 3. Phosphocellulose chromatography The material which did not bind to DEAE-cellulose was chromatographed on phosphocellulose as described in the text. After collection of the material which did not absorb to phosphocellulose (not shown) , the column was eluted with a gradient of NaCl as indicated. Aliquots of the individual fractions were diluted (1:100) in Buffer M plus 0.2 M NaCl, and 1 1 of the dilution was assayed for reverse transcriptase activity (♦——•) and RNase H activity ( Δ - - - -Δ) as described in the Experimental Procedures. 25 ^1 aliquot of the column eluant was assayed directly for total protein (0 - - - - o) as described in Experimental Procedures. Fig. 4. Polyribocytidvlic Acid-Aαarose Chromatography The bound fraction from the phosphocellulose column was pooled, diluted, and applied to a polyribocytidylic acid-agarose column as described in the text. The column was washed and eluted with a salt gradient as indicated. Fractions were diluted (1:200) as described in Fig. 3. Reverse transcriptase (• •) and RNase H (Δ- - -£>) activities were assayed as described in Ex¬ perimental Procedures in the presence of 1 j and 3 ul of the diluted enzyme, respectively. Total protein (0 - - - 0) was determined using 10 ul of the column eluant as described in the Experimental Procedures. Fig. 5. Glycerol gradient centrifuσation of reverse transcriptase fusion protein. 1445 units of pB6Bl5.23 reverse transcriptase (50 ul) was adjusted to final concentrations of 25 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 1 mM dithiothreitol, 0.1 mM PMSF, 10% glycerol, and 0.02% Nonidet P40. An aliquot (200 ul) was layered onto a 4.8 ml linear gradient of 15-35% glycerol in the same buffer. The gradient was centrifuged for 24 h at 48,000 rpm in a Sorvall AΞ-650 rotor. Fractions (180 ul) were collected from the bottom of the tube, diluted 1:20 in Buffer M plus 0.2 M NaCl, and assayed for reverse transcriptase activities (1 and 3^ιl/assay, respectively) as described in Exper¬ imental Procedures. Catalase, aldolase, and cytochrome C were sedimented in a parallel tube. The position of the markers were indicated by the arrows. Aliquots (5 ^Λ1) of fractions 13-21 were analyzed by electro- phoresis through a sodium dodecyl sulfate polyacryla- rnide gel; and the proteins were stained with silver as described (10) . The peptide composition of these frac¬ tions is shown beneath the graph. The position of migration and the M r of the marker proteins are indi¬ cated at the left and right of the gel. Detailed Description of the Invention A double-stranded DNA plasmid has been made which, when expressed in a suitable host cell, produces a polypep¬ tide having reverse transcriptase activity. The plas¬ mid includes in 5' to 3' order: a DNA sequence which includes an inducible promoter; a DNA sequence which includes an ATG initiation codon; the central portion of the Moloney murine leukemia virus (MuLV) pol gene, said central portion including a DNA sequence which encodes the polypeptide having reverse transcriptase activity; a DNA sequence which contains a gene associ¬ ated with a selectable or identifiable phenotypic trait such as drug resistance, e.g. ampicillin resis¬ tance, which is manifested when the vector is present in the host cell; and a DNA sequence which contains an origin of replication from a bacterial plasmid capable of autonomous replication in the host cell, e.g., Escherichia ££ll. In one embodiment the inducible promoter of the plasmid is one which is induced when the host cell is grown upon a medium deficient in one or more amino acids. Thus, the inducible promoter may be the Trp promoter of Escherichia cjli. and the medium deficient in the amino acid tryptophan. In another embodiment the inducible promoter is one which is in¬ duced when the host cell is subjected to increased temperature. The ATG initiation codon of the plasmid may be derived from the coding sequence of the Trp E protein of Escherichia coli. e.g. a DNA sequence derived from a 54 nucleotide long sequence encoding a portion of the Trp E protein of Escherichia cjjl . In one embodiment the origin of replication is derived from pBR322. The plasmid may comprise a circular double-stranded DNA sequence such as the plasmid identified as pB6B15.23, having the restriction map shown in Fig. 2 and deposit¬ ed in £. coli HB101 under ATCC No. 39939. The central portion of the MuLV pol gene of the plasmid may comprise the nucleotide sequence from about nucleo¬ tide 2574 to about nucleotide 4588. In one embodiment the 5' end of the central portion of the pol gene is 21 nucleotides from the start of the DNA sequence which encodes the polypeptide having reverse transcriptase activity. Methods used in preparing the DNA vector and transform¬ ing suitable cells to the production of the polypeptide having reverse transcriptase activity are known in the art and described more fully hereinafter under Experi¬ mental Details. Conventional cloning vehicles such as plas ids, e.g., pB__322, can be modified or engineered using known meth¬ ods so as to produce novel cloning vehicles which con¬ tain DNA encoding a non-naturally occurring polypeptide having reverse transcriptase activity. Similarly, such cloning vehicles can be modified or engineered so that they contain DNA sequences, i.e., inducible promoters (Trp promoter, etc.), involved in the regulation or expression of the sequences encoding a polypeptide having reverse transcriptase activity. The DNA mole¬ cules so inserted may be made by various methods in¬ cluding enzymatic or chemical synthesis. The resulting cloning vehicles are chemical entities which do not occur in nature and may only be created by the modern technology commonly described as recombinant DNA technology. These cloning vehicles, including the plasmid of this invention, may be introduced into a suitable host cell, either procaryotic, e.g., bacterial (£. coli or a. subtilis. etc.) or eucaryotic, e.g., yeast, using techniques known to those skilled in the art, such as transformation, transfection and the like. The one embodiment of this invention is the £.. coli HB101 strain containing the plasmid pB6B15.23 deposited under ATCC No. 39939. The cells into which the plasmid of this invention is introduced will thus contain DNA encoding a non-naturally occurring poly¬ peptide having reverse transcriptase activity. Fur¬ ther, the expression of the DNA encoding the non-natu¬ rally occurring polypeptide will be under the control of the Trp promoter. The resulting cells into which DNA encoding the non- naturally occurring polypeptide encoding reverse tran¬ scriptase activity and encoding the Trp promoter has been introduced may be grown under suitable conditions known to those skilled in the art so as to control and effect the expression of the genetic information encod¬ ed by the DNA and permitting the production of the polypeptide having reverse transcriptase activity and the recovery of the resulting polypeptide. Thus one embodiment of this invention concerns the polypeptide so prepared, e.g. the polypeptide having reverse tran¬ scriptase activity characterized by being encoded by the plasmid pB6Bl5.23. A further embodiment concerns a method for recovering the polypeptide of this invention from most cells in which it has been produced. The method comprises dis¬ rupting the cells, recovering soluble material containing the reverse transcriptase polypeptide from the disrupted cells and separately recovering the reverse transcriptase polypeptide from the soluble material in purified form. In a specific embodiment the separate recovery of the reverse transcriptase polypeptide comprises chromatography on phospho¬ cellulose followed by chromatography on poly- ribocytidylic acid-agarose. Still another embodiment of this invention is a method for reverse transcription of an RNA molecule. The method comprises contacting the RNA molecule with the polypeptide of this invention under suitable reverse transcribing conditions so as to produce a DNA molecule which is complimentary to the RNA molecule. EXPERIMENTAL DETAILS FIRST SERIES OF EXPERIMENTS Material and Methods Bacteria. Eschericia coli strain HB101 (rec A13 " , hsdR~, hsdM~, lacYl, SupE44) was used as the host for most experiments (11) . The DNA Polymerase I-deficient strain C2110 (his-, rha-, polAl-) used to eliminate the presence of endogenous DNA synthetic activity, was the kind gift of D. Figurski. Cells were transformed to aropicillin resistance as described. Pl smids. The trpE fusion vector pATHl, containing the 5' proximal part of the £ιp£ gene followed by a poly- linker sequence, was the generous gifts of T.J. Koerner and A. Tsagaloff. Plasmid pTll, containing ' a full- length copy of the M-MuLV genome was the source of the pol gene. Enzvmatic reactions. DNAs were digested with selected restriction enzymes (New England Biolabs) under condi¬ tions specified by the manufacturer. DNA fragments were purified by agarose gel electrophoresis and eluted by the glass powder method. DNA was treated with the enzymes exonuclease Bal31, nuclease SI, and T4 DNA ligase as described previously. Reverse transcriptase assays measured the incorporation of radioactive dTTP into homopoly er on synthetic templates as previously described. Product DNA was detected by measuring the radioactivity binding to DEAE paper (DE81; Whatman) by autoradiography or by scintillation counting in Aquasol (NEN) . DNA sequencing. The bases in plasmid pSHNB β flanking the site of deletion were determined by the procedure of Ma am and Gilbert (9). Plasmid DNA was cleaved with Hinc II, and the 5' ends of the fragments were labelled with polynucleotide kinase. The 240-bp fragment con¬ taining the site of fusion was purified, the label at one end was removed by cleavage with Rsa I, and the DNA was subjected to chemical degradation and gel electro¬ phoresis. Pre p aration of crude lysates. These procedures were modifications of the method of Kleid et al. (12). Cul¬ tures (0.5 ml) were grown to stationary phase in M9 medium (13) plus 0.5% casamino acids, thiamine (10 jig/ml), tryptophan (20 pg/iwl) and ampicillin (50 jig/ml) , diluted 1:10 into medium without tryptophan, and grown for 1 h at 30°C. The cells were induced by addition of indoleacrylic acid to 5 μq/ml , grown an additional 2 h, and harvested by centrifugation. Total cell protein for gel analysis was isolated by resus- pending the cells from 1 ml of culture in 50 ul of cracking buffer (6 M urea, 1% SDS, 1% SDS, 1% beta- mercaptoethanol, 10 mM sodium phosphate pH 7.2) at 37°C for 1 h. Preparation of total protein extracts for enzymatic assays, and the subsequent separation of proteins into soluble and insoluble fractions were carried out as described (12) . Extracts made by the dilute lysis procedure were prepared from small cul¬ tures lysed in 1/lOth volume, and large-scale extracts made by the concentrated lysis procedure were made from 500 ml cultures lysed in l/200th volume. Tmmunoprecipitations. Cells (7.5 ml cultures) were labelled by addition of 35 S-methionine to 40 uCi/ml at the time of induction, treated with lysozyme as above. and lysed with PLB buffer (1% Triton X100, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM sodium phosphate pH 7.5, and 0.1 M NaCl) for 15 min. at 0°C. After addition of fixed s. aureus cells (25 ^1 of a 1:1 suspension; Pan- sorbin, CalBiochero) the lysate was clarified (45,000 rpm, 90 min.), and 200 μl aliquots were incubated with antiserum (5 μl ) overnight. The immune complexes were adsorbed to fixed S. aureus for 1 h at 0°C, collected and analyzed by SDS polyacrylamide gel electrophoresis as described (14) . Partial purification of pSHl and pSHNBδ proteins. HB101 cells bearing pSHl or pSHNBδ were grown to sta¬ tionary phase at 37°C and induced as above. The cells were collected by centrifugation, washed, resuspended in l/200th volume of buffer (50 mM Tris-Cl pH 7.5, 0.5 mM EDTA, 0.3 M NaCl), and treated with lysozyme (1 mg/ml) at 0°C for 30 min. The cells were lysed with NP40 (0.2%), and the lysate was made 1M in NaCl, clari¬ fied at 8000xg for 30 min., and dialyzed against buffer B (50 mM Tris-Cl ph 8.0, 1 mM EDTA, 1 mM dithiothrei- tol, 10% glycerol) containing 0.1% NP40 and 25 mM NaCl. DNA was precipitated by the addition of 0.3 volumes of streptomycin sulfate solution (5% in buffer B contain¬ ing 25 mM NaCl) , and the supernatant was applied to a DEAE cellulose column (DE52; Whatman) equilibrated with buffer B plus 25 mM NaCl. The activity was eluted with buffer B plus 0.2 M NaCl. Ammonium sulfate fractionations. Activity eluted from DEAE cellulose columns was precipitated by the addition of solid ammonium sulfate to the appropriate concentra¬ tion. The solution was stirred for 1 h, and the pre¬ cipitate was pelleted (11,000 rpm, 30 min.) . Results Construction of TrpE-pol gene fusion. In our initial effort to express reverse transcriptase, we chose a fragment from the central portion of pol gene of the biologically active copy of the viral genome cloned in the plasmid pTll (15) . Sac I cleaves within the 5 » portion of the pol gene encoding the viral protease; a deletion mutation near this site does not effect re¬ verse transcriptase activity (16). Similarly, Hind III makes a single cleavage in the 3* portion of the pol gene; deletions at this site also did not affect pro¬ duction of the enzyme (17). The 2.5 kb fragment pro¬ duced by cleavage with Sac I plus Hind III was isolated and inserted into the polylinker sequence of the ex¬ pression vector pATHl (T.J. Koerner and A. Tzagoloff, Personal Communication.) The resulting plasmid, pSHl, contained the TrpE promoter, 326 codons of the trpE gene, and the coding region for the central portion of the pol gene appended in the correct reading frame (Figure 1). The gene product would contain 36,200 daltons of the trpE polypeptide joined to 87,7000 dal- tons of p ol protein; translational termination of the fusion protein would occur at an amber codon immedi¬ ately downstream of the pol sequences. In an attempt to form smaller protein products that would more closely resemble the authentic enzyme, we modified the initial construct. We removed the bulk of the trpE sequences and portions of the 5' end of the pol gene by creating a series of deletion mutations in the pSHl plasmid. The general scheme used to create the deletions is shown in Figure 1. pSHl DNA was cleaved near the 5 1 end of the JL∑LEE gene with Nru I, and treated with the exonuclease Bal31; the DNA was recleaved with Sac I at the 3' end of the tιp£ gene, and the termini were blunted with nuclease SI. The linear DNA was purified, recyclized with T4 DNA ligase, and used to transform HB101 cells to ampicillin resis¬ tance. Two-thirds of these clones should contain frameshift mutations; only one-third might encode im¬ proved levels of activity. Approximately 100 clones were recovered from this procedure. Analysis of the DNA from several colonies showed that varying amounts of the trpE and pol genes had indeed been removed (data not shown) . The TΓPE-POI fusions induce reverse transcriptase ac¬ tivity. Cells containing pATHl and pSHl were starved for tryptophan, harvested, and lysed, and the crude extracts were tested for reverse transcriptase activi¬ ty. The assay measured the incorporation of radioac¬ tive dTTP on a synthetic template (polyriboadenylate) primed with oligo dT and was similar to identical assay ' s previously used to detect the viral enzyme (18) . Extracts prepared from HB101 cells, or from cells bear¬ ing pATHl, showed significant basal activity in the assay. The bulk of this background activity is attrib¬ utable to the presence of DNA polymerase I in the ex¬ tracts; this enzyme is known to exhibit reverse tran¬ scriptase activity (19) . Cells bearing plasmid pSHl consistently showed four to six fold higher activity over the control cells (Table 1) . The level of activi¬ ty per ml in these crude extracts was considerably higher than that in viral harvests taken from infected NIH/3T3 cell lines. Recovery of the activity in the soluble fraction required the presence of nonionic detergent and high salt concentrations (data not shown) . TAB E 1 DNA synthesis on synthetic templates by bacterial extracts; sensitivity to NEM. pmol incorpo- Cells Plasmid Treatment ratedI per ul Expt 1: ΞB101 pATHl lysate 2.00 HB101 pATHl + NEM, then DTT 1.55 HBIOI pATHl + DTT, then NEM 1.40 HBIOI pSHl lysate 23.7 HBIOI pSHl + NEM, then DTT 2.48 HBIOI pSHl + DTT, then NEM 15.9 HBIOI pSHl + premixed DTT + NEM 16.9 Expt 2: HBIOI .pATH lysate 59.6 HBIOI pATHl + NEM, then DTT 45.7 HBIOI pSHl lysate 751 HBIOI pSHl + NEM, then DTT 98.2 HBIOI pSHNB6 lysate 2090 HBIOI pSHNBδ + NEM, then DTT 237 C2110 pSHl lysate 1590 C2110 pSHl + NEM, then DTT 164 Expt 3: HBIOI pSHl DEAE eluate 112 HBIOI pSHl + NEM, then DTT 27.3 HBIOI pSHl 40-70% AS fraction 140 HBIOI pSHl + NEM, then DTT 94.4 HBIOI pSHl 0-40 % AS fraction 252.6 HBIOI pSHl + NEM, then DTT 25.5 Lysates were prepared from the indicated bacterial Cells carrying the indicated plasmids, and assayed for reverse transcriptase after various treatments. . En¬ tries are the pmoles of P-dTTP incorporated into DNA per microliter of extract under standard conditions (see Methods) . Extracts for expt. 1 were made by the dilute lysis procedure, those for expt. 2 by the con¬ centrated lysis procedure, and those for expt 3. as in the text. Protein concentrations (mg/ml) were: expt. 1: 1.34 and 1.51; expt. 2: 3.82, 3.10, 3.53, and 3.6; expt. 3: 1.05, 2.39, and 0.59. To test whether the increased activity could be attrib¬ uted to an increase in the level of DNA polymerage I, the sensitivity of the activity to the sulfhydryl rea¬ gent N-ethyl maleiraide (NEM) was determined. The au- 5 thentic murine reverse transcriptase is exquisitely sensitive to the sulfhydryl reagent N-etheyl maleimide (NEM) , while the bacterial DNA poly erase I is resis¬ tant (20). Treatment of the extracts of HB101, or of HB101 carrying pATHl, with NEM had no effect on the 10 activity; but treatment of extracts of cells carrying pSHl reduced the high level of activity to that of the control extracts. This result suggested that the pSHl plasmid induced a novel reverse transcriptase activity with properties similar to those of the authentic en- 5 zyme. Further evidence that the additional activity was not due to elevated levels of DNA polymerase I was obtained by repeating the assays in a bacterial host carrying a 20 mutation in p_alΔ, the structural gene for the enzyme. Strain C2110 (po Δl-) was transformed to .ampicillin resistance with the plasmids pATHl and pSHl; because the polA gene is required for plasmid replication, these transformations occur at low frequency. The 25. plasmids are apparently maintained by recombination with the host chromosome. The continued presence of the polAl- mutation was confirmed (21) by testing the strains for sensitivity to methylmethane sulphonate (MMS) . Extracts of strain C2110, or of strain C2110 30 bearing the pATHl vector, showed no measurable reverse transcriptase activity in the assay, confirming that the background activity of HB101 was indeed due to DNA polymerase I. Reverse transcriptase assays of bacteri¬ al extracts of total proteins were performed. Aliquots 35 were incubated in a reaction cocktail containing la- belled precursors, and the products were spotted on DEAE paper, washed, and exposed to X-ray film. The extracts of HB101 cells carrying the indicated plasmid were prepared and either 0.1 microliters, 1 microliter, or 10 microliters were assayed. Cells carrying pSHl expressed nearly a 10-fold higher level of activity over control cells. Virus preparation from infected NIH/3T3 cells (10 microliters) was also performed. HB101 cells carrying the indicated plasmids were as¬ sayed using 1 microliter or 5 microliters of extract. C2110 cells carrying the indicated plasmids were as¬ sayed as before. Extracts of C2110 bearing pSHl showed the same high levels of activity seen in the HB101 host. Reverse transcriptase assays as a screen of cloned variants of the pSHl plasmid. Amounts of 0.1 ul total protein extract from each clone were assayed as described below. Cells carrying pATH-1 and pSHl were used as standards. One plasmid, pSHNBδ, showed significantly higher levels than the parent. Plasmid pSHNB63 showed a high level comparable to that of pSHNBδ. Although some constructs showed higher levels than pSHl, none equalled the levels of pSHNBδ and pSHNBδ3. These results suggest that the plasmid speci¬ fied considerable reverse transcriptase activity, inde¬ pendent of DNA polymerase I. It is noteworthy that the single copy of the gene fusion in the C2110 cells expressed as much activity as the multicopy genes in HB101. emoval __£ ______ trpg sequences results in increased enzyme activiy. Cultures containing each of the variant plasmids gener¬ ated by mutagenesis of pSHl were grown and starved for tryptophan; extracts were assayed for reverse trans- criptase activity as before. Approximately 100 inde¬ pendent clones were screened, and two were found to produce dramatically higher levels of activity than the parental pSHl plasmid. One of these clones, carrying plasmid pSHNBδ, was chosen for further study. Quanti¬ tative assays reproducibly showed that cells carrying the new plasmid expressed a 4-8 fold higher level of activity than cells carrying pSHl; the cells showed as much as a 35-fold increase in activity over cells car¬ rying the pATH-1 vector alone (Table 1) . Restriction analysis of the pSHNBδ DNA showed that the bulk of the trpE sequences had been successfully re¬ moved, and that only a short part of the 5' end.of the trpE gene was joined to the pol sequences. T05define the precise junction in this clone, the DNA sequence of this region was determined by the procedure of Maxam and Gilbert (9). The sequence (Figure 1) showed that 18 codons of trpE were joined, in the correct reading frame, to the pol gene; 17 bp had been removed from the pol gene by the SI treatment, leaving only 7 codons of pol sequence 5' to the start of the mature reverse transcriptase. This deletion in this active clone did not extend upstream of the Nru I site, in contrast to the deletions present in many less active clones. The plasmid in the second highly active clone, pSHNB63, also retained similar trpE coding sequences (data not shown) . These results suggest that the presence of the trpE codons upstream of this site may help to stabilize the protein product. Analysis of fusion proteins encoded by pSHl and pSHNBδ . We next analyzed the polypeptides synthesized in HBIOI cells carrying the pSHl and pSΞNBδ plasmids. Both the total proteins, and the proteins remaining insoluble after addition of detergent and high salt concentra¬ tions were isolated (12) . These fractions were sub¬ jected to SDS polyaerylamide gel electrophoresis, and the proteins were detected by Coo assie stain. Cells transformed with the pATHl vector, alone contained large amounts of a truncated trpE protein migrating at the position of a 37,000-dalton polypeptide; as previously reported, the majority of this protein was recovered in the insoluble fraction (22) . The sequence of the pSHl plasmid predicted the formation of a fusion protein containing both the £χp£ and paL polypeptides, of 124,000 daltons; cells carrying pSHl exhibited a major new protein migrating at approximately 120,000 dal¬ tons, in good agreement with the expected size. Virtu¬ ally all of this polypeptide was recovered in the in¬ soluble fraction. In addition, many smaller proteins specific to cells carrying pSHl were detected. These proteins, ranging in size from 110,000 to 52,000 dal¬ tons, represented a major portion of the total mass of the new protein induced by pSHl. We have found that extended formation of these smaller proteins, and that the addition of protease inhibitors during the lysis did not reduce the formation of these products*. The structure of the pSHNBδ plasmid predicted the syn¬ thesis of a protein of 89,000 daltons; examination of cells carrying pSHNBδ showed substantial amounts of a new protein of about 90,000 daltons, as well as lesser amounts of smaller proteins. SDS polyacrylamide gel electrophoresis of bacterial proteins from HB101 cells carrying various plasmids was performed. Proteins were analyzed by Coomassie blue staining. Experiments were performed on proteins of HB101 cells containing vector pATHl alone; total proteins of HBIOI cells containing plas id pSHl; insoluble fraction from cells carrying pATH; and insoluble fraction from cells carrying pSHl. Coomassie staining of insoluble proteins was performed on gels containing a) cells without plasmid, b) cells carrying pSHNBδ. A fluorogram of proteins etabolical- ly labelled after induction of the trp operon in cells carrying pSHNBδ was also performed. The major bands induced by pSHNBδ are visible at 90, 70, and 60 kilo- daltons. Immunoprecipitation of bacterial proteins was performed. Cultures were labelled with 35 S-methionine after induction of the trp operon, and the soluble proteins extracted with detergents (see Methods) . The extracts were incubated with various sera, and the immune complexes were collected with S. aureus cells. The bound proteins were analyzed by SDS gel electrophoresis and fluorography. The gels contained samples of proteins of control HB101 cells carrying the vector pATHl; proteins of HBIOI cells carrying pSHl; proteins of HB101 cells carrying pSHNB6; normal rabbit serum; rabbit serum specific for the trpE protein; NCI serum #775-424 specific for reverse transcriptase; and NCI serum #775-454 specific for reverse transcriptase. These proteins could be detected in total lysates, but there were high levels of other bacterial proteins in the same region of the gels. Analysis of the proteins labelled with S-methionine at the time of induction of the £j_pE. gene clearly revealed the 90,000 dalton protein and two major species at approximately 70,000 and 60,000 daltons. The identity of these proteins as products of the gene fusion was confirmed by immunoprecipitation with spe¬ cific antisera. Cells were labelled with S-methio¬ nine after induction of the trp operon. The proteins were extracted with strong detergents, immunoprecipi- tated with specific sera, and analyzed by electro¬ phoresis and fluorography. All cells tested showed high levels of a 60 kd protein which was nonspecifi- cally precipitated by all sera; cells carrying pATHl contained in addition high levels of trpE proteins in the range of 30 to 35 kd which were specifically pre¬ cipitated by serum prepared against the £J_E£ polypro¬ tein. These control cells contained no proteins reac¬ tive with sera raised against authentic murine reverse transcriptase. Cells carrying pSHl contained new proteins reactive with the specific sera. A protein of 120,000 daltons, and a number of smaller polypeptides ranging in size from 110,000 to 60,000 daltons were barely detectable with the trpE serum. A similar spectrum of proteins was readily seen with either of two sera reactive with reverse transcriptase (lanes 7 and 8) , demonstrating that the new proteins contain determinants of both trpE and reverse transcriptase. The soluble fractions were highly enriched for the smaller proteins relative to the full-length product, compared with the insoluble fraction, suggesting that the small proteins were se¬ lectively extracted. Immunoprecipitation of the proteins from cells carrying pSHNBδ with sera specific for viral reverse trans¬ criptase showed that a 90,000. dalton protein and at least one major smaller protein were also recognized by the sera. A higher proportion of the full-size pSHNBδ protein was recovered in the soluble fraction than with the corresponding pSHl protein. Thus, the removal of the bulk of the J∑LEE. sequences, and possibly part of the 5'-p2 sequences, resulted in the synthesis of a smaller protein with increased solubility, improved stability to proteolytic degradation, and exhibiting a higher level of reverse transcriptase activity. we have not determined whether the higher activity is due solely to an increase in the amount of soluble full- size protein recovered, or whether it is due in addi¬ tion to an increase in the specific activity of the enzyme. Partial purification of the pSHNBδ protein To characterize the new activities induced by the gene fusions, and to demonstrate that the DNA synthetic activity was not due to DNA Polymerase I, we partially purified the activities. Large-scale cell cultures were- prepared and starved for tryptophan as before. The cells were harvested, washed, and lysed by treat¬ ment with lysozyme and NP40 detergent, and the insolu¬ ble material removed by centrifugation (see Methods) . The salt concentration was reduced by dialysis, and the bulk of the DNA in the solution removed by precipita¬ tion with streptomycin sulphate; 80% of the activity remained soluble through this procedure. The material was applied to a DEAE cellulose column at a low ionic strength, and the activity was recovered by elution with buffer containing 0.2 M NaCl. Fractionation of the eluate from cells carrying pSHl by ammonium sulfate precipitation showed (Table 1) that most of the activity was recovered in the fraction precipitated by 40% saturated ammonium sulfate. The proportion of the activity that was sensitive to NEM was monitored as before. The bulk (90%) of the activi¬ ty in this fraction was sensitive to NEM, indicating that the activity was due to the pSHl enzyme. The rest of the initial activity was recovered in the 40-to-70% aπanoniu sulfate fraction, known to contain DNA poly¬ merase I (23) . This activity, as expected, was largely resistant to NEM treatment. The activity induced by pSHNBδ behaved similarly. After elution from DEAE cellulose, the bulk of this activity was precipitated by addition of ammonium sul¬ fate to 45% of saturation, and the fraction of the activity which was sensitive to NEM was monitored as before. The crude material was 23% resistant. The activity which was precipitated by ammonium sulfate was only 9.5% resistant, and was therefore enriched for reverse transcriptase-like activity; the activity which remained soluble was now 72% resistant, and therefore consisted largely of DNA polymerase I. Further purifi¬ cation of the pSHNBδ protein on phosphocellulose showed that the activity could be bound and eluted with buffer containing 0.1-0.2 M NaCl. Preliminary characteriza¬ tion of this activity showed that long DNA products could be synthesized, and that RNAse H activity had copurified through these steps. A more detailed de¬ scription of the purification and characterization of this and similar fusion proteins will follow. Discussion These experiments demonstrate that portions of the pol gene of a mammalian retrovirus can be expressed as a gene fusion with the bacterial trpE gene. The fusion proteins are sufficiently abundant and stable to be detected after electrophoresis of the total bacterial proteins, and are major proteins in an insoluble frac¬ tion of the lysate. Our crude estimate is that the Pfii-related products represent about 1% of the total protein after induction of the trp operon. Extracts containing these proteins show reverse transcriptase activity, as assayed by the synthesis of DNA on ribo- homopolymer templates. The level of activity is many- fold greater than the low activity due to the endo¬ genous DNA polymerase I, is independent of the polA gene in the host, and shows biochemical properties distinct from those of this enzyme. It is clear that the gene constructs lead to the forma¬ 0 tion of shorter products as well, probably formed by degradation of the primary translation product within the cell. The shorter products may be responsible for much of the detectable activity. Thus, many modifica¬ tions in the gene fusion which allow the direct forma- 5 tion of similar, smaller proteins might yield higher levels of recoverable activity. The increased activity seen for the pSHNBδ construct is consistent with this notion. Recently, further efforts to trim the size of the gene to its minimum have recently led to the syn¬ 0 thesis of products with increased solubility, stabili¬ ty, and activity. We believe that the expression of the murine reverse transcriptase in bacterial cells will lead to several _ important projects. Firstly, the availability of large quantities of the purified enzyme will allow extensive characterization of the enzyme. Secondly, mutations can be readily introduced into the cloned gene fusions, and large numbers of bacterial cultures can be screened -_ for the presence of rare variants exhibiting desirable changes in the activity. It may be possible, for exam¬ ple, to construct variants which do not express RNAse H activity. Thirdly, mutations such as temperature-sen¬ sitive mutations can also be generated, and a DNA frag¬ ment containing the alteration can be recovered and reinserted into the complete viral genome. In this way it may be possible to study the effects of many new mutations on the retroviral life cycle and determine new functions for the reverse transcriptase enzyme. SECOND SERIES QF EXPERIMENTS Materials and Enzymes ( Λ 32 P) dATP, ( < 32 P) TTP, and ( o 32 p) dCTP were pur- 3 32 chased from Amersham; ( J H) ATP and ( Y P) ATP were from ICN. DEAE-cellulose (DE52) , phosphocellulose (Pll) , and DEAE-cellulose paper (DE81) " were obtained from Whatman. Agarose-polycytidilic acid was either purchased from PL bioche icals or synthesized as de¬ scribed (24) . Polycytidilic acid was purchased from PL Biochemicals. Cyanogen bromide activated agarose, 3-indoleacrylic acid, and protein .molecular weight standards were purchased from Sigma, lysozyme from Worthington, and p-Toluenesulfonylflouride from Kodak. iLt. coli RNA polymerase was purified as previously de¬ scribed (25,26) . Bal 31 nuclease was purchased from IBI; T4 Polynucleotide Kinase from BRL; Calf Intesti¬ nal Alkaline Phosphatase from Boehringer Mannheim; and all restriction endonucleases from New England Bio- labs. T4 DNA ligase was a gift of J. van Oostrum, of this department. PolyA + RNA from human fetal muscle tissue was a gift of L. Saez, Albert Einstein College of Medicine, Bronx, N.Y. Total RNA from human reticu- locyte lysate was a gift of Dr. C. Dobkin, this insti¬ tution. Actinomycin D was a gift of Dr. S. Silverstein, this institution. Sera specific for the N-terminal 37,000 daltons of the TrpE protein was a gift of Dr. 0. Witte, University of California, Los Angeles. Sera #775-424 and #775-454 were raised in goat against Rauscher reverse transcriptase and ob¬ tained from the National Institutes of Health. The Rauscher reverse transcriptase was isolated by ion exchange chromatography and gradient centrifugation. The sera showed cross reactivity to the reverse tran¬ scriptase and PS 0 - 45 proteins. Buffers Hind III and Pvu I digestion buffer contained 10 mM Tris-HCl buffer, pH 7.5, 6 mM MgCl j , 0 Λ mM dithio- threitol, and 60 or 120 mM NaCl, respectively. Buffer M contained 50 mM Tris-HCl buffer, pH 7.0, 1 mM EDTA, 1 mM dithiothreitol, 0.1% nonidet P40, and 10% glycer¬ ol. Storage buffer contained 50 mM Tris-HCl buffer, pH 8.0, 1 mM EDTA, 5 mM dithiothreitol, 0.1% nonidet P40, 0.1M NaCl, and 50% glycerol. Growth <?f Bacteria A liter of HB101 cells containing plasmid pB6B15.23 was grown overnight at 37°C in supplemented media (see above) in the presence of tryptophan. The cells were diluted twelve fold in supplemented media lacking tryp¬ tophan, and grown at 30°C until the culture reached an O.D.CQO of 0 * . Indoleacrylic acid was then added to a concentration of 5 ^ μg/ l, and growth was continued to a final O.D.g 00 of 0.8-1.0. Cells were collected in a Sharpel centrifuge, washed in 50 mM Tris-HCl buffer, pH 7.5, 0.5 mM EDTA, and 0.15 M NaCl, spun at 3300xg for 20 minutes, resuspended (1:1 w/v) in 50 mM Tris-HCl buffer, pH 7.5, and 10% sucrose, and frozen in an etha- nol/dry ice bath. Reverse Transcriptase Assay RNA-dependent DNA polymerase activity was assayed as previously described (18) with the following modifica¬ tions: 1) assays (50 ^l) contained 10 g/ml oligo dT and 20 u_g/ml poly rA; 2) incubations were at 37°C for 15 minutes; 3) 40 μl aliquots were removed, spotted on DE81 chromatography paper, and washed three times in 0.3 M NaCl, 0.03 M NaCitrate (2X SSC) for 5 minutes each. Filters were washed in ethanol and the radioac¬ tivity was determined by liquid scintillation counting in Aquasol aqueous scintillant. Preparation of DNA-( 3 H) NA hybrid Reaction mixture (500 μ l) contained 40 mM Tris-HCl buffer, pH 7.9, 32 nmol of M13 single stranded circular DNA, 8 mM MgCl 2 , 2 mM dithiothreitol, 100 mM KC1, CTP, GTP, and UTP each at 115 μH, 55 H of ( 3 H) ATP (specif¬ ic activity*1142 cpm/pmol) , and 28 ^.g of i__ coli RNA polymerase. The mixture was incubated at 37°C for 75 minutes, the reaction was stopped by the addition of 10 M EDTA, and the products were extracted with an equal volume of phenol, ether extracted twice, and concen¬ trated by vacuum centrifugation. The fraction (50 Λ I) was loaded onto a G-50 column (1 x 22 cm) . The 32 P- labeled material which eluted in the void volume was pooled, concentrated by vacuum centrifugation, and precipitated with 2.5 volumes of ethanol in the pres¬ ence of 1 M ammonium acetate. The pellet was washed with 70% ethanol, and resuspended in 5 mM Tris-HCl buffer, pH 7.5, 0.5 mM EDTA (1 ml). RNase H assay Reaction mixtures (50 μ l) contained 40 mM tris-HCl buffer, pH 8.0, 2 mM dithiothreitol, 40 mM KC1, 1 mM MnCl 2 , and 10 pmol of ( 3 H) AMP incorporated into DNA- ( 3 H)RNA hybrid and enzyme as indicated. Mixtures were incubated for 30 minutes at 37°C, and the reactions were stopped by the addition of aPP^r pH 6.0, to final concentration of 0.05 M (50^x1) and 250 ug sonicated salmon sperm DNA. Precipitation of the residual RNA/DNA was carried out in the presence of 20 q bovine serum albumin by the addition of trichloroacetic acid to 8%. The mixture was kept on ice for 10 minutes and spun for 10 minutes in an Eppendorf microcentrifuge, and an 150 j l aliquot of the supernatant was counted for ( 3 H) radioactivity using Aquasol liquid scintil- lant. Protein determination Protein concentration was determined using the method described by Bradford (27) . Isolation of polvA+RNA 01igo(dT)-celluose column chromatography was performed as described (28) . 340 μq of total RNA from human reticulocyte lysate was loaded onto a column (200^1) and the bound fraction was collected (1.2 ml), precipi¬ tated in ethanol in the presence of 0.4 M NaCl, washed three times in 70% ethanol and once in 100% ethanol. The pellet was resuspended in 14 ul of 10 mM Tris-HCl buffer, pH 7.5 and ImM EDTA. Enzvmatic manipulations of DNA 11.5 μq of pSHNBδ plasmid prepared as described (29) was linearized with Hind III restriction enzyme (40 units) as described above. The reaction mixture was incubated for 2 hours at 37°C, extracted with an equal volume of phenol two times, ether extracted twice, and precipitated with 2.5 volumes of ethanol in the pres¬ ence of 0.5 M NaCl. Pellets were dried under vacuum and resuspended in 600 xl of Bal 31 buffer (20 mM Tris- HCl, pH 8.0, 600 mM NaCl, 12.5 CaCl 2 and 12.5 MM MgCl 2 ) . To each of six 100 μl aliquots, 1.8 units of Bal 31 exonuclease (IBI) was added and the mixtures were incubated at 30°C for either 0, 7.5, 15, 30, 45, or 60 minutes. Digestion was terminated by addition of sodium dodecyl sulfate and EDTA to a final concentra¬ tion of 0.1% and 12 mM, respectively. Reaction mix¬ tures were heated at 65°C for 15 minutes, phenol ex¬ tracted, ether extracted, and precipitated in ethanol as described above. The pellet was resuspended in PvuI buffer and digested with 8 units of Pvu I restriction enzyme. The reaction was quenched with electrophoresis sample buffer (10% glycerol, 0.1% sodium dodecyl sul¬ fate, 20 mM EDTA) and the DNA was fractionated by electrophoresis in a 0.8% agarose gel containing 1 ,g/ml ethidium bromide in TEA buffer (50 mM Tris-HCl, 1 mM EDTA, Acetic acid to pH 8.0). Inspection of the DNA under UV light indicated that the rate of digestion was 30 bases/minute and that the Pvu I digestion was not to completion. The predominant DNA species for each time point was isolated form the gel by the glass powder method (30) . In parallel, the parent plasmid pATHl, was in digested with Smal (20 units) in buffer (6 mM Tris-HCl buffer, pH 8.0), 20 mM KCL, 6 mM MgCl 2 , 1 mM dithiothreitol, and 100 g/ml bovine serum albumin for 2 hours at 37°C and subsequently with Pvu I (8 units) after the addition of 100 mM NaCl. The 780 base fragment was isolated from a 1.2% agarose gel as de¬ scribed above. The 780 base fragment isolated from pATHl and the approximately 5000 base long fragment isolated after Bal 31 digestion of pSHNBδ for either 7.5 or 15 minutes were mixed and treated as described (20) with 3.5^u.g of T4 DNA ligase at 15°C, overnight. Since the Pvu I digestion on pSHNBδ was to completion, two constructs could be formed. One would result from an intramolecular ligation of linear molecule which was bidirectionally digested with Bal 31 nuclease. The termination codon for the protein would appear randomly within the plasmid sequence depending on the extend of the nuclease digestion. Alternatively, in- termolecular joining of the two fragments could result in a unidirectional deletion. This would result in a known DNA sequence and defined terminator codons being placed 3' downstream of the coding region of the gene. Aliquots of the ligation reaction mixtures were used to transform I*. coli HB101 to ampicillan resistance (31) . Plasmids isolated from colonies (32) were screened for deletions using Sal I, Pvull, Bgl I, Bgl II, and Xmnl. Although two possible constructs could have been made, only the intermolecular ligation described above was detected. Screening deletion mutants for Reverse Transcriptase activ ty Crude extracts of colonies were made as described (see Bacterial strains and media) and assayed for reverse transcriptase activity, with the following modifica¬ tions: 1) Two hours after induction with indoleacry- lic acid, a 5 ml culture was spun in a Savant Speedvac concentrator in the absence of a vacuum. The pellet was washed in 1.25 ml of 50 mM Tris-HCl buffer, pH 7.5, 0.5 mM EDTA, and 0.15 M NaCl, transferred to 1.5 ml microfuge tubes and recentrifuged. 2) The time of lysozyme digestion and nonidet P40 treatment was in¬ creased to 30 and 15 minutes, respectively. 3) The soluble extract was assayed for activity. Immune Precipitation Extracts for immune precipitation were prepared as described for screening of deletion mutants (see above) with the following modifications. ( 35 S)-methionine was added to 40 ^Ci/ml (7.5 ml culture) at the time of indoleacrylic acid addition. Uninduced control cul¬ tures of pB6B15.23 were grown in the presence of sup¬ plemented M9 media plus (50jug/ml) ampicillin and 200 mg/ml tryptophan throughout; indoleacrylic acid was omitted. Cells lacking plasmids were grown in the absence of ampicillin. After digestion with lysozyme, the solution was made 1% Triton X100, 0.5% sodium de- oxycholate, 0.1% sodium dodecyl sulfate, 10 mM NaPi, pH 7.5, and 0.1 M NaCl (IX phospholysis buffer). The mixture was kept on ice for 15 minutes and 1.13 ml of IX phospholysis buffer and 25 ul of formalin-fixed, heat-killed Staphylococcus aureaus cells (Pansorbin resuspended 1:1 v/v in phospholysis buffer plus 1% bovine serum albumin) were added. The mixture was spun in a Ti50 rotor at 45,00Q rpm for 90 minutes, and 200 J.1 aliquots of the supernatant were incubated with each antibody (5 ixl) overnight. The complexes were absorbed to Pansorbin for 1 hour on ice, and collected by centrifugation. Pellets were washed twice in 500 ul of IX phospholysis buffer and resuspended in 0.125 M Tris-HCl buffer, pH 6.8, 2% sodium dodecyl sulfate, 20% glycerol, 0.01% bromophenol blue, 62 mM EDTA, and 2% - mercaptoethanol (50 ^1). Samples were boiled for 10 minutes and aliquots (30 ^Λ.1) were subjected to sodium dodecyl sulfate polyaerylamide gel electrophoresis (10% polyacrylamide separating gel, 6% polyacrylamide stack¬ ing gel) . The gel was washed 3 times for 5 minutes in H-O, and once in 1 M sodium salicylate for 30 minutes, dried and subjected to autoradiography. ATPase Assay Reaction mixtures ( 25 J .1) contained 50 mM Tris-HCl, pH 8.3, 2 mM DTT, 2 mM MgCl 2 , and 1 mM ( X 32 P) ATP (2 cpm/pmol) , and levels of enzyme varying between 29 and 145 units. The mixtures also contained either no DNA, xi74 single stranded circles (380 ng) , phage lambda DNA digested with Hind III (750 ng) , or a mix¬ ture of poly (rA) and oligo (dT) (1 and 0.5 g, respec¬ tively) . Reactions were incubated at 37°C for 30 min¬ utes, spotted on polyethyleneimine plates, chromato- graphed in 1 M KH 2 PO., pH 3.4, and subjected to autora¬ diography. Purification of Reverse Transcriptase Activity 7 grams (packed cell weight) of HBIOI cells containing pB6B15.23 induced as described above were thawed and made 50 mM Tris-HCl, pH 7.5, 10% sucrose, 0.3 M NaCl, 1 mM EDTA, and 1 mM PMSF to a final w/v ratio of 1:4. Lysozyme (1 mg/ml, final concentration) was added and the suspension was kept on ice for 10 minutes. Nonidet P40 was then added to a final concentration of 0.2%. The lysate was incubated an additional 5 minutes, was made 1 M with NaCl, and was centrifuged at 30,000 rpm in a Ti60 rotor for 30 minutes. The supernatant was dialyzed for 1 hour against Buffer M, and then diluted with Buffer M to a conductivity equivalent to that of buffer M plus 75 mM NaCl (total volume * 238 ml) . The fraction was loaded on to a DEAE column (4 x 16.5 cm; DE52, Whatman ) equilibrated with Buffer M + 75 mM NaCl. The column was washed with the same buffer. Most of the reverse transcriptase activity was not retained by the resin (see Table 2) . The flow through fraction was applied directly onto a phosphocellulose column (2.5 x 28 cm; P-ll, Whatman) equilibrated with Buffer M + 75 mM NaCl. The column was washed with 1 column volume of the same buffer and eluted with 650 ml linear gradient of 75-700 mM NaCl in Buffer M. Fractions (10 ml) were collected and assayed for reverse transcrip¬ tase and RNase H activities. As shown in Figure 3, the reverse transcriptase activity was completely re¬ tained on the column and eluted as a single peak be¬ tween 0.21-0.24 M NaCl. The predominant RNase H activ¬ ity was coincident with the reverse transcriptase ac¬ tivity. Phosphocellulose fractions 27-30 were pooled (42.5 ml). Aliquots (10 ml) were diluted in Buffer M to a conductivity equivalent to 50 mM NaCl in Buffer M and loaded individually onto an Agrarose-polyribocyti- dilic acid column (1.1 x 8.5 cm) equilibrated in Buff¬ er M containing 50 mM NaCl. The column was washed with 1 column volume of the same buffer and eluted with a linear (56 ml) gradient of 50 to 300 mM NaCl in Buffer M. The reverse transcriptase activity, the RNase H activity, and the total protein co-chromatographed and eluted in a coincident peak between 135-180 mM NaCl. Fractions 24-34 were pooled and concentrated by hy¬ droxylapatite column chromatography. The fraction was loaded on a column (1.3 x 3 cm) equilibrated in Buffer M containing 0.22 M NaCl, washed with 1 column volume of the same buffer, and eluted with Buffer M containing 0.2 M NaCl and 100 mM NaP^ pH 7.0. Fractions (1 ml) were collected; those containing reverse transcriptase activity were dialyzed for 7 hours against storage buffer and kept at -70°C in aliquots. RESULTS Construction of a Gene Fusion Expressing Stable Fusion Protein An outline of the scheme used to stably express the MuLV reverse transcriptase activity, is presented in Figure 2. The initial construct used as the starting material for further manipulation was the plasmid pSHIBδ , containing the region of M-MuLV from nucleotide position 2574 to 4893 (8) inserted in frame downstream of the first eighteen codons of the £_,. £L__ i trpE pro¬ tein (6) . Our earlier work demonstrated that extracts of £. coli strains bearing this plasmid contained high levels of reverse transcriptase activity (6) . Analysis of the proteins synthesized in these strains, however, indicated that the major product was broken down into smaller species; partial purification of the soluble reverse transcriptase also indicated that multiple species were active. The breakdown of the fusion pro¬ tein was not prevented by the addition of protease inhibitors in the lysis procedure (unpublished observa¬ tions) . An additional problem was that the majority of the fusion proteins partitioned into the insoluble fraction after cell lysis. In an attempt to stabilize and solubilize the protein, deletions were made at the 3* terminus of the cloned pol gene. The assumption was made that random Bal 31 deletions at this terminus might result in the forma¬ tion of a protein that more closely resembled the au¬ thentic cleavage product, and might improve its stabil¬ ity in £. coli. To make deletions, plasmid pSHNBβ was linearized with Hind III, digested with Bal 31 nuclease for varying lengths of time, religated with T4 DNA ligase and used to transform £. coli HB101. Colonies were selected, and crude extracts were prepared and assayed for reverse transcriptase. Table 2 summarizes the screening of colonies which were digested with Bal 31 nuclease for 7.5 and .15 minutes, producing an aver¬ age deletion size of 200 and 400 bp, respectively. Since the deletions were bidirectional, the average number of base pairs removed from within the pol gene would be half of these numbers. The soluble reverse transcriptase activity of each colony produced after Bal 31 digestion for 7.5 minutes was similar to that of the parent plasmid pSHNBδ. Colonies produced after digestion with Bal 31 nuclease for 15 minutes yielded a much larger range of activities. 10% of the colonies screened yielded no detectable reverse transcriptase activity, 45% of the colonies yielded less than 50% as much activity as the cells containing the parent pSHNBδ plasmid, and an additional 10% of the colonies yielded up to four-fold higher activity than the parent strain. The extracts from colonies which displayed enhanced reverse transcriptase activity were analyzed by poly¬ acrylamide gel electrophoresis. One colony was select¬ ed for further study (pB6B15.23) because of the follow¬ ing features: 1) The specific activity of extracts from these cells was 3.5-4 times that of pSHNBβ; 2) The level of induction was reproducible; 3) Coomassie blue staining of polyacrylamide gels indicated that a single species of M r =71,000 was highly and stably overpro¬ duced; 4) Comparison of the insoluble and soluble frac- tions by polyacrylamide gel electrophoresis indicated that at least 30% of the M r »71,000 band could be de¬ tected in the soluble fraction (data not shown) . The DNA sequence of this plasmid in the region of the deletion was determined and is shown at the bottom of Figure 2. The deletion resulted in the removal of 204 nucleotides of the M-MuLV pol gene sequence and 64 bases of pATHl. The first stop codon is found twenty- seven bases from the new junction between the MuLV coding sequence and the vector. The carboxy1 terminus of the fusion protein would contain nine novel amino acids encoded by the pBR322 sequence. TABLE 2 Summary of Bal 31 Deletion of pSHNB6 clone of MiiT.V Reverse Transcriptase Time Bal 31 Average No. of % of pSHNBδ reverse digestion deletion colonies transcriptase acti- (min) size screened vity 0-5 5-50 50-100 100 number of colonies 7.5 200 6 0 0 6 0 15 400 49 5 22 17 5 Summary of Bal 31 deletion of pSHNBδ clone of M-MuLV reverse transcriptase. Isolation of DNA fragments and subsequent Bal 31 nuclease digestion was as described in Experimental Procedures. The average deletion size was determined by electrophoresis of an aliquot of the reaction mixture on a 0.6% agarose gel in 90 mM Tris base, 90 mM Boric acid, and 2.5 mM EDTA. Extracts were prepared and assayed for reverse transcriptase as de¬ scribed in Experimental Procedures. Fractions were diluted (1:20) in Buffer M plus 0.2 M NaCl, assayed, and the level of activity was compared with the activi¬ ty of the parent pSHNBδ construct. The entries in the table summarize three separate assays. Analy sis of the Fusion Proteins by Immune Precipitation Analysis of crude extracts of cells car rying the PB6B15.23 plasmid by polyacrylamide gel electrophoresis and Coomassie bl ue stain indicated that a single stable fusion protein was synthesized. To determine if this was the only product made and if this product was structurally similar to the viral MuLV reverse tran¬ scriptase, immunoprecipitation was performed using 10 various sera. Cultures of HB101 alone , HB101 bearing the vector plasmid pATHl , or HB101 bearing pB6B15 .23 , were grown with and without induction of the trp oper¬ on and were then labelled with ( 35 S) methionine . Ex¬ tracts were prepared and were incubated with either , c normal goat - serum ; normal rabbit serum ; two sera pro¬ duced against authentic Rauscher MuLV reverse tran¬ scriptase, termed 775-424 and 775-454 ; and a serum specif ic for the N-terminus of the trpE protein . Immu¬ noprecipitation of S-labelled extracts with various 20 sera were pe rformed as follows : Preparation of 35 S-labelled extracts, precipitation of 35 S-labelled extracts, precipitation with various sera, and gel electrophoresis were as described in Experimen¬ 25 tal Procedures. The various sera used were as follows: Normal goat serum; Normal rabbit serum; Serum #775-424 (raised against Rauscher reverse transcriptase); Serum #775-454 (raised against Rauscher reverse transcrip¬ tase); Serum raised against the N-terminus of TRpE 30 protein. A protein of M =59,000 was precipitated non- specifically with all the sera and extracts teste). Both anti-Rauscher reverse transcriptase sera recog¬ nized a single protein species of M *71,000 in the extracts of cells expressing the cloned M-MuLV reverse transcriptase. This protein, was not present in HB101 35 or in HB101 bearing the parent plasmid pATHl. This unique protein species had the same electrophoretic mobility as the protein identified previously by Coomassie blue staining of crude extracts on polyacryl¬ amide gels. Analysis of cells grown under conditions repressing the trp operon (in the presence of trypto¬ phan and without the addition of indoleacrylic acid) indicated that lower levels of the M =71,000 species were being synthesized, although the protein could 10 still be detected. The serum specific for the trpE protein recognized a M =52,000 species in HB101, pre¬ sumed to be the £χs£ protein encoded by the chromosome (calculated M =57,524), as well as the truncated M =37,000 JtipE protein encoded by pATHl.. In extracts -.. of HB101 containing pB6B15.23 grown under conditions of induction, only the M r =71,000 fusion protein, which contains only the first eighteen amino acids of the trpE protein, was not recognized by this serum. Purification o_f Reverse Transcriptase and Associated 20 RNase H To characterize the reverse transcriptase activity induced in cells carrying the pB6B15.23 plasmid, and to 25 determine whether this activity did indeed reside in the novel ppl-related protein, the activity was puri¬ fied. The main assay for the enzyme measured the in¬ corporation of radiolabelled dTTP into polymeric form on a poly(rA) template primed with oligo(dT). The 30 crude extracts were prepared by detergent lysis after lysozyme treatment; the conditions of detergent and high salt utilized were important in solubilizing the activity. The presence of a nonionic detergent was required throughout the purification to prevent aggre¬ 35 gation and loss of activity. The purification of the pB6B15.23 reverse transcriptase is detailed in the Experimental Procedures, and is summarized in Table 3. The final procedure involved column chromatography on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, and hydroxylapatite. The DEAE-cellulose step was used to remove nucleic acids from the preparation. Since the fusion protein did not bind DEAE-cellulose, only a modest purification of the enzyme was obtained by passing the activity through the column in a low salt concentration. Phos¬ phocellulose column chromatography was the single most important step in the purification, resulting in a 6.6- fold increase in the specific activity of the prep¬ aration; the total recovery in this step was, unfortu¬ nately, rather low (24%) . Polyribocytidylic acid chro¬ matography was useful because very few proteins were capable of binding to the resin; the pB6B15.23 enzyme was essentially the only protein bound and eluted from the resin. Due to the high level of expression of the fusion protein in £_,. coli , the overall preparation of the reverse transcriptase activity required only a 22- fold purification. The M-MuLV reverse transcriptase has been reported to have an associated RNase H activity (4,5). To deter¬ mine if the region of the pol gene expressed in J a . coli encoded the RNase H function, fractions from each stage Table 3 Purification of pB6B!5.23 Reverse Transcriptase and Associated Activities RNase II Protein Reverse Transcriptase Fraction Specific Specific Activity Activity Units/mg Yield Units/mg Units/ Yield Protein Units/ml Units % Protein nd Units % mg DEAE load 2643 629,200 100% 737 225 53,500 100% 62.7 854 DEAE Flow through 1400 490,000 78% 972 31.6 11,060 21% 21.9 504 1 _ Phosphocellulose 2786 118,420 19% 6,440 52.2 2,218 4.1 121 18.4 Poly C Agarose 4918 56,510 9.0% 12,840 62.2 715 1.3 163 4.4 Hydroxylapatite 15120 52,920 8.4% 13,330 205 716 1.3 180 4.0 Dialysis 28910 50,590 8.0% 16,160 297 519 0.97 166 3.1 Purification of pB6Bl5.23 reverse transcriptase and associated activities. Reverse transcriptase, RNase H, and total protein were assayed as described in Experimental Procedures. One unit of reverse transcriptase is defined as that amount of enzyme which will incorporate one nmol of ( 32 P) dTMP into a form retained on DE81 paper in 15 min. at 37°C. One unit of HNase 11 activity Is defined as that amount of enzyme which will release one mnol of ( 3 H) AMP from an RNΛ.DNΛ hybrid into an acid soluble form in 30 min. at 37°C. of the purification were assayed for RNase H as well as reverse transcriptase activity. The profile of these two activities on phosphocellulose chromatography is shown in Figure 3. A substantial amount of RNAse H activity was eluted with low salt concentrations; in addition, a predominant peak of RNase H activity was coincident with the reverse transcriptase activity and eluted at 0.22 M NaCl. The RNase H activity eluting with the low salt concentration was not identified nor characterized further. The phosphocellulose fractions containing the reverse transcriptase activity and RNase H activity were further chromatographed on polyribo¬ cytidylic acid agarose (Figure 4) . The reverse tran¬ scriptase activity and the RNase H activity co-chro- matographed as a single peak on this resin. These two activities were also associated after hydroxylapatite column chromatography and after glycerol gradient cen¬ trifugation in 0.5 M NaCl (see below). These results suggest that the central portion of the pol gene of M- MuLV present in the construct encodes both reverse transcriptase and RNase H activities. Sodium Dodecvl Sulfate Gel Electrophoresis To assess the purity of the reverse transcriptase, the protein fractions from the various stages of the puri¬ fication were subjected to sodium dodecyl sulfate poly¬ acrylamide gel electrophoresis, and the proteins were visualized by the silver staining procedure (10) . pB6B15.23 reverse transcriptase was purified by sodium dodecyl sulfate-polyaerylamide gel electrophoresis. Samples from various stages of the purification were subjected to electrophoresis through a 10% polyacryl¬ amide gel containing a 6% stacking gel followed by staining with silver as described (10) . Samples were loaded which contained the following DEAE cellulose load; 18.1 ug protein, 10.6 units of enzyme. DEAE flow through fraction; 12.6 j g protein, 10.1 units of en¬ zyme; pooled phosphocellulose preparation; 2.15 jug protein, 11.1 units of enzyme. Pooled polyribocytidy¬ lic acid-agarose preparation; 0.95 j g protein, 10.0 units of enzyme; pooled hydroxylapatite preparation; 1.03j protein, 10.8 units of enzyme; and final prepa¬ ration after dialysis; 0.89 jug protein, 11.5 units of enzyme. The markers used were the-β, ι > <r and c*. subunit of £. coli RNA polymerase, myosin, /£> - galactosidase, albumin (bovine), albumin (egg), and carbonic anhydrase. The data presented the polypeptide composition of the material applied to the DEAE-cellulose column of the material that flowed through the DEAE-cellulose column of the pooled frac¬ tions after chromatography on phosphocellulose, polyribocytidylic acid-agarose, and hydroxylapatite; and of the final fraction. The analysis showed the presence of a single major band of M * 71,000 in the late stages of the purification. Although the largest loss of reverse transcriptase activity occurred during phosphocellulose chromatography, extensive purification of the M_,»71,000 protein was achieved by this step. The M =71,000 protein is greater than 95% pure after polyribocytidylic acid-agarose chromatography. The hydroxylapatite chromatography and dialysis steps were mainly useful as a means of concentrating the protein. Glycerol Gradient Centrifugation To determine the subunit structure of the purified pB6B15.23 reverse transcriptase, the enzyme was further characterized by glycerol gradient sedimentation (Fig¬ ure 5). The reverse transcriptase and RNase H activi- ties co-sedimented as a single peak with a sedimenta¬ tion coefficient of 4.65S. Based on this sedimentation coefficient, the molecular mass of the species was estimated to be about 65,000 daltons. The proteins from the gradient fractions surrounding this peak of activity were subjected to sodium dodecyl sulfate poly¬ acrylamide gel electrophoresis, and the proteins de¬ tected by silver stain. The results (Figure 5) showed that the presence and intensity of the M r » 71,000 spe¬ cies parallelled the reverse transcriptase and associ¬ ated RNase H profile. The purified fusion protein appears to behave as a monomer, as has been reported for the authentic M-MuLV reverse transcriptase (4,5). Requirements for Reverse - Transcriptase Activity of fusion Protein The assay used for the purification of the pB6B15.23 reverse transcriptase activity measured the incorpora¬ tion of dTMP on a poly (rA) template primed with oligo (dT) . The requirements and the optimal conditions for DNA synthesis by the purified enzyme on this synthetic substrate were determined (Table 4, Section A). Under optimal conditions, incorporation of dTMP occurred linearly with time from 2 minutes to up to 2 h. Maxi¬ mal DNA synthesis saturated at 500 pmols of dTMP incor¬ porated, equivalent to one third of the available sin¬ gle stranded template. Incorporation was almost total¬ ly dependent on the presence of the template -poly (rA) and primer oligo (dT) . In addition, DNA synthesis required a divalent cation; either Mn ++ or Mg ++ were capable of supporting synthesis. The maximal incorpo¬ ration occurred between 0.5-1.0 mM MnCl 2 and was great¬ ly inhibited at levels higher than 2 mM (data not shown) . Mg supported DNA synthesis to a much lower TABLE 4 Requirements for pB6B15.23 MuLV Reverse Transcriptase pmol ( 32 P) TMP incorporated A. Additions Complete 73 Omit DTT 80 Omit MnCl- 0.2 Omit MnCl,, Add MgCl, 1.5 Add MgCl 2 70 Omit oligo (dT) 1.3 Omit poly (rA) 0.2 B Preincufration Complete 50 • Heated 37°C,. 15 min. ' 46 Proteinase K 0.1 10 mM N-Ethylmaleimide, plus.50 mM DTT 0.5 50 mM DTT, plus 10 mM N-Ethylmaleimide 43 Heated 70°C, 15 min. 0.1 Experiment A. The complete reaction mixture contained 50 mM Tris-HCl, pH 8.3, 20 mM dithiothreitol, 0.5 mM MnCl 2 , 60 mM NaCl, 10 αg/ml oligo (dT) , 20 u.g/ml poly (rA), 20 M dTTP (818 cpm/pmol), 0.1% NP40, and 0.072 unit of enzyme. Individual components were omitted as indicated. MgCl 2 was added at 0.5 mM concentration. Experiment B. Complete reaction mixture was as de¬ scribed in experiment A. pB6B15.23 reverse transcrip¬ tase was diluted (1:400) in Buffer M plus 0.2 M NaCl and treated as described prior to the reverse tran¬ scriptase assay. 20 μl of enzyme was digested with proteinase K (5 g) at 37 C for 15 min. Treatment with N-ethyklmaleimiαe and dithiothreitol was performed on ice for 15 min. degree than Mn ++ , with optimal activity occurring at 0.5 mM; higher concentration of Mg ++ also inhibited the reaction (data not shown) . The addition of Mg ++ to a reaction mixture that already contained Mn + , however, did not inhibit the Mn ++ -dependent synthesis. Maximal DNA synthesis on poly (rA): oligo (dT) occurred in the presence of 60-80 mM NaCl; standard reaction mixtures contained 60 mM NaCl. The effect of higher ionic strength is shown in Table 5, Section A. Inhibi¬ tion of DNA synthesis occurred at 120 mM NaCl, with only 12% of the activity remaining at 240 mM NaCl. The enzyme activity was inhibited by inorganic phosphate at levels between 5 mM and 40 mM. The enzyme was extreme¬ ly sensitive to pyrophosphate (Table 5, Section B) ; considerable inhibition of dTMP incorporation was seen at 0.1 mM NaPPi. Similar results were found when the concentration of free Mn ++ was maintained at 0.5 mM or at 1.0 mM in the reaction mixtures. Omission of DTT from the reaction mixture appeared to slightly stimulate the reaction; however, the enzyme fraction contained 5 mM DTT, which may have partially compensated for this omission. The enzyme was almost completely inhibited in the presence of the sulfhydryl antagonist, N-ethylmaleimide (Table 4, Section B) . This inhibition was completely prevented by the prior addition of dithiothreitol. The final fraction was sensitive to preincubation with proteinase K as well as heating at 70°C for 15 minutes. Heating at 42°C for 15 minutes resulted in a 50% loss of activity (data not shown) . TABLE 5 Effects of Salt and Inhibitors of pB6B15.23 MuLV Re¬ verse Transcriptase A. Complete 125 Add 5 mM sodium phosphate 91 Add 20 mM sodium phosphate 58 Add 40 mM sodium phosphate 17 Add NaCl, total 120 mM 70 Add NaCl, total 180 mM 45 Add NaCl, total 240 mM 15 B Complete 99 Add 0.1 mM sodium pyrophosphate plus 0.1 mM MnCl 2 67 Add 0.5 mM sodium pyrophosphate plus 0.5 mM MnCl 2 50 Add 1.0 mM sodium pyrophosphate plus 1.0 mM MnCl, 6.4 Effect of salt and inhibitors of pB6B15.23 MuLV reverse transcriptase. Complete reaction mixture was as de¬ scribed in Table 4. Assay was performed as described in-experimental Procedures. The specific activity of (cv P) dTTP in Experiment A and B was 774 and 316 cpm/pmol, respectively. Conta inating Activities The purified enzyme preparation was assayed with ATPase activity in the absence and presence of single-and double-stranded DNA, and of poly rA: oligo dT, using 32 32 (7^ P) ATP. No release ( P) inorganic phosphate could be detected. Fidelity of DNA synthesis The assay measuring incorporation on the oligo (dT) and poly (rA) substrates was found to be totally dependent on template and primer (see above) . To determine the fidelity of the template-directed synthesis, the incor- poration of various (< 3 P) dNTPs was measured (Table 6) . (c * *P) dTTP was the only nucleotide with which significant incorporation could be detected. Since the authentic M-MuLV reverse transcriptase is capable of DNA-dependent DNA synthesis using an RNA primer, the possibility existed for the incorporation of dAMP: this would result from the use of the oligo (dT) as a template and the poly (rA) as the primer. The mea¬ sured level of ( 32 P) dAMP incorporation was small and accounted for less than 0.1% of that observed with 32 (cKr'P) dTTP. In the presence of unlabelled dTTP, the 32 level of misincorporation of ( P) dCMP decreased, 32 whereas a small increase of ( P) dAMP incorporation was seen. The increase in dAMP incorporation may re¬ flect a low level of second-strand DNA synthesis. Synthesis of Lone cDNA Products In the viral life cycle, reverse transcriptase must synthesize double stranded DNA products over 8 kb in length (1, 24, 25). The size and nature of the cDNA TABLS 6 Incorporation of various dNTPs pmol dNTP ( 32 P) dNMP incorporated ( 32 P) dTTP 144 (Λ 32 P) dCTP 0.01 (λ 32 P) dGTP 0.03 (S 32 P) dATP 0.16 (*- 32 P) dCTP plus TTP 0.01 (<*? 2 P) dGTP plus TTP 0.01 (Λ. 32 P) dATP plus TTP 0.19 Incorporation of various dNTPs. Assay was performed as described in Experimental Procedures. Labelled and unlabelled nucleoside triphosphates were added to 20 uM concentration. The specific activity of each (Λ. P) dNTP was: dTTP, 961 cpm/pmol; dCTP, 16,950 cpm/pmol; dGTP, 14,630 cpm/pmol; and dATP, 13,620 cpm/pmol. products of pB6B15.23 reverse transcriptase were char¬ acterized using various poly A + mRNA preparations primed with oligo (dT) . Reaction mixtures (30 u_l) contained 100 mM Tris-HCl, pH 8.3, 10 mM MgCl 2 , 2 mM dithiothreitol, l ug/ml oligo (dT) , 200JULM dATP, dGTP, dATP, and dCTP (2160 cpm/pmol), 150 mM KC1, 0.05% Nonidet P40, and 60 ng of HOPC1 poly A (+) RNA. PB6B15.23 reverse transcriptase was added as follows: 0.029 unit; 0.29 unit; 2.9 units; 29 units; 145 units; no enzyme addition; reaction in absence of RNA. Reac¬ tion mixtures were incubated at 37°C for 1 h. and stopped by the addition of 20j l of 2% sodium dodecyl ■ sulfate, 50 mM EDA. lO g of carrier tRNA was added, and the mixture was extracted with phenol (50 , back extracted with H 2 0 (25 ul), and ether extracted twice. Mixture was made 2 M in Ammonium acetate ' and DNA prod¬ ucts were precipitated after the addition of 2.5 vol¬ umes of ethanol. Pellets were resuspended in 2 M am¬ monium acetate (50 ul) and reprecipitate in 2.5 vol¬ umes of ethanol. Pellet was resuspended in 10 μl of 10 mM EDTA, and made 50 mM NaOH, 1 mM EDTA, 10% glycer¬ ol, in the presence of bromophenol blue, xylene cyanol, and bromocresol green indicator dyes. Samples were loaded onto a 1.2% agarose gel and electrophoresed for 14 h at 30 V in 30 mM NaOH, 1 mM EDTA. Gels were dried 32 and subjected to autoradiography. (Λ P) dNTPs were incorporated, and the DNA products were analyzed by electrophoresis through alkaline agarose gels and auto¬ radiography. A titration of the enzyme on 60 ng of poly A + RNA from HOPC myeloma cells, under conditions described for the avian reverse transcriptase, showed that the size of the DNA product depended dramatically on the amount of enzyme added. Little or no synthesis was detectable in the presence of 0.02-0.29 unit of enzyme. In the presence of 2.89 units of enzyme, the average DNA product was 365 bases long. When the amount of enzyme was increased ten fold, the DNA prod¬ ucts were between 315 and 1900 bases in length; the prominent 1 kb species corresponds to the cDNA copy of the light chain mRNA. In the presence of still higher levels of enzyme, the size of the DNA product remained constant, indicating the absence of contaminating RNase. No DNA synthesis was detected in the absence of enzyme or template RNA. Optimal conditions for synthesis with the pB6B15.23 reverse transcriptase were found by titrating the vari¬ ous components of the reaction. Both MgCl 2 and MnCl, could fulfill the divalent cation requirement at opti- mal concentrations of 10 mM and 6 mM, respectively. Maximal size and DNA synthesis occurred with a mixture of 8 mM MgCl 2 and 4 mM MnCl 2 . At this level of diva¬ lent cations, the addition of 60 mM NaCl inhibited the DNA synthesis (data not shown). Efficient DNA synthe¬ sis occurred in the presence of high concentrations of deoxynucleoside triphosphates; reaction mixtures con¬ tained 2 mM of each dNTP. Using the optimal conditions, cDNA was synthesized with an oligo (dT) primer on poly A + RNA isolated from human fetal muscle tissue. This RNA preparation was chosen because it is presumably enriched for the very large (7 kb) rπyosin heavy chain mRNA; the size distribution of the product would therefore not. be limited by the size of the RNA templates. Using a commercial preparation of the avian viral reverse transcriptase (20.8 units), the average size of the product was 1200-3300 bases. The product was analyzed using human fetal muscle poly A ' + ' RNA template. Reaction mixtures (30 u.1) con- tained 50 mM Tris-HCl, pH 8.3, 8 mM MgCl 2 , 4 mM MnCl 2 , 10 mM DTT, 1 jug/ml oligo (dT) , 2 mM dCTP (1132 cpm/pmol), dGTP, dATP, and dTTP, 0.01% nonidet P40, and lj g of poly A^ + ' RNA isolated from human fetal muscle. Reverse transcriptase was added as follows: AMV re¬ verse transcriptase, 21 units; pB6Bl5.23 reverse tran¬ scriptase 29 units; 87 units; 174 units; no enzyme addition; and 29 units in the absence of added RNA. Reactions were stopped and prepared for electrophoresis through 0.7% alkaline agarose gel as described in Fig. 3. The amount of sample and the exposure time of the individual lanes varied as followed: 40% of the sam¬ ple was electrophoresed and the gel was exposed for 48 h; 60% of the sample was electrophoresed, gel was ex- posed for 12 h. One sample contained 3 J 2 4 P-labeled Hindlll digest of phage DNA. The product produced with 28.9, 86.7, and 173 units of pB6B15.23 reverse tran¬ scriptase. DNA synthesis saturated with 86.7 units of enzyme, and the majority of the DNA products were be¬ tween 1.3 and 9.9 kilobases in length. The size dis¬ tribution was not changed by the presence of excess enzyme, indicating that RNase contamination was negli¬ gible. DNA synthesis was not detected in the absence of NA or reverse transcriptase. To determine whether the products were single-stranded copies of the mRNAs or double-stranded molecules re¬ sulting from hairpin loopbacks serving as primers, cDNA synthesis was performed on poly A mRNA isolated from human reticulocyte lysates. Product analysis using human reticulocyte poly A^ + ' RNA as template was performed as follows: Poly A^ + ' RNA from human reticulocyte was isolated as described in Experimental Procedures. Reaction mixtures containing approximately 0.2 ug of RNA were assembled as de¬ scribed in above. Actinomycin D (0.5 μΔ. in ethanol) was added to 100 μq/ml where indicated. Samples were electrophoresed through a 1.2% alkaline agarose gel. The samples were as follows: product of AMV reverse transcriptase; a) 2.1 units; b) 21 units; c) product of pB6B15.23 reverse transcriptase, d) 21 units; e) 87 units; f) 29 units plus actinomycin D, g) 87 units plus actinomycin D, h) no enzyme addition, and i) 29 units enzyme in absence of RNA. j) marker 32 P-labeled Hindlll fragments of phage. Exposure a) and b) was 12 h, and c)-d) exposed 24 h. The predominant species in these preparations are mRNAs of about 570 and 640 nu- cleotides, encoding the o and>β -globins. Synthesis with low levels of avian reverse transcriptase yielded a single major product approximately 600 bases in length, corresponding to the full-length single-strand¬ ed cDNA species. synthesis at high levels yielded predominantly two DNA species approximately 600 and 1200 bases in length, corresponding to the single- and double-stranded cDNAs; the major product was the small¬ er, single-stranded cDNA copy. Synthesis with low levels of the purified pB6B15.23 reverse transcriptase also yielded the single-stranded product. With high levels of the enzyme, the 1200 base products were the predominant species, and the single strand products were not detected as a discrete species, indicating that after the completion of the full-length first strand, the DNA was efficiently looped back and used as a primer for the second-strand synthesis. To confirm that the 1200-base products were in fact the result of second-strand synthesis, DNA synthesis was performed in the presence of actinomycin D. Actinomycin D binds preferentially to double-stranded nucleic acids and therefore inhibits the second strand synthesis. The major products of cDNA synthesis of reticulocyte polyA + RNA with pB6Bl5.23 reverse transcriptase in the pres¬ ence of actinomycin D were the 600 base species. No products larger than this species could be detected. As before, no products were synthesized in the absence of RNA or enzyme. Discussion These experiments show that a gene fusion containing a portion of the bacterial trpE gene and the central portion of the M-MuLV poJ gene can induce the synthesis of a stable protein with high levels of reverse tran¬ scriptase activity. A critical step in the successful expression of this activity was the screening of numer¬ ous variants of our initial gene constructs for the formation of maximum levels of stable, soluble protein. Although our initial clone did induce considerable reverse transcriptase activity (6) , the fusion protein was exceedingly unstable, and the large primary trans¬ lation product was reproducibly cleaved into several distinct proteolytic products. The distribution of those species into soluble and insoluble fractions showed that the smaller products were more soluble than the larger ones. The approach taken to counter these problems, therefore, was to generate deletions in the DNA which removed unnecessary codons, and to screen the variants for maximal activity. This procedure resulted in the isolation of a construct that overproduced an extremely stable, soluble, and active fusion protein. This approach may be of general use in maximizing solu¬ ble activity of a variety of proteins expressed in £_». coli. The reverse transcriptase fusion protein required a 22- fold purification to yield a nearly homogeneous enzyme preparation. The purification scheme involved multiple column chromatography steps, including polyribocytidy¬ lic acid-agarose. This resin has been used for the rapid method of purification of RNA dependent-DNA poly¬ merase from avian myeloblastosis virions (33). The results described in this paper showed that this affin¬ ity column was useful as well for the rapid purifica¬ tion of reverse transcriptase activities expressed in _____ £_2li. New information has been obtained about the various functional domains of the pol gene through its expres¬ sion in £jt ≤≤ϋ. The expression of. the pol. gene from nucleotide position 2574-4588 (8) confirms that both the reverse transcriptase and RNase H activities are encoded by this region and can coexist in a single protein species of M r =71,000. Neither activity re¬ quired the exact viral termini, since the N-terminus of the fusion protein is encoded by the trpE gene and the C-terminus contains nine random amino acids encoded by pBR322 sequences. Analysis of clones showed that a deletion 140 base pairs larger than that in pB6Bl5.23 (up to the Bglll site) still did not abolish reverse transcriptase activity. Due to the difficulty in obtaining large quantities of the authentic M-MuLV reverse ' transcriptase, direct comparison between the pB6B15.23 enzyme and the authen¬ tic enzyme could not be made. Data previously pub¬ lished on the viral enzyme indicates that the pB6Bl5.23 reverse transcriptase was identical to its viral coun¬ terpart in its optimal conditions for synthesis on poly (rA): oligo (dT) (4,18,34). The sedimentation coeffi- cients determined by glycerol gradient centrifugation also showed that both the viral and the cloned enzyme were monomers (4,5). The major difference detected between the viral protein and pB6B15.23 reverse tran¬ scriptase was the low activity of the pB6B15.23 enzyme on poly (rA) : oligo (dT) in the presence of Mn ++ vs. Mg ++ for the authentic viral protein was reported to be 3.5:1 (4), whereas for pB6Bl5.23 reverse transcriptase, this ratio was 49:1. The structure of the reverse transcriptase from avian retroviruses is quite different from that of the murine viruses. The predominant functional form of the avian enzyme is a heterodimer of two subunits (35) , alpha and beta; the larger beta subunit is cleaved in the virion to ' form the smaller alpha subunit and a third protein, pp32, exhibiting DNA endonuclease activity (36,37,38) . The enzymatic properties of the avian enzyme is also quite different from those of the pB6B15.23 enzyme. Published protocols, for example, have suggested that the addition of NaPPi (39) and synthesis at elevated temperatures (40) were suitable for the formation of full-length products using the avian enzyme; we found that the bacterial enzyme, in contrast, was very sensi¬ tive to NaPPi and lost 50% of its activity in 15 min¬ utes when incubated at 42°C. An additional difference between the avian and the pB6B15.23 enzyme was that the avian enzyme formed double-stranded cDNAs only poorly, while the bacterial reverse transcriptase was found to efficiently catalyze hairpin synthesis on DNA to form double stranded DNA products. Actinomycin D inhibited double stranded DNA synthesis and limited synthesis to the first strand, as with known DNA polymerases. On a natural RNA template, the size of the DNA product was found to increase with increasing concentration of the cloned M-MuLV reverse transcriptase; under optimal conditions, the enzyme could synthesize cDNAs up to 9.9 kb long. Maximal DNA synthesis using the pol A + mRNA from human fetal muscle tissue occurred when the pro¬ tein was present at a 4.5-fold excess over the RNA (w/w) , or approximately one molecule of protein every 48 nucleotides; it is not known, however, what fraction of the reverse transcriptase molecules are active. Further studies in this laboratory will focus on gener¬ ating temperature sensitive mutuants of the reverse transcriptase and RNase H activities. These mutants will be isolated by mutagenesis of the cloned MuLV reverse transcriptase; the effects of the mutations will be analyzed after transfer of the altered DNA back into the viral genome and recovery of virus. We hope that analysis of such mutants will result in a better understanding of the role of the activities of the enzyme in the viral life cycle and the interactions of the protein with the other viral gene products. References 1. Varmus, H. , and Swanstrom, R. (1982) in RNA Tumor Viruses (R. Weiss, N. Teich, H. Varmus, and J. Coffin, eds) pp. 369-512. Cold Spring Harbor, New York. 2. Witte, O.N., and Baltimore, D. (1978) J. Virol 26. 750-761. 3. Kopchik, J. J., Karshin, W.L., and Arlinghaus, R.B. (1979) JI. Virol 2ϋ, 610-623. 4. Verma, I.M. (1975) J. Virol 15. 843-854. ' 5. Gerard, G.F., and Grandgenett, D.P. 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Claims: What is Claimed is: 1. A double-stranded DNA plasmid which, when ex¬ pressed in a suitable host cell, produces a polypeptide having reverse transcriptase activity, the plasmid com¬ prising in 5' to 3' order: a DNA sequence which includes an inducible promoter; a DNA sequence which includes an ATG initiation codon; the central portion of the Moloney murine leukemia virus (MuLV) pol gene, said central portion including a DNA sequence which encodes the polypeptide having re- verse transcriptase activity; a DNA sequence which contains a gene associated with a selectable or identifiable phenotypic trait which is manifested when the vector is present in the host cell; and a DNA sequence which contains an origin of replication from a bacterial plasmid capable of autonomous replica¬ tion in the host cell. 2. A plasmid of claim 1, wherein the suitable host cell is Escherichia £21 . 3. A plasmid of claim 1, wherein the inducible pro¬ moter is one which is induced when the host cell is grown upon a medium deficient in one or more amino acids. 4. A plasmid of claim 3, wherein the inducible pro¬ moter is the Trp promoter of Escherichia coli and the medium is deficient in tryptophan. 5. A plasmid of claim 4, wherein the ATG initiation codon is derived from the coding sequence of the Trp E protein of Escherichia coli. 6. A plasmid of claim 5, wherein the ATG initiation codon is derived from a 54 nucleotide long sequence encoding a portion of the Trp E protein of Escherichia cp i. 7. A plasmid of claim 1, wherein the inducible pro¬ moter is one which is induced when the host cell is subjected to increased temperature. 8. A plasmid of claim 1, wherein the phenotypic trait is drug resistance. 9. A plasmid of claim 8, wherein the drug resistance is ampicillin resistance.. 10. A plasmid of claim 1, wherein the origin of repli¬ cation is derived from pBR322. 11. A plasmid of claim 1, wherein the double-stranded DNA is circular. 12. The plasmid of claim 1 identified as pB6Bl5.23 having the restriction map shown in Fig. 2 and deposit¬ ed in fi. coli HB101 under ATCC No. 39939. 13. A plasmid of claim 1, wherein the central portion of the MuLV poj gene comprises from about nucleotide 2574 to about nucleotide 4588. 14. A plasmid of claim 1, wherein the 5 1 end of the central portion of the pal gene is 21 nucleotides from the start of the DNA sequence which encodes the poly¬ peptide having reverse transcriptase activity. 15. A suitable host cell which comprises the plasmid of claim 1. 16. A bacterial host cell of claim 15. 17. A yeast host cell of claim 16. 18. A bacterial host cell which contains the plasmid of claim 1. 19. An £. coli HB101 strain of claim 18 deposited under ATCC No. 39939. 20. A method for producing a polypeptide having re¬ verse transcriptase activity which comprises growing the host cell of claim 15 under suitable conditions permitting production of the polypeptide and recovering the resulting polypeptide. 21. A method for producing a polypeptide having re¬ verse transcriptase activity which comprises growing the host cell of claim 18 under suitable conditions permitting production of the polypeptide and recovering the resulting polypeptide. 22. A method for producing a polypeptide having re¬ verse transcriptase activity which comprises growing the host cell of claim 19 under suitable conditions permitting production of the polypeptide and recovering the resulting polypeptide. 23. The polypeptide having reverse transcriptase ac¬ tivity prepared according to the method of claim 22. 24. A non-naturally occurring polypeptide having re¬ verse transcriptase activity characterized by being encoded by the plasmid pB6B15.23. 25. A method for recovering purified polypeptide of claim 24 from host cells in which it has been produced which comprises disrupting cells, recovering soluble material containing the reverse transcriptase polypeptide from the disrupted cells and separately recovering the reverse trascriptase polypeptide from the soluble material in purified form. 26. A method of claim 25, wherein the separate recovery of the reverse transcriptase polypeptide comprises chromatography on phosphocellulose followed by chromatography on polyribocytidylic acid-agarose. 27. A method for reverse transcription of an RNA mole¬ cule which comprises contacting the RNA molecule with the polypeptide produced in claim 22 under suitable reverse transcribing conditions so as to produce a DNA molecule which is complementary to the RNA molecule.

Inventors: GOFF STEPHEN P, ROTH MONICA J, TANESE NAOKO, GOFF, STEPHEN, P., ROTH, MONICA, J., TANESE, NAOKO
Applicants: UNIV COLUMBIA, THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF
Application year: 1986
Main classification: C12N1
IPC codes: C07K14, C12N9, C12N15