Bacterial 3-Galactosidase as a Marker of Rous ... - Europe PMC

MOLECULAR AND CELLULAR BIOLOGY, Feb. 1985, p. 281-290 0270-7306/85/020281-10$02.00/0 Copyright © 1985, American Society for Microbiology

Vol. 5, No. 2

Bacterial 3-Galactosidase as a Marker of Rous Sarcoma Virus Gene Expression and Replication PAMELA A. NORTON AND JOHN M. COFFIN* Department of Molecular Biology and Microbiology and Cancer Research Center, Tufts University School of Medicine, Boston, Massachusetts 02111 Received 17 September 1984/Accepted 14 November 1984

We have developed a convenient and sensitive assay of eucaryotic gene expression which uses the Escherichia coli lacZ gene product, j-galactosidase, as a nonselectable marker. This system has been applied to the analysis of Rous sarcoma virus replication and gene expression. Avian cells were transfected with plasmids encoding in-frame gene fusions of the N-terminal portion of the gag gene to a 'lacZ gene, which requires both transcriptional and translational initiation signals; these were supplied by the virus long terminal repeat and leader region. Readily detectable quantities of 0-galactosidase were synthesized in transfected cells; it was demonstrated that the levels of enzyme activity induced in such cultures increased linearly with the input DNA concentration and also correlated with mRNA levels. By using a Rous sarcoma virus-derived vector containing the src gene and a related virus as a helper, it was shown that lac sequences were compatible with all phases of the virus life cycle. gag-lacZ fusion proteins were immunoprecipitable from cultures which stably expressed lacZ as well as src. Virus rescued from stably transfected cultures resulted in continued lac and src expression in recipient cells. One particular construction was efficiently transmitted as virus, although it lacked sequences thought to be important for encapsidation of RNA into virions. The data presented here demonstrate the use of lacZ as a marker of retrovirus gene expression and replication.

Retrovirus replication and gene expression are interwoven in a complex fashion, as are the sequences which regulate these processes. As a consequence, an understanding of the viral genome requires not only dissection of isolated functional regions, but also analysis of the manner in which different regions interact, both structurally and functionally. Studies of deletion mutants have shown that the products encoded by the viral structural genes of gag, pol, and env may be supplied in trans, and have identified certain regions of the genome which act in cis. Regions required in cis for reverse transcription of genome RNA, integration of proviral DNA, and synthesis of RNA include the long terminal repeats (LTRs) and adjacent noncoding regions (45). The LTRs which flank an integrated DNA provirus are composed of sequences derived from both the 5' and 3' ends of the RNA genome (refered to as U5 and U3, respectively) and contain signals for transcription initiation and for generation of 3' ends of viral genome and mRNA. It is presumed that the genome contains sequences which modulate the splicing of mRNAs, as not all viral transcripts are spliced; still other sequences direct the encapsidation of full-length RNA genomes. There is genetic evidence that a sequence within the untranslated leader region near the 5' end of the genome is required for packaging of Rous sarcoma virus (RSV) genomes into virions (22, 39). A similar function has also been attributed to 115 bases near the 3' end of the genome (44), as well as to a 28-base near-perfect palindrome which lies within the gag coding sequence (35). However, it is difficult to assign specific functions to these regions, as any mutation might have pleiotropic effects. Although the expression of virtually any gene, viral or otherwise, can be monitored by directly measuring the accumulation of RNA transcripts after introduction into an appropriate cell type, it is often simpler to substitute an indicator gene which confers a readily detectable phenotype.

The Escherichia coli lacZ gene seemed a logical candidate to adapt to eucaryotic expression systems, as lac fusions have been extensively used to study gene expression in procaryotes, and many vectors exist with lac DNA flanked by convenient restriction sites (7). Furthermore, a colorimetric assay which is both simple and quantitative over a wide range exists for the gene product, P-galactosidase (30), and the enzyme can tolerate the substitution of many other sequences for its N terminus without loss of activity (6). In light of these properties, we began to utilize lacZ in analyses of the sequences required in cis for gene expression and replication of RSV in avian cells. It has previously been found that RSV sequences can direct the synthesis of 3-galactosidase as a gag-lacZ fusion protein in E. coli (29). gag is the viral structural gene located nearest to the 5' end of the genome (12). It was anticipated that viral transcriptional and translational control signals should effect the synthesis of a similar gag-lacZ fusion product in eucaryotic cells. Detection of P-galactosidase activity in cells into which DNA encoding such a fusion protein had been introduced would provide a simple assay for RSV gene expression. We also wished to determine whether lac operon sequences could be transduced in a retrovirus vector, that is, replicated by a helper virus, then stably integrated as proviral DNA. For the purposes of these studies, we developed a simple lysis and assay procedure for the detection of bacterial ,-galactosidase in avian cells and demonstrated that the assay quantitatively reflects the level of viral gene expression. To test for viral transmission of lac sequences, two plasmids were constructed which contained gag-lacZ fusions and the RSV src gene flanked by LTRs, so as to resemble proviruses. One of the two vectors was missing a portion of the palindromic sequence thought to be essential for packaging of genome RNA. Analysis of virus released from cells which had been transfected with either of these plasmids, along with a molecularly cloned helper virus,

* Corresponding author. 281

NORTON AND COFFIN

282 E

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plasmids require transcriptional as well as translational control signals to synthesize 3-galactosidase fusion proteins. Most of these plasmids are diagrammed (see Fig. 1 or 4). Enzymes and reagents. Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, and Si nuclease were obtained from either New England Biolabs or Bethesda Research Laboratories. o-Nitrophenyl ,B-D-galactoside (ONPG) and purified P-galactosidase were obtained from Sigma Chemical Co. DNase I was obtained from Worthington Diagnostics, and RNasin was from Promega Biotech. Calf intestinal alkaline phosphatase was from Boehringer Mannheim Corp., as was 5-chloro-4-bromo-3-indolyl-P-Dgalactoside (X-gal). Proteinase K was supplied by Calbiochem-Behring. Construction of plasmids. Restriction endonucleases and T4 DNA ligase were used in accordance with recommendations of the manufacturer. Plasmid pZ-1 resulted from the ligation of a lac'Z-containing EcoRI-SalI fragment of pMC1871 to pBR322 DNA which had been digested with the same two enzymes (Fig. 1). Also shown in Fig. 1 is the construction of pPN-7. A BglII (base 7736)-BamHI (base 532) fragment of pATV-8, containing a portion of src, LTR, leader, and gag sequences, was inserted at the BamHI site of pZ-1 upstream of lacZ (the numbers in parentheses refer to the published sequence of Pr-RSV-C; 36). The construction of pPN-10, pPN-11, and pPN-12 (diagrammed in Fig. 3) will be described in detail elsewhere (manuscript in prepa-

ration). 1kb

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FIG. 1. Structures of plasmids pZ-1 and pP:N-7. The lac'Z fragment derived from pMC1871 was removed as an EcoRI-SalI fragment and inserted into pBR322; there is an a dditional EcoRI site

within the lacZ-coding sequence. Not shown is an SmaI site which lies between the EcoRI and BamHI sites at the 5' end of lacZ; this SmaI site is unique within the plasmid. The frag contains the DNA which was inserted into pZ-1 to generate 3' portion of src, 3' nontranslated sequences, a cornmplete LTR, the 5' untranslated leader region, and 153 nucle otides of gag. The resulting gene fusion puts gag and lacZ into the same reading frame. Shaded bars represent lac sequences; open barrs, pBR322; and thin lines, viral sequences, with the exception of the LTR, which is boxed. The open box represents U3, and the shaded box denotes RU5. Only relevant restriction sites are indicatezd; sites in parentheses were destroyed by construction manipula tions. E, EcoRI; B, BamHI; S, SaIl; Bg, BglII.

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indicated that both constructions transduc ed 3-galactosidase activity and also induced foci of transfornned cells. Surprisingly, we also found that the palindromic sequence was not absolutely required for virus rescue, althc)ugh subtle effects on encapsidation efficiency cannot be ruleid out. MATERIALS AND METH(DDS

Bacterial strains and plasmids. The E coli K-12 strain RV200 (thi AlacX74 rpsL200), or a recA d(erivative of it, was used for all bacterial manipulations. Plas mids used for the constructions described in this paper iniclude pATV-8, a molecular clone of Pr-RSV-C permuted aIt a HindIII site in pol (21), and pRAV-1, a molecular clone oif RAV-1 permuted at the unique SstI site in the leader regic)n (38). Also used were pMC1871 (7) and pMC1403 and p tMC874 (6), all of which contain a lac'Z gene which is d eleted for the sequences encoding the eight N-terminal amiino acids. All three

The vectors pPN-1 and pPN-2 are shown (see Fig. 4); these contain viral sequences derived from pATV-8 and lac operon sequences from pMC1403 or the related plasmid pMC874. The lac operon fragments carried by these last two plasmids are identical but for a single-base change near the 3' end of the lacZ gene of pMC1403, which destroys an EcoRI recognition site (6). pPN-1 contains a viral BamHI fragment (bases 4715 to 532, with an internal XhoI deletion of bases 5258 to 6983; base 1 corresponds to the RNA cap site; 36) inserted upstream of lacZ and an XhoI-SstI fragment (bases 6983 to 255) of pATV-8 inserted downstream of lac sequences. The result is that the lacZ and src genes are flanked by LTRs and other noncoding sequences in a structure resembling a provirus. pPN-2 was generated from pPN-1 by replacement of the viral BamHI fragment with a BglII fragment (bases 7738 to 1630) of pATV-8. For construction convenience, both provirus-like units are flanked with duplications of certain viral and lac sequences; these plasmids were maintained in a recA host to minimize loss of sequences through homologous recombination. Plasmid DNAs were isolated from cleared bacterial cell lysates by centrifugation in cesium chloride-ethidium bromide gradients. The identity of all plasmids was verified by cleavage with the appropriate restriction enzymes, followed by agarose gel electrophoresis. Transfection of avian fibroblasts. Turkey embryo fibroblasts were prepared and maintained as previously described (8). Cells were used for transfection experiments after one to five passages in vitro. DEAE-dextran-mediated transfection was essentially as described (11). Briefly, cells were seeded at 2.5 x 105 per 35-mm well or at 5.0 x 105 per 60-mm dish 24 h before transfection. Immediately before transfection, monolayers were washed with phosphate-buffered saline, and then cells were exposed to a mixture of DEAE-dextran (1 mg/ml in Hanks buffered saline) and plasmid DNA. Unless otherwise noted, the DNA was not cleaved before transfection. The mixture was left in contact with the cells for 30 min at 37°C, and then growth medium

VOL. 5, 1985

37°C, fresh medium was added. For transient expression experiments, cells remained in the original dish for 3 days before enzyme assays were performed. For longer-term rescue experiments, cells were transferred to new plates 2 to 3 days after transfection. Assay for j(-galactosidase activity. The assay for galactosidase activity in avian cells was modified slightly from the assay described for bacteria by Miller (30). Cell monolayers were washed with phosphate-buffered saline and lysed for several minutes in 0.2 ml of 0.1% sodium dodecyl sulfate (SDS) in phosphate-buffered saline, and then 0.8 ml of PM-2 buffer (33 mM NaH2PO4, 66 mM Na2HPO4, 0.1 mM MnC12, 2 mM MgSO4, 40 mM P-mercaptoethanol; 34) was added. Lysates were transfered to transparent tubes, and 0.2 ml of ONPG, freshly dissolved at 4 mg/ml in PM-2 buffer, was added. Thoroughly mixed samples were incubated at 37°C until a yellow color was obvious (an absorbance at 420 nm of between 0.1 and 1.0 was optimal; this usually required 0.5 to 1.0 h). Normally, a lysate of mocktransfected cells was included as a negative control, and purified P-galactosidase was included as a positive control, if needed. The tubes were placed on ice, and enzyme reactions were terminated by the addition of 0.5 ml of 1 M Na2CO3; the length of time (in minutes) of each reaction was recorded. After samples were clarified (a cycle of freezing and thawing facilitated this) the amount of ONPG hydrolyzed was determined spectrophotometrically. Units of ,Bgalactosidase activity were calculated as follows: units = (the absorbance at 420 nm x 380)/minutes, where 380 is a constant, such that 1 unit is equivalent to 1 nmol of ONPG hydrolyzed per min. If large numbers of samples were to be screened, it was convenient to add the ONPG directly to the culture dish containing the cell lysate; the hydrolysis of the substrate could be observed readily, but the accuracy of the assay suffered, possibly due to incomplete mixing of assay components. Isolation of RNA, preparation of probe, and Si nuclease mapping. RNA was prepared essentially as described (17). Monolayers were lysed with 150 mM NaCl-10 mM Tris-hydrochloride (pH 7.5)-2 mM EDTA (pH 7.5)1.0% SDS and then incubated at 37°C for 60 min in the presence of 100 ,ug of proteinase K per ml. Nucleic acids were sheared, extracted with phenol-chloroform until the interface was clear, and then extracted once with chloroform alone before ethanol precipitation. The total nucleic acids were resuspended in 50 mM Tris-hydrochloride (pH 7.5-S5 mM MgCI2 and digested with 50 ng of DNAse I for 30 min at 37°C in the presence of 60 U of RNasin. The digested nucleic acid was extracted once with phenol-chloroform and once with chloroform and then reprecipitated. To prepare an end-labeled probe, 6 ,ug of plasmid DNA was digested with BamHI to completion. The DNA was dephosphorylated with calf intestinal alkaline phosphatase and then 5' end labeled by the action of T4 polynucleotide kinase (26). The DNA was then digested with SphI to generate a fragment with a single labeled 5' end; this fragment, which is diagrammed (see Fig. 3), was separated from other labeled fragments by electrophoresis on a 5% polyacrylamide gel. The fragment was eluted from the gel and precipitated three times with ethanol before use (28). Various amounts of RNA were mixed with a constant amount of end-labeled probe in preparation for the S1 mapping. DNA hybridization and S1 digestion conditions were essentially as described (29), except that S1 digestions were performed at 42°C. Digested samples were ethanol precipitated before electrophoresis under denaturing condi-

P-GALACTOSIDASE AS A MARKER OF RSV

283

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DNA (jig) FIG. 2. Dose-response of increasing amounts of transfected DNA. Turkey embryo fibroblasts were plated at 2.5 x 101 cells per 35-mm well 1 day before transfection. Each well was treated with DEAE-dextran, and the amount of uncleaved pPN-7 DNA added is shown on the abscissa; sheared salmon sperm DNA was added where necessary, so that all wells received 5 ,ug of total DNA. Cells were lysed and assayed 3 days posttransfection; each point represents the enzyme activity from a single well.

tions on a 4% polyacrylamide gel. Labeled marker DNAs were a gift of S. A. Herman. Labeling of proteins, immunoprecipitation, and gel elec-

trophoresis. Fibroblasts were labeled as previously described (8), with 250 ,uCi of [35S]methionine per 100-mm plate; labeled monolayers were washed three times with phosphate-buffered saline and then stored at -70°C. Bacteria were labeled with 200 ,uCi of [35S]methionine per ml essentially as described (29), but the cultures were at a lower cell density (absorbance at 600 nm = 0.3 to 0.5). After labeling, bacteria were chilled, washed with 10 mM Tris-hydrochloride, and stored as pellets at -70°C. Lysis of avian cells and bacteria, as well as immunoprecipitation, was essentially as described (8, 29). Each immunoprecipitation contained 1 x 106 to 3 x 106 acid-insoluble cpm. Rabbit anti-p-galactosidase was a gift from R. C. C. Huebner; anti-p27 serum was a gift from D. Bolognesi. In vitro cleavage of immunoprecipitates with disrupted avian myeloblastosis virus was as described previously (29). Samples were subjected to discontinuous SDS-polyacrylamide gel electrophoresis (29).

284

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