Transfection of a glycosylated phosphatidylinositol-anchored folate-binding protein complementary DNA provides cells with the ability to survive in low folate medium.

KB cells express a folate-binding protein that is anchored to the plasma membrane by a glycosylated phosphatidylinositol (GPI) tail and these cells can grow in medium containing a very low folate concentration (1 nM). In contrast, mouse 3T3 cells do not express a membrane-associated folate-binding protein and cannot grow under similar low folate conditions. In these studies, 3T3 cells were transfected with a vector containing the cDNA that codes for the KB cell folate-binding protein. In contrast to the wild-type 3T3 cells, the transfected 3T3 cells express a level of folate-binding protein similar to KB cells, 1 and 1.4 ng/micrograms protein, respectively. The capacity for binding [3H] folate to the surface of transfected 3T3 cells cultured in folate-deficient medium is 7.7 pmol/10(6) cells, and this is approximately 50% of the surface binding capacity of KG cells under similar culture conditions. Moreover, after treatment of the transfected 3T3 cells with phospholipase C specific for phosphatidylinositol, the binding of [3H] folate to the surface of these cells is reduced by 90%, indicating that, like the KB cells, the folate-binding protein is anchored to the plasma membrane by a GPI tail. Although the doubling time of wild-type 3T3 cells markedly increases after 13 d of culture in folate-deficient medium, the doubling time of both the transfected 3T3 cells and KB cells do not change. The results of these experiments indicate that the GPI-anchored folate-binding protein provides a mechanism to maintain a level of folate that permits the folate-dependent metabolic functions necessary for cell survival under low folate conditions.

Synthesis of functional mRNA in mammalian cells by bacteriophage T3 RNA polymerase.

We found that the 5' nontranslated leader sequence from encephalomyocarditis virus (EMCV) allowed transcripts that were synthesized by the T3 RNA polymerase in mammalian cells to be translated in a cap-independent fashion. Stable mouse cell lines that carry the T3 RNA polymerase gene expressed the chloramphenicol acetyltransferase (CAT) gene under the control of a phage promoter when the CAT gene was fused to the EMCV leader and introduced into the cells by transient DNA uptake. The level of gene expression in such cells was similar to or greater than that observed with a conventional transient expression vector that is dependent on transcription by the host RNA polymerase II. Expression of the EMCV-CAT fusion gene was stimulated by cotransfection of the cells with a gene that encodes the poliovirus protease 2A protein (which inhibits cap-dependent translation), demonstrating that the EMCV-CAT fusion gene was expressed in a cap-independent fashion. Introduction of both the T3 RNA polymerase gene and the EMCV-CAT fusion gene into a variety of cultured mammalian cell lines (HeLa, BSC40, Ltk-, NIH 3T3, and C127) demonstrated that the T3-EMCV expression system functions in a broad range of cell types.

Termination and slippage by bacteriophage T7 RNA polymerase.

We have examined the termination efficiency of T7 and T3 RNA polymerases (RNAPs) at a variety of termination signals. In agreement with previous investigators we find that termination occurs after the synthesis of an RNA product with a stable secondary structure followed by a run of U residues. Stem-loop structures that lack a 3' U-tract fail to terminate the phage enzyme. The distance (or the sequence) between the start site for transcription and the termination signal may also be important, as placing the terminator at different locations downstream from the promoter, or changing the promoter sequence, results in alterations in termination efficiency. We have explored termination at extended runs of homopolymers in the absence of an apparent stem-loop structure, and have observed that the enzyme terminates (inefficiently) when synthesizing U-rich transcripts, but not A- or C-rich transcripts. This is especially true at low concentrations of UTP. Strikingly, when an elongation complex (EC) encounters a dA-tract in the template strand it is able to slide on the template, resulting in the synthesis of products that have more or fewer U residues than predicted by the sequence of the DNA. This observation suggests that the formation of an RNA: DNA hybrid may be important to the lateral stability of the EC (its ability to maintain proper register with the DNA template). We have also explored the termination properties of a proteolytically nicked form of T7 RNAP. The nicked enzyme forms a less stable EC than the intact RNAP and dissociates more readily from the template in regions that encode inherently destabilizing RNAs (e.g. stem-loop structures, poly(U)-tracts). However, the nicked enzyme terminates less efficiently at the late T7 terminator (T7-T phi) or at a termination signal in the human preproparathyroid hormone gene. These results suggest that termination is a highly specific event, and not merely a consequence of decreased stability of the EC. Our observations are not consistent with previous models of termination by the phage RNAP and indicate that revisions to these models may be required.

Characterization of bacteriophage T7 RNA polymerase by linker insertion mutagenesis.

Thirty-four mutants of phage T7 RNA polymerase (RNAP) were generated by linker-insertion mutagenesis and characterized with respect to their ability to carry out various steps in the transcription cycle. A number of mutants with interesting biochemical properties were identified. These include: (1) Mutant RNAPs that are catalytically active but that bind weakly to a T7 promoter; one of these mutants is affected in a region of the RNAP that exhibits homology with the sigma subunit of Escherichia coli RNAP. Another is affected in a region that has been previously implicated in the discrimination of T7 versus T3 promoters (Joho, et al., 1990). (2) Mutant RNAPs that can bind to the promoter but are transcriptionally inactive; some of these RNAPs lack catalytic activity, others are catalytically active but are unable to initiate productive transcription at a T7 promoter. Among the latter class of mutants are enzymes that appear to be weakened in their ability to melt open (or to remain associated with) double-stranded DNA; these RNAPs make only abortive initiation products and are unable to proceed to the formation of a productive elongation complex. The mutations causing this phenotype affect regions of the RNAP that exhibit homology with the catalytic site of DNA polymerase I (Delarue et al., 1990). (3) A C-terminal insertion mutant with properties similar to a previously characterized "foot" mutant (Mookhtiar et al., 1991). This RNAP appears to be defective in the very early steps of transcription and may be unable to translocate and/or empty the active site. (4) A mutant that is transcriptionally active, but is unable to complement the growth of T7 gene 1- phage. This phenotype may result from disruption of a function of the RNAP that is distinct from its role in RNA synthesis.

Substitution of a single bacteriophage T3 residue in bacteriophage T7 RNA polymerase at position 748 results in a switch in promoter specificity.

The bacteriophage T3 and T7 RNA polymerases (RNAP) are closely related, yet exhibit high specificity for their own promoter sequences. In this work the primary determinant of T7 versus T3 promoter specificity has been localized to a single amino acid residue at position 748 in the T7 RNAP. Substitution of this residue (Asn) with the corresponding residue found in T3 RNAP (Asp) results in a switch in promoter specificity, and specifically alters recognition of the base pairs (bp) at positions -11 and, possibly, -10 in the promoter. A complementary mutation in T3 RNAP (T3-D749N) results in a similar switch in promoter preference for that enzyme. The hierarchy of bp preference by the mutant and wild-type enzymes for bp at -10 and -11, and the results of previous experiments, lead to a model for specificity in which it is proposed that N748 in T7 RNAP (and D749 in T3 RNAP) make specific hydrogen bonds with bases at -11 and -10 on the non-template strand in the major groove. The specificity determining region of T7 RNAP does not appear to exhibit homology to any known sequence-dependent DNA binding motif.

Interrupting the template strand of the T7 promoter facilitates translocation of the DNA during initiation, reducing transcript slippage and the release of abortive products.

We have explored the effects of a variety of structural and sequence changes in the initiation region of the phage T7 promoter on promoter function. At promoters in which the template strand (T strand) is intact, initiation is directed a minimal distance of 5 nt downstream from the binding region. Although the sequence of the DNA surrounding the start site is not critical for correct initiation, it is important for melting of the promoter and stabilization of the initiation complex. At promoters in which the integrity of T strand is interrupted by nicks or gaps between -5 and -2 the enzyme continues to initiate predominately at +1. However, under these conditions there is a decrease in the release of abortive products of 8-10 nt, a decrease in the synthesis of poly(G) products (which arise by slippage of the nascent transcript), and a defect in displacement of the RNA. We propose that unlinking the binding and initiation regions of the T strand changes the manner in which this strand is retained in the abortive complex, reducing or eliminating the need to pack or "scrunch" the strand into the complex during initiation and lowering a thermodynamic barrier to its translocation.

A mutant T7 RNA polymerase that is defective in RNA binding and blocked in the early stages of transcription.

We have identified a mutation (E148A) in T7 RNA polymerase (RNAP) that results in an enzyme which aborts transcription primarily when the nascent RNA achieves a length of 5 nt. This phenomenon is observed at a consensus promoter, but is even more strongly observed at promoters that are altered in the initiation region. Although the abortive product is of a fixed length (5 nt), the positions of the base substitutions in the initiation region that enhance this effect do not appear to be fixed, and we have observed the effect with a variety of initiation-region promoter variants. The phenomenon is also observed during promoter-independent transcription when transcribing a homopolymeric template such as poly(dC). Under conditions where the active site of the RNAP cannot extend beyond the third nucleotide in the template strand and the maximum length of the RNA:DNA hybrid cannot exceed three base-pairs (i.e. when synthesizing oligoG products due to transcript slippage at a promoter that initiates with the sequence +1 GGG...) the mutant RNAP gives rise to a normal spectrum of products 2 to 14 nt in length with no evidence of a block at 5 nt. Neither promoter binding nor promoter melting appears to be involved in this phenotype, as the mutant RNAP binds normally to promoter sequences and the behavior of the enzyme is unaffected by removal of the non-template strand in the initiation region of the promoter or on a supercoiled template. Importantly, the mutant RNAP is defective in binding single strand oligomers of RNA. These results suggest that the affected region of the RNAP may form part of the RNA product binding site and may be involved in the transition from an unstable initiation complex to a stable elongation complex, perhaps by sensing the presence of a nascent RNA and/or RNA:DNA hybrid.

Hierarchy of base-pair preference in the binding domain of the bacteriophage T7 promoter.

The activity of bacteriophage T7 RNA polymerase (RNAP) at a collection of T7 promoter mutants having all possible single base-pair substitutions in the region from -15 to -6 was determined by transcription in vitro, thus establishing a hierarchy of base-pair preference at each position. The tolerance of the RNAP for base-pair substitutions is not uniform across the binding domain. Under stringent conditions (20 mM-MgCl2), T7 RNAP is highly permissive for all base-pair substitutions at -13 and -12. The RNAP is partially permissive at -15, -14, -11, -10 and -6, and exhibits a clear pattern of base-pair preference at these positions. The RNAP is non-permissive for substitutions at -9 to -7, and will accept only the consensus base-pairs at these positions. Under lower stringency conditions (8 mM-MgCl2, or additionally in the presence of dimethylsulfoxide) a decrease in specificity is observed at most positions except -9. Analysis of these data suggests potential contacts that may be important for promoter function.

Discrimination between bacteriophage T3 and T7 promoters by the T3 and T7 RNA polymerases depends primarily upon a three base-pair region located 10 to 12 base-pairs upstream from the start site.

The bacteriophage T3 and T7 RNA polymerases are closely related, yet are highly specific for their own promoter sequences. To understand the basis of this specificity, T7 promoter variant that contain substitutions of T3-specific base-pairs at one or more positions within the T7 promoter consensus sequence were synthesized and cloned. Template competition assays between variant and consensus promoters demonstrate that the primary determinants of promoter specificity are located in the region from -10 to -12, and that the base-pair at -11 is of particular importance. Changing this base-pair from G.C, which is normally present in T7 promoters, to C.G, which is found at this position in T3 promoters, prevented utilization by the T7 RNA polymerase and simultaneously enabled transcription from the variant T7 promoter by the T3 enzyme. Substitution of T7 base-pairs with T3 base-pairs at other positions where the two consensus sequences diverge affected the overall efficiency with which the variant promoter was utilized by the T7 RNA polymerase, but these changes were not sufficient to permit recognition by the T3 RNA polymerase. Switching the -11 base-pair in the T3 promoter consensus to the T7 base-pair prevented utilization by the T3 RNA polymerase, but did not allow the T3 variant promoter to be utilized by the T7 RNA polymerase. This probably reflects a greater specificity of the T7 RNA polymerase for base-pairs at other positions where the promoter sequences differ, most notably at -15. The magnitude of the effects of base substitutions in the T7 promoter on promoter strength (-11C much greater than -10C greater than -12A) correlates with the affinity of the T7 polymerase for the promoter variants, suggesting that the discrimination of the phage RNA polymerases for their promoters is mediated primarily at the level of DNA binding, rather than at the level of initiation.

Identification of a region of the bacteriophage T3 and T7 RNA polymerases that determines promoter specificity.

Bacteriophages T7 and T3 encode DNA-dependent RNA polymerases that are 82% homologous, yet exhibit a high degree of specificity for their own promoters. A region of the RNA polymerase gene (gene 1) that is responsible for this specificity has been localized using two approaches. First, the RNA polymerase genes of recombinant T7 x T3 phage that had been generated in other laboratories in studies of phage polymerase specificity were characterized by restriction enzyme mapping. This approach localized the region that determines promoter specificity to the 3' end of the polymerase gene, corresponding to the carboxyl end of the polymerase protein distal to amino acid 623. To define more closely the region of promoter specificity, a series of hybrid T7/T3 RNA polymerase genes was constructed by in vitro manipulation of the cloned genes. The specificity of the resulting hybrid RNA polymerases in vitro and in vivo indicates that an interval of the polymerase that spans amino acids 674 to 752 (the 674 to 752 interval) contains the primary determinant of promoter preference. Within this interval, the amino acid sequences of the T3 and T7 enzymes differ at only 11 out of 79 positions. It has been shown elsewhere that specific recognition of T3 and T7 promoters depends largely upon base-pairs in the region from -10 to -12. An analysis of the preference of the hybrid RNA polymerases for synthetic T7 promoter mutants indicates that the 674 to 752 interval is involved in identifying this region of the promoter, and suggests that another domain of the polymerase (which has not yet been identified) may be involved in identifying other positions where the two consensus promoter sequences differ (most notably at position -15).

Characterization of two types of termination signal for bacteriophage T7 RNA polymerase.

The late bacteriophage T7 terminator (T7-T phi) encodes an RNA sequence that can form a stable stem-loop structure followed by a run of six uridylate residues; termination occurs at a 3' G residue just downstream of the U run. In this work, we have explored the features of this signal that are required for efficient termination by T7 RNA polymerase. Whereas replacement of the template-encoded 3' G residue with A, C, or U by site-directed mutagenesis had little effect, removal of the U-tract prevented termination. Deletion analysis indicates that the stem-loop and U-tract are not sufficient for termination, and that sequences upstream from the terminator have marked effects on the position and efficiency of termination. A sequence within the human preproparathyroid hormone (PTH) gene that encodes an interrupted run of six U residues, but lacks an apparent stem-loop structure, also serves as an efficient terminator for T7 RNA polymerase. We have mapped the primary site of termination in the PTH signal to a G residue that lies downstream of the U-rich run (UUUUCUUG). Deletion analysis indicates that the minimal region required for PTH terminator function extends only 23 bp upstream from the termination site, and subcloning of a 31 bp fragment that includes this region of the PTH signal provides efficient termination. A modified form of T7 RNA polymerase resulting from a single proteolytic cleavage between residues 178 and 179, or mutant polymerases that are altered in this region of the enzyme, fail to recognize the PTH signal while still terminating at T7-T phi.

Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability.

We have constructed a series of plasmid templates that allow T7 RNA polymerase (RNAP) to be halted at defined intervals downstream from its promoter in a preserved sequence context. While transcription complexes halted at +3 to +6 are highly unstable, complexes halted at +10 to +14 dissociate very slowly and gradually lose their capacity to extend transcripts. Complexes halted at +18 and beyond dissociate more readily, but the stability of the these complexes is enhanced significantly in the presence of the next incoming nucleotide. Unexpectedly, the stability of complexes halted at +14 and beyond was found to be lower on supercoiled templates than on linear templates. To explore this further, we used synthetic DNA templates in which the nature of the non-template (NT) strand was varied. Whereas initiation complexes are less stable in the presence of a complementary NT strand, elongation complexes are more stable in the presence of a complementary NT strand, and the presence of a non-complementary NT strand (a mismatched bubble) results in even greater stability. The results suggest that the NT strand plays an important role in displacing the nascent RNA, allowing its interaction with an RNA product binding site in the RNAP. The NT strand may also contribute to stabilization by interacting directly with the enzyme. A mutant RNAP that has a deletion in the flexible "thumb" domain responds to changes in template topology in a manner that is similar to that of the wild-type (WT) enzyme, but halted complexes formed by the mutant enzyme are particularly dependent upon the presence of the NT strand for stability. In contrast, an N-terminal RNAP mutant that has a decreased capacity to bind single-stranded RNA forms halted complexes with much lower levels of stability than the WT enzyme, and these complexes are not stabilized by the presence of the NT strand. The distinct responses of the mutant RNAPs to changes in template structure indicate that the N-terminal and thumb domains have quite different functions in stabilizing the transcription complex.

Mutant bacteriophage T7 RNA polymerases with altered termination properties.

We have identified mutants of bacteriophage T7 RNA polymerase (RNAP) that are altered in their ability to pause or terminate at a variety of signals. These signals include a terminator found fortuitously in the human preproparathyroid hormone (PTH) gene, a pause site found in the concatamer junction (CJ) of replicating T7 DNA, and termination signals that are also utilized by Escherichia coli RNAP (e.g. rrnB T1 and T2). Whereas the mutant enzymes terminate normally at the late terminator in T7 DNA (T(phi)) and rrnB T2, they fail to terminate at one of the termination sites of rrnB T1, and also fail to recognize the PTH and CJ signals. The mutant enzymes exhibit normal processivity on linear templates, but show a slightly reduced processivity on supercoiled templates and terminate more efficiently when synthesizing poly(U) tracts. The mutant enzymes also show a decreased tendency to produce aberrant transcription products from DNA templates having protruding 3' ends. T7 lysozyme (an inhibitor of T7 RNAP) has been shown to exert its action by preventing the transition of the RNAP from an unstable initiation complex (IC) to a stable elongation complex (EC). We have found that T7 lysozyme enhances recognition of CJ by wild-type T7 RNAP, and that mutant T7 RNAPs that show increased sensitivity to lysozyme show enhanced recognition of this signal, even in the absence of lysozyme. These results, together with the observation that the mutations that result in the termination-deficient phenotype affect a region of the RNAP that has been implicated in RNA binding and upstream promoter contacts, support the hypothesis that, in some cases, termination represents a reversal of the events that occur during initiation.

Pausing and termination by bacteriophage T7 RNA polymerase.

Two types of sites are known to cause pausing and/or termination by bacteriophage T7 RNA polymerase (RNAP). Termination at class I sites (typified by the signal found in the late region of T7 DNA, TPhi) involves the formation of a stable stem-loop structure in the nascent RNA ahead of the point of termination, and results in termination near runs of U. Class II sites, typified by a signal first identified in the cloned human preproparathyroid hormone (PTH) gene, generate no evident structure in the RNA but contain a conserved sequence ahead of the point of termination, and also contain runs of U. Termination at class I and class II sites may involve non-equivalent mechanisms, as mutants of T7 RNA polymerase have been identified that fail to recognize class II sites yet continue to recognize class I sites. In this work, we have analyzed pausing and termination at several class II sites, and variants of them. We conclude that the 7 bp sequence ATCTGTT (5' to 3' in the non-template strand) causes transcribing T7 or T3 RNA polymerase to pause. Termination 6 to 8 bp past this sequence is favored by the presence of runs of U, perhaps because they destabilize an RNA:DNA hybrid. The effects of T7 lysozyme on pausing and termination are consistent with the idea that termination involves a reversion of the polymerase from the elongation to the initiation conformation, and that lysozyme inhibits the return to the elongation conformation. A kinetic model of pausing and termination is presented that provides a consistent interpretation of our results.

Characterization of structural features important for T7 RNAP elongation complex stability reveals competing complex conformations and a role for the non-template strand in RNA displacement.

We have characterized the roles of the phage T7 RNA polymerase (RNAP) thumb subdomain and the RNA binding activity of the N-terminal domain in elongation complex (EC) stability by evaluating how disrupting these structures affects the dissociation rates of halted ECs. Our results reveal distinct roles for these elements in EC stabilization. On supercoiled or partially single-stranded templates the enzyme with a deletion of the thumb subdomain is exceptionally unstable. However, on linear duplex templates the polymerase which has been proteolytically cleaved within the N-terminal domain is the most unstable. The differences in the effects of these RNAP modifications on the stability of ECs on the different templates appear to be due to differences in EC structure: on the linear duplex templates the RNA is properly displaced from the DNA, but on the supercoiled or partially single-stranded templates an extended RNA:DNA hybrid makes a larger contribution to the conformational state of the EC. The halted EC can therefore exist either in a conformation in which the RNA is displaced from the DNA and forms an interaction with the RNAP, or in a conformation in which a more extended RNA:DNA hybrid is present and the RNA:RNAP interaction is less extensive. The partitioning between these competing conformations appears to be a function of the energetics of template reannealing and the relative strengths of the RNA:RNAP interaction and the RNA:DNA hybrid.

Randomised evaluation of alternative electrosurgical modalities to treat bladder outflow obstruction in men with benign prostatic hyperplasia.

OBJECTIVES: To compare and evaluate the clinical and cost-effectiveness of transurethral vaporisation of the prostate (TUVP), a new electrosurgical modality, with the standard treatment, transurethral resection of the prostate (TURP). DESIGN: A multicentre randomised controlled trial of pragmatic design with associated economic evaluation using cost minimisation. SETTING: Patients were recruited from four centres in south-east England. PARTICIPANTS: Men requiring surgery for lower urinary tract symptoms deemed to be due to benign prostatic hypertrophy. INTERVENTIONS: TURP was performed and subsequent management conducted according to the usual practice of the clinical team. TUVP was performed with the most promising available equipment using a technique described in the literature. Postoperative management after TUVP was left to the ward team, who were not necessarily informed to which treatment arm the patient had been allocated. For the purpose of the study, patients were assessed clinically, by questionnaire and investigation at baseline, 2 months and 6 months after randomisation. A long-term follow-up postal questionnaire was sent to each patient at 2 years. For the economic evaluation, direct costs from the NHS viewpoint were collected. MAIN OUTCOME MEASURES: A reduction of at least 5 from the International Prostate Symptom Score (IPSS) was taken as a satisfactory outcome. The IPSS quality of life (QoL) question provided disease-specific information about QoL. Secondary outcome measures included urinary flow rate, post-void urinary volume, prostate volume and pressure-flow urodynamics. Questionnaires used included SF-36, EuroQol and a sexual function section based on the International Continence Society 'Benign Prostatic Hyperplasia' (ICS-BPH) questionnaire. Measurement of full blood count and urea and electrolytes was made at baseline and at 24 hours. Adverse events were recorded during the hospital stay and at follow-up visits. RESULTS: TURP and TUVP were both effective in producing a clinically important reduction in IPSS and positive change in the IPSS QoL question. The success rate for relief of symptoms was 85% for TURP and 74% for TUVP. Neither the success of the treatment nor the change in aggregated IPSS was significantly different between the groups. The improvement was sustained to 24 months after treatment with no significant difference between the groups. The effectiveness of both treatments was also equivalent when assessed through improvement in objective measures of urinary tract function, reduction in prostate size and the change in health questions of SF-36. The absolute incidence of adverse events was similar between the two groups. The incidence of severe or prolonged bleeding was less with TUVP, as evidenced by the need for blood transfusion and the drop in haemoglobin level 24 hours postoperatively. TURP and TUVP are broadly equivalent in direct NHS resource use. This study did not show any significant difference in inpatient stay or use of outpatient resources between the groups. The disposable electrodes used for TUVP are more expensive than reusable TURP electrodes. CONCLUSIONS: TURP and TUVP are equivalently effective in improving the symptoms of benign prostatic enlargement over at least 2 years. TUVP is associated with less morbidity due to haemorrhage than TURP. Replacement of TURP by TUVP would not produce a significant cost benefit to the NHS unless a reduction hospital inpatient stay of at least 1 day could be secured. Further research is necessary to determine why patients stay in hospital after transurethral surgery to the prostate and how a reduction in the length of stay can be achieved. A much larger observational study/audit is required to assess the incidence of infrequently occurring adverse events after TUVP. Longer term follow-up is also needed.

Optimization of the hygromycin B resistance-conferring gene as a dominant selectable marker in mammalian cells.

The HyR gene, conferring resistance to hygromycin B (Hy), has been modified for optimal expression in mammalian cells. Modifications to the HyR gene and its expression cassette include: (1) removal of all upstream start codons, (2) conversion of the region around the start codon to the consensus sequence associated with efficient translation initiation, and (3) removal of downstream splice donor and acceptor sequences. The resulting HyR gene is an efficient dominant selectable marker that is useful for studies requiring resistance from a low-copy-number gene driven by a promoter of moderate strength. The HyR gene was also tested for its compatibility with BPV vectors. Mouse C127 cells harboring pHyR-BPV plasmids exhibited properties of BPV-transformed cells and were resistant to toxic levels of Hy. The vectors were stable as episomes and present in high copy. The HyR gene thus joins the NmR (neo) gene as the only dominant selectable markers that are known to be compatible with BPV replication.

Phage RNA polymerase vectors that allow efficient gene expression in both prokaryotic and eukaryotic cells.

We have developed expression vectors that direct the synthesis of proteins from a common set of signals in both prokaryotic and eukaryotic cells. To allow transcription from a common promoter the vectors rely upon a phage RNA polymerase (RNAP). To direct initiation of translation to the same start codon the vectors utilize an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) that has been modified to include a prokaryotic ribosome-binding site (RBS) at an appropriate distance upstream from the desired start codon. These vectors provide levels of expression in eukaryotic cells that exceed those of a conventional RNAP-II-based system by 7-fold, and expression in bacterial cells at levels comparable to other phage RNAP-based systems. Inclusion of a lac repressor and a phage promoter/lac operator fusion element allows tight regulation. Cotransfection of eukaryotic cells with the expression vector and a vector that encodes the phage RNAP provides high-level transient expression without the need to construct specialized stable cell lines.

Cloning and expression of the bacteriophage T3 RNA polymerase gene.

The gene that encodes the RNA polymerase of bacteriophage T3 (gene 1) has been cloned into a pBR322 derivative under the control of an inducible lacUV5 promoter. Large quantities of the protein are synthesized after induction of cells that carry this plasmid. RNA polymerase purified from these overproducing cells selectively initiates transcription from T3 promoter sequences as demonstrated by transcription of a dual promoter plasmid that carries both T3 and T7 promoters. Cells that carry the T3 RNA polymerase gene can complement amber mutants of T3 that are defective in gene 1 but not gene 1 amber mutants of T7, and vice versa; this experiment demonstrates the specificity of these enzymes in vivo.

Regulation of coliphage T3 and T7 RNA polymerases by the lac repressor-operator system.

The single-polypeptide RNA polymerases that are encoded by bacteriophage T7 and its relatives form the basis of highly specific and efficient transcription systems. Here, we describe the regulation of transcription from phage promoters by the lac repressor-operator system of Escherichia coli. A synthetic oligodeoxyribonucleotide that contains the core sequence of the lac operator (lacO) was cloned at various distances downstream from the transcription start point (tsp) of the T3 and T7 promoters. The ability of lac repressor to prevent transcription from the phage promoters in vitro was dependent on the position of the operator. Efficient repression was observed when the center of the operator was placed between +14 and +27 (+1 being the tsp), whereas the repressor had little effect when bound to operators centered at +64. For in vivo studies, the chloramphenicol acetyltransferase (CAT)-encoding reporter gene was placed under the control of various promoter-operator constructs, and introduced into bacterial cells containing the genes for the lac repressor and T3 or T7 RNA polymerase. As with in vitro studies, high levels of repression (greater than 4000-fold) of T3 and T7 RNA polymerase activity were achieved, and repression was reversed by the inducer isopropyl-beta-D-thiogalactopyranoside. When the T3 promoter-lacO constructs are used to regulate the expression of a target gene in combination with an inducible RNA polymerase gene under control of the lacUV5 promoter, the doubly regulated system provides extremely tight levels of repression, yet allows high levels of expression after induction. In such a system, we observed a greater than 10(5)-fold increase in CAT activity within 30 min after induction. This system should prove useful in cloning and expressing genes that are potentially toxic to the host cells.