Phage Lambda PL Promoter Research Articles

Anti-mRNA: specific inhibition of translation of single mRNA molecules.

A plasmid was constructed to generate RNA complementary to the beta-galactosidase mRNA under control of the phage lambda PL promoter. When this anti-mRNA was produced, synthesis of beta-galactosidase was dramatically inhibited (98%). Syntheses of galactoside permease and transacetylase, whose coding sequences are downstream of the beta-galactosidase coding region, are inhibited to a lesser degree, 80% and 55%, respectively. The generation of anti-mRNA that can be targeted to inhibit a single species of mRNA molecule within cells provides a potent mechanism by which specific transcripts can be translationally inactivated. This can be used to determine the function of proteins as well as to select cloned genes in a single rapid and convenient step.

Identification of the secY (prlA) gene product involved in protein export in Escherichia coli.

The gene secY (or prlA) is essential for protein export across the cytoplasmic membrane of Escherichia coli. The protein product of secY has been identified using the gene cloned under the control of the pL promoter of phage lambda in combination with the maxicell system. The protein was found to have some unusual properties. First, it is important not to heat the protein at 100 degrees C in the SDS sample buffer for its subsequent detection by gel electrophoresis. Second, migration of the protein in SDS-polyacrylamide gel electrophoresis is variable depending on the gel compositions. Gels with stronger sieving effect give higher apparent molecular weights. These properties are similar to those of hydrophobic proteins of the cytoplasmic membrane, such as the lactose permease. Finally, a major fraction of the protein synthesized from the overproducing plasmid is degraded rapidly in vivo. The altered protein from the secY24 mutant gene is even more unstable. These results provide information which is basic for the dissection of the protein export machinery of the bacterial cell.

Demonstration of two operator elements in gal: in vitro repressor binding studies.

Genetic and DNA base sequence analyses of cis-dominant mutations that derepress the gal operon of Escherichia coli suggested the existence of two operator loci needed for gal repression. One (OE) is located immediately upstream to the two overlapping gal promoters and the other (OI) is inside the first structural gene. We have investigated the ability of wild-type and mutant OE and OI DNA sequences to bind to gal repressor. The repressor has been purified from cells containing a multicopy plasmid in which the repressor gene is brought under the control of phage lambda PL promoter. The DNA-repressor interactions are detected by the change in electrophoretic mobility of labeled DNA that accompanies its complex formation with repressor protein. The purified repressor shows concentration-dependent binding to both O+E and O+I but not to OEc and OIc sequences. These results authenticate the proposed operator role of the two homologous gal DNA control elements and thereby establish that the negative control of the gal operon requires repressor binding at both OE and OI, which are separated by greater than 90 base pairs.

Expression of normal and transforming H-ras genes in Escherichia coli and purification of their encoded p21 proteins.

The H-ras gene of the BALB murine sarcoma virus (BALB-MSV) was placed under the transcriptional control of the tightly regulated PL promoter of bacteriophage lambda in the expression vectors pEV-vrf-1 and pRC23. Upon derepression of the PL promoter, large amounts (10-20% of total cellular protein) of the H-ras gene product p21 are synthesized in Escherichia coli. We constructed three H-ras gene expression vectors, designated pJCL-H5, pJCL-E30, and pJCL-33. pJCL-H5 directs the synthesis of p21, a fusion protein whose four amino-terminal residues are replaced by eight amino acids coded for by plasmid sequences. The 13 5' coding nucleotides of the BALB-MSV H-ras gene missing in pJCL-H5 were regenerated in pJCL-E30 by inserting a pair of complementary synthetic oligodeoxynucleotides. As a result, pJCL-E30 encodes a p21 protein, p21T, of sequence identical to that of the transforming p21 protein of BALB-MSV. pJCL-33 is a derivative of pJCL-E30 in which the 12th codon, AAA, a lysine codon, was replaced by GGA, a glycine codon. Thus, pJCL-33 directs the synthesis of a p21 protein, p21N, whose sequence corresponds to that of a normal cellular p21 protein. We report the purification of H-ras p21 proteins to apparent homogeneity by a method involving solubilization with chaotropic agents followed by reverse-phase high-performance liquid chromatography.

Isolation of gram quantities of EcoRI restriction and modification enzymes from an overproducing strain.

Structural genes for EcoRI restriction endonuclease and modification methylase have been inserted into the plasmid vector pKC30 (Shimatake, H., and Rosenberg, M. (1981) Nature (Lond.) 292, 128-132) downstream from the bacteriophage lambda pL promoter. Upon induction of pL expression in strains producing a thermolabile lambda cI857 repressor, synthesis of EcoRI polypeptides is enhanced to the extent that after 4 h they represent several per cent of the total cell protein. Purification of activities overproduced in this manner yields preparations of endonuclease and methylase which appear identical to those obtained from conventional sources, with overall yields corresponding to 0.5 to 0.9 g of each enzyme/kg of cell paste.

Phase variation and the Hin protein: in vivo activity measurements, protein overproduction, and purification.

The alternate expression of the Salmonella flagellin genes H1 and H2 is controlled by the orientation of a 995-base-pair invertible segment of DNA located at the 5' end of the H2 gene. The hin gene, which is encoded within the invertible region, is essential for the inversion of this DNA segment. We cloned the hin gene into Escherichia coli and placed it under the control of the PL promoter of bacteriophage lambda. These cells overproduced the Hin protein. In vivo inversion activity was measured by using a recombinant lambda phage which contains the H2 and lacZ genes under the control of the invertible region. Using this phage, we showed that the amount of inversion activity is proportional to the amount of Hin protein in the cell. An inactive form of the protein was purified by using the unusual solubility properties of the overproduced protein. The amino acid composition of the protein agreed with the DNA sequence of the hin gene. Antibodies were made to the isolated protein. These antibodies cross-reacted with two other unidentified E. coli proteins.

Overproduction and purification of a bacteriophage SPO1-encoded RNA polymerase sigma factor.

Gene 28 of bacteriophage SPO1 encodes an RNA polymerase sigma factor sigma gp28, which replaces the host Bacillus subtilis sigma subunit sigma 55, to alter the promoter recognition specificity of RNA polymerase. A fragment of SPO1 DNA containing gene 28 was placed under the control of the PL promoter of bacteriophage lambda in an Escherichia coli expression vector. When transcription of gene 28 was induced by derepression of PL, the sigma gp28 synthesized constituted several per cent of total cellular protein. Sigma gp28 purified from these cells was able to confer specificity for SPO1 middle gene promoters upon B. subtilis core RNA polymerase, and also enabled E. coli core RNA polymerase to recognize and initiate transcription from an SPO1 middle gene promoter.

High-level expression of human interferon gamma in Escherichia coli under control of the pL promoter of bacteriophage lambda

Several recombinant plasmids have been constructed which direct high-level synthesis of mature human interferon gamma (IFN-γ) in Escherichia coli using the inducible leftward promoter P L of phage λ followed by a translational initiator region derived either from the phage MS2 replicase gene or the E. coli tryptophan attenuator region. Under these conditions, IFN levels of up to 25 % of the total cellular protein can be achieved. The highest levels were obtained when a terminator of transcription was cloned downstream from the IFN-γ sequence. IFN-γ was almost entirely found in the initial pellet fraction and not in soluble extracts. Co-induction of the lysis genes derived from phage MS2 or from phage λ, inserted downstream from the IFN-γ sequence, did not enhance the biological activity present in the supernatant fraction.

Overproduction and purification of the connector protein of Bacillus subtilis phage phi 29.

A phi 29 DNA fragment containing genes 10 and 11, coding for the connector protein and the lower collar protein, respectively, has been cloned in the pBR322 derivative plasmid pKC30 under the control of the PL promoter of phage lambda. Two polypeptides with the electrophoretic mobility of proteins p10 and p11 were labelled with 35S-methionine after heat induction. The proteins were characterized as p10 and p11 by radioimmunoassay and they represented about 10% and 7%, respectively, of the total E. coli protein after 4 hours of induction. These proteins represent less than 1% of the B. subtilis protein in phi 29-infected cells. Protein p10 has been highly purified from the E. coli cells carrying the recombinant plasmid. Antibodies raised against the purified protein p10 reacted with the connector protein produced in phi 29-infected B. subtilis.

Analysis of the Escherichia coli proBA locus by DNA and protein sequencing.

A 2.9 kb DNA fragment carrying the Escherichia coli proBA region, which encodes the first two enzymes of the proline biosynthetic pathway, was subcloned onto an expression plasmid carrying both the bacteriophage lambda PL promoter (lambda PL) and the lambda gene encoding a thermolabile cI repressor protein (cI857). Derepression of the lambda PL promoter by thermal inactivation of the cI857 repressor protein resulted in the simultaneous overproduction of the proB (gamma-glutamyl kinase) and proA (gamma-glutamyl phosphate reductase) gene products. Nucleotide sequence analysis of the proBA locus allowed gene assignments consistent with the NH2 and COOH-terminal analyses and amino acid compositions of homogeneous preparations of the proB and proA proteins. The contiguous nature of the proB and proA genes suggests that the two genes constitute an operon in which proB precedes proA.

Temperature-inducible amber suppressor: construction of plasmids containing the Escherichia coli serU- (supD-) gene under control of the bacteriophage lambda pL promoter.

An Escherichia coli DNA fragment containing the structural gene serU132 for the nonsense suppressor tRNASer2am was identified and purified by being cloned into a plasmid vector. Information obtained from DNA sequence analysis was used to select a serU132 fragment for insertion downstream from the bacteriophage lambda pL promoter in two pBR322-lambda derivatives. In nonsense mutant strains bearing the resulting serU132 hybrid plasmids, the presence of the lambda cI857 repressor gene carried on the same plasmid or in a prophage genome permits thermal regulation of suppressor synthesis.

High-level expression in Escherichia coli of enzymatically active Harvey murine sarcoma virus p21ras protein.

The gene for the Harvey murine sarcoma virus (Ha-MuSV) p21ras protein was fused to the amino-terminal portion of the bacteriophage lambda cII gene on the expression vector pJL6. The fusion was such that transcription was controlled by the well-regulated phage lambda pL promoter, and translation initiated in the cII gene continued in frame into the ras gene sequences that code for p21. When the pL promoter was derepressed, the Escherichia coli cells harboring the fusion plasmid synthesized 23,000-dalton protein, which represented more than 10 percent of the total cellular protein. This protein was chimeric and contained 14 residues, which were specified by the vector; these residues were followed by all of the amino acids that make up Ha-MuSV p21ras except for four residues at the amino-terminal end. The protein appears similar to Ha-MuSV p21ras in that it undergoes immunoprecipitation by monoclonal antibodies directed toward that protein, binds guanosine diphosphate, and is capable of autophosphorylation.

Purification and properties of T4 phage thymidylate synthetase produced by the cloned gene in an amplification vector.

We have introduced the T4 thymidylate synthetase gene, resident in a 2.7-kilobase EcoRI restriction fragment, into an amplification plasmid, pKC30. By regulating expression of this gene from the phage lambda pL promoter within pKC30 in a thyA host containing a temperature-sensitive lambda repressor, the T4 synthetase could be amplified about 200-fold over that after T4 infection. At this stage, a 20-fold purification was required to obtain homogeneous enzyme, mainly by an affinity column procedure. The purified plasmid-amplified T4 synthetase appeared to be identical with the T2 phage synthetase purified from phage-infected Escherichia coli in molecular weight, amino end group analysis, and immunochemical reactivity. The individual nature of the phage and host proteins was revealed by the fact that neither the T2 nor the T4 enzyme reacted with antibody to the E. coli synthetase, nor did antibody to the phage enzymes react with the E. coli synthetase. These differences were corroborated by DNA hybridization experiments, which revealed the absence of apparent homology between the T4 and E. coli synthetase genes. The techniques and genetic constructions described support the feasibility of employing similar amplification methods to prepare highly purified thymidylate synthetases from other sources.

Molecular cloning of human interleukin 2 cDNA and its expression in E. coli.

A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes. Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA. A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal sequence of 20 amino acids. A mature polypeptide starting with either Met-Ala-Pro or Met-Pro was expressed in E. coli under control of the E. coli trp promoter or using a combination of the phage lambda PL promoter and a ribosome binding site derived from phage Mu. The bacterial IL2 polypeptide had a molecular weight of 15,000 daltons and accounted for more than 10% of the total E. coli proteins in fully induced cells; it was biologically active in the T-cell specific DNA synthesis assay, even after recovery from a SDS-containing polyacrylamide gel.

High-level synthesis in Escherichia coli of the SV40 small-t antigen under control of the bacteriophage lambda pl promoter

Several plasmids were constructed in which the SV40 small-t antigen gene was inserted in close proximity downstream from the thermoinducible leftward promoter ( p l ) of bacteriophage λ. Upon temperature induction the best of our constructions expressed a small-t-related 19000-dalton polypeptide in an amount corresponding to approx. 2.5% of total de novo protein synthesis. This 19000-dalton protein was identified as small-t by specific immunoprecipitation with anti-T serum and by two-dimensional fingerprint analysis. In addition to the 19 000-dalton product, representative plasmids expressed fairly large amounts (up to 7% of total de novo protein synthesis) of a protein with an apparent M r of 14500. This 14500-dalton polypeptide was shown to be related to authentic small-t. Presumably the secondary structure of the mRNA starting at is such that translation initiation at an internal AUG codon of the small-t gene is favored over initiation at the true initiating codon.

Escherichia coli delta 1-pyrroline-5-carboxylate reductase: gene sequence, protein overproduction and purification.

The sequence of the Escherichia coli proC gene which encodes for delta 1-pyrroline-5-carboxylate (PCA) reductase was determined. Overproduction of the proC gene product via an expression plasmid carrying the bacteriophage lambda PL promoter allowed the purification to homogeneity of PCA reductase by affinity adsorption chromatography. NH2 and COOH-terminal analysis and amino acid composition of the purified proC protein is consistent with the gene sequence reported. The molecular weight of the proC monomer is 28,112.

High level synthesis in Escherichia coli of the Bacillus subtilis phage phi 29 proteins p3 and p4 under the control of phage lambda PL promoter.

The Hind III G fragment from the Bacillus subtilis phage phi 29 DNA, inserted downstream from the bacteriophage lambda promoter PL carried by a pBR322 derivative plasmid (pPLc28), directed the synthesis in E. coli of two proteins of apparent molecular weight 27500 and 12500. With the use of the recombinants obtained with the DNA from mutants sus3(91) and sus4(56), the two proteins were identified as a modified p3 (p3'), the protein covalently linked to the 5' ends of phi 29 DNA, and p4, responsible for the phi 29 late transcription, respectively. Under the best conditions used, proteins p4 and p3' were produced in E. coli from the cloned DNA fragments in an amount corresponding to approximately 30% and 6% of total de novo protein synthesis, respectively.

Isolation and characterization of a human fibroblast interferon gene and its expression in Escherichia coli.

Human fibroblasts were induced for interferon synthesis, and the mRNA coding for interferon was partially purified. On this basis, double-stranded DNA copies were synthesized enzymatically and were inserted into cloning vehicles. A large collection of colonies containing chimeric plasmids was obtained. An RNA selection method was used for the identification of several individual clones containing the human fibroblast interferon (IFN-beta) cDNA. From the nucleotide sequence of the cloned structural gene, the primary structure of the mRNA and, hence, of the protein itself was deduced. IFN-beta is a polypeptide 166 amino acids long and contains a single site for N-glycosylation; IFM-beta is normally made as a preinterferon containing a signal sequence that is 21 amino acids long. The interferon gene was inserted into an expression plasmid under thermoinducible control of the phage lambda PL promoter. this allowed the synthesis of polypeptides in the bacteria, which for all physicochemical, biological, and immunologic properties tested closely resembled authentic IFN-beta.

Cloning of cDNA of major antigen of foot and mouth disease virus and expression in E. coli.

Double-stranded DNA copies of the single-stranded genomic RNA of foot and mouth disease virus have been cloned into the Escherichia coli plasmid pBR322. A restriction map of the viral genome was established and aligned with the biochemical map of foot and mouth disease virus. The coding sequence for structural protein VP1, the major antigen of the virus, was identified and inserted into a plasmid vector where the expression of this sequence is under control of the phage lambda PL promoter. In an appropriate host the synthesis of antigenic polypeptide can be demonstrated by radioimmunoassay.

Expression of human fibroblast interferon gene in Escherichia coli.

The human fibroblast interferon gene was inserted in a thermoinducible expression plasmid under control of the phage lambda PL promoter. The primary translation products predicted on the basis of the plasmid constructions were hybrid proteins starting with beta-lactamase or phage MS2 polymerase information followed by the total preinterferon. On induction, antiviral activity, whose physico-chemical, immunological and biological characteristics closely corresponded to those of authentic human fibroblast interferon, was synthesized. Processing to a size compatible with mature but unglycosylated authentic product was observed.