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At least 73 records · Page 4Linked to original sources
PubMed1992

Efficient expression of tum- antigen P91A by transfected subgenic fragments.

Mutagen treatment of mouse P815 tumor cells produces immunogenic mutants that express new transplantation antigens (tum- antigens) recognized by cytolytic T cells. The gene encoding tum- antigen P91A comprises 12 exons and a mutation located in exon 4 is responsible for the production of a new antigenic peptide. Transfection experiments showed that the expression of the antigen could be transferred not only by the entire gene but also by gene segments comprising only the mutated exon and parts of the surrounding introns. This was observed with subgenic regions that were not cloned in expression vectors. Antigen expression did not require the integration of the transfected gene segment into a resident P91A gene by homologous recombination. It also occurred when the subgenic segment was transfected without the usual selective gene, which comprises an eucaryotic promoter, and also without plasmid sequences, which are known to contain weak promoters. When a stop codon was introduced at the beginning of exon 4, the expression of the antigen was maintained and evidence was obtained that an ATG codon located in this region served as initiation site for the translation of the antigenic peptide. But we have not obtained evidence indicating that antigenic peptides are direct translation products rather than degradation products of entire proteins.

Amino Acid Sequence↗
PubMed1989

Cauliflower mosaic virus promoters direct efficient expression of a bacterial G418 resistance gene in Schizosaccharomyces pombe.

A system is presented for transformation of the fission yeast Schizosaccharomyces pombe to resistance against the antibiotic G418. The bacterial resistance gene of the transposon Tn5 is expressed under the control of promoters and transcription terminators from cauliflower mosaic virus (CaMV). The promoter of the S. pombe alcohol dehydrogenase gene has also been used. Transformants can be selected directly on medium containing G418 (up to 1 mg/ml) due to inactivation of G418 by the Tn5 gene product, the aminoglycoside 3'-phosphotransferase (II). The plant viral promoter 35S confers higher resistance to G418 than the 19S promoter. This corresponds to the relative strengths of these promoters in plant cells. The strong plant promoter 35S yields resistance comparable to that obtained with the strong S. pombe promoter from the alcohol dehydrogenase gene. The constructions with the two plant promoters have been used on multicopy shuttle plasmids that replicate autonomously in S. pombe and Escherichia coli. In addition the 35S and the 19S constructions have been inserted into the S. pombe genome where they confer G418 resistance as single copy genes. Since vector sequences are excluded in this case, all the necessary signals for expression of G418 resistance are contained within the DNA fragments containing the plant promoters, the resistance gene and the plant terminators. This transformation system is independent of S. pombe mutants. It may be useful for the transformation of other lower eukaryotes. The activity of the CaMV promoters in S. pombe may be exploited for the expression of plant genes in fission yeast.

Alcohol Dehydrogenase↗
PubMed1990

Evolutionary conservation of transcriptional machinery between yeast and plants as shown by the efficient expression from the CaMV 35S promoter and 35S terminator.

Complementation of fission yeast mutants by plant genomic libraries could be a promising method for the isolation of novel plant genes. One important prerequisite is the functioning of plant promoters and terminators in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Therefore, we studied the expression of the bacterial beta-glucuronidase (GUS) reporter gene under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and 35S terminator. We show here that S. pombe initiates transcription at exactly the same start site as was reported for tobacco. The 35S CaMV terminator is appropriately recognized leading to a polyadenylated mRNA of the same size as obtained in plant cells transformed with the same construct. Furthermore, the GUS-mRNA is translated into fully functional GUS protein, as determined by an enzymatic assay. Interestingly, expression of the 35S promoter in the budding yeast S. cerevisiae was found to be only moderate and about hundredfold lower than in S. pombe. To investigate whether different transcript stabilities are responsible for this enormous expression difference in the two yeasts, the 35S promoter was substituted by the ADH (alcohol dehydrogenase) promoter from fission yeast. In contrast to the differential expression pattern of the 35S promoter, the ADH promoter resulted in equally high expression rates in both fission and budding yeast, comparable to the 35S promoter in S. pombe. Since the copy number of the 35S-GUS constructs differs only by a factor of two in the two yeasts, it appears that differential recognition of the 35S promoter is responsible for the different transcription rates.

Alcohol Dehydrogenase↗
PubMed1989

A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae.

A homologous transformation system for Aspergillus oryzae is described. The system is based on an A. oryzae strain deficient in orotidine-5'-phosphate decarboxylase (pyrG) and the vector pAO4-2, which contains a functional A. oryzae pyrG gene as selection marker. Transformation of the A. oryzae pyrG mutant with circular pAO4-2 resulted in the appearance of Pyr+ transformants at a frequency of up to 20 per micrograms of DNA, whereas with linear pAO4-2 up to 200 transformants per micrograms DNA were obtained. In 75% of the Pyr+ transformants recombination events had occurred at the pyrG locus, most of which (90%) resulted in insertion of one or two copies of the vector and the others (10%) in a replacement of the mutant allele by the wild-type allele. Vector pAO4-2 is also capable of transforming a corresponding mutant of Aspergillus niger. This transformation system was used to introduce into A. oryzae the heterologous and non-selectable bacterial genes lacZ, encoding beta-galactosidase, and uidA, encoding beta-glucuronidase. Using the Aspergillus nidulans gpdA promoter to drive bacterial gene expression in A. oryzae, relatively high levels of activity, as well as protein per se, as judged by western blot analyses, were obtained.

Aspergillus↗
PubMed1987

Heat shock inactivates a supernatant factor(s) specifically required for efficient expression of the amp gene in Escherichia coli.

A 160,000 x g supernatant of E. coli extract prepared from cells grown at 30 degrees C stimulated specifically the expression of the amp gene on pBR322 and pBR328 in an in vitro gene expression system from E. coli. This activity of the supernatant was markedly reduced when the cells were exposed to 42 degrees C for 30 min prior to preparing the supernatant. These results are consistent with the view that heat shock-induced repression of the amp gene expression is due to inactivation of a supernatant factor(s) required for effective expression of the amp gene.

Ampicillin Resistance↗
PubMed1990

High-efficiency expression of rat protein kinase C-gamma in baculovirus-infected insect cells.

We have expressed rat protein kinase C-gamma in insect cells using a baculovirus vector. The yield of expressed protein kinase C-gamma is about 4% of total protein. The recombinant protein shows a prominent band at about 80 kDa on SDS-polyacrylamide gels, which can be identified as protein kinase C-gamma by Western blotting using monoclonal antibodies against protein kinase C-gamma. Upon incubation with [gamma-32P]ATP and in the presence of Ca2+, phosphatidylserine and diacylglycerol this protein autophosphorylates. Its enzyme activity shows the characteristic properties of mammalian protein kinase C.

Animals↗
PubMed1984

Efficient expression of cloned complementary DNAs for secretory proteins after injection into Xenopus oocytes.

Cloned complementary DNAs encoding chicken ovalbumin, chicken prelysozyme and calf preprochymosin, prochymosin and chymosin were inserted downstream from various viral promoters in modified recombinant "shuttle" vectors. Microinjection of the ovalbumin, prelysozyme and preprochymosin constructs into the nuclei of Xenopus laevis oocytes resulted in the synthesis, segregation in membranes and secretion into the extracellular medium of ovalbumin, lysozyme and prochymosin, respectively. Judging from molecular weight estimations, lysozyme and prochymosin were correctly proteolytically processed while ovalbumin, which lacks a cleavable signal sequence, was glycosylated. Injection of the DNA construct encoding prochymosin without its signal sequence resulted in synthesis of prochymosin protein that was localized exclusively in the oocyte cytoplasm. No immunospecific protein was detected after injection of the DNA encoding mature chymosin. In terms of protein expression in oocytes, the Herpes simplex thymidine kinase (TK) promoter was up to sevenfold more effective than the simian virus 40 (SV40) early promoter, and equally as effective as the Moloney murine sarcoma virus long terminal repeat element. Where tested, protein expression in oocytes was much reduced if DNA sequences encoding the SV40 small t intron and its flanking sequences were present in the constructs. S1 nuclease mapping of transcripts produced after injection of DNAs containing the TK promoter indicated that the majority of transcripts initiated at, or within, two bases of the known "cap" site. However, minor transcripts initiating upstream from this site were observed and one (or more) of these transcripts was responsible for the synthesis of an ovalbumin polypeptide containing a 51 amino acid N-terminal extension. This extended protein remained in the oocyte cytosol. When ovalbumin cDNA was inserted into the vectors with opposite polarity to the viral promoter, expression in oocytes resulted in the predominant synthesis and secretion of a variant ovalbumin with a 21 amino acid N-terminal extension, although some full-length ovalbumin was also synthesized and secreted. S1 mapping revealed the presence, in these oocytes, of transcripts of predicted polarity initiating 118 bases upstream from the wild type ovalbumin initiator ATG, at a previously unreported SV40 "promoter". No protein synthesis was detected after the injection of these reverse-orientation constructs into baby hamster kidney (BHK-21) cells.

Amino Acid Sequence↗
PubMed1985

The herpes simplex virus amplicon. IV. Efficient expression of a chimeric chicken ovalbumin gene amplified within defective virus genomes.

cDNA sequences of the chicken ovalbumin gene were fused to an alpha (immediate early) promoter of herpes simplex virus and to genomic ovalbumin 3'-flanking sequences. The chimeric alpha-ovalbumin gene was introduced into defective virus genomes which were stably propagated in serially passaged virus stocks in the presence of helper virus. Analyses of polypeptides synthesized in cells infected with the resultant defective virus stocks revealed the abundant expression of the chimeric alpha-ovalbumin gene. The presence of introns was not essential for this expression.

Carcinoma, Squamous Cell↗
PubMed1990

Both amino acid changes in nsP1 of Sindbis virusLM21 contribute to and are required for efficient expression of the mutant phenotype.

SVLM21 is a mutant of Sindbis virus which in contrast to the standard virus, SVSTD, is able to replicate in Aedes albopictus mosquito cells deprived of methionine. Previously, by making use of the infectious Toto plasmids, we had constructed recombinant viruses containing various SVLM21 sequences, and were thereby able to map the mutations associated with the SVLM21 phenotype to the gene for the nonstructural protein nsP1. Two mutations were found in the nsP1 gene of SVLM21. These led to predicted amino acid changes at residue 87 from Arg to Leu, and at residue 88 from Ser to Cys. In the work presented here, we assess the relative contributions of these two mutations to the SVLM21 phenotype using site-directed mutagenesis to obtain virus encoding only the change to Leu at residue 87 of nsP1 (SVMS319), or only the change to Cys at residue 88 (SVMS321). In addition we show that SVLM10, which was isolated during the selection procedure for SVLM21, encodes only the change at residue 88. In addition to its ability to grow in methionine-deprived mosquito cells, SVLM21 differs from SVSTD in two other respects: (1) it shows an increased sensitivity to neplanocin A (NPA) and (2) it generates increased levels of methyltransferase in infected cells. Whether we looked at resistance to low methionine, sensitivity to NPA, or levels of methyltransferase generated, SVMS319, SVMS321, and SVLM10 all expressed only a partial SVLM21 phenotype. Furthermore we were not able in these experiments to distinguish between these three viruses. We conclude therefore that both amino acid changes, i.e., at residues 87 and 88, are required to produce the full SVLM21 phenotype, and that both changes contribute equally.

Aedes↗
PubMed1984

Plasmid vectors designed for high-efficiency expression controlled by the portable recA promoter-operator of Escherichia coli.

A novel expression vector using the 236-bp promoter-operator fragment of the recA gene (recApo) of Escherichia coli has been constructed. This DNA fragment contains complete signals for the initiation of RNA synthesis, as well as for regulation by the lexA product, but lacks the coding sequence for the RecA protein. The strength of the recA promoter was examined by assaying beta-galactosidase activity expressed from a cro-lacZ fused gene placed downstream of the promoter. Under noninducing conditions, the promoter was regulated by the LexA protein, and the fused gene was expressed only weakly. Upon induction by nalidixic acid in a recA+ strain, high expression was observed for an extended period. After 5 h under inducing conditions, as much as 11% of the total cellular protein was cro-lacZ product. The expression level was higher than that from promoters of lac, trp, and lambda early genes.

Bacterial Proteins↗
PubMed1988

Vectors for efficient expression in mammalian fibroblastoid, myeloid and lymphoid cells via transfection or infection.

We have constructed two related types of multi-cloning mammalian expression vectors. The first, pMPSVEH/HE, carries the promoter of the myeloproliferative sarcoma virus (MPSV). This promoter was found to be stronger than both the SV40 early and the trans-activated human immunodeficiency virus promoters in many cell lines including human and rodent fibroblastoid, lymphoid or myeloid cells. The other, pBEH/HE, carries the simian virus 40 (SV40) early promoter and origin of replication. This offers the possibility of encapsidation in SV40 pseudovirions and subsequent gene transfer into, e.g., hemopoietic cells, via infection. The usefulness of the expression systems was tested with a number of genes and cell lines.

Animals↗
PubMed1988

Tandem gene amplification in vitro for rapid and efficient expression in animal cells.

Plasmid vectors pHSG293 and pHSG747, suitable for in vitro gene amplification for subsequent animal-cell expression, were developed. A cosmid vector pHSG293 confers Km resistance to Escherichia coli host cells and G418 resistance to animal cells and contains a single BstXI recognition/cleavage site, CCACGGGG/CTGG, near the cos site (the recognition site is underlined). The cassette vector plasmid pHSG747 contains a multiple cloning site (MCS) between the simian virus 40 early promoter and the poly(A) signal sequence flanked by the same BstXI sites and confers Cm resistance to E. coli host cells. After inserting a coding fragment for human protein C or its derivative in the appropriate orientation in the MCS of pHSG747, the BstXI expression unit fragment was purified, mixed with BstXI-digested pHSG293 DNA at a molecular ratio of 20 to 40:1 and ligated. This allowed for tandem gene amplification due to asymmetric cohesive ends. Ligation products were packaged in lambda phage particles, amplified in E. coli cells as large cosmid molecules, and then introduced into CHO cells. G418R transformants were found to produce and secrete recombinant protein molecules at a high level. The plasmid vectors developed in this work will provide a rapid screening system useful for protein engineering in animal cells.

Animals↗
PubMed1991

Broad host-range vector for efficient expression of foreign genes in gram-negative bacteria.

A broad host-range expression plasmid was constructed comprising the incQ replicon, the recA promoter from Escherichia coli and the g10-L ribosome binding site (RBS) derived from bacteriophage T7. The structural genes for porcine somatotropin (pst) and E. coli beta-galactosidase (lacZ) were used to monitor gene expression in a diverse collection of Gram-negative bacterial hosts: Escherichia coli, Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas testosteroni, Serratia marcescens and Erwinia herbicola. The E. coli recA promoter was functional in this wide range of hosts and was inducible by the addition of nalidixic acid. Moreover, the level of lacZ expression was often at least as high as that observed in E. coli. Previous studies had shown that the g10-L RBS was superior to a simple "consensus" RBS sequence for expression of foreign genes in E. coli. Here we demonstrate a 38 to 70 fold increase in expression in two Pseudomonas hosts using the g10-L RBS, indicating that the translational enhancer present in the g10-L RBS is also functional in other bacteria. The juxtaposition of these transcriptional and translational elements in a broad host-range vector provides a simple way to evaluate alternate hosts for recombinant protein production.

Cloning, Molecular↗
PubMed1983

Efficient expression of influenza virus NS1 nonstructural proteins in Escherichia coli.

RNA segment 8 of the influenza A virus genome codes for two nonstructural proteins, NS1 and NS2, for which the functions are unknown. Cloned cDNA copies of this gene from three different influenza A virus strains were inserted into an Escherichia coli plasmid expression vector, pAS1, carrying the strong regulatable lambda phage promoter, PL. After induction, the NS1 proteins were overproduced to levels of 20-25% of total cellular protein. This was surprising in that the codon composition for these eukaryotic genes is similar to that for weakly expressed proteins in E. coli. Thus, under the appropriate conditions, it appears that high level expression of genes containing a relatively large proportion of minor codons can be obtained. The NS1 protein produced in bacteria from a cloned cDNA copy of the A/PR/8/34 virus NS gene was purified to apparent homogeneity and used to generate a high-titer monospecific rabbit antiserum. Immunoprecipitation studies showed this antibody to be crossreactive against the NS1 proteins produced by several different influenza A virus strains. Immunofluorescence experiments in Madin-Darby canine kidney cells showed the NS1 proteins to be located in the nucleoplasm early in infection for all strains examined. With some of the strains, NS1-specific immunofluorescence was observed predominantly in the nucleoli later in infection. This technology can be used to obtain other viral proteins in pure form for structural, functional, and immunological studies.

Animals↗
PubMed1988

Design of vectors for efficient expression of human purine nucleoside phosphorylase in skin fibroblasts from enzyme-deficient humans.

Purine nucleoside phosphorylase (PNP; purine-nucleoside orthophosphate ribosyltransferase, EC 2.4.2.1) deficiency is an inherited disorder associated with a severe immune defect that is fatal. Enzyme replacement therapy is an attractive approach to treatment of this disease. To this aim we constructed retroviral vectors containing a human PNP cDNA and a selectable gene encoding neomycin phosphotransferase. PNP expression was controlled by either the early promoter from simian virus 40, the immediate early promoter from human cytomegalovirus, or the retroviral promoter. Cultured skin fibroblasts from two unrelated PNP-deficient patients that were infected with these vectors expressed mean PNP activities of 0.03, 0.74, and 5.9 mumol/hr per mg of protein, respectively. The latter infectants had PNP activities eight times the level of 0.74 mumol/hr per mg of protein observed in normal skin fibroblasts, enabling rapid metabolism of exogenous deoxyguanosine, the cytotoxic metabolite that accumulates in the plasma of PNP-deficient patients. These experiments indicate that viral long terminal repeat was the strongest promoter for expression of PNP and suggest the potential of human skin fibroblasts as vehicles for therapeutic gene expression.

Cells, Cultured↗
PubMed1984

Efficient expression in Escherichia coli of a mature and a modified human interferon-beta 1.

Ten recombinant plasmids were constructed which direct the synthesis of a mature human interferon-beta 1 (IFN-beta 1) under the control of the Escherichia coli tryptophan (trp) promoter. The spacing between the Shine-Dalgarno sequence of the trpL and the ATG initiation codon of the interferon gene was varied from 6 to 23 nucleotides by utilizing a Cla I site located within the spacer region of the plasmids. The optimal spacing for expression of IFN-beta 1 was determined to be 8-13 nucleotides from the results of interferon assay. The E. coli lipoprotein (lpp) promoter was also used for expression of IFN-beta 1 in E. coli. The results with an expression vector carrying a lpp-lac promoter showed that a modified IFN-beta 1 containing an additional 7 amino acids at the amino-terminus might be less active than the mature molecule.

Amino Acid Sequence↗