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Results for “expression efficiency”

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At least 19 recordsLinked to original sources
PubMed1985

Transfer of genes into embryonal carcinoma cells by retrovirus infection: efficient expression from an internal promoter.

The expression of retroviral genomes after infection of embryonal carcinoma (EC) cells is usually restricted. We have constructed a retroviral vector which has the potential to express efficiently in EC cells an inserted sequence, the selectable neomycin (neo) gene, from an internal thymidine kinase promoter. A second retrovirus vector contained in addition a v-myc oncogene, which in NIH 3T3 cells could be expressed from the viral long terminal repeat (LTR) through a subgenomic mRNA. 10-100% of the EC cells infected with recombinant virus gave rise to neo-resistant colonies following drug selection. The neo gene was expressed in both the non-permissive EC and the permissive NIH 3T3 cells at similar levels, and transcription of neo-specific RNA was initiated at the proper site in the thymidine kinase promoter. In contrast, v-myc mRNA was produced only in the NIH 3T3 cells and not in EC cells, even after induction of differentiation. Thus, genes inserted into retrovirus constructs can easily be introduced into EC cells by virus infection, even without drug selection, and they can be efficiently expressed when transcribed from promoters other than the viral LTRs.

Animals↗
PubMed1986

Efficient expression of unfused human alpha D-interferon in Escherichia coli using overlapping termination and initiation codons (TGATG) in its signal sequence.

A plasmid carrying the lambda PL promoter was constructed to express efficiently unfused human alpha D-interferon (HuIFN-alpha D) in Escherichia coli using a TGATG site in its signal sequence, which occurs also in the lambda DNA sequence. The unfused nature of HuIFN-alpha D expressed by pBV867 in E. coli (BMH 71-18) was confirmed by the following evidence: first, the purified IFN showed a single band of 19.5K in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); a 27K band, representing lambda N-HuIFN-alpha D fusion protein, was not detected. Second, the peak of IFN activity coincided with the 19.5K protein. Third, the peak of absorbent material for human leukocyte IFN antibody coincided with that of IFN activity. Finally, amino-terminal sequencing of purified IFN demonstrated an unfused HuIFN-alpha D. This suggests that E. coli is able to process the signal sequence of HuIFN-alpha D. Studies on the mechanism of "translational coupling" initiation of gene expression were carried out by the construction of two hybrid plasmids and titration of the IFN activities produced by them. The level of expression by the ATG-TGATG initiation mode was found to be six times higher than that of the single ATG mode.

Bacteriophage lambda↗
PubMed1989

Retroviral vector-mediated high-efficiency expression of adenosine deaminase (ADA) in hematopoietic long-term cultures of ADA-deficient marrow cells.

Two recombinant retroviral vectors encoding the cDNA of the human adenosine deaminase (ADA; EC 3.5.4.4) gene and the bacterial neomycin resistance (Neo) gene have been used to transduce bone marrow cells obtained from four patients affected by the ADA-deficient variant of severe combined immunodeficiency. By utilizing the long-term marrow culture system, freshly isolated bone marrow cells were subjected to multiple infection cycles with cell-free supernatants containing high titers of viral vector and then maintained in long-term marrow culture in the absence of any overt selection pressure. By using this experimental protocol, about 30-40% of the hematopoietic progenitors were productively transduced with the viral vector, as judged by the appearance of G418-resistant colonies derived from granulocyte/macrophage and multipotent hematopoietic progenitor cells. The vector-encoded human ADA gene was expressed efficiently in both the myeloid and lymphoid progeny of the cultured bone marrow cells, reaching levels between 15% and 100% as compared to the levels of ADA in normal bone marrow cells. The efficiency of gene transfer and ADA production was proportional to the number of infection cycles. Furthermore, transduction of the ADA vectors into the bone marrow cells derived from an ADA-deficient patient restored the capacity of the cells to respond to phytohemagglutinin and interleukin 2.

Adenosine Deaminase↗
PubMed1988

An EBV-based mammalian cell expression vector for efficient expression of cloned coding sequences.

The construction of a mammalian cell expression vector using human cytomegalovirus immediate early gene enhancer to initiate transcription of inserted coding sequences is described. The vector also carries Epstein-Barr virus EBNA-1 nuclear antigen gene, ori-P sequences and hygromycin B resistance gene hph from E. coli. The expression capacity of this construct was tested by inserting the chloramphenicol acetyltransferase (CAT) gene into the vector. The EBV-CAT construct was transfected into various cell lines and high levels of CAT activity were obtained in human and monkey cells. In these cells, the vector DNA also replicates as an extrachromosomal element having 1 to 20 copies per cell. In most cases, the vector copy number and the expression level of inserted gene was in positive correlation in different cell clones.

Acetyltransferases↗
PubMed1989

Expression of human erythropoietin cDNA in human lymphoblastoid Namalwa cells: the inconsistency of a stable expression level with transient expression efficiency.

Recombinant plasmids for the expression of human erythropoietin (EPO) cDNA in Namalwa cells were constructed. From the results of the EPO expression efficiency in transiently transfected cells, it was found that the simian virus 40 (SV40) early promoter directs EPO synthesis more efficiently in Namalwa cells than does the long terminal repeat promoter of Rous sarcoma virus and that the 3'-noncoding sequence including splice junction and polyadenylation site derived from the rabbit beta-globin gene are more effective than those of the SV40 early gene. However, in stable transformants, no simple relationship was found between the expression level of EPO cDNA and the structure of the introduced expression vectors.

Base Sequence↗
PubMed1989

High-efficiency expression of mammalian beta-adrenergic receptors in baculovirus-infected insect cells.

Infection of a clonal isolate of Spodoptera frugiperda cells (Sf9) with a baculovirus expression vector harboring the cDNA encoding the beta-adrenergic receptor resulted in a high efficiency expression. At 48 hr post-infection, the level of expression of beta-adrenergic receptors was approximately 12 million/cell. Specific activities of crude lysates of infected Sf9 cells were approximately 30 pmol/mg of protein, 5-fold greater than those of membranes of high-expressor Chinese hamster ovary cells stably transfected with an SV-40 expression vector. One liter of infected Sf9 cells expresses 20-40 nmol of receptor. Autoradiography of membranes incubated with the beta-adrenergic antagonist [125I]iodoazidobenzylpindolol, photolyzed, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed 46,000- (presumably unglycosylated) and 48,000-Mr peptides for Sf9 cells as compared to approximately 65,000-Mr for Chinese hamster ovary cells. The baculovirus Sf9 system provides high-efficiency expression of receptor sufficient to permit physicochemical analyses.

Animals↗
PubMed1985

Efficient expression of the Escherichia coli leuB gene in yeast.

Efficient expression of the Escherichia coli leuB (beta-isopropylmalate dehydrogenase) gene occurred in yeast after in vitro DNase digestion and religation of plasmid bound leuB and the yeast HIS3 DNA which placed the 5' end of the yeast HIS3 gene immediately adjacent to the coding region of the E. coli leuB gene. Two structurally distinct classes of gene fusions were constructed, each involved portions of the yeast HIS3 gene which contributed DNA sequences responsible for leuB expression in yeast. The first class involved fusion of the HIS3 coding region to bacterial DNA resulting in the production of a fusion protein with beta-isopropylmalate dehydrogenase activity. The second class consisted of bacterial DNA, including the leuB coding region, fused to the HIS3 promotor region with the absence of any portion of the HIS3 coding region. In both constructions the HIS3 promotor region is required for transcription, however, translation of the class two fusion is initiated at a bacterial DNA coded AUG, and the 5' end of the mRNA coded by the leuB gene mapped predominantly at bacterial DNA sequences. The DNA sequence responsible for the 5' end of the HIS3 mRNAs remain in the class two gene fusions but this did not preclude the initiation of transcription at bacterial DNA sequences. The pattern of mRNA initiation at bacterial DNA suggests that DNA sequences at, or adjacent to, the site of transcription initiation are involved in the determination of the sites of initiation, and perhaps the frequency at which initiation occurs.

3-Isopropylmalate Dehydrogenase↗
PubMed1988

Cloning of genomic DNA for tumor necrosis factor and efficient expression in CHO cells.

A genomic clone for human tumor necrosis factor (TNF-alpha) was isolated using synthetic oligonucleotide probes. The genomic DNA was cleaved to remove 5' regulatory sequences and cloned in a PSVE3 expression vector containing the SV40 early promoter. The plasmid was co-transfected with a selectable dihydrofolate reductase (DHFR) gene into DHFR-deficient Chinese hamster ovary cells. Efficient expression of TNF mRNA was established by Northern analysis. Expression of TNF protein was assayed for by cytotoxic activity for cycloheximide-treated SV80 fibroblasts. Selected transfected cultures secreted as much as 50,000 units of TNF activity/ml of culture medium. Synthesis of TNF protein was confirmed by immunofluorescence of transfected cells with a monoclonal antibody to TNF and immunoprecipitation of 17 kD protein from transfected CHO culture supernates. The efficient expression of TNF from genomic DNA in transfected mammalian cells may be advantageous for biologic uses.

Animals↗
PubMed1990

Efficient expression of the Saccharomyces cerevisiae glycolytic gene ADH1 is dependent upon a cis-acting regulatory element (UASRPG) found initially in genes encoding ribosomal proteins.

The glycolytic form of alcohol dehydrogenase (ADHI) is encoded by the ADH1 gene of Saccharomyces cerevisiae. We found that efficient expression of the ADH1 gene requires a sequence between bp -635 and -615 with respect to the +1 mRNA start point; removal of this sequence reduced ADH1 mRNA levels 25-fold but did not affect carbon-source regulation. DNaseI footprinting analysis of the ADH1 promoter revealed the specific protection of a perfect match to UASRPG at -630 to -615. UASRPG is thought to be responsible for activation of transcription, via binding of the translation upstream factor (TUF), of genes encoding components of the translational apparatus. In band retardation assays, the promoters for the elongation factor 1 alpha-encoding genes (TEF1 and TEF2) competed for binding of the protein to the copy of UASRPG in the ADH1 promoter. We conclude that TUF is probably involved in activation of the bulk of ADH1 transcription. Further, we propose that TUF has a role in the activation of many or most glycolytic genes. If so, it is essential for efficient expression of a wide variety of functionally disparate products that are required by yeast cells for rapid growth.

Alcohol Dehydrogenase↗
PubMed1988

Efficient expression of the yeast metallothionein gene in Escherichia coli.

The yeast metallothionein gene CUP1 was cloned into a bacterial expression system to achieve efficient, controlled expression of the stable, unprocessed protein product. The Escherichia coli-synthesized yeast metallothionein bound copper, cadmium, and zinc, indicating that the protein was functional. Furthermore, E. coli cells expressing CUP1 acquired a new, inducible ability to selectively sequester heavy metal ions from the growth medium.

Amino Acid Sequence↗
PubMed1992

High-efficiency expression and solubilization of functional T cell antigen receptor heterodimers.

The T cell receptor (TCR) zeta chain was attached to the TCR alpha and beta extracellular domains to induce efficient expression of alpha beta heterodimers that can recognize complexes of antigen with major histocompatibility complex (MHC) molecules. Chimeric constructs expressed in RBL-2H3 cells were efficiently transported to the cell surface uniquely as disulfide-linked heterodimers. Transfectants were activated by specific antigen-MHC complexes, which demonstrated that the expressed alpha beta was functional and that CD3 was not required for antigen-MHC binding. Constructs with thrombin cleavage sites were efficiently cleaved to soluble disulfide-linked heterodimers. Thus, attachment of TCR zeta domains and protease cleavage sites to TCR alpha and beta induces expression of demonstrably functional heterodimers that can be solubilized.

Amino Acid Sequence↗
PubMed1990

New vectors for the efficient expression of mammalian genes in cultured cells.

We have constructed a new pair of plasmid vectors for the efficient expression of mammalian genes. The first of the new plasmids, pAVE1, was derived from pCMVcat [Foecking and Hofstetter, Gene 45 (1986) 101-105] by replacing the chloramphenicol acetyltransferase-encoding sequences in the latter for a multiple cloning site. Since it possesses the powerful enhancer-promoter unit of the immediate early gene of human cytomegalovirus, pAVE1 is ideal for the expression of mammalian genes. The second expression vector, pAVE2, resulted when the 3'-end flanking region from the human growth hormone-encoding gene (hGH) was incorporated in pAVE1. This region provides sequences for 3'-end processing and polyadenylation of primary transcripts. Thus, pAVE2 is suitable for expression of cDNAs in cultured cells, where introns have little effect on gene expression. To test our new vectors, we inserted the structural region of the chromosomal hGH gene into pAVE1, and its cDNA into pAVE2. By independently transfecting the resulting recombinant plasmids into COS-7 cells, we have achieved high levels of hGH transient expression with both vectors.

Cells, Cultured↗
PubMed1990

Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system.

We have characterized the promoter region of the human elongation factor 1 alpha-encoding gene (EF-1 alpha) and developed a versatile expression system which has a wide host range and a high efficiency of gene expression. To identify the promoter region of the EF-1 alpha gene necessary for efficient gene expression, we constructed four pEF-CAT plasmids that have the bacterial cat gene fused to four different sites of the human EF-1 alpha gene: (i) ligated to the end of the TATA box (pEF220-CAT); (ii) ligated in exon 1 (pEF204-CAT and pEF233-CAT), and (iii) ligated in exon 2 (pEF321-CAT). All the pEF-CAT plasmids were highly expressed in all the cell types tested, including fibroblasts and lymphoid cells. Plasmid pEF321-CAT, which contains the first exon and the first intron, gave the highest level of cat expression. Plasmids pEF204- and pEF233-CAT, which contain part of the first exon but do not contain the first intron, were less efficient in cat expression than was pEF321-CAT. Plasmid pEF220-CAT, which lacks both the first exon and the first intron, was the least efficient. Plasmid pEF321-CAT was several- to 100-fold more efficient in cat expression than plasmid pSV2-CAT depending on the recipient cell types. The promoter of pEF321 plasmid also directed the stable expression of the bacterial neo gene more efficiently than the promoter of the simian virus 40 (SV40) early gene or the long terminal repeat of Rous sarcoma virus. Using this system, the SV40 early gene and the cDNA encoding human CD4 were also expressed efficiently.

Animals↗
PubMed1989

Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in vivo.

The dihydrofolate reductase (DHFR) gene encodes an enzyme important for metabolism and cell growth. We have found multiple DNA-protein interactions within the hamster DHFR gene promoter in vitro. These interactions occur over the consensus binding sites for two eucaryotic transcription factors. Sp1 and E2F. The DHFR E2F consensus site possesses a dyad symmetry and is unique in its location immediately 3' to the major transcription start site. The interaction of E2F with the DHFR promoter has been detected in HeLa nuclear extracts, confirmed by using partially purified E2F, and characterized by both enzymatic and chemical assays of the DNA-protein interaction. A mutation of the E2F recognition sequence which abolishes E2F binding to the DHFR promoter results in a two- to fivefold decrease of in vitro transcriptional activity and a fivefold reduction of DHFR promoter activity in transient-expression assays. Thus, the interaction of E2F with the DHFR promoter is required for efficient expression of the DHFR gene.

Animals↗
PubMed1986

Retroviral mutants efficiently expressed in embryonal carcinoma cells.

The myeloproliferative sarcoma virus (MPSV) is a unique member of the Moloney murine sarcoma virus family. Due to mutations in the U3 region of its long terminal repeat, MPSV has an expanded host range that includes cells of the hematopoietic compartment. Using a MPSV recombinant containing the gene for neomycin-resistance (NeoR-MPSV), we demonstrate that the host range of MPSV also includes undifferentiated F9 embryonal carcinoma cells. Transfer of G418-resistance with NeoR-MPSV to F9 cells is almost as efficient as G418-resistance transfer to fibroblasts, in contrast to G418-resistance transfer to PCC4 embryonal carcinoma cells, which is at least 3 orders of magnitude lower. To isolate NeoR-MPSV mutants that are efficiently expressed in PCC4 cells, G418-resistant PCC4 cell lines were induced to differentiate, and the provirus was rescued by superinfection with murine leukemia virus. Viral isolates (PCMV-5 and -6; PCMV = PCC4 cell-passaged NeoR-MPSV) were obtained and assayed for expression in embryonal carcinoma cells. The efficiency of NeoR transfer was equally as high in both F9 and PCC4 as in fibroblasts. mos oncogene expression was unaltered as judged by transformation capability. No gross alteration in the coding region and in the long terminal repeat was detectable by restriction enzyme analysis. NeoR-MPSV and its mutants PCMV-5 and -6 can thus be utilized as vectors for the efficient transduction of genes into embryonic cells.

Animals↗
PubMed1986

Efficient expression of the Saccharomyces cerevisiae PGK gene depends on an upstream activation sequence but does not require TATA sequences.

The Saccharomyces cerevisiae PGK (phosphoglycerate kinase) gene encodes one of the most abundant mRNA and protein species in the cell. To identify the promoter sequences required for the efficient expression of PGK, we undertook a detailed internal deletion analysis of the 5' noncoding region of the gene. Our analysis revealed that PGK has an upstream activation sequence (UASPGK) located between 402 and 479 nucleotides upstream from the initiating ATG sequence which is required for full transcriptional activity. Deletion of this sequence caused a marked reduction in the levels of PGK transcription. We showed that PGK has no requirement for TATA sequences; deletion of one or both potential TATA sequences had no effect on either the levels of PGK expression or the accuracy of transcription initiation. We also showed that the UASPGK functions as efficiently when in the inverted orientation and that it can enhance transcription when placed upstream of a TRP1-IFN fusion gene comprising the promoter of TRP1 fused to the coding region of human interferon alpha-2.

Base Sequence↗
PubMed1991

Herpes simplex virus transactivator ICP4 operationally substitutes for the cellular transcription factor Sp1 for efficient expression of the viral thymidine kinase gene.

The herpes simplex virus type 1 (HSV-1) ICP4 protein is a transcriptional activator of many eucaryotic RNA polymerase II promoters. The HSV-1 thymidine kinase gene (tk) promoter is induced by ICP4 and contains binding sites for the cellular transcription factors TFIID, Sp1, and CCAAT-binding proteins, each of which affects expression of the tk gene. In this study, the effects of mutations in these sites on the transcription of tk in the presence and absence of ICP4 were determined during viral infection. Only the TATA box was necessary for efficient expression in the presence of ICP4; however, ICP4 apparently can still induce tk transcription even when the TATA box is disrupted. Alteration of the Sp1 sites had a minor effect on ICP4-induced expression in comparison to a large effect in the absence of ICP4, indicating that ICP4 can operationally substitute for the function of the transcription factor Sp1. In addition, tk was still expressed with the kinetics of an early gene in the absence of binding sites for Sp1 and CCAAT-binding proteins.

Animals↗