Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “expression efficiency”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources
PubMed1991

Identification of cis sequences controlling efficient position-independent tissue-specific expression of human major histocompatibility complex class I genes in transgenic mice.

We previously reported that genomic major histocompatibility complex class I human leukocyte antigen (HLA)-B7 gene constructs with as little as 0.66 kb of 5'- and 2.0 kb of 3'-flanking DNA were expressed efficiently and appropriately in transgenic mice. To identify and characterize the relevant cis-acting regulatory elements in more detail, we have generated and analyzed a series of transgenic mice carrying native HLA-B7 genes with further 5' truncations or intronic deletions and hybrid constructs linking the 5'-flanking region of B7 to a reporter gene. We were unable to detect a specific requirement for sequence information within introns 2 to 7 for either appropriate constitutive or inducible class I expression in adult animals. The results revealed the presence of cis-acting regulatory sequences between -0.075 kb and -0.66 kb involved in driving efficient copy number-dependent constitutive and gamma interferon-enhanced tissue-specific expression. The region from -0.11 to -0.66 kb is also sufficient to prevent integration site-specific "position effects," because in its absence HLA-B7 expression is frequently detected at significant levels at inappropriate sites. Conserved sequence elements homologous to the H-2 class I regulatory element, or enhancer A, and the interferon response sequence are located between about -151 and -228 bp of the B7 gene. Our results also indicate the existence of sequences downstream of -0.11 kb which can influence the pattern of tissue-specific expression of the HLA-B7 gene and the ability of this gene to respond to gamma interferon.

Animals↗
PubMed1985

Promoter domains required for expression of plasmid-borne copies of the herpes simplex virus thymidine kinase gene in virus-infected mouse fibroblasts and microinjected frog oocytes.

A transient expression assay was used to measure the relative template activities of mutated tk genes in mouse L cells induced in trans by herpes simplex virus (HSV). In this assay, expression of the wild-type HSV type 1 tk gene is induced at least 200-fold by the superinfecting virus. Genetic lesions that were assayed include 5' deletions, clustered base substitutions, single base substitutions, intrapromoter inversions, and intrapromoter recombinants with the HSV type 2 tk gene. Roughly half of the mutations that were tested were found to weaken tk expression efficiency, and the remaining mutations did not alter expression. The spatial distribution of mutations that reduce expression efficiency in trans-induced mouse fibroblasts facilitated the construction of a map of promoter domains. The most gene-proximal promoter domain is located between 16 and 32 base pairs (bp) upstream of the tk mRNA cap site and contains a TATA homology. Two more distally located promoter domains were mapped to discrete locations upstream from the TATA homology. One of these distal domains is located between 47 and 79 bp upstream from the mRNA cap site, and the other is located between 84 and 105 bp upstream from the tk gene. The boundaries of these three promoter domains, with one exception, coincided with the set of domains delineated previously in a frog oocyte microinjection assay. The concordant behavior of tk promoter mutants in microinjected frog oocytes and trans-induced mouse fibroblasts leads us to propose that recognition and activation of the HSV tk promoter is mediated by cellular transcription factors that are common to frogs and mice.

Animals↗
PubMed1992

Expression of diphtheria toxin A-chain in mature B-cells: a potential approach to therapy of B-lymphoid malignancy.

Specifically targeted expression of a toxin gene potentially represents a novel approach to cancer therapy. With a view to the ablation of B-cell malignancies, we have constructed a plasmid, designated pTHA71, which expresses the A-chain of diphtheria toxin (DT-A) with high efficiency and specificity in transfected, mature B-lymphoid cells. The construction incorporated immunoglobulin (Ig) kappa light chain gene regulatory sequences, including a kappa promoter, small intron, partial constant region exon, and 3'-flanking sequence (but lacking a known enhancer). These sequences conferred substantially more efficient expression of DT-A in mature B-cells than was seen from constructs that included only Ig promoters and enhancers. When transfected into the 70Z/3 murine pre-B-cell line, pTHA71 was only expressed efficiently if the cells were induced to express their endogenous, rearranged Ig kappa gene by prior exposure to lipopolysaccharide. The insertion of the enhancer from the Ig kappa large intron into pTHA71, generating pTHA81, did not markedly influence the level of DT-A expression in 70Z/3 cells. The observed absence of expression in pre-B-cells suggests that DT-A constructs similar to pTHA71 might be used for the therapeutic ablation of malignant B-cells of mature stages, while sparing normal progenitor cells.

Animals↗
PubMed2026

Controlling GRF4-GIF1 expression for efficient, genotype-independent transformation across wheat cultivars.

Wheat is a staple crop critical for global food security, and its continuous genetic improvement is essential to meet the demands of a growing population. Efficient, genotype-independent transformation is a major bottleneck in wheat functional genomics and gene editing. The growth regulating factor (GRF)-GRF-interacting factor (GIF) fusion technology enhances regeneration efficiency and broadens the range of transformable cultivars, but constitutive expression can reduce fertility and spikelet number. Here, we present an optimised Agrobacterium-mediated wheat transformation protocol incorporating GRF4-GIF1, tested across multiple tetraploid and hexaploid cultivars. Transformation efficiency was improved through adjustments in selection pressure, zeatin concentration, and promoter choice, with GRF4-GIF1 consistently enabling successful transformation across genotypes. Tissue-specific promoters and heat-inducible excision strategies effectively minimised pleiotropic effects, such as reduced fertility, while maintaining high transformation rates. This refined system provides a robust and versatile platform for gene function studies and gene editing, advancing genotype-independent wheat transformation and supporting breeding efforts to improve crop productivity, resilience, and nutritional value.

Triticum↗
PubMedDate not supplied

The pAS vector system and its application to heterologous gene expression in Escherichia coli.

There are numerous proteins of biological interest which cannot be obtained from natural sources in quantities sufficient for detailed biochemical and physical analysis. The limited bioavailability of these molecules has made it impossible to consider their potential utilization as either pharmacological agents and/or targets. One solution to this problem has been the development of recombinant vector systems which are designed to achieve efficient expression of cloned genes in a variety of biological systems. This paper will describe the development and application of a particular set of vectors which have been designed to achieve efficient expression of essentially any gene coding sequence in Escherichia coli. The system utilizes efficient phage-derived transcriptional and translational regulatory signals and provides a strong regulatable promoter, an antitermination mechanism to ensure efficient transcription across any gene insert, high stability and, when appropriate, efficient translation initiation information. In addition, a wide variety of host strains have been developed in order to help control, stabilize and maximize expression of various cloned genes. The system has now been used to express efficiently more than 75 different prokaryotic and eukaryotic gene products. The application of this system to the expression and characterization of several oncogene products will be described.

Bacteriophage lambda↗
PubMed1990

Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells.

Baculoviruses are currently used as vectors for the transient high-level expression of foreign gene products in insect cells. In this study, we demonstrate that baculoviruses can also be made to continuously express a foreign gene product by using the promoter from IE1, an immediate early viral gene, to produce stably-transformed insect cells. This approach gave levels of foreign gene expression lower than those usually obtained with the lytic baculovirus expression vector system. Expression, however, was continuous and stable, and a complex human glycoprotein (tissue plasminogen activator) was processed more efficiently. We conclude that stable transformation is a feasible approach for baculovirus-mediated foreign gene expression in lepidopteran cells, particularly for products that are relatively poorly-expressed and/or processed in lytically infected cells.

Animals↗
PubMed1983

Cloning of a LEU gene and an ARS site of Candida maltosa.

Gene libraries of DNA from an n-alkane-assimilating yeast strain, Candida maltosa IAM12247, were constructed, using Escherichia coli plasmid vector pBR322. A LEU gene from C. maltosa was cloned, and found to complement leu- mutations in E. coli and Saccharomyces cerevisiae. In E. coli, the LEU gene in the cloned yeast DNA fragment was efficiently expressed when inserted into the vector in one orientation, while in the other orientation, it was expressed only weakly. In S. cerevisiae, the Candida LEU gene was efficiently expressed when inserted into a shuttle vector pRC3 in both orientations, suggesting that the isolated Candida DNA fragment contains a promoter sequence of Candida in front of the LEU gene, which is operative in S. cerevisiae but not in E. coli. In addition, our data suggest that the cloned LEU fragment also contains an ARS (autonomously replicating sequence) site of C. maltosa.

Candida↗
PubMed1990

High efficiency transient expression of eukaryotic genes: use of an HSV-1 immediate early promoter (ICP4).

We have compared the transcriptional efficiencies of a number of eukaryotic promoters following DNA-mediated transfection into cultured rat hepatoma cells. We find that the highest levels of expression for the bacterial chloramphenicol acetyltransferase (CAT) reporter gene are observed with a herpes simplex virus type 1 (HSV-1) immediate early promoter when co-transfected with an expression construct bearing the gene for the HSV-1 transcriptional activator protein VP16. This transactivation phenomenon is specific for the HSV-1 immediate early promoter and increases the expression of the reporter gene 7-fold. Expression from the ICP4 promoter is 2.5-fold greater than the other promoters tested. In addition, expression from the ICP4 promoter can be induced, at varying times following transfection, by infecting the cells with HSV-1 viral particles. Two plasmids have been constructed which contain the HSV-1 ICP4 promoter adjacent to a multiple cloning site. One of the plasmids also contains SV40 splicing and polyadenylation signals.

Animals↗
PubMed1992

Promoter constructions for efficient secretion expression in Streptomyces lividans.

Promoters from different Streptomyces genes were cloned in front of the Tendamistat gene from S. tendae, in order to study secretion-expression in S. lividans using a pIJ702 plasmid vector system. Besides the promoters we cloned a transcriptional terminator downstream of the Tendamistat gene to improve transcription efficiency. The promoters we selected were: (1) a synthetic Escherichia coli-like consensus promoter; (2) the aphI promoter of the neomycin resistance gene from S. fradiae; (3) an ermE-up promoter mutant from Saccharopolyspora erythraea; (4) the melC promoter of the tyrosinase operon from Streptomyces antibioticus. In addition, we tested the thiostrepton-inducible tipA promoter from S. lividans in our Tendamistat secretion system. The promoters were cloned upstream of the Tendamistat ribosome binding site in order to conserve the original translation initiation. The Tendamistat secretion mediated by the different promoter constructions above varied dramatically in up to 10 mg/l in the case of the synthetic promoter and the aph promoter, and up to 500 mg/l mediated by the ermE-up promoter. The melC promoter allowed about 200 mg/l Tendamistat secretion and the tipA promoter proved to be inducible from less than 0.5 mg/l up to 40 mg/l of Tendamistat secretion. Based on the amount of secreted Tendamistat and on the analysis of mRNA levels, we conclude that transcriptional activity regulates the efficiency of our secretion-expression system.

Biotechnology↗
PubMed1985

Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene.

Amber, ochre and opal suppressor tRNA genes have been generated by using oligonucleotide directed site-specific mutagenesis to change one or two nucleotides in a human serine tRNA gene. The amber and ochre suppressor (Su+) tRNA genes are efficiently expressed in CV-1 cells when introduced as part of a SV40 recombinant. The expressed amber and ochre Su+ tRNAs are functional as suppressors as demonstrated by readthrough of the amber codon which terminates the NS1 gene of an influenza virus or the ochre codon which terminates the hexon gene of adenovirus, respectively. Interestingly, several attempts to obtain the equivalent virus stock of an SV40 recombinant containing the opal suppressor tRNA gene yielded virus lacking the opal suppressor tRNA gene. This suggests that expression of an efficient opal suppressor derived from a human serine tRNA gene is highly detrimental to either cellular or viral processes.

Animals↗
PubMed1988

Bovine papillomavirus E2 gene regulates expression of the viral E5 transforming gene.

We have performed transient-expression experiments with CV1 monkey kidney cells to investigate the role of the bovine papillomavirus type 1 (BPV1) E2 gene in the regulation of the E5 transforming gene. Direct analysis of the 7-kilodalton open reading frame (ORF) E5 protein and measurements of the expression of an E5-chloramphenicol acetyltransferase fusion protein indicate that the efficient expression of ORF E5 requires the full-length E2 gene, which can be supplied in trans. The viral long control region is required in cis for this response to ORF E2, and it acts in a position- and orientation-independent fashion characteristic of a transcriptional enhancer. Deletion analysis suggests that the P2443 promoter is required for efficient expression of the E5 gene. The E2 repressor activity encoded in the 3' end of the E2 gene inhibits the expression of ORF E5. These effects define a major BPV1 regulatory circuit and appear to explain the transformation behavior of a variety of BPV1 mutants.

Acetyltransferases↗
PubMed1992

Chimeric prion protein expression in cultured cells and transgenic mice.

The efficient expression of exogenous prion protein (PrP) molecules in mouse neuroblastoma cells that are chronically infected with murine scrapie prions (ScN2a cells; Butler, D.A., et al., 1988, J. Virol. 62, 1558-1564) and in transgenic mice is described. This technology allows investigation of the PrP molecule for structural regions involved in determining species specificity, as well as ablation experiments designed to address the functionality of particular regions of the PrP molecule. Previous reports demonstrated that the PrP gene specifies the host range for susceptibility of transgenic animals to prions (Scott, M., et al., 1989, Cell 59, 847-857; Prusiner, S.B., et al., 1990, Cell 63, 673-686). Consistent with these results, we showed that Syrian hamster (SHa) PrP is ineligible for efficient conversion to PrPSc in ScN2a cells. By constructing a series of chimeric mouse (Mo)/SHaPrP genes, we developed an epitopically tagged functional variant of the MoPrP gene, which can efficiently form protease-resistant PrP molecules upon expression in ScN2a cells. The presence of a defined epitope for an SHa-specific monoclonal antibody allows the products of this chimeric gene to be discriminated from endogenous MoPrP and creates a useful reagent for exploring structure/function relationships via targeted mutagenesis. In addition, we developed a transgenic mouse expression vector by manipulation of an SHaPrP cosmid clone. This vector permits the efficient expression of foreign PrP genes in the brains of transgenic animals, enabling pathological consequences of in vitro mutagenesis to be studied.

Animals↗
PubMed1992

The Tn10-encoded tetracycline resistance mRNA contains a translational silencer in the 5' nontranslated region.

We performed a mutational analysis of the left half of Tn10-encoded tet operator O2, located in the 5' nontranslated region of the mRNA for the resistance protein TetA, and determined the importance of that region for translation efficiency and mRNA stability. Transcriptional fusions of 17 mutants to lacZ expressed the same amounts of beta-galactosidase, while translational fusions varied 35-fold in expression efficiency. The mRNA half-lives varied 24-fold, with 9.6 min for the most highly expressed mRNA and 0.4 min for the least efficiently expressed mRNA. Toeprint experiments were performed to distinguish whether these mutations define a determinant of mRNA stability or influence translation initiation. The highly expressed mRNA was 24-fold more efficient in forming the initiation complex in vitro than the low-expression mutant. It was concluded that this sequence, albeit located upstream of the ribosome-binding sequence, is an important determinant for efficient initiation of translation. Secondary-structure calculations of the mRNAs revealed no correlation of the potential to form double strands masking the ribosome-binding sequence with expression efficiency.

Base Sequence↗
PubMed1991

Targeting expression to the mammary gland: intronic sequences can enhance the efficiency of gene expression in transgenic mice.

We are studying the tissue-specific expression of the sheep milk-whey protein gene, beta-lactoglobulin. We have used sequences derived from this gene to target the expression of biomedical proteins into milk with the intention to exploit this technology in transgenic sheep as a means of protein production. In the present study, a series of beta-lactoglobulin hybrid genes and beta-lactoglobulin minigenes were evaluated for expression in the mammary gland of transgenic mice. In particular, we have assessed whether there is a requirement for introns for efficient transgene expression in the mammary gland, since the coding sequences of many candidate proteins are available only as cDNAs. The results suggest that the inclusion of natural introns in constructs can enhance the efficiency of transgene expression. Thus, a hybrid construct comprising 4.3 kb of the immediate 5' flanking sequences of beta-lactoglobulin fused to a genomic minigene encoding human alpha-antitrypsin (alpha 1AT) was expressed much more efficiently than an alpha 1AT-cDNA construct containing the same beta-lactoglobulin segment. Similarly, the intact beta-lactoglobulin gene was expressed more efficiently than the corresponding intronless beta-lactoglobulin minigene. This effect was not seen in transient expression experiments in baby hamster kidney cells when beta-lactoglobulin-alpha 1AT constructs were driven by SV40 enhancer sequences. The effect cannot be explained by a simple requirement for splicing, since the inclusion of the first beta-lactoglobulin intron into cDNA constructs encoding human alpha 1AT or beta-lactoglobulin itself failed to enhance the efficiency of transgene expression. It is concluded that sequence elements within introns may interact with the upstream 5' flanking sequences of beta-lactoglobulin and enable the latter to function efficiently in the mammary gland of transgenic mice.

Animals↗
PubMedDate not supplied

Construction of a broad host range shuttle vector for gene cloning and expression in Actinobacillus pleuropneumoniae and other Pasteurellaceae.

We have constructed a pair of broad host range expression vectors, pJFF224-NX and pJFF224-XN, based on plasmid RSF1010, which enable cloning and efficient expression of genes in Actinobacillus pleuropneumoniae and Pasteurella haemolytica and in Escherichia coli. The vectors consist of the minimal autonomous replicon of the broad host range plasmid RSF1010 and a type II chloramphenicol acetyl transferase gene for chloramphenicol resistance selection. In addition, they contain a gene expression cassette based on the E. coli bacteriophage T4 gene 32 promoter region and a transcription stop signal, which are separated by a segment of multiple cloning sites in both orientations. Electroporation and subsequent selection for chloramphenicol resistance was used for the introduction of the vectors in A. pleuropneumoniae and P. haemolytica. A promoterless xy/E gene from the Pseudomonas putida TOL plasmid was cloned onto pJFF224-NX. This plasmid enabled efficient expression of active catechol2,3oxygenase in A. pleuropneumoniae and P. haemolytica. It was stably maintained in A. pleuropneumoniae without antibiotic selection, showing less than 0.1% loss after 100 generations, while native RSF1010 and other RSF1010-based vectors were unstable in this host.

Actinobacillus pleuropneumoniae↗
PubMed1984

Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins.

Construction of a family of bacterial expression vectors, pEX1-3, is described. These vectors are derived from a cro-lacZ gene fusion plasmid which expresses large quantities of fusion protein under the control of the PR promoter of bacteriophage lambda. A polylinker has been engineered into the 3' end of the lacZ gene in all three translational reading frames, and stop signals for transcription and translation inserted, so that any open reading frame DNA may be expressed as a hybrid beta-galactosidase protein. cDNA fragments cloned in these vectors can be detected with an efficiency of greater than 1 in 3, thus enabling the detection of rare cDNA molecules. In addition, the low solubility of hybrid proteins leads to a rapid isolation procedure allowing antibodies of pre-determined specificity to be made against expressed regions of cloned DNA. We describe the cloning of albumin and complement C9 genes from a human cDNA library using polyclonal and monoclonal antibodies.

Bacteriophage lambda↗
PubMed1983

Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae.

We have constructed a high-efficiency expression vector to direct the synthesis of heterologous polypeptides in yeast. The vector is termed a sandwich expression vector as the heterologous gene is inserted between the 5' and 3' control regions of the efficiently expressed yeast PGK gene. We have used this vector to direct the expression of three derivatives of the calf chymosin cDNA gene; preprochymosin, prochymosin and chymosin. Prochymosin is synthesised to at least 5% of total yeast-cell protein and furthermore, it can be readily activated to produce an enzyme which has milk-clotting activity.

Animals↗
PubMed1988

Cloning of a LEU gene and an ARS site of Rhodotorula glutinis.

Genomic library of DNA from an extracellular protease-producing yeast strain, Rhodotorula glutinis K-24, was constructed, using Escherichia coli plasmid vector PBR322. A LEU gene from R. glutinis was cloned, and found to complement leu- mutations in E. coli and Saccharomyces cerevisiae. In E. coli, the LEU gene in the cloned yeast DNA fragment was efficiently expressed when inserted into the vector in one orientation. In S. cerevisiae, the R. glutinis LEU gene was efficiently expressed when inserted into a shuttle vector YEP13 in both orientations, suggesting that the isolated R. glutinis DNA fragment contains a promotor sequence of R. glutinis in front of the LEU gene. In addition, our data suggests that the cloned LEU fragment also contains an ARS (autonomously replication sequence) site of R. glutinis that could function in S. cerevisiae.

Blotting, Southern↗