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 1,171 records · Page 65Linked to original sources
PubMed2026

Research note: Development of a recombinant duck enteritis virus vector expressing DHAV-3 VP1 and DTMUV prM/TE genes.

Duck enteritis virus (DEV) is a promising viral vector for vaccine development. In a previous study, an HDR-CRISPR/Cas9-based strategy was used to generate a recombinant virus, rDEV-DHAV-VP1, by inserting the VP1 gene of duck hepatitis A virus type 3 (DHAV-3) into the UL27/UL26 intergenic region of DEV vaccine strain, resulting in good genetic stability and immunogenicity. In the present study, the same strategy was applied to insert the EGFP gene into the US7/US8 and LORF11/UL55 intergenic regions of a DEV vaccine strain. Among the evaluated insertion sites, the highest level of EGFP expression was observed at the US7/US8 locus, followed by the UL27/UL26 locus. Based on rDEV-DHAV-VP1, the pre-membrane (prM) and truncated envelope (TE) genes of duck Tembusu virus (DTMUV) were further inserted into the US7/US8 locus, resulting in a bivalent recombinant virus, rDEV-VP1-prM/TE. The recombinant virus exhibited growth kinetics comparable to those of the parental virus, while maintaining efficient expression and high genetic stability of the inserted genes. These findings indicate that the HDR-CRISPR/Cas9 system is an efficient strategy for generating stable DEV-based recombinant vectors and provides a promising platform for the development of multivalent vaccines against major duck viral diseases.

CRISPR/Cas9 genome editing↗
PubMed1979

Prolactin-mediated transcriptional and post-transcriptional control of casein gene expression.

The mechanism by which prolactin, a peptide hormone, regulates casein gene expression has been studied in mammary gland organ culture. After prolactin addition, a 2-4 fold increase in the rate of casein mRNA transcription was observed within 1 hr and maintained for at least 24 hr. This increased rate of transcription is not sufficient to account for the mass accumulation of casein mRNA. The half-life of casein mRNA is also increased 17-25 fold in the presence of prolactin. This change in casein mRNA half-life, coupled with a 2-4 fold increase in the rate of transcription, can account for the normal accumulation of casein mRNA observed after prolactin addition. This hormone-induced change in casein mRNA half-life appeared to be selective, since prolactin was found to exert only a slight effect (1-4 fold) on the half-life of poly(A) RNA determined under identical pulse-chase conditions. The hormonal regulation of casein gene expression thus does not app-ar to be an "all or none" process occurring only at the transcriptional or post-transcriptional levels, but rather may involve a coordinated response at several levels to permit the efficient expression of specialized differentiated functions.

Animals↗
PubMed2003

Design of catabolic cassettes for styrene biodegradation.

A broad-host range metabolic cassette has been designed that, under the control of the Ptac promoter, expresses the sytABCD catabolic genes from Pseudomonas sp. Y2, which are responsible for the transformation of styrene into phenylacetic acid (styrene upper pathway). This novel cassette confers to phenylacetic acid-degrading bacteria the ability to grow efficiently on styrene as the sole carbon and energy source. By combining both the sty cassette and the archetypal pWW0 TOL plasmid into the well-known Pseudomonas putida F1 aromatic biodegrader, we have constructed a novel derivative strain that shows one of the largest degradative potentials so far described for aromatic hydrocarbons, because it is able to use BTEX compounds (benzene, toluene, ethylbenzene and xylenes) and styrene as a source of carbon and energy. Furthermore, the sty cassette was engineered within a mini-transposon and endowed with a gene containment system, based on the toxic effect of the colicin E3 RNase, to reduce its lateral spread to other hosts. This contained cassette lacks defined transcriptional regulatory signals and, thus, it becomes an alternative strategy to select recombinant strains that efficiently express the desired phenotype from housekeeping regulatory elements.

Bacteria↗
PubMed1978

Photodynamic activity of dyes with different DNA binding properties I. free-radical induction in DNA.

The photosensitizing efficiencies of eight dyes have been compared; two acridines, two xanthene derivatives, one sulphur-containing dye and three chemotherapeutic agents. The analysed reaction was the photosensitized induction of free radicals in calf-thymus DNA at low temperature. The binding of these dyes to DNA was first measured. Both strong (process I) and weak (process II) binding, with different intensities, either alone or together, were observed as mode of fixation. Whatever the nature of their binding, all the dyes used revealed a photosensitizing power as inducers of peroxide radicals in DNA. Their relative efficiencies, expressed as a function of the amount of dye molecules bound to DNA, were found to be very different. Intercalation, however, appeared to favour the free-radical induction as the first strongly bound molecules were more efficient.

Acridines↗
PubMed1997

Mammalian cDNA and prokaryotic reporter sequences silence adjacent transgenes in transgenic mice.

The ovine beta-lactoglobulin gene is expressed efficiently and at high levels in the mammary gland of transgenic mice. In contrast, when this gene is linked to a second gene construct comprising a mammalian cDNA or a CAT reporter sequence it fails to be expressed in the majority of transgenic lines generated. We suggest that mammalian cDNAs and prokaryotic reporter sequences can serve as active foci for gene silencing in the mammalian genome.

Animals↗
PubMed1987

IS2 activates the ilvA gene of Pseudomonas cepacia in Escherichia coli.

The ilvA gene of Pseudomonas cepacia was expressed poorly in Escherichia coli. Insertion of IS2 upstream of the cloned gene dramatically increased its transcription, resulting in an 85-fold increase in threonine dehydratase (deaminase) specific activity. The results confirm earlier reports that IS2 promotes efficient expression of foreign genes in E. coli.

Cloning, Molecular↗
PubMed2005

Construction of prokaryotic expression system of TGF-beta1 epitope gene and identification of recombinant fusion protein immunity.

AIM: To insert the constructed TGF-beta(1) epitope gene into the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-beta(1) antigenicity in its prokaryotic expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti-TGF-beta(1) vaccine. METHODS: The TGF-beta(1) encoding epitope gene (the mature TGF-beta(1) from 78-109 amino acid residues, TGF-beta(1)(32)) was amplified by polymerase chain reaction from the recombinant pGEM-7z/ TGF-beta(1)(32) vector. The HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-beta(1)(32) and HBcAg89-144 into restrictive endonuclease enzyme and ligated with T(4) ligase. The fusion gene fragments HBcAg1-71-TGF-beta(1)(32)- HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E. coli BL21 (DE3) under induction of IPTG. After purification with Ni(+2)-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope. RESULTS: Enzyme digestion analysis and sequencing showed that TGF-beta(1) epitope gene was inserted into the el loop of C-terminus of truncated hepatitis B core antigen. SDS-PAGE analysis showed that relative molecular mass (Mr) of the expressed product by pET28a (+)/CTC was Mr 24,600. The output of the target recombinant protein was approximately 34.8% of the total bacterial protein, mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-beta(1) polyclonal IgG but not with anti-HBcAg. The purity of protein was about 90% and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-beta(1) antigenicity and could be used as anti-TGF-beta(1) vaccine. CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF-beta(1) epitope gene was successfully established. The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-beta(1). The expressed TGF-beta(1) epitope gene shows good immunogenicity and antigenicity.

Epitopes↗
PubMed1999

Clinical aspects of intratumoral gene therapy.

One of the major obstacles to the development of gene therapy for cancer is our inability to deliver genes to all targets within the body. Thus, effective methodology does not exist to deliver a gene intravenously with the expectation that it will selectively localize within the target tumor, will not localize in other tissues and will be expressed efficiently. While one can take advantage of tissue-specific promoters to activate the gene only in a given target tissue, only a small fraction of the vector will be taken up in the target tissue and expressed. Consequently, since accessible local or regional tumor masses are a major problem in many cancers, there has been a strong emphasis on clinical trials in intratumoral and peritumoral gene delivery.

Adenoviruses, Human↗
PubMed2004

[The anti-HBV effect and mechanism of C gene truncated mutant in vitro].

OBJECTIVE: To explore the effect and mechanism on HBV replication in C gene truncated mutant. METHODS: Protein expression of C gene truncated vector and wild C gene vector were assay by SDS-PAGE Western blot. Constructed C gene truncated expression vector was cotransfected with wild HBV genome; virus load was detected by PCR in the culture medium and the cell. The formation of core particle was assay by Native western blot. RESULTS: The recombinant vectors can efficiently express. Virus load of the cotransfected group by pcDNA3-deltaC and adwR9 was lower than that of control group in the culture medium and the cell. Protein band of the co-expressed group by pcDNA3-deltaC and pcDNA3-C showed slightly weaker than that of the co-expressed group by pcDNA3 and pcDNA3-C. CONCLUSION: C gene truncated mutant could interfere with the formation of core particle and reduce of HBV replication

Cell Line↗
PubMedDate not supplied

Alphaviruses as tools in neurobiology and gene therapy.

The broad host range and superior infectivity of alphaviruses have encouraged the development of efficient expression vectors for Semliki Forest virus (SFV) and Sindbis virus (SIN). The generation of high-titer recombinant alphavirus stocks has allowed high-level expression of a multitude of nuclear, cytoplasmic, membrane-associated and secreted proteins in a variety of different cell lines and primary cell cultures. Despite the viral cytopathogenic effects, functional assays on recombinant proteins are possible for a time-period of at least 24 hours post-infection. The high percentage (80-95%) of primary neurons infected with SFV has allowed localization and functional studies of recombinant proteins in these primary cell cultures. Through multiple infection studies the interaction of receptor and G protein subunits has become feasible. Establishment of efficient scale-up procedures has allowed production of large quantities of recombinant protein. Potential gene therapy applications of alphaviruses could be demonstrated by injection of recombinant SIN particles expressing beta-galactosidase into mouse brain. Tissue/cell specific infection has been achieved by introduction of an IgG-binding domain of protein A domain into one of the spike proteins of SIN. This enabled efficient targeting of infection to human lymphoblastoid cells.

Alphavirus↗
PubMed1999

Differential expression of Listeria monocytogenes virulence genes in mammalian host cells.

We have used RT-PCR and GFP-mediated fluorescence to analyse the regulation of PrfA-dependent virulence genes of Listeria monocytogenes during proliferation in mammalian host cells. Our data show that most of the PrfA-regulated virulence genes are more efficiently expressed, as measured by transcript levels, when L. monocytogenes is grown in macrophages and macrophage-like cells rather than in epithelial cells, hepatocytes or endothelial cells. The promoters for hly and plcA are predominantly activated within the phagosomal compartment, while those for actA and inlC are predominantly activated in the host cell cytosol. Expression of actA and plcB precedes that of inlC after infection of epithelial cells and macrophages. Little transcription of inlA or inlB is observed in epithelial cells and there is only slightly more in macrophages. In both cell types the level of transcription of the inlAB operon is lower than is seen under extracellular growth conditions in rich media, which is compatible with the assumption that InlA and InlB are not required during intracellular growth of the bacteria. Activation of the PrfA-independent iap promoter is also low during intracellular growth, although the gene product (p60) is required for cell viability. The levels of the PrfA-dependent virulence gene transcripts do not correlate with the amount of prfA transcript present, which is low under all intracellular conditions analysed, suggesting that the prfA transcript is either highly unstable in bacteria that are growing intracellularly, or that the small amount of PrfA produced is highly activated by additional component(s).

Animals↗
PubMed1987

Characterization of glucosyltransferase expressed from a Streptococcus sobrinus gene cloned in Escherichia coli.

The gene encoding a glucosyltransferase which synthesized water-insoluble glucan, gtfI, previously cloned from Streptococcus sobrinus strain MFe28 (mutans serotype h) into a bacteriophage lambda vector, was subcloned into the plasmid pBR322. The recombinant plasmid was stable in Escherichia coli and gtfI was efficiently expressed. The GTF-I expressed in E. coli was compared to the corresponding enzymes in S. sobrinus strains MFe28 (serotype h), B13 (serotype d) and 6715 (serotype g) and shown to resemble them closely in molecular mass and isoelectric point. The insoluble glucan produced by GTF-I from recombinant E. coli consisted of 1,3-alpha-D-glycosyl residues (approximately 90%). An internal fragment of the gtfI gene was used as a probe in hybridization experiments to demonstrate the presence of homologous sequences in chromosomal DNA of other streptococci of the mutans group.

Cloning, Molecular↗
PubMed1999

Reduction of syndecan-1 mRNA in cervical-carcinoma cells is involved with the 3' untranslated region.

Syndecan-1 is a transmembrane proteoglycan expressed predominantly in epithelial cells. Studies with immunohistochemistry have shown that syndecan-1 expression is reduced in carcinoma derived from human epidermis. Here we show that syndecan-1 mRNA, which is abundant in human primary keratinocyte (HK) and HaCaT spontaneous immortalized keratinocyte, is decreased in cervical-carcinoma cell lines. Further, in relation to a long and well-conserved 3' untranslated region (3' UTR) of syndecan-1 cDNA, we examined whether 3' UTR is involved with syndecan-1-mRNA reduction in cervical-carcinoma cells. A stable transfection experiment showed that addition of the 3' UTR does not affect expression in HaCaT, but that syndecan-1 cDNA containing the 3' UTR is not expressed efficiently selectively in cervical-carcinoma cell lines. The transient assay with CAT reporter plasmids linking the 3' UTR confirmed this, and indicated that the 3' end of the 3' UTR (nt 2285-2410) is required to influence expression in cervical-carcinoma cells. Further excessive expression of syndecan-1 suppressed growth in cervical-carcinoma cells. These results demonstrate that the reduction of syndecan-1 mRNA involved with the 3' untranslated region gives growth advantage to cervical-carcinoma cells.

Amino Acid Sequence↗
PubMed2003

Gene expression within cell-sized lipid vesicles.

Functional protein synthesis was observed in cell-sized lipid vesicles following encapsulation of a gene-expression system. Expression of rsGFP (red-shifted green fluorescent protein) within individual vesicles was observed by fluorescence microscopy. Interestingly, at the early stage of the reaction, the expression efficiency inside the vesicle was remarkably higher than that in the solution outside. The synthesized rsGFP in individual vesicles is safe from attack by proteinase K added to the external aqueous solution. Studies on cell-sized vesicles expressing protein should contribute to a fundamental understanding of certain aspects of living systems and will be useful for practical applications, such as the construction of microreactors.

Endopeptidase K↗
PubMed2001

Functional comparison of the alpha3A and alpha3B cytoplasmic domain variants of the chicken alpha3 integrin subunit.

Integrin alpha3beta1 can be alternatively spliced to generate alpha3A and alpha3B cytoplasmic domain variants that are conserved among vertebrates. To identify distinct functions of these variants, we transfected cells with intact alpha3 integrins or chimeric receptors. alpha3Abeta1 and alpha3Bbeta1 each localized to focal contacts in keratinocytes on an extracellular matrix rich in laminin-5, to which both are known to bind with high affinity. However, alpha3B accumulated intracellularly in keratinocytes on collagen, suggesting that laminin binding may stabilize alpha3Bbeta1 surface expression. Neither alpha3 cytoplasmic domain affected recruitment of chimeric alpha5 integrins to fibronectin-induced focal contacts, and either substituted for the alpha5 cytoplasmic domain in alpha5beta1-mediated cell migration. However, the alpha5/alpha3B chimera localized to cell-cell borders in MDCK or CHO cells to a lesser extent than did the alpha5/alpha3A chimera. To determine whether the alpha3 cytoplasmic domains conferred distinct localization to a nonintegrin protein, we transfected cells with interleukin-2 receptor (IL-2R) chimeras containing the alpha3 cytoplasmic domains. The IL-2R/alpha3A chimera was expressed efficiently on the cell surface, while the IL-2R/alpha3B chimera accumulated intracellularly. Our findings suggest that the alpha3B cytoplasmic domain harbors a retention signal that is regulated in an intact integrin and can alter cell surface expression and distribution of alpha3beta1.

Alternative Splicing↗
PubMed2005

Regulation of the nap operon encoding the periplasmic nitrate reductase of Paracoccus pantotrophus: delineation of DNA sequences required for redox control.

Expression of the nap operon, encoding the periplasmic nitrate reductase in Paracoccus pantotrophus, is maximal when cells are grown aerobically, but not anaerobically, with butyrate. Two promoters, termed P1 and P2, control operon expression and the operon-proximal P2 promoter is primarily responsible for increased nap expression in the presence of butyrate. A near-perfect palindromic sequence is centred at +7, relative to the P2 transcription start site. Mutation of this palindrome demonstrated that it is important for regulation of nap operon expression in response to both the redox and the oxidation state of the carbon substrate. A 5' deletion analysis of the nap promoter fused to lacZ revealed that full redox control of expression was retained when the DNA sequence up to position -49 bp, relative to the operon-distal P1 transcription start site, was removed. Encroaching beyond this position resulted in an approximately 4-fold reduction in expression when cells were grown aerobically with butyrate. Additionally, point mutations at position -38 and -45 relative to P1 also resulted in a reduction in expression during aerobic growth with butyrate. A GC-rich region of nap promoter DNA, centred on position -41 relative to the P1 transcription start site is thus proposed as a second DNA motif that is important for efficient expression of the nap operon.

Bacterial Proteins↗
PubMed1987

Identification of a promoter sequence in the plasmid pUL340 of Brevibacterium lactofermentum and construction of new cloning vectors for corynebacteria containing two selectable markers.

A strong promoter P1 has been found in plasmid pUL340, a cloning vector used to transform corynebacteria. This promoter is also expressed efficiently in Escherichia coli. A gene (cat) for chloramphenicol acetyltransferase from Streptomyces acrimycini and a gene (hyg) for hygromycin phosphotransferase from Streptomyces hygroscopicus were subcloned in different positions of the Brevibacterium lactofermentum plasmid pUL340. Both resistance genes are expressed in B. lactofermentum from their own promoters or from the endogenous promoter in pUL340. These genes provide useful screening markers for selecting transformants of B. lactofermentum together with the kanamycin-resistance gene from the transposon Tn5.

Brevibacterium↗
PubMed2000

Transcription factor BETA2 acts cooperatively with E2A and PDX1 to activate the insulin gene promoter.

The insulin gene is efficiently expressed only in pancreatic beta cells. Using reverse transcriptase-polymerase chain reaction analysis, we show that insulin mRNA levels are at least 10(5)-fold higher in beta cells than non-beta cells. To examine the underlying mechanisms, we expressed beta cell transcription factors by transfection of non-beta cells. Separate expression of BETA2, E2A, or PDX1 led to modest (<10-fold) activation of the insulin promoter, whereas co-expression of the three proteins produced synergistic, high level activation (160-fold). This level of activity is approximately 25% that observed in transfected beta cell lines. Of the three factors studied, BETA2 appears to play a dominant role. Efficient transcription required a C-terminal activation domain of BETA2 and an N-terminal region, which does not function as an independent activation domain. The myogenic basic helix-loop-helix (bHLH) protein MyoD was unable to bind and activate the promoter, even when its DNA binding region was replaced with that of BETA2. Our results demonstrate the central importance of BETA2 in insulin gene transcription and the importance of sequences outside the canonical DNA binding domain in permitting efficient DNA binding and cell-specific activity of the insulin gene promoter.

Animals↗