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Results for “neutral integration site”

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At least 19 recordsLinked to original sources

PubMed2026

A CRISPR-Cas9 Toolkit Enabling Tunable Integration and Transient Homologous Recombination Enhancement in Yarrowia lipolytica.

Although the oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory, its application remains constrained by inefficient homology-directed repair (HDR) and a lack of precise genomic integration tools. To address these limitations, we developed a comprehensive genetic toolkit featuring three synergistic advancements. First, we systematically identified 55 neutral integration sites with tunable expression profiles, enabling stable, position-independent gene integration with predictable transcriptional output across a 12.88-fold dynamic range. Second, we established a dual-readout high-throughput screening platform combining colony morphology analysis with hrGFP fluorescence. This approach accurately measures locus-specific homologous recombination (HR) efficiency while eliminating false positives by dominant non-homologous end joining (NHEJ). Third, we engineered a transient HR enhancement system by fusing the Sae2 exonuclease to Cas9 via a flexible (GGGGS)3 linker. This fusion significantly boosts HR efficiency and surpasses the cleavage activity of unmodified Cas9 without introducing permanent genomic modifications or compromising cellular fitness. Finally, HR efficiency for single-gene integration was increased from 46.5% to 77.5% while the dual-locus editing efficiency reached 64.1% when using 500-bp homology arms, and the engineered strains demonstrated improved genetic stability compared to those with constitutive HR enhancement.

Yarrowia↗

PubMed1997

Recombinant adeno-associated virus for muscle directed gene therapy.

Although gene transfer with adeno-associated virus (AAV) vectors has typically been low, transduction can be enhanced in the presence of adenovirus gene products through the formation of double stranded, non-integrated AAV genomes. We describe the unexpected finding of high level and stable transgene expression in mice following intramuscular injection of purified recombinant AAV (rAAV). The rAAV genome is efficiently incorporated into nuclei of differentiated muscle fibers where it persists as head-to-tail concatamers. Fluorescent in situ hybridization of muscle tissue suggests single integration sites. Neutralizing antibody against AAV capsid proteins does not prevent readministration of vector. Remarkably, no humoral or cellular immune responses are elicited to the neoantigenic transgene product E. coli beta-galactosidase. The favorable biology of rAAV in muscle-directed gene therapy described in this study expands the potential of this vector for the treatment of inherited and acquired diseases.

Animals↗

PubMed2000

Construction of promoter probe vectors for Synechocystis sp. PCC 6803 using the light-emitting reporter systems Gfp and LuxAB.

Two promoter probe vectors were constructed for the cyanobacterium Synechocystis sp. strain PCC 6803 using reporter genes, which can be easily detected and quantified in vivo by the ability of their encoded proteins to emit light. The vectors allow the transcriptional fusion of promoter sequences with the gfp and luxAB genes, respectively, and their stable integration into a neutral site of the Synechocystis chromosome. Functionality of these vectors was demonstrated by cloning the promoter of the isiAB operon into both promoter probe vectors and analyzing the stress-dependent emission of light by the obtained reporter strains. As was found before for the isiAB operon, the P(isiAB) reporter gene fusions were induced by iron starvation and high salt stress. Induction rates of mRNA of the wild type operon and the reporter gene fusions were found to be essentially the same, indicating that a promoter fragment containing all necessary regulatory elements has been cloned. However, using the gfp gene a slow increase of protein and fluorescence was found, while the luxAB reporter gene constructs led to a rapid increase in luminescence. The same was found after retransfer of cells back into control media, in which the Gfp protein disappeared slowly, while the LuxAB-based luminescence decreased rapidly. These experiments show that both reporter genes can be used in Synechocystis: the luxAB system seems to be favourable regarding reaction time, while the gfp system has the advantage of being independent from any substrate.

Aldehydes↗

PubMed2000

The Escherichia coli heat shock protein ClpB restores acquired thermotolerance to a cyanobacterial clpB deletion mutant.

In both prokaryotes and eukaryotes, the heat shock protein ClpB functions as a molecular chaperone and plays a key role in resisting high temperature stress. ClpB is important for the development of thermotolerance in yeast and cyanobacteria but apparently not in Escherichia coli. We undertook a complementation study to investigate whether the ClpB protein from E coli (EcClpB) differs functionally from its cyanobacterial counterpart in the unicellular cyanobacterium Synechococcus sp. PCC 7942. The EcClpB protein is 56% identical to its ClpB1 homologue in Synechococcus. A plasmid construct was prepared containing the entire E coli clpB gene under the control of the Synechococcus clpB1 promoter. This construct was transformed into a Synechococcus clpB1 deletion strain (deltaclpB1) and integrated into a phenotypically neutral site of the chromosome. The full-length EcClpB protein (EcClpB-93) was induced in the transformed Synechococcus strain during heat shock as well as the smaller protein (EcClpB-79) that arises from a second translational start inside the single clpB message. Using cell survival measurements we show that the EcClpB protein can complement the Synechococcus deltaclpB1 mutant and restore its ability to develop thermotolerance. We also demonstrate that both EcClpB-93 and -79 appear to contribute to the degree of acquired thermotolerance restored to the Synechococcus complementation strains.

Cyanobacteria↗

PubMed1988

Expression of neutral endopeptidase (enkephalinase) in heterologous COS-1 cells. Characterization of the recombinant enzyme and evidence for a glutamic acid residue at the active site.

Neutral endopeptidase (EC 3.4.24.11) is an integral membrane protein found in the plasma membrane of many cell types. The cDNA coding for the complete primary structure of neutral endopeptidase has recently been cloned and sequenced (Devault, A. Lazure, C., Nault, C., Le Moual, H., Seidah, N. G., Chretien, M., Kahn, P., Powell, J., Mallet, J., Beaumont, A., Roques, B. P., Crine, P., and Boileau, G. (1987) EMBO J. 6, 1317-1322). Comparison of the sequence of neutral endopeptidase with that of thermolysin, a bacterial Zn-metalloendopeptidase, suggests that Glu-584 in neutral endopeptidase probably corresponds to Glu-143 in thermolysin, which is an essential amino acid involved in catalysis. To test directly the importance of Glu-584 in the catalytic activity of neutral endopeptidase by site-directed metagenesis, we have constructed an expression vector in which the rabbit kidney cDNA encoding the entire neutral endopeptidase sequence is introduced downstream from the SV40 virus early promotor. After transfection in COS-1 monkey kidney cells, this vector was found to promote the expression of a protein with biochemical and catalytic properties identical to kidney neutral endopeptidase. Oligonucleotide-directed mutagenesis of Glu-584 to either valine or aspartic acid completely abolished the enzymatic activity of the recombinant protein without changing its affinity for the substrate-related tritiated inhibitor [3H]N-[(2R,2S)-3-hydroxyamino-carbonyl-2-benzyl-1-oxopropyl]-glycine. This observation clearly identifies Glu-584 as one of the important residues responsible for the catalytic activity of the enzyme.

Animals↗

PubMed1996

Molecular genetic methods for improving secondary-metabolite production in actinomycetes.

The practical applications of genetic engineering to improve secondary-metabolite production in actinomycetes are potentially numerous, but have been limited by: (1) restriction barriers, which can hinder the introduction of DNA into many actinomycetes; (2) self-replicating plasmid-cloning vectors, which generally inhibit secondary-metabolite production; and (3) recombinants containing heterologous DNA, which may be subject to additional regulatory hurdles. Recently, intergeneric conjugation has been used to circumvent host restriction, and integration of cloned DNA into neutral genomic sites prevents product inhibition by self-replicating plasmids, and has enabled construction of recombinant strains lacking heterologous DNA sequences. The rpsL system permits direct selection for gene replacements and gene insertions that can facilitate this process.

Actinomycetales↗

PubMed1995

Characterization of cis elements that regulate the expression of glnA in Synechococcus sp. strain PCC 7942.

The upstream noncoding region of the Synechococcus sp. strain PCC 7942 (hereafter referred to as Synechococcus 7942) glnA gene was fused to the cat gene in order to study the expression of glnA both in Synechococcus 7942 and in Escherichia coli. The lack of cat expression in E. coli indicated that the glnA promoter was not recognized by E. coli RNA polymerase. The fused construct was integrated into the Synechococcus 7942 chromosome at a neutral site. Expression of the cat reporter gene was regulated under various nitrogen conditions in a way similar to that of the glnA gene. A deletion introduced at the binding site of the NtcA regulatory protein abolished derepression of the glnA promoter during growth in nitrate and under nitrogen starvation. Deletion of the sequence between the transcription and translation start sites of glnA prevented the repression observed during growth in ammonium. These results indicate that the glnA promoter is subject to complex regulation that involves sequences upstream and downstream from the transcription start site.

Base Sequence↗

PubMed1997

Analysis of the interaction of the human immunodeficiency virus type 1 gp120 envelope glycoprotein with the gp41 transmembrane glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein interacts with the viral receptor (CD4) and with the gp41 transmembrane envelope glycoprotein. To study the interaction of the gp120 and gp41 envelope glycoproteins, we compared the abilities of anti-gp120 monoclonal antibodies to bind soluble gp120 and a soluble glycoprotein, sgp140, that contains gp120 and gp41 exterior domains. The occlusion or alteration of a subset of gp120 epitopes on the latter molecule allowed the definition of a gp41 "footprint" on the gp120 antibody competition map. The occlusion of these epitopes on the sgp140 glycoprotein was decreased by the binding of soluble CD4. The gp120 epitopes implicated in the interaction with the gp41 ectodomain were disrupted by deletions of the first (C1) and fifth (C5) conserved gp120 regions. These deletions did not affect the integrity of the discontinuous binding sites for CD4 and neutralizing monoclonal antibodies. Thus, the gp41 interface on the HIV-1 gp120 glycoprotein, which elicits nonneutralizing antibodies, can be removed while retaining immunologically desirable gp120 structures.

CD4 Antigens↗

PubMed1998

Engineering passive immunity in transgenic mice secreting virus-neutralizing antibodies in milk.

Protection against enteric infections can be provided by the oral administration of pathogen-neutralizing antibodies. To provide passive immunity, 18 lines of transgenic mice secreting a recombinant monoclonal antibody (Mab) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. The genes encoding a chimeric Mab with the variable modules of the murine TGEV-specific Mab 6A.C3 and the constant modules of a human IgG, isotype Mab were expressed under the control of regulatory sequences derived from the whey acidic protein, which is an abundant milk protein. The Mab 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species, which does not allow the selection of neutralization escape mutants. Antibody expression titers of 10(6) were obtained in the milk of transgenic mice that reduced TGEV infectivity 10(6)-fold. The antibody was synthesized at high levels throughout lactation. Integration of matrix attachment region sequences with the antibody genes led to a 20- to 10,000-fold increase in the antibody titer in 50% of the transgenic animals. Antibody expression levels were transgene copy number independent and related to the site of integration. The generation of transgenic animals producing virus neutralizing antibodies in milk could provide an approach to protection against neonatal infections of the enteric tract.

Animals↗

PubMed1991

Use of a lambda gt11 expression library to localize a neutralizing antibody-binding site in glycoprotein E2 of Sindbis virus.

The Sindbis virus envelope contains two species of integral membrane glycoproteins, E1 and E2. These proteins form heterodimers, and three dimeric units assemble to form spikes incorporated into the viral surface which play an important role in the specific attachment of Sindbis virus to host cells. To map the neutralization epitopes on the surface of the virus, we constructed a lambda gt11 expression library with cDNA inserts 100 to 300 nucleotides long obtained from randomly primed synthesis on Sindbis virus genomic RNA. This library was screened with five different neutralizing monoclonal antibodies (MAbs) specific for E2 (MAbs 50, 51, 49, 18, and 23) and with one neutralizing MAb specific for E1 (MAb 33). When 10(6) lambda gt11 plaques were screened with each antibody, four positive clones that reacted with E2-specific MAb 23 were found. These four clones contained overlapping inserts from glycoprotein E2; the domain from residues 173 to 220 of glycoprotein E2 was present in all inserts, and we concluded that this region contains the neutralization epitope recognized by the antibody. No clones that reacted with the other antibodies examined were found, and we concluded that these antibodies probably recognize conformational epitopes not present in the lambda gt11 library. We suggest that the E2 domain from residues 173 to 220 is a major antigenic determinant of Sindbis virus and that this domain is important for virus attachment to cells.

Amino Acid Sequence↗

PubMed1998

Lactogenic immunity in transgenic mice producing recombinant antibodies neutralizing coronavirus.

Protection against coronavirus infections can be provided by the oral administration of virus neutralizing antibodies. To provide lactogenic immunity, eighteen lines of transgenic mice secreting a recombinant IgG1 monoclonal antibody (rIgG1) and ten lines of transgenic mice secreting recombinant IgA monoclonal antibodies (rIgA) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. Genes encoding the light and heavy chains of monoclonal antibody (MAb) 6A.C3 were expressed under the control of regulatory sequences derived from the mouse genomic DNA encoding the whey acidic protein (WAP) and beta-lactoglobulin (BLG), which are highly abundant milk proteins. The MAb 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species. This MAb does not allow the selection of neutralization escaping virus mutants. The antibody was expressed in the milk of transgenic mice with titers of one million as determined by RIA, and neutralized TGEV infectivity by one million fold corresponding to immunoglobulin concentrations of 5 to 6 mg per ml. Matrix attachment regions (MAR) sequences were not essential for rIgG1 transgene expression, but co-microinjection of MAR and antibody genes led to a twenty to ten thousand-fold increase in the antibody titer in 50% of the rIgG1 transgenic animals generated. Co-microinjection of the genomic BLG gene with rIgA light and heavy chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and BLG genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of BLG co-integration. Antibody expression levels were transgene copy number independent and integration site dependent. The generation of transgenic animals producing virus neutralizing antibodies in the milk could be a general approach to provide protection against neonatal infections of the enteric tract.

Animals↗

PubMed1998

Nature of the elements transporting long-chain fatty acids through the red cell membrane.

Docosahexaenoic acid is found to be bound to three equivalent sites on albumin with the same affinities as palmitic acid at 0-38 degreesC, which demonstrates that ethene-1,2-diyl- and methylene-groups contribute equally to the affinity. The equilibrium dissociation constants (Kdms) for red cell membrane binding sites of linoleic- and docosahexaenoic acid at pH 7.3 are determined at temperatures between 0 and 37 degrees C. The temperature-independent capacities for binding are 12 +/- 1 and 25.4 +/- 3.0 nmoles g-1 ghosts respectively. Double isotope binding experiments reveal that the unsaturated fatty acids: arachidonic-, linoleic-, docosahexaenoic-, and oleic acid have partially shared capacities in ratios approximately 1:2:4:5, in contrast to the noncompetitive binding of palmitic acid. The observations suggest a two-tier binding limitation. One is the number of protein sites binding fatty acid anions electrostatically and the other is the number of suitable annular lipids adaptively selected among membrane lipids by the hydrocarbon chain. These competition conditions are confirmed by measurements of the tracer exchange efflux at near 0 degrees C from albumin-free and albumin-filled ghosts of linoleic- and docosahexaenoic acid, either alone or in the presence of arachidonic- and palmitic acid. Under equilibrium conditions, the calculated ratios of inside to outside membrane binding is below 0.5 for four unsaturated fatty acids. The unidirectional rate constants of translocation between the inside and the outside correlate with the number of double bonds in these fatty acids, which are also correlated with the dissociation rate constants of the complexes with albumin. The membrane permeation occurs presumably by binding of the anionic unsaturated fatty acids to an integral protein followed by channeling of the neutral form between opposite binding sites of the protein through annular lipids encircling the protein.

Binding, Competitive↗

PubMed2001

Genetic methods and strategies for secondary metabolite yield improvement in actinomycetes.

The foundation for any strain improvement program is efficient random chemically-induced mutagenesis coupled with highly reproducible fermentation and product assays. The broad spectrum of spontaneous mutations can be leveraged in some cases by direct selection of mutants with desired traits. Transposons containing outward-reading promoter activity might be used to enhance yields by inducing promoter fusions, disrupting negative regulatory elements, or disrupting genes involved in competing pathways. Transposons might also be used to identify and clone positive regulatory genes. As knowledge of the key elements in the fermentation process and secondary metabolite biosynthesis grows, gene cloning and targeted gene duplication becomes an important tool. Duplication of genes involved in rate limiting steps can be achieved to improve product yields by inserting the desired gene(s) into neutral sites in the chromosome by homologous recombination or by site-specific integration. The probabilities and frequencies of success of the molecular genetic approaches should increase with an increasing knowledge of key factors influencing product yields. This knowledge can be broadened dramatically by a combination of structural and functional genomics, gene disruption analysis and metabolic modeling. Protoplast fusion can be used to recombine beneficial traits from any of the other approaches.

Actinomycetales↗

PubMed2005

Sequence variation in G-protein-coupled receptors: analysis of single nucleotide polymorphisms.

We assessed the disease-causing potential of single nucleotide polymorphisms (SNPs) based on a simple set of sequence-based features. We focused on SNPs from the dbSNP database in G-protein-coupled receptors (GPCRs), a large class of important transmembrane (TM) proteins. Apart from the location of the SNP in the protein, we evaluated the predictive power of three major classes of features to differentiate between disease-causing mutations and neutral changes: (i) properties derived from amino-acid scales, such as volume and hydrophobicity; (ii) position-specific phylogenetic features reflecting evolutionary conservation, such as normalized site entropy, residue frequency and SIFT score; and (iii) substitution-matrix scores, such as those derived from the BLOSUM62, GRANTHAM and PHAT matrices. We validated our approach using a control dataset consisting of known disease-causing mutations and neutral variations. Logistic regression analyses indicated that position-specific phylogenetic features that describe the conservation of an amino acid at a specific site are the best discriminators of disease mutations versus neutral variations, and integration of all our features improves discrimination power. Overall, we identify 115 SNPs in GPCRs from dbSNP that are likely to be associated with disease and thus are good candidates for genotyping in association studies.

Amino Acid Substitution↗

PubMed2001

DNA integrity and total oxyradical scavenging capacity in the Mediterranean mussel, Mytilus galloprovincialis: a field study in a highly eutrophicated coastal lagoon.

In Mediterranean coastal lagoons, the combination of human impact and wide variability of natural environmental factors can lead to upsets in ecosystem homeostasis resulting in biodiversity decline. Oxidative damage has been causally linked to various kinds of environmental stress, both natural and artificial, the result being impairment of cellular functions. DNA damage and the efficiency of antioxidant defences in Mytilus galloprovincialis from the highly eutrophicated Orbetello Lagoon (Tuscany, Italy) were investigated, respectively by the single cell gel electrophoresis (or Comet test) and the total oxyradical scavenging capacity assay. Results showed significantly higher levels of DNA damage in mussels collected from the inner parts of the lagoon compared to specimens from more external sites. Specimens with the lower genetic integrity also exhibited a reduced efficiency in neutralizing three potent cellular oxidizing species, namely peroxyl radicals (ROO*), hydroxyl radicals (*OH) and peroxynitrite (HOONO), suggesting the involvement of reactive oxygen species in mediating the genetic damage. The analyzed biological parameters also showed a seasonal variability with a minimum of both DNA integrity and antioxidant scavenging efficiency during the warm months and an opposite trend in winter. The potential of analyzed techniques is discussed for the assessment of both anthropogenic and natural disturbance.

Animals↗

PubMed1998

Transgenic mice secreting coronavirus neutralizing antibodies into the milk.

Ten lines of transgenic mice secreting transmissible gastroenteritis coronavirus (TGEV) neutralizing recombinant monoclonal antibodies (rMAbs) into the milk were generated. The rMAb light- and heavy-chain genes were assembled by fusing the genes encoding the variable modules of the murine MAb 6A.C3, which binds an interspecies conserved coronavirus epitope essential for virus infectivity, and a constant module from a porcine myeloma with the immunoglobulin A (IgA) isotype. The chimeric antibody led to dimer formation in the presence of J chain. The neutralization specific activity of the recombinant antibody produced in transiently or stably transformed cells was 50-fold higher than that of a monomeric rMAb with the IgG1 isotype and an identical binding site. This rMAb had titers of up to 10(4) by radioimmunoassay (RIA) and neutralized virus infectivity up to 10(4)-fold. Of 23 transgenic mice, 17 integrated both light and heavy chains, and at least 10 of them transmitted both genes to the progeny, leading to 100% of animals secreting functional TGEV neutralizing antibody during lactation. Selected mice produced milk with TGEV-specific antibody titers higher than 10(6) as determined by RIA, neutralized virus infectivity by 10(6)-fold, and produced up to 6 mg of antibody per ml. Antibody expression levels were transgene copy number independent and integration site dependent. Comicroinjection of the genomic beta-lactoglobulin gene with rMAb light- and heavy-chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and beta-lactoglobulin genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of beta-lactoglobulin cointegration. This approach may lead to the generation of transgenic animals providing lactogenic immunity to their progeny against enteric pathogens.

Amino Acid Sequence↗

PubMed1999

A three-dimensional construction of the active site (region 507-749) of human neutral endopeptidase (EC.3.4.24.11).

A three-dimensional model of the 507-749 region of neutral endopeptidase-24.11 (NEP; E.C.3.4.24.11) was constructed integrating the results of secondary structure predictions and sequence homologies with the bacterial endopeptidase thermolysin. Additional data were extracted from the structure of two other metalloproteases, astacin and stromelysin. The resulting model accounts for the main biological properties of NEP and has been used to describe the environment close to the zinc atom defining the catalytic site. The analysis of several thiol inhibitors, complexed in the model active site, revealed the presence of a large hydrophobic pocket at the S1' subsite level. This is supported by the nature of the constitutive amino acids. The computed energies of bound inhibitors correspond with the relative affinities of the stereoisomers of benzofused macrocycle derivatives of thiorphan. The model could be used to facilitate the design of new NEP inhibitors, as illustrated in the paper.

Amino Acid Sequence↗

PubMed1999

Endothelial injuries of coronary arteries distal to thrombotic sites: role of adhesive interaction between endothelial P-selectin and leukocyte sialyl LewisX.

Intracoronary thrombus formation is associated with epicardial vasoconstriction distal to the thrombotic site. To investigate the mechanisms of abnormal vasomotor function of the artery distal to the thrombotic site, we studied coronary vessels in dogs with cyclic flow variations (CFVs) of the left anterior descending coronary artery (LAD) stenosis with endothelial injury. Coronary rings isolated from the LAD (proximal, stenotic, and distal sites) and control circumflex coronary arteries were tested for responsiveness to endothelium-dependent (acetylcholine and A23187) and endothelium-independent vasodilators (NaNO2). Endothelium-independent relaxation was intact in all 4 sites. Endothelium-dependent relaxation was intact in the control and proximal sites and impaired in the stenotic sites. Relaxations not only to acetylcholine and A23187 but also to serotonin, ADP, and thrombin were impaired in the distal sites after observing CFVs for 80 minutes. Electron microscopy revealed the loss of endothelial integrity with leukocyte adherence to the endothelium in the distal sites. Immunohistochemical expression of P-selectin on the endothelial cells was more upregulated in the distal site than in the proximal site, and P-selectin mRNA expression was significantly greater in the ischemic region distal to the thrombotic site than in the proximal nonischemic region. PB1.3, a neutralizing monoclonal antibody against P-selectin, and sialyl LewisX (SLeX)-containing oligosaccharide SLeX, a carbohydrate analogue of selectin ligand, preserved endothelial function without affecting CFVs. SLeX-containing oligosaccharide preserved endothelial integrity of the distal site and inhibited P-selectin expression of the distal site. Thus, the adhesive interaction between endothelial P-selectin and leukocyte SLeX may play an important role in endothelial injuries of the coronary artery distal to the thrombotic site.

Animals↗