1.
A new phage display system to construct multicombinatorial libraries of very large antibody repertoires.
Geoffroy, F.; Sodoyer, R.; Aujame, L.
Gene
vol. 151 issue 1-2 December 30, 1994. p. 109-113
► We present an easy and efficient technique for the construction of large phage-displayed…
(more)
▼ We present an easy and efficient technique for the construction of large phage-displayed antibody (Ab) repertoires through the recombination of two separate heavy (VH) and light (VL) chain gene libraries. Here, the system has been applied to the display of a chimpanzee anti-HIV gp160 Ab. The process, which makes use of λ phage att recombination sites, leads to the irreversible physical association between plasmid and phagemid vectors carrying, respectively, VL and VH sequences. The heat-inducible expression of the Int recombinase allows perfect control of recombination. Selection of the recombinant phagemid is made possible by the assembly, in vivo, of a genetic marker (chloramphenicol resistance) created only after the correct recombination event. Theoretically, all possible associations between the VL and VH sequences should be obtained, and it should be possible to generate multicombinatorial libraries of close to 10^1^2 clones.
Keywords: [abr] lox
DOI: 10.1016/0378-1119(94)90639-4. ISSN: 0378-1119.
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2.
Identification and expression
analysis of early cold-induced genes from cold-hardy Citrus
relative Poncirus trifoliata (L.) Raf.
Sahin-Cevik, M.
Gene
vol. 512 issue 2 January 10, 2013. p. 536-545
► Citrus is one of the most economically important fruit crops growing in subtropical…
(more)
▼ Citrus is one of the most
economically important fruit crops growing in subtropical and
tropical regions. Most commercially important Citrus varieties are
susceptible to cold; therefore, low and freezing temperatures are
the main limiting factors for citrus production in subtropical
areas. Since Poncirus trifoliata (L.) Raf. is a cold-hardy,
interfertile Citrus relative, it serves as a genetic resource for
improving cold tolerance in cold sensitive commercial Citrus
species. While gene induced in response to long-term cold
acclimation was previously identified in Poncirus, early response
of Poncirus to cold has not been explored in detail. To identify
early cold-responsive genes, a subtractive cDNA library was
constructed using 4-h cold-treated and untreated control Poncirus
seedlings in this study. A total of 210 randomly picked clones from
the subtracted library with cold-induced genes were sequenced. The
sequences obtained from the majority of these clones shared
homology with previously identified cold-induced and/or
environmental stress-regulated genes in other plants. Reverse
northern blot analysis of the expression of these cDNAs with
cold-treated and untreated control probes revealed that expression
of 64 cDNAs was increased two to 11 fold in response to 4-h cold
treatment. While the majority of these genes were related with cell
rescue, defense, cell death and aging, transcription, metabolism,
protein fate, energy, cellular communication and signal
transduction, transport facilitation and development, some of them
did not show homology with genes with known functions. Individual
expression analysis of nine selected genes by semi-quantitative
RT-PCR using mRNA from cold-treated and untreated control plants
confirmed that the expression of selected cDNAs was all induced in
response to cold. The results demonstrated that although a few
genes were commonly induced in response to both short and long-term
cold acclimation in Poncirus, majority of early cold-responsive
genes were different from previously identified late
cold-responsive genes in Poncirus.
Keywords: [abr] SSH; suppression subtractive
hybridization; [abr] CLT; citrus low temperature
genes; [abr] ABA; abscisic acid; [abr] GRP; glycine-rich protein; [abr] NBS-LRR; nucleotide-binding
site-leucine-rich repeat; [abr] LOX; lipoxygenase; [abr] CAF1; CCR4 associated factor
1; [abr] PR; pathogenesis-related; [abr] cDNA; complementary DNA; [abr] RT-PCR; reverse-transcription
polymerase chain reaction;…
DOI: 10.1016/j.gene.2012.09.084. ISSN: 0378-1119.
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3.
Recombinase-mediated cassette
exchange (RMCE) - A rapidly-expanding toolbox for targeted genomic
modifications.
Turan, S.; Zehe, C.; Kuehle, J.; Qiao, J.; Bode, J.
Gene
vol. 515 issue 1 February 15, 2013. p. 1-27
► Starting in 1991, the advance of Tyr-recombinases Flp and Cre enabled superior strategies…
(more)
▼ Starting in 1991, the advance
of Tyr-recombinases Flp and Cre enabled superior strategies for the
predictable insertion of transgenes into compatible target sites of
mammalian cells. Early approaches suffered from the reversibility
of integration routes and the fact that co-introduction of
prokaryotic vector parts triggered uncontrolled
heterochromatization. Shortcomings of this kind were overcome when
Flp-Recombinase Mediated Cassette Exchange entered the field in
1994. RMCE enables enhanced tag-and-exchange strategies by
precisely replacing a genomic target cassette by a compatible donor
construct. After ''gene swapping'' the donor cassette is safely
locked in, but can nevertheless be re-mobilized in case other
compatible donor cassettes are provided (''serial RMCE''). These
features considerably expand the options for systematic, stepwise
genome modifications. The first decade was dominated by the
systematic generation of cell lines for biotechnological purposes.
Based on the reproducible expression capacity of the resulting
strains, a comprehensive toolbox emerged to serve a multitude of
purposes, which constitute the first part of this review. The
concept per se did not, however, provide access to high-producer
strains able to outcompete industrial multiple-copy cell lines.
This fact gave rise to systematic improvements, among these certain
accumulative site-specific integration pathways. The exceptional
value of RMCE emerged after its entry into the stem cell field,
where it started to contribute to the generation of induced
pluripotent stem (iPS-) cells and their subsequent differentiation
yielding a variety of cell types for diagnostic and therapeutic
purposes. This topic firmly relies on the strategies developed in
the first decade and can be seen as the major ambition of the
present article. In this context an unanticipated, potent property
of serial Flp-RMCE setups concerns the potential to re-open loci
that have served to establish the iPS status before the site
underwent the obligatory silencing process. Other relevant options
relate to the introduction of composite Flp-recognition target
sites (''heterospecific FRT-doublets''), into the LTRs of
lentiviral vectors. These ''twin sites'' enhance the safety of iPS
re-programming and -differentiation as they enable the subsequent
quantitative excision of a transgene, leaving behind a single
''FRT-twin''. Such a strategy combines the established expression
potential of the common retro- and lentiviral systems with options
to terminate the process at will. The remaining genomic tag serves
to identify and characterize the insertion site with the goal to
identify genomic ''safe harbors'' (GOIs) for re-use. This is
enabled by the capacity of ''FRT-twins'' to accommodate any
incoming RMCE-donor cassette with a compatible design.
Keywords: [abr] attB/attP; recombinase-attachment sites
within the bacterial and phage genomes; [abr] ccc; covalently-closed
circular; [abr] Cre; cyclization recombinase/causes
recombination; [abr] DSB; double-strand break; [abr] ESC; embryonic stem cell; [abr] flexing; Flp-mediated excision; [abr] EUCOMM; European Conditional Mouse
Mutagenesis program; [abr] flirted; FRT-flanked; [abr] floxed; flanked…
DOI: 10.1016/j.gene.2012.11.016. ISSN: 0378-1119.
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4.
Recombinase-mediated cassette exchange (RMCE) - A rapidly-expanding toolbox for targeted genomic modifications.
Turan, S.; Zehe, C.; Kuehle, J.; Qiao, J.; Bode, J.
Gene
vol. 515 issue 1 February 15, 2013. p. 1-27
► Starting in 1991, the advance of Tyr-recombinases Flp and Cre enabled superior strategies…
(more)
▼ Starting in 1991, the advance of Tyr-recombinases Flp and Cre enabled superior strategies for the predictable insertion of transgenes into compatible target sites of mammalian cells. Early approaches suffered from the reversibility of integration routes and the fact that co-introduction of prokaryotic vector parts triggered uncontrolled heterochromatization. Shortcomings of this kind were overcome when Flp-Recombinase Mediated Cassette Exchange entered the field in 1994. RMCE enables enhanced tag-and-exchange strategies by precisely replacing a genomic target cassette by a compatible donor construct. After ''gene swapping'' the donor cassette is safely locked in, but can nevertheless be re-mobilized in case other compatible donor cassettes are provided (''serial RMCE''). These features considerably expand the options for systematic, stepwise genome modifications. The first decade was dominated by the systematic generation of cell lines for biotechnological purposes. Based on the reproducible expression capacity of the resulting strains, a comprehensive toolbox emerged to serve a multitude of purposes, which constitute the first part of this review. The concept per se did not, however, provide access to high-producer strains able to outcompete industrial multiple-copy cell lines. This fact gave rise to systematic improvements, among these certain accumulative site-specific integration pathways. The exceptional value of RMCE emerged after its entry into the stem cell field, where it started to contribute to the generation of induced pluripotent stem (iPS-) cells and their subsequent differentiation yielding a variety of cell types for diagnostic and therapeutic purposes. This topic firmly relies on the strategies developed in the first decade and can be seen as the major ambition of the present article. In this context an unanticipated, potent property of serial Flp-RMCE setups concerns the potential to re-open loci that have served to establish the iPS status before the site underwent the obligatory silencing process. Other relevant options relate to the introduction of composite Flp-recognition target sites (''heterospecific FRT-doublets''), into the LTRs of lentiviral vectors. These ''twin sites'' enhance the safety of iPS re-programming and -differentiation as they enable the subsequent quantitative excision of a transgene, leaving behind a single ''FRT-twin''. Such a strategy combines the established expression potential of the common retro- and lentiviral systems with options to terminate the process at will. The remaining genomic tag serves to identify and characterize the insertion site with the goal to identify genomic ''safe harbors'' (GOIs) for re-use. This is enabled by the capacity of ''FRT-twins'' to accommodate any incoming RMCE-donor cassette with a compatible design.
Keywords: [abr] loxP
DOI: 10.1016/j.gene.2012.11.016. ISSN: 0378-1119.
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5.
Escherichia coli genome targeting I. Cre-Zox-mediated in vitro generation of ori^- plasmids and their in vivo chromosomal integration and retrieval.
Hasan, N.; Koob, M.; Szybalsk, W.
Gene
vol. 150 issue 1 December 2, 1994. p. 51-56
► We have constructed a plasmid system designed for the insertion of cloned DNA…
(more)
▼ We have constructed a plasmid system designed for the insertion of cloned DNA (e.g., genes, gene fusions, regulatory elements, etc.) into the Escherichia coli genome. Its principal feature is the presence of two tandem lox sites on the plasmids, which upon Cre-mediated in vitro recombination resolve the plasmids into ori^- and ori^+ DNA circles. The non-replicating ori^- circles contain the λ, attP site, several unique restriction sites for cloning, a NotI site and Km^R, a kanamycin-resistance-encoding gene. The ori^+ circles carry the origin of DNA replication (ori) together with several cleavage sites not present in the ori^- circles, including the rare site for the very efficient I-SceI enzyme, that are used to inactivate the ori^+ circles and any unresolved plasmid DNA. We have used this system to insert cloned DNA into the host genome at (i) the attB site, by Int-mediated integration and (ii) at any predetermined sequence, as mediated by the Rec system(s) of the host. The genomes of the resulting transformants were analyzed by Nod digestion of the chromosomal DNA, embedded in agarose microbeads, followed by pulsed-field gel electrophoresis. A system for the retrieval of DNA fragments inserted at the attB site was also developed.
Keywords: [abr] loxP
DOI: 10.1016/0378-1119(94)90856-7. ISSN: 0378-1119.
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6.
The complete sequence of the rabbit erythroid cell-specific 15-lipoxygenase mRNA: comparison of the predicted amino acid sequence of the erythrocyte lipoxygenase with other lipoxygenases.
Fleming, J.; Thiele, B.J.; Chester, J.; O'Prey, J.; Janetzki, S.; Aitken, A.; Anton, I.A.; Rapoport, S.M.; Harrison, P.R.
Gene
vol. 79 issue 1 June 30, 1989. p. 181-188
► We report the complete sequence of the rabbit reticulocyte (RBC) 15-lipoxygenase (LOX) mRNA…
(more)
▼ We report the complete sequence of the rabbit reticulocyte (RBC) 15-lipoxygenase (LOX) mRNA as deduced from (i) sequencing cDNA recombinants isolated by screening cDNA libraries or polymerase-chain-reactions, and (ii) the sequence originating from the transcription start point obtained by primer extension-sequencing reactions. Like the human leukocyte 5-LOX mRNA, the RBC 15-LOX mRNA contains a very short 5 ' -untranslated region with a long 3 ' -untranslated region. But, unlike the human leukocyte 5-LOX mRNA, the RBC 15-LOX mRNA contains an intriguing repeated sequence (ten copies with the consensus sequence C4PuC3TCTTC4AAG) just after the translational stop codon, which may be involved in its regulation during reticulocyte maturation. Comparison of the RBC 15-LOX mRNA sequence with those of the previously published human 5-LOX mRNA and the soybean 3-LOX gene shows only a few short regions of sequence similarity. However, the predicted amino acid sequences of the encoded LOX enzymes show certain conserved regions that are presumably involved in their catalytic activity, in particular a cluster of five conserved histidines that we predict chelate the iron moiety involved in the active site.
Keywords: [abr] LOX; [abr] LOX
DOI: 10.1016/0378-1119(89)90103-0. ISSN: 0378-1119.
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7.
The promoter structure and complete sequence of the gene encoding the rabbit erythroid cell-specific 15-lipoxygenase.
O'Prey, J.; Chester, J.; Thiele, B.J.; Janetzki, S.; Prehn, S.; Fleming, J.; Harrison, P.R.
Gene
vol. 84 issue 2 December 14, 1989. p. 493-499
► We report the isolation and complete sequence of the gene encoding the rabbit…
(more)
▼ We report the isolation and complete sequence of the gene encoding the rabbit erythroid-cell-specific 15-lipoxygenase (RBC 15-LOX), containing 14 exons spanning 8.0 kb. The transcription start point was mapped by S1 nuclease-protection experiments and comparison with the sequence of the RBC 15-LOX mRNA, as defined previously by primer extension experiments. The promoter contains a TATA-like motif, but no CCAAT motif in the canonical position, and lies within a 'CpG-rich island'. Functional analysis of the immediate 5'-flanking DNA by transfection experiments shows that a 150 nucleotide (nt) 5'fragment linked to the chloramphenicol acetyltransferase gene acts as a functional promoter in both erythroid and nonerythroid cell lines and responds in an erythroid-specific manner to the enhancer from the Friend murine leukaemia virus long terminal repeat, whereas a 40-nt fragment is inactive. Intron 7 contains eight copies of a 54-nt repeat containing a region with homology to the simian virus 40/immunoglobulin gene enhancers.
Keywords: [abr] LOX; [abr] lox
DOI: 10.1016/0378-1119(89)90526-X. ISSN: 0378-1119.
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8.
Taking phage integration to the next level as a genetic tool for mycobacteria.
Huff, J.; Czyz, A.; Landick, R.; Niederweis, M.
Gene
vol. 468 issue 1-2 November 15, 2010. p. 8-19
► Genes must be stably integrated into bacterial chromosomes for complementation of gene deletion…
(more)
▼ Genes must be stably integrated into bacterial chromosomes for complementation of gene deletion mutants in animal infection experiments or to express antigens in vaccine strains. However, with currently available vectors it is cumbersome to create multiple, stable, unmarked chromosomal integrations in mycobacteria. Here, we have constructed a novel integration vector for mycobacteria that enables expression of genes from a cassette protected from transcriptional interference by bi-directional transcriptional terminators proven to be highly efficient in in vitro transcription termination assays. Removal of the integrase gene by a site-specific recombinase, easily identifiable by loss of a backbone reporter gene, stabilizes the integration cassette and makes this vector ideally suitable for infection experiments. This integration vector can be easily adapted to different mycobacteriophage attachment sites (attB) due to its modular design. Integration of a gfp expression cassette at the L5, Giles and Ms6 attB sites in the chromosomes of Mycobacterium smegmatis and Mycobacterium tuberculosis yielded identical gfp expression levels, indicating that none of these sites are compromised for gene expression. The copy number of pAL5000-based extrachromosomal plasmids is 23 in M. smegmatis as determined by quantitative real-time PCR and accounts for the previously observed drastic reduction of gene expression upon integration of plasmids into the chromosome of mycobacteria. Gfp expression and fluorescence of M. smegmatis and M. tuberculosis strains with multiple integrations of gfp increased concomitantly with the copy number demonstrating that these vectors can be used to generate stronger phenotypes and/or to analyze several genes simultaneously in vivo.
Keywords: [abr] loxP
DOI: 10.1016/j.gene.2010.07.012. ISSN: 0378-1119.
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9.
A phage T4 in vitro packaging system for cloning long DNA molecules.
Rao, V.B.; Thaker, V.; Black, L.W.
Gene
vol. 113 issue 1 April 1, 1992. p. 25-33
► Recombinant plasmid DNAs containing long DNA inserts that can be propagated in Escherichia…
(more)
▼ Recombinant plasmid DNAs containing long DNA inserts that can be propagated in Escherichia coli would be useful in the analysis of complex genomes. We tested a bacteriophage T4 in vitro DNA packaging system that has the capacity to package about 170 kb of DNA into its capsid for cloning long DNA fragments. We first asked whether the T4 in vitro system can package foreign DNA such as concatemerized λimm434 DNA and phage P1-pBR322 hybrid DNA. The data suggest that the T4 system can package foreign DNA as efficiently as the mature phage T4 DNA. We then tested the system for its ability to clone foreign DNA fragments using the P1-pBR322 hybrid vectors constructed by Sternberg [Proc. Natl. Acad. Sci. USA 87 (1990) 103-107]. E. coli genomic DNA fragments were ligated with the P1 vectors containing two directly oriented loxP sites, and the ligated DNA was packaged by the T4 in vitro system. The packaged DNA was then transduced into E. coli expressing the phage P1 cyclization recombination protein recombinase to circularize the DNA by recombination between the loxP sites situated at the ends of the transduced DNA molecule. Clones with long DNA inserts were obtained by using this approach, and these were maintained as single-copy plasmids under the control of the P1 plasmid replicon. Clones with up to about 122-kb size inserts were recovered using this approach.
Keywords: [abr] loxP
DOI: 10.1016/0378-1119(92)90666-D. ISSN: 0378-1119.
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10.
A selective λ phage cloning vector with automatic excision of the insert in a plasmid.
Maruyama, I.N.; Brenner, S.
Gene
vol. 120 issue 2 October 21, 1992. p. 135-141
► A bacteriophage λ cloning vehicle has been constructed for the generation of cDNA…
(more)
▼ A bacteriophage λ cloning vehicle has been constructed for the generation of cDNA libraries. The vector has the following properties. (1) It has a unique BamHI site engineered into the λ gam gene. Segments of DNA can be cloned into this site and clones with an insert can be selected by their ability to grow on an Escherichia coli host lysogenic for phage P2 (Spi^- phenotype). (2) When the recombinant phage infects a Cre-producing E. coli strain, a site-specific recombination event results in the excision of a plasmid replicon with the cloned insert. (3) Single-stranded DNAs can be recovered by growing helper M13 phages on bacteria harboring such plasmids. The vector, λMGU2, has been used to construct a nematode (Caenorhabditis elegans) cDNA library.
Keywords: [abr] loxP
DOI: 10.1016/0378-1119(92)90086-5. ISSN: 0378-1119.
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11.
Molecular evolution of the fibulins: Implications on the functionality of the elastic fibulins.
Segade, F.
Gene
vol. 464 issue 1-2 September 15, 2010. p. 17-31
► The fibulins form a family of secreted proteins associated with the basement membrane,…
(more)
▼ The fibulins form a family of secreted proteins associated with the basement membrane, cell adhesive structures, and elastic fibers characterized by the presence of a unique fibulin-like C-terminal domain preceded by a rod-like tandem array of calcium-binding EGF modules. We traced the origin of the fibulin gene family to the base of the metazoans. In invertebrates, Fibulin-1 and Hemicentin comprise the fibulin gene set. Diversification of the fibulins took place in the last common ancestor to the chordates by gene duplication of an ancestral Fibulin-1 gene. Further duplications at the vertebrate stem and in teleost fishes increased the number of fibulin genes to nine, including the novel Fibulin-8. Extensive gene loss has happened repeatedly, including Fibulin-8 in placental mammals and Fibulin-4 in birds. The Fibulin-3/4/5 clade of elastic fibulins branched out at the sequence level after relaxation of selective constraints immediately after the two gene duplication events in quick succession that originated the individual vertebrate Fibulin-3, -4, and -5 genes. Divergence took place mostly in the Fibulin-5 branch, at the atypical first EGF module and at the fibulin-like C-terminal region. Differentiation in gene expression further split Fibulin-5 from the other elastic fibulins, likely contributing to its nonredundant role in elastic fiber assembly.
Keywords: [abr] LOX
DOI: 10.1016/j.gene.2010.05.003. ISSN: 0378-1119.
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12.
Cloning of a rabbit erythroid-cell-specific lipoxygenase mRNA.
Thiele, B.J.; Fleming, J.; Kasturi, K.; O'Prey, J.; Black, E.; Chester, J.; Rapoport, S.M.; Harrison, P.R.
Gene
vol. 57 issue 1 1987. p. 111-119
► We report the isolation of cDNA recombinants representing part of the rabbit reticulocyte…
(more)
▼ We report the isolation of cDNA recombinants representing part of the rabbit reticulocyte (immature red blood cell, RBC) lipoxygenase (LOX) mRNA. One cDNA predicts an amino acid (aa) sequence matching exactly the unique N-terminal 30-aa sequence of the purified enzyme. Further, the reticulocyte mRNA, hybrid-selected by this recombinant, can be translated in vitro to give a polypeptide that comigrates with the purified reticulocyte LOX and is recognised by affinity-purified anti-RBC LOX polyclonal antibodies. Southern blotting experiments hybridising the RBC LOX cDNAs available to total rabbit genomic DNA digested with various restriction enzymes gives a fairly simple hybridisation pattern under moderate stringency conditions: moreover, the same pattern is obtained with a cloned fragment of genomic DNA containing the RBC LOX gene. This indicates that the RBC LOX gene is unique in the genome and seems not to be very closely related to the genes encoding the other tissue LOXs. We also show by Northern transfer/hybridisation experiments that the RBC LOX mRNA is expressed only in the red cell lineage but not in white blood cells (bone marrow or spleen) or in other non-erythroid cells tested (e.g., brain and lung).
Keywords: [abr] LOX; [abr] LOX
DOI: 10.1016/0378-1119(87)90182-X. ISSN: 0378-1119.
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13.
Lipoxygenase in Caragana jubata responds to low temperature, abscisic acid, methyl jasmonate and salicylic acid.
Bhardwaj, P.K.; Kaur, J.; Sobti, R.C.; Ahuja, P.S.; Kumar, S.
Gene
vol. 483 issue 1-2 September 1, 2011. p. 49-53
► Lipoxygenase (LOX) catalyses oxygenation of free polyunsaturated fatty acids into oxylipins, and is…
(more)
▼ Lipoxygenase (LOX) catalyses oxygenation of free polyunsaturated fatty acids into oxylipins, and is a critical enzyme of the jasmonate signaling pathway. LOX has been shown to be associated with biotic and abiotic stress responses in diverse plant species, though limited data is available with respect to low temperature and the associated cues. Using rapid amplification of cDNA ends, a full-length cDNA (CjLOX) encoding lipoxygenase was cloned from apical buds of Caragana jubata, a temperate plant species that grows under extreme cold. The cDNA obtained was 2952bp long consisting of an open reading frame of 2610bp encoding 869 amino acids protein. Multiple alignment of the deduced amino acid sequence with those of other plants demonstrated putative LH2/ PLAT domain, lipoxygenase iron binding catalytic domain and lipoxygenase_2 signature sequences. CjLOX exhibited up- and down-regulation of gene expression pattern in response to low temperature (LT), abscisic acid (ABA), methyl jasmonate (MJ) and salicylic acid (SA). Among all the treatments, a strong up-regulation was observed in response to MJ. Data suggests an important role of jasmonate signaling pathway in response to LT in C. jubata.
Keywords: [abr] LOX
DOI: 10.1016/j.gene.2011.05.014. ISSN: 0378-1119.
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14.
Intra- and intramolecular site-specific recombination in plant cells mediated by bacteriophage P1 recombinase.
Dale, E.C.; Ow, D.W.
Gene
vol. 91 issue 1 1990. p. 79-85
► A site-specific recombination system has many potential uses for rearranging genetic material in…
(more)
▼ A site-specific recombination system has many potential uses for rearranging genetic material in higher eukaryotic cells: for example, the control of gene expression by deletion or inversion of DNA segments, the clustering of transgenic constructs via site-specific integration, and the generation of chromosomal translocations. In this report, we describe a first step towards the application of a site-specific recombination system in plant cells. By use of a transient assay, we demonstrate that the bacteriophage P1 cre gene can be expressed as a functional recombinase in tobacco cells. Upon expression in tobacco protoplasts, Cre recognizes its target sites, lox, and mediates reciprocal genetic crossovers at these sites. When the lox sites are present in cis to one another, and arranged in either direct or inverted orientations, we detect Cre/lox-specific deletion and inversion events, respectively. The placement of lox sites in trans resulted in the co-integration of the substrates by Cre-mediated intermolecular recombination. These results indicate that the Cre/lox site-specific recombinaton system might be further developed as an additional tool for manipulating DNA in plant cells. Applications relevant to the genetic engineering of higher plants are discussed.
Keywords: [abr] lox
DOI: 10.1016/0378-1119(90)90165-N. ISSN: 0378-1119.
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15.
A novel phage λ replacement Cre-lox vector that has automatic subcloning capabilities.
Holt, C.L.; May, G.S.
Gene
vol. 133 issue 1 October 29, 1993. p. 95-97
► We have developed a novel phage λ replacement cloning vector, λpAn. λpAn allows…
(more)
▼ We have developed a novel phage λ replacement cloning vector, λpAn. λpAn allows one to automatically subclone the insert as a plasmid using the Cre-loxP site-specific recombination system. This eliminates the need to subclone insert fragments and permits the rapid structural analysis of insert DNA. λpAn is similar to other phage λ replacement vectors taking inserts ranging in size from 5 to 19 kb. We have placed the pyrG gene of Aspergillus nidulans on the vector as a nutritional selective marker for transformation. We have developed this vector as part of an overall plan to facilitate the cloning of dominant extragenic suppressor mutations from A. nidulans, but also know that it is a generally useful vector for the purposes of isolating genomic clones without the need to subclone from the phage λ vector.
Keywords: [abr] loxP
DOI: 10.1016/0378-1119(93)90230-Z. ISSN: 0378-1119.
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16.
Temporary effect of postharvest
UV-C irradiation on gene expression profile in tomato fruit.
Liu, C.; Cai, L.; Han, X.; Ying, T.
Gene
vol. 486 issue 1-2 October 15, 2011. p. 56-64
► To obtain an overall view on gene expression during the early stage (24h)…
(more)
▼ To obtain an overall view on
gene expression during the early stage (24h) of tomato fruit in
response to postharvest UV-C irradiation (4kJ/m^2), we performed a
microarray analysis by using Affymetrix Tomato Genechip. The
results showed that 274 and 403 genes were up- or down-regulated,
respectively, more than two folds in postharvest tomato fruit
irradiated with UV-C as compared with that in control fruit. The
up-regulated genes mainly involve in signal transduction, defense
response and metabolism. Conversely, genes related to cell wall
disassembly, photosynthesis and lipid metabolism were generally
down-regulated. These results opened ways to probe into the
molecular mechanisms of the effects of postharvest UV-C irradiation
on increased disease resistance, delayed softening, better quality
maintenance and prolonged postharvest life in tomato fruit.
Keywords: [abr] ACC;
1-Aminocyclopropane-1-carboxylic acid; [abr] EXP; expansin; [abr] GAPDH; Glyceraldehyde-3-phosphate
dehydrogenase; [abr] IAA; Indole-3-acetic acid; [abr] LOX; Lipoxygenase; [abr] PAL; Phenylalanine ammonia
lyase; [abr] PR; Pathogenesis-related; [abr] ROS; Reactive oxygen species; [abr] SAM; S-adenosyl-L-metionine; [abr] XTH; Xyloglucan
endotransglycosylase/hydrolase; Tomato fruit; UV-C; Gene expression; Temporary effect
DOI: 10.1016/j.gene.2011.07.001. ISSN: 0378-1119.
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17.
Temporary effect of postharvest UV-C irradiation on gene expression profile in tomato fruit.
Liu, C.; Cai, L.; Han, X.; Ying, T.
Gene
vol. 486 issue 1-2 October 15, 2011. p. 56-64
► To obtain an overall view on gene expression during the early stage (24h)…
(more)
▼ To obtain an overall view on gene expression during the early stage (24h) of tomato fruit in response to postharvest UV-C irradiation (4kJ/m^2), we performed a microarray analysis by using Affymetrix Tomato Genechip. The results showed that 274 and 403 genes were up- or down-regulated, respectively, more than two folds in postharvest tomato fruit irradiated with UV-C as compared with that in control fruit. The up-regulated genes mainly involve in signal transduction, defense response and metabolism. Conversely, genes related to cell wall disassembly, photosynthesis and lipid metabolism were generally down-regulated. These results opened ways to probe into the molecular mechanisms of the effects of postharvest UV-C irradiation on increased disease resistance, delayed softening, better quality maintenance and prolonged postharvest life in tomato fruit.
Keywords: [abr] LOX
DOI: 10.1016/j.gene.2011.07.001. ISSN: 0378-1119.
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18.
The Idefix enhancer-blocking insulator also harbors barrier activity.
Brasset, E.; Hermant, C.; Jensen, S.; Vaury, C.
Gene
vol. 450 issue 1-2 January 15, 2010. p. 25-31
► Chromatin insulators are cis-regulatory sequences participating in the regulation of gene expression. Their…
(more)
▼ Chromatin insulators are cis-regulatory sequences participating in the regulation of gene expression. Their presence within the genome is associated with two main functions. One of them is an enhancer-blocking function that blocks enhancer-promoter communication when the insulator is located in between. The second is a boundary or barrier function that insulates independent units of transcription. This latter is observed when two insulators flanking a gene and its regulatory sequences block the regulatory influences of surrounding chromatin. Some years ago, we reported the presence of an insulator within the retrotransposon Idefix from Drosophila melanogaster. This insulator displays an enhancer-blocking activity toward an enhancer located within a second retrotransposon called ZAM. Here, we show that this insulator is not specific to the ZAM enhancer but has the capacity to interfere in the communication established between a broad range of cis-regulatory enhancer and a promoter. Furthermore, we show that, if it is placed on both sides of a transgene, this insulator acts as a barrier able to isolate the transgene from its repressive or enhancing environment. Thus, the Idefix insulator carries both an enhancer-blocking and a barrier activity. According to these properties, the Idefix insulator might prove to be a useful tool to isolate artificial transgenes from positive or negative influences from their integration sites.
Keywords: [abr] lox
DOI: 10.1016/j.gene.2009.09.015. ISSN: 0378-1119.
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19.
Formation of small circular DNA molecules via an in vitro site-specific recombination system.
Hoess, R.; Wierzbicki, A.; Abremski, K.
Gene
vol. 40 issue 2-3 1985. p. 325-329
► The Cre-lox site-specific recombination system of bacteriophage P1 has been used to investigate…
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▼ The Cre-lox site-specific recombination system of bacteriophage P1 has been used to investigate the role of DNA flexibility in recombination. We have determined that a minimal distance of 82 bp must separate two loxP sites located on the same DNA molecule to allow these sites to undergo intramolecular recombination with one another. As a result of recombination, DNA circles as small as 116bp have been produced. In addition, we have demonstrated that the nuclease BAL 31 recognizes distortions in the DNA helix resulting from the formation of small DNA circles whose length is not a multiple of the helical repeat.
Keywords: [abr] lox
DOI: 10.1016/0378-1119(85)90056-3. ISSN: 0378-1119.
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