Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The yeast 2-micron circle plasmid encodes a protein, FLP, that mediates site-specific recombination across the two FLP-binding sites of the plasmid. We have used a novel technique, "exonuclease-treated substrate analysis," to determine the minimal duplex DNA sequence needed for this recombination event. A linear DNA containing two FLP sites in a direct orientation was treated with the double-strand specific 3'-exonuclease, exonuclease III, to generate molecules with a nested set of single-strand deletions that extended into one of the FLP sites. The DNA was then end-labeled at the sites of the deletions and used as a substrate for recombination in vitro. FLP-mediated recombination between two FLP sites excised a restriction endonuclease cleavage site from the DNA. Comparison of the fragments produced by restriction enzyme digestion of untreated and FLP-treated DNA showed to the nucleotide the duplex DNA sequence required for FLP-mediated recombination. To examine essential sequences in the opposite DNA strand, similar experiments were done using the 5'-exonuclease encoded by phage T7. The minimal essential duplex DNA sequence lies within the region of the FLP site that was previously shown to be protected from nuclease digestion in the presence of FLP. A modified form of this technique can be used to study the minimal sequence requirements of site-specific DNA binding proteins.
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PMID:Determination of DNA sequences essential for FLP-mediated recombination by a novel method. 299 71

A system of versatile insertion plasmids was constructed that permits efficient delivery of the target sites of an ultra-rare-cutting endonuclease and the recombinase FLP into preselected sites of the bacterial genome. With the help of this system, the pathogenicity island LEE of the Escherichia coli O157:H7 genome was excised and isolated in vitro, deleted in vivo, rescued as a plasmid, and transferred into another strain.
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PMID:Versatile insertion plasmids for targeted genome manipulations in bacteria: isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli O157:H7 genome. 920 66

We used a recently developed method to produce mutant alleles of five endogenous Drosophila genes, including the homolog of the p53 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-SceI endonuclease generates extrachromosomal linear DNA molecules in vivo. These molecules undergo homologous recombination with the corresponding chromosomal locus to generate targeted alterations of the host genome. The results address several questions about the general utility of this technique. We show that genes not near telomeres can be efficiently targeted; that no knowledge of the mutant phenotype is needed for targeting; and that insertional mutations and allelic substitutions can be easily produced.
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PMID:Targeted mutagenesis by homologous recombination in D. melanogaster. 1208 94

Ends-in and ends-out refer to the two arrangements of donor DNA that can be used for gene targeting. Both have been used for targeted mutagenesis, but require donors of differing design. Ends-out targeting is more frequently used in mice and yeast because it gives a straightforward route to replace or delete a target locus. Although ends-in targeting has been successful in Drosophila, an attempt at ends-out targeting failed. To test whether ends-out targeting could be used in Drosophila, we applied two strategies for ends-out gene replacement at the endogenous yellow (y) locus in Drosophila. First, a mutant allele was rescued by replacement with an 8-kb y(+) DNA fragment at a rate of approximately 1/800 gametes. Second, a wild-type gene was disrupted by the insertion of a marker gene in exon 1 at a rate of approximately 1/380 gametes. The I-SceI endonuclease component alone is not sufficient for targeting: the FLP recombinase is also needed to generate the extrachromosomal donor. When both components are used we find that ends-out targeting can be approximately as efficient as ends-in targeting, and is likely to be generally useful for Drosophila gene targeting.
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PMID:Ends-out, or replacement, gene targeting in Drosophila. 1258 26

Studies of regulatory systems in transgenic Drosophila are often compromised by possible genomic position effects on gene expression. As a result, it is desirable to be able to manipulate multiple regulatory elements in a single transgene construct. We developed an I-SceI endonuclease-based method to efficiently delete preassigned sequences from transgenes with the use of direct repeat sequences of just 126 nucleotides. This system can be used in combination with the existing cre-lox and FLP-FRT recombinational mechanisms in order to modify up to three regulatory regions in a given transgene. We validated the utility of our combination approach by demonstrating new properties of the Fab-7 insulator.
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PMID:Handling three regulatory elements in one transgene: combined use of cre-lox, FLP-FRT, and I-Scel recombination systems. 1638 6

We present a series of 14 binary vectors suitable for Agrobacterium-mediated transformation of dicotyledonous plants and adaptable for biolistic transformation of monocotyledonous plants. The vector size has been minimized by eliminating all non-essential elements from the vector backbone and T-DNA regions while maintaining the ability to replicate independently. The smallest of the vector series is 6.3 kb and possesses an extensive multiple cloning site with 21 unique restriction endonuclease sites that are compatible with common cloning, protein expression, yeast two-hybrid and other binary vectors. The T-DNA region was engineered using a synthetic designer oligonucleotide resulting in an entirely modular system whereby any vector element can be independently exchanged. The high copy number ColE1 origin of replication has been included to enhance plasmid yield in Escherichia coli. FRT recombination sites flank the selectable marker cassette regions and allow for in planta excision by FLP recombinase. The pORE series consists of three basic types; an 'open' set for general plant transformation, a 'reporter' set for promoter analysis and an 'expression' set for constitutive expression of transgenes. The sets comprise various combinations of promoters (P (HPL), P (ENTCUP2) and P (TAPADH)), selectable markers (nptII and pat) and reporter genes (gusA and smgfp).
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PMID:pORE: a modular binary vector series suited for both monocot and dicot plant transformation. 1727 15

Epstein-Barr virus (EBV) is an oncogenic herpesvirus associated with a variety of malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma (NPC). Functions of most EBV genes have not been determined. The use of bacterial artificial chromosome (BAC) to clone and modify the genome of EBV has enhanced the gene function study in the context of genome. Infectious clones of EBV were previously established by using EBV-BAC plasmid p2089. In order to further investigate EBV mutant biology, an easy and efficient method for gene modification in EBV-BAC was developed and detailed. The kanamycin gene (kan) flanked by recombinase FLP recognition targets (FRTs) was amplified from plasmid pKD13 and inserted into the vector of pcDNA3.1(+). Through the introduction of restriction endonuclease BsmB I in PCR primers, NPC-derived LMP1 gDNA containing the full-length ORF was then precisely ligated with kan on pcDNA3.1(+). The linear DNA segment of kan-LMP1 was transformed into E. coli DH10B cells containing p2089 and plasmid pKD46, homologous recombination was subsequently mediated by redalphabetagamma system from bacteriophage lambda. By this linear transformation and ET cloning, the full-length LMP1 in EBV-BAC (p2089) was replaced by the kan-LMP1. The introduced kan gene in EBV-BAC genome was eliminated specifically by the recombinase FLP when transformed by plasmid pCP20, leaving an FRT scar of 69 bp. The mutant could be identified by antibiotic screening and PCR amplification on bacteria medium. This method allows the gene of interest to be easily modified alone and then to be introduced into EBV-BAC genome. Following this example of gene substitution, other mutations such as deletion, insertion and point mutation become convenient work, and this improved method can be a potential use of gene modification in other BAC-based herpesvirus genome.
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PMID:[Gene modification in the genome of Epstein-Barr virus cloned as a bacterial artificial chromosome]. 1847 68

The UV hypersensitive CHO cell mutant UV41 is the archetypal XPF mammalian cell mutant, and was essential for cloning the human nucleotide excision repair (NER) gene XPF by DNA transfection and rescue. The ERCC1 and XPF genes encode proteins that form the heterodimer responsible for making incisions required in NER and the processing of certain types of recombination intermediates. In this study, we cloned and sequenced the CHO cell XPF cDNA, determining that the XPF mutation in UV41 is a +1 insertion in exon 8 generating a premature stop codon at amino acid position 499; however, the second allele of XPF is apparently unaltered in UV41, resulting in XPF heterozygosity. XPF expression was found to be several-fold lower in UV41 compared to its parental cell line, AA8. Using approaches we previously developed to study intrachromosomal recombination in CHO cells, we modified UV41 and its parental cell line AA8 to allow site-specific gene targeting at a Flp recombination target (FRT) in intron 3 of the endogenous adenine phosphoribosyltransferase (APRT) locus. Using FLP/FRT targeting, we integrated a plasmid containing an I-SceI endonuclease sequence into this site in the paired cell lines to generate a heteroallelic APRT duplication. Frequencies of intrachromosomal recombination between APRT heteroalleles and the structures of resulting recombinants were analyzed after I-SceI induction of site-specific double-strand breaks (DSBs) in a non-homologous insertion contained within APRT homology. Our results show that I-SceI induced a small proportion of aberrant recombinants reflecting DSB-induced deletions/rearrangements in parental, repair-proficient AA8 cells. However, in XPF mutant UV41, XPF heterozygosity is responsible for a similar, but much more pronounced genomic instability phenotype, manifested independently of DSB induction. In addition, gene conversions were suppressed in UV41 cells compared to wild-type cells. These observations suggest that UV41 exhibits a genomic instability phenotype of aberrant recombinational repair, confirming a critical role for XPF in mammalian cell recombination.
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PMID:Characterization of CHO XPF mutant UV41: influence of XPF heterozygosity on double-strand break-induced intrachromosomal recombination. 1854 76

A DNA fingerprinting method known as ALIS-FLP (amplified ligation selected fragment-length polymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs in that only one specific restriction enzyme (TspRI) is used. The cohesive ends of the DNA fragments are ligated with two types of oligonucleotide. A long oligonucleotide containing the primer site and the specific 9 nt 3 prime end, which is complementary to specific 9 nt, cohesive 3 prime end of the TspRI genomic DNA fragment, and a short, degenerated, oligonucleotide covering the remaining TspRI cohesive ends. Other cohesive ends are covered by a short degenerated oligonucleotide lacking the primer site. The ligation mixture is used as a template for amplification using a single primer corresponding to the 5 prime end of the long, specific oligonucleotide. The selection of TspRI digested genomic DNA fragments for amplification is achieved by sequence selective ligation of the specific long oligonucleotide carrying the primer site to both ends of the specific target fragment. This technique allows for differentiation of the organisms without previous knowledge of their DNA sequence. The usefulness of the method is confirmed by genotyping of 70 previously characterized clinical E. coli isolates. The grouping obtained was identical to the results of REA-PFGE. Versatility of the method is highlighted, i.e. its combining the advantages of the AFLP technique with a simple, rapid and cheap polymerase chain reaction product detection method.
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PMID:ALIS-FLP: amplified ligation selected fragment-length polymorphism method for microbial genotyping. 1860 79

We have demonstrated that targeted mutagenesis can be accomplished in maize plants by excision, activation, and subsequent elimination of an endonuclease in the progeny of genetic crosses. The yeast FLP/FRT site-specific recombination system was used to excise and transiently activate the previously integrated yeast I-SceI homing endonuclease in maize zygotes and/or developing embryos. An artificial I-SceI recognition sequence integrated into genomic DNA was analyzed for mutations to indicate the I-SceI endonuclease activity. Targeted mutagenesis of the I-SceI site occurred in about 1% of analyzed F1 plants. Short deletions centered on the I-SceI-produced double-strand break were the predominant genetic lesions observed in the F1 plants. The I-SceI expression cassette was not detected in the mutant F1 plants and their progeny. However, the original mutations were faithfully transmitted to the next generation indicating that the mutations occurred early during the F1 plant development. The procedure offers simultaneous production of double-strand breaks and delivery of DNA template combined with a large number of progeny plants for future gene targeting experiments.
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PMID:Targeted mutagenesis in the progeny of maize transgenic plants. 1946 65


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