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)

Efficient selection of the genetically modified cell population is a critical step to obtain the cells with desired properties. In this study, we propose an antigen-mediated genetically modified cell amplification (AMEGA) system employing an antibody/receptor chimera that triggers a growth signal in response to a non-toxic hapten dimer. An anti-fluorescein single-chain Fv fused to the extracellular D2 domain of erythropoietin receptor and transmembrane/intracellular domains of gp130 was expressed together with a model transgene, enhanced green fluorescent protein (EGFP) downstream of IRES sequence, by retroviral infection to IL-3-dependent Ba/F3 cells. Addition of fluorescein dimers connected by various oligo-DNA linkers induced selective growth of transfectants, thus leading to efficient expansion of EGFP-positive cell population. Also, digestion of the oligonucleotides by specific restriction endonuclease completely suppressed cell growth. Because these hapten dimers are not harmful for normal cells, the approach will be especially useful for reversible in vitro or in vivo expansion of genetically modified cell population employed for cell therapy and tissue engineering.
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PMID:Selection of genetically modified cell population using hapten-specific antibody/receptor chimera. 1501 36

Reciprocal translocations are common in cancer cells, but their creation is poorly understood. We have developed an assay system in Saccharomyces cerevisiae to study reciprocal translocation formation in the absence of homology. We induce two specific double-strand breaks (DSBs) simultaneously on separate chromosomes with HO endonuclease and analyze the subsequent chromosomal rearrangements among surviving cells. Under these conditions, reciprocal translocations via nonhomologous end joining (NHEJ) occur at frequencies of approximately 2-7 x 10(-5)/cell exposed to the DSBs. Yku80p is a component of the cell's NHEJ machinery. In its absence, reciprocal translocations still occur, but the junctions are associated with deletions and extended overlapping sequences. After induction of a single DSB, translocations and inversions are recovered in wild-type and rad52 strains. In these rearrangements, a nonrandom assortment of sites have fused to the DSB, and their junctions show typical signs of NHEJ. The sites tend to be between open reading frames or within Ty1 LTRs. In some cases the translocation partner is formed by a break at a cryptic HO recognition site. Our results demonstrate that NHEJ-mediated reciprocal translocations can form in S. cerevisiae as a consequence of DSB repair.
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PMID:Reciprocal translocations in Saccharomyces cerevisiae formed by nonhomologous end joining. 1502 Apr 64

Small interfering RNAs (siRNAs) potently silence expression of target genes. In principle siRNA libraries can be used to perform effective genome-scale functional genetic screens in mammalian cells, but their development has been hampered by the need to chemically synthesize thousands of oligonucleotides and to incorporate them into expression vectors. We have developed a technology to efficiently convert a double-stranded cDNA library into a retroviral siRNA library in which 21-base siRNAs are produced in infected cells at high levels and efficiently block expression of their target genes. The key steps are the generation of random cDNA fragments that are fused to a hairpin linker, cleavage with the MmeI endonuclease that creates 20- to 21-bp cDNA fragments, conversion to a double-stranded DNA that contains two copies of the cDNA insert in a head-to-head palindrome, and insertion of the construct downstream of a polymerase III promoter. We constructed a siRNA library with 3 x 10(6) clones from a mouse embryo cDNA library; siRNAs were found against many different genes; and multiple siRNAs can be generated from a single mRNA. We further showed that specific siRNAs were efficiently produced in stably infected mammalian cells and resulted in significant and specific reduction of their target mRNAs. Because no prior knowledge about target transcripts is needed, a cDNA-derived siRNA library will generate siRNAs against unknown transcripts and genes. Finally, cDNA-derived siRNA libraries can be readily generated from any cell type or species, enabling genome-wide functional screens in many biological systems.
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PMID:Small interfering RNA production by enzymatic engineering of DNA (SPEED). 1506 16

We assayed error-prone double-strand break (DSB) repair in wild-type and isogenic Mlh1-null mouse embryonic fibroblasts containing a stably integrated DSB repair substrate. The substrate contained a thymidine kinase (tk) gene fused to a neomycin-resistance (neo) gene; the tk-neo fusion gene was disrupted in the tk portion by a 22bp oligonucleotide containing the 18 bp recognition site for endonuclease I-SceI. Following DSB-induction by transient expression of I-SceI endonuclease, cells that repaired the DSB by error-prone nonhomologous end-joining (NHEJ) and restored the correct reading frame to the tk-neo fusion gene were recovered by selecting for G418-resistant clones. The number of G418-resistant clones induced by I-SceI expression did not differ significantly between wild-type and Mlh1-deficient cells. While most DSB repair events were consistent with simple NHEJ in both wild-type and Mlh1-deficient cells, complex repair events were more common in wild-type cells. Furthermore, genomic deletions associated with NHEJ events were strikingly larger in wild-type versus Mlh1-deficient cells. Additional experiments revealed that the stable transfection efficiency of Mlh1-null cells is higher than that of wild-type cells. Collectively, our results suggest that Mlh1 modulates error-prone NHEJ by inhibiting the annealing of DNA ends containing noncomplementary base pairs or by promoting the annealing of microhomologies.
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PMID:Modulation of error-prone double-strand break repair in mammalian chromosomes by DNA mismatch repair protein Mlh1. 1508 8

TP-PCR,a method developed for fusion gene construction without the use of endonuclease and ligase, was performed to construct a fused fpg gene. The TP-PCR reaction system contained three primers and two templates and resulting PCR product, fused fpg gene, consisted of three sections: pheB gene, which was responsible for catechol 2,3-dioxygenase, gfp gene for GFP protein and the intermediate ligation segment which was designed for the correct expression of the fusion gene. The result in this paper showed that the TP-PCR method is one of rapid and convenient methods for fused gene construction.
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PMID:[Construction of a fused fpg gene by using TP-PCR method]. 1564 55

We examined error-prone nonhomologous end joining (NHEJ) in Msh2-deficient and wild-type Chinese hamster ovary cell lines. A DNA substrate containing a thymidine kinase (tk) gene fused to a neomycin-resistance (neo) gene was stably integrated into cells. The fusion gene was rendered nonfunctional due to a 22-bp oligonucleotide insertion, which included the 18-bp I-SceI endonuclease recognition site, within the tk portion of the fusion gene. A double-strand break (DSB) was induced by transiently expressing the I-SceI endonuclease, and deletions or insertions that restored the tk-neo fusion gene's reading frame were recovered by selecting for G418-resistant colonies. Overall, neither the frequency of recovery of G418-resistant colonies nor the sizes of NHEJ-associated deletions were substantially different for the mutant vs. wild-type cell lines. However, we did observe greater usage of terminal microhomology among NHEJ events recovered from wild-type cells as compared to Msh2 mutants. Our results suggest that Msh2 influences error-prone NHEJ repair at the step of pairing of terminal DNA tails. We also report the recovery from both wild-type and Msh2-deficient cells of an unusual class of NHEJ events associated with multiple deletion intervals, and we discuss a possible mechanism for the generation of these "discontinuous deletions."
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PMID:A role for DNA mismatch repair protein Msh2 in error-prone double-strand-break repair in mammalian chromosomes. 1578 95

To express the extracellular fragement of hepatoma associated antigen HAbl8G(HAb18GEF) in E. coli efficiently in a non-fusing way, the cDNA of HAb18GEF gene was inserted into prokaryotic expression vector pET21a + . The secondary structure and codon adaptation of translational initiation region (TIR, from-30 to + 39) in mRNA of recombinant vector HAb18GEF/ pET21a + was predicted simultaneously by computer-aided design. Stable Stem-Loop structures and many low-usage codons were detected in mRNA-TIR of non-optimized recombinant HAb18GEF/pET21a + vector. The stability of mRNA-TIR in recombinant HAb18GEF/pET21a + vector was reduced with following methods: (1) optimization of secondary structure (2) optimization of codon adaptation. These optimization were realized by non-continual site-directed mutagenesis without changing any amino acid sequence in TIR. After being checked through restriction endonuclease digestion and confirmed through nucleotide sequencing, the pre-optimized and post-optimized recombinant vectors were transformed into competent E. coli JM109-DE3. The resulted recombinant clones were selected randomly and induced by IPTG at 37 degrees C. The induced production of these recombinants was analyzed by SDS-PAGE, indirect ELISA, Western blot, and cell fractionation assay. The amount of HAb18GEF mRNA was also detected by RNA dot blot between pre-optimized recombinant and post-optimized recombinant. The results revealed that recombinant non-fused vectors HAb18GEF/pET21a + were successfully constructed and optimized in the secondary structure and codon adaptation of TIR respectively. The HAb18GEF was expressed efficiently in a non-fusing way in recombinant E. coli by secondary structure optimization or codon adaptation optimization. Whereas, no expression of HAb18GEF was detected in pre-optimized recombinants. The non-fused expression products-HAb18GEF, mainly as inclusion body in E. coli, yielded highly above 29.3%. A trait of expression HAb18GEF was also detected both in intermembrane space and in culture medium due to over-expression and cell leakage. Difference in non-fused expression level of HAb18GEF between secondary structure optimization and codon adaptation optimization was negligible. No difference in amount of transcribed mRNA of HAb18GEF between the pre-optimized and the post-optimized recombinants was detected. To sum up, it's feasible to express hepatoma associated antigen HAb18GEF in a non-fusing way by reducing the stability of TIR in mRNA.
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PMID:[Non-fused expression of HAb18GEF by reducing stability of translational initiation region in mRNA]. 1596 4

The ORF of the Cr.psbA4 intron of Chlamydomonas reinhardtii mediates efficient intron homing, and contains an H-N-H and possibly a GIY-YIG motif. The ORF was over-expressed in Escherichia coli without non-native amino acids, but was mostly insoluble. However, co-over-expression of E. coli chaperonins GroEL/GroES solubilized approximately 50% of the protein, which was purified by ion-exchange and heparin-affinity chromatography. Biochemical characterization showed that the protein is a double-strand-specific endonuclease that cleaves fused psbA exon 4-exon 5 DNA, and was named I-CreII. I-CreII has a relatively relaxed divalent metal ion requirement (Mg(2+), Mn(2+), Ca(2+), and Fe(2+) supported cleavage), is insensitive to salt <350 mM, and is stabilized by DNA. Cleavage of target DNA occurs close (4 nt on the top strand) to the intron-insertion site, and leaves 2-nt 3'-OH overhangs, similar to GIY-YIG endonucleases. The boundaries of the recognition sequence span approximately 30 bp, and encompass the cleavage and intron-insertion sites. Cleavage of heterologous psbA DNAs indicates the enzyme can tolerate multiple, but not all, substitutions in the recognition site. This work will facilitate further study of this novel endonuclease, which may also find use in site-specific manipulation of chloroplast DNA.
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PMID:Expression, purification, and biochemical characterization of the intron-encoded endonuclease, I-CreII. 1609 17

A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E. coli DE3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic vaccine against the pathogen infection. The gene encoding PIB was amplified by PCR from Neisseria gonorrhoeae and cloned into prokaryotic expression plasmid pET-28a(+) to construct a pET-PIB recombinant, which was verified by restriction endonuclease and DNA sequencing. Protein PIB was expressed in E. coli DE3 induced with IPTG. The antigenicity of the expressed protein was evaluated by indirect ELISA. Rabbits were immunized with the protein and serum was collected after immunization. To assess the immunogenicity of the protein, the titer of serum to protein PIB was determined by ELISA. DNA sequence analysis showed that the nucleic acid sequence of PIB gene was 99.28% of homology compared with that (NGPIB18) published in GenBank. A 41 kD fused protein was detected by SDS-PAGE and was proven to have reactivity with anti-PIB polyclonal antibody from mouse. A polyclonal antibody to PIB of 1:4000 titer determined by indirect EISA was obtained from rabbit immunized with the purified product. Recombinant plasmid encoding PIB of Neisseria gonorrhoeae was constructed. Protein PIB with antigenicity and immunogenicity was successfully expressed.
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PMID:Construtcion of Neisseria gonorrhoeae porin B plasmid recombinant and its expression in E. coli. 1620 Dec 62

Strains of Ophiostoma ulmi, O. novo-ulmi subsp. americana, O. novo-ulmi subsp. novo-ulmi and O. himal-ulmi were examined for optional introns/insertions within the following mitochondrial genes: small subunit RNA gene (rns), large ribosomal subunit gene (rnl) and the cytochrome oxidase subunit I gene (coxI). Insertions were noted in the rns and coxI genes in strains of O. ulmi, the less aggressive species, but absent in strains of the more aggressive O. novo-ulmi subsp. americana. Strains of all species examined had a group I intron present in the U11 region of the mitochondrial-rnl gene. In all but two strains of O. novo-ulmi subsp. americana, this rnl-U11 intron was about 1.5 kb in length whereas a 2.6 kb version of this element was present in all strains representing O. ulmi, O. novo-ulmi subsp. novo-ulmi, and Ophiostoma himal-ulmi. Irrespective of size, this intron based on RNA folds is a class IA1 group I intron and it encodes a putative ORF for the rps3 ribosomal protein. The size variation of the rnl-U11 intron was examined in detail for two strains of O. novo-ulmi subsp. americana and sequence data suggests the presence of a complex ORF within the 2.6 kb version of this intron; here a homing endonuclease-like gene has been inserted in frame and fused to the carboxyl-terminus of the putative rps3 coding region. The mitochondrial optional introns/insertions in combination with nuclear markers might be useful in distinguishing among the various species and subspecies of the O. ulmi s. lat. complex.
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PMID:Optional mitochondrial introns and evidence for a homing-endonuclease gene in the mtDNA rnl gene in Ophiostoma ulmi s. lat. 1627 6


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