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)

We have developed a simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, we have constructed Chinese hamster cell lines that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure. Analysis of these cell lines with Southern blots confirmed the presence of a small number of restriction endonuclease fragments containing human DNA specifically. These cell lines represent a convenient and simple means to clone the human genomic sequences of interest.
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PMID:Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases. 634 61

The effect of mitochondrial DNA (mtDNA) on the phenotypic expression of tumorigenicity was examined by its cytoplasmic transmission from nontumorigenic cells to tumorigenic cells. Enucleated cells of the rat embryonic cell line 3Y1CAP, which are nontumorigenic and resistant to chloramphenicol, were fused with whole cells of the rat glioma C6BU-1 line, which are tumorigenic and resistant to 5-bromodeoxyuridine, and the cybrid colonies growing in selective medium with 5-bromodeoxyuridine (30 micrograms/ml) and chloramphenicol (50 micrograms/ml) were isolated clonally. Cytoplasmic transmission of 3Y1CAP mtDNA to C6BU-1 cells was confirmed by quantitative analysis of their mtDNA with restriction endonuclease. Subclones containing various amounts of mtDNA from 3Y1CAP cells were isolated from one cybrid clone, Y22, and their tumorigenicities were examined by inoculating 2 X 10(6) cells s.c. into nude mice. The tumorigenicities of these cybrid cubclones were almost identical to that of the nuclear donor C6BU-1 cells with respect to the tumor incidence (number of animals bearing tumors per number of animals inoculated), latent period, and growth rates of tumors. Moreover, analysis of chromosomes and mtDNAs of the cells recovered from the tumors obtained showed that the tumors were derived from the cells inoculated and that no selective overgrowth of segregants that had lost mtDNA from 3Y1CAP cells occurred in the nude mice. These observations suggest that expression of tumorigenicity of C6BU-1 cells was not suppressed by cytoplasmic transmission of nontumorigenic 3Y1CAP mtDNA.
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PMID:Effect of mitochondrial DNA transmitted cytoplasmically from nontumorigenic to tumorigenic rat cells on the phenotypic expression of tumorigenicity. 674 11

Using the promotor-cloning vehicle described by An and Friesen (J. Bacteriol. 140:400-410, 1979), Escherichia coli chromosomal deoxyribonucleic acid fragments derived from the lambda drifd18 transducing phage were cloned in one of several unique restriction endonuclease sites adjacent to tetracycline(tet) genes that lack their own promotor. One of these plasmids has been used to isolate nine variants having mutations that lie in a putative internal promoter which is located between rplL and rpoB. Deoxyribonucleic acid sequence analysis revealed that, in all nine mutants, a single base change, C to T, in the ribonucleic acid polymerase recognition site led to a large increase in promoter activity. Analysis of a variety of plasmids in which tet is fused to various promoters yielded the following results: (i) rplK and rplA, genes for ribosomal protein L11 and L1, respectively, were cotranscribed from a common promoter located upstream from rplK; (ii) there was a strong promoter in the region between the rplKA operon and rplJ, the gene for ribosomal protein L10; (iii) an attenuator region was located between rplL, the gene for ribosomal protein L12, and rpoB, the gene for ribonucleic acid polymerase subunit beta; (iv) transcription terminated immediately after rpoC, the gene for ribonucleic acid polymerase subunit beta'; (v) a gene coding for unknown protein U, which is located between tufB and the rplKA operon, had its own promoter; (vi) the tufB gene was separated from all of the genes described above and had its own promoter.
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PMID:Characterization of promoter-cloning plasmids: analysis of operon structure in the rif region of Escherichia coli and isolation of an enhanced internal promoter mutant. 700 14

Chemically synthesized leu-enkephalin gene was fused to a large Eco RI-Bam HI fragment of pBR322 along with a Eco RI fragment of Ch4A phage DNA carrying the promoter and most of the E.coli beta-galactosidase gene. The resulting recombinant DNA was used to transform E. coli cells. Transformants were screened for Tc-sensitivity, Am-resistance, and beta-galactosidase constitutional synthesis. Restriction endonuclease analysis combined with DNA sequencing of the plasmid DNAs revealed a complete nucleotide leu-enkephalin sequence and Eco RI lac-operon fragment in two possible orientations. Radioimmunoassay for leu-enkephalin activity in BrCN-treated bacterial extracts showed that in vivo leu-enkephalin is synthesized only in strains carrying plasmids with the proper lac-fragment orientation. About 5.10(4) molecules of the former are synthesized per single E. coli cell. One of the clones was used for leu-enkephalin purification. Using 100 g of cells it is possible to obtain about 2 mg of practically pure leu-enkephalin.Images
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PMID:Leu-enkephalin purification from E. coli cells carrying the plasmid with fused synthetic leu-enkephalin gene. 700 33

Hybrid plasmids that replicate in both Escherichia coli and Streptomyces lividans were constructed in vitro by joining the E. coli-derived plasmid pACYC184 or pACYC177, at their BamHI or PstI restriction site, respectively, to S. lividans plasmid pSLP111. After introduction of the composite replicons into S. lividans by transformation, chloramphenicol (Cm) resistance encoded by pACYC184 and kanamycin resistance encoded by pACYC177 were phenotypically expressed in the S. lividans host. A Sau3A restriction endonuclease-generated deoxyribonucleic acid fragment from pACYC184 containing the entire structural gene for the Cm acetyltransferase enzyme, but lacking the nucleotide sequence ordinarily serving as the Cm resistance gene promoter, also specified resistance to Cm when introduced in either orientation into the BamHI or BclI endonuclease cleavage site of pSLP111 or into corresponding sites of the analogous plasmid pSLP101. These findings make it unlikely that the biologically active CM acetyltransferase was being made in S. lividans as part of a fused protein, but instead indicate that the ATG start codon used for initiation of translation of the Cm resistance gene in E. coli was also utilized in S. lividans. In contrast, the synthesis of messenger ribonucleic acid that encodes the Cm acetyltransferase in S. lividans was, in at least one instance, apparently initiated at nucleotide sequences within the S. lividans plasmid vector, with resulting transcriptional read-through into the E. coli-derived deoxyribonucleic acid segment.
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PMID:Cloning and expression in streptomyces lividans of antibiotic resistance genes derived from Escherichia coli. 701 22

We have utilized RT-PCR to clone the porcine p55TNFR cDNA, encoding the 55-kDa tumor necrosis factor receptor (TNFR), encompassing the entire coding region and most of the 3' untranslated region. PCR was performed using total cellular RNA of porcine kidney cell line 15 [PK(15)] and primers for the human p55TNFR. Since the length of the entire fragment was over 2000 bp, we fused two amplified subfragments with the help of a restriction endonuclease. The entire fragment was cloned and its amino acid (aa) sequence was compared to the human, rat and mouse p55TNFR. This comparison revealed identities of 79, 71 and 72%, respectively. The highest identities of 90, 80 and 85% were detected in the so called "death domain" for the human, rat and mouse sequences, respectively. This domain is crucial for the cytotoxic signal transduction of p55TNFR.
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PMID:Cloning of the cDNA encoding the porcine p55 tumor necrosis factor receptor. 759 Feb 78

The polymerase-chain-reaction technique is used to produce fusion proteins via deletion of any intervening piece of DNA. Here a stretch of six histidine codons is fused to the 3'-terminus of any defined gene using a standard plasmid vector or a derivative thereof. The advantage over existing methods is that no other amino acids besides the six histidines are added to the protein terminus and only one oligonucleotide needs to be synthesized as special primer. Genes of interest must only be cloned in the correct orientation into a universal multilinker. Using just one specific primer derived from the 3'-terminus of the gene and one standard primer derived from the six histidine codons the fusion is performed by amplifying the entire vector system as described for inverse PCR. As an example, we report on the modification and purification of the restriction endonuclease HgiBI (GGWCC). Enzymatically active protein was obtained in a single step purification under nondenaturating conditions with a purity greater than 95% according to polyacrylamide gel electrophoresis.
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PMID:PCR directed preparation and single step purification of highly active histidine-tagged restriction endonuclease HgiBI (GGWCC). 783 56

In eukaryotes, horizontal gene transfer is a rare event. Here we show that the mitochondrial genome of a lower fungus, Allomyces macrogynus, has an extra DNA segment not present in a close relative, Allomyces arbusculus. This insert consists of the C terminus of a foreign gene encoding a subunit of the ATP synthetase complex (atp6) plus an open reading frame encoding an endonuclease. The inserted atp6 portion is fused in phase to the resident gene, resulting in expression of a hybrid atp6 gene and the displacement of the original C-terminal atp6 region. We present evidence that this insertion may have been acquired by interspecific transfer and we discuss the possible role of the endonuclease in this process.
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PMID:Interspecific transfer of mitochondrial genes in fungi and creation of a homologous hybrid gene. 799 39

Mobile group I introns are hypothesized to have arisen after invasion by endonuclease-encoding open reading frames (ORFs), which mediate their mobility. Consistent with an endonuclease-ORF invasion event, we report similarity between exon junction sequences (the recognition site for the mobility endonuclease) and intron sequences flanking the endonuclease ORF in the sunY gene of phage T4. Furthermore, we have demonstrated the ability of the intron-encoded endonuclease to recognize and cleave these intron sequences when present in fused form in synthetic constructs. These observations and accompanying splicing data are consistent with models in which the invading endonuclease ORF is provided safe haven within a splicing element. In turn the intron is afforded immunity to the endonuclease product, which imparts mobility to the intron.
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PMID:Evolution of mobile group I introns: recognition of intron sequences by an intron-encoded endonuclease. 799 69

To define the mechanisms of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transcription of the ornithine decarboxylase (ODC) gene, we isolated a genomic clone (hODC41B) of ODC from a human leukocyte genomic DNA library. The restriction endonuclease map, in comparison with the previously published sequences of the human ODC gene, indicated that hODC41B contained a 15.7-kb sequence that extended from the sixth exon to about 10 kb upstream of the ODC gene. A 2.5-kb genomic fragment containing the 5' flanking region and the first exon was subcloned and sequenced. Sequence analysis revealed multiple putative promoter/enhancer elements (a TATA box, a CAAT box, 17 GC boxes, and a cAMP-responsive element) but no consensus AP-1 sequences (TGAGTCA) in the 2.5-kb 5' flanking region. However, three AP-1 sequences were located in introns 3, 5, and 11. We constructed a series of chimeric genes containing part of the first exon and increasingly longer 5' flanking sequences of the ODC gene fused to either bacterial chloramphenicol acetyltransferase (CAT) or luciferase reporter genes. TPA inducibility was determined by transient transfection and measurement of CAT or luciferase expression in HeLa cells. The induction of CAT activity by TPA decreased with decreasing lengths of the 5' flanking sequences up to nt -82. The TPA induction from the construct -72 ODC CAT was threefold to sevenfold, and the TPA inducibility of the same fragment was about ninefold to 30-fold with the luciferase reporter gene. Further deletion analysis revealed TPA-responsive sequences in ODC nt -42 to +54. Gel mobility shift assays using alpha-32P-end labeled ODC nt -42 to +60 revealed that nt -42 to +60 specifically bound HeLa cell nuclear proteins. HeLa cell nuclear protein binding to ODC nt -42 to +60 could not be completely competed by AP-1-, AP-2-, AP-3-, or SP1-responsive sequences.
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PMID:Non-AP-1 tumor promoter 12-O-tetradecanoylphorbol-13-acetate-responsive sequences in the human ornithine decarboxylase gene. 804 98


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