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)

Fourteen human interleukin-2 (IL-2) analogs have been cloned and expressed in E. coli, starting from a chemically synthesized gene for human IL-2 optimized for expression in E. coli. These analogs were purified to greater than 95% purity as determined by SDS-PAGE, and were measured for biological activity in a 3H-thymidine incorporation assay using an IL-2 dependent murine T-cell line (CTLL). One analog was made which eliminated the N-terminal 23 amino acids from the protein by replacing one restriction endonuclease fragment with another. This analog, which begins at an internal methionine, had no detectable CTLL activity. Thirteen analogs were constructed using oligonucleotide site-directed mutagenesis. Four of these analogs were truncated at various residues near the C-terminus (residues 106, 116, 121 and 126). These analogs had at least 500-fold lower CTLL activities than the natural recombinant IL-2. The remaining nine analogs had substitutions at 1, 2, or all 3 of the three cysteine residues in the protein (residues 58, 105 and 125). Substituting an alanine, asparagine, aspartic acid, or serine at residue 125 resulted in highly active molecules with CTLL activities similar to that of the natural recombinant IL-2. The analogs with alanine and serine substitutions at residue 125 actually had slightly higher CTLL activities than the natural recombinant IL-2. Substituting alanine for cysteine at position 125 and serine for cysteine at either position 58 or 105 yielded analogs with about 150-fold lower CTLL activities than natural recombinant IL-2. Substituting an alanine for the cysteine at position 125 and serines for cysteines at both positions 58 and 105 resulted in an analog with 30-fold lower CTLL activity than the natural recombinant IL-2. The ten analogs with less than 1.0% of the CTLL activity of natural recombinant IL-2 were tested for competition with the natural recombinant IL-2 by mixing a 10-to 100- fold excess of the analog with the natural recombinant IL-2 and assaying the mixture in the CTLL assay. None of these analog mixtures resulted in a lower activity than mixing the natural recombinant IL-2 with buffer alone, implying that none of these analogs effectively competes with the natural recombinant IL-2 for binding to IL-2 receptors during incubation with the CTLL cells. If reduced binding does occur, it may be the direct cause of their lower activities.
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PMID:Construction, purification and biological activities of recombinant human interleukin-2 analogs. 306 70

The Eco RI endonuclease and methylase recognize the same hexanucleotide substrate sequence. We have determined the sequence of a fragment of DNA which encodes these enzymes using the chain-termination method of Sanger (Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467). The amino acid sequences of both enzymes were derived from the DNA sequence. The coding regions selected include the only open translational frames of sufficient length to accommodate the enzymes. They coincide with previously established gene boundaries and orientation. The predicted amino acid sequences correlate well with analyses of the purified protein. Comparison of the nucleotide and protein sequences reveals no homology between the endonuclease and methylase which might provide insight into the origin of the restriction-modification system or the mechanism of common substrate recognition. Based on secondary structure predictions, the two enzymes also have grossly different molecular architecture. The base composition of the sequence is 65% A + T, and the codon usage is significantly different from that observed in several Escherichia coli chromosomal genes. In some cases, frequently selected codons are recognized by minor tRNA species. A spontaneous mutation in the endonuclease gene was isolated. Serine replaces arginine at residue 187. In crude extracts, Eco RI specific cleavage is approximately 0.3% wild type.
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PMID:Sequence analysis of the DNA encoding the Eco RI endonuclease and methylase. 625 3

Four cosmid clones, each with an average insert size of 40 kilobase pairs and containing the factor B gene, were isolated from a human genomic DNA library. The clones were identified by hybridization with a 515-base-pair cDNA probe isolated by using a unique 17-base synthetic oligonucleotide probe from a human liver cDNA library. The cosmid clones were characterized by restriction endonuclease digestion and Southern blotting, and a partial restriction map of the DNA represented in the cosmids was constructed. The Bb portion of the factor B gene is about 4 kb in length. DNA sequence analysis has resulted in the determination of 3.3 kb of sequence at the 3' end of the gene. This region codes for amino acids 87-505 of Bb and includes the whole of the serine proteinase domain of the protein. The three active site residues of histidine, aspartic acid, and serine found at positions 267, 317, and 440 of the Bb sequence, respectively, lie on separate exons. Other functional regions within the serine proteinase domain are separated also by intervening sequences.
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PMID:Molecular cloning and characterization of the gene coding for human complement protein factor B. 630 26

We have identified a patient with mild diabetes, marked fasting hyperinsulinemia (89 to 130 microU of insulin per milliliter), and a reduced fasting C-peptide: insulin molar ratio of 1.11 to 1.50 (normal, greater than 4). The patient responded normally to exogenous insulin. However, her endogenous immunoreactive insulin showed reduced biologic activity during a glucose-clamp study with hyperglycemia and a reduced ability to bind to the insulin receptor and stimulate glucose transport in vitro. Family studies showed that five additional relatives in three generations had variable degrees of glucose intolerance, marked hyperinsulinemia, and a reduced peripheral C-peptide:insulin molar ratio. Restriction-endonuclease cleavage of DNA isolated from circulating leukocytes in the patient and in family members with hyperinsulinemia revealed loss of the MboII recognition site in one allele of the insulin gene--consistent with a point mutation at position 24 or 25 in the insulin B chain. Other studies using high-pressure liquid chromatography and detailed gene analysis have identified the defect as a serine for phenylalanine substitution at position 24 of the insulin B chain. The secretion of a structurally abnormal insulin should be considered in patients with hyperinsulinemia who respond normally to exogenous insulin and have a reduced C-peptide:insulin molar ratio. Glucose tolerance may range from relatively normal to overtly diabetic.
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PMID:Familial hyperinsulinemia due to a structurally abnormal insulin. Definition of an emerging new clinical syndrome. 637 26

Although multiple chromosomes occur in bacteria, much remains to be learned about their structural and functional interrelationships. To study the structure-function relationships of chromosomes I and II of the facultative photosynthetic bacterium Rhodobacter sphaeroides 2.4.1T, auxotrophic mutants were isolated. Five strains having transposon insertions in chromosome II showed requirements for p-aminobenzoic acid (pABA)-dihydroxybenzoic acid (dHBA), serine, thymine, uracil, or histidine. The His, Thy, and pABA-dHBA mutants reverted to prototrophy at low frequency and concordantly lost their transposon insertions from the genome. The Ser, Ura, and pABA-dHBA mutants were complemented by cosmids that carried the region of chromosome II where the transposon insertions were located. The cosmids used for complementation analysis were selected, on the basis of map position, from a set of overlapping clones that had been ordered by a combination of hybridization and restriction endonuclease mapping. These experiments provide the basis for detailed studies of the structure, function, and interaction between each chromosome, and they demonstrate at this early stage of investigation that no fundamental differences exist between each chromosome.
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PMID:Multiple chromosomes in bacteria: structure and function of chromosome II of Rhodobacter sphaeroides 2.4.1T. 800 95

Three chemically distinct serine, but not cysteine, protease inhibitors (phenylmethylsulphonyl fluoride, N-tosyl-L-phenylalanylchloromethyl ketone and 3,4-dichloroisocoumarin) prevented, in a dose-dependent manner, the characteristic apoptotic internucleosomal DNA cleavage (DNA ladder) typically observed in thymocytes in response to dexamethasone and teniposide VM-26. This effect was not the result of a direct inhibition of the Ca2+,Mg(2+)-dependent endonuclease, since oligonucleosomal DNA cleavage occurred in the presence of these inhibitors in isolated nuclei. The proteolytic step occurred at a very early stage of apoptosis, and preincubation of thymocytes with the inhibitors before dexamethasone or teniposide VM-26 were added irreversibly suppressed ladder formation. This implied that the cellular effector(s) of these compounds preexisted and were not resynthesized in response to the inducers of apoptosis. Serine protease inhibitors also suppressed apoptotic cell shrinkage and complete nuclear collapse, suggesting that these morphological changes were directly related to internucleosomal fragmentation of DNA. However, the serine protease inhibitors did not prevent high molecular weight DNA cleavage (> 50 kilobases) that preceded the ladder formation and thymocytes still died by apoptosis. This supported the view that internucleosomal DNA cleavage, considered to be the biochemical marker of apoptosis, might in fact be a late and dispensable step and that the newly described high molecular weight DNA cleavage might be a better indicator of apoptosis.
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PMID:Role of proteolysis in apoptosis: involvement of serine proteases in internucleosomal DNA fragmentation in immature thymocytes. 819 1

Escherichia coli RuvC protein is a specific endonuclease that resolves recombination intermediates into viable products. The structural features needed for RuvC activity were investigated by sequencing three ruvC mutations and relating the base pair changes identified to the activity of the mutant proteins. Each of the three mutations is a single base-pair substitution. ruvC51 converts glycine-15 to an aspartic acid residue. The product of ruvC51 was purified and shown to retain the ability to bind junctions, albeit with a slightly reduced affinity. However, it has lost the ability to resolve these structures by symmetrical cleavage. A multicopy ruvC51 plasmid confers sensitivity to UV light in a ruvC+ strain. The ruvC53 allele causes a glycine-17 to serine substitution while ruvC55 produces a stop codon. Neither of these genes produces a stable product. The results suggest that the N-terminal domain of RuvC may be concerned with cleavage of junctions.
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PMID:An E. coli RuvC mutant defective in cleavage of synthetic Holliday junctions. 839 86

Proteolysis is an early event of apoptosis which appears to be associated with activation of the endonuclease which is responsible for internucleosomal DNA cleavage. The present study was designed to reveal the possible role of proteolysis in other early events, such as chromatin condensation, nuclear breakdown, and destabilization of in situ DNA double-stranded structure. Apoptosis of human leukemic HL-60 cells and rat thymocytes was induced by different agents, including DNA topoisomerase inhibitors, an RNA antimetabolite, and the glucocorticosteroid, prednisolone. DNA degradation was evaluated by pulsed field and conventional gel electrophoresis and by the presence of in situ DNA strand breaks. DNA stability was estimated by the measure of its sensitivity in situ to denaturation. Chromatin condensation, nuclear breakdown, and other morphological changes were monitored by interference contrast and UV microscopy following cell staining with the DNA-specific fluorochrome 4',6-diamidino-2- phenylindole. Several irreversible or reversible serine protease inhibitors prevented internucleosomal DNA degradation, nuclear breakdown, and destabilization of DNA double-stranded structure. The effective inhibitors, however, did not prevent the onset of chromatin condensation, nor the loss of the fine structural framework, nor the initial step of DNA cleavage generating DNA fragments of >=50 kb in size. The data indicate that in both cell systems the activity of proteases sensitive to the inhibitors tested is needed for internucleosomal DNA cleavage to occur. The data also suggest that these proteases may be involved in dissolution of the nuclear envelope. Because nuclear matrix proteins and histones stabilize DNA in situ, and the decrease in DNA stability which occurs during apoptosis is precluded by the inhibitors, it is likely that serine proteases may degrade DNA stabilizing proteins. The activity of these proteases, however, appears needed neither for DNA cleavage to >=50-kb fragments nor for the onset of chromatin condensation which is associated with dissolution of the structural framework of the nucleus.
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PMID:Effect of protease inhibitors on early events of apoptosis. 860 14

Analysis of 94 kb of DNA, located between map positions 88 and 182 kb in the 330-kb chlorella virus PBCV-1 genome, revealed 195 open reading frames (ORFs) 65 codons or longer. One hundred and five of the 195 ORFs were considered major ORFs. Twenty-six of the 105 major ORFs resembled genes in the databases including three chitinases, a chitosanase, three serine/threonine protein kinases, two additional protein kinases, a tyrosine protein phosphatase, two ankyrins, an ornithine decarboxylase, a copper/zinc-superoxide dismutase, a proliferating cell nuclear antigen, a DNA polymerase, a fibronectin-binding protein, the yeast Ski2 protein, an adenine DNA methyltransferase and its corresponding DNA site-specific endonuclease, and an amidase. The genes for the 105 major ORFs were evenly distributed along the genome and, except for one noncoding 1788-nucleotide stretch, the genes were close together. Unexpectedly, a 900-bp region in the 1788-bp noncoding sequence resembled a CpG island.
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PMID:Analysis of 94 kb of the chlorella virus PBCV-1 330-kb genome: map positions 88 to 182. 861 77

We studied the role of proteases in apoptosis using a cell-free system prepared from a human leukemia cell line. HL60 cells are p53 null and extremely sensitive to a variety of apoptotic stimuli including DNA damage induced by the topoisomerase I inhibitor, camptothecin. We measured DNA fragmentation induced in isolated nuclei by cytosolic extracts using a filter elution assay. Cytosol from camptothecin-treated HL60 cells induced internucleosomal DNA fragmentation in nuclei from untreated cells. This fragmentation was suppressed by serine protease inhibitors. Serine proteases (trypsin, endoproteinase Glu-C, chymotrypsin A, and proteinase K) and papain by themselves induced DNA fragmentation in naive nuclei. This effect was enhanced in the presence of cytosol from untreated cells. Cysteine protease inhibitors (E-64, leupeptin, Ac-YVAD-CHO [ICE inhibitor]) did not affect camptothecin-induced DNA fragmentation. The apopain/Yama inhibitor, Ac-DEVD-CHO, and the proteasome inhibitor, MG-132, were also inactive both in the cell-free system and in whole cells. Interleukin-1 beta converting enzyme (ICE) or human immunodeficiency virus protease failed to induce DNA fragmentation in naive nuclei. Together, these results suggest that DNA damage activates serine protease(s) which in turn activate(s) nuclear endonuclease(s) during apoptosis in HL60 cells.
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PMID:DNA fragmentation induced by protease activation in p53-null human leukemia HL60 cells undergoing apoptosis following treatment with the topoisomerase I inhibitor camptothecin: cell-free system studies. 880 33


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