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 gene for L-lactate dehydrogenase (LDH) (EC 1.1.1.27) of Thermus caldophilus GK24 was cloned in Escherichia coli using synthetic oligonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of the LDH was deduced from the nucleotide sequence. The deduced amino acid sequence agreed with the NH2-terminal and COOH-terminal sequences previously reported and the determined amino acid sequences of the peptides obtained from trypsin-digested T. caldophilus LDH. The LDH comprised 310 amino acid residues and its molecular mass was determined to be 32,808. On alignment of the whole amino acid sequences, the T. caldophilus LDH showed about 40% identity with the Bacillus stearothermophilus, Lactobacillus casei and dogfish muscle LDHs. The T. caldophilus LDH gene was expressed with the E. coli lac promoter in E. coli, which resulted in the production of the thermophilic LDH. The gene for the T. caldophilus LDH showed more than 40% identity with those for the human and mouse muscle LDHs on alignment of the whole nucleotide sequences. The G + C content of the coding region for the T. caldophilus LDH was 74.1%, which was higher than that of the chromosomal DNA (67.2%). The G + C contents in the first, second and third positions of the codons used were 77.7%, 48.1% and 95.5% respectively. The high G + C content in the third base caused extremely non-random codon usage in the LDH gene. About half (48.7%) the codons in the LDH gene started with G, and hence there were relatively high contents of Val, Ala, Glu and Gly in the LDH. The contents of Pro, Arg, Ala and Gly, which have high G + C contents in their codons, were also high. Rare codons with U or A as the third base were sometimes used to avoid the TCGA sequence, the recognition site for the restriction endonuclease, TaqI. Two TCGA sequences were found only in the sequence of CTCGAG (XhoI site) in the sequenced region of the T. caldophilus DNA. There were three segments with similar sequences in the two 5' non-coding regions, probably the promoter and ribosome-binding regions, of the genes for the T. caldophilus LDH and the Thermus thermophilus 3-isopropylmalate dehydrogenase.
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PMID:Nucleotide sequence and characteristics of the gene for L-lactate dehydrogenase of Thermus caldophilus GK24 and the deduced amino-acid sequence of the enzyme. 353 39

From skin of Xenopus laevis, cDNA libraries were constructed and clones coding for the precursors of caerulein were isolated and sequenced. Using restriction endonuclease digestions, three different types of preprocaerulein cDNAs could be discerned. These were termed types I, III, and IV in accordance with the number of caerulein copies present in the sequence, the type III being the most abundant one. An incomplete copy of a fourth variant, termed type I', was also found. Besides deletions/insertions encompassing one or two caerulein sequences, these types also differ from each other by several point mutations. In the homologous precursor polypeptides deduced from the nucleotide sequence of these cloned cDNAs, the caerulein copies are flanked by complex processing sequences. These are Arg-Arg-Phe-Ala-Asp-Gly or Arg-Arg-Asp-Gly at the amino-terminal side and Gly-Arg-Arg at the carboxyl end. Between caerulein copies, highly homologous segments are present both at the polypeptide and cDNA level. This homology is evident both within a given precursor, where up to three such segments are present, and between the different types of precursors. We conclude that preprocaerulein cDNAs in the skin of X. laevis represent a small family, at least part of which is derived from different genes rather than being formed by alternative splicing of pre-mRNAs.
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PMID:Sequence of preprocaerulein cDNAs cloned from skin of Xenopus laevis. A small family of precursors containing one, three, or four copies of the final product. 375 78

The two apocytochrome c proteins of yeast are coded for by separate genes. Iso-2-cytochrome c differs from the iso-1 protein at 17 positions within a homologous sequence of 108 amino acids. The previously cloned iso-1-cytochrome c coding sequence has been used to identify lambda-yeast recombinant phage containing the gene for iso-2-cytochrome c. The latter protein contains the dipeptide Ala-Ala which is coded for by the nucleic acid sequence G-C-N-G-C-N. The recognition specificity of restriction endonuclease Fnu4HI for G-C-N-G-C provided a rapid means of locating the region of the cloned fragment which codes for iso-2-cytochrome c. The DNA sequence of this gene has been determined and compared with that of the iso-1-cytochrome c locus. There is no intervening sequence within the gene for iso-2-cytochrome c. At 45 of the 91 positions for which iso-1- and iso-2-cytochrome c have the same amino acid, the codons differ. Such third position variation does not occur within the region coding for amino acids 70-80, the protein sequence that is also most conserved among all eukaryotic cytochromes c.
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PMID:Isolation and sequence of the gene for iso-2-cytochrome c in Saccharomyces cerevisiae. 624 66

NH2- and COOH-terminal amino acid sequences of the Eco RI restriction and modification enzymes have been determined. The results allow localization of the coding regions within the DNA segment which controls activity of both enzymes. Processing of the endonuclease is limited to removal of NH2-terminal formylmethionine whereas, in the case of the methylase, formylMet-Ala is removed.
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PMID:Partial NH2- and cooh-terminal sequence analyses of Eco RI DNA restriction and modification enzymes. 625 2

The gene for the H-2K class I antigen of the bm1 variant was cloned and analyzed at the DNA level and compared with the previously cloned parent B6/Kh gene. Sequence determination and comparative restriction endonuclease studies indicate that Kbm1 is derived from the Kb gene. Seven nucleotide changes within a 13-nucleotide stretch distinguish the mutant from the parent gene and result in amino acid differences at positions 152, 155, and 156 in the antigen. The data confirm previously reported changes at amino acid positions 155 and 156 (arginine to tyrosine and leucine to tyrosine, respectively) and extend the altered region to include two nucleotides encoding a glutamate to alanine substitution at amino acid 152, a change not detected by the protein studies because of limitations of the methods used. The DNA sequence encoding this region of the Kbm1 glycoprotein is identical to the DNA sequence of at least one other known class I gene in the mouse, a finding consistent with the hypothesis that the mutation was not a random event but may be the result of a block transfer of information by a copy mechanism analogous to gene conversion. As the sequence analysis of the coding region for the first 273 amino acid residues shows identity between parent and mutant except for the seven nucleotide changes, all variant-parent functional differences must depend only on the cluster of three amino acid differences in the second domain of the Kb glycoprotein.
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PMID:Comparison of the cloned H-2Kbm1 variant gene with the H-2Kb gene shows a cluster of seven nucleotide differences. 630 Aug 87

Blood group antigens are structural variants in surface carbohydrate or amino acid polymorphisms on extracellular domains of membrane proteins. The red cell water channel-forming integral protein (Aquaporin CHIP) is a homotetramer with only one N-glycosylated subunit, however no CHIP-associated blood group antigens have yet been identified. Immunoblotting, monosaccharide composition analysis, and selective glycosidase digestions revealed that the CHIP-associated oligosaccharide contains ABH determinants and resembles a band 3-type glycan that cannot be cleaved from intact membranes by Peptide:N-glycosidase F. The molecular structure of the Colton antigens was previously unknown, but CHIP was selectively immunoprecipitated with anti-Coa or anti-Co(b). The DNA sequence from Colton-typed individuals predicted that residue 45 is alanine in the Co(a+b-) phenotype and valine in the Co(a-b+) phenotype. The nucleotide polymorphism corresponds to a PflMI endonuclease digestion site in the DNA from Co(a-b+) individuals. These studies have defined antigens within two blood group systems on CHIP: (a) an ABH-bearing polylactosaminoglycan attached to a poorly accessible site in the native membrane; and (b) the Colton antigen polymorphism which may permit the identification of rare individuals with defective water channel expression.
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PMID:Human red cell aquaporin CHIP. I. Molecular characterization of ABH and Colton blood group antigens. 752 82

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 alpha-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472-1481). To further probe the structure-function relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of alpha-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.
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PMID:Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding. 753 47

The catalytic center of the restriction endonuclease (ENase) EcoRI is structurally homologous to that of EcoRV, BamHI and PvuII. Each of these ENases contains a short motif of three to four amino acid (aa) residues which are positioned in a similar orientation to the scissile phosphodiester bond. We have mutated these aa (Pro90, Asp91, Glu111 and Lys113) in EcoRI to determine their individual roles in catalysis. The replacement of Asp91 and Lys113, respectively, by conservative mutations (Ala91, Asn91, Ala113, Gln113, His113 and Leu113) resulted in a reduction of binding affinity and complete loss of cleavage activity. Only Lys113-->Arg substitution still allows to cleave DNA, albeit with a rate reduced by at least four orders of magnitude. Lys113 seems to stabilize the structure of the wild-type (wt) ENase since all five ENase variants with mutations at this position show a strongly enhanced tendency to aggregate. The Ala and Gln mutants of Glu111 bind the recognition sequence slightly stronger than wt EcoRI and cleave it with a low, but detectable rate. Only the Glu111-->Lys mutant, in which the charge is reversed, shows neither binding nor cleavage activity. Pro90 is not important for catalysis, because the Ala90 mutant cleaves DNA with an only slightly reduced rate. Under star conditions, however, this mutant is even more active than wt EcoRI. Therefore, the charged aa Asp91, Glu111 and Lys113 are essential for catalytic activity of the EcoRI ENase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Site-directed mutagenesis in the catalytic center of the restriction endonuclease EcoRI. 760 70

HAP1 protein, the major apurinic/apyrimidinic (AP) endonuclease in human cells, is a member of a homologous family of multifunctional DNA repair enzymes including the Escherichia coli exonuclease III and Drosophila Rrp1 proteins. The most extensively characterised member of this family, exonuclease III, exhibits both DNA- and RNA-specific nuclease activities. Here, we show that the RNase H activity characteristic of exonuclease III has been conserved in the human homologue, although the products resulting from RNA cleavage are dissimilar. To identify residues important for enzymatic activity, five mutant HAP1 proteins containing single amino acid substitutions were purified and analysed in vitro. The substitutions were made at sites of conserved amino acids and targeted either acidic or histidine residues because of their known participation in the active sites of hydrolytic nucleases. One of the mutant proteins (replacement of Asp-219 by alanine) showed a markedly reduced enzymatic activity, consistent with a greatly diminished capacity to bind DNA and RNA. In contrast, replacement of Asp-90, Asp-308 or Glu-96 by alanine led to a reduction in enzymatic activity without significantly compromising nucleic acid binding. Replacement of His-255 by alanine led to only a very small reduction in enzymatic activity. Our data are consistent with the presence of a single catalytic active site for the DNA- and RNA-specific nuclease activities of the HAP1 protein.
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PMID:Site-directed mutagenesis of the human DNA repair enzyme HAP1: identification of residues important for AP endonuclease and RNase H activity. 778 8


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