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951.
Scanning mutagenesis is an attractive tool for protein structure-function correlation analysis. With one round of this method it is possible to obtain a library containing all possible single-residue mutants of the protein of interest. The practical application of this approach is currently limited by the large number and cost of the required 30-35mer oligonucleotides. As an alternative, we studied the ligation of shorter DNA oligonucleotides (6-11mer) containing a degenerate binding site and a desired mutation mismatch to a nested set of megaprimers annealed to the gene of interest. T4 DNA ligase was able to perform this task, and the obtained ligation products were elongated by DNA polymerase. The effectiveness of ligation depends on the length of the random binding site of the mutagenic oligonucleotide, on its molar excess over the template-primer complex and on the position of the mismatching tri-nucleotide insert with respect to the joining site. The secondary structure of the DNA template close to the joining site also influences the ligation yield. Mismatching oligonucleotides, protected by a 3'-phosphate group, were joined to a nested set of megaprimers, the latter being obtained by a novel procedure called reversible chain termination, i.e., termination of the dsDNA synthesis with ddNTP followed by the subsequent removal of the incorporated ddNMP with exonuclease III. T7 sequenase 2.0 DNA polymerase elongated the ligation products after the 3'-phosphate protection group was removed with T4 polynucleotide kinase, resulting in the incorporation of a specific tri-nucleotide mismatch into dsDNA. This sequence of reactions serves as the basis for a novel scanning mutagenesis procedure. 相似文献
952.
Lomax RB Camello C Van Coppenolle F Petersen OH Tepikin AV 《The Journal of biological chemistry》2002,277(29):26479-26485
We have studied the Ca(2+) leak pathways in the endoplasmic reticulum of pancreatic acinar cells by directly measuring Ca(2+) in the endoplasmic reticulum ([Ca(2+)](ER)). Cytosolic Ca(2+) ([Ca(2+)](C)) was clamped to the resting level by a BAPTA-Ca(2+) mixture. Administration of cholecystokinin within the physiological concentration range caused a graded decrease of [Ca(2+)](ER), and the rate of Ca(2+) release generated by 10 pm cholecystokinin is at least 3x as fast as the basal Ca(2+) leak revealed by inhibition of the endoplasmic reticulum Ca(2+)-ATPase. Acetylcholine also evokes a dose-dependent decrease of [Ca(2+)](ER), with an EC(50) of 0.98 +/- 0.06 microm. Inhibition of receptors for inositol 1,4,5-trisphosphate (IP(3)) by heparin or flunarizine blocks the effect of acetylcholine but only partly blocks the effect of cholecystokinin. 8-NH(2) cyclic ADP-ribose (20 microm) inhibits the action of cholecystokinin, but not of acetylcholine(.) The basal Ca(2+) leak from the endoplasmic reticulum is not blocked by antagonists of the IP(3) receptor, the ryanodine receptor, or the receptor for nicotinic acid adenine dinucleotide phosphate. However, treatment with puromycin (0.1-1 mm) to remove nascent polypeptides from ribosomes increases Ca(2+) leak from the endoplasmic reticulum by a mechanism independent of the endoplasmic reticulum Ca(2+) pumps and of the receptors for IP(3) or ryanodine. 相似文献
953.
Polyanichko AM Chikhirzhina EV Skvortsov AN Kostyleva EI Colson P Houssier C Vorob'ev VI 《Journal of biomolecular structure & dynamics》2002,19(6):1053-1062
We have studied structural changes in DNA/protein complexes using the CD spectroscopy, upon the interaction of HMG1-domains with calf thymus DNA at different ionic strengths. HMG1 protein isolated from calf thymus and recombinant HMG1-(A+B) protein were used. Recombinant protein HMG1-(A+B) represents a rat HMG1 lacking C-terminal acidic tail. At low ionic strength (15 mM NaCl) we observed similar behavior of both proteins upon interaction with DNA. Despite this, at higher ionic strength (150 mM NaCl) their interaction with DNA leads to a completely different structure of the complexes. In the case of HMG1-(A+B)/DNA complexes we observed the appearance of DNA fractions possessing very high optical activity. This could be a result of formation of the highly-ordered DNA structures modulated by the interaction with HMG1-domains. Thus the comparison studies of HMG1 and HMG1-(A+B) interaction with DNA show that negatively charged C-terminal tail of HMG1 modulates interaction of the protein with DNA. The striking difference of the behaviour of these two systems allows us to explain the functional role of multiple HMG1 domains in some regulatory and architectural proteins. 相似文献
954.
955.
The cyanine dyes Cy3 and Cy5 have proven valuable in numerous applications involving conjugation with proteins. Practical syntheses of lysine-selective, succinimidyl ester derivatives of these dyes have been published, and succinimidyl esters are commercially available. However, the published syntheses of cysteine-selective derivatives produce relatively low yields from expensive starting materials, or produce molecules with marginal water solubility for protein labeling. We report here facile syntheses (four steps, >50% overall yield) of iodoacetamide, sulfhydryl-reactive derivatives of the Cy3 and Cy5 fluorophores. These novel derivatives have good water solubility (>2.5 mM) and bear only one reactive side chain, reducing possible protein cross-linking encountered with previous derivatives. 相似文献
956.
957.
Yoshinori Nishita Alexei V. Abramov Takahiro Murakami Ryuichi Masuda 《Acta theriologica》2018,63(3):369-378
The sable (Martes zibellina) is a medium-sized mustelid inhabiting forest environments in Siberia, northern China, the Korean Peninsula, and Hokkaido Island, Japan. To further understand the molecular evolution of the major histocompatibility complex (MHC), we sequenced part of exon 2 in MHC class II DRB genes, including codons encoding the antigen binding site, from 33 individuals from continental Eurasia and Japan. We identified 16 MHC class II DRB alleles (Mazi-DRBs), some of which were geographically restricted and others broadly distributed, and eight putative pseudogenes. A single-breakpoint recombination analysis detected a recombination site in the middle of exon 2. A mixed effects model of evolution analysis identified five amino acid sites presumably under positive selection. These sites were all located in the region 3′ to the recombination site, suggesting that positive selection and recombination could be committed to the diversity of the M. zibellina DRB gene. In a Bayesian phylogenetic tree, all Mazi-DRBs and the presumed pseudogenes grouped within a Mustelidae clade. The Mazi-DRBs showed trans-species polymorphism, with some alleles most closely related to alleles from other mustelid species. This result suggests that the sable DRBs have evolved under long-lasting balancing selection. 相似文献
958.
An Archaeal Aerotaxis Transducer Combines Subunit I Core Structures of Eukaryotic Cytochrome c Oxidase and Eubacterial Methyl-Accepting Chemotaxis Proteins 总被引:5,自引:1,他引:4
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Alexei Brooun James Bell Tracey Freitas Randy W. Larsen Maqsudul Alam 《Journal of bacteriology》1998,180(7):1642-1646
Signal transduction in the archaeon Halobacterium salinarum is mediated by three distinct subfamilies of transducer proteins. Here we report the complete htrVIII gene sequence and present analysis of the encoded primary structure and its functional features. HtrVIII is a 642-amino-acid protein and belongs to halobacterial transducer subfamily B. At the N terminus, the protein contains six transmembrane segments that exhibit homology to the heme-binding sites of the eukaryotic cytochrome c oxidase. The C-terminal domain has high homology with the eubacterial methyl-accepting chemotaxis protein. The HtrVIII protein mediates aerotaxis: a strain with a deletion of the htrVIII gene loses aerotaxis, while an overproducing strain exhibits stronger aerotaxis. We also demonstrate that HtrVIII is a methyl-accepting protein and demethylates during the aerotaxis response. 相似文献
959.
Genetic Recombination in Bacillus subtilis 168: Effects of recU and recS Mutations on DNA Repair and Homologous Recombination
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Bacillus subtilis recombination-deficient mutants were constructed by inserting a selectable marker (cat gene) into the yppB and ypbC coding regions. The yppB:cat and ypbC:cat null alleles rendered cells sensitive to DNA-damaging agents, impaired plasmid transformation (25- and 100-fold), and moderately affected chromosomal transformation when present in an otherwise Rec+ B. subtilis strain. The yppB gene complemented the defect of the recG40 strain. yppB and ypbC and their respective null alleles were termed “recU” and “recU1” (recU:cat) and “recS” and “recS1” (recS:cat), respectively. The recU and recS mutations were introduced into rec-deficient strains representative of the α (recF), β (addA5 addB72), γ (recH342), and (recG40) epistatic groups. The recU mutation did not modify the sensitivity of recH cells to DNA-damaging agents, but it did affect inter- and intramolecular recombination in recH cells. The recS mutation did not modify the sensitivity of addAB cells to DNA-damaging agents, and it marginally affected recF, recH, and recU cells. The recS mutation markedly reduced (about 250-fold) intermolecular recombination in recH cells, and there were reductions of 10- to 20-fold in recF, addAB, and recU cells. Intramolecular recombination was blocked in recS recF, recS addAB, and recS recU cells. RecU and RecS have no functional counterparts in Escherichia coli. Altogether, these data indicate that the recU and recS proteins are required for DNA repair and intramolecular recombination and that the recF (α epistatic group), addAB (β), recH (γ), recU (), and recS genes provide overlapping activities that compensate for the effects of single mutation. We tentatively placed recS within a new group, termed “ζ.” 相似文献
960.
J. G. Zeikus Claire Vieille Alexei Savchenko 《Extremophiles : life under extreme conditions》1998,2(3):179-183
Recent findings on the biochemical and molecular features of the following thermozymes are presented, based on their biotechnological
use: α-amylase and amylopullulanase, used in starch processing; glucose isomerase, used in sweetener production; alcohol dehydrogenase,
used in chemical synthesis; and alkaline phosphatase, used in diagnostics. The corresponding genes and recombinant proteins
have been characterized in terms of sequence similarities, specific activities, thermophilicity, and unfolding kinetics. Site-directed
and nested deletion mutagenesis were used to understand structure–function relationships. All these thermozymes display higher
stability and activity than their counterparts currently used in the biotechnology industry.
Received: January 22, 1998 / Accepted: February 16, 1998 相似文献