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2.
Kei Watanabe Kenta Wada Tomoko Ohashi Saki Okubo Kensuke Takekuma Ryoichi Hashizume Jun-Ichi Hayashi Tadao Serikawa Takashi Kuramoto Yoshiaki Kikkawa 《PloS one》2012,7(11)
We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene. 相似文献
3.
Cloning and nucleotide sequence of the isoamylase gene from Pseudomonas amyloderamosa SB-15 总被引:3,自引:0,他引:3
A Amemura R Chakraborty M Fujita T Noumi M Futai 《The Journal of biological chemistry》1988,263(19):9271-9275
The gene (iam) coding for isoamylase (glycogen 6-glucanohydrolase) of Pseudomonas amyloderamosa SB-15 was cloned. Its nucleotide sequence contained an open reading frame of 2313 nucleotides (771 amino acids) encoding a precursor of secreted isoamylase. The precursor contained a signal peptide of 26 amino acid residues at its amino terminus and three regions homologous with those conserved in alpha-amylases (1,4-alpha-D-glucan 4-glucanohydrolase) of species ranging from prokaryotes to eukaryotes. These homologous regions were also found in another debranching enzyme, pullulanase (pullulan 6-glucanohydrolase) from Klebsiella aerogenes. Sequences of the isoamylase also showed significant homology with those between positions 300 and the carboxyl terminus of pullulanase. The regions required for the specificity of isoamylase were discussed on the basis of a comparison of its amino acid sequence with those of alpha-amylases, cyclomaltodextrin glucanotransferases, and pullulanase. 相似文献
4.
Takashi Saito Kensuke Futatsugi Daisuke Miki Hiroshi Suzuki Kiyoshi Yasukawa 《Biotechnology Techniques》1992,6(4):365-370
Summary Murine interleukin-6 (mIL-6) was expressed inEscherichia coli as human growth hormone (hGH) fusion protein. The products were cleaved by thrombin to liberate mIL-6. Monoclonal and polyclonal
antibodies specific to mIL-6 were prepared by immunizing rats with mIL-6 thus obtained. ELISA for the quantitation of mIL-6
was also established, which could detect mIL-6 in a quantity as low as 2 ng/ml. 相似文献
5.
A sequence of 10 amino acids (I-C-S-D-K-T-G-T-L-T) of ion motive ATPases such as Na+/K+-ATPase is similar to the sequence of the beta subunit of H+-ATPases, including that of Escherichia coli (I-T-S-T-K-T-G-S-I-T) (residues 282-291). The Asp (D) residue phosphorylated in ion motive ATPase corresponds to Thr (T) of the beta subunit. This substitution may be reasonable because there is no phosphoenzyme intermediate in the catalytic cycle of F1-ATPase. We replaced Thr-285 of the beta subunit by an Asp residue by in vitro mutagenesis and reconstituted the alpha beta gamma complex from the mutant (or wild-type) beta and wild-type alpha and gamma subunits. The uni- and multisite ATPase activities of the alpha beta gamma complex with mutant beta subunits were about 20 and 30% of those with the wild-type subunit. The rate of ATP binding (k1) of the mutant complex under uni-site conditions was about 10-fold less than that of the wild-type complex. These results suggest that Thr-285, or the region in its vicinity, is essential for normal catalysis of the H+-ATPase. The mutant complex could not form a phosphoenzyme under the conditions where the H+/K+-ATPase is phosphorylated, suggesting that another residue(s) may also be involved in formation of the intermediate in ion motive ATPase. The wild-type alpha beta gamma complex had slightly different kinetic properties from the wild-type F1, possibly because it did not contain the epsilon subunit. 相似文献
6.
M Setaka N Satoh T Kobayashi T Hongo T Kwan A Yamaguchi M Futai 《Journal of biochemistry》1986,99(3):777-783
A phospholipid bilayer membrane was spread from an organic solvent solution between a polyacrylamide gel surface and an aqueous buffer solution. The membrane was quite similar to the conventional black lipid membrane, but was of a large size and was stable since it was supported on the gel surface. Bacteriorhodopsin, impregnated into the membrane, generated membrane potential and current upon illumination. The induced current was large, and this was attributed to the large area of the present membrane. Remarkable responses of the light-induced potential and current were also observed with a thick layer of organic solvent containing phospholipids. The effects of applied membrane potential, carbonylcyanide-m-chlorophenyl hydrazone (CCCP) and gramicidin were examined on these photoresponses. Steady-state current, which is due to protons flowing through the membrane, was enormously enhanced by applying membrane potential opposite to the photopotential or by adding gramicidin to the membrane-forming solution. 相似文献
7.
Escherichia coli K-12 strains in the absence of the lactose carrier grew on the disaccharide melibiose as the sole source of carbon. The presence of 0.1 mM Li+ in the medium strongly inhibited growth of such cells, and Li+-resistant mutants appeared after several days of incubation. These mutants showed altered cation coupling to melibiose transport via the melibiose carrier. Cotransport between H+ and melibiose was lost in the mutants, although Na+-melibiose cotransport was retained. We observed no Li+-melibiose cotransport. Therefore, these mutants represent a new type of cation-coupling mutants of the melibiose carrier. 相似文献
8.
Sixteen mutants of Escherichia coli defective in H+-ATPase (proton-translocating ATPase) were tested for their ability to recombine with hybrid plasmids carrying various portions of the beta subunit cistron. Twelve mutations were mapped within the carboxyl half of the cistron corresponding to amino acid residues 279 to 459 (domain II), while four mutations were mapped within residues 17 to 278 (domain I). The biochemical properties of these mutants were analyzed in terms of the proton permeability of their membranes and the assembly properties of their F1F0 complex. The mutants were classified according to the properties into three types, I, II, and III. In 12 mutants of type I, proton conduction in membrane vesicles was blocked and little F1 was released from the membranes under conditions in which F1 could be released from wild-type membranes, suggesting that assembly of the F1F0 complex is structurally and functionally defective. F1 was partially purified with very low recovery from one of the type I mutants, KF16. ATPase activity was reconstituted from this F1 with the beta subunit of the wild type, confirming the genetic results. Only one mutant, KF38, was classified as type II. Its membranes were partially leaky to protons and its F1 was releasable, suggesting that the interaction of its F1 and F0 was unstable. Type III mutants, KF11 and KF43, had an F1F0 complex with very low activity, in which the structure of F1 was relatively similar to that of the wild type. F1 was purified as a single complex from KF43 in this study and from KF11 previously (H. Kanazawa, Y. Horiuchi, M. Takagi, Y. Ishino, and M. Futai (1980) J. Biochem. 88, 695-703). Reconstitution experiments in vitro showed that the F1's of both mutants were defective in the beta subunit. The properties of the altered F1 of KF43 differed from those of F1 of KF11, suggesting that the mutation sites of KF43 and KF11 were different. From the results of mapping mutation sites and the biochemical properties of the mutants, the correlation of structural domains with function of the beta subunit is discussed. Most type I and type II mutations except that of KF39 were mapped in domain II, while the type III mutations were mapped in domain I, suggesting that domain II is more important than domain I for the function of the beta subunit, especially in terms of proper assembly of the F1F0 complex. 相似文献
9.
Overproduction of truncated subunit a of H+-ATPase causes growth inhibition of Escherichia coli. 总被引:2,自引:1,他引:1
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Genes (uncB) for wild-type and mutant a subunits of Escherichia coli H+-ATPase (F0F1) were cloned into recombinant plasmids. The subunits were expressed under the control of a weak promoter of the unc operon at 30 degrees C and strong promoters of lambda phage at 42 degrees C. At 30 degrees C, the wild type and a truncated (Glu-269----end) a subunit complemented the defect of the a subunit mutant KF24A (Trp-111----end), whereas the other mutant subunits (Trp-111----end, Trp-231----end, Gln-252----end, and a subunit with a deletion of residues 21 to 227) did not. Three mutant subunits (Trp-231----end, Gln-252----end, and Glu-269----end) and the wild-type a subunit caused growth inhibition associated with cell elongation, an uneven distribution of membrane proteins, and an altered septum structure when they were expressed at 42 degrees C. These phenomena were not observed with the other mutant subunits, suggesting that overproduction of the middle region (between residues 111 and 230) of the a subunit causes growth inhibition. 相似文献
10.
M Tagaya T Yagami T Noumi M Futai F Kishi A Nakazawa T Fukui 《The Journal of biological chemistry》1989,264(2):990-994
Proline 17 in the glycine-rich region of adenylate kinase was replaced by Gly (the Gly-mutant) or Val (the Val-mutant) by site-directed mutagenesis. The mutant enzymes were purified to homogeneous states on sodium dodecyl sulfate-gel electrophoresis after solubilization of the proteins from the pellets of cell lysates of Escherichia coli. The apparent Km values of the Gly- and the Val-mutants for AMP increased approximately 7- and 24-fold, respectively, as compared with that of the wild-type enzyme. The apparent Km values for ATP also increased 7- and 42-fold in the Gly- and Val-mutants, respectively. In contrast, Vmax values of both mutant enzymes were comparable to that of the wild-type enzyme. These results suggest that Pro-17 plays an important role for the binding of substrates, but not for catalytic efficiency, although it does not directly interact with substrates. Adenosine diphosphopyridoxal, which specifically modifies Lys-21 in adenylate kinase (Tagaya, M., Yagami, T., and Fukui, T. (1987) J. Biol. Chem. 262, 8257-8261), inactivated the wild-type and mutant enzymes at almost the same rates. Interestingly, both mutant enzymes showed higher specificities for adenine nucleotides than the wild-type enzyme. Both mutant enzymes were less resistant than the wild-type enzyme against inactivation at elevated temperatures or by treatment with trypsin. It would appear that most of the properties of the mutant enzymes may be explained on the basis of a need for conformational flexibility of the loop which includes Pro-17 for substrate binding. 相似文献