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81.
HMG1-related DNA-binding protein isolated with V-(D)-J recombination signal probes. 总被引:8,自引:2,他引:6 下载免费PDF全文
M Shirakata K Hüppi S Usuda K Okazaki K Yoshida H Sakano 《Molecular and cellular biology》1991,11(9):4528-4536
In order to isolate cDNA clones for DNA-binding components of the V-(D)-J recombinase, phage libraries from a pre-B-cell line were screened with a radiolabeled probe containing recombination signal sequences (RSS). Among prospective clones, cDNA T160 was analyzed further. It produced a protein of 80.6 kDa which bound to DNA containing RSS but not to DNA in which the RSS had been mutated. A search of a data base revealed that the T160 protein has significant sequence homology (56%) to the nonhistone chromosomal protein HMG1 within the C-terminal region of 80 amino acids. DNA-binding analysis with truncated proteins showed that the HMG homology region is responsible for DNA binding. Using restriction fragment length polymorphisms, the T160 gene was mapped at the proximal end of mouse chromosome 2. Evidence was obtained for genetic linkage between the T160 gene and the recombination activator genes RAG-1 and RAG-2. 相似文献
82.
The nanosecond fluorescence depolarization method was applied to measure the fluorescence lifetime () and the rotational correlation time () of bovine serum albumin (BSA) labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl-Cl). Changes of and of dansyl BSA in the guanidine denaturation and in the thermal denaturation were examined. In parallel, the secondary structural change of dansyl BSA was followed by circular dichroism measurements. The magnitude of was almost unchanged between 1 and 2 M guanidine, where the secondary structure of the protein was predominantly disrupted; whereas that of began to increase before the disruption of secondary structure in the guanidine denaturation. In the thermal denaturation, in contrast, changes of both and occurred in a temperature range where the secondary structure was predominantly disrupted. The volume of equivalent sphere (V
e
) and the axial ratio () for the BSA were 3.6–3.8×10–19 cm3 and 3.6 at 2M guanidine as against 2.1×10–19 cm3 and 2.2 in the absence of guanidine (25°C), respectively. The magnitudes ofV
e
and were 4.9×10–19 cm3 and 4.5 at 65°C, respectively. Although the secondary structural change of dansyl BSA was irreversible in the thermal denaturation,V
e
and were reversible. 相似文献
83.
Association of Lps gene with natural resistance of mouse macrophages against Legionella pneumophila 总被引:3,自引:0,他引:3
Shin-ichi Yoshida Yoshitaka Goto Hiroshi Miyamoto Hironobu Fujio Yasuo Mizuguchi 《FEMS microbiology letters》1991,89(1):51-56
We have cloned a 1.6-kb region of chromosomal DNA from Thermoplasma acidophilum into Escherichia coli using as a probe part of the Methanococcus vannielii fus-gene. The sequence of the clone was highly homologous to part of the corresponding Methanococcus vannielii gene. By chromosome walking, a 4.7-kb EcoRI fragment containing the complete gene was isolated. Nucleotide sequencing revealed an open reading frame of 2196 nucleotides. The deduced amino acid sequence contains the known peptide sequence around the ADP-ribosylation site of T. acidophilum elongation factor 2, which unequivocally confirms that the fus-gene has been cloned. The amino acid sequence was compared to that of hamster and E. coli, as well as to known archaebacterial EF-2 sequences. 相似文献
84.
The depletion of an essential amino acid (aa), tryptophan, caused by interferon-gamma (IFN-gamma)-mediated induction of indoleamine 2,3-dioxygenase (IDO) in mouse allografted tumor cells, has been suggested as a reason for the allograft rejection. To elucidate the mechanism of this IDO induction, attempts were made to isolate cDNA clones encoding mouse IDO. In seven of 25 mouse cell lines, IDO was induced by IFN-gamma, and the highest IDO induction was observed in the case of rectal cancer (CMT-93) cells, which were further stimulated two- to threefold by the simultaneous addition of dibutyryl cyclic AMP (Bt2cAMP). A cDNA library was prepared from poly(A)+ RNA isolated from CMT-93 cells treated with IFN-gamma/Bt2cAMP. The cDNA clones were isolated using the cDNA encoding human IDO as a probe. The mouse IDO cDNA encodes a 407-aa protein with an Mr of 45,639. The deduced aa sequence agreed with partial aa sequences derived from endopeptidase digestion of purified mouse IDO and revealed 61% homology with that of human IDO. Transient expression of the mouse IDO cDNA in COS-7 cells yielded a high level of IDO activity in the cells. Northern hybridization analysis of RNA in CMT-93 cells indicated that IFN-gamma induced the IDO mRNA, and that the level of RNA was increased by simultaneous addition of Bt2cAMP, while Bt2cAMP itself had no effect on mRNA induction. 相似文献
85.
Bacillus subtilis inositol dehydrogenase-encoding gene (idh): sequence and expression in Escherichia coli. 总被引:2,自引:0,他引:2
The Bacillus subtilis inositol dehydrogenase (Idh)-encoding gene (idh) was cloned in the B. subtilis temperate phage, rho 11, and then in Escherichia coli plasmids (pBR322 and pUC118). The nucleotide sequence of the idh gene, which consists of 344 codons and whose product has an Mr of 38,351, was determined. E. coli, bearing pIOL05d15, in which expression of the idh gene is under the control of the lac promoter of pUC118, overproduced an active Idh to approx. 20% of total protein upon addition of isopropyl-beta-D-thiogalactopyranoside. This overproduced enzyme cross-reacted with an anti-Idh antibody, and exhibited the same Mr and substrate specificity as those of the B. subtilis enzyme. 相似文献
86.
K Inada S Endo K Takahashi M Suzuki T Narita T Yoshida H Suda T Komuro M Yoshida 《Microbiology and immunology》1991,35(4):303-314
We established a new method of plasma treatment for the removal of interfering factors in the plasma to allow detection of endotoxin by limulus test. The limulus test used was an endotoxin-specific chromogenic test, the Endospecy test. Perchloric acid (PCA) treatment and centrifugation (PCA method) is usually used to remove interfering factors from plasma, with the precipitate being discarded and the supernatant used to detect endotoxin. As the solubilized precipitates of endotoxin-spiked plasma and some patient plasma were found to contain the Endospecy activity, we have devised a new method assaying endotoxin in both the supernatant and precipitate. This study confirmed that the solubilized precipitate of endotoxin-spiked plasma had Endospecy activity and found that the precipitate had other endotoxin activities, such as lethality in galactosamine-sensitized mice and pyrogenicity in rabbits. We also confirmed that interfering factors were completely removed from plasma samples by this new method. The endotoxin level after the new PCA method was found to be about 8 times higher than that determined after PCA treatment and the new PCA method surpasses the conventional PCA method with regard to the positive rate of endotoxin contents in clinical samples. These results indicate that the new PCA method is superior to the PCA method as a plasma pretreatment method for limulus test. 相似文献
87.
New preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for fatty acids and derivatives 总被引:6,自引:0,他引:6
A preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for carboxylic acids is described. 9-Anthraldehyde hydrazone is oxidized with an organic oxidant, N-chlorosuccinimide, in an organic solvent such as ethyl acetate to give ADAM, and then the reaction mixture is directly used as the reagent solution for the derivatization of carboxylic acids. Both the oxidation and the derivatization reaction are carried out at room temperature, and an aliquot of the derivatization mixture is directly injected into a chromatograph. 9-Anthrylmethyl ester derivatives formed from ADAM and various carboxylic acids are sufficiently separated on a reversed-phase column and are sensitively detected fluorometrically. The present method was applied to the high-performance liquid chromatographic determination of long and short chain fatty acids, keto acids, and hydroxy acids. 相似文献
88.
Binding sites for mouse epidermal growth factor (EGF) were observed in purified nuclear fraction from rat liver. The binding data yielded a curvilinear Scatchard plot which was decomposed into two binding components with different binding affinities. The binding component with higher affinity (Kd approximately 10(-10) M) represented approximately 15% of the total binding, while the high affinity binding sites were less than 5% of the total in the microsomes. The ligand-dependent autophosphorylation, one of the characteristic features of EGF receptor in the plasma membrane, was not observed at the site of the receptor (170 KDa) in the nuclear fraction. The binding characteristics for EGF fluctuated during the course of liver regeneration after partial hepatectomy; the binding capacity in the nuclear fraction increased in contrast to the decrease in the microsomes. However, the binding sites in the nuclear fraction obtained in the early period after partial hepatectomy consisted only of low affinity ones. 相似文献
89.
Since its discovery more than 20 years ago the structure of a strongly fluorescent compound called "pleurodeles blue" has remained unknown. Isolation of this pigment has been carried out by successive column chromatographies including epichlorohydrin-triethanolamine-Sephadex and phospho-Sephadex. The structural elucidation of a novel pyridone N-glycoside, 1-beta-D-glucopyranosylpyrid-2(1H)-one-6-acetic acid, is based on a detailed study of its high-resolution mass spectra, 1H and 13C-NMR spectra and its hydrolysis to yield D-glucose. 相似文献