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21.
22.
Cloning of multiple copies of immunoglobulin variable kappa genes in cosmid vectors 总被引:1,自引:0,他引:1 下载免费PDF全文
The possibility of cloning large segments of DNA in cosmid vectors offers distinct advantages, in particular for the study of multigene families. Large size fragments of mouse embryo DNA were successfully cloned in the cosmid pHC 79. Twelve recombinants hybridizing specifically to an immunoglobulin kappa chain variable region probe were identified. In 9 of these recombinants, the size of the insert ranges from 30 to 43 kilobases. Factors affecting the cloning efficiency of a complex mammalian genome in cosmids were studied. The stability of these recombinant cosmids and the preparation of recombinant cosmid DNA are also discussed. 相似文献
23.
Two pure peroxidase isoenzymes B1 and D4 were isolated from the upper parts of 10-day-old wheat seedlings by means of gel and ion-exchange chromatography. Their MWs were 85000 and 24000 respectively. B1 was unstable and under various conditions it was converted to another isoenzyme, electrophoretically identical with D4. B1 contains about 40% of neutral sugars: 17.2% arabinose, 15.3% galactose, 5% glucose and traces of mannose. D4 is free of neutral sugars. None of the isoenzymes contained amino sugars. B1 oxidizes ferulic and p-coumaric acids. This oxidation has two pH optima of 4.4 and 5.4–5.6 and is inhibited by high concentrations of substrates, cyanide and azide. B1 oxidizes IAA in the presence of phenolic cofactor and Mn2+ ions. IAA oxidation has two pH optima of 4.5 and 5.6 and is inhibited by high substrate concentration, cyanide and azide, and by a number of indole derivatives. The main products of IAA oxidation are 3-methyleneoxindole and indole-3-methanol. o- and p- diphenols induce a lag period prior to IAA oxidation. Ferulic acid is oxidized during this lag period, probably to a dimer. B1 is able to produce H2O2 from oxygen. Mn2+ ions, a phenolic cofactor and an electron donor (IAA or NADH) are needed. B1 oxidizes α-keto-γ- methylmercaptobutyric acid to ethylene. D4 has a low peroxidatic activity and is inactive as an IAA oxidase. Thus B1 is probably an active cell wall-bound peroxidase isoenzyme, whereas D4 is its decomposition product. 相似文献
24.
Miloš Sedlák Jaromír Kaválek Vladimír Machácek Vojeslav Šterba 《Molecules and cells》1992,1(10):170-174
The cyclization reactions of N-methyl-N’-(2-methoxycarbonylphenyl)sulfamide (1a), N-methyl-N-(2-methoxycarbonylphenyl)-sulfamide (2a), and 2-methoxycarbonylphenylsulfamide (3a) were studied in aqueous amine buffers (butylamine, ethanolamine, morpholine, glycinamide). The dependences observed between the rate constants and buffer concentrations show that the reactions are subject to base catalysis in all the three cases, the decomposition of the tetrahedral intermediate being rate limiting. The ratio of the relative rate constants of the base catalyzed cyclizations reactions of the three derivatives is 1a: 2a: 3a = 1: 20000: 100. The logarithm of rate constants of the base catalyzed cyclization reactions was plotted against the pKa values of conjugated acids of the individual amines used as the buffers in the cyclization of compound 1a, and the value of the Brönsted coefficient obtained was about 0.1, which means that the proton transfer from the intermediate to the basic buffer component is thermodynamically favorable. The intermediate is a much weaker base, and the reaction is controlled by diffusion. The slope of an analogous dependence for compound 2a gradually decreases from values near to 0.5 to values near to zero, which means that the intermediate formed from compound 2a (pKa ≈ 9.3) has a pKa value comparable with that of the acid buffer component. 相似文献
25.
Expression of rat procathepsin B in yeast led to the secretion of both the latent and mature forms of the enzyme. Culture in the presence of a cysteine proteinase inhibitor prevented this processing. We have expressed and purified a mutant form of rat procathepsin B whose active-site cysteine residue has been changed to a serine, and which also lacks the glycosylation site in the mature region of the protein. This non-active mutant protein was secreted essentially in an unprocessed form. The purified protein has been incubated with a variety of proteinases, and results indicate that cathepsins D and L, as well as mature cathepsin B itself, can produce a processed (single-chain) form of cathepsin B from this precursor. Amino-terminal sequencing of these processed forms has revealed that they are all elongated by a few residues with respect to the mature form found in vivo. The action of a combination of cathepsin B with dipeptidylpeptidase I produced a single-chain form of cathepsin B with the correct amino terminus. This work has also shown that the processing of procathepsin B to a single-chain form can be an autocatalytic process, in at least an intermolecular manner. 相似文献
26.
J. T. P. Albrechtová J. Ullmann J. Krekule A. Blažková I. Machžčková Z. Vondráková J. Blažková S. Procházka V. Borkovec D. A. Baker F. Didehvar Z. Tesařová V. Czaková M. Zima K. Doležal T. Doleželová V. Psota M. Dundelová V. Reinöhl S. Prochàzka J. Mikulík M. Dvořěk J. Černohorskě O. Auxtová B. Cholvadová D. Lišková M. Kubačková S. Karácsonyi L. Bilisics 《Biologia Plantarum》1992,34(1):569-587
27.
The aim of our study was to determine if the oocytes of pregnant cattle are capable for undergoing embryonic growth following in vitro fertilization. The ovaries of nine heifers at 4 to 7 months of pregnancy were collected at an abattoir and transferred to the laboratory. A total 191 oocytes (10.6 per ovary) collected by aspiration were matured and fertilized by frozen-thawed semen. Embryos were co-cultured with granulosa cells in modified TCM 199 medium and 20% estrous cow serum. The cleavage rate of embryos was 48%, and 41% of of the cleaved embryos developed to the morula/blastocyst stage 7 days after insemination. Additionally, the ovaries of 10 nonpregnant heifers were also collected, yielding 213 oocytes (10.7 per ovary). The cleavage rate was 51%, and 35% of those which cleaved reached the morula/blastocyst stage. No significant differences were found between the two groups. The average number of transferable-stage embryos obtained from pregnant and nonpregnant animals was 4.1 and 3.7, respectively. Our results indicate that preganancy does not influence the meiotic competence of bovine oocytes, and transferable stage embryos can be obtained by the fertilization of oocytes derived from pregnant animals. 相似文献
28.
H Mach J A Thomson C R Middaugh R V Lewis 《Archives of biochemistry and biophysics》1991,287(1):33-40
We have employed near ultraviolet derivative absorption spectroscopy to study the microenvironments of phenylalanine residues in proteins. The use of second-derivative uv spectra in the 250- to 270-nm range effectively suppresses spectral contributions from tryptophan and tyrosine residues. Fitting a polynomial to the numerically calculated second-derivative spectrum allows precise determination of the position of the negative derivative peak near 258 nm. This position is shown to be correlated with the polarity of the microenvironments of phenylalanine residues. This approach allows monitoring of changes in the state of phenylalanine side chains during folding/unfolding of the proteins. In addition, this method permits perturbation of protein samples with ethylene glycol to be used to establish the relative degree of solvent exposure of protein phenylalanine. 相似文献
29.
30.
The plasma membrane of mammalian cells can mediate the cytotoxic and cytocidal effects of colicin E3. As little as 102 lethal units of purified colicin E3 per cell exert a pronounced cytocidal effect on human epithelial HeLa cells and as little
as 104 lethal units per cell also on line L mouse fibroblasts in tissue culture. Cells in complete monolayers are rapidly killed,
become spherical and shrink, they are detached from the support and finally autolyzed. The percentage of killed cells in both
lines is directly proportional to the multiplicity of colicin used. Theld
50 for HeLa cells is about 30 times lower than for L cells. At the multiplicity of 105 l.u., usually 100 % HeLa cells and 90 % L cells are killed in 2–3 days. Purified colicins E2 and D have no demonstrable cytological
effect on HeLa cells, although DNA synthesis in L cells appears to be partly inhibited by colicin E2. The profound effect
of colicin E3 on mammalian cells could be interpreted in a similar way as in bacteria,viz. as a specific cleavage of rRNA. 相似文献