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41.
To alleviate plasmid instability and to prolong the production phase of subtilisin, integrable plasmid and spore mutants are used. Compared with batch-type shake flask cultures, spore mutants' ability to produce subtilisin can be well pronounced in fed-batch and continuous cultures. Hence, the two culture methods make it possible to identify the peculiar characteristics of the spore mutants unobtainable in batch culture. Spore mutants can enhance subtilisin productivity and prolong subtilisin production time in fed-batch culture as well as enable us to use very low dilution rates (<0.1 h(-1)) without losing productivity in continuous culture, thereby improving the conversion yield of the nitrogen source. At 0.05 h(-1) the spollG mutant of Bacillus subtilis DB104 (Deltanpr Deltaapr) (Em(r)) spollG (Bim(r)):: pMK101 (Cm(r)) showed a subtilisin yield about ten times higher than that from wild-type DB104 (Deltanpr Deltaapr)::pMK101 (Cm(r)). (c) 1995 John Wiley & Sons, Inc. 相似文献
42.
Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana 总被引:23,自引:3,他引:20
Sung Aeong Oh Joon-Hyun Park Gyu In Lee Kyung Hee Paek Soon Ki Park Hong Gil Nam 《The Plant journal : for cell and molecular biology》1997,12(3):527-535
Four mutants that show the delayed leaf senescence phenotype were isolated from Arabidopsis thaliana . Genetic analyses revealed that they are all monogenic recessive mutations and fall into three complementation groups, identifying three genetic loci controlling leaf senescence in Arabidopsis . Mutations in these loci cause delay in all senescence parameters examined, including chlorophyll content, photochemical efficiency of photosystem II, relative amount of the large subunit of Rubisco, and RNase and peroxidase activity. Delay of the senescence symptoms was observed during both age-dependent in planta senescence and dark-induced artificial senescence in all of the mutant plants. The results indicate that the three genes defined by the mutations are key genetic elements controlling functional leaf senescence and provide decisive genetic evidence that leaf senescence is a genetically programmed phenomenon controlled by several monogenic loci in Arabidopsis . The results further suggest that the three genes function at a common step of age-dependent and dark-induced senescence processes. It is further shown that one of the mutations is allelic to ein2-1 , an ethylene-insensitive mutation, confirming the role of ethylene signal transduction pathway in leaf senescence of Arabidopsis . 相似文献
43.
Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B. 下载免费PDF全文
In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate. 相似文献
44.
The capability to expand human bone marrow mononuclear cells (BM MNC) in high density perfusion culture chambers (bioreactors) has recently been developed. In these bioreactors, total cell colony-forming unit-granulocyte/macrophage (CFU-GM), and long-term culture-initiating cell (LTC-IC) numbers increase significantly over a 14-day period. However, cell growth ceases after the 14-day period, possibly due to cell density limitations. Because of the remaining presence of early cells, it should be feasible to replate the cells and obtain continued expansion. In this study, we demonstrate that bioreactors generate cells, which upon replating into secondary bioreactors, lead to continued cell, CFU-GM, and LTC-IC8 (measured after 8 weeks of secondary culture) expansion. A two-stage protocol, involving the replating of cells on days 9 to 12 of culture into new bioreators at the original seeding density, yielded greater than 50-fold cell expansion from BM MNC in 25 days. CFU-GM were expanded inhibitory factor (LIF) had no significant effect on total cells, CFU-GM, or LTC-IC5 in this system. We conclude that two-stage bioreactor cultures are capable of supporting extended growth of human BM MNC, CFU-GM, and LTC-IC8. The continued expansion of these primitive cells in the second stage of culture suggests that primitive cells with significant proliferative potential were generated in this system, and previous data on LTC-IC5 expansion has now been extended to LTC-IC8 expansion. Further optimization of culture conditions is likely to improve on the results obtained here, thus making perfusion bioreactor culture correspondingly more attractive for expanding BM MNC for BM transplantation. 相似文献
45.
Ectomycorrhizas were synthesized in pots and growth pouches betweenQuercus serrata, Q. acutissima, and two ectomycorrhizal fungi,Pisolithus tinctorius andHebeloma cylindrosporum. Root morphology and the structure of the mantle and Hartig net were compared using light, fluorescence, scanning and transmission
electron microscopy.P. tinctorius initially colonized root cap cells, and eventually produced a highly branched lateral root system with a complete mantle,
whereasH. cylindrosporum promoted root elongation with few hyphae on the root apex surface indicating that interaction between roots differs with
fungal species. Hartig net structure and hyphal inclusions varied between all the combinations tested. There were structural
differences between mycorrhizas ofH. cylindrosporum/Q. acutissima grown in soil and growth pouches, which indicate that the growth pouch environment can induce artefacts in roots. Fruit bodies
ofH. cylindrosporum developed in pots withQ. acutissima. AlthoughP. tinctorius has been used to inoculate oak seedlings in the nursery, results of this study indicate thatH. cylindrosporum may also be an effective ectomycorrhizal fungus forQ. serrata andQ. acutissima. 相似文献
46.
The effects of chick brain–spinal cord extract on morphological development and cyclic nucleotide levels of cultured chick embryo skeletal muscle cells were determined. It had previously been shown that the extract stimulated morphological differentation, protein synthesis, and cholinesterase activity of muscle cells. Myoblasts fused earlier and an increase in number as well as diameter of myotubes were seen in the extract treated cultures. Cyclic nucleotides levels were higher (almost twice the controls for both adenosine 3′, 5′ -cyclic monophosphate and guanosine 3′, 5′ -cyclic monophosphate) and preceded their occurence in the control cultures. It was suggested that factor(s) in the extract interact with membrane receptor(s) to alter nucleotide levels which, in turn, allow the effects to be expressed. 相似文献
47.
Anabaena sp., isolated from a rice paddy, was investigated for its nitrogen fixation as measured by acetylene reduction activity (ARA)
in P-limited continuous and light-limited semi-continuous cultures. Growth rate (μ) under P limitation was a function of cell
P content (q
p). Both the photosynthetic capacity (Pmax) and photosynthetic efficiency (α) increased with μ when expressed per cell, but not per unit chla. The ARA of steady-state cells under P limitation increased with μ and was linearly related to C-fixation rate. This was
apparently a consequence of the control of C-fixation by P limitation. In light-limited cells, steady state ARA, both at the
culture light intensity and in the dark, increased asymptotically with μ, but the activity in the dark was only about 51%
of that in the light. When the light level of steady-state cells grown at a high in intensity was switched to a low level,
ARA decreased exponentially with time. Dark ARA activity also showed a similar decline, but at much lower levels. Thus, ARA
depended not only on light history, but also immediate photosynthesis. Steady-state ARA at the ambient intensity or in the
dark showed a strong correlation with14C-fixation rate. ARA of light-limited cells showed the same light-saturation characteristics as their14C-fixation, with the same initial saturation intensity,I
k. The ratios of Pmax to the maximum ARA (ARAmax), and α to the slope of ARA (αara) were identical. A comparison of gross to net photosynthesis and N2 fixation suggested that there was little leakage or excretion of fixed C or N. 相似文献
48.
Analysis of the Gal3 Signal Transduction Pathway Activating Gal4 Protein-Dependent Transcription in Saccharomyces Cerevisiae 总被引:12,自引:2,他引:10 下载免费PDF全文
The Saccharomyces cerevisiae GAL/MEL regulon genes are normally induced within minutes of galactose addition, but gal3 mutants exhibit a 3-5-day induction lag. We have discovered that this long-term adaptation (LTA) phenotype conferred by gal3 is complemented by multiple copies of the GAL1 gene. Based on this result and the striking similarity between the GAL3 and GAL1 protein sequences we attempted to detect galactokinase activity that might be associated with the GAL3 protein. By both in vivo and in vitro tests the GAL3 gene product does not appear to catalyze a galactokinase-like reaction. In complementary experiments, Escherichia coli galactokinase expressed in yeast was shown to complement the gal1 but not the gal3 mutation. Thus, the complementation activity provided by GAL1 is not likely due to galactokinase activity, but rather due to a distinct GAL3-like activity. Overall, the results indicate that GAL1 encodes a bifunctional protein. In related experiments we tested for function of the LTA induction pathway in gal3 cells deficient for other gene functions. It has been known for some time that gal3gal1, gal3gal7, gal3gal10, and gal3 rho- are incapable of induction. We constructed isogenic haploid strains bearing the gal3 mutation in combination with either gal15 or pgi1 mutations: the gal15 and pgi1 blocks are not specific for the galactose pathway in contrast to the gal1, gal7 and gal10 blocks. The gal3gal5 and gal3pgi1 double mutants were not inducible, whereas both the gal5 and pgi1 single mutants were inducible. We conclude that, in addition to the GAL3-like activity of GAL1, functions beyond the galactose-specific GAL1, GAL7 and GAL10 enzymes are required for the LTA induction pathway. 相似文献
49.
Ferrous Iron and Sulfur Oxidation and Ferric Iron Reduction Activities of Thiobacillus ferrooxidans Are Affected by Growth on Ferrous Iron, Sulfur, or a Sulfide Ore 总被引:4,自引:3,他引:1 下载免费PDF全文
Isamu Suzuki Travis L. Takeuchi Trin D. Yuthasastrakosol Jae Key Oh 《Applied microbiology》1990,56(6):1620-1626
Eight strains of Thiobacillus ferrooxidans (laboratory strains Tf-1 [= ATCC 13661] and Tf-2 [= ATCC 19859] and mine isolates SM-1, SM-2, SM-3, SM-4, SM-5, and SM-8) and three strains of Thiobacillus thiooxidans (laboratory strain Tt [= ATCC 8085] and mine isolates SM-6 and SM-7) were grown on ferrous iron (Fe2+), elemental sulfur (S0), or sulfide ore (Fe, Cu, and Zn). The cells were studied for their aerobic Fe2+ - and S0-oxidizing activities (O2 consumption) and anaerobic S0-oxidizing activity with ferric iron (Fe3+) (Fe2+ formation). Fe2+-grown T. ferrooxidans cells oxidized S0 aerobically at a rate of 2 to 4% of the Fe2+ oxidation rate. The rate of anaerobic S0 oxidation with Fe3+ was equal to the aerobic oxidation rate in SM-1, SM-3, SM-4, and SM-5, but was only one-half or less that in Tf-1, Tf-2, SM-2, and SM-8. Transition from growth on Fe2+ to that on S0 produced cells with relatively undiminished Fe2+ oxidation activities and increased S0 oxidation (both aerobic and anaerobic) activities in Tf-2, SM-4, and SM-5, whereas it produced cells with dramatically reduced Fe2+ oxidation and anaerobic S0 oxidation activities in Tf-1, SM-1, SM-2, SM-3, and SM-8. Growth on ore 1 of metal-leaching Fe2+-grown strains and on ore 2 of all Fe2+-grown strains resulted in very high yields of cells with high Fe2+ and S0 oxidation (both aerobic and anaerobic) activities with similar ratios of various activities. Sulfur-grown Tf-2, SM-1, SM-4, SM-6, SM-7, and SM-8 cultures leached metals from ore 3, and Tf-2 and SM-4 cells recovered showed activity ratios similar to those of other ore-grown cells. It is concluded that all the T. ferrooxidans strains studied have the ability to produce cells with Fe2+ and S0 oxidation and Fe3+ reduction activities, but their levels are influenced by growth substrates and strain differences. 相似文献
50.
Natural killer cell-mediated lysis of herpes simplex virus-infected fibroblasts: inability to detect soluble factors that contribute to lysis 总被引:1,自引:0,他引:1
We investigated the role of soluble factors in natural killer (NK) cell-mediated lysis of herpes simplex virus (HSV)-infected cells. Supernatants generated by incubating human peripheral blood mononuclear cells with HSV-infected human fibroblasts contained tumor necrosis factor (TNF) and lysed uninfected U937 cells, but not HSV-infected fibroblasts. U937 cells, but not HSV-infected fibroblasts, were lysed when exposed to recombinant TNF (rTNF) for 18 hr. NK cell-mediated lysis of HSV-infected fibroblasts was not inhibited by addition of anti-TNF or anti-lymphotoxin (LT) antibodies to cytotoxicity assays. Thus, a role for soluble factors, and in particular TNF and LT, in NK cell-mediated lysis of HSV-infected cells could not be demonstrated. 相似文献