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971.
A method was developed to provide a real-time measurement of intracellular adenosine 5'-triphosophate (ATP) concentrations in growing Escherichia coli. The bacteria to be monitored must first be modified by inserting the cDNA for firefly luciferase expressed from a constitutive promoter. Such a construct leads to constant specific activity of firefly luciferase during both the lag phase and exponential growth. When the luciferase substrate, D-luciferin, is added to the medium, ATP within the cells is utilized in the luciferase-catalyzed reaction that produces light. The light is carried from the bioreactor to a computer-based detector by an optical fiber. The detected per cell light emission varies during exponential growth. Analysis of cytoplasm extracts shows that this variance is related to changes in the ATP concentration, which ranges from 1 to 6 times the literature value for K(M). Experimental analyses demonstrated that inner filter effects are not a significant factor affecting the use of this system. The method was tested in a benchtop fermentor at cell densities above 13 g/L dry cell weight. A correction factor based on the accumulated light data is calculated and used in real time to account for consumption of luciferin from the culture broth by the light producing reaction. Dissolved oxygen concentrations must be kept above 15% of air saturation to ensure constant light output, but no detectable increase in oxygen demand is seen. The method does not significantly affect growth or production rates. (c) 1996 John Wiley & Sons, Inc. 相似文献
972.
Energy metabolism and ATP balance in animal cell cultivation using a stoichiometrically based reaction network 总被引:1,自引:0,他引:1
A metabolic reaction network is developed for the estimation of the stoichiometric production of adenosine triphosphate (ATP) in animal cell culture. By using the material balance data from fed-batch and batch cultures of hybridoma cells, the stoichiometric ATP productions are determined with estimated effective P/O ratios of 2 for NADH and 1.2 for FADH(2). A significant percentage of the ATP requirement (16-41%) in hybridoma cells is generated directly from free energy release without the participation of oxygen. The oxidative phosphorylation of NADH accounts for about 60% of the total ATP production in the fed-batch cultures and about 47% in the batch culture. The oxidative phosphorylation of FADH(2) accounts for less then 20% of the total ATP production in all cases.A fractional model is devised to analyze the contribution of each nutrient to the ATP production. Results show that a majority of the ATP is produced from glucose metabolism (60-76%). Less than 30% of the ATP is derived from glutamine, and less than 11% is derived from other essential amino acids. The analysis also shows that the glycolytic pathway generates more ATP in the batch (41%) than in the fed-batch (<27%) cultures. The TCA cycle provides 51-68% of the total ATP production. The calculated stoichiometric oxygen consumption differs among the batch and fed-batch cultures, depending on the glucose concentration. This result suggests that the relationship between the oxygen uptake rate (OUR) and cell growth may change with the culture conditions. However, the calculated respiratory quotient (RQ) is relatively constant in all cases.A linear relationship is obtained between the specific ATP production rate and the specific cell growth rate. The maximum ATP yield and the maintenance ATP requirement are determined based on this linear relationship. The biosynthetic ATP demand estimated from the dry cell weight and cell composition is significantly lower than that calculated from the maximum ATP yield, indicating that the non-growth-associated ATP demand may contain other factors than what is considered in the estimation of the biosynthetic ATP demand. (c) 1996 John Wiley & Sons, Inc. 相似文献
973.
Pei-Wen Chiang SuQing Wang Paul Smithivas Woo-Joo Song Saravanan Ramamoorthy Joseph Hillman Sheryl Puett Margaret L. Van Keuren Eric Crombez Arun Kumar Thomas W. Glover Diane E. Miller Chun-Hui Tsai C.Clare Blackburn Xiao-Ning Chen Zhiguang Sun Jan-Fang Cheng Julie R. Korenberg David M. Kurnit 《Genomics》1996,34(3):328
974.
975.
976.
The site-specific integration of the phage ?CTX genome, which carries the gene for a pore-forming cytotoxin, into the Pseudomonas aeruginosa chromosome was analysed. The 1,167 by integrase gene, int, located immediately upstream of the attachment site, attP, was characterized using plasmid constructs, harbouring the integration functions, and serving as an integration probe in both P. aeruginosa and Escherichia coli. The attP plasmids p1000/p400 in the presence of the int plasmid pIBH and attP-int plasmids pINT/pINTS can be stably integrated into the P. aeruginosa chromosome. Successful recombination between the attP plasmid p1000 and the attB plasmid p5.1, in the presence of the int plasmid pIBH in E. coli HB101 showed that the int gene is active in trans in E. coli. The int gene product was detected as a 43 kDa protein in E. coli maxicells harbouring pINT. Proposed integration arm regions downstream of attP are not necessary for the integration process. pINT and phage ?CTX could be integrated together into P. aeruginosa chromosomal DNA, yielding double integrates. 相似文献
977.
在0.5%葡萄糖-Mandels盐培养液中添加终浓度为0.5%的L-山梨糖,可使里斯木霉(Trichoderma reesei C 30)和拟康氏木霉(Trichoderma pseudokoningii S38)的内切和外切β-1,4葡聚糖酶合成速率在培养的2天内提高4倍。与此同时β-葡萄糖苷酶的合成没有明显变化。L-山梨糖能明显抑制菌丝生长,但对葡萄糖的吸收没有影响,对菌丝分泌纤维素酶的机制影响不大。其对酶合成的促进可能主要是通过降低了菌丝体的生长速率。 相似文献
978.
979.
Abstract The fatty acid (FA) compositions for total lipids from fat body, hemolymph and flight muscle of the armyworm moths, Mythirnna separata, at rest and after tethered flight for 1 h were determined by GC and GC-MS. The composition in these tissues comprises myristic acid (1%-2%), palmitic acid (more than 35%1, palmitoleic acid (9%-11%), stearic acid (less than 1%), oleic acid (about 32%), linoleic acid (12%-17%) and linolenic acid (3%-6%). After flight, FA level in the fat body, compared to that at rest, shows a significant decline at about 20 μg/mg tissue.h-1; the concentration of FAs in hemolymph rises evidently, but change of FA content in flight muscle appears to be small. From the changes of proportional composition of FAs in fat body, hemolymph and flight muscle, it is found that the FAs selectively utilized for flight in flight muscle are predominantly the palmitic acid and oleic acid. 相似文献
980.
籼稻232蜡质基因转录起始位点的鉴定 总被引:5,自引:0,他引:5
Northernblot杂交分析和蜡质基因cDNA的序列分析表明水稻蜡质基因的转录本可能延伸到翻译起始密码子(ATG)上游12kb处。据此设计了21Nt的寡核苷酸引物,并以籼稻232胚乳RNA为模板,以引物延伸法确定籼稻232蜡质基因的转录起始点,籼稻蜡质基因的转录起始旁邻顺序CTCACCA与高等植物基因的转录起始点一致顺序CTCATCA仅相差1个碱基。通过顺序比较,对东乡野生稻蜡质基因中的转录起始位点的位置,以及对此两稻种中TATA盒的可能顺序进行了讨论。 相似文献