Effect of alteration of the acetic acid synthesis pathway on the fermentation pattern of escherichia coli

The glucose metabolism of an Escherichia coli strain bearing mutations abolishing both acetyl phosphotransferase (PTA) and acetate kinase (ACK) activities was studied under aerobic and anaerobic conditions. These studies were conducted in a complex medium with the mutant carrying no plasmid, the mutant carrying the common cloning vector pUC19, and the mutant carrying a plasmid bearing the "pet" operon that encodes Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase activities. The mutant carrying no plasmid showed lower specific growth and glucose uptake rates relative to the parent wild-type strain (K-12), Lactic acid was produced at higher levels than the wild type, and considerable amounts of pyruvic acid were secreted as an unusual byproduct. Analysis of other fermentation products showed low but significant amounts of acetic acid, no accumulation of formic acid, and lower secretion of succinate and ethanol. The maintenance of the plasmid pUC19 in the mutant negatively affected metabolism. Expression of the pet operon overcame the metabolic stress caused by the plasmid, enhancing growth and glucose uptake rates to the values observed in the plasmidfree mutant. Also, expression of the pet operon allowed consumption of pyruvate accumulated during the first hours of fermentation.     

Influence of dilution rate and induction of cloned gene expression in continuous fermentations of recombinant yeast

The effects of growth rate on cloned gene product synthesis in recombinant Saccharomyces cerevisiae have been studied in continuous culture. The plasmid employed contains a yeast GAL10-CYC1 hybrid promoter directing expression of the E. coli lacZ gene. beta-Galactosidase production was therefore controlled by the yeast galactose regulatory circuit, and the induction process and its effects were studied at the various dilution rates. At all dilution rates plasmid stability decreased with induction of lacZ gene expression. In some instances, two induced "steady states" were observed, the first 10-15 residence times after induction and the second after 40-50 residence times. The second induced steady state was characterized by greater biomass concentration and lower beta-galactosidase specific activity relative to the first induced "steady-state." beta-Galactosidase specific activity and biomass concentration increased as dilution rate was reduced, and despite lower flow rate and plasmid stability, overall productivity (activity/L/hr) was substantially higher at low dilution rate. Important factors influencing all of the trends were the glucose and galactose (inducer) concentrations in the vessel and inducer metabolism.     

Molecular evolution of the psi eta-globin gene locus: gibbon phylogeny and the hominoid slowdown

An 8.4-kb genomic region spanning both the psi eta-globin gene locus and flanking DNA was sequenced from the common gibbon (Hylobates lar). In addition, sequencing of the entire orthologous region from galago (Galago crassicaudatus) was completed. The gibbon and galago sequences, along with published orthologous sequences from 10 other species, were aligned. These noncoding nucleotide sequences represented four human alleles, four apes (chimpanzee, gorilla, organgutan, and gibbon), an Old World monkey (rhesus monkey), two New World monkeys (spider and owl monkeys), tarsier, two strepsirhines (galago and lemur), and goat. Divergence and maximum parsimony analyses of the psi eta genomic region first groups humans and chimpanzees and then, at progressively more ancient branch points, successively joins gorillas, orangutans, gibbons, Old World monkeys, New World monkeys, tarsiers, and strepsirhines (the lemuriform-lorisiform branch of primates). This cladistic pattern supports the taxonomic grouping of all extant hominoids into family Hominidae, the division of Hominidae into subfamilies Hylobatinae (gibbons) and Homininae, the division of Homininae into tribes Pongini (orangutans) and Hominini, and the division of Hominini into subtribes Gorillina (gorillas) and Hominina (chimpanzees and humans). The additional gibbon and galago sequence data provide further support for the occurrence of a graded evolutionary-rate slowdown in the descent of simian primates, with the slowing rate being more pronounced in the great-ape and human lineages than in the gibbon or monkey lineages. A comparison of global versus local molecular clocks reveals that local clock predictions, when focused on a specific number of species within a narrow time frame, provide a more accurate estimate of divergence dates than do those of global clocks.     

Male-male behavior and sexual dimorphism of the ear of a zaprochiline tettigoniid (Orthoptera: Tettigoniidae)

The external anatomy of the auditory system of an undescribed zaprochiline tettigoniid (Genus nov. 22 Sp. 1, Australian National Insect Collection, Canberra) shows sexual dimorphism: the male appears to have no auditory spiracle equivalent to that seen in the female. Nocturnally active males aggregate around female required nectar sources in a random manner with regard to each other. There is limited evidence, either from song interaction or from their behavior in the field, that males respond to each other by acoustic cues. Laboratory trials, testing male phonotaxis, showed that movement was random with respect to a target group of caged calling males. In the field, the only signs of agonistic behavior consisted of an increased calling rate when males were close together. Taken together, these data suggest that the male may not preceive sound in the same way as the female.     

NMR spectroscopy of recombinant chinese hamster ovary (CHO) cells in a packed bed reactor

Summary Several clones of CHO cells, including recombinant cell lines expressing Hepatitis B surface antigen, were grown in macroporous collagen microspheres. These provided sufficient cell density in a packed bed recirculation system for phosphorus-31 nuclear magnetic resonance spectroscopic estimation of metabolite concentration. Intracellular nucleoside triphosphate as well as nucleoside tri- plus diphosphate levels were higher in the methotrexate-selected clones compared to the dhfr^– cell line.     

Effect of ammonium ion and extracellular pH on hybridoma cell metabolism and antibody production

The effects of NH(4)Cl addition on batch hybridoma cell growth at different external pH values (pH(e)) were investigated in a bioreactor at constant pH and dissolved oxygen concentration. In agreement with measurements in flasks, changes in pH(e) over the range 6.8-7.6 had minor effects on growth. Addition of 3 mM NH(4)Cl had little effect on cell growth while 10 mM NH(4)Cl caused a substantial growth inhibition, Measurements of the effects of pH(e) and NH(4)Cl concentration on cell metabolism gave similar results for cells grown in flasks in an incubator and in the bioreactor. As pH(e) decreases, the integral cell yield on glucose increases. There is a correlation between the effects of pH(e) on glycolysis and previous measurements of its effects on intracellular pH (pH(i)). Increases in NH(4)Cl concentration were previously determined to decrease pH(i) and are shown here to decrease the integral cell yield on glucose. At all pH(e) values in the absence of NH(4)Cl, glutamine is depleted at the time the maximum cell density is reached. Both pH(e) decreases and NH(4)Cl concentration increases lead to decreases in the integral cell yield on glutamine. Changes in pH(e) and in the NH(4)Cl concentration that cause growth inhibition have no effect on the specific antibody production rate for cells grown in flasks in an incubator or in the bioreactor. Changes in the NH(4)Cl concentration have no effect on the quality of the antibody produced, to a first level of characterization.     

Propagation of an amplifiable recombinant plasmid in Saccharomyces cerevisiae: flow cytometry studies and segregated modeling

Efficient expression of a foreign protein product by the yeastSaccharomyces cerevisiaerequires a stable recombinant vector present at a high number of copies per cell. A conditional centromere yeast plasmid was constructed which can be amplified to high copy number by a process of unequal partitioning at cell division, followed by selection for increased copy number. However, in the absence of selection pressure for plasmid amplification, copy number rapidly drops from 25 plasmids/cell to 6 plasmids/cell in less than 10 generations of growth. Copy number subsequently decreases from 6 plasmids/cell to 2 plasmids/cell over a span of 50 generations. A combination of flow cytometric measurement of copy number distributions and segregated mathematical modeling were applied to test the predictions of a conceptual model of conditional centromereplasmid propagation. Measured distributions of plasmid content displayed a significant subpopulation of cells with a copy number of 4-6, evenin a population whose mean copy number was 13.5. This type of copy number distribution was reproduced by a mathematical model which assumes that amaximum of 4-6 centromere plasmids per cell can be stably partitionedat cell division. The model also reproduces the observed biphasic kinetics of plasmid number instability. The agreement between simulation and experimental results provides support for the proposed model and demonstrates the utility of the flow cytometry/segregated modeling approach for the study of multicopy recombinant vector propagation.     

Electrochemical cells for voltammetry, coulometry, and protein activity assays of small-volume biological samples

Cell designs, experimental protocols, and results for electrochemical investigation of small quantitites of biological materials under anaerobic conditions are reported. Three types of electrochemical experiments are considered: (i) cyclic voltammetry of 20- to 100-microliters samples; (ii) direct coulometry of 0.5- to 1.5-ml samples; and (iii) an electrochemically initiated protein activity assay which includes provision for analysis of gaseous reaction products and correlation with electron flux. The first two procedures are illustrated by measurement of the formal electrode potential (E0') and number of electrons transferred (n) in redox reactions of small quantities of biological and inorganic materials. The third procedure is illustrated by assaying the activity of the MoFe protein plus Fe protein complex from Azotobacter vinelandii nitrogenase for reduction of C2H2 to C2H4.