Energizing cell-free protein synthesis with glucose metabolism

In traditional cell-free protein synthesis reactions, the energy source (typically phosphoenolpyruvate (PEP) or creatine phosphate) is the most expensive substrate. However, for most biotechnology applications glucose is the preferred commercial substrate. Previous attempts to use glucose in cell-free protein synthesis reactions have been unsuccessful. We have now developed a cell-free protein synthesis reaction where PEP is replaced by either glucose or glucose-6-phosphate (G6P) as the energy source, thus allowing these reactions to compete more effectively with in vivo protein production technologies. We demonstrate high protein yields in a simple batch-format reaction through pH control and alleviation of phosphate limitation. G6P reactions can produce high protein levels ( approximately 700 microg/mL of chloramphenical acetyl transferase (CAT)) when pH is stabilized through replacement of the HEPES buffer with Bis-Tris. Protein synthesis with glucose as an energy source is also possible, and CAT yields of approximately 550 mug/mL are seen when both 10 mM phosphate is added to alleviate phosphate limitations and the Bis-Tris buffer concentration is increased to stabilize pH. By following radioactivity from [U-(14)C]-glucose, we find that glucose is primarily metabolized to the anaerobic products, acetate and lactate. The ability to use glucose as an energy source in cell-free reactions is important not only for inexpensive ATP generation during protein synthesis, but also as an example of how complex biological systems can be understood and exploited through cell-free biology.     

Characterization of cross-linked hydroxycinnamic acid amides isolated from potato common scab lesions

Four feruloyl amides, N-trans-feruloyloctopamine (1), N-cis-feruloyloctopamine (2), N-trans-feruloyltyramine (3), N-cis-feruloyltyramine (4), a cross-linked N-trans-feruloyltyramine dimer (5), and a cross-linked N-cis-feruloyltyramine dimer (6) were isolated from potato common scab lesions. The compounds were purified by TLC and characterized by a combination of (1)H and (13)C NMR spectroscopic techniques. The presence of an accompanying minor complex of cross-linked dimers containing both feruloyltyramines and feruloyloctopamines was also demonstrated. This is the first characterization of cross-linked hydroxycinnamic acid amides associated with wound healing in potato (Solanum tuberosum) tubers.     

Lack of geographic variation in anonymous nuclear polymorphisms in the American oyster, Crassostrea virginica

Comparing geographic variation of noncoding nuclear DNA polymorphisms, which presumably are neutral to natural selection, with geographic variation of allozymes is potentially a good way to detect the effects of selection on allozyme polymorphisms. A previous study of four anonymous nuclear markers in the American oyster, Crassostrea virginica, found dramatic differences in allele frequency between the Gulf of Mexico and the Atlantic Ocean. In contrast, 14 allozyme polymorphisms were fairly uniform in frequency between the two areas. This led to the conclusion that all of the allozyme polymorphisms were kept uniform in frequency by balancing selection. To test the robustness of this pattern, six additional anonymous nuclear DNA polymorphisms were surveyed in oysters from Panacea, Fla, and Charleston, S.C. on the Gulf and Atlantic coasts, respectively. Unlike the previously studied DNA markers, the six DNA polymorphisms examined here show geographic variation that is not significantly greater than that of allozymes. The reason for the discrepancy between the two sets of DNA polymorphisms is unclear.      

Penetration of dioxygen into proteins studied by quenching of phosphorescence and fluorescence

Experiments were done to measure the ability of dioxygen to collisionally quench the phosphorescent and fluorescent tryptophans in alcohol dehydrogenase and alkaline phosphatase. In all cases, luminescence is quenched with rate constants close to 1 x 10(9) M-1 s-1. The rate of reaching the buried tryptophans is little affected by solvent viscosity due to added glycerol. Quenching by dioxygen is not due to a protein-opening reaction. It appears to be rate limited by internal protein diffusion rather than at the entry step. Dioxygen appears to enter the proteins directly, as in liquidlike diffusion, rather than through transiently forming channels that are only present a small fraction of the time. A high-pressure oxygen system is described that considerably facilitates fluorescence quenching experiments.     

Mapping of ilvO loci of Escherichia coli K-12 with bacteriophage lambda dilv.

A set of lambda dilv phage have been used in a deletion mapping procedure to determine the location of two previously characterized ilvO alleles. In contrast to earlier conclusions derived from three-factor crosses and episome-shortening techniques with phage P1, the order found is ilvG-ilvO-ilvEDA. A three-factor cross with phage P1 is described that is not consistent with this location for an ilvO allele. Further analysis of this particular three-factor cross revealed than an artifact attributable to a mutual syntrophism had skewed the apparent frequency of inheritance of the ilvO locus. The role of mutual syntrophism is discussed as a source of mapping errors for the ilvO locus. The value of this set of lambda dilv phage and this mapping procedure for obtaining comparatively unambiguous data on the locations of the ilv structural and regulatory genes is demonstrated.     

Patterns and functions of STAT activation during Drosophila embryogenesis

The JAK/STAT pathway mediates cytokine signaling in mammals and is involved in the function and development of the hematopoietic and immune systems. To investigate the biological functions of the JAK/STAT pathway during Drosophila development, we examined the tissue-specific localization of the tyrosine-phosphorylated, or activated form of Drosophila STAT, STAT92E. Here we show that during Drosophila embryonic development STAT92E activation is prominently detected in multiple tissues and in different developmental stages. These tissues include the tracheal pits, elongating intestinal tracks, and growing axons. We demonstrate that stat92E mutants are defective in tracheal formation, hindgut elongation, and nervous system development. Conversely, STAT92E overactivation caused premature development of the tracheal and nervous systems, and over-elongation of the hindgut. These results suggest that STAT activation is involved in proper differentiation and morphogenesis of multiple tissues during Drosophila embryogenesis.     

Regulation of Root-Associated Methanotrophy by Oxygen Availability in the Rhizosphere of Two Aquatic Macrophytes

The relative importance of oxygen for root-associated methanotrophy was examined by using sediment-free, intact freshwater marsh plants (Pontederia cordata and Sparganium eurycarpum) incubated in split chambers. The root medium contained approximately 100 (mu)M methane. Methane oxidation was calculated from the difference between methane loss from chambers in the presence and absence of 1 mM 1-allyl-2-thiourea, a methanotrophic inhibitor. When the root medium was oxic, methane oxidation accounted for 88 and 63% of the total methane depletion for S. eurycarpum and P. cordata, respectively; the remainder represented diffusional loss to the atmosphere via roots, stems, and leaves. Under suboxic conditions, methane oxidation was not detectable for S. eurycarpum but accounted for 68% of total methane depletion for P. cordata. The introduction of a biological oxygen sink, Pseudomonas aeruginosa, resulted in complete loss of methane oxidation in S. eurycarpum chambers under oxic conditions, while methane consumption continued (51.6% of total methane depletion) in P. cordata chambers. The differences between plant species were consistent with their relative ability to oxygenate their rhizospheres: during a suboxic incubation, dissolved oxygen decreased by 19% in S. eurycarpum chambers but increased by 232% for P. cordata. An in situ comparison also revealed greater methanotrophic activity for P. cordata than S. eurycarpum.