An efficient method for high-fidelity BAC/PAC retrofitting with a selectable marker for mammalian cell transfection.

Large-scale genomic sequencing projects have provided DNA sequence information for many genes, but the biological functions for most of them will only be known through functional studies. Bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs) are large genomic clones stably maintained in bacteria and are very important in functional studies through transfection because of their large size and stability. Because most BAC or PAC vectors do not have a mammalian selection marker, transfecting mammalian cells with genes cloned in BACs or PACs requires the insertion into the BAC/PAC of a mammalian selectable marker. However, currently available procedures are not satisfactory in efficiency and fidelity. We describe a very simple and efficient procedure that allows one to retrofit dozens of BACs in a day with no detectable deletions or unwanted recombination. We use a BAC/PAC retrofitting vector that, on transformation into competent BAC or PAC strains, will catalyze the specific insertion of itself into BAC/PAC vectors through in vivo cre/loxP site-specific recombination.

Hypermutation in derepressed operons of Escherichia coli K12.

This article presents evidence that starvation for leucine in an Escherichia coli auxotroph triggers metabolic activities that specifically target the leu operon for derepression, increased rates of transcription, and mutation. Derepression of the leu operon was a prerequisite for its activation by the signal nucleotide, guanosine tetraphosphate, which accumulates in response to nutritional stress (the stringent response). A quantitative correlation was established between leuB mRNA abundance and leuB- reversion rates. To further demonstrate that derepression increased mutation rates, the chromosomal leu operon was placed under the control of the inducible tac promoter. When the leu operon was induced by isopropyl-D-thiogalactoside, both leuB mRNA abundance and leuB- reversion rates increased. These investigations suggest that guanosine tetraphosphate may contribute as much as attenuation in regulating leu operon expression and that higher rates of mutation are specifically associated with the derepressed leu operon.

Flux Analysis of Glucose Metabolism in Rhizopus oryzae for the Purpose of Increasing Lactate Yields

A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found.

Models of metabolism in Rhizopus oryzae.

A flux analysis of glucose metabolism in Rhizopus oryzae was achieved using [14C]-labeled glucose and acetate. The rates of glucose utilization and end product production were estimated, and metabolite pool sizes and specific radioactivities were determined. These data were analysed using a specific radioactivity curve-matching program called TFLUX. The analysis is consistent with the existence of separate mitochondrial and cytosolic pools of pyruvate, malate and fumarate.

Absence of ocular pathology after repeated exposure of unanesthetized monkeys to 9.3-GHz microwaves.

Unfettered monkeys (Macaca mulatta) have been trained to expose the face and eyes to pulsed microwave radiation at a frequency of 9.31 GHz and an average power density of 150 mW/cm2. Performance of an operant response required the monkeys to maintain the head within the field of the radiation source. Twelve monkeys were individually irradiated during 30 to 40 sessions and then were observed for a period of one year. No deleterious effects such as cataracts have been observed.

Acquisition, discriminative stimulus control, and retention of increases/decreases in blood pressure of normotensive human subjects.

The aims of this study of 24 normotensive subjects were: to compare a free-operant with a discrete-trials training format; to determine the most effective training procedure by comparing instrumental conditioning with instructional set and a control; to see if both increases and decreases in blood pressure could be brought under discriminative control, and to examine the maintenance of acquired self-control of blood pressure. A 2 X 3 design was employed in which two trial formats (free operant and discrete trials) were factorially compared with three training conditions (instrumental conditioning, instructional set, and control). Instrumental conditioning was found superior to both the instructional set and control conditions in producing increases and decreases in mean diastolic blood pressure. The free-operant format led to a greater degree of learned BP control in that subjects were able to increase and decrease their blood pressure by 10% to 15% of basal value and to maintain the blood-pressure operant after contingent auditory feedback/reinforcement was removed. Training was discontinued when subjects in the other five groups failed to reach criterion after 10 consecutive acquisition sessions.