Expression of beta-lactamase by recombinant Escherichia coli strains containing plasmids of different sizes--effects of pH, phosphate, and dissolved oxygen

The characteristics of growth and synthesis of plasmid-encoded protein were studied for strains of recombinant E. coli JM103 which carried the beta-lactamase gene on plasmids of different sizes. The plasmids used included the vector pUC8 and its recombinant derivatives containing varying-sized inserts of Drosophila DNA (not expressed in E. coli). Luria broth (LB) and a minimal medium (M9) supplemented in some cases with additional inorganic phosphate were used as growth media. There was no evidence of segregational instability in these experiments, where no antibiotic selection pressure was employed. Responses of the recombinant strains to variations in environmental parameters including pH, phosphate concentration in the medium, and aeration rate were examined. While the cell growth rate in LB decreased with pH in the range 7.0-8.0, the bulk beta-lactamase activity was maximized at an intermediate pH. The recombinant cell growth rate decreases with increasing plasmid size in the minimal medium, while such decrease is not significant when a rich medium such as LB is used. There is an intermediate plasmid size in the range studied (2.7-8.7 kb), at which beta-lactamase activity is maximum. While reduction in aeration rate (which determines the dissolved oxygen level) is detrimental for cell growth, it is beneficial for beta-lactamase synthesis. The bulk beta-lactamase activity therefore exhibits a maximum with respect to aeration rate. Cell growth and beta-lactamase production are affected in a similar manner by phosphate concentration in the minimal medium and therefore both are maximized at the same phosphate concentration. This investigation demonstrates clearly how the production of a recombinant plasmid-encoded protein can be maximized by proper manipulation of culture conditions and how it is affected by plasmid size.     

Photoreceptor-specific efficiencies of β-carotene,zeaxanthin and lutein for photopigment formation deduced from receptor mutant Drosophila melanogaster

Summary Drosophila rearing media had only -carotene, zeaxanthin or lutein as precursors for photopigment chromophores. Zeaxanthin and lutein are potentially optimum sources of the 3-hydroxylated retinoids of visual and accessory photopigments. Mutants made the electroretinogram in white (w) eyes selective for compound eye photoreceptors R1–6, R7 and R8: R1–6 domiantes w's electroretinogram; R7/8 generates w;ora's (ora = outer rhabdomeres absent); R8 generates w sev;- ora's (sev = sevenless). Microspectrophotometry revealed R1-6's visual pigment. In w, all 3 carotenoids yielded monotonic dose-responses for sensitivity (Fig. 4) or visual pigment (Fig. 7). An ultraviolet sensitivity peak from R1-6's sensitizing pigment was present at high but not low doses (Fig. 1). In w;ora, all 3 carotenoids gave similar spectra dominated by R7's high ultraviolet sensitivity (Fig. 2). For w sev;ora, all spectra were the shape expected for R8, peaking around 510 nm (Fig. 3). The sensitivity dose-response was at its ceiling except for low doses in w;ora (Fig. 5) and zero supplementation in w sev;ora (Fig. 6). Hence, without R1-6, most of our dose range mediated maximal visual pigment formation. In Drosophila, -carotene, zeaxanthin and lutein mediate the formation of all major photopigments in R1-6, R7 and R8.Abbreviations ERG electroretinogram - MSP microspectrophotometry - HPLC high pressure liquid chromatography - n.a. numerical aperture - w, sev, ora Drosophila mutants - y, p, r marg types of R7 and R8     

Inhibition of the cellular response to interferons by products of the adenovirus type 5 E1A oncogene.

Expression of the E1A oncogene of adenovirus type 5 inhibits the response of interferon (IFN)-inducible constructs to Type I (alpha,beta) and II (gamma) IFNs in transient transfection assays. In human cell lines stably expressing E1A mRNA and protein acquisition of an antiviral state and the induction of a number of genes in response to alpha- and gamma-IFNs is inhibited. A short IFN-stimulable response element (ISRE) present in the 5' flanking region of a number of genes mediates induction by alpha- and gamma-IFNs. In cells expressing E1A there is a substantial reduction in the levels of the ISRE-binding factors E and M, inducible by alpha-IFN, and of factor G, inducible by gamma-IFN. In E1A-expressing cells the E alpha subunit of factor E is activated normally in response to alpha-IFN; the defect is in the production or activation of the E gamma subunit. The inhibitory activity of E1A is lost upon deletion of the CR1 domain. The induction of HLA class II genes by gamma-IFN, which involves a different DNA response element(s), and of beta-IFN mRNA in response to double-stranded RNA are also inhibited by E1A. An essential component(s) of a number of signalling pathways must, therefore, be subject, directly or indirectly, to inhibition by E1A.     

A small deletion and an adjacent base exchange in a potential stem-loop region of the neurofibromatosis 1 gene

Summary A single-strand conformational polymorphism found in the DNA of a patient with neurofibromatosis 1 (NF1) was shown to be caused by a deletion of a CCACC or CACCT sequence and an adjacent transversion, located about 500 base pairs downstream from the region that codes for a functional domain of the NF1 gene product. This mutation could also be detected in the patient and in his affected daughter by heteroduplex analysis. The deletion removes the proximal half of a small potential stem-loop and interrupts the reading frame in exon 1. A severely truncated protein with a grossly altered carboxy terminus lacking one third of its sequence is expected to be formed from the mutant allele.     

Construction of a cDNA to the hamster CAD gene and its application toward defining the domain for aspartate transcarbamylase.

cDNA complementary to hamster mRNA encoding the CAD protein, a multifunctional protein which carries the first three enzymes of pyrimidine biosynthesis, was constructed. The longest of these recombinants (pCAD142) covers 82% of the 7.9-kilobase mRNA. Portions of the cDNA were excised and replaced by a lac promoter-operator-initiation codon segment. The resultant plasmids were transfected into an Escherichia coli mutant defective in aspartate transcarbamylase, the second enzyme of the pathway. Complementation of the bacterial defect was observed with as little as 2.2 kilobases of cDNA sequence, corresponding to the 3' region of the mRNA. DNA sequencing in this region of the hamster cDNA reveals stretches which are highly homologous to the E. coli gene for the catalytic subunit of aspartate transcarbamylase; other stretches show no homology. The highly conserved regions probably reflect areas of protein structure critical to catalysis, while the nonconserved regions may reflect differences between the quaternary structures of E. coli and mammalian aspartate transcarbamylases, one such difference being that the bacterial enzyme in its native form is allosterically regulated and the mammalian enzyme is not.     

Regulation of end-expiratory lung volume during sleep in premature infants

To investigate the regulation of end-expiratory lung volume (EEV) in premature infants, we recorded airflow, tidal volume, diaphragm electromyogram (EMG), and chest wall displacement during sleep. In quiet sleep, EEV during breathing was 10.8 +/- 3.6 (SD) ml greater than the minimum volume reached during unobstructed apneas. In active sleep, no decrease in EEV was observed during 28 of 35 unobstructed apneas. Breaths during quiet sleep had a variable extent of expiratory airflow retardation (braking), and inspiratory interruption occurred at substantial expiratory flow rates. During active sleep, the expiratory flow-volume curve was nearly linear, proceeding nearly to the volume axis at zero flow, and diaphragm EMG activity terminated near the peak of mechanical inspiration. Expiratory duration (TE) and inspiratory duration (TI) were significantly shortened in quiet sleep vs. active sleep although tidal volume was not significantly different. In quiet sleep, diaphragmatic braking activity and shortened TE combined to maintain EEV during breathing substantially above relaxation volume. In active sleep, reduced expiratory braking and prolongation of TE resulted in an EEV that was close to relaxation volume. We conclude that breathing strategy to regulate EEV in premature infants appears to be strongly influenced by sleep state.     

Frequency characteristics of the saccadic eye movement

Using a piecewise linear approach, individual saccadic eye movements have been Fourier decomposed in an attempt to determine the effect of saccadic amplitude on frequency characteristics. These characteristics were plotted in the traditional Bode plot form, showing gain and phase as a function of frequency for various eye movement amplitudes. Up to about one octave beyond the -3 db gain frequency, the limiting system dynamics represented by the saccadic trajectory of a given amplitude may be considered linear and second order. The -3 db gain frequency was used as a measure of bandwidth, and the -90 degrees phase crossover frequency was used as a measure of undamped natural frequency. These two quantities were used to calculate the damping factor. Both bandwidth and undamped natural frequency decrease with increasing saccadic eye movement amplitude. The damping factor shows no trend with amplitude and indicates approximate critical damping. When compared with the normal variation of characteristics for a given movement, the frequency characteristics of fixed-amplitude saccades showed no generalized trends with changes in direction or DC operating level of movement.