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Overproduction from a cellulase gene with a high guanosine-plus-cytosine content in Escherichia coli 总被引:1,自引:0,他引:1
G P O'Neill D G Kilburn R A Warren R C Miller 《Applied and environmental microbiology》1986,52(4):737-743
A recombinant exoglucanase was expressed in Escherichia coli to a level that exceeded 20% of total cellular protein. To obtain this level of overproduction, the exoglucanase gene coding sequence was fused to a synthetic ribosome-binding site, an initiating ATG, and placed under the control of the leftward promoter of bacteriophage lambda contained on the runaway replication plasmid vector pCP3 (E. Remaut, H. Tsao, and W. Fiers, Gene 22:103-113, 1983). With the exception of an inserted asparagine adjacent to the initiating ATG, the highly expressed exoglucanase is identical to the native exoglucanase. The overproduced exoglucanase can be isolated easily in an enriched form as insoluble aggregates, and exoglucanase activity can be recovered by solubilization of the aggregates in 6 M urea or 5 M guanidine hydrochloride. Since the codon usage of the exoglucanase gene is so markedly different from that of E. coli genes, the overproduction of the exoglucanase in E. coli indicates that codon usage may not be a major barrier to heterospecific gene expression in this organism. 相似文献
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The abundance of an mRNA encoding an HMG1/2 protein from Pharbitis nil (HMG1) has been previously shown to be regulated by light and an endogenous rhythm in cotyledons. A second Pharbitis nil HMG cDNA (HMG2) was characterized. The sequence of HMG2 was 82% and 86% identical to HMG1 at the nucleotide and amino acid level, respectively. As with HMG1, HMG2 mRNA was detected in all vegetative tissues and was most abundant in roots. However, unlike HMG1, HMG2 mRNA abundance did not increase upon transfer of cotyledons to darkness and did not exhibit regulation by an endogenous circadian rhythm when maintained in continuous darkness over a 68 h period. Similarly, while the abundance of HMG1 mRNA during a dark period that induced photoperiodically controlled flowering was dramatically affected by brief light exposure (night break), this treatment had no effect on HMG2 mRNA abundance. Collectively, these data are consistent with a role of HMG1 in contributing to the circadian-regulated and/or dark-regulated gene expression with constitutive expression of HMG2 playing a housekeeping role in the general regulation of gene expression in Pharbitis nil cotyledons. 相似文献
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Physical and biological parameters that determine the fate of p-chlorophenol in laboratory test systems.
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Shake-flask and microcosm studies were conducted to determine the fate of para-chlorophenol (p-CP) in water and sediment systems and the role of sediment and nonsediment surfaces in the biodegradation process. Biodegradation of p-CP in estuarine water samples in shake flasks was slow over incubation periods of 300 h. The addition of detrital sediment resulted in immediate and rapid degradation evidenced by the production of 14CO2 from [14C]p-CP. The addition of sterile sediment, glass beads, or sand resulted in approximately four to six times more CO2 evolution than observed in the water alone. Densities of p-CP-degrading bacteria associated with the detrital sediment were 100 times greater than those enumerated in water. Bacteria in the water and associated with the sediment after preexposure of both water and sediment of p-CP demonstrated enhanced biodegradation. In some microcosms, p-CP was degraded completely in the top 1.0 cm of intact sediment beds. Sediment reworking activities by benthic invertebrates from one site were sufficient to mix p-CP deep into the sediment bed faster than biodegradation or molecular diffusion. p-CP was persistent at lower depths of the sediment, possibly a result of reduced oxygen conditions preventing aerobic biodegradation. 相似文献
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In vivo ionizing irradiations produce deletions in the hprt gene of human T-lymphocytes 总被引:6,自引:0,他引:6
Janice A. Nicklas J. Patrick O'Neill Timothy C. Hunter Michael T. Falta Malcolm J. Lippert David Jacobson-Kram Jerry R. Williams Richard J. Albertini 《Mutation research》1991,250(1-2):383-396
The hprt T-lymphocyte cloning assay, which detects mutations occurring in vivo in humans, has been used to examine mutants induced in patients receiving radioimmunoglobulin therapy (RIT) for cancer. Samples from 13 patients before treatment (controls) and 15 samples from 12 patients after treatment were studied for both mutant frequencies and molecular changes in the hprt mutant T-cell clones. Patients were studied up to 48 months after treatment. Post-RIT patients showed increased mutant frequencies as compared to pre-treatment values. T-cell receptor (TCR) gene analysis of mutant T-cell clones demonstrated that 84% arose independently, both pre- and post-treatment, which is the same proportion as seen in normal individuals. However, several individuals did show large sets of mutants with the same TCR gene rearrangement patterns. Molecular analysis of mutants demonstrated a greater proportion of mutations with hprt gene changes on Southern blots after RIT treatment than before (40% versus 20%). RIT increases the proportion of mutations with total rather than partial gene deletions or other gross structural changes compared to normal individuals or pre-treatment patients. These studies are defining the spectrum for radiation-induced hprt gene mutations in vivo in human T-lymphocytes. 相似文献