Overproduction from a cellulase gene with a high guanosine-plus-cytosine content in Escherichia coli

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.     

Familial interactions of male rhesus monkeys in a semi-free-ranging troop

Although the central and stable position of female rhesus monkeys in semi-free-ranging bands has been observed and described, the position of the developing male in the social group is not as well delineated. The present study is an attempt to describe interactions of 18 males with members of their families. Quantitative observations were made on one band (E) of rhesus monkeys on Cayo Santiago. Incidences of behavior recorded were joining, grooming, and threatening. The resulting data were analyzed by comparing the behavior of the males studied toward members of their genealogy as well as unrelated members of the social group. Behavior of family and non-relatives toward the males was also revealed. Statistical analysis revealed that the male subjects engaged in more positive social interactions within the matrifocal family than with other members of the genealogy, and in turn, more such behavior was observed within the genealogy than with unrelated monkeys. Threatening behavior within the genealogies was infrequent. Age effects were noted in that family members directed more interactions toward younger males and as males matured, they groomed more but joined family members less frequently. These data suggest mechanisms of socialization within the genealogy that may be different from those employed within the unrelated social group.     

NMR studies of the conformational change in human N-p21ras produced by replacement of bound GDP with the GTP analog GTP gamma S.

1H-Detected 15N-edited NMR in solution was used to study the conformational differences between the GDP- and GTP gamma S-bound forms of human N-p21ras. The amide protons of 15N-labeled glycine and isoleucine were observed. Resonances were assigned to residues of particular interest, glycines-60 and -75 and isoleucines-21 and -36, by incorporating various 13C-labeled amino acids in addition to [15N]glycine and [15N]iosleucine and by replacing Mg2+ by Co2+. When GTP gamma S replaced GDP in the active site of p21ras, only 5 of the 14 glycine amide resonances show major shifts, indicating that the conformational effects are fairly localized. Responsive glycines-10, -12, -13, and -15 are in the active site. Gly-75, located at the far end of a conformationally-active loop and helix, also responds to substitution of GTP gamma S for GDP, while Gly-77 does not, supporting a role for Gly-75 as a swivel point for the conformational change. The amide proton resonances of isoleucines-36 and -21 and a third unidentified isoleucine also undergo major shifts upon replacement of GDP by GTP gamma S. Thus, the effector-binding loop containing Ile-36 is confirmed to be involved in the conformational change, and the alpha-helix containing Ile-21 is also shown to be affected.     

A programmable environmental system for maintenance and observation of individually housed ticks

This paper describes an inexpensive, space-saving, and labor-efficient method for housing and making repeated observations of individual ticks held under specific photoperiodic, temperature, and humidity conditions. Using 96-well assay plates to house ticks, various developmental parameters, sex, survival, etc., of several thousand individual ticks can be recorded routinely in less than 1 hr. This design also permits more than 3,500 individual ticks to be maintained in 1 small (18.9-L) aquarium that serves as a humidity chamber. A small, unilluminated constant temperature incubator was converted inexpensively into a light and temperature programmable unit to contain the aquarium by adding a timing system, light fixture, and a second thermostat.     

Cytochrome P450cam-catalyzed oxidation of a hypersensitive radical probe.

trans-1-Phenyl-2-vinylcyclopropane, a hypersensitive radical probe, is oxidized by cytochrome P450cam (CYP101) to a diastereomeric mixture of the corresponding epoxide (81%), (trans-2-phenylcyclopropyl)acetaldehyde (6%), and trans-5-phenyl-2-penten-1,5-diol (13%). trans-5-Phenyl-2-penten-1-ol and (trans-2-phenylcyclopropyl)ethane-1,2-diol are not detectably formed. Authentic standards of all the products have been synthesized and used to establish the identities (or the absence) of the metabolites. Studies with [18O]H2O demonstrate that the oxygens at positions 1 and 5 in the rearranged diol derive from molecular oxygen and water, respectively. Catalytic turnover of the enzyme is required for product formation from the olefin, but incubation of the epoxide metabolite with the enzyme, or with buffer alone, yields both the aldehyde and the rearranged diol products. The absence of trans-5-phenyl-2-penten-1-ol implies that the lifetime of the putative radical intermediate is so short that its existence as a discrete entity is questionable. A cationic intermediate is unlikely but cannot be excluded because the same metabolites are formed in a secondary reaction, even at pH 8.0, from the epoxide. The results provide no evidence for the involvement of radicals or cations in the epoxidation reaction, in agreement with results on the oxidation of olefins in organic solvents by metalloporphyrin catalysts.     

Competition between native and monoacetylated polymyxin for charged adsorption sites

The native antibiotic polymyxin B, carrying five positive charges, displaces the monoacetylated on from its adsorbed state at the interface between negatively charged lipid layers ans water. A simulation model for asdorption, governed by electrostatic forces, is presented. The model adequently representes the competitive adsorption kinetics and equilibria.