mRNA stability and antibody production in CHO cells: Improvement through gene optimization

The productivity of stably transfected cell lines is of critical importance for the manufacturing of therapeutic proteins. Various methods have been successfully implemented to increase the production output of mammalian cell cultures. Increasing evidence suggests that optimization of the gene coding sequences of an expression vector can improve specific cell line yield of the recombinant protein. Here we demonstrate that gene optimization substantially enhances antibody production in Chinese hamster ovary cells. When gene optimization was applied to the heavy and light chain genes of a therapeutic antibody, we observed increased antibody production in transient transfection. Elevated heavy chain mRNA level was associated with the increase of antibody production. Further analysis suggested that the increased antibody expression is attributable to enhanced mRNA stability resulting from gene optimization. Gene optimization also led to increased antibody production in stable clones.     

Bacillus subtilis ribonucleases J1 and J2 form a complex with altered enzyme behaviour

Ribonucleases J1 and J2 are recently discovered enzymes with dual 5′‐to‐3′ exoribonucleolytic/endoribonucleolytic activity that plays a key role in the maturation and degradation of Bacillus subtilis RNAs. RNase J1 is essential, while its paralogue RNase J2 is not. Up to now, it had generally been assumed that the two enzymes functioned independently. Here we present evidence that RNases J1 and J2 form a complex that is likely to be the predominant form of these enzymes in wild‐type cells. While both RNase J1 and the RNase J1/J2 complex have robust 5′‐to‐3′ exoribonuclease activity in vitro, RNase J2 has at least two orders of magnitude weaker exonuclease activity, providing a possible explanation for why RNase J1 is essential. The association of the two proteins also has an effect on the endoribonucleolytic properties of RNases J1 and J2. While the individual enzymes have similar endonucleolytic cleavage activities and specificities, as a complex they behave synergistically to alter cleavage site preference and to increase cleavage efficiency at specific sites. These observations dramatically change our perception of how these ribonucleases function and provide an interesting example of enzyme subfunctionalization after gene duplication.     

Exogenous GA3 Application Can Compensate the Morphogenetic Effects of the GA-Responsive Dwarfing Gene Rht12 in Bread Wheat

The most common dwarfing genes in wheat, Rht-B1b and Rht-D1b, classified as gibberellin-insensitive (GAI) dwarfing genes due to their reduced response to exogenous GA, have been verified as encoding negative regulators of gibberellin signaling. In contrast, the response of gibberellin-responsive (GAR) dwarfing genes, such as Rht12, to exogenous GA is still unclear and the role of them, if any, in GA biosynthesis or signaling is unknown. The responses of Rht12 to exogenous GA₃ were investigated on seedling vigour, spike phenological development, plant height and other agronomic traits, using F_2∶3 and F_3∶4 lines derived from a cross between Ningchun45 and Karcagi-12 in three experiments. The application of exogenous GA₃ significantly increased coleoptile length and seedling leaf 1 length and area. While there was no significant difference between the dwarf and the tall lines at the seedling stage in the responsiveness to GA₃, plant height was significantly increased, by 41 cm (53%) averaged across the three experiments, in the GA₃-treated Rht12 dwarf lines. Plant height of the tall lines was not affected significantly by GA₃ treatment (<10 cm increased). Plant biomass and seed size of the GA₃-treated dwarf lines was significantly increased compared with untreated dwarf plants while there was no such difference in the tall lines. GA₃-treated Rht12 dwarf plants with the dominant Vrn-B1 developed faster than untreated plants and reached double ridge stage 57 days, 11 days and 50 days earlier and finally flowered earlier by almost 7 days while the GA₃-treated tall lines flowering only 1–2 days earlier than the untreated tall lines. Thus, it is clear that exogenous GA₃ can break the masking effect of Rht12 on Vrn-B1 and also restore other characters of Rht12 to normal. It suggested that Rht12 mutants may be deficient in GA biosynthesis rather than in GA signal transduction like the GA-insensitive dwarfs.     

Uptake of water from a Kandosol subsoil. II. Control of water uptake by roots

Aim

To test for the presence of an impediment to water flow at the soil-root interface.

Methods

Wheat plants were grown in repacked and undisturbed field soil. Their transpiration rate, E, was varied in several steps from low to high and then back to low again, while the hydrostatic pressure in the leaf xylem, ψ _xylem, was measured non-destructively and continuously. These measurements were compared to a mathematical model that calculated ψ _xylem by assuming that the hydraulic resistance across the plant was constant and that the radial flow of water to unit length of a typical plant root generated gradients in pressure in the soil water.

Results

For the repacked soil, the radial flow model could not match the experiment during the falling phase of E, unless it was assumed that either an additional, constant, interfacial resistance between the soil and the roots had developed when E was large and ψ _xylem was rapidly falling, or that the resistance within the plant had changed. For the undisturbed field soil, the radial flow model did not agree with the experiment. Plausible agreement was achieved when plant water uptake was accounted for using a distributed sink model in HYDRUS-1D, with E integrated across the rootzone. This approach was based on the measured large variation in the vertical distribution of roots.

Conclusions

There was no strong evidence of large drawdowns of soil water in the rhizosphere, even when ψ _xylem was falling rapidly when E was large and the soil was moderately dry. Thus, there seems to have been an additional impediment to water flow from soil to plant, either within the plant, or at the interface between the two.     

Hydroseeding tackifiers and dryland moss restoration potential

Tackifiers are long‐chain carbon compounds used for soil stabilization and hydroseeding and could provide a vehicle for biological soil crust restoration. We examined the sensitivity of two dryland mosses, Bryum argenteum and Syntrichia ruralis, to three common tackifiers—guar, psyllium, and polyacrylamide (PAM)—at 0.5×, 1.0×, and 2.0× of recommended (×) concentrations for erosion control and revegetation. We measured moss shoot, gemma, and protonema production as well as moss organic matter and bound sand masses as indicators of growth and soil holding ability. We tested sand and tackifier chemistry to investigate potential nutrient and toxicant potential on moss growth. Groups of 10 fragments from field‐collected mosses were grown on sand in open petri dishes arranged in a growth chamber in replicated blocks containing each tackifier and concentration combination plus a distilled water control. Bryum (n = 10) and Syntrichia (n = 9) growth were measured at the end of 6 and 5 weeks, respectively. Overall model tests yielded statistically significant results (p < 0.001) for every variable in each species. When compared to water, guar tended to decrease growth, psyllium tended to increase growth, and PAM's effects were generally neutral to positive. Within tackifier types, increasing concentrations of guar tended to decrease growth, while increasing concentrations of psyllium tended to increase growth. Changes in PAM concentrations had little effect on growth. Increases in guar and psyllium lowered pH and increased P and K. Psyllium and PAM yielded promising results as potential agents of dispersal and adherence of dryland mosses in field restoration.     

GA-Responsive Dwarfing Gene Rht12 Affects the Developmental and Agronomic Traits in Common Bread Wheat

Opportunities exist for replacing reduced height (Rht) genes Rht-B1b and Rht-D1b with alternative dwarfing genes, such as the gibberellin-responsive gene Rht12, for bread wheat improvement. However, a comprehensive understanding of the effects and mode of action of Rht12 is lacking. In the present study, the effects of Rht12 were characterized by analyzing its effects on seeding vigour, seedling roots, leaf and stem morphology, spike development and carbohydrate assimilation and distribution. This was carried out in the four genotypes of F_2:3 lines derived from a cross between Ningchun45 and Karcagi (12) in two experiments of autumn sowing and spring sowing. Rht12 significantly decreased stem length (43%∼48% for peduncle) and leaf length (25%∼30% for flag leaf) while the thickness of the internode walls and width of the leaves were increased. Though the final plant stature was shortened (40%) by Rht12, the seedling vigour, especially coleoptile length and root traits at the seedling stage, were not affected adversely. Rht12 elongated the duration of the spike development phase, improved the proportion of spike dry weight at anthesis and significantly increased floret fertility (14%) in the autumn sowing experiment. However, Rht12 delayed anthesis date by around 5 days and even the dominant Vrn-B1 allele could not compensate this negative effect. Additionally, grain size was reduced with the ability to support spike development after anthesis decreased in Rht12 lines. Finally, grain yield was similar between the dwarf and tall lines in the autumn sowing experiment. Thus, Rht12 could substantially reduce plant height without altering seeding vigour and significantly increase spikelet fertility in the favourable autumn sowing environment. The successful utilization of Rht12 in breeding programs will require careful selection since it might delay ear emergence. Nonetheless, the potential exists for wheat improvement by using Rht12.     

Molecular basis for the recognition and cleavage of RNA by the bifunctional 5'-3' exo/endoribonuclease RNase J

RNase J is a key member of the β-CASP family of metallo-β-lactamases involved in the maturation and turnover of RNAs in prokaryotes. The B.?subtilis enzyme possesses both 5'-3' exoribonucleolytic and endonucleolytic activity, an unusual property for a ribonuclease. Here, we present the crystal structure of T.?thermophilus RNase J bound to a 4 nucleotide RNA. The structure reveals an RNA-binding channel that illustrates how the enzyme functions in 5'-3' exoribonucleolytic mode and how it can function as an endonuclease. A second, negatively charged tunnel leads from the active site, and is ideally located to evacuate the cleaved nucleotide in 5'-3' exonucleolytic mode. We show that B.?subtilis RNase J1, which shows processive behavior on long RNAs, behaves distributively for substrates less than 5 nucleotides in length. We propose a model involving the binding of the RNA to the surface of the β-CASP domain to explain the enzyme's processive action.     

Consequences of thermal acclimation for the mating behaviour and swimming performance of female mosquito fish

The mating system of eastern mosquito fish (Gambusia holbrooki) is dominated by male sexual coercion, where all matings are forced and females never appear to cooperate and actively avoid all attempts. Previous research has shown that male G. holbrooki offer a model system for examining the benefits of reversible thermal acclimation for reproductive success, but examining the benefits to female avoidance behaviour has been difficult. In this study, we examined the ability of non-male-deprived female G. holbrooki to avoid forced-coercive matings following acclimation to either 18 or 30 degrees C for six weeks (12h light:12h dark photoperiod). Thermal acclimation of burst and sustained swimming performance was also assessed, as these traits are likely to underlie their ability to avoid forced matings. There was no influence of thermal acclimation on the burst swimming performance of female G. holbrooki over the range 18-30 degrees C; however, sustained swimming performance was significantly lower in the warm- than the cool-acclimation group. For mating behaviour, we tested the hypothesis that acclimation would enhance the ability of female G. holbrooki to avoid forced matings at their host acclimation temperature relative to females acclimated to another environment. However, our hypothesis was not supported. The rate of copulations was almost three times greater for females acclimated to 30 degrees C than 18 degrees C when tested at 30 degrees C, indicating that they possess the ability to alter their avoidance behaviour to 'allow' more copulations in some environments. Coupled with previous studies, female G. holbrooki appear to have greater control on the outcome of coercive mating attempts than previously considered and can alter their propensity to receive forced matings following thermal acclimation. The significance of this change in female mating-avoidance behaviours with thermal acclimation remains to be explored.