SV40 large T antigen hexamer structure: domain organization and DNA-induced conformational changes

Simian Virus 40 replication requires only one viral protein, the Large T antigen (T-ag), which acts as both an initiator of replication and as a replicative helicase (reviewed in ). We used electron microscopy to generate a three-dimensional reconstruction of the T-ag hexameric ring in the presence and absence of a synthetic replication fork to locate the T-ag domains, to examine structural changes in the T-ag hexamer associated with DNA binding, and to analyze the formation of double hexamers on and off DNA. We found that binding DNA to the T-ag hexamer induces large conformational changes in the N- and C-terminal domains of T-ag. Additionally, we observed a significant increase in density throughout the central channel of the hexameric ring upon DNA binding. We conclude that conformational changes in the T-ag hexamer are required to accommodate DNA and that the mode of DNA binding may be similar to that suggested for some other ring helicases. We also identified two conformations of T-ag double hexamers formed in the presence of forked DNA: with N-terminal hexamer-hexamer contacts, similar to those formed on origin DNA, or with C-terminal contacts, which are unlike any T-ag double hexamers reported previously.     

Effectiveness of assessment of vigour and productivity in young vs old bushes and mature clones of tea (Camellia sinensis)

The effectiveness and validity of assessment of bush vigour as expressed by bush area, shoot number and yield in selecting for high yielding clones in tea was tested by comparing the field performance of 50 source bushes when they were 4 yr old in 1977/78 with that of the same bushes when they were 13 yr old in 1986/87 and also with that of the 8 yr old vegetatively propagated plants (clones) derived from them and transplanted to the field in December 1979. Apparently vigorous bushes produced the highest yield. The three characters measured in mature source bushes and, to a lesser extent, clonal derivatives were positively correlated with measurements in young source bushes. Seven of the best yielding source bushes and five of the best clones in 1986/87 were present in the 10 highest yielding source bushes identified in the 1977/78 season. Assessment of bush area and shoot number was confirmed as an effective and valid method of identifying a small, highest yielding group of source bushes from a large population of seedling tea which should go for rooting assessment and further more thorough screening of yield.     

Application of a noninhibitory growth model to predict the transient response in a chemostat

A method of adapting a kinetic model based on steady-state chemostat data to predict the transient performance of a chemostat culture is presented. The proposal provides for a time delay which can be considered equivalent to a period of reduced activity of the organism subsequent to the introduction of a step change in operating conditions. The adapted kinetic model gives substantially better performance in predicting the transient response of an experimental system than the unmodified kinetic model.     

Patterns of isotype commitment in human B cells: limiting dilution analysis of Epstein Barr virus-infected cells

The frequency of Epstein Barr virus- (EBV) inducible IgM, IgG, and IgA-secreting cells in human peripheral blood and tonsil was determined by performing limiting dilution experiments in suspension culture. We devised a method of propagating small numbers of EBV-infected B cells with irradiated umbilical cord blood cells as a feeder layer. Precursor cell frequencies can be derived from these experiments; we have shown by statistical analysis that they conform to the single hit model of the Poisson distribution. By using this technique, a significant percentage of surface immunoglobulin-bearing lymphocytes are activated to secrete immunoglobulin in vitro. On the average, 8 to 38% of B cells in peripheral blood and tonsil can be propagated and the secreted immunoglobulin from the clonal progeny can be analyzed. Neither the EBV immune status of the donor nor the source of the umbilical cord blood feeder layer could account for the variations in cloning efficiency observed among donors. A surprisingly high frequency of B cells secreted IgA in vitro and we have shown that a small proportion of B cell clones in tonsil and peripheral blood secrete both IgM and IgA during the 4-wk culture period. Other B cells, including all those that produce IgG, appear to be committed to the secretion of a single isotype. Thus, these studies establish methodology for the analysis of the secreted products of human B cells at the single cell level and demonstrate that the progeny of at least some clones can secrete more than one isotype in vitro.     

Halogenated alcohols as solvents for proteins: FTIR spectroscopic studies.

Fourier transform infrared (FTIR) spectroscopy has been applied to investigate the secondary structure of proteins and polypeptides in halogenated alcohols. Each alcohol studied was able, as a pure liquid, to induce conversion of the beta-sheet protein concanavalin A into a predominantly alpha-helical configuration. In 2H2O/alcohol mixtures, helicogenisis was also apparent, decreasing in the order dichloroethanol greater than bromoethanol greater than trifluoroethanol greater than chloroethanol greater than fluoroethanol. At concentrations below those found to be helicogenic, disruption of the protein secondary structure by the alcohols resulted in pronounced aggregation. At concentrations insufficient to cause noticeable disruptions of the secondary structure at room temperature, the thermal stability of the protein was greatly reduced. We suggest the helicogenic effect exhibited by halogenated alcohols to be related to a combination of a relatively low dielectric constant and a high dipole moment, the latter causing disruption of the internal hydrogen bond networks and the former causing refolding to a helical configuration. The results presented here highlight the risk of using halogenated alcohols, both as solvents for proteins and as a test of the intrinsic capacity of proteins and peptides to adopt helical secondary structures.     

When and how did the terrestrial mid-Permian mass extinction occur? Evidence from the tetrapod record of the Karoo Basin,South Africa

A mid-Permian (Guadalupian epoch) extinction event at approximately 260 Ma has been mooted for two decades. This is based primarily on invertebrate biostratigraphy of Guadalupian–Lopingian marine carbonate platforms in southern China, which are temporally constrained by correlation to the associated Emeishan Large Igneous Province (LIP). Despite attempts to identify a similar biodiversity crisis in the terrestrial realm, the low resolution of mid-Permian tetrapod biostratigraphy and a lack of robust geochronological constraints have until now hampered both the correlation and quantification of terrestrial extinctions. Here we present an extensive compilation of tetrapod-stratigraphic data analysed by the constrained optimization (CONOP) algorithm that reveals a significant extinction event among tetrapods within the lower Beaufort Group of the Karoo Basin, South Africa, in the latest Capitanian. Our fossil dataset reveals a 74–80% loss of generic richness between the upper Tapinocephalus Assemblage Zone (AZ) and the mid-Pristerognathus AZ that is temporally constrained by a U–Pb zircon date (CA-TIMS method) of 260.259 ± 0.081 Ma from a tuff near the top of the Tapinocephalus AZ. This strengthens the biochronology of the Permian Beaufort Group and supports the existence of a mid-Permian mass extinction event on land near the end of the Guadalupian. Our results permit a temporal association between the extinction of dinocephalian therapsids and the LIP volcanism at Emeishan, as well as the marine end-Guadalupian extinctions.     

Effects of adenine nucleotides on the Ca2+-gated cation channel in sarcoplasmic reticulum vesicles

The effects of nucleotides on the Ca2+-gated cation channel in sarcoplasmic reticulum (SR) vesicles were studied by measuring choline influx. The choline influx was measured by following the change in scattered light intensity using the stopped flow technique. ATP enhanced the Ca2+-induced choline influx. The activation followed a single-site titration curve with a dissociation constant of 1.0 +/- 0.5 mM, independent of the Ca2+ concentration. ATP seems to increase the pore radius or number of channels without affecting the gating mechanism of the Ca2+-gated cation channel. ADP, AMP, and adenine enhanced the choline transport in a manner similar to ATP, but cAMP, ITP, UTP, CTP, and GTP did not. The apparent dissociation constants and the maximal activations were as follows: ATP 1.0 mM, 28-fold; ADP 0.9 mM, 18-fold; AMP 0.6 mM, 7-fold, and adenine 0.4 mM, 4-fold. Adenine and AMP behaved as a competitive inhibitor for the activation by ATP. These results are consistent with the Ca2+-induced Ca2+ release observed in skinned muscle fiber and isolated SR.