Pea (Pisum sativum) cells arrested in G2 have nascent DNA with breaks between replicons and replication clusters

DNA fiber autoradiography and alkaline sucrose sedimentation of DNA of cultured pea-root cells (Pisum sativum) arrested in G2 by carbohydrate starvation demonstrated that nascent DNA molecules of replicon (16–27 × 10⁶ D) and apparent cluster (~330 × 10⁶ D) size were not joined. That the arrested cells were in G2 was confirmed by single-cell autoradiography and cytophotometry. In pea there are about 18 replicons per average cluster, 4.2 × 10³ clusters, and 7.7 × 10⁴ replicons per genome.     

Replication of the rRNA and legumin genes in synchronized root cells of pea (Pisum sativum): evidence for transient EcoR I sites in replicating rRNA genes

Summary The temporal pattern of replication of the rRNA and legumin genes differs in synchronized pea root cells. The relative number of rRNA genes replicated hourly during the first five hours of S phase ranges between 5 and 10 percent. In late S phase, during hours six through nine, the number of rRNA genes replicated increases reaching a maximum of about 25 percent at the ninth hour. Unlike the rRNA genes, the legumin genes have a wave-like pattern of replication peaking in early S phase at the third hour and again in late S phase at the eighth hour.Replicating rDNA, isolated by benzoylated naphthoylated DEAE-column chromatography, has EcoR I restriction sites that are absent in non-replicating rDNA sequences. The cleavage of these sites is independent of the time of rDNA replication. The transient nature of the EcoR I sites suggests that they exist in a hemimethylated state in parental DNA.The two Hind III repeat-size classes of rDNA of var. Alaska peas are replicated simultaneously as cells progress through S phase. Thus, even if the 9.0 kb and 8.6 kb repeat classes are located on different chromosomes, their temporal order of replication is the same.     

Replication termini in the rDNA of synchronized pea root cells (Pisum sativum)

In synchronized root cells of Pisum sativum (cv. Alaska) the joining of nascent replicons is delayed until cells reach the S-G(2) boundary or early G(2) phase. To determine if the delayed ligation of nascent chains occurs at specific termination sites, we mapped the location of arrested forks in the ribosomal DNA (rDNA) repeats from cells in late S and G(2) phases. Two-dimensional (neutral-alkaline) agarose electrophoresis and Southern blot hybridization with specific rDNA sequences show that only cells located at the S-G(2) boundary and early G(2) phase produce alkali-released rDNA fragments of discrete size. The released fragments are from a particular restriction fragment, demonstrating that the replication forks stop non-randomly within the rDNA repeats. Indirect end-labeling with probes homologous to one or the other end of the fork-containing restriction fragment shows that there are two termination regions, T(1) and T(2), where forks stop. T(1) is located in the non-transcribed spacer and T(2) is at the junction between the non-transcribed spacer and the 18S gene. The two termini are separated by 1.3 kb. Replication forks stop at identical sites in both the 8.6- and 9.0-kb rDNA repeat size classes indicating that these sites are sequence determined.     

The quality and diagnostic outcome of postbronchoscopic sputum

In patients with a malignant lung process (146 patients with primary lung cancer and 21 patients with a metastatic lung malignancy), the quality and the percentage of true-positive diagnoses of sputum produced directly after bronchoscopy were evaluated. The quality of prebronchoscopic and postbronchoscopic sputum samples differed significantly, the former being of better quality for cytologic diagnosis. When calculated for the whole group, the quality of postbronchoscopy sputum was not related to any specific anamnestic or clinical patient data. Overall, there was no statistically significant difference between the percentages of true-positive diagnoses in prebronchoscopic and postbronchoscopic sputum specimens. However, the percentage of true-positive diagnoses was significantly higher in postbronchoscopic sputum in comparison with the results of sputum cytology prior to bronchoscopy when the results were calculated separately for patients with low forced expiratory volume values (less than 50% of the vital capacity) or for patients without a history of previous sputum production. In patients who produced bloody sputum, the percentage of true-positive diagnoses in prebronchoscopic sputum was significantly higher than in postbronchoscopic sputum.     

Measurement and prediction of digestibility of forage maize in The Netherlands

Results of digestibility trials on fresh and ensiled maize (aerial parts) were analysed and compared with laboratory data.

Digestibility of the organic matter of ears was almost constant at 84–85%. Digestibility of organic matter of stover showed great variation. Variation between animals in apparent digestibility of organic matter was greater for maize diets than for diets based on grass products. Most of the differences between animals were caused by differences in the digestion of cell walls. When the true digestibility of cell contents was assumed to be complete, endogenous excretion was, on average, only 7% with respect to organic matter intake, and seemed dependent on digestibility of organic matter.

Prediction of apparent digestibility was more accurate with digestibility in vitro than with crude fibre, although for maize silage, significant relationships were found between crude fibre content and digestibility of organic matter. Standard deviation of organic matter digestibility between lots of fresh and ensiled maize grown on commercial farms amounted to only about 1.2 –2%. As this was only twice the accuracy of digestibility in vivo with four animals, fixed values will be presented to farmers, possibly corrected for year, hybrid selection and cropping practice.     

Habitat availability does not explain the species richness patterns of European lentic and lotic freshwater animals

Aim In Europe, the relationships between species richness and latitude differ for lentic (standing water) and lotic (running water) species. Freshwater animals are highly dependent on suitable habitat, and thus the distribution of available habitat should strongly influence large‐scale patterns of species richness. We tested whether habitat availability can account for the differences in species richness patterns between European lentic and lotic freshwater animals. Location Europe. Methods We compiled occurrence data of 1959 lentic and 2445 lotic species as well as data on the amount of lentic and lotic habitats across 25 pre‐defined biogeographical regions of European freshwaters. We used the range of elevation of each region as a proxy for habitat diversity. We investigated the relationships between species richness, habitat availability and habitat diversity with univariate and multiple regression analyses. Results Species richness increased with habitat availability for lentic species but not for lotic species. Species richness increased with elevational range for lotic species but decreased for lentic species. For both groups, neither habitat availability nor diversity could account for previously reported latitudinal patterns in species richness. For lotic species, richness declined with latitude, whereas there was no relationship between habitat availability and latitude. For lentic species, richness showed a hump‐shaped relationship with latitude, whereas available habitat increased with latitude. Main conclusions Habitat availability and diversity are poor predictors of species richness of the European freshwater fauna across large scales. Our results indicate that the distributions of European freshwater animals are probably not in equilibrium and may still be influenced by history, namely the recurrent European glaciations and possible differences in post‐glacial recolonization. The distributions of lentic species appear to be closer to equilibrium than those of lotic species. This lends further support to the hypothesis that lentic species have a higher propensity for dispersal than lotic species.     

Structural and functional characterization of a triple mutant form of S100A7 defective for Jab1 binding

S100A7 (psoriasin) is a calcium‐ and zinc‐binding protein implicated in breast cancer. We have shown previously that S100A7 enhances survival mechanisms in breast cells through an interaction with c‐jun activation domain binding protein 1 (Jab1), and an engineered S100A7 triple mutant (Asp⁵⁶Gly, Leu⁷⁸Met, and Gln⁸⁸Lys—S100A7₃) ablates Jab1 binding. We extend these results to include defined breast cancer cell lines and demonstrate a disrupted S100A7₃/Jab1 phenotype is maintained. To establish the basis for the abrogated Jab1 binding, we have recombinantly produced S100A7₃, demonstrated that it retains the ability to form an exceptionally thermostable dimer, and solved the three dimensional crystal structure to 1.6 Å. Despite being positioned at the dimer interface, the Leu⁷⁸Met mutation is easily accommodated and contributes to a methionine‐rich pocket formed by Met¹², Met¹⁵, and Met³⁴. In addition to altering the surface charge, the Gln⁸⁸Lys mutation results in a nearby rotameric shift in Tyr⁸⁵, leading to a substantially reorganized surface cavity and may influence zinc binding. The final mutation of Asp⁵⁶ to Gly results in the largest structural perturbation shortening helix IV by one full turn. It is noteworthy that position 56 lies in one of two divergent clusters between S100A7 and the functionally distinct yet highly homologous S100A15. The structure of S100A7₃ provides a unique perspective from which to characterize the S100A7‐Jab1 interaction and better understand the distinct functions between S100A7, and it is closely related paralog S100A15.