Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways^1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time^3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein⁶. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured^7-9.The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.     

Comparative metabolic diversity in two solar salterns

The purpose of this study was to compare the metabolic diversity of the whole microbial community in an oligotrophic saltern (Eilat, Israel) and in a saltern with a more enriched source water (Newark, California). Between 1993 and 1998 water samples were taken from selected locations within the solar salterns of the Cargill Solar Salt Plant, Newark, California, and the Israel Salt Co. in Eilat, Israel. To examine the whole community metabolic diversity, we used the 96-well BIOLOG GN^{{ TM}} plates which contain 95 different carbon sources and a control well. Plates from samples containing greater than 15% salt were excluded from the final analyses because of a lack of reproducibility when multiple plates were inoculated with the same sample. The data were analyzed by simple matching coefficient and principal component analysis. Both methods gave similar results. Two major clusters were formed. These could be subdivided into 10 sub-clusters with only three samples from the Newark saltern in December 1997 joining at the 95% similarity level. Most of the inlet and lower salinity samples from the Cargill samples comprised one large subcluster. Several carbon sources were used by 85% of the microbial community from the California samples, while 85% of the Eilat samples had no commonly used carbon sources. These results suggest that ponds in different geographic locations may have communities with different microbial populations despite the similarities in salt content and processing for salt production.     

The cost of migration: spoonbills suffer higher mortality during trans-Saharan spring migrations only

Explanations for the wide variety of seasonal migration patterns of animals all carry the assumption that migration is costly and that this cost increases with migration distance. Although in some studies, the relationships between migration distance and breeding success or annual survival are established, none has investigated whether mortality during the actual migration increases with migration distance. Here, we compared seasonal survival between Eurasian spoonbills (Platalea leucorodia leucorodia) that breed in The Netherlands and migrate different distances (ca 1000, 2000 and 4500 km) to winter in France, Iberia and Mauritania, respectively. On the basis of resightings of individually marked birds throughout the year between 2005 and 2012, we show that summer, autumn and winter survival were very high and independent of migration distance, whereas mortality during spring migration was much higher (18%) for the birds that wintered in Mauritania, compared with those flying only as far as France (5%) or Iberia (6%). As such, this study is the first to show empirical evidence for increased mortality during some long migrations, likely driven by the presence of a physical barrier (the Sahara desert) in combination with suboptimal fuelling and unfavourable weather conditions en route.     

On the role of phylogeny in determining activity patterns of rodents

Evolutionary plasticity is limited, to a certain extent, by phylogenetic constraints. We asked whether the diel activity patterns of animals reflect their phylogenies by analyzing daily activity patterns in the order Rodentia. We carried out a literature survey of activity patterns of 700 species, placing each in an activity time category: diurnal, nocturnal, or active at both periods (a-rhythmic). The proportion of rodents active at these categories in the entire order, was compared to the activity patterns of species of different families for which we had data for over ten species each: Dipodidae, Echimyidae, Geomyidae, Heteromyidae, Muridae, and Sciuridae. Activity times of rodents from different habitat types were also compared to the ordinal activity time pattern. We also calculated the probability that two random species (from a particular subgroup: family, habitat, etc.) will be active in the same period of the day and compared it to this probability with species drawn from the entire order. Activity patterns at the family level were significantly different from the ordinal pattern, emphasizing the strong relationship between intra-family taxonomic affiliation and daily activity patterns. Large families (Muridae and Sciuridae) analyzed by subfamilies and tribes showed a similar but stronger pattern than that of the family level. Thus it is clear that phylogeny constrains the evolution of activity patterns in rodents, and may limit their ability to use the time niche axis for ecological separation. Rodents living in cold habitats differed significantly from the ordinal pattern, showing more diurnal and a-rhythmic activity patterns, possibly due to physiological constraints. Ground-dwelling rodents differed significantly, showing a high tendency towards a-rhythmic activity, perhaps reflecting their specialized habitat. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Isolation of a DNA Fragment Containing Replication Functions from IncP2 Megaplasmid pMG2

Replication functions with IncP2 specificity were identified on a 3-kilobase DNA fragment isolated from the 400-kilobase Pseudomonas megaplasmid pMG2.     

Chromatin Assembly Factor I Mutants Defective for PCNA Binding Require Asf1/Hir Proteins for Silencing

Chromatin assembly factor I (CAF-I) is a conserved histone H3/H4 deposition complex. Saccharomyces cerevisiae mutants lacking CAF-I subunit genes (CAC1 to CAC3) display reduced heterochromatic gene silencing. In a screen for silencing-impaired cac1 alleles, we isolated a mutation that reduced binding to the Cac3p subunit and another that impaired binding to the DNA replication protein PCNA. Surprisingly, mutations in Cac1p that abolished PCNA binding resulted in very minor telomeric silencing defects but caused silencing to be largely dependent on Hir proteins and Asf1p, which together comprise an alternative silencing pathway. Consistent with these phenotypes, mutant CAF-I complexes defective for PCNA binding displayed reduced nucleosome assembly activity in vitro but were stimulated by Asf1p-histone complexes. Furthermore, these mutant CAF-I complexes displayed a reduced preference for depositing histones onto newly replicated DNA. We also observed a weak interaction between Asf1p and Cac2p in vitro, and we hypothesize that this interaction underlies the functional synergy between these histone deposition proteins.