Investigation into the nature of a Bacillus promoter cloned into a promoter-probe plasmid

The alpha-amylase-coding gene (amy) of Bacillus amyloliquefaciens NCP1 was cloned into the Bacillus subtilis promoter probe vector pPL603b.1, using a BglII digest of chromosomal DNA. The resulting plasmid, pVC102, was shown to have a BglII site within the insert. It was determined that this was the result of the fortuitous co-cloning of 2.88-kb and 0.92-kb BglII fragments separated in NCP1 DNA by approx. 3 kb. Unexpectedly, this co-cloning was readily repeated. Subcloning showed that while the 2.88-kb amy-bearing fragment was sufficient for amylase production, it might not have been capable of promoting sufficient levels of chloramphenicol resistance under the conditions used in the cloning experiments. The promoter on the 0.92-kb BglII fragment was more efficient, although its sequence differed from the canonical promoter sequence recognised by B. subtilis RNA polymerase E.sigma 43. As other promoter-bearing fragments from NCP1 DNA operated equally efficiently when cloned into pPL603b.1, the reason for the repeated co-cloning of the 2.88-kb and 0.92-kb NCPI BglII fragments may well be due to structural parameters, whereby certain nucleotide sequences are more readily cloned than others.     

Phylogenetic,Metabolic, and Taxonomic Diversities Shape Mediterranean Fruit Fly Microbiotas during Ontogeny

The Mediterranean fruit fly (medfly) (Ceratitis capitata) lays eggs in fruits, where larvae subsequently develop, causing large-scale agricultural damage. Within its digestive tract, the fly supports an extended bacterial community that is composed of multiple strains of a variety of enterobacterial species. Most of these bacteria appear to be functionally redundant, with most strains sustaining diazotrophy and/or pectinolysis. At least some of these bacteria were shown to be vertically inherited, but colonization, structural, and metabolic aspects of the community''s dynamics have not been investigated. We used fluorescent in situ hybridization, metabolic profiling, plate cultures, and pyrosequencing to show that an initial, egg-borne, diverse community expands throughout the fly''s life cycle. While keeping “core” diazotrophic and pectinolytic functions, it also harbors diverse and fluctuating populations that express varied metabolic capabilities. We suggest that the metabolic and compositional plasticity of the fly''s microbiota provides potential adaptive advantages to the medfly host and that its acquisition and dynamics are affected by mixed processes that include stochastic effects, host behavior, and molecular barriers.     

Endoplasmic Reticulum Stress Induces a Caspase-dependent N-terminal Cleavage of RBX1 Protein in B Cells

Endoplasmic reticulum (ER) stress develops when the ER is overloaded with too many proteins to fold. This elicits a signaling pathway called the unfolded protein response. The unfolded protein response is physiologically required for the terminal development of B cells into antibody-secreting plasma cells. Ring Box Protein 1 (RBX1) is a 14-kDa protein necessary for ubiquitin ligation activity of the multimeric cullin ring ubiquitin ligases (CRLs). As RBX1 is shared by a large number of CRLs, alterations in its activity may lead to global changes in protein stability. We discovered that RBX1 is cleaved in the course of LPS-induced plasma cell differentiation and in multiple myeloma cell lines upon induction of pharmacological ER stress. The cleavage is executed by several caspase proteases that cleave RBX1 eight amino acids from the N terminus. To address the possible implication of RBX1 cleavage for CRL activity, we replaced the endogenous RBX1 homolog of the yeast Saccharomyces cerevisiae, Roc1, with the wild type or the N-terminal Δ8 mutant human RBX1. We show that yeast expressing the cleaved RBX1 are hypersensitive to ER stress and are impaired in CRL-mediated ubiquitination and degradation. We propose a model by which N-terminal cleavage of RBX1 impairs its activity and promotes susceptibility to ER stress induction.     

The Claustrum Supports Resilience to Distraction

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Persistence to anti-cancer treatments in the stationary to proliferating transition

The heterogeneous responses of clonal cancer cells to treatment is understood to be caused by several factors, including stochasticity, cell-cycle dynamics, and different micro-environments. In a tumor, cancer cells may encounter fluctuating conditions and transit from a stationary culture to a proliferating state, for example this may occur following treatment. Here, we undertake a quantitative evaluation of the response of single cancerous lymphoblasts (L1210 cells) to various treatments administered during this transition. Additionally, we developed an experimental system, a “Mammalian Mother Machine,” that tracks the fate of thousands of mammalian cells over several generations under transient exposure to chemotherapeutic drugs. Using our developed system, we were able to follow the same cell under repeated treatments and continuously track many generations. We found that the dynamics of the transition between stationary and proliferative states are highly variable and affect the response to drug treatment. Using cell-cycle markers, we were able to isolate a subpopulation of persister cells with distinctly higher than average survival probability. The higher survival rate encountered with cell-cycle phase specific drugs was associated with a significantly longer time-till-division, and was reduced by a non cell-cycle specific drug. Our results suggest that the variability of transition times from the stationary to the proliferating state may be an obstacle hampering the effectiveness of drugs and should be taken into account when designing treatment regimens.     

Partial Substitution of Serum in Hematopoietic Cell Line Media by Synthetic Polymers

Several synthetic polymers (hydroxyethyl starch, sodium carboxymethyl cellulose, polyvinylpyrrolidone) markedly improved the growth of three human lymphocyte cell lines [Roswell Park Memorial Institute (RPMI) 1348, 1788, and 8098]. Growth was stimulated when each of these polymers was added to RPMI 1640 medium supplemented with only 2% fetal bovine serum. Dextran T-40, T-70, and T-110 varied in their effect on the growth of these cell lines. Dextran T-250 and Haemaccel did not improve cell yields when partially substituted for the serum. The successful partial substitution of polymers for serum was specific for individual cell lines.     

Large scale production of human lymphoblastoid (Namalva) interferon

Summary Large scale production of human lymphoblastoid (Namalva) interferon (IF) is described. Cell propagation, in up to 50 1 culture volume, was carried out in a low cost medium by a semi-continuous cultivation method. IF was induced by Sendai virus, testing two induction methods. The yield of crude IF varied in the range of 12 – 100 × 10³ IF units.ml^-1. A weekly production output of 1 – 5 × 10⁸ units crude IF was obtained.     

Abscisic Acid and cytokinin contents of leaves in relation to salinity and relative humidity

The question is raised whether the hormonal modifications in a plant exposed to osmotic root stress result directly from the decrease in water potential of the root environment or from disturbances of the plant's water balance.