Plasmid rescue - a tool for reproducible recovery of genes from transfected mammalian cells?

Summary The efficient rescue of plasmids containing the thymidine kinase gene (tk) of Herpes simplex virus type I from genetically transformed mouse cells by transformation of bacteria is described. Rescued plasmids contain insertions of calf DNA used as a carrier in the transfection but usually lack portions of plasmid DNA. Deletions generally concern the region spanning from around the PvuII site of pBR322 to within the tetracycline resistance coding sequence, whereas the extent of tk sequence deletion varies, depending on the site of its integration (BamHI or PvuII) into the plasmid. Modelling the rescue process by transformation of bacteria with a mixture of original plasmids and sheared mouse cell DNA clearly demonstrates that deletions are caused by the presence of the mammalian DNA and they probably occur during re-transformation of bacteria before the onset of tetracycline gene expression. Plasmids lacking the Tc^r region are reproducibly rescuable without deletion. Methods for reproducible re-isolation of transferred genes from mammalian cells are discussed.     

A selection mosaic in the facultative mutualism between ants and wild cotton

In protection mutualisms, one mutualist defends its partner against a natural enemy in exchange for a reward, usually food or shelter. For both partners, the costs and benefits of these interactions often vary considerably in space because the outcome (positive, negative or neutral) depends on the local abundance of at least three species: the protector, the beneficiary of protection and the beneficiary's natural enemy. In Gossypium thurberi (wild cotton), ants benefit nutritionally from the plant's extrafloral nectaries and guard plants from herbivores. Experimentally altering the availability of both ants and extrafloral nectar in three populations demonstrated that the mutualism is facultative, depending, in part, on the abundance of ants and the level of herbivore damage. The species composition of ants and a parasitic alga that clogs extrafloral nectaries were also implicated in altering the outcome of plant-ant interactions. Furthermore, experimental treatments that excluded ants (the putative selective agents) in combination with phenotypic selection analyses revealed that selection on extrafloral nectary traits was mediated by ants and, importantly, varied across populations. This work is some of the first to manipulate interactions experimentally across multiple sites and thereby document that geographically variable selection, mediated by a mutualist, can shape the evolution of plant traits.     

TRPC3 mediates T-cell receptor-dependent calcium entry in human T-lymphocytes

Stimulation of the T-cell receptor (TCR) activates Ca2+ entry across the plasma membrane, which is a key triggering event for the T-cell-associated immune response. We show that TRPC3 channels are important for the TCR-dependent Ca2+ entry pathway. The TRPC3 gene was found to be damaged in human T-cell mutants defective in Ca2+ influx. Mutations of the TRPC3 gene were accompanied by changes of TRPC3 gene expression. Introduction of the complete human TRPC3 cDNA into those mutants rescued Ca2+ currents as well as TCR-dependent Ca2+ signals. Our data provide the initial step toward understanding the molecular nature of endogenous Ca2+ channels participating in T-cell activation and put forward TRPC3 as a new target for modulating the immune response.     

Administration of antiprogestin J956 for contraception in bears: a pharmacological study

Effective and reversible control of reproduction in bears is highly desirable for conservation and management programs in zoos to establish genetically variable ex-situ populations of bears within the constraints of limited space in captivity. The reproductive physiology of bears is characterized by two main traits--seasonality and delayed implantation, which is progesterone dependent. This offers the opportunity to interrupt early pregnancy by short-term administration of antiprogestins. The aim of the present study was to investigate the pharmacological characteristics of antiprogestin J956 to establish an efficient contraceptive protocol for administration of J956 in captive bears. The J956 binds to the uterine progesterone receptor of bears (n = 2) with almost the same relative binding affinity (1.25) as progesterone. The blood serum level of J956 after oral (on four consecutive days) and single parenteral administration was determined by a modified progestin receptor assay. The relative bioavailability of J956 after oral administration was approximately 10% of the parenteral administration. The estimated half-life was 12 to 16 hours after oral administration. Parenteral treatment of J956 (10 mg/kg body mass) led to sustain plasma concentrations (6.4 +/- 1.3 ng/mL) in one black bear and in five brown bears. The plasma level lasted for almost 2 months. Oral and low dosage parenteral (1 mg/kg body mass) administration of J956 had no effect on ongoing pregnancies in bears. Whereas single parenteral administration with higher dosages of J956 (7.5 to 10 mg/kg body mass) efficiently prevented implantation of early embryos in eight female captive bears.     

Improving the performance of Rosetta using multiple sequence alignment information and global measures of hydrophobic core formation

This study explores the use of multiple sequence alignment (MSA) information and global measures of hydrophobic core formation for improving the Rosetta ab initio protein structure prediction method. The most effective use of the MSA information is achieved by carrying out independent folding simulations for a subset of the homologous sequences in the MSA and then identifying the free energy minima common to all folded sequences via simultaneous clustering of the independent folding runs. Global measures of hydrophobic core formation, using ellipsoidal rather than spherical representations of the hydrophobic core, are found to be useful in removing non-native conformations before cluster analysis. Through this combination of MSA information and global measures of protein core formation, we significantly increase the performance of Rosetta on a challenging test set. Proteins 2001;43:1-11.     

The "A" rule revisited: polymerases as determinants of mutational specificity

Organisms control the specificity and frequency with which they mutate via their complement of proteins. The mismatch repair (MMR) proteins correct errors after they are made. The DNA polymerases of the cell determine the response to damaged DNA which has not been repaired by excision. Polymerase action can be considered as consisting of three main steps: addition of a base, proofreading of the added nucleotide and elongation. Each of these steps is kinetically complex and can be modulated. The modulation accounts for different behaviors of organisms in response to stress. The recent findings of DNA polymerases with properties appropriate for dealing with damaged DNA may help to account for the phenomenon of spontaneous mutation and for the hypermutability associated with immunoglobulin maturation and carcinogenesis.     

The distribution of 3-hydroxy oxylipins in fungi

One of the best-kept secrets by fungi especially yeast is the function of the different shapes and surface structures of their vegetative and sexual cells. They definitely do not produce these shapes (e.g. round, elongated, kidney, needle, hat, saturnoid, etc.) and surfaces (e.g. smooth, rough, hairy, warty, etc.) for our curiosity or to be classified, but surely produce these for their own benefit. This mini-review will show that a large variety of 3-hydroxy oxylipins are widely distributed in the fungal domain and closely associated with these surface ornamentations. In concert with nano-scale surface structures, they probably play a role in cell aggregation as well as spore release from sexual structures such as asci.