Cyclophilin 40: An Hsp90-cochaperone associated with apo-steroid receptors

Cyclophilin 40, a divergent loop cyclophilin first identified in association with the estrogen receptor α, contains a C-terminal tetratricopeptide repeat domain through which it shares structural identity with FK506-binding protein 52 (FKBP52) and other partner cochaperones in steroid receptor-heat shock protein 90 (Hsp90) complexes. By dynamically competing for Hsp90 interaction, the cochaperones allow the receptors to establish distinct Hsp90-chaperone complexes, with the potential to exert tissue-specific control over receptor activity. Cyclophilin 40 regulates Hsp90 ATPase activity during receptor-Hsp90 assembly. Functional deletion of the cyclophilin 40 yeast homologue, Cpr7, adversely affected estrogen receptor α and glucocorticoid receptor activity that could be fully restored, either with wild type Cpr7 or Cpr7 with a cyclophilin domain lacking isomerase activity. We draw parallels with the mechanism already established for FKBP52 and propose that the cyclophilin 40 divergent loop interfaces with a contact surface on the steroid receptor ligand-binding domain to achieve an optimal orientation for receptor activity.     

A solid-phase screen for protein kinase substrate selectivity.

Cassette mutagenesis was used to synthesize an Escherichia coli expression library of unique phosphorylation sites. The cassette encodes a central serine residue surrounded by every combination of Ala, Arg, Gln, Glu, Gly, and Pro residues over a 7-residue segment (a total of 6(7) approximately 2.8 x 10(5) sequences). The cassette was inserted into the gene of a suitable carrier protein and expressed in E. coli with the T7 expression system, and the resultant library was subjected to solid-phase protein phosphorylation assays on nitrocellulose filters. When the library was screened with TPK1 delta, the modified catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase, individual colonies that expressed substrates for this kinase were identified. By DNA sequencing through the cassette region of positive clones, the consensus recognition sequence for TPK1 delta was deduced and found to conform with the well-established substrate selectivity of its mammalian homolog (Arg-Arg-Xaa-Ser). Because a large number of clones can be sequenced rapidly, and the positions of invariant residues composing a recognition site identified, this approach may be useful as a general screen of protein kinase substrate selectivity.     

The influence of lethal mutations on the quantification of radiation transformation frequencies

Transformation frequencies for gamma irradiated C3H 10T1/2 cells have been analysed, taking account of the occurrence of lethal mutations in these cells. Lethal mutations already noted by these authors in primary thyroid and established CHO K1 cells occur at high levels in C3H 10T1/2 cells and lead, therefore, to considerable underestimates of transformation frequency, particularly at high doses where this is expressed on a per surviving cell basis. The results may help to provide an explanation for the dose response plateau which is seen when these cells are irradiated and transformed foci per surviving cell are scored.     

An inquiry into the evolutionary status and apparent identity of San hunter-gatherers

An attempt is made to reconcile a popular view of the Bushmen or San of southern Africa with reality. Following an analysis of the assumed relationships of living hunter-gatherers with their Pleistocene forebears and modem neighbors, the identity of the San is explored using archaeological and historical evidence. Finally an alternative view of modern San, consistent with this evidence, is proposed.     

Genome-wide analysis of substrate specificities of the Escherichia coli haloacid dehalogenase-like phosphatase family

Haloacid dehalogenase (HAD)-like hydrolases are a vast superfamily of largely uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Using a representative set of 80 phosphorylated substrates, we characterized the substrate specificities of 23 soluble HADs encoded in the Escherichia coli genome. We identified small molecule phosphatase activity in 21 HADs and beta-phosphoglucomutase activity in one protein. The E. coli HAD phosphatases show high catalytic efficiency and affinity to a wide range of phosphorylated metabolites that are intermediates of various metabolic reactions. Rather than following the classical "one enzyme-one substrate" model, most of the E. coli HADs show remarkably broad and overlapping substrate spectra. At least 12 reactions catalyzed by HADs currently have no EC numbers assigned in Enzyme Nomenclature. Surprisingly, most HADs hydrolyzed small phosphodonors (acetyl phosphate, carbamoyl phosphate, and phosphoramidate), which also serve as substrates for autophosphorylation of the receiver domains of the two-component signal transduction systems. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, YniC. Many of the secondary activities of HADs might have no immediate physiological function but could comprise a reservoir for evolution of novel phosphatases.     

Sequence‐level comparative analysis of the Brassica napus genome around two stearoyl‐ACP desaturase loci

We conducted a sequence‐level comparative analyses, at the scale of complete bacterial artificial chromosome (BAC) clones, between the genome of the most economically important Brassica species, Brassica napus (oilseed rape), and those of Brassica rapa, the genome of which is currently being sequenced, and Arabidopsis thaliana. We constructed a new B. napus BAC library and identified and sequenced clones that contain homoeologous regions of the genome including stearoyl‐ACP desaturase‐encoding genes. We sequenced the orthologous region of the genome of B. rapa and conducted comparative analyses between the Brassica sequences and those of the orthologous region of the genome of A. thaliana. The proportion of genes conserved (～56%) is lower than has been reported previously between A. thaliana and Brassica (～66%). The gene models for sets of conserved genes were used to determine the extent of nucleotide conservation of coding regions. This was found to be 84.2 ± 3.9% and 85.8 ± 3.7% between the B. napus A and C genomes, respectively, and that of A. thaliana, which is consistent with previous results for other Brassica species, and 97.5 ± 3.1% between the B. napus A genome and B. rapa, and 93.1 ± 4.9% between the B. napus C genome and B. rapa. The divergence of the B. napus genes from the A genome and the B. rapa genes was greater than anticipated and indicates that the A genome ancestor of the B. napus cultivar studied was relatively distantly related to the cultivar of B. rapa selected for genome sequencing.