Structural characterization of yeast acidic ribosomal P proteins forming the P1A-P2B heterocomplex

Acidic ribosomal P proteins form a distinct lateral protuberance on the 60S ribosomal subunit. In yeast, this structure is composed of two heterocomplexes (P1A-P2B and P1B-P2A) attached to the ribosome with the aid of the P0 protein. In solution, the isolated P proteins P1A and P2B have a flexible structure with some characteristics of a molten globule [Zurdo, J., et al. (2000) Biochemistry 39, 8935-8943]. In this report, the structure of P1A-P2B heterocomplex from Saccharomyces cerevisiae is investigated by means of size-exclusion chromatography, chemical cross-linking, circular dichroism, light scattering, and fluorescence spectroscopy. The circular dichroism experiment shows that the complex could be ranked in the tertiary class of all-alpha proteins, with an average alpha-helical content of approximately 65%. Heat and urea denaturation experiments reveal that the P1A-P2B complex, unlike the isolated proteins, has a full cooperative transition which can be fitted into a two-state folding-unfolding model. The behavior of the complex in the presence of 2,2,2-trifluoroethanol also resembles a two-state folding-unfolding transition, further supporting the idea that the heterocomplex contains well-packed side chains. In conclusion, the P1A-P2B heterocomplex, unlike the isolated proteins, has a well-defined hydrophobic core. Consequently, the complex can put up its structure without additional ribosomal components, so the heterodimeric complex reflects the intrinsic properties of the two analyzed proteins, indicating thus that this is the only possible configuration of the P1A and P2B proteins on the ribosomal stalk structure.     

Developmental expression of zebrafish emx1 during early embryogenesis

Emx family homeobox genes, Emx1 and Emx2, play an essential role in rostral brain development in mammalian embryos. Here we report a zebrafish emx family gene, emx1, which is more similar to the mouse Emx1 gene than the previously reported zebrafish emx1 gene; we propose to rename that gene emx3. The expression of emx1 is first detected around the 10-somite stage in the pineal gland (epiphysis) primodium in the developing anterior brain and in the pronephric primodium within the intermediate mesoderm. emx1 expression in the epiphysis has not been reported in other species. Expression in the epiphysis is suppressed at 23 h post-fertilization (hpf) in the floating head (flh) mutant, in which development of the epiphysis is impaired. Subsequently, emx1 is expressed in the telencephalon, as reported in mammals, and can be detected in the olfactory placode and in a small group of cells in the forebrain at 25 hpf. In the mesoderm, emx1 expression is gradually concentrated in the posterior pronephric duct during somitogenesis, and becomes expressed predominantly in the urogenital opening at 25 hpf. Thus, emx1 displays a unique expression pattern that is distinct from the patterns of emx2 and emx3.     

Probabilistic expert systems for DNA mixture profiling

We show how probabilistic expert systems can be used to structure and solve complex cases of forensic identification involving DNA traces that might be mixtures of several DNA profiles. In particular, this approach can readily handle cases where the number of contributors to the mixture cannot be regarded as known in advance. The flexible modularity of the networks used also allows us to handle still more complex cases, for example where the finding of a mixed DNA trace is compounded by such features as missing individuals or the possibility of unobserved alleles.     

The subcellular distribution of the human ribosomal "stalk" components: P1, P2 and P0 proteins

The ribosomal "stalk" structure is a distinct lateral protuberance located on the large ribosomal subunit in prokaryotic, as well as in eukaryotic cells. In eukaryotes, this ribosomal structure is composed of the acidic ribosomal P proteins, forming two hetero-dimers (P1/P2) attached to the ribosome through the P0 protein. The "stalk" is essential for the ribosome activity, taking part in the interaction with elongation factors.In this report, we have shown that the subcellular distribution of the human P proteins does not fall into standard behavior of regular ribosomal proteins. We have used two approaches to assess the distribution of the P proteins, in vivo experiments with GFP fusion proteins and in vitro one with anti-P protein antibodies. In contrast to standard r-proteins, the P1 and P2 proteins are not actively transported into the nucleus compartment, remaining predominantly in the cytoplasm (the perinuclear compartment). The P0 protein was found in the cytoplasm, as well as in the nucleus; however, the nucleoli were excluded. This protein was scattered around the nuclei, and the distribution might reflect association with the so-called nuclear bodies. This is the first example of r-proteins that are not actively transported into the nucleus; moreover, this might imply that the "stalk" constituents are assembled onto the ribosomal particle at the very last step of ribosomal maturation, which takes part in the cell cytoplasm.     

A statistical treatment of biases affecting the estimation of mutation rates

We consider the estimation of mutation rates, using family data obtained from disputed paternity cases. It is shown how to take appropriate account of a number of complicating features-in particular, hidden mutation, differential mutation, and uncertain paternity-which can necessitate large corrections to simple estimates.     

The arginine-facing amino acid residue of the rat aquaporin 1 constriction determines solute selectivity according to its size and lipophilicity

Aquaporins (AQP) are transmembrane channels for small, predominantly uncharged solutes. Their selectivity is partly determined by the aromatic/arginine constriction. Ammonia is similar in size and polarity to water, yet a subset of aquaporins distinguishes between the two. We mutated the constriction of water-selective rat AQP1 to mimic that of the ammonia-permeable human AQP8 by replacing Phenylalanine 56 with histidine, Histidine 180 with isoleucine, and Cysteine 189 with glycine, alone and in combination. Only AQP1 mutants including the H180I exchange increased the ammonia and methylamine tolerance of yeast. In a second set of mutations, we replaced Histidine 180 with alanine, leucine, methionine, phenylalanine, asparagine or glutamine. AQP1 H180A was equivalent to AQP1 H180I. AQP1 H180L increased ammonia but not methylamine tolerance of yeast. AQP1 mutants with methionine, phenylalanine, asparagine or glutamine in place of Histidine 180, increased neither ammonia nor methylamine tolerance of yeast. All mutants conducted water, as judged by osmotic assays with yeast sphaeroplasts. We propose that the arginine-facing amino acid residue is the most versatile selector of aquaporin constrictions, excluding Escherichia coli glycerol facilitator-type aquaporins.