The evolutionary significance of genetic variation at enzyme synthesizing loci in the teleost Fundulus heteroclitus

Using the teleost Fundulus heteroclitus as an experimental model, we demonstrate how a multi-disciplinary approach to problems of intraspecies genetic variation can provide a better understanding of complex biodiversity problems than can be addressed by a more monolithic approach. We emphasize the importance of starting with simple molecular systems and making predictions that can be tested by experimentation at a higher level of biological complexity-leading from molecules to cells to organ systems to organisms and eventually laboratory and field selection experiments. Using this interdisciplinary approach, we address the classical 'selectionisti neutralist' controversy.     

Proligands with protease‐regulated binding activity identified from cell‐displayed prodomain libraries

A general method was developed for the discovery of protease‐activated binding ligands, or proligands, from combinatorial prodomain libraries displayed on the surface of E. coli. Peptide libraries of candidate prodomains were fused with a matrix metalloprotease‐2 substrate linker to a vascular endothelial growth factor‐binding peptide and sorted using a two‐stage flow cytometry screening procedure to isolate proligands that required protease treatment for binding activity. Prodomains that imparted protease‐mediated switching activity were identified after three sorting cycles using two unique library design strategies. The best performing proligand exhibited a 100‐fold improvement in apparent binding affinity after exposure to protease. This method may prove useful for developing therapeutic and diagnostic ligands with improved systemic targeting specificity.     

Cloning and Functional Characterisation of Two Novel Nicotinic Acetylcholine Receptor α Subunits from the Insect Pest Myzus persicae

Abstract: Nicotinic acetylcholine receptors play a major role in excitatory neurotransmission in insect CNSs and constitute an important target for insecticides. Here, we report the isolation and functional characterisation of two cDNAs encoding nicotinic acetylcholine receptor α subunits from a major insect pest, the peach-potato aphid Myzus persicae . These two subunits, termed Mpα1 and Mpα2, are respective structural homologues of the Drosophila Dα2/ Schistocerca gregaria αL1 α-subunit pair and the Drosophila ALS α subunit. Xenopus oocyte expression confirmed that each Myzus subunit can form functional acetylcholine- or nicotine-gated channels. However, some electrophysiological and pharmacological properties of the Myzus subunits were distinct from those encoded by the corresponding Drosophila subunits. Coexpression of the Myzus subunits with the chick β2 subunit revealed other differences from the Drosophila system, as only very limited potentiation of agonist-induced currents was observed with Mpα2 and none with Mpα1. Available data therefore indicate that structurally homologous insect nicotinic acetylcholine receptor α subunits from different species can exhibit distinctive physiological and pharmacological characteristics.     

Evidence for a precursor molecule of Brazil nut 2 S seed proteins from biosynthesis and cDNA analysis

Summary Seed storage proteins were extracted from Brazil nut (Bertholletia excelsa H.B.K.) seed embryos at various maturation stages. Salt-soluble and water-soluble proteins (globulins and albumins) were separated by gel chromatography and exhaustive dialysis against water. Both fractions were analysed by one- and two-dimensional polyacrylamide gel electrophoresis. Amino acid analysis revealed that both fractions are unusually high in methionine. The albumins consist of a family of low molecular weight polypeptides that are heterogeneous with respect to pI and are identical to the high methionine 2 S proteins described by Youle and Huang (1981). The biosynthesis of this class of proteins in maturing embryos was followed by in vivo labelling combined with immunological studies. Western blotting with monospecific antibodies against purified 2 S albumins and sequencing of a nearly complete cDNA clone revealed that they are synthesized via a precursor polypeptide.     

The sequence of a porcine cDNA encoding α-lactalbumin

A cDNA clone encoding porcine α-lactalbumin (αLA) was isolated and sequenced. The longest clone was 688 nucleotides (nt) long and encoded a preprotein of 141 amino acids (aa) including a leader peptide of 19 aa. The porcine cDNA exhibited a nt similarity of between 72.2%–83.5% to other αLA cDNAs and an aa similarity of between 50.8%–85.2% with other αLA aa sequences. The derived aa sequence varied at three positions from a previously reported sequence for porcine αLA obtained by direct aa sequencing.     

Identification of GABAA Receptor Subunits in Rat Retina: Cloning of the Rat GABAA Receptor ρ2-Subunit cDNA

Abstract: We identified GABA_A receptor subunits in rat retina using PCR. The high degree of conservation among previously described members of ligand-gated anion channels in transmembrane domains was used to design degenerate sense and antisense oligonucleotides. These oligonucleotides were used as primers for PCR, which was applied to the rat retina cDNA. Analysis of clones derived from the PCR amplification identified the GABA_Aα₁, β₁, β₃, and γ₂ subunits and the glycine α₁ subunit. In addition, two clones closely related to the human GABA_Aρ-subunit class were obtained. Molecular cloning revealed one of them as the rat counterpart of the human ρ₂ subunit. Northern blot analysis demonstrated the expression of mRNAs for ρ subunits in retina. These results further support the hypothesis that bicuculline-insensitive GABA channels in rat retina are comprised of ρ subunits.