Nucleotide polymorphism at the alcohol dehydrogenase locus of pocket gophers, genus Geomys

Using the strictly neutral model as a null hypothesis, we tested for deviations from expected levels of nucleotide polymorphism at the alcohol dehydrogenase locus (Adh-1) within and among four species of pocket gophers (Geomys bursarius major, G. knoxjonesi, G. texensis llanensis, and G. attwateri). The complete protein-encoding region was examined, and 10 unique alleles, representing both electromorphic and cryptic alleles, were used to test hypotheses (e.g., the neutral model) concerning the maintenance of genetic variation. Nineteen variable sites were identified among the 10 alleles examined, including 9 segregating sites occurring in synonymous positions and 10 that were nonsynonymous. Several statistical methods, including those that test for within-species variation as well as those that examine variation within and among species, failed to reject the null hypothesis that variation (both within and between species of Geomys) at the Adh locus is consistent with the neutral theory. However, there was significant heterogeneity in the ratio of polymorphism to divergence across the gene, with polymorphisms clustered in the first half of the coding region and fixed differences clustered in the second half of the gene. Two alternative hypotheses are discussed as possible explanations for this heterogeneity: an old balanced polymorphism in the first half of the gene or a recent selective sweep in the second half of the gene.      

On the inhibition of muscle membrane chloride conductance by aromatic carboxylic acids

25 aromatic carboxylic acids which are analogs of benzoic acid were tested in the rat diaphragm preparation for effects on chloride conductance (G(Cl)). Of the 25, 19 were shown to reduce membrane G(Cl) with little effect on other membrane parameters, although their apparent K(i) varied widely. This inhibition was reversible if exposure times were not prolonged. The most effective analog studied was anthracene-9-COOH (9-AC; K(i) = 1.1 x 10(-5) M). Active analogs produced concentration-dependent inhibition of a type consistent with interaction at a single site or group of sites having similar binding affinities, although a correlation could also be shown between lipophilicity and K(i). Structure-activity analysis indicated that hydrophobic ring substitution usually increased inhibitory activity while para polar substitutions reduced effectiveness.

These compounds do not appear to inhibit G(Cl) by altering membrane surface charge and the inhibition produced is not voltage dependent. Qualitative characteristics of the I-V relationship for Cl(-) current are not altered. Conductance to all anions is not uniformly altered by these acids as would be expected from steric occlusion of a common channel. Concentrations of 9-AC reducing G(Cl) by more than 90 percent resulted in slight augmentation of G(I). The complete conductance sequence obtained at high levels of 9-AC was the reverse of that obtained under control conditions. Permeability sequences underwent progressive changes with increasing 9-AC concentration and ultimately inverted at high levels of the analog. Aromatic carboxylic acids appear to inhibit G(Cl) by binding to a specific intramembrane site and altering the selectivity sequence of the membrane anion channel.

     

Barley aleurone cells contain two types of vacuoles. Characterization Of lytic organelles by use of fluorescent probes

Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence microscopy using pH-sensitive fluorescent probes and a fluorogenic substrate for cysteine proteases showed that both protein storage vacuoles and secondary vacuoles are acidic, lytic organelles. Ratio imaging showed that the pH of secondary vacuoles was lower in aleurone protoplasts incubated in gibberellic acid than in those incubated in abscisic acid. Uptake of fluorescent probes into intact, isolated protein storage vacuoles and secondary vacuoles required ATP and occurred via at least two types of vanadate-sensitive, ATP-dependent tonoplast transporters. One transporter catalyzed the accumulation of glutathione-conjugated probes, and another transported probes not conjugated to glutathione.     

Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under free-living conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).     

Hormonally regulated programmed cell death in barley aleurone cells

Cell death was studied in barley (cv Himalaya) aleurone cells treated with abscisic acid and gibberellin. Aleurone protoplasts incubated in abscisic acid remained viable in culture for at least 3 weeks, but exposure to gibberellin initiated a series of events that resulted in death. Between 4 and 8 days after incubation in gibberellin, >70% of all protoplasts died. Death, which occurred after cells became highly vacuolated, was manifest by an abrupt loss of plasma membrane integrity followed by rapid shrinkage of the cell corpse. Hydrolysis of DNA began before death and occurred as protoplasts ceased production of alpha-amylase. DNA degradation did not result in the accumulation of discrete low molecular weight fragments. DNA degradation and cell death were prevented by LY83583, an inhibitor of gibberellin signaling in barley aleurone. We conclude that cell death in aleurone cells is hormonally regulated and is the final step of a developmental program that promotes successful seedling establishment.     

Expression of the alpha 1-proteinase inhibitor gene family during evolution of the genus Mus

alpha 1-Proteinase inhibitors (alpha 1-PIs) are members of the serpin superfamily of proteinase inhibitors, and are important in the maintenance of homeostasis in a wide variety of animal taxa. Previous studies have shown that in mice (genus Mus), evolution of alpha 1-PIs is characterized by gene amplification, region-specific concerted evolution, and rapid accumulation of amino acid substitutions. The latter occurs primarily in the reactive center, which is the region of the alpha 1-PI molecule that determines the inhibitor's specificity for target proteinases. The P1 residue within the reactive center, which is methionine in so-called orthodox alpha 1-PIs and an amino acid other than methionine in unorthodox alpha 1-PIs, is a primary determinant of inhibitor specificity. In the present study, we find that the expression of mRNAs encoding unorthodox alpha 1-PIs is polymorphic within Mus species, i.e., among individuals or inbred strains. This is in striking contrast to mRNAs that encode orthodox alpha 1-PIs, whose concentrations are relatively invariant. The intraspecies variations in mRNA expression represent polymorphisms in the structure of the alpha 1- PI gene family. The results, taken together with previously described aspects of alpha 1-PI evolution, indicate that the dissimilar levels of polymorphism exhibited by orthodox and unorthodox alpha 1-PIs, which likely have distinct physiological functions, may reflect different levels of selective constraint. The significance of this finding to the evolution of gene families is discussed.