Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Sequence dependence of protein isoprenylation

Several proteins have been shown to be post-translationally modified on a specific C-terminal cysteine residue by either of two isoprenoid biosynthetic pathway metabolites, farnesyl diphosphate or geranylgeranyl diphosphate. Three enzymes responsible for protein isoprenylation were resolved chromatographically from the cytosolic fraction of bovine brain: a farnesyl-protein transferase (FTase), which modified the cell-transforming Ras protein, and two geranyl-geranyl-protein transferases, one (GGTase-I) which modified a chimeric Ras having the C-terminal amino acid sequence of the gamma-6 subunit of heterotrimeric GTP-binding proteins, and the other (GGTase-II) which modified the Saccharomyces cerevisiae secretory GTPase protein YPT1. In a S. cerevisiae strain lacking FTase activity (ram1), both GGTases were detected at wild-type levels. In a ram2 S. cerevisiae strain devoid of FTase activity, GGTase-I activity was reduced by 67%, suggesting that GGTase-I and FTase activities derive from different enzymes but may share a common genetic feature. For the FTase and the GGTase-I activities, the C-terminal amino acid sequence of the protein substrate, the CAAX box, appeared to contain all the critical determinants for interaction with the transferase. In fact, tetrapeptides with amino acid sequences identical to the C-terminal sequences of the protein substrates for FTase or GGTase-I competed for protein isoprenylation by acting as alternative substrates. Changes in the CAAX amino acid sequence of protein substrates markedly altered their ability to serve as substrates for both FTase and GGTase-I. In addition, it appeared that FTase and GGTase-I had complementary affinities for CAAX protein substrates; that is, CAAX proteins that were good substrates for FTase were, in general, poor substrates for GGTase-I, and vice versa. In particular, a leucine residue at the C terminus influenced whether a CAAX protein was either farnesylated or geranylgeranylated preferentially. The YPT1 C terminus peptide, TGGGCC, did not compete or serve as a substrate for GGTase-II, indicating that the interaction between GGTase-II and YPT1 appeared to depend on more than the 6 C-terminal residues of the protein substrate sequence. These results identify three different isoprenyl-protein transferases that are each selective for their isoprenoid and protein substrates.     

Structural homology among mammalian and Saccharomyces cerevisiae isoprenyl-protein transferases

Farnesyl-protein transferase (FTase) purified from rat or bovine brain is an alpha/beta heterodimer, comprised of subunits having relative molecular masses of approximately 47 (alpha) and 45 kDa (beta). In the yeast Saccharomyces cerevisiae, two unlinked genes, RAM1/DPR1 (RAM1) and RAM2, are required for FTase activity. To explore the relationship between the mammalian and yeast enzymes, we initiated cloning and immunological analyses. cDNA clones encoding the 329-amino acid COOH-terminal domain of bovine FTase alpha-subunit were isolated. Comparison of the amino acid sequences deduced from the alpha-subunit cDNA and the RAM2 gene revealed 30% identity and 58% similarity, suggesting that the RAM2 gene product encodes a subunit for the yeast FTase analogous to the bovine FTase alpha-subunit. Antisera raised against the RAM1 gene product reacted specifically with the beta-subunit of bovine FTase, suggesting that the RAM1 gene product is analogous to the bovine FTase beta-subunit. Whereas a ram1 mutation specifically inhibits FTase, mutations in the CDC43 and BET2 genes, both of which are homologous to RAM1, specifically inhibit geranylgeranyl-protein transferase (GGTase) type I and GGTase-II, respectively. In contrast, a ram2 mutation impairs both FTase and GGTase-I, but has little effect on GGTase-II. Antisera that specifically recognized the bovine FTase alpha-subunit precipitated both bovine FTase and GGTase-I activity, but not GGTase-II activity. Together, these results indicate that for both yeast and mammalian cells, FTase, GGTase-I, and GGTase-II are comprised of different but homologous beta-subunits and that the alpha-subunits of FTase and GGTase-I share common features not shared by GGTase-II.     

Calls,colours, shape,and genes: a multi‐trait approach to the study of geographic variation in the Amazonian frog Allobates femoralis

Evolutionary divergence in behavioural traits related to mating may represent the initial stage of speciation. Direct selective forces are usually invoked to explain divergence in mate‐recognition traits, often neglecting a role for neutral processes or concomitant differentiation in ecological traits. We adopted a multi‐trait approach to obtain a deeper understanding of the mechanisms behind allopatric divergence in the Amazonian frog, Allobates femoralis. We tested the null hypothesis that geographic distance between populations correlates with genetic and phenotypic divergence, and compared divergence between mate‐recognition (acoustic) and ecological (coloration, body‐shape) traits. We quantified geographic variation in 39 phenotypic traits and a mitochondrial DNA marker among 125 individuals representing eight populations. Geographic variation in acoustic traits was pronounced and tracked the spatial genetic variation, which appeared to be neutral. Thus, the evolution of acoustic traits tracked the shared history of the populations, which is unexpected for pan‐Amazonian taxa or for mate‐recognition traits. Divergence in coloration appeared uncorrelated with genetic distance, and might be partly attributed to local selective pressures, and perhaps to Batesian mimicry. Divergence in body‐shape traits was low. The results obtained depict a complex evolutionary scenario and emphasize the importance of considering multiple traits when disentangling the forces behind allopatric divergence. ©2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 826–838.     

Catalytic properties of mutant 23 S ribosomes resistant to oxazolidinones

Kinetic analysis of ribosomal peptidyltransferase activity in a methanolic puromycin reaction with wild type and drug-resistant 23 S RNA mutants was used to probe the structural basis of catalysis and mechanism of resistance to antibiotics. 23 S RNA mutants G2032A and G2447A are resistant to oxazolidinones both in vitro and in vivo with the latter displaying a 5-fold increase in the value of Km for initiator tRNA and a 100-fold decrease in Vmax in puromycin reaction. Comparison of the Ki values for oxazolidinones, chloramphenicol, and sparsomycin revealed partial cross-resistance between oxazolidinones and chloramphenicol; no cross-resistance was observed with sparsomycin, a known inhibitor of the peptidyltransferase A-site. Inhibition of the mutants using a truncated CCA-Phe-X-Biotin fragment as a P-site substrate is similar to that observed with the intact initiator tRNA, indicating that the inhibition is substrate-independent and that the peptidyltransferase itself is the oxazolidinone target. Mapping of all known mutations that confer resistance to these drugs onto the spatial structure of the 50 S ribosomal subunit allows for docking of an oxazolidinone into a proposed binding pocket. The model suggests that oxazolidinones bind between the P- and A-loops, partially overlapping with the peptidyltransferase P-site. Thus, kinetic, mutagenesis, and structural data suggest that oxazolidinones interfere with initiator fMet-tRNA binding to the P-site of the ribosomal peptidyltransferase center.     

Evolution of base composition in the insulin and insulin-like growth factor genes

The genomes of homeothermic (warm-blooded) vertebrates are mosaic interspersions of homogeneously GC-rich and GC-poor regions (isochores). Evolution of genome compartmentalization and GC-rich isochores is hypothesized to reflect either selective advantages of an elevated GC content or chromosome location and mutational pressure associated with the timing of DNA replication in germ cells. To address the present controversy regarding the origins and maintenance of isochores in homeothermic vertebrates, newly obtained as well as published nucleotide sequences of the insulin and insulin-like growth factor (IGF) genes, members of a well-characterized gene family believed to have evolved by repeated duplication and divergence, were utilized to examine the evolution of base composition in nonconstrained (flanking) and weakly constrained (introns and fourfold degenerate sites) regions. A phylogeny derived from amino acid sequences supports a common evolutionary history for the insulin/IGF family genes. In cold- blooded vertebrates, insulin and the IGFs were similar in base composition. In contrast, insulin and IGF-II demonstrate dramatic increases in GC richness in mammals, but no such trend occurred in IGF- I. Base composition of the coding portions of the insulin and IGF genes across vertebrates correlated (r = 0.90) with that of the introns and flanking regions. The GC content of homologous introns differed dramatically between insulin/IGF-II and IGF-I genes in mammals but was similar to the GC level of noncoding regions in neighboring genes. Our findings suggest that the base composition of introns and flanking regions is determined by chromosomal location and the mutational pressure of the isochore in which the sequences are embedded. An elevated GC content at codon third positions in the insulin and the IGF genes may reflect selective constraints on the usage of synonymous codons.      

Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.