Extensive copy-number variation of the human olfactory receptor gene family

As much as a quarter of the human genome has been reported to vary in copy number between individuals, including regions containing about half of the members of the olfactory receptor (OR) gene family. We have undertaken a detailed study of copy-number variation of ORs to elucidate the selective and mechanistic forces acting on this gene family and the true impact of copy-number variation on human OR repertoires. We argue that the properties of copy-number variants (CNVs) and other sets of large genomic regions violate the assumptions of statistical methods that are commonly used in the assessment of gene enrichment. Using more appropriate methods, we provide evidence that OR enrichment in CNVs is not due to positive selection but is because of OR preponderance in segmentally duplicated regions, which are known to be frequently copy-number variable, and because purifying selection against CNVs is lower in OR-containing regions than in regions containing essential genes. We also combine multiplex ligation-dependent probe amplification (MLPA) and PCR to assay the copy numbers of 37 candidate CNV ORs in a panel of ~50 human individuals. We confirm copy-number variation of 18 ORs but find no variation in this human-diversity panel for 16 other ORs, highlighting the caveat that reported intervals often overrepresent true CNVs. The copy-number variation we describe is likely to underpin significant variation in olfactory abilities among human individuals. Finally, we show that both homology-based and homology-independent processes have played a recent role in remodeling the OR family.     

Molecular defense responses in roots and the rhizosphere against Fusarium oxysporum

Plants face many different concurrent and consecutive abiotic and biotic stresses during their lifetime. Roots can be infected by numerous pathogens and parasitic organisms. Unlike foliar pathogens, root pathogens have not been explored enough to fully understand root-pathogen interactions and the underlying mechanism of defense and resistance. PR gene expression, structural responses, secondary metabolite and root exudate production, as well as the recruitment of plant defense–assisting “soldier” rhizosphere microbes all assist in root defense against pathogens and herbivores. With new high-throughput molecular tools becoming available and more affordable, now is the opportune time to take a deep look below the ground. In this addendum, we focus on soil-borne Fusarium oxysporum as a pathogen and the options plants have to defend themselves against these hard-to-control pathogens.     

The control of seasonal changes in the pigmentation of lime aphid nymphs, Eucallipterus tiliae

Lime aphid nymphs may be found with or without bands of black pigment on the dorsal surfaces of the head, thorax and abdomen. The proportion of pigmented nymphs in a population varies during a season, although first generation nymphs are always unpigmented. The appearance of the black pigment is found to be induced by both crowding and changes in the leaves associated with leaf maturity. Since the crowding stimulus is also found to be transmitted via the leaf, this suggests that pigmentation may be ultimately controlled by a single plant-borne factor, possibly the nutritional quality of the leaves. The possible significance of this response in the life of the aphid is discussed.

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zusammenfassung Bei der Lindenblattlaus können Larven mit und ohne schwarz pigmentierten Bändern auf der Rückenseite des Kopfs, des Thorax und des Abdomens gefunden werden. Der Anteil pigmentierter Larven in einer Population schwankt während einer Saison, indessen sind die Larven der ersten Generation nie pigmentiert. Das schwarze Pigment wird induziert durch Crowding und durch Veränderungen im Blatt, welche mit der Blattreifung zusammenhängen. Da auch der Crowdingstimulus durch das Blatt übertragen wird, dürfte die Pigmentierung letztlich durch einen einzigen in der Pflanze befindlichen Faktor, möglicherweise die Nahrungsqualität der Blätter, gesteuert werden. Die mögliche Bedeutung dieses Zusammenhangs im Leben der Blattlaus wird diskutiert.

     

Purification and characterization of two Cl- -activated aminopeptidases hydrolysing basic termini from human skeletal muscle

Two aminopeptidases (I and II), hydrolysing basic termini, were purified to homogeneity (as judged by polyacrylamide gel electrophoresis) from human quadriceps muscle by anion-exchange chromatography and preparative electrophoresis. The electrophoretic migration rate of II was approximately 80% of that of I. Both enzymes had the following properties: optimum activity was at pH 6.5; addition of 0.15 M Cl- or Br- anions resulted in a 20-fold or 10-fold increase in activity respectively. There was little or no increase in activity on the addition of other anions, or divalent cations (0.05-5mM). Approximately 50% inhibition of activity was obtained in the presence of bestatin (0.1 microM), rho-hydroxymercuriphenylsulphonic acid (0.1 microM), EDTA (10 mM), 1,10-phenanthroline (100 microM), N-ethylmaleimide (1 mM) and But-Thr-Phe-Pro (0.5 mM). The molecular mass was 72 000 Da (gel filtration). Only the arginyl and lysyl 7-amino-4-methylcoumarin (Amc) derivatives were appreciably hydrolysed; approximate Km values for the reaction of I and II with these substrates (10-250 microM) were estimated as follows: Arg-Amc, KmI = 70 microM, KmII = 270 microM; Lys-Amc KmI = 280 microM, KmII = 400 microM. Both enzymes hydrolysed dipeptides with Arg or Lys as the NH2-terminal amino acid, however this was not an absolute requirement for dipeptide hydrolysis. The action of I and II on physiologically active oligopeptides was very restricted, with only bradykinin, proangiotensin and neurotensin being appreciably degraded. The breakdown of these peptides did not occur by classical aminopeptidase action (i.e. hydrolysis of the NH2-terminal residues), but via cleavage of internal peptide bonds. These results suggest that I and II may be isoenzymes of a Cl- -requiring, thiol-type aminopeptidase, which hydrolyses basic termini. These enzymes may act primarily as dipeptidases, with a very restricted mode of action in the degradation of naturally occurring oligopeptides.     

PKC epsilon is associated with myosin IIA and actin in fibroblasts

Proteins coimmunoprecipitating with protein kinase C (PKC) epsilon in fibroblasts were identified through matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF m/s). This method identified myosin IIA in PKC epsilon immunoprecipitates, as well as known PKC epsilon binding proteins, actin, beta'Cop and cytokeratin. Myosin is not a substrate for PKC epsilon. Immunofluorescence analysis showed that PKC epsilon is colocalised with actin and myosin in actomyosin stress fibers in fibroblasts. Inhibitors of PKC and myosin ATPase activity, as well as microfilament-disrupting drugs, all inhibited spreading of fibroblasts after passage, suggesting a role for a PKC epsilon-actin-myosin complex in cell spreading.     

Cell surface expression of 4β-galactosyltransferase accompanies rat parotid gland acinar cell transition to growth

Rat parotid gland acinar cells stimulated to divide by a chronic regimen of isoproterenol demonstrate a dramatic increase in the synthesis of the glycosyltransferase 4β-galactosyltransferase. A plasma membrane localization for much of the increase in 4β-galactosyltransferase was determined by density gradient membrane fractionation. Golgi-enriched fractions showed no increase in specific activity, while plasma membrane activity increased 40-fold. This selective increase at the cell surface was confirmed by immunofluorescence of intact, nonpermeabilized cells from treated glands, using a monospecific antibody prepared against the purified bovine milk transferase. In detergent-permeabilized cells staining of nontreated cells was seen only as groups of perinuclear vesicles, presumed to be Golgi apparatus. In isoproterenol-treated and permcabilized cells both presumptive Golgi and cell surface staining was apparent. Enzyme assays performed on intact cells established that the enzyme's active site was oriented to the exterior of the cells. The transferase could be detected as early as 3 hr after the primary challenge with isoproterenol. Pretrcatment of rats with cycloheximide prevented its appearance.     

Dietary zinc-methionine enhances mononuclear-phagocytic function in young turkeys

The ability of dietary zinc-methionine (Zn-Met) to enhance mononuclear-phagocytic function againstSalmonella arizona andenteritids was investigated in young turkeys. Feed/gain and body wt gain at 21 d of age were not affected by Zn-Met. The addition of 30 or 45 ppm Zn from Zn-Met to a Zn adequate diet significantly increased cutaneous basophil hypersensitivity to phytohemagglutinin-P. The clearance of intravenously administeredS. enteritidis from blood was not affected by 30 ppm of supplemental Zn from Zn-Met. However, 30 ppm Zn from Zn-Met increased the reduction of intravenously administeredS. arizona from spleen. Percentages of myeloid and mononuclear-phagocytic cells before and afterS. enteritidis infection were not affected by supplemental Zn-Met. Turkeys supplemented with Zn-Met showed enhanced in vitro phagocytosis ofS. enteritidis by Sephadex-elicited abdominal exudate cells. The phagocytosis ofS. arizona was unaffected by Zn-Met.     

Phycobilins of cryptophycean algae. Occurrence of dihydrobiliverdin and mesobiliverdin in cryptomonad biliproteins.

Structures of the open-chain tetrapyrrole (bilin) prosthetic groups of the cryptophycean biliproteins phycocyanin 645 (Cr-PC 645; from strain UW374), phycoerythrin 566 (Cr-PE 566; from strain Bermani) and phycoerythrin 545 (Cr-PE 545; from Proteomonas sulcata Hill & Wetherbee) were examined by absorption, 1H NMR spectroscopy, and mass spectrometry. These biliproteins carry the following covalently attached bilins: Cr-PC 645 (alpha subunit) has one mesobiliverdin, (beta subunit), two phycocyanobilins and a doubly linked 15,16-dihydrobiliverdin; Cr-PC 566 (alpha), bilin 584, (beta), phycoerythrobilin and two bilin 584 chromophores (Wedemayer, G.J., Wemmer, D.E., and Glazer, A.N. (1991) J. Biol. Chem. 266, 4731-4741); Cr-PE 545 (alpha) has one 15,16-dihydrobiliverdin and (beta), only phycoerythrobilins. This is the first report of naturally occurring biliproteins carrying either 15,16-dihydrobiliverdin or mesobiliverdin chromophores. Native cryptomonad phycobiliproteins have been classified on the basis of the position of their long wavelength absorption maxima. However, comparison of the bilins of Cr-PE 566 from strain Bermani with those of Cr-PE 566 of strain CBD shows that the two proteins carry different bilins on the alpha subunit. Consequently, the identity of the bilin prosthetic groups on cryptophycean phycobiliproteins cannot be unambiguously inferred from simple inspection of the visible absorption spectra.