Rapid amplification and cloning of Tn5 flanking fragments by inverse PCR

A simple approach is described to efficiently amplify DNA sequences flanking transposon Tn5 insertions. The method involves: (i) digestion with a restriction enzyme that cuts within Tn5; (ii) self-ligation under conditions favouring the production of monomeric circles; (iii) four parallel PCR reactions using primers designed to amplify left or right flanking sequences, and to distinguish target amplicons from non-specific products. This reveals the number of Tn5 insertions and the size of flanking genomic restriction fragments, without Southern blot analysis. The amplified product contains restriction sites that facilitate cohesive-end cloning. This rapid method is demonstrated using Tn5 and Tn5-Mob tagged DNA sequences involved in albicidin biosynthesis in Xanthomonas albilineans. It is generally applicable for efficient recovery of DNA sequences flanking transposon Tn5 derivatives in insertional mutagenesis studies.     

Transgenic expression of trehalulose synthase results in high concentrations of the sucrose isomer trehalulose in mature stems of field‐grown sugarcane

Sugarcane plants were developed that produce the sucrose isomers trehalulose and isomaltulose through expression of a vacuole‐targeted trehalulose synthase modified from the gene in ‘Pseudomonas mesoacidophila MX‐45’ and controlled by the maize ubiquitin (Ubi‐1) promoter. Trehalulose concentration in juice increased with internode maturity, reaching about 600 mm , with near‐complete conversion of sucrose in the most mature internodes. Plants remained vigorous, and trehalulose production in selected lines was retained over multiple vegetative generations under glasshouse and field conditions.     

Effect of calcium ions on the processing of pro-opiomelanocortin by bovine intermediate lobe pro-opiomelanocortin-converting enzyme.

The effect of Ca2+ on the extent and pattern of processing of pro-opiomelanocortin and an N-terminal fragment by a purified pituitary secretory vesicle, soluble aspartic endoprotease, was studied. Ca2+ stimulated the first cleavage of pro-opiomelanocortin by pro-opiomelanocortin-converting enzyme to yield 21-23 kDa adrenocorticotropin and beta-lipotropin, but its effect was minimal. The production of adrenocorticotropin from the 21-23 kDa intermediate was stimulated approximately 2.3-fold in the presence of 10 mM Ca2+, and processing of beta-lipotropin to beta-endorphin was stimulated about 1.3-1.4-fold by 5-10 mM Ca2+. The production of gamma-melanotropin-immunoreactive material from bovine N-pro-opiomelanocortin(1-77) was stimulated approximately 1.3-fold at both 100 microM and 1.5-2.0 mM Ca2+. Further characterization of the gamma-melanotropin-immunoreactive material by HPLC demonstrated that the major products were gamma 3-[Lys]melanotropin and gamma 3-melanotropin at both Ca2+ concentrations. These results indicate that pro-opiomelanocortin-converting enzyme is stimulated by Ca2+.     

L-phenylisopropyladenosine (L-PIA) diminishes halothane anesthetic requirements and decreases noradrenergic neurotransmission in rats

The effect of L-phenylisopropyladenosine (L-PIA), the A1 adenosine agonist, on the depth of anesthesia was investigated in halothane-anesthetized rats. L-PIA treatment reduced the minimum anesthetic concentration (MAC) of halothane that prevented 50% of animals from moving in response to a painful stimulus by 49%. MAC experiments performed with L-PIA given in conjunction with A1 adenosine receptor antagonists which either permeate the blood-brain barrier (8-phenyltheophylline [8-PT] or do not (8-sulphophenyltheophylline [8-So-PT]) indicate that central mechanisms are involved. Noradrenergic neurotransmission was diminished following L-PIA administration in halothane-anesthetized rats in all brain regions. These data suggest that acute L-PIA treatment decreases central noradrenergic neurotransmission and may represent the mechanism for the decrease in halothane dose to achieve an anesthetic endpoint anesthetic response to halothane.     

An investigation of the molecular properties and stability of intermediates of proenkephalin in isolated bovine adrenal medullary chromaffin granules

An antiserum to a synthetic peptide corresponding to residues 95-117 of bovine proenkephalin recognizes all the major intermediates of this prohormone in bovine adrenal medulla (Birch, N. P. and Christie, D. L. (1986) J. Biol. Chem. 261, 12213-12221). This antiserum enabled an investigation of the stability and molecular properties of intermediates in the processing of proenkephalin in bovine adrenal medullary chromaffin granules. Intact and hypotonic lysates of chromaffin granules were incubated at 37 degrees C and the stability of intermediates assessed by gel filtration followed by radioimmunoassay and gel electrophoresis in combination with immunoblotting. Processing was slow in intact granules compared with incubations of hypotonic lysates which resulted in the selective cleavage of an Mr 27,000 intermediate and increases in the amounts of immunoreactivity of lower molecular weight. Protease inhibitors increased the stability of the 27-kilodalton intermediate, the most effective being p-chloromercuribenzoate. Preliminary evidence was obtained for the regulation of the processing of this intermediate by soluble factors present in chromaffin granules. It appears that membrane-associated intermediates of proenkephalin are relatively stable, although analysis of soluble immunoreactivity released during the incubation of chromaffin granule membranes showed a decrease in the 27-kilodalton intermediate and increased amounts of lower molecular weight intermediates. Analysis of hypotonic lysates by two-dimensional gel-electrophoresis showed that proenkephalin intermediates exhibit significant microheterogeneity. It will be important to compare the products of proenkephalin generated by purified proteases with a putative role in the processing of this prohormone with the properties of endogenous intermediates as revealed in this study.     

Best5: a novel interferon-inducible gene expressed during bone formation.

Regulation of bone formation is important in the pathogenesis of many conditions such as osteoporosis, fracture healing, and loosening of orthopedic implants. We have recently identified a novel rat cDNA (best5) by differential display PCR that is regulated during osteoblast differentiation and bone formation in vitro and in vivo. Expression of best5 mRNA is induced in cultures of osteoblasts by both interferon-alpha (IFN-alpha) or IFN-gamma. Whereas IFN-alpha induced a rapid, transient induction of best5 expression peaking at 4-6 h poststimulation, IFN-gamma elicited a more prolonged induction of best5 expression, which remained elevated 48 h poststimulation. A polyclonal antibody generated to a peptide derived from the best5 coding region recognized a 27 kDa protein on Western blot analysis of osteoblast lysates. We localized BEST5 protein in osteoblast progenitor cells and mature osteoblasts in sections of rat tibiae and in sections of bones loaded in vivo to induce adaptive bone formation. Best5 may therefore be a fundamental intermediate in the response of osteoblasts to stimuli that modulate proliferation/differentiation, such as interferons or mechanical loading. These findings highlight the close interactions between the immune system and bone cells and may open new therapeutic avenues in modulating bone mass.     

Generation of Lys-gamma 3-melanotropin from pro-opiomelanocortin 1-77 by a bovine intermediate lobe secretory vesicle membrane-associated aspartic protease and purified pro-opiomelanocortin converting enzyme

The ability of bovine intermediate lobe secretory vesicle membrane-associated enzyme(s) and purified, soluble paired basic residue-specific, pro-opiomelanocortin converting enzyme (Loh, Y.P., Parish, D. C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205) to cleave bovine NH2-terminal pro-opiomelanocortin1-77 (N-POMC 1-77) was investigated. Purified pro-opiomelanocortin converting enzyme and an enzyme activity associated with the secretory vesicle membrane were shown to cleave bovine N-POMC1-77 to two major products: N-POMC1-49 and Lys-gamma 3-melanotropin (MSH), and one minor product, gamma 3-MSH. These products were identified by their retention times on high performance liquid chromatography, immunological characteristics, and for Lys-gamma 3-MSH, amino acid composition. The products generated indicate cleavage preferentially between Arg 49-Lys 50 of bN-POMC1-77 (where b indicates bovine), which is identical to the processing pattern found in the bovine intermediate lobe in situ. The membrane converting activity was shown to be stimulated by 5 mM Ca2+ and has a pH optimum of 4-5 and an inhibitor profile characteristic of an aspartic protease. This suggests that the membrane-associated enzyme involved is very similar or identical to the purified, soluble pro-opiomelanocortin converting enzyme, which has previously been reported to be an acidic, aspartic protease responsible for the initial steps of POMC processing. The results of this study lead to the proposal that the lack of processing of the Arg49-Lys50 site in POMC in the anterior lobe versus the intermediate lobe of the pituitary in vivo may be due to other regulatory mechanisms rather than invoking the existence in the intermediate lobe of another enzyme specific for this site, different from pro-opiomelanocortin converting enzyme.