Expression of a gene encoding a scorpion insectotoxin peptide in yeast, bacteria and plants.

The nucleotide sequence encoding the scorpion insectotoxin I5A was chemically synthesized and expressed in yeast, bacteria and tobacco. The I5A peptides produced in these organisms were purified using an immunoaffinity chromatography procedure. I5A produced using the bacterial secretion system was efficiently secreted and released into the culture medium. In contrast, only a trace amount of I5A was detected in bacterial cytosols when expressed from a direct expression vector, suggesting that I5A was unstable in bacterial cells. I5A secreted from yeast using an alpha-factor signal sequence was shown to have an N-terminal (Glu-Ala)2 extension, indicating incomplete processing of the secreted peptide by dipeptidyl aminopeptidase A. In tobacco, a nonsecreted form of the protein was produced. No measurable insect toxicity was observed when insect larvae were assayed, regardless of whether I5A was produced in yeast, bacteria or tobacco. The lack of toxicity is almost certainly the result of improper folding due to incorrect disulfide bond formation. The inability to produce a biologically active peptide must be overcome before scorpion toxins might be used for the genetic engineering of plants for insect resistance. The yeast and bacterial expression systems described here may be useful for further studies on the problem of expressing a biologically active peptide.

Volatile constituents in juice and oil of Australian wild lime (Microcitrus inodora).

Fifty-three volatile constituents from the juice and twenty from the peel oil of Microcitrus inodora have been identified by gas chromatographic and mass spectral analysis. All except seven had been reported earlier as citrus constituents. Since M. inodora is used as a parent for production of new citrus hybrids, this information will be useful to horticulturists, plant breeders and phytochemists.

Dehydrothalebanin: a source of resistance from Glycosmis pentaphylla against the citrus root weevil Diaprepes abbreviatus.

Roots of a citrus relative, Glycosmis pentaphylla (orangeberry), were shown to inhibit the growth and survival of larvae of the citrus root weevil Diaprepes abbreviatus. Roots of G. pentaphyllaincorporated into the diet of D. abbreviatus increasingly inhibited the growth of neonate larvae with increased concentration of roots, while roots from citrus rootstocks produced little inhibition. The diet-incorporation assay was used to guide fractionation of an active acetone extract of G.pentaphylla roots. Three major fractions from silica open-column liquid chromatography were active, and these were purified using semipreparative normal-phase HPLC. A single active HPLC subfraction was isolated from each of the three liquid chromatography fractions, and two active compounds were isolated and identified by GC-MSD. GC-MSD and NMR identified one compound as the amide dehydrothalebanin B, and the other was identified by GC-MSD as dieldrin, a chlorinated hydrocarbon insecticide whose origin in our samples is uncertain.

Identification of sources of plant resistance to Diaprepes abbreviatus (Coleoptera: Curculionidae) by three bioassays.

Host plant resistance to the root weevil Diaprepes abbreviatus (L.) was assessed for 3 citrus rootstock cultivars, 5 promising hybrid rootstocks, and 3 citroid fruit trees using 3 bioassay methods: a pot bioassay with 1-yr seedlings; a new, 21-cm plastic cell bioassay with 5-mo seedlings; and a diet incorporation bioassay. The plastic cell bioassay is a more rapid screening method and is capable of evaluating a larger number of entries in a shorter period compared with current methods. The 3 bioassays yielded similar results. Larval growth was inhibited by 2 of the remote citroid fruit trees, Murraya koenigii (L.) Sprengel and Glycosmis pentaphylla (Retzius) Correa, compared with growth on commercial rootstock cultivars. Specifically, larvae allowed to feed on roots of M. koenigii or G. pentaphylla gained less weight compared with larvae fed on the commercial rootstock cultivar 'Swingle' [Citrus paradisi Macfayden x Poncirus trifoliata (L.) Rafinesque-Schmaltz]. The resistance of G. pentaphylla confirms previous reports. M. koenigii is a new source of resistance to D. abbreviatus.

cDNA sequence and catalytic properties of a chick embryo alcohol dehydrogenase that oxidizes retinol and 3beta,5alpha-hydroxysteroids.

This study was undertaken to identify the cytosolic 40-kDa zinc-containing alcohol dehydrogenases that oxidize all-trans-retinol and steroid alcohols in fetal tissues. Degenerate oligonucleotide primers were used to amplify by polymerase chain reaction 500-base pair fragments of alcohol dehydrogenase cDNAs from chick embryo limb buds and heart. cDNA fragments that encode an unknown putative alcohol dehydrogenase as well as the class III alcohol dehydrogenase were identified. The new cDNA hybridized with two messages of approximately 2 and 3 kilobase pairs in the adult chicken liver but not in the adult heart, muscle, testis, or brain. The corresponding complete cDNA clones with a total length of 1390 base pairs were isolated from a chicken liver lambdagt11 cDNA library. The open reading frame encoded a 375-amino acid polypeptide that exhibited 67 and 68% sequence identity with chicken class I and III alcohol dehydrogenases, respectively, and had lower identity with mammalian class II (55-58%) and IV (62%) isozymes. Expression of the new cDNA in Escherichia coli yielded an active alcohol dehydrogenase (ADH-F) with subunit molecular mass of approximately 40 kDa. The specific activity of the recombinant enzyme, calculated from active site titration of NADH binding, was 3.4 min-1 for ethanol at pH 7.4 and 25 degrees C. ADH-F was stereospecific for the 3beta,5alpha- versus 3beta,5beta-hydroxysteroids. The Km value for ethanol at pH 7.4 was 17 mM compared with 56 microM for all-trans-retinol and 31 microM for epiandrosterone. Antiserum against ADH-F recognized corresponding protein in the chicken liver homogenate. We suggest that ADH-F represents a new class of alcohol dehydrogenase, class VII, based on its primary structure and catalytic properties.