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Butenyl-spinosyns, a natural example of genetic engineering of antibiotic biosynthetic genes.

Spinosyns, a novel class of insect active macrolides produced by Saccharopolyspora spinosa, are used for insect control in a number of commercial crops. Recently, a new class of spinosyns was discovered from S. pogona NRRL 30141. The butenyl-spinosyns, also called pogonins, are very similar to spinosyns, differing in the length of the side chain at C-21 and in the variety of novel minor factors. The butenyl-spinosyn biosynthetic genes (bus) were cloned on four cosmids covering a contiguous 110-kb region of the NRRL 30141 chromosome. Their function in butenyl-spinosyn biosynthesis was confirmed by a loss-of-function deletion, and subsequent complementation by cloned genes. The coding sequences of the butenyl-spinosyn biosynthetic genes and the spinosyn biosynthetic genes from S. spinosa were highly conserved. In particular, the PKS-coding genes from S. spinosa and S. pogona have 91-94% nucleic acid identity, with one notable exception. The butenyl-spinosyn gene sequence codes for one additional PKS module, which is responsible for the additional two carbons in the C-21 tail. The DNA sequence of spinosyn genes in this region suggested that the S. spinosa spnA gene could have been the result of an in-frame deletion of the S. pogona busA gene. Therefore, the butenyl-spinosyn genes represent the putative parental gene structure that was naturally engineered by deletion to create the spinosyn genes.

Anti-Bacterial Agents↗

Small is useful in endocrine disrupter assessment--four key recommendations for aquatic invertebrate research.

As we enter the 21st "biocentury", with issues such as biodiversity and biotechnology growing in public profile, it is important to reflect on the immense ecological, medical and economic importance of invertebrates. Efforts to understand the diverse biology of invertebrates come from many directions, including Nobel Prize winning developmental biology, research to control insects that threaten human health and food supplies, aquaculture opportunities and also within ecotoxicology. In the latter context, this special journal volume highlights the importance of addressing endocrine disruption in aquatic invertebrates, from molecular and cellular biomarkers to population-relevant adverse effects. The contributors to this special volume have provided an excellent assessment of both the fundamental endocrinology and applied ecotoxicology of many aquatic invertebrate groups. On the premise that reproductive success is ultimately the vital population parameter, this chapter gives a personal view of key gaps in knowledge in invertebrate reproductive and developmental endocrinology and ecotoxicology. Based on current knowledge, there are four key issues that need to be prioritised within aquatic ecotoxicology: (1) a wider assessment of the reproductive status of invertebrates in both freshwater and coastal ecosystems; (2) prioritisation of laboratory studies in OECD and other regulatory test organisms, including basic endocrinology and ADME (absorption, distribution, metabolism and excretion) research; (3) development and validation of mechanistic biomarkers that can be used as "signposts" to help prioritise species and chronic test endpoint selection, and help link data from laboratory and field studies; and (4) develop a comparative invertebrate toxicology database utilising the prioritised reference chemicals from the EDIETA workshop, encompassing the diverse modes-of-action pertinent to endocrine disrupter testing in both aquatic arthropod and non-arthropod invertebrates.

Animals↗

Stable transformation of the cotton plastid genome and maternal inheritance of transgenes.

Chloroplast genetic engineering overcomes concerns of gene containment, low levels of transgene expression, gene silencing, positional and pleiotropic effects or presence of vector sequences in transformed genomes. Several therapeutic proteins and agronomic traits have been highly expressed via the tobacco chloroplast genome but extending this concept to important crops has been a major challenge; lack of 100 homologous species-specific chloroplast transformation vectors containing suitable selectable markers, ability to regulate transgene expression in developing plastids and inadequate tissue culture systems via somatic embryogenesis are major challenges. We employed a 'Double Gene/Single Selection (DGSS)' plastid transformation vector that harbors two selectable marker genes (aph A-6 and npt II) to detoxify the same antibiotic by two enzymes, irrespective of the type of tissues or plastids; by combining this with an efficient regeneration system via somatic embryogenesis, cotton plastid transformation was achieved for the first time. The DGSS transformation vector is at least 8-fold (1 event/2.4 bombarded plates) more efficient than 'Single Gene/Single Selection (SGSS)' vector (aph A-6; 1 event per 20 bombarded plates). Chloroplast transgenic lines were fertile, flowered and set seeds similar to untransformed plants. Transgenes stably integrated into the cotton chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. Cotton is one of the most important genetically modified crops (120 billion US dollars US annual economy). Successful transformation of the chloroplast genome should address concerns about transgene escape, insects developing resistance, inadequate insect control and promote public acceptance of genetically modified cotton.

Blotting, Southern↗

Bancroftian filariasis in coastal north Trinidad: the effects of mass chemotherapy using spaced doses of diethylcarbamazine citrate on human microfilaraemias and vector infection rates.

The effects of a mass chemotherapy programme using spaced doses of diethylcarbamazine citrate on Wuchereria bancrofti microfilaraemias and vector infection rates were studied in an isolated rural community of 650 people in north Trinidad where a microfilaria rate of 15% had previously been recorded. A single oral dose of 6 mg/kg body weight was given at monthly intervals for one year. After 6 months, 79% (52/66) of persons previously microfilaraemic had become negative. After 12 months the negative proportion had increased to 90% (63/70). The geometric mean microfilaria density of those still microfilaraemic was reduced from 19.7 to 4.6 per 100 mm3 of blood. Of 147 initially negative persons, none had become positive following treatment. Filarial infection rates in Culex quinquefasciatus from randomly sampled houses fell from 6.4% to zero; in houses where occupants with microfilaraemia had been identified before treatment, the rate was reduced from 15.8% to zero. In a supplementary study, however, weekly collections from one initially positive house showed a rapid decline in the vector infection rate from 43% to 2% after three months but an additional 3 months elapsed before the index reached zero. There was negligible microfilarial uptake by the vectors from residual low-density microfilaraemias (less than 10 microfilariae per 100 mm3 of blood) after chemotherapy.

Animals↗