Use of an electrostatic sprayer for control of anopheline mosquitoes

The Electrodyn sprayer was compared with a compression sprayer (Hudson X-pert) for residual application of cypermethrin, a pyrethroid insecticide, to control the malaria vectors Anopheles arabiensis Patton and An. funestus Giles in experimental huts at Magugu in Tanzania. The time taken for hut spraying, 2-2.5 min per hut, was similar for both types of sprayer. Two or three huts were treated internally with cypermethrin at 40 or 80 mg a.i./m2 using Electrodyn formulation for comparison with 80 mg a.i./m2 using wettable powder formulation. Each of the twelve huts (including five untreated controls) was fitted with window exit traps and either louvre or verandah traps for mosquito sampling. The Electrodyn sprayer was fitted with a pair of elbowed deflectrodes to direct the positively charged spray droplets onto walls and ceiling. All treatments gave 94-100% mortality-rates of indoor-resting anophelines throughout the evaluation period of 11 weeks post-spray. Reductions of An.arabiensis and An.funestus females by 10-42% and 62-91%, respectively, in rooms and by 72% and 51% in exit traps indicated that cypermethrin deterred mosquitoes from entering the huts. Overall mortality-rates of mosquitoes were 66% of both species in huts treated with 40 mg/m2 Electrodyn, 43% An.funestus and 71% An.arabiensis due to 80 mg/m2 Electrodyn formulation and 49% An.funestus and 64% An.arabiensis due to 80 mg/m2 WP formulation (no significant differences). It is concluded that the Electrodyn sprayer with deflectrodes is a convenient and effective means of residual house-spraying with pyrethroid insecticide for malaria vector control.     

Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was experessed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded -casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plamid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.Abbreviations cfu colony forming units - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] Dedicated to Prof. Dr. G. Drews on the occasion of his 65th birthday     

Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive rDNA sequences

An attempt was made to transform Alternaria alternata protoplasts using a plasmid vector, pDH25, bearing the Escherichia coli hygromycin B (Hy) phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter. Transformants arose on a selective medium containing 100 μg Hy/ml. There were two types of transformants, forming large and small colonies on the selective medium. Transformation with one μg of the vector produced an average of 4.5 large colonies and 600 small ones. In large-colony transformants, the vector often integrated into the recipient chromosome in the form of highly rearranged tandem arrays. To increase transformation efficiency, fragments of the highly repetitive ribosomal RNA gene cluster (rDNA) of A. alternata were used to construct four new vectors for homologous recombination system. Use of these vectors gave higher transformation efficiency than the original plasmid. The best vector, pDH25r1a, gave rise to large-colony transformants at a frequency 20 times higher than pDH25. Transformation events in A. alternata with pDH25r1a occured by homologous recombination as a single crossover between the plasmid-borne rDNA segment and its homologue in the chromosome, often giving rise to tandemly repeated vector DNA.     

三个与agropine合成相关的基因在大肠杆菌微细胞中的表达

pTiAch 5 TR区与在植物中积累agropine相关的三个基因片段已导入pINIIA或pACYC184,在大肠杆菌微细胞中表达出杂合蛋白。三个基因片段的pINIIA重组质粒的表达得到稳定蛋白、不稳定蛋白和在两个相表达出相似蛋白的三种结果。基因2'HindⅢ片段上的UGAA序列是在两个相表达相似蛋白的原因。从产生的杂合蛋白分子量和读码方式看,pTiAch 5中基因2'和基因1'的结构与pTi 15955近似,但由基因0'片段产生的杂合蛋白分子量比根据pTi15955 DNA序列数据推测的稍大。从表达强度和微细胞操作看,这个系统还不适应从大肠杆菌中方便地纯化杂合蛋白的需要。     

Synthesis of the biologically active β-subunit of human nerve growth factor in Escherichia coli

The gene(NGFB) encoding the β subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters p_R and p_L, and expressed in Escherichia coli. The recombinant protein represented approximately 3% of the total cellular protein. Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule. Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons. These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E. coli.     

An expression vector inhibits gene expression in Xenopus embryos by antisense RNA

Summary An expression vector was constructed containing the entire bovine papilloma virus (BPV-1) genome and part of the a-actin gene of Xenopus laevis cloned in the antisense orientation into the neomycin resistance gene under the control of the herpes simplex virus (HSV) thymidine kinase (TK) promoter. When this vector is microinjected into X. laevis embryos it replicates extrachromosomally, at least up to the tadpole stage, and a fusion RNA is synthesized after the mid blastula transition (MBT). The expression of the antisense gene results in a morphological abnormality of somites demonstrating that antisense RNA generated by an episomal replicating expression vector can inhibit the expression of a selected gene during early embryogenesis of X. laevis.     

The acetycholinesterase gene ofAnopheles stephensi

1. The acetylcholinesterase (AChE) gene from the important malaria vector Anopheles stephensi has been isolated by homology to the Drosophila acetylcholinesterase gene. 2. The complete sequence and intron-exon organization has been determined. The encoded protein has 69% identity to Drosophila AChE and 38 and 36% identity to Torpedo AChE and human butyrylcholinesterase, respectively.     

The genetics and expression of an esterase locus inAnopheles gambiae

The main polymorphic system of esterase isoenzymes in adults of the G3 laboratory strain ofAnopheles gambiae consists of two to five major bands of activity per individual. The bands are designated 5S, 5F, 13, 14, and 15. In genetic crosses, the genes which coded for the bands assorted as three codominant alleles, Est A, Est B, and Est C, at a single autosomal locus. Homozygotes for the Est C allele were significantly underrepresented among backcross progeny. The developmental pattern of esterase expression was examined. Esterase gene expression in embryos was first detectable between 2 and 12 hr after oviposition. The initiation or termination of expression of some of the bands corresponded to boundaries between developmental stages. Most of the esterase fractions were not specifically localized within the tissues tested, with the exception of a series of bands which were restricted largely to adult male testes.     

Non-oncogenic plant vectors for use in the agrobacterium binary system

Summary Agrobacterium strains harbouring the T-region and the virulence-region of the Ti plasmid on separate replicons still display efficient T-DNA transfer to plants. Based on this binary vector strategy we have constructed T-region derived gene vectors for the introduction of foreign DNA into plants. The vectors constructed can replicate in E. coli, thus the genetic manipulations with them can be performed with E. coli as a host. They can be transferred to Agrobacterium as a cointegrate with the wide host range plasmid R772. Their T-regions are transferred to plant cells from Agrobacterium strains conferring virulence functions.The plasmid pRAL 3940 reported here is 11.5 kb large, contains a marker to identify transformed plant cells and unique restriction sites for direct cloning of passenger DNA, flanked by the left- and right-hand border fragments of the T-region (including the 25 bp border repeats). The plasmid is free of onc-genes. Therefore, is does not confer tumorigenic traits on the transformed plant cells and mature, fertile plants can thus be regenerated from them.