Duplication of theAce.1 locus inCulex pipiens mosquitoes from the Caribbean

InCulex pipiens mosquitoes, AChE1 encoded by the locusAce.1 is the target of organophosphorus and carbamate insecticides. In several resistant strains homozygous forAce.1 ^RR , insensitive AChE1 is exclusively found. An unusual situation occurs in two Caribbean resistant strains where each mosquito, at each generation, displays a mixture of sensitive and insensitive AChE1. These mosquitoes are not heterozygotes,Ace.1 ^RS , as preimaginal mortalities cannot account for the lethality of both homozygous classes. This situation is best explained by the existence of twoAce.1 loci, coding, respectively, a sensitive and an insensitive AChE1. Thus, we suggest that in the Caribbean a duplication of theAce.1 locus occurred before the appearance of insecticide resistance at one of the two copies.     

几种杀虫药剂敏感蚊类酯酶多态性的研究

通过蚊虫酯酶蛋白的淀粉凝胶电泳分析和基因组DNA的限制性酶切片段长度多态性(RFLPs)比较, 对尖音库蚊Culex pipiens、三带喙库蚊Culex tritaeniorhynchus和中华按蚊Anopheles sinensis有机磷杀虫药剂敏感种群的酯酶蛋白和结构基因的多态性进行分析。发现在蛋白质水平上,三带喙库蚊敏感种群(n=54)在酯酶α和β位点分别存在2个和3个等位基因,在DNA水平上有2.9%的个体具有与酯酶β1¹基因1.3 kb Cdna片段同源的1.3 kb单拷贝带存在。发现中华按蚊敏感种群 (n= 50)中具有低活性的非特异性酯酶存在,在蛋白质水平上,酯酶α和β位点各有一个等位基因;在DNA水平上,通过对单个蚊虫基因组DNA的研究未发现有与酯酶β1¹基因同源的酯酶编码基因的存在。对尖音库蚊北京敏感种群(n= 64)的研究发现,在酯酶α和β位点都存在5个等位基因,在DNA水平上,使用一个限制性内切酶(EcoRI),15只蚊虫的样本在酯酶β位点发现了5个等位基因,说明在尖音库蚊北京敏感种群的酯酶β基因周围存在着较大的中性多态性,在有机磷杀虫剂的选择下,这些中性多态性可能会成为基因扩增的潜在因素。     

Molecular comparisons of the Culex pipiens (L.) complex esterase gene amplicons

The amplification of carboxylesterase genes is a mechanism of organophosphate resistance in Culex mosquitoes. Amplified carboxylesterase genes from an insecticide resistant Culex pipiens strain collected in Cyprus were analysed and compared to other Culex amplified carboxylesterase alleles. A 12 kb section of genomic DNA containing two gene loci coding for carboxylesterase alleles A5 and B5 was cloned and sequenced. A comparison between this amplicon and one from a strain with co-amplified carboxylesterase alleles A2 and B2 revealed a number of differences. The intergenic spacer was 3.7 kb in length in the A5-B5 amplicon (2.7 kb in A2-B2) and contained putative Juan and transposable elements upstream of B5. A fragment of a gene with high homology to aldehyde oxidase was also present immediately downstream of A5. The comparison revealed no differences that would explain the successful spread of the A2-B2 amplicon worldwide whilst the A5-B5 amplicon is restricted to the Mediterranean.     

Genetic polymorphism of esterase in plasma of American mink (Mustela vison L.)

Plasma samples of 412 minks, including 20 families and representing 15 lines, have been investigated by isoelectric focusing for the enzyme esterase (ES). The observed variation of the enzyme may be explained as a result of one locus with at least seven codominant alleles. The segregation of six alleles assumed for the locus in 20 families supports this genetic model. Genetic divergence among the lines is observed and may be due to founder effect and/or selection.     

香菇B交配型位点的分子遗传学结构研究II.一个香菇单核体的B位点结构的分子解析

在先前的工作中,曾经运用简并PCR和染色体步行的方法从香菇中获得了1个信息素受体编码基因和1个信息素前体编码基因。根据香菇135菌株的原生质体单核体的全基因组测序信息,设计了4对引物,用于扩增香菇苏香菌株的原生质体单核体SUP2中的信息素受体编码基因STE-3的同源物及其侧翼保守基因。实验结果共获得了33,655bp的DNA序列,运用BlastX搜索对所获得的序列进行同源性分析后,发现了7个推定基因,其中有3个为信息素受体编码基因。再根据信息素前体所具有的保守基序特征,在2个信息素受体编码基因附近发现了4个信息素前体编码基因。首次对香菇的B交配型位点的分子遗传学结构有了比较全面的了解。     

Identification of two distinct E-NTPDases in liver of goldfish (Carassius auratus L.)

We have recently reported the existence of ATPase activity capable of hydrolyzing extracellular ATP and localized at the external cell membrane of goldfish hepatocytes [Am. J. Physiol. (1998) 274 R1031]. In the present study, we investigated whether one or more enzymes of the ATP diphosphohydrolase family (called E-NTPDases) are responsible for the hydrolysis of extracellular ATP and other nucleotides. Using soluble extracts from goldfish liver, enzyme activity was detected in the presence of ATP (32.1±4.0 nmol Pi liberated mg protein⁻¹ min⁻¹), ADP (20.7±3.3 nmol Pi liberated mg protein⁻¹ min⁻¹) and UTP (20.7±1.2 nmol Pi liberated mg protein⁻¹ min⁻¹). In line with the presence of this hydrolytic activity, liver samples separated by non-denaturing gel electrophoresis and subsequently exposed to either ATP, ADP or UTP yielded a single band with enzyme activity and similar electrophoretic mobility. Subsequent SDS-PAGE electrophoresis of the active bands resulted in the appearance of two protein bands with molecular masses of 70 and 64 kDa. Inmunoblotting of soluble extracts and microsomes obtained from goldfish liver, using a monoclonal antibody against CD39 (a well-known E-NTPDase), detected a single 97-kDa protein. The enzyme activity measured in solution and in native gels, together with structural information from denaturing gels plus immunoblots, points to the existence, in goldfish liver, of at least two different E-NTPDases.     

Genetics of two tissue esterase polymorphisms (Est-4 and Est-5) in the rabbit

Two polymorphic esterase systems were found after electrophoresis of rabbit tissue homogenates. Each of these systems is controlled by an autosomal locus with two alleles. Est-4 determines the absence (Est-4^a) or presence (Est-4^b) of two bands of esterase activity with intermediate anodal mobility and broad substrate specificity. This polymorphism was found to be present in liver, small intestine, and spleen but not in kidney, heart, and testis. Est-5 is coding for cathodally migrating esterases which differ in mobility (Est-5^a and Est-5^b). This polymorphism was found only in kidney and testis homogenates. Est-5 esterases are more active against -naphthyl acetate than against -naphthyl acetate and have no activity against -naphthyl butyrate. Linkage analysis indicated that Est-4 is localized on rabbit LG VI as part of a cluster of esterase loci, whereas Est-5 segregates independently. Rabbits from two inbred and nine partly inbred strains were tested for these polymorphisms.This investigation was supported in part by Public Health Service Research Grant RR-00251 from the Division of Research Resources and by funds from the University of Utrecht. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Interaction of the B locus and the GVHR-selected lines in the graft-versus-host reaction in chickens

Genetic differences affecting the degree of splenomegaly in the graft-versus-host reaction (GVHR) of chickens were studied. Two B genotypes, B⁹B⁹ and B¹¹B¹¹, and two GVHR-selected lines, H and L, were examined. The degree of splenomegaly of B⁹ B⁹→ B¹¹B¹¹ was significantly higher than that of B¹¹B¹¹→ B⁹B⁹ for all line combinations. In contrast, the inoculation of H into L gave consistently higher splenomegaly than that of L into H. This suggested that the effects of B locus were higher in hosts than in donors, while those of the GVHR-selected lines were higher in donors than in hosts.
The analysis of variance revealed that both the differences between the reciprocal combination of B genotypes and between the GVHR line combinations were statistically highly significant. Furthermore, the interaction of B genotypes and GVHR lines was also highly significant.     

Differential expression of esterase and MDH isozymes during in vitro culture in maize (Zea mays L.)

Isozyme patterns of esterase and malate dehydrogenase were analyzed at different stages of in vitro culture of immature embryos and glumes of Zea mays L. viz. explant, callus formation, root formation and shoot formation. Significant changes in isoenzyme patterns of esterase and MDH were observed besides the appearance of specific and new isozymes. Specific fast migrating isozymes were noted in differentiating calli of embryo and glume calli which were absent at other stages suggesting a possible association of these isozyme patterns with in vitro differentiation.     

Reduced productivity of Culex pipiens and Cx. restuans (Diptera: Culicidae) mosquitoes in parking area catch basins in the northeast Chicago metropolitan area

From June to September, 2016, 100 catch basins in eight parking areas were monitored weekly for the presence of mosquito pupae in the operational area of the North Shore Mosquito Abatement District (NSMAD) located just north of Chicago, IL, U.S.A. Weekly results from these basins were compared to weekly samples taken from residential street catch basins, the most common type of catch basin treated seasonally by the NSMAD with larvicides. Over the 17 study weeks, residential street basins had a mean rate of productivity (pupae per basin‐visit) 12 times that of parking area catch basins. The two parking area sites with the highest mean rate of productivity were associated with county forest preserves. Productivity in both street and parking area basins was positively associated with the presence of three or more deciduous trees within 20 m of basins and if they were located directly adjacent to curbs. Alternatively, productivity was negatively associated with the proportion of impervious surface within 10 m of basins and weekly rainfall. Findings suggest that reduced catch basin larvicide applications may be appropriate in many parking area sites.     

Chromosome landing at the Mla locus in barley (Hordeum vulgare L.) by means of high-resolution mapping with AFLP markers

 The complex Mla locus of barley determines resistance to the powdery mildew pathogen Erysiphe graminis f. sp. hordei. With a view towards gene isolation, a population consisting of 950 F₂ individuals derived from a cross between the near-isogenic lines ‘P01’ (Mla1) and ‘P10’ (Mla12) was used to construct a high-resolution map of the Mla region. A fluorescence-based AFLP technique and bulked segregant analysis were applied to screen for polymorphic, tightly linked AFLP markers. Three AFLP markers were selected as suitable for a chromosome-landing strategy. One of these AFLP markers and a closely linked RFLP marker were converted into sequence-specific PCR markers. PCR-based screening of approximately 70 000 yeast artificial chromosome (YAC) clones revealed three identical YACs harbouring the Mla locus. Terminal insert sequences were obtained using inverse PCR. The derived STS marker from the right YAC end-clone was mapped distal to the Mla locus. Received: 17 July 1998 / Accepted: 9 August 1998     

Cytochalasin B affects the structural polarity of statocytes from cress roots (Lepidium sativum L.)

Summary The effect of cytochalasin B (CB; 25 ·ml^–1 in 1% dimethylsulfoxide, DMSO) upon the structural polarity of statocytes in cress roots is demonstrated. If normal, vertically grown roots are incubated in CB, the structural polarity of the statocytes is altered according to the developmental stage of the root. Statocytes from young roots (13 or 17 hours, additionally 7 hours CB) are characterized by proximal ER cisternae and a sparsely developed distal ER-complex. Statocytes from older roots (24 hours, additionally 7 hours CB) still accumulate distal ER, as in control roots, but at the proximal cell pole in the vicinity of the nucleus additional ER is found. These effects are reversed by washing out the drug in DMSO. Growth of the roots under a continuous supply of CB yields statocytes with sedimented nuclei, proximal ER and almost no distal ER. Together with quantitative data from morphometric studies, a dynamic model of the expression of inherent cell polarity in structural polarity is proposed.Abbreviations CB cytochalasin B - DMSO dimethylsulfoxide - ER endoplasmic reticulum Preliminary results were presented at the joint Annual Meeting of the Belgian and German Society for Cell Biology, Bonn, 18–22 March 1985; Eur. J. Cell Biol. 36 (Suppl. 7), 1985, 25.Dedicated to Professor Dr. A.Betz on the occasion of his 65th birthday.     

Molecular characterization of two novel esterase genes from carmine spider mite,Tetranychus cinnabarinus (Acarina: Tetranychidae)

Abstract Two novel esterase complementary DNAs were identified and cloned from the insecticide-susceptible strain of Tetranychus cinnabarinus (Boisduval) (Acarina: Tetranychidae), which were designated as TCE1 and TCE2, respectively. The cDNA of TCE1 gene contained an open reading frame (ORF) of 1701 bp encoding 567 amino acids, and a predicted molecular weight of 62.75 kDa, the cDNA of TCE2 contained an ORF of 1680 bp encoding 560 amino acids, and a predicted molecular weight of 63.14 kDa. TCE1 and TCE2 were submitted to GenBank, accession number EU130461 and EU130462. The well-conserved sequence motif, GXSXG, used as a signature pattern in the esterase family are present in both TCE1 and TCE2 (GQSAG in TCE1, whereas GESAG in TCE2), indicating that these two genes are predicted to be esterases. Comparison of the deduced amino acid sequence with the published mite esterase sequence coming from Boophilus microplus showed that TCE1 shares 33.98% identity and TCE2 shares 33.46% identity. TCE1 and TCE2 share 46.4% identity. Quantitative real-time polymerase chain reaction revealed that expression level of the TCE2 gene was relatively higher than that of the TCE1 in all instars examined except the protonymph, and the expression level of these two esterase genes in adults of T. cinnabarinus was significantly higher than that in any other instars, respectively. T. cinnabarinus is an important agricultural mite pest and esterases are important in the metabolisms of insects and mites; the genomic information obtained in this study will contribute to esterase molecular biological study on mite pest species.     

Characterisation of a cluster of TRIM-B30.2 genes in the chicken MHC B locus

We have identified and characterised a cluster of six TRIM-B30.2 genes flanking the chicken BF/BL region of the B complex. The TRIM-B30.2 proteins are a subgroup of the TRIM protein family containing the tripartite motif (TRIM), consisting of a RING domain, a B-box and a coiled coil region, and a B30.2-like domain. In humans, a cluster of seven TRIM-B30.2 genes has been characterised within the MHC on Chromosome 6p21.33. Among the six chicken TRIM-B30.2 genes two are orthologous to those of the human MHC, and two (TRIM41 and TRIM7) are orthologous to human genes located on Chromosome 5. In humans, these last two genes are adjacent to GNB2L1, a guanine nucleotide-binding protein gene, the ortholog of the chicken c12.3 gene situated in the vicinity of the TRIM-B30.2 genes. This suggests that breakpoints specific to mammals have occurred and led to the remodelling of their MHC structure. In terms of structure, like their mammalian counterparts, each chicken gene consists of five coding exons; exon 1 encodes the RING domain and the B-box, exons 2, 3 and 4 form the coiled-coil region, and the last exon represents the B30.2-like domain. Phylogenetic analysis led us to assume that this extended BF/BL region may be similar to the human extended class I region, because it contains a cluster of BG genes sharing an Ig-V like domain with the BTN genes (Henry et al. 1997a) and six TRIM-B30.2 genes containing the B30.2-like domain, shared with the TRIM-B30.2 members and the BTN genes.     

Isolation and Identification of Two 2,3-Unsubstituted Chromones from Glasswort (Salicornia europaea L.)

Two 2,3-unsubstituted chromones were isolated from the reddish leaves and stems of glasswort (Salicornia europaea L.) and, on the basis of chemical and spectral evidences and syntheses of both of the compounds, they were identified to be 6,7-methylenedioxychromone and 6,7-dimethoxychromone, respectively. This is the first report which shows the natural occurrence of these two chromones.     

Identification of three Wx proteins in wheat (Triticum aestivum L.)

Nullisomic analysis of waxy (Wx) protein of hexaploid wheat (Triticum aestivum L.) cv. “Chinese Spring” using two-dimensional polyacrylamide gel electrophoresis revealed that threeWx loci,Wx-A1, Wx-B1, andWx-D1, located on chromosome arms 7AS, 4AL, and 7DS, produce three distinct Wx subunit groups, subunit group-A (SGA), SGB, and SGD, respectively. SGA has a higher molecular weight and a more basic isoelectric point (pI) than the other two. SGB and SGD have the same molecular weight but a slightly different pI range. Owing to the detection of these three subunit groups, we were able to identify the expression of three waxy genes in wheat endosperm and to find two types of mutants among Japanese wheat cultivars, one lacking SGA and the others SGB. These results suggest the possibility of breeding a waxy wheat.     

Structural homology of storage proteins coded by the Hor-1 locus of barley (Hordeum vulgare L.)

Three C hordein fractions were prepared by ion-exchange chromatography of a total hordein preparation on carboxymethyl cellulose at pH 4.6 Polyacrylamide gel electrophoresis at pH 3.2 and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) at pH 8.9 showed that each fraction contained a single major band. The apparent molecular weights of these were determined by SDS-PAGE as 58, 57, and 54,000. When compared by isoelectric focusing, however, the 58 and 57,000 components each separated into two major bands and the 54,000 component into four. Amino acid analysis showed that although the three fractions had similar compositions with high glutamate+glutamine (38–39%), proline (30–32%) and phenylalanine (8–9%) contents, some differences were present, notably in the relative content of lysine. The three fractions had identical amino acid sequences for the first ten residues at the N-terminal end. They also had identical sequences for the first five residues at the C-terminal end, with the exception that a mixture of two amino acids were released from position 4 of the 58,000 fraction only. Peptide mapping with three enzymes (trypsin, chymotrypsin and V8 protease) indicated that the 58 and 57,000 fractions were more closely related to each other than to the 54,000 fraction. It is suggested that the 57 and 58,000 fractions and the 54,000 fraction constitute two families of closely related polypeptides which are coded by genes derived from the duplication and divergence of a single ancestral gene.     

Identification of DNA amplification fingerprinting (DAF) markers close to the symbiosis-ineffective sym31 mutation of pea (Pisum sativum L.)

 We demonstrate efficient genome mapping through a combination of bulked segregant analysis (BSA) with DNA amplification fingerprinting (DAF). Two sets of 64 octamer DAF primers, along with two PCR programs of low- and high-annealing temperatures (30°C and 55°C, respectively), appeared to be enough to locate molecular markers within 2–5 cM of a gene of interest. This approach allowed the rapid identification of four BSA markers linked to the pea (Pisum sativum L.) Sym31 gene, which is responsible for bacteroid and symbiosome differentiation. Three of these markers are shown to be tightly linked to the sym31 mutation. Two markers flanking the Sym31 gene, A21-310 and B1-277, cover a 4–5 cM interval of pea linkage group 3. Both markers were converted to sequence-characterized amplified regions (SCARs). The flanking markers may be potential tools for marker-assisted selection or for positional cloning of the Sym31 gene. Received: 2 July 1998 / Accepted: 8 October 1998