Characterization of novel esterases in insecticide-resistant mosquitoes

In the mosquito Culex pipiens complex (Diptera: Culicidae), the amplification of carboxylesterase genes is an important mechanism providing resistance to organophosphate insecticides. Various amplified alleles at the Ester locus have been identified over the world. In this study, two newly detected Ester alleles, Ester(B10) and Ester(11) (including associated Ester(A11) and Ester(B11)), coding for esterases B10 and A11-B11, respectively, are characterized qualitatively and quantitatively. A high molecular identity is observed both at the nucleotide level and at the deduced amino acid level among the known Ester alleles. Real-time quantitative PCR results suggest 2.5-fold amplification of the Ester(B10) allele, 36.5-fold amplification of the Ester(A11) allele, and 19.1-fold amplification of the Ester(B11) allele. The ca. 2-fold difference in amplification level between Ester(A11) and Ester(B11) may indicate a new model for the esterase amplification. Bioassays show that these two resistant Ester alleles only can confer moderate or low resistance to the tested organophosphate insecticides.     

球形芽孢杆菌C3—41二元毒素基因在苏云金芽孢杆菌以色列亚种中的？…

球形芽孢杆菌Ｃ３－４１是我国分离的一株对蚊幼虫有毒杀作用的高毒力菌株,对库蚊、按蚊幼虫的毒性高于２３６２菌株,Ｓｏｕｔｈｅｒｎ杂交证明Ｃ３－４１总ＤＮＡ中３．５ＫｂＨｉｎｄＩＩＩ片段上带有４１．９和５１．４ｋＤ二元毒素基因。     

球形芽孢杆菌C3-41二元毒素基因在苏云金芽孢杆菌以色列亚种中的克隆和表达

球形芽孢杆菌C3-41是我国分离的一株对蚊幼虫有毒杀作用的高毒力菌株,对库蚊、按蚊幼虫的毒性高于2362菌株,Southern杂交证明C\-3\|41总DNA中35Kb HindIII片段上带有419和514kD二元毒素基因,该片段由3479个核苷酸组成,核苷酸序列同2362菌株的二元毒素基因序列完全相同。含二元毒素基因的重组质粒pCW\|1和pCW\|2能在大肠杆菌中表达产生二元毒蛋白,但表达量低,重组子杀蚊毒性低。无晶体型苏云金芽孢杆菌以色列亚种重组子在其芽孢形成中能产生以晶体形式存在的二元毒素蛋白,其全发酵液和纯化晶体蛋白的杀蚊活性与C\-3\|41相近。     

Structural organization of the estalpha3(1) gene in a Colombian strain of Culex quinquefasciatus differs from that in Cuba

In Culex mosquitoes (Diptera: Culicidae), the most common mechanism for resistance to organophosphorus (OP) insecticides involves amplification of one or more esterases. Two esterase loci are often involved, with different allelic forms co-amplified. Estalpha3(1) is co-amplified with estbeta1 in a Colombian (COL) strain of Culex quinquefasciatus Say. These two alleles co-migrate on acrylamide gels, often leading to misscoring of the phenotype as elevation of a single estbeta enzyme. By sequencing COL genomic DNA, we determined the estalpha3(1) gene length is 1623 nucleotides. The open reading frame of estalpha3(1) encodes a 540 amino acid protein, as for estalpha2(1) in strain Pel RR from Sri Lanka. The intron/exon boundaries of estalpha3(1) are identical to those of estalpha2(1), suggesting that they are alleles of the same locus. The COL estalpha3(1) gene differs from estaalpha3(2) in strain MRES from Cuba, although they have equivalent electrophoretic mobility, showing that these two strains contain distinct resistance-associated amplicons. Twenty nucleotide differences were scored between the MRES partial 495 bp sequence and that in the COL strain, with two amino acid changes, demonstrating distinct estalpha enzymes. Our sequencing data show 95% identity between the three estalpha genes (each has six introns and seven exons) in OP-resistant Cx. quinquefasciatus. Amplified estalpha3(1) and estbeta1 are at least 10kb apart in temephos-selected COL and 2.7kb apart in Pel RR, whereas these non-amplified genes are only 1.7kb apart in the nonselected parental COL stock, as in Pel SS (susceptible Sri Lankan strain), demonstrating that this region of the genome is susceptible to expansion and contraction.     

Cloning and nucleotide sequence of a gene from Lactobacillus sake Lb706 necessary for sakacin A production and immunity.

Sakacin A is an antilisterial bacteriocin produced by Lactobacillus sake Lb706. In order to identify genes involved in sakacin A production and immunity, the plasmid fraction of L. sake Lb706 was shotgun cloned directly into a sakacin A-nonproducing and -sensitive variant, L. sake Lb706-B, by using the broad-host-range vector pVS2. Two clones that produced sakacin A and were immune to the bacteriocin were obtained. A DNA fragment of approximately 1.8 kb, derived from a 60-kb plasmid of strain Lb706 and present in the inserts of both clones, was necessary for restoration of sakacin A production and immunity in strain Lb706-B. The sequence of the 1.8-kb fragment from one of the clones was determined. It contained one large open reading frame, designated sakB, potentially encoding a protein of 430 amino acid residues. Hybridization and nucleotide sequence analyses revealed that the cloned sakB complemented a mutated copy of sakB present in strain Lb706-B. The sakB gene mapped 1.6 kb from the previously cloned structural gene for sakacin A (sakA) on the 60-kb plasmid. The putative SakB protein shared 22% amino acid sequence identity (51% similarity if conservative changes are considered) to AgrB, the deduced amino acid sequence of the Staphylococcus aureus gene agrB. The polycistronic agr (accessory gene regulator) locus is involved in the regulation of exoprotein synthesis in S. aureus. Similar to the AgrB protein, SakB had some features in common with a family of transmembrane histidine protein kinases, involved in various adaptive response systems of bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonallelic histone gene clusters of individual sea urchins (Lytechinus pictus): Polarity and gene organization

We have analyzed the histone genes from the sea urchin Lytechinus pictus. Examination of native DNA from individuals reveals four major Eco RI restriction endonuclease histone gene DNA fragments which have been labeled A (6.0 kb), B (4.1 kb), C (3.1 kb) and D (1.2 kb). The fragments A, B and C have been cloned into E. coli plasmids (pLpA, pLpB and pLpC). These histone gene fragments display length and sequence heterogeneity in different individuals. The plasmid pLpA contains the coding regions for H1, H4, H2B and H3 histones, and we determined that the DNA fragment D is tandem to A in native DNA and that it contains the H2A gene. The plasmids pLpB and pLpC contain the histone genes H2A-H1-H4 and H2B-H3, respectively, and together contain the sequences for the five major histones. Restriction analysis of native L. pictus DNA reveals that B and C are tandem to each other but not intermingled with the A-D-type repeat units, and are thus in separate clusters with a repeat length of 7.2 kb. Since the two cluster types do not segregate, they are not alleles. Hybridization of histone mRNA to exonuclease III-digested linear DNA demonstrated an identical polarity of the histone genes in the A-D- and B-C-type repeat units. This result revealed that the L. pictus histone genes have a polarity which is the same as other sea urchin histone genes examined to date—that is, 3′ H1-H4-H2B-H3-H2A 5′. Restriction endonuclease cleavage patterns of the cloned segments indicate that considerable sequence heterogeneity exists between the two types of histone gene repeat units.     

Esterases A5-B5 in organophosphate-resistant Culex pipiens from Italy

Abstract. Culex pipiens mosquitoes from Lignano city, Udine province, northeast Italy, were found to carry over-produced non-specific esterases Al, A2-B2 and A4-B4 or A5-B5, detected by starch gel electrophoresis, giving multiple resistance to organophosphorus insecticides. In order to differentiate between A4-B4 and A5-B5 esterases, the latter known only from Cyprus whereas the former is widespread in Italy and elsewhere, restriction fragment length polymorphism (RFLP) analysis was performed at the esterase B locus. Both B4 and B5 haplotypes were found. This is the first record of A5-B5 esterase-mediated resistance in continental Europe.     

被动迁移在抗性进化中的作用

为了明确迁移和基因交流在杀虫剂抗性基因进化中的作用,我们从四个不同的地区采集有机磷抗性的库蚊野生种群,利用淀粉电泳鉴定了各种群中存在的已知过量产生酯酶的分布频率,并通过5个假定的中性位点的电泳多态性分析了种群间的遗传多样性。结果表明种群间的基因交流是存在的,遗传分化与地理位置存在一定关系,而抗性等位基因A2一B2的分布却与种群间的遗传分化不一致。对这种差异的解释是：被动迁移(铁路运输等)加速了抗性基因的交流,而当抗性基因以自然迁飞的方式向周围地区扩散时,却是一个相对缓慢的过程。     

Identification of cis sequences controlling efficient position-independent tissue-specific expression of human major histocompatibility complex class I genes in transgenic mice.

We previously reported that genomic major histocompatibility complex class I human leukocyte antigen (HLA)-B7 gene constructs with as little as 0.66 kb of 5'- and 2.0 kb of 3'-flanking DNA were expressed efficiently and appropriately in transgenic mice. To identify and characterize the relevant cis-acting regulatory elements in more detail, we have generated and analyzed a series of transgenic mice carrying native HLA-B7 genes with further 5' truncations or intronic deletions and hybrid constructs linking the 5'-flanking region of B7 to a reporter gene. We were unable to detect a specific requirement for sequence information within introns 2 to 7 for either appropriate constitutive or inducible class I expression in adult animals. The results revealed the presence of cis-acting regulatory sequences between -0.075 kb and -0.66 kb involved in driving efficient copy number-dependent constitutive and gamma interferon-enhanced tissue-specific expression. The region from -0.11 to -0.66 kb is also sufficient to prevent integration site-specific "position effects," because in its absence HLA-B7 expression is frequently detected at significant levels at inappropriate sites. Conserved sequence elements homologous to the H-2 class I regulatory element, or enhancer A, and the interferon response sequence are located between about -151 and -228 bp of the B7 gene. Our results also indicate the existence of sequences downstream of -0.11 kb which can influence the pattern of tissue-specific expression of the HLA-B7 gene and the ability of this gene to respond to gamma interferon.     

羧酸酯酶A2在大肠杆菌中的表达及特征分析

本研究采用RT-PCR法从抗性蚊虫（Culex quinquefasciatus)中克隆了羧酸酯酶A2的全长cDNA,对其进行了序列测定。并构建了融合表达质粒pET-ESTA2,转化大肠杆菌BL21后,在异丙基硫代半乳糖苷（IPTG）的诱导下,使羧酸酯酶A2在大肠杆菌内得到表达,表达产物经亲和层析纯化获得了1条带的重组蛋白,与报道的从蚊虫体内纯化的羧酸酯酶相比,从表达产物中纯化的羧酸酯酶Km与其一致,但从表达产物纯化的羧酸酯酶的Vm比蚊虫中纯化的羧酸酯酶的Vm高,表明用亲和层析纯化的羧酸酯酶A2比从蚊虫中提取的纯度高,羧酸酯酶A2表达纯化及特征分析为其应用奠定了基础。     

Distribution and dynamics of esterase alleles in Culex pipiens complex in China

To investigate insecticide resistance and dynamic changes of carboxylesterase polymorphism in mosquitoes with time in the Culex pipiens complex (Diptera: Culicidae), nine field mosquito populations were collected in China. The resistance levels of fourth-instar larvae to organophosphate (dichlorvos, parathion, and chlorpyrifos), carbamate (fenobucarb and propoxur), and pyrethroid (permethrin, deltamethrin and tetramethrin) insecticides were determined by bioassay. Larvae had more resistance to organophosphate insecticides than to carbamate insecticides. A low but significant resistance was observed for carbamate insecticides. The resistance to pyrethroid insecticides varied from sensitive to high. Starch gel electrophoresis revealed the presence of the overproduced esterases B1, A2B2, A8B8, A9B9, B10 and A11B11. The frequency of each overproduced esterases varied depending on its regional localities. Compared with published surveys, the C. pipiens complex, which exhibited a high polymorphism of applied esterase alleles in China, showed dynamic evolution over time under local specific insecticide selection. The results are discussed in the context of recent alterations to insecticide campaigns, and in the evolution of resistance genes in Chinese C. pipiens populations.     

羧酸酯酶A2在大肠杆菌中的表达及特征分析

本研究采用RT-PCR法从抗性蚊虫(Culex quinquefasciatus)中克隆了羧酸酯酶A2的全长cDNA,对其进行了序列测定,并构建了融合表达质粒pET-ESTA2。转化大肠杆菌BL21后,在异丙基硫代牛乳糖苷(IPTG)的诱导下,使羧酸酯酶A2在大肠杆菌内得到表达。表达产物经亲和层析纯化获得了1条带的重组蛋白。与报道的从蚊虫体内纯化的羧酸酯酶相比,从表达产物中纯化的羧酸酯酶Km与其一致,但从表达产物纯化的羧酸酯酶的Vm比蚊虫中纯化的羧酸酯酶的Vm高。表明用亲和层析纯化的羧酸酯酶A2比从蚊虫中提取的纯度高。羧酸酯酶A2表达纯化及特征分析为其应用奠定了基础。     

Cloning and characterization of the ribosomal protein L3 (<Emphasis Type="Italic">RPL3</Emphasis>) gene family from <Emphasis Type="Italic">Triticum aestivum</Emphasis>

Plant pathogenic fungi of the genus Fusarium can cause severe diseases on small grain cereals and maize. The contamination of harvested grain with Fusarium mycotoxins is a threat to human and animal health. In wheat production of the toxin deoxynivalenol (DON), which inhibits eukaryotic protein biosynthesis, is a virulence factor of Fusarium, and resistance against DON is considered to be part of Fusarium resistance. Previously, single amino acid changes in RPL3 (ribosomal protein L3) conferring DON resistance have been described in yeast. The goal of this work was to characterize the RPL3 gene family from wheat and to investigate the potential role of naturally existing RPL3 alleles in DON resistance by comparing Fusarium-resistant and susceptible cultivars. The gene family consists of three homoeologous alleles of both RPL3A and RPL3B, which are located on chromosomes 4A (RPL3-B2), 4B (RPL3-B1), 4D (RPL3-B3), 5A (RPL3-A3), 5B (RPL3-A2) and 5D (RPL3-A1). Alternative splicing was detected in the TaRPL3-A2 gene. Sequence comparison revealed no amino acid differences between cultivars differing in Fusarium resistance. While using developed SNP markers we nevertheless found that one of the genes, namely, TaRPL3-A3 mapped close to a Fusarium resistance QTL (Qfhs.ifa-5A). The potential role of the RPL3 gene family in DON resistance of wheat is discussed.     

HLA gene and haplotype frequencies in Uruguay

A total of 604 individuals living in Montevideo and other places in Uruguay were studied in relation to three I HLA loci. The most common alleles observed (percentages in parentheses) were A2(24), A9(15), A19(11), B12(12), B35(12), and C4(16). The most marked departures from linkage equilibrium (all numbers multiplied by 10⁵) were B35-C4(636), A2-B5(590), A2C3(515), A2B14(494), and A19-B12(485). These findings do not contradict the hypothesis that while most of the Uruguayan population is of Caucasoid origin, significant African and Amerindian genes may exist in its gene pool.     

Analysis of the duplicated human C4/P450c21/X gene cluster

Gene duplications, deletions and rearrangements occur with an unusually high frequency in the region of the P450c21 genes encoding 21-hydroxylase. In the human genome, the locus contains at least 6 genes, oriented 5′ C4A, P450c21A, XA, C4B, P450c21B, XB 3′. Sequence analysis of the XA gene, of the 5′ flanking DNA of the C4A gene, and of part of the XB gene revealed that this gene cluster was duplicated by nonhomologous recombination at a CAAG tetranucleotide. The location of this duplication suggests that it may have occurred after mammalian speciation. The XA gene is abundantly expressed in the human adrenal as a stable 2.6 kb RNA, but it is not known if that RNA serves a biological function. Knowledge of the anatomy of the XA gene facilitates genetic analysis of disease-causing lesions in the P450c21B gene. Southern blotting data show that about 76% of disordered P450c21B alleles bear gene microconversions that resemble point mutations; the remaining alleles are equally distributed between gene deletions and large gene conversions.     

The immunoglobulin heavy-chain variable region in insulin-dependent diabetes mellitus: affected-sib-pair analysis and association studies.

We have analyzed immunoglobulin heavy-chain variable-region (VH) polymorphisms and genetic susceptibility to insulin-dependent diabetes mellitus (IDDM) by using a set of polymorphic loci that span approximately 1,000 kb of the VH region on chromosome 14q32. One hundred one Finnish families with at least two children affected with IDDM were studied. Conventional RFLPs determined by hybridization were used, since no microsatellite repeat markers have been available for this gene region. No evidence for linkage between the VH genes and IDDM could be obtained from haplotype-sharing analysis among the 133 diabetic sib pairs. The frequencies of various VH genotypes were also compared between 101 familial IDDM cases and 114 controls derived from the Finnish background population. The distribution of the genotypes at the VH2-B5 locus was significantly different between these groups (P=.004), the 3.4/3.4 genotype being less common in the IDDM cases. In addition, a different genotype distribution at the VH5-B2 locus was observed in the diabetic subjects (P = .022). When the IDDM cases were stratified by presence or absence of the high-risk HLA-DQB1*0302 allele, no differences in VH genotype frequencies were observed between the 0302-positive and 0302-negative cases. In the transmission test for linkage disequilibrium (TDT), no differences were found between the expected and observed frequencies of the transmitted alleles at the VH2-B5 or VH5-B2 locus. In conclusion, significant differences in VH genotype distributions were observed between the familial IDDM cases and the controls, but the observed associations could not be confirmed by the TDT. Haplotype sharing analysis provided no evidence for genetic linkage between the VH gene region and IDDM.     

Effect of pKM101 plasmid on lethal and mutagenic damage in UV-irradiated E. coli strains

Introduction of the R-factor plasmid pKM101 increased resistance to UV-killing in uvr lexA(Ind-) recA+ strains of E. coli K12 as well as B, while their UV mutability was not affected. Similar effects were also observed in those strains when the 18-B plasmid (a pBR322 derivative carrying the region (about 5 kb) of the 35.4 kb pKM101 plasmid) was introduced. The muc genes which are considered to be involved in error-prone repair are contained in 18-B. These results suggest the possibility that the pKM101 effect requires the host recA gene and a common genetic region, including the muc genes, in both plasmids and is associated with some unmutable repair systems.     

中国汉族个体HLA-A、-B基因全长序列的测定及调控区多态性

文章利用20个中国汉族个体样本建立了稳定精确的HLA-A、-B基因全长序列的克隆测序方法, 获得HLA-A 10个等位基因4.2 kb序列, HLA-B 6个等位基因3.7 kb序列, 序列涵盖了两个基因的所有外显子、所有内含子、5′启动子区以及3′非翻译区(3′UTR)。A*1153是文章发现的一个新等位基因, B*151101的内含子序列、5个HLA-A以及2个HLA-B等位基因的5′启动子序列和3′UTR序列为国际上首次报道, 其他等位基因均延伸了IMGT/HLA数据库中释放的全长序列。文章首次在中国汉族个体中测定了IMGT/HLA数据库中没有覆盖的HLA-A、-B基因的上游5′启动子以及下游3′UTR区域的多态性模式。HLA-A基因5′启动子延伸区域共发现26个SNPs和一处3 bp(AAA/-)的插入/缺失, 3′UTR延伸区域共发现14个SNPs; HLA-B基因5′启动子延伸区域共发现5个SNPs和一处1 bp(T/-)的插入/缺失, 3′UTR延伸区域共发现8个SNPs。通过对两个基因的5′启动子、外显子以及3′UTR的系统发育树分析, 发现两个基因调控区与外显子的进化关系有所不同, HLA-A基因除A*24020101外, 其他等位基因两端调控区与外显子连锁比较紧密, HLA-B基因两端调控区与外显子之间则发生了较为频繁的重组事件。