The DSC1 channel,encoded by the smi60E locus,contributes to odor-guided behavior in Drosophila melanogaster

Previously, we generated P-element insert lines in Drosophila melanogaster with impaired olfactory behavior. One of these smell-impaired (smi) mutants, smi60E, contains a P[lArB] transposon in the second intron of the dsc1 gene near a nested gene encoding the L41 ribosomal protein. The dsc1 gene encodes an ion channel of unknown function homologous to the paralytic (para) sodium channel, which mediates neuronal excitability. Complementation tests between the smi60E mutant and several EP insert lines map the smell-impaired phenotype to the P[lArB] insertion site. Wild-type behavior is restored upon P-element excision. Evidence that reduction in DSC1 rather than in L41 expression is responsible for the smell-impaired phenotype comes from a phenotypic revertant in which imprecise P-element excision restores the DSC1 message while further reducing L41 expression. Behavioral assays show that a threefold decrease in DSC1 mRNA is accompanied by a threefold shift in the dose response for avoidance of the repellent odorant, benzaldehyde, toward higher odorant concentrations. In situ hybridization reveals widespread expression of the dsc1 gene in the major olfactory organs, the third antennal segment and the maxillary palps, and in the CNS. These results indicate that the DSC1 channel contributes to processing of olfactory information during the olfactory avoidance response.     

Functional genomics of odor-guided behavior in Drosophila melanogaster

The avoidance response to repellent odorants in Drosophila melanogaster, a response essential for survival, provides an advantageous model for studies on the genetic architecture of olfactory behavior. Transposon tagging in a highly inbred strain of flies in combination with a rapid and simple statistical behavioral assay enables the identification of not only large phenotypic effects, but also small aberrations from wild-type avoidance behavior. The recent completion of the sequence of the Drosophila genome facilitates the molecular characterization of transposon-tagged genes and correlation between gene expression and behavior in smell-impaired (smi) mutant lines. Quantitative genetic analyses of a collection of smi lines in a co-isogenic background revealed an extensive network of epistatic interactions among genes that shape the olfactory avoidance response. Candidate genes for several of these transposon-tagged smi loci implicate genes that mediate odorant recognition, including a novel odorant binding protein; signal propagation, including a voltage-gated sodium channel; and a protein containing multiple leucine rich repeats and PDZ domains likely to be involved in postsynaptic organization in the olfactory pathway. Several novel genes of unknown function have also been implicated, including a novel tyrosine-regulated protein kinase. The discovery and characterization of novel gene products that have major, hitherto unappreciated effects on olfactory behavior will provide new insights in the generation and regulation of odor-guided behavior. The identification and functional characterization of proteins encoded by smi genes that form part of the olfactory subgenome and correlation of polymorphisms in these genes with variation in odor-guided behavior in natural populations will advance our understanding of the genetic architecture of chemosensory behavior.     

P Transposon-Induced Dominant Enhancer Mutations of Position-Effect Variegation in Drosophila Melanogaster

P transposon induced modifier mutations of position-effect variegation (PEV) were isolated with the help of hybrid dysgenic crosses (π2 strain) and after transposition of the mutator elements pUChsneory(+) and P[lArB]. Enhancer mutations were found with a ten times higher frequency than suppressors. The 19 pUChsneory(+)- and 15 P[lArB]-induced enhancer mutations can be used for cloning of genomic sequences at the insertion sites of the mutator elements via plasmid rescue. Together with a large sample of X-ray-induced (48) and spontaneous (93) enhancer mutations a basic genetic analysis of this group of modifier genes was performed. On the basis of complementation and mapping data we estimate the number of enhancer genes at about 30 in the third chromosome and between 50 and 60 for the whole autosome complement. Therefore, enhancer of PEV loci are found in the Drosophila genome as frequently as suppressor genes. Many of the enhancer mutations display paternal effects consistent with the hypothesis that some of these mutations can induce genomic imprinting. First studies on the developmentally regulated gene expression of PEV enhancer genes were performed by β-galactosidase staining in P[lArB] induced mutations.     

Effects of Single P-Element Insertions on Bristle Number and Viability in Drosophila Melanogaster

Single P-element mutagenesis was used to construct 1094 lines with P[lArB] inserts on all three major chromosomes in an isogenic background previously free of P elements. The effects of insertions on bristle number and on viability were assessed by comparison to 392 control lines. The variance and effects of P-element inserts on bristle number and viability were larger than those inferred from spontaneous mutations. The distributions of effects on bristle number were symmetrical and highly leptokurtic, such that a few inserts with large effects caused most of the increase in variance. The distribution of effects on viability were negatively skewed and platykurtic. On average, the effects of P-element insertions on bristle number were partly recessive and on viability were completely recessive. P-element inserts with large effects on bristle number tended to have reduced viability, but the correlation between the absolute value of the effects on bristle number and on viability was not strong. Fifty P-element inserts tagging quantitative trait loci (QTLs) with large effects on bristle number were mapped cytogenetically. Two P-element-induced scabrous alleles and five extramacrochaetae alleles were generated. Single P-element mutagenesis is a powerful method for identifying QTLs at the level of genetic locus.     

Olfactory neurons are interdependent in maintaining axonal projections

In mice, individual olfactory neurons express one of the thousand distinct olfactory receptor genes [1] [2] [3]. Neurons that express a given receptor converge on distinct loci in the olfactory bulb to form structures called glomeruli [4] [5] [6]. The olfactory receptor is involved in an instructive manner in this axonal convergence [6] [7] but little is known about the mechanisms involved in maintaining convergence. We have previously created a transgenic olfactory receptor locus that functions independently of the endogenous loci [8]. Here, we show that, although the projections of neurons expressing this ectopic transgenic olfactory receptor always converge in newborn mice, surprisingly, in adult mice, convergence is not always maintained. Moreover, in adult mice there is a positive correlation between the number of neurons expressing the transgenic receptor and the probability of maintaining convergence. These observations, taken together with the variability observed in wild-type [4] [6] and genetically manipulated mice ([6] and our unpublished observations), suggest that olfactory neurons require the presence of other similar axons to maintain a glomerulus. We call this phenomenon interdependence.     

Quantitative Genetic Variation of Odor-Guided Behavior in a Natural Population of Drosophila Melanogaster

Quantitative genetic variation in behavioral response to the odorant, benzaldehyde, was assessed among a sample of 43 X and 35 third chromosomes extracted from a natural population and substituted into a common inbred background. Significant genetic variation among chromosome lines was detected. Heritability estimates for olfactory response, however, were low, as is typical for traits under natural selection. Furthermore, the loci affecting naturally occurring variation in olfactory response to benzaldehyde were not the same in males and females, since the genetic correlation between the sexes was low and not significantly different from zero for the chromosome 3 lines. Competitive fitness, viability and fertility of the chromosome 3 lines were estimated using the balancer equilibrium technique. Genetic correlations between fitness and odor-guided behavior were not significantly different from zero, suggesting the number of loci causing variation in olfactory response is small relative to the number of loci causing variation in fitness. Since different genes affect variation in olfactory response in males and females, genetic variation for olfactory response could be maintained by genotype X sex environment interaction. This unusual genetic architecture implies that divergent evolutionary trajectories for olfactory behavior may occur in males and females.     

Asymmetric exchange is associated with P element induced male recombination in Drosophila melanogaster

Spontaneous meiotic recombination events do not normally occur in the male germ line of Drosophila melanogaster. However, such events are induced in males when a P transposable element or a source of P element encoded transposase protein is present in its genome. This report concerns a molecular analysis of the meiotic exchanges that were induced in the male Drosophila by P elements within a genetically marked region of the third chromosome. The marked region also harbors a single P-element called P(lArB). Fifty-six percent of the P(lArB) region crossovers indicated some alterations in the P element 5' fragment. Such alterations appear to be related to asymmetric or unequal genetic exchanges. Finally, P(lArB) excision was found to be independent of P(lArB) region crossover events.     

Epistatic partners of neurogenic genes modulate Drosophila olfactory behavior

The extent to which epistasis affects the genetic architecture of complex traits is difficult to quantify, and identifying variants in natural populations with epistatic interactions is challenging. Previous studies in Drosophila implicated extensive epistasis between variants in genes that affect neural connectivity and contribute to natural variation in olfactory response to benzaldehyde. In this study, we implemented a powerful screen to quantify the extent of epistasis as well as identify candidate interacting variants using 203 inbred wild‐derived lines with sequenced genomes of the Drosophila melanogaster Genetic Reference Panel (DGRP). We crossed the DGRP lines to P[GT1]‐element insertion mutants in Sema‐5c and neuralized (neur), two neurodevelopmental loci which affect olfactory behavior, and to their coisogenic wild‐type control. We observed significant variation in olfactory responses to benzaldehyde among F₁ genotypes and for the DGRP line by mutant genotype interactions for both loci, showing extensive nonadditive genetic variation. We performed genome‐wide association analyses to identify the candidate modifier loci. None of these polymorphisms were in or near the focal genes; therefore, epistasis is the cause of the nonadditive genetic variance. Candidate genes could be placed in interaction networks. Several candidate modifiers are associated with neural development. Analyses of mutants of candidate epistatic partners with neur (merry‐go‐round (mgr), prospero (pros), CG10098, Alhambra (Alh) and CG12535) and Sema‐5c (CG42540 and bruchpilot (brp)) showed aberrant olfactory responses compared with coisogenic controls. Thus, integrating genome‐wide analyses of natural variants with mutations at defined genomic locations in a common coisogenic background can unmask specific epistatic modifiers of behavioral phenotypes.     

Dynamic genetic interactions determine odor-guided behavior in Drosophila melanogaster

Understanding the genetic architecture of complex traits requires identification of the underlying genes and characterization of gene-by-gene and genotype-by-environment interactions. Behaviors that mediate interactions between organisms and their environment are complex traits expected to be especially sensitive to environmental conditions. Previous studies on the olfactory avoidance response of Drosophila melanogaster showed that the genetic architecture of this model behavior depends on epistatic networks of pleiotropic genes. We performed a screen of 1339 co-isogenic p[GT1]-element insertion lines to identify novel genes that contribute to odor-guided behavior and identified 55 candidate genes with known p[GT1]-element insertion sites. Characterization of the expression profiles of 10 p[GT1]-element insertion lines showed that the effects of the transposon insertions are often dependent on developmental stage and that hypomorphic mutations in developmental genes can elicit profound adult behavioral deficits. We assessed epistasis among these genes by constructing all possible double heterozygotes and measuring avoidance responses under two stimulus conditions. We observed enhancer and suppressor effects among subsets of these P-element-tagged genes, and surprisingly, epistatic interactions shifted with changes in the concentration of the olfactory stimulus. Our results show that the manifestation of epistatic networks dynamically changes with alterations in the environment.     

Advantages of a P-element construct containing MtnA sequences for the identification of patterning and cell determination genes in Drosophila melanogaster

The P[MTW] transposon carries a functional MtnA (metallothionein) gene and a miniwhite reporter gene. When P[MTW] was transformed into Drosophila, many lines were found to show position-dependent expression patterns of the miniwhite or the MtnA transgene. Identification of several of the target genes indicated that this construct behaves as an enhancer or silencer trap. For instance, expression of at least one reporter transgene was shown to correlate with that of the endogenous gene in the case of insertions in Ultrabithorax, four-jointed, and the iroquois complex. The frequency of patterns recovered with P[MTW] is higher than that reported for P[LacW], suggesting that P[MTW] has unusual properties. The possibility of biased insertion of P[MTW] was assayed by screening a sample of 66 MTW lines for modifiers of the extra sex comb phenotype caused by a hypomorphic allele of polyhomeotic. Seven modifiers were recovered, which could be ranked in two classes: genes involved in leg morphogenesis (including four-jointed and spitz), and genes of the Polycomb- or trithorax-Group, including trithorax and batman, a new gene which encodes a product with a BTB/POZ domain. Taken together, these results indicate that P[MTW] allows the tagging of patterning and cell determination genes, and thus provides a useful tool for identifying new developmental functions.     

A screen for genes expressed in the olfactory organs of Drosophila melanogaster identifies genes involved in olfactory behaviour

Background

For insects the sense of smell and associated olfactory-driven behaviours are essential for survival. Insects detect odorants with families of olfactory receptor proteins that are very different to those of mammals, and there are likely to be other unique genes and genetic pathways involved in the function and development of the insect olfactory system.

Methodology/Principal Findings

We have performed a genetic screen of a set of 505 Drosophila melanogaster gene trap insertion lines to identify novel genes expressed in the adult olfactory organs. We identified 16 lines with expression in the olfactory organs, many of which exhibited expression of the trapped genes in olfactory receptor neurons. Phenotypic analysis showed that six of the lines have decreased olfactory responses in a behavioural assay, and for one of these we showed that precise excision of the P element reverts the phenotype to wild type, confirming a role for the trapped gene in olfaction. To confirm the identity of the genes trapped in the lines we performed molecular analysis of some of the insertion sites. While for many lines the reported insertion sites were correct, we also demonstrated that for a number of lines the reported location of the element was incorrect, and in three lines there were in fact two pGT element insertions.

Conclusions/Significance

We identified 16 new genes expressed in the Drosophila olfactory organs, the majority in neurons, and for several of the gene trap lines demonstrated a defect in olfactory-driven behaviour. Further characterisation of these genes and their roles in olfactory system function and development will increase our understanding of how the insect olfactory system has evolved to perform the same essential function to that of mammals, but using very different molecular genetic mechanisms.     

酿酒酵母乙酸耐受性相关的微卫星分子标记筛选

【目的】筛选与酵母乙酸耐受性状紧密相关的微卫星分子标记。【方法】以两株表型差异菌株YHA和YLA作为亲本构建F2代菌株共计160株,选取15个微卫星位点通过PCR方法在40株子代中扩增产物,利用SPSS 11.5软件分析耐酸性状与微卫星序列间的相关性。【结果】找到3个与乙酸耐受性性状相关的微卫星位点,其中位点14P2与酵母乙酸耐受性状有极显著的正相关性(P<0.01),15P2和15P3与酵母乙酸耐受性具有显著的负相关性(P<0.01和P<0.05);此外对于微卫星位点14P2,耐酸亲本YHA在该位点的基因片段(344 bp)在子代耐酸菌株中出现频率达到70.6%,而不耐酸亲本YLA的基因片段(331 bp)在子代不耐酸菌株中出现的频率达91.3%。【结论】微卫星14P2的等位基因在子代菌株中的遗传具有明显的偏好性,该微卫星位点与某种耐酸基因存在一定的连锁遗传,为酵母分子标记辅助育种提供了有价值的遗传标记。     

2009～2010年北京儿童腹泻中轮状病毒新VP4基因亚型的研究

P[8]b基因亚型是国内外新近发现的A组人轮状病毒(HRV)VP4基因的一种新亚型,本研究旨在建立有效鉴别HRV P[8]a和P[8]b基因亚型及P[4]和P[6]基因型的VP4基因打点杂交分型方法,并运用此方法对2009～2010年首都儿科研究所附属儿童医院门诊及住院腹泻患儿中P[8]b基因亚型HRV的流行情况及其G/P基因组合情况进行研究。通过对GenBank序列数据库可检索到的国内外HRV各种P基因型及亚型的VP4基因序列应用相关软件进行基因分析,在不同基因型别间核苷酸变异密集而相同P基因型内核苷酸高度保守的的位置设计各型别探针,并分别以本实验室上传GenBank的北京HRV地方株P[4]和P[6]基因型及P[8]a和P[8]b亚型的VP4基因作为相应型别探针的合成引物的设计模板及探针经PCR合成的合成模板,合成地高辛素标记的DNA探针。经测序验证所建立的VP4基因杂交分型方法结果可靠。对门诊88例(55%,88/160)及住院79例(70.5%,79/112)HRV腹泻患儿的P分型结果显示P[8]a亚型仍为主要型别,前者为96.6%(85/88),而后者为62.0%(49/79);P[8]b亚型在住院HRV感染腹泻患儿中占较高比例(27.9%,22/79),虽然其在门诊HRV感染患儿中也存在,但仅占2.3%(2/88);另外单纯P[4]基因型HRV感染仅在住院腹泻患儿中检测到1例(1.3%,1/79),而P[6]基因型在门诊及住院HRV感染腹泻患儿中均未检测到;本组标本中HRV P[8]b亚型主要与G9基因型组合。本研究表明G9P[8]b型HRV在北京腹泻儿童中有流行。     

副溶血弧菌海产品分离株tdh基因及其临近区域结构分析

摘要：【目的】初步探索副溶血弧菌海产品分离株tdh基因区域的结构特征。【方法】采用长距离PCR和基因步移技术进行tdh基因侧翼序列扩增,测序验证后拼接成疑似毒力基因片段,将所获序列与NCBI数据库进行比较,初步明确tdh基因侧翼序列的结构与功能。【结果】海产品分离株ZS34与参考菌株 RIMD2210633的tdh基因区域（VPA1310-VPA1327）结构基本一致,核苷酸同源性达98.3%;而FJ14与WZ64株基因组中的tdh基因均与tdh3的同源性最高,在基因组中的位置也不同于ZS34株和参考菌株     

Using the P[wHy] hybrid transposable element to disrupt genes in region 54D-55B in Drosophila melanogaster

Understanding the function of each gene in the genome of a model organism such as Drosophila melanogaster is an important goal. The development of improved methods for uncovering the mutant phenotypes of specific genes can accelerate achievement of this goal. The P[wHy] hybrid transposable element can be used to generate nested sets of precisely mapped deletions in a given region of the Drosophila genome. Here we use the P[wHy] method to generate overlapping, molecularly defined deletions from a set of three P[wHy] insertions in the 54E-F region of chromosome 2. Deletions that span a total of 0.5 Mb were identified and molecularly mapped precisely. Using overlapping deletions, the mutant phenotypes of nine previously uncharacterized genes in a 101-kb region were determined, including identification of new loci required for viability and female fertility. In addition, the deletions were used to molecularly map previously isolated lethal mutations. Thus, the P[wHy] method provides an efficient method for systematically determining the phenotypes of genes in a given region of the fly genome.     

Physical mapping in the region of human Xq12-21.1 using pulsed field gel electrophoresis

A number of human disease genes have been localised to Xq12-21.1. A genetic map of this region has previously been constructed using family linkage studies and has been complemented by physical mapping studies using hybrid and deletion cell lines. We have constructed a preliminary long-range physical map of the region, which incorporates thirteen polymorphic and non-polymorphic probes, using pulsed field gel electrophoresis. The order of loci that can be inferred from all the genetic and physical mapping data is: cen-DXS133-[DXS153, DXS159]-DXS132-DXS135-[DXS131, DXS162]-[DXS325, DXS-347, DXS441]-PGKl-DXS447-DXS72-tel. The detection of several large non-overlapping MluI fragments by these probes implies that the minimum extent of the genomic DNA containing these loci is 16Mb. This information should be useful in the eventual identification and isolation of the genes responsible for diseases that map to this region.