Comparative study of molecular characteristics of X and Y nucleolar organizers of various lines of Drosophila melanogaster

We have determined for the X chromosomes of 10 laboratory strains and the Y chromosomes of 4 of them both the total number of ribosomal units and the relative percentages of uninterrupted (ins-), type 1 (ins1: with distinction between small ins1S and large ins1L) and type 2 (ins2) interrupted ribosomal units. These studies were made with the DNA extracted from third instar larval diploid tissues (brains and imaginal discs) of X/X female lines or XNO-/Y male lineages (devoid of X ribosomal genes) whose members possess copies of the same initial X or Y chromosome. Between the X chromosomes as well as the Y chromosomes an approximately equal to 2-fold variation was observed in the total number of ribosomal genes: from approximately equal to 200 to 420 for the X chromosomes and from approximately equal to 150 to 330 for the Y ones. The Y chromosomes are devoid of insertion 1 interrupted units, but one can observe some variation in the percentage and hence the absolute numbers of uninterrupted and insertion 2 interrupted units. Among the X chromosomes a very large variation exists between the percentage and absolute number of all the ribosomal unit types; it is to be noted especially that the number of uninterrupted units, which are the only kind of ribosomal genes actively transcribed, can vary from about 20 to 140 without any differences in the development of the different strains.     

DNA polymerase activity in developmental stages of Drosophila melanogaster

An assay procedure was developed that allowed the first reproducible measurement of DNA polymerase activity in all developmental stages of Drosophila melanogaster. Evidence is presented that the same enzymatic species is present in extracts of embryos, pupae, and adults of both sexes and that this activity has many properties similar to vertebrate α-polymerases. Polymerase activity per individual is low in embryos and rises steadily through larval instars, reaches a peak in early pupae, declines through the late pupal period, and remains low in newly eclosed adults of both sexes. A dramatic increase is observed in adult females as mature oocytes are formed. This pattern of enzyme activity is completely coincident with changes in DNA levels during development, and suggests that the Drosophila enzyme, like vertebrate α-polymerases, functions in cellular DNA replication. Two mutagen-sensitive mutants, deficient in both replication on undamaged templates and postreplication repair, were found to have normal levels of this α-polymerase activity. Our results suggest that a single enzymatic species of α-polymerase holoenzyme exists in Drosophila and is common to all developmental stages of this organism.     

Forty years of solitude: life-history divergence and behavioural isolation between laboratory lines of Drosophila melanogaster

The study of the early stages of speciation can benefit from examination of differences between populations of known history that have been separated for a short time, such as a few thousands of generations. We asked whether two lines of Drosophila melanogaster that were isolated more than 40 years ago have evolved differences in life-history characters, or have begun to evolve behavioural or postzygotic isolation. One line, which is resistant to DDT, showed lower egg production and a shorter lifespan than a susceptible line. These differences are not a pleiotropic effect of resistance because they are not attributable to the chromosome that contains the resistance factors. The two lines have begun to become behaviourally isolated. Again, the isolation is not attributable to genes on the chromosome that contains resistance factors. The lines show only prezygotic isolation; there is no evidence of reduced fitness of F1 or F2 hybrids. These lines and others like them, should be excellent subjects for analyses of genetic changes that could lead to speciation.     

Directed alterations of the X-chromosomal ribosomal gene repeats in mutant sc8 of Drosophila melanogaster

The patterns of the ribosomal DNA (rDNA) repeat units in seven Drosophila melanogaster inversional mutants have been studied. Among them, only the In(1)sc⁸ and its deletional derivative Df(1)mal¹² female rDNAs exibited significant reduction in the size of nearly all units, compared to the wild-type females (Canton S, Oregon R). Further investigation shows that each kind of repeat (insertion-free, insertion-containing) in the Xsc⁸ rDNA array is highly enriched with short (reduced to 4 kilobases) intergenic spacers (IGSs). We revealed two main types of rearrangements. Only part of the 4 kb IGSs display variable length deletions (0.2–0.6 kb) at the 5′ ends, within the so-called ‘1900’ base pair (bp) region, recognizable by restriction endonuclease AluI. The presence of additional 100–150 bp DNA in the start portion of this region has also been demonstrated. In contrast, the 3′ end spacer regions, corresponding to the external transcribed spacer, do not show any changes in size. These data indicate how reductions of approximately 1.1 kb DNAs in sc⁸ IGSs, carrying both the rearranged and non-rearranged ‘1900’ sequences, are achieved: the fixed decrease of a number of 240 bp AluI subrepeats, clustered in the central IGS portion, also contribute. None of the other similar inversional mutants examined has so many IGS variants. Therefore, alterations in the Xsc⁸ rRNA gene cluster seem not to be dependent on its inversional status. This revised version was published online in July 2006 with corrections to the Cover Date.     

黑腹果蝇抗真菌肽Drosomycin同系物的免疫原性和抗真菌活性

通过黑腹果蝇 Drosophila melanogaster抗真菌肽Drosomycin（Drs）及其同系物Drs-lC和Drs-lE的抗体制备及Western blotting 结果,分析了Drs同系物的免疫原性与其抗真菌活性的关系。研究采用了2种技术路线,分别将Drs、Drs-lC和Drs-lE 基因构建成与细胞生长因子基因 afgf 融合的重组表达质粒 pET-afgf-Drs、pET-afgf-C和pET-afgf-E,以及通过基因同向串连获得重组表达质粒 pRSET-2Drs、4Drs、6Drs 和 pRSET-2E、4E、6E,并将这些重组表达质粒转化到BL21（DE3）plysS受体菌进行诱导表达。分离纯化后的融合蛋白afgf-Drs、afgf-C和afgf-E 以及串连蛋白 4 Drs、4 Drs-lE分别免疫小白鼠获得相应的抗血清。Western blotting免疫原性检测结果表明,Drs及其同系物与各自的抗血清具有强的免疫反应,同时相互间也有交叉免疫反应,提示它们具有相似的主要抗原决定簇,这些抗原决定簇可能与抗真菌活性无关。同系物之间抗真菌活性的差异可能来源于某些细微结构上的差异。     

Genes required for embryonic muscle development in Drosophila melanogaster A survey of the X chromosome

We have begun a genetic analysis to dissect the process of myogenesis by surveying the X chromosome of Drosophila melanogaster for mutations that affect embryonic muscle development. Using polarised light microscopy and antibody staining techniques we analysed embryos hemizygous for a series of 67 deletion mutations that together cover an estimated 85% of the X chromosome, or 16.5% of the genome. Whereas the mature wild type embryo has a regular array of contractile muscles that insert into the epidermis, 31 of the deletion mutants have defects in muscle pattern, contractility or both, that cannot be attributed simply to epidermal defects and identify functions required for wild type muscle development. We have defined mutant pattern phenotypes that can be described in terms of muscle absences, incomplete myoblast fusion, failure of attachment of the muscle to the epidermis or mispositioning of attachment sites. Thus muscle development can be mutationally disrupted in characteristic and interpretable ways. The areas of overlap of the 31 deletions define 19 regions of the X chromosome that include genes whose products are essential for various aspects of myogenesis. We conclude that our screen can usefully identify loci coding for gene products essential in muscle development.     

生活中潜在诱变剂对果蝇存活率及遗传变异的影响

用紫外线、抗氧化剂、化妆品等多种生活中潜在的诱变剂处理野生型黑腹果蝇,观察果蝇的存活率及性状遗传变异情况,进而分析这些诱变剂对于果蝇的影响。实验结果表明,随紫外线照射时间的增加,果蝇生活力降低,子代果蝇突变率增加;随培养基中抗氧化剂浓度的增加,果蝇突变率与死亡率均呈上升趋势;不同化妆品也对果蝇造成了明显伤害,造成亲代个体死亡,后代出现突变型果蝇。     

ADH enzyme activity and Adh gene expression in Drosophila melanogaster lines differentially selected for increased alcohol tolerance

In Drosophila melanogaster, alcohol dehydrogenase (ADH) activity is essential for ethanol tolerance, but its role may not be restricted to alcohol metabolism alone. Here we describe ADH activity and Adh expression level upon selection for increased alcohol tolerance in different life-stages of D. melanogaster lines with two distinct Adh genotypes: Adh(FF) and Adh(SS). We demonstrate a positive within genotype response for increased alcohol tolerance. Life-stage dependent selection was observed in larvae only. A slight constitutive increase in adult ADH activity for all selection regimes and genotypes was observed, that was not paralleled by Adh expression. Larval Adh expression showed a constitutive increase, that was not reflected in ADH activity. Upon exposure to environmental ethanol, sex, selection regime life stage and genotype appear to have differential effects. Increased ADH activity accompanies increased ethanol tolerance in D. melanogaster but this increase is not paralleled by expression of the Adh gene.     

Native Molecular Forms of Head Acetylcholinesterase from Adult Drosophila melanogaster: Quaternary Structure and Hydrophobic Character

The native molecular forms of acetylcholinesterase (AChE) present in adult Drosophila heads were characterized by sedimentation analysis in sucrose gradients and by nondenaturing electrophoresis. The hydrophobic properties of AChE forms were studied by comparing their migration in the presence of Triton X100, 10-oleyl ether, or sodium deoxycholate, or in the absence of detergent. We examined the polymeric structure of AChE forms by disulfide bridge reduction. We found that the major native molecular form is an amphiphilic dimer which is converted into hydrophilic dimer and monomer on autolysis of the extracts, or into a catalytically active amphiphilic monomer by partial reduction. The latter component exists only as trace amounts in the native enzyme. Two additional minor native forms were identified as hydrophilic dimer and monomer. Although a significant proportion of AChE was only solubilized in high salt, following extractions in low salt, this high salt-soluble fraction contained the same molecular forms as the low salt-soluble fractions: thus, we did not detect any molecular form resembling the asymmetric forms of vertebrate cholinesterases.     

Cytological location and further characterization of the Third chromosome resistance gene in Drosophila melanogaster

Mutations in the Third chromosome resistance (Tcr; 3-39.6) gene confer dominant resistance to α-methyl dopa and suggest the gene is involved in catecholamine metabolism. Evidence for involvement in catecholamine metabolism comes from the three phenotypes associated with the mutant Tcr chromosomes dominant resistance, dominant rescue of partially complementing l(2)amd alleles, and recessive lethal phenotypes. Only dominant resistance to αs-methyl dopa, however, was mapped to the Tcr locus. Both recessive lethality and dominant rescue of l(2)amd alleles have now been mapped to the Tcr gene and, through the isolation of a new deletion in the region, we demonstrate these phenotypes are due to a loss of Tcr function. This deletion places the Tcr gene in the 69B4-5 to 69C8-11 region. Additionally, we have tested and verified three predictions of the biochemical model proposed by Bishop, Sherald, and Wright (1989) for the function of the Tcr protein. This revised version was published online in July 2006 with corrections to the Cover Date.     

重金属镉胁迫对果蝇dDnmt2、dMBD2/3表达量的影响

【目的】探明重金属镉(Cadmium,Cd)对黑腹果蝇Drosophila melanogaster DNA甲基化修饰相关基因表达的影响,初步分析镉胁迫可能导致果蝇的表观遗传变异及其可遗传性。【方法】收集8 h内羽化未交配的雌、雄果蝇,在添加不同质量浓度(0、0.9375、1.875、3.75、7.5、15.0、30.0、60.0 mg/kg)Cd的培养基中培养,以Real-time PCR定量检测亲代(F0)果蝇生殖系统、去生殖系统体细胞、整体表达量变化趋势及解除胁迫的子代(F1)果蝇DNA甲基化修饰系统相关基因(dDnmt2、dMBD2/3)在mRNA水平的表达变化。【结果】重金属镉胁迫诱导了果蝇卵巢、精巢、去卵巢雌果蝇、去精巢雄果蝇、完整雌果蝇、完整雄果蝇的dDnmt2、dMBD2/3在mRNA水平的表达上调,呈现一定剂量依赖性及雌、雄组织差异性,且这种表达变化持续至子一代。【结论】研究结果揭示了重金属镉胁迫可诱导果蝇dDnmt2、dMBD2/3表达量上调,其可能与果蝇的DNA甲基化修饰过程相关联,导致表观遗传变异并可能向子代传递。     

黑腹果蝇种组的核型与进化研究

观察了国内黑腹果蝇种组34种果蝇的有丝分裂中期核型,其中首次描述了一些新核型。系统地分析了黑腹果蝇种组8个种亚组之间的核型进化关系及种间亲缘关系。结果是:elegans种亚组的核型为A型;eugracilis、melanogaster和ficusphila种亚组的核型为C型;takahashii和suzukii种亚组的核型为C型和D型;montium种亚组的核型为B、C、C’、D、D’、和E型;ananassae种亚组的核型为F、G和H型。从核型分化的角度可以将黑腹果蝇种组分为5个谱系:elegans,eugracilis-melanogaster-ficusphila,takkahashii-suzukii,montium,ananassae。这与2004年Yang等的观点基本一致,正好从核型进化的角度验证了Yang通过DNA序列分析所得到的结果。差别只在于elegans种亚组,作者把它单独列为一支,认为是祖先种亚组。通过选取同一种果蝇的几个不同地域单雌系的核型分析,结果表明:同一种果蝇的核型存在地域差异。这种差异可能是由于不同生境造成,也可能是本身进化程度的差异,或是两种因素相互作用的结果。     

Thermal evolution of rate of larval development in Drosophila melanogaster in laboratory and field populations

The duration of Drosophila melanogaster larval and pupal periods was measured in laboratory thermal lines and in populations collected along a latitudinal transect in eastern Australia. In replicated laboratory lines kept for 9 years at 16.5° C or 25° C the duration of larval development had continued to diverge compared with 4 and 5 years previously, with more rapid larval development, and hence reduced total duration of pre-adult development, in the low temperature lines at both experimental temperatures. After 4 years of separate evolution, lines derived from the 25° C lines and subsequently cultured at 29° C showed no evidence of significant divergence in the duration of any part of the pre-adult period. The geographic populations showed a decrease in the duration of larval development, and hence of the total pre-adult period, with increasing latitude. In both laboratory and field populations, evolution at lower temperature was associated with more rapid larval development to a larger adult body size, the opposite genetic correlation between these traits to that found within a single temperature. The indications are that lower temperatures may be permissive of more efficient growth in D. melanogaster. It will be important to discover if evolution in response to temperature induces similar correlations in other ectotherms.     

Exploring the physiology and pathology of aging in the intestine of Drosophila melanogaster

The gastrointestinal tract, due to its role as a digestive organ and as a barrier between the exterior and interior milieus, is critically impacted by dietary, environmental, and inflammatory conditions that influence health and lifespan. Work in flies is now uncovering the multifaceted molecular mechanisms that control homeostasis in this tissue, and establishing its central role in health and lifespan of metazoans. The Drosophila intestine has thus emerged as a productive, genetically accessible model to study various aspects of the pathophysiology of aging. Studies in flies have characterized the maintenance of regenerative homeostasis, the development of immune senescence, the loss of epithelial barrier function, the decline in metabolic homeostasis, as well as the maintenance of epithelial diversity in this tissue. Due to its fundamental similarity to vertebrate intestines, it can be anticipated that findings obtained in this system will have important implications for our understanding of age-related changes in the human intestine. Here, I review recent studies exploring age-related changes in the fly intestine, and their insight into the regulation of health and lifespan of the animal.     

Inferring the effects of demography and selection on Drosophila melanogaster populations from a chromosome-wide scan of DNA variation

Identifying regions of the Drosophila melanogaster genome that have been recent targets of positive Darwinian selection will provide evidence for adaptations that have helped this species to colonize temperate habitats. We have begun a search for such genomic regions by analyzing multiple loci (about 250) dispersed across the X chromosome in a putatively ancestral population from East Africa and a derived European population. For both populations we found evidence for past changes in population size. We estimated that a major bottleneck associated with the colonization of Europe occurred about 3,500-16,000 years ago. We also found that while this bottleneck can account for most of the reduction in variation observed in the European sample, there is a deficit of polymorphism in some genomic regions that cannot be explained by demography alone.     

霍氏肠杆菌对黑腹果蝇生长和发育的影响

【背景】共生菌群可以影响宿主的生理、代谢以及神经行为,黑腹果蝇是研究宿主与共生菌作用机制的优秀遗传模型。【目的】分离和鉴定黑腹果蝇体内的共生菌,并研究其对果蝇生长和发育的影响。【方法】利用YG固体培养基分离果蝇肠道菌;革兰氏染色、生化鉴定和16S rRNA基因比对鉴定菌种;体内定殖和世代传递实验验证细菌与果蝇的共生关系;建立无菌和悉菌模型,通过发育历期和生长速率来验证其促生长作用;RT-PCR检测促生长分子基因表达;免疫荧光染色检测果蝇肠道细胞的增殖;葡萄糖氧化酶法检测葡萄糖浓度。【结果】分离到一株能够明显促进果蝇生长发育的细菌,经鉴定为霍氏肠杆菌(Enterobacter hormaechei),该菌可以稳定定殖于黑腹果蝇肠道以及培养基内,而且可以世代垂直传递。该菌可明显地促进果蝇的生长,并通过加速促胸腺激素的分泌来发挥促生长作用,同时可以促进果蝇肠道细胞的增殖。霍氏肠杆菌相较于无菌果蝇可以降低体内葡萄糖水平。【结论】霍氏肠杆菌是黑腹果蝇的有益共生菌,实验证实其可以促进果蝇的生长和发育。     

An X chromosome locus in Drosophila melanogaster that enhances survival of the triplo-lethal genotype,Dp-(Tpl)

Only a single locus (Tpl) is known in the Drosophila melanogaster genome that leads to early lethality when present as a heterozygous duplication (three doses) or deficiency (one dose). We report the recovery of third instar larvae (and of occasional adults) carrying a duplication for the triplo-lethal locus, Dp(Tpl). Karyotype analysis of the larvae showed that the individuals surviving were almost entirely 3X;2A metafemales. We examined the question of whether the entire X or a single X locus was a major factor permitting survival. X-Y translocations were used to produce females hyperploid for different portions of the X and carrying Dp(Tpl). Analysis of metaphase chromosomes by quinacrine fluorescence pattern indicates that the X chromosome region between 6D and 7DE must be present in an extra copy to enhance the survival of Tpl duplication-bearing females. Another type of experiment suggests that it is the region between 7C and 7DE which is essential.     

冻干胡蜂蜂蛹对果蝇寿命和繁殖性能的影响

本实验通过研究胡蜂蜂蛹冻干品对果蝇寿命和繁殖力的影响,证明胡蜂蜂蛹具有延缓衰老和增强繁殖性能的作用。采用乙醚麻醉法,收集8h（小时）内羽化的成虫,选择个体大小相近的雌雄果蝇,在不同浓度的培养基中饲养,每两天观察统计一次果蝇死亡数,直到全部死亡。结果表明,雌雄果蝇在5．0％实验组与对照组的平均寿命比较均具有极显著差异性（P〈0．01）,雌雄果蝇在0．2％和1．0％实验组与对照组比较平均寿命差异性显著（P〈0．05）,由此可见胡蜂蜂蛹可延长果蝇平均寿命;0．2％,1．0％组的果蝇子代数显著高于对照组果蝇子代数（P〈0．05）,5．0％组果蝇子代数极显著高于对照组果蝇子代数（P〈0．01）,由此可见冻干胡蜂蜂蛹可增强果蝇的繁殖能力。