Adaptation to a Starch Environment and Regulation of alpha-Amylase in Drosophila

The adaptation to glucose and starch foods insix species, D. melanogaster, D.virilis, D. saltans, D. funebris,D. levanonensis and D. americana, wasstudied by measuring productivity. D.melanogaster and D. virilis adapted more to thestarch environment than to the glucose environment,while D. saltans adapted more to the glucoseenvironment than to the starch environment. D.funebris, D. levanonensis, and D. americana did not distinctlyadapt to either environment. In addition, the regulationof amylase in the six species was investigated bymeasuring the levels of amylase activity with glucoseand starch food environments. The levels of amylaseactivity in D. levanonensis and D.saltans were substantially low, indicating thatthese species cannot utilize starch as a carbon source.The starch-adapted species, D. melanogaster and D.virilis, showed higher levels of amylase activitywith the starch environment and higher inducibility.These results suggest that changing the regulation ofamylase is important for the adaptation to a starch environment inDrosophila.     

Isolation of Protease-Free Alcohol Dehydrogenase (ADH) from Drosophila simulans and Several Homozygous and Heterozygous Drosophila melanogaster Variants

The enzyme alcohol dehydrogenase (ADH) fromseveral naturally occurring ADH variants ofDrosophila melanogaster and Drosophilasimulans was isolated. Affinity chromatography withthe ligand Cibacron Blue and elution with NAD⁺ showed similarbehavior for D. melanogaster ADH-FF, ADH-71k,and D. simulans ADH. Introduction of a secondCibacron Blue affinity chromatography step, withgradient elution with NAD⁺, resulted in pure and stable enzymes. D.melanogaster ADH-SS cannot be eluted from theaffinity chromatography column at a high concentrationof NAD⁺ and required a pH gradient for itspurification, preceded by a wash step with a high concentration ofNAD⁺. Hybrid Drosophila melanogasteralcohol dehydrogenase FS has been isolated fromheterozygous flies, using affinity chromatography withfirst elution at a high concentration NAD⁺, directlyfollowed by affinity chromatography elution with a pHgradient. Incubation of equal amounts of pure homodimersof Drosophila melanogaster ADH-FF and ADH-SS,in the presence of 3 M urea at pH 8.6, for 30 min at roomtemperature, followed by reassociation yielded activeDrosophila melanogaster ADH-FS heterodimers. Noproteolytic degradation was found after incubation ofpurified enzyme preparations in the absence or presenceof SDS, except for some degradation of ADH-SS after verylong incubation times. The thermostabilities of D.melanogaster ADH-71k and ADH-SS were almostidentical and were higher than those of D.melanogaster ADH-FF and D. simulans ADH. Thethermostability of D. melanogaster ADH-FS waslower than those of D. melanogaster ADH-FF andADH-SS. D. melanogaster ADH-FF and ADH-71k have identical inhibition constantswith the ligand Cibacron Blue at pH 8.6, which are twotimes higher at pH 9.5. The K_i values forD. simulans ADH are three times lower at bothpH values. D. melanogaster ADH-SS and ADH-FS havesimilar K_i values, which are lower than thosefor D. melanogaster ADH-FF at pH 8.6. But at pH9.5 the K_i value for ADH-FS is the same as atpH 8.6, while that of ADH-SS is seven times higher. Kinetic parameters ofDrosophila melanogaster ADH-FF, ADH-SS, andADH-71k and Drosophila simulans ADH, at pH 8.6and 9.5, showed little or no variation inK_m ^eth values. TheK_m ^NAD values measured at pH 9.5for Drosophila alcohol dehydrogenases are alllower than those measured at pH 8.6. The rate constants(k_cat) determined for all fourDrosophila alcohol dehydrogenases are higher at pH 9.5 than at pH 8.6. D.melanogaster ADH-FS showed nonlinear kinetics.     

果蝇P转座因子的研究进展

果蝇P因子是DNA转座子,在近几十年里受到很大关注。可用于确认有关基因,克隆基因以及安置基因回到基因组。P因子的高易动性及其保持和对内部序列强烈的修饰作用也是P因子的本质特征。P因子的另一重要用途是用于产生转基因果蝇。目的基因置于质粒内P因子中可在转座酶的作用下插入前胚盘胚。携带目的基因的P因子可从质粒转座到任意染色体上。据报道,在典型实验中,插入可育果蝇的10％～20％可产生转化体后裔。但是以这种可动DNA片段作为载体尚存在转移基因的不稳定性及与内源跳跃基因的相互影响。本文介绍了果蝇P转座因子的一些研究进展。这些因子的遗传可动性也使它们适用于建造载体产生转基因生物。若如此,载体导入外源基因组的遗传稳定性问题将是一个重要课题。 Abstract：P elements in D.melanogaster are DNA transposons and received greater attention within the last few decades.P elements are used for identifying genes of interest,for cloning them,and for placing them back into the genome.The high mobility of P elements and their retention of this mobility and drastic modiffications to their internal sequences are also essential features.Another most important use of P elements is that of making transgenic flies.Desired gene is placed between P-element ends,usually within a plasmid,and injected into preblastoderm embryos in the presence of transposase.This P element then transposes from the plasmid to a random chromosomal site.Reported in a typical experiment,10％～20％ of the fertile injected flies produce transformant progeny.But the instability of the transferred gene carried on a piece of mobile DNA as a vector and its interaction with endogenous jumping genes.This paper introduced the studies advances of P transposable element in Drosophila.The genetic mobility of these elements can also make them suitable for the construction of vectors to create transgenic organisms.If so,the genetic stability of the vectors introduced to a foreign genome should be a important subject.     

Regulation of synthesis of the larval serum proteins of Drosophila melanogaster

We have determined the relative amounts of subunits of larval serum proteins (LSPs) 1 and 2 during larval development in Drosophila melanogaster. These results indicate that synthesis of polypeptide subunits of LSP-1 and LSP-2 is coordinate: the proteins are first detected at the same time; they accumulate in a coordinate fashion; their RNAs are first detected at the same time; the RNAs also accumulate in similar relative amounts. Analyses of fat body polypeptides and fat body RNA indicate that synthesis of LSP-1 declines at a time when there are still substantial quantities of LSP-1 RNA in the cytoplasm. Cessation of LSP-1 subunit synthesis occurs before cessation of LSP-2 synthesis, indicating that at late times the genes (or mRNAs) for these two proteins are subject to different "switch-off" controls.     

Genetic load and viability studies in Korean natural populations of Drosophila melanogaster

Further studies about the amount of genetic load in the Korean Anyang and Cheju (Sughipo) Island populations of Drosophila melanogaster were performed. In total 1630 second chromosomes were extracted from the Anyang opulation between 1983 and 1985; 19.0 % of the chromosomes proved lethal, 8.8 % semilethal in homozygous condition. From the island oulation, 504 wild second chromosomes were analysed in 1986; 24.2 % were lethal, 4.6 % subleiaf A slight increase of lethal and semilethal frequencies between 1976 and 1986 can be observed. Mean viabilities of “all homozy-gotes”, “quasinormal-homozygotes”, and “random heterozygotes” were estimated from crossin experiments with marker strains. Random heterozygotes were always more viable than quasinormal homozygotes. An analysis for correlation between random heterozgous and homozygous viabilities gave values significantly different from zero only for the 1985 kyang sample (r =—0.4625, P < 0.01), but no significances could be observed for all other Anyang samples from 1983, 1984, and 1986, respectively. he effective population sizes were estimated to be between 2000 and 6300 individuals for the Anyan and 4200 individuals for the island population, using Nel's formula (1968). It is sugested that baknced natural selection is mainly responsible for the maintenance of genetic load in the Anyang natural populations of D. melanogaster.     

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.     

Divergence and heterogeneity of the histone gene repeating units in the Drosophila melanogaster species subgroup

The repeating units of the histone gene cluster containing the H1, H2A, H2B and H4 genes were amplified by PCR from the Drosophila melanogaster species subgroup, i.e., D. yakuba, D. erecta, D. sechellia, D. mauritiana, D. teissieri and D. orena. The PCR products were cloned and their nucleotide sequences of about 4.6-4.8kbp were determined to elucidate the mechanism of molecular evolution of the histone gene family. The heterogeneity among the histone gene repeating units was 0.6% and 0.7% for D. yakuba and D. sechellia, respectively, indicating the same level of heterogeneity as in the H3 gene region of D. melanogaster. Divergence of the genes among species even in the most closely related ones was much greater than the heterogeneity among family members, indicating a concerted mode of evolution for the histone gene repeating units. Among the species in the D. melanogaster species subgroup, the histone gene regions as well as 3rd codon position of the coding region showed nearly the same GC contents. These results suggested that the previous conclusion on analysis of the H3 gene regions, the gene family evolution in a concerted fashion, holds true for the whole histone gene repeating unit.     

Divergence of water balance mechanisms in two melanic Drosophila species from the western Himalayas

Drosophila busckii is more abundant under colder and drier montane habitats in the western Himalayas as compared to Drosophila melanogaster but the mechanistic basis of such climatic adaptations is largely unknown. We tested the hypothesis whether genetic variation or phenotypic plasticity of cuticular traits confer adaptive protection against desiccation stress in two melanic Drosophila species living under drier montane localities. For D. melanogaster, changes in melanisation are known to be associated with reduced water loss but there are no data on D. busckii. We investigated changes in body melanisation, cuticular lipids, desiccation resistance, water loss, extractable hemolymph volume (%), and dehydration tolerance in six sympatric populations of D. busckii and D. melanogaster over an altitudinal range of 640-2236 m. D. busckii is a melanic species but changes in cuticular water loss are negatively correlated with cuticular lipid mass and not with body melanisation. In D. melanogaster, there are no plastic effects (14-28 °C) for cuticular lipid mass but variation in body melanisation is associated with desiccation-related traits. Effects of organic solvents (hexane or chloroform: methanol), developmental plasticity and seasonal variation in cuticular lipids affect body water loss in D. busckii but no such changes occur in D. melanogaster. Thus, sympatric populations of D. busckii and D. melanogaster have evolved different water balance mechanisms under shared environmental conditions in the western Himalayas. Multiple measures of desiccation resistance in these species show clinal variation with altitude, consistent with adaptation to increased desiccation stress.     

肠道微生物群加重黑腹果蝇盐胁迫反应

【背景】高盐饮食目前引起普遍关注,肠道微生物与盐胁迫的相互作用正成为研究热点之一。【目的】以黑腹果蝇为宿主模型,探讨肠道微生物对果蝇盐胁迫反应的影响与潜在机理。【方法】利用平板计数法和定量PCR法检测果蝇肠道载菌量;利用存活率和运动能力测定装置测定果蝇适合度;用化学试剂和抗生素处理建立无菌果蝇,测定肠道菌对果蝇盐胁迫反应的影响;利用亮蓝食用色素染料渗透性实验检测果蝇肠道屏障的完整性;应用实时定量PCR检测先天免疫系统的活性。【结果】高盐处理引起果蝇肠道菌群失调,导致其肠道载菌量显著增加。此外,高盐饮食(high salt diet,HSD)降低了黑腹果蝇成虫的存活率和运动能力。经0.75 mol/L NaCl处理,雌性GF (germ-free)果蝇存活率比对照组升高了11%,同时混合抗生素有效地提高了高盐处理后果蝇的存活率。肠道微生物加剧了肠道屏障功能损伤,雌性GF果蝇出现染料渗透性实验现象的百分率比对照组降低了8%。在分子水平上,盐胁迫下雌性GF果蝇体内Attacin-C、Duox基因表达水平分别是CR(conventionally reared)果蝇的2.5倍和1.7倍。【结论】肠道微生物加重果蝇盐胁迫反应,引发高盐诱导的肠屏障功能紊乱,并且抑制高盐诱导的先天免疫活性。     

Historical effective size and the level of genetic diversity in Drosophila melanogaster and Drosophila pseudoobscura

We report the results of a sequential gel electrophoretic study of protein variation in Drosophila melanogaster and its comparison with D. pseudoobscura. The number of alleles and mean heterozygosity were lower in D. melanogaster than in D. pseudoobscura. On the other hand, geographical populations of Drosophila melanogaster have been shown to be much more differentiated than those of D. pseudoobscura. The results suggest that in D. melanogaster low-frequency alleles have been lost during the colonization process and that major alleles have become differentiated among populations. Population bottlenecks, due to various causes, appear to have played a significant role in the shaping of genetic variation in natural populations of many species. It is proposed that a comparison of genetic variation at homologous gene loci between related species can bring out effects of historical bottlenecks and provide an alternative approach for analyzing causes of genetic variation in natural populations.We thank the Natural Science and Engineering Research Council of Canada for financial support (Grant A0235 to R.S.S.).     

Sex combs,allometry, and asymmetry in Drosophila

There has been recent debate about the expected allometry of sexually‐selected traits. Although sexually‐selected traits exhibit a diversity of allometric patterns, signalling characters are frequently positively allometric. By contrast, insect genitalia tend to be negatively allometric, although the allometry of nongenital sexually‐selected characters in insects is largely unknown (with some notable exceptions). It has also been suggested that there should be a negative association between the asymmetry and size of bilaterally‐paired, sexually‐selected traits, although this claim is controversial. We assessed the allometry and asymmetry (fluctuating asymmetry, FA) of a nongenital contact–courtship structure, the sex comb, in replicate populations of three species of Drosophila (we also measured wing FA). Sex combs are sexually‐selected characters used to grasp the female's abdomen and genitalia and to spread her wings prior to and during copulation. Although species differed in the size of the sex combs, all combs were positively allometric, and comb allometry did not generally differ significantly between species or populations. Comb and wing asymmetry did vary across species, although not across populations of the same species. However, FA was trait specific and was never negatively associated with trait size. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 923–934.     

β-Hydroxy-α-ketobutyric acid in Drosophila melanogaster,with reference to biosynthesis of drosopterins

A substance designated as compound D, which reacts spontaneously with 7,8-dihydropterin to give drosopterins, is found in Drosophila melanogaster. The compound was partially purified from the extract of flies by column chromatography and identified as β-hydroxy-α-ketobutyric acid by analysis of its 2,4-dinitrophenylhydrazone, mass spectrometry and reactivity with 7,8-dihydropterin. A highly significant correlation (r = 0.969, p < 0.001) was found between the amounts of the compound and drosopterins in the eye-pigment mutants of Drosophila. Changes of the compound during development of flies were also closely related to those of drosopterins. Based on these observations, a role of the compound in biosynthesis of drosopterins has been discussed.     

Accumulation of Transposable Elements in the Heterochromatin and on the Y Chromosome of Drosophila simulans and Drosophila melanogaster

The elements of the transposon families G, copia, mdg 1, 412, and gypsy that are located in the heterochromatin and on the Y chromosome have been identified by the Southern blotting technique in Drosophila simulans and D. melanogaster populations. Within species, the abundance of such elements differs between transposon families. Between species, the abundance in the heterochromatin and on the Y chromosome of the elements of the same family can differ greatly suggesting that differences within a species are unrelated to structural features of elements. By shedding some new light on the mechanism of accumulation of transposable elements in the heterochromatin, these data appear relevant to the understanding of the long-term interaction between transposable elements and the host genome. Received: 8 August 1997 / Accepted: 11 December 1997     

Latitudinal Adh allozymic variation and ethanol tolerance in Indian populations of Drosophila melanogaster

Ten Indian geographical populations of D. melanogaster were assayed electrophoretically for Adh genic variation. The Indian geographical populations of D. melanogaster revealed significant clinal variation (3 % for 1 d? latitude) at Adh locus and Adh^F allelic frequency correlated significantly with increase in latitude. It was suggested that the abundance of secondary alcohols in the southern Indian tropical and humid environment might exert selective pressure favouring higher frequency of Adh^S allele. Patterns of ethanol utilization as well as ethanol tolerance were analyzed in larval and adult individuals of six geographical populations of D. melanogaster. Latitudinal variation in ethanol tolerance was observed in D. melanogaster populations from India. The parallel occurrence of latitudinal variation it Adh locus as well as ethanol tolerance in Indian geographical populations of D. melanogaster could be maintained by balancing natural selection varying spatially along the north-south axis of the Indian sub-continent.     

Contrasting patterns of geographic variation in the cosmopolitan sibling species Drosophila melanogaster and Drosophila simulans

An electrophoretic study was carried out to compare the geographic pattern of genetic variation in Drosophila simulans with that of its sibling species, Drosophila melanogaster. An identical set of 32 gene-protein loci was studied in four geographically distant populations of D. simulans and two populations of D. melanogaster, all originating from Europe and Africa. The comparison yielded the following results: (1) tropical populations of D. simulans were, in terms of the number of unique alleles, average heterozygosity per locus, and percentage of loci polymorphic, more variable than conspecific-temperate populations; (2) some loci in both species showed interpopulation differences in allele frequencies that suggest latitudinal clines; and (3) temperate-tropical genetic differentiation between populations was much less in D. simulans than in D. melanogaster. Similar differences between these two species have previously been shown for chromosomal, quantitative, physiological, and middle-repetitive DNA variation. Estimates of N _m (number of migrants per generation) from the spatial distribution of rare alleles suggest that both species have similar levels of interpopulation gene flow. These observations lead us to propose two competing hypotheses: the low level of geographic differentiation in D. simulans is due to its evolutionarily recent worldwide colonization and, alternatively, D. simulans has a narrower niche than D. melanogaster. Geographic variation data on different genetic elements (e.g., mitochondrial DNA, two-dimensional proteins, etc.) are required before these hypotheses can be adequately tested.We thank the Natural Science and Engineering Research Council of Canada for financial support (Grant A0235 to R.S.S.).