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。     

Defective neuroblast commitment in mutants of the achaete-scute complex and adjacent genes of D. melanogaster

Loss of function mutations in genes of the achaete-scute complex (ASC) or in the gene vnd of D. melanogaster result in neural hypoplasia. Two types of defects contribute to the development of the neural hypoplasic phenotype: a lower than normal proportion of neuroblasts delaminate from the neuroectoderm, and there is abundant cell death in the neural primordium during later stages. In addition, we found that increasing the copy number of ASC wild-type alleles leads to effects opposite to those caused by their deletion. All of these results indicate that the function of these genes is required for the commitment of neuroectodermal cells as neuroblasts and that the loss of these genetic functions causes the cells either to take on an epidermal fate or to die.     

Maternal expression of genes that regulate the bithorax complex of Drosophila melanogaster

A relatively large number of genes have been described that are required for the normal spatial expression of the genes of the bithorax complex. Most of these regulators appear to act negatively and are required to prevent indiscriminate expression of bithorax complex (BX-C) functions. In this report we examine five negative BX-C regulators to determine whether these are maternally expressed in germ-line derived cells. The genes studied include Additional sex combs (Asx), Polycomblike (Pcl), Sex comb extra (Sce), Sex comb on midleg (Scm), and lethal(4)29 [l(4)29]. The maternal germ-line dependent expression of each of these genes is assessed by comparison of zygotes from mothers whose functional germ cells carry no wild-type alleles to zygotes from mothers whose germ cells contain one wild-type allele. Because mutant alleles of each of the genes studied are recessive lethals, mosaic females with homozygous or hemizygous mutant germ lines were produced by pole cell transplantation. The results demonstrate that all of the negative regulators tested are expressed in the maternal germ line and all play important roles in the regulation of BX-C activities during embryogenesis. The absence of maternally supplied products from all of the genes studied except l(4)29 can be largely or completely compensated for by the activity in the zygote of a paternally contributed wild-type allele. It is argued that, with the exception of l(4)29, the genes studied in this report are qualitatively similar in function to the previously described BX-C regulators Pc, esc, and sxc. The available evidence indicates that genes within this group have functions that are not restricted to the regulation of genes that control segmental identity.     

Transcriptional organization of the Drosophila melanogaster ribosomal RNA genes.

Five distinct DNA replicating intermediates have been separated from lysates of bacteriophage G4-infected cells pulse-labelled during the period of replicative form synthesis using propidium diiodide/caesium chloride gradients. These are a partially single-stranded theta structure that is labelled in both the viral and complementary DNA strands; partially single-stranded circles, some with an unfinished viral DNA strand (25%) and some with an unfinished complementary DNA strand (75%); replicative form II(RFII) and replicative form I(RFI) DNA labelled only in the complementary DNA strand. To explain the pulse-label data a model is proposed in which G4 replicative form replication takes place by a displacement mechanism in which synthesis of the new viral DNA strand displaces the old viral DNA strand as a single-stranded DNA loop (D-loop) and when the displacement reaches half way round the molecule (the origin of synthesis of the G4 viral and complementary DNA strands are on opposite sides of the genome, Martin &; Godson 1977) synthesis of the complementary DNA strand starts, but in the opposite direction. Strand separation of the parent helix runs ahead of DNA synthesis, releasing two partially single-stranded circles from the replicating structure which then complete their replication as free single-stranded DNA circles. No evidence was found to support a rolling circle displacement mechanism of G4 replicative form synthesis.     

Chromatin structure of the histone genes of D. melanogaster

We have examined the chromatin structure of the histone gene repeat of D. melanogaster using an indirect end-labeling technique. Our results show that each DNA segment of the repeat is packaged into a precisely defined and characteristic structure, as follows: the nontranscribed spacers display a "normal" chromatin arrangement, with each nucleosome precisely positioned on the underlying DNA sequence; the 5' ends of all five histone genes are in an exposed configuration, highly sensitive to both micrococcal nuclease and DNAase I; and the genes have an "altered" chromatin structure, as indicated by the weak and irregularly spaced nuclease cuts. This well-defined chromatin arrangement is established early in development and is stably maintained throughout the remainder of the D. melanogaster life cycle.     

Courtship song recognition in the Drosophila melanogaster complex: heterospecific songs make females receptive in D. melanogaster, but not in D. sechellia

Abstract. The courtship song emitted by male wing vibration has been regarded as one of the most important signals in sexual isolation in the species of the Drosophila melanogaster complex. Inter- and intraspecific crosses were observed using males whose wings were removed (mute) or females whose aristae were removed (deaf). Females of D. melanogaster, D. simulans , and D. mauritiana mated with heterospecific males in the song-present condition (cross between normal females and winged males) more often than in the no-song condition (cross between normal females and wingless males or between aristaless females and winged males) or they showed no preference between the two conditions. It is possible that in these females heterospecific courtship songs play a role as if they were conspecific. In contrast, the females of D. sechellia mated with D. melanogaster or D. simulans males in the no-song condition more often than in the song-present condition, suggesting that they reject males with heterospecific song. Female mate recognition depending on the courtship song in D. melanogaster, D. simulans , and D. mauritiana is considered to be relatively broader and that in D. sechellia narrower.     

Two non-linked genes for specific virulence of Leptopilina boulardi against Drosophila melanogaster and D. yakuba

The developmental success of Leptopilina boulardi parasitoids within host species of the melanogaster subgroup is determined mainly by their ability to suppress the host immune reaction (virulence). Host resistance and parasitoid virulence are genetically variable in both partners. A gene for specific resistance against L. boulardi (Rlb) has been identified in Drosophila melanogaster, and a gene for the immune suppression (IS) of D. melanogaster has been identified in L. boulardi. To understand the evolution of the IS gene, we determined its specificity regarding potential hosts of the melanogaster subgroup. It did not affect the virulence against any other species of the melanogaster subgroup and was called ISm for immune suppression of D. melanogaster. Another gene (ISy), non-linked to the gene ISm, was characterized for the specific immune suppression of D. yakuba. These results suggesting that natural selection for virulence against one host species does not influence the evolution of virulence against another will allow us to develop pertinent hypotheses concerning the evolution of this character which is expected to drive the evolution of the parasitoid toward narrow host specialization. This revised version was published online in July 2006 with corrections to the Cover Date.     

Control of start codon choice on a plant viral RNA encoding overlapping genes.

The signals that control initiation of translation in plants are not well understood. To dissect some of these signals, we used a plant viral mRNA on which protein synthesis initiates at two out-of-frame start codons. On the large subgenomic RNA (sgRNA1) of barley yellow dwarf virus-PAV serotype, the coat protein (CP) and overlapping 17K open reading frames (ORFs) are translated beginning at the first and second AUG codons, respectively. The roles of bases at positions -3 and +4 relative to the AUG codons in efficiency of translation initiation were investigated by translation of sgRNA1 mutants in a cell-free extract and by expression of a reporter gene from mutant sgRNA1 leaders in protoplasts. The effects of mutations that disrupted and restored secondary structure encompassing the CP AUG independently of, and in combination with, changes to bases -3 and +4 were also examined. Partial digestion of the 5' end of the sgRNA1 leader with structure-sensitive nucleases gave products that were consistent with the predicted secondary structure. Secondary structure had an overall inhibitory effect on translation of both ORFs. In general, the "Kozak rules" of start codon preference predominate in determining start codon choice. Unexpectedly, for a given CP AUG sequence context, changes that decreased initiation at the downstream 17K AUG also reduced initiation at the CP AUG. To explain this observation, we propose a new model in which pausing of the ribosome at the second AUG allows increased initiation at the first AUG. This detailed analysis of the roles of primary and secondary structure in controlling translation initiation should be of value for understanding expression of any plant gene and in the design of artificial constructs.     

The influence of junction conformation on RNA cleavage by the hairpin ribozyme in its natural junction form

In the natural form of the hairpin ribozyme the two loop-carrying duplexes that comprise the majority of essential bases for activity form two adjacent helical arms of a four-way RNA junction. In the present work we have manipulated the sequence around the junction in a way known to perturb the global folding properties. We find that replacement of the junction by a different sequence that has the same conformational properties as the natural sequence gives closely similar reaction rate and Arrhenius activation energy for the substrate cleavage reaction. By comparison, rotation of the natural sequence in order to alter the three-dimensional folding of the ribozyme leads to a tenfold reduction in the kinetics of cleavage. Replacement with the U1 four-way junction that is resistant to rotation into the antiparallel structure required to allow interaction between the loops also gives a tenfold reduction in cleavage rate. The results indicate that the conformation of the junction has a major influence on the catalytic activity of the ribozyme. The results are all consistent with a role for the junction in the provision of a framework by which the loops are presented for interaction in order to create the active form of the ribozyme.     

Developmental control of transduced dopa decarboxylase genes in D. melanogaster

Summary Seventeen new euchromatic integration sites of the dopa-decarboxylase gene (Ddc) have been generated using p-mediated transduction. The developmental expression of the integrated genes was examined by monitoring the embryonic induction of dopa decarboxylase enzyme activity (DDC) and by monitoring the developmental pattern of DDC activity from late third instar to eclosion. The majority of inserts are regulated correctly within about 30% of controls. Several cases of multiple insertion events were recovered and these show correspondingly elevated levels of activity and are regulated normally. The pattern of expression of one insert (15C) falls outside the normal range. Multiple copies of transduced Ddc genes are used to test for effects of elevated gene dose on levels of expression. One insert on the X chromosome shows little or no dosage compensation. Possible reasons for the differences between the regulation of transduced genes in Drosophila and the regulation of transformed genes in mammalian systems are discussed.     

Bicarbonate stabilizes isolated D1/D2/cytochrome b 559 complex of photosystem 2 against thermoinactivation

It has been shown that thermoinactivation of the isolated D1/D2/cytochrome b ₅₅₉ complex (RC) of photosystem 2 (PS-2) from pea under anaerobic conditions at 35°C in 20 mM Tris-HCl buffer (pH 7.2) depleted of HCO₃⁻, with 35 mM NaCl and 0.05% n-dodecyl-β-maltoside, results in a decrease in photochemical activity measured by photoreduction of the PS-2 primary electron acceptor, pheophytin (by 50% after 3 min of heating), which is accompanied by aggregation of the D1 and D2 proteins. Bicarbonate, formate, and acetate anions added to the sample under these conditions differently influence the maintenance of photochemical activity: a 50% loss of photochemical activity occurs in 11.5 min of heating in the presence of bicarbonate and in 4 and 4.6 min in the presence of formate and acetate, respectively. The addition of bicarbonate completely prevents aggregation of the D1 and D2 proteins as opposed to formate and acetate (their presence has no effect on the aggregation during thermoinactivation). Since the isolated RCs have neither inorganic Mn/Ca-containing core of the water-oxidizing complex nor nonheme Fe²⁺, it is supposed that bicarbonate specifically interacts with the hydrophilic domains of the D1 and D2 proteins, which prevents their structural modification that is a signal for aggregation of these proteins and the loss of photochemical activity.