Expression Pattern Diversity and Functional Conservation Between Retroposed PRAT Genes from Drosophila melanogaster and Drosophila virilis

Gene duplication by retrotransposition duplicates only the coding and untranslated regions of a gene and, thus, biases retroduplicated genes toward having different expression patterns from their parental genes. As such, genes duplicated by retrotransposition are more likely to develop novel expression domains. To explore this idea further, we used the Prat/Prat2 gene duplication in Drosophila as a case study to examine the aftermath of a retrotransposition event that resulted in both the parent and the child gene becoming essential for survival. We used the Gal4-UAS transgene system with EGFP as a reporter to determine the developmental expression patterns of Prat and Prat2 from D. melanogaster (DmPrat and DmPrat2) and Prat from D. virilis (DvPrat). We also tested the functional equivalence of the protein products of DmPrat and DmPrat2. We found that each of the proteins could rescue DmPrat mutations, showing that the requirement for both Prat and Prat2 in Drosophila is not simply due to differences in protein function. In contrast, we found that the DmPrat and DmPrat2 genes have developed nonoverlapping patterns of expression, which correlate with their respective loss-of-function phenotypes. We further found that DvPrat expression is similar to DmPrat during development but differs in adult gonads. Thus, the function of the Prat retrogene has not diverged in the D. melanogaster and D. virilis lineages, while some aspects of its expression pattern have evolved. Finally, we have identified promoter elements, conserved upstream of DmPrat and DvPrat, that this retrogene has acquired to drive its expression.     

Regulation and metamorphosis of the abdominal histoblasts ofDrosophila melanogaster

Summary The development of the adult abdomen ofDrosophila melanogaster was analyzed by histology, microcautery, and genetic strategies. Eight nests of diploid histoblasts were identified in the newly hatched larva among the polytene epidermal cells of each abdominal segment: pairs of anterior dorsal, posterior dorsal, and ventral histoblast nests and a pair of spiracular anlagen. The histoblasts do not divide during larval life but begin dividing rapidly 3 h after pupariation, doubling every 3.6 h. Initially they remain confined to their original area, but 15 h after pupariation the nests enlarge, and histoblasts replace adjacent epidermis cell by cell. The histoblasts cover half the abdomen by 28 h after pupariation and the rest by 36 h. Polytene epidermal cells of the intersegmental margin are replaced last. Cautery of the anterior dorsal nest caused deletion of the whole corresponding hemitergite, whereas cautery of the posterior dorsal nest caused the deletion of the macrochaetae of the posterior of the hemitergite. Cautery of the ventral nest deleted the hemisternite and the pleura, whereas cautery of the spiracular anlagen deleted the spiracle. Results of cautery also revealed that no macrochaetae formed on the tergite in the absence of adjacent microchaetae. Clonal analysis revealed that there were no clonal restrictions within a hemitergite at pupariation. Cautery of polytene epidermal cells other than those of the intersegmental margin failed to affect tergite development. However, cautery of polytene epidermal cells of the intersegmental margin adjacent to either dorsal histoblast nest caused mirror-image duplications of the anterior or posterior of the hemitergite in 10% of the hemitergites. Forty percent of the damaged presumptive hemitergites formed complete hemitergites, indicating extensive pattern regulation and regeneration. Pattern duplication and regeneration were accounted for in terms of intercalation and a model of epimorphic pattern regulation (French et al., 1976). Histoblasts in adjacent segments normally develop independently, but if they are enabled to interact by deleting the polytene epidermal cells of the intersegmental margin, they undergo intercalation which results in duplication or regeneration. The possible role of the intersegmental margin cells of insects in development was analyzed.     

In vitro induced pinocytotic activity by a juvenile hormone analogue in oocytes of Drosophila melanogaster

Summary Pinocytotic activity has been analyzed in Drosophila oocytes following either in vivo or in vitro exposure to horseradish peroxidase. The enzyme tracer gains access to the yolk spheres only when supplied to the oocyte in vivo. In oocytes cultured in vitro, peroxidase remains restricted to the residual coated vesicles and to the tubular profiles formed in excess in the cortical ooplasm.In an attempt to induce peroxidase uptake by oocytes cultured in vitro, various incubations were tested. Among these, hemolymph from both sexes is capable of promoting peroxidase uptake up to a level comparable to that detectable in vivo. On the other hand, fat body extracts fail to promote such cellular activity. Finally, the juvenile hormone analogue ZR-515 is shown to be the only factor required to promote pinocytotic activity under the experimental conditions tested. The observations are interpreted to indicate that vitellogenin has no inductive role on pinocytosis but simply acts by adhering to the forming coated vesicles which in turn are produced by the oolemma in response to the action of juvenile hormone.     

Juvenile hormone stimulation of ornithine decarboxylase activity during vitellogenesis in Drosophila melanogaster

Summary The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, becomes elevated in intact female Drosophila melanogaster shortly after adult eclosion. This activity reaches a peak at 24 h following eclosion, and then drops to lower levels by 48 h. This pattern is not observed in males, consistent with the hypothesis that polyamine synthesis is involved in ovarian maturation in Drosophila. Abdomens isolated within 2 h of adult eclosion do not display elevated ODC activity or ovarian maturation. However, a 250-ng dose of the juvenile hormone analog methoprene (ZR-515) applied in acetone to these abdomens, recovers ovarian maturation and causes a 5–10 fold increase in enzyme activity over controls treated with acetone alone. The same dose of the inactive precursor methyl farnesoate caused no such increase, whereas a 500-ng dose of the newly discovered natural Drosophila JHB₃ stimulated a four-fold response. The response to methoprene was dose-dependent, showing stimulatory activity at a dose as low as 10 ng. This stimulation by JHA is rapid, occurring between 1 and 3 h following hormone treatment, reminiscent of JH induction of fat body vitellogenin synthesis in Drosophila. Elevated ODC activity appeared to be localized in the adult fat body. During embryogenesis, ODC activity remained undetectable until just prior to hatching, when a large increase was detected. We postulate that JH may, either directly or indirectly, regulate polyamine biosynthesis in vivo, and that this synthesis may be required for the production of macromolecules during Drosophila vitellogenesis or embryogenesis.Abbreviations JH juvenile hormone - JHA juvenile hormone analog - ODC ornithine decarboxylase - SAMDC S-adenosyl-methionine decarboxylase - JHB ₃ juvenile hormone III bisepoxide     

Analysis of twin of eyeless regulation during early embryogenesis in Drosophila melanogaster

The Drosophila Pax6 homolog twin of eyeless (toy) is so far the first zygotically expressed gene involved in eye morphogenesis in Drosophila. The study of its expression during embryogenesis is therefore informative of the initial events of eye development in Drosophila. We have analyzed how the initial expression domain of toy at cellular blastoderm is regulated. We show that the three maternal patterning systems active in the cephalic region (the anterior, terminal and dorsal-ventral systems) cooperate with zygotically activated gap genes to shape the initial expression domain of toy. Whereas Bicoid, Dorsal and Torso signaling synergistically act as activators, Hunchback, Knirps and Decapentaplegic act as repressors.     

The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila melanogaster

Seminal proteins from the Drosophila male accessory gland induce post-mating responses (PMR) in females. The PMR comprise behavioral and physiological changes that include increased egg laying, decreased receptivity to courting males, and changes in the storage and use of sperm. Many of these changes are induced by a “sex peptide” (SP) and are maintained by SP’s binding to, and slow release from, sperm. The accessory gland contains two secretory cell types with distinct morphological and developmental characteristics. Products of these “main” and “secondary” cells work interdependently to induce and maintain the PMR. To identify individual genes needed for the morphology and function of secondary cells, we studied iab-6^cocu males, whose secondary cells have abnormal morphology and fail to provide products to maintain the PMR. By RNA-seq, we identified 77 genes that are downregulated by a factor of >5× in iab-6^cocu males. By functional assays and microscopy, we tested 20 candidate genes and found that at least 9 are required for normal storage and release of SP in mated females. Knockdown of each of these 9 genes consequently leads to a reduction in egg laying and an increase in receptivity over time, confirming a role for the secondary cells in maintaining the long-term PMR. Interestingly, only 1 of the 9 genes, CG3349, encodes a previously reported seminal fluid protein (Sfp), suggesting that secondary cells may perform essential functions beyond the production and modification of known Sfps. At least 3 of the 9 genes also regulate the size and/or abundance of secondary cell vacuoles, suggesting that the vacuoles’ contents may be important for the machinery used to maintain the PMR.     

巴西橡胶树两个蔗糖合成酶基因的克隆和表达分析

为了解蔗糖合成酶在巴西橡胶树(Hevea brasiliensis)生长和发育过程中的功能,利用RACE技术从巴西橡胶树中克隆了蔗糖合成酶基因,并对基因的表达特征进行了分析。结果表明,从巴西橡胶树中克隆了两个蔗糖合成酶基因(HbSS1和HbSS2),HbSS1全长2864 bp,编码806个氨基酸;HbSS2全长2815 bp,编码811个氨基酸。两个基因编码的蛋白具有典型的植物蔗糖合成酶结构特征,包含1个磷酸化位点和两个保守的功能域。半定量RT-PCR分析表明,HbSS1和HbSS2在各组织器官中均有表达,其中HbSS1在叶中的表达量最高,HbSS2在树皮中的表达量最高,这说明HbSS1和HbSS2可能参与了各组织的生长和代谢过程,且功能有所分化。     

Regulation of Krüppel expression in the anlage of the Malpighian tubules in the Drosophila embryo

The expression of most Drosophila segmentation genes is not limited to the early blastoderm stage, when the segmental anlagen are determined. Rather, these genes are often expressed in a variety of organs and tissues at later stages of development. In contrast to the early expression, little is known about the regulatory interactions that govern the later expression patterns. Among other tissues, the central gap gene Krüppel is expressed and required in the anlage of the Malpighian tubules at the posterior terminus of the embryo. We have studied the interaction of Krüppel with other terminal genes. The gap genes tailles and huckebein, which repress Krüppel in the central segmentation domain, activate Krüppel expression in the posterior Malpighian tubule domain. The opposite effect on the posterior Krüppel expression is achieved by the interposition of another factor, the homeotic gene fork head, which is not involved in the control of the central domain. In addition, Krüppel activates different genes in the Malpighian tubules than in the central domain. Thus, both the regulation and the function of Krüppel in the Malpighian tubules differ strikingly from its role in segmentation.     

Fine structure of flight muscles in different Notch mutants of Drosophila melanogaster reared at different temperatures

Summary The fine structure of the indirect flight muscles was studied by electron microscopy in the following Notch locus mutants of Drosophila melanogaster reared at 18° C or 29° C for 6 days after eclosion: Ax ¹⁶¹⁷²/Ax¹⁶¹⁷², Ax²⁸/ Ax²⁸, l(1)N^ts1/l(1)N^ts1,l(1)N^ts1/Y and in wild-type controls. The flies were raised up to eclosion at 25° C or 18° C. It was observed that the l(1)N^ts1 flies gradually became flightless within a few days if reared at 29° C as adults, and gross changes in the fine structure of the flight muscles were also observed in flies of this genotype. Peripheral myofilaments of myofibrils were disarranged and the mitochondria diminutive. At 18° C the flight muscles remained normal. In all of the Abruptex (Ax) combinations the flight muscles remained similar to the wild-type controls at both 18° C and 29° C, i.e. they were normal. The results suggest that the Notch gene is active in adult flies in addition to its activity during embryonic, larval and pupal stages, and is directly or indirectly involved in the adult development of the muscle tissue.     

鹅掌楸LcUGE基因的克隆和组织表达分析

为了解鹅掌楸(Liriodendron chinense)的UGE基因功能,采用RACE和EPIC-PCR技术克隆到2个UGE基因,命名为LcUGE1和LcUGE2。结果表明,LcUGE1基因的c DNA全长为1 531 bp,包含1 050 bp的开放阅读框,编码349个氨基酸, gDNA长度为11 920 bp;LcUGE2基因的c DNA长度为1 378 bp,包含1 056 bp的开放阅读框,编码351个氨基酸,g DNA长度为6544 bp。LcUGE1和LcUGE2基因均含有9个外显子和8个内含子,且外显子长度和内含子剪切位点序列几乎一致,但内含子片段长度存在显著差异。编码的LcUGE1和LcUGE2蛋白高度保守,保守性达到82%。LcUGE1基因在雄蕊中表达量最高,而LcUGE2基因则在花萼中表达量最高。这表明LcUGEs基因可能参与鹅掌楸的生殖发育过程。     

Phylogenetic incongruence in the Drosophila melanogaster species group

Drosophila melanogaster and its close relatives are used extensively in comparative biology. Despite the importance of phylogenetic information for such studies, relationships between some melanogaster species group members are unclear due to conflicting phylogenetic signals at different loci. In this study, we use twelve nuclear loci (eleven coding and one non-coding) to assess the degree of phylogenetic incongruence in this model system. We focus on two nodes: (1) the node joining the Drosophila erecta-Drosophila orena, Drosophila melanogaster-Drosophila simulans, and Drosophila yakuba-Drosophila teissieri lineages, and (2) the node joining the lineages leading to the melanogaster, takahashii, and eugracilis subgroups. We find limited evidence for incongruence at the first node; our data, as well as those of several previous studies, strongly support monophyly of a clade consisting of D. erecta-D. orena and D. yakuba-D. teissieri. By contrast, using likelihood based tests of congruence, we find robust evidence for topological incongruence at the second node. Different loci support different relationships among the melanogaster, takahashii, and eugracilis subgroups, and the observed incongruence is not easily attributable to homoplasy, non-equilibrium base composition, or positive selection on a subset of loci. We argue that lineage sorting in the common ancestor of these three subgroups is the most plausible explanation for our observations. Such lineage sorting may lead to biased estimation of tree topology and evolutionary rates, and may confound inferences of positive selection.     

Regulation of amylase activity in Drosophila melanogaster: Variation in the number of enzyme molecules produced by different amylase genotypes

Purified amylases from high- and low-activity variants of Drosophila melanogaster showed identical specific activities. Immunoelectrophoresis of crude larval homogenates showed severalfold differences between strains in the amounts of cross-reacting material. Control of amylase activity is trans-acting in heterozygotes between high- and low-activity variants. These results suggest the existence of polymorphic regulatory genes affecting the production levels of amylase protein in D. melanogaster.This work was supported by Grant GM-21279 from the Institute of General Medical Science of the NIH to R. C. Lewontin and by an Operating Grant from the Natural Sciences and Engineering Research Council Canada to D. A. Hickey.     

Early Development of the Secretory Cavity of Peltate Glands in Humulus lupulus L. (Cannabaceae)

Early development of the secretory cavity of chemically fixed peltate glands in Humulus lupulus L. showed secretions with different densities, light, gray and dark, in the cytoplasm of disc cells and in the periplasmic space adjacent to the developing secretory cavity. Secretions were detected in the disc cell wall and subsequently in the developing secretory cavity under the subcuticular wall of the sheath. Light and gray secretions in the cavity possessed a membrane-like surface feature. Secretions were in contact with the irregular inner surface of the cuticle. Secretions contributed to the thickening of the cuticle, whereas the membrane-like surface feature contributed to a network of Cannabis striae distributed throughout the cuticle. This study supports an early development and organization of the secretory cavity in H. lupulus, parallel to those in Cannabis, and may represent common features for lipophilic glands in angiosperms.     

Effects of Polycomb Group Mutations on the Expression of Ultrabithorax in the Drosophila Visceral Mesoderm

The Polycomb group (PcG) genes encode repressors of many developmental regulatory genes including homeotic genes and are known to act by modifying chromatin structure through complex formation. We describe how Ultrabithorax (Ubx) expression is affected by the PcG mutants in the visceral mesoderm. Mutant embryos of the genes extra sex combs (esc), Polycomb (Pc), additional sex combs (Asx) and pleiohomeotic (pho) were examined. In each mutation, Ubx was ectopically expressed outside of their normal domains along the anterior-posterior axis in the visceral mesoderm, which is consistent with the effect of PcG proteins repressing the homeotic genes in other tissues. All of these four PcG mutations exhibit complete or partial lack of midgut constriction. However, two thirds of esc mutant embryos did not show Ubx expression in parasegment 7 (PS7). Even in the embryos showing ectopic Ubx expression, the level of Ubx expression in the PcG mutations was weaker than that in normal embryos. We suggest that in PcG mutations the ectopic Ubx expression is caused by lack of PcG repressor proteins, while the weaker or lack of Ubx expression is due to the repression of Ubx by Abd-B protein which is ectopically expressed in PcG mutations as well.     

LTR Retrotransposon-Gene Associations in Drosophila melanogaster

Thirty-three percent (228/682) of all long terminal repeat (LTR) retrotransposon sequences (LRSs) present in the sequenced Drosophila melanogaster genome were found to be located in or within 1000 bp of a gene. Recently inserted LTR retrotransposons are significantly more likely to be located in or within genes than are older, fragmented LTR retrotransposon sequences, indicating that most LRS-gene associations are selected against over evolutionary time. LRSs associated with conserved genes (homologenes) are especially prone to negative selection. In contrast, fragmented LRSs that have persisted in the genome over long spans of evolutionary time are preferentially associated with genes involved in signal transduction and other newly evolved functions. Reviewing Editor: Dr. Juergen Brosius