Nonrecombining genes in a recombination environment: the Drosophila "dot" chromosome

Rate of recombination is a powerful variable affecting several aspects of molecular variation and evolution. A nonrecombining portion of the genome of most Drosophila species, the "dot" chromosome or F element, exhibits very low levels of variation and unusual codon usage. One lineage of Drosophila, the willistoni/saltans groups, has the F element fused to a normally recombining E element. Here, we present polymorphism data for genes on the F element in two Drosophila willistoni and one D. insularis populations, genes previously studied in D. melanogaster. The D. willistoni populations were known to be very low in inversion polymorphism, thus minimizing the recombination suppression effect of inversions. We first confirmed, by in situ hybridization, that D. insularis has the same E + F fusion as D. willistoni, implying this was a monophyletic event. A clear gradient in codon usage exists along the willistoni F element, from the centromere distally to the fusion with E; estimates of recombination rates parallel this gradient and also indicate D. insularis has greater recombination than D. willistoni. In contrast to D. melanogaster, genes on the F element exhibit moderate levels of nucleotide polymorphism not distinguishable from two genes elsewhere in the genome. Although some linkage disequilibrium (LD) was detected between polymorphic sites within genes (generally <500 bp apart), no long-range LD between F element loci exists in the two willistoni group species. In general, the distribution of allele frequencies of F element genes display the typical pattern of expectations of neutral variation at equilibrium. These results are consistent with the hypothesis that recombination allows the accumulation of nucleotide variation as well as allows selection to act on synonymous codon usage. It is estimated that the fusion occurred ～20 Mya and while the F element in the willistoni lineage has evolved "normal" levels and patterns of nucleotide variation, equilibrium may not have been reached for codon usage.     

Persistence of oviposition rhythm in individuals of Drosophila melanogaster reared in an aperiodic environment for several hundred generations

The oviposition rhythm of individual flies of Drosophila melanogaster from a population maintained in an aperiodic environment (with light, temperature, humidity, and other factors which could provide time cues, kept constant) for several hundred generations was assayed in constant light (LL), in light/dark (LD 12:12 hr) cycle, and in constant darkness (DD). More than 50% of the flies assayed exhibited rhythmicity in oviposition in all three light regimes. The results indicate that the phenomenon of egg laying is rhythmic in individual D. melanogaster females and is controlled by an endogenous time keeping mechanism. The persistence of the oviposition rhythm in a large proportion of individuals in the population after several hundred generations of rearing in a constant environment strengthens the view that possessing biological clocks may confer some intrinsic fitness advantage even to organisms living in aperiodic environments. J. Exp. Zool. 290:541-549, 2001.     

Social interactions of monkeys reared in a nuclear family environment versus monkeys reared with mothers and peers

Four-year-old laboratory-born rhesus monkeys that had been reared in a nuclear family social environment consisting of mothers, fathers, siblings, peers, and other adults of both sexes were permitted to interact in various combinations with equal-aged monkeys that had been reared in an environment consisting of only mothers and peers. It was found that in most interaction sessions nuclear family subjects exhibited significantly higher levels of dominance and activity behaviors and significantly lower levels of submissive and passive behaviors than the mother-peer-reared subjects. These differences were not evident when subjects were tested within their own rearing groups. The significance of the results with respect to previous and future studies of social development in differential social environments is discussed.This research was supported by grant No. MH-11894 from the National Institute of Mental Health to the University of Wisconsin Primate Laboratory, by the Grant Foundation, and by grant No. RR-0167 from the National Institutes of Health to the University of Wisconsin Primate Research Center.     

Entrainment of eclosion rhythm in Drosophila melanogaster populations reared for more than 700 generations in constant light environment

In this paper, we report the results of our extensive study on eclosion rhythm of four independent populations of Drosophila melanogaster that were reared in constant light (LL) environment of the laboratory for more than 700 generations. The eclosion rhythm of these flies was assayed under LL, constant darkness (DD) and three periodic light-dark (LD) cycles (T20, T24, and T28). The percentage of vials from each population that exhibited circadian rhythm of eclosion in DD and in LL (intensity of approximately 100 lux) was about 90% and 18%, respectively. The mean free-running period (τ) of eclosion rhythm in DD was 22.85 ± 0.87 h (mean ± SD). Eclosion rhythm of these flies entrained to all the three periodic LD cycles, and the phase relationship (ψ) of the peak of eclosion with respect to “lights-on” of the LD cycle was significantly different in the three periodic light regimes (T20, T24, and T28). The results thus clearly demonstrate that these flies have preserved the ability to exhibit circadian rhythm of eclosion and the ability to entrain to a wide range of periodic LD cycles even after being in an aperiodic environment for several hundred generations. This suggests that circadian clocks may have intrinsic adaptive value accrued perhaps from coordinating internal metabolic cycles in constant conditions, and that the entrainment mechanisms of circadian clocks are possibly an integral part of the clockwork.     

Ecdysteroid-inducible polypeptides in a Drosophila cell line

In the Drosophila melanogaster cell line Kc-H, ecdysteroid hormone treatment causes increased relative synthesis of three ecdysteroid-inducible polypeptides (EIPs), named according to their molecular weights (in kilodaltons) EIP 40, EIP 29 and EIP 28. Increased synthesis of the EIPs is detectable within 45 min (EIP 28) or 75 min (EIPs 40 and 29), is maximal at 4-8 hr and continues for almost 2 days. During this period no other major changes in protein synthesis are discernible using one-dimensional gels. At maximum, EIP 28 synthesis is elevated at least 10 fold above its basal level, and EIPs 40 and 29 somewhat less. EIP induction is ecdysteroid-specific and is detectable in the presence of 10(-8) M 20-hydroxyecdysone. It does not occur in hormone-resistant cells. Apparently identical polypeptides are inducible in another ecdysteroid-responsive cell line, Schneider's line 3. Because EIP synthesis is an early and substantial response to ecdysteroids, this is a promising system for the study of steroid hormone action.     

Structural organization of the "zipper line" in Drosophila species with giant spermatozoa

The "zipper line" of Drosophila melanogaster and of Drosophila species characterized by giant spermatozoa (D. hydei, D. kanekoi and D. bifurca) was studied by electron microscopy using conventional thin-sections, lectin labeling and freeze-fracture replicas. In cross sections the membrane specializations are located either at the level of the short cistern close to the large mitochondrial derivative where a small tuft of glycocalyx is visible or, in species characterized by long spermatozoa, along a cistern beneath the plasma membrane. In correspondence of such cistern, the plasma membrane exhibits a thick and extended glycocalyx. At this level, as well as at the short tuft of D. melanogaster, alpha-mannose residues were detected. The "zipper" of D. melanogaster consists of rows of intramembrane particles longitudinally disposed along the sperm tail and associated with the external face of the plasma membrane. On the protoplasmatic face a narrow ribbon of transversal grooves is visible. Freeze-fracture replicas have revealed, in the region characterized by extended glycocalyx, the presence of a large ribbon of intramembrane particles disposed in parallel transversal rows, associated with the protoplasmatic membrane face. On the complementary external face a ribbon of parallel transversal grooves was observed. It is suggested that membrane specializations are mechanical devices to protect spermatozoa from torsion and bending in the seminal vesicles and then in the female storage organ.     

Mobilization of a Hobo-related Sequence in the Genome of Drosophila simulans

The hobo transposable element can occur under three forms in the Drosophila genome: as a complete element (also called canonical), as internally deleted copies, or as hobo-related sequences (relics). Some evidence indicated that canonical elements and internally deleted copies are recent acquisitions of Drosophila genomes, while the “relics” are old components, normally degenerated and immobile. Here we present the characterization of a hobo-related sequence, found in the genome of a hypermutable strain of D. simulans, which insertion into the white locus raised a de novo white mutation. It is a shorter hobo related element presenting, overall, roughly 18% of divergence at the DNA level from the canonical hobo, with many indels that make clear this element is defective. However, its ITRs and flanking regions are extremely conserved. This is the first hobo “relic” showed to be mobilizable. We suggest, and point up some evidences, toward the idea that this sequence could have been mobilized by the canonical element. The presence of a similar “relic” element in D. sechellia allows us to suggest that these elements have been maintained mobilizable since the time of divergence between these species.     

Transgenerational Inheritance of Diet-Induced Genome Rearrangements in Drosophila

Ribosomal RNA gene (rDNA) copy number variation modulates heterochromatin formation and influences the expression of a large fraction of the Drosophila ge-nome. This discovery, along with the link between rDNA, aging, and disease, high-lights the importance of understanding how natural rDNA copy number variation arises. Pursuing the relationship between rDNA expression and stability, we have discovered that increased dietary yeast concentration, emulating periods of dietary excess during life, results in somatic rDNA instability and copy number reduction. Modulation of Insulin/TOR signaling produces similar results, indicating a role for known nutrient sensing signaling pathways in this process. Furthermore, adults fed elevated dietary yeast concentrations produce offspring with fewer rDNA copies demonstrating that these effects also occur in the germline, and are transgenera-tionally heritable. This finding explains one source of natural rDNA copy number variation revealing a clear long-term consequence of diet.     

Effects of ecdysone on a Drosophila cell line

When treated by ecdysone, Drosophila cells of the line Kc start aggregating in clumps 24 h later. Different early events occur before this morphological process: DNA synthesis and mitosis stop after 8–10 h; RNA synthesis after a transient stimulation is progressively inhibited.     

Antimicrobial activity of lactic acid bacteria from sour milk products "Narine", "Karine", and "Matsun"

We studied antimicrobial properties of lactic acid bacteria from sour milk products Narine, Karine, and Matsun. The whey of sour milk products included two major fractions of sugars and L-lactic acid and its sodium and calcium salts. Antimicrobial activity of Narine, Karine, and Matsun was related to the presence of L-lactic acid and its sodium and calcium salts.     

Genome manipulation by homologous recombination in Drosophila.

By utilising a cell's recombinational machinery, researchers in many different model organisms have been able to perform gene targeting experiments in which specific sequence alterations are introduced into virtually any endogenous gene. Not only can functional knock-outs be generated by gene targeting, interesting alleles with mutations encoding specific amino acid replacements can also be made. A practical gene targeting method has only recently become available for Drosophila. This article reviews the Drosophila gene targeting method, with emphases placed on different approaches that are being used to generate different mutations.     

Protochlorophyllide oxidoreductase: "dark" reactions of a light-driven enzyme

The light-driven enzyme NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a key regulatory step in the chlorophyll biosynthesis pathway. As POR is light activated, it allows intermediates in the reaction pathway to be observed by initiating catalysis with illumination at low temperatures, a technique that has recently been used to study the initial photochemistry. Here, we use low-temperature spectroscopy to show that the catalytic mechanism of POR involves two additional steps, which do not require light and have been termed the "dark" reactions. The first of these involves the conversion of the product of the initial light-driven reaction, a nonfluorescent radical species, into a new intermediate that has an absorbance maximum at 681 nm and a fluorescence peak at 684 nm. During the second dark step this species gradually blue shifts to yield the product, Chlide. The temperature dependence for each of these two processes was measured; the data revealed that these steps could only occur close to or above the "glass transition" temperature of proteins, suggesting that domain movements and/or reorganization of the protein are required for these stages of the catalytic mechanism.     

A Toolkit of CRISPR-Based Genome Editing Systems in Drosophila

The last couple of years have witnessed an explosion in development of CRISPR-based genome editing technologies in cell lines as well as in model organisms. In this review, we focus on the applications of this popular system in Drosophila. We discuss the effectiveness of the CRISPR/Cas9 systems in terms of delivery, mutagenesis detection, parameters affecting efficiency, and off-target issues, with an emphasis on how to apply this powerful tool to characterize gene functions.     

Development of orientation selectivity in the primary visual cortex of normally and dark reared kittens

Kinetics of the development of orientation tuning are inferred from quantitative analysis of extracellular recordings in the primary visual cortex of normally and dark reared kittens. 712 visual cells were classified in three functional groups: a) non-specific cells, and b) immature cells which are not as orientation selective as c) specific cells. Power regression and covariance analysis indicate that the "critical period" begins before 19 days and that the kinetics of the immature pool are the same in both rearing conditions. A catenary process of development of orientation selectivity is proposed, the immature compartment being a transit pool between non-specific and specific cells. Two sequential stages occur: 1) the realisation of an intrinsic programme of maturation, by which cortical specificity appears at eye opening and increases independently of visual experience 2) a phase of "epigenesis" beginning at 19 days, during which functional modification depends on visual experience.     

Development of orientation selectivity in the primary visual cortex of normally and dark reared kittens

The development of orientation selectivity in the primary visual cortex is described by first-order kinetics between three functional compartments chained in a catenary mode. A first model is presented, in which two unidirectional kinetics with constant exchange coefficients, symmetrical in their effects, function in an alternating mode depending on the presence or absence of visual experience. The failure of this model to simulate the modifications induced by a delayed visual experience, when the exchange coefficients are identified to fit normal and dark rearing, supports the hypothesis that the maturation process consequent to interaction with visual environment is dependent on the date at which it is allowed to take place. A second model is then proposed, in which exchange coefficients are piecewise linear functions of time. In order to correctly predict the functional effects of restricted visual experience following prior dark rearing, it is assumed that visuomotor experience during the critical period permits the expression of a non-linear modifiability gradient which may have been masked up to this point by the absence of vision or eye movements.