Euchromatic and heterochromatic domains at Drosophila telomeres.

Noncoding repetitive sequences make up a large portion of eukaryotic genomes, but their function is not well understood. Large blocks of repetitive DNA-forming heterochromatin around the centromeres are required for this region to function properly, but are difficult to analyze. The smaller regions of heterochromatin at the telomeres provide an opportunity to study their DNA and protein composition. Drosophila telomere length is maintained through the targeted transposition of specific non-long terminal repeat retrotransposons to chromosome ends, where they form long tandem arrays. A subterminal telomere-associated sequence (TAS) lies immediately proximal to the terminal-retrotransposon array. Here, we review the experimental support for the heterochromatic features of Drosophila telomeres, and provide evidence that telomeric regions contain 2 distinct chromatin subdomains: TAS, which exhibits features that resemble beta heterochromatin; and the terminal array of retrotransposons, which appears euchromatic. This organization is significantly different from the telomeric organization of other eukaryotes, where the terminal telomerase-generated repeats are often folded in a t-loop structure and become part of the heterochromatin protein complex.     

Impact of Multiple Insertions of two Retroelements, ZAM and Idefix at an Euchromatic Locus

Transposable elements represent a large fraction of eukaryotic genomes and they are thought to play an important role in chromatin structure. ZAMand Idefixare two LTR-retrotransposons from Drosophila melanogastervery similar in structure to vertebrate retroviruses. In all the strains where their distribution has been studied, ZAMappears to be present exclusively in the intercalary heterochromatin while Idefixcopies are mainly found in the centromeric heterochromatin with very few copies in euchromatin. Their distribution varies in a specific strain called RevI in which the mobilization of ZAMand Idefixis highly induced. In this strain, 15 copies of ZAMand 30 copies of Idefixare found on the chromosomal arms in addition to their usual distribution. Amongst the loci where new copies are detected, a hotspot for their insertion has been detected at the whitelocus where up to four elements occurred within a 3-kb fragment at the 5′ end of this gene. This property of ZAMand Idefixto accumulate at a defined site provides an interesting paradigm to bring insight into the effect exerted by multiple insertions of transposable elements at an euchromatic locus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transposon Defense by Endo-siRNAs,piRNAs and Somatic pilRNAs in Drosophila: Contributions of Loqs-PD and R2D2

Transposable elements are a serious threat for genome integrity and their control via small RNA mediated silencing pathways is an ancient strategy. The fruit fly Drosophila melanogaster has two silencing activities that target transposons: endogenous siRNAs (esiRNAs or endo-siRNAs) and Piwi-interacting small RNAs (piRNAs). The biogenesis of endo-siRNAs involves the Dicer-2 co-factors Loqs-PD, which acts predominantly during processing of dsRNA by Dcr-2, and R2D2, which primarily helps to direct siRNAs into the RNA interference effector Ago2. Nonetheless, loss of either protein is not sufficient to produce a phenotype comparable with a dcr-2 mutation. We provide further deep sequencing evidence supporting the notion that R2D2 and Loqs-PD have partially overlapping function. Certain transposons display a preference for either dsRBD-protein during production or loading; this appeared to correlate neither with overall abundance, classification of the transposon or a specific site of genomic origin. The endo-siRNA biogenesis pathway in germline operates according to the same principles as the existing model for the soma, and its impairment does not significantly affect piRNAs. Expanding the analysis, we confirmed the occurrence of somatic piRNA-like RNAs (pilRNAs) that show a ping-pong signature. We detected expression of the Piwi-family protein mRNAs only barely above background, indicating that the somatic pilRNAs may arise from a small sub-population of somatic cells that express a functional piRNA pathway.     

Enhancement and Suppression of a Euchromatic Position Effect at NOTCH in Drosophila

The recessive visible rough-eye mutant facet-strawberry, fa^swb, is caused by the deletion of 0.8 kb of base sequences from the 5' end of the Notch locus. Visible deficiencies adjacent to fa^swb suppress this mutant effect of the Notch locus, and in the same region (between salivary bands 3C1 and 3C7), we have demonstrated the presence of at least one partial suppressor and one enhancer of the fa^swb position effect at Notch.—The enhancer seems to be a small inversion approximately equal to the salivary-band doublet 3C2, 3, and the partial suppressor lies between the inversion in 3C2, 3 and the small deletion in fa^swb immediately distal to 3C7. Neither the enhancer, e(fa^swb), nor the partial suppressor, su(fa^swb), can be detected except when linked in cis to fa^swb. The e(fa^swb) and the su(fa^swb), in unison, act antagonistically on the fa^swb position effect.—The fa^swb mutant is interpreted to be a nonvariegating position effect at the Notch locus resulting from a novel euchromatic—euchromatic association of base sequences caused by the small deletion.     

Drosophila Transposon Insertions as Unknowns for Structured Inquiry Recombination Mapping Exercises in an Undergraduate Genetics Course

Structured inquiry approaches, in which students receive a Drosophila strain of unknown genotype to analyze and map the constituent mutations, are a common feature of many genetics teaching laboratories. The required crosses frustrate many students because they are aware that they are participating in a fundamentally trivial exercise, as the map locations of the genes are already established and have been recalculated thousands of times by generations of students. We modified the traditional structured inquiry approach to include a novel research experience for the students in our undergraduate genetics laboratories. Students conducted crosses with Drosophila strains carrying P[lacW] transposon insertions in genes without documented recombination map positions, representing a large number of unique, but equivalent genetic unknowns. Using the eye color phenotypes associated with the inserts as visible markers, it is straightforward to calculate recombination map positions for the interrupted loci. Collectively, our students mapped 95 genetic loci on chromosomes 2 and 3. In most cases, the calculated 95% confidence interval for meiotic map location overlapped with the predicted map position based on cytology. The research experience evoked positive student responses and helped students better understand the nature of scientific research for little additional cost or instructor effort.INQUIRY-BASED learning in which students are engaged in open-ended, student-centered, hands-on activities is an important tool for training undergraduates to think like scientists (Colburn 2000; Handelsman et al. 2004). With this approach, students learn scientific subjects by interpreting and discussing experimental results in a fashion similar to that used by scientific researchers (NRC 2003). There are three main approaches to instruction via inquiry. In open inquiry, students formulate their own problem, as well as the procedures to investigate the problem. In guided inquiry, the instructor provides the problem and necessary materials, but the students devise an experimental procedure to investigate the problem. Finally, in structured inquiry, the instructor provides the problem, the materials, and the procedure, but the students are required to gather and interpret the experimental data independently, coming to their own conclusions (Welch et al. 2006). In each case, the instructor does not provide “the answer” to the problem. In the ideal case, the instructor does not even know what the answer will be prior to the student experiment, forcing the students to grapple with the information themselves. Inquiry-based laboratories can even be extended so that students are participating in novel research as part of their coursework (DebBurman 2002; Buckner et al. 2007), which been shown to improve undergraduate retention and student performance in biology lecture courses (Marcus et al. 2009).The process of inquiry has been identified as central to training students to understand fundamental approaches used in the field of genetics such as the design of controlled crosses and interpretation of experimental data (Cartier and Stewart 2000). Pukkila (2004) discusses effective methods by which inquiry-based learning can be incorporated into undergraduate genetics lecture courses with large enrollments and into recitation sections. However, the implementation of inquiry-based approaches in undergraduate genetics laboratories has not been discussed extensively.Teaching laboratories offer some advantages for inquiry learning because they generally contain small groups of students, facilitating a flexible and intimate learning environment with many interactions between students and the instructor, as well as among classmates. However, teaching laboratories associated with large lecture courses also offer some challenges, in particular how to deliver substantially similar experiences to laboratory sections taught by multiple instructors, as well as how to provide inquiry-based learning in a logistically manageable and cost-effective manner. For these reasons, most inquiry-based genetics laboratory exercises have used the structured inquiry approach, for example, using many Drosophila melanogaster strains with similar mutant phenotypes (e.g., white eyes and black bodies), but a variety of genotypes, in a series of standardized genetic mapping crosses to familiarize students with the collection and interpretation of classical genetic data (MacIntyre 1974; Pye 1980). The difficulty with contrived genetic unknowns carrying well-mapped genetic mutations is that many students become frustrated that their hard work evaluating the crosses over a period of several months is devoted to a fundamentally trivial exercise, as the recombination map locations of the genes are already established in the scientific literature and have been recalculated thousands of times by generations of genetics students.We have expanded upon the structured inquiry approach to genetics to include novel research experiences for the students in our undergraduate genetics laboratories. They conduct mapping crosses with Drosophila strains carrying P-element transposon insertions in genes without documented recombination map positions. The stock centers maintain very large collections of P-element transposon stocks with known insertion sites on the cytological and genome maps (Spradling et al. 1999). However, in spite of the cytology to recombination map equivalence table available in FlyBase (2009), very few of the transposon inserts have been formally placed on the recombination map. By using the eye color phenotypes associated with many transposon inserts as visible markers in genetic crosses (Marcus 2003), it is straightforward to calculate recombination map positions for the interrupted loci. The stock collections contain many stocks with identical transposons inserted at different chromosomal locations, providing a large number of unique, but equivalent genetic unknowns that can be used for recombination mapping exercises. At the same time, this approach provides students with the opportunity to map genes that have never been mapped before, allowing them to make small but useful contributions to the field of Drosophila genetics.     

P Element Insertions and Rearrangements at the Singed Locus of Drosophila Melanogaster

DNA from the singed gene of Drosophila melanogaster was isolated using an inversion between a previously cloned P element at cytological location 17C and the hypermutable allele singed-weak. Five out of nine singed mutants examined have alterations in their DNA maps in this region. The singed locus is a hotspot for mutation during P-M hybrid dysgenesis, and we have analyzed 22 mutations induced by P-M hybrid dysgenesis. All 22 have a P element inserted within a 700-bp region. The precise positions of 10 P element insertions were determined and they define 4 sites within a 100-bp interval. During P-M hybrid dysgenesis, the singed-weak allele is destabilized, producing two classes of phenotypically altered derivatives at high frequency. In singed-weak, two defective P elements are present in a "head-to-head" or inverse tandem arrangement. Excision of one element results in a more extreme singed bristle phenotype while excision of the other leads to a wild-type bristle phenotype.     

Insertions of the Transposon Tn1 into the PSEUDOMONAS AERUGINOSA Chromosome

The transposon Tn1 has been translocated to the chromosome of Pseudomonas aeruginosa from plasmid R18, following hydroxylamine mutagenesis of the plasmid. Twelve insertions were mapped to six distinct sites distal to 55 min of the origin of chromosome transfer by the plasmid FP2. These map locations were confirmed by host chromosome mobilization tests mediated by plasmids R18 or R91-5, due to Tn1 homology between plasmid and host chromosome. All the Tn1 chromosomal inserts were retransposable to other plasmids (Sa, R931 and R38). The behavior of Tn1 in P. aeruginosa was very similar to its behavior in Escherichia coli with respect to regional specificity, orientation of insertion and in serving as regions of homology for host chromosome mobilization by plasmids. This last property has permitted the demonstration that Tn1 on R18 and R91-5 is in opposite orientation with respect to the origin of transfer (oriT) of the two plasmids.     

Recombination in the Vicinity of Insertions of Transposon Tn 5 in MYXOCOCCUS XANTHUS

To test genetic recombination in the vicinity of insertions of the transposon Tn5, crosses were performed by transduction between M. xanthus strains carrying different insertions of Tn5. One member of each pair carried resistance to kanamycin (Tn5-Km); the other carried resistance to tetracycline (Tn5-Tc). The distance between each pair of Tn5 insertions was also measured by restriction mapping. The physical distance corresponding to each recombination frequency was calculated from the transductional linkage and compared with distance on the restriction map. A good correspondence between the two measures of distance was obtained for a pair of Tn5 insertions near the cglB locus and for another pair near the mgl locus. Correspondence between the two measurements of distance, the observed allelic behavior of Tn5-Km and Tn5 -Tc at the same locus and the finding of the same frequencies of recombinants in reciprocal crosses implied that recombination in the vicinity of Tn 5 was normal.     

Insertions of a Novel Class of Transposable Elements with a Strong Target Site Preference at the R Locus of Maize

The r locus of maize regulates anthocyanin synthesis in various tissues of maize through the production of helix-loop-helix DNA binding proteins capable of inducing expression of structural genes in the anthocyanin biosynthetic pathway. The complex r variant, R-r:standard (R-r), undergoes frequent mutation through a variety of mechanisms including displaced synapsis and crossing over, and intrachromosomal recombination. Here we report a new mechanism for mutation at the R-r complex: insertion of a novel family of transposable elements. Because the elements were first identified in the R-p gene of the R-r complex, they have been named P Instability Factor (PIF). Two different PIF elements were cloned and found to have identical sequences at their termini but divergent internal sequences. In addition, the PIF elements showed a marked specificity of insertion sites. Six out of seven PIF-containing derivatives examined had an element inserted at an identical location. Two different members of the PIF element family were identified at this position. The seventh PIF-containing derivative examined had the element inserted at a distinct position within r. Even at this location, however, the element inserted into a conserved target sequence. The timing of PIF excision is unusual. Germinal excision rates can range up to several percent of progeny. Yet somatic sectors are rare, even in lines exhibiting high germinal reversion rates.     

Studying Patterns of Recent Evolution at Synonymous Sites and Intronic Sites in Drosophila melanogaster

Most previous studies of the evolution of codon usage bias (CUB) and intronic GC content (iGC) in Drosophila melanogaster were based on between-species comparisons, reflecting long-term evolutionary events. However, a complete picture of the evolution of CUB and iGC cannot be drawn without knowledge of their more recent evolutionary history. Here, we used a polymorphism dataset collected from Zimbabwe to study patterns of the recent evolution of CUB and iGC. Analyzing coding and intronic data jointly with a model which can simultaneously estimate selection, mutational, and demographic parameters, we have found that: (1) natural selection is probably acting on synonymous codons; (2) a constant population size model seems to be sufficient to explain most of the observed synonymous polymorphism patterns; (3) GC is favored over AT in introns. In agreement with the long-term evolutionary patterns, ongoing selection acting on X-linked synonymous codons is stronger than that acting on autosomal codons. The selective differences between preferred and unpreferred codons tend to be greater than the differences between GC and AT in introns, suggesting that natural selection, not just biased gene conversion, may have influenced the evolution of CUB. Interestingly, evidence for non-equilibrium evolution comes exclusively from the intronic data. However, three different models, an equilibrium model with two classes of selected sites and two non-equilibrium models with changes in either population size or mutational parameters, fit the intronic data equally well. These results show that using inadequate selection (or demographic) models can result in incorrect estimates of demographic (or selection) parameters.     

Cis-Effects of Heterochromatin on Heterochromatic and Euchromatic Gene Activity in Drosophila Melanogaster

Chromosomal rearrangements that juxtapose heterochromatin and euchromatin can result in mosaic inactivation of heterochromatic and euchromatic genes. This phenomenon, position effect variegation (PEV), suggests that heterochromatic and euchromatic genes differ in their regulatory requirements. This report describes a novel method for mapping regions required for heterochromatic genes, and those that induce PEV of a euchromatic gene. P transposase mutagenesis was used to generate derivatives of a translocation that variegated for the light(+) (lt(+)) gene and carried the euchromatic white(+) (w(+)) gene on a transposon near the heterochromatin-euchromatin junction. Cytogenetic and genetic analyses of the derivatives showed that P mutagenesis resulted in deletions of several megabases of heterochromatin. Genetic and molecular studies showed that the derivatives shared a euchromatic breakpoint but differed in their heterochromatic breakpoint and their effects on seven heterochromatic genes and the w(+) gene. Heterochromatic genes differed in their response to deletions. The lt(+) gene was sensitive to the amount of heterochromatin at the breakpoint but the heterochromatic 40Fa gene was not. The severity of variegated w(+) phenotype did not depend on the amount of heterochromatin in cis, but varied with local heterochromatic environment. These data are relevant for considering mechanisms of PEV of both heterochromatic and euchromatic genes.     

Insertions and deletions trigger adaptive walks in Drosophila proteins

Maps that relate all possible genotypes or phenotypes to fitness--fitness landscapes--are central to the evolution of life, but remain poorly known. An insertion or a deletion (indel) of one or several amino acids constitutes a substantial leap of a protein within the space of amino acid sequences, and it is unlikely that after such a leap the new sequence corresponds precisely to a fitness peak. Thus, one can expect an indel in the protein-coding sequence that gets fixed in a population to be followed by some number of adaptive amino acid substitutions, which move the new sequence towards a nearby fitness peak. Here, we study substitutions that occur after a frame-preserving indel in evolving proteins of Drosophila. An insertion triggers 1.03 ± 0.75 amino acid substitutions within the protein region centred at the site of insertion, and a deletion triggers 4.77 ± 1.03 substitutions within such a region. The difference between these values is probably owing to a higher fraction of effectively neutral insertions. Almost all of the triggered amino acid substitutions can be attributed to positive selection, and most of them occur relatively soon after the triggering indel and take place upstream of its site. A high fraction of substitutions that follow an indel occur at previously conserved sites, suggesting that an indel substantially changes selection that shapes the protein region around it. Thus, an indel is often followed by an adaptive walk of length that is in agreement with the theory of molecular adaptation.     

Selection and Maintenance of Sexual Identity in the Drosophila Germline

Unlike sex determination in the soma, which is an autonomous process, sex determination in the germline of Drosophila has both inductive and autonomous components. In this paper, we examined how sexual identity is selected and maintained in the Drosophila germline. We show that female-specific expression of genes in the germline is dependent on a somatic signaling pathway. This signaling pathway requires the sex-non-specific transformer 2 gene but, surprisingly, does not appear to require the sex-specific genes, transformer and doublesex. Moreover, in contrast to the soma where pathway initiation and maintenance are independent processes, the somatic signaling pathway appears to function continuously from embryogenesis to the larval stages to select and sustain female germline identity. We also show that the primary target for the somatic signaling pathway in germ cells can not be the Sex-lethal gene.     

Association of the Production of Phenylpropanoid Compounds at the Infection Sites of Corynespora cassiicola with Soybean Resistance against Target Spot

This study investigated, at the microscopic level, whether the differential defence responses of soybean cultivars that are resistant (Fundacep 59) and susceptible (TMG 132) to target spot, caused by Corynespora cassiicola, could be associated with an increase in the production of phenolics, flavonoids and lignin at the infection sites. Many larger necrotic lesions with yellow halos were noticed on the leaves of plants from cultivar TMG 132, in contrast to the leaves of plants from cultivar Fundacep 59. Necrotic lesions also developed on the petioles of leaves of plants from cultivar TMG 132, while on the petioles and veins of leaves of plants from cultivar Fundacep 59, the lesions were of purple colour. The growth of fungal hyphae was reduced on the leaves of plants from cultivar Fundacep 59, and an apparently high density of trichomes was found in comparison with the leaves of plants from cultivar TMG 132. An appressorium‐like structure was produced at one or both extremities of the conidium of C. cassiicola, preferentially at the major and minor veins on the adaxial leaf surface of plants from both cultivars. Most cells on the leaves of plants from cultivar Fundacep 59 reacted against C. cassiicola infection by accumulating phenolic‐like compounds, which contributed to the death of many fungal hyphae and a greater maintenance of cell integrity. In contrast, fungal hyphae grew without any impedance in the leaf cells of plants from cultivar TMG 132, which was associated with signs of intense leaf tissue disorganization. Stronger autofluorescence and deposition of lignin and flavonoids were found in the cells of leaves of plants from cultivar Fundacep 59, in contrast to cultivar TMG 132. It can be concluded that soybean resistance to target spot is probably dependent on the activation of the phenylpropanoid pathway.     

Obervations on the Induction of Position Effect Variegation of Euchromatic Genes in Drosophila Melanogaster

In the T(1;2)dor(var7) translocation, the 1A-2B7-8 segment of the X chromosome is brought to the vicinity of 2R-chromosome heterochromatin resulting in position effect variegation of dor, BR-C and more distal genes, as well as compaction of chromatin in this segment. By irradiation of T(1;2)dor(var7), nine reversions (rev) to a normal phenotype were recovered. In two cases (rev27, rev226), the 1A-2B7-8 section is relocated to the 19A region of the X chromosome, forming free duplications (1A-2B7-8/19A-20F-X-het). Modifiers of position effect do not change the normal expression of the BR-C and dor genes in these duplications. In five reversions (rev3, rev40, rev60, rev167, rev175), free duplications have formed from the 1A-2B7-8 fragment and X chromosome heterochromatin. In these rearrangements, modifiers of position effect (low temperature, removal of Y and 2R-chromosome heterochromatin and a genetic enhancer (E-var(3)201) induce position-effect again. Two reversions (rev45 and rev110) are associated with additional inversions in the original dor(var7) chromosomes. The inversions relocate part of the heterochromatin adjacent to the 1A-2B7-8 section into new positions. In T(1;2)dor(rev45), position-effect is seen in the 2B7-8-7A element as compaction spreading from 2B7-8 proximally in some cases as far as the 5D region. Thus, in rev45 the pattern of euchromatin compaction is reciprocal to that of the initial dor(var7) strain. Apparently, it is due to the same variegation-evoking center near the 2R centromere in both cases. In all nine revertants, weakening or complete disappearance of the position-effect is observed despite retention of the 20- kb heterochromatic segment adjacent to the 1A-2B7-8 region. Thus, a 20-kb heterochromatic sequence does not inactivate euchromatin joined to it.