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Obviously there is no 100% exact number, but I came across this on flybase, the gold standard for annotation. I am confused now. "Genes located to the genome", is that what I am looking for? If so, what does "Genes not located to the genome" mean?
Personal correspondence to representative at FlyBase.org
In FlyBase we have been gathering information about genes and phenotypes for over 20 years including information from papers and resources older than that. We annotate gene models on the Drosophila melanogaster genome assembly and when possible associate those annotations with other information (eg. phenotype, function etc) that is known about the gene. However, in some cases, especially for genes and phenotypes that were described in the older literature before the genome was sequenced we still have some information about those genes but have been unable to associate that information with a genome annotation. Those are what we call unlocalized genes (i.e. those without a annotation on the genome).
Drosophila melanogaster as a Model System to Study Mitochondrial Biology
Mitochondria play an essential role in cellular homeostasis. Although in the last few decades our knowledge of mitochondria has increased substantially, the mechanisms involved in the control of mitochondrial biogenesis remain largely unknown. The powerful genetics of Drosophila combined with a wealth of available cell and molecular biology techniques, make this organism an excellent system to study mitochondria. In this chapter we will review briefly the opportunities that Drosophila offers as a model system and describe in detail how to purify mitochondria from Drosophila and to perform the analysis of developmental gene expression using in situ hybridization.
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By Mark D. Adams , Susan E. Celniker , Robert A. Holt , Cheryl A. Evans , Jeannine D. Gocayne , Peter G. Amanatides , Steven E. Scherer , Peter W. Li , Roger A. Hoskins , Richard F. Galle , Reed A. George , Suzanna E. Lewis , Stephen Richards , Michael Ashburner , Scott N. Henderson , Granger G. Sutton , Jennifer R. Wortman , Mark D. Yandell , Qing Zhang , Lin X. Chen , Rhonda C. Brandon , Yu-Hui C. Rogers , Robert G. Blazej , Mark Champe , Barret D. Pfeiffer , Kenneth H. Wan , Clare Doyle , Evan G. Baxter , Gregg Helt , Catherine R. Nelson , George L. Gabor , Miklos , Josep F. Abril , Anna Agbayani , Hui-Jin An , Cynthia Andrews-Pfannkoch , Danita Baldwin , Richard M. Ballew , Anand Basu , James Baxendale , Leyla Bayraktaroglu , Ellen M. Beasley , Karen Y. Beeson , P. V. Benos , Benjamin P. Berman , Deepali Bhandari , Slava Bolshakov , Dana Borkova , Michael R. Botchan , John Bouck , Peter Brokstein , Phillipe Brottier , Kenneth C. Burtis , Dana A. Busam , Heather Butler , Edouard Cadieu , Angela Center , Ishwar Chandra , J. Michael Cherry , Simon Cawley , Carl Dahlke , Lionel B. Davenport , Peter Davies , Beatriz de Pablos , Arthur Delcher , Zuoming Deng , Anne Deslattes Mays , Ian Dew , Suzanne M. Dietz , Kristina Dodson , Lisa E. Doup , Michael Downes , Shannon Dugan-Rocha , Boris C. Dunkov , Patrick Dunn , Kenneth J. Durbin , Carlos C. Evangelista , Concepcion Ferraz , Steven Ferriera , Wolfgang Fleischmann , Carl Fosler , Andrei E. Gabrielian , Neha S. Garg , William M. Gelbart , Ken Glasser , Anna Glodek , Fangcheng Gong , J. Harley Gorrell , Zhiping Gu , Ping Guan , Michael Harris , Nomi L. Harris , Damon Harvey , Thomas J. Heiman , Judith R. Hernandez , Jarrett Houck , Damon Hostin , Kathryn A. Houston , Timothy J. Howland , Ming-Hui Wei , Chinyere Ibegwam , Mena Jalali , Francis Kalush , Gary H. Karpen , Zhaoxi Ke , James A. Kennison , Karen A. Ketchum , Bruce E. Kimmel , Chinnappa D. Kodira , Cheryl Kraft , Saul Kravitz , David Kulp , Zhongwu Lai , Paul Lasko , Yiding Lei , Alexander A. Levitsky , Jiayin Li , Zhenya Li , Yong Liang , Xiaoying Lin , Xiangjun Liu , Bettina Mattei , Tina C. McIntosh , Michael P. McLeod , Duncan McPherson , Gennady Merkulov , Natalia V. Milshina , Clark Mobarry , Joe Morris , Ali Moshrefi , Stephen M. Mount , Mee Moy , Brian Murphy , Lee Murphy , Donna M. Muzny , David L. Nelson , David R. Nelson , Keith A. Nelson , Katherine Nixon , Deborah R. Nusskern , Joanne M. Pacleb , Michael Palazzolo , Gjange S. Pittman , Sue Pan , John Pollard , Vinita Puri , Martin G. Reese , Knut Reinert , Karin Remington , Robert D. C. Saunders , Frederick Scheeler , Hua Shen , Bixiang Christopher Shue , Inga Sidén-Kiamos , Michael Simpson , Marian P. Skupski , Tom Smith , Eugene Spier , Allan C. Spradling , Mark Stapleton , Renee Strong , Eric Sun , Robert Svirskas , Cyndee Tector , Russell Turner , Eli Venter , Aihui H. Wang , Xin Wang , Zhen-Yuan Wang , David A. Wassarman , George M. Weinstock , Jean Weissenbach , Sherita M. Williams , Trevor Woodage , Kim C. Worley , David Wu , Song Yang , Q. Alison Yao , Jane Ye , Ru-Fang Yeh , Jayshree S. Zaveri , Ming Zhan , Guangren Zhang , Qi Zhao , Liansheng Zheng , Xiangqun H. Zheng , Fei N. Zhong , Wenyan Zhong , Xiaojun Zhou , Shiaoping Zhu , Xiaohong Zhu , Hamilton O. Smith , Richard A. Gibbs , Eugene W. Myers , Gerald M. Rubin , J. Craig Venter
Immunofluorescent staining indicates that the D. virilis dot chromosome is largely euchromatic, in contrast to the heterochromatic D. melanogasterdot chromosome
The dot chromosome of D. melanogaster is largely heterochromatic, with some interspersed domains of euchromatin . Immunofluorescent staining of D. melanogaster polytene chromosomes using HP1 antibody shows a banded pattern on the dot chromosome. Many species in the Drosophila genus closely related to D. melanogaster share this staining pattern, including D. simulans, D. yakuba, and D. pseudoobscura (data not shown). In D. melanogaster, staining with an antibody against histone H3 methylated at lysine 9 (anti-H3K9me) coincides with the HP1 staining, at a level slightly less than seen in the pericentric heterochromatin  (Figure 1a). In contrast, the dot chromosome of D. virilis does not stain with either anti-HP1 or anti-H3K9me (Figure 1b), supporting the inference that the banded portion of the dot chromosome of D. virilis is generally euchromatic.
Identification of fosmids from the dot chromosome of D. virilis
The chromosomes of D. virilis tend to map to corresponding portions of the chromosomes of D. melanogaster . We compared the recently posted genomic sequence for D. pseudoobscura [37, 40] with the D. melanogaster dot chromosome genes to look for regions of sufficient sequence similarity to act as conserved hybridization probes. The desired probes (see Materials and methods) were radiolabeled and used to screen a D. virilis genomic library (BDVIF01 fosmids, Tucson strain 15010-1001.10, available spotted on a single filter) at low stringency. Positive clones were verified and characterized by in situ hybridizations to the polytene chromosomes from third instar larval salivary glands of D. virilis. Sample results are shown in Figure 2. Eleven fosmids were recovered with homology to the dot chromosome of D. virilis, and seven fosmids were recovered with homology to the major chromosome arms. Based on the in situ hybridization results, the order of the fosmid clones on the dot chromosome is as follows: contigs 30, 103, and 106 appear to cluster near the centromere contigs 67, 72, and 91 are in the middle of the chromosome and contigs 50 and 113 hybridize near the telomere. There is also a minor signal with the contig 30 probe near the telomere this may be the result of a repetitive element present in multiple regions in the chromosome.
In situ hybridizations of fosmids to D. virilis polytene chromosomes. Fosmid DNA was labeled and used for in situ hybridization on denatured polytene chromosomes from D. virilis. Three examples are shown (left to right: contigs 106, 72, 113) demonstrating hybridization to a specific band on the dot chromosome (arrowhead). In some cases, signal is associated with the chromocenter, presumably due to repetitive sequences shared with the band on the dot. In situ hybridizations were performed with at least one fosmid from every contig from the dot chromosome with similar results (data not shown). See Table 1 for the chromosome locations of the other fosmids.
Fosmid sequencing and annotation
The 18 fosmids recovered from the screen were sequenced in collaboration with the Genome Sequencing Center at Washington University School of Medicine. Plasmid subclone libraries were prepared and approximately 600 subclones from each fosmid were end sequenced. The sequences were assembled and finished to high quality by Washington University undergraduate students in the Bio 4342 'Research Explorations in Genomics' course, using phred, phrap, and consed [41–43]. Finished sequences had an estimated error rate of less than 0.01%, and showed in silico restriction digests that matched digests obtained from the starting fosmid with a minimum of two enzymes. Students annotated the finished sequences by looking for genes, repetitive elements, and other features as described in Materials and methods. Four pairs of fosmids have significant sequence overlap each pair was collapsed into a single contig of non-redundant sequence (contigs 30, 50, 67, and 80).
Initial annotation focused on gene finding. D. virilis is evolutionarily close enough to D. melanogaster that the protein coding regions are well conserved. Gene prediction algorithms and local alignment search tools (such as GENSCAN and BLAST see Materials and methods) were used to annotate genes and determine intron-exon boundaries. In most cases, it was possible to identify the entire coding region of the gene, but the high level of sequence divergence made defining untranslated regions impossible . Comparison of the D. virilis contigs with homologous regions of the D. melanogaster dot chromosome identified specific regions where synteny has been maintained, as well as those regions where inversions have occurred. Figure 3 shows a comparison of two D. virilis contigs with the homologous regions from the D. melanogaster chromosomes. Detailed annotation results and comparisons between the other individual D. virilis fosmids and their homologous regions in D. melanogaster are available as Additional data file 1 (dot chromosome sequences) and Additional data file 2 (non-dot chromosome sequences). Note that the strain of D. virilis used here is a different strain from that recently sequenced (by Agencourt Bioscience Corporation, Beverly, MA, USA). The two strains differ by about 1% base substitutions, with numerous insertions or deletions (indels), but show similar organization at the gene level (CDS, unpublished observation). The clone-based sequencing used here results in more accurate inferences in regions that are highly repetitive the sequences most likely to be missed in whole genome shotgun techniques are the repeats .
Map for two sample contigs from D. virilis (Dv) in comparison with homologous regions of the D. melanogaster (Dm) genome. Shown are two contigs from D. virilis with the corresponding regions from D. melanogaster. Coding sequences (dark blue boxes) are indicated above each diagram. In the case of D. melanogaster, the thick dark blue bar indicates open reading frames (ORFs), and the thin aqua bar indicates UTRs only ORFs are identified for D. virilis. Repeat sequences are shown below: red boxes are DNA transposon fragments, while other repetitive elements are represented as yellow boxes. (a) Contig 112 represents a clone from one of the large chromosomes of D. virilis. While the orientations of Egfr and CG10440 are the same with respect to each other, there is a large tandem repeat between the two genes in D. virilis, but not in D. melanogaster. (b) Contig 67 represents a clone from the dot chromosome of D. virilis. The structure of the genomic region is similar to the corresponding region in D. melanogaster, but there is more intergenic space in D. virilis, whereas in D. melanogaster, there are more transposable elements in the introns. All of the fosmids described here with homologous regions in D. melanogaster have been annotated in a similar manner the maps are available in the Additional data files. Scale: one division equals 5 kb.
Table 1 shows all contigs sequenced, giving their total sizes, listing annotated genes, and providing clone names (BACPAC Center). In situ hybridization results identified the fosmids as either on the dot chromosome or on a major D. virilis chromosome. In parentheses following each gene is the chromosome position of the gene in the genome of D. melanogaster. Figure 4 maps the contigs from the dot chromosome of D. virilis to the dot chromosome of D. melanogaster based on the presence of orthologous genes. Three of the contigs (67, 106, and 113) are completely syntenic with respect to the D. melanogaster dot chromosome. One contig, 103, is completely syntenic with respect to its genes from the dot chromosome, but also contains CG5367, a gene from the second chromosome of D. melanogaster. Four contigs (30, 72, 50, and 91) contain genes that are exclusively from the dot chromosome of D. melanogaster but show evidence of a high number of inversions with respect to the D. melanogaster chromosome. For example, contig 30 contains both pan and Caps, genes that come from opposite sides of the banded portion of the D. melanogaster dot chromosome. (This rearrangement was also observed in earlier studies .) Of the 28 genes identified in the D. virilis dot chromosome clones, only one lies elsewhere in the D. melanogaster genome. In the D. virilis contigs from major chromosomes, four (contigs 13, 112, 121, 122) are completely syntenic compared to homologous gene regions from D. melanogaster, and two (contigs 11 and 80) show inversions within the chromosomes. Only one major chromosome contig (80) contains a gene that is found on the dot chromosome in D. melanogaster. Contig 80 maps to a major arm of D. virilis it contains D. melanogaster dot chromosome gene CG1732 flanked by several genes from D. melanogaster chromosome 3. In total, the fosmids sequenced represent 372,650 bp of sequence from the dot chromosome of D. virilis and 273,110 bp of sequence from the major chromosomes. D. virilis contigs 72 and 91 from the dot chromosome and 11 and 80 from the major arms showed so much rearrangement that it was impossible to define precise homologous area(s) from D. melanogaster. These contigs were not used in comparisons for intron size, percent DNA transcribed, or in any of the repeat density calculations. Maps representing locations and sizes of genes and repeats in each contig are available in Additional data files 1 and 2.
Map of the D. virilis (Dv) dot chromosome contigs in relation to the dot chromosome of D. melanogaster (Dm). Shown at the bottom is a map of the genes on the D. melanogaster dot chromosome. Colored bars with labels represent genes for which we have identified a (complete or partial) homologue in the D. virilis fosmids sequenced. Colored boxes above the scale bar are schematic (not to scale) representations of the D. virilis contigs. Immediately above the scale bar is a representation of those sequenced contigs that contain syntenic regions from D. virilis, where genes are in the same order and orientation as in D. melanogaster. In the uppermost portion of the figure are the contigs mapping to the D. virilis dot chromosome that are rearranged with respect to the D. melanogaster dot chromosome. Boxes are color-coded to represent the genes present in the contig, with dashed lines connecting to show the extent of rearrangement. Notably, contig 30 contains both pan and Caps, which lie on opposite sides of the banded portion of the D. melanogaster dot chromosome.
Average intron size and percent DNA transcribed
While centromeric regions are rich in satellite DNA and relatively gene poor , gene density (defined as the number of genes per Mb) in the banded portion of the dot chromosome is similar to the major chromosomes of D. melanogaster  (66.5 genes/Mb for the dot and 74.6 genes/Mb for the major chromosomes for the regions analyzed here). This is also true for the regions of the D. virilis genome we have sequenced (62.2 genes/Mb for the dot and 67.3 genes/Mb for major chromosomes). Observation of those few heterochromatic genes that have been cloned and sequenced (for example, light ) suggests that these genes may have larger introns on average, and this has been reported for D. melanogaster dot chromosome genes . Average intron size, defined as total intron length divided by total number of introns, is 448 bp (± 126 bp) for our sample from the major D. virilis chromosomes and 405 bp (± 110 bp) for the corresponding regions of D. melanogaster. D. virilis dot chromosome genes in our sample have an average intron length of 890 bp (± 179 bp) in homologous regions of the D. melanogaster genome, it is 859 bp (± 115 bp). Figure 5 shows a graph that compares the intron size cumulative distribution functions of the dot chromosomes with the major chromosomes. Due to the non-normal distribution of intron sizes, the non-parametric Kolmogorov-Smirnov (KS) test is used to evaluate the statistical significance in the pairwise comparisons. The KS test indicates that the difference in the distribution of intron sizes between the two dot chromosomes is not statistically significant (D = 0.1237, p = 0.2816). However, the distribution of intron sizes for the dot chromosomes is significantly different from those for the major chromosomes for both species (D = 0.223, p = 0.0496 and D = 0.245, p = 0.0291 for D. virilis and D. melanogaster, respectively).
Distribution of intron sizes in D. virilis compared to D. melanogaster. Introns from all D. virilis and D. melanogaster genes in the contigs studied were separated into groups based on size. The number on the x axis represents the minimal intron size an intron is counted in that bin if it has that many bases or fewer. The y axis tallies the percent of total introns that fall into that bin. The two dot chromosomes have significantly similar intron size distributions, which differ significantly from those of the major chromosome arms.
Percent DNA transcribed, defined as primary transcript length over total sequence length, is more similar between the homologous chromosomes than between the dot chromosomes and the major chromosomes. (In this instance, 5' and 3' untranslated regions (UTRs) were not scored in calculations of percent DNA transcribed, as these regions could not be identified in the putative D. virilis genes.) The sequenced regions of the D. virilis and comparable regions of the D. melanogaster dot chromosomes have transcript densities of 58.7% and 51.0%, respectively, while transcript densities of the major chromosomes are 22.2% for D. virilis and 25.9% for D. melanogaster. The difference in percent DNA transcribed between the dot and non-dot contigs reflects the larger average size of introns in the dot chromosome genes.
(dC-dA)·(dG-dT) dinucleotide repeat frequency
One marker of euchromatin is the presence of abundant (dC-dA)·(dG-dT) dinucleotide repeats, also known as CA/GT repeats. In situ hybridization shows that these repeats are widely distributed in euchromatin, but that the dot chromosome of D. melanogaster has a much lower density of these repeats . The dot chromosome of D. virilis has a CA/GT repeat frequency similar to its major autosomes, as shown by in situ hybridization . Dinucleotide repeat analysis of the sequences from the D. virilis fosmids in comparison with the homologous regions of the D. melanogaster genome supports the in situ hybridization results. The fosmids from the dot chromosome of D. virilis have CA/GT repeats with an average length of 36 bp and a total density of 0.15%. Regions of the D. melanogaster dot chromosome homologous to these fosmids have only one CA/GT repeat, which is 21 bp long, giving a total CA/GT density of 0.0069%. In the D. virilis clones mapping to major chromosomes, 0.96% of the DNA is made up of CA/GT, with the average repeat being 32 bp long. In homologous regions of the D. melanogaster genome, 0.32% of the DNA is CA/GT, with the average length of dinucleotide regions being 24 bp. Thus, while the D. virilis dot chromosome has a lower level of CA/GT than the major chromosome arms (about six-fold less than D. virilis and about two-fold less than D. melanogaster), it has a approximately 20-fold higher level of this repeat than is found in the dot chromosome of D. melanogaster.
Initial analysis of known repetitive elements in the D. virilis contigs was performed using RepeatMasker . RepBase 8.12 [46, 47] contains previously characterized repeats from the D. virilis species group. As a simple initial approach we searched for de novo repeats by comparing the fosmid sequences to each other, looking for regions of high similarity by BLASTN . Most apparently novel repeated sequences identified by this technique were immediately adjacent to known repeats identified by RepeatMasker and were, therefore, assumed to be unmasked extensions of those repeats. A few novel repeats were identified that were not similar to any other known repetitive element, expressed sequence tag (EST), or protein sequence. Using this simple technique, novel repeats constituted less than 1% of the total repetitive DNA however, given the small size of our dataset (0.65 Mb) it is possible that repetitive elements could be missed.
Figure 6a shows the repeat density of different classes of repetitive elements in the D. virilis contigs and the comparable regions of the D. melanogaster genome using RepeatMasker/RepBase (Drosophila default parameters) plus this simple de novo BLASTN technique. While there is some variation in repeat density between the contigs of a given region (dot chromosome or major chromosome), the totals appear to represent an average value of the contigs studied. Using this analysis, the overall repeat density of the D. virilis dot chromosome contigs is 14.6% the average of the individual repeat densities is 15.4% ± 7.9%. The overall repeat density of the homologous D. melanogaster regions is 25.3% the average of the individual repeat densities is 24.7% ± 5.4%. Fosmids from the dot chromosome of D. melanogaster show a consistently higher density of DNA transposons and DINE-1 elements than do the fosmids from the dot chromosome of D. virilis. Comparison of the sample from the dot chromosome of D. melanogaster analyzed here to the entire banded portion of the dot chromosome (using RepeatMasker and RepBase 8.12) shows very similar results (Figure 6a). In contrast, the euchromatic arms of the large chromosomes of D. melanogaster and D. virilis have similar repeat densities, with approximately 6% of the sequence classified as repetitive. (Quesneville et al.  estimate the total repeat density of D. melanogaster to be 5.3%.) Other repeat types differed between the two species as well. In our sample from these chromosome arms, D. virilis has more simple repeats and D. melanogaster has more retroelements. Overall, these results suggest that both the higher repeat density and the overrepresentation of DNA transposons contribute to heterochromatin formation on the D. melanogaster dot chromosome. However, because D. virilis is not as well studied as D. melanogaster, it is possible that this approach misses some uncharacterized repeats. To address this issue, we undertook several different strategies.
Repeat analysis of D. virilis contigs compared to the D. melanogaster genome. The repeat density, defined as the percentage of total sequence (in base-pairs) that has been annotated as repetitive has been calculated using the D. virilis fosmid sequence obtained in this study and homologous regions from D. melanogaster (see Materials and methods). D. melanogaster and D. virilis have a very similar low repeat density on the major chromosome arms, and a similar but much higher repeat density on the dot chromosomes. (a) Percent repeat for each type identified by RepeatMasker using RebBase 8.12 with additional repeats identified in a BLASTN all-by-all comparison of the fosmid sequences presented here. (b) Percent repeat for each type identified by RepeatMasker using the Superlibrary (see text for description). The dot chromosome of D. melanogaster has about three times more DNA transposon sequence than does the D. virilis dot chromosome. 'Unknown' repeats are those from both RebBase 8.12 and the D. virilis PILER-DF library that have not been classified as to type.
Recent investigations have developed multiple search tools for de novo identification of novel repetitive sequences in genome assemblies [50, 51]. Using such tools, we created a 'Superlibrary' in which we added sequences from species-specific libraries from both D. melanogaster and D. virilis to the RebBase 8.12 Drosophila transposable element (TE) library to generate a library with as little bias as possible. The additional repeats came from three sources. Two novel repetitive elements that were identified in D. melanogaster using the PILER-TR program were added . We also added a complete set of 66 elements from D. virilis identified by PILER-DF analysis (C Smith and G Karpen, personal communication) of the posted D. virilis whole genome assembly . Finally, a recently identified sequence of DINE-1 from D. yakuba was added .
All of the D. virilis and D. melanogaster sequences used in this study were then analyzed for repetitive DNA using RepeatMasker with this Superlibrary. This approach identified a total repeat density of the D. virilis contigs from the dot chromosome of 22.8%, while homologous regions of the D. melanogaster dot chromosome have 26.5% repetitive DNA (Figure 6b). Using the same Superlibrary, the segments from the major chromosomes of D. virilis have a total repeat density of 8.4%, compared to D. melanogaster major chromosomes, which have a density of 6.8%. This analysis shows that the overall density of repeats on the D. virilis and D. melanogaster dot chromosome fosmids is similar, and significantly higher than the density of repeats on the major chromosomes from either species. Other analysis techniques used to assess the difference between the D. virilis and D. melanogaster sequences, including a TBLASTX comparison using a RebBase 8.12 library from which invertebrate sequences had been removed [49, 54], and a Repeat Scout library assembly , also showed little difference in the total amount of repetitive sequence found in the D. virilis and D. melanogaster dot sequences (not shown). Thus, all of the follow-up techniques applied indicate that the sequences from the dot chromosomes of both D. virilis and D. melanogaster are enriched for repetitive sequences compared to the sequences derived from the major chromosomes of both species. The analysis of each contig as well as the total representation of each type of repeat is presented in Table 2 and in Figure 6b. The contrast between the results shown in Figure 6a and those shown in Figure 6b illustrates the problem posed by biased repeat libraries, an issue that must be carefully considered in studies of this type. The observation that three different analyses (discussed above) support the results shown in Figure 6b lends confidence to the conclusions derived here.
While the overall density of repetitious elements is similar, there is a major difference in the density of DNA transposons (Table 2). Of the D. melanogaster dot chromosome DNA from our sample, 18.6% consists of remnants of DNA transposons, including sequences from 1360 elements, P elements (artifacts and related fragments), Tc1 elements and DINE-1. Only 6.4% of these regions from the dot chromosome of D. virilis consists of remnants of DNA transposons, about a three-fold reduction. The bulk of the repetitive sequence in the D. virilis dot fosmids tentatively classified as DNA transposons are the dvir.16.2 centroid and the dvir.16.17 centroid, sequences identified in the PILER-DF analysis. Table 3 shows the repeat element and class of the most common repeats in the D. virilis and D. melanogaster dot chromosome contigs studied here, as identified by RepeatMasker/Superlibrary. DNA transposon families are preferentially represented in D. melanogaster, while retroelements (LINEs and LTRs) are more common in D. virilis. Examination of the quantitative results in Table 2 suggests that the dot chromosome of D. virilis has an increase in retroelements (9.1%) in comparison with homologous regions of D. melanogaster (4.2%). However, this difference appears to be due to sample bias, as RepeatMasker/RebBase 8.12 classifies 8.7% of the whole D. melanogaster dot chromosome as retroelements.
DINE-1, also known as DNAREP-1 or INE-1, is a repetitive element that is very common in the genome of D. melanogaster . The density of DINE-1 elements is especially high on the dot chromosome of D. melanogaster, more so than on the major chromosome arms or on the dot chromosome of D. virilis [17, 56]. Using our Superlibrary and repeat identification process, RepeatMasker identifies 0.1% of the D. virilis contigs as sequences with significant similarity to DINE-1 elements, while in the homologous regions of the D. melanogaster dot the density is 10.5%. (The entire D. melanogaster dot has a 9.2% incidence of DINE-1 elements, assessed using RepeatMasker/Superlibrary.) There has been considerable debate as to the origin of DINE-1 elements [56, 57]. Kapitonov and Jurka  have recently suggested that DINE-1 is a retrotransposon based on homology to a D. virilis Penelope GenBank accession, but sequences with homology to DINE-1 in this accession fall outside of the canonical Penelope sequence  (C Bergman, personal communication). Analysis of DINE-1 elements in D. yakuba suggests a relatively recent burst of transposition in that species. A consensus sequence based on these recent DINE-1 elements contains no long terminal repeats nor a poly-A tail (suggestive of a retroelement), but does have a terminal 12 bp perfect repeat, a characteristic of transposons . Thus, while we have provided separate statistics for this class, we consider DINE-1 elements to be DNA transposon remnants. Separate statistics are also provided in Table 2 for the 1360 DNA transposon fragments, as this class is of particular interest as a potential target for heterochromatin formation, as discussed above. Again, this family is significantly enriched in D. melanogaster dot chromosome fosmids, making up 4.1% of the sample DNA, in comparison to 0.8% in D. virilis dot chromosome fosmids and 0% in the samples from the major chromosome arms.
Transposable elements are much more prevalent in the introns of heterochromatic genes than in the introns of euchromatic genes  this may contribute to the evolution and structure of genes in heterochromatin. Maintaining a focus on total repeat density (and not repeat type), we analyzed the introns of all of the contigs with a repeat database generated by combining the RepeatScout output from both the D. melanogaster and the D. virilis whole genome assemblies. Using RepeatMasker with this library (omitting low complexity and simple repeats), one finds that introns of the D. virilis dot chromosome genes studied here contain 27.0% repetitive elements, while in homologous regions of the D. melanogaster dot, 33.1 % of the introns are made up of repetitive elements. Analysis of the contigs from the major chromosomes of D. virilis and the homologous regions from D. melanogaster did not find any recognizable transposable elements in the introns. Thus the two dot chromosomes are in this respect more similar to each other than they are to the major chromosomes from either species.
Comparing Figures 6a and 6b, it is apparent that the two repeat-finding strategies represented gave very different results. D. melanogaster and D. virilis are fairly close together phylogenetically, but use of the previously defined RepBase library, which has good representation of D. melanogaster repeats, was insufficient to find all of the D. virilis repeats, particularly on the dot chromosome. This result stresses the importance of using techniques such as PILER to find species-specific repeats as new species are sequenced, even when repeat sequences are available from a well-studied nearby species. Relying on existing repeat databases can lead to erroneously low estimates of repeat content.
The genetic basis of female pheromone differences between Drosophila melanogaster and D. simulans
Chemical signals are one means by which many insect species communicate. Differences in the combination of surface chemicals called cuticular hydrocarbons (CHCs) can influence mating behavior and affect reproductive isolation between species. Genes influencing three CHC compounds have been identified in Drosophila melanogaster. However, the genetic basis of other CHC compounds, whether these genes affect species differences in CHCs, and the genes' resulting effect on interspecies mating, remains unknown. We used fine-scale deficiency mapping of the third chromosome to identify 43 genomic regions that influence production of CHCs in both D. melanogaster and Drosophila simulans females. We identified an additional 23 small genomic regions that affect interspecies divergence in CHCs between females of these two species, one of which spans two genes known to influence the production of multiple CHCs within D. melanogaster. By testing these genes individually, we determined that desat1 also affects interspecific divergence in one CHC compound, while desat2 has no effect on interspecific divergence. Thus, some but not all genes affecting intraspecific amounts of CHCs also affect interspecific divergence, but not all genes or all CHCs. Lastly, we find no evidence of a relationship between the CHC profile and female attractiveness or receptivity towards D. melanogaster males.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Creation of female offspring used…
Creation of female offspring used for deficiency mapping to locate genes contributing to…
Mirrored CHC profiles for female…
Mirrored CHC profiles for female D. melanogaster ( a ) and D. simulans…
Variation among deficiency lines and…
Variation among deficiency lines and genotypes in relative concentration of ( a )…
Biochemical pathway overview ( a…
Biochemical pathway overview ( a ) and specific steps ( b ) for…
The Scientific Importance of Drosophila Melanogaster
Drosophila melanogaster, or the fruit fly as it is more commonly called, has played an important part in science. It has aided scientists in the discovery of many different principles. Its importance continues today.
The fruit fly has been used for approximately a century in scientific research, according to the University of Michigan Museum of Zoology. It has played an especially large role in the study of genetics. Thomas H. Morgan utilized the fruit fly to prove the chromosomal theory of inheritance. This showed that chromosomes carry genetic information. The fruit fly was vital in discoveries regarding gene mapping, multiple alleles, spistatis and sex-linked inheritance. Research continued on Drosophila melanogaster by H. Sturtevant. He created genetic maps of the fruit fly.
There are various reasons why Drosophila melanogaster has been such an ideal subject for studies in genetics and biology. First, it can reproduce very easily in captivity. It is very easy to breed many different subjects for use in studies.
The fruit fly has a very short lifespan. In seven days it matures into an adult. Because of this, researchers could study many generations in a short span of time. This is especially useful for genetic research.
It is simple to care for fruit flies. It is also inexpensive. Fruit flies reproduce very quickly, with one female creating a hundred eggs every single day. It is easy to tell the difference between males and females, as well as identify virgin females. There are only four pairs of chromosomes, and this makes it very easy to study them. Meiotic recombination does not occur in males. Also, because they have been studied so extensively, their entire genome was sequenced, allowing for easy research. All of these traits make Drosophila melanogaster the ideal subject to use in scientific research.
Despite the simplicity of Drosophila melanogaster, scientists can learn a great deal about genetics and biology from it. Many of the same principles about genetics are the same for fruit flies and other animals, including humans.
Drosophila melanogaster is even used to teach children about the importance of science. The same benefits that make it a great subject for established research scientists make it easy to use for high school and college students, according to the biology department at the University of Arizona. Thus they can help teach the next generation of scientists.
Although Drosophila melanogaster are simple creatures, their contribution to science through the years has been nothing short of amazing. They continue to be studied all over the world.
Invertebrate Learning and Memory
Thomas Riemensperger , André Fiala , in Handbook of Behavioral Neuroscience , 2013
Introduction: Strategies to Determine Neuronal Substrates Underlying Learning and Memory
Neuroscientific research on learning and memory ultimately aims to reveal neuronal structures and cellular mechanisms through which experience-dependent information is acquired, stored, and retrieved by neuronal circuitries of the brain. 1 To gain access to the neuronal circuits and biophysical mechanisms mediating changes in behavior, two general strategies are possible: (1) observation of neuronal processes in correlation with learning or memory retrieval and (2) experimental interference with neuronal processes to systematically manipulate learning and memory formation. To facilitate experiments, ‘simplified systems’ have often been chosen as preferred objects of research. The reasons for using a particular model system, be it an entire animal or a piece of nervous tissue, are usually due to technical advantages. The nervous system of marine snails consists of large neurons that can easily be impaled with recording and stimulation electrodes. 2 Honeybees exhibit a remarkable complexity in their learning capabilities, and electrophysiological approaches, optical imaging techniques, or pharmacological interventions are possible, for which the relative diminutiveness of the brain compared to that of mammals provides clear advantages. 3 Rodent model systems are attractive objects of study because of their relative evolutionary proximity to humans compared to invertebrates. Here, often ‘simplified systems’ are extracted by using isolated circuits, such as hippocampus slices. 2 For a long time, the fruit fly Drosophila melanogaster has been a favorite organism for geneticists, mainly due to the fast reproduction cycle and the large number of offspring that facilitates the screen for mutants. 4 Whereas this has been an excellent model organism for genetic studies, for many decades it was not useful for physiological investigations: Its small neurons and fine neurites are not advantageous for electrophysiological analyses of individual neurons, and electrophysiological techniques have long been restricted mainly to extracellular sensillum recordings 5 and recordings from neuromuscular preparations of the larval body wall. 6 However, two developments have made Drosophila amenable for physiological studies. First, germline transformation 7 and the versatility of binary expression systems 8 provide the possibility to target transgenes to distinct and defined populations of neurons. 9 Second, new proteins as molecular tools have been designed that can be transgenically expressed. DNA-encoded fluorescence probes can be targeted to defined cells in order to observe diverse parameters of cellular functions, such as calcium influx, second messenger-dependent signaling, and synaptic transmission, 10 and the discovery of light-sensitive cation channels has made it possible to manipulate the membrane potentials of neurons simply by illumination—a technology that is generally termed ‘optogenetics.’ 11,12 Because these molecular techniques—optical imaging of cellular processes using DNA-encoded probes and optical activation of neurons using light-gated cation channels—resemble the use of recording and stimulation electrodes in electrophysiology, we refer to these in combination as optophysiological approaches.
Understanding biochemical and physiological mechanisms that mediate learning and memory formation requires some knowledge about where in the brain those changes may happen that are causative for it. This classical ‘localization problem’ is not easy to solve, and it cannot be solved with a single experimental approach. 13 To determine whether distinct changes in neuronal activity (here subsuming all possible neuronal processes that can potentially be altered during learning, such as changes in synaptic transmitter release, postsynaptic excitation, or excitability of circuits in general) are indeed the biophysical substrates through which learning and memory observed in behavior are manifested, several experimental tests have been formulated. 13–16 Although experiments to determine whether neuronal substrates are necessary and sufficient to promote a certain type of learning differ slightly among researchers, 13–16 they generally include (1) disruptive alterations of neuronal functions, (2) detectability of changes in correlation with experience-dependent changes in behavior, and (3) artificial mimicry of changes in neuronal function that can substitute for a natural change in behavior. Here, we summarize how the use of optophysiological tools, among others, may contribute to such experiments.
To illustrate the technical approaches, we restrict ourselves to differential associative olfactory learning and the formation of short-term memory in D. melanogaster, 17,18 a learning paradigm that is widely used ( Figure 6.1A ). In this classical conditioning procedure, one odor as conditioned stimulus (CS+) is presented to a group of fruit flies in temporal coincidence with an electric shock as unconditioned stimulus (US). A second odor is presented without any punishment (CS–). In a subsequent test situation, the animals can chose between the two arms of a T-maze that contain either the CS+ or the CS–. Learning and short-term memory are assessed by calculating the proportion of animals avoiding the odor that has been associated with the electric shock in comparison to the total number of flies. 18 The neuronal pathways through which olfactory information is processed in the Drosophila brain have been characterized to some extent ( Figures 6.1B and 6.1C ). 20–22 Fruit flies perceive odors by olfactory sensory neurons housed within sensillae of various morphological types that are located on the third antennal segments and the maxillary palps. 23 Olfactory sensory neurons project via the antennal nerves to the antennal lobes, the primary olfactory neuropils of the insect brain, and ramify their terminal aborizations in spherical structures called glomeruli. 24 The basic logic of connectivity is simple: Those sensory neurons that express a given specific olfactory receptor target one or very few identifiable glomeruli. 24–26 Excitatory and inhibitory local interneurons that interconnect glomeruli process the odor information with the antennal lobe, 27–29 and olfactory projection neurons convey the odor information further to the lateral protocerebrum and the mushroom body. 30–32 The logic of odor coding is crucially different between the antennal lobe and the mushroom body. Whereas odors are represented at the level of the antennal lobes in terms of spatiotemporal patterns of overlapping glomerular activity, 33,34 the intrinsic mushroom body neurons (Kenyon cells) show a sparse response to particular odor stimuli—that is, only a small fraction of the approximately 2500 Kenyon cells per hemisphere selectively respond to a particular odor with the generation of very few action potentials. 35,36 This particular coding scheme appears favorable for selectively assigning positive or negative values to a given odor representation through associative learning ( Figure 6.1C ). 21,37 Modulatory neurons that release biogenic amines as transmitters (e.g., dopamine and octopamine) and that broadly innervate mushroom bodies are assumed to carry the value information evoked by the US. 21,37 The following sections summarize the experimental approaches that have been used to test this idea.
Figure 6.1 . Classical olfactory conditioning in Drosophila.
(A) Schematic depiction of a differential conditioning paradigm. 18 During training, flies are sequentially exposed to a non-reinforced odor (CS–) and, with a delay, an odor (CS+) that is temporally paired with an electric shock (unconditioned stimulus). Subsequently, the flies are transferred to a T-maze in which they approach or escape either of the two presented odors. (B) Illustration of the olfactory pathway in the Drosophila brain. Odors are perceived by receptors located on the antennae (AN) and conveyed by olfactory sensory neurons (yellow) to the antennal lobes (AL). Olfactory projection neurons (green) convey the information to the mushroom bodies (MB) and the lateral horn (LH). (C) Hypothetical neuronal circuit mediating olfactory associative learning. Odor stimuli are encoded at the level of the antennal lobes as combinatorial glomerular activity patterns and conveyed to the intrinsic mushroom body neurons (Kenyon cells). Here, olfactory information is represented as sparse neuronal activity. Punishment is mediated by dopaminergic neurons, and in coincidence with odor-evoked activity transmission by Kenyon cell output synapses is modified. (D) Temperature-dependent suppression of neurotransmitter release using shibire ts , a temperature-sensitive variant of the protein dynamin. 19 A temperature shift from the permissive (22°C) toward the restrictive (30°C) temperature suppresses synaptic vesicle recycling, ultimately causing a disruption of synaptic transmission.
Male-Male Courtship Pattern Shaped By Emergence Of A New Gene In Fruit Flies
When a young gene known as sphinx is inactivated in the common fruit fly, it leads to increased male-male courtship, scientists report in the May 27, 2008, issue of the Proceedings of the National Academy of Sciences. High levels of male-male courtship are widespread in many fly species, but not in Drosophila melanogaster, the tiny insect that has been a mainstay of genetic research for more than a century.
In 2002, the research team of Manyuan Long, professor of ecology and evolution at the University of Chicago, and colleagues discovered that D. melanogaster possessed the sphinx gene--and other fly species did not.
In order to study the function of this two million-year-old gene, Hongzheng Dai and Ying Chen--former graduate students in Long's lab and first authors of this study--created flies with a suppressed version of the sphinx gene, which is expressed in male reproductive glands. Loss of the gene produced no apparent changes.
"The flies looked normal," Long said. But when the researchers put two males that lacked the sphinx gene together, they noticed that the males were "interested in other males."
They repeated the experiment many times, Long said. It consistently produced the same results. Males without sphinx pursued each other more than 10 times longer than did males with a working copy of the gene. They performed all stages of normal male-female courtship--orienting, tapping, singing, licking, attempting--except for copulating.
"Male-male courtship might have been common in the ancestral D. melanogaster population," Long said. "Sphinx appears to have evolved to reduce this in one single species." By silencing this gene, the researchers may have generated an ancestral genotype that existed before sphinx originated.
D. melanogaster separated from related species about three million years ago, the researchers say. Male-male courtship could have been common among the fly's ancestors before that separation up to at lease 25-30 million years ago.
"Species that don't have this gene show more male-male courtship behavior than those that do have it," Long said. "The absence or presence of the sphinx gene appears to regulate the diversity of male-male courtship behavior among flies. This suggests that the genetic control of male courtship is an evolving system, which can recruit new genetic components and change courtship behaviors."
"This is the genetic interpretation," Long said. "Of course other factors, like the environment, are also likely to have an influence."
The scientists also noticed that groups of males without a working copy of sphinx tended to behave differently, often forming chains of flies positioned behind each other. This is a typical male-male courtship behavior, Long said, not seen in male-female relations.
Female flies without sphinx, on the other hand, did not show any changes in reproductive behavior compared to females with sphinx. This is not surprising, the authors say, since the sphinx gene is not expressed in female reproductive tissues.
Normal females were not able to attract the attentions of sphinxless males, which were more interested in each other than in females. But when these males could not complete the copulation process with other males, they would return to the females, Long said.
"Sphinx is not a protein-coding gene, but an RNA gene," Long said. "So, the question is: How do RNA genes interact and regulate other genes" We are exploring this in our lab."
The study was supported by the National Institutes of Health, the National Science Foundation, the David and Lucile Packard Foundation and the Leukemia and Lymphoma Society. Additional authors are Sidi Chen, Qiyan Mao, David Kennedy, Patrick Landback and Wei Du from the University of Chicago and Adam Eyre-Walker from the University of Sussex, Brighton, U.K.
Materials provided by University of Chicago Medical Center. Note: Content may be edited for style and length.
Lethal Gene Keeps Fly Species Separate
Research uncovers new information about the biological processes that help ensure that two fly species don't interbreed.
Pour some cold cream into a cup of hot, black coffee, and you end up with a drink that&rsquos midway between the two ingredients in color, temperature, and flavor. A similar kind of blending can occur if members of closely related species frequently mate with each other, but many species have mechanisms to prevent such mixing. Now, Howard Hughes Medical Institute scientists have discovered one of the few genes that ensures that two species don&rsquot interbreed.
HHMI investigators Harmit Malik of the Fred Hutchinson Cancer Research Center and Jay Shendure of the University of Washington and their colleagues devised a new mutagenesis-based approach to investigate the molecular genetic basis of speciation. They used this approach to identify a gene that kills male offspring that result from mating between two fruit fly species. &ldquoIt&rsquos a pretty big step in our understanding of what is the biological process that leads to incompatibility&rdquo between different species, says Malik. He and his colleagues reported their findings on December 18, 2015, in the journal Science.
More than 150 years ago, Charles Darwin noted that interbreeding between species could blur their differences. &ldquoSpecies within the same country could hardly have kept distinct had they been capable of crossing freely,&rdquo he wrote in On the Origin of Species. A variety of obstacles can prevent species from intermixing, producing what scientists term reproductive isolation. The species may reproduce at different times of the year, for example, or their progeny may die or be sterile, as is the case for mules, the offspring of mating between horses and donkeys. Although researchers have been searching for genes that keep species separate, Malik notes that so far, &ldquothere are very few cases in which we can say, &lsquothis gene is mediating reproductive isolation.&rsquo&rdquo
Two of the genes scientists have uncovered enable the fruit fly Drosophila melanogaster to remain reproductively isolated from the similar fly species D. simulans. Both species live throughout much of the world, giving them plenty of opportunities to interbreed in the wild. Yet when a female D. melanogaster and a male D. simulans mate, only their female offspring live all of the males die shortly after hatching.
Researchers aren&rsquot sure why the female offspring don&rsquot perish, but they&rsquove shown that the genes Hmr and Lhr are partly responsible for the deaths of the hybrid males. Evidence suggested that a third gene was also involved, but nobody had identified it.
Nitin Phadnis, who was a postdoctoral researcher in Malik&rsquos lab and is now on the faculty at the University of Utah, designed an ingenious experiment to ferret out this elusive gene. The team fed male D. simulans flies a chemical that triggers mutations. The researchers hoped that in some flies these alterations would occur in the gene they were searching for and disable it. Although these flies would be extremely rare, the scientists would be able to recognize them because their sons would survive. After nearly two years of crossbreeding flies and sorting through more than 330,000 of their progeny, Malik and colleagues tallied only six living male offspring.
But that was enough to pinpoint the gene. When the scientists sequenced the genomes of these flies, they discovered that all six carried mutations in gfzf, a gene that helps orchestrate cell division. To demonstrate that gfzf was the third killer gene, the researchers engineered female D. melanogaster flies to produce a type of RNA molecule that shuts down the D. simulans version of gfzf. The team then bred the engineered females with male D. simulans flies. Some sons from these crosses inherited their mothers&rsquo ability to shut down the D. simulans version of gfzf. These lucky males didn&rsquot die young, confirming the team&rsquos identification of gfzf.
Malik and colleagues determined that gfzf kills male offspring when they are starting to transform from larvae into adults. Only a small fraction of the cells in a larva produce all of the cells in an adult fly, and these larval cells need to divide rapidly. However, gfzf curtails cell reproduction in hybrid male offspring, preventing them from generating the new cells required to form an adult body.
&ldquoIt&rsquos so difficult to identify these genes&rdquo that produce reproductive isolation, says Malik. &ldquoI&rsquom hopeful that the strategy we identify in this paper will give us a bounty of genes in the future.&rdquo Malik adds that there&rsquos suggestive evidence that a fourth gene helps the two fly species remain reproductively isolated, and he and his colleagues have begun looking for it.