Melanogaster Genome Research Articles

MGEScan-non-LTR: computational identification and classification of autonomous non-LTR retrotransposons in eukaryotic genomes

Computational methods for genome-wide identification of mobile genetic elements (MGEs) have become increasingly necessary for both genome annotation and evolutionary studies. Non-long terminal repeat (non-LTR) retrotransposons are a class of MGEs that have been found in most eukaryotic genomes, sometimes in extremely high numbers. In this article, we present a computational tool, MGEScan-non-LTR, for the identification of non-LTR retrotransposons in genomic sequences, following a computational approach inspired by a generalized hidden Markov model (GHMM). Three different states represent two different protein domains and inter-domain linker regions encoded in the non-LTR retrotransposons, and their scores are evaluated by using profile hidden Markov models (for protein domains) and Gaussian Bayes classifiers (for linker regions), respectively. In order to classify the non-LTR retrotransposons into one of the 12 previously characterized clades using the same model, we defined separate states for different clades. MGEScan-non-LTR was tested on the genome sequences of four eukaryotic organisms, Drosophila melanogaster, Daphnia pulex, Ciona intestinalis and Strongylocentrotus purpuratus. For the D. melanogaster genome, MGEScan-non-LTR found all known ‘full-length’ elements and simultaneously classified them into the clades CR1, I, Jockey, LOA and R1. Notably, for the D. pulex genome, in which no non-LTR retrotransposon has been annotated, MGEScan-non-LTR found a significantly larger number of elements than did RepeatMasker, using the current version of the RepBase Update library. We also identified novel elements in the other two genomes, which have only been partially studied for non-LTR retrotransposons.

Evolutionary conservation and changes in insect TRP channels

BackgroundTRP (Transient Receptor Potential) channels respond to diverse stimuli and thus function as the primary integrators of varied sensory information. They are also activated by various compounds and secondary messengers to mediate cell-cell interactions as well as to detect changes in the local environment. Their physiological roles have been primarily characterized only in mice and fruit flies, and evolutionary studies are limited. To understand the evolution of insect TRP channels and the mechanisms of integrating sensory inputs in insects, we have identified and compared TRP channel genes in Drosophila melanogaster, Bombyx mori, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, and Pediculus humanus genomes as part of genome sequencing efforts.ResultsAll the insects examined have 2 TRPV, 1 TRPN, 1 TRPM, 3 TRPC, and 1 TRPML subfamily members, demonstrating that these channels have the ancient origins in insects. The common pattern also suggests that the mechanisms for detecting mechanical and visual stimuli and maintaining lysosomal functions may be evolutionarily well conserved in insects. However, a TRPP channel, the most ancient TRP channel, is missing in B. mori, A. mellifera, and N. vitripennis. Although P. humanus and D. melanogaster contain 4 TRPA subfamily members, the other insects have 5 TRPA subfamily members. T. castaneum, A. mellifera, and N. vitripennis contain TRPA5 channels, which have been specifically retained or gained in Coleoptera and Hymenoptera. Furthermore, TRPA1, which functions for thermotaxis in Drosophila, is missing in A. mellifera and N. vitripennis; however, they have other Hymenoptera-specific TRPA channels (AmHsTRPA and NvHsTRPA). NvHsTRPA expressed in HEK293 cells is activated by temperature increase, demonstrating that HsTRPAs function as novel thermal sensors in Hymenoptera.ConclusionThe total number of insect TRP family members is 13-14, approximately half that of mammalian TRP family members. As shown for mammalian TRP channels, this may suggest that single TRP channels are responsible for integrating diverse sensory inputs to maintain the insect sensory systems. The above results demonstrate that there are both evolutionary conservation and changes in insect TRP channels. In particular, the evolutionary processes have been accelerated in the TRPA subfamily, indicating divergence in the mechanisms that insects use to detect environmental temperatures.

Domains of Heterochromatin Protein 1 Required forDrosophila melanogasterHeterochromatin Spreading

Centric regions of eukaryotic genomes are packaged into heterochromatin, which possesses the ability to spread along the chromosome and silence gene expression. The process of spreading has been challenging to study at the molecular level due to repetitious sequences within centric regions. A heterochromatin protein 1 (HP1) tethering system was developed that generates "ectopic heterochromatin" at sites within euchromatic regions of the Drosophila melanogaster genome. Using this system, we show that HP1 dimerization and the PxVxL interaction platform formed by dimerization of the HP1 chromo shadow domain are necessary for spreading to a downstream reporter gene located 3.7 kb away. Surprisingly, either the HP1 chromo domain or the chromo shadow domain alone is sufficient for spreading and silencing at a downstream reporter gene located 1.9 kb away. Spreading is dependent on at least two H3K9 methyltransferases, with SU(VAR)3-9 playing a greater role at the 3.7-kb reporter and dSETDB1 predominately acting at the 1.9 kb reporter. These data support a model whereby HP1 takes part in multiple mechanisms of silencing and spreading.

Graded Dorsal and Differential Gene Regulation in the Drosophila Embryo

A gradient of Dorsal activity patterns the dorsoventral (DV) axis of the early Drosophila melanogaster embryo by controlling the expression of genes that delineate presumptive mesoderm, neuroectoderm, and dorsal ectoderm. The availability of the Drosophila melanogaster genome sequence has accelerated the study of embryonic DV patterning, enabling the use of systems-level approaches. As a result, our understanding of Dorsal-dependent gene regulation has expanded to encompass a collection of more than 50 genes and 30 cis-regulatory sequences. This information, which has been integrated into a spatiotemporal atlas of gene regulatory interactions, comprises one of the best-understood networks controlling any developmental process to date. In this article, we focus on how Dorsal controls differential gene expression and how recent studies have expanded our understanding of Drosophila embryonic development from the cis-regulatory level to that controlling morphogenesis of the embryo.

Massively parallel resequencing of the isogenic Drosophila melanogaster strain w1118; iso-2; iso-3 identifies hotspots for mutations in sensory perception genes

We used the Illumina reversible-short sequencing technology to obtain 17-fold average depth (s.d.~8) of ~94% of the euchromatic genome and ~1-5% of the heterochromatin sequence of the Drosophila melogaster isogenic strain w1118; iso-2; iso-3. We show that this strain has a ~9 kb deletion that uncovers the first exon of the white (w) gene, ~4 kb of downstream promoter sequences, and most of the first intron, thus demonstrating that whole-genome sequencing can be used for mutation characterization. We chose this strain because there are thousands of transposon insertion lines and hundreds of isogenic deficiency lines available with this genetic background, such as the Exelixis, Inc., and the DrosDEL collections. We compared our sequence to Release 5 of the finished reference genome sequence which was made from the isogenic strain y1; cn1 bw1 sp1 and identified ~356,614 candidate SNPs in the ~117 Mb unique sequence genome, which represents a substitution rate of ~1/305 nucleotides (~0.30%). The distribution of SNPs is not uniform, but rather there is a ~2-fold increase in SNPs on the autosome arms compared with the X chromosome and a ~7-fold increase when compared to the small 4th chromosome. This is consistent with previous analyses that demonstrated a correlation between recombination frequency and SNP frequency. An unexpected finding was a SNP hotpot in a ~20Mb central region of the 4th chromosome, which might indicate higher than expected recombination frequency in this region of this chromosome. Interestingly, genes involved in sensory perception are enriched in SNP hotspots and genes encoding developmental genes are enriched in SNP coldspots, which suggests that recombination frequencies might be proportional to the evolutionary selection coefficient. There are currently 12 Drosophila species sequenced, and this represents one of many isogenic Drosophila melanogaster genome sequences that are in progress. Because of the dramatic increase in power in using isogenic lines rather than outbred individuals, the SNP information should be valuable as a test bed for understanding genotype-by-environment interactions in human population studies.

Molecular variation of the testes-specific βNACtes genes in the Drosophila melanogaster genome

The βNACtes gene family of the Drosophila melanogaster genome provides a model for investigating the mechanisms of the molecular evolution of recently evolved genes. The βNACtes genes code for proteins that are homologous to the subunit of the nascent polypeptide-associated complex (NAC), are expressed exclusively in the testis, and are localized on the X chromosome as two-gene clusters and one separate copy. Population polymorphism of the βNACtes genes was studied using several wild-type D. melanogaster stocks, and βNACtes paralogs were compared with each other. A heterogeneous pattern was observed for βNACtes polymorphism: the 3′ genes of the two-gene clusters were low polymorphic, whereas, separate, the βNACtes1 gene was the most variable. The 5′ βNACtes copies of the two-gene tandems were practically identical, whereas the 3′ βNACtes copies were highly diverged. Hence, local gene conversion was assumed to provide for the selective homogenization of the 5′ genes. A comparison of the βNACtes paralogs showed that the majority of amino acid differences were in the N-terminal region, containing the βNAC domain. The McDonald-Kreitman test was used to analyze the divergence of βNACtes paralogs and implicated positive selection in the evolution of the βNACtes gene family.

Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions

Insulators are protein-bound DNA elements that are thought to play a role in chromatin organization and the regulation of gene expression by mediating intra- and interchromosomal interactions. Suppressor of Hair-wing [Su(Hw)] and Drosophila CTCF (dCTCF) insulators are found at distinct loci throughout the Drosophila melanogaster genome and function by recruiting an additional protein, Centrosomal Protein 190 (CP190). We performed chromatin immunoprecipitation (ChIP) and microarray analysis (ChIP-chip) experiments with whole-genome tiling arrays to compare Su(Hw), dCTCF, boundary element-associated factor (BEAF), and CP190 localization on DNA in two different cell lines and found evidence that BEAF is a third subclass of CP190-containing insulators. The DNA-binding proteins Su(Hw), dCTCF, and BEAF show unique distribution patterns with respect to the location and expression level of genes, suggesting diverse roles for these three subclasses of insulators in genome organization. Notably, cell line-specific localization sites for all three DNA-binding proteins as well as CP190 indicate multiple levels at which insulators can be regulated to affect gene expression. These findings suggest a model in which insulator subclasses may have distinct functions that together organize the genome in a cell type-specific manner, resulting in differential regulation of gene expression.

Drosophila Genomics; Telomerases

BioTechniquesVol. 46, No. 6 NIH Grant WatchOpen AccessDrosophila Genomics; TelomerasesKristie Nybo & Bruce PerryKristie NyboSearch for more papers by this author & Bruce PerrySearch for more papers by this authorPublished Online:25 Apr 2018https://doi.org/10.2144/000113167AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail BioTechniques and NIH Sales reviewed grants made by the National Institutes of Health during the last 30 days. Here are some highlights from among the largest R01 and R03 grants awarded during March 2009.Drosophila Population Genomics $799,999(2R01HG002942-05, National Human Genome Research Institute)Charles Langley (University of California, Davis, CA)Goal: To completely sequence 50 Drosophila melanogaster genomes and annotate the genomic polymorphisms according to their diverse functions. In the course of the study, an appropriate resequencing technology will be developed, validated, and sustained, and the value of genomic studies of population polymorphisms will be emphasized.Telomerase in Development, Senescence, and Neoplasia $535,923(5R01CA084628-17, National Cancer Institute)Ronald Depinho (Dana Farber Cancer Institute, Boston, MA)Goal: To explore the roles of telomeres, telomerase, and p53 in tumor progression and metastases in a telomerase knockout mouse strain. This study aims to investigate how the telomere and p53 pathways interact to govern tissue stem cell survival, transformation, and evolution into malignant disease. This will be accomplished through studies where telomere function is restored through inducible alleles for telomerase reverse transcriptase and p53; genetic screening to define the telomere checkpoint response; investigation of telomerase and p53 contributions to tumor progression; and the actions of established tumors in response to termination of telomere activity.Regulation of Cell Growth and Proliferation by Myc $530,193(5R01CA020525-33, National Cancer Institute)Robert Eisenman (Fred Hutchinson Cancer Research Center, Seattle, WA)Goal: To link Myc's developmental functions with its ability to induce chromatin modifications. To accomplish this, the cytokine signaling role of N-myc in the developing mouse nervous system will be studied using targeted gene deletions and a mouse tumor model. Secondly, N-myc will be deleted with temporal and locational specificity. Finally, the mechanism, nature, and extent of chromatin modifications in response to Myc will be explored.Transsynaptic Tracing of Olfactory Bulb Input to the Olfactory Cortex $74,475(5R03DC008874-03, National Institute on Deafness and Other Communication Disorders)David Willhite (Yale University, New Haven, CT)Goal: To map olfactory bulb input patterns to the olfactory cortex using transsynaptic tracing. The study will provide the first quantitative evidence of regional olfactory bulb input segregations, the degree of input as a function of distance, and whether input patterns are conserved between individuals.FiguresReferencesRelatedDetails Vol. 46, No. 6 Follow us on social media for the latest updates Metrics Downloaded 169 times History Published online 25 April 2018 Published in print May 2009 Information© 2009 Author(s)PDF download

Anopheles Fibrinogen-related Proteins Provide Expanded Pattern Recognition Capacity against Bacteria and Malaria Parasites

The fibrinogen-related protein family (FREP, also known as FBN) is an evolutionarily conserved immune gene family found in mammals and invertebrates. It is the largest pattern recognition receptor gene family in Anopheles gambiae, with as many as 59 putative members, while the Drosophila melanogaster genome has only 14 known FREP members. Our sequence and phylogenetic analysis suggest that this remarkable gene expansion in the mosquito is the result of tandem duplication of the fibrinogen domain. We found that the majority of the FREP genes displayed immune-responsive transcription after challenge with bacteria, fungi, or Plasmodium, and these expression patterns correlated strongly with gene phylogeny and chromosomal location. Using RNAi-mediated gene-silencing assays, we further demonstrated that some FREP members are essential factors of the mosquito innate immune system that are required for maintaining immune homeostasis, and members of this family have complementary and synergistic functions. One of the most potent anti-Plasmodium FREP proteins, FBN9, was found to interact with both Gram-negative and Gram-positive bacteria and strongly co-localized with both rodent and human malaria parasites in the mosquito midgut epithelium, suggesting that its defensive activity involves direct interaction with the pathogen. Interestingly, FBN9 formed dimers that bound to the bacterial surfaces with different affinities. Our findings indicate that the A. gambiae FREP gene family plays a central role in the mosquito innate immune system and provides an expanded pattern recognition and anti-microbial defense repertoire.

Big Genomes Facilitate the Comparative Identification of Regulatory Elements

The identification of regulatory sequences in animal genomes remains a significant challenge. Comparative genomic methods that use patterns of evolutionary conservation to identify non-coding sequences with regulatory function have yielded many new vertebrate enhancers. However, these methods have not contributed significantly to the identification of regulatory sequences in sequenced invertebrate taxa. We demonstrate here that this differential success, which is often attributed to fundamental differences in the nature of vertebrate and invertebrate regulatory sequences, is instead primarily a product of the relatively small size of sequenced invertebrate genomes. We sequenced and compared loci involved in early embryonic patterning from four species of true fruit flies (family Tephritidae) that have genomes four to six times larger than those of Drosophila melanogaster. Unlike in Drosophila, where virtually all non-coding DNA is highly conserved, blocks of conserved non-coding sequence in tephritids are flanked by large stretches of poorly conserved sequence, similar to what is observed in vertebrate genomes. We tested the activities of nine conserved non-coding sequences flanking the even-skipped gene of the teprhitid Ceratis capitata in transgenic D. melanogaster embryos, six of which drove patterns that recapitulate those of known D. melanogaster enhancers. In contrast, none of the three non-conserved tephritid non-coding sequences that we tested drove expression in D. melanogaster embryos. Based on the landscape of non-coding conservation in tephritids, and our initial success in using conservation in tephritids to identify D. melanogaster regulatory sequences, we suggest that comparison of tephritid genomes may provide a systematic means to annotate the non-coding portion of the D. melanogaster genome. We also propose that large genomes be given more consideration in the selection of species for comparative genomics projects, to provide increased power to detect functional non-coding DNAs and to provide a less biased view of the evolution and function of animal genomes.

A Drosophila Protein Family Implicated in Pheromone Perception Is Related to Tay-Sachs GM2-Activator Protein

Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons.

Epigenetic Investigations in Drosophila for Identifying Genes Responsive to Different Stress Conditions

Long-lived organisms tend to be more resistant to various forms of environmental stress. For a while authors reported the absence of methylated cytosine residues in Drosophila melanogaster genome, but recently there have been published several studies which proved that Drosophila genome contain methylated cytosines. With respect to these studies, we conducted an epigenetic screening, trying to test in different Drosophila strains, the response of several genes to unlike stress condition. Four single-copy genes were monitored for their status of methylation: Hsp70, Hsp22, CG3706 and Indy, in three different Drosophila strains, named so that to correspond with the polluted regions from Romania chosen for investigation: Bucharest-Polycolor (B), Seaca (Tr), Braila (Br). For each selected gene we investigated mainly the promoter region. All the results were related to that obtained with the control isogenised strain from the lab, the iso strain. The methods used for investigation were bisulfite sequencing along with molecular cloning and MSP (Methylation Specific PCR). The results demonstrated that the selected genes for this screening are not silenced by methylation, irrespective of the stock selected from different polluted areas. Our results are not so surprisingly if is taken into account that it have been previously shown that in Drosophila only about 0,1-0,6% of all cytosine are methylated, and nowadays are still lots of questions for a specific role of the DNA methylation in flies.

Using whole genome presence/absence data to untangle function in 12 Drosophila genomes

The Drosophila 12 genome data set was used to construct whole genome, gene family presence/absence matrices using a broad range of E value cutoffs as criteria for gene family inclusion. The various matrices generated behave differently in phylogenetic analyses as a function of the e-value employed. Based on an optimality criterion that maximizes internal corroboration of information, we show that values of e-105 to e-125 extract the most internally consistent phylogenetic signal. Functional class of most genes and gene families can be accurately determined based on the D. melanogaster genome annotation. We used the gene ontology (GO) system to create partitions based on gene function. Several measures of phylogenetic congruence (diagnosis, consistency, partitioned support , hidden support) for different higher and lower level GO categories, were used to mine the data set for genes and gene families that show strong agreement or disagreement with the overall combined phylogenetic hypothesis. We propose that measures of phylogenetic congruence can be used as criteria to identify loci with related GO terms that have a significant impact on cladogenesis.

High Rate of Recent Transposable Element–Induced Adaptation in Drosophila melanogaster

Although transposable elements (TEs) are known to be potent sources of mutation, their contribution to the generation of recent adaptive changes has never been systematically assessed. In this work, we conduct a genome-wide screen for adaptive TE insertions in Drosophila melanogaster that have taken place during or after the spread of this species out of Africa. We determine population frequencies of 902 of the 1,572 TEs in Release 3 of the D. melanogaster genome and identify a set of 13 putatively adaptive TEs. These 13 TEs increased in population frequency sharply after the spread out of Africa. We argue that many of these TEs are in fact adaptive by demonstrating that the regions flanking five of these TEs display signatures of partial selective sweeps. Furthermore, we show that eight out of the 13 putatively adaptive elements show population frequency heterogeneity consistent with these elements playing a role in adaptation to temperate climates. We conclude that TEs have contributed considerably to recent adaptive evolution (one TE-induced adaptation every 200–1,250 y). The majority of these adaptive insertions are likely to be involved in regulatory changes. Our results also suggest that TE-induced adaptations arise more often from standing variants than from new mutations. Such a high rate of TE-induced adaptation is inconsistent with the number of fixed TEs in the D. melanogaster genome, and we discuss possible explanations for this discrepancy.

Testing the palindromic target site model for DNA transposon insertion using the Drosophila melanogaster P-element

Understanding the molecular mechanisms that influence transposable element target site preferences is a fundamental challenge in functional and evolutionary genomics. Large-scale transposon insertion projects provide excellent material to study target site preferences in the absence of confounding effects of post-insertion evolutionary change. Growing evidence from a wide variety of prokaryotes and eukaryotes indicates that DNA transposons recognize staggered-cut palindromic target site motifs (TSMs). Here, we use over 10 000 accurately mapped P-element insertions in the Drosophila melanogaster genome to test predictions of the staggered-cut palindromic target site model for DNA transposon insertion. We provide evidence that the P-element targets a 14-bp palindromic motif that can be identified at the primary sequence level, which predicts the local spacing, hotspots and strand orientation of P-element insertions. Intriguingly, we find that the although P-element destroys the complete 14-bp target site upon insertion, the terminal three nucleotides of the P-element inverted repeats complement and restore the original TSM, suggesting a mechanistic link between transposon target sites and their terminal inverted repeats. Finally, we discuss how the staggered-cut palindromic target site model can be used to assess the accuracy of genome mappings for annotated P-element insertions.

The Correlation Between Recombination Rate and Dinucleotide Bias in Drosophila melanogaster

Revealing how recombination affects genomic sequence is of great significance to our understanding of genome evolution. The present paper focuses on the correlation between recombination rate and dinucleotide bias in Drosophila melanogaster genome. Our results show that the overall dinucleotide bias is positively correlated with recombination rate for genomic sequences including untranslated regions, introns, intergenic regions, and coding sequences. The correlation patterns of individual dinucleotide biases with recombination rate are presented. Possible mechanisms of interaction between recombination and dinucleotide bias are discussed. Our data indicate that there may be a genome-wide universal mechanism acting between recombination rate and dinucleotide bias, which is likely to be neighbor-dependent biased gene conversion.

Sequence relationships among C. elegans, D. melanogaster and human microRNAs highlight the extensive conservation of microRNAs in biology.

microRNAs act in a prevalent and conserved post-transcriptional gene regulatory mechanism that impacts development, homeostasis and disease, yet biological functions for the vast majority of miRNAs remain unknown. Given the power of invertebrate genetics to promote rapid evaluation of miRNA function, recently expanded miRNA identifications (miRBase 10.1), and the importance of assessing potential functional redundancies within and between species, we evaluated miRNA sequence relationships by 5′ end match and overall homology criteria to compile a snapshot overview of miRNA families within the C. elegans and D. melanogaster genomes that includes their identified human counterparts. This compilation expands literature documentation of both the number of families and the number of family members, within and between nematode and fly models, and highlights sequences conserved between species pairs or among nematodes, flies and humans. Themes that emerge include the substantial potential for functional redundancy of miRNA sequences within species (84/139 C. elegans miRNAs and 70/152 D. melanogaster miRNAs share significant homology with other miRNAs encoded by their respective genomes), and the striking extent to which miRNAs are conserved across species—over half (73/139) C. elegans miRNAs share sequence homology with miRNAs encoded also in both fly and human genomes. This summary analysis of mature miRNA sequence relationships provides a quickly accessible resource that should facilitate functional and evolutionary analyses of miRNAs and miRNA families.

Delineating Slowly and Rapidly Evolving Fractions of the Drosophila Genome

Evolutionary conservation is an important indicator of function and a major component of bioinformatic methods to identify non-protein-coding genes. We present a new Bayesian method for segmenting pairwise alignments of eukaryotic genomes while simultaneously classifying segments into slowly and rapidly evolving fractions. We also describe an information criterion similar to the Akaike Information Criterion (AIC) for determining the number of classes. Working with pairwise alignments enables detection of differences in conservation patterns among closely related species. We analyzed three whole-genome and three partial-genome pairwise alignments among eight Drosophila species. Three distinct classes of conservation level were detected. Sequences comprising the most slowly evolving component were consistent across a range of species pairs, and constituted approximately 62-66% of the D. melanogaster genome. Almost all (>90%) of the aligned protein-coding sequence is in this fraction, suggesting much of it (comprising the majority of the Drosophila genome, including approximately 56% of non-protein-coding sequences) is functional. The size and content of the most rapidly evolving component was species dependent, and varied from 1.6% to 4.8%. This fraction is also enriched for protein-coding sequence (while containing significant amounts of non-protein-coding sequence), suggesting it is under positive selection. We also classified segments according to conservation and GC content simultaneously. This analysis identified numerous sub-classes of those identified on the basis of conservation alone, but was nevertheless consistent with that classification. Software, data, and results available at www.maths.qut.edu.au/-keithj/. Genomic segments comprising the conservation classes available in BED format.

The sex chromosome that refused to die

Chromosomes that harbor dominant sex determination loci are predicted to erode over time--losing genes, accumulating transposable elements, degenerating into a functional wasteland and ultimately becoming extinct. The Drosophila melanogaster Y chromosome is fairly far along this path to oblivion. The few genes on largely heterochromatic Y chromosome are required for spermatocyte-specific functions, but have no role in other tissues. Surprisingly, a recent paper shows that divergent Y chromosomes can substantially influence gene expression throughout the D. melanogaster genome.1 These results show that variation on Y has an important influence on the deployment of the genome.

Characterization of a Novel Caenorhabditis elegans Prolyl 4-Hydroxylase with a Unique Substrate Specificity and Restricted Expression in the Pharynx and Excretory Duct

Collagen prolyl 4-hydroxylases (C-P4Hs) have a critical role in collagen synthesis, since 4-hydroxyproline residues are necessary for folding of the triple-helical molecules. Vertebrate C-P4Hs are alpha(2)beta(2) tetramers in which the beta subunit is identical to protein-disulfide isomerase (PDI). Three isoforms of the catalytic alpha subunit, PHY-1, PHY-2, and PHY-3, have been characterized from Caenorhabditis elegans, PHY-1 and PHY-2 being responsible for the hydroxylation of cuticle collagens, whereas PHY-3 is predicted to be involved in collagen synthesis in early embryos. We have characterized transcripts of two additional C. elegans alpha subunit-like genes, Y43F8B.4 and C14E2.4. Three transcripts were generated from Y43F8B.4, and a polypeptide encoded by one of them, named PHY-4.1, assembled into active (PHY-4.1)(2)/(PDI-2)(2) tetramers and PHY-4.1/PDI-2 dimers when coexpressed with C. elegans PDI-2 in insect cells. The C14E2.4 transcript was found to have a frameshift leading to the absence of codons for two residues critical for P4H catalytic activity. Thus, C. elegans has altogether four functional C-P4H alpha subunits, PHY-1, PHY-2, PHY-3, and PHY-4.1. The tetramers and dimers containing recombinant PHY-4.1 had a distinct substrate specificity from the other C-P4Hs in that they hydroxylated poly(l-proline) and certain other proline-rich peptides, including ones that are expressed in the pharynx, in addition to collagen-like peptides. These data and the observed restricted expression of the phy-4.1 transcript and PHY-4.1 polypeptide in the pharyngeal gland cells and the excretory duct suggest that in addition to collagens, PHY-4.1 may hydroxylate additional proline-rich proteins in vivo.