Comparative genomics of the eukaryotes

Comparative Genomics of the Eukaryotes

Gerald M. Rubin1, Mark D. Yandell3, Jennifer R. Wortman3, George L. Gabor Miklos4,Catherine R. Nelson2, Iswar K. Hariharan5, Mark E. Fortini6, Peter W. Li3, Rolf Apweiler7,Wolfgang Fleischmann7, J. Michael Cherry8, Steven Henikoff9, Marian P. Skupski3, SimaMisra2, Michael Ashburner7, Ewan Birney7, Mark S. Boguski10, Thomas Brody11, PeterBrokstein2, Susan E. Celniker12, Stephen A. Chervitz13, David Coates14, Anibal Cravchik3,Andrei Gabrielian3, Richard F. Galle12, William M. Gelbart15, Reed A. George12, LawrenceS. B. Goldstein16, Fangcheng Gong3, Ping Guan3, Nomi L. Harris12, Bruce A. Hay17, RogerA. Hoskins12, Jiayin Li3, Zhenya Li3, Richard O. Hynes18, S. J. M. Jones19, Peter M.Kuehl20, Bruno Lemaitre21, J. Troy Littleton22, Deborah K. Morrison23, Chris Mungall12,Patrick H. O'Farrell24, Oxana K. Pickeral10, Chris Shue3, Leslie B. Vosshall25, JiongZhang10, Qi Zhao3, Xiangqun H. Zheng3, Fei Zhong3, Wenyan Zhong3, Richard Gibbs26, J.Craig Venter3, Mark D. Adams3, and Suzanna Lewis21Howard Hughes Medical Institute, Berkeley Drosophila Genome Project, University of California,Berkeley, CA 94720, USA2Department of Molecular and Cell Biology, Berkeley Drosophila Genome Project, University ofCalifornia, Berkeley, CA 94720, USA3Celera Genomics, Rockville, MD, 20850 USA4GenetixXpress, 78 Pacific Road, Palm Beach, Sydney, Australia 21085Massachusetts General Hospital Cancer Center, Building 149, 13th Street, Charlestown, MA02129 USA6Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104,USA7EMBL-EBI, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK8Department of Genetics, Stanford University, Palo Alto, CA 94305, USA9Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, WA 98109,USA10National Center for Biotechnology Information, National Library of Medicine, National Institutes ofHealth, Bethesda, MD 20894, USA11Neurogenetics Unit, Laboratory of Neurochemistry, National Institute of Neurological Disordersand Stroke, National Institutes of Health, Bethesda, MD 20892, USA12Berkeley Drosophila Genome Project, Lawrence Berkeley National Laboratory, Berkeley, CA94720, USA13Neomorphic, 2612 Eighth Street, Berkeley, CA 94710, USA14School of Biology, University of Leeds, Leeds LS2 9JT, UK15Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge,MA 02138, USA16Departments of Cellular and Molecular Medicine and Pharmacology, Howard Hughes MedicalInstitute, University of California–San Diego, La Jolla, CA 92093, USA17Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA

NIH Public AccessAuthor ManuscriptScience. Author manuscript; available in PMC 2009 September 29.

Published in final edited form as:Science. 2000 March 24; 287(5461): 2204–2215.

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18Howard Hughes Medical Institute, Massachusetts Institute of Technology (MIT), Cambridge, MA02139, USA19Genome Sequence Centre, BC Cancer Research Centre, 600 West 10th Avenue, Vancouver,BC, V52 4E6, Canada20Molecular and Cell Biology Program, University of Maryland at Baltimore, Baltimore, MD 21201,USA21Centre de Génétique Moléculaire, CNRS, 91198 Gif-sur-Yvette, France22Center for Learning and Memory, MIT, 77 Massachusetts Avenue, Cambridge, MA 02139, USA23Regulation of Cell Growth Laboratory, Division of Basic Sciences, National Cancer Institute–Frederick Cancer Research and Development Center, National Institutes of Health, Frederick, MD21702, USA24Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143,USA25Center for Neurobiology and Behavior, Columbia University, New York, NY 10032, USA26Baylor College of Medicine Human Genome Sequencing Center, Department of Molecular andHuman Genetics, Baylor College of Medicine, Houston, TX 77030, USA

AbstractA comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, andSaccharomyces cerevisiae—and the proteins they are predicted to encode—was undertaken in thecontext of cellular, developmental, and evolutionary processes. The nonredundant protein sets offlies and worms are similar in size and are only twice that of yeast, but different gene families areexpanded in each genome, and the multidomain proteins and signaling pathways of the fly and wormare far more complex than those of yeast. The fly has orthologs to 177 of the 289 human diseasegenes examined and provides the foundation for rapid analysis of some of the basic processesinvolved in human disease.

With the full genomic sequence of three major model organisms now available, much of ourknowledge about the evolutionary basis of cellular and developmental processes will derivefrom comparisons between protein domains, intracellular networks, and cell-cell interactionsin different phyla. In this paper, we begin a comparison of D. melanogaster, C. elegans, andS. cerevisiae. We first ask how many distinct protein families each genome encodes, how thegenes encoding these protein families are distributed in each genome, and how many genesare shared among flies, worms, yeast, and mammals. Next we describe the composition andorganization of protein domains within the proteomes of fly, worm, and yeast and examine therepresentation in each genome of a subset of genes that have been directly implicated ascausative agents of human disease. Then we compare some fundamental cellular anddevelopmental processes: the cell cycle, cell structure, cell adhesion, cell signaling, apoptosis,neuronal signaling, and the immune system. In each case, we present a summary of what wehave learned from the sequence of the fly genome and how the components that carry out theseprocesses differ in other organisms. We end by presenting some observations on what we havelearned, the obvious questions that remain, and how knowledge of the sequence of theDrosophila genome will help us approach new areas of inquiry.

The “Core Proteome”How many distinct protein families are encoded in the genomes of D. melanogaster, C.elegans, and S. cerevisiae (1), and how do these genomes compare with that of a simple

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prokaryote, Haemophilus influenzae? We carried out an “all-against-all” comparison of proteinsequences encoded by each genome using algorithms that aim to differentiate paralogs—highlysimilar proteins that occur in the same genome—from proteins that are uniquely represented(Table 1). Counting each set of paralogs as a unit reveals the “core proteome”: the number ofdistinct protein families in each organism. This operational definition does not includeposttranslationally modifed forms of a protein or isoforms arising from alternate splicing.

In Haemophilus, there are 1709 protein coding sequences, 1247 of which have no sequencerelatives within Haemophilus (2). There are 178 families that have two or more paralogs,yielding a core proteome of 1425. In yeast, there are 6241 predicted proteins and a coreproteome of 4383 proteins. The fly and worm have 13,601 and 18,424 (3) predicted protein-coding genes, and their core proteomes consist of 8065 and 9453 proteins, respectively. It isremarkable that Drosophila, a complex metazoan, has a core proteome only twice the size ofthat of yeast. Furthermore, despite the large differences between fly and worm in terms ofdevelopment and morphology, they use a core proteome of similar size.

Gene DuplicationsMuch of the genomes of flies and worms consists of duplicated genes; we next asked how theseparalogs are arranged. The frequency of local gene duplications and the number of theirconstituent genes differ widely between fly and worm, although in both genomes most paralogsare dispersed. The fly genome contains half the number of local gene duplications relative toC. elegans (4), and these gene clusters are distributed randomly along the chromosome arms;in C. elegans there is a concentration of gene duplications in the recombinogenic segments ofthe autosomal arms (1). In both organisms, approximately 70% of duplicated gene pairs are onthe same strand (306 out of 417 for D. melanogaster and 581 out of 826 for C. elegans). Thelargest cluster in the fly contains 17 genes that code for proteins of unknown function; the nextlargest clusters both consist of glutathione S-transferase genes, each with 10 members. Incontrast, 11 of 33 of the largest clusters in C. elegans consist of genes coding for seventransmembrane domain receptors, most of which are thought to be involved in chemosensation.Other than these local tandem duplications, genes with similar functional assignment in theGene Ontology (GO) classification (5) do not appear to be clustered in the genome.

We next compared the large duplicated gene families in fly, worm, and yeast without regardto genomic location. All of the known and predicted protein sequences of these three genomeswere pooled, and each protein was compared to all others in the pool by means of the programBLASTP. Among the larger protein families that are found in worms and flies but not yeastare several that are associated with multicellular development, including homeobox proteins,cell adhesion molecules, and guanylate cyclases, as well as trypsinlike peptidases and esterases.Among the large families that are present only in flies are proteins involved in the immuneresponse, such as lectins and peptidoglycan recognition proteins, transmembrane proteins ofunknown function, and proteins that are probably fly-specific: cuticle proteins, peritrophicmembrane proteins, and larval serum proteins.

Gene SimilaritiesWhat fraction of the proteins encoded by these three eukaryotes is shared? Comparativeanalysis of the predicted proteins encoded by these genomes suggests that nearly 30% of thefly genes have putative orthologs in the worm genome. We required that a protein showsignificant similarity over at least 80% of its length to a sequence in another species to beconsidered its ortholog (6). We know that this results in an underestimate, because the lengthrequirement excludes known orthologs, such as homeodomain proteins, which have littlesimilarity outside the homeodomain. The number of such fly-worm pairs does not decreasemuch as the similarity scores become more stringent (Table 2A), which strongly suggests that

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we have indeed identified orthologs, which may share molecular function. Nearly 20% of thefly proteins have a putative ortholog in both worm and yeast; these shared proteins probablyperform functions common to all eukaryotic cells.

We also compared the proteins of fly, worm, and yeast to mammalian sequences. Mostmammalian sequences are available as short expressed sequence tags (ESTs), so we dispensedwith the requirement for similarity over 80% of the length of the proteins. Table 2B presentsthese data. Half of the fly protein sequences show similarity to mammalian proteins at a cutoffof E < 10−10 (where E is expectation value), as compared to only 36% of worm proteins. Thisdifference increases as the criteria become more stringent: 25% versus 15% at E < 10−50 and12% versus 7% at E < 10−100. Because many of the comparisons are with short sequences, itis likely that many of these sequence similarities reflect conserved domains within proteinsrather than orthology. However, it does suggest that the Drosophila proteome is more similarto mammalian proteomes than are those of worm or yeast.

Protein Domains and FamiliesProteins are often mosaic, containing two or more different identifiable domains, and domainscan occur in different combinations in different proteins. Thus, only a portion of a protein maybe conserved among organisms. We therefore performed a comparative analysis of the proteindomains composing the predicted proteomes from D. melanogaster, C. elegans, and S.cerevisiae using sequence similarity searches against the SWISS-PROT/TrEMBLnonredundant protein database (7), the BLOCKS database (8), and the InterPro database (9).The 200 most common fly protein families and domains are listed in Table 3, and the 10 mosthighly represented families in worm and yeast are shown in Table 4. InterPro analyses plusmanual data inspection enabled us to assign 7419 fly proteins, 8356 worm proteins, and 3056yeast proteins to either protein families or domain families. We found 1400 different proteinfamilies or domains in all: 1177 in the fly, 1133 in the worm, and 984 in yeast; 744 familiesor domains were common to all three organisms.

Many protein families exhibit great disparities in abundance, and only the C2H2-type zincfinger proteins and the eukaryotic protein kinases are among the top 10 protein familiescommon to all three organisms. There are 352 zinc finger proteins of the C2H2 type in the flybut only 138 in the worm; whether this reflects greater regulatory complexity in the fly is notknown. The protein kinases constitute approximately 2% of each proteome. Curation of thegenomic data revealed that Drosophila has approximately 300 protein kinases and 85 proteinphosphatases, around half of which had previously been identified. In contrast, there areapproximately 500 kinases and 185 phosphatases in the worm; the difference is largely due tothe worm-specific expansion of certain families such as the CK1, FER, and KIN-15 families.There are currently approximately 600 kinases and 130 phosphatases in humans, and it isexpected that these figures will rise to 1100 and 300, respectively, when the sequence of thehuman genome is completed (10). Of the proteins uncovered in this analysis, over 70% exhibitsequence similarity outside the kinase or phosphatase domain to proteins in other species. Inthe kinase group, approximately 75% are serine/threonine kinases, and 25% are tyrosine ordual-specificity kinases. Over 90% of the newly discovered kinases are predicted tophosphorylate serine/threonine residues; this group includes the first atypical protein kinase Cisoforms identified in Drosophila. In addition, we found counterparts of the mammaliankinases CSK, MLK2, ATM, and Peutz-Jeghers syndrome kinase, and additional members ofthe Drosophila GSK3B, casein kinase I, SNF1-like, and Pak/STE20-like kinase families. Inthe fly protein phosphatase group, approximately 42% are predicted to be serine/threoninephosphatases; 48% are tyrosine or dual-specificity phosphatases. Among the newly discoveredphosphatases, 35% are serine/threonine phosphatases, most of which are related to the proteinphosphatase 2C family, and 65% are tyrosine or dual-specificity phosphatases. The fly and

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worm both contain close relatives to many of the known mammalian lipid kinases andphosphatases; however, no SH2-containing inositol 5′ phosphatase SHIP is apparent. Finally,it has been found that the assembly of kinase signaling complexes in vertebrate cells is aidedby the presence of scaffolding and adaptor molecules, many of which contain phosphoproteinbinding domains; we found 85 such proteins in the fly, including counterparts to IRS, VAV,SHC, JIP, and MP1.

Two remarkable findings emerge from the peptidase data that may reflect different approachesto growth and development in flies, worms, and humans. The pattern and distribution ofpeptidase types are similar between the fly and the worm: there are approximately 450peptidases in the fly and 260 in the worm. The difference is due almost entirely to the expansionor contraction of a single class of trypsin-like (S1) peptidases. C. elegans has seven of thisclass and yeast has one, but the fly has 199. Of these, 163 are small proteins of approximately250 amino acids containing single trypsin domains; very few are mosaic proteins. Theremainder have either multiple trypsin-like domains or long stretches of amino acids with noreadily identifiable motif, usually at the NH2-terminus. In humans, trypsin-like peptidasesperform diverse functions in digestion, in the complement cascade, and in several othersignaling pathways (11), and flies may have a similarly wide range of uses for these proteins.The extensively characterized members of this family, which include Snake, Easter, Nudel,and Gastrulation-defective, are all key members of a regulatory cascade that controlsdorsoventral patterning in the fly (12). In addition, flies have only two members of the M10class of peptidases, which include the matrix metalloproteases, collagenases, and gelatinasesthat are essential for tissue remodeling and repair in vertebrates.

The number of identifiable multidomain proteins is similar in the fly and the worm: 2130 and2261, respectively. Yeast has only 672 (Table 5). Part of this difference is accounted for byproteins with extracellular domains involved in cell-cell and cell-substrate contacts (13), suchas the immunoglobulin domain–containing proteins, which are more abundant in flies than inworms (153 versus 70) and are nonexistent in yeast. Two other common extracellular domainsoccur in similar numbers in fly and worm: EGF (110 versus 109, respectively) and fibronectintype III (46 versus 43) but are rare or absent in yeast. Extracellular regions of proteins oftencontain a variety of repeated domains (14), and so these proteins may account for our findingthat flies have a larger number of proteins with multiple InterPro domains than either wormsor yeast (2107 versus 1747 and 525, respectively) (Table 6). Some multidomain proteins ofthe fly are particularly heterogeneous: Two low-density lipoprotein receptor–related proteinshave 75 InterPro domains each. Another protein of unknown function has 62 InterPro domains;the most heterogeneous worm and yeast proteins [SWISS-PROT/TrEMBL accession numbers(AC), Q04833 and P32768, respectively] have 61 and 18 InterPro domains, respectively. Therecan be extensive repetition of the same domain within a protein; for example, animmunoglobulin-like domain is repeated 52 times within one protein of unknown function inthe fly. The large worm protein UNC-89 contains 48 immunoglobulin-like domains (SWISS-PROT/TrEMBL AC, Q17362). In contrast, the largest number of repeats in yeast, of a C2H2-type zinc finger domain, occurs nine times in the transcription factor TFIIIA (SWISS-PROT/TrEMBL AC, P39933).

The heterotrimeric GTP-binding protein (G protein)–coupled receptors (GPCRs) are a largeprotein family in flies, worms, and vertebrates whose members are involved in synapticfunction, hormonal physiology, and the regulation of morphological movements duringgastrulation and germ band extension (15). There are predicted to be at least 700 GPCRs inthe human genome (16) and roughly 1100 GPCRs in C. elegans (17). We found approximately160 GPCR genes in the Drosophila genome, 57 of which appear to be olfactory receptors.Drosophila, C. elegans, and vertebrates each have diverse families of odorant receptors that,although recognizable as GPCRs, are unrelated by sequence and therefore apparently evolved

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independently. The number of odorant receptors in vertebrates ranges from around 100 inzebrafish and catfish to approximately 1000 in the mouse; C. elegans also has approximately1000. In the fly, as in zebrafish and mouse, there is a correlation between the number of odorantreceptors and the number of discrete synaptic structures called glomeruli in the olfactoryprocessing centers of the brain (16,18). In the mouse, each glomerulus is dedicated to receivingaxonal input from neurons expressing a particular odorant receptor (16). Therefore, thecorrelation between number of odorant receptors and number of glomeruli may reflect aconservation in the organizational logic of odor recognition in insect and vertebrate brains.Although the fly odorant receptors are extremely diverse, there are a number of subfamilieswhose members share 50 to 65% sequence identity. The distribution of odorant receptor genesis different among these organisms as well. Unlike C. elegans or vertebrate odorant receptors,which are in large linked arrays, the fly odorant receptor genes are distributed as single genesor in arrays of two or three. Vertebrate receptors are encoded by intronless genes, but both flyand worm receptor genes have multiple introns. These distinctions suggest that in addition todifferences in the sequences of the odorant receptors of the different organisms, the processesgenerating the families of receptors may have differed among the lineages that gave rise toflies, worms, and vertebrates.

The data suggest conservation of hormone receptors between flies and vertebrates;nevertheless, there is a greater diversity of hormone receptors in both C. elegans and vertebratesthan in Drosophila. Insects are subject to complex hormonal regulation, but no apparenthomologs of vertebrate neuropeptide and hormone precursors were identified. However, manyreceptors with sequence similarity to vertebrate receptors for neurokinin, growth hormonesecretagogue, leutotropin (follicle-stimulating hormone and luteinizing hormone), thyroid-stimulating hormone, galanin/allatostatin, somatostatin, and vasopressin were identified. OtherGPCRs include a seventh Drosophila rhodopsin and homologs of adenosine, metabotropicglutamate, γ-aminobutyric acid (GABA), octopamine, serotonin, dopamine, and muscarinicacetylcholine receptors. In addition, there are GPCRs that are unique to Drosophila, otherswith sequence similarity to C. elegans and human orphan receptors, and an insect diuretichormone receptor that is closely related to vertebrate corticotropin-releasing factor receptor.Finally, we found several atypical seven-transmembrane domain receptors, including 10Methuselah (MTH)–like proteins and four Frizzled (FZ)–like proteins. A mutation in mthincreases the fly's life-span and its resistance to various stresses (19); the FZ-like proteinsprobably serve as receptors for different members of the Wingless/Wnt family of ligands.

Human Disease GenesStudies in model organisms have provided important insights into our understanding of genesand pathways that are involved in a variety of human diseases. In order to estimate the extentto which different types of human disease genes are found in flies, worms, and yeast, wecompiled a set of 289 genes that are mutated, altered, amplified, or deleted in a diverse set ofhuman diseases and searched for similar genes in D. melanogaster, C. elegans, and S.cerevisiae, as described in the legend to Fig. 1. Of these 289 human genes, 177 (61%) appearto have an ortholog in Drosophila (Fig. 1). Only proteins with similar domain structures wereconsidered to be orthologs; this judgment was made by human inspection of the InterProdomain composition of the fly and human proteins. The importance of human inspection, aswell as consideration of published information, is underscored by the fact that some sequenceswith extremely high similarity scores to proteins encoded by fly genes, such as LCK andMyotonic Dystrophy 1, were judged not to be orthologous, but others with relatively low scores,such as p53 and Rb1, were considered to be orthologs. We attempted this additional level ofanalysis only for the fly proteins, as the lower overall level of similarity of worm and yeastproteins made these subjective judgments even more difficult. Some of the human diseasegenes that are absent in Drosophila reflect clear differences in physiology between the two

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organisms. For instance, none of the hemoglobins, which are mutated in thalassemias, haveorthologs in Drosophila. In flies, oxygen is delivered directly to tissues via the tracheal systemrather than by circulating erythrocytes. Similarly, several genes required for normalrearrangement of the immunoglobulin genes do not have Drosophila orthologs.

Of the cancer genes surveyed, 68% appear to have Drosophila orthologs. In addition topreviously described proteins, these searches identified clear protein orthologs for menin(MEN; multiple endocrine neoplasia type 1), Peutz-Jeghers disease (STK11), ataxiatelangiectasia (ATM), multiple exostosis type 2 (EXT2), a second bCL2 family member, asecond retinoblastoma family member, and a p53-like protein. Despite its relatively lowsequence similarity to the human genes, the Drosophila gene encoding p53 was considered anortholog because it shows a conserved organization of functional domains, and its DNA bindingdomain includes many of the same amino acids that appear to be hot spots for mutations inhuman cancer. Comparison of the fly p53-like protein with the human p53, p63, and p73proteins suggests that it may represent a progenitor of this entire family. In mammalian cells,levels of p53 protein are tightly regulated in vivo by its interaction with the Mdm2 protein,which in turn binds to p19ARF (20). This mode of regulation, which modulates the activity ofp53 but probably not of p63 or p73 (21), may not apply to the Drosophila protein, because wehave not been able to identify orthologs of either Mdm2 or p19ARF in Drosophila.Interestingly, likely orthologs of the breast cancer susceptibility genes BRCA1 and BRCA2were not found in Drosophila. In most instances, cancer genes that have a Drosophila orthologalso have an ortholog in C. elegans, although the extent of sequence similarity to the wormgene is lower. In a minority of instances, a C. elegans ortholog was clearly absent. Cancergenes with orthologs in Drosophila and apparently not in C. elegans include p53 andneurofibromatosis type 1 (22), the two genes implicated in tuberous sclerosis (TSC1 andTSC2) (23), and MEN. The two TSC gene products are thought to bind to each other and mayfunction in a pathway that is conserved between humans and Drosophila but is absent in C.elegans and S. cerevisiae. However, the limitations of this type of analysis are clearly illustratedby our inability to find a bCL2 ortholog in C. elegans using these search parameters. The C.elegans ced-9 gene has been shown to function as a bCL2 homolog, and its protein is 23%identical to the human protein over its entire length (24).

Numerous orthologs of neurological genes are also found in the Drosophila genome. Some,such as Notch (CADASIL syndrome), the beta amyloid protein precursorlike gene, andPresenilin (Alzheimer's disease), were already known from previous studies in the fly. Thegenome sequencing effort has uncovered several additional genes that are likely to be orthologsof human neurological genes, such as tau (frontotemporal dementia with Parkinsonism), theBest macular dystrophy gene, neuroserpin (familial encephalopathy), genes for limb girdlemuscular dystrophy types 2A and 2B, the Friedreich ataxia gene, the gene for Miller-Diekerlissencephaly, parkin (juvenile Parkinson's disease), and the Tay-Sachs and Stargardt's diseasegenes. Several genes implicated in expanded polyglutamine repeat diseases, includingHuntington's and spinal cerebellar ataxia 2 (SCA2), are found in the fruit fly. Most humanneurological disease genes surveyed were also detected in C. elegans, and some were evenfound in yeast, although a few examples are apparently present only in Drosophila, such asthe Parkin and SCA2 orthologs.

Among genes implicated in endocrine diseases, those functioning in the insulin pathway aremostly conserved. In contrast, members of pathways involving growth hormone,mineralocorticoids, thyroid hormone, and the proteins that regulate body mass in vertebrates,such as those encoding leptin, do not appear to have Drosophila orthologs. Surprisingly, aprotein that shows significant sequence similarity to the luteinizing hormone receptor is presentin Drosophila (25). The physiological ligand for this receptor is not known. A number of genesthat have been implicated in human renal disorders have orthologs in Drosophila, despite the

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differences between human kidneys and insect Malpighian tubules. In many instances, thesegene products are involved in fluid and electrolyte transport across epithelia. Not surprisingly,most disease genes that function in intracellular metabolic pathways appear to haveDrosophila orthologs.

Developmental and Cellular ProcessesDevelopmental strategies in various phyla are overtly very different, from the fixed cell lineageof C. elegans to the syncytial embryogenic development of the fly, to early embryogenesis inamphibians and mammals. A number of major processes—cell division, cell shape, signalingpathways, cell-cell and cell-substrate adhesion, and apoptosis— determine the developmentaloutcomes of these very different embryos. Although there are many more, such as the processesthat determine embryonic gradients, cell polarities, and cell movement, here we examine thefirst five, beginning with cell cycle components, and examine what new insights have beengained from the genomic data that affect our knowledge of the evolution of developmentalprocesses. We then discuss the processes of neuronal signaling and innate immunity.

Cell cycleDespite conservation of the mechanisms regulating cell cycle progression, many of thefunctions governing this progression are encoded by gene families whose individual membersare not conserved between vertebrates and yeast. For example, the cyclins of S. cerevisiae canbe divided into a G1 class (Cln1, Cln2, and Cln3) and an S/G2 class (Clb1 through Clb6); it isnot possible to identify orthologs of individual vertebrate cyclins. Consequently, analysis ofthe roles of particular vertebrate cell cycle genes benefits from a genetic model in whichparallels are more evident. Analysis of the Drosophila genome sequence supports and extendsprevious suggestions of strong parallels between fly and human cell cycle regulators. Orthologsof vertebrate cell cycle cyclins—cyclin A (CycA), CycB, CycB3, CycE, and CycD—have beenidentified in Drosophila, as have orthologs of cyclins that appear to have roles in transcription:CycC, CycH, CycK, and CycT. Apparent orthologs of these cyclins can be also be found inC. elegans; however, the level of similarity to the vertebrate members is invariably substantiallyless. Indeed, BLAST comparisons suggest that vertebrate and Drosophila CycA and CycBshare more sequence similarity with yeast than with proposed C. elegans orthologs.Examination of other cell cycle regulators confirms that quite precise comparisons can be madebetween vertebrates and flies; parallels with yeast are looser. For example, like vertebrates,Drosophila uses several different cyclin-dependent kinases (Cdks) to regulate different aspectsof the cell cycle; S. cerevisiae and Schizosaccharomyces pombe use only one. Cloning effortsand the genome sequence revealed Drosophila orthologs of vertebrate Cdk1 (cdc2) and Cdk2(cdc2c), as well as a single Drosophila Cdk (Cdk4/6) with close similarity to both Cdk4 andCdk6. As in vertebrates, Drosophila has two distinct kinases that add inhibitory phosphate toCdk1, the previously identified Wee, and a recently recognized homolog of Myt1, which wasinitially identified as a membrane-associated inhibitory kinase in Xenopus (26). C. elegans alsohas two homologs of these kinases (Wee1.1 and Wee1.3); however, similarity scores do notplace these into distinct Wee1 and Myt1 subtypes. Each of these genes appears to be presentin a single copy, a factor that simplifies genetic interpretations.

The retinoblastoma gene product pRb is a crucial cell cycle regulator in mammals and is thoughtto modulate S-phase entry via its interactions with the transcriptional regulator E2F and itsdimerization partner (DP). This important mode of regulation is not found in yeast, but manycomponents of the Rb pathway have been identified and studied in Drosophila (27). Thesequencing effort uncovered a second Rb-related gene in Drosophila and confirmed theexistence of only two E2F family members and a single DP ortholog. C. elegans also has anRb-related gene, isolated in a genetic screen for mutations affecting cell fate decisions (28),but it has not been shown to play a direct role in cell cycle regulation. Also evident from the

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sequence are eight skp-like genes and six cullin-related genes. The Skp and Cullin proteinsfunction in a complex that mediates the degradation of specific target proteins during crucialcell cycle transitions. Further exploration of the genome sequence should define orthologs tomost vertebrate cell cycle genes and lead to genetic tests of their regulation and function.

CytoskeletonA large number of proteins link events at the cell surface with cytoskeletal networks andintracellular messengers (13). We found approximately 230 genes (approximately 2% of thepredicted genes) that encode cytoskeletal structural or motor proteins; these represent mostmajor families found in other invertebrates and vertebrates (29). The fraction of theDrosophila genome devoted to cytoskeletal functions appears to be somewhat smaller thanthat found in C. elegans (5%) (30); whether this reflects a true biological difference or adifference in classification criteria remains to be discovered. Of the Drosophila cytoskeletalgenes, 90 encode proteins belonging to the kinesin, dynein, or myosin motor superfamilies, oraccessory or regulatory proteins known to interact with the motor protein subunits.Approximately 80 genes encode actin-binding proteins, including proteins belonging to thespectrin/α-actinin/dystrophin superfamily of membrane cytoskeletal and actin–cross-linkingproteins. Twenty genes encode proteins that are likely to bind microtubules, based on theirsimilarity to microtubule-binding proteins found in other organisms. Fourteen genes encodemembers of the actin superfamily, 12 encode members of the tubulin superfamily, and 5 encodeseptins. Overall, the representation of predicted cytoskeletal protein types and families issimilar to what has been found for C. elegans, although Drosophila has many more dyneins,probably because C. elegans lacks motile cilia and flagella.

Among this collection of cytoskeletal genes are several interesting and in some cases long-sought genes. One gene encodes a protein with striking homology to proteins of the tau/MAP2/MAP4 family that share a characteristic repeated microtubule-binding domain. Two encodenew tubulins; one appears most closely related to α-tubulin, and the other appears most closelyrelated to β-tubulin, both with approximately 50% identity. Neither new tubulin has greatersimilarity to the other, more divergent members of the tubulin superfamily, such as γ-, δ-, orε-tubulin (31). Thus, both Drosophila and C. elegans appear to lack δ- and ε-tubulin, eventhough δ-tubulin is highly conserved between Chlamydomonas and humans. There are alsothree new members of the central motor domain family of kinesins that encode nonmotorproteins that regulate microtubule dynamics (32). There are clear homologs of the dystrophincomplex and of dystrobrevin. Finally, the fly lacks cytoplasmic intermediate filament proteins,other than nuclear lamins, although other invertebrates, including C. elegans, appear to havegenes encoding these (33). Drosophila and C. elegans both also appear to lack a gene encodingkinectin, the proposed receptor for kinesin and cytoplasmic dynein on vesicles and organelles(34). Flies and worms must thus use different proteins to link microtubule motors to vesiclesand organelles.

Cell adhesionCell-cell adhesion and cell-substrate adhesion molecules have been crucial to the developmentof multicellular organisms and the evolution of complex forms of embryogenesis (13). Thetransmembrane extracellular matrix-cytoskeleton linkage via integrins is ancient. There arefive α and two β integrins in the fly, two α and one β in C. elegans, and at least 18 α and eightβ in vertebrates. Integrin-associated cytoplasmic proteins (talin, vinculin, α-actinin, paxillin,FAK, p130CAS, and ILK) are encoded by single-copy fly genes, as are tensin and syndecan.

Two genes for type IV collagen subunits and genes for the three subunits of laminin werealready known in the fly. Analysis of the genome revealed no more laminin genes and onlyone more collagen, which is closest to types XV and XVIII of vertebrates. A counterpart of

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this collagen is found in C. elegans, which has on the order of 170 collagens. Most important,it appears that the core components of basement membranes (two type IV collagen subunits,three laminin subunits, entactin/nidogen, and one perlecan), are all present in flies. Thisconstitution of basement membranes was clearly established early in evolution and has beenwell conserved in metazoans; remarkably, the fly preserves the linked head-to-headorganization of vertebrate type-IV collagen genes. In contrast to this conservation, many well-known vertebrate integrin (ECM) ligands are absent from the fly: fibronectin, vitronectin,elastin, von Willebrand factor, osteopontin, and fibrillar collagens are all missing.

The fly has three classic cadherins, two of which are closely linked, but no protocadherins ofthe type found in vertebrates as clusters with common cytoplasmic domains (35). Vertebrateshave three such clusters encoding over 50 protocadherins and close to 20 classical cadherins.The fly has no reelin, an ECM ligand for CNR-type protocadherins in vertebrates (36).However, there are other fly proteins with cadherin repeats, including the previously knownFat, Dachsous, and Starry night, and a new very large protein related to Fat. C. elegans has 15genes containing cadherin repeats; the number in humans is now 70 and will undoubtedly rise(13).

Cell signalingComponents of known signaling pathways in the fly and worm have largely been uncoveredby examinations of developmental systems. It is a tribute to the previous genetic analyses donein these organisms that only a modest number of new components of the known signalingpathways were revealed by analysis of the genomic sequence. The core components definedin flies and worms have been used in modified and expanded forms in vertebrates (37). Thepredominant pathways—transforming growth factor–β (TGF-β), receptor tyrosine kinases,Wingless/Wnt, Notch/lin-12, Toll/IL1, JAK/STAT/cytokine, and Hedgehog (HH) signalingnetworks—all have largely conserved fly and vertebrate components. The worm, by contrast,does not appear to possess the HH or Toll/IL1 pathways, nor does it have all of the componentsof the Notch/lin-12 network (38). Two new proteins of the TGF-β superfamily were identified,bringing the total to seven; all seven are members of the bone morphogenetic protein (BMP)or β-activin subfamilies. We detected no representatives of the other branches of thissuperfamily, namely the TGF-β, α-inhibin, and Mullerian inhibiting substance (MIS)subfamilies. Three new members of the Wingless/Wnt family were identified, bringing thetotal to seven. Each of these proteins has sequence similarity to a different vertebrate Wntprotein; this ancient family clearly underwent much of its expansion before the divergence ofthe arthropod and chordate lineages. There is only one member of the Notch and HH families,in contrast to the many members of these families in vertebrates.

ApoptosisThe core apoptotic machinery of Drosophila shares many features in common with that ofmammals. Many apoptosis-inducing signals lead to activation of members of the caspasefamily of proteases. These proteases function in apoptotic processes as cell death signaltransducers and death effectors, and in nonapoptotic processes in flies and mammals (39).Drosophila contains genes encoding 8 caspases, as compared to 4 in the worm and at least 14in mammals. Three of the fly caspases contain long NH2-terminal prodomains of 100 to 200amino acids that are characteristic of caspases that function as signal transducers. Theseprodomains are thought to mediate caspase recruitment into signaling complexes in whichactivation occurs in response to oligomerization. In one pathway described in mammals butnot in worms, death signals cause the release of proteins, including cytochrome c and theapoptosis-inducing factor (AIF), from mitochondria (40). The human protein Apaf-1, inconjunction with cytochrome c, activates CARD domain–containing caspases (41).Drosophila has an Apaf-1 counterpart, a CARD domain–containing caspase, and AIF;

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Drosophila also has counterparts to the caspase-activated DNAse CAD/CPAN/DFF40, itsinhibitor ICAD/DFF45, and the chromatin condensation factor Acinus (42).

Pro- and anti-apoptotic BCL2 family members regulate apoptosis at multiple points (43).Drosophila encodes two BCL2 family proteins, though more divergent family members mayexist. Fifteen BCL2 family proteins have been identified in mammals and two in the worm. Inaddition, inhibitor of apoptosis (IAP) family proteins negatively regulate apoptosis (44). Theyare defined by the presence of one or more NH2-terminal repeats of a BIR domain, a motif thatis essential for death inhibition. Drosophila has four proteins with this motif, as compared toseven identified thus far in mammals. There are several BIR domain–containing proteins inC. elegans and yeast, but none has been implicated in cell death regulation. Reaper (RPR),Wrinkled (W), and Grim are essential Drosophila cell death activators (45). Orthologs havenot been identified in other organisms, but they are likely to exist because RPR, W, and Griminduce apoptosis in vertebrate systems and physically interact with apoptosis regulators thatinclude IAPs and the Xenopus protein Scythe (46), for which there is a predicted Drosophilahomolog.

Neuronal signalingThe neuronal signaling systems in flies, worms, and vertebrates reveal extensive conservationof some components, as well as extreme divergence, or the total absence, of others. There isno voltage-activated sodium channel in the worm (17); flies and vertebrates generate sodium-dependent action potentials. The fly genome encodes two pore-forming subunits for sodiumchannels (Para and NaCP60E), and also four voltage-dependent calcium channel α subunits,including one T-type/α1G, one L-type/α1D (Dmca1D), one N-type/α1A (Dmcα1A), and oneprotein that is more similar to an outlying C. elegans protein than to known vertebrate calciumchannels. Additional fly calcium channel subunits include one (β, one γ 2, and three α 2subunits.

The worm genome encodes over 80 potassium channel proteins (17); the fly genome has only30. The extent to which these different family sizes contribute to the establishment of uniqueelectrical signatures is unknown. The fly potassium channel family includes five Shaker-likegenes (Shaker, Shab, Shal, and two Shaws); a large conductance calcium-activated channelgene (slowpoke); a slack subunit relative; three members of the eag family (eag, sei, and elk);one small conductance calcium-regulated channel gene; one KCNQ channel gene; and fourcyclic nucleotide–gated channel genes. In addition, there are 50 TWIK members in the worm,but only 11 fly members of the two-pore/TWIK family with four transmembrane domains.There are also three fly members of the inward rectifier/two transmembrane family. Finally,neither the fly nor the worm has discernible relatives of a number of mammalian channel-associated subunits such as minK and miRP1.

There are also major differences postsynaptically. C. elegans has approximately 100 membersof a family of ligand-gated ion channels (17); flies have about 50. The worm has 42 nicotinicacetylcholine receptor subunits and 37 GABA(A)-like receptor subunits; the fly contains only11 nicotinic receptor subunit genes and 12 GABA(A)/glycine-like receptor subunit genes. Incontrast, there are 30 members of the excitatory glutamate receptor family in the fly but only10 in the worm. These include subtypes of the AMPA, kainate, NMDA, and delta families. Inaddition, the fly genome contains a large number of PDZ-containing genes, approximately adozen of which encode proteins that have high sequence similarity to mammalian proteins thatinteract with specific subsets of ion channels. We also found a number of additional ion channelfamilies, including three voltage-dependent chloride channels, 14 Trp-like channels, 24amiloride-sensitive/degenerin-like sodium channels, one ryanodine receptor, one IP3 (inositol1,4,5-trisphosphate) receptor, eight innexins, and two porins. C. elegans is missing a nitricoxide synthase gene, copies of which occur in fly and vertebrate genomes.



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A large array of proteins mediates specific aspects of synaptic vesicle trafficking andcontributes to the conversion of electrical signals to neurotransmitter release. Thesecomponents of exocytosis and endocytosis are relatively well conserved with respect to bothdomain structures and amino acid identities (50 to 90%). The fly has enzymes for the synthesisof the neurotransmitters glutamate, dopamine, serotonin, histamine, GABA, acetylcholine, andoctopamine, and a family of conserved transporters is likely to be involved in loading vesicleswith these neurotransmitters. The conserved vesicular trafficking proteins, with 50 to 80%amino acid identity, include members of the Munc-18, SCAMP, synaptogyrin, HRS2,tomosyn, cysteine string protein, exocyst (SEC 5, 6, 7, 8, 10, 13, 15, EXO 70, and EXO84),synapsin, rab-philin-3A, RIM, rab-3, CAPS, Mint, Munc-13, NSF, α and γ SNAP, DOC-2B,latrophilin, Veli, CASK, VAP-33, Snapin, SV2, and complexin families. Generally, there isonly one homolog in Drosophila for every three to four isoforms in mammals. However, thereare eight fly synaptotagmin-like genes, making this the largest family of vesicle proteins inDrosophila (47). However, there is no homolog of synaptophysin, an early candidate for avesicle fusion pore, which indicates a nonessential role in exocytosis for this particular proteinacross phyla.

Membrane trafficking also requires interactions between compartment-specific vesicular andtarget membrane proteins (v-SNAREs and t-SNAREs, respectively), whose subcellulardistribution and combinatorial binding patterns are predicted to define organelle identity andtargeting specificity (48). The completed fly genome allows us to address whether there is anycorrelation between the increased developmental complexity of multicellular organisms and alarger number of SNAREs than that found in unicellular organisms. In the fly, we find sixsynaptobrevins, three SNAP-25s, 10 syntaxins, and four additional t-SNAREs (membrin,BET1, UFE1, and GOS28), and the number of SNAREs is similar between yeast (49) andDrosophila. Thus, basic subcellular compartmentalization and membrane trafficking to andbetween these various compartments has not changed dramatically in multicellular versusunicellular organisms. Dynamin, clathrin, the clathrin adapter proteins, amphiphysin,synaptojanin, and a number of additional genes that encode proteins with defined endocytoticmotifs are all present.

In contrast to the conservation of the synaptic vesicle trafficking machinery, the few identifiedproteins present at mammalian active zones, namely aczonin, bassoon, and piccolo, do nothave relatives in Drosophila. There are, however, numerous proteins in the fly withcombinations of C2 domains, PDZ domains, zinc fingers, and proline-rich domains, indicatingthat the precise protein composition of active zones is likely to vary among metazoans. Inaddition, Drosophila contains a neurexin III gene and four neuroligin genes that may be partof a neurexin-neuroligin complex that has been widely proposed to provide a synaptic scaffoldfor linking pre- and postsynaptic structures in mammals (50). Potential agrin and Musk genesare also present, though the overall sequence similarity is low.

ImmunityMulticellular organisms have elaborate systems to defend against microbial pathogens. Onlyvertebrates have an acquired immune system, but both vertebrates and invertebrates share amore primitive innate immune system. Innate immunity is based on the detection of commonmicrobial molecules such as lipopolysaccharides and peptidoglycans by a class of receptorsknown as pattern recognition receptors (51). We identified a large family of genes encodinghomologs of receptors that are involved in microbial recognition in other organisms. Theseinclude two new homologs of the Drosophila Scavenger Receptors (dSR-CI), nine membersof the CD36 family, 11 members of the peptidoglycan recognition protein (PGRP) family,three Gram-negative binding protein (GNBP) homologs, and several lectins (52).



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The recognition of infection by immuno-responsive tissues induces a battery of defense genesvia Toll/nuclear factor kappa B (NF-κB) pathways in both Drosophila and mammals (53). TheToll receptor was initially discovered as an essential component of the pathway that establishesthe dorsoventral axis of the Drosophila embryo. Recent genetic studies now reveal that Tollsignaling pathways are key mediators of immune responses to fungi and bacteria in bothDrosophila and mice (53). We found seven additional homologs of Toll proteins inDrosophila, all of which are more similar to each other than to their mammalian counterparts.Some of these other Toll proteins, like 18-wheeler, will probably mediate innate immuneresponses. In Drosophila, infection by at least some microbes induces a proteolytic cascadethat leads to the processing of Spaetzle (SPZ), a cytokine-like protein, which then activatesToll (53). We found two proteins related to SPZ with similarities that include most or all ofthe cysteine residues of SPZ. Given the presence of multiple Toll-like receptors inDrosophila, these new SPZ-like proteins may also function in the immune system. With theexception of the two I-κB kinase homologs and the three rel proteins (Dorsal, Dif, and Relish),the Drosophila genome appears to contain only single copies of the genes encodingintracellular components of the Toll pathway: Tube, Pelle, and Cactus. How do the differentToll receptors trigger specific immune responses using the same intracellular intermediates?One explanation is that additional signaling components remain unidentified; anotherexplanation is crosstalk with other signaling pathways. In contrast, a Toll ortholog has not beenidentified in C. elegans, although there are some Toll-like receptors. C. elegans, in addition,does not possess homologs of NF-κB/dorsal transcriptional activators that functiondownstream of Toll. Although it is probable that the worm has retained parts of the innateimmunity network, there is no clear evidence of an inducible host defense system in the worm.

One of the most potent innate immune responses in insects is the transcriptional induction ofgenes encoding antimicrobial peptides (53). In contrast to Metchnikowin, Drosocin, andDefensin peptides, which are encoded by single genes, the sequence data indicate that, like thepreviously identifed cecropin clusters, several antimicrobial peptides are encoded by genefamilies that are larger than previously suspected. Four genes appear to encode antifungalpeptide Drosomycin isoforms, and two genes each code for the antibacterial proteins Attacinand Diptericin. These additional genes may generate peptides with slightly different spectra ofantimicrobial activity or may simply amplify the antimicrobial response.

Concluding RemarksWhat have we learned about the proteins encoded by the three sequenced eukaryotic genomes?Some information emerges readily from the comparison of the fly, worm, and yeast genomes.First, the core proteome sizes of flies and worms are similar and are only twice the size of thatof yeast. This is perhaps counterintuitive, because the fly, a multicellular animal withspecialized cell types, complex development, and a sophisticated nervous system, looks morethan twice as complicated as single-celled yeast. The lesson is that the complexity apparent inthe metazoans is not achieved by sheer number of genes (54). Second, there has been aproliferation of bigger and more complex proteins in the two metazoans relative to yeast,including, not surprisingly, more proteins with extracellular domains involved in cell-cell andcell-substrate interactions. Finally, the population of multidomain proteins is somewhat largerand more diverse in the fly than in the worm. There is presently no practical way to quantifydifferences in biological complexity between two organisms, however, so it is not possible tocorrelate this increased domain expansion and diversity in the fly with differences indevelopment and morphology.

The availability of the annotated sequence of the Drosophila genome enhances the fly'susefulness as an experimental organism. By greatly facilitating positional cloning, the genomesequence will increase the efficiency of genetic screens that seek to identify genes underlying



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many complex processes of cell biology, development, and behavior. Such screens have beenthe mainstay of Drosophila research and have contributed enormously to our knowledge ofmetazoan biology. The genome sequencing effort has revealed a number of previouslyunknown counterparts to human genes involved in cancer and neurological disorders; forexample, p53, menin, tau, limb girdle muscular dystrophy type 2B, Friedrich ataxia, andparkin. All of these fly genes are present in a single copy in the genome and can be geneticallyanalyzed without uncertainty about redundant copies. More genetic screens are important inorder to uncover interacting network members. Orthologs of these network members can thenbe sought in the human genome to determine if alterations in any of them predispose humansto the disease in question, an experimental paradigm that has already been successfullyexecuted in several cases. Flies can also play an important role in exploring ways to rectifydisease phenotypes. For example, at least 10 human neurodegenerative diseases are caused byexpansion of polyglutamine repeats (55). Human proteins containing expanded polyglutaminerepeats have been expressed in flies, resulting in the formation of nuclear inclusions that containthe protein as well as other shared components (56), just as in humans. It has been shown thatdirected expression of the human HSP70 chaperone in the fly can totally suppressneurodegeneration resulting from expression of the human spinocerebellar ataxia type 3 protein(57). The power and speed of this in vivo system are unparalleled, and we anticipate theincreased use of such “humanized” fly models.

Knowing the complete genomic sequence also allows new experimental approaches to long-standing problems. For example, it makes it possible to study networks of genes rather thanindividual genes or pathways. Assaying the level of transcription of every gene in the genomemakes it at least theoretically possible to monitor the expression of an entire network of genessimultaneously. One problem that is approachable this way is the combinatorial control of genetranscription. The fly genome appears to encode only about 700 transcription factors, andmutations in over 170 have already been isolated and characterized. The techniques areavailable to measure the changes in expression of every gene in individual cell types as aconsequence of loss or overexpression of each transcription factor. We can look for commonsequence elements in the promoters of coregulated genes and perform chromatin immuno-precipitation to identify the in vivo binding sites of individual factors. For the first time, wecan envision obtaining the data needed to understand the behavior of a complex regulatorynetwork. Of course, collecting these data is a massive task, and developing methods to analyzethe data is even more daunting. But it is no longer ludicrous to try.

How big is the core proteome of humans? Vertebrates have many gene families with three orfour members: the HOX clusters, calmodulins, Ezrins, Notch receptors, nitric oxide synthases,syndecans, and NF1 transcription factor genes are some examples (58). This is evidence fortwo genome doublings during mammalian evolution, superimposed on which were theamplifications and contractions over evolutionary time that uniquely characterize each lineage(59). The human genome, with 80,000 or so genes, is likely to be an amplified version of avery much smaller genome, and its core proteome may not be much larger than that of the flyor worm; that is, the more complex attributes of a human being are achieved using largely thesame molecular components. The evolution of additional complex attributes is essentially anorganizational one; a matter of novel interactions that derive from the temporal and spatialsegregation of fairly similar components.

Finally, approximately 30% of the predicted proteins in every organism bear no similarity toproteins in its own proteome or in the proteomes of other organisms. In other words, sequencesimilarity comparisons consistently fail to give us information about nearly a third of thecomponents that make every organism uniquely itself. What does this mean with respect to theevolution and function of these proteins? Does each genome contain a sub-population of veryrapidly evolving genes? One-third of randomly chosen cDNA clones do not cross-hybridize



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between D. melanogaster and Drosophila virilis (60). Even though these are distantly relatedspecies, they are developmentally and morphologically very similar. Crystallographic data willbe needed to determine whether these proteins that have diverged in primary sequence havemaintained their three-dimensional structures or have diverged so far that new folds anddomains have formed.

Our first look at the annotated fly genome provokes these and other questions. Access to thegenomic sequence will help us design the experiments needed to answer them. The relativesimplicity and manipulability of the fly genome means that we can address some of thesebiological questions much more readily than in vertebrates. That is, after all, what modelorganisms are for.

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54. Miklos GLG. J Am Acad Arts Sci 1998;127:197.55. Perutz M. Trends Biochem Sci 1999;24:58. [PubMed: 10098399]56. Warrick JM, et al. Cell 1998;93:939. [PubMed: 9635424]Jackson GR, et al. Neuron 1998;21:633.

[PubMed: 9768849]57. Warrick JM, et al. Nature Genet 1999;23:425. [PubMed: 10581028]58. Spring J. FEBS Lett 1997;400:2. [PubMed: 9000502]59. Aparicio S. Trends Genet 2000;16:54. [PubMed: 10652527]60. Schmid KJ, Tautz D. Proc Natl Acad Sci USA 1997;94:9746. [PubMed: 9275195]61. Chervitz SA, et al. Science 1998;282:2022. [PubMed: 9851918]62. See www.sciencemag.org/feature/data/1049664.shl for complete protein domain analysis.63. Paralogous gene families (Table 1) were identified by running BLASTP. A version of NCBI-BLAST2

optimized for the Compaq Alpha architecture was used with the SEG filter and the effective searchspace length (Y option) set to 17,973,263. Each protein was used as a query against a database of allother proteins of that organism. A clustering algorithm was then used to extract protein families fromthese BLASTP results. Each protein sequence constitutes a vertex; each HSP between proteinsequences is an arc, weighted by the BLAST Expect value. The algorithm identifies protein familiesby first breaking all arcs with an E value greater than some user-defined value (1 × 10−6 was usedfor all of the analyses reported here). The resulting graph is then split into subgraphs that contain atleast two-thirds of all possible arcs between vertices. The algorithm is “greedy”; that is, it arbitrarilychooses a starting sequence and adds new sequences to the subgraph as long as this criterion is met.An interesting property of this algorithm is that it inherently respects the multidomain nature ofproteins: For example, two multidomain proteins may have significant similarity to one another butshare only one or a few domains. In such a case, the two proteins will not be clustered if the unshareddomains introduce a large number of other arcs.

64. An NxN BLASTP analysis was performed for each fly, worm, and yeast sequence in a combined dataset of these completed proteomes. The databases used are as follows: Celera-BDGP, 14,195 predictedprotein sequences (1/5/2000); WormPep18, Sanger Centre, 18,424 protein sequences; and SGD, 6246protein sequences (1/7/2000). BLASTP analysis was also performed against known mammalianproteins (2/1/2000, GenBank nonredundant amino acid, Human, Mouse, and Rat, 75,236 proteinsequences), and TBLASTN analysis was performed against a database of mammalian ESTs (2/1/00,GenBank dbEST, Human, Mouse, and Rat). A version of NCBI-BLAST2 optimized for the CompaqAlpha architecture was used with the SEG filter and the effective search space length (Y option) setto 17,973,263.

65. The many participants from academic institutions are grateful for their various sources of support.Participants from the Berkeley Drosophila Genome Project are supported by NIH grant P50HG00750(G.M.R.) and grant P4IHG00739 (W.M.G.).



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http://www.sciencemag.org/feature/data/1049664.shl



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Fig. 1.Fly (F), worm (W), and yeast (Y) genes showing similarity to human disease genes. Thiscollection of human disease genes was selected to represent a cross section of humanpathophysiology and is not comprehensive. The selection criteria require that the gene isactually mutated, altered, amplified, or deleted in a human disease, as opposed to having afunction deduced from experiments on model organisms or in cell culture. Due to redundancyin gene and protein sequence databases, a single reference sequence for each gene had to bechosen. Most reference sequences represent the longest mRNA of several alternatives inGenBank. Authoritative sources in the literature and electronic databases [Online MendelianInheritance in Man (OMIM)] were also consulted. In all, 289 protein sequences met thesecriteria. These were used as queries to search a database consisting of the sum total of geneproducts (38,860) found in the complete genomes of fly, worm, and yeast. 12,953 was used asthe effective database size (the z parameter in BLAST). BLASTP searches were conducted asdescribed for full genome searches, except for the z parameter. To control for potentialframeshift errors in the Drosophila genome sequence, searches against a six-frame translationof the entire genome (using TBLASTN) were also conducted with the disease gene sequencesusing the z parameter above. Only two cases in which matches to genomic sequence were betterthan to the predicted protein were found, and these were manually corrected to reflect the betterTBLASTN scores in the table. Results are scaled according to various levels of statisticalsignificance, reflecting a level of confidence in either evolutionary homology or functionalsimilarity. White boxes represent BLAST E values >1 × 10−6, indicating no or weak similarity;light blue boxes represent E values in the range of 1 × 10−6 to 1 × 10−40; purple boxes representE values in the range of 1 × 10−40 to 1 × 10−100; and dark blue boxes represent E values <1 ×10−100, indicating the highest degree of sequence conservation. Actual E values can be found



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in the Web supplement to this figure (62), where links to OMIM and GenBank may also befound. A plus sign indicates our best estimate that the corresponding Drosophila gene productis the functional equivalent of the human protein, based on degree of sequence similarity,InterPro domain composition, and supporting biological evidence, when available. A minussign indicates that we were unable to identify a likely functional equivalent of the humanprotein.



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Table 1Numbers of distinct gene families versus numbers of predicted genes and their duplicated copies in H. influenzae, S.cerevisiae, C. elegans, and D. melanogaster. Row one shows the total number of genes in each species. Row two showsthe total number of all genes in each genome that appear to have arisen by gene duplication. Row three is the totalnumber of distinct gene families for each genome. Each proteome was compared to itself using the same parametersas described in (63).

H. influenzae S. cerevisiae C. elegans D. melanogaster

Total no. of predictedgenes

1709 6241 18424 13601

No. of genes duplicated 284 1858 8971 5536

Total no. of distinctfamilies

1425 4383 9453 8065


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Rubin et al. Page 22Ta

ble

2T

able

2A

. Sim

ilarit

y of

sequ

ence

s in

pred

icte

d pr

oteo

mes

of D

. mel

anog

aste

r, S.

cere

visi

ae, a

nd C

. ele

gans

. To

be sc

ored

as a

sim

ilarit

y,ea

ch p

airw

ise

sim

ilarit

y w

as re

quire

d to

ext

end

over

mor

e th

an 8

0% o

f the

leng

th o

f the

que

ry s

eque

nce

at a

n E

valu

e le

ss th

an th

atin

dica

ted.

For

exa

mpl

e, in

“Fl

y pr

otei

ns in

Fly

-yea

st,”

the

colu

mn

labe

led

E <

10−1

0 sh

ows

the

num

ber a

nd p

erce

ntag

e of

fly

prot

eins

that

mat

ch y

east

pro

tein

s at

this

E v

alue

or l

ess

and

for w

hich

mor

e th

an 8

0% o

f the

leng

th o

f the

fly

prot

ein

is a

ligne

d w

ith th

e ye

ast

prot

ein.

Eac

h se

t of

pairs

was

ana

lyze

d w

ithou

t con

side

ratio

n of

the

third

pro

teom

e. T

he r

ows

labe

led

“Fly

-wor

m-y

east

” re

port

the

com

posi

tion

of an

inde

pend

ent c

lust

erin

g in

whi

ch o

nly

grou

ps co

ntai

ning

a m

embe

r fro

m al

l thr

ee p

rote

omes

wer

e cou

nted

. The

num

bers

are

slig

htly

hig

her f

or th

e “F

ly-w

orm

-yea

st”

coun

ts th

an fo

r the

“Fl

y-ye

ast”

or “

Wor

m-y

east

” co

unts

bec

ause

of s

eque

nce

brid

ging

; tha

tis

, not

all

sequ

ence

s with

in a

gro

up n

eces

saril

y ha

ve a

sign

ifica

nt m

atch

to a

ll ot

her m

embe

rs o

f tha

t gro

up. S

ee (6

) for

det

ails

.

E <

10−1

0E

< 10

−20

E <

10−5

0E

< 10

−100

(n)

(%)

(n)

(%)

(n)

(%)

(n)

(%)

Fly

prot

eins

in:

Fl

y-ye

ast

2345

16.5

1877

13.2

1036

7.3

433

3.1

Fl

y-w

orm

4998

35.2

4212

29.7

2442

17.2

1106

7.8

Fl

y-w

orm

-yea

st33

0323

.324

2817

.111

137.

843

53.

1

Wor

m p

rote

ins i

n:

W

orm

-yea

st21

8411

.817

689.

593

35.

037

42.

0

Fl

y-w

orm

4795

25.8

4004

21.6

2403

12.9

1092

5.9

Fl

y-w

orm

-yea

st32

2917

.424

3913

.111

156.

041

92.

3

Yea

st p

rote

ins i

n:

Fl

y-ye

ast

1856

29.4

1567

24.8

891

14.1

376

6.0

W

orm

-yea

st17

0427

.014

2522

.680

212

.733

55.

3

Fl

y-w

orm

-yea

st18

3329

.115

2524

.283

113

.235

25.

6

Tab

le 2

B. A

com

paris

on o

f D. m

elan

ogas

ter,

C. e

lega

ns, a

nd S

. cer

evis

iae

prot

ein

sequ

ence

s to

each

oth

er a

nd to

mam

mal

ian

sequ

ence

s (64

). Th

is ta

ble

repo

rts th

e nu

mbe

r and

per

cent

of f

ly, w

orm

,or

yea

st q

uery

sequ

ence

s with

sim

ilarit

ies l

ess t

han

the

indi

cate

d E

valu

e cu

toff

s. Fo

r exa

mpl

e, in

the

“Fly

vs.

Yea

st”

com

paris

on, 3

986

or 2

8.1%

of f

ly p

rote

ins h

ave

a si

mila

rity

with

a y

east

pro

tein

with

an

E va

lue

less

than

1 ×

10−

10. E

ST E

val

ues a

re n

ot d

irect

ly c

ompa

rabl

e to

pro

tein

E v

alue

s, be

caus

e th

e re

sulti

ng a

lignm

ents

are

shor

ter.

No

sim

ilarit

y E

> 10

−4E

< 10

−10

E <

10−2

0E

< 10

−50

E <

10−1

00

(n)

(%)

(n)

(%)

(n)

(%)

(n)

(%)

(n)

(%)

Fly

vs.

Y

east

8177

57.6

3986

28.1

2677

18.9

1266

8.9

504

3.6


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E <

10−1

0E

< 10

−20

E <

10−5

0E

< 10

−100

(n)

(%)

(n)

(%)

(n)

(%)

(n)

(%)

W

orm

5110

36.0

6743

47.5

5180

36.5

2832

19.9

1197

8.4

M

amm

alia

n58

3341

.170

3249

.558

3741

.135

8025

.217

7212

.5

M

amm

alia

n ES

Ts53

8637

.973

2951

.653

5237

.717

7512

.511

00.

8

Wor

m v

s.

Y

east

1254

168

.035

8219

.423

7812

.911

066.

040

12.

2

Fl

y86

0346

.771

3838

.854

2829

.528

8015

.612

296.

7

M

amm

alia

n10

152

55.1

6550

35.6

4999

27.1

2782

15.1

1211

6.6

M

amm

alia

n ES

Ts10

354

56.2

6005

32.6

4000

21.7

1170

6.4

680.

4

Yea

st v

s.

Fl

y26

1441

.925

6441

.019

1030

.610

2116

.440

86.

5

W

orm

2762

44.2

2358

37.8

1730

27.7

882

14.1

348

5.6

M

amm

alia

n32

3051

.723

4037

.518

0228

.999

215

.942

96.

9

M

amm

alia

n ES

Ts31

0649

.723

1937

.115

5324

.950

38.

118

0.3


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Rubin et al. Page 24Ta

ble

3N

umbe

r of p

rote

ins i

n D

. mel

anog

aste

r (F)

, C. e

lega

ns (W

), an

d S.

cer

evis

iae

(Y) c

onta

inin

g th

e 20

0 m

ost f

requ

ently

occ

urrin

g pr

otei

ndo

mai

ns in

D. m

elan

ogas

ter.

Dom

ain

iden

tifie

rs a

re f

rom

Int

erPr

o (9

), a

new

dat

abas

e th

at h

as b

egun

to in

tegr

ate

the

inde

pend

ent

data

base

s of l

ocal

ized

pro

tein

sequ

ence

pat

tern

s int

o a

sing

le re

sour

ce. T

he b

eta

rele

ase

used

incl

udes

PR

OSI

TE, P

RIN

TS, a

nd P

FAM

.In

terP

ro c

onsi

ders

a si

gnat

ure

to b

e tru

e if

its sc

ore

is a

bove

a th

resh

old

spec

ified

for t

hat s

igna

ture

by

the

indi

vidu

al d

atab

ase.

Res

ults

of th

e In

terP

ro a

naly

sis

may

diff

er fr

om re

sults

obt

aine

d ba

sed

on h

uman

cur

atio

n of

pro

tein

fam

ilies

, due

to th

e lim

itatio

ns o

f lar

ge-

scal

e aut

omat

ic cl

assi

ficat

ions

. In

som

e ins

tanc

es, d

iffer

ent I

nter

Pro

dom

ains

corr

espo

nd to

diff

eren

t fea

ture

s of p

rote

ins w

ithin

the s

ame

fam

ily; f

or e

xam

ple,

IPR

0016

50 a

nd IP

R00

1410

(26

and

42 in

the

tabl

e). S

ee (6

2) fo

r liv

e lin

ks to

the

Inte

rPro

dat

abas

e.

Acc

. No.

FW

YIn

terp

ro D

omai

n N

ame

1.IP

R00

0694

579

398

40Pr

olin

e-ric

h re

gion

2.IP

R00

0822

352

138

47Zi

nc fi

nger

, C2H

2 ty

pe

3.IP

R00

0719

249

388

119

Euka

ryot

ic p

rote

in k

inas

e

4.IP

R00

1254

199

131

Serin

e pr

otea

ses,

tryps

in fa

mily

5.IP

R00

1314

178

50

Chy

mot

ryps

in se

rine

prot

ease

fam

ily (S

1)

6.IP

R00

1680

167

9590

G-p

rote

in b

eta

WD

-40

repe

ats

7.IP

R00

0504

160

9255

RN

A-b

indi

ng re

gion

RN

P-1

(RN

A re

cogn

ition

mot

if)

8.IP

R00

0495

153

700

Imm

unog

lobu

lins &

maj

or h

isto

com

patib

ility

com

plex

pro

tein

s

9.IP

R00

0345

145

177

Cyt

ochr

ome

c fa

mily

hem

e-bi

ndin

g si

te

10.

IPR

0003

7914

011

238

Este

rase

/lipa

se/th

ioes

tera

se

11.

IPR

0022

9013

817

111

0Se

rine/

Thre

onin

e pr

otei

n ki

nase

s act

ive-

site

12.

IPR

0020

4813

079

16EF

-han

d fa

mily

13.

IPR

0013

5611

388

10H

omeo

box

dom

ain

14.

IPR

0005

6111

010

90

EGF-

like

dom

ain

15.

IPR

0016

1110

848

7Le

ucin

e-ric

h re

peat

16.

IPR

0018

4110

511

335

Zinc

fing

er, C

3HC

4 ty

pe (R

ING

fing

er)

17.

IPR

0023

5610

033

50

G-p

rote

in c

oupl

ed re

cept

ors,

rhod

opsi

n fa

mily

18.

IPR

0010

6697

5446

Suga

r tra

nspo

rter

19.

IPR

0011

2894

733

Cyt

ochr

ome

P450

enz

yme

20.

IPR

0021

1090

7719

Ank

yrin

-rep

eat

21.

IPR

0006

1887

00

Inse

ct c

utic

le p

rote

in

22.

IPR

0012

4587

630

Tyro

sine

kin

ase

cata

lytic

dom

ain

23.

IPR

0014

4082

4634

TPR

repe

at

24.

IPR

0001

3079

198

Neu

tral z

inc

met

allo

pept

idas

es, z

inc-

bind

ing

regi

on


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Rubin et al. Page 25A

cc. N

o.F

WY

Inte

rpro

Dom

ain

Nam

e

25.

IPR

0023

8078

4122

Tran

sfor

min

g pr

otei

n P2

1 R

AS

26.

IPR

0016

5076

6675

DN

A/R

NA

hel

icas

e do

mai

n (D

EAD

/DEA

H b

ox)

27.

IPR

0016

1772

5632

AB

C tr

ansp

orte

rs fa

mily

28.

IPR

0018

4971

6727

PH d

omai

n

29.

IPR

0014

7869

602

PDZ

dom

ain

(als

o kn

own

as D

HR

or G

LGF)

30.

IPR

0014

8869

85

Myc

-type

, hel

ix-lo

op-h

elix

dim

eriz

atio

n do

mai

n si

gnat

ure

31.

IPR

0010

5167

6138

ATP

-bin

ding

tran

spor

t pro

tein

, 2nd

P-lo

op m

otif

32.

IPR

0019

9367

4335

Mito

chon

dria

l ene

rgy

trans

fer p

rote

ins

33.

IPR

0007

3466

94

Lipa

se

34.

IPR

0002

1064

103

1B

tb/tt

k do

mai

n

35.

IPR

0005

7563

5436

ATP

/GTP

-bin

ding

site

mot

if A

(P-lo

op)

36.

IPR

0014

5263

5525

Src

hom

olog

y 3

(SH

3) d

omai

n

37.

IPR

0010

9261

388

Hel

ix-lo

op-h

elix

DN

A-b

indi

ng d

omai

n

38.

IPR

0021

9861

6313

Shor

t-cha

in d

ehyd

roge

nase

/redu

ctas

e (S

DR

) sup

erfa

mily

|

39.

IPR

0021

0658

1417

Am

inoa

cyl-t

rans

fer R

NA

synt

heta

ses c

lass

-II

40.

IPR

0018

0651

4623

Ras

fam

ily

41.

IPR

0023

4750

221

Glu

cose

/ribi

tol d

ehyd

roge

nase

fam

ily

42.

IPR

0014

1046

4348

DEA

D/D

EAH

box

hel

icas

e

43.

IPR

0017

7746

432

Fibr

onec

tin ty

pe II

I dom

ain

44.

IPR

0001

6943

221

Euka

ryot

ic th

iol (

cyst

eine

) pro

teas

es a

ctiv

e si

tes

45.

IPR

0005

2142

446

Glu

tath

ione

S-tr

ansf

eras

e

46.

IPR

0016

2242

911

Pota

ssiu

m c

hann

el

47.

IPR

0025

5742

60

Chi

tin b

indi

ng d

omai

n

48.

IPR

0000

5140

3821

SAM

(and

som

e ot

her n

ucle

otid

e) b

indi

ng m

otif

49.

IPR

0021

7240

320

Low

den

sity

lipo

prot

ein

(LD

L)-r

ecep

tor c

lass

A (L

DLR

A) d

omai

n

50.

IPR

0000

6338

3212

Thio

redo

xin

fam

ily

51.

IPR

0016

2338

2922

Dna

J dom

ain

52.

IPR

0020

1838

440

Car

boxy

lest

eras

es ty

pe-B

53.

IPR

0013

0437

165

0C

-type

lect

in d

omai

n

54.

IPR

0003

8736

8312

Tyro

sine

spec

ific

prot

ein

phos

phat

ase

55.

IPR

0002

1535

90

Serp

ins


NIH


NIH


NIH



cc. N

o.F

WY

Inte

rpro

Dom

ain

Nam

e

56.

IPR

0010

0535

1619

Myb

DN

A b

indi

ng d

omai

n

57.

IPR

0014

1235

1514

Am

inoa

cyl-t

rans

fer R

NA

synt

heta

ses c

lass

-I

58.

IPR

0019

3935

2729

AA

A-p

rote

in (A

TPas

es a

ssoc

iate

d w

ith v

ario

us c

ellu

lar a

ctiv

ities

)

59.

IPR

0019

6535

2216

PHD

-fin

ger

60.

IPR

0000

0834

349

Prot

ein

kina

se C

2 do

mai

n

61.

IPR

0006

0834

1816

Ubi

quiti

n-co

niug

atin

g en

zym

es

62.

IPR

0017

8134

334

LIM

dom

ain

63.

IPR

0009

8033

431

Src

hom

olog

y 2

(SH

2) d

omai

n

64.

IPR

0022

1333

590

UD

P-gl

ucor

onos

yl &

UD

P-gl

ucos

yl tr

ansf

eras

es

65.

IPR

0003

0132

190

Tran

smem

bran

e 4

fam

ily

66.

IPR

0009

3431

5621

Serin

e/th

reon

ine

spec

ific

prot

ein

phos

phat

ase

fam

ily

67.

IPR

0012

5131

166

CR

AL/

TRIO

dom

ain

68.

IPR

0018

8131

340

Cal

cium

-bin

ding

EG

F-lik

e do

mai

n

69.

IPR

0021

7331

42

PfkB

fam

ily o

f car

bohy

drat

e ki

nase

s

70.

IPR

0001

9430

522

ATP

synt

hase

alp

ha &

bet

a su

buni

ts

71.

IPR

0002

1729

224

Tubu

lin fa

mily

72.

IPR

0008

7329

2311

AM

P-bi

ndin

g do

mai

n

73.

IPR

0000

7328

1716

Alp

ha/b

eta

hydr

olas

e fo

ld

74.

IPR

0001

5228

280

Asp

artic

aci

d &

asp

arag

ine

hydr

oxyl

atio

n si

te

75.

IPR

0004

0828

63

Reg

ulat

or o

f chr

omos

ome

cond

ensa

tion

(RC

C1)

76.

IPR

0008

3428

91

Zinc

car

boxy

pept

idas

es, c

arbo

xype

ptid

ase

A m

etal

lopr

otea

se (M

14) f

amily

77.

IPR

0017

1528

223

Cal

poni

n ho

mol

ogy

(CH

) dom

ain

78.

IPR

0020

8628

1313

Ald

ehyd

e de

hydr

ogen

ase

fam

ily

79.

IPR

0022

1928

361

Phor

bol e

ster

s/di

acyl

glyc

erol

bin

ding

dom

ain

80.

IPR

0004

8327

70

Leuc

ine

rich

repe

at C

-term

inal

dom

ain

81.

IPR

0008

8627

811

Endo

plas

mic

retic

ulum

targ

etin

g se

quen

ce

82.

IPR

0011

7527

810

Neu

rotra

nsm

itter

-gat

ed io

n-ch

anne

l

83.

IPR

0002

1926

175

Dbl

dom

ain

(dbl

/cdc

24 rh

oGR

F fa

mily

)

84.

IPR

0006

2626

279

Ubi

quiti

n do

mai

n

85.

IPR

0006

2926

2220

ATP

-dep

ende

nt h

elic

ase,

DEA

D-b

ox su

bfam

ily

86.

IPR

0008

5926

550

CU

B d

omai

n


NIH


NIH


NIH



cc. N

o.F

WY

Inte

rpro

Dom

ain

Nam

e

87.

IPR

0009

5826

216

KH

dom

ain

88.

IPR

0017

5226

226

Kin

esin

mot

or d

omai

n

89.

IPR

0020

6726

116

Mito

chon

dria

l car

rier p

rote

in

90.

IPR

0002

0525

2210

NA

D b

indi

ng si

te

91.

IPR

0002

9925

130

Ban

d 4.

1 fa

mily

92.

IPR

0004

4925

108

Ubi

quiti

n-as

soci

ated

dom

ain

93.

IPR

0009

1025

158

HM

G1/

2 (h

iqh

mob

ility

gro

up) b

ox

94.

IPR

0010

5425

321

Gua

nyla

te c

ycla

se

95.

IPR

0012

0225

175

WW

/rsp5

/WW

P do

mai

n

96.

IPR

0005

9524

192

Cyc

lic n

ucle

otid

e-bi

ndin

g do

mai

n

97.

IPR

0008

3224

100

G-p

rote

in c

oupl

ed re

cept

ors f

amily

2 (s

ecre

tin-li

ke)

98.

IPR

0011

4024

3010

AB

C tr

ansp

orte

r tra

nsm

embr

ane

regi

on

99.

IPR

0012

1424

276

SET-

dom

ain

of tr

ansc

riptio

nal r

egul

ator

s (TR

X, E

Z, A

SH1

etc)

100.

IPR

0018

7124

1815

bZIP

(Bas

ic-le

ucin

e zi

pper

) tra

nscr

iptio

n fa

ctor

fam

ily

101.

IPR

0020

4923

160

Lam

inin

-type

EG

F-lik

e (L

E) d

omai

n

102.

IPR

0021

1123

212

Cat

ion

chan

nels

, 6TM

regi

on (t

rans

ient

rece

ptor

pot

entia

l sub

type

)

103.

IPR

0000

4822

162

IQ c

alm

odul

in-b

indi

ng d

omai

n

104.

IPR

0013

5322

1214

Mul

tispe

cific

pro

teas

es o

f the

pro

teas

ome

105.

IPR

0018

1022

215

11F-

box

dom

ain

106.

IPR

0022

2322

340

Panc

reat

ic tr

ypsi

n in

hibi

tor (

Kun

itz) f

amily

107.

IPR

0007

1821

290

Nep

rilys

in m

etal

lopr

otea

se (M

13) f

amily

108.

IPR

0009

6421

153

Ster

ile-a

lpha

mod

ule

(SA

M) d

omai

n

109.

IPR

0013

1121

130

Solu

te b

indi

ng p

rote

in/g

luta

mat

e re

cept

or d

omai

n

110.

IPR

0013

9421

2418

Ubi

quiti

n ca

rbox

yI-te

rmin

al h

ydro

lase

s fam

ily 2

111.

IPR

0015

9421

136

DH

HC

-type

Zn-

finge

r

112.

IPR

0016

2821

224

0C

4-ty

pe st

eroi

d re

cept

or z

inc

finge

r

113.

IPR

0020

1721

193

Spec

trin

repe

at

114.

IPR

0021

1321

64

Ade

nine

nuc

leot

ide

trans

loca

tor 1

115.

IPR

0021

2621

150

Cad

herin

dom

ain

116.

IPR

0001

9520

1712

Rab

GA

P/TB

C d

omai

n

117.

IPR

0001

9820

1910

Rho

GA

P do

mai

n


NIH


NIH


NIH



cc. N

o.F

WY

Inte

rpro

Dom

ain

Nam

e

118.

IPR

0007

9520

1715

GTP

-bin

ding

elo

ngat

ion

fact

or

119.

IPR

0019

3020

114

Mem

bran

e al

anyl

dip

eptid

ase,

fam

ily M

1

120.

IPR

0024

2220

147

Perm

ease

s for

am

ino

acid

s & re

late

d co

mpo

unds

, fam

ily II

121.

IPR

0001

6619

3316

His

tone

-fol

d/TF

IID

-TA

F/N

F-Y

dom

ain

122.

IPR

0006

9019

87

RN

A-b

indi

ng p

rote

in C

2H2

Zn-f

inge

r dom

ain

123.

IPR

0017

6619

194

Fork

hea

d do

mai

n

124.

IPR

0021

3019

178

Cyc

loph

iIin-

type

pep

tidyl

-pro

lyl c

is-tr

ans i

som

eras

e

125.

IPR

0022

9319

1625

Perm

ease

s for

am

ino

acid

s & re

late

d co

mpo

unds

, fam

ily I

126.

IPR

0001

7518

120

Sodi

um:n

eure

trans

mitt

er sy

mpo

rter f

amily

127.

IPR

0003

3018

2017

SNF2

& o

ther

s N-te

rmin

al d

omai

n

128.

IPR

0007

4218

90

EGF-

like

dom

ain,

subt

ype

2

129.

IPR

0009

6118

2410

Prot

ein

kina

se C

term

inal

dom

ain

130.

IPR

0011

7318

174

Gly

cosy

l tra

nsfe

rase

, fam

ily 2

131.

IPR

0002

4217

763

Tyro

sine

spec

ific

prot

ein

phos

phat

ases

132.

IPR

0004

6717

114

D11

1 do

mai

n

133.

IPR

0006

3617

221

Cat

ion

chan

nels

, 6TM

regi

on (n

on-li

gand

gat

ed)

134.

IPR

0007

1717

138

Dom

ain

in c

ompo

nent

s of t

he p

rote

asom

e, C

OP9

-com

plex

&el

F3 (P

CI)

135.

IPR

0009

5317

152

Chr

omo

dom

ain

136.

IPR

0010

7117

00

Alp

ha-to

coph

erol

tran

spor

t pro

tein

137.

IPR

0011

6317

1116

Smal

l nuc

lear

ribo

nucl

eopr

otei

n (S

m p

rote

in)

138.

IPR

0013

2717

44

FAD

-dep

ende

nt p

yrid

ine

nucl

eotid

e re

duct

ase

139.

IPR

0013

9517

116

Ald

o/ke

to re

duct

ase

fam

ily

140.

IPR

0017

3417

31

Sodi

um:s

olut

e sy

mpo

rter f

amily

141.

IPR

0017

5717

2217

E1-E

2 A

TPas

es p

hosp

hory

latio

n si

te

142.

IPR

0017

9117

160

Lam

inin

-G d

omai

n

143.

IPR

0018

7317

220

Am

ilorid

e-se

nsiti

ve so

dium

cha

nnel

144.

IPR

0019

6917

842

Euka

ryot

ic &

vira

l asp

arty

l pro

teas

es a

ctiv

e si

te

145.

IPR

0000

8716

166

0C

olla

gen

tripl

e he

lix re

peat

146.

IPR

0002

5316

616

Fork

head

-ass

ocia

ted

(FH

A) d

omai

n

147.

IPR

0005

3616

880

Liga

nd-b

indi

ng d

omai

n of

nuc

lear

hor

mon

e re

cept

or

148.

IPR

0013

2016

100

Liga

nd-g

ated

ion

chan

nel


NIH


NIH


NIH



cc. N

o.F

WY

Inte

rpro

Dom

ain

Nam

e

149.

IPR

0014

8716

1310

Bro

mod

omai

n

150.

IPR

0020

2716

1124

Am

ino

acid

per

mea

se

151.

IPR

0020

4616

11

SAR

1 G

TP-b

indi

ng p

rote

in fa

mily

152.

IPR

0000

1415

81

Gen

eral

ized

PA

S do

mai

n

153.

IPR

0001

7215

10

GM

C o

xido

redu

ctas

es

154.

IPR

0002

5115

127

AD

P-rib

osyl

atio

n fa

ctor

s fam

ily

155.

IPR

0005

6915

55

HEC

T-do

mai

n (U

biqu

itin-

trans

fera

se)

156.

IPR

0007

7215

120

Lect

in d

omai

n of

rici

n b-

chai

n, 3

cop

ies

157.

IPR

0012

2315

341

Gly

cosy

l hyd

rola

ses f

amily

18

158.

IPR

0016

0915

205

Myo

sin

head

(mot

or d

omai

n)

159.

IPR

0018

2815

190

Rec

epto

r fam

ily li

gand

bin

ding

regi

on

160.

IPR

0021

2915

71

Pyrid

oxal

-dep

ende

nt d

ecar

boxy

lase

fam

ily

161.

IPR

0024

6515

10

Gro

wth

fact

or &

cyt

okin

e re

cept

or fa

mily

sign

atur

e 2

162.

IPR

0001

5914

112

Ras

-ass

ocia

ted

(Ral

GD

S/A

F-6)

dom

ain

163.

IPR

0002

2514

62

Arm

adill

o/pl

akog

lobi

n A

RM

repe

at

164.

IPR

0002

7914

108

Act

in

165.

IPR

0005

6614

60

Lipo

calin

& c

ytos

olic

fatty

-aci

d bi

ndin

g pr

otei

n

166.

IPR

0005

7714

32

Car

bohy

drat

e ki

nase

, FG

GY

fam

ily

167.

IPR

0007

4614

00

Pher

omon

e/ge

nera

l odo

rant

bin

ding

pro

tein

, PB

P/G

OB

P fa

mily

168.

IPR

0008

8414

270

Thro

mbo

spon

din

type

I do

mai

n

169.

IPR

0011

0014

53

Pyrid

ine

nucl

eotid

e-di

sulfi

de o

xido

redu

ctas

e, c

lass

I

170.

IPR

0011

5914

92

Dou

ble-

stra

nded

RN

A b

indi

ng (D

sRB

D) d

omai

n

171.

IPR

0011

9914

85

Cyt

ochr

ome

B5

172.

IPR

0013

5714

2111

BR

CT

dom

ain

173.

IPR

0015

8914

81

Act

inin

-type

act

in-b

indi

ng d

omai

n

174.

IPR

0017

5314

103

Enoy

l-CoA

hyd

rata

se/is

omer

ase

175.

IPR

0018

7814

249

Zn-f

inge

r CC

HC

type

176.

IPR

0019

5214

01

Alk

alin

e ph

osph

atas

e fa

mily

177.

IPR

0022

1614

171

Ion

trans

port

prot

ein

178.

IPR

0024

6414

98

DEA

H-b

ox su

bfam

ily A

TP-d

epen

dent

hel

icas

e

179.

IPR

0001

0713

83

SPR

Y d

omai

n


NIH


NIH


NIH



cc. N

o.F

WY

Inte

rpro

Dom

ain

Nam

e

180.

IPR

0004

2513

86

MIP

fam

ily

181.

IPR

0005

0813

23

Sign

al p

eptid

ase

182.

IPR

0007

2713

1415

t-SN

AR

E co

iled-

coil

dom

ain

183.

IPR

0009

0113

67

Car

bam

oyl-p

hosp

hate

synt

hase

184.

IPR

0014

6113

167

Peps

in (A

1) a

spar

tic p

rote

ase

fam

ily

185.

IPR

0015

0613

360

Ast

acin

(Pep

tidas

e fa

mily

M12

A) f

amily

186.

IPR

0015

2313

110

‘Pai

red

box’

dom

ain

187.

IPR

0018

2713

2|0

‘Hom

eobo

x’ a

nten

nape

dia-

type

pro

tein

188.

IPR

0018

7613

71

Zn-f

inge

r in

ranb

p &

oth

ers

189.

IPR

0024

2313

89

TCP-

1 (T

aille

ss c

ompl

ex p

olyp

eptid

e)/c

pn60

cha

pero

nin

fam

ily

190.

IPR

0028

9313

81

MY

ND

fing

er

191.

IPR

0004

6112

48

Alp

ha a

myl

ase

192.

IPR

0007

9812

40

Ezrin

/radi

xin/

moe

sin

fam

ily

193.

IPR

0010

2312

1314

Hea

t sho

ck p

rote

in h

sp70

194.

IPR

0015

0812

10

NM

DA

rece

ptor

195.

IPR

0016

8312

915

PX (B

em1/

NC

F1/P

I3K

) dom

ain

196.

IPR

0019

17I

126

4A

min

otra

nsfe

rase

s cla

ss-I

I

197.

IPR

0019

3212

98

Prot

ein

phos

phat

ase

2C

198.

IPR

0000

5011

80

Phos

phot

yros

ine

inte

ract

ion

dom

ain

(PID

)

199.

IPR

0001

8211

99

cety

ltran

sfer

ase

(GN

AT)

fam

ily

200.

IPR

0002

4311

27

Prot

easo

me

B-ty

pe su

buni

t


NIH


NIH


NIH



Table 4The 10 InterPro protein domains occurring in the largest number of differentproteins in S. cerevisiae and C. elegans.

Acc. no. InterPro domain name No. of proteins

S. cerevisiae

IPR000719 Eukaryotic protein kinase 119

IPR001680 G-protein beta WD-40 repeats 90

IPR001650 DNA/RNA helicase domain (DEAD/DEAH box) 75

IPR001138 Fungal transcriptional regulatory protein, N-terminus 60

IPR001042 TYA transposon protein 57

IPR000504 RNA-binding region RNP-1 (RNA recognition motif) 55

IPR001410 DEAD/DEAH box helicase 48

IPR000822 Zinc finger, C2H2 type 47

IPR001066 Sugar transporter 46

IPR001969 Eukaryotic and viral aspartyl proteases active site 42

C. elegans

IPR000168 7-Helix G-protein coupled receptor, nematode (probably olfactory) family 545

IPR000694 Proline-rich region 398

IPR000719 Eukaryotic protein kinase 388

IPR002356 G-protein–coupled receptors, rhodopsin family 335

IPR001628 C4-type steroid receptor zinc finger 224

IPR001810 F-box domain 215

IPR000087 Collagen triple helix repeat 166

IPR001304 C-type lectin domain 165

IPR002900 Domain of unknown function 142

IPR000822 Zinc finger, C2H2 type 138


NIH


NIH


NIH



Table 5Proteins in D. melanogaster, C. elegans, and S. cerevisiae with more than one InterPro domain. These numbers representthe total number of recognizable domains within a single protein, no matter whether they are multiple copies of thesame domain or different domains.

InterPro domains perprotein

D. melanogaster(number of proteins)

C. elegans(number of proteins)

S. cerevisiae(number of proteins)

2 920 1236 410

3 388 458 121

4 219 182 58

5 163 98 26

6 101 72 17

7 92 53 15

8 58 27 7

9 42 25 4

10 22 18 7

11–15 73 43 6

16–20 18 17 1

21–30 22 22 0

31–50 8 5 0

51–75 4 5 0


NIH


NIH


NIH



Table 6Proteins in D. melanogaster, C. elegans, and S. cerevisiae with multiple different InterPro domains. Individual InterProdomains are counted only once per protein, regardless of how many times they occur in that protein.

Unique InterPro domainsper protein

D. melanogaster(number of proteins)

C. elegans(number of proteins)

S. cerevisiae(number of proteins)

2 1474 1248 402

3 413 335 95

4 156 114 23

5 52 38 4

6 8 9 1

7 or more 4 3 0


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Comparative genomics of the eukaryotes

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