Protein functions and biological contexts.

The availability of a rough draft of the predicted human proteome allows an evaluation of the extent to which the predicted and biochemical functions of proteins are in alignment, and the roles of different technologies and approaches to understanding human diseases and instantiating therapeutics. Microarray technologies at the transcriptomic and proteomic levels can be high throughput and excellent for diagnostic purposes, but their informational outputs are inferior in quality to those emerging from the co- and post-translational levels and from antibody-based molecular anatomy. It is now abundantly clear that data transfer between the transcriptome and proteome is not straightforward, and that increasing emphasis needs to be placed on pure proteomic approaches at the structural, quantitative, cell biological and phenomic levels, with special focus on embryogenic and foetal processes. Finally, the precision genetic engineering that is required to evaluate the functional significance of context-dependent protein interactions underpinned by post-translational modifications and proteolytic cleavage events, is still too time consuming and rudimentary to be implemented on a large scale in the mouse, and basic principles and first order networks will need to be sorted out in even simpler model systems such as Drosophila.

Integrating molecular medicine with functional proteomics: realities and expectations.

We analyze key proteomic issues and cutting-edge technologies that will spearhead inroads into functional interpretations of human diseases and their therapeutic rectification, following the availability of the predicted human proteome. We contrast the distinctions between high quality data that are low throughput, (e.g., 3-D proteomic reconstructions in embryogenic and nervous system contexts, and multigenerational transgenic studies), versus automated data harvesting that is more distant from human disease phenotypes and currently fulfills a diagnostic role, (e.g., molecular portraits of human diseases via transcriptomic analyses). We examine the extent to which these approaches impinge upon a realistic understanding of human diseases, namely how close they come to revealing the causal events involved in the initiation of disease. While tissue sources from human embryogenesis, foetal development and the brain remain the absolute priority, the pragmatic approaches utilize judicious data integration from selected proteomic studies of model organisms. The role of genome-wide disease-related screens, "humanized" transgenic analyses, multigenerational gene interference methods, and analyses of post-translational modifications in epigenetic contexts from Drosophila will be crucial, since these avenues are far too slow and transgenically cumbersome in mammals. Finally, the implementation of multi compartment electrolyzers (MCE) and multi photon detection (MPD) systems will be pivotal for the proteomic profiling of human tissue samples.

The sequence of the human genome.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

Comparative genomics of the eukaryotes.

A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

Data transferability from model organisms to human beings: insights from the functional genomics of the flightless region of Drosophila.

At what biological levels are data from single-celled organisms akin to a Rosetta stone for multicellular ones? To examine this question, we characterized a saturation-mutagenized 67-kb region of the Drosophila genome by gene deletions, transgenic rescues, phenotypic dissections, genomic and cDNA sequencing, bio-informatic analysis, reverse transcription-PCR studies, and evolutionary comparisons. Data analysis using cDNA/genomic DNA alignments and bio-informatic algorithms revealed 12 different predicted proteins, most of which are absent from bacterial databases, half of which are absent from Saccharomyces cerevisiae, and nearly all of which have relatives in Caenorhabditis elegans and Homo sapiens. Gene order is not evolutionarily conserved; the closest relatives of these genes are scattered throughout the yeast, nematode, and human genomes. Most gene expression is pleiotropic, and deletion studies reveal that a morphological phenotype is seldom observed when these genes are removed from the genome. These data pinpoint some general bottlenecks in functional genomics, and they reveal the acute emerging difficulties with data transferability above the levels of genes and proteins, especially with complex human phenotypes. At these higher levels the Rosetta stone analogy has almost no applicability. However, newer transgenic technologies in Drosophila and Mus, combined with coherency pattern analyses of gene networks, and synthetic neural modeling, offer insights into organismal function. We conclude that industrially scaled robogenomics in model organisms will have great impact if it can be realistically linked to epigenetic analyses of human variation and to phenotypic analyses of human diseases in different genetic backgrounds.

An essential cell division gene of Drosophila, absent from Saccharomyces, encodes an unusual protein with tubulin-like and myosin-like peptide motifs.

Null mutations at the misato locus of Drosophila melanogaster are associated with irregular chromosomal segregation at cell division. The consequences for morphogenesis are that mutant larvae are almost devoid of imaginal disk tissue, have a reduction in brain size, and die before the late third-instar larval stage. To analyze these findings, we isolated cDNAs in and around the misato locus, mapped the breakpoints of chromosomal deficiencies, determined which transcript corresponded to the misato gene, rescued the cell division defects in transgenic organisms, and sequenced the genomic DNA. Database searches revealed that misato codes for a novel protein, the N-terminal half of which contains a mixture of peptide motifs found in alpha-, beta-, and gamma-tubulins, as well as a motif related to part of the myosin heavy chain proteins. The sequence characteristics of misato indicate either that it arose from an ancestral tubulin-like gene, different parts of which underwent convergent evolution to resemble motifs in the conventional tubulins, or that it arose by the capture of motifs from different tubulin genes. The Saccharomyces cerevisiae genome lacks a true homolog of the misato gene, and this finding highlights the emerging problem of assigning functional attributes to orphan genes that occur only in some evolutionary lineages.

The dodo gene family encodes a novel protein involved in signal transduction and protein folding.

Recent studies in yeast, Drosophila and humans have revealed the existence of a highly conserved gene encoding a novel protein, Dodo, comprised of four modules: a WW domain, involved in protein-protein interactions, a peptidyl-prolyl cis-trans isomerase (PPIase) domain belonging to a recently described third family of PPIases involved in protein folding and unfolding, a nuclear localization motif and finally, a long, surface-exposed alpha-helix that is likely to be involved in binding to a cell cycle serine/threonine kinase. The genetic, molecular, biochemical and structural data are reviewed in the context of the potential biological properties of this new protein family.

The Drosophila melanogaster dodo (dod) gene, conserved in humans, is functionally interchangeable with the ESS1 cell division gene of Saccharomyces cerevisiae.

We have sequenced the region of DNA adjacent to and including the flightless (fli) gene of Drosophila melanogaster and molecularly characterized four transcription units within it, which we have named tweety (twe), flightless (fli), dodo (dod), and penguin (pen). We have performed deletion and transgenic analysis to determine the consequences of the quadruple gene removal. Only the flightless gene is vital to the organism; the simultaneous absence of the other three allows the overriding majority of individuals to develop to adulthood and to fly normally. These gene deletion results are evaluated in the context of the redundancy and degeneracy inherent in many genetic networks. Our cDNA analyses and data-base searches reveal that the predicted dodo protein has homologs in other eukaryotes and that it is made up of two different domains. The first, designated WW, is involved in protein-protein interactions and is found in functionally diverse proteins including human dystrophin. The second is involved in accelerating protein folding and unfolding and is found in Escherichia coli in a new family of peptidylprolyl cis-trans isomerases (PPIases; EC 5.2.1.8). In eukaryotes, PPIases occur in the nucleus and the cytoplasm and can form stable associations with transcription factors, receptors, and kinases. Given this particular combination of domains, the dodo protein may well participate in a multisubunit complex involved in the folding and activation of signaling molecules. When we expressed the dodo gene product in Saccharomyces cerevisiae, it rescued the lethal phenotype of the ESS1 cell division gene.

Molecular and mutational analysis of a gelsolin-family member encoded by the flightless I gene of Drosophila melanogaster.

The flightless locus of Drosophila melanogaster has been analyzed at the genetic, molecular, ultrastructural and comparative crystallographic levels. The gene encodes a single transcript encoding a protein consisting of a leucine-rich amino terminal half and a carboxyterminal half with high sequence similarity to gelsolin. We determined the genomic sequence of the flightless landscape, the breakpoints of four chromosomal rearrangements, and the molecular lesions in two lethal and two viable alleles of the gene. The two alleles that lead to flight muscle abnormalities encode mutant proteins exhibiting amino acid replacements within the S1-like domain of their gelsolin-like region. Furthermore, the deduced intron-exon structure of the D. melanogaster gene has been compared with that of the Caenorhabditis elegans homologue. Furthermore, the sequence similarities of the flightless protein with gelsolin allow it to be evaluated in the context of the published crystallographic structure of the S1 domain of gelsolin. Amino acids considered essential for the structural integrity of the core are found to be highly conserved in the predicted flightless protein. Some of the residues considered essential for actin and calcium binding in gelsolin S1 and villin V1 are also well conserved. These data are discussed in light of the phenotypic characteristics of the mutants and the putative functions of the protein.

The human homologue of the Drosophila melanogaster flightless-I gene (flil) maps within the Smith-Magenis microdeletion critical region in 17p11.2.

The Smith-Magenis syndrome (SMS) appears to be a contiguous-gene-deletion syndrome associated with a proximal deletion of the short arm of chromosome 17 in band p11.2. The spectrum of clinical findings includes short stature, brachydactyly, developmental delay, dysmorphic features, sleep disturbances, and behavioral problems. The complex phenotypic features suggest deletion of several contiguous genes. However, to date, no protein-encoding gene has been mapped to the SMS critical region. Recently, the Drosophila melanogaster flightless-I gene, fliI, and the homologous human cDNA have been isolated. Mutations in fliI result in loss of flight ability and, when severe, cause lethality due to incomplete cellularization with subsequent abnormal gastrulation. Here, we demonstrate that the human homologue (FLI) maps within the SMS critical region. Genomic cosmids were used as probes for FISH, which localized this gene to the 17p11.2 region. Somatic-cell hybrid-panel mapping further localized this gene to the SMS critical region. Southern blot analysis of somatic-cell hybrids and/or FISH analysis of lymphoblastoid cell lines from 12 SMS patients demonstrates the deletion of one copy of FLI in all SMS patients analyzed.

The Drosophila melanogaster flightless-I gene involved in gastrulation and muscle degeneration encodes gelsolin-like and leucine-rich repeat domains and is conserved in Caenorhabditis elegans and humans.

Mutations at the flightless-I locus (fliI) of Drosophila melanogaster cause flightlessness or, when severe, incomplete cellularization during early embryogenesis, with subsequent abnormalities in mesoderm invagination and in gastrulation. After chromosome walking, deficiency mapping, and transgenic analysis, we have isolated and characterized flightless-I cDNAs, enabling prediction of the complete amino acid sequence of the 1256-residue protein. Data base searches revealed a homologous gene in Caenorhabditis elegans, and we have isolated and characterized corresponding cDNAs. By using the polymerase chain reaction with nested sets of degenerate oligonucleotide primers based on conserved regions of the C. elegans and D. melanogaster proteins, we have cloned a homologous human cDNA. The predicted C. elegans and human proteins are, respectively, 49% and 58% identical to the D. melanogaster protein. The predicted proteins have significant sequence similarity to the actin-binding protein gelsolin and related proteins and, in addition, have an N-terminal domain consisting of a repetitive amphipathic leucine-rich motif. This repeat is found in D. melanogaster, Saccharomyces cerevisiae, and mammalian proteins known to be involved in cell adhesion and in binding to other proteins. The structure of the maternally expressed flightless-I protein suggests that it may play a key role in embryonic cellularization by interacting with both the cytoskeleton and other cellular components. The presence of a highly conserved homologue in nematodes, flies, and humans is indicative of a fundamental role for this protein in many metazoans.

Molecules and cognition: the latterday lessons of levels, language, and lac. Evolutionary overview of brain structure and function in some vertebrates and invertebrates.

The characteristics of the nervous systems of a number of organisms in different phyla are examined at the recombinant DNA, protein, neuroanatomic, neurophysiological, and cognitive levels. Among the invertebrates, special attention is paid to the advantages as well as the shortcomings of the fly Drosophila melanogaster, the worm Caenorhabditis elegans, the honey bee Apis mellifera, the sea hare Aplysia californica, the octopus Octopus vulgaris, and the squid Loligo pealei. Among vertebrates, the focus is on Homo sapiens, the mouse Mus musculus, the rat Rattus norvegicus, the cat Felis catus, the macaque monkey Macaca fascicularis, the barn owl Tyto alba, and the zebrafish Brachydanio rerio. Vertebrate nervous systems have also been compared in fossil vs. extant organisms. I conclude that complex nervous systems arose in the Early Cambrian via a big bang that was underpinned by a modular method of construction involving massive pleiotropy of gene circuits. This rapidity of construction had enormous implications for the degrees of freedom that were subsequently available to evolving nervous systems. I also conclude that at the level of neuronal populations and interactions of neuropiles there is no model system between phyla except at the basic macromolecular level. Further, I argue that to achieve a significant understanding of the functions of extant nervous systems we need to concentrate on fewer organisms in greater depth and manipulate genomes via transgenic technologies to understand the behavioral outputs that are possible from an organism. Finally, I analyze the concepts of "perceptual categorization" and "information processing" and the difficulties involved in the extrapolation of computer analogies to sophisticated nervous systems.

The sluggish-A gene of Drosophila melanogaster is expressed in the nervous system and encodes proline oxidase, a mitochondrial enzyme involved in glutamate biosynthesis.

Certain gene mutations in Drosophila melanogaster cause sluggish motor activity. We have localized the transcription unit of the sluggish-A gene to a 14.7-kb region at the base of the X chromosome and have cloned corresponding cDNAs. The predicted protein product has significant sequence similarity to Saccharomyces cerevisiae proline oxidase (EC 1.5.99.8), a mitochondrial enzyme which catalyzes the first step in the conversion of proline to glutamate. In the mutant fly, mitochondrial proline oxidase activity is reduced and has kinetic properties different from those of the wild type, providing further evidence that the gene encodes proline oxidase. Indeed, the free proline level in mutant flies is elevated. When the mutant is rescued by transformation, the proline oxidase and free proline levels, as well as the motor and phototactic behavior, are restored to normal. During embryonic development the sluggish-A transcript is predominantly expressed in the nervous system. Significantly, it has previously been reported that a mouse mutant, PRO/Re, which has reduced proline oxidase activity and elevated free proline levels, also exhibits sluggish behavior.

Analysis of a cDNA from the neurologically active locus shaking-B (Passover) of Drosophila melanogaster.

We have isolated and sequenced a cDNA from the shaking-B locus of Drosophila melanogaster. The cDNA contains an open reading frame with extensive homology to another D. melanogaster gene, l(1)ogre. This suggests the existence of a new family of proteins required for the development and maintenance of the D. melanogaster nervous system.

The evolution of protein domains and the organizational complexities of metazoans.

There is an increasingly heated debate on the very existence of a 'universe of exons' and on the types of genomes that existed after the RNA world. What has been lost in the excitement are the biological issues that relate to the rapid emergence of phenotypic novelties. These issues can be examined by integrating data on protein domains and genomic evolution with the geochemical and palaeontological records.

Molecular cloning and analysis of small optic lobes, a structural brain gene of Drosophila melanogaster.

Mutations in the small optic lobes (sol) gene of Drosophila melanogaster cause specific cells to degenerate in the developing optic lobes, resulting in the absence of certain classes of columnar neurons. These neuronal defects lead to specific alterations in behavioral characteristics, particularly during flight and walking maneuvers. We have isolated the wild-type sol locus by microcloning and chromosomal walking and have established its genetic and molecular limits. Two major transcripts of 5.8 and 5.2 kilobases are produced from this locus by alternative splicing and are present throughout the entire life cycle. Sequence analyses of cDNAs corresponding to these two classes of transcripts predict two proteins of 1597 and 395 amino acids. The first shows similarity in its carboxyl-terminal region to the catalytic domain of a vertebrate calcium-activated neutral protease (calpain), whereas its amino-terminal region contains several zinc-finger-like repeats of the form WXCX2CX10-11CX2C. The second predicted protein contains only the first two of the zinc-finger-like repeats and is missing the calpain domain. By constructing transgenic flies carrying a single wild-type copy of the sol gene in a homozygous sol mutant background, we have restored the normal neuroanatomical phenotype to individuals that would have developed mutant brains.

Molecular and cytogenetic analysis of the heterochromatin-euchromatin junction region of the Drosophila melanogaster X chromosome using cloned DNA sequences.

We have used three cloned DNA sequences consisting of (1) part of the suppressor of forked transcription unit, (2) a cloned 359-bp satellite, and (3), a type I ribosomal insertion, to examine the structure of the base of the X chromosome of Drosophila melanogaster where different chromatin types are found in juxtaposition. A DNA probe from the suppressor of forked locus hybridizes exclusively to the very proximal polytenized part of division 20, which forms part of the beta-heterochromatin of the chromocenter. The cloned 359-bp satellite sequence, which derives from the proximal mitotic heterochromatin between the centromere and the ribosomal genes, hybridizes to the under replicated alpha-heterochromatin of the chromocenter. The type I insertion sequence, which has major locations in the ribosomal genes and in the distal mitotic heterochromatin of the X chromosome, hybridizes as expected to the nucleolus but does not hybridize to the beta-heterochromatic division 20 of the polytene X chromosome. Our molecular data reveal that the suppressor of forked locus, which on cytogenetic grounds is the most proximal ordinary gene on the X chromosome, is very close to the junction of the polytenized and non-polytenized region of the X chromosome. The data have implications for the structure of beta-heterochromatin-alpha-heterochromatin junction zones in both mitotic and polytene chromosomes, and are discussed with reference to models of chromosome structure.