"Extended Fitness" hypothesis: a link between individual and group selection.

What are the targets of natural selection remains a controversial issue in Biology. Here I propose the "Extended Fitness" hypothesis, in which extended phenotypes emerge as a link between individual and group selection. The basic premise of the extended fitness hypothesis is that extended phenotypes can be used by members of the same group since they are adapted to use them. Thus, extended phenotypes can also contribute to the fitness of members of the same species. Group selection emerges as a natural consequence of the shared use of extended phenotypes, which allow the evolutionary forces acting on individuals to become effective at the group level. The extended fitness hypothesis is supported by several observations found in nature. The hypothesis presented here provides a theoretical framework for the design of both modeling and experimental approaches to studying the role of extended phenotypes in group selection.

Bioinformatics construction of the human cell surfaceome.

Cell surface proteins are excellent targets for diagnostic and therapeutic interventions. By using bioinformatics tools, we generated a catalog of 3,702 transmembrane proteins located at the surface of human cells (human cell surfaceome). We explored the genetic diversity of the human cell surfaceome at different levels, including the distribution of polymorphisms, conservation among eukaryotic species, and patterns of gene expression. By integrating expression information from a variety of sources, we were able to identify surfaceome genes with a restricted expression in normal tissues and/or differential expression in tumors, important characteristics for putative tumor targets. A high-throughput and efficient quantitative real-time PCR approach was used to validate 593 surfaceome genes selected on the basis of their expression pattern in normal and tumor samples. A number of candidates were identified as potential diagnostic and therapeutic targets for colorectal tumors and glioblastoma. Several candidate genes were also identified as coding for cell surface cancer/testis antigens. The human cell surfaceome will serve as a reference for further studies aimed at characterizing tumor targets at the surface of human cells.

What can digital transcript profiling reveal about human cancers?

Important biological and clinical features of malignancy are reflected in its transcript pattern. Recent advances in gene expression technology and informatics have provided a powerful new means to obtain and interpret these expression patterns. A comprehensive approach to expression profiling is serial analysis of gene expression (SAGE), which provides digital information on transcript levels. SAGE works by counting transcripts and storing these digital values electronically, providing absolute gene expression levels that make historical comparisons possible. SAGE produces a comprehensive profile of gene expression and can be used to search for candidate tumor markers or antigens in a limited number of samples. The Cancer Genome Anatomy Project has created a SAGE database of human gene expression levels for many different tumors and normal reference tissues and provides online tools for viewing, comparing, and downloading expression profiles. Digital expression profiling using SAGE and informatics have been useful for identifying genes that have a role in tumor invasion and other aspects of tumor progression.

pp-Blast: a "pseudo-parallel" Blast.

We have developed a software called pp-Blast that uses the publicly available Blast package and PVM (parallel virtual machine) to partition a multi-sequence query across a set of nodes with replicated or shared databases. Benchmark tests show that pp-Blast running in a cluster of 14 PCs outperformed conventional Blast running in large servers. In addition, using pp-Blast and the cluster we were able to map all human cDNAs onto the draft of the human genome in less than 6 days. We propose here that the cost/benefit ratio of pp-Blast makes it appropriate for large-scale sequence analysis. The source code and configuration files for pp-Blast are available at http://www.ludwig.org.br/biocomp/tools/pp-blast.

An international database and integrated analysis tools for the study of cancer gene expression.

Researchers working collaboratively in Brazil and the United States have assembled an International Database of Cancer Gene Expression. Several strategies have been employed to generate gene expression data including expressed sequence tags (ESTs), serial analysis of gene expression (SAGE), and open reading-frame expressed sequence tags (ORESTES). The database contains six million gene tags that reflect the gene expression profiles in a wide variety of cancerous tissues and their normal counterparts. All sequences are deposited in the public databases, GenBank and SAGEmap. A suite of informatics tools was designed to facilitate in silico analysis of the gene expression datasets and are available through the NCI Cancer Genome Anatomy Project web site (http://cgap.nci.nih.gov).

ExInt: an Exon Intron Database.

The Exon/Intron Database (ExInt) stores information of all GenBank eukaryotic entries containing an annotated intron sequence. Data are available through a retrieval system, as flat-files and as a MySQL dump file. In this report we discuss several implementations added to ExInt, which is accessible at http://intron.bic.nus.edu.sg/exint/newexint/exint.html.

Intron distribution difference for 276 ancient and 131 modern genes suggests the existence of ancient introns.

o introns delineate elements of protein tertiary structure? This issue is crucial to the debate about the role and origin of introns. We present an analysis of the full set of proteins with known three-dimensional structures that have homologs with intron positions recorded in GenBank. A computer program was generated that maps on a reference sequence the positions of all introns in homologous genes. We have applied this program to a set of 665 nonredundant protein sequences with defined three-dimensional structures in the Protein Data Bank (PDB), which yielded 8,217 introns in 407 proteins. For the subset of proteins corresponding to ancient conserved regions (ACR), we find that there is a correlation of phase-zero introns with the boundary regions of modules and no correlation for the phase-one and phase-two positions. However, for a subset of proteins without prokaryotic counterparts (131 non-ACR proteins), a set of presumably modern proteins (or proteins that have diverged extremely far from any ancestral form), we do not find any correlation of phase-zero intron positions with three-dimensional structure. Furthermore, we find an anticorrelation of phase-one intron positions with module boundaries: they actually have a preference for the interior of modules. This finding is explicable as a preference for phase-one introns to lie in glycines, between G/G sequences, the preference for glycines being anticorrelated with the three-dimensional modules. We interpret this anticorrelation as a sign that a number of phase-one introns, and hence many modern introns, have been inserted into G/G "protosplice" sequences.

Alternative spliced transcripts as cancer markers.

Eukaryotic mRNAs are transcribed as precursors containing their intronic sequences. These are subsequently excised and the exons are spliced together to form mature mRNAs. This process can lead to transcript diversification through the phenomenon of alternative splicing. Alternative splicing can take the form of one or more skipped exons, variable position of intron splicing or intron retention. The effect of alternative splicing in expanding protein repertoire might partially underlie the apparent discrepancy between gene number and the complexity of higher eukaryotes. It is likely that more than 50% form. Many cancer-associated genes, such as CD44 and WT1 are alternatively spliced. Variation of the splicing process occurs during tumor progression and may play a major role in tumorigenesis. Furthermore, alternatively spliced transcripts may be extremely useful as cancer markers, since it appears likely that there may be striking contrasts in usage of alternatively spliced transcript variants between normal and tumor tissue than in alterations in the general levels of gene expression.

Generation of a database containing discordant intron positions in eukaryotic genes (MIDB).

MOTIVATION: Intron sliding is the relocation of intron-exon boundaries over short distances and is often also referred to as intron slippage or intron migration or intron drift. We have generated a database containing discordant intron positions in homologous genes (MIDB--Mismatched Intron DataBase). Discordant intron positions are those that are either closely located in homologous genes (within a window of 10 nucleotides) or an intron position that is present in one gene but not in any of its homologs. The MIDB database aims at systematically collecting information about mismatched introns in the genes from GenBank and organizing it into a form useful for understanding the genomics and dynamics of introns thereby helping understand the evolution of genes. RESULTS: Intron displacement or sliding is critically important for explaining the present distribution of introns among orthologous and paralogous genes. MIDB allows examining of intron movements and allows mapping of intron positions from homologous proteins onto a single sequence. The database is of potential use for molecular biologists in general and for researchers who are interested in gene evolution and eukaryotic gene structure. Partial analysis of this database allowed us to identify a few putative cases of intron sliding. AVAILABILITY: http://intron.bic.nus.edu.sg/midb/midb.html

Human semaphorin 6B [(HSA)SEMA6B], a novel human class 6 semaphorin gene: alternative splicing and all-trans-retinoic acid-dependent downregulation in glioblastoma cell lines.

We have identified a novel human gene related to the class 6 semaphorin family of axon guidance molecules, termed human semaphorin 6B or (HSA)SEMA6B. Two splicing variants of this gene were identified by RT-PCR: (HSA)SEMA6B.1 (short isoform) and (HSA)SEMA6B.2 (longer isoform). Computational analysis suggests that these isoforms correspond to putative secreted and transmembranous semaphorins, respectively. The levels of (HSA)SEMA6B expression were evaluated by Northern blot analysis in different tissues and in some pathological and pharmacological conditions. We observed that (HSA)SEMA6B is highly expressed in human brain and at lower levels in a variety of other tissues. Interestingly, the (HSA)SEMA6B transcript was downregulated in two different human glioblastoma cell lines (T98G and A172) upon prolonged treatment with all-trans-retinoic acid, an anti-tumor and differentiation-inducing agent.

Definition of the gene content of the human genome: the need for deep experimental verification.

Based on the analysis of the drafts of the human genome sequence, it is being speculated that our species may possess an unexpectedly low number of genes. The quality of the drafts, the impossibility of accurate gene prediction and the lack of sufficient transcript sequence data, however, render such speculations very premature. The complexity of human gene structure requires additional and extensive experimental verification of transcripts that may result in major revisions of these early estimates of the number of human genes.

Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags.

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1, 181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html).

NABC1 (BCAS1): alternative splicing and downregulation in colorectal tumors.

We have identified a new splicing variant of the gene "novel amplified in breast cancer 1," NABC1 (HGMW-approved symbol BCAS1). This variant, which we call NABC1_5B, uses a previously unidentified 135-bp exon. Also in this report, we confirm that NABC1 is overexpressed in breast tumors and show that both NABC1 and NABC1_5B are downregulated in colorectal tumors.

Shotgun sequencing of the human transcriptome with ORF expressed sequence tags.

Theoretical considerations predict that amplification of expressed gene transcripts by reverse transcription-PCR using arbitrarily chosen primers will result in the preferential amplification of the central portion of the transcript. Systematic, high-throughput sequencing of such products would result in an expressed sequence tag (EST) database consisting of central, generally coding regions of expressed genes. Such a database would add significant value to existing public EST databases, which consist mostly of sequences derived from the extremities of cDNAs, and facilitate the construction of contigs of transcript sequences. We tested our predictions, creating a database of 10,000 sequences from human breast tumors. The data confirmed the central distribution of the sequences, the significant normalization of the sequence population, the frequent extension of contigs composed of existing human ESTs, and the identification of a series of potentially important homologues of known genes. This approach should make a significant contribution to the early identification of important human genes, the deciphering of the draft human genome sequence currently being compiled, and the shotgun sequencing of the human transcriptome.

ExInt: an Exon/Intron database.

The Exon/Intron (ExInt) database incorporates information on the exon/intron structure of eukaryotic genes. Features in the database include: intron nucleotide sequence, amino acid sequence of the corresponding protein, position of the introns at the amino acid level and intron phase. From ExInt, we have also generated four additional databases each with ExInt entries containing predicted introns, introns experimentally defined, organelle introns or nuclear introns. ExInt is accessible through a retrieval system with pointers to GenBank. The database can be searched by keywords, locus name, NID, accession number or length of the protein. ExInt is freely accessible at http://intron.bic.nus.edu.sg/exint/exint.html

IE-Kb: intron exon knowledge base.

SUMMARY: IE-Kb (Intron Exon-Knowledge base) illustrates the intron-exon dynamics in eukaryotic genes. We have developed three different knowledge sets, namely 'Non-redundant ExInt', 'Non-redundant Pfam-ExInt complement' and 'Non-redundant GenBank eukaryotic subdivisional sets' to understand this phenomenon. Statistical analysis is performed on each knowledge set and the results are made available online. The entries in knowledge sets are ranked based on their intron length, exon length and protein length with relational hyper-links to the corresponding intron phase, intron position, intron sequence, gene definition and parent GenBank entry.

Centripetal modules and ancient introns.

We have created an algorithm which instantiates the centripetal definition of modules, compact regions of protein structure, as introduced by Go and Nosaka (M. Go and M. Nosaka, 1987. Protein architecture and the origin of introns. Cold Spring Harbor Symp. Quant. Bio. 52, 915-924). That definition seeks the minima of a function that sums the squares of C-alpha carbon distances over a window around each amino acid residue in a three-dimensional protein structure and identifies such minima with module boundaries. We analyze a set of 44 ancient conserved proteins, with known three-dimensional structures, which have intronless homologues in bacteria and intron-containing homologues in the eukaryotes, with a corresponding set of 988 intron positions. We show that the phase zero intron positions are significantly correlated with the module boundaries (p = 0.0002), while the intron positions that lie within codons, in phase one and phase two, are not correlated with these 'centripetal' module boundaries. Furthermore, we analyze the phylogenetic distribution of intron positions and identify a subset of putatively 'ancient' intron positions: phase zero positions in one phylogenetic kingdom which have an associated intron either in an identical position or within three codons in another phylogenetic kingdom (a notion of intron sliding). This subset of 120 'ancient' introns lies closer to the module boundaries than does the full set of phase zero introns with high significance, a p-value of 0.008. We conclude that the behavior of this set of introns supports the prediction of a mixed theory: that some introns are very old and were used for exon shuffling in the progenote, while many introns have been lost and added since.

Toward a resolution of the introns early/late debate: only phase zero introns are correlated with the structure of ancient proteins.

We present evidence that a well defined subset of intron positions shows a non-random distribution in ancient genes. We analyze a database of ancient conserved regions drawn from GenBank 101 to retest two predictions of the theory that the first genes were constructed by exon shuffling. These predictions are that there should be an excess of symmetric exons (and sets of exons) flanked by introns of the same phase (positions within the codon) and that intron positions in ancient proteins should correlate with the boundaries of compact protein modules. Both these predictions are supported by the data, with considerable statistical force (P values < 0.0001). Intron positions correlate to modules of diameters around 21, 27, and 33 A, and this correlation is due to phase zero introns. We suggest that 30-40% of present day intron positions in ancient genes correspond to phase zero introns originally present in the progenote, while almost all of the remaining intron positions correspond to introns added, or moved, appearing equally in all three intron phases. This proposal provides a resolution for many of the arguments of the introns-early/introns-late debate.

Relationship between "proto-splice sites" and intron phases: evidence from dicodon analysis.

The coding sequence at the boundaries of exons flanking nuclear introns shows some degree of conservation. To the extent that such sequences might be recognized by the splicing machinery, this conservation may be a derived result of evolution for efficient splicing. Alternatively, such conserved sequences might be remnants of proto-splice sites, which might have existed early in eukaryotic genes and served as the targets for the insertion of introns, as has been proposed by the introns-late theory. The distribution of intron phases, the position of the intron within a codon, is biased with an over-representation of phase 0 introns. Could any distribution of proto-splice sites account for today's intron phase distribution? Here, we examine the dicodon usage in six model organisms, based on current sequences in the GenBank database, and predict the phase distribution that would be expected if introns had been inserted into proto-splice sites. However, these predictions differ between the various model organisms and disagree with the observed intron phase distributions. Thus, we reject the hypothesis that introns are inserted into hypothetical proto-splice sites. Finally, we analyze the sequences around the splice sites of introns in all six of the species to show that the actual conservation of sequence in exon regions near introns is very small and differs considerably between these species, which is inconsistent with a general proto-splice sites model.