Given that gene duplication is a major driving force of evolutionary change and the key mechanism underlying the emergence of new genes and biological processes, this study sought to use a novel genome-wide approach to identify genes that have undergone lineage-specific duplications or contractions among several hominoid lineages. Interspecies cDNA array-based comparative genomic hybridization was used to individually compare copy number variation for 39,711 cDNAs, representing 29,619 human genes, across five hominoid species, including human. We identified 1,005 genes, either as isolated genes or in clusters positionally biased toward rearrangement-prone genomic regions, that produced relative hybridization signals unique to one or more of the hominoid lineages. Measured as a function of the evolutionary age of each lineage, genes showing copy number expansions were most pronounced in human (134) and include a number of genes thought to be involved in the structure and function of the brain. This work represents, to our knowledge, the first genome-wide gene-based survey of gene duplication across hominoid species. The genes identified here likely represent a significant majority of the major gene copy number changes that have occurred over the past 15 million years of human and great ape evolution and are likely to underlie some of the key phenotypic characteristics that distinguish these species. This genome-wide analysis reports the major lineage-specific gene copy number changes that have occurred over the past 15 million years of human and great ape evolution The evolution of genomes has been primarily driven by single basepair mutation, chromosomal rearrangement, and gene duplication , with thOver the past few years, as the human genome sequence has become available, it has become apparent that recent segmental duplications in the human genome are far more frequent than originally believed, comprising approximately 5% of the available sequence . DuplicaThe assessment of DNA copy number changes between different human genomes has been aided by the development of comparative genomic hybridization (CGH), which originally involved cohybridizing differentially labeled test and reference genomic DNAs to normal metaphase chromosomes . A cytogWhile cDNA microarrays, containing sequences derived from tens of thousands of genes, have been used extensively to profile mRNA expression levels , their uUntil now, cDNA aCGH studies have been limited to only within-species comparisons, partly due to concerns related to the increased sequence divergence that would come into play with interspecies applications. Such sequence divergence may produce differential hybridization signals that would be difficult to distinguish from those that arose from copy number changes. Fortunately, despite their significant anatomical and physical differences, hominoid species show a strikingly high degree of similarity at the genome sequence level, with the average sequence divergence values estimated as 1.24%, 1.62%, and 1.63% for human–chimp, human–gorilla, and chimp–gorilla, respectively, and orangutan showing approximately 3.1% sequence divergence when compared to human, chimp, or gorilla .Because of this close sequence conservation, we reasoned that it may be possible to use cDNA aCGH to directly compare the cross-species hybridization signatures of human genes to those of the great apes and to identify genes that have alterations in copy number and/or significant changes in exonic sequence between human and other hominoid species. After we initiated such a cDNA aCGH study, two interhominoid aCGH reports appeared that used arrays containing either cloned or amplified genomic DNAs . While thttp://rana.lbl.gov/EisenSoftware.htm) was used for visualization of aCGH data for each gene as it occurred in the genome, permitting a “gene-by-gene” survey of the data and allowing for easy detection of interspecies copy number variations, whether they occur as single isolated genes or as multigene blocks.Interhominoid cDNA aCGH was carried out in a series of pairwise comparisons using microarrays containing 39,711 human cDNAs, representing the majority of all human genes . The paiResults of the distribution of lineage-specific (LS) aCGH signatures for different individual hominoid species are presented in Homo/Pan split, showed fewer LS signatures (92) than did human (140), even though they represent the same evolutionary age. As mentioned below, this is due in large part to the significant number of LS gene copy number increases found in human.Several interesting features were evident from these data. First, when increases and decreases were scored separately or combined, the number of LS signatures was generally in proportion to the evolutionary age of that lineage, although not in all cases. Bonobo and chimpanzee, from the time since the Second, while all lineages showed more gene copy number increases than decreases, this was most pronounced in humans, with 134 cDNAs representing increases and only six representing decreases. This increase-to-decrease ratio (22.3:1) was significantly greater than that of any of the great apes, which showed ratios ranging from 2.75:1 (chimpanzee) to 1.18:1 (orangutan). It is worth noting that only genes found in the human genome are represented on the cDNA arrays, and if there are genes that are absent in human but present in the great apes, e.g., genes that were lost as the human lineage emerged, those genes would not be part of this analysis. So, while it is likely that the complete loss of both copies of a gene in an LS manner is a rare event, the number of genes identified here as having a reduced copy number specifically in the human lineage may be an underestimate of the true total.Third, as mentioned above, for all lineages tested, the number of genes showing LS increases was greater than those showing LS decreases. Determination as to whether this is due to some, as yet unknown, ascertainment bias of the method or whether this is a real evolutionary tendency favoring gene duplication over gene loss will require further investigation. The favoring of gains over losses is even more striking when two additional factors are considered. (1) The fact that the cDNAs were only from human, while likely to be important to the low number of genes showing human lineage-specific (HLS) losses previously mentioned, does not help explain why, for all lineages tested, the number of LS genes showing increases was greater than the number showing decreases. To the contrary, if there were genes not on the microarray because they were only found in one or more of the great ape lineages, inclusion of such genes would be expected to add to the total number of LS increases, making the disparity between increased and decreased LS genes even greater. (2) If human/great ape sequence divergence was responsible for some of the LS aCGH signals that were obtained, it would, if anything, produce a falsely elevated number of LS decreases.Homo/Pan split, 12; gorilla, 17.3; and orangutan, 17.1.Fourth, while only orangutan had more LS gene copy number increases (222) than did human (134), when the number of genes showing copy number increases was measured as a function of the evolutionary age of the lineage, human showed the greatest number of expansions of any hominoid. When measured as copy number increases per million years of age, the following values were obtained: human, 26.8; bonobo and chimpanzee since the Pan/gorilla, 16 genes; Pan/orangutan, 27, and gorilla/orangutan, 45. For gene increases, this trend continued, with gorilla/orangutan (17) showing more changes than Pan/orangutan (nine). Interestingly, Pan/gorilla showed a departure from this trend with 28 increased genes, suggesting that gene expansion may have been particularly active in the African great apes as a group. There were also a number of more complex gene copy number changes in the five hominoid lineages, with some species showing an increase relative to human for a particular gene and others showing a decrease. These changes are likely due to more than one event, which may be indicative of a genomic region that is relatively unstable and/or of genes whose copy numbers have been influenced by different selection pressures. We identified 190 genes that showed copy number changes in multiple lineages, bringing the total number of LS genes identified to 1,005, which represents 3.4% of the total number of genes tested on the microarrays. Given the relatively conservative selection criteria used  genome assembly lists multiple Chromosome 5 locations for some of the HLS cDNAs contained on the positive BAC (e.g., BIRC1) and therefore it is likely that the multiple, closely spaced signals seen in some of the human interphase spreads , as well as a set of flanking genes not shown to be increased in human (control probe), were evaluated by interphase and metaphase fluorescence in situ hybridization (FISH) using bacterial artificial chromosome (BAC) probes see . The FIS spreads A reflectMetaphase FISH showed both the test probe and the flanking probe to be located in the human 5q13 band. Both probes were located in the proximal q arm of the orangutan (PPY) Chromosome 4 and in the p arms of the bonobo (PPA) and chimpanzee (PTR) Chromosomes 4. In the gorilla (GGO), both probes were located on the gorilla Chromosome 19. All of these primate locations are consistent with described evolutionary chromosomal rearrangements, with the orangutan Chromosome 4 considered to be the ancestral Chromosome V . These rIt is of interest that, considering the orangutan Chromosome 4 as the ancestral Chromosome V, rearrangements at this site have occurred in all of the other three great ape species (pericentric inversion in bonobo and chimpanzee, translocation in gorilla) and in the human (gene duplication). This region is also involved in spinal muscular atrophy (SMA), which is characterized by deletions of one or more genes in this region . Taken tFGF7-like genes. Some genes we identified as having LS aCGH signatures have been previously studied by others using different methods, which provides a means of independently checking the accuracy of the cDNA aCGH data presented here. One such gene, the FGF7 gene on Chromosome 15, was studied by morpheus on Chromosome 16 was reported and shown to exhibit gene copy number variation between several hominoid species (morpheus genes to be 15, 25–30, 17, and nine for human, chimp, gorilla, and orangutan, respectively. In order to provide an independent test of the accuracy of the interspecies cDNA aCGH data we generated, the aCGH signatures of morpheus-like cDNAs were assembled for the same hominoids (2 ratios for these cDNAs indicated that chimpanzee had the most copies, gorilla was slightly higher than human, and orangutan clearly had the fewest, results that are in very good agreement (correlation = 0.96) with the copy number estimates reported independently by Recently the identification of a multimember gene family named  species . Using aominoids B. The avCXYorf1 gene family. They found values of seven, two, three, and one for human, chimpanzee, gorilla, and orangutan, respectively. These values closely mirrored the aCGH values that were obtained (obtained C (correlFLJ22004 gene shows the greatest gorilla-specific copy number increase (average log2 ratio = 3.94). This gene resides near the fusion region on Chromosome 2q14.1 (see below) and is contained within BAC RP11-432G15. Consistent with the aCGH data, two independent interhominoid FISH studies, by our lab (Based on aCGH data, the  our lab  and by FHomo/Pan split (less than 5–6 MYA) and therefore should represent a subset of the segmental duplications found in the WSSD dataset. Results of this analysis confirmed this expectation . The regions near the C-band regions on 16 (15 cDNAs) and Y (14 cDNAs) also showed an enrichment of LS genes, although to a lesser extent. These regions, as well as the pericentromeric regions of the acrocentric chromosomes, which showed enrichment for LS genes, are known to contain highly repetitive DNA, which may make them especially prone to recombination and duplication.Genes identified as having a variation in copy number specific for one or more hominoid lineages occurred either as single isolated genes or as clusters of genes. This latter category likely reflects LS copy number changes that involved blocks of contiguous genes. In addition, certain specific regions of the genome, while not necessarily composed of contiguously positioned LS genes, showed a marked enrichment for LS genes. Surveying the genome for regions containing contiguous gene clusters of LS genes or for regions highly enriched in LS genes  identified 23 prominent sites ; Table 2Previous reports have shown that recent (less than 40 MYA) segmental duplications in the human genome are positionally biased and found more frequently in pericentromeric and subtelomeric regions . ConsistBIRC1 gene, which we show is amplified uniquely in humans, are sometimes found in SMA patients. This region and another at 5p14.3–5p13.3 that also contains a cluster of LS genes are near the breakpoint sites of a pericentric inversion that occurred during hominoid speciation  maps to Chromosome 5q13 and was elevated specifically in the human lineage. NAIP has been implicated in delaying neuronal programmed cell death (Of the 140 genes showing HLS variation in copy number, 134 represented human gene increases and six represented decreases . While rll death  and is kll death . If an iSLC6A13), a leucine zipper-containing gene highly expressed in brain (KIAAA0738), α7 cholinergic receptor/Fam7 fusion gene (CHRFAM7A), a p21-activated kinase (PAK2), a Rho GTPase-activating protein (SRGAP2), a Rho guanine nucleotide exchange factor (ARHGEF5) that is a member of the rhodopsin-like G protein-coupled receptor family, and Rho-dependent protein kinase (ROCK1). Inhibition of ROCK1 has been shown to prevent long-term memory, and ROCK1, together with a RhoGEF and RhoGAP, have been recently implicated in a model of long-term memory based on fear conditioning   at Chromosome 19q13 that is significantly elevated in human relative to most hominoids map to the two chromosomal regions giving the highest LOD scores in a recent genome-wide scan related to specific language impairment (Another gene showing an HLS copy number increase, c growth . Overexp pattern , raisingpairment .AQP7), which is thought to be involved in water transport across membranes, shows an HLS increase in copy number, while the genes immediately flanking it (NFX1 and AQP3) do not show HLS aCGH signals. Similarly, AQP7 gene has been recently (less than 40 MYA) duplicated several times while flanking regions show no recent duplication. These data suggest that a series of HLS segmental duplications occurred that focused primarily on the AQP7 gene, which spans 17 kb of the 22 kb duplication. This observation, together with the fact that several of the additional AQP7 copies appear to be potentially functional (see below), raises the possibility that significant selection pressure may have been exerted on AQP7-like genes specifically in the human lineage.The aquaporin 7 gene  and, in bonobos and chimps, the FCER2 gene, encoding a lymphocyte IgE receptor, and the IL1RL1 gene encoding an interleukin receptor 1-like protein. Also, it has been shown that chimpanzees can synthesize a form of sialic acid while humans cannot, owing to the loss of function in humans of a specific sialic acid hydroxylase  encodes the fibroblast growth factor receptor 3 (FGFR3), which when disrupted in humans causes achondroplasia, the most frequent form of short-limb dwarfism. The SET8 gene is also significantly elevated in copy number only in gorilla (average log2 ratio = 2.65) and also related to development. The gene encodes a transcription factor and appears to be homologous (protein similarity of 43% over 110 amino acids) to the Drosophila trithorax gene, which functions in segmentation determination through interaction with bithorax and antennapedia complex genes, suggesting that it may serve a role in gorilla-specific development. There were a significant number of genes (28) showing increased copy numbers specifically in the African great apes (bonobo, chimpanzee, and gorilla). Among these were the MSTP028 gene, encoding a voltage gated potassium channel; the PLA2G4B gene, encoding phospholipase A2β, which shows high brain and (in particular) cerebellar expression; and the SPTBN5 gene, which encodes a nonerythroid spectrin. SPTBN5 is immediately adjacent to PLA2G4B at Chromosome 15q15.1 in the genome and, like PLA2G4B, shows high cerebellar expression, raising the possibility that their function(s) in the African great apes may be linked. Finally, while the HLS and LS genes mentioned above have interesting biological implications related to human and great ape differences, each should be viewed as tentatively HLS or LS until the interhominoid copy number differences for these genes are confirmed by independent methods.Another gene specifically increased in gorilla (average logClassification of HLS and LS genes according to predicted molecular function was carried out by Gene Ontology (GO) analysis. For the great majority of functional categories, both HLS and LS gene groups gave GO distributions similar to that found with all known genes (UniGene collapsed set), with ligand binding, catalytic activity, signal transducer activity, and transporter activity being the four most highly represented functional categories . This anAQP7 gene predicts that of seven closely related (greater than 90%) copies in the genome, at least four appear to be potentially functional. In contrast, the FLJ13263 gene had four closely related sequences, and these all appear to be pseudogene-like. Finally, the fact that it has been shown that pseudogenes can play important functional roles . Using this comparison, we have determined that genes that gave aCGH signatures indicative of copy number increase specifically in the human lineage, showed a 7-fold increase in the frequency of gaps and absent sequence homology in the chimpanzee draft compared to a randomly selected gene (EST) set  database's best reciprocal alignment of the July 2003 human genome and the November 2003 Arachne 4X chimpanzee draft genome (EST) set . Such a However, a limitation of only comparing the human and chimpanzee genomes is that no out-group analysis is provided, preventing discrimination of ancestral and derived forms and limiting the ability to identify gene copy number changes unique to a specific hominoid lineage. In contrast, the interhominoid aCGH studies described here provide reliable genome-wide data for out-group analysis across five primate species, allowing easy identification of LS copy number differences.each of the four great apes studied). Of the 353 genes that were “human > chimp,” 200 were not found in the HLS set, indicating that over half (57%) of the “human > chimp” genes were not HLS.In order to provide some perspective on the importance of out-group data when trying to identify LS gene changes, a comparison was carried out between two aCGH clone sets. One set contained 153 genes we identified by cDNA aCGH that were specifically increased in copy number in the human lineage when compared to each of the four great ape lineages (i.e., HLS). The other clone set, while derived from the same aCGH experiments using the same cutoff values, contained 353 genes that showed aCGH signals in which the human copy number was greater than the chimpanzee (i.e., “human > chimp”). Comparison of these two datasets allows one to determine how frequently a “human > chimp” gene is also HLS (i.e., human copy number is greater than PLA2G4B and SPTBN5) specifically elevated in the African great apes, showed that, in chimpanzee, signals were widely distributed among many chromosomes, while in gorilla the signals were restricted to two sites, one single copy and the other multicopy  and cDNAs that mapped to multiple genomic locations (multiple locations). In this latter category, we also included a minority of cDNAs that had no assignable location in the genome assembly. We identified HLS cDNAs that showed stronger hybridization with human DNA  and determined how many of these occurred in each of the two mapping categories. HLS signatures were found for 0.185% of unique location cDNAs (66/35,680) and 2.88% of multiple location cDNAs (116/4,031), a frequency difference of more than an order of magnitude (approximately 1:16).Such a strong enrichment, in the multiple location category, of genes showing increased human aCGH signals specific to the human lineage would be expected if such genes were present as multiple closely related copies with distinct genome locations and, as a result, were placed in the multiple location group. No such gene distribution bias would be expected if the LS signatures were mainly due to sequence divergence.Additionally, we estimated what fraction of LS cDNAs in each species were cDNAs with multiple human map positions. Values of 59%, 10%, 13%, 14%, 10%, and 20% were obtained for human, bonobo, chimp, bonobo/chimp total, gorilla, and orangutan, respectively, providing further support that the increased  HLS aCGH signatures that were obtained are likely due to gene copy number increases specific to the human lineage.We also carried out interhominoid FISH using a BAC probe (RP11-93K3) containing a gene (IMAGE 1882505) that gave a reduced signal specifically in the orangutan lineage, which is the lineage where sequence divergence might have its greatest artifactual contribution. Resulting FISH data see  showed 1Cot-1 DNA  indicated that there was no correspondence between genes hybridizing more strongly to Cot-1 and genes that are LS.Lastly, to address the possibility that such signals might be due to highly repetitive sequences associated with LS genes that were not effectively blocked during hybridization, we examined the cDNA sequences of five cDNAs that showed stronger hybridization with human DNA. In all cases no repeats were found that would account for the HLS aCGH data. In addition, hybridization using labeled DNAs that were used for this study were derived from human , bonobo , chimpanzee , gorilla , and orangutan . Human and chimpanzee genomic DNA samples were isolated from blood cells using Super Quick-Gene kits from the Analytical Genetic Testing Center (Denver, Colorado, United States). One gorilla and two bonobo samples were isolated from cell lines using DNeasy Tissue kits from Qiagen . An orangutan sample and a gorilla sample were isolated from blood by other laboratories. Remaining DNAs (one bonobo, one gorilla, and two orangutan) were obtained from the Coriell Institute (Camden, New Jersey, United States) and originally derived from primary fibroblast cell lines.http://image.llnl.gov) and spotting them onto Corning GAPSII aminosilane slides using a custom-built robotic arrayer (http://cmgm.stanford.edu/pbrown/mguide/index.html). The labeling of genomic DNA and hybridization to cDNA microarrays were performed as previously described (Cot-1 DNA (Invitrogen), 100 μg of yeast tRNA (Invitrogen), and 20 μg of poly(dA-dT) (Sigma, St. Louis, Missouri, United States) to block hybridization to nonspecific and repetitive elements in genomic DNA. We adjusted the final hybridization volume (40 μl) to contain 3.5× SSC and 0.3% SDS. Following sample denaturation (2 min at 100 °C) and a Cot-1 preannealing step (20 min at 37 °C), we cohybridized test and reference samples to a cDNA microarray containing 39,711 nonredundant cDNA clones, representing 29,619 different human genes. Samples were hybridized at 65 °C for 16 h. Following hybridization, arrays were washed in 2× SSC, 0.03% SDS for 5 min at 65 °C, followed by successive washes in 1× and 0.2× SSC for 5 min each at room temperature.DNA microarrays used in this study were fabricated by PCR-amplifying IMAGE clones (escribed . In briehttp://genome-www5.stanford.edu) for analysis. For each experiment, fluorescence ratios were normalized by setting the average log2 fluorescence ratio for all array elements equal to 0. We included for analysis only those genes that were reliably measured (i.e., fluorescence intensity/background of greater than 1.4 in the reference channel) in greater than or equal to 50% of samples. Genes not meeting these criteria were viewed as absent. Map positions for cDNA clones on the array were assigned using the UCSC GoldenPath assembly (http://genome.ucsc.edu/), November 2002 freeze. Gene copy number ratios were visualized in log2 colorimetric scale with the genes ordered by chromosomal position using TreeView version 1.6 (http://rana.lbl.gov/EisenSoftware.htm). To provide the most accurate depiction of chromosomal gene distribution, cDNAs with multiple genome map positions (more than 1 Mb apart) were represented in TreeView at each assigned map location.Individual microarrays were imaged with a GenePix 4000B scanner  and fluorescence intensities were extracted using GenePix Pro 3.0 software and uploaded into the Stanford Microarray Database (SMD) . Second, for a gene copy number change to be considered unique to a particular species, at least half of the absolute values of comparisons within that species had to meet or exceed a threshold of 0.5 with all such values in the same direction, i.e., either all positive or all negative, and at least half of the absolute values of comparisons within each of the remaining species had to be below a threshold of 0.5. For example, for a gorilla LS gene, at least half of the gorilla comparisons had to meet or exceed the 0.5 threshold, while at least half of the comparisons within each of the remaining species had to be below the threshold. Third, in order to compensate for missing (i.e., “absent”) values for a given cDNA of all “present” values within each species, no more than one could fall below the threshold (0.5) for each species. Fourth, to ensure sufficiently high signal-to-noise in the identification of altered ratios, for a given cDNA and given great ape species, each absolute value of the average of the species versus human comparison for that species had to be at least 2.5-fold greater than the absolute value of each remaining species average, including human versus human comparisons. For HLS genes, the absolute value of each species average of the great ape versus human comparisons had to be at least 2.5-fold greater than the average of the absolute value of the human versus human comparisons.For selection of LS cDNAs, the values considered were the logFor cDNAs in which the copy number was either increased or decreased in two or more hominoids relative to all the other hominoids, the same criteria were used as before, except the cDNA would have to meet or exceed the 0.5 threshold selection criteria for more than one species.2 value = 0.39). In a similar manner, we took advantage of the fact the one of the human-to-human comparisons used in our experiments was between a male and female. In this context, X chromosome genes in the female should be present as two copies while in the male will exist as one copy. Calculation of the average aCGH ratios of 957 such genes in the male/female comparison yielded a log2 value of 0.21. The different values obtained in these two tests may reflect the fact that in the male/female comparison a Y chromosome was present, while this was not true in the other study, which used XO cell lines. The presence of sequences on the Y that are shared with the X could have produced a compression of aCGH fluorescence ratio values, accounting for the difference in X chromosome-related log2 ratios described above. Similar compression effects on X chromosome ratios have recently been reported  and from great ape fibroblast cell lines, obtained from Coriell. The four great ape species studied were bonobo (Pan paniscus [PPA], Coriell #AG05253A), chimpanzee (Pan troglodytes [PTR], Coriell #AG06939A), lowland gorilla (Gorilla gorilla [GGO], Coriell #AG05251B), and Sumatran orangutan (Pongo pygmaeus [PPY], Coriell #AG12256).Using standard procedures, metaphase spreads and interphase nuclei were prepared from human lymphocytes (OCLN, GTF2H2, and BIRC1 was selected as a probe for the region with increased copy number in human. A second BAC clone (RP11-1077O1) flanking the region amplified in human and containing portions of the RAD17 gene was selected as a control probe. BAC clones were obtained from BACPAC Resources at the Children's Hospital Oakland Research Institute and from Research Genetics. Whole-cell PCR was done to verify that the OCLN, GTF2H2, and BIRC1 genes were on BAC CTD-2288G5 and that the RAD17 gene was on BAC RP11-1077O1. BAC DNAs were prepared using Large Construct Kits (Qiagen). BAC probes were directly labeled with Spectrum Green (Vysis, Downers Grove, Illinois, United States) and Spectrum Orange (Vysis) using the Vysis Nick Translation Kit and protocol.One BAC clone (CTD-2288G6) containing all or portions of the coding regions for Cot-1 DNA was used to block cross-hybridization of high-copy repeat sequences. In each experiment, dual-color hybridization was performed using a probe carrying genes with a predicted increase in copy number specifically in the human lineage (CTD-2288G6 or CTC-790E5) and a flanking probe (RP11-1077O1 or RP11-1113N2) containing a gene not predicted to show an HLS increase in copy number. For each species, two separate hybridizations were performed: one with the probe containing the genes showing increased human copy number labeled with Spectrum Green and the flanking probe with Spectrum Orange, and the other in which the dyes were reversed. For each probe combination for each species, a minimum of 200 interphase nuclei and ten metaphase spreads were examined. A whole chromosome painting probe for human Chromosome 5  was used to confirm the gorilla Chromosome 19 to be syntenic with the human Chromosome 5 for the region of interest.FISH analyses with the BAC probes were performed using standard techniques. 0. Nuclei that were in S/G2 demonstrated four copies of the control probe and the test probes were proportionately in multiple copies of the number established in the nonproliferating cells. Similar experimental conditions were used for the additional BAC FISH analyses described.The hominoid cell lines used for FISH analysis were grown asynchronously in monolayer culture. Metaphase spreads and nuclei were obtained from a shake-off preparation and thus were somewhat selected for proliferative activity. Similarly, human lymphocyte cultures stimulated with the mitogen phytohemagglutinin contain cells in various stages of the cell cycle. In order to judge the replication state of the nuclei scored, dual-color FISH assays included probes both for DNA sequences that by aCGH showed copy number difference between test and reference DNA and for sequences on the same human chromosome that had the same (diploid) number of copies. Nuclei that showed diploid copy number of this control probe were assessed to be in Ghttp://www.ncbi.nlm.nih.gov/Entrez) sequence retrieval tool and saved locally in FASTA format. Likewise, the random IMAGE clone sequences were obtained by first downloading GI numbers for all human IMAGE clones and then using a random number generator to pick approximately 200 random IMAGE clones from the list of GI numbers. These random IMAGE clone sequences were then downloaded from Entrez in a similar fashion. The April 2002 WSSD dataset was downloaded from the Segmental Duplication Database website . The two IMAGE clone sequence datasets were formatted and “BLASTed” against the WSSD sequences locally using NCBI's stand-alone BLAST executables for Windows. BLASTs were limited to an expect value of e−20 and then the best match was reported by a Perl (http://activestate.com/) script for each query. No restrictions on percent identity of the match or match length were imposed.Sequences of IMAGE clones for each HLS gene were obtained using NCBI's Entrez (http://genome.ucsc.edu/cgi-bin/hgGateway). The BLAT hits were parsed such that only hits with a percent identity greater than or equal to 90% were reported. Furthermore, only hits with a match coverage (match length/query length) greater than or equal to 50% were reported.The HLS gene IMAGE clone sequences see  were comhttp://genome.ucsc.edu/goldenPath/14nov2002/database/). Using the position data obtained from the BLAT alignments along with a 50 kb buffer on both sides of the alignments, the relative repeat content was determined for each HLS gene region using a Perl script. As a comparison, the relative repeat content was determined for the entire genome. Annotated gaps within the regions and the human genome were subtracted from the percent content calculation so that these content values were not skewed by gaps. Only long interspersed nuclear element (LINE), long terminal repeat (LTR), short interspersed nuclear element (SINE), Simple, and Satellite classes of repeats were included in the analysis.Repeat annotation was downloaded from UCSC . Likewise, the Satellite repeat content for the LS genes within the 23 clusters was also determined in a similar fashion.http://apps1.niaid.nih.gov/david/upload.asp)  (oad.asp) . Analysehttp://apps1.niaid.nih.gov/david/)  , a progrGenBank accession numbers were used for the HLS and LS analysis due to nearly half of the genes lacking UniGene numbers, thus making GenBank accession numbers more inclusive of the entire HLS and LS dataset analysis. Alternatively, UniGene numbers were used for the the genome-wide analysis because they provide a nonredundant dataset which is a much closer estimate to the number of genes (33,655) in the human genome versus the human RefSeq accession numbers. When subtracting all computer-based models from human RefSeq, only 20,850 RefSeq accession numbers were available for analysis.http://genome.ucsc.edu/goldenPath/gbdDescriptions.html). From the all_est file, 200 random IMAGE clones were picked to ensure that at least one EST per IMAGE clone would map to the human genome. The EST sequences for both the HLS and random datasets were downloaded from GenBank and compared to the July 2003 human genome via a locally installed version of BLAT. BLAT output was parsed so that hits with a score greater than 200 and percent identity greater than 90% were examined for chimpanzee homology. The score and percent identity calculations mimic the calculations performed with the Web-based version of BLAT (http://genome.ucsc.edu/cgi-bin/hgBlat); the formula for these calculations was provided by Donna Karolchik.The HLS dataset is identical to that previously described. The random dataset chosen for this analysis was determined from UCSC's all_est annotation (http://genome.ucsc.edu/goldenPath/hg16/versusPt0/). For each BLAT hit, each block of alignment was compared to the chimpanzee versus human best chain annotation and was scored as follows: “chimp positive” indicates the block is entirely homologous to chimp; “chimp partial” indicates the block is partially homologous to chimp but there are gaps in the homology; “chimp gap” indicates the block is within a gap of the chimp homology; “chimp negative” indicates the block has no homology to chimpanzee. The summary numbers are based on all of the blocks of alignments and how they are scored in reference to chimpanzee homology.The BLAT hits, as defined as one or more blocks of alignment within score and percent identity cutoffs, were compared to the chimpanzee versus human reciprocal best chain alignment annotation . The following primer/probe sequences were used: PLA2G4B F 5′-GCAGGTCTGGGTGAGGGTT-3′, PLA2G4B R 5′-GCTGCACCTGATCCCCACT-3′, and the probe 5′-VIC-CAGGAAGTTGCCACACAGGTGAGCA-TAMRA-3′; FLJ31659 F 5′-GCTCAGACATCCAGGGACGA-3′, FLJ31659 R 5′-CGCTTCTCCCAGGATTGGT-3′, and the probe 5′-VIC-CACATTCGTCCAACAGCGGTCGC-TAMRA-3′; BC040199 F 5′-GAGGAAGGTTGGGTGTGGAG-3′, BC040199 R 5′-ACTGGGTGTCCTGCTGGCT-3′, and the probe 5′-VIC-TTGCTTGCTGTGGCCCCAAGCT-TAMRA-3′; CFTR F 5′-CGCGATTTATCTAGGCATAGGC-3′, CFTR R 5′-TGTGATGAAGGCCAAAAATGG-3′, and the probe 5′-6FAM-TGCCTTCTCTTTATTGTGAGGACACTGCTCC-TAMRA-3′.RT-PCR analysis of interhominoid DNA copy number variation was carried out using an ABI Prism 7700 sequence detector  . Exon-sp2, 900 nM forward primer, 300 nM reverse primer, 200 nM probe, 1.25 U AmpliTaq Gold DNA polymerase (PE/ABI), and the template genomic DNA. Thermal cycling conditions were as follows: activation of TaqGold at 95 °C for 10 min followed by 40 cycles of amplification at 95 °C for 15 s and 60 °C for 1 min.Amplification reactions were performed in MicroAmp optical tubes (PE/ABI) in a 50 μl mix containing 8% glycerol, 1× TaqMan buffer A (500 mM KCl, 100 mM Tris-HCl, 0.1 M EDTA, 600 nM passive reference dye ROX [pH 8.3 at room temperature]), 300 μM each of dATP, dGTP, and dCTP and 600 μM dUTP, 5.5 mM MgClt), which represents the PCR cycle at which an increase in reporter fluorescence above a baseline signal can first be detected. The starting copy number of each test gene was quantified in the ape samples by determining the Ct of the test gene and using a standard curve for copy number. The standard curve for each gene was generated using the fluorescent data from five serial dilutions of human genomic DNA and calculating a single copy of each gene per haploid human genome, as annotated in the current genome build. Copy numbers of the test genes in ape samples were normalized to the copy number of the CFTR gene, which serves as a control representative of a single gene per haploid genome  Normal human control, PHA-stimulated lymphocytes. Multiple (10–12) signals present: 9p12, 9q12, 4q arm, and two acrocentric p arm regions. The Chromosome 9 signals appear to flank the 9q heterochromatin and centromere regions, with the p arm signal a double signal.(B) Bonobo fibroblast culture. Multiple (10–12) signals.(C) Chimpanzee fibroblast culture. Multiple (10–12) signals.(D) Gorilla fibroblast culture. Multiple (12–15) signals.(E) Orangutan fibroblast culture. Two signals present on a pair of homologues (i.e., single copy in haploid genome). Also shown is a TreeView image  for IMAGE cDNA clone 1882505.(3.29 MB EPS).Click here for additional data file.Figure S2Figure shows a TreeView image of blocks of HLS genes selected using increasingly relaxed selection criteria. The top-most group represents HLS genes selected using the standard 0.5 cutoff value described in (1.9 MB EPS).Click here for additional data file.Figure S3(A) Normal human control, PHA-stimulated lymphocytes. Signal at Chromosome 2q13 and 22qtel.(B) Bonobo fibroblast culture. Four signals.(C) Chimpanzee fibroblast culture. Four signals.(D) Gorilla fibroblast culture. More than 30 signals. Hybridization to most subtelomeric regions.FLJ22004 cDNAs.(E) Orangutan fibroblast culture. No apparent red signal. Probe BAC RP11-1007701 in green included as internal hybridization control. Also shown are aCGH TreeView images  for three (6.94 MB EPS).Click here for additional data file.Figure S4Pie graphs showing the distribution of GO molecular function categories within HLS, LS, and whole genome gene lists. The top 22 categories are named in the legend in descending order of representation for all three groups. Categories were ranked by normalizing each category value for HLS and LS lists to the genome-wide list and then ranking the sum of these values for each category. Less well-represented categories were omitted from the graphs in order to enhance legibility, and zero values are not listed.(1.1 MB EPS).Click here for additional data file.Figure S52 ratios for each cDNA clone and the copy number ratio of the test gene to the CFTR (control) gene, as determined by RT-PCR, are shown for the indicated species. Also shown are TreeView images of interhominoid aCGH results for the indicated cDNAs and a graphical depiction of the correlation between aCGH signal and copy number ratio to CFTR (RT-PCR).RT-PCR was used to provide an independent confirmation of interspecies cDNA aCGH data for three genes in which aCGH signals were different in the African great apes compared to human and orangutan. The chromosomal location, IMAGE clone ID, and GenBank accession numbers are provided for each cDNA. The species average logPLA2G4B cDNA clone located on human Chromosome 15 using the UCSC November 2002 human genome assembly. The correlation between RT-PCR and aCGH-based copy number estimates is 0.94.(A) FLJ31659 cDNA clone located on human Chromosome 4 using the UCSC November 2002 human genome assembly. As in (A), the correlation between RT-PCR and aCGH data is 0.97.(B) BC040199 transcript located on human Chromosome 7 using the UCSC November 2002 human genome assembly. As in (A), the correlation between RT-PCR and aCGH data is 0.97.(C) (1.29 MB EPS).Click here for additional data file.Figure S6(A) Normal human control, PHA-stimulated lymphocytes. Two signals localized to Chromosome 15q15.1.(B) Chimpanzee fibroblast culture. Two signals on the chromosome syntenic to human Chromosome 15 (at arrows). Multiple additional signals in the subtelomeric regions.(C) Gorilla fibroblast culture. Two signals on the chromosome syntenic to Chromosome 15 (at arrows). Two additional signals on a large metacentric chromosome, which in interphase appear as amplified signals.(D) Orangutan fibroblast culture. Two signals on the chromosome syntenic to human Chromosome 15.(4.39 MB EPS).Click here for additional data file.Protocol S1(2 KB TXT).Click here for additional data file.Table S12 aCGH values are listed for the human versus human samples (n = 5), human versus bonobo samples (n = 3), human versus chimpanzee (n = 4), human versus gorilla (n = 3), and human versus orangutan (n = 3). This table is tab-delimited and can be opened in Microsoft Excel to view the raw numbers or can be browsed using TreeView (see Protocol S1). Column B provides information regarding IMAGE clone number, chromosome, and nucleotide position (UCSC November 2002 freeze), Unigene ID, EST accession numbers, and known gene information.Values are provided for genes (cDNAs) queried by interhominoid aCGH. For each IMAGE clone queried, log(12.84 MB TXT).Click here for additional data file.Table S2–20 was used and the best hit is reported in the table. Query refers to the HLS gene EST sequences; subject refers to the WSSD sequences. Score, expect value, and percent identity (ID) are reported for the best BLAST hit, while the start and stop positions and length for both query and subject are also reported.For each IMAGE clone of the HLS genes, one or more EST sequences were used as a query for a BLAST search against the WSSD dataset. An expect value cutoff of e(434 KB DOC).Click here for additional data file.Table S3For each of the 23 LS gene clusters, Satellite repeat subclass analysis was performed. The table lists the cluster's cytogenetic region, the chromosome and start and stop positions, and the adjusted length after accounting for gaps in the genomic sequence. The percent content for 24 subclasses of Satellite repeats is listed for each of the 23 gene clusters. Summary information includes the average content of the 24 subclasses of Satellite repeats for all of the clusters as well as the average for the entire human genome. The difference and fold change are calculated based on comparing the cluster averages to the entire human genome averages.(111 KB DOC).Click here for additional data file.Table S4Similar to Table S1, but only IMAGE clones with LS characteristics are listed, and each is ranked based on average fluorescence signal (highest to lowest) within each lineage.(269 KB TXT).Click here for additional data file.Table S5(52 KB DOC).Click here for additional data file.Table S6Presented are pertinent data from GO analysis with DAVID, including numbers of classified and unclassified genes in each gene list, as well as the data returned for each of the 22 most represented molecular function categories. Listed are GO identification numbers (GOIDs) and names for each of the top 22 categories, as well as raw values and relative percent values for HLS, LS, and genome classifications. Relative percent columns are taken as the ratio of the number of classifications in each category to the number of genes classified in the list. The average percent is also provided as the average of these relative percent values across the three groups. This is intended as a metric to help gauge deviations in group relative percent values from the combined average value.(81 KB DOC).Click here for additional data file.Table S7The table has three sections: a summary showing the percentages of blocks in each respective chimpanzee homology scoring class; a table with the HLS versus chimpanzee data; and a table with the random versus chimpanzee data. The HLS versus chimpanzee and random versus chimpanzee tables have columns derived from both parsing the BLAT PSL data and from the chimpanzee homology comparison. The table lists the IMAGE clone and the EST accession number used as a query, the hit number, the score and percent identities, the start and stop positions in the query, the chromosome and chromosome start and stop positions, the number of blocks of alignment for the hit, the numbers of blocks that fall into each chimpanzee homology scoring class, and finally the respective chimpanzee scaffold(s) for each hit, if available.(3.58 MB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/) for FGF7 is NM_002009 and for morpheus is AF132984.The GenBank accession number (


IEC-18 cells are a non-transformed, immortal cell line derived from juvenile rat ileal crypt cells. They may have experimental advantages over tumor-derived gastrointestinal lineages, including preservation of phenotype, normal endocrine responses and retention of differentiation potential. However, their proclivity for spontaneous differentiation / transformation may be stereotypical and could represent a more profound experimental confounder than previously realized. We hypothesized that IEC-18 cells spontaneously diverge towards a uniform mixture of epigenetic fates, with corresponding phenotypes, rather than persist as a single progenitor lineage.IEC-18 cells were cultured for 72 hours in serum free media (SFM), with and without various insulin-like growth factor agonists to differentially boost the basal rate of proliferation. A strategy was employed to identify constitutive genes as markers of divergent fates through gene array analysis by cross-referencing fold-change trends for individual genes against crypt cell abundance in each treatment. We then confirmed the cell-specific phenotype by immunolocalization of proteins corresponding to those genes. The majority of IEC-18 cells in SFM alone had a loss in expression of the adenomatous polyposis coli (APC) gene at the mRNA and protein levels, consistent with adenoma-like transformation. In addition, a small subset of cells expressed the serotonin receptor 2A gene and had neuroendocrine-like morphology.IEC-18 cells commonly undergo a change in cell fate prior to reaching confluence. The most common fate switch that we were able to detect correlates with a down regulation of the APC gene and transformation into an adenoma-like phenotype. In the last quarter century, more than five hundred published manuscripts contained experiments using the popular rat crypt cell lines IEC-6 and IEC-18 originally established by Quaroni and Isselbacher in 1978 and 1981 . These lOne of the more exciting aspects of IEC-18 cells is their capacity to retain the crypt cell phenotype as well as the continued potential for differentiation/maturation into enterocytes -5. HowevThis is a fairly crucial experimental question because serum-restricted conditions allow the tightest experimental rigor but may drive IECs towards altered cell fates, potentially introducing uncontrolled experimental confounders. Until recently, we did not have the necessary tools with which to determine more subtle epigenetic divergences in IECs. Our current investigation uses a novel strategy to demonstrate that spontaneous cell fate divergence is not only highly prevalent in IEC-18 cells, but it is fairly uniform and therefore a predictable confounder for gene array studies in this cell line.We have recently discovered a novel antigen localization pattern by using anti-carboxyl IGF binding protein-2 (IGFBP-2) antibody  to demonstrate a multivesicular pattern in some IEC-18 cells but not in others that are immediately adjacent . Both stress fibers and microvilli are heavily stained and appear to have high turnover rates in cells that have C2 staining, but not in those without it Figure . We noteWhen IEC-18 cells were plated at variable densities in 10% fetal bovine serum (FBS), we saw that all crypt cells were initially positive for C2 staining, but with increasing density there were increasing numbers of C2 negative cells Figure . We alsoTo determine if the C2 positive and negative IEC-18 cells were each capable of phenotype preservation during proliferation, IGFBP-2 stained cells on coverslips were searched for mitotic cells in the stage of cytokinesis and their images were captured by digital photomicroscopy (n = 6 cell pairs for each phenotype). In each case, all daughter cells had the same phenotype as their sister, confirming that both the C2 positive and C2 negative phenotypes are conserved in subsequent cycles of mitosis  while none of the treatments significantly altered the abundance of C2 negative cells. This strategy allowed us to systematically skew the cell composition and use gene array analysis to determine whether C2 positive cells were epigenetically divergent.Because IEC-18 cells grow best in the presence of high dose insulin, we suspected that crypt cell proliferation was dependent upon IGF receptor stimulation. In general, differentiated and benignly transformed epithelial cells are less likely to proliferate upon reaching confluence, so we sought to preferentially drive crypt cell proliferation in a graded fashion using different IGF receptor agonists Figure . IGF-II Eleven genes met our criteria for a significant fold change, four were positively correlated with crypt cell abundance and seven were inversely correlated Figure . Of thesWestern blots using antibodies against APC and 5-HT2A in IEC-18 cell lysates revealed staining of the appropriate sized band for each Figure . In the Immunolocalization for APC revealed that C2 positive cells also had intense APC staining whereas C2 negative cells either had limited or no APC staining Figure . This fiin vivo [Immunolocalization for 5-HT2A demonstrated a second cell phenotype Figure  and 5D. in vivo . The celDouble immunolabeling with overlay technique (using C2 and actin antibodies) demonstrated that C2 negative cells have a paucity of microvilli in comparison to C2 positive cells Figure . AdditioIn this manuscript we have taken advantage of an IEC immunolocalization marker that we call C2 to demonstrate two forms of cell fate divergence within IEC-18 culture. Using a combination of gene array screening and immunolocalization, we found that C2 is lost in over half the cells by the time of confluence and its loss is also associated with a down regulation of the APC gene, decreased APC protein abundance, decreased actin filament turn over, and reduced microvillar density. In short, there is an adenoma-like phenotype that fits with this genotype and this fits well with what is known about the function of the APC protein -15. In aWhile we think our findings have important implications for the existing IEC literature, the more important aspect of this manuscript may be in the methodology we have piloted. In many ways, cell culture, whether primary or immortalized, transformed or not, is by definition a model in flux. The progenitor lineage that a researcher starts with is rarely the hodge podge they end up with after a limited number of passages and it is common, if not expected that epigenetic drift will occur with each cell culture passage. However, what we describe in this manuscript is different in that IEC-18 cells are displaying a uniform trend in cell fate divergence – a trend that can be modulated with IGF. Many if not most gut epithelial cell lines are IGF (or high dose insulin) dependent for proliferation and thus are potentially vulnerable to this biological confounder. It will remain to be seen if other epithelial cell types have similar behaviors when examined in this fashion. Conversely, there is also a positive light to our findings; IEC-18 cells could be a compelling model for spontaneous adenomatous transformation because these adenoma-like cells are arising from a genetically competent progenitor prior to reaching confluence. To our knowledge, no such model with this property has been previously defined.Our study has notable weaknesses and strengths. First, we have identified two divergent cell fates but have only partially characterized them because we were focused on developing a viable screening methodology (hence the ubiquitous use of the word "like" in this manuscript). Second, we are using a rat gene array chip that has approximately 9000 non-EST genes per chip. This is not an exhaustive survey of the rat genome and it is possible that there are other cell fates present in IEC-18 culture that we did not detect. Third, our phenotype assays are based on immunolocalization, which is a semi-quantitative technique with regards to assessing protein abundance. However, in this case we are actually combining cell-to-cell differences in protein abundance with distinguishing morphologic characteristics (e.g. loss of microvilli, flattening, bipolar shape, dendritic arbors, etc.) to delineate the phenotypes between adjacent cells. In short, what we are quantifying, in the case of adenoma-like cells, is the percentage of cells with a given phenotype. For this purpose, blinded immunolocalization is simple, quantitative and exceedingly efficient. The combined methodologies we chose result in a highly accurate technique for assessing divergence and their specificity can be bolstered by comparative studies of co-divergent markers (as we demonstrated with C2 and APC). As for other positives, the methodology is relatively rapid and can detect low prevalence phenotypes (as demonstrated by the anti-5-HT2A antibody). Additionally, we have demonstrated that a transcriptional marker is not required to create an effective screen. What is required, and what should probably prompt a researcher to employ this methodology, is a probe, a phenotype, or a pleiotropism that results in a consistently heterogeneous and quantifiable pattern (as C2 proved to be for us).In closing, we point out one last caveat. Gene array investigation is an evolving science but there remain three potential pitfalls for every new application: experimental design flaws, data integrity issues and biological misassumptions -20. In tWe have demonstrated a novel methodology for detecting and characterizing cell fate divergence in cell cultures derived from a common progenitor. The majority of IEC-18 cells are transformed into adenoma-like cells in SFM. IGF agonists reduce the rate of transformation by driving proliferation of the progenitor phenotype but do not prevent it. We also detected a very low incidence of differentiation toward a neuroendocrine-like cell type in these same cultures.-6M NBI 31772 , as a means of boosting crypt cell proliferation. We point out that crypt cells can be driven to proliferate despite confluence whereas more differentiated epithelial cell types are resistant to IGF-driven proliferation upon reaching confluence. Alternatively, IEC-18 cells were diluted to 1/8, 1/4 and 1/2 the original seeding density and plated in DMEM with 10% FBS for 24 hours as a means to evaluate cell fate divergence in the presence of serum.Rat ileal epithelial cells (IEC-18 – American Type Culture Collection, Rockville, MD) from aliquots of passage numbers 6–8 were grown to confluence in DMEM with 10% fetal bovine serum (FBS) and .1% insulin. The time period for complete epithelial cell confluence was 24–48 hours. Confluent cells were incubated for 72 hours in DMEM to establish a serum free period either alone or with 10iego, CA ), 0.5 × Cell lysate samples were collected from 150 mm dishes, washed with PBS and recovered in 700 uL of lysis buffer by scraping the dish. Cells were spun down for 10 minutes in a pre-chilled centrifuge. The supernatant was diluted 4:1 with loading buffer and run on 12 or 15% acrylamide gels. After the gel had been transferred onto a nitrocellulose membrane, 0.5% Ponceau S stain was applied to confirm equitable protein transfer across all lanes.Western blot membranes were incubated in 5% milk block for 1 hour at RT. Goat polyclonal primary antibody for APC and 5-HT2A were applied for 1 hour at RT. Primary antibody was washed off with TBS-Tween and secondary antibody was placed on the membranes for one hour, then washed again. ABC (Elite Series, Vector Labs) was placed on the membrane for an hour and then washed and visualized with SuperSignal chemiluminescent substrate (Pierce, Rockford, IL), each was prepared per the manufacturer's instructions.ell Culture methods and then utilized to screen for specific cell fate markers. Total RNA was harvested for each treatment condition in triplicate experiments and then used for gene chip analysis per the manufacturer's protocol (rat gene chip # 230A, Affymetrix, Santa Clara, CA). All available non-EST tags were searched within the R3-IGF-I data set and those with significantly different fold changes when compared to SFM cells were selected as potential candidates for further analysis . To further refine the list of potential candidates, the fold changes for NBI 31772 and IGF-II analog, which had step-wise reductions in effects upon crypt cell proliferation, were used to determine if there was a positive or inverse trend between a selected gene's mRNA abundance and crypt cell abundance. In this way, we sought to select constitutively expressed genes, whose primary differences in abundance would be due to differences in cell composition. Our statistical test for determining significance was a two-tailed paired t-test .Immunolocalization in IEC-18 cells was performed for IGFBP-2 (C2), f-actin, IGF receptor type 2 and villin as well as two proteins that were screened out by our gene array analysis (APC and 5-HT2A) in a minimum of three separate experiments for each. In brief, the cells were fixed in formalin overnight, washed in DIG buffer (4% 1M Tris Base, 6% 5M NaCl, 16% 1M Tris HCl) 5 times then put in blocking solution (1% wt/vol BSA in DIG buffer) for 1 hour at room temperature. This was followed by a one-hour incubation with goat polyclonal primary antibody  in antibody diluent solution (5% 1M Tris HCl, 1% 1 M Tris Base, 9% NaCl, 3.3% Triton-X 100). Primary antibody was washed off with DIG × 5 and secondary antibody (donkey anti-goat [Jackson Immunoresearch Labs, West Grove, PA]) was placed on the slides for one hour. ABC (Elite Series, Vector Labs) was prepared and applied per the manufacturer's instructions, washed as above and visualized with DAB solution (Sigma, St. Louis, MO) in parallel. The slides were counterstained with hematoxylin, cover-slipped, photomicrographed and representative images were chosen for publication. With respect to the actin visualization, detection was performed such that colored precipitate was observed under the microscope and stopped when stress fibers and microvillus cores were evident and before the high background of the total cell f-actin began to stain efficiently. In the crypt cell quantification experiments, six 10X images were taken at random, printed on color paper at maximum size, blinded, and all cells were assessed as C2 positive or C2 negative and their means and standard deviations calculated for each treatment condition.To objectively test our observations that C2 positive cells have a distinctive colocalization pattern when compared to C2 negative cells, C2 double labeling experiments with f-actin and with APC were performed as overlay experiments using digital microscopy. In brief, C2 immunolocalization was performed as described above except that a grid was drawn on the wet mount slide, allowing digital photomicrographs to be taken at 40X of C2 staining while still covered with stop solution and then a second antibody, either for f-actin or APC was applied and the same process repeated before being counter-stained with hematoxylin and cover-slipped. The same exact images were then re-mapped using the grid and the stored digital images were used for precise position verification. The overlaid double-labeled image was then taken and contrasted to the original image to allow comparison of differential localization of the two antigens.IGF, insulin-like growth factor; IGFBP-2, IGF binding protein-2; IGF-II analog, synthetic truncated IGF-II; R3-IGF-I, synthetic long arginine IGF-I; NBI 31772, an alpha-numeric designation for a non-biologic compound that best displaced IGFs from IGF binding proteins in a large bioassay screen; SFM, serum-free media; APC, adenomatous polyposis coli; 5-HT2A, serotonin receptor 2A; C2, IGFBP-2 carboxyl fragmentPG designed the study, participated in the immunolocalization studies, and performed data analyses. JP carried out the immunolocalization, cell count, and Western blot studies. NF maintained the IEC cells lines and expertly provided uniformly confluent cells. PG and JP produced the figures and drafted the manuscript. All authors read and approved the final manuscript.


Human T-cell leukemia virus type I (HTLV-I) Tax protein is a transcriptional regulator of viral and cellular genes. In this study we have examined in detail the determinants for Tax-mediated transcriptional activation.Whereas previously the LTR enhancer elements were thought to be the sole Tax-targets, herein, we find that the core HTLV-I TATAA motif also provides specific responsiveness not seen with either the SV40 or the E1b TATAA boxes. When enhancer elements which can mediate Tax-responsiveness were compared, the authentic HTLV-I 21-bp repeats were found to be the most effective. Related bZIP factors such as CREB, ATF4, c-Jun and LZIP are often thought to recognize the 21-bp repeats equivalently. However, amongst bZIP factors, we found that CREB, by far, is preferred by Tax for activation. When LTR transcription was reconstituted by substituting either κB or serum response elements in place of the 21-bp repeats, Tax activated these surrogate motifs using surfaces which are different from that utilized for CREB interaction. Finally, we employed artificial recruitment of TATA-binding protein to the HTLV-I promoter in "bypass" experiments to show for the first time that Tax has transcriptional activity subsequent to the assembly of an initiation complex at the promoter.Optimal activation of the HTLV-I LTR by Tax specifically requires the core HTLV-I TATAA promoter, CREB and the 21-bp repeats. In addition, we also provide the first evidence for transcriptional activity of Tax after the recruitment of TATA-binding protein to the promoter. In eukaryotes, transcription by RNA polymerase II requires the orderly recruitment of basal transcription factors and activators to the core promoter and enhancers, respectively ,2. The cHuman T-cell leukemia virus type I (HTLV-I) Tax protein is a unique transcriptional regulator . Tax canTo date, the molecular mechanisms for Tax trans-activation have been well studied. Due to its pleiotropic activities, there are likely nuances to Tax's activity which remain unrevealed. Currently, we understand Tax to harbor a minimal activation domain , to inteIn mammalian cells, the artificial recruitment of TBP sufficiently activates transcription from some promoters -24. It iHere, we have constructed a series of chimeric enhancer-TATAA reporters to analyze the functional roles of these transcription elements in Tax-mediated activation. We observed that Tax activates the HTLV-I 21-bp repeats more potently than other enhancer elements. Analysis of ten mutants of Tax revealed that Tax utilizes different domains to target different cellular factors. We also found that multiple bZIP transcription factors including the newly-identified LZIP are involved in Tax activation of HTLV-I LTR. Finally, two other salient findings are that optimal Tax-responsiveness is specified by the HTLV-I-specific TATAA element, and that Tax synergizes with artificially recruited, DNA-tethered, TBP in a phase of transcription after the assembly of an initiation complex at the promoter.Tax can activate transcription through 21-bp repeats, CRE, κB site or SRE -9. HowevEach reporter contains two copies of enhancer motifs (21-bp repeats, CRE, AP1, Sp1, κB or SRE) and a minimal HTLV-I TATAA promoter Fig. . BecauseWhen the reporters were tested in the presence of Tax, a different pattern emerged. Transcription from the 21-bp repeats was stimulated approximately 70-fold  of Tax function with respect to TBP recruitment, we constructed a series of reporter plasmids Fig.  with twoHere, we have delineated functional requirements for both the TATAA promoter and the 21-bp enhancer elements in HTLV-I Tax mediated activation of the viral LTR. To date Tax has been considered solely to initiate transcription. Our study shows for the first time that Tax has a transcriptional role after assembly of an initiation complex at the promoter.HTLV-I is etiologically associated with adult T-cell leukemia ,39. ExprII28 [Beyond the requirement for the 21-bp enhancer, our experiments revealed that the HTLV-I TATAA is also specifically preferred by Tax Fig. . This fiII28  might prA provocative notion which emerges from our study is that Tax can further activate a promoter at which TBP has already been artificially tethered Fig. . ExperimAll the transcriptional assays in the present study were based on transiently transfected reporters. We noted that transiently transfected and stably integrated promoters might behave differently ,56. ObviChloramphenicol acetyltransferase (CAT) reporter plasmid pG5CAT was from Clontech. CAT plasmid pU3RCAT containing the HTLV-I LTR has been previously described . Other CSequences of canonical CRE, Sp1, AP1 and κB motifs in the reporter plasmids have been described ,58,59. HExpression plasmids for wild type and mutant Tax have been described elsewhere ,61. pIEXExpression plasmid pRSV-KCREB for the dominant-negative CREB protein KCREB  was kind5 cells/well into six-well culture plates and transfected using calcium phosphate method as described [14C-labeled chloramphenicol (Amersham) and acetyl coenzyme A  for CAT reaction. CAT activities were detected using thin-layer chromatography and quantified by phosphorimager (Molecular Dynamics). For transfection of cells, each well received the same dose of plasmids. The empty vector or pUC19 was added to compensate for the different amounts of plasmids when necessary.HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with fetal calf serum and antibiotics, seeded at 5 × 10escribed . Jurkat escribed . BrieflyNone declared.


To determine the prevalence, types and demographic features of child labour among school children in Nigeria.A cross-sectional interview study of 1675 randomly selected public primary and secondary school pupils aged 5 to less than 18 years was conducted in the Sagamu Local Government Area of Ogun State, Nigeria from October 1998 to September 1999.The overall prevalence of child labour was 64.5%: 68.6% among primary and 50.3% among secondary school pupils. Major economic activities included street trading (43.6%), selling in kiosks and shops (25.4%) and farming (23.6%). No child was involved in bonded labour or prostitution. Girls were more often involved in labour activities than boys (66.8% versus 62.1%, p = 0.048): this difference was most obvious with street trading (p = 0.0004). Most of the children (82.2%) involved in labour activities did so on the instruction of one or both parents in order to contribute to family income. Children of parents with low socio-economic status or of poorly educated parents were significantly involved in labour activities (p = 0.01 and p = 0.001 respectively). Child labour was also significantly associated with increasing number of children in the family size (p = 0.002). A higher prevalence rate of child labour was observed among children living with parents and relations than among those living with unrelated guardians.It is concluded that smaller family size, parental education and family economic enhancement would reduce the pressure on parents to engage their children in labour activities. Child labour covers all economic activities carried out by children regardless of their occupational status . It has Most of child labour takes place in Asia, the Pacifics and Africa -9. In moThis presentation is part of a broader study on child labour among school children in public primary and junior secondary schools in Sagamu Local Government Area of Ogun State, Nigeria. The study was conducted between October 1998 and September 1999 (inclusive). It was approved by the Ethical Committee of the Olabisi Onabanjo University Teaching Hospital Sagamu (OOUTH).Sagamu Local Government Area (SLGA) is one of the 15 Local Government Areas in the State. It is predominantly rural and semi-urban with a land space of 68.4 sq km. The estimated population for the year 1997 is 177,514. Administratively, SLGA is made up of 11 wards. The total school enrolment in the 50 primary and 16 junior secondary schools (JSS) for the year 1997 was 30,597, comprising 21,476 primary and 9,121 JSS pupils. Excluding 6,762 new entrants who did not have academic records for the preceding year left 23,835 pupils (17,891 primary and 5,944 junior secondary school pupils). Thus the ratio of primary to junior secondary school pupil population was about 3 to 1. This ratio was considered while selecting the subjects for the study.The survey sample was drawn randomly from all 50 public primary and 16 secondary schools in the LGA. Using ballot papers, one primary and one secondary school were randomly selected from each administrative ward. However, one ward had no primary school and four wards had no secondary school. Thus, a total of ten primary and seven secondary schools were selected. There were five classes in each primary school and two in each junior secondary after excluding primary 1 and JSS1 pupils because they were new entrants in their respective schools and therefore did not have academic records for the preceding year. Each class was made up of at least two arms.One arm of each eligible class in the selected schools was randomly selected, also using ballot papers. Altogether, 64 classes were selected: 50 classes from 10 primary schools and 14 classes from 7 secondary schools. Thirty pupils were randomly selected from each class to form the study school. The pupils were interviewed one at a time between the hours of 8.00 am and 2.00 pm on Monday through Friday using a structured, close-ended questionnaire. The interviews were conducted by author MBF with the help of previously trained research assistants. Relevant background information on age, sex, family background, involvement or otherwise in after-school economic activities were obtained. Information on parental education, occupation/income was obtained through requests accompanying letters to parents and guardians asking for their consent. The occupation/income and educational attainments of the parents were used to determine socio-economic index scores of the children using modified criteria described by Oyedeji .Each parent was given two index scores: one for occupation/income and the other for educational attainment. The scores were rated on a scale of 1 to 5, from the more educated and more highly placed occupation/income groups to the least advantaged. The mean of the four scores for both parents was calculated and the value became the assigned socioeconomic group of the family/child. Where the calculated mean was not a whole number, the next higher integer was used.Child labour was defined as any type of economic task, paid, unpaid, or exploitative, engaged in by a child less than 18 years of age, which places the interests of the beneficiary well above those of the child and is detrimental to the physical mental, social, educational and moral development of the child [For the purpose of analysis the study children were grouped into two; those involved in child labour and those who were not. Chi-square analysis was used to compare proportions and probability; (p) values less than 5% (0.05) were accepted as statistically significant.A total of 1675 day pupils were recruited into the study. The male/female ratio was 1:1.02. The majority (86.4%) of respondents were of Yoruba extract. Their ages ranged between 5 and less than 18 years. Those in primary schools were 1299 while 376 were in junior secondary schools as shown in Table As shown in Figure 2 = 5.22, p <0.025). The prevalence of child labour did not vary significantly among children living with different types of relatives (p > 0.05). A significant rising trend of involvement in child labour with increasing number of children in the family (χ2 for trend = 9.13, p = 0.0025) was observed.Over 80% of the study children lived with one or both parents, 15.2% with other relatives and very few (0.3%) with unrelated guardians. Children living with parents (both or single) and relatives had higher prevalence rates of child labour. The number of children living with unrelated guardians was relatively small but this group had less than 50% the prevalence of child labour of their counterparts living with relatives  declare that they have no competing interests.MBF conceived of the study, participated in its design, collection, statistical analysis and interpretation of data. FON contributed to the statistical analysis, interpretation of the data and has been involved in drafting and revising the manuscript.AOO participated in coordination of the study and revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: