Andrew Fortna; Young Kim; Erik MacLaren; Kriste Marshall; Gretchen Hahn; Lynne Meltesen; Matthew Brenton; Raquel Hink; Sonya Burgers; Tina Hernandez-Boussard; Anis Karimpour-Fard; Deborah Glueck; Loris McGavran; Rebecca Berry; Jonathan Pollack; James M Sikela

“ It 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 ( [18] ) . Taken together these data suggest this region is one of high genomic instability that is relevant to both disease and evolutionary processes. ”

“ 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. ”

“ 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. ”

“ 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 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.
It has been pointed out that the human genome is a mosaic composed of some regions more closely related to chimpanzee and, less frequently, others more closely related to gorilla ( [41] ) . ”

“ Human genomic DNA represents at least a 20-fold increase in complexity compared to human cellular mRNA, and the cDNA array elements represent a smaller (e.g., less than 2 kb) , generally more discontinuous hybridization target for a genomic DNA sample. These technical issues notwithstanding, highly reproducible aCGH signals can be obtained using human genomic DNA against high-density human cDNA microarrays, and gene changes as small as an increase or decrease of a single copy can be detected ( [9] ) . ”

“ Interestingly, gorillas more than any other hominoid are folivorous. They eat leaves primarily, but also (like other hominoids) fruit, foods that contain energy-rich cellulose and pectin. This fact, together with the observation that FLJ22004 is highly amplified only in the gorilla lineage, raises the possibility that amplification of this gene provides enhanced cellulase and pectinase capabilities, which in turn would facilitate utilization of the two key dietary staples of this species. ”

“ 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. ”

“ Results/Discussion Identification of Lineage-Specific Gene Duplication and Contraction
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 (Table S1) . The pairwise comparisons involved using a great ape (or human control) as the test genomic DNA sample (Cy5 red dye) and a sex-matched human as the reference genomic DNA sample (Cy3 green dye) in all comparisons. ”

“ The changes identified likely represent most of the major LS gene-associated copy number changes that have occurred over the past 15 MY of human and great ape evolution. Further analyses of this dataset, of which only a fraction has been highlighted here, should provide additional insights into gene duplication and genome evolution, the relationship of genome instability, evolutionary adaptation, and disease, and the genes that underlie the phenotypic differences among human and great ape species. ”

“ While these studies provided useful insights into hominoid DNA copy variations, they afforded little direct knowledge of changes in individual gene copy number and covered only limited sections of the genome. In contrast, interhominoid aCGH using human cDNA microarrays, representing more than 29,000 different genes, would allow a level of genomic resolution not previously obtainable and also provide direct data regarding the recent evolutionary history of a significant majority of human and great ape genes. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ Repeat annotation was downloaded from UCSC (http://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. ”

“ FISH Analysis
Using standard procedures, metaphase spreads and interphase nuclei were prepared from human lymphocytes (Homo sapiens [HSA] ) 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) . ”

“ After collapsing the LS dataset by UniGene cluster to remove redundant cDNAs corresponding to the same gene, 815 different genes were identified that gave aCGH signatures unique to a specific hominoid lineage. Each respective lineage and the numbers of genes identified that showed LS copy number change (increases/decreases) are as follows: human: 134/6; bonobo: 23/17; chimpanzee: 11/4; bonobo/chimpanzee pre-split: 26/11; gorilla: 121/52 ”

“ 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. ”

“ GenBank accession numbers were used as unique identifiers, and gene lists were annotated and functionally characterized using DAVID (Database for Annotation Visualization and Integrated Discovery) (http://apps1.niaid.nih.gov/david/upload.asp) ( [43] ) . Analyses were performed at level one for DAVID and at a threshold cutoff of 1, which provides high coverage but relatively low specificity and considers all classifications. Analysis was carried out on both lists, first using those genes with GenBank accession numbers, and then only those genes with known gene symbols. ”

“ The chromosomal location, IMAGE clone ID, and GenBank accession numbers are provided for each cDNA. The species average log2 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) . (A) PLA2G4B cDNA clone located on human Chromosome 15 using the UCSC November 2002 human genome assembly. ”

“ G (–) ; CG (–) O (+) ; C (–) GO (+) ; and CO (+) G (–) .FISH Confirmation of a Human-Specific Duplication of a Gene Cluster on Chromosome 5q13.3 Detected by Interspecies cDNA aCGH (A) Human duplication of a cluster of genes at Chromosome 5q13.3. is shown by two separate, and sometimes multiple, red BAC probe (CTD-2288G5) signals in interphase cells, with only one green BAC probe signal (RP11-1077O1) for a flanking region. Metaphase FISH shows both probes at band 5q13. ”

“ Orangutan fibroblast culture. Two signals on the chromosome syntenic to human Chromosome 15. (4.39 MB EPS) .
File:Pbio.0020207.sg006.epsClick here for additional data file. Protocol S1 (2 KB TXT) .
File:Pbio.0020207.sd001.txtClick here for additional data file. Table S1 Values are provided for genes (cDNAs) queried by interhominoid aCGH. For each IMAGE clone queried, log2 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) . ”

“ Table 1 "Comparison of HLS Gene and WSSD Datasets"File:Pbio.0020207.t001The complete HLS clone-by-clone comparison to the WSSD dataset can be found in Table S1 Table 2 "Genome Distribution and Repeat Content of Clusters of LS Genes"File:Pbio.0020207.t002Inspection of the whole genome aCGH dataset identified clusters of genes that showed LS signatures. ”