Animal Breeding and Genetics Group, Wageningen Institute of Animal Sciences, Wageningen, The Netherlands;
Department of Genetics, Case Western Reserve University School of Medicine
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Abstract |
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Introduction |
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Comparison of gene and cytogenetic/molecular cytogenetic maps between different extant species can be used to infer the chromosomal constitution of the last common ancestor between these species. For example, chromosomal reconstruction of the ancestral karyotype of primates suggests that 1820 human chromosomes have remained unchanged during evolution and that the rest have had but a single exchange each (O'Brien and Stanyon 1999
). Nevertheless, different exchanges have occurred in the lineages leading to distinct primate families and genera. In contrast, based on the cumulative mapping of 223 genes in the chicken genome, the predicted number of conserved autosomal fragments between chickens and humans is 96, and for the chicken-mouse comparison this number is 152 (Burt et al. 1999
).
Comparative maps between mammals suggest that the number of chromosomal rearrangements in these species can vary from 1 to 10 per million years (O'Brien et al. 1999
). The observed number of conserved segments in chicken, in comparison with that in human or mouse, therefore indicates an unexpected low number of rearrangements between the two lineages that led to the development of the avian and mammalian species. These results, however, are based on relatively low density chicken-human and chicken-mouse comparative maps. Therefore, gene mapping to a higher resolution would be expected to identify additional chromosome rearrangements over evolutionary time. Although a small improvement in the chicken-human comparative map has been realized through the mapping of genes in chicken by both cytogenetics (Suzuki et al. 1999
) and linkage analysis within reference pedigrees (Groenen et al. 2000
), there has been relatively little further progress.
To obtain a high-density comparative map, regional mapping of sufficient numbers of coding sequences in the species of interest is necessary. The recent construction of arrayed genomic libraries of large insert clones such as bacterial artificial chromosomes (BACs) for many species, including chicken (Crooijmans et al. 2000
), are powerful tools with which to perform comparative and physical mapping. Large insert clones are used as cytogenetic probes and for direct sequencing and therefore are also useful in identifying orthologous genes to known genes in other species. So far, only eight genes that are located on human chromosome 15 have been mapped in the chicken. Seven of these genes map to chicken chromosome 10, whereas the RYR3 gene maps to chromosome 5 (Jones et al. 1997
; Smith and Cheng 1998
; Groenen et al. 1999
; Morton et al. 1999
; Crooijmans et al. 2000
). In this study, we describe the generation of the first detailed comparative map between human chromosome Hsa15, its mouse counterparts on chromosomes Mmu7, Mmu2, and Mmu9, and the homologous regions on the three chicken chromosomes Gga10, Gga5, and Gga1 through the identification and mapping of almost 100 genes in the chicken. These results indicate the occurrence of multiple inter- and intrachromosomal rearrangements during the evolution of these chromosomes between the two species.
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Materials and Methods |
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Additional BACs were isolated using STS markers developed from chicken sequences present in GenBank. The sequences used were selected based on the preliminary chicken-human comparative map (Morton et al. 1999
; Groenen et al. 2000
). Toward this end, human sequences from genes and ESTs known to be located on Hsa15 (www.ncbi.nlm.gov; Homo sapiens genome view build 22 [June 18, 2001]) were used to identify sequences from homologous chicken genes present in GenBank.
BAC-End Sequencing
BAC DNA was isolated with the REAL Prep 96 plasmid kit (Qiagen) or as described by Crooijmans et al. (2000)
and dissolved in 32 µl 5 mM Tris-HCl (pH 8.0). PCR Sequencing was performed in 40 µl, consisting of 16 µl of BAC DNA, 8 µl Half Big Dye terminator (Genpak Ltd.), 8 µl Big Dye Terminator Rrmix (Perkin-Elmer), and 8 µl of M13 forward or M13 reverse sequence primer (10 pmol/µl). The amplification reactions were as follows: 5 min at 96°C, followed by 45 cycles of 30 s at 96°C, 20 s at 50°C, and 4 min at 60°C. The amplification product was precipitated with isopropanol and finally dissolved in 3µl 83% deionized formamide and 17% loading buffer (Perkin-Elmer). The sequences were analyzed on a 4.75% Long Ranger Gel (FMC) on an automated ABI377 sequencer (Perkin-Elmer). Electrophoresis was performed for 7 h on 36-cm gels, and the results were analyzed using sequence software (ABI, Perkin-Elmer).
Sample Sequencing of BACs
EcoRI-digested BAC DNA was ligated into the EcoRI site of pTZ18R and transformed to DH5. Twelve subclones per BAC clone were selected, and plasmid DNA was isolated (Qiaprep 96 miniprep kit; Qiagen). The PCR sequence reaction was performed in 10 µl, with 200500 ng plasmid DNA, 2 µl Half Big Dye terminator (Genpak Ltd.), 2 µl Big Dye Terminator Rrmix (Perkin-Elmer), and 1 µl of M13 forward or M13 reverse sequence primer (0.8 pmol/µl). PCR was performed according to ABI (Perkin-Elmer), and the excess dye terminator was removed by precipitation with isopropanol. Sequence reactions were analyzed on a 96-well 36-cm 4.75% denaturing Long Ranger Gel (FMC) according to ABI (Perkin-Elmer). All sequences obtained were first analyzed with PREGAP4 of the STADEN software package, and vector, Escherichia coli, and low-quality sequences were removed. The resulting sequences were compared with sequences deposited in the public databases using the network BLAST client software of the NCBI (blastcl3). We inferred homology from DNA-level BLAST expectation values (E) less than 1.0 x 10-7. In the case of observed sequence identity to gene families, the human conserved map position enabled the identification of the orthologous gene.
Fluorescent In Situ Hybridization
Two-color fluorescent in situ hybridization (FISH) was performed according to Trask et al. (1991)
. EcoRI-digested BAC DNA was labeled by random priming either with biotin-16-dUTP or with digoxigenin-11-dUTP (Boehringer Mannheim) (Ruyter-Spira et al. 1998
). BAC clones were isolated from markers known to be located on specific chromosomes (Groenen et al. 2000
). The BAC clones used as FISH markers to identify the specific chromosome were BAC bw016D10 from marker ADL0038 and BAC bw008K20 from marker MCW0228 for GGA10, BAC bw009B13 from marker ADL0298 and BAC bw037H20 from marker MCW0263 for GGA5, and BAC bw038E08 from marker MCW0107 and BAC bw030P07 from marker MCW0248 for GGA1.
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Results and Discussion |
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Seventy different BAC clones derived from Gga10 were selected and used for sample sequencing. All sequences obtained by BAC-end sequencing and sample sequencing were compared with sequences in the nucleotide database (BLAST). In most cases, the observed homology (E values >10-7) to human genes, in combination with the human map location, enabled the unambiguous identification of the orthologous gene. Often, several different exons of the same gene were identified. For example, six subclones from BAC bw046J07 (MCW0194) identified seven different exons of the gene MLSN1. Six different exons of the gene IQGAP1 were identified with six subclones from BAC bw098O14 (ADL0112). More difficulties occur when homology is detected with several genes belonging to a gene family. This occurred after shotgun sequencing of a BAC clone derived from marker ABR0012 mapped to Gga10. Gene identity was found with transducin-like enhancer protein family; TLE1 (83% identity), TLE2 (77%), TLE3 (88%), and TLE4 (83%). TLE1 and TLE4 are located on Hsa9, TLE2 is located on Hsa19, and TLE3 is located on Hsa15 (15q22; 67.767.7 Mb). According to the gene identity and human chromosome location of the TLE gene family, we assume TLE3 is located on Gga10.
Blocks with similar gene orders in chickens and humans were identified in several contigs, such as the contig of the mapped marker ADL0038. The gene/EST order, identified with the sequences of chicken BAC clones from this block, was FLJ20086 (53.953.9), TCF12 (54.254.6), and ALDH1A2 (55.255.3), with the sequence notation in megabases in parentheses. A similar gene order in chickens and humans was also identified with sequence homology within one BAC clone. For example, with BAC clone bw094I14, within the chicken contig of MCW0035, the two genes BTBD1 and HOMER-2B were identified, which are both mapped in humans to Hsa15 (81.681.7 and 81.481.4 Mb).
The sequence results, together with the genes mapped by FISH, revealed sequence identity to almost 120 human, mouse, rat, and chicken genes and ESTs. The homologs of several chicken genes and ESTs, such as the epsilon adaptin gene (ADTE), have not been mapped in humans yet. The chicken ADTE gene, found after sample sequencing of a BAC clone of marker MCW0357 (CYP19), belongs to the adaptin family. Of this family, beta 1, delta, and gamma are mapped to human chromosomes 22, 19, and 16, respectively. Since human CYP19 maps to Hsa15 (fig. 1 ), the human ADTE gene is predicted to be on Hsa15. Besides homology found with genes predominantly mapped to Hsa15, sequence homology was occasionally observed with human genes that mapped to other human chromosomes (fig. 1 ). For example, the HK1 gene maps to Hsa10, the GNRHR gene maps to Hsa4, and the ENC1 gene maps to Hsa5. These observed homologies either may indicate the presence of gene families of which one member has not yet been mapped to Hsa15 or might indicate the presence of small regions of homology to other human chromosomes. However, in the latter case, one would expect that more genes from those regions would have been identified in the current study. The former hypothesis is further strengthened by the fact that for the chicken genes that have related human genes located on different human chromosomes, on the same chicken BAC clone a gene has been identified whose ortholog does map to Hsa15 (i.e., the HK1 and RAB11A genes on BAC bw017J21, and the GNRHR and PUNC genes on BAC bw091J11, respectively).
The human chromosome 15-chicken comparative map consists of 91 genes and ESTs and is shown in figure 1
. The genes that are located in humans on chromosome 15 are located in chickens in seven regions of conserved synteny on three different chromosomes, Gga1, Gga5, and Gga10. The majority of these genes, however, are mapped in chicken to chromosome 10. In mice, four conserved chromosome segments are also observed, in the order Mmu7, Mmu2, Mmu9, and Mmu7. In an attempt to reconstruct a common ancestor of humans, mice, and chickens, three time nodules in evolution have been described (Burt et al. 1999
). The first time nodule is 300 MYA, when birds diverged from mammals; the second is 100 MYA, when the mouse diverged; and the third is 65 MYA, when the common ancestor of the primates lived. The reconstructed ancestor of all primates has a chromosome 6 consisting of a rather unchanged human chromosome 15 and chromosome 14 (O'Brien and Stanyon 1999
). This reconstructed chromosome is based on the comparison of chromosome paints of primates in which the gene order of the conserved segments was not considered. The comparison of Hsa15 with chicken and of Hsa15 with mouse chromosomes indicates the occurrence of six and three interchromosomal rearrangements (interCRs) during evolution between each pair of species (fig. 2 ). Only one of these rearrangements appears to be at the same location in the chicken and the mouse; if so, this would indicate that this translocation occurred in the lineage leading to humans after the mouse and human lineages diverged, between 100 and 65 MYA. The other five interchromosomal rearrangements involving segments on Gga1-Gga10 (twice) and Gga5-Gga10 (three times) probably occurred before the human and mouse lineages diverged, either in a predecessor of mammals or in the chicken lineage. In the mouse, the other two interCRs between the segments on Mmu2-Mmu9 and Mmu9-Mmu7 probably occurred within the mouse lineage during evolution starting 100 MYA.
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Based on the rates of chromosomal change observed in mammals (Andersson et al. 1996
; O'Brien, Weinberg, and Lyons 1997
), one can calculate that the number of conserved segments between chickens and humans would be on the order of 100600. Based on the comparative mapping data of 223 genes, Burt et al. (1999)
suggested that the number of conserved segments between chickens and humans would be in the lower part of this range. However, our data on the detailed comparison between human chromosome 15 and that of the chicken indicates that this was probably an underestimate and that the number of conserved segments is at least 600 (19 conserved segments between Gga10 and Hsa15, with a size of Gga10 of approximately 3%4% of the chicken genome).
In this paper, we clearly demonstrate the importance of high gene densities in comparative mapping in identification of both inter- and intrachromosomal rearrangements. The development of complete physical maps either as BAC contigs or as the complete sequence will further aid in the detailed reconstruction of rearrangements during evolution which resulted in the chromosomes in the different species as we know them today. A detailed comparative map as described in this paper will be of much value in the identification and further characterization of candidate genes in quantitative trait loci studies in the chicken as well as in the analysis of complex traits in humans and other vertebrate species.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Present address: Center for Neurobiology and Behavior, Department of Psychiatry, University of Pennsylvania.
2 Keywords: genes
chicken
human
rearrangements
FISH
BACs
3 Address for correspondence and reprints: Richard P. M. A. Crooijmans, Wageningen Institute of Animal Sciences, Animal Breeding and Genetics Group, Wageningen Agricultural University, Postbox 338, 6700 AH Wageningen, The Netherlands. richard.crooijmans{at}alg.vf.wau.nl
.
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