Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Santiago, Galicia, Spain
Correspondence: E-mail: bnjgm{at}usc.es.
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Abstract |
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Key Words: minisatellite evolution phylogenetic footprint primates human minisatellite MsH42
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Introduction |
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The human minisatellite MsH42 (MsH42) is a low-polymorphic locus (three alleles have been identified in the human populations) that is localized in the chromosome 15q25.1 inside intron 5, between exons 5 and 6, of the gene Q9ULM1 (fig. 1A). This GC-rich minisatellite is able to interact specifically with nuclear proteins (Boán et al. 1997). Moreover, MsH42, with its proximal flanking sequences (the MsH42 region), is able to enhance in vitro intramolecular homologous recombination, promoting high rates of equal crossovers (Boán et al. 1998, 2002).
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Materials and Methods |
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PCR Amplifications and DNA Sequencing
Primer pairs PS1/PS2 and P1/P2 were used to amplify the MsH42 region. Primer pairs E5.1/E5.2 and E6.1/E6.2 were used to amplify exons 5 and 6 of the Q9ULM1 gene (www.ensembl.org/Homo_sapiens), respectively. Primers PC1 and PC2 were designed based on two highly conserved motifs situated near MsH42 and were employed to amplify the minisatellite and its proximal flanking sequences. The sequences of the primers are PS1 (5'-CTGCAGCAATGGACTCAAAA-3'), PS2 (5'-CTGCAGACTCCAAATCCTAA-3'), P1 (5'-CTTGGGCACTCTAGGACACC-3'), P2 (5'-CACAGCTCTGGCTACAAGAG-3'), E5.1 (5'-TTTGCTCTGGGATTTAAGGC-3'), E5.2 (5'-CAACAAGCCATTGGAGCCAT-3'), E6.1 (5'-ATCAAGGACGTTGT GGGCTA-3'), E6.2 (5'-TTGCAG TCTTGCCTGGGCTT-3'), PC1 (5'-GGGCAGTGTTGAGAGTGAGC-3'), and PC2 (5'-TATCTTCATGAACTCACACT-3'). The general conditions for all amplifications and the cycling conditions for PS1/PS2 and P1/P2 were as described elsewhere (Boán et al. 1997, 2000). The denaturing, annealing, and extension steps for the new combinations of primers were PC1/PC2: 95°C 1 min, 56°C 40 s, and 72°C 30 s; E5.1/E5.2: 95°C 1 min, 58°C 15 s, and 72°C 20 s; E6.1/E6.2: 95°C 1 min, 60°C 15 s, and 72°C 20 s; E5.1/PS2: 95°C 1 min, 54°C 40 s, and 72°C 2 min. PCR products were cycle sequenced using the 377 DNA Automated Sequencer (Applied Biosystems).
Footprinting Analysis
The probes for footprinting experiments were the Fp1 and Fp2 DNA fragments (fig. 1A). The Fp1 fragment was synthesized by PCR with primers PS1 and Pf1 (5'-GCCTCTCCCAGCTCTCCCAGCCCT-3') as follows: denaturing 95°C 1 min, annealing 70°C 30 s, and extension 72°C 50 s. The Fp2 fragment was obtained by DdeI digestion of the P1/P2 amplification fragment of the MsH42 region. These fragments were cloned in the pGemT-easy vector (Promega Inc.) in both orientations, and the probes were obtained by digestion with SacII and NotI. The NotI end of both strands (upper and lower) was end-labeled with radioactive nucleotides yielding specific activities of 5 x 108 cpm/µg. The nuclear extracts were prepared from testis, brain, and liver of 3-month-old Sprague-Dawley rats (Boán et al. 1998). The DNase I footprinting reactions were performed with the SureTrack Footprinting kit (Amersham Pharmacia Biotech) by mixing 50 µg of nuclear extracts, 3 µg of poly(dI-dC)·(dI-dC), and 0.5 µg of calf thymus DNA in the binding buffer with 0.1 ng of radioactive probe. The DNase I digestions were set up as follows: 0.1 U for 1.5 min in the reaction with BSA instead of protein extract (control reactions), 1.5 U for 1.5 min for reactions with testes extract, and 0.1 U for 1 min in experiments with brain or liver extracts. The purified DNAs were analyzed on 6% sequencing gels.
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The GenBank accession numbers for the DNA sequences in this study are AY270192 to AY270201 for the minisatellite region, AY268962 to AY268968 for exon 5, and AY268969 to AY268975 for exon 6.
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Results and Discussion |
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We performed amplifications with E5.1/PC2 to determine if the MsH42 region was also localized within an intron in primates. The results of these PCRs demonstrated the intronic localization of the MsH42-related minisatellite in primates (data not shown). On the other hand, we sought for the presence of the MsH42 region in the mouse, rat, zebrafish, and Drosophila genomic databases (www.ensembl.org). The result of these searches did not reveal any homologous sequences to the MsH42 region in those genomes. Because the genomes of both murine species contain the exons that flank MsH42, and there is no trace of the minisatellite, it is reasonable to conclude that the MsH42 minisatellite was originated during primate evolution.
To analyze the organization of the MsH42 region in the primates, the PCR products were sequenced. Figure 2 shows the alignment of a representative allele from each species in relation to the human MsH42 short allele. The tamarin version of the MsH42 locus has two repeat variants (A and C) common to humans, intercalated with other similar repeats, reminiscent of a pre-MsH42 region. In the Cercopithecoidea monkeys, macaque and mandrill, the minisatellite contains seven repeat units with the same arrangement in both species except for a G/A transition; this result agrees with the close phylogenetic relationship between these monkeys. Furthermore, in these primates, a constant characteristic of MsH42 emerges for the first time in all species: A is the first variant repeat and C1 is the last one (from 5' to 3'). In the gibbon DNA, the organization of the minisatellite is rather unusual and consists of 16 repeats, four of them restricted to this species (A5, C3, C4, D). The orangutan minisatellite has a repeat composition that is very similar to humans, and among its 13 repeats, only B4 is unique to this species. The panorama in the African great apes changes dramatically and the organization of MsH42 becomes highly homologous to the human locus. Thus, the analysis of gorilla samples revealed the existence of three alleles (table 1) with only one variant repeat (C2) absent in the human alleles. All gorilla alleles share a group of nine repeats at the 5' end of the minisatellite and another group of five repeats at the 3' end. Consequently, the differences among these alleles lie in the central portion of the minisatellite. The chimpanzees alleles (table 1) show an organization very similar to the human short allele, with the exception of two rare repeats (C2 and B3). It is worth noting that the GOR1 and CHI1 alleles are highly homologous to the human short allele, pointing towards their common origin from an ancestral array.
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Genomic sequence comparisons among distant species are a useful tool to identify regulatory elements present in the noncoding fraction of the genome. For example, sequence-specific conservation of noncoding DNA may imply functional constraint on these sequences and slower rates of molecular evolution (Ludwig 2002). Close examination of the sequences flanking MsH42 revealed an important conservation among primates (fig. 2). In particular, there are two highly conserved stretches (HCS) at both sides of the minisatellite, denoted as 5'HCS and 3'HCS. The 5'HCS comprises 29 bp with only two nucleotide changes in the tamarin, whereas the 3'HCS comprises 26 bp and shows only two transitions (A/G) in the macaque. It is likely that mandrill has both HCS because its genomic DNA was amplified with primers PC1/PC2, whose sequence is inside such regions. Phylogenetic footprinting (Tagle et al. 1988; Gumucio et al. 1992) and phylogenetic shadowing (Boffelli et al. 2003) have been used to identify putative regulatory elements, exploiting alignments across numerous distantly related or closely related species. We have previously detected protein-DNA interactions in this area by band-shifting experiments (Boán et al. 1997). Therefore, we carried out footprinting analysis to determine if these HCS were specifically recognized by proteins, thereby reflecting a function within the genome. For this purpose, we used the probes Fp1 and Fp2 (fig. 1). Fp1 contains the first 127 bp of the MsH42 region, including the 5'HCS and the first nine repeats. Fp2 has the last nine repeats of MsH42 and the next 175 bp downstream MsH42, including the 3'HCS. The results obtained with both orientations (upper and lower strands) of Fp1, using nuclear extracts from liver and brain, demonstrated the presence of two protected zones, 5f1 and 5f2, localized in the 5' flanking region of MsH42 (fig. 3). In the experiments performed with the Fp2 probe, we found two protections, 3f1 and 3f2, with the liver extract (fig. 3). Noteworthy, the 5f1 and 3f1 footprints include the 5'HCS and the 3'HCS, respectively, demonstrating the existence of a protein(s) that specifically interact with these conserved sequences, resembling phylogenetic footprints. Testes nuclear extract did not show any protected zone (fig. 3), indicating that testes may not express the protein(s) that recognizes such conserved zones. Taking into account that the MsH42 locus is situated within an intron, it is tempting to speculate that these HCS could play a role as regulatory elements of the Q9ULM1 gene. It should be mentioned that the footprinting experiments were done using rat nuclear extracts. Given that the rat genome does not contain the MsH42 region, it is reasonable to conclude that the sequences involved in the generation of footprints are present elsewhere in the rat genome and, hence, highly conserved.
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A phylogenetic analysis using the DNA sequences from the minisatellite region and exons 5 and 6 was performed. The results from the incongruence-length difference test indicate a significant character congruence (P = 1) that allows us to reconstruct a neighbor-joining topology from the total data set (data not shown). The phylogenetic tree so obtained is identical to the standard hypothesis about the phylogenetic relationships of primates (Miyamoto, Slightom, and Goodman 1987). Under the parsimony criterion, the total and the minisatellite flanking data sets also reflect the widely accepted primate tree, whereas alternative topologies were recovered from the exons and minisatellite sequences (data not shown). However, the consistency-index values as well as the Kishino-Hasegawa test indicate that these sequences make a consistent statement with respect to the standard primate topology. These results indicate that the mutational events observed in the minisatellite MsH42 are in agreement with the primate evolutionary history described for other noncoding sequences (Saitou and Ueda 1994; Apoil and Blancher 2000). Moreover, the estimation of parameter (
= 0.256) from population data suggests a mutation rate in the range of those estimated for other intron sequences in human genome (Huang et al. 1998).
The results presented here provide strong evidence that MsH42 was originated within an intron from a progenitor sequence, without a well-defined minisatellite structure, that experienced mutations leading to the formation of the first MsH42 repeat variants (fig. 4). Such progenitor sequence could have been originated by slipped-strand mispairing and unequal crossing-over between noncontiguous repeats formed by chance (Levinson and Gutman 1987; Haber and Louis 1998; Taylor and Breden 2000). The existence of the MsH42-like region in tamarin indicates its ancient generation during the earliest primate lineage evolution, before the divergence between Old World and New World monkeys about 40 MYA (Goodman 1999). This belief is strongly supported by the lack of amplifications in three prosimian species and because MsH42 homologous sequences could not be retrieved from the genomic databases of other organisms.
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The theory that modern humans originated in Africa is strongly supported by analysis of genetic variation in people today and from fossil discoveries (Stinger 2003). The existence of a highly homologous MsH42 allele in humans and African great apes, together with the fact that the frequency of the short allele in African humans (0.48 ± 0.07) is three times higher than the observed frequency in Europeans (0.16 ± 0.02) (Boán et al. 2002), supports the "Out of Africa" hypothesis. What is the origin of human MsH42 polymorphism? According to our results, the sequence of events that generated the present MsH42 polymorphism could have been as follows: First, there was the short allele coming from an evolutionary ancestor common to the great apes, second, a single duplication of a repeat block produced the long allele, third, the long allele generated the middle one by a deletion (fig. 4). The maintenance of the repeat arrangement in the three alleles points towards the mispairing of repeat blocks as the most probable mutational events in the human MsH42, as it has been proposed for many minisatellites (Charlesworth, Sniegowski, and Stephan 1994).
A useful approach to study human evolution at the molecular level is to consider our genome as a mosaic in which each DNA segment has its own evolutionary history (Pääbo 2003). Our results allowed us to figure out when the human MsH42 began to exist early in primates, the birth of the minisatellite, and how this locus evolved to become MsH42 in humans. We believe that the present work is a worthwhile contribution to the knowledge of the low-variability minisatellites origin and that the evolutionary analysis of the MsH42 region is a small step toward the understanding of how the human genome has been made.
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Acknowledgements |
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Footnotes |
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