Conservation Within Artiodactyls of an AATA Interrupt in the IGF-I Microsatellite for 19–35 Million Years

Mohammad Reza Shariflou1, and Chris Moran

Department of Animal Sciences, University of Sydney, New South Wales, Australia

Abstract

Occurrence of an AATA interrupt in the IGF-I microsatellite was investigated in a number of Artiodactyl species, namely pigs, camels, deer, cattle, goats, and sheep. Comparison of DNA sequences in the 5' flank of the microsatellite in these species revealed that the interrupt within the microsatellite is conserved in deer, cattle, sheep, and goats but is absent from camels and pigs. The interrupt was introduced into the Artiodactyl phylogeny after the divergence of the Camelidae but before the divergence of the Cervidae, and thus its time of origin can be estimated to be 19–35 MYA. In contrast to the repeat units which are hypermutable, the interrupt has been conserved for a very long time and may even have suppressed microsatellite variation by inhibiting replication slippage. A 12-bp deletion in the 5' flank of the microsatellite in camels corresponds to a consensus reversed repeat in deer, cattle, sheep, and goats with unknown functional significance. Apart from this deletion, the 5' flank of the microsatellite is highly conserved in Artiodactyl species.

Introduction

Microsatellites are runs of tandemly repeated DNA with repeat units of 1–6 bp. These tandem repeats are highly abundant in mammalian genomes (Tautz 1989Citation ; Buchanan et al. 1993Citation ), hyperpolymorphic (Weber 1990Citation ), and easily detectable using PCR. These features of microsatellites have enhanced their utility as genetic markers, in constructing genome maps (Love et al. 1990Citation ; Barendse et al. 1997Citation ), in mapping QTL (Georges et al. 1995Citation ; Henry et al. 1998Citation ), and in characterizing breed differentiation (Bowcock et al. 1994Citation ; Pihkanen, Vainola, and Varvio 1996Citation ). Conservation of dinucleotide microsatellites has been reported among different groups of species, e.g., cattle/sheep (Gortari et al. 1997Citation ) and cattle/sheep/goats (Kossarek et al. 1995Citation ), enabling comparative genetic studies and the design of consensus PCR primers to amplify template DNA from different species (Kirkpatrick 1992Citation ; Gortari et al. 1997Citation ).

In some microsatellites, the repeat units are interrupted by nonrepeat sequences, producing imperfect microsatellites. The presence of imperfect microsatellites has been reported in several species, e.g., humans (Chung et al. 1993Citation ), goats (Pepin et al. 1995Citation ), pigs (Wintero, Fredholm, and Andersson 1994Citation ), honeybees (Estoup et al. 1995Citation ), and salmonid fish (Angers and Bernatchez 1997Citation ). Studies on goats (Pepin et al. 1995Citation ) and humans (Weber 1990Citation ) showed that the number of alleles is decreased in interrupted dinucleotide repeats. However, the evolutionary implications and conservation of interrupts within microsatellites have not yet been well studied.

A (CA)n microsatellite exists in the 5' flank of the IGF-I gene (about 1 kb from the transcription start site) in a number of species, namely goats (GenBank accession number D26116), sheep (Dickson, Saunders, and Gilmour 1991Citation ), pigs (Weller et al. 1993Citation ), horses (Caetano and Bowling 1998Citation ), humans (Rotwein et al. 1986Citation ), and rats (Shimatsu and Rotwein 1987Citation ). Given the close proximity of the microsatellite to the transcription site and the ability of the CA repeats to form a Z-DNA structure (Nordheim and Rich 1983Citation ), a potential enhancer element (Hamada et al. 1984Citation ), there might be an important functional role for this microsatellite. Furthermore, Casas-Carillo et al. (1997)Citation reported that the IGF-I microsatellite is associated with average daily gain in pigs. Therefore, these features of the IGF-I microsatellite have made it attractive for study in mammalian species. The objective of this study was to investigate the conservation of the IGF-I microsatellite in a number of Artiodactyl species. Of particular interest was the occurrence of an AATA interrupt within the (CA)n repeat units. Conservation of this interrupt in a number of Artiodactyl species allowed estimation of its time of origin.

Materials and Methods

Source of DNA Sequence Data
Microsatellite regions were amplified and sequenced for cattle, camels, and deer, using genomic DNA from a Holstein-Friesian sire (Bos taurus), a pet female camel (Camelus dromedarius), and a red deer (Cervus elaphus), respectively (see following section for primers and PCR conditions). DNA sequences for goats (accession number D26116), sheep (accession number X17229), pigs (accession number X64400), horses (accession number U83589), humans (accession numbers M12659 and M77496), and rats (accession number J02743) were obtained from the GenBank database.

Primers, PCR Conditions, and Sequencing
Primers 5'-GGGTATTGCTAGCCAGCT-3' and 5'-CATATTTTTCTGCATAACTTGAACCT-3' were from Kirkpatrick (1992)Citation . PCR reactions were performed in a total volume of 25 µl containing 1.5 mM MgCl2, 200 µm each dNTP, 0.4 µM each primer, 1 x PCR buffer, 0.5 U Taq DNA Polymerase (Promega), and 100 ng genomic DNA. The thermal cycling program consisted of predenaturation at 94°C for 3 min, 36 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 45 s, followed by a final extension at 72°C for 5 min. PCR products were extracted from agarose gels and sequenced using a DNA sequencing kit (Promega). In each case, both strands were sequenced, using P32-labeled forward or reverse primer.

Results

A single pair of primers (Kirkpatrick 1992Citation ) produced successful amplification of a fragment, using genomic DNA from cattle, deer, and camels. Sequencing the amplified fragments revealed the presence of (CA)n repeats in these species. Sequence data were submitted to GenBank with the accession numbers AF174576 for cattle, AF174577 for deer, and AF174578 for camels. The DNA sequences, obtained in the present study, were aligned with corresponding published data for goats (accession number D26116), sheep (accession number X17229), pigs (accession number X64400), horses (accession number U83589), humans (accession numbers M12659 and M77496), and rats (accession number J02743), with horses, humans, and rats used as outgroups. Sequence alignment was performed using the multiple-alignment option in ECLUSTALW software in ANGIS. Some sequences in the microsatellite region causing artifactual alignments in flanking DNA were deleted from human and rat sequences to anchor primer sequences in all species (primer sequences are not shown) and to obtain a more meaningful alignment of the sequences flanking the microsatellite repeat (fig. 1 ). Nucleotide sequences from 1 to 37 (37 nucleotides) showed 89% homology among all Artiodactyl species, including cattle, deer, and camels (fig. 1 , Align1), 89% homology between Artiodactyls and horses (fig. 1 , Align2), and 86% homology between Artiodactyls and humans (fig. 1 , Align3). The homology decreased to 59% for rats (fig. 1 , Align4), which is a high conservation of a noncoding sequence in such distantly related species.



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Fig. 1.—Alignment of nucleotide sequences of the IGF-I microsatellite in different species.

 
Nucleotides from 38 to 49, preceding the microsatellite, while absent from camels, are highly conserved in Artiodactyl species. The consensus sequence of this region consists of 5'-ATGCACTCACGT-3'. Apart from the initial A nucleotide, the sequence is a reversed repeat rotated about a central T nucleotide. However, in the second half, a C-to-T transition for cattle (5'-TGCACTCATGT-3') and a G-to-A transition for sheep (5'-TGCACTCACAT-3') has occurred, disturbing the reversed repeat in these species. Whether this unusual structure at the 5' end of the microsatellite has any function remains unknown. Also, the mechanism and reason for its deletion from camels is unclear.

Of the greatest interest is the conservation of an AATA interruption in the microsatellite in some species within the Artiodactyl. That is, the interrupt exists in goats, sheep, cattle, and deer ((CA)6AATA(CA)n) and does not exist in camels and pigs. It is also absent from the outgroup species, horses, humans, and rats. Using published phylogenetic data (Miyamoto et al. 1993Citation ; Honeycutt et al. 1995Citation ), the time of origin of the interrupt was estimated to be 19–35 MYA (fig. 2 ).



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Fig. 2.—Artiodactyl phylogeny showing time of origin of the AATA Interrupt. This phylogeny is based on data from Miyamoto et al. (1993) and Honeycutt et al. (1995)

 
Discussion

The present study expands the conservation of the (CA)n repeat in the IGF-I microsatellite to cattle, deer, and camels. In general, the results presented here showed almost complete conservation of the 5' flank of this microsatellite not only in Artiodactyl species, but also in one of the outgroup species, horses. There are also considerable similarities with the other distantly related outgroup species, humans and rats. This is probably an indication of an important functional role in this region. In particular, the nucleotide identities of the 5' flank are progressively reduced when Artiodactyl species are separately compared among themselves (89%; fig. 1 , Align1), then with humans (86%; fig. 1 , Align2), and then with rats (59%; fig. 1 , Align3). This decline is in accordance with the evolutionary relationships among these species and is consistent with similar homology patterns in other species, e.g., conservation of flanking sequences of a (GT)n repeat in humans/chimps/monkeys (100%), mice/rats (71%), and humans/rodents (30%) reported by Stallings et al. (1991)Citation .

The sequence of nucleotides 25 to 30 clusters Artiodactyls into three groups, i.e., camels/pigs, containing TTGGAG, cattle/deer, containing TGGGGG, and goats/sheep, containing GGGGGG. Information in this region produced a phylogenetic tree (data not shown) similar to that expected from large data sets (Miyamoto et al. 1993Citation ; Honeycutt et al. 1995Citation ). This information might be useful for evolutionary studies of the IGF-I microsatellite, similar to that in salmonid fish (Angers and Bernatchez 1997Citation ) and horseshoe crabs (Orti, Pearse, and Avise 1997Citation ). However, it is noted that horses, an outgroup species from the order Perissodactyla, are also classified with pigs and camels. Therefore, information in this region is not enough to distinguish horses from Artiodactyls.

The interspecific pattern of occurrence of the AATA interrupt is consistent with known Artiodactyl phylogeny, as illustrated in figure 2 . Published phylogenetic data (Miyamoto et al. 1993Citation ; Honeycutt et al. 1995Citation ) indicate that pigs (family Suidea) diverged about 55 MYA from the base of the Artiodactyl lineage. The camel (family Camelidae) diverged about 35 MYA. Later, the deer (family Cervidae) branched off (about 19 MYA). The Bovidae (cattle, sheep, and goats) branched off more recently. Therefore, it can be concluded that the interrupt was introduced into the Artiodactyls sometime between the evolutionary divergence of camels and deer, i.e., between 19 and 35 MYA. This implies that the interrupt originated about halfway through the evolution of the Artiodactyls and has been conserved in later species, i.e., in the family Bovidae, including cattle (subfamily Bovinae), sheep, and goats (subfamily Caprinae). Therefore, this interrupt has been conserved over a remarkably long period of evolutionary history. This is all the more remarkable given the hypermutability of repeat number in (CA)n microsatellites.

The number of CA repeats in the 5' flank of the AATA interrupt is smaller (6 repeats) and is the same for all interrupt-containing species, while it is larger and varied (8–10 repeats) in the 3' flank of the interrupt. This suggests that the interrupt, as a type of anchor point, impedes replication slippage in its 5' flank, preventing the variation of repeat number in this block. However, the larger and variable number of repeat units in the 3' flank of the interrupt suggests that replication slippage can occur downstream of the interrupt.

Overall, the interrupt has contributed to a lower level of variation in the interrupt-containing species. That is, the number of alleles in both sheep (Adams and Maddox 1994Citation ) and cattle (Kirkpatrick 1992Citation ; Shariflou 1997Citation ), which contain the AATA interruption, is 3, while in pigs (Kirkpatrick 1992Citation ; Muladno 1994Citation ) and horses (Caetano and Bowling 1998Citation ), both lacking the interruption, the numbers are at least 6 and 5, respectively. Therefore, the interrupt has also caused considerable suppression of variability of allele size as a result of the stability of repeat number. This is in agreement with other interruptions, such as TT within a (GT)n motif in Apis mellifera (Estoup et al. 1995Citation ) and CAT in a (CAG)n motif in humans (Chung et al. 1993Citation ). Stability of repeat number, because of the presence of an interrupt, is an important issue in preventing a disease arising from SCA1 locus in humans (Chung et al. 1993Citation ).

Given that the mutation rate of repeat number is so high (Shibata et al. 1994Citation ), it is virtually impossible to recognize identity by descent of microsatellite alleles between closely related species and often within species. Despite this plasticity in repeat number, this AATA interrupt has been conserved for at least 19 Myr. Given that both the repeat and the interrupt are located in a noncoding region, it is remarkable that the interrupt has been spared from substitution or deletion mutations, unless, of course, it has some cryptic functional significance.

Acknowledgements

We would like to thank Chris Taylor from the Victorian Institute of Animal Science (VIAS), Victoria, Australia, for donating the cattle DNA sample, Jessica Moran and Andrew Moran for donating a camel blood sample, Judy Broom from the Department of Biochemistry, University of Otago, New Zealand, for donating a deer DNA sample. M.R.S. is grateful to the MCHE, Iran, for financial support while on leave from the Department of Animal Sciences, University of Tehran, Iran. This work was partially supported by a DRDC grant to C.M. in 1992–1994.

Footnotes

Claudia Kappen, Reviewing Editor

1 Present address: Plant Breeding Institute, University of Sydney, New South Wales, Australia. Back

1 Keywords: Artiodactyl IGF-I microsatellite AATA interruption Back

2 Address for correspondence and reprints: Mohammad Reza Shariflou, Plant Breeding Institute, C44, University of Sydney, PMB 11, COBBITTY NSW 2570, Australia. E-mail: msharifl{at}pop.usyd.edu.au Back

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Accepted for publication December 14, 1999.