Department of Biological Sciences, Texas Tech University, Lubbock
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Abstract |
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Key Words: heteroplasmy Crocodylus mitochondrial control region VNTR Crocodylia
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Introduction |
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The most commonly invoked model for explaining the change in number of repeat units in the control region is the illegitimate elongation model of Buroker et al. (1990). This model uses competitive displacement of the H-strand by the D-loop to insert and remove repeats and provides a reasonable explanation for this process in and around the ETAS domain of the control region. Unfortunately, by relying on this competition, the applicability of the model is limited to tandem repeats in the ETAS domain. Two other models have been suggested that are more appropriate to repeated units at the 3' end of the control region. Broughton and Dowling (1997) offered two models to explain duplications in the control regionthe heavy strand and light strand models. Both invoke improper upstream initiation of replication and duplication of the "extra" sequence just before termination to explain the presence of repeats in this part of the genome. By contrast, the model of Mundy, Winchell, and Woodruff (1996) suggests that secondary structure characterizing the neighboring 12S rRNA or tRNA genes contributes to the formation of tandem repeats by causing strand slippage in the nascent heavy strand. Other authors have proposed models of mitochondrial recombination for increasing or decreasing the number of repeats (Lunt and Hyman 1997), but this is considered rare at best (Erye-Walker and Awadalla 2001; Ladoukakis and Zouros 2001).
The study of repeated sequences is important for several reasons. Most obviously, they lend insight into the processes involved in the evolution of the nuclear and mitochondrial genomes. They may also provide information on genome function. For example, in Adelie penguins (Pygoscelis adeliae), repeated sequences contain the transcriptional promoters for both the mitochondrial heavy and light strands (Ritchie and Lambert 2000). As a result, we should expect to find transcripts of varying sizes possibly requiring differential processing. Several human diseases have been linked to mitochondrial heteroplasmy, and animal model systems may provide insights into the dynamics of transmission and development of these diseases (Tamura et al. 1999; Bianchi, Bianchi, and Richard 2001; Sanchez-Cespedes et al. 2001). Studying tandem repeats in the animal mitochondrial genome may also be useful from a population level perspective. A number of researchers have attempted to employ these types of repeats to study population dynamics (Rand and Harrison 1989; Arnason and Rand 1992; Brown, Beckenbach, and Smith 1992; Hoelzel, Hancock, and Dover 1993; Cesaroni et al. 1997) with mixed results.
Herein, we describe tandem repeats in the mitochondrial control region of 11 species in the family Crocodylidae, including interindividual and intraindividual variation in Crocodylus moreletii. We also comment on similar repeat units in the control region of Caiman crocodilus, Alligator mississippiensis, and A. sinensis.
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Materials and Methods |
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The primers CR2L (5'-CGT TAT ACA TAT TAC TCT TTA ATT AGG CCC CC-3') and 12SH1 (5'-GTT GAG CAG TAG CTA ATA ATA AGG TCA GGA-3') were used to amplify the 3' end of the D-loop in 100 µl reactions under the following conditions: 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.1% Triton 100, 1 mM MgCl2, 0.15 µM of each dNTP, 0.2 µM of each primer, and 1 U Taq polymerase. A thermal profile of 3 cycles of 95°C for 1 min, 58°C for 1 min, 72°C for 1.5 min, followed by 30 cycles of 94°C for 15 sec, 59°C for 20 sec, and 65°C for 2 min was used. This was followed by a final elongation of 20 min at 72°C. The reduced elongation temperature was used to try and increase the fidelity of the enzyme while allowing the addition of a terminal A nucleotide for TA cloning provided by Taq polymerase.
PCR products were checked for heteroplasmy and polymorphism on a 2.0% agarose gel stained with ethidium bromide. Amplification products were cleaned using the Qiagen PCR purification kit (Qiagen Inc., Valencia, Calif.) after which, the fragments were inserted into the pCR-4 cloning vector using the TOPO-TA cloning kit from Invitrogen (Carlsbad, Calif.). Plasmids were chemically transformed into TOP10 (Invitrogen) competent cells using the protocols provided. At least 10 colonies from each individual were screened for inserts via PCR using the primers M13F and M13R under standard conditions. Colonies were then selected on the basis of size differences. The number of colonies sequenced from each individual was dependent on the number of different size fragments observed, but in each case, at least two colonies were selected. Plasmids from selected colonies were isolated using the Qiagen miniprep kit and sequenced using an ABI 310 genetic analyzer employing the sequencing primers M13F and M13R. Sequences were analyzed and edited using the program BioEdit (Hall 1999). Analysis for secondary structure was performed with RNAstructure3.71 (Mathews et al. 1999) using the default settings. Sequences from each clone examined have been deposited with GenBank (accession numbers AY138864 to AY138894).
Most products from the region under investigation ranged between 550 and 750 bp in length. This sometimes presented a problem for sequencing analysis because the ABI 310 genetic analyzer typically yields readable sequences of only 400 to 500 bp. The presence of the repeated sequences did not allow for the use of internal primers for either PCR or sequencing. We were, therefore, forced to use information on fragment size from gel electrophoresis and sequence overlap to determine the sequence for the entire fragment. Fragments exceeding 800 bp (seen primarily in C. palustris and C. porosus) had little overlap when sequenced from both directions, and, therefore, base identity in the central regions was less certain.
While size variation in the tandem repeat region among and within individuals was observed in several species of the Crocodylidae (Crocodylus intermedius, C. palustris, C. niloticus, C. mindorensis, C. siamensis, and C. porosus) via direct sequencing, we chose to concentrate our efforts on a more detailed analysis of repeats in Morelet's crocodile (C. moreletii). This was accomplished by modifying the primer 12SH1 to include a HEX label and amplifying the tandem repeat region from 19 individuals of C. moreletii. After amplification, PCR products were analyzed on an ABI 310 genetic analyzer using Genescan software (ABI).
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Results |
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In all members of Crocodylidae a distinct poly-A tract, beginning approximately 30 bp downstream from the presumed CSB-3 sequence (Ray and Densmore 2002) was observed. The poly-A region varied among sequenced clones but ranged from 53 bp (Crocodylus palustris) to 83 bp (C. rhombifer). In some clones, the poly-A region was followed by a shorter but distinct tract of nucleotides dominated by thymine residues. This pattern was characteristic of New World crocodiles (C. acutus, C. moreletii, C. rhombifer, and C. intermedius) and several sequences obtained from C. niloticus and C. mindorensis (the Nile and Philippine crocodiles, respectively). Tandemly repeated sequences immediately followed the poly-A or poly-T tracts in all clones and made up a large portion (40% to 64%) of the CSB domain.
Tandem Repeat Primary Structure
Within Crocodylidae, the sequences of repeats were similar and appear to be derived from the same ancestral sequence. They show a high A/T bias (> 78%) with runs of up to 8 nt each. In all Old World species examined, the longest single repeat was 63 bp (Crocodylus siamensis). In New World species, the longest single repeat was 74 bp (C. acutus). Generally, Old World species showed less variation in repeat length as the sequence proceeded from 5' to 3'. With the exception of C. siamensis, these species exhibited a pattern in which the repeats varied by only 1 or 2 nt among fully repeated sequences. The main array (three to 10 repeats) was usually followed by a truncated version of the full sequence (10 to 38 bp).
New World crocodile species tended to be highly variable along the entire length of the repeat region. These species typically showed a pattern of shortening as they proceeded from 5' to 3' along the light strand. For example, in one individual of Crocodylus moreletii, clone 3557.3 (fig. 2), the initial sequence (copy 1) is 70 bp in length. Copy 1 is imperfectly repeated in the adjacent sequence as a 70-bp sequence with seven nucleotide substitutions and two indels. Copies 3, 4, and 5 are perfectly repeated sequences of 51 bp each. They differ from copy 2 by one substitution and a single large deletion. The final copy of the repeated sequence is truncated to 33 bp and is a perfect repeat of the first 35 bp of copies 3, 4, and 5. This pattern is repeated in the other New World species studied, whereas in the Old World species the progressive shortening was either not observed or was seen only in some clones.
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Intraindividual and Interindividual Variation
Nine size classes ranging in size from 536 bp to 678 bp were observed among the 29 Crocodylus moreletii sampled (table 2). Three heteroplasmic individuals were identified, cm2664, cm2465, and cm3433. All three contained two haplotypes, 592/645 bp in cm2664, 593/645 bp in cm2465, and 536/678 bp in cm3433.
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Discussion |
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As noted in figure 3, the poly-A portions located 5' of the tandem repeats form stable secondary structures in all species considered. This being the case, the model of Mundy, Winchell, and Woodruff (1996) is applicable if one assumes that the nascent light strand (not the heavy strand as described in their model) is allowed to "slip" immediately after this region is copied (fig. 5). This need only happen once, after which the standard model of slipped-strand mispairing can explain expansions and contractions of the tandem array (see below). Assuming this is the correct model, the original duplication would be 3' of and adjacent to the poly-A tract.
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One of the most commonly invoked models to explain the expansion and contraction of repeated sequences in the control region is that of Buroker et al. (1990). However, their model is not useful for explaining changes in copy number for the repeats described here. Although it has yet to be precisely located, the origin of replication in crocodilian mtDNA is probably not immediately adjacent to the 12S rRNA gene (Ray and Densmore 2002). Thus, the generation of repeats is most likely not due to D-loop/H-strand competition. Instead, slipped-strand mispairing (Levinson and Gutman 1987) could occur during heavy-strand and/or light-strand replication. At these times, one strand of the nascent double helix can presumably slip easily and fold into the proposed secondary structure (fig. 3). Depending on which strand slips and is mispaired, arrays can be increased or decreased in size.
The Alligator mississippiensis array is interesting in that there are two different sets of tandem arrays adjacent to one another (fig. 6). The first array consists of three identical sequences. The second array is made up of 14 repeats that are identical with the exception of a single-base deletion in repeat number 12. Even more interesting are the chimeric aspects of the region. The first three repeats share the initial 14 bp of sequence (shaded in fig. 6) with the second set of repeats. Furthermore, each repeat in the second array contains a direct copy of the 15-bp nucleotide sequence (lowercase in fig. 6) immediately preceding the first array. These features lend clues as to how such an interesting pattern could have arisen via slipped-strand mispairing, although involving a more complicated mechanism than described above. Slippage during replication could explain the pattern if pairing were to occur between the sequence before the first repeat and the nascent chain of the last sequence of the first tandem array via the common sequence, ATATTATA. Alligator sinensis tandem repeats follow a similar heterogeneous pattern (GenBank sequence AF511507).
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Intraindividual and Interindividual Variation and Heteroplasmy
Two types of variation among and within individuals were detected. The first type is variation due to single or multiple nucleotide insertion/deletion events in the poly-A tract. The second type is insertion/deletion of entire tandem repeats. For example, in Crocodylus moreletii there were nine size classes found. The nine distinctly sized fragments (table 2) can be grouped into classes of 536, 59x, 64x, and 678. The 59x and 64x size classes vary by 2 and 3 bp, respectively. The smaller size differences were due to variation in the poly-A tract, and larger differences resulted from variation in the number of tandem repeats. For example, the repeat region from one individual (cm2664) exhibited two haplotypes derived from both types of variation (fig. 7). First, in the poly-A tract of clone 2664.7 (592 bp), there are two deletions of 1 and 3 A nt, respectively, at the 5' end of the sequence when compared with the second sequenced clone 2664.2 (645 bp). The second difference is the complete deletion of one tandem repeat (49 bp).
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Finally, it will be interesting to assess the structure of any possible repeated sequences in the two gharials, Gavialis gangeticus, and Tomistoma schlegelli. These two crocodilians are at the center of a continuing conflict between morphology-based and molecule-based hypotheses about the relationships within the Crocodylia (Brochu and Densmore 2001). Most studies employing morphology of both extant and fossil crocodilians find that the true gharial (Gavialis) is an ancient taxon whose ancestors diverged from the rest of the group in the late Cretaceous or early Tertiary. In these studies, the false gharial (Tomistoma) is usually found to have an affinity with the family Crocodylidae, a much more recent group. By contrast, all biochemically/molecularly-based analyses to date have found the two gharials to be closely related, usually forming a sister clade to the Crocodylidae (see Brochu and Densmore 2001 for a review). Eventual characterization of any repeats present in the control region of these species may provide evidence for both divergence timing and relative placement of these taxa with regard to the other crocodylian lineages.
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Supplementary Materials |
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Acknowledgements |
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Footnotes |
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