Journal of Heredity Advance Access originally published online on August 31, 2005
Journal of Heredity 2005 96(5):603-606; doi:10.1093/jhered/esi095
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Brief Communication |
Localization of Repetitive DNA Sequences on in vitro Xenopus laevis Chromosomes by Primed in situ Labeling (PRINS)
From the University of Illinois, Department of Crop Sciences, 1201 W. Gregory Dr., 320 ERML, Urbana, IL 61801
Address correspondence to A. Lane Rayburn at the address above, or e-mail: arayburn{at}uiuc.edu.
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Abstract |
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Xenopus laevis is an important reference model organism used in many vertebrate studies. Gene mapping in X. laevis, in comparison to other reference organisms, is in its early stages. Few studies have been conducted to localize DNA sequences on X. laevis chromosomes. Primed in situ labeling (PRINS) is a recently developed innovative tool that has been used to locate specific DNA sequences in various organisms. PRINS has been reported to have increased sensitivity compared to other in situ hybridization techniques. In the present study, PRINS was first used to label the location of telomeres at the ends of in vitro X. laevis chromosomes. The terminal location was as expected from in vivo reports, however, the overall amount seemed to decrease in the in vitro chromosomes. Once the PRINS technique was optimized, this technique was used to determine the chromosomal location of the satellite 1 repetitive sequence, which is an important sequence in X. laevis development. The sequence was observed on the interstitial regions of the majority of the chromosomes similar to the in vivo locations reported. In contrast to the telomeric sequence, the amount of sequence appeared to increase in the satellite 1 sequence. PRINS was found to be useful in the localization of repetitive DNA sequences in the X. laevis genome.
Xenopus laevis is allopolyploid and is regarded as a tetraploid with 36 chromosomes, even though it is a functional diploid (Tymowska 1991). X. laevis has become an important species in developmental studies, but a genetic map for this model organism is lacking. Unlike the gene localization of the human and mouse genome, X. laevis chromosome mapping is in its infancy. One important chromosomal feature is the location of specific repetitive DNA sequences.
Few studies have been conducted to locate specific DNA sequences in X. laevis chromosomes. In earlier studies by Hummel et al. (1984), traditional in situ hybridization was successful in locating three different repeat families in X. laevis chromosomes. More recently, fluorescence in situ hybridization (FISH) has been used to locate sequences in X. laevis (Krylov et al. 2003; Schmid 2001). Two sequences located on X. laevis chromosomes are the telomeric sequences and satellite (sat) 1.
The telomeric sequence is a conserved sequence among vertebrates, including fish, reptiles, birds, mammals, and amphibians (Meyne et al. 1989). The telomeric region located at the end of chromosomes is required for replication and stability of the chromosome (Lavoie et al. 2003). Bassham et al. (1998) characterized the telomeric region of X. laevis and reported the similarities and differences of this region with human telomeres. Telomeres in X. laevis were also investigated in vivo by Cohen and Mechali (2002) to further characterize the presence of telomeres in X. laevis development.
The sat 1 sequence, also referred to as the oocyte activation in Xenopus (OAX) sequence, is a highly repetitive 741 bp DNA that exists as dispersed tandem clusters (Lam and Carroll 1983a; Whitford et al. 2000). Satellite DNAs are often confined to the heterochromatin present around centromeric or telomeric regions, suggesting that satellite DNA may have some structural function in chromosomes (Pasero et al. 1993). Sat 1 is believed to have been derived from a transfer RNA (tRNA) gene and the function of the sequence is reported to be important in X. laevis development (Engelke 1988; Whitford et al. 2000).
Primed in situ labeling (PRINS) is an innovative tool that has recently been developed to detect or locate specific sequences on chromosomes. PRINS has several advantages over the more traditional method of in situ hybridization, in that the procedure is faster, higher specificity is obtained, and background is reduced. PRINS has been applied to locate specific DNA sequences in plants, mammalian cells, and human cells (Kubalakova et al. 2001; Musio and Rainaldi 1997; Reiter et al. 1999).
In the present study, PRINS was used to localize repetitive sequences on X. laevis chromosomes obtained from cell culture. While cell culture provides an efficient means of developing molecular mapping techniques, the issue of chromosomal changes also needed to be addressed. The PRINS technique was optimized for both the telomeric and sat 1 sequence. The location and signal intensity was noted for both sequences and compared to the reported location and intensity on chromosomes obtained in vivo.
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Materials and Methods |
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Slide Preparation and Chromosome Characterization
Xenopus laevis (A6) kidney cells were obtained from ATCC (CCL-102). The X. laevis cells were maintained in NCTC-109 media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO) at 26°C at 5% carbon dioxide. Slides were prepared by placing cells in 0.1 µg/ml colcemid. After 4 h the cells were washed in 1x phosphate buffered saline (PBS) and the cells were detached from the flask by trypsin treatment to obtain the greatest quantity of metaphase chromosomes. Cells were centrifuged at 4°C for 6 min at 900 rpm and resuspended in residual media. A hypotonic solution of 0.067 M KCl was added. After hypotonic treatment the cells were fixed in Carnoy's fixative (3 methanol:1 glacial acetic acid). The cells were then dropped onto slides (MJ Research, Reno, NV) and allowed to dry for at least 2 days. To characterize the chromosomes, slides were stained in 0.2 µg/ml propidium iodide (PI) for 30 min. One slide from five separate flasks was made. Ten spreads per slide were examined using an Olympus BX61 microscope. The number of chromosomes and chromosome morphology were recorded.
PRINS Reaction
Slides were first dehydrated in an ethanol series (70%, 90%, and 100%) for 3 min each and then denatured for 2 min at 70°C in 70% formamide, 2x SSC (pH 7.0). The ethanol dehydration series was repeated and the slides were allowed to air dry. Fifty microliters of a reaction mix was added to each slide. The reaction mix contains 1x Taq buffer (Eppendorf, Westbury, NY), 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.02 mM dTTP, 0.02 mM fluorescein 12-dUTP, 1x self-seal reagent (MJ Research, Reno, NV), 0.01% BSA, 1 U Taq polymerase (Eppendorf, Westbury, NY), and 3 µg primer. For the labeling of the telomeres, the primer sequence (CCCTAA)7 was used. The sat 1 primer was GGCAGGGCTGGAAGTCAATCT. Both primers were made by the W. M. Keck Center for Comparative and Functional Genomics (University of Illinois at Urbana-Champaign). Coverslips were placed on each slide and the reaction ran on an Omnigene Thermocycler equipped with a hybridization block. The single PRINS program for the telomeric primer was 4 min at 94°C for denaturation, 3 min at 60°C for annealing, and 30 min at 70°C for extension. The single PRINS program for the sat 1 primer was 4 min at 94°C, 3 min at 55°C, and 30 min at 60°C. Upon completion of the reaction the coverslips were removed and slides placed in stop buffer (0.5 M NaCl and 0.05 M EDTA) for 1 min at 65°C. The slides were then washed three times in 4x SSC, 0.05% Tween 20 for 5 min each. The slides were counterstained with PI at 0.2 µg/ml for 30 min. Following counterstaining, the slides were drained and Vectashield antifade solution (Vector Laboratory, Burlingame, CA) was added. A coverslip was then placed on each slide.
Fluorescent labeling was detected on the chromosomes using an Olympus BX61 microscope with Omega fluorescent filters. The first filter combination had an excitation wavelength of 475 nm with a 40 nm wave half-height width and an emission signal of 535 nm with a 45 nm wave half-height width to observe the fluorescein signal. The second filter combination had an excitation wavelength of 525 nm with a 45 nm wave half-height width and an emission signal of 565 nm long-pass to observe the counterstaining of the chromosomes with PI. Pictures were taken using an Olympus Magnafire digital color camera and software. Pictures were produced using the color merge option. One slide was prepared from each of five separate flasks. Ten spreads per slide were analyzed and the number of chromosomes labeled and the position of the label were recorded.
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Results and Discussion |
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The X. laevis A6 cell line is a hyperdiploid stemline with a reported average of 44 chromosomes (product description sheet, A6 [Kidney, South African clawed toad, Xenopus laevis] ATCC CCL-102). The number of chromosomes most commonly present in all spreads in this study was 44 (Figure 1A). There were an average of 2 telocentric, 8 acrocentric, 24 submetacentric, and 10 metacentric chromosomes. The reported karyotype obtained from live X. laevis is 36 chromosomes with 14 acrocentric, 16 submetacentric, and 6 metacentric chromosomes (Tymowska 1991). The differences in centromeric location and chromosome number between the cell line and the live frogs may be due to multiple chromosomal aberrations such as translocations, inversions, deletions, or duplications and chromosome misdivision that occur during cell culture.
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Primed in situ labeling is a technique that was developed to locate specific DNA sequences on chromosomes as an alternative to traditional in situ hybridization. The advantages of using PRINS over traditional in situ hybridization include rapidity of the procedure, the high specificity of chromosome labeling, and the reduced background due to the short reaction time and specificity of the primers (Musio and Rainaldi 1997). In the current study, PRINS proved to be a rapid and reliable method for detecting the specific location of the telomeric sequence and sat 1 sequence on X. laevis chromosomes.
Primed in situ labeling has commonly been used to detect the telomeric sequence (TTAGGG)n in mammalian chromosomes, including Chinese hamster cell lines, mice, and humans (Lavoie et al. 2003; Musio and Rainaldi 1997; Serakinci and Koch 2000; Therkelsen et al. 1995). In this study, the telomeric sequence was detected routinely at the end of chromosomes on about 65–75% of the X. laevis chromosomes using PRINS (Figure 2A). The low intensity of the telomere signal on the X. laevis chromosomes is similar to the weak signal of immortal hamster cell telomeres seen by Serakinci and Koch (2000). They reported that shortened telomeres were responsible for the weak signal observed in an immortalized cell line. Observations by Meyne et al. (1989) of labeled X. laevis telomeres from live frogs obtained by FISH indicate more copies of the sequence than seen in this study. The hypothesis is that the sequence is present at lower copy number in X. laevis chromosomes isolated from the immortalized A6 cell line compared to live frogs in a manner similar to what has been observed in other cell culture systems. It should be noted, however, that the lower intensity of the telomere signal could be due to the sensitivity of the assay.
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A total of 10 spreads on five separate slides were analyzed for the sat 1 sequence. There was an average of 64%, or 28 chromosomes in a 44 chromosome spread, labeled for the sat 1 sequence (Figures 1B and 2B). The sat 1 sequence on the majority of chromosomes was located in the interstitial region on the long arm. An average of two chromosomes per spread was double labeled on both the long and short arm. The double signals were located on the longer chromosomes. The sat 1 sequence signal, which has been reported to be present at a high copy number, was much stronger in comparison to the telomeric signal. In the present study, 64% of the chromosomes were labeled, compared to 44% of the chromosomes labeled by FISH [as shown by Schmid (2001)] on chromosomes isolated from live frogs. Thus, unlike the telomere sequence, the sat 1 sequence appears to increase in copy number within cell culture.
The sat 1 sequence or other similar sequences have not been found in other Xenopus species, including X. mulleri and X. borealis, and thus the sequence is not conserved among closely related species (Lam and Carroll 1983b). The sat 1 sequence is reported to encode for a collection of RNA polymerase III transcripts (Lund and Dahlberg 1992). Reports on the expression of the sequence have ranged from no expression (Lam and Carroll 1983a) to expression primarily in oocytes (Wolffe 1989) to transient expression at the midblastula transition (Lund and Dahlberg 1992). Most recently, Whitford et al. (2000) reported expression of the sat 1 sequence during gastrula stages, by tailbud stage embryos, in developing somites and differentiating skeletal muscles, and in the dorsal aspect of the neural tube. The complete function of sat 1 DNA in vivo is not yet completely known, but it is hypothesized that the sat 1 sequence may be important only at certain developmental stages and that expression is very highly regulated. That the sat 1 sequence is conserved and actually appears to increase in copy number per cell in vitro may be an indication of potential additional functions of the sat 1 region in X. laevis.
Primed in situ labeling was found to be a useful technique in detecting the location of specific DNA sequences in X. laevis chromosomes obtained in vitro. The telomeric sequence was detected at the ends of the majority of chromosomes, as expected, but at a lower signal intensity than was observed in chromosomes obtained in vivo. The sat 1 DNA was most commonly located in the interstitial region of the chromosomes on the long arm, with more signal per cell in vitro than observed in chromosomes obtained in vivo. Overall, PRINS has the potential to be an important tool in the comparative physical mapping of X. laevis chromosomes.
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Acknowledgments |
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We thank Laura E. Guest of the W. M. Keck Center for Comparative and Functional Genomics, University of Illinois at Urbana-Champaign, for help with primer design.
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Footnotes |
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Corresponding Editor: Hector Seuanez
Received December 10, 2004
Accepted May 26, 2005
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References |
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