Molecular Biology Reports 28: 119–122, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Nucleotide sequence of BamHI family satellite DNA and its unit lengthpolymorphism in bluegill sunfish Lepomis macrochirus

Tsutomu Takahashi1, Yuya Kawamura1, Nobukazu Sakata1, Gamal E. Elmesiry1, YasuhiroTakemon1, Kazumi Tanida1, Shinsei Minoshima2, Nobuyoshi Shimizu2 & Mikio Kato1,∗1Department of Life Sciences, College of Integrated Arts and Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai 599-8531, Japan; 2Department of Molecular Biology, Keio University School of Medicine 35Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; ∗Author for correspondence (Phone & Fax: +81 72 2549746; E-mail mkato@el.cias.osakafu-u.ac.jp)

Accepted 23 October 2001

Key words: bluegill sunfish, local population, repetitive DNA, satellite DNA, unit length

Abstract

We have isolated and sequenced a member of tandem repetitive DNA containing BamHI site (BamHI familysatellite DNA) from bluegill sunfish Lepomis macrochirus. PCR amplification with specific primers was performedto define the size of unit length repeat of the BamHI family satellite DNA, revealing that there were two distinct sizeof DNA fragments (0.9 kb and 1.3 kb) in the PCR products. The longer fragment (1.3 kb) consisted of internal sub-duplication of shorter fragment (0.9 kb). We have compared the size of PCR products among four fish populations,and found that both fragments co-existed in one population whereas the longer fragment was dominant in otherthree populations. The results may reflect ongoing homogenization of satellite DNA type over a short evolutionarytime scale.

Introduction

Tandem arrayed repetitive DNA sequences, knownas satellite DNA, commonly exist in the centromericregions of vertebrate chromosomes. Although theyare known to participate in the construction of func-tional centromeres [1–5], their nucleotide sequencesare highly variable. Because of their higher sequencediversity among closely related species, satellite DNAsequences are utilized for phylogenetic and taxonomicstudies [6–9]. Tandem repeat DNA sequences are sub-jected to concerted evolution that homogenizes mem-ber sequences within species [10]. Elder and Turner[11] showed that sequence homogenization events oc-cur very frequently in pupfish, and the homogenizedsegments are rapidly fixed in the respective localpopulations. In the present work, we report the nu-cleotide sequence of a member of satellite DNA andthe occurrence of the unit length polymorphism in thepopulations of bluegill sunfish Lepomis macrochirus.

Materials and methods

Nucleotide sequences

The fish individuals of L. macrochirus were collectedby angling at the small ponds in the central Japan in1999 (Kyoto population) and in 2000 (Sakai and Mat-suyama populations). We have obtained 17 individualsfrom Sounoike Pond in Sakai City, 6 individuals fromMizorogaike Pond in Kyoto City, and 8 individualsfrom Nishinagatoike Pond (Matsuyama-N) and 7 in-dividuals from Yamadaike Pond (Matsuyama-Y) inMatsuyama City. High molecular genomic DNA wasprepared from liver tissues as described [12]. Thegenomic DNA isolated from a Sakai individual wasdigested with excess amount of EcoRI restriction en-donuclease in the presence of 10% glycerol, and theDNA fragments were fractionated by electrophoresison 1.4% agarose gel. The agarose gel block con-taining the DNA fragments ranging from 500 bp to1000 bp was cut and the DNA fragments were ex-

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Figure 1. Nucleotide sequence of BamHI family satellite DNA in bluegill sunfish L. macrochirus. The sequence data appears in Gen-Bank/EMBL/DDBJ International Databases under the accession number AF214109. BamHI restriction site is underlined. Two primer DNA,BG16R2 and BG16F1, are indicated by arrows, which were used for PCR analysis (see Figure 2).

tracted by the Sephaglas BandPrep Kit (Pharmacia).They were cloned into the EcoRI site of pUC19 vec-tor. The recombinant plasmids having multicopy DNAsequences were probed by colony hybridization withthe digoxigenin-labeled total genomic DNA as de-scribed previously [13]. Nucleotide sequences of posi-tive clones were determined by a model ABI373 DNASequencer and Dye Terminator Cycle Sequencing Kit(Applied Biosystems) with synthetic DNA primers(Kurabo).

PCR amplification

PCR was performed in 25 µl of the reaction mixturescontaining 1 ng of genomic DNA with 0.4 µM DNAprimers BG16R2 and BG16F1, 0.2 mM each of dNTP,1× buffer supplied by the manufacturer (TOYOBO)and 0.5 unit of rTaq DNA polymerase (TOYOBO).Nucleotide sequences of BG16R2 and BG16F1 are in-

dicated in Figure 1. A series of incubation at 94 ◦C for30 sec/60 ◦C for 30 sec/72 ◦C for 2 min was repeatedfor 35 cycles.

Southern blot analysis

The genomic DNA was treated with BamHI restrictionendonuclease (TOYOBO). The DNA fragments wereelectrophoresed on 0.7% agarose gel and transferred toHybond-N membrane (Amersham). They were probedby pBG16 DNA that was labeled with digoxigenin byDIG DNA Labeling and Detection Kit (Boehringer).

Results and discussion

In the first instance, we have identified three pos-itive clones. The clone pBG4 contains an invertedrepeat region in 1 kb inserted DNA (GenBank ac-cession number AB046703), pBG6 contains (CA)25

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Figure 2. PCR analysis of BamHI family satellite DNA. (A) Ampli-fication with BG16R2 and BG16F1 primers. PCR products obtainedfrom two typical individuals are shown on 1% agarose gel. Elec-trophoretic patterns were classified into two types: one has anintense band of 0.9 kb (type S), and another has longer DNA bands(type L). Lane M, 100 bp ladder DNA size marker (New EnglandBiolab); lane 1, PCR product from type L individual; lane 2, PCRproduct from type S individual; lane 3, BamHI-digest of type L PCRproduct; lane 4, BamHI-digest of type S PCR product. An additional0.4 kb DNA band in lane 3 is indicated by asterisk. The signal ratioof upper (0.6 kb) and lower (0.3 kb) DNA bands in lane 3 is largerthan that in lane 4, probably due to incomplete digestion of theDNA fragments containing internal duplication. (B) Effect of thenumber of cycles in polymerase chain reaction. PCR amplificationwas performed for various cycles of incubation as indicated at thetop. DNA size marker of 100 bp ladder was loaded on the lane M.

repeat and (GTAA)5 repeat sequences in 1.1 kb in-serted DNA (AF234178), and pBG16 contains about0.9 kb inserted DNA that is a member of tandem re-peat sequence (AF214109). Nucleotide sequence ofthe inserted DNA of pBG16 is shown in Figure 1.

We have performed PCR amplification with DNAprimers BG16R2 and BG16F1, and found two dif-ferent electrophoretic patterns of the PCR productsdepending upon the template genomic DNA (Fig-ure 2A, lanes 1 and 2). One group of individuals

Figure 3. Southern blotting analysis of BamHI-digested genomicDNA. Genomic DNA from a type L individual (chosen from Kyotopopulation) was digested with various amount of BamHI restrictionendonuclease. Dots at the left of panel indicate the electrophoreticmobility of HindIII-digested λ DNA size marker. Genomic DNA(3 µg) was treated with 10 units (lane 1), 5 units (lane 2), 2.5 units(lane 3) or 1.25 units (lane 4) of BamHI for 15 min at 37 ◦C. Eachlane containes 1.5 µg of DNA fragments.

showed an intense DNA band on the agarose gel atthe monomer size (about 0.9 kb) as expected by thepBG16 sequence (hereafter, type S). Another group ofindividuals showed longer DNA bands (about 1.3 kband 1.7 kb) more intense than the 0.9 kb band (here-after, type L). BamHI digestion of the PCR productssuggested that the 1.3 kb fragment contained 0.4 kbsequence having BamHI site in addition to 0.9 kbsequence (Figure 2A, lane 3). Difference in PCRproducts observed here was tested by examining thenumbers of incubation cycles in PCR (Figure 2B).When the incubation was cycled for 25 times, fourDNA bands were observed (0.9 kb, 1.3 kb, 1.7 kb andlonger) in the type S individual, whereas two DNAbands (1.3 kb and 1.7 kb) were major in the type Lindividuals. The 0.9 kb band became more intensein the type S individual and the 1.3 kb band became

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more intense in the type L individual, according to theincrement of PCR cycles. The results indicated that0.9 kb DNA sequence was much less abundant in thegenome of the type L individuals than in that of thetype S individuals.

Southern blot hybridization analysis of the type Lindividual probed by pBG16 sequence has shown apossibility that there were two different monomericunits of the tandem repeat (Figure 3). In the lane 1of Figure 3, a strong signal was observed at 0.9 kb andfaint signals were also observed at 0.4 kb and 1.3 kb.The multiples of both 0.9 kb and 1.3 kb fragmentswere observed in the partially-digested DNA (lanes 3and 4 in Figure 3). The DNA signals with the lengthsof these multiples plus 0.4 kb were also observed (i.e.,the bands of 2.2 kb, 3.0 kb, etc.). The results ofPCR and Southern hybridization together may indi-cate that the repeating unit of 0.9 kb is divided intotwo subregions of 0.5 kb and 0.4 kb sequences. Thelatter, containing BamHI site, has duplicated tandem,making up the 1.3 kb unit.

L. macrochirus is one of the alien fishes in Japan.It was introduced from North America in 1960, andhas been widespread in Japan by artificial release [14].Characterization of local populations by molecularmarkers will be required for tracing the route of expan-sion of habitancy. We have examined the PCR prod-ucts of 38 individuals in four populations, and foundthat type S individuals appeared in Sakai population(9 out of 17), but in other populations, type L individ-uals are dominant (Kyoto, 5 out of 6; Matsuyama-Y,6 out of 7; Matsuyama-N, all of eight individuals).The occurrence of type L individuals in Sakai popu-lation is significantly lower than the other populations(p =6C0(8/17)6+6C1(9/17)(8/17)5 = 0.084 for Ky-oto; p =7C0(8/17)7+7C1(9/17)(8/17)6 = 0.045for Matsuyama-Y; p =8C0(8/17)8 = 0.002 forMatsuyama-N). The populations in Kyoto and Mat-suyama may be in the process of homogenizationtoward type L. There is a possibility, however, thatcertain group of individuals (type L) has been solelyintroduced to the ponds of Kyoto and Matsuyama. Itis more plausible that expanded repeating unit (1.3 kb)is being major in natural populations, or a shorter unit(0.9 kb) is being lost from the genome. As theorizedpreviously [15], the satellite DNA may have evolvedthrough the stepwise expansion of repeating unit byinternal sub-duplication, and it will be rapidly fixed inpopulation.

Acknowledgements

The authors are grateful to Mr. Masanori Yoshida forpreparation of plasmid DNA and the members of theStudy Group of Mizorogaike Aquatic Organisms forcollecting fish samples. This work was supported inpart by a fund for ‘Research for the Future’ Programfrom the Japan Society for the Promotion of Science(JSPS).

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