Cloning and characterization of a rat-specific repetitive DNA sequence

87 Gene, 45 (1986) 87-93

Elsevier

GENE 1658

Short Communications

Cloning and characterization of a rat-specific repetitive DNA sequence

(Recombinant DNA; transfection assay; hybridization probe; Southern blot; rat LINE family)

Scott K. Shore,* Lee T. Bacheler, ** J. Kimball de Riel, Louis R. Barrows** and Mark Lynch**

Fels Research Institute, Temple University School of Medicine, Temple University, Philadelphia, PA 19140 (U.S.A.) Tel. (215)-

221-4300

(Received July 19th, 1985)

(Revision received March 27th, 1986)

(Accepted April 17th, 1986)

SUMMARY

A 2.1-kb EcoRI fragment of rat DNA has been cloned and sequenced. This fragment contained a repetitive

element which was highly specific for rat DNA and widely dispersed throughout the rat genome. The repetitive

element is homologous to a sequence found in the 3’ end of the rat LINE family. Because of its high degree

of species specificity and its heterodisperse distribution, this sequence provided a useful marker for rat DNA

in DNA transfection experiments into mouse host cells.

INTRODUCTION

The genomes of most vertebrate species contain

repetitive DNA sequences, i.e., multiple copies of

more or less closely related DNA elements which

* To whom correspondence and reprint requests should be

addressed.

** Present addresses: (L.T.B.) E.I. DuPont de Nemours and Co.

Inc., Central Research and Development Department Experi-

mental Station, Wilmington, DE 19898 (U.S.A.) Tel. (302)772-

7096; (L.R.B.) Department of Pharmacology, George Washing-

ton University, Washington, DC 20037 (U.S.A.) Tel. (202)676-

2917; (M.L.) Department of Cell Biology, Smith Kline and

French Laboratories, Swedeland, PA 19479 (U.S.A.)

Tel. (215) 270-4938.

Abbreviations: bp, pase pair(s); EtdBr, ethidium bromide; kb,

kilobases or 1000 bp; LINE, long interspersed nt element; nt,

nucleotide(s); SINE, short interspersed nt element; SSC, 0.15 M

NaCl,0.015 M Na,.citrate,pH 7.6; SSPE, 0.18 M NaCl,O.Ol M

NaH,PO,, 1 mM EDTA, pH 7.4.

form families of cross-hybridizing sequences (Britten

and Kohne, 1968; Singer, 1982). These repetitive

DNA sequence families can vary in size from a small

number of members, 10-100, to over 100000 copies

per haploid genome. Two general classes of organi-

zation of these repetitive elements have been recog-

nized. Some repetitive DNA sequences are tandemly

reiterated, in long stretches of contiguous DNA.

Such reiterated sequences can often be resolved as

‘satellites’ distinct from the bulk of the chromosomal

DNA in isopycnic buoyant density gradients

(Szybalski, 1968), or as distinct bands after restric-

tion enzyme digestion (Philippsen et al., 1974).

Other repetitive DNA elements, several hundred

bases long (SINES) or several kb long (LINES) are

dispersed throughout the genomic DNA as unlinked

single copies of a DNA sequence (Jelinek and

Schmid, 1982; Singer and Skowronski, 1985).

The presence of such interspersed reiterated DNA

sequences has been used to follow the fate of donor

0378-l I19/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

88

DNA sequences in DNA transfection experiments. member of the AZu family (Schmid and Jelinek, 1982) For example, DNA samples from human tumors was used to demonstrate the presence of acquired have been used to transfect mouse NIH 3T3 cells human DNA sequences in the resulting trans- resulting in the induction of transformed foci (Peru- formants. cho et al., 1981; Shih et al., 1981). Hybridization We have studied the activation of cellular proto- with a cloned human repetitive DNA sequence, a oncogenes in rat tumors by a similar approach, and

A b

23.6 -

9.6-

6.6 -

4.3-

2.3-

2.0-

0.5-

6 C a b c

Fig. 1. Distribution and specificity of pRS21 sequences. (A) Rat DNA was digested with EcoRI, fractionated in an agarose gel, and

transferred to nitrocellulose filters (Southern, 1975). Lanes: (a) EtdBr-stained gel and (b) the corresponding Southern blot probed with

nick-translated insert of pRS21. (B) Hybridization of pRS21 insert to Southern blots of&‘coRI-digested samples of genomic DNA from

human, rabbit, mink CC1 cells, guinea pig, BHK hamster cells, wild mouse SC-l, mouse NIH 3T3 cells, and rat. (C) Southern blot of

pRS21 insert hybridized to EcoRI-digested DNAs. Lanes: (a) T9a1, a transformed NIH 3T3 cell line derived by transfection of DNA

from a rat kidney Sbrosarcoma induced by dimethylnitrosamine; (b) NIH 3T3 cells; (c) MNUBIIal, a transformed NIH 3T3 cell line

derived by transfection of DNA from a rat mammary carcinoma induced by N-methyl-nitrosourea. 10 ng of DNA was digested per lane

in all three panels. Hybridizations were performed under stringent conditions (42”C, 50% formamide, 5% SSPE) for 16-20 h. Blots

were washed to a final stringency of 0.1 x SSC at 65°C. Size markers (in kb) are 1 DNA digested with HindHI.

have sought to demonstrate that transformed foci of ization, which paralleled the general distribution of

mouse NIH 3T3 cells which arose had indeed EcoRI fragments in the digest (Fig. 1A). These

acquired rat DNA sequences. Because of the close results suggested that the pRS21 insert contained a

evolutionary relationship between rats and mice, it highly repeated DNA sequence which was widely

seemed unlikely that total rat repetitive DNA would dispersed in the rat genome. We observed no local-

be able to discriminate between rat and mouse ization of hybridization to the satellite bands

genomic DNA. We therefore isolated and cloned, for observed in the EtdBr staining pattern of EcoRI-cut

use as a hybridization probe, a copy of a rat repetitive rat DNA, suggesting that the pRS21 repetitive DNA

DNA sequence which was highly reiterated, widely sequence was distinct from the repeated DNA

interspersed in the rat genome, and specific for rat sequences which comprise these EcoRI satellite

DNA sequences. bands.

EXPERIMENTAL AND DISCUSSION

(a) Identification of a rat repetitive DNA clone

A 2 to 2.5kb agarose-gel fraction of EcoRI-cut Sprague-Dawley rat liver DNA was cloned into pBR328 (Maniatis et al., 1982). The resulting col- onies were screened with labeled rat DNA under conditions in which only repetitive DNA sequences would be expected to hybridize (Shen and Maniatis, 1980). Plasmids isolated from three positively hybridizing clones were labeled and hybridized to EcoRI-cut rat and mouse DNA. The clone which showed the strongest differential hybridization to rat DNA was designated pRS21 and further character- ized. Hybridization of the excised and radiolabeled insert of pRS2 1 to a Southern blot of EcoRI-digested total rat DNA revealed an intense smear of hybrid-

A

4

89

(b) Species specificity of pRS21 sequences

The representation of a pRS21-like DNA sequence in the genomes of a number of rodent species was examined by hybridization to blots of EcoRI-cut cellular DNA. Under stringent hybridization and washing conditions, the pRS21 probe hybridized significantly only to rat and hamster DNA (Fig. 1B). The hybridization of pRS21 to hamster DNA gave a less intense signal but showed the same heterodisperse distribution of related DNA fragments as did hybridization to the rat DNA. The pRS21 probe did not hybridize to the DNA of guinea pig, mink, rabbit, or human cells. Interestingly, the pRS21 probe also did not hybridize significantly to mouse DNA, even though these two species are closely related. This observation suggested to us that the pRS21 sequences of rat DNA might be useful for following the acquisition of rat DNA sequences by mouse NIH

5 ! I I

- 100 bp

Hinf I

Pvu II

Hpa II

Fig. 2. Restriction map of pRS21 and localization of the repetitive element. (A) Restriction map locations were determined from either

EtdBr-stained agarose gels or 3ZP-end-labeled fragments of restriction enzyme digests of pRS21. The fragments were then compared

with marker fragments of known M,. Bg, EglI; Bm, BumHI; E, EcoRI; Hf, Hi&I; Hp, HpaII; Ps, &I; Pv, PvuII; Xh, XhoI. (B) Solid

lines indicate fragments of pRS21 digests which hybridized to nick-translated high M, rat DNA on a Southern blot. Hybridization

conditions were as described in Fig. 1.

90

3T3 cells following transfection. The results of such an experiment are shown in Fig. 1C. Mouse NIH 3T3 DNA shows essentially no hybridization while T9al and MNUBIIal, two transformed mouse cell lines derived by transfection with rat tumor DNA, have acquired a distinct subset of rat DNA sequences. These results demonstrate that the repetitive DNA sequence contained in the pRS21 insert can be used as a species-specific marker for rat DNA sequences in DNA transfection experiments into mouse cells.

(c) Characterization of the repetitive DNA element

To localize the repetitive DNA element within the pRS21 sequence, a restriction map of the cellular DNA insert was made and Southern blots of restriction enzyme-digested plasmid DNA were hybridized with nick-translated total rat DNA (Fig. 2). The repetitive DNA element spans a 39%bp HpaII fragment in the lefthand portion of the DNA insert, between nt 360 and 758. Filter hybridization of pRS21 to dilutions of total rat DNA yielded an estimate of approx. lo5 copies of the repetitive element per haploid genome (not shown).

(d) Sequence analysis

The sequence of the entire 2127-bp insert in pRS21 was determined by the method of Maxam and Gilbert (1980) (Fig. 3). Computer-aided analysis of the sequence of the pRS21 insert was carried out on a VAX-11/780 computer (Digital Equipment Corp.) at the Institute for Cancer Research in Philadelphia, using the ICR sequence program package compiled and integrated by P. Young, H. Gael, and J. Lipton. Comparison with published DNA sequences indicates that the repetitive sequence in pRS21 consists of the complement of the 3’-terminal 600 bp of the rat LINE family, a long interspersed element of approx. 6.7 kb related to the evolutionarily conserved Ll family (Burton et al., 1986). A full-length copy of one member of the rat LINE family (LINE 3) has been sequenced (D’Ambrosio et al., 1986). The complement of the pRS21 insert over nt 896-297 is homologous to nt 6457-7058 of the LINE 3 sequence. The overall homology is 91%. The LINE element in pRS21 is adjacent to a run of short repeat DNA with the

sequence (GTT),, and includes a region of C-rich DNA (nt 370-416). Blot analysis of rat DNA with probes from the LINE 3 sequence have suggested that most copies of this element are full-length (D’Ambrosio et al., 1986). On the other hand, chal- lenge of the Los Alamos rodent database with the sequence from pRS21 indicates that several truncated copies of the 3’ terminus of the LINE sequence are located near known rat coding genes, including a sequence flanking the 3 ’ end of a rat serum albumin gene (Sargent, 1981), within intron A of the rat y- casein gene (Yu-Lee and Rosen, 1983) and within intron D of the rat prolactin gene (Gubbins et al., 1980; Cooke and Baxter, 1982). Additionally, the sequence published by Scarpulla (1985) of a 1.3-kb truncated rat LINE element also shares the same approx. 3’ end as the LINE 3 repeat. These observa- tions suggest that truncated copies of the 3’ end of the LINE repeat may be fairly abundant in the rat genome as has been reported for the mouse (Fanning, 1983; Voliva et al., 1983).

We also found significant but weaker homology between a short section of the pRS21 repeat and

members of the mouse R repeat (Gebhard et al., 1982; Wilson and Storb, 1983). The observed homology was around 70% over a span of 80 bp, presumably not enough to permit cross-recognition under normal conditions of hybridization. This relatively weak homology between the pRS21 insert repeat and mouse sequences contrasts with the much stronger homology (85-95% ; J.K.d.R., unpublished observation) between elements of the murine Ll repeat (Jahn et al., 1980; Brown and Piechaczyk, 1983; Fanning et al., 1983; Mason et al., 1983; Voli- va et al., 1983; Martin et al., 1984; Meunier-Rotival and Bernardi, 1984) and sequences present in both LINE 3 (D’Ambrosio et al., 1986) and the truncated 1.3-kb LINE element described by Scarpulla (1985). Rat LINE elements longer than 850 bp are thus much more likely to cross-hybridize with mouse LINE sequences. Because sequences at the 3’ end of the LINE repeat are relatively species-specific and may be more widely distributed in the rat genome than other portions of the LINE repeat, pRS21 or other short 3’ rat LINE elements are preferable candidates for discriminating rat from mouse DNA.

91

A SAATTCATTA ATTAAAATGA AAATGCTTTA TAAATGCAAG CCTAAGTATT GACTCAAAGG TTTTGACTTG TGAAATTCAT GTTCAAAAGA ATGACCAGAT

ATGTAAATTT AAGAATGTTG CTTGCTGACT CTTCCATGAA GCCAGATTTC TATGTGTAAT ATGTAAAATA ATCAAGAAGT ATTTCAACTT AATACCTGTG

AAAGAAAAWI AAAAAGCCAC GTTGGACAGA MAGGTTGAA CTAAAATTCA GCAGAGGAGT TTGTTGTTGT TGTTGTTGTT GTTGTTGTTG TTGTTGTTAT

TTTTAATTAA CTTWIGTATT TCTTATATAC ATTTCGAGTG TTATTCCCTT TCCCGGTTTC CGGGCAAACA TCCCCCTAAT CCCTCCCCCT TCCCCTCCCC

TCCCCATCCT CCCCCCATTG CCGCTCTCCC CCCAACAATC TTGTTCACTG GGGGTTCAGT CTTAGCAGGA CCCAGGGCTT CCCCTTCTAC TGGTGATCTT 500

ATTAGGATAT TCATTGCTAC CTATGGGGTC AGAGTCAATG GTCAGTCCAT GTATAGTCTT TAGGTAGTTG CTTAGTCCCT GGAAGCTCTG GTTGCTTGGC

ATTGTTGTAC ATATGGGGTC TCGAGCCCCT TCAAGCTCTT CCAGTTCTTT CTCTGATTCC TTCAACGGGG ATCCTATTCT CAGTTCAGTG GTTTGCTGCT

6GCATTCGCC TCTGTATTTG CTGTATTCTG GCTGTGTCTC TCAGGAGffiA TCTATATCCG GCTCCTGTCG GCCTGCCCTT CTTTGCTTCA TCCATCTCGT

CTAATTGGAT MCTGTATGT GTATGGGCCA CATGTGGGGC AGGCTCTGAA TGGGTGTTCC TTCTGTGTCT GTTTTAATCT TTGCCTCTCT ATTCCCCAGC

AGAGIIAGTTT TAAGCATAGA ACATGCCTCC TGAACTCCTT 66AATTAGAA AAGGCACACA TCCAAGGCAC ATCACTGGGC TTGGCCATTA ACTGTTAAAT 1000

GTCCTATGAA AM6ATAT6A TCTWATGT GGTGGCTTTC TTATGGGTGA GGCAATTCCT 6AATGTGGCT AACATCCCCA AACCATTTGT G6AACAGCCC

TGCCACAGCT GACAAACGTT ACCTCTATTA TTGAGCAGCA CATCCCAACC GTCTACACTG TTACCCACTT GTTATGTAGA AAATGAATGA ACTAACATCA

ACTCACATTA GTTTCTAAAT 6ASATT6TCT TGAGAAAATA GATAGATAAG ATGAGGATGA 'LAATATTCAA TAACTATGTG ACATGCAGGA ACAATTTCCC

CASATCAWZ CTUSACTCAA TAATTAAAGA TAGTCAAAAC TCCAGCTACT GGGTGGTGCT GGGTCATTCT AAGTTGATGG CAAACAAAAG TCTTTGTATC

ATCTGA66CT TCTCTGAGCA 6A6ATG6ATT AAAAAAGAAA TAATAAGGCA TCTTCTTGAT CTTCTTGGAA TATGTAATTT GAAGAAGTTA AAATATCAAA 1500

AGTTGACTTC TGTGTCTGTA TTTTGTATAA TAGATATTGT GATCATTTCC AAAGAGACCA TGGGGGTCTG GCAAATCCTA GTAAAAGTAA TATATTAGAA

TCCTATTCA6 TTATGCATCA GCAACTCTM TTTTCTTTCT ACTTTTTCCT GAGTGAATTA GGGAAGACAC AACTAGGCCA TGTTGGCCTG CCCAGATGCT

CTAAGCATTG GTGCTCCTGC ATTGAGATTT TTTGTTCTCC CTGTGTATGC TCCTGCCTCG TTTTTATTCC TTTCTTTTGC TTTCTTGCAT TTCT6AAAGT

AGCTAGCTGC ACT6CTT6GT TTCATAATAT GTCATACCAA TCAAAAGAAA CCAAACCCAG CATTATTTGG AGCCCTTAAT GCCACTGTTA TTCCCTCTTT

64A6GTCAGA TAGTCAATGA TAGGTCTCCT GTGGGGTTTT GGACCATMT CACAGGGGTT AGGTCTGTTG AGCACCTCTT GTCCACAATT AATAGAGTGC 2000

TGCAGTGGTC ATAGCTACAG CTTGGTMTC GGTTAAAAAC CAGGGAGGGC AGACTGTCTA lTGGGG6TTT CAGTGTTATT CCACACAGTT TAAMGTGAT

GTTAAAATCA AACCACTAGG AGAATTC

6 E W-WV ~HP AC I

Av E

I I I I I I

HfHf Hf Hf Hf Hf

I I

200 bp

Fig. 3. Nucleotide sequence of pRS21 insert. (A) The nt sequence of the entire pRS21 EcoRI insert. (B) Strategy used to derive the nt

sequences for both strands. AC, AccI; Av, AvaII; Bm, BamHI; E, EcoRI; Hf, Hinfl; Hp, HpaII.

92

ACKNOWLEDGEMENTS

Supported in part by grants from the National Institutes of Health CA-19519 and BRSG 307- RR05417 to L.T.B., grant l-899 from the March of Dimes Birth Defects Foundation to J.K.d.R., and grants from the National Foundation for Cancer Research and the Samuel S. Fels Fund of Philadelphia to Peter N. Magee in whose laboratory S.K.S. is and L.R.B. was a postdoctoral fellow. S.K.S. and M.L. were supported by a training grant from the National Cancer Institute, CA-09214. Cathy Bruno provided expert technical assistance. We thank T. Sargent for providing a copy of the sequence of the rat repetitive DNA element flanking the rat serum albumin gene.

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Communicated by S.T. Case.

Documents

Cloning and characterization of a rat-specific repetitive DNA sequence