Cloning and characterization of a rat-specific repetitive DNA sequence

87

Gene, 45 (1986) 87-93 Elsevier GENE

1658

Short Communications Cloning and characterization DNA;

(Recombinant

of a rat-specific repetitive DNA sequence

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.) 221-4300 (Received

July 19th, 1985)

(Revision

received

(Accepted

March

Tel. (215)-

27th, 1986)

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.

form families of cross-hybridizing

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:

Inc., Central mental

(L.T.B.) E.I. DuPont

de Nemours

and Co.

and Development

Department

Experi-

Research

Station,

Wilmington,

7096; (L.R.B.) Department ton University, 2917;

(M.L.)

French

DE 19898 (U.S.A.) of Pharmacology,

Washington, Department

Laboratories,

Tel. (302)772-

George

DC 20037 (U.S.A.) of Cell Biology, Swedeland,

PA

Washing-

Tel. (202)676-

Smith

Kline

19479

and

(U.S.A.)

Tel. (215) 270-4938. Abbreviations: kilobases

bp, pase pair(s);

or 1000 bp; LINE,

nucleotide(s);

EtdBr,

SINE, short interspersed

NaCl,0.015

M Na,.citrate,pH

NaH,PO,,

1 mM EDTA,

0378-l I19/86/$03.50

0

ethidium

long interspersed

bromide;

kb,

nt element;

nt,

nt element;

SSC, 0.15 M

7.6; SSPE, 0.18 M NaCl,O.Ol

M

pH 7.4.

1986 Elsevier

Science Publishers

B.V. (Biomedical

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 organization of these repetitive elements have been recognized. 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 restriction 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 Division)

88

DNA sequences in DNA transfection experiments. For example, DNA samples from human tumors have been used to transfect mouse NIH 3T3 cells resulting in the induction of transformed foci (Perucho et al., 1981; Shih et al., 1981). Hybridization with a cloned human repetitive DNA sequence, a

member of the AZufamily (Schmid and Jelinek, 1982) was used to demonstrate the presence of acquired human DNA sequences in the resulting transformants. We have studied the activation of cellular protooncogenes in rat tumors by a similar approach, and

A

C

6 b

a

23.6

b

c

-

9.66.6 -

4.3-

2.32.0-

0.5-

Fig. 1. Distribution transferred

and specificity

to nitrocellulose

of pRS21

filters (Southern,

sequences.

nick-translated

insert of pRS21. (B) Hybridization

human,

mink CC1 cells, guinea pig, BHK hamster

rabbit,

pRS21 insert hybridized

to EcoRI-digested

from a rat kidney Sbrosarcoma derived by transfection in all three panels. were washed

induced

DNAs.

to a final stringency

of pRS21 insert to Southern

were performed

fractionated

blots of&‘coRI-digested

in an agarose

Southern

samples

NIH 3T3 cell line derived

(b) NIH 3T3 cells; (c) MNUBIIal, induced

under stringent

by N-methyl-nitrosourea.

of genomic

conditions

Size markers

(42”C,

(in kb) are 1 DNA digested

blot of of DNA

NIH 3T3 cell line

10 ng of DNA was digested

50% formamide,

with

DNA from

by transfection

a transformed

gel, and

blot probed

cells, wild mouse SC-l, mouse NIH 3T3 cells, and rat. (C) Southern

carcinoma

of 0.1 x SSC at 65°C.

with EcoRI,

gel and (b) the corresponding

Lanes: (a) T9a1, a transformed

by dimethylnitrosamine;

of DNA from a rat mammary

Hybridizations

(A) Rat DNA was digested

1975). Lanes: (a) EtdBr-stained

per lane

5% SSPE) for 16-20 h. Blots with HindHI.

89

ization, which paralleled the general distribution of EcoRI fragments in the digest (Fig. 1A). These results suggested that the pRS21 insert contained a highly repeated DNA sequence which was widely dispersed in the rat genome. We observed no localization of hybridization to the satellite bands observed in the EtdBr staining pattern of EcoRI-cut rat DNA, suggesting that the pRS21 repetitive DNA sequence was distinct from the repeated DNA sequences which comprise these EcoRI satellite bands.

have sought to demonstrate that transformed foci of mouse NIH 3T3 cells which arose had indeed acquired rat DNA sequences. Because of the close evolutionary relationship between rats and mice, it seemed unlikely that total rat repetitive DNA would be able to discriminate between rat and mouse genomic DNA. We therefore isolated and cloned, for use as a hybridization probe, a copy of a rat repetitive DNA sequence which was highly reiterated, widely interspersed in the rat genome, and specific for rat DNA sequences.

(b) Species specificity EXPERIMENTAL

of pRS21 sequences

AND DISCUSSION

(a) Identification

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

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 colonies 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 characterized. 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 hybridA

4

5 I

!

I

-

100 bp

Hinf I Pvu

II

Hpa II

Fig. 2. Restriction

map of pRS21 and localization

EtdBr-stained

agarose

with marker

fragments

lines indicate conditions

fragments

gels or 3ZP-end-labeled

of the repetitive

fragments

of known M,. Bg, EglI; Bm, BumHI; of pRS21

were as described

digests

in Fig. 1.

element. (A) Restriction

of restriction

which hybridized

E, EcoRI;

enzyme

map locations

digests of pRS21.

Hf, Hi&I; Hp, HpaII;

to nick-translated

were determined

The fragments

Ps, &I;

from either

were then compared

Pv, PvuII; Xh, XhoI. (B) Solid

high M, rat DNA on a Southern

blot. Hybridization

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, challenge 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 ycasein 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 observations 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; Voliva 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

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

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

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

GGTTAAAAAC

CAGGGAGGGC

AGACTGTCTA

lTGGGG6TTT

CAGTGTTATT

CCACACAGTT

TAAMGTGAT

TGCAGTGGTC

ATAGCTACAG

CTTGGTMTC

GTTAAAATCA

AACCACTAGG

AGAATTC

500

1000

1500

2000

6 E

W-WV

I I

HfHf

I

AC

~HP

Av

I

I

I

I

Hf

Hf

Hf

Hf

E I

I

200 bp Fig. 3. Nucleotide sequences

sequence

for both strands.

of pRS21 insert. (A) The nt sequence AC, AccI; Av, AvaII;

Bm, BamHI;

of the entire pRS21 EcoRI insert. (B) Strategy E, EcoRI;

Hf, Hinfl; Hp, HpaII.

used to derive the nt

92

ACKNOWLEDGEMENTS

Jelinek,

W.R. and Schmid,

ryotic

Supported in part by grants from the National Institutes of Health CA-19519 and BRSG 307RR05417 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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