Proc. NatL Acad. Sci. USAVol. 79, pp. 6123-6127, October 1982Biochemistry

Mouse EcoRi satellite DNA contains a sequence homologous to thelong terminal repeat of the intracisternal A particle gene

(DNA hybridization/repetitive DNA/retrovirus)

ANNE BROWN AND Ru CHIH C. HUANG*The Johns Hopkins University, Baltimore, Maryland 21218

Communicated by James Bonner, June 16, 1982

ABSTRACT EcoRI restriction endonuclease fragments fromthe mouse 1,350-base-pair EcoRI satellite sequence were clonedin pBR322 plasmid. One of these fragments, SAT-1, was hybrid-ized to various restriction fragments from cloned mouse intracis-ternal type A particle (LP)' genes and to mouse total genomicDNA. Hybridization occurred between the SAT-1 sequence andeach of the JAP clones. The common region of hybridization ineach of the clones was the long terminal repeat region of the UAPgenes.

A moderately repeated 1,350-base-pair (bp) satellite DNA se-quence has been identified in mouse BALB/c, F9, 3T3, S107,and Friend cell lines after cleavage of total genomic DNA withEcoRI restriction enzyme (1-3). The satellite was found to makeup 0.5-3.0% of the genome with a repeat frequency of approx-imately 104 in a Friend erythroleukemia cell line. The satellitewas shown to consist of related but nonidentical sequences,-which are arranged.in a dispersed fashion in the genome (2).

Recently, we (4-6) and others (7, 8) have done extensive stud-ies on the structural organization of the genes coding for intra-cisternal A particle (IAP) RNA. IAPs are noninfectious retro-virus-like particles that are seen within the endoplasmicreticulum in several mouse tumor cell. lines and in normalpreimplantation mouse embryos (9-12). There are approxi-mately 1,000 IAP genes dispersed throughout the genome,where they. make up over 0.2% of the total DNA (4, 13). In-dividual IAP genes show a divergence of sequences and a di-versity ofsequence arrangements. We have previously reportedthat IAP genes, like those coding for type B and type C retro-viruses, contain long terminal repeats (LTRs) flanking the IAPstructural gene (5), and also that both the 5' and the 3' ends ofIAP RNA are encoded within the LTR.(6).We report here that sequence homology exists between sat-

ellite sequences and a portion of the IAP genome. The homol-ogy is confined to the LTR region, the region to 'which the ter-mini of IAP RNA have been mapped (6).

MATERIALS AND METHODSPreparation ofTissue, Plasmid, and Phage DNAs. DNA was

isolated from BALB/c mouse liver, kidney, spleen, and brainby the procedure of Marmur (14). DNA was isolated from ter-atoma F9 (15), MOPC 315, and parietal yolk sac carcinoma celllines (16) by the procedure of Moshier and Huang as describedin ref. 4. Derivatives ofACH4A bacteriophage containing IAPinserts were propagated in liquid culture and DNA was pre-pared according to Enquist et aL (17). Individual EcoRI restric-tion fragments from IAP genes were previously subcloned inpBR322 plasmid (4). The recombinant plasmids in Escherichia

coli strain HB101 cells were grown in L broth and the DNA wasprepared by a modification of the procedure of Birnboim andDoly (18) or as described in ref. 19.

Restriction enzymes were purchased from Bethesda Re-search Laboratories and assay' conditions were from the sup-plier's.manual. DNA restriction fragments were fractionated byelectrophoresis in agarose gels in 0.1 M Tris borate, pH 8.3/0.002 M Na2EDTA.

Preparation of Probes for Nick-Translation. One milligramof recombinant pBR322 plasmid was digested with the appro-priate restriction enzyme. The digest was applied to a 20.0 x20.0 cm agarose gel in a well 20.0 X 1.0 X 0.3 cm and electro-phoresed in 0.1 M Tris borate, pH 8.3/0.002 M Na2EDTA at200 V. The desired DNA band was visualized by ultraviolet lightafter ethidium bromide staining and was recovered by elec-troelution. The DNA was applied to a DEAE-cellulose column(1.0 x 0.4 cm), washed with 0.05 M Tris'HCI, pH 7.5/0.1 MNaCI, and eluted with-3.0 ml of 1.0 M NaCI/0.05 M Tris-HCI,pH 7.5. Nick-translation of the eluted DNA fragments was per-formed according to Rigby et aL (20). The final specific activitywas 0.5-1.0 X 108 cpm//zg of DNA.

Hybridization. DNA restriction fragments on agarose gelswere transferred to a nitrocellulose filter by the method ofSouthern (21). Filters were incubated in hybridization buffer(0.45 M NaCI/0.045 M sodium citrate/0. 1% NaDodSO4/0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02 M potassiumphosphate, pH 6.8/100 Mzg of yeast tRNA per ml) for 24 hr at67TC. Filters were incubated with the radioactive probe (0.5X 106 cpm/ml) in hybridization buffer for 24 hr at 670C. Theywere washed twice in 0.45 M NaCI/0.045 M sodium citrate/0.1% NaDodSO4/0.02 M potassium phosphate, pH 6.8, for 30min at 67TC. The filters were washed twice under stringentconditions (0.015 M NaCl/0.0015 M sodium citrate/0. 1%NaDodSO4/0.02 M potassium phosphate, pH 6.8, for 30 minat 67C). Filters were dried and exposed at -70°C with Du PontCronex High Plus intensifying screens.

Preparation of the 1,350-bp Satellite Fragment. Six hun-dred micrograms of mouse strain BALB/c liver DNA was di-gested with EcoRI and fractionated by electrophoresis on a 1.0%agarose gel 20.0 x 43.0 x 1.0 cm, well size 18.0 X 1.0 X 0.3cm, as described above. The gel was stained with ethidium bro-mide, the satellite DNA band was excised, and the DNA wasremoved from the gel by electroelution. The DNA was appliedto a DEAE-cellulose column, washed, eluted, and precipitatedas described above. Approximately 2.0 ,Ag of material was re-covered, inserted into pBR322 at the EcoRI site, and propa-gated in E. coli strain HB101 (22-24). Fifty-milliliter culturesof tetracycline-resistant transformant colonies were grown. The

Abbreviations: bp, base pair; IAP, intracisternal A particle; LTR, longterminal repeat.* To whom reprint requests should be addressed.

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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bacteria were harvested, the plasmid DNA was prepared anddigested with EcoRI, and the digest was subjected to electro-phoresis in an agarose gel. Colonies containing plasmid DNAwith a 1,350-bp EcoRI insert band were retained for furtherstudy.

RESULTSHybridization of TAP DNA-to EcoRI-Digested Mouse DNA.

DNA from various mouse tissues was digested with EcoRI re-

striction endonuclease. The resulting restriction fragmentswere separated by electrophoresis on long agarose gels andqtninprl with Pthifliiim hrnmidp Wp vnnlictpntlv nhcarvprd A

Strongly- r-i-uo-r-es-cmg- Dana at a posinoh corresponding to ap-

proximately 1,350 bp (Fig. 1). This band was also observed inEcoRI-digested DNA from tissue types in addition to thoseshown in Fig. 1, including mouse brain, kidney, spleen, andteratoma PYS-1. We hybridized the total EcoRI-digested mouseliver DNA with 32P-labeled IAP DNA probes derived fromthree different regions of the IAP genome. The three IAPprobes are indicated in Fig. 2 A and B in the areas which are

underscored by black bars. The 1,250-bp fragment (Fig. 2B)occurs in six of seven cloned IAP genes (4); it is adjacent to butdoes not include the LTR. When the 1,250-bp fragment was

hybridized to Southern blots of EcoRI-digested mouse liverDNA, several strongly hybridizing bands appeared (Fig. 3A),but none corresponded to the 1,350-bp satellite band.The 800-bp probe (Fig. 2A) is present in all seven IAP clones.

It is found near the LTR (4). Hybridization of this fragment toa Southern blot of EcoRI-digested mouse liver DNA is shownin Fig. 3, lane B. Several discrete bands hybridized to the probebut none of these corresponded to the 1,350-bp satellite band.

The third IAP DNA probe, derived from a Pst I/EcoRT-digestion of 19B (Fig. 2B), contains approximately 400 nucleo-tides that are part of the LTR sequence (unpublished DNA se-

quence analysis). We reported earlier that most of the LTR se-

quence in the 400-bp fragment hybridized to the 5' end of IAPRNA (6). Fig. 3, lane C, shows the results of hybridization ofthe 400-bp fragment to a Southern blot of EcoRI-digestedmouse liver DNA. There was a strong background of hybrid-ization to the many dispersed TAP genes containing LTR se-

quences with a diversity of cellular flanking sequences. There

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FIG. 1. Ethidium bromide-stained gel of EcoRT restriction frag-ments of DNA from mouse strains BALB/c and Swiss Webster. LaneA, solid myeloma. MOPC 315 tumor grown in BALB/c mice; lane B,liver from adult BALB/c mice; lane C, teratoma F9 tissue culture cells;lane D, Swiss Webster neonatal mice. DNA (5 Mug) was digested withDEcoRT and the resulting fragments were separated by electrophoresisin a 1% agarose gel (20 x 43 cm, horizontal). The arrow designates theposition of satellite DNA sequences, approximately 1,350 bp in size,after digestion with EcoRI. AC1857-DNA digested with HindE wasused to estimate molecular weights as shown in bp in the extreme leftlane.

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FIG. 2. Restriction enzyme maps of two IAP-containing ACH4A recombinant DNA clones, A81 (A) and A19 (B). For each clone, the first line(1) shows the nomenclature and sizes in kbp of the mouse-derived EeoRl restriction fragments in each of the recombinant-TAP ACH4A clones. The.second line (2) shows a detailed-restriction enzyme map of each clone. The areas underscored by black bars designate the fragments that were usedas hybridization probes and their sizes in kbp. The third line (3) is a simple schematic of the complete TAP gene bounded by two terminal repeats(boxes) containing sequences from both the 3' end and the 5' end of the TAP RNA (fourth line, B). The TAP gene boundaries have been determinedby electron microscopy (5) and the repeat has been characterized by DNA sequence analysis (unpublished data). The sequence homology betweenthe IAP genes of 81 and 19 is described elsewhere (6). S1 nuclease mapping of RNA termini is also described in ref. 6.

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Proc. Nati Acad. Sci. USA 79 (1982) 6125

were, additionally, several discrete signals which were visibleabove the background; the strongest of these was found at aposition corresponding to the i,350-bp EcoRI satellite band inFig. 1. These results suggested that the 1,350-bp EcoRI satelliteDNA shares sequence homology with the LTR sequence, butnot with internal adjacent sequences of IAP genes.

Hybridization of Cloned Satellite DNA (SAT-1) to DigestedMouse Genomic DNA. In order to define the relationship ofthe 1,350-bp satellite band to IAP DNA sequences, we clonedpurified satellite fragments in pBR322 as described in Materialsand Methods. One of the clones was designated pSAT-1. TheDNA insert (SAT-1) was isolated, labeled with 32p, and used asa probe in the hybridization study below.Mouse liver DNA was digested' with Xba I, Sst I, Pst I, or

EcoRl. The hybridization pattern of the SAT-1 fragment to di-gested total mouse DNA is shown in Fig. 4. The most prominentsignal was seen at the satellite band of EcoRI-digested DNAshown in lane 4. This information indicates that the SAT-I frag-ment contains sequences that are repeated in the satellite band.There were several other bands that hybridized well over back-ground, an indication that sequences homologous to the SAT-1sequence are found in other repeated arrangements in the ge-nome. The background hybridization consistently observed waspossibly due to hybridization ofthe SAT-1 fragment to the manydispersed LTR-containing IAP genes.

Hybridization of the SAT-i Fragment with Digested DNAfrom Individual LAP Clones. Seven ACH4A recombinant clonescontaining IAP genes have been described (4). These clones

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were characterized by heteroduplex mapping and were foundto contain some common gene regions with nonhomologousflanking cellular regions. P-Labeled SAT-1 insert or 400-bpLTR-containing DNA fragment from i9B were hybridized toSouthern blots of EcoRI-digested IAP clones. The results areshown in Fig. 5. The 400-bp and the SAT-1 probes hybridizedto the same fragments (Fig. 5 B and C), and each of the hy-bridizing fragments contains a LTR sequence (ref. 6 and un-published data). Some ofthe bands resulting from A81, A62, andA19 digestion, such as 81B, 62A, and 19A (Fig. 2), showed asomewhat weaker signal than the other bands. We have foundthat the LTR in 62 has undergone base changes, deletions, andinsertions when compared to the LTRs of 19B and 19D (Fig.2), which are identical (unpublished sequence data).

Position of the Region Within the LTR of the TAP Gene thatShares Sequence Homology with the Satellite DNA SAT-1. Todefine the region of homology between SAT-1 and the LTRsequence, the LTR region was digested with several differentrestriction enzymes and the resulting fragments were hybrid-ized to the 3P-labeled satellite DNA SAT-1 probe. A detailedmap of the restriction sites is shown in Fig. 6. We observedhybridization ofthe SAT-i probe to the 400-bp LTR-containingfragment (Pst I/EcoRI digested) from 19B (Fig. 7A). Becausethe 400-bp fragment contains approximately 100 bp ofa cellularflanking sequence (Fig. 6B, region C2) outside the LTR region,the possibility did exist that the homology between the 400-bpfragment and SAT-i was with the flanking region and not withthe LTR region. To distinguish between the two possibilities,we compared the hybridization of SAT-1 to DNA fragments

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FIG. 3. Hybridization analysis of strain BALB/c mouse liverDNAdigested with EcoRT. Eachlane contained 5 Mg of total liver DNA from

BALB/c mice. After electrophoresis (1% agarose, 20 x 43 cm) andDNA transfer, the nitrocellulose filter was incubated with 32P-labeled,nick-translated, denatured DNA probes from different regions ofcloned IAP genes as diagrammed in Fig. 2: 1,250 bp from the EcoRIsite to the BamHI site of 19B, IAP internal sequence (lane A), 800 bpfrom theBamHI to theHindHI site of 81A, TAP internal sequence (laneB), and 400 bp from the Pst I site to the EcoRT site, 19B LTR sequenceof the IAP gene (lane C). The arrow indicates the position of the EcoRIsatellite DNA band' as in Fig. 1. Specific activity of the probes andhybridization conditions are described in the text.

FIG. 4. Hybridization of mouse BALB/c liver DNA restriction en-donuclease fragments to cloned satellite DNA probe. DNA (5 pg) wasdigested with Xba I (lane 1), Pst I (lane 2), Sst I (lane 3), orEcoRI (lane4). The resulting fragments were separated by electrophoresis on a 1%agarose gel, transferred to nitrocellulose filters, and hybridized to a1,350-bp cloned satellite DNA fragment. The. arrow indicates the po-sition of the satellite DNA band as in Fig. 1.

Biochemistry: Brown and Huang

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6126 Biochemistry: Brown and Huang

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FIG. 5. Hybridization analysis of EcoRI re-striction fragments of cloned IAP genes. One mi-crogram of DNA from recombinant bacterio-phage A IAP clones (4) was digested with EcoRI.The restriction fragments were subjected to elec-trophoresis in two 1% agarose gels, stained withethidium bromide at 1 pg/ml, and photographed(A). DNA fragments were transferred to nitro-cellulose filters. Hybridization was with the nick-translated 400-bp l9B Pst I/EcoRI LTR sequence

(B) or with nick-translated 1,350-bp cloned SAT-1 DNA (C).

isolated from 19D and i9B. The only homologous sequences

shared by 19D and i9B are in the LTR region (6). Therefore,hybridization of the SAT-1 probe to both 19B and 19D wouldbe the result ofhomology with the LTR and not with the flank-ing cellular regions. For this study pi9D, the pBR322 recom-binant'plasmid containing the mouse 1,400-bp insert, was di-gested with EcoRI and the 1,400-bp insert (19D) was purifiedfrom an agarose gel. The insert was digested with several dif-ferent restriction enzymes and the restriction fragments were

separated on a gel, transferred to nitrocellulose, and hybridizedto SAT-1 probe.

Digestion of the pi9D insert with BstNI generated four re-

striction fragments (Fig. 6). There is a BstNI restriction siteapproximately 100 bp to the left of the Pst I site. Digestion withthe enzyme generated a 320-bp fragment containing the se-

quence flanking the LTR. A 420-bp fragment containing LTRsequences was also generated' by digestion with BstNI.We observed strong hybridization of the SAT-i1fragment to

the 420-bp fragment containing the LTR, but we did not detecthybridization to the 320-bp fragment immediately adjacent to

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FIG. 6., Restriction maps of LTR-containing regions of two LATgenes. EcoRI restriction fragments B and D of A19- (Fig. 2) were sub-cloned in pBR322, and the B and D fragments from the recombinantplasmids were digested with various restriction enzymes. For each sub-clone, the top line shows the restriction sites and the lines beneathshow the sizes of the restriction fragments,obtained with each enzymeplus EcoRI. The heavy line delineates the boundaries of the LTR asdetermined by electron microscopy. C1 and C2 refer to cellular -se-quences flanking the LTR and- the dashed line refers to the internal1AP gene region. 19B and 19D contain the two ends of an IAP- gene.

the LTR or to the 600-bp BstNI fragment proximal to the 320-bp fragment (Fig. 7B). This result indicates that there are no

homologous sequences between SAT-i and the cellular flankingDNA in 19D and supports the conclusion that it is only the LTRregion in the 400-bp Pst I/EcoRI fragment of i9B that hybrid-ized to the SAT-1 satellite DNA.To position the region of homology within the LTR, the

1,400-bp 19D insert was digested with Pst I to yield an 1,100-bpfragment and a 300-bp fragment, and the SAT-1 probe was hy-bridized to a Southern blot of these fragments. There was little,if any, hybridization between SAT-1 DNA and the 1,100-bpfragment (Fig. 7C). There was strong hybridization between the300-bp fragment and SAT-1 DNA. The 1,100-bp fragment con-

tains about 100 bp of the LTR and- a cellular flanking region(C1, in Fig. 6) (5). The 300-bp Pst I/EcoRI fragment contains

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FIG. 7. Hybridization analysis of the LTR DNA in 19D and 19B.Lanes labeled 1 show the ethidium bromide-stained restriction frag-ments on an agarose gel. Lanes labeled 2 show the autoradiograms ofthe gels after Southern blotting and, hybridization to 32P-labeledSAT-1 DNA. (A) The,400-bp DNA fragment purified from 19B (Pst Ito EcoRI). (B) The 1,400-bp EcoRI mouse insert, 19D, digested withBstNI. (C) The 1,400-bp EcoRI mouse insert, 19D, digested with Pst I.(D) The 400-bp DNA fragment purified from 19B (Pst I to EcoRI) di-gested with Hinfl. pBR322 HinfI markers are included. Undigested19D is shown in lane 2 of C (top band). In D, the 150-bp fragment isbarely visible in the stained gel. An enlarged diagram of the restrictionsites in 19B and in 19D is shown in Fig. 6.

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Proc. Nad Acad. Sci. USA 79 (1982)

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Proc. Nati. Acad. Sci. USA 79 (1982) 6127

only LTR sequences. We therefore conclude that the homologybetween SAT-1 and the LTR extends from just left of the PstI site to near the EcoRI site within the LTR region. To deter-mine the extent of this homology, the 400-bp Pst I/EcoRI frag-ment from 19B was digested with Hinfl, yielding a 250-bp anda 150-bp fragment (Fig. 6). When these fragments were hy-bridized to SAT-1 probe, we observed a positive signal at 250bp and a somewhat weaker signal at around 150 bp (Fig. 7D).The 150-bp HinfI fragment was very faint in the original gel,but the autoradiographic signal corresponded closely to the 154-bp pBR322 Hinfl marker and also to a 19D Sst I/EcoRI 140-bpfragment (Fig. 6B) (Sst I data not shown). Because fragmentsof less than 200 bp do not transfer to the filter as efficiently aslarger fragments, the signal at 150 may be underestimated bythe Southern blot result.From these studies we conclude that the regions of homology

between SAT-1 and 19D is approximately 250 bp positionedat the Pst I site and extending to the right of the HinfI site with-in the LTR sequence. The SAT-1 fragment is not homologousto the cellular flanking sequence in 19D and homology to the100-bp portion of the LTR left of the Pst I site is not extensive.

DISCUSSIONHorz et al. (1) discovered that the mouse genome contains a fam-ily of highly reiterated DNA sequences that, when digestedwith EcoRI, yield 1,350-bp fragments. Individual restrictionfragments from this family were cloned by Cheng and Schild-kraut (2), Heller and Arnheim (3), and more recently in our lab-oratory. We found that the cloned DNA shares sequence ho-mology with IAP genes, which we (4) and others (7, 8, 13) havepreviously described. The cloned satellite fragment, SAT-1,was hybridized to restriction fragments from seven cloned IAPgenes, and the region ofhomology was determined. Our resultsindicate that the region of homology is not in IAP internal se-quences but is located in the LTR. The LTR region is significantbecause the IAP RNA 5' and 3' termini have been mapped tothis region and RNA transcription was shown to initiate in theLTR (ref. 6 and unpublished data).How are the high numbers of copies of satellite sequences

generated in BALB/c mice? Several speculations may be of-fered in light of the present study. The first possibility is thatsatellite sequences derive from an endogenous retrovirus thatshares a sequence with the IAP LTR. If, however, satellite se-quences are highly reiterated cellular sequences, how and whendid the amplification of these sequences occur? We can pos-tulate that the satellite sequences are nonmobile or mobile ge-netic elements; as nonmobile elements, the satellite sequencescould be derived via the precise integration of IAP genes at areiterated cellular sequence. If the integrated structure con-tains two conserved EcoRI sites, one in the cellular sequenceand the other in the IAP gene next to the LTR, digestion withEcoRI would generate a reiterated EcoRI restriction fragmentcontaining a cellular sequence and an IAP LTR. Most of our IAPclones do contain an EcoRI site next to the LTR sequence (4).Although we did not find sequence homology between SAT-1and cellular regions of 19D, this theory remains a strong pos-sibility. The possibility can be tested by searching for homologybetween cloned satellite sequences and cellular sequencesflanking IAP genes.

SAT-1 may be considered as part of a mobile genetic elementcapable of amplification. It has been recently found that the1.35-kbp fragment is part of a larger unit (25). Integration of anIAP gene within the unit and subsequent excision of the non-LTR sequences may have generated a structure like SAT-1.

Lone terminal repeats have been observed for yeast 8 se-quences (26) and for retroviruses in chickens (27).

The satellite sequences may also be part of a novel transpos-able element created by the duplication of a LTR and a cellularflanking sequence that creates a unique DNA sequence bound-ed by two LTRs. This structure could then undergo amplifica-tion by reintegrations.The IAP genes are the only mouse retroviral genes that re-

semble the 1,350-bp EcoRI satellite DNA with respect to highcopy numbers. It is tempting to speculate that the IAP LTRsequence is a "super" transposon containing a sequence thatfacilitates a high rate of transposition. If one assumes that theLTR is a very efficient promoter, it follows that large amountsof RNA are potentially available for reverse transcription andintegration. A mechanism to explain the high copy numbers of1,350-bp EcoRI satellite DNA and IAP genes found in themouse genome is thus suggested. These events are heritableand may very well take place early in embryogenesis when IAPgenes are normally expressed.We thank Dr. Jeanette Felix for providing the PYS-1 and F9 teratoma

cells that were used in these experiments. This work was supported byNational Institutes of Health Grants CA13953 and 5T32 AG00069.

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2. Cheng, S. M. & Schildkraut, C. L. (1980) Nucleic Acids Res. 8,4075-4090.

3. Heller, R. & Arnheim, N. (1980) Nucleic Acids Res. 8, 5031-5042.4. Ono, M., Cole, M. D., White, A. T. & Huang, R. C. C. (1980)

Cell 21, 465-473.5. Cole, M. D., Ono, M. & Huang, R. C. C. (1981)J. Virol. 38, 680-

687.6. Cole, M. D., Ono, M. & Huang, R. C. C. (1981) J. ViroL, in

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BioL 96, 171-184.23. Mishima, Y., Kominami, R., Hongo, T. & Muramatsu, M. (1980)

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