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

Mouse EcoRi DNA contains a sequence homologous to the 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 from coli strain HB101 cells were grown in L broth and the DNA was the mouse 1,350-base-pair EcoRI satellite sequence were cloned prepared by a modification of the procedure of Birnboim and in pBR322 plasmid. One of these fragments, SAT-1, was hybrid- Doly (18) or as described in ref. 19. ized to various restriction fragments from cloned mouse intracis- Restriction enzymes were purchased from Bethesda Re- ternal type A particle (LP)' genes and to mouse total genomic search Laboratories and assay' conditions were from the sup- DNA. Hybridization occurred between the SAT-1 sequence and plier's.manual. DNA restriction fragments were fractionated by each of the JAP clones. The common region of hybridization in electrophoresis in agarose gels in 0.1 M Tris borate, pH 8.3/ each of the clones was the long terminal repeat region of the UAP 0.002 M Na2EDTA. genes. Preparation of Probes for Nick-Translation. One milligram A moderately repeated 1,350-base-pair (bp) satellite DNA se- of recombinant pBR322 plasmid was digested with the appro- quence has been identified in mouse BALB/c, F9, 3T3, S107, priate restriction enzyme. The digest was applied to a 20.0 x and Friend cell lines after cleavage oftotal genomic DNA with 20.0 cm agarose gel in a well 20.0 X 1.0 X 0.3 cm and electro- EcoRI restriction enzyme (1-3). The satellite was found to make phoresed in 0.1 M Tris borate, pH 8.3/0.002 M Na2EDTA at up 0.5-3.0% ofthe genome with a repeat frequency ofapprox- 200 V. The desired DNA band was visualized by ultraviolet light imately 104 in a Friend erythroleukemia cell line. The satellite after ethidium bromide staining and was recovered by elec- was shown to consist of related but nonidentical sequences,- troelution. The DNA was applied to a DEAE-cellulose column which are arranged.in a dispersed fashion in the genome (2). (1.0 x 0.4 cm), washed with 0.05 M Tris'HCI, pH 7.5/0.1 M Recently, we (4-6) and others (7, 8) have done extensive stud- NaCI, and eluted with-3.0 ml of 1.0 M NaCI/0.05 M Tris-HCI, ies on the structural organization ofthe genes coding for intra- pH 7.5. Nick-translation ofthe eluted DNA fragments was per- cisternal A RNA. formed according to Rigby et aL (20). The final specific activity particle (IAP) IAPs are noninfectious retro- was 0.5-1.0 X 108 cpm//zg of DNA. virus-like particles that are seen within the endoplasmic Hybridization. DNA restriction fragments on agarose gels reticulum in several mouse tumor cell. lines and in normal were transferred to a nitrocellulose filter by the method of preimplantation mouse embryos (9-12). There are approxi- Southern (21). Filters were incubated in hybridization buffer mately 1,000 IAP genes dispersed throughout the genome, (0.45 M NaCI/0.045 M sodium citrate/0. 1% NaDodSO4/ where they. make up over 0.2% of the total DNA (4, 13). In- 0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02 M potassium dividual IAP genes show a divergence of sequences and a di- phosphate, pH 6.8/100 Mzg of yeast tRNA per ml) for 24 hr at versity ofsequence arrangements. We have previously reported 67TC. Filters were incubated with the radioactive probe (0.5 that IAP genes, like those coding for type B and type C retro- X 106 cpm/ml) in hybridization buffer for 24 hr at 670C. They viruses, contain long terminal repeats (LTRs) flanking the IAP were washed twice in 0.45 M NaCI/0.045 M sodium citrate/ structural gene (5), and also that both the 5' and the 3' ends of 0.1% NaDodSO4/0.02 M potassium phosphate, pH 6.8, for 30 IAP RNA are encoded within the LTR.(6). min at 67TC. The filters were washed twice under stringent We report here that sequence homology exists between sat- conditions (0.015 M NaCl/0.0015 M sodium citrate/0. 1% ellite sequences and a portion of the IAP genome. The homol- NaDodSO4/0.02 M potassium phosphate, pH 6.8, for 30 min ogy is confined to the LTR region, the region to 'which the ter- at67C). Filters were dried and exposedat -70°C with Du Pont mini of IAP RNA have been mapped (6). Cronex High Plus intensifying screens. Preparation of the 1,350-bp Satellite Fragment. Six hun- MATERIALS AND METHODS dred micrograms of mouse strain BALB/c liver DNA was di- Preparation ofTissue, Plasmid, and Phage . DNA was gested withEcoRI and fractionated by electrophoresis on a 1.0% isolated from BALB/c mouse liver, kidney, spleen, and brain agarose gel 20.0 x 43.0 x 1.0 cm, well size 18.0 X 1.0 X 0.3 by the procedure of Marmur (14). DNA was isolated from ter- cm, as described above. The gel was stained with ethidium bro- atoma F9 (15), MOPC 315, and parietal yolk sac carcinoma cell mide, the satellite DNA band was excised, and the DNA was lines (16) by the procedure ofMoshier and Huang as described removed from the gel by electroelution. The DNA was applied in ref. 4. Derivatives of ACH4A bacteriophage containing IAP to a DEAE-cellulose column, washed, eluted, and precipitated inserts were propagated in liquid culture and DNA was pre- as described above. Approximately 2.0 ,Ag of material was re- pared according to Enquist et aL (17). Individual EcoRI restric- covered, inserted into pBR322 at the EcoRI site, and propa- tion fragments from IAP genes were previously subcloned in gated in E. coli strain HB101 (22-24). Fifty-milliliter cultures pBR322 plasmid (4). The recombinant plasmids in Escherichia oftetracycline-resistant transformantcolonies were grown. The The publication costs ofthis article were defrayed in part bypage charge Abbreviations: bp, base pair; IAP, intracisternal A particle; LTR, long payment. This article must therefore be hereby marked "advertise- terminal repeat. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 6123 Downloaded by guest on October 1, 2021 Al QA nvA UIInvfr P&-n,- An*1AA sndQ TrTVA 70 .QORO -I,- _

bacteria were harvested, the plasmid DNA was prepared and A B C D

digested with EcoRI, and the digest was subjected to electro- _. phoresis in an agarose gel. Colonies containing plasmid DNA _o with a 1,350-bp EcoRI insert band were retained for further study. RESULTS Hybridization of TAP DNA-to EcoRI-Digested Mouse DNA. DNA from various mouse tissues was digested with EcoRI re- striction endonuclease. The resulting restriction fragments were separated by electrophoresis on long agarose gels and qtninprl 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 in 2 250- EcoRI-digested DNA from tissue types in addition to those shown in Fig. 1, including mouse brain, kidney, spleen, and ;930 - teratoma PYS-1. We hybridized the totalEcoRI-digested mouse liver DNA with 32P-labeled IAP DNA probes derived from three different regions of the IAP genome. The three IAP probes 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 but does not include the LTR. When the 1,250-bp fragment was hybridized to Southern blots of EcoRI-digested mouse liver DNA, 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 ofthis fragment to a Southern blot of EcoRI-digested mouse liver DNA is shown in Fig. 3, lane B. Several discrete bands hybridized to the probe but none ofthese corresponded to the 1,350-bp satellite band. The third IAP DNA probe, derived from a Pst I/EcoRT- FIG. 1. Ethidium bromide-stained gel of EcoRT restriction frag- digestion of 19B (Fig. 2B), contains approximately 400 nucleo- ments of DNA from mouse strains BALB/c and Swiss Webster. Lane tides that are part ofthe LTR sequence (unpublished DNA se- A, solid myeloma. MOPC 315 tumor grown in BALB/c mice; lane B, quence analysis). We reported earlier that most ofthe LTR se- liverfrom adult BALB/c mice; lane C, teratoma F9tissue culture cells; quence in the 400-bp fragment hybridized to the 5' end ofIAP lane D, Swiss Webster neonatal mice. DNA (5 Mug) was digested with RNA (6). Fig. 3, lane C, shows the results of hybridization of DEcoRT and the resulting fragments were separated by electrophoresis in a 1% agarose gel (20 x 43 cm, horizontal). The arrow designates the to a the 400-bp fragment Southern blot of EcoRI-digested position of satellite DNA sequences, approximately 1,350 bp in size, mouse liver DNA. There was a strong background of hybrid- after digestion with EcoRI. AC1857- DNA digested with HindE was ization to the many dispersed TAP genes containing LTR se- used to estimate molecular weights as shown in bp in the extreme left quences with a diversity ofcellular flanking sequences. There lane.

A _, D _, A 81 (I) 5.0 3.3 5 6.8

(2) )'R e =4-f I t -T AL 0.80 (3) 3 5' 7.2 3' e

B 19 (1) C D a A 2.0 1.45 4..5 5.1

4 _m w + HI (2) * * 1.25 0.40 (3) 3. 5 4.8 3051 'EcoRu I 5-I - - -3 * MYELOMA A-PARTICLE BamHI RNA PstI o 1.0 kbp Hindill + Sst

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 used as 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 determined by electron microscopy (5) and the repeat has been characterized by DNA sequence analysis (unpublished data). The sequence homology between the IAP genes of 81 and 19 is described elsewhere (6). S1 nuclease mapping of RNA termini is also described in ref. 6. Downloaded by guest on October 1, 2021 Biochemistry: Brown and Huang Proc. Nati Acad. Sci. USA 79 (1982) 6125 were, additionally, several discrete signals which were visible were characterized by heteroduplex mapping and were found above the background; the strongest of these was found at a to contain some common gene regions with nonhomologous position corresponding to the i,350-bp EcoRI satellite band in flanking cellular regions. P-Labeled SAT-1 insert or 400-bp Fig. 1. These results suggested thatthe 1,350-bp EcoRI satellite LTR-containing DNA fragment from i9B were hybridized to DNA shares sequence homology with the LTR sequence, but Southern blots of EcoRI-digested IAP clones. The results are not with internal adjacent sequences of IAP genes. shown in Fig. 5. The 400-bp and the SAT-1 probes hybridized Hybridization ofCloned Satellite DNA (SAT-1) to Digested to the same fragments (Fig. 5 B and C), and each of the hy- Mouse Genomic DNA. In order to define the relationship of bridizing fragments contains a LTR sequence (ref. 6 and un- the 1,350-bp satellite band to IAP DNA sequences, we cloned published data). Some ofthe bands resultingfrom A81, A62, and purified satellite fragments in pBR322 as described in Materials A19 digestion, such as 81B, 62A, and 19A (Fig. 2), showed a and Methods. One of the clones was designated pSAT-1. The somewhat weaker signal than the other bands. We have found DNA insert (SAT-1) was isolated, labeled with 32p, and used as that the LTR in 62 has undergone base changes, deletions, and a probe in the hybridization study below. insertions when compared to the LTRs of 19B and 19D (Fig. Mouse liver DNA was digested' with Xba I, Sst I, Pst I, or 2), which are identical (unpublished sequence data). EcoRl. The hybridization pattern ofthe SAT-1 fragment to di- Position ofthe Region Within the LTR ofthe TAP Gene that gested total mouse DNA is shown in Fig. 4. The most prominent Shares Sequence Homology with the Satellite DNA SAT-1. To signal was seen at the satellite band of EcoRI-digested DNA define the region of homology between SAT-1 and the LTR shown in lane 4. This information indicates that the SAT-I frag- sequence, the LTR region was digested with several different ment contains sequences that are repeated in the satellite band. restriction enzymes and the resulting fragments were hybrid- There were several other bands that hybridized well over back- ized to the 3P-labeled satellite DNA SAT-1 probe. A detailed ground, an indication that sequences homologous to the SAT-1 map of the restriction sites is shown in Fig. 6. We observed sequence are found in other repeated arrangements in the ge- hybridization ofthe SAT-i probe to the 400-bp LTR-containing nome. The background hybridization consistently observed was fragment (Pst I/EcoRI digested) from 19B (Fig. 7A). Because possibly due to hybridization ofthe SAT-1 fragment to the many the 400-bp fragment contains approximately 100 bp ofa cellular dispersed LTR-containing IAP genes. flanking sequence (Fig. 6B, region C2) outside the LTR region, Hybridization of the SAT-i Fragment with Digested DNA the possibility did exist that the homology between the 400-bp from Individual LAP Clones. Seven ACH4A recombinant clones fragment and SAT-i was with the flanking region and not with containing IAP genes have been described (4). These clones the LTR region. To distinguish between the two possibilities, we compared the hybridization of SAT-1 to DNA fragments A e 1 2 3 4

I.. 250- -_ I I

. 4.S ~- | * ~~I

DO- 1u. I --21-

0:,q _

FIG. 3. Hybridization analysis of strain BALB/c mouse liver DNA digested with EcoRT. Eachlane contained 5 Mg of total liver DNA from BALB/c mice. After electrophoresis (1% agarose, 20 x 43 cm) and DNA transfer, the nitrocellulose filter was incubated with 32P-labeled, nick-translated, denatured DNA probes from different regions of cloned IAP genes as diagrammed in Fig. 2: 1,250 bp from the EcoRI FIG. 4. Hybridization of mouse BALB/c liver DNA restriction en- site to the BamHI site of 19B, IAP internal sequence (lane A), 800 bp donuclease fragments to cloned satellite DNA probe. DNA (5 pg) was from theBamHI to theHindHI site of81A, TAP internal sequence (lane digested withXba I (lane 1), PstI (lane 2), SstI (lane 3), orEcoRI (lane B), and 400 bp from thePst I site to the EcoRT site, 19B LTR sequence 4). The resulting fragments were separated by electrophoresis on a 1% of the IAP gene (lane C). The arrow indicates the position of the EcoRI agarose gel, transferred to nitrocellulose filters, and hybridized to a satellite DNA band' as in Fig. 1. Specific activity of the probes and 1,350-bp cloned satellite DNA fragment. The. arrow indicates the po- hybridization conditions are described in the text. sition of the satellite DNA band as in Fig. 1. Downloaded by guest on October 1, 2021 6126 Biochemistry: Brown and Huang Proc. Nad Acad. Sci. USA 79 (1982)

a r- ^ Rit, '. 4 E I?9 X. 0 s62 A,17 A',2 x81 %:4 xA*: 2X'} x62 x:7 .% 8 ix. :4-: '.,- Q, ". 21 -"-) ."A r:.,) 'A -7

9400 1 6:>00 _Pf _:. FIG. 5. Hybridization analysis of EcoRI re- i_ ~~~~~~~~~~~~~~~~~~~~...Ad45.00, striction fragments of cloned IAP genes. One mi- I...% .~~~~~~~~~~~~~~~ A:&. crogram of DNA from recombinant bacterio- I~~~~~~~~~~~~~~~~~ was 2?250 I At .. t0 phage A IAP clones (4) digested with EcoRI. 1960 -, The restriction fragments were subjected to elec- trophoresis in two 1% agarose gels, stained with ethidium bromide at 1 pg/ml, and photographed (A). DNA fragments were transferred to nitro- cellulose filters. Hybridization was with the nick- translated 400-bp l9BPst 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 the LTR or to the 600-bp BstNI fragment proximal to the 320- shared by 19D and i9B are in the LTR region (6). Therefore, bp fragment (Fig. 7B). This result indicates that there are no hybridization of the SAT-1 probe to both 19B and 19D would homologous sequences between SAT-i and the cellular flanking be the result ofhomology with the LTR and not with the flank- DNA in 19D and supports the conclusion that it is only the LTR ing cellular regions. For this study pi9D, the pBR322 recom- region in the 400-bp Pst I/EcoRI fragment of i9B that hybrid- binant'plasmid containing the mouse 1,400-bp insert, was di- ized to the SAT-1 satellite DNA. gested with EcoRI and the 1,400-bp insert (19D) was purified To position the region of homology within the LTR, the from an agarose gel. The insert was digested with several dif- 1,400-bp 19D insert was digested with Pst I to yield an 1, 100-bp ferent restriction enzymes and the restriction fragments were fragment and a 300-bp fragment, and the SAT-1 probe was hy- separated on a gel, transferred to nitrocellulose, and hybridized bridized to a Southern blot ofthese fragments. There was little, to SAT-1 probe. if any, hybridization between SAT-1 DNA and the 1, 100-bp Digestion of the pi9D insert with BstNI generated four re- fragment (Fig. 7C). There was strong hybridization between the striction fragments (Fig. 6). There is a BstNI restriction site 300-bp fragment and SAT-1 DNA. The 1, 100-bp fragment con- approximately 100 bp to the left ofthe Pst I site. Digestion with tains about 100 bp of the LTR and- a cellular flanking region the enzyme generated a 320-bp fragment containing the se- (C1, in Fig. 6) (5). The 300-bp Pst I/EcoRI fragment contains quence flanking the LTR. A 420-bp fragment containing LTR

sequences was also generated' by digestion with BstNI. A C D We observed strong hybridization of the SAT-i1fragment to 2 2 the 420-bp fragment containing the LTR, but we did not detect hybridization to the 320-bp fragment immediately adjacent to

_ _ 63 z _0 (aZ _r 0 A e _oc (A p 19D In I I PBR3221_. I I I --- 1.11322 | f1i LTR )L.L

U1 I Bst NI 50 600 bp 320 bp 420 bp Sst1

350 bp 900 bp 160 bp 517-

I ___ I Pst i 1100 bp 300 bp £. B 344- 0. 298 .p 19 B .< 5~C) _.. --i- A P P83 g ~~~~~~I__ __ 1 BR22 21 IR3!H22 LTR C2

400 bp l Sst I 140 bp 260 bp l Hinf 1 250 bp 150 bp FIG. 7. Hybridization analysis of the LTR DNA in 19D and 19B. Lanes labeled 1 show the ethidium bromide-stained restriction frag- FIG. 6., Restriction maps of LTR-containing regions of two LAT ments on an agarose gel. Lanes labeled 2 show the autoradiograms of genes. EcoRI restriction fragments B and D of A19- (Fig. 2) were sub- the gels after Southern blotting and, hybridization to 32P-labeled cloned in pBR322, and the B and D fragments from the recombinant SAT-1 DNA. (A) The,400-bp DNA fragment purified from 19B (Pst I plasmids were digested with various restriction enzymes. Foreachsub- to EcoRI). (B) The 1,400-bp EcoRI mouse insert, 19D, digested with clone, the top line shows the restriction sites and the lines beneath BstNI. (C) The 1,400-bp EcoRI mouse insert, 19D, digested with Pst I. show the sizes of the restriction fragments,obtained with each enzyme (D) The 400-bp DNA fragment purified from 19B (Pst I to EcoRI) di- plus EcoRI. The heavy line delineates the boundaries of the LTR as gested with Hinfl. pBR322 HinfI markers are included. Undigested determined by electron microscopy. C1 and C2 refer to cellular -se- 19D is shown in lane 2 of C (top band). In D, the 150-bp fragment is quences flanking the LTR and- the dashed line refers to the internal barely visible in the stained gel. An enlarged diagram of the restriction 1AP gene region. 19B and 19D contain the two ends of an IAP- gene. sites in 19B and in 19D is shown in Fig. 6. Downloaded by guest on October 1, 2021 Biochemistry: Brown and Huang Proc. Nati. Acad. Sci. USA 79 (1982) 6127 only LTR sequences. We therefore conclude that the homology Lone terminal repeats have been observed for yeast 8 se- between SAT-1 and the LTR extends from just left of the Pst quences (26) and for retroviruses in chickens (27). I site to near the EcoRI site within the LTR region. To deter- The satellite sequences may also be part ofa novel transpos- mine the extent ofthis homology, the 400-bp Pst I/EcoRI frag- able element created by the duplication ofa LTR and a cellular ment from 19B was digested with Hinfl, yielding a 250-bp and flanking sequence that creates a unique DNA sequence bound- a 150-bp fragment (Fig. 6). When these fragments were hy- ed by two LTRs. This structure could then undergo amplifica- bridized to SAT-1 probe, we observed a positive signal at 250 tion by reintegrations. bp and a somewhat weaker signal at around 150 bp (Fig. 7D). The IAP genes are the only mouse retroviral genes that re- The 150-bp HinfI fragment was very faint in the original gel, semble the 1,350-bp EcoRI satellite DNA with respect to high but the autoradiographic signal corresponded closely to the 154- copy numbers. It is tempting to speculate that the IAP LTR bp pBR322 Hinfl marker and also to a 19D Sst I/EcoRI 140-bp sequence is a "super" transposon containing a sequence that fragment (Fig. 6B) (Sst I data not shown). Because fragments facilitates a high rate of transposition. If one assumes that the of less than 200 bp do not transfer to the filter as efficiently as LTR is a very efficient promoter, it follows that large amounts larger fragments, the signal at 150 may be underestimated by of RNA are potentially available for reverse transcription and the Southern blot result. integration. A mechanism to explain the high copy numbers of From these studies we conclude that the regions ofhomology 1,350-bp EcoRI satellite DNA and IAP genes found in the between SAT-1 and 19D is approximately 250 bp positioned mouse genome is thus suggested. These events are heritable at the Pst I site and extending to the right ofthe HinfI site with- and may very well take place early in embryogenesis when IAP in the LTR sequence. The SAT-1 fragment is not homologous genes are normally expressed. to the cellular flanking sequence in 19D and homology to the 100-bp portion of the LTR left of the Pst I site is not extensive. We thank Dr. Jeanette Felix for providing the PYS-1 and F9 teratoma cells that were used in these experiments. This work was supported by DISCUSSION National Institutes of Health Grants CA13953 and 5T32 AG00069. Horz et al. (1) discovered that the mouse genome contains a fam- 1. Horz, W., Hess, T. & Zachau, H. G. (1974) Eur. J. Biochem. 45, ily of highly 501-512. reiterated DNA sequences that, when digested 2. Cheng, S. M. & Schildkraut, C. L. (1980) Nucleic Acids Res. 8, with EcoRI, yield 1,350-bp fragments. Individual restriction 4075-4090. fragments from this family were cloned by Cheng and Schild- 3. Heller, R. & Arnheim, N. (1980) Nucleic Acids Res. 8, 5031-5042. kraut (2), Heller and Arnheim (3), and more recently in our lab- 4. Ono, M., Cole, M. D., White, A. T. & Huang, R. C. C. (1980) oratory. We found that the cloned DNA shares sequence ho- Cell 21, 465-473. mology with IAP genes, which we (4) and others (7, 8, 13) have 5. Cole, M. D., Ono, M. & Huang, R. C. C. (1981)J. Virol. 38, 680- previously described. The cloned satellite 687. fragment, SAT-1, 6. Cole, M. D., Ono, M. & Huang, R. C. C. (1981) J. ViroL, in was hybridized to restriction fragments from seven cloned IAP press. genes, and the region ofhomology was determined. Our results 7. Leuders, K. K. & Kuff, E. L. (1980) Proc. Nati Acad. Sci. USA indicate that the region of homology is not in IAP internal se- 77, 3571-3575. quences but is located in the LTR. The LTR region is significant 8. Kuff, E. L., Smith, L. A. & Leuders, K. K. (1981) Mol. Cell. Biol because the IAP RNA 5' and 3' termini have been mapped to 3, 216-227. 9. Calarco, P. G. & Szollosi, D. (1973) Nature (London) New Biol. this region and RNA transcription was shown to initiate in the 243, 91-93. LTR (ref. 6 and unpublished data). 10. Chase, D. G. & Piko, L. (1973) J. Natl. Cancer Inst. 51, 1971- How are the high numbers of copies of satellite sequences 1973. generated in BALB/c mice? Several speculations may be of- 11. Biczysko, W., Pienkowski, M., Solter, D. & Koprowsky, H. fered in light of the present study. The first possibility is that (1973)J. Natl Cancer Inst. 51, 1041-1050. satellite sequences derive from an that 12. Wivel, N. A. & Smith, G. K. (1971) Int.J. Cancer 7, 167-175. 13. Leuders, K. K. & Kuff, E. L. (1977) Cell 12, 963-972. shares a sequence with the IAP LTR. If, however, satellite se- 14. Marmur, J. (1961)J. Mol. Biol 3, 208-218. quences are highly reiterated cellular sequences, how and when 15. Nicholas, J. F., Avner, P., Gaillard, J., Gurenet, J. L., Jakob, H. did the amplification of these sequences occur? We can pos- & Jacob, F. (1976) Cancer Res. 36, 4224-4231. tulate that the satellite sequences are nonmobile or mobile ge- 16. Pierce, G. B., Bullock, W. K. & Huntington, R. (1970) Cancer netic elements; as nonmobile elements, the satellite sequences 25, 644-658. could be derived via the precise integration of IAP genes at a 17. Enquist, L., Tiemeier, D., Leder, P., Weisbert, R. & Sternberg, N. (1976) Nature (London) 259, 596-598. reiterated cellular sequence. If the integrated structure con- 18. Birnboim, M. & Doly, R. (1979) Nucleic Acids Res. 7, 6-13. tains two conserved EcoRI sites, one in the cellular sequence 19. Schibler, U., Marcu, K. B. & Perry, R. P. (1978) Cell 15, and the other in the IAP gene next to the LTR, digestion with 1495-1509. EcoRI would generate a reiterated EcoRI restriction fragment 20. Rigby, P. W., Dieckmann, R., Rhodes, C. & Berg, P. (1977) J. containing a cellular sequence and an IAP LTR. Most ofour IAP Mol Biol 113, 237-251. clones do contain an EcoRI site next to the LTR sequence (4). 21. Southern, E. M. (1975)J. Mol Biol 98, 503-517. 22. Dugaiczyk, A., Boyer, H. W. & Goodman, H. M. (1975)J. Mol Although we did not find sequence homology between SAT-1 BioL 96, 171-184. and cellular regions of 19D, this theory remains a strong pos- 23. Mishima, Y., Kominami, R., Hongo, T. & Muramatsu, M. (1980) sibility. The possibility can be tested by searching for homology Biochemistry 19, 3780-3786. between cloned satellite sequences and cellular sequences 24. Cohen, S. N., Chang, A. C. Y. & Hsu, L. (1972) Proc. Natl Acad. flanking IAP genes. Sci. USA 69, 2110-2114. SAT-1 may be considered as part ofa mobile genetic element 25. Meunier-Rotival, M., Soriano, P., Cuny, G., Strauss, F. & Ber- nardi, G. (1982) Proc. Natl Acad. Sci. USA 79, 355-359. capable of amplification. It has been recently found that the 26. Hughes, S. H., Toyoshima, K., Bishop, J. M. & Varmus, H. E. 1.35-kbp fragment is part ofa larger unit (25). Integration of an (1981) Virology 108, 189-207. IAP gene within the unit and subsequent excision of the non- 27. Neiman, P., Beemon, K. & Luce, J. A. (1981) Proc. Natl Acad. LTR sequences may have generated a structure like SAT-1. Sci. USA 78, 1896-1900. Downloaded by guest on October 1, 2021