Restriction endonuclease map of endogenous mouse mammary tumor virus loci in GR, DBA, and NFS mice

VIROLOGY

(1986)

148,23’7-242

Restriction Endonuclease Map of Endogenous Mouse Mammary Tumor Virus Loci in GR, DBA, and NFS Mice DOUGLAS

A. GRAY,*

EDWIN C. M. LEE CHAN,* JANET AND VINCENT L. MORRIS*

I. MACINNES,?

fDepartwwnt of Microbiology and Immu?wm, University of Western Ontario, London, Ontario N6A 5C1, Canada and TDepartment of Veterinary Mimokdwy and Immunolngy, University of GueLph, Gwlph, Ontario NlG 2 Wl, Can&a Received

Mouse

mammary

tumor

August

virus

5, 1985;

accepted

October

7, 1985

(MMTV)

is integrated in the genome of most mice as an proviral loci (Mtv-1 and Mtv-2) are associated with virus expression and tumorigenicity. We prepared restriction endonuclease maps of the endogenous MMTV proviruses in two strains, DBA and GR, which contain the Mtv-2 and Mtv-2 loci, plus a third strain, NFS, which has a low mammary tumor incidence. We find that all these mouse strains have certain MMTV loci in common even though their origins are widely divergent. We also find that some integrated MMTV proviruses appear to have undergone alterations or deletions when compared with MMTV exogenous proviral DNA. We have thus made a comprehensive characterization of MMTV loci in these mouse strains which could serve as a basis for the study of their differences in expression.

endogenous provirus. Two of these MMTV

63 1986 Academic

Press, Inc.

Mouse mammary tumor virus (MMTV) is an RNA tumor virus that is associated with mammary carcinogenesis. MMTV can be transmitted vertically in the germline as a stable hereditable element (r-4). The MMTV proviral loci Mtv-1 and Mtv-0 are associated with MMTV production in the milk (5). In addition, Mtv-1 in C3H mice and Mtv-2 in GR mice are associated with mammary tumor development in the absence of milk-borne virus (5-r). However, there are differences in the expression of these two loci. Mtv-1 is associated with relatively low incidence (22-52%) of late mammary tumors which occur at 18-24 months (1, 8). In contrast, Mtv-2 is associated with a high mammary tumor incidence (97%) and early development of mammary tumors (3-13 months; personal communication of Dr. J. Holben, Institute for Medical Research, Camden, N. J.). Expression of the other endogenous proviruses in GR and DBA mice cannot be detected, and they are presumably silent (5). The transcription of endogenous MMTV proviral sequences can also vary. For example, the transcription of endogenous 237

MMTV proviral loci in BALB/c mammary tumors is less than 1% of that observed in GR mammary tumors (9). The study of these loci is therefore important to gain a better understanding of the regulation of expression of genes associated with the induction of cancer. Work has begun in several laboratories to organize MMTV specific EcoRI and BamHI restriction endonuclease fragments into MMTV loci (IO-IS’). However, in some cases MMTV restriction endonuclease fragments have been incorrectly assigned (11). While restriction endonuclease maps have been prepared for a few isolated loci (13-X), no comprehensive characterization of the endogenous MMTV loci has been made. We have prepared restriction endonuclease maps to characterize eight MMTV loci, including Mtv-1 and Mtv-2. In addition we have assigned MMTV restriction endonuclease fragments to two newly defined MMTV loci. In the current study MMTV 3’ and 5’ specific probes and single and double digestion of DNA samples with restriction endonucleases were used to orient the MMTV loci. 0042~6622/86 Copyright All righta

$3.00 Q 1996 by Academic Preen. Inc. of reproduction in any form reserved.

238

SHORT COMMUNICATIONS

BumHI, EcoRI, and Sac1 were used to produce restriction endonuclease maps for the endogenous MMTV proviral loci in GR, NFS, and DBA mouse strains (Fig. 1). EcoRI cleaves MMTV proviral DNA at a single site and therefore generates two unique cell-virus junction fragments for each complete proviral locus (16,~). Similarily, BumHI, which cleaves MMTV genomes at one or two sites, gives rise to two

cell-virus junction fragments for each complete MMTV locus and, for some proviruses, an additional l.l-kb internal fragment (16, 17). However, internal BamHI and EcoRI sites may vary with individual MMTV loci. Sac1 sites are of interest because Sac1 cleaves the LTR of exogenous MMTV proviral DNA but not the LTR of integrated endogenous MMTV which are not expressed (18, 19). We refer to the en-

B

114-6.9-0.9

F

16.7 - 11.7 S

S

E

6

10 - 6.3 .4

i!6 - 6.7

(Unit

f

B

II)

lp 9 - 5.6 b

6.5 - 4.5 hw-I)

A 0

10

20

30

40

kb

FIG. 1. Restriction endonuclease map of endogenous MMTV loci. The MMTV specific 3 probe was a 1.6-kb PstI fragment containing the MMTV av gene; the 5’ probe was a l.l-kbPstI fragment containing the MMTV gag gene. The location of these probes on the integrated MYTV provirus is indicated. The closed boxes represent MMTV long terminal repeat sequences. The restriction endonuclease sites are represented as follows: E-EcoRI, S--saCI, B--BarnHI, and P-FstI.

SHORT

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dogenous MMTV loci by the sizes of their MMTV specific EcoRI fragments (e.g., the 16.7-11.7 locus is that proviral copy which produces 16.7- and ll.?-kb MMTV-specific fragments after digestion with EcoRI). We have previously correlated some of the EcoRI fragments containing MMTV proviral DNA sequences with their corresponding BamHI fragments for three different strains of mice-GR, DBA, and NFS (11). We have now prepared restriction endonuclease maps for the MMTV loci present in these three strains (Fig. 1). The current studies used genetic crosses and somatic cell hybrid lines (SCH) which contain only one or a few MMTV loci and probes which are specific for the MMTV env sequences (3’ probe) and gag sequences (5’ probe) (see Fig. 1 for a further description of the 3’ and 5’ probes). Using Southern transfer analysis and MMTV 3’ and 5’ probes, we first determined the 3’ and 5’ orientation of the MMTV specific restriction endonuclease fragments. These results are summarized in Table 1. The backcross animals and somatic cell hybrid lines, DNA extraction, and restriction endonuclease analysis have been previously described (11). Our previous results indicate that the GR, DBA, and NFS mice contain MMTV restriction endonuclease fragments which comigrate even after prolonged electrophoresis (15). We have now determined that these loci have the same size 3’ and 5’ junction fragments (Table 1). It is of partitular interest that while previous results show that the 7.8 and 6.7-kb EcoRI fragments (unit II) corn&ate for GR, DBA, and NFS (11); we found that this locus also had the same restriction endonuclease pattern in C57BL and BALB/c mice (Fig. 1; 14). Both the 108.3 and 7.8-6.7 loci contain only one BamHI fragment (II). The 17.8-kb BamHI fragment (from the 10-8.3 locus) and the 14.4-kb BamHI fragment (from the 7.8-6.7 locus) each annealed with both the 3’ and 5’ MMTV probes (Table 1). Using the 3’ and 5’ MMTV probes, we have also determined correlations between EcoRI fragments and BamHI fragments not previously reported. By using prolonged times of electrophoresis, we deter-

239

mined that a 13-kb BamHI fragment correlated with the 16-kb EcoRI MMTV fragment observed in DBA mice (Table 1). There was complete cosegregation of these fragments in the 28 backcross mice we examined. Six of these backcross animals lacked the 16.7-11.7 locus but still produced both the 16.7-kb EcoRI and the 13-kb BamHI fragments. Ten backcross mice had the 16.7-kb fragment without the 13-kb fragment because in these animals the 16.7kb fragment has previously been shown to be part of a separate locus, the 16.7-11.7 locus (II). We determined that digestion of the 17.4-6.9-0.9 locus with BamHI resulted in a fragment with a molecular weight of approximately 13.5 kb (Table 1). DNA from a backcross mouse, which only contained a 17.4-6.9-0.9 GR MMTV locus, only produced a 13.5-kb BamHI MMTV fragment that was also produced by GR parental DNA. Results were similar for both the 3’ and 5’ probes. This 13.5-kb fragment also comigrated with a similar size fragment found in BALB/c mice. Using a similar analysis we identified the BamHI fragments (11.4 and 14.4 kb) which were derived from the 11.4-7.0 locus (Table 1). A previous report (11) describes a 17.2kb BamHI fragment in the NFS strain. We have subsequently determined that this fragment is not present in all NFS mice obtained from the NIH (data not shown). This result could be due to incomplete inbreeding of this strain or due to an acquisition of a novel provirus by an individual breeding mouse. An example of such an acquisition was observed by Cohen and Varmus (4). We will not consider this locus further. An internal EcoRI site is present in exogenous MMTV DNA 3.5 kb from the 3’ end of the integrated provirus; similarily exogenous MMTV DNA has BamHI sites 2.2 and 3.3 kb from the 3’ end (16,17’). However, some of the endogenous MMTV loci we have examined differ from these exogenous MMTV proviruses. For example the 16.711.7 and the 7.8-6.7 loci only have one BamHI site (11). The exogenous MMTV proviruses contain a BamHI site in the 1.6kb PstI fragment and in the 4.1/5.3-kb PstI fragments (see Fig. 1). To determine which

240

SHORT COMMUNICATIONS TABLE ORGANIZATION

OF

MMTV

SPECIFIC

RESTRICTION

17.4-6.9-0.9 16.7-11.7 16.7 15.0-5.8 11.4-7.0 10.0-8.3 9.0-5.8 7.8-6.7 6.5-4.5 1.4

Mtv-18d Mtv-F Mtv-lsd Mtv-ii, -12j Mtv-2 Mtv-ld( Mtv-13 Mtv-8 Mtv-1 Mtv-14

5*

3’O

17.4” 16.7” 16.7 15.0 7.v 10.0” 9.0 7.8’ 6.5

6.9’ 11.7@ h

1

ENDONUCLEASE

FRAGMENTS

BornHI (kb)

EcoRI (kb) LOCUS0

1

5.8 11.4e 8.3” 5.8 6.7’ 4.5

5’

3’ 13.5’

8.0 13.0 23.4 14.4

1

5’

3’

17.0 17.2 h 4.2 11.4

17.8f 17.2 14.4 8.0

Sac1 (kb)

4.4 14.4 4.4

1.6 19.5f I I I

5.1 8.0 or 11.8 9.7 10.5

1

11.5 9.4 5.1 17.2

a The endogenous MMTV loci are designated by the sizes of their EcoRI fragments in kilobases. For comparison we also give the corresponding Mtv locus designations which are reviewed by Traina-Dorge and Cohen (20). We have indicated where our recent findings revise previous locus assignments. * 5’ restriction endonuclease junction fragment. ’ 3’ restriction endonuclease junction fragment. d Locus that we have newly assigned. e 3’ and 5’ assignments of these EcoRI fragments agree with previously published results (21). f For this restriction endonuclease, only a single fragment spans the integrated MMTV DNA copy at this locus. No restriction endonuclease site is located in this integrated MMTV DNA copy. rA 16.7-kb EcoRI fragment has previously been assigned to this locus (20). Our work has shown that a 11.7kb EwRI fragment is also part of this locus (11). ‘No restriction endonuclease fragments that anneal with the 3’ MMTV probe have been identified. ‘Not determined. jPrevious reports assign the 15.0-kb EcoRI fragment to locus Mb-12 and the 5.8-kb EcoRI fragment to locus Mb-11 (20). Our work indicates that these two fragments combine to form one locus (11). ‘Previous published reports have incorrectly suggested that the 11.7- and lO.O-kb EwRI fragments form a locus (Mtv-IO) (2&). We have now shown that the 11.7-kb EwRI fragment is part of the Mb-7 locus and that the lO.O-kb EcoRI fragment together with the 8.3-kb EwRI fragment forms a separate locus. The Mb10 locus is therefore redefined to contain the lO.O-8.3-kh EcoRI fragments (22). r This MMTV fragment does not anneal with 3 or 5’ MMTV probes.

of these sites is present in the 16.7-11.7 and 7.8-6.7 MMTV loci, we did Southern transfer analysis. DNA from a backcross mouse with only the 16.7-11.7 GR locus was digested with P&I or PstI and BamHI. The internal 1.6-kb PstI fragment from this locus, which includes the sequences we use for the 3’probe, was not digested by BamHI (data not shown). SCH 19 produces a PstI fragment of 4.1 kb from the 16.7-11.7 GR locus (11); this fragment was digested by BamHI to produce a 3.7-kb fragment (data not shown). Similarily, SCH 14 DNA, containing only the 16.7-11.7 GR locus (U), produced a 5.2-kb PstI fragment which was digested with BamHI to produce a 4.8-kb

fragment (data not shown); the other PstI fragments were not digested by BamHI. Thus the 16.7-11.7 locus has a BamHI site in the internal 4.1-kb PstI fragment, and the 7.8-6.7 locus has the BamHI site in the 5.2-kb PstI fragment (Fig. 1). The sizes of the PstI-BamHI digestion fragments were consistent with the placement of the BumHI site 3.3 kb from the 3’ end of the proviruses (Fig. 1). We found that the MMTV sequences in the 17.4-6.9-0.9 locus had two internal EcoRI sites which produced the 0.9-kb MMTV fragment. Restriction endonuclease double digestions were used, and the results were consistent with the placement

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of EcoRI sites 2.6 and 3.5 kb from the 3’ end of the integrated MMTV DNA (Fig. 1). The 10.8-8.3, 17.4-6.9-0.9, and ‘7.8-6.7 loci only produced one MMTV specific BumHI fragment (Table 1). This situation could be produced by an internal BumHI site and two junction fragments of identical size. Alternatively, the MMTV sequences could have no internal BamHI sites, but the BamHI fragment could span the entire MMTV sequence. P&I-BumHI double digestions revealed that the 7.8-6.7 locus had a BumHI site in the internal 4.1-kb PstI fragment (Fig. 1). Similar analysis indicated that the 10-8.3 and 17.4-6.9-0.9 loci did not contain internal BumHI sites (Fig. 1). In both cases the BumHI fragments are of sufficient length to span the integrated MMTV DNA sequences. The 1.4-kb EcoRI fragment and its corresponding 7.3-kb BumHI fragment were previously reported in GR, NFS, and DBA mice (11). These fragments annealed with the MMTV LTR probe but not with our 3’ or 5’ probes (data not shown). This locus thus likely consists mainly of LTR sequences. All published restriction endonuclease maps of endogenous and exogenous MMTV proviruses have placed an EcoRI site approximately 3.5 kb from the 3’ end of the provirus (1%18,Z.Y) We find that all of the complete integrated MMTV proviruses we examined had at least one internal EcoRI site (Table 1). Furthermore, our double restriction endonuclease digestion data were all consistent with the published placement of an EcoRI site 3.5 kb from the 3’ end of the provirus. This EcoRI site thus appears to be a constant feature in all complete MMTV proviruses. In addition, published data indicate that internal PstI sites approximately 1.3,2.9, and 8.1 kb from the 3’ end of the MMTV provirus are also a constant feature (H-18, 23). Previously published results (1%18,23) and double digestions with BumHI and PstI or EcoRI allowed the assignment of an internal BumHI site at 3.3 kb from the 3’ end of all of the MMTV proviral loci examined except for the 17.4-6.9-0.9 and 10-8.3 loci. The data in Table 1 and in the text of the previous sections and the fixing of an internal EcoRI and BumHI site thus allowed the construc-

241

tion of the restriction endonuclease map shown in Fig. 1. Where a locus lacked an internal BumHI site, double digestions with BumHI and PstI or EcoRI were used to assign the BumHI sites in the mouse flanking sequences. The Sac1 sites were assigned using techniques similar to those described for BumHI and EcoRI. Endogenous MMTV DNA sequences are of great interest in understanding mouse mammary neoplasia. Mtv-1 in C3H mice and Mtv-2 in GR mice are associated with mammary tumor development in the absence of milk-borne virus (5-7). Our previous results characterized MMTV loci (including Mtv-1 and Mtv-2) in terms of the BumHI and EcoRI fragments that were produced (11). In this report we have prepared restriction endonuclease maps for the endogenous MMTV proviral loci in GR, NFS, and DBA mouse strains (Fig. 1). We find internal alterations and deletions in these endogenous MMTV DNA copies. These findings differ from what was reported by Peterson and co-workers for C57BL/6 (14). Therefore, either the multiple copies of MMTV in C57BL/6 originated from infections by similar MMTV proviruses, the multiple copies resulted from amplification of one of the integrated proviruses, or the integrated proviruses are more stable in C57BL/6 than in GR, NFS, and DBA. The internal restriction endonuclease sites of Mtv-2, which is associated with a high incidence of early mammary tumors, closely resemble the sites in exogenous MMTV proviruses. They both produce internal MMTV specific 4.1-kb PstI and l.lkb BumHI fragments; in addition they both have Sac1 sites in their LTR sequences (Fig. 1; 11,13,16,17). In contrast the Mtv-1 locus, which is only associated with a relatively low incidence of late mammary tumors, does not produce the 4.1-kb PstI fragment and does not have a Sac1 site in its LTR sequences (Fig. 1; 11). The significance of these observations is currently under investigation. It is thus hoped that our determination of the organization of Mtv-1, Mtv-2, and other endogenous MMTV loci in the GR, DBA, and NFS mouse strains will assist in determining the reasons for the

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differences in the expression and tumorigenicity associated with different endogenous proviral loci. ACKNOWLEDGMENTS We thank Dr. Christine Koaak, NIH, Bethesda, Maryland for assistance in preparing the somatic cell hybrid lines and for providing tissues from the NFS backcross mice. We also thank her for reading the manuscript and offering useful suggestions. We thank Dr. E. Y. Lasfargnes, Dr. S. Dales, Dr. J. E. Majors, and Dr. H. E. Varmus for providing the materials mentioned in the text. MMTV (C3H) and AMV pelymerase was supplied by the Office of Program Reeources and Logistics, National Cancer Institute. This work was supported by a National Cancer Institute of Canada grant awarded to V.L.M. A Medical Research Council of Canada studentship was awarded to D.A.G. REFERENCES 1.

6. 3. 4 5. 6.

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Z VAN NIE, R., VERSTRAETEN, A. A., and DE MOES, J., Iti J Cancer 19,383~399 (1977). Biophya Acta 365,2368. BENTVELZEN, P., Biochim 259 (1974). 9. VARMUS, H. E., QUINTREU, N., MEDEIROS, E.. BISHOP, J. M., NOWINSKI, R. C., and SARKAR, N. H.. J. Md Bid 79,663-679 (1973). 10. TRAINA, V. L., TAYLOR, B. A., and COHEN. J. C., J. Vird 49,735-744 (1981). 11. MACINNES. J. I., MORRIS, V. L., FLINTOFF, W. F., and KOZAK, C. A., Vi&tag9 132,12-25 (1984). 19. CALLAHAN, R., GWAHAN, D., and KOZAK, C., J. ViroL 49,1C@5-1008 (1984). 1s. MICHALIDES, R., WAGJZNAAR, E., and WELTERS, P., Mel and CelL BioL 6,823~830 (1985). 14 PETERSON, D. O., KRIZ, K. G., MARICH, J. E., and TOOHEY, Id. G., J. ViroL 54,525~531 (1985). 15. PRAKASH, O., KOZAK, C., and SARKAR, N. H., J. ViroL 54,2X&294 (1985). 16. SHANK, P. R.. COHEN. J. C., VARMUS, H. E., YAMAMOTO, K. R., and RINGOLD, G. M., Pvoc Nut1 Acad Sti USA 75,2112-2116 (1978). 17. COHEN, J. C., SIUNK. P. R., MORRIS. V. L., CAIUXFF, R., and VARMUS, H. E., CeU 16,333-345 (1979). 18. DONEHOWER, L. A., HUANG, A. L., and HAGER, G. L., .J. ViroL 37.226-238 (1981). 19. DONEHOWER. L. A., FLEURDELYS, B., and HAGER G. L., J. V$rol 45,941~949 (1983). 60. TRAINA-DORGE, V., and COHEN, J. C., Cuw. Top Microbid Immwwl 166,35-56 (1983). 21. GRONER, B., B~EZTI, E., DIGGELMANN, H., and HINES, N. E., J. ViroL 36.734-745 (1989). 62. POPKO, B. J., and PAULEY. R. J., virus Rea 2,231243 (1985). 2.9. COHEN, J. C., MAJORS, J. E., and VARMUS, H. E., J. vird 32,4&3-496 (1979).