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Restriction endonuclease mapping of the proviral DNA of the exogenous RIII murine mammary tumor virus
Abstract:
Cellular DNA containing integrated murine mammary tumor virus (MuMTV) was isolated from FeI/C6 feline kidney cells and CCL64 mink lung cells infected with milkborne RIII MuMTV. By using restriction enzyme HpaI, intact RIII MuMTV provirus (length, 8.7 kilobases [kb]) was excised from the cellular DNA. Subsequent restriction endonuclease analysis of this HpaI fragment with KpnI, HindIII, EcoRI, BamHI, BglII, PstI, SstI, SalI, and XhoI enabled us to construct a map of the RIII virus genome. A comparison of this map with the maps of the GR and C3H MuMTV's revealed that there are greater sequence differences between the RIII virus and the GR and C3H MuMTV proviruses than there are between the GR and C3H proviruses. The following are features of the restriction map unique to the RIII provirus: the presence of three BamHI and two EcoRI cleavage sites, a HpaI cleavage site in the terminal 3'-5' repeat unit of the provirus, and the absence of an XhoI cleavage site. Another distinguishing feature of the RIII provirus is that the sizes of some of the restriction fragments produced by cleavage of the RIII provirus with PstI are different from the sizes of the fragments obtained by PstI cleavage of the GR and C3H proviruses. Like the GR proviral DNA, the RIII proviral DNA has three SstI (SacI) cleavage sites, whereas the C3H provirus has only two SstI sites. HpaI digestion of MuMTV-infected mink lung cell DNA revealed only one class of provirus (an 8.7-kb fragment); however, we observed several minor classes of RIII proviral DNA in addition to the major class of provirus DNA in infected cat kidney cells. PstI digestion of the HpaI 8.7-kb fragments from both feline and mink cells generated a 3.7-kb DNA fragment identical in size to a PstI-generated fragment that has been found in GR and C3H milkborne virus-infected cells. Although a fragment similar in size to the milkborne 3.7-kb PstI fragment has been found as an endogenous component in many C3H and GR mouse tissues, we did not observe such an endogenous fragment in the RIII mouse strain. Therefore, the 3.7-kb fragment may be useful as a marker for the milkborne RIII MuMTV provirus in RIII mice.
Insights
Researchers mapped the RIII murine mammary tumor virus (MuMTV) genome, revealing unique genetic sequences compared to GR and C3H MuMTV strains. This analysis identified a specific 3.7-kb DNA fragment as a potential marker for milkborne RIII MuMTV in RIII mice.
Area of Science:
- Virology
- Molecular Biology
- Genomics
Background:
- Murine mammary tumor virus (MuMTV) is a retrovirus known to cause mammary tumors in mice.
- Different MuMTV strains, such as RIII, GR, and C3H, exhibit variations in their genetic makeup and oncogenic potential.
- Understanding these genetic differences is crucial for studying MuMTV pathogenesis and developing diagnostic tools.
Purpose of the Study:
- To construct a detailed restriction map of the milkborne RIII MuMTV provirus.
- To compare the RIII MuMTV genome with those of GR and C3H MuMTV strains.
- To identify unique genetic markers for the RIII MuMTV provirus.
Main Methods:
- Isolation of cellular DNA containing integrated MuMTV from infected feline and mink cells.
- Excising intact RIII MuMTV provirus using the restriction enzyme HpaI.
- Performing restriction endonuclease analysis with multiple enzymes (KpnI, HindIII, EcoRI, BamHI, BglII, PstI, SstI, SalI, XhoI) to map the viral genome.
- Comparing restriction maps of RIII, GR, and C3H MuMTV proviruses.
Main Results:
- A restriction map of the 8.7-kilobase (kb) RIII MuMTV provirus was constructed.
- The RIII MuMTV genome exhibits greater sequence divergence from GR and C3H MuMTV compared to the differences between GR and C3H.
- Unique features of the RIII map include specific numbers of BamHI and EcoRI sites, a HpaI site in the 3' repeat, and the absence of an XhoI site.
- PstI digestion yielded fragments of different sizes for RIII MuMTV compared to GR and C3H.
- A 3.7-kb PstI fragment, found in GR and C3H infected cells and endogenous mouse tissues, was identified in RIII MuMTV but not as an endogenous component in RIII mice.
Conclusions:
- The RIII MuMTV provirus possesses distinct genetic features compared to GR and C3H strains.
- The identified 3.7-kb PstI fragment serves as a potential diagnostic marker for milkborne RIII MuMTV in RIII mice.
- Comparative genomic analysis provides insights into MuMTV strain diversity and evolution.