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Characterization of endogenous and exogenous mouse mammary tumor virus proviral DNA with site-specific molecular
Abstract:
Restriction fragments of the mouse mammary tumor virus (MMTV) proviral DNA were obtained by molecular cloning procedures. A 4-kilobase fragment delimited by two PstI sites was isolated from unintegrated, linear MMTV DNA and amplified in the pBr322 plasmid vector. EcoRI fragments of proviral DNA, integrated into the genome of a GR mammary tumor cell line, were isolated as lambda recombinant molecules. Five different recombinant phages which contained the 3' region of the MMTV proviral DNA and adjacent host DNA sequences were isolated. Heteroduplex analysis and S1 nuclease digestion suggested that there is no extensive sequence homology in the host DNA flanking the different proviral genes. The cloned DNA was fractionated into site-specific restriction fragments which served as molecular probes in the analysis of the endogenous MMTV proviral copies of C3H, GR, BALB/c, and feral mice. This allowed the correlation of MMTV-specific EcoRI fragments obtained from genomic DNA of these strains with the 5' and 3' ends of the proviral gene. Restriction fragments of two clones which contained the proviral sequences adjacent to the flanking host DNA as well as 1 to 2 kilobases of host DNA were used as hybridization probes, and the results allow the following conclusions: the proviral DNA of both clones contains nucleotide sequences complementary to the 5' and 3' ends of proviral DNA; and the host DNA flanking one clone belongs to the unique class of genomic DNA, whereas the DNA flanking the second clone is reiterated at least 15 times within the mouse genome.
Insights
Researchers cloned mouse mammary tumor virus (MMTV) proviral DNA fragments. They analyzed MMTV DNA integrated into mouse genomes, revealing unique and reiterated host DNA sequences flanking proviral genes.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Mouse mammary tumor virus (MMTV) is a retrovirus associated with mammary tumors in mice.
- Understanding the integration and structure of MMTV proviral DNA in host genomes is crucial for studying viral pathogenesis and host-viral interactions.
Purpose of the Study:
- To molecularly clone and characterize MMTV proviral DNA fragments.
- To analyze the host DNA sequences flanking integrated MMTV proviral DNA in different mouse strains.
Main Methods:
- Molecular cloning of MMTV proviral DNA fragments using PstI and EcoRI restriction enzymes.
- Amplification of cloned DNA in plasmid vectors (pBr322) and lambda phage.
- Heteroduplex analysis and S1 nuclease digestion to assess sequence homology.
- Hybridization studies using cloned DNA fragments as probes to analyze endogenous MMTV proviral copies.
Main Results:
- Successfully isolated and amplified a 4-kilobase PstI fragment of unintegrated MMTV DNA.
- Isolated five lambda recombinant phages containing the 3' region of integrated MMTV proviral DNA and adjacent host DNA.
- Demonstrated no extensive sequence homology in host DNA flanking different proviral genes.
- Correlated MMTV-specific EcoRI fragments from genomic DNA with proviral gene ends.
- Identified host DNA flanking one MMTV clone as unique, while the other is reiterated.
Conclusions:
- Cloned MMTV proviral DNA contains sequences complementary to both 5' and 3' ends of the proviral DNA.
- The integration pattern of MMTV proviral DNA involves both unique and reiterated host genomic sequences.
- These findings provide insights into the integration mechanisms and potential host interactions of MMTV.