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Published on: June 25, 2014
Cloning and characterization of an envelope-specific probe from xenotropic murine leukemia proviral DNA
Abstract:
An 8.9-kilobase EcoRI restriction fragment was cloned from mink cells chronically infected with NFS-Th-1 xenotropic murine leukemia virus by using a lambda phage host vector system. After its transfer into pBR322, the EcoRI DNA insert was characterized and found to contain 6.7 kilobases of proviral DNA sequences and 2.2 kilobases of mink cellular DNA flanking the 5' end of the viral genome. A 500-base pair fragment which was located at the 3' terminus of the cloned DNA insert and which mapped to the env region of xenotropic proviral DNA was subcloned into pBR322. This xenotropic envelope proviral DNA segment did not hybridize to ecotropic murine leukemia proviruses but did anneal to representative alpha and beta xenotropic and seven different mink cell focus-inducing proviral DNAs. The cloned xenotropic envelope-specific probe was also used in blot hybridization experiments to analyze the arrangement of related sequences in preparations of different mouse liver DNAs.
Insights
Researchers cloned xenotropic murine leukemia virus DNA from infected mink cells. A specific DNA segment targeting the env region was identified and used to analyze viral sequences in mouse DNA.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Murine leukemia viruses (MLVs) are retroviruses implicated in various cancers.
- Xenotropic MLVs (X-MLVs) exhibit a broad host range, including non-murine species.
- Understanding X-MLV integration and host interactions is crucial for cancer research.
Purpose of the Study:
- To clone and characterize xenotropic murine leukemia virus (X-MLV) proviral DNA from infected mink cells.
- To develop a specific DNA probe for the X-MLV env gene.
- To investigate the presence and arrangement of X-MLV related sequences in mouse DNA.
Main Methods:
- Cloning of an 8.9-kilobase EcoRI restriction fragment from mink cells infected with NFS-Th-1 X-MLV using a lambda phage host vector system.
- Subcloning of a 500-base pair fragment from the 3' terminus of the cloned DNA, mapping to the env region of X-MLV proviral DNA.
- Hybridization studies using the subcloned fragment to analyze X-MLV related sequences in various viral DNAs and mouse liver DNA.
Main Results:
- Successfully cloned and characterized an 8.9-kb fragment containing X-MLV proviral DNA and flanking mink cellular DNA.
- Developed a 500-bp probe specific to the X-MLV env region, which did not hybridize to ecotropic MLVs.
- The probe hybridized to alpha and beta xenotropic MLVs and mink cell focus-inducing (MCF) proviral DNAs, indicating sequence homology.
- Blot hybridization revealed the arrangement of related sequences in different mouse liver DNA preparations.
Conclusions:
- A specific DNA probe for the xenotropic murine leukemia virus env gene was successfully generated.
- This probe facilitates the detection and analysis of X-MLV and related sequences in various host genomes.
- The findings contribute to understanding MLV diversity and host-virus interactions, relevant for oncogenesis research.
