Doctoral Dissertations

Date of Award

8-1987

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Wen K. Yang

Committee Members

Ray Popp, Salil Niyogi, Kai-Len Lee, Julian Preston

Abstract

The genome of the RFM/Un mouse harbors many copies of elements related to marine leukemia viruses (MuLVs). Not all of these elements are capable of producing infectious virus particles without undergoing some modification or rearrangement. Nonetheless, these non-producing elements may have a profound effect upon the host organism.

Two MuLV-related elements have been characterized in detail. These elements were found in the chromosomal DNA of the RFM/Un mouse as solitary long terminal repeats (LTRs). Southern blotting experiments demonstrated that both solitary LTRs and the unique-sequence regions on either side of them were present in most inbred mouse strains examined; the flanking sequences were represented in several feral Mus species and Mus musculus subspecies, although the solitary LTRs were not necessarily present in these mice. Restriction of genomic DNA from liver and spleen of two inbred mouse strains with PstI and the methylation-sensitive endonuclease Smal, followed by hybridization of the resulting DNA fragments to the unique-sequence probes from the region 3' to each solitary LTR, revealed that neither element is detectably methylated at its LTR Smal site in at least one tissue in both mouse strains. Neither solitary LTR, however, appeared to be particularly sensitive to DNAse I digestion, leaving unresolved the possibility that such elements may be promoting the transcription of adjacent cellular sequences.

A similar approach was employed in the determination of DNAse I sensitivity and methylation status of MuLV LTR-related elements in DNA obtained from the spleen and liver of the RFM/Un mouse. Although there was no correlation between hypomethylation at the LTR Smal site and DNAse I sensitivity. Southern blotting experiments demonstrated a correlation between hypomethylation as detected by viral structural gene probes and the presence of an insertion into the U3 region of the LTR of MCF-type proviruses. Hence most of the MCF-type proviruses that harbor 170-200-bp insertions into their LTR U3 regions were relatively insensitive to Smal restriction as determined from use of the LTR probe; use of gag, gag-pol, and env gene probes, however, revealed that some of these MCF-type elements, unlike most endogenous ecotropic and xenotropic proviruses, were relatively hypomethylated at Smal sites within the structural genes. Although several members of this class of endogenous proviruses are known to have suffered deletions in their structural genes, it may be that they are responsible for a substantial portion of the MuLV-related transcripts observed in the RFM/Un mouse.

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