Doctoral Dissertations

Chin-Yih Ou

Date of Award

8-1982

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Wen-Kuang Yang

Committee Members

Francis T. Kenney, Fred Hartman, Raymond Tennant, Arthur Brown

Abstract

High molecular weight EcoR-I fragments of normal Balb/c mouse DNA, containing murine leukemia virus-related sequences, have been molecularly cloned in lambda Charon 9 vector. Of 14 independent isolates, the AL-10 clone with an insert of 10.5-kbp has been extensively characterized. It contains an MuLV-related sequence of 6.8-kbp in length which includes the left LTR, gag, pol and a portion of env genes. The sequence of thirty-four hundred nucleotides of this DNA has been determined. The MuLV provirus in the AL-10 DNA may represent a xenotropic provirus; however structural analysis suggests that it is not the iododeoxyuridine-inducible xenotropic or ectotropic locus of the Balb/c mouse.

AL-10 has a relatively large LTR of 695-bp; the U5 region is relatively conserved (77%), whereas the U3 region is highly variable as compared with the LTR sequence of Moloney MuLV. The 75-bp enhancer sequence present in Moloney MuLV LTR has significant alterations in AL-10 LTR and is not duplicated. A 165-bp sequence, flanked by 10-bp repeats, is located between the promoter and enhancer sequences of the U3 region. This sequence is not found in all the other known LTRs of ecotropic MuLVs. In vitro transcription assay demonstrates that the promoter of this LTR is functional; however, its activity is lower than that of WN1802N strain of MuLV. The sequence of the tRNA binding site for reverse transcription is not complementary to the 3' terminal sequence of mouse proline tRNA.

The coding sequence for the gag polyprotein ends with a TAG codon prior to the coding sequence for pol gene. As compared to the gag protein of Moloney MuLV, p30 is the most conserved protein in amino acid sequence, with only 11 changes out of 263 amino acids; p15 and p12 are highly variable with changes of 23% and 38% respectively, while p10 has a moderate change of 11%.

The sequence of a tropism-associated oligonucleotide of WN1802B virus, reported by Hopkins and coworkers, is located within the part of genome that determines p30 of AL-10. Base substitution in WN1802N virus results in 2 amino acid changes at the position of 109 and 110 of p30. This observation supports the hypotheses that the p30 sequence of WN1802B virus is derived from an endogenous xenotropic provirus and that the tropism conversion is caused by a subtle change in a stretch of a few amino acids in p30.

A 299-bp interspersed, repetitive sequence is present 127-bp upstream from the LTR. The 5' half of this sequence is related to the consensus sequence of the mouse repetitive sequence (B1) with a low degree of homology (60%). The 3' half of this sequence is also interspersed, repetitive in the mouse genome; however, it is not related to the B1 consensus sequence.

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