Doctoral Dissertations

Date of Award

8-1986

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Wen K. Yang

Committee Members

Francis T. Kenney, Julian Preston, Kai-Lin Lee, Lawrence R. Boone

Abstract

The structures of specific regions of the murine leukemia virusrelated proviral DNA important in regulation of transcription, replica tion and host-range type specificity were determined by nucleotide sequence analysis in several endogenous MuLV-related sequences isolated from the RFM/Un mouse. Transcriptional activities and infectious properties of selected molecular clones of these sequences were also examined after transfection into a heterologous host cell line. Restriction endonuclease map analyses of 5 recombinant clones showed that only 5' portion of proviruses were present in pRFM#l and pRFM#9; that pRFM#16 and pRFM#17 were defective at least in the pol gene and that pRFM//6 harbored a full-length MuLV-related provirus. Analysis of the region which corresponds to the site of tRNA primer binding for initiation of (-) strand DNA synthesis in functional retroviruses demonstrated that 4 out of 5 clones contained binding sites with perfect complementary matches (18/18) with the 3'-terminal nucleotide sequence of a major rat glutamine tRNA isoacceptor. Furthermore, these sites showed dispersed 5 base mismatches with the 3'-terminal 18 nucleotide sequence of proline tRNA which serves as a primer in all ecotropic MuLV strains examined thus far. Structural analysis of the Integration site of pRFM#6 provirus showed that each inverted repeat at the terminus of the provirus was linked to a short direct repeat of cellular origin, a structure similar to those of integrated infectious proviruses or transposable gene elements. The 5' and 3' LTRs of pRFM#6 provirus were iv identical except for 2 base transitions and 1 nucleotide deletion/ addition in the U3 region. When compared with the infectious RFV provirus LTR, most of the transcriptional regulatory elements were identical and occurred at nearly the same distances from the mRNA cap site. While strong homologies were observed in the U5 and R regions, the U3 of pRFM#6 LTR contained a novel =170 bp sequence segment which was absent in RFV LTR. Sequence analysis of the endonuclease gene, predicted a polypeptide sequence of 390 amino acids for pRFM#6 provirus which was 15 residues shorter than the predicted endonuclease peptide sequence of AKV, an endogenous type C retrovirus of AKR mouse. Two polypeptides showed 86% sequence homology. Both by DNA gel blot analysis and nucleotide sequence analyses, the 5'-env gene of pRFM#6 provirus was found to be similar to that of the mink cell focus forming (MCF)-type murine leukemia virus but significantly different from those of ecotropic and xenotropic MuLVs. The cellular DNA site harboring the pRFM#6 provirus in RFM/Un mice, also harbors the provirus in genomes of BALB/c, AKR, C3H and CBA but not in those of C37BL/6 and NFS/N mice. The human HT1080 fibrosarcoma cells transfected with pRFM#6 and pRFM#16 DNAs, contained low amounts of MuLV-specific transcripts. A similar splicing mechanism was found in human and murine cells as evidenced by the presence of a 24S transcript encoding the env proteins in RNAs from both systems.

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