Title

Characterization of the mouse strain carbon(3)hafnium/selenium germinally-transmitted mtv proviruses: Analysis of the transcriptional regulatory region and identification of mtv-1 as the specific provirus template of mammary tissue mtv RNA transcripts

Date of Award

1987

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Microbiology and Immunology

First Committee Member

Robert J. Pauley, Committee Chair

Abstract

The C3Hf/Se mouse strain contains three full-length endogenous mouse mammary tumor virus proviruses in germline DNA, designated mtv-1, mtv-8, and mtv-11. C3Hf/Se mtv germline proviruses were analyzed on the DNA level for transcriptional activity in mammary tissues. The 3$\sp\prime$ EcoR I fragment of mtv-1, the C3Hf/Se endogenous provirus, implicated in tumorigenesis, was cloned into lambda gt 10. The 4.5 kbp insert contained 2.9 kbp of 3$\sp\prime$ mtv structural gene sequences, the 1.3 kbp terminal repeat (LTR) and 0.3 kbp of host DNA. For sequence homology and diversity between the U3 region of mtv-1 LTR, mtv-8 U3 region, a 1.5 kbp Pst I-EcoR I fragment containing mtv-1 LTR and host sequences was subcloned into the M13 sequencing vectors. The 1.3 kbp LTR of mtv-1 was sequenced using synthetic oligonucleotides. No sequence modifications of the mtv-1 LTR explained its transcriptional activity. Other mtv proviruses remain cryptic. Characterization of mtv-1 LTR revealed sequence heterogeneity compared to the same region in mtv-8 LTR. Optimal sequence alignment of mtv-1 to mtv-8 LTR sequence revealed 123 bases in mtv-8 LTR was replaced with 116 bases within mtv-1 in which there were 73 unaligned bases compared to mtv-8, due to base mismatches, additions or deletions. This heterogeneity located 5$\sp\prime$ to the transcriptional regulatory sequences in mtv-1 altered the open reading frame (orf) of mtv-1 LTR. Computer translation of mtv-1 orf showed total disruption of COOH-terminus of putative orf proteins. Mtv-11 LTR transcriptional regulatory region was compared to mtv-8 LTR sequence. Promoter and regulatory regions were similar to mtv-8 and mtv-1 DNA sequence. The area 5$\sp\prime$ to the transcriptional regulatory sequences in mtv-11 U3 region unlike sequence alterations in mtv-1 LTR. Mtv-11 sequences of the LTR were 100% homologous to mtv-8 LTR sequences. The 116 base pair sequence alteration in the U3 region of mtv-1 LTR identified the mtv provirus template(s) for mtv RNA transcripts that accumulate in C3Hf/Se mammary tissues by S1 nuclease mapping. Results from the S1 nuclease mapping experiments demonstrated that mtv-1 is transcriptionally active in C3Hf/Se mammary tumors whereas mtv-8 and mtv-11 are either not transcriptionally active or are expressed at levels too low to be detected by the S1 method. (Abstract shortened with permission of author.)

Keywords

Biology, Microbiology

Link to Full Text

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