Abstract
Expression of mouse mammary tumor virus in T lymphocytes appears to be required for accession of horizontally transmitted virus to the mammary gland. Further, deletions in the long terminal repeat which relax constraints on viral transcription promote T cell lymphoma. We have identified a polypurine transcriptional repressor element (NRE1) that is deleted from viruses that induce T cell lymphoma. NRE1 binding activity in nuclear extracts proved to be related to a growth inhibitory activity that represses c-myc expression in mature B cells. Mobility shift, DNA footprinting, and UV cross-linking identified three factors that interacted preferentially with double-stranded NRE1 or the separated single strands. NRE1 binding factor (NBF) (80 kDa) bound double and upper strand NRE1, apparently in concert with NRE1 associated factor (NAF) (95 kDa), which interacted directly with DNA only on the upper strand. NRE1 lower strand binding factor (NLF) (50 kDa) cross-linked specifically to lower strand NRE1. On sucrose gradients NBF, NAF, and NLF binding activities cosedimented at 8 S, implying an in vitro association of the 50-, 80-, and 95-kDa factors which precedes DNA binding. Therefore, NRE1 appears to be the site of action of a complex transcriptional repressor comprised of at least three factors that interact differentially with each DNA strand to repress steroid hormone-induced transcription of mouse mammary tumor virus in T cells.
Highlights
From the Departmentsof $Medicine and §Biochemistry, University of Ottawa, Loeb Institute for Medical Research, Ottawa Civic Hospital, Ottawa, OntarioK l Y 4E9, Canada
Expressionofmousemammarytumor virus in T plasmacytoma releases a block on c-myc expression, in part lymphocytes appears to be required for accession of by separating the c-myc promoter from a binding sitefor horizontally transmittedvirus to the mammary gland. plasmacytoma repressor factor (PRF),’ a growth inhibitory
Nuclear extracts were prepared from two tissue culture cell lines: Jurkat T cells, in which deletion of the mammary tumor virus (MMTV) long terminal repeat (LTR) between -530 and -180 has been shown previously to result in astrong induction of transcription [33]; and CAC-E1A cells, an MMTV transformed mammary epithelial cell line in which transcription of endogenousvirus has ceased [47].Both nuclear extracts formed three complexes on the DNA, but the major complex derived from the CAC-E1A extract hada distinctly slower mobility than the comparable Jurkat complex
Summary
Oligonucleotides and Phmids-Oligonucleotides were synthesized stranded MTV DNA was prepared by extension of a 6-nucleotide on an Applied Biosystems DNA synthesizer (University of Ottawa primer (5'-AACTGA-3'), hybridized to lower strand MTV, with. Plasmid pHCWT was by SDS-polyacrylamide gel electrophoresis, 0.2 ng of specific DNAs cloned by isolating the 130-bp HindIIIISacI (-237/-107) fragment were incubated in binding buffer with 1-12 pg of double- or singlefrom pHCWT [28] and introducing it into the HindIII/SacI site of stranded calf thymus DNA, 0.5pg of BSA, and 10 pgof nuclear pMMTVCAT [38]. Tions were performed by a modification of the electroporation protocol For cross-linking followed bymobility shift, the quantity of specific of Ustav and Stenlund [39]. Electroporation conditions in a directly into wells of 10% SDS-polyacrylamidegels with an extended cell porator electroporation apparatus (Life Technologies, Inc.) were 6%stacking gel and electrophoresed for 2400 V/h, dried, and exposed as follows: Jurkat, 240 V, 1180p F CAC-ElA, 340V, 800 NMuMG; as described above. DNA binding assays were performed using 0.5 ng of 32P-kinasedspecific DNA as indicated, in
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