Negative regulatory element associated with potentially functional promoter and enhancer elements in the long terminal repeats of endogenous murine leukemia virus-related proviral sequences

Abstract

Three series of recombinant DNA clones were constructed, with the bacterial chloramphenicol acetyltransferase (CAT) gene as a quantitative indicator, to examine the activities of promoter and enhancer sequence elements in the 5' long terminal repeat (LTR) of murine leukemia virus (MuLV)-related proviral sequences isolated from the mouse genome. Transient CAT expression was determined in mouse NIH 3T3, human HT1080, and mink CCL64 cultured cells transfected with the LTR-CAT constructs. The 700-base-pair (bp) LTRs of three polytropic MuLV-related proviral clones and the 750-bp LTRs of four modified polytropic proviral clones, in complete structures either with or without the adjacent downstream sequences, all showed very little or negligible activities for CAT expression, while ecotropic MuLV LTRs were highly active. The MuLV-related LTRs were divided into three portions and examined separately. The 3' portion of the MuLV-related LTRs that contains the CCAAC and TATAA boxes was found to be a functional promoter, being about one-half to one-third as active as the corresponding portion of ecotropic MuLV LTRs. A MboI-Bg/II fragment, representing the distinct 190- to 200-bp inserted segment in the middle, was found to be a potential enhancer, especially when examined in combination with the simian virus 40 promoter in CCL64 cells. A PstI-MboI fragment of the 5' portion, which contains the protein-binding motifs of the enhancer segment as well as the upstream LTR sequences, showed moderate enhancer activities in CCL6 cells but was virtually inactive in NIH 3T3 cells and HT1080 cells; addition of this fragment to the ecotropic LTR-CAT constructs depressed CAT expression. Further analyses using chimeric LTR constructs located the presence of a strong negative regulatory element within the region containing the 5' portion of the enhancer and the immediate upstream sequences in the MuLV-related LTRs.

Alignment of U3 region sequences of mammalian type C viruses: identification of highly conserved motifs and implications for enhancer design

“…The GRE site was retained in most viruses. Finally, as noted previously by others, many endogenous viruses have a 190-bp insert between their enhancer and promoter; this element may in itself provide an enhancer function (8,36). Thus, the enhancer regions of these viruses were frequently distinct from those of the exogenous viruses.…”

Origins of enhancer sequences of recombinant murine leukemia viruses from spontaneous B- and T-cell lymphomas of CWD mice

“…Perhaps a more plausible explanation is that, unlike Bxv-1, the donor of the CWM-T-15 enhancer core region sequences contains defective or inhibitory U3 region sequences that flank the boundaries of the enhancer core region. In support of this hypothesis are reports from other investigators that the endogenous polytropic proviruses (which appear to be closely related to the donor of the CWM-T-15 enhancer sequences) contain substitutions, deletions, insertions, and negative regulatory elements in the U3 region that are absent in the allelic sequences of replication-competent MuLVs (5,14,42,43,45). Thus, viruses that acquire a large segment of the U3 region of the CWM-T-15 donor and the associated defective sequences may be subject to a negative selection.…”

DNA breaks and chromosomal aberrations arise when replication meets base excision repair

“…MPG-overexpressing T02-M5 and T02-M9 cells were obtained using the expression vector pEZ-1 that was constructed from a pGB4 vector and the human MPG cDNA sequence in the plasmid pPG23 (Chakravarti et al, 1991;Ibeanu et al, 1992). pGB4 is a high-level expression vector for mammalian cells under the control of LTR promoter of WNB5 ectopic mouse leukemia virus (Ch'ang et al, 1989). MPG / MEFs were generated from MPG knockout mouse embryos.…”