Identification of the SL3-3 virus enhancer core as a T-lymphoma cell-specific element.

Transient expression assays were used to determine the sequences within the long terminal repeat (LTR) that define the high activity in T-lymphoma cells of the leukemogenic SL3-3 virus in comparison with that of the nonleukemogenic Akv virus. Each of these viruses contains sequences related to the consensus element, the enhancer core. The SL3-3 and Akv enhancer cores differ at a single base pair. Substitution of the Akv core element into the SL3-3 LTR decreased expression in T-lymphoma cells but not in other cell types. Likewise, substitution of the SL3-3 core sequence into the Akv LTR increased expression in T-lymphoma cells but not in other types of hematopoietic cells. These data indicate that the SL3-3 enhancer core sequence functions better than that of Akv in T-lymphoma cells, but in other hematopoietic cell types the two are approximately equivalent. Competition DNA-protein binding assays were used to assess what nuclear factors from T-lymphoma lines and non-T lines bound to the SL3-3 and Akv core elements. Factors were detected that bound specifically to either the SL3-3 or Akv core but not to the other. Another factor was detected that bound equally well to both. However, none of these factors was specific to T-lymphoma cells.     

A single point mutation activates the Moloney murine leukemia virus long terminal repeat in embryonal stem cells.

The expression of Moloney murine leukemia virus (Mo-MuLV) and Mo-MuLV-derived vectors is restricted in undifferentiated mouse embryonal carcinoma and embryonal stem (ES) cells. We have previously described the isolation of retroviral mutants with host range properties expanded to embryonal cell lines. One of these mutants, the murine embryonic stem cell virus (MESV), is expressed in ES cell lines. Expression of MESV in these cells relies on DNA sequence motifs within the enhancer region of the viral long terminal repeat (LTR). Here we show that replacement of the Mo-MuLV enhancer region by sequences derived from the MESV LTR results in the activation of the Mo-MuLV LTR in ES cells. The enhancer regions of MESV and Mo-MuLV differ by seven point mutations. Of these, a single point mutation at position -166 is sufficient to activate the Mo-MuLV LTR and to confer enhancer-dependent expression to Mo-MuLV-derived retroviral vectors in ES cells. This point mutation creates a recognition site for a sequence-specific DNA-binding factor present in nuclear extracts of ES cells. This factor was found by functional assays to be the murine equivalent to human Sp1.     

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

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.     

An enhancer at the 3'' end of the mouse immunoglobulin heavy chain locus.

A tissue-specific enhancer (E mu) lies between the joining (JH) and mu constant region (C mu) gene segments of the immunoglobulin heavy chain (IgH) locus. Since mouse endogenous IgH genes are efficiently transcribed in its absence, the normal function of this enhancer remains ill-defined. Recently, another lymphoid-specific enhancer of equal strength has been identified 3' of the rat IgH locus. We have isolated an analogous sequence from mouse and have mapped it 12.5 kb 3' of the 3'-most constant region gene (C alpha-membrane) of the BALB/c mouse locus. The mouse and rat sequences are 82% homologous and share with other enhancers several DNA sequence motifs capable of binding protein. However, in transient transfection assays, the mouse sequence behaves as a weaker enhancer. The role of this distant element in the expression of endogenous IgH genes, both in E mu-deficient, Ig-producing cell lines and during normal B cell development, is discussed.