The enhancer elements from either simian virus 40 or murine sarcoma virus activate the expression of a transfected rat insulin 1 (r11) gene when placed within 2.0 kilobases or less of the rl1 gene cap site. Inclusion of 4.0 kilobases of upstream r11 sequence, however, results in a substantial reduction in the enhancer-dependent insulin gene expression. These observations suggested that a negative transcriptional regulatory element was present between 2.0 and 4.0 kilobases of the r11 sequence. To test this notion, we employed a heterologous enhancer-dependent transcription assay in which the simian virus 40 72-base-pair repeat is linked to a human f8-globin gene. Addition of the upstream rI1 element to this system decreased the level of enhancer-dependent fl-globin transcription by a factor of 5 to 15. This rIb "silencer" element functions in a manner relatively independent of position and orientation and requires a cis-dependent relationship to the transcription unit on which it acts. Thus, the silencer sequence seems to have a number of the characteristics of enhancer elements, and we suggest that it may function by the converse of the enhancer mechanism. The rIb silencer sequence was identified as a member of a long interspersed rat repetitive family. Thus, a potential role for certain repetitive sequences interspersed throughout the eukaryotic genome may be to regulate gene expression by retaining transcriptional activity within defined domains.Enhancers are cis-dependent DNA sequences that activate the transcription of viral and cellular genes in a manner relatively independent of position and orientation (1,2). This transcriptional activation can occur over distances as great as 6 kilobases (kb) (3, 4) from either the 5' or 3' end of the gene. What appears to be more important than the distance between an enhancer and the promoter being assayed for transcription are the specific sequences that intervene. Thus, for example, if an enhancer is flanked on both sides by active promoters, its effect on more distal promoter elements is likely to be significantly reduced (4-6).In studying the sequences upstream of the rat insulin 1 (rIb) gene, we have identified an element that appears to suppress enhancer-dependent transcriptional activity by a distinct mechanism. In fact, this negative regulatory element, which we will refer to as a silencer (see ref. 7), appears to have many of the characteristics of enhancers including position-and orientation-independent function on a heterologous gene. The identification of this rI, silencer element as a member of a family of long interspersed rat repetitive sequences (LINES; refs. 8 and 9) suggests a potential role for some of these repetitive elements in gene regulation. MATERIALS AND METHODSPlasmid Constructions. The recombinant plasmids used in this study were constructed according to methods that use standard recombinant DNA technology (10). The parental vector irSVHPA128 (kindly provided by M. Green and T.Maniatis) contains the human P3-globin gene with its prom...

A simian virus 40 genomic fragment containing the genes coding for the large T and small t antigens was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the strong polyhedrin promoter. Infection of eucaryotic Spodopterafrugiperda (SF9) cells with this recombinant virus produced significant amounts of small t antigen and little or no large T protein. Analysis by Northern blotting and S1 nuclease digestion revealed correct and preferential utilization of the small t splicing signals.