cDNA clones corresponding to an Mr approximately 80,000 receptor (type I receptor) for interleukin‐1 (IL‐1) have been isolated previously by mammalian expression. Here, we report the use of an improved expression cloning method to isolate human and murine cDNA clones encoding a second type (Mr approximately 60,000) of IL‐1 receptor (type II receptor). The mature type II IL‐1 receptor consists of (i) a ligand binding portion comprised of three immunoglobulin‐like domains; (ii) a single transmembrane region; and (iii) a short cytoplasmic domain of 29 amino acids. This last contrasts with the approximately 215 amino acid cytoplasmic domain of the type I receptor, and suggests that the two IL‐1 receptors may interact with different signal transduction pathways. The type II receptor is expressed in a number of different tissues, including both B and T lymphocytes, and can be induced in several cell types by treatment with phorbol ester. Both IL‐1 receptors appear to be well conserved in evolution, and map to the same chromosomal location. Like the type I receptor, the human type II IL‐1 receptor can bind all three forms of IL‐1 (IL‐1 alpha, IL‐1 beta and IL‐1ra). Vaccinia virus contains an open reading frame bearing strong resemblance to the type II IL‐1 receptor.

Sequences that comprise the 244-base-pair polyomavirus enhancer region are also required in cis for viral DNA replication (Tyndall et al., Nucleic Acids Res. 9:6231-6250, 1981). We have studied the relationship between the sequences that activate replication and those that enhance transcription in two ways. One approach, recently described by de Villiers et al. (Nature [London], 312:242-246, 1984), in which the polyomavirus enhancer region was replaced with other viral or cellular transcriptional enhancers suggested that an enhancer function is required for polyomavirus DNA replication. The other approach, described in this paper, was to analyze a series of deletion mutants that functionally dissect the enhancer region and enabled us to localize four sequence elements in this region that are involved in the activation of replication. These elements, which have little sequence homology, are functionally redundant. Element A (nucleotides 5108 through 5130) was synthesized as a 26-mer with XhoI sticky ends, and one or more copies were introduced into a plasmid containing the origin of replication, but lacking the enhancer region. Whereas one copy of the 26-mer activated replication only to 2 to 5% of the wild-type level, two copies inserted in either orientation completely restored replication. We found that multiple copies of the 26-mer were also active as a transcriptional enhancer by measuring the l-globin mRNA levels expressed from a plasmid that contained either the polyomavirus enhancer or one or more copies of the 26-mer inserted in a site 3' to the 13-globin gene. We observed a correlation between the number of inserted 26-mers and the level of 3-globin RNA expression.Transcriptional enhancers were originally identified within the genomes of DNA tumor viruses simian virus 40 (SV40) (2, 38) and polyomavirus (8, 9) as DNA segments that stimulate the transcription of linked genes in an orientationindependent manner and that function over long distances (16,26). Many other viral (19,20,22,[27][28][29][30][31][32] and several cellular (1, 14, 15, 41) enhancers have since been described. Although the general importance of enhancers in the regulation of transcription has been clearly demonstrated, numerous questions about their structure and function remain unanswered. There are striking DNA sequence homologies shared among several enhancers, but no unique consensus sequence common to all of them has emerged. Some enhancers contain direct tandem repeats of sequences about 70 base pairs (bp) in length. The repeats represent redundant copies of the same signal, because deletion of either does not alter transcriptional efficiency (2). Other enhancers lack direct repeats, but still involve elements dispersed over 100 to 300 bp that appear to be in some way functionally redundant. The polyomavirus enhancer was the first example of the latter situation (9, 46). This enhancer occurs within a 244-bp DNA fragment that also includes cis-acting elements essential for viral DNA replication. The sequences within the polyomavir...

The Epstein-Barr virus (EBV) genome becomes established as a multicopy plasmid in the nucleus of infected B lymphocytes. A cis-acting DNA sequence previously described within the BamHI-C fragment of B95-8 strain, encoding the EBV-associated nuclear antigen EBNA-1), the cis-acting sequence from the BamHI-C fragment, and a dominant selectable marker gene encoding G-418 resistance in animal cells. After being transfected into HeLa cells, this plasmid persisted extrachromosomally at a low copy number, with no detectable rearrangements or deletions. Two mutations in the BamHI-K-derived portion of p410+, a large in-frame deletion and a linker insertion frameshift mutation, both of which alter the carboxy-terminal portion of EBNA-1, destroyed the ability of the plasmid to persist extrachromosomally in HeLa cells. A small in-frame deletion and linker insertion mutation in the region encoding the carboxy-terminal portion of EBNA-1, which replaced 19 amino acid codons with 2, had no effect on the maintenance of p410+ in HeLa cells. These observations indicate that EBNA-1, in combination with a cis-acting sequence in the BamHI-C fragment, is in part responsible for extrachromosomal EBV-derived plasmid maintenance in HeLa cells. Two additional activities have been localized to the BamHI-C DNA fragment: (i) a DNA sequence that could functionally substitute for the simian virus 40 enhancer and promoter elements controlling the expression of G-418 resistance and (ii) a DNA sequence which, although not sufficient to allow extrachromosomal plasmid maintenance, enhanced the frequency of transformation to G-418 resistance in EBV-positive (but not EBV-negative) cells. These findings suggest that the BamHI-C fragment contains a lymphoid-specific or EBV-inducible promoter or enhancer element or both.

Sequences which activate polyoma virus DNA replication are located within a region that also includes the transcriptional enhancer. We demonstrate a cis involvement of enhancers in DNA replication by showing that this region can be replaced by other enhancers, in a position- and orientation-independent manner, and that an immunoglobulin gene enhancer confers tissue-specific replicatory ability.

Epstein-Barr virus (EBV) genome contains two cis-acting elements which are required for stable extrachromosomal plasmid maintenance in latently infected cells. The first consists of 20 30-base-pair (bp) repeats, each of which contains a DNA-binding site for EBV nuclear antigen 1 (EBNA-1), the transacting factor required for plasmid persistence. The second element is composed of a 65-bp dyad symmetry, containing four EBNA-1-binding sites. Deletion mutants were constructed which reduce the number of EBNA-1-binding sites in the 30-bp repeats, alter the number of EBNA-1-binding sites in the dyad region, or truncate the dyad element. The effect of the deletion mutations on plasmid maintenance was examined by transfecting recombinant plasmids, containing both the mutated EBV sequences and a drug resistance marker, into D98-Raji cells. The plasmids were tested for their ability to generate drug-resistant D98-Raji cell colonies and their capacity to be maintained in an extrachromosomal form without undergoing extensive rearrangements. EBV plasmids with 12 or 15 copies of the 30-bp repeats were wild type in both assays. Plasmids with just two or six copies of these repeated elements failed to generate drug-resistant colonies at a normal level, and normal episomal plasmids were not detected in the resulting colonies. Rare colonies of cells resulting from transfection of these twoor six-copy mutants contained rearranged, episomal forms of the input plasmids. The rearrangements most often produced head-to-tail oligomers containing a minimum of eight 30-bp repeated elements. The rearranged plasmids were shown to be revertant for plasmid maintenance in that they yielded wild-type or greater numbers of drug-resistant colonies and persisted at the wild-type or a greater episomal copy number. By use of an EBV plasmid that contained no 30-bp elements, no revertants could be isolated. One to five copies of a synthetic linker corresponding to a consensus 30-bp repeated element inserted into a plasmid with no 30-bp elements now permitted the generation of oligomeric, episomal forms of the mutant test plasmid. These experiments demonstrate a requirement for a minimal number (six to eight copies) of the 30-bp repeated element. Deletions in the 65-bp dyad region had little or no effect upon the ability to generate enhanced numbers of drug-resistant D98-Raji colonies, indicating that the 30-bp repeated element is predominantly required for this phenotype. Plasmids bearing deletions in the 5' side of the dyad sequence retained the capacity to produce stimulated numbers of drug-resistant colonies, but the extrachromosomal plasmids in these cells all contained structural rearrangements. In contrast, deletions impinging on the 3' side of the dyad element completely abolished extrachromosomal plasmid maintenance. Simian virus 40 enhancer sequences placed adjacent (3' side) to the 65-bp dyad also abolished the ability of the EBV plasmids to be maintained in an extrachromosomal state. These studies identify the regions of the 65-bp dyad that ar...