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The IL-1R antagonist protein (IRAP) is a competitive inhibitor of IL-1, which is predominantly synthesized by monocytes. We show that this molecule is also expressed in human synovial fibroblasts and dermal fibroblasts (CRL 1445). IRAP mRNA was regulated in a time- and dose-dependent manner by IL-1 alpha, TNF-alpha, LPS, and PMA. Maximal induction of IRAP mRNA was observed between 8 and 16 h after stimulation with IL-1 alpha (1 U/ml), TNF-alpha (10 U/ml), LPS (50 ng/ml), and PMA (10 ng/ml). Their relative efficacy was as follows: PMA > LPS > IL-1 alpha > TNF-alpha. Potentiation was observed when fibroblasts were treated with IL-1 alpha plus basic fibroblast growth factor and IL-1 alpha plus platelet-derived growth factor-BB homodimer. Although LPS and PMA were the best inducers of IRAP mRNA, quantitation of the IRAP protein revealed that its synthesis and release were differentially regulated. Immunoprecipitation and SDS-PAGE of culture supernatant from LPS-treated cells and cell lysates of fibroblasts treated with LPS or PMA showed a single IRAP band with a molecular mass of approximately 22 kDa. Very little IRAP was detected in culture supernatants of cells treated with PMA. Quantitation of IRAP revealed that LPS induced the synthesis of secreted IRAP that was released, whereas the majority of the protein induced by PMA remained cell-associated. Reverse transcriptase-polymerase chain reaction amplification demonstrated that although LPS and PMA induced both transcripts, LPS preferentially induced secreted IRAP, whereas PMA differentially induced intracellular IRAP mRNA. Fibroblasts synthesize at least two different forms of IRAP depending on the inducing signal, and may regulate the inflammatory response by dampening the proinflammatory effects of IL-1 via a negative feedback mechanism with IRAP. The relative importance of fibroblast sIRAP vs intracellular IRAP in regulating the inflammatory response by the connective tissue remains to be determined.

The IL-1R antagonist protein (IRAP) is a competitive inhibitor of IL-1, which is predominantly synthesized by monocytes. We show that this molecule is also expressed in human synovial fibroblasts and dermal fibroblasts (CRL 1445). IRAP mRNA was regulated in a time- and dose-dependent manner by IL-1 alpha, TNF-alpha, LPS, and PMA. Maximal induction of IRAP mRNA was observed between 8 and 16 h after stimulation with IL-1 alpha (1 U/ml), TNF-alpha (10 U/ml), LPS (50 ng/ml), and PMA (10 ng/ml). Their relative efficacy was as follows: PMALPSIL-1 alphaTNF-alpha. Potentiation was observed when fibroblasts were treated with IL-1 alpha plus basic fibroblast growth factor and IL-1 alpha plus platelet-derived growth factor-BB homodimer. Although LPS and PMA were the best inducers of IRAP mRNA, quantitation of the IRAP protein revealed that its synthesis and release were differentially regulated. Immunoprecipitation and SDS-PAGE of culture supernatant from LPS-treated cells and cell lysates of fibroblasts treated with LPS or PMA showed a single IRAP band with a molecular mass of approximately 22 kDa. Very little IRAP was detected in culture supernatants of cells treated with PMA. Quantitation of IRAP revealed that LPS induced the synthesis of secreted IRAP that was released, whereas the majority of the protein induced by PMA remained cell-associated. Reverse transcriptase-polymerase chain reaction amplification demonstrated that although LPS and PMA induced both transcripts, LPS preferentially induced secreted IRAP, whereas PMA differentially induced intracellular IRAP mRNA. Fibroblasts synthesize at least two different forms of IRAP depending on the inducing signal, and may regulate the inflammatory response by dampening the proinflammatory effects of IL-1 via a negative feedback mechanism with IRAP. The relative importance of fibroblast sIRAP vs intracellular IRAP in regulating the inflammatory response by the connective tissue remains to be determined.

We constructed mouse dihydrofolate reductase (DHFR) minigenes (dhfr) that had 1.5 kilobases of 5' flanking sequences and contained either none or only one of the intervening sequences that are normally present in the coding region. They were greater than or equal to 3.2 kilobase long, about one-tenth the size of the corresponding chromosomal gene. Both of these minigenes complemented the DHFR deficiency in Chinese hamster ovary dhfr-1-cells at a high frequency after DNA-mediated gene transfer. The level of DHFR enzyme in various transfected clones varied over a 10-fold range but never was as high as in wild-type Chinese hamster ovary cells. In addition, the level of DHFR in primary transfectants did not vary directly with the copy number of the minigene, which ranged from fewer than five to several hundred per genome. The minigenes could be amplified to a level of over 2,000 copies per genome upon selection in methotrexate, a specific inhibitor of DHFR. In one case, the amplified minigenes were present in a tandem array; in two other cases, a rearranged minigene plasmid and its flanking chromosomal DNA sequence were amplified. Thus, the mouse dhfr minigenes could be transcribed, expressed, and amplified in Chinese hamster ovary cells, although the efficiency of expression was generally low. The key step in the construction of these minigenes was the generation in vivo of lambda phage recombinants by overlapping regions of homology between genomic and cDNA clones. The techniques used here for dhfr should be generally applicable to any gene, however large, and could be used to generate novel genes from members of multigene families.

The use of murine dihydrofolate reductase (dhfr) gene amplification mutants enabled us to identify important structural and functional features of the dhfr promoter region. We found another transcription unit, at least 14 kilobases in size, which initiates within 130 base pairs of the major dhfr transcript and is transcribed divergently. The 5' ends of both transcripts were analyzed and found to have multiple initiation sites. The major dhfr transcript and the divergent transcript appear to share the same promoter region; the longer transcripts of the dhfr gene overlap with the divergent transcripts and use a different promoter region. The divergent transcript appears to code for a protein; an homologous sequence to its first exon is found in the corresponding location near the human dhfr gene.

The use of murine dihydrofolate reductase (dhfr) gene amplification mutants enabled us to identify important structural and functional features of the dhfr promoter region. We found another transcription unit, at least 14 kilobases in size, which initiates within 130 base pairs of the major dhfr transcript and is transcribed divergently. The 5' ends of both transcripts were analyzed and found to have multiple initiation sites. The major dhfr transcript and the divergent transcript appear to share the same promoter region; the longer transcripts of the dhfr gene overlap with the divergent transcripts and use a different promoter region. The divergent transcript appears to code for a protein; an homologous sequence to its first exon is found in the corresponding location near the human dhfr gene.

We constructed a gene library from a murine cell line with amplified dihydrofolate reductase (dhfr) genes by inserting random segments of DNA into lambda Ch4A. From this library, the dhfr gene and 30 kilobase pairs of surrounding DNA were cloned, and the restriction map was determined. All of the coding regions were sequenced and show that the gene spans a total of 31 kilobase pairs and has five intervening sequences in the coding portion of the gene. In addition, two classes of variant dhfr genes were found in the amplified line, which were amplified and present at levels of 10 to 30% of the normal dhfr genes. Numerous repeated sequences were located throughout the gene region, some of which share homology with previously defied families of repeats.

Terminal deoxynucleotidyl transferase (TdT) is uniquely associated with the major thymocyte population and a minor bone marrow population which is separable on bovine serum albumin gradients. The expression of TdT in the bone marrow subpopulation is under thymic regulation in that this population is significantly reduced in nude mice or is lost with time after thymectomy. The expression of TdT in athymic mice can be induced both in vivo and in vitro by thymosin fraction 5 or by beta3, a peptide purified from thymosin fraction 5. The optimal concentration of beta3 in the in vitro induction of TdT is approximately 10 ng/ml. These results demonstrate that thymic hormones control the early differentiation of prothymocytes in the bone marrow.

The distribution of terminal deoxynucleotidyl transferase (TdT) peaks I and II, in single cell suspensions of thymuses, bone marrow, and peripheral lymphoid organs fractionated in discontinuous bovine serum albumin gradients, was examined in a variety of mouse strains and Fischer 344 rats to relate the normal patterns of thymocyte differentiation to the leukemic process. TdT peaks I and II were found in fractions A (10 to 23%), B (23 to 26%), and C (26 to 29%) of the thymus of both normal and leukemic C57BL/6 mice, whereas only peak I was found in the same fractions of AKR mice. TdT in bone marrow was found mainly in fraction A in both normal and leukemic mice. The specific activity of TdT in this fraction, which comprises only 1 to 5% of the total bone marrow cell population, was similar to that of the thymus. The cell population of fraction A of the bone marrow was found to increase (10 to 15-fold) in leukemic mice. Only low levels of TdT activity were found in either whole or fractionated bone marrow of athymic NIH Swiss (nu/nu) mice.

We constructed a gene library from a murine cell line with amplified dihydrofolate reductase (dhfr) genes by inserting random segments of DNA into lambda Ch4A. From this library, the dhfr gene and 30 kilobase pairs of surrounding DNA were cloned, and the restriction map was determined. All of the coding regions were sequenced and show that the gene spans a total of 31 kilobase pairs and has five intervening sequences in the coding portion of the gene. In addition, two classes of variant dhfr genes were found in the amplified line, which were amplified and present at levels of 10 to 30% of the normal dhfr genes. Numerous repeated sequences were located throughout the gene region, some of which share homology with previously defied families of repeats.