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A human myelomonocytic cell line, U937, produced an interleukin-1 (IL-1) receptor antagonist protein (IRAP) which was purified and partially sequenced. A complementary DNA coding for IRAP was cloned and sequenced. The mature translation product of the cDNA has been expressed in Escherichia coli and was an active competitive inhibitor of the binding of IL-1 to the T-cell/fibroblast form of the IL-1 receptor. Recombinant IRAP specifically inhibited IL-1 bioactivity on T cells and endothelial cells in vitro and was a potent inhibitor of IL-1 induced corticosterone production in vivo.

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Human platelet heparanase has been purified to homogeneity and shown to consist of two, non-covalently associated polypeptide chains of molecular masses 50 and 8 kDa. Protein sequencing provided the basis for determination of the full-length cDNA for this novel protein. Based upon this information and results from protein analysis and mass spectrometry, we propose a scheme to define the structural organization of heparanase in relation to its precursor forms, proheparanase and pre-proheparanase. The 8-and 50-kDa chains which make up the active enzyme reside, respectively, at the NH 2 -and COOH-terminal regions of the inactive precursor, proheparanase. The heparanase heterodimer is produced by excision and loss of an internal linking segment. This paper is the first to suggest that human heparanase is a two-chain enzyme.

The rat ROMK gene encodes inwardly rectifying, ATP-regulated K+ channels [K. Ho, C. G. Nichols, W. J. Lederer, J. Lytton, P. M. Vassilev, M. V. Kanazirska, and S. C. Hebert. Nature Lond. 362: 31-38, 1993; H. Zhou, S. S. Tate, and L. G. Palmer. Am. J. Physiol. 266 (Cell Physiol. 35): C809-C824, 1994], and mRNA encoding these channels is widely expressed in distal cortical and outer medullary nephron segments [see companion study; W.-S. Lee and S. C. Hebert. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F1124-F1131, 1995]. Using approaches based on homology to ROMK1, we have identified two additional ROMK isoforms, ROMK2b and ROMK3. Analysis of the nucleotide sequences of the ROMK isoforms indicates that molecular diversity of ROMK transcripts is due to alternative splicing at both the 5'-coding and 3'-noncoding regions. The splicing at the 5' end of ROMK gives rise to channel proteins with variable-length NH2 termini containing different initial amino acid sequences. Functional expression of these isoforms in Xenopus oocytes showed that they form functional Ba(2+)-sensitive K+ channels. The nephron distribution of mRNAs encoding alternatively spliced isoforms of ROMK (ROMK1-ROMK3) was investigated by reverse transcription-polymerase chain reaction (RT-PCR) of nephron segments dissected from rat kidney. Nondegenerate PCR primer pairs were designed to span at least one intron and to amplify specific alternatively spliced forms of ROMK.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations in the dystrophin gene are responsible for Duchenne and Becker muscular dystrophy (DMD/BMD). Studies of dystrophin expression and function have benefited from use of the mdx mouse, an animal model for DMD/BMD. Here we characterized mutations in three additional strains of mdx mice, the mdx2cv, mdx4cv and mdx5cv alleles. The mutation in the mdx2cv mouse was found to be a single base change in the splice acceptor sequence of dystrophin intron 42. This mutation leads to a complex pattern of aberrant splicing that generates multiple transcripts, none of which preserve the normal open reading frame. In the mdx5cv allele, the dystrophin mRNA contains a 53 bp deletion of sequences from exon 10. Analysis of the genomic DNA uncovered a single A to T transversion in exon 10. Although this base change does not alter the encoded amino acid, a new splice donor was created (GTGAG) that generates a frameshifting deletion in the processed mRNA. In the mdx4cv allele, direct sequencing revealed a C to T transition in exon 53, creating an ochre codon (CAA to TAA). The differential location of these mutations relative to the seven known dystrophin promoters results in a series of mdx mouse mutants that differ in their repertoire of isoform expression, such that these mice should be useful for studies of dystrophin expression and function. The mdx4cv and mdx5cv strains may be of additional use in gene transfer studies due to their low frequency of mutation reversion.

Tissue-and developmental stage-specific expression of the human 13-like globin genes is regulated by a combination of ubiquitous and erythroid-restricted trans factors that bind to cis elements near each of the five active genes. Additional interactions of these cis and trans factors with sequences located in the far 5' end of the cluster occur by as yet obscure mechanisms. Because of the complexity of this regulatory puzzle, precise identification of the determinants that control hemoglobin switching has proven difficult. Phylogenetic footprinting is an evolutionary approach to this problem which is based on the supposition that the basic mechanisms of switching are conserved throughout mammalian phylogeny. Alignment of the 5' flanking regions of the 'y genes of several species allows the identification of footprints of 100%/ conserved sequence. We have now tested oligomers spanning 13 such phylogenetic footprints and find that 12 are bound by nuclear proteins. One conserved element located at -1086 from the 'y genes exhibits repressor activity in transient transfection studies. The protein that binds this element, CSBP-1 (conserved sequence-binding protein 1), also binds at three sites within a silencer element upstream from the £ globin gene. Further analysis reveals that the CSBP-1 binding activity is identical to that of a recently cloned zinc finger protein that has been shown to act as a repressor in other systems. The binding of CSPB-1 to silencer sequences in the £ and 'y globin genes may be important in the stage-specific silencing of these genes.