Influence of the R(61,2)- and S(61,2)-alpha-(N6-adenyl)styrene oxide adducts on the A.C mismatched base pair in an oligodeoxynucleotide containing the human N-ras codon 61.

Conformational studies of R- and S-alpha-(N6-adenyl)styrene oxide adducts mismatched with deoxycytosine at position X6 in d(CGGACXAGAAG).d(CTTCTCGTCCG), incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, are described. These were the R- and S(61,2)C adducts. The S(61,2)C adduct afforded a stable solution structure, while the R(61,2)C adduct resulted in a disordered structure. Distance restraints for the S(61, 2)C adduct were calculated from NOE data using relaxation matrix analysis. These were incorporated as effective potentials into the total energy equation. The structures were refined using restrained molecular dynamics calculations which incorporated a simulated annealing protocol. The accuracy of the emergent structures was evaluated by complete relaxation matrix methods. The structures refined to an average rms difference of 1.07 A, determined by pairwise analysis. The experimentally determined structure was compared to NOE intensity data using complete relaxation matrix back-calculations, yielding an R1x value of 11.2 x 10(-)2. The phenyl ring of the styrene in the S(61,2)C adduct was in the major groove and remained oriented in the 3'-direction as observed for the corresponding S(61,2) adduct paired with thymine [Feng, B., Zhou, L., Pasarelli, M., Harris, C. M., Harris, T. M., and Stone, M. P. (1995) Biochemistry 34, 14021-14036]. A shift of the modified adenine toward the minor groove resulted in the styrenyl ring stacking with nucleotide C5 on the 5'-side of the lesion, which shifted toward the major groove. Unlike the unmodified A.C mismatch, neither the S(61,2)C nor the R(61,2)C adduct formed protonated wobble A.C hydrogen bonds. This suggests that protonated wobble A.C pairing need not be prerequisite to low levels of alpha-SO-induced A --> G mutations. The shift of the modified adenine toward the minor groove in the S(61,2)C structure may play a more important role in the genesis of A --> G mutations. The disordered structure of the R(61,2)C adduct provides a potential explanation as to why that adduct does not induce A --> G mutations.

Molecular characterization of the human interleukin (IL)-17 receptor.

Human interleukin 17 (hIL-17) is a T-cell derived cytokine that exhibits 63% amino acid sequence identity to mouse IL-17 (mIL-17) and 57% identity to a viral protein encoded by the herpesvirus saimiri (HSV) gene 13 (HVS13). The IL-17 family of proteins binds to a unique mouse receptor (mIL-17R). Using nucleic acid hybridization techniques, a cDNA encoding a human homologue of the mIL-17R (hIL-17R) was isolated from a human T cell library. The predicted amino acid sequence of the hIL-17R is 69% identical to the mIL-17R, shares no homology with previously identified cytokine receptor families, and exhibits a broad tissue distribution. The hIL-17R gene was localized to chromosome 22. Monoclonal antibodies (mAbs) generated against the hIL-17R were able to block the IL-17-induced production of cytokine from human foreskin fibroblast (HFF) cells. Binding studies suggest that recombinant hIL-17 binds to the hIL-17R with low affinity.

Human IL-17: a novel cytokine derived from T cells.

A cDNA encoding human IL-17 (hIL-17) was cloned from a CD4+ T cell library. The predicted 155-amino acids sequence contains an N-terminal signal peptide and exhibits 72% amino acid identity with HVS13, an open reading frame from a T-lymphotropic Herpesvirus saimiri, and 63% with murine CTLA8. High levels of hIL-17 were induced from primary peripheral blood CD4+ T cells upon stimulation. When expressed in CV1/EBNA cells, recombinant hIL-17 was secreted in both glycosylated and nonglycosylated forms. A hIL-17.Fc fusion protein and supernatants from cells transfected with hIL-17 induced IL-6 and IL-8 production and enhanced the surface expression of the intracellular adhesion molecule-1 (ICAM-1) in human fibroblasts.

Herpesvirus Saimiri encodes a new cytokine, IL-17, which binds to a novel cytokine receptor.

Herpesvirus Saimiri gene 13 (HVS13) exhibits 57% identity with the predicted sequence of a T cell-derived molecule termed CTLA8. Recombinant HVS13 and CTLA8 stimulate transcriptional factor NF-kappa B activity and interleukin-6 (IL-6) secretion in fibroblasts, and costimulate T cell proliferation. An HVS13.Fc fusion protein was used to isolate a cDNA encoding a novel receptor that also binds CTLA8. This receptor is unrelated to previously identified cytokine receptor families. A recombinant soluble receptor inhibited T cell proliferation and IL-2 production induced by PHA, concanavalin A (conA), and anti-TCR MAb. These results define CTLA8 and HVS13 as novel cytokines that bind to a novel cytokine receptor. We propose to call these molecules IL-17, vIL-17, and IL-17R, respectively.

Genomic organization of the type I and type II IL-1 receptors.

Genomic clones spanning the human type I and type II interleukin-1 receptor loci have been isolated. Approximately 75 kb of genomic DNA is required to encode the type I receptor, and approximately 38 kb to encode the type II receptor. In each case, the receptor coding region is contained in only about 20 kb of DNA, with most of the rest of each locus representing the distance between the two (type II receptor) or three (type I receptor) presumptive promoters which drive transcription. The virtually identical location of introns within the ligand-binding portion of the coding regions reinforces the presumption that the two receptors derive from a common ancestor. Yeast artificial chromosomes (YACs) have been isolated which contain the two IL-1 receptors. One of the YACs also contains the T1/ST2 IL-1 receptor-related gene. The relative map position of these three genes has been determined.