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Notes: Abstract The human IFI16 gene is a member of an interferon-inducible family of mouse and human genes closely linked on syntenic regions of chromosome 1. Expression of these genes is largely restricted to hemopoietic cells, and is associated with the differentiation of cells of the myeloid lineages. As a prelude to defining the mechanisms governing IFI16 expression, we have deduced its genomic organization using a combination of genomic cloning and polymerase chain reaction amplification of genomic DNA. IFI16 consists of ten exons and nine intervening introns spanning at least 28 kilobases (kb) of DNA. The reiterated domain structure of IFI16 protein is closely reflected in its intron/exon boundaries, and may represent the evolutionary fusion of several independent functional domains. Thus, exon 1 consists of 5' untranslated (UT) sequences and contains sequence motifs that may confer interferon-inducibility, and exon 2 encodes the lysine-rich amino-terminal (“K”) region, which possesses DNA-binding activity. Exon 3 codes for a domain which is poorly conserved between family members, except for a strongly retained basic motif likely to provide nuclear localization. The first of two 200 amino acid repeat domains that are the hallmark of this family (domain A) is represented jointly on exons 4 and 5, which are reiterated as exons 8 and 9, respectively, to encode the second 200 amino acid domain (B). Two intervening serine-threonine-rich domains (C and C'), unique to IFI16, are each encoded by single exons of identical length (exons 5 and 6). These domains are predicted to encode semi-rigid “spacer” domains between the 200 amino acid repeats. The reiterated nature of exons 4 to 6 and the insertion of introns into a single reading frame strongly suggest that IFI16 and related genes arose by a series of exon duplications, some of which antedated speciation into mouse and humans. Several alternative mRNA cap sites downstream of a TATA consensus sequence were defined, using primer extension analysis of mRNA. Sequencing of ~1.7 kb of DNA upstream of this region revealed no recognizable consensus elements for induction by interferon-α (interferon-α/ß-stimulated response elements), but two motifs resembling interferon-γ activation sites were located. IFNs α and γ both induce IFI16 mRNA expression in myeloid cells. Interferon-α inducibility of IFI16 may be regulated by an interferon-α/ß-stimulated response consensus element in the 5' UT exon, as a similar motif is conserved in the corresponding position in the related myeloid cell nuclear differentiation antigen gene. An interferon-γ-activation site consensus was also located in this region of IFI16.

Notes: Abstract A cluster of at lest six interferon-γ (IFNγ)-inducible genes designated Ifi201-204 and located on mouse chromosome 1 has recently been described. Here , we report a human IFN-γ-inducible gene, IFI 16, which has nucleotide sequence similarity with portions of two of the mouse genes, Ifi202 and Ifi204. A full-length cDNA clone derived from IFI 16 [2.709 kilobases (kb)] contained a single open reading frame of 2.187 kb which encoded a putative polypeptide of 729 amino acids and a predicted non-glycosylated M r of 80020. IFI 16 mRNA was found to be constitutively expressed in lymphoid cells and in cell lines of both the T and B lineages. By contrast, the mRNA was not expresed by the cell lines HL-60, U937, and K562, which represent early stages of myeloid development, but was strongly inducible in HL-60 and U937 with IFN-γ. The IFI 16 protein demonstrated a putative domain structure with patchy similarity to the proteins expressed from gene Ifi202 and Ifi204. The mouse and human proteins each contain two analogous ≈200 amino acid domains which are imperfect copies, but IFI 16 demonstrated additional unique regions, including a Lys-rich N-terminal portion and a “spacer” region between the reiterated domains, analogous to spacer regions in the CD5 and CD8α molecules. Using a panel of inter-species somatic cell hybrid cell lines, IFI 16 was localized to the chromosomal region 1q12→1qter, a region systenic between mouse an man. DNA blotting indicated that, in contrast to the mouse, IFI 16 is present as a single copy gene in the human genome. The authors are pleased to make the cDNA clones described in this paper available to interested investigators.

Notes: Upon interaction with target cells, cytotoxic T lymphocytes and natural killer cells vectorially secrete highly specialized cytoplasmic granules containing perforin and a family of serine proteases (granzymes). This granule exocytosis mechanism of cytolysis is of patho-physiological importance, and usually results in target cell DNA fragmentation. Neither perforin nor granzymes possess inherent nuclease activity, but in combination they can induce target cell apoptosis. Perforin forms transmembrane pores in the target cell, thereby enabling granzymes to access target cell substrates. The target cell substrates of granzymes are unknown, but granzyme A binding and cleavage of the nuclear shuttle protein nucleolin in target cells demonstrates that granzymes may act on nuclear substrates. Furthermore, the presence of granzyme B and other granzyme activities in the nucleus of cytotoxic lymphocytes indicates that granzymes can be transported from the cytoplasm to the nucleus.It is hypothesized that perforin enables effector granzymes to enter the target cell cytoplasm and following their transport into the nucleus, granzymes cleave specific target cell nuclear proteins to activate autolytic endonucleases that fragment DNA. In cytotoxic effectors, these nuclear substrates are normally protected from granzymes by endogenous inhibitors.