Cloning a novel member of the human interferon-inducible gene family associated with control of tumorigenicity in a model of human melanoma.

Chromosome 6-mediated suppression of tumorigenicity in malignant melanoma cell lines provides a model system to identify genes associated with the reversion of the tumorigenic phenotype. Using subtractive cDNA selection, we recently identified a series of novel genes which are differentially expressed in association with chromosome 6-mediated suppression. We now report the molecular characterization of a novel gene termed AIM2 for (Absent In Melanoma), which represents a 1485 bp cDNA. An open reading frame of 1032 base pairs, corresponding to 344 amino acid residues, is predicted. The predicted protein shares a conserved sequence domain of approximately 200 amino acids with known interferon-inducible genes of both human and mouse. We demonstrate that the AIM2 gene encodes a transcript of approximately 2 kb which is expressed in spleen, small intestine, and peripheral blood leukocytes. In addition, we have localized AIM2 to the long arm of human chromosome 1 (band q22) in a highly conserved region which also contains the known interferon-inducible genes IFI16 and MNDA. We have also demonstrated that, like IFI16 and MNDA, AIM2 is induced in HL60 cells by interferon gamma. Our findings support the existence of a family of genes in this region similar to the well-characterized mouse Ifi200 gene family.

A non-directed, hydroxylamine-generated suppressor mutation in the P3 pairing region of the bacteriophage T4 td intron partially restores self-splicing capability.

Hydroxylamine (HA) mutagenesis of an HA-induced splicing-defective bacteriophage T4 td intron mutant with a mutation in the intron P3 RNA pairing region was used to generate pseudorevertants. Because HA can only cause GC to AT transitions, the original mutant (H104A) could not undergo true reversion, yet the compensatory mutation on the opposite side of the P3 helix, which was complementary to the original H104A mutation, could occur. A pseudorevertant was isolated that contained both the original H104A mutation and the compensatory mutation HS9. By phenotypic and molecular genetic criteria, this double mutant (H104A-HS9) was shown to be able to undergo significant RNA splicing, thus confirming the existence and functional importance of the long-range P3 pairing region in this phage intron. The second-site suppressor mutation (HS9) was isolated by phage cross and also exhibited some self-splicing ability. A correlation exists between the strength of P3 helix Watson-Crick base pairing and the apparent level of splicing when wild-type, H104A, HS9, and H104A-HS9 are compared. This suggests that the primary role of the P3 RNA pairing region in the T4 td intron is structural in contributing to the critical RNA secondary structure.