Molecular Cloning and Expression of an Interferon‐Inducible Protein Encoded by Gene 203 from the Gene 200 Cluster

Abstract

We report here the complete coding sequence of a 203 cDNA, a member of the interferon-inducible Ifi 200 gene family. By combining reverse-transcriptase PCR and rapid amplification of cDNA ends (RACE) techniques we have obtained a 3.8-kb cDNA corresponding to a 203 mRNA. When used as a probe in northern analysis, its 3' segment hybridized to a 3.8-kb interferon-inducible mRNA, whereas the S'-end additionally hybridized to a less abundant interferon-inducible 1 .8-kb mRNA. Nucleotide sequence analysis revealed that the two mRNAs share the 5'-untranslated region and the same open reading frame, which encodes a hydrophilic protein composed of 408 amino acids. The difference between them is due to a 3'-untranslated region extended by alternative polyadenylation site selection. Furthermore, 203 mRNA was found to be inducible by interferon-a in various murine cell lines. Using polyclonal antibodies raised against a segment specific for the 203 protein, we established that p203 protein levels increase on treatment with interferon-a in murine fibroblasts and that p203 is located in the nucleus. MATERIALS AND METHODSCells and culture conditions. Balb/c 3T3, NIH 3T3 and BLKS cells (murine fibroblasts) were grown as monolayers in Dulbecco's modified Eagle's medium (GibcoiBRL) supplemented with 10% calf serum (Gibco/BRL; Balb/c 3T3 and NIH 3T3 cells) or 10% fetal calf serum (Gibco/BRL; BLKS cells). L12R4 (murine T lymphoma cell line) and L1210 (murine pre-B lymphoma cell line) cells were grown in RPMI 1640 me-

Transcription and Growth Regulatory Functions of the HIN-200 Family of Proteins

“…The size of the spacer region in IFI 16 is regulated by mRNA splicing and can contain one, two, or three copies of a highly conserved 56-amino-acid S/T/P domain encoded by distinct exons (23). In contrast, MNDA, AIM-2, p203, and D3 contain only one 200-amino-acid domain (3,17,18,20). The amino acid composition of the A domains expressed in different HIN-200 proteins is highly conserved, as are the B domains expressed in different family members.…”

“…The HIN-200 family of proteins consists of a number of highly homologous human and murine proteins with similar primary amino acid sequences and biological characteristics. The human HIN-200 family members include IFI 16 (39), the myeloid nuclear differentiation antigen (MNDA) (2,18), and AIM-2 (absent in melanoma) (17), while the mouse HIN-200 family members include p202 (3), p203 (20), p204 (3), and D3 (38). There are a number of reviews that elegantly outline the biochemical characteristics of these proteins and their patterns of expression (15,27,30).…”

The retinoblastoma protein is an essential mediator that links the interferon-inducible 204 gene to cell-cycle regulation

“…Although these results indicate that p204 is probably involved in the control of the cell cycle throughout the pRB-pathway, they do not shed light on the mechanisms it employs. For this purpose in a previous study we generated both p204 deletion mutants lacking one of the two domains, and chimeric proteins bearing the a domain from p204 and the b domain from p203, another member of the I®200 family (Gribaudo et al, 1997(Gribaudo et al, , 1999. The results of transfection in sensitive cells clearly demonstrated that to exert its antiproliferative activity, accumulate cells at the G1/S border, and inhibit the E2F-mediated transcriptional activity p204 needs both domains.…”

The murine homolog of the HIN 200 family, Ifi 204, is constitutively expressed in myeloid cells and selectively induced in the monocyte/macrophage lineage

“…However, despite few well-established examples, the exact function of the IFN-induced proteins that are ultimately the mediators of their biological activity are still quite unknown [3]. One protein family (p200) consists of at least four members designated p202, p203, p204, and D3, encoded by a cluster of genes located in the q21-23 region of mouse chromosome 1 (Ifi 200 genes) [4][5][6][7][8][9]. All these proteins share one or two partially conserved 200-amino acid segments, designated as the type a and the type b domain.…”