His-1 is a novel gene implicated in myeloid leukemia based upon its common activation in murine retrovirus-induced myeloid leukemias. The gene is highly conserved in mammalian evolution, and it is rarely expressed in mature tissues. Based upon the absence of an extensive open reading frame (orf) in the His-1 cDNA, we hypothesize that the product of the His-1 gene is a non-coding RNA molecule, and that retroviral activation of this unusual gene plays a role in myeloid leukemogenesis. The proposed research will study the His-1 gene in detail, and examine its transforming potential in myeloid cells.
The specific aims of the investigation are: 1) Identification of the coding potential of the His-1 gene:
This aim will focus on discriminating whether one of the small orfs is translated, or if the gene encodes a non-coding RNA. Since the mouse gene is highly conserved in humans, this will be approached by sequencing the human homology to identify orfs that are present in the regions of maximal homology. Antibodies will be raised against any conserved peptides to determine if the protein is expressed in cells that express His-1 RNA. If no conserved orf is identified we will assume that the RNA is non- coding and study its association with ribosomes by Northern blot analysis of subcellular fractions. 2) Analysis of His-1 structure/function: Regardless of the outcome of AIMI, available evidence indicates that His-1 expression has transforming activity in myeloid cells. We therefore propose studying the biological consequences of His-1 expression by gene transfer into myeloid cell lines and normal bone marrow progenitor cells in vitro. Conserved regions identified in AIMI will be deleted and the effects of deletion mutants will be compared to those of the normal gene. The leukemogenicity of His-1 expression in vivo will be studied by constructing a His-1 expression retrovirus, and examining its ability to accelerate the rate of tumor induction by Moloney murine leukemia virus embryogenesis by in situ hybridization, in addition to defining the transcriptional start site and the minimal promoter region of the gene.
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