Resolving the genetic regulatory mechanism(s) and biochemical factors involved in human beta-globin gene switching is the goal of research outlined in this application. Switching is the process of temporal expression of beta-globin-like polypeptides during the developmental transition from fetal to postnatal life. Of particular interest is the molecular switch from fetal hemoglobin to adult hemoglobin, presumably, as a result of derepression (or induction) of beta-globin chain production and suppression of gamma-globin transcription upon birth. The clinical importance of this step lies in the fact that elevated amounts of fetal hemoglobin can provide considerable protection to patients suffering from sickle cell anemia. Cell fusion studies involving two erythroleukemia cell lines, one human-derived, K562, and one derived from mouse, MEL, has shown the switch from gamma-globin to Beta-globin chain production depends on trans-acting factors. The two cell lines differ, therefore, in the type of hemoglobin they can produce upon induction; fetal for the human cell line and adult for the mouse cell line. The research proposed will focus on utilizing novel methods to directly clone the gene(s) encoding the regulatory factors involved in the transcriptional regulation of the beta-globin gene through positive selection methods. A modified version of the Okayama-Berg plasmid has been constructed. The new plasmid can be selected for when carrying the cDNA of a beta-globin gene trans-activator. The selection of the plasmid is dependent on the expression of a bacterial aminoglycoside 3'- phosphotransferase gene under the transcriptional control of the human beta-globin promoter. We will construct total cDNA libraries from one cell line (gamma-globin off, beta-globin on) in our plasmid. This library will subsequently be introduced into another cell line (gamma-globin on, Beta-globin off) to identify clones which express trans-acting factors that actIvate the transcription of the Beta-globin gene. The known sequences flanking The cDNA cloning site of the plasmid will be used to retrieve the insert from the human cells utilizing PCR techniques. We will reclone the amplified cDNA into bacterial vectors to be used as a probe to recover the chromosomal copy of the gene. We will sequence both the native and the cDNA clones. We will purify the transfactor(s) by over expressing the gene in E. coiL The purified protein will be used in structure-function analysis experiments. These studies will produce considerable insight into the switching phenomenon which ultimately could provide new approaches to management of sickle cell anemia.
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