Identification of iron responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions.
The goal of the present study is to identify genes that respond to iron availability. Suppression subtraction hybridization (SSH) was used to generate cDNA libraries from iron loaded and control human astrocytoma cells (SW1088). The cDNA libraries were screened with antisense cDNA probes obtained from mRNA isolated from astrocytoma cells exposed to three conditions: (i) normal media (control), (ii) deferox-amine treated (iron deficient) or (iii) iron loaded. The screening of the cDNA libraries with antisense probes from the three conditions enhanced the screening efficiency and decreased the number of false positives. Positive clones were identified and sequenced. The genes of interest were further analyzed by determining changes in hybridization signal on northern blots from astrocytoma cells exposed to iron or deferoxamine over different time intervals. Our analysis identified cDNAs corresponding to known iron responsive genes such as L-chain ferritin, but also revealed a number of mRNAs with novel sequences and mRNAs previously not known to be responsive to iron such as one of the ABC transporters and Thy-1 glycoprotein. Thus our results suggest that the expression of a number of genes may be influenced by changes in iron availability.
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Department of Neuroscience and Anatomy and George M. Leader Family Laboratory for Alzheimer's Disease Research, PO Box 850, Pennsylvania State University College of Medicine, M. S. Hershey Medical Center, Hershey, PA 17033, USA.
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JR
D001483:Base Sequence; D015152:Blotting, Northern; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007501:Iron / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D013382:Subtraction Technique; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D007501:Iron