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 ava ilability, Suppression subtraction hybridization (SSH) was used to generate cDNA libraries from iron loaded and control human astrocytoma cells (SW108 8), The cDNA libraries were screened with antisense cDNA probes obtained fr om mRNA isolated from astrocytoma cells exposed to three conditions: (i) no rmal media (control), (ii) deferoxamine treated (iron deficient) or (iii) i ron loaded. The screening of the cDNA libraries with antisense probes from the three conditions enhanced the screening efficiency and decreased the nu mber of false positives, Positive clones were identified and sequenced. The genes of interest were further analyzed by determining changes in hybridiz ation signal on northern blots from astrocytoma cells exposed to iron or de feroxamine over different time intervals, Our analysis identified cDNAs cor responding to known iron responsive genes such as L-chain ferritin, but als o 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.