STUDIES ON THE REGULATORY FUNCTIONS OF A NUCLEIC ACID BINDING PROTEIN (RF-36) ISOLATED FROM LENS TISSUE (DNA STRUCTURE, TOPOISOMERASE I

A nucleic acid binding protein has been isolated from a calf lens and purified to apparent homogeneity by DNA-cellulose affinity chromatography. Its molecular weight was determined by SDS/PAGE to be 36,000 and designated as RF-36.;The aims of this study were focused on the problems associated with the regulation of gene expression in lens tissue. (1) Whether RF-36 cooperatively induces high-order conformational changes of structure at the superhelical and chromatin DNA level? (2) What roles does RF-36 play in the process of chromatin activation? (3) What is the nature of the regulatory binding domain?;The results show that RF-36 binds to DNA and unwinds double-stranded supercoiled DNA thus facilitating topoisomerase I action; RF-36 unwinds nucleosome DNA as revealed by an increase in the efficiency of nuclease S1 digestion; RF-36 attaches to the linker DNA of chromatin to protect chromatin against micrococcal nuclease digestion. The transcriptional efficiency of wheat germ pol II and Hela cell transcription systems was enhanced when RF-36 was introduced to chromatin template or cloned plasmid DNA (p(alpha)Ac) template containing genomic (alpha)-crystallin DNA. However, linearized p(alpha)Ac fragments without the promoter elements, do not exhibit such transcriptional stimulation by RF-36. The transcripts synthesized from RF-36 induced-chromatin templates were found to be homogeneous in size. The results that transcripts are derived from lens gene families and further studies revealed that they are loosely-related to the (alpha)-crystallin gene were obtained from molecular hybridization. Footprint studies on p(alpha)Ac fragment suggest that a unique DNA region immediately following the TATA sequence was protected.;These data demonstrate that the molecular mechanism of RF-36 action is associated with cooperatively induced changes in DNA conformation. These changes relate to the regulatory region of DNA is evidenced by the enhanced RNA-polymerase transcription. RF-36 binding and transcriptional specificity were observed since the (alpha)-crystallin gene transcript was produced. Crystallins are the predominant genes expressed in vivo, thus, the affect RF-36 role in the in vitro transcriptional studies is consistent with gene expression in the lens.