Characterization of a repressor element and purification of its cognate DNA-binding protein for the transcription of the genes for the antifreeze proteins in wolffish (Anarhichas lupus)

• Main Author: Stefánsson, Stefán Einar
• Format: Thesis
• Language: English
• Published: 1997
• Subjects: Antarctic; Antarc*
• Online Access: http://hdl.handle.net/1807/10619; http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq27734.pdf

grantor: University of Toronto Many marine fish secrete antifreeze proteins (AFP) into their sera to protect them selves from freezing in icy sea water. Four different types of AFPs have been characterized, antifreeze glycoprotein and AFP type I, II and III. The transcriptional control for the Type III Antifreeze protein (AFP) genes is poorly understood. In this present study, all known Type III genes and flanking DNA sequences were compared, revealing that the Type III AFP genes from wolffish (Anarhichas lupus), ocean pout (Macrozoarces americanus) and Antarctic eel pout (Lycodichthys dearborni) have common core sequences. These core segments are intervened by unique regions, which are present in some, but not in all of the genes aligned. A region from wolffish AFP genes, located between $-$220 to $-$90 bp upstream from the transcription start site, was previously characterized to repress transcription. This segment falls into two different core regions, with a unique segment between. Two out of the three characterized wolffish sequences, have two 12 base pair repeated elements in tandem, called "b" elements within this described repressor region. The first repeated "b" element, is within a core region, and therefore is present in all Type III AFP flanking sequences. The second repeated "b" element is unique to the two wolffish sequences. The $-$220 to $-$90 bp fragment is shown to interact with proteins from nuclear extract of various sources with strongest interaction in liver tissues. Several proteins appear with the $-$220 to $-$90 bp DNA segment. The "b" elements also interact with nuclear proteins from the liver. Furthermore, the repressor region is shown here to be due to the two "b" elements, which mediate a strong repression of transcription, presumably by interacting with the general transcription factors. Several additional DNA-binding sites are also identified within this segment. To further distinguish the nature of the repressor element, its putative binding protein(s) was purified and characterized. Three proteins of different size appear to interact with the "b" element in liver tissue. These proteins, termed pBB for protein binding to "b" element, are between 34 and 38.5 kDa in size. The size of the purified proteins is in good agreement with cross-linking experiment performed on the crude nuclear extract. All three proteins appear to be rich in glutamine and glutamic acid residues, which are commonly found in repression and activation domains of trans-acting factors. Finally, all three proteins have identical N-terminal sequences, raising the possibility that the difference in molecular weight might be due to micro-heterogeneity, alternative splicing of the mRN. Both the elements and the DNA-binding proteins described here appear to be novel, and have not been previously characterized. Ph.D.