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A protein which binds specifically to MspI8 (a 454 bp long repeated sequence highly homologous to the 5’ untranslated region (5'UTR) of the LINE 1 sequence) was found and identified in nuclear extracts of rat liver cells. This protein was detected using the electrophoretic mobility shift assay (EMSA) and was purified by Q-Sepharose and DNA affinity chromatography. Its molecular mass was estimated by SDS electrophoresis as 29 kDa. The possibility that this protein (p29) is the rat analogue of human L1PBP-A, specific for the human LINE sequence LPE1, is discussed.
Proteins recognizing DNA damaged by the chemical carcinogen N- acetoxy-acetylaminofluorene (AAAF) were analyzed in nuclear extracts from rat tissues, using a 36 bp oligonucleotide as the substrate and an electrophoretic mobility shift assay. Two major proteins that form complexes with DNA damaged by AAAF were detected; one of them also bound DNA damaged by cis-diammine-dichloroplatinum. The complex specific for AAAF-damaged DNA contained protein loosely attached to nuclear components. It was extracted with 0.1 M NaCl. The amount of this protein was estimated at about 105 copies per liver cell nucleus, and its probable size was about 42 kDa as detected by the Southwestern blotting assay. Its affinity for DNA damaged by AAAF was ~10-fold higher than that for undamaged DNA. Analogous AAAF- DDB (damaged-DNA-binding) proteins were also detected in extracts from rat brain, testis and kidney tissue. The levels of such proteins were not affected in rats treated with the carcinogen 2-acetylaminofluorene.
Soluble tropoelastin is a precursor of elastin. It is transported by elastin binding protein (EBP) into intercellular space. The EBP-tropoelastin complex undergoes disintegration in the intercellular space. EBP returns to the cell and tropoelastin joins with microfibril proteins. Lysine residues of tropoelastin undergo oxidative deamination. Desmosine, isodesmosine and cross bonds arise. Thus tropoelastin loses solubility and transforms to elastin. The elastic fiber created consists of microfibrils on the periphery and an elastin core inside. Elastin provides blood vessels, lungs, cartilage and skin with the ability to reverse deformations. A decrease of elastin content or changes in this protein structure results in pathological conditions such as aneurysms and pulmonary emphysema.
The C-terminal end of the fragile X mental retardation protein contains a stretch of amino acid residues that are enriched in arginine and glycine. Recent studies using recombinant FMRPs have demonstrated that this region participates in RNA binding in vitro, with calculated KdS ranging from 1-10 nM depending on the RNA. It is known that other arginine glycine-rich proteins are subject to site-specific methylation by protein arginine methyltransferases (PRMTs) that are particularly abundant in most cells. We have demonstrated that the interaction of homoribopolymer mimetic RNAs with human FMRP (hFMRP) made in PRMT-containing cell-free lysates is more sensitive to increasing salt concentrations than recombinant hFMRP expressed in bacteria. We have also shown that blocking methylation with adenosine-2', 3'-dialdehyde (AdOx) alters homoribopolymer binding and hFMRP target mRNA binding; both increases and decreases are observed as a function of methylation. These data suggest that changes in PRMT activity that occur during development, or arise via signal transduction may be a means of regulating the binding of hFMRP to mRNA in vivo.
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