Rat Nuclear Extracts Research Articles

Generation of DNA Double-strand Breaks by Two Independent Enzymatic Activities in Nuclear Extracts

We have reported the existence in rat nuclear extracts of a specific cleavage activity on a DNA fragment containing the human minisatellite MsH42 region (minisatellite plus its flanking sequences). Here, we have developed a system to analyse the nature of the cleavage products from the MsH42 region generated by the nuclear extracts. Our results demonstrated the formation of DNA double-strand breaks (DSB) in the MsH42 region by two different enzymatic activities, and that their distribution along this fragment changes depending on the presence of Mg2+. In the assays with Mg2+, the DSB were located in the minisatellite and its 3'-flanking region, showing preference for G-rich stretches. Oligonucleotide mutagenesis analysis confirmed that this enzymatic activity has a strong preference for G-tracts and that the recognition site is polarized towards the 3' end. Moreover, this activity cuts GC palindromes efficiently. In contrast, in the experiments without Mg2+, most DSB were mapped within the minisatellite flanking sequences. The analysis with oligonucleotides showed that G-tracts are recognized by this endonuclease activity, but with differences in the cleavage behaviour with respect to the reactions observed with Mg2+. The existence of two separate activities (Mg2+-dependent and Mg2+-independent) for the production of DSB was confirmed by analysing the effect of EGTA, N-ethyl maleimide, ionic strength, and by preincubations of the nuclear extracts at different temperatures. The tissue distribution of both DSB-producing activities was also different. The in vitro system used in the present work may be a useful tool for studying the formation of DSB and for investigation of the mechanisms of DNA repair.

Sequence analysis of the rat phenylalanine hydroxylase gene promoter.

We have characterized the 5'-end (3218 bp) of the rat phenylalanine hydroxylase (PAH) gene. Within this PAH promoter sequence, we have identified a number of putative regulatory sites analogous to those present in the human and murine PAH promoters. In particular, potential HNF 1 binding sites and a CRE have been identified. These sequences respectively bind HNF1 and CREB transcription factors present in rat nuclear extracts and may be significant in the tissue-specific and hormonal control of PAH expression.

The role of CCAAT/enhancer-binding protein alpha and a protein that binds to the activator-protein-1 site in the regulation of liver-specific expression of the winter flounder antifreeze protein gene.

Winter flounder, Pleuronectes americanus, produces antifreeze proteins (AFPs) to avoid freezing during winter. The AFP in the blood is synthesized by the liver and its annual level is regulated by environmental factors and somatotropin. The only intron (+106 to +602) of the AFP gene contains putative binding sites for liver-enriched transcription activators. Due to the lack of an appropriate fish liver cell line, a mammalian model system was used to demonstrate the liver-specific enhancer activity of the intron. Deletion analysis of the intron indicated nucleotide sequences between +192 bp and +334 bp were important for the enhancer activity. Further refinement showed that the element B (+303 to +322 bp) resided in this region interacted with proteins from rat nuclear extracts by DNase I footprinting and mobility shift analysis. Gel retardation with competition and supershift experiments defined element B to include binding sites for CCAAT/enhancer binding protein a (C/EBP alpha), a liver-enriched transcription factor, and a novel activator protein-1 (AP-1) site binding protein, designated as antifreeze enhancer binding protein (AEP). Residues important to DNA-protein interaction in element B were mapped by methylation interference. Mutations in element B by site-directed mutagenesis destroyed the enhancer activity of the intron. Ultraviolet crosslinking experiments using element B revealed the specific binding of two proteins of approximately 43 and 80 kDa. The present findings provide a mechanism(s) by which the tissue specificity of AFP gene expression can be achieved. Integration of the present information with the somatotropin-mediated signal transduction pathways has generated a working model for the hormonal regulation and seasonal expression of the AFP gene.

Characterization of a Silencer Regulatory Element in the Rat PAP I Gene Which Confers Tissue-Specific Expression and Is Promoter-Dependent

Previous analysis of the rat PAP I promoter indicated that the region between nt −180 and −81 possessed silencer activity in cells that did not express PAP I. Based on this finding, we performed a series of experiments to characterize functionally that region and analyze the nuclear proteins interacting with it. Transient transfection assays were conducted in the fibroblast Rat2 cell line, in which PAP I is not expressed, and in the pancreatic cell line AR-42J, expressing PAP I, using the CAT gene as reporter. Experiments in Rat2 cells revealed that the sequence with silencer activity was located within the rep27 region (position −180/−153). Suppressor activity was observed when rep27 was inserted upstream from the core PAP I promoter, in both orientations. By contrast, inserting the rep27 region in front of the promoters of SV40 or thymidine kinase did not affect or weakly enhanced CAT activity. Suppressor activity is therefore position-independent and promoter-dependent. In pancreatic AR-42J cells, rep27 act as a positive element but did not alter CAT expression when inserted in front of the core PAP I promoter or heterologous promoters. Electrophoretic mobility shift assays allowed identification of specific DNA–protein complexes. The shifted complex migrated at the same position with both Rat2 and AR-42J nuclear extracts. Moreover, similar band shifts were obtained with rat nuclear extracts from healthy pancreas, pancreas with acute pancreatitis, liver, kidney, spleen, and small intestine. Results suggest that the rep27cis-acting element contributes to the tissue specific expression of the PAP I gene. That activity could be mediated by the synergistic action of several transcription factors, one of which being present in all cells.

Demonstration of Trans-acting Factors Binding to the Promoter Region of the Testis-Specific Rat Protamine P1 Gene

Potential regulatory DNA elements within the rat protamine P1 promoter region have been identified using gel retardation assays and DNase I footprinting analysis with rat nuclear extracts obtained from different tissues. Distinctive gel shift bands are generated by rat nuclear extracts from mature testis, kidney, brain, spleen and liver, which bind to the cis-acting SRE (Serum response element) and Prot1C (Protamine 1 Consensus) oligonucleotide sequences [Queralt R. and Oliva R. Gene. 133 (1993) 197-204]. In vitro DNase I footprinting analysis demonstrates protection of the SRE region at positions −124 to −115. In addition, we have detected protection in two new regions adjacent to the SRE that we called SAP (SRE Adjoining Protection; nt −153 to −141) and SEP (SRE Extended Protection; nt −114 to −100), respectively. These sequences, SAP and SEP, show no apparent consensus homology with cis-acting elements of other known transcription factors. Two additional DNase I protected regions have also been found at positions +27 to +46 and +61 to +88, the first of which contains the sequence +42 TCGNNNNNGCCAA +30 recognized by nuclear factor I (NFI).

Sp1 DNA binding efficiency is highly reduced in nuclear extracts from aged rat tissues.

To explore the role of transcriptional factors in the genesis of the senescent phenotype, nuclear extracts from 4- and 30-month-old rat brains were analyzed for the presence of DNA-binding proteins able to interact with double-stranded oligonucleotides containing recognition sites for sequence-specific DNA-binding factors. Gel shift assays revealed that the DNA-binding efficiency of Sp1 is significantly reduced in aged animals compared to young ones, whereas CTF/NF1 and AP1 from young and old rat nuclear extracts bind their DNA targets with the same efficiency. The quantitative analysis of Sp1 by immunoblotting indicated that equivalent quantities and degrees of heterogeneity of Sp1 protein are present in both nuclear extracts, suggesting that the observed difference is not due to a different expression of this transcriptional factor. DNase I footprinting of the heavy chain ferritin gene promoter, which contains a Sp1 binding site, demonstrated that the nuclear extract from 30-month-old rat brain does not protect the region involved in the regulation of the H ferritin gene by Sp1. This results in a reduction of about 50% of the expression of the H ferritin mRNA in aged rat brains. Furthermore, the Sp1 binding sites present in the SV40 early promoter are not protected in a DNase I footprinting assay where a nuclear extract from 30-month-old rat brain was used as a source of DNA binding proteins. Liver nuclear extracts prepared from young and aged rats demonstrated that a decrease of Sp1 binding efficiency is similarly present in this tissue.