The McrB restriction system of Escherichia coli K-12 is responsible for the biological inactivation of foreign DNA that contains 5-methylcytosine residues (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986). Within the McrB region of the chromosome is the mcrB gene, which encodes a protein of 51 kilodaltons (kDa) (T. K. Ross, E. C. Achberger, and H. D. Braymer, Gene 61:277-289, 1987), and the mcrC gene, the product of which is 39 kDa (T. K. Ross, E. C. Achberger, and H. D. Braymer, Mol. Gen. Genet., in press). The nucleotide sequence of a 2,695-base-pair segment encompassing the McrB region was determined. The deduced amino acid sequence was used to identify two open reading frames specifying peptides of 455 and 348 amino acids, corresponding to the products of the mcrB and mcrC genes, respectively. A single-nucleotide overlap was found to exist between the termination codon of the mcrB gene and the proposed initiation codon of the mcrC gene. The presence of an additional peptide of 33 kDa in strains containing various recombinant plasmids with portions of the McrB region has been reported by Ross et al. (Gene 61:277-289, 1987). The analysis of frameshift and deletion mutants of one such hybrid plasmid, pRAB-13, provided evidence for a second translational initiation site within the McrB open reading frame. The proposed start codon for translation of the 33-kDa peptide lies 481 nucleotides downstream from the initiation codon for the 51-kDa mcrB gene product. The 33-kDa peptide may play a regulatory role in the McrB restriction of DNA containing 5-methylcytosine.
Recombinant 1,25-dihydroxyvitamin D3 receptor from a baculovirus expression system requires a mammalian-derived nuclear accessory protein for binding to a vitamin D response element (DRE). This was established by electrophoretic mobility shift analyses using radiolabeled DNA probes consisting of DREs from two vitamin D-responsive genes. Mammalian nuclear extract was also required for the binding of wild-type porcine vitamin D receptor to a DRE. Surprisingly, the accessory factor-dependent formation of receptor-DRE complex was independent of exogenous 1,25-dihydroxyvitamin D3. A 59-to 64-kDa accessory protein from porcine intestinal nuclear extract was identified by sizeexclusion chromatography. Nuclear extracts from rat liver and kidney contained accessory factor, whereas smaller amounts were detected in heart muscle. Spleen and skeletal muscle contained no detectable accessory factor.An understanding of the biological actions of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] requires a comprehensive study of its intracellular receptor (1, 2). It is through this receptor that 1,25-(OH)2D3 exhibits hormonal activities that include stimulation of calcium absorption in the intestine, calcium mobilization from bone, and calcium reabsorption in kidney (1). A probable mechanism for these activities involves an interaction between hormonally altered receptor protein and chromatin, influencing the expression of genes whose products are involved in those functions (2).The molecular cloning of cDNAs encoding human (3) and rat (4) vitamin D receptors has permitted a structural analysis based on their deduced amino acid sequences. Similarities in structural domains of these receptor proteins include a cysteine-rich region at the amino terminus, characteristic of a DNA binding motif, and a hydrophobic pocket near the carboxyl terminus that is thought to form the site of ligand binding. The domains were more clearly defined within the human vitamin D receptor protein using deletion analysis and expression studies of the cDNA (5). These structural determinations have resulted in the inclusion of the 1,25-(OH)2D3 receptor as a member ofthe steroid, thyroid, and retinoic acid superfamily of receptor proteins (6,7).Several recent advances have provided tools by which the interaction between the vitamin D receptor and nucleic acid could be examined. For example, the realization of cis-acting nucleotide sequence response elements that confer vitamin D responsiveness within the transcriptional control regions of the mouse osteopontin gene (8) and the human (9, 10) and rat (11) osteocalcin genes has provided target sequences for in vitro studies of 1,25-(OH)2D3 receptor binding. Studies using these vitamin D response elements (DREs) have defined a transcriptional effect of 1,25-(OH)2D3 on homologous and heterologous promoter activities (10). Other progress has resulted from the production of elevated levels of recombinant vitamin D receptor from a yeast expression system using the human cDNA (12) and from a baculovirus expression sy...
The McrB restriction system in Escherichia coli K12 causes sequence-specific recognition and inactivation of DNA containing 5-methylcytosine residues. We have previously located the mcrB gene near hsdS at 99 min on the E. coli chromosome and demonstrated that it encodes a 51 kDa polypeptide required for restriction of M.AluI methylated (A-G-5mC-T) DNA. We show here, by analysis of maxicell protein synthesis of various cloned fragments from the mcrB region, that a second protein of approximately 39 kDa is also required for McrB-directed restriction. The new gene, designated mcrC, is adjacent to mcrB and located distally to hsdS. The McrB phenotype has been correlated previously with restriction of 5-hydroxy-methyl-cytosine (HMC)-containing T-even phage DNA that lacks the normal glucose modification of HMC, formally designated RglB (for restriction of glucoseless phage). This report reveals a difference between the previously correlated McrB and RglB restriction systems: while both require the mcrB gene product only the McrB system requires the newly identified mcrC-encoded 39-kDa polypeptide.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.