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Query: EC:2.4.2.7 (adenine phosphoribosyltransferase)
 692 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The functional human adenine phosphoribosyltransferase (APRT) gene is less than 2.6 kilobases in length and contains five exons. The amino acid sequences of APRTs have been highly conserved throughout evolution. The human enzyme is 82%, 90%, and 40% identical to the mouse, hamster, and Escherichia coli enzymes, respectively. The promoter region of the human APRT gene, like that of several other "housekeeping" genes, lacks "TATA" and "CCAAT" boxes but contains five GC boxes that are potential binding sites for the Sp1 transcription factor. The distal three, however, are dispensable for gene expression. Comparison between human and mouse APRT gene nucleotide sequences reveals a high degree of homology within protein coding regions but an absence of significant homology in 5' flanking, 3' untranslated, and intron sequences, except for similarly positioned GC boxes in the promoter region and a 26-base-pair region in intron 3. This 26-base-pair sequence is 92% identical with a similarly positioned sequence in the mouse gene and is also found in intron 3 of the hamster gene, suggesting that its retention may be a consequence of stringent selection. The positions of all introns have been precisely retained in the human and both rodent genes, as has an unusual AG/GC donor splice site in intron 2. Particularly striking is the distribution of CpG dinucleotides within human and rodent APRT genes. Although the nucleotide sequences of intron 1 and the 5' flanking regions of human and mouse APRT genes have no substantial homology, they have a frequency of CpG dinucleotides that is much higher than expected and nonrandom considering the G + C content of the gene. Retention of an elevated CpG dinucleotide content, despite loss of sequence homology, suggests that there may be selection for CpG dinucleotides in these regions and that their maintenance may be important for APRT gene function.
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PMID:Comparative anatomy of the human APRT gene and enzyme: nucleotide sequence divergence and conservation of a nonrandom CpG dinucleotide arrangement. 355 38

Human nuclear poly(ADP-ribosyl) transferase (ADPRT) protein content in cells suggests that ADPRT expression is stringently controlled. Analysis of the 3 kb promoter sequence, which is required for high level expression, revealed an extraordinary architecture: several Sp1 motifs are located in the vicinity of the first exon but the closest CCAAT/TATA boxes are several hundred basepairs away. Four Alu type repetitive sequences are in the promoter structure. Within these Alu sequences there exist inverted repeat elements, which could form two mutually exclusive types of DNA tertiary structure consisting of quadruplex DNA and loops resembling rackets. Thereby, a CCAAT/TATA element would be moved to spatial vicinity of the Sp1 site activating the promoter. Deletion analysis showed the functional significance of these racket elements. We also obtained evidence for DNA racket structures when we studied mutational mechanisms in a human adenine phosphoribosyltransferase (APRT) deficient patient. One of his alleles harbours a novel complex type of deletion/insertion mutation. Based on several highly informative sequence features in this genomic region a model is proposed for the generation of this unusual type of mutation involving two steps: an initial targeting step and a subsequent complex rearrangement. This process includes the formation of a DNA racket structure, which resembles that of the ADPRT promoter. Thus we conclude that DNA racket structures seem to be of general importance in nature.
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PMID:Regulation of the human poly(ADP-ribosyl) transferase promoter via alternative DNA racket structures. 757 33

Transcriptional regulation of the Chinese hamster ovary (CHO) adenine phosphoribosyltransferase-encoding gene (APRT) was studied. The 5' region of the CHO APRT is G + C-rich, but lacking TATA or CCAAT boxes. RNase protection assays indicate that it contains multiple transcription start points (tsp). A tsp 64 bp upstream from the translation start codon is denoted as +1. Linker-scanning (LS) mutation analysis indicates that the -33 to +19 region is important in regulating APRT transcription. Mutations in the -23 to -14 region abolish transcription initiated from the -23 downstream region. An unidentified protein complex binds to this region. Three Sp1-binding sites are found in the APRT promoter; however, mutations of the Sp1-binding sites do not reduce APRT transcription. Mutations at two putative GCF-binding sites increase levels of transcription.
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PMID:Identification of the cis-elements required for transcriptional control of the Chinese hamster ovary APRT gene. 762 45

In an attempt to find the mechanism by which CpG islands remain free of methylation we have undertaken a detailed examination of the mouse adenine phosphoribosyltransferase (aprt) gene. This housekeeping gene has a CpG island that extends over the gene promoter and includes the first two exons. We show that the island is free of methylation at all CpGs, whereas the flanks are methyated. Detailed patterns of methylation beyond the boundaries of the CpG island vary between cells. In vivo footprinting across the island region shows that three GC boxes clustered at the 5' edge of the CpG island are occupied, most probably by Sp1. No other footprints are detected within the island region. Deletion or mutagenesis of the Sp1 sites causes de novo methylation of the CpG island in a transgenic mouse assay. Thus, the peripherally located Sp1 sites are necessary to keep the aprt island methylation free.
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PMID:Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. 795 95

Animal somatic cell DNA is characterized by a bimodal pattern of methylation: tissue-specific genes are methylated in most cell types whereas housekeeping genes have 5' CpG islands which are constitutively unmethylated. Because methyl moieties derived from the gametes are erased in the morula and early blastula, this profile must be re-established in every generation; this is apparently accomplished by a wave of non-CpG island de novo methylation that occurs at implantation. Using transfection into embryonic stem cells and transgenic mice as a model system, we now show that Sp1 elements play a key role in protecting a CpG island in the adenine phosphoribosyltransferase (APRT) gene from de novo methylation. This recognition mechanism represents a critical step in embryogenesis, as it is responsible for setting up the correct genome methylation pattern which, in turn, is involved in regulating basal gene expression in the organism.
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PMID:Sp1 elements protect a CpG island from de novo methylation. 809 Feb 26

The promoter region of the mouse adenine phosphoribosyltransferase (aprt) gene contains one non-consensus Sp1 binding site at its 5' end followed by three consensus Sp1 binding sites. The two 3'-most binding sites are sufficient for maximal expression of aprt , suggesting that the non-consensus and consensus binding sites at the 5' end are redundant. However, the two 3' sites are not sufficient to block epigenetic inactivation, which led to the hypothesis that the redundant consensus and/or non-consensus 5' Sp1 binding sites are required to block inactivation events. To test this hypothesis, promoter region constructs were made in which the two 5' Sp1 binding sites were mutated alone or in tandem, and then each construct was tested for its ability to withstand epigenetic inactivation. A cis -acting methylation center that is normally located 1.2 kb upstream of the promoter was used to induce inactivation. The results demonstrate that the presence of the redundant consensus Sp1 binding site is required to block methylation-associated gene inactivation. Therefore, the Sp1 binding sites comprising the mouse aprt promoter have evolved two distinct functions, one to promote transcription and the other to block epigenetic inactivation.
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PMID:The primary function of a redundant Sp1 binding site in the mouse aprt gene promoter is to block epigenetic gene inactivation. 980 14

DNA in somatic tissue is characterized by a bimodal pattern of methylation, which is established in the animal through a series of developmental events. In the mouse blastula, most DNA is unmethylated, but after implantation a wave of de novo methylation modifies most of the genome, excluding the majority of CpG islands, which are mainly associated with housekeeping genes. This genomic methylation pattern is broadly maintained during the life of the organism by maintenance methylation, and generally correlates with gene expression. Experiments both in vitro and in vivo indicate that methylation inhibits transcription. It has not yet been possible, however, to determine the role of DNA methylation on specific sequences during normal development. Cis-acting regulatory elements and trans-acting factors appear to be involved in both stage- and tissue-specific demethylation processes. Sp1-like elements have a key role in protecting the CpG island of Aprt (encoding adenine phosphoribosyltransferase) from de novo methylation, and when these elements are specifically mutated, the Aprt CpG island becomes methylated in transgenic mice. We have now characterized an embryo-specific element from the CpG island sequence upstream of Aprt that can protect itself from de novo methylation in transgenic mice as well as reduce methylation of flanking sequences. We placed this element on a removable cassette adjacent to a human HBB (encoding beta-globin) reporter and generated a transgene whose methylation pattern can be switched in vivo. Analysis of globin transcription in this system showed that methylation in cis inhibits gene expression in a variety of tissues, indicating that DNA modification may serve as a global genomic repressor.
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PMID:DNA methylation represses transcription in vivo. 1036 68

We have utilized the Escherichia coli lac repressor-operator system to test whether protein binding can interfere with de novo DNA methylation in mammalian cells. We find that a DNA binding protein can protect sites on the episome as well as in the genome from the de novo methylation activity of Dnmt3a. Transcriptional machinery moving through the binding sites does not affect the de novo methylation of these sites, and it does not affect the binding protein protection of these sites from de novo methylation. This study and previous studies provide a possible mechanism for the observation that an Sp1 site can serve as a cis-acting signal for demethylation and for preventing de novo methylation of the CpG island upstream of the mouse adenine phosphoribosyltransferase (Aprt) gene. These findings also support the hypothesis that protein binding may play a crucial role in changes of CpG methylation pattern in mammalian cells.
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PMID:Protein binding protects sites on stable episomes and in the chromosome from de novo methylation. 1131 67

Unmethylated CpG islands are known to keep adjacent promoters transcriptionally active. In the CpG island adjacent to the adenosine phosphoribosyltransferase gene, the protection against transcriptional silencing can be attributed to the short CpG-rich core element containing Sp1 binding sites. We report here the insertion of this CpG island core element, IE, into the long terminal repeat of a retroviral vector derived from Rous sarcoma virus, which normally suffers from progressive transcriptional silencing in mammalian cells. IE insertion into a specific position between enhancer and promoter sequences led to efficient protection of the integrated vector from silencing and gradual CpG methylation in rodent and human cells. Individual cell clones with IE-modified reporter vectors display high levels of reporter expression for a sustained period and without substantial variegation in the cell culture. The presence of Sp1 binding sites is important for the protective effect of IE, but at least some part of the entire antisilencing capacity is maintained in IE with mutated Sp1 sites. We suggest that this strategy of antisilencing protection by the CpG island core element may prove generally useful in retroviral vectors.
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PMID:The core element of a CpG island protects avian sarcoma and leukosis virus-derived vectors from transcriptional silencing. 1855 Jun 62