Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

Using simple linear fragments of the Chinese hamster adenine phosphoribosyltransferase (APRT) gene as targeting vectors, we have investigated the homology dependence of targeted recombination at the endogenous APRT locus in Chinese hamster ovary (CHO) cells. We have examined the effects of varying either the overall length of targeting sequence homology or the length of 5' or 3' flanking homology on both the frequency of targeted homologous recombination and the types of recombination events that are obtained. We find an exponential (logarithmic) relationship between length of APRT targeting homology and the frequency of targeted recombination at the CHO APRT locus, with the frequency of targeted recombination dependent upon both the overall length of targeting homology and the length of homology flanking each side of the target gene deletion. Although most of the APRT+ recombinants analyzed reflect simple targeted replacement or conversion of the target gene deletion, a significant fraction appear to have arisen by target gene-templated extension and correction of the targeting fragment sequences. APRT fragments with limited targeting homology flanking one side of the target gene deletion yield proportionately fewer target gene conversion events and proportionately more templated extension and vector correction events than do fragments with more substantial flanking homology.
Mol Cell Biol 1994 Oct
PMID:Homology dependence of targeted recombination at the Chinese hamster APRT locus. 793 85

The nature of multilocus deletions eliminating the adenine phosphoribosyltransferase (aprt) gene was analyzed in a CHO cell strain heterozygous for this locus. These deletions arose at a high frequency, spanning an estimated average length of 4250 kb. To detect breakpoints participating in their formation, a 200-kb region surrounding aprt was screened for novel fragments. Seven novel fragments were detected, five of which were clustered around the aprt gene itself. Despite the existence of at least eight Alu-equivalent repeats in this region, no breakpoints fell within these elements. Two deletions were characterized in more detail by cloning and sequencing their junction fragments. The novel DNA detected at one junction was unique, whereas that situated at the junction of the other deletion was of a repetitive nature, consisting of a truncated intracisternal-A particle gene. The contrasting nature of these junctions may imply that multilocus deletions of aprt can occur by one of several mechanisms.
Somat Cell Mol Genet 1994 Jul
PMID:Molecular characterization of multilocus deletions at a diploid locus in CHO cells: association with an intracisternal-A particle gene. 797 4

A 21-bp deletion in the third exon of the APRT gene in Chinese hamster ovary (CHO) cells was corrected by transfection with a plasmid containing hamster APRT sequences. Targeted correction frequencies in the range of 0.3-3.0 x 10(-6) were obtained with a vector containing 3.2 kb of APRT sequence homology. To examine the influence of vector configuration on targeted gene correction, a double-strand break was introduced at one of two positions in the vector prior to transfection by calcium phosphate-DNA coprecipitation or electroporation. A double-strand break in the region of APRT homology contained in the vector produced an insertion-type vector, while placement of the break just outside the region of homology produced a replacement-type vector. Gene targeting with both linear vector configurations yielded equivalent ratios of targeted recombinants to nontargeted vector integrants; however, targeting with the two different vector configurations resulted in different distributions of targeted recombination products. Analysis of 66 independent APRT+ recombinant clones by Southern hybridization showed that targeting with the vector in a replacement-type configuration yielded fewer targeted integrants and more target gene convertants than did the integration vector configuration. Targeted recombination was about fivefold more efficient with electroporation than with calcium phosphate-DNA coprecipitation; however, both gene transfer methods produced similar distributions of targeted recombinants, which depended only on targeting vector configuration. Our results demonstrate that insertion-type and replacement-type gene targeting vectors produce similar overall targeting frequencies in gene correction experiments, but that vector configuration can significantly influence the yield of particular recombinant types.
Somat Cell Mol Genet 1993 Jul
PMID:Targeting vector configuration and method of gene transfer influence targeted correction of the APRT gene in Chinese hamster ovary cells. 810 43

Expression of a recessive phenotype can occur by a number of different mechanisms, such as chromosomal deletion, recombination, and intragenic frameshift mutation or base substitution. To examine the contribution of different mutational events, we isolated and characterized a human fibroblast cell line heterozygous at the adenine phosphoribosyltransferase (APRT) locus. Cells that subsequently lost APRT activity were selected, cloned, and analyzed for the mechanisms contributing to the loss of APRT activity. Loss of APRT activity occurred at a rate of 7.8 x 10(-5) per allele per cell generation. Molecular analysis of DNA from 21 independent APRT- clones demonstrated that 62% of mutants had lost the functional allele and that the rest had incurred intragenic mutations. Loss of the functional allele was frequently accompanied by loss of the proximal marker D16S77 but not the more distant proximal marker D16S4, indicating that a high frequency of mitotic recombination or deletion occurred at the region between D16S77 and D16S4 on chromosome 16. Loss of APRT activity in the remaining 38% of the clones was predominantly due to point mutations. These data demonstrate that the mechanisms for loss of heterozygosity at the APRT locus are similar to those found in retinoblastoma and other tumors. The autosomal location of the APRT gene and the ease with which its phenotype can be selected make this gene useful for modeling mutational events at loci important to carcinogenesis.
Mol Carcinog 1993
PMID:Loss of heterozygosity: the most frequent cause of recessive phenotype expression at the heterozygous human adenine phosphoribosyltransferase locus. 821 32

The synthesis of proteins necessary for the respiratory reduction of nitrate to dinitrogen is induced in most denitrifying bacteria by a shift to anaerobiosis. A homolog of the fur gene, which encodes a redox-active transcriptional activator in Escherichia coli, was isolated from Pseudomonas stutzeri by using the anr gene of Pseudomonas aeruginosa as the hybridization probe (R. G. Sawers, Mol. Microbiol. 5:1469-1481, 1991). The coding region was located on a 3-kb SmaI fragment. An open reading frame of 735 nucleotides, designated fnrA, had the coding potential for a protein of 244 amino acids (M(r) = 27,089) with 51.2% positional identity to the Fnr protein of E. coli and 86.1% to the Anr protein of P. aeruginosa. The fnrA gene gave a single transcript of 0.85 kb and complemented nitrate-dependent anaerobic growth of an fnr deletion mutant of E. coli. An open reading frame immediately downstream of fnrA encoded adenine phosphoribosyltransferase (EC 2.4.2.7). Mutations in fnrA were generated in vitro by insertional mutagenesis followed by gene replacement. Gene inactivation was shown by loss of the fnrA transcript and detection of an arginine deiminase (EC 3.5.3.6)-negative phenotype in the mutants. However, neither the enzymatic activities nor the levels of anaerobic expression of the respiratory enzymes nitrate reductase (EC 1.7.99.4), nitrate reductase (EC 1.9.3.2), NO reductase (EC 1.7.99.7), and N2O reductase (EC 1.7.99.6) were changed in fnrA mutants versus the P. stutzeri wild type. A promoter-probe vector for Fnr-dependent transcription was activated anaerobically in the fnrA mutants, suggesting the existence of a second Fnr homolog in the same bacterium. The Fnr-binding motifs, apparent in the promoter region of genes encoding denitrification components of P. stutzeri, are likely to be recognized by this second Fnr homolog. Preliminary evidence indicates also the presence of the catabolite activator protein, Crp, in P. stutzeri.
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PMID:Anaerobic control of denitrification in Pseudomonas stutzeri escapes mutagenesis of an fnr-like gene. 822 70

Using a strategy based on reverse transcription and the polymerase chain reaction, we have determined the order of splicing of the four introns of the endogenous adenine phosphoribosyltransferase (aprt) gene in Chinese hamster ovary cells. The method involves a pairwise comparison of molecules that retain one intron and have either retained or spliced another intron(s). A highly preferred order of removal was found: intron 3 > 2 > 4 = 1. This order did not represent a linear progression from one end of the transcript to the other, nor did it correlate with the conformity of the splice site sequences to the consensus sequences or to the calculated energy of duplex formation with U1 small nuclear RNA. By using actinomycin D to inhibit RNA synthesis, the in vivo rate of the first step in splicing was estimated for all four introns; a half-life of 6 min was found for introns 2, 3, and 4. Intron 1 was spliced more slowly, with a 12-min half-life. A substantial amount of RNA that retained intron 1 as the sole intron was exported to the cytoplasm. In the course of these experiments, we also determined that intron 3, but not intron 4, is spliced before 3'-end formation is complete, probably on nascent transcripts. This result is consistent with the idea that polyadenylation is required for splicing of the 3'-most intron. We applied a similar strategy to determine the last intron to be spliced in a very large transcript, that of the endogenous dihydrofolate reductase (dhfr) gene in Chinese hamster ovary cells (25 kb). Here again, intron 1 was the last intron to be spliced.
Mol Cell Biol 1993 Oct
PMID:Order of intron removal during splicing of endogenous adenine phosphoribosyltransferase and dihydrofolate reductase pre-mRNA. 841 21

A 2.1-kilobase pair region located just upstream of the mouse aprt (adenine phosphoribosyltransferase) gene has a methylation pattern that is conserved in mouse tissues and culture cell lines. This upstream region includes four HpaII/MspI sites. Two of these sites are fully methylated, one is partially methylated, and one is unmethylated. Transfection experiments have demonstrated that the conserved methylation pattern can be reproduced in a mouse embryonal carcinoma stem cell line via de novo methylation (Turker, M.S., Mummaneni, P., and Bishop, P.L. (1991) Somat. Cell Mol. Genet. 17, 151-157). To examine the molecular basis of the conserved methylation pattern, a plasmid-based deletion analysis was conducted by removing and rearranging specific portions of the upstream region. Unmethylated versions of these plasmid constructs were then transfected into the mouse stem cell line and the methylation status of the remaining HpaII/MspI sites determined with a Southern blot analysis. By using this approach, a cis-acting sequence within the upstream region of approximately 0.8 kilobase pairs was identified which appears responsible for the conserved methylation pattern. We use the term "de novo methylation center" to denote this sequence. Based on the results obtained, a model is offered to explain the formation of the conserved methylation pattern in the upstream region.
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PMID:A cis-acting element accounts for a conserved methylation pattern upstream of the mouse adenine phosphoribosyltransferase gene. 841 60

The usefulness of adenovirus type 5 as a vector for homologous recombination was examined in CHO cells by using the adenine phosphoribosyltransferase (aprt) gene. Infection of a hemizygous CHO APRT- cell line containing a 3-bp deletion in exon 5 of the aprt gene with a recombinant adenovirus containing the wild-type gene resulted in restoration of the APRT+ phenotype at a frequency of 10(-5) to 10(-6) per infected cell. A relatively high frequency (approximately 6 to 20%) of the transductants appears to result from a homologous recombination event. The mutation on the chromosomal aprt gene is corrected in the homologous recombinants, and APRT expression is restored to a normal hemizygous level. Neither adenovirus nor exogenous promoter sequences are detected in the homologous recombinants. The remaining transductants result from random integration of the aprt gene with the adenovirus sequence. A number of adenovirus vectors containing different promoter sequences linked to the hamster aprt gene were constructed. A possible role for the promoter region in the homologous recombination event was indicated by the lack of homologous recombination in constructs lacking an active promoter.
Mol Cell Biol 1993 Feb
PMID:Correction of a deletion mutant by gene targeting with an adenovirus vector. 842 11

We have sequenced homologous DNA fragments of 2.7 and 2.8 kbp derived from the closely related mouse species Mus musculus domesticus (M. domesticus) and Mus musculus musculus (M. musculus), respectively. These two species diverged approximately 1 million years ago. Each DNA fragment contains 1.35 kbp of the 3' end of the constitutively expressed 2.2-kbp aprt (adenine phosphoribosyltransferase) gene and a similarly sized nontranscribed region downstream of the aprt gene. The aprt gene region contains protein coding sequences (0.35 kbp), intronic sequences (0.75 kbp), and a 3' nontranslated sequence (0.25 kbp). Both the M. domesticus and M. musculus downstream regions share three partial copies of the B1 repetitive element with the M. musculus downstream region containing an additional complete copy of this element. A comparison of the 2.7- and 2.8-kbp DNA fragments revealed a total of 63 molecular alterations (i.e., mutations) that were approximately fourfold more abundant in the nontranscribed downstream region than in the transcribed aprt gene. Of the 11 mutations observed in the transcribed region, 7 were found in introns, 3 in the 3' untranslated sequence, and 1 was a synonymous change in an exon. A comparison of the human and M. domesticus aprt genes has previously revealed no homology in either the intronic or 3' nontranslated regions with the exception of a 26-bp sequence in intron 3 and sequences at the exon/intron boundaries necessary for correct mRNA splicing (Broderick et al., Proc. Natl. Acad. Sci. USA, 84:3349, 1987). Therefore, there does not appear to be selective pressure for sequences within these regions. We conclude that there is a lower rate of accumulation of "silent" mutations in the transcribed mouse aprt gene than in a contiguous nontranscribed downstream region. A possible molecular mechanism involving preferential DNA repair for the transcribed region is discussed.
J Mol Evol 1993 Jan
PMID:Region-specific rates of molecular evolution: a fourfold reduction in the rate of accumulation of "silent" mutations in transcribed versus nontranscribed regions of homologous DNA fragments derived from two closely related mouse species. 843 77

The Aprt locus of Drosophila encodes the structural gene for the purine salvage enzyme adenine phosphoribosyltransferase. Aprt is autosomal and enzyme activity is gene-dose-dependent in Drosophila melanogaster. However, Aprt is X-linked and dosage compensated in Drosophila pseudoobscura, as shown here. The Aprt genes of both Drosophila species contain a DNA sequence associated with nuclear matrix attachment sites and these Aprt sequences specifically bind to nuclear matrix in vitro. Putative promoter sequences positioned upstream of the predicted transcriptional start site in the two Aprt genes have a similar structure of direct repeats with an overlapping dyad symmetry, but the DNA sequence of these motifs is not conserved between the two species. Biological features in mutants of Aprt as well as natural variants suggest that dosage compensation of this gene in Drosophila pseudoobscura is due to a general control mechanism on X-linked genes rather than a gene-specific mechanism.
Mol Gen Genet 1993 Apr
PMID:Adenine phosphoribosyltransferase genes in two Drosophila species: dosage compensation, a nuclear matrix attachment site, and a novel intron position. 849 6


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