Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A complete deficiency of the purine salvage enzyme, hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8), in man results in the Lesch-Nyhan (LN) syndrome. Two unrelated patients with the full LN syndrome showed no evidence of a major alteration to the gene encoding HPRT (HPRT) by restriction endonuclease analysis, but exhibited negligible levels of HPRT mRNA on Northern blots. DNA from these patients was characterised further. Amplification, by the polymerase chain reaction (PCR), of individual HPRT-exon fragments from genomic DNA followed by nucleotide (nt) sequence analysis using automated technology, revealed single-base mutations in each patient. One patient has an insertion of a T within exon-2, which places a stop codon in frame, presumably resulting in premature termination of translation of the HPRT mRNA. The other patient has a G----A base substitution at the 5' end of intron-6, at the junction of exon-6 and intron-6. Although dot blot analysis indicated negligible HPRT mRNA in lymphoblast cells from both patients, we were successful in amplifying HPRT cDNA using PCR. Direct nt sequence analysis of the amplified cDNA confirmed the insertion of a T in exon-2 in the one patient and revealed a complete deletion of exon-6 in the other patient, the latter event presumably arising due to aberrant splicing of primary message. Both mutations were also confirmed by hybridisation of amplified genomic DNA with allele-specific oligodeoxyribonucleotide probes. This study illustrates two approaches for analysing DNA mutations at the molecular level and demonstrates the power of PCR technology in the study of genetic diseases.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The molecular characterisation of HPRT CHERMSIDE and HPRT COORPAROO: two Lesch-Nyhan patients with reduced amounts of mRNA. 184 May 49

Three-allele restriction fragment length polymorphisms (RFLPs) for the restriction endonuclease Bam HI are known at the hypoxanthine-guanine phosphoribosyltransferase (HPRT, E.C.2.4.2.8.) gene locus. The alleles are expressed phenotypically on Southern blots as three distinct pairs of fragments that hybridize to HPRT cDNA: i) a 22-kilobase (kb)/25-kb pair, ii) a 12-kb/25-kb pair, and iii) a 22-kb/18-kb pair. Allele frequencies in 119 unrelated Japanese people were 0.38 for the 22-kb/25-kb allele, 0.43 for the 12-kb/25-kb allele, and 0.19 for the 22-kb/18-kb allele, an average heterozygosity of 66% in Japanese females, a higher rate than in Caucasian females. Five out of nine carriers of partial or complete HPRT deficiency showed heterozygous patterns for Bam HI RFLPs.
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PMID:Bam HI restriction fragment length polymorphisms for hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of carriers and controls of HPRT deficiency in Japan. 197 Feb 11

The CHO-UV-1 mutant, a Chinese hamster ovary cell with defective postreplication recovery of DNA, is 2- to 4-fold more sensitive than its wild-type counterpart (CHO-77256) to the lethal effects of ethylating agents and UV radiation; it is also hypersensitive (10- to 20-fold) to some DNA-methylating and -cross-linking agents. We studied the CHO-UV-1 mutant further to define its phenotype in terms of DNA damage induction and repair, methyltransferase activity, and effects of caffeine on mutational and lethal responses. Both wild-type and CHO-UV-1 cells incurred similar levels and types of damage when exposed to UV radiation, N-methyl-N'-nitro-N-nitrosoguanidine, or N-methyl-N-nitrosourea. The rate and extent of repair of Micrococcus luteus endonuclease-sensitive sites after UV irradiation or treatment with N-methyl-N'-nitro-N-nitrosoguanidine were also equivalent in these two cell types. Twenty % of the initial endonuclease-sensitive sites induced in either cell line remained at 18 h after UV irradiation; approximately 8% of the sites after N-methyl-N'-nitro-N-nitrosoguanidine exposure were present in both parental and CHO-UV-1 cells after a 17-h repair period. Moreover, the ability of CHO-UV-1 to resynthesize and ligate DNA during excision repair was similar to that of its parent. Neither CHO-UV-1 nor CHO-77256 had appreciable levels of O6-methylguanine-DNA methyltransferase activity which ameliorates the cytotoxicity of alkylating agents. Caffeine, a known inhibitor of postreplication repair, decreased the frequency of mutation induction at the hypoxanthine-guanine phosphoribosyltransferase locus by 40-55% in CHO-77256 but not in CHO-UV-1. These results rule out defective excision repair as a factor in the hypersensitivity of the CHO-UV-1 mutant to DNA-damaging agents. Hence, this cell line appears to derive from a mutation affecting nonexcision repair processes and should be useful in clarifying the mechanism(s) of postreplication recovery of DNA in mammalian cells.
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PMID:Genetic and biochemical characterization of the CHO-UV-1 mutant defective in postreplication recovery of DNA. 231 21

DNA sequences of the X-chromosome-linked hypoxanthine phosphoribosyltransferase (HPRT) and glucose 6-phosphate dehydrogenase (G6PD) genes have revealed the presence of clusters of CpG dinucleotides, raising the possibility that such clusters are involved in the control of expression of these genes, which are expressed in all tissues. Although CpG clusters are not exclusive features of the X chromosome, the analysis of X-linked genes provides the means to determine whether CpG clusters are control elements; one of the two homologous X loci in female mammals is not expressed, so that active and inactive versions of the gene can be compared. In fact, it has been shown that these CpG clusters are undermethylated when the gene is active and extensively methylated when the gene is inactive. In addition to hypomethylation, chromatin hypersensitivity to endonuclease digestion is a known hallmark of regulatory sequences in eukaryotic genes. We report here that the CpG clusters of the active hprt and g6pd genes are not only undermethylated, but also hypersensitive to MspI, DNase I and S1 nuclease, further supporting the suggestion that they are involved in the control of expression of these genes.
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PMID:Clusters of CpG dinucleotides implicated by nuclease hypersensitivity as control elements of housekeeping genes. 298 78

The organization of the X-linked gene for human hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8.) has been determined by a combination of restriction endonuclease mapping, heteroduplex analysis and DNA sequence analysis of overlapping genomic clones. The entire gene is 42 kilobases in length and split into 9 exons. The sizes of the 7 internal exons and the exon-intron boundaries are identical to those of mouse HPRT gene. The 5' end of the gene lacks the prototypical 5' transcriptional regulatory sequence elements but contains extremely GC-rich sequences and five GC hexanucleotide motifs (5'-GGCGGG-3'). These structural features are very similar to those found in the mouse HPRT gene and to some of the regulatory signals common to a class of constitutively expressed "housekeeping" genes. Several transcriptional start sites have been identified by nuclease protection studies. Extensive sequence homology between the mouse and human genes is found in the 3' non-coding portion of the gene.
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PMID:The organization of the human HPRT gene. 300 6

The restriction endonuclease Alu I induces chromosomal aberrations and mutations in the hypoxanthine phosphoribosyltransferase (HPRT) locus as measured by 6-thioguanine resistance (TGr) in V79 hamster cells. Alu I does not induce mutations in the Na+/K+ ATPase locus as measured by ouabain resistance (OUAr). The data are interpreted to mean that most if not all Alu I-induced TGr mutations represent chromosomal aberrations.
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PMID:The restriction endonuclease Alu I induces chromosomal aberrations and mutations in the hypoxanthine phosphoribosyltransferase locus, but not in the Na+/K+ ATPase locus in V79 hamster cells. 301 99

Using cloned cDNA sequences of murine and human hypoxanthine phosphoribosyltransferase (HPRT: IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), we have identified and characterized a three-allele restriction-fragment-length polymorphism for the restriction endonuclease BamHI at the human HPRT locus. The alleles are expressed phenotypically on Southern blots as three distinct pairs of fragments that hybridize to HPRT cDNA: (i) a 22-kilobase (kb)/25-kb pair, (ii) a 12-kb/25-kb pair, and (iii) a 22-kb/18-kb pair. In addition to fragments from the HPRT locus, sequences recognized by both HPRT cDNA probes are also present on at least two autosomes in the human genome. Allele frequencies in an unselected Caucasian population are 0.77 for the 22-kb/25-kb allele. 0.16 for the 12-kb/25-kb allele, and 0.07 for the 22-kb/18-kb allele, resulting in an average heterozygosity of 38% in females in this population. This polymorphism should facilitate gene mapping by linkage in this region of the human X chromosome.
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PMID:A three-allele restriction-fragment-length polymorphism at the hypoxanthine phosphoribosyltransferase locus in man. 630 59

In somatic mammalian cells, homologous recombination is a rare event. To study the effects of chromosomal breaks on frequency of homologous recombination, site-specific endonucleases were introduced into human cells by electroporation. Cell lines with a partial duplication within the HPRT (hypoxanthine phosphoribosyltransferase) gene were created through gene targeting. Homologous intrachromosomal recombination between the repeated regions of the gene can reconstruct a functioning, wild-type gene. Treatment of these cells with the restriction endonuclease Xba I, which has a recognition site within the repeated region of HPRT homology, increased the frequency or homologous recombination bv more than 10-fold. Recombination frequency was similarly increased by treatment with the rare-cutting yeast endonuclease PI-Sce I when a cleavage site was placed within the repeated region of HPRT. In contrast, four restriction enzymes that cut at positions either outside of the repeated regions or between them produced no change in recombination frequency. The results suggest that homologous recombination between intrachromosomal repeats can be specifically initiated by a double-strand break occurring within regions of homology, consistent with the predictions of a model.
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PMID:Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases. 862 83

To investigate the effects of in vivo genomic DNA double-strand breaks on the efficiency and mechanisms of gene targeting in mouse embryonic stem cells, we have used a series of insertion and replacement vectors carrying two, one, or no genomic sites for the rare-cutting endonuclease I-SceI. These vectors were introduced into the hypoxanthine phosphoribosyltransferase (hprt) gene to produce substrates for gene-targeting (plasmid-to-chromosome) or intrachromosomal (direct repeat) homologous recombination. Recombination at the hprt locus is markedly increased following transfection with an I-SceI expression plasmid and a homologous donor plasmid (if needed). The frequency of gene targeting in clones with an I-SceI site attains a value of 1%, 5,000-fold higher than that in clones with no I-SceI site. The use of silent restriction site polymorphisms indicates that the frequencies with which donor plasmid sequences replace the target chromosomal sequences decrease with distance from the genomic break site. The frequency of intrachromosomal recombination reaches a value of 3.1%, 120-fold higher than background spontaneous recombination. Because palindromic insertions were used as polymorphic markers, a significant number of recombinants exhibit distinct genotypic sectoring among daughter cells from a single clone, suggesting the existence of heteroduplex DNA in the original recombination product.
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PMID:Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double-strand breaks in mouse embryonic stem cells. 963 91

The CRISPR-Cas9 system uses guide RNAs to direct the Cas9 endonuclease to cleave target sequences. It can, in theory, target essentially any sequence in a genome, but the efficiency of the predicted guide RNAs varies dramatically. If no targeted cells are obtained, it is also difficult to know why the experiment fails. We have developed a transient transfection based method to enrich successfully targeted cells by co-targeting the hypoxanthine phosphoribosyltransferase (HPRT) gene. Cells are transfected with two guide RNAs that target respectively HPRT and the gene of interest. HPRT targeted cells are selected by resistance to 6-thioguanine (6-TG) and then examined for potential alterations to the gene targeted by the co-transfected guide RNA. Alterations of many genes, such as AAVS1, Exo1 and Trex1, are highly enriched in the 6-TG resistant cells. This method works in both HCT116 cells and U2OS cells and can easily be scaled up to process multiple guide RNAs. When co-targeting fails, it is straightforward to determine whether the target gene is essential or the guide RNA is ineffective. HPRT co-targeting thus provides a simple, efficient and scalable way to enrich gene targeting events and to identify the cause of failure.
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PMID:Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene. 2613 Jul 22


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