Human Hypoxanthine Phosphoribosyltransferase Research Articles

Technical Report: A Simple and Robust Real-Time Quantitative PCR Method for the Detection of Radiation-Induced Multiple Exon Deletions of the Human HPRT Gene.

The hypoxanthine-phosphoribosyltransferase (HPRT) mutation assay has been widely used to investigate gene mutations induced by radiation. Here, we developed a novel method detecting deletions of multiple exons of the HPRT gene based on real-time quantitative PCR (qPCR). Immortalized normal human fibroblasts (BJ1-hTERT) were irradiated at various doses with γ rays, subjected to the 6-thioguanine (6-TG) selection, and more than one hundred 6-TG-resistant (6-TGR) clones were isolated. High-molecular-weight genomic DNA was extracted, and real-time qPCR was performed with the nine exon-specific primers. Optimization of the primer concentration, appropriate selection of PCR enzyme and refinement of the reaction profiles enabled simultaneous quantitative amplification of each exon. We were able to identify 6-TGR clones with total deletions, which did not show any amplification of the nine exons, and partial deletion mutants, in which one or some of the nine exons were missing, within a few days. This novel technique allows systematic determination of multiple deletions of the HPRT exons induced by ionizing radiation, enabling high-throughput and robust analysis of multiple HPRT mutants.

Prolonged cultured human hepatocytes as an in vitro experimental system for the evaluation of potency and duration of activity of RNA therapeutics: Demonstration of prolonged duration of gene silencing effects of a GalNAc-conjugated human hypoxanthine phosphoribosyl transferase (HPRT1) siRNA

We report here the evaluation of a novel in vitro experimental model, prolonged cultured human hepatocytes (PCHC), as an experimental system to evaluate the potency and duration of effects of oligonucleotide therapeutics. A novel observation was made on the redifferentiation of PCHC upon prolonged culturing based on mRNA profiling of characteristic hepatic differentiation marker genes albumin, transferrin, and transthyretin. Consistent with the known de-differentiation of cultured human hepatocytes, decreases in marker gene expression were observed upon culturing of the hepatocytes for 2 days. A novel observation of re-differentiation was observed on day 7 as demonstrated by an increase in expression of the marker genes to levels similar to that observed on the first day of culture. The expression of the differentiation marker genes was highest on day 7, followed by a gradual decrease but remained higher than that on day 2 for up to the longest culture duration evaluated of 41 days. The redifferentiation phenomenon suggests that PCHC may be useful for the evaluation of the duration of effects of oligonucleotide therapeutics on gene expression in human hepatocytes. A proof of concept study was thereby conducted with PCHC with a GalNAc-conjugated siRNA targeting human hypoxanthine phosphoribosyl transferase1 (HPRT1). HPRT1 mRNA expression in siRNA-treated cultures decreased to 21% of that in untreated hepatocytes on day 1, <10% from days 2 to 12, <20% from days 16 to 33, and eventually recovered to 64% by day 41. Our results suggest that PCHC represent a clinically-relevant cost- and time-efficient experimental tool to aid in the evaluation of GalNAc-siRNA silencing activity, providing information on both efficacy and duration of efficacy. PCHC may be applicable in the drug development setting as a species- and cell type-relevant experimental tool to aid the development of oligonucleotide therapeutics.

Reduced levels of dopamine and altered metabolism in brains of HPRT knock-out rats: a new rodent model of Lesch-Nyhan Disease.

Lesch-Nyhan disease (LND) is a severe neurological disorder caused by loss-of-function mutations in the gene encoding hypoxanthine phosphoribosyltransferase (HPRT), an enzyme required for efficient recycling of purine nucleotides. Although this biochemical defect reconfigures purine metabolism and leads to elevated levels of the breakdown product urea, it remains unclear exactly how loss of HPRT activity disrupts brain function. As the rat is the preferred rodent experimental model for studying neurobiology and diseases of the brain, we used genetically-modified embryonic stem cells to generate an HPRT knock-out rat. Male HPRT-deficient rats were viable, fertile and displayed normal caged behaviour. However, metabolomic analysis revealed changes in brain biochemistry consistent with disruption of purine recycling and nucleotide metabolism. Broader changes in brain biochemistry were also indicated by increased levels of the core metabolite citrate and reduced levels of lipids and fatty acids. Targeted MS/MS analysis identified reduced levels of dopamine in the brains of HPRT-deficient animals, consistent with deficits noted previously in human LND patients and HPRT knock-out mice. The HPRT-deficient rat therefore provides a new experimental platform for future investigation of how HPRT activity and disruption of purine metabolism affects neural function and behaviour.

Modulation of nociceptin and its receptor by phorbol-12-myristate-13-acetate via specific signal transduction kinases

• MM6 cells cultured with different concentrations (10-2-102 ng/ml) of phorbol-12-myristate-13-acetate (PMA) for 48 hrs. • Cells were treated with / without PMA 10 ng/ml for 0, 3, 6, 12, 24, 48 hrs. • mRNA of NOP and the nociceptin precursor prepronociceptin (PNoc) detected by quantitative RT-PCR.2 mRNA expression is presented as ratio of NOP or PNoc / reference gene HPRT (human hypoxanthine phosphoribosyl-transferase) normalized by the ratio of the calibrator (normalized ratio). • Intervention study: MM6 cells pretreated with PD98509 (PD) 30 μM, SP600125 (SP) 10 μM , SB203580 (SB) 10 μM or Bay 117821 (Bay) 3 μM for 1 hr to inhibit specific MAPKK/MEKs, JNK, p38, or NF-κB signaling pathway; followed by a co-culture with PMA 10 ng/ml for 6 or 48 hrs. • Intracellular phosphorylated kinases detected by phospho-flow. • Statistics: Medians with interquartile ranges (IQR); Mann-Whitney U test, subsequent post hoc analysis; level of significance: p<0.05. Figure 1: Time course of NOP and PNoc mRNA expression MM6 cells cultured with / without PMA 10 ng/ml.

Bacterial delivery of large intact genomic-DNA-containing BACs into mammalian cells

Efficient delivery of large intact vectors into mammalian cells remains problematical. Here we evaluate delivery by bacterial invasion of two large BACs of more than 150 kb in size into various cells. First, we determined the effect of several drugs on bacterial delivery of a small plasmid into different cell lines. Most drugs tested resulted in a marginal increase of the overall efficiency of delivery in only some cell lines, except the lysosomotropic drug chloroquine, which was found to increase the efficiency of delivery by 6-fold in B16F10 cells. Bacterial invasion was found to be significantly advantageous compared with lipofection in delivering large intact BACs into mouse cells, resulting in 100% of clones containing intact DNA. Furthermore, evaluation of expression of the human hypoxanthine phosphoribosyltransferase (HPRT) gene from its genomic locus, which was present in one of the BACs, showed that single copy integrations of the HPRT-containing BAC had occurred in mouse B16F10 cells and that expression of HPRT from each human copy was 0.33 times as much as from each endogenous mouse copy. These data provide new evidence that bacterial delivery is a convenient and efficient method to transfer large intact therapeutic genes into mammalian cells.

Human Gene Targeting Favors Insertions Over Deletions

Gene targeting is a powerful technique for manipulating the human genome, but few studies have directly compared the targeting frequencies of various types of vector constructs. Here we show that similar targeting constructs are able to insert nucleotides at the homologous chromosomal target locus more efficiently than they can delete nucleotides, and combination insertion/deletion vectors appear to target at intermediate frequencies. This holds true for deletions ranging from 1 to 334 bp and insertions ranging from 1 to 1332 bp. In addition, vectors designed to inactivate the human hypoxanthine phosphoribosyltransferase gene (HPRT) by deleting nucleotides often produced rearrangements at the target locus that in many cases were due to insertions of multimerized vector constructs, effectively converting a deletion vector into an insertion vector. These findings were obtained when adeno-associated virus vectors were used to efficiently deliver single-stranded DNA targeting constructs, but the same phenomenon was also observed when transfecting linearized double-stranded plasmids. Thus human cells distinguish between deletion and insertion vectors and process their recombination intermediates differently, presumably at the heteroduplex stage, with implications for the design of gene-targeting vectors and the evolution of human genomes.

The Role for Glutamic Acid at Position 196 in Human Hypoxanthine Phosphoribosyltransferase (HPRT) as Investigated Using Site-Directed Mutagenesis

The crystal structure of human HPRT reveals the involvement of E196 side chain at the A-B dimer interface. Interference by valine substitution at this position (E196V), as identified in patients with Lesch-Nyhan disease, nearly abolishes enzymatic activity. Kinetic analysis of the active mutants (E196A, E196D, E196Q, and E196R) suggests that interaction between K68 and E196 side chains contributes to stabilization of cis-configuration during the catalytic cycle. The study also provides further insight into the role of A-B dimer interactions relating to K68 in the regulation of cis-trans isomerization that potentially governs the rate-limiting steps in the HPRT reaction.

Stochastic properties of radiation-induced DSB: DSB distributions in large scale chromatin loops, the HPRT gene and within the visible volumes of DNA repair foci

Purpose: We computed probabilities to have multiple double-strand breaks (DSB), which are produced in DNA on a regional scale, and not in close vicinity, in volumes matching the size of DNA damage foci, of a large chromatin loop, and in the physical volume of DNA containing the HPRT (human hypoxanthine phosphoribosyltransferase) locus.Materials and methods: The model is based on a Monte Carlo description of DSB formation by heavy ions in the spatial context of the entire human genome contained within the cell nucleus, as well as at the gene sequence level.Results: We showed that a finite physical volume corresponding to a visible DNA repair focus, believed to be associated with one DSB, can contain multiple DSB due to heavy ion track structure and the DNA supercoiled topography. A corrective distribution was introduced, which was a conditional probability to have excess DSB in a focus volume, given that there was already one present. The corrective distribution was calculated for 19.5 MeV/amu N ions, 3.77 MeV/amu α-particles, 1000 MeV/amu Fe ions, and X-rays. The corrected initial DSB yield from the experimental data on DNA repair foci was calculated. The DSB yield based on the corrective function converts the focus yield into the DSB yield, which is comparable with the DSB yield based on the earlier PFGE experiments. The distribution of DSB within the physical limits of the HPRT gene was analyzed by a similar method as well.Conclusion: This corrective procedure shows the applicability of the model and empowers the researcher with a tool to better analyze focus statistics. The model enables researchers to analyze the DSB yield based on focus statistics in real experimental situations that lack one-to-one focus-to-DSB correspondance.

Cloning of Rabbit &amp;lt;italic&amp;gt;HPRT&amp;lt;/italic&amp;gt; Gene Using the Recombineering System

Hypoxanthine phosphoribosyltransferase (HPRT) plays an important role in the metabolic salvage of purines, and been used as an alternative pathway for mutant selection in many studies. To facilitate its application in rabbits, we have cloned the cDNA and genomic DNA of the rabbit HPRT gene using an approach that combines bioinformatics and recombineering methods. The cDNA is comprised of 1449 bp containing a coding sequence for a protein of 218 amino acids. The deduced amino acid sequence of the rabbit HPRT gene shares 98%, 97%, 98% and 94% identity with human, mouse, pig and cattle HPRT genes, respectively. Reverse transcription-polymerase chain reaction analysis showed that this gene is ubiquitously expressed in tissues of adult rabbit. The rabbit HPRT gene spans approximately 48 kb in length and consists of nine exons. The cloning of the rabbit HPRT gene shows the usefulness of the recombineering system in cloning genes of large size. This system may facilitate the subcloning of DNA from bacterial artificial chromosomes for cloning genes of large size or filling big gaps in genomic sequencing.

Dual Subcellular Localization in the Endoplasmic Reticulum and Peroxisomes and a Vital Role in Protecting against Oxidative Stress of Fatty Aldehyde Dehydrogenase Are Achieved by Alternative Splicing

Fatty aldehyde dehydrogenase (FALDH, ALDH3A2) is thought to be involved in the degradation of phytanic acid, a saturated branched chain fatty acid derived from chlorophyll. However, the identity, subcellular distribution, and physiological roles of FALDH are unclear because several variants produced by alternative splicing are present in varying amounts at different subcellular locations. Subcellular fractionation experiments do not provide a clear-cut conclusion because of the incomplete separation of organelles. We established human cell lines heterologously expressing mouse FALDH from each cDNA without tagging under the control of an inducible promoter and detected the variant FALDH proteins using a mouse FALDH-specific antibody. One variant, FALDH-V, was exclusively detected in peroxisomal membranes. Human FALDH-V with an amino-terminal Myc sequence also localized to peroxisomes. The most dominant form, FALDH-N, and other variants examined, however, were distributed in the endoplasmic reticulum. A gas chromatography-mass spectrometry-based analysis of metabolites in FALDH-expressing cells incubated with phytol or phytanic acid showed that FALDH-V, not FALDH-N, is the key aldehyde dehydrogenase in the degradation pathway and that it protects peroxisomes from oxidative stress. In contrast, both FALDHs had a protective effect against oxidative stress induced by a model aldehyde for lipid peroxidation, dodecanal. These results suggest that FALDH variants are produced by alternative splicing and share an important role in protecting against oxidative stress in an organelle-specific manner.

A novel expression system for genomic DNA loci using a human artificial chromosome vector with transformation-associated recombination cloning

Following the recent completion of the human genome sequence, genomics research has shifted its focus to understanding gene complexity, expression, and regulation. However, in order to investigate such issues, there is a need to develop a practical system for genomic DNA expression. Transformation-associated recombination (TAR) cloning has proven to be a convenient tool for selective isolation of a genetic locus from a complex genome as a circular YAC using recombination in yeast. The human artificial chromosome (HAC) vector containing an acceptor loxP site has served as a platform for the reproducible expression of transgenes. In this study, we describe a system that efficiently expresses a genetic locus in mammalian cells by retrofitting a TAR-YAC with the donor loxP site and loading it onto the HAC vector by the Cre/loxP system. In order to demonstrate functional expression of genomic loci, the entire human hypoxanthine phosphoribosyl transferase (HPRT) locus contained in a 100 kb YAC was loaded onto the HAC vector and was shown to complement the genetic defect in Hprt-deficient CHO cells. Thus, the combination of TAR cloning and the HAC vector may serve as a powerful tool for functional genomic studies.

826. The Risk of Integration: Characterization and Testing of Cell Lines Engineered for the Study of Insertional Gene Activation

The potential therapeutic effectiveness of gene transfer in many cases requires more extended duration of expression, most reliably achieved by the use of integrating vectors such as retroviruses, lentiviruses, or transposons. Recently there has been considerable interest in and concern about the safety of integrating vectors for gene therapy and the risk of insertional oncogene activation. At last year's meeting of the ASGT (abstract |[num]|7, 2004), we reported the establishment of a series of cell lines we call |[ldquo]|HT-FH|[rdquo]|, derived from human HT1080 cells, containing an inactive selectable marker (a human hypoxanthine phosphoribosyltransferase [HPRT] cDNA regulated by a minimal fos promoter), designed to screen for insertional activation events by selection in HAT (hypoxanthine, amethopterin, thymidine) medium. Here we describe further characterization and testing of these cell lines for insertional activation studies.

A survey of splice variants of the human hypoxanthine phosphoribosyl transferase and DNA polymerase beta genes: products of alternative or aberrant splicing?

Errors during the pre-mRNA splicing of metazoan genes can degrade the transmission of genetic information, and have been associated with a variety of human diseases. In order to characterize the mutagenic and pathogenic potential of mis-splicing, we have surveyed and quantified the aberrant splice variants in the human hypoxanthine phosphoribosyl transferase (HPRT) and DNA polymerase beta (POLB) in the presence and the absence of the Nonsense Mediated Decay (NMD) pathway, which removes transcripts with premature termination codons. POLB exhibits a high frequency of splice variants (40-60%), whereas the frequency of HPRT splice variants is considerably lower (approximately 1%). Treatment of cells with emetine to inactivate NMD alters both the spectrum and frequency of splice variants of POLB and HPRT. It is not certain at this point, whether POLB and HPRT splice variants are the result of regulated alternative splicing processes or the result of aberrant splicing, but it appears likely that at least some of the variants are the result of splicing errors. Several mechanisms that may contribute to aberrant splicing are discussed.

Genetic Interactions between BLM and DNA Ligase IV in Human Cells

BLM has been implicated in DNA double-strand break (DSB) repair, but its precise role remains obscure. To explore this, we generated BLM(-/-) and BLM(-/-)LIG4(-/-) cells from the human pre-B cell line Nalm-6. BLM(-/-) cells exhibited retarded growth, increased mutation rates, and hypersensitivity to agents that block replication fork progression. Interestingly, these phenotypes were significantly suppressed by deletion of LIG4, suggesting that nonhomologous end-joining (NHEJ) is unfavorable for integrity and survival of cells lacking BLM. We propose that the absence of BLM leads to accumulation of replication-associated, one-ended DSBs, which are deleterious to cells and lead to genomic instability when repaired by NHEJ. In addition, the NHEJ pathway per se was marginally affected by BLM deficiency, as evidenced by x-ray sensitivity and I-SceI-based DSB repair assays. More intriguingly, however, these experiments revealed the presence of an alternative, DNA ligase IV-independent end-joining pathway, which was significantly affected by the loss of BLM. Collectively, our results provide the first evidence for genetic interactions between BLM and NHEJ in human cells.

Steady-state kinetics of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi

The kinetic mechanism for the reaction catalyzed by the hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi was analyzed to determine the feasibility of designing a parasite-specific mechanism-based inhibitor of this enzyme. The results show that the HPRT from T. cruzi follows an essentially ordered bi–bi reaction, and like its human counterpart also likely forms a dead end complex with purine substrates and the product pyrophosphate. Computational fitting of the kinetics data to multiple initial velocity equations gave results that are consistent with the dead end complex arising when the hypoxanthine- or guanine-bound form of the enzyme binds pyrophosphate rather than the phosphoribosylpyrophosphate substrate of the productive forward reaction. Limited proteolytic digestion was employed to provide additional support for formation of the dead end complex and to estimate the K d values for substrates of both the forward and reverse reactions. Due to similarities with the kinetic mechanism of the human HPRT, the results reported here for the HPRT from T. cruzi indicate that the design of a mechanism-based inhibitor of the trypanosomal HPRT, that would not also inhibit the human enzyme, may be difficult. However, the results also show that a potent selective inhibitor of the trypanosomal HPRT might be achieved via the design of a bi-substrate type inhibitor that incorporates analogs of moieties for a purine base and pyrophosphate.

Structural and functional analysis of mutations at the human hypoxanthine phosphoribosyl transferase (HPRT1) locus.

Hypoxanthine phosphoribosyl transferase (HPRT, also known as HGPRT) is an often-used genetic marker in eukaryotic cells. The gene is conserved from bacteria to human, with retained catalytic activity, although substrate specificity may have changed, and the enzyme is essential in malaria-causing protozoans. Inherited mutations in the human HPRT1 gene result in three different phenotypes: Lesch-Nyhan syndrome (LNS or LND), LND variants, and HPRT-related hyperuricemia (HRH). In cultured cells, loss of HPRT activity gives rise to 6-thioguanine (6-TG) resistance. In general, cells from LND patients are also 6-TG resistant, whereas cells from HRH patients are not, with some interesting exceptions. Using modeling methods, we have studied the correlation between the mutable and nonmutated amino acid residues on one hand, and sequence conservation and predicted phenotypic effects on the other hand. Our results demonstrate that most of the mutations are explainable by the predicted effect on protein structure and function. They are also consistent with sequence conservation. Moreover, the mutational profiles of TG-resistant cells and LND overlap to a great extent, while most of the mutations in HRH are unique to that condition. We have also noticed a strong correlation between mutations in the tetramer interfaces and observed phenotypes, suggesting a functional role for a tetramer transition during catalysis.

Nucleosomes are translationally positioned on the active allele and rotationally positioned on the inactive allele of the HPRT promoter.

Differential chromatin structure is one of the hallmarks distinguishing active and inactive genes. For the X-linked human hypoxanthine phosphoribosyltransferase gene (HPRT), this difference in chromatin structure is evident in the differential general DNase I sensitivity and hypersensitivity of the promoter regions on active versus inactive X chromosomes. Here we characterize the nucleosomal organization responsible for the differential chromatin structure of the active and inactive HPRT promoters. The micrococcal nuclease digestion pattern of chromatin from the active allele in permeabilized cells reveals an ordered array of translationally positioned nucleosomes in the promoter region except over a 350-bp region that is either nucleosome free or contains structurally altered nucleosomes. This 350-bp region includes the entire minimal promoter and all of the multiple transcription initiation sites of the HPRT gene. It also encompasses all of the transcription factor binding sites identified by either dimethyl sulfate or DNase I in vivo footprinting of the active allele. In contrast, analysis of the inactive HPRT promoter reveals no hypersensitivity to either DNase I or a micrococcal nuclease and no translational positioning of nucleosomes. Although nucleosomes on the inactive promoter are not translationally positioned, high-resolution DNase I cleavage analysis of permeabilized cells indicates that nucleosomes are rotationally positioned over a region of at least 210 bp on the inactive promoter, which coincides with the 350-bp nuclease-hypersensitive region on the active allele, including the entire minimal promoter. This rotational positioning of nucleosomes is not observed on the active promoter. These results suggest a model in which the silencing of the HPRT promoter during X chromosome inactivation involves remodeling a transcriptionally competent, translationally positioned nucleosomal array into a transcriptionally repressed architecture consisting of rotationally but not translationally positioned nucleosomal arrays.

Optimization of a versatile in vitro transcription assay for the expression of multiple start site TATA-less promoters.

Previous work from our laboratory has allowed for the subdivision of RNA polymerase II TATA-less promoters into two classes: those that initiate at a single start site (SSS) and those that initiate at multiple start sites (MSS). MSS promoters are defined by the lack of a TATA box and the presence of a transcription initiation window and a downstream MED-1 element (GCTCCC/G) [Ince, T. A., and Scotto, K. W. (1995) J. Biol. Chem. 270, 30249-30252]. Further insight into the mechanisms regulating TATA-less MSS promoters has been hampered by the lack of an in vitro transcription assay in which multiple start sites can be reproduced. In the present study, we describe the development of a versatile in vitro transcription system optimized for the expression of MSS promoters, termed the multiple promoter comparison (MPC) assay. By alteration of assay parameters including template length, cation and nucleotide concentrations, and RNA isolation method, the accurate and robust transcription of two MSS promoters, pgp1 (hamster P-glycoprotein class I homologue) and HPRT (human hypoxanthine phosphoribosyltransferase), was accomplished. Moreover, both TATA-containing and TATA-less single start site promoters were also transcribed in the MPC assay, making this the first general in vitro transcription system for the simultaneous analysis of all three classes of RNA polymerase II genes.

The Assembly of Large BACs by in Vivo Recombination

We have developed a method for recombining bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) containing large genomic DNA fragments into a single vector using the Cre–lox recombination system from bacteriophage P1 in vivo. This overcomes the limitations of in vitro methods for generating large constructs based on restriction digestion, ligation, and transformation of DNA into Escherichia coli cells. We used the method to construct a human artificial chromosome vector of 404 kb encompassing long tracts of α satellite DNA, telomeric sequences, and the human hypoxanthine phosphoribosyltransferase gene. The specificity of Cre recombinase for loxP sites minimizes the possibility of intramolecular rearrangements, unlike previous techniques using general homologous recombination in E. coli, and makes our method compatible with the presence of large arrays of repeated sequences in cloned DNA. This methodology may also be applied to retrofitting PACs or BACs with markers and functional sequences.

Deletion in the hypoxanthine phosphoribosyltransferase gene caused by Alu-Alu recombination in two Japanese patients with Lesch-Nyhan syndrome.

Lesch-Nyhan (LN) syndrome is caused by a severe genetic deficiency of hypoxanthine phosphoribosyltransferase (HPRT), which is characterized by neuropsychological disorders (including self-mutilation) and hyperuricemia. The human HPRT gene is located on the X chromosome and its length is 57 kb1. The coding sequence of 654 bp is divided into 9 exons. Although there is great heterogeneity in the known hprt mutations associated with LN syndrome, approximately 85% of LN mutations are located within the cording region or the splicing sequences of the HPRT gene, which can be easily predicted by examination of HPRT cDNA. However, the remaining 15% are large genomic alterations such as total or partial gene deletions and duplications, which often does not produce HPRT mRNA, and it is difficult to determine mutational breakpoints due to large size of introns. According to our strategy for analysis of LN mutations (Fig. l), we have identified three independent base substitutions in three Japanese LN patients and one identical large genomic deletion in two unrelated Japanese