Identification of Reference Genes for Circadian Studies on Brain Microvessels and Choroid Plexus Samples Isolated from Rats.

Delivery of putative compounds of therapeutic value to the brain is limited by brain barriers: the blood-brain barrier located in the endothelium of the brain microvessels (BrMVs) and the blood-cerebrospinal fluid barrier located in the epithelium of the choroid plexus (ChP). Understanding their function and modulation by the circadian clock may enhance the efficacy of brain-targeting therapies. The aim of the present study was to evaluate the stability of 10 reference genes in the BrMV and ChP, isolated from male and female rats at six time points (ZT1, 5, 9, 13, 17, and 21). Gene evaluations were performed by qPCR, analyzed by RefFinder tool, and verified by analyzing the expression of the brain and muscle ARNT-like 1 (Bmal1) using the qPCR and digital PCR methods. We identified as the most stable genes for circadian studies tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and apolipoprotein E (Apoe) for BrMV, and beta actin (Actb) and hypoxanthine-guanine phosphoribosyltransferase (Hprt1) for ChP. After verification, ribosomal protein (Rps18) was also included as a sufficient reference gene. Additionally, the observed gender difference in the Bmal1 oscillations in both BrMV and ChP suggests that separate studies for each gender are recommended.

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.

CRISPR/Cas9-Mediated Scanning for Regulatory Elements Required for HPRT1 Expression via Thousands of Large, Programmed Genomic Deletions.

The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion ("ScanDel"). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder. Altogether, we programmed 4,342 overlapping 1 and 2 kb deletions that tiled 206 kb centered on HPRT1 (including 87 kb upstream and 79 kb downstream) with median 27-fold redundancy per base. We functionally assayed programmed deletions in parallel by selecting for loss of HPRT function with 6-thioguanine. As expected, sequencing gRNA pairs before and after selection confirmed that all HPRT1 exons are needed. However, HPRT1 function was robust to deletion of any intergenic or deeply intronic non-coding region, indicating that proximal regulatory sequences are sufficient for HPRT1 expression. Although our screen did identify the disruption of exon-proximal non-coding sequences (e.g., the promoter) as functionally consequential, long-read sequencing revealed that this signal was driven by rare, imprecise deletions that extended into exons. Our results suggest that no singular distal regulatory element is required for HPRT1 expression and that distal mutations are unlikely to contribute substantially to Lesch-Nyhan syndrome burden. Further application of ScanDel could shed light on the role of regulatory mutations in disease at other loci while also facilitating a deeper understanding of endogenous gene regulation.

Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence.

Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies.

Morin, a Bioflavonoid Suppresses Monosodium Urate Crystal-Induced Inflammatory Immune Response in RAW 264.7 Macrophages through the Inhibition of Inflammatory Mediators, Intracellular ROS Levels and NF-κB Activation.

Our previous studies had reported that morin, a bioflavanoid exhibited potent anti-inflammatory effect against adjuvant-induced arthritic rats. In this current study, we investigated the anti-inflammatory mechanism of morin against monosodium urate crystal (MSU)-induced inflammation in RAW 264.7 macrophage cells, an in vitro model for acute gouty arthritis. For comparison purpose, colchicine was used as a reference drug. We have observed that morin (100-300 µM) treatment significantly suppressed the levels of inflammatory cytokines (TNF-α, IL-1ß, IL-6, MCP-1 and VEGF), inflammatory mediators (NO and PEG2), and lysosomal enzymes (acid phosphatase, ß-galactosidase, N-acetyl glucosamindase and cathepsin D) in MSU-crystals stimulated macrophage cells. The mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, and MCP-1), inflammatory enzymes (iNOS and COX-2), and NF-κBp65 was found downregulated in MSU crystal stimulated macrophage cells by morin treatment, however, the mRNA expression of hypoxanthine phospho ribosyl transferse (HPRT) was found to be increased. The flow cytometry analysis revealed that morin treatment decreased intracellular reactive oxygen species levels in MSU crystal stimulated macrophage cells. The western blot analysis clearly showed that morin mainly exerts its anti-inflammatory effects by inhibiting the MSU crystal-induced COX-2 and TNF-α protein expression through the inactivation of NF-κB signaling pathway in RAW 264.7 macrophage cells similar to that of BAY 11-7082 (IκB kinase inhibitor). Our results collectively suggest that morin can be a potential therapeutic agent for inflammatory disorders like acute gouty arthritis.

Measuring recombination proficiency in mouse embryonic stem cells.

A method is presented to measure homologous recombination in mouse embryonic stem cells by both gene targeting and short-tract gene conversion of a double-strand break (DSB). A fluorescence-based reporter is first gene targeted to the Hprt locus in a quantifiable way. A homing endonuclease expression vector is then introduced to generate a DSB, the repair of which is also quantifiable.

A modified TALEN-based system for robust generation of knock-out human pluripotent stem cell lines and disease models.

BACKGROUND: Transcription activator-like effector nucleases (TALENs) have emerged as a tool for enabling targeted gene editing and disruption in difficult systems, such as human pluripotent stem cells (hPSCs). The modular architecture of TAL effectors theoretically enables targeting of any genomic locus and several cloning systems for custom TALEN assembly have recently been established. However, there is a lack of versatile TALEN expression systems applicable to hPSCs. RESULTS: Here, we extend an existing TALE assembly system by a dual set of expression vectors for efficient application of TALEN technology in hPSCs. This is characterized by improved TALEN architecture as well as antibiotic resistance and fluorescent reporter cassettes, thus enabling enrichment for transfected cells. Improved functionality of the combined system was demonstrated by targeted disruption of the HPRT1 gene to create isogenic disease models of Lesch-Nyhan-Syndrome. Using female hPSCs, homozygous disruption of HPRT1 occurred at efficiencies of up to 15%. Differentiating isogenic knock-out cells both into central nervous system (CNS) as well as into sensory-like neurons recapitulated previously described phenotypes based on patient-specific induced PSCs and extended these findings to non-CNS neurons, respectively. CONCLUSION: The combined vector system allows for flexible and affordable generation of knock-out hPSCs lines, thus enabling investigation of developmental processes as well as the generation of isogenic disease models without the need for patient material.

Reference genes in the developing murine brain and in differentiating embryonic stem cells.

OBJECTIVES: Gene expression analysis via quantitative real-time PCR (qPCR) is a key approach in biological and medical research. Here, variations between runs and samples are compensated for by in-parallel analysis of reference genes, which require a most stable expression throughout all samples and experimental procedures to function as internal standards. In reality, there is no universal reference gene; but rather, assumed reference genes vary widely among various cell types. This demands an evaluation of reference genes for each specific experimental purpose, especially in the case of developmental studies. The aim of the present study was to identify suitable reference genes for gene expression analysis in the developing murine brain neocortex in vivo and in mouse embryonic stem cells (mESC) throughout differentiation in vitro. METHODS: The five candidate genes Actb, 18s, Gapdh, Hprt, and RpII were analyzed throughout development in vivo and in vitro using the quartiles of C(q) values, fold change, coefficient of variation (CV) and the difference between maximum minus twofold standard deviation and mean as the criteria to evaluate their expression stability. RESULTS: We found that RpII was the most stable expressed gene in mESC throughout differentiation, while in the developing murine neocortex Gapdh showed the highest expression stability. CONCLUSIONS: Based on our results, we suggest for gene expression analysis in the context of neurodevelopment the usage of RpII as a reference gene for mESC and Gapdh or Hprt for the murine neocortex.

Nuclear factor-κB mediated lipopolysaccharide-induced mRNA expression of hepcidin in human peripheral blood leukocytes.

Hepcidin is a known key modulator of iron homeostasis and an innate immune molecule secreted by the liver. The transcriptional mechanism of hepcidin in hepatocytes during inflammation is mediated via the IL-6/STAT3 pathway. Recently, hepcidin demonstrated an anti-inflammatory function in endotoxic mice, and a TLR4-dependent inducible expression of hepcidin was detected in myeloid cells. In this study, we explored the expression and signaling mechanism regulating hepcidin mRNA expression in peripheral blood leukocytes. The mRNA levels of hepcidin in peripheral blood leukocytes from patients with severe sepsis (n = 14) was significantly higher than those in healthy controls (n = 16;0.286 ± 0.065 vs 0.068 ± 0.025; P < 0.05). Ex vivo studies found hepcidin mRNA can be highly induced by challenge of 100 ng/ml LPS or 20 ng/ml TNF-α in peripheral blood leukocytes rather than IL-6, IL-1 and IFN-γ. Anti-TNF-α antibody significantly decreased the levels of hepcidin mRNA induced by LPS. Inhibitor of nuclear factor (NF)-κB rather than that of STAT3 completely abolished the inducibility of hepcidin mRNA in PBMCs and neutrophils. These results indicate that hepcidin mRNA expression in peripheral blood leukocytes induced by LPS depends on NF-κB, and TNF-α may be a key mediator in this procedure.

Variable expression of HPRT deficiency in 5 members of a family with the same mutation.

BACKGROUND: Lesch-Nyhan disease is an inborn error of purine metabolism that results from deficiency of the activity of hypoxanthine phosphoribosyltransferase (HPRT). In the classic disease, the activity of the enzyme is completely deficient; the patient has mental retardation, spasticity, dystonia, and self-injurious behavior, as well as elevated concentrations of uric acid in blood and urine and its consequences of nephropathy, urinary tract calculi, and tophaceous gout. The HPRT gene is located on the X chromosome, and its expression is usually X-linked recessive. There are variant HPRT enzymes with some activity, and milder clinical expression, but the rule has been that each mutation produces a stereotypical pattern of clinical disease. OBJECTIVE: To document a family in which a single mutation has led to 3 different phenotypes in 5 individuals. DESIGN: Case reports. Settings A foundation devoted to the investigation and care of patients with rare diseases and a university-based biochemical genetics laboratory. MAIN OUTCOME MEASURES: Clinical and biochemical observations of predominantly 1 generation of a family. RESULTS: A mutation (IVS6 + 2) led to deletion of exon 6. In 1 patient, the phenotype was that of classic Lesch-Nyhan syndrome, while the patient's brother and uncle had a much milder disease, which was difficult to distinguish from good health; 2 cousins had an intermediate phenotype. CONCLUSION: It is no longer true that a given mutation in the HPRT gene will lead to a reproducible pattern of clinical expression.

Deep subcutaneous adipose tissue: a distinct abdominal adipose depot.

OBJECTIVE: Abdominal visceral (VAT) and subcutaneous adipose tissue (SAT) display significant metabolic differences, with VAT showing a functional association to metabolic/cardiovascular disorders. A third abdominal adipose layer, derived by the division of SAT and identified as deep subcutaneous adipose tissue (dSAT), may play a significant and independent metabolic role. The aim of this study was to evaluate depot-specific differences in the expression of proteins key to adipocyte metabolism in a lean population to establish a potential physiologic role for dSAT. RESEARCH METHODS AND PROCEDURES: Adipocytes and preadipocytes were isolated from whole biopsies taken from superficial SAT (sSAT), dSAT, and VAT samples obtained from 10 healthy normal weight patients (7 women and 3 men), with a mean age of 56.4 +/- 4.04 years and a mean BMI of 23.1 +/- 0.5 kg/m2. Samples were evaluated for depot-specific differences in insulin sensitivity using adiponectin, glucose transport protein 4 (GLUT4), and resistin mRNA and protein expression, glucocorticoid metabolism by 11beta-hydroxysteroid dehydrogenase type-1 (11beta-HSD1) expression, and alterations in the adipokines leptin and tumor necrosis factor-alpha (TNF-alpha). RESULTS: Although no regional differences in expression were observed for adiponectin or TNF-alpha, dSAT whole biopsies and adipocytes, while intermediary to both sSAT and VAT, reflected more of the VAT expression profile of 11beta-HSD1, leptin, and resistin. Only in the case of the intracellular pool of GLUT4 proteins in whole biopsies was an independent pattern of expression observed for dSAT. In an evaluation of the homeostatic model, dSAT 11beta-HSD1 protein (r = 0.9573, p = 0.0002) and TNF-alpha mRNA (r = 0.8210, p = 0.0236) correlated positively to the homeostatic model. DISCUSSION: Overall, dSAT seems to be a distinct abdominal adipose depot supporting an independent metabolic function that may have a potential role in the development of obesity-associated complications.

Mutagenicity of stereochemical configurations of 1,2-epoxybutene and 1,2:3,4-diepoxybutane in human lymphblastoid cells.

The carcinogenicity of 1,3-butadiene (BD) is related to its bioactivation to several DNA-reactive metabolites; accumulating evidence suggests that the stereochemistry of these BD intermediates may play a significant role in the mutagenic and carcinogenic actions of the parent compound. The objective of this study was to evaluate the cytotoxicity and mutagenicity of stereochemical forms of 1,2-epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB), two genotoxic BD metabolites, in a human lymphoblastoid cell line, TK6. Cytotoxicity was measured by comparing cloning efficiencies in chemical-exposed cells versus those in control cells. The hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK) mutant frequencies (MFs) were measured using a cell cloning assay. HPRT mutants collected from cells exposed to the three forms of DEB were analyzed by PCR to characterize large genetic alterations. All the three stereoisomers of DEB caused increased HPRT and TK MFs compared to the concurrent control samples. There were no significant differences in cytotoxicity or mutagenicity among the three isomers of DEB in TK6 cells. Molecular analysis of HPRT mutants revealed similar distributions of types of mutations among the three isomers of DEB. There were also no statistically significant differences in mutagenic efficiencies between the two isomers of EB in TK6 cells. These results were consistent with the in vivo findings that there was little difference in the mutagenic efficiencies of racemic-DEB versus meso-DEB in rodents. Thus, in terms of mutagenic efficiency, stereochemical configurations of EB and DEB are not likely to play a significant role in the mutagenicity and carcinogenicity of BD.

Impaired transport as a mechanism of resistance to thiopurines in human T-lymphoblastic leukemia cells.

In order to better understand the mechanisms of resistance to thiopurines, we studied two sublines of the MOLT4 T-lymphoblastic leukemia cell line, resistant to 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). We found that the underlying mechanism of resistance in both resistant cell lines was a markedly reduction in initial transport of 6-MP (3- and 5-fold, respectively, in 6-MP- and 6-TG-resistant cells). No significant alteration of activities of hypoxanthine-guanine phosphoribosyl transferase, thiopurine methyltransferase or inosine monophosphate dehydrogenase, the key enzymes involved in the metabolism of thiopurines was detected. We conclude that defected initial transport of thiopurines by cells may very well explain their resistance to these drugs.

Leishmania amazonensis: participation of regulatory T and B cells in the in vitro priming (PIV) of CBA/J spleen cells susceptible response.

CBA/J mice are resistant to Leishmania major and susceptible to Leishmania amazonensis. Early events determine infection outcome. Until now, PIV (in vitro priming) immune response to L. amazonensis has not been assessed. Herein, we have shown that compared to L. major, L. amazonensis induced higher parasite burden associated to similar IL-4, IFN-gamma, and TNF-alpha mRNA expressions and IFN-gamma and IL-10 levels. Although similar amounts of IL-10 were detected, the frequency of intracellular IL-10 positive B cells was enhanced in spleen cells stimulated with anti-CD3/anti-CD28, or anti-CD3/anti-CD28 and L. amazonensis, compared to L. major-stimulation. Interestingly, IL-10- producing B cells were reduced in response to anti-CD3/anti-CD28 stimulation combined with L. major compared to the other groups. L. amazonensis may favor T regulatory cell development, since 40% of all the CD4+CD25+ were CD25(high) cells. These data suggest that in PIV, susceptibility to L. amazonensis is not related to Th cell polarization, but to the presence and activity of regulatory T and B cells.

In vivo mutagenesis induced by benzo[a]pyrene instilled into the lung of gpt delta transgenic mice.

Benzo[a]pyrene (B[a]P) is a ubiquitous airborne pollutant whose mutagenicity has been evaluated previously by oral and intraperitoneal administration to experimental animals. In this study, mutagenesis in the lungs, the target organ of air pollutants, was examined after a single intratracheal instillation of 0-2 mg B[a]P into gpt delta transgenic mice. Intratracheal injection of B[a]P resulted in a statistically significant and dose-dependent increase in gpt mutant frequency as measured by 6-thioguanine selection. The mutant frequencies at B[a]P doses of 0.5, 1, and 2 mg were 2.8, 4.2, and 6.8 times higher than the frequency seen in nontreated mice (0.60 +/- 0.13 x 10(-5)). The most frequent mutations induced by B[a]P treatment were G:C-->T:A transversions, which are characteristic of B[a]P mutagenesis in other models, and single-base deletions of G:C base pairs. To characterize the hotspots of B[a]P-induced mutations in the gpt gene, we analyzed sequences adjacent to the mutated G:C base pairs. Guanine bases centered in the nucleotide sequences CGT, CGA, and CGG were the most frequent targets of B[a]P. Our results indicate that intratracheal instillation of B[a]P into gpt delta mice causes a dose-dependent increase in gpt mutant frequency in the lung, and that the predominant mutation induced is G:C-->T:A transversion.

Bioassay for detection of methotrexate in serum.

OBJECTIVE: A bioassay is developed for the measurement of methotrexate (MTX) in serum. METHODS: The assay is based on MTX inhibition of the proliferation of hypoxanthine-guanosine phosphoribosyl transferase (HGPRT) negative mouse B-cells (B9.H). HGPRT negative cells cannot use the salvage pathway of nucleotide synthesis to overcome inhibition by MTX. RESULTS: When B9.H cells are cultured with serial dilutions of serum, inhibition of proliferation is a measure of the amount of MTX in the serum. Circulating folates do not interfere with the assay. CONCLUSION: This simple assay can detect low concentrations of MTX in serum: it is therefore useful for following the pharmacodynamics of functional MTX after low-dose MTX treatment.

Single-cell quantitative RT-PCR analysis of Cpt1b and Cpt2 gene expression in mouse antral oocytes and in preimplantation embryos.

Fatty acids represent an important energy source for preimplantation embryos. Fatty acids oxidation is correlated with the embryo oxygen consumption which remains relatively constant up to the 8-cell stage, but suddenly increases between the 8-cell and morula stages. The degradation of fatty acids occurs in mitochondria and is catalyzed by several carnitine acyl transferases, including two carnitine palmitoyl transferases, CPT-I and CPT-II. We have carried out a study to determine the relative number of transcripts of Cpt1b and Cpt2 genes encoding for m-CPT-I and CPT-II enzymes, during mouse preimplantation development. Here we show that Cpt1b transcripts are first and temporally detected at the 2-cell stage and reappear at the morula and blastocyst stage. Cpt2 transcripts decrease following fertilization to undetectable levels and are present again later at the morula stage. These results show that transcription of both Cpt1b and Cpt2 is triggered at the morula stage, concomitantly with known increasing profiles of oxygen uptake and fatty acids oxidation. Based on the number of Cpt2 transcripts detected, we could discriminate the presence of two groups of embryos with high and low number of transcripts, from the zygote throughout preimplantation development. To further investigate if the establishment of these two groups of embryos occurs prior to fertilization, we have analyzed the relative number of transcripts of both genes in antral and ovulated MII oocytes. As for preimplantation embryos, MII oocytes show two groups of Cpt2 expression. Antral oocytes, classified according to their chromatin configuration in SN (surrounded nucleolus, in which the nucleolus is surrounded by a rim of Hoechst-positive chromatin) and NSN (not surrounded nucleolus, in which this rim is absent), show three groups with different numbers of Cpt2 transcripts. All NSN oocytes have a number of Cpt2 transcripts doubled compared to that of the group of MII oocytes with high expression. Instead, SN oocytes could be singled out into two groups with high and low numbers of Cpt2 transcripts, similar to those found in MII oocytes. The results of this study point out a correlation between the timing of fatty acids oxidation during preimplantation development and the expression of two genes encoding two enzymes involved in the oxidative pathway. Furthermore, although the biological meaning for the presence of two groups of oocytes/embryos with different levels of Cpt2 transcripts remains unclear, the data obtained suggest a possible correlation between the levels of Cpt2 expression and embryo developmental competence.

Selection of HPRT primers as controls for determination of mRNA expression in dogs by RT-PCR.

Reliable housekeeping gene controls are critical for measuring and comparing gene expression at the transcription level by Northern blot and RT-PCR. In order to develop such controls for studying cytokine mRNA expression in dogs, DNA sequence encoding a full-length canine HPRT protein has been obtained. Numerous primer pairs derived from the canine HPRT sequence have been tested on canine genomic DNA as well as cDNA. The data from the present study suggest that there may be processed HPRT pseudogenes in dogs. Three pairs of canine HPRT primers designed and tested in the present study were able to differentiate between cDNA and genomic DNA under specific PCR conditions. These primers would be useful controls for measurement of mRNA expression by RT-PCR in the dog.

Protein preparation, crystallization and preliminary X-ray crystallographic studies of a thermostable hypoxanthine-guanine phosphoribosyltransferase from Thermoanaerobacter tengcongensis.

Native and His-tagged mutant (L160I) hypoxanthine-guanine phosphoribosyltransferase (HGPRT) from Thermoanaerobacter tengcongensis were cloned, expressed in Escherichia coli and purified. Both proteins were crystallized with polyethylene glycol as the main precipitant at 293 K using the hanging-drop vapour-diffusion method. The crystal of native HGPRT belongs to space group C222(1), with unit-cell parameters a = 65.77, b = 137.73, c = 95.27 A, and diffracted to 2.2 A resolution on an in-house X-ray generator. The crystal of the His-tagged mutant (L160I) HGPRT belongs to the space group I222, with unit-cell parameters a = 52.21, b = 88.36, c = 93.03 A, and diffracted to 1.7 A resolution in-house.