Expression Of HPRT Research Articles

Fine-needle aspiration technique under endoscopic ultrasound guidance: A technical approach for RNA profiling of pancreatic neoplasms.

Early diagnosis of pancreatic ductal adenocarcinoma (PDAC) has been a longstanding challenge. The prognosis of patients with PDAC depends on the stage at diagnosis. It is necessary to identify biomarkers for the detection and differentiation of pancreatic tumors and optimize PDAC sample preparation procedures for DNA and RNA analysis. Most molecular studies are done using paraffin-embedded blocks; however, the integrity of DNA and RNA is often compromised in this format. Moreover, RNA isolated from human pancreatic tissue samples is generally of low quality, in part, because of the high concentration of endogenous pancreatic RNAse activity present. To assess the potential of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) to obtain specimens from pancreatic neoplasms for subsequent RNA molecular profiling, including next-generation sequencing (NGS). Thirty-four EUS-FNA samples were included in this study: PDAC (n = 15), chronic pancreatitis (n = 5), pancreatic cysts (n = 14), mucinous cysts (mucinous cystic neoplasia/intraductal papillary mucinous neoplasia) n = 7, serous cystic neoplasms n = 5, and pseudocysts n = 2. Cyst material consisted of cyst fluid and cyst wall samples obtained by through-the-needle biopsy (TTNB). Samples were stored at -80 °C until analysis. RNA purity (A260/230, A260/280 ratios), concentration, and integrity (RIN) were assessed. Real-time polymerase chain reaction was conducted on all samples, and small RNA libraries were prepared from solid mass samples. RNA was successfully extracted from 29/34 (85%) EUS-FNA samples: 100% pancreatic adenocarcinoma samples, 100% chronic pancreatitis samples, 70% pancreatic fluid cyst samples, and 50% TTNB samples. The relative expression of GAPDH and HPRT were obtained for all successfully extracted RNA samples (n = 29) including low-quality RNA specimens. Low concentration and nonoptimal RIN values (no less than 3) of RNA extracted from EUS-FNA samples did not prevent NGS library preparation. The suitability of cyst fluid samples for RNA profiling varied. The quality of RNA extracted from mucinous cyst fluid had a median RIN of 7.7 (5.0-8.2), which was compatible with that from solid neoplasms [6.2 (0-7.8)], whereas the quality of the RNA extracted from all fluids of serous cystic neoplasms and TTNB samples had a RIN of 0. The results demonstrate the high potential of EUS-FNA material for RNA profiling of various pancreatic lesions, including low-quality RNA specimens.

HPRT promotes proliferation and metastasis in head and neck squamous cell carcinoma through direct interaction with STAT3

Increasing effort has been put into finding novel molecular pathways to improve the efficiency of EGFR inhibitors against head and neck squamous cell cancer (HNSCC). In this study, we performed data mining and bioinformatically analysed RNA-Seq data downloaded from TCGA and confirmed that higher expression of HPRT in HNSCC tissue was related to poor prognosis of patients. Then, we conducted in vitro and in vivo loss- and gain-of-function experiments to demonstrate the role of HPRT in HNSCC cell lines. Overexpression of HPRT increased the gene expression of epithelial mesenchymal transition markers via direct interaction with STAT3. Knocking down HPRT significantly decreased tumour growth and enhanced the anticancer effect of EGFR inhibitors against HNSCC xenografts. In conclusion, HPRT is a binding partner of STAT3 that promotes EMT and proliferation. Our findings support HPRT as a promising prognostic indicator and potential therapeutic target for HNSCC.

Examination of Hypoxanthine Guanine Phosphoribosyltransferase as a biomarker for colorectal cancer patients

ABSTRACTThe aim of this study is to investigate these enzymes as possible biomarkers in two colorectal cancer cell lines: HT29, SW480, SW620, and Colo205. With 1,168,929 individuals currently diagnosed with colorectal cancer in the United States, there remains a need to find biomarkers to improve diagnosis and expand treatment options for patients. Due to their role in proliferation and cell cycle regulation, we hypothesized an increase in salvage pathway enzyme (APRT, DCK, and HPRT) expression and possible presentation within colon cancer cells. Enzyme surface localization was assessed utilizing confocal microscopy, flow cytometry, and scanning electron microscopy. General protein expression was evaluated utilizing immunohistochemistry and Western blot analysis. While we found no statistically significant presence of either APRT or DCK on the membranes of SW620, Colo205, and HT29 cells, but found significant expression of HPRT on the surface of HT29, SW480, and SW620 cells. The average population fluorescence increased by 28%, 58%, and 40% in HT29, SW620, and SW480 cells, respectively, when compared to isotype controls. Confocal microscopy images revealed direct overlap between SW620 cells stained with a membrane dye and anti-HPRT antibody, indicating co-localization on the plasma membrane. In addition, cells treated with gold labelled HPRT antibody experienced significant changes in gold weight percentage on both SW620 and HT29 cells when compared to isotype controls. When evaluating expression within normal tissue, there was insignificant levels of HPRT binding. These data collectively suggest that HPRT may be a possible biomarker target for the identification and treatment of colorectal cancer.

Abstract 2149: Unusual expression of HPRT on the surface of the colorectal cancer cell lines HT29 and SW620

Abstract Colorectal cancer is one of the most common cancers in the world, affecting nearly 1.2 million people in the United States alone. The aim of this study is to investigate the salvage pathway enzyme HPRT as a possible biomarker in two colorectal cancer cell lines: HT29 and SW620. HPRT is a transferase in the purine salvage pathway that functions primarily by catalyzing the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. Because of its role in proliferation and cell cycle regulation, we hypothesized an increase in HPRT expression within cancer cells, which could potentially lead to presentation on the surface of the cell. HPRT surface localization was assessed utilizing confocal microscopy, flow cytometry, and scanning electron microscopy. These techniques allowed us to visualize HPRT on the plasma membrane and quantify expression. There was statistically significant expression of HPRT on the surface of both HT29 and SW620 cells with a 28% and 58% fluorescent shift in the population, respectively. Confocal microscopy images revealed overlap between cells stained with a membrane dye and anti-HPRT FITC antibody, which indicates a direct relationship between HPRT and the plasma membrane of SW620 cells. To visualize the location of HPRT on the plasma membrane, anti-HPRT antibodies were labelled with gold and protein presence was quantified using an electron microscope, which measured the gold elemental weight percentage of each sample. When exposed to anti-HPRT antibody, the gold weight percentage of the samples significantly increased with 12.4% gold in SW620 cells and 11.2% gold in HT29 cells, indicating significant HPRT surface presence. The observed localization of HPRT to the plasma membrane was variable between the two different cancer cell lines. SW620 cells, which are faster growing and more aggressive, had significantly higher HPRT presentation that HT29 cells. These data collectively suggest that HPRT may be a possible biomarker target for the identification and possible treatment of colorectal cancer cells. Citation Format: Evita G. Weagel, Michelle H. Townsend, Michael D. Anderson, Edwin J. Velazquez, K Scott Weber, Richard A. Robison, Kim L. O'Neill. Unusual expression of HPRT on the surface of the colorectal cancer cell lines HT29 and SW620 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2149. doi:10.1158/1538-7445.AM2017-2149

Abstract 4660: HPRT as a biomarker for tumor classification and its variability of expression in breast cancer

Abstract The purpose of this study is to investigate the differential expression of HRPT between various breast cancer tissues in comparison to healthy breast tissue in order to evaluate its potential use as a diagnostic tool. Breast cancer is the most common malignancy diagnosed in women and remains incurable in the metastatic setting requiring accurate and reliable tools for early diagnosis. We analyzed HPRT, a purine salvage pathway enzyme responsible for synthesizing 90% of the available GMP and IMP in cellular maintenance. Due to its critical role in maintaining necessary nucleotides for cell proliferation, we hypothesized an increase in the expression of HPRT in cancer. Tissues from 48 patients with breast carcinoma were stained using standard immunohistochemistry (IHC) techniques. We examined infiltrating ductal carcinoma, margin of carcinoma, hyperplasia, atypical hyperplasia, adenosis, collagen fibers tissues, and normal breast tissue in order to determine expression between various breast cancers. Briefly, tissues were treated with a polyclonal anti-HPRT antibody along with a GADPH positive control and a universal negative control. Tissues were incubated with an HRP-polymer conjugated antibody, and DAP substrate which reacts to antibody-antigen binding sites and results in a color change. Adenosis and hyperplasia are benign breast conditions and are early signs of breast carcinoma development. Hyperplasia tissues, which are in a state of rapid proliferation, showed significant upregulation of HPRT and adenosis tissues showed approximately 42% upregulation. This HPRT upregulation in pre-stage 1 tissues indicates that it may be used as a biomarker to detect early stages of breast cancer. Expression of HPRT was also found to be upregulated in 65% of infiltrating duct carcinoma showing high expression in active malignant areas. In marginal tissue located on the perimeter of primary tumors only 31% of tissues show upregulation of the protein. Because the levels of HPRT in marginal tissue is significantly lower than those found in primary tumors, HPRT may serve as a useful diagnostic tool when evaluating successful tumor removal. These data suggests HPRT as a useful diagnostic tool when identifying tissue with a high proliferation rate indicative of potential cancer development. Additionally, HPRT may also be used as a tool for pathologists to evaluate the success of tumor removal. Citation Format: Michelle H. Townsend, Abigail M. Felsted, Jacob Clarke, Richard A. Robison, Kim L. O'Neill. HPRT as a biomarker for tumor classification and its variability of expression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4660. doi:10.1158/1538-7445.AM2017-4660

HMGA2 expression distinguishes between different types of postpubertal testicular germ cell tumour

The group of postpubertal testicular germ cell tumours encompasses lesions with highly diverse differentiation – seminomas, embryonal carcinomas, yolk sac tumours, teratomas and choriocarcinomas. Heterogeneous differentiation is often present within individual tumours and the correct identification of the components is of clinical relevance. HMGA2 re‐expression has been reported in many tumours, including testicular germ cell tumours. This is the first study investigating HMGA2 expression in a representative group of testicular germ cell tumours with the highly sensitive method of quantitative real‐time PCR as well as with immunohistochemistry. The expression of HMGA2 and HPRT was measured using quantitative real‐time PCR in 59 postpubertal testicular germ cell tumours. Thirty specimens contained only one type of tumour and 29 were mixed neoplasms. With the exception of choriocarcinomas, at least two pure specimens from each subgroup of testicular germ cell tumour were included. In order to validate the quantitative real‐time PCR data and gather information about the localisation of the protein, additional immunohistochemical analysis with an antibody specific for HMGA2 was performed in 23 cases. Expression of HMGA2 in testicular germ cell tumours depended on the histological differentiation. Seminomas and embryonal carcinomas showed no or very little expression, whereas yolk sac tumours strongly expressed HMGA2 at the transcriptome as well as the protein level. In teratomas, the expression varied and in choriocarcinomas the expression was moderate. In part, these results contradict data from previous studies but HMGA2 seems to represent a novel marker to assist pathological subtyping of testicular germ cell tumours. The results indicate a critical role in yolk sac tumours and some forms of teratoma.

P090 Differential effects of sulphasalazine, azathioprine and prednisolone on the MAP-kinases gene expression in the TNBS-induced intestinal inflammation

elucidated but is currently presumed to result from a complex interplay among genetic, environmental, microbial and immune factors. Animal models of intestinal inflammation have given researchers a better understanding of the mechanisms involved in the pathogenesis of inflammatory bowel disease. Based on this, the aim of this study was to investigate the profile of NFkB, heparanase, HSP70 and HPRT gene expression in intestinal inflammation induced by TNBS (trinitrobenzenesulphonic acid) and to evaluate the effects of sulphasalazine, prednisolone and azathioprine on these gene expressions. Methods: Intestinal inflammation was induced in rat by intracolonic instillation of TNBS. Rats orally received 50mg/kg of sulphasalazine, 2mg/kg of prednisolone or 2mg/kg of azathioprine at 96, 72, 48, 24 and 2 hours before colitis induction. Animals were killed 48 h after colitis induction, colonic segments were obtained after laparotomy for macroscopic (colon weight and length and macroscopic damage score) and biochemical evaluation of myeloperoxidase (MPO) and alkaline phosphatase (AP) activities and glutathione (GSH) level. Gene expression analysis of NF-kB, heparanase, HSP70 and HPRT were performed on 100mg of colon tissue using qRT-PCR. Results: All gene expressions were increased in colitic animals when compared to healthy animals, indicating the involvement of these genes in intestinal inflammatory process induced by TNBS. Treatment of healthy animals with sulphasalazine and prednisolone did not affect gene expression, but it was observed that azathioprine enhanced HSP70 gene expression in healthy animals. On the other hand, treatment of colitic animals with sulphasalazine restored heparanase, HPRT and NFkB gene expression, while azathioprine restored the heparanase and HPRT gene expression and prednisolone only restored gene expression of the HPRT and HSP70. Conclusions: Gene expression of NF-kB, heparanase, HSP70 and HPRT participate on the intestinal inflammatory process induced by TNBS in rats and lead to new molecular markers or therapeutic targets. Furthermore, sulphasalazine, prednisolone and azathioprine produced differential pharmacological effects on NF-kB, heparanase, HSP70 and HPRT gene expression. Financial support: FAPESP and CAPES.

Selection of Endogenous Control Genes for Normalization of Gene Expression Analysis after Experimental Brain Trauma in Mice

Quantitative measurements of gene expression require correction for tissue sample size, RNA quantity, and reverse transcription efficiency. This can be achieved by normalization with control genes. The study was designed to identify candidates not altered after brain trauma. Male C57Bl/6 mice were anesthetized with isoflurane, and a pneumatic brain trauma was induced by controlled cortical impact (CCI) on the right parietal cortex. Brains were removed at 15 min, and 3, 6, 12 and 24 h after CCI and from naive animals (n = 6 each). Absolute copies of six control genes (beta-2-microglobin [B2M], cyclophilin A, beta-actin, hypoxanthine ribosyltransferase [HPRT], porphobilinogen deaminase [PBGD], and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and one example target gene (iNOS) were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR; Lightcycler) in the traumatic focus and contralateral tissue. Control gene expression was stable until 12 h after CCI. At 24 h after CCI expression of B2M, cyclophilin A and HPRT remained stable in the contusion, while expression of beta-actin, GAPDH, and PBGD increased. Due to variations between animals (+/-85%), increases in beta-actin (+64%) and GAPDH (+59%) did not reach the level of significance. In non-contused tissue, expression of all genes dropped 24 h after CCI (range, -17% to -61%). Due to low variations between animals and stable expression after CCI, B2M and cyclophilin A seem to be suitable to serve as single normalizer. Normalization of the example target gene iNOS resulted in varying relative expression extending from onefold (PBDG) to 10-fold (HPRT). The results suggest that the knowledge of the temporal profile of control genes is essential to properly interpret results of mRNA quantification.

260 COMPARISON OF HOUSEKEEPING GENE EXPRESSION IN INDIVIDUAL 8-CELL STAGE MOUSE EMBRYOS PRODUCED UNDER DIFFERENT CONDITIONS

Real-time PCR has the potential to accurately quantify the mRNA level of selected genes in single cells and individual pre-implantation-stage embryos. The goal of our study was to examine the variations in gene expression within individual embryos of the same stage and between embryos of the same stage but from different sources. In our study, we determined expression level of the 7 most commonly used housekeeping genes in 8-cell-stage mouse embryos produced under different culture conditions. Messenger RNA of 6 embryos each that was derived in vivo, or cultured in vitro from the zygote stage, or derived from oocytes activated parthenogenetically and developed in vitro were extracted individually followed by reverse transcription into cDNA. Optimized real-time PCR was performed for cytoplasmic beta-actin (Actb), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), H2A histone family, member Z (H2afz), hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1), ubiquitin C (Ubc), peptidylprolyl isomerase A (cyclophilin A) (Ppia), and eukaryotic translation elongation factor 1 epsilon 1 (Eef1e1) genes. The results were analyzed, and the percentage standard error of the mean relative expression value was compared for all genes. All 7 genes were presented above the detection limit in all samples. One or two individual embryos showed 2- to 4-fold higher mRNA levels than the average for all genes in the group. The embryos cultured in vitro showed much higher expression levels of H2afz, Ppia, and Eef1e1 genes than those in the in vivo group. The parthenogenetic group was similar to the in vivo group in expression of Actb, H2afz, Hprt, and Eef1e1 genes, but showed significant differences (P < 0.05; Student's t-test) compared to the in vitro group (Table 1). The percent standard error of the mean decreased gradually as the number of samples was increased. The 6 individual embryos in similar groups showed relatively low variability compared to embryos at similar stage but produced in different conditions. Interestingly, the parthenogenetic embryos showed a level of gene expression comparable to that of the in vivo ones, notwithstanding their culture in vitro. In conclusion, morphological observations and similarity in developmental stage alone cannot guarantee the uniformity of embryo samples, and a minimum of 4–6 replicates per treatment is needed. Moreover, we showed that culture condition itself has an effect on housekeeping gene expression, which, if neglected, might result in misinterpretation of data. Table 1.Relative expression values of the different culture groups (mean ±SE; n =6 embryos) This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No. 070246), and Hungarian National Science Fund (OTKA) (Grant No. T046171).

Transgenes Targeted to the HPRT Locus and Expressed from a Tetracycline-Inducible Promoter Are Silenced during Embryonic Stem Cell Differentiation.

Genetic modification of embryonic stem (ES) cells provides a powerful system for the correlation of in vitro and in vivo gene function, an issue relevant to transplantation, gene therapy and tissue engineering. ES cells also present unique obstacles to genetic manipulation, particularly with regard to epigenetic modulation of transgene expression. Strategies used to circumvent problems associated with methylation, histone deactylation, and chromatin remodeling include modification of vector sequences, insulators, and targeting transgenes to sites of “open” chromatin structure. Systems combining gene targeting with non-viral promoters have shown particular promise. In these studies, a variety of transgenes were introduced into Ainv15 ES cells and doxycycline (dox)-induced expression evaluated by flow cytometry and quantitative PCR. Ainv ES cells express a reverse tetracycline transactivator (rtTA) from the ROSA26 locus and allow Cre-mediated insertion of transgenes into the HPRT locus adjacent to a tetracycline response element (TRE). As expected, transgene expression in undifferentiated ES cells could be regulated by dose and time of exposure to dox. However, expression levels varied dramatically for different transgenes; variability was transgene-specific and did not directly correlate with transgene size or the presence of YFP sequences. In addition, regardless of initial expression level, all transgenes were transcriptionally silenced during ES cell differentiation as embryoid bodies (EBs). Distinct patterns of silencing occurred when cells were induced for 24 hours immediately preceding analysis or continuously from the start of EB differentiation. In both cases, a pronounced loss of expression occurred between days 4–7; however, EBs treated with dox for 24 hours maintained a population of cells capable of maximal induction through day 6, whereas continuously treated cells showed a marked decline in this population by day 4. These results suggested two distinct processes: a primary loss of inducibility independent of dox exposure and a secondary refractoriness of expressing cells to reinduction. The reproducible patterns further suggested that loss of TRE activity or general silencing of the HPRT locus were responsible. To distinguish between these possibilities, expression of HPRT and NeoR, which flank transgenes in targeted Ainv cells, were evaluated. In contrast to the loss of transgene expression, HPRT and NeoR expression remained stable or increased between EB day 3–7, indicating that silencing was not due to general chromatin remodeling within the locus. Expression of rtTA was also stable, excluding the possibility that silencing of the ROSA26 locus was responsible. We conclude that silencing in this system is due to a loss of TRE activity between EB days 4–7, and speculate that inhibitory factors produced by ES cells at this stage of differentiation are responsible. The findings underscore the complexities of gene regulation in ES cells, and raise the possibility that the capacity of cells to express transgenes may in part reflect intrinsic cell potential. In addition, if cells of particular lineages produce factors that interfere with TRE activity, there are obvious implications for using the Tet-On system for inducing gene expression in those lineages.

RT-PCR su singola cellula per lo studio dell’espressione genica durante lo sviluppo preimpianto di topo

The study of gene expression during preimplantation embryonic development is useful to understand which are the molecular mechanisms involved in the progressive passage from a maternal to a zygotic regulation of gene expression. It is our laboratory main concern to develop techniques that would allow a more precise characterisation of the timing of transcription and amount of transcripts of genes that are well known to play an important role in the early events of preimplantation development. In this study we illustrate the use of a very sensitive protocol of semiquantitative single-cell RT-PCR that we have developed. This technique allows the simultaneous analysis of up to eight genes on a single cell. Here we show the experiments performed to validate this novel assay. As an example we have analysed the quantitative expression ofHprt, Bax, Bex-1, Cpt-1, Cpt-2, Oct-4, Tsix andXist on single blastocysts.

Sex-chromosome linked gene expression in in-vitro produced bovine embryos.

The expression of XIST, G6PD, HPRT, ZFX and ZFY were investigated in in-vitro produced bovine embryos. Transcripts of these genes were assayed by RT-PCR in pools of pre-compaction stage embryos and sexed pools of morulae and blastocysts. The expression of XIST, G6PD, HPRT and ZFX in female and male morulae and blastocysts were compared using a semi-quantitative RT-PCR. G6PD, HPRT and ZFX transcripts were noted in all pre-compaction stage embryos and in female and male blastocysts. ZFY transcripts were detected in unsexed pools of 8-16-cell stage embryos and in male blastocysts. XIST transcripts were detected in unsexed pools at the 8-16 cell stage, in male and female morulae, and in female blastocysts. The level of XIST RNA was significantly higher in female morulae than in males. Levels of G6PD and HPRT RNA were also higher in female morulae and blastocysts than in males, but only G6PD levels were significantly different between the sexes. The expression of ZFX was also significantly higher in female than in male blastocysts. These results show sexually dimorphic expression of sex chromosome linked genes prior to the blastocyst stage in in-vitro produced bovine embryos.

Sequence, expression and characterization of HPRTMoose Jaw: a point mutation resulting in cooperativity and decreased substrate affinities.

We have sequenced and studied the expressed protein of an HPRT mutation characterized by 5-12% residual erythrocyte activity, for which affected males exhibit hyperuricemia, arthritis and renal disease but are without severe neurological involvement. The HPRTMoose Jaw mutation is due to a single C to G transversion at nucleotide 582 relative to initiation of translation corresponding to substitution of aspartate 194 by glutamate. The mutant and wild type proteins were expressed and purified using the bacterial expression vector, pMAL-c2. The Km for hypoxanthine was increased 12-fold from 0.94 +/- 0.26 to 11.5 +/- 1.3 microM for control and mutant respectively. The apparent Km for PP-ribose-P was increased 44-fold from 6.8 +/- 0.6 to 295 +/- 7 microM for control and mutant respectively. Although the kcat of the mutant protein was equivalent to wild type, the catalytic efficiency, kcat/Km, of the purified mutant protein was only 6 and 3% of wild type with hypoxanthine and PP-ribose-P respectively. The mutant protein also exhibited positive cooperativity with PP-ribose-P, having a Hill coefficient of 2.3. The decreased substrate affinities and PP-ribose-P associated cooperativity of HPRTMoose Jaw provide additional evidence for the influence of carboxy-terminal residues of HPRT in specific catalytic functions.

Inhibition of Plasmodium falciparum hypoxanthine-guanine phosphoribosyltransferase mRNA by antisense oligodeoxynucleotide sequence

Comparative biochemical studies between the malaria parasite, Plasmodium falciparum, and its host have revealed differences in a number of metabolic pathways. For example, the parasite has enzymes to carry out de novo pyrimidine synthesis but is not capable of de novo synthesis of purine nucleotides. The parasite must salvage purines from its host to supply the requirement for purine nucleotides. The enzyme required for this salvage is hypoxanthine-guanine phosphoribosyltransferase This enzyme has been suggested as a target for chemotherapy by the design of analogue substrate inhibitors of the parasite enzyme. An alternate strategy, and one which we have been exploring, is the inhibition of expression of the parasite HPRT using antisense oligodeoxynucleotides.

High level expression in Escherichia coli of soluble, enzymatically active schistosomal hypoxanthine/guanine phosphoribosyltransferase and trypanosomal ornithine decarboxylase.

The bacterial alkaline phosphatase (phoA) promoter and signal peptide have been used previously to control recombinant expression and secretion of eukaryotic proteins in Escherichia coli. Other reports have shown that this expression system can generate relatively modest levels of active hypoxanthine/guanine phosphoribosyltransferase (HPRT; hypoxanthine phosphoribosyltransferase; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), which carries part of the signal peptide but remains in the cytosol of the bacteria. Herein, the phoA promoter without its associated signal peptide is used to regulate expression of the HPRT of Schistosoma mansoni and the ornithine decarboxylase (ODC; L-ornithine carboxy-lyase, EC 4.1.1.17) of Trypanosoma brucei, two enzymes that have been identified as potential targets for antiparasitic chemotherapy. The levels of recombinant expression range from 20% to 60% of the total bacterial protein, and the majority of both recombinant enzymes was soluble. The specific activity for the recombinant trypanosomal ODC was one-third to two-thirds that of the authentic native enzyme and yields were predicted to be 15-30 mg of active enzyme per liter of bacterial culture. The specific activity for the recombinant schistosomal HPRT was equivalent to that for the native enzyme purified from schistosomes and up to 10 mg of enzymatically active HPRT has been purified from a 0.5-liter culture of treated bacteria. These results represent a break-through in recombinant expression of HPRT and ODC.

Expression of human HPRT mRNA in brains of mice infected with a recombinant herpes simplex virus-1 vector

Complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, the Lesch-Nyhan syndrome. This disorder has been identified as a candidate for initial attempts at somatic cell gene therapy. We have previously reported the construction of a recombinant herpes simplex virus type 1 (HSV-1) vector containing human hprt cDNA sequences under the regulatory control of the viral thymidine kinase gene ( tk) [Palella et al., Mol. Cell. Biol. 8 (1988) 457–460]. Infection of HPRT - cultured rat neuronal cells with these vectors resulted m transient expression of human hprt. In this paper, we report the expression of human hprt mRNA transcripts in the brains of mice infected in vivo with this vector by direct intracranial inoculation. Human hprt transcripts were distinguished from endogenous mouse transcripts by RNase A mapping using riboprobes transcribed from human hprt cDNA. These initial studies demonstrate the transfer and transcription of a human gene in brain cells by direct in vivo infection with recombinant HSV-1 vectors.

Assignment of genes for human monoamine oxidases A and B to the X chromosome.

Monoclonal antibodies that immunoprecipitate human monoamine oxidase (MAO) A or human MAO B, but not the corresponding mouse enzymes, were used to assay for the presence of immunoprecipitable MAO A or MAO B (presumably coded by the respective human genes) in mouse-human hybrid somatic cell lines containing small numbers of human chromosomes. The results were as follow: Extracts of a human lymphoblastoid x mouse hepatoma hybrid line that retained the human X chromosome contained immunoprecipitable MAO B, while a similar hybrid line that contained the same human chromosomes, except for the human X, did not. Extracts of a human fibroblast x mouse neuroblastoma hybrid cell line, whose human chromosomal material consisted solely of the X, contained both immunoprecipitable MAO A and MAO B. Extracts of a related hybrid line, whose human chromosomal material consisted solely of an autonomous fragment and a fragment translocated to a mouse chromosome, contained immunoprecipitable MAO A. However, the level of immunoprecipitable MAO B activity in extracts of this hybrid was low or undetectable. Among extracts of 33 human fibroblast x mouse hepatoma hybrids that had been selected for expression of the X-linked human enzyme HPRT, 60% contained immunoprecipitable MAO B. This figure was comparable to the 58% that expressed the X-linked human isozyme for glucose-6-phosphate dehydrogenase (G6PD). When 11 of these hybrid lines, which contained immunoprecipitable MAO B and human HPRT, were selected for loss of HPRT, all lost immunoprecipitable MAO B in addition to HPRT. These data demonstrate that genes controlling the expression of MAO A and MAO B, which can be immunoprecipitated with the human-specific monoclonal antibodies, are located on the human X chromosome. Properties of the immunological epitopes recognized by the monoclonal antibodies suggest that the X-linked genes detected in this study are probably structural genes for the enzymes.

Analysis of the expression of chicken and rat gene products in myoblast × myoblast cell hybrids

Hybrid cells were isolated by fusing primary chicken myoblasts to HPRT-deficient rat L6 myoblasts and incubating the cells in medium containing HAT and ouabain. All hybrid clones contained both rat and chicken chromosomes and expressed a number of gene products characteristic of both species. Although all clones were capable of fusing spontaneously to form myofibers, immunofluorescence and isoenzyme analysis revealed only the rat forms of skeletal muscle myosin and MM-creatine kinase. No differentiated gene products of chicken origin were detected. Analysis of the expression of chicken HPRT revealed that some hybrid clones were capable of modulating this enzyme activity when switched from HAT medium into thioguanine medium and back into HAT, even though HPRT is normally a constitutively expressed enzyme. Parental control cells were incapable of this modulation phenomenon.

Somatic cell genetic analysis of transgenome integration.

The site of association of the human transgenome and host murine chromosomes was determined in several subclones of a stable human/mouse transformed cell line. Chromosomes were transferred from each of three transformed subclones into Chinese hamster recipient cells, and selection was applied for the expression of human transgenome-encoded HPRT. A series of trispecific microcell hybrids was isolated and characterized for each subclone. Evidence is presented that, within a given transformed subclone, only a single host (murine) chromosome was associated with the human transgenome. This contrasts with previous results which utilized a newly stabilized transformed cell line as the microcell donor and in which a variety of chromosomal sites of association existed. The results presented here support the view that the heterogeneity of transgenome association (integration) sites in newly stabilized transformants was due to the fact that these populations were multiclonal mixtures resulting from independent stabilization events. The initial heterogeneity in the population was subsequently reduced upon prolonged cultivation, as a subset of the original population became predominant.