Expression Of Hypoxanthine-guanine Phosphoribosyltransferase Research Articles

Brain purines in a genetic mouse model of Lesch-Nyhan disease.

Mice carrying a mutation in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) have recently been produced to provide an animal model for Lesch-Nyhan disease. The current studies were conducted to characterize the consequences of the mutation on the expression of HPRT and to characterize potential changes in brain purine content in these mutants. Our results indicate that the mutant animals have no detectable HPRT-immunoreactive material on western blots and no detectable HPRT enzyme activity in brain tissue homogenates, confirming that they are completely HPRT deficient (HPRT-). Despite the absence of HPRT-mediated purine salvage, the animals have apparently normal brain purine content. However, de novo purine synthesis, as measured by [14C]formate incorporation into brain purines, is accelerated four- to fivefold in the mutant animals. This increase in the synthesis of purines may protect the HPRT- mice from potential depletion of brain purines despite complete impairment of HPRT-mediated purine salvage.

Utility of isoelectric focusing techniques for detection of induced in vivo mammalian microlesions

We have recently developed a novel and highly efficient strategy that exclusively uses the purine analog 6-thioguanine (6TG) for both pretransplantation conditioning and post-transplantation chemoselection of hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient bone marrow (BM). In a mouse BM transplantation model, combined 6TG preconditioning and in vivo chemoselection consistently achieved >95% engraftment of HPRT-deficient donor BM and long-term reconstitution of histologically and immunophenotypically normal hematopoiesis in both primary and secondary recipients, without significant toxicity and in the absence of any other cytotoxic conditioning regimen. To translate this strategy for combined 6TG conditioning and chemoselection into a clinically feasible approach, it is necessary to develop methods for genetic modification of normal hematopoietic stem cells (HSC) to render them HPRT-deficient and thus 6TG-resistant. Here we investigated a strategy to reduce HPRT expression and thereby confer protection against 6TG myelotoxicity to primary murine BM cells by RNA interference (RNAi). Accordingly, we constructed and validated a lentiviral gene transfer vector expressing short-hairpin RNA (shRNA) that targets the murine HPRT gene. Our results showed that lentiviral vector-mediated delivery of HPRT-targeted shRNA could achieve effective and long-term reduction of HPRT expression. Furthermore, in both an established murine cell line as well as in primary murine BM cells, lentiviral transduction with HPRT-targeted shRNA was associated with enhanced resistance to 6TG cytotoxicity in vitro. Hence this represents a translationally feasible method to genetically engineer HSC for implementation of 6TG-mediated preconditioning and in vivo chemoselection.

Hypoxanthine guanine phosphoribosyl transferase expression in early mouse development.

Dosage compensation for X-linked genes occurs by the process of X-chromosome inactivation (XCI) in mammalian somatic cells (Lyon 1972). The inactivation of one X chromosome in females takes place early in development, although the exact time is unknown. One method of ascertaining the time of XCI is to determine the activity of the X chromosome at different stages of development as measured by the activity of an X-coded enzyme. Prior to XCI, and in the absence of other dosage compensating mechanisms, the activity for an embryonically expressed X-coded enzyme should be twice as high in female embryos with two X chromosomes, as in male embryos with one X chromosome. The distribution of enzyme activities for single embryos from a litter would have two equal-sized peaks that are separated by a factor of two. The convergence of the two peaks into one would indicate that XCI had occurred.

Reversion in expression of hypoxanthine-guanine phosphoribosyltransferase in 6-thioguanine resistant neuroblastoma: evidence for reduced enzyme levels associated with unaltered catalytic activity.

Five clones of mouse neuroblastoma cells able to grow in hypoxanthine-aminopterin-thymidine containing medium were isolated from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8; IMP: pyrophosphate phosphoribosyltransferase) deficient cell line. These hypoxanthine-aminopterin-thymidine resistant revertant clone had 45-55% of wild-type cell HGPRT activity. Kinetic studies indicated that the HGPRT in revertant clones had a reduced maximal velocity as compared to wild type cells based on cell protein. Apparent Km values of HGPRT for hypoxanthine and 5-phosphoribosyl-1-pyrophosphate were similar in wild-type and revertant cells. Heat inactivation studies demonstrated a similar heat lability for HGPRT in revertant and wild-type cells. An antibody fraction prepared from serum of rabbits immunized with HGPRT partially purified from mouse liver was used to measure the amount of cross-reacting material in normal and revertant clones. The revertant clones had one-half the amounth of cross-reacting material present in wild-type cells, based on a given amount of cell protein. These data indicate that the revertant cells may contain fewer HGPRT molecules with unaltered catalytic activity.

Reversion in expression of hypoxanthine-guanine phosphoribosyl transferase following cell hybridization.

Hybridization of mutant cell lines deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT; E.C.: 2.4.2.8) from a variety of established rodent sources with HGPRT plus human cells yielded progeny cells which grew in selective medium containing hypoxanthine, aminopterin and thymidine (HAT). The same result was obtained when the human cell used was an HGPRT minus transformed line derived from a patient with the Lesch-Nyhan syndrome. Electrophoretic analysis indicated that all HAT-resistant progeny clones contained an active HGPRT enzyme which was indistinguishable from the wild type enzyme of the corresponding normal rodent cells. In contrast, no HAT-resistant cells have been obtained when the same HGPRT minus rodent cells were subjected to fusion processes in the absence of human cells or when they fused with similarly derived HGPRT minus mutant cells of other rodents. Reversion in expression of the rodent gene for HGPRT was detected in clones which retained one or more human chromosomes and in clones which contained no detectable human chromosomal material. The observed re-expression of rodent HGPRT in HAT-resistant clones suggests that HGPRT plus as well as HGPRT minus human cells contributed a factor which determined the expression of respective rodent structural genes for HGPRT. In contrast, HGPRT minus rodent cells were unable to induce the synthesis or normal HGPRT in the cells derived from the patient with the Lesch-Nyhan syndrome.