Expression of human and Chinese hamster hypoxanthine-guanine phosphoribosyltransferase cDNA recombinants in cultured Lesch-Nyhan and Chinese hamster fibroblasts

The results presented in this communication demonstrate that hypoxanthine-guanine phosphoribosyltransferase (HPRT) cDNA can be expressed in both Chinese hamster and human fibroblasts deficient in the endogenous gene product at levels permitting normal growth of the transformants. All the elements necessary for this expression are present in a pBR322-derived plasmid containing HPRT cDNA coding sequence and a retroviral long terminal repeat. These molecules function in both species investigated and, at least in the case of the Chinese hamster transformants, are efficient at the single copy level. Although the effects of the presence of intron sequences and a polyadenylation signal within the plasmids have yet to be evaluated, these studies demonstrate that neither is an absolute requirement for expression of HPRT cDNA sequences in cultured mammalian cells. We describe the construction of recombinant plasmids containing wild type human or Chinese hamster HPRT cDNA sequences in tandem with a retroviral LTR which confer the HPRT+ phenotype in HPRT-deficient V79 and Lesch-Nyhan fibroblasts. Both stable and unstable transformants, that expressed HPRT mRNA and protein, were isolated at high frequency.     

黄鳝Hprt基因的克隆及表达分析

次黄嘌呤鸟嘌呤磷酸核糖转移酶(Hprt)参与嘌呤核苷酸的补救合成。采用RACE技术克隆了黄鳝的次黄嘌呤鸟嘌呤磷酸核糖转移酶基因,它的全长cDNA 为1 452 bp,预测编码218个氨基酸,与人类、小鼠、鸡和斑马鱼等脊椎动物Hprt氨基酸序列之间的同源性超过76.7％。基于该基因氨基酸序列构建了进化树,显示与斑马鱼Hprt基因更同源。RT-PCR表明黄鳝Hprt基因在多种组织中广谱表达,表明黄鳝该基因在功能和进化上的保守性。      

Screening for mutations in human HPRT cDNA using the polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis (CDGE).

Previously, we reported the modification of denaturing gradient gel electrophoresis called constant denaturant gel electrophoresis (CDGE). CDGE separates mutant fragments in specific melting domains. CDGE seems to be a useful tool in mutation detection. Since the hypoxanthine phosphoribosyltransferase (HPRT) gene is widely used as target locus for mutation studies in vitro and in vivo, we have examined the approach of analyzing human HPRT cDNA by polymerase chain reaction (PCR) and CDGE. All nine HPRT exons are included in a 716-bp cDNA fragment obtained by PCR using HPRT cDNA as template. When the full-length cDNA fragment was examined by CDGE, it was possible to detect mutations only in the last part of exon 8 and exon 9. However, digestion of the cDNA fragment with the restriction enzyme AvaI prior to CDGE enabled us to detect point mutations in most of exon 2, the beginning of exon 3, the last part of exon 8 and exon 9. With the use of two internal primer sets, including a GC-rich clamp on one of the primers in each pair, a region containing most of exon 3 through exon 6 was amplified and we were able to resolve fragments with point mutations in this region from wild-type DNA. The approach described here allows for rapid screening of point mutations in about two thirds of the human HPRT cDNA sequence. In a test of this approach, we were able to resolve 12 of 13 known mutants. The mutant panel included one single-base deletion, one two-base deletion and 11 single-base substitutions.     

Characterisation of cDNA clones for hypoxanthine-guanine phosphoribosyltransferase from the human malarial parasite, Plasmodium falciparum: comparisons to the mammalian gene and protein.

The isolation of cDNA clones for hypoxanthine-guanine phosphoribosyltransferase (HPRT) from the human malarial parasite, Plasmodium falciparum, is described. Northern analysis indicates that P. falciparum HPRT mRNA is the same size as that coding for mammalian HPRT. The predicted amino acid sequence of the P. falciparum HPRT protein shows extensive homology to the mammalian enzyme. Homology between the two proteins occurs in distinct blocks and a putative catalytic binding domain in the centre of the protein is also conserved. Five out of the seven characterised mammalian HPRT missense mutations map to regions which are conserved in the P. falciparum protein.     

Human hypoxanthine-guanine phosphoribosyltransferase: a single nucleotide substitution in cDNA clones isolated from a patient with Lesch-Nyhan syndrome (HPRTMidland)

We have determined the molecular basis for hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency in a patient, J.H., with Lesch-Nyhan syndrome. Radioimmunoassay of lysates of erythrocytes or cultured B-lymphoblasts showed that this patient had no detectable HPRT enzyme activity or HPRT protein. HPRT-specific mRNA levels were normal by Northern analysis. We created a cDNA library from mRNA isolated from cultured lymphoblasts derived from this patient. Nucleotide sequencing of full-length HPRT cDNA clones revealed a single nucleotide (nt) substitution: a T-to-A transversion at nt 389. We have designated this variant HPRTMidland. The predicted amino acid (aa) substitution in HPRTMidland is a valine to aspartic acid at aa 130. This substitution is within 2 aa of the amino acid substitution in a previously defined HPRT variant, HPRTAnn Arbor. Both mutations are within a highly conserved sequence in the putative 5-phosphoribosyl-1-pyrophosphate-binding domain. The amino acid substitution in HPRTMidland causes a significant perturbation in the predicted secondary structure of this region. The HPRTMidland mutation affects a different domain of HPRT than the HPRTFlint mutation located at 167 nt away.     

Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells.

The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.     

Disruption of the Hypoxanthine‐Guanine Phosphoribosyl‐Transferase Gene Caused by a Translocation in a Patient with Lesch‐Nyhan Syndrome

In this study, we have identified a novel mechanism of mutation involving translocation between the HPRT1 loci and other loci on the X chromosome. In HRT‐25's cDNA obtained from a patient with Lesch‐Nyhan syndrome, the upstream region of exon 3 was amplified, but the full‐length region was not amplified. The use of 3′ rapid amplification of cDNA ends polymerase chain reaction (3′RACE‐PCR) for HRT‐25 revealed part of intron 3 and an unknown sequence which have not identified the HPRT1 gene starting at the 3′ end of exon 3. We analyzed HPRT1 genomic DNA in order to confirm the mutation with the unknown sequence in the genomic DNA. Unknown sequence compared through BLAST analysis of human genome (NCBI; http://www.ncbi.nlm.nih.gov/BLAST/) showed that at least 0.5 to 0.6‐Mb telomeric to HPRT1 on chromosome Xq where located near LOC340581. This study provides the molecular basis for the involvement of genomic instability in germ cells.     

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency: Identification of point mutations in Japanese patients with Lesch-Nyhan syndrome and hereditary gout and their permanent expression in an HPRT-deficient mouse cell line

Two different single nucleotide transitions of hypoxanthine-guanine phosphoribosyltransferase (HPRT) were identified in a Japanese patient with Lesch-Nyhan syndrome (LNS) and a patient with hereditary gout. HPRT enzyme activities in the two patients were severely deficient, but the size and amount of mRNA were normal according to Northern analysis. Entire coding regions of HPRT cDNAs were amplified by PCR and sequenced. A G-to-A substitution at base 208 in exon 3, which predicted glycine 70 to arginine, was detected in the LNS patient (identical mutation with HPRT_Utrecht). A C-to-A substitution at base 73 in exon 2, which predicted proline 25 to threonine, was detected in the gout patient (designated HPRT_Yonago). We transfected normal HPRT cDNA, mutant cDNA with HRPT_Utrecht or mutant cDNA with HPRT_Yonago, respectively, to HPRT-deficient mouse cells and isolated permanent expression cell lines. The HPRT-deficient mouse cells had no detectable HPRT activity and a very low amount of HPRT mRNA. When the HPRT-deficient mouse cells were transfected with normal human cDNA, HPRT enzyme activity increased to 21.8% that of normal mouse cells. The mouse cells transfected with HPRT_Utrecht showed no increase in HPRT activity; however, when the mouse cells were transfected with HPRT_Yonago, the activity increased to 2.4% that of normal activity. The proliferative phenotypes of these cells in HAT medium and in medium containing 6-thioguanine were similar to those of skin fibroblasts from the patients. This series of studies confirmed that each of the two point mutations was responsible for the decreases in HPRT enzyme activity, and the proliferative phenotypes in HAT medium and medium containing 6-thioguanine.     

Molecular cloning of p53 cDNA of Mongolian gerbil and establishment of yeast p53 functional assay system

Background. Epidemiological studies have shown a correlation between Helicobacter pylori infection and human gastric carcinogenesis. A Mongolian gerbil model has demonstrated that H. pylori infection induced gastric carcinoma. However, the disadvantage of this animal model is a lack of information regarding the cellular genes involved in oncogenesis. Mutation of the p53 gene is one of the most common steps in gastric carcinogenesis. In this study, we aimed to clone the p53 gene of the Mongolian gerbil and detect the functional mutations in H. pylori‐infected animals. Materials and Methods. The p53 complementary DNA (cDNA) of Mongolian gerbil was cloned by the methods of reverse‐transcribed polymerase chain reaction and rapid amplification of cDNA ends. Results. The p53 cDNA of Mongolian gerbil has a 78.8% homology to that of humans. A novel yeast p53 assay system was established and enabled to detect the functional mutations of the p53 gene in the stomach of the Mongolian gerbil. Conclusions. This is the first report of the complete sequence of wild‐type p53 cDNA of the Mongolian gerbil. This genetic information and an assay system designed to detect the functional mutations of the p53 gene are useful for further investigations of gastric oncogenesis in this animal model.     

Radiation-induced HPRT mutations resulting from misrejoined DNA double-strand breaks

DNA double-strand breaks (DSBs) are the most severe lesions induced by ionizing radiation, and unrejoined or misrejoined DSBs can lead to cell lethality, mutations and the initiation of tumorigenesis. We have investigated X-ray- and alpha-particle-induced mutations that inactivate the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene in human bladder carcinoma cells and in hTERT-immortalized human fibroblasts. Fifty to 80% of the mutants analyzed exhibited partial or total deletions of the 9 exons of the HPRT locus. The remaining mutants retained unaltered PCR products of all 9 exons but often displayed a failure to amplify the HPRT cDNA. Hybridization analysis of a 2-Mbp NotI fragment spanning the HPRT gene with a probe 200 kbp distal to the HPRT locus indicated altered fragment sizes in most of the mutants with a wild-type PCR pattern. These mutants likely contain breakpoints for genomic rearrangements in the intronic sequences of the HPRT gene that allow the amplification of the exons but prevent HPRT cDNA amplification. Additionally, mutants exhibiting partial and total deletions of the HPRT exons also frequently displayed altered NotI fragments. Interestingly, all mutations were very rarely associated with interchromosomal exchanges analyzed by FISH. Collectively, our data suggest that intrachromosomal genomic rearrangements on the Mbp scale represent the prevailing type of radiation-induced HPRT mutations.     

Variable stability of a selectable provirus after retroviral vector gene transfer into human cells.

Human lymphoblasts deficient in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) were infected with an amphotropic helper-free retroviral vector expressing human HPRT cDNA. The stability and expression of the HPRT provirus in five cell lines with different proviral integration sites were examined by determining HPRT mutation and reversion frequencies and by blot hybridization studies. Mutation to the HPRT-negative phenotype occurred at frequencies of approximately 4 X 10(-5) to 3 X 10(-6) per generation. Most mutations in each of the five cell lines were associated with partial or complete deletions or rearrangements of the provirus. Several mutants retained a grossly intact HPRT provirus, and in one such mutant HPRT shutdown resulted from a revertible epigenetic mechanism that was not associated with global changes in proviral methylation. Therefore, mutation and shutdown of the HPRT provirus in human lymphoblasts result from mechanisms similar to those reported for several other avian and mammalian replication-competent retroviruses.     

Identification of a single nucleotide change in a mutant gene for hypoxanthine-guanine phosphoribosyltransferase (HPRTAnn Arbor)

Summary HPRT_{Ann Arbor} is a variant of hypoxanthine (guanine) phosphoribosyl-transferase (HPRT: EC 2.4.2.8), which was identified in two brothers with hyperuricemia and nephrolithiasis. In previous studies, this mutant enzyme was characterized by an increased K_m for both substrates, a normal V_max, a decreased intracellular concentration of enzyme protein, a normal subunit molecular weight and an acidic isoelectric point under native isoelectric focusing conditions. We have cloned a full-length cDNA for HPRT_{Ann Arbor} and determined its complete nucleotide sequence. A single nucleotide change (TG) at nucleotide position 396 has been identified. This transversion predicts an amino acid substitution from isoleucine (ATT) to methionine (ATG) in codon 132, which is located within the putative 5-phosphoribosyl-1-pyrophosphate (PRPP)-binding site of HPRT.