Localization of the gene coding for adenine phosphoribosyltransferase on the genetic map of chromosome 8 in the mouse

Polymorphism of electrophoretic mobility of adenine phosphoribosyltransferase (APRT) was found in a population of domestic mice, Mus musculus bactrianus. The Aprt gene was mapped using two markers: plasma esterase 1 coded by the gene Es-1 situated at the distance of 26 morgans from the centromere, and a Robertsonian translocation Rb (8.17) 1 Iem which marks the centromere. The results of linkage analysis permitted to localize the gene Aprt at 51 morgans from the centromere, and 25 morgans distal from the gene Es-1 on the genetic map of chromosome 8. It is found that emotional stress does not alter the recombination rate at chromosome 8, when spermatocytes are at the pachytene stage.     

Role of the mismatch repair gene, Msh6, in suppressing genome instability and radiation-induced mutations

Mismatch repair (MMR) is critical for preserving genomic integrity. Failure of this system can accelerate somatic mutation and increase the risk of developing cancer. MSH6, in complex with MSH2, is the MMR protein that mediates DNA repair through the recognition of 1- and 2-bp mismatches. To evaluate the effects of MSH6 deficiency on genomic stability we compared the frequency of in vivo loss of heterozygosity (LOH) between MSH6-proficient and deficient, 129S2xC57BL/6 F1 hybrid mice that were heterozygous for our reporter gene Aprt. We recovered mutant cells that had functionally lost APRT protein activity and categorized the spectrum of mutations responsible for the LOH events. We also measured the mutant frequency at the X-linked gene, Hprt, as a second reporter for point mutation. In Msh6-/-Aprt+/- mice, mutation frequency at Aprt was elevated in both T cells and fibroblasts by 2.5-fold and 5.7-fold, respectively, over Msh6+/+Aprt+/- littermate controls. While a modest increase in mitotic recombination (MR) was observed in MSH6-deficient fibroblasts compared to wild type controls, point mutation was the predominant mechanism leading to APRT deficiency in both cell types. Base substitution, consisting of multiple types of transitions, accounted for all of the point mutations identified within the Aprt coding region. We also assessed the role of MSH6 in preventing mutations caused by a common environmental mutagen, ionizing radiation (IR). In Msh6-/-Aprt+/- mice, 4Gy of X-irradiation induced a significant increase in point mutations at both Aprt and Hprt in T cells, but not in fibroblasts. These findings indicate that MutS alpha reduces spontaneous and IR-induced mutation in a cell type-dependant manner.     

Plasmid, phage, and genomic DNA-mediated transfer and expression of prokaryotic and eukaryotic genes in cultured human cells

Transfection of mammalian cells with genomic DNA and cloned genes is now relatively routine. However, the vast majority of studies have used rodent cells as recipients. Here we describe efficient transfection of two human cell lines, the hypoxanthine guanine phosphoribosyltransferase (HPRT)-deficient HeLa line, D98/AH-2, and the adenine phosphoribosyltransferase (APRT)-deficient HT1080 line, HTD114. D98/AH-2 cells were transfected with the pSV2-gpt plasmid of Mulligan and Berg, which contains the E. coli xanthine-guanine phosphoribosyltransferase (gpt) gene, and Gpt + transfectants were selected in HAT medium. HTD114 cells were transfected with (1) genomic hamster DNA, and ouabain resistant transfectants were selected in 5 X 10(-7)M ouabain; (2) with hamster and mouse genomic DNA, and Aprt + cells were selected in AAA medium; (3) with plasmids containing either the cloned hamster or mouse APRT genes, and Aprt + cells were selected; and (4) with phage particles containing a cloned mouse APRT gene, and Aprt + cells were selected. Transfection efficiencies ranged from 0.25 to 1.5 X 10(3) transfectants per microgram DNA, and in certain cases secondary transfections were done. Foreign DNA in recipients was detected by blot hybridization, and the expression of foreign genes was detected by cell growth in selective media and the expression of enzymes characteristic of the species of the donor DNA. The majority of transfectants showed stable expression of the transgenome.     

A novel selectable system for detecting expansion of CAG.CTG repeats in mammalian cells

CAG.CTG repeat expansions cause more than a dozen neurodegenerative diseases in humans. To define the mechanism of repeat instability in mammalian cells we developed a selectable assay to detect expansions of CAG.CTG triplet repeats in Chinese hamster ovary (CHO) cells. We showed previously that long tracts of CAG.CTG repeats, embedded in an intron of the APRT gene, kill expression of the gene, rendering the cells APRT-. By contrast, tracts with fewer than 34 repeats allow sufficient expression to give APRT+ cells. Although it should be possible to use APRT+ cells with short repeats to assay for expansion events by selecting for APRT- cells, we find that APRT+ cells with 31 repeats are not killed by the standard APRT- selection protocol, most likely because they produce too little Aprt to incorporate sufficient 8-azaadenine into their adenine pool. To overcome this problem, we devised a new selection, which increases the proportion of the adenine pool contributed by the salvage pathway by partially inhibiting the de novo pathway. We show that APRT- CHO cells with 61 or 95 CAG.CTG repeats survive this selection, whereas cells with 31 repeats die. Using this selection system, we can select for expansion to as few as 39 repeats. Thus, this assay can monitor expansions across the critical boundary from the longest lengths of normal alleles to the shortest lengths of disease alleles.     

Assignment of a processed mouse Aprt pseudogene to the same chromosome as the functional gene

A novel genetic system has been used to demonstrate that a processed adenine phosphoribosyltransferase (Aprt) pseudogene is located on mouse chromosome 8, which is the same chromosome that carries the functional Aprt gene. A restriction fragment length polymorphism associated with the pseudogene was found to segregate concordantly with chromosome 8 in APRT- mutants of a near-diploid cell line that had lost one copy of the chromosome.     

Loss of heterozygosity and base substitution at the APRT locus in mismatch-repair-proficient and -deficient colorectal carcinoma cell lines.

We determined the nature of mutations occurring at the autosomal APRT locus in mismatch-repair-proficient and -deficient colorectal carcinoma cell lines. The analysis of mutations that result in APRT deficiency in a mismatch-repair-deficient strain of DLD-1 heterozygous for this locus enabled us to measure the rate of loss of the wild-type gene through deletion, recombination, or gene conversion as well as the rate of point mutation. The overall rate of mutation at the APRT locus in DLD-1 was elevated 100-fold compared with the mismatch-repair-proficient colorectal carcinoma cell line SW620. Loss of heterozygosity (LOH) at APRT accounted for only 4 to 9% of mutant strains derived from DLD-1, indicating a rate for these types of events of 4 x 10(-7) to 9 x 10(-7). In SW620 the rate of LOH at APRT was about 10-fold higher. LOH was not found at polymorphic markers within the same chromosome subband as APRT, indicating that only a limited portion of the chromosome was affected by these alterations. Chromosome painting of SWS620 mutants revealed that the loss of APRT occurred together with a substantial portion of the long arm of chromosome 16. Differences in the nature of base substitutions at APRT (e.g., the proportion of mutations resulting from transitions or transversions) in these tumor cell lines were also detected. There was also an important similarity---the presence of a mutant APRT gene with multiple base substitutions that may be the result of some sort of error-prone DNA synthesis.     

Homology dependence of targeted recombination at the Chinese hamster APRT locus.

Using simple linear fragments of the Chinese hamster adenine phosphoribosyltransferase (APRT) gene as targeting vectors, we have investigated the homology dependence of targeted recombination at the endogenous APRT locus in Chinese hamster ovary (CHO) cells. We have examined the effects of varying either the overall length of targeting sequence homology or the length of 5' or 3' flanking homology on both the frequency of targeted homologous recombination and the types of recombination events that are obtained. We find an exponential (logarithmic) relationship between length of APRT targeting homology and the frequency of targeted recombination at the CHO APRT locus, with the frequency of targeted recombination dependent upon both the overall length of targeting homology and the length of homology flanking each side of the target gene deletion. Although most of the APRT+ recombinants analyzed reflect simple targeted replacement or conversion of the target gene deletion, a significant fraction appear to have arisen by target gene-templated extension and correction of the targeting fragment sequences. APRT fragments with limited targeting homology flanking one side of the target gene deletion yield proportionately fewer target gene conversion events and proportionately more templated extension and vector correction events than do fragments with more substantial flanking homology.     

Human alpha-globin gene expression following chromosomal dependent gene transfer into mouse erythroleukemia cells.

We have used the genetic marker, adenine phosphoribosyl transferase (APRT), an enzyme known to be on human chromosome 16, to establish a method for the transfer of human α-globin genes into mouse erythroleukemia cells. Mouse erythroleukemia cells devoid of detectable levels of APRT were fused with fractions of human marrow enriched in human erythroid cells. The hybrid cells arising from this fusion were isolated in medium supplemented with aminopterin and thymidine, and used adenine as the sole purine source. This population of hybrid cells was dominated by cells (80%) in which human chromosome 16 was present. Human chromosomes 4, 5 and 6 were also found in these cells. The hybrid cells were then placed in medium supplemented with diaminopurine (DAP), which is lethal for cells containing APRT. Greater than 95% of the DAP-selected hybrid cells lacked human chromosome 16. Cytoplasmic RNA was extracted from the two hybrid cell populations and assayed by molecular hybridization for sequences coding for human α-globin. Carboxymethyl cellulose chromatography was used to study the level of synthesis of human a-globin in the hybrids. The original hybrid cell, which contained a high frequency of human chromosome 16, also contained high levels of human a-globin mRNA and human α-globin chains. Hybrid cells counter-selected in DAP and thus lacking human chromosome 16 were devoid of detectable levels of human APRT, human α-globin mRNA and human α-globin chains. This work shows that transfer of human chromosome 16 into the MEL cell is possible using a chromosomedependent, APRT-mediated method of gene transfer. Using this system in which expression of the human α-globin gene occurs, we were also able to confirm our earlier assignment of the human α-globin gene to human chromosome 16. This system may be of further use in identifying genetic elements governing expression of the human α-globin gene which can be carried with human chromosome 16 as it is donated to the mouse erythroleukemia cell by donor cells of different epigenotypes.     

水稻基因APRT的克隆及其与温敏核雄性不育的关系

在拟南芥中腺嘌呤磷酸核糖转移酶基因(APRT)突变导致植株雄性不育.本文首次报道从水稻(Oryza sativa subsp.indica)中克隆了基因APRT(GenBank登录号AY238894),并将其定位于水稻第4染色体的一个BAC克隆(AL606604)的58 000 bp至63 000 bp区域.该基因长4 220 bp(起始密码子至终止密码子),含7个外显子、6个内含子,编码的APRT蛋白长212个氨基酸残基,与其他物种来源的APRT序列存在很高的同源性.与大麦、小麦、拟南芥1型及其2型的该蛋白同源性分别为54.9%、54.9%、49.6%和59.5%.经保守结构域搜索发现该蛋白中存在APRT催化结构域.从DNA、mRNA两个水平分析了该基因与水稻温敏核雄性不育(TGMS)的关系,结果表明:受温度诱导,水稻"安农S-1"APRT基因的表达变化可能与温敏核雄性不育表现型具相关性.     

Common characteristics of mutant adenine phosphoribosyltransferases from four separate Japanese families with 2,8-dihydroxyadenine urolithiasis associated with partial enzyme deficiencies

Summary 2,8-Dihydroxyadenine urolithiasis associated with partial deficiencies of adenine phosphoribosyltransferase (APRT) has been found only among Japanese families. All Caucasian patients with the same lithiasis are completely deficient in this enzyme. Partially purified APRT from one of the Japanese families with the lithiasis associated with a partial deficiency of APRT had a reduced affinity for 5-phosphoribosyl-1-pyrophosphate (PRPP). In the present investigations, we have shown that this characteristic is common in mutant enzymes from all the four separate Japanese urolithiasis families associated with partial APRT deficiencies so far tested. The mutant enzymes also had several other characteristics in common including increased resistance to heat in the absence of PRPP and reduced sensitivity to the stabilizing effect of PRPP. These data suggest that these families have a common mutant allele (APRT ^* J) at the APRT gene locus.     

Determination of spontaneous loss of heterozygosity mutations in Aprt heterozygous mice.

A mouse model was generated to investigate loss of heterozygosity (LOH) events in somatic cells. The adenine phosphoribosyltransferase ( Aprt ) gene was disrupted in embryonic stem cells using a conventional gene targeting approach and subsequently Aprt hetero-zygous and homozygous mice were derived. Aprt homozygous deficient animals were viable though the mendelian inheritance pattern was skewed. On average these mice died at 6 months of age from severe renal failure. In T-lymphocytes of Aprt heterozygous mice the mean spontaneous mutant frequency at the Aprt locus was 8.7 x 10(-6) while the frequency was 0.8 x 10(-6) at the hypoxanthine phosphoribosyltransferase locus. In order to determine whether LOH events contribute to the high spontaneous mutant frequency at the Aprt locus, 140 Aprt mutant T-lymphocyte clones were expanded and analysed by allele-specific PCR. In 97 (69%) of these clones the wild-type allele had been lost. Nine of the mutant clones were characterized in more detail using dual-coloured fluorescence in situ hybridization analysis. Five out of six of the mutant clones which arose from an LOH event, based on the PCR assay, contained a duplication of the targeted allele. Therefore, mitotic recombination or chromosome loss followed by duplication of the remaining homologue appears to be the predominant mechanism for the in vivo generation of Aprt mutant T-lymphocytes.     

A new location for the human adenine phosphoribosyltransferase gene (APRT) distal to the haptoglobin (HP) and fra(16)(q23)(FRA16D) loci

The human adenine phosphoribosyltransferase gene (APRT) was mapped with respect to the haptoglobin gene (HP) and the fragile site at 16q23.2 (FRA16D). A subclone of APRT and a cDNA clone of HP were used for molecular hybridization to DNA from mouse-human hybrid cell lines containing specific chromosome 16 translocations. The APRT subclone was used for in situ hybridization to chromosomes expressing FRA16D. APRT was found to be distal to HP and FRA16D and was localized at 16q24, making the gene order cen-FRA16B-HP-FRA16D-APRT-qter.     

A mouse-human hybrid cell panel for mapping human chromosome 16

A mouse-human hybrid cell panel for human chromosome 16 was constructed from human cell lines with breakpoints on chromosome 16 at p13.11, q13, q22 and q24. Fusions with the human fibroblast line GM3884, t(X;16)(q26;q24) allowed the isolation of clones with either the derivative X or the derivative 16 as the only human chromosome. This was a consequence of both the genes APRT and HPRT being involved in the translocation. The breakpoints of the line GM3884 were confirmed by aphidicolin induction of the common fragile site at 16q23. The results of the fusions with this line suggest a localisation of the APRT gene at 16q24 and confirm the localisation of HPRT to Xq26 to Xq27.3. These hybrid cell lines enable the localisation of genes and DNA fragments to six clearly defined regions. Further localisation within three of these regions is possible by use of the three fragile sites on chromosome 16. In situ hybridisation with the probe pBLUR confirmed that of three lines tested all contained a single human chromosome.     

The Locus for Human Adenine Phosphoribosyltransferase on Chromosome No. 16

Evidence for assigning the locus determining the structure of adenine phosphoribosyltransferase (APRT) to human chromosome No. 16 is presented. Hybrids of APRT-deficient mouse cells and of human fibroblasts having normal APRT were isolated by fusing the parental cells with Sendai virus, blocking de novo purine nucleotide synthesis with azaserine and selecting for hybrids that could use exogenous adenine. The hybrid clones that were studied had only APRT activity that was indistinguishable from human APRT with regard to electrophoretic migration and reaction with antibodies against the partially purified human enzyme. No. 16 was the only human chromosome consistently present in all of the clones, and in one clone, it was the only human chromosome detected. Selection against hybrid cells with 2,6-diaminopurine (DAP) yielded DAP-resistant survivors that lacked chromosome No. 16. One hybrid that originally had an intact No. 16 yielded adenine-utilizing subclones that lacked No. 16 but had a new submetacentric chromosome. The distribution of centromere-associated heterochromatin and the fluorescence pattern indicated that this chromosome consisted of a mouse telocentric chromosome and the long arm of No. 16. Cells having the submetacentric chromosome had human APRT. Both the enzyme and the chromosome were absent in DAP-resistant derivatives. These results suggest that the structure of APRT is defined by a locus on the long arm of human chromosome No. 16.     

Expression of recessive Aprt- mutations in mouse CAK cells resulting from chromosome loss and duplication

Karyotypes of recessive mutants at the autosomal adenine phosphoribosyltransferase (Aprt) locus in a clone of the near-diploid mouse CAK cell line have been analyzed. The Aprt located on chromosome 8. One copy of chromosome 8 was morphologically abnormal in the parental clone (CAK-B3-Toyr13) from which Aprt- mutants were isolated. Among 22 mutants, there were ten in which one copy of chromosome 8 had been lost. Four of these were monosomic, and in the others duplication of the remaining homolog had occurred. These findings indicate that newly induced recessive mutations in cultured mammalian cells can be expressed as the result of loss of one chromosome carrying a wild-type allele with or without duplication of the homolog carrying the mutant allele. Loss and duplication would not be detected in cell lines lacking morphologically marked chromosomes.     

Cloning the complete human adenine phosphoribosyl transferase gene

We have isolated a clone from a human genomic lambda library which cross-hybridises with the cloned hamster adenine phosphoribosyl transferase gene (aprt). After restriction mapping and further hybridisation to the hamster gene, a series of putative human aprt-containing fragments has been isolated and tested for ability to transform adenine phosphoribosyl transferase-deficient (aprt-) strains of Chinese hamster ovary (CHO) cells to APRT proficiency. Transforming activity was detected in a 48-kb lambda clone, the 17.4-kb EcoRI insert, and an 8.6-kb HincII fragment. Smaller fragments have thus far shown no transforming activity. Transformants appear to be stable for the APRT+ phenotype, and human aprt DNA sequences are present in the hamster transformants. The 8.6-kb HincII fragment has been subcloned and the insert mapped. Nonrepetitive regions of this subclone have been identified, and should prove valuable for chromosome walking studies on human chromosome 16, familial studies of a human aprt- trait, the analysis of restriction fragment length polymorphisms (RFLPs) in the area surrounding the aprt gene, and the fine structure mapping of the mutations induced by chemical carcinogens and alkylating agents.     

Chromosomal and regional localization of the genes for UMPH2, APRT, PEPD, PEPS, PSP, and PGP in mink: comparison with man and mouse

Segregation of mink biochemical markers uridine 5'-monophosphate phosphohydrolase-2 (UMPH2), adenine phosphoribosyltransferase (APRT), phosphoserine phosphatase (PSP), phosphoglycolate phosphatase (PGP), peptidases D (PEPD) and S (PEPS), as well as mink chromosomes, was investigated in a set of mink x mouse hybrid clones. The results obtained allowed us to make the following mink gene assignments: UMPH2, chromosome 8; PEPD and APRT, chromosome 7; PEPS, chromosome 6; and PSP and PGP, chromosome 14. The latter two genes are the first known markers for mink chromosome 14. For regional mapping, UMPH2 was analyzed in mouse cell clones transformed by means of mink metaphase chromosomes (Gradov et al., 1985) and also in mink x mouse hybrid clones carrying fragments of mink chromosome 8 of different sizes. Based on the data obtained, the gene for UMPH2 was assigned to the region 8pter----p26 of mink chromosome 8. The present data is compared with that previously established for man and mouse with reference to the conservation of syntenic gene groups and G-band homoeologies of chromosomes in mammals.     

Analysis of Aprt deficient mutants of Friend erythroleukaemia cells

Cytogenetic analysis of mutants of the Friend Erythroleukaemia cell line deficient in adenine phosphoribosyl transferase (Aprt) was undertaken to ascertain whether non-disjunctional events were involved in the production of this mutation. All mutant clones examined were found to carry two copies of chromosome 8, the chromosome to which Aprt maps. Since the two homologues are distinguishable by silver staining, it was also clear that no mutants contained two copies of one homologue having lost the other. Treatment of mutants with 5-azacytidine failed to reactivate the Aprt locus.