Transformation of the gene for hypoxanthine phosphoribosyltransferase.

Purified DNA from wild-type Chinese ovary (CHO) cells has been used to transform three hypoxanthine phosphoribosyltransferase (HPRT) deficient murine cell mutants to the enzyme positive state. Transformants appeared at an overall frequency of 5 x 10(-8) colonies/treated cell and expressed CHO HPRT activity as determined by electrophoresis. One gene recipient, B21, was a newly isolated mutant of LMTK- deficient in both HPRT and thymidine kinase (TK) activities. Transformation of B21 to HPRT+ occurred at 1/5 the frequency of transformation to TK+; the latter was, in turn, an order of magnitude lower than that found in the parental LMTK- cells, 3 x 10(-6). Thus both clonal and marker-specific factors play a role in determining transformability. The specific activity of HPRT in transformant extracts ranged from 0.5 to 5 times the CHO level. The rate of loss of the transformant HPRT+ phenotype, as measured by fluctuation analysis, was 10(-4)/cell/generation. While this value indicates stability compared to many gene transferents, it is much greater than the spontaneous mutation rate at the indigenous locus. The ability to transfer the gene for HPRT into cultured mammalian cells may prove useful for mutational and genetic mapping studies in this well-studied system.     

Elevated levels of erythrocyte hypoxanthine phosphoribosyltransferase associated with allelic variation of murine Hprt

Murine stocks with wild-derived hypoxanthine phosphoribosyltransferase (HPRT) A alleles (Hprt a) have erythrocyte HPRT activity levels that are approximately 25-fold (Mus musculus castaneus) and 70-fold (Mus spretus) higher than those of laboratory strains of mice with the common Hprt b allele (Mus musculus: C3H/HeHa or C57B1/6). Since the purified HPRT A and B enzymes have substantially similar maximal specific activities (64 and 46 units/mg of protein, respectively), we infer that these HPRT activity levels closely approximate the relative levels of HPRT protein in these cells. Red blood cells of HPRT A and B mice have similar levels of adenine phosphoribosyltransferase activity (APRT; EC 2.4.2.7) and reticulocyte percentages, which suggests that the elevated levels of HPRT in erythrocytes of HPRT A mice are not secondary consequences of abnormal erythroid cell development. The HPRT activity levels in reticulocytes of HPRT B mice are approximately 35-fold higher than the levels in their erythrocytes and approach the HPRT activity levels in reticulocytes of HPRT A mice. Thus, the marked differences in the levels of HPRT protein in erythrocytes of HPRT A and B mice result from differences in the extent to which the HPRT A and B proteins are retained as reticulocytes mature to erythrocytes. The substantial and preferential loss of HPRT B activity from reticulocytes is paralleled by an equivalent loss of HPRT immunoreactive protein (i.e., CRM) from that cell, and we infer that the HPRT B protein is degraded or extruded as reticulocytes mature to erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequence analysis of human hypoxanthine phosphoribosyltransferase (HPRT) gene deletions.

We have determined the nucleotide sequences of 10 intragenic human HPRT gene deletion junctions isolated from thioguanine-resistant PSV811 Werner syndrome fibroblasts or from HL60 myeloid leukemia cells. Deletion junctions were located by fine structure blot hybridization mapping and then amplified with flanking oligonucleotide primer pairs for DNA sequence analysis. The junction region sequences from these 10 HPRT mutants contained 13 deletions ranging in size from 57 bp to 19.3 kb. Three DNA inversions of 711, 368, and 20 bp were associated with tandem deletions in two mutants. Each mutant contained the deletion of one or more HPRT exon, thus explaining the thioguanine-resistant cellular phenotype. Deletion junction and donor nucleotide sequence alignments suggest that all of these HPRT gene rearrangements were generated by the nonhomologous recombination of donor DNA duplexes that share little nucleotide sequence identity. This result is surprising, given the potential for homologous recombination between copies of repeated DNA sequences that constitute approximately a third of the human HPRT locus. No difference in deletion structure or complexity was observed between deletions isolated from Werner syndrome or from HL60 mutants. This suggests that the Werner syndrome deletion mutator uses deletion mutagenesis pathway(s) that are similar or identical to those used in other human somatic cells.     

Tissue of origin determines cancer-associated CpG island promoter hypermethylation patterns

Background

Aberrant CpG island promoter DNA hypermethylation is frequently observed in cancer and is believed to contribute to tumor progression by silencing the expression of tumor suppressor genes. Previously, we observed that promoter hypermethylation in breast cancer reflects cell lineage rather than tumor progression and occurs at genes that are already repressed in a lineage-specific manner. To investigate the generality of our observation we analyzed the methylation profiles of 1,154 cancers from 7 different tissue types.

Results

We find that 1,009 genes are prone to hypermethylation in these 7 types of cancer. Nearly half of these genes varied in their susceptibility to hypermethylation between different cancer types. We show that the expression status of hypermethylation prone genes in the originator tissue determines their propensity to become hypermethylated in cancer; specifically, genes that are normally repressed in a tissue are prone to hypermethylation in cancers derived from that tissue. We also show that the promoter regions of hypermethylation-prone genes are depleted of repetitive elements and that DNA sequence around the same promoters is evolutionarily conserved. We propose that these two characteristics reflect tissue-specific gene promoter architecture regulating the expression of these hypermethylation prone genes in normal tissues.

Conclusions

As aberrantly hypermethylated genes are already repressed in pre-cancerous tissue, we suggest that their hypermethylation does not directly contribute to cancer development via silencing. Instead aberrant hypermethylation reflects developmental history and the perturbation of epigenetic mechanisms maintaining these repressed promoters in a hypomethylated state in normal cells.     

山羊克隆胎儿不同组织中H19基因CpG岛甲基化模式及表达量检测

表观重编程异常是核移植胚胎发育异常的重要原因。为了研究克隆山羊胎儿不同组织中H19基因CpG岛甲基化水平和相对表达量,本实验运用亚硫酸盐法和荧光实时定量PCR法分别检测了死亡克隆山羊胎儿和同期普通山羊胎儿(对照组)肝脏、胎盘、肾脏、肺脏和心脏组织中H19基因CpG岛甲基化水平和mRNA的相对表达量。结果表明,克隆山羊胎儿胎盘组织中H19基因第5个CpG岛的甲基化水平显著高于对照组(70%vs49.41%,P0.05),H19基因相对表达量显著低于对照组(883.3vs1264.5,P0.05);肺脏组织甲基化水平显著低于对照组(63.53%vs88.24%,P0.05),相对表达量显著高于对照组(1003.4vs515.5,P0.05);其他各组差异不显著(P0.05)。结果说明,H19基因在克隆山羊胎儿部分组织中DNA甲基化重编程异常,而且这种异常影响H19基因的正常表达,这也可能是导致克隆动物死亡的重要因素之一。     

Study of Alu sequences at the hypoxanthine phosphoribosyltransferase (hprt) encoding region of man

The hypoxanthine phosphoribosyltransferase (hprt) encoding region of man is considered rich in Alu sequences; with 49 sequences present within 57 kilobases. Subfamily classification of the Alu sequences and identification of flanking direct repeats has been carried out to detect past rearrangements associated with their insertion into the region. Members of the Alu-J and three Alu-S subfamilies are present, along with the existence of free left arm sequences. Using available data, a comparison is made of the Alu subfamilies present at different gene regions. The heterogeneity in the number of each subfamily present at different genes shows that no one particular subfamily attained saturation in the genome. Several adjacent insertions of Alu sequences are seen at the hprt region. Furthermore two novel sequences are described, there is an incident where one Alu sequence has inserted into the middle poly(A) tract of an existing sequence at the hprt region; while another resulted from an Alu/Alu cross-over event elsewhere in the genome, before insertion into the hprt region. Once inserted, the Alu sequences are rarely subject to loss or rearrangement.     

Molecular structure and genetic stability of human hypoxanthine phosphoribosyltransferase (HPRT) gene duplications.

We have determined the genetic stability of three independent intragenic human HPRT gene duplications and the structure of each duplication at the nucleotide sequence level. Two of the duplications were isolated as spontaneous mutations from the HL60 human myeloid leukemia cell line, while the third was originally identified in a Lesch-Nyhan patient. All three duplications are genetically unstable and have a reversion rate approximately 100-fold higher than the rate of duplication formation. The molecular structures of these duplications are similar, with direct duplication of HPRT exons 2 and 3 and of 6.8 kb (HL60 duplications) or 13.7 kb (Lesch-Nyhan duplication) of surrounding HPRT sequence. Nucleotide sequence analyses of duplication junctions revealed that the HL60-derived duplications were generated by unequal homologous recombination between clusters of Alu repeats contained in HPRT introns 1 and 3, while the Lesch-Nyhan duplication was generated by the nonhomologous insertion of duplicated HPRT DNA into HPRT intron 1. These results suggest that duplication substrates of different lengths can be generated from the human HPRT exon 2-3 region and can undergo either homologous or nonhomologous recombination with the HPRT locus to form gene duplications.     

hMLH1基因启动子CpG岛甲基化致MSI与肿瘤的关系

通过人类错配修复基因( hMLHl)启动子CpG岛甲基化与微卫星不稳定性(MSI)的分析,探讨癌症发病的机制.错配修复基因hMLH1启动子CpG岛甲基化是hMLH1基因失活的重要机制,而hMLH1的表达失活则可导致MSI的产生,促进癌症的发生.根据一系列研究得出结论,在肿瘤组织中hMLH1基因启动子CpG岛甲基化和微卫星不稳定(MSI)有显著相关性,并在癌症早期发生、发展过程中起重要作用.因此临床检测hMLH1基因启动子CpG岛甲基化及微卫星不稳定可能成为癌症鉴别诊断、评价预后、指导化疗的分子标志物之一.     

The mouse L-histidine decarboxylase gene: structure and transcriptional regulation by CpG methylation in the promoter region

To investigate the regulation of mouse L-histidine decarboxylase (HDC) gene expression, we isolated genomic DNA clones encoding HDC. Structural analysis revealed that the mouse HDC gene was composed of 12 exons, spanning approximately 24 kb. Northern blotting analysis indicated that, among the cell lines examined, a high level of HDC gene expression was restricted to mature mast cell lines and an erythroblastic cell line. The gene was induced strongly in the mouse immature mast cell line P815 after incubation in the peritoneal cavity of BDF1 mice. We observed that the promoter region was demethylated in the HDC-expressing cell lines and in induced P815 cells. Interestingly, forced demethylation by 5-azacytidine (5-azaC) treatment induced high expression of HDC mRNA in P815 cells. The activity of a mouse HDC promoter-reporter construct stably transfected in P815 cells was repressed by in vitro patch-methylation. This low promoter activity of the patch-methylated reporter construct was restored after 5-azaC treatment, which demethylated the patch-methylated promoter. These results indicate that DNA methylation state of the promoter region controls HDC gene expression.     

Novel mutational spectrum induced by N-methyl-N'-nitro-N-nitrosoguanidine in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene in diploid human fibroblasts

The kinds and locations of mutations in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene of 75 independent mutants, derived from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated normal human fibroblasts, were characterized by direct sequencing of mRNA-polymerase chain reaction (mRNA-PCR)-amplified cDNA. Treatment of human cells with low (6 or 8 microM) or high (10 or 12 microM) doses of MNNG resulted in 35-fold or 150-fold average increases in mutation frequency, respectively. A high frequency of mutants lacking a complete exon was observed in both groups. Further characterization of half of these mutants by DNA-PCR amplification of intron-exon boundaries showed that they contained base substitutions. The kinds of base substitutions differed distinctly between these two groups. In the low dose group, a broad mutational spectrum was observed: ten out of the 31 base substitutions were A.T to G.C transitions, six contained G.C to A.T transitions, and the other 15 exhibited transversions. In contrast, the majority (84%) of base substitutions among the high dose group were G.C to A.T transitions; the others (16%) were transversions. All of the 32 G.C to A.T transitions were located on the non-transcribed strand, assuming that the causative premutational lesion was O6-methylguanine. These results indicate preferential repair of lesions located on the transcribed strand. In addition, G.C to A.T and A.T to G.C transitions preferentially occurred at positions with guanine and thymine at the adjacent 5' position, respectively.     

Tissue-specific methylation of a CpG island in transgenic mice.

Clustering of CpG dinucleotides in CpG-rich islands is a characteristic feature of mammalian genomes. Such CpG islands are frequently associated with genes and usually hypomethylated, regardless of the gene activity. This is the case for the CpG island of the murine Thy-1 gene. A transgenic line containing multiple copies of a truncated, concatemeric CpG island from the Thy-1.1 allele (Thy-1.2 background) showed that a stable fraction (approx. 0.20) became fully methylated in somatic tissues of homozygous mice with respect to testable restriction sites, while the remaining copies were methylation-free, i.e., this methylation appears to be an 'all-or-none' phenomenon. DNA from extraembryonic tissues (placenta and yolk sac) and epididymal sperm showed, however, an even higher degree of methylation in two distinct patterns. In the extraembryonic tissue, partial methylation of each copy was seen, whereas in sperm a high degree of 'all-or-none' methylation (greater than 0.35) was observed.     

De novo DNA methylation at the CpG island of the zebrafish no tail gene

The zebrafish no tail gene (ntl) is indispensable for the formation of the notochord and the tail structure. Here we showed that de novo DNA methylation occurred at the CpG island of ntl. The methylation started at the segmentation stage and continued after the larval stage. However, it occurred predominantly between 14 and 48 h postfertilization, which overlaps the period in which ntl expression disappears in the notochord and the tailbud. This inverse correlation, together with the methylation-associated formation of an inaccessible chromatin structure at the ntl CpG island region, suggested the involvement of the de novo methylation in ntl repression. Since no changes in methylation patterns were observed at the CpG islands of four other zebrafish genes, there must be a mechanism in zebrafish for specific methylation of the ntl CpG island.     

Classification of DNA methylation patterns in tumor cell genomes using a CpG island microarray

Our group has initiated experiments to epigenetically profile CpG island hypermethylation in genomic DNA from tissue specimens of head and neck squamous cell carcinoma (HNSCC) using a microarray of 12,288 CpG island clones. Our technique, known as a methylation-specific restriction enzyme (MSRE) analysis, is a variation of the differential methylation hybridization (DMH) technique, in that it is not an array comparison of two DNA samples using methylation-specific restriction enzymes. Instead, it is a comparison of a single DNA sample's response to a methylation-sensitive restriction enzyme (HpaII) and its corresponding methylation-insensitive isoschizomer (MspI). Estimation of the reproducibility of this microarray assay by intraclass correlation (ICC) demonstrated that in four replicate experiments for three tumor specimens, the ICC observed for a given tumor specimen ranged from 0.68 to 0.85 without filtering of data. Repeated assays achieved 87% concordance or greater for all tumors after filtering of array data by fluorescence intensity. We utilized hierarchical clustering on a population of 37 HNSCC samples to cluster tumor samples with similar DNA methylation profiles. Supervised learning techniques are now being utilized to allow us to identify associations between specific epigenetic signatures and clinical parameters. Such techniques will allow us to identify select groups of CpG island loci that could be used as epigenetic markers for both diagnosis and prognosis in HNSCC.     

Mutations in the hypoxanthine guanine phosphoribosyltransferase gene (HPRT1) in Asian HPRT deficient families

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase, (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. We have identified 34 mutations in 28 Japanese, 7 Korean, and 1 Indian families with the patients manifesting different clinical phenotypes, including two rare cases in female subjects, by the analysis of all nine exons of HPRT from the genomic DNA and reverse transcribed mRNA using PCR technique coupled with direct sequencing.     

CpG island methylation in precursors of gastrointestinal malignancies

Gastrointestinal malignancies account for about 20% of all cancers worldwide. It is widely accepted that cancer evolves through several stepwise morphological stages such as the adenoma-carcinoma and hyperplastic polyp-serrated adenoma-carcinoma sequences in colorectal cancers, and the metaplasia-dysplasia-carcinoma sequences in esophageal and gastric cancers. The morphological progression is associated with the accumulation of multiple genetic and epigenetic events. It is now recognized that epigenetic silencing of gene expression by CpG island methylation is an important alternative mechanism of inactivating tumor suppressor genes. Inflammatory conditions of the gastrointestinal and pancreaticobiliary tracts and liver such as Barrett esophagus, Helicobacter pylori gastritis, inflammatory bowel disease and viral hepatitis, are associated with increased frequency of malignancies and CpG methylation. In addition, CpG methylation is present in aberrant crypt foci and pancreatic intraepithelial neoplasia that are considered putative precursors of colon and pancreatic carcinomas, respectively. Understanding of these early genetic and epigenetic changes allows for the discoveries of potential screening, monitoring and therapeutic strategies. Targeting of the epigenetic changes that occur before the development of frank malignancy offers a potential chemopreventive strategy.