Chromosome-mediated transfer and amplification of an altered mouse dihydrofolate reductase gene

We have conferred methotrexate resistance on mouse 3T6 fibroblasts by chromosome-mediated transfer of an altered dihydrofolate reductase gene encoding a highly methotrexate-insensitive enzyme. The methotrexate-resistant 3T6 cell line from which the chromosomes were prepared contains multiple copies of the altered dihydrofolate reductase gene, all of which appear to reside on double-minute chromosomes. Transformants selected at 0.2 M methotrexate contain 10–20 times more of the transferred altered gene than of the resident normal gene. The altered genes are associated with double-minute chromosomes and are permanently lost following growth of the transformants in the absence of methotrexate. Growth of the transformants in increasing concentrations of methotrexate leads to the emergence of cells which have accumulated double-minute chromosomes and which have amplified only the transferred dihydrofolate reductase gene.     

Pseudogenes of the human HPRT1 gene

Entrez Gene lists four HPRT1 gene pseudogenes (HPRTP1, HPRTP2, HPRTP3, and HPRTP4) mapping to chromosomes 3, 5, 11q, and 11q, respectively, as originally reported by Patel et al. in 1984 (Patel PI, et al. 1984 Somat Cell Mol Genet 10:483-493). However, the Entrez Gene reports for three of the four pseudogenes (HPRTP1, HPRTP3, and HPRTP4) are currently empty. A BLAST search of both GenBank (Homo sapiens) and the human genome found the chromosome 5 associated HPRTP2 sequence and a single chromosome 11q sequence (HPRTP3 or HPRTP4?). This chromosome 11 sequence had a unique 7.2 kb insert, which may explain why it originally appeared to be two separate pseudogenes. No evidence of a chromosome 3 associated sequence was found; however, a sequence highly homologous to HPRT1 was located on chromosome 4. All of these sequences are intronless processed pseudogenes. Lastly, a sequence highly homologous to HPRT1 exon 8 was found on chromosome 10. This homologous sequence was exactly exon 8 of a gene designated PRTFDC1, for phosphoribosyl transferase domain containing 1. This gene with unknown function is almost completely homologous to HPRT1 in exon structure (except for a 21 bp (seven amino acid) insertion in exon 1) and 68% homologous in amino acid sequence.     

Investigation of chromosome-mediated gene transfer using the HPRT region of the human X chromosome as a model

A panel of over 50 hybrid cells containing varying portions of the long arm of the human X chromosome have been obtained by chromosome-mediated gene transfer (CMGT) of human chromosomes to mouse cells deficient in HPRT. This panel is used to investigate the size and integrity of transfected human chromosome fragments and also to examine the effect of including a selectable DNA plasmid in the transfection mix. Chromosomal rearrangements are found to be generated in the chromosome transfer process, and the human X centromeric region is detected in the transfected cells at an unusually high frequency. Extensive lengths of X chromosome DNA are transferred intact, suggesting potential uses of CMGT in cloning large genes and loci for which only the chromosomal map position is known.     

Organization of the HPRT gene and related sequences in the human genome

Comparative Southern hybridization of cDNA probes to DNA from cells carrying either one or four X chromosomes has been used to distinguish sequences derived from the functional locus for hypoxanthine-guanine phosphoribosyltransferase (HPRT) on the X chromosome from four independent HPRT-like autosomal sequences in the human genome. Subfragments of cDNA were then used to orient fragments from the HPRTlocus with respect to the mRNA sequence. The chromosomal origin of each of the autosomal sequences was determined by Southern analysis using DNA from a panel of human-Chinese hamster somatic cell hybrids. Two of the HPRT-like sequences were localized to chromosome 11, the third to chromosome 3, and the fourth to the region between p13 and q11 on chromosome 5. Three of these four autosomal sequences were isolated from genomic recombinant libraries and subcloned fragments from each were used as probes to study restriction fragment length polymorphisms (RFLP) at these loci. A RFLP for MspIwas found at the HPRT-like locus on chromosome 5 with a 1.3-kb major allele (frequency=0.8) and a 3.6-kb minor allele (frequency=0.2).     

Characteristics of the interferon system of an embryonal carcinomal cell are recessive in intraspecific somatic cell hybrids

We have obtained hybrids of PCC4-aza 1, a mouse embryonal carcinoma stem cell line, and two different thymidine kinase deficient mouse cell lines. We have examined the ability of the parental and hybrid cells to produce interferon after infection with the Newcastle Disease virus and to enter the antiviral state when treated with mouse interferon. The interferon system of PCC4-aza 1 is inactive; this characteristic is recessive in the hybrids obtained.     

Breakpoints and junctional regions of intragenic deletions in the HPRT gene in human T-cells

DNA sequences of the deletion breakpoints of 24 human T-lymphocytehprt gene mutations are reported. These independent deletions ranged in size from 18 to 15655 base pairs. Seven of the 21 in vivo mutations arose in normal adults, three in normal children, eight in radioimmunotherapy patients and three in platinum chemotherapy patients. One in vitro mutation was isolated after 93cGy radon exposure and two after 300cGy radiation. The breakpoints were found to be non-random and a cluster of small deletions in exon 6 is reported. Ten of the mutations has 2–5bp direct repeats at the breakpoints. There was no excess of deletion-associated motifs over that expected by chance. Some breakpoints do occur at consensus topoisomerase II cleavage sites and the centromeric end of a Donehower sequence occurs exactly at a telomeric breakpoint. Three mutants had breakpoints at hairpins expected by the model of Glickman and Ripley (1).     

Horizontal gene transfer in human pathogens

Abstract

Horizontal gene transfer has a tremendous impact on the genome plasticity, adaptation and evolution of bacteria. Horizontally transferred mobile genetic elements are involved in the dissemination of antibiotic resistance and virulence genes, thus contributing to the emergence of novel “superbugs”. This review provides update on various mechanisms of horizontal gene transfer and examines how horizontal gene transfer contributes to the evolution of pathogenic bacteria. Special focus is paid to the role horizontal gene transfer plays in pathogenicity of the emerging human pathogens: hypervirulent Clostridium difficile and Escherichia coli (including the most recent haemolytic uraemic syndrome outbreak strain) and methicillin-resistant Staphylococcus aureus (MRSA), which have been associated with largest outbreaks of infection recently.     

Gene targeting using a mouse HPRT minigene/HPRT-deficient embryonic stem cell system: Inactivation of the mouseERCC-1 gene

A convenient system for gene targeting that uses hypoxanthine phosphoribosyltransferase (HPRT) minigenes as the selectable marker in HPRT-deficient mouse embryonic stem (ES) cells is described. Improvements to the expression of HPRT minigenes in ES cells were achieved by promoter substitution and the provision of a strong translational initiation signal. The use of minigenes in the positive-negative selection strategy for gene targeting was evaluated and the smaller minigenes were found to be as effective as a more conventional marker—the herpes simplex virus thymidine kinase gene. Minigenes were used to target the DNA repair gene ERCC-1 in ES cells. A new HPRT-deficient ES cell line was developed that contributes with high frequency to the germ line of chimeric animals. The ability to select for and against HPRT minigene expression in the new HPRT-deficient ES cell line will make this system useful for a range of gene-targeting applications.     

Nitric oxide-induced mutations in the HPRT gene of human lymphoblastoid TK6 cells and in Salmonella typhimurium

Characterization of mutations induced by NO in different experimental systems will facilitate elucidation of mechanisms underlying its genotoxicity. The mutagenic specificity of NO in human cells is of particular interest in view of its potential role in inflammation-associated carcinogenesis. We compared mutagenesis in human lymphoblastoid TK6 cells and in Salmonella typhimurium induced by exposure to NO delivered into the medium at rates approximating its production by activated macrophages. Exposure of TK6 cells continuously for 60 min decreased viability by 88%, and survivors exhibited a sixfold increase in mutant fraction in the hprt gene. Independent mutants were isolated and mutations characterized by RT-PCR and DNA sequencing. Among a total of 68 mutants analyzed, RT-PCR products were obtained in 41 (60%), and cDNA sequencing revealed that 26 (63%) of them contained mutations located in the hprt coding region. Base substitutions were present in 18 mutants, 12 occurring at A:T base pairs. Seven mutants contained deletions of 1-27 bp and one a 13-bp insertion; the 15 remaining RT-PCR products contained whole-exon deletions, 14 involving single exons. Six tester strains of S. typhimurium, each containing one of the six possible point mutations in the target codon of a gene in the histidine biosynthetic pathway, were similarly treated with NO and induction of mutation was detected by reversion to histidine auxotrophy. Significant increases were observed in frequencies of each of the six possible base mutations, with the highest occurring in G:C --> A:T transitions. The pattern of NO-induced hprt mutations in TK6 cells was similar to a recently published spectrum in spontaneous mutants, suggesting that reactive species derived from NO may contribute to spontaneous mutagenesis of the endogenous hprt gene in human cells.     

人脂联素基因在昆虫杆状病毒表达系统中的表达

目的利用杆状病毒表达系统探讨人脂联素(ADPN)基因在Sf9昆虫细胞中的高效表达。方法利用PCR方法扩增人脂联素基因,与pFastBac1质粒连接,转化入含有穿梭载体bacmid的大肠杆菌DH10Bac中,筛选发生转座作用的重组穿梭载体Bacmid-ADPN并转染Sf 9昆虫细胞,使其表达重组杆状病毒,经SDS-PAGE、Western blot检测表达产物。结果重组杆状病毒感染的Sf 9细胞形态变化明显,能够表达出与脂联素多克隆抗体相结合的蛋白,蛋白分子质量约为30 ku。结论人脂联素基因在真核细胞中成功得到表达,为进一步研究其生物学活性和作用机制奠定基础。     

Efficient gene transfer into the human natural killer cell line, NKL, using the Amaxa nucleofection system

Natural killer (NK) cell lines are useful for studying facets of NK cell biology. Such cell lines are notoriously difficult to transfect by traditional methods, a fact that has hampered NK cell biology studies for a long time. To overcome this, we investigated the use of the Amaxa nucleofection system that directly transfers DNA into the nucleus of the cell. This technology has revolutionized transfection studies with heretofore relatively transfection resistant cell types such as T cells, B cells and dendritic cells. Despite these advances, NK cells and NK cell lines have remained relatively resistant to transfection, including nucleofection. In this study we employed cDNA for SHP1 and various Rab proteins cloned in enhanced green/yellow fluorescent protein (EGFP/EYFP) expression plasmids for transient transfections into NKL cells. The expression of EGFP/EYFP fusion proteins was analyzed by flow cytometry, immunoblot and confocal microscopic analyses. We achieved 40-70% transfection efficiency with high levels of expression in this cell line with 85-90% viability. The method used in this report proves to be far superior to existing methods for delivering DNA into this well studied NK cell line and, consequently, provides new experimental opportunities.     

Molecular analysis of HPRT1(+) somatic cell hybrids derived from a carrier of an HPRT1 mutation responsible for Lesch-Nyhan syndrome

Heterozygous carriers of HPRT1 mutations responsible for Lesch-Nyhan syndrome can be detected by analysis of somatic cell hybrids derived from peripheral blood lymphocytes and Hprt1-negative cells of rodent origin followed by selection in culture medium containing hypoxanthine, aminopterine, and thymidine (HAT). The parental origin of the X chromosome containing the normal HPRT1 allele in HPRT1(+) hybrid cell lines can be determined by molecular haplotyping attributable to highly polymorphic X-linked markers. We used this procedure to study a presumed carrier whose paternal active X chromosome always segregated in the cell hybrids derived from her. Conversely, her maternal X chromosome was systematically absent in most cell hybrids, or when present, it was inactive and coexisted with an active, paternal X chromosome. These results clearly demonstrated that the proband was a heterozygous carrier of a mutation responsible for HPRT1 deficiency.     

Retroviral transfer of HSV1-TK gene into human lung cancer cell line

We used a recombinant retrovirus as one of the potential vectors for human gene therapy to transfer a drug sensitivity gene into human lung cancer cells. The gene encoding the thymidine kinase (TK) of herpes simplex virus type 1 (HSV1) was used as the drug sensitivity gene. The antiherpes drugs acyclovir (ACV) and ganciclovir (GCV) were chosen to test the HSV1-TK activity transferred into the human lung cancer cell lines. The rationale for this approach was that ACV and GCV are nucleoside analogs specifically converted by HSV1-TK to a toxic form capable of inhibiting DNA synthesis or disrupting cellular DNA replication. The results obtained from our experiments demonstrate that the retroviral vector-mediated HSV1-TK gene transfer leads to ACV- and GCV-dependent cytotoxicity in human lung cancer cell lines, including both small-cell carcinoma and nonsmall-cell carcinoma. Although the gene transfer of HSV1-TK gene into tumor cells would be one model for gene therapy to control lung cancer, further investigations are necessary for the proper choice of the therapeutic gene and vector targeting such as tumor cell specific delivery of the gene or tumor cell specific expression of the transduced gene.Abbreviations ACV Acyclovir - GCV Ganciclovir - HSV1 Herpes simplex virus type 1 - LTR Long terminal repeat - TK Thymidine kinase     

Transduction of the CHOaprt gene into mouse L cells using an adeno-5/APRT recombinant virus

An adenovirus-5 recombinant virus Adapt1 carrying the Chinese hamster ovary (CHO) adenine phosphoribosyltransferase (aprt) gene was constructed by insertion of a 2.5-kb fragment containing the complete CHOaprt structural gene linked to a Moloney murine sarcoma virus (MSV) promoter into the E3 region of adenovirus-5. The CHOaprt gene was in the opposite orientation to the adenovirus E3 promoter. Mouse Lapt^– tk^– (LAT) cells expressed the CHOaprt gene when infected with the virus, even at low MOI (O.1). APRT activity was detectable from approximately 20 h postinfection. At a low frequency, LAT cells were transformed to aprt⁺, and four stable transductants were selected in adenine, azaserine (AA) medium. Such cells expressed APRT at 50% wild-type activity and the enzyme was shown to be CHO APRT by starch gel electrophoresis. DNA was isolated from the transductants and probed with CHOaprt-specific DNA and with viral DNA probes. The results indicated that the CHOaprt gene was integrated into the LAT cells at a site other than mouseaprt. Although neighboring viral sequences were integrated and maintained in the transductants, viral sequences further upstream and downstream of theaprt gene were absent.F.L.G. is a Terry Fox Cancer Research Scientist.     

Adenoviral vector-mediated gene transfer for human gene therapy

Human gene therapy promises to change the practice of medicine by treating the causes of disease rather than the symptoms. Since the first clinical trial made its debut ten years ago, there are over 400 approved protocols in the United States alone, most of which have failed to show convincing data of clinical efficacy. This setback is largely due to the lack of efficient and adequate gene transfer vehicles. With the recent progress in elucidating the molecular mechanisms of human diseases and the imminent arrival of the post genomic era, there are increasing numbers of therapeutic genes or targets that are available for gene therapy. Therefore, the urgency and need for efficacious gene therapies are greater than ever. Clearly, the current fundamental obstacle is to develop delivery vectors that exhibit high efficacy and specificity of gene transfer. Recombinant adenoviruses have provided a versatile system for gene expression studies and therapeutic applications. Of late, there has been a remarkable increase in adenoviral vector-based clinical trials. Recent endeavors in the development of recombinant adenoviral vectors have focused on modification of virus tropism, accommodation of larger genes, increase in stability and control of transgene expression, and down-modulation of host immune responses. These modifications and continued improvements in adenoviral vectors will provide a great opportunity for human gene therapy to live up to its enormous potential in the second decade.     

Efficient modification of the APRT gene by FLP/FRT site-specific targeting

The FLP/FRT site-specific recombination system was established and characterized at the APRT gene in CHO cells. Targeting frequencies with FLP-stimulation were about 1 to 5×10^–5, which were 6–22-fold above gene targeting frequencies in the absence of FLP. Fifty two APRT⁺ cell lines were analyzed by Southern blotting: 56% were FLP-targeted integrants; 33% were APRT target convertants; 11% gave undefined patterns. In separate experiments we first enriched for integrants by screening for two additional markers carried on the targeting vector; 18 of 19 (95%) of the resulting cell lines were integrants. Intrachromosomal site-specific recombination was tested by reexposing integrants to FLP. Intrachromosomal popouts were stimulated over 200-fold, while homologous recombination in an adjacent interval was unchanged. The utility of this system was demonstrated by one-step FLP targeting to generate chromosomal substrates for homologous recombination, and by a two-step, FLP-and-run procedure to construct a chromosomal substrate for illegitimate recombination.     

Molecular analysis of APRT deficiency in mouse P19 teratocarcinoma stem cell line

We have used four gene probes specific for mouse chromosome 8, including adenine phosphoribosyltransferase (aprt), to demonstrate that the P19 teratocarcinoma stem cell line contains two disinct chromosome 8 homologs. One represents the common laboratory mouse C3H (Mus musculus domesticus) homolog while the second homolog was presumably contributed by a feralMus musculus musculus animal. Six cell lines with APRT heterozygous deficiencies were isolated from P19 subclones. A molecular analysis of these heterozygotes demonstrated that three arose by deletion of theMus musculus musculus aprt allele and three arose byaprt gene inactivation. APRT homozygous deficient cell lines were isolated from both classes of heterozygote; most contained little or no detectable APRT activity. When the heterozygous deficiency was due to deletion of theMus musculus musculus aprt allele, the most frequent event yielding homozygous deficient cell lines was associated with loss of heterozygosity for all tested markers on theMus musculus domesticus homolog indicating chromosome los. In contrast, when the initial event resulting in APRT heterozygous deficiency was gene inactivation, homozygotes arose predominantly from gene deletion or a second inactivation event. These results suggest a potential relationship between the first- and second-step events resulting in APRT deficiencies.     

Normal HPRT coding region in complete and partial HPRT deficiency

Lesch-Nyhan syndrome is an X-linked recessive inborn error of metabolism due to a virtually complete lack of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity (OMIM 300322). Partial deficiency of HPRT (OMIM 300323) is characterized by the effects of excess uric acid synthesis and a continuum spectrum of neurological manifestations, without the manifestations of full-blown Lesch-Nyhan syndrome. Both diseases have been associated with mutations in the HPRT gene. These mutations are heterogeneous and disperse throughout the entire HPRT gene. In 2005 Dawson et al. described, for the first time, an individual with gout in whom HPRT deficiency appeared to be due to a defect in gene regulation. In the present study we present four patients with partial HPRT deficiency and one patient with Lesch-Nyhan syndrome who showed a normal HPRT coding sequence and markedly decreased HPRT mRNA expression. This is the first report of a patient with Lesch-Nyhan syndrome due to a defect in HPRT gene expression regulation.     

Southern blot analysis of T-cell receptor gene rearrangements in cynomolgus monkeys,and identification of a progenitor cell HPRT mutation

Increases in peripheral blood T-lymphocyte HPRT mutant frequency may reflect either a number of independent HPRT gene mutational events or clonal proliferation of a single HPRT mutant. Sequence analysis of HPRT mutations in conjunction with T-cell receptor (TCR) gene rearrangement pattern analysis can distinguish these possibilities. Our laboratory previously characterized a nonhuman primate model for in vivo mutation studies using the clonal HPRT mutation assay. In the present study we report the use of probes for human TCR B and y genes to characterize TCR rearrangements in cynomolgus monkeys. Together, these methods were used to examine a monkey which exhibited a mean spontaneous HPRT mutant frequency (MF) of 16.4 × 10^?6, compared to the normal mean MF of 3.03 × 10^?6. The elevated MF resulted from the occurrence of a single HPRT mutation in a lymphocyte progenitor cell or stem cell, since T-cell clones isolated from the monkey exhibited a G to T transversion at base pair 539 in the HPRT coding region, and had unique rearrangements of TCR y along with an apparent germline TCR B configuration. In a preliminary in vivo mutation study, the animal was treated with the investigational potent mutagen and antitumor agent adozelesin (U-73975). No increase in HPRT mutant frequency was observed. The HPRT mutant clones isolated after treatment showed rearrangement of both TCR y and B genes. Possible explanations for these findings are discussed. © 1995 Wiley-Liss, Inc.