Amplification Of Specific Genes Research Articles

Duplex real-time SYBR green PCR assays for detection of 17 species of food- or waterborne pathogens in stools.

A duplex real-time SYBR Green LightCycler PCR (LC-PCR) assay with DNA extraction using the QIAamp DNA Stool Mini kit was evaluated with regard to detection of 8 of 17 species of food- or waterborne pathogens in five stool specimens in 2 h or less. The protocol used the same LC-PCR with 20 pairs of specific primers. The products formed were identified based on a melting point temperature (T(m)) curve analysis. The 17 species of food- or waterborne pathogens examined were enteroinvasive Escherichia coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enterotoxigenic E. coli, enteroaggregative E. coli, Salmonella spp., Shigella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis, Campylobacter jejuni, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Aeromonas spp., Staphylococcus aureus, Clostridium perfringens, and Bacillus cereus. No interference with the LC-PCR assay was observed when stool specimens were artificially inoculated with each bacterial species. The detection levels were approximately 10(5) food- or waterborne pathogenic bacteria per g of stool. The protocol for processing stool specimens for less than 10(4) food- or waterborne pathogenic bacteria per g of stool requires an overnight enrichment step to achieve adequate sensitivity. However, the rapid amplification and reliable detection of specific genes of greater than 10(5) food- or waterborne pathogenic bacteria per g in samples should facilitate the diagnosis and management of food- or waterborne outbreaks.

Characterisation of Leishmania donovani promastigotes resistant to hexadecylphosphocholine (miltefosine)

Leishmania donovani promastigote lines resistant to hexadecylphosphocholine (HePC, miltefosine) at 2.5, 5.0, 10.0, 20.0 and 40.0 μM were developed in vitro by continuous step-wise drug pressure. The 40 μM line was 15 times more resistant to HePC than the wild-type clone and showed cross-resistance to the ether lipid ET-18-OCH 3 (edelfosine) but not to the standard anti-leishmanial drugs. Resistance was stable up to 12 weeks in drug-free culture medium. No amplification of specific genes, including the multidrug resistance P-glycoprotein gene, could be detected in the resistant parasites.

Homogeneously staining regions in 223 breast carcinomas: cytogenetic and clinicopathological correlations.

A correlation analysis was performed on 223 breast carcinomas to assess the relationships between gene amplification, karyotypic and clinicopathological features. Homogeneously staining region (HSR) is the most frequent form of amplification found in breast cancer. HSR-containing tumours accounted for 60% of the cases. Although up to 40% of tumours with slightly altered karyotype contained HSRs, an excess of HSRs was found within the tumours whose karyotype showed the highest rates of rearranged chromosomes. HSRs were also found to be particularly frequent in small tumours of high histological grade and with a low expression of progesterone receptors. An excess of HSRs seems to be observed in younger patients, however, significant correlation could be demonstrated only for patients below 55 years and below 60 years, compared with older ones. With a 120-month follow-up for 152 patients, a significant association between the presence of HSRs and a shortened overall survival was observed. Altogether, the presence of HSRs appears to be a good indicator of poor prognosis. Further studies are needed to determine whether amplification of specific genes or cell ability to amplify is the most important parameter for tumour progression.

A technique for eliminating allele specific amplification failure during DNA amplification of heterozygous cells for preimplantation diagnosis.

Advances in techniques of molecular biology have made possible the amplification of specific genes from single cells. This has a major clinical application in preimplantation diagnosis of monogenic disorders. However, the incidence of allele specific amplification failure (allele drop out) in heterozygous single cells can lead to misdiagnosis and the transfer of affected embryos. Few studies have been done to investigate the actual cause of allele drop out, although some investigators have succeeded in reducing but not eliminating it. Here we report the efficiency of amplifying both alleles in heterozygous cells lysed according to two different protocols. A total of 177 heterozygous cells from carriers of cystic fibrosis (CF) and haemoglobin C (HbC) were lysed using two different lysis buffers. Interestingly none of the cells that were lysed with sodium dodecyl sulphate/proteinase K showed any example of allele specific amplification failure whereas in those lysed by KOH/dithiothreitol it was present in 17.6 and 4.7% of the CF and HbC cells respectively. Our results suggest that the phenomenon of allele specific amplification failure is at least in part dependent on the lysis buffer used.

Fluorescence in situ hybridization

Spectacular advances in the use of fluorescence in situ hybridization (FISH) for the visualisation of specific DNA sequences in metaphase chromosomes and interphase cells have been made over the last few years making the technique a useful tool in clinical research. One of the biggest impacts has been in the field of detection and diagnosis of human malignancies. Chromosomal translocations, deletions, amplification of specific genes and changes in chromosome number can all be detected in the non-dividing interphase nucleus using probes ranging from whole chromosome 'paints' to individual gene specific probes. Gene mapping has also benefited from advances in FISH technology. Target sequences ranging from one to several hundred kilobases can be visualised on metaphase chromosomes and spatial resolution in interphase cells permits the ordering of two probes over a distance as small as 1000 base pairs. The potential uses of FISH continue to increase with each new technical innovation.

Polymerase chain reaction in situ: Intracellular amplification and detection of HIV-1 proviral DNA and other specific genes

The ability to detect a single copy of a specific gene in situ has many advantages and multiple applications in molecular biology, pathology and cell biology. We report here a unique, highly sensitive and specific technique which can be utilized to detect a single copy of human immunodeficiency virus type I (HIV-1) provirus and other genes, at the single cell level, by in situ amplification of a portion of a gene sequence. In this method, a polymerase chain reaction (PCR) can be carried out in situ, in fixed cells, on specially designed glass slides. After amplification one can detect the amplified signals by the in situ hybridization method, utilizing either biotinylated probes or 32P-labelled probes. The early molecular events in the retroviral life-cycle of HIV-1, in specific target cells, are demonstrated utilizing in situ PCR. The techniques utilized in this procedure and various potential uses of this methodology are described.

Clonal variation of expression of the genes coding for plasminogen activators, their inhibitors and the urokinase receptor in HT1080 sarcoma cells.

The human sarcoma cell line HT1080 was found, by in situ hybridization, to consist of cells expressing various levels of urokinase (uPA) and tissue type (tPA) plasminogen activator (PA) suggesting clonal variation of expression of these genes. Colonies originating from single HT1080 cells were, therefore, established and screened for PA activity using a fibrin agarose overlay. Colonies inducing lysis (clone C+ and H+) or no lysis (clones B- and M-) were isolated and tested for mRNA levels of uPA, tPA, uPA receptor (uPAR) and the 3 PA inhibitors (PAI), PAI-1, PAI-2 and protease-nexin I. The different clones revealed considerable variation of expression of the different PA and PAI genes, with lysis-inducing clones expressing mainly the PA genes, whereas non-lysing clones demonstrated higher expression of the PAI genes. Amplification or loss of specific genes was excluded by Southern blotting. The protein levels of cellular and secreted PA and PAI determined by ELISA and Western blots demonstrated a pattern similar to that observed for PA and PAI mRNA concentrations, suggesting clonal differences either on the level of transcription or in RNA processing and/or stability. Due to complex interactions between PA and PAI, neither mRNA nor protein levels of the different genes were predictive for the amount of functional PA activity present in the supernatant or on the cell surface of the different clones. Receptor-bound uPA activity was found to be considerably higher in lysis-inducing than in non-lysing clones and the activity was dependent on neutralization by PAI-1 rather than on the level of uPAR mRNA.

DNA sequences amplified in cancer cells: an interface between tumor biology and human genome analysis

There is growing evidence that amplification of specific genes is associated with tumor progression. While several proto-oncogenes are known to be activated by amplification, it is clear that not all the genes involved in DNA amplification in human tumors have been discovered. Our approach to the identification of such genes is based on the ‘reverse genetics’ methodology. Anonymous amplified DNA fragments are cloned by virtue of their amplification in a given tumor. These sequences are mapped in the normal genome and hence define a new genetic locus. The amplified domain is isolated by long-range cloning and analyzed along three lines of investigation: new genes are sought that can explain the biological significance of the amplification; the structure of the domain is studied in normal cells and in the amplification unit in the cancer cell; attempts are made to identify molecular probes of diagnostic value within the amplified domain. This application of genome technology to cancer biology is demon-strated in our study of a new genomic domain at chromosome 10q26 which is amplified specifically in human gastric carcinomas.

DNA amplification in genetic toxicology

Chemical compounds can cause amplifications of specific DNA sequences. DNA amplification may result in an enhanced production of gene products which help cells to cope with the chemicals. This may lead to a resistance of the cells toward the agent. Additionally, initiation of transformation or progression of transformed cells to tumorigenicity may also involve DNA amplification. Therefore, it is of interest to study the potential of chemicals to induce DNA amplification. This report focuses on the investigation of a variety of chemicals in 2 systems with which the amplification of viral DNA is measured within cells in culture. One model system comprises the measurement of SV40 DNA content in an SV40-transformed Chinese hamster cell line following chemical treatment. Antitumor agents as well as genotoxic and non-genotoxic compounds were studied in this system as a first step to determine the DNA amplification-inducing potential of a variety of differently acting chemical compounds. Also, a novel assay based on adeno-associated virus infection of cells is described. This system may offer the possibility of studying DNA amplification in a variety of different target cells. For the future, the need is stressed to develop and analyze versatile to study amplification of specific target genes in untransformed cells and in tumor cells.

Over-replication of DNA in S phase Chinese hamster ovary cells after DNA synthesis inhibition.

Agents that inhibit DNA synthesis increase the frequency of methotrexate resistance and gene amplification in cultured mammalian cells. Chinese hamster ovary cells blocked with hydroxyurea rereplicated dihydrofolate reductase gene sequences within a single cell cycle upon release from the block (Mariani, B.D., and Schimke, R.T. (1984) J. Biol. Chem. 259, 1901-1910). Perturbation of DNA synthesis was postulated to result in misfiring of replicon initiation, subsequent over-replication of DNA sequences, and amplification of specific genes. To test this hypothesis, we have exposed Chinese hamster ovary cells pulsed with bromodeoxyuridine to three agents that inhibit DNA synthesis and enhance gene amplification: UV irradiation, hydroxyurea, and aphidicolin. After release from the block, the progression of cells throughout the cell cycle was analyzed by flow cytometry through simultaneous measurement of total cellular DNA content and bromodeoxyuridine-labeled DNA. Although the cell cycle effects varied depending on the agent used for the block, in all cases a subset of cells that were in S phase at the time of the block exhibited DNA histograms with greater than 4C DNA content at various times after release and prior to cell division. Cells with the excess DNA were approximately 10-fold more resistant to methotrexate compared to treated cells with normal DNA content or untreated cells. Therefore, cells in S phase at the time of the block produce excess DNA per cell prior to division, and this over-replicated DNA may be relevant to gene amplification and drug resistance.

Oncogene Amplification in Tumor Cells

Publisher Summary This chapter summarizes the third mechanism of oncogene activation—oncogene amplification. It illustrates the various ways by which the oncogenic potential of different proto-oncogenes can be activated. Because of the involvement of myc oncogenes in amplifications in a variety of tumors, other lesions that also activate the cellular oncogene c-myc and the aspects of the normal regulation of this oncogene are also described. Since its discovery in drug-resistant eukaryotic cells, somatic amplification of specific genes has been implicated in an increasing variety of adaptive responses of cells to environmental stresses. Experimental work on drug-resistant cells has shown that in the absence of a selection pressure (drug), double minute chromosomes (DMINs) and the amplified genes that they contain are lost, whereas amplified DNA in the form of homogeneously staining regions (HSRs) is retained in the cells. If DMINs and HSRs contain amplified genes that encode drug-resistant or growth-stimulating protein products, it would follow that the more stable chromosomal form, the HSR, confers a greater selective growth advantage for cells. Although DMINs and HSRs have been described predominantly in tumor cells selected for the resistance to cytotoxic drugs, it is also clear that DMINs and HSRs may be present in cancer cells before the start of therapy.

Acquisition of multiple nuclei and the activity of DNA polymerase alpha and reinitiation of DNA replication in terminally differentiated adult cardiac muscle cells in culture.

Terminally differentiated ventricular cardiac muscle cells isolated from the adult rat and maintained in cell culture have been observed to acquire multiple nuclei. In one cultured myocyte as many as 10 nuclei have been counted. Apparently, these multiple nuclei are formed by DNA replication followed by karyokinesis; the cells must then fail to complete mitosis and divide. To investigate whether DNA synthesis was occurring, the cells were cultured in the presence of [3H]thymidine and then processed for autoradiography. Mononucleated, binucleated, and multinucleated cells incorporate [3H]thymidine into DNA as evidenced by the high concentration of silver grains over their nuclei. Peak periods of incorporation were observed to occur at 10- to 12-day intervals; at 11, 23, and 33 days after initially placing the cells in culture. When the cells were maintained in the presence of [3H]thymidine continuously from Day 7 to Day 17 of culture, 23% of the cells became labeled. If the cells were cultured continuously for 30 days in the presence of [3H]thymidine, from Day 10 to Day 40, 56% of the cells were labeled. Isopycnic gradient analysis indicates that this thymidine incorporation was into DNA that was being replicated semiconservatively; these experiments did not eliminate the possibility, however, that this incorporation was due to amplification of specific genes, such as those coding for the contractile proteins. The activity of DNA polymerase alpha also returns to these cells. These studies demonstrate that the terminally differentiated mammalian ventricular cardiac muscle cell, previously thought to have permanently lost the capacity to replicate DNA during early development, is able to reinitiate semiconservative DNA replication when grown in culture.

DNA-puffing patterns in the salivary glands of Trichosia pubescens (Diptera-Sciaridae)

The puffing pattern in polytene chromosomes from salivary glands of fourth-instar larvae of Trichosia pubescens was studied. It was found that the puffing pattern is rather constant during most of larval life but changes continuously in a precise sequential order during the period preceding pupation. During this period, characterized by drastic changes in the puffing pattern, amplification of specific genes and expansion of the DNA-puffs occur. The pattern of protein synthesis in the salivary gland has also been studied by means of SDS-polyacrylamide gel electrophoresis and fluorography. It changes drastically and continuously during the phases preceding pupation. These changes in protein synthesis could be correlated with the changes in the puffing pattern, essentially with the activity of the DNA-puffs.

Regulated expression of mammalian genes: globin and immunoglobulin as model systems.

Most general models put forward to explain the molecular basis of differentiation invoke either the amplification of specific genes, their transcriptional activation, some form of post-transcriptional control, or a combination of these mechanisms. We wished to distinguish between these experimentally and have selected two models for this purpose, erythropoiesis and immunogenesis. Both are derived from the hematopoietic system. Erythropoiesis, as observed in the adult, represents a late stage in the process of differentiation. This process is continuous during the lifetime of the individual. Protein synthesis during the penultimate stage in the development of the erythrocyte, the reticulocyte, involves the almost exclusive synthesis of globin, providing a convenient biochemical marker for the isolation of a specific mRNA and gene product. Immunogenesis, on the other hand, appears to be a far more complex process. An especially intriguing and unique feature of the immunogenic response is the necessity of accounting, in genetic terms...