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PubMed2005

Selection of control genes for quantitative RT-PCR based on microarray data.

Use of internal reference gene(s) is necessary for adequate quantification of target gene expression by RT-PCR. Herein, we elaborated a strategy of control gene selection based on microarray data and illustrated it by analyzing endomyocardial biopsies with acute cardiac rejection and infection. Using order statistics and binomial distribution we evaluated the probability of finding low-varying genes by chance. For analysis, the microarray data were divided into two sample subsets. Among the first 10% of genes with the lowest standard deviations, we found 14 genes common to both subsets. After normalization using two selected genes, high correlation was observed between expression of target genes evaluated by microarray and RT-PCR, and in independent dataset by RT-PCR (r = 0.9, p < 0.001). In conclusion, we showed a simple and reliable strategy of selection and validation of control genes for RT-PCR from microarray data that can be easily applied for different experimental designs and tissues.

Algorithms↗
PubMed2005

The maize Knotted1 gene is an effective positive selectable marker gene for Agrobacterium-mediated tobacco transformation.

We have assessed the use of a homeobox gene knotted1 (kn1) from maize as a selectable marker gene for plant transformation. The kn1 gene under the control of cauliflower mosaic virus 35S promoter (35S::kn1) was introduced into Nicotiana tabacum cv. Xanthi via Agrobacterium-mediated transformation. Under nonselective conditions (without antibiotic selection) on a hormone-free medium (MS), a large number of transgenic calli and shoots were obtained from explants that were infected with Agrobacterium tumefaciens LBA4404 harboring the 35S::kn1 gene. On the other hand, no calli or shoots were produced from explants that were infected with an Agrobacterium strain harboring pBI121 (nptII selection) or from uninfected controls cultured under identical conditions. Relative to kanamycin selection conferred by nptII, the use of kn1 resulted in a 3-fold increase in transformation efficiency. The transgenic status of shoots obtained was confirmed by both histochemical detection of GUS activity and molecular analysis. The results presented here suggest that kn1 gene could be used as an effective alternative selection marker with a potential to enhance plant transformation efficiency in many plant species. With kn1 gene as a selection marker gene, no antibiotic-resistance or herbicide-resistance genes are needed so that potential risks associated with the use of these traditional selection marker genes can be eliminated.

Caulimovirus↗
PubMed2005

Targeting gene expression selectively in cancer cells by using the progression-elevated gene-3 promoter.

One impediment to effective cancer-specific gene therapy is the rarity of regulatory sequences targeting gene expression selectively in tumor cells. Although many tissue-specific promoters are recognized, few cancer-selective gene promoters are available. Progression-elevated gene-3 (PEG-3) is a rodent gene identified by subtraction hybridization that displays elevated expression as a function of transformation by diversely acting oncogenes, DNA damage, and cancer cell progression. The promoter of PEG-3, PEG-Prom, displays robust expression in a broad spectrum of human cancer cell lines with marginal expression in normal cellular counterparts. Whereas GFP expression, when under the control of a CMV promoter, is detected in both normal and cancer cells, when GFP is expressed under the control of the PEG-Prom, cancer-selective expression is evident. Mutational analysis identifies the AP-1 and PEA-3 transcription factors as primary mediators of selective, cancer-specific expression of the PEG-Prom. Synthesis of apoptosis-inducing genes, under the control of the CMV promoter, inhibits the growth of both normal and cancer cells, whereas PEG-Prom-mediated expression of these genes kills only cancer cells and spares normal cells. The efficacy of the PEG-Prom as part of a cancer gene therapeutic regimen is further documented by in vivo experiments in which PEG-Prom-controlled expression of an apoptosis-inducing gene completely inhibited prostate cancer xenograft growth in nude mice. These compelling observations indicate that the PEG-Prom, with its cancer-specific expression, provides a means of selectively delivering genes to cancer cells, thereby providing a crucial component in developing effective cancer gene therapies.

Animals↗
PubMedDate not supplied

Microarray data simulator for improved selection of differentially expressed genes.

The development of microarray technology has allowed researchers to measure expression levels of thousands of genes simultaneously. Analysis of these data requires the best normalization and statistical approaches to account for the biological and technical variability inherent in the technique. To approach this problem we have developed a publicly available simulator of microarray hybridization experiments that can be used to help assess the accuracy of bioinformatic tools in discovering significant genes. After analyzing microarray hybridization experiments from over 50 samples, an estimate of various degrees of technical and biological variability was obtained. This information was used to develop a simulator of microarray hybridization data which modeled "normal tissue samples" and "diseased tissue samples" with known, defined, changes in gene expression (a "gold standard"). The data derived from the simulator were then used to evaluate the true positive and false negative rates of several normalization procedures and gene selection techniques. We found that the type of normalization approach used was an important aspect of data analysis. Global normalization was the least accurate approach. Evaluation of gene selection techniques showed that "Significance analysis of microarrays" (SAM) and "Patterns of Gene Expression" (PaGE) were more accurate than simple t-test analysis. We provide access to the microarray hybridization simulator as a public resource for biologists to further test new emerging genomic bioinfomatic tools.

Computer Simulation↗
PubMed1995

Blasticidin S deaminase gene (BSD): a new selection marker gene for transformation of Arabidopsis thaliana and Nicotiana tabacum.

Arabidopsis thaliana and Nicotiana tabacum were transformed to blasticidin S (BS) resistance with BSD (the BS deaminase gene from Aspergillus terreus) using the Agrobacterium-mediated transformation method. Expression of BSD allowed direct selection of transformants by the fungicide, and both kinds of transgenic plants showed high level of resistance phenotype at 100 ppm of BS sprayed on the leaves. Using Botrytis cinerea, the causal fungus of gray mold disease, it was exemplified that application of BS could control the disease in transgenic tobacco with negligible phytotoxicity.

Aminohydrolases↗
PubMed1997

Selective growth hormone/placental lactogen gene transcription and hormone production in pre- and postmenopausal human ovaries.

In addition to effects of pituitary-derived gonadotropins, human GH modulates and regulates intraovarian reproductive processes in a dose-dependent manner via the endocrine GHRH/GH/insulin-like-growth-factor I (IGF-I) axis. Based on increasing evidence that ovarian regulation involves a complex system of putative para/autocrine factors, we investigated the possibility of gene-selective intraovarian GH/placental lactogen (PL) hormone production, with emphasis on differences between pre- and postmenopause. Analysis of both premenopausal (n = 8) and postmenopausal (n = 10) ovarian-derived messenger ribonucleic acid by reverse transcription-PCR, which amplifies all major gene products of the five-member GH/PL gene cluster GH-N, GH-V, PL-A/B, and PL-L, revealed specific transcripts in all specimens. Their share in gene selective expression by analytical restriction enzyme digestion was determined. The expression pattern of GH/PL messenger ribonucleic acid shows PL-A/B > GH-N, which sets it apart from those of pituitary and placenta. Local production of the respective protein hormones was verified by two time-resolved immunofluorometric assays for human PL-A/B and GH-N; significant amounts of these hormones were detected in cytosolic extracts of premenopausal (n = 6; 555.5 +/- 171 ng PL-A/B and 0.8 +/- 0.6 ng GH-N/g tissue wet wt) and postmenopausal (n = 6; 5.2 +/- 2.7 ng PL-A/B and 0.9 +/- 0.6 ng GH-N/g tissue wet wt) ovaries. No difference was observed between pre- and postmenopausal ovarian GH-N contents, but PL values were 2-3 orders of magnitude lower in postmenopausal tissue (P < 0.001). Serum levels of healthy premenopausal (n = 21) and postmenopausal (n = 16) women were less than 0.02 ng PL/mL. In summary, ovarian-derived GH-N and PL-A/B synthesis correlates well with the established local cascade of GHRH, GHRH receptor, GH receptor, IGF-I, and IGF-I receptor as a putative para/autocrine regulator of ovarian reproductive function.

Adult↗
PubMed2003

Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation failure and infertility.

Endometriosis is clinically associated with pelvic pain and infertility, with implantation failure strongly suggested as an underlying cause for the observed infertility. Eutopic endometrium of women with endometriosis provides a unique experimental paradigm for investigation into molecular mechanisms of reproductive dysfunction and an opportunity to identify specific markers for this disease. We applied paralleled gene expression profiling using high-density oligonucleotide microarrays to investigate differentially regulated genes in endometrium from women with vs. without endometriosis. Fifteen endometrial biopsy samples (obtained during the window of implantation from eight subjects with and seven subjects without endometriosis) were processed for expression profiling on Affymetrix Hu95A microarrays. Data analysis was conducted with GeneChip Analysis Suite, version 4.01, and GeneSpring version 4.0.4. Nonparametric testing was applied, using a P value of 0.05, to assess statistical significance. Of the 12,686 genes analyzed, 91 genes were significantly increased more than 2-fold in their expression, and 115 genes were decreased more than 2-fold. Unsupervised clustering demonstrated down-regulation of several known cell adhesion molecules, endometrial epithelial secreted proteins, and proteins not previously known to be involved in the pathogenesis of endometriosis, as well as up-regulated genes. Selected dysregulated genes were randomly chosen and validated with RT-PCR and/or Northern/dot-blot analyses, and confirmed up-regulation of collagen alpha2 type I, 2.6-fold; bile salt export pump, 2.0-fold; and down-regulation of N-acetylglucosamine-6-O-sulfotransferase (important in synthesis of L-selectin ligands), 1.7-fold; glycodelin, 51.5-fold; integrin alpha2, 1.8-fold; and B61 (Ephrin A1), 4.5-fold. Two-way overlapping layer analysis used to compare endometrial genes in the window of implantation from women with and without endometriosis further identified three unique groups of target genes, which differ with respect to the implantation window and the presence of disease. Group 1 target genes are up-regulated during the normal window of implantation but significantly decreased in women with endometriosis: IL-15, proline-rich protein, B61, Dickkopf-1, glycodelin, N-acetylglucosamine-6-O-sulfotransferase, G0S2 protein, and purine nucleoside phosphorylase. Group 2 genes are normally down-regulated during the window of implantation but are significantly increased with endometriosis: semaphorin E, neuronal olfactomedin-related endoplasmic reticulum localized protein mRNA and Sam68-like phosphotyrosine protein alpha. Group 3 consists of a single gene, neuronal pentraxin II, normally down-regulated during the window of implantation and further decreased in endometrium from women with endometriosis. The data support dysregulation of select genes leading to an inhospitable environment for implantation, including genes involved in embryonic attachment, embryo toxicity, immune dysfunction, and apoptotic responses, as well as genes likely contributing to the pathogenesis of endometriosis, including aromatase, progesterone receptor, angiogenic factors, and others. Identification and validation of selected genes and their functions will contribute to uncovering previously unknown mechanism(s) underlying implantation failure in women with endometriosis and infertility, mechanisms underlying the pathogenesis of endometriosis and providing potential new targets for diagnostic screening and intervention.

Blotting, Northern↗
PubMed2003

Selection of normalizer genes in conducting relative gene expression analysis of embryos.

BACKGROUND: In relative gene expression analysis, a reference gene for sample normalization is required for determining target expression changes among experimental treatment groups. Since some developmental toxicants secondarily cause general growth retardation and/or other general biological changes, commonly used housekeeping genes may not serve as accurate normalizers. METHODS: We conducted real-time polymerase chain reaction (PCR) with normalization to calculate relative target transcriptional change, using housekeeping and structure-specific expression genes as normalizers. Relative levels of Hoxb1 expression were measured in cultured rodent embryos at 24 hr post retinoic acid (RA) administration. Transcriptional response was also evaluated using two novel compounds that produced posterior axial and growth defects in rat whole-embryo culture. Embryos treated with these compounds were evaluated for general biological processes, and their respective biological states were considered in the context of the relative gene expression change calculated with the housekeeping normalizers. RESULTS: Normalized RA-induced Hoxb1 expression demonstrated that only some reference genes accurately quantitated the expected 1.5- to 2-fold increase in Hoxb1 expression. Evaluation of the test compounds demonstrated that only normalization with the spatially-restricted hindbrain gene, Krox-20, calculated significant expression decreases of T-gene, a gene known to be functionally relevant in posterior axial development. Reduction in T-gene expression was confirmed qualitatively by whole-mount in situ hybridization. CONCLUSIONS: Prudent reference gene selection is important in evaluating relative gene expression in embryos. An experimental control design is proposed to facilitate the identification of normalizing genes that will accurately calculate relative gene expression change in treated embryos.

Animals↗
PubMed1999

Molecular analysis of the Cryptococcus neoformans ADE2 gene, a selectable marker for transformation and gene disruption.

Cryptococcus neoformans is an important fungal pathogen of man. The incidence of cryptococcal disease has increased dramatically in patients immunocompromised because of HIV infection, organ transplantation, or treatment with cytotoxic chemotherapy or corticosteroids. This organism is an excellent model for molecular dissection of fungal pathogenesis and virulence factors. Here we report the nucleotide sequence of the C. neoformans serotype D genomic ADE2 gene, which encodes a phosphoribosylaminoimidazole carboxylase required for purine biosynthesis. Importantly, this version of the ADE2 gene has been used as the selectable marker for virtually all gene disruptions by transformation and homologous recombination in C. neoformans. We compare the nucleotide and amino acid sequences of the ADE2 gene and product to other highly related adenine biosynthetic genes and enzymes from other yeasts and fungi. We also describe a series of convenient ADE2 cassettes for gene disruption construct preparation. Finally, we have identified the ade2 mutations in strains M001 and M049, adenine auxotrophic mutants derived from the serotype A strain H99. These mutant strains have served as recipients for targeted gene disruptions using the ADE2 gene. These studies should facilitate transformation and gene disruption approaches using the ADE2 selectable marker in this important human fungal pathogen.

Amino Acid Sequence↗
PubMed2004

Selection for gene clustering by tandem duplication.

In prokaryotic genomes, related genes are frequently clustered in operons and higher-order arrangements that reflect functional context. Organization emerges despite rearrangements that constantly shuffle gene and operon order. Evidence is presented that the tandem duplication of related genes acts as a driving evolutionary force in the origin and maintenance of clusters. Gene amplification can be viewed as a dynamic and reversible regulatory mechanism that facilitates adaptation to variable environments. Clustered genes confer selective benefits via their ability to be coamplified. During evolution, rearrangements that bring together related genes can be selected if they increase the fitness of the organism in which they reside. Similarly, the benefits of gene amplification can prevent the dispersal of existing clusters. Examples of frequent and spontaneous amplification of large genomic fragments are provided. The possibility is raised that tandem gene duplication works in concert with horizontal gene transfer as interrelated evolutionary forces for gene clustering.

Benzoates↗
PubMed1996

Potent and selective inhibition of gene expression by an antisense heptanucleotide.

Factors that govern the specificity of an antisense oligonucleotide (ON) for its target RNA include accessibility of the targeted RNA to ON binding, stability of ON/RNA complexes in cells, and susceptibility of the ON/RNA complex to RNase H cleavage. ON specificity is generally proposed to be dependent on its length. To date, virtually all previous antisense experiments have used 12-25 nt-long ONs. We explored the antisense activity and specificity of short (7 and 8 nt) ONs modified with C-5 propyne pyrimidines and phosphorothioate internucleotide linkages. Gene-selective, mismatch sensitive, and RNase H-dependent inhibition was observed for a heptanucleotide ON. We demonstrated that the flanking sequences of the target RNA are a major determinant of specificity. The use of shorter ONs as antisense agents has the distinct advantage of simplified synthesis. These results may lead to a general, cost-effective solution to the development of antisense ONs as therapeutic agents.

Animals↗
PubMed1998

Identification of a novel class of genomic DNA-binding sites suggests a mechanism for selectivity in target gene activation by the tumor suppressor protein p53.

There are two response elements for p53 in the promoter of the gene for the cyclin-dependent kinase inhibitor p21. The binding of p53 to the 5' site was enhanced by incubation with monoclonal antibody 421, whereas the binding of p53 to the 3' site was inhibited. Mutational analysis showed that a single-base change caused one element to behave like the other. A response element in the human cdc25C promoter is bound by p53 with properties similar to the 3' site. These results identify two classes of p53-binding sites and suggest a mechanism for target gene selectivity by p53.

Animals↗
PubMed2004

Clusters of co-expressed genes in mammalian genomes are conserved by natural selection.

Genes that belong to the same functional pathways are often packaged into operons in prokaryotes. However, aside from examples in nematode genomes, this form of transcriptional regulation appears to be absent in eukaryotes. Nevertheless, a number of recent studies have shown that gene order in eukaryotic genomes is not completely random, and that genes with similar expression patterns tend to be clustered together. What remains unclear is whether co-expressed genes have been gathered together by natural selection to facilitate their regulation, or if the genes are co-expressed simply by virtue of their being close together in the genome. Here, we show that gene expression clusters tend to contain fewer chromosomal breakpoints between human and mouse than expected by chance, which indicates that they are being held together by natural selection. This conclusion applies to clusters defined on the basis of broad (housekeeping) expression, or on the basis of correlated transcription profiles across tissues. Contrary to previous reports, we find that genes with high expression are not clustered to a greater extent than expected by chance and are not conserved during evolution.

Animals↗
PubMed1991

Cotransformation and gene targeting in mouse embryonic stem cells.

We have investigated cotransformation in mammalian cells and its potential for identifying cells that have been modified by gene targeting. Selectable genes on separate DNA fragments were simultaneously introduced into cells by coelectroporation. When the introduced fragments were scored for random integration, 75% of the transformed cells integrated both fragments within the genome of the same cell. When one of the cointroduced fragments was scored for integration at a specific locus by gene targeting, only 4% of the targeted cells cointegrated the second fragment. Apparently, cells that have been modified by gene targeting with one DNA fragment rarely incorporate a second DNA fragment. Despite this limitation, we were able to use the cotransformation protocol to identify targeted cells by screening populations of colonies that had been transformed with a cointroduced selectable gene. When hypoxanthine phosphoribosyltransferase (hprt) targeting DNA was coelectroporated with a selectable neomycin phosphotransferase (neo) gene into embryonic stem (ES) cells, hprt-targeted colonies were isolated from the population of neo transformants at a frequency of 1 per 70 G418-resistant colonies. In parallel experiments with the same targeting construct, hprt-targeted cells were found at a frequency of 1 per 5,500 nonselected colonies. Thus, an 80-fold enrichment for targeted cells was observed within the population of colonies transformed with the cointroduced DNA compared with the population of nonselected colonies. This enrichment for targeted cells after cotransformation should be useful in the isolation of colonies that contain targeted but nonselectable gene alterations.

Animals↗
PubMed2004

Generation of double gene disruptions in Dictyostelium discoideum using a single antibiotic marker selection.

Gene targeting is a powerful molecular genetic technique that has been widely used to understand specific gene function in vivo. This technique allows the ablation of an endogenous gene by recombination between an introduced DNA fragment and the homologous target gene. However, when multiple gene disruptions are needed, the availability of only a limited number of marker genes becomes a complication. Here we describe a new approach to perform double gene disruptions in Dictyostelium discoideum by simultaneous transfection of two gene targeting cassettes followed by performing clonal selection against only one marker gene. The subsequent PCR-based screens of blasticidin-resistant clones revealed the integration of both the selected and the nonselected targeting cassettes at their original respective loci creating complete gene disruptions. For the genes we have tested in these studies (myosin heavy chain kinases B and C), the efficiency of the double gene targeting event is found in the range of 2%-5% of all blasticidin-resistant colonies following the transfection step. This approach for the simultaneous disruptions of multiple genes should prove to be a valuable tool for other laboratories interested in creating multiple gene disruptants in Dictyostelium or other organisms where a limited number of selectable markers are available.

Animals↗
PubMed1986

Alpha 4, the major regulatory protein of herpes simplex virus type 1, is stably and specifically associated with promoter-regulatory domains of alpha genes and of selected other viral genes.

Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. However, the mechanism by which alpha 4 regulates gene expression remained unknown. We report that labeled DNA fragments containing promoter-regulatory domains of three alpha (alpha 0, alpha 4, and alpha 27) and a gamma 2 gene form stable complexes with proteins from infected-cell lysates as detected by a gel electrophoresis binding assay. The protein(s) exhibits sequence specificity since autologous DNA fragments but not heterologous DNA fragments, synthetic polydeoxynucleotide chains, or salmon sperm DNA competitively displace the DNA probe from the complexes. Murine monoclonal antibody to alpha 4 protein added before or after DNA-protein complex formation further retarded the electrophoretic mobility of the complexes whereas monoclonal antibody to alpha 0, alpha 27, or to a viral glycoprotein had no effect. Complexes consisting of the promoter-regulatory domain of the beta-class thymidine kinase gene and infected cell proteins were low in abundance and could be detected only in the presence of antibody to alpha 4 protein. The alpha 4 protein, therefore, forms stable complexes with promoter-regulatory domains of alpha genes and of selected other herpes simplex virus type 1 genes either alone or in combination with other proteins.

DNA, Viral↗
PubMed2006

Selecting "significant" differentially expressed genes from the combined perspective of the null and the alternative.

In the search for genes associated with disease, statistical analysis yields a key towards reproducible results. To avoid a plethora of type I errors, classical gene selection procedures strike a balance between magnitude and precision of observed effects in terms of p-values. Protecting false discovery rates recovers some power but still ranks genes according to classical p-values. In contrast, we propose a selection procedure driven by the concern to detect well-specified important alternatives. By summarizing evidence from the perspective of both the null and such an alternative hypothesis, genes line up in a substantially different order with different genes yielding powerful signals. A cutoff point for a measure of relative evidence which balances the standard p-value, p0, with its counterpart, p1, derived from the perspective of the target alternative, determines our gene selection. We find the cutoff point that maximizes an expected specific gain. This yields an optimal decision which exploits gene-specific variances and thus involves different type I and type II errors across genes. We show the dramatic impact of this alternative perspective on the detection of differentially expressed genes in hereditary breast cancer. Our analysis does not rely on parametric assumptions on the data.

Biometry↗
PubMed2004

Bystander-mediated regression of murine neuroblastoma via retroviral transfer of the HSV-TK gene.

Selective introduction of genes conferring chemosensitivity into proliferating tumor cells may be used to treat cancer. We investigated the bystander effect of retrovirus-mediated gene transfer of herpes simplex virus thymidine kinase (HSV-TK) gene to murine neuroblastoma cell line (neuro-2a) in vitro and in vivo, and we examined whether the mechanism of bystander effect in neuroblastoma would also depend on connexin-dependent gap junction and/or immune response. A strong bystander effect was observed in vitro, whereby nontransduced tumor cells in proximity to transduced cells acquired susceptibility to ganciclovir (GCV) killing. Implanted mixtures of wildtype cells and HSV-TK transduced cells showed a potent bystander effect upon administration of GCV in A/J mice. HSV-TK/GCV system in murine neuroblastoma induced systemic immunity. Immunohistochemical staining showed many CD4+ and CD8+ cell infiltration but did not show anti-connexin 43+ cells. In conclusion, a strong bystander effect was observed in vitro and in vivo. The bystander effect in murine neuroblastoma might be dependent on immune response and/or on other mechanism such as protein phosphorylation or transfer of apoptotic vesicle, rather than connexin-dependent gap junction.

Animals↗