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Results for “gene expression variability”

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PubMed2004

Selection of appropriate control genes to assess expression of tumor antigens using real-time RT-PCR.

Real-time reverse transcription PCR (RT-PCR) is a sensitive and accurate method to monitor gene expression and is often used to profile the expression of putative tumor antigens in the context of immunotherapy. However, this technique consists of several steps, including cell processing, RNA extraction, RNA storage, assessment of RNA concentration, and cDNA synthesis prior to PCR. To compensate for potential variability introduced in this procedure, the expression of housekeeping genes is commonly assessed in parallel with the expression of the gene of interest. In this study, the expression of a variety of housekeeping genes in a panel of 26 different human tumor and embryonal cell lines was assessed using real-time RT-PCR. For some control genes, the variability in expression was significant between different cell lines, despite the equalization of quantities of input RNA. The greatest variability was found for GAPDH. The lowest variability was found for beta-glucuronidase (GUS) and 18S rRNA. While real-time RT-PCR is a powerful tool for gene expression analysis, these results suggest that the choice of control genes to normalize the expression of the gene of interest is critical to the interpretation of experimental results and should be tailored to the nature of the study.

Animals↗
PubMed2004

The Global Error Assessment (GEA) model for the selection of differentially expressed genes in microarray data.

MOTIVATION: Microarray technology has become a powerful research tool in many fields of study; however, the cost of microarrays often results in the use of a low number of replicates (k). Under circumstances where k is low, it becomes difficult to perform standard statistical tests to extract the most biologically significant experimental results. Other more advanced statistical tests have been developed; however, their use and interpretation often remain difficult to implement in routine biological research. The present work outlines a method that achieves sufficient statistical power for selecting differentially expressed genes under conditions of low k, while remaining as an intuitive and computationally efficient procedure. RESULTS: The present study describes a Global Error Assessment (GEA) methodology to select differentially expressed genes in microarray datasets, and was developed using an in vitro experiment that compared control and interferon-gamma treated skin cells. In this experiment, up to nine replicates were used to confidently estimate error, thereby enabling methods of different statistical power to be compared. Gene expression results of a similar absolute expression are binned, so as to enable a highly accurate local estimate of the mean squared error within conditions. The model then relates variability of gene expression in each bin to absolute expression levels and uses this in a test derived from the classical ANOVA. The GEA selection method is compared with both the classical and permutational ANOVA tests, and demonstrates an increased stability, robustness and confidence in gene selection. A subset of the selected genes were validated by real-time reverse transcription-polymerase chain reaction (RT-PCR). All these results suggest that GEA methodology is (i) suitable for selection of differentially expressed genes in microarray data, (ii) intuitive and computationally efficient and (iii) especially advantageous under conditions of low k. AVAILABILITY: The GEA code for R software is freely available upon request to authors.

Algorithms↗
PubMed1987

Novel genetic components controlling invertase production in Saccharomyces cerevisiae.

Low levels of invertase (EC 3.2.1.26) activity were observed in most diploid strains of S. cerevisiae used in this work. There was no effect of mating type on invertase levels, and cell surface was not a limiting factor, because an increase in ploidy did not cause further decrease in specific invertase activity. Finally, some diploids showed the activity expected from the additive effects of different SUC genes, and haploid strains possessing two SUC genes expressed very variable invertase activities depending on the strain. This suggested the existence of one or more additional genes which control the levels of invertase. Genetic analysis of SUC5 strains provided evidence of the existence of a new gene, RPS5, which drastically reduced the specific invertase activity in strains possessing active SUC alleles. The recessive allele of this gene (rps5) allows expression of higher levels of invertase. We suggest that genes similar RPS5 are responsible for the low levels of invertase activity observed in diploid strains of S. cerevisiae.

Diploidy↗
PubMed1996

Non random usage of T cell receptor alpha gene expression in atopy using anchored PCR.

The T cell receptor (TCR) alpha beta heterodimer recognises antigenic peptide fragments presented by Class II MHC. This interaction initiates T cell activation and cytokine release with subsequent recruitment of inflammatory cells. Previous work from our group suggests a qualitative difference in variable alpha gene expression in atopy as compared to non atopic controls. In this study we examine TCR alpha repertoire using anchored PCR to provide a quantitative assessment of the V alpha and J alpha repertoire. One atopic (DRB1*0701,DRB1*15: DRB4*0101, DRB5*01: DQB1* 0303, DQB1*601/2) and one non-atopic (DRB1*0701,DRB1*03011/2: DRB4*01, DRB3*0x: DQB1* 0303, DQB1*0201/2) control were studied. Variable gene usage was markedly limited in the atopic individual. V alpha 1, 3, 8 accounted for 60% and J alpha 12, 31 30% of the gene usage. There was evidence of preferential V alpha-J alpha gene pairing and clonal expansion. We conclude that there is a marked non random TCR alpha gene distribution in atopy using both V alpha family and anchored PCR. This may be due in part to antigen driven clonal expansion.

Gene Expression↗
PubMed1969

A model relating gene replicas and gene repression to phenotypic expression and variability.

It is suggested that the development of a dominant character in higher organisms starts with the activation of a major gene. This gene replicates; the number of replicas is controlled by a second gene or set of genes, which also ensure that the most suitable allele, if more than one is present, makes the replicas. The major gene replicas then make messenger RNA and are reinforced in this by minor genes. The joint product of major gene replicas and minor genes activates a fourth gene or set of genes which produce a repressor that damps down the activity of the major gene replicas. The evidence for this scheme, which comes from studies of selection experiments in populations, is summarized and the evidence from studies of DNA and RNA metabolism is referred to in this paper.

DNA Replication↗
PubMed2002

Differential expression of genes encoding immunomodulatory proteins in response to amphotericin B in human mononuclear cells identified by cDNA microarray analysis.

Amphotericin B (AMB) is an antifungal agent that possesses immunomodulatory properties that may contribute to its infusion-related toxicity and activity. It has previously been shown to induce the expression of genes encoding the cytokines interleukin (IL)-1 beta and tumour necrosis factor (TNF)-alpha and the chemokines IL-8 and macrophage inflammatory protein (MIP)-1 beta in the human monocytic cell line THP-1. In an effort to identify additional AMB-responsive genes, the gene expression profiles of both THP-1 cells and human peripheral blood mononuclear cells (hPBMCs) on exposure to AMB were assessed using cDNA microarray analysis. In addition to genes known to be AMB responsive, we found the genes encoding IL-1 alpha and MIP-1 alpha to be AMB responsive in both THP-1 cells and hPBMCs. Increases in MIP-1 alpha and MIP-1 beta were also observed in the supernatants of hPBMCs exposed to AMB. The expression of several genes in response to AMB was unique to either cell type. Furthermore, variability in gene expression in hPBMCs was observed between donors. These genes and respective gene products may have significance in the infusion-related toxicity and activity of AMB.

Adjuvants, Immunologic↗
PubMed1993

Immunophenotypic and idiotypic characterisation of the leukaemic B-cells from patients with prolymphocytic leukaemia: evidence for a selective expression of immunoglobulin variable region (IGV) gene products.

B-cell prolymphocytic leukaemia (B-PLL) is a rare chronic lymphoproliferative disease characterised by a massive splenomegaly associated with a mild or no lymphadenopathy and a high leukocyte count, mostly representing prolymphocytic features. We have studied membrane expression of certain Ig VK and VH gene products in five patients with B-PLL using a panel of monoclonal anti-subgroup and anti-cross-reactive idiotype (CRI) antibodies. Membrane expression of leukocyte-associated markers has also been investigated. The leukaemic cells from four patients expressed VKIII and VKIIIb subgroup and sub-subgroup kappa light chains. The VKIIIb and VHI-associated CRI identified by the monoclonal antibodies (MoAb) 17-109 and G8 were co-expressed in one patient. No B-cells from the patients expressed the VHIII-associated CRI. The same pattern of CRI expression was observed in a serum paraprotein collected from one of the patients. These results suggest a biased selection for the IG VKIII and VKIIIb light chains in B-PLL.

Aged↗
PubMed2002

beta-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels.

BACKGROUND: The use of reverse transcription-polymerase chain reaction (RT-PCR) to measure mRNA levels has led to the common use of beta-actin and GAPDH housekeeping genes as denominators for comparison of samples. Expression of these genes is assumed to remain constant, so normalising for variations in processing and signal quantitation. However, it is well documented that beta-actin and GAPDH expression is upregulated with proliferation, activation, and differentiation. We hypothesised that airway samples which differ in their cellular profiles and activation status have different levels of expression of GAPDH and beta-actin. METHODS: The mRNA for beta-actin, GAPDH, and interleukin (IL)-2 was measured in bronchoalveolar lavage (BAL) fluid cells and endobronchial biopsy tissue by competitive RT-PCR in a cross sectional study of 26 normal controls and 92 asthmatic subjects. RESULTS: For both BAL fluid cells and biopsy tissue, asthmatics overall had reduced expression of GAPDH and beta-actin mRNA. In asthmatic subjects not using inhaled corticosteroids (ICS), GAPDH mRNA levels in both BAL fluid and biopsy tissue, and beta-actin mRNA in BAL fluid cells were 10 times lower than samples from both normal controls and from asthmatic subjects using ICS. beta-Actin mRNA in biopsy specimens showed the same pattern of expression, but asthmatic subjects not using ICS were not significantly different from those receiving ICS treatment. IL-2 mRNA levels did not differ between the subject or treatment groups but, when expressed as a ratio with beta-actin, significant differences were seen. CONCLUSIONS: beta-Actin and GAPDH used as denominators of gene expression quantitation in asthma research can cause confounding. Housekeeping genes need careful validation before their use in such quantitative mRNA assays.

Actins↗
PubMed1996

Differential binding avidities of human IgM for staphylococcal protein A derive from specific germ-line VH3 gene usage.

Human IgM that express the variable region genes of the VH3 family bind staphylococcal protein A (SPA). We previously reported that the SPA-binding IgM can be divided into two groups based on the differential binding avidities for solid-phase SPA. To study the molecular basis for these differences, we cloned B cells from human blood by EBV transformation. The nucleotide sequences of the expressed Ig heavy chain genes were determined on 20 B cell clones that produce SPA-binding IgM. The germ-line VH3 gene usage in IgM with high avidities for SPA were distinct from the germ-line VH3 genes used in IgM with low avidities for SPA. There was no correlation in the usage of D or JH genes or in the usage of light chains in IgM according to the SPA binding avidity. These results suggest that the differential binding avidities for SPA are at least partly due to specific germ-line VH3 gene usage. An investigation of direct binding of SPA to the synthetic peptides corresponding to the portions of the variable regions of SPA-binding and non-SPA-binding IgM showed that the peptides corresponding to the VH3 family specific framework region 3 sequences had significant SPA binding capacities, while the peptides corresponding to the other subdomains and those corresponding to framework region 3 of the reported VH3 sequences from non-SPA-binding IgM showed little or no binding. It is of interest that the Ig-framework region 3 subdomain corresponds to the fourth hypervariable region, which in the TCR-beta chain has been implicated as a critical site for T cell superantigen binding.

Adult↗
PubMed1990

Molecular studies on LAK cells.

We have developed a culture system for long-term growth of human LAK cells exhibiting an elevated, wide-spectrum anti-tumor cytotoxicity. The phenotypic and molecular properties of the final LAK cell populations indicated that they consist of three main types: a) NK-like lymphocytes (type I): NKH-1+, Ti alpha/beta-, Ti gamma/delta-, CD3-lymphocytes carrying the germline configuration of all TCR genes and expressing variable amount of the 1.0 beta mRNA and variably sized T delta transcripts; b) gamma/delta-like T lymphocytes (type II) NKH-1+, Ti alpha/beta-, Ti gamma/delta+, CD3+ lymphocytes carrying polyclonal rearrangements of the gamma and delta genes and expressing high levels of mature gamma and delta transcripts; c) alpha/beta-like T lymphocytes (type III): NKH-1+, Ti alpha/beta+, Ti gamma/delta-, CD3+ lymphocytes carrying rearrangements of all TCR genes and expressing high levels of mature alpha and beta transcripts. We took advantage of the high number of available LAK cells to clarify: 1) the origin of the NK-LAK delta transcripts. delta gene expression in LGL, NK clones and type I LAK cultures revealed six delta transcripts (3.5, 3.1, 2.2, 2.0, 1.5 and 1.3 kb), which varied in number and relative abundance in the different samples. None of the six known V delta was expressed and the delta locus was retained in its germline configuration suggesting that the delta expression is due to a partially rearranged or germline transcripts; 2) the origin of the NK-LAK truncated T beta transcript. We isolated two different clone types from a type I LAK cell cDNA library: a) J-C clones consisting of one of three J beta regions and the corresponding C beta 1 or C beta 2 regions; b) X-J-C and C-X clones, containing as yet unidentified (X) sequences. The presence of RSSs in J-C clones suggests that they derive from mRNAs transcribed from a promoter in the 5'J. Nucleotide analysis demonstrated that only one of the isolated clones had the potential to code a short T beta protein.

Amino Acid Sequence↗
PubMed1990

Trypanosome variable surface glycoproteins: composite genes and order of expression.

Combinatorial processes increase the diversity of variable surface glycoproteins (VSGs) expressed by Trypanosoma equiperdum. We show here that a single telomeric pseudogene provides the 3' portion of three distinct T. equiperdum VSG genes by recombination with different 5' donor pseudogenes. Regions of sequence homology among the pseudogenes determine the sites of recombination in the formation of the expressed copies. This suggests that the recombination between any given basic copy (BC) and the expression-linked copy (ELC) depends on their sharing homology. We present evidence that this is the case and propose that such rules account for the order of expression of the VSGs. These results demonstrate how homologous recombination can generate an ordered sequence of gene expression.

Animals↗
PubMed2002

Molecular and genetic characterizations of five pathogenic and two non-pathogenic monoclonal antiphospholipid antibodies.

Antiphospholipid syndrome (APS) is an autoimmune disease that is characterized by thrombosis, recurrent fetal loss and thrombocytopenia. Antiphospholipid antibodies, detected by enzyme-linked immunoabsorbent assays (aCL) and/or in vitro blood clotting assays (LAC) are strongly associated with APS. Both the molecular structures used by pathogenic antiphospholipid antibodies and the genetic mechanisms leading to their production are unknown. We describe here the variable region genes of seven IgG antiphospholipid antibodies derived from two APS patients. Of these, five are pathogenic as defined in a mouse model of thrombosis and two are not. Analyses of the expressed variable region genes show no preferential V gene usage. However, similar to anti-DNA antibodies, pathogenic antiphospholipid antibodies contain an increased number of arginine residues in the third complimentarity-determining region (CDR3) of their H chains. The increased accumulation of arginine residues in the V(H) CDR3 may act to enhance antigen binding, promote disease and point to the importance of the H chain in the pathogenic potential of certain antiphospholipid antibodies.

Adolescent↗
PubMed1995

Characterization of human AMP deaminase 2 (AMPD2) gene expression reveals alternative transcripts encoding variable N-terminal extensions of isoform L.

AMP deaminase (AMPD) is a highly regulated enzymic activity and multiple isoforms of this enzyme are coded for by a multigene family in mammalian species, including man. Isoform L (liver) is the main activity present in adult human liver and is the protein product of the AMPD2 gene, which is widely expressed in non-muscle tissues and cells. A previous report described almost the full-length cDNA sequence and part of the human AMPD2 gene and also presented Northern blot evidence for multiple transcripts in brain. This study was performed to further characterize the AMPD2 gene and its expression in human tissues. AMPD2 genomic and human cerebellum cDNA clones were isolated, sequenced and used as probes in RNase protection analyses which together demonstrated the following: (1) an intervening sequence near the 5'-end of the published AMPD2 cDNA, which affects the predicted N-terminal amino acid sequence of isoform L; (2) alternative transcripts resulting from exon shuffling at, or near, the 5'-end of the AMPD2 gene that exhibit tissue-specific patterns of relative abundance; (3) predicted usage of three different initiation codons to confer variable N-terminal extensions on isoform L polypeptides; and (4) an extension of a 3' untranslated sequence in some AMPD2 transcripts. In addition, reverse transcriptase PCR and additional RNase protection analyses were used to map the 5'-ends of two mutually-exclusive exon 1 sequences, both of which contain multiple transcription-initiation sites. These results are discussed in relation to predicted isoform L diversity across human tissues and cells.

AMP Deaminase↗
PubMed2004

Array-based analysis of gene expression in childhood acute lymphoblastic leukemia.

Gene expression profiles of 10 children with acute lymphoblastic leukemia (ALL) were detected using cDNA arrays. Total RNAs were isolated from peripheral blood leukocytes of the patients at diagnosis. For detection of expression profiles we used Atlas Human Cancer cDNA Arrays (Clontech) with 588 genes. Although the study revealed variability of gene expression in many genes, we identified a number of genes with the same expression changes (up-regulation: PCNA, ERCC1; down-regulation: jun-B, BCL-2 related protein A1, CRAF-1, PBP) in most examined patients. Our objective was to identify genes that were differentially expressed in ALL and might contribute to development (and characterization) of the disease.

Adolescent↗
PubMed1996

Contrasting effects of the SATB1 core nuclear matrix attachment region and flanking sequences of the keratin 18 gene in transgenic mice.

The 2.3 kb and 3.5 kb of DNA that flank the human keratin 18 (K18) gene and synthetic nuclear matrix attachment regions (MAR) composed of the binding sites for the SATB1 nuclear protein were fused to a reporter gene that utilizes the mouse metallothionein promoter and the human growth hormone gene (MThGH). Transgenic mice were generated from both constructions and the control MThGH gene to test K18 and SATB1 MAR sequences for the ability to insulate the reporter gene from integration site-specific position effects. The MThGH control gene was variably expressed in brain, heart, intestine, kidney, liver, and testes, confirming previous studies. In contrast, the MThGH gene insulated by the K18 flanking sequences was expressed in the same tissues of four independent transgenic animals at levels correlated with the copy number except for intestine. The average level of expression on a per gene basis of the K18 insulated gene was from 9- to 49-fold higher than the control. The MThGH gene linked to the SATB1 MAR sequences was completely repressed in the brains and kidneys of all six transgenic mice. However, expression was nearly as efficient in testes as the K18-insulated gene. Both the SATB1 MAR and the K18 flanking sequences confer position-independent transcriptional status on the reporter gene in some or many tissues. However, the effects are stimulatory for the K18 elements and generally suppressive for the SATB1 MAR elements.

Animals↗
PubMed1994

Autosomal dominant transmission of Pallister-Hall syndrome.

A mother and daughter with Pallister-Hall syndrome are described. This documented parent-to-child transmission supports the hypothesis of autosomal dominant inheritance of this condition. The mother survived childhood and became pregnant without exogenous endocrine supplements, demonstrating that the gene manifests variable expressivity.

Adult↗