Abstract: BACKGROUND: Campylobacter jejuni is widespread in the environment and is the major cause of bacterial gastroenteritis in humans. In the present study we use microarray-based comparative genomic hybridizations (CGH), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to analyze closely related C. jejuni isolates from chicken and human infection. RESULTS: With the exception of one isolate, the microarray data clusters the isolates according to the five groups determined by PFGE. In contrast, MLST defines only three genotypes among the isolates, indicating a lower resolution. All methods show that there is no inherit difference between isolates infecting humans and chicken, suggesting a common underlying population of C. jejuni. We further identify regions that frequently differ between isolates, including both previously described and novel regions. Finally, we show that genes that belong to certain functional groups differ between isolates more often than expected by chance. CONCLUSION: In this study we demonstrated the utility of 70-mer oligonucleotide microarrays for genotyping of Campylobacter jejuni isolates, with resolution outperforming MLST.
Abstract: BACKGROUND: Various types of amplification techniques have been developed in order to enable microarray gene expression analysis when the amount of starting material is limited. The two main strategies are linear amplification, using in vitro transcription, and exponential amplification, based on PCR. We have evaluated the performance of a linear and an in-house developed exponential amplification protocol that relies on 3' end tag sequences. We used 100 ng total RNA as starting material for amplification and compared the results with data from hybridizations with unamplified mRNA and total RNA. RESULTS: Preservation of expression ratios after amplification was examined comparing log(2) ratios obtained with amplification protocols to those obtained with standard labelling of mRNA. The Pearson correlations were 0.61 and 0.84, respectively, for the two linear amplification replicates and 0.76 and 0.80 for the two exponential amplification replicates. The correlations between repeated amplifications was 0.82 with the exponential method and 0.63 with the linear, indicating a better reproducibility with the PCR-based approach. CONCLUSION: Both amplification methods generated results in agreement with unamplified material. In this study, the PCR-based method was more reproducible than in vitro transcription amplification. Advantages with the in-house developed method are the lower cost since it is non-commercial and that the PCR generated product offers compatibility with both sense and antisense arrays.
Abstract: The extent to which duplications and deletions occur in the Plasmodium falciparum genome, outside of the subtelomeres, and their contribution to the virulence of the malaria parasite is not known. Here we show the presence of multiple genome wide copy number polymorphisms (CNPs) covering 82 genes, the most extensive spanning a cumulative size of 110kilobases. CNPs were identified in both laboratory strains and fresh clinical isolates using a 70-mer oligonucleotide microarray in conjunction with fluorescent in situ hybridizations and real-time quantitative PCR. The CNPs were found on all chromosomes except on chromosomes 6 and 8 and involved a total of 50 genes with increased copy numbers and 32 genes with decreased copy numbers relative to the 3D7 parasite. The genes, amplified in up to six copies, encode molecules involved in cell cycle regulation, cell division, drug resistance, erythrocyte invasion, sexual differentiation and unknown functions. These together with previous findings, suggest that the malaria parasite employs gene duplications and deletions as general strategies to enhance its survival and spread. Further analysis of the impact of discovered genetic differences and the underlying mechanisms is likely to generate a better understanding of the biology and the virulence of the malaria parasite.
Abstract: There is increasing evidence that tumors are heterogeneous and that a subset of cells act as cancer stem cells. Several proto-oncogenes and tumor suppressors control key aspects of stem cell function, suggesting that similar mechanisms control normal and cancer stem cell properties. We show here that the prototypical tumor suppressor p53, which plays an important role in brain tumor initiation and growth, is expressed in the neural stem cell lineage in the adult brain. p53 negatively regulates proliferation and survival, and thereby self-renewal, of neural stem cells. Analysis of the neural stem cell transcriptome identified the dysregulation of several cell cycle regulators in the absence of p53, most notably a pronounced downregulation of p21 expression. These data implicate p53 as a suppressor of tissue and cancer stem cell self-renewal.
Abstract: BACKGROUND: Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types. RESULTS: Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development. CONCLUSION: Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal/differentiation of HSCs, and function of Lhx2 in organ development and stem/progenitor cells of non-hematopoietic origin.
Abstract: Stem cells generally reside in a stem cell microenvironment, where cues for self-renewal and differentiation are present. However, the genetic program underlying stem cell proliferation and multipotency is poorly understood. Transcriptome analysis of stem cells and their in vivo microenvironment is one way of uncovering the unique stemness properties and provides a framework for the elucidation of stem cell function. Here, we characterize the gene expression profile of the in vivo neural stem cell microenvironment in the lateral ventricle wall of adult mouse brain and of in vitro proliferating neural stem cells. We have also analyzed an Lhx2-expressing hematopoietic-stem-cell-like cell line in order to define the transcriptome of a well-characterized and pure cell population with stem cell characteristics. We report the generation, assembly and annotation of 50,792 high-quality 5'-end expressed sequence tag sequences. We further describe a shared expression of 1065 transcripts by all three stem cell libraries and a large overlap with previously published gene expression signatures for neural stem/progenitor cells and other multipotent stem cells. The sequences and cDNA clones obtained within this framework provide a comprehensive resource for the analysis of genes in adult stem cells that can accelerate future stem cell research.
Abstract: BACKGROUND: Transcriptional profiling using microarrays has developed into a key molecular tool for the elucidation of gene function and gene regulation. Microarray platforms based on either oligonucleotides or purified amplification products have been utilised in parallel to produce large amounts of data. Irrespective of platform examined, the availability of genome sequence or a large number of representative expressed sequence tags (ESTs) is, however, a pre-requisite for the design and selection of specific and high-quality microarray probes. This is of great importance for organisms, such as Arabidopsis thaliana, with a high number of duplicated genes, as cross-hybridisation signals between evolutionary related genes cannot be distinguished from true signals unless the probes are carefully designed to be specific. RESULTS: We present an alternative solid-phase purification strategy suitable for efficient preparation of short, biotinylated and highly specific probes suitable for large-scale expression profiling. Twenty-one thousand Arabidopsis thaliana gene sequence tags were amplified and subsequently purified using the described technology. The use of the arrays is exemplified by analysis of gene expression changes caused by a four-hour indole-3-acetic (auxin) treatment. A total of 270 genes were identified as differentially expressed (120 up-regulated and 150 down-regulated), including several previously known auxin-affected genes, but also several previously uncharacterised genes. CONCLUSIONS: The described solid-phase procedure can be used to prepare gene sequence tag microarrays based on short and specific amplified probes, facilitating the analysis of more than 21,000 Arabidopsis transcripts.
Abstract: BACKGROUND: Neural stem cells (NSCs) can be isolated from the adult mammalian brain and expanded in culture, in the form of cellular aggregates called neurospheres. Neurospheres provide an in vitro model for studying NSC behaviour and give information on the factors and mechanisms that govern their proliferation and differentiation. They are also a promising source for cell replacement therapies of the central nervous system. Neurospheres are complex structures consisting of several cell types of varying degrees of differentiation. One way of characterising neurospheres is to analyse their gene expression profiles. The value of such studies is however uncertain since they are heterogeneous structures and different populations of neurospheres may vary significantly in their gene expression. RESULTS: To address this issue, we have used cDNA microarrays and a recently reported tag cDNA amplification method to analyse the gene expression profiles of neurospheres originating from separate isolations of the lateral ventricle wall of adult mice and passaged to varying degrees. Separate isolations as well as consecutive passages yield a high variability in gene expression while parallel cultures yield the lowest variability. CONCLUSIONS: We demonstrate a low technical amplification variability using the employed amplification strategy and conclude that neurospheres from the same isolation and passage are sufficiently similar to be used for comparative gene expression analysis.
Abstract: BACKGROUND: The recently discovered adult neural stem cells, which maintain continuous generation of new neuronal and glial cells throughout adulthood, are a promising and expandable source of cells for use in cell replacement therapies within the central nervous system. These cells could either be induced to proliferate and differentiate endogenously, or expanded and differentiated in culture before being transplanted into the damaged site of the brain. In order to achieve these goals effective strategies to isolate, expand and differentiate neural stem cells into the desired specific phenotypes must be developed. However, little is known as yet about the factors and mechanisms influencing these processes. It has recently been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neural stem cell proliferation both in vivo and in vitro. RESULTS: We used cDNA microarrays with the aim of analysing the transcriptional changes underlying PACAP induced proliferation of neural stem cells. The primary neural stem/progenitor cells used were neurospheres, generated from the lateral ventricle wall of the adult mouse brain. The results were compared to both differentiation and proliferation controls, which revealed an unexpected and significant differential expression relating to withdrawal of epidermal growth factor (EGF) from the neurosphere growth medium. The effect of EGF removal was so pronounced that it masked the changes in gene expression patterns produced by the addition of PACAP. CONCLUSION: Experimental models aiming at transcriptional analysis of induced proliferation in primary neural stem cells need to take into consideration the significant effect on transcription caused by removal of EGF. Alternatively, EGF-free culture conditions need to be developed.
Abstract: Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.
Abstract: Monitoring of differential gene expression is an important step towards understanding of gene function. We describe a comparison of the representational difference analysis (RDA) subtraction process with corresponding microarray analysis. The subtraction steps are followed in a quantitative manner using a shotgun cloning and sequencing procedure that includes over 1900 gene sequences. In parallel, the enriched transcripts are spotted onto microarrays facilitating large scale hybridization analysis of the representations and the difference products. We show by the shotgun procedure that there is a high diversity of gene fragments represented in the iterative RDA products (92-67% singletons) with a low number of shared sequences (<9%) between subsequent subtraction cycles. A non redundant set of 1141 RDA clones were immobilized on glass slides and the majority of these clones (97%) gave repeated good fluorescent signals in a subsequent hybridization of the labelled and amplified original cDNA. We observed only a low number of false positives (<2%) and a more than twofold differential expression for 32% (363) of the immobilized RDA clones. In conclusion, we show that by random sequencing of the difference products we obtained an accurate transcript profile of the individual steps and that large-scale confirmation of the obtained transcripts can be achieved by microarray analysis.
Abstract: BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR) gene polymorphism may cause hyperhomocysteinemia, which affects the vascular endothelium and may induce occlusive vascular disease (OVD). Hypertension thickens small-sized arterial walls and attenuates intramural blood flow. Such OVD can be studied in retinal angiograms as a decrease in the arterio-venous ratio (AVR). Diabetes, by altering microvascular structure and function, in many ways modifies this AVR. OBJECTIVE: To assess whether MTHFR gene polymorphism (C677T) by causing hyperhomocysteinemia affects the retinal AVR in type 2 diabetic and non-diabetic subjects. METHODS: Eighty-four recently diagnosed (< 1 year) type 2 diabetic and 115 non-diabetic subjects were included in the study. Retinal fluoresceine angiograms were recorded and the mean AVR was calculated by measuring transverse vessel diameters at 6 locations. The mean AVR was used as a marker of OVD. The MTHFR VV, VA and AA genotypes were determined by PCR and plasma homocysteine by high-pressure liquid chromatography. RESULTS: In the diabetic subjects with the VV, VA and AA genotypes, the plasma homocysteine levels were 16.5 +/- 7, 12.5 +/- 4.6 and 11.3 +/- 4.9 microM, respectively (p = 0.008, ANCOVA). The corresponding values in controls were 14.6 +/- 3.8, 13.7 +/- 5.7 and 11.6 +/- 4.4 (p = 0.08). Correspondingly, in the diabetic subjects, the AVR values were 0.71 +/- 0.07, 0.75 +/- 0.07 and 0.73 +/- 0.1 (p = NS, ANOVA) and in the control subjects they were 0.8 +/- 0.14, 0.81 +/- 0.12 and 0.76 +/- 0.09 (p = NS, ANOVA). Multiple linear regression analysis (best model chi2 = 18.2, R2 = 0.10, p < 0.001) showed that AVR was related to diastolic blood pressure (t = -3.7, p < 0.001) and GFR (t = -2.2, p = 0.03). There was no relation between the AVR and plasma homocysteine levels. CONCLUSION: In the present study of recently diagnosed type 2 diabetic and non-diabetic subjects, MTHFR gene polymorphism (C677T mutation) slightly affected the plasma homocysteine level but did not alter the arterio-venous ratio.
Abstract: BACKGROUND: The C677T mutation of the methylenetetrahydrofolate reductase (MTHFR) gene leads to C/C, C/T and T/T genotypes, which affect the plasma homocysteine concentration in humans. In mini-pigs, high serum homocysteine levels are associated with defects in the internal elastic lamina (IEL) of the artery wall, which are apparently related to the migration of smooth muscle cells into the intima during atherogenesis. We studied the association between the MTHFR genotypes and the number of gaps in the IEL in the wall of the five major abdominal arteries. MATERIALS AND METHODS: The autopsy study included 123 subjects (90 males and 33 females) aged 18-93. For the light microscopy, a 0.5 cm circular segment of the coeliac, the superior mesenteric, the inferior mesenteric and the renal arteries were cut and embedded in paraffin blocks. The circumference of the IEL, the thickness of the intima and the number of the gaps per millimetre in the IEL were measured by MOP 3 image analysis. RESULTS: The T-allele carriers (C/T and T/T) of the MTHFR gene had significantly less gaps in the IEL than the subjects with the C/C genotype in the superior mesenteric and in the left renal arteries (2.02 +/- 2.25 vs. 2.53 +/- 1.89, P < 0.04 and 0.56 +/- 1.09 vs. 1.82 +/- 2.66, P < 0.02, respectively). The trend was similar for the coeliac and the right renal arteries. CONCLUSIONS: Our result suggests that MTHFR polymorphism may be involved in the fragmentation of the IEL.
Abstract: OBJECTIVES: To analyze the early gene expression in macrophages accompanying the phenotypic changes into foam cells upon exposure to oxidized low-density lipoprotein. To identify candidate genes and markers for further studies into the pathogenesis of atherosclerosis. METHODS: Cells of the monocytic cell line THP-1 were activated by PMA and exposed to oxidized low-density lipoprotein. Gene expression profiles were investigated after 24 h, using a solid phase cDNA representational difference analysis (RDA) method and shotgun sequencing. Results were verified by microarray hybridization, and analyzed in the virtual chip display of a novel software tool for transcript profile exploration. RESULTS: By comparing transcript profiles of exposed/unexposed cells, 1,984 transcript sequences, representing a total of 921 genes with altered expression levels in response to oxidized low-density lipoprotein exposure, were identified. Genes that are central to cell cycle control and proliferation, inflammatory response, and of pathways not previously implicated in atherosclerosis were identified. The data obtained is also made available on-line at http:// biobase.biotech.kth.se/thp1a for further exploration. CONCLUSION: The identification of new candidate genes for atherosclerotic disease through RDA-based transcript profiling facilitates further functional genomic studies in coronary artery disease. Candidate genetic polymorphism markers of potential clinical relevance can be identified by filtering information in genome variation databases through the virtual chip analysis of the transcript profiles and subsequently tested in association studies.
Abstract: The missense mutation in the 677th nucleotide (C677T) of methylenetetrahydrofolate reductase gene causes substitution of valine (V) for alanine (A) resulting in three genotypes VV, VA and AA. The VV genotype causes hyperhomocysteinemia and may be a risk factor for coronary artery disease. We determined genotypes by polymerase chain reaction and subsequent restriction fragment length analysis and compared them in 84 patients with type 2 diabetes and in 115 non-diabetic subjects with and without coronary disease. Fractional urinary excretion rate of albumin was assessed by nephelometry. The VV, VA, and AA frequencies in the diabetic and in the control groups were 0.095, 0.357, 0.548 and 0.061, 0.417, 0.522, respectively (p = NS, diabetic vs. controls, chi2 test). Genotype frequencies did not differ in either diabetic or control subjects between those with or those without coronary disease (chi2 test). The fractional urinary excretion rate of albumin (mean +/-SD) in diabetic patients with the VV genotype i.e. 1.59 +/-0.71 was lower (Kruskall-Wallis test p = 0.002) than in the other genotypes i.e. VA 5.98 +/-9.75 and AA 3.75 +/-4.77, respectively (post-hoc Mann-Whitney test VV vs. VA p = 0.005 and VV vs. AA p = 0.054, respectively). We found that in patients with type 2 diabetes the methylenetetrahydrofolate reductase VV genotype was associated with a low urinary albumin excretion but not with coronary artery disease or diabetes per se.
