LNA-modified oligodeoxynucleotide hybridization with DNA microarrays printed on nanoporous membrane slides

We report a robust method for the detection of hybridization events using a microarray-based assay on a nanoporous membrane platform. The technique is characterized by a hybridization time of only 1 hour and uses Cy5- labeled, 7-mer oligodeoxynucleotide probes modified with locked nucleic acid (LNA) nucleotides. We show that the volume of the DNA spotted onto a nanomembrane can be reduced to ∼4 nL with detectable signal intensity. Moreover, the amount of the DNA target could be reduced to 4 fmol. The described approach could dramatically increase the throughput of techniques based on sequencing by hybridization, such as oligofingerprinting, by decreasing the total number of probes that are needed for analysis of large clone sets and reduction of the sample/reagent consumption. The method is particularly advantageous when numerous hybridization-based assays must be performed for characterization of sample sets of 100,000 or more

Screening of human gene promoter activities using transfected-cell arrays

Promoters are the best characterized transcriptional regulatory sequences in complex genomes because of their predictable location immediately upstream of transcription start sites. Despite a substantial body of literature describing transcriptional promoters, the identification of true start sites for all human transcripts is far from complete. The same is true of the key structural and functional elements responsible for promoter action in different cell types. In order to identify elements responsible for promoter activity, we applied transfected-cell array technology to functionally evaluate promoters for genes involved in inflammatory bowel disease. Seventy-four promoters were examined by reverse transfection of a promoter-fluorescent reporter constructs into a human embryonic kidney cell line (HEK293T). Sixteen (21.6%) promoters were found to be active in HEK293 T cells. Correlations between promoter activity and endogenous transcript level were calculated, and 75% of active promoters were found to be associated with transcriptional activity of their gene counterparts. These results provide experimental evidence of promoter activity, which may aid in understanding the regulation of gene expression. Moreover, this is the first large-scale functional study of regulatory sequences to use a high-throughput transfected-cell array technique

Functional analysis and identification of cis-regulatory elements of human chromosome 21 gene promoters

Given the inherent limitations of in silico studies relying solely on DNA sequence analysis, the functional characterization of mammalian promoters and associated cis-regulatory elements requires experimental support, which demands cloning and analysis of putative promoter regions. Focusing on human chromosome 21, we cloned 182 gene promoters of 2500 bp in length and conducted reporter gene assays on transfected-cell arrays. We found 56 promoters that were active in HEK293 cells, while another 49 promoters could be activated by treatment of cells with Trichostatin A or depletion of serum. We observed high correlations between promoter activities and endogenous transcript levels, RNA polymerase II occupancy, CpG islands and core promoter elements. Truncation of a subset of 62 promoters to ∼500 bp revealed that truncation rarely resulted in loss of activity, but rather in loss of responses to external stimuli, suggesting the presence of cis-regulatory response elements within distal promoter regions. In these regions, we found a strong enrichment of transcription factor binding sites that could potentially activate gene expression in the presence of stimuli. This study illustrates the modular functional architecture of chromosome 21 promoters and helps to reveal the complex mechanisms governing transcriptional regulatio

Washing scaling of GeneChip microarray expression

BACKGROUND Post-hybridization washing is an essential part of microarray experiments. Both the quality of the experimental washing protocol and adequate consideration of washing in intensity calibration ultimately affect the quality of the expression estimates extracted from the microarray intensities. RESULTS We conducted experiments on GeneChip microarrays with altered protocols for washing, scanning and staining to study the probe-level intensity changes as a function of the number of washing cycles. For calibration and analysis of the intensity data we make use of the 'hook' method which allows intensity contributions due to non-specific and specific hybridization of perfect match (PM) and mismatch (MM) probes to be disentangled in a sequence specific manner. On average, washing according to the standard protocol removes about 90% of the non-specific background and about 30-50% and less than 10% of the specific targets from the MM and PM, respectively. Analysis of the washing kinetics shows that the signal-to-noise ratio doubles roughly every ten stringent washing cycles. Washing can be characterized by time-dependent rate constants which reflect the heterogeneous character of target binding to microarray probes. We propose an empirical washing function which estimates the survival of probe bound targets. It depends on the intensity contribution due to specific and non-specific hybridization per probe which can be estimated for each probe using existing methods. The washing function allows probe intensities to be calibrated for the effect of washing. On a relative scale, proper calibration for washing markedly increases expression measures, especially in the limit of small and large values. CONCLUSIONS Washing is among the factors which potentially distort expression measures. The proposed first-order correction method allows direct implementation in existing calibration algorithms for microarray data. We provide an experimental 'washing data set' which might be used by the community for developing amendments of the washing correction.This publication is supported by the Leipzig Interdisciplinary Research Cluster of Genetic Factors, Clinical Phenotypes and Environment (LIFE Center, Universität Leipzig) and an Australian Academy of Science Visits to Europe grant. LIFE is funded by means of the European Union, by the European Regional Development Fund (ERFD) and by means of the Free State of Saxony within the framework of the excellence initiative

Functional analysis and identification of cis-regulatory elements of human chromosome 21 gene promoters

Given the inherent limitations of in silico studies relying solely on DNA sequence analysis, the functional characterization of mammalian promoters and associated cis-regulatory elements requires experimental support, which demands cloning and analysis of putative promoter regions. Focusing on human chromosome 21, we cloned 182 gene promoters of 2500 bp in length and conducted reporter gene assays on transfected-cell arrays. We found 56 promoters that were active in HEK293 cells, while another 49 promoters could be activated by treatment of cells with Trichostatin A or depletion of serum. We observed high correlations between promoter activities and endogenous transcript levels, RNA polymerase II occupancy, CpG islands and core promoter elements. Truncation of a subset of 62 promoters to ∼500 bp revealed that truncation rarely resulted in loss of activity, but rather in loss of responses to external stimuli, suggesting the presence of cis-regulatory response elements within distal promoter regions. In these regions, we found a strong enrichment of transcription factor binding sites that could potentially activate gene expression in the presence of stimuli. This study illustrates the modular functional architecture of chromosome 21 promoters and helps to reveal the complex mechanisms governing transcriptional regulation

Yeast

An increase in the mitochondrial rho- mutagenesis is a well-known response of yeast cells to mutations in numerous nuclear genes as well as to various kinds of stress. Despite extensive studies for several decades, the biological significance of this response is still not fully understood. The genetic approach to solving this enigma includes a study of genes that are required for the high incidence of spontaneous rho- mutants. We have obtained mutations of a few nuclear genes of that sort and found that mutations in certain genes, including CDC28, the central cell-cycle regulation gene, result in a decrease in spontaneous rho- mutability and simultaneously affect the maintenance of the yeast chromosomes and plasmids. Two more genes resembling CDC28 in this respect are identified in the present work as a result of the characterization of four new mutants. These two genes are NET1 and HFI1 which mediate important regulatory protein-protein interactions in the yeast cell. The effects of four mutations, including net1-srm and hfi1-srm, on the maintenance of the yeast mitochondrial genome, chromosomes and plasmids, as well as on the cell's sensitivity to ionizing radiation, are also described. The data presented suggest that the pleiotropic srm mutations determining coordinate changes in the fidelity of mitotic transmission of chromosomes, plasmids and mtDNA molecules identify genes that most probably operate high up in the hierarchy of the general genetic regulation of yeast