Quantitative comparison of cDNA-AFLP, microarrays, and GeneChip expression data in Saccharomyces cerevisiae

cDNA-AFLP is a genome-wide expression analysis technology that does not require any prior knowledge of gene sequences. This PCR-based technique combines a high sensitivity with a high specificity, allowing detection of rarely expressed genes and distinguishing between homologous genes. In this report, we validated quantitative expression data of 110 cDNA-AFLP fragments in yeast with DNA microarrays and GeneChip data. The best correlation was found between cDNA-AFLP and GeneChip data. The cDNA-AFLP data revealed a low number of inconsistent profiles that could be explained by gel artifact, overexposure, or mismatch amplification. In addition, 18 cDNA-AFLP fragments displayed homology to genomic yeast DNA, but could not be linked unambiguously to any known ORF. These fragments were most probably derived from 5' or 3' noncoding sequences or might represent previously unidentified ORFs. Genes liable to cross hybridization showed identical results in cDNA-AFLP and GeneChip analysis. Three genes, which were readily detected with cDNA-AFLP, showed no significant expression in GeneChip experiments. We show that cDNA-AFLP is a very good alternative to microarrays and since no preexisting biological or sequence information is required, it is applicable to any species.     

Gene expression profiling of human diseases by serial analysis of gene expression

Until recently, the approach to understanding the molecular basis of complex syndromes such as cancer, coronary artery disease, and diabetes was to study the behavior of individual genes. However, it is generally recognized that expression of a number of genes is coordinated both spatially and temporally and that this coordination changes during the development and progression of diseases. Newly developed functional genomic approaches, such as serial analysis of gene expression (SAGE) and DNA microarrays have enabled researchers to determine the expression pattern of thousands of genes simultaneously. One attractive feature of SAGE compared to microarrays is its ability to quantify gene expression without prior sequence information or information about genes that are thought to be expressed. SAGE has been successfully applied to the gene expression profiling of a number of human diseases. In this review, we will first discuss SAGE technique and contrast it to microarray. We will then highlight new biological insights that have emerged from its application to the study of human diseases.     

The organization of the histone genes in the genome of Xenopus laevis.

We have studied the organization of the histone genes in the DNA from several individuals of Xenopus laevis. For that purpose, Southern blots of genomic DNA, that was digested with several restriction enzymes, were hybridized with radioactively labeled DNA fragments from clone X1-hi-1 (14), containing genes for Xenopus histones H2A, H2B, H3 and H4. In the DNA of all animals that were screened we found a major repeating unit of 14 kilobasepairs, which contains genes for histones H2A, H2B, H3 and H4 (H1 not tested) and is represented up to 30 times in the genome. The order of the genes in this major repeating unit is H4 - H3 - H2A - H2B. This order is different from that in the histone DNA of clone X1-hi-1, i.e. H3 - H4 - H2A - H2B. In addition to the genes in the major repeating unit, histone genes are present in unique restriction fragments in numbers that vary from one animal to another. The restriction patterns for the histone genes in these unique fragments were found to be different for all eight Xenopus individuals that were screened. The cloned Xenopus histone gene fragment X1-hi-1 represents such a unique fragment and is not present in the DNA of each single individual. The total number of genes coding for each of the nucleosomal histones is 45-50 per haploid genome.     

Genomic microarrays in the spotlight

Microarray-based comparative genomic hybridization (array-CGH) has emerged as a revolutionary platform, enabling the high-resolution detection of DNA copy number aberrations. In this article we outline the use and limitations of genomic clones, cDNA clones and PCR products as targets for genomic microarray construction. Furthermore, the applications and future aspects of these arrays for DNA copy number analysis in research and diagnostics, epigenetic profiling and gene annotation are discussed. These recent developments of genomic microarrays mark only the beginning of a new generation of high-resolution and high-throughput tools for genetic analysis.     

伤寒沙门菌基因组DNA芯片的制备与基因表达谱分析应用

伤寒沙门菌是一种具有鞭毛的革兰阴性人类肠道致病菌,也是一种重要的原核生物研究用模式菌．基因组芯片能够系统、全面且高效地观察生物的基因表达及进行基因组结构比较．利用伤寒沙门菌现有的全基因组序列,以Ty2菌株的基因组为基准,选取CT18菌株和z66阳性菌株的特异性蛋白编码基因,设计特异性引物,经PCR有效扩增出4 201个基因,产物纯化后点样于多聚赖氨酸玻片制备伤寒沙门菌基因组DNA芯片,并验证了芯片样点位次与效果．通过对基因表达谱分析的各种条件进行优化,建立相应的表达谱分析方法,并用于比较伤寒沙门菌野生株在高渗、低渗条件下的基因表达差异,结果与以前的报道基本一致．结果表明,成功建立了伤寒沙门菌基因组DNA芯片及表达谱分析方法,可为有关伤寒沙门菌基因表达调控及致病性机理、进化和基因多样性等方面的深入研究提供有效的技术支持．     

Regression as a method to predict copy numbers in comparative genomic hybridization studies on bacteria

Comparative genomic hybridizations (CGH) using microarrays are performed with bacteria in order to determine the level of genomic similarity between various strains. The microarrays applied in CGH experiments are constructed on the basis of the genome sequence of one strain, which is used as a control, or reference, in each experiment. A strain being compared with the known strain is called the unknown strain. The ratios of fluorescent intensities obtained from the spots on the microarrays can be used to determine which genes are divergent in the unknown strain, as well as to predict the copy number of actual genes in the unknown strain. In this paper, we focus on the prediction of gene copy number based on data from CGH experiments. We assumed a linear connection between the log2 of the copy number and the observed log2-ratios, then predictors based on the factor analysis model and the linear random model were proposed in an attempt to identify the copy numbers. These predictors were compared to using the ratio of the intensities directly. Simulations indicated that the proposed predictors improved the prediction of the copy number in most situations. The predictors were applied on CGH data obtained from experiments with Enterococcus faecalis strains in order to determine copy number of relevant genes in five different strains.     

The SphS-SphR two component system is the exclusive sensor for the induction of gene expression in response to phosphate limitation in synechocystis

Living organisms respond to phosphate limitation by expressing various genes whose products maintain an appropriate range of phosphate concentrations within each cell. We identified previously a two component system, which consists of histidine kinase SphS and its cognate response regulator SphR, which regulates the expression of the phoA gene for alkaline phosphatase under phosphate-limiting conditions in the cyanobacterium Synechocystis sp. PCC 6803. In the present study, we used DNA microarrays to investigate the role of SphS and SphR in the regulation of the genome-wide expression of genes in response to phosphate limitation. In wild-type cells, phosphate limitation strongly induced the expression of 12 genes with induction factors greater than 7. These genes were included in three clusters of genes, namely, the pst1 and pst2 clusters that encode phosphate transporters; the phoA gene and the nucH gene for the extracellular nuclease. Phosphate limitation strongly repressed the expression of only the urtA gene with induction factors below 0.2. Inactivation of either of SphS or SphR completely eliminated the phosphate limitation-inducible expression of the 12 genes and the phosphate limitation-repressible expression of the urtA gene. These results suggest that the SphS-SphR two component system in Synechocystis sp. PCC 6803 is the dominant sensory system that controls gene expression in response to phosphate limitation.     

Experimental analysis of recently transposed insertion sequences in the cyanobacterium Synechocystis sp. PCC 6803.

The genome DNA of the cyanobacterium Synechocystis sp. PCC 6803 carries a number of insertion sequences (Kaneko, T. et al. 1996, DNA Res., 3, 109-136). We analyzed one of the abundant ISs (ISY203 group of IS4 family) in the common three substrains of Synechocystis and found that the four ISs with identical nucleotide sequences were present only in the "Kazusa" strain, whose complete genome sequence had been determined, while absent in ancestral strains (the original strain from Pasteur Culture Collection and its glucose-tolerant derivative). Three of these ISs were found in the genomic sequence as transposase genes of sll1474, sll1780 and slr1635. The fourth was on the plasmid, pSYSM. On the other hand, all three strains had a novel IS (denoted ISY203x), of which the nucleotide sequence was totally identical to the four ISs found only in the Kazusa strain. Since the flanking regions of ISY203x did not match any part of the genome or of the known plasmids of Synechocystis, it is presumably located on a yet uncharacterized plasmid. These suggest that the four ISs in Kazusa strain were recently transposed from ISY203x. Apparently, the transposition inactivated four preexisting genes, of which modified forms are presented as putative genes (sll1473, sll1475, slr1862, slr1863, slr1635 and ssl2982) in the list of the complete genome (CyanoBase: http://www.kazusa.or.jp/cyano/cyano.html). The possible effects of transposition of ISs in Synechocystis are discussed in relation to phenotypic mutations and microevolution.     

Comparative genomics and experimental promoter analysis reveal functional liver-specific elements in mammalian hepatic lipase genes

Background

Array-based comparative genomic hybridization (aCGH) is a high-throughput method for measuring genome-wide DNA copy number changes. Current aCGH methods have limited resolution, sensitivity and reproducibility. Microarrays for aCGH are available only for a few organisms and combination of aCGH data with expression data is cumbersome.

Results

We present a novel method of using commercial oligonucleotide expression microarrays for aCGH, enabling DNA copy number measurements and expression profiles to be combined using the same platform. This method yields aCGH data from genomic DNA without complexity reduction at a median resolution of approximately 17,500 base pairs. Due to the well-defined nature of oligonucleotide probes, DNA amplification and deletion can be defined at the level of individual genes and can easily be combined with gene expression data.

Conclusion

A novel method of gene resolution analysis of copy number variation (graCNV) yields high-resolution maps of DNA copy number changes and is applicable to a broad range of organisms for which commercial oligonucleotide expression microarrays are available. Due to the standardization of oligonucleotide microarrays, graCNV results can reliably be compared between laboratories and can easily be combined with gene expression data using the same platform.     

Hybridization of genomic DNA to microarrays: a challenge for the analysis of environmental samples

The use of DNA microarrays for detection and identification of bacteria and genes of interest from various environments (e.g. soil, sediment, water column...) is a major challenge for microbiologists working on functional diversity. So far, most of the genomic methods that have been described rely on the use of taxonomic markers (such as 16S rRNA) that can be easily amplified by PCR prior to hybridization on microarrays. However, taxonomical markers are not always informative on the functions present in these bacteria. Moreover, genes for which sequence database is limited or that lack any conserved regions will be difficult to amplify and thus to detect in unknown samples. Furthermore, PCR amplification often introduces biases that lead to inaccurate analysis of microbial communities. An alternative solution to overcome these strong limitations is to use genomic DNA (gDNA) as target for hybridisation, without prior PCR amplification. Though hybridization of gDNA is already used for comparative genome hybridization or sequencing by hybridization, yet to the high cost of tiling strategies and important data filtering, its adaptation for use in environmental research poses great challenges in terms of specificity, sensitivity and reproducibility of hybridization. Considering the very faint number of publications that have described hybridization of gDNA to microarrays for environmental applications, we confront in this review the different approaches that have been developed so far, and propose alternative strategies that may contribute to improve the development of microarrays for studying the microbial genetic structure and composition of samples of high environmental and ecological value.     

Mathematical design of prokaryotic clone-based microarrays

Background  

Clone-based microarrays, on which each spot represents a random genomic fragment, are a good alternative to open reading frame-based microarrays, especially for microorganisms for which the complete genome sequence is not available. Since the generation of a genomic DNA library is a random process, it is beforehand uncertain which genes are represented. Nevertheless, the genome coverage of such an array, which depends on different variables like the insert size and the number of clones in the library, can be predicted by mathematical approaches. When applying the classical formulas that determine the probability that a certain sequence is represented in a DNA library at the nucleotide level, massive amounts of clones would be necessary to obtain a proper coverage of the genome.     

Genomic changes during chronic Helicobacter pylori infection

The gastric pathogen Helicobacter pylori shows tremendous genetic variability within human populations, both in gene content and at the sequence level. We investigated how this variability arises by comparing the genome content of 21 closely related pairs of isolates taken from the same patient at different time points. The comparisons were performed by hybridization with whole-genome DNA microarrays. All loci where microarrays indicated a genomic change were sequenced to confirm the events. The number of genomic changes was compared to the number of homologous replacement events without loss or gain of genes that we had previously determined by multilocus sequence analysis and mathematical modeling based on the sequence data. Our analysis showed that the great majority of genetic changes were due to homologous recombination, with 1/650 events leading to a net gain or loss of genes. These results suggest that adaptation of H. pylori to the host individual may principally occur through sequence changes rather than loss or gain of genes.     

Genomic DNA microarray analysis: identification of new genes regulated by light color in the cyanobacterium Fremyella diplosiphon

Many cyanobacteria use complementary chromatic adaptation to efficiently utilize energy from both green and red regions of the light spectrum during photosynthesis. Although previous studies have shown that acclimation to changing light wavelengths involves many physiological responses, research to date has focused primarily on the expression and regulation of genes that encode proteins of the major photosynthetic light-harvesting antennae, the phycobilisomes. We have used two-dimensional gel electrophoresis and genomic DNA microarrays to expand our understanding of the physiology of acclimation to light color in the cyanobacterium Fremyella diplosiphon. We found that the levels of nearly 80 proteins are altered in cells growing in green versus red light and have cloned and positively identified 17 genes not previously known to be regulated by light color in any species. Among these are homologs of genes present in many bacteria that encode well-studied proteins lacking clearly defined functions, such as tspO, which encodes a tryptophan-rich sensory protein, and homologs of genes encoding proteins of clearly defined function in many species, such as nblA and chlL, encoding phycobilisome degradation and chlorophyll biosynthesis proteins, respectively. Our results suggest novel roles for several of these gene products and highly specialized, unique uses for others.