Using high-density exon arrays to profile gene expression in closely related species

Global comparisons of gene expression profiles between species provide significant insight into gene regulation, evolutionary processes and disease mechanisms. In this work, we describe a flexible and intuitive approach for global expression profiling of closely related species, using high-density exon arrays designed for a single reference genome. The high-density probe coverage of exon arrays allows us to select identical sets of perfect-match probes to measure expression levels of orthologous genes. This eliminates a serious confounding factor in probe affinity effects of species-specific microarray probes, and enables direct comparisons of estimated expression indexes across species. Using a newly designed Affymetrix exon array, with eight probes per exon for approximately 315 000 exons in the human genome, we conducted expression profiling in corresponding tissues from humans, chimpanzees and rhesus macaques. Quantitative real-time PCR analysis of differentially expressed candidate genes is highly concordant with microarray data, yielding a validation rate of 21/22 for human versus chimpanzee differences, and 11/11 for human versus rhesus differences. This method has the potential to greatly facilitate biomedical and evolutionary studies of gene expression in nonhuman primates and can be easily extended to expression array design and comparative analysis of other animals and plants.     

Exon arrays provide accurate assessments of gene expression

We have developed a strategy for estimating gene expression on Affymetrix Exon arrays. The method includes a probe-specific background correction and a probe selection strategy in which a subset of probes with highly correlated intensities across multiple samples are chosen to summarize gene expression. Our results demonstrate that the proposed background model offers improvements over the default Affymetrix background correction and that Exon arrays may provide more accurate measurements of gene expression than traditional 3' arrays.     

Complete nucleotide sequence of the high molecular weight human IGF-I mRNA

IGF-I gene expression in mammals typically results in multiple mRNA species ranging in size between 0.7 and 7.6 kb. The smaller mRNA species have largely been characterized by the analysis of nearly full-length cDNAs. This report describes the first complete sequence of the prominent high molecular weight (7.6 kb) IGF-I mRNA species. Isolation and nucleotide sequence analysis of cDNA clones from human adult liver and uterus leiomyoma cDNA libraries resulted in a 7236 bp long sequence followed by a poly(A) tail. The sequence data, in combination with structural analysis of the human IGF-I gene, show that the 7.6 kb human IGF-I mRNA contains 6611 bp of untranslated 3' terminal sequence derived from a single exon. Alternate employment of two polyadenylation signals within the sequence transcribed from this exon generates two mRNAs of 1.1 and 7.6 kb.     

A sequence-oriented comparison of gene expression measurements across different hybridization-based technologies

Over the last decade, gene expression microarrays have had a profound impact on biomedical research. The diversity of platforms and analytical methods available to researchers have made the comparison of data from multiple platforms challenging. In this study, we describe a framework for comparisons across platforms and laboratories. We have attempted to include nearly all the available commercial and 'in-house' platforms. Using probe sequences matched at the exon level improved consistency of measurements across the different microarray platforms compared to annotation-based matches. Generally, consistency was good for highly expressed genes, and variable for genes with lower expression values as confirmed by quantitative real-time (QRT)-PCR. Concordance of measurements was higher between laboratories on the same platform than across platforms. We demonstrate that, after stringent preprocessing, commercial arrays were more consistent than in-house arrays, and by most measures, one-dye platforms were more consistent than two-dye platforms.     

Using DNA microarrays to study gene expression in closely related species

MOTIVATION: Comparisons of gene expression levels within and between species have become a central tool in the study of the genetic basis for phenotypic variation, as well as in the study of the evolution of gene regulation. DNA microarrays are a key technology that enables these studies. Currently, however, microarrays are only available for a small number of species. Thus, in order to study gene expression levels in species for which microarrays are not available, researchers face three sets of choices: (i) use a microarray designed for another species, but only compare gene expression levels within species, (ii) construct a new microarray for every species whose gene expression profiles will be compared or (iii) build a multi-species microarray with probes from each species of interest. Here, we use data collected using a multi-primate cDNA array to evaluate the reliability of each approach. RESULTS: We find that, for inter-species comparisons, estimates of expression differences based on multi-species microarrays are more accurate than those based on multiple species-specific arrays. We also demonstrate that within-species expression differences can be estimated using a microarray for a closely related species, without discernible loss of information. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Transcriptional signatures of normal human mammary epithelial cells in response to benzo[a]pyrene exposure: a comparison of three microarray platforms

Microarrays are used to study gene expression in a variety of biological systems. A number of different platforms have been developed, but few studies exist that have directly compared the performance of one platform with another. The goal of this study was to determine array variation by analyzing the same RNA samples with three different array platforms. Using gene expression responses to benzo[a]pyrene exposure in normal human mammary epithelial cells (NHMECs), we compared the results of gene expression profiling using three microarray platforms: photolithographic oligonucleotide arrays (Affymetrix), spotted oligonucleotide arrays (Amersham), and spotted cDNA arrays (NCI). While most previous reports comparing microarrays have analyzed pre-existing data from different platforms, this comparison study used the same sample assayed on all three platforms, allowing for analysis of variation from each array platform. In general, poor correlation was found with corresponding measurements from each platform. Each platform yielded different gene expression profiles, suggesting that while microarray analysis is a useful discovery tool, further validation is needed to extrapolate results for broad use of the data. Also, microarray variability needs to be taken into consideration, not only in the data analysis but also in specific probe selection for each array type.     

Molecular Evolution of Vertebrate Goose-Type Lysozyme Genes

We have found that mammalian genomes contain two lysozyme g genes. To better understand the function of the lysozyme g genes we have examined the evolution of this small gene family. The lysozyme g gene structure has been largely conserved during vertebrate evolution, except at the 5' end of the gene, which varies in number of exons. The expression pattern of the lysozyme g gene varies between species. The fish lysozyme g sequences, unlike bird and mammalian lysozyme g sequences, do not predict a signal peptide, suggesting that the encoded proteins are not secreted. The fish sequences also do not conserve cysteine residues that generate disulfide bridges in the secreted bird enzymes, supporting the hypothesis that the fish enzymes have an intracellular function. The signal peptide found in bird and mammalian lysozyme g genes may have been acquired as an exon in the ancestor of birds and mammals, or, alternatively, an exon encoding the signal peptide has been lost in fish. Both explanations account for the change in gene structure between fish and tetrapods. The mammalian lysozyme g sequences were found to have evolved at an accelerated rate, and to have not perfectly conserved the known active site catalytic triad of the bird enzymes. This observation suggests that the mammalian enzymes may have altered their biological function, as well.     

Organization of telomere sequences in birds: evidence for arrays of extreme length and for in vivo shortening

Telomeres are the specialized ends of chromosomes consisting of highly conserved repeat (5'-TTAGGG-3')(n) sequences. Lack of information regarding the existence of an in vivo telomere clock function in birds, conflicting data regarding telomere array length in the chicken model, and the paucity of molecular telomere information for other avian species led us to study telomere array organization within and among 18 species and subspecies of birds. Most of the species contained between 2% and 4% telomere sequence per diploid genome. Arrays spanning 0.5-10 kb (Class I) and 10-40 kb (Class II) were observed in all of the species studied. Extremely long arrays, ranging from hundreds of kilobases to 1-2 Mb (Class III) were observed in all except two raptor species, the northern goshawk and American bald eagle. In chicken, there was evidence for shortening of the Class II arrays in vivo, based on intraindividual comparisons of somatic versus germline tissues in birds of different ages; terminally differentiated erythrocyte arrays were, on average, 2.3 kb shorter than sperm (germline) arrays. This study provides the first evidence for the existence of telomere arrays significantly larger than have been described for any vertebrate species to date and for developmentally programmed in vivo telomere shortening in the Aves taxa. The novel finding of megabase-sized telomere arrays may be an important feature of avian karyotypes that contain a large number of very small genetic units, the microchromosomes.     

A method for cross-species gene expression analysis with high-density oligonucleotide arrays

DNA microarrays have been widely used in gene expression analysis of biological processes. Due to a lack of sequence information, the applications have been largely restricted to humans and a few model organisms. Presented within this study are results of the cross-species hybridization with Affymetrix human high-density oligonucleotide arrays or GeneChip® using distantly related mammalian species; cattle, pig and dog. Based on the unique feature of the Affymetrix GeneChip® where every gene is represented by multiple probes, we hypothesized that sequence conservation within mammals is high enough to generate sufficient signals from some of the probes for expression analysis. We demonstrated that while overall hybridization signals are low for cross-species hybridization, a few probes of most genes still generated signals equivalent to the same-species hybridization. By masking the poorly hybridized probes electronically, the remaining probes provided reliable data for gene expression analysis. We developed an algorithm to select the reliable probes for analysis utilizing the match/mismatch feature of GeneChip®. When comparing gene expression between two tissues using the selected probes, we found a linear correlation between the cross-species and same-species hybridization. In addition, we validated cross-species hybridization results by quantitative PCR using randomly selected genes. The method shown herein could be applied to both plant and animal research.