Cross-species Microarray Research Articles

Microarray and real-time PCR analyses reveal mating type-dependent gene expression in a homothallic fungus

Sordaria macrospora is a homothallic ascomycete which is able to form fertile fruiting bodies without a mating partner. To analyze the molecular basis of homothallism and the role of mating products during fruiting body development, we have deleted the mating type gene Smta-1 encoding a high-mobility group domain (HMG) protein. The DeltaSmta-1 deletion strain is morphologically wild type during vegetative growth, but it is unable to produce perithecia or ascospores. To identify genes expressed under control of Smta-1, we performed a cross-species microarray analysis using Neurospora crassa cDNA microarrays hybridized with S. macrospora targets. We identified 107 genes that are more than twofold up- or down-regulated in the mutant. Functional classification revealed that 81 genes have homologues with known or putative functions. Comparison of array data from DeltaSmta-1 with those from three phenotypically similar mutants revealed that only a limited set of ten genes is deregulated in all mutants. Remarkably, the ppg2 gene encoding a putative lipopeptide pheromone is 500-fold down-regulated in the DeltaSmta-1 mutant while in all other sterile mutants this gene is up-regulated.

Cross-species microarray hybridization to identify developmentally regulated genes in the filamentous fungus Sordaria macrospora

The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies that protect the developing ascospores and ensure their proper discharge. Several regulatory genes essential for fruiting body development were previously isolated by complementation of the sterile mutants pro1, pro11 and pro22. To establish the genetic relationships between these genes and to identify downstream targets, we have conducted cross-species microarray hybridizations using cDNA arrays derived from the closely related fungus Neurospora crassa and RNA probes prepared from wild-type S. macrospora and the three developmental mutants. Of the 1,420 genes which gave a signal with the probes from all the strains used, 172 (12%) were regulated differently in at least one of the three mutants compared to the wild type, and 17 (1.2%) were regulated differently in all three mutant strains. Microarray data were verified by Northern analysis or quantitative real time PCR. Among the genes that are up- or down-regulated in the mutant strains are genes encoding the pheromone precursors, enzymes involved in melanin biosynthesis and a lectin-like protein. Analysis of gene expression in double mutants revealed a complex network of interaction between the pro gene products.

Cross-species hybridisation of pig RNA to human nylon microarrays

BackgroundThe objective of this research was to investigate the reproducibility of cross-species microarray hybridisation. Comparisons between same- and cross-species hybridisations were also made. Nine hybridisations between a single pig skeletal muscle RNA sample and three human cDNA nylon microarrays were completed. Three replicate hybridisations of two different amounts of pig RNA, and of human skeletal muscle RNA were completed on three additional microarrays.ResultsReproducibility of microarray hybridisations of pig cDNA to human microarrays was high, as determined by Spearman and Pearson correlation coefficients and a Kappa statistic. Variability among replicate hybridisations was similar for human and pig data, indicating the reproducibility of results were not compromised in cross-species hybridisations. The concordance between data generated from hybridisations using pig and human skeletal muscle RNA was high, further supporting the use of human microarrays for the analysis of gene expression in the pig. No systematic effect of stripping and re-using nylon microarrays was found, and variability across microarrays was minimal.ConclusionThe majority of genes generated highly reproducible data in cross-species microarray hybridisations, although approximately 6% were identified as highly variable. Experimental designs that include at least three replicate hybridisations for each experimental treatment will enable the variability of individual genes to be considered appropriately. The use of cross-species microarray analysis looks promising. However, additional validation is needed to determine the specificity of cross-species hybridisations, and the validity of results.

Upregulation of collagens detected by gene array in a model of flow-induced pulmonary vascular remodeling.

We recently reported localized increased pulmonary arterial resistance, neointimal lesions, and medial thickening induced by aortopulmonary anastomosis in young pigs. This model was used to investigate changes in expression of genes potentially involved in pulmonary vascular remodeling employing a high throughput Atlas Human Cardiovascular Array carrying approximately 600 cardiovascular-related cDNA sequences. Data were confirmed by Northern analysis, Western blots, and histological examination. With the use of lower stringency conditions for hybridization, 56% of the 588 human genes on the array showed visible signal after autoradiography. Approximately 10% of the genes with visible hybridization were altered by shunt-induced high flow. Extracellular matrix and cell adhesion molecules were the most highly represented group of upregulated genes. To our knowledge, our data are the first to demonstrate flow-induced changes in gene expression using a combination of cross species cDNA arrays, homologous hybridization, immunospecific protein, and histology. Our observations expand the list of genes as putative candidates in pulmonary vascular remodeling and support the utility of cross-species microarray analysis in such applications.