Applications of DNA and protein microarrays in comparative physiology

DNA microarrays have revolutionized gene expression studies and made large-scale parallel measurement of whole genome expression a feasible technique in model species where genomes are well characterized. Such studies are perfectly suited to unraveling the complex regulation and/or interaction of both genes and proteins likely involved in most physiological processes. Gene expression profiles are currently being used to identify genes underlying a range of physiological responses. Characterization of these genes will help to elucidate the pathways and processes regulating physiological processes. Expanding the use of DNA microarrays to non-model species that have been critical in elucidating certain physiological pathways will be valuable in determining the genes associated with these processes. Approaches that do not require complete genome information have recently been applied to "non-model" organisms. As whole genomes are sequenced for non-model organisms, the application of DNA microarrays to comparative physiology will expand even further. The recent development of protein microarrays will be critical in understanding the regulation of physiological processes not accounted for at the genomic level. Together, DNA and protein microarrays provide the most thorough and efficient method of understanding the molecular basis of physiological processes to date. In turn, classical physiological approaches will be vital in characterizing and verifying the function of the novel genes identified by microarray experiments. Ultimately, DNA and protein microarray expression profiles may be used to predict physiological responses.     

Measuring and analyzing tissue specificity of human genes and protein complexes

Proteins and their interactions are essential for the survival of each human cell. Knowledge of their tissue occurrence is important for understanding biological processes. Therefore, we analyzed microarray and high-throughput RNA-sequencing data to identify tissue-specific and universally expressed genes. Gene expression data were used to investigate the presence of proteins, protein interactions and protein complexes in different tissues. Our comparison shows that the detection of tissue-specific genes and proteins strongly depends on the applied measurement technique. We found that microarrays are less sensitive for low expressed genes than high-throughput sequencing. Functional analyses based on microarray data are thus biased towards high expressed genes. This also means that previous biological findings based on microarrays might have to be re-examined using high-throughput sequencing results.     

Transcriptional regulation of meiosis in yeast

The genes required for meiosis and sporulation in yeast are expressed at specific points in a highly regulated temporal pathway. Recent experiments using DNA microarrays to examine gene expression during meiosis and the identification of many regulatory factors have provided important advances in our understanding of how genes are regulated at the different stages of meiosis.     

Clinical and functional target validation using tissue and cell microarrays

Expression levels of thousands of genes or proteins can be readily determined using microarray techniques. However, this represents only the first step in understanding the biological and medical significance of these molecules. New high-throughput techniques, such as tissue and cell microarrays, will facilitate clinical and functional analysis of molecular targets.     

Identification of genes responsive to low-intensity pulsed ultrasound stimulations

This study was designed to compare the temporal changes of gene expression profile in osteoblastic cell lines (SaOS-2) treated with low-intensity pulsed ultrasound stimulation (LIPUS) using complementary DNA (cDNA) microarrays. SaOS-2 cells were treated with LIPUS for 20 min. Thereafter, cells were harvested and RNA was extracted twice at 4 and 24 h, respectively. Using cDNA microarrays, 7488 genes with changes in expression in SaOS-2 cells were identified for comparison. Microarray analysis revealed a total of 165 genes in SaOS-2 cells were regulated at 4 and 24 h after LIPUS treatment. Except for 30 known LIPUS-regulated genes, our study demonstrated for the first time that over 100 genes were related to the underlying molecular mechanism of LIPUS and suggested that LIPUS might regulate a transient expression of numerous critical genes in osteoblastic cells. These results provide further understanding of the role of LIPUS in the regulation of osteoblastic gene expression potentially involved in the molecular mechanism of osteogenesis in fracture repair.     

Identification of changes in gene loci potentially associated with cervical cancer using NotI microarrays

The investigation of the cancer-associated structural and epigenetic changes in cell genome is a major approach for understanding mechanisms of cancerogenesis. To investigate these genome changes, novel technique of microarrays comprising NotI-linking genome clones was developed. Twenty eight samples from patients with cervical cancer were analyzed using NotI microarrays of human chromosome 3. Deletions, amplifications and methylation were detected for 109 out of 182 NotI clones with different frequency. Notably, 17 NotI-linking clones showed genomic changes in more than 35% of tumor samples investigated, which suggests involvement of genes associated with these clones in development of cervical cancer.     

Microarray-based genomic surveying of gene polymorphisms in <Emphasis Type="Italic">Chlamydia trachomatis</Emphasis>

By comparing two fully sequenced genomes of Chlamydia trachomatis using competitive hybridization on DNA microarrays, a logarithmic correlation was demonstrated between the signal ratio of the arrays and the 75-99% range of nucleotide identities of the genes. Variable genes within 14 uncharacterized strains of C. trachomatis were identified by array analysis and verified by DNA sequencing. These genes may be crucial for understanding chlamydial virulence and pathogenesis.     

Identification of genes responsive to sodium butyrate in colonic epithelial cells

We identified genes responsive to sodium butyrate (SB) in colonic epithelial cells using cDNA microarrays. Treatment with 2 mM SB of colonic epithelial cells (MCE301), which was derived from transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen, arrested cell growth and showed a differentiated phenotype accompanying an increase in alkaline phosphatase activity. Of the approximately 900 genes analyzed, SB down-regulated 25 genes and up-regulated 88 genes by a factor of 2.0 or greater. Northern blot or TaqMan and Western blot analyses confirmed that the mRNA and protein levels of cyclin D1 and the level of proliferating cell nuclear antigen decreased, whereas the levels of integrin beta1 and osteopontin increased. The present results regarding the changes in gene expression, arrived at using microarrays, will provide a basis for a further understanding of the molecular mechanisms of cell growth arrest and differentiation in response to SB in colonic epithelial cells.     

DNA微阵列技术及其在肿瘤生物学中的应用

人类基因组测序工作的完成使人们可以方便地调用任何基因序列,但仅有基因序列并不能解释众多的生物学问题,这要求发展一种高通量的技术用于研究基因的生物学功能以及基因的相互作用。DNA微阵列技术以其高通量的特点,已经在肿瘤生物学的研究中逐渐被采用。由于癌症是源于基因表达谱改变的基因疾病,通过DNA微阵列技术研究癌症细胞和对应的正常细胞的基因表达差异,将会使人们更好地了解肿瘤的形成和发展过程。     

Generalist genes and cognitive neuroscience

Multivariate genetic research suggests that a single set of genes affects most cognitive abilities and disabilities. This finding already has far-reaching implications for cognitive neuroscience, and will become even more revealing when this - presumably large - set of generalist genes is identified. Similar to other complex disorders and dimensions, molecular genetic research on cognitive abilities and disabilities is adopting genome-wide association strategies. These strategies involve very large samples to detect DNA associations of small effect size using microarrays that simultaneously assess hundreds of thousands of DNA markers. When this set of generalist genes is identified, it can be used to provide solid footholds in the climb towards a systems-level understanding of how genetically driven brain processes work together to affect diverse cognitive abilities and disabilities.     

The next generation of microarray research: applications in evolutionary and ecological genomics

Microarray technology is one of the key developments in recent years that has propelled biological research into the post-genomic era. With the ability to assay thousands to millions of features at the same time, microarray technology has fundamentally changed how biological questions are addressed, from examining one or a few genes to a collection of genes or the whole genome. This technology has much to offer in the study of genome evolution. After a brief introduction on the technology itself, we then focus on the use of microarrays to examine genome dynamics, to uncover novel functional elements in genomes, to unravel the evolution of regulatory networks, to identify genes important for behavioral and phenotypic plasticity, and to determine microbial community diversity in environmental samples. Although there are still practical issues in using microarrays, they will be alleviated by rapid advances in array technology and analysis methods, the availability of many genome sequences of closely related species and flexibility in array design. It is anticipated that the application of microarray technology will continue to better our understanding of evolution and ecology through the examination of individuals, populations, closely related species or whole microbial communities.     

Environmental genomics: exploring the unmined richness of microbes to degrade xenobiotics

Increasing pollution of water and soils by xenobiotic compounds has led in the last few decades to an acute need for understanding the impact of toxic compounds on microbial populations, the catabolic degradation pathways of xenobiotics and the set-up and improvement of bioremediation processes. Recent advances in molecular techniques, including high-throughput approaches such as microarrays and metagenomics, have opened up new perspectives and pointed towards new opportunities in pollution abatement and environmental management. Compared with traditional molecular techniques dependent on the isolation of pure cultures in the laboratory, microarrays and metagenomics allow specific environmental questions to be answered by exploring and using the phenomenal resources of uncultivable and uncharacterized micro-organisms. This paper reviews the current potential of microarrays and metagenomics to investigate the genetic diversity of environmentally relevant micro-organisms and identify new functional genes involved in the catabolism of xenobiotics.     

Analysis of host responses to microbial infection using gene expression profiling

Gene expression profiling offers new opportunities for understanding host-cell responses to microbial pathogens and their products. Current strategies involve either first identifying mRNAs that differ in their expression status under different experimental conditions and later defining the identity of the respective genes (for example, differential display or serial analysis of gene expression), or alternatively assessing changes in the expression of already defined genes (for example, cDNA or oligonucleotide microarrays). Early studies indicate the power of gene expression profiling for providing new insights into groups of genes whose expression is altered during the course of host-microbe interactions, and for the discovery of cellular genes that were not previously recognized to be regulated by infection.     

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.     

Human genomics and microarrays: implications for the plastic surgeon

The Human Genome Project was launched in 1989 in an effort to sequence the entire span of human DNA. Although coding sequences are important in identifying mutations, the static order of DNA does not explain how a cell or organism may respond to normal and abnormal biological processes. By examining the mRNA content of a cell, researchers can determine which genes are being activated in response to a stimulus.Traditional methods in molecular biology generally work on a "one gene: one experiment" basis, which means that the throughput is very limited and the "whole picture" of gene function is hard to obtain. To study each of the 60,000 to 80,000 genes in the human genome under each biological circumstance is not practical. Recently, microarrays (also known as gene or DNA chips) have emerged; these allow for the simultaneous determination of expression for thousands of genes and analysis of genome-wide mRNA expression.The purpose of this article is twofold: first, to provide the clinical plastic surgeon with a working knowledge and understanding of the fields of genomics, microarrays, and bioinformatics and second, to present a case to illustrate how these technologies can be applied in the study of wound healing.     

Application of microarray technology in primate behavioral neuroscience research

Gene expression profiling of brain tissue samples applied to DNA microarrays promises to provide novel insights into the neurobiological bases of primate behavior. The strength of the microarray technology lies in the ability to simultaneously measure the expression levels of all genes in defined brain regions that are known to mediate behavior. The application of microarrays presents, however, various limitations and challenges for primate neuroscience research. Low RNA abundance, modest changes in gene expression, heterogeneous distribution of mRNA among cell subpopulations, and individual differences in behavior all mandate great care in the collection, processing, and analysis of brain tissue. A unique problem for nonhuman primate research is the limited availability of species-specific arrays. Arrays designed for humans are often used, but expression level differences are inevitably confounded by gene sequence differences in all cross-species array applications. Tools to deal with this problem are currently being developed. Here we review these methodological issues, and provide examples from our experiences using human arrays to examine brain tissue samples from squirrel monkeys. Until species-specific microarrays become more widely available, great caution must be taken in the assessment and interpretation of microarray data from nonhuman primates. Nevertheless, the application of human microarrays in nonhuman primate neuroscience research recovers useful information from thousands of genes, and represents an important new strategy for understanding the molecular complexity of behavior and mental health.     

Genes that code for T cell signaling proteins establish transcriptional regulatory networks during thymus ontogeny

Gene expression profiling by cDNA microarrays during murine thymus ontogeny has contributed to dissecting the large-scale molecular genetics of T cell maturation. Gene profiling, although useful for characterizing the thymus developmental phases and identifying the differentially expressed genes, does not permit the determination of possible interactions between genes. In order to reconstruct genetic interactions, on RNA level, within thymocyte differentiation, a pair of microarrays containing a total of 1,576 cDNA sequences derived from the IMAGE MTB library was applied on samples of developing thymuses (14-17 days of gestation). The data were analyzed using the GeneNetwork program. Genes that were previously identified as differentially expressed during thymus ontogeny showed their relationships with several other genes. The present method provided the detection of gene nodes coding for proteins implicated in the calcium signaling pathway, such as Prrg2 and Stxbp3, and in protein transport toward the cell membrane, such as Gosr2. The results demonstrate the feasibility of reconstructing networks based on cDNA microarray gene expression determinations, contributing to a clearer understanding of the complex interactions between genes involved in thymus/thymocyte development.