Current Genomics - Volume 4, Issue 7, 2003

Annotating the noncoding portion of the human genome and identifying functional regulatory elements embedded in its sequence creates a continuing challenge. Historically, the functional characterization of regulatory elements has been slow, labor-intensive and inadequate to keep up with the demands of whole-genome analysis. Recently, there has been an explosion of computational techniques and tools available to assist in the annotation of noncoding DNA and improve the accurate prediction of regulatory sequences. Here, we review some of the bioinformatic strategies and computational tools that are increasingly being used to analyze large genomic data sets and to facilitate the high-throughput identification of candidate regulatory sequences in whole genomes.

Gene Ontology facilitates biomedical knowledge representation and efficient information management. The systematic representation and hierarchical structure of Gene Ontology bring forth great potential to examine data and information across the broad spectrum of biology. This article briefly discusses GO annotation and three interesting areas in Gene Ontology-facilitated genome analysis.

The decline in functional performance and restriction of adaptability represents the hallmark of skeletal muscle pathologies. The characteristic loss in muscle mass, coupled with a decrease in strength and force output, has been associated with a selective activation of apoptotic pathways and a general reduction in survival mechanisms. Aging and genetic diseases, such as muscular dystrophies, amyotrophic lateral sclerosis, cancer and AIDS, are characterized by alterations in metabolic and physiological parameters, progressive weakness in specific muscle groups, modulation in muscle-specific transcriptional mechanisms and persistent protein degradation. The inability to regenerate and repair the injured muscle is another serious complication in muscle pathologies. Tissue remodeling is therefore an important physiological process, which allows skeletal muscle to respond to environmental demands and to undergo adaptive changes in cytoarchitecture and protein composition, in response to a variety of stimuli. Alterations in extracellular agonists, receptors, protein kinases, intermediate molecules, transcription factors and tissue-specific gene expression compromise the functionality of skeletal muscle tissue, leading to muscle degeneration. Although considerable information has accumulated regarding the physiopathology of muscle diseases, the associated molecular mechanisms are still poorly understood. In this review, we will discuss the molecular basis of muscle atrophy, wasting and regeneration and the current gene and cell therapeutic approaches to attenuate atrophy and frailty associated with muscle diseases.

The ability to silence specific genes of choice consistently and efficiently has always been a major goal for scientists. The emerging field of RNA interference (RNAi), a process in which target mRNAs are degraded by small interfering RNA (siRNA), may indeed provide this long sought after tool. The importance of this technology has been highlighted recently by Science, which has voted the RNAi discoveries as the “&;Breakthrough of the Year” in 2002. Essentially, RNAi involves an initiation and an effector step whereby introduced dsRNA is digested into 19-21 duplex siRNA by cleavage with Dicer and siRNA binds to an RNA-induced silencing complex (RISC). Activation of RISC targets the homologous sequence (transcript) and results in the cleavage of mRNA. The models of this RNAi mechanism and its applications, derived from biochemical and genetic approaches, are described in this minireview. So far, RNAi has proven to be a useful technique for genomic studies in C.elegans, D.melanogaster and various plants, for example. The use of RNAi has also been invaluable in studies in which morphological and developmental variability between species was investigated. Targeted, sequence specific siRNAs that suppress or silence gene expression have the potential to be in great demand as tools for for the treatment of human disease. There have already been several studies that have utilized siRNA to inhibit HIV-1 infection and replication, for example. The expansion of RNAi for biomedical therapeutics seems inevitable. This minireview analytically summarizes the advantages and the current and future potential of RNAi technology whilst simultaneously investigating any shortfalls or difficulties. Importantly, in vitro and in vivo applications in the laboratory and in human disease models are also described.

There is increasing evidence both from, animal models and clinical investigations that luminal bacteria play a pivotal role in the pathogenesis of Crohn's disease (CD). In almost all murine models for spontaneous colitis, animals stay healthy as long as they are kept under germ free conditions. Antibacterial treatment reduces disease severity in some patients with CD. This aforementioned hypothesis is further supported by the identification of the first susceptibility gene for CD. Single nucleotide polymorphisms in the caspase recruitment domain 15 gene (CARD15, formerly known as NOD2) have been identified recently. The respective CARD15 alleles increase the risk of developing CD. The CARD15 protein is located in the cytoplasm of mononuclear cells where it is involved in the recognition of bacterial components leading to subsequent activation of nuclear factor kB. As rare alleles of the SNPs are not found in all patients with CD and as these alleles are found in healthy persons as well, variations in the CARD15 gene are neither necessary nor sufficient for the genetic predisposition to CD. Further genes are supposed to play a crucial role in the pathogenesis of CD. Disturbed activation of the innate immune system by bacterial antigens seems to be crucial in a subgroup of patients with CD. Genes involved in pathways of the innate immune system represent good candidate genes for the predisposition to CD. Abundant polymorphisms in these genes have been described. The putative role of these polymorphisms in the genetic predisposition to CD is revisited for this multifactorial model disease.

Our ability to study thousands of genes simultaneously over the last few years owing not only to the development of DNA microarray technology, but also to large-scale yeast two-hybrid and other methods, has brought about a paradigmatic shift in biology. Armed with the complete sequences of several genomes including humans, more and more biomedical investigators are now interested in understanding the functions of gene products, or proteins, on a global scale. In this article, the promises and pitfalls of two methods used for proteomics studies - protein microarrays and mass spectrometry - towards achieving this goal are discussed.

Microarray expression profiling is a complex experimental methodology comprising sequential steps: fluorescent or radioactive labelling of extracted RNA, hybridisation to the array, washing, scanning, and image processing of the array and data analysis. Each of these steps is a possible source of artefacts that potentially can influence the final result, and hence the evaluation of differential gene expression. In addition, the quality of expression profiling experimental data also depends upon the design and quality of the arrays themselves. Here we review specific problems and pitfalls that may occur during expression profiling experiments and suggest possible remedies.

The Agouti Related Protein (AgRP), Neuropeptide Y (NPY), Cocaine and Amphetamine-Regulated Transcript (CART), and Proopiomelanocortin hormone (POMC) are four essential neuropeptides that play an import role in regulation of food intake and energy homeostasis in mammals. Each of the two pairs of neuropeptides, AgRP / NPY and CART / POMC, are co-expressed in distinctly separate neurons in the hypothalamus. The four neuropeptides are also expressed widely in peripheral tissues with the adrenal gland being the major site of expression except in the case of NPY that is expressed predominantly in the prostate. There is abundant information regarding the physiological roles of these genes but there is limited information regarding their transcriptional regulation in the brain and the periphery. We employed multiple in silico methods from genome-wide applications and genomic sequence from promoter regions to predict common and unique regulatory patterns of transcription for these genes. hCART and hPOMC are phylogenetically closely related in terms of the common transcription factors (TFs) that were predicted for the two promoters, whereas hAgRP is a distant relative to hNPY. We also identified short regions (20 nucleotides or less) in the promoters of pairs of these genes that were 100% identical and are candidate TF binding sites. These studies provide a paradigm for analogous applications to other co-regulated genes and could lead to the identification of functional promoter targets for interventional therapies.