Serial Analysis of Gene Expression: Applications in Human Studies

Serial analysis of gene expression (SAGE) is a powerful tool, which provides quantitative and comprehensive expression profile of genes in a given cell population. It works by isolating short fragments of genetic information from the expressed genes that are present in the cell being studied. These short sequences, called SAGE tags, are linked together for efficient sequencing. The frequency of each SAGE tag in the cloned multimers directly reflects the transcript abundance. Therefore, SAGE results in an accurate picture of gene expression at both the qualitative and the quantitative levels. It does not require a hybridization probe for each transcript and allows new genes to be discovered. This technique has been applied widely in human studies and various SAGE tags/SAGE libraries have been generated from different cells/tissues such as dendritic cells, lung fibroblast cells, oocytes, thyroid tissue, B-cell lymphoma, cultured keratinocytes, muscles, brain tissues, sciatic nerve, cultured Schwann cells, cord blood-derived mast cells, retina, macula, retinal pigment epithelial cells, skin cells, and so forth. In this review we present the updated information on the applications of SAGE technology mainly to human studies

Nucleolin: a multifunctional major nucleolar phosphoprotein

Nucleolin is a major protein of exponentially growing eukaryotic cells where it is present in abundance at the heart of the nucleolus. It is highly conserved during evolution. Nucleolin contains a specific bipartite nuclear localization signal sequence and possesses a number of unusual structural features. It has unique tripartite structure and each domain performs a specific function by interacting with DNA or RNA or proteins. Nucleolin exhibits intrinsic self-cleaving, DNA helicase, RNA helicase and DNA-dependent ATPase activities. Nucleolin also acts as a sequence-specific RNA binding protein, an autoantigen, and as the component of a B cell specific transcription factor. Its phosphorylation by cdc2, CK2, and PKC-zeta modulate some of its activities. This multifunctional protein has been implicated to be involved directly or indirectly in many metabolic processes such as ribosome biogenesis (which includes rDNA transcription, pre-rRNA synthesis, rRNA processing, ribosomal assembly and maturation), cytokinesis, nucleogenesis, cell proliferation and growth, cytoplasmic-nucleolar transport of ribosomal components, transcriptional repression, replication, signal transduction, inducing chromatin decondensation and many more (see text). In plants it is developmentally, cell-cycle, and light regulated. The regulation of all these functions of a single protein seems to be a challenging puzzle

Prokaryotic and eukaryotic DNA helicases

DNA helicases are ubiquitous molecular motor proteins which harness the chemical free energy of ATP hydrolysis to catalyze the unwinding of energetically stable duplex DNA, and thus play important roles in nearly all aspects of nucleic acid metabolism, including replication, repair, recombination, and transcription. They break the hydrogen bonds between the duplex helix and move unidirectionally along the bound strand. All helicases are also translocases and DNA-dependent ATPases. Most contain conserved helicase motifs that act as an engine to power DNA unwinding. All DNA helicases share some common properties, including nucleic acid binding, NTP binding and hydrolysis, and unwinding of duplex DNA in the 3' to 5' or 5' to 3' direction. The minichromosome maintenance (Mcm) protein complex (Mcm4/6/7) provides a DNA-unwinding function at the origin of replication in all eukaryotes and may act as a licensing factor for DNA replication. The RecQ family of helicases is highly conserved from bacteria to humans and is required for the maintenance of genome integrity. They have also been implicated in a variety of human genetic disorders. Since the discovery of the first DNA helicase in Escherichia coli in 1976, and the first eukaryotic one in the lily in 1978, a large number of these enzymes have been isolated from both prokaryotic and eukaryotic systems, and the number is still growing. In this review we cover the historical background of DNA helicases, helicase assays, biochemical properties, prokaryotic and eukaryotic DNA helicases including Mcm proteins and the RecQ family of helicases. The properties of most of the known DNA helicases from prokaryotic and eukaryotic systems, including viruses and bacteriophages, are summarized in tables

Unraveling DNA repair in human: molecular mechanisms and consequences of repair defect

Cellular genomes are vulnerable to an array of DNA-damaging agents, of both endogenous and environmental origin. Such damage occurs at a frequency too high to be compatible with life. As a result cell death and tissue degeneration, aging and cancer are caused. To avoid this and in order for the genome to be reproduced, these damages must be corrected efficiently by DNA repair mechanisms. Eukaryotic cells have multiple mechanisms for the repair of damaged DNA. These repair systems in humans protect the genome by repairing modified bases, DNA adducts, crosslinks and double-strand breaks. The lesions in DNA are eliminated by mechanisms such as direct reversal, base excision and nucleotide excision. The base excision repair eliminates single damaged-base residues by the action of specialized DNA glycosylases and AP endonucleases. Nucleotide excision repair excises damage within oligomers that are 25 to 32 nucleotides long. This repair utilizes many proteins to remove the major UV-induced photoproducts from DNA, as well as other types of modified nucleotides. Different DNA polymerases and ligases are utilized to complete the separate pathways. The double-strand breaks in DNA are repaired by mechanisms that involve DNA protein kinase and recombination proteins. The defect in one of the repair protein results in three rare recessive syndromes: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. This review describes the biochemistry of various repair processes and summarizes the clinical features and molecular mechanisms underlying these disorders

Serial Analysis of Gene Expression: Applications in Malaria Parasite, Yeast, Plant, and Animal Studies

The serial analysis of gene expression (SAGE) method is based on the isolation of unique sequence tags from individual transcripts and concatenation of tags serially into long DNA molecules. SAGE is an innovative technique that offers the potential of cataloging both the identity and relative frequencies of mRNA transcripts in a given RNA preparation. It can quantify low-abundance transcripts and reliably detect relatively small differences in transcript abundance between cell populations. SAGE data can be used to complement studies in cases where other gene expression methods may be more convenient or efficient. SAGE can be used in a wide variety of applications to identify disease-related genes, to analyze the effect of drugs on tissues, and to provide insights into the disease pathways. The most important application of SAGE is the identification of differentially expressed genes. In this review, we describe various applications of this powerful technology in malarial parasite, yeast, plant, and animal systems

Unraveling DNA helicases: motif, structure, mechanism and function

DNA helicases are molecular 'motor' enzymes that use the energy of NTP hydrolysis to separate transiently energetically stable duplex DNA into single strands. They are therefore essential in nearly all DNA metabolic transactions. They act as essential molecular tools for the cellular machinery. Since the discovery of the first DNA helicase in Escherichia coli in 1976, several have been isolated from both prokaryotic and eukaryotic systems. DNA helicases generally bind to ssDNA or ssDNA/dsDNA junctions and translocate mainly unidirectionally along the bound strand and disrupt the hydrogen bonds between the duplexes. Most helicases contain conserved motifs which act as an engine to drive DNA unwinding. Crystal structures have revealed an underlying common structural fold for their function. These structures suggest the role of the helicase motifs in catalytic function and offer clues as to how these proteins can translocate and unwind DNA. The genes containing helicase motifs may have evolved from a common ancestor. In this review we cover the conserved motifs, structural information, mechanism of DNA unwinding and translocation, and functional aspects of DNA helicases

Rice SUV3 is a bidirectional helicase that binds both DNA and RNA

BACKGROUND: Helicases play crucial role in almost all the nucleic acid metabolism including replication, repair, recombination, transcription, translation, ribosome biogenesis and splicing and these processes regulate plant growth and development. It is suggested that helicases play essential roles in stabilizing growth in plants under stress because their presence in the stress-induced ORFs has been identified. Moreover in a recent study we have reported that SUV3 helicase from Oryza sativa (OsSUV3) functions in salinity stress tolerance in transgenic rice by improving the antioxidant machinery. SUV3 helicase has been identified and characterized from yeast and human systems but the properties and functions of plant SUV3 are poorly understood. RESULTS: In this study, the purification and extensive characterization of recombinant OsSUV3 protein (67 kDa) is presented. OsSUV3 binds to DNA and RNA and exhibits DNA as well as RNA-dependent ATPase activities. It also contains the characteristic DNA and RNA helicase activity. OsSUV3 can use mainly ATP or dATP as energy source for the unwinding activity and it cannot unwind the blunt-end duplex DNA substrate. It is interesting to note that OsSUV3 unwinds DNA in both the 5’-3’ and 3’-5 directions and thus its activity is bipolar in vitro. The Km values of OsSUV3 are 0.51 nM and 0.95 nM for DNA helicase and RNA helicase, respectively. CONCLUSIONS: This study is the first direct evidence to show the bipolar DNA helicase activity of OsSUV3 protein. The unique properties of OsSUV3 including its dual helicase activity imply that it could be a multifunctional protein involved in biologically significant process of DNA and RNA metabolisms. These results should make significant contribution towards better understanding of SUV3 protein in plants. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12870-014-0283-6) contains supplementary material, which is available to authorized users

Nitric Oxide as a Unique Bioactive Signaling Messenger in Physiology and Pathophysiology

Nitric oxide (NO) is an intra- and extracellular messenger that mediates diverse signaling pathways in target cells and is known to play an important role in many physiological processes including neuronal signaling, immune response, inflammatory response, modulation of ion channels, phagocytic defense mechanism, penile erection, and cardiovascular homeostasis and its decompensation in atherogenesis. Recent studies have also revealed a role for NO as signaling molecule in plant, as it activates various defense genes and acts as developmental regulator. In plants, NO can also be produced by nitrate reductase. NO can operate through posttranslational modification of proteins (nitrosylation). NO is also a causative agent in various pathophysiological abnormalities. One of the very important systems, the cardiovascular system, is affected by NO production, as this bioactive molecule is involved in the regulation of cardiovascular motor tone, modulation of myocardial contractivity, control of cell proliferation, and inhibition of platelet activation, aggregation, and adhesion. The prime source of NO in the cardiovascular system is endothelial NO synthase, which is tightly regulated with respect to activity and localization. The inhibition of chronic NO synthesis leads to neurogenic and arterial hypertensions, which later contribute to development of myocardial fibrosis. Overall, the modulation of NO synthesis is associated with hypertension. This review briefly describes the physiology of NO, its synthesis, catabolism, and targeting, the mechanism of NO action, and the pharmacological role of NO with special reference to its essential role in hypertension

Cold- and salinity stress-induced bipolar pea DNA helicase 47 is involved in protein synthesis and stimulated by phosphorylation with protein kinase C

Helicases are involved in the metabolism of nucleic acid; this is very sensitive to the abiotic stresses that reduce plant growth and productivity. However, the molecular targets responsible for this sensitivity have not been well studied. Here we report on the isolation and characterization of cold- and salinity stress-induced pea DNA helicase 47 (PDH47). The transcript of PDH47 was induced in both shoots and roots under cold (4°C) and salinity (300 mm NaCl) stress, but there was no change in response to drought stress. Tissue-specific differential regulation was observed under heat (37°C) stress. ABA treatment did not alter expression of PDH47 in shoots but induced its mRNA in roots, indicating a role for PDH47 in both the ABA-independent and ABA-dependent pathways in abiotic stress. The purified recombinant protein (47 kDa) contains ATP-dependent DNA and RNA helicase and DNA-dependent ATPase activities. With the help of photoaffinity labeling, PDH47 was labeled by [α-32P]-ATP. PDH47 is a unique bipolar helicase that contains both 3' to 5' and 5' to 3' directional helicase activities. Anti-PDH47 antibodies immunodeplete the activities of PDH47 and inhibit in vitro translation of protein. Furthermore, the PDH47 protein showed upregulation of protein synthesis. The activities of PDH47 are stimulated after phosphorylation by protein kinase C at Ser and Thr residues. Western blot analysis and in vivo immunostaining, followed by confocal microscopy, showed PDH47 to be localized in both the nucleus and cytosol. The discovery of cold- and salinity stress-induced DNA helicase should make an important contribution to a better understanding of DNA metabolism and stress signaling in plants. Its bipolar helicase activities may also be involved in distinct cellular processes in stressed conditions

A pea homologue of human DNA helicase I is localized within the dense fibrillar component of the nucleolus and stimulated by phosphorylation with CK2 and cdc2 protein kinases

DNA helicases catalyse the transient opening of duplex DNA during nucleic acid transactions. Here we report the isolation of a second nuclear DNA helicase (65 kDa) from Pisum sativum (pea) designated pea DNA helicase 65 (PDH65). The enzyme was immunoaffinity purified using an antihuman DNA helicase I (HDH I) antibody column. The purified PDH65 showed ATP- and Mg2+-dependent DNA and RNA unwinding activities, as well as ssDNA-dependent ATPase activity. The direction of DNA unwinding was 3' to 5' along the bound strand. Antibodies against HDH I recognized the purified PDH65, and immunodepletion with these antibodies removed the DNA and RNA unwinding and ATPase activities from purified preparations of PDH65. The DNA and RNA unwinding activities were upregulated after phosphorylation of PDH65 with CK2 and cdc2 protein kinases. By incorporation of BrUTP into pea root tissue, followed by double immunofluorescence labelling and confocal microscopy, PDH65 was shown to be localized within the dense fibrillar component of pea root nucleoli in the regions around the rDNA transcription sites. These observations suggest that PDH65 may be involved both in rDNA transcription and in the early stages of pre-rRNA processing