Current Genomics - Volume 7, Issue 4, 2006

Spatial and temporal control of proteome expression is critical for cellular homeostasis. The ability to regulate polypeptide synthesis allows the cell to rapidly respond to changes in its environment. Under stress conditions, capdependent translation initiation is downregulated and alternative mechanisms of translation initiation are favoured for the production of critical proteins that ultimately determine whether the cell is able to overcome the stress. One such alternative mechanism of translation initiation is mediated by sequence elements located downstream of the 5' cap structure that are able to directly recruit ribosomes to a region proximal to the translation start site. Identifying the eukaryotic mRNAs that contain such internal ribosome entry sites (IRESes) is an important first step in cataloguing the cellular complement of proteins whose expression is translationally regulated during cellular stress and understanding how cells regulate translation under stress conditions. To date, no consensus sequence motif or structure has been identified as a signature of cellular IRES activity, making it difficult to identify the full complement of eukaryotic IRESes. This review will underscore the challenges faced in identifying IRESes on a genomic scale and potential solutions will be presented.

Brain system is composed of enormous numbers of diversified single neurons. Therefore assessing epigenetic and genetic regulation in the nucleus of single neurons is a new challenge for understanding neuronal commitment, differentiation and maturation. As differentiated neurons are, in nature, postmitotic, neither the genome nor its epigenetic modifications are easy to evaluate fully. The cloning of mammalian cells, which has been used mainly in the fields of assisted reproduction and regenerative medicine, can be applied to propagating the entire genome of single neurons. In addition, embryonic stem (ES) cells derived from embryos cloned from single neuronal nuclei provide an “eternal” resource for assessing the genetic content of individual neurons. Here, we discuss the possible genetic/epigenetic regulation of gene expression in the nuclei of single neurons, and the utility of cloning by neuronal nuclear transfer to assess the genomic constitution of the single nucleus of differentiated neurons during development. This use of cloning technology may be a fruitful approach for analyzing the entire genome of individual single neurons.

Biology as a whole has entered a new era in which data analysis plays a prominent role; but in the field of evolutionary genomics, data analysis has so far yielded little of value. This relative failure has been due in large part to methodological problems. Frequently, researchers have not sufficiently considered alternative hypotheses, leading to a kind of “computer-assisted storytelling”. Moreover, there has been widespread use of model-based statistical methods that depend heavily on assumptions regarding evolutionary processes of which we have little knowledge. The field of evolutionary genomics would benefit from a greater use of “sturdy statistics” that are model-free and make few assumptions about processes we do not understand.

Most proteins required for chloroplast function are encoded in the nuclear genome and have to be translocated into the organelle upon synthesis on cytosolic ribosomes. The translocation is facilitated by a proteinaceous machinery located in the outer and inner chloroplast membrane, the Toc and Tic complexes (translocon at the outer/inner chloroplast membrane). In the past years, many components of these complexes -including receptors, channels and regulatory proteins - have been isolated and characterized biochemically. Recently, the functional analysis of these proteins was complemented by characterization of corresponding loss of function mutants in Arabidopsis thaliana as a model system. Here, we will discuss these in vivo data and the results of expression profiling in the context of current biochemical models.

Glutamate receptors are the principal neurotransmitter receptors in the central nervous system and categorized into ionotopic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). iGluRs internally contain ligand-gated ion-channels, and mGluRs are a class of G-protein coupled receptors (GPCRs) that possess a seven transmembrane region. iGluRs are considered to diverge as ligand-gated channel proteins from ancestral potassium channels. A prokaryotic iGluR, GluR0, has been found to bind glutamate, form potassium-selective channels and be related in amino-acid sequence to both eukaryotic iGluRs and potassium channels. While, the class III GPCRs including the sensory receptors, the GABAβ receptors (GABAβRs) and mGluRs are considered to diverge from common ancestral GPCRs. mGluR orthologs have been identified in Drosophila, C. elegans and higher organisms. We have screened the Dictyostelium genome database using ligand binding domain of rat mGluR1 as a bait, and identified a receptor, DdmGluPR (Dictyostelium metabotropic glutamate precursor receptor), belonging to the mGluR family. The residues of mGluRs involved in the binding of the α-carboxylic and α-amino groups of glutamate are well conserved in DdmGluPR but the residues interacting with the γ-carboxylic group of glutamate are not. The phylogenetic analysis suggests that DdmGluPR diverged after the mGluR family-GABAβ receptors split but before mGluR family divergence. GABA induces but glutamate acts as a competitive inhibitor of GABA for the efficient induction of encapsulation through DdmGluPR in Dictyostelium. DdmGluPR has a hybrid structure with extracellular region similar to mGluRs and transmembrane region similar to GABAβRs. We propose that DdmGluPR is evolutionary precursor to mGluRs.

Antigenic differences between normal and malignant cells of the cancer patient form the rationale for clinical immunotherapeutic strategies. Because the antigenic phenotype of neoplastic cells varies widely among different cells within the same malignant cell-population, immunization with a vaccine that stimulates immunity to the broad array of tumor antigens expressed by the cancer cells is likely to be more efficacious than immunization with a vaccine for a single antigen. A vaccine prepared by transfer of DNA from the tumor into a highly immunogenic cell line can encompass the array of tumor antigens that characterize the patient's neoplasm. Poorly immunogenic tumor antigens, characteristic of malignant cells, can become strongly antigenic if they are expressed by highly immunogenic cells. A DNA-based vaccine was prepared by transfer of genomic DNA from a breast cancer that arose spontaneously in a C3H/He mouse into a highly immunogenic mouse fibroblast cell line, where genes specifying tumor-antigens were expressed. The fibroblasts were modified in advance of DNA-transfer to secrete an immune augmenting cytokine and to express allogeneic MHC class Ideterminants. In an animal model of breast cancer metastatic to the brain, introduction of the vaccine directly into the tumor bed stimulated a systemic cellular anti-tumor immune response measured by two independent in vitro assays and prolonged the lives of the tumor-bearing mice. Furthermore, using antibodies against the various T-cell subsets, it was determined that the systemic cellular anti-tumor immunity was mediated by CD8+, CD4+ and NK/LAK cells. The application of DNA-based genomic vaccines for the treatment of a variety of brain tumors is being explored.

The molecular biology of aging has always been of extreme interest to researchers. Recent advances made through studies of model systems have greatly increased efforts to elucidate the mechanisms involved in aging. It now appears that in yeast there are two pathways controlling aging: i) the generation of extrachromosomal rDNA circles (ERCs) through the antagonistic action of Sir2p and Fob1p; and ii) the stress response pathway, regulated by the Sch9p and Tor1p kinases. The stress response pathway in higher eukaryotes is inhibited by the insulin-signaling pathway. Mutation to the AKT kinase disrupts insulin signaling and increases longevity. Yeast express orthologues of the insulin-signaling pathway, yet insulin is not a natural substrate for yeast. The yeast Sch9p is structurally and functionally related to the AKT kinase, but the remainder of the putative yeast AKT pathways is uncharacterized. Further mystery revolves around downstream targets that are directly required for longevity. Yeast provide an opportunity to investigate the function of the yeast AKT pathway at a molecular level. A potential downstream target has been identified in yeast that links the glucose and stress response pathways with lifespan: the Anaphase Promoting Complex (APC). This review will discuss the role the APC plays in established aging pathways in yeast and the implications on higher eukaryotes.