Current Genomics - Volume 5, Issue 1, 2004

The genocentric view of disease that has dominated biomedical science for the past 25 years has proven insufficient for determining causation of most diseases. The technology arising from genomic efforts, however, provides powerful tools for developing the more meaningful cellular and molecular understanding of disease causation, particularly as it is affected by environmental and behavioral parameters. The Environmental Genome Project, the Mouse Genetic Variation Mapping Initiative, and the Comparative Toxicogenomics Initiative are discussed as projects that aim to capitalize on genomic technology in ways that better define the environmental and genetic underpinnings of chronic diseases and disorders.

The repair of musculoskeletal tissues has posed a constant challenge for orthopaedic surgeons, and the occurrence of bone and cartilage injuries is expected to increase with the aging of the world population. To overcome the limitations of current treatments, tissue engineering enhanced through gene therapy is garnering significant interest as a promising new alternative. This paper reviews the essential factors involved in tissue engineering, including the appropriate cell source, inductive agents, scaffolds, and mechanical stimulation. Particular emphasis is placed on the use of muscle-derived stem cells that can be genetically engineered to deliver growth factors to the site of injury and initiate the formation of new bone and cartilage. These same gene-carrying cells may also serve as a source of progenitor cells for bone and cartilage formation, making muscle-based gene therapy and tissue engineering a potential treatment for cartilage and bone defects.

Lentiviral vector systems continue to be developed and investigated for possible use in clinical gene transfer studies and for application as potential gene therapies. These vector systems, as a new technology and potentially novel medical therapy, raise a number of safety concerns, both real and perceived. Careful and thorough characterization of the lentiviral vector systems and of how they might interact with cellular genomes and other pathogens is necessary in order to maximize the safety of this technology as it is developed and the safety of both individuals undergoing gene transfer procedures and the general population. Identification of potential safety concerns should not prevent or unnecessarily delay the development of lentiviral vectors for use in clinical gene transfer, but suggest areas of investigation where it would be wise to allocate resources and effort.

As more and more genomes have been sequenced, the hotspot of biological science is moving from the study on nucleic acids to that on proteins. One of the most representative affairs in this era is the launching of projects focusing on the high throughput determination of the protein three dimensional structures in a genome scale, named structural genomics projects. The common objective of the pilot projects is construction of a platform to clone hundreds to thousands of targets, and purify, at the first stage, the highly expressed and soluble proteins, then solve dozens to hundred of structures by means of X-ray crystallography or NMR. The first bottleneck in this pipeline is obtaining manipulable quantity (milligram level according to the necessity of the current crystallographic or NMR technology) of soluble proteins, which are properly folded. For this purpose, a series of methodologies have been established. His-tag makes it possible to purify the desired protein from the crude extract of the host cells or the mix of in vitro expression system in a single step of affinity purification. Beyond His-tag and other short affinity tags, a series of fusion tags have been developed for the purpose of solubility enhancement. Many expression hosts based on bacterium Escherichia coli or yeast Pichia pastoris have been constructed to express heterologous proteins. The influences of temperature during induction and co-expression with chaperones are systematically investigated. The effects of the N-terminal tags, either small or big ones, are examined and compared with those carrying a tag at the C-terminus. Some techniques of in vitro evolution are transferred and applied to increase the expression level and solubility of the targets. These efforts are helpful, at least at one hand, for speeding up the production of folded and concentrated proteins, the sample feeding the crystallographers and NMR spectroscopists. Furthermore three newly determined structures by the pilot structural genomics projects were shown to demonstrate how to decipher biological function from the 3-D structure of a novel protein and how to interpret the structure-function relationship and its application on drug design. In present paper, we review the popular techniques applied in the current structural genomics projects, their effects and possible improvements in the future, especially those for protein preparation and function interpretation.

Faithful genome transmission requires the cooperation of a network of pathways including cell cycle checkpoint, DNA replication, repair and recombination. The different DNA repair pathways must also be coordinated as function of the type of damage, the cell cycle and differentiation. DNA double-strand breaks (DSBs) are highly toxic lesions, which can be produced by physiological cell processes, such as meiosis or V(D)J recombination or by genotoxic stresses, such as ionizing radiation or replication inhibition. In mammalian cells, two major classes of processes can repair DSBs: non-homologous end-joining (NHEJ) or homologous recombination (HR). It has been proposed that the two processes can compete, via the binding to the broken DNA ends by the Ku80-Ku70 heterodimer (NHEJ) versus RAD52 protein (HR). Consistent with the competition for the DNA ends model, mammalian NHEJ defective cells show increased HR, induced by DSB generating treatment; this stimulation is specific to DSB since neither spontaneous nor UV-C-induced HR are stimulated. However the regulation could be more complex since different cell situations can affect the choice between NHEJ and HR, such as the stage of embryonic development, the persistence and / or accumulation of DSBs. The phase of the cell cycle has also been proposed to affect this channeling. In addition, despite the cell cycle regulation, the two processes can cooperate or act sequentially. After describing the molecular mechanisms of NHEJ and HR processes, their regulation is presented and discussed in this review. A model of regulation of DSB repair with respect to the cell cycle is proposed.

The availability of complete genome sequences has created the need for comprehensive methods to explore gene function. S. cerevisiae is the first eukaryotic organism for which the DNA sequence became available, and exhibits many characteristics, such as amenable genetics and controllable physiology, which make it an ideal experimental system for the functional analysis of novel genes. The most direct way to determine gene function involves the disruption of the unknown genes followed by phenotypic analysis. In the last eight years, different systematic approaches have been employed to produce collections of mutant strains in which every putative ORF was erased from the yeast genome. Moreover, because of the high level of redundancy found in the yeast genome, new vectors and efficient methods have been created to perform multiple deletions in the same strain background. Such a collection of tools and mutants provide a major resource for both conventional functional analysis, and for the new whole-genome approaches aiming to characterise gene function via large-scale competition experiments.

Molecular genetic mechanisms show diversity during prostate carcinogenesis due to extensive tumor heterogeneity and gene complexity. Understanding the molecular mechanisms involved in initiation and progression of prostate cancer will lead to the development of strategies for early detection, prevention and therapeutic intervention. This review focuses on inheritable and sporadic prostate cancer genes involved in proliferation, metastasis, angiogenesis, tumor suppression, apoptosis, and cellular stress responses.