Current Genomics - Volume 4, Issue 3, 2003

Cardiovascular disease is the most common cause of death in today's society. Heart transplantation is the only available treatment for patients suffering from end-stage congestive heart failure (CHF). Causes underlying the development of CHF are still unknown, but it has been suggested that proinflammatory cytokines play an important role.In the context of transplantation, proinflammatory T-helper 1 cytokines like TNF-α and IL-2 that mediate the cellular immune response are believed to be involved in acute graft rejection. On the other hand, Th2 cytokines, like IL-4, IL-6 and IL-10, induce tolerance, by down-regulating the Th1 response and cytokine production. Cytokine release by macrophages, lymphocytes and other cell types in the microenvironment of the graft, especially the balance between Th1 and Th2, is thought to be critical for the development of acute rejection. Production of cytokines has been shown to be under genetic control of single nucleotide polymorphisms (SNP) in mainly the promoter regions of cytokine genes. Genotypes of these SNP were shown to control the differential production of these cytokines, leading to a wide variety of cytokine patterns among individuals. Since levels of these cytokines affect the Th1 / Th2 balance, genotypes may be related to acute allograft rejection. Many studies have shown an association between cytokine gene polymorphisms and the development of several infections, allergies and autoimmune diseases. Also, associations between SNP in different cytokine genes and transplant rejection have been extensively studied. In our lab, SNP in the genes of TNF-α, TGF-β, IL- 4 and IL-10 were studied in a panel of 70 cardiac transplant patients and 61 of their donors. These data are discussed in the context of literature data.

For historical reasons, bioinformatics has been focused on the analysis of coding sequences. Genome annotation mainly consists in the localization of putative open reading frames, and assignment of a function to their product. Non-coding sequences are, however, an essential part of the information contained in a genome. In particular, these sequences mediate transcriptional regulation, which is crucial to many aspects of life: metabolic regulation, embryonic development, cell cycle, immune response, etc. In silico analysis of non-coding sequences can provide important information about gene function, and about the way genes interact with each other to form molecular networks. Different algorithms are required for the prediction of regulatory elements than for the analysis of coding and protein sequences. This paper discusses several recent attempts to analyze the non-coding fraction of whole genomes, and emphasizes upon different ways by which comparative genomics has been used to improve the prediction of regulatory elements.

While a gene family by definition will have homologies in sequence, the “functional genomics” or characterization of the functionality of siblings can reveal significant differences. One gene family that shares an ATPbinding cassette (ABC) sequence motif is comprised of members named “ABC Transporters.” Until 1989, the members of this family were primarily ATPases that pumped compounds out of cells and many were linked to multidrug resistance (MDR). Hence, at that time the function of the members of the ABC gene family appeared equally homologous to their structure. Since then the discovery of new members, like the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel and the sulfonylurea receptor (SUR), increased diversity and the homologous structure seemed to no longer link to homologous function. In this mini-review we will introduce two parallel investigations, separated by 10 years, where researchers re-examined the functionality of the human ABC transporters, CFTR and MDR, under the hypothesis that the structurally similar ABC transporters must have similarities in function.

RNA trafficking within the cell represents an important component of gene expression in a variety of organisms. This process directs RNA to select regions of the cell and has functional consequences to cell physiology and function. Intranuclear RNA trafficking can also influence RNA maturation including 5'- and 3'-end-processing, splicing and nuclear export. Within the cytosol, RNA trafficking influences the localization and levels of gene expression of the protein product to prevent ectopic expression, for example. For most cellular RNAs this process usually depends on an accurate and precisely timed sequence of events where each step depends on that which precedes it, usually initiated by the specific binding of trans-acting proteins to cognate RNA cis sequences to dictate their fate. This is also true for retroviral RNAs (see below).Human immunodeficiency virus type 1 (HIV-1) is a major affliction of the 21st century and it is estimated that more than 45 million people worldwide are infected. Although no cure is available, regimented antiviral drug therapy has become the golden standard for care. Accompanied with lifestyle changes both can not only bring virus to near undetectable levels but can also dramatically increase the lifespan and maintain a quality of life for a person living with the virus. As an intracellular parasite HIV-1 uses host cell proteins and machinery in most -if not all- of its replication steps including viral entry, integration, transcription and viral assembly. The understanding of their involvement in these steps will contribute in the treatment and perhaps the eventual eradication of HIV-1 infection. For this -the next generation of HIV-1 therapeutics- we will require a profound understanding of the biological role of these factors and, specifically, how they interact with the virus. This information will eventually lead to the development of specific inhibitors, which will likely be used in combination with existing retroviral therapeutic approaches.This review serves to update the reader on particular aspects of nucleocytoplasmic trafficking of HIV-1 RNAs involving HIV-1 Rev, Gag and associated host cell co-factors. It will update the reader on the current body of knowledge on host and viral proteins and mechanisms involved in the movement of HIV-1 RNAs out of the nucleus, within the cytosol and eventually to the sites of viral assembly. The novelty will be in its examination of the recent developments that implicate cellular RNA-binding proteins involved in HIV-1 RNA localization and cytosolic RNA trafficking. It is expected that host cell proteins and viral proteins will ultimately prove to be critical for these late steps of the viral lifecycle.

Whole genome sequences of microbial pathogens present new opportunities for clinical application. Presently, genome sequencing of the human protozoan parasite Leishmania major is in progress. The driving forces behind the genome project are to identify genes with key cellular functions and new drug targets, to increase knowledge on mechanisms of drug resistance and to favor technology transfer to scientists from endemic countries. Sequencing of the genome is also aimed at the identification of genes that are expressed in the infectious stages of the parasite and in particular in the intracellular form of the parasite. Several protective antigens of Leishmania have been identified. In addition to these antigens, lysosomal cysteine proteinases (CPs) have been characterized in different strains of Leishmania and Trypanosoma, as new target molecules. Recently, we have isolated and characterized Type I (CPB) and Type II (CPA) cysteine proteinase encoding genes from L. major. The exact function of cysteine proteinases of Leishmania is not completely understood, although there are a few reports describing their role as virulence factors. One specific feature of CPB in Leishmania and other trypanosomatids, is the presence of a Cterminal extension (CTE) which is possibly indicative of conserved structure and function. Recently, we demonstrated that DNA immunization of genetically susceptible BALB / c mice, using a cocktail of CPB and CPA genes, induced long lasting protection against L. major infection. This review intends to give an overview of the current knowledge on genetic vaccination used against leishmaniasis and the importance of CP genes for such an approach.

Paramyxoviruses are enveloped viruses containing a non-segmented negative strand RNA genome, and many of them are associated with traditional diseases of childhood and severe disease of humans and animals. In the last two decades, previously unidentified paramyxoviruses have emerged as the cause of serious disease outbreaks in a number of animal species including humans. Hendra and Nipah viruses which recently emerged from their natural fruit bat hosts to infect livestock animals and subsequently man with disastrous consequences are only the latest of a series of novel paramyxoviruses which have emerged to cause disease in livestock and marine animal species. The genetic characterization not only of disease-causing paramyxoviruses but also of recently isolated paramyxoviruses not associated with disease and viruses isolated many years ago and provisionally classified as paramyxoviruses, has provided a golden opportunity for systematic and comparative studies of this important group of viruses at a molecular level. The genomic RNA of negative strand RNA viruses serves as a template for the synthesis of mRNA during transcription and the production of antigenome (+) strands during replication. The genome organization and the structure are highly conserved among this class of viruses. In this review, we will discuss a novel strategy for the rapid characterization of genomes of novel paramyxoviruses and summarize some of the interesting and startling genomic features that our studies have revealed. These include a much wider range of genome sizes than previously acknowledged for members of the Paramyxoviridae, novel genome terminal sequences and intergenic regions, the presence of extensive non-translated regions in certain members of this ever increasing virus family, novel coding patterns of the P gene and the use of different nucleotides for transcription initiation. The molecular studies described provide fresh insight into paramyxovirus genome diversity and evolution.

Currently rational drug design is limited to using protein targets in the design and production of therapeutic agents. However, many genetic and infectious diseases may not be adequately treated with this approach. In these circumstances the DNA sequence of an offending gene is itself a potential target for rational drug development. Genetargeted therapeutic strategies require the development of ligands that can recognize and bind unique DNA targets sequence specifically. Several approaches have been described for the development of sequence-specific DNA targeting agents. These include synthetic polyamides that recognize and bind to DNA in the minor groove, peptide nucleic acids which can penetrate the DNA duplex and form a P-loop, or a triple-helical structure with one of its strands, and triplehelix (triplex) forming oligonucleotides which bind to the major groove of duplex DNA at polypyrimidine / polypurine sequences. Of these, Triplex-Forming Oligonucleotides (TFOs) are the most extensively characterized synthetic ligands capable of recognizing and binding sequence specifically to duplex DNA. Consequently, they have been the focus of a new gene therapy strategy that we call ‘anti-gene radiotherapy’. This strategy employs TFOs labeled with Auger-electronemitting radionuclides to produce sequence-specific DNA double strand breaks that ultimately lead to gene inactivation following repair. Anti-gene radiotherapy is made possible by the highly localized damage produced by decay of an Augeremitter, such as 125I, and the sequence specific positioning of DNA damage made possible by TFOs. This report will address recently described strategies that employ these gene-targeting methods to alter target gene expression or structure, with particular emphasis being paid to the use of TFOs in anti-gene radiotherapy.