Applications of DNA shuffling to pharmaceuticals and vaccines

DNA shuffling is a practical process for directed molecular evolution which uses recombination to dramatically accelerate the rate at which one can evolve genes. Single and multigene traits that require many mutations for improved phenotypes can be evolved rapidly. DNA shuffling technology has been significantly enhanced in the past year, extending its range of applications to small molecule pharmaceuticals, pharmaceutical proteins, gene therapy vehicles and transgenes, vaccines and evolved viruses for vaccines, and laboratory animal models.     

Implications of sequencing bacterial genomes for pathogenesis and vaccine development

Improvements in homology search methodology and functional predictions are being complemented by the increase in the volume of sequence data with which comparative analyses can be performed. The experimental methods needed for investigation of gene function and expression in a variety of model systems of infection continue to develop. The identification of surface-exposed microbial structures and their conservation in natural populations of pathogenic species offers prospects for developing novel vaccines. A major challenge is the development of efficient screening methods to select the most promising candidates, such as immunisation with DNA.     

Chemical approaches toward understanding base excision DNA repair

Despite the importance of DNA repair in protecting the genome, the molecular basis for damage recognition and repair remains poorly understood. In the base excision repair pathway (BER), DNA glycosylases recognize and excise damaged bases from DNA. This review focuses on the recent development of chemical approaches that have been applied to the study of BER enzymes. Several distinctive classes of noncleavable substrate analogs that form stable complexes with DNA glycosylases have recently been designed and synthesized. These analogs have been used for biochemical and structural analyses of protein—DNA complexes involving DNA glycosylases, and for the isolation of a novel DNA glycosylase. An approach to trap covalently a DNA glycosylase-intermediate complex has also been used to elucidate the mechanism of DNA glycosylases.     

The mycobacterial cell wall: structure, biosynthesis and sites of drug action

Structural analysis has been successfully implemented recently to obtain valuable information on the mycobacterial cell wall components, many of which have formed the basis for biosynthesis and functional studies towards developing better drugs and possible vaccines. The highly complex and well organized structure unique to mycobacteria, represents the best target for novel antimycobacterial agents. Until recently, our knowledge of the enzymes responsible for the biogenesis of the cell wall components was almost negligible. The pathways are now being elucidated in several laboratories. Highlights of this review include significant advances in the structure and biochemistry of the major cell wall components and potenital targets for generation of new drugs.     

Bacterial ghosts--biological particles as delivery systems for antigens, nucleic acids and drugs

Despite the exponential rate of discovery of new antigens and DNA vaccines resulting from modern molecular biology and proteomics, the lack of effective delivery technology is a major limiting factor in their application. The bacterial ghost system represents a platform technology for antigen, nucleic acid and drug delivery. Bacterial ghosts have significant advantages over other engineered biological delivery particles, owing to their intrinsic cellular and tissue tropic abilities, ease of production and the fact that they can be stored and processed without the need for refrigeration. These particles have found both veterinary and medical applications for the vaccination and treatment of tumors and various infectious diseases.     

Insights into the mechanisms of homologous recombination from the structure of RuvA

The recent structure determination of RuvA has provided the first insights into the structural basis for its interaction with Holliday junction DNA. Multiple copies of a helix-hairpin-helix motif which line the four grooves between the monomers in the tetrameric structure are thought to be involved in the interaction of the protein with its DNA target. This suggests that the four arms of the junction are held by RuvA in a fourfold symmetric arrangement and has fuelled ideas on the way in which components of the Ruv complex combine to catalyse the process of homologous recombination     

Agents that target telomerase and telomeres

Telomeres are guanine-rich regions that are located at the ends of chromosomes and are essential for preventing aberrant recombination and protecting against exonucleolytic DNA degradation. Telomeres are maintained by telomerase, an RNA-dependent DNA polymerase. Because telomerase is known to be expressed in tumor cells, which concurrently have short telomeres, and not in most somatic cells, which usually have long telomeres, telomerase and telomere structures have been recently proposed as attractive targets for the discovery of new anticancer agents. The most exciting current strategies are aimed at specifically designing new drugs that target telomerase or telomeres and new models have been formulated to study the biological effects of inhibitors of telomerase and telomeres both in vitro and in vivo.     

Vicious circles: a mechanism for yeast aging

The past year has confirmed the great potential of the yeast Saccharomyces cerevisiae as a model to study aging. Ground breaking papers have revealed similarities between aging in yeast and in mammals, and have identified genetic instability of the ribosomal DNA array as the first known cause of aging in yeast cells.     

The new generation of recombinant viral subunit vaccines

There have been two major focal points for the development of recombinant viral subunit vaccines. Expression strategies for the candidate antigens have focused increasingly on producing more natural antigenic structures, for example, virus-like particles. Also, new adjuvants have been used in order to increase immunogenicity.     

Xenogeneic organ transplantation

Over the past few years, several major advances have occurred in the understanding of how the humoral and cellular immune system of humans recognizes and destroys transplant cells, tissues and organs derived from animal sources. Consequently, armed with this new knowledge, several laboratories have now developed novel immunoprotective technologies that may allow xenotransplantation to be clinically feasible.     

Bioinformatics: from genome data to biological knowledge

Recently, molecular biologists have sequenced about a dozen bacterial genomes and the first eukaryotic genome. We can now obtain answers to detailed questions about the complete set of genes of an organism. Bioinformatics methods are increasingly used for attaching biological knowledge to long lists of genes, assigning genes to biological pathways, comparing the gene sets of different species, identifying specificity factors, and describing sets of highly conserved proteins common to all domains of life. Substantial progress has recently been made in the availability of primary and added-value databases, in the development of algorithms and of network information services for genome analysis. The pharmaceutical industry has greatly benefited from the accumulation of sequence data through the identification of targets and candidates for the development of drugs, vaccines, diagnostic markers and therapeutic proteins.     

Generating sexually differentiated songs

The past year has witnessed increased confusion as to the role of gonadal hormones in the development of neuroeffectors for sexually differentiated vocalizations in several species. Are sex differences in levels of circulating gonadal hormones robust enough to account for the full spectrum of male/female differences? Understanding how vocal behaviors are generated has improved, permitting greater insights into how differences in cell number and type contribute to male- and female-specific songs in frogs and birds.     

Artificial dinuclear phosphoesterases

Many elegant dinuclear transition metal and lathanide complexes that efficiently hydrolyze phosphate esters including RNA and DNA have been reported recently. In most cases, the dinuclear complexes are much more reactive than the corresponding mononuclear metal complexes. Furthermore, structural and kinetic data indicate that substantial rate acceleration for phosphate diester cleavage is obtained by bridging the dinuclear metal centers with the two phosphoryl oxygens. Interestingly, such bridging structures have recently been implicated in several metalloenzyme catalyzed phosphate hydrolyses.     

Recognition of cationic species with synthetic receptors

In the past year progress in the study of cationic species has been made, particularly in our understanding of the factors which control the selective recognition of biologically important cations such as ammonium, alkali and alkaline earth metal ions, and of metal ions used in biomedicine such as lanthanides and iron(III). Based on this knowledge, several new hosts with improved transport, photophysical and biological properties have been designed.     

Out of Africa? What do genes tell us?

Genetic diversity patterns in nuclear versus mitochondrial systems and in low versus high mutation rate systems do not support the hypothesis of a recent African origin for all of humanity following a split between Africans and non-Africans 100,000 years ago, nor do genetic distance data. Geographical analyses of nuclear and mitochondrial gene trees do not support the hypothesis of a recent global replacement of humans coming out of Africa, although a local replacement event in Europe is indicated by these analyses and recent studies on Neandertal DNA. The gene tree analyses instead indicate that genetic interchanges have ensured that all of humanity has evolved as a single evolutionary lineage with no major splits.     

Ionic currents, transmitters and models of motor pattern generators

Recent work, combining direct study of ion channels and synapses with pharmacological manipulations and realistic computer simulations, has deepened our understanding of how motor circuits produce rhythmic outputs. In several preparations, both the roles of some key ionic currents in circuit operation and the mechanisms by which circuit operation may be modulated have been identified.     

The dynamics of cyclin dependent kinase structure

In the past year, several new crystal structures have provided exciting insights into the conformational changes underlying the regulation of cyclin-dependent kinases. We now understand the structural basis of many of the mechanisms by which cyclin-dependent kinases are regulated, including activation by cycling binding and phosphorylation, inhibition by the inhibitor p27, and binding by the CKS proteins.     

Mechanisms of DNA bending

Genome packaging and gene regulation require DNA bending. Recent developments in the elucidation of the mechanisms involved in DNA bending include new X-ray structures (most notably that of the mammalian nucleosome) wherein DNA is bent, controversy surrounding interpretation of DNA-bending experiments with basic-leucine zipper proteins, studies of electrostatic effects in DNA bending, and the design of artificial DNA-bending ligands.