Synthetic genomics: potential and limitations

Technologies to synthetically assemble chromosome sized fragments of DNA as well as to enable making thousands of simultaneous changes to existing genomes are now available. These capacities are collectively termed synthetic genomics. The implications of synthetic genomics extend beyond the limited pathway and gene engineering of the past to include the engineering or whole metabolisms, regulatory networks, and even ecosystems. However, in order for those potentials to be met, certain limitations and barriers must be overcome. These barriers no longer include DNA modification and assembly, but instead are based in the limited organisms that many synthetic genomics methods function in, and the limited software for designing custom genomic sequences.     

Comparative genomics in salt tolerance between Arabidopsis and aRabidopsis-related halophyte salt cress using Arabidopsis microarray

Salt cress (Thellungiella halophila), a halophyte, is a genetic model system with a small plant size, short life cycle, copious seed production, small genome size, and an efficient transformation. Its genes have a high sequence identity (90%-95% at cDNA level) to genes of its close relative, Arabidopsis. These qualities are advantageous not only in genetics but also in genomics, such as gene expression profiling using Arabidopsis cDNA microarrays. Although salt cress plants are salt tolerant and can grow in 500 mm NaCl medium, they do not have salt glands or other morphological alterations either before or after salt adaptation. This suggests that the salt tolerance in salt cress results from mechanisms that are similar to those operating in glycophytes. To elucidate the differences in the regulation of salt tolerance between salt cress and Arabidopsis, we analyzed the gene expression profiles in salt cress by using a full-length Arabidopsis cDNA microarray. In salt cress, only a few genes were induced by 250 mm NaCl stress in contrast to Arabidopsis. Notably a large number of known abiotic- and biotic-stress inducible genes, including Fe-SOD, P5CS, PDF1.2, AtNCED, P-protein, beta-glucosidase, and SOS1, were expressed in salt cress at high levels even in the absence of stress. Under normal growing conditions, salt cress accumulated Pro at much higher levels than did Arabidopsis, and this corresponded to a higher expression of AtP5CS in salt cress, a key enzyme of Pro biosynthesis. Furthermore, salt cress was more tolerant to oxidative stress than Arabidopsis. Stress tolerance of salt cress may be due to constitutive overexpression of many genes that function in stress tolerance and that are stress inducible in Arabidopsis.     

Comparative genomics reveals expansion of the FLC region in the genus Arabidopsis

Mechanisms of genome evolution are poorly understood although recent genome sequencing is providing the tools to begin to illuminate such mechanisms. Using high-resolution molecular cytogenetic tools, we examined the structural evolution of 790 kb surrounding the evolutionarily important FLC locus of Arabidopsis thaliana in three of its relatives, Arabidopsis halleri, Arabidopsis neglecta and Arabidopsis arenosa. Sequenced BACs from A. thaliana were used as heterologous probes across these species and genome expansion was found in all three species relative to A. thaliana, ranging from 16 to 27%. Expansion was seen along the length of the entire region but molecular analyses revealed no characteristic pattern of either intra- or intergenic expansion among these species. Mapping of BACs on DNA fibers from A. thaliana revealed one possible error, ~14 kb missing from the reported sequence, indicating that for comparative studies it is important to confirm the reference sequence to which comparison will be made.     

DNA sequencing: present limitations and prospects for the future

As the human genome program gets under way, we examine the progress made since the first human genome meeting in Santa Cruz in 1985. The lessons of the last 5 years demonstrate that progress has been much slower than anticipated. The new technology being developed in 1985 was fluorescent sequencing and multiplexing. These techniques are now established, but they still have to produce a substantial sequence to rival those determined by conventional technology. Inspection of the EMBL and GenBank databases shows few large sequences have been determined and that there is a large discrepancy between what is theoretically possible and what has been achieved so far.     

Adult stem cell transplantation in stroke: its limitations and prospects

A growing number of studies have demonstrated stem cell-based therapy provides a feasible, realistic approach to the restoration of lost brain function after stroke. Moreover, adult stem cells may provide more appropriate clinical strategies. Leading candidate sources include bone marrow, peripheral blood, adipose tissue, skeletal muscle, and olfactory mucosa, which act as central repositories for multipotent stem cells that can repopulate neural tissues. The medical society is currently enthusiastic concerning the clinical applications of autologous adult stem cells in stroke, based on promising results obtained during experimental studies and initial clinical trials. However, before embracing clinical applications, several essential precautions must be properly addressed. For example, the regenerative potentials of adult stem cells decline with age, and stem cells isolated from aged patients may retain dysfunctional characteristics. Are the natures and amounts of available autologous cells appropriate for therapeutic application in stroke? Do transplanted cells remain functional in the diseased brain, and if so what are the optimal injection routes, cell doses, and timings? Thus, we believe that success in future clinical trials will depend on careful investigation at the experimental level, to allow us to understand not only the practicalities of stem cell use, but also the underlying biological principles involved. Here, we review the advantages and disadvantages of the different adult stem cell sources and discuss the challenges that must be negotiated to achieve transplantation success.     

Comparative genomics and bioenergetics.

Bacterial and archaeal complete genome sequences have been obtained from a wide range of evolutionary lines, which allows some general conclusions about the phylogenetic distribution and evolution of bioenergetic pathways to be drawn. In particular, I searched in the complete genomes for key enzymes involved in aerobic and anaerobic respiratory pathways and in photosynthesis, and mapped them into an rRNA tree of sequenced species. The phylogenetic distribution of these enzymes is very irregular, and clearly shows the diverse strategies of energy conservation used by prokaryotes. In addition, a thorough phylogenetic analysis of other bioenergetic protein families of wide distribution reveals a complex evolutionary history for the respective genes. A parsimonious explanation for these complex phylogenetic patterns and for the irregular distribution of metabolic pathways is that the last common ancestor of Bacteria and Archaea contained several members of every gene family as a consequence of previous gene or genome duplications, while different patterns of gene loss occurred during the evolution of every gene family. This would imply that the last universal ancestor was a bioenergetically sophisticated organism. Finally, important steps that occurred during the evolution of energetic machineries, such as the early evolution of aerobic respiration and the acquisition of eukaryotic mitochondria from a proteobacterium ancestor, are supported by the analysis of the complete genome sequences.     

Advances in maize genomics: the emergence of positional cloning

Positional cloning has been and remains a powerful method for gene identification in Arabidopsis. With the completion of the rice genome sequence, positional cloning in rice also took off, including the cloning of several quantitative trait loci. Positional cloning in cereals such as maize whose genomes are much larger than that of rice was considered near impossible because of the vast amounts of repetitive DNA. However, conservation of synteny across the cereal genomes, in combination with new maize resources, has now made positional cloning in maize feasible. In fact, a chromosomal walk is usually much faster than the more traditional method of gene isolation in maize by transposon tagging.