Comparative genomics of the restriction-modification systems in Helicobacter pylori

Helicobacter pylori is a Gram-negative bacterial pathogen with a small genome of 1.64–1.67 Mb. More than 20 putative DNA restriction-modification (R-M) systems, comprising more than 4% of the total genome, have been identified in the two completely sequenced H. pylori strains, 26695 and J99, based on sequence similarities. In this study, we have investigated the biochemical activities of 14 Type II R-M systems in H. pylori 26695. Less than 30% of the Type II R-M systems in 26695 are fully functional, similar to the results obtained from strain J99. Although nearly 90% of the R-M genes are shared by the two H. pylori strains, different sets of these R-M genes are functionally active in each strain. Interestingly, all strain-specific R-M genes are active, whereas most shared genes are inactive. This agrees with the notion that strain-specific genes have been acquired more recently through horizontal transfer from other bacteria and selected for function. Thus, they are less likely to be impaired by random mutations. Our results also show that H. pylori has extremely diversified R-M systems in different strains, and that the diversity may be maintained by constantly acquiring new R-M systems and by inactivating and deleting the old ones.

Toward a plant genomics initiative: Thoughts on the value of cross-species and cross-genera comparisons in the grasses

Comparative genomics offers unparalleled opportunities to integrate historically distinct disciplines, to link disparate biological kingdoms, and to bridge basic and applied science. Cross-species, cross-genera, and cross-kingdom comparisons are proving key to understanding how genes are structured, how gene structure relates to gene function, and how changes in DNA have given rise to the biological diversity on the planet. The application of genomics to the study of crop species offers special opportunities for innovative approaches for combining sequence information with the vast reservoirs of historical information associated with crops and their evolution. The grasses provide a particularly well developed system for the development of tools to facilitate comparative genetic interpretation among members of a diverse and evolutionarily successful family. Rice provides advantages for genomic sequencing because of its small genome and its diploid nature, whereas each of the other grasses provides complementary genetic information that will help extract meaning from the sequence data. Because of the importance of the cereals to the human food chain, developments in this area can lead directly to opportunities for improving the health and productivity of our food systems and for promoting the sustainable use of natural resources.

Complete sequencing of the Fugu WAGR region from WT1 to PAX6: Dramatic compaction and conservation of synteny with human chromosome 11p13

The pufferfish Fugu rubripes has a genome ≈7.5 times smaller than that of mammals but with a similar number of genes. Although conserved synteny has been demonstrated between pufferfish and mammals across some regions of the genome, there is some controversy as to what extent Fugu will be a useful model for the human genome, e.g., [Gilley, J., Armes, N. & Fried, M. (1997) Nature (London) 385, 305–306]. We report extensive conservation of synteny between a 1.5-Mb region of human chromosome 11 and <100 kb of the Fugu genome in three overlapping cosmids. Our findings support the idea that the majority of DNA in the region of human chromosome 11p13 is intergenic. Comparative analysis of three unrelated genes with quite different roles, WT1, RCN1, and PAX6, has revealed differences in their structural evolution. Whereas the human WT1 gene can generate 16 protein isoforms via a combination of alternative splicing, RNA editing, and alternative start site usage, our data predict that Fugu WT1 is capable of generating only two isoforms. This raises the question of the extent to which the evolution of WT1 isoforms is related to the evolution of the mammalian genitourinary system. In addition, this region of the Fugu genome shows a much greater overall compaction than usual but with significant noncoding homology observed at the PAX6 locus, implying that comparative genomics has identified regulatory elements associated with this gene.

Genetic footprinting with mariner-based transposition in Pseudomonas aeruginosa

The complete DNA sequence of Pseudomonas aeruginosa provides an opportunity to apply functional genomics to a major human pathogen. A comparative genomics approach combined with genetic footprinting was used as a strategy to identify genes required for viability in P. aeruginosa. Use of a highly efficient in vivo mariner transposition system in P. aeruginosa facilitated the analysis of candidate genes of this class. We have developed a rapid and efficient allelic exchange system by using the I-SceI homing endonuclease in conjunction with in vitro mariner mutagenesis to generate mutants within targeted regions of the P. aeruginosa chromosome for genetic footprinting analyses. This technique for generating transposon insertion mutants should be widely applicable to other organisms that are not naturally transformable or may lack well developed in vivo transposition systems. We tested this system with three genes in P. aeruginosa that have putative essential homologs in Haemophilus influenzae. We show that one of three H. influenzae essential gene homologs is needed for growth in P. aeruginosa, validating the practicality of this comparative genomics strategy to identify essential genes in P. aeruginosa.

RefSeq and LocusLink: NCBI gene-centered resources

Thousands of genes have been painstakingly identified and characterized a few genes at a time. Many thousands more are being predicted by large scale cDNA and genomic sequencing projects, with levels of evidence ranging from supporting mRNA sequence and comparative genomics to computing ab initio models. This, coupled with the burgeoning scientific literature, makes it critical to have a comprehensive directory for genes and reference sequences for key genomes. The NCBI provides two resources, LocusLink and RefSeq, to meet these needs. LocusLink organizes information around genes to generate a central hub for accessing gene-specific information for fruit fly, human, mouse, rat and zebrafish. RefSeq provides reference sequence standards for genomes, transcripts and proteins; human, mouse and rat mRNA RefSeqs, and their corresponding proteins, are discussed here. Together, RefSeq and LocusLink provide a non-redundant view of genes and other loci to support research on genes and gene families, variation, gene expression and genome annotation. Additional information about LocusLink and RefSeq is available at http://www.ncbi.nlm.nih.gov/LocusLink/.

Genomic analysis of orthologous mouse and human olfactory receptor loci

Olfactory receptor (OR) genes represent ≈1% of genomic coding sequence in mammals, and these genes are clustered on multiple chromosomes in both the mouse and human genomes. We have taken a comparative genomics approach to identify features that may be involved in the dynamic evolution of this gene family and in the transcriptional control that results in a single OR gene expressed per olfactory neuron. We sequenced ≈350 kb of the murine P2 OR cluster and used synteny, gene linkage, and phylogenetic analysis to identify and sequence ≈111 kb of an orthologous cluster in the human genome. In total, 18 mouse and 8 human OR genes were identified, including 7 orthologs that appear to be functional in both species. Noncoding homology is evident between orthologs and generally is confined within the transcriptional unit. We find no evidence for common regulatory features shared among paralogs, and promoter regions generally do not contain strong promoter motifs. We discuss these observations, as well as OR clustering, in the context of evolutionary expansion and transcriptional regulation of OR repertoires.