Genome-wide mutant collections: toolboxes for functional genomics

The sequencing of entire genomes has led to the identification of many genes. A future challenge will be to determine the function of all of the genes of an organism. One of the best ways to ascertain function is to disrupt genes and determine the phenotype of the resulting organism. Novel large-scale approaches for generating gene disruptions and analyzing the resulting phenotype are underway in the budding yeast Saccharomyces cerevisiae and other organisms including flies, Mycoplasma, worms, plants and mice. These approaches and mutant collections will be extremely valuable to the scientific community and will dramatically alter the manner in which science is performed in the future.     

Genome-wide survey of prokaryotic O-protein phosphatases

Complex and diverse signal transduction circuits are responsible for the efficient functioning of cellular network. Protein kinases and O-protein phosphatases are primarily responsible for propagating such stimuli within a eukaryotic cell. However, there is limited understanding of O-protein phosphatases in the prokaryotic genomes. The availability of complete genome sequence information for several prokaryotes permits a genome-wide survey of O-protein phosphatases. The distribution of the various protein phosphatase families has been observed to be mosaic, with the exception of the members of the phospho protein family P (PPP), which is consistent with previous studies. The PPP family is ubiquitous in the prokaryotic world and undergoes the highest sequence divergence within a genome amongst phosphatases studied. The co-occurrence of low molecular mass tyrosine phosphatase (LMWPc) and PPP domain in a single polypeptide suggests that the protein present in Archaeoglobus fulgidus might represent the progenitor for all protein phosphatases. The curation of data on prokaryotic protein phosphatases provides a convenient framework for the analysis of domain architectures and for characterising structural and functional properties of this important family of signalling proteins.     

Patterns of nucleotide substitution in pseudogenes and functional genes

Summary The pattern of point mutations is inferred from nucleotide substitutions in pseudogenes. The pattern obtained suggests that transition mutations occur somewhat more frequently than transversion mutations and that mutations result more often in A or T than in G or C. Our results are discussed with respect to the predictions from Topal and Fresco's model for the molecular basis of point (substitution) mutations (Nature 263:285–289, 1976). The pattern of nucleotide substitution at the first and second positions of codons in functional genes is quite similar to that in pseudogenes, but the relative frequency of the transition CT in the sense strand is drastically reduced and those of the transversions CG and GC are doubled. The differences between the two patterns can be explained by the observation that in the protein evolution amino acid substitutions occur mainly between amino acids with similar biochemical properties (Grantham, Science 185:862–864, 1974). Our results for the patterns of nucleotide substitutions in pseudogenes and in functional genes lead to the prediction that both the coding and non-coding regions of protein coding genes should have high frequencies of A and T. Available data show that the non-coding regions are indeed high in A and T but the coding regions are low in T, though high in A.     

Genome-wide survey of alternative splicing in the grass Brachypodium distachyon: a emerging model biosystem for plant functional genomics

A draft sequence of the genome of Brachypodium distachyon, the emerging grass model, was recently released. This represents a unique opportunity to determine its functional diversity compared to the genomes of other model species. Using homology mapping of assembled expressed sequence tags with chromosome scale pseudomolecules, we identified 128 alternative splicing events in B. distachyon. Our study identified that retention of introns is the major type of alternative splicing events (53%) in this plant and highlights the prevalence of splicing site recognition for definition of introns in plants. We have analyzed the compositional profiles of exon-intron junctions by base-pairing nucleotides with U1 snRNA which serves as a model for describing the possibility of sequence conservation. The alternative splicing isoforms identified in this study are novel and represent one of the potentially biologically significant means by which B. distachyon controls the function of its genes. Our observations serve as a basis to understand alternative splicing events of cereal crops with more complex genomes, like wheat or barley.     

A genome-wide survey of human thioredoxin and glutaredoxin family pseudogenes

The thioredoxin/glutaredoxin family consists of small heat-stable proteins that have a highly conserved CXXC active site and that participate in the regulation of many redox reactions. We have searched the human genome sequence to find putative pseudogenes (non-functional copies of protein-coding genes) for all known members of this family. This survey has resulted in the identification of seven processed pseudogenes for human Trx1 and two more for human Grx1. No evidence for the presence of processed pseudogenes has been found for the remaining members of this family. In addition, we have been unable to detect any non-processed pseudogenes derived from any member of the family in the human genome. The seven thioredoxin pseudogenes can be divided into two groups: Trx1-psi2, -psi3, -psi4, -psi5 and -psi6 arose from the functional ancestor, whereas Trx1-psi1 and -psi7 originated from Trx1-psi2 and -psi6, respectively. In all cases, the pseudogenes originated after the human/rodent radiation as shown by phylogenetic analysis. This is also the case for Grx1-psi1 and Grx1-psi2, which are placed between rodent and human sequences in the phylogenetic tree. Our study provides a molecular record of the recent evolution of these two genes in the hominid lineage.     

Genome-wide survey and expression profiling of CCCH-zinc finger family reveals a functional module in macrophage activation

Previously, we have identified a novel CCCH zinc finger protein family as negative regulators of macrophage activation. To gain an overall insight into the entire CCCH zinc finger gene family and to evaluate their potential role in macrophage activation, here we performed a genome-wide survey of CCCH zinc finger genes in mouse and human. Totally 58 CCCH zinc finger genes in mouse and 55 in human were identified and most of them have not been reported previously. Phylogenetic analysis revealed that the mouse CCCH family was divided into 6 groups. Meanwhile, we employed quantitative real-time PCR to profile their tissue expression patterns in adult mice. Clustering analysis showed that most of CCCH genes were broadly expressed in all of tissues examined with various levels. Interestingly, several CCCH genes Mbnl3, Zfp36l2, Zfp36, Zc3h12a, Zc3h12d, Zc3h7a and Leng9 were enriched in macrophage-related organs such as thymus, spleen, lung, intestine and adipose. Consistently, a comprehensive assessment of changes in expression of the 58 members of the mouse CCCH family during macrophage activation also revealed that these CCCH zinc finger genes were associated with the activation of bone marrow-derived macrophages by lipopolysaccharide. Taken together, this study not only identified a functional module of CCCH zinc finger genes in the regulation of macrophage activation but also provided the framework for future studies to dissect the function of this emerging gene family.     

Genome-wide high-throughput screens in functional genomics

The availability of complete genome sequences from many organisms has yielded the ability to perform high-throughput, genome-wide screens of gene function. Within the past year, rapid advances have been made towards this goal in many major model systems, including yeast, worms, flies, and mammals. Yeast genome-wide screens have taken advantage of libraries of deletion strains, but RNA-interference has been used in other organisms to knockdown gene function. Examples of recent large-scale functional genetic screens include drug-target identification in yeast, regulators of fat accumulation in worms, growth and viability in flies, and proteasome-mediated degradation in mammalian cells. Within the next five years, such screens are likely to lead to annotation of function of most genes across multiple organisms. Integration of such data with other genomic approaches will extend our understanding of cellular networks.     

Genome-wide survey for genes horizontally transferred from cellular organisms to baculoviruses

The phylogeny of 13 viral species in the genera Granulovirus and Nucleopolyhedrovirus (family Baculoviridae) was reconstructed on the basis of 22 conserved protein families shared by all species, and a comprehensive homology search and phylogenetic analysis of the complete genomes of these viruses was used to test for horizontal gene transfer from cellular organisms. Statistically significant evidence of horizontal transfer was found in the case of six protein families (DNA ligase, ribonucleotide reductase 1, SNF2 global transactivator, inhibitor of apoptosis, chitinase, and UDP-glucosyltransferase). Three of these families are known to play key roles in the infection of insect hosts by these viruses. There was evidence that two of these (inhibitor of apoptosis and UDP-glucosyltransferase) were derived from the insect host. By contrast, the gene encoding chitinase in these viruses was evidently derived from a group of bacteria (the gamma subdivision of proteobacteria), which use chitinase to break down fungal chitins.     

Genome-wide association and functional follow-up reveals new loci for kidney function

Chronic kidney disease (CKD) is an important public health problem with a genetic component. We performed genome-wide association studies in up to 130,600 European ancestry participants overall, and stratified for key CKD risk factors. We uncovered 6 new loci in association with estimated glomerular filtration rate (eGFR), the primary clinical measure of CKD, in or near MPPED2, DDX1, SLC47A1, CDK12, CASP9, and INO80. Morpholino knockdown of mpped2 and casp9 in zebrafish embryos revealed podocyte and tubular abnormalities with altered dextran clearance, suggesting a role for these genes in renal function. By providing new insights into genes that regulate renal function, these results could further our understanding of the pathogenesis of CKD.     

Vertebrate pseudogenes

Pseudogenes are commonly encountered during investigation of the genomes of a wide range of life forms. This review concentrates on vertebrate, and in particular mammalian, pseudogenes and describes their origin and subsequent evolution. Consideration is also given to pseudogenes that are transcribed and to the unusual group of genes that exist at the interface between functional genes and non-functional pseudogenes. As the sequences of different genomes are characterised, the recognition and interpretation of pseudogene sequences will become more important and have a greater impact in the field of molecular genetics.     

Genome-wide analysis of pseudogenes reveals HBBP1’s human-specific essentiality in erythropoiesis and implication in β-thalassemia

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Microbial survey of a full-scale, biologically active filter for treatment of drinking water

The microbial community of a full-scale, biologically active drinking water filter was surveyed using molecular techniques. Nitrosomonas, Nitrospira, Sphingomonadales, and Rhizobiales dominated the clone libraries. The results elucidate the microbial ecology of biological filters and demonstrate that biological treatment of drinking water should be considered a viable alternative to physicochemical methods.