Agrobacterium-mediated transformation as a tool for functional genomics in fungi

In the era of functional genomics, the need for tools to perform large-scale targeted and random mutagenesis is increasing. A potential tool is Agrobacterium-mediated fungal transformation. A. tumefaciens is able to transfer a part of its DNA (transferred DNA; T-DNA) to a wide variety of fungi and the number of fungi that can be transformed by Agrobacterium-mediated transformation (AMT) is still increasing. AMT has especially opened the field of molecular genetics for fungi that were difficult to transform with traditional methods or for which the traditional protocols failed to yield stable DNA integration. Because of the simplicity and efficiency of transformation via A. tumefaciens, it is relatively easy to generate a large number of stable transformants. In combination with the finding that the T-DNA integrates randomly and predominantly as a single copy, AMT is well suited to perform insertional mutagenesis in fungi. In addition, in various gene-targeting experiments, high homologous recombination frequencies were obtained, indicating that the T-DNA is also a useful substrate for targeted mutagenesis. In this review, we discuss the potential of the Agrobacterium DNA transfer system to be used as a tool for targeted and random mutagenesis in fungi.     

Comparative and functional genomics of closteroviruses

The largest extant RNA genomes are found in two diverse families of positive-strand RNA viruses, the animal Coronaviridae and the plant Closteroviridae. Comparative analysis of the viruses from the latter family reveals three levels of gene conservation. The most conserved gene module defines RNA replication and is shared with plant and animal viruses in the alphavirus-like superfamily. A module of five genes that function in particle assembly and transport is a hallmark of the family Closteroviridae and was likely present in the ancestor of all three closterovirus genera. This module includes a homologue of Hsp70 molecular chaperones and three diverged copies of the capsid protein gene. The remaining genes show dramatic variation in their numbers, functions, and origins among closteroviruses within and between the genera. Proteins encoded by these genes include suppressors of RNA silencing, RNAse III, papain-like proteases, the AlkB domain implicated in RNA repair, Zn-ribbon-containing protein, and a variety of proteins with no detectable homologues in the current databases. The evolutionary processes that have shaped the complex and fluid genomes of the large RNA viruses might be similar to those that have been involved in evolution of genomic complexity in other divisions of life.     

Comparative genomics as a tool to reveal functional equivalences between human and mouse dendritic cell subsets

Summary: During evolution, vertebrates have developed an adaptive immune system able to cope with a variety of pathogens. Dendritic cells (DCs) are central to this process. DCs integrate information derived from pathogens or endogenous danger signals and convey them to T lymphocytes. Most of the present knowledge on DCs was generated in mice or by using human DCs differentiated in vitro from monocytes. In both species, several DC subsets have been identified in vivo based on differences in their phenotypes, anatomical locations or functions. In mice, protective immunity against intracellular pathogens or tumors can be induced most efficiently by targeting antigens to the CD8α⁺ DCs, a subset of DCs which resides in lymphoid tissues and is especially efficient at cross-presenting exogenous antigens to CD8⁺ T lymphocytes. In contrary, harnessing human DC subsets for medical purposes is currently hampered by insufficient knowledge about these cells. To overcome this cognitive gap, we are using comparative genomics as a tool for designing hypotheses and experiments to further characterize DC subset functions and their molecular control, including the investigation of the functional equivalences that might exist between human and mouse DC subsets.     

Comparative genomics as a tool to understand evolution and disease

When the human genome project started, the major challenge was how to sequence a 3 billion letter code in an organized and cost-effective manner. When completed, the project had laid the foundation for a huge variety of biomedical fields through the production of a complete human genome sequence, but also had driven the development of laboratory and analytical methods that could produce large amounts of sequencing data cheaply. These technological developments made possible the sequencing of many more vertebrate genomes, which have been necessary for the interpretation of the human genome. They have also enabled large-scale studies of vertebrate genome evolution, as well as comparative and human medicine. In this review, we give examples of evolutionary analysis using a wide variety of time frames—from the comparison of populations within a species to the comparison of species separated by at least 300 million years. Furthermore, we anticipate discoveries related to evolutionary mechanisms, adaptation, and disease to quickly accelerate in the coming years.The human genome project pioneered not only the bacterial artificial chromosome (BAC)-based sequencing of a mammalian-sized genome (International Human Genome Sequencing Consortium 2001), but also the methodology of whole-genome shotgun (WGS) sequencing (Venter et al. 2001). WGS sequencing was further improved and applied to the mouse genome (Mouse Genome Sequencing Consortium 2002) and then became the technique of choice for many vertebrate genomes (International Chicken Genome Sequencing Consortium 2004; Lindblad-Toh et al. 2005; Mikkelsen et al. 2007; Warren et al. 2008). This methodology has two advantages: It allows a relatively unbiased approach to sequencing a genome and it has the ability to be automated and hence cost effective. Thus, it revolutionized the study of comparative genomics of vertebrate genomes. New sequencing technologies have further reduced the cost of WGS sequencing, making vertebrate genome sequencing even more popular (Li et al. 2010).Prior to whole-genome sequencing of many vertebrates, the ENCODE project had selected a representative ∼1% on the human genome to be systematically sequenced in a BAC-by-BAC approach across mammals and some vertebrates. The comparative ENCODE project demonstrated the presence of widespread orthology between species, high levels of conservation within genes, as well as extensive signals of conservation outside genes. Noncoding features lacking experimental validation, however, were harder to interpret than protein-coding genes (Margulies et al. 2007).The human genome sequence described many of the features of the human genome such as transposable elements (TEs), segmental duplications, genes, and their promoters. The human gene count predicted at approximately 40,000 (International Human Genome Sequencing Consortium 2001) was a huge refinement from the previously cited estimate of 100,000 genes. Nonetheless, it was far above the current tally of somewhere close to 22,000 human genes (Clamp et al. 2007).For many scientists, the comparison of the mouse and human genomes came as a strong confirmation that large-scale comparative genomics is essential for understanding the human genome. Comparison of these two mammals refined the mammalian gene count to ∼30,000 and allowed the first genome-wide estimate of the minimum fraction of the human genome that is conserved across placental mammals and is hence functional: a full 5%, much more than the ∼1.2% occupied by protein-coding sequence (Mouse Genome Sequencing Consortium 2002).     

Comparative evolutionary genomics of androgen-binding protein genes

Allelic variation within the mouse androgen-binding protein (ABP) alpha subunit gene (Abpa) has been suggested to promote assortative mating and thus prezygotic isolation. This is consistent with the elevated evolutionary rates observed for the Abpa gene, and the Abpb and Abpg genes whose products (ABPbeta and ABPgamma) form heterodimers with ABPalpha. We have investigated the mouse sequence that contains the three Abpa/b/g genes, and orthologous regions in rat, human, and chimpanzee genomes. Our studies reveal extensive "remodeling" of this region: Duplication rates of Abpa-like and Abpbg-like genes in mouse are >2 orders of magnitude higher than the average rate for all mouse genes; synonymous nucleotide substitution rates are twofold higher; and the Abpabg genomic region has expanded nearly threefold since divergence of the rodents. During this time, one in six amino acid sites in ABPbetagamma-like proteins appear to have been subject to positive selection; these may constitute a site of interaction with receptors or ligands. Greater adaptive variation among Abpbg-like sequences than among Abpa-like sequences suggests that assortative mating preferences are more influenced by variation in Abpbg-like genes. We propose a role for ABPalpha/beta/gamma proteins as pheromones, or in modulating odorant detection. This would account for the extraordinary adaptive evolution of these genes, and surrounding genomic regions, in murid rodents.     

Comparative genomics on Wnt5a and Wnt5b genes

Canonical WNTs (WNT2, WNT2B, etc) activate the beta-catenin-TCF pathway to induce carcinogenesis, while non-canonical WNTs (WNT5A, WNT11, etc) activate the planar cell polarity (PCP) pathway to induce cell motility and metastasis. WNT5A gene at chromosome 3p14.3 and WNT5B gene at chromosome 12p13.33 are paralogs within the human genome. Here, we identified and characterized rat Wnt5a and Wnt5b genes by using bioinformatics. Rat Wnt5a and Wnt5b genes, consisting of five exons, were identified within AC095764.5 and AC112027.3 genome sequences, respectively. Rat Wnt5a (380 aa) and Wnt5b (359 aa) were secreted proteins with 24 conserved Cys residues and four Asn-linked glycosylation sites, which showed 75.8% total-amino-acid identity. Nucleotide position 182586-183836 of AC095764.5 genome sequence and nucleotide position 161044-159886 of AC121764.2 genome sequence were identified as evolutionarily conserved rat Wnt5a and human WNT5A promoters, respectively. Nucleotide identity between rat Wnt5a and human WNT5A promoters was 72.5%. E47 and NKX2-5-binding sites were evolutionarily conserved among rat Wnt5a, mouse Wnt5a, and human WNT5A promoters. On the other hand, rodent Wnt5b promoters and human WNT5B promoter were significantly divergent. Up-regulation of Wnt5b during rodent adipocytic differentiation does not simply indicate the implication of WNT5B in human adipogenesis. Real susceptibility gene for type 2 diabetes, associated with SNP within intron 3 of human WNT5B gene (IMS-JST024404), remains to be identified. This is the first report on rat Wnt5a and Wnt5b genes as well as on comparative genomics for Wnt5a and Wnt5b orthologs.     

Comparative genomics analysis of five families of antimicrobial peptide-like genes in seven ant species

Ants, as eusocial insects, live in dense groups with high connectivity, increasing the risk of pathogen spread and possibly driving the evolution of their antimicrobial immune system. Draft genomes of seven ant species provide a new source to undertake comparative study of their antimicrobial peptides (AMPs), key components of insect innate immunity. By using computational approaches, we analyzed five AMP families that include abaecins, hymenoptaecins, insect defensins, tachystatins, and crustins in ants, which comprise 69 new members. Among them, a new type of proline-rich abaecins was recognized and they are exclusively present in ants. Hymenoptaecins, a family of glycine-rich AMPs from Hymenoptera and Diptera, exhibit variable numbers of intragenic tandem repeats in a lineage-specific manner and all hymenoptaecins in ants have evolved an acidic C-terminal propeptide. In some ant species, insect defensins with the cysteine-stabilized α-helical and β-sheet (CSαβ) fold and tachystatin-like AMPs with the inhibitor cysteine knot (ICK) fold have undergone gene expansion and differential gene loss. Moreover, extensive sequence diversity exists in the C-termini of the defensins and the ICK-type peptides and the n-loop of the defensins. Also, we identified for the first time a crustin-type AMP in ants, which are only known in crustaceans previously. These ant crustins evolutionarily gain an aromatic amino acid-rich insertion when compared with those of crustaceans. Our work not only enlarges the insect AMP resource, but also sheds light on the complexity and dynamic evolution of AMPs in ants.     

Identification of rheumatoid arthritis causal genes using functional genomics

Over the past decade, genome‐wide association studies have contributed a wealth of knowledge to our understanding of polygenic disorders such as rheumatoid arthritis. As the size of sample cohorts has improved so too have the computational and experimental methods used to robustly define variants associated with disease susceptibility. The challenge now remains to translate these findings into improved understanding of disease aetiology and patient care. Whilst much of the focus of translating the findings of genome‐wide association studies has been on global analysis of all variants identified, careful functional study of individual disease susceptibility loci will be required in order to refine our understanding of how individual variants contribute to disease risk. Here, we present the argument behind such an approach and describe some of the novel tools being used to investigate risk loci. This includes the use of chromosomal conformation capture techniques and modifications of the CRISPR‐Cas9 system, with several examples of their implementation being described.     

Comparative genomics analysis of Mycobacterium NrdH-redoxins

NrdH is one of the intracellular redoxins exist in Mycobacterium and other microorganisms lacking the common glutathione system. NrdH seems a chimeric protein of glutaredoxin amino acid sequence and thioredoxin activity. It serves as a hydrogen donor for ribonucleotide reductase. Mycobacterium tuberculosis might take advantage of this enzyme to persist in the highly oxidized host mononuclear phagocytes and maintain the balance of intracellular redox potential. Physiological role of NrdH and comparison of its genomic context organization were summarized here. The most intriguing feature might be its new drug targets potential.     

Comparative genomic analysis as a tool for biological discovery

The recent completion of the human genome sequence has enabled the identification of a large fraction of our gene catalogue and their physical chromosomal position. However, current efforts lag at defining the cis-regulatory sequences that control the spatial and temporal patterns of each gene's expression. This task remains difficult due to our lack of knowledge of the vocabulary controlling gene regulation and the vast genomic search space, with greater than 95% of our genome being noncoding. Recent comparative genomic-based strategies are beginning to aid in the identification of functional sequences based on their high levels of evolutionary conservation. This has proven successful for comparisons between closely related species such as human–primate or human–mouse, but also holds true for distant evolutionary comparisons, such as human–fish or human–bird. In this review we provide support for the utility of cross-species sequence comparisons by illustrating several applications of this strategy, including the identification of new genes and functional non-coding sequences. We also discuss emerging concepts as this field matures, such as how to properly select which species for comparison, which may differ significantly between independent studies.     

Genomic analysis of allergen genes in Aspergillus spp: the relevance of genomics to everyday research.

Full genomic sequencing of Aspergillus fumigatus and other genomes has allowed correction of Aspergillus allergen gene sequences and requires revision of Genbank and IUIS sequences of allergens. In addition allergens in other fungal species may be found in the aspergilli. We compared the published sequences of numerous allergens with recently available genome sequences. This analysis suggests that Aspf 56KD, Asp f 15 and Asp f 16 should be removed from the approved allergen list and that Asp f 17 is a larger protein than published. Additionally we propose likely gene candidates for Asp f GST (Afu6g09690) and Asp o lipase (AO090701000644). We suggest that the heat shock allergens should be re-classified: Asp f 12 should be classified as HSP90 (Asp f 12), HSP88 (Mala s 10) and HSP70 (Alt a 3, Cla h 4 and Pen c 19) according to human gene nomenclature. Comparison of fungal allergen databases with genome sequences suggests the presence of a core set of allergen - like proteins in all fungi. We also analysed allergens in the 3 sequenced aspergilli to look for internal homologies and this suggests that multi gene families may produce numerous cross-reactive allergens.     

Citrate synthase gene sequence: a new tool for phylogenetic analysis and identification of Ehrlichia

The sequence of the citrate synthase gene (gltA) of 13 ehrlichial species (Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia muris, an Ehrlichia species recently detected from Ixodes ovatus, Cowdria ruminantium, Ehrlichia phagocytophila, Ehrlichia equi, the human granulocytic ehrlichiosis [HGE] agent, Anaplasma marginale, Anaplasma centrale, Ehrlichia sennetsu, Ehrlichia risticii, and Neorickettsia helminthoeca) have been determined by degenerate PCR and the Genome Walker method. The ehrlichial gltA genes are 1,197 bp (E. sennetsu and E. risticii) to 1,254 bp (A. marginale and A. centrale) long, and GC contents of the gene vary from 30.5% (Ehrlichia sp. detected from I. ovatus) to 51.0% (A. centrale). The percent identities of the gltA nucleotide sequences among ehrlichial species were 49.7% (E. risticii versus A. centrale) to 99.8% (HGE agent versus E. equi). The percent identities of deduced amino acid sequences were 44.4% (E. sennetsu versus E. muris) to 99.5% (HGE agent versus E. equi), whereas the homology range of 16S rRNA genes was 83.5% (E. risticii versus the Ehrlichia sp. detected from I. ovatus) to 99.9% (HGE agent, E. equi, and E. phagocytophila). The architecture of the phylogenetic trees constructed by gltA nucleotide sequences or amino acid sequences was similar to that derived from the 16S rRNA gene sequences but showed more-significant bootstrap values. Based upon the alignment analysis of the ehrlichial gltA sequences, two sets of primers were designed to amplify tick-borne Ehrlichia and Neorickettsia genogroup Ehrlichia (N. helminthoeca, E. sennetsu, and E. risticii), respectively. Tick-borne Ehrlichia species were specifically identified by restriction fragment length polymorphism (RFLP) patterns of AcsI and XhoI with the exception of E. muris and the very closely related ehrlichia derived from I. ovatus for which sequence analysis of the PCR product is needed. Similarly, Neorickettsia genogroup Ehrlichia species were specifically identified by RFLP patterns of RcaI digestion. If confirmed this technique will be useful in rapidly identifying Ehrlichia spp.     

Comparative and functional genomics of the innate immune system in the malaria vector Anopheles gambiae

Summary: In much of Africa, the mosquito Anopheles gambiae is the major vector of human malaria, a devastating infectious disease caused by Plasmodium parasites. Vector and parasite interact at multiple stages and locations, and the nature and effectiveness of this reciprocal interaction determines the success of transmission. Many of the interactions engage the mosquito's innate immunity, a primitive but very effective defense system. In some cases, the mosquito kills the parasite, thus blocking the transmission cycle. However, not all interactions are antagonistic; some represent immune evasion. The sequence of the A. gambiae genome revealed numerous potential components of the innate immune system, and it established that they evolve rapidly, as summarized in the present review. Their rapid evolution by gene family expansion diversification as well as the prevalence of haplotype alleles in the best‐studied families may reflect selective adaptation of the immune system to the exigencies of multiple immune challenges in a variety of ecologic niches. As a follow‐up to the comparative genomic analysis, the development of functional genomic methodologies has provided novel opportunities for understanding the immune system and the nature of its interactions with the parasite. In this context, identification of both Plasmodium antagonists and protectors in the mosquito represents a significant conceptual advance. In addition to providing fundamental understanding of primitive immune systems, studies of mosquito interactions with the parasite open unprecedented opportunities for novel interventions against malaria transmission. The generation of transgenic mosquitoes that resist malaria infection in the wild and the development of antimalarial ‘smart sprays’ capable of disrupting interactions that are protective of the parasite, or reinforcing others that are antagonistic, represent technical challenges but also immense opportunities for improvement of global health.     

AnoEST: toward A. gambiae functional genomics

Here, we present an analysis of 215,634 EST and cDNA sequences of a major vector of human malaria Anopheles gambiae structured into the AnoEST database. The expressed sequences are grouped into clusters using genomic sequence as template and associated with inferred functional annotation, including the following: corresponding Ensembl gene prediction, putative orthologous genes in other species, homology to known proteins, protein domains, associated Gene Ontology terms, and corresponding classification into broad GO-slim functional groups. AnoEST is a vital resource for interpretation of expression profiles derived using recently developed A. gambiae cDNA microarrays. Using these cDNA microarrays, we have experimentally confirmed the expression of 7961 clusters during mosquito development. Of these, 3100 are not associated with currently predicted genes. Moreover, we found that clusters with confirmed expression are nonbiased with respect to the current gene annotation or homology to known proteins. Consequently, we expect that many as yet unconfirmed clusters are likely to be actual A. gambiae genes. [AnoEST is publicly available at http://komar.embl.de, and is also accessible as a Distributed Annotation Service (DAS).].     

Functional genomics approach to the identification of virulence genes involved in Edwardsiella tarda pathogenesis

Edwardsiella tarda is an important cause of hemorrhagic septicemia in fish and also of gastro- and extraintestinal infections in humans. Here, we report the identification of 14 virulence genes of pathogenic E. tarda that are essential for disseminated infection, via a genome-wide analysis. We screened 490 alkaline phosphatase fusion mutants from a library of 450,000 TnphoA transconjugants derived from strain PPD130/91, using fish as an infection model. Compared to the wild type, 15 mutants showed significant decreases in virulence. Six mutants had insertions in the known virulence-related genes, namely, fimA, gadB, katB, pstS, pstC, and ssrB. Some mutants corresponded to known genes (astA, isor, and ompS2) that had not been previously shown to be involved in pathogenesis, and three had insertions in two novel genes. In vivo infection kinetics experiments confirmed the inability of these attenuated mutants to proliferate and cause fatal infection in fish. Screening for the presence of the above-described virulence genes in six virulent and seven avirulent strains of E. tarda indicated that seven of the genes were specific to pathogenic E. tarda. The genes identified here may be used to develop vaccines and diagnostic kits as well as for further studying the pathogenesis of E. tarda and other pathogenic bacteria.