The multiple facets of homology and their use in comparative genomics to study the evolution of genes, genomes, and species

The incredible development of comparative genomics during the last decade has required a correct use of the concept of homology that was previously utilized only by evolutionary biologists. Unhappily, this concept has been often misunderstood and thus misused when exploited outside its evolutionary context. This review brings back to the correct definition of homology and explains how this definition has been progressively refined in order to adapt it to the various new kinds of analysis of gene properties and of their products that appear with the progress of comparative genomics. Then, we illustrate the power and the proficiency of such a concept when using the available genomics data in order to study the evolution of individual genes, of entire genomes and of species, respectively. After explaining how we detect homologues by an exhaustive comparison of a hundred of complete proteomes, we describe three main lines of research we have developed in the recent years. The first one exploits synteny and gene context data to better understand the mechanisms of genome evolution in prokaryotes. The second one is based on phylogenomics approaches to reconstruct the tree of life. The last one is devoted to reminding that protein homology is often limited to structural segments (SOH=segment of homology or module). Detecting and numbering modules allows tracing back protein history by identifying the events of gene duplication and gene fusion. We insist that one of the main present difficulties in such studies is a lack of a reliable method to identify genuine orthologues. Finally, we show how these homology studies are helpful to annotate genes and genomes and to study the complexity of the relationships between sequence and function of a gene.     

The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins

The von Recklinghausen neurofibromatosis locus, NF1, encodes a protein with homology restricted to the catalytic region of the RAS GTPase-activating protein, GAP, and with extensive homology to the IRA1 and IRA2 gene products of the yeast S. cerevisiae. A segment of the NF1 cDNA gene, expressed in yeast, can complement loss of IRA function and can inhibit both wild-type and mutant activated human H-ras genes that are coexpressed in yeast. Yeast expressing the NF1 segment have increased H-ras GTPase-stimulating activity. These studies indicate that the NF1 gene product can interact with RAS proteins and demonstrate structural and functional similarities and differences among the GAP, IRA1, IRA2, and NF1 proteins.     

Insect segmentation: Genes, stripes and segments in "Hoppers".

Recent work has revealed that orthologues of several segmentation genes are expressed in the grasshopper embryo, in patterns resembling those shown in Drosophila. This suggests that, despite great differences between the embryos, a hierarchy of gap/pair-rule/segment polarity gene function may be a shared and ancestral feature of insect segmentation.     

Chicken orthologues of mammalian imprinted genes are clustered on macrochromosomes and replicate asynchronously

In the chicken genome, most orthologues of mouse imprinted genes are clustered on macrochromosomes. Only a few orthologues are located in the microchromosome complement. Macrochromosomal and, to a lesser extent, microchromosomal regions containing imprinted gene orthologues exhibit asynchronous DNA replication. We conclude that highly conserved arrays of imprinted gene orthologues were selected during vertebrate evolution, long before these genes were recruited for parent-specific gene expression by genomic imprinting mechanisms. Evidently, the macrochromosome complement provides a better chromatin environment for the establishment of asynchronous DNA replication and imprinted gene expression later in evolution than microchromosomes.     

The novel Cladosporium fulvum lysin motif effector Ecp6 is a virulence factor with orthologues in other fungal species

During tomato leaf colonization, the biotrophic fungus Cladosporium fulvum secretes several effector proteins into the apoplast. Eight effectors have previously been characterized and show no significant homology to each other or to other fungal genes. To discover novel C. fulvum effectors that might play a role in virulence, we utilized two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to visualize proteins secreted during C. fulvum –tomato interactions. Three novel C. fulvum proteins were identified: CfPhiA, Ecp6 and Ecp7. CfPhiA shows homology to proteins found on fungal sporogenous cells called phialides. Ecp6 contains lysin motifs (LysM domains) that are recognized as carbohydrate-binding modules. Ecp7 encodes a small, cysteine-rich protein with no homology to known proteins. Heterologous expression of Ecp6 significantly increased the virulence of the vascular pathogen Fusarium oxysporum on tomato. Furthermore, by RNA interference (RNAi)-mediated gene silencing we demonstrate that Ecp6 is instrumental for C. fulvum virulence on tomato. Hardly any allelic variation was observed in the Ecp6 coding region of a worldwide collection of C. fulvum strains. Although none of the C. fulvum effectors identified so far have obvious orthologues in other organisms, conserved Ecp6 orthologues were identified in various fungal species. Homology-based modelling suggests that the LysM domains of C. fulvum Ecp6 may be involved in chitin binding.     

Structural organization of the 3'' half of the rat thyroglobulin gene

We report the structural organization of an 80 Kb segment of rat DNA, which encodes for about 40% of Thyroglobulin mRNA at the 3' end. The codogenic information included in this segment is splitted in 17 exons of homogeneous size (about 200 bp). The seven exons at the extreme 3' end have been precisely defined by DNA sequence analysis. No clear sequence homology is found among the exons, even though their coding capacity is quite similar, from 55 to 63 aminoacids residues. We located 2 hormonogenic (T4 forming) sites on the extreme 3' end of the gene in different exons. The DNA sequence coding for these functional sites shows a 70% homology in a 50 nucleotides segment. In addition we found a remnant of this sequence in other exons of the gene. Two large introns have been found on the 3' end of the gene: one is 17 Kb and the other one is more than 30 Kb long. On the basis of these findings and of preliminary studies on the remaining 5' end of the gene, we can predict that the minimum length of the rat TGB gene will be 150 Kb, which makes this gene the largest so far identified eukaryotic gene. We propose in addition that the 3' end exons arose by duplication of a common ancestor.     

The<Emphasis Type="Italic">Escherichia fergusonii iucABCD iutA</Emphasis> genes are located within a larger chromosomal region similar to pathogenicity islands

Three strains of Escherichia fergusonii (EF873, EF1496, EF939) of 50 strains tested produced the hydroxamate siderophore aerobactin. Screening of a cosmid library of the strain EF873 chromosomal DNA (in aerobactin nonproducing Escherichia coli VCS257) for aerobactin production identified iucABCD and iutA gene orthologues. The predicted IucABCD and IutA proteins showed 59-65% identity to the corresponding proteins of Shigella flexneri and E. coli. Aerobactin molecules synthesized by E. fergusonii and E. coli strains stimulated growth of aerobactin indicator strains harboring either E. coli or E. fergusonii iutA genes. In the 12 kb upstream and 17 kb downstream regions of the iuc and iut genes, 20 additional ORFs were identified. Their gene products showed homology to proteins from E. coli, S. flexneri, Klebsiella aerogenes, Pseudomonas aeruginosa and Vibrio cholerae. Probes recognizing DNA sequences from a region of more than 25 kb, which included the iucABCD and iutA genes, hybridized with chromosomal DNA of two aerobactin-producing strains (EF873 and EF939), but not with other nonproducing E. fergusonii strains tested. These data, together with the genetic organization of this region, suggest that E. fergusonii iucABCD iutA genes are a portion of a larger segment of DNA similar to pathogenicity islands of other bacteria.     

Using RNAi to investigate orthologous homeotic gene function during development of distantly related insects

Gene product distribution is often used to infer developmental similarities and differences in animals with evolutionarily diverse body plans. However, to address commonalties of developmental mechanisms, what is really needed is a method to assess and compare gene function in divergent organisms. This requires mutations eliminating gene function. Such mutations are often difficult to obtain, even in organisms amenable to genetic analysis. To address this issue we have investigated the use of double-stranded RNA interference to phenocopy null mutations. We show that RNA interference can be used to phenocopy mutations of the Deformed orthologues in Drosophila and Tribolium. We discuss the possible use of this technique for comparisons of developmental mechanisms in organisms with differing ontogenies.     

The region of the IncN plasmid R46 coding for resistance to beta-lactam antibiotics, streptomycin/spectinomycin and sulphonamides is closely related to antibiotic resistance segments found in IncW plasmids and in Tn21-like transposons.

The nucleotide sequence of a 2.5 kb segment of the pKM101 (R46) genome has been determined. The 1.3 kb from a BamHI site at 153 to base 1440 differs by only 2 bases from a part of the published sequence of the aadB (gentamicin resistance) gene region including the coding region for the N-terminal 70 amino acids of the predicted aadB product. The same sequence has been found 5'-to the dhfrII gene of R388 and to the aadA gene of Tn21 (R538-1). Three open reading frames are located in this region, two on the same strand as the resistance genes and one on the complementary strand. The latter predicts a polypeptide of 337 amino acids, whose N-terminal segment is 40% homologous to the predicted product of an open reading frame of 179 amino acids located next to the dhfrI gene of Tn7. The oxa2 (oxacillin resistance) gene predicts a long polypeptide commencing with (the N-terminal) 70 amino acids of the aadB product. A similar arrangement is found in the aadA gene of R538-1. The N-terminal segment of an aadA gene is located 3'- to oxa2, separated by 36 bases. Sequences surrounding the BamHI site are identical to sequences 5'- to the tnpM gene of Tn21 and homology ceases where homology between Tn21 and Tn501 commences. The possibility that this antibiotic resistance segment is a discrete mobile DNA element is discussed.     

Evolution and molecular characterization of a beta-globin gene from the Australian Echidna Tachyglossus aculeatus (Monotremata).

Coinciding with a period in evolution when monotremes, marsupials, and eutherians diverged from a common ancestor, a proto-beta-globin gene duplicated, producing the progenitors of mammalian embryonic and adult beta-like globin genes. To determine whether monotremes contain orthologues of these genes and to further investigate the evolutionary relationships of monotremes, marsupials, and eutherians, we have determined the complete DNA sequence of an echidna (Tachyglossus aculeatus) beta-like globin gene. Conceptual translation of the gene and sequence comparisons with eutherian and marsupial beta-like globin genes and echidna adult beta-globin indicate that the gene is adult expressed. Phylogenetic analyses do not clearly resolve the branching pattern of mammalian beta-like globin gene lineages and it is therefore uncertain whether monotremes have orthologues of the embryonic beta-like globin genes of marsupials and eutherians. Four models are proposed that provide a framework for interpreting further studies on the evolution of beta-like globin genes in the context of the evolution of monotremes, marsupials, and eutherians.     

Comparative genomics in teleost species: Knowledge transfer by linking the genomes of model and non-model fish species

Comparative genomics is a powerful tool to transfer knowledge coming from model fish species to non-model fish species of economic or/and evolutionary interest. Such transfer is of importance as functional studies either are difficult to perform with most non-model species. The first comparative map constructed using the human and the chimpanzee genome allowed the identification of putative orthologues. Although comparative mapping in teleosts is still in its infancy, five model teleost genomes from different orders have been fully sequenced to date and the sequencing of several commercially important species are also underway or near completion. The accessibility of these whole genome sequences and rapid developments in genomics of fish species are paving the way towards new and valuable research in comparative genetics and genomics. With the accumulation of information in model species, the genetic and genomic characterization of non-model, but economically, physiologically or evolutionary important species is now feasible. Furthermore, comparison of low coverage gene maps of non-model fish species against fully sequenced fish species will enhance the efficiency of candidate gene identification projected for quantitative trait loci (QTL) scans for traits of special interest.     

Nucleotide sequence of the sucA gene encoding the 2-oxoglutarate dehydrogenase of Escherichia coli K12

The nucleotide sequence of a 3180-base-pair segment of DNA, containing the sucA gene encoding the 2-oxoglutarate dehydrogenase component (E1o) of the 2-oxoglutarate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method. The sucA structural gene contains 2796 base pairs (932 codons, excluding the initiation codon AUG) and encodes a polypeptide having a glutamine residue at the amino terminus, a glutamate residue at the carboxy-terminus and a calculated Mr = 104905. The predicted amino acid composition is in good agreement with published information obtained by hydrolysis of the purified enzyme. There is a striking lack of sequence homology between the 2-oxoglutarate dehydrogenase (E1o) and the corresponding pyruvate dehydrogenase (E1p), which suggests that the two components are not closely related in evolutionary terms. The location and polarity of the sucA gene, relative to the restriction map of the corresponding segment of DNA, are consistent with it being the proximal gene of the suc operon, as defined in previous genetic and post-infection labelling studies, but it could also form part of a more complex regulatory unit. The sucA gene is preceded by a segment of DNA that contains many substantial regions of hyphenated dyad symmetry including an IS-like sequence of the type that is thought to function as an intercistronic regulatory element. This segment also contains three putative RNA polymerase binding sites and a good ribosome binding site.     

Gene replacement with one-sided homologous recombination.

Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.     

Dichotomy in the NRT gene families of dicots and grass species

A large proportion of the nitrate (NO(3)(-)) acquired by plants from soil is actively transported via members of the NRT families of NO(3)(-) transporters. In Arabidopsis, the NRT1 family has eight functionally characterised members and predominantly comprises low-affinity transporters; the NRT2 family contains seven members which appear to be high-affinity transporters; and there are two NRT3 (NAR2) family members which are known to participate in high-affinity transport. A modified reciprocal best hit (RBH) approach was used to identify putative orthologues of the Arabidopsis NRT genes in the four fully sequenced grass genomes (maize, rice, sorghum, Brachypodium). We also included the poplar genome in our analysis to establish whether differences between Arabidopsis and the grasses may be generally applicable to monocots and dicots. Our analysis reveals fundamental differences between Arabidopsis and the grass species in the gene number and family structure of all three families of NRT transporters. All grass species possessed additional NRT1.1 orthologues and appear to lack NRT1.6/NRT1.7 orthologues. There is significant separation in the NRT2 phylogenetic tree between NRT2 genes from dicots and grass species. This indicates that determination of function of NRT2 genes in grass species will not be possible in cereals based simply on sequence homology to functionally characterised Arabidopsis NRT2 genes and that proper functional analysis will be required. Arabidopsis has a unique NRT3.2 gene which may be a fusion of the NRT3.1 and NRT3.2 genes present in all other species examined here. This work provides a framework for future analysis of NO(3)(-) transporters and NO(3)(-) transport in grass crop species.