A different function for a member of an ancient and highly conserved cytochrome P450 family: from essential sterols to plant defense

CYP51 sterol demethylases are the only cytochrome P450 enzymes with a conserved function across the animal, fungal, and plant kingdoms (in the synthesis of essential sterols). These highly conserved enzymes, which are important targets for cholesterol-lowering drugs, antifungal agents, and herbicides, are regarded as the most ancient member cytochrome P450 family. Here we present a report of a CYP51 enzyme that has acquired a different function. We show that the plant enzyme AsCYP51H10 is dispensable for synthesis of essential sterols and has been recruited for the production of antimicrobial compounds (avenacins) that confer disease resistance in oats. The AsCyp51H10 gene is synonymous with Sad2, a gene that we previously had defined by mutation as being required for avenacin synthesis. In earlier work, we showed that Sad1, the gene encoding the first committed enzyme in the avenacin pathway (beta-amyrin synthase), had arisen by duplication and divergence of a cycloartenol synthase-like gene. Together these data indicate an intimate evolutionary connection between the sterol and avenacin pathways. Sad1 and Sad2 lie within 70 kb of each other and are expressed specifically in the epidermal cells of the root tip, the site of accumulation of avenacins. These findings raise intriguing questions about the recruitment, coevolution, and regulation of the components of this specialized defense-related metabolic pathway.     

Formation of plant metabolic gene clusters within dynamic chromosomal regions

In bacteria, genes with related functions often are grouped together in operons and are cotranscribed as a single polycistronic mRNA. In eukaryotes, functionally related genes generally are scattered across the genome. Notable exceptions include gene clusters for catabolic pathways in yeast, synthesis of secondary metabolites in filamentous fungi, and the major histocompatibility complex in animals. Until quite recently it was thought that gene clusters in plants were restricted to tandem duplicates (for example, arrays of leucine-rich repeat disease-resistance genes). However, operon-like clusters of coregulated nonhomologous genes are an emerging theme in plant biology, where they may be involved in the synthesis of certain defense compounds. These clusters are unlikely to have arisen by horizontal gene transfer, and the mechanisms behind their formation are poorly understood. Previously in thale cress (Arabidopsis thaliana) we identified an operon-like gene cluster that is required for the synthesis and modification of the triterpene thalianol. Here we characterize a second operon-like triterpene cluster (the marneral cluster) from A. thaliana, compare the features of these two clusters, and investigate the evolutionary events that have led to cluster formation. We conclude that common mechanisms are likely to underlie the assembly and control of operon-like gene clusters in plants.     

Partial mRNA sequences for human A alpha, B beta, and gamma fibrinogen chains: evolutionary and functional implications.

Using rat cDNA and genomic probes to screen a human liver cDNA library, we have isolated clone of 2,274, 855, and 736 base pairs (bp) coding for the A alpha, B beta and gamma chains of human fibrinogen. Sequence analysis reveals a hitherto unrecognized extension of 15 amino acids at the carboxyl terminus of the A alpha chain, the terminal residue of which is proline. This brings the known length of the human A alpha chain to 625 amino acids. The 13-amino-acid repeated region in the midportion of the A alpha chain clearly has arisen through an 8-fold duplication of a 39-bp genetic element, which itself appears to have been constructed from smaller 6-bp repeating units. Greater than 50% sequence homology between B beta- and gamma-chain coding regions confirms postulates that these genes have arisen by duplication and subsequent divergence of an ancestral gene. A comparison of human and rat gamma-chain cDNAs shows more than 88% sequence homology over the carboxyl-terminal 162 amino acids, implying strong selective pressures on these portions of the gamma-chain gene.     

A new class of oxidosqualene cyclases directs synthesis of antimicrobial phytoprotectants in monocots

Many plants synthesize antimicrobial secondary metabolites as part of their normal program of growth and development, often sequestering them in tissues where they may protect against microbial attack. These include glycosylated triterpenoids (saponins), natural products that are exploited by man for a variety of purposes including use as drugs [Hostettmann, K. & Marston, A. (1995) Saponins (Cambridge Univ. Press, Cambridge, U.K.)]. Very little is known about the genes required for the synthesis of this important family of secondary metabolites in plants. Here we show the novel oxidosqualene cyclase AsbAS1 catalyzes the first committed step in the synthesis of antifungal triterpenoid saponins that accumulate in oat roots. We also demonstrate that two sodium azide-generated saponin-deficient mutants of oat, which define the Sad1 genetic complementation group, are defective in the gene encoding this enzyme and provide molecular genetic evidence indicating a direct link between AsbAS1, triterpenoid saponin biosynthesis, and disease resistance. Orthologs of AsbAS1 are absent from modern cereals and may have been lost during selection, raising the possibility that this gene could be exploited to enhance disease resistance in crop plants.     

Breakup of a homeobox cluster after genome duplication in teleosts

Several families of homeobox genes are arranged in genomic clusters in metazoan genomes, including the Hox, ParaHox, NK, Rhox, and Iroquois gene clusters. The selective pressures responsible for maintenance of these gene clusters are poorly understood. The ParaHox gene cluster is evolutionarily conserved between amphioxus and human but is fragmented in teleost fishes. We show that two basal ray-finned fish, Polypterus and Amia, each possess an intact ParaHox cluster; this implies that the selective pressure maintaining clustering was lost after whole-genome duplication in teleosts. Cluster breakup is because of gene loss, not transposition or inversion, and the total number of ParaHox genes is the same in teleosts, human, mouse, and frog. We propose that this homeobox gene cluster is held together in chordates by the existence of interdigitated control regions that could be separated after locus duplication in the teleost fish.     

Elephant shark (Callorhinchus milii) provides insights into the evolution of Hox gene clusters in gnathostomes

We have sequenced and analyzed Hox gene clusters from elephant shark, a holocephalian cartilaginous fish. Elephant shark possesses 4 Hox clusters with 45 Hox genes that include orthologs for a higher number of ancient gnathostome Hox genes than the 4 clusters in tetrapods and the supernumerary clusters in teleost fishes. Phylogenetic analysis of elephant shark Hox genes from 7 paralogous groups that contain all of the 4 members indicated an ((AB)(CD)) topology for the order of Hox cluster duplication, providing support for the 2R hypothesis (i.e., 2 rounds of whole-genome duplication during the early evolution of vertebrates). Comparisons of noncoding sequences of the elephant shark and human Hox clusters have identified a large number of conserved noncoding elements (CNEs), which represent putative cis-regulatory elements that may be involved in the regulation of Hox genes. Interestingly, in fugu more than 50% of these ancient CNEs have diverged beyond recognition in the duplicated (HoxA, HoxB, and HoxD) as well as the singleton (HoxC) Hox clusters. Furthermore, the b-paralogs of the duplicated fugu Hox clusters are virtually devoid of unique ancient CNEs. In contrast to fugu Hox clusters, elephant shark and human Hox clusters have lost fewer ancient CNEs. If these ancient CNEs are indeed enhancers directing tissue-specific expression of Hox genes, divergence of their sequences in vertebrate lineages might have led to altered expression patterns and presumably the functions of their associated Hox genes.     

Paralogous vitamin D receptors in teleosts: transition of nuclear receptor function

The availability of multiple teleost (bony fish) genomes is providing unprecedented opportunities to understand the diversity and function of gene duplication events using comparative genomics. Here we describe the cloning and functional characterization of two novel vitamin D receptor (VDR) paralogs from the freshwater teleost medaka (Oryzias latipes). VDR sequences were identified through mining of the medaka genome database in which gene organization and structure was determined. Two distinct VDR genes were identified in the medaka genome and mapped to defined loci. Each VDR sequence exhibits unique intronic organization and dissimilar 5' untranslated regions, suggesting they are not isoforms of the same gene locus. Phylogenetic comparison with additional teleosts and mammalian VDR sequences illustrate that two distinct clusters are formed separating aquatic and terrestrial species. Nested within the teleost cluster are two separate clades for VDRalpha and VDRbeta. The topology of teleost VDR sequences is consistent with the notion of paralogous genes arising from a whole genome duplication event prior to teleost radiation. Functional characterization was conducted through the development of VDR expression vectors including Gal4 chimeras containing the yeast Gal4 DNA binding domain fused to the medaka VDR ligand binding domain and full-length protein. The common VDR ligand 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)(2)D(3)] resulted in significant transactivation activity with both the Gal4 and full-length constructs of medaka (m) VDRbeta. Comparatively, transactivation of mVDRalpha with 1alpha,25(OH)(2)D(3) was highly attenuated, suggesting a functional divergence between these two nuclear receptor paralogs. We additionally demonstrate through coactivator studies that mVDRalpha is still functional; however, it exhibits a different sensitivity to 1alpha,25(OH)(2)D(3), compared with VDRbeta. These results suggest that in mVDRalpha and VDRbeta have undergone a functional divergence through a process of sub- and/or neofunctionalization of VDR nuclear receptor gene pairs.     

Hox cluster duplications and the opportunity for evolutionary novelties

Hox genes play a key role in animal body plan development. These genes tend to occur in tightly linked clusters in the genome. Vertebrates and invertebrates differ in their Hox cluster number, with vertebrates having multiple clusters and invertebrates usually having only one. Recent evidence shows that vertebrate Hox clusters are structurally more constrained than invertebrate Hox clusters; they exclude transposable elements, do not undergo tandem duplications, and conserve their intergenic distances and gene order. These constraints are only relaxed after a cluster duplication. In contrast, invertebrate Hox clusters are structurally more plastic; tandem duplications are common, the linkage of Hox genes can change quickly, or they can lose their structural integrity completely. We propose that the constraints on vertebrate Hox cluster structure lead to an association between the retention of duplicated Hox clusters and adaptive radiations. After a duplication the constraints on Hox cluster structure are temporarily lifted, which opens a window of evolvability for the Hox clusters. If this window of evolvability coincides with an adaptive radiation, chances are that a modified Hox cluster becomes recruited in an evolutionary novelty and then both copies of duplicated Hox clusters are retained.     

Chloroplast small heat shock proteins: evidence for atypical evolution of an organelle-localized protein

Knowledge of the origin and evolution of gene families is critical to our understanding of the evolution of protein function. To gain a detailed understanding of the evolution of the small heat shock proteins (sHSPs) in plants, we have examined the evolutionary history of the chloroplast (CP)-localized sHSPs. Previously, these nuclear-encoded CP proteins had been identified only from angiosperms. This study reveals the presence of the CP sHSPs in a moss, Funaria hygrometrica. Two clones for CP sHSPs were isolated from a F. hygrometrica heat shock cDNA library that represent two distinct CP sHSP genes. Our analysis of the CP sHSPs reveals unexpected evolutionary relationships and patterns of sequence conservation. Phylogenetic analysis of the CP sHSPs with other plant CP sHSPs and eukaryotic, archaeal, and bacterial sHSPs shows that the CP sHSPs are not closely related to the cyanobacterial sHSPs. Thus, they most likely evolved via gene duplication from a nuclear-encoded cytosolic sHSP and not via gene transfer from the CP endosymbiont. Previous sequence analysis had shown that all angiosperm CP sHSPs possess a methionine-rich region in the N-terminal domain. The primary sequence of this region is not highly conserved in the F. hygrometrica CP sHSPs. This lack of sequence conservation indicates that sometime in land plant evolution, after the divergence of mosses from the common ancestor of angiosperms but before the monocot-dicot divergence, there was a change in the selective constraints acting on the CP sHSPs.     

Structure of homeobox-leucine zipper genes suggests a model for the evolution of gene families.

Homeobox genes are present in both plants and animals. Homeobox-leucine zipper genes, however, have been identified thus far only in the small mustard plant Arabidopsis thaliana. This observation suggests that homeobox-leucine zipper genes evolved after the divergence of plants and animals, perhaps to mediate specific regulatory events. To better understand this gene family, we isolated several sequences containing the homeobox-leucine zipper motif and carried out a comparative analysis of nine homeobox-leucine zipper genes (HAT1, HAT2, HAT3, HAT4, HAT5, HAT7, HAT9, HAT14, and HAT22). Gene structures, sequence comparisons, and chromosomal locations suggest a simple model for the evolution of these genes. The model postulates that a primordial homeobox gene acquired a leucine zipper by exon capture. The nascent homeobox-leucine zipper gene then appears to have undergone a series of gene duplication and chromosomal translocation events, leading to the formation of the HAT gene family. This work has general implications for the evolution of regulatory genes.     

Evolution of mouse immunoglobulin lambda genes.

The mouse has four C lambda and two V lambda genes. We have isolated Charon 4A clones that contain all six lambda genes from a BALB/c germ-line library. We present here the DNA sequences of the C lambda 2, C lambda 3, and C lambda 4 genes and also correct what are apparently errors in previous reports of C lambda 1 protein and DNA sequences. In addition, we have analyzed cloned DNAs by restriction mapping and electron microscopy to determine the relationships among the various lambda genes. By heteroduplex analysis, two gene clusters containing JC lambda 3--JC lambda 1 and JC lambda 2--JC lambda 4 show homology extending from the J regions 5' of C lambda 3/C lambda 2 to just 3' of C lambda 1/C lambda 4. Other than the region between the genes, very little homology exists in the C lambda flanking regions. In contrast, V lambda 1 and V lambda 2 genes show considerable homology extending into the 5' flanking regions. Large inverted repeats are found in the 5' flanking regions of V lambda 1 and C lambda 3, as well as in the 3' flanking regions of both C lambda gene clusters. DNA sequence divergences between the C lambda genes indicate that an ancestral JC lambda x--JC lambda g gene cluster arose at about the time of the first mammals by duplication of a primordial JC lambda gene. The data further suggest that the JC lambda x--JC lambda gene cluster duplicated after the speciation of mouse and man and subsequently diverged into the present day JC lambda 3--JC lambda 1 and JC lambda 2--JC lambda 4 gene clusters. C lambda 4, a pseudogene, became inactive at about the time of duplication of the ancestral JC lambda x--JC lambda y cluster. Comparison of DNA sequence divergence between the V lambda 1 and V lambda 2 genes demonstrates an anomaly. The percentage of amino acid replacement changes is approximately the same for V lambda 1/V lambda 2 as for C lambda 3/C lambda 2, implying that the ancestral V lambda gene was duplicated at the same time, and possibly together with, the JC lambda x--JC lambda y cluster. However, there are fewer silent changes than amino acid replacement changes between the V lambda 1/V lambda 2 genes, suggesting either that a selective pressure acted on the silent sites or that V lambda genes have only recently been duplicated. We also consider the possibility of a gene conversion event subsequent ot a more ancient duplication.     

An orphaned mammalian beta-globin gene of ancient evolutionary origin

Mammals possess multiple, closely linked beta-globin genes that differ in the timing of their expression during development. These genes have been thought to be derived from a single ancestral gene, by duplication events that occurred after the separation of the mammals and birds. We report the isolation and characterization of an atypical beta-like globin gene (omega-globin) in marsupials that appears to be more closely related to avian beta-globin genes than to other mammalian beta-globin genes, including those previously identified in marsupials. Phylogenetic analyses indicate that omega-globin evolved from an ancient gene duplication event that occurred before the divergence of mammals and birds. Furthermore, we show that omega-globin is unlinked to the previously characterized beta-globin gene cluster of marsupials, making this the first report of an orphaned beta-like globin gene expressed in a vertebrate.     

The evolution of plant nuclear genes

We analyze the evolutionary dynamics of three of the best-studied plant nuclear multigene families. The data analyzed derive from the genes that encode the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS), the gene family that encodes the enzyme chalcone synthase (Chs), and the gene family that encodes alcohol dehydrogenases (Adh). In addition, we consider the limited evolutionary data available on plant transposable elements. New Chs and rbcS genes appear to be recruited at about 10 times the rate estimated for Adh genes, and this is correlated with a much smaller average gene family size for Adh genes. In addition, duplication and divergence in function appears to be relatively common for Chs genes in flowering plant evolution. Analyses of synonymous nucleotide substitution rates for Adh genes in monocots reject a linear relationship with clock time. Replacement substitution rates vary with time in a complex fashion, which suggests that adaptive evolution has played an important role in driving divergence following gene duplication events. Molecular population genetic studies of Adh and Chs genes reveal high levels of molecular diversity within species. These studies also reveal that inter- and intralocus recombination are important forces in the generation allelic novelties. Moreover, illegitimate recombination events appear to be an important factor in transposable element loss in plants. When we consider the recruitment and loss of new gene copies, the generation of allelic diversity within plant species, and ectopic exchange among transposable elements, we conclude that recombination is a pervasive force at all levels of plant evolution.     

A new hemoglobin gene from soybean: a role for hemoglobin in all plants.

We have isolated a new hemoglobin gene from soybean. It is expressed in cotyledons, stems of seedlings, roots, young leaves, and in some cells in the nodules that are associated with the nitrogen-fixing Bradyrhizobium symbiont. This contrasts with the expression of the leghemoglobins, which are active only in the infected cells of the nodules. The deduced protein sequence of the new gene shows only 58% similarity to one of the soybean leghemoglobins, but 85-87% similarity to hemoglobins from the nonlegumes Parasponia, Casuarina, and barley. The pattern of expression and the gene sequence indicate that this new gene is a nonsymbiotic legume hemoglobin. The finding of this gene in legumes and similar genes in other species strengthens our previous suggestion that genomes of all plants contain hemoglobin genes. The specialized leghemoglobin gene family may have arisen from a preexisting nonsymbiotic hemoglobin by gene duplication.     

Positive selection driving diversification in plant secondary metabolism

In Arabidopsis thaliana and related plants, glucosinolates are a major component in the blend of secondary metabolites and contribute to resistance against herbivorous insects. Methylthioalkylmalate synthases (MAM) encoded at the MAM gene cluster control an early step in the biosynthesis of glucosinolates and, therefore, are central to the diversification of glucosinolate metabolism. We sequenced bacterial artificial chromosomes containing the MAM cluster from several Arabidopsis relatives, conducted enzyme assays with heterologously expressed MAM genes, and analyzed MAM nucleotide variation patterns. Our results show that gene duplication, neofunctionalization, and positive selection provide the mechanism for biochemical adaptation in plant defense. These processes occur repeatedly in the history of the MAM gene family, indicating their fundamental importance for the evolution of plant metabolic diversity both within and among species.     

Molecular evolution of the HoxA cluster in the three major gnathostome lineages

The duplication of Hox clusters and their maintenance in a lineage has a prominent but little understood role in chordate evolution. Here we examined how Hox cluster duplication may influence changes in cluster architecture and patterns of noncoding sequence evolution. We sequenced the entire duplicated HoxAa and HoxAb clusters of zebrafish (Danio rerio) and extended the 5' (posterior) part of the HoxM (HoxA-like) cluster of horn shark (Heterodontus francisci) containing the hoxa11 and hoxa13 orthologs as well as intergenic and flanking noncoding sequences. The duplicated HoxA clusters in zebrafish each house considerably fewer genes and are dramatically shorter than the single HoxA clusters of human and horn shark. We compared the intergenic sequences of the HoxA clusters of human, horn shark, zebrafish (Aa, Ab), and striped bass and found extensive conservation of noncoding sequence motifs, i.e., phylogenetic footprints, between the human and horn shark, representing two of the three gnathostome lineages. These are putative cis-regulatory elements that may play a role in the regulation of the ancestral HoxA cluster. In contrast, homologous regions of the duplicated HoxAa and HoxAb clusters of zebrafish and the HoxA cluster of striped bass revealed a striking loss of conservation of these putative cis-regulatory sequences in the 3' (anterior) segment of the cluster, where zebrafish only retains single representatives of group 1, 3, 4, and 5 (HoxAa) and group 2 (HoxAb) genes and in the 5' part of the clusters, where zebrafish retains two copies of the group 13, 11, and 9 genes, i.e., AbdB-like genes. In analyzing patterns of cis-sequence evolution in the 5' part of the clusters, we explicitly looked for evidence of complementary loss of conserved noncoding sequences, as predicted by the duplication-degeneration-complementation model in which genetic redundancy after gene duplication is resolved because of the fixation of complementary degenerative mutations. Our data did not yield evidence supporting this prediction. We conclude that changes in the pattern of cis-sequence conservation after Hox cluster duplication are more consistent with being the outcome of adaptive modification rather than passive mechanisms that erode redundancy created by the duplication event. These results support the view that genome duplications may provide a mechanism whereby master control genes undergo radical modifications conducive to major alterations in body plan. Such genomic revolutions may contribute significantly to the evolutionary process.     

Duplication of large genomic regions during the evolution of vertebrate homeobox genes

The phylogenetic relationships of 21 murine Antp-class (Drosophila mutation Antennapedia-type class) homeobox genes have been analyzed, and several groups of related genes have been identified. The murine Antp-class homeobox genes are localized within four gene clusters. The similar structural organization of the four gene clusters strongly suggests that genes within a group of related Antp-class homeobox genes are derived from duplications of large genomic regions. After the duplication, the gross structures of the homeobox gene clusters have been maintained over a long period of evolutionary time, indicating that the specific organization of genes within a cluster may be of functional importance.