A gene cluster for secondary metabolism in oat: implications for the evolution of metabolic diversity in plants

The evolution of the ability to synthesize specialized metabolites is likely to have been key for survival and diversification of different plant species. Oats (Avena spp.) produce antimicrobial triterpenoids (avenacins) that protect against disease. The oat beta-amyrin synthase gene AsbAS1, which encodes the first committed enzyme in the avenacin biosynthetic pathway, is clearly distinct from other plant beta-amyrin synthases. Here we show that AsbAS1 has arisen by duplication and divergence of a cycloartenol synthase-like gene, and that its properties have been refined since the divergence of oats and wheat. Strikingly, we have also found that AsbAS1 is clustered with other genes required for distinct steps in avenacin biosynthesis in a region of the genome that is not conserved in other cereals. Because the components of this gene cluster are required for at least four clearly distinct enzymatic processes (2,3-oxidosqualene cyclization, beta-amyrin oxidation, glycosylation, and acylation), it is unlikely that the cluster has arisen as a consequence of duplication of a common ancestor. Although clusters of paralogous genes are common in plants (e.g., gene clusters for rRNA and specific disease resistance), reports of clusters of genes that do not share sequence relatedness and whose products contribute to a single selectable function are rare [Gierl, A. & Frey, M. (2001) Planta 213, 493-498]. Taken together, our evidence has important implications for the generation of metabolic diversity in plants.     

Genes for the peptidoglycan synthesis pathway are essential for chloroplast division in moss

The general consensus is that a cyanobacterium phagocytosed by a host cell evolved into the plastids of red and green algae, land plants, and glaucophytes. In contrast to the plastids of glaucophytes, which retain a cyanobacterial-type peptidoglycan layer, no wall-like structures have been detected in plastids from other sources. Although the genome of Arabidopsis thaliana contains five genes that are essential for peptidoglycan synthesis, MurE, MurG, two genes for D-Ala-D-Ala ligase (Ddl), and the gene for translocase I (MraY), their functions have not been determined. We report that the moss Physcomitrella patens has nine homologous genes related to peptidoglycan biosynthesis: MurA, B, C, D, E, and F, Ddl, genes for the penicillin-binding protein Pbp, and dd-carboxypeptidase (Dac). Corroborating a computer prediction, analysis of the GFP fusion proteins with the N terminus of PpMurE or of PpPbp suggests that these proteins are located in the chloroplasts. Gene disruption of the PpMurE gene in P. patens resulted in the appearance of macrochloroplasts both in protonema and in leaf cells. Moreover, gene knockout of the P. patens Pbp gene showed inhibition of chloroplast division in this moss; however, no Pbp gene was found in A. thaliana.     

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.     

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.     

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.     

Transposable elements and small RNAs contribute to gene expression divergence between Arabidopsis thaliana and Arabidopsis lyrata

Transposable elements (TEs) are often the primary determinant of genome size differences among eukaryotes. In plants, the proliferation of TEs is countered through epigenetic silencing mechanisms that prevent mobility. Recent studies using the model plant Arabidopsis thaliana have revealed that methylated TE insertions are often associated with reduced expression of nearby genes, and these insertions may be subject to purifying selection due to this effect. Less is known about the genome-wide patterns of epigenetic silencing of TEs in other plant species. Here, we compare the 24-nt siRNA complement from A. thaliana and a closely related congener with a two- to threefold higher TE copy number, Arabidopsis lyrata. We show that TEs--particularly siRNA-targeted TEs--are associated with reduced gene expression within both species and also with gene expression differences between orthologs. In addition, A. lyrata TEs are targeted by a lower fraction of uniquely matching siRNAs, which are associated with more effective silencing of TE expression. Our results suggest that the efficacy of RNA-directed DNA methylation silencing is lower in A. lyrata, a finding that may shed light on the causes of differential TE proliferation among species.     

Evolution of olfactory receptor genes in the human genome

Olfactory receptor (OR) genes form the largest known multigene family in the human genome. To obtain some insight into their evolutionary history, we have identified the complete set of OR genes and their chromosomal locations from the latest human genome sequences. We detected 388 potentially functional genes that have intact ORFs and 414 apparent pseudogenes. The number and the fraction (48%) of functional genes are considerably larger than the ones previously reported. The human OR genes can clearly be divided into class I and class II genes, as was previously noted. Our phylogenetic analysis has shown that the class II OR genes can further be classified into 19 phylogenetic clades supported by high bootstrap values. We have also found that there are many tandem arrays of OR genes that are phylogenetically closely related. These genes appear to have been generated by tandem gene duplication. However, the relationships between genomic clusters and phylogenetic clades are very complicated. There are a substantial number of cases in which the genes in the same phylogenetic clade are located on different chromosomal regions. In addition, OR genes belonging to distantly related phylogenetic clades are sometimes located very closely in a chromosomal region and form a tight genomic cluster. These observations can be explained by the assumption that several chromosomal rearrangements have occurred at the regions of OR gene clusters and the OR genes contained in different genomic clusters are shuffled.     

Multiple GAL pathway gene clusters evolved independently and by different mechanisms in fungi

A notable characteristic of fungal genomes is that genes involved in successive steps of a metabolic pathway are often physically linked or clustered. To investigate how such clusters of functionally related genes are assembled and maintained, we examined the evolution of gene sequences and order in the galactose utilization (GAL) pathway in whole-genome data from 80 diverse fungi. We found that GAL gene clusters originated independently and by different mechanisms in three unrelated yeast lineages. Specifically, the GAL cluster found in Saccharomyces and Candida yeasts originated through the relocation of native unclustered genes, whereas the GAL cluster of Schizosaccharomyces yeasts was acquired through horizontal gene transfer from a Candida yeast. In contrast, the GAL cluster of Cryptococcus yeasts was assembled independently from the Saccharomyces/Candida and Schizosaccharomyces GAL clusters and coexists in the Cryptococcus genome with unclustered GAL paralogs. These independently evolved GAL clusters represent a striking example of analogy at the genomic level. We also found that species with GAL clusters exhibited significantly higher rates of GAL pathway loss than species with unclustered GAL genes. These results suggest that clustering of metabolic genes might facilitate fungal adaptation to changing environments both through the acquisition and loss of metabolic capacities.     

Nucleotide polymorphism in colicin E1 and Ia plasmids from natural isolates of Escherichia coli.

We examined DNA sequence polymorphism for the colicin gene clusters of seven ColE1 and six ColIa plasmids obtained from natural isolates of Escherichia coli. These gene clusters harbor levels of nucleotide diversity ranging from 0.006 (ColIa) to 0.054 (ColE1). This level of diversity is similar to that observed for chromosomally encoded E. coli genes. However, the variance associated with these estimates is severalfold higher for the plasmid-encoded genes. This increased variance may be due to the differing plasmid population sizes. The pattern of colicin gene cluster polymorphism suggests that the two colicins are evolving in different fashions. ColE1 accumulates polymorphism at an elevated rate in the central domain of the colicin protein, while ColIa polymorphism is distributed evenly along the gene cluster. Comparison of the patterns of divergence between colicin and related proteins of ColIa and Ib and patterns of polymorphism within ColIa suggest that this gene cluster is not evolving in a neutral fashion. These data lend support to the hypothesis that colicin gene clusters may evolve under the influence of diversifying selection.     

Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase

Galacturonosyltransferases (GalATs) are required for the synthesis of pectin, a family of complex polysaccharides present in the cell walls of all land plants. We report the identification of a pectin GalAT (GAUT1) using peptide sequences obtained from Arabidopsis thaliana proteins partially purified for homogalacturonan (HG) alpha-1,4-GalAT activity. Transient expression of GAUT1 cDNA in the human embryonic kidney cell line HEK293 yielded uridine diphosphogalacturonic acid:GalAT activity. Polyclonal antibodies generated against GAUT1 immunoabsorbed HG alpha-1,4-GalAT activity from Arabidopsis solubilized membrane proteins. blast analysis of the Arabidopsis genome identified a family of 25 genes with high sequence similarity to GAUT1 and homologous genes in other dicots, in rice, and in Physcomitrella. Sequence alignment and phylogenetic Bayesian analysis of the Arabidopsis GAUT1-related gene family separates them into four related clades of GAUT and GAUT-like genes that are distinct from the other Arabidopsis members of glycosyltransferase family 8. The identification of GAUT1 as a HG GalAT and of the GAUT1-related gene family provides the genetic and biochemical tools required to study the function of these genes in pectin synthesis.     

Operons and SL2 trans-splicing exist in nematodes outside the genus Caenorhabditis

The genomes of most eukaryotes are composed of genes arranged on the chromosomes without regard to function, with each gene transcribed from a promoter at its 5′ end. However, the genome of the free-living nematode Caenorhabditis elegans contains numerous polycistronic clusters similar to bacterial operons in which the genes are transcribed sequentially from a single promoter at the 5′ end of the cluster. The resulting polycistronic pre-mRNAs are processed into monocistronic mRNAs by conventional 3′ end formation, cleavage, and polyadenylation, accompanied by trans-splicing with a specialized spliced leader (SL), SL2. To determine whether this mode of gene organization and expression, apparently unique among the animals, occurs in other species, we have investigated genes in a distantly related free-living rhabditid nematode in the genus Dolichorhabditis (strain CEW1). We have identified both SL1 and SL2 RNAs in this species. In addition, we have sequenced a Dolichorhabditis genomic region containing a gene cluster with all of the characteristics of the C. elegans operons. We show that the downstream gene is trans-spliced to SL2. We also present evidence that suggests that these two genes are also clustered in the C. elegans and Caenorhabditis briggsae genomes. Thus, it appears that the arrangement of genes in operons pre-dates the divergence of the genus Caenorhabditis from the other genera in the family Rhabditidae, and may be more widespread than is currently appreciated.     

Identification of 10 murine homeobox genes.

In Drosophila a number of genes important in establishing segmentation patterns and in determining segment identities have been shown to carry the homeobox sequence. Over 30 murine homeobox genes have been cloned, many on the basis of sequence homology to Drosophila prototypes. Here we report the cloning and sequencing of 10 new and 6 previously known homeobox genes by screening a murine genomic library with a 768-fold degenerate oligonucleotide corresponding to the most conserved 8-amino acid motif in the recognition helix of the homeodomain. Eight of these new homeobox genes have been chromosomally mapped. Four genes do not belong to any of the known homeobox gene clusters but instead map to new locations on chromosome 1 (single gene) and chromosome 5 (three genes). Sequence comparisons indicate that two of these are very closely related and represent a distinct new category of homeobox genes. The remaining four mapped genes reside in previously established murine homeobox gene clusters. Specifically, two map to the cluster HOX-1 on chromosome 6 and one each to HOX-3 and HOX-4 on chromosome 15 and 2, respectively. The ratio of newly identified homeobox genes to the previously characterized murine homeobox genes suggests that there remain several uncharacterized homeobox genes in the murine genome.     

Identification of transferred DNA insertions within Arabidopsis genes involved in signal transduction and ion transport.

The transferred DNA (T-DNA) of Agrobacterium tumefaciens serves as an insertional mutagen once integrated into a host plant's genome. As a means of facilitating reverse genetic analysis in Arabidopsis thaliana, we have developed a method that allows one to search for plants carrying F-DNA insertions within any sequenced Arabidopsis gene. Using PCR, we screened a collection of 9100 independent T-DNA-transformed Arabidopsis lines and found 17 T-DNA insertions within the 63 genes analyzed. The genes surveyed include members of various gene families involved in signal transduction and ion transport. As an example, data are shown for a T-DNA insertion that was found within CPK-9, a member of the gene family encoding calmodulin-domain protein kinases.     

Dispersal of NK homeobox gene clusters in amphioxus and humans

The Drosophila melanogaster genome has six physically clustered NK-related homeobox genes in just 180 kb. Here we show that the NK homeobox gene cluster was an ancient feature of bilaterian animal genomes, but has been secondarily split in chordate ancestry. The NK homeobox gene clusters of amphioxus and vertebrates are each split and dispersed at two equivalent intergenic positions. From the ancestral NK gene cluster, only the Tlx-Lbx and NK3-NK4 linkages have been retained in chordates. This evolutionary pattern is in marked contrast to the Hox and ParaHox gene clusters, which are compact in amphioxus and vertebrates, but have been disrupted in Drosophila.