Phylogenetic analysis of the UDP-glycosyltransferase multigene family of Arabidopsis thaliana

A class of UDP-glycosyltransferases (UGTs) defined by the presence of a C-terminal consensus sequence is found throughout the plant and animal kingdoms. Whereas mammalian enzymes use UDP-glucuronic acid, the plant enzymes typically use UDP-glucose in the transfer reactions. A diverse array of aglycones can be glucosylated by these UGTs. In plants, the aglycones include plant hormones, secondary metabolites involved in stress and defense responses, and xenobiotics such as herbicides. Glycosylation is known to regulate many properties of the aglycones such as their bioactivity, their solubility, and their transport properties within the cell and throughout the plant. As a means of providing a framework to start to understand the substrate specificities and structure-function relationships of plant UGTs, we have now applied a molecular phylogenetic analysis to the multigene family of 99 UGT sequences in Arabidopsis. We have determined the overall organization and evolutionary relationships among individual members with a surprisingly high degree of confidence. Through constructing a composite phylogenetic tree that also includes all of the additional plant UGTs with known catalytic activities, we can start to predict both the evolutionary history and substrate specificities of new sequences as they are identified. The tree already suggests that while the activities of some subgroups of the UGT family are highly conserved among different plant species, others subgroups shift substrate specificity with relative ease.     

Comparative genomic analysis of the mouse and rat amylase multigene family

The rat and mouse amylase gene families were characterized using sequence data from the UCSC genome assembly. We found that the rat genome contains one amylase-1 and two amylase-2 genes, lying close to one another on the same chromosome. Detailed analysis revealed at least six additional amylase pseudogenes in the rat genome in the region adjacent to the amylase-2 genes. In contrast, the mouse has one amylase-1 gene and five amylase-2 genes; the latter are tandemly and systematically arranged on the same chromosome and were generated by segmental duplication. Detailed analysis revealed that the mouse has two amylase pseudogenes, located 5' to the five amylase-2 segments. Thus, the amylase genes of mouse and rat tend to be amplified; the sequences of some of them are fixed while others have become pseudogenes during evolution. This is the second report of amylase genomic organization in mammals and the first in the rodents.     

Comparative analysis of gnathostome Otx gene expression patterns in the developing eye: implications for the functional evolution of the multigene family

We have performed a detailed analysis of the expression pattern of the three gnathostome Otx classes in order to gain new insights into their functional evolution. Expression patterns were examined in the developing eye of a chondrichthyan, the dogfish, and an amniote, the chick, and compared with the capacity of paralogous proteins to induce a pigmented phenotype in cultured retina cells in cooperation with the bHLH-leucine zipper protein Mitf. This analysis indicates that each Otx class is characterized by highly specific and conserved expression features in the presumptive RPE, where Otx1 and Otx2, but not Otx5, are transcribed at optic vesicle stages, in the differentiating neural retina, where Otx2 and Otx5 show a conserved dynamic expression pattern, and in the forming ciliary process, a major site of Otx1 expression. Furthermore, the paralogous proteins of the dogfish and the mouse do not display any significant difference in their capacity to induce a pigmented phenotype, suggesting a functional equivalency in the specification and differentiation of the RPE. These data indicate that specific functions selectively involving each Otx orthology class were fixed prior to the gnathostome radiation and highlight the prominent role of regulatory changes in the functional diversification of the multigene family.     

Predicted expansion of the claudin multigene family

Claudins (Cldn) are essential membrane proteins of tight junctions (TJs), which form the paracellular permselective barrier. They are produced by a multi-gene family of 24 reported members in mouse and human. Based on a comprehensive search combined with phylogenetic analyses, we identified three novel claudins (claudin-25, -26, and -27). Quantitative RT-PCR revealed that the three novel claudins were expressed in a tissue- and/or developmental stage-dependent manner. Claudins-25 and -26, but not claudin-27, were immunofluorescently localized to TJs when exogenously expressed in cultured MDCK and Eph epithelial cell lines. These findings expand the claudin family to include at least 27 members.     

The olfactory multigene family.

A novel multigene family has been identified that is likely to encode odorant receptors on olfactory sensory neurons. Further studies on this gene family are likely to shed light on the molecular mechanisms underlying information coding in the mammalian olfactory system. This review is also published in Current Opinion in Genetics and Development 1992, 2:467-473.     

The olfactory multigene family.

A novel multigene family has been identified that is likely to encode odorant receptors on olfactory sensory neurons. Further studies on this gene family are likely to shed light on the molecular mechanisms underlying information coding in the mammalian olfactory system. This review is also published in Current Opinion in Neurobiology 1992, 2:282-288.     

Birth-and-death evolution of the internalin multigene family in Listeria

The birth-and-death model of multigene family evolution describes patterns of gene origination, diversification and loss within multigene families. Since it was first developed in the 1990s, the model has been found to characterize a large number of eukaryotic multigene families. In this paper, we report for the first time a bacterial multigene family that undergoes birth-and-death evolution. By analyzing the evolutionary relationships among internalins, a relatively large and diverse family of genes associated with key virulence functions in Listeria, we demonstrate the importance of birth-and-death evolution in the diversification of this important bacterial pathogen. We also detected two instances of lateral gene transfer within the internalins, but the estimated frequency would have been much higher had it not been analyzed within the context of birth-and-death evolutionary dynamics and a phenomenon that we term "paralog-sorting", which involves the unequal transmittal of gene duplicates during or subsequent to the speciation process. As such, in addition to providing the first demonstration of birth-and-death evolution within a bacterial multigene family, our results indicate that the extent of lateral transfer in bacterial multigene families should be re-examined in the light of birth-and-death evolution.     

Structure and evolution of the apolipoprotein multigene family

We present the complementary DNA and deduced amino acid sequence of rat apolipoprotein A-II (apoA-II), and the results of a detailed statistical analysis of the nucleotide and amino acid sequences of all the apolipoprotein gene sequences published to date: namely, those of human and rat apoA-I, apoA-II and apoE, rat apoA-IV, and human apoC-I, C-II and C-III. Our results indicate that the apolipoprotein genes have very similar genomic structures, each having a total of three introns at the same locations. Using the exon/intron junctions as reference points, we have obtained an alignment of the coding regions of all the genes studied. It appears that the mature peptide regions of these genes are almost completely made up of tandem repeats of 11 codons. The part of mature peptide region encoded by exon 3 contains a common block of 33 codons, whereas the part encoded by exon 4 contains a much more variable number of internal repeats of 11 codons. These genes have apparently evolved from a primordial gene through multiple partial (internal) and complete gene duplications. On the basis of the degree of homology of the various sequences, and the pattern of the internal repeats in these genes, we propose an evolutionary tree for the apolipoprotein genes and give rough estimates of the divergence times between these genes. Our results show that apoA-II has evolved extremely rapidly and that apoA-I and apoE also have evolved at high rates but some regions are better conserved than the others. The rate of evolution of individual regions seems to be related to the stringency of their functional requirements.     

Genetic analysis of the ADGF multigene family by homologous recombination and gene conversion in Drosophila

Many Drosophila genes exist as members of multigene families and within each family the members can be functionally redundant, making it difficult to identify them by classical mutagenesis techniques based on phenotypic screening. We have addressed this problem in a genetic analysis of a novel family of six adenosine deaminase-related growth factors (ADGFs). We used ends-in targeting to introduce mutations into five of the six ADGF genes, taking advantage of the fact that five of the family members are encoded by a three-gene cluster and a two-gene cluster. We used two targeting constructs to introduce loss-of-function mutations into all five genes, as well as to isolate different combinations of multiple mutations, independent of phenotypic consequences. The results show that (1) it is possible to use ends-in targeting to disrupt gene clusters; (2) gene conversion, which is usually considered a complication in gene targeting, can be used to help recover different mutant combinations in a single screening procedure; (3) the reduction of duplication to a single copy by induction of a double-strand break is better explained by the single-strand annealing mechanism than by simple crossing over between repeats; and (4) loss of function of the most abundantly expressed family member (ADGF-A) leads to disintegration of the fat body and the development of melanotic tumors in mutant larvae.     

The actin multigene family in Populus: organization, expression and phylogenetic analysis

Despite the significance of actin in plant growth and development, little is known of the structure, expression and evolution of the actin gene family in woody plants. In this study, we systematically examined the diversification of the actin gene family in Populus by integrating genomic organization, expression, and phylogeny data. Genome-wide analysis of the Populus genome indicated that actin is a multigene family consisting of eight members, all predicted to encode 377-amino acid polypeptides that share high sequence homology ranging from 94.2 to 100% identity. Microarray and real-time PCR expression analysis showed that the PtrACT family members are differentially expressed in different tissues, exhibiting overlapping and unique expression patterns. Of particular interest, all PtrACT genes have been found to be preferentially expressed in the stem phloem and xylem, suggesting that poplar PtrACTs are involved in the wood formation. Gene structural and phylogenetic analyses revealed that the PtrACT family is composed of two main subgroups that share an ancient common ancestor. Extremely high intraspecies synonymous nucleotide diversity of π_syn = 0.01205 was detected, and the π_non-syn/π_syn ratio was significantly less than 1; therefore, the PtACT1 appears to be evolving in Populus, primarily under purifying selection. We demonstrated that the actin gene family in Populus is divided into two distinct subgroups, suggesting functional divergence. The results reported here will be useful in conducting future functional genomics studies to understand the detailed function of actin genes in tree growth and development.     

Molecular evolution of the toll-like receptor multigene family in birds

Toll-like receptors (TLR) are membrane-bound sensors of the innate immune system that recognize invariant and distinctive molecular features of invading microbes and are also essential for initiating adaptive immunity in vertebrates. The genetic variation at TLR genes has been directly related to differential pathogen outcomes in humans and livestock. Nonetheless, new insights about the impact of TLRs polymorphism on the evolutionary ecology of infectious diseases can be gained through the investigation of additional vertebrate groups not yet investigated in detail. In this study, we have conducted the first characterization of the entire TLR multigene family (N = 10 genes) in non-model avian species. Using primers targeting conserved coding regions, we aimed at amplifying large segments of the extracellular domains (275-435 aa) involved in pathogen recognition across seven phylogenetically diverse bird species. Our analyses suggest avian TLRs are dominated by stabilizing selection, suggesting that slow rates of nonsynonymous substitution help preserve biological function. Overall, mean values of ω (= d(n)/d(s)) at each TLR locus ranged from 0.196 to 0.517. However, we also found patterns of positive selection acting on specific amino acid sites that could be linked to species-specific differences in pathogen-associated molecular pattern recognition. Only 39 of 2,875 (～1.35%) of the codons analyzed exhibited significant patterns of positive selection. At least one half of these positively selected codons can be mapped to putative ligand-binding regions, as suggested by crystallographic structures of TLRs and their ligands and mutagenic analyses. We also surveyed TLR polymorphism in wild populations of two bird species, the Lesser Kestrel Falco naumanni and the House Finch Carpodacus mexicanus. In general, avian TLRs displayed low to moderate single nucleotide polymorphism levels and an excess of silent nucleotide substitutions, but also conspicuous instances of positive directional selection. In particular, TLR5 and TLR15 exhibited the highest degree of genetic polymorphism and the highest occurrence of nonconservative amino acid substitutions. This study provides critical primers and a first look at the evolutionary patterns and implications of TLR polymorphism in non-model avian species and extends the list of candidate loci for avian eco-immunogenetics beyond the widely employed genes of the Major Histocompatibility Complex (MHC).     

Comparative analysis of thermoregulation in the nests of certain species of social insects

Studies have been made on thermal regulation in the nests of families of the honey bee Apis mellifera, wasp Dolihovespula silvestris and bumblebees Bombus terrestris, B. agrorum and B. lapidaris during their maximum development. It was shown that thermoregulation significantly stimulated the brood in the nest. Among the species investigated, the highest thermoregulatory capacities are exhibited by honey bees, this fact being associated with the large number of individuals in their colonies. During cooling, bees group around the brood and their bodies make a cover of thermal insulation. Sheltering of the nests plays the main role in heat preservation of wasps and bumblebees. The latter, using contact method of heating of cells with the brood, provide rather constant temperature for its development. Honey bees, wasps and bumblebees react to overheating in the nests essentially in a similar way, i. e. by active aeration of their nests by vigorous wing beatings. The frequency of beatings increases with the increase in temperature.     

Identification of members of the P-glycoprotein multigene family.

Overproduction of P-glycoprotein is intimately associated with multidrug resistance. This protein appears to be encoded by a multigene family. Thus, differential expression of different members of this family may contribute to the complexity of the multidrug resistance phenotype. Three lambda genomic clones isolated from a hamster genomic library represent different members of the hamster P-glycoprotein gene family. Using a highly conserved exon probe, we found that the hamster P-glycoprotein gene family consists of three genes. We also found that the P-glycoprotein gene family consists of three genes in mice but has only two genes in humans and rhesus monkeys. The hamster P-glycoprotein genes have similar exon-intron organizations within the 3' region encoding the cytoplasmic domains. We propose that the hamster P-glycoprotein gene family arose from gene duplication. The hamster pgp1 and pgp2 genes appear to be more closely related to each other than either gene is to the pgp3 gene. We speculate that the hamster pgp1 and pgp2 genes arose from a recent gene duplication event and that primates did not undergo this duplication and therefore contain only two P-glycoprotein genes.     

Characterization of the carp myosin heavy chain multigene family

We isolated partial coding sequences for 29 carp myosin heavy chain genes (MyoHCs) and determined the nucleotide sequences around the region encoding the loop 2 of the myosin molecule. The predicted amino acid sequences from the isolated genes all showed very high similarity to those of skeletal and cardiac muscles from higher vertebrates, but not to those of smooth and non-muscle counterparts. Among all clones isolated, carp MyoHC10, MyoHCI-1-3 and MyoHC30 showed exon-nucleotide sequences identical to those of cDNAs encoding the loop 2 region of the 10 degrees C-, intermediate- and 30 degrees C-type fast skeletal isoforms [Hirayama and Watabe, Euro. J. Biochem. 246 (1997) 380-387]. The loop 2 of 28 types of carp MyoHCs was encoded by two exons separated by an intron corresponding to that of the 16th in higher vertebrate MyoHCs, whilst this intron was not found in carp MyoHC30. Although carp MyoHC30 had a gene organization different from those of higher vertebrates and other carp MyoHCs, its predicted amino acid sequence for loop 2 showed the highest homology to those of higher vertebrates among carp MyoHCs. In the 28 carp MyoHCs containing the intron, a combination of different nucleotide sequences for the two resulted in 14 distinct series for the combined coding sequence. These different nucleotide sequences encoded nine distinct amino acid sequences. Phylogenetic analysis for the present loop 2 and light meromyosin previously reported for carp MyoHCs [Imai et al., J. Exp. Biol. 200 (1997) 27-34] revealed that carp MyoHCs have recently diverged and are more closely related to each other than to MyoHCs from other species.     

Autophosphorylation activity of the Arabidopsis ethylene receptor multigene family

Receptors for the gaseous phytohormone ethylene show sequence similarity to bacterial two-component histidine kinases. These receptors are encoded by a multigene family that can be divided into subfamilies 1 and 2. It has been previously shown that a subfamily 1 Arabidopsis thaliana ethylene receptor, ETR1, autophosphorylates in vitro on a conserved histidine residue (1). However, sequence comparisons between the five ethylene receptor family members suggest that subfamily 2 members do not have all the motifs necessary for histidine kinase activity. Further, a tobacco subfamily 2 receptor, NTHK1, autophosphorylates on serines and threonines in vitro (2). Here we show that all five Arabidopsis ethylene receptor proteins autophosphorylate in vitro. We analyzed the nature of the phosphorylated amino acids by acid/base stability and bi-dimensional thin layer electrophoresis and demonstrated that unlike ETR1 all other ethylene receptors autophosphorylate predominantly on serine residues. ERS1, the only other subfamily 1 receptor, is able to phosphorylate on both histidine and serine residues in the presence of Mn2+. However, histidine autophosphorylation is lost when ERS1 is assayed in the presence of both Mg2+ and Mn2+, suggesting that this activity may not occur in vivo. Furthermore, mutation of the histidine residue conserved in two-component systems does not abolish serine autophosphorylation, eliminating the possibility of a histidine to serine phosphotransfer. Our biochemical observations complement the recently published genetic data that histidine kinase activity is not necessary for ethylene receptor function in plants and suggest that ethylene signal transduction does not occur through a phosphorelay mechanism.     

A multigene family encoding R-SNAREs in the ciliate Paramecium tetraurelia

SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane interactions and are conventionally divided into Q-SNAREs and R-SNAREs according to the possession of a glutamine or arginine residue at the core of their SNARE domain. Here, we describe a set of R-SNAREs from the ciliate Paramecium tetraurelia consisting of seven families encoded by 12 genes that are expressed simultaneously. The complexity of the endomembrane system in Paramecium can explain this high number of genes. All P. tetraurelia synaptobrevins (PtSybs) possess a SNARE domain and show homology to the Longin family of R-SNAREs such as Ykt6, Sec22 and tetanus toxin-insensitive VAMP (TI-VAMP). We localized four exemplary PtSyb subfamilies with GFP constructs and antibodies on the light and electron microscopic level. PtSyb1-1, PtSyb1-2 and PtSyb3-1 were found in the endoplasmic reticulum, whereas PtSyb2 is localized exclusively in the contractile vacuole complex. PtSyb6 was found cytosolic but also resides in regularly arranged structures at the cell cortex (parasomal sacs), the cytoproct and oral apparatus, probably representing endocytotic compartments. With gene silencing, we showed that the R-SNARE of the contractile vacuole complex, PtSyb2, functions to maintain structural integrity as well as functionality of the osmoregulatory system but also affects cell division.