Polymorphism of genes encoding drug-metabolizing and inflammation-related enzymes for susceptibility to cholangiocarcinoma in Thailand.

BACKGROUND: Cholangiocarcinoma (CCA) is an intractable cancer, and its incidence in northeastern Thailand is the highest worldwide. Infection with the liver fluke Opisthorchis viverrini (OV) has been associated with CCA risk. However, animal experiments have suggested that OV alone does not induce CCA, but its combination with a chemical carcinogen like nitrosamine can cause experimentally induced CCA in hamsters. Therefore, in humans, other environmental and genetic factors may also be involved. AIM: To examine relations between risk for CCA and genetic polymorphisms in carcinogen-metabolizing and inflammation-related genes. METHODS: This hospital-based case-control study enrolled 95 case-control pairs matched by age (± 5 years) and sex. We examined relations between risk for CCA and genetic polymorphisms in carcinogen-metabolizing and inflammation-related genes, serum anti-OV, alcohol consumption, and smoking. Polymorphisms of CYP2E1, IL-6 (-174 and -634), IL-10 (-819), and NF-κB (-94) and their co-occurrence with polymorphisms in the drug-metabolizing enzyme gene GSTT1 or GSTM1 were also analyzed. RESULTS: Although CCA risk was not significantly associated with any single polymorphism, persons with the GSTT1 wild-type and CYP2E1 c1/c2 + c2/c2 genotype had an increased risk (OR = 3.33, 95%CI: 1.23-9.00) as compared with persons having the GSTT1 wild-type and CYP2E1 c1/c1 wild genotype. The presence of anti-OV in serum was associated with a 7- to 11-fold increased risk, and smoking level was related to an OR of 1.5-1.8 in multivariable analyses adjusted for each of the seven genetic polymorphisms. CONCLUSION: In addition to infection with OV, gene-gene interactions may be considered as one of the risk factors for CCA development.

Geography-Dependent Horizontal Gene Transfer from Vertebrate Predators to Their Prey.

Horizontal transfer (HT) of genes between multicellular animals, once thought to be extremely rare, is being more commonly detected, but its global geographic trend and transfer mechanism have not been investigated. We discovered a unique HT pattern of Bovine-B (BovB) LINE retrotransposons in vertebrates, with a bizarre transfer direction from predators (snakes) to their prey (frogs). At least 54 instances of BovB HT were detected, which we estimate to have occurred across time between 85 and 1.3 Ma. Using comprehensive transcontinental sampling, our study demonstrates that BovB HT is highly prevalent in one geographical region, Madagascar, suggesting important regional differences in the occurrence of HTs. We discovered parasite vectors that may plausibly transmit BovB and found that the proportion of BovB-positive parasites is also high in Madagascar where BovB thus might be physically transported by parasites to diverse vertebrates, potentially including humans. Remarkably, in two frog lineages, BovB HT occurred after migration from a non-HT area (Africa) to the HT hotspot (Madagascar). These results provide a novel perspective on how the prevalence of parasites influences the occurrence of HT in a region, similar to pathogens and their vectors in some endemic diseases.

Ancient and recent gene duplications as evolutionary drivers of the seed maturation regulators DELAY OF GERMINATION1 family genes.

The DELAY OF GERMINATION1 (DOG1) family genes (DFGs) in Arabidopsis thaliana are involved in seed dormancy, reserve accumulation, and desiccation tolerance. Decoding the molecular evolution of DFGs is key to understanding how these seed programs evolved. This article demonstrates that DFGs have diverged in the four lineages DOG1, DOG1-LIKE4 (DOGL4), DOGL5 and DOGL6, whereas DOGL1, DOGL2 and DOGL3 arose separately within the DOG1 lineage. The systematic DFG nomenclature proposed in this article addresses the current issues of inconsistent DFG annotation and highlights DFG genomic synteny in angiosperms. DFG pseudogenes, or collapsed coding sequences, hidden in the genomes of early-diverging angiosperms are documented here. They suggest ancient birth and loss of DFGs over the course of angiosperm evolution. The proposed models suggest that the origin of DFG diversification dates back to the most recent common ancestor of living angiosperms. The presence of a single form of DFG in nonflowering plants is discussed. Phylogenetic analysis of gymnosperm, lycophyte, and liverwort DFGs and similar genes found in mosses and algae suggests that DFGs diverged from the TGACG motif-binding transcription factor genes before the divergence of the bryophyte lineage.

Ancient Memories of Seeds: ABA-Dependent Growth Arrest and Reserve Accumulation.

Genetic studies of seed maturation regulators, combining transcriptomics and network analysis, suggest the significance of genetic diversification for maturation programs, particularly in seed plants. By contrast, analogs of the maturation programs, such as dormancy and desiccation tolerance, are also found in non-seed plants. It is thus conceivable that seeds recalled or renovated ancient programs of stress-induced growth arrest that were already present in ferns, bryophytes, and possibly in algae. This opinion article discusses the origins and genetic diversification of the abscisic acid (ABA)-dependent seed maturation programs by addressing questions provoked by recent findings about the DELAY OF GERMINATION1 (DOG1) family proteins, which regulate reserve accumulation, dormancy, and/or desiccation tolerance in seeds in a gene-specific or redundant manner.

Genetic Diversity among Clouded Salamanders (Hynobius nebulosus) in Shiga Prefecture.

The Japanese clouded salamander (Hynobius nebulosus) is a lentic-breeding species distributed throughout western Japan. Threats, such as habitat loss, have led to it being categorized as a vulnerable species. To explore the phylogeographic features and population differentiation among clouded salamanders in Shiga prefecture, we analyzed sequences of the mitochondrial cytochrome b gene. DNA samples were collected from 29 distinct breeding sites, and 53 cytochrome b haplotypes were identified. On the basis of comparison of the composition and frequency of haplotypes in each breeding site, salamanders in each habitat appeared to have distinct characteristics. Significant genetic differentiation was observed in 93.3% of possible pairs of habitats in Shiga prefecture, and 67.7% of habitat pairs within the same locality were found to be significantly different. These results suggest that the salamanders' poor locomotion combined with topographic effects may have contributed to the diversity of locally distributed salamanders in Shiga. Phylogenetic analysis showed that haplotypes of H. nebulosus in Shiga can be divided into five groups (the Nagahama-Maibara group, Otsu group, Konan group 1, Konan group 2, and Takashima group), each with a distinct geographical distribution. Haplotypes of the Otsu group, however, were exceptionally widely distributed. The results of the present study will contribute to the future of H. nebulosus conservation management in Shiga.

Transcriptional activation of a chimeric retrogene PIPSL in a hominoid ancestor.

Retrogenes are a class of functional genes derived from the mRNA of various intron-containing genes. PIPSL was created through a unique mechanism, whereby distinct genes were assembled at the RNA level, and the resulting chimera was then reverse transcribed and integrated into the genome by the L1 retrotransposon. Expression of PIPSL RNA via its transcription start sites (TSSs) has been confirmed in the testes of humans and chimpanzee. Here, we demonstrated that PIPSL RNA is expressed in the testis of the white-handed gibbon. The 5'-end positions of gibbon RNAs were confined to a narrow range upstream of the PIPSL start codon and overlapped with those of orangutan and human, suggesting that PIPSL TSSs are similar among hominoid species. Reporter assays using a luciferase gene and the flanking sequences of human PIPSL showed that an upstream sequence exhibits weak promoter activity in human cells. Our findings suggest that PIPSL might have acquired a promoter at an early stage of hominoid evolution before the divergence of gibbons and ultimately retained similar TSSs in all of the lineages. Moreover, the upstream sequence derived from the phosphatidylinositol-4-phosphate 5-kinase, type I, alpha 5' untranslated region and/or neighboring repetitive sequences in the genome possibly exhibits promoter activity. Furthermore, we observed that a TATA-box-like sequence has emerged by nucleotide substitution in a lineage leading to humans, with this possibly responsible for a broader distribution of the human PIPSL TSSs.

Cross-Kingdom Commonality of a Novel Insertion Signature of RTE-Related Short Retroposons.

In multicellular organisms, such as vertebrates and flowering plants, horizontal transfer (HT) of genetic information is thought to be a rare event. However, recent findings unveiled unexpectedly frequent HT of RTE-clade LINEs. To elucidate the molecular footprints of the genomic integration machinery of RTE-related retroposons, the sequence patterns surrounding the insertion sites of plant Au-like SINE families were analyzed in the genomes of a wide variety of flowering plants. A novel and remarkable finding regarding target site duplications (TSDs) for SINEs was they start with thymine approximately one helical pitch (ten nucleotides) downstream of a thymine stretch. This TSD pattern was found in RTE-clade LINEs, which share the 3'-end sequence of these SINEs, in the genome of leguminous plants. These results demonstrably show that Au-like SINEs were mobilized by the enzymatic machinery of RTE-clade LINEs. Further, we discovered the same TSD pattern in animal SINEs from lizard and mammals, in which the RTE-clade LINEs sharing the 3'-end sequence with these animal SINEs showed a distinct TSD pattern. Moreover, a significant correlation was observed between the first nucleotide of TSDs and microsatellite-like sequences found at the 3'-ends of SINEs and LINEs. We propose that RTE-encoded protein could preferentially bind to a DNA region that contains a thymine stretch to cleave a phosphodiester bond downstream of the stretch. Further, determination of cleavage sites and/or efficiency of primer sites for reverse transcription may depend on microsatellite-like repeats in the RNA template. Such a unique mechanism may have enabled retroposons to successfully expand in frontier genomes after HT.

CENP-B box, a nucleotide motif involved in centromere formation, occurs in a New World monkey.

Centromere protein B (CENP-B) is one of the major proteins involved in centromere formation, binding to centromeric repetitive DNA by recognizing a 17 bp motif called the CENP-B box. Hominids (humans and great apes) carry large numbers of CENP-B boxes in alpha satellite DNA (AS, the major centromeric repetitive DNA of simian primates). Only negative results have been reported regarding the presence of the CENP-B box in other primate taxa. Consequently, it is widely believed that the CENP-B box is confined, within primates, to the hominids. We report here that the common marmoset, a New World monkey, contains an abundance of CENP-B boxes in its AS. First, in a long contig sequence we constructed and analysed, we identified the motif in 17 of the 38 alpha satellite repeat units. We then sequenced terminal regions of additional clones and found the motif in many of them. Immunostaining of marmoset cells demonstrated that CENP-B binds to DNA in the centromeric regions of chromosomes. Therefore, functional CENP-B boxes are not confined to hominids. Our results indicate that the efficiency of identification of the CENP-B box may depend largely on the sequencing methods used, and that the CENP-B box in centromeric repetitive DNA may be more common than researchers previously thought.

Combined effects of polymorphisms of DNA-repair protein genes and metabolic enzyme genes on the risk of cholangiocarcinoma.

OBJECTIVE: Although Opisthorchis viverrini is a risk factor for cholangiocarcinoma, not all the infected individuals develop cholangiocarcinoma. We investigated whether the base excision repair enzyme gene polymorphisms with differentiated repair capacities of inflammation-related deoxyribonucleic acid damage may play a key role and such possible effects from those genes may be increased or diminished in co-existence of polymorphisms of metabolic enzymes, including glutathione-S-transferases mu 1 and glutathione-S-transferases θ1. METHODS: We genotyped five non-synonymous single-nucleotide polymorphisms of three genes, including the human homolog of the 8-oxoguanine glycosylase 1 Ser326Cys, X-ray repair cross-complementing protein 1 Arg194Trp, Arg280His and Arg399Gln and poly (adenosine diphosphate ribose) polymerase 1 Val762Ala in 87-94 matched case-control pairs, and examined relations between those polymorphisms and the risk of cholangiocarcinoma. RESULTS: Any single polymorphism did not have a measurable association with the risk of cholangiocarcinoma. However, when considering glutathione-S-transferases mu 1 polymorphism together, the human homolog of the 8-oxoguanine glycosylase 1 codon 326 polymorphism was related to the decreased risk; odds ratios were 1.00 (reference), 0.06 (95% confidence interval 0.01-0.53), 0.06 (0.01-0.54) and 0.14 (0.02-1.08) for persons with human homolog of the 8-oxoguanine glycosylase 1 Ser/Ser and glutathione-S-transferases mu 1 wild, ones with Ser/Ser and glutathione-S-transferases mu 1 null, ones with Ser/Cys or Cys/Cys and glutathione-S-transferases mu 1 wild and ones with Ser/Cys or Cys/Cys and glutathione-S-transferases mu 1 null, respectively (P for interaction <0.01). Further adjustment for the presence of anti-Opisthorchis viverrini antibody, smoking and alcohol drinking did not change the decreased risk. Other combinations of deoxyribonucleic acid-repair gene polymorphism and glutathione-S-transferases were not associated with the risk of cholangiocarcinoma. CONCLUSIONS: The present findings suggested that decreased capacity of deoxyribonucleic acid-repair gene, human homolog of the 8-oxoguanine glycosylase 1, may be related to decreased risk if much damaged cells die before malignant transformation.

RNA-Mediated Gene Duplication and Retroposons: Retrogenes, LINEs, SINEs, and Sequence Specificity.

A substantial number of "retrogenes" that are derived from the mRNA of various intron-containing genes have been reported. A class of mammalian retroposons, long interspersed element-1 (LINE1, L1), has been shown to be involved in the reverse transcription of retrogenes (or processed pseudogenes) and non-autonomous short interspersed elements (SINEs). The 3'-end sequences of various SINEs originated from a corresponding LINE. As the 3'-untranslated regions of several LINEs are essential for retroposition, these LINEs presumably require "stringent" recognition of the 3'-end sequence of the RNA template. However, the 3'-ends of mammalian L1s do not exhibit any similarity to SINEs, except for the presence of 3'-poly(A) repeats. Since the 3'-poly(A) repeats of L1 and Alu SINE are critical for their retroposition, L1 probably recognizes the poly(A) repeats, thereby mobilizing not only Alu SINE but also cytosolic mRNA. Many flowering plants only harbor L1-clade LINEs and a significant number of SINEs with poly(A) repeats, but no homology to the LINEs. Moreover, processed pseudogenes have also been found in flowering plants. I propose that the ancestral L1-clade LINE in the common ancestor of green plants may have recognized a specific RNA template, with stringent recognition then becoming relaxed during the course of plant evolution.

Parallel relaxation of stringent RNA recognition in plant and mammalian L1 retrotransposons.

L1 elements are mammalian non-long terminal repeat retrotransposons, or long interspersed elements (LINEs), that significantly influence the dynamics and fluidity of the genome. A series of observations suggest that plant L1-clade LINEs, just as mammalian L1s, mobilize both short interspersed elements (SINEs) and certain messenger RNA by recognizing the 3'-poly(A) tail of RNA. However, one L1 lineage in monocots was shown to possess a conserved 3'-end sequence with a solid RNA structure also observed in maize and sorghum SINEs. This strongly suggests that plant LINEs require a particular 3'-end sequence during initiation of reverse transcription. As one L1-clade LINE was also found to share the 3'-end sequence with a SINE in a green algal genome, I propose that the ancestral L1-clade LINE in the common ancestor of green plants may have recognized the specific RNA template, with stringent recognition then becoming relaxed during the course of plant evolution.

Approaches to understanding adaptations of skin color variation by detecting gene-environment interactions.

Genetic and environmental factors are both part of an elaborate feedback mechanism whereby the human adaptive form reacts to environmental stimuli via internal adjustments. Human survival may ultimately depend on understanding two important components of future environmental adaptation. First, we must elucidate the dynamics of the human genome underpinning the complex human phenotype. Second, we must understand how the environment pressures and affects the genome, helping to determine human traits. This article reviews current approaches to detecting the natural selection of skin color variation in human populations. We include statistical methods for clarifying gene-environment interactions applicable to the interactions with UV radiation levels. We recommend spatial data mining as an efficient approach that applies environmental association rules, extending our knowledge of adaptation to the environment.

Inference for the initial stage of domain shuffling: tracing the evolutionary fate of the PIPSL retrogene in hominoids.

Domain shuffling has provided extraordinarily diverse functions to proteins. Nevertheless, how newly combined domains are coordinated to create novel functions remains a fundamental question of genetic and phenotypic evolution. Previously, we reported a unique mechanism of gene creation, whereby new combinations of functional domains are assembled from distinct genes at the RNA level, reverse transcribed, and integrated into the genome by the L1 retrotransposon. The novel gene PIPSL, created by the fusion of phosphatidylinositol-4-phosphate 5-kinase (PIP5K1A) and 26S proteasome subunit (S5a/PSMD4) genes, is specifically transcribed in human and chimpanzee testes. We present the first evidence for the translation of PIPSL in humans. The human PIPSL locus showed a low nucleotide diversity within 11 populations (125 individuals) compared with other genomic regions such as introns and overall chromosomes. It was equivalent to the average for coding sequences or exons from other genes, suggesting that human PIPSL has some function and is conserved among modern populations. Two linked amino acid-altering single-nucleotide polymorphisms were found in the PIPSL kinase domain of non-African populations. They are positioned in the vicinity of the substrate-binding cavity of the parental PIP5K1A protein and change the charge of both residues. The relatively rapid expansion of this haplotype might indicate a selective advantage for it in modern humans. We determined the evolutionary fate of PIPSL domains created by domain shuffling. During hominoid diversification, the S5a-derived domain was retained in all lineages, whereas the ubiquitin-interacting motif (UIM) 1 in the domain experienced critical amino acid replacements at an early stage, being conserved under subsequent high levels of nonsynonymous substitutions to UIM2 and other domains, suggesting that adaptive evolution diversified these functional compartments. Conversely, the PIP5K1A-derived domain is degenerated in gibbons and gorillas. These observations provide a possible scheme of domain shuffling in which the combined parental domains are not tightly linked in the novel chimeric protein, allowing for changes in their functional roles, leading to their fine-tuning. Selective pressure toward a novel function initially acted on one domain, whereas the other experienced a nearly neutral state. Over time, the latter also gained a new function or was degenerated.

Comprehensive screening of human genes with inhibitory effects on yeast growth and validation of a yeast cell-based system for screening chemicals.

To evaluate yeast as a high-throughput cell-based system for screening chemicals that may lead to drug development, 10,302 full-length human cDNAs (~50% of the total cDNAs) were introduced into yeast. Approximately 5.6% (583 clones) of the cDNAs repressed the growth of yeast. Notably, ~25% of the repressive cDNAs encoded uncharacterized proteins. Small chemicals can be readily surveyed by monitoring their restorative effects on the growth of yeast. The authors focused on protein kinases because protein kinases are involved in various diseases. Among 263 protein kinase cDNAs (~50% of the total) expressed in yeast, 60 cDNAs (~23%), including c-Yes, a member of the Src tyrosine kinase family, inhibited the growth of yeast. Known inhibitors for protein kinases were examined for whether they reversed the c-Yes-induced inhibition of the yeast growth. Among 85 inhibitors tested, 6 compounds (PP2, PP1, SU6656, purvalanol, radicicol, and geldanamycin) reversed the inhibition, indicating a high specificity sufficient for validating this screening system. Human c-Yes was found to interact with Hsc82, one of the yeast chaperones. Radicicol and geldanamycin probably exerted their actions through interactions with Hsc82. These results indicate that when human proteins requiring molecular chaperones for their activities are subjected to the yeast screening system, 2 groups of chemicals may be found. The actions of one group are exerted through direct interactions with the human proteins, whereas those of the other group are mediated through interactions with chaperones.

A novel testis ubiquitin-binding protein gene arose by exon shuffling in hominoids.

Most new genes arise by duplication of existing gene structures, after which relaxed selection on the new copy frequently leads to mutational inactivation of the duplicate; only rarely will a new gene with modified function emerge. Here we describe a unique mechanism of gene creation, whereby new combinations of functional domains are assembled at the RNA level from distinct genes, and the resulting chimera is then reverse transcribed and integrated into the genome by the L1 retrotransposon. We characterized a novel gene, which we termed PIP5K1A and PSMD4-like (PIPSL), created by this mechanism from an intergenic transcript between the phosphatidylinositol-4-phosphate 5-kinase (PIP5K1A) and the 26S proteasome subunit (PSMD4) genes in a hominoid ancestor. PIPSL is transcribed specifically in the testis both in humans and chimpanzees, and is post-transcriptionally repressed by independent mechanisms in these primate lineages. The PIPSL gene encodes a chimeric protein combining the lipid kinase domain of PIP5K1A and the ubiquitin-binding motifs of PSMD4. Strong positive selection on PIPSL led to its rapid divergence from the parental genes PIP5K1A and PSMD4, forming a chimeric protein with a distinct cellular localization and minimal lipid kinase activity, but significant affinity for cellular ubiquitinated proteins. PIPSL is a tightly regulated, testis-specific novel ubiquitin-binding protein formed by an unusual exon-shuffling mechanism in hominoid primates and represents a key example of rapid evolution of a testis-specific gene.

Assessing the monophyly of chlorophyll-c containing plastids by multi-gene phylogenies under the unlinked model conditions.

Recent multi-gene phylogenetic analyses of plastid-encoded genes have recovered a robust monophyly of chlorophyll-c containing plastids (Chl-c palstids) in cryptophytes, haptophytes, photosynthetic stramenopiles, and dinoflagellates. However, all the plastid multi-gene phylogenies published to date utilized the "linked" model, which ignores the heterogeneity of sequence evolution across genes in alignments. Both empirical and simulation studies show that, compared to the linked model, the "unlinked" model, which accounts for gene-specific evolution, can greatly improve multi-gene estimations. Here we newly sequenced 46 genes of Chl-c plastids, and examined the Chl-c plastid evolution by multi-gene analyses under the unlinked model. Unexpectedly, Chl-c plastid monophyly received only low to medium support in our analyses based on multi-gene data sets including up to 4829 alignment positions. Although we systematically surveyed and excluded the genes that could mislead estimation, the (inconclusive) support for Chl-c plastid monophyly was not significantly altered. We conclude that the estimates from the current plastid-encoded gene data are insufficient to resolve Chl-c plastid evolution with confidence, and are highly affected by genes subjected to the analyses, and methods for tree reconstruction applied. Thus, future data analyses of larger multi-gene data sets, preferentially under the unlinked model, are required to conclusively understand Chl-c plastid evolution.

Whole-genome screening indicates a possible burst of formation of processed pseudogenes and Alu repeats by particular L1 subfamilies in ancestral primates.

BACKGROUND: Abundant pseudogenes are a feature of mammalian genomes. Processed pseudogenes (PPs) are reverse transcribed from mRNAs. Recent molecular biological studies show that mammalian long interspersed element 1 (L1)-encoded proteins may have been involved in PP reverse transcription. Here, we present the first comprehensive analysis of human PPs using all known human genes as queries. RESULTS: The human genome was queried and 3,664 candidate PPs were identified. The most abundant were copies of genes encoding keratin 18, glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein L21. A simple method was developed to estimate the level of nucleotide substitutions (and therefore the age) of PPs. A Poisson-like age distribution was obtained with a mean age close to that of the Alu repeats, the predominant human short interspersed elements. These data suggest a nearly simultaneous burst of PP and Alu formation in the genomes of ancestral primates. The peak period of amplification of these two distinct retrotransposons was estimated to be 40-50 million years ago. Concordant amplification of certain L1 subfamilies with PPs and Alus was observed. CONCLUSIONS: We suggest that a burst of formation of PPs and Alus occurred in the genome of ancestral primates. One possible mechanism is that proteins encoded by members of particular L1 subfamilies acquired an enhanced ability to recognize cytosolic RNAs in trans.

V-SINEs: a new superfamily of vertebrate SINEs that are widespread in vertebrate genomes and retain a strongly conserved segment within each repetitive unit.

We have identified a new superfamily of vertebrate short interspersed repetitive elements (SINEs), designated V-SINEs, that are widespread in fishes and frogs. Each V-SINE includes a central conserved domain preceded by a 5'-end tRNA-related region and followed by a potentially recombinogenic (TG)(n) tract, with a 3' tail derived from the 3' untranslated region (UTR) of the corresponding partner long interspersed repetitive element (LINE) that encodes a functional reverse transcriptase. The central domain is strongly conserved and is even found in SINEs in the lamprey genome, suggesting that V-SINEs might be approximately 550 Myr old or older in view of the timing of divergence of the lamprey lineage from the bony fish lineage. The central conserved domain might have been subject to some form of positive selection. Although the contemporary 3' tails of V-SINEs differ from one another, it is possible that the original 3' tail might have been replaced, via recombination, by the 3' tails of more active partner LINEs, thereby retaining retropositional activity and the ability to survive for long periods on the evolutionary time scale. It seems plausible that V-SINEs may have some function(s) that have been maintained by the coevolution of SINEs and LINEs during the evolution of vertebrates.