Host Nuclear Genes Research Articles

Direct RNA sequencing approach to compare non-model mitochondrial transcriptomes: An application to a cephalopod host and its mesozoan parasite.

To identify non-protein coding as well as truncated or premature RNA sequences expressed and obtain more complete transcriptome information, we combined the MinION direct RNA-sequencing of a conventional poly(A) RNA purification method with poly(A)-tagging of the non-coding RNA (ncRNA) fraction. This approach was applied to transcriptome sequencing of the dicyemid mesozoan, Dicyema misakiense, which has minicircular mitochondrial DNA molecules where each molecule encodes a single gene, as well as the host. Using informatics analysis, we distinguished dicyemid RNAs from those of the host squid. The poly(A) RNAs were assigned to host mitochondrial genes, host nuclear protein-coding genes, Dicyema nuclear protein-coding genes, and Dicyema mitochondrial genes in the decreasing order. Our poly(A)-tailing method recovered significantly more ncRNAs from the host compared with the sequencing of poly(A) RNAs. Furthermore, our method captured various lengths of squid mitochondrial DNA (mtDNA) transcripts at different steps of maturation including a read of 3,500bp, which covers 21% of the squid mitochondrial genome, possibly a premature host RNA product. In contrast, shorter and less abundant reads were recovered from the dicyemid mitochondrial RNAs (mtRNAs). Even the longest read was 307bp covering only a part of a minicircle. This study revealed significantly different modes of the mitochondrial transcription between a mesozoan and the host. Our approach to perform direct RNA-sequencing combined with the poly(A)-tailing reaction can be an effective method to fully capture non-poly(A) transcripts in a wide range of organisms.

An NAD<sup>+</sup> dependent/sensitive transcription system: Toward a novel anti-cancer therapy

Cancer is widely known as a “genetic disease” because almost all cancers involve genomic mutations. However, cancer could also be referred to as a metabolic disease because it mainly depends on glycolysis to produce adenosine triphosphate (ATP) and is frequently accompanied by dysfunctions in the mitochondria, which are the primary organelle required in all eukaryotic cells. Importantly, almost all (99%) mitochondrial proteins are encoded by host nuclear genes. Not only cancer but also aging-related diseases, including neurodegenerative diseases, are associated with a decline in mitochondrial functions. Importantly, the nicotinamide adenine dinucleotide (NAD+) level decreases with aging. This molecule not only plays important roles in metabolism but also in the DNA repair system. In this article, we review 5′-upstream regions of mitochondrial function-associated genes to discuss the possibility of whether NAD+ is involved in transcriptional regulations. If the expression of these genes declines with aging, this may cause mitochondrial dysfunction. In this regard, cancer could be referred to as a “transcriptional disease”, and if so, novel therapeutics for cancer will need to be developed.

Regulation of chloroplast and nucleomorph replication by the cell cycle in the cryptophyte Guillardia theta

The chloroplasts of cryptophytes arose through a secondary endosymbiotic event in which a red algal endosymbiont was integrated into a previously nonphotosynthetic eukaryote. The cryptophytes retain a remnant of the endosymbiont nucleus (nucleomorph) that is replicated once in the cell cycle along with the chloroplast. To understand how the chloroplast, nucleomorph and host cell divide in a coordinated manner, we examined the expression of genes/proteins that are related to nucleomorph replication and chloroplast division as well as the timing of nuclear and nucleomorph DNA synthesis in the cryptophyte Guillardia theta. Nucleus-encoded nucleomorph HISTONE H2A mRNA specifically accumulated during the nuclear S phase. In contrast, nucleomorph-encoded genes/proteins that are related to nucleomorph replication and chloroplast division (FtsZ) are constantly expressed throughout the cell cycle. The results of this study and previous studies on chlorarachniophytes suggest that there was a common evolutionary pattern in which an endosymbiont lost its replication cycle-dependent transcription while cell-cycle-dependent transcriptional regulation of host nuclear genes came to restrict the timing of nucleomorph replication and chloroplast division.

Host nuclear genotype influences phenotype of a conditional mutualist symbiont.

Arthropods commonly carry maternally inherited intracellular bacterial symbionts that may profoundly influence host biology and evolution. The intracellular symbiont Rickettsia sp. nr. bellii swept rapidly into populations of the sweetpotato whitefly Bemisia tabaci in the south-western USA. Previous laboratory experiments showed female-bias and fitness benefits were associated with Rickettsia infection, potentially explaining the high frequencies of infection observed in field populations, but the effects varied with whitefly genetic line. Here, we explored whether host extranuclear or nuclear genes influenced the variation in the Rickettsia-host phenotype in two genetic lines of the whitefly host, each with Rickettsia-infected and uninfected sublines. Introgression between the Rickettsia-infected subline of one genetic line and the Rickettsia-uninfected subline of the other was used to create two new sublines, each with the maternally inherited extranuclear genetic lineages of one line (Rickettsia, two other symbionts and the mitochondria) and the nuclear genotype of the other. Performance assays comparing the original and new lines showed that in addition to Rickettsia, the interaction of Rickettsia infection with host nuclear genotype influenced development time and the sex ratio of the progeny, whereas the extranuclear genotype did not. Host nuclear genotype, but not extranuclear genotype, also influenced thetitre of Rickettsia. Our results support the hypothesis that differences in host nuclear genotype alone may explain considerable within-population variation in host-symbiont phenotype and may contribute to the observed variation in Rickettsia-whitefly interactions worldwide.

Selective sweep of Wolbachia and parthenogenetic host genomes - the example of the weevil Eusomus ovulum.

Most parthenogenetic weevil species are postulated to have originated via hybridization, but Wolbachia has also been speculated to play a role via the induction of parthenogenesis. Here, we examine the molecular diversity of Wolbachia and parthenogenetic host genomes. The host species studied here, Eusomus ovulum, is known to be exclusively parthenogenetic and triploid. The E. ovulum populations that we examined had a low genetic diversity of mitochondrial (cytochrome oxidase I gene) and nuclear markers (internal transcribed spacer 2 and elongation factor 1-α gene), and they all were infected by only single bacteria strains (genotyped for five genes according to the multilocus sequence typing system). We found significant signs of linkage disequilibrium and a lack of recombination amongst all of the examined genomes (bacteria and host), which strongly indicates a selective sweep. The lack of heterozygosity in host nuclear genes, missing bisexual populations and selective sweep between the parthenogenetic host and bacteria genomes suggest that parthenogenesis in this species could have originated as a result of infection rather than hybridization. However, the finding that highly similar Wolbachia strains are also present in other parthenogenetic weevils from the same habitat suggests the opposite scenario: bacteria may have infected the already parthenogenetic lineage and taken advantage of the host's unisexual reproduction.

Implication of bidirectional promoters containing duplicated GGAA motifs of mitochondrial function-associated genes

Mitochondria are well known as the primary required organelle in all eukaryotic cells. They have their own mtDNA containing genes that encode tRNAs, rRNAs and a set of functional proteins required for energy (ATP) production. However, almost all (99%) of mitochondrial proteins are encoded by host nuclear genes. Therefore, expression of mitochondrial protein-encoding genes should be regulated similarly to genes that are present in the host nuclear chromosomes. Interestingly, from genomic database assisted surveillance, it was revealed that a lot of mitochondrial function associated protein-encoding genes are oppositely linked in a head-head manner. If the two head-head conjugated genes are regulated by the same transcription factor(s), their expression would be dependent on the direction of transcription machinery that contains RNA polymerase II to execute mRNA synthesis. In this article, we will focus on several examples of the mitochondrial and the partner gene sets and discuss putative functions of transcription factor binding elements in the bidirectional promoters of mitochondrial function-associated genes in chromosomes.

THE ROLES OF HOST EVOLUTIONARY RELATIONSHIPS (GENUS: NASONIA) AND DEVELOPMENT IN STRUCTURING MICROBIAL COMMUNITIES

The comparative structure of bacterial communities among closely related host species remains relatively unexplored. For instance, as speciation events progress from incipient to complete stages, does divergence in the composition of the species' microbial communities parallel the divergence of host nuclear genes? To address this question, we used the recently diverged species of the parasitoid wasp genus Nasonia to test whether the evolutionary relationships of their bacterial microbiotas recapitulate the Nasonia phylogenetic history. We also assessed microbial diversity in Nasonia at different stages of development to determine the role that host age plays in microbiota structure. The results indicate that all three species of Nasonia share simple larval microbiotas dominated by the γ-proteobacteria class; however, bacterial species diversity increases as Nasonia develop into pupae and adults. Finally, under identical environmental conditions, the relationships of the microbial communities reflect the phylogeny of the Nasonia host species at multiple developmental stages, which suggests that the structure of an animal's microbial community is closely allied with divergence of host genes. These findings highlight the importance of host evolutionary relationships on microbiota composition and have broad implications for future studies of microbial symbiosis and animal speciation.

Comparisons of host mitochondrial, nuclear and endosymbiont bacterial genes reveal cryptic fig wasp species and the effects of Wolbachiaon host mtDNA evolution and diversity

BackgroundFigs and fig-pollinating wasp species usually display a highly specific one-to-one association. However, more and more studies have revealed that the "one-to-one" rule has been broken. Co-pollinators have been reported, but we do not yet know how they evolve. They may evolve from insect speciation induced or facilitated by Wolbachia which can manipulate host reproduction and induce reproductive isolation. In addition, Wolbachia can affect host mitochondrial DNA evolution, because of the linkage between Wolbachia and associated mitochondrial haplotypes, and thus confound host phylogeny based on mtDNA. Previous research has shown that fig wasps have the highest incidence of Wolbachia infection in all insect taxa, and Wolbachia may have great influence on fig wasp biology. Therefore, we look forward to understanding the influence of Wolbachia on mitochondrial DNA evolution and speciation in fig wasps.ResultsWe surveyed 76 pollinator wasp specimens from nine Ficus microcarpa trees each growing at a different location in Hainan and Fujian Provinces, China. We found that all wasps were morphologically identified as Eupristina verticillata, but diverged into three clades with 4.22-5.28% mtDNA divergence and 2.29-20.72% nuclear gene divergence. We also found very strong concordance between E. verticillata clades and Wolbachia infection status, and the predicted effects of Wolbachia on both mtDNA diversity and evolution by decreasing mitochondrial haplotypes.ConclusionsOur study reveals that the pollinating wasp E. verticillata on F. microcarpa has diverged into three cryptic species, and Wolbachia may have a role in this divergence. The results also indicate that Wolbachia strains infecting E. verticillata have likely resulted in selective sweeps on host mitochondrial DNA.

Methylation of HPV16 genome CpG sites is associated with cervix precancer and cancer

Objective.Invasive cervix cancer (ICC) is the second most common malignant tumor in women. Human papillomavirus 16 (HPV16) causes more than 50% of all ICC and is a major cause of cervix intraepithelial neoplasia (CIN). DNA methylation is a covalent modification predominantly occurring at CpG dinucleotides. Such epigenetic modifications are associated with changes in DNA–protein interactions and gene activation. This study examined the association of viral and host genomic methylation patterns and cervix neoplasia. Methods.Exfoliated cervical lavage samples positive for HPV16 from women with and without cytomorphic changes of infection (n=46), CIN2 (n=12), and CIN3+ (n=27) were used to interrogate the methylation patterns of the HPV16 L1 gene and upstream regulatory region (URR), five host nuclear genes (TERT, RARB, DAPK1, MAL, and CADM1), and mitochondrial DNA (mtDNA). DNA isolated from exfoliated cervicovaginal cells was treated with bisulfite, specific regions of the viral and host genome were PCR amplified and CpG methylation was quantified using EpiTYPER and pyrosequencing. Results.Methylation at 14 of the tested CpG sites within the HPV16 L1 region were significantly higher in CIN3+ compared to HPV16 genomes from women without CIN3+. In contrast, 2/16 CpG sites in HPV16 URR, 5/5 in TERT, 1/4 in DAPK1 and 1/3 mtDNA, and 2/5 in RARB were associated with increased methylation in CIN3+. Conclusions.These results indicate that increased methylation of CpG sites in the HPV16 L1 ORF is associated with CIN3+ and, thus, may constitute a potential biomarker for precancerous and cancerous cervix disease.

Codon Usage Biases of Transposable Elements and Host Nuclear Genes in Arabidopsis thaliana and Oryza sativa

Transposable elements (TEs) are mobile genetic entities ubiquitously distributed in nearly all genomes. High frequency of codons ending in A/T in TEs has been previously observed in some species. In this study, the biases in nucleotide composition and codon usage of TE transposases and host nuclear genes were investigated in the AT-rich genome of Arabidopsis thaliana and the GC-rich genome of Oryza sativa. Codons ending in A/T are more frequently used by TEs compared with their host nuclear genes. A remarkable positive correlation between highly expressed nuclear genes and C/G-ending codons were detected in O. sativa (r=0.944 and 0.839, respectively, P<0.0001) but not in A. thaliana, indicating a close association between the GC content and gene expression level in monocot species. In both species, TE codon usage biases are similar to that of weakly expressed genes. The expression and activity of TEs may be strictly controlled in plant genomes. Mutation bias and selection pressure have simultaneously acted on the TE evolution in A. thaliana and O. sativa. The consistently observed biases of nucleotide composition and codon usage of TEs may also provide a useful clue to accurately detect TE sequences in different species.

MODES OF ACQUISITION OFWOLBACHIA: HORIZONTAL TRANSFER, HYBRID INTROGRESSION, AND CODIVERGENCE IN THENASONIASPECIES COMPLEX

Wolbachia are maternally inherited bacteria that infect a large number of insects and are responsible for different reproductive alterations of their hosts. One of the key features of Wolbachia biology is its ability to move within and between host species, which contributes to the impressive diversity and range of infected hosts. Using multiple Wolbachia genes, including five developed for Multi-Locus Sequence Typing (MLST), the diversity and modes of movement of Wolbachia within the wasp genus Nasonia were investigated. Eleven different Wolbachia were found in the four species of Nasonia, including five newly identified infections. Five infections were acquired by horizontal transmission from other insect taxa, three have been acquired by hybridization between two Nasonia species, which resulted in a mitochondrial-Wolbachia sweep from one species to the other, and at least three have codiverged during speciation of their hosts. The results show that a variety of transfer mechanisms of Wolbachia are possible even within a single host genus. Codivergence of Wolbachia and their hosts is uncommon and provides a rare opportunity to investigate long-term Wolbachia evolution within a host lineage. Using synonymous divergence among codiverging infections and host nuclear genes, we estimate Wolbachia mutation rates to be approximately one-third that of the nuclear genome.

Multiple Gene Phylogenies Support the Monophyly of Cryptomonad and Haptophyte Host Lineages

Cryptomonad algae acquired their plastids by the secondary endosymbiotic uptake of a eukaryotic red alga. Several other algal lineages acquired plastids through such an event [1], but cryptomonads are distinguished by the retention of a relic red algal nucleus, the nucleomorph [2]. The nucleomorph (and its absence in other lineages) can reveal a great deal about the process and history of endosymbiosis, but only if we know the relationship between cryptomonads and other algae, and this has been controversial. Several recent analyses have suggested a relationship between plastids of cryptomonads and some or all other red alga-containing lineages [3-6], but we must also know whether host nuclear genes mirror this relationship to determine the number of endosymbiotic events, and this has not been demonstrated. We have carried out an expressed sequence tag (EST) survey of the cryptomonad Guillardia theta. Phylogenetic analyses of 102 orthologous nucleus-encoded proteins (18,425 amino acid alignment positions) show a robust sister-group relationship between cryptomonads and the haptophyte algae, which also have a red secondary plastid. This relationship demonstrates that loss of nucleomorphs must have taken place in haptophytes independently of any other red alga-containing lineages and that the ancestor of both already contained a red algal endosymbiont.

Fighting back against male-killers

Male-killing endosymbionts create a genetic black hole into which host nuclear genes vanish. In a recent paper, Hornett et al. transferred male-killing Wolbachia between different strains of the butterfly Hypolimnas bolina through hybridization and backcrossing. Their results provide unambiguous evidence of genetic variation for resistance to male-killers. A possible consequence of such variation is that male-killing might appear and disappear quickly on an evolutionary timescale.

EXPRESSION AND MODULATION OF EMBRYONIC MALE-KILLING IN DROSOPHILA INNUBILA: OPPORTUNITIES FOR MULTILEVEL SELECTION

Organisms and the symbionts they harbor may experience opposing forces of selection. In particular, the contrasting inheritance patterns of maternally transmitted symbionts and their host's nuclear genes can engender conflict among organizational levels over the optimal host offspring sex ratio. This study uses a male-killing Wolbachia endosymbiont and its host Drosophila innubila to experimentally address the potential for multilevel selection in a host-symbiont system. We show that bacterial density can vary among infected females, and that females with a higher density have a more female-biased offspring sex ratio. Furthermore, bacterial density is an epigenetic and heritable trait: females with a low bacterial load have daughters with a lower-than-average bacterial density, whose offspring then experience less severe male-killing. For infected sons, the probability of embryonic mortality increases with the bacterial density in their mothers. The frequency distribution of Wolbachia density among individual D. innubila females, and therefore the dynamics of infection within populations of these flies, results both from processes affecting the growth and regulation of bacterial populations within cytoplasmic lineages and from selection among cytoplasmic lineages that vary in bacterial density. Estimates of effective population size of Wolbachia within cytoplasmic lineages and of D. innubila at the host population level suggest that selection among cytoplasmic lineages is likely to overwhelm the results of selection within lineages.

EXPRESSION AND MODULATION OF EMBRYONIC MALE-KILLING IN DROSOPHILA INNUBILA: OPPORTUNITIES FOR MULTILEVEL SELECTION

Organisms and the symbionts they harbor may experience opposing forces of selection. In particular, the contrasting inheritance patterns of maternally transmitted symbionts and their host's nuclear genes can engender conflict among organizational levels over the optimal host offspring sex ratio. This study uses a male-killing Wolbachia endosymbiont and its host Drosophila innubila to experimentally address the potential for multilevel selection in a host-symbiont system. We show that bacterial density can vary among infected females, and that females with a higher density have a more female-biased offspring sex ratio. Furthermore, bacterial density is an epigenetic and heritable trait: females with a low bacterial load have daughters with a lower-than-average bacterial density, whose offspring then experience less severe male-killing. For infected sons, the probability of embryonic mortality increases with the bacterial density in their mothers. The frequency distribution of Wolbachia density among individual D. innubila females, and therefore the dynamics of infection within populations of these flies, results both from processes affecting the growth and regulation of bacterial populations within cytoplasmic lineages and from selection among cytoplasmic lineages that vary in bacterial density. Estimates of effective population size of Wolbachia within cytoplasmic lineages and of D. innubila at the host population level suggest that selection among cytoplasmic lineages is likely to overwhelm the results of selection within lineages.

Evolutionarily Stable Infection by a Male-Killing Endosymbiont in Drosophila innubilaSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY541089, AY541237 and AY552552, AY552553.

Maternally inherited microbes that spread via male-killing are common pathogens of insects, yet very little is known about the evolutionary duration of these associations. The few examples to date indicate very recent, and thus potentially transient, infections. A male-killing strain of Wolbachia has recently been discovered in natural populations of Drosophila innubila. The population-level effects of this infection are significant: approximately 35% of females are infected, infected females produce very strongly female-biased sex ratios, and the resulting population-level sex ratio is significantly female biased. Using data on infection prevalence and Wolbachia transmission rates, infected cytoplasmic lineages are estimated to experience a approximately 5% selective advantage relative to uninfected lineages. The evolutionary history of this infection was explored by surveying patterns of polymorphism in both the host and parasite genomes, comparing the Wolbachia wsp gene and the host mtDNA COI gene to five host nuclear genes. Molecular data suggest that this male-killing infection is evolutionarily old, a conclusion supported with a simple model of parasite and mtDNA transmission dynamics. Despite a large effective population size of the host species and strong selection to evolve resistance, the D. innubila-Wolbachia association is likely at a stable equilibrium that is maintained by imperfect maternal transmission of the bacteria rather than partial resistance in the host species.

Host genotype determines cytoplasmic incompatibility type in the haplodiploid genus Nasonia.

In haplodiploid species, Wolbachia-induced cytoplasmic incompatibility (CI) can be expressed in one of two ways: as a "conversion" of diploid fertilized eggs into haploid males or as embryonic mortality. Here we describe CI-type variation within the parasitic wasp genus Nasonia and genetically analyze the basis of this variation. We reach four main conclusions: (i) CI is expressed primarily as conversion in N. vitripennis, but as embryonic mortality in the sibling species N. giraulti and N. longicornis; (ii) the difference in CI type between N. giraulti (mortality) and N. vitripennis (conversion) is determined by host nuclear genotype rather than by Wolbachia differences; (iii) N. vitripennis "conversion genes" are recessive in hybrid females; and (iv) a difference in CI level between the sibling species N. giraulti and N. longicornis is due to the different Wolbachia infections in the species rather than to the host genotype. These results show that host nuclear genes can influence the type of CI present in a species. On the basis of these findings, we propose a model for how different CI types evolve in haplodiploids due to selection on nuclear genes modifying CI.

Increased male fertility in Tribolium confusum beetles after infection with the intracellular parasite Wolbachia.

The cytoplasmically inherited microorganism Wolbachia pipientis behaves like a sexually selected trait in its host, the flour beetle Tribolium confusum, enhancing male fertility at the expense of female fecundity. Here we show that infected females have fewer offspring than uninfected females but infected males have a large fertility advantage over uninfected males within multiply-inseminated infected or uninfected females. The male fertility effect accelerates the spread of the Wolbachia through the host population and expands the initial opportunity for hitch-hiking of host nuclear genes. Sperm competition in a host, mediated by endosymbionts, has not been previously described.