P450 Gene Family Research Articles

Dynamics of copy number variation in host races of the pea aphid.

Copy number variation (CNV) makes a major contribution to overall genetic variation and is suspected to play an important role in adaptation. However, aside from a few model species, the extent of CNV in natural populations has seldom been investigated. Here, we report on CNV in the pea aphid Acyrthosiphon pisum, a powerful system for studying the genetic architecture of host-plant adaptation and speciation thanks to multiple host races forming a continuum of genetic divergence. Recent studies have highlighted the potential importance of chemosensory genes, including the gustatory and olfactory receptor gene families (Gr and Or, respectively), in the process of host race formation. We used targeted resequencing to achieve a very high depth of coverage, and thereby revealed the extent of CNV of 434 genes, including 150 chemosensory genes, in 104 individuals distributed across eight host races of the pea aphid. We found that CNV was widespread in our global sample, with a significantly higher occurrence in multigene families, especially in Ors. We also observed a decrease in the gene probability of being completely duplicated or deleted (CDD) with increase in coding sequence length. Genes with CDD variants were usually more polymorphic for copy number, especially in the P450 gene family where toxin resistance may be related to gene dosage. We found that Gr were overrepresented among genes discriminating host races, as were CDD genes and pseudogenes. Our observations shed new light on CNV dynamics and are consistent with CNV playing a role in both local adaptation and speciation.

Identification of a novel cytochrome P450 gene, CYP321E1 from the diamondback moth, Plutella xylostella (L.) and RNA interference to evaluate its role in chlorantraniliprole resistance.

Insect cytochrome P450 monooxygenases (P450s) play an important role in catalysis of many reactions leading to insecticides resistance. Our previous studies on transcriptome analysis of chlorantraniliprole-resistant development in the diamondback moth, Plutella xylostella revealed that up-regulation of cytochrome P450s are one of the main factors leading to the development of chlorantraniliprole resistance. Here, we report for the first time a novel cytochrome P450 gene CYP321E1, which belongs to the cytochrome P450 gene family CYP321. Real-time quantitative PCR (RT-qPCR) analyses indicated that CYP321E1 was expressed at all developmental stages of P. xylostella but was highest in the fourth-instar larvae; furthermore, the relatively high expression was observed in the midgut of the fourth-instar larvae, followed by fat bodies and epidermis. The expression of CYP321E1 in P. xylostella was differentially affected by three representative insecticides, including alphamethrin, abamectin and chlorantraniliprole. Among them, the exposure to chlorantraniliprole resulted in the largest transcript level of this cytochrome P450 gene. The findings suggested potential involvement of CYP321E1 in chlorantraniliprole resistance of P. xylostella. To assess the functional link of CYP321E1 to chlorantraniliprole resistance, RNA interference (RNAi)-mediated gene silencing by double stranded RNA (dsRNA) injecting was used. Results revealed that injection delivery of dsRNA can greatly reduce gene expression after 24h. As a consequence of RNAi, a significant increment in mortality of larvae injected CYP321E1 dsRNA was observed after 24h of exposure to chlorantraniliprole. These results strongly support our notion that this novel cytochrome P450 gene plays an important role in chlorantraniliprole detoxification in the diamondback moth and is partly responsible for its resistance.

RNA-Seq analysis implicates detoxification pathways in ovine mycotoxin resistance.

Mycotoxin induced hepatoxocity has been linked to oxidative stress, resulting from either an increase in levels of reactive oxygen species (ROS) above normal levels and/or the suppression of antioxidant protective pathways. However, few detailed molecular studies of mycotoxicoses in animals have been carried out. This study use current RNA-seq based approaches to investigate the effects of mycotoxin exposure in a ruminant model. Having first assembled a de novo reference transcriptome, we use RNA-Seq technology to define in vivo hepatic gene expression changes resulting from mycotoxin exposure in relationship to pathological effect. As expected, characteristic oxidative stress related gene expression is markedly different in animals exhibiting poorer outcomes. However, expression of multiple genes critical for detoxification, particularly members of the cytochrome P450 gene family, was significantly higher in animals exhibiting mycotoxin tolerance (‘resistance’). Further, we present novel evidence for the amplification of Wnt signalling pathway activity in ‘resistant’ animals, resulting from the marked suppression of multiple key Wnt inhibitor genes. Notably, ‘resistance’ may be determined primarily by the ability of an individual to detoxify secondary metabolites generated by the metabolism of mycotoxins and the potentiation of Wnt signalling may be pivotal to achieving a favourable outcome upon challenge.

The molecular evolution of cytochrome P450 genes within and between drosophila species.

We map 114 gene gains and 74 gene losses in the P450 gene family across the phylogeny of 12 Drosophila species by examining the congruence of gene trees and species trees. Although the number of P450 genes varies from 74 to 94 in the species examined, we infer that there were at least 77 P450 genes in the ancestral Drosophila genome. One of the most striking observations in the data set is the elevated loss of P450 genes in the Drosophila sechellia lineage. The gain and loss events are not evenly distributed among the P450 genes—with 30 genes showing no gene gains or losses whereas others show as many as 20 copy number changes among the species examined. The P450 gene clades showing the fewest number of gene gain and loss events tend to be those evolving with the most purifying selection acting on the protein sequences, although there are exceptions, such as the rapid rate of amino acid replacement observed in the single copy phantom (Cyp306a1) gene. Within D. melanogaster, we observe gene copy number polymorphism in ten P450 genes including multiple cases of interparalog chimeras. Nonallelic homologous recombination (NAHR) has been associated with deleterious mutations in humans, but here we provide a second possible example of an NAHR event in insect P450s being adaptive. Specifically, we find that a polymorphic Cyp12a4/Cyp12a5 chimera correlates with resistance to an insecticide. Although we observe such interparalog exchange in our within-species data sets, we have little evidence of it between species, raising the possibility that such events may occur more frequently than appreciated but are masked by subsequent sequence change.

Neither Cytochrome P450 Family Genes nor Neuroendocrine Factors could Independently Predict the SSRIs Treatment in the Chinese Han Population

This study was intended to explore the relationship between the genetic polymorphisms of the 8 single nucleotide polymorphisms (SNPs) at CYP genes, neuroendocrine factors and the response to selective serotonin reuptake inhibitors (SSRIs) in Chinese Han depressive patients. This was a 6-week randomized controlled trial consisting of 290 Chinese Han depressive patients treated with SSRIs. 8 SNPs of CYP450 genes and 7 neuroendocrine factors were detected. Allele and genotype frequencies were compared between responders and non-responders. The relationships between neuroendocrine factors and treatment response were also analyzed. No significant differences were found in clinical features between 2 groups at the baseline. No statistical correlation was found between either the genotype or allele frequencies of SNPs in CYP1A2, CYP2C19, or CYP2D6 gene and the -efficacy of SSRIs. There were strong linkage disequilibria between rs4986894, rs1853205, and rs12767583 of CYP2C19 genes, and rs2472299, rs2472300 of CYP1A2 genes. No associations were found between the above haplotypes and the antidepressant response. No neuroendocrine factor was a significant predictor for a response to SSRI antidepressants independently. The combination of neuroendocrine factors, however, predicted the response by 76.1%. There were no significant associations between the 6 SNPs of CYP gene polymorphisms and SSRI response. Neither cytochrome P450 family genes nor neuroendocrine factors independently predict the patients' response to the antidepressants separately. A combination of neuroendocrine factors, however, does have the potential to predict the response.

Role of glutathione S-transferase and cytochrome P-450 polymorphisms in the clinical outcome of osteosarcoma patients.

10535 Background: Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. Glutathione S-transferase (GST) and Cytochrome P-450 (CYP) family genes are involved in almost all anticancer drugs metabolism. The polymorphisms in these genes are associated with anticancer drugs resistance and toxicity events. This study aims to investigate the genotype frequencies of GSTM1, GSTT1, GSTM3, CYP1A2, CYP2C9 and CYP3A5 genes in OS patients and the influence of these polymorphisms in their clinical outcome. Methods: We investigated the peripheral blood DNA of 70 OS patients (25 metastatic and 45 nonmetastatic at diagnosis), following the GLATO - Latin American Group of Osteosarcoma Treatment - 2006. GSTM1 and GSTT1 deletion polymorphisms were examined through a multiplex-PCR and the GSTM3 polymorphism of three base pair-deletion using PCR-RFLP method. CYP1A2, CYP2C9 and CYP3A5 single nucleotide polymorphisms (SNPs) were investigated through real time PCR using TaqMan probe. Results: We found that GSTM1 null genotype was correlated with relapse occurrence (p=0,031) in patients that received high doses of chemotherapy. The CYP1A2*F allele was associated with lung metastasis (p=0,032), lung relapse (p=0,018) and high grade of ototoxicity (p=0,039). The CYP3A5*3 allele was associated with high grade of hepatotoxicity (p=0,010). The presence of at least one GSTM3*B allele was associated with better overall survival (p=0,045). The presence of CYP3A5*3 homozygous genotype was associated with better overall survival (p=0,043) in metastatic patients at diagnosis. The genotype GSTM3*B/ GSTM1 present, GSTM3*B/ GSTT1 present and GSTM3*B/CYP3A5*3 in metastatic patients at diagnosis were associated with better survival (p=0,048; p=0,004; p=0,012, respectively). Conclusions: The findings of this study suggest that GST and CYP polymorphisms may have a role in treatment response and may be an important marker in the future to personalized therapy in OS. Furthermore, CYP1A2*F allele is correlated with risk of lung metastasis and GSTM3*B homozygous genotype in combination with GSTM1 present, GSTT1 present and CYP3A5*3 were associated with better survival.

De Novo Assembly, Functional Annotation and Comparative Analysis of Withania somnifera Leaf and Root Transcriptomes to Identify Putative Genes Involved in the Withanolides Biosynthesis

Withania somnifera is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicine systems due to bioactive molecules known as withanolides. As genomic information regarding this plant is very limited, little information is available about biosynthesis of withanolides. To facilitate the basic understanding about the withanolide biosynthesis pathways, we performed transcriptome sequencing for Withania leaf (101L) and root (101R) which specifically synthesize withaferin A and withanolide A, respectively. Pyrosequencing yielded 8,34,068 and 7,21,755 reads which got assembled into 89,548 and 1,14,814 unique sequences from 101L and 101R, respectively. A total of 47,885 (101L) and 54,123 (101R) could be annotated using TAIR10, NR, tomato and potato databases. Gene Ontology and KEGG analyses provided a detailed view of all the enzymes involved in withanolide backbone synthesis. Our analysis identified members of cytochrome P450, glycosyltransferase and methyltransferase gene families with unique presence or differential expression in leaf and root and might be involved in synthesis of tissue-specific withanolides. We also detected simple sequence repeats (SSRs) in transcriptome data for use in future genetic studies. Comprehensive sequence resource developed for Withania, in this study, will help to elucidate biosynthetic pathway for tissue-specific synthesis of secondary plant products in non-model plant organisms as well as will be helpful in developing strategies for enhanced biosynthesis of withanolides through biotechnological approaches.

Multiple P450 genes: Identification, tissue-specific expression and their responses to insecticide treatments in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidea)

The oriental fruit fly Bactrocera dorsalis is an economically important pest, yet little research has focused on the mechanisms underlying the increasing insecticide resistance of this species. In this study, 12 cytochrome P450 genes were isolated from B. dorsalis, and alignments of the deduced amino acid sequences confirmed that they contained the conserved motifs of the P450 superfamily. Phylogenetic analyses indicated relationships with CYP3, CYP4, and mitochondrial cytochrome P450 gene families. Expression profiles of the P450 genes were compared between control and insecticide-treated adult flies using real-time quantitative PCR (RT-qPCR) methods. Results showed that five, four, and seven P450s were up-regulated following exposure to LD50 doses of malathion, abamectin, and beta-cypermethrin, respectively, and that transcriptional levels reached a maximum at 36h post-exposure. In addition, several genes did not show significant changes in expression, and several down-regulated genes were also noted. Most of the P450s (except CYP28F1) in untreated adults showed higher levels of transcription in the midgut, fat body, and/or Malpighian tubule than in the head. Taken together, these findings showed that multiple P450s are up-regulated in response to insecticide exposure and have various levels in different tissues. Further studies are needed to determine their possible role in the insecticide metabolism.

Chronic Ethanol Exposure Increases Cytochrome P‐450 and Decreases Activated in Blocked Unfolded Protein Response Gene Family Transcripts in Caenorhabditis elegans

Ethanol is a widely consumed and rapidly absorbed toxin. While the physiological effects of ethanol consumption are well known, the underlying biochemical and molecular changes at the gene expression level in whole animals remain obscure. We exposed the model organism Caenorhabditis elegans to 0.2 M ethanol from the embryo to L4 larva stage and assayed gene expression changes in whole animals using RNA-Seq and quantitative real-time PCR. We observed gene expression changes in 1122 genes (411 up, 711 down). Cytochrome P-450 (CYP) gene family members (12 of 78) were upregulated, whereas activated in blocked unfolded protein response (ABU) (7 of 15) were downregulated. Other detoxification gene family members were also regulated including four glutathione-S-transferases and three flavin monooxygenases. The results presented show specific gene expression changes following chronic ethanol exposure in C. elegans that indicate both persistent upregulation of detoxification response genes and downregulation of endoplasmic reticulum stress pathway genes.

Draft genome of the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major forest pest.

BackgroundThe mountain pine beetle, Dendroctonus ponderosae Hopkins, is the most serious insect pest of western North American pine forests. A recent outbreak destroyed more than 15 million hectares of pine forests, with major environmental effects on forest health, and economic effects on the forest industry. The outbreak has in part been driven by climate change, and will contribute to increased carbon emissions through decaying forests.ResultsWe developed a genome sequence resource for the mountain pine beetle to better understand the unique aspects of this insect's biology. A draft de novo genome sequence was assembled from paired-end, short-read sequences from an individual field-collected male pupa, and scaffolded using mate-paired, short-read genomic sequences from pooled field-collected pupae, paired-end short-insert whole-transcriptome shotgun sequencing reads of mRNA from adult beetle tissues, and paired-end Sanger EST sequences from various life stages. We describe the cytochrome P450, glutathione S-transferase, and plant cell wall-degrading enzyme gene families important to the survival of the mountain pine beetle in its harsh and nutrient-poor host environment, and examine genome-wide single-nucleotide polymorphism variation. A horizontally transferred bacterial sucrose-6-phosphate hydrolase was evident in the genome, and its tissue-specific transcription suggests a functional role for this beetle.ConclusionsDespite Coleoptera being the largest insect order with over 400,000 described species, including many agricultural and forest pest species, this is only the second genome sequence reported in Coleoptera, and will provide an important resource for the Curculionoidea and other insects.

Using Drosophila melanogaster to validate metabolism-based insecticide resistance from insect pests

Identifying molecular mechanisms of insecticide resistance is important for preserving insecticide efficacy, developing new insecticides and implementing insect control. The metabolic detoxification of insecticides is a widespread resistance mechanism. Enzymes with the potential to detoxify insecticides are commonly encoded by members of the large cytochrome P450, glutathione S-transferase and carboxylesterase gene families, all rapidly evolving in insects. Here, we demonstrate that the model insect Drosophila melanogaster is useful for functionally validating the role of metabolic enzymes in conferring metabolism-based insecticide resistance. Alleles of three well-characterized genes from different pest insects were expressed in transgenic D. melanogaster : a carboxylesterase gene (αE7) from the Australian sheep blowfly Lucilia cuprina, a glutathione S-transferase gene (GstE2) from the mosquito Anopheles gambiae and a cytochrome P450 gene (Cyp6cm1) from the whitefly Bemisia tabaci. For all genes, expression in D. melanogaster resulted in insecticide resistance phenotypes mirroring those observed in resistant populations of the pest species. Using D. melanogaster to assess the potential for novel metabolic resistance mechanisms to evolve in pest species is discussed.

Additional Selection for Insecticide Resistance in Urban Malaria Vectors: DDT Resistance in Anopheles arabiensis from Bobo-Dioulasso, Burkina Faso

In the city of Bobo-Dioulasso in Burkina Faso, Anopheles arabiensis has superseded Anopheles gambiae s.s. as the major malaria vector and the larvae are found in highly polluted habitats normally considered unsuitable for Anopheles mosquitoes. Here we show that An. gambiae s.l. adults emerging from a highly polluted site in the city centre (Dioulassoba) have a high prevalence of DDT resistance (percentage mortality after exposure to diagnostic dose = 65.8% in the dry season and 70.4% in the rainy season, respectively). An investigation into the mechanisms responsible found an unexpectedly high frequency of the 1014S kdr mutation (allele frequency = 0.4), which is found at very low frequencies in An. arabiensis in the surrounding rural areas, and an increase in transcript levels of several detoxification genes, notably from the glutathione transferase and cytochrome P450 gene families. A number of ABC transporter genes were also expressed at elevated levels in the DDT resistant An. arabiensis. Unplanned urbanisation provides numerous breeding grounds for mosquitoes. The finding that Anopheles mosquitoes adapted to these urban breeding sites have a high prevalence of insecticide resistance has important implications for our understanding of the selective forces responsible for the rapid spread of insecticide resistant populations of malaria vectors in Africa.

The involvement of heat shock protein and cytochrome P450 genes in response to UV-A exposure in the beetle Tribolium castaneum

Sunlight is an important environmental factor that affects all living organisms on Earth. Ultraviolet A (UV-A) is one of the many frequency bands found in sunlight. Many animals use UV-A to attain visual cues, for example, in foraging and mate selection. However, UV-A can also induce damage, such as oxidative stress, DNA lesions and apoptosis. In the present study, we investigated the effects of UV-A on the survival, fecundity and expression profiles of several stress-responsive genes belonging to the heat shock protein (Hsp) and the cytochrome CYP6BQ families from the adult red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). The results showed that short-term UV-A exposure (365nm, <4h) did not influence the survival or fecundity of the beetles; however, Hsp27, Hsp68, Hsp83, CYP6BQ4 and CYP6BQ8 mRNA levels significantly increased during the first 2h of UV-A exposure. Among them, Hsp68 was the most highly up-regulated, increasing by 8.9-fold. These results indicate that these genes may participate in the defense against harmful UV-A radiation. In addition, we investigated the potential transcription factor binding motifs (TFBMs) in the promoter sequences of genes induced in similar pattern from the Hsp and P450 gene families; the results indicated that, these motifs are highly homologous to environmental stress transcription factor binding sites in mammals. Our experiments revealed that UV-A irradiation could influence the expression profile of stress-responsive genes, such as Hsps and P450s, which have universal TFBMs, and that these genes may be involved in reducing the ecological challenges posed by irradiation.

Identification of two new cytochrome P450 genes and RNA interference to evaluate their roles in detoxification of commonly used insecticides in Locusta migratoria

Cytochrome P450 monooxygenases (cytochrome P450s), found in virtually all living organisms, play an important role in the metabolism of xenobiotics such as drugs, pesticides, and plant toxins. We have previously evaluated the responses of the oriental migratory locust (Locusta migratoria) to the pyrethroid insecticide deltamethrin and revealed that increased cytochrome P450 enzyme activity was due to increased transcription of multiple cytochrome P450 genes. In this study, we identified for the first time two new cytochrome P450 genes, which belong to two novel cytochrome P450 gene families. CYP409A1 belongs to CYP409 family whereas CYP408B1 belongs to CYP408 family. Our molecular analysis indicated that CYP409A1 was mainly expressed in fatbodies, midgut, gastric caecum, foregut and Malpighian tubules of the third- and fourth-instar nymphs, whereas CYP408B1 was mainly expressed in foregut, hindgut and muscle of the insects at all developmental stages examined. The expression of these two cytochrome P450 genes were differentially affected by three representative insecticides, including carbaryl (carbamate), malathion (organophosphate) and deltamethrin (pyrethroid). The exposure of the locust to carbaryl, malathion and deltamethrin resulted in reduced, moderately increased and significantly increased transcript levels, respectively, of the two cytochrome P450 genes. Our further analysis of their detoxification roles by using RNA interference followed by deltamethrin bioassay showed increased nymph mortalities by 21.1% and 16.7%, respectively, after CYP409A1 and CYP408B1 were silenced. These results strongly support our notion that these two new cytochrome P450 genes play an important role in deltamethrin detoxification in the locust.

마늘유래 Cytochrome P450 유전자의 변이 분석

Wound inducible P450-Esg cDNA, one of cytochrome P450 gene family, was isolated from shoot of Euiseong garlic cultivar. P450-Esg cDNA possesses highly conserved heme-binding domain in the nucleotide sequence, and 1,419 bp of open reading frame (ORF) coding of 473 amino acids. Based on the nucleotide sequence analysis of P450-Esg homologous from twelve garlic cultivars, two domains, one domain between 472 to 510 bp, and the other between 1,210 to 1,249 bp from start codon (ATG), showed various nucleotide polymorphism among cultivars. Sequence of heme-binding domain in P450-Esg homologous, which is located at the domain between 1,210 to 1,240 bp from start codon, showed various nucleotide polymorphism as well as amino acid sequence polymorphism among twelve garlic cultivars. Anther domain, between 472 to 510 bp from start codon, showed exactly same amino acid sequence in the twelve garlic cultivars, but there were various single nucleotide polymorphism to the cultivars.

Identification and expansion of a unique stem cell population from adult mouse gallbladder

The identification of resident stem cells in the mouse gallbladder is, to date, unexplored. In addition, the relationship between adult gallbladder stem cells and intrahepatic bile duct (IHBD) cells is not well understood. The aim of this study was to isolate stem cells from an adult mouse gallbladder and determine whether they were unique, compared to IHBD cells. By limiting dilution analyses and index sorts, we found that an EpCAM(+) CD49f(hi) epithelial cell subpopulation from primary gallbladder is enriched in colony-forming cells, compared to EpCAM(+) CD49f(lo) cells. EpCAM(+) CD49f(hi) cells expressed cluster of differentiation (CD)29, CD133, and stem cell antigen-1, but were negative for lineage markers CD31, CD45, and F4/80. Using a novel feeder cell-culture system, we observed long-term (>passage 20) and clonal expansion of the EpCAM(+) CD49f(hi) cells in vitro. In a matrigel differentiation assay, EpCAM(+) CD49f(+) cells expanding in vitro underwent organotypic morphogenesis forming ductular structures and cysts. These structures are similar to, and recapitulate a transport function of, primary gallbladder. EpCAM(+) CD49f(+) cells also engraft into the subcutaneous space of recipient mice. We compared primary gallbladder and IHBD cells by flow cytometry and found phenotypic differences in the expression of CD49f, CD49e, CD81, CD26, CD54, and CD166. In addition, oligonucleotide microarrays showed that the expanded EpCAM(+) CD49f(+) gallbladder cells and IHBD cells exhibit differences related to lipid and drug metabolism. Notable genes that were different are cytochrome P450, glutathione S-transferase, Indian hedgehog, and solute carrier family genes. We have isolated an epithelial cell population from primary mouse gallbladder with stem cell characteristics and found it to be unique, compared to IHBD cells.

Molecular analysis of Italian patients with congenital glaucoma.

Purpose: Congenital glaucoma (CG) results from poorly understood developmental abnormalities of the aqueous drainage structures and is clinically characterized by high intraocular pressure (IOP), epiphora, corneal oedema, photophobia, blepharospasm and ocular enlargement. To date, more than 50 pathogenic mutations in the CYP1B1 gene, a member of the cytochrome P450 gene family, have been reported in CG patients. The aim of this paper was to determine the genetic defects underlying CG in Italian patients. Methods: Molecular analysis of CYP1B1 was performed on the DNA of 120 patients and the transcript region of the MYOC/TIGR gene was also studied in all the patients bearing only one mutation in CYP1B1 gene. Previously described CYP1B1 polymorphisms (R48G, A119S, L432V, D449D and N453S) were also analyzed in our patients. Results: Seventeen different variations of CYP1B1 were found in 30 of the 120 (25.0%) CG patients. Twelve of these changes had been identified in previous reports as disease-causing mutations, while L26R, P52L, A106D, A237E and F440L are described here for the first time. F440L has always been found in cis with P52L, both in patients and healthy carriers, suggesting its role as a rare polymorphism linked to the P52L, while the other new variations we found could possibly play a pathogenetic role. G61E and 1775-1801dup27 are the most frequent mutations in our patients. Two amino acid variations (A447V and R76K) were identified in MYOC/TIGR analysis. Conclusions: Our results confirm the major role of the CYP1B1 gene in congenital glaucoma and also suggest an autosomal recessive role of MYOC/TIGR in a digenic inheritance model.

A De Novo Expression Profiling of Anopheles funestus, Malaria Vector in Africa, Using 454 Pyrosequencing

Background Anopheles funestus is one of the major malaria vectors in Africa and yet there are few genomic tools available for this species compared to An. gambiae. To start to close this knowledge gap, we sequenced the An. funestus transcriptome using cDNA libraries developed from a pyrethroid resistant laboratory strain and a pyrethroid susceptible field strain from Mali.ResultsUsing a pool of life stages (pupae, larvae, adults: females and males) for each strain, 454 sequencing generated 375,619 reads (average length of 182 bp). De novo assembly generated 18,103 contigs with average length of 253 bp. The average depth of coverage of these contigs was 8.3. In total 20.8% of all reads were novel when compared to reference databases. The sequencing of the field strain generated 204,758 reads compared to 170,861 from the insecticide resistant laboratory strain. The contigs most differentially represented in the resistant strain belong to the P450 gene family and cuticular genes which correlates with previous studies implicating both of these gene families in pyrethroid resistance. qPCR carried out on six contigs indicates that these ESTs could be suitable for gene expression studies such as microarray. 31,000 sites were estimated to contain Single Nucleotide Polymorphisms (SNPs) and analysis of SNPs from 20 contigs suggested that most of these SNPs are likely to be true SNPs. Gene conservation analysis confirmed the close phylogenetic relationship between An. funestus and An. gambiae.ConclusionThis study represents a significant advance for the genetics and genomics of An. funestus since it provides an extensive set of both Expressed Sequence Tags (ESTs) and SNPs which can be readily adopted for the design of new genomic tools such as microarray or SNP platforms.

Comparative Analysis of Human Embryonic Stem Cell and Induced Pluripotent Stem Cell-Derived Hepatocyte-Like Cells Reveals Current Drawbacks and Possible Strategies for Improved Differentiation

Hepatocytes derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) could provide a defined and renewable source of human cells relevant for cell replacement therapies and toxicology studies. However, before patient-specific iPSCs can be routinely used for these purposes, there is a dire need to critically compare these cells to the golden standard--hESCs. In this study, we aimed at investigating the differences and similarities at the transcriptional level between hepatocyte-like cells (HLCs) derived from both hESCs and iPSCs. Two independent protocols for deriving HLCs from hESCs and iPSCs were adopted and further characterization included immunocytochemistry, real-time (RT)-polymerase chain reaction, and in vitro functional assays. Comparative microarray-based gene expression profiling was conducted on these cells and compared to the transcriptomes of human fetal liver and adult liver progenitors. HLCs derived from hESCs and human iPSCs showed significant functional similarities, similar expression of genes important for liver physiology and common pathways. However, specific differences between the 2 cell types could be observed. For example, among the cytochrome P450 gene family, CYP19A1, CYP1A1, and CYP11A1 were enriched in hESC-derived HLCs, and CYP46A1 and CYP26A1 in iPSC-derived HLCs. HLCs derived from hESCs and human iPSCs exhibited broad similarities but as well meaningful differences. We identified common upregulated transcription factors, which might serve as a source for generating a cocktail of factors able to directly transdifferentiate somatic cells into HLCs. The findings may be vital to the refinement of protocols for the efficient derivation of functional patient-specific HLCs for regenerative and toxicology studies.

Zingiber zerumbet CYP71BA1 catalyzes the conversion of α-humulene to 8-hydroxy-α-humulene in zerumbone biosynthesis.

Plant cytochrome P450s are involved in the biosynthesis of various classes of secondary metabolites. To elucidate the biosynthesis of zerumbone, a sesquiterpenoid with multiple potential anticancer properties, a family of P450 genes expressed in rhizomes of Zingiber zerumbet Smith, were cloned using a PCR-based cloning strategy. After functional expression in yeast, one of these P450s was found to convert α-humulene into 8-hydroxy-α-humulene, a proposed intermediate of zerumbone biosynthesis. This P450 has been designated CYP71BA1, a new member of the CYP71 family. CYP71BA1 transcripts were detected almost exclusively in rhizomes and showed a similar expression pattern to ZSS1 transcripts during rhizome development. Coexpression of a gene cluster encoding four enzymes of the mevalonate pathway with CYP71BA1 and ZSS1 in Escherichia coli leads to the production of 8-hydroxy-α-humulene in the presence of mevalonate, suggesting the possibility of microbial production of this zerumbone intermediate from a relatively simple carbon source by metabolic engineering.