Transcriptomic dataset of the development and maturation of the Rhipicephalus microplus ovary.

To conduct differential gene expression analysis, ovaries from the cattle tick Rhipicephalus microplus were dissected at three distinct developmental stages (preingurgitated, immature ingurgitated, and mature ingurgitated). Additionally, undissected intact mature males and complete ingurgitated female ticks without ovaries (carcasses) were also collected to serve as reference samples for analysis. To perform total RNA purification, tissue from ten individuals representing each of the five previously described conditions was pooled. mRNA was isolated from the purified total RNA using the oligo (dT) method. Following fragmentation, double stranded cDNA was synthesized and ligated to sequencing adapters. Suitable-sized fragments were subsequently used for PCR amplification. Libraries were analyzed and quantified using an Agilent 2100 Bioanalyzer and an ABI StepOnePlus Real-Time PCR System. A total of 45.64 Gb bases were sequenced using the Illumina HiSeq sequencing platform. After assembling the samples and correcting for abundance, we obtained 82,877 unigenes. The total length, average length, N50, and GC content of the unigenes were 89,754,828 bp,1,082 bp,2,068 bp and 49.04 % respectively. For functional annotation, the unigenes were aligned with 7 functional databases. The number of unigenes identified in the functional databases were as follows: 32,518 (NR:39.24 %), 10,259 (NT:12.38 %), 23,624 (Swissprot:28.50 %), 22,203 (KOG:26.79 %), 25,072 (KEGG:30.25 %), 17,435(GO:21.04 %), and 23,220 (InterPro:28.02 %). Unigene candidate coding regions (CDS) among the unigenes were predicted using TransDecoder software and 42,143 CDS were detected. We also detected 10,522 simple sequence repeats (SSRs) distributed on 8,126 unigenes, and predicted 4,672 transcription factors (TF) coding unigenes. Our data can be used to identify genes that are important for male and female tick and arachnid reproduction and tick general physiology.

Escargot a Snail superfamily member and its multiple roles in Drosophila melanogaster development.

The Snail superfamily of transcription factors plays a crucial role in metazoan development; one of the most important vertebrate members of this family is Snai1 which is orthologous to the Drosophila melanogaster esg gene. This review offers a comprehensive examination of the roles of the esg gene in Drosophila development, covering its expression pattern and downstream targets, and draws parallels between the vertebrate Snai1 family proteins on controlling the epithelial-to-mesenchymal transition and esg. This gene regulates stemness, ploidy, and pluripontency. esg is expressed in various tissues during development, including the gut, imaginal discs, and neuroblasts. The functions of the esg include the suppression of differentiation in intestinal stem cells and the preservation of diploidy in imaginal cells. In the nervous system development, esg expression also inhibits neuroblast differentiation, thus regulating the number of neurons and the moment in development of neuronal differentiation. Loss of esg function results in diverse developmental defects, including defects in intestinal stem cell maintenance and differentiation, and alters imaginal disc and nervous system development. Expression levels of esg also play a role in regulating longevity and metabolism in adult stages. This review provides an overview of the current understanding of esg's developmental role, emphasizing cellular and tissue effects that arise from its loss of function. The insights gained may contribute to a better understanding of evolutionary conserved developmental mechanisms and certain metabolic diseases.

Parkinsonian phenotypes induced by Synphilin-1 expression are differentially contributed by serotonergic and dopaminergic circuits and suppressed by nicotine treatment.

Synphilin-1 is a protein encoded by the human SNCAIP gene whose function has yet to be fully understood. However, it has been linked to familial Parkinson's disease (PD). Synphilin-1 is a major component of the Lewy bodies found in neurons in the substantia nigra pars compacta of PD patients. Synphilin-1 expression in serotonergic and/or dopaminergic neurons of Drosophila melanogaster induces neurodegeneration, as well as motor and non-motor PD like symptoms. In this work, we examined the contribution of the serotonergic and dopaminergic circuits in the development of PD-like phenotypes. We found that olfactory and visual symptoms are majorly contributed by the serotonergic system, and that motor symptoms and reduction in survival are mainly contributed by the dopaminergic system. Chronic nicotine treatment was able to suppress several of these symptoms. These results indicate that both the serotonergic and dopaminergic systems contribute to different aspects of PD symptomatology and that nicotine has beneficial effects on specific symptoms.

The BE (2)-M17 cell line has a better dopaminergic phenotype than the traditionally used for Parkinson´s research SH-SY5Y, which is mostly serotonergic.

SH-SY5Y is a cell line derived from human neuroblastoma. It is one of the most widely used in vitro models to study Parkinson's disease. Surprisingly, it has been found that it does not develop a dopaminergic phenotype after differentiation, questioning its usefulness as a Parkinson's model. There are other in vitro models with better dopaminergic characteristics. BE (2)-M17 is a human neuroblastoma cell line that differentiates when treated with retinoic acid. We compared the dopaminergic and serotonergic properties of both cell lines. BE (2)-M17 has higher basal levels of dopaminergic markers and acquires a serotonergic phenotype during differentiation while maintaining the dopaminergic phenotype. SH-SY5Y has higher basal levels of serotonergic markers but does not acquire a dopaminergic phenotype upon differentiation.

A Single Nucleotide Polymorphism Translates into a Radical Amino Acid Substitution at the Ligand-Binding Site in Fasciola hepatica Carboxylesterase B.

Fasciola hepatica anthelmintic resistance may be associated with the catalytic activity of xenobiotic metabolizing enzymes. The gene expression of one of these enzymes, identified as carboxylesterase B (CestB), was previously described as inducible in adult parasites under anthelmintic treatment and exhibited a single nucleotide polymorphism at position 643 that translates into a radical amino acid substitution at position 215 from Glutamic acid to Lysine. Alphafold 3D models of both allelic sequences exhibited a significant affinity pocket rearrangement and different ligand-docking modeling results. Further bioinformatics analysis confirmed that the radical amino acid substitution is located at the ligand affinity site of the enzyme, affecting its affinity to serine hydrolase inhibitors and preferences for ester ligands. A field genotyping survey from parasite samples obtained from two developmental stages isolated from different host species from Argentina and Mexico exhibited a 37% allele distribution for 215E and a 29% allele distribution for 215K as well as a 34% E/K heterozygous distribution. No linkage to host species or geographic origin was found in any of the allele variants.

Transcriptome-Based Identification of a Functional Fasciola hepatica Carboxylesterase B.

Bioinformatics analysis of the complete transcriptome of Fasciola hepatica, identified a total of ten putative carboxylesterase transcripts, including a 3146 bp mRNA transcript coding a 2205 bp open reading frame that translates into a protein of 735 amino acids, resulting in a predicted protein mass of 83.5 kDa and a putative carboxylesterase B enzyme. The gene coding for this enzyme was found in two reported F. hepatica complete genomes stretching 23,230 bp, containing two exons of 1282 and 1864 bp, respectively, as well as a 20,084 bp intron between the exons. The enzymatic activity was experimentally assayed on F. hepatica protein extracts by SDS-PAGE zymograms using synthetic chromogenic substrates, confirming both the theoretical molecular weight and carboxylesterase enzymatic activity. Further bioinformatics predicted that this enzyme is an integral component of the cellular membrane that should be active as a 167 kDa homodimer complex and polyacrylamide gel electrophoresis (PAGE) zymograms experiments confirmed the analysis. Additional bioinformatics analysis showed that DNA sequences that code for this particular enzyme are highly conserved in other parasitic trematodes, although they are labeled hypothetical proteins.

Nicotine suppresses Parkinson's disease like phenotypes induced by Synphilin-1 overexpression in Drosophila melanogaster by increasing tyrosine hydroxylase and dopamine levels.

It has been observed that there is a lower Parkinson's disease (PD) incidence in tobacco users. Nicotine is a cholinergic agonist and is the principal psychoactive compound in tobacco linked to cigarette addiction. Different studies have shown that nicotine has beneficial effects on sporadic and genetic models of PD. In this work we evaluate nicotine's protective effect in a Drosophila melanogaster model for PD where Synphilin-1 (Sph-1) is expressed in dopaminergic neurons. Nicotine has a moderate effect on dopaminergic neuron survival that becomes more evident as flies age. Nicotine is beneficial on fly survival and motility increasing tyrosine hydroxylase and dopamine levels, suggesting that cholinergic agonists may promote survival and metabolic function of the dopaminergic neurons that express Sph-1. The Sph-1 expressing fly is a good model for the study of early-onset phenotypes such as olfaction loss one of the main non-motor symptom related to PD. Our data suggest that nicotine is an interesting therapeutic molecule whose properties should be explored in future research on the phenotypic modulators of the disease and for the development of new treatments.

Transcriptome assembly dataset of anthelmintic response in Fasciola hepatica.

Fasciola hepatica is a worldwide distributed zoonotic parasitic trematode, which causes a severe liver disease clinically known as fasciolasis in a large number of wild animals, several livestock species as well as humans, prevention and control of fasciolasis is made by massive use of anthelmintic compounds on livestock and inevitably this practice has led to the emergence of anthelmintic resistant Fasciola hepatica and there is a great scientific effort to elucidate the molecular basis of anthelmintic resistance of parasitic helminths in general and of Fasciola hepatica in particular that may lead to improved anthelmintic compounds. In our project, we sequenced the transcriptomes obtained from the anthelmintic response to Triclabendazole and Albendazole on four samples from sensitive and resistant strains of Fasciola hepatica on Illumina HiSeq 4000 Platform and generated about 10.03 Gb per sample. The average genome-mapping rate is 81.29% and the average gene-mapping rate is 62.81%. 30,105 genes were identified in which 28,669 of them are known genes and 1,237 of them are novel genes from novel coding transcripts without any known features, 20,743 novel RNA transcripts were identified of which 14,293 of them are previously unknown splicing event for known genes but no alternative splicing was detected, the remaining 5,213 transcripts were found to be long noncoding RNA.

Proteasome Subunits Involved in Neurodegenerative Diseases.

The ubiquitin-proteasome system is the major pathway for the maintenance of protein homeostasis. Its inhibition causes accumulation of ubiquitinated proteins; this accumulation has been associated with several of the most common neurodegenerative diseases. Several genetic factors have been identified for most neurodegenerative diseases, however, most cases are considered idiopathic, thus making the study of the mechanisms of protein accumulation a relevant field of research. It is often mentioned that the biggest risk factor for neurodegenerative diseases is aging, and several groups have reported an age-related alteration of the expression of some of the 26S proteasome subunits and a reduction of its activity. Proteasome subunits interact with proteins that are known to accumulate in neurodegenerative diseases such as α-synuclein in Parkinson's, tau in Alzheimer's, and huntingtin in Huntington's diseases. These interactions have been explored for several years, but only until recently, we are beginning to understand them. In this review, we discuss the known interactions, the underlying patterns, and the phenotypes associated with the 26S proteasome subunits in the etiology and progression of neurodegenerative diseases where there is evidence of proteasome involvement. Special emphasis is made in reviewing proteasome subunits that interact with proteins known to have an age-related altered expression or to be involved in neurodegenerative diseases to explore key effectors that may trigger or augment their progression. Interestingly, while the causes of age-related reduction of some of the proteasome subunits are not known, there are specific relationships between the observed neurodegenerative disease and the affected proteasome subunits.

Rpt2 proteasome subunit reduction causes Parkinson's disease like symptoms in Drosophila.

The dysfunction of the proteasome-ubiquitin system is commonly reported in several neurodegenerative diseases. Post mortem samples of brains of patients with Parkinson´s disease present cytoplasmic inclusions that are rich in proteins such as ubiquitin, Tau, and α-synuclein. In Parkinson´s disease, a specific reduction of some of the proteasome subunits has also been reported. However, the specific role of the different proteasome subunits in dopaminergic neuron degeneration has not been thoroughly explored. In this work, we used the Gal4/UAS system to test fourteen Drosophila melanogaster RNAi lines from the Bloomington Drosophila Stock Center. Each of these lines targets a different proteasome subunit. To identify the strains that were able to induce neurodegeneration, we drove the expression of these lines to the eye and cataloged them as a function of the extent of neurodegeneration that they induced. The targeted proteasomal subunits are conserved in mammals and therefore may be relevant to study proteasome related diseases. The RNAi line among the regulatory subunits with the most penetrant phenotype targeted the proteasomal subunit Rpt2 and we decided to further characterize its phenotypes. Rpt2 knockdown in the Drosophila central nervous system reduced the activity of the proteasome, augmented the amount of insoluble ubiquitinated protein, and elicited motor and non-motor phenotypes that were similar to the ones found in Drosophila and other models for Parkinson's disease. When Rpt2 is silenced pan-neurally, third instar larvae have locomotion dysfunctions and die during pupation. Larval lethality was avoided using the Gal80-Gal4 system to induce the expression of the Rpt2 RNAi to dopaminergic neurons only after pupation. The reduction of Rpt2 in adult dopaminergic neurons causes reduced survival, hyperactivity, neurodegeneration, and sleep loss; probably recapitulating some of the sleep disorders that Parkinson's disease patients have before the appearance of locomotion disorders.

Physiological evidence that three known mutations in the para-sodium channel gene confer cypermethrin knockdown resistance in Rhipicephalus microplus.

BACKGROUND: Acaricide resistance is a central problem for the control of the cattle tick Rhipicephalus microplus. The physiological effects and phenotypes of the mutations that cause acaricide resistance are not always well understood or characterized. Single nucleotide polymorphisms (SNPs) that confer cypermethrin knockdown resistance (kdr) have been reported in R. microplus. These SNPs have been associated and correlated with pyrethroid resistance although there is no direct physiological evidence that their presence does confer kdr in this organism. METHODS: Resistant and susceptible strain resistance profiles were obtained using the larval packet discriminating dose assay. The relevant genomic regions of the para-sodium channel were amplified using standard PCR; SNPs were detected by sequencing the corresponding amplicons. Ovary response to cypermethrin exposure/treatment was evaluated using videometrical analysis. RESULTS: We found that the pyrethroid resistance trait is stable in a resistant reference strain after years without selection, suggesting that the resistance conferring mutations are fixed in the population. In this strain, a change in the structure of the pre-synaptic para-sodium channel caused by the G184C, the C190A and the T2134A SNPs appears to confer resistance. These mutations are absent in the susceptible strain used as control. We demonstrate that cypermethrin blocks ovary contraction in cypermethrin-susceptible ticks. We also show that ovaries from organisms that carry the kdr associated SNPs still contract at cypermethrin concentrations that completely block ovary contraction in the susceptible strain. The configuration of the experimental system excludes a xenobiotic detoxification mechanism. CONCLUSIONS: This is the first report that presents physiological evidence that the presence of the G184C, the C190A, and the T2134A mutations in the para-sodium channel correlates with maintaining muscle contractility in R. microplus exposed to cypermethrin. These SNPs may confer cypermethrin resistance in this organism by avoiding presynaptic blockage, inhibiting the flaccid muscle paralysis characteristic of this acaricide. The videometric assay that we previously validated can be used to detect more rapidly than other assays that involve larval mortality kdr-like cypermethrin resistant tick strains, permitting to directly assay adult pre-engorged females after they are collected on the field without waiting until eggs are laid and larvae eclose.

Adrenergic ligands that block oviposition in the cattle tick Rhipicephalus microplus affect ovary contraction.

The tyraminergic/octopaminergic system is central for the control of arthropod oviposition. Previous works demonstrated that the pharmacological perturbation of this system inhibits oviposition in the cattle tick Rhipicephalus microplus. In this work, we describe a physiologically active whole-mount preparation of the contractile tick ovary that allows the quantitative videometrical analysis of ovary contraction in response to different compounds. Eight adrenergic ligands known to inhibit oviposition, including octopamine and tyramine were tested. These compounds exhibited antagonistic effects; octopamine relaxes the ovary preparation while tyramine induces a very strong contraction. The other adrenergic compounds tested were classified as able to contract or relax ovary muscle tissue. Isoprotenerol has a stronger relaxative effect than octopamine. Tyramine induces the biggest contraction observed of all the compounds tested, followed, in descending amount of contraction, by salbutamol, prazosin, epinastine, clonidine and the acaricide amitraz. The effect of these adrenergic ligands on the ovary preparation, explains why these molecules inhibit tick oviposition and suggest a regulatory mechanism for ovary contraction and relaxation during oviposition. Our results also provide a physiological explanation of the egg-laying inhibition effect of amitraz when used on the cattle tick.

The Esg Gene Is Involved in Nicotine Sensitivity in Drosophila melanogaster.

In humans, there is a strong correlation between sensitivity to substances of abuse and addiction risk. This differential tolerance to drugs has a strong genetic component. The identification of human genetic factors that alter drug tolerance has been a difficult task. For this reason and taking advantage of the fact that Drosophila responds similarly to humans to many drugs, and that genetically it has a high degree of homology (sharing at least 70% of genes known to be involved in human genetic diseases), we looked for genes in Drosophila that altered their nicotine sensitivity. We developed an instantaneous nicotine vaporization technique that exposed flies in a reproducible way. The amount of nicotine sufficient to "knock out" half of control flies for 30 minutes was determined and this parameter was defined as Half Recovery Time (HRT). Two fly lines, L4 and L70, whose HRT was significantly longer than control´s were identified. The L4 insertion is a loss of function allele of the transcriptional factor escargot (esg), whereas L70 insertion causes miss-expression of the microRNA cluster miR-310-311-312-313 (miR-310c). In this work, we demonstrate that esg loss of function induces nicotine sensitivity possibly by altering development of sensory organs and neurons in the medial section of the thoracoabdominal ganglion. The ectopic expression of the miR-310c also induces nicotine sensitivity by lowering Esg levels thus disrupting sensory organs and possibly to the modulation of other miR-310c targets.

Physical and functional interactions between Drosophila homologue of Swc6/p18Hamlet subunit of the SWR1/SRCAP chromatin-remodeling complex with the DNA repair/transcription factor TFIIH.

The multisubunit DNA repair and transcription factor TFIIH maintains an intricate cross-talk with different factors to achieve its functions. The p8 subunit of TFIIH maintains the basal levels of the complex by interacting with the p52 subunit. Here, we report that in Drosophila, the homolog of the p8 subunit (Dmp8) is encoded in a bicistronic transcript with the homolog of the Swc6/p18(Hamlet) subunit (Dmp18) of the SWR1/SRCAP chromatin remodeling complex. The SWR1 and SRCAP complexes catalyze the exchange of the canonical histone H2A with the H2AZ histone variant. In eukaryotic cells, bicistronic transcripts are not common, and in some cases, the two encoded proteins are functionally related. We found that Dmp18 physically interacts with the Dmp52 subunit of TFIIH and co-localizes with TFIIH in the chromatin. We also demonstrated that Dmp18 genetically interacts with Dmp8, suggesting that a cross-talk might exist between TFIIH and a component of a chromatin remodeler complex involved in histone exchange. Interestingly, our results also show that when the level of one of the two proteins is decreased and the other maintained, a specific defect in the fly is observed, suggesting that the organization of these two genes in a bicistronic locus has been selected during evolution to allow co-regulation of both genes.

Perturbation of tyraminergic/octopaminergic function inhibits oviposition in the cattle tick Rhipicephalus (Boophilus) microplus.

The cattle tick Rhipicephalus microplus, is one of the most damaging livestock ectoparasites. Tropical tick infestation limits the introduction of high-yield bovine varieties because they do not have immunity to the diseases transmitted by these ectoparasites. This tick is usually controlled with chemical acaricides but their indiscriminate use has created resistant populations. The discovery of new molecules that can be used for tick control is urgent. Based on the knowledge that octopamine, a biogenic amine analog to epinephrine, is central to the regulation of oviposition in several studied arthropods and that an imbalance in octopamine release causes sterility in a Drosophila model. Tyramine, octopamine and epinastine and 83 adrenergic compounds classified by their effect in the vertebrate systems were screened for their ability to block oviposition in Rhipicephalus microplus. Of these molecules, we found that 10 alpha-agonists, 3 alpha-antagonists, 5 beta-adrenergic agonists, 7 beta-antagonists and Norepinephrine were able to inhibit oviposition in this tick at pharmacological concentrations. Surprisingly, tyramine appears to be more potent than octopamine. The probable physiological causes of this inhibition are discussed. Our results suggest that although there are alpha adrenergic-like receptors in the tick, they do not behave in a manner completely analogous to their vertebrate counterparts.

Synphilin suppresses α-synuclein neurotoxicity in a Parkinson's disease Drosophila model.

Parkinson's disease (PD) is the second most common neurodegenerative disorder in humans. It affects 1% of the population over 65-years old. Its causes are environmental and genetic. As the world population ages, there is an urgent need for better and more detailed animal models for this kind of disease. In this work we show that the use of transgenic Drosophila is comparable to more complicated and costly animal models such as mice. The Drosophila model behaves very similar to the equivalent transgenic mice model. We show that both Synphilin-1 and α-synuclein are toxic by themselves, but when co-expressed, they suppress their toxicity reciprocally. Importantly, the symptoms induced in the fly can be treated and partially reverted using standard PD pharmacological treatments. This work showcases Drosophila as a detailed and multifaceted model for Parkinson's disease, providing a convenient platform in which to study and find new genetic modifiers of PD. genesis 49:392-402, 2011.

Drosophila p53 is required to increase the levels of the dKDM4B demethylase after UV-induced DNA damage to demethylate histone H3 lysine 9.

Chromatin undergoes a variety of changes in response to UV-induced DNA damage, including histone acetylation. In human and Drosophila cells, this response is affected by mutations in the tumor suppressor p53. In this work, we report that there is a global decrease in trimethylated Lys-9 in histone H3 (H3K9me3) in salivary gland cells in wild type flies in response to UV irradiation. In contrast, flies with mutations in the Dmp53 gene have reduced basal levels of H3K9me3, which are then increased after UV irradiation. The reduction of H3K9me3 in response to DNA damage occurs preferentially in heterochromatin. Our experiments demonstrate that UV irradiation enhances the levels of Lys-9 demethylase (dKDM4B) transcript and protein in wild type flies, but not in Dmp53 mutant flies. Dmp53 binds to a DNA element in the dKdm4B gene as a response to UV irradiation. Furthermore, heterozygous mutants for the dKdm4B gene are more sensitive to UV irradiation; they are deficient in the removal of cyclobutane-pyrimidine dimers, and the decrease of H3K9me3 levels following DNA damage is not observed in dKdm4B mutant flies. We propose that in response to UV irradiation, Dmp53 enhances the expression of the dKDM4B histone demethylase, which demethylates H3K9me3 preferentially in heterochromatin regions. This mechanism appears to be essential for the proper function of the nucleotide excision repair system.

p8/TTDA overexpression enhances UV-irradiation resistance and suppresses TFIIH mutations in a Drosophila trichothiodystrophy model.

Mutations in certain subunits of the DNA repair/transcription factor complex TFIIH are linked to the human syndromes xeroderma pigmentosum (XP), Cockayne's syndrome (CS), and trichothiodystrophy (TTD). One of these subunits, p8/TTDA, interacts with p52 and XPD and is important in maintaining TFIIH stability. Drosophila mutants in the p52 (Dmp52) subunit exhibit phenotypic defects similar to those observed in TTD patients with defects in p8/TTDA and XPD, including reduced levels of TFIIH. Here, we demonstrate that several Dmp52 phenotypes, including lethality, developmental defects, and sterility, can be suppressed by p8/TTDA overexpression. TFIIH levels were also recovered in rescued flies. In addition, p8/TTDA overexpression suppressed a lethal allele of the Drosophila XPB homolog. Furthermore, transgenic flies overexpressing p8/TTDA were more resistant to UV irradiation than were wild-type flies, apparently because of enhanced efficiency of cyclobutane-pyrimidine-dimers and 6-4 pyrimidine-pyrimidone photoproducts repair. This study is the first using an intact higher-animal model to show that one subunit mutant can trans-complement another subunit in a multi-subunit complex linked to human diseases.

DNA repair and transcriptional deficiencies caused by mutations in the Drosophila p52 subunit of TFIIH generate developmental defects and chromosome fragility.

The transcription and DNA repair factor TFIIH is composed of 10 subunits. Mutations in the XPB, XPD, and p8 subunits are genetically linked to human diseases, including cancer. However, no reports of mutations in other TFIIH subunits have been reported in higher eukaryotes. Here, we analyze at genetic, molecular, and biochemical levels the Drosophila melanogaster p52 (DMP52) subunit of TFIIH. We found that DMP52 is encoded by the gene marionette in Drosophila and that a defective DMP52 produces UV light-sensitive flies and specific phenotypes during development: organisms are smaller than their wild-type siblings and present tumors and chromosomal instability. The human homologue of DMP52 partially rescues some of these phenotypes. Some of the defects observed in the fly caused by mutations in DMP52 generate trichothiodystrophy and cancer-like phenotypes. Biochemical analysis of DMP52 point mutations introduced in human p52 at positions homologous to those of defects in DMP52 destabilize the interaction between p52 and XPB, another TFIIH subunit, thus compromising the assembly of the complex. This study significantly extends the role of p52 in regulating XPB ATPase activity and, consequently, both its transcriptional and nucleotide excision repair functions.

TFIIH trafficking and its nuclear assembly during early Drosophila embryo development.

We present the first analysis of the dynamics of the transcription DNA-repair factor TFIIH at the onset of transcription in early Drosophila development. TFIIH is composed of ten polypeptides that are part of two complexes - the core and the CAK. We found that the TFIIH core is initially located in the cytoplasm of syncytial blastoderm embryos, and that after mitotic division ten and until the cellular blastoderm stage, the core moves from the cytoplasm to the nucleus. By contrast, the CAK complex is mostly cytoplasmic during cellularization and during gastrulation. However, both components are positioned at promoters of genes that are activated at transcription onset. Later in development, the CAK complex becomes mostly nuclear and co-localizes in most chromosomal regions with the TFIIH core, but not in all sites, suggesting that the CAK complex could have a TFIIH-independent role in transcription of some loci. We also demonstrate that even though the CAK and the core coexist in the early embryo cytoplasm, they do not interact until they are in the nucleus and suggest that the complete assembly of the ten subunits of TFIIH occurs in the nucleus at the mid-blastula transition. In addition, we present evidence that suggests that DNA helicase subunits XPB and XPD are assembled in the core when they are transported into the nucleus and are required for the onset of transcription.