Publications by authors named "Petr Vojta"

16 Publications

  • Page 1 of 1

Establishing community reference samples, data and call sets for benchmarking cancer mutation detection using whole-genome sequencing.

Nat Biotechnol 2021 09 9;39(9):1151-1160. Epub 2021 Sep 9.

CCR Collaborative Bioinformatics Resource (CCBR), Office of Science and Technology Resources, Center for Cancer Research, Bethesda, MD, USA.

The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses.
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http://dx.doi.org/10.1038/s41587-021-00993-6DOI Listing
September 2021

Toward best practice in cancer mutation detection with whole-genome and whole-exome sequencing.

Nat Biotechnol 2021 09 9;39(9):1141-1150. Epub 2021 Sep 9.

European Infrastructure for Translational Medicine, Amsterdam, the Netherlands.

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.
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http://dx.doi.org/10.1038/s41587-021-00994-5DOI Listing
September 2021

Prognostic value of tumor-infiltrating lymphocytes (TILs) and their association with PD-L1 expression and DNA repair protein RAD51 in patients with resected non-small cell lung carcinoma.

Lung Cancer 2020 09 23;147:30-38. Epub 2020 Jun 23.

Department of Medical Oncology and Hematology, Cantonal Hospital, CH-9007, St. Gallen, Switzerland.

Objectives: DNA repair proteins have emerged as potential predictors for immunotherapy response alongside PD-L1 expression, tumor-infiltrating lymphocytes (TILs) and tumor mutational burden. We analyzed expression of PD-L1, TILs count and expression of the homologous recombination (HR) protein RAD51, as potential prognostic factors in patients with resected non-small-cell lung carcinoma (NSCLC).

Materials And Methods: Discovery set included 96 NSCLC patients from the University Hospital Olomouc (Czech Republic) and a replication set included 1109 NSCLC patients from University Hospital Zurich (Switzerland). Tissue microarrays (TMAs) were stained using the automated staining platform Ventana Benchmark Ultra with antibodies against RAD51,CD3, CD8, CD68 and PD-L1.

Results: Loss of nuclear RAD51 protein was associated with high TILs (r=-0.25, p = 0.01) and PD-L1 status (10.6 vs. 2.4 %, p = 0.012) in patients receiving neoadjuvant chemo-/radiotherapy (CT/RT). In silico analysis from the TCGA data set showed a negative relationship between RAD51 mRNA expression and CD45 (r = ‒0.422, p < 0.0001), CD68 (r = ‒0.326, p < 0.001), CD3 (r = ‒0.266, p < 0.001) and CD8 (r = ‒0.102, p < 0.001). RAD51 low/PD-L1 high patients were clustered as separate entity in the replication set and in TCGA dataset. High TILs status was significantly associated with improved OS in the replication set (unadjusted HR = 0.57, 95 % CI 0.42-0.76, p < 0.001). Similar results have been seen for CD3, CD8 and CD68.

Conclusions: In conclusion, RAD51 nuclear loss is weakly associated with increased TILs and high PD-L1 at the time of surgery in curatively resected NSCLC and after prior exposure to neoadjuvant chemo- or radiotherapy. Both high TILs and RAD51 nuclear loss were confirmed as independent prognostic factors in curatively resected NSCLC.
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http://dx.doi.org/10.1016/j.lungcan.2020.06.025DOI Listing
September 2020

Czech and Slovak Diamond-Blackfan Anemia (DBA) Registry update: Clinical data and novel causative genetic lesions.

Blood Cells Mol Dis 2020 03 11;81:102380. Epub 2019 Nov 11.

Department of Pediatrics, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, I. P. Pavlova 6, 77900 Olomouc, Czech Republic. Electronic address:

Diamond-Blackfan anemia (DBA) is a rare congenital erythroid aplasia, underlied by haploinsufficient mutations in genes coding for ribosomal proteins (RP) in approximately 70% of cases. DBA is frequently associated with somatic malformations, endocrine dysfunction and with an increased predisposition to cancer. Here we present clinical and genetic characteristics of 62 patients from 52 families enrolled in the Czech and Slovak DBA Registry. Whole exome sequencing (WES) and array comparative genomic hybridization (aCGH) were employed to identify causative mutations in newly diagnosed patients and in cases with previously unrecognized molecular pathology. RP mutation detection rate was 81% (50/62 patients). This included 8 novel point mutations and 4 large deletions encompassing some of the RP genes. Malignant or predisposing condition developed in 8/62 patients (13%): myelodysplastic syndrome in 3 patients; breast cancer in 2 patients; colorectal cancer plus ocular tumor, diffuse large B-cell lymphoma and multiple myeloma each in one case. These patients exclusively harbored RPL5, RPL11 or RPS19 mutations. Array CGH is beneficial for detection of novel mutations in DBA due to its capacity to detect larger chromosomal aberrations. Despite the importance of genotype-phenotype correlation in DBA, phenotypic differences among family members harboring an identical mutation were observed.
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http://dx.doi.org/10.1016/j.bcmd.2019.102380DOI Listing
March 2020

Corrigendum: Modification of Barley Plant Productivity Through Regulation of Cytokinin Content by Reverse-Genetics Approaches.

Front Plant Sci 2018;9:1996. Epub 2019 Feb 4.

Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czechia.

[This corrects the article DOI: 10.3389/fpls.2018.01676.].
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http://dx.doi.org/10.3389/fpls.2018.01996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6369263PMC
February 2019

Modification of Barley Plant Productivity Through Regulation of Cytokinin Content by Reverse-Genetics Approaches.

Front Plant Sci 2018 27;9:1676. Epub 2018 Nov 27.

Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czechia.

Barley is one of the most important cereals, which is used for breweries, animal and human feeds. Genetic manipulation of plant hormone cytokinins may influence several physiological processes, besides others stress tolerance, root formation and crop yield. , endogenous cytokinin status is finely regulated by the enzyme cytokinin dehydrogenase (EC 1.5.99.12; CKX), that irreversible degrades the side chain of adenine-derived isoprenoid cytokinins. Increasing grain yield by mean of manipulation of endogenous cytokinin content was assayed by the silencing of the gene. Moreover, to elucidate the putative role of gene on grain production, knocked-out mutant plants were generated using the RNA-guided Cas9 system. Homozygote transgenic plants with silenced gene and azygous knock-out mutants have been selected and analyzed. Both reduced expression of gene and CKX activity were measured in different stages of barley grain development. Phenotyping of the transgenic lines revealed reduced root growth, however, plants produced more tillers and grains than azygous wild-type controls and the total yield was increased up to 15 per cent. Although plant productivity was increased, total grain biomass was decreased to 80% of WT grains. RNA-seq analysis of knock-down transgenic lines revealed that several important macronutrient transporters were downregulated in the stage of massive starch accumulation. It suggests that local accumulation of cytokinins negatively affected nutrients flow resulting in reduced grain biomass. Obtained results confirmed the key role of HvCKX1 for regulation of cytokinin content in barley.
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http://dx.doi.org/10.3389/fpls.2018.01676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277847PMC
November 2018

Integrated physical map of bread wheat chromosome arm 7DS to facilitate gene cloning and comparative studies.

N Biotechnol 2019 Jan 8;48:12-19. Epub 2018 Mar 8.

Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, 783 71 Olomouc, Czech Republic. Electronic address:

Bread wheat (Triticum aestivum L.) is a staple food for a significant part of the world's population. The growing demand on its production can be satisfied by improving yield and resistance to biotic and abiotic stress. Knowledge of the genome sequence would aid in discovering genes and QTLs underlying these traits and provide a basis for genomics-assisted breeding. Physical maps and BAC clones associated with them have been valuable resources from which to generate a reference genome of bread wheat and to assist map-based gene cloning. As a part of a joint effort coordinated by the International Wheat Genome Sequencing Consortium, we have constructed a BAC-based physical map of bread wheat chromosome arm 7DS consisting of 895 contigs and covering 94% of its estimated length. By anchoring BAC contigs to one radiation hybrid map and three high resolution genetic maps, we assigned 73% of the assembly to a distinct genomic position. This map integration, interconnecting a total of 1713 markers with ordered and sequenced BAC clones from a minimal tiling path, provides a tool to speed up gene cloning in wheat. The process of physical map assembly included the integration of the 7DS physical map with a whole-genome physical map of Aegilops tauschii and a 7DS Bionano genome map, which together enabled efficient scaffolding of physical-map contigs, even in the non-recombining region of the genetic centromere. Moreover, this approach facilitated a comparison of bread wheat and its ancestor at BAC-contig level and revealed a reconstructed region in the 7DS pericentromere.
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http://dx.doi.org/10.1016/j.nbt.2018.03.003DOI Listing
January 2019

Acute lymphoblastic leukemia in a child with Leri-Weill syndrome and complete SHOX gene deletion: A Case Report.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2018 Mar 21;162(1):65-70. Epub 2018 Feb 21.

Department of Pediatrics, Faculty of Medicine and Dentistry, Palacky University Olomouc and University Hospital Olomouc, Czech Republic.

Background: Leri-Weill syndrome (LWS) ranks among conditions with short stature homeobox gene (SHOX) haploinsufficiency. Data on possible association of SHOX aberrations with malignant diseases are scarce.

Methods And Results: We report a unique case of an 8-year-old girl who was successfully treated for acute lymphoblastic leukemia (pre-B ALL, intermediate risk) and was subsequently diagnosed with LWS due to characteristic clinical appearance (short disproportionate stature, Madelung deformity of the wrist) and molecular genetic examination (complete deletion of SHOX). An identical SHOX deletion was identified also in the patient's mother. Leukemic cells of the patient were retrospectively examined by array comparative genomic hybridization (aCGH), which revealed five regions of deletions at chromosome X, including the SHOX gene locus.

Conclusion: Growth retardation in children with hemato-oncologic malignancies cannot always be attributed to cytotoxic treatment and should be carefully evaluated, especially with regards to growth hormone therapy.
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http://dx.doi.org/10.5507/bp.2018.002DOI Listing
March 2018

Newly designed 11-gene panel reveals first case of hereditary amyloidosis captured by massive parallel sequencing.

J Clin Pathol 2018 Aug 17;71(8):687-694. Epub 2018 Feb 17.

Department of Haematooncology, University Hospital Ostrava, Ostrava, Czech Republic.

Aims: Amyloidosis is caused by deposition of abnormal protein fibrils, leading to damage of organ function. Hereditary amyloidosis represents a monogenic disease caused by germline mutations in 11 amyloidogenic precursor protein genes. One of the important but non-specific symptoms of amyloidosis is hypertrophic cardiomyopathy. Diagnostics of hereditary amyloidosis is complicated and the real cause can remain overlooked. We aimed to design hereditary amyloidosis gene panel and to introduce new next-generation sequencing (NGS) approach to investigate hereditary amyloidosis in a cohort of patients with hypertrophic cardiomyopathy of unknown significance.

Methods: Design of target enrichment DNA library preparation using Haloplex Custom Kit containing 11 amyloidogenic genes was followed by MiSeq Illumina sequencing and bioinformatics identification of germline variants using tool VarScan in a cohort of 40 patients.

Results: We present design of NGS panel for 11 genes (, , , , , , , , , , ) connected to various forms of amyloidosis. We detected one mutation, which is responsible for hereditary amyloidosis. Some other single nucleotide variants are so far undescribed or rare variants or represent common polymorphisms in European population.

Conclusions: We report one positive case of hereditary amyloidosis in a cohort of patients with hypertrophic cardiomyopathy of unknown significance and set up first panel for NGS in hereditary amyloidosis. This work may facilitate successful implementation of the NGS method by other researchers or clinicians and may improve the diagnostic process after validation.
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http://dx.doi.org/10.1136/jclinpath-2017-204978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204976PMC
August 2018

Dataset for transcriptional response of barley (Hordeum vulgare) exposed to drought and subsequent re-watering.

Data Brief 2016 Sep 30;8:334-41. Epub 2016 May 30.

Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University in Olomouc, Czech Republic.

Barley (Hordeum vulgare) is an economically important species, which can be cultivated in environmentally adverse conditions due to its higher tolerance in contrast to other cereal crops. The draft of H. vulgare genome is available already for couple of years; however its functional annotation is still incomplete. All available databases were searched to expand current annotation. The improved annotation was used to describe processes and genes regulated in transgenic lines showing higher tolerance to drought in our associated article, doi:10.1016/j.nbt.2016.01.010 (Vojta et al., 2016) [1]. Here we present whole transcriptome response, using extended annotation, to severe drought stress and subsequent re-watering in wild-type barley plants in stem elongation phase of growth. Up- and down-regulated genes fall into distinct GO categories and these enriched by stress and revitalization are highlighted. Transcriptomic data were evaluated separately for root and aerial tissues.
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http://dx.doi.org/10.1016/j.dib.2016.05.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4908270PMC
September 2016

Whole transcriptome analysis of transgenic barley with altered cytokinin homeostasis and increased tolerance to drought stress.

N Biotechnol 2016 Sep 11;33(5 Pt B):676-691. Epub 2016 Feb 11.

Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University in Olomouc, Šlechtitelů 27, 783 71 Olomouc, Czech Republic. Electronic address:

Cytokinin plant hormones have been shown to play an important role in plant response to abiotic stresses. Herein, we expand upon the findings of Pospíšilová et al. [30] regarding preparation of novel transgenic barley lines overexpressing cytokinin dehydrogenase 1 gene from Arabidopsis under the control of mild root-specific promotor of maize β-glycosidase. These lines showed drought-tolerant phenotype mainly due to alteration of root architecture and stronger lignification of root tissue. A detailed transcriptomic analysis of roots of transgenic plants subjected to revitalization after drought stress revealed attenuated response through the HvHK3 cytokinin receptor and up-regulation of two transcription factors implicated in stress responses and abscisic acid sensitivity. Increased expression of several genes involved in the phenylpropanoid pathway as well as of genes encoding arogenate dehydratase/lyase participating in phenylalanine synthesis was found in roots during revitalization. Although more precursors of lignin synthesis were present in roots after drought stress, final lignin accumulation did not change compared to that in plants grown under optimal conditions. Changes in transcriptome indicated a higher auxin turnover in transgenic roots. The same analysis in leaves revealed that genes encoding putative enzymes responsible for production of jasmonates and other volatile compounds were up-regulated. Although transgenic barley leaves showed lower chlorophyll content and down-regulation of genes encoding proteins involved in photosynthesis than did wild-type plants when cultivated under optimal conditions, they did show a tendency to return to initial photochemical activities faster than did wild-type leaves when re-watered after severe drought stress. In contrast to optimal conditions, comparative transcriptomic analysis of revitalized leaves displayed up-regulation of genes encoding enzymes and proteins involved in photosynthesis, and especially those encoded by the chloroplast genome. Taken together, our results indicate that the partial cytokinin insensitivity induced in barley overexpressing cytokinin dehydrogenase contributes to tolerance to drought stress.
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http://dx.doi.org/10.1016/j.nbt.2016.01.010DOI Listing
September 2016

Quantitative and qualitative transcriptome analysis of four industrial strains of Claviceps purpurea with respect to ergot alkaloid production.

N Biotechnol 2016 Sep 28;33(5 Pt B):743-754. Epub 2016 Jan 28.

Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University in Olomouc, Šlechtitelů 27, 783 71 Olomouc, Czech Republic.

The fungus Claviceps purpurea is a biotrophic phytopathogen widely used in the pharmaceutical industry for its ability to produce ergot alkaloids (EAs). The fungus attacks unfertilized ovaries of grasses and forms sclerotia, which represent the only type of tissue where the synthesis of EAs occurs. The biosynthetic pathway of EAs has been extensively studied; however, little is known concerning its regulation. Here, we present the quantitative transcriptome analysis of the sclerotial and mycelial tissues providing a comprehensive view of transcriptional differences between the tissues that produce EAs and those that do not produce EAs and the pathogenic and non-pathogenic lifestyle. The results indicate metabolic changes coupled with sclerotial differentiation, which are likely needed as initiation factors for EA biosynthesis. One of the promising factors seems to be oxidative stress. Here, we focus on the identification of putative transcription factors and regulators involved in sclerotial differentiation, which might be involved in EA biosynthesis. To shed more light on the regulation of EA composition, whole transcriptome analysis of four industrial strains differing in their alkaloid spectra was performed. The results support the hypothesis proposing the composition of the amino acid pool in sclerotia to be an important factor regulating the final structure of the ergopeptines produced by Claviceps purpurea.
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http://dx.doi.org/10.1016/j.nbt.2016.01.006DOI Listing
September 2016

Transgenic barley overexpressing a cytokinin dehydrogenase gene shows greater tolerance to drought stress.

N Biotechnol 2016 Sep 7;33(5 Pt B):692-705. Epub 2016 Jan 7.

Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University in Olomouc, Šlechtitelů 27, 783 71 Olomouc, Czech Republic. Electronic address:

Together with auxins, cytokinins are the main plant hormones involved in many different physiological processes. Given this knowledge, cytokinin levels can be manipulated by genetic modification in order to improve agronomic parameters of cereals in relation to, for example, morphology, yield, and tolerance to various stresses. The barley (Hordeum vulgare) cultivar Golden Promise was transformed using the cytokinin dehydrogenase 1 gene from Arabidopsis thaliana (AtCKX1) under the control of mild root-specific β-glucosidase promoter from maize. Increased cytokinin degradation activity was observed positively to affect the number and length of lateral roots. The impact on morphology depended upon the recombinant protein's subcellular compartmentation. While assumed cytosolic and vacuolar targeting of AtCKX1 had negligible effect on shoot growth, secretion of AtCKX1 protein to the apoplast had a negative effect on development of the aerial part and yield. Upon the application of severe drought stress, all transgenic genotypes maintained higher water content and showed better growth and yield parameters during revitalization. Higher tolerance to drought stress was most caused by altered root morphology resulting in better dehydration avoidance.
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http://dx.doi.org/10.1016/j.nbt.2015.12.005DOI Listing
September 2016

Whole genome amplification induced bias in the detection of KRAS-mutated cell populations during colorectal carcinoma tissue testing.

Electrophoresis 2015 Mar 19;36(6):937-40. Epub 2015 Feb 19.

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic.

Whole genome amplification replicates the entire DNA content of a sample and can thus help to circumvent material limitations when insufficient DNA is available for planned genetic analyses. However, there are conflicting data in the literature whether whole genome amplification introduces bias or reflects precisely the spectrum of starting DNA. We analyzed the origins of discrepancies in KRAS (Kirsten rat sarcoma viral oncogene homolog gene) mutation detection in six of ten samples amplified using the GenomePlex® Tissue Whole Genome Amplification kit 5 (WGA5; Sigma-Aldrich, St. Louis, MO, USA) and KRAS StripAssay® (KRAS SA; ViennaLab Diagnostics, Vienna, Austria). We undertook reextraction, reamplification, retyping, authentication, reanalysis, and reinterpretation to determine whether the discrepancies originated during the preanalytical, analytical, and/or interpretative phase of genotyping. We conclude that a combination of glass slide/sample heterogeneity and biased amplification due to stochastic effects in the early phases of whole genome amplification (WGA) may have adversely affected the results obtained. Our findings are relevant for both forensic genetics testing and massively parallel sequencing using preamplification.
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http://dx.doi.org/10.1002/elps.201400136DOI Listing
March 2015

Diversity, phylogeny and expression patterns of Pou and Six homeodomain transcription factors in hydrozoan jellyfish Craspedacusta sowerbyi.

PLoS One 2012 30;7(4):e36420. Epub 2012 Apr 30.

Department of Genomics and Bioinformatics, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

Formation of all metazoan bodies is controlled by a group of selector genes including homeobox genes, highly conserved across the entire animal kingdom. The homeobox genes from Pou and Six classes are key members of the regulation cascades determining development of sensory organs, nervous system, gonads and muscles. Besides using common bilaterian models, more attention has recently been targeted at the identification and characterization of these genes within the basal metazoan phyla. Cnidaria as a diploblastic sister group to bilateria with simple and yet specialized organs are suitable models for studies on the sensory organ origin and the associated role of homeobox genes. In this work, Pou and Six homeobox genes, together with a broad range of other sensory-specific transcription factors, were identified in the transcriptome of hydrozoan jellyfish Craspedacusta sowerbyi. Phylogenetic analyses of Pou and Six proteins revealed cnidarian-specific sequence motifs and contributed to the classification of individual factors. The majority of the Craspedacusta sowerbyi Pou and Six homeobox genes are predominantly expressed in statocysts, manubrium and nerve ring, the tissues with sensory and nervous activities. The described diversity and expression patterns of Pou and Six factors in hydrozoan jellyfish highlight their evolutionarily conserved functions. This study extends the knowledge of the cnidarian genome complexity and shows that the transcriptome of hydrozoan jellyfish is generally rich in homeodomain transcription factors employed in the regulation of sensory and nervous functions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0036420PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340352PMC
September 2012
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