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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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2019. Published by Elsevier Inc.)

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., (Copyright © 2018. Published by Elsevier B.V.)

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. In planta , 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 HvCKX1 gene. Moreover, to elucidate the putative role of HvCKX1 gene on grain production, knocked-out Hvckx1 mutant plants were generated using the RNA-guided Cas9 system. Homozygote transgenic plants with silenced HvCKX1 gene and azygous knock-out Hvckx1 mutants have been selected and analyzed. Both reduced expression of HvCKX1 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.

Immunoglobulin light chain amyloidosis (AL amyloidosis - ALA) is a monoclonal gammopathy characterized by presence of aberrant plasma cells producing amyloidogenic immunoglobulin light chains. This leads to formation of amyloid fibrils in various organs and tissues, mainly in heart and kidney, and causes their dysfunction. As amyloid depositing in target organs is irreversible, there is a big effort to identify biomarker that could help to distinguish ALA from other monoclonal gammopathies in the early stages of disease, when amyloid deposits are not fatal yet. High throughput technologies bring new opportunities to modern cancer research as they enable to study disease within its complexity. Sophisticated methods such as next generation sequencing, gene expression profiling and circulating microRNA profiling are new approaches to study aberrant plasma cells from patients with light chain amyloidosis and related diseases. While generally known mutation in multiple myeloma patients (KRAS, NRAS, MYC, TP53) were not found in ALA, number of mutated genes is comparable. Transcriptome of ALA patients proves to be more similar to monoclonal gammopathy of undetermined significance patients, moreover level of circulating microRNA, that are known to correlate with heart damage, is increased in ALA patients, where heart damage in ALA typical symptom.Key words: amyloidosis - plasma cell - genome - transcriptome - microRNA.

Multiple myeloma is a plasma cell dyscrasia. It is the second most common hematological malignancy which is characterized by proliferation of clonal plasma cells producing harmful monoclonal immunoglobulin. Despite treatment modalities greatly evolved during the last decade, small amount of aberrant residual cells reside in patients after therapy and can cause relapse of the disease. Characterization of the residual, resistant clones can help to reveal important therapeutic targets for application of effective and precious treatment. We use CD38, CD45, CD56 and CD19 sorted aberrant plasma cells to perform next generation sequencing of their exome. Among the 213 genes in which at least one variant was present, the most interesting was found gene NRAS, one of the most often mutated gene in multiple myeloma, and homologs of 88 gene panel previously used for multiple myeloma sequencing among which was a gene previously identified as gene meaningful in bortezomib resistance. Nevertheless, the results of next generation exome sequencing need to be interpreted with caution, since they rely on bioinformatical analysis, which is still being optimized. The results of next generation sequencing will also have to be confirmed by Sanger sequencing. Final results supported by larger cohort of patients will be published soon.Key words: multiple myeloma - minimal residual disease - exome - next generation sequencing.

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

We have cloned the cDNA for the eighth human DNA polymerase, DNA polymerase &theta;. The human cDNA encodes a putative DNA polymerase of 1762 amino acids with a calculated molecular mass of 198 kDa. The derived protein sequence is homologous to the Drosophila melanogaster mus308 protein product, a putative DNA polymerase-helicase involved in repair of interstrand crosslinks. The C-terminal region contains the canonical DNA polymerase motifs A, B, and C found in the family A type of DNA polymerases, which includes Escherichia coli polymerase I. The N-terminal region contains a putative ATP binding domain but not motifs for a helicase. The gene was mapped by radiation hybrid analysis to chromosome 3q within an interval flanked by proximal marker D3S1303 and distal marker D3S3576 and, based on proximity to a gene that has been mapped cytogenetically, within band 3q13.31., (Copyright 1999 Academic Press.)

The thymidine kinase locus (Tk1) in Tk(+/-)-3.7.2C mouse lymphoma cells is widely used to identify mutagenic agents. Because Trp53 (the mouse homolog of human TP53) is located with Tk1 on chromosome 11 and is critical in regulating cellular responses following exposure to DNA damaging agents, we wanted to determine if these mouse lymphoma cells harbor mutations in Trp53. Single-stranded conformation polymorphism (SSCP) analysis of PCR-amplified exons 4-9 of Trp53 indicated mutations in both exons 4 and 5. We sequenced exons 4-9 from isolated clones of Tk(+/-)-3.7.2C cells and a Tk-/- mutant (G4). Mutant G4 has two copies of the chromosome carrying the Tk1- allele and no copy of the chromosome carrying the Tk1+ allele and thus could establish linkage of the individual Trp53 and Tk1 alleles. DNA sequence analysis revealed no mutations in exons 6-9 in any Tk(+/-)-3.7.2C or G4 clones. As suggested by SSCP, there was a nonsense mutation in exon 4 at bp 301 (codon 101) in one Trp53 allele. Tk(+/-)-3.7.2C clones have both mutant and wild-type sequences at bp 301; G4 clones have wild-type exon 4 sequence. These data allow assignment of the Trp53 exon 4 mutated allele to chromosome 11 carrying the Tk1+ allele. The exon 4 mutation leads to a stop codon early in translation, thus functionally deleting the Trp53 allele on the Tk1(+)-bearing chromosome. As previously reported, we find a missense mutation in exon 5 at bp 517 (codon 173) in one Trp53 allele. Using the G4 clones we determined that the exon 5 mutation is linked to the Tk1- allele. Thus the Tk +/-(-)3.7.2C mouse lymphoma cells have two mutant Trp53 alleles, likely accounting for their rapid cell growth and contributing to their ability to detect the major types of mutational damage associated with the etiology of tumor development. This ability to integrate across the mutational events seen in the multiple stages of tumor development further supports the use of the assay in chemical and drug safety studies and its recommendation as part of the required screening battery for regulatory agency submissions.

Microcell-mediated introduction of a neo-tagged human chromosome 1 (HC-1-neo) into several immortal cell lines has previously been shown to induce growth arrest and phenotypic changes indicative of replicative senescence. Somatic cell hybridization studies have localized senescence activity for immortal hamster 10W-2 cells to a cytogenetically defined region between 1q23 and the q terminus. Previous microcell-mediated chromosome transfer experiments showed that a chromosome 1 with an interstitial q-arm deletion (del-1q) lacks senescence inducing activity for several immortal human cell lines that are sensitive to an intact HC-1-neo. In contrast, our studies reveal that the del-1q chromosome retains activity for 10W-2 cells, indicating that there are at least two senescence genes on human chromosome 1. Sequence-tagged site (STS) content analysis revealed that the q arm of the del-1q chromosome has an interstitial deletion of approximately 63 centimorgans (cM), between the proximal STS marker DIS534 and distal marker DIS412, approximately 1q12 to 1q31. This deletion analysis provides a candidate region for one of the senescence genes on 1q. In addition, because this deletion region extends distally beyond 1q23, it localizes the region containing a second senescence gene to approximately 1q31-qter, between DIS422 and the q terminus. STS content analysis of a panel of 11 10W-2 microcell hybrid clones that escaped senescence identified 2 common regions of loss of 1q material below the distal breakpoint of del-1q. One region is flanked by markers DIS459 and ACTN2, and the second lies between markers WI-4683 and DIS1609, indicating that the distal 1q senescence gene(s) localizes within 1q42-43.

Human normal cells have been shown to undergo a limited number of cell doublings, a phenomenon termed cellular senescence. Human chromosome 1 has been implicated in this process, and several lines of evidence indicate that there is a senescence-inducing gene or genes on human chromosome 1q. Our approach to analyze the senescence-inducing effect of chromosome 1 includes the use of somatic cell hybrid revertants. We show here that fusion of a hypoxanthine phosphoribosyl transferase-negative mouse cell line (A9) containing a human neo-tagged chromosome 1 with an immortal hamster cell line (10W-2) results in cell hybrids that senesce after a few population doublings. Rare revertants that had escaped senescence were obtained after one large fusion experiment. Thirty-five nonsenescent hybrids were obtained from a total of approximately 1 million hybrids, and 25 of these were subjected to further analysis. The presence of a single copy of human chromosome 1 in the revertant hybrids was confirmed by fluorescence in situ hybridization analysis using a chromosome 1-specific painting probe. No visible translocations or deletions of chromosome 1 were observed in any of the hybrids. Deletion mapping revealed that 11 (56%) of the hybrids analyzed had lost one or more markers on chromosome 1q. Two regions with deletions were detected, one of which has been shown to be implicated in the senescence-inducing effect exerted by chromosome 1 following monochromosome transfer (P. J. Vojta et al., manuscript submitted for publication). The present study suggests that two separate loci on human chromosome 1q may be of importance for the induction of senescence. Moreover, this set of nonsenescent revertants could be useful for future detailed analyses of the senescence-inducing loci.

SDI1 is an inhibitor of DNA synthesis that we isolated by expression screening cDNAs prepared from senescent, terminally nondividing human cells. Other groups then cloned this gene as a cyclin-dependent kinase (cdk)-interacting protein (CIP1, p21) that inhibits cdks; the gene was also isolated by screening for genes transactivated by p53 (WAF1). p53 levels are low in senescent and quiescent contact-inhibited or serum-deprived normal human cells, which we have found express high levels of SDI1 mRNA. This indicates that alternate pathways for upregulation of message level of this gene may exist. We therefore proceeded with the study presented here, treating human cells with a variety of growth-arrest-inducing agents, including some that damaged DNA, and found that RNA levels of SDI1 were increased in all cases that resulted in growth inhibition. More important, with the exception of gamma-radiation, most of these agents were able to elevate SDI1 message levels in cells lacking wild-type p53. At least two distinct kinetic profiles for RNA induction were observed, one that implicated p53 transactivation and occurred early enough to cause arrest, and another that clearly was p53 independent and suggested a role for the SDI1 gene product in the maintenance rather than in the cause of inhibition of DNA synthesis.

Based on the previously reported sequence, we isolated an independent cDNA clone encoding a binding component of the human type I interferon receptor (IFN-R). This cDNA is identical to the published sequence except that it lacks 62 bases of 5' untranslated sequence and terminates at the first of two potential polyadenylation sites. In Northern blot analyses of poly(A)+RNAs from both IFN-sensitive and IFN-resistant Daudi cells, this cloned cDNA hybridized to a predominant mRNA of 2.4 kb, as well as to mRNAs of 1.8, 4.8, and 5.6 kb, and occasionally 6.9 kb. These various transcripts, which were also observed at similar levels in Raji B cells and two T-cell lines, Jurkat and MOLT-4, were detected after high-stringency washes, and by alternate probes corresponding to subfragments of the cDNA. In contrast, only the 4.8- and 5.6-kb transcripts hybridized to a polymerase chain reaction (PCR)-derived probe that corresponded to genomic sequences immediately down-stream from the second polyadenylation site. These results indicate that the latter transcripts arise from the same gene as the predominant 2.4-kb mRNA due to incomplete transcription termination at either of the known polyadenylation sites. Finally, Northern blot analysis of total RNAs revealed the presence of the predominant 2.4-kb type I IFN-R transcript in numerous tissues from second trimester human fetuses, suggesting that the type I IFN-R gene is constitutively expressed in multiple cell types.