Specific quantification of root-colonizing arbuscular mycorrhizal fungi (AMF) by quantitative real-time PCR is a high-throughput technique, most suitable for determining abundances of AMF species or isolates in previously characterized experimental systems. The principal steps are the choice and validation of an appropriate assay to specifically amplify a gene fragment of the target AMF, preparation of templates from root samples, and quantification of the fungal gene copy numbers in these templates. The use of a suitable assay is crucial for a correct data collection but also highly specific for each experimental system and is therefore covered by general recommendations. Subsequently, specific steps are described for the validation of the assay using a standard dilution series, the determination of appropriate dilutions of DNA extracts from roots, and the quantification of the gene copy numbers in samples including calculations.

• Klíčová slova
• Abundance, Arbuscular mycorrhiza, Community, Glomeromycota, Inoculation, Quantification, Quantitative real-time PCR, Root colonization,
• MeSH
• DNA fungální genetika   izolace a purifikace   MeSH
• genová dávka genetika   MeSH
• kořeny rostlin genetika   mikrobiologie   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• mykorhiza genetika   izolace a purifikace   MeSH
• půda MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA fungální MeSH
• půda MeSH

Once believed to be restricted only to endotherms (mammals and birds), several poikilothermic amniote lineages have recently been documented to possess long-term evolutionary stability in their sex chromosomes. However, many important lineages were not included in these tests. Previously, based on molecular evidence, we documented the homology of well-differentiated sex chromosomes among seven families of iguanas (Pleurodonta), with basilisks (Corytophanidae) being the only exception, as the tested genes linked to X, but missing on the Y chromosome, in other iguanas were autosomal or pseudoautosomal in basilisks. In this study, we test the homology of sex chromosomes in the remaining, previously unstudied iguana families (Hoplocercidae, Leiosauridae, Liolaemidae, Polychrotidae) and in the basilisk genus Corytophanes. Our results show that 12 currently recognized families of iguanas share X-specific gene content conserved from the common ancestor living in the Cretaceous period. However, the results in the genus Corytophanes indicate the loss of the ancestral differentiated sex chromosomes from the ancestor of basilisks. Our new data further confirm the extensive stability of sex chromosomes in iguanas, thus enabling molecular sexing based on the comparison of the number of X-specific genes by quantitative PCR (qPCR) in all but one family of this widely diversified clade.

• Klíčová slova
• Dedifferentiation, Evolution, Sex chromosomes, X-linked genes, qPCR,
• MeSH
• chromozom X genetika   MeSH
• fylogeneze MeSH
• genová dávka genetika   MeSH
• karyotyp MeSH
• kvantitativní polymerázová řetězová reakce veterinární   MeSH
• leguáni genetika   MeSH
• pohlavní chromozomy genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: As up to 60 % of triple negative breast carcinomas are reported to express EGFR, the receptor is a potential target for biological therapy. The exact role EGFR plays in triple negative breast carcinoma (TNBC) biology, however, remains uncertain. We aimed to discover associations between EGFR protein expression as well as gene copy number changes and clinico-pathologic TNBC characteristics. METHODS: We performed an immunohistochemical and dual in situ hybridization study on a set of 52 archive cases of pre-treatment TNBC in order to detect EGFR protein expression and EGFR gene copy number changes. Clinico-pathologic and follow up data were compared with EGFR status for determining possible links between EGFR and tumor characteristics and/or behavior. RESULTS: 88.5 % of our cases showed EGFR expression. We found no significant links between EGFR expression and tumor grade (p = 0.204), lymph node status (p = 0.514) or p53 status (p = 0.078). Though EGFR gene amplification (EGFR gene:chromosome 7 ratio 2) was rare (1.9 % of all cases), a high gene copy number ( 4 copies per cell) was observed in 15.4 % of all cases. High EGFR gene copy number appeared to be more common in non-ductal, special-type carcinomas than in ductal carcinomas. Neither EGFR expression nor EGFR gene copy number was associated with event-free survival. CONCLUSION: EGFR changes do not appear to be associated with markers of aggressive behavior in TNBC. Further studies with much larger sample sizes are essential in understanding the role EGFR plays in TNBC biology in order to identify the patients that could benefit from EGFR targeted therapy.

• Klíčová slova
• EGFR - triple negative breast cancer - prognosis - gene amplification.,
• MeSH
• dospělí MeSH
• erbB receptory analýza   genetika   metabolismus   MeSH
• genová dávka genetika   MeSH
• imunohistochemie MeSH
• kohortové studie MeSH
• lidé středního věku MeSH
• lidé MeSH
• prognóza MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• triple-negativní karcinom prsu chemie   diagnóza   metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• erbB receptory MeSH

16S ribosomal RNA currently represents the most important target of study in bacterial ecology. Its use for the description of bacterial diversity is, however, limited by the presence of variable copy numbers in bacterial genomes and sequence variation within closely related taxa or within a genome. Here we use the information from sequenced bacterial genomes to explore the variability of 16S rRNA sequences and copy numbers at various taxonomic levels and apply it to estimate bacterial genome and DNA abundances. In total, 7,081 16S rRNA sequences were in silico extracted from 1,690 available bacterial genomes (1-15 per genome). While there are several phyla containing low 16S rRNA copy numbers, in certain taxa, e.g., the Firmicutes and Gammaproteobacteria, the variation is large. Genome sizes are more conserved at all tested taxonomic levels than 16S rRNA copy numbers. Only a minority of bacterial genomes harbors identical 16S rRNA gene copies, and sequence diversity increases with increasing copy numbers. While certain taxa harbor dissimilar 16S rRNA genes, others contain sequences common to multiple species. Sequence identity clusters (often termed operational taxonomic units) thus provide an imperfect representation of bacterial taxa of a certain phylogenetic rank. We have demonstrated that the information on 16S rRNA copy numbers and genome sizes of genome-sequenced bacteria may be used as an estimate for the closest related taxon in an environmental dataset to calculate alternative estimates of the relative abundance of individual bacterial taxa in environmental samples. Using an example from forest soil, this procedure would increase the abundance estimates of Acidobacteria and decrease these of Firmicutes. Using the currently available information, alternative estimates of bacterial community composition may be obtained in this way if the variation of 16S rRNA copy numbers among bacteria is considered.

• MeSH
• délka genomu genetika   MeSH
• DNA bakterií genetika   MeSH
• ekosystém * MeSH
• fylogeneze MeSH
• genetická variace * MeSH
• genom bakteriální genetika   MeSH
• genová dávka genetika   MeSH
• počítačová simulace MeSH
• půdní mikrobiologie MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• stromy mikrobiologie   MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• RNA ribozomální 16S MeSH

BACKGROUND: Tandemly arranged nuclear ribosomal DNA (rDNA), encoding 18S, 5.8S and 26S ribosomal RNA (rRNA), exhibit concerted evolution, a pattern thought to result from the homogenisation of rDNA arrays. However rDNA homogeneity at the single nucleotide polymorphism (SNP) level has not been detailed in organisms with more than a few hundred copies of the rDNA unit. Here we study rDNA complexity in species with arrays consisting of thousands of units. METHODS: We examined homogeneity of genic (18S) and non-coding internally transcribed spacer (ITS1) regions of rDNA using Roche 454 and/or Illumina platforms in four angiosperm species, Nicotiana sylvestris, N. tomentosiformis, N. otophora and N. kawakamii. We compared the data with Southern blot hybridisation revealing the structure of intergenic spacer (IGS) sequences and with the number and distribution of rDNA loci. RESULTS AND CONCLUSIONS: In all four species the intragenomic homogeneity of the 18S gene was high; a single ribotype makes up over 90% of the genes. However greater variation was observed in the ITS1 region, particularly in species with two or more rDNA loci, where >55% of rDNA units were a single ribotype, with the second most abundant variant accounted for >18% of units. IGS heterogeneity was high in all species. The increased number of ribotypes in ITS1 compared with 18S sequences may reflect rounds of incomplete homogenisation with strong selection for functional genic regions and relaxed selection on ITS1 variants. The relationship between the number of ITS1 ribotypes and the number of rDNA loci leads us to propose that rDNA evolution and complexity is influenced by locus number and/or amplification of orphaned rDNA units at new chromosomal locations.

• MeSH
• diploidie * MeSH
• DNA rostlinná genetika   MeSH
• genetická variace genetika   MeSH
• genetické lokusy genetika   MeSH
• genová dávka genetika   MeSH
• mezerníky ribozomální DNA genetika   MeSH
• ribozomální DNA genetika   MeSH
• rostlinné geny genetika   MeSH
• sekvenční analýza DNA MeSH
• Southernův blotting MeSH
• tabák genetika   MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• mezerníky ribozomální DNA MeSH
• ribozomální DNA MeSH

Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is characterized by accumulation of autofluorescent storage material in neural tissues and neurodegeneration and has an age of onset in the third decade of life or later. The genetic and molecular basis of the disease has remained unknown for many years. We carried out linkage mapping, gene-expression analysis, exome sequencing, and candidate-gene sequencing in affected individuals from 20 families and/or individuals with simplex cases; we identified in five individuals one of two disease-causing mutations, c.346_348delCTC and c.344T>G, in DNAJC5 encoding cysteine-string protein alpha (CSPα). These mutations-causing a deletion, p.Leu116del, and an amino acid exchange, p.Leu115Arg, respectively-are located within the cysteine-string domain of the protein and affect both palmitoylation-dependent sorting and the amount of CSPα in neuronal cells. The resulting depletion of functional CSPα might cause in parallel the presynaptic dysfunction and the progressive neurodegeneration observed in affected individuals and lysosomal accumulation of misfolded and proteolysis-resistant proteins in the form of characteristic ceroid deposits in neurons. Our work represents an important step in the genetic dissection of a genetically heterogeneous group of ANCLs. It also confirms a neuroprotective role for CSPα in humans and demonstrates the need for detailed investigation of CSPα in the neuronal ceroid lipofuscinoses and other neurodegenerative diseases presenting with neuronal protein aggregation.

• MeSH
• dominantní geny genetika   MeSH
• dospělí MeSH
• exony genetika   MeSH
• genetická vazba MeSH
• genová dávka genetika   MeSH
• lidé MeSH
• lipoylace MeSH
• lyzozomy metabolismus   ultrastruktura   MeSH
• membránové proteiny genetika   MeSH
• molekulární sekvence - údaje MeSH
• mozek metabolismus   patologie   ultrastruktura   MeSH
• mutace genetika   MeSH
• neuronální ceroidlipofuscinózy epidemiologie   genetika   patologie   MeSH
• neurony metabolismus   patologie   ultrastruktura   MeSH
• proteiny tepelného šoku HSP40 genetika   MeSH
• regulace genové exprese MeSH
• rodina MeSH
• rodokmen MeSH
• segregace chromozomů genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• transport proteinů MeSH
• věk při počátku nemoci MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cysteine string protein MeSH Prohlížeč
• membránové proteiny MeSH
• proteiny tepelného šoku HSP40 MeSH