The monoclonal antibody TU-20 and its scFv fragment were radiolabeled with 125 I in order to develop new imaging agents against the specific neuronal marker III beta-tubulin. The reaction via chloramine-T using thiosulfate as a stopping reductant was determined as the most convenient way for radioiodination. The preserved immunological properties of radioiodinated species were estimated by ELISA, electrophoresis, and immunohistochemistry with autoradiography. Biodistribution studies revealed a different behavior of radioiodinated TU-20 and its scFv.

• MeSH
• Autoradiography MeSH
• Financing, Organized MeSH
• Immunoglobulin Fragments diagnostic use   chemistry   immunology   MeSH
• Immunohistochemistry MeSH
• Immunoconjugates MeSH
• Isotope Labeling MeSH
• Antibodies, Monoclonal MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Peripheral Nervous System Diseases metabolism   MeSH
• Iodine Radioisotopes diagnostic use   chemistry   MeSH
• Tissue Distribution MeSH
• Tubulin immunology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Animals MeSH

Většinu eukaryotického genomu představují DNA sekvence, které nekódují proteiny. Tyto sekvence jsou přepisovány buď podle vývojového programu daného organizmu nebo v rámci odpovědi na vnější signály. Výsledkem transkripce takových sekvencí je pak velké množství dlouhých nekódujících RNA (lncRNA). Celogenomové studie předpokládají existenci více než 3 300 lncRNA. Dlouhé nekódující RNA jsou definovány jako molekuly nekódujících RNA o délce více než 200 nukleotidů. Vzhledem k vysoké míře komplexnosti a rozmanitosti těchto sekvencí byl nárůst poznání v této oblasti relativně pomalý. Ačkoli bylo dosud funkčně charakterizováno pouze omezené množství lncRNA, jejich regulační potenciál je již dnes evidentní. LncRNA hrají klíčové role jak v transkripčních, tak v post-transkripčních regulačních drahách. U mnoha nádorových onemocnění dochází k deregulaci lncRNA, což společně s jejich funkčními vlastnostmi naznačuje jejich významný potenciál v procesech maligní transformace. Tento přehledový článek je zaměřen na shrnutí nedávno objevených skupin lncRNA, popis jejich biologických funkcí a zejména na jejich význam v nádorové biologii a translačním onkologickém výzkumu.

A major portion of the eukaryotic genome is occupied by DNA sequences; transcripts of these sequences do not code for proteins. This part of the eukaryotic genome is transcribed in a developmentally regulated manner or as a response to external stimuli to produce large numbers of long non-coding RNAs (lncRNAs). Genome-wide studies indicate the existence of more than 3,300 lncRNAs. Long non-coding RNAs are tentatively defined as molecules of ncRNAs that are more than two hundred nucleotides long. Due to the complexity and diversity of their sequences, progress in the field of lncRNAs has been very slow. Nonetheless, lncRNAs have emerged as key molecules involved in the control of transcriptional and posttranscriptional gene regulatory pathways. Although limited numbers of functional lncRNAs have been identified so far, the immense regulatory potential of lncRNAs is already evident, emphasizing that a genome-wide characterization of functional lncRNAs is needed. The fact that many lncRNAs are deregulated in various human cancers, together with their functional characteristics, implies their eminent role in carcinogenesis. In this review, we summarize novel classes of lncRNAs, describe their biological functions emphasizing their roles in tumor biology and translational oncology research.

• Keywords
• lincRNA, T-UC,
• MeSH
• 3' Untranslated Regions physiology   genetics   immunology   MeSH
• 5' Untranslated Regions physiology   genetics   immunology   MeSH
• Financing, Organized MeSH
• Genetic Markers genetics   MeSH
• Genetic Structures MeSH
• Genome, Human physiology   genetics   immunology   MeSH
• Carcinoma, Hepatocellular diagnosis   genetics   MeSH
• Humans MeSH
• RNA, Small Untranslated genetics   isolation & purification   MeSH
• MicroRNAs genetics   isolation & purification   MeSH
• Prostatic Neoplasms diagnosis   genetics   MeSH
• Breast Neoplasms diagnosis   genetics   MeSH
• Neoplasms diagnosis   etiology   genetics   MeSH
• RNA, Untranslated diagnostic use   genetics   isolation & purification   MeSH
• Untranslated Regions physiology   genetics   immunology   MeSH
• Telomere-Binding Proteins genetics   MeSH
• Pseudogenes physiology   genetics   immunology   MeSH
• Translational Research, Biomedical methods   trends   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Review MeSH

Nonsense mutations turn a coding (sense) codon into an in-frame stop codon that is assumed to result in a truncated protein product. Thus, nonsense substitutions are the hallmark of pseudogenes and are used to identify them. Here we show that in-frame stop codons within bacterial protein-coding genes are widespread. Their evolutionary conservation suggests that many of them are not pseudogenes, since they maintain dN/dS values (ratios of substitution rates at non-synonymous and synonymous sites) significantly lower than 1 (this is a signature of purifying selection in protein-coding regions). We also found that double substitutions in codons-where an intermediate step is a nonsense substitution-show a higher rate of evolution compared to null models, indicating that a stop codon was introduced and then changed back to sense via positive selection. This further supports the notion that nonsense substitutions in bacteria are relatively common and do not necessarily cause pseudogenization. In-frame stop codons may be an important mechanism of regulation: Such codons are likely to cause a substantial decrease of protein expression levels.

• MeSH
• Bacteria classification   genetics   MeSH
• Bacterial Proteins classification   genetics   MeSH
• Point Mutation MeSH
• Phylogeny MeSH
• Models, Genetic MeSH
• Evolution, Molecular MeSH
• Codon, Nonsense * MeSH
• Open Reading Frames genetics   MeSH
• Prokaryotic Cells metabolism   MeSH
• Pseudogenes genetics   MeSH
• Base Sequence MeSH
• Sequence Homology, Nucleic Acid MeSH
• Selection, Genetic MeSH
• Codon, Terminator genetics   MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Species within the angiosperm genus Silene contain the largest mitochondrial genomes ever identified. The enormity of these genomes (up to 11 Mb in size) appears to be the result of increased non-coding DNA, which represents >99 % of the genome content. These genomes are also fragmented into dozens of circular-mapping chromosomes, some of which contain no identifiable genes, raising questions about if and how these 'empty' chromosomes are maintained by selection. To assess the possibility that they contain novel and unannotated functional elements, we have performed RNA-seq to analyze the mitochondrial transcriptome of Silene noctiflora. RESULTS: We identified regions of high transcript abundance in almost every chromosome in the mitochondrial genome including those that lack any annotated genes. In some cases, these transcribed regions exhibited higher expression levels than some core mitochondrial protein-coding genes. We also identified RNA editing sites throughout the genome, including 97 sites that were outside of protein-coding gene sequences and found in pseudogenes, introns, UTRs, and transcribed intergenic regions. Unlike in protein-coding sequences, however, most of these RNA editing sites were only edited at intermediate frequencies. Finally, analysis of mitochondrial small RNAs indicated that most were likely degradation products from longer transcripts, but we did identify candidates for functional small RNAs that mapped to intergenic regions and were not associated with longer RNA transcripts. CONCLUSIONS: Our findings demonstrate transcriptional activity in many localized regions within the extensive intergenic sequence content in the S. noctiflora mitochondrial genome, supporting the possibility that the genome contains previously unidentified functional elements. However, transcription by itself is not proof of functional importance, and we discuss evidence that some of the observed transcription and post-transcriptional modifications are non-adaptive. Therefore, further investigations are required to determine whether any of the identified transcribed regions have played a functional role in the proliferation and maintenance of the enormous non-coding regions in Silene mitochondrial genomes.

• MeSH
• RNA Editing MeSH
• Genome, Mitochondrial * MeSH
• Genome, Plant * MeSH
• DNA, Intergenic MeSH
• RNA, Messenger MeSH
• Open Reading Frames MeSH
• Pseudogenes MeSH
• RNA MeSH
• Genes, Plant MeSH
• Sequence Analysis, RNA MeSH
• Silene genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Altering amounts of a protein in a cell has become a crucial tool for understanding its function. In many organisms, including the protozoan parasite Trypanosoma brucei, protein overexpression has been achieved by inserting a protein-coding sequence into an overexpression vector. Here, we have adapted the PCR only based system for tagging trypanosome proteins at their endogenous loci such that it in addition enables a tetracycline-inducible T7 RNA polymerase-mediated protein overexpression. Hence, this approach bypasses the need for molecular cloning, making it rapid and cost effective. We validated the approach for ten flagellum-associated proteins with molecular weights ranging from 40 to over 500 kDa. For a majority of the recombinant proteins a significant (3-50 fold) increase in the cellular amount was achieved upon induction of overexpression. Two of the largest proteins studied, the dynein heavy chains, were significantly overexpressed, while two were not. Our data suggest that this may reflect the extent of the T7 RNA polymerase processivity on the trypanosome genomic DNA. We further show that the overexpression is informative as to cellular functions of the studied proteins, and that these cultures can serve as an excellent source for purification of the overexpressed proteins. We believe that this rapid in locus overexpression system will become a valuable tool to interrogate cellular functions and biochemical activities of trypanosome proteins.

• MeSH
• DNA-Directed RNA Polymerases metabolism   MeSH
• Dyneins biosynthesis   MeSH
• Gene Expression MeSH
• Genes, Protozoan MeSH
• Protozoan Proteins biosynthesis   isolation & purification   MeSH
• Recombinant Proteins biosynthesis   isolation & purification   MeSH
• Trypanosoma brucei brucei * genetics   metabolism   MeSH
• Viral Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Telomerase is essential for proper functioning of telomeres in eukaryotes. We cloned and characterised genes for the protein subunit of telomerase (TERT) in the allotetraploid Nicotiana tabacum (tobacco) and its diploid progenitor species Nicotiana sylvestris and Nicotiana tomentosiformis with the aim of determining if allopolyploidy (hybridisation and genome duplication) influences TERT activity and divergence. Two of the three sequence variants present in the tobacco genome (NtTERT-C/s and NtTERT-D) revealed similarity to two sequence variants found in N. sylvestris and another variant (NtTERT-C/t) was similar to TERT of N. tomentosiformis. Variants of N. sylvestris origin showed less similarity to each other (80.5 % in the genomic region; 90.1 % in the coding sequence) than that between the NtTERT-C/s and NtTERT-C/t variants (93.6 and 97.2 %, respectively). The NtTERT-D variant was truncated at the 5' end, and indels indicated that it was a pseudogene. All tobacco variants were transcribed and alternatively spliced sequences were detected. Analysis of gene arrangements uncovered a novel exon in the N-terminal domain of TERT variants, a feature that is likely to be commonly found in Solanaceae species. In addition, species-specific duplications were observed within exon 5. The putative function, copy number and evolutionary origin of these NtTERT sequence variants are discussed.

• MeSH
• Alternative Splicing MeSH
• Exons MeSH
• Transcription, Genetic MeSH
• Genetic Loci MeSH
• Genome, Plant MeSH
• Gene Rearrangement MeSH
• Introns MeSH
• RNA Isoforms MeSH
• Evolution, Molecular MeSH
• Molecular Sequence Data MeSH
• Gene Order MeSH
• Pseudogenes MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Sequence Alignment MeSH
• Nicotiana genetics   MeSH
• Telomerase genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The division cycle of CNS cells was arrested in G0/G1 (86.6%) and G2 (12.8%) phases in diapausing larvae of Chymomyza costata. A two-step response was observed when the diapause was induced by transferring the 3rd instar larvae from long-day to short-day conditions: first, the proportion of G2-arrested cells increased rapidly within a single day after transfer; and second, the increase of G0/G1-arrested cells started with a delay of 5 days after transfer. The changes of relative mRNA levels of seven different genes, which code for important cell cycle regulatory factors [Cyclins D and E, kinases Wee1 and Myt1, phosphatase Cdc25 (String), Dacapo (p27), and PCNA] were followed using qRT-PCR technique. Two reference genes (Rp49 and ss-tubulin) served as a background. Significant transcriptional responses to photoperiodic transfer were observed for two genes: while the relative levels of dacapo mRNA increased during the rapid entry into the G2 arrest, the pcna expression was significantly downregulated during the delayed onset of G0/G1 arrest. In addition, moderate transcriptional upregulations of the genes coding for two inhibitory kinases, wee1 and myt1 accompanied the entry into diapause. The other genes were expressed equally in all photoperiodic conditions.

• MeSH
• Cell Cycle physiology   MeSH
• Central Nervous System cytology   MeSH
• Drosophilidae cytology   physiology   MeSH
• Photoperiod MeSH
• Transcription, Genetic physiology   MeSH
• Insect Proteins genetics   metabolism   MeSH
• Cloning, Molecular MeSH
• Larva cytology   physiology   MeSH
• Life Cycle Stages MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Polyploidization is considered one of the main mechanisms of plant genome evolution. The presence of multiple copies of the same gene reduces selection pressure and permits sub-functionalization and neo-functionalization leading to plant diversification, adaptation and speciation. In bread wheat, polyploidization and the prevalence of transposable elements resulted in massive gene duplication and movement. As a result, the number of genes which are non-collinear to genomes of related species seems markedly increased in wheat. RESULTS: We used new-generation sequencing (NGS) to generate sequence of a Mb-sized region from wheat chromosome arm 3DS. Sequence assembly of 24 BAC clones resulted in two scaffolds of 1,264,820 and 333,768 bases. The sequence was annotated and compared to the homoeologous region on wheat chromosome 3B and orthologous loci of Brachypodium distachyon and rice. Among 39 coding sequences in the 3DS scaffolds, 32 have a homoeolog on chromosome 3B. In contrast, only fifteen and fourteen orthologs were identified in the corresponding regions in rice and Brachypodium, respectively. Interestingly, five pseudogenes were identified among the non-collinear coding sequences at the 3B locus, while none was found at the 3DS locus. CONCLUSION: Direct comparison of two Mb-sized regions of the B and D genomes of bread wheat revealed similar rates of non-collinear gene insertion in both genomes with a majority of gene duplications occurring before their divergence. Relatively low proportion of pseudogenes was identified among non-collinear coding sequences. Our data suggest that the pseudogenes did not originate from insertion of non-functional copies, but were formed later during the evolution of hexaploid wheat. Some evidence was found for gene erosion along the B genome locus.

• MeSH
• Brachypodium genetics   MeSH
• Chromosomes, Plant genetics   MeSH
• DNA, Plant genetics   MeSH
• Gene Duplication MeSH
• Phylogeny MeSH
• Genetic Loci genetics   MeSH
• Genome, Plant genetics   MeSH
• Mutagenesis, Insertional MeSH
• Contig Mapping MeSH
• Evolution, Molecular * MeSH
• Polyploidy MeSH
• Triticum genetics   MeSH
• Pseudogenes genetics   MeSH
• Oryza genetics   MeSH
• Sequence Analysis, DNA MeSH
• Chromosomes, Artificial, Bacterial MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

• MeSH
• Genetics MeSH

• Conspectus
• Obecná genetika. Obecná cytogenetika. Evoluce
• NML Fields
• genetika, lékařská genetika

Cystatin C /CysC/ je nízkomolekulární neglykovaný bazický protein, jehož hladina je řízena konstantní produkcí jadernými buňkami a glomerulární filtrací /GFR/. Je reabsorbován a degradován v pars convoluta proximálního tubulu a nemá extraglomerulární sekreci. CysC proto představuje citlivý marker GFR, jehož hladina není ovlivněna věkem, pohlavím ani svalovou hmotou, a při jeho stanovení nedochází k analytickým interferencím. Cílem práce bylo zjistit vztah mezi sérovou hladinou CysC a kreatininu /S-cr/ a mezi hladinou CysC a clearencí endogenního kreatininu /C-cr/ u diabetiků s nefropatií a prokázat, že zvláště v počátečních stadiích nefropatie je CysC citlivý marker stupně GFR. CysC byl stanovován z ranního odběru krve soupravou Cystatin C PET Kit Code K 0071 fy DAKO A/S na automatickém analyzátoru Konelab K 60. Na témže přístroji a ze stejného vzorku krve byly stanovovány hladiny kreatininu. Soubor tvořilo 41 diabetiků, 20 žen a 21 mužů, v průměrném věku 64,5 ±15,9 let. Kontrolní soubor představovalo 58 nediabetiků s nefropatií, 32 žen a 26 mužů, v průměrném věku 60,6 ±14,3 let. Při srovnání hladiny CysC a S-cr byla u diabetiků zjištěna významná kladná korelace (85 %), nebyl zjištěn významný rozdíl ve srovnání s nediabetiky (korelační koeficient 88 %). Významná záporná korelace byla naopak zjištěna při srovnání hladiny CysC a hodnoty C- cr a opět nebyl nalezen významný rozdíl mezi souborem diabetiků (79 %) a nediabetiků (77 %). Při srovnání hla-diny S-cr a CysC při počínajícím snížení GFR (C-cr 1,0-1,20 ml/s) byla častěji nalezen zvýšený CysC při ještě normální hodnotě S-cr, ale soubor těchto pacientů není zatím dostatečně reprezentativní. Závěr: 1) CysC dobře koreluje s hodnotou S-cr i C-cr, 2) nebyly zjištěny signifikantní rozdíly v korelaci mezi diabetiky a nediabetiky, 3) stanovení CysC je vhodná metoda pro posouzení GFR, na rozdíl od stanovení S-cr a C-cr není ovlivněna potravou, extraglomerulární sekrecí, přesností sběru moče a analytickými interferencemi.

• MeSH
• Cystatins blood   MeSH
• Diabetic Nephropathies metabolism   MeSH
• Glomerular Filtration Rate MeSH
• Humans MeSH
• Check Tag
• Humans MeSH
• Publication type
• Congress MeSH