RESUMO
BACKGROUNDS: Despite recent advances, many cancers are still detected too late for curative treatment. There is, therefore, a need for the development of new diagnostic methods and biomarkers. One approach may arise from the detection of extrachromosomal circular DNA (eccDNA), which is part of cell-free DNA in human plasma. AIMS: First, we assessed and compared two methods for the purification of eccDNA from plasma. Second, we tested for an easy diagnostic application of eccDNA liquid biopsy-based assays. MATERIALS & METHODS: For the comparison we tested a solid-phase silica purification method and a phenol/chloroform method with salt precipitation. For the diagnostic application of eccDNA we developed and tested a qPCR primer-based SNP detection system, for the detection of two well-established cancer-causing KRAS mutations (G12V and G12R) on circular DNA. This investigation was supported by purifying, sequencing, and analysing clinical plasma samples for eccDNAs containing KRAS mutant alleles in 0.5 mL plasma from 16 pancreatic ductal adenocarcinoma patients and 19 healthy controls. RESULTS: In our method comparison we observed, that following exonuclease treatment a lower eccDNA yield was found for the phenol/chloroform method (15.7%-26.7%) compared with the solid-phase purification approach (47.8%-65.9%). For the diagnostic application of eccDNA tests, the sensitivity of the tested qPCR assay only reached ~10-3 in a background of 105 wild type (wt) KRAS circular entities, which was not improved by general amplification or primer-based inhibition of wt KRAS amplification. Furthermore, we did not detect eccDNA containing KRAS in any of the clinical samples. DISCUSSION: A potential explanation for our inability to detect any KRAS mutations in the clinical samples may be related to the general low abundance of eccDNA in plasma. CONCLUSION: Taken together our results provide a benchmark for eccDNA purification methods while raising the question of what is required for the optimal fast and sensitive detection of SNP mutations on eccDNA with greater sensitivity than primer-based qPCR detection.
RESUMO
Bioconversion of hemicelluloses into simpler sugars leads to the production of a significant amount of pentose sugars, such as d-xylose. However, efficient utilization of pentoses by conventional yeast production strains remains challenging. Wild yeast strains can provide new industrially relevant characteristics and efficiently utilize pentose sugars. To explore this strategy, we isolated gut-residing yeasts from the termite Macrotermes bellicosus collected in Comoé National Park, Côte d'Ivoire. The yeasts were classified through their Internal Transcribed Spacer/Large Subunit sequence, and their genomes were sequenced and annotated. We identified a novel yeast species, which we name Barnettozyma botsteinii sp. nov. 1118T (MycoBank: 833563, CBS 16679T and IBT 710) and two new strains of Kurtzmaniella quercitrusa: var. comoensis (CBS 16678, IBT 709) and var. filamentosus (CBS 16680, IBT 711). The two K. quercitrusa strains grow 15% faster on synthetic glucose medium than Saccharomyces cerevisiae CEN.PKT in acidic conditions (pH = 3.2) and both strains grow on d-xylose as the sole carbon source at a rate of 0.35 h-1. At neutral pH, the yeast form of K. quercitrusa var. filamentosus, but not var. comoensis, switched to filamentous growth in a carbon source-dependent manner. Their genomes are 11.0-13.2 Mb in size and contain between 4888 and 5475 predicted genes. Together with closely related species, we did not find any relationship between gene content and ability to grow on xylose. Besides its metabolism, K. quercitrusa var. filamentosus has a large potential as a production organism, because of its capacity to grow at low pH and to undergo a dimorphic shift.
Assuntos
Isópteros , Animais , DNA Fúngico , Isópteros/genética , Técnicas de Tipagem Micológica , Filogenia , Análise de Sequência de DNARESUMO
Circular DNA can arise from all parts of eukaryotic chromosomes. In yeast, circular ribosomal DNA (rDNA) accumulates dramatically as cells age, however little is known about the accumulation of other chromosome-derived circles or the contribution of such circles to genetic variation in aged cells. We profiled circular DNA in Saccharomyces cerevisiae populations sampled when young and after extensive aging. Young cells possessed highly diverse circular DNA populations but 94% of the circular DNA were lost after â¼15 divisions, whereas rDNA circles underwent massive accumulation to >95% of circular DNA. Circles present in both young and old cells were characterized by replication origins including circles from unique regions of the genome and repetitive regions: rDNA and telomeric Y' regions. We further observed that circles can have flexible inheritance patterns: [HXT6/7circle] normally segregates to mother cells but in low glucose is present in up to 50% of cells, the majority of which must have inherited this circle from their mother. Interestingly, [HXT6/7circle] cells are eventually replaced by cells carrying stable chromosomal HXT6 HXT6/7 HXT7 amplifications, suggesting circular DNAs are intermediates in chromosomal amplifications. In conclusion, the heterogeneity of circular DNA offers flexibility in adaptation, but this heterogeneity is remarkably diminished with age.
Assuntos
Senescência Celular/genética , Replicação do DNA , DNA Circular/química , Saccharomyces cerevisiae/genética , DNA Circular/análise , Variação Genética , Padrões de Herança , Proteínas de Transporte de Monossacarídeos/genética , Sequências Repetitivas de Ácido Nucleico , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Amino acids can induce yeast cell adhesion but how amino acids are sensed and signal the modulation of the FLO adhesion genes is not clear. We discovered that the budding yeast Saccharomyces cerevisiae CEN.PK evolved invasive growth ability under prolonged nitrogen limitation. Such invasive mutants were used to identify amino acid transporters as regulators of FLO11 and invasive growth. One invasive mutant had elevated levels of FLO11 mRNA and a Q320STOP mutation in the SFL1 gene that encodes a protein kinase A pathway regulated repressor of FLO11. Glutamine-transporter genes DIP5 and GNP1 were essential for FLO11 expression, invasive growth and biofilm formation in this mutant. Invasive growth relied on known regulators of FLO11 and the Ssy1-Ptr3-Ssy5 complex that controls DIP5 and GNP1, suggesting that Dip5 and Gnp1 operates downstream of the Ssy1-Ptr3-Ssy5 complex for regulation of FLO11 expression in a protein kinase A dependent manner. The role of Dip5 and Gnp1 appears to be conserved in the S. cerevisiae strain ∑1278b since the dip5 gnp1 ∑1278b mutant showed no invasive phenotype. Secondly, the amino acid transporter gene GAP1 was found to influence invasive growth through FLO11 as well as other FLO genes. Cells carrying a dominant loss-of-function PTR3(647::CWNKNPLSSIN) allele had increased transcription of the adhesion genes FLO1, 5, 9, 10, 11 and the amino acid transporter gene GAP1. Deletion of GAP1 caused loss of FLO11 expression and invasive growth. However, deletions of FLO11 and genes encoding components of the mitogen-activated protein kinase pathway or the protein kinase A pathway were not sufficient to abolish invasive growth, suggesting involvement of other FLO genes and alternative pathways. Increased intracellular amino acid pools in the PTR3(647::CWNKNPLSSIN)-containing strain opens the possibility that Gap1 regulates the FLO genes through alteration of the amino acid pool sizes.