Biology subjects

Fraczek, M.

Publications and source records attributed to Fraczek, M..

3 recordsLinked to original sources

bioRxiv2025

Impact of Sperm Fractionation on Chromosome Positioning, Chromatin Integrity, DNA Methylation and Hydroxymethylation Level

BackgroundSperm chromosomes are non-randomly organized in the cell nucleus, which plays an important role in the regulation of early embryo development. It is determined by the specific localisation of sperm chromosomal regions carrying genes with expression crucial at the first contact with ooplasm during fertilization. Thus, the aim of this study was to determine whether the application of selective methods providing high-quality spermatozoa with good motility and/or morphology, may increase the frequency of gametes with the specific positioning of chromosomes. For the first time we have used the sequential staining algorithm for consecutive analyses of the same individual sperm cells with a fixed position, what enables to achieve a full and detailed documentation at the single cell level. MethodsSemen samples from 5 normozoospermic males were collected and processed for fractionation via swim up (to select viable and motile spermatozoa) or Percoll density gradient (90/47%; for good sperm motility and morphology). Sperm chromatin protamination was assessed by aniline blue (AB) staining, while DNA fragmentation by acridine orange (AO) (ssDNA fragmentation) or TUNEL assay (ssDNA and dsDNA fragmentation). Then, sequential staining and analyses of the same individual sperm cell with a fixed position on a slide were performed, in the following order: (i) fluorescence in situ hybridization (FISH) for determination of positioning of chromosomal centromeres: 4, 7, 8, 9, 18, X and Y, with so-called: linear and radial estimations applied, followed by distance measurements between selected pairs of chromosomes; and (ii) immunofluorescent (IF) measurement of global sperm DNA methylation (5mC) and hydroxymethylation (5hmC) levels, what added additional data about epigenetic layer of the sperm chromosomes positioning. ResultsOur study demonstrated that high-quality sperm selection methods significantly: (i) increased frequency of spermatozoa with good chromatin protamination (+[~]25%) and 5mC and 5hmC DNA levels (+[~]9.5%), and (ii) reduced rate of spermatozoa with ssDNA fragmentation (-[~]65%). Motile and morphologically normal spermatozoa showed distinct chromosome repositioning with sex chromosomes shifted to the nuclear periphery, a key chromosomal region of the initial interaction with the ooplasm during fertilization process. Evaluated autosomes revealed various patterns of repositioning. Conclusions Our findings underline the validity of methods used for selection of high-quality spermatozoa in assisted reproductive technologies (ART), also in the context of the sperm chromosomal topology and chromatin integrity, crucial at the first steps during fertilization.

genetics↗

bioRxiv2022

Telomere to telomere sequence of model Aspergillus fumigatus genomes

The pathogenic fungus Aspergillus fumigatus is a major etiological agent of fungal invasive and chronic diseases affecting tens of millions of individuals worldwide. A high-quality reference genome is a fundamental resource to study its biology, pathogenicity and virulence as well as to discover better and more effective treatments against diseases caused by this fungus. Here, we used PacBio Single Molecule Real-Time (SMRT) and Oxford Nanopore sequencing for de novo genome assembly of two laboratory reference strains of A. fumigatus,CEA10 and A1160. We generated full length chromosome assemblies and a comprehensive telomere to telomere coverage for these two strains including ribosomal repeats and the sequences of centromeres, which we discovered to be composed of long transposon elements.

genomics↗

bioRxiv2022

Analysis of synthetic genetic interactions of non-coding RNAs in Saccharomyces cerevisiae

Non-coding RNAs (ncRNAs) with unknown function have been found to be responsible for fitness changes in yeast. With the help of synthetic genetic array (SGA) methodology, genetic interaction screenings between protein-coding genes have contributed to the understanding of complex genetic pathways in eukaryotes. By extending this approach to the ncRNAs, we created more than 15,000 ncRNA double mutants and scored their epistasis in different environments. More than 90% of ncRNA interactions did not correlate with the epistasis of their neighbouring protein-coding genes, showing that the ncRNA and protein interaction networks are largely independent. In the presence of different stressors, epistatic changes were observed with an overall increase in negative interactions. Such network plasticity supports the notion that these ncRNAs may have environmentally dependent functions and can contribute to phenotypic adaptation. The U3 paralogs, SNR17A and SNR17B, share most interactions in rich media, as expected, however, in stressful conditions, there was an increase of paralog-unique interactions, suggesting that SNR17A and SNR17B may have diverged cellular roles under different environmental pressures and may have neo-functionalised after genome duplication. Further investigation of the interaction networks between SNR17 and protein-coding genes revealed the emergence of paralog-specific interactions under stress conditions. Gene Ontology enrichment analysis suggested a novel potential role of SNR17B in chromatin structural remodelling and DNA topology under osmotic and oxidative stress. Overall, this genome-wide study uncovers the level of epistasis involving ncRNA mutants aiding functional assignment and expanding our fundamental knowledge of these genetic elements.

genetics↗