1,N 6 -α-hydroxypropanoadenine, the acrolein adduct to adenine, is a substrate for AlkB dioxygenase

1,N6-α-hydroxypropanoadenine (HPA) is an exocyclic DNA adduct of acrolein – an environmental pollutant and endocellular oxidative stress product. Escherichia coli AlkB dioxygenase belongs to the superfamily of α-ketoglutarate (αKG)- and iron-dependent dioxygenases which remove alkyl lesions from bases via an oxidative mechanism, thereby restoring native DNA structure. Here, we provide in vivo and in vitro evidence that HPA is mutagenic and is effectively repaired by AlkB dioxygenase. HPA generated in plasmid DNA caused A → C and A → T transversions and, less frequently, A → G transitions. The lesion was efficiently repaired by purified AlkB protein; the optimal pH, Fe(II), and αKG concentrations for this reaction were determined. In vitro kinetic data show that the protonated form of HPA is preferentially repaired by AlkB, albeit the reaction is stereoselective. Moreover, the number of reaction cycles carried out by an AlkB molecule remains limited. Molecular modeling of the T(HPA)T/AlkB complex demonstrated that the R stereoisomer in the equatorial conformation of the HPA hydroxyl group is strongly preferred, while the S stereoisomer seems to be susceptible to AlkB-directed oxidative hydroxylation only when HPA adopts the syn conformation around the glycosidic bond. In addition to the biochemical activity assays, substrate binding to the protein was monitored by differential scanning fluorimetry allowing identification of the active protein form, with cofactor and cosubstrate bound, and monitoring of substrate binding. In contrast FTO, a human AlkB homolog, failed to bind an ssDNA trimer carrying HPA

Branchpoint translocation by fork remodelers as a general mechanism of R-loop removal.

Co-transcriptional R loops arise from stalling of RNA polymerase, leading to the formation of stable DNA:RNA hybrids. Unresolved R loops promote genome instability but are counteracted by helicases and nucleases. Here, we show that branchpoint translocases are a third class of R-loop-displacing enzyme in vitro. In cells, deficiency in the Fanconi-anemia-associated branchpoint translocase FANCM causes R-loop accumulation, particularly after treatment with DNA:RNA-hybrid-stabilizing agents. This correlates with FANCM localization at R-loop-prone regions of the genome. Moreover, other branchpoint translocases associated with human disease, such as SMARCAL1 and ZRANB3, and those from lower organisms can also remove R loops in vitro. Branchpoint translocases are more potent than helicases in resolving R loops, indicating their evolutionary important role in R-loop suppression. In human cells, FANCM, SMARCAL1, and ZRANB3 depletion causes additive effects on R-loop accumulation and DNA damage. Our work reveals a mechanistic basis for R-loop displacement that is linked to genome stability

Platysetosus occultus gen. nov., sp. nov., a new genus and species of mite from Tasmania (Acari: Uropodina)

Dylewska, M., Błoszyk, J., Halliday, R. B. (2006): Platysetosus occultus gen. nov., sp. nov., a new genus and species of mite from Tasmania (Acari: Uropodina). Zootaxa 1223: 55-64, DOI: 10.5281/zenodo.17260

Molecular markers used in selection of breeding animals

Selekcja i dobór osobników do kojarzeń jest istotnym etapem pracy hodowlanej. Jednym z najstarszych i najbardziej powszechnych kryteriów decydujących o wyborze zwierząt do dalszych kojarzeń są cechy fenotypowe. W celu osiągnięcia lepszych efektów hodowlanych, klasyczną selekcję coraz częściej uzupełnia się wynikami badań wykorzystującymi markery molekularne powiązane z cechami użytkowymi. Pierwotnie stosowane markery były oparte na polimorfizmie losowo amplifikowanych fragmentów DNA i polimorfizmie długości fragmentów restrykcyjnych. Rozwój technik molekularnych i genomiki pozwoliły na pełniejsze zrozumienie podłoża genetycznego cech użytkowych i tym samym na wprowadzenie bardziej informatywnych technik opartych na polimorfizmie sekwencji mikrosatelitarnych, jak i wielkoskalowych analizach genomowych wielu tysięcy polimorficznych nukleotydów. W pracy przedstawiono najczęściej wykorzystywane markery molekularne, scharakteryzowano zasadę ich oznaczania oraz przedstawiono przykłady ich zastosowania w naukach zootechnicznych.Selection plays a crucial role in animal breeding. The oldest and still used methods of selection of animals to further breeding are often based on the phenotypic traits. This approach allows to improve results of breeding in relatively slowly pace and only in narrow degree. More effective way of obtaining progress is involving of molecular techniques and markers into breeding programs. There are many types of molecular markers associated with traits that are important from the viewpoint of the breeders as well as consumers. The most primary molecular markers are based on the proteins but their effectiveness is not sufficient. Therefore polymorphisms present in the genetic material were consider as a better way of enhancing of breeding results. The milestone in molecular biology was developing of PCR technique. Amplification of genetic material open new possibilities in many branches of science but also industry and agriculture. In this way such techniques as RAPD – Random Amplification of Polymorphic DNA, or RFLP – Restriction Fragment Length Polymorphism, were established. Whereas RAPD is mostly used to analysis of genetic diversity between populations, RFLP technique enabled investigating of association between particular alleles and the productive traits. Constant progress in molecular biology brings even more informative methods from polymorphic microsatellite markers up to high-throughput sequencing revealing whole genomes. Microsatellite polymorphism is based on the STR sequences (Short Tandem Repeats) that exhibit considerable variation between individuals, therefore they are great tool to monitoring of genetic structure of breeding population, designing of breeding programs and protecting against the inbreeding depression. Sequencing techniques are focused on the SNP polymorphism (Single Nucleotide Polymorphism), appearing between genomes. Sanger sequencing is limited to analysis of relatively small sequences, whereas NGS techniques allow to screen whole genomes in searching of polymorphic nucleotides involved in expression of desire traits. Results of sequencing are used to designing and development of SNP panels, that enable simultaneous screening of huge number of polymorphic nucleotide. Modern breeding programs are often supplemented by the results of genomic analysis, that brings meaningful insight into genetic background of such traits as meat quality, milk production or reproduction traits. Constant development of technology is followed by the decreasing of costs and therefore it seems that breeding programs assisted by molecular markers will be more widely introduced into the common usage

Effect of modified starches on texture and meltability of processed cheese analogues

Celem pracy było wyprodukowanie analogów serów topionych, w których kazeinę kwasową częściowo zastąpiono modyfikowaną skrobią z kukurydzy woskowej (CH20) lub skrobią modyfikowaną z tapioki (V60T i VA85T) oraz określenie tekstury i topliwości otrzymanych produktów. Teksturę analogów serów topionych oznaczono za pomocą analizatora tekstury TA-XT2i. Przy użyciu próbnika cylindrycznego o średnicy 15 mm wykonano profilową analizę tekstury (TPA), w której zmierzono przylegalność, spójność i sprężystość analogów serowych. Natomiast w teście przebijania analogów serów topionych zastosowano próbnik cylindryczny o średnicy 10 mm. Pomiary lepkości analogów wykonano za pomocą reometru rotacyjnego Brookfield DV II+ przy użyciu przystawki Helipath (F). Topliwość określono zmodyfikowanym testem Schreibera. Zastosowanie skrobi modyfikowanych wpłynęło na teksturę i topliwość analogów serów topionych. Zwiększenie dodatku skrobi powodowało zwiększenie twardości oraz zmniejszenie topliwości w porównaniu z próbkami kontrolnymi. Nie zaobserwowano istotnych różnic w spójności i sprężystości pomiędzy próbkami kontrolnymi a próbkami zawierającymi modyfikowaną skrobię z kukurydzy woskowej CH20 oraz próbkami z dodatkiem skrobi modyfikowanych z tapioki (z wyjątkiem analogu z 5-procentowym dodatkiem skrobi modyfikowanej z tapioki VA85T). Zdecydowanie największą lepkością spośród wszystkich analogów charakteryzowały się próbki otrzymane z dodatkiem modyfikowanej skrobi z kukurydzy woskowej CH20, a w szczególności analogi z 3- i 4-procentową zawartością tej skrobi. Lepkość próbek z dodatkiem skrobi z tapioki VA85T zmniejszała się w miarę zwiększania w nich zawartości skrobi.The objective of this study was to manufacture processed cheese analogues, in which acid casein was partially replaced by a modified waxy maize starch (CH20) or a modified tapioca starch (V60T and VA85T), and to determine the texture and meltability of the products manufactured. The texture of processed cheese analogues was analysed using a TA-XT2i Texture Analyser. With a cylindrical sampler of 15 mm diameter, a Texture Profile Analysis (TPA) was performed and the adhesiveness, cohesiveness, and springiness of processed cheese analogues were measured. In the puncture test used to analyze the processed cheese analogues, a 10 mm dia cylindrical sampler was applied. The viscosity of the processed cheese analogues was measured using a Brookfield DV II+ rotational viscometer with a Helipath Stand (F). The meltability of the processed cheese analogues was determined using a modified Schreiber test. The application of modified starches impacted the textural properties and meltability of processed cheese analogues. The addition of an increased amount of starch caused the hardness to increase and the meltability to decrease compared to the control samples. No significant differences were reported in the cohesiveness and springiness of the control samples, samples containing the modified waxy-maize starch CH20, and samples with the addition of the modified tapioca starch (except for the analogue with the added 5 % of the modified tapioca starch VA85T). The samples obtained with the addition of the modified waxy-maize starch CH20 were characterized by definitely the highest viscosity of all the analogues, in particular the analogues containing 3 and 4 % of that starch. The viscosity of the samples with the addition of VA85T tapioca starch decreased along with the increasing content of starch therein