Molecular phylogenetic analysis of Tulipa (Liliaceae) from Aksu-Zhabagly Nature Reserve

Barcodes are conserved sequences of genomic, plastid and mitochondrial DNA that can be utilized to uniquely identify an unidentified specimen to its species when conventional identification methods are inapplicable. Among prokaryotic and eukaryotic species, nuclear ribosomal internal transcribed spacer (ITS) sections are one of the most often utilized DNA markers in DNA barcoding and phylogenetic research. In addition to the ribosomal genes, the plastid genes are the most suitable for identifying plant species. The Aksu-Zhabagly Nature Reserve is the oldest nature reserve in Central Asia and is home to 1,312 vascular plant species, 44 of which are categorized as threatened or endangered in Kazakhstan's red data book. In this study, a collection of specimens of uncommon tulip species was compiled, along with their morphological identification and DNA barcoding. The ITS region and parts of the matK and ycf1b genes of tulip plastid DNA were sequenced. The evolutionary link between species of tulips was investigated. Phylogenetic study predicted two Tulipa subclades. Tulipa species have substantially preserved MatK genes. Tulips' ycf1b gene has evolved more slowly than other Liliaceae family members. Nuclear and plastid DNA sequences investigated Tulipa species evolutionary relationships. The findings about the ITS region of nuclear DNA were more definite. Overall, our work shows that genetic data will be important in determining species concepts in this genus, however, even with a molecular perspective pulling apart closely related taxa can be extremely challenging

Isolation of <i>Bacillus</i> sp. A5.3 Strain with Keratinolytic Activity

Environmental safety and economic factors necessitate a search for new ways of processing poultry farm feathers, which are 90% β-keratin and can be used as a cheap source of amino acids and peptones. In this study, feather-decomposing bacteria were isolated from a site of accumulation of rotten feathers and identified as Bacillus. Among them, the Bacillus sp. A5.3 isolate showed the best keratinolytic properties. Scanning electron microscopy indicated that Bacillus sp. A5.3 cells closely adhere to the feather surface while degrading the feather. It was found that Bacillus sp. A5.3 secretes thermostable alkaline proteolytic and keratinolytic enzymes. Zymographic analysis of the enzymatic extract toward bovine serum albumin, casein, gelatin, and β-keratin revealed the presence of proteases and keratinases with molecular weights 20–250 kDa. The proteolytic and keratinolytic enzymes predominantly belong to the serine protease family. Proteome analysis of the secreted proteins by nano-HPLC coupled with Q-TOF mass spectrometry identified 154 proteins, 13 of which are proteases and peptidases. Thus, strain Bacillus sp. A5.3 holds great promise for use in feather-processing technologies and as a source of proteases and keratinases

The major Arabidopsis thaliana apurinic/apyrimidinic endonuclease, ARP is involved in the plant nucleotide incision repair pathway

International audienc

Obtaining of Recombinant Camel Chymosin and Testing Its Milk-Clotting Activity on Cow&rsquo;s, Goat&rsquo;s, Ewes&rsquo;, Camel&rsquo;s and Mare&rsquo;s Milk

In the cheese-making industry, commonly chymosin is used as the main milk-clotting enzyme. Bactrian camel (Camelus bactrianus) chymosin (BacChym) has a milk-clotting activity higher than that of calf chymosin for cow&rsquo;s, goat&rsquo;s, ewes&rsquo;, mare&rsquo;s and camel&rsquo;s milk. A procedure for obtaining milk-clotting reagent based on recombinant camel chymosin is proposed here. Submerged fermentation by a recombinant yeast (Pichia pastoris GS115/pGAPZ&alpha;A/ProchymCB) was implemented in a 50 L bioreactor, and the recombinant camel chymosin was prepared successfully. The activity of BacChym in yeast culture was 174.5 U/mL. The chymosin was concentrated 5.6-fold by cross-flow ultrafiltration and was purified by ion exchange chromatography. The activity of the purified BacChym was 4700 U/mL. By sublimation-drying with casein peptone, the BacChym powder was obtained with an activity of 36,000 U/g. By means of this chymosin, cheese was prepared from cow&rsquo;s, goat&rsquo;s, ewes&rsquo;, camel&rsquo;s and mare&rsquo;s milk with a yield of 18%, 17.3%, 15.9%, 10.4% and 3%, respectively. Thus, the proposed procedure for obtaining a milk-clotting reagent based on BacChym via submerged fermentation by a recombinant yeast has some prospects for biotechnological applications. BacChym could be a prospective milk-clotting enzyme for different types of milk and their mixtures

Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways

International audienceOxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O$_2$. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) $Nucleic$ $Acids$ $Res.$ 40, 2506-2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3'-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins

Aberrant repair initiated by the adenine-DNA glycosylase does not play a role in UV-induced mutagenesis in Escherichia coli

International audienceBackground: DNA repair is essential to counteract damage to DNA induced by endo-and exogenous factors, to maintain genome stability. However, challenges to the faithful discrimination between damaged and non-damaged DNA strands do exist, such as mismatched pairs between two regular bases resulting from spontaneous deamination of 5-methylcytosine or DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved the mismatch-specific DNA glycosylases that can recognize and remove regular DNA bases in the mismatched DNA duplexes. The Escherichia coli adenine-DNA glycosylase (MutY/MicA) protects cells against oxidative stress-induced mutagenesis by removing adenine which is mispaired with 7,8-dihydro-8-oxoguanine (8oxoG) in the base excision repair pathway. However, MutY does not discriminate between template and newly synthesized DNA strands. Therefore the ability to remove A from 8oxoG•A mispair, which is generated via misincorporation of an 8-oxo-2′-deoxyguanosine-5′-triphosphate precursor during DNA replication and in which A is the template base, can induce A•T/C•G transversions. Furthermore, it has been demonstrated that human MUTYH, homologous to the bacterial MutY, might be involved in the aberrant processing of ultraviolet (UV) induced DNA damage. Methods: Here, we investigated the role of MutY in UV-induced mutagenesis in E. coli. MutY was probed on DNA duplexes containing cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproduct (6-4PP). UV irradiation of E. coli induces Save Our Souls (SOS) response characterized by increased production of DNA repair enzymes and mutagenesis. To study the role of MutY in vivo, the mutation frequencies to rifampicin-resistant (RifR) after UV irradiation of wild type and mutant E. coli strains were measured. Results: We demonstrated that MutY does not excise Adenine when it is paired with CPD and 6–4PP adducts in duplex DNA. At the same time, MutY excises Adenine in A•G and A•8oxoG mispairs. Interestingly, E. coli mutY strains, which have elevated spontaneous mutation rate, exhibited low mutational induction after UV exposure as compared to MutY-proficient strains. However, sequence analysis of RifR mutants revealed that the frequencies of C/T transitions dramatically increased after UV irradiation in both MutY-proficient and -deficient E. coli strains. Discussion: These findings indicate that the bacterial MutY is not involved in the aberrant DNA repair of UV-induced DNA damag

Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis

International audienc

Mechanism of stimulation of DNA binding of the transcription factors by human apurinic/apyrimidinic endonuclease 1, APE1

International audienc