Nuclease
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A rapid test for the detection of staphylococcal thermostable nuclease (TNase) is described. The procedure consists of heat inactivation of solid cultures of staphylococci and microslide agar diffusion in toluidine blue agar containing deoxyribonucleic acid. Using this method the results are obtained about 1 d sooner than with the conventional method.
The single-strand-specific S1 nuclease from Aspergillus oryzae is an archetypal enzyme of the S1-P1 family of nucleases with a widespread use for biochemical analyses of nucleic acids. We present the first X-ray structure of this nuclease along with a thorough analysis of the reaction and inhibition mechanisms and of its properties responsible for identification and binding of ligands. Seven structures of S1 nuclease, six of which are complexes with products and inhibitors, and characterization of catalytic properties of a wild type and mutants reveal unknown attributes of the S1-P1 family. The active site can bind phosphate, nucleosides, and nucleotides in several distinguished ways. The nucleoside binding site accepts bases in two binding modes-shallow and deep. It can also undergo remodeling and so adapt to different ligands. The amino acid residue Asp65 is critical for activity while Asn154 secures interaction with the sugar moiety, and Lys68 is involved in interactions with the phosphate and sugar moieties of ligands. An additional nucleobase binding site was identified on the surface, which explains the absence of the Tyr site known from P1 nuclease. For the first time ternary complexes with ligands enable modeling of ssDNA binding in the active site cleft. Interpretation of the results in the context of the whole S1-P1 nuclease family significantly broadens our knowledge regarding ligand interaction modes and the strategies of adjustment of the enzyme surface and binding sites to achieve particular specificity.
- MeSH
- aminokyseliny metabolismus MeSH
- Aspergillus oryzae enzymologie metabolismus MeSH
- endonukleasy specifické pro jednořetězcové nukleové kyseliny metabolismus MeSH
- fungální proteiny metabolismus MeSH
- katalytická doména fyziologie MeSH
- katalýza MeSH
- kinetika MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- substrátová specifita MeSH
- vazebná místa fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases). SHR ova were microinjected with constructs pTAL438/439 coding for a sequence-specific endonuclease that binds to target sequence in the first coding exon of the Plzf gene. Out of 43 animals born after microinjection, we detected a single male founder. Sequence analysis revealed a deletion of G that resulted in frame shift mutation starting in codon 31 and causing a premature stop codon at position of amino acid 58. The Plzftm1Ipcv allele is semi-lethal since approximately 95% of newborn homozygous animals died perinatally. All homozygous animals exhibited manifestations of a caudal regression syndrome including tail anomalies and serious size reduction and deformities of long bones, and oligo- or polydactyly on the hindlimbs. The heterozygous animals only exhibited the tail anomalies. Impaired development of the urinary tract was also revealed: one homozygous and one heterozygous rat exhibited a vesico-ureteric reflux with enormous dilatation of ureters and renal pelvis. In the homozygote, this was combined with a hypoplastic kidney. These results provide evidence for the important role of Plzf gene during development of the caudal part of a body-column vertebrae, hindlimbs and urinary system in the rat.
- MeSH
- alely MeSH
- DNA vazebné proteiny nedostatek genetika metabolismus MeSH
- exony MeSH
- genotyp MeSH
- genový targeting MeSH
- heterozygot MeSH
- homozygot MeSH
- krysa rodu rattus MeSH
- lokus kvantitativního znaku MeSH
- mnohočetné abnormality genetika patologie veterinární MeSH
- ocas abnormality MeSH
- polydaktylie genetika patologie veterinární MeSH
- posunová mutace MeSH
- potkani inbrední SHR MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- TALENs genetika metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The endonuclease TBN1 from Solanum lycopersicum (tomato) was expressed in Nicotiana benthamiana leaves and purified with suitable quality and in suitable quantities for crystallization experiments. Two crystal forms (orthorhombic and rhombohedral) were obtained and X-ray diffraction experiments were performed. The presence of natively bound Zn2+ ions was confirmed by X-ray fluorescence and by an absorption-edge scan. X-ray diffraction data were collected from the orthorhombic (resolution of 5.2 Å) and rhombohedral (best resolution of 3.2 Å) crystal forms. SAD, MAD and MR methods were applied for solution of the phase problem, with partial success. TBN1 contains three Zn2+ ions in a similar spatial arrangement to that observed in nuclease P1 from Penicillium citrinum.
- MeSH
- deoxyribonukleasy chemie genetika MeSH
- ionty chemie MeSH
- konformace proteinů MeSH
- krystalizace MeSH
- krystalografie rentgenová MeSH
- molekulární sekvence - údaje MeSH
- rekombinantní proteiny chemie genetika MeSH
- rostlinné proteiny chemie genetika MeSH
- Solanum lycopersicum chemie genetika MeSH
- zinek chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Approximately 35 % of the mouse genes are indispensable for life, thus, global knock-out (KO) of those genes may result in embryonic or early postnatal lethality due to developmental abnormalities. Several KO mouse lines are valuable human disease models, but viable homozygous mutant mice are frequently required to mirror most symptoms of a human disease. The site-specific gene editing systems, the transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs) and the clustered regularly interspaced short palindrome repeat-associated Cas9 nuclease (CRISPR/Cas9) made the generation of KO mice more efficient than before, but the homozygous lethality is still an undesired side-effect in case of many genes. The literature search was conducted using PubMed and Web of Science databases until June 30th, 2020. The following terms were combined to find relevant studies: "lethality", "mice", "knock-out", "deficient", "embryonic", "perinatal", "rescue". Additional manual search was also performed to find the related human diseases in the Online Mendelian Inheritance in Man (OMIM) database and to check the citations of the selected studies for rescuing methods. In this review, the possible solutions for rescuing human disease-relevant homozygous KO mice lethal phenotypes were summarized.
- MeSH
- CRISPR-Cas systémy genetika MeSH
- editace genu metody MeSH
- fenotyp MeSH
- myši knockoutované MeSH
- myši MeSH
- nukleasy s motivem zinkových prstů genetika MeSH
- TALENs genetika MeSH
- ztráta embrya genetika prevence a kontrola MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
