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Articles by Irina V. Getun in JoVE
Purificação de Citometria de Fluxo de células de camundongo meiótica
Irina V. Getun1, Bivian Torres2, Philippe R.J. Bois1
1Genome Plasticity Laboratory, Department of Cancer Biology, The Scripps Research Institute, 2Flow Cytometry Core, The Scripps Research Institute
Um método eficiente para obter altamente purificada viável frações meiótica de testículo do rato é descrito, que combina uma refinada célula dissociação protocolo com fluorescentes separação de células ativadas (FACS). Este método tira vantagem de diferenças no conteúdo de DNA nuclear e densidade de frações discretas meiótica.
Other articles by Irina V. Getun on PubMed
Native-like Conformations Are Sampled by Partially Folded and Disordered Variants of Bovine Pancreatic Trypsin Inhibitor
Biochemistry. Feb, 2004 | Pubmed ID: 14769035
Partially folded conformational ensembles of bovine pancreatic trypsin inhibitor (BPTI) are accessed by replacing Cys 5, 30, 51, and 55 by alpha-amino-n-butyric acid (Abu) while retaining the disulfide between Cys 14 and 38; the resultant variant is termed [14-38](Abu). Two new analogues with modifications in the beta-turn, P26D27[14-38](Abu) and N26G27K28[14-38](Abu), are compared to partially folded [14-38](Abu), as well as to [R](Abu), the unfolded protein with all six Cys residues replaced by Abu. Structural features of the new analogues of [14-38](Abu) have been determined by circular dichroism (CD), one-dimensional (1)H NMR, and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence experiments. Both analogues are more disordered than the parent [14-38](Abu), but while P26D27[14-38](Abu) has a small population of native-like conformations observed by NMR, no ordered structure is detected for N26G27K28[14-38](Abu). Trypsin inhibition assays were carried out using a modified rat trypsin, C191A/C220A, that minimizes cleavage of unfolded peptides. Both [14-38](Abu) and P26D27[14-38](Abu) significantly inhibit modified trypsin. N26G27K28[14-38](Abu) has low but measurable inhibitor activity, while [R](Abu) has no activity even when in very high molar excess relative to trypsin. ANS fluorescence is enhanced by [14-38](Abu) and by both variants but not by [R](Abu). We conclude that partially folded ensembles of BPTI, even those with little or no CD- or NMR-detectable structure, contain minor populations of native-like conformations. Partially folded [14-38](Abu) and both variants, as well as [R](Abu), have enhanced negative ellipticity in CD spectra acquired in the presence of the osmolyte trimethylamine N-oxide (TMAO). TMAO-induced structure is formed cooperatively, as indicated by thermal unfolding curves. Inhibitor activity as a function of TMAO concentration implies that the osmolyte-induced structure is native-like for [14-38](Abu) and P26D27[14-38](Abu) and is probably native-like for N26G27K28[14-38](Abu). [R](Abu) also shows increased CD-detected structure in the presence of TMAO, but such structure is likely to be collapsed and non-native.
Partially Folded Bovine Pancreatic Trypsin Inhibitor Analogues Attain Fully Native Structures when Co-crystallized with S195A Rat Trypsin
Journal of Molecular Biology. Jan, 2008 | Pubmed ID: 18054043
Crystal structures, at 1.7 A resolution, were solved for complexes between each of two chemically synthesized partially folded analogues of bovine pancreatic trypsin inhibitor (BPTI) with the proteolytically inactive rat trypsin mutant S195A. The BPTI analogue termed [14-38](Abu) retains only the disulfide bond between Cys14 and Cys38, while Cys5, Cys30, Cys51, and Cys55 are replaced by isosteric alpha-amino-n-butyric acid residues. The analogue K26P,A27D[14-38](Abu) contains two further replacements, by statistically favored residues, in the type I beta-turn that has been suggested to be a main site for initiation of BPTI folding. As a control, the structure of the complex between S195A trypsin and wild-type BPTI was also solved. Despite significant differences in the degree of structure detected among these three BPTIs in solution by several biophysical techniques, their tertiary folds once bound to S195A trypsin in a crystalline lattice are essentially superimposable.
Nucleic Acids Research. Apr, 2010 | Pubmed ID: 20081202
Genome-wide analyses have suggested thousands of meiotic recombination hot spots across mammalian genomes. However, very few hot spots have been directly analyzed at a sub-kb scale for crossover (CO) activity. Using recombinant inbred strains as a CO library, here we report the identification and detailed characterization of seven new meiotic hot spots on mouse chromosome 19, more than doubling the number of currently available mouse hot spots. Although a shared feature is the narrow 1.5-2.5-kb width of these recombinogenic sites, these analyses revealed that hot spots have diverse sequence attributes and distinct symmetric and asymmetric CO profiles. Interestingly, CO molecules with discontinuous conversion tracts are commonly observed, contrasting with those found in human. Furthermore, unlike human hot spots, those present in the mouse do not necessarily have a quasi-normal CO distribution but harbor CO repulsion zones within recombinogenic cores. We propose a model where local chromatin landscape directs these repulsion zones.
EMBO Reports. Jul, 2010 | Pubmed ID: 20508641
During meiosis, paternal and maternal homologous chromosomes recombine at specific recombination sites named hotspots. What renders 2% of the mammalian genomes permissive to meiotic recombination by allowing Spo11 endonuclease to initiate double-strand breaks is largely unknown. Work in yeast has shown that chromatin accessibility seems to be important for this activity. Here, we define nucleosome profiles and dynamics at four mouse recombination hotspots by purifying highly enriched fractions of meiotic cells. We found that nucleosome occupancy is generally stable during meiosis progression. Interestingly, the cores of recombination hotspots have largely open chromatin structure, and the localization of the few nucleosomes present in these cores correlates precisely with the crossover-free zones in recombinogenic domains. Collectively, these high-resolution studies suggest that nucleosome occupancy seems to direct, at least in part, how meiotic recombination events are processed.