组蛋白不同乙酰化位点突变对酵母细胞生长和Ssa3、Gal1基因表达的影响

组蛋白乙酰化对基因表达和细胞生长非常重要.为揭示组蛋白H3K14和H4K8的乙酰化修饰对不同条件下细胞生长和Ssa3、Gal1基因表达的重要性及二者功能差异.构建了H3K14、H4K8分别突变为精氨酸的单突变株S14、S8及二者同时突变的双突变株D814,并对其在正常、高温、咖啡因存在等条件下生长及Ssa3、Gal1表达进行比较.结果表明,所有突变株对咖啡因敏感性增加;D814对温度敏感,且在供试条件下其生长及Ssa3和Gal1激活均明显慢于野生型和单突变株;除半乳糖和葡萄糖为单一碳源,30℃时两单突变株差别不大外,其它条件下S8生长及Ssa3和Gal1激活均慢于S14.表明H3K14、H4K8乙酰化对细胞生长和适应不利环境非常重要,而且在对不利条件的快速适应方面,H4K8的乙酰化修饰可能更为重要.组蛋白突变株的表型缺陷是因该条件下细胞生存所必需的基因激活延迟所致.     

Dose and Time‐Dependent Effects of Cyanide on Thiosulfate Sulfurtransferase, 3‐Mercaptopyruvate Sulfurtransferase,and Cystathionine λ‐Lyase Activities

We assessed the dose‐dependent effect of potassium cyanide (KCN) on thiosulfate sulfurtransferase (TST), 3‐mercaptopyruvate sulfurtransferase (3‐MPST), and cystathionine λ‐lyase (CST) activities in mice. The time‐dependent effect of 0.5 LD50 KCN on cyanide level and cytochrome c oxidase (CCO), TST, 3‐MPST, and CST activities was also examined. Furthermore, TST, 3‐MPST, and CST activities were measured in stored mice cadavers. Hepatic and renal TST activity increased by 0.5 LD50 KCN but diminished by ≥2.0 LD50. After 0.5 LD50 KCN, the elevated hepatic cyanide level was accompanied by increased TST, 3‐MPST, and CST activities, and CCO inhibition. Elevated renal cyanide level was only accompanied by increased 3‐MPST activity. No appreciable change in enzyme activities was observed in mice cadavers. The study concludes that high doses of cyanide exert saturating effects on its detoxification enzymes, indicating their exogenous use during cyanide poisoning. Also, these enzymes are not reliable markers of cyanide poisoning in autopsied samples.     

Positioning of a Nucleosome on Mouse Satellite DNA Inserted into a Yeast Plasmid Is Determined by Its DNA Sequence and an Adjacent Nucleosome

It has earlier been shown that multiple positioning of nucleosomes on mouse satellite DNA is determined by its nucleotide sequence. To clarify whether other factors, such as boundary ones, can affect the positionings, we modified the environment of satellite DNA monomer by inserting it into a yeast plasmid between inducible GalCyc promoter and a structural region of the yeast FLP gene. We have revealed that the positions of nucleosomes on satellite DNA are identical to those detected upon reconstruction in vitro. The positioning signal (GAAAAA sequence) of satellite DNA governs nucleosome location at the adjacent nucleotide sequence as well. Upon promoter induction the nucleosome, translationally positioned on the GalCyc promoter, transfers to the satellite DNA and its location follows the positioning signal of the latter. Thus, the alternatives of positioning of a nucleosome on satellite DNA are controlled by its nucleotide sequence, though the choice of one of them is determined by the adjacent nucleosome.     

Spc98p Directs the Yeast γ-Tubulin Complex into the Nucleus and Is Subject to Cell Cycle-dependent Phosphorylation on the Nuclear Side of the Spindle Pole Body

In the yeast Saccharomyces cerevisiae, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope. Microtubule organization requires the γ-tubulin complex containing the γ-tubulin Tub4p, Spc98p, and Spc97p. The Tub4p complex is associated with cytoplasmic and nuclear substructures of the SPB, which organize the cytoplasmic and nuclear microtubules. Here we present evidence that the Tub4p complex assembles in the cytoplasm and then either binds to the cytoplasmic side of the SPB or is imported into the nucleus followed by binding to the nuclear side of the SPB. Nuclear import of the Tub4p complex is mediated by the essential nuclear localization sequence of Spc98p. Our studies also indicate that Spc98p in the Tub4p complex is phosphorylated at the nuclear, but not at the cytoplasmic, side of the SPB. This phosphorylation is cell cycle dependent and occurs after SPB duplication and nucleation of microtubules by the new SPB and therefore may have a role in mitotic spindle function. In addition, activation of the mitotic checkpoint stimulates Spc98p phosphorylation. The kinase Mps1p, which functions in SPB duplication and mitotic checkpoint control, seems to be involved in Spc98p phosphorylation. Our results also suggest that the nuclear and cytoplasmic Tub4p complexes are regulated differently.     

Insertion of the cytochrome b5 heme-binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

Under native conditions, apocytochrome b(5) exhibits a stable core and a disordered heme-binding region that refolds upon association with the cofactor. The termini of this flexible region are in close proximity, suggesting that loop closure may contribute to the thermodynamic properties of the apocytochrome. A chimeric protein containing 43 residues encompassing the cytochrome loop was constructed using the cyanobacterial photosystem I accessory protein E (PsaE) from Synechococcus sp. PCC 7002 as a structured scaffold. PsaE has the topology of an SH3 domain, and the insertion was engineered to replace its 14-residue CD loop. NMR and optical spectroscopies showed that the hybrid protein (named EbE1) was folded under native conditions and that it retained the characteristics of an SH3 domain. NMR spectroscopy revealed that structural and dynamic differences were confined near the site of loop insertion. Variable-temperature 1D NMR spectra of EbE1 confirmed the presence of a kinetic unfolding barrier. Thermal and chemical denaturations of PsaE and EbE1 demonstrated cooperative, two-state transitions; the stability of the PsaE scaffold was found only moderately compromised by the insertion, with a DeltaT(m) of 8.3 degrees C, a DeltaC(m) of 1.5 M urea, and a DeltaDeltaG degrees of 4.2 kJ/mole. The data implied that the penalty for constraining the ends of the inserted region was lower than the approximately 6.4 kJ/mole calculated for a self-avoiding chain. Extrapolation of these results to cytochrome b(5) suggested that the intrinsic stability of the folded portion of the apoprotein reflected only a small detrimental contribution from the large heme-binding domain.