Purification and Identification of Uracil and 5-Methylcytosine from Chick Embryo DNA

It was found that minor components were approximately 1% of the total deoxyribosides in the analysis for base composition of chick embryo DNA. In order to identify the minor components, this work was done. The separation of the minor components from the major components in a DNA hydrolysate was carried out by celite column chromatography and the final purification was accomplished by paper chromatography. For detection of minor components at the deoxyriboside level, the microbial assay method with Lactobacillus leichmannii ATCC 7830 was applied. Thus, two minor deoxyribosides of the DNA were identical with deoxyuridine and 5-methyldeoxycytidine in spectral, electrophoretic and chromatographic properties, respectively. Furthermore, uracil and 5-methylcytosine were purified and identified from perchloric acid hydrolysate of the DNA.     

Relationship between Endogenous Factor and Decrease in Color Formation of Low Quality Pork

To clarify the reason for the decrease in CFR (color forming ratio) of pale, soft and exudative (PSE) porcine muscle, the effect of simulation of the PSE conditions on cooked cured meat color was investigated in fractionated components from porcine skeletal muscle, 24 hr postmortem. Although the solubility of total proteins and heme pigments markedly decreased in sarcoplasm by this treatment, no decline in CFR of the sarcoplasm could be observed when compared to the controls. A similar tendency was observed in both high- and low-molecular weight fractions of sarcoplasm. On the other hand, myofibrils simulated the PSE conditions in the presence of myoglobin significantly decreased CFR, and the extent of this decline was nearly consistent with the case of whole muscle. The reasons for the decrease in CFR of PSE muscle are discussed.     

Physiological modelling of the response of Kocuria rosea exposed to changing water activity

Physiological effects of NaCl concentration (equivalent water activities, a_w, 1 to 0.87) were investigated with the moderately halophilic and piezotolerant bacterium, Kocuria rosea (formerly Micrococcus roseus), grown in bacteriological peptone/yeast extract broth. This bacterium, which was isolated from open shallow seawater, can grow in 150 g NaCl l^–1 (optimum NaCl concentration: 30 g l^–1, a_w=0.984) and under 207 MPa of hydrostatic pressure. The effects of water activity on _m can be quantitatively predicted, to a high level of accuracy by application of the Aiba/Edwards, and the Levenspiel-type unstructured inhibition-type kinetic models.     

基于内源III-B型CRISPR-Cas系统构建超嗜热火球菌Pyrococcus yayanosii A1的基因敲降系统

【目的】亚氏火球菌(Pyrococcusyayanosii)A1菌株的最适生长温度为98°C,最适生长压力为5.2×104Pa,是研究此类严格厌氧、超嗜热和兼性嗜压古菌的环境适应性机制的良好材料。本研究基于P. yayanosii A1基因组中III-B型CRISPR-Cas系统,旨在建立可应用于此类古菌的基因表达敲降系统(geneknock-downsystem)。【方法】人工构建的mini-CRISPR簇,由内源性CRISPR簇Group1的重复序列(repeats)和待敲降的淀粉酶基因(PYCH_13690)中的原间隔序列(protospacer)组成。将该mini-CRISPR簇插入到具有辛伐他汀抗性的穿梭载体p LMOShhp中,使之转录与目的基因m RNA匹配的cr RNA,引导Cmr复合物对其进行切割。【结果】使用高静水压(HHP)诱导型启动子Phhp诱导mini-CRISPR簇表达时,淀粉酶基因的m RNA数量在5.2×104Pa静水压时下调到原来的67.05%,在1.0×102Pa静水压时下调为原来的49.69%;而使用组成型启动子Phmtb诱导mini-CRISPR簇表达时,淀粉酶基因的m RNA数量在5.2×104Pa静水压时下调为原来的58.48%,在1.0×102Pa静水压时下调为原来的23.97%。使用鲁戈氏碘液显色法对这两类基因表达敲降突变菌株的培养物中残留的淀粉含量进行分析,结果显示突变菌株的淀粉降解能力明显降低。【结论】在P. yayanosii A1中建立了基于内源Ⅲ-B型CRISPR-Cas系统的基因敲降系统,可以用于抑制此类超嗜热嗜压古菌体内基因的表达。