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31.
32.
Kawarabayasi Yutaka; Tanaka Ayako; Ohara Osamu; Arakawa Taku; Oka Masanori; Kato Hisako; Morita Miyo; Fujisawa Hisao 《DNA research》1994,1(6):289-296
To sequence a DNA segment inserted into a cosmid vector underthe directed sequencing strategy, we established a simple andrapid method for generating nested deletions which uses thein vitro packaging system of bacteriophage T3 DNA. The principleis based on the previous finding that this system can translocateany linear double-stranded DNA up to 40 kb into the phage capsidin a time-dependent manner and the encapsulated DNA becomesDNase-resistant. For this purpose, we constructed a cosmid vectorthat carries two different antibiotic selection markers at bothsides of the multiple cloning site, and after insertion of aDNA segment, the clone was linearized by -terminase at the cossite. After the packaging reaction in vitro followed by DNasetreatment, the encapsulated DNA was introduced into Escherichiacoli cells to give clones with unidirectional deletions by differentialantibiotic selection. Restriction and sequence analyses of deletionclones demonstrated that an ordered set of clones with nesteddeletions, ranging from less than 1 kb to 25 kb, was createdfrom either the end of the DNA segment. Thus, nested deletionclones that cover the entire region of a 40-kb cosmid insertcan be obtained by a single packaging reaction, and its restrictionmap can be simultaneously obtained. 相似文献
33.
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Full-grown amphibian oocytes that had been arrested at meiotic prophase I contained an activity that prevented the cell cycle from progressing beyond a G2 -like stage. Injection of the contents of germinal vesicles (GV-content) or cytoplasm obtained from oocytes of the frog Rana rugosa prevented fertilized eggs of Cynops pyrrhogaster or Bufo japonicus from cleaving. The nuclei in the arrested eggs consisted of thin chromosomes and nucleolus-like particles enclosed within clear nuclear membrane and their volume increased as a function of time after injection. Cycling of maturation-promoting factor (MPF) did not occur in the injected eggs, but DNA synthesis was not disturbed. The injection of exogenous MPF into the eggs induced the reinitiation of the cell cycle with progression to the M phase and subsequent cleavage. Furthermore, the injection into the full-grown oocytes of Bufo inhibited induction of the maturation of oocytes by progesterone. These results demonstrate that a factor that arrests the cell cycle either at a G2 -like stage of mitosis or at prophase in meiosis is present both in the GV and cytoplasm of frog oocytes. We refer to this factor as a G2 -specific cytostatic factor (G2 -CSF). G2 -CSF may play an important role not only in the physiological arrest at prophase I in meiosis, but also in regulation of the G2 /M transition in the cell cycle of early embryonic cells. 相似文献
35.
We cloned and sequenced cDNAs encoding calponin (Calp) and SM22 (smooth muscle-specific 22-kDa protein) from rat aorta (RaA) smooth muscle (Smu) cells. The 1504-bp calp cDNA contains a single open reading frame (ORF) which encodes 297 amino acids (aa) (Mr 33 342). The 1186-bp SM22 cDNA contains a single ORF which encodes 201 aa (Mr 22 601). There were 43% identical aa in a 181-aa overlap between RaA Calp and SM22. Especially for the C-terminal region of SM22 and for the first repeat motif of Calp, 70% identity was observed. Northern blot analysis revealed that the calp and SM22 mRNAs were expressed in RaA Smu, but not in rat cardiac and skeletal muscles. SM22 mRNA was much more abundant than calp mRNA in RaA (3- to 4-fold). The expression levels of the calp and SM22 mRNAs in RaA showed a significant increase for 5 to 15 week old rats (1.5- to 3-fold) with vascular development and blood pressure elevation. No significant differences were observed in the expression of the RaA calp and SM22 mRNAs between normotensive (Wistar Kyoto) and spontaneously hypertensive rats (SHR). 相似文献
36.
37.
Efficient production of IGG human monoclonal antibodies by lymphocytes stimulated by lipopolysaccharide,pokeweed mitogen,and interleukin 4 总被引:1,自引:0,他引:1
Kaoru Yoshinari Kenji Arai Hideki Kimura Kunio Matsumoto Yutaka Yamaguchi 《In vitro cellular & developmental biology. Animal》1996,32(6):372-377
Summary Extensive screening of the mitogens lipopolysaccharide (LPS), pokeweed mitogen (PWM), andStaphylococcus aureus Cowan I (SAC I), alone and in combination and with and without interleukin (IL) was performed forin vitro activation of regional lymph node lymphocytes from lung cancer patients for the production of human IgG, IgM, and IgA. As
assessed by electrofusion of the lymphocytes following their exposure to these agents with mouse myeloma cells and incubation
of the fused hybridoma, a remarkable stimulatory effect was shown by LPS and particularly by LPS plus IL-4, which was substantially
greater than that of either SAC I or PWM with or without various IL. Optimization studies indicated that the addition of PWM
to LPS and IL-4 in the culture medium further stimulated the human antibody (Ab) production, and that the optimal formulation
for stimulations of human IgG production was a culture medium containing 20 μg/ml of LPS, 1/500 of PWM, and 100 u/ml of IL-4. 相似文献
38.
Carotenogenic mutants ofPhycomyces, which accumulate excess β-carotene or its intermediates, always failed in zygospore development. No improvement occurred
when such mutants were mated together with a helper wild type of the same mating type against the wild type of the opposite
mating type. Addition of excess synthesized pheromone, trisporin B, also failed to improve the zygospore development, though
the mating response was significantly activated in the early stages and abundant zygophores were formed. Exceptional acceleration
of the zygospore development under these experimental conditions occurred in a regulatory albino mutant (carA), which does not accumulate excess intermediate carotenoids. Chemically- or genetically-induced ovarproduction of β-carotene
or lycopene also inhibited the zygospore development. These results imply that the zygospore development ofPhycomyces is maximal when the intracellular amount of β-carotene is optimal (=wild type), and that pheromones act mainly in the early
stages of mating, while other factors such as the cell-to-cell recognition system may also be involved in the later stages.
Intracellular accumulation of excess β-carotene or its intermediates probably disturb such later-stage factors. 相似文献
39.
Taro Suzuki Anatoly A. Tsygankov Jun Miyake Yutaka Tokiwa Yasuo Asada 《Biotechnology letters》1995,17(4):395-400
Summary Effect of pH of culture media on intracellular accumulation of poly-(hydroxybutyrate) (PHB) by a non-sulfur photosynthetic bacterium, Rhodobacter sphaeroides strain RV was studied in pH-stat cultures. Sub-optimal pH for growth, 8.0 or 8.5 gave the higher content of PHB rather than optimal pH 7.5 for growth. These results show that growth and PHB accumulation of the bacteria can be controlled by pH of culture media. 相似文献
40.
The addition reaction to N-methyl-(S)-alanine or N-methyl-(S)-phenylalanine N-car-boxyanhydride (NCA) of 3-methyl-5-substituted hydantoin (HDT) catalyzed by a tertiary amine was investigated as a model reaction for the propagation reaction of NCA according to the activated-NCA mechanism. Several activated HDTs having the (S)-configuration of the asymmetric carbon atom were found to react more rapidly than their activated enantiomers. This experimental result indicates that the enantiomer selection by terminal-unit control takes place in the propagation reaction according to the activated-NCA mechanism in which an activated NCA is added to a terminal acylated NCA ring of the growing chain. The enantiomer excess of the HDT recovered from the reaction mixture of N-methyl-(S)-phenylalanine NCA and racemic HDTs activated by a tertiary amine was determined. The extent of the enantiomer selection in the polymerization was found to be 3–10 times as large as that in the model reaction. From these results, it was concluded that the chirality of the penultimate unit, as well as that of the terminal NCA ring, plays an important role in determining the enantiomer selection in the NCA polymerization. 相似文献