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31.
Summary A cDNA clone in pBR322 that cross-hybridizes with a mouse carbonic anhydrase form II (CAII) probe has been sequenced and identified as mouse carbonic anhydrase form I (CAI). The 1224-base-pair clone encodes the entire 260-amino-acid protein and appears to contain an Alu-like element in the 3 untranslated region. The deduced amino acid sequence exhibits 77% homology to human CAI and contains 17 of the 20 residues that are considered unique to and invariant for all mammalian CAI isozymes. The results of a detailed comparison of the nucleic acid sequences spanning the coding regions of mouse CAI and rabbit CAI have been used to calibrate an evolutionary clock for the carbonic anhydrases (CAs). These data have been applied to a comparison of the mouse CAI and CAII nucleic acid sequences to calculate the divergence time between the two genes. The divergence-time calculation provides the first estimation of the evolutionary relationship between CAs based entirely on nucleotide sequence comparison. 相似文献
32.
Christina Scharnhorst Hartmut Heinze Gabriele Meyer Waldemar Kolanus Klaus Bartsch Susanne Heinrichs Thomas Gudschun Margret Möller Frank Herzfeld 《Plant molecular biology》1985,4(4):241-245
Summary cDNA clones were isolated for a chloroplast protein, the mRNA of which is induced to maximum levels within 2–4 h after onset
of illumination in five day old, etiolated pea seedlings.
The cDNA library was constructed from poly(A)+-mRNA which was isolated from 4 h illuminated seedlings. The extremely short induction period of the early light induced protein(ELIP)-mRNA
established the basis of our screening procedure. Colony hybridization experiments were performed with32P-labelled cDNA probes, synthesized from RNA of seedlings which had been exposed to different programs of illumination. Plasmid
DNAs were isolated from colonies showing strong hybridization signals exclusively with cDNA corresponding to the 4 h-mRNA.
Hybrid released translation of preselected plasmids p 17/C2 and p17/C4 revealed a peptide of Mr 24 000. After posttranslational importin vitro, the processed product of Mr 17 000 appears in the chloroplast. Using these clones, the expression of the ELIP-mRNA was investigated by DOT-hybridization.
The ELIP-mRNA reaches maximum levels within 2–4 hours after onset of illumination. Our results correspond precisely to thein vivo characteristics and indicate positive identification of the sought clones. 相似文献
33.
The araBAD operon of Salmonella typhimurium LT2. I. Nucleotide sequence of araB and primary structure of its product, ribulokinase 总被引:6,自引:0,他引:6
Hybrid plasmids containing the araBAD operon of Salmonella typhimurium LT2 were characterized by Southern blot and genetic analyses. The nucleotide sequence of araB was determined. The araB gene product, ribulokinase (EC 2.7.1.16), was purified and the results of amino acid composition analysis and partial amino acid sequence are in agreement with predictions from the DNA sequence. Ribulokinase is 569 amino acid residues long and has a calculated Mr of 61 793. Ribulokinase shares significant homology with xylulose kinase from Escherichia coli. Codon usage in the araB gene does not favor those codons which have intermediate codon-anticodon binding energy. 相似文献
34.
Abstract 3 new shuttle cloning vectors for gene transfer into Escherichia coli and Anacystis nidulans have been constructed by utilizing the cyanobacterial origin of replication of the small plasmid pANS from A. nidulans . 2 of these new vectors, pXB7 (pDPL13 derivative) and pECAN8 (pUC8 derivative), convey ampicillin resistance, and transform A. nidulans with relatively high frequencies. Vector pXB7 has 10 unique cloning sites; pECAN8 contains 4 cloning sites within the lacZ gene permitting rapid detection of DNA inserts in the presence of Xgal. The third vector, pKBX, has a lower transformation frequency but adds kanamycin resistance as a selectable gene for shuttle vectors of cyanobacteria. 相似文献
35.
Abstract Listeriolysin, an SH-activated haemolysin probably involved in Listeria pathogenicity, has been cloned into the cosmid vector pHC79 and was expressed in Escherichia coli HB101 cells. Chromosomal DNA of Listeria monocytogenes serovar 1/2 was partially digested with Mbo I and ligated to the Bam HI cleaved cosmid. From 2000 recombinant clones examined, 12 (0.6%) produced haemolysin in solid and liquid media. All of them contained chromosome fragments of Listeria of about 40 kb. The cloning of the listeriolysin determinant will lead to a better understanding of the basis of Listeria pathogenicity. 相似文献
36.
Koji Yamada Masumi Ohtsu Genki Kimura 《In vitro cellular & developmental biology. Plant》1985,21(8):428-432
Summary Sodium butyrate causes proliferation arrest with a G2 (4C) DNA content and induces formation of tetraploid cells upon removal
of the inhibitor, in rat 3Y1 diploid fibroblasts. We isolated tetraploid clones from the butyrate-treated 3Y1 cells with high
efficiency; among 21 clones randomly isolated, 5 were pure diploid, 7 were mainly tetraploid with a small contaminating diploid
population, and 7 were pure tetraploid. Among the pure tetraploid clones, two showed doubled chromosome numbers with slightly
broader distributions than that seen in parental 3Y1 cells. Butyrate further induced polyploid formation in the tetraploid
cells thus produced, but octaploid cells that resulted could not be maintained for prolongeed, cultivation. We found no difference
between the tetraploid and the (parental and parallel isolated) diploid clones in terms of colony-forming ability, proliferation
rate, and sensitivity to density-dependent inhibition of proliferation. These results suggest that doubling of chromosome
number by itself does not cause a change in proliferation property. The tetraploid clones had lower average saturation densities
possibly due to enlargement of cell size represented by higher cellular protein content. 相似文献
37.
Robert T. Przygoda K. Takayama Karl A. Traul A. Tummey 《In vitro cellular & developmental biology. Plant》1985,21(1):32-38
Summary An atmosphere containing 10% CO2 has been generally accepted as optimal for the growth of Syrian hamster embryo cells in a clonal transformation assay. Data
presented in this paper show that 10% CO2 may not be the optimum environment for this assay.
Using 10 or 20% (analytically measured) CO2 in air (1 atm pressure), hamster embryo cell pools were examined for clonal growth characteristics and transformability using
five known carcinogens and a single noncarcinogenic compound. At 10% CO2, only 2 of 11 pools weee transformed by the five carcinogens but not by the noncarcinogen. At 20% CO2, six of seven pools were transformed by the five carcinogens and not by the noncarcinogen. Further, the transformation frequencies
were found to be greater in cultures incubated in an atmosphere consisting of 20% CO2 in air. The data also show that 20% CO2 increased the cloning efficiency of these cells.
A comparison of the 10 and 20% CO2 data to results reported from other conflicting interlaboratory results with this assay system may be due, in part, to variations
of CO2 concentrations. In some instances, the CO2 levels indicated by incubator flow meters vary considerably from analytically determined CO2 values. To prevent these CO2 discrepancies and their resultant effects on transformation and cloning efficiency, methods for monitoring the CO2 environment other than flow meters are recommended.
The observation of increased cloning efficiencies and transformation rates strongly suggests that culture incubation at 20%
CO2 is a preferred environment for the conduct of this assay. 相似文献
38.
Improved clonal and nonclonal growth of human,rat and bovine adrenocortical cells in culture 总被引:2,自引:0,他引:2
Jan M. McAllister Peter J. Hornsby 《In vitro cellular & developmental biology. Plant》1987,23(10):677-685
Summary This report describes the development of a culture system for long-term growth and cloning of human fetal adrenocortical cells.
Optimal conditions for stimulating clonal growth were determned by testing the efficacy of horse serum (HS), fetal bovine
serum (FBS), fibroblast growth factor (FGF), epidermal growth factor (EGF), fibronectin, and a combination of growth factors,
UltroSer G, in stimulating growth from low density. Optimal conditions for clonal growth were achieved using fibronectin-coated
dishes and DME/F12 medium with 10% FEBS, 10% HS, 2% UltroSer G, and 100 ng/ml FGF or 100 pM EGF. Conditions for growth at clonal density were found to be optimal for growth of early passage, nonclonal cultures at
higher densities. The improved growth conditions used for cloning were shown to allow continued long-term growth of nonclonal
human adrenocortical cells without fibroblasts overgrowth. All cells in cultures grown in HS, FBS, and UltroSer G had morphologic
characteristics of adrenocortical cells, whereas cells grown in FBS only rapidly became overgrown with fibroblasts. Clonal
and nonclonal early passage human adrenocortical cells had smilar mitogenic responses to FGF and EGF. Whereas FGF, EGF, and
UltroSer G showed similar stimulation of DNA synthesis and clonal growth in human adrenocortical cells and human adrenal gland
fibroblasts, the tumor promoter 12-O-teradecanoylphorbol-13-acetate stimulated growth only in adrenocortical cells and was strongly inhibitory to growth in fibroblasts.
In both cell types, forskolin inhibited DNA synthesis. Human adrenocortical cell cultures were functional and synthesized
cortisol, dehydroepiandrosterone, and dehydroepiandrosterone sulfate. The improved growth conditions for clonal growth of
human adrenocortial cells also provided optimal conditions for long-term growth of cultured rat adrenocortical cells and ncreased
the cloning efficiency of cultured bovine adrenocortical cells.
This work was supported by Research grants AG-00936 and AG-06108 from the National Institute on Aging, Bethesda, MD. 相似文献
39.
油菜细胞质雄性不育系叶绿体DNA特异片段的分子克隆 总被引:7,自引:0,他引:7
采用高离子浓度缓冲液法分别提取油菜不育系及保持系的叶绿体DNA。经Sepharcse 4B柱层析纯化后,得到具有较高纯度的叶绿体DNA样品。将其分别用Eco RI、Bam HI、HimdHI、PstI和XhoI 5种限制性内切酶酶解,得到5种限制性内切酶图谱。其中除PstI图谱外,其它4种酶谱均显示出明显的两系间叶绿体DNA结构差异。以pBR 322为载体,分别克隆了不育系Bam HI图谱上的3个特异片段。得到的3个克隆,经克隆杂交及电泳分析后,证实分别带有上述3个目的片段。这些特异片段的特性及其与花粉育性的关系尚在研究中。 相似文献
40.
Summary Earlier studies found that cotton (Gossypium hirsutum L.) cotyledons contain several mRNAs which are more abundant during late embryogenesis than in mid-embryogenesis or early germination. They are here termed Late embryogenesis-abundant mRNAs, encoded by Lea loci. Complementary DNA clones for 18 such mRNA sequences, defined at a hybridization criterion of Tm-15°C, were identified in a mature embryo cDNA library by differential cDNA hybridization. At a lower hybridization criterion, some sequence homology was found within several of these cloned Lea mRNA sequences. Each Lea mRNA sequence comprises 0.04–1.3% of mature embryo poly(A)+ mRNA, a level ten-fold to several hundred-fold higher than in young embryo or 24 h seedling poly(A)+ mRNA. Of 18 Lea mRNA sequences examined in cultured young embryos, the level of at least 13 are specifically increased by exogenous abscisic acid (ABA), several to a level near that in normal mature embryos. However, the abundance of several of the sequences does not appear to be significantly modulated by ABA. The LEA polypeptides encoded by 10 Lea mRNA sequences were identified by hybrid-arrested translation. They include most of the late embryogenesis-abundant, ABA-inducible, polypeptides previously identified. Preliminary results suggest that many of the individual Lea mRNA sequences are transcribed from 1–3 genes in each of cotton's two subgenomes. 相似文献