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941.
Transformation of mammalian cells with genes from procaryotes and eucaryotes. 总被引:398,自引:0,他引:398
M Wigler R Sweet G K Sim B Wold A Pellicer E Lacy T Maniatis S Silverstein R Axel 《Cell》1979,16(4):777-785
We have stably transformed mammalian cells with precisely defined procaryotic and eucaryotic genes for which no selective criteria exist. The addition of a purified viral thymidine kinase (tk) gene to mouse cells lacking this enzyme results in the appearance of stable transformants which can be selected by their ability to grow in HAT. These biochemical transformants may represent a subpopulation of competent cells which are likely to integrate other unlinked genes at frequencies higher than the general population. Co-transformation experiments were therefore performed with the viral tk gene and bacteriophage ΦX174, plasmid pBR322 or the cloned chromosomal rabbit β-globin gene sequences. Tk+ transformants were cloned and analyzed for co-transfer of additional DNA sequences by blot hybridization. In this manner, we have identified mouse cell lines which contain multiple copies of 4)X, pBR322 and the rabbit β-globin gene sequences. The ΦX co-transformants were studied in greatest detail. The frequency of co-transformation is high: 15 of 16 tk+ transformants contain the ΦX sequences. Selective pressure was required to identify co-transformants. From one to more than fifty ΦX sequences are integrated into high molecular weight nuclear DNA isolated from independent clones. Analysis of subclones demonstrates that the ΦX genotype is stable through many generations in culture. This co-transformation system should allow the introduction and stable integration of virtually any defined gene into cultured cells. Ligation to either viral vectors or selectable biochemical markers is not required. 相似文献
942.
Wolfgang Weber Hans Zähner Johannes Siebers Karsten Schröder Axel Zeeck 《Archives of microbiology》1979,121(2):111-116
Zusammenfassung
Streptomyces glaucescens, Stamm Tü 49=ETH 22794, produziert neben Hydroxystreptomycin die Tetracenomycine, ein Gemisch lipophiler Antibiotica, von denen das mengenmäßig vorherrschende und zugleich das aktivste das Tetracenomycin C ist. Das blaßgelbe, kristallisierte Tetracenomycin C hat die Summenformel C23H20O11 und ähnelt chemisch den Tetracyclinen und den Anthracyclinonen.Tetracenomycin C ist wirksam gegen einige Grampositive Bakterien und vor allem gegen Streptomyceten; Gram-negative Bakterien werden nicht gehemmt. Aufgrund der Unterschiede bei den funktionellen Gruppen, des Wirkungsspektrums und den bislang bekannten Daten zur Wirkungsweise kann Tetracenomycin C weder zu den Tetracyclinen noch zu den Anthracyclinonen gerechnet werden.Der Erfolg der Suche nach neuen Antibiotica hängt in starkem Maße von dem verwendeten Screening-Modell ab. Ein erfolgversprechendes Modell sollte unter anderen zwei Bedingungen erfüllen:Erstens sollen ungewöhnliche, bisher nicht erfaßte Aktivitäten sichtbar werden und zweitens sollen bekannte Substanzen weitgehend ausgeschlossen werden. Ein Screening-Modell, das solche Ansprüche erfüllt, ist die Suche nach Engspektrum-Antibiotica (Zähner, 1974, 1977), die nur wenige Testkeime hemmen. Wie bei einigen anderen nichtklassischen Screening-Methoden wird hier auf eine Korrelation zwischen erstem Test und späterer Anwendung verzichtet; der erste Test dient nur als Leitschiene für die Auffindung und Isolierung eines neuen Metaboliten. Erst auf einer zweiten Stufe wird die Frage der Einsatzbarkeit des neuen Metaboliten geprüft. Neuartige Screeningmethoden werden erst seit relativ kurzer Zeit eingesetzt. Es schien uns deshalb lohnend, auch ältere, gut charakterisierte Antibioticaproduzenten auf die Bildung bisher übersehener Sekundärmetaboliten anzusehen.
Streptomyces glaucescens (Stamm Tü 49=ETH 22794) ist schon seit längerer Zeit als Produzent von Hydroxy-Streptomycin bekannt (Hütter, 1967). Das Hauptinteresse an diesem Stamm gilt jedoch seiner Genetik, insbesondere den genetischen Grundlagen der Melaninbildung (Lerch u. Ettlinger, 1972; Baumann et al., 1974; Baumann u. Kocher, 1976). Bei der Prüfung dieses Stammes auf Bildung von Engspektrum-Antibiotica fanden wir die Tetracenomycine, ein Gemisch lipophiler Antibiotica. Hauptkomponente ist Tetracenomycin C, das nachstehend beschrieben werden soll.
Metabolic products of microorganisms. 175. Tetracenomycin C
Streptomyces glaucescens, strain Tü 49=ETH 22794, produces hydroxystreptomycin as well as the tetracenomycins, a mixture of several lipophilic antibiotics. The main component and the most active one is tetracenomycin C. Tetracenomycin C has a molecular formula C23H20O11 and is chemically related to tetracyclines and anthracyclinones.The pale yellow antibiotic is active against some gram-positive bacteria, especially against streptomycetes. Gram-negative bacteria and fungi are not inhibited. In considering the differences of biological activity and the functional groups of the molecule, tetracenomycin C is not a member of the tetracycline or anthracyclinone group of antibiotics.
174. Mitteil.: W. Keller-Schierlein und H.-V. Naegeli. Synthese von enantio-Ferrichrom. Helv. Chim. Acta (im Druck) 相似文献
943.
The landing response of tethered flying housefliesMusca domestica elicited by motion of periodic gratings is analysed. The field of view of the compound eyes of a fly can be subdivided into a region of binocular overlap and a monocular region. In the monocular region the landing response is elicited by motion from front to back and suppressed by motion from back to front. The sensitivity to front to back motion in monocular flies (one eye covered with black paint) has a maximum at an angle 60°–80° laterally from the direction of flight in the equatorial plane. The maximum of the landing response to front to back motion as a function of the contrast frequencyw/ is observed at around 8 Hz. In the region of binocular overlap of monocular flies the landing response can be elicited by back to front motion around the equatorial plane if a laterally positioned pattern is simulataneously moved from front to back. 40° above the equatorial plane in the binocular region the landing response in binocular flies is elicited by upward motion, 40° below the equatorial plane in the binocular region it is elicited by downward motion. The results are interpreted as an adaptation of the visual system of the fly to the perception of a flow field having its pole in the direction of flight. 相似文献
944.
Leon Axel 《Bulletin of mathematical biology》1976,38(6):671-677
The Kedem-Katchalsky equations for fluid flux across membranes may not be adequate for large solvent flows. In particular,
for an example of two membranes in series, it is argued that they would predict physically unreasonable behavior. An alternate
equation for solute flow is proposed for a simple sieving membrane. For the same example, this equation predicts more physically
reasonable results. 相似文献
945.
We have analyzed the efficiency with which specific nucleotide sequences within nucleosomes are recognized and cleaved by DNA restriction endonucleases. A system amenable to this sort of analysis is the cleavage of the bovine genome with the restriction endonuclease EcoRI. Bovine satellite I comprises 7% of the genome and is tandemly repetitious with an EcoRI site at 1400 base pair (bp) intervals within this sequence. The ease with which this restriction fragment can be measured permits an analysis of the accessibility of this sequence when organized in a nucleosomal array.Initial studies indicated that satellite I sequences are organized in a nucleosomal structure in a manner analogous to that observed for total genomic DNA. We then examined the accessibility of the EcoRI cleavage sites in satellite to endonucleolytic cleavage in intact nuclei. We find that whereas virtually all the satellite I sequences from naked DNA are cleaved into discrete 1400 bp fragments, only 33% of the satellite I DNA is liberated as this fragment from intact nuclei. These data indicate that 57% of the EcoRI sites in nuclei are accessible to cleavage and that cleavage can occur within the core of at least half the nucleosomal subunits. Analysis of the products of digestion suggests a random distribution of nucleosomes about the EcoRI sites of satellite I DNA.Finally, the observation that satellite sequences can be cleaved from nuclei to 1400 bp length fragments with their associated proteins provides a method for the isolation of specific sequences as chromatin. Using sucrose gradient velocity centrifugation, we have isolated a 70% pure fraction of satellite I chromatin. Nuclease digestion of this chromatin fraction reveals the presence of nucleosomal subunits and indicates that specific sequences can be isolated in this manner without gross disorganization of their subunit structure. 相似文献
946.
Anti human M2 type and anti human L type pyruvate kinase sera allowed us to distinguish two groups of pyruvate kinase in man. Erythrocyte and liver (L type) enzymes on the one hand were inhibited by anti L and not all by anti M2 serum; pyruvate kinase from all the other tissues on the other hand were inhibited by anti M2 and not at all by anti L serum. This latter group represent the M type pyruvate kinase isozymes. The M type isozymes have been studied by electrofocusing in thin layer acrylamide-ampholine gel. In adult tissues 4 types of isozymes were found, designated, from acid to alkaline pH, as M2 (predominant form in spleen, leukocytes, lung...), M3, M4 and M1 (predominant form in muscle and brain). In foetal tissues an extra band M2, called M2f, more anodic than M2, was added to the previously described isozymes. Except in brain (in which the isozymes M2, M3, M4 and M1 were found), the most anodic bands (M2f, M2 and M3) were predominant in all the foetal tissues. The isozymes M2f and M2 seem therefore to be the original M type pyruvate kinase forms from which the other isozymes issue. The rate of each isozyme seems to depend on tissue factors characterizing the state of differentiation of some tissues, as indicated by the ability of adult muscle extracts to change the isozymes M2 and M3 into more cathodic forms. 相似文献
947.
Mariano J. Levin Dominique Daegelen Marie-Claire Meienhofer Jean-Claude Dreyfus Axel Kahn 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,699(2):77-83
A study was performed to determine whether M1 and M2 pyruvate kinases were synthesized under the direction of one or two messenger RNAs. We compared M1 and M2 pyruvate kinases purified from fresh tissues with those neosynthesized under the direction of messenger RNAs from tissues synthesizing either M1 or M2. RNA was isolated from rat muscle, lung, spleen and kidney by ethanol precipitation in 7 M guanidium chloride, translated in rabbit reticulocyte system and newly-synthesized pyruvate kinase subunits were purified by microimmunoaffinity chromatography. Pyruvate kinase from fresh muscle and spleen was purified in one step by a similar process. Muscle and spleen RNA directed the synthesis of M subunits with molecular weights of approx. 61000 and 62000, respectively, the same as those of the corresponding fresh tissue monomers. In addition, peptide maps obtained by partial digestion of neosynthesized M1 and M2 with V8 protease from Staphylococcus aureus confirmed that these polypeptides were clearly different. 相似文献
948.
Klaus Hausen 《Biological cybernetics》1982,45(2):143-156
The three horizontal cells of the lobula plate of the blowflyCalliphora erythrocephala were studied anatomically and physiologically by means of cobalt impregnations and intracellular recordings combined with Procion and Lucifer Yellow injections. The cells are termed north, equatorial and south horizontal cell (HSN, HSE, HSS) and are major output neurons of the optic lobe. 1. The dendritic arborizations of the HSN, HSE, HSS reside in a thin anterior layer of the lobula plate and extend over the dorsal, equatorial and ventral parts of this neuropil, respectively. Due to the retinotopic organization of the optic lobe, these parts correspond anatomically to respective regions of the ipsilateral visual field. Homologue horizontal cells in both lobula plates of the same animal and in different animals are highly variable with respect to their individual dendritic branching patterns. They are extraordinarily constant, on the other hand, with regard to the position and size of their dendritic fields as well as their dendritic branching density distributions. Each cell covers about 40% of the total area of the lobula plate and shows the highest dendritic density near the lateral margin of the neuropil which subserves the frontal eye region. The axons of the horizontal cells are relatively short and large in diameter; they terminate in the posterior ventrolateral protocerebrum. 2. The horizontal cells are directionally selective motion sensitive visual interneurons responding preferentially to progressive (front to back) motion in the ipsilateral visual field with graded depolarization of their axons and superimposed action potentials. Stimulation with motion in the reverse direction leads to hyperpolarizing graded responses. The HSE and HSN are additionally activated by regressive motion in the contralateral visual field. 相似文献
949.
Summary Closed circular DNAs have been isolated from Oenothera and analysed by gel electrophoresis. A number of different size classes could be observed in the supercoiled fraction from the density gradient. The five smallest size classes of 6.3, 7.0, 8.2, 9.9, and 13.5 kilobases linear length were purified and their restriction fragment sizes compared. These five mtDNA species appear to be distinct homologous populations with no detectable large homologies between thedifferent classes. Hybridization data confirm this result; the smallest species hybridizes to itself and very weakly to some of the larger DNA molecules indicating only very little common sequence arrangement between the different circular DNAs. A restriction map of the smallest size class was constructed and confirms the circular arrangement of this DNA.Abbreviations used
kb
kilobases or kilobasepairs
-
mtDNA
mitochondrial DNA
-
EthBr
Ethidium bromide
-
SSC
150 mM NaCl, 15 mM sodium citrate (pH 7.0) 相似文献
950.
Cornelius Hammer Axel Temming Hans-Joachim Schubert 《International Review of Hydrobiology》1994,79(3):385-396
Measurements of the swimming activity of a group of roach (12–19 cm TL, average) in a circular swimming chamber revealed two distinct activity patterns: a diurnal and a nocturnal one. The experiments showed that, having the choice, two factors stimulated the rhythmicity of the swimming behaviour of the fish, i.e. light intensity and the presence of a current field in the proximity of the fish. During daytime (bright light conditions) the fish moved into the current field and swam on average at 0.4 BL/s (resting swimming). The roach remained swimming at this speed even if no current field was established, however, then distributed evenly in the basin. By contrast, during night (dim light conditions) the fish predominantly chose the still water section but swam on average with a cruising speed of 1.6 BL/s (night swimming). Accordingly, they did not seek the still water section for night swimming if the light was kept on. Then again, the fish distributed more or less evenly in the basin. The results support the hypothesis that the fish migrate during night-time and do this preferably in still water. 相似文献