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排序方式: 共有173条查询结果,搜索用时 11 毫秒
1.
G G Hillebrand A H McCluskey K A Abbott G G Revich K L Beattie 《Nucleic acids research》1984,12(7):3155-3171
A method has been developed for simultaneous comparison of the propensity of a DNA polymerase to misincorporate at different points on a natural template-primer. In this method elongation of a [5'-32P] primer, annealed to a bacteriophage template strand, is carried out in the presence of only three dNTPs (highly purified by HPLC). Under these conditions the rate of primer elongation (monitored by gel electrophoresis/autoradiography) is limited by the rate of misincorporation at template positions complementary to the missing dNTP. Variations in the rate of elongation (revealed by autoradiographic banding patterns) reflect variations in the propensity for misincorporation at different positions along the template. The effect on primer elongation produced by addition of a chemically modified dNTP to 'minus' reactions reveals the mispairing potential of the modified nucleotide during DNA synthesis. By use of this electrophoretic assay of misincorporation we have demonstrated that the fidelity of E. coli DNA polymerase I varies greatly at different positions along a natural template, and that BrdUTP and IodUTP can be incorporated in place of dCTP during chain elongation catalyzed by this enzyme. 相似文献
2.
Influence of template primary and secondary structure on the rate and fidelity of DNA synthesis 总被引:5,自引:0,他引:5
High resolution gel electrophoresis was used to monitor the successive addition of dNMP residues onto the 3'-OH ends of discrete 5'-32P-primers, during DNA synthesis on natural templates. Resulting autoradiographic banding patterns revealed considerable variation in the relative rates of incorporation at different positions along the template. The pattern of "pause sites" along the template was unique for each of three different DNA polymerases (polymerase I (the "large fragment" form of Escherichia coli), T4 polymerase (encoded by bacteriophage T4), and AMV polymerase (DNA polymerase of avian myeloblastosis virus]. Most pause sites were not caused by attenuation of polymerization at regions of local secondary structure in the template. Assays of the accuracy of incorporation at different positions along the template (in which elongation was monitored in the presence of only 3 of the 4 2'-deoxynucleoside 5'-triphosphates) strongly suggested that the relative fidelity of DNA synthesis catalyzed by different polymerases depends on the position on the template at which the comparison is made. Primer-templates were constructed that permitted comparison of elongation during synthesis on a single-stranded template with that during polymerization through a double-stranded region (wherein elongation required concomitant displacement of a strand annealed adjacent to the 5'-32P-primer). Although strand displacement DNA synthesis catalyzed by polymerase I occurred approximately ten times more slowly than synthesis in the same region of a single-stranded viral template, most of the pause sites were the same in the presence or absence of "tandem" primer. Electrophoretic assays of the fidelity of DNA synthesis suggested that an increased tendency toward misincorporational "hotspots" occurred when elongation required concomitant strand displacement. 相似文献
3.
Template-dependent variation in the relative fidelity of DNA polymerase I of Escherichia coli in the presence of Mg2+ versus Mn2+ 总被引:2,自引:0,他引:2
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The fidelity of E. coli DNA polymerase I in the presence of Mg2+ vs Mn2+ was examined at many positions along natural DNA templates, by use of an electrophoretic assay of misincorporation. Although there was an overall greater tendency for misincorporation to occur in Mn2+-activated chain elongation, some specific sites on the template were more prone to misincorporation with Mg2+ and others with Mn2+. This sequence-dependent effect was seen in spite of the finding that the relative rate of incorporation of the correct nucleotide at different positions on the template was essentially the same with Mg2+ and Mn2+. In agreement with previous studies, the fidelity of E. coli pol I was higher at activating, than at inhibiting, concentrations of Mg2+. The results reveal new complexities regarding the role of divalent cation in the control of fidelity in DNA synthesis and attest to the dynamic nature of interactions between DNA polymerase, its substrates and divalent metal activator during the course of polymerization on natural templates. 相似文献
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5.
BIOVOLUME CALCULATION FOR PELAGIC AND BENTHIC MICROALGAE 总被引:17,自引:0,他引:17
Helmut Hillebrand Claus-Dieter Dürselen David Kirschtel Utsa Pollingher Tamar Zohary 《Journal of phycology》1999,35(2):403-424
Microalgal biovolume is commonly calculated to assess the relative abundance (as biomass or carbon) of co-occurring algae varying in shape and/or size. However, a standardized set of equations for biovolume calculations from microscopically measured linear dimensions that includes the entire range of microalgal shapes is not available yet. In comparison with automated methods, the use of microscopical measurements allows high taxonomic resolution, up to the species level, and has fewer sources of error. We present a set of geometric shapes and mathematical equations for calculating biovolumes of >850 pelagic and benthic marine and freshwater microalgal genera. The equations are designed to minimize the effort of microscopic measurement. The similarities and differences between our proposal for standardization and previously published proposals are discussed and recommendations for quality standards given. 相似文献
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7.
Eric M. Lind Elizabeth Borer Eric Seabloom Peter Adler Jonathan D. Bakker Dana M. Blumenthal Mick Crawley Kendi Davies Jennifer Firn Daniel S. Gruner W. Stanley Harpole Yann Hautier Helmut Hillebrand Johannes Knops Brett Melbourne Brent Mortensen Anita C. Risch Martin Schuetz Carly Stevens Peter D. Wragg 《Ecology letters》2013,16(4):513-521
Plant growth can be limited by resource acquisition and defence against consumers, leading to contrasting trade‐off possibilities. The competition‐defence hypothesis posits a trade‐off between competitive ability and defence against enemies (e.g. herbivores and pathogens). The growth‐defence hypothesis suggests that strong competitors for nutrients are also defended against enemies, at a cost to growth rate. We tested these hypotheses using observations of 706 plant populations of over 500 species before and following identical fertilisation and fencing treatments at 39 grassland sites worldwide. Strong positive covariance in species responses to both treatments provided support for a growth‐defence trade‐off: populations that increased with the removal of nutrient limitation (poor competitors) also increased following removal of consumers. This result held globally across 4 years within plant life‐history groups and within the majority of individual sites. Thus, a growth‐defence trade‐off appears to be the norm, and mechanisms maintaining grassland biodiversity may operate within this constraint. 相似文献
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9.
Vorwerk S Biernacki S Hillebrand H Janzik I Müller A Weiler EW Piotrowski M 《Planta》2001,212(4):508-516
Three of the nitrilase isoenzymes of Arabidopsis thaliana (L.) Heynh. are located on chromosome III in tandem and these genes (NIT2/NIT1/NIT3 in the 5′→3′ direction) encode highly similar polypeptides. Copy DNAs encompassing the entire coding sequences for all three
nitrilases were expressed in Escherichia coli as fusion proteins containing a C-terminal hexahistidine extension. All three nitrilases were obtained as enzymatically active
proteins, and their characteristics were determined, including a detailed comparative analysis of their substrate preferences.
All three nitrilases converted indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA), albeit, compared to the most effective
substrates found, phenylpropionitrile (PPN), allylcyanide, (phenylthio)acetonitrile and (methylthio)acetonitrile, with low
affinity and velocity. The preferred substrates are either naturally occurring substrates, which may originate from glucosinolate
breakdown, or they are close relatives of these. Thus, a major function of NIT1, NIT2 and NIT3 is assigned to be the conversion
to carboxylic acids of nitriles from glucosinolate turnover or degradation. While all nitrilases exhibit a similar pH optimum
around neutral, and NIT1 and NIT3 exhibit a similar temperature optimum around 30 °C independent of the substrate analyzed
(IAN, PPN), NIT2 showed a remarkably different temperature optimum for IAN (15 °C) and PPN (35–40 °C). A potential role for
NIT2 in breaking seed dormancy in A. thaliana by low temperatures (stratification), however, was ruled out, although NIT2 was the predominantly expressed nitrilase isoform
in developing embryos and in germinating seeds, as judged from an analysis of β-glucuronidase reporter gene expression under
the control of the promoters of the four isogenes. It is possible that NIT2 is involved in supplying IAA during seed development
rather than during stratification.
Received: 13 May 2000 / Accepted: 14 August 2000 相似文献
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