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71.
利用PCR技术将本室克隆到的强启动功能片段取代麦迪霉素丙酰化酶基因(mpt)的启动子或与mpt基因自身启动子串连,获得含mPt重组质粒pCHFPE3和pCHFPE2。用含有这两个质粒的Streptomyces lividans TK24对螺旋霉素进行微生物转化,结果表明,与含有原启动子的mpt.S.lividans TK24(p.WFPE)相比,丙酰螺旋霉素的组分比例分别提高了89.02%和58.53%。含重组质粒pCHFPE2的螺旋霉素产生菌S.spiramyceticus发酵产物中丙酰螺旋霉素的组分也有较大辐度的提高。说明利用该强启动功能片段可以提高麦迪霉素丙酰化酶基因的表达。 相似文献
72.
Genomic sequencing by ligation-mediated PCR 总被引:8,自引:0,他引:8
Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited due
to the complexity of the mammalian genome. Ligation-mediated PCR (LMPCR) is a sensitive genomic sequencing procedure that
generates high quality, reproducible sequence ladders starting with only 1 μg of uncloned mammalian DNA per reaction. This
genomic sequencing procedure can be adapted for various methylation, in vivo footprinting and DNA adduct mapping procedures.
We provide a detailed protocol for genomic sequencing by LMPCR and discuss the principles and applications of the method. 相似文献
73.
B. Arnholdt-Schmitt S. Herterich K. -H. Neumann 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1995,91(5):809-815
Investigations were performed on growth phase-dependent EcoRII site-specific DNA methylation of the carrot genome during primary culture to elucidate physiological aspects of genome DNA variability in tissue culture. While DNA methylation of the root cambium and the secondary phloem and petioles of carrot leaves were strikingly different, the methylation level of the secondary phloem seemed to be independent of cultivar origin, the age of the plants and the extent of secondary root growth. As was shown earlier a change in the differentiated state of the secondary phloem by tissue culture leads to changes in genome modification. Whereas de novo methylation was observed during the first 2 weeks of growth initiation, the results presented demonstrate genome de-methylation during the transition to stationary growth indicating differential nome methylation during different phases of culture. The presence of kinetin in the nutrient medium of the primary culture was found to be antagonistic to changes in genome modification in general. De novo methylation and subsequent de-methylation of the carrot genome are discussed as gross changes obviously essential to molecular genome differentiation during tissue culture. 相似文献
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Clare Gough Pascale Hemon Maurice Tronchet Christophe Lacomme Yves Marco Dominique Roby 《Molecular & general genetics : MGG》1995,247(3):323-337
A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5 flanking DNA sequence from the str246C gene fused to the -glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized. histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5 deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 by was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.Joint first authors 相似文献
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The glucan-binding lectin (GBL) ofStreptococcus sobrinus is cell associated, enabling the bacteria to be aggregated by -1,6 glucans. Glucans, such as amylose, pullulan, laminarin and nigeran, have no affinity for the lectin. High molecular weight -1,6 glucans (dextrans) readily aggregate the bacteria, whereas low molecular weight glucans inhibit the aggregation brought about by the high molecular weight species. Methylated glucan T-2000 (an -1,6 glucan with an average molecular weight of 2 × 106 Da) aggregated the bacteria very poorly when the extent of methylation (DS, or degree of substitution) was high, and less poorly when the DS was low. Similarly, methylated low molecular weight -1,6 glucan was a poor inhibitor of aggregation induced by the high molecular weight glucan T-2000. Because the methylation occurred primarily on the hydroxyl of C-2, it is suggested that the hydroxyl is needed for formation of the lectin-glucan complex. It appears that the GBL is not only stereospecific in interaction with glucans, but also regiospecific, interacting only with the underivatized -1,6-glucan. 相似文献
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