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HEMIPARASITIC NUTRITION IN ANGIOSPERMS 总被引:14,自引:6,他引:8
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Discrimination Amongst Leishmania by Polymerase Chain Reaction and Hybridization with Small Subunit Ribosomal DNA Derived Oligonucleotides 总被引:1,自引:0,他引:1
SILVIA R. B. ULIANA KIMBERLYN NELSON STEPHEN M. BEVERLEY ERNEY P. CAMARGO LUCILE M. FLOETER-WINTER 《The Journal of eukaryotic microbiology》1994,41(4):324-330
ABSTRACT. A method for discriminating among Leishmania is described, based upon small subunit ribosomal DNA sequence differences. The method was to amplify the entire 2.2 kb small subunit rDNA by polymerase chain reaction using conserved primers specific for the 5' and 3' termini of the small subunit ribosomal RNA, and then hybridize the product dotted onto nylon membranes with labeled oligonucleotides. The design of the hybridization probes was based upon complete small subunit rDNA sequences from L. amazonensis, L. major and L. guyanensis and partial sequences of L. mexicana, L. braziliensis, L. tropica and L. chagasi. A high degree of sequence similarity (> 99%) among species was found. However, sufficient sequence divergence occurred to permit the design of internal oligonucleotide probes specific for species complexes. This procedure successfully discriminated amongst a wide range of Leishmania isolates. The method detected as few as 10 cultured organisms and detected parasites in tissue samples from experimentally infected animals. Non-radioactive labeling showed the same specificity and sensitivity as radioactive probes. 相似文献
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Nucleoside diphosphate kinase (NDP kinase) from Paramecium was purified to homogeneity. The native enzyme was 80 kDa (by gel filtration), with subunits of 18 and 20 kDa. Near the amino terminus, 15 of 20 residues were identical with those in human NDP kinase, and 17 of 20 with the awd gene product from Drosophila. NDP kinase bound α-labeled ATP and GTP, and a photoreactive GTP analog labeled both subunits. Purified NDP kinase underwent autophosphorylation on a histidine and a serine residue using either ATP or GTP as a substrate. The enzyme also catalyzed acid-stable phosphorylation of casein and phosvitin. This protein kinase activity is distinct from the histidine phosphorylation that is part of the NDP kinase catalytic cycle. Antiserum against the purified protein from Paramecium cross-reacted with 16- to 20-kDa proteins in most species tested, and with a larger protein (44 kDa) in Paramecium, Xenopus, and two human lines. The multiple forms (20 and 44 kDa) of the NDP kinase in Paramecium and its protein kinase activity, suggest that the protein is more than a housekeeping enzyme; it may have regulatory roles such as those of the NDP kinase-like awd protein of Drosophila and Nm23 protein of humans. 相似文献
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G. E. N. NELSON 《Journal of applied microbiology》1979,46(1):93-96
l -Serine dehydratase (SD) was produced to the extent of only 0·04 international unit (iu) per mg dry weight by Aeromonos punctata NRRL B-928 when grown on a chemically defined medium. Addition of 2% (w/v) l -serine to this medium increased SD ten-fold or more, indicating a significant inductive effect. d -Serine was toxic to the organism, making weights and enzyme titres uncertain, but dl -serine induced a specific activity of SD at least equal to that from the l enantiomorph. With complex media containing beef extract, the toxic effect of d -serine was partially overcome, and sufficient growth was obtained to permit reliable analytical figures; d -serine induced slightly better specific activities than the l isomer. dl -Serine at 2% concentration induced a specific activity of 1·2 iu/mg, twice that of l -serine at that level. 相似文献