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31.
32.
Polymerase chain reaction (PCR) technology has revolutionized the process of isolating and amplifying segments of DNA. One powerful application of PCR is its use in precise site-directed mutagenesis (SDM). SDM provides an elegant tool for scientists and engineers to explore biocatalytic mechanisms and processes to understand the structural-functional relationships of enzymes and other proteins. This article reviews techniques and methodology used in site-directed mutagenesis of genes by PCR. 相似文献
33.
An inflatable minirhizotron system for root observations with improved soil/tube contact 总被引:3,自引:1,他引:2
Commonly used minirhizotrons consisting of a transparent tube inserted into the soil seldom attain good contact between the tube and the soil, which leads to root growth occurring in a gap rather than in the soil. A new system is described involving an inflatable flexible rubber wall, made from a modified motorcycle tube. Pressure ensures a proper tube/soil contact so that the environmental circumstances for root growth along the tube more closely correspond to those in the undisturbed soil. Before the endoscope slide is introduced into the minirhizotron for taking pictures, the inflatable tube is removed, so that there is no-often opaque-wall between the endoscope and the roots. This improves the picture quality and facilitates the analysis of root images. 相似文献
34.
Rapid and simple isolation of pure photosystem II core and reaction center particles from spinach 总被引:2,自引:2,他引:0
Peter J. van Leeuwen Maaike C. Nieveen Erik Jan van de Meent Jan P. Dekker Hans J. van Gorkom 《Photosynthesis research》1991,28(3):149-153
Pure and active oxygen-evolving PS II core particles containing 35 Chl per reaction center were isolated with 75% yield from spinach PS II membrane fragments by incubation with n-dodecyl--D-maltoside and a rapid one step anion-exchange separation. By Triton X-100 treatment on the column these particles could be converted with 55% yield to pure and active PS II reaction center particles, which contained 6 Chl per reaction center.Abbreviations Bis-Tris
bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane
- Chl
chlorophyll
- CP29
Chl a/b protein of 29 kDa
- Cyt b
559
cytochrome b
559
- DCBQ
2,5-dichloro-p-benzo-quinone
- LHC II
light-harvesting complex II, predominant Chl a/b protein
- MES
2-[N-Morpholino]ethanesulfonic acid
- Pheo
pheophytin
- PS H
photosystem II
- QA
bound plastoquinone, serving as the secondary electron acceptor in PS II (after Pheo)
- SDS
sodiumdodecylsulfate 相似文献
35.
A model is presented describing the relationship between chlorophyll fluorescence quenching and photoinhibition of Photosystem (PS) II-dependent electron transport in chloroplasts. The model is based on the hypothesis that excess light creates a population of inhibited PS II units in the thylakoids. Those units are supposed to posses photochemically inactive reaction centers which convert excitation energy to heat and thereby quench variable fluorescence. If predominant photoinhibition of PS II and cooperativity in energy transfer between inhibited and active units are presumed, a quasi-linear correlation between PS II activity and the ratio of variable to maximum fluorescence, FVFM, is obtained. However, the simulation does not result in an inherent linearity of the relationship between quantum yield of PS II and FVFM ratio. The model is used to fit experimental data on photoinhibited isolated chloroplasts. Results are discussed in view of current hypotheses of photoinhibition.Abbreviations FM
maximum total fluorescence
- F0
initial fluorescence
- FV
maximum variable fluorescence
- PS
Photosystem
- QA, QB
primary and secondary electron acceptors of Photosystem II 相似文献
36.
Universal primers for amplification of three non-coding regions of chloroplast DNA 总被引:90,自引:0,他引:90
Six primers for the amplification of three non-coding regions of chloroplast DNA via the polymerase chain reaction (PCR) have been designed. In order to find out whether these primers were universal, we used them in an attempt to amplify DNA from various plant species. The primers worked for most species tested including algae, bryophytes, pteridophytes, gymnosperms and angiosperms. The fact that they amplify chloroplast DNA non-coding regions over a wide taxonomic range means that these primers may be used to study the population biology (in supplying markers) and evolution (inter- and probably intraspecific phylogenies) of plants. 相似文献
37.
Jac M. M. J. G. Aarts Jan G. J. Hontelez Peter Fischer Ruud Verkerk Albert van Kammen Pim Zabel 《Plant molecular biology》1991,16(4):647-661
With a view to cloning the root-knot nematode resistance gene Mi in tomato by chromosome walking, we have developed a molecular probe for the tightly linked acid phosphatase-1 (Aps-1) locus. The acid phosphatase-1 allozyme (APS-11), encoded by the Aps-1
1 allele originating from Lycopersicon peruvianum, was purified to apparent homogeneity from tomato roots and suspension cells. Microsequencing of CNBr and tryptic peptides generated from APS-11 provided a partial amino acid sequence, which accounted for approximately 23% of the protein and revealed two stretches of homology with soybean proteins KSH3 and VSP27, comprising 22 matches within 26 amino acid residues. The partial amino acid sequence information enabled us to isolate a 2.4 kb genomic Aps-1
1 sequence by means of the polymerase chain reaction (PCR), primed by degenerate pools of oligodeoxyribonucleotides, synthesized on the basis of the amino acid sequences. Synthesis of the 2.4 kb PCR product was specific for genomic templates carrying the L. peruvianum Aps-1
1 allele. Crucial to the priming specificity and the synthesis of the 2.4 kb genomic sequence was the use of degenerate primer pools in which the number of different primer species was limited by incorporating deoxyinosine phosphate residues at three and four base ambiguities. In using cDNA as a template, a 490 bp sequence was obtained, indicating a high proportion of intron sequences in the 2.4 kb genomic Aps-1
1 sequence. The Aps-1
1 origin of the PCR product was confirmed by RFLP (restriction fragment length polymorphism) analysis, using both a chromosome 6 substitution line and a pair of nearly isogenic lines, differing for a small chromosomal region around the Aps-1/Mi loci. 相似文献
38.
Ignacio R. Rodriguez Pedro Gonzales J. Samuel Zigler Teresa Borrás 《生物化学与生物物理学报:疾病的分子基础》1992,1180(1):44-52
A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens diseases at the molecular level. ζ-crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several ζ-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3′ end of the coding region. The deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic ζ-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the α-crystallin gene disclosed a dinucleotide delection of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered ζ-crystallin protein. This if the first time a genomic mutation in an enzyme/crytallin gene has been directly linked to a congenital cataract. 相似文献
39.
Summary
Agapanthus umbelatus pollen tubes (PTs) display a number of different growth patterns when germinated in an electric field of 750 mV· mm–1. When pollen is germinated near the cathode (82.44% of orientation to the cathode side) or near the anode (55.35% of orientation to the anode), growth is oriented parallel to the applied field but when germinated at an intermediate position, there is random growth. An increase and decrease in the orientation rates as well as reversion of the polarized growth were observed when the growth conditions were systematically altered. These findings reflect the influence of different ionic currents present in the germination medium. These ionic currents induce the formation of ionic gradients, which were monitored by ion-HPLC. The individual omission of Ca2+, K+, Mg2+ and Cl– suppresses or alters the oriented growth pattern. The presence of ionic gradients is not by itself suficient to trigger the polarization of tube growth as the presence of an electric field which drives the ionic currents is essential for this to occur.Abbreviations PT
Pollen tube
- DNS
3,5-dinitro salycilic acid;
- TP
transient polarization
- HPLC
high precision liquid chroma tography
- DC
direct current 相似文献
40.