【正文】 they are, in reality, only onetenth this diameter. (Courtesy of P. Sammeh and G. Borisy.) Figure 359. Methods to introduce a membraneimpermeant substance into a cell. (A) the substance is injected through a micropipette. (B) the cell membrane is made transiently permeable to the substance by disrupting the membrane structure with a brief but intense electric shock. (C) membranebounded vesicles are loaded with the desired substance and then induced to fuse with the target cells. Figure 364. Indirect immunocytochemistry. The method is very sensitive because the primary antibody is itself recognized by many molecules of the secondary antibody. The secondary antibody is covalently coupled to a marker molecule that makes it readily detectable. Commonly used marker molecules include fluorescein or rhodamine dyes, the enzyme horseradish peroxidase or colloidal gold spheres, and the enzymes alkaline phosphatase or peroxidase. Figure 365. Preparation of hybridomas that secrete monoclonal antibodies against a particular antigen (X). The selective growth medium used contains an inhibitor (aminopterin) that blocks the normal biosynthetic pathways by which nucleotides are made. The cells must therefore use a bypass pathway to synthesize their nucleic acids, and this pathway is defective in the mutant cell line to which the normal B lymphocytes are fused. Because neither cell type used for the initial fusion can grow on its own, only the hybrid cells survive. 7. Monoclonal Antibodies 8. Gene Knockout mice Mario Capecchi (Late 1980s) (University of Utah) embryonic stem cells in inner cell mass as target cells 1/104 cells undergo a process of homologous rebination. 9. The technique for the take apart and gather up of cell, and microscope manipulation ?Preparation and reform of karyoplast and cytoplast ?Transgenic animals and plants Transgenic mice 10 weeks 44g and 29g THANKS! 。 C, adapted from original provided by B. Furugren.) 5. Protein structure A. Xray crystallography Figure 349. NMR spectroscopy. (A) An example of the data from an NMR machine. This is a twodimensional NMR spectrum derived from the carboxylterminal domain of the enzyme cellulase. The spots represent interactions between hydrogen atoms that are near neighbors in the protein and hence their distance apart. Complex puting methods, in conjunction with the known amino acid sequence, enable possible patible structures to be derived. In (B) 10 structures, which all satisfy the distance constraints equally well, are shown superimposed on one another, giving a good indication of the probable threedimensional structure. (Courtesy of P. Kraulis.) B. NMR spectroscopy Figure 720. In situ hybridization for RNA localization in tissues. Autoradiograph of a section of a very young Drosophila embryo that has been subjected to in situ hybridization using a radioactive DNA probe plementary to a gene involved in segment development. The probe has hybridized to RNA in the embryo, and the pattern of autoradiographic silver grains reveals that the RNA made by the gene (ftz) is localized in alternating stripes across the embryo that are three or four cells wide. At this stage of development (cellular blastoderm), the embryo contains about 6000 cells. (E. Hafen et al, Cell 37:833841, 1984.) 6. Tracing and Assaying Molecules Inside Cells Figure 351. Electronmicroscopic autoradiography. The results of a pulsechase experiment in which pancreatic beta cells were fed 3Hleucine for 5 minutes followed by excess unlabeled leucine (the chase). The amino acid is largely incorporated into insulin, which is destined for secretion. After a 10minute chase the labeled protein has moved from the rough ER to the Golgi stacks (A), where its position is revealed by the black silver grains in the photographic emulsion. After a further 45minute chase the labeled protein is found in electrondense secretory granules (B).(Courtesy of L. Orci, from Diabetes 31:538565) Figure 357. Visualizing intracellular Ca2+ concentrations using a fluorescent indicator. The branching tree of dendrites of the Purkinje cell in the cerebellum receives more than 100,000 synapses from other neurons. The output from the cell is conveyed along the single axon seen leaving the cell body at the bottom of the picture. This image of the intracellular calcium concentration in a single Purkinje cell was taken using a lowlight camera and the Ca2+sensitive fluorescent indictor fura2. The concentration of free Ca2+ is represented by different colors, red being the highest and blue the lowest. (Courtesy of . Tank et al.) Figure 358. Fluorescent analogue cytochemistry. Fluorescence micrograph of the leading edge of a living fibroblast that has been injected with rhodaminelabeled tubulin. The microtubules throughout the cell have incorporated the labeled tubulin molecules. Thus individual microtubules can be detected and their dynamic behavior followed using puterenhanced imaging, as shown here. Although the microtubules appear to be about 181。Farrell.) Figure 346. Western blotting or immunoblotting. The total proteins from dividing tobacco cells in culture are first separated by twod