H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 101
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Cells containing TK convert thiscompound into 5-bromodeoxyuridine monophosphate,which is then converted into a nucleoside triphosphate byother enzymes. The triphosphate analog is incorporated byDNA polymerase into DNA, where it exerts its toxic effects.This pathway is blocked in TK mutants, and thus they areresistant to the toxic effects of 5-bromodeoxyuridine. Similarly, cells lacking the HGPRT enzyme, such as the HGPRTmyeloma cell lines used in producing hybridomas, can be isolated because they are resistant to the otherwise toxic guanine analog 6-thioguanine.Normal cells can grow in HAT medium because eventhough the aminopterin in the medium blocks de novo synthesis of purines and TMP, the thymidine in the medium istransported into the cell and converted into TMP by TK andthe hypoxanthine is transported and converted into usablepurines by HGPRT.
On the other hand, neither TK norHGPRT cells can grow in HAT medium because each lacksan enzyme of the salvage pathway. However, hybrids formedby the fusion of these two mutants will carry a normal TKgene from the HGPRT parent and a normal HGPRT genefrom the TK parent. The hybrids will thus produce bothfunctional salvage-pathway enzymes and will grow on HATmedium.KEY CONCEPTS OF SECTION 6.7Growth and Use of Cultured CellsGrowth of vertebrate cells in culture requires rich media containing essential amino acids, vitamins, fatty acids,and peptide or protein growth factors; the last are frequently provided by serum.■Most cultured vertebrate cells will grow only when attached to a negatively charged substratum that is coatedwith components of the extracellular matrix.■Primary cells, which are derived directly from animal tissue, have limited growth potential in culture and may giverise to a cell strain.
Transformed cells, which are derivedfrom animal tumors or arise spontaneously from primarycells, grow indefinitely in culture, forming cell lines (seeFigure 6-37).■The fusion of an immortal myeloma cell and a singleB lymphocyte yields a hybrid cell that can proliferateindefinitely, forming a clone called a hybridoma (seeFigure 6-38).
Because each individual B lymphocyteproduces antibodies specific for one antigenic determinant (epitope), a hybridoma produces only the mono-■clonal antibody synthesized by its original B-lymphocyteparental cell.HAT medium is commonly used to isolated hybridomacells and other types of hybrid cells.■PERSPECTIVES FOR THE FUTUREA deeper understanding of the integration of cells into tissuesin complex organisms will draw on insights and techniquesfrom virtually all subdisciplines of molecular cell biology: biochemistry, biophysics, microscopy, genetics, genomics, proteomics, and developmental biology. An important set ofquestions for the future deals with the mechanisms by whichcells detect mechanical forces on them and the extracellularmatrix, as well as the influence of their three-dimensionalarrangements and interactions. A related question is how thisinformation is used to control cell and tissue structure andfunction.
Shear stresses can induce distinct patterns of gene expression and cell growth and can greatly alter cell metabolismand responses to extracellular stimuli. Future research shouldgive us a far more sophisticated understanding of the roles ofthe three-dimensional organization of cells and ECM components in controlling the structures and activities of tissues.Numerous questions relate to intracellular signaling fromCAMs and adhesion receptors. Such signaling must be integrated with other cellular signaling pathways that are activated by various external signals (e.g., growth factors) sothat the cell responds appropriately and in a single coordinated fashion to many different simultaneous internal andexternal stimuli. How are the logic circuits constructed thatallow cross-talk between diverse signaling pathways? Howdo these circuits integrate the information from these pathways? How is the combination of outside-in and inside-outsignaling mediated by CAMs and adhesion receptors mergedinto such circuits?The importance of specialized GAG sequences in controlling cellular activities, especially interactions betweensome growth factors and their receptors, is now clear.
Withthe identification of the biosynthetic mechanisms by whichthese complex structures are generated and the developmentof tools to manipulate GAG structures and test their functions in cultured systems and intact animals, we can expecta dramatic increase in our understanding of the cell biologyof GAGs in the next several years.A structural hallmark of CAMs, adhesion receptors,and ECM proteins is the presence of multiple domains thatimpart diverse functions to a single polypeptide chain. Itis generally agreed that such multidomain proteins aroseevolutionarily by the assembly of distinct DNA sequencesencoding the distinct domains.
Genes encoding multipledomains provide opportunities to generate enormous sequence and functional diversity by alternative splicing andthe use of alternate promoters within a gene. Thus, evenAnalyze the Datathough the number of independent genes in the humangenome seems surprisingly small in comparison with otherorganisms, far more distinct protein molecules can be produced than predicted from the number of genes. Such diversity seems especially well suited to the generation ofproteins that take part in specifying adhesive connectionsin the nervous system, especially the brain. Indeed, severalgroups of proteins expressed by neurons appear to havejust such combinatorial diversity of structure.
They includethe protocadherins, a family of cadherins with as many as70 proteins encoded per gene; the neurexins, which comprise more than 1000 proteins encoded by three genes; andthe Dscams, members of the IgCAM superfamily encodedby a Drosophila gene that has the potential to express38,016 distinct proteins owing to alternative splicing. Acontinuing goal for future work will be to describe and understand the molecular basis of functional cell–cell andcell–matrix attachments—the “wiring”—in the nervoussystem and how that wiring ultimately permits complexneuronal control and, indeed, the intellect required to understand molecular cell biology.2.
Cadherins are known to mediate homophilic interactions between cells. What is a homophilic interaction, andhow can it be demonstrated experimentally for E-cadherins?3. What is the normal function of tight junctions? Whatcan happen to tissues when tight junctions do not functionproperly?4. What is collagen, and how is it synthesized? How do weknow that collagen is required for tissue integrity?5. You have synthesized an oligopeptide containing anRGD sequence surrounded by other amino acids.
What is theeffect of this peptide when added to a fibroblast cell culturegrown on a layer of fibronectin absorbed to the tissue culturedish? Why does this happen?6. Blood clotting is a crucial function for mammalian survival. How do the multiadhesive properties of fibronectinlead to the recruitment of platelets to blood clots?7. Using structural models, explain how integrins mediateoutside-in and inside-out signaling.8.
How do changes in molecular connections between theextracellular matrix (ECM) and cytoskeleton give rise toDuchenne muscular dystrophy?KEY TERMSadhesion receptor 199anchoring junction 202basal lamina 202cadherin 199cell-adhesionmolecule (CAM) 198cell line 236cell strain 236cell wall 231connexin 231dystrophin glycoproteincomplex (DGC) 227epithelium 201extracellular matrix(ECM) 199fibril-associatedcollagen 217fibrillar collagen 217fibronectin 220glycosaminoglycan(GAG) 213241HAT medium 238hyaluronan 217hybridoma 238immunoglobulincell-adhesion molecule(IgCAM) 227integrin 199laminin 211monoclonal antibody 237multiadhesive matrixprotein 209paracellular pathway 208plasmodesma 233proteoglycan 209RGD sequence 221selectin 199syndecan 214tight junction 202REVIEW THE CONCEPTS1.
Using specific examples, describe the two phenomenathat give rise to the diversity of adhesive molecules.9. What is the difference between a cell strain, a cell line,and a clone?10. Explain why the process of cell fusion is necessary toproduce monoclonal antibodies used for research.A N A LY Z E T H E DATAResearchers have isolated two E-cadherin mutant isoformsthat are hypothesized to function differently from that of thewild-type E-cadherin.
An E-cadherin negative mammary carcinoma cell line was transfected with the mutant E-cadheringenes A (part a in the figure) and B (part b) (diamonds) andthe wild-type E-cadherin gene (black circles) and comparedto untransfected cells (open circles) in an aggregation assay.In this assay, cells are first dissociated by trypsin treatmentand then allowed to aggregate in solution over a period ofminutes. Aggregating cells from mutants A and B arepresented in panels a and b respectively. To demonstrate thatthe observed adhesion was cadherin-mediated, the cellswere pretreated with a nonspecific antibody (left panel) or afunction-blocking anti-E-cadherin monoclonal antibody(right panel).a. Why do cells transfected with the wild-type E-cadheringene have greater aggregation than control, nontransfectedcells?b.
From these data, what can be said about the function ofmutants A and B?242CHAPTER 6 • Integrating Cells into TissuesNonspecificAggregation (%)(a)Anti-E-cadherin808060604040202000−200204060−200Time (min)204060Time (min)Aggregation (%)(b)808060604040202000−200204060Time (min)c. Why does the addition of the anti-E-cadherin monoclonal antibody, but not the nonspecific antibody, blockaggregation?d. What would happen to the aggregation ability of the cellstransfected with the wild-type E-cadherin gene if the assaywere performed in media low in Ca2?REFERENCESCell–Cell and Cell–Matrix Adhesion:An OverviewGumbiner, B. M. 1996.
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