- Email: [email protected]
Manual labours
TI BS 15 -
MARCH1990
BOOKREVIEWS The many layers of Franklin Ben Franklin Stilled the Waves by Charles Tanford, Duke University Press, 1989, £31.85 (227 pages) ISBN 0 8223 0876 2 This is a book which combines erudition with entertainment. Its theme is the development of surface chemistry, from Franklin's studies on 'pouring oil on troubled waters' in 1773 through to Grendel and Gorter's work in 1925 in which they demonstrated with neat simplicity that a typical cell membrane is essentially a bilayer of lipid, two molecules thick. Somewhat strangely, acceptance and refinement of this bilayer model did not occur until the 1970s; the author discusses, among other matters, some possible reasons for this delay. Charles Tanford is Professor Emeritus of Physiology at Duke University, a former president of the Biochemical Society, a distinguished researcher on biological membranes and now resident in England. An enthusiast also for the history of his science, the book develops its theme by diverting, but always relevant, accounts of the personalities of the key men and women centrally involved in the path to the bilayer concept. They include: Pliny the Elder, whose account of the effect of oil on sea waves had interested young Franklin; Joseph Priestley, whose friendship with Franklin led to Priestley's remarkable work in chemistry; the physicists Lord Rayleigh, von Helmholtz and Lord Kelvin; the versatile and brilliant Thomas Young; and, nearer our own century, J. W. Gibbs and Agnes Pockels. Later still, Irving Langmuir, Charles Overton and Evert Gorter are the central figures. All are interesting and unusual people, with Franklin the most interesting of all. Born in Boston and trained as a printer, Franklin was successful as a journalist and publisher, swimmer, politician, statesman and physicist. At 27 he published Poor Richard's Almanac, which was a great success; he 'filled all the little spaces which occurred between the remarkable days of the calendar with proverbial sentences...' concocted by himself. Many are trite platitudes of the 'virtue is its own reward' kind, but he also used the sly and salty wit for which he is familiar, and which deflated some of the preciousness of the age (samples include
his advice to young men to take older mistresses 'because they are so grateful', and to be not deterred from marrying money by a plain face; 'all cats are grey in the dark'). His franchises in printing and other businesses made him rich, and in 1746, when he was nearly 40, he became interested in electricity. At that time this yielded only parlour tricks, but at least the dry air of Philadelphia made these more reproducible than in damp Europe. Franklin's experiments and ideas turned the tricks into a science; made him the best-known scientist of his day; and through his suggestion that lightning was a gigantic electric spark and could by a pointed rod be drawn 'silently out of a cloud before it came nigh enough to strike' he devised the lightning conductor to protect buildings. In this way he showed, probably for the first time, that pure research could lead to practical uses. On another front, as a diplomat, he tried hard to prevent the Anglo-American war; failing in this, he was one of the five men who drafted the Declaration of Independence in 1776, and as ambassador there he secured a valuable alliance with France in 1778. Thereafter, as an urbane raconteur and elder statesman, he maintained his interest in science and politics; and despite being an anglophile, a socialite and womanizer and a tippler, he was seen as a true sample of the virtuous home-spun American sage, complete with rustic fur cape over his own hair (he was unique in the Versailles court in his wiglessness) and with the bifocal spectacles he invented. Wide-
Manual labours Plant Molecular Biology: A Practical Approach edited by C. H. Shaw, IRL Press, 1988. £19.00/US$38.00 (xx + 313 pages) ISBN 1 85221 057 5
Methods in Plant Molecular Biology by M. A. Schuler and R. E.. Zielinski, Academic Press, 1989. £19.00 (spiralbound) (xv + 171 pages) ISBN 0 12 632340 2 One of my favourite sayings is 'ask five molecular biologists how to do something, and you'll get ten different
ranging as ever in his interests, he had records made of the temperature of the North Atlantic and so mapped the Gulf Stream for the first time; present at the first ascents of hydrogen-filled balloons in the 1780s, he foresaw future work on the physics of the atmosphere and even aerial warfare. Along with that other extraordinary adventurer, Benjamin Thompson (Count Rumford) he was to be virtually the only man born in America to generate ideas in science of high novelty and distinction until J. W. Gibbs created chemical thermodynamics, in the late 19th century. Rightly, Tanford's book keeps its focus on Franklin mainly directed to his work on surface effects, but in linking the story of the bilayer membrane with him and the other central figures, it gives a highly readable account of this important biochemical concept. Like a good party, one would wish it to go on longer. Its errors are few (e.g.J.J. Thomson is firmly but erroneously claimed never to have married, which is odd; he and his son G. P. Thomson are among the few father-son pairs of Nobel laureates) and its bibliography is useful, despite some unexpected omissions, such as Franklin's autobiography, and the recent biographies of him by R. W. Clark (1983) and E. Wright (1986). Undoubtedly, this is a book to enjoy.
IAN T. MILLAR Department of Chemistry, The University, Keele, Staffordshire ST5 5BG, UK.
answers'. Everyone, it seems, has their own customized method - and a number of tricks which they use to get things to work. This variation is gradually being translated into an ever increasing number of laboratory manuals, each reflecting a different personal approach. At present, it seems to be the turn of plant molecular biologists to put pen to paper, with at least ten different books being published during the last year. Nevertheless, there is something to be said for having a reference work to consult, and to discover the basis for the individual techniques. If you have some experience in molecular biology, but want to increase your repertoire, or apply your knowledge to plants, then a good book to try is Plant Molecular Biology, A Practical Approach, another in the excellent stepby-step series published by IRL Press. There are chapters on general topics, 119
TIBS 15-MARCH1990 such as analysis of gene expression and structure, or the isolation of mitochondriai DNA, but the emphasis is on the particular difficulties posed by plants - the abundance of nucleases for instance, or the need to break the cell wall. Other chapters cover topics unique to plants and include chloroplast molecular biology, transformation with Agrobacterium and protoplast preparation. The book ends with chapters on three specialized subjects: plant viruses, Chlamydomonas and cyanobacteria, all of which, although amenable to most techniques, nonetheless have their own particular vagaries, about which it is useful to know. Throughout the book, the usual format of the 'Practical Approach' series is taken, with tables of detailed step-by-step protocols interspersed in the more discursive text. The latter, although not essential in order to follow the procedures, provides the rationale behind the techniques, and also considers the limitations, so that adapting them for your own system is much easier. One
aspect that is different from most of the earlier books in the series is that it is available in spiralbound format - much more 'practical' for the bench than paperback. The intended audience for Methods in Plant Molecular Biology is quite different. It is specifically designed for use as a manual for an undergraduate laboratory course, and is based on one which is run at the University of Illinois. The experiments described introduce a wide range of different techniques, starting from basic cloning and analysis of DNA, through translation in vitro by isolated chloroplasts and the wheat germ system, and concluding with dideoxynucleotide DNA sequencing and transformation with Agrobacterium. This course is meant to be done two days a week over a 15-week semester, but it is possible to pick out small groups of experiments to illustrate more specific techniques, such as basic cloning or characterization of a genomic DNA clone, which could be done in shorter periods. Each experiment has an introduction which explains the overall
procedure, and the rationale for doing it. The protocols themselves are clearly presented and easy to follow, and include interesting snippets of information, such as why chloroform is often included in a phenol extraction, or why different proteins give different responses with the Lowry test. In addition, there are highlighted warnings to alert the students to potential dangers to themselves, or to their experimental results, and notes for instructors to facilitate the setting up and running of the practicals. Its value, however, is likely to be confined to someone thinking of instituting a practical course in this area, rather than as a research laboratory manual. In the end, whichever book you buy, I bet that within a short period of time you will be developing your own way of doing things - and then you can write your own 'definitive' manual!
Shedding light on living cells
the light microscope, is being used increasingly to study the dynamics of cellular processes. This renaissance of the light microscope has come about partly because of the development of techniques that facilitate the non-invasive observation of live cells. Phase and Nomarski optics convert small refractive index differences within a sample into intensity changes. More recently, electronic contrast enhancement has pushed the limits of what can be detected by Nomarski optics to the extent that individual microtubules with a diameter of one-twentieth the wavelength of visible light may be clearly discerned in favourable circumstances. Powerful though these techniques are, they are undiscriminating in that all structures that have a different refractive index from their immediate environment will be imaged. Stains with specific affinities have long been used as a method of discriminating a structure of interest from a complex background in fixed cells. Fluorescent stains have become increasingly used because of the high detection sensitivities that may be obtained. Immunofluorescence techniques have been developed to the extent that a specific stain may be constructed for practically any cellular component that possesses a unique hapten. In recent years a number of research groups have developed methods for using selective fluorescent staining techniques in living cells. These methods open up the exciting possibility of being able to follow
the fate of a particular cellular component during normal cellular processes. Furthermore, fluorochromes have been developed that alter their fluorescence emission characteristics when they are bound to a specific ion species; others respond to changes in membrane potential. It is therefore possible to use these indicator fluorochromes to measure important physiological parameters optically, such as free calcium levels, cellular pH and membrane potential. The application of fluorescence microscopy to living cells is often not a straightforward matter. Problems such as low fluorescent intensity and phototoxicity often arise. Given the state of the field and the potential power of these techniques it is timely that the American Society for Cell Biology has devoted Volumes 29 and 30 of their series Methods in Cell Biology to the subject of fluorescence microscopy of living cells in culture. The editors are to be commended for producing an excellent, informative reference work which will be of great use to all who are either using these techniques, or contemplating using them. All the contributors seem to have done their best to help and inform rather than to dazzle and intimidate. There are many hints and tips included that are not present in the original papers. The topics range from basic fluorescence microscopy to methods for evaluating the performance of optical instruments; the incorporation of macromolecules into cells to fluorescent analogue
Methods in Cell Biology, Vols 29 and 30; Fluorescence Microscopy of Living Cells in Culture, Parts A and B edited by Yu-I.i Wang and D. Lansing Taylor, Academic Press, 1989. Part A $59.00 (xi + 333 pages) ISBN 0 12 564129 X Part B $94.00 (xiv + 503 pages) ISBN 0 12 564130 3 Even the most die-hard biochemist or molecular biologist now appreciates that a cell is not simply an oily bag containing a complex mixture of reacting chemicals, but rather it is an intricate, dynamic, selfassembling machine with a rich variety of component parts. Many basic cellular functions such as karyokinesis, cytokinesis, locomotion and secretion operate by the assembly and disassembly of spatially ordered mechanisms. This, in turn, requires the vectored intracellular transport of specific subcellular components. In order to understand how such a machine works, it is essential to have both information on the structure and organization of the component parts, together with a knowledge of how these structures change over time. The electron microscope has given us detailed, highresolution snapshots of cellular components but its humble father, 120
ALISON SMITH Universityof Cambridge,Departmentof Botany, Downing Street, CambridgeCB2 3EA, UK.
MARCH1990
BOOKREVIEWS The many layers of Franklin Ben Franklin Stilled the Waves by Charles Tanford, Duke University Press, 1989, £31.85 (227 pages) ISBN 0 8223 0876 2 This is a book which combines erudition with entertainment. Its theme is the development of surface chemistry, from Franklin's studies on 'pouring oil on troubled waters' in 1773 through to Grendel and Gorter's work in 1925 in which they demonstrated with neat simplicity that a typical cell membrane is essentially a bilayer of lipid, two molecules thick. Somewhat strangely, acceptance and refinement of this bilayer model did not occur until the 1970s; the author discusses, among other matters, some possible reasons for this delay. Charles Tanford is Professor Emeritus of Physiology at Duke University, a former president of the Biochemical Society, a distinguished researcher on biological membranes and now resident in England. An enthusiast also for the history of his science, the book develops its theme by diverting, but always relevant, accounts of the personalities of the key men and women centrally involved in the path to the bilayer concept. They include: Pliny the Elder, whose account of the effect of oil on sea waves had interested young Franklin; Joseph Priestley, whose friendship with Franklin led to Priestley's remarkable work in chemistry; the physicists Lord Rayleigh, von Helmholtz and Lord Kelvin; the versatile and brilliant Thomas Young; and, nearer our own century, J. W. Gibbs and Agnes Pockels. Later still, Irving Langmuir, Charles Overton and Evert Gorter are the central figures. All are interesting and unusual people, with Franklin the most interesting of all. Born in Boston and trained as a printer, Franklin was successful as a journalist and publisher, swimmer, politician, statesman and physicist. At 27 he published Poor Richard's Almanac, which was a great success; he 'filled all the little spaces which occurred between the remarkable days of the calendar with proverbial sentences...' concocted by himself. Many are trite platitudes of the 'virtue is its own reward' kind, but he also used the sly and salty wit for which he is familiar, and which deflated some of the preciousness of the age (samples include
his advice to young men to take older mistresses 'because they are so grateful', and to be not deterred from marrying money by a plain face; 'all cats are grey in the dark'). His franchises in printing and other businesses made him rich, and in 1746, when he was nearly 40, he became interested in electricity. At that time this yielded only parlour tricks, but at least the dry air of Philadelphia made these more reproducible than in damp Europe. Franklin's experiments and ideas turned the tricks into a science; made him the best-known scientist of his day; and through his suggestion that lightning was a gigantic electric spark and could by a pointed rod be drawn 'silently out of a cloud before it came nigh enough to strike' he devised the lightning conductor to protect buildings. In this way he showed, probably for the first time, that pure research could lead to practical uses. On another front, as a diplomat, he tried hard to prevent the Anglo-American war; failing in this, he was one of the five men who drafted the Declaration of Independence in 1776, and as ambassador there he secured a valuable alliance with France in 1778. Thereafter, as an urbane raconteur and elder statesman, he maintained his interest in science and politics; and despite being an anglophile, a socialite and womanizer and a tippler, he was seen as a true sample of the virtuous home-spun American sage, complete with rustic fur cape over his own hair (he was unique in the Versailles court in his wiglessness) and with the bifocal spectacles he invented. Wide-
Manual labours Plant Molecular Biology: A Practical Approach edited by C. H. Shaw, IRL Press, 1988. £19.00/US$38.00 (xx + 313 pages) ISBN 1 85221 057 5
Methods in Plant Molecular Biology by M. A. Schuler and R. E.. Zielinski, Academic Press, 1989. £19.00 (spiralbound) (xv + 171 pages) ISBN 0 12 632340 2 One of my favourite sayings is 'ask five molecular biologists how to do something, and you'll get ten different
ranging as ever in his interests, he had records made of the temperature of the North Atlantic and so mapped the Gulf Stream for the first time; present at the first ascents of hydrogen-filled balloons in the 1780s, he foresaw future work on the physics of the atmosphere and even aerial warfare. Along with that other extraordinary adventurer, Benjamin Thompson (Count Rumford) he was to be virtually the only man born in America to generate ideas in science of high novelty and distinction until J. W. Gibbs created chemical thermodynamics, in the late 19th century. Rightly, Tanford's book keeps its focus on Franklin mainly directed to his work on surface effects, but in linking the story of the bilayer membrane with him and the other central figures, it gives a highly readable account of this important biochemical concept. Like a good party, one would wish it to go on longer. Its errors are few (e.g.J.J. Thomson is firmly but erroneously claimed never to have married, which is odd; he and his son G. P. Thomson are among the few father-son pairs of Nobel laureates) and its bibliography is useful, despite some unexpected omissions, such as Franklin's autobiography, and the recent biographies of him by R. W. Clark (1983) and E. Wright (1986). Undoubtedly, this is a book to enjoy.
IAN T. MILLAR Department of Chemistry, The University, Keele, Staffordshire ST5 5BG, UK.
answers'. Everyone, it seems, has their own customized method - and a number of tricks which they use to get things to work. This variation is gradually being translated into an ever increasing number of laboratory manuals, each reflecting a different personal approach. At present, it seems to be the turn of plant molecular biologists to put pen to paper, with at least ten different books being published during the last year. Nevertheless, there is something to be said for having a reference work to consult, and to discover the basis for the individual techniques. If you have some experience in molecular biology, but want to increase your repertoire, or apply your knowledge to plants, then a good book to try is Plant Molecular Biology, A Practical Approach, another in the excellent stepby-step series published by IRL Press. There are chapters on general topics, 119
TIBS 15-MARCH1990 such as analysis of gene expression and structure, or the isolation of mitochondriai DNA, but the emphasis is on the particular difficulties posed by plants - the abundance of nucleases for instance, or the need to break the cell wall. Other chapters cover topics unique to plants and include chloroplast molecular biology, transformation with Agrobacterium and protoplast preparation. The book ends with chapters on three specialized subjects: plant viruses, Chlamydomonas and cyanobacteria, all of which, although amenable to most techniques, nonetheless have their own particular vagaries, about which it is useful to know. Throughout the book, the usual format of the 'Practical Approach' series is taken, with tables of detailed step-by-step protocols interspersed in the more discursive text. The latter, although not essential in order to follow the procedures, provides the rationale behind the techniques, and also considers the limitations, so that adapting them for your own system is much easier. One
aspect that is different from most of the earlier books in the series is that it is available in spiralbound format - much more 'practical' for the bench than paperback. The intended audience for Methods in Plant Molecular Biology is quite different. It is specifically designed for use as a manual for an undergraduate laboratory course, and is based on one which is run at the University of Illinois. The experiments described introduce a wide range of different techniques, starting from basic cloning and analysis of DNA, through translation in vitro by isolated chloroplasts and the wheat germ system, and concluding with dideoxynucleotide DNA sequencing and transformation with Agrobacterium. This course is meant to be done two days a week over a 15-week semester, but it is possible to pick out small groups of experiments to illustrate more specific techniques, such as basic cloning or characterization of a genomic DNA clone, which could be done in shorter periods. Each experiment has an introduction which explains the overall
procedure, and the rationale for doing it. The protocols themselves are clearly presented and easy to follow, and include interesting snippets of information, such as why chloroform is often included in a phenol extraction, or why different proteins give different responses with the Lowry test. In addition, there are highlighted warnings to alert the students to potential dangers to themselves, or to their experimental results, and notes for instructors to facilitate the setting up and running of the practicals. Its value, however, is likely to be confined to someone thinking of instituting a practical course in this area, rather than as a research laboratory manual. In the end, whichever book you buy, I bet that within a short period of time you will be developing your own way of doing things - and then you can write your own 'definitive' manual!
Shedding light on living cells
the light microscope, is being used increasingly to study the dynamics of cellular processes. This renaissance of the light microscope has come about partly because of the development of techniques that facilitate the non-invasive observation of live cells. Phase and Nomarski optics convert small refractive index differences within a sample into intensity changes. More recently, electronic contrast enhancement has pushed the limits of what can be detected by Nomarski optics to the extent that individual microtubules with a diameter of one-twentieth the wavelength of visible light may be clearly discerned in favourable circumstances. Powerful though these techniques are, they are undiscriminating in that all structures that have a different refractive index from their immediate environment will be imaged. Stains with specific affinities have long been used as a method of discriminating a structure of interest from a complex background in fixed cells. Fluorescent stains have become increasingly used because of the high detection sensitivities that may be obtained. Immunofluorescence techniques have been developed to the extent that a specific stain may be constructed for practically any cellular component that possesses a unique hapten. In recent years a number of research groups have developed methods for using selective fluorescent staining techniques in living cells. These methods open up the exciting possibility of being able to follow
the fate of a particular cellular component during normal cellular processes. Furthermore, fluorochromes have been developed that alter their fluorescence emission characteristics when they are bound to a specific ion species; others respond to changes in membrane potential. It is therefore possible to use these indicator fluorochromes to measure important physiological parameters optically, such as free calcium levels, cellular pH and membrane potential. The application of fluorescence microscopy to living cells is often not a straightforward matter. Problems such as low fluorescent intensity and phototoxicity often arise. Given the state of the field and the potential power of these techniques it is timely that the American Society for Cell Biology has devoted Volumes 29 and 30 of their series Methods in Cell Biology to the subject of fluorescence microscopy of living cells in culture. The editors are to be commended for producing an excellent, informative reference work which will be of great use to all who are either using these techniques, or contemplating using them. All the contributors seem to have done their best to help and inform rather than to dazzle and intimidate. There are many hints and tips included that are not present in the original papers. The topics range from basic fluorescence microscopy to methods for evaluating the performance of optical instruments; the incorporation of macromolecules into cells to fluorescent analogue
Methods in Cell Biology, Vols 29 and 30; Fluorescence Microscopy of Living Cells in Culture, Parts A and B edited by Yu-I.i Wang and D. Lansing Taylor, Academic Press, 1989. Part A $59.00 (xi + 333 pages) ISBN 0 12 564129 X Part B $94.00 (xiv + 503 pages) ISBN 0 12 564130 3 Even the most die-hard biochemist or molecular biologist now appreciates that a cell is not simply an oily bag containing a complex mixture of reacting chemicals, but rather it is an intricate, dynamic, selfassembling machine with a rich variety of component parts. Many basic cellular functions such as karyokinesis, cytokinesis, locomotion and secretion operate by the assembly and disassembly of spatially ordered mechanisms. This, in turn, requires the vectored intracellular transport of specific subcellular components. In order to understand how such a machine works, it is essential to have both information on the structure and organization of the component parts, together with a knowledge of how these structures change over time. The electron microscope has given us detailed, highresolution snapshots of cellular components but its humble father, 120
ALISON SMITH Universityof Cambridge,Departmentof Botany, Downing Street, CambridgeCB2 3EA, UK.