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- - T a b l e 3,
(a) Time to product introduction (months) Method of transformation
Test engineered phenotype (R1 plants)
First largescale field testing
Product introduction
5
17
41
8
48
72
No regeneration through tissue culture Regeneration
(Agrobacterium)
(b) Transformation of elite varieties through particle acceleration
Time (months) Number of seeds Acres needed
Generation R3 R4
R1
R2
5 10 0
13
17
29
10 3
10 5
0.1
1
10 7 10 2
109 10 4
9
R5
Field test for product quality
order to increase the nutritional v a l u e of this i m p o r t a n t crop.
Acknowledgements We t h a n k T. Ford, A. A n d e r s o n , L. Amerson, B. Cohen, B. H a m m e r and A. F e l d m e i e r for excellent technical assistance, K. Barton for p r o v i d i n g Figure 3 and W. Brill for p r o v i d i n g Table 3. []
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Commercialization 41 >1011 >106
Product introduction
References 1 Hoskin, R. L. (1987) USDA Econom. Res. Service 1-35 2 Christou, P., Murphy, J. E. and Swain, W. F. (1987) Proc. Natl Acad. Sci. USA 84, 3962-3966 3 Lazzeri, P. A., Hildebrand, D. F. and Collins, G. B. (1985) Plant 54oi. Biol. Reporter 3, 160-167 4 Faccioti, D., O'Neal, J. K., Lee, J. and Shewmaker, C. K. (1985) Bio/Tech[]
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Scanning tunnelling microscopy in biotechnology Patricia G. Arscott and Victor A. Bloomfield The scanning tunnelling microscope (STM) is capable of atomic resolution of highly conductive materials. Whether biological molecules can be visualized to the same extent remains an open question, but remarkable progress in the past year confirms the possibility of seeing the fine structure of nucleic acids, proteins, membranes and viruses, and provides evidence that their dynamic interactions can be monitored under conditions approximating to those of the native environment. T h i r t y years ago, Professor Richard F e y n m a n d e l i v e r e d a s p e e c h before the A m e r i c a n Physical Society enP. G. Arscott and V. A. B]oomfield are at the Department of Biochemistry, University of Minnesota, 1479 Gortner Avenue, St Paul MN.55108, USA.
titled ' T h e W o n d e r s That Await a Micro-Microscope'. 1 The questions he raised c o u l d not have b e e n m o r e provocative: w h a t good w o u l d it do to see atoms distinctly? Could w e t h e n not easily answer some of the most central and f u n d a m e n t a l
© 1990, Elsevier Science Publishers Ltd (UK) 0167 - 9430/90/$2.00
nology 3, 241-246 5 Owens, L. and Cress, D. E. (1985) Plant Physiol. 77, 87-94 6 Hinchee, M. A. W., Connor-Ward, D. V., Newell, C. A., McDonnell, R. E., Sato, S. J., Gasser, C. S., Fischhoff, D. A., Re, D. B., Fraley, R. T. and Horsch, R. B. (1988) Bio/ Technology 6, 915-922 7 Christou, P., McCabe, D. E. and Swain, W. F. (1988) Plant Physiol. 87, 671-674 8 Klein, T. M., Wolf, E. D., Wu, R. and Sanford J. C. (1987) Nature 327, 70-73 9 Klein, T. M., Harper, E. C., Svab, Z., Sanford, J. C., Fromm, M. E. and Maliga, P. (1988) Proc. Natl Acad. Sci. USA 85, 8502-8505 10 Christou, P. and Yang, N-S. (1989) Ann. Bot. 64, 225-234 11 Jefferson, R. A. (1987) Plant Mol. Biol. Rep. 5, 387-405 12 Christou, P. Ann. Bot. (in press) 13 McCabe, D. E., Swain, W. F., Martinell, B. J. and Christou, P. (1988) Rio~Technology 6, 923-926 14 Christou, P., Swain, W. F., Yang, N-S. and McCabe, D. E. (1989) Proc. Natl Acad. Sci. USA 86, 7500-7504 15 Christou, P. and Swain, W. F. (1990) Theor. App]. Genet. 79, 337-341 16 Bennett, M. D. and Smith, J. B. (1976) Phil. Trans. R..Soc. Lond. B 274, 227-274
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questions of biology? A n d c o u l d we not change the a r r a n g e m e n t of atoms and m a n i p u l a t e the genes and molecules t h e y specify to n e w and useful p u r p o s e ? At the time, although r e s o l u t i o n on material samples to 2A or 3A was possible, the highest r e s o l u t i o n obt a i n e d for biological s p e c i m e n s w i t h the electron m i c r o s c o p e was about l o A - barely sufficient to distinguish single-stranded from double-stranded DNA. Optics and t e c h n i q u e s of sample preparation i m p r o v e d over the next t w e n t y years, but the necessity of exposing delicate structures to h i g h e r energy electron beams imp o s e d severe constraints on attaining higher resolution. D e v e l o p m e n t s that finally led to major breakt h r o u g h s in m i c r o s c o p y and biotechnology occurred independently, at about the same time. Routine use of r e c o m b i n a n t DNA t e c h n i q u e s and the first scanning t u n n e l l i n g microscope were i n t r o d u c e d in the late 1970s and early 1980s.
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~Fig.
1
Scanning tunnelling microscope The STM is capable of resolving features that are an atom wide and 1/100 of an atom high on a metal surface 2. It is not a new and improved electron microscope, but an entirely different type of instrument modelled on the topografiner, a scanning probe profilometer that gives three-dimensional line plots of surface topography. The imaging system of the STM depends on the p h e n o m e n o n of electron tunnelling: electrons in a Conductive material face a barrier of infinite width at the material's surface. However, in response to an applied voltage and the close proximity of another conductor, electrons can be induced to tunnel across to the second conductor; the smaller the gap, the greater the current. The imaging system uses, typically, currents and voltages of - l n A and 100 mV which, applied across a gap of 1-2 nm and confined to a small cross-sectional area, can generate high current density. However, due to variations in geometry and local electron density at the tip and sample surface, it is difficult to calculate the field strength or current density at the sample surface, and the risk of dielectric breakdown or beam damage cannot be realistically assessed. Although it may seem surprising that biological materials such as DNA (which are essentially insulators or poor semiconductors), can be examined without damage in the absence of a protective coating, images of apparently intact molecules can be obtained, with dimension measurements close to crystallographic values. The advantages of scanning profilometry had not been fully realized until G. Binnig and H. Rohrer, in studying surface properties of metals and semi-conductors at IBM, Zurich, found a way to damp vibrations and so keep the probe from crashing w h en scanning at very close range. In conjunction with the use of piezoelectric crystals that may be stably positioned on the subatomic scale, this enabled a scan to be maintained within a few &ngstrSms of a surface, low enough to permit a current of electrons to tunnel between the tip of the probe and the surface, i.e., to pass through regions of nonconductivity despite the energy barrier. Using the strength of the current as
--
Scan Head containing piezoelectric crystal z
Electrical connection Probe
Scan Head rests on screws to hold it above the sample
~ x
Sample on ubstrate
Base Cable to the control unit
Suspended platform or table designed to damp vibrations
The scanning tunnelling microscope (STM) and its mode of operation. The scanning process is entirely computer-controlled. A specimen is positioned beneath a needle-like probe that moves back and forth in a raster pattern a few &ngstrSms above the surface. The probe is at the base of a scan head containing a piezoelectric crystal that contracts and expands in response to voltage applied to the control elements for lateral (x, y) and vertical (z) movement. Voltage to the z element is supplied through a feedback loop that adjusts the gain to maintain either a constant distance or a constant signal from the surface. The set point, i.e., the level o f current detected by the probe, is typically about 1 nA and the applied voltage about 100 mV. Scan sizes and rates are variable. The x, y and z co-ordinates are digitized, stored, and m a y be displayed as grey- or color-scale images on the computer screen. Diagram not drawn to scale.
a gauge of gap distance, they showed that they could trace the intricate contours of a surface by holding the current constant and recording the vertical movement of the probe as it is scanned. The basic design and functioning of an STM is given in Fig. 1. The success of the STM sparked a series of spin-offs that allow different aspects of a surface to be examined and compared. The first of these instruments, the atomic force microscope (AEM), was invented by Binnig and colleagues 3 to circumvent one of the main limitations of the STM: a specimen cannot be visualized if it does not adequately conduct electrons. They modified the prototype STM design by mounting a diamond-tipped probe on a cantilever and scanning the specimen so as to lightly touch the tip and deflect the cantilever toward an electrode a few fingstr6ms away. The gap dis-
tance between the cantilever and the electrode, determined by the strength of a tunnelling current as in the STM, gave a measure of interatomic forces and hence the arrangement of atoms at the specimen surface. Although their A r M offered considerable advantages, it was a more difficult instrument to operate and not as sensitive as the STM. Newer models use optical interferometry to detect deflection of the cantilever and have a m u c h lighter touch than the original ( - 1 0 -10 N). Both the STM and A r M are commercially available, and magnetic force, optical, ion conductance, thermal, and acoustic microscopes are being developed along similar lines 4.
Advantages over other methods of obtaining structural information It is not yet known h o w high a resolution of biomolecular structure can be attained with any of the
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--Fig. 2,
Poly(dG-me5dC) . poly(dG-me5dC) in the left-handed Z form. Reprinted, with permission, from Ref. 6.
scanning probe microscopes, but it is already evident that the STM yields detailed structural information that cannot presently be obtained by electron microscopy or other means. DNA, RNA and proteins have been successfully imaged without shadow or stain. Physical methods such as optical diffraction and circular dichroism average over many molecules, whereas STMs may examine single molecules in a heterogeneous population and detect localized, intramolecular variations in structure. Such specific information is invaluable in a wide range of applications; for example, assaying the specificity of enzyme activity or antibody and fluorescent labeling, testing the therapeutic and toxicological effects of drugs, monitoring biodegradation processes, and developing new methods of genetic engineering.
Progress in imaging biological molecules Our STM images of DNA 5,6 and RNA 5 are sufficiently well resolved to permit reliable measurements of helical periodicity to be obtained,
enabling comparison of the conformation and hydration state of these molecules under different conditions. Images of DNA in the lefthanded Z form 6 are shown in Fig. 2. The handedness is evident, and in certain orientations the zigzag backbone and grooves in the molecule can be clearly discerned. Periodicities agree with the values obtained by fiber diffraction and indicate that the specimens are not significantly denatured. In Dunlap and Bustamante's images of single-stranded polydeoxyadenylate7, the imidazole and pyrimidine rings of the bases are clearly outlined against the substrate. Their dimensions and spacing are consistent with crystallographic data. Cricenti and co-workers 8 may have succeeded in visualizing the phosphate atoms in DNA fixed with tris(1-aziridinyl) phosphine oxide, a compound that has an affinity for sugars. When the STM image is aligned with a model of B-DNA, bright spots adjacent to the sugars appear at the positions of phosphates in the helical backbone. Interpretation of the image is somewhat
equivocal in that similar spots in the minor groove appear to correspond to bases; unfortunately, none is present in the major groove to confirm this. Can STM be used to sequence nucleic acids? Dunlap and Bustamante's results 7 indicate the feasibility of differentiating purines from pyrimidines, but at this stage it is not clear whether all four bases can be identified without specific labels. Attempts to sequence DNA by electron microscopy failed for at least two reasons. Single-stranded DNAs and polynucleotides tend to fold back on themselves so that label ligands are superimposed or clumped, and when bombarded by heavy doses of electrons, the ligands dissociate. The higher resolution of the STM should make it easier to trace the configuration of single strands and to see individual labels, but better spreading techniques are needed for optimal results. The lower electron energies used in the STM should diminish the problem of labels dissociating. Sequencing methods based on the specificity of enzyme cleavage are well developed
154
and unlikely to be supplanted. Nevertheless, it would seem worthwhile to take advantage of the fact that a wider variety of labels can be used in STM experiments than in electron microscopy; heavy metals are not needed for contrast. Smaller ligands with a higher specificity could make it possible to label the bases in the grooves of double-stranded DNA and to identify accessible sites for other types of interactions. Studies of individual amino acids 9 may open the way for sequencing proteins too. Current work is focused mainly on secondary and tertiary structure. DNA, RNA 5-8,1°-19 and the other biological molecules that have been examined all have a recognizable size a n d shape, or form extensive two-dimensional arrays on the substrate; these include a DNAprotein complex 9,2°,21, the enzymes phosphorylase 22, kinase 22, and catalase 23, fibrous aggregates of collagen 24, fibrinogen 25 and tubulin 26, albumin 27, ferritin 28, bacterial proteins and membranes 29-32, and whole bacteriophages 33,34. For some of these, the specimens were coated with platinum or platinum/carbon, and it was not possible to resolve features smaller than the grain size of the metal.
Resolution, contrast, and interpretation of the image The quality of the image obtained depends primarily on the stability of the system; on how closely the probe can approach the specimen and how stationary the specimen remains under the force of the probe. In contrast to metals and semiconductors, biological molecules are flexible and mobile. Clusters of sorbic acid deposited on graphite have been observed to move away from the scan area at the rate of i A/min 35. Even larger adsorbates are known to diffuse across the surface, and it has been noticed that some of the best STM images are obtained where such movements are limited, as at steps on the substrate or in closely packed aggregates. Comparisons of images taken at different scan rates indicate that small-scale movements of sidechains and domains can cause an image to blur. There is thus some disadvantage to being able to examine naked molecules; the stains and shadows applied routinely in electron microscopy, and which may be
I - I B T E C H - J U N E 1990 [Vol. 8]
used in STM to enhance contrast, also confer stability to the specimen. No matter how stable the system, one cannot hope to see atoms in the specimen or in the substrate unless the tip of the probe consists of a single atom. This is not as difficult as might be expected; STM probes are typically made of platinum/iridium or tungsten wire, and may be cut with scissors and sharpened to a fine point by electrochemical etching or machine lathing, or left as they are. A crude cut has jagged edges and points as sharp as any tip that can be produced with more expense and effort, but there is a fairly high probability of getting a tunnelling current from more than one point. Probes are routinely tested on a flat substrate of known pattern and dimensions before the specimen is scanned. However, there is no guarantee of a single tunnelling tip on a rough surface. The tunnelling current is proportional to the effective radius of the tip plus the surface curvature beneath the tip (1/Reff=l/Rtip+l/Rsurf), multiplied by the exponential of the gap distance times the square root of the work function 36. The latter term relates to the conductivity of the specimen, and is usually an unknown quantity in experiments with biological molecules. The reported values for resistivity and activation energy in the few molecules that have been assayed are probably inaccurate. DNA and RNA, for example, have been characterized as semiconductors in stretched fibers and gels 37,38. Given the likelihood of sequence-dependent variations in conductivity along the helices, as well as localized differences in hydration, ionization, and orientation, one expects that open pathways for electron transfer must be in register for measurements across several molecules to give information about individual conductivity. Neither the specimen's thickness nor the presence of specific residues is a particularly good indicator of whether it is an insulator or a conductor. The probability of electron tunnelling through a specimen in many small steps may be greater than the probability of tunnelling in one long step 16. Computer simulations based on Feynman's path integral method of defining the tunnelling pathway in proteins suggest that the strategic placement
of internal cavities is as important as the amino acid or base composition of a specimen 39. Contrast in the image depends on there being sufficient variation in current flow to give some range to the vertical movement of the probe. Regions of peak conductivity are readily identified (see Fig. 1) but not so easily interpreted without some knowledge of the geometric or electronic structure of the specimen. With double-stranded DNA, which is about 20 A in diameter, the peaks are 9 to 30 A high 5,15,16, and with various proteins, they are a quarter to a half the physical height of the molecules 21,22,34. There is evidence that the force of the probe may transiently flatten the specimen and give anomalously low values in the vertical dimension 14. Only a limited amount of information can be gained by comparing STM images to electron micrographs, and most researchers rely on computer models constructed from crystallographic coordinates. While this is satisfactory in some cases, it is not in others, and the ultimate goal is to compare STM and AFM images made under the same conditions.
Additional advantages and potential applications The phenomenon of electron tunnelling is well documented in photosynthesis and respiration, and may play a significant role in many b i o logical processes involving enzyme catalysis, signal transduction and the long range transfer of energy. These are not active areas of STM research at present, but are mentioned to illustrate the usefulness of the instrument in studying the electrical properties of biological materials. There is no way other than by scanning tunnelling microscopy to measure the flow of electrons through unaggregated specimens. The STM and AFM can be operated in air, vacuum, oil, water, or polyelectrolyte solutions, and at varying temperatures and humidities, with only minor adjustments in instrumentation. The time it takes to record an image is in the order of seconds. This versatility is extremely advantageous, since it allows the dynamic interactions of biological molecules to be closely monitored under conditions approximating to their native environ-
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ment; a series of AFM images showing t h r o m b i n catalyzing the conversion of fibrinogen to fibrin has been p r o d u c e d 4°. Structural details are less well defined than in dry scans, due to the greater mobility of specim e n s in liquid t h a n in air. L i n d s a y and colleagues 12'14'18'19 have d e v e l o p e d electroplating techniques to deposit and h o l d specim e n s to the substrate for studying n u c l e i c acids u n d e r water. Their STM and AFM images of nucleosomal DNA are sufficiently resolved to reveal kinks in the m o l e c u l e s and to enable d e t e r m i n a t i o n of helical periodicities. The d i m e n s i o n s obtained are close to the e x p e c t e d d i m e n s i o n s of hydrated, B-form DNA, but the m o l e c u l e s a p p e a r to be m u c h p l u m p e r and s m o o t h e r than m o d e l s w o u l d predict. This is probably a c o n s e q u e n c e of small blurring motions, w h i c h m a y c o n t i n u e to be a limiting factor in obtaining high resolution u n d e r solution conditions. W h e t h e r or not atomic r e s o l u t i o n is achieved, the results so far r e p r e s e n t i m p o r t a n t advances in microscopy. In addition, these experiments demonstrate the practicality of e x a m i n i n g the d y n a m i c properties and interactions of small molecules at the s u r f a c e - l i q u i d interface. It s h o u l d n o w be possible to d e t e r m i n e the b i n d i n g specificity of a protein to a solid phase s u p p o r t for example, or to d e t e r m i n e the inertness of i m p l a n t materials s u c h as heart valves and prostheses in b o d y fluids. Recent d e v e l o p m e n t s suggest that the STM m a y have preparative as well as analytical use in biology and biotechnology. In e x a m i n i n g phthalates on graphite, Foster et al. 41 f o u n d t h e y c o u l d pin s p e c i m e n s to the substrate, and r e m o v e them, by r a p i d l y varying the voltage during the course of a scan. T h e y also s h o w e d that a 100 n a n o s e c o n d pulse w i t h the probe directly over the s p e c i m e n resulted in cleavage of the b e n z e n e ring from the m o l e c u l a r structure. Garfunkel and colleagues 42 s u b s e q u e n t l y r e p o r t e d using the STM probe to make holes and etch lines on a c o n d u c t i n g oxide, Rbo.3MoO3 (a process they called 'nanolithography'). As these experi m e n t s indicate, the ability to see molecules' in atomic d i m e n s i o n s and t h e n to be able to m a n i p u l a t e t h e m p h y s i c a l l y is no longer a distant prospect.
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Acknowledgements This research has b e e n s u p p o r t e d b y NIH a n d NSF. The authors are grateful for the collaborative efforts of G. Lee and D. F. Evans of the Center for Interfacial Engineering, University of Minnesota, USA.
References 1 Feynman, R. P. (1960) Saturday Review 43, 45-47 2 Lang, N. D. (1986) Phys. Rev. Lett. 56, 1164-1167 3 Binnig, G., Quate, C. F. and Gerber, C. H. (1986) Phys. Rev. Lett. 56, 930 4 Wickramasinghe, H. K. (1989) Sci. Amer. 261, 98-105 5 Lee, G., Arscott, P. G., Bloomfield, V. A. and Evans, D. F. (1989) Science 244, 475-477 6 Arscott, P. G., Lee, G., Bloomfield, V. A. and Evans, D. F. (1989) Nature 119,484-486 7 Dunlap, D. D. and Bustamante, C. (1989) Nature 342,204-206 8 Cricenti, A., Selci, S., Felici, A. C., Generosi, R., Gori, E., Djaczenko, W. and Chiarotti, G. (1989) Science 245, 1226-1227 9 Feng, L., Hu, C. Z. and Andrade, J. D. (1988) J. Microscopy 152,811-816 10 Binnig, G. and Rohrer, H. (1984) in Trends in Physical (Janta, J. and Pantoflicek, J., eds), pp. 38-46, European Physical Society, The Hague 11 Travaglini, G., Rohrer, It., Amrein, M. and Gross, H. (1987) Surf. Sci. 181, 380-390 12 Lindsay, S. M. and Barris, B. (1988) J. Vac. Sci. Technol. A 6, 544-547 13 Barris, B., Knipping, U., Lindsay, S. M., Nagahara, L. and Thundat, T. (1988) Biopolymers 27, 1691-1696 14 Lindsay, S. M., Thundat, T. and Nagahara, L. (1988) in Biological and Artificial Intelligence Systems (Clementi, E. and Chin, S., eds), pp. 125-141, ESCOM Scientific Publications 15 Beebe, T. P., Jr, Wilson, T. E., Ogletree, F., Katz, J. E., Balhorn, R., Salmeron, M. B. and Siekhaus, W. J. (1989) Science 243,370-372 16 Keller, D., Bustamante, C. and Keller, R. W. (1989) Proc. Natl Acad. Sci. USA 86; 5356-5360 17 Lindsay, S. M., Thundat, T., Nagahara, L., Knipping, U. and Rill, R. L. (1989) Science 244, 1063-1064 18 Lindsay, S. M., Nagahara, L. A., Thundat, T., Knipping, U., Rill, R. L., Drake, B., Prater, C. B., Weisenhorn, A. L., Gould, S. A. C. and Hansma, P. K. (1989) J. Biomol. Struct. Dyn. 7, 279-287 19 Lindsay, S. M., Nagahara, L. A.,
Thundat, T. and Oden, P. (1989) J. Biomol. Struct. Dyn. 7, 289-299 20 Amrein, M., Stasiak, A., Gross, H., Stoll, E. and Travaglini, G. (1988) Science 240, 514-516 21 Amrein, M., Durr, R., Stasiak, A., Gross, H. and Travaglini, G. (1989) Science 243, 1708-1711 22 Edstrom, R. D., Meinke, M. H., Yang, X., Yang, R. and Evans, D. F. (1989) Biochemistry 28, 4939-4942 23 Hansma, P. K., Elings, V. B., Marti, O. and Bracker, C. E. (1988) Science 211, 2O9-242 24 Voelker, M. A., Hameroff, S. R., He, J. D., Dereniak, E. L., McCuskey, R. S., Schneiker, C. W., Chvapil, T. A., Bell, L. S. and Weiss, L. B. (1988) J. Microscopy 152, 557-566 25 Gould, So A. C., Drake, B., Prater, C. B., Weisenhorn, A. L., Lindsay, S. M. and Hansma, Po K. (1989) in Proceedings of the 47th Annual Meeting of the Electron Microscopy Society of America (Bailey, G. W., ed.), pp. 32-33, San Francisco Press 26 Simic-Krstic, Y., Kelley, M., Schneiker, C., Krasovich, M., McCuskey, R. D., Koruga, D. and Hameroff, S. (1989) FASEB J. 3, 2184-2188 27 Feng, L., Hu, C. Z. and Andrade, J. D. (1988) J. Colloid Interface Sci. 126, 650-653 28 Laegsgaard, E., Besenbacher, F., Mortensen, K. and Stensgaard, I. (1988) J. Microscopy 152,663-669 29 Garcia, R., Keller, D., Panitz, J. A., Bear, D. G. and Bustamante, C. (1989) Ultramicroscopy 27, 367-374 30 Guckenberger, R., Wiegrabe, W. and Baumeister, W. (1989) J. Microscopy 152, 795-802 31 Stemmer, A., Reichelt, R., Engel, A., Rosenbusch, J. P., Ringger, M., Hidber, H. R. and Guntherodt, H-J. (1987) Surf. Sci. 181,394-402 32 Fisher, K. A., Whitfield, S., Thomson, R. E., Yanagimoto, K., Gustafsson, M. and Clarke, J. (1989) in Proceedings of the 47th Annual Meeting of the Electron Microscopy Society of America (Bailey, G. W., ed.), pp. 14-15, San Francisco Press 33 Dahn, D. C., Watanabe, M. O., Blackford, B. L., Jericho, M. H. and ~Beveridge, T. J. (1988) J. Vac. Sci. Technol. A 6,548-552 34 Baro, A. M., Miranda, R., Alaman, J., Garcia, N., Binnig, G., Rohrer, H., Gerber, C. and Carrascosa, J. L. (1985) Nature 315,253-254 35 Smith, D. P. E., Kirk, M. D. and Quate, C. F. (1987) J. Chem. Phys. 86, 6034-6038 36 Garcia, N. and Flores, F. (1984) Physica 127B, 137-142 37 Eley, D. D. and Spivey, D. I. (1962) Trans. Faraday Soc. 58, 411-415 38 Snart, R. S. (1968) Biopolymers 6,
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293-297 39 Kuki, A. and Wolynes, P. G. (1987) Science 236, 1647-1652 40 Drake, B., Prater, C. B., Weisenhorn, A. L., Gould, S. A. C., Albrecht, T. R.,
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Quate, C. F., Cannell, D. S., Hansma, H. G. and Hansma, P. K. (1989) Science 243, 1586-1589 41 Foster, J. S., Frommer, J. E. and Arnett, P. C. (1989) Nature 331,
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Manipulation of methionine-rich protein genes in plant seeds Susan B. Altenbach and Robert B. Simpson Animal feeds based on soybean and corn meals are often deficient in the sulfur-containing amino acids, methionine and cysteine. At present, this deficiency is remedied by amino acid supplementation of the diet. A number of methionine-rich seed proteins have been identified; seed-specific expression of genes encoding these proteins is expected to improve the protein quality in feed seed and thereby eliminate the need for supplementation. Increased demand for animal protein in human diets has caused a shift towards intensive rearing of farm animals. This has necessitated greater emphasis on developing optimal nutritional formulations and feeding regimes for monogastric livestock to ensure maximal growth of the animals. The requirements for protein, carbohydrate and fat are determined through feeding trials and are dependent on both the type of animal and the stage of development. When assessing protein quality of feed formulations, the amounts of ten essential amino acids present in the food (amino acids that cannot be synthesized by the animal and must be provided in the diet) are compared to the individual requirements of the animal. Plant seeds, in particular soybean and corn, provide the majority of protein consumed by farm animals. However, the amounts of certain essential amino acids in the seed proteins of soybean and corn fall somewhat short of those required by most farm animals for maximum growth. The problem of protein quality in these meals stems from the amino S. B. Altenbach and R. B. Simpson are at the Plant Cell Institute, 6560 Trinity Court, Dublin, CA 94568. USA. @ 1990, Elsevier Science Publishers Ltd (UK)
acid composition of the most abundant proteins accumulated in the seeds. The major seed proteins of soybean are deficient in the sulfurcontaining amino acids, methionine and cysteine, while the seed proteins of corn are deficient in lysine and tryptophan. Even with the common practice of combining corn and soybean for animal feeds, the resultant meal is deficient and must be
324-326 42 Garfunkel, E., Rudd, G., Novak, D., Wang, S., gbert, G., Greenblatt, M., Gustafsson, T. and Garofalini, S. H. (1989) Science 246, 99-100
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supplemented with amino acids. In recent years, much has been learned about the biosynthesis and deposition of the major seed storage proteins and the regulation of the genes that encode these proteins. A number of seed proteins that contain high concentrations of methionine, one of the key essential amino acids added to both poultry and swine feeds, have been identified. The cloning of DNA sequences encoding these proteins may permit the methionine deficiencies of seed proteins to be remedied using a biotechnological approach (Fig. 1), thereby reducing or eliminating the need for supplementation of animal feed formulations with methionine. In this paper, we describe methionine-rich seed proteins and assess the prospects of using genes encoding these proteins to improve the methionine content of plant seed proteins. By increasing the levels of methionine in soybean seed proteins, a nutritionally complete protein source could be generated; additional methionine can also compensate for cysteine deficiencies since cysteine can be synthesized from methionine.
~Fig. 1
identification of I Methionine-Rich Proteins .
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Improved protein quality L-Normal agronomic traits
improved Livestock Feed
Steps for improving nutritional quality of plant seeds: enhancing the methionine content of seed proteins for u s e in livestock feed.
0167 - 9430/90/$2.00
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- - T a b l e 3,
(a) Time to product introduction (months) Method of transformation
Test engineered phenotype (R1 plants)
First largescale field testing
Product introduction
5
17
41
8
48
72
No regeneration through tissue culture Regeneration
(Agrobacterium)
(b) Transformation of elite varieties through particle acceleration
Time (months) Number of seeds Acres needed
Generation R3 R4
R1
R2
5 10 0
13
17
29
10 3
10 5
0.1
1
10 7 10 2
109 10 4
9
R5
Field test for product quality
order to increase the nutritional v a l u e of this i m p o r t a n t crop.
Acknowledgements We t h a n k T. Ford, A. A n d e r s o n , L. Amerson, B. Cohen, B. H a m m e r and A. F e l d m e i e r for excellent technical assistance, K. Barton for p r o v i d i n g Figure 3 and W. Brill for p r o v i d i n g Table 3. []
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Commercialization 41 >1011 >106
Product introduction
References 1 Hoskin, R. L. (1987) USDA Econom. Res. Service 1-35 2 Christou, P., Murphy, J. E. and Swain, W. F. (1987) Proc. Natl Acad. Sci. USA 84, 3962-3966 3 Lazzeri, P. A., Hildebrand, D. F. and Collins, G. B. (1985) Plant 54oi. Biol. Reporter 3, 160-167 4 Faccioti, D., O'Neal, J. K., Lee, J. and Shewmaker, C. K. (1985) Bio/Tech[]
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Scanning tunnelling microscopy in biotechnology Patricia G. Arscott and Victor A. Bloomfield The scanning tunnelling microscope (STM) is capable of atomic resolution of highly conductive materials. Whether biological molecules can be visualized to the same extent remains an open question, but remarkable progress in the past year confirms the possibility of seeing the fine structure of nucleic acids, proteins, membranes and viruses, and provides evidence that their dynamic interactions can be monitored under conditions approximating to those of the native environment. T h i r t y years ago, Professor Richard F e y n m a n d e l i v e r e d a s p e e c h before the A m e r i c a n Physical Society enP. G. Arscott and V. A. B]oomfield are at the Department of Biochemistry, University of Minnesota, 1479 Gortner Avenue, St Paul MN.55108, USA.
titled ' T h e W o n d e r s That Await a Micro-Microscope'. 1 The questions he raised c o u l d not have b e e n m o r e provocative: w h a t good w o u l d it do to see atoms distinctly? Could w e t h e n not easily answer some of the most central and f u n d a m e n t a l
© 1990, Elsevier Science Publishers Ltd (UK) 0167 - 9430/90/$2.00
nology 3, 241-246 5 Owens, L. and Cress, D. E. (1985) Plant Physiol. 77, 87-94 6 Hinchee, M. A. W., Connor-Ward, D. V., Newell, C. A., McDonnell, R. E., Sato, S. J., Gasser, C. S., Fischhoff, D. A., Re, D. B., Fraley, R. T. and Horsch, R. B. (1988) Bio/ Technology 6, 915-922 7 Christou, P., McCabe, D. E. and Swain, W. F. (1988) Plant Physiol. 87, 671-674 8 Klein, T. M., Wolf, E. D., Wu, R. and Sanford J. C. (1987) Nature 327, 70-73 9 Klein, T. M., Harper, E. C., Svab, Z., Sanford, J. C., Fromm, M. E. and Maliga, P. (1988) Proc. Natl Acad. Sci. USA 85, 8502-8505 10 Christou, P. and Yang, N-S. (1989) Ann. Bot. 64, 225-234 11 Jefferson, R. A. (1987) Plant Mol. Biol. Rep. 5, 387-405 12 Christou, P. Ann. Bot. (in press) 13 McCabe, D. E., Swain, W. F., Martinell, B. J. and Christou, P. (1988) Rio~Technology 6, 923-926 14 Christou, P., Swain, W. F., Yang, N-S. and McCabe, D. E. (1989) Proc. Natl Acad. Sci. USA 86, 7500-7504 15 Christou, P. and Swain, W. F. (1990) Theor. App]. Genet. 79, 337-341 16 Bennett, M. D. and Smith, J. B. (1976) Phil. Trans. R..Soc. Lond. B 274, 227-274
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questions of biology? A n d c o u l d we not change the a r r a n g e m e n t of atoms and m a n i p u l a t e the genes and molecules t h e y specify to n e w and useful p u r p o s e ? At the time, although r e s o l u t i o n on material samples to 2A or 3A was possible, the highest r e s o l u t i o n obt a i n e d for biological s p e c i m e n s w i t h the electron m i c r o s c o p e was about l o A - barely sufficient to distinguish single-stranded from double-stranded DNA. Optics and t e c h n i q u e s of sample preparation i m p r o v e d over the next t w e n t y years, but the necessity of exposing delicate structures to h i g h e r energy electron beams imp o s e d severe constraints on attaining higher resolution. D e v e l o p m e n t s that finally led to major breakt h r o u g h s in m i c r o s c o p y and biotechnology occurred independently, at about the same time. Routine use of r e c o m b i n a n t DNA t e c h n i q u e s and the first scanning t u n n e l l i n g microscope were i n t r o d u c e d in the late 1970s and early 1980s.
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~Fig.
1
Scanning tunnelling microscope The STM is capable of resolving features that are an atom wide and 1/100 of an atom high on a metal surface 2. It is not a new and improved electron microscope, but an entirely different type of instrument modelled on the topografiner, a scanning probe profilometer that gives three-dimensional line plots of surface topography. The imaging system of the STM depends on the p h e n o m e n o n of electron tunnelling: electrons in a Conductive material face a barrier of infinite width at the material's surface. However, in response to an applied voltage and the close proximity of another conductor, electrons can be induced to tunnel across to the second conductor; the smaller the gap, the greater the current. The imaging system uses, typically, currents and voltages of - l n A and 100 mV which, applied across a gap of 1-2 nm and confined to a small cross-sectional area, can generate high current density. However, due to variations in geometry and local electron density at the tip and sample surface, it is difficult to calculate the field strength or current density at the sample surface, and the risk of dielectric breakdown or beam damage cannot be realistically assessed. Although it may seem surprising that biological materials such as DNA (which are essentially insulators or poor semiconductors), can be examined without damage in the absence of a protective coating, images of apparently intact molecules can be obtained, with dimension measurements close to crystallographic values. The advantages of scanning profilometry had not been fully realized until G. Binnig and H. Rohrer, in studying surface properties of metals and semi-conductors at IBM, Zurich, found a way to damp vibrations and so keep the probe from crashing w h en scanning at very close range. In conjunction with the use of piezoelectric crystals that may be stably positioned on the subatomic scale, this enabled a scan to be maintained within a few &ngstrSms of a surface, low enough to permit a current of electrons to tunnel between the tip of the probe and the surface, i.e., to pass through regions of nonconductivity despite the energy barrier. Using the strength of the current as
--
Scan Head containing piezoelectric crystal z
Electrical connection Probe
Scan Head rests on screws to hold it above the sample
~ x
Sample on ubstrate
Base Cable to the control unit
Suspended platform or table designed to damp vibrations
The scanning tunnelling microscope (STM) and its mode of operation. The scanning process is entirely computer-controlled. A specimen is positioned beneath a needle-like probe that moves back and forth in a raster pattern a few &ngstrSms above the surface. The probe is at the base of a scan head containing a piezoelectric crystal that contracts and expands in response to voltage applied to the control elements for lateral (x, y) and vertical (z) movement. Voltage to the z element is supplied through a feedback loop that adjusts the gain to maintain either a constant distance or a constant signal from the surface. The set point, i.e., the level o f current detected by the probe, is typically about 1 nA and the applied voltage about 100 mV. Scan sizes and rates are variable. The x, y and z co-ordinates are digitized, stored, and m a y be displayed as grey- or color-scale images on the computer screen. Diagram not drawn to scale.
a gauge of gap distance, they showed that they could trace the intricate contours of a surface by holding the current constant and recording the vertical movement of the probe as it is scanned. The basic design and functioning of an STM is given in Fig. 1. The success of the STM sparked a series of spin-offs that allow different aspects of a surface to be examined and compared. The first of these instruments, the atomic force microscope (AEM), was invented by Binnig and colleagues 3 to circumvent one of the main limitations of the STM: a specimen cannot be visualized if it does not adequately conduct electrons. They modified the prototype STM design by mounting a diamond-tipped probe on a cantilever and scanning the specimen so as to lightly touch the tip and deflect the cantilever toward an electrode a few fingstr6ms away. The gap dis-
tance between the cantilever and the electrode, determined by the strength of a tunnelling current as in the STM, gave a measure of interatomic forces and hence the arrangement of atoms at the specimen surface. Although their A r M offered considerable advantages, it was a more difficult instrument to operate and not as sensitive as the STM. Newer models use optical interferometry to detect deflection of the cantilever and have a m u c h lighter touch than the original ( - 1 0 -10 N). Both the STM and A r M are commercially available, and magnetic force, optical, ion conductance, thermal, and acoustic microscopes are being developed along similar lines 4.
Advantages over other methods of obtaining structural information It is not yet known h o w high a resolution of biomolecular structure can be attained with any of the
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--Fig. 2,
Poly(dG-me5dC) . poly(dG-me5dC) in the left-handed Z form. Reprinted, with permission, from Ref. 6.
scanning probe microscopes, but it is already evident that the STM yields detailed structural information that cannot presently be obtained by electron microscopy or other means. DNA, RNA and proteins have been successfully imaged without shadow or stain. Physical methods such as optical diffraction and circular dichroism average over many molecules, whereas STMs may examine single molecules in a heterogeneous population and detect localized, intramolecular variations in structure. Such specific information is invaluable in a wide range of applications; for example, assaying the specificity of enzyme activity or antibody and fluorescent labeling, testing the therapeutic and toxicological effects of drugs, monitoring biodegradation processes, and developing new methods of genetic engineering.
Progress in imaging biological molecules Our STM images of DNA 5,6 and RNA 5 are sufficiently well resolved to permit reliable measurements of helical periodicity to be obtained,
enabling comparison of the conformation and hydration state of these molecules under different conditions. Images of DNA in the lefthanded Z form 6 are shown in Fig. 2. The handedness is evident, and in certain orientations the zigzag backbone and grooves in the molecule can be clearly discerned. Periodicities agree with the values obtained by fiber diffraction and indicate that the specimens are not significantly denatured. In Dunlap and Bustamante's images of single-stranded polydeoxyadenylate7, the imidazole and pyrimidine rings of the bases are clearly outlined against the substrate. Their dimensions and spacing are consistent with crystallographic data. Cricenti and co-workers 8 may have succeeded in visualizing the phosphate atoms in DNA fixed with tris(1-aziridinyl) phosphine oxide, a compound that has an affinity for sugars. When the STM image is aligned with a model of B-DNA, bright spots adjacent to the sugars appear at the positions of phosphates in the helical backbone. Interpretation of the image is somewhat
equivocal in that similar spots in the minor groove appear to correspond to bases; unfortunately, none is present in the major groove to confirm this. Can STM be used to sequence nucleic acids? Dunlap and Bustamante's results 7 indicate the feasibility of differentiating purines from pyrimidines, but at this stage it is not clear whether all four bases can be identified without specific labels. Attempts to sequence DNA by electron microscopy failed for at least two reasons. Single-stranded DNAs and polynucleotides tend to fold back on themselves so that label ligands are superimposed or clumped, and when bombarded by heavy doses of electrons, the ligands dissociate. The higher resolution of the STM should make it easier to trace the configuration of single strands and to see individual labels, but better spreading techniques are needed for optimal results. The lower electron energies used in the STM should diminish the problem of labels dissociating. Sequencing methods based on the specificity of enzyme cleavage are well developed
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and unlikely to be supplanted. Nevertheless, it would seem worthwhile to take advantage of the fact that a wider variety of labels can be used in STM experiments than in electron microscopy; heavy metals are not needed for contrast. Smaller ligands with a higher specificity could make it possible to label the bases in the grooves of double-stranded DNA and to identify accessible sites for other types of interactions. Studies of individual amino acids 9 may open the way for sequencing proteins too. Current work is focused mainly on secondary and tertiary structure. DNA, RNA 5-8,1°-19 and the other biological molecules that have been examined all have a recognizable size a n d shape, or form extensive two-dimensional arrays on the substrate; these include a DNAprotein complex 9,2°,21, the enzymes phosphorylase 22, kinase 22, and catalase 23, fibrous aggregates of collagen 24, fibrinogen 25 and tubulin 26, albumin 27, ferritin 28, bacterial proteins and membranes 29-32, and whole bacteriophages 33,34. For some of these, the specimens were coated with platinum or platinum/carbon, and it was not possible to resolve features smaller than the grain size of the metal.
Resolution, contrast, and interpretation of the image The quality of the image obtained depends primarily on the stability of the system; on how closely the probe can approach the specimen and how stationary the specimen remains under the force of the probe. In contrast to metals and semiconductors, biological molecules are flexible and mobile. Clusters of sorbic acid deposited on graphite have been observed to move away from the scan area at the rate of i A/min 35. Even larger adsorbates are known to diffuse across the surface, and it has been noticed that some of the best STM images are obtained where such movements are limited, as at steps on the substrate or in closely packed aggregates. Comparisons of images taken at different scan rates indicate that small-scale movements of sidechains and domains can cause an image to blur. There is thus some disadvantage to being able to examine naked molecules; the stains and shadows applied routinely in electron microscopy, and which may be
I - I B T E C H - J U N E 1990 [Vol. 8]
used in STM to enhance contrast, also confer stability to the specimen. No matter how stable the system, one cannot hope to see atoms in the specimen or in the substrate unless the tip of the probe consists of a single atom. This is not as difficult as might be expected; STM probes are typically made of platinum/iridium or tungsten wire, and may be cut with scissors and sharpened to a fine point by electrochemical etching or machine lathing, or left as they are. A crude cut has jagged edges and points as sharp as any tip that can be produced with more expense and effort, but there is a fairly high probability of getting a tunnelling current from more than one point. Probes are routinely tested on a flat substrate of known pattern and dimensions before the specimen is scanned. However, there is no guarantee of a single tunnelling tip on a rough surface. The tunnelling current is proportional to the effective radius of the tip plus the surface curvature beneath the tip (1/Reff=l/Rtip+l/Rsurf), multiplied by the exponential of the gap distance times the square root of the work function 36. The latter term relates to the conductivity of the specimen, and is usually an unknown quantity in experiments with biological molecules. The reported values for resistivity and activation energy in the few molecules that have been assayed are probably inaccurate. DNA and RNA, for example, have been characterized as semiconductors in stretched fibers and gels 37,38. Given the likelihood of sequence-dependent variations in conductivity along the helices, as well as localized differences in hydration, ionization, and orientation, one expects that open pathways for electron transfer must be in register for measurements across several molecules to give information about individual conductivity. Neither the specimen's thickness nor the presence of specific residues is a particularly good indicator of whether it is an insulator or a conductor. The probability of electron tunnelling through a specimen in many small steps may be greater than the probability of tunnelling in one long step 16. Computer simulations based on Feynman's path integral method of defining the tunnelling pathway in proteins suggest that the strategic placement
of internal cavities is as important as the amino acid or base composition of a specimen 39. Contrast in the image depends on there being sufficient variation in current flow to give some range to the vertical movement of the probe. Regions of peak conductivity are readily identified (see Fig. 1) but not so easily interpreted without some knowledge of the geometric or electronic structure of the specimen. With double-stranded DNA, which is about 20 A in diameter, the peaks are 9 to 30 A high 5,15,16, and with various proteins, they are a quarter to a half the physical height of the molecules 21,22,34. There is evidence that the force of the probe may transiently flatten the specimen and give anomalously low values in the vertical dimension 14. Only a limited amount of information can be gained by comparing STM images to electron micrographs, and most researchers rely on computer models constructed from crystallographic coordinates. While this is satisfactory in some cases, it is not in others, and the ultimate goal is to compare STM and AFM images made under the same conditions.
Additional advantages and potential applications The phenomenon of electron tunnelling is well documented in photosynthesis and respiration, and may play a significant role in many b i o logical processes involving enzyme catalysis, signal transduction and the long range transfer of energy. These are not active areas of STM research at present, but are mentioned to illustrate the usefulness of the instrument in studying the electrical properties of biological materials. There is no way other than by scanning tunnelling microscopy to measure the flow of electrons through unaggregated specimens. The STM and AFM can be operated in air, vacuum, oil, water, or polyelectrolyte solutions, and at varying temperatures and humidities, with only minor adjustments in instrumentation. The time it takes to record an image is in the order of seconds. This versatility is extremely advantageous, since it allows the dynamic interactions of biological molecules to be closely monitored under conditions approximating to their native environ-
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ment; a series of AFM images showing t h r o m b i n catalyzing the conversion of fibrinogen to fibrin has been p r o d u c e d 4°. Structural details are less well defined than in dry scans, due to the greater mobility of specim e n s in liquid t h a n in air. L i n d s a y and colleagues 12'14'18'19 have d e v e l o p e d electroplating techniques to deposit and h o l d specim e n s to the substrate for studying n u c l e i c acids u n d e r water. Their STM and AFM images of nucleosomal DNA are sufficiently resolved to reveal kinks in the m o l e c u l e s and to enable d e t e r m i n a t i o n of helical periodicities. The d i m e n s i o n s obtained are close to the e x p e c t e d d i m e n s i o n s of hydrated, B-form DNA, but the m o l e c u l e s a p p e a r to be m u c h p l u m p e r and s m o o t h e r than m o d e l s w o u l d predict. This is probably a c o n s e q u e n c e of small blurring motions, w h i c h m a y c o n t i n u e to be a limiting factor in obtaining high resolution u n d e r solution conditions. W h e t h e r or not atomic r e s o l u t i o n is achieved, the results so far r e p r e s e n t i m p o r t a n t advances in microscopy. In addition, these experiments demonstrate the practicality of e x a m i n i n g the d y n a m i c properties and interactions of small molecules at the s u r f a c e - l i q u i d interface. It s h o u l d n o w be possible to d e t e r m i n e the b i n d i n g specificity of a protein to a solid phase s u p p o r t for example, or to d e t e r m i n e the inertness of i m p l a n t materials s u c h as heart valves and prostheses in b o d y fluids. Recent d e v e l o p m e n t s suggest that the STM m a y have preparative as well as analytical use in biology and biotechnology. In e x a m i n i n g phthalates on graphite, Foster et al. 41 f o u n d t h e y c o u l d pin s p e c i m e n s to the substrate, and r e m o v e them, by r a p i d l y varying the voltage during the course of a scan. T h e y also s h o w e d that a 100 n a n o s e c o n d pulse w i t h the probe directly over the s p e c i m e n resulted in cleavage of the b e n z e n e ring from the m o l e c u l a r structure. Garfunkel and colleagues 42 s u b s e q u e n t l y r e p o r t e d using the STM probe to make holes and etch lines on a c o n d u c t i n g oxide, Rbo.3MoO3 (a process they called 'nanolithography'). As these experi m e n t s indicate, the ability to see molecules' in atomic d i m e n s i o n s and t h e n to be able to m a n i p u l a t e t h e m p h y s i c a l l y is no longer a distant prospect.
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Acknowledgements This research has b e e n s u p p o r t e d b y NIH a n d NSF. The authors are grateful for the collaborative efforts of G. Lee and D. F. Evans of the Center for Interfacial Engineering, University of Minnesota, USA.
References 1 Feynman, R. P. (1960) Saturday Review 43, 45-47 2 Lang, N. D. (1986) Phys. Rev. Lett. 56, 1164-1167 3 Binnig, G., Quate, C. F. and Gerber, C. H. (1986) Phys. Rev. Lett. 56, 930 4 Wickramasinghe, H. K. (1989) Sci. Amer. 261, 98-105 5 Lee, G., Arscott, P. G., Bloomfield, V. A. and Evans, D. F. (1989) Science 244, 475-477 6 Arscott, P. G., Lee, G., Bloomfield, V. A. and Evans, D. F. (1989) Nature 119,484-486 7 Dunlap, D. D. and Bustamante, C. (1989) Nature 342,204-206 8 Cricenti, A., Selci, S., Felici, A. C., Generosi, R., Gori, E., Djaczenko, W. and Chiarotti, G. (1989) Science 245, 1226-1227 9 Feng, L., Hu, C. Z. and Andrade, J. D. (1988) J. Microscopy 152,811-816 10 Binnig, G. and Rohrer, H. (1984) in Trends in Physical (Janta, J. and Pantoflicek, J., eds), pp. 38-46, European Physical Society, The Hague 11 Travaglini, G., Rohrer, It., Amrein, M. and Gross, H. (1987) Surf. Sci. 181, 380-390 12 Lindsay, S. M. and Barris, B. (1988) J. Vac. Sci. Technol. A 6, 544-547 13 Barris, B., Knipping, U., Lindsay, S. M., Nagahara, L. and Thundat, T. (1988) Biopolymers 27, 1691-1696 14 Lindsay, S. M., Thundat, T. and Nagahara, L. (1988) in Biological and Artificial Intelligence Systems (Clementi, E. and Chin, S., eds), pp. 125-141, ESCOM Scientific Publications 15 Beebe, T. P., Jr, Wilson, T. E., Ogletree, F., Katz, J. E., Balhorn, R., Salmeron, M. B. and Siekhaus, W. J. (1989) Science 243,370-372 16 Keller, D., Bustamante, C. and Keller, R. W. (1989) Proc. Natl Acad. Sci. USA 86; 5356-5360 17 Lindsay, S. M., Thundat, T., Nagahara, L., Knipping, U. and Rill, R. L. (1989) Science 244, 1063-1064 18 Lindsay, S. M., Nagahara, L. A., Thundat, T., Knipping, U., Rill, R. L., Drake, B., Prater, C. B., Weisenhorn, A. L., Gould, S. A. C. and Hansma, P. K. (1989) J. Biomol. Struct. Dyn. 7, 279-287 19 Lindsay, S. M., Nagahara, L. A.,
Thundat, T. and Oden, P. (1989) J. Biomol. Struct. Dyn. 7, 289-299 20 Amrein, M., Stasiak, A., Gross, H., Stoll, E. and Travaglini, G. (1988) Science 240, 514-516 21 Amrein, M., Durr, R., Stasiak, A., Gross, H. and Travaglini, G. (1989) Science 243, 1708-1711 22 Edstrom, R. D., Meinke, M. H., Yang, X., Yang, R. and Evans, D. F. (1989) Biochemistry 28, 4939-4942 23 Hansma, P. K., Elings, V. B., Marti, O. and Bracker, C. E. (1988) Science 211, 2O9-242 24 Voelker, M. A., Hameroff, S. R., He, J. D., Dereniak, E. L., McCuskey, R. S., Schneiker, C. W., Chvapil, T. A., Bell, L. S. and Weiss, L. B. (1988) J. Microscopy 152, 557-566 25 Gould, So A. C., Drake, B., Prater, C. B., Weisenhorn, A. L., Lindsay, S. M. and Hansma, Po K. (1989) in Proceedings of the 47th Annual Meeting of the Electron Microscopy Society of America (Bailey, G. W., ed.), pp. 32-33, San Francisco Press 26 Simic-Krstic, Y., Kelley, M., Schneiker, C., Krasovich, M., McCuskey, R. D., Koruga, D. and Hameroff, S. (1989) FASEB J. 3, 2184-2188 27 Feng, L., Hu, C. Z. and Andrade, J. D. (1988) J. Colloid Interface Sci. 126, 650-653 28 Laegsgaard, E., Besenbacher, F., Mortensen, K. and Stensgaard, I. (1988) J. Microscopy 152,663-669 29 Garcia, R., Keller, D., Panitz, J. A., Bear, D. G. and Bustamante, C. (1989) Ultramicroscopy 27, 367-374 30 Guckenberger, R., Wiegrabe, W. and Baumeister, W. (1989) J. Microscopy 152, 795-802 31 Stemmer, A., Reichelt, R., Engel, A., Rosenbusch, J. P., Ringger, M., Hidber, H. R. and Guntherodt, H-J. (1987) Surf. Sci. 181,394-402 32 Fisher, K. A., Whitfield, S., Thomson, R. E., Yanagimoto, K., Gustafsson, M. and Clarke, J. (1989) in Proceedings of the 47th Annual Meeting of the Electron Microscopy Society of America (Bailey, G. W., ed.), pp. 14-15, San Francisco Press 33 Dahn, D. C., Watanabe, M. O., Blackford, B. L., Jericho, M. H. and ~Beveridge, T. J. (1988) J. Vac. Sci. Technol. A 6,548-552 34 Baro, A. M., Miranda, R., Alaman, J., Garcia, N., Binnig, G., Rohrer, H., Gerber, C. and Carrascosa, J. L. (1985) Nature 315,253-254 35 Smith, D. P. E., Kirk, M. D. and Quate, C. F. (1987) J. Chem. Phys. 86, 6034-6038 36 Garcia, N. and Flores, F. (1984) Physica 127B, 137-142 37 Eley, D. D. and Spivey, D. I. (1962) Trans. Faraday Soc. 58, 411-415 38 Snart, R. S. (1968) Biopolymers 6,
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293-297 39 Kuki, A. and Wolynes, P. G. (1987) Science 236, 1647-1652 40 Drake, B., Prater, C. B., Weisenhorn, A. L., Gould, S. A. C., Albrecht, T. R.,
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Quate, C. F., Cannell, D. S., Hansma, H. G. and Hansma, P. K. (1989) Science 243, 1586-1589 41 Foster, J. S., Frommer, J. E. and Arnett, P. C. (1989) Nature 331,
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Manipulation of methionine-rich protein genes in plant seeds Susan B. Altenbach and Robert B. Simpson Animal feeds based on soybean and corn meals are often deficient in the sulfur-containing amino acids, methionine and cysteine. At present, this deficiency is remedied by amino acid supplementation of the diet. A number of methionine-rich seed proteins have been identified; seed-specific expression of genes encoding these proteins is expected to improve the protein quality in feed seed and thereby eliminate the need for supplementation. Increased demand for animal protein in human diets has caused a shift towards intensive rearing of farm animals. This has necessitated greater emphasis on developing optimal nutritional formulations and feeding regimes for monogastric livestock to ensure maximal growth of the animals. The requirements for protein, carbohydrate and fat are determined through feeding trials and are dependent on both the type of animal and the stage of development. When assessing protein quality of feed formulations, the amounts of ten essential amino acids present in the food (amino acids that cannot be synthesized by the animal and must be provided in the diet) are compared to the individual requirements of the animal. Plant seeds, in particular soybean and corn, provide the majority of protein consumed by farm animals. However, the amounts of certain essential amino acids in the seed proteins of soybean and corn fall somewhat short of those required by most farm animals for maximum growth. The problem of protein quality in these meals stems from the amino S. B. Altenbach and R. B. Simpson are at the Plant Cell Institute, 6560 Trinity Court, Dublin, CA 94568. USA. @ 1990, Elsevier Science Publishers Ltd (UK)
acid composition of the most abundant proteins accumulated in the seeds. The major seed proteins of soybean are deficient in the sulfurcontaining amino acids, methionine and cysteine, while the seed proteins of corn are deficient in lysine and tryptophan. Even with the common practice of combining corn and soybean for animal feeds, the resultant meal is deficient and must be
324-326 42 Garfunkel, E., Rudd, G., Novak, D., Wang, S., gbert, G., Greenblatt, M., Gustafsson, T. and Garofalini, S. H. (1989) Science 246, 99-100
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supplemented with amino acids. In recent years, much has been learned about the biosynthesis and deposition of the major seed storage proteins and the regulation of the genes that encode these proteins. A number of seed proteins that contain high concentrations of methionine, one of the key essential amino acids added to both poultry and swine feeds, have been identified. The cloning of DNA sequences encoding these proteins may permit the methionine deficiencies of seed proteins to be remedied using a biotechnological approach (Fig. 1), thereby reducing or eliminating the need for supplementation of animal feed formulations with methionine. In this paper, we describe methionine-rich seed proteins and assess the prospects of using genes encoding these proteins to improve the methionine content of plant seed proteins. By increasing the levels of methionine in soybean seed proteins, a nutritionally complete protein source could be generated; additional methionine can also compensate for cysteine deficiencies since cysteine can be synthesized from methionine.
~Fig. 1
identification of I Methionine-Rich Proteins .
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Gene Isolation
b Corn b Rice -- Brazil nut Marketing
Expressionin
I Other Ptant Seeds
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Regulation Government approval
Public acceptance Segregation from commodity seeds
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Product Development
Improved protein quality L-Normal agronomic traits
improved Livestock Feed
Steps for improving nutritional quality of plant seeds: enhancing the methionine content of seed proteins for u s e in livestock feed.
0167 - 9430/90/$2.00
