Making your own gene library

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This work was supported in part by C U H K grant No 2030142 and R G C grant No 2130054.

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show the binding mechanism of the P -- O group to the enzyme. With organophosphorus compound, the bound organophosphorus compound also undergoes cleavage to release the corresponding thiol or alcohol, leaving phosphorylated enzyme. Nevertheless, the phosphorylated enzyme is only hydrolyzed very slowly. Hence, the active site of the enzyme is permanently blocked, and the inhibition remains unchanged in the in vitro test of the enzyme over a period of study. The toxicity depends on the affinity of the enzyme for the inhibitor. This experiment could provide useful information on the nature of the organophosphorus inhibitors.

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Making Your Own Gene Library JOSl~ E PI~REZ-ORTiN, 1,2 M A R C E L Li DEL OLMO, 1'2 E M I L I A M A T A L L A N A ~a and V I C E N T E T O R D E R M

21nstituto de Agroquimica y Tecnologia de Alimentos CSIC, Aptdo Correos 73 E- 46100 Burjassot Spain

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1Departamento de Bioquimica y Biologia Molecular Facultades de Ciencias Universitat de Valencia Avda Dr Moliner 50 E-46100 Burjassot Spain

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1 Holmstadt B. (1959) Pharmacol. Rev. 11, 567-688 2 Hobbiger F. (1956) Br. J. Pharmacol. 11, 295-303 3 Heath, D. F. (1961) OrganophosphorusPoisons• Pergamon Press, New York 4 Aldridge, W. N. and Reiner, E. (1972) Interaction of esterases with esters of organophosphorus and carbamic acids. In Frontiersof Biology, Vol. 26, pp. 1-328• North Holland, Amsterdam 5 Berman H. A., Yguerabide J. and Taylor P. (1980) Biochemistry 19, 2226-2235 6 Quinn D. M. (1987) Chem. Rev. 87, 955-979 7 Lockridge O., Bartels C. F., Vaughan T. A., Wong C. K., Norton S. E. and Johnson L. L. (1987) J. Biol. Chem. 262, 549-557 8 Kissel J. A., Fontaine R. N., Turck C. W•, Brockman H. L• and Hui D. Y. (1989)Biochim. Biophys.Acta 1005, 177-182 9 Murphy, S. D. (1986) Toxic effects of pesticides. In Toxicology,3rd edn, ed. D. Casarett and D. Doull, pp. 519-581. Macmillan, New York 10 Cuatrecasas P. and Anfinsen C. B• (1971) Meth. Enzymol. 22, 345-378 11 Hodgson A. J. and Chubb I. W. (1983)J. Neurochem. 41, 654-662 12 Ralston J. S., Rush R. S., Doctor B. P. and Wolfe A. D. (1985) J. Biol. Chem. 260, 4312-4318 13 Doctor B. P., Chapman T. C., Christner C. E., Deal C. D., De La Hoz D. M., Gentry M• K., Ogert R. A., Rush R. S., Smyth K. K. and Wolfe A. D. (1990) FEBS Lett. 266, 123-127 14 De La Hoz D., Doctor B. P., Ralston J. S., Rush R. S• and Wolfe A. D. (1986) Life Sciences 39, 195-199

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B I O C H E M I C A L E D U C A T I O N 25(4) 1997

Genetic engineering, recombinant D N A technology, DNA manipulation, gene cloning, etc, are becoming words in common use rather than being scientific terms limited to experts in molecular biology. All of them, although not always properly used, are frequently found in publications devoted to the spreading of science and even in the non-specialized press• For this reason, there is a recent tendency to include recombinant DNA experiments in basic or intermediate courses, such as biology, chemistry, pharmacy or medicine. Such experiments afford students initial practice for more advanced courses in molecular biology, but they are also very interesting for people with other professional activities, such as those of physicians,

238 teachers, etc, because they can improve scientific knowledge related to their tasks. Although some of these techniques are very simple and require little equipment, they are highly illustrative. Here we propose an experiment aimed at constructing a genomic library. It can be carried out over a week, in four or five consecutive sessions, and it is specially suitable to become familiar with Genetic Engineering because it includes a wide variety of techniques. It also helps students to understand some concepts more clearly, such as donor and vector DNAs, construction of recombinant DNA, host strain, experiments in gene cloning, etc.

Theoretical background First, it is necessary to define the concept of a genomic D N A library. Simply outlined, a good genomic D N A library consists of a collection of microorganisms or clones, usually E. coli, each carrying a molecule of vector D N A which has been ligated to a fragment of foreign or donor D N A from a different organism. The higher the number of different microorganisms in the collection, the higher the probability of having a copy of any given gene from the donor genome in the library. Such probability is also dramatically increased when the average insert size in the library is large (see below). In the ideal genomic library, the size of foreign D N A present in each clone has to be large enough to contain one or more complete genes. The Clarke and Carbon equation 1 provides a way of calculating what size a library should have to obtain a given probability (P) of including any particular sequence of the foreign DNA: N = ln(1 - P)/ln(1 - f ) where N is the total number of recombinant microorganisms andfis the quotient between the insert fragment size (in bp) and the genome size of the foreign organism. The construction of a genomic D N A library requires a series of steps and each of these has to be optimized in order to obtain a high number of clones. These steps are: fractionation of donor DNA, ligation with vector DNA, transformation of E. coli cells with recombinant D N A and growth of the genomic D N A library. Fragmentation of donor D N A is an important step. Thus, a completely random fragmentation is required yielding D N A fragments of suitable size (depending on the vector to be used) and carrying overlapping terminal sequences. Standard fragmentation techniques include controlled mechanical shearing or partial cleavage with a four-base cutter restriction enzyme (see references 2-4 for details). When the genome size is not very large, it is also possible to use a six-base cutter restriction enzyme, allowing an average fragment size of 46 (4096 bp). The choice of vector D N A is directly dependent on the size of foreign D N A fragments. Some special vectors, such as yeast artificial chromosomes (YACs), can accept inserts of up to 1000 kb. Other more usual vectors, such as cosmids and bacteriophage P1, can carry inserts of up to 40 and 100 kb, respectively. It is also possible to use bacteriophage lambda derived vectors, mainly the class known as replacement vectors, able to accept inserts in the 10-22 kb range. Finally, there are plasmid vectors accepting inserts of usually less than 10 kb which are easily handled and widely used for subcloning or generation of 'sub-libraries' from cosmid clones. Plasmids are used in other cases as vectors for 'primary' libraries. For instance, when working with yeast 5 or with prokaryotes it can be useful to make a genomic library directly in a shuttle plasmid vector because the screening can be done by phenotypic assay in the appropriate mutant strain of the donor organism. All the subsequent steps (vector and donor D N A ligation, transformation of E. coli with recombinant D N A and growth of the culture) are methodo-

BIOCHEMICAL EDUCATION 25(4) 1997

logically different depending on the chosen vector. The last step of the experiment is, however, the same: plating the cells on selective medium and calculation of the library size. Here, we propose a practical protocol, including all the important stages, which allows the students to construct their own yeast genomic library in an extremely simple way, but without impairing the goal of the experiment. Yeast and vector D N A s are fragmented by the student with two different restriction enzymes. This allows one to obtain recombinant D N A in only one day. The choice of a plasmid vector makes it very easy to introduce recombinant D N A into previously prepared competent E. coli cells by heat-shock-based transformation. pBluescript plasmid is very suitable for this experiment because it is widely distributed, easily available and it codifies for two selection systems: Ampicillin resistance of the transformed cells and development of white-blue colony colour depending on whether an insert is present. These properties make it easier to count the clones and identify colonies carrying recombinant plasmids directly. In any case, pBluescript plasmid can be substituted by other equally suitable plasmids, such as those of the pUC family. Finally, we take advantage of the lag-times during the experiment, to explain and discuss the potential of this technology, the problem of biohazard, or other related subjects with the students.

Experimental protocol Day 1: Library construction The first step in library construction is to perform restriction digestions of pBluescript vector and yeast DNAs. These DNAs should be obtained previously by the teacher in order to simplify the experiment. Both are easily prepared following standard protocols in amounts large enough to be used for one or two years in practical classes. Plasmid D N A can be prepared as maxi- or midi-prep following the protocols described 4. Yeast D N A of high quality can be obtained by using CsC16 or with longer protocols which do not need ultracentrifugation 7. Approx 1 #g yeast D N A and 100 ng vector D N A are separately digested with HindIII (2u) and EcoRI (2u) in a final volume of 20 #1 in 0.5 ml Eppendorf tubes. Reaction mixtures are prepared with 1 #1 vector D N A or 5 #1 yeast DNA, 1 #1 of each enzyme, 2 #1 of appropriated buffer 10 × (supplied with enzymes) and H20 to 20 #1. Digestions are carried out at 37°C for 90 min. During incubation, a 0.7% agarose gel in 0.5 x TBE buffer (TBE 0.5 x: 44.5 mM Tris, 44.5 mM borate, 0.5 mM EDTA, pH 8.3) containing 0.4 #g/ml of ethidium bromide is prepared as described elsewhere 4. CAUTION: Ethidium bromide is both an irritant and a potent mutagen. Protective gloves should always be worn while using solutions of the dye and handling gels that have been stained with it. A face mask should also be used when weighing out the pure compound. Methods for disposal of this mutagen are described elsewhere 4. After digestion, 4 #1 aliquots of each sample are mixed with 1 #1 of 6 × loading buffer (30% glycerol, 6 x TBE, 0.25% bromophenol blue, 0.25% xylene cyanol) on a piece of parafilm and loaded onto the gel. Undigested vector and yeast DNAs are used as controls. Electrophoresis is carried out at 150 V for approx 1 h and the gel is examined and photographed under UV light (302 nm) using protective eyeglasses. CAUTION: safety glasses should be worn when observing gels illuminated with UV light. Remaining samples (16/A) are precipitated with 32 #1 of cold E t O H and kept at - 2 0 ° C for 30 min. Precipitated D N A is collected by centrifugation at 12000 rev/min for 15 min and washed with cold 70% EtOH. After spinning again at 5000 rev/min for 5 min, pellets are dried and resuspended in 4 #1 H20.

239 Ligation reaction is prepared by mixing 4 #1 vector DNA, 4 #1 yeast DNA, 1/A ligation buffer 10 x (supplied with the enzyme) and I/~1 (lu) of T4 D N A ligase. The composition of this mixture can be determined in previous test experiments in each lab course. Incubation is carried out at 14°C overnight. Day 2 Transformation of competent E coli cells. E coli DH5c~ competent cells for transformation are prepared previously by standard methods of treatment with calcium chloride 4 and tested for efficiency after freezing at -80°C. Usually, 1 0 6 transformants per/zg of D N A is adequate efficiency for this experiment. As competent cell preparation is a time consuming and tricky protocol, we recommend having them previously prepared and tested, although it is also possible to include a rapid protocol like the one recently described ~ for transformation with purified plasmids. In this case an additional day is necessary to grow E. coli cells. 100 #1 aliquots of competent cells are used for each transformation. After allowing them to thaw on ice, the ligation mix is added and tubes are kept on ice for 20 min. Then cells are heat-shocked for 2 min at 37°C and transferred again to ice for 1 min. Addition of 0.2 ml of LB (1% Triptone, 0.5% yeast extract, 1% NaCI) and incubation at 37°C for 1 h allows for cell recovery. After incubation, appropriate dilutions of transformed cells in sterile water are prepared and 100 #1 are spread on plates of selective medium. These plates are prepared with LB medium (LB plus 2% agar) containing 50 pg/ml Ampicillin and 40/~g/ml 5-bromo-4-chloro-3-indolyl-fi-D-galaetopyranoside (X-Gal). Twenty minutes after plating, the plates are inverted and incubated overnight at 37°C. Day 3 The total number of colonies in the plates of transformants are counted and the number of clones in the library is averaged. To determine the number of true recombinant plasmids, the percentages of white and blue colonies have to be calculated. Isolation of recombinant plasmids In order to determine the average insert size in the library, recombinant plasmids are isolated from white colonies. Two tubes containing 3 ml of LB+Ampicillin (50 ~g/ml) are inoculated with 1 or 3 to 5 white colonies from plates containing X-Gal and incubated in a shaker at 37°C overnight. Comments on using these two methods are included below. Day 4 The alkaline lysis method is used to obtain plasmid D N A in a miniprep scale as previously described 4'9. Cells from 1.5 ml of each culture are collected by filling up 1.5 ml Eppendorf tubes and spinning at 5000 rev/min for 3 rain. After removing supernatants, cell pellets are resuspend in 100 pl of GTE solution (50 mM Glucose, 10 mM EDTA, 25 mM Tris-HC1, pH 8.0) and incubated 5 rain on ice. Cells have to be completely resuspended for efficient breakage. 200 ~1 of freshly prepared NaOH-SDS solution (0.2M NaOH, 1% SDS) are added and, after mixing, samples are incubated again on ice for 5 rain. Finally, samples are neutralized by adding 150 #1 of potassium acetate buffer (5M KCH3COO, pH 4.8) and incubated on ice for 5 rain. Cell debris is sedimented by centrifuging at 12000 rev/min for 5 min and 0.35 ml of supernatants are quickly transferred to new 1.5 ml Eppendorf tubes. Pellets are discarded. Plasmid D N A is precipitated with 0.7 ml of cold ethanol and kept at --20°C for 10rain. Then, samples are centrifuged at 12000 rev/min for 5 min and D N A pellets are washed with 500 ~1 of cold 70% EtOH. After washing, E t O H is eliminated and the pellets are dried and resuspended in 25/zl of TE (10 mM Tris-HC1 pH 7.5, 1raM EDTA) containing RNase A (10 ~g/ml). Restriction analysis of recombinant plasmids The next step is a restriction digestion of recombinant plasmid in order to release the inserts. 5/A aliquots of isolated plasmid D N A are

BIOCHEMICAL EDUCATION 25(4) 1997

mixed with 1.5 pl of appropriate restriction buffer (supplied with the enzymes) and digested with 1/11 (2u) of EcoRI and 1 #1 (2u) of HindIII in a final volume of 15/A. Restriction samples are incubated at 37°C for 90 min. For the electrophoretic analysis of these samples, 3/d of 6 x loading buffer for agarose gel electrophoresis are added to each sample or, alternatively, samples can be precipitated with 30 #1 of cold EtOH, washed with cold 70% E t O H and resuspended in 7 #1 of 1 x loading buffer. Day 5 Restriction samples are analysed by agarose gel electrophoresis in a 0.7% agarose gel prepared as described above and using pBluescript as control and 2-HindIII as size marker. Electrophoresis is carried out at 150 V for approx 1.5 h and the gel is examined and photographed under UV light. Using the size marker, the size of all the inserts is calculated and the insert size in the library is averaged. The total number of white colonies, the average insert size and the size of the cloned genome are then used to calculate the probability of having any particular D N A sequence in the library.

Results Digestion of plasmid DNA and yeast genomic DNA with restriction endonucleases Figure 1 shows the result of a typical experiment in which the two DNAs needed for the construction of the library are digested with the restriction endonucleases EcoRI and HindIII. In the case of the vector (compare lanes 1 and 3) a unique band corresponding to the linear form of the plasmid is detected. In the case of the D N A to be introduced into the vector, it is possible to see just a smear (compare lanes 2 and 4) indicating the progress of the digestion. In this sample the enzymes have cut the whole yeast D N A randomly in some of their recognition sequences and therefore it is not possible to detect discrete bands.

Ligation of the two DIVAs and transformation of the bacterial strain After ligation and transformation ofE. coli strain DH5~, the total number of clones and the number of white clones are determined. As an average of the results obtained by many groups in our laboratory classes, we have found a total of 1886 clones, 606 (32%) white. The total number of clones depends greatly on the efficiency of transformation of the bacterial cells and the amount of D N A used for the ligation reaction and these two aspects should be controlled to get this result at least. The relatively low percentage of white colonies is due to the protocol followed. In order to make it easy, cheap and as short as possible, the band corresponding to the linear form of the vector is not purified from an agarose gel. For this reason, linear DNA can be contaminated with small traces of the circular form of the plasmid (not detected in the agarose gel in Fig 1) and with the short fragment of the vector corresponding to the polylinker region between the recognition sites for EcoRI and HindlII. If this step is carried out the number of white colonies relative to blue colonies increases, but this extends time in the laboratory by a day.

Analysis of the average insert size in the recombinant plasmids To get all the information about the quality of the library, students should determine the average size of the inserts in the recombinant plasmids. Two approaches have been tested for this purpose. The first one consists of isolating plasmid D N A from individual white colonies after growth in LB+Ampicillin liquid medium. The second one consists of isolating plasmid D N A from several colonies (3-5) grown together in the same culture. This latter approach is preferred because allows for the identification of a higher number of inserts and hence the statistical data are more significant. To get a similar number of inserts with the former approach each student should carry out 3-5

240 minipreps instead of one, which increases the time required for the experiment (specially for students without experience in this procedure) and the number of agarose gels required for analysing the results. In both cases, DNAs obtained from the minipreps are digested with EcoRI and HindlII to release the insert from the vector and samples are then analysed on an agarose gel which also includes a size marker.

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Figure 2 shows the result of a typical experiment in which both methods have been followed. In the first part of the Figure (lanes 1-4) the second method has been used. In all samples, it is possible to detect the band corresponding to the vector and a few bands corresponding to each one of the inserts cloned into the plasmids isolated from each culture. Lanes 7-10 show the result when the first strategy is followed. In this case we always detect the band of the vector and one band smaller in size corresponding to the insert of the plasmid isolated from the culture. The total number of inserts identified in lanes 1-4 is 12 and in lanes 7-10 is four so the statistical significance of the results is different. It is worth noting that in a laboratory class with 12 students in which each one makes a miniprep from a mixed culture of 5 colonies, the number of inserts analysed in just one agarose gel can be increased to 60. In the experiment shown in Fig 2 the average size of the insert using any of the strategies is 1.08 kb. This figure is determined by making a plot in which log bp of the fragments corresponding to the size marker is represented against the migrated distance (in mm). Then the distance corresponding to each insert allows size determination by interpolation in the plot. With the information on the average size of the insert, it is possible to analyse the representativeness of the library.

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Figure 1 DIVAs for the ligation. (A) Simplified map of the pBluescript plasmid vector. Restriction sites for E c o R / ( E ) and HindlII (H) are shown. Ampicillin resistance and lacZ' genes are also indicated. (B) Analysis of the digestion of the two DNAs to be ligated with the restriction endonucleases E c o R / and HindlII. Lane 1 shows the native pBluescript plasmid. Two bands corresponding to supercoiled (s) and circular relaxed (c) DNA appear. Lane 2 contains yeast genomic DNA. In lane 3, 4 pl of the restriction mixture corresponding to the vector were loaded. A unique band corresponding to linear DNA (lin) is detected. Lane 4 contains the same amount of the restriction mixture of yeast DATA. Samples have been analysed on a O.7% agarose gel containing ethidium bromide (0. 4 pg/ml of gel)

BIOCHEMICAL EDUCATION 25(4) 1997

Figure 2 Determination of the average size of the inserts. Lanes 1-4 correspond to EcoRI-HindlII restriction of plasmid minipreps obtained from cultures in which several colonies were inoculated. Sizes of the molecular weight marker 2/HindlII shown on lane 5 are indicated on the left. In lane 6, bands corresponding to supercoiled, circular and polymeric forms of native pBluescript plasmid are seen from bottom to top. Lanes 7-10 correspond to EcoRI-HindIII restrictions of plasmid miniprcps obtained from cultures in which just one colony was inoculated. The arrow points to the vector fragment present in all the restriction samples. The whole restriction mixture was applied in the lanes 1 to 4 and 7 to 10. Samples were analysed on a O.7% agarose gel containing ethidium bromide (0.4 I~g/ml of gel)

241 Considering 606 white clones with an average insert size of 1.08 kb, we have introduced a total of 655 kb of yeast genome in the vector. Taking into account that the yeast genome contains 13 × 106 bp, this means just 0.050 genome equivalents. This number would, of course, be too low if we wanted to use this library for anything else than teaching purposes. The probability that any particular sequence is present in the library can be determined from the Clarke and Carbon equation ~. In our case, N is 606 a n d f i s the quotient between the insert size (1080 bp) and the genome size (13 × 106 bp). With our data we found a probability (P) of 0.051. According to this formula we would need 3.6 × 105 white colonies to get a 95% probability of including any particular sequence in the library if the average size of the insert is 1.08 kb.

Discussion Cloning genes is one of the most important goals of genetic engineering. For this purpose the construction of gene libraries, both genomic or from cDNA, is almost unavoidable. Theoretical and practical knowledge about gene libraries is, therefore, an essential requisite for a good education in genetic engineering or molecular biology. Although construction of gene libraries is common in advanced lab courses for graduate students or postdocs, it is difficult to adapt the long, demanding and expensive techniques of such experiments for the use of less skilled students. Recently, a very simple set of experiments for students of an even lower formation level has been published 8, including bacterial transformation and phenotypic characterization of clones but no recombinant D N A construction is performed. The practical protocol we propose here has been developed to overcome all the problems mentioned above. The experimental steps have been kept to a minimum in order to avoid possible mistakes and also to reduce the working days to five, compatible with a week-long intensive schedule. The longest steps, such as ligation, selecting transformants and growing transformed bacteria, have been placed at the end of the three first days in order to allow overnight incubations. The two last sessions can be combined in one day, if needed, because no overnight incubation is required. Using two six-base cutting enzymes ( E c o R I and HindIII) avoids vector religation. This could have been accomplished by using one enzyme and dephosphorylating the vector, but this step is long and very error-prone so we have decided not to include it in the experimental protocol. The use of six-base cutters as cloning enzymes and complete digestion instead of four-base cutters and partial digestion is a quality-limiting step for the library. Many genes could be cut by the selected enzyme(s). It would not be a problem if the purpose of the library is not to obtain full-length genes. This is even a minor problem if the organism used as a source of genomic D N A contains none or, few introns: eg bacteria or lower eukaryotes. However, this limitation does not affect how representative the library is, given that such a calculation does not take into account the kind of D N A fragments, but only the number of clones and the size of the inserts. A more important problem is, however, that the use of two different enzymes with non-compatible cutting sites means that half of the generated D N A fragments are unclonable in the E c o R I - H i n d l I l cut plasmid. This also reduces the quality of the library without affecting the theoretical representativeness, as calculated by the Clarke and Carbon formula 1.

BIOCHEMICAL EDUCATION 25(4) 1997

An important point to emphasize is that the amount of yeast and plasmid D N A and, hence, the amount of competent cells, used here is one or two orders of magnitude below that usually employed in most molecular biology labs. This greatly affects the expected amount of clones and, therefore, the quality and representativeness of the library. We have observed that the best values students obtain are 2700 clones, 75 % with insert. We wish to point out that, although these are not good results for a 'real' scientist, they are good enough for students because they work on this experiment in the same way they would work if the amount of materials were higher and protocols longer or more sophisticated. One must also consider that the cost and simplicity of the described protocols allow the gene library construction to be adapted to the student lab. It is necessary to point out that many eukaryotic genomic libraries are made using cosmids or ~.vectors. However, libraries in plasmids are also common for lower eukaryotes or prokaryotes and, in any case, protocols are similar. We have chosen yeast because genomic libraries for this organism are in shuttle plasmids 5:°. Moreover, its small genome size improves the representativeness of the library obtained. However, we wish to point out that the experiment presented here can be done exactly as it is written if another D N A source is used instead of yeast. In these cases commercially available DNA, such as salmon sperm, calf thymus, E. coli, etc, or D N A isolated following the appropriate protocols may be used. Of course, the value used for the genome size in the Clarke and Carbon formula should be changed accordingly. We have worked with 8-16 students per group. It is convenient for students to work in pairs because it greatly reduces the error rate. Our experience over eight years is that many of the students obtain a library and, therefore, experience this important aspect of molecular biology for themselves. Adapting non-radioactive probing methods to screening their own libraries could be the next step in practical learning of hunting genes.

Acknowledgements The authors acknowledge the input provided by many undergraduate students during eight years of practical classes.

References 1 Clarke L. and Carbon J. (1976) A colony bank containing synthetic ColE1 hybrid plasmids representative of the entire E. coli genome. Cell 9, 91-99 2 Old, R. W. and Primrose, S. B. (1985) Principles of Gene Manipulation. Blackwell Scientific, Oxford 3 Winnacker, E. L. (1987) From Genes to Clones: Introduction to Gene Technology. VCH, Weinheim 4 Sambrook, J., Fristch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, USA 5 Rose M.D. and Broach J.R. (1991) Cloning genes by complementation in yeast. Methods Enzymol. 194, 195-230 6 Kaiser, C., Michaelis, S. and Mitchell, A. (1994) Methods in Yeast Genetics, 2nd edn. Cold Spring Harbor Laboratory Press, USA 7 Philippsen P., Stotz A. and Scherf C. (1991) DNA of Saccharomyces cerevisiae. Methods EnzymoL 194, 169-182 8 P6rez-Pons J.A. and Querol E. (1996) A laboratory class experiment illustrating basic principles of DNA cloning and molecular biology techniques. Biochem. Educ. 24, 54-56

242 9 P6rez-Ortln J.E., Ram6n D., Ferrer S. and Tordera V. (1986) Rapid plasmid isolation. A laboratory experiment for intermediate and advanced students. Biochern. Educ. 14, 142-144 10 Carlson M. and Botsein D. (1982) Two differentially regulated mRNAs with different 3' ends encode seceted and intracellular forms of yeast invertase. Cell 28, 145-154

Letter to the Editor From Donald E Nicholson

Metabolic Maps Dear Sir, A critical response to an article is generally to be welcomed, both by editor and author, since it is evidence of having been read and may make changes possible. There have been several comments and criticisms of my article in the April issue of Biochemical Education (25, 62-70) and of aspects of my Metabolic Pathways Chart which accompanied that issue, to which I am happy to respond. The letter from Professor Bentley pointed out four structural errors in the map, such as quinone with only one oxygen, or a benzene ring with only two double bonds. His criticisms are especially deserved in the light of my reiterated plea for accuracy which I made in the article. In slight mitigation, it was proofread by many people, and I displayed a copy of the map for several weeks in a leading British Biochemistry Department (together with an offer of £5 for every mistake found--which cost me very little). Although that offer has now expired, I shall be very pleased to receive details of further errors--and indeed any other comments. Professor Bentley's stricture on my use of horizontal carbohydrate structures are, of course, fully justified. This has always been a problem, and in a book on Metabolic Pathways 1 written years ago with Stan Dagley (a really great teacher of Biochemistry) we wrote 'It is important to realise that the linear structures of sugars and their related metabolites must be rotated through 90 ° before they become Fischer projections'. With his plea for the use of chemical rather than trivial names, I am less convinced. A major object of the map is to encourage students to 'explore'--and the discovery of recognisable names

BIOCHEMICAL EDUCATION 25(4) 1997

like Vitamin K or E is likely to have a more satisfying impact than would phylloquinone or c~ tocopherol--particularly to medical students who need all the encouragement we can offer. A major problem which has resulted in several critical comments has been the persistent use in my article (but not in the map) of the abbreviations for the pyridine nucleotides: NAD/NADH2 and NADP/NADPH2 rather than the much more usual convention N A D + / N A D H + H + and NADPH+/ N A D P H + H +. In fact, both conventions are validated by the IUBMB Committee on Nomenclature with perhaps a preference for the former, partly on the grounds that the charge on the N A D molecule is actually negative. I recognise my inconsistency in using one convention for the article and the other for the map. In fact, at the proof stage of the map, I changed conventions in deference to proof readers who insisted that most students would more easily recognise and understand the more traditional presentation. But neither is really chemically valid! Dr Candlish raises the question as to whether a map of metabolic pathways has ever been made available to students sitting examinations. I remember this was tried out at Leeds University many years ago and I later discussed the matter with several students. The response was generally pretty mature. They realised that they had lost the ability to impress the examiners by accurate memorisation of pathways, but they had gained something much more important--more time and 'brain-space' for better understanding of how the pathways worked--of interrelationships, co-factors, compartmentalisation, regulation, and so on. I am sure that this problem is much more acute now than it was then--particularly for medical students. Professor Campbell wonders whether potential Biochemistry students in schools are not put off Biochemistry by the complexity of the maps. This could be true if it is assumed that they would have to be memorised, as perhaps occurred in the past. I would like to think, however, that the result of their display is to provoke curiosity and then to stimulate explorat i o n - w h i c h is surely a worthy start to biochemical education and, indeed, any intellectual adventure. Donald E Nicholson

References 1. Dagley, S. and Nicholson, D. E. (1970) An Introduction to Metabolic" Pathways.