Appendix: Methods of Studying Nucleic Acids

Appendix: methods of studying nucleic acids

This appendix describes some of the most ribose or deoxyribose, or purine and py• important general methods used in the study rimidine. The tissue is treated with cold of nucleic acids not considered elsewhere in dilute trichloroacetic or perchloric acid to this book. The emphasis is on the principles precipitate nucleic acid and protein, and underlying the methods, and no attempt lipid is removed by extraction with organic is made to provide laboratory protocols, solvent. In the procedure of Schneider [ 11] which are now available in abundance, total nucleic acid (DNA + RNA) can be and to which reference will be made. It is separated from protein by hydrolysis in hot inevitable that some of the methods dealt acid, the liberated products being soluble with here will be superseded in the lifetime (section 2.7.3). These latter are then assayed of this edition. The reader wishing inform• for ribose and deoxyribose. In the procedure ation about current methodologies is there• of Schmidt and Thannhauser [12] the DNA fore advised to consult the most recent texts. and RNA are separated by alkaline hydro• However, at the time of writing the cited lysis of the latter followed by acidification references are recommended for more (Fig. A.l). detailed information on principles [ 1-5] and Analysis of nucleic acid phosphorus in practice [6-10]. such fractions usually employs a colour reaction involving the formation of a phos• phomolybdate complex [13, 14]. Following A.1 OCCURRENCE AND CHEMICAL depurination (section 2.7.2), deoxyribose ANALYSIS can be determined by a colour reaction with diphenylamine (section 2. 7.1) [ 15]. This A.1.1 Chemical determination of nucleic reaction is specific for DNA. In contrast, acids in tissues the orcinol colour reaction employed for depurinated RNA also occurs to a lesser The usual approach to the determination of extent with DNA [16]. The spectral prop• nucleic acids in tissues involves fractionation erties of the purine and pyrimidine bases of to remove lipids and proteins etc., followed such hydrolysed nucleic acids may also be by assay on the basis of either phosphorus, used as a basis of nucleic acid assay. These 594 Appendix: methods of studying nucleic acids

Tissue (section 2.7.4). Protein also absorbs at • t Cold dilu~e TCA or PCA 260 nm, although to a much lesser extent, Organic solven~s dependent on the content of the aromatic amino acids phenylalanine, tryptophan and tyrosine. Because the latter two of these Acid -soluble Nucleic acid + + have an absorption maximum at 280 nm it lipid prorein is possible to obtain an approximate estim• frachon residue Ho~ TCA or PCA ation of total nucleic acid and protein in Alkaline diges~ion Acidifica~ion a mixture by employing the following formulae based on [17]:

Soluble ex~rad Residue Soluble ex~rac~ Residue con~aining conrnining con~aining (pro~ein) Nucleic acid = 0.064 A260 - 0.031 A280 RNA DNA RNA+ DNA (mg/ml) Fig. A. I Extraction and fractionation of nucleic acids from tissues. *TCA - trichloroacetic acid, Protein (mg/ml) = 1.45 A280 - 0.74A260 tPCA- perchloric acid.

Typical values for the nucleic acid and bases have absorption maxima in the region protein contents of some rat tissues are of 260 nm (Fig. A.2). The spectral properties given in Table A.l. These may be expressed of double-stranded and single-stranded per gram tissue wet weight, but it is often DNA have already been discussed (section useful to express them in terms of tissue 2.4.1 ), as has the effect of pH thereon protein, which may be easily estimated

16 M 'o

l( 14

Ql u c 0 .D L. ~ 6 .D 0 L. 0 4 0 ~ 2

200 320

Fig. A.2 Ultraviolet absorption spectra for purine and pyrimidine bases at pH7. Occurrence and chemical analysis 595

Table A.l Typical values for the concentrations of nucleic acids in different rat tissues

Tissue RNA (mglg protein) DNA (mglg protein) RNA/DNA

Brain 40 15 2.7 Kidney 43 18 2.4 Liver 54 12 4.5 Skeletal muscle 62 13 4.8 Spleen 58 58 1.0 Thymus 30 93 0.3

Table A.2 Molar percentages of bases in RNAs from various sources

Type Adenine Guanine Cytosine Uracil

Ribosomal* E. coli 16SrRNA 25.2 31.6 22.9 20.3 23SrRNA 26.2 31.4 22.0 20.4 5SrRNA 19.2 34.2 30.0 16.7 Rat 18S rRNA 22.5 29.2 26.5 21.8 28S rRNA 16.6 35.5 31.6 16.2 5S rRNA 18.3 32.5 27.5 21.7

Messengert E. coli Outer-membrane lipoprotein 28.3 24.2 22.7 24.8 Rabbit P-globin 23.6 27.2 23.0 26.2 Rat a-actin 21.7 26.4 30.6 21.3

Viral* Bacteriophage MS2 23.4 26.0 26.1 24.5 Poliomyelitis virus 29.6 23.0 23.3 24.1 Rous sarcoma virus 23.8 28.8 25.3 22.1 Tobacco mosaic virus 29.1 24.2 19.1 27.6

* Ignoring base modifications. t Ignoring 3'poly(A) tails.

[18, 19]. Values of haploid DNA content for scribed for DNA in section 2.2.2. Typical various organisms are given in Table 3.1. values for DNA are given in Table 2.3 and the variation in percentage GC content in different genomes can be found in Table A.1.2 Analysis of base composition and 2.6. The principles of determining the base nearest-neighbour frequency composition of RNA molecules are similar to those for DNA, and values for some The molar proportions of bases in nucleic types of RNA (actually based on sequence acids are determined by hydrolysis, sep• analysis) are given in Table A.2. Transfer aration and spectral quantification, as de- RNA is not included because of the large 596 Appendix: methods of studying nucleic acids

proportion of modified bases (sections 2.1.2 sedimentation rate [25], electron microscopy and 11.7.2). In the case of rRNA (section [26], and radioautography [27]. Generally a 11.6.3) the modified bases have been combination of two or more methods has ignored in Table A.2. It may be remarked been used [28]. that, although it was stated in chapter 12 Although measurement of the sedimen• that rRNA is GC-rich, this need only be tation rate can be used to estimate the true in relation to the overall base com• molecular weight of an RNA molecule position of the genome, rather than in (section A.2.2), this method is not readily absolute terms. The examples of mRNAs in applicable to DNA molecules because of Table A.2 are rather arbitrary, and dramatic their extremely large size and asymmetric variations in the base compositions of shape. The theory behind the calculations different mRNAs are seen in the same depends on a knowledge of diffusion co• orgamsm. efficients but these are equally difficult to The determination of nearest-neighbour obtain either by sedimentation analysis frequencies (i.e. the relative frequency of or optical mixing spectroscopy [29-31]. occurrence of the sixteen different dinu• However, empirical formulae relating cleotides, NpM) in DNA, although to a sedimentation coefficient (sfo.w) and mole• considerable extent superseded by nucleo• cular weight (M) have been derived by tide sequence determination (section A.5), comparing the sedimentation rates of still has applications. It is described in molecules of known size. For example, the section 6.4.3. equation for linear, double-stranded DNA is

0 79 A.1.3 Estimation of the molecular sfo. w = 2.8 + 0.00834 M .4 [28, 32] weight of DNA On sedimentation of DNA to equilibrium Methods of estimating the size of small in density gradients (usually of CsCl - DNA molecules (up to lOkb) using electro• section A.2.1) the width of the band of phoresis on acrylamide and agarose gels are DNA is inversely proportional to the square described in section A.2.1. Such methods root of the molecular weight. As with light can be accurately calibrated using DNA scattering (see below) the result is depen• molecules of known sequence. DNA mole• dent on the concentration of the DNA and cules of up to even 1000 kb in length can be a series of values must be obtained and analysed by pulsed field gel electrophoresis extrapolated to zero concentration [33]. (section A.2.1). This is the most widely used, fundamental Of the more classical approaches, the method of estimating the size of DNA best absolute methods of determining molecules and is applicable to molecules the molecular weight of DNA are light of up to 108 daltons (150 kb ). scattering on low-angle instruments [20-22], Light scattering was widely used in the equilibrium analytical ultracentrifugation in 1950s to measure molecular weights of DNA gradients of caesium chloride [23], and up to 5000 kb. This technique, together with viscoelastic relaxation [24]. Most other conventional analytical velocity centri• methods have an empirical basis and rely for fugation to determine sedimentation co• their calibration on the few light-scattering efficients, should lead to reliable absolute experiments that have been performed. determination of molecular weight [34]. But These more empirical methods include the only since the introduction of the laser and measurement of intrinsic viscosity [25] and the production of machines to enable study Occurrence and chemical analysis 597

of scattering at low angles and changes in has been switched off. This viscoelastic the Doppler shift in the wavelength of the effect is caused by the stretched DNA scattered laser light has the method been molecules relaxing to the unstressed con• extended to molecules of larger size [29-32]. figuration. The theory derived by Zimm However, at low angles the scattering by to explain this effect has been used to dust particles becomes a major problem and measure the size of the DNA in Drosophila with large molecules different regions of the chromosomes (each of which contains one same molecule serve as scattering centres. DNA molecule [24]) showing the method The above methods all suffer from the to be applicable to DNA molecules of disadvantage that they are badly affected by molecular weight up to 79 x 109 (over any heterogeneity in the size of the DNA 105 kb). The method has the major advan• molecules. The problem is greatest when tage that it measures the size of the largest the intention is to prepare whole DNA DNA molecules in the solution [36]. molecules since high-molecular-weight DNA Two 'visual' methods of measuring the is very susceptible to shearing forces and to size of DNA molecules involve electron the action of contaminating nucleases. Thus microscopy and autoradiography. With all molecular-weight determinations of electron microscopy the Kleinschmidt an unknown DNA must be carried out in technique involves spreading the DNA on nuclease-free conditions (achieved by heat• grids coated with polylysine or polystyrene- sterilization), without pipetting or any other 4-vinylpyridine [37]. A drop of DNA sol• manipulation which puts shear stress on the ution in 1 M ammonium acetate containing DNA. It is extremely difficult to prepare 0.1 mg/ml denatured cytochrome cis applied high molecular-weight DNA intact from to the surface of a solution of 0.2 M am• chromosomes and these methods tend to monium acetate. A film of cytochrome c reflect the size of the smaller molecules. spreads across the surface, binding to the Attempts to relate molecular weight DNA which is disentangled and spread out. to viscosity involve subjecting the DNA A grid is touched to the surface and the solution to shear forces which tend to break sample is dehydrated in ethanol. It may long DNA molecules, and must be used with be stained with uranyl acetate or by evap• caution. However, Zimm developed a low• oration of platinum on to the surface. In the shear viscometer consisting of a slowly latter process the grid is rotated so as to rotating tube (the Cartesian diver) immersed cause the metal to pile up against all surfaces in a DNA solution under pressure, and a of the DNA, which stands out from the relationship has been established between grid [30]. intrinsic viscosity (17) and molecular weight Double-stranded DNA is readily visual• (M) of linear duplex DNA similar to that ized by this technique but single-stranded between sedimentation coefficient and DNA (or RNA) molecules require the molecular weight: presence of formamide to prevent their collapse [38]. Even so they appear as rather 0.665 log M = 2.863 + log (17 + 5) [35] wispy threads and may be stretched out to different extents. The incorporation of A more important observation is that, proteins which bind strongly to single• when the Zimm viscometer is used to stranded DNA (e.g. bacteriophage T4 gene measure the size of very large DNA mole• 32 protein: section 6.5.2) causes these cules, the Cartesian diver rotates in the regions to assume a profile thicker even reverse direction for a while after the power than the native DNA regions and they now 598 Appendix: methods of studying nucleic acids

have a regular mass per unit length. The deoxyribonucleases) and RNA, and the presence, on the same grid, of marker DNA avoidance of mechanical shearing. For molecules of known length (e.g. SV40 DNA eukaryotic cells a combination of pronase or in the relaxed circular form) allows the size protease K, a detergent and extraction with of an unknown DNA molecule to be found phenol can be used directly to disrupt the by measuring the relative contour lengths cells and remove protein. For the cells of on electron micrographs. This method can Gram-negative bacteria such as E. coli readily be used with DNA molecules of up gentle lysis can be effected using lysozyme. to about 103 kb, but larger molecules cannot Phenol extraction is used widely in the iso• be accommodated in a single frame. lation of both DNA and RNA, which are In denaturation mapping (section 6.8.1) retained in the aqueous phase whereas double-stranded DNA is partially denatured denatured protein collects at the interface (usually by alkali treatment) and the un• between this and the phenol phase, which paired bases reacted with formaldehyde to contains the lipids. RNA may be removed prevent their reannealing. The duplex DNA from the DNA by using pure pancreatic molecule now exhibits single-stranded ribonuclease, or by isopycnic ultracen• bubbles in the (A + T)-rich regions to give trifugation in a gradient of CsCl, which a characteristic map. is, in any case, a useful step for further When DNA is labelled in vivo with purification. [ 3H]thymidine and the cells lysed on a In isopycnic ultracentrifugation a density microscope slide to release the intact DNA gradient is established which encompasses molecules their position can be visualized by the buoyant densities of the molecules to be radioautography. The radioactive DNA on separated. Molecules will assume a position the slide is cqvered with a layer of photo• on the gradient corresponding to their own graphic film which is activated by the P• buoyant density, after which no further particles produced by the decay of the 3H separation can occur. In this method an to produce black grains. As with electron equilibrium is established, and the method micrographs, the size of the DNA can be is sometimes referred to as 'equilibrium obtained by measuring the contour length ultracentrifugation' to distinguish it from of the radioautographic image. This method the rate-zonal method (section A.2.2). As uses light microscopy and so the field size is already mentioned (section 2.4.3), the much larger than with electron microscopy. buoyant density of double-stranded DNA Cairns applied it to the study of the E. coli varies with the mole fraction of ( G + C). In chromosome, which is 3000 kb long [39]. general, however, RNA has a much higher buoyant density (about 1.9 g/ml) than double-stranded DNA (about 1.7 g/ml), A.2 ISOLATION AND SEPARATION OF which, in turn, is higher than that of pro• NUCLEIC ACIDS tein (about 1.3 g/ml). Single-stranded DNA is of slightly higher buoyant density than A.2.1 Isolation and separation of DNA double-stranded DNA (e.g. for a double• stranded DNA of buoyant density 1. 703 g/ The main problems to be overcome in iso• ml, the value for the single-stranded form lating high-molecular-weight chromosomal is 1.717 g/ml). DNA from bacterial [40] or eukaryotic cells Isopycnic ultracentrifugation is also used [41] are the removal of protein (especially to separate small bacterial plasmids from Isolation and separation of nucleic acids 599

chromosomal DNA. The basis of separation in this case is not a difference in GC contents (these are, in general, similar) but the fact that the chromosomal DNA is linear (normal methods do not extract the large circular bacterial chromosome intact) whereas the plasmids are circular. This Chromosomal difference is exploited by the addition of DNA saturating amounts of the intercalating fluorescent dye, ethidium bromide. The intercalation requires that the DNA strands be forced apart, with concomitant decrease in buoyant density and partial unwinding of the double helix. This latter process is hindered in the closed-circular plasmid molecules with the result that they bind less ethidium bromide and have a higher buoyant density than the linear chromosomal (and open-circular and linearized plasmid) molecules (Fig. A.3). Before attempting to separate plasmid and chromosomal DNA it is advisable to enrich the preparation in plasmid DNA. The most frequently em• Fig. A.3 Separation of dosed-circular DNA of ployed method for achieving this in pre• plasmid pBR322 from E. coli chromosomal parative work involves the use of an alkaline DNA by isopycnic centrifugation in a CsCI density gradient in the presence of ethidium pH during lysis and extraction [42]. This bromide. The band marked 'chromosomal denatures the DNA, causing strand-separ• DNA' may also contain nicked plasmid DNA ation in the case of linear molecules. On molecules. neutralization the latter will not renature, whereas the closed-circular plasmid DNA will and can be separated from the insoluble somal DNA consist of sheared fragments, denatured DNA. For small-scale isolation these do not in general separate on isopycnic of plasmid DNA, a boiling step is usually ultracentrifugation because the fragmen• used in place of the treatment with alkali tation is random and the base compositions [43]. of individual fragments are generally similar. Ethidium/CsCl gradients are also used in Exceptions do occur in the case of regions of the purification of small animal viruses (e.g repetitive DNA in eukaryotes (sections 3.2.2 SV 40) which contain circular genomes. In and 7. 7) which may be generated as quite this case the initial purification involves homogeneous species in relatively large removal of most of the high-molecular• amounts if the fragments are of an appro• weight cellular DNA as a precipitate ad• priate size. Should the base composition of sorbed to sodium dodecyl sulphate, leaving such a repeated DNA be markedly different the virus DNA in the so-called Hirt extract from the average base composition of the [44]. chromosomal DNA, resolution from the Although most preparations of chromo- bulk of the latter will occur. Such DNA 600 Appendix: methods of studying nucleic acids e 3000 1 2 Nicked __ _ 2000 --Origin circ l es ~ Lineors -- . .,-• ." - 2319 ~ 1000 Supercorls c: - 1 250 Q) 560 E Ol ·"\. 234 .....~ 500 ..... 0 $:"_ ~ Ol c: Q) • _J (a) "' 100;------,------~------~ 0 10 20 30 Disrance of migrat-ion (mm) (b)

Fig. A.4 Agarose gel electrophoresis of DNA. (a) Separation of: 1, different forms of DNA of plasmid pBR322; 2, fragments of DNA (lengths indicated in kb) derived from plasmid pBR322 by double-digestion with restriction endonuclease BamH I and Bgl I; (b) Plot of length of DNA fragment (log scale) against distance of migration (linear scale) of data from (a)2, illustrating linear relationship.

was originally given the descriptive name reaction (section A.7), and the separation 'satellite DNA' (section 3.2.2(b}}, a de• of such fragments is a common requirement signation which is sometimes applied to in recombinant DNA manipulations. Most all repetitive DNA, regardless of base• frequently this is achieved by horizontal composition or function. Such loose ter• agarose gel electrophoresis (46). This sep• minology is to be discouraged. It is also arates different linear DNA molecules possible to isolate the tandemly repeated according to size (the mobility is inversely ribosomal RNA and histone genes of certain proportional to the log 10 of the molecular organisms by this method [45). weight}, as the charge per unit length of Small-scale column chromatography has different molecules is identical and the been used for more rapid separation of larger molecules will be retarded by the gel plasmid and chromosomal DNA. The matrix (Fig. A.4) . The DNA is 'visualized' chromedia employed include anion• by use of ethidium bromide. The percentage exchange resins, gel permeation resins and of agarose can be increased to separate finely divided glass, and proprietary columns smaller-sized DNA fragments, but for very are available in which the separation can be small species polyacrylamide gel electro• further accelerated by centrifugal elution. phoresis (which is often used preparatively Separation of DNA ~y isopycnic ultra• for fragments up to 1 kb) must be used [47]. centrifugation or chromatography is largely This method is also employed in rapid confined to the different chromosomal nucleic acid sequencing methods (section species that one might find in a given cell. A.5), in which case 8 M urea is included to Precise fragments of such DNAs can be ensure denaturation. generated by restriction endonucleases As agarose gel electrophoresis is fre• (section A.6) or by the polymerase chain quently used to analyse preparations of Isolation and separation of nucleic acids 601

kb kb

600 550 600 550 420 420 • 320 320 220 220 ,

Fig. A.S Pulsed field gel electrophoresis of fragments of human leukocyte DNA. The restriction endonucleases indicated, which cleave vertebrate DNA infrequently, were used to generate large fragments which were subjected to 62 s pulses at 290 V for 39 h in 0.8% agarose using a hexagonal arrangement of electrodes. (a) DNA stained with ethidium bromide, (b) autoradiograph after hy• bridization to a radioactive probe derived from a highly polymorphic region of DNA located down• stream of the human a-globin gene complex. (Illustration kindly provided by Dr A. M. Frischauf.)

plasmid DNA which may be slightly nicked , introduction of the technique of pulsed field it is worth mentioning that the closed• gel electrophoresis [49, 50], in overcoming circular supercoiled form of the plasmid this limitation, has made it possible to migrates more rapidly than the less compact separate DNA molecules of about 1000 kb relaxed-circular form produced by single• and above (Fig. A.5). In this technique stranded nicks. Any linearized plasmid the electric field is alternated at intervals molecules generally migrate at an inter• between the forward direction and one at mediate position between these two, a result an angle to this (it may even be directly that is perhaps a little unexpected (Fig. A.4). opposite - there are many variations). Agarose gel electrophoresis in the presence Although the basis of the separation can be of varying amounts of ethidium bromide can envisaged simplistically in terms of different be used to determine the number of super• rates of reorientation of the DNA molecules helical turns in a closed-circular DNA, as as the direction of the field changes, there is the interchelating ethidium bromide pro• still debate regarding a rigorous physical gressively reduces the number of super• explanation [51] . The resolving power of helical turns, with a consequent effect on this method is such that it was necessary to the rate of electrophoretic migration [48]. devise new methods of preparing DNA that Very large molecules of DNA (greater avoided shear breakage. The technique than about 100 kb) cannot be separated by usually employed for this purpose is to conventional agarose gel electrophoresis in embed the live cells in molten agarose, an electric field of constant direction. The allowing it to solidify into blocks on cooling, 602 Appendix: methods of studying nucleic acids

after which the DNA can be treated in situ separating RNA molecules is rate-zonal by allowing reagents to diffuse into (and out ultracentrifugation employing sucrose density of) the blocks. Pulsed field gel electro• gradients [58]. In this method the separation phoresis can be performed on the gel blocks, of RNA is based primarily on size (in prin• and in this way the separation of intact yeast ciple, molecular shape can also have an chromosomes has been achieved [49]. influence, but with RNAs differences in shape make no significant contribution to A.2.2 Isolation and separation of RNA the separation). It should be emphasized that the density range of sucrose solutions In principle the methods used to extract does not extend to that of RNA species and, RNA from cells are generally similar to in contrast to isopycnic ultracentrifugation those for DNA. Degradation of RNA by involving CsCl gradients (section A.2.1), no ribonuclease is a greater threat than that of equilibrium is reached. The species to be deoxyribonuclease degradation of DNA, separated are applied in a narrow zone at especially in the case of mRNA, but there the top of the sucrose gradient, the main are a variety of methods to counter this. purpose of which is to prevent diffusion The major separation problem is posed by of the zones of individual species during the large number of different RNA species separation (Fig. A.6). lsopycnic ultracen• in eukaryotes, and to study a particular trifugation in gradients of dense sucrose species it is necessary to separate it from (often employing a discontinuous or 'step the others. gradient') may be used to separate 'free' One preliminary that may be useful in ribosomes from those bound to the mem• particular cases is subcellular fractionation brane of the endoplasmic reticulum (section [52]. Thus, the nuclei may be removed by 12.8.1) [59]. low-speed centrifugation (700 g for 5 min) Rate-zonal ultracentrifugation in sucrose after gentle disruption of the cells in the density gradients is also used to separate presence of sucrose (to preserve the osmo• different size classes of polyribosomes larity), the mitochondria may be removed (section 12.1) and to separate the large by further centrifugation (lOOOOg for and small subunits of ribosomes after their 10min), and the ribosomes separated from dissociation (section 12.6). soluble RNA species such as tRNA by Rate-zonal ultracentrifugation is clearly ultracentrifugation (e.g. 100 000 g for inappropriate for the separation of different 90 min). It may of course be necessary to species of tRNA, which are broadly similar purify further the subcellular fraction in in size. Methods involving separation on which one is interested, and methods for the basis of charge and hydrophobicity nuclei [53], nucleoli [54], mitochondria [55], differences (e.g. chromatography on BD• and ribosomes [56] are available. It is worth cellulose and RPC5) may be employed for mentioning that the most appropriate this purpose [60]. method for isolating an organelle, such as Although mRNA may be isolated from the nucleus, for subsequent RNA extraction purified polysomes, direct extraction from (where purity is of greatest importance) tissues is more frequently employed to may be quite different from the method minimize degradation by ribonuclease. The which will yield the most biologically active use of high concentrations of the chaotropic material [57]. salt, guanidinium thiocyanate [61], is per• One of the most widely used methods of haps more widespread than the methods Isolation and separation of nucleic acids 603

(a) (b) (c) (d)

centrifuge

Construct Appl y Ioyer of RNA zones gradient RNA to top of after gradient centri fug ation

(e) 285

t Froct ion number t Bottom of Top of gradient gradient

Fig. A.6 Rate zonal centrifugation of RNA through a sucrose density gradient. A sucrose density gradient is constructed in a centrifuge tube (a) and the RNA solution applied on top (b). During ultracentrifugation the main components of the RNA separate into zones, primarily on the basis of molecular weight (c). These zones may be recovered by puncturing the bottom of the tube and collecting different fractions in separate tubes (d). The separated RNA may be visualized and quantified by measurement of the absorbance at 260nm (e). Steps (d) and (e) may be conveniently combined by pumping the gradient through the flow-cell of a recording spectrophotometer. involving phenol and detergents, mentioned smaller than that for oligo(dT)"cellulose above for DNA. Another alternative is (about 6-10 residues, compared with 15 guanidine hydrochloride in combination residues), and although the latter material with organic solvents [7]. Inhibitors of is in more widespread use, the former has ribonuclease, such as placental ribonuclease applications to mRNAs having very short inhibitor protein [62], vanadyl ribonu• poly(A) tails. A mixture of different cleoside complexes [63], or macaloid [64] , mRNAs can be separated to a certain extent are sometimes used in preparing mRNA. on the basis of size, either by sucrose density The major problem to be overcome in the gradient centrifugation or, on a smaller isolation of mRNA is purification from other scale, by agarose gel electrophoresis - species of RNA, which are approximately broadly as for DNA but generally under 20 times more abundant. Affinity chroma• denaturing conditions (section A.2.1). Both tography using oligo(dT)-cellulose (65] or agarose gel electrophoresis and polyacryl• poly(U)-Sepharose (66], materials that bind amide gel electrophoresis have been applied the poly(A) tails of mRNAs, is the basis of to other types of RNA (67, 68]. The isolation the separation of mRNA from other species of individual mRNA species is usually of RNA. The minimum size of poly(A) tail achieved by recombinant DNA techniques required for binding to poly(U)-Sepharose is (section A.8). 604 Appendix: methods of studying nucleic acids

Agarose gel of DNA fragments Alkali Neutralize denahJre t t t t . f--t--/-/-/lI Nitrocellulose 'i > Tissues \ Filter paper Ylfl?ffdZ/lZJ Gel Bake Hybridization 1 j t::£1 ~ I ,_____== _l~!I ..---- Autoradiography Transfer assembly ------J Nitrocellulose Photographic film Fig. A. 7 Southern blotting. Fragments of DNA are fractionated by electrophoresis on agarose, the DNA denatured in alkali and, after neutralization, transferred (blotted) from the gel to nitrocellulose membrane by capillary action at relatively high ionic strength in the transfer assembly shown. The transferred DNA is baked onto the nitrocellulose, after which it can be hybridized in solution with a radioactive probe and the hybridizing bands visualized by autoradiography. In the hypothetical example illustrated only one of the original four bands has hybridized to the probe.

Nucleic acids may be precipitated with heteroduplexes can be most useful in a ethanol, 2.5 volumes in the presence of variety of other situations. However, for 0.3 M sodium acetate, being frequently most routine work heteroduplexes are employed. This is useful for purification detected by the radioactivity of one of their and concentration purposes. components. (Both radioactive and non• radioactive labelled nucleic acid 'probes' may be used (section A.4), but in the A.3 HYBRIDIZATION OF NUCLEIC following we refer only to radioactively ACIDS labelled probes for economy of expression). Originally, in order to identify which The denaturation and renaturation of component of DNA fractionated by agarose homoduplex DNA molecules has been dealt gel electrophoresis hybridized to a particular with in section 2.4, where the effects of base radioactively labelled probe one had to composition, temperature and ionic strength perform time-consuming and inconvenient were mentioned. The application of DNA multiple hybridizations in solution. To renaturation to determining the copy overcome this problem Southern [69] number of different portions of eukaryotic devised a method of transferring the DNA genomes ( C0 t value analysis) has been from the fragile gel to a solid nitrocellulose described in section 3.2.2. An important membrane by means of simple capillary technique in molecular biology is the form• action (Fig. A.7), although this can be ation of heteroduplexes between different performed more rapidly with the aid of a DNA molecules or between DNA and vacuum [70]. The DNA must be denatured RNA. The application of electron micro• with alkali before transfer, and immobilized scopic visualization of such heteroduplexes on the membrane afterwards. This latter formed in solution to the determination of can be achieved by baking at 80°C (in a the positions of introns (R-Loop mapping) vacuum oven to prevent the nitrocellulose has already been described (section 8.2), igniting) or, in the case of the subsequently and the electron microscopic analysis of introduced nylon membranes, by ultraviolet Hybridization of nucleic acids 605

produced by restriction enzyme digestion of total genomic DNA (Fig. A.8(b)), and in fact the method was originally applied to uncloned genomic and subgenomic DNAs. Such genomic Southern blotting has become a routine tool in screening for human genetic disorders by the detection of restriction fragment length polymorphisms (section 3.1.4(d)) [71). The conditions of washing the membranes depend on the degree of homology between the two members of the heteroduplex. (The homology need not be 100% as hybridiza• (a) ® (b) tion may be between a variety of related but non-identical sequences.) When the two Fig. A.8 Example of results of Southern blot• ting experiment. (a) Cloned DNA from a mouse/ members of the heteroduplex are identical, bacteriophage lambda recombinant (cf. Fig. the washing is usually performed at l2°C A.24), or (b) chromosomal DNA from mouse below the melting temperature (Tm), which liver, were digested with (different) restriction in a solution 0.2 M in Na + is related to the endonucleases, subjected to electrophoresis on mole percentage of (G +C) by the equation: agarose gels and Southern blotting performed as in Fig. A.7. Hybridization ~as to a 32P-labelled mouse actin eDNA clone (cf. Fig. A.21). S: Tm = 69.3 + 0.41 (G + C) [72] ethidium bromide stained gels photographed under illumination with ultraviolet light. A: autoradiographs of the nitrocellulose. The effect of ionic strength (p) on the melting temperature is given by the equation [73]: light. This method of transfer is commonly called Southern blotting, after its originator. The membrane is usually hybridized with the radioactive DNA probe in a minimum volume of solution in a sealed polythene bag at 68°C and conditions of relatively high Thus, to allow for the fact that the Tm of ionic strength that promote heteroduplex duplex DNA decreases by 1oc per 1-1.5% formation. (If 50% formamide is included mismatch [74, 75] the 'stringency' of the the hybridization is performed at 42oC.) hybridization can be decreased by lowering The membrane is washed under suitable the temperature of the wash and/or by conditions (see below), dried, and subjected increasing the ionic strength of the washing to autoradiography. This method is most buffer. easily applied to detecting which restriction Southern's method for the transfer fragment of a larger piece of cloned DNA of DNA from gels to a solid membrane contains sequences homologous to a par• has been extended to RNA, where it ticular probe (Fig. A.8(a)). However, the has acquired the rather illogical name of sensitivity of the method is such that it can Northern blotting. (There is a third point detect individual fragments in the continuum on the compass, 'Western blotting', the 606 Appendix: methods of studying nucleic acids electrophoretic transfer of proteins from DNA, which is reacted with an antibiotin polyacrylamide gels to nitrocellulose.) The antibody and then with a second antibody methodology of Northern blotting is dif• coupled to an enzyme such as horseradish ferent from that of Southern blotting peroxidase or alkaline phosphatase. Ori• because RNA does not bind to nitrocellulose ginally the enzyme was used to catalyse a paper under conditions in which DNA colour reaction to allow visualization of the does, and is hydrolysed by alkali. To over• DNA, but more recently detection has been come this problem, diazobenzyloxymethyl improved by using chemiluminescent sub• (DBM)-cellulose paper, which binds RNA strates for alkaline phosphatase [81], or (and DNA), was introduced [76]. However, by coupling the peroxidase reaction to a the use of this has declined since it was photochemical reaction. discovered that RNA is, in fact, immobilized on nitrocellulose membranes if it has first been subjected to electrophoresis under A.4.1 General labelling methods denaturing conditions. This may be achieved by including formaldehyde [77], methyl When the objective of labelling a nucleic mercuric hydroxide [78], or glyoxal and acid is merely that of allowing its detection, dimethyl sulphoxide [79] in the agarose gel. methods that cause the incorporation of Hybridization of immobilized nucleic radioactivity throughout the molecule are acids on membrane supports is not restricted suitable, as well as the specific end-labelling to material transferred from agarose gels. methods described in section A.4.2. His• It can be used for detecting recombinant torically, RNA and DNA were made DNA in bacterial colonies or bacteriophage radioactive from 3H-, 14C- or 32P-Iabelled plaques after transfer from petri dishes precursors in vivo, but methods involving (section A.8) or for multiple samples of total the incorporation of radioactive label in vitro cellular RNA applied directly to the nitro• are more convenient for recombinant DNA cellulose (the so-called dot-blot technique). work and produce material of higher specific activity. One highly efficient method of labelling A.4 METHODS OF LABELLING double-stranded DNA in vitro is by 'nick NUCLEIC ACIDS translation' (Fig. A.9) using [a-32P]dNTPs [82]. In this method nicks are introduced There are many circumstances in which the into the DNA with DNase and the 5'• detection of DNA is only possible if the phosphate ends produced can serve as DNA is labelled in some way. Furthermore, substrates for the 5' ~ 3' exonucleolytic certain techniques require DNA labelled activity of E. coli DNA polymerase I, which specifically at one end. By far the most at the same time repairs the gaps by addition common method of achieving such labelling of the [a- 32P]dNTPs to the 3' -OH end of the is with radioactivity (usually 32P; less com• 'nick' (section 6.4.2). In equally widespread monly 35S or 3H), and for economy of use for labelling double-stranded DNA expression the discussion below will be to high specific activity is the method of in terms of radioactively labelled probes. 'random-priming' [83, 84]. A random However, non-radioactive labelling methods mixture of oligonucleotides (6-12 mers) is are increasingly being used. Most frequently used to prime the synthesis of copies of the biotin-11-dUTP [80] is used to label the DNA template, which must be denatured Methods of labelling nucleic acids 607

5' ••.• G p G p T p C p T pAp G p A •.•• control of a bacteriophage promoter [86], (a) 31 •••• C p C p A p G p A p T p C p T ••.• and generate radioactively labelled RNA copies of the DNA by transcription in vitro with the bacteriophage RNA polymerase 1DNa"' I and [a-32P]NTPs. Systems based on bac• Nick teriophages SP6, T7 and T3 have been used 1 1 3 ' 5 for this purpose, as their RNA polymerases 51 •••• G p G HpT p C p T p A p G p A •... (b) 0 are highly specific for their own promoters. 3' ••.• C p C p A p G p A p T p C p T .•.. An advantage of generating single-stranded labelled RNA, rather than DNA, probes is p,;Ns DNA polymerose I that they are easier to remove from the ! DNA template, which has the potential to ------Nick interfere in subsequent hybridizations. 1 I * * * * 3'' 5 (c) 5 . . • • G p G p T p C p T p A0 H pG p A • • · · Small oligonucleotides used as probes are 3' •..• C p C p A p G p A p T p C p T •... usually made radioactive by end-labelling (section A.4.2), but procedures have been +pT, pC,pA +pp devised for producing uniformly labelled probes in situations where a higher specific Fig. A.9 Nick translation. Double-stranded activity is required [87]. DNA (a) is treated with a low concentration of pancreatic DNase producing occasional nicks It is possible to label mRNA directly with 3'-0H groups (b) on which DNA poly• in vitro by certain end-labelling methods merase can start polymerization. The use of (section A.4.2) but a copying method is [a-32P]dNTPs produces a radioactive phos• generally preferred because it produces a phodiester bond pand the 5' ~ 3' exonuclease more stable product of higher specific activ• action of the enzyme successively cleaves further 32 non-radioactive phosphodiester bonds which are ity. This involves the use of [a- P]dNTPs replaced by radioactive ones (c). In the course and reverse transcriptase to produce a of this replacement the position of the nick 32P-labelled single-stranded eDNA copy undergoes vectorial translation, as indicated. (section A.8.2). The labelling of tRNA in vitro is im• portant in sequence determination in cases for this purpose. The Klenow fragment of where insufficient material is available for DNA polymerase I is used to avoid de• spectral analysis and it is not possible to struction of the primers and products by the label with 32P in vivo. As well as enzymic 5' ~ 3' exonucleolytic activity of the intact end-labelling methods (see below) chemical enzyme, and [a-32P]dNTPs are again used labelling using [ 3H]borohydride has been as labelled precursors. employed [88, 89]. For certain purposes it is necessary to prepare single-stranded labelled probes. One method of doing this [85] is by cloning A.4.2 End-labelling methods the DNA into the single-stranded phage vector M13, and copying it in vitro using an End-labelling does not produce DNA oligonucleotide primer and [a-32P]dNTPs probes of such a high specific activity as (cf. section A.5.1). An alternative method do methods of uniform labelling; but, of producing single-stranded probes is to nevertheless, this is necessary for certain clone into a vector that puts it under the specific applications. These include se- 608 Appendix: methods of studying nucleic acids

(a) t w-GpGpApTpCpCp~ Bam HI 5' pGpApTpCpCp- ~CpCpTpApGpGp~ 3'Ho GP-• ~ Alkalinet phosPhatase 5' *pGpApTpCpCp"""" PPPrA* 5'Ho GpAp T p C p C p- 3'Ho Gp.....,_ T4 Polynucleotide kinase 3'Ho GP-

(b) t ~GpGpApTpCpCp--- Bam HI 5'pGpApTpCpCp- --CpCpTpApGpGp--- 3'HoG P- t Klenow fragment of DNA polymerase I

~GpApCpGpTpCp~ 3 I HO Ap c p Gp T p c p- + T4 DNA ~ polymerase 5'p GP- T4 DNA polymerase 3'oH-

Fig. A.lO Methods for end-labelling DNA fragments. (a) use of polynucleotide kinase and [y- 32P]A TP for 5' -labelling of cohesive ends with 5' -overhang produced by restriction endonuclease such as BamH I; (b) use of Klenow fragment of DNA polymerase I and [a-32P)dNTPs for 3'-labelling of cohesive ends with 5'-overhang produced by restriction endonuclease such as BamH I. In this case any of the (a-32P]dNTPs could have been used for labelling if the other non-radioactive dNTPs had been included to allow complete fill-in; (c) use of phage T4 DNA polymerase and [a-32P]dNTPs for 3'-labelling of cohesive ends with 3'-overhang produced by restriction endonuclease such as Pst I. An excess of the other three non-radioactive dNTPs prevents the exonuclease activity proceeding further. Methods (a) and (c) may also be applied to blunt ends produced by restriction endonucleases such as Sma I. N.B. In each case the other end of the fragment (not illustrated) will also be labelled if it is similar, and a second restriction endonuclease digestion or strand-separation will be required to obtain a fragment of DNA labelled at one end only. Determination of nucleic acid sequences 609

quence analysis by the method of Maxam Several methods for end-labelling are and Gilbert (section A.5.1), mapping of available when the nucleic acid is only to RNA transcripts (section A.9.1), and 'DNA be used as a probe. With DNA, extension footprinting' (section A.9.2). It is also the of 3' -overhanging ends using terminal most usual way of labelling oligonucleotide transferase and [a-32P]dNTP [92] is an probes. alternative to the use of DNA polymerase. The DNA to be labelled is generated by For mRNA, extension of the 3'-end with cleavage with a restriction endonuclease E. coli poly(A) polymerase can be used (section A.6) and the method of labelling for labelling with [a- 32P]A TP, or depends on whether flush ends, or 'sticky' [a- 32P]cordycepin (3' -deoxy ATP) if the ends (in which there is a 5' -P04 or 3' -OH addition of only a single nucleotide is overhang) are produced. Ends with a 5' -P04 required [93]. An alternative to covalent overhang are easily labelled by either of two incorporation as a method of end-labelling, methods (Fig. A.10(a) and (b)), in one useful in certain restricted circumstances, is of which the 5'-P04 is removed by alka• hybridization of a labelled fragment to line phosphatase and replaced by the y• suitable single-stranded regions. In the case phosphate of [y-32P]A TP in a reaction of eukaryotic mRNAs, [ 3H]poly(U) can be catalysed by bacteriophage-T4 polynucleo• hybridized to the poly(A) tails [94]. tide kinase (section 4.7.1) [90]. In the other the Klenow fragment of E. coli DNA polymerase I (section 6.4.3) is usually A.5 DETERMINATION OF NUCLEIC used to fill the gap in the strand with the ACID SEQUENCES recessed 3' -OH end, using an appropriate [a- 32P]dNTP, although bacteriophage T4 A.5.1 Determination of DNA sequences DNA polymerase may also be used (Fig. A.10(b)). (The 5' ~ 3' exonuclease of the There are two rapid methods in use for complete E. coli enzyme would result sequencing large fragments of DNA. in degradation of the template for this Although the method of Maxam and Gilbert reaction.) Thus, these two methods com• [95]) has been largely replaced by that of plement each other in labelling different Sanger [96] for general purposes, we shall strands. describe it here as it is still important in The polynucleotide kinase method may particular situations. A description of a third be adapted for labelling the 5' -P04 of flush method (the 'Wandering Spot' or 'Mobility ends by effecting local denaturation of the Shift' method), which still finds application latter, but cannot be applied efficiently to small oligonucleotides, can be found where there is a 3'-0H overhang. The 3' ~ in [97]. Both the method of Maxam and 5' exonuclease and 5' ~ 3' polymerase Gilbert and that of Sanger involve the activities of the Klenow fragment of DNA generation of a 'ladder' of fragments of polymerase I can be used to label flush ends different sizes, but with one common end. or 3' -OH protruding ends by a replacement The basis of these methods is to generate synthesis method (Fig. A.10(c)), although specific sets of radioactively labelled frag• bacteriophage-T4 DNA polymerase (which ments, each set terminating at a particular has a more powerful 3' ~ 5' exonuclease base (in the ideal case), so that by using activity and also no 5' ~ 3' exonuclease) high-resolution polyacrylamide gels [98, is more effective [91]. 99] fragments differing by only a single 0 J N HN ~ .., 0 H NAN )

'\.IIO-P-O-CH2 2 I_ 5' ~0 0 3' 0 I -0-P=O I 0\

0 OH H "' , - I I Q;~r-"\ 0-P-0. + H2C = C- CH = CH -C=N\...... J b- +

(a)

0 '\ II - 0-P-0 "l 0 b- O-~-O-CH2 ~ ~- 5 1 ~0H HJ='\_j + OH- 3'H' ------0I 0-P=O I + 0\ 0

(b) Fig. A.ll Base-specific cleavage reactions ust:d in the sequencing method of Maxam and Gilbert. (a) Dimethyl sulphate reaction for guanine residues; (b) Hydrazine reaction for pyrimidine residues (thymine illustrated). For further details see text and reference [95], from which this figure is adapted, with permission. Determination of nucleic acid sequences 611

nucleotide may be resolved and the se• in section A.4.2. Such methods, in fact, quence can be deduced. In both cases generally label both ends of a piece of cloned DNA is normally used, although duplex DNA, and fragments with a single• the method of Maxam and Gilbert can be labelled end are generated by restriction applied to uncloned genomic DNA. endonuclease digestion of this labelled As sequences accumulate, it becomes DNA followed by separation on poly• necessary to use computer programs to acrylamide gels. Less commonly, single handle and analyse them. Pioneers in end-labelled molecules are obtained by developing such programs were Staden strand separation of the DNA. An example in Cambridge [100], and a group at the of a Maxam-Gilbert sequencing gel and its University of Wisconsin in the USA [101]. interpretation is given in Fig. A.12. Because the advantage of the Sanger (a) The method of Maxam and Gilbert [95] method of sequencing depends on cloning DNA into appropriate vectors and copying The essence of this method is the chemical it in vitro, it is in circumstances where this is fragmentation of either single- or double• either impossible or undesirable that the stranded DNA by base-specific reactions. method of Maxam and Gilbert finds its most The reactions most commonly used are frequent use today. Such circumstances those absolutely specific for guanine residues include DNA that is unstable when cloned, or for cytosine residues, and those that are and DNA with regions of secondary struc• specific only for purinf;s or pyrimidines. ture (generally arising from a high (G + C)• (There are some other, less frequently used, content) that prevent the DNA polymerase reactions [102, 103].) Cleavage at guanine used in the Sanger method from completing residues is effected by methylation with its copying. However, the main current dimethyl sulphate at the N7 position, application of the method is for sequencing leading to instability of the glycosidic link• uncloned genomic DNA to study either its age which is then hydrolysed by piperidine, methylation state or its interaction with followed by {J-elimination of both phos• proteins. The latter application (genomic phates from the sugar (Fig. A.ll(a)). Purine footprinting) is described in section A. 9 .2. nucleotide linkages are hydrolysed with acid The study of DNA methylation by the (section 2.7.2), again followed by piperidine method of Maxam and Gilbert is based on treatment. Pyrimidine residues are hydro• the fact that, under the conditions normally lysed by hydrazine (Fig. A.ll(b)), a reaction used, cleavage by hydrazine does not occur which, in the case of thymine, can be in• at 5-methylcytosine residues, causing a gap hibited by 2M NaCl, thus allowing specific in the sequencing ladder [104]. It is nec• cleavage at cytosine residues. essary to analyse the methylation state of Conditions of chemical cleavage are the genomic DNA in situ, as this is altered generally adjusted to try to obtain a single after cloning into prokaryotic vectors. scission per DNA molecule. Even so, each Genomic sequencing was devised by scission would produce two fragments. In Church and Gilbert [105], and the method order to visualize on polyacrylamide gels has subsequently undergone refinement only fragments of increasing length ema• [106]. The genomic DNA is digested with nating from one end of the DNA, a single an appropriate restriction endonuclease to end of the DNA must be radioactively generate a fragment containing the region labelled using one of the methods described of interest with suitable end points for A T 32.p CCTGTATGCCA ....

Chemical I cleavage before I G before I A+ G before I T + C c 32p 32p 32p c 32p c 32p c c 32p C C T 32 pCCT 32pCCTG 32 pCCTGT 32 pCCTGTA 32p C C T G TAT 32pCCTGTAT 32 pCCTGTATG 32 pCCTGTATG 32pCCTGTATGC 32 pCCTGTATGC 32p C C T G TAT G C C

(a) (b)

Fig. A.l2 Example of DNA sequence determined by the method of Maxam and Gilbert. (a) The 32P-end-labelled fragments derived from chemical cleavage of 5'-end-labelled DNA. These are set out in increasing order of size to allow comparison with (b) the autoradiograph of the part of the gel on which these (and larger fragments) were separated on the basis of size. It can be seen that bands appearing in both G and G +A tracks are assigned toG whereas those only appearing in the G + A track are assigned to A. C and Tare similarly differentiated. (This is, in fact, a simplified example, and the extreme 5'-fragments are normally quite difficult to read.) Determination of nucleic acid sequences 613

sequencing, and the base-specific reactions [a- 35S]dNTP in the reactions, and base• are performed. Because the restriction specific chain termination is effected by fragment of interest cannot be purified from the addition of the appropriate 2' ,3'• all the other fragments generated, it is not dideoxynucleoside triphosphate ( ddNTP), possible to end-label it covalently as for which, lacking a 3'-0H, cannot be ex• conventional sequencing. Instead, the ends tended further. An example of a Sanger of individual strands of the restriction sequencing gel and its interpretation is fragment are labelled after the chemical given in Fig. A.13. cleavages have been performed. Initially Initially, the widespread application of this was done indirectly by hybridization this method was prevented by the require• to suitable single-stranded radioactively ment for a single-stranded template and the labelled probes following gel electrophoresis need for a separate oligonucleotide primer and transfer to a nitrocellulose membrane. for each piece of DNA to be sequenced. (This, of course, presupposes some prior However, these limitations were overcome knowledge of the sequence from analysis of when the single-stranded bacteriophage, cloned DNA.) Such indirect end-labelling M13, was adapted for use as a sequencing had a number of disadvantages [106], but vector [108, 109]. The DNA to be sequenced the most severe was the weakness of the is subcloned into one of a variety of adjacent radioactive 'signal' obtained. However, sites in the double-stranded replicative form more recently the polymerase chain reaction of the bacteriophage vector' and can be (section A. 7), primed unidirectionally by an sequenced in the easily prepared single• oligonucleotide hybridized to the common stranded DNA using oligonucleotide end of the ladder of fragments, has been primers complementary to regions of the used to generate more highly labelled copies bacteriophage DNA flanking the cloning of the fragments, which can be subjected to sites (Fig. A.14). electrophoresis and autoradiography in the This development led to a dramatic normal manner [107]. increase in the use of the Sanger method, for which a number of refinements have (b) The Sanger dideoxy method [98] been made. These include the replacement of the Klenow fragment by chemically The essence of this method is the primed modified bacteriophage T7 DNA poly• synthesis of partial copies of the DNA to merase to extend the size range of the copies be sequenced, with random base-specific generated [110], or by the thermostable premature termination of the copying pro• DNA polymerase from Thermus aquaticus ducing ladders of different length fragments (Taq DNA polymerase) to allow the copying terminating in each of the four different reaction to be performed at a temperature bases. The copying is catalysed by the at which regions of DNA secondary struc• Klenow fragment of E. coli DNA poly• ture no longer cause premature termination. merase I, which requires a primer and that The effect of DNA secondary structure the template (the DNA to be copied) be causing anomalous migration during gel in the single-stranded form. (The Klenow electrophoresis (so-called 'compressions') fragment, lacking the 5' ~ 3' exonuclease, can be eliminated or decreased by replacing is used to prevent attack on the 5' -end of dGTP by 7-deaza-2'-dGTP. Sanger se• the primer.) The copies are made radio• quencing using M13 vectors always entails active by inclusion of an [a-32P]dNTP or a starting the copying from a similar position the

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strand. primer Determination of nucleic acid sequences 615

to facilitate this process, although there is also a more recent variant of the method in which the need for subcloning is actually bypassed [112].

A.5.2 Determination of RNA sequences

Before the advent of rapid DNA sequencing methods, considerable effort was expended on developing techniques for sequencing RNA. The availability of partially or totally Primer hybridization site base-specific endonucleases allowed em• ployment of approaches formally rather similar to those used for sequencing pro• teins, but requiring different methods for separating and analysing the oligoribo• nucleotides. Although the sequencing of a DNA copy must now be the method of choice for sequencing any RNA, this approach may not be applicable to small RNAs or oligonucleotides, and in any case does not identify modified nucleotides. In Fig. A.14 The bacteriophage vector M13mp18. these cases (especially for tRNAs) direct The double-stranded replicative form of this methods are still employed [113). single-stranded DNA phage is shown with Rapid sequencing methods involving authentic genes 1-X indicated. The multiple cloning sequence interrupts the E. coli lacZ' base-specific cleavage and separation of (truncated P-galactosidase) gene, and the fragments on polyacrylamide gels have been position of hybridization of one of the primers introduced for 5'-end-labelled RNA [114, that may be used for sequencing by the Sanger 115). The cleavage is effected enzymically dideoxy method is shown (cf. Fig. A.13). with the following ribonucleases (section 4.3): in the multiple-cloning site near to the universal primer. Therefore, different frag• Ribonuclease T1 : cleaves 3' of G ments of the sequence of interest must be Ribonuclease U2 : cleaves 3' of A subcloned into the vector to obtain their sequences. This process has been acce• Ribonuclease Phy M : cleaves 3' of A and U lerated by a technique in which successive B. cereus ribonuclease : cleaves 3' of U and C deletions of increasing length generate sub• clones in which different portions of a long sequence of interest are brought into the In other respects the method and the inter• proximity of the multiple-cloning site [111] pretation of the results are formally similar to (Fig. A.15). Special vectors are available that of Maxam and Gilbert, described in with suitable restriction endonuclease sites section A.5.l(a). 616 Appendix: methods of studying nucleic acids

Insert DNA

B:site giving 3' overhang

Site complementary to sequencing ~er

(i) + Digestion with restriction endonucleases A and B A B 5' --.r~------...... ,,------r 3' f s Qi) + Digestion with exonuclease Ill lor dillerenttimes

f Digestion with 51 nuclease (ii) f Treatment with Klenow fragment ol DNA polymerase I f Recircularizalion with DNA ligase 0

Fig. A. IS Generation of unidirectional deletions with exonuclease lii to facilitate sequencing by the Sanger dideoxy method. The DNA to be sequenced is cloned into the double-stranded form of a vector in which there are two unique restriction endonuclease sites separating it from the site to which the sequencing primer hybridizes. (i) Digestion at the site proximal to the insert (A) generates a 5' -overhang, whereas digestion at the site proximal to the primer site (B) generates a 3' -overhang. (ii) Digestion with the exonuclease (which is specific for 3'-0H ends in double-stranded DNA) only occurs at site A, causing deletions the size of which depend on the time of digestion. (iii) The single-stranded regions are removed with Sl nuclease, any remaining 5'-overhangs 'filled' using the Klenow fragment of E. coli DNA polymerase I, and the DNA religated to bring the new end-points of the deleted insert in the proximity of the primer site. After transformation of E. coli and preparation of templates, sequencing can be performed from these new end points, which correspond to internal positions in the original insert. For further details see [111 ]. Restriction mapping of DNA 617

{ i) {ii) E1 {i) {ii) (iii) {iv) A A' Gr------. ---10 kb----.: -4kb--6kb- t=10 kb - I 9 kb -- t--6 kb (iii) (iv) - t--4 kb E2 E2 E1 - - 1--3 kb I Ai--1+------.....o!A' I AI t t 1A' ----9kb-"""""":"'"• -3kb-6kb- or 1kb E2 or 1kb E1 E2 ---1 kb II Al-l ------....~.o!-tiA' II A I t t lA' ~--9kb--- -+-4kb-+-+-5kb- @'------' 1 kb 1kb (a) (b) Fig. A.16 Example of restriction mapping. The objective here is to determine the relative positions of the recognition sites for two restriction endonucleases, E1 and E2, along a hypothetical10 kb fragment of DNA. (a) If the 10 kb fragment (i) is cleaved by E1 to give 4 kb and 6 kb fragments the two ends of the DNA can be distinguished as A and A' (ii). This then defines two possible positions of cleavage (I and II) of a second enzyme E2, which generates 1 kb and 9 kb fragments (iii). The two alternatives I and II may be distinguished by the different sized fragments they predict in the case of double-digestion with Eland E2 (iv); (b) diagrams of the results of agarose gel electrophoresis of (i)- (iv). The result in lane (iv) is only consistent with alternative I, and hence (iv)/I represents an (albeit limited) restriction map of the 10 kb fragment.

A.6 RESTRICTION MAPPING OF DNA method in its simplest form involves digesting the DNA separately and in combination with When studying specific regions of genomic different restriction endonucleases, sep• or cloned DNA it is necessary to have some arating the resulting fragments by agarose sort of 'map' to differentiate one area from (or sometimes polyacrylamide) gel electro• another. There are two different types of phoresis (section A.2.1), visualizing these map. A genetic map can be constructed (in under ultraviolet light after staining with suitable organisms) in which the positions of ethidium bromide, and estimating their sizes functional genes contributing to particular in relation to standards [116]. If there are not phenotypes can be interrelated by studying too many recognition sites for the enzymes it is the frequency of genetic recombination possible to deduce the position of these, as is between normal and mutated alleles. A illustrated in Fig. A.16. physical map, on the other hand, can be It can be appreciated that for enzymes constructed in the absence of genetic data or with many recognition sites it is both difficult knowledge of any function the DNA might and tedious to construct a restriction map by have, and uses purely physical techniques. this procedure. In the case of large pieces of These include the formation of hetero• DNA (e.g. those cloned in bacteriophage duplexes between standard DNA molecules lambda or cosmid vectors - section A.8.4) and the DNA under investigation, and re• most restriction endonucleases will have striction mapping, the subject of this section. many recognition sites, so that it is difficult A restriction map indicates the positions to circumvent this problem by choice of along a piece of DNA at which there are enzyme. In any case it is often useful to have recognition sites for particular type II re• a restriction map for enzymes that cleave a striction endonucleases (section 4.5.3). The piece of DNA at many positions. An alter- 618 Appendix: methods of studying nucleic acids

E3 E3 E3 E3 S~ain Au~orad ( i ) AI I I I I I A' e ""-----v---''--v-'~ '-v-' 3 2·5 1· 5 1 2 kb r-9 kb r-7 kb End-label* - ! - r--4·5 kb ( i i ) - -3 kb I* A' -..... 1--2 kb 1. Diges~ wi~h E2 !2. lsola~e 9 kb frogmen~ E3 E3 E3 E3 (iii) 1-----'-t__ fL---..L.f ...lfl----ti* A' (±'JL.....-_ _.._ _ ___.

Par~ial diges~ (b) 1wr~h E3 .l...!!L* 3 kb * ( iv) 4·5 kb * 7 kb * 9 kb * Labelled produc~s (a) Fig. A.l7 Example of partial digestion restriction mapping. (a)(i) The objective here is to deter• mine the relative positions of the four recognition sites for the restriction endonuclease E3 on the hypothetical DNA fragment of Fig. A.16. This is end-labelled (ii), digested with enzyme E2, which is known to cleave close to one end, and the larger (9kb) fragment isolated (iii). This is subjected to partial digestion with E3 giving a mixture of end-labelled fragments together with non-labelled fragments not illustrated (iv); (b) diagram of the results of electrophoresis of the partial digestion products. The distances of the E3 recognition sites from end A' can be read off the autoradiography ladder using molecular size markers {cf. Fig. A.4(b)). This allows the restriction map of (a)(i) to be deduced. native approach was devised involving end• strategy, for application to DNA cloned into labelling of the DNA and partial digestion bacteriophage or cosmid vectors, replaces with single restriction endonucleases [117). end-labelling by hybridization of a specific In essence the strategy is similar to that for 32P-Iabelled oligonucleotide to either the Maxam and Gilbert DNA sequencing using right or left cohesive end of the molecule a single base-specific reaction, except that [118). This avoids the need for secondary analysis of the larger fragments usually cleavage and gel fractionation to produce a employs the standard agarose gels. Although fragment of DNA labelled at one end. the pattern of stained fragments is complex, the autoradiograph of the dried gel reveals a A.7 THE POLYMERASE CHAIN ladder of fragments, the increasing sizes of REACTION which represent increasing distances from the labelled end. This is illustrated schema• The standard method for purifying in• tically in Fig. A.l7. A modification of this dividual genes or mRNAs is by cloning in The polymerase chain reaction 619

Primer2

3 ' ~ 5'

Target DNA 5' •••• • ••• 3' 3' •••• • ••• 5' I I I 5' ...... 3' Denature, Anneal, Cycle 1 Primer 1 Polymerize

• •••• 5' 3' a • •• 5' 0 Primer 1 - .....•••• -- •••• --- •••• Denature, Cycle2 Anneal, Polymerize 5' - • •••• 31 5' Primer2 - Dena lure, - Cycle 3 Anneal, Polymerize

3' 5' s· - • Primer 1 - Fig. A.l8 The polymerase chain reaction. The figure shows three cycles of polymerization, with only the products of one strand being shown for simplicity at each cycle. For details see text. bacteria, as described in section A.8 below. in Fig. A.l8. Two oligonucleotides are However, it is now possible to use an chosen to encompass the region of DNA to enzymic reaction, the polymerase chain be amplified. Each must be complementary reaction (PCR) , to amplify minute quantities to a different strand in such a way that their of specific regions of DNA without the 3'-ends are directed towards one another. intermediary of bacteria [119]. The poly• The double-stranded template DNA is merase chain reaction is not to be viewed denatured and then allowed to anneal to as a substitute for cloning, rather it is an the oligonucleotides. These are then used extremely powerful technique that can be to prime the copying of the DNA template used in concert with cloning methods to by bacterial DNA polymerase in vitro, study and manipulate nucleic acids. producing multiple copies, but with random The principle of the method is illustrated end-points. For clarity, Fig. A.l8 shows 620 Appendix: methods of studying nucleic acids

extension from just one of the primers in responding to proteins for which sequence this first reaction, and in subsequent steps. information is available has been greatly The DNA is again denatured and the daugh• facilitated and accelerated (section A.8), ter molecules allowed to reanneal and a as has the process of mutagenesis in vitro second copying reaction performed. The (section A.9.4). It is now much easier to daughter molecules generated from the excise precise regions of DNA, without first reaction will be primed by the second being dependent on the presence of suitable oligonucleotide, and the copies generated restriction endonuclease sites; and the will all terminate at the common end of the inclusion of suitable restriction endonu• daughter molecules corresponding to the clease sites at the 5' -ends of the primers first oligonucleotide primer. Successive allows equally precise insertion into, for reactions with alternate primers will gen• example, expression vectors ( cf. section erate a geometrically increasing number A.9.3). The method has also been applied of molecules of DNA of uniform length. to genomic DNA sequencing (section Using 25-45 cycles of reaction it is poss• A.5.1(a)) and chromosome walking (section ible to amplify DNA a million times or A.8), and novel applications of the method more. Initially the method was tedious and continue to be reported. costly because the denaturation step in• The current limitation of the polymerase activated the Klenow fragment of E. coli chain reaction is the size of the region of DNA polymerase at each cycle, and the pro• DNA that can be amplified (above about ducts were very heterogeneous. However, 1 kb, the efficiency declines); and its major the introduction of the thermostable DNA problem is cross-contamination: a con• polymerase from T. aquaticus allowed re• sequence of the exquisite sensitivity that is peated denaturation steps to be performed its major strength. One should also mention at 94°C without loss of enzyme activity, that errors are introduced into the amplified and the elongation reaction could also be DNA with a frequency that requires the performed at a higher temperature with exercise of considerable caution. However, resulting greater specificity [120]. these problems are being actively addressed The outstanding feature of the polymerase [214], and it is clear that the polymerase chain reaction is its sensitivity, allowing the chain reaction and its various adaptations detection of a single mRNA molecule per will continue to exert a profound influence cell. Thus, the most spectacular applications on developments in biology. ofthe method (reviewed in [121]) have been of a general nature: the analysis of DNA A.8 CLONING DNA from mummified museum specimens in the field of evolutionary biology, from single A.8.1 The principles hair roots for forensic purposes, and from trace amounts of viruses in experimental The study of individual mRNAs and of medicine. The method has likewise found particular regions of genomic DNA was application in the diagnosis of genetic hampered historically by two problems. The diseases and in determining the sex of fer• first was the difficulty of devising chemical tilized embryos. However, the application methods to isolate one particular nucleic of the method to the study of nucleic acids acid species from, perhaps, tens of thousands has, in its way, produced equally remark• of chemically similar distinct species. The able results. The cloning of cDNAs cor- second was that, even if a chemical separ- Cloning DNA 621

ation method had been found, the amount chimeric DNA produced by the insertion of nucleic acid isolated would in most cases of the DNA to be cloned is a DNA re• have been too small to allow subsequent combinant. The two main types of vector study. The cloning of DNA employs a are bacterial plasmids and bacteriophages biological, rather than a chemical, strategy (section A.8.2). (In theory one could to overcome both these problems. actually integrate the foreign DNA into This biological strategy can be illustrated the bacterial chromosome, but in practice by first considering how one can study the it is generally preferable to use extra• properties of a single mutant bacterium, chromosomal vectors.) present in a population of a million similar 2. The DNA (or a DNA copy of the but non-mutant ('wild-type') bacteria. To do mRNA) must be inserted into the vector this one spreads all the bacteria out on agar in a manner which allows its subsequent plates so thinly that each is separated from removal. In all cases this is achieved by its neighbour. Each bacterium gives rise to a the use of restriction endonucleases. visible colony, which is, in fact, composed of 3. The vector must be introduced into the identical bacteria derived from the division bacterium. In the case of plasmid vectors of the original one. Such an assembly of this process is called transformation, and genetically identical individuals is called a requires the cell wall of the bacteria to be clone. In the case of the clone derived from made permeable to the plasmid. In the the mutant bacterium, one could also say case of bacteriophage lambda this is that one had cloned the mutant DNA. Even achieved through the normal process by though this mutant DNA originally existed which the phage infects the bacteria - as a single chromosome in minute amount, referred to as transfection in this context. one could obtain large quantities of the 4. One must be able to distinguish bacteria DNA from a bulk liquid culture seeded by a containing the vector from those which single colony. The basis of the purification do not. This is relatively simple in the and isolation of an mRNA or a chromo• case of bacteriophage, which usually pro• somal DNA segment using a biological duce visible lysis of the bacteria. In the strategy is to introduce them into individual case of plasmids it is achieved by incor• bacteria so that they may be cloned in an porating a gene for antibiotic resistance analogous way to the hypothetical mutant into the vector. chromosomal DNA considered above. For 5. Bacteria containing recombinant DNAs this reason the process is often referred to must be distinguished from those contain• as molecular cloning. ing non-recombinant vector. This may There are several problems to be over• be achieved by a variety of chemical or come before this strategy can be realized in genetic stratagems (selection), or by more practice. These are enumerated below, and tedious physical analysis. the next section considers different ways in 6. In many cases it is also necessary to be which they are tackled. able to identify which one (if any) of thousands of clones contains the par• 1. The foreign DNA must be presented as ticular DNA of interest (screening). part of a molecule that can replicate The methods used in screening will be along with the bacterial (or other host) considered specifically in relation to chromosome. Such vehicles for introduc• eDNA and genomic cloning (section ing the DNA are called vectors, and the A.8.3). 622 Appendix: methods of studying nucleic acids

The discussion of molecular cloning above endonuclease sites into which foreign DNA has assumed that E. coli will be used as the may be inserted without disabling the biological host for the foreign DNA. This is plasmid, a gene (ApR in Fig. A.19, although because, whatever the source of the DNA, its correct genetic designation is bla) for it is easier to perform initial cloning in resistance to the antibiotic ampicillin per• bacteria. It may subsequently be of interest mitting the selection of bacteria which have to introduce the cloned DNA into other taken up the plasmid, and a fragment types of cells, e.g. those of yeast, higher of the lac operon (the lac operator and animals, or plants. This is discussed in promoter regulatory regions and the N• section A.9.3 in relation to higher animals terminal portion - a-fragment - of the and a specific use of yeast is described in ,8-galactosidase gene) which are included section A.8.3. However, the reader is to allow one to distinguish between re• directed elsewhere for further details of combinants and non-recombinants. cloning in other systems [3, 5, 122, 123). In the simplest case a fragment of foreign DNA generated from a larger molecule by digestion with a restriction endonuclease A.8.2 The major cloning vectors (e.g. EcoR I) is ligated with DNA ligase (section 6.5.1) to pUC18 DNA that has As already stated, the cloning of a piece of been digested with the same enzyme, foreign DNA in E. coli requires its intro• generating the chimeric molecule shown in duction into a suitable vector. Here we Fig. A.19(b). To enable the plasmid DNA outline the way in which the two main types to enter the E. coli cells (transformation) of cloning vector, plasmids and bacterio• these are generally pretreated with calcium phages, are used, considering separately chloride which, together with a short ex• bacteriophage lambda and bacteriophage posure to elevated temperature, appears to M13. act by altering the structure of the cell wall (126]. Transformed cells are selected by (a) Plasmid vectors plating the bacteria on agar containing the antibiotic, ampicillin (a derivative of peni• Plasmids are self-replicating double• cillin), which will prevent the growth of stranded circular DNA molecules found in cells that do not contain any plasmid. It is certain bacteria (section 3.6). Especially possible to use a chemical strategy to select useful are those that occur in multiple copies for recombinants: pretreating the linearized per cell, but natural plasmids had to be plasmid (but not the foreign DNA) with modified extensively before they could serve alkaline phosphatase will prevent re-ligation as efficient cloning vehicles. The most well of plasmid to itself. However, in many known of the plasmid vectors is probably cases some of the transformed bacteria will pBR322 [124), but this has been superseded contain plasmid with no insert. Colonies by more sophisticated derivatives, and it is of such bacteria may be identified because one of these, pUC18 [125), that has been the cloning region of the vector is at the end chosen to illustrate the use of plasmids as of the lacZ' gene, specifying the a-fragment vectors (Fig. A.19). The essential features of ,8-galactosidase. Normally, if the plasmid of pUC18 are an origin of replication that lacZ' gene is induced with IPTG (isopro• allows the plasmid to replicate as the bac• pylthio-,8-galactoside) in an E. coli strain teria divide and multiply, unique restriction with a ,8-galactosidase gene lacking the Cloning DNA 623

---- _:-.------(a) EcoR I ~) + fcoRI

WAn DNA ligase + (ii~

~i) t EcoR I EcoR I EcoR I

Eci:IA I fecfll (b) Foreign DNA

Fig. A.l9 Use of the plasmid vector pUC18. (a) The gene for ampicillin resistance (ApR), the origin of replication (ori), and the lac· fragment containing the E. coli /acZ' (truncated P-galactosidase) gene interrupted by the multiple cloning sequence (cf. Fig. A.l4) ofpUC18 are shown; (b) to clone into e.g. the EcoR I site in the multiple cloning region, the vector DNA is digested with this enzyme (i), as is the foreign DNA (ii), and these are ligated together (iii), among the products being the desired recom• binant, shown, in which the lacZ' gene has been inactivated by insertion of the foreign DNA.

N-terminal portion (actually carried on contain plasmid with inserts, whereas the the F' episome), complementation of the blue colonies generally will not. two peptides will occur giving enzymically Plasmid vectors are small enough that active P-galactosidase (the inclusion of the they are relatively easy to construct with multiple cloning sites does not alter the specific unique restriction endonuclease reading frame or result in inactivation of sites. They were originally much employed the P-galactosidase). The p-galactosidase for eDNA cloning, but more recently this can be detected by the blue colour produced use has declined dramatically. Currently when 'X-gal' (5-bromo-4-chloro-3-indolyl-P• they are used for subcloning larger pieces o-galactoside) is present as a substrate. of cloned DNA, and as the basis of more However, in most cases the insertion of sophisticated vectors, especially those used foreign DNA into the multiple cloning sites for the expression of the genetic information at the extreme N-terminal region of P• in the DNA (section A.9.3). galactosidase will cause inactivation (so• called 'insertional inactivation'). Thus, (b) Bacteriophage M13 if the transformed bacteria are plated on agar containing IPTG and X-gal (as well as Vectors based on bacteriophage M13 have ampicillin) the white colonies will generally already been mentioned in relation to the 624 Appendix: methods of studying nucleic acids

Replaceable region • • • -i · · · -1 38.5 52kb

Packagable size range

Fig. A.20 Potential for bacteriophage lambda to be adapted as a cloning vector. The region marked 'replaceable' contains genes that are not essential for the lytic growth cycle. Some of the restriction endonuclease sites shown for EcoR I, BamH I and Sal I in wild-type bacteriophage lambda must be removed before these can be used as cloning sites in the lambda vectors /cgtlO and /cgtll (Fig. A.22) and EMBL3 (Fig. A.24). For further details see text.

Sanger dideoxy method of sequencing DNA (c) Bacteriophage lambda (section A.5.l(b)), and this is their primary use. Fig. A.14 illustrates the cloning vector, The use of derivatives of bacteriophage M13mp18, a member of the family of lambda as cloning vectors is governed by M13mp vectors, for which the lac fragment somewhat different considerations than the of the pUC vectors was initially engineered use of plasmids. In plasmids there is no [127]. Although this is technically a single• restriction on the size of the DNA that may stranded phage vector, the double-stranded be inserted (although larger inserts often replicative form illustrated is formally transform less efficiently and cause slower analogous to a plasmid, and the method of growth), but in bacteriophage lambda there cloning, transformation, and detection of is a size restriction of 38.5-52 kb for the recombinants is analogous to that described DNA in order for it to be 'packageable' for pUC18, above. The main difference into phage heads. The size of wild-type from cloning in pUC18 is that there is bacteriophage lambda is about 49 kb, which no gene for antibiotic resistance: identi• would not give much scope for its use as a fication of cells that have taken up the vector, were it not that a continuous portion vector is provided by the visible formation of the phage genome of about 16 kb is not of 'plaques' (in fact areas of more slowly required for lytic growth (Fig. A.20). Thus, growing bacteria, as this bacteriophage does lambda vectors can be designed to allow not lyse cells). replacement of this region of DNA by As already mentioned, the clustering of foreign DNA (replacement vectors), or they the restriction endonuclease sites together can be truncated in the dispensable region to in M13mp18 not only allows identification of a size at which they are still packageable, but recombinants by insertional inactivation of which allows the insertion of an appreciable P-galactosidase, but also allows a single amount of foreign DNA (insertion vectors). oligonucleotide to be used to prime copying Examples of these types of vector are of the single-stranded form of the recom• described in sections A.8.3 and A.8.4. binant, irrespective of the nature of the The use of bacteriophage lambda as a insert being sequenced. cloning vector involves the same initial use Cloning DNA 625

of restriction endonucleases as for plasmids. mANA However, to introduce the recombinants s· 3' into the cell they are first 'packaged' into ~) ~ Oigo(dT) phage particles in vitro , using an extract from infected cells containing the necessary S i -- 3· ~Mm·~ +Reverse b'anscriptase enzymes [128], and the bacteria are then ~i) d TPs infected (transfected) with the packaged recombinant. Clones of bacteriophage are

identified as plaques: clear areas of the ~ii) +RNase H bacterial 'lawn' where the infection has s· 3' produced lysis. There is no single common

way of distinguishing recombinant and non• f1V) ~ Klenow ~agment ol DNA pdymerase I recombinant phage, and indeed the most fcd1 1melhytase useful vectors are those in which some method is available to prevent infection ~ Add fcoR I 6nkers D (v) CJ by the non-recombinants. Examples are DNA ligase mentioned later. One feature of using cloning vectors (VIl based on bacteriophage lambda is that +fcoR I packaging in vitro followed by transfection results in a higher efficiency of transform• ation (number of transformants per mole Fig. A.21 Preparation of eDNA for insertion into cloning vectors. Poly(A)+ mRNA is primed of DNA) than does the transformation of with oligo(dT) (i) and a eDNA copy synthesized plasmids by the calcium chloride procedure. (ii). After RNase H treatment to nick the mRNA strand of the hybrid (iii), the Klenow fragment of DNA polymerase I is used to fill the gaps and A.8.3 eDNA cloning replace the RNA by DNA, and the DNA is methylated with EcoR I methylase (iv). EcoR I linkers are then ligated to the ends of the double• For cloning eDNA - DNA copies of mRNA stranded eDNA (v) and cleaved with EcoR I (vi) - it is now usual to employ one of two ready for insertion into the EcoR I site of an insertion vectors based on bacteriophage appropriate vector (Fig. A.22). lambda, A.gt10 or A.gt11, depending on what screening procedure one intends to adopt. We shall first describe how double-stranded ing the second strand relied on the self• DNA for cloning is prepared from poly(A)• priming that occurs when (after removing containing (poly(At) mRNA (Fig. A.21). the mRNA) the single-stranded eDNA folds The poly(A)+ mRNA is usually purified on back on itself. However, the Sl nuclease an affinity column of oligo( dT)-cellulose used to destroy the loop had the disadvan• (section A.2.2) and reverse-transcribed tage that it destroyed the extreme 5' end of using retroviral reverse transcriptase (sec• the eDNA. For this reason the use of Sl tion 6.4.8) primed by oligo( dT) hybridized nuclease has been superseded by methods to the poly(A) tail of the mRNA. This re• that allow production of full-length eDNA sults in a single-stranded eDNA , comp• copies of mRNAs. Although a variety of lementary to the mRNA. For a number of solutions to the problem of priming the years the usual procedure for synthesiz- second strand have been employed [129, 626 Appendix: methods of studying nucleic acids

130], currently a method based on the use of case of A.gtlO) or on there being antibodies E. coli RNase H (section 4.3.4) is most available against the protein (in the case of widespread [131]. RNase H nicks the mRNA A.gtll). in the hybrid, and DNA polymerase (the With A.gtlO (Fig. A.22(a)) the strategy is Klenow fragment or the enzyme from to screen the plaques with a 32P-labelled bacteriophage T4) is used to perform re• oligonucleotide probe corresponding to the placement synthesis of DNA from the nucleotide sequence predicted by a part of 3'-0H groups generated. The ends of such the amino acid sequence. Because of the double-stranded DNA produced must be degeneracy of the genetic code (section modified so that they can be inserted into 12.2.2) such nucleotide sequences cannot be the EcoR I cloning sites in A.gtlO and A.gt11. predicted with certainty. It is necessary to This is achieved by 'blunt-end' ligation of select sequences containing amino acids of 'linkers' to them using DNA ligase (section minimal codon degeneracy, to consider 6.5.1). Such linkers are double-stranded what bias in codon usage might exist in the oligodeoxynucleotides containing a restric• organism from which the mRNA is derived tion endonuclease site which is cleaved after (section 12.2.4), and to employ enough the ligation. This generates 'cohesive ends' different oligonucleotides of sufficient that may be specifically ligated to the cor• length to allow for mistakes. Such probing responding cohesive ends in the digested with oligonucleotides was initially by the vector. (This is more efficient than blunt-end technique of plaque hybridization [133], a ligation directly into a blunt-ended site. variant of the earlier technique of colony Blunt-end ligation is much less efficient per hybridization [134], and is illustrated in se than ligation of fragments with cohesive Fig. A.23. It involves transferring duplicate ends. However, the high molar concen• copies of the DNA in the plaques from a tration of the small linkers can drive this 'master' agar plate to nitrocellulose mem• reaction in a manner impossible with the branes (replica plating) and then immo• larger molecules.) The use of EcoR I linkers bilizing it, as in Southern blotting (section raises the problem of preventing cleavage A.3). It is the lower background of bacterial of any EcoR I sites in the eDNA. This is DNA in plaques, compared with colonies, overcome by prior methylation of the that is one of the reasons that lambda eDNA with EcoR I methylase. vectors are now preferred to plasmid vectors Tlie choice between A.gt10 and A.gt11 [132] for eDNA cloning. Nevertheless plaque for eDNA cloning relates to the problem, hybridization with oligonucleotides can be not so far discussed, of identifying the tedious and result in false 'positives', so that eDNA corresponding to the mRNA of since the advent of the polymerase chain interest among the large number of different reaction it has become usual to screen a clones (the eDNA 'library') that will be eDNA library (or single-stranded uncloned generated (screening the library). In general, eDNA) by PCR using two oligonucleotides, such identification depends on particular clone the amplified DNA, check its se• information about the protein encoded by quence, and use the cloned DNA to probe the mRNA. Although many different the library by plaque hybridization for a screening procedures have been used, the full-length eDNA clone. two vectors under consideration rely on The specific advantage of using A.gt10 for there being either partial amino acid se• eDNA cloning is that it allows discrimination quence information for the protein (in the against non-recombinants. This is by virtue Cloning DNA 627

EcoR I (a) A.gt10 43 kb cl

EcoR I

(b) Agt 11 c:: ======;::='===::;:;======3' - 5' 44 kb lacZ I Digest with EcoR I TUgat e prepared eDNA -

EcoR I £coR I ~ I

~+ mANA

C=N+ Fusion Protein Fig. A.22 A.gtlO and A.gtll eDNA cloning vectors. (a) A.gtlO, showing the EcoR I cloning site in the cl gene. (b) A.gtll , showing the Eco R I cloning site in the lacZ gene (enlarged detail below the vector) used to generate a P-galactosidase fusion protein. For details see text.

~ Denature DNA (alkali) ~

Transfer DNA in .J~ -8-a-ke-to-im-m-ob-il-iz_e_ _.. ~ plaques w nitrocellulosety Hybridize to \ ash, dry, and labelled probe expos~ to X-ray f1lm

Plate with plaques

Autoradiographic image Fig. A.23 Plaque hybridization. Bacteriophage DNA in plaques is immobilized on nitrocellulose and hybridization to a suitable 32P-Iabelled probe (see text) allows identification of bacteriophage carrying inserts of interest. In the diagram the number of plaques has been greatly decreased for clarity. 628 Appendix: methods of studying nucleic acids

of the fact that the EcoR I cloning site is in conceived for the use of A.gtll [135], but this the ci gene, which is required for lysogeny was unreliable and was abandoned in favour (section 10.1.6). By transfecting the pack• of the more direct approach [132]. Although aged phage into E. coli bearing the high there are plasmid vectors with a similar frequency lysogeny mutant HflA150, one cloning site which are useful for producing ensures that only those phage in which the fusion protein against cDNAs already ci gene is inactivated - the recombinants - cloned, ).,gtll is preferred for screening form plaques. with antibodies as there is less potentially ).gtll (Fig. A.22(b)) is a vector especially interfering protein in the plaques than in designed to allow the eDNA to be expressed bacterial colonies. as protein for immunological screening. The Confirmation that the clone one has problem with expression of eukaryotic isolated is the one desired can be either by cDNAs in E. coli is that they lack the DNA sequencing, if the sequence of the requisite bacterial promoter and Shine and protein is known, or by 'indirect expression'. Dalgarno sequence (section 12.4.1). The By indirect expression is meant the ex• solution to this problem is to put the eu• pression of the mRNA after selection by karyotic DNA under the influence of bac• DNA-RNA hybridization. The cloned terial regulatory signals. In fact the reader DNA is immobilized on a nitrocellulose may have realized that if an incomplete filter, and an mRNA preparation (consisting eukaryotic eDNA clone (i.e. one lacking of a mixture of species) from an appropriate sequences corresponding to the 5' -untrans• tissue or cell is passed through the filter lated portion of the mRNA) is cloned into [136]. Only the mRNA complementary to the pUC or M13 vectors (section A.8.2), the particular immobilized DNA clone will there is a one~in-six chance of it being form a hybrid and be retained on the filter. inserted in the right orientation and with It can then be released (washed off at low maintenance of the reading frame, and of ionic strength) and translated in a cell-free this leading to the synthesis of a fusion system (section A.ll) [137]. This is known protein comprising the N-terminal amino as hybrid-selection or hybrid-release trans• acids of p-galactosidase and a part of the lation. Alternatively, the denatured cloned protein corresponding to the eDNA clone. DNA can be mixed with the mRNA pre• Such an expressed fusion protein would paration, the mixture added to the cell-free most likely have antigenic determinants system and the loss of a particular product that would be recognized by a polyclonal detected (hybrid-arrest translation [138]). In antibody to the eukaryotic protein. In fact, both cases the product may be identified pUC or M13mp vectors are not very suitable by its electrophoretic mobility, immuno• for production of fusion proteins because logically (if this was not the original basis the eukaryotic polypeptide is for some of selection) or, in particular cases, by its reason often unstable in E. coli, and suffers biological activity [139]). proteolysis. It has been found that if the fusion protein contains a much longer N-terminal portion of bacterial protein it is A.8.4 Genomic cloning generally much more stable, and for this reason in A.gtll the foreign DNA is cloned Other vectors than those described above near the C-terminus of the P-galactosidase are used when the objective is to prepare a gene. Originally a lysogenic strategy was eukaryotic genomic library; i.e. a collection Cloning DNA 629 - :I:- ii- ~i~ Ill&!~

EMBL3 4-1 4-1 43kb -4-Replaceab~ :I:- - -e - - ~- ~&1! i:I: &1!~ y ]~ ~'! ... i lJ ~ y - - ::e:I: "1&1! - - i~ y ~ ~~ I -'! I :I:- :I:- ,... •,...,.DNA digested with BamH I (etc.) !f f -:I: :I:- ~i i~ ~ ~ 38.5-52 kb Recombinant of lambda and genomic DNA Fig. A.24 Bacteriophage lampda vector EMBL3 for cloning genomic DNA. The figure shows the cloning of a BamH I fragment of genomic DNA between the BamH I sites of the vector, with digestion by EcoR I being used to prevent religation of the central fragment of the vector. If genomic DNA digested with Sau3A I rather than BamH I is used (see text) the recombinant will generally lack the BamH I sites indicated. However, the insert can be recovered using Sal I. Only the desired product of ligation is illustrated. of clones in which the whole DNA of the ment vectors, that can accept 15-20kb genome of a particular eukaryotic organism of DNA that are preferred for genomic or cell is represented. The main reason for cloning, and a representative vector, EMBL this is that the large size of eukaryotic 3, will be considered to illustrate their use. genomes requires vectors with the largest EMBL 3 [141], is shown in Fig. A.24. capacity for foreign DNA. For example, to This has been engineered so that restriction be 99% certain that a library of 15 kb inserts endonuclease digestion with BamH I, EcoR is representative of the 3 x 106 kb mouse I or Sal I cuts out a non-essential piece of genome, 106 clones are required. Plasmid DNA of 14kb, which can then be replaced vectors have the capacity for large inserts, by foreign DNA (up to about 23kb) with but the efficiency of transformation of compatible cohesive ends. To prevent calcium-treated cells by the resulting larger religation of the lambda 'centre' when using DNA is low. Lambda insertion vectors can replacement vectors, the lambda 'arms' be transfected efficiently into cells, but, can either be first purified, or use made of although sometimes used in genomic cloning the fact that bacteria that are lysogens for (e.g. Charon 16A [140]), do not have bacteriophage P2 do not support growth of optimal capacity. It is the lambda replace- phages which contain the red and gamma 630 Appendix: methods of studying nucleic acids

genes which reside in the fragment replaced Possible cloning sites in the recombinant. It may be mentioned that the compatible cohesive ends generated in the fragments of genomic DNA need not be (and generally are not) produced using the same restriction endonuclease as used in cleaving the vector. Thus, cleavage of DNA with the enzyme Sau3A I (which has the 4-base recognition sequence 'GATC) pro• pHC79 duces identical cohesive ends to cleavage with BamH I (which has the 6-base re• lambda cognition sequence G'GATCC). If high• molecular-weight chromosomal DNA is subjected to partial digestion with Sau3A I one is more likely to obtain clonable cos fragments of any particular area of the Fig. A.25 Cosmid vector pHC79. This is a genome than by complete (or partial) simple cosmid vector containing an ampicillin digestion with BamH I, for which there resistance gene, origin of replication and unique cloning sites derived from the plasmid pBR322 are fewer recognition sites. (the precursor of pUC18 - Fig. A.l9), together Because of the large size of the introns in with a portion of circularized bacteriophage many eukaryotic genes, a 20 kb insert in a lambda DNA containing the cos site recognized particular lambda genomic clone may not by the lambda packaging system. The cleaved contain the whole of a given gene of interest. vector is ligated to large restriction fragments of genomic DNA that allow the formation of Vectors have therefore been developed linear assemblies containing an insert flanked by two which, although more difficult to handle, molecules of linearized cosmid to provide the will accept even larger inserts. One such two cos sites required for packaging in vitro. class of vector is the cosmid, a simple representative of which, pHC79 [142], is be considered as being formerly analogous shown in Fig. A.25. Cosmids are based on to cosmids, although they take advantage of bacteriophage lambda but retain only the a natural plasmid stage in the life cycle cohesive ends (cos) of the bacteriophage, of bacteriophage Pl. two of which are sufficient to allow pack• To encompass a length of genome greater aging in vitro. This may allow insertion of up than the capacity of the vector containing to 45 kb of foreign DNA, but the packaged the genomic clones it is necessary to per• recombinant DNA is no longer infective form the operation known as chromosome after it penetrates the host cell. For this walking, in which a fragment of unique• reason the cosmid vector has an origin of sequence DNA near the extremity of the replication and antibiotic resistance gene clone is used as a probe to re-screen the that allow it to behave like a plasmid, and genomic library for an overlapping clone selection of transformed colonies can be containing the adjoining area of the genome. made on antibiotic plates. More recently With the start of efforts to determine the vectors based on bacteriophage P1 have sequence of complete genomes it has been developed that can accept up 100 kb of become necessary to construct overlapping DNA [143]. These vectors, the genetics of sets of clones covering such distances that which are too complex to discuss here, can even the vectors just mentioned are in- Analysis and manipulation of cloned DNA 631

BamH I I BamH I ' EccA I TEL TRP ARS1 CEN4 URA3 TEL

..,.,...__..,.. _ __.____....___..----~.I II II I_ _...,'I DNAChromosoma tigated herel IIJ-lL----...1.---L--~ I I ... Ecdl I EccAI

Fig. A.26 Yeast artificial chromosome vector p Y AC4. The plasmid form, maintained in E. coli, is digested with BamH I and EcoR I generating two vector arms, each with a yeast telomere (TEL) at one end. Large fragments of chromosomal DNA produced by partial digestion with EcoR I can be ligated between these, producing artificial chromosomes which can be maintained in yeast. ARSl and CEN4 are a yeast replication origin and centromere, respectively, and TRP, SUP4 and URA3 yeast selectable markers. For further details see text. adequate. A major breakthrough in this pYAC4 is in the SUP4 gene, encoding a respect has been the development of yeast suppressor tRNA (section 12.9.6) which artificial chromosomes (Y ACs) [144, 145], is inactivated by the insertion of foreign which, using a eukaryotic microorganism DNA. Recombinants can be detected on as host, also allow the cloning of certain the basis of the colour of the colonies when eukaryotic sequences that are unstable in a host with an ade2 ochre mutation is used. a bacterial environment. The essential Because the Y ACs exist in the same en• features for the replication of a yeast vironment as natural chromosomes, the chromosome are the centromere, the recombinants could, in principle, be as large telomeres and an origin of replication; and as the latter. In practice, the cloning of these ( CEN4, TEL, and ARSJ, respectively) inserts of 1000 kb (1Mb) into these vectors are the essential constituents of p Y AC4 has been achieved. (Fig. A.26). Markers must be included to select for transformants after introduction of the DNA (by permeablization with A.9 ANALYSIS AND MANIPULATION calcium chloride and ethylene glycol) into OF CLONED DNA yeast spheroplasts (yeast lacking their cell wall). In pYAC4 these are provided by the There are many different manipulations that TRPJ and URA3 genes, which allow growth an investigator might wish to perform on a on medium lacking tryptophan and uracil in cloned nucleic acid. This section is restricted a yeast mutant in which these genes are to describing the principles underlying the non-functional. The EcoR I cloning site of most common of these. 632 Appendix: methods of studying nucleic acids

TATAA ATG

(i) I I

Bg/11 Bam HI

Cleave I s' end-label t G (ii) A T *-=====- .. G• Isolate antisense T strand - yA Hybridiz.e to m RNA .. (iii) 5, .. •• ---

51 '"'""" j ( iv) -* (a) - (b) Fig. A.27 Example of nuclease Sl mapping to determine a transcriptional start point. (a) A cloned fragment of genomic DNA hybridizing to a particular mRNA has been sequenced and the likely initiation codon and TATAA box identified (i). A restriction fragment that is likely to contain the transcriptional start point is isolated from this region and labelled at the S' -end (ii), the anti-sense strand is isolated and hybridized to mRNA (iii), and the single-stranded regions of the DNA (and mRNA) not involved in the hybrid is digested with Sl nuclease leaving the hybrid (iv); (b) the size of the protected DNA in (iv) is determined on a polyacrylamide sequencing gel. In this case Maxam and Gilbert sequencing reactions have been performed on the digested and undigested portion of (iii) and have been subjected to parallel electrophoresis to allow exact definition of the transcriptional start point in the sequence (the G indicated by the arrow). The position of the TATAA box is also indicated on the sequencing gel. (Data relating to the rnRNA for the small subunit of herpes simplex virus 1 ribonucleotide reductase, kindly provided by Dr Barklie Clements.)

A.9.1 Mapping of RNA transcripts mRNA encoded by the cloned DNA and the complementary single strand of the latter. One general problem related to cloned This is possible by judicious choice of genomic DNA encoding a protein is to hybridization temperature, because of the determine precisely which parts of the DNA greater stability of the DNA:RNA hybrids are represented in the final processed RNA compared with the original DNA:DNA transcript. The main technique for deter• hybrid. The endonuclease S1 (section 4.2.1) mining the positions of intron splice sites is used to digest the non-hybridized, single• and the points at which transcription of stranded regions of the DNA, leaving the mRNA starts and finishes is nuclease Sl regions of DNA complementary to the mapping [146). The principle of this method mRNA intact. The size of the undigested is to form a DNA:RNA hybrid between the DNA can be determined by electrophoresis Analysis and manipulation of cloned DNA 633

I> .q. I. 1 A ·--• "----• •----- ·---- ·----- ·---- ·----- :~ ·--- ·---- u ••~ '] ·-- ·-- •'·~J ·-· cGel ,] ·- t ] : I 1 I ... G DNase t I ... - : J • ------•

( ll ( 11) n ( a ) 1 c~

( b )

Fig. A.28 Example of DNase footprinting. (a) Partial digestion of a piece of end-labelled DNA to whi~h a protein (R) is bound (ii) results in the absence of the end-labelled fragments having a cleavage point in this region, although these are found in the digest (i) of the unprotected DNA; (b) the results of separation of the products of such an experiment on a polyacrylamide sequencing gel. The example is of the lac operator DNA and the lac repressor protein (R). I is IPTG (0.3 M) , which does not prevent the binding of a mutant repressor used in this study. C,T, and A,G represent the results of a Maxam• Gilbert C + T and A+ G reactions on the undigested end-labelled fragment. Adapted from [149], with permission. on alkaline agarose gels (to hydrolyse the nuclease Sl. Exonuclease VII will digest RNA), and different fragments are ident• single-stranded ends protruding from a ified by hybridizing to appropriate 32P• mRNA/DNA hybrid but, unlike the endo• labelled probes. For a precise determination nuclease S 1, cannot digest a single-stranded of the transcription boundaries an appro• intron looped out from such a hybrid. priate 32P-end-labelled restriction fragment For determination of the 5' -ends of of the cloned DNA is used in the hybridiza• mRNAs, nuclease S1 mapping is usually tion, and this can subsequently be subjected used in combination with primer extension to electrophoresis on a polyacrylamide [148]. This latter technique involves a small sequencing gel alongside suitable standards restriction fragment or oligonucleotide of the restriction fragment in question known to lie near to, but 3' of, the 5' -end of (Fig. A.27). the mRNA; and this is used to prime copying In determining the position of introns of the mRNA with reverse transcriptase. If it can be helpful to employ exonuclease the primer is 32P-labelled at its 5' -end or VII [147] (section 4.5.2) in addition to the the DNA labelled during synthesis, the 634 Appendix: methods of studying nucleic acids size of the product can be determined by as a hole or gap on the sequencing gel (Fig. polyacrylamide gel electrophoresis. Primer A.28). Unprotected DNA subjected to extension can also be used in eDNA cloning sequencing reactions is analysed by electro• to 'extend' eDNA clones already isolated phoresis on the same gel to allow the easy which do not reflect the full extent of the identification of the region of the DNA mRNA to its 5'-end. protected. It is possible to use alternative cleavage reagents to DNase I which allow one to A.9.2 Identification of regions of DNA obtain more precise information about the that interact with proteins residues that interact with the protein. For example, chemical methylation and The regulation of gene expression is achieved 5-bromodeoxyuridine substitution with sub• through the interaction of proteins with sequent cleavage by ultraviolet light have DNA; hence when a particular piece of been used to address the role of specific genomic DNA has been cloned it is often purines and thymine residues, respectively of interest to identify the regions of DNA [150). involved in such interactions. One general The development of sequencing in vivo method of studying such interactions is by (section A.5) allowed the footprinting tech• DNA footprinting [149]. This involves form• nique to be extended to the study of inter• ing a complex between the protein and a actions between uncloned DNA and protein fragment of the double-stranded DNA, in vivo [106, 151 ]. One approach has been to radioactively end-labelled (section A.4.2) irradiate nuclei with ultraviolet light, pro• at one end only, as for Maxam-Gilbert ducing pyrimidine dimers (section 7.2.5). sequencing (section A.5.1(a)). It is then The resulting saturation of the 5,6-double subjected to digestion with bovine DNase I bond allows ring opening through reduction (section 4.5.1) and the region of DNA in with sodium borohydride [152). Alter• contact with the protein is protected by the natively, it is possible to apply the use of latter from digestion. If complete digestion dimethyl sulphate for the methylation of occurred the protected fragment would need purines to intact systems such as E. coli cells to be isolated and analysed. However, if the [153] and mammalian nuclei [154). digestion is performed under conditions that The objective of the footprinting tech• on average result in a single endonucleolytic nique is to identify the regions of DNA cut per molecule (cf. the use of similar con• involved in interaction with proteins. In ditions for chemical cleavage) an easier a few cases this has been followed by X• analysis is possible. This involves subjecting ray crystallography of complexes formed the DNA to electrophoresis in the type of between purified proteins and chemically denaturing polyacrylamide gel used for synthesized oligonucleotides corresponding sequencing. If no protein is present during to their binding sites (e.g. cover illustration the DNase digestion a ladder of end-labelled [155]). However, it is often the case that the oligonucleotides encompassing all possible identity of the protein components of the sizes is produced on autoradiography. How• individual interactions are unknown, and ever, when the DNA-protein complex is in this situation the technique of band• subjected to DNase treatment no scission retardation [156] (also known as band-shift, occurs in the protected region and no end• gel-retardation and mobility-shift) may be labelled oligonucleotides terminating in undertaken to aid the identification and this region will be generated. This appears purification of such proteins. The basis of Analysis and manipulation of cloned DNA 635

(i) (ii) (iii) (iv) this technique is the lower mobility during gel electrophoresis of a protein-DNA com• plex compared with the DNA alone. If one a-TIF complex optimizes conditions to avoid dissociation of the complex, one is able to see a diminution in the intensity of the free DNA and the OCT-1 complex appearance of a slower-moving species (Fig. A.29). The technique can be applied to quite small DNA fragments and synthetic oligo• nucleotides, and so can also be used as a complement to footprinting in studying regions of DNA that bind proteins.

A.9.3 Expression

After having cloned a eDNA or gene en• coding a particular protein one may well wish to use the clone to express the protein. However, there are quite different ob• jectives one might have in seeking to do this, and the precise objective will determine the appropriate method to adopt. In this section we shall consider three possible objectives and corresponding expression systems. The objective may be to obtain large amounts of protein, in which case one is most likely to attempt to over-express a cloned eDNA from a bacterial or yeast plasmid or from a eukaryotic viral vector; one may merely wish to detect some pro• duct, or be interested primarily in the Unbound non-coding parts of a eukaryotic genomic oligonucleotide

Fig. A.29 Band retardation assay of complexes infected with herpes simplex virus. It can be seen of protein transcription factors with regulatory that a complex of similar mobility to that formed DNA sequences. The figure shows an auto• with purified OCT-1 (i) is formed with nuclear radiograph obtained from a non-denaturing extract from both uninfected (iii) and infected polyacrylamide gel to which had been applied a (iv) cells, and that an additional complex (with a 32P-Iabelled oligonucleotide (a 29-mer containing virus transcriptional regulatory protein known as the promoter of the herpes simplex virus 1 a-TIF) is formed with nuclear extract from immediate-early gene a-0) incubated with various infected cells only. Lanes (i) and (ii) are from a protein fractions: (i) pure OCT-1 transcription separate experiment to that of lanes (iii) and (iv), factor (section 10.5), (ii) pure OCT-1 transcrip• which have been exposed for a longer time to tion factor preincubated with an anti-serum allow visualization of the a-TIF complex. The against it, (iii) nuclear extracts from uninfected data for this figure were kindly provided by Drs HeLa cells, (iv) nuclear extracts from HeLa cells Frances Purves and Bernard Roizman. 636 Appendix: methods of studying nucleic acids

RBS Nco ! AGGA CCATGG [ • AGGAAACAGACCATGG ...1 - ATG '-

Fig. A.30 Bacterial plasmid expression vector pKK-233-2. An exploded view is shown of the Nco I cloning site containing an ATG initiation codon 3' to a Shine and Dalgarno ribosome binding site (RBS). Transcription is driven by the 'tac' promoter (Ptac) and terminated by the 5S rRNA transcrip• tional terminator (T). The vector also contains the ampicillin resistance gene (ApR) and origin of replication (ori) of pUC18 (Fig. A.l9). For further details see text.

clone that regulate its transcription, m clones (there is no way to surmount the which case transient expression may be splicing problem), and typically contain a most appropriate; or one may wish to study translational initiation codon at or near a the expression of a eukaryotic gene in a cloning site preceded at the optimal distance cellular context, in which case it is necessary by a strong bacterial promoter and a strong to attempt to integrate it into the host Shine and Dalgarno ribosome-binding site. genome. In some vectors the position of cloning will result in a fusion protein containing a non• (a) Over-expression from vectors authentic N-terminus, which must either be tolerated or removed by a specific protease. The expression of a bacterial gene from However, other vectors such as pKK 233- 2 a bacterial vector is reasonably straight• (157] (Fig. A.30) are adapted for the precise forward, but the expression of the protein positioning within the context of the pro• encoded by a eukaryotic eDNA or gene karyotic signals of cDNAs in which the from a bacterial vector presents more initiation codon is contained in a Nco I site, problems. These relate to the fact that CCATGG. This may at first sight seem bacteria do not contain the splicing system of limited applicability, but in fact this necessary to process correctly genes that sequence conforms well to the Kozak con• contain introns, and that the signals for the sensus for initiation of eukaryotic translation start of transcription and translation are (section 12.5.1) and many eukaryotic in• quite different in eukaryotes and pro• itiation codons either lie within such Nco I karyotes (chapters 9 and 12). Because of sites or can easily be made to do so by site• the ease with which bacteria can be handled directed mutagenesis (section A.9.4). The and the large scale on which they can be vector pKK 233-2 contains quite a strong grown, special bacterial vectors have been E. coli promoter, but it has been found designed for expression of eukaryotic that even greater expression can be obtained proteins. These are designed for eDNA using vectors such as the pET series [158], in Analysis and manipulation of cloned DNA 637

which the DNA is cloned into a site con• bination with the foreign DNA cloned into a trolled by a promoter for bacteriophage 17 suitable plasmid; and there must be some and is then expressed in a strain of bacteria means of selecting for recombinants. In the which can synthesize the highly specific T7 baculovirus vector it is necessary to isolate RNA polymerase. non-viable cells (as indicated by staining) Although some eukaryotic proteins can that do not contain polyhedrin protein be expressed from such vectors (which are inclusion bodies. also useful for over-expression of those The baculovirus system is technically bacterial proteins which are normally only rather demanding, and other virus vectors weakly expressed), in many cases little have been developed that are easier to protein is obtained. The major reason for handle. The greatest promise seems to be this seems to be that many eukaryotic offered by sophisticated vectors [ 162] based proteins are unstable in a bacterial en• on vaccinia virus, a lytic virus that infects a vironment, as already mentioned in relation variety of mammalian cells. One vector to the design of Jcgtll (section A.8.3). based on this virus, Vac/Opm, contains Indeed, if the objective in expressing a the gene for bacteriophage 17 RNA poly• protein is to raise antibodies, it is better to merase, the expression of which is inducible generate fusion proteins using Jcgt11 or (constitutive expression is detrimental to conceptually similar, and perhaps more this virus) and under the indirect control of easily handled, plasmid vectors such as the strong vaccinia late promoter Pll. (It is those of the pUEX series [159]. However, if the lac/ gene that is under the direct control the objective is to obtain authentic func• of the vaccinia promoter, and the 17 poly• tional protein, eukaryotic vectors may be merase gene is under the control of the the only alternative. One possibility is to lac operator, and hence can be induced by use yeast vectors (e.g. [160]), but formam• IPTG.) The foreign gene of interest is malian proteins yeast still may not be a cloned into a site where it is under the suitable environment. It is for this reason control of a strong bacteriophage T7 pro• that certain viruses of higher eukaryotes moter, and hence can be expressed at high have been employed as vectors for over• levels. Indeed, much more mRNA is made expression of eukaryotic proteins. One than can be 'capped', and for this reason virus that has been much exploited is the the cloning site of the vector is preceded baculovirus, Autographa califarnica mono• by the 5' -leader sequence of encephalo• nuclear polyhedrosis virus, in insect cells myocarditis (EMC) virus, which, like polio, (Spodoptera frigiperda; Sf9) [161]. This allows cap-independent translation (sections virus has an extremely abundant late gene• 12.5.1 and 12.9.5). The eDNA to be ex• product, polyhedrin, that can constitute up pressed is initially cloned into a plasmid, to 50% of the total cellular protein. Because optimally into an Nco I site (as for pKK this protein is not essential for production of 233-2, above), and this is flanked by se• infectious extracellular virus, foreign genes quences for the vaccinia thymidine kinase can be cloned in front of the polyhedrin gene (tk), to allow recombination and promoter in place of the polyhedrin gene selection for tk- virus after introducing the and over-expressed. In this, and other DNA into cells using the calcium phosphate vectors based on large eukaryotic viruses, it technique (A.9.3(b)). The selection is done is necessary to introduce the foreign DNA by using tk- cells (the tk gene is not essential into the virus vector by genetic recom- for either cells or virus in culture) and 638 Appendix: methods of studying nucleic acids growing on bromodeoxyuridine (BrUdr), unable to form virus particles, and they which can only be incorporated into DNA are referred to as viral mini-rep/icons. Nor by tk+ virus in which thymidine kinase can they be maintained permanently in cells converts BrUdr to BrdUMP. Ultraviolet in the way in which bacterial plasmids can, light will fragment the DNA of those viruses as the massive replication makes the cells in which thymine has been replaced by unviable. bromouracil (cf. section A.9.2), killing them For vectors based on SV40 to retain the and hence allowing selection of recombinant ability of the viral DNA to replicate they tk- virus. require the presence of an origin of replica• tion and the large T antigen, although the (b) Transient expression in eukaryotic cells latter is usually provided by the use of a transformed COS cell line [167]. The vectors For some experimental purposes it is suf• also contain bacterial plasmid sequences for ficient to obtain low level expression of a cloning and propagation, and hence may be eukaryotic gene. One way of obtaining referred to as shuttle vectors (they 'shuttle' transient transcription of a cloned gene is microinjection into the nucleus of frog oocytes [163]. It is more usual to inject mRNA, rather than DNA, and this can be generated by transcription in vitro of DNA cloned into a vector that puts it under the control of a promoter for bacteriophage 17 [164]. Less specialized methods of intro• ducing DNA into cells are also available, however. Most widespread is the technique of calcium phosphate co-precipitation of DNA onto tissue culture cells [165], which take up the precipitate, apparently by a process of phagocytosis. An alternative is electroporation, in which the cell membrane is made permeable by a brief pulse of Fig. A.3l The CAT vector pSVO-cat [169]. The prokaryotic portion of the vector contains the high-voltage electricity [166]. ampicillin resistance gene (ApR) and origin of The amount of DNA introduced into cells replication (ori) of pUC18 (Fig. A.19) for by calcium phosphate co-precipitation or propagation in E. coli. The eukaryotic portion electroporation is normally too small to derives predominantly from the early region of produce detectable amounts of protein SV40 into which the E. coli chloramphenicol transacetylase gene (cat - strictly speaking emf) It is for this reason the DNA is per se. has been inserted in an orientation that allows usually cloned into a vector containing a it to use the polyadenylation/processing site eukaryotic origin of replication, so that indicated (polyA). The SV40 promoters have after 24 or 48 h sufficient DNA is generated been removed in this vector (and also the SV40 to give detectable quantities of transcripts origin of replication) so that insertion of a III site is and their products. The most frequently eukaryotic promoter into the Hind needed to produce transcription of the cat gene used of such vectors are based on the small after transfection into eukaryotic cells. Similar circular DNA virus, SV40; although it is vectors with a greater nUI{lber of possible cloning important to stress that such vectors are sites are also available [170]. Analysis and manipulation of cloned DNA 639

between prokaryotic and eukaryotic hosts). advantages in cloning the DNA into a vector A variety of such SV 40 vectors is available containing both the selectable marker and for different purposes. If one merely wishes sequences that will enhance the expression to demonstrate that a DNA sequence of the integrated gene. Markers that allow encodes a particular protein, the vector can selection in eukaryotic cells include gpt, the facilitate or optimize expression of this by gene for E. coli xanthine-guanine phos• providing promoter, enhancer and poly• phoribosyltransferase (which confers adenylation signal (e.g. [168]). Alternatively resistance to mycophenolic acid), and neo, one may wish to study the regulatory regions the gene for bacterial aminoglycoside associated with a cloned gene, and require a phosphotransferase (which confers re• vector Jacking promoter or enhancer or sistance to aminoglycoside antibiotics such both. In such a case it is advantageous for as neomycin, kanamycin and geneticin). the regulatory sequences to be able to drive Sequences included to enhance expression the transcription of a sequence coding for a include the SV 40 enhancer and retrovirus protein that can be detected and quantified LTRs. The vectors (e.g. [171]) generally more easily than the authentic product of include the origin of replication and am• the gene, and it is for this purpose that the picillin resistance gene of pUC18 for cloning CAT vectors were developed. These allow and propagation in E. coli. In some cases the 5' -non-coding and untranslated regions the expression is under the control of a of a gene to be linked to the coding region promoter inducible by glucocorticoids or for the bacterial enzyme chloramphenicol heavy metals (e.g. [172]). The SV40 origin acetyltransferase (CAT). CAT assays, as of replication may also be present. in these they are called, are extremely sensitive and vectors, which are often also used for are performed using [14C]chloramphenicol transient expression; but it should be as a substrate, and separating the acetylated stressed that this origin has no role when chloramphenicol products from unreacted the vector is used for stable integration. substrate by thin-layer chromatography This latter point is underlined by the fact [169]. An example of a CAT vector is that with these vectors it is generally pre• presented in Fig. A.31. ferable to linearize the DNA before trans• fection. This is not the case, however, for (c) Expression after integration into a vectors based on defective retroviruses, cellular eukaryotic genome which use the natural integration of retro• viral DNA into the chromosome of the host A small proportion of eukaryotic cells cell to achieve cleaner and more efficient transfected with DNA actually become integration (and perhaps better expression) 'transformed': the DNA is integrated into [173). the chromosome. If such transformed cells A limitation of systems in which cloned are selected from the vast majority of un• genes are stably integrated into tissue transformed cells, cell lines can be obtained culture cells arises when the major interest in which to study the expression of stably is in the tissue-specific expression of a integrated exogenous genes. It is not strictly particular gene. A technique in which this necessary to incorporate the selectable problem is overcome involves the micro• marker into the same piece of DNA as the injection of cloned DNA into fertilized gene to be expressed, and co-transfection is mouse eggs to produce so-called transgenic often performed. However, there are mice [174]. The foreign DNA is integrated 640 Appendix: methods of studying nucleic acids

at such an early stage that it is generally present in all daughter cells, and certainly in the progeny of those mice that pass it on through the germ line to the next generation. It is thus possible, using suitable E1 constructs, to study whether a particular (a) tissue-specific gene is expressed in a tissue• specific manner in its new location, and to Linearize with enzyme E1 investigate the regions of DNA which confer ! tissue-specificity of expression [175).

(b) A.9.4 Mutagenesis in vitro

In studies of cloned genes of the type de• scribed in sections A.9.2 and A.9.3, con• clusions regarding the importance of re• gulatory regions of DNA can be tested (c) and extended if it is possible to produce mutations in the regions of interest. Such Add hnkec• !Red,cula,lze mutations are difficult to produce in higher eukaryotes in vivo, but powerful techniques are available to produce them in cloned DNA in vitro. The technique of mutagenesis in vitro, especially when directed to specific nucleotides (see below) can also be applied (d) to the coding regions of genes, the products E2 of which it is possible to express, purify and Fig. A.32 Use of exonuclease Bal 31 to gen• subject to structural or functional studies erate bidirectional deletions. A hypothetical [176]. Some of the methods used to generate plasmid (a) is illustrated in a region of which such mutations are described below. (solid shading) it is desired to obtain deletions. A Deletion mutagenesis may be performed neighbouring restriction endonuclease site, El, using the enzyme Bal 31 nuclease (section is used to linearize the plasmid (b) and this is digested for different times generating a 4.2.1 ). This enzyme has exonuclease activity spectrum of deleted molecules (c). These are against double-stranded DNA. Hence, if recircularized using linkers for a restriction a plasmid containing the cloned gene is endonuclease, E2, giving plasmids such as (d). linearized by restnctJon endonuclease digestion at a point in the region of interest, Bal 31 nuclease will digest it in a way that employed to fill in any overhangs) are ligated produces a deletion emanating from this to linkers (section A.8.3), cleaved, and point in both directions. The size of the ligated together with DNA ligase (Fig. deletion is determined by the time allowed A.32) [177). One problem with such deletion for digestion, after which the blunt ends mutants when used to assess the importance (treatment with the Klenow fragment of of deleted sequences is that they alter the E. coli DNA polymerase and dNTPs is relative proximity of the remaining regions Analysis and manipulation of cloned DNA 641

of DNA. This disadvantage is overcome in ss M 13mp recombinant the technique of linker scanning mutagenesis in which small regions of DNA may be replaced by a different sequence of the same length [178]. Descriptions of this method 0 and a more convenient alternative strategy for achieving the same end [179] are, Hyb,;d;ze to ~ -+- however, beyond the scope of this book. It is most desirable to be able to produce mutations at specific sites (site-directed mutagenesis) and this can be achieved using synthetic oligonucleotides. The classic Q manner for doing this [180] involves sub• DNA _I_ DNA cloning the DNA into a bacteriophage M13 polymerase ' ligase vector (Fig. A.14) that allows the generation of a single-stranded circular recombinant DNA. An oligonucleotide (e.g. 10-12 mer) is synthesized corresponding to the target of mutation and surrounding nucleotides, except that it contains the desired base• 0 change. This is annealed to the single• Transform E coli stranded circular DNA,. which is then converted into the replicative double• stranded form in vitro using the Klenow fragment of DNA polymerase I and DNA ligase. When E. coli are transfected with this DNA, phage containing both wild-type and mutant single-strands will result and give rise to 'plaques'. These can be dis• tinguished by blotting onto nitrocellulose and hybridization to the 32P-Iabelled oligo• Plaque nucleotide originally used, under conditions hybridize with that enable a perfect match (the desired mutant) to be distinguished from a single mismatch (the wild type). This is shown Identify mutants diagrammatically in Fig. A.33. More soph• Fig. A.33 Site-directed mutagenesis. The base isticated strategies (e.g. [181]) that optimize to be altered is shown as an open circle and the yield and selection of mutants are now the mismatch (mutation) in the chemically available. A modification of the procedure synthesized oligonucleotide as a solid circle. just described [182] may be used to generate For further details see text. short, specific deletions of a type hardly possible with Bat 31 nuclease. In this case single-stranded circular DNA if the area to the oligonucleotide synthesized is a hybrid be deleted loops out, and the desired mutant of the sequences directly flanking the area second strand can then be synthesized to be deleted. This can hybridize to the in vitro. More recently the polymerase 642 Appendix: methods of studying nucleic acids

Fully orotecte~ 110nooer

D11 TOC~2 R tal l ), '------5' 11 3 // 0 N-'1 I j P' Cli30" n + ~~

2nocycle

(0) lei

5'HO RJR

3' 1 Fig. A.34 The phosphite triester method of oligonucleotide synthesis. (a) The protected phos• phoramidite monomer is activated in the 3'-phosphate position by a suitable weak acid (e.g. tetrazole) allowing condensation (b) with 5' -activated monomer attached via its 3' -0 to a solid support. The phosphite is then oxidized to a phosphate (c). Removal of the 5'-protecting DMT group of the dinucleotide with, for example, dichloroacetic acid (d) prepares this for the next cycle of condensation (e). R == benzoyl adenine, benzoyl cytosine, isobutyryl guanine or thymine. chain reaction has been employed for the synthesis of oligonucleotides is now avail• production of a variety of mutations by able commercially, as are machines for 'in strategies that do not involve cloning into house' synthesis. The chemistry involved Ml3 [183]. derives from that developed by Khorana, the application of which culminated in the synthesis of a tRNA gene [184]. The method A.10 CHEMICAL SYNTHESIS OF at present in greatest use , the phosphite OLIGONUCLEOTIDES triester method (Fig. A.34), will be outlined here. Further details can be found elsewhere The importance of chemically synthesized [185]. oligonucleotides for PCR (section A.7), in Synthesis of oligonucleotides involves labelling DNA (section A.4.1 ), as primers the formation of successive diester bonds for sequencing in M13 (section A.5.1(b )), between the 5' -OH group of one nucleotide for producing mutagenesis in vitro (section derivative and 3' -OH of a second nucleotide A.9.4) and in studying specific interactions derivative. In order to simplify the synthetic between nucleic acids and proteins (section procedure the first nucleotide is directly A.9.2) has already been mentioned. Custom linked to a solid support (e.g. silica gel) Cell-free systems 643

packed in a column. It is necessary to oligonucleotides containing more than activate one of the components for the 150 bases. reaction, and this requires that other po• tentially reactive groups elsewhere in the molecules be 'protected' by reversible A.11 CELL-FREE SYSTEMS FOR chemical modification. The reactive com• TRANSCRIPTION AND ponent is the free monomer in which a TRANSLATION [8] 3'-0P04 has been substituted by dialkyl phosphoamidite (one of a variety of the The transcription and translation of cloned possible dialkyl substitutions of the N is genes in intact cells has already been dis• illustrated), and, after activation, this reacts cussed in section A.9.3. Here we consider with the free 5'-0H group of the bound methods for studying transcription and nucleotide or oligonucleotide to give a translation in cell-free systems, with par• phosphite triester. This must be oxidized ticular reference to eukaryotes. (e.g. with aqueous iodine) to a stable phos• Systems for performing transcription of photriester before the next synthetic step. eukaryotic genes in vitro are somewhat The 3'-0H of the immobilized reactant is limited. It is possible to use extracts of HeLa protected by virtue of its attachment to cell nuclei to study the transcription of genes the solid support, the 5'-0H of the free by pol III [186], but for genes requiring pol monomer is protected by a dimethoxytrityl II the method is more difficult to apply group, a methyl group is usually employed [187]. In the latter case it is more common to protect the hydroxyl on the 3'-0P04, and to study the elongation of already initiated the individual bases are also protected. transcripts in isolated nuclei, using the After reaction excess reagents are then so-called nuclear run-off assay [188] (also washed off the column, any unreacted referred to as nuclear 'run-on'). 5'-0H groups are blocked or 'capped' As regards translation, the intact-cell using acetic anhydride, and the 5'-0- system of microinjection into frog oocytes dimethoxytrityl protecting group is removed (or eggs) has the advantage that protein by 80% acetic acid to allow the extended synthesis may proceed for several days bound oligomer to react with another [189]. Cell-free systems, however, are activated monomer in the next round of of greater utility to the non-specialized condensation. At the end of the synthesis laboratory, even though these are not as the oligonucleotide must be chemically simple to establish in eukaryotes as in E. coli released from the column and deprotected. (see below), and activity is easily lost during Removal of the oligonucleotide chain from cell fractionation. Necessary components the solid support depends, of course, on the of the system may be lost with nuclei and nature of the linkage. Ester linkage via mitochondria or adhere to intercellular succinyl groups is quite common, in which structures. Furthermore, the disruption of case cleavage occurs at the same time as the cytoskeleton may be a factor contri• deprotection of the bases. buting to loss of activity. It is therefore This phosphite triester method, which is perhaps not coincidental that the two most used by most automatic synthesizers ('gene widely used systems for cell-free translation machines'), is relatively rapid (condensation are derived from rather specialized cells. By is completed in about 2 min), and has a high far the most popular system is that derived enough efficiency to allow the synthesis of from rabbit reticulocytes using hypotonic 644 Appendix: methods of studying nucleic acids

Table A.3 Some inhibitors of DNA synthesis

Category Examples Reference

1. Reagents interacting with DNA* (a) Alkylating agents Dimethyl sulphate MitomycinC Nitrogen and sulphur mustards section 7.2.2 MNNGandNMS (b) Intercalating agents Acridine dyes section 7.2.3 Actinomycins section 7.2.3 Adriamycin [198] Anthracenes section 7.2.2 Benzpyrene Ethidium bromide section 7.2.3 Propidium diiodide (c) Intertwining agents Distamycin [199] Netropsin [199]

2. Analogues of bases, Acyclovir section 3.7.7(c) nucleosides etc. t Adenine P-1-o-arabinoside section 7.2.1 Amethopterin sections 5.2 and 5.7 Aminopterin sections 5.2 and 5.7 2-aminopurine section 7.2.1 Aphidicolin section 6.4.7 8-azaguanine section 7.2.1 Azaserine section 5.2 6-azauracil section 7.2.1 2' azido-2' -deoxynucleosides [201] 5-bromodeoxycytidine [197] Cytosine P-1-o-arabinoside section 7.2.1 Diazooxynorleucine section 5.2 Dideoxynucleosides section 6.4. 7 andA.5.1(b) 5-fluorodeoxycytidine [197] 5-fluorodeoxyuridine section 5.2 5-fluorouracil section 7.2.1 Hydroxyurea section 5.2 6-mercaptopurine section 7.2.1

3. Inhibitors of topoisomerases Coumermycin section 6.5.4 Nalidixic acid Novobiocin Oxolinic acid

4. Inhibitors of cell division Colee mid [202] Colchicine Vinblastine Vincristine

• These may be mutagens and/or agents that interfere with replication or transcription. t These may interfere with nucleoside biosynthesis or, following incorporation into DNA, block further chain elongation or transcription. Cell-free systems 645

Table A.4 Some inhibitors of RNA synthesis

Inhibitor Remarks Reference ------Actinomycin D Inhibits both prokaryotic and section 9.2.3 eukaryotic transcription by complexing with deoxyguanosine residues. Hence transcription of rRNA is more sensitive than that of mRNA a-Amanitin and Inhibits eukaryotic RNA polymerase section 9.3 other fungal amatoxins II specifically (also RNA polymerase III at very high concentration) Cordycepin Inhibits poly( A) polymerase section 11.5 (3' -deoxyadenosine) responsible for 3'-polyadenylation of eukaryotic mRNAs Dichlororibofuranosyl Inhibits appearance of eukaryotic [203] benzimidazole mRNA in the cytoplasm, although only partially inhibiting synthesis of hnRNA Rifampicin and streptovaricin Bind toP subunit of E. coli section 9.2.3 RNA polymerase, inhibiting initiation Streptolydigin Bind top subunit of E. coli section 9 .2.1 RNA polymerase, inhibiting elongation

lysis followed by low-speed centrifugation Prokaryotic cell-free systems for trans• to remove the cell membranes. This rabbit lation were developed very early on; the reticulocyte lysate has been further modified ribosomes, tRNAs and soluble factors for to allow more efficient utilization of ex• translation of exogenous mRNA being ogenous mRNA. The endogenous globin present in the supernatant after centri• mRNA is degraded by the Ca2+ -dependent fugation of disrupted cells at 30 000 g for micrococcal nuclease that can subsequently c. 30 min. This system was originally devel• be inactivated by chelation with EGTA oped by Nirenberg and Matthaei in their [137]. Such nuclease-treated lysates are work on the genetic code and was sub• commercially available. The other cell-free sequently developed for use in studying the system still in general use is that derived translation of the stable mRNAs of the RNA from the post-microsomal supernatant of coliphages [192]. The low endogenous wheat germ [190], which has an intrinsically mRNA activity of such systems is a reflec• low content of endogenous mRNA. One tion of the very short half-lives of bacterial limitation of the reticulocyte lysate and mRNAs, which make it difficult to isolate wheat-germ systems (but not of the frog bacterial mRNAs and study their translation oocyte) is the lack of endoplasmic reticulum directly. To overcome this problem a cell• for correct processing of translation products free system for translation was devised that contain signal peptides (section 12.8.1 ). in which the mRNA could be continually This may be overcome by the addition of a generated by simultaneous transcription microsomal membrane fraction, e.g. from of the gene. This coupled transcription• dog pancreas [191]. translation system [193] was originally 646 Appendix: methods of studying nucleic acids

Table A.5 Some inhibitors of protein synthesis

Prokaryotic/ Inhibitor eukaryotic Remarks Reference

Abrin E as for ricin (below) (211] Aurin tricarboxylic P,E Inhibits initiation by preventing (195, 196] acid binding of mRNA to ribosome Chloramphenicol p Inhibits elongation at peptidyl section 12.6.2 transferase. Resistant mitochondria have altered large rRNA Colicin E3 p Specific nuclease for site on (197, 208) 16SrRNA Cycloheximide E Inhibits elongation and initiation, (195] 'freezing' ribosomes on polysomes. Resistant yeast have altered 60S subunits Diphtheria toxin E Inhibits elongation by section 12.5.2 inactivating EF-2 by ADP ribosylation Edeine A P,E Inhibits initiation at either mRNA- (195, 196] or initiator tRNA-binding step Emetine E Inhibits elongation at translocation (195, 207] step. Resistant hamster cells have altered protein S14 Erythromycin p Inhibits elongation at section 12.6.2 transpeptidation step Ethionine P,E Causes synthesis of abnormal (210) proteins with ethionine replacing methionine Fluoride (P?), E Inhibits initiation in intact cells (205) 5-fluorotryptophan P,E Prevents activation of tRNA Trp [210) by competitive inhibition of synthetase Fusidic acid P,E Inhibits elongation, preventing (195, 196) release of EF-G. GDP complex from ribosome. Resistant bacteria have altered EF-G. Guanylyl methylene P,E Non-hydrolysable analogues section 12.5.1 diphosphonate and ofGTP and 12.4.2 Guanylyl imidodiphosphate Kanamycin P,E Inhibits elongation and causes [195, 196] misreading Kasugamycin p Inhibits initiation, section 12.6.2 m~Am~A sequence of 16S rRNA involved Kirromycin p Inhibits elongation, preventing section 12.6.2 release of EF-Tu. GDP complex from ribosome 0-methyl threonine P,E Causes synthesis of abnormal [210) proteins with 0-methyl threonine replacing threonine Cell-free systems 647

Table A.S (continued)

Prokaryotic! Inhibitor eukaryotic Remarks Reference

Modeccin E as for ricin (below) [211] Neomycin P,E Causes misreading and inhibits section A.9.3(c) initiation and elongation. Used as a selectable DNA marker in eukaryotic cells Norvaline P,E Prevents activation of tRNA Trp [210] Pactamycin P,E Inhibits initiation, was used to [204] determine gene order of proteins derived from picornaviral polyproteins Paromomycin p Inhibits initiation. Resistant section 12.6.2 mitochondria have altered small rRNA Puromycin P,E Inhibits elongation by binding section 12.4 to A-site and reacting with peptidyl-tRNA Ricin E Inhibits elongation by specific [211] N-glycosidase action on native 28SrRNA a-sarcin (P),E Specific nuclease for 28S rRNA [209] in ribosomes (non-specific with naked rRNA). Inactive against intact E. coli, but specifically cleaves 23S rRNA in ribosomes. Shiga toxin E As for ricin (above) [211] Showdomycin P,E Inhibits initiation at the stage of [195] ternary complex formation Sparsomycin P,E Inhibits elongation at peptidyl [195, 196] transferase step Spectinomycin p Inhibits elongation at [195, 196] transpeptidation. Resistant ribosomes have altered protein S5 Streptomycin p Binds to 30S subunit to cause section 12.6.3 misreading and (at higher concentrations) inhibition of initiation Tetracycline p Inhibits elongation by blocking [206] binding of aminoacyl-tRNA to the A-site on the 30S ribosomal subunit Thiostrepton p Inhibits elongation, preventing section 12.6.2 binding of EF-G . GTP complex to ribosome Trimethoprim p Prevents formation offMet-tRNA [210] by inhibition of synthesis of 10 N -formyl-H4-folate 648 Appendix: methods of studying nucleic acids

Table A.6 Some E. coli genes relevant to nucleic acid metabolism

Gene symbol Explanation Reference ada Repair enzyme for removal of section 7 .3.1 methyl groups alaS Alanyl-tRNA synthetase section 12.3.2 alaT-W Alanyl-tRNAs section 12.3.1 apaH ApppppA hydrolase section 12.10.3 argS Arginyl-tRNA synthetase section 12.3.2 argT-X Arginyl-tRNAs section 12.3.1 asnS Asparaginyl-tRNA synthetase section 12.3.2 asnT-V Asparaginyl-tRNAs section 12.3.1 aspS Aspartyl-tRNA synthetase section 12.3.2 aspT-V Aspartyl-tRNAs section 12.3.1 altA Integration site for bacteriophage section 7.5.1 lambda (similarly att¢80 etc.) cca tRNA nucleotidyl transferase section 11.7 .1 em/A Chloramphenicol acetyltransferase section A.9.3(b) cysS Cysteinyl-tRNA synthetase section 12.3.2 cysT Cysteinyl-tRNA section 12.3.1 dam DNA adenine methylase section 4.6.1 dcm DNA cytosine methylase section 4.6.1 dnaA (8, C, etc.) Enzymes of DNA biosynthesis Tables 6.3 and 6.5 dut (dnaS) dUTPase section 6.3.2 endA DNA endonuclease I Table4.3 ffh Component of signal recognition particle section 12.8.1 ffs 4.5SRNA section 12.8.1 ftsY Receptor of signal recognition particle section 12.8.1 fusA Protein synthesis factor EF-G section 12.4.2 ginS Glutaminyl-tRNA synthetase section 12.3.2 glnU-V Glutaminyl-tRNAs section 12.3.1 gltT-W Glutamyl-tRNAs section 12.3.1 gltX Glutamyl-tRNA synthetase section 12.3.2 glyS Glycyl-tRNA synthetase section 12.3.2 glyT-W Glycyl-tRNAs section 12.3.1 gpp Guanosine pentaphosphatase section 11.12 groEL, ES Chaperone proteins section 12.8 gyrA, B (cou, natA) DNA gyrase (two subunits) section 6.5.4 hfl High frequency of lysogenization by section A.8.2 bacteriophage lambda hisR Histidinyl-tRNA section 12.3.1 hisS Histidinyl-tRNA synthetase section 12.3.2 hsdM, R, S Host restriction/modification system section 4.5.3 (methylase, endonuclease and specificity components of EcoB and EcoK in Band K strains, respectively) ileS Isoleucyl-tRNA synthetase section 12.3.2 ileT-V, X Isoleucyl-tRNAs section 12.3.1 infA, B, C Protein synthesis factors IF-1, section 12.4.1 IF-2 and IF-3 lacA, /, Y, Z Components of the lac operon section 10.1.1 lepB Signal peptidase I section 12.8.1 leuS Leucyl-tRNA synthetase section 12.3.2 leuT -X, Z Leucyl-tRNAs section 12.3 .1 Cell-free systems 649

Table A.6 (continued)

Gene symbol Explanation Reference

lig (dnaL) DNA ligase Table6.5 /spA Signal Peptidase II (specific for section 12.8.1 pro lipoprotein) lysS Lysyl-tRNA synthetase section 12.3.2 lysT, V Lysyl-tRNAs section 12.3.1 lysU Lysyl-tRNA synthetase (inducible) section 12.3.2 metG Methionyl-tRNA synthetase section 12.3.2 metT, Y, Z Methionyl-tRNAs section 12.3.1 mutD (dnaQ) DNA polymerase III (e) Table6.5 mutH, L, S Uncharacterized proteins involved in section 7.3.3 DNA repair nrdA, B (dnaF) ribonuclease reductase Table6.5 (two subunits) nusA, B Proteins involved in termination of section 9.2.4 transcription nusC, D, E = rpoB, rho and rpsJ, respectively section 9.2 oriC Origin of DNA replication section 6.10.1 pheS, T Phenylalanyl-tRNA synthetase section 12.3.2 (two subunits) pheU, V Phenylalanyl-tRNAs section 12.3.1 phr Deoxyribodipyrimidine photolyase section 7 .3.1 pnp Polynucleotide phosphorylase section 4.4 po/A, B, C DNA polymerases I, II and III (dna£) Tables 6.2 and 6.5 prfA, B Protein synthesis factors RF-1 and RF-2 section 12.4.3 pr/A, C, D, F, G Proteins affecting protein export section 12.8.1 prmA, B Ribosomal protein methylases section 12.6 proS Prolyl-tRNA synthetase section 12.3.2 proK-M Prolyl-tRNAs section 12.3.1 purA-N Enzymes of purine biosynthesis section 5.2 pyrA-1 Enzymes of pyrimidine biosynthesis section 5.4 recA Protein involved in recombination section 7 .4.1 and repair recB, C, D DNA exonuclease V (three subunits) Table 4.4 and section 7.4.2 recE DNA exonuclease VIII section 4.5.2 reeF, J, N, 0, Q Proteins involved in DNA recombination section 7.4 and repair re/A Stringent factor section 12.10 rep DNA helicase section 6.5.3 rho Transcriptional termination factor rho section 9.2.4 rimB-L Ribosomal protein modification enzymes section 12.6.1 (methylases, acetylases, etc.) rna-rnh Ribonucleases I-III, D, E and H Table 4.1 rnpA-B Ribonuclease P (protein and nucleic section 11.3.4 acid components) rp/A- Y, rpmA -J 50S subunit ribosomal proteins Ll-L36 section 12.6.1 rpoA-D, H, N RNA Eolymerase subunits a, p, P', section 8.1.1 r/0 , a· and a61, rpsA-U 30S subunit ribosomal proteins S1-S21 section 12.6.1 rrfA-E, G, H 5S rRNA genes of operons rrnA-H section 8.4.4 rr/A-E, G, H 23S rRNA genes of operons rrnA-H section 8.4.3 650 Appendix: methods of studying nucleic acids

Table A.6 (continued)

Gene symbol Explanation Reference rrnA-E, G, H rRNA operons section 8.4.3 rrsA-E, G, H 16S rRNA genes of operons rrnA-H section 8.4.3 sbcB DNA exonuclease I Table 4.4 secA, B, D-F, Y Proteins affecting protein export section 12.8.1 (secA, E, Y = pr/D, G, A, respectively) serS Seryl-tRNA synthetase section 12.3.2 serT-X Seryl-tRNAs section 12.3.1 spoT Guanosine tetraphosphatase section 12.10 ssb DNA-binding protein Table 6.5 tdk Thymidine kinase section 5.8 thrS Threonyl-tRNA synthetase section 12.3.2 thrT-W Threonyl-tRNAs section 12.3.1 thy A Thymidylate synthetase section 5.6 top A Topoisomerase I Table6.5 tral Helicase I (plasmid encoded) section 6.5.3 trmA-F tRNA ·methylases section 11.7 .2 trpS Tryptophanyl-tRNA synthetase section 12.3.2 trpT Tryptophanyl-tRNA section 12.3.2 tsf Protein synthesis factor EF-Ts section 12.4.2 tufA, B Protein synthesis factor EF-Tu section 12.4.2 (two genes) tyrS Tyrosyl-tRNA synthetase section 12.3.2 tyrT,U Tyrosyl-tRNAs section 12.3.1 umuC,D Proteins involved in error-prone DNA section 7.3.4 repair ung Uracil-DNA-glycolase section 6.3.2 uvrA-C AP endonuclease (three components) Table 4.3 and section 7.3.2 uvrD Helicase II section 6.5.3 va/S Valyl-tRNA synthetase section 12.3.2 va/T-W Valyl-tRNAs section 12.3.1 xthA DNA exonuclease III section 4.5.2 xseA, B DNA exonuclease VII (two subunits) Table4.4

applied to genes cloned by more traditional the convenience of the reader the most genetic techniques using specialized trans• common of these are summarized in Tables ducing phages. A.3 to A.5. Many inhibitors of transcription and translation are specific for eukaryotic or prokaryotic systems, and this specificity A.12 THE USE OF INHIBITORS IN is indicated in Tables A.4 and A.5. Most THE STUDY OF GENE EXPRESSION inhibitors of DNA synthesis (Table A.3) do not discriminate between eukaryotes and In the course of this book reference has prokaryotes; however, the dideoxynu• been made to the use of antibiotics and other cleosides, on conversion into the triphos• inhibitors of macromolecular synthesis. For phates, do not inhibit eukaryotic DNA References 651

polymerase a. In contrast, a fairly specific symbols the reader is advised to consult inhibition of eukaryotic DNA polymerase the E. coli linkage map [212]. Finally, it is a is obtained with aphidicolin. Further worth mentioning that sequences have been general [194-197] and specific [198-211] determined corresponding to many of details may be found elsewhere. these genetic loci [213].

A.13 E. COLI GENES RELEVANT TO REFERENCES NUCLEIC ACID METABOLISM 1 Cantoni, G. L. and Davies, D. R. (1971) Much of the study of nucleic acid meta• Procedures in Nucleic Acid Research, vol. 2, Harper and Row, New York. bolism has been conducted in E. coli, and 2 Birnie, G. D. and Rickwood, D. (1978) many of the components involved were Centrifugal Separations in Molecular and identified by mutations in their genes. Cell Biology, Butterworths, London. Because of this, and in view of the wide• 3 Old, R. W. and Primrose, S. B. (1989) spread use of specialized E. coli strains Principles of Gene Manipulation (4th edn), Blackwell, Oxford. among non-geneticists undertaking re• 4 Walker, J. M. and Gaastra, W. (1983) combinant DNA work, we felt it might be Techniques in Molecular Biology, Croom useful to provide a summary of the names Helm, London. given to some selected genetic loci of E. coli 5 Kingsman, S. M. and Kingsman, A. J. (Table A.6). (1988) Genetic Engineering, Blackwell, Oxford. A few words of caution are necessary 6 Habel, K. and Salzman, N. P. (1969) regarding Table A.6. Because genetic loci Fundamental Techniques in Virology, were often defined before the corresponding Academic Press, New York. gene-product was known, there exist alter• 7 Sambrook, J., Fritsch, E. F. and Maniatis, natives to some of the names listed. In some T. (1989) Molecular Cloning, a Laboratory Manual (2nd edn), Cold Spring Harbor cases names have merely been rationalized Laboratory, New York. to conform to a pattern such as dna, rpo, 8 Hames, B. D. and Higgins, S. J. (1984) rps; and older nomenclature based on Transcription and Translation: a Practical antibiotic resistance, for example, may still Approach, IRL Press, Oxford. be found, especially in descriptions of geno• 9 Berger, S. L. and Kimmel, A. R. (1987) types. Thus, bacteria with a totally inactive Guide to Molecular Cloning Techniques, Academic Press, New York. gene for ribosomal protein S12 may be 10 Wu, R., Grossman, L. and Moldave, K. designated rpsL-, whereas bacteria with (1989) Recombinant DNA Methodology, the mutation in the rpsL gene that conveys Academic Press, New York. dependence on the antibiotic streptomycin 11 Schneider, W. C. (1945)1. Bioi. Chern., 161, (section 12.6.3) are generally designated 293. 12 Schmidt, G. and Thannhauser, S. J. (1945) 0 Sm , the gene being originally called J. Bioi. Chern., 161, 83. strA. Also worth mentioning are the sup 13 Fiske, C. and Subbarow, Y. (1929) J. Bioi. genotypes, which define mutations in Chern., 81, 629. tRNA genes (e.g. supF is in tyrT) causing 14 Berenblum, I. and Chain, E. (1958) a particular termination codon to be read Biochem. J., 82, 286. 15 Burton, K. (1956) Biochem. J., 62, 315. as an amino acid codon (suppression - 16 Ceriotti, G. (1955) J. Bioi. Chern., 214, 59. section 12.9.6). 17 Warburg, 0. and Christian, W. (1942) For fuller details of these and other gene Biochem. Z., 310, 384. 652 Appendix: methods of studying nucleic acids

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208 Boon, T. (1972) Proc. Nat/. Acad. Sci. USA, 212 Bachmann, B. J. (1990) Microbial. Rev., 69, 549. 54, 130. 209 Wool, I. G. (1984) Trends Biochem. Sci., 213 Rudd, K. E., Miller, W., Ostell, J. and 9, 14. Benson, D. A. (1990) Nucleic Acids Res., 210 Nierhaus, K. H. and Wittmann, H. G. 18, 313. (1980) Naturwiss., 67, 234. 214 Erlich, H. A., Gelfand, D. and Sninsky, J. 211 Endo, Y., Mitsui, K., Motizuki, M. and (1991) Science, 252, 1643. Tsurugi, K. (1987) J. Bioi. Chem., 262, 5908. Index

Abrin646 structure, 74, 81 ribonucleotide (AI CAR) 136 Accuracy, translational534, 539, transcription, 359 ll-Aminolevulinate, synthesis 584 558-9 Adenylosuccinic acid 136, 137 Aminopeptidase 519 Acetylaminofluorene (AAF) 262 ADP ribosylation 547,561 Aminopterin 136, 144 Acetylcytosine 491, 522 Adriamycin 644 2-Aminopurine 259 Acetylpyridine thiosemicarbazone Affinity chromatography 384, 453 Ampicillin resistance 622-3, 639 143 Aflatoxin 261 Amplification of genes 48, 227-9 Acridine dye 261 Agarose gel electrophoresis see Analogues, of bases and nucleosides Actin Electrophoresis of nucleic 259-60,644 genes,291,321,322 acids Androgen 420 pseudogenes, 307 AIDS virus see Human Aneuploid cell43 Actinomycin 261-2,347 immunodeficiency virus Angiogenin 104 Activation domain Alarmone 582-3, 584 Anisomycin 561 acidic,431-2 Alkaline Antennapedia see Mutants; Gene glutamine-rich, 432 gradient electrophoresis see Anthracene 261 proline-rich, 432 Electrophoresis, alkaline Anti conformation 15-17 Activator element 301-2 gradient Anti-association factor 542, 543, 547 Activator protein 177, 212, 406 hydrolysis of RNA see RNA, Anti-repression loop 380 Active chromatin 432 alkaline hydrolysis Antibiotic resistance 296, 555, 558, DNase sensitivity, 432-3 phosphatase see Phosphatase, 621-2,630,651 matrix association, 437-8 alkaline Antibiotics 542,547,557,558,561, methylation, 432, 433-4 Alkylating agents 260, 644 562,639,650-1 nucleosome phasing, 432, 435-6 Allantoin 147 see also Individual entries proteins, 434-5 Allolactose 383 Antibody see Immunoglobulin supercoiling, 432 Allopurinol148 Anticodon 475,515,518,519-24, Z-DNA,438 Alu family 50, 52, 295, 305-6 525,526,528-30,531-4,536, Acyclovir 84 a-Amanitin 211, 351, 645 537,539,543,549,558,560, Adenine deaminase 146 Amatoxins 351 563,580-1 Adenine, structure 5, 6 Amber mutant and codon 79, 518, see also Transfer RNA Adenoassociated virus (AAV) 81, 233 540,570,580 Antimutator mutation 170 Adenosine deaminase 146,260 Amethopterin (Methotrexate) 136, Antioncogene 87 Adenosine, structure 8 144,228 Antisense RNA 210,217,399,438-9 S-Adenosyl-methionine Aminoacyl-tRNA synthetase 530, Antitermination 350, 399 capping, 480 531-4,547,579,581,583-4 complex, 401 co-repressor, 389, 394 proofreading, 534 N-protein, 400-1 DNA modification, 121, 122 reaction, 531 Q-protein, 400-2 S-Adenosyl-homocysteine, and structure, 532-4 Antiviral therapy 84 capping480 3-(3-Aminocarboxypropyl)-1-methyl AP endonuclease 265,266 Adenovirus pseudouridine 491 Ap4A see Diadenosine mRNA translation, 578 5-Aminoimidazole ribonucleotide tetraphosphate oncogenic potential, 427 (AIR) 135-7 Aphidicolin 176, 651 replication, 218-19 5-Aminoimidazole-4-carboxamide AraBAD operon see Operon 658 Index

Arabinoside 260 terminase, 233 chemical reactions, 32-36 AraCfP 176 vectors, 624-30 composition Archae bacteria 560-1 Xis protein, 279 DNA, 12,13 initiator tRNA, 561 ~S2,371,452,569-70 RNA,7 introns, 320, 475 mRNA, 84, 371, 569 exchange in RNA and DNA, 126 ribosomes, 561 replicase, 371 insertion, 496-8 ribosomal proteins, 553, 561 translation, 524, 569 methylation see Nucleotide ribosomal RNA, 561 ~13 methylation ARS see Autonomous replicating as vector, 607,613-15,623-4, modification, 5, 7, 82,344,457, sequence 641 491-2, 495-6 Artificial chromosome see Yeast replication, 196-8,202-5 nucleotides artificial chromosome ~u,86,297,298-9 see also RNA modified Aspartate carbamoyltransferase 139 N4, 369 molar proportions, 12, 13, 595 ATPase, translational543, 566-8, PI, 86,209,630 pairing, 14, 15,519-22,527-30 569 X174, 83, 197,317 stacking, 15-17, 521 Att sites 277-9 polynucleotide kinase, 124-5 sugar linkage, 15-17,235,268 Attenuation 385,396-9,527 ()p,371,452,569,579,580 tautomerism, 5, 259 AU-rich sequence 574 R17 transposition, 495-6 Aurin tricarboxylic acid 646 amber mutant, 540 BD-cellulose 602 Autogenic control389, 391, 571-3 mRNA,452 Bentonite, and RNase inhibition 108 Autonomous replicating sequence replicase, 371 Benzpyrene 261 (ARS) 224-5,631 retronphage R73, 305 Berk-Sharp technique see Nuclease Sl Autosome 503 RNA polymerase, 369, 607 mapping Auxotroph 68 SP01,403,405,468 Biotin-11-dUTP 606 Avian infectious bronchitis virus SP6, 369,607 Biotyi-A~P 389 (IBV) 581 structure, 73-5 Bisulphite 33 Avian sacrcoma virus 426 temperate, 86 Bithorax see ~utants Azacytidine 123-259 T even (T2, T4) Bleomycin 261 8-Azaguanine 259 assembly, 77 Branch migration 272 Azaserine 136, 140 base composition of DNA, 81, 82 5-Bromodeoxycytidine 644 Azathioprine 138 DNA polymerase, 216,609 5-Bromodeoxyuridine 259, 634, 638 6-Azaruacil 259 gene 32 protein, 183, 184, 597 BSE (Bovine spongiform Azidothymidine 84 introns, 468 encephalopathy) 88 polynucleotide phosphorylase, Budding yeast see Yeast; Budding Bl family 52,306 124-5 Buoyant density 24-6, 598-9 Bacillus subtilis RNA polymerase, modification, Bypass replication 269-270 phage infection, 402-5 406-7 sigma factors, 402-5 structure, 74 cA~P see Cyclic A~P sporulation, 402-5 transcription, 406-7 cJ gene see Bacteriophage lambda Bacteriophage T3, 607 C value paradox 47 f2 T4, 406-7 CAAT box 353-4, 408 mRNA, 452, 570 gene 60 mRNA, 582 Caesium chloride, ultracentrifugation replicase, 371 T5,81 325,596 fd, 197-8 n,369,453 Cairns structure 202, 203 G4, 23, 197, 198 initiation of replication, 213-15 Calcitonin information content, 83-5 DNA polymerase, 613 gene, 501 lambda RNA polymerase, 213,369,607, gene-related peptide, 421, 501 anti-terminators, 399-402 637 Calcium-binding protein 418 att sites, 277-9 terminal repetition, 80 Calcium phosphate co-precipitation c/gene,86,391,628 termination of replication, 231-3 637,638 ci mRNA, 535 virulent, 86 Calladine 's rules 19 cohesive ends, 80, 232, 618, 630 see also Viruses; Individual entries Calmodulin Cro gene, 86, 391-3 Baculovirus 637 genes regulated by, 421 Cro protein, 86, 392-3,400 Bal31 nuclease 99,640 pseudogene, 307 Gamma gene, 629 Baltimore classification 75 Camptothecin 189 initiation of replication, 208, 209 Bam HI family see L1 family Cancer see Oncogene integration, 277-9 Bam island 363 CAP see Catabolite activator protein lysogeny, 86,391 Bam 5 family see L1 family 5'-Cap lytic cycle, 391, 399-402 Band-retardation 354, 634 function, 479, 543 packaging in vitro, 78, 625 Band-shift see Band-retardation mitochondrial absence, 332 repressor, 391-3 Base myxovirus use as primer, 373 Red genes, 629 analogues, 259, 644 mRNA, 478-81, 543, 579, 637 Index 659

SL RNA, 465, 480 Chiasma270 puff, 65, 418 snRNA, 456, 480 Chloramphenicol542, 547, 557, 561 scaffold, 42, 61 viral, 370, 373 Chloramphenicol acetyltransferase segmentation, 45 Capsomere 73 (CAT) 639 size measurement, 44, 597-8,601 Carbamoylphosphate 139 Chloroplast terminal repetition, 80 5-Carboxymethoxy uracil522-3 4.5S rRNA, 550 translocation, 289,290,291,295 5-Carboxymethylaminomethyl uracil DNA,67,563 walking, 620, 630 522-3 genes,67,331-5,368-9 X inactivation, 123 Carcinogenesis 260 genetic code, 525, 563 Circular dichroism 21 Carcinogen 257-263,426 promoters, 368-9 Circular permutation 81215 Cascade protein biosynthesis, 563, 584 Cis-acting element 339 developmental see Gene, cascade protein targetting, 568 Class switching immunoglobulin 288- (developmental) ribosomal proteins, 333, 563 9 lytic see Lytic cycle of RNA polymerase, 333 Clay derivatives as RNase inhibitors bacteriophage rRNA genes, 333-4 108 Cassette model281 transcription, 368-9 Clonal selection 289 CAT (Chloramphenicol acetyl tRNA genes, 563 Cloning, DNA 620-31 transferase) assay 639 Christmas tree effect 65 Codon515-6 Catabolism of purines and Chromatid 43 amino acid, 517,519 pyrimidines 145-149 Chromatin context, 580 Catabolite activator protein (CAP) chemical cleavage, 59, 60 initiation, 518,519,534-7,543,546 385-7,394,437 cross-linking, 58 termination, 518, 524, 540-2, 547 complex with cAMP, 386 domains, 415 usage, 526-7 Catabolite repression 385-7 DNase sensitivity, 55-58,432-3 Cohesive ends 80, 208, 232, 618, 626, Catalytic RNA see RNA, catalytic hypomethylation, 432-4 629,630 Catenation of DNA 186, 187 matrix-association, 61, 437-8 Col El 73, 216, 217, 439 Cauliflower mosaic virus 180, 222 nucleosome phasing, 60, 432, 435- Colcemid 644 CCA terminus of tRNA 495, 528-9, 6 Colchicine 644 538,558,585 proteins, 53, 434-5 Colicin 73,646 see also Pre-tRNA; Transfer RNA reconstitution, 60 Colony hybridization 626 cdc2 protein kinase 43, 211 replication, 236-238 Complementary DNA (eDNA,) 180, eDNA see Complementary DNA solenoid, 61 306,318,607,620,623,625-8, Cell structure, 53-64 634,635-7 diploid,43 supercoiling, 26, 61-63 Complementation haploid,43 torsional stress, 27 assay in vitro, 195, 196 hybrid, 45 transcriptional control, role in, 407 assay in vivo, 79 synchronized, 43, 143 viral, 83 Computer analysis 291, 611 Cell cycle Z-DNA,438 Concatamer 187,232 animal,42 Chromatosome 58 Conditional lethal mutant 79, 193 bacterial, 69 Chromosome Conjugal transfer 72, 205 Cell lysate (for DNA replication) 194 acrocentric, 44 Conjugation 72,217 Cell-free system artificial see Yeast artificial Consensus sequence transcription, 352,356,360,387, chromosome CAP-cAMP-binding, 386 643 bacterial, 68 definition, 339 translation banding, 44, 65 enhancer,418,419,421,426 E. coli,524,534,645 cohesive ends, 80 eukaryotic transcriptional reticulocyte lysate, 645 condensation, 42, 44 termination, 363-6 wheat germ, 645 double minute, 228 eukaryotic translational initiation, Cellophane disc assay 194 eukaryotic, 43 545,636 Centimorgan 45 gene allocation, 45-47 histone gene, 484-5 Central dogma 2, 496 gene locations, 324,325,329,409- intron splice site, 458, 462-5 Centriole 43 10,414 mitochondrial promoter, 368 Centromere 44, 45 isolation, 46 polyadenylation, 481 CO-deficiency 268 jumping,47 pre-mRNA 3' -processing, 481-5 Chaperonin 566-8, 583 lampbrush, 64 prokaryotic promoter, 342-3,369, Chargaffs rule 13 maps, 45, 47,317 402,407 Chelate effect 387 metacentric, 44 RNA polymerase II promoter, Chemical reactions metaphase, 43 353-4 bases, 32-4 organization, 43 transcription factor-binding, 418, ribose and deoxyribose, 32 Philadelphia, 289 419,421 Chi (x) site 274 polytene, 65, 418, 483 transcriptional termination, Chi (x) structure 272 pseudoautosomal, 290 vaccinia, 370 660 Index

Constitutive enzyme 382 Deoxyadenosine inhibition of see also Anti-association factor Copia 302-3, 304, 307 ribonucleotide reductase 143 Distamycin 644 Copolymerase In 174, 185 Deoxyribonuclease (DNase) DNA Cordycepin 609, 645 AP endonucleases, 112,265-260 A,BandCforms, 17,18 Corepressor 385-9 DNasel (pancreatic), 109 base composition, 12, 13 Coronavirus 581 footprinting, 634 bending, 18, 20, 433, 436-7 COS cell line 211, 638 hypersensitivity, 432-3 binding Cosmid see Vector nick translation, 606 motifs, 17,427-31 cos site 233, 630 sensitivity of chromatin, 58 of proteins, 17, 19,392-6,424, Cot ( Ccl) analysis 24, 48, 321, 604 DNasellllO 427-32, 634-5 Coumermycin 187 endonuclease see Endonuclease breathing, 22 CpG island see Methylation, HTF exonuclease see Exonuclease buoyant density, 24-6,596,598-9 island phage-induced endonucleases, 264, catenation, 186, 187 Cro gene see Bacteriophage lambda 274 chloroplast, 67, 563 Cro protein see Bacteriophage Deoxyribonucleotide cloning, 620-31 lambda biosynthesis, 141-3 complexity, 24 Crossing-over 270 Deoxyribose complementary (eDNA), 180,306, unequal, 292-3, 294 biosynthesis, 141-3 318,607,620,623,625-8,634, Cruciform 27,274 chemical reactions, 32 635-7 Cryptogene 496 structure, 6 Cot ( C0t) analysis, 24, 25, 321, 604 CTP-synthetase 140 Depurination 32, 593 cruciform, 27,28 Cyclic AMP (cAMP) 9, 386,421 Diadenosine tetraphosphate (Ap4A) curved,20 transcriptional activation, 421 583-4 denaturation, 22 receptor protein see Catabolite 6-Diazo-5-oxonorleucine (DON) 136, DNase sensitivity in chromatin, activator protein 140 432-3 response element (CRE), 421, 427 Diazobenzyloxymethyl (DBM)• dynamic structure, 22 Cyclin 43, 177 cellulose paper 606 electron microscopy, 26-8, 156-8, Cyclobutane dimer 263, 264 Dichlororibofuranosyl benzimidazole 201,596-8,604 Cycloheximide 646 645 electrophoresis, 23, 27,600-2,603, Cytidine Dideoxy sequencing method 613-5, 606,613,617,632-5 addition, 499 624 enzymes of synthesis, 162-189 structure, 8 2' ,3' -Dideoxynucleoside triphosphate foldback, 28, 52 Cytidine deaminase 148 613 footprinting, 354, 417, 634 Cytopathic effect 78 2' ,3' -Dideoxynucleosidcs 650 G + C content, 13,598,605,611 Cytosine, structure 6 2',3'-Dideoxythymidine 176 gyrase, 187,188,347 Cytosine arabinoside 264 Diethyl pyrocarbonate and RNase haploid content (C value), 47 Cytosine deaminase 148 inhibition 108 helicase, see Helicase Differential processing of pre-mRNA heteronomous, 20 dam 121,267 321' 499-504 interspersed repetitive, 50-52 D loop see Displacement loop Dihydro-orotase 139 isolation, 598-602 (DBM)-cellulose see Dihydrofolate reductase (DHFR) 144 junk,47 Diazobenzyloxymethyl• gene amplification, 223, 228 kinetoplast, 67 cellulose Dihydrothymine 148 kinase, 126 dcm 121,268 Dihydrouracil 148 kinks, 20 Dda helicase 185 Dihydrouridine 528-9 ligase, 181-3,200,622,626 dCMP deaminase 144 I ,25-Dihydroxy vitamin D 418 light scattering, 596-7 Deamination 258, 268 Dimer linker, 55 7-Deaza-2' -dGTP 613 cyclobutane, 263, 264 looping, 118,199,210,287,288, Deformylase 519 thymine, 263-6 385,387-8 Degeneracy see Genetic code Dimethyl sulphate 260,611,634 loops, 223,225-6, 387-8,396, Deletion mutagenesis see Mutants Dimethylnitrosamine 260 436-7 Demethylation Diphenylamine, reaction 32, 593 major groove, 15-16 azacytidine, 123 Diphtheria toxin 547,561 melting, 22, 436, 605 passive, 124 Diploid ce1143 methylase, 121 programmed, 124, 433 Direct repeat see Repeated DNA methylation, 23, 12-4, 207, 433-4, progressive, 433 Discontinuous replication 158 491-2,611,626,634 see also Methylation Displacement loop (D loop) methyltransferase, 121 Denaturation mapping 158, 598 in DNA replication, 202, 218 minor groove, 15-16 Denaturation of DNA 22-4 in recombination, 272 mitochondrial, 65, 562 Density of DNA see DNA, buoyant in transcription, 368 modification, 117 density Dissociation element 301-302 molecular weight, 14, 596-8 3'-Deoxy ATP609, 645 Dissociation factor, translational, 547 nucleosome-free, 432, 435-6 Index 661

packaging, 53-60 translations see Proofreading applications, 120,609,617-8, palindromic sequences, 27, 28, 52, Editosome 497-8 621 384,386,389,391,419,484 EF-1, EF-2, EF-3, EF-Ts, EF-G, EF• uvrABC,266 polarity of strands, 15, 168 Tu see Elongation factors of viral, 113, 373 polymerase, see Polymerase, DNA protein biosynthesis see also Nuclease; primase, see Primase Egg white protein 418 Deoxyribonuclease; propeller twist, 19 Egg yolk precursor protein 418 Ribonuclease recombinant technology, see DNA eiF-1 etc. see Initiation of protein Endoplasmic reticulum 564, 602, 645 cloning biosynthesis lumenal retention of proteins, 567 relative undermethylation, 433-4 Electron translocation of proteins across, 564 renaturation, 24 diffraction, 548 Endoribonuclease repetitive, 48-52 microscopy forming 3' -phosphate groups, 100- replication, see Replication chromatin, 55-7,61-3 106 restriction, 117 DNA, 26-8, 156-8,201,318, forming 5' -phosphate groups, 106 restriction mapping, 617-8 415,596-8,604 see also Ribonuclease right-handed, 15 immune,554 Enhancer339,370,408-9 roll, 19 Electrophoresis of nucleic acids cAMP-regulated, 421-2,427,434 satellite, 48-50, 293, 600 agarose gel, 318, 596, 600-1,603, consensus sequences, 418,419,421, secondary structure, 14 606,617 426 sedimentation, 596 alkaline gradient, 23 distinction from promoter, 409 sequence determination, 609-615 polyacrylamide gel, 596, 600, 603, GAL,410-11 slide, 19 611,615,633-4 globin, 413-5 supercoils (superhelices), 26, 27, pulsed-field, 46,596,601-2 hormone-regulated, 418-20 347 Electroporation 638 immunoglobulin, 417-8,431 topoisomerase, 185-9,347,433 Elongation, factors of protein inducible, 418-22 torsional stress, 347 biosynthesis intron, 408,417 triple-stranded helix, 29 archaebacterial, 561 light-regulated, 421-2 twist, 19 chloroplast, 563 LTR,374,436 unique,48 EF-1,547 multiplicity, 409 unwinding protein see Helicase EF-2, 547,578 rDNA,361 wrinkled, 20 EF-3, 547 steroid hormone-activated, 418-20 writhe, 27 EF-G, 538,539,540,542,555-6, SV40, 411-13,639 Z-form, 21, 438 560 targets for regulatory protein, 409 dna mutants 193 EF-Tu,538,539,556,560,573,583 tissue-specific, 415,417 DnaA protein 205-7 EF-Ts, 536, 583 Enhancon 408 DNase see Deoxyribonuclease eukaryotic, 547 Episome72 Docking protein 564-5 mitochondrial, 562 Epitope tagging 351 Domains 320, 424 prokaryotic, 538-9 Epstein-Barr virus 225 Domain control region see Locus selanocysteinyl-tRNA, 581 Error-prone repair 269, 270, 289 control region Elongation, of RNA 346-7 Errors DON see 6-Diazo-5-oxonorleucine EMBL 3, 629-30 rates in DNA polymerase, 190-3 Dosage compensation 503 Embryogenesis 422-5 translational, 534, 559-60 Dot-blot 606 Emetine646 see also Accuracy; Proofreading; Double minute 228 Encephalomyocarditis (EMC) virus Suppression Downstream 340 637 Erythromycin 557 Downstream element 408 End-labelling, of nucleic acids, see Ethidium bromide 261,559,600,601, Drosophila Labelling methods, for nucleic 617 embryonic development, 422-5 acids Ethionine 646 sex determination, 503-4 Endo configuration 15-17 Euchromatin 53 Drug resistance see Antibiotic Endonuclease Evolution resistance AP endonuclease, 112 aminoacyl-tRNA synthetases, 532 Duplication, gene see Gene assay, 109 concerted, 294-5 duplication cap-dependent, 373 genetic codes, 524-5 dut mutants 158 endonucleases ofT4, 113, 264, 274 globin gene, 291,294,321 dUTPase 142, 158 HOof yeast, 281 intron role, 320-21 Dye-binding 261 of E. coli, 110, 111 mitochondrial, 333, 562 restriction,l07,117-20, 609,611, transposition and, 296 E1A protein 427 617-8,621 Excision repair 264-7 Edeine646 type I, 117-8 Exinuclease 266, 267 Editing type II, 118-20 Exo configuration 15-17 replicational see Proofreading type III, 120-21 Exon 318 RNA,496-9 nomenclature, 120 cassette, 502 662 Index

domain encoding, 320-21 gag 180 families, 290-1, 321, 425 evolution of proteins, 320-21 GAL genes 409-411, 436 frequency, 48 ligation, 463, 466, 472 activation, 410 G-protein, 322 mini,464 catabolite repression, 411 GAL,410-11 mutually exclusive, 502 nucleosome phasing, 436 gap,423 shuffling, 320-21 operators, 411 globin, 291-4,318,321,363,413-7 Exonuclease 106-7 organization, 409-11 glutamine synthetase, 405-6 associated with E. coli DNA promoter, 410 gpt,639 polymerase I, 116, 170, 171, proteins, 409-11,428,430,431 gryA and B, 187 265 ~-Galactosidase 381-4, 516, 622-4, haptoglobin, 293 E. coli, 113, 114 628 heat-shock, 405 exonuclease III, 114 ~-Galactoside histone, 53, 318, 320-24 exonuclease VII, 273,274,633 permease, 382 homeotic, 423-5 see also Nuclease; transacetylase, 382 housekeeping,353,434 Deoxyribonuclease; Gametogenesis 122 hunchback, 423 Ribonuclease Gamma (y) radiation 262 hypomethylation, 433-4 Expression, induction of gene- see Gap junction 145 immunoglobulin, 321,417-8,431, Induction of gene expression GCN4 regulatory protein 546 464,501 Expression, of cloned genes 635-40 Gel-retardation see Band-retardation insulin,307 Expression-linked copy of VSG 282 Gene interferon, 318 External transcribed spacer 230, 325- 55 RNA, 322, 329-30 jun, 421,426-7 7,361,489-91 A of lj>X174, 203 keratin, 322 acid phosphatase, 436 knirps, 423 F-factor 71 actin, 291,321,322 kriippel, 423 F' -factor 72 active, 434-5, 437 lac, 381-5 Feeder cells 260 activation, 410 light-regulated, 421 Ferritin, control of synthesis 573 ada,264 low-density lipoprotein receptor ,321 Fibre autoradiography 222 amplification, 48, 325-6 machine, 642-3 Fibrillarin 490-91 antennapedia, 424-5 maternal, 423 Fidelity apocytochrome, 332, 471 membrane-transporter, 322 ofreplication, 190-2 araBAD, 387-9 mitochondrial, 331-5 of translation see Accuracy; Errors; ATPase,332 multicopy, 321-35 Proofreading; Suppression bicoid (bed), 423 mut, 268 Fingerprint 50 calcitonin, 501 myoglobin, 321 Fluorescence activated cell sorter calmodulin-regulated, 421,425 myosin, 500-501 (FACS) 46, 47 cascade (developmental), 423, 425 NADH dehydrogenase, 332 Fluoride 646 chorion, 228, 322 neo,639 Fluoro-dUMP 14,259 chloroplast, 331-5 nitrogen fixation, 279,406 Fluorodeoxycytidine 644 cloning see Cloning numbers, 317 Fluorodeoxyuridine 145,259 clustering, 322-31,425 omp,439 Fluorotryptophan 646 cob, 332 onco-, see Oncogene Fluorouracil259 collagen,319,321,322 oskar,425 Foldback DNA 28, 52 conversion, 270,293,294-5,307 ovalbumin, 318 Footprinting, DNA 354,417,634 cop,210 ovomucoid, 321 Formaldehyde 33, 569-70, 598, 606 correction, 293,294-5 pair rule, 423 Formamide 597, 605 crystallin, 322 plant, 421 Formamidopyrimidine 262, 264, 265 cytochrome oxidase, 332, 333, 471 pol, 180 Formate dehydrogenase, synthesis dam,267 polarity, 423 581 dcm,268 protein-encoding, 332 Formic acid 35 dhfr, 224,228 pseudo-, 293-4, 306-8, 322, 324 N-Formylmethionyl-tRNA 518-9, dna, 193-5 pyruvate kinase, 321 534-6,538,540,562 dihydrofolate reductase, 439 quintets, 323 see also Initiation; Transfer RNA discontinuous, 318 RAG,287 N10-Formyltetrahydrofolic acid 136, duplication, 290-5, 307, 321 ribosomal protein, 333, 463, 526, 519 dystrophin, 319 553,561,571 Fragment reaction 538,541,547 embryonic, 422-5 rRNA, 223-4,227-30,317,321, Frameshift 180, 262, 296, 302, 570, engrailed, 423 322, 324-9, 333-4 581-2 evolution, 320 rearrangement, 277-89 mutation see Mutants and Mutation expression see Induction of gene receptor protein, 322 Fusidic acid 646 expression regulator, 339 a-foetoprotein, 322 ribulose-1 ,5 bisphosphate G-band44 fos,426-1 carboxylase, 333, 421 Index 663

serine protease, 321 Guanosine deaminase, 146 High frequency lysogeny (hfl) 628 serum albumin, 322 Guanosine 5' -diphosphate 3'• High mobility group (HMG) proteins snRNA, 359-60, 364 diphosphate (ppGpp) 582-3 55,435 structural, 382-3, 385 Guanosine 5' -triphophate 3'• Histone structure, 52 diphosphate (pppGpp) 582-3 acetylation, 54, 435 tRNA, 322, 330-31, 334-5, 562 5'-Guanylylmethylene diphosphonate ADP ribosylation, 54, 435 tubulin, 321, 322 536,538 classification, 53 U6RNA,359 Gyrase 187, 188, 347 development-specific, 54 ultrabithorax, 424-5 genes,53,318,320-24 umu,270 Haemin 573, 576 H1, 53, 61,435 vitellogenin, 319 Haemoglobin H2A, 53, 54, 55 VSG, 282, 283 anti-Lepore, 292 H2B,53, 55 wingless, 424-5 control of synthesis, 413-7, 576-8 H3, 53,54 xis, 279 genes, 291 H4, 53,54 see also individual genes (globin, Lepore,293 H5,54 insulin, etc.) mRNA, 571,574 in active chromatin, 54,434-5 Gene 32 protein see Bacteriophage, T polysomes, 576 methylation, 54, 435 even see also Globin modification, 54, 435 Genetic code 516-526 Hairpin loops in RNA 29-32, 216, mRNA,574 chloroplast, 525,563 217,348,468,472,473,485, phosphorylation, 54, 435 ciliated protozoan, 524 492,552,558 synthesis, 237 degeneracy, 519-24 Hammerhead structure 473 transcriptional termination, 364-5 determination, 516 Haploid DNA content (C value) 43, ubiquitination, 54, 435 evolution, 524-5, 527 44,47 variants, 54, 323 mitochondrial, 524-5 Haploid cells 43 HIV see Human immunodeficiency mycoplasmal, 524 Haptoglobin genes 293 virus standard, 516-9 HAT medium 144 HMG proteins see High mobility variation, 524-6 Heat shock group proteins Genetic engineering see Cloning nucleotides, 583-4 HO endonuclease 281 Genetic loci in E. coli 648-651 gene expression induced by, 418 Holliday model271-3 Genome41 proteins, 405,420,566,567 Holopolymerase III 173-5 Genomic sequencing see Sequence regulon, 405 Homeobox 424-5, 427-8 determination sigma factor, 405 Homeotic genes 423-5 Globin Helical twist angle 19 Homogeneously staining region 228 gene Helicase 184, 185,267,349,364 Hoogsteen base interaction 31,33 control elements, 413-7 dda, 185 Housekeeping gene see Genes, family, 291-5,321,324 a-Helix and DNA binding 430-31 houskeeping tissue-specific expression, 413-7 Helix-coil transition 22 Hox protein 425 processed pseudogene, 307,324 Helix-destabilizing (HD) protein 183 Hormone response elements 419-421 see also Haemoglobin Helix-loop-helix motif 431 HTF island 434 Glucocorticoid response element Helix-tum-helix motif392-4, 424, HU protein 207 419-21 427-8 Human immunodeficiency virus Glucosyltransferase 215,216 Heparin, and RNase inhibition 108 (HIV) 76, 578, 581 Glutamine synthetase 405-6 Hepadnavirus 581 Hybrid cells 45 Glutathione peroxidase, synthesis 581 Hepatitis B virus 180, 222 Hybrid-arrest translation 628 Glycinamide ribonucleotide (GAR) Hepatitis delta virus 88 Hybrid-release translation 628 135 Herpes simplex virus (HSV) Hybrid-selection 628 Glycosylase 265, 266 base composition, 26 Hybridization in situ 46 Glyoxal606 genome structure, 81 Hybridization of nucleic acids 604-6, Goldberg/Hogness box 353 inhibition of host protein synthesis, 628,632 Gout 148 576 Hydrazine 34, 611 Gratuitous inducer 383-4 inhibition of replication, 143 Hydroxyapatite 24 GT I AG rule 458 Hershey-Chase experiment 78 Hydroxylamine 34 GTPase, translational539, 555-6, Heterochromatin 49, 53, 413 Hydroxymethylase 144,215 565 Heteronomous DNA 20 Hydroxymethylcytosine 144, 215,216 Guanidine hydrochloride 603 Heterogeneous nuclear (hn) RNA Hydroxyphenylazouracil (HPUra) Guanidinium thiocyanate, and RNase 453 175 inhibition 108, 602 Heterogeneous nuclear Hydroxyurea 143 Guanine structure 5, 6 ribonucleoprotein (hnRNP) Hyperchromic effect 22 Guanosine 465 Hypersensitive site 414, 432-3 addition, 499 Heterokaryon 45 Hypomethylation of active genes structure, 8 Hfr68, 72 432-4 664 Index

Hypoxanthine, structure 7 IF-3,534-536,537,540,542 electron micrograph of, 318, 457 tRNA, 518-9, 534-6, 538, 540, function, 320-21 I element see Transposable element 543,562,577 group I, 319, 332, 466-71 IBV see Avian infectious bronchitis Initiation, of transcription group II, 320,471-2 virus eukaryotic, 352-63 group III, 320 IF-1, IF-2, IF-3see Initiation of complex, 354-5,358-9,362 L19 IVS, 466-70 protein biosynthesis control, 381-96,407-38 maturases, 471 Immune electron microscopy see in vitro systems, 352, 356, 360 mitochondrial genes, 332, 470-71 Electron microscopy factors, 354-5,357-60,362 mobility, 321, 471 Immunity mitochondrial, 368 mutation, 295, 458 bacteriophage, 86 multiple sites, 353, 363, 479 non-consensus, 464 transposon, 299-300 RNA polymerase I, 360-63 organization, 52 Immunoglobulin RNA polymerase II, 353-5 origin, 320-21 allelic exclusion, 286 RNA polymerase III, 355-60 plant, 464 genes284-9, 321,417-8 prokaryotic, 341-6 prokaryotic, 319-20,468 controlled expression, 417-8, complex, 341-6 retained, 500 501 inhibitors, 341, 344 Rloops,318,455,457,604 enhancers, 417-8 start site, 340, 344 rRNA genes, 327-9,466-71 promoters, 417-8 Inosine splice sites, 457-8 rearrangement, 284-9, 501 anticodon, 521,522 splicing, 301, 319 somatic mutation, 289 insertion into tRNA, 126 transposition, 319, 472 switch, 288,289 structure, 8-9 tRNA genes, 475-8 membrane and secreted forms, 501, Inosine monophosphate (IMP) 136, Invasion, single strand 274-7 566 137 Inversion of DNA 280, 281 Imuran 138 Insert see Intron Inverted repeat see Repeated DNA In see Integrase Insertion sequence (in rONA) 326-8, IPTG see Isopropylthio-~- Incompatability 209, 210, 220 466-71 galactoside) Induction of gene expression see also lntron; Transposable Iron-responsive element (IRE) 573 calcium-binding protein, 418 element IS element see Transposable elements egg white protein, 418 Insulin lsogenic strains 45 egg yolk precursor protein, 418 genes,307 Isopropylthio-~-galactoside (IPTG) growth factor-stimulated, 421 phosphorylation of EF-2, 578 383-4, 622-3, 637 heavy metal-stimulated, 418 Intasome 279 Isopycnic ultracentrifugation 598-9, methylation-stimulated, 124 Integrase 278, 302, 304, 392 602 light-stimulated, 421-2 Integration host factor 278 Isoschizomer 120, 433 prokaryotic, 381-9 Integration protein see lntegrase steroid hormone-stimulated, 418- Intercalating agent 599 Junk DNA see DNA, junk 21 Interferon 574-5, 576-8 touch-stimulated, 421-2 Internal control region 356-9 Kanamycin 561,639,646 virus-stimulated, 418 Internal guide sequence 469-70 Karyotype 43 Inhibitors 650-651 see also specific Internal transcribed spacer 325-7, Kasugamycin 558 entries (chloramphenicol, 361,489-91 Kinase (nucleoside and nucleotide) hydroxyurea etc.) Internally eliminated sequence (IES) 145 Initiation, of protein biosynthesis 283 see also Polynucleotide kinase; codons employed, 518,519,534-7, lnternucleotide linkage 10 Nucleic acid kinase; Protein 543,546 Interphase 10 kinase; Thymidine kinase eukaryotic factors, 543-544 Interspersed repetitive DNA 50, 52 Kinetic proofreading see Proofreading eiF-2, 543-4, 576-8 Intervening sequence see Intron Kinetoplast DNA 67, 218 eiF-28, 544,577-8 Intracisternal A-particle 303, 307 Kirromycin 646 eiF-3, 543, 547 Intron 318-20,454-72 Kleinschmidt technique 597 e1F-4A, 543, 570-1 archaebacterial, 320, 475 Klenow fragment of E. coli DNA eiF-48, 543, 571 autocatalytic splicing, 320 polymerase I 169,607,609, e1F-4C, 543 boundaries, 457-8 613,620,626,640,641 eiF-40, 544 branch point, 459-63 Knotting of DNA 186, 187 e1F-4E, 543, 578 class I see Intron, group I Kornberg enzyme 166-72 eiF-4F, 543,546,571,579 class II see lntron, group II Kozak model see Scanning model eiF-5, 543 classical, 319 eiF-6. 547 deletion, 306-7,321 L1 family 52, 304-5 mitochondrial, 562 differential processing, 321, 499- Labelling methods, for nucleic acids prokaryotic factors, 534-8 504 end, 607-9 IF-1,534-6,540,542 discordant, 321 general, 606-7 IF-2, 534-6, 538, 539 enhancer,408,417 non-radioactive, 606 Index 665 lac operon see Operon, lac Melting temperature ( Tm) 22, 605 5-Methoxycarbonylmethyl uracil522- Lactose, and induction 381-5 Membrane protein synthesis 566-7 3 Lag phase (of virus growth) 77 6-Mercaptopurine 135, 259 5-Methoxycarboxymethoxy uracil Lagging strand 158 Meselson and Radding model 272 522-3 Lambda see Bacteriophage lambda Meselson and Stahl experiment 154, 5-Methoxyuracil522-3 Lamin42 155 Methyl mercuric hydroxide 606 Lampbrush chromosome 64 Messenger RNA (mRNA) 1-Methyladenine 260-7 Lariat, structure of excised introns 3' non-coding region, 396-9, 481- 3-Methyladenine 260 459,472 5, 573-4, 632 6-Methyladenine 121, 207 Leader, ofmRNA 396-9,464-5, 5' non-coding region, 396-9, 452, 5-Methylaminomethyl-2-thiouridine 535,546 480-1,535,546,633 495 Leading strand 158 abundance,350,452-3 Methylation Lesch-Nyhan syndrome 138 cap,370,373,453,478-81,543, DNA, 121 Leucine zipper 426, 430-31 579,637 changes during gene expression, Leukaemia virus 76 see also Moloney crystallin, 453 122 murine leukaemia virus decoding, 558 dam, 121,207 LexA protein 270 dihydrofolate reductase, 439 dcm, 121 A.gt10626-8 discovery, 451 de novo, 123 A.gt11628 eukaryotic, 453, 545, 570 HTF island, 434 Ligase ferritin, 573 hypo-, 433-4 DNA see DNA ligase fibroin, 453, 526 maintenance, 122, 123 RNA, 476-8, 497 gal, 452 methyltransferase (methylase), Light scattering 596-7 GCN4 regulatory protein, 546 121-4 Light-regulated genes 421 haemoglobin, 455,458,571,574 mismatch repair, 267-9 LINEs 50, 52, 293, 303, 304-5, 307 half life, 452, 453, 485, 573-4 see also Demethylation Linker histone, 484-5574 RNA, 124,491-2,495-6 nucleosome, 55, 58,60 immunoglobulin, 453 see also individual RNA species oligodeoxynucleotide, 626, 640 lac, 452 2' -0-Methylcytidine 522 Linker-scanning 641 leader sequences, see 5' non-coding 5-Methylcytosine 121,433,522,611 Linking number27, 186-8 region deamination, 124, 268 Locus control region (LCR) 414-7 maternal, 571, 574 structure, 6 Long terminal repeat (L TR) 302, 374, maturation, 451-66, 478-86,499- JVS, N10-Methylenetetrahydrofolic 436,639 504 acid 135, 144 Lysidine (2-lysylcytosine) 522, 532 monocistronic, 454, 543, 545 Methylguanine methyltransferase Lysogeny 85, 86, 391, 628 nascent, 350, 364 (Ada) 264 Lysosome retention of proteins in 567 ovalbulin, 453 2-Methylguanosine 495 Lysozyme 42, 78, 598 poly( At, 481-4, 574, 625 2' -0-Methylguanosine 522 Lytic cycle of bacteriophage 76-78, poly( A)-, 484-6,574 7-Methylguanosine 495 391 poly( A) tail, 332, 453, 481-4, 573- Methylmethanesulphonate (MMS) 4,603,625 260 Ml RNA 472,493 polyadenylation, 332, 481-4, 499- Methylnitronitrosoguanidine MI3 see Bacteriophage MI3 504,574 (MNNG)260 Ml3mp18 614,623-4 polycistronic, 381,383,452,454, 2-Methylribose 492 Macaloid, and RNase inhibition 108, 564,571-2 2-Methylthio-~- 603 precursor, 452-66,478-86 isopentenyladenosine 496 Macronucleus of ciliates 283, 284, 326 processing, 451-66, 478-86, 499- 0-Methylthreonine 646 Major groove 15,393,428 504 Methyltransferase of DNA 121-4 Malachite green 25 prokaryotic, 451-3, 571-3, 645 Micrococcal nuclease 98, 645 Mapping purification, 453, 602-3 Microinjection see Xenopus oocyte denaturation, 158, 598 RNA phage, 371-4, 452, 569-70, Microbodies, translocation of genetic, 79, 317 645 proteins to 568-9 restriction, 120,617-8 secondary structure, 452, 543, 569- Micronucleus of ciliates 283-4, 326 Rloop,604 71 MIF-1 family see Ll family transcript, 632-4 structure, 452, 454 Mini-replicon, viral638 MAT locus 281-3 subgenomic, 302, 545 Minichromosome 55-83 Mating type 281-3 tubulin, 576 Minisatellite 49 Matrix, nuclear42, 64,366-7,437-8, transport, 505 Minor bases 5, 7 465-6 vitellogenin, 453 see also Modified bases Matrix attachment region (MAR) 64, Metaphase 44 Minor groove 15, 393 224,367,415,437 Methotrexate see Amethopterin Minute virus of mouse (MVM) 81, Maxam and Gilbert sequencing 5-Methoxycarbonylmethyl-2- 233 method 3, 609, 611-3 thiouracil 522-3 Mismatch repair 267-9 666 Index

Misreading see Accuracy; Errors; dilute coat colour, 303 Non-radioactive labelling 606 Fidelity dna,193 Non-transcribed spacer 230, 326-8, Missense mutation see Mutant dut, 158 360-61 Mitochondria frameshift, 193,262,294 Nonsense mutation see Mutants D loop, 66, 368 homeotic, 423 Northern blotting 605 DNA,65,562 in vitro, 640-2 Norvaline 647 DNA polymerase, 178, 218 intron, 295, 458 Novobiocin 187 evolutionary origin, 562 lac/, 382 Nuclear- see Nucleus genes,66,331-5,368 linker-scanning, 641 Nuclear magnetic resonance (NMR) genetic code, 524-5, 562-3 mut, 267, 268 18,22,424 genome, 66, 67,331-5 mutator, 170 Nuclease introns, 319-20,332,468-71 nonsense, see amber; ochre; opal Bal3!, 99,640 origin of replication, 67, 368 ochre, 518 classification, 97 polyadenylation, 332 opal, 518 micrococcal, 55-9, 98, 645 promoters, 368 petite, 66 mung bean, 99 protein targeting, 567-8 pseudogene, 293-4 N. crassa, 98 replication, 217, 218 selection, 68 PI, 98 ribosomes, 333,562 site-directed, 641 recBCD,274 RNA polymerase, 368 sof, 158 Sl, 98,25 rRNA,333 somatic, 289 mapping, 623-3 rRNA genes, 66,317,333-4 spo,402 uvrABC endonuclease 266 splicing in, 468-71 suppressor,518,542,560,631 see also Deoxyribonuclease; transcription, 66, 367-8 temperature-sensitive, 79, 193 Exonuclease; Ribonuclease tRNA genes, 66, 334-5, 562 transpositional, 295, 301, 303 Nucleic acid tRNA, 334, 562-3 number,518 base-composition, 12-14, 595-6 Mitomycin C 260 ung, 159 chemical determination, 32,593-5 Mitosis 42, 43 Mutator mutation 170 computer analysis, 291, 611 MMS see Methylmethanesulphonate Mycophenolic acid 136, 137, 39 hydrolysis, 12, 592 MNNGsee Mycoplasma, genetic code 524 kinases, 124-6 Methylnitronitrosoguanidine Myeloma ce11453 labelling methods 606-9 Mobility-shift see Band-shift Myosin phosphatases, 124-6 Modification of DNA 121-4, 215-6 alternative processing, 500-1 primary structure, 9-14 Modeccin 647 genes, 291, 500-1 sequence determination, 609-16 Modified bases polysomes, 516 tissue contents, 594-5 rRNA, 491-2, 550 Myxoviruses, replication of373 see also DNA; RNA; individual tRNA, 126,495-6,521,522,523, nucleic acids 524,528,529,532,537-8,558, n (primosome protein) 197-9 Nuclein I 563,580 N-protein utilization site (Nut site) Nucleoid 68 Molecular weight of DNA 14, 596-8 400-1 Nucleolus Moloney murine leukaemia virus N-utilization substance (Nus) 350, 401 isolation, 602 579-80 N. crassa nuclease 98 organizer, 44,325 Monocistronic mRNA see mRNA, Nalidixic acid 187 pre-rRNA, 360, 489 monocistronic Nearest-neighbour analysis 167, 168 processing, 489-93 Monoclonal antibody 145 Negative control systems 219-20,385 ribonucleoprotein, 489 Mouse hepatitis virus 581 Neomycin 639 RNA polymerase, 489 Mouse mammary tumour virus 418, Netropsin 644 snRNAs, 490-91 436,581 Neutron Nucleoplasm 350 MS2 see Bacteriophage, MS2 diffraction, 58 Nucleoplasmin 237 msDNA element 305 scattering, 553 Nucleoside analogues 259 Mu see Bacteriophage, Mu Nick translation 116, 164, 166, 171, Nucleoside deoxyribosyl transferase Mung bean nuclease 99 606 126 Mustards 44, 65,260 Nitrogen, assimilation 405-6 Nucleoside phosphorylase 138 Mutagen 257-63,391 Nitrogen fixation genes (nin 279-80, Nucleoside 8 Mutants and Mutation 406 Nucleoskeleton 42, 366-7,437-8 3'-codon, 519 Nitrogen mustard 260 Nucleosome 55-60 amber,190-1,518,540,570,580 Nitrous acid 32, 258 assembly, 237, 238 aminoacyl-tRNA synthetase, 533 NMR see Nuclear Magnetic core, 58,59 antennapedia, 423-5 Resonance phasing, 60, 435-6 antimutator, 170 Non-histone chromatin protein 55, regions of DNA, lacking, 60, 238, bithorax, 423-5 435 432,436 conditional lethal, 79, 193 Non-polyadenylated mRNA 484-5 segregation, 236, 237 deletion, 640 see also Polyl!denylated mRN A spacing, 55,60 Index 667

Nucleotide 9 pseudo-, 383 pET vectors 636-7 derivatives, as RNase inhibitors, repressor binding, 384-5,392-5 Petite mutants 66, 218 108 sequence, 383-4 Phage see Bacteriophages; Viruses metabolism, 135-9 trp, 385,394 Phasing of nucleosomes 60-61, 435-6 methylation see Methylation of Operon 317,381 pHC79630 DNA arabinose (araBAD), 387-9,394 Phenol red 25 Nucleus biotin ( bio), 389 Philadelphia chromosome 289 isolation, 602 definition, 381 Phleomycin 260 lamina,42 galacotose (gal), 386, 396 Phosphatase matrix, 42, 61,366-7,415,437-8, histidine (his), 398 alkaline, 606,609,622 465-6,505 ilvGMEDA, 398 exonuclease III, 114 matrix-associated regions, 64, 367, lactose (lac), 381-7,452 polynucleotide kinase, 124, 125 437 leucine (leu), 398 Phosphite triester method 642-3 nuclear run-off 643 phenylalanine (phe), 398 Phosphodiester bond nuclear run-on, 643 pyrBJ, 399 2' -5'' 459. 575 pore complex, 41,42 pyr£,399 3'-5',10 protein targetting, 41, 569 rRNA (rm), 324-5 cleavage, 34 scaffold, 42, 61, 64, 188, 366-7, 437 tRNA,330 Phosphodiesterase skeleton, see Nucleoskeleton theory of, 382 venom, 99 Numbering of purine and pyrimidine threonine (thr), 398 spleen, 99 rings 6 tryptophan (trp), 385,396-8,452 Phosphonoacetic acid 179 Nus seeN-utilization substance Orcinol reaction 32, 593 Phosphonoacetyi-L-aspartate 140 Nut site seeN-protein utilization site Origin of replication see Replication 5-Phosphoribosylamine (PRA) 135 Orotic acid 139 5-Phosphoribosylpyrophosphate Ochre mutant and codon 518 Orotidine monophosphate (OMP) (PRPP) 135-7 Oestrogen response element 419-20 139, 140 amidotransferase, 135-7 Okazaki piece (fragment) 157-9 Ovalbumin gene 318 synthetase, 135 initiation, 159-162 Ovomucoid gene 321 5-Phosphoribosyltransferase Oligo(dT) cellulose 453,603,625 Oxolinic acid 187 adenine (APRT), 138 2', 5' -Oiigoadenylate (2' -5' A) 105, hyoxanthine, guanine (HGPRT), 573 P factors and elements 301, 500 138 Oligonucleotide p34 protein kinase 43 Phosphorylation linker, 626,640 p53 212 aminoacyl-tRNA synthetase, 579 primer, 552,606,607,613,615, Pactamycin 647 EF-2, 578 620,633 Palindromic sequence 28, 384, 386, eiF-2, 576-8 probes,607,609,626 389,391,419,484 eiF-4E, 578 sequencing, 609 Pancreatic deoxyribonuclease see eukaryotic acidic ribosomal site-directed mutagenesis, 641 Deoxyribonuclease proteins,553,578 synthesis, 642-3 Pancreatic ribonuclease see eukaryotic ribosomal protein S6, X-ray crystallography, 19-21 Ribonuclease 579 Oncogenes 425-7 Papovavirus 210-13 histone, 54, 435 eiF-4E gene, 543 Paromomycin 559 HMG proteins, 435 ets, 427 Parvovirus 81, 233 RNA polymerase, 352 fos, 426-7,574 Paternity testing 50-51 Tag,211,212 jun, 421,426-7 Pausing transcription factors, 406, 427 rnyc,5427,43l, 74 of ribosomes, 396-8, 581-2 Photochemistry of bases 36 rnyb, 427 of RNA polymerase, 347,350,364, Photoreactivation 264 rei, 427 399-401 Picornavirus role in cancer, 88, 290, 426-7 pBR322 73, 622 translation, 575 Onion skin replication 228-9 PCR see Polymerase chain reaction replication, 371 Oocytes see Xenopus oocytes Pedigree analysis 50-51 see also individual viruses Opal mutant and codon 518 Pentose sugars 6-7 Pilus 71-2,77 Open chromatin domain 415 Peptidase pKK 233-2 636 Open complex, for transcriptional amino-, 519 Plasmid initiation 344-5,387 signal, 564-5,566 2~ of yeast, 224-5 Operator 339 Peptidyltransferase 515,538,541-2, classification, 71 GAL,410-11 547,557-8 Col Et, 439 isolation, 384 Peptidylglycan, synthesis 584 copy number, 209,210 lac, 382-5 Perchloric acid 12, 35, 593 DNA isolation, 598-9 lambda, 390-3 Periodate 32 incompatible, 210,217 palindromic nature, 384,386,389, Permeable cells 161, 193, 194 oriC, 205-8 391 Permissive conditions 79, 193 Pl, 209 668 Index

replication, 209,210,216,217 mitochondrial, 178 Polytene chromosome 65, 283, 418 structure, 71 mutator mutants, 170 Poly(U) Sepharose 453, 603 see also Vector; individual plasmids processi vi ty, 17 4 Polyvinylsuphate, and RNase Pneumococcus 69 pyrophosphorolysis, 177 inhibition 108 Poliovirus reverse transcriptase, 179, 180, Pore complex (nuclear) 41, 42,569 genome structure, 80 302-7,607,625,633 Post-replication repair 269-70 inhibition of host protein synthesis, 1,174,581 Post-translational modification 518, 579 T. aquaticus (Taq), 175,613,620 553,567 mRNA translation, 546, 579 terminal transferase, 179,288,609 Pox virus see Vaccinia virus replication, 371-3 Polymerase, RNA POU domain 428 Poly( A) polymerase active sites, 345 ppGpp see Guanosine 5'-diphosphate E. coli, 609 ADP ribosylation, 406 3'-diphosphate cytoplasmic, 574 bacterial, 340-41 pppGpp see Guanosine 5'• nuclear, 481-4 bacteriophage, 370-71 triphosphate 3' -diphosphate Polyacrylamide gel electrophoresis SP6, 369,607 Pre-mRNA see Electrophoresis of nucleic T3,369, 607 alternative processing, 499-504 acids T7,369,607,613,637 calcitonin, 501 Polyadenylation of mRNA 323, 453- chloroplast, 368-9 calcitonin-related peptide, 501 4,481-4 core enzyme, 340 capping, 478-9 alternative, 499-504 DNA binding, 341-4,352 control of processing, 499-504 assays, 482 elongation by, 346-7 Drosophila sex determination, complex, 483-4 eukaryotic, 350-52 503-4 control, 499-504 I, 325, 350-52 globin, 456, 458 function, 484, 573-4 11,304,322,350-55,370,399 immunoglobulin, 501 mechanism, 481-4 III, 306, 330, 333, 350-52, 370 kininogen, 502 mitochondrial, 332 genes,526,573 leader of, 480-81 phases, 482 holoenzyme, 340 myosin, 500 sites, 481-3 homology, 341,351-2 nascent, 350, 364 Polyanions, and RNase inhibition 108 inhibition, 340,347, 351 ovalbumin, 457 Polycistronic mRNA see mRNA, initiation by, 341-6,352-63 ovomucoid, 457 polycistronic nuclear matrix association, 438 processing, 451-66, 478-86, 499- Poly d(A-T) 172 metal ion requirements, 340, 350 504 Poly(dG).Poly(dC) 172 mitochondrial, 217,368 T-antigen, 500 Polygene transcript 465 modification, 352, 406 transport, 505 Polymerase chain reaction (PCR) 47, molecules per cell, 341 tropomysin, 502 600,613,618-20,626,641-2 nuclear, 350-52 vitellogenin, 457. Polymerase, DNA 162-180 nusA protein binding, 401 Pre-rRNA a, 176, 650-1 pausing, 347, 350, 364 eukaryotic, 489-93 B. subtilis polymerases, 175 phosphorylation, 352 maturation, see processing p, 178 prokaryotic, 340-41 processing, 466-71,486-493 bacteriophage T4, 175,609,626 promoters, 340-44, 353-4, 356- prokaryotic, 486-9 E. coli polymerase I (Kornberg 61,368,70 ribonucleoproteins, 492-3 enzyme), 166-72, 199,299, rate of transcription, 347 splicing, 466-70 609,626 reconstitution, 351 Pre-tRNA structure, 166, 169 relative molecular mass, 340, 351- base modification, 495-6 mechanism, 163-6 2,370 eukaryotic, 493-6 active centre, 169-70 RNA-dependent (replicase), 371- processing, 472-3, 475-8, 493-6 associated nucleases, 169-71 4,569-71,573,583 prokaryotic, 493 repair, 267 sigma factor, 340-45, 402-6 splicing, 475-8 role in vivo, 171, 172 subunit structure, 340-41, 350-52 synthesis of CCA terminus, 495 E. coli polymerase II, 172, 173 termination by, 347-50 Preprimosome 197-9 E. coli polymerase III, 173-5, 199, ternary complex, 346 Preproinsulin see Insulin 269,270 viral, 347, 369-74 Presynaptic filament 276 li, 177 Polymorphism 45 Pribnow box 342 E, 177 Polynucleotide kinase 124, 125, 609 Primary structure of nucleic acids 9- error rates, 170, 190, 192 Polynucleotide phosphorylase 108-9 14 eukaryotic, 176-9 Polyoma virus 14, 85 Primase 159, 177,189,526 y, 174,178,218,581 Polypeptide formation see Protein Primer 159, 177, 189,306-8,373, herpes simplex virus, 179 biosynthesis 374,552,584,606,607,613, Klenow fragment, 607,609,613, Polyprotein 545,546,579 615,620,625,633 620,626,640,641 Polyribosome (polysome) 516,547, Primer-extension 633-4 mechanism, 163-6 602 Primosome 199 Index 669

Prion88 upstream elements, 353-4, 408 pUEX637 Processing variant sigma factors, 402-6 Puff, chromosome 228,418 3'-,481-6 viral, 369-74 Pucker,ofribose 15-17 5'-, 478-81 Proofreading Pulsed-field gel electrophoresis 596, control, 499-504 kinetic, 190, 560 601-2 differential, 499-504 replication, 170, 190 Purine pre-mRNA, 451-66,478-86 translation biosynthesis, 135-8 pre-rRNA, 466-71,486-93 aminoacylation, 534 catabalism, 145-8 pre-tRNA, 475-8, 493-6 decoding, 560 insertion, 126 protein, 545, 567 Propeller twist 19 numbering, 6 Proflavine 261 Prophage 85-6,391 preformed, 138 Progesterone 420 Propium diiodide 644 structure, 5-6 Pro head 77-78 Protozoa, ciliated 524 tautomerism, 5, 527 Proliferating cell nuclear antigen Protease Puromycin 536, 538-9, 549 (PCNA) 177 cleavage of p220, 579 X174 (bacteriophage) Promoter MAS,568 information content, 82-4 5S RNA gene, 355-60 recA,270 replication, 196-8,202-5 -100 elements, 354 see also Peptidase pYAC4631 adenovirus, 427 Protein A of X174 203 Pyrimidine alternative, 500-01 Protein biosynthesis biosynthesis, 139-40 ara, 387-8 archaebacterial, 560-1 catabalism, 145-8 bio, 389 chloroplast, 563 numbering, 6 blocking by repressor proteins, 383 direction, 515 structure, 5-6 CAAT box, 353-4, 408 eukaryotic, 542-8 tautomerism, 5, 257 chloroplast, 368-9 mitochondrial, 562-3 Pyrophosphatase 164 closed, 344-5, 385, 406 prokaryotic, 534-42 5' -5' pyrophosphate bridge 478-9 consensussequence,342,353,369, rate, 516 402,407 translational regulation, 569-82 Q-band 44, 65 differentially responsive, 500-04 see also Elongation; Initiation; Queuosine (Q) 495,522,580 distinction from enhancers, 408 Termination insertion into tRNA, 126 divergent, 389 Protein kinase 43,211,421,576-8, Quinacrine mustard 44, 65 down mutants, 342 579 duplications, 361 Protein phosphatase 212 R-factor 72 E. coli consensus, 342 Protein phosphorylation see R family see L1 family factors for, 354-5,357-60,362 Phosphorylation R loops 318-9, 455, 457 GAL,410-ll Protein synthesis see Protein Random-priming 606-7 GC-rich, 353-4, 408 biosynthesis Rate-zonal ultracentrifugation 598, globin, 413-7 Protein targetting 563-9 602 immunoglobulin, 417-8 Protein-nucleic acid interaction 122, Reading frame 83-5,296,546,579- internal, 357-9 392-6,427-32,553-5,571-2, 82,623,628 lac, 383,386 see also specific protein motifs Rearrangement of genes lambda, 391,400 (leucine zipper, zinc finger immunoglobulin, 284-9,417 LTR, 374, 436 etc.) macronucleus, 283-4 mitochondrial, 368 Prototroph 68 nitrogen-fixation, 279-80 mouse mammary tumour virus PRPP-amidotransferase 135 pilus, 280 (MMTV),436 PRPP-synthetase 135, 136 T cell receptor, 286 open,344-5,385,406 pSC101,73 yeast mating type, 280-2 Pribnow box, 342 Pseudo gene VSG,282-3 prokaryotic, 342-5 5S rRNA, 294, 328-9 RecA protein 270, 275, 391 RNA polymerase I, 360-63 7SRNA,308 RecBCD nuclease 274 RNA polymerase II, 322, 353-4 actin, 307 see also Nuclease RNA polymerase III, 355-60 calmodulin, 307 Receptor proteins for steroid RNA polymerase binding, 341-4 duplicative, 293-4, 322 hormones 419-20 rRNA gene, 360-63 globin, 293-4,307,324,414 Recombinant DNA see Cloning, split, 356-9 immunoglobulin, 307 DNA sporulation, 403 insulin, 307 Recombinase 286, 287 strength, 342 processed, 306-8 Recombination 270-9 TATA box, 353-5,408 snRNA,308 Holliday model, 271 tRNA,358-9 Pseudoknot31,32,558,573,561 illegitimate, 271 trp, 385 Pseudouridine ('1'), 9, 492, 529, 550, meiotic, 270, 271 up mutants, 344 562 Meselson and Radding model, 272 upstream, 359-60 pUC18 622, 624, 626 rec system, 274-7 670 Index

reciprocal, 292-3 origin sequestration, 207 Reticulocyte lysate 578, 577, 645 site-specific, 277 origin strategy, 202 Retinoic acid 418,420 transposable elements, 299 PI, 209 Retronphage cjJR73 305 see also Cloning papillomavirus, 210-3 Retroposon 303 Red genes of bacteriophage lambda phagelambda,208,209 Retrovirus 179,303,307,579,581, 275,629 phage T4, 215, 216 584-5,639 Regulator gene 339 phage T7, 213-5 endogenous303,307 lac, 383 polyoma, 210-3 oncogenic 87, 425-6 araC,387-8 retrovirus, 220, 221 replication ofDNA 220-3,374 trp, 385 rhabdovirus, 373 see also Reverse transcriptase, Release factor see Termination, rRNA genes, 223-4 individual entries protein biosynthesis role of methylation, 207,209,210 Reverse transcriptase 180, 199, 302- Renaturation of DNA 22-4 run away, 213 7,318,607,625,633 Reovirus SV40,210-3 Reverse transcription 180, 199, 220- genome structure, 81 yeast, 224-5 2,302,304,306-8,552,558 replication of, 374 in vitro systems, 193-6, 200 RF replication 202-5 Rep204 lagging strand synthesis, !58, 197, RFLP see Restriction fragment length RepA209 200 polymorphism Repair of DNA leading strand synthesis, !58, 200 Rhabdovirus 373 error-prone, 269, 270 onion skin, 229 Rho factor 349-350 excision, 264-7 rate, 202-5, 223 Ribonuclease (RNase) mismatch, 267-9 reconstitution in vitro, 196 2' -5' A dependent see RNase L post-replication, 269,270 RF, 203-5 B. cereus, 105-6,615 reversal of damage, 269, 270 RNA virus, 371-4 bovine semen, 102-3 Repeated DNA 48 rolling circle, 202 double-stranded RNA specific, 107 direct target, 280- 1, 295, 298, 301, semiconservative, 153-5 inhibitor proteins, 108, 603 302,303,304,306,308 single-stranded DNA phage, 196- non-secretory, 104 interspersed, 50 200 pancreatic see RNase A inverted, 280-1, 296, 301, 302, 303 termination, 230-6 RNase I, 101, 105 tandem, 48-50,291-3 cyclic chromosomes, 231 RNase II, 101, 106-7, 109 terminal, 218-9,231-3296,303 phage T7, 231-3 RNase III, 106, 453, 483, 493 Repeating elements, in rONA 230, telomere, 234-6 RNase A (pancreatic), 100-104, 361 timing, 227 598 Repetitive DNA see Repeated DNA transcriptional activation, 207,415 mechanism of action, 100-102 Replica plating 626 unidirectional, 201,216,217 structure, 100 Replicase see Polymerase, RNA, Replicon 222-7, 231 assay, 102 RNA -dependent Replisome 226 RNase BN, 493 Replication Replitase 144, 177 RNase D, 493 bidirectional, 201,222,223 Repression RNase E, 487 chromatin, 236-8 catabolite, 385-7 RNase H, 107,207,216,217,626 competition with transcription, 230, loop, 387-8 RN ase H activity of reverse 231 trp operon, 385 transcriptase, 107, 180 complex, 216,226 Repressor RNase L, 105, 575 conservative, 154 ara, 387-9 RNase M5, 487-9 continuous, 162 bio, 389, 394 RNase P, 101, 106,472-3,487, direction of chain growth, 159 complex with corepressor, 385, 389 493,540 discontinuous, 158 dimer, 391,393-5 RNase Phy I, II, M, 105-6,615 dispersive, 154 DNA binding, 384-5, 392-4 RNase Q, 493 factor, 212 haem-controlled (HCR), 577 RNase S, 100 fidelity, 190-2 isolation, 384-92 RNase T, 493,615 fork, 155, 157 lac, 383-5,394,526 RNase T1, 105 initiation, 201-220 lambda (cl protein), 391-3,431, RNase T2, 105 adenovirus, 218-9 535 RNase U2, 105,615 ColE1,216-7 met, 389, 394 RNase Y, 493 complex systems, 222-7 protein, 383 Type I, 103-4 control, 219,220 trp, 385, 393-4, 535 Type II, 104 dhfr gene, 224 Resolvase 297, 298, 299 Ribonucleoprotein E. coli, 205-8 Restriction endonuclease 117-21, heterogeneous (hnRNP), 465 matrix attachment, 224-6 318,609,611,617-8,621 ribosome precursor, 492-3 mitochondria, 217-8 Restriction fragment length small nuclear (snRNP), 460-66 oriC, 205-8 polymorphism (RFLP) 46, 605 U-series snRNA-containing origin location, 201, 225 Restriction mapping 120, 617-8 (snurps), 460-66 Index 671

Ribonucleotide reductase 141-3,259 Xenopus, 227,327-8 P-site, (peptidyl site), 515-6, gene amplification, 143 Ribosomal proteins 549-50, 552-3, 535-6, 538-9, 540, 556, 55S Lactobacillus, 141 55,557 R-site (recognition site), 539 Ribose archaebacterial, 561 structure, 548-5S chemical reactions, 32 chloroplast, 333, 563 subunits, 492-3, 534-43, 547,548- methylation, 478-9, 492, 550 mitochondrial, 562 55,558,561,577,57S,5S3 structure, 6, 7 eukaryotic, 550,553,578-9,583 tunnel,54S Ribosomal DNA see Ribosomal RNA genes,322,463,526 Ribosylthymine 495 genes LI,573 Ribosyltransferase 126 Ribosomal RNA (rRNA) L2,554,557 Ribozyme 3, 466, 557 2S,550 L3,554 Ribulose- I ,5 bisphosphate 5S,324,329,549,550,552,554, L4,554 carboxylase-oxygenase 333, 562,563 L5,554 421 5.8S, 325-6, 489-92, 550, 552 L6,552,554 Ricin647 16S,324,535,542,549-50,554, L7nL12,552,553,555,561,57S Rifampicin 205,341,344,347 558,561,563,572 L10,552,553,555,561,573 RF, RF-1, RF-2, RF-3, RRF see 18S,324,489-92,543,549-50,561 LII, 555 Termination of protein 23S,324,549-50,552,554,555-6, LIZ, see L7/L12 biosynthesis 558,572 L13, 554 RNA 285,324,489-92,549-50,552 LI6, 554, 557 2S RNA see Ribosomal RNA, 2S chloroplast, 4.5S, 550 LIS, 554 4.5S RNA, 565 genes see Ribosomal RNA genes L20,554 5S RNA see Ribosomal RNA, 5S mitochondrial, 562-3 L23,554 5.SS RNA see Ribosomal RNA, modified nucleotides, 550 L24,554 5.8S precursors, 466-71, 486-93 L25,554 7.1S RNA, 456 processing, 466-71, 486-93 L27,557 7.2SRNA,456 structure, 552 see also secondary L35, 553 7.3S RNA, 456 structure, RNA L36,553 7LRNA,456 U-series see small nuclear RNA mRNA,571-3 7S RNA, 306,456,564-5 Ribosomal RNA genes RNA-binding, 554, 571-3 processed pseudogenes, 308 5SrRNA SI,536,553,5S3 7SKRNA,456 chromosomal location, 329 S3,553 7SLRNA,456 eukaryotic, 329, 355-60 54,554,560,571,573 7SMRNA,456 heterogeneity, 330,360 S5,553 16S RNA see Ribosomal RNA, 16S multiple, 322 S6 (prokaryotic), 553 ISS RNA see Ribosomal RNA, ISS oocyte, 329,360 S6 (mammalian), 579 23S RNA see Ribosomal RNA, 23S prokaryotic, 329 S7,554,571 2SS RNA see Ribosomal RNA, 2SS promoters, 355-60 SS,554,571,573 alkaline hydrolysis, 35,593,563 repeating unit, 328-9 510,553 antisense, 210, 217, 300,438-9 somatic, 329-60 S12, 559-60 biosynthesis see Transcription 16S, ISS, 28S and 23S rRNA S14,553 catalytic amplification, 227, 325-6 SI5,554 group I introns, 466-7 chloroplast, 333-4 516,553 group II introns, 471-2 Drosophila, 326-8 S17,553,554 Ml RNA, 472, 493 enhancers, 361-2 S19,553 mitochondrial, 215, 217 eukaryotic, 325-9 520,552,554 RNase P, 472-3,493 extrachromosomal, 328-9 S21,526,55S rRNA,557 inserts, 328 S27a (mammalian), 583 satellite RNA, 473 intergenic spacer, 326-8,360-61 synthesis, 492,571-3 telomerase, 234-6, 473-4 introns, 328 Ribosome use as enzymes, 470-471 mitochondrial, 333-4 archaebacterial, 561 virusoids, 88, 473 multiple, 322, 324-9 binding site see Shine and Dalgarno depurination, 593 palindromic, 327-8 sequence double-stranded, 30,575-8 promoter, 360-63 chloroplast, 333,563 editing, see Editing, RNA prokaryotic, 324-5 mitochondrial, 333, 562 elongation see Elongation of RNA replication, 230 pausing, 396-8,581-2 genomes (viral), 371-4 sequence, 328 precursor, 492-3 guide, 469-70, 497-S spacers, 324-9 purification, 602 hairpin loop, 30, 348, 468, 472, 473, transcriptional termination, 325 sites 485,492,552,558 Tetrahymena, 227, 327-8 A-site (aminoacyl site), 515-6, hammerhead structure, 473 transcription units, 230, 326 53S-9, 556, 582 hnRNA, 453-4 unusual arrangements, 327-8 E-sites (exit sites), 539-40 isolation, 602-4 672 Index

kinase, 124-6 rate, 596 Small nuclear ribonucleoprotein ligase, in tRNA splicing, 475-8 see also Ultracentrifugation (snRNP) Ml,472-93 Segmented genome 81 in 3'-processing, 364,485 messenger see Messenger RNA Selanocysteine 518,580-1 in pre-rRNA processing, 490 methylation, 124,491-2,495-6 Selection, of DNA recombinants 621, in spliceosome formation, 460-66 see also Ribosomal RNA, 622,630,637-8,639 Ul,459 methylation, etc. Semiconservative replication 153-5 U2,460-62 nascent, 350, 364 Sequence determination U3,490 nucleolar see Nucleolar RNA DNA, 609-615 U4/U6, 460-61 polymerase see Polymerase, RNA genomic, 434, 611-2 U5,460 pseudoknot, 32 RNA,615 U7, 364,485 ribosomal see Ribosomal RNA Sex determination, Drosophila 503-4 Sodium dodecyl sulphate 108, 599 secondary structure, 29-32,329, Shiga toxin 647 So[ mutants 158 462,468,472,473,475,528-9, Shine and Dalgarno sequence Solenoid 61 535,543,546,552,556,569- base-pairing, 535 Somatic 71,573,581 chloroplasts, 563 cell genetics, 45 sequence determination, 615 cloning vectors, 628, 636 mutation, 289 SL RNA, 464-5 eukaryotes, 543 SOS functions 270, 391 small nuclear see Small nuclear mitochondria, 562 Southern blotting 605 RNA mRNA, 452,535-6,581-2 Spacer 323,326-9,360-61 synthesis see Transcription 16S rRNA, 535-6,558 Sparsomycin 542, 547 tertiary structure, 521, 530, 552, Shorthand notation 10-11 Specificity factor 482-4 558,573,585 Shortpatch repair 266, 267 Spectinomycin 647 transfer, see Transfer RNA Shuttle vectors 638-9 Spine of hydration 16 transport, 505 Sigma factor 340-46 Spleen phosphodiesterase see triple-stranded regions, 31 expression, 526 Phosphodiesterase U-series see Small nuclear RNA variants, 402-6 Spliceosome 319, 459-64 VAl RNA, 359,578 Signal Splicing 318 world,585 peptide, 564-6 alternative, 499-504 see also Ribosomal RNA etc. peptidase, 564-5, 566 autocatalytic, 320, 466-72 RNasin 108 recognition particle (SRP), 564-5 branch site, 459-63 Rolling circle 20-5,233 recognition particle receptor, 564 auxiliary factors, 460, 465 Rous sarcoma virus 88,303,581 Simian virus 40 (SV 40) consensussequence,458,462-3 Run-off, nuclear 643 alternative splicing of mRNA, 500 control, 499-504 Run-on, nuclear 643 DNA isolation, 599 cryptic, 458 enhancer,639 differential, 499-504 enhancer-promoter complex, 411- donor sites, 458 Sl nuclease see Nuclease Sl 3 fungi, 463-4 Salvage pathway 138, 140, 145 genome structure, 85 in vitro, 459,475 Sanger sequencing method 3, 613-5, minichromosome, 59,83 matrix, role of, 465-6 624 regulatory element, 411-3 maturases, 470-71 a-Sarcin 556, 647 replication initiation, 210-3,639 mitochondrial genes, 471 Sarcoma virus see Moloney murine T-antigen, 500 models, 460-61 sarcoma virus; Rous sarcoma vectors, 638-9 plant, 464 virus; Avian sarcoma virus Sinbis virus 545, 579, 580 pre-rRNA, 466-71 Satellite SINEs 50-2,293,303,305-6,308 pre-tRNA, 475-8 DNA, 48, 293, 600 Single-strand invasion 274-7 sites on pre-mRNA, 457-8 RNA,473 Single-stranded DNA binding protein snRNA, role of, 459-65 Satellite virus 81, 88 seeSSB summary of mechanisms, 476 Scaffold attachment sequence (SAR) Site-directed mutagenesis 641 trans, 464-5 64,367,437 Sleeping sickness 282 yeast, 463-4 Scanning model543-6 Small nuclear RNA (snRNA) 455-6 Sporulation 402-5 Screening, of clones 621,625-6,628 Ul,459 SSB (single-stranded DNA binding Secondary structure U2,459-63 protein) 183 DNA, 14-22 U4-Ul4, 364, 459, 460, 490 Stacking interaction 17, 521, 529, 530, RNA, 329, 462, 468, 472, 473, 475, U7,485 537 528-9,535,543,546,552,556, caps,459,465 Stem-loop structures 27-31,347, 569-71,573,581 genes,359-60,364 348,528-9,570,578,581 Secretion, protein 564-6 in nucleolus, 490 Steroid hormone Sedimentation processed pseudogenes, 308 gene expression, induced by, 418- coefficient, 325, 596-7 3' processing, 485 21 equilibrium, 24-6 splicing, 459-66 receptor proteins, 419-20,428-30 Index 673

Strand displacement 164, 166, 218-9 factor-independent, 325, 348-9, Tissue-specific gene expression 413-8 Streptolydigin 341, 347 396 TMV see, Tobacco mosaic virus Streptomycin 558-60 identification, 348, 363 Tn transposon see Transposable Streptovaricin 347 lambda bacteriophage, 399-402 elements Stringent response 582-3 NusA and, 350 Tobacco mosaic virus (TMV) 74, 78, Subcellular components, separation pausing of RNA polymerase, 579-80,584 of602 348,350 Topoisomerase 64, 185-9, 226, 231, Subtilisin action on E. coli DNA premature, 347 347,433 polymerase I 169 prokaryotic, 347-50 Toluene catabolism 406 Sucrose density gradient rate, 348 Torsion angle 15-17 centrifugation 602, 603 recognition site, 348, 349, 363, Touch-activated genes 421 Sugar-phosphate backbone of DNA 364 tra gene 72, 184 17 regulation, 396-402 Trans-acting factor 339 Sulphur mustard 260 rho factor-dependent, 349-50, Trans-splicing 464-5 Supercoil26, 344,347,601 399,400,407 Transcribed spacer 326-328, 360-61, Superhelix density 27, 601 RNA polymerase I, 365-6 489-91 Suppression 302, 518, 542, 560, 579- RNA polymerase II, 363-5 Transcription 81 RNA polymerase III, 365 5S RNA, 355-60 SV 40 see Simian Virus 40 vaccinia, 370 chloroplast genes, 368-9 Switch sequences 288, 289 translation cloned genes, 352 Syn conformation 17,235,268 codons,518,524,540-2,546 competition with replication, 230-1 Synaptonemal complex 270 eukaryotic, 547-8 complex, 354-5,358-9 Synchronized cells 43, 143 factors (RFs), 524,540-2,547, control, 381-439 Synthetic oligonucleotide see 581-2 definition, 339 Oligonucleotide mitochondrial, 525 elongation, 346-7 prokaryotic, 540-2 see also Elongation factor see also Supression factor T-antigen 84, 87,211-3,412,500 Ternary complex acidic domains, 431-2 T-cell receptor genes 28, 286 transcriptional, 346 activation domains, 431-2 T-even phage structure 74 translational, 536,538-9,543,547, API family, 415,430 T phage see Bacteriophage 560 associated terms, 339-40, 408 Taq polymerase see DNA polymerase Tertiary structure of RNA 521, 530, conventions, 339-40 Targetting, of proteins 563-9 552,558,573,585 CRE-binding, 421, 427 TATA box 322,353-5,359 Tetracycline 647 dimerization, 426-7,430 Tautomerization of bases 5, 257 Tetrahymena DNA-binding domains, 392-6, Telomerase 245, 235, 473-4, 585 macronucleus, 283, 326 424,427-32 Telomere 307,584-5 micronucleus, 283, 326 E1A,427 Temperature-sensitive mutant 79 mitochondrial tRNAs, 563 erythroid-specific, 415-7 Template 163 palindromic rRNA, 327-8 ligand-binding, 419-20 Temporal-specific gene expression rONA, 65-6, 227 negative, 411,415,418 413-8 rRNA amplification, 326 Oct, 418 Teniposide 189 splicing of rRNA, 466-70 oncogenic, 425-7 ter sequence 231 telomere, 234 phosphorylation, 421 Terminal protein (of adenovirus) 218, Tetranucleotide hypothesis 2 positive, 415 219 Tetraploid cell 43 silencing, 416 Terminal repetition 218,231-3,296, Tetraloop 31 SL, 362 303 TFI, II, III see Transcription factor Sp1, 432,434 see also Repeated DNA Thalassaemia 363, 413, 458, 481 steroid-inducible, 419-21,428- Terminal transferase 179,288,609 Theta (9) structure 202, 203 30 Terminase 233 Thioredoxin 142 TFI, 362 Termination Thiostrepton 555, 556 TFII, 354-5 replication, 230-6 4-Thiouridine 495 TFIII, 355-60, 437 cyclic chromosomes, 231 Thymidine tissue-specific, 362,415,418 linear chromosomes, 231-3 excess, 143 UBF,362 telomeres, 234-6 structure, 8 viral, 370 transcription, 363-6 Thymidine kinase 144, 145 in vitro, 352, 356, 355-63 anti-termination, 350, 399-402 Thymidylate synthetase 144 initiation, 341-6, 355-63 attenuation, 385, 396-9 Thymine see also Initiation consensus sequence, 363-6 biosynthesis, 143, 144 mitochondrial genes, 368 eukaryotic, 363-6 dimer, 263 start site, 340, 344, 353 factors, 349, 364-6 structure, 6 termination, 347-50,363-6 factor-dependent, 349-50, 399 Thyroxin 418, 420 see also Termination 674 Index

tRNA,358-9 Transformylase 519,543 Unwinding protein see Helicase unit, 339 Transgenic mice 639-40 Upstream viral genes, 369-74 Transition temperature see Melting activating sequence, 408, 414 Xenopus oocytes, 352-6 temperature definition, 340 Transcription/translation unit 451 Translation see Protein biosynthesis promoter element, 353-4, 359, 408 Transcriptional element see Translocation Uracil N-glycosylase 159 Enhancon of peptidyl-tRNA from A- toP• Uracil structure 6 Transduction 85, 86 site, 539,547,556 Uric acid 146 Transfer (tra) genes 72 of proteins to subcellular Uricase 147 Transfer RNA (tRNA) compartments, 563-9 Uri dine aminoacylation, 531-4 Transplantation antigen 87 insertion and deletion, 496-8 11-aminolevulinate synthesis, 584 Transposable elements 296-305 structure, 8 analagous structures in plant Activator, 301-2 Uridylate transferase 497 viruses, 584 Cin4, 305 UV (ultraviolet) radiation 263,391, anticodon, 475,515,518,519-524, Copia, 302-3, 304, 307 604,617,634,638 525, 526, 528-30, 531-4, 536, eukaryotic, 301-3 uvrABC endonuclease 266 537,539,543,549,558,560, I element, 304-5 563,580-1 ingi3, 305 Vaccinia virus archaebacterial, 561 IS element, 296-7,299,438-9 base composition, 26 attenuation and, 396-8 Mu, 297, 298-9 genes,370 base modification, 5-6, 495-6, P factor (P element), 301,500 promoters, 370 521,522,523,528-9,532,538, prokaryotic, 296-301 RNA polymerase, 370 562,580,581 retronphage cpR73, 305 transcription, 370 CCA terminus, 495,528-9,538, Tn transposons, 73, 296-7, 298, translation of mRNA, 578 558,585 299-300, 438-9 vectors, 576 chloroplast, 368, 563 Ty,302-3,304,307 VAI RNA 359, 578 determinants of identity, 532-4 Transposase 296-8, 299-300, 301, Vanadyl-ribonucleoside, RNase E. coli tRNA 01", 532-4 302,438-9 inhibition by 108 E. coli tRNAtt"', 518, 530, 537-8 Transposition 295-308 Variable number tandem repeat E. coli tRNA~"', 518 intron, 319, 472 (VNTR)49 eukaryotic tRNA'(I•', 543 mechanism (prokaryotic), 297-9 Variant surface glycoprotein (VSG) genes regulation, 299-301 282,283 chloroplast, 334-5 see also Transposable element Vector clusters, 330-1, 334-5 Transposon see Transposable element bacteriophage lambda, 624-30 eukaryotic, 330-31 Trimethoprim 647 bacteriophage Ml3, 623-4 mitochondrial, 334-5,368 Tritium exchange 22 bacteriophage Pl, 630 multiple copy, 322 Tropomyosin 502 baculovirus, 637 prokaryotic, 330 tRNA guanine ribosyl transferase 126 CAT,638-9 promoters, 358-9" tRNA guanine transglycosylase 126 cosmid,630 tandem repeats, 331 tRNA nucleotidyltransferase 584 EMBL 3, 629-30 iso-accepting, 526 trp operon see Operon, trp J..gt10, 626-8 mitochondrial, 522-4,562-3 Trypanosome 234, 282, 283, 464-5, J..gtll, 628 peptidylglycan synthesis, 584 496-8 pBR322, 73, 622 precursors, 475-8, 493-6 Tubulin, control of synthesis 576 pET,636-7 promoter, 357-9 Tumour antigen 84, 87 pHC79,630 primer for reverse transcriptase, Tumour virus 87, 425-7 pKK 233-2, 636 180,220-1,374,584 see also Retrovirus; individual plasmid, 73, 622-3 processing see Pre-tRNA viruses pSC101, 73 purification, 602 Tus231 pUC18, 622, 624, 626 redundant, 518 Twist27 pUEX,637 structure, 29, 30,358,527-30 Two-out-of-three hypothesis 524 pYAC4,631 suppressor,518,542,560,580,6J1 Ty element 302-3, 304, 307 shuttle, 638-9 Tetrahymena, 563 SV40, 638-9 yeast tRNAA•p, 530,533-4 Ubiquitin 435, 583, 584 vaccinia virus, 576 yeast tRNAPhc, 521,529-30, 533, Ultracentrifugation Venom phosphodiesterase see 563 isopycnic, 598-9,602 Phosphodiesterase Transfection 86, 621, 625 rate-zonal, 598, 602 Very short patch repair 268 Transferrin receptor, control of Ultraviolet radiation see UV radiation Vesicular stomatitis virus 373 synthesis 573 Umber mutant and codon 518 Vinblastine 644 Transformation Unassigned reading frame 66 Vincristine 644 animal cell, 71, 87, 639 ung mutants 159 Virion proteins 84 bacterial, 69,621,622,624,629 Unique DNA 48 Viroid473 Index 675

Virus VP16189 Xanthine, structure 7 adsorption, 76 Vpg peptide 371 Xanthosine monophosphate (XMP) animal, 77 VSG see Variant surface glycoprotein 146 assembly, 77 Xenopus bacterial see Bacteriophage Wandering-spot sequencing 609 egg, 222-5, 226 classification, 75 Wild-type68 oocyte, 352, 571 coat, 74 Wobble 519-24,526,529,562-3 oocyte transcription system, 352, DNA, 75 Writhe27 356,638 DNA modification, 117, 121,215-6 Wybutosine 4, 6, 496,521 oocyte translation system, 643, 645 early and late function, 84,369, Wyosine see Wybutosine pattern of DNA interspersion, 52 400,405,406,412 rONA, 326-8 early proteins, 77, 369, 400, 405, X-chromosome Xylene catabolism 406 406,412 dosage compensation, 503 evolution, 317 inactivation, 123 Y-RNA465 life cycle, 76, 399-402 X-gal (5-bromo-4-chloro-3-indolyl- Y AC see Yeast artificial chromosome mutants, 79 beta-D-galactoside) 623 Yeast nucleic acid, 79 Xis279 artificial chromosome (Y A C), 45, oncogenic, 425-7 X-irradiation 262 631 packaging, 77 X-ray budding,44 penetration, 76 crystallography mating type, 280-2 peptides, 84 aminoacyl-tRNA synthetase, plasmid, 224-5 release, 75 532-4 pre-mRNA splicing, 463 replication, 371-4 chromatin, 58 rDNA,230 RNA, 75,371-4 DNA-protein complex, 384, structure, 73-5 393,424,428,429,623 Z-DNA21 tail fibre, 74 EF-Tu, 538 Zimm viscometer 597 transcription, 369-70 e1ongator tRNA, 521,529-30, Zinc tumour, 84, 87,374,425-7 534 binding domains, 358, 428-30 tumour antigens, 84 initiator tRNA, 530, 536 finger, 357-8,420,428-30 see also Bacteriophage; individual oligodeoxynucleotide, 19-21, factor TFIIIA, 358, 428 entries, e.g. Herpes virus 523 gal4 proteins, 428, 430 Virusoid 88, 473 ribosomal protein, 553 steriod hormone receptor, 420, Viscoelastic relaxation 597 transcription factor, 424 428-30 Vm26!89 fibre diffraction of DNA, 14, 18,61 RNA polymerase and, 340,351 VNTR see Variable number tandem scattering, 548 Zipper,leucine 430-31,426 repeat Xanthine oxidase 146