Denaturation map of bacteriophage T7 DNA and direction of DNA transcription

J. Nol. Bid. (1972) 70,239-251

Denaturation

Map of Bacteriophage T7 DNA and Direction of DNA Transcription BEATEI~ G~MEZ~ AIND D. tia Divihn of Biology, University of Team P.O. Box 30365, Dcdh, Tems 75230, U.S.A. (Received 1 Februay

1972)

After partial den&u&ion of bacteriophage T7 DNA by heat or alkctli, electron microscopy showed preferential strand separation at A + T-rich sitea which map asymmetrically et the following fractione3. lengths: 0.01, 0.16, 0.21, O-26, 0.33, O-42, 0.46,0*67, 0+33,0~66,0~86,0~91 and 0.99. This map physically distinguishes between the two molecular ends of T7 DNA. In uitro synthesis of RNA by Escheridie c& RNA-polymemse with T7 DNA as a template and subsequent partial heat den&u&ion of the complexed DNA permitted simultaneous localization of RNA-synthesis sites and of strand-separation sites on individual moleoules. Since the enzyme is known to initiate olose to the genetically defined left-end of T7 DNA, it ~88 found that the fraotional length 0 of the den&n&ion map is equivalent to the left-end of the genetic mep and that at least 90% of RNA synthesis is initiated cloee to the left-end of T7 DNA.

1. Introduction Transoription of T7 DNA in vitro starts at a specific initiation site close to one end of the DNA molecule and proceeds toward the other end, as shown by electron microscopy of the transcription complex (Davis t Hymen, 1970). Similar experiments indicated that in our hands some additional initiation sites, possibly of artificial origin, were present (Lang, 1971). It could not be decided by electron microsoopy alone, whether transcription always starta from the same molecular end or from either end. An independent distinotion between the two ends was needed to distinguish between these possibilities, and also to serve as reference for future experiments by providing a physical definition of “left” and ‘kight”. The two ends of a linear DNA molecule can be distinguished by electron mioroscopy if there is an observable and consistent difFerence or asymmetry between the two halves. The nucleotide sequence is asymmetric but cannot be seen. However, after partial denaturation of duplex DNA by heat or alkali, sites of strand separation along DNA molecules which are rich in adenine * thymine (A . T) nuoleotide pairs, have been seen and their positions mapped according to Inman, 1966,1967; Follett & Crawford, 1967,1968; Bourguignon, 1968; Inman & Bertani, 1969; Inman & S&n&, 1970; Doerkler & Kleinschmidt, 1970; S&n% & Inman, 1970,1971; Wensink t Brown, 1971). In all cues, the map of A + T-rich sites was found to be asymmetric, thus permitting distinction between the two terminal regions. t Present eddress: Department Eoologia Humene, Faculted de Medioine, Universidad Neaional Autonoms de Mexioo, Mexioo 20. D.F. 239

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Some difficulties in constructing the deaeturation map of T7 DNA were expected. Its nucleotide sequence is unique (Ritchie, Thomas, MacHattie & Wensink, 1967) but the melting curve is one of the steepest known for natuml DNA’s (Crothers, 1968). This signifies that there are only small fluctuations in A + T density along the DNA molecule. In contrast, bacteriophage X DNA has a broad melting curve and since the sites denaturing at the lower temperatures appear within the same half of the DNA (Inman, 1966), there is no orientation problem for pattern recognition. It was of interest to learn whether a pattern in T7 DNA could be found at all. If so, denaturation analysis would probably be possible with any natural duplex DNA. Indeed, a deneturation pattern for T7 DNA was detected which is asymmetric and characteristic, as shown below. The next experiment w&sto synthesize RNA using Escherichia coli RNA polymerase with T7 DNA as a template, followed by interruption of synthesis and partial heatdenaturation of the DNA. Subsequent electron microscopy revealed the positions of both the transcription site and A + T-rich sites along individual DNA molecules. A simple comparison with the previously established denaturation map showed that at least 99% of the observed in vitro transcription starts from the same molecular end.

2. Materials and Methods (a) DNA

T7M bacteriophage, a gift of I. Rubenstein and originally obtained from A. D. Hershey, wss grown in liquid cultures of E. coli B. Final purification was carried out by CsCl density-gredient equilibrium centrifugation. The DNA wss extracted twice with redistilled phenol; it was dialyzed against and stored in 0.01 M-N~H,PO~ buffer, containing 0.001 MEDTA and adjusted to pH 7-8 with NeOH. Additional DNA’s, used for hyperohromicity measurements, were also purified by phenol extraction: calf thymus DNA (Worthington) and DNA from bacteriophages h and TS. curves (b) Mel&g Four stoppered l-cm quartz cuvettes were used, two of which contained fractions of the DNA in question, one contained a different DNA with minimal 6ne structure in its melting curve suoh as oalf thymus DNA and the fourth ouvette contained guauine and thymine nuoleotides as a reference (Szybalski L Mennigmenn, 1962). Phosphate buffer (see section (a) above) and optical density (about 0.7, measured at room temperature and after evacuation for degassing) was equal in all cuvettes. Optical densities were automatically measured at 3 cyoles/min with a Beckman DU-Gilford 2000 recording spectrophotometer at 260 nm wavelength. Full scale deflection of the recorder pen was adjusted to an O.D. window of O-7 to 1.0. A linear temperature increase of 0.4 deg. C/mm was provided by a programmed ws,ter thermostat. Evaporation wss about O6o/o per 4 hr in all cuvettes. The printed melting curves were checked, point by point, using the following internal controls: (i) the two curves for the unknown DNA should be similer in every detail and (ii) an apparent fine structure in these two curves should be absent in the calf thymus DNA reference curve. These criteria indicate the lack of artifacts caused by random errors or by gas bubbles possibly generated during the run. For amplification of fine structure, melting curves were graphically differentiated (Falkow & Cowie, 1968) with the help of a halftransparent mirror, mounted perpendioular to the graph. (c) RNA Byntheais DNA-dependent RNA polymersse preparations from E. coli B, containing sigma but no rho factor, were gifts of Drs H. Bremer, K. Miiller L P. Witonsky. The enzyme activities were in the order of 10 nmoles ribonucleotides synthesized/ml./16 pg/Ei min in 0.2 ~-Kc1

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at excess DNA. 4 or 40 ~1. of 0.4 mg polymerase/ml. were added to a 0*4-ml. solution, pH 7.5, containing 200 mu-KCl, 5 mm-Mg a+, 2 maa-Mn2+, 50 mu-sodium cacodylate, 12 rnM-2-mercttptoethanol, 0.4 rnM each of nucleotide triphosphates ATP, GTP and UTP, and 12 w T7M DNA/ml. After 2 min incubation at 37°C for initiation of synthesis, 4 pg rifampicin (Mann) were added in some experiments (Results, section (c)) to inactivate polymerase which hed not yet initiated an RNA chain (Wehrli, Kntisel, S&mid L Staehelin, 1968). After 2 more min, addition of the fourth nucleotide, 0.4 rnx-CTP, allowed the synthesis of RNA chains to begin. The reaction was stopped by chilling and dilution at varying times between 1 and 15 min. At this stage grids were prepared for electron microscopy either directly or after partial denatumtion of the DNA in the transcription complex. (d) Partial denutwation of DNA Procedures were essentially those reported by Inman (1967) and S&n% L Inman (1970). (i) By heat To 0.21 ml. of the above reaction mixture containing the transcription complex were added, at O”C, 1.01 ml. of 0.01 M-phosphate buffer (pH 7*8),0*50 ml. dimethylsulfoxide (to reduce the melting temperature) and 0.28 ml. 37% formaldehyde (to stabilize strand separation). This solution was heated to about 49°C for partial denaturation, kept there for 6 min, and then shifted to 37°C for 25 min. For partial denatumtion of control DNA, the polymerase and the nucleotide triphosphates in the above O-21-ml. reaction mixture were replaced by their solvents. (ii) By al&i To 400 ml. of a stock solution cont&ning 0.05 M-NazCOe, 10% formaldehyde and 0.005 MEDTA were added 52 d. 1 N-NaOH to raise the pH to about 11. Then either 10 ~1 or 210 M T7M DNA/ml. solution or 1 ~1. (O-03 O.D .aeounit) of T7M bacteriophage suspension were added. Fractions were taken out at different times between 1 and 60 min and diluted without neutralization for immediate sampling by electron microscopy. Suitable preparations were usually found among samples taken at intermediate times. (e) Electron microscopy Specimen grids were prepared by the micro-version of the spontaneous adsorption method (Lang L Mitani, 1970). Droplets of 40 ~1. containing about 0.1 pg partiallydenatured free or transcribed DNA/ml., 0.15 M-rtmmOb~~~ acetake, 2 pg cytochrome c/ ml., and 0.07 M-formaldehyde, were deposited on a Teflon surface. After 10 to 15 min, carbon-coated grids (Siemens-type) were briefly touched face-down to the droplets, then touched for 10 set to ethanol, dried on filter paper and shadowed with platinum at a 7’ angle from two directions. Microgmphs on Kodak Electron Image plates obtained with 8 Siemens IA electron microscope at a magnification of 10,000 were projeoted and the DNA traced on paper. The position of synthesized RNA, appearing as “bushes” and the position and length of strand separations (eyes) along the DNA were determined with a map measurer. The smallest eyes clearly detectable were about 200 A long. The lengths of single-stranded portions of DNA were multiplied by a factor obtained from Fig. 2 of Bujard’s paper (1970) to correct for ionic-strength dependence of the linear density of single-stranded DNA. This factor is 1.39 et O-15 ionic strength and its application gives the length which the single strand would have when in a double-stranded form. With this correction, the partially denatured T7M DNA had a contour length of 12-4 pm & @6 pm as compared to the control of 12.15 pm & 0.25 pm for native untreated T7M DNA (Lang, 1970). The errors are sample standard deviations. The total length of each DNA molecule was then normalized to 1 and any positions along it were given in fractional lengths. Most preparations of the undenatured transcription complex were sampled for electron microscopy by the diffusion method in 0.15 x-ammonium aaetate (Lang, Bujard, Wolff & Russell, 1967).

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3. Results (a) iWelting of DNA The normalized melting curves shown in Figure 1 show that the melting transition of T7M DNA is steeper than that of other DNA’s. All curves have a reproducible fine structure which is amplified by differentiation as shown for T7M DNA in Figure 2 Fine structure may be expected to indicate local fluctuations in A + T composition

FIQ. 1. Normalized melting curves for phenol-extra&ad DNA from bacteriophages h, T5, T7M and from calf thymus cells. Plotted is h, the percentage of total hyperchromioity, ver%w) the differenoe between temperature (T) and melting temperature (T,,,, at h = 0.6). AU T, values were similar, between 69 and 75°C. -O-m-, h DNA; -Q-n--, calf thymua DNA; -O-O--, TS DNA; -m-m--, T’7 DNA. G+C(%) 30,

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FIG. 2. Graphio differentiation of the T7M DNA melting curve gives dh/dT (-O--O--). showing st least two fractions (nos 3 and 4, . . . .) centered around 43 and 48% G + C content. The original melting curve (h) ia also indiceted (- . . . -) in arbitrary ordinate units. The G + C soale, upper abscissa, has been caloulated according to Owen, Hill & Lapage (1969). based on an aver8ge G + C-content of T7 DNA of 43% (Wyatt & Cohen, 1963).

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whether the melting curve was obtained at equilibrium a linear increase of O-4 deg. C/minute as used.

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for each temperature or during

(b) Denuturation mp of T7M DNA After the experiments with partial heat denaturation, it became evident that eyes along T7 DNA molecules, even on the same specimen grid, varied with regard to number, position and length to such an extent that no two molecules, out of 84, were precisely congruent. This is probably a reflection of relatively small fluctuations of the local A + T density along the DNA as suggested by Figure 1. In addition, the distribution of eyes along the DNA was not sufficiently asymmetric to permit unambiguous orientation of all molecules for the detection of the denaturation map. The following was considered to overcome this difficulty. (i) A search by computer for the denaturation map which differs maximally from a random distribution of denatured sites among all possible left-right combinations was rejected because 84 molecules can be ordered in as many as 284 different ways. (ii) Denatured sites were marked on strips of paper representing DNA molecules. A denaturation map was constructed by trial and error. The quickest procedure is to find groups of molecules which have a few sites at similar positions when properly oriented and then to compare and orient these subgroups. Most of the remaining molecules may then easily be added in a best-fitting orientation. This procedure has been repeated three times with different subgroups, resulting in identical maps. (iii) Each position of a denaturation site, expressed as fractional length, x, of a molecule, was marked on graph paper twice, at x and at the mirror position 1 -x. The number distribution of these positions, which is symmetric with respect to x = 05, showed several peaks. The most significant peak was found at x = 0.16. About 50% of the molecules had a site at or close to this position. These molecules were then reoriented so that the site at x = O-16 was toward the same side in each case, and all the mirror-image sites were erased. This established an intermediate denaturation map to which the remaining molecules could be fitted. Procedures (ii) and (iii) led to the same map. Figures 3 and 4 represent oriented T7M DNA molecules after partial heat or alkali denaturation, respectively. These data were combined by dividing the abscissa in 200 intervals of 0906 fractional length each and countii all molecules showing strand separation in each interval. The resulting denaturation maps are shown in Figures 5 and 6. The following oan be concluded from Figures 3 to 6. (i) It is possible to detect a consistent denaturation map of T7M DNA. Denaturation by controlled heat or alkali treatment result in essentially similar maps. Differences in fine structure are probably not signiiicant for the reasons mentioned at the beginning of section (b) above. (ii) Both maps are asymmetric with respect to fractional length 05 and show major A + T-rich sites at fractional lengths O-01, O-16, O-21, O-26, O-26, O-33, O-42, 0.46, 0.57,0*63,0*66,0-86,0*91 and O-99, as briefly reported earlier (Lang, 1971,15th Annual MeetinglBiophysical Society, 1971. Abstracts, p. 220a). Only major peaks have been listed because in both cases (Figs 3 and 4) the orientation of 6 molecules happened to be equivocal. (iii) The fluctuation of the position of a site around its average is larger than expected from experimental fluctuations in molecular length. This was found also for DNA from adenovirus type 2 by Doerfler t Kleinschmidt (1970). (iv) It appears that pattern recognition is easier after alkali treatment than after

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FIGS 3 and 4. Colleation of T7M DNA moleoulee after perti den&u&ion, in the preaenae of formaldehyde. by heat (Fig. 3, 84 moleoules) and by alkali (Fig. 4, 103 moleculee). Each line represents one moleoule, normalized to a fractional length 1. Sites of strand separation are indioated aa boxes or termine 1 forks. The number of sites per moleoule inoreaees from bottom to top; the averages are 3.9 (Fig. 3) and 10.4 (Fig. 4), reepeotively. The overage length appearing denatured per moleoule is 6.2% (Fig. 3) and 16.5% (Fig. 4). The left-right orientations shown hnve been ohosen aa desoribed in Results, e&ion (b).

heating, although the patterns are not strictly comparable since the DNA solvents and the average number of denatured sites per molecule differ, being 3.9 and 10.4 in Figurea 3 and 4, respectively. (c) The transcription complex As a result of in vitro synthesis of RNA in O-2 M-KC1 by DNA-dependent RNA polymerase along a T7M DNA template, “bushes” can be seen attached to the DNA. The shape of a bush is irregular and its size very difficult to measure, but it seems that the size increases with synthesis time. A bush is evidently RNA by the following

0.5 Fmctionol length Fm.5. 1

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FIG.& Fros 5 and 6. Mapa showing sites of partial den&w&ion from Fig. 3) and by alknli (Fig. 6, obtained from Fig. 4).

of T7M DNA by heat (Fig, 5, obtained

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criteria : bushes do not appear in the absence of either Mg2 + , or the four nucleotides, or CTP alone, or polymerase, nor after incubation with ribonuclease. When rifampicin was added, after initiation of RNA synthesis but before adding the fourth nuoleotide (CTP), then a bush contains probably only one RNA strand and one polymerase molecule. Two-thirds of all bushes appeared to be attached to DNA and the rest to be free, independent of the polymerase to DNA ratio. Figure 7 shows that the distribu-

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Fro. 7. Distribution of the number of bushes found attached per T7M DNA molecule after 5 min RNA synthesis in the presence of rifampiain. The average for the 422 DNA mole&es aounted is 0.974 bu&/moleoule. The measured distribution (oircles) is nearly identioal with a Poisson distribution (broken line) for the came average.

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Fro. 8. Distribution of the position Fig. 7) as meanare d by the frequency the nearer DNA end. Only moleoules (solid linee; 102 moleoules) and 6 min

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of a bush with T7M DNA moleoules (same preparation as in of observed fractional contour length8 between 8 bush and with one bush were used. RNA synthesis times were 1 min (broken lines: 136 moleoules).

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tion of bushes among different DNA molecules (circles), with an average of 0.974 bush per DNA molecule, closely resembles a Poisson distribution (broken line) around the same average. This suggests random selection of identical DNA molecules for initiation of RNA synthesis and also that one-third of the complexes had dissociated at random. In contrast, the position of a bush along an individual DNA molecule was non-random (Lang, 1971). The distribution of the distances from a bush to the nearer end of the DNA molecule to which it was attached showed a maximum which shifts toward the center of the DNA molecule when the synthesis time is increased from 1 to 15 minutes, con6rming results obtained by Davis & Hyman (1970). An example is shown in Figure 8. The average RNA chain growth-rate is estimated to be 23 nucleotides per second per polymerase moleoule from the shift of average position with time. The heterogeneity of the position of a bush along a DNA molecule was consideraMe in some preparations, even after only 1 minute synthesis: 67% of the bushes were attached close to a DNA end, within one-tenth of the total

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Fro. 9. Collection of 96 T7M DNA molecules after 6 min of in v&o RNA syntheeia (without rifampiciu) and subsequent partial heat denaturation. Attachment sitas of bushen (RNA) are indicated by dote, denaturetion sites by boxes or forks. In this case, the molecules heve been oriented such th& ell bushes 8p~ on the wme aide. The avemge number of den&m-&ion aitea per molecule is 6.8.

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DNA length, but the remaining 33% were found elsewhere. This and the fact that about 25% of the T7M DNA molecules had more than one bush attached at the high polymerase concentration seem to indicate that specific or non-specific starting sites are present in our system, in addition to the specific site close to a DNA end. Singlestrand breaks in DNA could possibly create such non-specific starting sites. Omission of rifampicin resulted in larger bushes (Plate I), indicating that multiple transcription had occurred. (d) Correlation of transcription and denaturation map RNA was synthesized for 5 minutes in the absence of rifampicin as described in Materials and Methods, section (c), and the DNA was partially denatured by heating the reaction mixture to about 49°C. This heating apparently did not change the morphology of the bushes. Micrographs were then made of all complexes which showed denaturation sites and, on the same DNA molecule, one or more bushes attached unambiguously to one DNA half. Plate I is an example. Figure 9 shows the collected and normalized molecules which, in this case, were oriented in such a way that all bushes are on one side. The next step was to compare the denaturation pattern as obtained from Figure 9 with the denaturation map established for free DNA as depicted in Figures 5 and 6. The result is that the denaturation pattern of DNA in the transoription complex oriented with all bushes on one side does fit the denaturation map in the orientation shown in Figure 10. However, 10 out of the 95 molecules in Figure 9 would fit the denaturation pattern equally well or better if inverted. The conolusion is, therefore, that the synthesis of at least 90% of the observed RNA molecules originated at a specific initiation site close to one particular DNA end which is characterized by a major denaturation site at a fractional length of O-16, measured from this end.

4. Discussion The evidence leading to the conclusion that sites of strand separation in partially denatured DNA represent A + T-rich sequences, as well as a detailed discussion, have been given by Inman (1966). These conclusions apply equally to T7 DNA, except for the additional fact that the proper orientation, necessary in constructing a denaturation map from these molecules, is not obvious. This is due, on one hand, to the failme of denaturation sites to appear preferentially in one-half of the molecule. On the other hand, the number and position of these sites fluctuate considerably among molecules taken from a single preparation. The question therefore arises as to whether the positions of denaturation sites are unique or random. In this work, several different approaches converged to essentially the same map, suggesting its uniqueness. In addition, one can show by analogy that construction of a map from a random distribution of events is nearly impossible. Using a table of 100 lines of random digits, each line of 100 digits representing one molecule, the positions of groups 00, 11 and 22 were marked; on the average there were about 3 such events per line. The resulting map showing random fluctuation was then refined by selecting the highest three peaks and orienting as many lines as possible to fit the position of these three peaks. After 50 lines were incorporated in this manner, the map apparently showed non-randomness which, however, disappeared after trying to fit the remaining 50 lines. This is rather trivial, but illustrates the requirement that in constructing a denaturation map

PLATE I. A bacteriophage T7M DNA molecule after in vilro synthesis of RNA and subsequent partial heat denaturation. The RNA synthesis site, upper right-hand comer, and a few small sites of strand separation (arrows) can be located in this way on the same DNA molecule. Magnificat,ion 43,500; t,ho bar represents 1 pm.

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b) I

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FIG. 10. (a) Mapped sites of partial heat den&m&ion obtained after orienting all molecules with their ettihed buehes on one side 88 drawn in Fig. 9. Denaturation between 0 and O-2 fractional length is under-represented because some sites were obscured by bushes. The inset shows the distribution of bushes. (b) The denaturation map of Fig. 6, with inverted ordinata and oriented aa shown, fits the upper map. Opposite orientation, by inversion of the lower abscissa would be a misfit. from real molecules with no obvious orientation, all molecules, including those with ambiguous orientation, have to be used in order to exclude the possibility that the resulting map is an artifact. Most of the heat-denaturation experiments were done at temperatures where about four small strand-separations per molecule became visible, in the hope that their positions would be more precise. This did not prove correct; rather, an average of 5 to 15 sites per molecule, as shown in Figure 4, seems to optimize pattern recognition. An inspection of Figures 3 and 4 suggests th8t denaturation of T7M DNA in the presence of formaldehyde is a multmucleated process, similar to that for X DNA. However, when formaldehyde is absent or at very low ionic strength (09016), heatdenaturation of X DNA w8s reported by Fuke, Wada & Tomizawa (1970) to progress inward from one particular end of the molecule in a zipper-like ftbshion. The results of this work, based on electron microscopy alone, permit the following stetement :

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At least 90% of an vitro synthesized RNA is initiated close to one specific end of the linear T7 DNA molecule. Defining this end to be at fractional length 0 and the other end at 1, then the A + T-rich sites of strand separation which appear after partial heat- or alkali-denaturation in the presence of formaldehyde are found at average fractional lengths O-01, O-16, 0.21, O-26, O-33, O-42, 0*46,0-57, 063, O-66, O-86,O-91 and O-99. The terms “left” and “right” are convenient abbreviations of a deteotable asym metric feature and have been arbitrarily committed to the genetio map. The genetio map of T7 is transcribed from left to right (Summers & Szybalski, 1968; Studier & Maizel, 1969). The left end of the genetic map is thus equivalent to the fractional length 0. Compared with physical mapping by deletion mutants, denaturation mapping requires no second (mutant) DNA. In Figure 11 the results of this work are summarized together with information on initiation and termination of early RNA synthesis in vivo and in vitro (Davis & Hyman, 1970; Hyman, 1971), on the DNA strand which serves as a template and on the direction of transcription after infection (Summers & Szybalski, 1968 ; Studier t Maizel, 1969) and on terminal redundancy (Ritohie et al., 1967). A + T-rich sites I

III

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initiation site FractIonal I 00 I-

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Fxa. 11. A model of the T7M DNA moleoule showing some 8vaileble infornwtion (see text) inoluding the position of A + T-rioh sites reported here. Prominent site8 8re indiccrted by heevy lines.

A partial-denaturation map of T7 DNA essentially similar to ours has been established independently by Wolfson, Dressier & Magazin (1971). They applied it to show that the Y-shaped T7 DNA molecules found in infected E. wli cells are intermediates of replication but not of recombination. In a subsequent paper (Dressler, Wolfson & Magazin, 1972), evidence was given that replication proceeds bidirectionally and originates within the left-half of the T7 DNA at fractional length 0.17. This location is probably indistinguishable from the major A + T-rich site at fractional length O-16. We thank &I& B. Bruton and Mr L. Lewis, Jr. for their careful technical 88&&nce, Drs H. Bremer, K. MiiUer, and P. Witoneky for helpful discussions and generous gifts of

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RNA polymerase and Dr H. Hendrickson for TS DNA. This work was supported by National Science Foundation researoh grants GB6837 and GB14094 and by U.S. Public Health Service grants, Career Development Award GM34964 and reseclrch grant GM13234.

REFERENCES Bourguignon, M. F. (1968). B&him. bioplvya. Acta, 166, 242. Bujard, H. (1970). J. MO,!. Bid. 49, 126. Crothers, D. M. (1908). Biq3oZymm3, 6, 1391. Davis, R. W. & Hyman, R. W. (1970). Cold Sp. Harb. Symp. Quant. BioZ. 33,269. Doerfder, W. & Kleinschmidt, A. K. (1970). J. Mol. BioZ. SO, 679. Dressier, D., Wolfson, J. & Magazin, M. (1972). Proc. Nat. Acad. Sci., Wmh. 69, 998. Fslkow, S. & Cowie, D. B. (1968). J. Bad. 96,777. Follett, E. A. Cl. & Crawford, L. V. (1967). J. Mol. B&Z. 28,461. Follett, E. A. C. & Crewford, L. V. (1968). J. Mol. BioZ. 34, 565. Puke, M., Wsda, A. & Tomizawa, J-I. (1970). J. Mot. BioZ. 51, 255. Hyman, R. W. (1971). J. Mol. BioZ. 61, 369. Inman, R. B. (1966). J. Mol. BioZ. 18, 464. liman, R. B. (1967). J. Mol. BioZ. 28, 103. Inman, R. B. & Bertani, G. (1969). J. Mol. BioZ. 44, 533. Inman, R. B. & S&n&, M. (1970). J. Mol. BioZ. 49, 93. Lang, D. (1970). J. Mol. BioZ. 54, 557. Lang, D. (1971). Phil. Tram. Roy. Sot. B, 261, 161. Lang, D., Bujard, H., Wolff, B. t Russell, D. (1967). J. Mol. BioZ. 23, 163. Lang, D. AZMitani, M. (1970). Biqwlynwrs, 9, 373. Owen, R. J., Hill, L. R. & Lapage, S. P. (1969). BiopoZymm, 7, 503. Ritchie, D. A., Thomas, C. A., Jr., MacHattie, L. A. & Wensink, P. C. (1967). J. Mol. Biol. 23, 365. S&n6s, M. & liunsn, R. B. (1970). J. Mol. Biol. 51, 61. Schn&, M. & Inmm, R. B. (1971). J. Mol. BioZ. 55,31. Studier, F. W. & Msizel, J. V., Jr. (1969). FiroZogy, 89, 575, Summers, W. C. & Szybalski, W. (1968). Virology, 34, 9. Ssybalski, W. & Mennigmam, H.-D. (1962). An&t. B&hem, 3,267. Wehrli, W., Kniisel, F., S&mid, K. & Staehelin, M. (1968). Pmt. Nat. Acud. Sci., Wmh. 61, 667. Wensink, P. C. L Brown, D. D. (1971). J. Mol. B&Z. 60,235. Wolfson, J., Dressier, D. & Megasin, M. (1971). Pmt. Nat. Acud. Sci., Wwh. 09, 499. Wyatt, G. R. & Cohen, S. S. (1963). Biochm. J. 66, 774.