New map of bacteriophage lambda DNA.

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(1)Vol. 33, No. 1 JOURNAL OF VIROLOGY, Jan. 1980, p. 390-400 0022-538X/80/01-0390/11$02.00/0 New Map of Bacteriophage Lambda DNAt DONNA L. DANIELS, JEFFREY R. DE WET,4 AND FREDERICK R. BLATTNER* Laboratory of Genetics, University of Wisconsin-Madison, Madison, Wisconsin 53706 Restriction enzymes have proved to be useful for the physical dissection of the genomes of organisms ranging from bacteriophages to mammals. These endonucleases have been used to map and compare genomes, to produce DNA fragments for DNA sequencing, and to construct recombinant DNA molecules. Maps are useful both for the isolation of defined fragments of the genome and in the analysis of hybrid chromosomes and deletion mutants. Cleavage maps of bacteriophage A have been published for restriction endonucleases cutting with 14 different recognition specificities (1-4, 17, 18, 21, 23, 26, 31, 33, 36, 38, 51, 56), and incomplete mapping information is available for a number of others (5, 8, 23, 48). Attempts to collate the various maps led to discrepancies, inconsistencies, and uncertainties. This was not unexpected, since the different maps were made in different laboratories with different methods of measuring fragment sizes and different length standards. To resolve these problern and produce a single consistent map, it was necessary to obtain more fragment measurements and new information from multiple enzyme digestions. In this paper we present a unified map of phage lambda, which includes 11 enzymes for which maps have been published previously tion on the relative order and precise locations of cut sites that was not previously available. This map of lambda formed an indispensable basis for the mapping of the Charon vectors in the accompanying paper (9). MATERIALS AND METHODS DNA. DNA was prepared by phenol extraction of lambda phage that had been purified by two steps of centrifugation in CsCl gradients (55). OX174 RF DNA was purchased from New England Biolabs. Plasmid DNA was prepared as described previously (20). Ac72 was obtained from W. Szybalski. Restriction endonucleases. EcoRI isolation was as described previously (55). All other restriction endonucleases were purchased from New England Biolabs. DNA was digested to completion at 370C in 6 mM MgCl2-6 mM Tris-hydrochloride (pH 7.4)-6 mM ,B-mercaptoethanol. BamHI and KpnI digestions were done at 50 mM NaCl. Sall and PvuI digestions were done at 100 mM NaCl. Double digestions were done at the conditions of the enzyme requiring the most salt. Gel electrophoresis. Agarose gel electrophoresis was done on horizontal slabs as described by Shinnick et al. (46). Various agarose concentrations were employed (0.7 to 1.2%). Vertical acrylamide slab gels (5, 7.5, or 10%) were employed to resolve low-molecularweight DNA fragments, as described previously (27). Fragment size measurement. Three different mixture size standards were used in this work. (AvaI, BamHI, BglIl, EcoRI, HindIII, KpnI, marker 1 contained EcoRI digests of three phage SmaI, SalI, SstI, XbaI, and XhoI) and 1 new Mixture DNAs and one plasmid DNA (Acb2, AcIKHlOOnin5, PvuI. enzyme, Charon 10, pKDT3) (Fig. 1); mixture 2 contained a To produce this map, 41 different restriction HpaII-EcoRI double digest and a HpaII digest of enzyme target sites were precisely located rela- pKDT3 (8); and mixture 3 contained PstI, HpaII, and tive to one another. Although all of these enzyme HpaI digests of 4X174 RF DNA. The calibration of cut sites had been mapped previously except for the size standards is described below. Distances of the PvuI sites, our new map contains informa- migration of marker bands were plotted versus the log of the length of the marker. In linear regions a best-fit t Paper no. 2385 from the Laboratory of Genetics, College line was determined by the least-squares method. Sizes of Agricultural and Life Sciences, University of Wisconsinof unknown fragments were computed from the regresMadison. : Present address: Department of Biology, University of sion line or, in nonlinear regions, read from the plot. Map construction. Map coordinates were chosen California, San Diego, CA 92037. 390 Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest A map of bacteriophage lambda was constructed, including accurate positions for all 41 cut sites made by 12 different restriction enzymes. Over 100 fragments from single, multiple, and partial enzyme digestions were measured versus standards that were calibrated with respect to DNA molecules of known sequence. The data were subjected to least-squares analysis to assign map coordinates. In no case did a fragment size predicted from the map differ from the measurement of the fragment by more than ±5%. This low error rate was consistent in all size ranges of fragments. The total length of lambda was calculated as 49,133 nucleotide pairs. This probably is accurate to within 500 base pairs.

(2) MAP OF BACTERIOPHAGE X VOL. 33, 1980 391 ORIGIN A A B pKDT3 Xcb2 L L K - C IOI 0 D - A . G E J E F G H . L - - J CHARON 10 L I H NM184 F L - M B N 0 ORIGIN B iiA oo _ - pKDT3 1L (CXpF,IKNR.STUV) RI A lH I I 0 D L _~t RI RI I imm434ori?A pVH51 J I I 0mm op Q R T -u FIG. 1. DNA electrophoresis size standards. Mixture 1 is a mixture of EcoRI digests of three phage DNAs (Acb2, AcIKHIOXnin5, and Charon 10) and one plasmid DNA (pKDT3). (A) EcoRI restriction map of the DNAs and a stimulation of the positions of bands on a 1% agarose gel of mixture 1. The dotted lines indicate the corresponding EcoRI sites in these lambda derivatives. Mixture 2 is a mixture of a HpaII digest and an EcoRI-HpaII double digest ofpKDT3 DNA. (B) Map of EcoRI and HpaII sites on pKDT3 and a stimulation of a 7.5% acrylamide gel of mixture 2. Fragments B, D, H, L, and 0 of mixture 2 have been sequenced. so as to minimize the sum of the squares of the fractional deviations of measured fragment sizes from sizes predicted by the map coordinates. The computer program has been described previously (43). RESULTS The complete restriction map of Ac72 for 12 enzymes, along with other genes and sites on the A molecule, is shown in Fig. 2. Derivation of this involved five steps: (i) establishment of calibration standards for measurement of DNA fragment sizes by gel electrophoresis, (ii) careful measurement of a large number of fragments from single and multiple digestions of lambda DNA, (iii) determination of the order of the restriction sites, (iv) assignment of map coordinates, and (v) correlation of the restriction map with the genetic map of lambda. map Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest ~lK I ~~l L XKHIOOnn5 L

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(4) MAP OF BACTERIOPHAGE A VOL. 33, 1980 * -0 w CO) 0 n o mobility of the sequenced 4X174 and pBR322 DNAs was linear for 31 fragments in the range from 67 to 603 base pairs. There was some scatter in the data, with nine fragments deviating from the regression line by 5 to 10%. This scatter may represent compositional effects or random variation, but use of the regression line avoids introducing systematic errors into the calibration of the marker mixture. Table 1 shows the sizes adopted for mixture 2 as a calibration standard, as computed from the regression line. Several of the components of mixture 2 have been sequenced recently (8, 16, 28, 44) (Table 1). The actual sizes of the H, 0, D, and L fragments agree with our calibration within 5%. The B fragment, however, definitely runs anomalously on gels. The deviation of 13.5% is reproducibly greater than we have observed for any other fragment. This fragment contains the X origin of replication. We found that several small fragments containing this region run as though they are 70 to 100 base pairs larger. There is apparently something about this region that lowers the mobility in acrylamide. No such effect on agarose gels was noted for the larger fragments containing this region (e.g., fragment 0 of mixture 1). When mixture 2 was used, the pseudosize of 504 base pairs was used for fragment B instead of its actual size (436 base pairs), or else the fragment was left out of the plot. pKDT3 MARKER MIX - c-Lr Y CL I be C IX Hae III pBR322 - -- oX MARKER 5386 Pst I 4994 HPa I 3730 Hpa I 2748 -_ - 872 527 461 403 309 --603 _ _ - HpaII 1697 Hpa II 1264 Hpa I 1353 1078 -- R2?Hpall 110 90 310 281 -271 --234 -194 - 76 118 -72 67-- 34 392 Hpa I 26-- - A FIG. 3. Calibration of sizes offragments in the markers. EcoRI digests ofAcb2, AcIKH100nin5, and pKDT3 were mixed with mixture 3 (a mixture of PstI, HpaI, and HpaII digests of OXl 74 DNA) and electrophoresed through 1% agarose gels. Sizes of fragments in mixture 3 are precisely known from sequence and are shown in (A). Sizes of bands H, I, J, K, L, M, N, and 0 of mixture I were determined from this gel as described in the text. (B) 7.5% acrylamide gels of an EcoRI-HpaII digest ofpBR322, a HaeIII digest of <X1 74 RF, and mixture 2. pBR322 and pX174 have been completely sequenced. Sizes of fragments in mixture 2 uere determined as described in the text. Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest Calibration of standards. At the time this work was begun, well-calibrated absolute-length standards for gel electrophoresis spanning the needed 100- to 20,000-base pair range were not available. We therefore chose to include a large number of well-resolved uncalibrated marker fragments with every gel, assuming that they could be calibrated eventually. Two marker mixtures were devised; mixture 1 covered large ranges, and mixture 2 covered small ranges of fragment size (Fig. 1). Mixture 1 was generally used on agarose gels, and mixture 2 was used on acrylamide gels. The gel systems which we chose were the high-salt-agarose system of Shinnick et. al. (46) and the 25% glycerol-moderate-salt-acrylamide gel system of Moore et. al. (27). Low-voltage gradients were used, and the temperature of the gels did not rise perceptibly above room temperature. With these precautions we did not have serious problems with composition dependence of fragment measurements, as has been described for another gel system (36). Calibration of mixture 2 was possible when the 4X174 and pBR322 DNA sequences became available (15, 39, 40, 50). Figure 3 shows one of the calibration gels. In this experiment restriction enzyme digests of (AX174 and pBR322 DNAs were electrophoresed versus mixture 2. The plot of the log of chain length versus electrophoretic 393

(5) 394 DANIELS, DE WET, AND BLATTNER J. VIROL. TABLE 1. Summary of sizes of standards in marker mixtures 1 and 2' Marker mixture 1 Fragof of mtx- Method Length by qe bynseh q byqse- 21,485 19,800 12,400 9,570 7,560 6,760 5,950 5,690 4,925 4,590 3,900 3,600 3,110 1,990 1,310 a a a a b a c c c c c c c c 21,728 21,569 19,800 12,400 9,800 7,469 7,125 7,536 5,917 5,480 4,753 5,617 5,127 4,612 5,901 5,681 4,845 3,298 1,280' 3,807 3,601 A+H B+G A B C D E F G H I J K L M N 0 P Q R S T U V 931 741 709 504 416 401 361 304 237 223 199 162 145 128 113 105 100 89 71 65 50 44 34 26 c,d c, d c c c c 436f 403 c c c c c 217' c c c c 123' c c c c c 95' c c c c a Sizes of fragments in the mixtures used as standards in this work are shown, and the methods of calibration are indicated. b Fragments are identified as in Fig. 1. Sizes were obtained as described in the text. For comparison, the sizes obtained by sequencing or by independent measurement by two different methods are indicated for some of the fragments. d Methods of calibration: a, mapping studies; b, measurement on agarose gels relative to other fragments of the marker; c, measurement on acrylamide or agarose gels relative to digests of 4X174 or pBR322 DNA; d, sum of subfragments. Lengths by measurements on gels, using multiples of 4X174 DNA as standards. f Electron microscope measurements relative to lambda (x485 base pairs per 1% of X) (6, 12, 49, 52). g See reference 28. h See references 16, 28, 34, 37, 41, and 44. 'See reference 16. Mixture 1 contains eight fragments small enough to be compared directly to restriction digests of 45X174, namely fragments H, I, J, K, L, M, N, and 0. This calibration gel is shown in Fig. 3. Recently, the sequence of fragment 0 has been completed (16, 28, 44), showing that our estimated size is accurate within 3%. Fragment N is also nearly completely sequenced and will provide additional calibration of mixture 1. The larger fragments of mixture 1 could not be calibrated by direct comparison to sequenced molecules since such molecules are not available. For calibration of these markers, three approaches were available: (i) summation of subfragments, (ii) electrophoretic comparison with concatenated sequenced molecules, and (iii) electron microscopy. The primary calibration of these fragments was done by the first method and verified by the other methods (Table 1). What was done was to measure subfragments versus calibrated standards in the small size range and to combine the measurements by the least-squares mapping technique. In this way sizes for fragments E and G were obtained by measuring 23 subfragments of AM72 DNA obtained by EcoRI, BamHI, XhoI, BglII, and HindIII digestions. Fragments B, C, and D were sized by combining measurements of 35 subfragments of Charon 10 DNA obtained by BglII, BamHI, KpnI, EcoRI, SstI, XbaI, and Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest A B C D E F G H I J K L M N 0 Initial ofbracalicalibration size' tion Marker mixture 2 Gel MeasureFragment'bInitial measure- ment by Measure- Revised of mixture cali- Method Length ment vs Thomas bracaliment 2 tion ofbramultiples and Allet by et lengthiby t of size tion q Davis . (3) (bp) ,X174e (45)f

(6) VOL. 33, 1980 395 ment on different gels usually agreed within 2%. The greatest variability occurred with the BamHI left end (fragment 1-4). Seven different measurements gave a mean of 5,627 bp (standard deviation, 0.033). This variability seems to be a function of that particular fragment rather than of the gel system. A typical fragment (fragment 39-42) gave a mean of 5,279 bp standard deviation, 0.008) when measured on six different gels. The longest fragment that we measured was BamHI-KpnI fragment 4-6. This fragment was measured by using a partial EcoRI digest of lambda DNA as well as mixture 1 as standard. Assignment of order of sites. Published maps for 11 of the 12 enzymes served as the starting point for our map. The order of most of the restriction sites for the 12 enzymes could be determined by inspection from fragment sizes. Whenever sites of two enzymes were closer together than 300 base pairs, we used a third enzyme to establish the order. For example, the BamHI and BglII sites (Fig. 2, sites 11 and 12) were shown by electrophoresis of a BamHIBglII double digest to be 91 base pairs apart. To determine the order, EcoRI-BamHI and EcoRIBglII double digests were electrophoresed in adjacent channels. The EcoRI-BamHI fragment was definitely shorter; thus, the orientation was determined to be EcoRI, BamHI, BglII. Once the order of all cut sites was known, each site was assigned a number from 1 (left end) to 43 (right end). Site 21 and sites 32 through 38 are within sequenced regions. Site 33 is the HindIII site introduced by the ind- mutation, whose location is known from DNA sequences of the cI ind+ and ind- proteins (R. T. Sauer and M. Ptashne, personal communication). Although Ac72 does not contain this site, it was included in this analysis because several of the Charon phage vectors contain it (9). Assignment of the map coordinates. The final restriction map is presented in Fig. 2. The positions of restriction sites were assigned from fragment sizes by minimization of the sum of the squares of the fractional deviations between measured fragment sizes and sizes predicted by subtracting map coordinates. This was done with a computer program (43) whose input and output are shown in Table 2. In addition to the map coordinates of each site presented in Fig. 2, the program listed the measured size of each fragment, the computed size of the map interval, and the percent change needed to accommodate the best fit. None of these adjustments exceeded 5%. The fact that the percent deviations are not correlated with fragment size indicates that the marker mixture calibration is not skewed with respect to size range. In the case of sequenced Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest PvuI digestions. Fragment F was measured by interpolation between fragments D and E. Sizes of fragments B, C, and D were confirmed by comparison on agarose gels with multiple 4)X174 RF DNA molecules. To obtain these markers, a preparation of OX174 RFII DNA was digested with EcoRI and then ligated at a high concentration to produce multiple-length 4X molecules. Agreement was better than ±3% (Table 1). Fragments A, E, G, H, I, and L of mixture 1, which are the EcoRI fragments of lambda, have been measured previously by Thomas and Davis (51) and Allet et al. (3) by electron microscopy. Table 1 shows a comparison of the electron micrographic and gel-derived sizes for these fragments after conversion of the former from percent lambda to base pairs. Agreement between the average of the electron microscope measurement and the gel-based calibration is +5% for all fragments. For fragment L, the right end of A, the Thomas-Davis measurement is 7% lower, whereas the measurement of Allet et al. is 7% higher than the gel-derived measurement, indicating, perhaps, that this fragment is difficult to measure by microscopy. Thomas and Davis pointed out the discrepancy between their electron micrographic and gel-based measurements for that fragment, but failure to take account of this has been a major cause of discrepancies in many published maps of DNA molecules in which the EcoRI digest of lambda, as calibrated by electron microscopy by Thomas and Davis, served as the marker. In summary, we are satisfied that mixtures 1 and 2, calibrated as in Table 1, form an adequate set of standards for gel measurements spanning the wide size range needed to map lambda. Clearly, the markers can be improved by refinement, especially in the large range. For example, the EcoRI fragment sizes of lambda obtained by subtracting coordinates on the final map (Table 1) are slightly different from the initial calibration. These differences, however, are minor. Measurement of fragments for the lambda map. To derive the lambda map, 120 fragments were measured by gel electrophoresis. Most measurements were on digests of Ac72 DNA. Digestion with some enzymes produced bands which were unresolvable. In these cases, the fragments were measured from digests of deletion or substitution mutants of lambda or of lambda fragments cloned in pBR322. All measurements are shown in Table 2. Fragments are identified by the site numbers of their endpoints in Fig. 2. Individual measurements on gels were quite reproducible. Measurements of the same frag- MAP OF BACTERIOPHAGE A

(7) 396 DANIELS, DE WET, AND BLATTNER J. VIROL. TABLE 2. Measured fragment sizesa Computed % Change MeuredCr% Fragment Change Fragment size Fragment_ __size __Change_ _Fragment_size size__ Measured 482 4,810 5,627 4,965 716 6,550 11,550 5,420 9,300 1,529 2,760 3,850 4,000 7,980 1,695 1,830 3,020 3,630 225 1,380 6,910 1,130 1,230 1,870 3,330 3,500 4,520 4,967 82 810 3,750 5,550 5,410 745 3,670 5,310 1,933 263 695 1,625 1,700 402 1,130 1,310 765 970 402 1,130 1,310 765 970 2,340 217 1,580 1,360 1,870 1,885 5,600 5,690 482 4,810 5,526 5,044 716 6,481 11,765 5,284 9,562 1,521 2,757 3,891 3,973 7,749 1,637 1,861 2,995 3,805 224 1,358 6,909 1,134 1,216 1,944 3,223 3,488 4,629 4,845 82 810 3,711 5,551 5,641 728 3,629 5,559 1,945 265 666 1,622 1,769 401 1,141 1,357 740 956 401 1,141 1,357 740 956 2,286 216 1,546 1,330 1,840 1,930 5,552 5,681 0.1 0.0 -1.8 1.6 0 -1.1 1.8 -2.6 2.7 -0.5 -0.1 1.0 -0.7 -3.0 -3.5 1.7 -0.8 4.6 -0.2 0.6 0 0.3 -1.2 3.8 -3.3 -0.4 2.3 -2.5 -0.1 0 -1.1 0 4.1 -2.4 -1.1 4.5 0.6 0.7 -4.4 -0.2 3.9 -0.3 1.0 3.5 -3.3 -1.4 -0.3 1.0 3.5 -3.3 -1.4 -2.3 -0.3 -2.2 -2.2 -1.7 2.4 -0.9 -0.1 19-22 19-24 19-27 19-30 20-21 20-22 20-23 20-24 20-25 20-26 20-27 21-22 21-23 22-23 22-24 22-28 23-24 23-27 23-28 23-31 24-25 24-26 24-27 24-28 24-29 24-30 24-31 25-26 25-31 25-38 26-27 26-28 26-31 26-38 27-28 27-30 27-31 27-35 27-38 28-29 28-31 28-35 28-38 28-39 28-40 29-31 I 29-34 30-38 31-32 32-39 32-40 35-39 37-40 38-39 38-40 38-41 39-40 39-41 39-42 1,730 5,510 7,350 9,500 517 610 4,030 4,360 5,370 5,910 6,000 91 3,835 3,765 3,750 6,487 130 1,955 3,020 5,205 1,028 1,545 1,845 2,880 4,055 4,140 5,170 506 4,200 6,580 238 1,290 3,730 5,980 964 2,300 3,450 4,787 5,690 1,240 2,425 3,760 4,750 5,300 7,280 1,225 2,460 3,440 585 2,405 4,400 1,695 3,000 710 2,690 5,000 1,875 4,143 5,279 1,783 5,534 7,335 9,636 510 600 4,222 4,351 5,399 5,911 6,151 90 3,712 3,622 3,751 6,556 130 1,930 2,934 5,371 1,048 1,560 1,801 2,805 4,030 4,102 5,242 512 4,194 6,488 241 1,245 3,682 5,976 1,004 2,301 3,441 4,781 5,735 1,225 2,437 3,777 4,731 5,448 7,346 1,212 2,441 3,434 585 2,426 4,324 1,671 2,969 717 2,615 5,055 1,898 4,338 5,185 3.0 0.5 -0.2 1.4 -1.5 -1.7 4.5 -0.2 0.5 0 2.5 -0.3 -3.3 -4.0 0 1.0 0 -1.3 -2.9 3.1 1.9 0.9 -2.5 -2.7 -0.6 -0.9 1.4 1.1 -0.1 -1.4 1.4 -3.6 -1.3 -0.1 4.0 0 -0.3 -0.1 0.8 -1.2 0.5 0.4 -0.4 2.7 0.9 -1.0 -0.8 -0.2 -0.1 0.9 -1.8 -1.4 -1.0 1.0 -2.9 1.1 1.2 4.5 -1.8 Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest 1-2 1-3 1-4 2-4 3-4 4-5 4-6 5-6 5-10 6-7 7-10 7-11 7-12 7-19 8-9 8-10 8-11 8-13 9-10 9-11 9-21 10-11 10-12 10-13 10-14 10-15 10-17 10-18 11-12 11-13 11-18 11-21 11-22 12-13 12-18 12-22 13-16 14-15 14-16 14-18 14-19 15-16 15-17 15-18 16-17 16-18 15-16 15-17 15-18 16-17 16-18 16-20 17-18 17-20 18-20 18-21 18-22 18-23 18-24

(8) MAP OF BACTERIOPHAGE A VOL. 33, 1980 397 TABLE 2-Continued Fragment Measured size 2,430 3,370 7,120 Computed size 2,440 3,287 % Change Fragment Measured size Computed % Change size 630 1,170 1,230 1,584 0.4 33-35 -2.5 -3.4 0.8 1.8 0.6 33-36 33-37 33-38 regions, we input enough redundant, perfectly consistent data to avoid adjustment of these fragment sizes (Table 2). Although previous lambda maps have employed percent or fractional coordinates, we decided for this map to switch to nucleotide pair coordinates. This simplifies the integration of DNA sequence data with other types of mapping data. A problem, however, is the assignment of a coordinate reference. Rather than assigning coordinate 0 to the poorly mapped left end, the EcoRI site in gene O (Fig. 2, site 38) was designated 40,000. This site is in the area of lambda that has been mapped most intensively and is within the longest section that has been sequenced. As mapping of the rest of lambda is improved, renumbering of this sequenced region will not be required. On the other hand, the current position of the left end becomes 369, which is inconvenient for some purposes. This will certainly change a number of times as mapping improves. Eventually, when the A sequence is complete, the molecule can be renumbered. The accuracy of the map depends on the accuracy of individual fragment measurements, as mentioned above, and on the number of different fragments measured which impinge on each map coordinate. The most accurate region of the map is in the middle of the genome. Every restriction interval between sites 10 and 38 is spanned by at least 10 measurements, and most regions are spanned by many more than this. The least accurate region of the map is in the left arm between sites 4 and 6. The interval 630 1,170 between sites 4 and 5 is spanned by only two measurements, and the interval between sites 5 and 6 is spanned by only three. Since these are all large measurements employing the largesized region of the marker mixture, the absolute error may be as much as 400 base pairs. Although we report map coordinates to single base pairs, it should be emphasized that this does not indicate the accuracy of each coordinate. Rather, in using this map, one should consider the accuracy of the difference between coordinates. It would be conservative to assume that these differences reflect the actual size of fragments within 10% for any region of this map. If the map coordinate of site 1 is subtracted from the map coordinate of site 43, a size of 49,133 bp is obtained for that fragment, which in this case is the total size of lambda. Because the marker mixture calibrations rest ultimately on sequenced DNA fragments, this can be viewed as an absolute (although indirect) measurement of X. Perhaps fortuitously, the measurement agrees well with recent estimates for the size of lambda; values of 48,100 ± 1.8% (52), 48,300 (49), 49,400 + 300 (6), and 48,000 (13) have been obtained by electron microscope measurements relative to OX174 RF DNA. This agreement lends support to our conclusions about the overall accuracy of the map. Correlation with the genetic map. A few restriction sites have been shown to be within A structural genes by DNA sequence. All of cI, cro, cII, and 0 have been sequenced (8, 19, 28, 33, 34, 41). These genes contain sites 32 and 33 (cI), 34 and 35 (cro), and 37 and 38 (0). Sites 21 Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest 0 0 0 6,887 1,230 840 847 0 1,584 34-35 0 111 1ll 4,367 4,447 34-36 651 651 4,900 0 4,929 -0.3 711 34-37 711 0 3,613 3,600 125 125 0 34-38 1,065 1,065 0 644 644 0 35-36 540 540 0 755 755 0 35-37 600 600 0 0 35-38 954 954 1,295 1,295 0 0 36-37 60 60 1,355 0 1,355 0 36-38 414 414 1,709 1,709 0 0 354 354 519 0 519 37-38 a Each fragment measured is identified by the site numbers of its endpoints (Fig. 2). Also shown are measured fragment sizes from digests of Xc72 DNA, sizes of the fragments as obtained by subtraction from the map, and the percent deviations of the sizes obtained by subtraction from the map from the measured fragment sizes. If a measured fragment size has more than three significant digits, it is the average of measurements from two or more different gels. The last 21 entries are from a sequenced region between sites 32 and 38 (8, 19,28, 34,41,44, 45), and measured size is that determined by sequence. 40-41 40-42 40-43 41-42 41-43 41- 2 42-43 32-33 32-34 32-35 32-36 32-37 32-38 33-34

(9) 398 DANIELS, DE WET, AND BLATTNER points based on protein sizing is at present quite tenuous, we have not presented actual coordinates for gene boundaries in Fig. 2. DISCUSSION The map presented here can be viewed as a map of wild-type lambda, since Ac72 is a clear point mutant of Apapa derived in a single step by Kaiser (22). Among geneticists Apapa is perhaps the most generally accepted wild-type strain (10). We have verified that our sample of Xc72 has the same buoyant density as Apapa (12). Comparison of our data on Ac72 with data from other laboratories showed no obvious strain differences for the 12 enzymes mapped in Fig. 2. However, one should be alert to the possibility of strain differences, since several strains have been called wild type (10) and various derivatives of these strains have been used as a wild type in experiments (12, 42, 47). One of the strains frequently used for restriction mapping studies is AcI857S7, which was derived from Apapa by a series of crosses and steps of mutagenesis, including a significant contribution from the heavily mutagenized stock Y10(X) (10). Nichols and Donelson found 10% sequence divergence between the right ends of AcI857prmll6S7 and XcI857S7 (30). Williams and Blattner found a new EcoRI site arising apparently by unselected spontaneous mutation in A Charon 4A (54). It is not generally possible to decide which strain differences can be ascribed to recent divergence and which can be traced to the progenitors of lambda. Another source of variation in restriction patterns is the state of methylation of the DNA. It is well known, for example, that EcoRII and MboI are particularly affected by adventitious methylation of DNA grown in Escherichia coli strains (27). However, we were surprised to find a single new HpaI site located at about position 15,500 when A phage was grown on strain K803, which lacks the K methylation activity, as compared with X phage grown on strain K802, which has the K methylation activity. We note that the proposed K recognition site, AACXXXXXXGTGC (24), can overlap with the HpaI recognition site, GTTAAC (14), so as to share a methylated adenine residue. If this interpretation is correct, it may serve to map one of the K sites on A. This solves a dfficult mapping problem since the K system is of class I and thus the enzyme cuts at a site far from the restriction site. These points illustrate some of the difficulties and caveats that go with any restriction map of Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest and 22 are in the int gene (R. Hoess and A. Landy, personal communication). Site 36 may also be in gene 0, but this is not clear since it is unknown which of two possible ribosome binding sites is the beginning of the 0 gene. At a lower level of certainty, sites 6 and 7 are probably within J: since this is such a large peptide, such assignments are within the resolution of the mapping techniques. Site 24 is probably within gene exo (51). An extensive map of lambda has been prepared by Szybalski and Szybalski; this map is based on an electron microscopic analysis of heteroduplexes between lambda and its deletion and substitution mutants (E. H. Szybalski and W. Szybalski, Gene, in press). Echols and Murialdo have also recently published a map showing their best estimates for the endpoints of structural genes based on genetic data, electron micrographic data, and sizes of protein products (11). We have attempted in Fig. 2 to integrate these two maps with the new restriction map. The first step was reconciliation of the scales of the three maps, especially the locations of landmarks att and imm434 relative to the left and right ends. The map of Szybalski and Szybalski and the Echols-Murialdo map by convention assigned att as 57.3%, the left and right boundaries of imm434 as 73.5 and 79.3%, respectively, and the ends as 0 and 100%. All of these points can be located precisely on the restriction map. The att site has been sequenced, and the sequenced region includes AvaI site 21. Thus, the center of the core of att was assigned coordinate 28,466. The left end of imm434 is known to be within a few base pairs of PL and within a sequenced region including BglII site 29. Thus, we assigned it coordinate 36,363. The right end of imm434 was assigned 39,076, based on the direct sequence analysis of X and Ximm434 DNA (16, 37, 44). These landmarks plus the two ends gave five points of correspondence among the three maps. We then assigned the other points by linear interpolation. The scale factors required for the various intervals ranged from 488 to 496 base pairs per 1% of A. These are all within the range of measurements of the total length of lambda. Assignment of the proteins on the left arm of lambda was based directly on the EcholsMurialdo map except, in accordance with recent data (H. Murialdo and J. Shaw, personal communication), Nu3 was placed within C, and of course the length of lambda was increased to currently accepted values. These changes go a long way toward alleviating the gene crowding discussed by Echols and Murialdo. However, since the possibility of overlapping genes has not been explored thoroughly and placement of end- J. VIROL.

(10) MAP OF BACTERIOPHAGE A VOL. 33, 1980 large DNA molecule. In another paper (9) extend the mapping to the Charon phages. a we ACKNOWLEDGMENTS We thank Katherine Denniston-Thompson and Pat AuYeung for running gels of HpaI digests, John L. Schroeder for feeding the computer, and E. Szybalski and W. Szybalski for their electron microscope maps and many helpful discussions. This research was supported in part by Public Health Service training grant GM01733 from the National Institutes of Health (to D.L.D.) and by Public Health Service grant GM12182 from the National Institutes of Health (to F.R.B.). 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 5:4105. 16. Grosscheld, R., and E. Schwartz. 1979. Nucleotide sequence of the cro-cII-oop region of bacteriophage 434 DNA. Nucleic Acids Res. 6:867. 17. Haggerty, D., and F. R. Schleif. 1976. Location in bacteriophage lambda DNA of cleavage sites of the sitespecific endonuclease from Bacillus amyloliquefaciens H. J. Virol. 18:659-663. 18. Hughes, S. G. 1977. A map of the cleavage sites for endonucleases AvaI in the chromosome of bacteriophage lambda. J. Biochem. 163:503-509. 19. Humayan, M. Z. 1977. DNA sequence at the end of the cI gene in bacteriophage lambda. Nucleic Acids Res. 4: 2137. 20. Humphreys, G. C., G. A. Willshaw, and E. S. Anderson. 1975. A simple method for the preparation of large quantities of pure plasmid DNA. Biochim. Biophys. Acta 383:457-463. 21. James, P. M., D. Sens, W. Natter, and S. K. Moore. 1976. Isolation and characterization of the specialized transducing bacteriophages OdargF and lambda h8OcI857dargF: specific cleavage of arginine transducing deoxyribonucleic acid by the endonucleases EcoRI and SmaR. J. Bacteriol. 126:487-500. 22. Kaiser, A. P. 1957. Mutations in a temperate bacteriophage affecting its ability to lysogenize Escherichia coli. Virology 3:42-61. 23. Kamp, D., R. Kahmann, D. Zipser, and R. J. Roberts. 1977. Mapping of restriction sites in the attachment site region of bacteriophage lambda. Mol. Gen. Genet. 154: 231-248. 24. Kan, N. C., J. A. Lautenberger, M. H. Edgell, and C. A. Hutchinson M. 1979. The nucleotide sequence recognized by the Escherichia coli K12 restriction and modification enzymes. J. Mol. Biol. 130:191-209. 25. Landy, A., and W. Ross. 1977. Viral integration and excision: structure of the lambda att sites. Science 197: 1147-1159. 26. McParland, R. H., L. R. Brown, and G. D. Pearson. 1976. Cleavage of lambda DNA by a site-specific endonuclease from Serratia marcescens. J. Virol. 19:10061011. 27. Moore, D. D., K. Denniston-Thompson, M. E. Furth, B. G. Williams, and F. R. Blattner. 1977. Construction of chimeric phages and plasmids containing the origin of replication of bacteriophage X. Science 198: 1041-1046. 28. Moore, D. D., K. Denniston-Thompson, K. E. Kruger, M. E. Furth, G. B. Williams, D. L. Daniels, and F. R. Blattner. 1978. Dissection and comparative anatomy of the origins of replication of lambdoid phages. Cold Spring Harbor Symp. Quant. Biol. 43:155-163. 29. Murray, K., S. G. Hughes, J. S. Brown, and S. A. Bruce. 1976. Isolation and characterization of two sequence-specific endonucleases from Anabaena variabilis. Biochem. J. 159:317-322. 30. Nichols, P. R., and J. E. Romelson. 1978. 178-Nucleotide sequence surrounding the cos site of bacteriophage lambda DNA J. Virol. 26:429-434. 31. Nosikov, V. V., E. A. Braga, and B. Sain. 1978. Physical mapping of bacteriophage lambda DNA with restriction endonuclease HpaI. Gene 4:69-84. 32. Perricaudet, M., and P. Tiollais. 1975. Defective bacteriophage lambda chromosome: potential vector for DNA fragments obtained after cleavage by Bacillus amyloliquefaciens endonuclease (BamI). FEBS Lett. 57:7-11. 33. Pirrotta, V. 1976. Two restriction endonucleases from Bacillus globiggi. Nucleic Acids Res. 3:1747-1760. 34. Ptashne, M., K. Bachman, M. Z. Humayan, A. Jeffrey, P. Maurer, B. Meyer, and R. T. Sauer. 1976. Autoregulation and function of a repressor in bacterio- Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest LITERATURE CITED I. Bukhari. 1975. Analysis of bacteriophage mu and lambda-mu hybrid DNAs by specific endonucleases. J. Mol. Biol. 92:529-540. Allet, B., P. G. N. Jeppesen, K. J. Katagiri, and H. Delius. 1973. Mapping the DNA fragments produced by cleavage of lambda DNA with endonuclease RI. Nature (London) 241:120-123. Allet, B., K. J. Katagiri, and R. F. Gesteland. 1973. Characterization of polypeptides made in vitro from bacteriophage lambda DNA. J. Mol. Biol. 78:589-600. Allet, B., and R. Solem. 1974. Separation and analysis of promoter sites in bacteriophage lambda DNA by specific endonucleases. J. Mol. Biol. 85:475-484. Blattner, F. R., M. Fiandt, K. K. Hass, P. A. Twose, and W. Szybalski. 1974. Deletions and insertions in the immunity region of coliphage lambda: revised measurement of the promoter-startpoint distance. Virology 62:458-471. Blattner, F. R., B. G. Williams, A. E. Blechl, K. Denniston-Thompson, H. E. Faber, L. A. Furlong, D. J. Grunwald, D. 0. Kiefer, D. D. Moore, J. W. Schumm, E. L. Sheldon, and 0. Smithies. 1977. Charon phages: safer derivatives of phage lambda for DNA cloning. Science 196:161-169. Davidson, N., and W. Szybalski. 1971. Physical and chemical characteristics of lambda DNA, p. 45-82. In A. D. Hershey (ed.), The bacteriophage lambda. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Denniston-Thompson, K., D. D. Moore, K. E. Kruger, M. E. Furth, and F. R. Blattner. 1977. The physical structure of the replication origin of bacteriophage lambda. Science 198:1051-1056. de Wet, J. R., D. L. Daniels, J. L. Schroeder, B. G. Williams, K. Denniston-Thompson, D. D. Moore, and F. R. Blattner. 1980. Restriction maps for twentyone Charon vector phages. J. Virol. 33:401-410. Dove, W. F. 1969. Strains of phage lambda in current use. Virology 38:349-351. Echols, H., and H. Murialdo. 1978. Genetic map of bacteriophage lambda. Microbiol. Rev. 42:577-591. Fiandt, M., A. Honigman, E. G. Rosenvold, and W. Szybalski. 1977. Precise measurement of the b2 deletion in coliphage lambda. Gene 2:289-298. Fiandt, M., Z. Hradecna, H. A. Lozeron, and W. Szybalski. 1971. Electron micrographic mapping of deletions, insertions, inversions, and homologies in the DNAs of coliphages lambda and phi 80, p. 329-354. In A. D. Hershey (ed.), The bacteriophage lambda. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Garfin, D. E., and H. M. Goodman. 1974. Nucleotide sequences of the cleavage sites of two restriction endonucleases from Hemophilus parainfluenzae. Biochem. Biophys. Res. Commun. 59:108-116. Gingeras, T. R., J. P. Milazzo, and R. J. Roberts. 1978. A computer assisted method for the determination of restriction enzyme recognition sites. Nucleic Acids Res. 1. Allet, B., and A. 399

(11) 400 DANIELS, DE WET, AND BLATTNER Hutchison HI, P. M. Slocombe, and M. Smith. 1978. The nucleotide sequence of bacteriophage phi X174. J. Mol. Biol. 125:225-246. 41. Sauer, R. T. 1978. DNA sequence of the bacteriophage lambda cI gene. Nature (London) 276:301-302. 42. Schnoes, M., and R. B. Inman. 1970. The positions of branch points in replicating lambda DNA. J. Mol. Biol. 51:61-73. 43. Schroeder, J. L., and F. R. Blattner. 1978. Least squares method for restriction mapping. Gene 4:167174. 44. Schwartz, E., G. Scherer, G. Hobom, and H. Kossel. 1978. Nucleotide sequence of cro, cII and part of the 0 gene in phage lambda DNA. Nature (London) 272:410414. 45. Sherer, G. 1978. Nucleotide sequence of the 0 gene and of the origin of replication in bacteriophage lambda DNA. Nucleic Acids Res. 5:3141-3155. 46. Shinnick, T. M., E. Lund, 0. Smithies, and F. R. Blattner. 1975. Hybridization of labeled RNA to DNA agarose gels. Nucleic Acids Res. 2:1911-1929. 47. Simon, M. N., R. W. Davis, and N. Davidson. 1971. Heteroduplexes of DNA molecules of lambdoid phages: physical mapping of their base sequence relationships by electron microscopy, p. 313-327. In A. D. Hershey (ed.), The bacteriophage lambda. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 48. Smith, D. I., F. R. Blattner, and J. Davies. 1976. The isolation and partial characterization of new restriction endonuclease from Providencia stuarti. Nucleic Acids Res. 3:343-353. 49. Stuber, D., and H. Bujard. 1977. Electron microscopy of DNA: determination of absolute molecular weights and linear density. Mol. Gen. Genet. 154:299-303. 50. Sutcliffe, G. 1978. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp. Quant. Biol. 43:77-90. 51. Thomas, M., and R. W. Davis. 1975. Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease. J. Mol. Biol. 91:315-328. 52. Vollenweider, H. J., A. James, and W. Szybalski. 1978. Discrete length classes of DNA depend on mode of dehydration. Proc. Natl. Acad. Sci. U.S.A. 75:710714. 53. Wellauer, P. K., R. H. Reeder, D. Carroll, D. D. Brown, A. Duetch, T. Higashinakagawa, and I. B. Dawid. 1974. Amplified ribosomal DNA from Xenopus laevis has heterogeneous spacer lengths. Proc. Natl. Acad. Sci. U.S.A. 71:2823-2827. 54. Williams, B. G., and F. R. Blattner. 1978. Construction and characterization of the hybrid bacteriophage lambda Charon vector phages. J. Virol. 29:555-575. 55. Williams, B. G., F. R. Blattner, S. R. Jaskunas, and M. Nomura. 1977. Insertion DNA from the spc-fus region of E. coli into Charon vector phages. J. Biol. Chem. 252:7344-7354. 56. Wilson, G. A., and F. E. Young. 1975. Isolation of sequence-specific endonuclease (BamI) from Bacillus amyloliquefaciens H. J. Mol. Biol. 97:123-125. Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest phage lambda. Science 194:156-161. 35. Roberts, R. J. 1976. Restriction endonucleases. Crit. Rev. Biochem. 4:123-164. 36. Robinson, L. H., and A. Landy. 1977. HindII, HindIII and HpaI restriction fragment maps of bacteriophage lambda DNA. Gene 2:1-31. 37. Rosenberg, M., D. Court, H. Shimatake, C. Brady, and D. L. Wulff. 1978. The relationship between function and DNA sequence in an intercistronic regulator region in phage lambda. Nature (London) 272:414-421. 38. Rosenvold, E. C., and A. Honigman. 1977. Mapping of AvaI and XmaI cleavage sites in bacteriophages lambda DNA including a new technique of DNA digestion in agarose gels. Gene 2:273-288. 39. Sanger, F., G. M. Air, B. G. Barrell, N. L. Brown, A. R. Coulson, J. C. Fiddes, C. A. Hutchison III, P. M. Slocombe, and M. Smith. 1977. Nucleotide sequence of bacteriophage phi X174 DNA. Nature (London) 265: 687-695. 40. Sanger, F., A. R. Coulson, T. Friedmann, G. M. Air, B. G. Barrell, N. L. Brown, J. C. Fiddes, C. A. J. VIROL.

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